THE AGEING HEARTTHE AGEING HEART

CLINICAL THERAPEUTIC STRATEGIES FOR CARDIAC STRESS CLINICAL THERAPEUTIC STRATEGIES FOR CARDIAC STRESS

AND AND

QUEST FOR MITOCHONDRIAL MARKERS QUEST FOR MITOCHONDRIAL MARKERS

OF IMPAIRED FUNCTIONAL RECOVERY AFTER STRESSOF IMPAIRED FUNCTIONAL RECOVERY AFTER STRESS

THE AGEING HEARTTHE AGEING HEART

CLINICAL THERAPEUTIC STRATEGIES FOR CARDIAC STRESS CLINICAL THERAPEUTIC STRATEGIES FOR CARDIAC STRESS

AND AND

QUEST FOR MITOCHONDRIAL MARKERS QUEST FOR MITOCHONDRIAL MARKERS

OF IMPAIRED FUNCTIONAL RECOVERY AFTER STRESSOF IMPAIRED FUNCTIONAL RECOVERY AFTER STRESS

Franklin Rosenfeldt, Salvatore Pepe, Francis Miller,Franklin Rosenfeldt, Salvatore Pepe, Francis Miller,

Phillip Nagley, Anthony Linnane, Anthony HadjPhillip Nagley, Anthony Linnane, Anthony Hadj

Cardiac Surgical Research Unit, Cardiac Surgical Research Unit,

Alfred Hospital and Baker Medical Research Institute and Alfred Hospital and Baker Medical Research Institute and

Department of Biochemistry and Molecular Biology, Monash Department of Biochemistry and Molecular Biology, Monash

Melbourne AustraliaMelbourne Australia

IN THE AGEING HEARTIN THE AGEING HEART THE THE RESPONSE RESPONSE TO MAJOR STRESS IS IMPAIREDTO MAJOR STRESS IS IMPAIRED

IN THE AGEING HEARTIN THE AGEING HEART THE THE RESPONSE RESPONSE TO MAJOR STRESS IS IMPAIREDTO MAJOR STRESS IS IMPAIRED

Why?Why?

•myocardial infarctionmyocardial infarction•coronary angioplasty coronary angioplasty •coronary bypass surgery coronary bypass surgery

that in the < 70 year old age groupthat in the < 70 year old age group

In >70 year old patients theIn >70 year old patients the mortality aftermortality after

is is three timesthree times

IN THE AGEING HEARTIN THE AGEING HEART THE THE RESPONSE TO RESPONSE TO MAJOR STRESS IS IMPAIREDMAJOR STRESS IS IMPAIRED

Why?Why?

Is there a Is there a ““fatal flaw”fatal flaw” in the ageing heart which reduces its in the ageing heart which reduces its ability to respond to stress?ability to respond to stress?

•myocardial infarctionmyocardial infarction•coronary angioplasty coronary angioplasty •coronary bypass surgery coronary bypass surgery

that in the < 70 year old age groupthat in the < 70 year old age group

In >70 year old patients theIn >70 year old patients the mortality aftermortality after

is is three timesthree times

RESPONSE OF THE YOUNG HEART TO CARDIAC SURGERYRESPONSE OF THE YOUNG HEART TO CARDIAC SURGERY Cardiac SurgeryCardiac Surgery

Young HeartYoung Heart Low mortalityLow mortalityFew complications Few complications

RESPONSE OF THE AGEING HEART TO CARDIAC SURGERYRESPONSE OF THE AGEING HEART TO CARDIAC SURGERY

Cardiac SurgeryCardiac Surgery

Young HeartYoung Heart Low mortalityLow mortalityFew complications Few complications

BiologicallyBiologically Ageing Heart Ageing Heart High mortalityHigh mortalityMany complications Many complications

Cardiac SurgeryCardiac Surgery

RESPONSE OF THE AGEING HEART TO CARDIAC SURGERYRESPONSE OF THE AGEING HEART TO CARDIAC SURGERY

Cardiac SurgeryCardiac Surgery

Young HeartYoung Heart Low mortalityLow mortalityFew complications Few complications

BiologicallyBiologically Ageing Heart Ageing Heart Higher mortalityHigher mortalityMore complications More complications

Cardiac SurgeryCardiac Surgery

Senesence-specific therapies ?Senesence-specific therapies ?

