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Heart Mitochondrial Cardiolipin Oxidation
during Ischemia in the Aged Rat
E. Lesnefsky, P. Minkler,
Charles Hoppel
Case School of Medicine
Louis Stokes VA Medical Center
Edward Lesnefsky, M.D.
Paul Minkler
Shadi Moghaddas, Ph.D.
Bernard Tandler, Ph.D.
Qun Chen, M.D., Ph.D.
Medhat Hassan, M.D.
Edwin Vazquez
Alessandro Riva, M.D., Ph.D.
Funding:
NIA PO1 AG15885
Aging Heart: Basis of Increased Ischemic/Reflow Injury
Heart Mitochondrial Cardiolipin Oxidation
during Ischemia in the Aged Rat
• Introduction
• Mitochondria in aging heart
• Mitochondrial Cardiolipin – ischemia in aging
heart
• Summary
Increased Injury in Aging Heart
• Aging hearts showed increased injury:
– Decreased hemodynamic recovery
– Increased CPK release-increased tissue damage
• Aging myocardium has an increased intrinsic
susceptibility to damage during ischemia and
reperfusion
Heart Mitochondrial Cardiolipin Oxidation
during Ischemia in the Aged Rat
• Introduction
• Mitochondria in aging heart
• Mitochondrial Cardiolipin – ischemia in aging
heart
• Summary
Cardiac Mitochondria Exist in Two Populations:
Subsarcolemmal Mitochondria (SSM)
and
Interfibrillar Mitochondria (IFM)
Cardiomyocyte
Subsarcolemmal Mitochondria under cell membrane
contractile
proteins
nucleus
Interfibrillar
Mitochondria in
contractile regions
Palmer et al. J Biol Chem 1977;252:8731-8738
Heart Mitochondrial Cardiolipin Oxidation
during Ischemia in the Aged Rat
• Problem
• Mitochondria in aging heart
• Mitochondrial Cardiolipin – ischemia in aging
heart
• Summary
Quantification and Characterization of Cardiolipin
Folch
Extract
Silica
Gel
Series of iso -octane:
ethyl acetate: HoAC
washes
Lipids
100% MEOH
Phospholipids
(PE, PI, CL , PC)
Normal Phase
HPLC
Phospholipids
(PE, PI, CL, PC)
NP-HPLC separation of PPL
PE
PI
CL
PC
= phosphatidylethanolamine
= phosphatidylinositol
= cardiolipin
= phosphatidylcholine
Reverse Phase-HPLC
separation of CL
PE
OD 206 nm
800
600
CL
400
200
PC
PI
0
10
20
30
40
50
Time, min
60
70
80
90
100
mAbs
100
RP-HPLC
separation of CL
1448
80
60
40
UV
20
0
0
5
10
15
20
25
30
35
Time (min)
Adult, 6mo IFM
9.91
1448
Relative Abundance
100
4 C18:2
TIC
3 C18:2
1 C18:1
50
3 C18:2
1 C18:0
1450
1452
0
2
4
6
8
10
12
14
16
18
20
Time (min)
22
24
26
28
30
Moghaddas et al.
J Gerontol Biol Sci
2002: 57:B22-28.
57:B22-28.
32
34
Aging Does not Alter Content of Cardiolipin
P=NS vs 6mo,  SEM
nmol/mg protein
60
SSM
IFM
50
40
30
20
10
0
Moghaddas et al. J Gerontol Biol Sci 2002: 57:B22-28.
Aging Does Not Alter Composition of Cardiolipin
RP-HPLC followed by Electrospray Ionization Mass Spectrometry
Separation of Cardiolipin Molecular Species
9.86
1448
100
(B) Aged, 24mo IFM
(A) Adult, 6mo IFM
9.91
1448
100
Relative Abundance
4 C18:2
50
3 C18:2
1 C18:1
1450
50
3 C18:2
1 C18:0
1452
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
18
20
22
1450
1452
0
2
4
6
8
10
12
14
16
Time (min)
24
26
28
30
32
Moghaddas et al. J
34
Gerontol Biol Sci 2002: 57:B22-28 .
