DATA SHEET

Title: Molecular mechanisms regulating oxygen transport and consumption in high

altitude and hibernating mammals

Subtitle: PhD Dissertation

Author: Inge Grønvall Revsbech

Affiniation: Section for Zoophysiology, Department of Bioscience, Aarhus University

Publisher: Aarhus University

Publication year: 2016

Cite as: Revsbech IG. (2016) Molecular mechanisms regulating oxygen transport and

consumption in high altitude and hibernating mammals.

PhD dissertation. Department of Bioscience, Aarhus University, Denmark

Illustrations: Illustrations by the author unless otherwise noted

Keywords: Hemoglobin; hibernator; high altitude; adaptation; acclimatization Bohr effect;

2,3-diphosphoglycerate; hydrogen sulfide; nitric oxide

Supervisor: Professor Angela Fago, Zoophysiology, Department of Bioscience, Aarhus

University, Denmark

Printed by: SunTryk

Number of pages: 106

Circulations: 15

References from boxes to be written in white here to get included in reference list for Zotero (Zotero

does not include textboxes):

(Daan et al., 1991; Dausmann et al., 2004; Dausmann et al., 2005; Heldmaier et al., 1993; Heldmaier et

al., 2004; Srere et al., 1992; Tøien et al., 2015; Tyuma et al., 1971; Wang, 1979)

CONTENTS

Preface ............................................................................................................................................................... 1

Acknowledgements ........................................................................................................................................... 2

English summary ............................................................................................................................................... 3

Dansk Resumé (Summary in Danish) ............................................................................................................... 6

CHAPTER I: INTRODUCTORY REVIEW .............................................................................................. 10

Oxygen means energy – for some, a little can go a long way ..................................................................... 12

Organismal responses: Acclimation, acclimatization and adaptation ................................................. 12

The environment sets the bar, or the body does - either way, you have to cope ................................. 13

Hemoglobin - the oxygen transporter .......................................................................................................... 16

Cooperativity of oxygen binding to hemoglobin ................................................................................. 16

Oxygen affinity adjustments ................................................................................................................ 17

Hemoglobin – amino acid substitutions and cofactor variation at play ....................................................... 22

Environmentally low oxygen selects for high hemoglobin-O2 affinity ............................................... 22

Temporary low oxygen linked with plastic adaptations in Hb-O2 affinity .......................................... 23

Fine-tuned regulators of O2 delivery and consumption: Introduction to NO and H2S ................................ 27

Nitric oxide – a short introduction ....................................................................................................... 28

Hydrogen sulfide – a short introduction .............................................................................................. 30

Fine- tuned regulators of delivery and consumption at play: NO and H2S ................................................. 34

On the perfusion side: NO and H2S affect blood vessel diameter ....................................................... 34

The balance of tissue oxygen consumption: possible involvement of H2S and NO ............................ 35

Conclusion and perspectives ....................................................................................................................... 41

Ongoing and future research........................................................................................................................ 44

References ................................................................................................................................................... 50

CHAPTER II: PAPERS ................................................................................................................................ 64

Paper I: Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in

the hibernating brown bear Ursus arctos ........................................................................................................ 66

Paper II: Phenotypic plasticity in blood–oxygen transport in highland and lowland deer mice .................... 76

Paper III: Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with

different altitudinal ranges ............................................................................................................................... 86

Paper IV: Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging brown bears and their

potential roles in hibernation ........................................................................................................................... 96

PREFACE

This dissertation represents the partial fulfilment of the requirements for the degree of Doctor of Philosophy

(PhD) at the Faculty of Science and Technology, Aarhus University. The work described in this dissertation

was carried out at the section of Zoophysiology, Department of Bioscience, Aarhus University under the

supervision of Angela M. Fago. Part of this work, including field expeditions was conducted in collaboration

with Jay F. Storz, University of Nebraska, Lincoln, and with the Scandinavian Brown Bear Research Project.

The overall aim of this project was to investigate molecular mechanisms of oxygen uptake, delivery and

consumption in animals living at extremes, with focus on hemoglobin oxygen affinity changes and allosteric

effectors as well as concentration changes and possible effects of the signaling molecules nitric oxide and

hydrogen sulfide.

My first informed contact with hibernator blood was at a drenched cemetery in Lincoln Nebraska 2010. I had

just flown over after spending half a year as an exchange student in Seattle, Washington, and literally walked

from arrival hall to rainy cemetery and lie-in-wait 13-lined ground squirrel hunt armed with our bait of

peanut butter and oats. Peanut butter in stages of dissolving everywhere (my pockets included) does leave a

lasting impression in a European olfactory memory. During my studies of high and low altitude ground

squirrels for what became Paper III, I became captivated by hibernators. When we got contacted by the

Scandinavian Brown Bear Research Project, and I later was taken on as the blood specialist of the field team,

I had to skip aside to spend some time just jumping up and down. Six years later, I am only the more

fascinated by these animals, and still eager to learn and understand more.

Chapter I in this dissertation consists of a broad review-like introduction to molecular mechanisms

controlling oxygen transport and consumption, discussing the work of this thesis in relation to the literature.

The introduction finalizes by a conclusion and perspectives paragraph as well as future experiments,

including two ongoing projects. Chapter II consists of four peer reviewed papers, published in international

journals. I am first-author on three of these papers, having been the major contributor to all fases of the

research, and contributed majorly to planning, data collection and writing of paper II. Paper III won the 2013

Outstanding Paper Prize in Journal of Experimental Biology.

Aarhus, January 2016

Inge G. Revsbech

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ACKNOWLEDGEMENTS

There are many people I wish to thank for being important parts of my past 6 years as a PhD-student on the

3+5 model, plus a maternal leave.

First and foremost, I wish to truly thank my supervisor Angela Fago, for believing and investing in the young

yet bachelor student who stumbled upon her door those six years ago. Angela has been a great advisor and

support and has shared my enthusiasm as well as helped me see the positive in the - at first hand - less than

ideal experiment outcomes. She even took a turn with the bears when I was field work incapable, tackling of

snowshoes being tricky due to advancing pregnancy. Angela also unusually quickly planned around a fast

idea of an especially interesting conference mid-teaching, both of us departing within a few weeks. For all

investments in me and my project, thank you.

I also wish to thank our collaborator Jay F. Storz at the University of Nebraska, Lincoln. During the first

years of my PhD, it was much due to Jay and wonderful people of his lab, that I got a running start, including

exciting early morning field work at 4000 meters. Our labs are in close collaboration and I have enjoyed

inputs from visits, especially from Post. Doc. Joana Projecto-Garcia.

A large and heartfelt thanks goes to everyone in the Scandinavian Brown Bear Research Project. Especially

to my “bear-boss” Ole Frøbert and his partner-in-crime, Johan Josefsson at Örebro University Hospital,

Sweden, as well as to Alina Evans, Peter Godsk Jørgensen, Jon Arnemo, Jon Swenson and Sven Brunberg. It

has been much through my involvement in this project that my passion for hibernators grew and flourished.

Thank you goes out to our good collaborators Christopher G. Kevil and Xinggui Shen at LSU heath Sciences

Center, Shreveport Louisiana for being willing to ‘jump in the bear cave’ with us and for many good

hydrogen sulfide discussions.

Also thank you to Tom Brittain and his kind lab for hosting me during two months at Auckland University,

New Zealand.

There has been a whole bunch of people involved more or less directly in my work during the years at

Zoophysiology. I wish to thank all of the staff at our department, all of them being ready with a helping hand

in their field. A special thanks goes to Hans Malte, Tobias Wang and Hans Gesser for assisting with ideas for

experimental planning. As well as to Nini Skovgaard and Nina Iversen for being part of planning and

conducting myography experiments. Huge thanks goes to our lab-techs Elin and Kirsten, who are just

wonderful people. Last but not least at all, a big thank you to our workshops and especially John Svane and

Niels Skyberg for kind help with quickly fixing all kinds of things.

Next, I wish to thank all students at Zoophysiology. Without a good work-community, work gets less fun.

Special thanks to former and present office mates, Frants Jensen, Cristian Bech Christensen, Christian

Damsgaard, Amanda Bundgård, Lærke Reinholdt, Mikkel Thomsen and to my PhD colleague and friend

Signe Helbo. Also thanks for shared teaching, courses and interests to Jonas, Anders, Anete and Catherine.

And thank you to everyone I forgot.

Lastly, I thank my good friends and family. My exceptional friends Marie and Sunna and “Sis” Emma. My

parents and my brother Lars for believing in and supporting me. Thank you to my patient husband Henrik

who has aided me in stress and exhilaration, 2-hour mice-recaptures in university basements and feedback on

countless talks and illustrations. Thank you for always being there to bring me back home, also in my head.

And finally, thank you to our bandit son Mikkel for making every day a brighter place – e.g. by cheerfully

uttering “fart!”

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ENGLISH SUMMARY

The aim of this thesis is to broaden the knowledge of molecular mechanisms of adjustment in oxygen (O2)

uptake, conduction, delivery and consumption in mammals adapted to extreme conditions. For this end, I

have worked with animals living at high altitude as an example of environmental hypoxia, and hibernating

mammals, as an example of closely balanced internal low O2. Studies have had two main focus points.

Firstly, I have investigated variations in hemolysate and hemoglobin (Hb) O2 affinity, working to pinpoint

whether and how functional changes in intrinsic affinity or cofactor sensitivity of the Hb molecule compares

to amino acid substitutions in the molecule, i.e., can be characterized as evolved genetic adaptation.

Phenotypic acclimatization in Hb- O2 affinity responses involves changes in cofactor to Hb tetramer ratio.

Secondly, I have worked with (in a cardiovascular perspective) fine-tuners of delivery and consumption: the

signaling molecules nitric oxide (NO) and hydrogen sulfide (H2S) that are ubiquitously produced in

mammalian tissues. These molecules and their potential effects seem particularly relevant to hibernators.

My dissertation consists of two chapters. Chapter I contains an introductory review to my field of study, and

chapter II consists of four published, peer-reviewed papers.

The introductory review deals firstly with the background for animal adaptation and acclimatization to

challenging conditions via changes in Hb-O2 affinity. Elevated blood O2 affinity is one of the repeatedly

found adaptive traits in animals living at high altitude and in hibernating mammals during hibernation

compared with the active state. Factors that affect O2 affinity of Hb include temperature, H+/CO2 via the Bohr

effect as well as Cl- and organic phosphates, in mammals mainly 2,3-diphosphoglycerate, DPG. I here show

how my results fit into the current knowledge. Secondly, I review the current literature on cardiovascular

effects of NO and H2S and discuss their potential effects in mainly hibernating mammals.

SUMMARY OF IMPORTANT FINDINGS OF THE INCLUDED PAPERS TO BE FOUND AT THE END OF

THIS DISSERTATION:

Paper I: Revsbech IG, Malte H, Fröbert O, Evans A, Blanc S, Josefsson J, Fago A. Decrease in the red cell

cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in the hibernating brown bear Ursus

arctos. Am J Physiol Regul Integr Comp Physiol 304: R43–R49, 2013.

Paper I: Revsbech et al., 2013a. Paper I in this thesis concerns variations in the red blood cell cofactor DPG

with hibernation in the brown bear and how this affects hemolysate and Hb O2 affinity. Decreases in

allosteric cofactor DPG has been observed in several small hibernators, but only one study in hedgehogs

have found a correlation with the observed hibernation-induced increases in blood O2 affinity. Bear

hibernation is in some aspects physiologically different from that of smaller hibernators (warmer body

temperature, no arousals). We found the DPG/Hb tetramer ratio to be reduced to about half of summer levels

in hibernating brown bear, and the winter hemolysate to indeed show increased O2 affinity when measured at

physiologically relevant temperatures (37 vs 30˚C). The difference in temperature was necessary but not

sufficient to induce this change. We then demonstrated the change in DPG to be responsible by adding the

measured amount of DPG to purified Hb from the individual bears. Modeling moreover showed that the

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winter decrease in DPG (and thus changes in affinity P50 and cooperativity n50) related to a somewhat lower

venous PO2 than in summer. If not for these changes, winter venous PO2 would have been appreciably

higher than summer due to an increase in hematocrit, which would likely have led to high reactive oxygen

species production.

Paper II: Tufts DM, Revsbech IG, Cheviron ZA, Weber RE, Fago A, Storz JF: Phenotypic plasticity in

blood–oxygen transport in highland and lowland deer mice, J Exp Bio 216: 216, 1167-1173, 2013

Paper II: Tufts et al., 2013. Paper II also concerns variation in DPG in the red blood cells, here measured in

high versus low altitude deer mice populations. Elevations in erythrocyte DPG, hematocrit, Hb

concentration, lower mean corpuscular Hb concentration and smaller red blood cells were found in a high

altitude deer mouse population. By conducting a common garden experiment over six weeks (housing the

animals at the same conditions at low altitude), these changes were reversed. Our group and collaborators

have published on the genetic adaptation of deer mouse Hb to high altitude, involving low cofactor

sensitivity. The reversal of these traits in the common garden experiment proves them to be acclimatization

responses that are part of a remarkable phenotypic plasticity, that we provide evidence can also be found in a

population in other aspects considered high altitude adapted.

Paper III: Revsbech IG, Tufts DM, Projecto-Garcia J, Moriyama H, Weber RE, Storz JF, Fago A.

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different

altitudinal ranges, J Exp Bio 216: 4264-4271, 2013.

Paper III: Revsbech et al., 2013b. The third paper to be published was actually the first study of my thesis,

however data collection was conducted over several years. This project concerned a thorugh comparison of

six species of high and low altitude marmotine ground squirrel Hb function and sequence data. Globin

sequencing by our collaborators showed highly unusual substitutions, according to the human HbA model

expected to affect the Bohr effect of the ground squirrel Hbs. In particular the β146His→Gln of the golden

mantled ground squirrel was expected to be detrimental to the Bohr effect of this Hb. Functional analysis in

our lab showed general high similar Hb-O2 affinity for all species, with suppressed Cl- and DPG sensitivities,

regardless of native altitude. Aditionally the 13-lined and golden mantled ground squirrel Hbs had substantial

Bohr effects, despite their substitutions. Taken together, these ground squirrels do not conform to the human

HbA model of Hb function, but rather the mentioned substitutions have a different effect on the genetic

background of these animals. Instead the collective number of surface histidines seems to decide the Bohr

effect. In addition, the hibernation-induced decrease in DPG observed in ground squirrels may not have any

appreciable direct allosteric effect,despite the fact that the DPG binding site was conserved. To our surprise

and delight, this paper won the Outstanding Paper Prize 2013 in the Journal of Experimental Biology.

Paper IV: Revsbech IG, Shen X, Chakravarti R, Jensen FB, Thiel B, Evans A, Kindberg J, Fröbert O,

Stuehr DJ, Kevil CG, Fago A. Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging

brown bears and their potential roles in hibernation. F Rad Biol Med 73: 349–357, 2014.

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Paper IV: Revsbech et al., 2014. The fourth and final paper included in this thesis was our entrance into H2S

biology. In this paper we provide the, to our knowledge, first measurements of plasma and red blood cell H2S

metabolites in the hibernating versus awake state of a hibernating animal. We investigated levels and

metabolites of both ubiquitous gaseous signaling molecules NO and H2S that can potentially inhibit

mitochondrial cytochrome c oxidase, and contribute to metabolic reduction during hibernation. H2S can be

devided into three major pools. I: acid-labile sulfur, containing mainly Fe-S clusters and persulfides that will

be released under acidic conditions. II: bound sulfane sulfur pool containing mainly thiosulfate and

polysulfides, converted into H2S under reducing conditions. And III: free H2S, at physiological pH mainly in

the form of HS-. To our surprise we did not find any increase in free H2S, but rather a shift in the pools of

H2S. During winter hibernation we observed a decrease in bound sulfane sulfur, indicating that this pool in

the low O2 environment is utilized for H2S production. The available cysteine, substate for enzymatic H2S

production, can then be allocated for production of glutathione, a major antioxidant also found in high levels

in red blood cells of hibernating bears. In contrast, changes in nitrite taken as a marker for NO production as

well as SNO-modification of a key glycolytic enzyme, was not significantly different. We thus propose

mainly H2S, and likely not NO, to be involved in hibernation, as has also been indicated by its ability to

induce a hibernation-like state in mice.

In conclusion the results of these studies shows that, when it comes to O2 transport, phenotypic plasticity and

in particular variations in red blood cell DPG levels plays large roles in acclimatizing animals that are in

other aspects considered adapted. However, not all hibernators necessarily utilize falls in DPG during

hibernation, and small hibernators may rely more on fall in body temperature to elevate blood O2 affinity.

Additionally, our studies indicate a role for H2S during hibernation, possibly as part of the metabolic

downregulation. Finally, results from this dessertaion support the growing theory that not necessarily only a

few amino acids are paramount to protein function, but rather their roles can be substituted by the cumulative

action of other amino acid substitutions working on the specific genetic background.

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DANSK RESUMÉ (SUMMARY IN DANISH)

Denne ph.d.-afhandlings formål er at øge vores viden om de molekylære mekanismer, som justerer ilts optag,

transport, levering og forbrug i pattedyr, der er tilpasset ekstreme vilkår. Jeg har arbejdet med dyr, som er

tilpasset til at leve i høje bjerge som eksempler på tilpasning til hypoxi i miljøet, samt dyr, der sover

vintersøvn som eksempel på kontrolleringen af den hårfine balance af lavt ilt indeni et dyr, der skal spare på

”madpakken”. Mine studier har haft to primære fokuspunkter. Jeg har undersøgt variationer i hæmolysat (en

opløsning af lyserede røde blodceller) og hæmoglobins (Hb) iltaffinitet. Jeg har arbejdet på at udpege hvilke

og hvordan funktionelle ændringer i iboende ilt affinitet af Hb molekylet i sig selv eller Hbs sensitivitet

overfor kofaktorer har rødder i aminosyre udskiftninger i molekylet, og derfor kan betragtes som en

genotypisk nedarvet tilpasning. Fænotypisk tilpasning i Hb iltaffinitet er til gengæld styret af plastiske eller

foranderlige ændringer, og involverer typisk ændringer i kofaktor til Hb tetramer ratioen. Mit andet

fokuspunkt har været arbejde med signalmolekylerne nitrogenoxid (NO) og hydrogen sulfid (H2S), der

konstant produceres i pattedyrs væv. Disse molekyler kan i et cardiovaskulært perspektiv fin-justere

iltlevering og forbrug, hvilket især synes relevant for et dyr i vintersøvn.

Min afhandling består af to hovedkapitler. Kapitel I udgøres af et introducerende review til mit

forskningsfelt, og kapitel II består af fire udgivne peer-reviewede artikler.

Det introducerende review omhandler i første del baggrunden for dyrs tilpasning gennem genetisk tilpasning

og fænotypisk tilpasning via ændringer i Hb iltaffinitet. Øget iltaffinitet af blodet er en af de ofte fundne træk

i dyr, som lever i højder samt i dvale tilstanden sammenlignet med den vågne, aktive tilstand. Faktorer som

påvirker iltaffiniteten af Hb inkluderer temperatur, H+/CO2 via Bohr effekten samt Cl- og organiske fosfater, i

pattedyr hovedsageligt 2,3-difosfoglycerat, DPG. Jeg diskuterer og sætter mine resultater i perspektiv til den

forhåndenværende litteratur på området. Den anden del af introduktionen omhandler og diskuterer literaturen

samt mine resultater omhandlende cardiovaskulære niveauer af NO og H2S og deres potentielle effekter især

i dyr, som sover vintersøvn.

SAMMENDRAG AF VIGTIGE RESULTATER I MINE PUBLICEREDE ARTIKLER (PAPERS), SOM

FINDES SIDST I AFHANDLINGEN:

Paper I: Revsbech IG, Malte H, Fröbert O, Evans A, Blanc S, Josefsson J, Fago A. Decrease in the red cell

cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in the hibernating brown bear Ursus

arctos. Am J Physiol Regul Integr Comp Physiol 304: R43–R49, 2013.

Paper I: Revsbech et al., 2013a. Paper I i denne afhandling omhandler variationer i de røde blodcellers Hb

kofaktor DPG under vintersøvn i den brune bjørn, og hvordan dette påvirker hæmolysat og Hb iltaffinitet.

Fald i den allosteriske kofaktor DPG har været set i adskillige mindre dyr, som sover vintersøvn, men en

sammenhæng med den observerede stigning i blodets iltaffinitet er hidtil kun set hos pindsvin. Vintersøvn i

brune bjørne er i nogle aspekter fysiologisk anderledes fra det, vi ser i små dyr (varmere kropstemperatur,

ingen opvågninger). Vi fandt en halvering i forhold til niveauet om sommeren i DPG/Hb tetramer ratioen i

6

den hibernerende brune bjørn, samt en øget iltaffinitet i vinter hæmolysaterne når de blev målt ved

fysiologisk relevante temperaturer (37 vs 30˚C). Forskellen i temperatur var nødvendig, men ikke

tilstrækkelig til at forårsage ændringerne i iltaffiniteten. Vi demonstrerede ved et forsøg, hvor vi tilsatte den

nøjagtige målte mængde DPG til oprenset Hb for hver bjørn, at ændringerne i DPG var årsagen til den

yderligere ændring i iltaffiniteten. Yderligere viste matematisk modelering, at vinterens fald i de rød

blodcellers DPG niveau (og derfor ændringer i iltaffinitet P50 og cooperativitet n50), var relateret til en lidt

lavere venøs ilttension PO2 om vinteren end om sommeren i bjørnens blod. Hvis de DPG-relaterede

ændringer i iltbinding ikke var sket, ville den venøse PO2, grundet en højere hæmatokrit i blodet om

vinteren, omvendt have været en del højere end om sommeren. Høj venøs PO2 ville have ført til skadelige

niveauer af frit ilt. Altså bidrager faldet i DPG til en lavere venøs PO2, hvilket bidrager til en lavere

produktion af frie iltradikaler.

Paper II: Tufts DM, Revsbech IG, Cheviron ZA, Weber RE, Fago A, Storz JF: Phenotypic plasticity in

blood–oxygen transport in highland and lowland deer mice, J Exp Bio 216: 216, 1167-1173, 2013

Paper II: Tufts et al., 2013. Paper II omhandler også variationen af DPG i de røde blodceller, her målt i der

mus populationer tilpasset høj versus lav højde. I populationen tilpasset høj højde fandt vi et øget indhold af

DPG i de røde blodceller, øget hæmatocrit, øget Hb koncentration, lavere mean coposcular Hb concentration

og mindre røde blodceller. Disse ændringer forsvandt efter et seks ugers common garden forsøg, hvor

musene blev holdt ved de samme betingelser ved lav højde. Vores gruppe og vores samarbejdspartnere har

tidligere publiceret fund af genetisk tilpasning af deer mus Hb inklusive en lav sensitivitet overfor

kofaktorer. At de fundne ændringer forsvandt i common garden forsøget viser, at de er en del af en

midlertidig tilpasningsevne, som hører under fænotypisk plasticitet. Fænotypisk plasticitet kan altså også

udgøre noget af tilpasningen til en, ellers i andre aspeketer, genetisk højdetilpasset population.

Paper III: Revsbech IG, Tufts DM, Projecto-Garcia J, Moriyama H, Weber RE, Storz JF, Fago A.

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different

altitudinal ranges, J Exp Bio 216: 4264-4271, 2013.

Paper III: Revsbech et al., 2013b. Den tredje artikel som blev publiceret var det første studie jeg tog del i

under min ph.d., men data indsamlingen blev udført over flere år. Dette projekt omhandler en dybtgående

sammenligning af Hb funktion og aminosyresekvens fra seks arter af relaterede murmeldyr og jordegern.

Globin sekventeringen udført af vore samarbjedspartnere viste højest usædvanlige substitutioner, som efter

den humane model for Hb funktion forventedes at påvirke Bohr effekten af jordegern Hb. Især substitutionen

β146His→Gln i golden mantled jordegern var forventet at påvirke Bohr effeketen af dette Hb stærkt.

Funktionelle analyser i vores laboratorium viste en generel høj og enslignende Hb iltaffinitet med lav Cl- and

DPG sensitivitet for alle seks arter uanset oprindelig højde. På trods af de nævnte substitutioner målte vi en

normal Bohr effekt i 13-lined og golden mantled jordegern Hb.

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Samlet set passer Hb funktionen af disse jordegern ikke med den humane HbA model for Hb funktion. I

stedet har de nævnte substitutioner en anden effekt i den genetiske baggrund for et jordegern, end i et

menneske. Det samlede antal histidiner på proteinets overflade ser i stedet ud til at udgøre den normale Bohr

effekt for disse hæmoglobiner. Desuden kan vi konkludere, at den vintersøvns-inducerede nedgang i røde

blodcellers DPG niveau observeret i jordegern, sandsynligvis ikke har en nævneværdig direkte allosterisk

effekt, på trods af at alle bindingsstederne for DPG findes konserveret i jordegern Hb. Til vores overraskelse

og glæde vandt denne artikel Outstanding Paper Prize 2013 i Journal of Experimental Biology.

Paper IV: Revsbech IG, Shen X, Chakravarti R, Jensen FB, Thiel B, Evans A, Kindberg J, Fröbert O,

Stuehr DJ, Kevil CG, Fago A. Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging

brown bears and their potential roles in hibernation. F Rad Biol Med 73: 349–357, 2014.

Paper IV: Revsbech et al., 2014. Den fjerde og sidste artikel inkluderet i denne afhandling var vores første

bekendtskab med H2S biologi. I denne artikel rapporterer vi, efter mit kendskab, de første målinger af plasma

og røde blodcellers niveauer af frit H2S og metabolitter i et hibernerende versus vågent stadie af et dyr, som

sover vintersøvn. Vi undersøgte niveauer og metabolitter af begge de fysiologisk allestedsværende gasser

NO og H2S, som potentielt kan inhibere mitokondriel cytocrom c oxidase og bidrage til metabolsk

depression under vintersøvn. H2S kan opdeles groft i tre store pools. I: Acid-labile sulfur, der består af

mestendels Fe-S-samlinger og persulfider, og som frigøres under forsurede forhold. II: bundet sulfur, der

hovedsagelig består af thiosulfat og polysulfider, og som frigøres til fri H2S i et reducerende miljø. Og III:

Fri H2S, ved fysiologisk pH primært på formen HS-. Til vores overraskelse fandt vi ingen øgning i frit H2S

under hibernering, men i stedet et skift i H2S pools. Under vintersøvnen så vi et fald i den bundne sulfur

pool, hvilket indikerede, at denne pool i det lave iltmiljø bliver brugt til produktion af frit H2S. Den

tilgængelige cystein, som er substrat for enzymatisk H2S produktion, kan så allokeres til produktionen af

glutathion, en vigtig antioxidant som vi også fandt høje niveauer af i røde blodceller. Til gengæld fandt vi

ingen signifikante ændringer i nitrit eller i SNO-modifikation af et nøgle-enzym i glycolysen, som blev brugt

som markør for NO biotilgængelighed. Vi foreslår derfor, at H2S er involveret i vintersøvn i brune bjørne,

som også indikeret ved dets evne til at forårsage et stadie lig vintersøvn i mus.

I konklusion viser resultaterne fra mine ph.d.- studier, at fænotypisk plasticitet og særligt variation i røde

blodcellers DPG niveauer i et ilttransport perspektiv kan spille en væsentlig rolle i yderligere at tilpasse dyr

til deres givne miljø. Til gengæld kan vi også konkludere, at ikke alle dyr som sover vintersøvn

tilsyneladende har nytte af en direkte allosterisk effekt af faldet i DPG i de røde blodceller. I stedet må det

store fald i kropstemperatur i mindre dyr være tilstrækkeligt til alene at øge Hbs iltaffinitet under

vintersøvnen. Desuden indikerer mine studier at H2S spiller en rolle under vintersøvn, muligvis som et led i

den metabolske nedjustering. Slutteligt støtter resultater fra denne afhandling den voksende teori, at ikke

nødvendigvis kun få aminosyrer er afgørende for et proteins funktion, men at hele den genetiske baggrund og

indbyrdes påvirkninger kan træde i stedet.

8

9

CHAPTER I: INTRODUCTORY REVIEW

10

ABBREVIATIONS USED IN THIS INTRODUCTION:

Hb: hemoglobin; Mb: myoglobin; NO: nitric oxide; H2S: hydrogen sulfide; CcOx: Cytochrome c oxidase,

complex IV of the electron transport chain; NOS: nitric oxide synthase; CSE:cystathionine -lyase; CBS:

cystathionine β-synthase; MST: 3-mercaptopyruvate sulfurtransferase; PO2: partial pressure of oxygen; P50:

oxygen tension at half-saturation; n50: Hill’s cooperativity coefficient at half-saturation; KT and KR: O2

association equilibrium constants of Hb in the deoxygenated (tense T) state and oxygenated (relaxed R)

states, respectively; DPG: 2,3-diphosphoglycerate; BMR: basic metabolic rate; Tb: body temperature. T:

temperature. GSH: gluthathione; GSSG: gluthathione disulfide; NADPH: nicotineamide adenine

dinucleotide-phosphate; NADH: nicotineamide adenine dinucleotide; FADH2: flavin adenine dinucleotide;

ROS: reactive oxygen species; GTP: guanosine triphosphate.

11

OXYGEN MEANS ENERGY – FOR SOME, A LITTLE CAN GO A LONG WAY

Mammals are as endotherms inherently slaves of a high energy demand, and thus a high oxygen (O2)

consumption most often requiring a high O2 environment. Some mammalian species are, however, to some

extent able to work around these constraints. Some mammals can cope well with low O2 conditions, and

some utilize periods of low O2 consumption. Whether accomplished by more permanent genetic adaptation

or by temporary plastic acclimatization responses, the means to go beyond the usual mammalian O2 -linked

constraints are intriguing to unravel. During my thesis I have dealt with molecular scale physiological

responses to the environmental setting of living at high altitude as well as the physiological mechanisms

enabling an accurate control of O2 inside a hibernating animal. The inherent appeal in this subject lay for me

first in mere fascination of the physiology. Secondly my captivation has come to lie in understanding these

responses and wishing to play part in the basic research that is paramount to potential human application.

Findings could well be relevant for conditions where O2 lack and reintroduction are central, as in ischemia-

reperfusion injury of the heart and brain.

Most multicellular life utilizes O2 as electron acceptor in the mitochondrial electron transport chain enabling

ATP production. Because O2 is of paramount importance for the production of energy, conditions limiting

the O2 supply to an animal is a challenging state. The animal in question must respond appropriately, or

energy deficiency will lead to loss of membrane potentials over cell and mitochondrial membranes,

ultimately resulting in cell death.

ORGANISMAL RESPONSES: ACCLIMATION, ACCLIMATIZATION AND ADAPTATION

A living being can cope with changes in its environment by responding. Responses over timespans of hours

and longer are usually classified as one of three: Responding to a single changed variable such as

temperature in an artificial or laboratory setting is termed acclimation. Responding to a changed natural

environment involving multiple variables such as allocation to high altitude is termed acclimatization.

Finally species living at e.g. high altitude often over multiple generations evolve changes in their genome

that adapts the population to this environment, known as adaptation (Gatten et al., 1988; Giordano, 2013).

Responses can happen on different physiological levels: Firstly, by optimizing the four linked mechanisms

of O2 transport: ventilation, pulmonary diffusion, circulation and tissue diffusion (Lenfant and Sullivan,

1971). Secondly, by reducing consumption; aerobic energy metabolism or shut-down of parts of the

organism in order to conserve available O2 for vital organs such as the brain. Metabolism and thus O2

consumption is tightly controlled in a hibernator, but the details of this control are yet unclear. Thirdly, some

species manage by upregulating glycolysis, which in most vertebrates (except from the crucian carp and

freshwater turtles) takes place primarily during short term need, yielding potentially troublesome lactic acid.

One of the highly repeated features observed across extensively divergent species adapted to hypoxia, is in

12

the first category: increased O2 affinity of blood hemoglobin (Hb) (Bunn, 1980). High Hb-O2 affinity may

enhance pulmonary O2 uptake in hypoxia whilst defending arterial O2 saturation, thus maintaining an O2

pressure gradient that drives the diffusion steps of O2 transport from lung epithelia to mitochondria

(Mairbäurl and Weber, 2012). Consequently, faced with hypoxia, increases in Hb-O2 affinity can play an

important part of coping responses (Mairbäurl, 1994; Turek et al., 1973; Turek et al., 1978a). Other

adjustments are often hematocrit and capillary density alterations as well as lung volume increases (Arieli

and Ar, 1979; Avivi et al., 2005; Monge and León-Velarde, 1991; Turek et al., 1978b). Employing an

increase of Hb-O2 affinity may be the case in both high altitude and hibernating mammals, although not

necessarily achieved in the same manner, or for the same effects, as we shall see in this introduction.

In this introduction, I will discuss physiologic adaptations in O2 transport and consumption found in animals

living and coping with hypoxia. I have mainly worked with two general varieties of low O2; high altitude

environmental hypoxia along with the low fluctuating O2 delivery and consumption of hibernators. The first

half of the introduction deals with conditions of low O2 and O2 delivery, introducing the blood O2 carrier Hb,

and discussing the molecular adaptations in and around Hb in achieving sufficient O2 delivery in hypoxic

conditions. The second half focuses on control of O2 consumption as ideally studied in a hibernating animal

downregulating metabolism and thus O2 utilization, compared with the same animal in its summer active

state. The endogenous gaseous signaling molecules nitric oxide and hydrogen sulfide may be part of

metabolic control, as they are both potential inhibitors of complex IV of the mitochondrial electron transport

chain, along with other effects. The introduction is finalized by a conclusion and perspectives section, as well

as future experiments where I will discuss future avenues of further understanding the mechanisms

controlling O2 delivery and consumption.