RESPONSE OF THE AGEING HEART TO CARDIAC SURGERYRESPONSE OF THE AGEING HEART TO CARDIAC SURGERY

Cardiac SurgeryCardiac Surgery

Young HeartYoung Heart Low mortalityLow mortalityFew complications Few complications

BiologicallyBiologically Ageing Heart Ageing Heart Higher mortalityHigher mortalityMore complications More complications

Cardiac SurgeryCardiac Surgery

Markers of biological age and predictors impaired responsiveness ?Markers of biological age and predictors impaired responsiveness ?

TARGETED METABOLIC CARDIOPROTECTION FOR STRESSED

AGEING HEART

Normal HeartNormal HeartNormalNormal MetabolismMetabolism

No ChangeNo Change

Coenzyme Q10

TARGETED METABOLIC CARDIOPROTECTION FOR STRESSED

AGEING HEART

Normal HeartNormal HeartNormalNormal MetabolismMetabolism

No ChangeNo Change

Stressed Heart with Stressed Heart with Disturbed MetabolismDisturbed Metabolism

Stresse.g. Cardiac Surgery

TARGETED METABOLIC CARDIOPROTECTION

Normal HeartNormal Heart No ChangeNo Change

Stressed Heart with Stressed Heart with Disturbed Energy Metabolism Disturbed Energy Metabolism

Normalised MetabolismNormalised MetabolismImproved FunctionImproved Function

Cardiac surgery = aerobic and ischaemic stresses

Coenzyme Q10

HYPOTHESESHYPOTHESES

1.1. In the ageing human myocardium In the ageing human myocardium there is an impaired myocardial response there is an impaired myocardial response to aerobic and ischaemic stress which to aerobic and ischaemic stress which impairs the response to major surgery impairs the response to major surgery

2. CoQ2. CoQ10 10 has an specific protective has an specific protective effect in the ageing hearteffect in the ageing heart

RAT STUDY OF SENESCENT HEARTRAT STUDY OF SENESCENT HEART AEROBIC STRESS AND Co QAEROBIC STRESS AND Co Q1010

•Young (5 months) and senescent rats (3 years)Young (5 months) and senescent rats (3 years)

•Treated for 6 weeks with intraperitoneal injections of Treated for 6 weeks with intraperitoneal injections of CoQCoQ10 10 (4 mg/kg) or placebo vehicle(4 mg/kg) or placebo vehicle

•Hearts removed and placed on Isolated working heartHearts removed and placed on Isolated working heart apparatusapparatus

•Paced at 510 beats/min for 2 hours = maximum aerobic Paced at 510 beats/min for 2 hours = maximum aerobic (oxygen demanding) stress(oxygen demanding) stress

•Recovery of pump function and efficiency expressed Recovery of pump function and efficiency expressed as percent of pre-pacing levelsas percent of pre-pacing levels

AEROBIC PACING STRESS IN WORKING RAT HEARTAEROBIC PACING STRESS IN WORKING RAT HEART

**

0

20

40

60

80

100Work MVO2 Efficiency

***

NS

Vehicle CoQ

% o

f Pre

-Pa

cin

g S

tre

ss

n=8 per group

Y S Y S

Vehicle CoQ

Y S Y S

Vehicle CoQ

Y S Y S

NS

NS**

Rowland MA, Rosenfeldt FL, et al Cardiovasc.Res 1998 40:165

EFFECT OFEFFECT OF ISCHAEMIC ISCHAEMIC STRESS IN HUMAN TISSUES STRESS IN HUMAN TISSUES

PROTECTIVE EFFECT OF CoQ PROTECTIVE EFFECT OF CoQ1010 AND VITAMIN E AND VITAMIN E

• Atrial tissue incubated with 400 micro molar Atrial tissue incubated with 400 micro molar CoQCoQ1010, 1 mM alpha tocopherol (Vit.E) or vehicle , 1 mM alpha tocopherol (Vit.E) or vehicle

for 30 min for 30 min

• Simulated ischaemia for 1 hour inducedSimulated ischaemia for 1 hour induced

• Post-ischaemic recovery measuredPost-ischaemic recovery measured

RESPONSE TO ISCHAEMIA/REPERFUSION (NO FLOW) STRESSRESPONSE TO ISCHAEMIA/REPERFUSION (NO FLOW) STRESS IN HUMAN HEART TISSUEIN HUMAN HEART TISSUE

