AGEING HEART

1
AGEING HEART VESSELS Melbourne,
Australia August 3-5, 2004
www.ishr.edu.au / ishr
Current Understanding, New Research and the
Challenge of Reducing the Health Care Impact of
Age-Related Cardiovascular Disease
2
Risk Factors for Cardiovascular Disease

• hypertension
• LDL/HDL cholesterol
• diabetes
• family history of CAD, gender
• excess alcohol, tobacco use
• physical inactivity
• obesity
• stress
• diet
• advanced age

3
Aging Involves Cardiac Remodeling
With Functional Adaptation
Independent of Disease

• Cell enlargement
• Cell rigidity
• Slower electrical properties
• More vulnerable to nutrient deprivation
  (ischemia)
• More vulnerable to heart attack (arrhythmia)
• Membrane protein and lipid function changed
• (receptors, enzymes and signalling
  intermediates)
• More free radical injury, less protective
  antioxidants
• (SOD, catalase, glutathione, vit E, Coenzyme
  Q10...)
• Less capable of hard work or recovery from stress
• Cell loss (death)

4
Consequences of Membrane Modification
Qualitative changes to lipids, proteins,
lipid-protein interactions - receptors,
channels, enzymes, - intracellular signaling -
ion homeostasis - energy metabolism -
altered membrane function efficiency -
augmented perturbation after stress lost
reserve
Carrier, i.e., ADP Translocase
Channels
5
Diet Is a Key Factor Involved in the Reduced
Incidence of Death From Age-associated
Diseases From epidemiological studies of
Mediterranean, Japanese, Eskimo other
populations
Fish-rich (omega-3 fatty acids) versus Animal-rich
(omega-6 fatty acids)
Dietary Antioxidants
6
Polyunsaturated fatty acids, PUFA

• long carbon chain (Cgt12, up to 24)
• degree of unsaturation (double bonds)
• 0 saturated, 1monounsaturated
• 2 or more polyunsaturated
• PUFA increased membrane fluidity, lower
  melting point
• omega-3 vs omega-6 position of methyl group,
• between 3 4 vs 6 7
• also affects fluidity and conformation
• precursors of lipid peroxides and eicosanoids
• signaling role

?
O
..n..
2
3
1
C
OH
7
Fish or ? -3 PUFA Intake Reduces Heart Disease
Risk

• Dart Study Lancet 1989 757-761
• MRFIT Proc Soc Exp Biol Med 1992 200
  177-82
• Indian Heart Study Br Med J 1992 304
  1015-19
• Lyon Diet Heart Study Lancet 1994 343 1454-9
• 
  Circulation 1999 99 779-85
• Health Professionals Study N Eng J Med 1995
  332 977-82
• Primary Cardiac Arrest JAMA 1995 274
  1363-1367
• Honolulu Heart Program Circulation 1996 94
  952-56
• Western Electric Study N Eng J Med 1997 336
  1046-53
• US Physicians Health Study JAMA 1998 279
  23-28
• GISSI Prevenzione Trial Circulation 2002
  105 1897-1903

8
GISSI PREVENZIONE TRIAL
n5666, 850mg omega-3 PUFA, 3.5 years
100
-15
-20
80
-45
60
of Patients
40
20
0
NO OMEGA-3
CV
Heart
Total
OMEGA-3
Events
Attack
Mortality
Circulation 2002 105 1897-1903
Marchioli R et al. , Lancet 1999 354 457-455
9
Omega-3 PUFA Attenuates the Rise in Arterial
Blood Pressure due to Age or Omega-6 PUFA

