THE AGEING HEART CLINICAL THERAPEUTIC STRATEGIES FOR CARDIAC STRESS AND QUEST FOR MITOCHONDRIAL MARK

1
THE AGEING HEART CLINICAL THERAPEUTIC
STRATEGIES FOR CARDIAC STRESSAND QUEST FOR
MITOCHONDRIAL MARKERS OF IMPAIRED FUNCTIONAL
RECOVERY AFTER STRESS
Franklin Rosenfeldt, Salvatore Pepe, Francis
Miller, Phillip Nagley, Anthony Linnane, Anthony
Hadj Cardiac Surgical Research Unit, Alfred
Hospital and Baker Medical Research Institute and
Department of Biochemistry and Molecular
Biology, Monash Melbourne Australia
2
IN THE AGEING HEART THE RESPONSE TO MAJOR
STRESS IS IMPAIRED
3
IN THE AGEING HEART THE RESPONSE TO MAJOR
STRESS IS IMPAIRED
In gt70 year old patients the mortality after

• myocardial infarction
• coronary angioplasty
• coronary bypass surgery

is three times
that in the lt 70 year old age group

• Why?

4
IN THE AGEING HEART THE RESPONSE TO MAJOR
STRESS IS IMPAIRED
In gt70 year old patients the mortality after

• myocardial infarction
• coronary angioplasty
• coronary bypass surgery

is three times
that in the lt 70 year old age group

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

5
RESPONSE OF THE YOUNG HEART TO CARDIAC SURGERY

• Cardiac Surgery

Young Heart
Low mortality Few complications
6
RESPONSE OF THE AGEING HEART TO CARDIAC SURGERY

• Cardiac Surgery

Young Heart
Low mortality Few complications
Cardiac Surgery
Biologically Ageing Heart
High mortality Many complications
7
RESPONSE OF THE AGEING HEART TO CARDIAC SURGERY

• Cardiac Surgery

Young Heart
Low mortality Few complications
Cardiac Surgery
Biologically Ageing Heart
Higher mortality More complications
Senesence-specific therapies ?
8
RESPONSE OF THE AGEING HEART TO CARDIAC SURGERY

• Cardiac Surgery

Young Heart
Low mortality Few complications
Cardiac Surgery
Biologically Ageing Heart
Higher mortality More complications
Markers of biological age and predictors impaired
responsiveness ?
9
TARGETED METABOLIC CARDIOPROTECTION FOR STRESSED
AGEING HEART

Normal Heart Normal Metabolism
No Change
Coenzyme Q10
10
TARGETED METABOLIC CARDIOPROTECTION FOR STRESSED
AGEING HEART

Normal Heart Normal Metabolism
No Change
Stress e.g. Cardiac Surgery
Stressed Heart with Disturbed Metabolism
11
TARGETED METABOLIC CARDIOPROTECTION

Normal Heart
No Change
Cardiac surgery aerobic and ischaemic stresses

Coenzyme Q10
Stressed Heart with Disturbed Energy
Metabolism
Normalised Metabolism Improved Function
12
HYPOTHESES 1. In the ageing human myocardium
there is an impaired myocardial response to
aerobic and ischaemic stress which impairs the
response to major surgery 2. CoQ10 has an
specific protective effect in the ageing heart

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RAT STUDY OF SENESCENT HEART AEROBIC STRESS AND
Co Q10

• Young (5 months) and senescent rats (3 years)
• Treated for 6 weeks with intraperitoneal
  injections of
• CoQ10 (4 mg/kg) or placebo vehicle
• Hearts removed and placed on Isolated working
  heart
• apparatus
• Paced at 510 beats/min for 2 hours maximum
  aerobic (oxygen demanding) stress
• Recovery of pump function and efficiency
  expressed
• as percent of pre-pacing levels

15
AEROBIC PACING STRESS IN WORKING RAT HEART
Rowland MA, Rosenfeldt FL, et al Cardiovasc.Res
1998 40165
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EFFECT OF ISCHAEMIC STRESS IN HUMAN TISSUES
PROTECTIVE EFFECT OF CoQ10 AND VITAMIN E

• Atrial tissue incubated with 400 micro molar
  CoQ10, 1 mM alpha tocopherol (Vit.E) or vehicle
• for 30 min
• Simulated ischaemia for 1 hour induced
• Post-ischaemic recovery measured

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RESPONSE TO ISCHAEMIA/REPERFUSION (NO FLOW)
STRESS IN HUMAN HEART TISSUE
Excised RAA
Force Transducer
Trace Recorder