+-

Voltage Duration Frequency

Stimulator

ForceTransducer

Water-jacketedbath (37 C)

ElectrodeBlock O2/CO2Humidified N2/CO2

Trace Recorder

o

Excised RAA

NormoxiaSimulatedIschemia

perfusatefree

TrabeculaeSeals

0

20

40

60

80

100

Vehicle CoQ10

p<0.01 vs <70yrs + p<0.01 vs Vehicle*

56.4±1.7

< 70 > 70

56.3±1.9 75.6±0.6 75.4±0.8

Age (years)

57n=

38 28 20

*

++

Trabecular CoQ10 ContentTrabecular CoQ10 ContentC

oenz

yme

Q10

(g

/g w

w)

Vehicle

Tocopherol1mM

CoQ10

400uM

0

20

40

60

80

Age (years)

Dev

elo

ped

Fo

rce

(% R

eco

very

) ** +

28 13 27 20 9 16

+

Effect of Co Q10 and Tocopherol

on Post-ischemic Recovery Effect of Co Q10 and Tocopherol

on Post-ischemic Recovery

< 70 > 70

Rosenfeldt FL, Pepe S et al Ann NY Acad Sci 2002 959: 355.

CONCLUSIONSCONCLUSIONS

The ageing heart has reduced capacity The ageing heart has reduced capacity to recover from aerobic andto recover from aerobic and ischaemic stressischaemic stress

Co QCo Q1010 has an senesence-specific has an senesence-specific

protective effect against both aerobic protective effect against both aerobic andand ischaemic stressischaemic stress

• Randomised, double blind placebo - controlled trialRandomised, double blind placebo - controlled trial..

• Elective primary cardiac surgical patientsElective primary cardiac surgical patients

• Patients (n=62) received Patients (n=62) received CoQCoQ1010 (300mg) or placebo (300mg) or placebo

(n=59) daily for (14 days average) prior to surgery(n=59) daily for (14 days average) prior to surgery

CLINICAL TRIAL OF CLINICAL TRIAL OF CoQCoQ1010 IN IN

CARDIAC SURGERYCARDIAC SURGERY CLINICAL TRIAL OF CLINICAL TRIAL OF CoQCoQ1010 IN IN

CARDIAC SURGERYCARDIAC SURGERY

Rosenfeldt FL, Pepe S et al Biogerontology 2002 959:355

0

2

4

6

8

10

12 Placebo

150mg/day

300mg/day

Oral TherapyOral Therapy

**

*P<0.001 vs Placebo: n=11 or 150mg/day: n=4

300mg/day: n=11

Coe

nzym

e Q

Coe

nzym

e Q

1010

g/m

g m

ito p

rote

ing/

mg

mito

pro

tein

MITOCHONDRIAL COENZYME QMITOCHONDRIAL COENZYME Q1010MITOCHONDRIAL COENZYME QMITOCHONDRIAL COENZYME Q1010

0

0.5

1

1.5

2

2.5

1 2

ADP:O EFFICIENCY RATIOADP:O EFFICIENCY RATIO

CoQCoQ1010

(n=11)(n=11)

PlaceboPlacebo(n=9)(n=9)

State III oxygen consumption of isolated human State III oxygen consumption of isolated human mitochondria at 37mitochondria at 37º C by oxidation of pyruvate (5mM)º C by oxidation of pyruvate (5mM)

AD

P:O

AD

P:O

** p=0.0002p=0.0002

0

10

20

30

40

50

60

70

1 2

RECOVERY OF DEVELOPED FORCE IN RECOVERY OF DEVELOPED FORCE IN MUSCLE STRIPS AFTER HYPOXIA MUSCLE STRIPS AFTER HYPOXIA P

erce

nt R

ecov

ery

Per

cent

Rec

over

y

PlaceboPlacebo(n=67)(n=67)