180

160
140

120
6 mo
100
24 mo
Systolic Pressure (mmHg)
80
n8 SD rats
60
40
20
0
Omega-3 PUFA
Omega-6 PUFA
PEPE, et al, 1999
10
Mitochondrial Membrane PUFA
? -6 PUFA ? -3 PUFA Age (mo)
6 24 6
24 160 10.00.29 14.00.69
9.50.35 11.00.64 180 23.01.15
26.01.73 10.10.72A
12.00.81A 161 0.220.05 0.30.12
0.70.10 0.80.05 181
9.00.81 9.00.43 9.50.40
10.00.58 182 (? -6) LA 17.01.21
11.01.44 12.00.58A
9.00.69 204 (? -6) AA 24.01.44
32.01.79 9.20.64A 12.00.58A 224 (?
-6) 1.10.18 1.60.15 0.10.03
0.10.01 183 (? -3) 0.20.03
0.20.03 1.80.06 2.00.17 205 (?
-3) EPA 0.10.03 0.10.05 6.00.17A
5.20.29A 225 (? -3) 2.00.12
0.80.12 2.20.05
5.00.58A 226 (? -3) DHA 9.00.64 2.00.43
27.01.01A 30.01.15A ? ? -6
42.10.85 44.61.58
21.30.50A 1.10.33A ? ? -3
11.30.69 3.10.44 37.11.1A
42.21.35A
Values (mol/100mol) are presented as mean SEM,
n5. Plt0.05 vs 6 mo A Plt0.05 vs n-6 PUFA
PEPE, et al, 1999
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?-3 ?-6 PUFA
A
A
2
1.5
6mo
24mo
?-3 ?-6 PUFA
1

0.5
0
Omega-3 PUFA
Omega-6 PUFA
plt0.05 vs 6mo A plt0.05 vs?-6 PUFA n5
PEPE, et al, 1999
12
?
?
-
-
6 PUFA
3 PUFA
6
24
6
24
Age (mo)

DPG
A
121.0
8.50.9
150.9
140.8

PC
461.5
531.7
421.8
421.6
A
PE
351.8
331.4
381.9
401.8
PS
2.40.7
2.50.5
2.20.2
2.40.3
PI
1.30.2
1.40.3
1.10.4
1.20.6
PEPE, et al
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Cardiolipin (DPG)
20
6mo
24mo
A
15

10
Total Phospholipids
5
0
Omega-3 PUFA
Omega-6 PUFA
plt0.05 vs 6mo Aplt0.05 vs?-6 PUFA n5
PEPE, et al
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CARDIOLIPIN Phosphatidylglycerol Unique ability
to interact with proteins - physico-chemical
- sensitive to oxidation Low
or absent ? activity of respiratory chain
complexes ? mitochondrial membrane
potential Inner membrane Intermembrane
space ADP-ATP carrier creatine
kinase phosphate carrier cytochrome
C pyruvate carrier carnitine carrier NADH
dehydrogenase succinate dehydrogenase complex
III cytochrome C oxidase ATP synthase
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Cardiolipin
Q
ANT
O
O
Krebs Cycle (PDH, KGDH)
The Inner Mitochondrial Membrane
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The Post-Ischemic Decline in Contractile
Recovery is Attenuated by Omega-3 PUFA
100
I/R
A
A
A
I/RRR
80
A

60

Control PSP (Normoxia)
40
20
0
6
24
6
24
Omega-3 PUFA
Omega-6 PUFA
plt0.05 vs 6mo A plt0.05 vs ? -6 PUFA n6
PEPE, et al, 1999
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Increased Omega-3 PUFA Incorporation into
Cardiac Membranes Augments
Cardiac Efficiency AND prevents Fatal VF
Incidence of Ventricular Arrhythmia
Myocardial
Oxygen Consumption
7.5
100
5.0
2
(mL/min/g dry wt) normalised per unit work
Ventricular
50
Fibrillation
MVO
2.5
0.0
0
omega-6 PUFA
omega-3 PUFA
Pepe McLennan, 2002
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Mitochondrial Calcium
2.5
C
2
I/R
I/RRR
1.5
Total Ca 2 nmol/mg protein
1
0.5
0
6
24
6
24
Omega-3 PUFA
Omega-6 PUFA
PEPE, et al, 1999
19
Pyruvate Dehydrogenase Activation
100
90
C
80
I/R
70
I/RRR
60
PDHA ( Total PDH)
50
40
30
20
10
0
6
24
6
24
Omega-6 PUFA
Omega-3 PUFA
PEPE, et al, 1999
20
ADPO Efficiency Ratio
A
A
2
1.5

