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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)