Content of Cardiolipin is Unaltered by Ischemia and
Reperfusion
in Adult and Aged Hearts
nmol/mg protein
60
SSM
IFM
p=NS, SEM
50
40
30
20
10
0
6mo
n=5
24mo
n=7
6mo
n=5
24mo
n=7
Ischemia-Reperfusion Increases Relative
Amount of Oxidized Cardiolipin in Aged Heart
m/z
1448
105
Relative Abundance
100
95
UV, OD 206nm
90
85
80
75
Control, IFM
I-R, IFM
70
65
m/z
1496
60
55
50
45
40
35
30
25
20
15
10
5
6
7
8
9
1
Ratio peak Area @OD206
6mo, n=4
0.8
10
11
12
13
14
15
Time (min)
*p<0.05vs 6mo, SD
24mo, n=6
0.6
*
*
0.4
0.2
0
SSM
IFM
• OD 206 peak area ratio =
1496 peak /1448 peak
• Peaks identified by
subsequent MS
Ischemia Alone Increases Relative Amount of
Oxidized Cardiolipin in Aged Heart
6mo, I-R n=4
Ratio Peak Area @OD206
0.8
24mo, I-R n=6
24mo, Isch n=4
0.6
*p<0.05vs 6mo, SD
*
0.4
*
*
*
0.2
0
SSM
IFM
MSn Analysis of Cardiolipin
-
O
1486.0
100
O
1448.0
80
MS
60
Relative Abundance
40
20
Native
0
22
P
OH
O
O
O P
H
O
O
O
O
869.5
15
MS21486m/z
10
C81H140O
H
O
O
O
O
O
O
O
2.5
2.0
1.5
1.0
0.5
0.0
12P2
= 869.4
C24H42O
11P2
= 607.2
C21H36O6P = 415.2
415.3
MS31486,869.5m/z
607.2
278 400
600
•
•
•
•
•
800
1000
m/z
1200
1400
1600
1800
= 1448
C42H72O
5
0
3.4
17P2
2000
m/z 1448: Intact cardiolipin (1496: K+ adduct)
m/z 869: glycero-phosphatidic acid fragment
m/z 607: phosphatidic acid
m/z 415: lyso-phosphatidic acid
m/z 278, 280: acyl-groups
MS Analysis of Oxidized Cardiolipin
80
60
-
1534 (1496+K)
100
O
1496.0
Oxidized
O P
MS
3
869.5
O
917.4
415.3
326
453
MS3
655.1
607.4
400
600
800
1000
m/z
1200
1400
1600
1800
2000
O
HOO
•
•
MSn localizes the +48 amu to one acyl-group
m/z 1496: Intact oxidized cardiolipin (1448+48)
m/z 917: oxidized glycero-phosphatidic acid
(869+48)
m/z 655: oxidized phosphatidic acid (607+48)
m/z 453: oxidized lyso-phosphatidic acid (415+48)
• m/z 326: oxidized linoleic acid (278+48)
O
O
H
O
O
O
O
MS2
1
•
•
O P
O
2
0
O
O
20
4
OK
H
40
0
30
25
20
15
10
5
0
5
OH
O
O
MS Analysis of Oxidized Cardiolipin:
Evidence of Linoleic Acid Oxidation
Oxidized
Native
-
O
O
O
-
P
OH
OK
O
O
O
P
O
O
O
O
H
O
O
O
O
O
O
O
O
O
O
O P
H
H
O
O
OK
O
O
H
O
O P
O
OH
O
O
oxidation
O
HOO
4 C18:2 = m/z 1448
4 C18:2 + 3 O = m/z 1496
Ischemia Increases Production of Oxidized
Cardiolipin in Aged Heart
100
95
90
HPLC/MS 1496 m/z RIC
85
80
75
Relative Abundance
70
65
24mo 25min Ischemia, IFM
60
55
6mo 25min Ischemia, IFM
50
45
40
24mo IFM
35
30
6mo IFM
25
20
15
10
5
0
2
4
6
8
10
12
14
16
18
Time (min)
20
22
24
26
28
30
32
34
Heart Mitochondrial Cardiolipin Oxidation
during Ischemia in the Aged Rat
• Problem
• Mitochondria in aging heart
• Mitochondrial Cardiolipin – ischemia in aging
heart
• Summary
Conclusions
Oxidative damage to cardiolipin occurs in the ischemicreperfused aged, but not the adult heart.
Ischemia alone is sufficient to induce oxidative damage
to cardiolipin in the aged rat heart.
Cardiolipin modification provides evidence of
enhanced oxidative damage to mitochondria during
ischemia in the aged heart.