The Sarcoplasmic Reticulum and SERCA: a Nexus for(i.e. coupling ratio of 2:1). Several SERCA...

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The Sarcoplasmic Reticulum and SERCA: a Nexus for Muscular Adaptive Thermogenesis

Journal: Applied Physiology, Nutrition, and Metabolism

Manuscript ID apnm-2019-0067.R1

Manuscript Type: Review

Date Submitted by the Author: 23-Apr-2019

Complete List of Authors: Gamu, Daniel; University of Waterloo, KinesiologyJuracic, Emma Sara; University of Waterloo, KinesiologyHall, Karlee; University of Waterloo, KinesiologyTupling, A. Russell; University of Waterloo, Kinesiology

Novelty bullets: points that summarize the key findings in

the work:

Keyword:

body mass maintenance, energy balance < energy regulation, skeletal muscle metabolism < metabolism, skeletal muscle function < skeletal muscle function, obesity < obesity, muscle physiology < muscle physiology

Is the invited manuscript for consideration in a Special

Issue? :Not applicable (regular submission)

https://mc06.manuscriptcentral.com/apnm-pubs

Applied Physiology, Nutrition, and Metabolism

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The Sarcoplasmic Reticulum and SERCA: a Nexus for Muscular Adaptive Thermogenesis

Daniel Gamu, Emma Sara Juracic, Karlee J. Hall, and A. Russell Tupling

Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada

Corresponding Author: Dr. A. Russell Tupling

University of Waterloo

200 University Ave. West

Waterloo Ontario, Canada

N2L 3G1

Phone: (519) 888-4567, ext. 33652

Fax: (519) 885-0470

e-mail: [email protected]




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mailto:[email protected]




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Abstract

We are currently facing an 'obesity epidemic' worldwide. Promoting inefficient

metabolism in muscle represents a potential treatment for obesity and its complications.

Sarco(endo)plasmic reticulum (SR) Ca2+-ATPase (SERCA) pumps in muscle are

responsible for maintaining low cytosolic [Ca2+] through the ATP-dependent pumping of

Ca2+ from the cytosol into the SR lumen. SERCA activity has the potential to be a

critical regulator of body mass and adiposity given that it is estimated to contribute

upwards of 20% of daily energy expenditure. More interestingly, this fraction can be

modified physiologically in the face of stressors like ambient temperature and diet,

through its physical interaction with several regulators known to inhibit Ca2+-uptake and

muscle function. In this review, we discuss advances in our understanding of Ca2+-

cycling thermogenesis within skeletal muscle, focusing on SERCA and its protein

regulators, which were thought previously to only modulate muscular contractility.

Novelty

ATP consumption by SERCA pumps comprises a large proportion of resting energy

expenditure in muscle and is dynamically regulated through interactions with small

SERCA regulatory proteins

SERCA efficiency correlates significantly with resting metabolism, such that

individuals with a higher resting metabolic rate (RMR) have less energetically

efficient SERCA Ca2+ pumping in muscle (i.e. lower coupling ratio)

Futile Ca2+ cycling is a versatile heat generating mechanism utilized by both skeletal

muscle and beige fat

Keywords: body mass maintenance; energy balance; skeletal muscle metabolism; skeletal

muscle function; obesity; muscle physiology

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Introduction

Obesity development can be understood from a thermodynamic perspective.

Body mass is maintained when caloric intake is matched by caloric expenditure (i.e.

energy balance). However, when caloric intake exceeds that of expenditure (i.e. positive

energy balance), the resulting surplus of calories are stored as fat in adipose tissue depots,

and when this surplus continues for a prolonged period of time, obesity and associated

metabolic complications can arise. Conversely, body mass can be reduced when caloric

expenditure exceeds that of intake (i.e. negative energy balance). Mathematically, this

model of obesity is simple to understand, however, the physiological determinants of

energy balance are dynamic (i.e. altered by environmental factors), and incompletely

understood.

Exploiting naturally occurring mechanisms that govern metabolic rate to increase

energy expenditure has long been a conceptually palatable idea to reduce obesity and

associated comorbidities. Although modifying dietary and activity patterns are a critical

component in solving the obesity epidemic, long-term weight-loss management can be

difficult for some individuals to achieve through lifestyle modifications, owing to

problems of adherence. Thus, finding ways to increase tissue-specific energy

expenditure, even slightly, may help to reduce adiposity and improve metabolism. In this

regard, numerous studies in rodents have shown that enhancing the thermogenic activity

of brown and beige adipocytes (described below) can mitigate obesity. Similar to

thermogenic adipocytes, mechanisms of energy wasting have now been identified within

skeletal muscle. Specifically, cycling of Ca2+ across the sarco(endo)plasmic reticulum

(SR) has been shown to make a quantitatively important fraction of skeletal muscle’s

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energy consumption. More interestingly, this fraction can be modified physiologically in

the face of stressors like ambient temperature and diet. In this review, we discuss

advances in our understanding of Ca2+-cycling thermogenesis within skeletal muscle,

focusing on the SR Ca2+-ATPase (SERCA) and its protein regulators.

Shades of Thermogenic Fat: Brown and Beige

Adaptive (or facultative) thermogenesis refers to a modifiable and regulated

change in energy expenditure in response to alterations in ambient temperature or food

availability (Cannon and Nedergaard 2004; Lowell and Spiegelman 2000), allowing

maintenance of physiological function during thermal or dietary stress. Classically,

adaptive thermogenesis has been studied in response to cold exposure (Cannon and

Nedergaard 2004). In this context, shivering thermogenesis refers to heat production

originating from continual muscular contraction in order to maintain body temperature

during acute cold exposure. However, contraction cannot continue indefinitely and

shivering eventually subsides, although body temperature continues to be maintained.