THE ENVIRONMENT SETS THE BAR, OR THE BODY DOES - EITHER WAY, YOU HAVE TO

COPE

In high altitude life, the by far most important stressor is the direct effect of height: hypobaric hypoxia. The

reduction in ambient PO2 exposes all animals living at height to a degree of chronic environmental hypoxia.

No short term solution is feasible. The native high altitude mammal generally follows one major strategy;

increase in O2 supply and transport capabilities, including blood O2 affinity (Bunn, 1980).

Examples of utilizing short term adaptations are the hibernating animals switching from euthermic state to 5-

7 months of drastically reduced metabolism every year. Hibernation is generally induced by periods of food

limitations rather than alone by the cold environmental temperatures per se (Milsom and Jackson, 2011;

Ortmann and Heldmaier, 2000). During bear hibernation energy is supplied by burning of fat (Robbins et al.,

2012). Decrease in body temperature and resultant Q10-effects on metabolic rate (Q10 being the rate

coefficient for a 10°C change in temperature) play a large but not sufficient role in sustaining metabolic

13

depression. During entrance and arousal, metabolism is the regulated variable, falling before body

temperature in entrance as seen in the dormouse (Heldmaier et al., 2004) and in some remaining low for

weeks at normal body temperature after arousal, as seen in black bears (Tøien et al., 2011). The extensive

metabolic reduction entails a drastic reduction of O2 consumption. During hibernation, basal metabolic rate

(BMR) will drop substantially; metabolism is down-regulated to between 0.02 to 0.06 ml O2 g-1 h-1 for all

hibernators, regardless of body size and euthermic BMR (Heldmaier et al., 2004; Milsom and Jackson, 2011;

Ortmann and Heldmaier, 2000; Tøien et al., 2015). For 33 species of small hibernating mammals the

metabolic reduction is to around 1-10% of BMR, with a mean of 4% (Geiser and Ruf, 1995). In bears, the

reduction is to 25% of summer BMR (Tøien et al., 2011).

Of organismal O2 consumption ~ 90% is utilized directly as electron acceptor in the mitochondrial electron

transport chain during ATP production. Both delivery to and consumption at the mitochondria are areas of

potential downregulation in order to achieve the reduced metabolic demand needed to survive all winter. In

addition hibernators are likely to experience mismatches in supply to consumption of O2 especially at arousal

from hibernation when body temperatures and metabolism return to euthermic states. Elevated O2 affinities

during hibernation could work to protect blood O2 saturation during arousals and apneas of 30-60 min as

seen in some smaller hibernators (Heldmaier et al., 2004) where PO2’s might become low.

14

BOX 1: HIBERNATION IS AN UNSTEADY STEADY STATE:

MISMATCHES IN O2 DELIVERY TO CONSUMPTION ARE LIKELY

All known hibernators except the bears and the Madagascar lemurs do not hibernate continuously during

their hibernation period, but rather express torpor bouts of stable and low body temperature (Tb) and

metabolism of up to 20 days, interrupted by 1-2 day arousals back to euthermic temperature, then start

over, as in the alpine marmot, figure 1 (Heldmaier et al., 2004). This behavior is exceedingly expensive

energetically, thus requiring an estimated 72% of all energy reserves expended during the hibernation

season of a marmot (Heldmaier et al., 1993). Despite including costly arousals, energy expenditure during

hibernation is reduced to ~15 % of normothermic expenditure (Wang, 1979). It has been speculated that

obtaining normal sleep as well as enabling protein production may be the background necessitating these

costly returns to normothermia (Srere et al., 1992; Daan et al., 1991). Regardless, hibernators are likely to

have evolved a suite of adaptations to handle inevitable mismatches in O2 delivery to consumption

resulting from this behavior.

Figure 1. Continuous versus interrupted hibernation. Parts A shows traces from an Alpine marmot. Part B,C shows traces from a black bear in hibernation MR is metabolic rate. Ta ambient temperature. Part A from Heldmaier et al., 2004. Parts B,C modified from Tøien et al., 2011

In comparison the larger bears do not

arouse between bouts of deep hibernation,

but instead show multiday cyclic

oscillations in body temperature (Tøien et

al., 2011). The rises in Tb are induced by

shivering (Tøien et al., 2015), and thus

metabolic rate also increases albeit very

transiently during warming fases (figure 1).

In black bears the cycles in Tb seem to

come at no metabolic cost and with an

unknown effect besides defending a lower

critical body temperature ~ 30°C (Tøien et

al., 2015). Around 30°C also seems to be

the critical temperature for the African fat-

tailed dwarf lemur that only exhibits

periodic arousals if not passively warmed

(by the sun) to above 30°C on a daily basis

(Dausmann et al., 2004; Dausmann et al.,

2005). Thus also bears are likely to be

adapted to handle sudden variations in O2

consumption during hibernation, and

especially in preparation for the final

spring arousal.

MR

C

A

B

15

HEMOGLOBIN - THE OXYGEN TRANSPORTER

Hemoglobin Hb is the blood O2 carrier and thus Hb and the erythrocyte environment surrounding it are the

main sites of molecular adaptation of O2 transport. All vertebrate Hbs are composed of four heme-carrying

globins, two α or α-like subunits, and two β- or β-like subunits. Each heme group contains a porphyrin ring

with an iron atom in the ferrous state (Fe2+) able to reversibly bind a single O2 molecule. In the major adult

human Hb, HbA, as well as most vertebrate Hbs the α-globin is 141 amino acid residues long and β-globin is

146 residues long. The structure of each globin subunit is characterized by its ‘globin-fold’ consisting of

seven to eight α-helices (named A to H) linked by non-helical segments, with amino and carboxyl termini

extensions (named NA and HC, respectively). During the oxygenation-deoxygenation cycle the Hb

undergoes a change in quaternary structure as the two αβ dimers rotate from another by 15°, switching from

the low affinity deoxy T (tense) state stabilized by salt bridges and hydrogen bonds, to the high affinity

oxygenated R (relaxed) state, where some of these bonds are broken (Perutz, 1972; Perutz, 1989). The

change in quaternary structure with oxygenation forms the background for Hb-O2 transport to be modified by

allosteric binding of cofactors stabilizing the T-state, as well as is the structural basis for cooperative binding

of O2.

COOPERATIVITY OF OXYGEN BINDING TO HEMOGLOBIN

The tetrameric Hb exhibits cooperative binding of O2, giving rise to an S-shaped O2 binding curve (figure

2B). How exactly the cooperative binding has roots in protein structure has been the gnawing bone of studies

for decades. Two models have been promoted in particular: the first work by Pauling and later Koshlad to

elucidate Hb cooperativity established the sequential model, arguing for the idea that each O2 binding

induces conformational change in the protein by direct heme-heme interaction (Koshland et al., 1966;

Pauling, 1935). Later the allosteric model or the MWC was put forward (Monod et al., 1965), proposing that

cooperativity arises from differences in quaternary structure of the low affinity T state and the high affinity R

state. Thus rising O2 tensions shifts the equilibrium from most in low affinity T-state to a higher population

of R-state quaternary structure, but each heme O2 binding is independent of the total number of O2 bound in

the tetramer. Perutz then elegantly demonstrated how much fitted together by describing how the T structure

is stabilized by salt bridges and how O2 binding to heme displaces iron causing helix movement, salt bridge

breaks and release of protons, collectively causing destabilization of the tetramer at the sliding interface of

αβ dimers, and shifting the quaternary equilibrium toward the R-state (Eaton et al., 1999; Perutz, 1970). The

Perutz/allosteric model is the essential base of our understanding of Hb-O2 binding today (Eaton et al.,

1999). The often described heme-heme interactions essential to Hb cooperativity seems a relic of the pure

sequential model, and is an imprecise way to describe the background for cooperativity. The details are not

yet fully agreed upon, but Hb does certainly bind O2 in a cooperative fashion that can be assessed by

measuring the Hill or cooperativity coefficient, n, the slope of a Hill-plot (figure 2C). Besides mere O2

16

affinity of a given Hb, the degree of cooperative binding logically would be another likely factor under

evolutionary pressure in adaptations to low O2 conditions. Cooperativity coefficient n was indeed found to

significantly differ between hibernating and summer active bear hemolysates, by our modeling playing an

important role in achieving a suitable venous PO2 despite the high winter hematocrit as will be discussed in

more detail later (Paper I: Revsbech et al., 2013a).

OXYGEN AFFINITY ADJUSTMENTS

Adjustments in Hb-O2 affinity may be mediated through changes in the intrinsic interaction between heme

groups in a dimer, affecting the inherent O2 affinity. Affinity can also be affected by amino acid substitutions

changing the sensitivity towards effectors that modify Hb-O2 affinity, or simply though changes in the

concentration of effectors inside the red blood cell (RBC). Allosteric effectors bind specific sites in the Hb,

increase T-state stabilization and thus increase the O2 tension required for half-saturation of the Hb, the P50

as well as may affect cooperativity, n (figure 2B), (Nikinmaa, 2001; Weber and Fago, 2004). The major

allosteric effectors for regulating Hb-O2 affinity in mammals are protons and CO2, both giving rise to the

Bohr effect, and the anions 2,3-diphosphoglycerate (DPG) and chloride. Cl- ions is in Human Hb are thought

to bind at two sites: between residues α1Val and α131Ser as well as β82Lys and β1Val (O’Donnell et al.,

1979; Riggs, 1988).

17

Figure 2. Details of oxygen binding

A: Heat forms part of the T-R equilibrium.

B: Allosteric effectors right shifts the Hb-O2 binding curve.

C: Schematic Hill plot depicting the cooperativity coefficient n50, this

slope is typically around 2.8 for human Hb.

D: Schematic Hill plots depicting the change in specificly KT with effector

binding.

Figure redrawn and modified from Weber and Fago 2004.

BOX 2: HEAT OF OXYGENATION AND HILL PLOTS

As discussed in the text, oxygen binding to hemoglobin is affected by

allosteric effectors H+, Cl-, organic phosphates P- and then temperature

T. The binding of O2 is an exothermic reaction, whereas the

dissociations of bound allosteric effectors are endothermic reactions,

consuming heat (figure 2A). The resultant heat of oxygenation is thus

diminished by the number of allosteric effectors bound (figure 2A).

The apparent heat of oxygenation ∆H’, i.e. the enthalpy here depicted

is calculated by use of the Van’t Hoff equation:

∆H=2.303R(∆logP50) / ((1/T1)-(1/T2))

Where R is the gas constant (1.987cal K-1mol-1), T1 and T2 are the

absolute temperatures (K), and ∆logP50 is the corresponding difference

in logP50. ∆H’ values are then corrected for heat of O2 in solution (-3.0

kcal mol-1) (Antonini and Brunori, 1971). The slight endothermic

component of the conformational shift T→R is constant and not

considered here. Thus ∆H’ is depicting how sensitive Hb-O2 binding

will be to variations in temperature. Accordingly high temperature is

amongst the factors causing a right shifting of the Hb-O2 binding curve

(figure 2B), causing the blood to require a higher PO2 to get 50%

saturated (P50).

Hill plots are used for interpreting O2-binding of a given Hb sample.

figure 2C is a schematic of a Hill plot, log (oxyHb/deoxy Hb) or

logY/(1-Y) vs. log PO2, where Y is fractional saturation. An estimate

of n50, the cooperativity coefficient for Hb O2 binding, is given by the

slope of the hill plot at 50% saturation of the Hb, that is, when log

(oxy/deoxy) is log1= 0 on the Y-axis. This value moreover

corresponds to logP50 on the x-axis. In Fig. 2D the upper and lower

asymptotes depict effector-induced decrease in O2 affinity of the T-

state (equilibrium constant for T state, KT) and less so that of the R-

state (KR) resulting in increased cooperativity, n50, the slope at line

Y=0.

18

AFFINITY ADJUSTMENTS: DPG

The organic phosphate DPG is produced as a side product of red cell glycolysis. DPG has been shown to

have a direct right-shifting effect on the Hb-O2 dissociation curve in humans (figure 2B), (Benesch and

Benesch, 1967; Benesch and Benesch, 1969). The effect is believed mediated through asymmetric binding

directly or through water molecules to seven positively charged sites in the central cavity between the two β-

chains of the deoxy T-state Hb molecule (figure 3). Thus DPG binds with β2His, β82Lys (via different

interactions) and β143His (weakly) of both β chains, and β1Val of one chain though water molecules, the

other β1Val binds back to its own chain β78Leu (Richard et al., 1993). The major difference from a previous

model being that DPG does not directly bind both β1Val as previously assumed (Arnone, 1972). The binding

of DPG is inhibited in the R-state where the central cavity is narrowed (Lukin and Ho, 2004; Perutz, 1970).

As a nondiffusable polyanion, DPG furthermore indirectly affects intracellular proton concentration,

affecting the Donnan equilibrium across the erythrocyte membrane. High intracellular DPG leads to an

influx of H+, which via the Bohr effect (below) may affect Hb-O2 binding (Duhm, 1971). We suggest DPG

affecting Hb O2 affinity through the Donnan equilibrium to be likely in ground squirrel Hbs that are largely

insensitive to DPG, as will be discussed further (Paper III: Revsbech et al., 2013b).

AFFINITY ADJUSTMENTS: TEMPERATURE AND BOHR EFFECT

Temperature and pH/pCO2 are other central factors affecting O2 binding to Hb. O2 binding is an exothermic

reaction, and dissociation of ligands is endothermic (figure 2A). Therefore a rise in temperature causes a fall

in Hb-O2 affinity (see box). The alkaline Bohr effect takes place between pH ~ 6-9 covering the

physiological range for a mammal, and consists of both direct CO2 binding as well as H+ binding to Hb. The

Bohr effect below pH 6.3 is termed the acid Bohr effect (Imai, 1982), but is unlikely to be applicable in a

mammalian physiological setting and therefore not considered further here. CO2 forms carbamates with the

uncharged NA termini of both α- and β chains, giving rise to the direct CO2 Bohr effect (Imai, 1982). The

fixed acid Bohr effect arises when H+ binding reduces Hb-O2 affinity at low pH as in working muscles, and

Figure 3. The DPG binding site in the central cavity of human Hb. Collectively, the whole cationic field is believed to contribute to DPG-Hb interactions. Modified from Stryer et al., 2002.

DPG

19

A B

Figure 4. Important amino acid substitutions likely to affect the Bohr effect in the sciurid species investigated in Paper III. A: the α-chain C-termini substitution α141Arg→Cys (top to bottom) found in all four tested sciurids. B: the β146 His→Gln (top to bottom) substitution found in one chain of Golden Mantled ground squirrel only. For details on function of each substitution, see text. From Jay Storz, personal communication (Allison J, Runck A M, Moriyama H, Bell K, Demboski J, Storz J).

H+ dissociation at respiratory surfaces increases affinity. The size of the Bohr effect for a given Hb can be

evaluated by estimating the Bohr coefficient given the change in logP50 upon a change in pH by 𝜑 =𝛿𝑙𝑜𝑔𝑃50

𝛿𝑝𝐻.

In the blood a local drop in pH would promote unloading of O2 at metabolically active tissues and loading of

O2 is facilitated when CO2 and protons dissociate from Hb at the lung alveoli, the latter mostly being relevant

in low O2 environments. Early studies have favored the view that the Bohr effect generally for all vertebrate

Hbs originates from a few key cationic groups that raise their pKa in the T→R transition as they form salt

bridges with chloride, phosphates or carboxylates in the T state and are free in the R state (Perutz, 1983).

Especially histidines and in particular one, 146His of both β chains, has been and is considered responsible

for approx. 40-60% of the fixed acid Bohr effect, (Berenbrink, 2006; Busch et al., 1991; Perutz, 1983; Shih

et al., 1993) (figure 4A). According to this theory, at low pH the Bohr protons will primarily protonate α-

amino termini and the C-terminal His (146His) of the β-chain thus stabilizing the deoxy T-state by formation

of a salt bridge with Asp94 of the same β chain (Perutz, 1970; Perutz, 1983; Van Beek and De Bruin, 1980).

The vicinity of the negatively-charged Asp raises the pKa of the β146His imidazole ring (Im) (𝑝𝐾𝑎 =

− log 𝐾𝑎 = − 𝑙𝑜𝑔[𝐼𝑚−][𝐻+]

[𝐻𝐼𝑚]) to a value substantially higher than in the oxy form, making H+ binding more

likely in deoxy T state Hb.

Furthermore, the α-chain C-termini α141Arg (figure 4B) has been attributed to stabilize the Hb deoxy C-

terminus by binding a Cl- ion together with α1Val (Perutz, 1970). Oxygenation-induced conformational

change breaks the ionic bond and induces deprotonation of the C-termini back to the NH2-form. This residue

thus has been considered to contribute to the Bohr effect in the presence of Cl- (Lukin and Ho, 2004). Newer

reports attributing the Bohr effect to almost exclusively histidine residues also estimates the β146His residue

A B

20

to be responsible of approximately 60% of the human HbA Bohr effect in the presence of 0.1M Cl-

(Berenbrink, 2006; Lukin and Ho, 2004).

The alkaline Bohr effect has historically largely been considered ascribable to a difference in pKa of

specific amino acid residues, from high pKa (and high affinity for H +) in the unliganded deoxy T form to low

pKa (and low affinity for H+) in the liganded R-state (Perutz, 1970; Perutz, 1983; Shih et al., 1993). Addition

of anions will also affect the contribution of each residue involved, and the Bohr effect is sensitive to

position and interactions of all hydrogen and anion binding sites in the molecule (Busch and Ho, 1990; Lukin

and Ho, 2004; Sun et al., 1997). Collectively the presence or absence of substitutions at or near a few

number of sites at first hand seems to determine Hb-O2 affinity, aligning with the neutral theory of molecular

evolution (Kimura, 1968; Perutz, 1983). However, as we shall also see in this thesis and Paper III, some

species retain “normal” Bohr coefficients although lacking what is still considered key residues (Paper III:

Revsbech et al., 2013b).

For all allosteric effects in Hb, it has for several species been shown that a palette of different substitutions at

a far distance from the ligand binding sites can also modify function of Hb, whereby small contributions

from many mutations may produce cumulative effects on function, again depending on the presence or

absence of other mutations, a phenomenon linked to genetic epistasis (Natarajan et al., 2013; Natarajan et al.,

2015; Tufts et al., 2015).

All in all, low in vivo levels or decreased Hb sensitivity of one or more of the mentioned effectors will cause

a higher O2 affinity of Hb, but will also limit the regulatory potential of this affinity. Readily reversed

regulations of the erythrocyte concentrations of effectors, e.g. the transient rise in human red cell DPG upon

translocation to high altitude favoring O2 unloading (Lenfant et al., 1968), is considered acclimatization

restrained by the phenotypic plasticity of the individual. On the other hand, regulations of intrinsic affinity or

sensitivity towards cofactors through structural changes in the Hb, is considered genotypic adaptation. Either

type of regulation may adapt an animal to its environment. Animals native to hypoxic environments like high

altitude are often characterized by a strategy of structural changes in the Hb protein chains affecting O2

affinity, thus making them genetically adapted to their environment (Bunn, 1980; Monge and León-Velarde,

1991; Weber, 2007).

21

HEMOGLOBIN – AMINO ACID SUBSTITUTIONS AND COFACTOR

VARIATION AT PLAY

Adjustments in blood O2 delivery with altitude is a product of organismal adaptations (e.g. higher capillary

density, higher lung diffusive conductance) and cellular to molecular adaptation. Changes can be at the level

of concentration differences of effectors affecting Hb-O2 binding, as well as changes in the concentration,

function and structure of the Hb molecule itself, affecting intrinsic O2 affinity or efficacy of effectors. In low

altitude human subjects translocated into moderate altitude below 2000m a rise in erythrocyte DPG is part of

the response, largely because stimulated erythropoiesis with height yields a greater fraction of young

erythrocytes in circulation. Young RBCs have a higher glycolytic rate and therefore a higher formation of

DPG (Lenfant et al., 1968; Mairbäurl, 1994). The reduced Hb-O2 affinity improves overall O2 delivery

despite a slight loss in arterial saturation (Mairbäurl, 1994; Turek et al., 1973; Weber, 2007).

At moderate altitude ventilatory compensated hypoxia leads to alkalosis and the decreased pH will via the

Bohr effect push Hb P50 within normal sea level values, compensating for the effects of DPG. It is not before

extreme altitudes that a right shifted O2 binding curve becomes counterproductive and a higher Hb O2

affinity favors O2 delivery. The limit is theoretically above 5000 meters in man (Samaja et al., 1986), but

will vary between species according to specific physiological limitations. High Hb-O2 affinity compared to

conspecifics is often observed in species adapted to high altitude, for mammals examples are the Andean

Chinchilla brevicaudata resident in 3000-5000m (Ostojic et al., 2002) and in North America the deer mouse

Peromyscus maniculatus resident at sealevel-4300m at least (Snyder, 1982; Storz et al., 2007). I will in the

following consider first genetic adaptation to altitude and in the next paragraph give examples of phenotypic

plasticity responses.

ENVIRONMENTALLY LOW OXYGEN SELECTS FOR HIGH HEMOGLOBIN-O2 AFFINITY

In high altitude native animals, genotypic specialization in Hb-O2 affinity is predominant. A classic example

is the bar-headed goose that flies over the Himalayas at 9000 m and can tolerate extreme hypoxia. This

tolerance is thought to be due mainly to one Hb substitution compared to the lowland graylag goose;

α1119Pro→Ala that deletes a contact to β155 and thereby destabilizes the α1β1 interface T-structure of HbA

(birds coexpress two Hbs, a major HbA and a minor HbD), giving rise to high O2 affinity (Jessen et al.,

1991; Liang et al., 2001; Perutz, 1983). In addition, two substitutions on the α chain of HbD may affect

binding of the avian organic phosphate, inositol pentaphosphate (McCracken et al., 2010), which could be

expected to lead to an even higher O2 affinity of the high affinity avian Hb D isoform. In a different part of

the world, the Andean goose also exhibiting high Hb-O2 affinity shows a substitution deleting the exact same

contacts (β55Leu→Ser) (Jessen et al., 1991). However, it was later found that this last substitution is not

unique to altitude adapted species (McCracken et al., 2010).

22

High altitude Andean camelids llama and guanacho have Hbs with lower DPG sensitivities compared to

most mammals (Petschow et al., 1977; Scott et al., 1977). In the llama this is considered due to an

β2His→Asn substitution that eradicates two out of the usual DPG contacts in the Hb tetramer yielding a

threefold lower binding constant of DPG to llama Hb compared to camel Hb (Bauer et al., 1980). An array of

mammals expresses multiple isoHbs. Alpaca are expressing 55% fetal Hb with a higher O2 affinity

(Reynafarje et al., 1975). Yaks have two major fetal Hb components and two to four major adult Hb

components, that via different O2 affinities extend the altitudinal range of the animals (Weber et al., 1988).

Deer mice are amongst the more intricate, expressing a complex Hb polymorphism with two α-globin genes

and two β-globin genes (Storz et al., 2007; Storz et al., 2009). The Hb composition of highland mice yields

slightly higher intrinsic O2 affinities, that, in combination with lowered DPG sensitivity and lowered Cl-

sensitivities, results in an elevated blood O2 affinity of high compared with low altitude mice (Storz et al.,

2009; Storz et al., 2010). Deer mice do at the same time show curious elevations of erythrocyte DPG

concentrations in high altitude populations compared with low altitude populations (Snyder, 1982), as was

also later found by our group to be part of the phenotypic plasticity of these animals (Paper II: Tufts et al.,

2013) and discussed in the following paragraph.

TEMPORARY LOW OXYGEN LINKED WITH PLASTIC ADAPTATIONS IN HB-O2 AFFINITY

High altitude adapted deer mice populations possess genetically adapted Hbs with higher O2-affinities.

Concurrently, high altitude mice have elevated erythrocyte DPG. High DPG would reduce Hb-O2 affinity

and thus seems counterintuitive in an adapted population, albeit the significance may be small due to the low

DPG sensitivity of highland mice Hb. Interestingly, we found DPG levels of high altitude mice to be lowered

upon 6 week translocation to low altitude (Paper II: Tufts et al., 2013). This implies that diverse DPG levels

inside the RBCs are part of the phenotypic plasticity of deer mice, even further broadening their abilities to

handle very different environments. One can speculate that highland mice may have a constant higher red

cell turnover and that the elevated levels of DPG of highland mice red cells may be an unavoidable side-

effect of the increased red cell glycolysis of young erythrocytes, as first proposed by Snyder (Snyder, 1982).

High DPG levels are then further compensated by the evolution of a low sensitivity toward this allosteric

effector and thus high altitude adapted deer mice retain high Hb-O2 affinity. When translocated to low

altitude, the mice acclimatize and DPG levels are no longer elevated (Paper II: Tufts et al., 2013). Or perhaps

the elevated DPG has roots in population admixture (Storz et al., 2010), retaining it as an acclimatization

response. Variations in organic phosphates to accommodate needs of increased O2 affinity that are not

permanent are also known in fish exposed to hypoxia (Lykkeboe and Weber, 1978; Weber and Lykkeboe,

1978). As well as in hibernating mammals (Burlington R.F. and Whitten, 1971; Harkness et al., 1974;

Kramm et al., 1975; Maginniss and Milsom, 1994; Tempel and Musacchia, 1975) as we also found in the

large hibernator, the brown bear (Paper I: Revsbech et al., 2013a)

23

Blood hematocrit and Hb concentration will also affect O2 capacity, but have not consistently been measured

to rise or fall with hibernation, perhaps due to sampling at very different periods of the hibernating state, as

observed in woodchucks (Harkness et al., 1974). Elevated hematocrits have been observed in translocated

humans (Monge and León-Velarde, 1991), in high altitude in the Russian wood mouse and in the deer mouse

(Hock, 1964; Kalabuchov, 1937; Paper II: Tufts et al., 2013), decreasing again with low altitude adaptation

(Reynafarje et al., 1975; Paper II: Tufts et al., 2013), thus classifying these changes as part of phenotypic

plasticity. It is intriguing to see the exact same plastic acclimations as in humans persist as part of the

phenotype of a high altitude adapted deer mouse population.

HIBERNATORS UTILIZE TEMPORARY PHENOTYPIC PLASTICITY IN OXYGEN TRANSPORT

Hibernators downregulate metabolism to extremely reduced levels (see box 1), implying also a much

reduced rate of breathing and heart beats, in bears breathing is reduced to 1-2 breaths/min and heart rate to ~

14 beats per min from ~55 beats per min in summer resting state (Tøien et al., 2011). This implies, that even

though the external environment has plenty of available O2, the internal environment of the bear does not,

and also must not (Paper I: Revsbech et al., 2013a). Hibernation is a temporary state, in many species further

interrupted by arousals (see box 1), and thus adaptations in O2 transport need to be reversible to optimize

delivery in both the hibernating and the normothermic states. Species like the hibernating hedgehog, the 13-

lined and the golden mantled ground squirrel, as well as our investigated brown bear have been shown to

increase their blood O2 affinities during hibernation compared to the euthermic state (Bartels et al., 1969;

Clausen and Ersland, 1968; Maginniss and Milsom, 1994; Musacchia and Volkert, 1971; Paper I: Revsbech

et al., 2013a).

Elevations in Hb-O2 affinities are highly attributable to direct effects of fall in body temperature on the Hb-

O2 equilibrium curve, but a temperature independent component has also been proposed, namely DPG. In

13-lined and golden-mantled ground squirrels, woodchucks and golden hamsters, a clear reduction in DPG in

the range 39-45% has been detected during hibernation (Burlington R.F. and Whitten, 1971; Harkness et al.,

1974; Kramm et al., 1975; Maginniss and Milsom, 1994; Tempel and Musacchia, 1975) and some studies

including work from our group on the brown bear (Paper I: Revsbech et al., 2013a) have proposed a direct

coupling between decreased DPG and rise in Hb-O2 affinity (Kramm et al., 1975; Paper I: Revsbech et al.,

2013a). In contrast are our studies in smaller hibernators: In six marmotine ground squirrels investigated in

Paper III, and perhaps general to rodent Hbs (Clementi et al., 2003; Runck et al., 2010; Storz et al., 2012),

sensitivity of Hb to the anion DPG is generally low (Paper III: Revsbech et al., 2013b). A low DPG

sensitivity makes the hibernation-induced observed DPG decreases across a large range of smaller

hibernating rodents unlikely to have any substantial effect on Hb-O2 affinity. Thus our work with small

rodent hibernators does not support the hypothesis of a direct relationship between decreases in allosteric

effector DPG and increases in Hb-O2 affinity during hibernation in rodents (Burlington R.F. and Whitten,

1971; Maginniss and Milsom, 1994; Tempel and Musacchia, 1975).

24

PERTURBATION OF THE DONNAN DISTRIBUTION OF IONS

Instead of a direct effect of decreases in red cell cofactor DPG on Hb- O2 affinity in the marmotine ground

squirrels investigated in Paper III, we suggest the possibility of an indirect relationship; namely that

decreases in the anion DPG via perturbation of the Donnan equilibrium of ions across the erythrocyte

membrane may still effect a decrease in P50 during hibernation. Fall in intraerythrocytic nondiffusable

polyanion DPG will cause an equal decrease in intracellular H+ and thus a rise in pH. In Paper III we found a

normal Bohr effect of the investigated Hbs, implying that the observed fall in DPG during hibernation may

This modeling is an example of the importance

of not only changes in Hb-O2 affinity in

acclimatizing an animal to control low O2.

Rather changes in the red cell environment

affects multiple parameters and they all interact

in the adaptation or acclimatization to the

conditions.

Figure 5. Modelled oxygen content curve with arterial and mixed venous points.

Modified from Paper I: Revsbech et al., 2013.

BOX 3: HEMOGLOBIN COOPERATIVITY SAVES THE DAY – OR THE WINTER

In the hibernating brown bear, another factor crucial for the nature of O2 transport is altered during

winter hibernation, namely the cooperativity coefficient, n. The decrease in cooperativity of winter

hemolysate observed by our group (Paper I: Revsbech et al., 20013a) seems to have its roots mainly

in the decreased DPG content (Tyuma et al., 1971), as n50 of hemolysate did not differ between

temperatures, but only between summer and winter state. This was furthermore reproducible in

purified Hb added the measured DPG content from summer and winter.

The difference in n50 between hibernating and summer active bear hemolysates, did by our modeling

play an important role in vivo in achieving a suitable venous PO2 despite the high winter hematocrit

(Paper I: Revsbech et al., 20013a). As can be visualized from the oxygen content curve (figure 5)

below, the changes in P50 and n50 collectively shifted the curve from a theoretical winter PvO2

substantially higher than the summer level, to one very close to the summer PvO2. In hibernation it

must be crucial for the bear to closely balance O2 delivery, and thus a high venous tension may have

provoked untimely O2 delivery and thus production of reactive oxygen species and oxidative stress.

25

have this indirect effect on Hb-O2 affinity, although the fall in Tb is likely to be the prominent effector (Paper

III: Revsbech et al., 2013b). However, for the larger and warmer hibernator, the brown bear (see box 1),

DPG also decreases during hibernation and this decrease together with the limited fall in Tb is in this

hibernator sufficient to explain the observed elevation of bear O2 affinity of hemolysate (Paper I: Revsbech

et al., 2013a). Taken together, decrease in DPG works directly in some hibernators like hedgehogs and bears

towards aiding a high Hb O2 affinity (Kramm et al., 1975; PaperI: Revsbech et al., 2013a), but may in others,

like grounds squirrels, marmots and woodchucks contribute only little or none (Harkness et al., 1974; Paper

III: Revsbech et al., 2013b).

THE WHOLE PICTURE VERSUS FEW IMPORTANT BINDING SITES

In the above mentioned study our group compared amino acid substitutions with functional data and found

highly unusual substitutions involving an expectedly detrimental substitution β146 His→Gln in the major Hb

β chain of the golden mantled ground squirrel. β146 His is considered responsible for ~ 60% of the Bohr

effect for all Hbs (Berenbrink, 2006). However, functional analysis showed a normal Bohr effect of this Hb.

Likewise, the observed low DPG sensitivity coincided in all six species with contained cationic residues

considered implicated in DPG binding in human Hb (Paper III: Revsbech et al., 2013b), namely β-chain

1Val, 2His, 82Lys and 143His (Richard et al., 1993). Conclusively, not all species seem to conform to the

human model of Hb function, or to theory dictating only a few amino acid residues to be responsible for

overall function. Instead, our study on ground squirrels amongst others (Natarajan et al., 2013; Natarajan et

al., 2015; Paper III: Revsbech et al., 2013b; Tufts et al., 2015) suggest that the general genetic background

and multiple substitutions rather than only a few key residues has a major impact on the Hb-O2 affinity.

26

FINE-TUNED REGULATORS OF O2 DELIVERY AND CONSUMPTION:

INTRODUCTION TO NO AND H2S

Oxygen arrives to tissues by bulk transport bound to Hb inside the RBCs, as described earlier in this

introduction. Part of fine-tuning both O2 delivery and consumption, amongst a cascade of other signaling

effects, are the endogenously produced gaseous messengers hydrogen sulfide, H2S, and nitric oxide or

nitrogen monoxide, NO. Both NO and H2S are ubiquitous cellular signaling molecules naturally synthesized

and degraded in peripheral tissues by endogenous enzymes. NO and H2S are now recognized as signaling

molecules invoking physiological function. Together with carbon monoxide (CO), they are collectively

referred to as gasotransmitters. Both NO and H2S are highly toxic at high concentrations, but have signaling

effects in lower concentrations. Their physiological functions involved in O2 delivery and consumption range

from control of blood vessel dilation (NO and H2S in systemic arteries) or contraction (H2S in pulmonary

vessels) to cytoprotection and inhibition of respiration. Albeit normally produced in a O2- dependent manner,

all production is not lost at low O2 conditions, as both NO and H2S can be reconstructed from oxidated end

products during particular conditions (Lundberg et al., 2008; Olson et al., 2013).

