Transcript No Slide Title

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
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
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)
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
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
Polyunsaturated fatty acids, PUFA
• long carbon chain (C>12, up to 24)
O
1
• degree of unsaturation (double bonds)
C
• 0= “saturated”, 1=“monounsaturated”
O
2 or more = “polyunsaturated”
2
3
..n..
CH 2 CH 2 (CH2)n
H
• 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

CH 3
Fish or  -3 PUFA Intake
Reduces Heart Disease Risk
•
•
•
•
•
•
•
•
•
•
Dart Study
MRFIT
Indian Heart Study
Lyon Diet Heart Study
Lancet 1989; : 757-761
Proc Soc Exp Biol Med 1992; 200: 177-82
Br Med J 1992; 304: 1015-19
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
GISSI PREVENZIONE TRIAL
n=5666, 850mg omega-3 PUFA, 3.5 years
% of Patients
100
-15%
-20%
80
-45%
60
40
20
NO OMEGA-3 0
OMEGA-3
CV
Events
Heart
Attack
Marchioli R et al. , Lancet 1999 354: 457-455
Total
Mortality
Circulation 2002; 105: 1897-1903
Omega-3 PUFA Attenuates the Rise in
Arterial Blood Pressure due to Age or Omega-6 PUFA
Systolic Pressure (mmHg)
180
*
160
*
140
*
120
80
6 mo
24 mo
60
n=8 SD rats
100
40
20
0
Omega-6 PUFA
PEPE, et al, 1999
Omega-3 PUFA
Mitochondrial Membrane PUFA
 -6 PUFA
Age (mo)
 -3 PUFA
6
24
6
24
16:0
10.0±0.29
14.0±0.69*
9.5±0.35
11.0±0.64
18:0
23.0±1.15
26.0±1.73
10.1±0.72A
12.0±0.81A
16:1
0.22±0.05
0.3±0.12
0.7±0.10
0.8±0.05
18:1
9.0±0.81
9.0±0.43
9.5±0.40
10.0±0.58
17.0±1.21
11.0±1.44*
12.0±0.58A
24.0±1.44
32.0±1.79*
9.2±0.64A
12.0±0.58A
1.1±0.18
1.6±0.15
0.1±0.03
0.1±0.01
0.2±0.03
0.2±0.03
1.8±0.06
2.0±0.17
0.1±0.03
0.1±0.05
6.0±0.17A
5.2±0.29A
2.0±0.12
0.8±0.12*
2.2±0.05
5.0±0.58A
9.0±0.64
2.0±0.43*
27.0±1.01A
30.0±1.15A
18:2 ( -6) LA
20:4 ( -6) AA
22:4 ( -6)
18:3 ( -3)
20:5 ( -3) EPA
22:5 ( -3)
22:6 ( -3) DHA
  -6
42.1±0.85
  -3
11.3±0.69
PEPE, et al, 1999
44.6±1.58
3.1±0.44*
9.0±0.69
21.3±0.50A 1.1±0.33A
37.1±1.1A 42.2±1.35A
Values (mol/100mol) are presented as mean ±SEM, n=5.
*= P<0.05 vs 6 mo; A = P<0.05 vs n-6 PUFA
-3 : -6 PUFA
-3 : -6 PUFA
A
A
2
1.5
6mo
24mo
1
0.5
*
0
Omega-6 PUFA
PEPE, et al, 1999
Omega-3 PUFA
*=p<0.05 vs 6mo; A p<0.05 vs-6 PUFA; n=5
Mitochondrial Membrane
Phospholipids
 -6 PUFA
Age (mo)
6
DPG 12±1.0
 - 3 PUFA
24
8.5±0.9
*
*
6
24
15±0.9
14±0.8 A
42±1.8
42±1.6 A
PC
46±1.5
53±1.7
PE
35±1.8
33±1.4
38±1.9
40±1.8
PS
2.4±0.7
2.5±0.5
2.2±0.2
2.4±0.3
PI
1.3±0.2
1.4±0.3
1.1±0.4
1.2±0.6
*=p<0.05 vs 6mo; A = p<0.05 vs  -6 PUFA
PEPE, et al
mean +/- SD; n=5; % of total mitochondrial phospholipids
Cardiolipin (DPG)
20
% Total Phospholipids
6mo
24mo
A
15
10
*
5
0
Omega-6 PUFA
PEPE, et al
Omega-3 PUFA
*=p<0.05 vs 6mo; A=p<0.05 vs-6 PUFA; n=5
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
ADP-ATP carrier
phosphate carrier
pyruvate carrier
carnitine carrier
NADH dehydrogenase
succinate dehydrogenase
complex III
cytochrome C oxidase
ATP synthase
Intermembrane space
creatine kinase
cytochrome C
Cardiolipin
Q
ANT
O•
O•
Krebs Cycle (PDH, KGDH)
The Inner Mitochondrial Membrane
The Post-Ischemic Decline in Contractile
Recovery is Attenuated by Omega-3 PUFA
% Control PSP (Normoxia)
100
80
I/R
I/R+RR
A
A
A
*
60
40
A
*
20
0
6
24
Omega-6 PUFA
PEPE, et al, 1999
6
24
Omega-3 PUFA
*=p<0.05 vs 6mo; A p<0.05 vs  -6 PUFA; n=6
Increased Omega-3 PUFA Incorporation into
Cardiac Membranes Augments
Cardiac Efficiency AND prevents Fatal VF
Incidence of
Ventricular Arrhythmia
100
7.5
5.0
2.5
% Ventricular
Fibrillation
(mL/min/g dry wt)
normalised per unit work
MVO 2
Myocardial
Oxygen Consumption
0.0
0
omega-6 PUFA
Pepe & McLennan, 2002
50
omega-3 PUFA
Mitochondrial Calcium
2.5
C
I/R
I/R+RR
Total [Ca 2+]
nmol/mg protein
2
1.5
1
0.5
0
6
24
Omega-6 PUFA
PEPE, et al, 1999
6
24
Omega-3 PUFA
Pyruvate Dehydrogenase Activation
100
PDHA (% Total PDH)
90
C
I/R
I/R+RR
80
70
60
50
40
30
20
10
0
6
24
Omega-6 PUFA
PEPE, et al, 1999
6
24
Omega-3 PUFA
ADP:O Efficiency Ratio
A
A
2
ADP:0
1.5
1
*
6 mo
24 mo
p<0.01 vs 6mo
*A p<0.01 vs  -6
0.5
0
Omega-6 PUFA
PEPE, et al, 1999
Omega-3 PUFA
37C by oxidation of pyruvate (5mM)+ malate (0.5mM)
during complete conversion of 0.5mM ADP to ATP
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 [Ca]2+
values are higher after omega -6 PUFA-rich diet and is
augmented by age.