Non-shivering thermogenesis refers to the ability to maintain continual heat production in

the absence of shivering; activation mechanisms that replace shivering help prevent

muscle fatigue and preserve muscular function despite continued thermal stress. In

homeothermic endotherms, mitochondrial uncoupling in brown adipose tissue (BAT) is

the best characterized mechanism of adaptive non-shivering thermogenesis. Unlike white

fat, BAT is specialized for heat production, owing to the presence of the inner

mitochondrial membrane protein uncoupling protein (UCP)-1. In response to

thermogenic stress (e.g. cold), UCP-1 acts as a proton channel, dissipating the

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mitochondrial H+ gradient at the expense of ATP production; heat is produced as protons

flow down their electrochemical gradient from the intermembrane space into the

mitochondrial matrix, and from the obligate consumption of energy substrates that is

required to maintain the H+ gradient in the face of continual proton leak.

While historically believed to be present only in human neonates, sensitive in vivo

imaging techniques (Hany et al. 2002; Nedergaard et al. 2007), coupled with histological

evidence from adipose biopsies (Cypess et al. 2009; Saito et al. 2009; van Marken

Lichtenbelt et al. 2009; Zingaretti et al. 2009), has confirmed the presence of BAT in

adult humans. Interestingly, human BAT activity/mass (as measured by tissue

flurodeoxyglucose uptake) is negatively associated with both adiposity and the presence

of type 2 diabetes (Cypess et al. 2009; Ouellet et al. 2011; Saito et al. 2009), suggesting it

has an important metabo-regulatory role. However, reduced BAT activity in obesity/type

2 diabetes may be more representative of BAT insulin resistance rather than impaired

thermogenesis per se (Blondin et al. 2015; Orava et al. 2013).

In addition to the distinct anatomical regions of classical BAT, UCP-1 positive

adipocytes can also be found interspersed within white adipose tissue. While

morphologically similar to brown adipocytes, these inducible thermogenic cells are

derived from a distinct cellular lineage to that of BAT (Harms and Seale 2013), and are

referred to as beige or brite (brown in white) adipocytes.

Although thermogenic adipocytes may be a major regulator of energy balance in

rodents (Enerback et al. 1997; Feldmann et al. 2009), the absolute amount of BAT

possessed by humans and its functional activity in both healthy and diseased states is not

completely resolved (Muzik et al. 2013; Porter et al. 2015; Saito et al. 2009; Warner and

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Mittag 2016; Zingaretti et al. 2009). Furthermore, mice lacking UCP-1 are able to

survive the cold when gradually adapted to a lower ambient temperature (Ukropec et al.

2006), through recruitment of UCP-1-independent mechanisms of non-shivering heat

production (Monemdjou et al. 2000; Rowland et al. 2015). Thus, discovery of other

adaptive thermogenic mechanisms that regulate body mass and systemic metabolism has

been of particular interest to the field. Specifically, mechanisms within skeletal muscle

may play a major role in the development of obesity (Bal et al. 2012; Bombardier et al.

2013a; Bombardier et al. 2013b; Gamu et al. 2015; Gamu et al. 2014; MacPherson et al.

2016).

Major Sites of Skeletal Muscle Energy Consumption During Contraction

Skeletal muscle has the capacity to contribute considerably to daily energy

expenditure because it comprises ~40% of adult body mass (Rolfe and Brown 1997).

Thus, small changes in myocellular energy consumption have the potential to

significantly impact daily energy expenditure. In response to depolarization of the

sarcolemmal and t-tubular membranes, Ca2+ is rapidly released from the SR lumen into

the cytosol (Dulhunty 2006). Upon release, Ca2+ ions bind to the thin filament regulatory

protein troponin C, causing a conformational change in tropomyosin and exposing

myosin binding sites along the actin filament (Dulhunty 2006). Once exposed, myosin

then interacts with actin, forming cross-bridges, and the hydrolysis of ATP by the myosin

ATPase provides the energy required for sarcomere shortening. In order for the muscle

fibre to then relax, the cytosolic Ca2+ must be pumped back into the SR lumen, an ATP-

dependent process mediated by the SERCA pumps (Tupling 2004). Furthermore, the

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electrochemical gradients of Na+ and K+ are returned by the action of the Na+/K+-ATPase

to allow for continual depolarization of the muscle fiber (Clausen 2003). During

isometric contraction, the contributions of the myosin ATPase, SERCA, and Na+/K+-

ATPase are estimated to contribute ~65%, ~30%, and ~5% towards ATP consumption,

respectively (Barclay 2017). While the processes of muscle contraction and resetting of

ion gradients inevitably contribute to shivering thermogenesis, mounting evidence

indicates that the energy consumed by SERCAs is a dynamically regulated process,

controlling core temperature and body mass in response to environmental stress, even in

resting (i.e. non-contracting) skeletal muscle (Gamu et al. 2014).

SERCA Activity: a Major Source of Muscular Energy Consumption

SERCAs are 110 kDa SR integral membrane proteins of the P-type ATPase

family. They contain a large cytoplasmic headpiece with three domains (nucleotide-

binding, phosphorylation and actuator), along with a short stalk region connecting its

headpiece to a transmembrane domain comprised of 10 α-helicies, of which 2 Ca2+

binding sites are situated within helices 4-6 and 8 (Toyoshima 2008). Based on its

structural configuration, SERCAs optimally transport 2 Ca2+ for every ATP hydrolyzed

(i.e. coupling ratio of 2:1).