Reaction products of both NO and H2S may modify proteins at reactive cysteine residues and radically alter

function, causing major downstream effects (Paulsen and Carroll, 2013). Redox signaling through site-

specific and reversible cysteine modifications enables an oxidant signal to be turned into an appropriate

biological response within and between cells. Both H2S and NO are proving increasingly interesting to study

as they appear involved in multiple adaptations and responses to hypoxia. Both alleviate

ischemia/reperfusion damage mainly through respiratory inhibition of cytochrome c oxidase (CcOx) in the

electron transport chain of mitochondria. NO plays a key role in anoxia and hypoxia adaptations from anoxia

in red-eared slider turtles and crucian carps in the natural overwintering habitat of frozen ponds (Jacobsen et

al., 2012; Jensen et al., 2014; Sandvik et al., 2012) to high altitude adaptations in Tibetan highland human

residents (Erzurum et al., 2007). H2S has the ability to induce an artificial hibernation-like state (Blackstone

et al., 2005), making it a molecule of special interest to study in natural hibernators. The current paragraph

serves to introduce NO and H2S as physiological players. In the following I will focus on mechanisms and

occurrence of selected NO and H2S mediated changes in physiology that could be relevant to mainly

hibernators. Cardiocascular origins, fates and effects of the two molecules are summarized in figure 6 at the

end of this paragraph. Some applications may also be relevant in high altitude adaptation as I will discuss

under future research.

27

NITRIC OXIDE – A SHORT INTRODUCTION

NO is a well-established gaseous signaling molecule with a palette of different effects on blood flow and

metabolism. NO is a free radical and stabilizes its unpaired electron by binding other compounds with

unpaired electrons. It also interacts with d-orbitals of transition metals of both non-heme and heme irons

(Thomas et al., 2003). In particular, NO has a high affinity for ferrous heme. One effect is through binding to

the heme of soluble guanylate cyclase in the endothelium causing an induction of a vasodilatory cascade

(Moncada and Higgs, 1993). Another effect is transient inhibition of respiration by binding to cytochrome c

oxidase (CcOx), complex IV in the mitochondrial electron transport chain (Cleeter et al., 1994). NO plays an

important role in alleviating ischemia/reperfusion injury through metabolic inhibition and additionally,

through binding to a reactive cysteine on complex I by S-nitrosation, suppresses reactive oxygen species

(ROS) generation in the mitochondria (Chouchani et al., 2013). All effects will be discussed below.

NO PRODUCTION AND LIFETIME

A family of NO synthesizing enzymes, NOS enzymes, generates NO endogenously by catalyzing an

NADPH and O2-dependent oxidation of L-arginine to L-citrulline and NO by the help of a number of

cofactors (Alderton et al., 2001; Li and Poulos, 2005). The isoform endothelial NOS, eNOS was first found

in the vascular endothelium, but since also discovered in epithelial cells, cardiac myocytes and neurons

(Dudzinski and Michel, 2007). The form eNOS can be upregulated by sheer stress of vasculature blood flow,

enabling close coupling of eNOS regulation to forces of flow (Balligand et al., 2009). Neuronal NOS, nNOS,

is found in neurons, epithelial cells and skeletal muscle. Inducible NOS, iNOS, can generate the highest

levels of NO in response to inflammatory stimuli in a great variety of tissues. All NOS isoforms are

regulated in activity and location by Ca2+ and calmodulin in addition to posttranslational modifications,

cofactor availability and substrate levels (Kone et al., 2003).

NO has a half-life of 2 ms to 2 seconds in vasculature, in effect keeping NO signaling effects local (Thomas

et al., 2001). NO is consumed by reaction with oxygenated Hb or Mb, to create nitrate, NO3-, (Moncada and

Higgs, 1993; M. P. Doyle and J. W. Hoekstra, 1981). In reaction with freely dissolved O2, NO is transformed

into nitrite, NO2-. A major avenue of O2 –independent production of NO away from site-specific NOS is by

reduction of the metabolite nitrite, NO2-, to NO (Lundberg et al., 2008). Heme proteins such as deoxygenated

hemoglobin (Cosby et al., 2003) and myoglobin (Shiva et al., 2007a) can exhibit high nitrite reductase

activity. This enables what was former considered a product alone (NO2-) to regenerate the active form NO

under hypoxic conditions. Nitrate can additionally be converted to nitrite by one of two ways to uphold

nitrite levels. One way is via the ubiquitous enzyme xanthine oxidoreductase, that also converts nitrite to

NO, and is able to convert nitrate to nitrite although the latter reaction is much slower (Jansson et al., 2008).

The other avenue is by a bacterial symbiosis with oral bacteria converting dietary and recycled nitrate

excreted in the salivary glands to nitrite, later taken up in the gut (Lundberg et al., 2008). Dietary

28

supplementation with nitrite or nitrate thus seems to alleviate ischemia/reperfusion injury in various settings

(Lundberg et al., 2009).

NO PRODUCTS AND FUNCTIONS

NO induces and regulates a broad range of biological functions, typically by binding either to ferrous iron in

heme proteins creating iron nitrosyl compounds, FeNO, or by binding to protein thiol groups, S-nitrosation,

SNO, or to aromatic rings, causing nitration (Hill et al., 2010).

The signaling action of NO depends strongly on the presence of other free radicals. Once reacted with

superoxide, O2-, NO forms peroxynitrite ONOO-, a nitrating and oxidating agent that may modify protein,

lipids, and DNA, but no longer interact with ferrous heme (McAndrew et al., 1997). Some target specificity

is achieved by location of O2- production, as superoxide is not freely diffusible across cell membranes (Hill

et al., 2010). Targets are cysteine and methionine (S-containing amino acids) as well as their incorporated

forms in protein thiols, with the latter reaction being much slower (Hill et al., 2010). Aromatic rings (amino

acids tryptophan and tyrosine) are also prone to nitration (Hill et al., 2010). Nitration of proteins can also

happen through other avenues. Nitrogen dioxide, NO2 and dinitrogen trioxide, N2O3 are byproducts of the

reaction of NO with O2, and may also cause nitration or S-nitrosation alone or in combination with ROS

radicals (Hill et al., 2010). In hypoxia an avenue of N2O3 production requires acidic conditions and high

concentrations of nitrite, as nitrous acid, HNO2 is in equilibrium with N2O3 in water (see Fago and Jensen,

2015). All protein modifications via NO are likely to be part of complex NO signaling.

In hypoxic conditions NO generated from nitrite reduction by deoxy hemoglobin will in vasculature bind

soluble guanylate cyclase, causing an increase in local vasodilation and blood flow in accord with hypoxia as

explained in detail further below (Moncada and Higgs, 1993). Nitrite is found in tenfold higher

concentrations in intracellular compartments in the hypoxic heart of crucian carp, in goldfish and in the red-

eared slider turtle, possibly by transfer from extracellular location or increased NOS expression (Hansen and

Jensen, 2010; Jensen et al., 2014; Sandvik et al., 2012). In hypoxic organs, NO binding to ferrous heme iron

in mitochondrial CcOx is competitive with O2 binding (Cleeter et al., 1994). Thus NO-induced metabolic

inhibition as well as vasodilation is most prevalent in low O2 conditions as during ischemia, where local

deoxy myoglobin will also supply NO generation from nitrite (Totzeck et al., 2012). During ischemia,

Complex I and III of the respiration chain become highly reduced because of electron loading from reducing

equivalents, causing a high potential for superoxide and other ROS production upon reoxygenation. NO has

the ability to prevent reverse electron transport by keeping complex I in an inactive state due to SNO

modification (Babot and Galkin, 2013). The reactant is likely N2O3 that will react with a specific cysteine

residue on complex I, which will be inhibited by the resulting S-nitrosation, thus blocking this main site of

ROS generation when reperfusion takes place (Babot and Galkin, 2013; Chouchani et al., 2013; see Fago and

Jensen, 2015). In low O2 conditions the available NO will largely stem from nitrite reduction, explaining

why additional nitrate or nitrite supplementation decrease ROS production (Shiva et al., 2007b). Tissue

29

specific increases of FeNO and SNO compounds can be used to indicate areas of NO production from nitrite

during hypoxia (Sandvik et al., 2012).

HYDROGEN SULFIDE – A SHORT INTRODUCTION

Hydrogen sulfide (H2S) is easily recognized by the rotten egg odor and is known as a potentially lethal

acquaintance if inhaled in too large doses. However H2S has within the last two decades been recognized as a

signaling molecule invoking physiological function akin to other gaseous species such as NO. H2S is

endogenously synthesized and is a small molecule that easily diffuses across cell membranes (Mathai et al.,

2009) with potential to be involved as a signaling agent in a plural of physiological processes. Possible H2S

involvement has been documented ranging from vasocontrol to neuromodulation, cardio protection,

metabolic inhibition and anti-inflammatory activities (Kimura, 2009). Research in possible involvement of

H2S and its metabolites in physiology have literally exploded in the past two decades and resulted in a wide

range of sulfide-signaling now known of in smaller or greater detail. Despite the great interest in H2S,

uncertainty still exists of what form or forms of H2S exhibits biological activity. Under physiological

conditions (37°C and pH 7.4) only 18.5% of free hydrogen sulfide exists as H2S, while the remainder

dissociates to HS- and H+ with trace amounts of S2- (Dombkowski et al., 2004; Hughes et al., 2009). H2S, HS-

, or even products of H2S combining with proteins may be the active form or forms of H2S signaling. Or

perhaps many forms have each their special reactions and activity. The field still refers to sulfide signaling as

H2S signaling, and thus, so will this review.

PRODUCTION AND POOLS OF SULFIDE

At least three endogenous enzymes produce H2S. H2S is created from cystathionine -lyase (CSE),

cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MST), from substrate L-cysteine

and homocysteine as well as 3-mercaptopyruvate, in the case of MST. 3-mercaptopyruvate is produced from

cysteine and α-ketoglutarate by cysteine aminotransferase (Shibuya et al., 2008). In humans the enzymes are

mostly site-specific in their distribution with CSE found predominantly in the smooth muscles of

vasculature, MST in vasculature endothelial cells, RBCs and in mitochondria, and CBS in brain, liver and

kidney (Calvert et al., 2009; Kabil et al., 2014) Kimura 2014, Szabó 2007, Vitvitsky et al 2015). Only the

zinc-dependent protein MST is found in mitochondria as well as the cytosol of cells, whereas CSE and CBS

are heme proteins with primary cytosolic location.

Hydrogen sulfide is constantly produced and oxidized at a tissue-specific rate, enabling an efficient control

of H2S tissue levels (Vitvitsky et al., 2012a). The exponential decay of H2S with time is highest under

aerobic conditions as it primarily occurs via a mitochondrial sulfide oxidation pathway involving an O2

consuming step (Vitvitsky et al., 2012b). A mechanism for O2-dependent methemoglobin (MetHb) catalyzed

H2S turnover in RBCs has moreover recently been found (Vitvitsky et al., 2015). MetHb is the state of Hb

where the heme iron is oxidized to the ferric state, Fe3+ instead of normal ferrous Fe2+ state, rendering the Hb

30

non-functional for O2 transport until reduced by methemoglobin reductase. The oxidation of H2S by metHb

seems also mostly O2 dependent, but ironbound polysulfides (e.g. FeII-S-S-SH) may be created under low O2

conditions (Vitvitsky et al., 2015). This finding indicates that other massively present heme proteins around

the body, with myoglobin Mb as the likely best candidate, also may play a role in H2S equilibrium and

creation of reactive metabolites, much akin to deoxy Hb and Mb regenerating NO from nitrite. Estimated

free H2S half-life is between 2 and 10 minutes in bovine liver, kidney and brain homogenates (Vitvitsky et

al., 2012a). Normal steady-state levels of free H2S are debated as measurements depend highly on techniques

that have only recently become precise enough. In the order of 14-17nM as found in whole tissue liver and

brain of mice (Furne et al., 2008) up to a few micro molar (µM) as found by our group in bears seems within

a realistic range of endogenous H2S (Paper IV: Revsbech et al., 2014). All H2S products can be divided into

pools: (1) free sulfide, containing mainly freely dissolved H2S and HS-; (2) Bound sulfane sulfur (BSS), the

products of H2S oxidation, containing thiosulfate S2O32- and polysulfides (e.g. RSSSR) that can be converted

to H2S under reducing conditions; (3) acid-labile sulfur, containing mainly Fe-S clusters and persulfides (eg.

RSSH), that are considered convertible into H2S only under rare acidic conditions (Kolluru et al., 2013).

SULFUR BINDING TO PROTEIN THIOLS AND HEME GROUPS CAN ALTER FUNCTION

H2S can be stored as bound sulfane sulfur (BSS) on protein cysteine (thiol) residues. BSS is created by a yet

not fully determined range of reactive sulfur species combining preferably with a previously oxidized thiol

species, e.g. cysteine disulfide (R-SSR) or sulfenic acid (R-SOH) by the process of sulfuration (also known

as S-sulfhydration ) to form persulfides (RSSH or HSSH) and polysulfides (e.g. RSSSR) (Francoleon et al.,

2011; Mustafa et al., 2009; Park et al., 2015). It should here be noted that the number of active or available

cysteine thiols in selected proteins may be background for species differences. Any addition of cysteines

yield sites for posttranslational modifications like S-nitrosation and sulfuration and can potentially affect

function of proteins and alter enzymatic capability as part of redox signaling. This yields a great potential for

downstream signaling. Hyper-activation is the result of sulfuration in cysteine, CysSSH as explained further

below (Ida et al., 2014) as well as increased catalytic activity for glyceraldehyde-3-phosphate dehydrogenase

GAPDH. Oppositely, inhibition following persulfidation has been observed for glucose 6-phoshate

dehydrogenase and other erythrocyte enzymes (Valentine et al., 1987). NO-induced S-nitrosation of proteins

and enzymes on the other hand often inactivates function, as seen for GAPDH-induced incorporation of

hemes into proteins (Chakravarti et al., 2010).

Per- and polysulfides have recently been seen to elicit highly interesting reactions. In brain tissue a sizable

proportion of the sulfur pool is in the bound state, which may serve to keep free H2S low in order to avoid

toxicity and to function as a readily released storage (Ishigami et al., 2009). Persulfides are now emerging as

highly potent compounds themselves, and recently it was shown that CBS and CSE can generate substantial

amounts of CysSSH when utilizing cystine (CysS-SCys) rather than singular cysteine (Ida et al., 2014).

CysSSH appear to react with endogenous glutathione (GSH) forming GSSH persulfides and polysulfides

with high occurrence as well as uniquely potent nucleophilic and antioxidant/reducing activities in mammals

31

(Ida et al., 2014). Where parental H2S is mainly involved in reduction of cysteine disulfide bonds (Kimura,

2015), Polysulfides (R-Sn-R) have also recently been shown to be potential signaling molecules acting by the

addition of sulfur to cysteine (thiol) residues on target proteins, generating BSS by sulfuration (Kimura,

2015). It is likely that several species currently considered H2S products have their distinct targets and

reactive functions, with some likely overlapping and contributing to one another.

H2S is primarily toxic owing to the fact that it binds to the binuclear Cu-heme iron complex in CcOx,

causing reversible inhibition of the whole terminal complex (CcOx, complex IV) of the electron transport

chain (Nicholls and Kim, 1982). Heme proteins exist in a great variety in cells and many of them could

potentially bind H2S. Proteins like myoglobin and Hb typically have a low affinity for H2S binding in

mammals (Nguyen et al., 1998), but could potentially still play a role due to their massive presence alone. To

prove this point stands the recent finding that the low levels of metHb normally present in blood (1-3% of

total Hb), are sufficient to bind and oxidize H2S to thiosulfate and heme –iron bound polysulfates in RBCs,

thus clearing H2S from circulation (Vitvitsky et al., 2015). OxyHb is then speculated to be recreated by

MetHb reductase releasing the ironbound polysulfides (Vitvitsky et al., 2015).

32

Figure 6. Overview of H2S and NO origin, principal fates and effects with focus on the cardiovascular (CV) system. For references, see text. CcOx: cytochrome c oxidase, complex IV of the electron transport chain.

33

FINE- TUNED REGULATORS OF DELIVERY AND CONSUMPTION AT

PLAY: NO AND H2S

In this paragraph I will focus on mechanisms and occurrence of selected NO and H2S mediated changes in

physiology that could be relevant to mainly hibernators. As I have worked with these gaseous signaling

molecules in potentially regulating delivery and consumption in hibernating animals (see Paper IV and

ongoing projects), I will focus on the following effects: vasodilation, antioxidant effects and metabolic

inhibition.

ON THE PERFUSION SIDE: NO AND H2S AFFECT BLOOD VESSEL DIAMETER

NO primarily mediates vasodilation in larger conductance vessels and has been recognized as a major

endothelium-derived relaxing factor (Nagao et al., 1992). Endothelium-derived hyperpolarizing factors also

control vessel tone and are dominant in the smaller resistance blood vessels that generally determine blood

pressure. H2S has been shown to be a major relaxing factor of the latter kind (Mustafa et al., 2011; Yang et

al., 2008). Via activation of three channels, the ATP-sensitive K+ as well as the intermediate and small

conductance Ca2+ sensitive K+ channels, H2S facilitates vasodilation in systemic arteries (Mustafa et al.,

2011; Zhao et al., 2001). In case of the ATP-sensitive K+ channel, the activation is now known to be due to

sulfuration or sulfhydration (addition of an –SH group) to a cysteine residue in the subunit region where ATP

binding would normally reduce activity (Mustafa et al., 2011). NO works through a different avenue. In the

vasculature, cholinergic stimulation (release acetylcholine) or physical shear stress in the vessel wall induces

Ca2+ dependent activation of eNOS that lead to NO production. NO freely diffuse into nearby smooth muscle

of the vessel wall to activate the heme-containing soluble guanylate cyclase, which by cGMP generation

cause vasorelaxation (Moncada and Higgs, 1993; Waldman and Murad, 1988). Possibly, a synergistic effect

of NO and H2S in combination may also exist on systemic vascular smooth muscle relaxation (Coletta et al.,

2012; Hosoki et al., 1997).

Localized vasorelaxation and resulting dilation of the vessel will increase blood flow to specific organs or

tissues, unless countered by other stimuli. Differences in utilized pools, but not in total quantity, of

endogenous H2S metabolites have been observed by our group during summer active and winter hibernating

states of brown bears, suggesting consumption of free H2S during hibernation (Paper IV: Revsbech et al.,

2014). During hibernation the sympathetic nervous system is responsible for keeping a marked

vasoconstriction in peripheral blood vessels, Likely foremost to keep blood pressure at reasonable levels

despite the decrease in heart rate (Lyman and O’Brien, 1963; Lyman et al., 1960; Tøien et al., 2011) Also,

diving mammals are capable of a similar response during breath-hold dives (Bron et al., 1966). The strong

adrenergic tone seen in peripheral vessels of many hibernators and divers would most likely prevent any

general vasodilatory effect of H2S and NO. This would serve to uphold the peripheral vasoconstriction that

34

may, besides keeping a stable blood pressure, be part of a conserved adaptive trait aiding metabolic

allocation to important organs during hibernation and breath-hold dives.

Nitrite in the circulation will surely be converted to NO in low O2 environments because of a higher quantity

of deoxy hemoglobin and myoglobin. It has been shown that sensitivity to NO donor-induced vasodilation is

unaltered in hibernation compared to euthermic state (Karoon et al., 1998). Given the high peripheral

vasoconstriction, NO-induced dilation of large conducting vessels would further enable these to contain most

of the cardiac output (Fago and Jensen, 2015). It is still plausible, that some degree of e.g. critical organ

(heart, brain) microcirculation regulation in the hibernating animal is regulated to avoid hypoxia by

compounds such as NO and H2S during hibernation. Even more so as mammalian RBCs may be able to

regulate their own H2S levels, as just discovered in human RBCs (Vitvitsky et al., 2015). Besides the bear,

differentiating endogenous H2S production in a natural hibernator has been reported only in one other

species: In lung tissue of the Syrian hamster torpor increased CBS expression and thus H2S production

twofold with H2S playing a role in hibernation linked lung remodeling (Talaei et al., 2011a; Talaei et al.,

2012).

Opposite to the relaxation response in systemic arteries, H2S in pulmonary arteries of most mammals can

enhance hypoxic vasoconstriction (Olson et al., 2006; Olson et al., 2013). Hypoxic vasoconstriction may in

the lungs of a hibernator serve to optimize lung efficiency even more than recognized in non-hibernators. On

one hand arterial O2 saturation is kept high as only parts of the lung still containing high PO2 is perfused. On

the other hand, when not inflated at the normal frequency, lung segments may be restricted in blood flow and

thus less energy is consumed in an organ that is not working at normal capacity during deep hibernation: the

Syrian hamster breathes 5-7 times per minute in hibernation versus 84± 20 times per minute in awake state

(Rubin et al., 1978; Talaei et al., 2011a). The golden-mantled ground squirrel will be in apnea for about 17

minutes between breathing bouts in deep hibernation (Milsom and Jackson, 2011). Yet again, in some divers

H2S can induce hypoxic vasodilation (Olson et al., 2010).

THE BALANCE OF TISSUE OXYGEN CONSUMPTION: POSSIBLE INVOLVEMENT OF H2S

AND NO Hibernators go through an extensive metabolic reduction entailing a drastic reduction of O2 consumption. Of

organismal O2 consumption 90% is utilized directly as electron acceptor in the mitochondrial electron

transport chain during ATP production. Of particular interest is therefore the ability of both H2S and NO to

reversibly inhibit mitochondrial CcOx of the electron transport chain, impeding mitochondrial O2

consumption and energy production (Cleeter et al., 1994; Collman et al., 2009; Cooper and Brown, 2008;

Hill et al., 1984). H2S of ~80ppm given to mice induce a hibernation-like state with reversible, dose-

dependent and temperature independent reductions in O2 consumption, ventilation, CO2 production and body

temperature (Blackstone and Roth, 2005). This ability showcases H2S as a possible key element in

substantial metabolic reduction. All pools of H2S (free sulfide, bound sulfane sulfur and the acid labile pool

35

(see figure 6) are relevant to assess when evaluating the status of sulfide signaling. In our studies on brown

bears, we did not, as first expected, see a increase in concentration of free or total sulfide when evaluated in

plasma and RBCs, but rather, we saw a constant concentration but a shift in composition; a shift in the pools

(Paper IV: Revsbech et al., 2014). During hibernation, remodeling of H2S metabolism was visible in blood

plasma as a significantly reduced quantity of free sulfide and BSS, and changes in red cells approached

significance as compared to the summer active state. Erythrocytes showed higher CSE activity in winter.

From that data we propose that the plasma pool of BSS is utilized during hibernation to generate free H2S in

the RBCs, which then may diffuse to nearby tissues. At low tissue O2 levels as are potentially the case in

hibernation (see below), the generated H2S may aid metabolic depression by inhibiting mitochondrial O2

consumption (Paper IV: Revsbech et al. 2014). Our study highlights the importance of evaluating all forms

of sulfide, especially under varying metabolic demands that may induce low O2 conditions and/or changes in

acidity that could affect the bound pools of H2S. In contrast we did not detect any significant differences in

nitrite of hibernating versus awake brown bears, suggesting NO metabolism is less involved (Paper IV:

Revsbech et al., 2014).

The strong metabolic suppression of hibernating bears implies a reduced O2 consumption. This is balanced

by small O2 supply from blood with a higher affinity (Paper I: Revsbech et al., 2013a). A steady state of

reduced supply and consumption invoking a maintained tissue PO2 is theoretically possible. However, it is

also logical that a lower but sufficient O2 tension in the tissues could be the outcome, as is the case at least in

brain tissue of the hibernating arctic ground squirrel (Ma and Wu, 2008). This latter situation is additionally

supported in the brown bear by our finding of sulfide recycling that is favored by local hypoxia (Paper IV:

Revsbech et al., 2014), as well as a lower arterial PO2 measured in hibernating, anesthetized bears (Evans et

al., 2012) and in hibernating 13-lined ground squirrel (Musacchia and Volkert, 1971). Further, we have

modeled an estimate on venous PO2 in the hibernating bear and found a slightly lower value than summer

venous PO2 (Paper I: Revsbech et al., 2013a). Our modeling assumed the same arterial PO2 in summer and

winter bears, and thus our results only understate the result that hibernating bears likely operate at a lower

internal PO2, but still sufficient to sustain aerobic metabolism.

The reduced PO2 of the blood required for full saturation following a markedly higher Hb-O2 affinity in the

hibernating brown bear (Paper I: Revsbech et al., 2013a) will result in a smaller PO2 diffusion gradient from

blood to tissues. This may perhaps aid in achieving or maintaining the new metabolic state directly by

limiting O2 delivery for oxidative phosphorylation, whilst still enabling it where it is needed. The creation of

free H2S from the bound sulfane sulfur pool has until now only been observed in neurons (Ishigami et al.,

2009) as well as in isolated arteries in strong hypoxia (PO2 <5mmHg) and deoxygenated homogenized

tissues (Olson et al., 2013). In the hibernating bear we propose the same mechanism (Paper IV: Revsbech et

al., 2014), but likely occurring at only slight hypoxia in line with our hypothesis of a reduced PO2 diffusion

gradient (Paper I: Revsbech et al., 2013a).

36

There are currently to my knowledge no reports of NO involved in inducing hibernation-like states, and thus

far levels of red cell and plasma nitrite in brown bears have not revealed any significant differences between

summer and winter states (Paper IV: Revsbech et al., 2014). Instead, slightly higher circulating nitrite levels,

possibly due to elevated NOS activity, seem to be a mark of hypoxia adapted ectotherms (Hansen and

Jensen, 2010; Jensen, 2009; Sandvik et al., 2012), as well as in at least one diving mammal (Soegaard et al.,

2012) and as mentioned earlier even in humans living in high altitude hypoxia in Tibet (Beall et al., 2012;

Erzurum et al., 2007). It is still conceivable that NO could play some role in hibernation.

CYTOPROTECTIVE AND ANTIOXIDANT EFFECTS OF H2S AND NO

Despite the low O2 intake and transport levels, hibernators are not continuously hypoxic, but remain aerobic

at a low steady state. However the steady state is in small hibernators interrupted by repeated arousals, and in

bears by cyclic oscillations in Tb and metabolism (see box 1). Thus mismatches in O2 delivery to

consumption are expected. It is likely that the electron transport chain of the inner mitochondrial membrane

is reduced during the low O2 flux of hibernation. A sudden surplus of available O2, largest at the resumed

euthermic breathing and heart rate at arousal, is therefore expected to yield a pulse of reactive oxygen

species, ROS. In particular organs that drastically change their activity level, like the heart, must be under

stress. Yet, a hibernating bear is fully able to engage in fight or flight at any disturbance. Clearly hibernators

have adapted to handle all parts of hibernation and arousal well, and an efficient antioxidant system must be

involved.

The major antioxidant effect of NO lies in the inhibition of oxidant production as the production of ROS can

be inhibited by S-nitrosation of complex I of the electron transport chain, a major site of ROS generation. In

hibernating Syrian hamsters, upregulation of H2S production seems to protect against hypothermia-induced

cell apoptosis, ROS formation and acidosis (Talaei et al., 2011b; Talaei et al., 2012). Cytoprotective and

antioxidant effects have been attributed to varying concentrations of H2S and its products or downstream

signaling. H2S protects neurons against ischemia-reperfusion-injury and oxidative stress primarily by

recovering levels of the major intracellular antioxidant glutathione (Kimura and Kimura, 2004; Kimura et al.,

2009). Excessive neurotransmitter glutamate buildup following ischemic events suppresses cystine import

into cells where cystine reduction yields cysteine, the substrate for the production of major endogenous

antioxidant glutathione (GSH). This is the background for oxidative glutamate toxicity (see Kimura and

Kimura, 2004). H2S increases the activity of a rate-limiting enzyme in thiol-containing GSH production (-

glutamylcysteine synthetase) as well as reduces the cysteine/glutamate antiporter inhibition by glutamate and

thus increase cystine import (Kimura and Kimura, 2004). More importantly, H2S directly reduces cystine to

cysteine in the extracellular space and a more efficient transporter imports the cysteine, contributing

dominantly to GSH production (Kimura et al., 2009). Together, these two mechanisms ensure a markedly

higher GSH production in the presence of H2S.

37

Additionally, H2S facilitates GSH redistribution to mitochondria where GSH can be in the first line of

defense to scavenge produced ROS (Kimura et al., 2009).. As a reducing substance H2S may also exhibit

direct ROS scavenging in the mitochondria, however this direct role is minor compared to that of GSH

(Kimura et al., 2009). Although these roles of H2S in antioxidant defense has been established for neurons,

similar mechanisms could be envisaged to take place in other cells as GSH is a ubiquitous antioxidant and

H2S readily crosses cell membranes (Cuevasanta et al., 2012; Riahi and Rowley, 2014).

GSH reaction with ROS yields oxidized GSSG, which needs to be reduced back by NADPH-dependent GSH

reductase, or actively exported from the cell (Raftos et al., 2010). Thus enough reducing power needs to be

present to reduce GSSG to GSH during mismatches in oxygen supply to consumption and resultant ROS

generation. During brown bear hibernation our group found indications that erythrocyte glycolysis is

downregulated substantially, as the side product of glycolysis, DPG was reduced about 50% (Paper I:

Revsbech et al., 2013a). Thus both aerobic respiration in surrounding tissues as well as red cell glycolysis are

operating at reduced rates during hibernation, therefore also yielding less NADPH. However, ROS

generation must be temporally restricted due to very transient increases in metabolic rate, especially in bears

(see box 1). It is therefore not unlikely that a hibernator may possess enough glucose to generate sufficient

additional NADPH by the pentose phosphate pathway, or possibly by other avenues like NADP-dependent

isocitrate dehydrogenase (Jo et al., 2001).

In the hibernating brown bear we did indeed see a large increase in RBC total GSH (Paper IV: Revsbech et

al., 2014). Using correlations, we found the high RBC GSH to correlate closely with plasma and RBC

cysteine availability, as also evidenced in humans where GSH synthesis of RBC is rate limited by plasma

and RBC cysteine (Raftos et al., 2010). Furthermore, it is interesting to note that in bear RBCs, the high GSH

occurs concurrently with what we hypothesize is H2S formation from thiosulfate as evidenced by a depleted

BSS pool during hibernation. Our hypothesis is that the hibernator in this way allocates the majority of

available cysteine to fuel GSH production, whilst still upholding a sufficient H2S production by regeneration

from oxidation products (Paper IV: Revsbech et al., 2014).

METABOLIC INHIBITION: NOT A LIGAND BANDING BATTLE

Depressed mitochondrial function can be induced by moderate to high levels of H2S (see below). Reversibly

inhibiting mitochondrial CcOx implies reducing metabolic demand for O2 and therefore reducing metabolism

as a whole. Inhalation of ̴ 80ppm of H2S has induced reversible suspended animation in mice involving all

the hallmarks of natural hibernation; reductions in O2 consumption and CO2 production and therefore in

metabolic rate as well as reductions in lung ventilation and body temperature (Blackstone et al., 2005). The

ability of H2S to induce a form of suspended animation or hibernation-like state has been attributed to

organismal mitochondrial inhibition (Blackstone and Roth, 2007; Blackstone et al., 2005; Cooper and

Brown, 2008; Li et al., 2012).

38

H2S-induced protection against ischemia-reperfusion injury in myocardia associated with preserved

mitochondrial structure, function and inhibition of respiration has been identified (Chen et al., 2007; Elrod et

al., 2007). Blockade of electron transport as well as a degree of mitochondrial uncoupling decreases

oxidative phosphorylation and causes reduced accumulation of reducing equivalents in the electron transport

chain (Chen et al., 2007). Most publications agree that the binding of H2S to CcOx heme is uncompetitive

with that of O2, in effect meaning O2 concentration does not affect binding directly (Cooper and Brown,

2008). Affinity of binding is weak with half-inhibition of respiration rate, Ki of 0.2-12.5µM (Collman et al.,

2009; Cooper and Brown, 2008). In comparison NO binding is competitive with O2 binding with an Ki of

reduced CcOx for NO of about 0.2 nM in total absence of O2, 60nM NO at 30 µM O2 (typical O2

concentration of tissues) up to 270nM at 145µM O2 (typical O2 concentration of arterial blood) (Brown and

Cooper, 1994; Cooper and Brown, 2008). The two ligands are not in direct competition as they do not inhibit

the same redox state of the enzyme; NO inhibits the reduced state (as well as interacts with the oxidized state

at CuB, see below) and sulfide binds to the oxidized states of the binuclear centre (Cooper and Brown, 2008).

Mitochondrial inhibition by H2S and NO is thus favored in low O2 conditions, precisely as would be the case

in hibernators (Revsbech et al., 2013a). Low O2 conditions are also the conditions where thiosulfate may

work as a pool to regenerate H2S (Olson et al., 2013), as data suggested to happen in bears (Paper IV:

Revsbech et al., 2014).

SUBSTRATE OR INHIBITOR – WHY NOT BOTH?

Due to the weak binding of H2S to CcOx, the response is highly concentration-dependent: Only moderate to

high H2S concentrations (>10-50µM, dependent on tissue and species) will inhibit CcOx O2 consumption

(Collman et al., 2009; Goubern et al., 2007; Nicholls and Kim, 1982; Yong and Searcy, 2001) On the other

hand, low H2S concentrations will function as a reducing agent and therefore as a substrate, donating

electrons to the electron transport chain and quite oppositely stimulate O2 consumption (Goubern et al.,

2007; Yong and Searcy, 2001). H2S can reduce both the oxidized CcOx active site as well as reduce

cytochrome c, the electron carrier normally reduced by electrons from the electron transport chain, originally

donated from food metabolism derived NADH or FADH2 at complex I and II (Collman et al., 2009).