-
Voltage
Duration
Frequency
Trabeculae
Stimulator
Seals
Water-jacketed bath (37 C)
perfusate free
o
Normoxia
Electrode Block
Simulated Ischemia
O2/CO2
Humidified N2/CO2
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Effect of Co Q10 and Tocopherol on Post-ischemic
Recovery

80


Vehicle

60
Tocopherol 1mM
Developed Force ( Recovery)
40
CoQ10 400uM
20
0
20 9 16
28 13 27
lt 70 gt 70
Age (years)
Rosenfeldt FL, Pepe S et al Ann NY Acad Sci 2002
959 355.
20
CONCLUSIONS
The ageing heart has reduced capacity to recover
from aerobic and ischaemic stress Co Q10 has an
senesence-specific protective effect against both
aerobic and ischaemic stress
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CLINICAL TRIAL OF CoQ10 IN CARDIAC SURGERY

• Randomised, double blind placebo - controlled
  trial.
• Elective primary cardiac surgical patients
• Patients (n62) received CoQ10 (300mg) or placebo
  (n59) daily for (14 days average) prior to
  surgery

Rosenfeldt FL, Pepe S et al Biogerontology 2002
959355
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MITOCHONDRIAL COENZYME Q10

Placebo
12
150mg/day
10
300mg/day
8
Coenzyme Q10 ?g/mg mito protein
6

Plt0.001 vs Placebo n11 or 150mg/day
n4 300mg/day n11
4
2
0
Oral Therapy
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ADPO EFFICIENCY RATIO

p0.0002
ADPO
Placebo (n9)
CoQ10 (n11)
State III oxygen consumption of isolated human
mitochondria at 37º C by oxidation of pyruvate
(5mM)
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RECOVERY OF DEVELOPED FORCE IN MUSCLE STRIPS
AFTER HYPOXIA

p0.0005
Percent Recovery
Placebo (n67)
CoQ10 (n98)
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CARDIAC FUNCTION IMPROVEMENT IN RESPONSE TO CoQ10
36
35
CoQ10
34
p0.002
LV Stroke Work Index (g.mm2)
33
Placebo
32
31
11
13
12
Pulmonary Capillary Wedge Pressure (mm Hg)
26
CONCLUSIONS Preoperative CoQ10 therapy in
elderly patients

• 1. Increases CoQ10 content of cardiac
  mitochondria
• 2. Improves cardiac mitochondrial efficiency
• 3. Increases tolerance to stress at a myocardial
  level
• 4. Improves post-operative cardiac pump function

27
NEGATIVE RISK FACTORS FOR MAJOR SURGERY THAT ALSO
CONTRIBUTE TO BIOLOGICAL AGE

• Physical unfitness
• Anxiety and depression

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PREOPERATIVE PREPARATION FOR HIGH RISK AND
ELDERLY CARDIAC SURGERY PATIENTSThe MPM Program

• Metabolic protection
• Physical Exercise program
• Mental relaxation stress reduction and
  meditation

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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 40165)
• Magnesium antagonises excess calcium and
  reduces reperfusion injury
• Omega-3-fatty Acids (3g)
• Improve membrane function, antiarrhythmic
• (Daily dosage)

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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

31
Change in health-related quality of life due to
exercise programme before CABG Hamilton Health
Science Surgical Centre, Ontario Canada
N.S.
Treated
P lt 0.004
Control
Annals Internal Medicine 2000253-262
32
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

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STRESS MANAGEMENT IN PATIENTS WITH ISCHAEMIC
HEART DISEASE

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

Relative Risk 1.0
Stress management was also associated with
reduced ischemia induced by mental stress and
ambulatory ischemia.
Blumenthal, J Wei Jiang, M Babyak, M et al.,
Archives of Internal Medicine 19971572213-222
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PRELIMINARY RESULTS OF MPM PROGRAMMEIN ELDERLY
PATIENTSUNDERGOING HEART SURGERY
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EFFECT ON QUALITY OF LIFE OF 1 MONTH ON MPM
PROGRAM BEFORE SURGERYAlfred Hospital

Physical
40
30
SF-36 Composite Score
p0.042 n 11
20
10
After Program
Before Program

Mental
50
SF-36 Composite Score
40
p0.027 n 11
30
20
10
Before Program
After Program
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EFFECT OF MPM PROGRAM AND SURGERY ON PHYSICAL
QUALITY OF LIFE VS USUAL CARE CONTROL
(Myles et al, 1998)
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EFFECT OF MPM PROGRAM AND SURGERY ON MENTAL
QUALITY OF LIFE VS USUAL CARE CONTROL
(Myles et al, 1998)
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CONCLUSION

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

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CONCLUSION

• But which particular patients will benefit most
  from such a programme?
• Patients who are biologically old!