Several SERCA isoforms exist, arising from developmental and tissue-specific

alternative splicing of the ATP2A1-3 genes (Hovnanian 2007). Traditionally, SERCA1a

is the major isoform co-expressed with myosin heavy chain type II comprising the fast-

twitch muscle phenotype, while SERCA2a is co-expressed with myosin heavy chain type

I comprising the slow-twitch muscle phenotype (Wu and Lytton 1993). However, we

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have shown in human single skeletal muscle fibres that this phenotypic expression pattern

is not as dichotomous as previously believed, with some type I fibres co-expressing

SERCA1a and some type II fibres co-expressing SERCA2a (Fajardo 2013). Despite

subtle distinctions in gene and amino acid sequence (Wuytack et al. 2002), no intrinsic

kinetic differences exist between SERCA2a and SERCA1a (Lytton et al. 1992). Instead,

the higher protein density of SERCA1a within fast-twitch fibres accounts for their faster

rates of Ca2+ uptake and thus, relaxation (Wu and Lytton 1993).

In addition to initiating muscular relaxation, SERCAs are the major protein

responsible for maintaining a low basal cytosolic free Ca2+ concentration ([Ca2+]f ),

despite a Ca2+ gradient >104 across the SR, favoring Ca2+ efflux (MacLennan 1990;

Toyoshima 2008). Ca2+ pumping occurs through a series of complex conformational

changes as SERCAs progress through their catalytic cycle (Figure 1; reactions 1-6).

SERCAs exist in one of two major conformational states, namely E1 or E2. In the E1

state: 1) ATP binds to the nucleotide-binding domain in the presence or absence of Ca2+;

2) SERCAs two Ca2+ binding sites face the cytosol with a high affinity, and when bound

to Ca2+; 3) the nucleotide-binding domain moves towards the phosphorylation domain so

that the γ-phosphate of ATP is transferred to Asp351; and 4) the cytoplasmic gate of the

Ca2+ binding sites closes and the two Ca2+ ions are occluded in the transmembrane

binding sites (Toyoshima 2009) (Figure 1; reactions 1-2). Once phosphorylated,

SERCAs transition to their E2 state, which is associated with: 1) release of ADP; 2) large

conformational changes of the transmembrane helices and destruction of the Ca2+ binding

sites; 3) opening of the luminal gate; and 4) releasing the Ca2+ into the SR lumen

(Toyoshima 2009) (Figure 1; reaction 3). After inorganic phosphate within the

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phosphorylation domain is exchanged for H2O, the luminal gate closes and the pump

transitions back to the E1 state (Toyoshima 2007), completing its catalytic cycle (Figure

1; reactions 4-6). Although the theoretical optimal coupling ratio is 2:1, the presence of

alternate reactions of the catalytic cycle can lower this ratio (described below); that is to

say, Ca2+ pumping can become less energetically efficient.

When luminal [Ca2+]f is high (e.g. during rest or relaxation), luminal Ca2+ may

remain bound to the E2 state and be carried back into the cytosol when SERCAs

transition to their E1 conformation; this loss of luminal Ca2+ occurs without the re-

synthesis of expended ATP, and is called uncoupled Ca2+ efflux or passive leak (Berman

2001; de Meis 2001) (Figure 1; reactions 7-9). Similarly, under high luminal [Ca2+]f , the

transition rate from E1 to E2 may be slowed, increasing the number of SERCA pumps in

the Ca2+-bound E1 state and promoting Pi release without transporting the bound Ca2+

ions, called uncoupled ATPase activity (Berman 2001; de Meis 2001) (Figure 1; reaction

10). Additionally, Ca2+ may fall off SERCAs during the E1 to E2 transition, following

transfer of the γ-phosphate of ATP to Asp351 and prior to closing of the cytoplasmic

gate, called slippage (Berman 2001; Smith et al. 2002) (Figure 1; reaction 11). Slippage

may be a feature of the physical interaction of SERCA with its regulatory protein

sarcolipin (discussed below) (Bal et al. 2012; Bombardier et al. 2013a; Bombardier et al.

2013b; Mall et al. 2006; Smith et al. 2002). These alternate reactions all result in a

continued need to pump cytosolic Ca2+, placing a greater ATP demand on SERCA pumps

to maintain a low cytosolic [Ca2+]f. As a result, SERCA’s theoretical optimal coupling is

lowered (i.e. coupling ratio < 2:1).

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Given the large SR gradient favoring Ca2+ efflux and that alternate reactions exist

that reduce SERCA pumping efficiency, it is not surprising then that ATP consumption

by SERCAs comprises a large proportion of resting (i.e. non-contracting) energy

expenditure by muscle (Chinet et al. 1992; Decrouy et al. 1993; Dulloo et al. 1994; Smith

et al. 2013). Our group has used intact mouse skeletal muscles, along with indirect

calorimetry, to determine the contribution of SERCA-mediated Ca2+ pumping to resting

energy expenditure of isolated muscle (Smith et al. 2013). By indirectly inhibiting

SERCA activity by blocking Ca2+ release from the ryanodine receptor (RyR) with high

Mg2+, we found that SERCA function accounts for ~42-48% of the basal metabolic rate

of both slow- and fast-twitch skeletal muscles (Smith et al. 2013). Because we were

unable to use direct inhibitors of the SERCA pump (Smith et al. 2013), RyR blockade

likely underestimates this percentage contribution because SERCAs themselves are a site

of SR Ca2+ leakage (Murphy et al. 2009). Our findings suggest there is a constant

requirement to pump Ca2+ across the SR, even when muscles are idle. When extrapolated

to the whole body level, SERCA activity may account for ~7-10% of whole-body basal

energy expenditure and ~15-20% of total daily energy expenditure in mammals (Smith et

al. 2013). What’s more, mounting evidence shows that SERCA’s contribution to muscle

and whole-body energy expenditure is dynamically regulated through its interaction with

small SR regulatory proteins.