Recently, Módis et al (2013) working in mammalian mitochondria found that low 1µM H2S stimulated

mitochondrial function synergistically with succinate (yielding FADH2 through Krebs cycle), however no

stimulation of mitochondrial electron flow was seen when succinate was absent (Módis et al., 2013). Thus at

low levels H2S can be a source of electrons and an inorganic energy substrate in a mammalian cell, but

seemingly only so in the presence of an active Krebs cycle. At higher levels however, H2S can indeed inhibit

mitochondrial respiration.

A similar situation to that of H2S is at play for NO, as NO noncompetitively binds the copper (CuB) of the

heme:copper binuclear center of oxidized CcOx, reducing the enzyme whilst oxidizing NO to nitrite. Thus

NO is acting like a substrate and an inhibitor simultaneously (Cooper, 2002; Torres et al., 2000). Oxidized

CuB does not bind O2 and NO binding to the CuB is thought to happen mainly in settings of low electron flux

39

(Cooper, 2002). Biased as I am, I cannot help but speculate here: NO-induced inhibition of CcOx at CuB

could be a potential avenue of mitochondrial respiration reduction in a fully aerobic environment with low

electron flux due to reduced metabolism. Could NO be both a substrate contributing to electron delivery and

a regulator of metabolism and ROS production in hibernators? It would require the affinity of CuB for NO to

be high enough compared with the reaction of NO and O2 to produce nitrate, and currently the precise

affinity is unknown (see Cooper, 2002). Despite our recent inconclusive findings with regard to changes in

nitrite levels during hibernation (Paper IV: Revsbech et al., 2014), NO and metabolites still hold potential for

the hibernation story.

NITRATE REDUCTASE ACTIVITY INCREASES WITH HEMOGLOBIN OXYGEN AFFINITY

It seems a vertebrate trend that higher O2 affinity of Hb is combined with a higher nitrite reductase activity of

the deoxy (R) form in hypoxia, as the R state of Hb has the higher nitrite reductase activity over the T-state

(Fago and Jensen, 2015; Jensen, 2009). We know that DPG levels fall drastically during hibernation in

several hibernating species, including our investigated brown bears (Kramm et al., 1975; Maginniss and

Milsom, 1994; Paper I: Revsbech et al., 2013a), as well as is the case for ATP or GTP in teleost fish exposed

to hypoxia (Jensen, 2004). This high O2 affinity of Hb translates directly into increased nitrite reductase

activity due to the shift of the T-R equilibrium towards the R-state as the T state is less stabilized by

phosphates (Paper I: Revsbech et al., 2013a). Thus a slightly higher capacity for nitrite reductase activity in a

hypoxic milieu can potentially be at play in blood of hibernators, enabling local vasodilation and perhaps

release of NO to aid CcOx inhibition as well as plug ROS production from complex I. Whilst NO may

inhibit respiration, it does not decrease contractility of heart muscle in some ectotherms; the hypoxic muscle

has been reported to contract equally well or even with a up to 60% higher force per O2 consumed, as seen in

the hypoxia tolerant turtle Trachemys scripta (Imbrogno et al., 2014; Misfeldt et al., 2009). The effect can be

attributed to increased mitochondrial coupling as the leak current of H+ over the inner mitochondrial

membrane is diminished, yielding more ATP per O2 consumed, thus increasing muscle efficiency (Larsen et

al., 2011). In mild hypoxia (9% O2) trout Mb is more efficient as a nitrite reductase than hypoxia-tolerant

high affinity goldfish Mb, as a larger part is deoxygenated at any given PO2 (Pedersen et al., 2010). Nitrite-

derived NO may be crucial in keeping the heart of a hypoxia-adapted animal pumping even in anoxia

(Stecyk et al., 2004), and the mechanism behind may well apply for hibernators where energy stores, and

thus the reducing equivalents creating the H+ gradient in the first place, must be stretched throughout the

winter. Also of interest is the ability of known hypoxia-tolerant ectotherms to translocate nitrite from plasma

and extracellular stores into tenfold higher concentrations in intracellular compartments in the hypoxic heart

of crucian carp as well as in goldfish and the red-eared slider turtle (Hansen and Jensen, 2010; Jensen et al.,

2014; Sandvik et al., 2012). We do not yet know if any part or how these adaptations to hypoxia seen in the

exothermic specialists, may also apply for the more energy demanding mammalian hibernators faced with a

similar, albeit less extreme task of coping with low O2.

40

CONCLUSION AND PERSPECTIVES

HYDROGEN SULFIDE AND NITRIC OXIDE: NEWCOMERS IN HIBERNATION SCIENCE

In this thesis I report the first work on a whole body scale measurement of the signaling molecules hydrogen

sulfide and nitric oxide in a hibernating animal like the brown bear. Until papers included in this thesis were

published, endogenous H2S in natural hibernation had only been reported for lung tissue of Syrian hamsters

(Talaei et al., 2011a; Talaei et al., 2012). A very recent paper (Dugbartey et al., 2015) reports plasma free

H2S levels to go up as well as increased renal expression of CBS, CSE and 3-MST under an artificially

induced hibernation-like state using 5’- Adenosine monophosphate (5’-AMP) in Syrian hamsters. However,

injection of the non-specific inhibitor of H2S production, amino-oxyacetic acid, did not affect induction of

the torpor-like state albeit did hinder H2S production, leading to the conclusion that H2S is not necessary for

artificial 5’AMP-induced hibernation (Dugbartey et al., 2015). However, our group did find changes in H2S

during natural hibernation in the brown bear (Paper IV: Revsbech et al., 2014).

In the blood of hibernating brown bears we did not see increases in total H2S as first expected, but instead a

fine-tuned remodeling of H2S metabolism, recycling oxidation products and thus conserving amino acid

cysteine for other puposes. We propose that hibernators prioritize the available free Cys for the production of

antioxidant glutathione, GSH, found in high levels in winter erythrocytes (Paper IV: Revsbech et al., 2014).

High antioxidant defenses are especially important during arousal induced mismatches in delivery and

consumption. Blood H2S is conveniently distributed throughout the body and thus ready to effect local

systemic vasodilation where needed in important organs as the heart and the brain, and to aid metabolic

suppression in tissues by inhibiting CcOx of the electron transport chain. We remain to see evidence of NO

and H2S affecting key glycolytic enzyme activities during hibernation, but SNO-GAPDH levels as well as

other enzyme activities could be relevant to reevaluate in smaller hibernmators. Clearly more studies are

needed to evaluate the possible involvement of H2S in natural hibernation. Perhaps studies can even

contribute towards future incuduction of a state of reduced metabolism in organs for transplants or in wider

perspectives, in humans after serious trauma.

CONSEQUENCES OF 2,3-DIPHOSPHOGLYCERATE VARIATIONS FROM SMALL TO LARGE

MAMMALS

This thesis also reports the first comparison and elucidation of mechanisms behind O2 transport in

hibernating versus euthermic states of the large hibernator the brown bear. As I hope made clear during this

thesis, bear hibernation is not a direct expansion of hibernation in small mammals, but involves a larger

degree of active metabolic reduction due to an only slight reduction in body temperature Tb as well as a more

continuous mode of hibernation. Nevertheless, as reported before in small hibernators, we found erythrocytes

to about half their DPG content during hibernation (Paper I: Revsbech et al., 2013a). We proved the decrease

in DPG to via a direct allosteric action affect Hb-O2 affinity. In concord with the decrease in Tb, the DPG

41

decrese thus yields a substantially higher hemolysate O2 affinity during winter compared with summer state.

In contrast, we found that six small rodent hibernators likely have no noteworthy effect of the decreases in

DPG (Paper III: Revsbech et al., 2013b), but probably rely more on their strong decrease on Tb to effect a

rise in Hb-O2 affinity (see box 1). As decreases in DPG will also affect the Donnan distribution of anions

across the erythrocyte membranes, an indirect effect on Hb-O2 affinity may take place through the Bohr

effect (Paper III: Revsbech et al., 2013b).

Additionally, decreases in DPG likely will affect the cooperativity of Hb-O2 binding, n, through less

stabilization of the reduced affinity of only the T state and thus a less steep Hill plot (see box 2) around 50%

saturation. We modeled this DPG reduction-induced decrease in n50 to likely be of substantial significance in

vivo in hibernating bears, as the reduction in cooperativity in consort with the elevated Hb-O2 affinity

yielded a sensible venous PO2 despite the high winter hematocrit (Paper I: Revsbech et al., 2013a). The

substantially higher venous PO2 if not for these changes in n and P50, would lead to untimely O2 delivery to

tissues and major ROS production (see Box 3). Also high altitude deer mice engage variations in DPG in

their adaptation to altitude. However, unlike elevated Hb-O2 affinity and reduced sensitivity to cofactors in

high altitude deer mice, the elevations in DPG, hematocrit and other factors are readily reversed with

translocation to low altitude. This indicates the plastic changes as part of a perhaps necessary impressive

phenotypic plasticity of these mice (Paper II: Tufts et al., 2013). The phenotypic plasticity of deer mice can

play part also in high altitude adaptation, although normally categorized as an acclimatization response.

In this thesis we provide support for the growing theory (Berenbrink, 2006; Natarajan et al., 2013; Natarajan

et al., 2015; Tufts et al., 2015) that not necessarily only specific amino acid residues are important for protein

function, but rather that the entire genetic background and multiple substitutions far away from the active

sites yields the protein phenotype (Paper III: Revsbech et al., 2013b). In Paper III we found the Bohr effect

unaffected by an expectedly detrimental amino acid substitution β146 His→Gln, at the same time as

sensitivity towards DPG was low in spite of the entire DPG-binding site being retained (Paper III: Revsbech

et al., 2013b). Additional work from our group on pika Hb reports how substitution sequence or chronology

on a genetic background has a major impact on protein function (Tufts et al., 2015); my own contribution to

this study was minor although I am a co-author, and the paper not included here as the deeper genetics are

outside the scope of this thesis.

INTERACTIONS OF MECHANISMS REGULATING OXYGEN TRANSPORT AND CONSUMPTION

For the ease of maintaining a logical overview, this thesis has largely been structured into discussing

modifications in oxygen transport involving Hb-O2 affinity adjustments via phenotypic adaptation or via

genotypic adaptation, separated from adjustments in oxygen delivery and consumption involving the

signaling molecules H2S and NO. In the integrated physiology of a high altitude animal or a hibernator,

modifications and interactions in transport and consumption obviously may occur. As decribed earlier in this

thesis, the high O2 affinity of Hb during hibernation will increase the nitrite reductase activity of Hb during

42

hibernation, potentially enabling higher NO production at low O2. Changes between summer and winter

nitrite, taken as a marker of NO-metabolism, was not statistically significant in our study (Paper IV:

Revsbech et al., 2014), but with P=0.043, they likely will be in a different study comparing fewer

parameters. I here speculate further: Given that sufficient NO is not converted to nitrite in the meeting with

free O2, or with localized production, NO-induced inhibition of CcOx at CuB (see paragraph on substrate or

inhibitor) could be a potential avenue of sustaining reduced mitochondrial respiration. And it would be

possible in a fully aerobic environment with low electron flux due to otherwise actively reduced metabolism.

This speculation would be difficult to test, but surely NO has interesting effects, also in a hibernator

perspective. For H2S, it has been recently shown that RBCs are able not only to produce H2S enzymatically,

but also to oxidize it to thiosulfate by reaction with ferric hemoglobin, also known as MetHb (Vitvitsky et

al., 2015). This opens up the avenue of all heme complexes in their ferric form to potentially play some role

in H2S homeostasis. In addition, on the vascular side a synergistic effect of NO and H2S in combination may

also exist on systemic vascular smooth muscle relaxation (Coletta et al., 2012; Hosoki et al., 1997).

Production of DPG inside the erythrocyte happens as a side product of glycolysis, and is thus directly

proportional to the level of glycolysis. In the context of hibernation, a downregulation of RBC glycolysis,

and thus of DPG production as found in the brown bear (~50%) fit in well (Paper I: Revsbech et al., 2013a).

However, as for the general reduction in metabolism, it is interesting to search for the cause, although one

must appreciate that the search may be a question of who came first. Mammalian erythrocytes possess no

mitochondria, but NO and H2S may still affect cell turnover by S-nitrosation and/or sulfuration of key

enzymes, likely upstream of DPG in glycolysis. The enzyme Glyceraldehyde 3-phosphate dehydrogenase,

GAPDH, catalyzing the sixth step of glycolysis is known to be S-nitrosated (Chakravarti et al., 2010).

GAPDH catalyzes the conversion of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate, the precursor to

DPG. We therefore set out to test the hypothesis of whether the decrease in DPG in winter could be linked

with a hibernation induced increase in GAPDH-SNO. In Paper IV we investigated activity and by biotin

switch S-nitrosation of GAPDH, but found no significant changes using the applied, admittedly strict (P<

0.002), statistical method (Paper IV: Revsbech et al., 2014). Applying an altered setup with less tested

parameters and more stable base levels of in-house kept animals may well provide a different outcome.

The molecular mechanisms of an endotherm body at low metabolism and limited O2 supply may give us new

knowledge of physiology at extremes and, if we can learn just parts of what aids metabolic suppression in a

hibernator, perhaps even the tools to prolong the window of opportunity for large operations as well as organ

transplants.

43

ONGOING AND FUTURE RESEARCH

ONGOING: MUSCLE AND ADIPOSE TISSUE HYDROGEN SULFIDE LEVELS OF

HIBERNATING BEARS

In a previous study, we found seasonal changes in the plasma levels of selected H2S metabolites, suggesting

that hydrogen sulfide, H2S, may be one of the temperature independent factors implicated in the metabolic

depression of hibernating brown bears (Paper IV: Revsbech et al., 2014). Specifically, our data analysis

indicated remodeling of H2S metabolism during hibernation and that the plasma pool of BSS may be utilized

during hibernation to generate free H2S in the RBCs. We proposed that H2S may then freely diffuse to

perfused tissues, where at the low O2 levels of hibernating tissues (Paper I: Revsbech et al., 2013a) it may aid

metabolic depression by inhibiting mitochondrial O2 consumption. The question then arises as to whether

changes in H2S plasma metabolites during hibernation are reflected by changes of the same or other

metabolites in other body parts. As muscle and adipose tissue are dominant tissues of a hibernating bear, and

in addition relatively simple to sample with minimal risk to the bear, we set out to investigate H2S

metabolites in these tissues of hibernating versus summer active bears.

A few organs are considered producers of the majority of endogenous H2S: Liver and kidney produce the

highest levels in most species (Kabil et al., 2011). In the rat, levels of H2S in adipose tissue are within the

range of other major H2S producing organs as kidney and liver (Feng et al., 2009). In humans skeletal muscle

levels of H2S producing enzymes CSE and CBS are comparable to that found in liver and kidney, however

expression levels of H2S generating enzymes are highly species specific (Chen et al., 2010; Du et al., 2013;

Veeranki and Tyagi, 2015). Collectively, both adipose tissue as well as skeletal muscle in hibernating versus

summer active brown bears may or may not have high expression patterns of H2S generating enzymes, and

thus until now unknown H2S metabolite profiles.

SKELETAL MUSCLE

Several studies have found skeletal muscle mass and morphology to be preserved during hibernation in

ground squirrel following specific changes in the mammalian target of raptamyosin (mTOR) pathway,

mTOR being a key kinase that upregulates muscle protein synthesis and limits fasting-induced proteolysis

and muscle degradation (Andres‐Mateos et al., 2013; Nowell et al., 2010; Wu and Storey, 2012), However

muscle maintenance is not necessarily static, but rather hibernation can be associated with an initial slight

skeletal muscle loss, stabilization and then recovery and return to summer protein synthesis levels in late

hibernation, prior to emergence, at least in the 13-lined ground squirrel (Hindle et al., 2015). In hibernating

bears lean body mass seems preserved due to low turnover and recycling of urea into amino acids as well as

de novo protein synthesis of selected proteins only (Nelson, 1980; Nelson et al., 1973; Stenvinkel et al.,

2013).

44

ADIPOSE TISSUE

During bear hibernation energy is supplied by burning of fat (Robbins et al., 2012), a process that – albeit at

lower rates – still requires O2. Hibernating bears do not accumulate lactate and are not hypoxic (Paper I:

Revsbech et al., 2013a). Adipose tissue is therefore metabolically active during hibernation. Adipose tissue

may also act as an insulin-sensitive organ and regulate blood glucose (Feng et al., 2009). Endogenous H2S

production by the enzyme CSE has been shown to inhibit glucose uptake in rat adipose tissue (Feng et al.,

2009), suggesting that H2S may be a novel insulin resistance regulator of adipose tissue (Feng et al., 2009).

Skeletal muscle and adipose tissue are both relevant to hibernation. Skeletal muscle is the largest organ in

terms of weight and would then contribute the most to the whole animal metabolic depression, especially

during inactivity, while adipose tissue is essential due to its role in maintaining energy metabolism during

hibernation. Therefore in principle H2S metabolites could differ between the two organs and between

seasons, aligning with different winter and summer metabolic activities and requirements.

Our aim in this project is to better understand the physiological role of H2S in natural hibernation and to this

end we have quantified composition of H2S metabolites in skeletal muscle and adipose tissue compared to

that of plasma and RBCs in winter hibernating and summer active wild brown bears. This study included

free-ranging subadult brown bears (2-4 years old), 7 in winter (February 2014, 4 females, 3 males), of which

5 were recaptured in summer (June 2014, 3 females, 2 males). We are additionally in the process of

obtaining measurements for CSE enzyme activity in adipose tissue and skeletal muscle during winter

hibernation and summer active state of these bears.

The results of this study are in the analysis and writing process, while awaiting more data.

ONGOING: NITRIC OXIDE MEDIATED REGULATION OF ARTERIAL FLOW IN A HIGH

ALTITUDE ADAPTED ANIMAL

NO causes vasodilation and thereby increased local perfusion trough activation of smooth muscle guanylyl

cyclase producing cyclic GMP. Additionally, an O2-independent formation of NO can happen by nitrite

reduction by deoxyHb, yielding NO in hypoxia independently of the endothelium.

Our collaborators have observed genetic differences in Hb-O2 affinity between high-and low altitude deer

mice (Storz et al., 2007; Storz et al., 2009; Storz et al., 2010). We now wish to expand this knowledge on

divergent O2 delivery by uncovering possible differences in the function of blood perfusion regulation

between high and low altitude adapted mice. It has before been observed that major differences in NO

metabolites and localized blood flow can exist between populations adapted to high and low altitude as the

study by Erzurum et al., comparing Tibetans (4,200m) and low-altitude American residents (206m) (Beall et

45

al., 2012; Erzurum et al., 2007). We aim to integrate this knowledge into understanding possible variation

and feedback in blood pressure response to acute hypoxia exposure in the same deer mice cohort.

Microscale blood pressure control studies have been performed by wire myography on mesenteric arteries.

Mesenteric arterial NO-mediated perfusion regulation responses were evaluated using noradrenaline as

contractant, acetylcholine (Ach) as an invoker of endogenous NO-production, sodium nitroprusside (SNP) as

an exogenous NO-donor, and N-nitro-L-arginine methyl ester hydrochloride (L-NAME) as a blocker of

endogenously produced NO, respectively. Representative traces from performed wire myography

experiments on first order mesenteric arteries are shown below; the top line was incubated 20 min with L-

NAME (10-5M, as negative controls) before the ACh protocol was performed. Experiments have been

performed on six mice from each cohort and data are to be evaluated.

Figure 7. Representative traces of relaxation responses from a low- and high altitude mouse 1st order mesenteric arteries. Arteries (2 per mouse) were precontracted with noradrenaline and relaxation in response to NO, produced endogenously by the endothelium eNOS by addition of Ach was evaluated. Intervals were of 2 minutes between each addition to the chamber. The top line of each mouse was incubated for 20 min with L-NAME (10-5M) prior to additions to attempt blocking endogenously produced NO. These mice seemed not to respond as expected to L-NAME, however the function of eNOS seemed necessary to archive relaxation.

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46

Preliminary data analysis gives the picture that high altitude mice seem to be more transient NO generators

than low altitude mice, as they show recontraction during each 2 min period between chamber additions. On

the other hand low altitude mice show more relaxation with endogenous produced NO. In both cases L-

NAME did not exclude NO production, however loss of eNOS function seemed to delay full relaxation. For

the cumulative SNP addition, both high and low altitude mice show continuous relaxation with increasing

concentration (traces not shown). Data awaits further analysis.

The aim of this experiment is to evaluate possible differences in the regulation of blood pressure and

perfusion between high and low altitude populations of deer mice, in response to hypoxia and to NO. To gain

this insight we are collaborating closely with Tobias Wang and Post Doc Nini Skovgaard from the

Zoophysiology department at Aarhus University as well as Nina Kerting Iversen, PhD from the same

department and currently Post Doc at Department of Clinical Medicine - Center for Functionally Integrative

Neuroscience, Aarhus University, for the experiments performed in Aarhus. Externally we are collaborating

with Jay Storz, University of Nebraska.

FUTURE: HYDROGEN SULFIDE METABOLITES MEASUREMENT METHOD SETUP

USING UNISENSE MICROSENSORS

The major obstacle to studying H2S in physiology is to obtain correct measurements. As also discussed in

this thesis, H2S and derived metabolites have been measured using several different methods, and it is only

recently that expected in vivo concentration ranges have been agreed on. As recently recognized, and also

evident from Paper IV (Paper IV: Revsbech et al., 2014), it is of great importance to evaluate all pools of

H2S (free sulfide, bound sulfane-sulfur and acid-labile sulfane sulfur). However, the only applicable method

so far consists of a series of complicated and equipment-heavy procedures using the monobromobimane

method (Kolluru et al., 2013). H2S is a volatile gas with a half-life of approximately 2-10 minutes in a

physiological environment (Vitvitsky et al., 2012a). It is also light sensitive and best measured under

anaerobic conditions (Kolluru et al., 2013).

In this project we aspire to set up a simple new measuring method of H2S and metabolites applying a

Unisense microsensor as well as a gas tight MicroRespiration chamber from the same company in which we

can conduct experiments under controlled conditions. The Unisense H2S sensor is an amperometric sensor

with an internal reference, a sensing and a guard anode. Both anodes are polarized against an internal

reference. H2S from the environment penetrates the membrane at the sensor tip according to the external

partial pressure. Inside the tip is an alkaline electrolyte, where formed HS- ions are immediately oxidized by

ferricyanide, producing sulfur and ferrocyanide. The signal is then generated indirectly by the re-oxidation of

ferrocyanide at the anode (Jeroschewski et al., 1996). The internal guard electrode contributes to the low

47

zero-current. As is the case for use of other very small microsensors, the Unisense H2S microsensor will

cause a minute, nonsignificant consumption of H2S in the sample.

Our aim is to evaluate the free H2S created from the major bound pools, I. by adding a reducing agent, e.g.

Dithiothreitol, DTT, to reduce the bound sulfane sulfur pool to free H2S, II. by adding an acidic buffer (e.g.

tested with 10mM Hepes, 0.5 mM etthylenediaminetetraacetic acid (EDTA) pH 3.4 at 20°C) to the sample,

thereby releasing the acid-labile pool. Obviously, constraints of volume and concentrations to obtain full

liberation of the bound pools will occur, and will be solved as we go. During a three day company workshop

at Unisense I have tested a coarse version of this method using human blood and was successful in obtaining

a signal. From that I can conclude that a microsensor built to high sensitivity is likely sufficiently sensitive

for mammalian measurements (1-5µM H2S) (Revsbech et al., 2014; Shen et al., 2012). Free sulfide H2S in

the blood samples before manipulation may, however, be too low to quantify alone. If measurable, the free

H2S will depend on the equilibrium constant at the given temperature as the majority of H2S at pH >7 will be

in HS- form (Dombkowski et al., 2004; Hughes et al., 2009), and only H2S will cross the micro sensor tip

membrane. The total free sulfide can then be back calulated from the given pKa at that temperature. The pKa

of sulfide is also susceptible to high salinity, however we will not work at salinities much higher than

physiological solution (0.9% NaCl) and thus the given error will be of no great consequence for the

measurement.

Once running we plan to transport the setup to places where it will be possible to work with fresh blood

samples. Samples can be taken from hibernators, and if possible, with modifications of the method also on

tissue homogenate. This method is best suited for immediate measurement of samples, and thus sample

processing for storage will also be an area of investigation, as no method has yet been routinely reported.

FUTURE: NITRITE AND HYDROGEN SULFIDE METABOLITES AND GLYCOLYTIC

ENZYME MODIFICATIONS IN A SMALL HIBERNATOR

Some of the limitations in applying wild brown bears would certainly be alleviated in studies on small

hibernators kept in controlled animal fascilities and used to some degree of handling, hopefully limiting the

individual variations that we see in wild-caught, and in summer helicopter-hunted bears. Small hibernators

have more stable states of reduction in metabolism, whereas multiday oscillations in Tb and metabolism may

have blurred results taken from hibernating bears. Until now we have worked with bears following the

argument of natural hibernation in a natural environment and a large animal, from which repeated and

sizeable sampling is (relatively) straightforward. It has also been hypothesized that bear physiology is more

akin to human physiology than that of rodents, which particularly holds true for weight specific metabolism.

On the other hand, however, especially when it comes to volatile gasotransmitters, the concentration changes

needed in a smaller hibernator to downregulate a much higher weight specific metabolism (Schmidt-Nielsen,

48

1984), are likely more pronounced. In the future, it would certainly be interesting to look at H2S and NO

metabolites, their distribution and possible enzyme modifications in organs like heart, liver and kidney taken

from small hibernating animals during deep hibernation. Hibernation does not entail hypoxia per se, but

hypoxia coping mechanisms could be envisaged to play a substantial role in protecting crucial organs like the

heart and the brain at inevitable mismatches in O2 supply and consumption with animal arousal from

hibernation. This is specifically the case in the smaller hibernators where ~ 6 months of hibernation is

interrupted by arousals to normal temperature and metabolism about once every or every second week (see

box 1).

Our group is in contact with Kelly Drew at the University of Alaska Fairbanks working with arctic ground

squirrels, Spermophilus parryii. We further aim to establish contacts at the 15th international Hibernation

symposium, to be held August 2016 in Las Vegas. We hope to conduct measurements of H2S and derived

pools in house using the above pilot method, but we will verify this method in collaboration with the Kevil

lab, LSU Health Sciences Center Shreveport, Louisiana. Nitrite measurements will be conducted in house or

in collaboration with Frank Jensen, University of Southern Denmark.

49

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CHAPTER II: PAPERS

64

65

Paper I

Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen

affinity in the hibernating brown bear Ursus arctos

Revsbech IG, Malte H, Fröbert O, Evans A, Blanc S, Josefsson J, Fago A

66

67

Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobinoxygen affinity in the hibernating brown bear Ursus arctos

Inge G. Revsbech,1 Hans Malte,1 Ole Fröbert,2 Alina Evans,3 Stéphane Blanc,4 Johan Josefsson,2

and Angela Fago1

1Zoophysiology, Department of Bioscience, Aarhus University, Aarhus C, Denmark; 2Department of Cardiology, ÖrebroUniversity Hospital, Örebro, Sweden; 3Department of Forestry and Wildlife Management, Campus Evenstad, HedmarkUniversity College, Elverum, Norway; and 4Institut Pluridisciplinaire Hubert Curien, Departement d’Ecologie Physiologie etEthologie, UMR 7178, Centre National Recherche Scientifique, Strasbourg, France

Submitted 21 September 2012; accepted in final form 15 November 2012

Revsbech IG, Malte H, Fröbert O, Evans A, Blanc S, Josefsson J,Fago A. Decrease in the red cell cofactor 2,3-diphosphoglycerate in-creases hemoglobin oxygen affinity in the hibernating brown bear Ursusarctos. Am J Physiol Regul Integr Comp Physiol 304: R43–R49, 2013.First published November 21, 2012; doi:10.1152/ajpregu.00440.2012.—During winter hibernation, brown bears (Ursus arctos) reduce basal O2

consumption rate to �25% compared with the active state, while bodytemperature decreases moderately (to �30°C), suggesting a temper-ature-independent component in their metabolic depression. To estab-lish whether changes in O2 consumption during hibernation correlatewith changes in blood O2 affinity, we took blood samples from thesame six individuals of hibernating and nonhibernating free-rangingbrown bears during winter and summer, respectively. A single hemo-globin (Hb) component was detected in all samples, indicating noswitch in Hb synthesis. O2 binding curves measured on red blood celllysates at 30°C and 37°C showed a less temperature-sensitive O2

affinity than in other vertebrates. Furthermore, hemolysates fromhibernating bears consistently showed lower cooperativity and higherO2 affinity than their summer counterparts, regardless of the temper-ature. We found that this increase in O2 affinity was associated witha significant decrease in the red cell Hb-cofactor 2,3-diphosphoglyc-erate (DPG) during hibernation to approximately half of the summervalue. Experiments performed on purified Hb, to which DPG had beenadded to match summer and winter levels, confirmed that the lowDPG content was the cause of the left shift in the Hb-O2 equilibriumcurve during hibernation. Levels of plasma lactate indicated thatglycolysis is not upregulated during hibernation and that metabolismis essentially aerobic. Calculations show that the increase in Hb-O2

affinity and decrease in cooperativity resulting from decreased red cellDPG may be crucial in maintaining a fairly constant tissue oxygentension during hibernation in vivo.

metabolic suppression; body temperature; oxygen binding curves;heat of oxygenation; hibernation

A DISTINCT TRAIT OF MAMMALIAN hibernation is the highly con-trolled decrease in body temperature and metabolic rate. Ahibernating brown bear (Ursus arctos) routinely spends 5–7mo per year in continuous dormancy with no food or waterintake, no urination, and no defecation (19, 36). During thistime, bears appear to be resistant to loss of muscle mass,strength, or bone density (18, 24, 29, 43, 14, 44). Duringhibernation bear body temperature is downregulated onlyslightly, fluctuating from �37°C to a minimum of 30°C, asfound in brown and in black bears (Ursus americanus) (17, 26,

27, 36, 43), whereas O2 consumption rate is downregulated by75% (43). In comparison, in most smaller hibernators, such asground squirrels and marmots, body temperature drops dramat-ically to values close to ambient temperatures (32, 37, 48), witha consequent strong Q10-induced depression of metabolic rate(where Q10 is the rate coefficient for a 10°C change in tem-perature). In spite of substantial downregulation of ventilationand heart rate, most hibernators likely experience only slight orno hypoxia and in some ground squirrels arterial O2 tension(PO2) is normal during torpor (16). As opposed to smallerhibernators that exhibit periods of spontaneous arousals back tonormothermic temperature and metabolic rate (33, 31), bearsdo not arouse to normothermic temperature during winter buthave shallow multiday cyclic (1.6–7.3 days) fluctuations inbody temperature (43). Nevertheless, as demonstrated in arecent study (43), bears exhibit a strong active aerobic meta-bolic depression and a weight-specific metabolic rate similar tothat of smaller hibernators (20, 25, 43), and for this reason,they are now recognized as true hibernators, even though theydo not show the dramatic drop in body temperature andarousals typical of small hibernators (43). Whether and howthe blood O2 transport of bears adapts to a decreased O2 supplyto tissues during hibernation is however, still unknown.

Earlier studies have found that in small hibernating mam-mals, blood O2 affinity increases markedly during hibernation(4, 9, 32). Potentially, blood O2 affinity can be affected in twoways: by structural changes in the blood O2 carrier hemoglobin(Hb) that lead to changes in the protein sensitivity to allostericcofactors and temperature, or by changes in the concentrationof allosteric cofactors inside the red blood cell. Such allostericcofactors include organic phosphates that bind to the centralcavity of the Hb tetramer and decrease O2 affinity by shiftingthe allosteric T-R equilibrium between the low-affinity (T) andthe high-affinity (R) protein conformation toward the T state.In most mammalian Hbs, the main anionic cofactors are theorganic phosphate 2,3-diphosphoglycerate (DPG) and Cl�,that in bear Hb have a large synergistic effect in regulating O2

binding (11). DPG binds allosterically to the low-affinityT-state conformation of mammalian Hbs and, thereby, de-creases Hb-O2 affinity (5). An increase in erythrocytic DPGdecreases Hb-O2 affinity, and, conversely, a decrease in DPGincreases Hb-O2 affinity.

An increase in blood O2 affinity has been observed in severalsmall hibernating animals during dormancy (4, 34), but has notyet been measured in bears. Although the molecular mecha-nisms for a hibernation-induced increase in blood O2 affinityhave not been much investigated, reductions in DPG levels

Address for reprint requests and other correspondence: A. Fago, Zoophysi-ology, Dept. of Bioscience, Aarhus Univ., C.F. Møllers Allé 3, DK-8000Aarhus C, Denmark (e-mail: angela.fago@biology.au.dk).

Am J Physiol Regul Integr Comp Physiol 304: R43–R49, 2013.First published November 21, 2012; doi:10.1152/ajpregu.00440.2012.

0363-6119/13 Copyright © 2013 the American Physiological Societyhttp://www.ajpregu.org R4368

have been found to be involved in the hedgehog (28) andpossibly in some hibernating rodents (23, 32).