CoQCoQ1010

(n=98)(n=98)

p=0.0005p=0.0005

**

Pulmonary Capillary Wedge Pressure (mm Hg)

LV Stroke Work Index (g.mm2)

11 12 13

31

32

33

34

35

36

p=0.002

Placebo

CoQ10

CARDIAC FUNCTION IMPROVEMENT IN RESPONSE TO CoQCARDIAC FUNCTION IMPROVEMENT IN RESPONSE TO CoQ1010

CONCLUSIONSCONCLUSIONS

Preoperative CoQPreoperative CoQ1010 therapy in elderly patients therapy in elderly patients

1. Increases CoQ1. Increases CoQ1010 content of cardiac mitochondria content of cardiac mitochondria

2. Improves cardiac mitochondrial efficiency2. Improves cardiac mitochondrial efficiency

3. Increases tolerance to stress at a myocardial 3. Increases tolerance to stress at a myocardial levellevel

4. Improves post-operative cardiac pump function4. Improves post-operative cardiac pump function

NEGATIVE RISK FACTORS FOR MAJOR SURGERY THAT ALSO CONTRIBUTE TO BIOLOGICAL AGE

• Physical unfitness

• Anxiety and depression

PREOPERATIVE PREPARATION FOR HIGH RISK AND ELDERLY CARDIAC SURGERY

PATIENTS

The MPM Program

• Metabolic protection

• Physical Exercise program

• Mental: relaxation stress reduction and meditation

AUGMENTED METABOLIC THERAPY

• Co-enzyme Q10 (300mg)

•Alpha-Lipoic Acid (150mg) Potent antioxidant that regenerates reduced Co-Q10, Vit E and Vit C Involved in mitochondrial energy metabolism.

•Magnesium Orotate (1.2g) Orotate: pyrimidine precursor that increases the level of energy substrates in heart (Rosenfeldt et al Cardiovasc Drugs and Ther 1998 40:165) Magnesium: antagonises excess calcium and reduces reperfusion injury

Omega-3-fatty Acids (3g) Improve membrane function, antiarrhythmic

(Daily dosage)

PHYSICAL EXERCISE PROGRAMME

Modest pre-operative exercise program before surgery for at least two weeks under the

supervision of a physiotherapist

Including– Treadmill walking– Stationary bicycle– Light Weights– Gentle stretching exercises– Light aerobic exercises

Change in health-related quality of life due to exercise programme before CABG

Hamilton Health Science Surgical Centre, Ontario Canada

Treated

ControlP < 0.004

N.S.

Annals Internal Medicine 2000:253-262

MENTAL PREPARATIONRELAXATION AND STRESS REDUCTION

• Patient taught techniques of stress reduction, meditation and relaxation

• Techniques learned in one or two instruction sessions then done in own home

STRESS MANAGEMENT IN PATIENTS STRESS MANAGEMENT IN PATIENTS WITH ISCHAEMIC HEART DISEASEWITH ISCHAEMIC HEART DISEASE

Effect of stress-management or exercise on the risk of having a major Effect of stress-management or exercise on the risk of having a major cardiac event over a 5 year periodcardiac event over a 5 year period. .

Stress management was also associated with reduced ischemiaStress management was also associated with reduced ischemia induced by mental stress and ambulatory ischemia.induced by mental stress and ambulatory ischemia.

Blumenthal, J; Wei Jiang, M; Babyak, M; et al., Archives of Internal Medicine 1997;157:2213-222Blumenthal, J; Wei Jiang, M; Babyak, M; et al., Archives of Internal Medicine 1997;157:2213-222

Relative RiskRelative Risk 1.0 1.0

P=0.41P=0.41P valueP value -- P=0.03P=0.03

0.680.68

Odds ratioOdds ratio 11

Exercise groupExercise group Control groupControl group Stress management groupStress management group