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The Regulatory Protein Sarcolipin Promotes Thermogenesis by Uncoupling

SERCAs

SERCA activity is regulated through its physical interactions with a growing list

of small proteins, the best characterized of which are sarcolipin (SLN) and

phospholamban (PLN). Historically, SLN and PLN are believed to be functional

homologues (MacLennan et al. 2003); both proteins are found endogenously within

oxidative skeletal muscle of mice, and when bound to SERCAs, slow the rate of muscular

relaxation by inhibiting Ca2+ uptake (Bombardier et al. 2013a; Fajardo et al. 2015; Gamu

et al. 2019; Slack et al. 1997; Tupling et al. 2011). Their inhibition of SERCA is reduced

when [Ca2+]f increases, likely dissociating them from the pump. However, cross-linking

studies show SLN to be more resistant to the dissociative effects of [Ca2+], as it can still

bind to SERCA at high [Ca2+] whereas PLN cannot (Bal et al. 2012; Sahoo et al. 2013;

Sahoo et al. 2015). While either protein may function to fine-tune SERCA activity and

SR Ca2+ load, the question of why such inhibitors of muscular function have evolved

remain incompletely understood. Mounting evidence suggests that, at least for SLN, there

is a role in muscular thermogenesis.

Using reconstituted lipid vesicles, increasing the molar ratio of SLN to SERCA

was found to decrease luminal Ca2+ accumulation without an affect on SERCA ATP

hydrolysis rate (Smith et al. 2002), in addition to increasing the amount of heat released

per mol of ATP consumed by SERCA during Ca2+ pumping (Mall et al. 2006). This

uncoupling of SERCA Ca2+ transport from ATP hydrolysis was attributed to SLN’s

ability to promote “slippage” of Ca2+ from the SERCA pump (Smith et al. 2002).

Together, these two in vitro studies were the first to demonstrate that the SERCA/SLN

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relationship is thermogenic in nature. SLN/SERCA1 co-transfection studies using HEK-

293 cells have also yielded similar results (Sahoo et al. 2013).

In agreement with these in vitro studies, our group has used SLN knockout mice

(Sln-/-) to show that endogenous levels of skeletal muscle SLN does in fact uncouple

SERCA function in vivo (Bombardier et al. 2013a). In the presence of a vesicular Ca2+

gradient (i.e. when luminal Ca2+ is able to accumulate within SR vesicles derived from

limb muscles, similar to intact muscle), SERCA’s apparent coupling ratio within

oxidative muscle was higher (i.e. more efficient) in Sln-/- mice relative to wild-type (WT)

controls due their lower rates of ATP usage (Bombardier et al. 2013a). Consequently, the

contribution of SERCA activity to resting energy expenditure of isolated muscle was

~13% lower in Sln-/- mice (Bombardier et al. 2013a). Furthermore, Sln-/- mice expend less

energy during sub-maximal treadmill exercise and are unable to maintain body

temperature following acute cold-exposure (Bal et al. 2012; Bombardier et al. 2013b;

Sahoo et al. 2013), indicating that SLN uncouples SERCA function even during exercise

and shivering thermogenesis. Physiologically, this means that more ATP is required to

pump Ca2+ across the SR when SERCA is bound to SLN. Thus, energy is “wasted” in

the presence of SLN as it is diverted away from that needed to translocate Ca2+ into the

SR lumen, regardless if muscle is active or idle. Not surprisingly, our group (Bombardier

et al. 2013b; Gamu et al. 2015; MacPherson et al. 2016) and others (Bal et al. 2012;

Rowland et al. 2016) have shown that Sln-/- mice develop diet-induced obesity resulting

from efficient SERCA metabolism, whereas mice over-expressing SLN are resistant to

diet-induced obesity (Maurya et al. 2015).

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It has become increasingly more appreciated that skeletal muscle and brown fat

are capable of communicating with various tissues, including each other (Bostrom et al.

2012; Rao et al. 2014; Stanford et al. 2018), through the release of myokines and

adipokines. In line with their established roles in thermoregulation, muscle-based

thermogenesis appears reciprocally regulated to that of brown fat in rodents. When

interscapular BAT is surgically removed in mice, skeletal muscle SLN protein content

markedly increases with prolonged cold-exposure, presumably to allow for the

maintenance of core body temperature (Bal et al. 2016). One important observation of

UCP-1 deficient mice is that, despite becoming hypothermic when acutely cold-exposed

(Enerback et al. 1997), they are capable of tolerating a gradual reduction in ambient

temperature (Ukropec et al. 2006), suggesting that alternate thermogenic mechanism(s)

are activated when BAT becomes defective. Indeed, skeletal muscle SLN protein content

is higher in cold-adapted UCP-1 knockout mice (Rowland et al. 2015). This raises the

possibility that organisms with relatively small amounts of brown fat may rely more on

SLN/SERCA for thermogenesis, a scenario that may be relevant to human adults given

that BAT mass, activity and recruitment can vary (Yoneshiro et al. 2013).