Here, we report O2 binding curves of red blood cell hemo-lysates and purified Hb from free-ranging radio-collared brownbears during summer activity and winter hibernation. Curveswere measured at temperatures close to the lowest measuredbody temperature of hibernating bears and the normothermictemperature of nonhibernating bears, 30°C and 37°C, respec-tively (26, 43), to take into account the effect of temperature onHb oxygenation. We also examined Hb multiplicity, concen-tration levels of the allosteric cofactor DPG present in the redcells, and of plasma lactate to evaluate possible differences inglycolytic activity for hibernating and nonhibernating bears.

MATERIALS AND METHODS

Blood sample collection and preparation. Samples of blood weretaken from the same six free-ranging 2- to 3-yr-old Eurasian brownbears, Ursus arctos, three females and three males captured duringwinter hibernation (February: females 35, 57, and 59 kg; males 21, 25and 58 kg) and summer (June: females, 28, 72, and 47 kg; males, 27,51, and 22 kg) in Dalarna county, Sweden, as described previously(17). The bears were immobilized by darting in the den duringFebruary 2011 and again by darting from a helicopter during June.Bears were anesthetized as described in detail in a previous study (17).Briefly, in winter, a mixture of tiletamine-zolazepam (1.1 mg/kg,except 2.5 mg/kg in one male bear, 25 kg), medetomidine (0.03mg/kg) and ketamine (1.3 mg/kg, except 3 mg/kg in one male bear, 25kg) was used, and in summer, a mixture of tiletamine-zolazepam (4.7mg/kg) and medetomidine (0.09 mg/kg) was used. Doses were basedon body mass and time of year (due to differences in expectedmetabolism), as previously reported (17). Blood samples were takenwithin �20 min from darting. All animal handling and sampling wascarried out under approval of the Swedish Ethical Committee onanimal research (C212/9). The performed procedure was in compli-ance with Swedish laws and regulations. In the field, blood samples(�1 ml) were taken from the jugular vein of anesthetized animals intosyringes containing 50 �l of 200 mM EDTA as anticoagulant. At thetime of sampling, rectal temperature of the bears were 33.8 � 1.5°Cduring winter sampling and 39.1 � 1.4°C during summer sampling.Plasma pH was unchanged (7.25 � 0.09 for winter bears and 7.25 �0.06 for summer bears), and slightly acidic, probably due to anesthe-sia, as previously reported (17). Blood samples were centrifuged witha portable centrifuge in Eppendorf tubes immediately after collectionto separate plasma and red blood cells (RBCs). Plasma and RBCsfrom each individual were immediately frozen on dry ice and shippedto Aarhus University, Denmark.

In the laboratory, samples were processed individually. Aliquots offrozen RBCs were added to 0.2 M HEPES buffer (pH 7.40) at a 1:1volume ratio, and cell debris was removed by centrifugation (7,000 g,5 min, 4°C). Levels of oxidized (met) Hb were negligible in allsamples, as judged form the absorbance ratios at 577 and 541 nm(A577/A541 � 1).

DPG, hemoglobin, and chloride. DPG concentration was assessedspectrophotometrically in all individual hemolysates using the DPGassay kit (Roche Diagnostics, Mannheim, Germany; cat no. 10 148334 001), where concentration of DPG in the reaction assay isstoichiometrically coupled to the decrease of NADH to NAD� thatwas followed at 340 nm (extinction coefficient 6.22 mM�1 cm�1)using a Uvikon 923 B double-beam UV/Vis spectrophotometer (Kon-tron Instruments, Milan, Italy) in 1-cm cuvettes. Deproteinization with0.6 M perchloric acid and neutralization of the supernatant using 2.5M K2CO3 was carried out with one tenth of the prescribed volumes.In the protocol used, 100 �l of hemolysate was added to 500 �lice-cold 0.6 M perchloric acid, mixed and centrifuged (2,000 g, 10min), 400 �l of the clear supernatant was then neutralized with 50 �l

of ice-cold 2.5 M K2CO3, and left on ice for �70 min. Samples werecentrifuged again to spin down precipitate (2,000 g, 5 min, 4°C), and100 �l of supernatant was used for the spectrophotometric assayfollowing the instructions provided by the manufacturer. One blankserved as reference for every 4–6 samples. The reaction was com-pleted after 25 min, whereafter the final absorbance did not changenoticeably. The method was validated in control experiments madewith fresh human blood and frozen RBCs. These experiments yieldedDPG values for human blood (4.25 � 0.54 mmol/l RBC) equivalentto those reported by the kit manufacturer (4.83 � 0.15 mmol/l RBC).

To determine the DPG to tetrameric Hb ratios in individual sam-ples, Hb concentration was measured using Drabkin’s method (15,42). Absorbance was read at 540 nm using a Uvikon 923 B DoubleBeam UV/Vis spectrophotometer (Kontron instruments; Milan, Italy)and the hemoglobin concentration determined from the cyanmethe-moglobin extinction coefficient at 540 nm of 10.99 mM�1 cm�1

(heme basis) (49).Hemoglobin multiplicity. To evaluate Hb multiplicity, individual

winter and summer hemolysates were analyzed on an isoelectricfocusing (IEF; pH range 3–9) polyacrylamide gels (Phastgel GEHealthcare Biosciences AB, Uppsala Sweden). A heme concentrationof 200 �M in the samples diluted in milliQ water yielded visible redbands. Adult human hemolysate was used for comparison.

Oxygen binding measurements of hemolysates. O2 equilibriumcurves were determined at constant temperatures of 30 and 37°C (�0.2°C). These temperatures were chosen as they are near to the lowestbody temperature measured in hibernating bears and the normaltemperature in summer active state, respectively (26, 36, 43). Curveswere determined using a modified diffusion chamber, as describedpreviously (40, 45) at a heme concentration of 1 mM in 0.1 M HEPESbuffer, pH 7.4. A pH of 7.4 was chosen, as it is close to normalmammalian blood pH. Two HEPES buffer stock solutions (1 M)differing slightly in pH were used to achieve the same final pH of 7.4of the sample at the two chosen temperatures. For each sample, pHwas measured using a Radiometer BMS2 Mk2 microelectrode assem-bly coupled to a Radiometer PHM64 pH meter. In each experiment, athin layer (� 0.01 mm) of sample was equilibrated with humidifiedgases of varying O2 tensions supplied by two cascaded Wösthoff(Bochum, Germany) gas-mixing pumps mixing pure (99.998%) N2

and air. Changes in absorbance at 436 nm upon stepwise increases inPO2 within the chamber were monitored to determine changes in O2

saturation as a function of changes in PO2. Zero and 100% O2

saturation were obtained from equilibration with pure N2 and O2,respectively. For each equilibration step, absorbance was obtainedusing the in-house made data acquisition software Spectrosampler(available on request). The O2 partial pressure required to achieve50% saturation of the Hb (P50) and cooperativity coefficient at 50%saturation (n50) were calculated from the zero intercept and slope,respectively, of Hill plots, log[Y/(1-Y)] vs. logPO2, where Y is frac-tional saturation. Hill plots were based on at least 4 saturation stepsbetween 0.3 and 0.7.

The temperature dependence of O2 binding expressed as the ap-parent heat of oxygenation (kcal/mol, 1 kcal � 4.184 kJ/mol) wascalculated by the van’t Hoff equation: �H � �4.57 [T1T2/(T1 � T2)] �logP50/1,000 kcal/mol, where T1 and T2 are the absolute tempera-tures (Kelvin) and �logP50 is the corresponding difference in logP50

at the two temperatures. The �H values presented have been correctedfor heat of O2 in solution (�3.0 kcal/mol) (1).

Oxygen binding measurements of purified hemoglobin. Hb waspurified from three winter and three summer individual hemolysates.Bear Hb was stripped from DPG by gel filtration by passing theindividual hemolysates (1 ml sample) through a Sephadex G-25M,PD-10 column (GE Healthcare, New York, NY) equilibrated with 10mM HEPES, pH 7.6, at 4°C. To facilitate removal of DPG, NaCl wasadded to the hemolysate samples to a final concentration of 0.2 Mbefore loading on column. Stripped Hb samples were then concen-trated by ultrafiltration at 4°C using Amicon ultra 0.5 ml 10 K

R44 HEMOGLOBIN OXYGEN AFFINITY IN HIBERNATING BROWN BEAR

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00440.2012 • www.ajpregu.org69

(Millipore) spin tubes. O2 binding curves for all Hb samples weremeasured at 30°C and 37°C, as described above (0.1 M HEPES, pH7.4, 1 mM heme) in the presence of added DPG and Cl�. For each Hbsample, summer or winter, DPG was added to obtain the same Hbtetramer to DPG ratio as that measured in the (unstripped) hemoly-sates. Chloride (Cl�) was added to the same final concentration as thatmeasured in the hemolysates (10 mM) using 1 M NaCl. Cl� concen-tration was measured using a Sherwood MKII model 926S chlorideanalyzer (Sherwood Scientific), requiring 5 �l per assessment (mea-surements were done in duplicates).

Plasma lactate concentration. To assess for changes in anaerobicmetabolism, concentration of lactate was determined enzymatically inthe plasma of winter and summer blood samples, following a previ-ously described method (38). This method utilizes the stoichiometricconversion of NAD� and lactate to NADH and pyruvate by lacticdehydrogenase (LDH), reaction 1 in the scheme below. The reactionis kept shifted to the right by the constant removal of pyruvate,which reacts with glutamate in the presence of glutamic pyruvatetransaminase (GPT), reaction 2, as shown below. Production ofNADH (equivalent to the lactate present) is quantified from theincrease in absorbance at 340 nm using the extinction coefficient of6.22 mM�1 cm�1.

Lactate � NAD� ↔LDH

Pyruvate � NADH � H� (1)

Pyruvate � glutamate¡GPT

alanine � � � ketoglutamate (2)

The original method of Passonneau and Lowry was slightly mod-ified. Thawed plasma (35–50 �l) was used directly for the assay (1 mltotal volume) in 1-cm cuvettes. Final concentrations in the cuvettewere: 50 mM 2-amino-2-methylpropanol buffer (ICN Biomedicals,Santa Ana, CA), pH 9.9; 50 mM glutamate (L-glutamic acid mono-sodium salt; ICN Biomedicals), pH 9.8; 3 mM NAD� (NAD� freeacid, grade II, 98%; Roche Diagnostics); 100 �g/ml LDH (frombovine heart, type XVII; Sigma-Aldrich), 100 �g/ml GPT (RocheDiagnostics). Glutamate and NAD� solutions were prepared each dayand kept on ice until used. Absorbance of samples was recordedimmediately after addition of GPT and again after 30-min incubationat room temperature until absorbance was stable. A blank (containingmilliQ water instead of plasma) was run in parallel every 1–4samples. A standard curve was constructed from 0.1 to 2 mM lactate(sodium L-lactate; Sigma-Aldrich). Greatest linearity was observed inthe lower end of this concentration range (0.1 to 0.5 mM lactate).Applying the indicated plasma volumes, the measured lactate valueswere in the most sensitive range of the calibration curve.

Estimation of venous PO2. Estimations of the venous O2 partialpressure (PvO2) in brown bears during activity and hibernation werecarried out applying physiological data obtained in a recent study onblack bears (43) that reports matching data for body weight, heart rate,and O2 consumption during activity and hibernation in the same,undisturbed individuals, under the assumption that the respiratory andcardiovascular physiology of resting brown and black bears is overallthe same. We first calculated the mixed venous O2 concentration(Cv�O2) from the Fick equation for hibernating and summer activebears:

C v�O2� CaO2

�V̇O2

Q̇(3)

where CaO2 is the arterial blood O2 concentration, V̇O2 is the O2

consumption rate, and Q̇ is the cardiac output (i.e., heart rate strokevolume). At any given PO2, O2 concentration in either arterial orvenous blood (CO2) is given by the sum of the physically dissolved O2

(�O2, PO2) and that bound to Hb, which, in turn, is the product of thetetrameric Hb concentration (4CHb) and the fractional O2 saturationgiven by the Hill equation, as indicated below:

CO2� �O2

· PO2� 4CHb

PO2

n

PO2

n � P50n (4)

where n is Hill’s cooperativity coefficient (n50) and P50 is the half-saturation partial pressure and �O2 is the physical solubility of O2 (8).We used n50 and P50 values measured here at 37°C and 30°C forsummer and winter samples, respectively (see Table 1 under RESULTS).CaO2 was first calculated from Eq. 4 assuming an arterial PaO2 of 100torr and an intracellular tetrameric Hb concentration of 5 mM andusing reported values of hematocrit (Hct) of 45 and 56.8% for brownbears measured during summer and winter, respectively (2). O2

consumption rates (V̇O2) of 0.30 and 0.069 ml O2·g�1·h�1 and heartrates of 55 bpm and 14.4 bpm were taken from previously publishedvalues (43) for summer and winter black bears, respectively. Cardiacoutput was calculated from the heart rate using a stroke volume of0.06 liter as expected for a 60-kg bear (i.e., the mean weight of thebears used in the study of 43), as described by Schmidt-Nielsen (39).After obtaining CaO2, the Cv�O2 value for summer active and hibernat-ing bears was obtained by Eq. 3, and was then used in Eq. 4 to obtainPvO2 values. Calculations were performed by iteration using a Math-ematica script (Wolfram Research, Champaign, IL).

Statistics. Values are presented as mean � SD. Paired t-test wasused for the statistical comparison between summer and winterindividual samples. Comparisons were statistically significant withP � 0.001. One comparison of P50 of hemolysates at summer 37 and30°C (Fig. 2B) was found to be significant by the Wilcoxon signedrank test, with P � 0.031.

RESULTS

There was no indication of variation in Hb isoforms betweensummer and winter blood samples, as determined by IEF gels(Fig. 1). Bears expressed a single Hb with an isoelectric pointsimilar to that of human HbA.

Oxygen binding curves of summer and winter hemolysatesmeasured at 30°C and 37°C, and the corresponding changes inP50 are shown in Fig. 2. When measured at the respectivephysiological temperatures, O2 affinity of winter hemolysatesmeasured at 30°C was significantly higher (i.e., P50 was sig-nificantly lower) than that of summer hemolysates measured at37°C (Fig. 2, Table 1). Although a significant effect of tem-perature on P50 values was observed (Fig. 2B), the temperaturechange alone was not enough to provide the observed shift inthe O2 equilibrium curve (Fig. 2A), and P50 values of winterand summer samples measured at identical temperatures weresignificantly different (Fig. 2B), suggesting changes in a solu-ble red cell allosteric cofactor. The cooperativity coefficient n50

was also significantly lower in winter compared with summer(Table 1), regardless of the temperature of measurement, indi-cating a change in the overall allosteric equilibrium between Tand R state. The heat of oxygenation (�H) was similar in summer and

Table 1. O2 affinity (P50), cooperativity coefficients (n50)measured at 30°C and 37°C and derived heat of oxygenation(�H) in brown bear hemolysates during winter and summer

Summer 37°C Summer 30°C Winter 37°C Winter 30°C

P50, torr 15.4 � 0.6 10.0 � 0.4 11.4 � 0.8* 7.3 � 0.6*n50 2.4 � 0.1 2.4 � 0.1 1.9 � 0.2* 1.7 � 0.2*�H, kcal/mol �8.5 � 0.8 �8.7 � 1.5

*Significant differences (P 0.001) from summer values (means �SD); n � 6.

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AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00440.2012 • www.ajpregu.org70

winter samples, with values of �8.5 � 0.8 kcal/mol and �8.7 �1.5 kcal/mol, respectively.

The red cell hemolysate DPG concentration was signifi-cantly lower in winter compared with summer hemolysates(Table 2). O2 equilibria are largely affected by DPG to Hbtetramer ratios rather than by DPG concentrations alone. In thewinter samples this ratio was �1:1, whereas in the summer

samples it increased significantly to �2:1. Plasma lactate didnot vary significantly between winter and summer (Table 2).

To evaluate whether the observed left shift in the O2 equi-librium curve found for the hemolysate of hibernating bears(Fig. 2) was due to a decrease in DPG, we removed endoge-nous DPG from the hemolysate from three bears, added exog-enous DPG to the same DPG:Hb tetramer ratio as in theuntreated hemolysate (i.e., 2:1 for summer samples and 1:1 forwinter samples) and measured O2 equilibria at the two tem-peratures. Cl� was added to the same final concentration asmeasured in the untreated hemolysates (10 mM Cl�). The P50

and n50 values (means � SD, n � 3) obtained in these sampleswere for winter bears 6.6 � 0.5 torr, and 1.8 � 0.04 (30°C),9.5 � 0.9 torr and 1.7 � 0.02 (37°C), respectively. For summerbears, the same parameters were 9.2 � 0.2 torr and 2.0 � 0.18(30°C), 12.6 � 0.2 torr, and 2.0 � 0.09 (37°C), respectively.As shown in Fig. 3, the change in P50 between summer andwinter samples obtained with purified Hb added to DPG wasnot significantly different from that obtained with the untreatedRBC lysates, at both temperatures (Fig. 3), demonstrating thatthe change in DPG concentration was responsible for theleft-shifted O2 equilibrium curves of hibernating bears reportedin Fig. 2.

When assuming similar heart rates and O2 consumption as inundisturbed hibernating and active black bears (43), the O2

tension of mixed venous blood (that approximates that existingin tissues) in hibernating and active brown bears can be

Fig. 1. Representative isoelectric focusing gel performed on three individualbrown bear summer and winter hemolysates, indicating the presence of a singlehemoglobin isoform as in human hemolysate. Identical results were obtainedwith the remaining individual bear hemolysates (not shown).

Fig. 2. A: representative Hb-O2 binding curves from winter and summerhemolysates of one individual brown bear measured at 30°C and at 37°C.B: P50 values (means � SD) for summer and winter hemolysates at 30°C and37°C (n � 6). *Significant differences (P 0.001) in P50 between 37°C and30°C. **Significant differences (P 0.001) between summer and winter. Onecomparison of P50 values (summer 37°C and 30°C, left-hand columns) wassignificant by Wilcoxon signed rank test (P � 0.031). Conditions were 1 mMheme, 0.1 M HEPES buffer, pH 7.4.

Table 2. DPG concentrations and DPG to hemoglobintetramer molar ratios in red blood cells and plasma lactateconcentrations in brown bear hemolysates during summerand winter

RBC DPG, mM [DPG]/[Hb4] Plasma Lactate, mM

Summer 4.41 � 0.50 2.12 � 0.39 5.81 � 2.77Winter 2.37 � 0.39* 0.99 � 0.21* 3.05 � 1.32

*Significant differences (P 0.001) from summer values (means � SD);n � 6. RBC, red blood cells; DPG, 2,3-diphosphoglycerate.

Fig. 3. Changes in P50 (torr) for brown bear winter and summer hemolysates(n � 6) and for samples of purified Hbs containing the same DPG/Hb tetramermolar ratios (n � 3) as in summer and winter hemolysates (Table 2), measuredat 30°C and 37°C. NS, not significant.

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predicted from the Fick equation when knowing values for P50,cooperativity coefficients n50 and blood Hb concentration, asdescribed in detail under MATERIALS AND METHODS. Figure 4shows the predicted O2 equilibrium curves for winter andsummer animals along with the estimated arterial and venousO2 saturation and respective PO2 values. In the calculations, P50

and n50 values were those measured at the physiologicaltemperatures of 37°C and 30°C for summer and winter sam-ples, respectively (Table 1). As evident from Fig. 4, winter O2

content of the blood is considerably elevated because of theincrease in Hct occurring during hibernation (2). If the O2

binding curve had remained unchanged during winter (dottedline, Fig. 4), venous PO2 would have been substantially ele-vated (�21.8 torr). However, a left-shift in the Hb-O2 bindingcurve may avoid this situation and maintain PvO2

of hibernatingbrown bears relatively unchanged (summer �14.8 and winter�11.5 torr) (Fig. 4).

DISCUSSION

In this study of free-ranging brown bears, we found amarked left-shift of the O2 equilibrium curve during hiberna-tion, which was associated with an increase in the Hb-O2

affinity and a decrease in cooperativity. We demonstrated thatthe differences between hibernation and active state can beexplained by a decrease in the red cell cofactor DPG, a majorallosteric cofactor of Hb, whereas the reduction in body tem-perature during hibernation alone cannot account for the ob-served shift in Hb oxygenation. In concert with decreasedventilation and heart rate, a left-shifted O2 equilibrium curvewould maintain the necessary O2 supply to tissues underconditions of a prolonged depressed O2 consumption rate, asduring hibernation. Such long-term effects are typically medi-ated by changes in the levels of organic phosphates, fromhigh-altitude mammals (46) to hypoxic fish (47). A similardecrease in DPG levels as found here has been demonstrated in

other hibernating mammals, such as the 13-lined ground squir-rel, the golden-mantled ground squirrel, the woodchuck, andthe hedgehog (7, 23, 28, 32). However, only in the hedgehoghas the DPG decrease been demonstrated to be directly coupledwith the increase in blood O2 affinity during hibernation (28).By contrast, high O2 consumption rates, as during intensemuscle exercise, are typically associated with a transient rightshift of the O2 binding curve, as, for example, due to the Bohreffect. In this study, we found that during hibernation, adepressed O2 consumption rate was not compensated for byincreased glycolysis, as plasma lactate levels did not increasein winter samples, in agreement with previous measurements(17). Levels of plasma lactate found in winter (3.05 � 1.32mM) were comparable to normal human values (22, 30, 41).The higher variation of plasma lactate concentration in thesummer samples may reflect a more variable activity of theindividual bears at the time of sampling (Table 2). This furtherconfirms that energy metabolism in hibernating bears is essen-tially aerobic, as also indicated by the use of fatty acids as theprimary energy fuel (35, 36).

When in the presence of anionic allosteric effectors, the Hbfrom brown bear, similar to that from other polar and cold-tolerant mammals, shows a reduced temperature sensitivity(i.e., a less negative �H) compared with other mammals,including humans (3, 11, 12, 21), a feature that has beenattributed to the presence of an additional allosteric Cl� bind-ing site on the � subunit between residues Lys8� and Lys76�(11, 12). Limited effect of temperature on Hb-O2 bindingfacilitates O2 delivery to poorly insulated body parts, includingcold extremities in contact with ice, a feature that has beeninterpreted as an energy-saving adaptive mechanism (10, 13).In bears, the comparatively small decrease in body temperatureduring hibernation would then cause a slight (albeit significant,Fig. 2B) effect on the O2 binding curve, as shown here by usand earlier by others (11). The calculated heat of oxygenation(�H) values (�8.5 � 0.8 summer and �8.7 � 1.5 kcal/molwinter) were consistent with previous �H data reported forbear Hb, with values ranging from �7 to �8.5 kcal/mol (6,11). Temperature sensitivity of O2 binding to Hb was overallsimilar in summer and winter hemolysates, indicating that afall in body temperature alone is necessary but not sufficientfor the decrease in P50 of hibernating bear hemolysates. Wefound that a significant part of the observed shift of the O2

equilibrium curve in hibernating bears is due to a decrease inred blood cell DPG levels (Figs. 2 and 3). No indication ofswitch in Hb expression with synthesis of new isoHb compo-nents with a higher O2 affinity was found in the brown bears(Fig. 1).

By binding to a specific site (including His2�, Lys82�, andHis143� in bear Hbs) in the central cavity of the T-stateconformation, DPG is a potent allosteric cofactor of brownbear Hb acting synergistically with Cl� ions (11). The overalleffect of a DPG decrease is an increase in the Hb-O2 affinityand a decrease in cooperativity. These effects are explainedby a destabilized T state due to a decrease in the DPG to Hbtetramer ratio, with the consequent shift of the T-R equilib-rium toward the high-affinity R state. We demonstrated thatDPG is the allosteric cofactor responsible for the affinitychanges, as these were reproducible when purified Hb andDPG were mixed in exactly the same ratios as found insummer and winter samples. In addition, higher DPG to Hb

Fig. 4. Predicted hemolysate O2 equilibrium curves of summer and winterbrown bears with the estimated arterial and mixed venous point plotted on thecurves (�). Curves are calculated from the Fick’s equation using mean valuesof P50 and n50 measured in summer hemolysates at 37°C (P50 � 15.4 torr,n50 � 2.4) and winter hemolysates at 30°C (P50 � 7.3 torr, n50 � 1.7) (Table1), using summer and winter Hcts of 45% and 56.8%, respectively (2). Dottedline indicates the theoretical curve having high winter Hct (56.8%) and P50 andn50 values of summer hemolysate, thus causing an increased PvO2 (Œ). Otherdetails are described in the MATERIALS AND METHODS section.

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tetramer ratios in summer bears (�2:1) than in humans(�1:1) may indicate some differences in the regulation ofDPG as a red blood cell metabolite.

Calculations show that the hibernation-associated elevationin blood O2 affinity and decrease in cooperativity of O2 bindingmay be crucial for maintaining mixed venous PO2 at a physi-ologically reasonable level in spite of the strong elevation ofHct (2) (Fig. 4). In the case of an unaltered O2 binding curve,the high Hct would have elevated winter PvO2

substantially(Fig. 4) and cause a potentially detrimental increase in dis-solved O2. The same conclusion is reached when applyingphysiological data from hibernating and active brown bearsunder anesthesia (17), although the sedative and disturbance-induced elevation, particularly in the heart rate, poses limita-tions to their use. Interestingly, P50 values calculated from thewhole blood saturation data of anesthetized winter and summerbears (19.7 and 32.1 torr, respectively) (17), also show asimilar left-shifted O2 binding curve during hibernation andconfirm the results here obtained on diluted hemolysates.

In black bears during and after hibernation, evidence hasbeen presented that changes in body temperature and metabolicrate expressed as rate of O2 consumption are to a certain extentindependent from each other (43). Bears emerging from theirdens show a rapid recovery of their body temperature to normallevels, whereas their basal metabolic rate seems to remainsuppressed and recovers only slowly. These findings indicate astrong temperature-independent remodeling of their metabo-lism during hibernation. Our results in brown bears furtherindicate that such remodeling may play a role in regulatingHb-O2 binding during hibernation.

Perspectives and Significance

The physiology of brown bear hibernation is inherentlyintriguing as bears seem less dependent on reductions in bodytemperature to aid metabolic regulation compared with other(smaller) hibernators, but rather may rely more heavily onactive aerobic metabolic suppression.

Here, we found indications that a side product of glycolysis,DPG, is substantially downregulated during hibernation, ineffect increasing blood O2 affinity substantially. The describedchanges in O2 affinity and cooperativity of Hb-O2 binding maybe crucial for defending internal PO2 during the hibernationperiod. Clearly, further studies will be needed to identify themechanisms involved in metabolic suppression in hibernatingbears and to establish whether the observed decrease in eryth-rocyte DPG, besides its role in adapting the blood O2 equilib-rium curve, is part of this metabolic remodeling. In this case,regulation of glycolytic enzymes upstream of DPG productionby pH, temperature, or other factors could be involved. Suchfindings could be of relevance to identify pharmacologicalmanipulation of energy metabolism that would preserve tissueintegrity in critically ill human patients. Further studies onhibernators such as the brown bear may reveal details of howthe substantial metabolic regulation is achieved.

ACKNOWLEDGMENTS

We thank the Scandinavian Bear Project for financial support, sampleavailability, and field logistics. We thank Roy Weber for useful comments andAnny Bang for technical assistance (Aarhus). This work was also funded byThe Danish Council for Independent Research, Natural Sciences.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: I.G.R., H.M., O.F., and A.F. conception and design ofresearch; I.G.R., O.F., A.E., S.B., and J.J. performed experiments; I.G.R. andH.M. analyzed data; I.G.R., H.M., and A.F. interpreted results of experiments;I.G.R., H.M., and A.F. prepared figures; I.G.R. and A.F. drafted manuscript;I.G.R., H.M., O.F., A.E., S.B., and A.F. edited and revised manuscript; I.G.R.,H.M., O.F., A.E., S.B., J.J., and A.F. approved final version of manuscript.

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75

Paper II

Phenotypic plasticity in blood–oxygen transport

in highland and lowland deer mice

Tufts DM, Revsbech IG, Cheviron ZA,

Weber RE, Fago A, Storz JF

76

77

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INTRODUCTIONIn air-breathing vertebrates living at high altitude, the reduced O2tension of inspired air requires compensatory physiologicaladjustments to ensure an adequate tissue O2 supply. Some of theseadjustments involve reversible changes in O2 transport by red bloodcells (RBCs), which is a function of the O2-carrying capacity of theblood (regulated by erythropoiesis) and blood–O2 affinity [regulatedby allosteric effectors of hemoglobin (Hb) function]. Mammals thatare native to high-altitude environments are typically characterizedby a suite of hematological and vascular traits that includes anelevated blood–O2 affinity, a normal or slightly increased hematocrit(Hct), and increased muscle capillary density (Hall et al., 1936;Chiodi, 1962; Bullard et al., 1966; Bullard, 1972; Monge and León-Velarde, 1991; Weber, 1995; Weber, 2007; Storz et al., 2010b;Mairbäurl and Weber, 2012). In comparisons between species orconspecific populations that are native to different elevational zones,common-garden and/or reciprocal-transplant experiments arerequired to assess whether observed physiological differencesreflect fixed, genetically based differences or environmentallyinduced acclimatization responses (phenotypic plasticity). In thelatter case, the acclimatization response may involve irreversiblechanges in phenotype (reflecting developmental plasticity) and/orreversible changes during adulthood (physiological plasticity or

‘phenotypic flexibility’) (Piersma and Drent, 2003). Regardless ofthe ontogenetic stage at which the plasticity is manifest, it isgenerally an open question as to whether a given change inphenotype represents an adaptive acclimatization response that hasevolved under the influence of natural selection (Kingsolver andHuey, 1998; Huey et al., 1999; Woods and Harrison, 2002;Ghalambor et al., 2007).

If physiological differences between highland and lowland nativesare attributable to adaptive phenotypic plasticity, then it promptsthe question of why traits that are characteristic of hypoxia-toleranthigh-altitude species are often not congruent with the typicalacclimatization response to hypoxia in lowland species (Monge andLeón-Velarde, 1991; Storz et al., 2010b). Lowland mammals thatare not genetically adapted to environmental hypoxia typicallyrespond to chronic O2 deprivation with an increased erythropoieticactivity (resulting in correlated increases in Hb concentration andHct) and a decreased blood–O2 affinity [mediated by an increasedRBC concentration of 2,3-diphosphoglycerate (DPG), an allostericeffector that reduces Hb–O2 affinity) (Lenfant et al., 1968; Baumannet al., 1971; Duhm and Gerlach, 1971; Mairbäurl et al., 1993;Quatrini et al., 1993). DPG directly reduces Hb–O2 affinity bypreferentially binding and stabilizing deoxygenated Hb, therebyshifting the allosteric equilibrium in favor of the low-affinity ‘T-

SUMMARYIn vertebrates living at high altitude, arterial hypoxemia may be ameliorated by reversible changes in the oxygen-carrying capacityof the blood (regulated by erythropoiesis) and/or changes in blood–oxygen affinity (regulated by allosteric effectors ofhemoglobin function). These hematological traits often differ between taxa that are native to different elevational zones, but it isoften unknown whether the observed physiological differences reflect fixed, genetically based differences or environmentallyinduced acclimatization responses (phenotypic plasticity). Here, we report measurements of hematological traits related toblood–O2 transport in populations of deer mice (Peromyscus maniculatus) that are native to high- and low-altitude environments.We conducted a common-garden breeding experiment to assess whether altitude-related physiological differences wereattributable to developmental plasticity and/or physiological plasticity during adulthood. Under conditions prevailing in theirnative habitats, high-altitude deer mice from the Rocky Mountains exhibited a number of pronounced hematological differencesrelative to low-altitude conspecifics from the Great Plains: higher hemoglobin concentrations, higher hematocrits, highererythrocytic concentrations of 2,3-diphosphoglycerate (an allosteric regulator of hemoglobin–oxygen affinity), lower meancorpuscular hemoglobin concentrations and smaller red blood cells. However, these differences disappeared after 6weeks ofacclimation to normoxia at low altitude. The measured traits were also indistinguishable between the F1 progeny of highland andlowland mice, indicating that there were no persistent differences in phenotype that could be attributed to developmentalplasticity. These results indicate that the naturally occurring hematological differences between highland and lowland mice areenvironmentally induced and are largely attributable to physiological plasticity during adulthood.

Supplementary material available online at http://jeb.biologists.org/cgi/content/full/216/7/1167/DC1

Key words: physiological plasticity, high altitude, hemoglobin, hematocrit, hypoxia, Peromyscus maniculatus, red blood cell.

Received 5 September 2012; Accepted 22 November 2012

The Journal of Experimental Biology 216, 1167-1173© 2013. Published by The Company of Biologists Ltddoi:10.1242/jeb.079848

RESEARCH ARTICLE

Phenotypic plasticity in blood–oxygen transport in highland and lowland deer mice

Danielle M. Tufts1,*, Inge G. Revsbech2, Zachary A. Cheviron1,3, Roy E. Weber2, Angela Fago2 and Jay F. Storz1

1School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA, 2Zoophysiology, Department of Bioscence, Aarhus University, Denmark and 3Department of Animal Biology, University of Illinois, Urbana-Champaign, IL 61801, USA

*Author for correspondence (dmtufts11@yahoo.com)

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state’ quaternary structure. The hypoxia-induced increase in themolar DPG/Hb ratio also indirectly reduces Hb–O2 affinity byaltering the Donnan equilibrium across the RBC membrane, as theincreased intracellular concentration of non-diffusible anions leadsto an associated influx of hydrogen ions, thereby enhancing the Bohreffect (Duhm, 1971; Samaja and Winslow, 1979).