0.260.26

PRELIMINARY RESULTS OF MPM PROGRAMME

IN ELDERLY PATIENTSUNDERGOING HEART

SURGERY

EFFECT ON QUALITY OF LIFE OF 1 MONTH ON MPM PROGRAM BEFORE SURGERYAlfred Hospital

After ProgramBefore Program

10

20

30

40

* p=0.042

n= 11

*

Before Program

10

20

30

40* p=0.027

n= 11

*50

After Program

SF-36 Composite

Score

SF-36 Composite

Score

Physical

Mental

EFFECT OF MPM PROGRAM AND SURGERY ON EFFECT OF MPM PROGRAM AND SURGERY ON PHYSICALPHYSICAL QUALITY OF LIFE QUALITY OF LIFE VSVS USUAL CARE CONTROL USUAL CARE CONTROL

(Myles et al, 1998)(Myles et al, 1998)

**

2020

4040

6060

8080

* * p=0.0023p=0.0023

(n= 6)(n= 6)

Before Before SurgerySurgery

1010

20203030

4040

* p=0.05; * p=0.05;

n= 64n= 64

**5050

1 month 1 month after surgeryafter surgery

MPM GroupMPM Group

Control GroupControl Group

Physical Physical SF-36 SF-36

Composite Composite ScoreScore

Physical Physical SF-36 SF-36

Composite Composite ScoreScore

EFFECT OF MPM PROGRAM AND SURGERY ON EFFECT OF MPM PROGRAM AND SURGERY ON MENTAL MENTAL QUALITY OF LIFE QUALITY OF LIFE VSVS USUAL CARE CONTROL USUAL CARE CONTROL

(Myles et al, 1998)

*

20

40

60

80

* p=0.03

(n= 6)

10

20

30

40

* p=0.05;

(n= 64)

*50

Before Surgery

Before Surgery

1 month after surgery

1 month after surgery

MPM Group

Control Group

Mental SF-36

Composite Score

Mental SF-36

Composite Score

*

CONCLUSIONCONCLUSION

A programme of combined metabolic, mental A programme of combined metabolic, mental and physical therapy shows promise as an and physical therapy shows promise as an inexpensive, holistic preparation in inexpensive, holistic preparation in senescent patients undergoing major senescent patients undergoing major surgerysurgery

CONCLUSIONCONCLUSION

But which particular patients will benefit But which particular patients will benefit most from such a programme? most from such a programme?

Patients who are Patients who are biologicallybiologically old! old!

MITOCHONDRIAL CAUSES MITOCHONDRIAL CAUSES

AND PREDICTORS OF BIOLOGICAL AGEAND PREDICTORS OF BIOLOGICAL AGE

MITOCHONDRIAL CAUSES MITOCHONDRIAL CAUSES

AND PREDICTORS OF BIOLOGICAL AGEAND PREDICTORS OF BIOLOGICAL AGE

•Mitochondrial oxidative phosphorylation declines with ageMitochondrial oxidative phosphorylation declines with age

•Mitchondrial mutations accumulate with ageingMitchondrial mutations accumulate with ageing

MITOCHONDRIAL CAUSES AND MARKERS OF CARDIAC AGEINGMITOCHONDRIAL CAUSES AND MARKERS OF CARDIAC AGEING

MITOCHONDRIAL CAUSES AND MARKERS OF CARDIAC AGEINGMITOCHONDRIAL CAUSES AND MARKERS OF CARDIAC AGEING

•Mitochondrial oxidative phosphorylation declines with ageMitochondrial oxidative phosphorylation declines with age

•Mitchondrial mutations accumulate with ageingMitchondrial mutations accumulate with ageing

•Could the abundance of mtDNA mutations or the magnitude of the cellular DNA content be clinically useful predictors of reduced tolerance to stress in the ageing heart?

MATERIALMATERIAL

Right atrium harvested from patients Right atrium harvested from patients undergoing cardiac surgeryundergoing cardiac surgery

METHODSMETHODS

11) Measure recovery of contractile function after stress) Measure recovery of contractile function after stress

2) Quantitate the abundance of MtDNA2) Quantitate the abundance of MtDNA49774977 deletion deletionand cellular content of mtDNAand cellular content of mtDNA

3) Correlate function with mtDNA3) Correlate function with mtDNA49774977 deletion abundance deletion abundance and MtDNA content and MtDNA content