Skeletal Muscle SERCA Efficiency and SLN: Relevance to Human Metabolism

Skeletal muscle energy expenditure can explain, in part, individual variation in

human basal metabolic rate (Zurlo et al. 1990). However, it is not clear what components

of muscle-specific energy consumption may account for this. Skeletal muscle

mitochondrial uncoupling contributes significantly towards energy expenditure (Rolfe

and Brown 1997), and enhanced mitochondrial uncoupling is a feature of successful

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weight loss (Harper et al. 2002). Based on our findings that SERCA activity comprises a

significant proportion of resting energy expenditure in muscle (Smith et al. 2013), we

have hypothesized that Ca2+ pumping efficiency may help explain variation in energy

expenditure and susceptibility to weight gain. By measuring SERCA coupling ratio in

homogenates from biopsied human vastus lateralis, we have found that SERCA

efficiency correlates significantly with resting metabolism, such that individuals with a

higher resting metabolic rate (RMR) have less energetically efficient SERCA Ca2+

pumping in muscle (i.e. lower coupling ratio) (Figure 2a). Interestingly, those with a

higher RMR also have a higher ratio of SERCA1a to SERCA2a (Figure 2b), which is

significant because at the single muscle fibre level, SERCA1a is highly co-expressed

with SLN in humans (Fajardo 2013). Mechanistically, it is possible that SLN-mediated

uncoupling of SERCA1a may be responsible for lowering Ca2+ pumping efficiency and

subsequently increasing energy expenditure.

To date, few studies have examined SLN-mediated thermogenesis in humans. In

overweight males with type 2 diabetes, vastus lateralis SLN protein expression has been

reported to correlate significantly with nonshivering thermogenesis following repeated

cold acclimation (Hanssen et al. 2015), although SLN expression remained unchanged by

repeated cold exposure. The only study to directly assess SLN-mediated thermogenesis in

human muscle utilized cultured myocytes harvested from vastus lateralis biopsies of

severely obese individuals (BMI > 40 kg/m2) (Paran et al. 2015). Although SLN

expression was elevated in primary muscle cells from obese compared to lean controls

(BMI < 25 kg/m2), SLN’s specific contribution to myocyte energy expenditure was

paradoxically impaired with obesity (Paran et al. 2015). This suggests that a failure of

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SLN-mediated thermogenesis may be a causal factor leading to weight gain in humans.

Furthermore, it appears that the amount of SLN protein is not the sole determinant of

SERCA uncoupling. It should be noted that the SR lipid profile was altered in myocytes

from obese individuals (Paran et al. 2015), which may impede SLN’s physical interaction

with SERCA, thus, preventing uncoupling of the pump.

Classical and Emerging SERCA Regulatory Proteins

PLN is perhaps the most well characterized regulatory protein of SERCAs.

Historically, PLN and SLN have been considered homologous regulators of the

Ca2+ATPase because they are structurally similar, which allows each to bind within the

same SERCA transmembrane region to reduce SERCAs Ca2+-affinity and inhibit

muscular contractility (Periasamy et al. 2008). However, it is becoming increasingly

appreciated that PLN and SLN may each serve unique physiological roles within skeletal

muscle. Unlike SLN, PLN has a larger cytosolic N-terminus containing 2 residues

(serine-16 and threonine-17) that are phosphorylated by protein kinase A and

Ca2+/calmodulin-dependent kinase II, respectively; PLN phosphorylation results in its

physical dissociation from the pump to enhance Ca2+ uptake and muscular relaxation

(Bhupathy et al. 2007; Periasamy et al. 2008). One critical feature of SLN allowing it to

regulate thermogenesis is its ability to bind to various kinetic states of SERCA’s reaction

cycle, even when the pump is occupied by Ca2+ ions, whereas binding of PLN to the

pump is mutually exclusive with Ca2+ (Sahoo et al. 2013). This feature of PLN, in

addition to it being reversibly regulated by sympathetic activation, likely makes it

incompatible as a “slippage”-induced uncoupler of SERCA like that of SLN (Figure 1).

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We have recently taken a physiological approach to test whether PLN is involved

in adaptive diet-induced thermogenesis using PLN-deficient mice (Pln-/-). Unlike our

previous work with Sln-/- mice (Bombardier et al. 2013a; Bombardier et al. 2013b), PLN

had no effect on skeletal muscle SERCA pumping efficiency, nor were Pln-/- mice

susceptible to an excessive diet-induced obesity phenotype (Gamu et al. 2019). These

findings are consistent with a previous report showing that only Sln-/- mice are susceptible

to hypothermia when acutely cold-exposed (Sahoo et al. 2013). Thus, only the physical

interaction of SLN with SERCA is thermogenic in nature.

The past several years have seen the discovery of a handful of new SERCA

regulatory proteins, some bearing structural resemblance to SLN. In particular,

myoregulin (MLN) has recently been described as a SERCA inhibitor found within

mammalian skeletal muscle that regulates Ca2+ homeostasis and contractility (Anderson

et al. 2015). Consistent with its ability to reduce SERCA Ca2+-affinity, MLN knockout

mice display enhanced running performance (Anderson et al. 2015). Although an

improved ability for Ca2+ uptake can explain the exercise performance phenotype of

MLN knockout mice, so too could a reduction in the energy demand for Ca2+ cycling.

However, it is currently unknown whether MLN is capable of uncoupling SERCA

function to promote thermogenesis like that of SLN. Recently, thyroid adenoma

associated (THADA) has been shown to uncouple SERCA in skeletal muscle, with

THADA knockout flies being obese, hyperphagic, and intolerant to cold (Moraru et al.