Within limits, an increased Hb concentration may enhance tissueO2 delivery under hypoxia because the associated increase in arterialO2 content can compensate for a reduced O2 saturation. However,results of several empirical and theoretical studies suggest thatincreasing the Hb concentration of partially saturated blood is notan ideal long-term solution to the problem of chronic hypoxemiabecause the associated increase in blood viscosity produces anelevated peripheral vascular resistance that can compromise cardiacoutput (Guyton and Richardson, 1961; Bullard, 1972; McGrath andWeil, 1978; Winslow and Monge, 1987; Monge and León-Velarde,1991; Connes et al., 2006; Schuler et al., 2010; Storz, 2010). Studiesof humans at high altitude have suggested that the optimal Hbconcentration at rest and at exercise may actually be quite close tothe typical sea level value (Winslow, 1988; Villafuerte et al., 2004),or perhaps only slightly higher (Reeves and León-Velarde, 2004),and it is well documented that excessive polycythemia is a causalfactor in the development of chronic mountain sickness (Winslowet al., 1985; Winslow and Monge, 1987; Monge and León-Velarde,1991; Rivera-Ch et al., 2007).

The adaptive significance of hypoxia-induced reductions inblood–O2 affinity depends on the severity of hypoxia as well as thepulmonary O2 diffusion capacity and numerous other taxon-specificphysiological attributes. Under conditions of severe hypoxia,theoretical and experimental results indicate that an increase in RBCDPG concentration and the concomitant decrease in Hb–O2 affinitywill generally have detrimental effects on tissue oxygenationbecause the reduced arterial O2 saturation more than offsets anybenefit of increased O2 unloading in the peripheral circulation (Tureket al., 1973; Eaton et al., 1974; Bencowitz et al., 1982; Willford etal., 1982).

Some of the best opportunities for examining plasticity in traitsassociated with hypoxia tolerance are provided by studies ofpopulation-level variation in species that are distributed across steepaltitudinal gradients. One such species is the deer mouse(Peromyscus maniculatus), which has the broadest altitudinaldistribution of any North American mammal (Hock, 1964). As deermice are continuously distributed from sea level to elevations above4300m, this species has proven to be an exemplary subject forresearch on mechanisms of adaptation and acclimatization to high-altitude hypoxia. An extensive body of work has documented thatdeer mice native to high altitude have elevated blood–O2 affinitiesrelative to lowland conspecifics, and these differences are largelyattributable to allelic differences in Hb–O2 affinity (Snyder, 1982;Snyder et al., 1982; Snyder, 1985; Chappell and Snyder, 1984;Chappell et al., 1988; Storz, 2007; Storz et al., 2009; Storz et al.,2010a). Additional evidence that allelic variation in Hb functioncontributes to local adaptation is provided by tests of whole-organism physiological performance involving wild-derived strainsthat express different Hb variants (Chappell and Snyder, 1984;Chappell et al., 1988; Hayes and Chappell, 1990) and populationgenetic analyses of variation in the underlying globin genes (Snyderet al., 1988; Storz, 2007; Storz et al., 2007; Storz and Kelly, 2008;Storz et al., 2007; Storz et al., 2009; Storz et al., 2012a). In lightof this evidence for adaptive, genetically based differences in Hb–O2affinity between deer mouse populations that are native to differentelevational zones, it is also of interest to assess the role of phenotypic

plasticity in modulating aspects of blood–O2 transport. Here, wereport measurements of hematological traits related to blood–O2transport capacity in populations of deer mice that are native to high-and low-altitude environments. The main objectives were (i) tocharacterize altitude-related differences in hematological traits, and(ii) to assess what fraction of the observed trait differences areattributable to phenotypic plasticity.

MATERIALS AND METHODSAnimals

In July–August of 2010 and 2011, we collected a total of 118 deermice, P. maniculatus (Wagner 1845), from a high-altitude localityin the Southern Rocky Mountains and a low-altitude locality in theGreat Plains, 770km to the East. We collected 58 highland micefrom the summit of Mount Evans (Clear Creek County, CO, USA;39°35′18″N, 105°38′38″W, elevation 4350m above sea level) and60 lowland mice from the prairie grassland of eastern Nebraska(Nine Mile Prairie, Lancaster County, NE, USA; 40°52′12″N,96°48′20.3″W, elevation 430m above sea level). All mice werecaptured using Sherman live traps baited with peanut butter andoats. We drew approximately 200μl of blood from the maxillaryvein of each mouse using a 5mm Goldenrod lancet (MEDIpointInc., Mineola, NY, USA).

A subset of 46 mice (24 highland and 22 lowland) weretransferred to a common-garden environment at the Animal ResearchFacility at the University of Nebraska (elevation 300m) where theywere allowed to acclimate for 6weeks. All mice were maintainedat a constant temperature (25°C) and on a standard light:dark cycle(12h:12h) for the duration of the experiment. During the 6weekacclimation period all mice were offered a standard diet ad libitum(Harlan Teklad Rodent Chow, Indianapolis, IN, USA). After theacclimation period, we again drew blood from each mouse asdescribed above, for repeat measurements of the same hematologicaltraits. We also conducted crosses between wild-caught mice fromthe same collection locality and we reared the resultant F1 progenyat the University of Nebraska and the University of Illinois underthe same common-garden conditions as described above. A total of71 F1 mice (N=48 and 23 descendants of highland and lowlandnatives, respectively) were phenotyped after they reached aminimum age of 66days. All experimental protocols were approvedby the Institutional Animal Care and Use Committees at theUniversity of Nebraska (IACUC no. 522) and the University ofIllinois (IACUC no. 10244).

Measurement of hematological traitsWe measured Hb concentration in whole blood using a HemoCueHb 201+ analyzer, following the manufacturer’s protocol (HemoCueAB, Ängelholm, Sweden). We measured Hct as the volume ofpacked RBCs relative to total blood volume in a heparinizedcapillary tube that was spun at 13,600g for 5min in a ZIPocritcentrifuge (LW Scientific Inc., Lawrenceville, GA, USA). We alsocalculated mean cell Hb concentration, MCHC (=Hbconcentration×100/Hct). To measure RBC size, we used a ZeissAxioplan 2 imaging microscope (Carl Zeiss, Gottingen, Germany)to measure the diameter of 10 RBCs per sample.

RBC DPG concentrations were determinedspectrophotometrically using a DPG kit, following themanufacturer’s protocol (Roche Applied Science, Indianapolis, IN,USA), with the exception that we used sample volumes of 100μlwhole blood rather than 1ml. We drew blood from a subset of 21mice (N=10 and 11 highland and lowland mice, respectively). Bloodwas collected in chilled heparinized capillary tubes and was

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immediately deproteinized with 500μl of cold 0.6moll–1 perchloricacid (HClO4). Samples were centrifuged at 5000r.p.m. for 10min,and 400μl of clear supernatant was then removed and neutralizedwith 50μl of 2.5moll–1 potassium carbonate (K2CO3). Samples werekept on ice for at least 60min and were then centrifuged again at5000r.p.m. for 10min. The supernatant was removed andimmediately frozen in liquid nitrogen prior to the spectrophotometricanalysis. The concentration of DPG in whole blood was estimatedfrom the coupled reduction of NADH to NAD+ in the reaction assayat 340nm (UVIKON 923 B Double Beam UV/VISSpectrophotometer, Kontron Instruments, Milan, Italy). The reactionwas completed after 25min and absorbance did not changenoticeably in later readings. Negative controls were run with eachanalysis and positive controls were performed with human blood.

Statistical analysisFor measures of Hb concentration in wild-caught mice (phenotypedat the site of capture) and for measures of all hematological traitsin the F1 progeny of wild-caught/lab-acclimated mice, we tested foraltitude-related differences in phenotype using standard t-tests. Forthe subset of mice that were included in the common-gardenacclimation experiment, we compared pre- and post-acclimation traitvalues using a repeated measures two-way ANOVA with nativealtitude (high versus low) and time (pre- versus post-acclimation)included as independent variables. In the analysis of trait variationin the common-garden F1 mice, we controlled for variation inindividual age (66–454days) by performing an ANCOVA withnative altitude as an independent variable and age as a covariate.We did not detect any significant differences between the sexes forany of the hematological traits, so data from males and females werepooled in all analyses. Similarly, we did not detect significant traitvariation among sibships within each set of F1 mice from high- andlow-altitude, so data from all families were pooled.Kolmogorov–Smirnov (K–S) tests did not reveal any significantdeviations from normality in any of the trait-specific data sets. Allstatistical analyses were conducted using the SAS software package(SAS Institute Inc., 2009) or VassarStats online statistical calculator(vassarstats.net).

RESULTSUnder the conditions prevailing in their natural habitats, the highlandP. maniculatus exhibited a significantly higher Hb concentrationthan the lowland P. maniculatus [mean ± 1 s.d., 2.83±0.28 versus2.33±0.32mmoll–1, respectively (Hb molecular mass 64.45kDa);t116=9.079, P<0.0001; Fig.1). However, after 6weeks of acclimationto normoxia in the common-garden laboratory environment, themean Hb concentration of the highland mice dropped by 8.8% to2.54±0.21mmoll–1 and was statistically indistinguishable from thatof the lab-acclimated lowland mice (2.55±0.20mmoll–1; Table1,Fig.2A). The F1 progeny of highland and lowland P. maniculatusalso had Hb concentrations that were statistically indistinguishable(2.33±0.21 versus 2.29±0.24mmoll–1, respectively; t69=0.739,P=0.489). Similar to the repeated-measures analysis of Hbconcentration (Fig.2A), an analysis of pre- and post-acclimationmeasures of Hct, MCHC, RBC size and RBC DPG concentrationfor the same subset of highland and lowland mice revealed similardegrees of plasticity (Fig.2B–E). Highland deer mice exhibited asignificantly higher Hct relative to lowland mice (59.67±4.83versus 48.94±4.82%, respectively; Table1, Fig.2A), but after6weeks of acclimation to normoxia, the average Hct of the highlandmice dropped by 17% such that post-acclimation values of thehighland and lowland mice were statistically indistinguishable

(49.77±3.35 versus 49.96±3.78%, respectively; Fig.2B). Relativeto the lowland mice, the highland mice had a lower MCHC(4.74±0.28 versus 5.05±0.45mmoll–1, respectively) and a smalleraverage RBC size (5.38±0.12 versus 5.72±0.41μm, respectively),but these differences disappeared after acclimation to normoxia(Fig.2C,D). The highland mice exhibited a significantly higher RBCDPG concentration (2.16±0.44 versus 1.47±0.39mmoll–1; Table 1,Fig.2E), but as with MCHC and RBC size, this differencedisappeared after acclimation to normoxia. For each of thehematological traits, the ‘in situ’ measurements of wild-caught micewere consistent with data from previous studies of Peromyscus mice(Fig.3).

Similar to the case with Hb concentration, the F1 progeny ofhighland and lowland parents did not exhibit significant differencesin Hct (49.20±3.21 versus 50.00±4.26%; t50=0.733, P=0.528),MCHC (4.81±0.34 versus 4.84±0.44mmoll–1; t50=–0.228, P=0.841),

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Fig.1. Hemoglobin (Hb) concentrations (mean ± 1 s.e.m.) of highland andlowland Peromyscus maniculatus measured at their native altitudes.Significant differences in mean values (P<0.05) are denoted by differentletters.

Table1. Differences in hematological traits between groups of deermice (high- versus low-altitude) and between pre- and post-

acclimation time points

Trait Factor d.f. F P

[Hb] Altitude 1,43 14.58 0.0004Time 1,44 2.33 0.1344

Altitude × time 1,44 15.54 0.0003Hct Altitude 1,43 29.99 <0.0001

Time 1,44 29.21 <0.0001Altitude × time 1,44 44.24 <0.0001

MCHC Altitude 1,43 2.17 0.1483Time 1,44 8.43 0.0058

Altitude × time 1,44 3.17 0.0821RBC size Altitude 1,18 0.37 0.549

Time 1,19 22.83 0.0001Altitude × time 1,19 9.82 0.0055

DPG/Hb4 Altitude 1,18 7.29 0.0147Time 1,19 27.6 <0.0001

Altitude × time 1,19 16.69 0.0006

Tests were carried out using a repeated-measures, two-way ANOVA.Hb, hemoglobin; Hct, hematocrit; MCHC, mean corpuscular Hb

concentration; RBC, red blood cell; DPG, 2,3-diphosphoglycerate.

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RBC size (5.57±0.28 versus 5.45±0.21μm; t42=1.380, P=0.126) orDPG/Hb ratio (2.45±0.51 versus 2.24±0.61; t23=0.858, P=0.458).These results indicate that the naturally occurring hematologicaldifferences between highland and lowland mice are environmentallyinduced and are largely attributable to physiological plasticity duringadulthood.

DISCUSSIONUnder conditions prevailing in their native habitats, highland deermice from the summit of Mount Evans exhibited a number ofpronounced hematological differences relative to lowlandconspecifics from the Great Plains. In comparison with lowlandmice, the highland mice exhibited higher Hb concentrations, higherHcts, higher DPG/Hb ratios, lower MCHC values and smaller RBCs.However, these differences disappeared after 6weeks of acclimationto normoxia at low altitude (Fig.2). The measured traits were alsoindistinguishable between the F1 progeny of highland and lowlandmice, indicating that there were no persistent, irreversible differencesin phenotype that could be attributed to developmental plasticity orfixed, genetically based differences. Studies of other physiologicaltraits in deer mice have documented similar degrees of plasticity inresponse to cold and/or hypoxic stress (Hammond et al., 1999;Hammond et al., 2001; Hammond et al., 2002; Rezende et al., 2004;Chappell et al., 2007; Russell et al., 2008; Rezende et al., 2009;Cheviron et al., 2013).

In the case of Hb concentration and Hct, the native characterstates of the highland mice were largely congruent with the typicalacclimatization response to hypoxia exhibited by lowland mammals

that have no known evolutionary history of residence at high altitude(Monge and León-Velarde, 1991). The elevated Hb concentrationsand Hcts observed in the highland deer mice are consistent withresults of previous studies of P. maniculatus (Gough and Kilgore,1964; Hock, 1964; Sealander, 1964; Dunaway and Lewis, 1965;Thompson et al., 1966; Sawin, 1970; Snyder, 1982; Snyder et al.,1982; Wyckoff and Frase, 1990; Hammond et al., 1999; Hammondet al., 2001) (supplementary material TableS1; Fig.3). By contrast,most rodent species that are native to high altitudes appear to haveHb concentrations and Hcts that are substantially lower than thoseof hypoxia-acclimated laboratory rats or house mice (Hall et al.,1936; Chiodi, 1962; Morrison et al., 1963a; Morrison et al., 1963b;Bullard et al., 1966) and hematological traits in the majority of high-altitude Andean rodents studied by Morrison and colleaguesremained unaltered after acclimation to normoxic conditions at sealevel (Morrison et al., 1963a; Morrison et al., 1963b).

In humans, highlanders that are indigenous to the Tibetan andEthiopian plateaus exhibit low Hb concentrations relative toAndean highlanders that are resident at comparable altitudes(Beall and Reischsman, 1984; Winslow et al., 1989; Beall et al.,1990; Beall et al., 1998; Beall et al., 2002; Garruto et al., 2003;Wu et al., 2005; Beall, 2006; Beall, 2007; Beall et al., 2010;Simonson et al., 2010). The high Hb concentrations of Andeanhighlanders mirror the expected acclimatization response tohypoxia in lowland natives, whereas the Tibetans and Ethiopiansappear to have evolved a blunted erythropoietic response toenvironmental hypoxia. Andeans also suffer a much higherincidence of chronic mountain sickness (Winslow and Monge,

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Fig.2. Changes in hematological traits (mean ± 1 s.e.m.) in highland and lowland P. maniculatus induced by acclimation to normoxia over a 6week period.(A)Hb concentration, (B) hematocrit (Hct), (C) mean corpuscular Hb concentration (MCHC), (D) red blood cell (RBC) size and (E) 2,3-diphosphoglycerate(DPG)/Hb4 ratio. Measures of Hb concentration, Hct and MCHC are based on 24 highland mice and 22 lowland mice, whereas measures of RBC size andDPG/Hb4 ratio were based on a subset of 10 highland mice and 11 lowland mice. A repeated-measures ANOVA was used to test for differences in meanvalues between treatment groups and between pre- and post-acclimation time points. Significant differences in mean values (P<0.05) are denoted bydifferent letters.

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1987). The elevated Hb concentrations of highland deer micemirror the acclimatization response to hypoxia in lowland species(e.g. Baumann et al., 1971; Duhm and Gerlach, 1971), suggestingthat the highland deer mice have a hematological profile that ismore similar to that of Andean humans than to that of Tibetansor Ethiopians. However, additional experiments are required toassess whether highland and lowland mice differ in theiracclimation responses to hypoxia, and it is an open question asto whether the elevated Hb concentration of high-altitude deermice represents an example of adaptive or maladaptive plasticity.

In addition to the potentially adverse effects of increased bloodviscosity on cardiac output, an increased Hct can also diminishplasma transport of free fatty acids and other metabolic fuels(McClelland, 2004). This is because the fuel transport capacity ofblood plasma is determined by the product of plasma flow and fuelconcentration, and plasma flow is an inverse function of Hct. Indeer mice living at high altitude, the effects of elevated Hct onplasma fuel transport may be especially significant because deermice rely heavily on fatty acid oxidation to fuel shiveringthermogenesis (Cheviron et al., 2012).

The elevated RBC DPG concentrations observed in highlanddeer mice are mostly consistent with previous reports. Snydermeasured RBC DPG concentrations in deer mice sampled fromlocalities ranging from 2590 to 4340m in the White Mountainsof eastern California (Snyder, 1982). Although the DPG/Hb ratiowas somewhat reduced in the high-altitude sample of mice from4340m, there was no monotonic altitudinal trend, as DPG/Hbratios were highest in mice from intermediate elevations. Similarto the results reported by Snyder (Snyder, 1982), our common-garden experiment revealed a reversal in relative DPGconcentrations such that the highland mice actually exhibited aslightly lower baseline DPG/Hb ratio after acclimation tonormoxia (Fig.2E). If the reduced baseline DPG/Hb ratio andelevated Hb–O2 affinity of highland deer mice are construed asadaptive mechanisms for maintaining an elevated blood–O2affinity under hypoxia, then the hypoxia-induced increase in RBCDPG would seem to be counterproductive (Storz et al., 2010b).In considering this seemingly maladaptive acclimatizationresponse, Snyder suggested that physiological constraints ofRBC energy metabolism may prevent evolutionary modificationsof intracellular DPG levels as the formation of DPG is stimulatedby overall glycolytic activity (Snyder, 1982). However, given thatthe optimal blood–O2 affinity is a non-linear function of ambientPO2 (Turek et al., 1973; Eaton et al., 1974; Bencowitz et al., 1982;Willford et al., 1982), empirical performance curves are needed

to evaluate whether hypoxia-induced reductions in the DPG/Hbratio are advantageous or disadvantageous at a given altitude. Aschanges in the DPG/Hb ratio also affect intracellular pH and Cl–

concentration, and as the adult Hbs of deer mice and other muroidrodents have ligand affinities that are more strongly modulatedby Cl– ions than by DPG (Storz et al., 2009; Storz et al., 2010a;Storz et al., 2012b; Runck et al., 2010), the net effects on blood–O2affinity are difficult to predict. Moreover, if deer mice have ahyperventilatory response to hypoxia, as in other mammals, theresultant increase in plasma pH (respiratory hypocapnia) mayeffectively counterbalance the inhibitory effects of DPG onHb–O2 affinity (Mairbäurl and Weber, 2012).

In contrast to the hematological changes that are typicallyassociated with the acclimatization response to hypoxia in lowlandmammals, genetically based changes in Hb structure that increaseintrinsic O2 affinity or that suppress sensitivity to allosteric effectorsare generally thought to make more important contributions tohypoxia tolerance in species that are high-altitude natives (Bunn,1980; Monge and León-Velarde, 1991; Storz, 2007; Weber, 1995;Weber, 2007; Storz and Moriyama, 2008; Storz et al., 2010b;Mairbäurl and Weber, 2012). High-altitude deer mice seem to defyMonge and León-Velarde’s proposed dichotomy between thehypoxia response strategies of highland and lowland natives (Mongeand León-Velarde, 1991), as they possess a set of hematologicaltraits that appear to fit both profiles. On the one hand, highland deermice exhibit elevated Hb concentrations, Hcts and RBC DPGconcentrations relative to lowland mice (Hock, 1964; Sawin, 1970;Snyder, 1982; Snyder et al., 1982), characteristics that seem to fitthe profile of a hypoxia-acclimated lowland species. On the otherhand, highland deer mice possess structurally distinct Hbs withslightly elevated O2 affinity (Storz et al., 2009; Storz et al., 2010a),which fits the profile of a genetically adapted, hypoxia-toleranthighland species.

In the case of highly labile hematological traits like Hbconcentration, Hct and RBC DPG concentration that are responsiveto minute changes in arterial PO2 and acid–base balance, it isprobably often the case that altitude-related trait differences betweenspecies or between conspecific populations are purely attributableto physiological plasticity, reflecting hypoxia-induced regulatorychanges in erythropoietic activity and RBC metabolism. The highdegree of plasticity that we have documented for hematological traitsin deer mice highlights the importance of using common-gardenexperiments to assess whether physiological differences betweenspecies or conspecific populations represent reversible, constitutivelyexpressed traits or irreversible, fixed differences.

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Fig.3. Scatterplot of mean Hb concentration (A)and mean Hct (B) against native altitude forpopulation samples of P. maniculatus and otherPeromyscus species from this and other studies.Filled symbols denote data points for populationsamples of P. maniculatus and open symbolsdenote data points for other species ofPeromyscus (P. boylii, P. crinitus, P. leucopus,P. nuttalli and P. polionotus). Raw data areprovided in supplementary material TableS1.

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ACKNOWLEDGEMENTSWe thank J. Projecto-Garcia for assistance with fieldwork, C. Brassil and P.Chernyavskiy for statistical advice, and G. B. McClelland, G. R. Scott and twoanonymous reviewers for helpful suggestions.

FUNDINGThis work was funded by grants from the National Institutes of Health/NationalHeart, Lung, and Blood Institute [grant nos R01 HL087216 and HL087216-S1],the National Science Foundation [grant no. IOS-0949931] and the Faculty ofScience and Technology, Aarhus University. Deposited in PMC for release after12 months.

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Paper III

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae)

with different altitudinal ranges

Revsbech IG, Tufts DM, Projecto-Garcia J, Moriyama H, Weber RE, Storz JF, Fago A.

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INTRODUCTIONFossorial and semi-fossorial mammals face challenges to respiratorygas transport associated with the chronic hypoxia and hypercapniaof underground burrows (Boggs et al., 1984). At high altitude, thehypoxic conditions of underground burrows are further compoundedby the reduced partial pressure of atmospheric O2 (PO2). In mountainranges of western North America, burrow-dwelling rodents in thesquirrel family (Sciuridae) are common denizens of the alpine andsubalpine zones, most notably the golden-mantled ground squirrel(Callospermophilus lateralis) and the yellow-bellied marmot(Marmota flaviventris), which occur at the highest possibleelevations in the contiguous United States (Armstrong, 1972;Chappell and Dlugosz, 2009). In addition to the hypoxic challengesassociated with life underground and life at high altitude, manysciurid rodents face additional gas transport challenges associatedwith depressed ventilation and episodic breathing during winterhibernation (Milsom, 1992; Milsom and Jackson, 2011; Webb andMilsom, 1994). In hibernating ground squirrels, prolonged periodsof apnea can result in wide fluctuations in arterial PO2 and plasmapH (Maginniss and Milsom, 1994; Malan et al., 1973; Musacchiaand Volkert, 1971).

In mammals adapted to life underground or life at high altitude,an elevated blood–O2 affinity can enhance pulmonary O2 loadingto compensate for the reduced PO2 of inspired air (Eaton, 1974;Eaton et al., 1974; Turek et al., 1973). Likewise, in hibernatingmammals, a left-shifted blood–O2 equilibrium curve can help

safeguard arterial O2 saturation during apneic periods and preventunfavorable O2 release to tissues (Maginniss and Milsom, 1994;Milsom and Jackson, 2011; Revsbech et al., 2013). An increase inblood–O2 affinity can be achieved by changes in the intrinsic O2affinity of hemoglobin (Hb), changes in the sensitivity of Hb toallosteric effectors and/or changes in the concentrations of allostericeffectors within the erythrocyte. In mammalian red cells, the mainorganic phosphate effector, 2,3-diphosphoglycerate (DPG),decreases Hb–O2 affinity by binding stereochemically at specificpositively charged residues in the central cavity of deoxygenatedHb: β1Val, β2His, β82Lys and β143His (Arnone, 1972). Theresultant stabilization of deoxy (T-state) Hb shifts the allostericequilibrium between the low-affinity T-state and the high-affinityR-state in favor of the low-affinity quaternary structure (Perutz,1970). Other factors that modulate Hb–O2 affinity are Cl– ions(Chiancone et al., 1972) and temperature (Antonini and Brunori,1971), as well as protons and CO2, which also bind preferentiallyto deoxy-Hb and promote O2 unloading in the tissue capillaries viathe Bohr effect (Perutz, 1983; Weber and Fago, 2004). Increases inblood–O2 affinity during winter hibernation have been documentedin a number of mammals, and such changes are generally attributableto reductions in red cell DPG, increases in pH and/or decreasedbody temperature (Burlington and Whitten, 1971; Maginniss andMilsom, 1994; Musacchia and Volkert, 1971; Revsbech et al., 2013;Tempel and Musacchia, 1975). To assess the relative efficacy ofdifferent mechanisms for increasing blood–O2 affinity during

SUMMARYSemi-fossorial ground squirrels face challenges to respiratory gas transport associated with the chronic hypoxia and hypercapniaof underground burrows, and such challenges are compounded in species that are native to high altitude. During hibernation,such species must also contend with vicissitudes of blood gas concentrations and plasma pH caused by episodic breathing.Here, we report an analysis of hemoglobin (Hb) function in six species of marmotine ground squirrels with different altitudinaldistributions. Regardless of their native altitude, all species have high Hb–O2 affinities, mainly due to suppressed sensitivities toallosteric effectors [2,3-diphosphoglycerate (DPG) and chloride ions]. This suppressed anion sensitivity is surprising given thatall canonical anion-binding sites are conserved. Two sciurid species, the golden-mantled and thirteen-lined ground squirrel, haveHb–O2 affinities that are characterized by high pH sensitivity and low thermal sensitivity relative to the Hbs of humans and othermammals. The pronounced Bohr effect is surprising in light of highly unusual amino acid substitutions at the C-termini that areknown to abolish the Bohr effect in human HbA. Taken together, the high O2 affinity of sciurid Hbs suggests an enhanced capacityfor pulmonary O2 loading under hypoxic and hypercapnic conditions, while the large Bohr effect should help to ensure efficientO2 unloading in tissue capillaries. In spite of the relatively low thermal sensitivities of the sciurid Hbs, our results indicate that theeffect of hypothermia on Hb oxygenation is the main factor contributing to the increased blood–O2 affinity in hibernating groundsquirrels.

Key words: adaptation, allostery, globin, hibernation, hypoxia.

Received 21 May 2013; Accepted 7 August 2013

The Journal of Experimental Biology 216, 4264-4271© 2013. Published by The Company of Biologists Ltddoi:10.1242/jeb.091397

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges

Inge G. Revsbech1, Danielle M. Tufts2, Joana Projecto-Garcia2, Hideaki Moriyama2, Roy E. Weber1, Jay F. Storz2,* and Angela Fago1,*

1Zoophysiology, Department of Bioscience, Aarhus University, C.F. Møllers Allè 3, DK-8000 Aarhus C, Denmark and 2School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA

*Authors for correspondence (jstorz2@unl.edu; angela.fago@biology.au.dk)

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hibernation, experiments on purified Hbs are required to evaluatethe independent and joint effects of DPG, pH and temperature onHb–O2 affinity under controlled conditions.

Here, we report an examination of the structural and functionalproperties of Hb in six species of marmotine ground squirrels(subfamily Xerinae, tribe Marmotini): golden-mantled groundsquirrel (C. lateralis) (Say 1823), yellow-bellied marmot (M.flaviventris) (Audubon and Bachman 1841), hoary marmot (M.caligata) (Eschscholtz 1829), thirteen-lined ground squirrel(Ictidomys tridecemlineatus) (Mitchill 1821), Uinta chipmunk(Tamias umbrinus) (Allen 1890) and least chipmunk (T. minimus)(Bachman 1839). All six species are semi-fossorial, winterhibernators that are native to North America. These six speciesinhabit a range of different elevations (Fig.1). For example, theyellow-bellied marmot is a strictly montane species that mainlyoccurs at elevations of 2000–4300m in the Rocky Mountains, theSierra Nevada, and other mountain ranges in western North America(Armstrong, 1972). By contrast, the closely related hoary marmotis mostly restricted to elevations <2500m in Alaska and westernCanada (Braun et al., 2011). The golden-mantled ground squirrel,Uinta chipmunk and least chipmunk are most common in themontane and subalpine zones, whereas the thirteen-lined groundsquirrel is mainly restricted to low-elevation grassland (Fig.1)(Armstrong, 1972; Streubel and Fitzgerald, 1978).

The objectives of this study were (i) to test the hypothesis thatthe elevated blood–O2 affinities of burrow-dwelling, hibernatingground squirrels are attributable to genetically based changes in Hbfunction (i.e. an increase in intrinsic Hb–O2 affinity and/or a decreasein sensitivity to allosteric effectors), (ii) to evaluate the efficacy ofdifferent allosteric effectors in regulating Hb–O2 affinity, and (iii)to test whether interspecific variation in Hb–O2 affinity is associatedwith native altitude.

MATERIALS AND METHODSSampling

We collected blood samples from wild-caught specimens of eachspecies in July–August 2010 and 2011. Golden-mantled groundsquirrels (N=6) were collected from two localities in the Front Rangeof the southern Rocky Mountains: Boulder County, CO, USA(40°01′45″N, 105°50′77″W; 3103m above sea level) and GrandCounty, CO, USA (38°84′37″N, 105°75′12″W; 2397m). Yellow-bellied marmots (N=5) were collected from near the summit ofMount Evans, Clear Creek County, CO, USA (39°15′24″N,106°10′54″W; 4300m), and hoary marmots (N=6) were collectedfrom two localities in central Alaska, viz., Eagle Summit, Yukon-

Koyukuk County, AK, USA (65°29′8″N, 145°24′0″W; 1119m) andWickersham Dome, Fairbanks North Star County, AK, USA(65°12′40″N, 148°3′37″W; 915m). Uinta chipmunks (N=2) and leastchipmunks (N=6) were collected from Park County, CO, USA(39°34′71″N, 105°63′19″W; 3013m) in addition to the same GrandCounty locality mentioned above. Thirteen-lined ground squirrels(N=2) were live-trapped in Lancaster County, NE, USA(40°52′12″N, 96°48′20″W; 430m).

Animals were handled in accordance with guidelines approvedby the University of Nebraska Institutional Animal Care and UseCommittee (IACUC no. 07-07-030D and 519) and the NationalInstitutes of Health (NIH publication no. 78-23). The thirteen-linedground squirrels were anesthetized using isoflurane and blood wasdrawn from the femoral vein. Blood was collected in cryotubes,snap-frozen in liquid nitrogen, and stored at −80°C. Frozen bloodsamples were used as a source of Hb for experimental studies andreticulocytes served as a source of globin mRNA for cDNAsequencing. The animals were released at the site of capture afterthey had regained consciousness. In the remaining species, bloodwas collected via cardiac puncture immediately after each animalwas killed and the blood samples were processed and stored asdescribed above. Additionally, bone marrow was collected from thefemurs of each animal as a source of globin mRNA. Bone marrowwas preserved in RNAlater (Qiagen, Valencia, CA, USA) and storedat −80°C prior to RNA extraction. With the exception of the thirteen-lined ground squirrels, all animals were prepared as voucheredmuseum specimens and were deposited in the vertebrate collectionsof the Denver Museum of Nature and Science (Denver, CO, USA;catalog numbers 11959–11962, 11967–11969, 11979–11983,11989–11990), the University of Alaska Museum of the North(Fairbanks, AK, USA; catalog numbers AF71385, AF71391–AF71395), and the University of Nebraska State Museum (Lincoln,NE, USA; catalog numbers NP1203–NP1207).

Globin sequencingAfter annotating the α- and β-globin genes from the genomeassembly of the thirteen-lined ground squirrel and other rodents(Hoffmann et al., 2008a; Hoffmann et al., 2008b; Opazo et al., 2008),we designed locus-specific primers for 5′ and 3′ RACE (rapidamplification of cDNA ends; Invitrogen Life Technologies,Carlsbad, CA, USA) to obtain cDNA sequence for the 5′ and 3′untranslated regions (UTRs) of each adult-expressed globin gene.After designing paralog-specific PCR primers with annealing sitesin the 5′ and 3′ UTRs, complete cDNAs were synthesized for eachgene by reverse-transcriptase PCR (RT-PCR) using the OneStep

Fig.1. Phylogenetic relationships andelevational ranges of six marmotine groundsquirrels included in the analysis of hemoglobin(Hb) function. Phylogenetic relationships arebased on a consensus of previous studies(Harrison, 2003; Helgen et al., 2009; Herron etal., 2004). In each terminal branch, the heightof the triangle is proportional to the elevationalrange limit of the corresponding species, andthe demarcation between the red and whiteparts of the triangle denotes the lower rangelimit. Elevational ranges (from Armstrong,1972; Braun et al., 2011; Streubel andFitzgerald, 1978) depicted in this figure arespecific to the geographic region in which eachspecies was sampled.