6 mo
1
ADP0
24 mo

plt0.01 vs 6mo
0.5
plt0.01 vs ? -6
A
0
Omega-3 PUFA
Omega-6 PUFA
37?C by oxidation of pyruvate (5mM) malate
(0.5mM) during complete conversion of 0.5mM ADP
to ATP
PEPE, et al, 1999
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Summary

• omega -6/ omega -3 ratio is increased with age
• Cardiolipin is significantly decreased with age
  whereas PC is increased with age.
• For the same level of contractile work, PDHA, and
  mito Ca2 values are higher after omega -6
  PUFA-rich diet and is augmented by age.
• omega-3 PUFA -rich diet attenuates ALL of these
  effects.

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Conclusions

• Manipulating cardiac membrane phospholipids and
  the omega-3/ omega -6 PUFA ratio alters the flux
  of Ca2 across the mitochondrial membrane and
  this may markedly impact intramitochondrial
  Ca2-dependent processes.

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Conclusions
? Evidence for thermodynamic inefficiency in
omega -6 PUFA or aging compared to omega -3 PUFA
? Potential for Ca overload after ischemia
is greater and thus more rapid onset of
MPT opening and cell de-energization in
omega -6 PUFA or with aging vs omega -3 PUFA . ?
Age-related reduction of cardiolipin augments
this effect.
2
24
4-hydroxy-2-nonenal, 4-HNE ? Specific aldehydic
product of oxygen free-radical induced lipid
peroxidation of Omega-6 PUFAs ? 4-HNE -a
second toxic messenger cytotoxic, mutagenic,
genotoxic, chemotactic
ROS
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Consequences of 4-HNE formation
? Michael-addition reactions with proteins at
sulfhydryl gp of cysteine, imidazole N of
histidine, ?-amine of lysine ?
Proarrhythmic changes to membrane
excitability ? -Activates PLC -Inhibits
Na-K ATPase -Inactivates glucose-6-phosphatase
-Induces heat shock protein synthesis -Inacti
vates glutathione peroxidase (inhibits
glutathione recycling) -Apolipoprotein B
adducts in atherosclerosis
26
Omega-3 PUFA Reduces Post-ischemic Coronary
Release of HNE
Omega-6 PUFA
Omega-3 PUFA
Reperfusion Time (min)
PEPE, et al
After 30 min global ischemia
27
Omega-3 PUFA Reduces Post-Ischemic Mitochondrial
4-HNE
Omega-3 PUFA
Omega-6 PUFA
PEPE, et al
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What Governs Mitochondrial Survival?
Mitochondrial Membrane Permeability Transition
and the MPT pore
? Ca-dependent opening of a high-conductance
channel in the inner mitochondrial membrane
-regulates membrane permeability may cause
loss of membrane potential ATP
31
Mitochondrial Membrane Permeability Transition
and the MPT pore
? Both regulated by High CaM Pi, long chain
acyl CoA, spermine, ADP, ROS, protein
sulfhydryl and thiol group modification (after
peroxidation) AND CARDIOLIPIN! ? Collapse of
the membrane potential leads to mitochondrial
swelling, rapid MPT and pore opening, Loss of
ATP production results in cell run down and
death. ? MPT pore opening releases
mitochondrial contents -incl cytochrome c
and can trigger caspase activation and the
apoptotic cascade.
32
Mitochondrial Membrane Permeability
Transition Confocal measurement of in situ
mitochondrial membrane potential and ROS using
TMRE and DCF in real time
33
TMRM fluorescence (mitochondrial membrane
potential) Isolated Mitochondria
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Modulators of Mean Time to Mito Membrane
Permeability Transition
SCAVENGING ROS INHIBITS THE MPT
35
MITOCHONDRIA OF SENESCENT CARDIOCYTES ARE MORE
SUSCEPTIBLE TO ROS RESCUE BY CoQ10
36
Total Coenzyme Q10 Concentration in Cardiac
Myocytes from 6 and 24 mo S-D rats
37
Reversal of membrane aging requires
Omega-3 Omega-6 PUFA Intrinsic Antioxidant
Systems
for improved mitochondrial response to stress
that matches the response of young hearts