2017); although promising, THADA’s role in mammalian energy balance and skeletal

muscle adaptive thermogenesis is unclear. Lastly, small ankryin 1 (sAnk1) is another

newly described inhibitor of SERCA that also binds to SLN (Desmond et al. 2015;

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Desmond et al. 2017). Interestingly, sAnk1 impairs the inhibitory action of SLN on

SERCA when all three proteins are co-expressed together in a heterologous cell system

(Desmond et al. 2017). Currently, it is unclear whether sAnk1 blocks SLN’s ability to

uncouple SERCA in skeletal muscle.

Unlike the SERCA regulators described above, dwarf open reading frame

(DWORF) is the first known SERCA activating protein, which enhances Ca2+-uptake by

physically displacing SLN, PLN, and MLN (Nelson et al. 2016). DWORF protein is

expressed within oxidative skeletal muscle (Nelson et al. 2016), although it is not known

what the co-expressed pattern is with other SERCA regulators at the muscle fiber level in

vivo. While enhancing Ca2+ uptake will benefit pathological states of contractile

dysfunction, the physical displacement of SLN from SERCA by DWORF may impair

muscular adaptive thermogenesis. In fact, enhanced DWORF binding to SERCA could

explain the previously observed impairment of SLN-mediated uncoupling in obese

human muscle, despite their higher SLN protein expression compared with lean controls

(Paran et al. 2015). Future studies should be directed at teasing apart the complex

interplay of these SERCA regulators under states requiring the activation of muscular

adaptive thermogenesis. Figure 3 provides a summary of the physical and functional

interaction between SERCA and its classical and emerging regulatory proteins.

SR Ca2+ Release and Reuptake: a Versatile Heat Generating Mechanism

Muscular contraction and relaxation generates heat, which is important for

shivering during cold exposure. The contribution of the SR itself to thermogenesis is

evident from the inherited disorder malignant hyperthermia, which can result from the

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mutation of several Ca2+-handling genes, causing excessive SR Ca2+ leak while

individuals are under anesthesia (Maclennan and Zvaritch 2011). Severe muscular

contracture results in life-threatening hyperpyrexia, requiring treatment with the RyR

antagonist dantrolene to prevent Ca2+ release and indirectly stop ATP usage by the

myosin and Ca2+-ATPases. While this is a pathological state, several similar examples of

futile Ca2+-cycling exist in nature, with the purpose of regulating tissue temperature.

Some deep-sea diving fishes possess an ocular “heater organ”, consisting of dense SR

and t-tubular networks devoid of myofiliments that continually release and re-uptake

Ca2+ ions to regulate brain and eye temperature (Morrissette et al. 2003). A similar

mechanism may also exist within the flexor digitorum brevis (FDB) muscle of mice,

which does not participate in shivering thermogenesis (Aydin et al. 2008; Bruton et al.

2010). Prolonged cold exposure (4°C for 4-5 weeks) causes a dissociation of the channel

stabilizing subunit calstabin-1 (also known as FKBP12) from RyR1 (Aydin et al. 2008),

resulting in Ca2+ leak that would subsequently need to be sequestered by SERCA. Cold-

adapted FDB muscles show increases in markers of mitochondrial biogenesis (Bruton et

al. 2010), possibly resulting from activation of Ca2+-dependent signaling pathways in

response to RyR destabilization. Recently, mild SR Ca2+ leak resulting in mitochondrial

biogenesis has been shown to occur with voluntary exercise in mice, resulting from such

RyR destabilization (Ivarsson et al. 2019).

It is becoming increasingly apparent that thermogenic adipose tissues possess

UCP1-independent mechanisms of heat generation (Bertholet et al. 2017), and it appears

that the endoplasmic reticulum (ER) also has a role in non-muscular tissues. Recently,

such a futile Ca2+-cycling mechanism has been reported within beige adipose tissue

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(Ikeda et al. 2017). Expanding the beige adipocyte pool in the absence of UCP-1

enhances the propensity for Ca2+ cycling by increasing the expression of SERCA2b, and

thus the thermogenic response to norepinephrine (Ikeda et al. 2017). Additionally, in

pigs, which lack functional UCP-1, norepinephrine-induced thermogenesis of beige fat is

dependent on SERCA2b-mediated Ca2+-cycling (Ikeda et al. 2017). Thus, UCP-1 is not

the sole heat generating mechanism in beige/brown fat, which raises the possibility that

some individuals may rely on SR/ER-mediated thermogenesis to control energy balance.

These studies beg the question of whether muscle fibers that do not express SLN possess

alternate modes of SR-mediated thermogenesis.

Regulation of Muscular Ca2+-Cycling by Thyroid Hormones

Thyroid hormones are known to regulate both skeletal muscle and cardiac Ca2+-

handling by controlling the expression of various SR proteins, including SERCA

isoforms and PLN (Carr and Kranias 2002). Because states of thyroid dysfunction alter

whole-body energy expenditure (Freake and Oppenheimer 1995), changes in Ca2+-

cycling may be a contributing factor. Experimental induction of hyperthyroidism (a

hypermetabolic state) by the administration of L-thyroxine (T4) or triiodothyronine (T3)

decreases the expression of PLN within both cardiac and skeletal muscle of experimental

animals (Arai et al. 1991; Holt et al. 1999; Jiang et al. 2004; Ketzer et al. 2009;

Minamisawa et al. 2006; Ojamaa et al. 2000; Trivieri et al. 2006). Furthermore, T3/T4

injection decreases SLN mRNA in both the atria and SOL of mice (Minamisawa et al.