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RT-PCR kit (Qiagen). We performed RT-PCR on RNA templateto amplify α- and β-globin cDNAs, which were then cloned andsequenced. Total RNA was extracted from reticulocytes orhematopoeitic bone marrow cells using the RNeasy Plus Mini Kit(Qiagen), and cDNA was generated from 1μg RNA by reversetranscription. For RT-PCR, we used the SuperScript III PlatinumOne-Step RT-PCR system with Platinum Taq DNA polymeraseHigh Fidelity (Invitrogen, Carlsbad, CA, USA). PCR cyclingconditions were as follows: one cycle at 50°C for 10min, followedby 40 cycles at 94°C for 15s, 55°C for 30s, and 68°C for 2min,and a final extension cycle at 68°C for 7min. We then cloned gel-purified RT-PCR products into pCR4-TOPO vector using theTOPO TA cloning kit (Invitrogen). For each individual groundsquirrel specimen, we sequenced a total of 20 clones containingproducts of paralog-specific RT-PCR (10 cloned α-globin cDNAsand 10 cloned β-globin cDNAs per individual). Thus, full-lengthinserts representing cDNAs of the adult-expressed α- and β-globingenes were sequenced at 10× coverage. PCR and RT-PCR primersfor all species are available upon request. All DNA sequences weredeposited in GenBank under the accession numbers KF010591–KF010606, KF153033–KF153050.

Analysis of Hb isoform compositionHemolysates were prepared by adding a 3- to 5-fold volume of ice-cold 10mmoll−1 Hepes pH7.8 to frozen blood. The red supernatantwas cleared by centrifugation and stripped from endogenous organicphosphates using a mixed-bed resin (MB-1, AG501-X8, Bio-Rad,Hercules, CA, USA) (Storz et al., 2012). The Hb isoform (isoHb)composition of hemolysates from each specimen was analyzed byisoelectric focusing (IEF, pH5–8) using polyacrylamide gels(Phastgel, GE Healthcare Biosciences AB, Uppsala, Sweden). Afterseparation of isoHbs by means of IEF, bands excised from each gelwere digested with trypsin, and the resultant peptides were identifiedby means of tandem mass spectrometry (MS/MS). The peak listsof the MS/MS data were generated by Distiller (Matrix Science,London, UK) using the charge state recognition and de-isotopingwith default parameters for quadrupole time-of-flight data. Databasesearches of the resultant MS/MS spectra were performed usingMascot v1.9.0 (Matrix Science). Specifically, peptide massfingerprints derived from the MS/MS analysis were used to querya customized database of α- and β-globin amino acid sequences(based on conceptually translated DNA sequences) from each ofthe six sciurid species included in the study, including the fullcomplement of pre- and post-natally expressed globin genes fromthe thirteen-lined ground squirrel genome assembly. The followingsearch parameters were used: no restriction on protein molecularweight or isoelectric point, and methionine oxidation allowed as avariable peptide modification. Mass accuracy settings were 0.15Dafor peptide mass and 0.12Da for fragment ion masses. We identifiedall significant protein hits that matched more than one peptide withP<0.05.

O2 equilibrium measurementsFor all species, O2 equilibria were measured on 3μl samples ofpurified hemolysate (0.2–0.3mmoll−1 heme, 0.1moll–1 Hepes) atpH7.4 and 37°C using a thermostatted thin-layer chambertechnique (Sick and Gersonde, 1969; Weber, 1992). Curves weremeasured for each sample in the absence (stripped hemolysate)and presence of DPG (DPG/Hb tetramer ratio=2.0), Cl– ions(0.1moll–1 KCl) and of both anionic effectors as describedpreviously (Storz et al., 2010a; Storz et al., 2012). As a measureof Hb–O2 affinity the PO2 at 50% heme saturation (P50) and the

corresponding cooperativity coefficient (n50) were calculated bylinear regression (r2≥0.99) from the zero intercept and slope,respectively, of Hill plots {log[Y/(1–Y)] versus log PO2, where Yis the fractional saturation} fitted to at least four saturation stepsbetween 20 and 80%.

The sensitivity of Hb–O2 affinity to changes in pH (Bohr effect)and temperature were examined in the golden-mantled groundsquirrel and in the thirteen-lined ground squirrel. These species werechosen to compare the functional consequences of unusual aminoacid substitutions (see Results) found in the β-chain Hbs of thegolden-mantled ground squirrel (a highland native) and the thirteen-lined ground squirrel (a lowland native). For the golden-mantledground squirrel Hb, O2-binding curves were also measured induplicate in the absence (stripped) and presence of DPG as describedabove at three different pH values (range 7.0–8.0, 37°C) (to assessthe Bohr effect) and three different temperatures (range 23–37°C,pH7.4; to measure the temperature effect). For thirteen-linedsquirrels, only one of the two individual hemolysates was suitablefor functional studies (N=1) and, because of limited sampleavailability, measures of pH and temperature sensitivity for thestripped Hbs of this species were based on two pH values (7.0 and7.4) and two temperatures (23 and 37°C). The Bohr effect wasquantified as φ=ΔlogP50/ΔpH (Bohr factor) by linear regression(r2≥0.99 for golden-mantled ground squirrel Hb) of the logP50 versuspH plot. The temperature dependence of O2 binding, expressed asthe apparent heat of oxygenation (ΔH, kcalmol−1; 1kcal=4.184kJ,was derived by linear regression (r2≥0.99 for golden-mantledground squirrel Hb) of the van’t Hoff plot ∆H=2.303R(∆logP50)/∆(1/T), where R is the gas constant (1.987calK−1mol−1) and T isthe absolute temperature (K). The ∆H values here reported werecorrected for heat of O2 in solution (−3.0kcalmol−1) (Antonini andBrunori, 1971).

RESULTSThe IEF analysis revealed that each of the marmotine groundsquirrels expressed one to three isoHbs, with one major isoHb(isoelectric point=7.2–7.4) that typically accounted for 50–65% ofthe total (Fig.2). Combined results of the MS/MS analysis andcDNA sequence analysis revealed that isoHb heterogeneity isattributable to the expression of two to three structurally distinct β-globin chains (except for the Uinta chipmunk, which co-expressestwo distinct α-chain isoforms) (Fig.3). Multiple alignments of α-and β-globin amino acid sequences from each of the study species(Fig.3) revealed unusual substitutions at highly conserved residuepositions. Specifically, the golden-mantled ground squirrel, thirteen-lined ground squirrel, Uinta chipmunk and least chipmunk share anunusual substitution at the C-terminus of the α-chain,α141Arg→Cys, and the major β-chain of the golden-mantledground squirrel has an unusual substitution at the C-terminus,β146His→Gln (Fig.3). Both C-terminal residues are known tocontribute to the pH sensitivity of Hb–O2 binding (the Bohr effect)in human Hb (Perutz, 1970; Weber and Fago, 2004; Perutz, 1983;Berenbrink, 2006; Fang et al., 1999; Lukin and Ho, 2004). Thegolden-mantled ground squirrel also has a highly unusualsubstitution at another highly conserved β-chain position,β55Met→Ile (Fig.3). In each species, the α- and β-chain trypticpeptides of the major isoHb yielded highly significant matches tothe translated cDNA sequences from the same individual specimens.The MS/MS analysis confirmed the presence of the unusual C-terminal residues mentioned above. The MS/MS analysis did notreveal the presence of any major subunit isoforms that were notalready characterized at the DNA level.

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The experimental measures of Hb function performed on thehemolysates under physiologically relevant conditions revealed thatthe six sciurid species have overall similar Hb–O2 affinities (i.e. P50values) in the presence and absence of allosteric effectors (Table1).Sensitivities to DPG and Cl– ions, as indexed by the difference inlogP50 values for stripped samples in the presence both effectors,were in the range 0.13–0.35, with the exception of thirteen-linedsquirrel Hb from a single individual, which showed a greatersensitivity of 0.45 (Table1). Under similar experimental conditions,the measured anion sensitivity of human HbA was reported as∆logP50(DPG+KCl)–stripped≈0.4 (Weber, 1992). Thus, Hbs of themarmotine ground squirrels have generally low anion sensitivitiesrelative to human HbA (see also Table1). The Hill coefficients (n50)were in the range 1.8–2.9 under all conditions, indicating cooperativeO2 binding and intact homotropic interactions (Table1).

Measures of the pH sensitivity of Hb–O2 affinity revealedsubstantial Bohr effects for the Hbs of the golden-mantled and thirteen-lined ground squirrels, with Bohr factors (φ=∆logP50/∆pH) of −0.5and −0.8 (stripped) and −0.6 and −0.8 (with DPG added), respectively.Thus, in spite of the β146His→Gln substitution in the major isoHbof the golden-mantled ground squirrel, the measured Bohr coefficient

was similar to that of the thirteen-lined ground squirrel Hb. The smallchanges in the Bohr factor observed upon addition of DPG areconsistent with the suppressed anion sensitivity of Hb–O2 affinity inthese species (Table1). Because the Bohr factor represents thenumber of additional protons bound per O2 molecule (i.e. the numberof additional protons released upon oxygenation if the Bohr factor isnegative), these values indicate the presence of two to four O2-linkedproton-binding sites per Hb tetramer in the T-state. For comparison,under similar experimental conditions, the Bohr coefficient forstripped human HbA has been reported to be −0.56 (Weber, 1992).The temperature sensitivity of stripped Hb oxygenation at pH7.4 (datanot shown), expressed as the heat of oxygenation (ΔH, i.e. enthalpy)was −6.8kcalmol−1 in the golden-mantled ground squirrel Hb and−3.3kcalmol−1 in the thirteen-lined ground squirrel Hb, whereas it is−14.3kcalmol−1 in human HbA (Weber, 1992). In the golden-mantled ground squirrel, the enthalpy of Hb oxygenation decreasedin magnitude to −4.4kcalmol−1 in the presence of DPG, revealingthe endothermic contribution of oxygenation-linked DPG dissociation.Representative O2-binding curves for golden-mantled ground squirrelHb measured at different pH values and temperatures are shown inFig.4.

D

B

7.35

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7.35

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M M M

M M

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Fig.2. Isoelectric focusing gels (pH5–8) ofnative Hbs from specimens of marmotineground squirrels. (A)Golden-mantled groundsquirrel (Callospermophilus lateralis), (B)least chipmunk (Tamias minimus), (C) Uintachipmunk (Tamias umbrinus), (D) thirteen-lined ground squirrel (Ictidomystridecimlineatus), (E, first three lanes) yellow-bellied marmot (Marmota flaviventris) and (E,last three lanes) hoary marmot (Marmotacaligata). In each panel, marker proteins areindicated by M and the correspondingisoelectric points are reported on the left sideof the gels.

A 8 13 17 55 57 65 71 73 78 80 82 114 115 131 141Human T A V V G A A V N L A P A S RHouse mouse S ∙ I ∙ ∙ ∙ G L G ∙ ∙ ∙ ∙ ∙ ∙Golden-mantled ground squirrel ∙ ∙ I I ∙ ∙ ∙ ∙ G ∙ ∙ ∙ ∙ ∙ CHoary marmot ∙ ∙ I I ∙ ∙ ∙ ∙ S ∙ ∙ S ∙ ∙ ∙Yellow-bellied marmot ∙ ∙ I I ∙ ∙ ∙ ∙ S ∙ ∙ Q ∙ ∙ ∙Thirteen-lined ground squirrel N C I ∙ ∙ ∙ ∙ ∙ G ∙ T ∙ ∙ ∙ CLeast chipmunk ∙ C ∙ ∙ A V G L S M ∙ ∙ ∙ T CUinta chipmunk I ∙ C ∙ ∙ ∙ ∙ G L T ∙ ∙ ∙ G T CUinta chipmunk II ∙ C ∙ ∙ A ∙ G L S M ∙ ∙ ∙ T C

B 9 11 12 13 14 19 20 21 22 23 25 27 33 43 44 55 56 58 68 80 104 109 110 112 114 121 125 126 128 135 142 146Human S V T A L N V D E V G A V E S M G P L N R V L C L E P V A A A HHouse mouse A ∙ S C ∙ ∙ S ∙ ∙ ∙ ∙ ∙ ∙ D ∙ ∙ ∙ A I S ∙ M I I ∙ D A A ∙ ∙ ∙ ∙Golden-mantled ground squirrel I A I N ∙ A ∙ A ∙ ∙ ∙ ∙ ∙ ∙ D ∙ I ∙ ∙ I ∙ ∙ ∙ I I ∙ D Q ∙ ∙ T ∙ QGolden-mantled ground squirrel II A I N ∙ A ∙ A ∙ ∙ ∙ ∙ ∙ ∙ D ∙ I ∙ ∙ I ∙ ∙ ∙ I I ∙ D Q ∙ ∙ T S ∙Hoary marmot N I S T A ∙ A A D I A ∙ ∙ T ∙ ∙ ∙ D I ∙ K M I I M D E T ∙ ∙ ∙ ∙Yellow-bellied marmot N I S T A ∙ T A D I A ∙ ∙ T ∙ ∙ ∙ D I D K M I I ∙ D A A ∙ ∙ ∙ ∙Thirteen-lined ground squirrel I N I S T A ∙ A A ∙ ∙ A S ∙ D A ∙ ∙ A I ∙ K M I I M D E A ∙ ∙ S ∙Thirteen-lined ground squirrel II N I S T A ∙ A ∙ A I A S I D ∙ ∙ ∙ D I ∙ ∙ I I I ∙ D E A ∙ ∙ ∙ ∙Thirteen-lined ground squirrel III N I S T V D ∙ ∙ A ∙ A ∙ I D ∙ ∙ ∙ A I ∙ ∙ ∙ I I ∙ D E A ∙ ∙ ∙ ∙Least chipmunk I ∙ ∙ A ∙ ∙ ∙ T ∙ ∙ ∙ ∙ ∙ ∙ D ∙ ∙ S ∙ F ∙ K ∙ ∙ V ∙ ∙ Q ∙ S T ∙ ∙Least chipmunk II ∙ ∙ A ∙ ∙ ∙ T ∙ ∙ ∙ ∙ ∙ ∙ D ∙ ∙ S ∙ F ∙ K ∙ ∙ V ∙ ∙ Q ∙ S T S ∙Uinta chipmunk I ∙ ∙ A ∙ ∙ ∙ T ∙ ∙ ∙ ∙ ∙ ∙ D ∙ ∙ S ∙ F ∙ K ∙ ∙ V ∙ ∙ Q ∙ S T ∙ ∙Uinta chipmunk II ∙ ∙ A ∙ ∙ ∙ T ∙ ∙ ∙ ∙ ∙ ∙ D ∙ ∙ S ∙ F ∙ K ∙ ∙ V ∙ ∙ Q ∙ S T S ∙

Fig.3. Variable residue positions in a multiple alignment of globin sequences for marmotine ground squirrels: golden-mantled ground squirrel (C. lateralis),hoary marmot (M. caligata), yellow-bellied marmot (M. flaviventris), thirteen-lined ground squirrel (I. tridecemlineatus), least chipmunk (T. minimus) and Uintachipmunk (T. umbrinus), with orthologous sequences from human (Homo sapiens) HbA and mouse (Mus musculus) Hb included for comparison. Variableamino acid sites in the α- and β-globin sequences are shown in A and B, respectively. Sites in helical domains are shaded in the human referencesequence. Multiple sequences from the same species represent tandemly duplicated genes. For each species, αI and βI represent subunits of the major Hbisoform (isoHb; see Results for details).

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Although Hb–O2 affinities are often increased in mammals thatare native to high altitude (Storz, 2007; Storz et al., 2010b; Weber,2007), we found that the yellow-bellied marmot (a highland species)and hoary marmot (a predominantly lowland species) have Hbs withvery similar O2 affinities (Table1). For the six sciurid species thatwe examined, linear regressions revealed a suggestive but non-significant positive relationship between Hb–O2 affinity and nativealtitude (i.e. a negative relationship between P50 and altitude,measured under physiological conditions in the presence of KCland DPG): P50(KCl+DPG) versus midpoint of elevational range,y=12.471–5.649x, r2=0.151 (P=0.446); P50(KCl+DPG) versuselevational range limit, y=12.150–3.035x, r2=0.050 (P=0.670).Linear regressions based on phylogenetically independent contrastswere also non-significant (data not shown).

DISCUSSIONMarmotine ground squirrels have markedly increased blood–O2affinities in comparison with non-burrowing sciurids and otherrodents (Hall, 1965; Hall, 1966; Bullard et al., 1966), and the resultsof our experiments suggest that these differences largely stem fromgenetically based changes in Hb function. In the presence of thephysiological anionic effectors Cl– and DPG at pH7.4, Hb–O2affinities of the six sciurid species that we examined wereappreciably higher than those of muroid rodents measured underidentical experimental conditions. In the presence of allostericeffectors (DPG and Cl–), P50 values for the sciurid Hbs ranged from9.3 to 11.9Torr (where 1Torr≈133Pa), with thirteen-lined groundsquirrel Hb from a single individual reaching 13.1Torr (Table1),whereas those for three species of house mice (Mus caroli, M.domesticus and M. musculus) ranged from 16.9 to 17.5Torr (Runcket al., 2010; Storz et al., 2012;) and those for deer mice (Peromyscusmaniculatus) ranged from 11.7 to 15.8Torr (Storz et al., 2010a).Similarly, intrinsic Hb–O2 affinities, as indexed by P50 values forstripped hemolysates of the sciurids, ranged from 5.2 to 7.1Torr(Table1), whereas comparable measurements for house mice anddeer mice were 7.6–8.8Torr (Storz et al., 2012) and 7.4–7.9Torr(Storz et al., 2010a), respectively. These comparisons suggest thatthe elevated Hb–O2 affinities of the marmotine ground squirrels are

attributable to a generalized suppression of anion sensitivitycombined with a high intrinsic Hb–O2 affinity (i.e. of the strippedHb). In conjunction with results from other recent studies (Clementiet al., 2003; Natarajan et al., 2013; Storz et al., 2009; Storz et al.,2010a; Storz et al., 2012; Weber, 1992), the data summarized inTable1 indicate that rodent Hbs are generally far less responsive toDPG than the Hbs of humans and many other mammals.

In hibernating golden-mantled ground squirrels and thirteen-linedground squirrels, red cell DPG concentrations are reduced by nearly50% compared with levels in non-hibernating individuals of thesame species (Burlington and Whitten, 1971; Maginniss andMilsom, 1994). However, as the Hbs of these two species have suchlow anion sensitivities, the dramatic reductions in intracellular DPGmay not have important direct effects on the regulation of Hb–O2affinity during hibernation. Instead, the reduced DPG concentrationmay indirectly affect Hb–O2 affinity by altering the Donnanequilibrium of protons across the red cell membrane, as the reducedintracellular concentration of non-diffusible anions produces acorresponding reduction in proton concentration (Duhm, 1971). Theresulting increase in intracellular pH at a given plasma pH wouldthen increase Hb–O2 affinity via the pronounced Bohr effect of thesciurid Hbs (Fig.4).

The elevated Hb–O2 affinities of the marmotine ground squirrelsshould enhance pulmonary O2 loading under hypoxia, but the trade-off is a reduced PO2 gradient for O2 diffusion from capillary bloodto the perfused tissue. As suggested previously (Maginnis andMilsom, 1994), the strong Bohr effect should compensate for thehigh Hb–O2 affinity and promote O2 unloading in tissue capillarybeds (where tissue pH is decreased) while simultaneouslycontributing to O2 loading at the pulmonary surfaces (where pH isincreased). The large Bohr effect in the Hbs of marmotine groundsquirrels may also play a role in pulmonary CO2 excretion, whereBohr protons liberated upon Hb oxygenation drive the formationof gaseous CO2 from plasma bicarbonate. Thus, a pronounced Bohreffect could be beneficial for tissue O2 delivery as well as pulmonaryCO2 excretion not only at high altitudes but also in hypercapnicburrows and during hibernation when CO2 accumulates duringprolonged apneic periods (Heldmaier et al., 2004; Milsom and

Table1. Mean Hb–O2 affinities, cofactor sensitivities and cooperativity coefficients of purified hemolysates from six sciurid species

Golden-mantled Hoary Yellow-bellied Thirteen-lined Least Uinta ground squirrel marmot marmot ground squirrel chipmunk chipmunk

(N=6) (N=4) (N=4) (N=1) (N=6) (N=2)

P50 (Torr)Stripped 7.1±1.4 5.2±0.3 6.0±0.4 7.9 5.2±0.3 6.0±0.4KCl 9.2±0.8 10.8±0.9 12.2±1.5 11.1 8.7±0.4 11.2±1.8DPG 9.8±0.9 12.2±0.8 10.7±0.5 11.2 9.0±0.5 10.1±2.3KCl+DPG 9.8±0.9 11.7±0.8 11.9±1.4 13.1 9.3±0.6 11.3±2.2

∆logP50KCl–stripped 0.13±0.04 0.32±0.02 0.31±0.03 0.38 0.10±0.02 0.18±0.07DPG–stripped 0.15±0.04 0.37±0.01 0.25±0.04 0.39 0.12±0.02 0.13±0.10(KCl+DPG)–stripped 0.16±0.04 0.35±0.01 0.30±0.03 0.45 0.13±0.03 0.18±0.08

n50Stripped 2.1±0.3 2.1±0.1 2.2±0.0 2.1 1.8±0.1 2.2± 0.2KCl 2.2±0.3 2.4±0.2 2.3±0.2 2.7 2.0±0.1 2.4±0.1DPG 2.3±0.2 2.9±0.3 2.6±0.2 2.1 2.0±0.2 2.2±0.5KCl+DPG 2.1±0.2 2.6±0.1 2.5±0.1 2.3 2.1±0.1 2.4±0.3

Data are means (± s.d.) from golden-mantled ground squirrel (Callospermophilus lateralis), hoary marmot (M. caligata), yellow-bellied marmot (Marmotaflaviventris), thirteen-lined ground squirrel (Ictidomys tridecimlineatus), least chipmunk (T. minimus) and Uinta chipmunk (Tamias umbrinus). Curves weremeasured at 0.2–0.3mmoll–1 heme in 0.1moll–1 Hepes buffer at pH7.4, 37°C, in the absence (stripped) and presence of added KCl (0.1moll–1) and/or 2,3-diphosphoglycerate (DPG; DPG/Hb tetramer ratio, 2.0), as indicated. The sample size in parentheses (N) refers to the number of individuals analysedper species.

P50, Hb–O2 affinity (where 1Torr≈133Pa); ∆logP50(x-stripped), cofactor-induced change in logP50 (where x is the cofactor); n50, cooperativity coefficient.

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Jackson, 2011). Overall blood PCO2 of the golden-mantled groundsquirrel does not increase during hibernation, possibly due toeffective CO2 excretion and/or high Hb buffer capacity, as suggestedby high plasma bicarbonate (Maginniss and Milsom, 1994).

Similar to other mammals that are native to arctic or subarcticenvironments (Brix et al., 1989; De Rosa et al., 2004; Weber andCampbell, 2011), temperature changes have little effect on Hb–O2affinity in the thirteen-lined and golden-mantled ground squirrels(Fig.4). In regionally heterothermic organisms, this thermalinsensitivity of Hb–O2 affinity helps to ensure O2 delivery to coldextremities while simultaneously reducing heat loss from the body(di Prisco et al., 1991; Coletta et al., 1992). In hibernating mammals,a low enthalpy of oxygenation would also reduce temperature-induced fluctuations in O2 delivery to tissues during periodic cyclesof hypothermia and rapid rewarming upon arousal (Milsom andJackson, 2011), thereby minimizing oxidative stress arising frommismatches between O2 supply and O2 consumption. Despite theremarkably low value of the oxygenation enthalpy in the golden-mantled ground squirrel Hb (−6.8kcalmol−1) compared with thatof human HbA (−14.3kcalmol−1) (Weber, 1992), the strong decreasein body temperature of this hibernating species (>30°C) appears tobe a major contributor to the left-shifted blood–O2 equilibrium curve

(Maginniss and Milsom, 1994). This inference is supported by thefact that when subtracted for the heat of O2 solubilization(−3.0kcalmol−1), ΔH values measured in the blood of hibernatingand summer-active golden-mantled ground squirrels (−2.9 and−2.7kcalmol−1, respectively) (Maginniss and Milsom, 1994) weresimilar to those measured here on purified Hb in the presence ofDPG (−4.4kcalmol−1). In the brown bear (Ursus arctos), bycontrast, changes in temperature and DPG jointly modulate Hboxygenation during hibernation to prevent inappropriate O2 deliveryto tissues (Revsbech et al., 2013).

Given our current understanding of the structural basis of Hballostery, the Hbs of marmotine ground squirrels are highly unusualin two respects: (i) they have markedly reduced anion sensitivitiesin spite of the fact that they retain each of the cationic residues thathave been directly implicated in DPG and Cl– binding; and (ii) theyhave large Bohr effects in spite of substitutions that eliminate highlyconserved C-terminal residues that mediate the Bohr effect in humanHbA.

With respect to the Bohr effect, studies of human HbA havedemonstrated that H+ ions mainly reduce Hb–O2 affinity byprotonating the α-amino termini and the C-terminal β146His, whichstabilizes the deoxy T-state through the formation of additional saltbridges within and between subunits (Berenbrink, 2006; Fang etal., 1999; Lukin and Ho, 2004; Perutz, 1970; Perutz, 1983; Weberand Fago, 2004). The C-terminal α141Arg does not directlycontribute to the Bohr effect [the pKa (acid dissociation constant)of Arg is 12.0, so it is overwhelmingly protonated at pH7.4], butit contributes indirectly to the Cl–-dependent Bohr effect byfacilitating the Cl–-assisted charge stabilization of the free-NH2terminus of α1Val. Accordingly, human HbA mutants that have thesame residues as the golden-mantled ground squirrel (Hb Nunobiki,α141Arg→Cys and Hb Kodaira, β146His→Gln) are characterizedby a marked reduction in the alkaline Bohr effect (Harano et al.,1992; Kwiatkowski and Noble, 1987; Shimasaki, 1985). In the caseof the β-chain carboxy terminus, the positive charge on β146His isstabilized in the T-state by the negative charge of β94Asp and, atleast in human HbA, it has been estimated that the oxygenation-linked deprotonation of β146His accounts for most of the Bohr effect(Fang et al., 1999; Lukin and Ho, 2004; Perutz, 1983; Perutz et al.,1985). In Hb Kodaira (and presumably in the major isoHb of thegolden-mantled ground squirrel that also possesses β146Gln),replacement of the β94–β146 salt bridge by an unionizable H-bondwould theoretically result in a diminished Bohr effect. The fact thatthe Bohr coefficient of golden-mantled ground squirrel Hb exceedsthat of normal human HbA (with α141Arg and β146His) suggeststhat the C-terminal substitutions have different effects in thesequence context (or ‘genetic background’) of golden-mantledground squirrel Hb than in human Hb. For example, the loss of theβ146His Bohr group may be partly compensated for by theα113Leu→His substitution in marmotine ground squirrels, whichresults in the gain of two titratable His residues per tetramer relativeto human HbA. The same α113Leu→His substitution was alsosuggested to contribute to a large Bohr effect in mole and shrewHb (Campbell et al., 2012). Detailed mutagenesis andcrystallographic studies will be required to illuminate thestructure–function relationships of golden-mantled ground squirrelHb. So far, our data support the view that the pH sensitivity of Hb–O2affinity stems from many surface His residues with individuallyminor effects, rather than small numbers of key residues (e.g.β146His) with major effects.

The high Hb–O2 affinities of marmotine ground squirrels areconsistent with data from other fossorial and semi-fossorial

0 5 10 15 20PO2 (Torr)

0

0.2

0.4

0.6

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tiona

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urat

ion

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0 5 10 15 20 25

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�

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Fig.4. Representative O2-binding curves for golden-mantled ground squirrelHb measured (A) at 37°C and pH7.0, 7.4 and 8.0 in the presence of 2,3-diphosphoglycerate (DPG; DPG/Hb tetramer ratio 2.0) and 0.1moll–1

KCl, and (B) at pH7.4 and 37, 30 and 23°C in the presence of DPG(DPG/Hb tetramer ratio 2.0) and 0.1moll–1 KCl. Fitting of data according tothe sigmoidal Hill equation [Y=PO2

n50/(P50n50+ PO2

n50)] is shown by solidlines. The same O2-binding curve measured at pH7.4 and 37°C in thepresence of DPG and KCl (filled circles, thick line) is shown in both panelsto illustrate the effects of changes in pH (A) or temperature (B).

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mammals (Hall, 1965; Hall, 1966; Jelkmann et al., 1981; Lechner,1976; Campbell et al., 2010). Comparisons among the six speciesthat we examined revealed a suggestive (but non-significant)positive relationship between Hb–O2 affinity and native altitude.However, the yellow-bellied marmot and hoary marmot exhibitedno appreciable difference in Hb–O2 affinity in spite of theirdramatically different elevational ranges. As ground squirrels areadapted to the respiratory gas transport challenges associated withsemi-fossorial habits and winter hibernation, it may be that theirability to survive and function at high altitude does not requireany additional modifications of Hb function. Consistent with thishypothesis, there are no appreciable differences in blood–O2affinity between highland and lowland populations of burrowingmole rats (Cryptomys hottentotus mahali) (Broekman et al., 2006).Similarly, pocket gophers of the genus Thomomys have elevatedblood–O2 affinities relative to non-fossorial rodents, but there areno apparent differences in Hb function between closely relatedpocket gopher species that are native to different elevational zones(Lechner, 1976).

CONCLUSIONThe Hbs of marmotine ground squirrels are surprisingly insensitiveto the modulating effects of DPG and Cl– ions, but are characterizedby large Bohr coefficients and low thermal sensitivities relative tothe Hbs of humans and other mammals. The suppressed anionsensitivity of the sciurid Hbs investigated here is unexpected in lightof the fact that they retain all the canonical organic phosphate- andCl–-binding sites in the central cavity, and the pronounced Bohreffect of golden-mantled ground squirrel Hb is surprising in lightof two highly unusual substitutions, α141Arg→Cys andβ146His→Gln, that are known to abolish the Bohr effect in humanHbA. The elevated blood–O2 affinity of ground squirrels suggestsenhanced capacities for pulmonary O2 loading under hypoxic andhypercapnic conditions, while the large Bohr effect should help toensure efficient O2 unloading in tissue capillaries.

LIST OF SYMBOLS AND ABBREVIATIONSDPG 2,3-diphosphoglycerateHb hemoglobinisoHb hemoglobin isoformn50 cooperativity coefficient of Hb–oxygen bindingP50 partial pressure of oxygen required to saturate Hb by 50%PO2 partial pressure of oxygenΔH enthalpy of Hb oxygenationφ Bohr factor

ACKNOWLEDGEMENTSWe thank G. Bachman, K. Bell, Z. Cheviron, A. Gunderson, L. Olson and A.Runck for assistance with fieldwork, J. Demboski and K. McCracken for logisticalassistance in the field, J. Allison, A. Bang, M. B. Hemmingsen and K. Williams forassistance with lab work, and F. Hoffmann and C. Natarajan for assistance withgene annotation and sequence alignments.

AUTHOR CONTRIBUTIONSI.G.R. and D.M.T. contributed equally to this work. The research was conceivedand designed by I.G.R., D.M.T., J.F.S. and A.F.; I.G.R., D.M.T., J.P.-G., H.M.,R.E.W., J.F.S. and A.F. executed and interpreted the results of experiments;I.G.R., J.F.S. and A.F. drafted the manuscript; all authors edited, revised andapproved the manuscript.

COMPETING INTERESTSNo competing interests declared.

FUNDINGThis work was funded by grants from the National Institutes of Health/NationalHeart, Lung, and Blood Institute [grant numbers R01 HL087216, HL087216-S1];

the National Science Foundation [IOS-0949931]; the Danish Council forIndependent Research, Natural Sciences [10-084565]; the Faculty of Science andTechnology, Aarhus University; The American Society for Mammalogists, and aUniversity of Nebraska Special Funds Research Grant. Deposited in PMC forrelease after 12 months.

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with Ligands. Amsterdam: North-Holland.Armstrong, D. M. (1972). Distribution of mammals in Colorado. Monograph of the

Museum of Natural History, University of Kansas 3, 1-415.Arnone, A. (1972). X-ray diffraction study of binding of 2,3-diphosphoglycerate to

human deoxyhaemoglobin. Nature 237, 146-149. Berenbrink, M. (2006). Evolution of vertebrate haemoglobins: histidine side chains,

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THE JOURNAL OF EXPERIMENTAL BIOLOGY 95

Paper IV

Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging brown bears and their

potential roles in hibernation

Revsbech IG, Shen X, Chakravarti R, Jensen FB, Thiel B, Evans A, Kindberg J, Fröbert O, Stuehr DJ, Kevil CG, Fago A

96

97

Original Contribution

Hydrogen sulfide and nitric oxide metabolites in the bloodof free-ranging brown bears and their potential roles in hibernation

Inge G. Revsbech a,1, Xinggui Shen b,1, Ritu Chakravarti c, Frank B. Jensen d, Bonnie Thiel e,Alina L. Evans f, Jonas Kindberg g, Ole Fröbert h, Dennis J. Stuehr c, Christopher G. Kevil b,Angela Fago a,n

a Department of Bioscience, Aarhus University, 8000 Aarhus C, Denmarkb Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USAc Department of Pathobiology, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195, USAd Department of Biology, University of Southern Denmark, 5230 Odense M, Denmarke Department of Medicine, Case Western Reserve University, Cleveland, OH 44106, USAf Department of Forestry and Wildlife Management, Faculty of Applied Ecology and Agricultural Sciences,Hedmark University College, Campus Evenstad, 2418 Elverum, Norwayg Department of Wildlife, Fish, and Environmental Studies, Swedish University of Agricultural Sciences, 90183 Umeå, Swedenh Department of Cardiology, Örebro University Hospital, 70362 Örebro, Sweden

a r t i c l e i n f o

Article history:Received 24 March 2014Received in revised form19 May 2014Accepted 30 May 2014Available online 5 June 2014

Keywords:Metabolic depressionAdaptationHypothermiaThiosulfatePolysulfideFree radicals

a b s t r a c t

During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating whiletheir basal metabolism is largely suppressed. To understand the underlying mechanisms of metabolicdepression in hibernation, we measured type and content of blood metabolites of two ubiquitousinhibitors of mitochondrial respiration, hydrogen sulfide (H2S) and nitric oxide (NO), in winter-hibernating and summer-active free-ranging Scandinavian brown bears. We found that levels of sulfidemetabolites were overall similar in summer-active and hibernating bears but their composition in theplasma differed significantly, with a decrease in bound sulfane sulfur in hibernation. High levels ofunbound free sulfide correlated with high levels of cysteine (Cys) and with low levels of bound sulfanesulfur, indicating that during hibernation H2S, in addition to being formed enzymatically from thesubstrate Cys, may also be regenerated from its oxidation products, including thiosulfate andpolysulfides. In the absence of any dietary intake, this shift in the mode of H2S synthesis would helppreserve free Cys for synthesis of glutathione (GSH), a major antioxidant found at high levels in the redblood cells of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation ofglyceraldehyde-3-phosphate dehydrogenase, taken as markers of NO metabolism, did not changeappreciably. Our findings reveal that remodeling of H2S metabolism and enhanced intracellular GSHlevels are hallmarks of the aerobic metabolic suppression of hibernating bears.