omega-3 PUFA -rich diet attenuates ALL of these effects.
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.
Conclusions
 Evidence for thermodynamic inefficiency in omega -6 PUFA
or aging compared to omega -3 PUFA
2+
 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.
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
PUFA
ROS
alkanes
conjugated dienes
alkoxyl radicals
aldehydes
(MDA
4HNE)
lipid hydroperoxides
peroxyl radicals
isoprostanes
hydroxylated FA
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
-Inactivates glutathione peroxidase
(inhibits glutathione recycling)
-Apolipoprotein B adducts in atherosclerosis
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
Omega-3 PUFA Reduces
Post-Ischemic Mitochondrial 4-HNE
Omega-6 PUFA
PEPE, et al
Omega-3 PUFA
Conclusions
 4-HNE a marker of free radical-induced peroxidation of
omega-6 PUFA is increased in the post-ischemic
myocardium of senescent rats vs young, due to the agerelated differences in total omega-6 PUFA.
Conclusions
 Replacing membrane omega-6 PUFA with omega-3
PUFA reduces the amount of 4-HNE formed during
ischemia and reperfusion, abolishing age-associated
differences in 4HNE formation.
This may impact pathogenesis and the consequences of
cardiac ischemia.
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
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.
Mitochondrial Membrane Permeability Transition
Confocal measurement of in situ mitochondrial
membrane potential and ROS using TMRE and DCF
in real time
TMRM fluorescence (mitochondrial membrane potential)
Isolated Mitochondria
Modulators of Mean Time to Mito Membrane
Permeability Transition
SCAVENGING ROS INHIBITS THE MPT
MITOCHONDRIA OF SENESCENT CARDIOCYTES ARE
MORE SUSCEPTIBLE TO ROS; RESCUE BY CoQ10
Total Coenzyme Q10 Concentration in Cardiac
Myocytes from 6 and 24 mo S-D rats
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