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MITOCHONDRIAL CAUSES AND PREDICTORS OF
BIOLOGICAL AGE

• Mitochondrial oxidative phosphorylation declines
  with age
• Mitchondrial mutations accumulate with ageing

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MITOCHONDRIAL CAUSES AND MARKERS OF CARDIAC
AGEING

• Mitochondrial oxidative phosphorylation declines
  with age
• Mitchondrial 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?

42
MATERIAL Right atrium harvested from patients
undergoing cardiac surgery
METHODS 1) Measure recovery of contractile
function after stress 2) Quantitate the
abundance of MtDNA4977 deletion and cellular
content of mtDNA 3) Correlate function with
mtDNA4977 deletion abundance and MtDNA content

In the same tissue sample
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RECOVERY OF FUNCTION DECLINES WITH AGE
90
80
70
60
50
Recovery Developed Force ()
40
N 52 R2 0.21 p lt 0.001
30
20
10
0
0
20
40
60
80
100
Age (years)
44

• mtDNA4977 DELETION
• Common Deletion
• Can cause mitochondrial diseases such as
• Kearn-Sayre Syndrome

Genes lost ATPase 8, ATPase6 ATP
6,COIII tRNAGly, ND3, tRNAArg, ND4L, ND4,
tRNAHis, tRNASer, tRNALeu, ND5
4977 Common deletion
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mtDNA4977 ABUNDANCE INCREASES WITH AGE IN
MYOCARDIUM
0
-2
-4
log (mtDNA4977 abundance)
-6
N 35 R2 0.78 P lt 0.001
-8
-10
Absolute value at 60 to 80 years is lt 0.01
-12
-14
0
20
40
60
80
100
Age (years)
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RECOVERY DEVELOPED FORCE DECLINES WITH mtDNA4977
ABUNDANCE
90
80
70
60
50
Recovery developed force ()
40
N 52 R2 0.09 P 0.036
30
20
10
0
-14
-12
-10
-8
-6
-4
-2
0
Log (mtDNA4977 abundance)
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mt DNA FORCE RECOVERY VS COPY NUMBER IN
MYOCARDIUM
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MULTIVARIATE ANALYSIS Predictors of Recovery of
Force
MtDNA 4977 Copy No Age P value 0.86
0.73 0.0001 Slope -0.36
-1.04 -0.76
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CONCLUSIONS In human cardiac tissue 1. Response
to ischaemia/reperfusion stress declines with age
2. Compared to chronological age MtDNA4977
deletion abundance and MtDNA copy number are not
useful predictors of the response to
ischaemia/reperfusion stress
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CLINICAL IMPLICATIONS

• These data from the ageing human heart do not
  support a major role for mtDNA mutations as a
  cause for or predictor of clinical dysfunction in
  the senescent heart
• Combined metabolic, mental and physical therapy
  shows promise as an inexpensive, holistic
  preparation in senescent patients undergoing
  major stress that may engineer negligible
  senesence

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ACKNOWLEDGMENTS
Salvatore Pepe, Ruchong Ou, Deahne Quick Baker
Heart Research Institute. Anthony Hadj, Robyn
Ascham, William Lyon, Silvana Marasco, and
Cardiac Surgeons Alfred Hospital Frank Miller,
Phillip Nagley, Anthony Linnane Department of
Biochemistry and Molecular Biology Monash
University Centre for Molecular
Biology and Medicine, Epworth Hospital
Melbourne, Australia
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THE END
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QUANTITATIVE PCR TO MEASURE ABUNDANCE OF
mtDNA4977
Standards Tissue DNA
5Kb
pCZ21 Standard
Zhang et al 1996
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MEASUREMENT OF mtDNA COPY NUMBER PER CELL To
prepare DNA standards a dual insert plasmid
constructed pFM11
Segment of b-globin gene Segment of mtDNA genome
(cytochrome b)
Miller FJ, Rosenfeldt FL, Nagley P, Linnane AW,
Zhang C Nucleic Acids Research 2003 31e61
55
Two separate PCRs performed comparing serially
diluted inputs of standard pFM11 with tissue
aliquot performed for 1) MtDNA template
abundance 2) b-Globin template abundance
Enabled accurate measurement ofnumber of mtDNA
genomes per b-globin gene Coefficient of
variation 7 Miller FJ, Rosenfeldt FL, Nagley P,
Linnane AW, Zhang C Nucleic Acids Research 2003
31e61
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MYOCARDIUM mtDNA COPY NUMBER DID NOT CHANGE WITH
AGE
N 35 R2 0.01 P 0.7 Av. Copy No 6970