& 2014 Elsevier Inc. All rights reserved.

Hibernating bears lie in dens for almost half a year withouteating or drinking while relying on body fat reserves before theyemerge relatively unharmed in the spring [1–3]. During winterhibernation, bears become essentially self-containing units withlittle or no exchange with the environment, and, to prolong bodyenergy reserves, they reach a profound hypometabolic state, with

lowered body temperatures and minimum metabolic rates downto �25% of the basal levels [3,4]. During hibernation, loweredheart and ventilation rates [4,5] and increased blood O2 affinity,due in part to the reduced body temperature and in part toreduced levels of red cell 2,3-diphosphoglycerate (DPG) [6], reduceO2 supply, thus matching the reduced tissue O2 consumption.Because of these adjustments, hibernators most likely remainessentially aerobic and experience little or no hypoxia [2].Whereas in small hibernators body temperature drops to only afew degrees above zero [3], bears hibernate at much less reducedbody temperatures (i.e., with regular oscillations between �30and 36 1C) [4,7], despite having the same weight-specific lowmetabolic rate as small hibernators [3]. This suggests a significanttemperature-independent component in the metabolic depressionof hibernating bears [4].

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/freeradbiomed

Free Radical Biology and Medicine

http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.0250891-5849/& 2014 Elsevier Inc. All rights reserved.

Abbreviations: CSE, cystathionine γ-lyase; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; RBC, red blood cell; DTPA, diethylenetriaminepentaacetic acid;SNO, S-nitrosothiol; ROS, reactive oxygen species; GSH, reduced glutathione; GSSG,oxidized glutathione; BSS, bound sulfane sulfur; SBD-F, 4-fluoro-7-sulfobenzofurazan; HPLC, high-performance liquid chromatography

n Corresponding author.E-mail address: angela.fago@biology.au.dk (A. Fago).1 These authors contributed equally to this work.

Free Radical Biology and Medicine 73 (2014) 349–357

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The remarkable ability of bears and other mammalian speciesto hibernate has remained poorly understood in terms of theunderlying mechanisms. After some early attempts to identify acirculating “trigger” molecule in the blood from hibernators [8,9],a first clue to understanding the key to metabolic depression inhibernation came from experiments [10] showing that miceinhaling �80 ppm hydrogen sulfide (sulfane, H2S) underwentdrastic but fully reversible reductions in metabolic rate, bodytemperature, lung ventilation, O2 consumption, and CO2 produc-tion. The dramatic changes observed, albeit artificial, were strik-ingly similar to those of natural hibernators. This hypometaboliceffect has been ascribed to the known ability of H2S to reversiblyinhibit mitochondrial cytochrome c oxidase when present at lowlevels [10–12]. However, it was not known whether levels of H2Sand of its physiological in vivo metabolites in fact change innatural hibernators such as bears.

Suppression of O2 consumption in hibernation necessarily ori-ginates from the mitochondria, where �90% of whole-animal O2

consumption takes place [13]. In principle, other signaling mole-cules capable of reversible inhibition of cytochrome c oxidase couldalso be involved in the metabolic suppression of hibernators. Onesuch molecule is nitric oxide (nitrogen monoxide, NO). H2S and NOare ubiquitous signaling molecules synthesized by naturally occur-ring enzymes (including cystathionine γ-lyase for H2S and nitricoxide synthases for NO) with profound physiological effects onmitochondrial respiration, blood pressure regulation, and cytopro-tection [14,15]. Because of their reactivity, both these signalingmolecules generate in vivo a broad range of oxidative products,each with distinctive biological activities. The complex in vivoeffects of NO and its products, in particular nitrite and S-nitro-sothiols (SNOs; formed when Cys thiols are modified by NO), areknown in good detail [16,17] because highly sensitive (e.g. chemi-luminescence and biotin switch) methods have been available forsome time for the detection of their low-nanomolar in vivo levels[18,19]. These methods have revealed important roles for circulatingnitrite as a storage pool of NO, from where NO can be regeneratedduring hypoxia and contribute to vasodilation and cytoprotection[16,20,21], and for S-nitrosation as a site-specific redox-dependentprotein modification in mammals [17] and in ectotherm vertebrates[22–24]. In contrast, the biological roles of H2S and its metabolitesin vivo have remained more elusive owing to technical limitationsfor their detection [25,26] and new methods are being currentlydeveloped to obtain reliable measures of physiological levels of H2Sand related compounds [14,27].

As fluctuations in respiratory rates are associated with oxidativestress, physiological metabolic suppression is tightly linked withantioxidant capacity. Hibernating bears most likely possess enhancedtolerance against oxidative stress and regenerative capacity as knownfor other animals capable of prolonged metabolic suppression [2,28].Enhanced oxidative stress typically occurs whenever mitochondrialactivity varies independent of available O2 and potentially damagingreactive oxygen species (ROS) are generated as a product [29]. Theubiquitous tripeptide glutathione (GSH) is a key element in the thiol-dependent cellular defense against ROS and redox imbalance. Forinstance, ectotherms experiencing seasonal periods of prolongedhypoxia and severe oxidative stress at arousal are known to possessmuch higher levels of GSH compared to their hypoxia-intolerantcounterparts [28].

In this study, we report measurements of a large number (23 intotal) of blood parameters taken from winter-hibernating andsummer-active free-ranging brown bears (the same individualsin winter and summer), with the intent to identify which para-meters could be involved in hibernation. Specifically, we examinedthe circulating levels of major H2S and NO metabolites; the activityof the enzyme cystathione γ-lyase (CSE), an important enzymecatalyzing the production of H2S from L-Cys in the circulation; and

the levels of free L-Cys and GSH and other thiols. We have alsoinvestigated levels, activity, and S-nitrosation of the enzymeglyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key gly-colytic enzyme known to undergo S-nitrosation [30], as a markerof targeted S-nitrosation-dependent control of energy metabolismand potentially involved in the reduction of downstream 2,3-diphosphoglycerate in RBCs during hibernation [6]. All of theinvestigated parameters were subjected to a stringent statisticalanalysis to test for significant differences and mutual correlationsbetween hibernating and summer active individual bears. Thefindings of this exploratory study unveil distinct potential roles ofH2S and NO-dependent signaling in physiological metabolicsuppression.

Materials and methods

Animals and blood samples

Animal handling and sampling were approved by the SwedishEthics Committee on Animal Research (C212/9) and the SwedishEnvironmental Protection Agency. Blood samples were collectedfrom seven 3- to 5-year-old (two males and five females) free-ranging anesthetized Eurasian brown bears (Ursus arctos) inDalarna and Gävleborgs Counties, Sweden. The bears were pre-viously equipped with global-positioning system collars as well asradio transmitters for tracking. Bears were immobilized by dartingin the den during winter (February 2013) and the same bears againby darting from a helicopter during summer (June 2013). Anes-thetics used in winter were a mixture of tiletamine–zolazepam(1.1 mg/kg), medetomidine (0.03 mg/kg), and ketamine (1.3 mg/kg) and in summer a mixture of tiletamine–zolazepam (4.7 mg/kg)and medetomidine (0.09 mg/kg) [7]. The medetomidine wasantagonized with 5 mg antisedan for each milligram of medeto-midine after the procedures were finished and after placing thebears back into the dens in winter [6,7]. Blood was taken from thejugular vein using heparinized vacuum tubes and immediatelycentrifuged in the field (4 min, 9000g) to separate plasma fromRBCs. For each individual, RBC aliquots were immediately frozen indry ice for later measurement of GAPDH activity or treated beforefreezing as described below under H2S products and NO products.All processing and freezing of blood samples were done in the fieldwithin 10 min of blood sample collection. Samples were protectedfrom light during processing. All chemicals were from Sigma–Aldrich unless otherwise stated.

H2S products

Biochemical forms of H2S were measured using the HPLCmonobromobimane (MBB) assay as previously reported [27,31].Aliquots of RBCs and plasma from individual bears were immedi-ately diluted 1:5 in rubber cap-sealed anaerobic Eppendorf vialscontaining previously degassed 100 mM Tris–HCl buffer, pH 9.5,0.1 mM diethylenetriamine pentaacetic acid (DTPA) and frozen indry ice for later measurement of H2S products, total GSH, cysteine,and homocysteine concentrations [27,31,32]. Additional RBC andplasma samples were frozen without further treatment immedi-ately after centrifugation for measurement of CSE activity. Allsamples were stored in liquid N2 and analyzed within 2 weeks ofcollection [33].

Measurement of total GSH, Cys, and homocysteine

Total GSH, Cys, and homocysteine were measured after thiolreduction and derivatization with 4-fluoro-7-sulfobenzofurazan(SBD-F) as described [32,34]. Briefly, samples were reduced by

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incubation with 30 mM tris(2-carboxyethyl)phosphine hydro-chloride at room temperature for 30 min. Trichloroacetic acid(100 mg/ml) was added to precipitate proteins. After centrifuga-tion for 10 min, the supernatants were derivatized with 90 mMSBD-F and the fluorescent thiol derivatives were separated on aC18 column by reverse-phase high-performance liquid chromato-graphy and detected by fluorescence (extinction 385 nm, emission515 nm).

CSE activity

CSE activity was measured as previously reported [35]. Briefly,tissue lysates were mixed with 2 mM cystathionine, 0.25 mM pyr-idoxal 50-phosphate in 100 mM Tris–HCl buffer and incubated for60 min at 37 1C. Trichloroacetic acid (10%) was added to the reactionmixture and spun down. The supernatant was then mixed with 1%ninhydrin reagent and incubated for 5 min in a boiling-water bath.The solution was then cooled on ice for 2 min and the absorbancemeasured at 455 nm using a Smart Spect Plus spectrophotometer(Bio-Rad). CSE activity was expressed as nanomoles of cystathionineconsumed per milligram of total protein per hour of incubation.

NO products

Aliquots of RBCs from individual bears were diluted 1:5 with aSNO/nitrite-stabilizing solution containing 4 mM ferricyanide(K3FeIII(CN)6), 10 mM N-ethylmaleimide, 0.1 mM DTPA [18] andfrozen in dry ice. Thawed hemolysate and plasma were vortexedand centrifuged (2 min, 16,000g, 4 1C), and supernatants wereimmediately measured. NO metabolites were measured by reduc-tive chemiluminescence using a Sievers (Boulder, CO, USA) nitricoxide analyzer (Model 280i) and previously described procedures[18,36]. Levels of SNO, iron–nitrosyl, and N-nitrosyl compoundswere below the detection limit (approximately 10 nM at thevolume and dilution used). Nitrite was subsequently determinedon samples deproteinized with ice-cold ethanol (1:1). Nitritepeaks were integrated with Origin software (OriginLab Corp.,Northampton, MA, USA).

Biotin switch

RBC samples were stored at �80 1C in stabilization solutioncontaining 10 mM N-ethylmaleimide. The biotin switch assay wasperformed as described [19,37] and pull-downs from Neutravidinresin (Pierce, Rockford, IL, USA) were probed for the presence ofGAPDH by Western blotting. Total GAPDH was tested in RBCsamples before processing samples for biotin switch.

GAPDH activity

GAPDH activity was assessed at 25 1C in the absence andpresence of 0.3 mM dithiothreitol by monitoring the time-dependent decrease in NADH absorbance at 340 nm (extinctioncoefficient 6.22 mM�1 cm�1) following the Sigma enzymatic assayprotocol (EC 1.2.1.12, Sigma–Aldrich) [37]. The assay proceeds intwo steps, catalyzed by 3-phosphoglyceric phosphokinase (step 1)and GAPDH (step 2):

3-phosphoglyceric acidþATP-1,3-DPGþADP, (1)

1,3-DPGþNADH-glyceraldehyde-3PþNADþþPi. (2)

Reactions were completed after 150 s. For each sample, theGAPDH activity reported is expressed as units per milligram oftotal hemoglobin, measured at 540 and 576 nm using knownextinction coefficients [38].

Statistical analysis

Statistical differences between plasma and RBC parameters ofthe same seven individual winter-hibernating and summer-activebears were assessed by parametric paired t test with a significancelevel set at P o 0.002 to account for multiple tests. Because of thehigh number of parameters measured on the same samples, a lowP value of o0.002 (�0.05/23) is required to validate that differ-ences between parameters measured in hibernating and summer-active bears are significant. Nonparametric paired Wilcoxon signedrank tests produced similar results (significance level P o 0.016).To search for relationships between pairs of individual parametersmeasured in winter-hibernating and summer-active bears, pair-wise Pearson correlations were calculated. The most interestingpairs were determined based on the strength (r 4 0.7 or r o�0.7) and significance (P o 0.05) of the correlation. Statisticalanalyses were performed with SAS software, version 9.3 (SASInstitute, Cary, NC, USA).

Results

We used the recently developed MBB method for the detection ofphysiological levels of various H2S metabolites [31] in plasma andRBCs isolated from the same individual hibernating and summer-active brown bears. H2S metabolites can be divided into (1) acid-labile sulfur, which mainly contains Fe–S clusters and persulfides andis converted into H2S under acidic conditions; (2) bound sulfanesulfur (BSS), which contains thiosulfate and polysulfides and isconverted into H2S under reducing conditions; and (3) free sulfide,containing mainly freely dissolved H2S and HS� [14]. We found thatin winter-hibernating bears, plasma contained significantly (P o0.002) lower BSS and free sulfide and significantly (P o 0.002)higher acid-labile sulfane than in summer-active bears (Fig. 1A;Supplementary Tables S1 and S2). Marked changes were also foundwhen measuring thiols. Levels of plasma Cys (the most abundantplasma thiol) and total GSH decreased significantly (P o 0.002)during hibernation (Fig. 1B, Supplementary Tables S1 and S2). Lesspronounced changes were found in other plasma parameters, such asan increase in CSE activity (P¼0.033) (Fig. 1C) and a decrease in totalsulfane sulfur (P¼0.0035) and nitrite (P¼0.043) (SupplementaryTables S1 and S2), all approaching significance. Conversely, in RBCs,all parameters examined remained relatively constant with theexception of total GSH, which increased significantly (P o 0.002)during hibernation (Figs. 2A and B; Supplementary Tables S3 and S4).CSE activity was higher in RBCs than in plasma and possiblyincreased in hibernating bears (Fig. 2C; P¼0.05). RBC nitrite alsoincreased during hibernation, albeit not significantly (P¼0.05;Supplementary Tables S3 and S4). Despite the level of total SNOcompounds being below the detection limit, S-nitrosated GAPDHwas detected in RBCs by the biotin switch method (Fig. 3A), but itslevels did not change significantly during hibernation and showed alarge individual variation (Fig. 3B, Supplementary Tables S3 and S4).Consistently, GAPDH activity was similar in hibernating and summer-active bears (Supplementary Tables S3 and S4). Changes approachingstatistical significance were found in RBC bound and total sulfanesulfur (P¼0.0072 and 0.0087, respectively), total homocysteine(P¼0.03), and GAPDH content (P¼0.0032) (Supplementary TablesS3 and S4).

We then analyzed all pairwise Pearson correlations for plasmaand RBC variables (Supplementary Figs. S1 and S2) within individualhibernating and summer-active bears to search for significant patterns(r higher than 0.7 for direct correlations and less than �0.7 for inversecorrelations). Selected correlations are shown in Fig. 4. In hibernatingbears plasma, BSS, and free sulfide were inversely correlated(r¼�0.77; Fig. 4A), whereas Cys was positively correlated with GSH

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(r¼0.89; Fig. 4B) and free sulfide (r¼0.87; Fig. 4C). Within RBCs, GSHwas positively correlated with Cys (r¼0.82) in hibernating bears(Fig. 4D). None of these parameters were significantly correlated insummer (Figs. 4A–D). These correlations suggest that during hiberna-tion plasma Cys availability is important for the generation of freesulfide and GSH (as also in RBCs) and that plasma BSS is used as asource of free sulfide. Furthermore, nitrite and SNO–GAPDH levelswere not correlated (Supplementary Fig. S2), suggesting a nitrite-independent mechanism for S-nitrosation in this enzyme. Othercorrelations, albeit significant, are not clearly interpretable in termsof seasonal patterns of their variations. We note, however, that astrong correlation between two parameters indicates that they areequivalent in what they are measuring. For example, in both hibernat-ing and summer-active bears, total and bound sulfane sulfur weretightly correlated (r¼0.91 in plasma and 0.94 and 0.96 in RBCs;Supplementary Figs. S1 and S2), meaning that the amount of variationin one variable is largely due to the other variable (as given by r2). Inother words, in these samples measuring total sulfane sulfur is largelyequivalent to measuring bound sulfane sulfur.

Discussion

How brown bears and other mammalian hibernators arecapable of drastically reducing their metabolic rate for longperiods of time while still preserving organ integrity is largelyunknown. A major finding of this study is that hibernation in free-ranging brown bears is associated with highly significant changesin plasma H2S metabolites and enhanced intracellular GSH levels.

The possible origin of H2S in hibernation

Overall, total sulfide did not change significantly in eitherplasma (Fig. 1A) or RBCs (Fig. 2A) upon hibernation, indicatingthat the balance between H2S generation and consumption islargely the same. Plasma values of �5 mM total sulfide in bears areabout the same as those found in mice (�4.5–4.8 mM) [31]. Theseresults indicate that it is not a general increase in H2S levels that isassociated with hibernation, but rather a shift in the way it isproduced and consumed. Consistent with this interpretation, the

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Fig. 2. RBC concentrations of H2S metabolites and thiols and CSE activity in summer-active and winter-hibernating free-ranging brown bears. RBC concentrations(means7SEM) of (A) sulfide (free sulfide, acid labile sulfide, bound sulfane sulfur and total sulfide) and (B) total thiols (Cys, homocysteine, and GSH). (C) CSE activity in thesame summer-active and winter-hibernating bears (n¼7). Significant differences between summer and winter values are indicated (*P o 0.002). (A) Whereas H2Smetabolites did not change, (B) total GSH increased significantly in RBCs upon hibernation. (C) CSE activity increased, but not significantly (P¼0.05).

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Fig. 1. Plasma concentrations of H2S metabolites and thiols and CSE activity in summer-active and winter-hibernating free-ranging bears. Concentrations (means7SEM) of(A) sulfide (free sulfide, acid labile sulfide, bound sulfane sulfur, and total sulfide) and (B) total thiols (Cys, homocysteine, and total GSH). (C) CSE activity in plasma of thesame summer-active and winter-hibernating bears (n¼7). Significant differences between summer and winter values are indicated (*P o 0.002). (A) The composition of H2Smetabolites but not of total sulfide and (B) the levels of Cys and total GSH changed significantly upon hibernation, whereas (C) CSE activity increased, albeit not significantly(P¼0.033).

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relative composition of H2S metabolites changed markedly inplasma (Fig. 1A) but not in RBCs (Fig. 2A), with more sulfidepresent in the plasma as the acid-labile fraction and less as freesulfide or BSS (Fig. 1A). The significant decrease in the plasma BSSpool (Fig. 1A) and the negative correlation between BSS and free

sulfide found in hibernating bears (Fig. 4A) are interesting, asthese results suggest that H2S is generated at the expense of theBSS pool, whereas in summer bears, there is no obvious correla-tion between these two parameters. Polysulfides and thiosulfate(S2O3

�) are major products of H2S oxidation contained in the BSS

Fig. 3. (A) RBC S-nitrosated GAPDH was pulled down by Neutravidin resin after lysates were treated with biotin-HDPD in the presence and absence of ascorbate. Eluates andcell lysates were probed for GAPDH. GAPDH was observed in the eluates from ascorbate-treated lysates but not in the absence of ascorbate. Cell lysates showed similarGAPDH content in summer-active and winter-hibernating bears. (B) Densitometric analysis of the Western blots showed not significantly different S-nitrosated GAPDHnormalized to total GAPDH between summer-active and winter-hibernating bears.

Fig. 4. Correlations between selected pairs of parameters in individual winter-hibernating and summer-active free-ranging brown bears. Pairwise Pearson correlations wereselected based on the strength (r 4 0.7 or r o �0.7) and significance (P o 0.05). (A) Plasma bound sulfane sulfur and free sulfide (r¼�0.77); (B) plasma Cys and GSH(r¼0.89); (C) plasma Cys and free sulfide (r¼0.87); (D) RBC Cys and GSH (r¼0.82). The pairs of variables shownwere significantly correlated in hibernating bears (shown bycontinuous lines) but not in summer-active ones. All other pairwise correlations are reported in Supplementary Figs. S1 and S2.

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fraction [14,25,39] that can be recycled back to H2S under reducingconditions [14,25], and enzymes catalyzing the conversion of thiosul-fate to H2S, including a ubiquitous GSH-dependent thiosulfate reduc-tase [25] and mitochondrial rhodanase and 3-mercaptopyruvatesulfur transferase [40], have been identified. A recent study [40]has reported H2S formation from thiosulfate and various reducingagents in tissue homogenates, indicating a biological role forthiosulfate in its reduction to H2S. Although future studies willbe needed to identify the BSS source of H2S in hibernating bears,the regeneration of H2S from one or more of its oxidative productswould be of particular physiological importance for the hibernat-ing bear, as it would help preserve levels of Cys for proteinand GSH synthesis during hibernation. This strategy would alsocontribute to preservation of body nitrogen stores and sustainprotein synthesis despite the absence of dietary intake of aminoacids [9,41].

Fig. 5 shows a plausible model for H2S origin and fate consistentwith our findings. In the blood of summer-active bears, H2S generatedin RBCs from the CSE-catalyzed conversion of Cys freely diffuses outinto plasma and is rapidly metabolized to generate thiosulfate andother oxidized products [14,39]. Because of its propensity to becomeoxidized [42,43], at normal O2 levels most H2S generated would beinactivated before reaching cytochrome c oxidase in the mitochondriaof perfused tissues. During hibernation, part of the plasma BSS pool istransferred to the RBC, where it is converted to H2S, a reaction that isfavored by reduced GSH [25]. The low arterial O2 tension [7] and highhemoglobin O2 affinity [6] in hibernating brown bears indicateconditions of low O2, under which H2S would be able to diffuse intonearby cells and contribute to suppressing mitochondrial respiration.It can be envisaged that similar O2-linked processes could also takeplace in cells and tissues other than blood.

Generation of H2S from Cys

Although the BSS may function as an alternative source of H2S,the positive correlation between plasma free sulfide and Cys(Fig. 4C) indicates that H2S is still produced by erythrocytic CSE,possibly even functioning at higher rates (Fig. 2C), whereas thelower CSE activity in plasma (Fig. 1C) would reflect a release fromhepatocytes and endothelium [44]. Other enzymes, including

cystathionine β-synthase, 3-mercaptopyruvate sulfurtransferase,and cysteine aminotransferase, may also synthesize H2S from Cysin various tissues and cellular compartments [45], and CSE mayeven translocate to mitochondria and improve ATP production invascular muscle cells [46], a process that may well occur in vivo inhibernating animals. Taken together, these results indicate thatplasma may contain circulating available pools of BSS and Cys foruptake into RBCs and tissues where they can be used in H2Ssynthesis and for mitochondrial function during hibernation.

Effects of H2S on mitochondrial respiration

Several studies have consistently reported lower mitochondrialrespiration rates in several hibernating ground squirrel species,especially in the liver [47,48] and in the skeletal muscle in 13-linedground squirrels [49]. In this species, the suppressed O2 consump-tion was not due to phosphorylation of respiratory complexes [50],thus supporting that a soluble factor, such as H2S, could beinvolved. Interestingly, mitochondrial respiration in cardiac mus-cle and brain was not depressed in hibernating ground squirrels[51], suggesting that during winter hibernation energy resourcesare preferentially allocated to these two vital organs.

H2S is a weak reversible inhibitor of the ferrous heme of cyto-chrome c oxidase in mitochondria, with estimated affinity constants inthe physiological micromolar range (0.2–12.5 mM) [11,12]. In bindingto cytochrome c oxidase with low affinity, it is readily displaced bystronger ligands, such as O2. Inhalation of �80 ppm gaseous H2S thatinduced suspended animation in mice [10] corresponds to �1 mMH2S[39], levels that are compatible with those found in this study (1.08and 2.00 mM free sulfide for winter-hibernating and summer-activebears, respectively; Supplementary Table S1). The results of this studyextend the original conclusion that H2S is involved in hibernation [10]by showing that in hibernating brown bears H2S may in part originatefrom plasma BSS.

Effects of H2S on the circulation

In addition to inhibiting mitochondrial respiration, H2S also hasmarked effects on the circulation, by acting as a hypoxic vasoconstric-tor or vasodilator in the pulmonary and systemic circulation,

Cys H2S H2SBSSCSE

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Fig. 5. Proposed role of blood H2S in the control of metabolic rate in summer-active and winter-hibernating brown bears. Cys enters the RBC through a Naþ-dependentmembrane transporter and is converted to GSH and to H2S by the enzyme cystathionine γ-lyase (CSE). In summer-active bears (left), H2S freely diffuses through membranesand is largely inactivated (discontinuous arrow) before reaching cytochrome c oxidase in the inner membrane of perfused tissues mitochondria, whereby O2 consumption isnot inhibited. H2S is also oxidized to bound sulfane sulfur (BSS) and exported to the plasma. In winter-hibernating bears (right), RBC H2S originates in part from the reductionof plasma BSS, a reaction that is favored by reduced GSH, and in part from the CSE-mediated conversion of Cys. Plasma BSS may then function as an available pool of H2Sbioactivity. The low substrate Cys concentration in the RBCs of winter-hibernating bears available for the CSE-catalyzed reaction (discontinuous arrow) suggests that Cys ispreferentially used to generate high levels of GSH. At low tissue O2 levels durSing hibernation, H2S generated (from either BSS or Cys) may inhibit mitochondrial O2

consumption and contribute to metabolic depression during hibernation.

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respectively [42]. Although H2S is a potent vasodilator of isolatedsystemic vessels [52], its effect in living hibernating animals wouldprobably be overwhelmed by a strong adrenergic tone that mayconstrict peripheral systemic blood vessels. In the systemic circulation,an increase in the peripheral resistance would then maintain adequateblood pressure despite the reduced cardiac output and prolong bodyenergy stores by redistributing blood flow to the most demandingorgans, representing a conserved adaptive trait in diving and hibernat-ing mammals [1,2]. Conversely, in the lung circulation, H2S maycontribute to hypoxic vasoconstriction [42] thereby helping to main-tain a high arterial O2 saturation at the low ventilation rates occurringduring hibernation [2,4]. Understanding the physiological processesoccurring in hibernating bears, including the effects of H2S onmetabolism and circulation, will help in improving therapeuticapplications of hypothermia and hypometabolism in human diseasesand preventing organ damage during cardiac arrest [53], immunesuppression [54,55], and major surgery [56].

GSH and hibernation

Another important finding of this study is the large increase inerythrocytic total GSH found in hibernating bears (Fig. 2B). TotalGSH was highly dependent on the availability of Cys in plasma andRBCs (Figs. 4B and D) and, consistent with these results, the rate ofsynthesis of GSH is rate limited by the levels of Cys present inhuman plasma and RBCs, which Cys enters through a Naþ-dependent transporter [57]. Thiol-containing GSH is an essentialcomponent of the defense against oxidative stress in that it reactswith ROS to generate oxidized GSSG, which is then reduced backto GSH via NADPH-dependent GSH reductase or actively exportedfrom the erythrocyte [57]. Although relying on aerobic metabo-lism, hibernating animals undergo periodic oscillations in theirmetabolic rates, with inevitable mismatches between local O2

supply and consumption and resulting generation of potentiallydamaging ROS [4,28]. Although we could not measure oxidized vsreduced GSH because of the time delay in collection and analysisof samples from the free-ranging bears, a large pool of total GSHavailable in RBCs (Fig. 2B) and probably in other tissues during thehibernation period would help limit periodic oxidative damage.We note that reduction of any oxidized GSSG back to GSH predictsthat sufficient NADPH is available as a reducing agent, wherebyglucose reserves (not the primary energy fuel during hibernation)might be diverted away from glycolysis to fuel the NADPH-generating pentose–phosphate pathway. In this process, reversibleinactivation of phosphofructokinase, a key enzyme of glycolysis,mediated by low temperature and pH may well play a role inhibernating bears as found in a small hibernating rodent [58]. Thiswould also explain our earlier finding of a substantial reduction inRBC 2,3-diphosphoglycerate, a side product of glycolysis, duringhibernation in brown bears [6].

H2S and NO: a comparison between two signaling molecules

Perhaps surprisingly, we did not find significant changes in themajor NO metabolite nitrite in plasma or RBCs nor in S-nitrosationlevels or activity of erythrocytic GAPDH, although we cannot ruleout that some of the changes (for example plasma nitrite withP¼0.043; Supplementary Table S2) may become statistically sig-nificant in separate studies with a lower number of parametersinvestigated (where P o 0.05 instead of P o 0.002 is sufficient).Other parameters that were not investigated here may also reveala role for NO in the control of hibernation, such as changes in NOSactivity, nitrate, or targeted S-nitrosation of key proteins orenzymes in blood or other tissues. Previous studies have shownthat H2S and NO share some important characteristics: they bothoriginate enzymatically from amino acids (L-Cys and L-Arg,

respectively) [59,60], can be regenerated from their respectiveoxidative products [40,61], and interact in the control of vasodila-tion [62] and in cytoprotection [63]. However, results from thisand previous investigations suggest that these two signalingmolecules may operate in different physiological and pathologicalcontexts [64]. Under hypoxia, the enzymatic rate of NO synthesisfrom L-Arg decreases (as O2 is a cosubstrate), whereas conversionof nitrite to NO increases, a process also favored by acidicconditions. Such conditions are present during acute exerciseand heart ischemia in mammals or even during prolonged accli-mation to extreme hypoxic and anoxic conditions as achieved bysome fish and turtles [28]. Accordingly, in these hypoxia-tolerantectotherms, levels of plasma nitrite are constitutively higher thanin mammals and hypoxia-intolerant species, and plasma nitrite isshifted to tissues and used for NO synthesis during hypoxia andanoxia [22,36,65]. Conversely, mammalian hibernators use storedfat as the major energy fuel to sustain a hypometabolic state inwhich little O2 is consumed and supplied, without becominghypoxic. As a result, blood pH and lactate remain relatively stableunder hibernation in brown bears [6,7]. Although the role of NO inmammalian hibernation is less clear than that of H2S, we speculatethat H2S-dependent inhibition may prevail in aerobic metabolicsuppression, as it occurs in hibernating bears, whereas NO-dependent inhibition may be dominant in hypoxic or even anoxicmetabolic suppression, as it occurs in a few ectotherm species,such as crucian carp [22] and turtle [23,65], that overwinter in atotal lack of O2. These complementary abilities of H2S and NO toinduce controlled and reversible hypometabolic states and toprotect cells and organs against O2 deprivation would have far-reaching consequences in biology and medicine.

Conclusion

In summary, our study is the first to show that in a hibernatingspecies in its natural environment, hibernation is associated with(1) a significant remodeling of H2S metabolism consistent withgeneration of H2S from both BSS and Cys and (2) a large increase inthe intracellular GSH pool available. Although the role of NO inhibernation remains to be conclusively established, these findingshighlight the emerging importance of sulfane metabolism inmetabolic depression and antioxidant defense and provide a raresnapshot into the physiological processes underlying the fascinat-ing phenomenon of mammalian hibernation.

Acknowledgments

We thank Dr. Rakesh P. Patel for helpful input on researchdesign. We thank Mr. Sven Brunberg and the personnel of theScandinavian Brown Bear Project, for organizing captures of thebears and logistics during the fieldwork, and Mr. John Glawe forsample processing. This study was supported by research grantsfrom the Danish Council for Independent Research, NaturalSciences (10-084565 to A.F.), National Institutes of Health(GM097041 to D.J.S and HL113303 to C.G.K.), Nordforsk (44042to O.F.), and the Lundbeck Foundation (R126-2012-12408 to O.F.).The Scandinavian Brown Bear Research Project is funded by theSwedish Environmental Protection Agency, the Norwegian Envir-onment Agency, the Swedish Association for Hunting and WildlifeManagement, Austrian Science Foundation, and the ResearchCouncil of Norway. This is paper no. 165 from the ScandinavianBrown Bear Research Project.

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Appendix A. Supplementary Information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.025.

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