2006; Trivieri et al. 2006). Together, these studies suggest that the physical interaction of

both SLN and PLN is diminished by T3/T4 signaling, and the resulting increase in

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metabolism with hyperthyroidism is likely not caused by uncoupling of SERCA function

through SLN’s physical interaction with the pump. Although the influence of thyroid

hormones on metabolism of peripheral tissues is extensive, they do directly impact

SERCA thermogenesis. Surprisingly, T4 administration increases the amount of heat

released from SR vesicles during Ca2+-pumping (Arruda et al. 2003; Ketzer et al. 2009),

this despite a presumed reduction in the interaction of the pump with its inhibitors.

Conversely, propylthiouracil-induced hypothryroidism (a hypometabolic state) increases

the expression of both PLN and SLN within the heart (Ketzer et al. 2009; Trivieri et al.

2006), while reducing the amount of heat released from the SR during Ca2+ pumping

(Ketzer et al. 2009). Again, the blunting of SERCA thermogenesis while simultaneously

increasing the expression of SLN/PLN with hypothyroidism is surprising in light of

SLN’s ability to uncouple Ca2+ transport from ATP hydrolysis (Bombardier et al. 2013a;

Frank et al. 2000; Mall et al. 2006; Sahoo et al. 2015; Smith et al. 2002).

As discussed above, several alternate reactions of SERCA’s catalytic cycle could

explain an increase in the heat released by the SR while pumping Ca2+ if SERCAs

physical interaction with its regulators is diminished, specifically uncoupled ATPase

activity and/or passive Ca2+ leak (Figure 1), both of which require luminal [Ca2+]f to be

high. Given that hyperthyroidism improves Ca2+-handling in part by reducing SERCA’s

inhibition by SLN or PLN, this may consequently increase SR Ca2+ load and

subsequently drive SERCA’s alternate reactions, particularly passive Ca2+ leak. Both

SLN (Asahi et al. 2004; Babu et al. 2007; Babu et al. 2006; Xie et al. 2012) and PLN

(Davia et al. 1999; Kadambi et al. 1996; Wolska et al. 1996) reduce the peak amount of

Ca2+ released by the SR during cardiac muscle activation, resulting from a lower SR Ca2+

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load due to inhibition of SERCA pumping by either protein (Davia et al. 1999; Huser et

al. 1998; Lukyanenko et al. 2001; Santana et al. 1997; Xie et al. 2012). Not surprisingly

then, SLN/PLN double knock-out (DKO) mice (i.e. Sln-/-/Pln-/-) have higher atrial and

ventricular SR Ca2+ content compared to WT control animals (Shanmugam et al. 2011).

While a greater SR Ca2+ load may benefit contractile force production, a functional

consequence in vivo may be an increased Ca2+ gradient across the SR favoring its loss.

Indeed, Ca2+ release events in the form of spontaneous Ca2+ waves, Ca2+-sparks, or SR

Ca2+ leak are increased in frequency and or/amplitude in the absence of SERCA

inhibition by either PLN (Aschar-Sobbi et al. 2012; Chan et al. 2015; Huser et al. 1998;

Lukyanenko et al. 2001; Santana et al. 1997; Sirenko et al. 2014; Wellman et al. 2001;

Zhang et al. 2010) or SLN (Xie et al. 2012). Consequently, ATP consumption by

SERCAs must increase to continually sequester Ca2+ ions lost from the SR to prevent its

cytotoxic accumulation, a mechanism of futile Ca2+ cycling akin to that of ocular heater

organs of fish or FDB fibres of mice described above. Thus, it is possible that either SLN

or PLN could indirectly contribute to energy metabolism in a distinct manner other than

slippage-induced uncoupling of SERCA. Future studies are needed to assess if such

mechanisms are active within muscle fibers that do not express SLN or other yet to be

identified SERCA uncouplers.

Conclusions

Although adaptive non-shivering thermogenesis is the major role of brown and

beige adipose tissue, skeletal muscle is now recognized to possess such function through

the action of SLN. By uncoupling Ca2+-transport from ATP hydrolysis within oxidative

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skeletal muscles, SLN promotes a futile Ca2+ cycle for the purpose of heat generation,

which helps regulate body temperature during cold exposure and protects against an

excessive diet-induced obesity phenotype. Given that brown fat mass/activity is variable

in human adults (Cypess et al. 2009; Vijgen et al. 2012), activating Ca2+-cycling

thermogenesis within skeletal muscle through SLN or other SERCA-based routes may be

a viable option to reduce adiposity and improve systemic metabolism.

Funding

This work was supported by research grants from the Canadian Institutes of Health

Research (CIHR; MOP 86618 and MOP 47296 to A.R.T.).

Conflict of Interest

The authors have no conflicts of interest to report.

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Figure Legends

Figure 1. Schematic representation of the catalytic cycle of SERCA illustrating both coupled and uncoupled reactions. Briefly, transport begins with the SERCA pump in its E1 conformational state with the sequential binding of Ca2+ to its two high-affinity sites on the cytoplasmic face of the transmembrane domain and the binding of ATP to the ATP binding site in the nucleotide binding domain (reaction 1). ATP hydrolysis and phosphorylation of SERCA on Asp351 (reaction 2) triggers a change in conformation to the E2 state (reaction 3). This state exhibits reduced Ca2+ affinity and thus Ca2+ dissociates from the pump and enters the SR lumen (reaction 4). After inorganic phosphate within the phosphorylation domain is exchanged for H2O, the luminal gate closes and the pump transitions back to the E1 state (reactions 5-6), completing its catalytic cycle. Under optimal conditions, the pump can transport two Ca2+ at the expense of one ATP molecule. In conditions of high luminal [Ca2+], a process known as passive leak may occur (reactions 7-9), in which one or two luminal Ca2+ remain bound to the pump in the E2 state and are transported out to the cytosol during SERCA transition to the E1 conformation. Moreover, high luminal [Ca2+] can also induce uncoupled ATPase activity (reaction 10), as it promotes the release of Pi without the transport of Ca2+. Slippage, as represented by reaction 11, involves the dissociation of one or two Ca2+ from SERCA during the E1 to E2 transition. Reactions 7-9, 10 and 11 are examples of alternate reactions which can act to reduce SERCA’s theoretical optimal coupling ratio as they induce a greater demand for SERCA ATP consumption in order to maintain a basal cytosolic [Ca2+].