In the In the same same tissue tissue samplesample

RECOVERY OF FUNCTION DECLINES WITH AGERECOVERY OF FUNCTION DECLINES WITH AGE

N = 52N = 52

RR2 2 = 0.21= 0.21

p < 0.001p < 0.001

00

1010

2020

3030

4040

5050

6060

7070

8080

9090

00 2020 4040 6060 8080 100100

Age (years)Age (years)

Re

cove

ry D

eve

lope

d F

orc

e (%

)R

eco

very

De

velo

ped

Fo

rce

(%)

mtDNAmtDNA49774977 DELETION DELETION• “ “Common Deletion ” Common Deletion ” • Can cause mitochondrial diseases such asCan cause mitochondrial diseases such as Kearn-Sayre SyndromeKearn-Sayre Syndrome

49774977““Common Common deletion”deletion”

Genes lost :Genes lost :ATPase 8, ATPase6 ATP ATPase 8, ATPase6 ATP 6,COIII6,COIIItRNAtRNAGlyGly, ND3, tRNA, ND3, tRNAArgArg,,ND4L, ND4, tRNAND4L, ND4, tRNAHisHis,,tRNAtRNASerSer, tRNA, tRNALeuLeu, ND5, ND5

mtDNAmtDNA49774977 ABUNDANCE INCREASES WITH AGE ABUNDANCE INCREASES WITH AGE

IN MYOCARDIUMIN MYOCARDIUM

N = 35N = 35

RR22 = 0.78 = 0.78

P < 0.001P < 0.001

-14-14

-12-12

-10-10

-8-8

-6-6

-4-4

-2-2

00

00 2020 4040 6060 8080 100100

Age (years)Age (years)

log

(m

tDN

A49

77 %

ab

un

dan

ce)

log

(m

tDN

A49

77 %

ab

un

dan

ce)

Absolute value at 60 to 80 years is < 0.01%Absolute value at 60 to 80 years is < 0.01%

RECOVERY DEVELOPED FORCE DECLINESRECOVERY DEVELOPED FORCE DECLINES

WITH mtDNAWITH mtDNA49774977 ABUNDANCE ABUNDANCE

N = 52N = 52

RR22 = 0.09 = 0.09

PP = 0.036 = 0.036

00

1010

2020

3030

4040

5050

6060

7070

8080

9090

-14-14 -12-12 -10-10 -8-8 -6-6 -4-4 -2-2 00

Log (mtDNA4977 abundance%)Log (mtDNA4977 abundance%)

Re

cove

ry d

eve

lop

ed fo

rce

(%

)R

eco

very

dev

elo

ped

forc

e (

%)

mt DNA FORCE RECOVERY VS mt DNA FORCE RECOVERY VS COPY NUMBERCOPY NUMBER IN MYOCARDIUM IN MYOCARDIUM

N = 39N = 39

RR22 = 0.01 = 0.01

PP = 0.46 = 0.46

00

1010

2020

3030

4040

5050

6060

7070

8080

9090

00 1010 2020 3030 4040

MtDNA copy number / nucleus (x1000)MtDNA copy number / nucleus (x1000)

Re

cove

ry d

eve

lop

ed fo

rce

(%

)R

eco

very

dev

elo

ped

forc

e (

%)

MtDNA MtDNA 4977 4977 Copy NoCopy No AgeAge

P valueP value 0.860.86 0.73 0.73 0.00010.0001

Slope Slope -0.36-0.36 -1.04 -1.04 -0.76-0.76

MULTIVARIATE ANALYSISMULTIVARIATE ANALYSISPredictors of Recovery of ForcePredictors of Recovery of Force

CONCLUSIONSCONCLUSIONS

In human cardiac tissue:In human cardiac tissue:

1. Response to ischaemia/reperfusion stress declines with age 1. Response to ischaemia/reperfusion stress declines with age

2. Compared to chronological age MtDNA2. Compared to chronological age MtDNA4977 4977 deletion abundance deletion abundance and MtDNA copy number are not useful predictors of the response and MtDNA copy number are not useful predictors of the response to ischaemia/reperfusion stress to ischaemia/reperfusion stress

CLINICAL IMPLICATIONSCLINICAL IMPLICATIONS

These data from the ageing human heart do not support a major These data from the ageing human heart do not support a major role for mtDNA mutations as a cause for or predictor of clinical role for mtDNA mutations as a cause for or predictor of clinical dysfunction in the senescent heartdysfunction in the senescent heart