Figure 2. Relationship between resting metabolic rate (RMR) and SERCA properties in human vastus lateralis. A total of 25 young adults, 6 females and 19 males, age 22.2 ± 3.6 yrs, BMI 23.9 ± 6.2 (range 17.1 – 38.5) were recruited for a study to examine the relationship between RMR and the efficiency of SERCA pumps in human skeletal muscle. Briefly, indirect calorimetry (Vmax, Vyaire Medical) was used to determine RMR. Participants came into the lab fasted between 6:30 am -8:30 am and were told to refrain from strenuous physical activity. Breath-by-breath O2 consumption and CO2 production rates were measured while participants were awake in a prone position and the most stable 10 minutes of collection were used to calculate RMR according to the Weir equation (kcal/min = [(3.941 x VO2 x 1000) + (1.106 x VCO2 x 1000)]). RMR was normalization to lean mass as measured by dual X-ray absorptiometry. SERCA coupling ratios were measured in vitro from homogenized samples of biopsied vastus lateralis, and are defined as the rate of Ca2+-dependent Ca2+-uptake divided by the rate of Ca2+-dependent ATP hydrolysis (Bombardier et al. 2013a). SERCA isoform expression from biopsied vastus lateralis homogenates was measured by standard Western blotting techniques and densitometry values were normalized to the loading control α-actin as we have previously done (Tupling et al. 2011). A) Correlation of RMR and SERCA coupling ratio: the correlation coefficient is -0.21 (p=0.024). Individuals with a higher RMR have a lower SERCA coupling ratio (i.e. lower SERCA efficiency). B) Correlation of RMR and SERCA1a/SERCA2a ratio: the correlation coefficient is 0.18 (p=0.04). Individuals with a higher RMR have a higher ratio of SERCA1a to SERCA2a.

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Figure 3: A graphical summary of the physical and functional interaction between SERCA and its known protein regulators. Sarcolipin (SLN), phosholamban (PLN) and myoregulin (MLN) inhibit SERCAs by lowering their apparent Ca2+ affinity, as indicated by an increase in KCa, whereas endogenous levels of these regulatory proteins do not alter SERCA Vmax. *Dwarf open reading frame (DWORF) counteracts the inhibitory effects of SLN, PLN and MLN by displacing these peptides from SERCA in a dose dependent manner; however, expression of DWORF alone does not directly stimulate SERCA activity. ☨Similarly, small ankryin 1 (sAnk1) alone does not alter SERCA activity; however, the interaction between sAnk1 and SLN suppresses the inhibitory effect of SLN on SERCA. The SERCA coupling ratio is reduced by SLN, which is a mechanism for muscle thermogenesis, but not PLN. The effects of MLN, DWORF and sAnk1 on SERCA coupling ratio and muscle thermogenesis have not been investigated. These inhibitory peptides can physiologically interact with either SERCA isoform; however, the preferential isoform for each respective peptide is highlighted in bold. Please refer to the text for more detailed information and citations.

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CYTOSOLH2OPi Uncoupled

ATPase Activity

(3)(6)(8)

2 Ca2+

ATP

E1 E1Ca2+

ADP

E1Ca2+ ~ P

Slippage

E1Ca2+

ADPATP

(1) (2)(9)

E2Ca2+ ~ PE2 ~ PE2 LUMENE2Ca2+

(4)(5)(7)

Passive Leak

(10)

(11)

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A B

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DraftSERCA Vmax No Change No Change No Change No Change No Change

SERCA KCa Increased Increased Increased Recovered* Recovered☨

Coupling Ratio Decreased No Change Unknown Unknown Unknown

Tissue Distribution

Cardiac (Atria) and Oxidative (mouse) or Glycolytic (Human)

Skeletal Muscle

Cardiac (Left Ventricle)and Oxidative Skeletal

Muscle

All Skeletal Muscle (Predominantly Glycolytic

Skeletal Muscle)

Cardiac and Oxidative Skeletal Muscle Oxidative Skeletal Muscle

SERCA IsoformAssociation SERCA1a/SERCA2a SERCA1a/SERCA2a SERCA1a/SERCA2a SERCA1a/SERCA2a SERCA1a

SLN PLN MLN DWORF sAnk1

Heat

ATP ADP + Pi

Ca2+

Ca2+

ME

RS T

QE

NVTL

LFVVM

FT

I

LLW

I

LL

V R SYQY

ATP ADP + Pi

Ca2+

Ca2+

ME

K VQYLT RSAI RRA STI

E M PQ

QARQ N l Q

NN

LFI FC LILI CLL LICIV

M L L

ATP ADP + Pi

Ca2+

Ca2+

ATP ADP + Pi

Ca2+

Ca2+

ATP ADP + Pi

Ca2+

Ca2+

EN

VT

L

LF

VV

M

FT

I

LL

W

I

L

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