Combined metabolic, mental and physical Combined metabolic, mental and physical therapy shows promise as an inexpensive, therapy shows promise as an inexpensive, holistic preparation in senescent patients holistic preparation in senescent patients undergoing major stress that may undergoing major stress that may “engineer negligible senesence”“engineer negligible senesence”

ACKNOWLEDGMENTSACKNOWLEDGMENTS

ACKNOWLEDGMENTSACKNOWLEDGMENTS

Salvatore Pepe, Ruchong Ou, Deahne QuickSalvatore Pepe, Ruchong Ou, Deahne Quick

Baker Heart Research InstituteBaker Heart Research Institute..

Anthony Hadj, Robyn Ascham, William Lyon, Silvana Anthony Hadj, Robyn Ascham, William Lyon, Silvana Marasco, and Cardiac SurgeonsMarasco, and Cardiac Surgeons Alfred Hospital Alfred Hospital

Frank Miller, Phillip Nagley, Anthony LinnaneFrank Miller, Phillip Nagley, Anthony Linnane

Department of Biochemistry and Molecular Biology Monash University Centre Department of Biochemistry and Molecular Biology Monash University Centre for Molecular Biology and Medicine, Epworth Hospitalfor Molecular Biology and Medicine, Epworth Hospital

Melbourne, AustraliaMelbourne, Australia

THE END

QUANTITATIVE PCR TO MEASURE QUANTITATIVE PCR TO MEASURE ABUNDANCE OF ABUNDANCE OF mtDNAmtDNA49774977

5Kb5Kb

Standards Tissue DNA

m tDNA 7901 m tDNA 13650

m tDNA 8470/13447

Zhang et al 1996

pCZ21 pCZ21 StandardStandard

MEASUREMENT OF mtDNA COPY NUMBER PER CELLMEASUREMENT OF mtDNA COPY NUMBER PER CELL

To prepare DNA standards a dual insert plasmid constructed : pFM11To prepare DNA standards a dual insert plasmid constructed : pFM11

mtDNA insert

-globin insert

nt14820 15253 439 84

LacZ’LacI

Segment of Segment of -globin gene-globin gene

Segment of mtDNA genome (cytochrome b)Segment of mtDNA genome (cytochrome b)

Miller FJ, Rosenfeldt FL, Nagley P, Linnane AW, Zhang C Miller FJ, Rosenfeldt FL, Nagley P, Linnane AW, Zhang C

Nucleic Acids Research 2003 31:e61Nucleic Acids Research 2003 31:e61

Standards Sample (tissue DNA)

Two separate PCRs performed comparing serially diluted inputs of Two separate PCRs performed comparing serially diluted inputs of standard pFM11 with tissue aliquot performed for:standard pFM11 with tissue aliquot performed for:

1) MtDNA template abundance1) MtDNA template abundance

2) 2) -Globin template abundance-Globin template abundance

Enabled accurate measurement ofnumber of mtDNA genomes per Enabled accurate measurement ofnumber of mtDNA genomes per -globin gene-globin gene

Coefficient of variation 7%Coefficient of variation 7%

Miller FJ, Rosenfeldt FL, Nagley P, Linnane AW, Zhang C Miller FJ, Rosenfeldt FL, Nagley P, Linnane AW, Zhang C

Nucleic Acids Research 2003 31:e61Nucleic Acids Research 2003 31:e61

MYOCARDIUM: mtDNA COPY NUMBER DID NOT MYOCARDIUM: mtDNA COPY NUMBER DID NOT CHANGE WITH AGECHANGE WITH AGE

N = 35N = 35

RR22 = 0.01 = 0.01

PP = 0.7 = 0.7

Av. Copy No = 6970Av. Copy No = 6970

00

55

1010

1515

2020

2525

3030

3535

00 2020 4040 6060 8080 100100

Age (years)Age (years)

MtD

NA

Cop

y N

um

ber

per

Nu

cle

us

MtD

NA

Cop

y N

um

ber

per

Nu

cle

us

(x10

00)

(x10

00)