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Heart Mitochondrial Cardiolipin Oxidation during Ischemia in the Aged Rat

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.

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Heart Mitochondrial Cardiolipin Oxidation during Ischemia in the Aged Rat

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  1. 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

  2. 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

  3. Heart Mitochondrial Cardiolipin Oxidation during Ischemia in the Aged Rat • Introduction • Mitochondria in aging heart • Mitochondrial Cardiolipin – ischemia in aging heart • Summary

  4. 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

  5. Heart Mitochondrial Cardiolipin Oxidation during Ischemia in the Aged Rat • Introduction • Mitochondria in aging heart • Mitochondrial Cardiolipin – ischemia in aging heart • Summary

  6. Cardiac Mitochondria Exist in Two Populations: Subsarcolemmal Mitochondria (SSM) and Interfibrillar Mitochondria (IFM) Cardiomyocyte Subsarcolemmal Mitochondria under cell membrane contractileproteins Interfibrillar Mitochondria in contractile regions nucleus Palmer et al. J Biol Chem 1977;252:8731-8738

  7. Heart Mitochondrial Cardiolipin Oxidation during Ischemia in the Aged Rat • Problem • Mitochondria in aging heart • Mitochondrial Cardiolipin – ischemia in aging heart • Summary

  8. Quantification and Characterization of Cardiolipin Quantification and Characterization of Cardiolipin Folch Silica Series of iso -octane: ethyl acetate: HoAC Extract Gel washes Lipids 100% MEOH Phospholipids (PE, PI, CL , PC) Normal Phase Normal Phase HPLC HPLC

  9. Aging Does not Alter Content of Cardiolipin Aging Does not Alter Content of Cardiolipin ± ± P=NS vs 6mo, SEM P=NS vs 6mo, SEM 60 60 SSM IFM 50 50 40 40 nmol/mg protein nmol/mg protein 30 30 20 20 10 10 0 0 6mo(n=9) 6mo(n=9) 24mo(n=10) 6mo(n=9) 24mo(n=10) 6mo(n=9) 24mo(n=10) 24mo(n=10) Moghaddas et al. J Gerontol Biol Sci 2002: 57:B22-28.

  10. Aging Does Not Alter Composition of Cardiolipin RP-HPLC followed by Electrospray Ionization Mass Spectrometry Separation of Cardiolipin Molecular Species 9.86 4 C18:2 1448 100 (B) Aged, 24mo IFM (A) Adult, 6mo IFM 9.91 1448 100 50 3 C18:2 1 C18:1 3 C18:2 1450 Relative Abundance 1 C18:0 50 1452 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 1450 1452 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Time (min) Moghaddas et al. J Gerontol Biol Sci 2002: 57:B22-28 .

  11. Content of Cardiolipin is Unaltered by Ischemia and Reperfusion in Adult and Aged Hearts p=NS, SEM IFM SSM 60 50 40 nmol/mg protein 30 20 10 0 24mo n=7 24mo n=7 6mo n=5 6mo n=5

  12. Ischemia-Reperfusion Increases Relative Amount of Oxidized Cardiolipin in Aged Heart 1 *p<0.05vs 6mo, SD 6mo, n=4 0.8 24mo, n=6 * 0.6 * Ratio peak Area @OD206 0.4 0.2 0 SSM IFM • OD 206 peak area ratio = 1496 peak /1448 peak • Peaks identified by subsequent MS

  13. 6mo, I-R n=4 24mo, I-R n=6 24mo, Isch n=4 Ischemia Alone Increases Relative Amount of Oxidized Cardiolipin in Aged Heart *p<0.05vs 6mo, SD 0.8 * * 0.6 * * 0.4 Ratio Peak Area @OD206 0.2 0 SSM IFM

  14. Oxidized Oxidized - O O H O K - O O H O K O P O O P O O P O O P O O O O O H H H H O O O O O O O O O O O O O O O O oxidation oxidation O H O O 4 C18:2 + 3 O = m/z 1496 4 C18:2 + 3 O = m/z 1496 MS Analysis of Oxidized Cardiolipin: MS Analysis of Oxidized Cardiolipin: Evidence of Evidence of Linoleic Linoleic Acid Oxidation Acid Oxidation Native Native 4 C18:2 = m/z 1448 4 C18:2 = m/z 1448

  15. 100 95 90 85 80 75 24mo 25min Ischemia, IFM 70 65 60 55 Relative Abundance 6mo 25min Ischemia, IFM 50 45 24mo IFM 40 35 30 6mo IFM 25 20 15 10 5 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Time (min) Ischemia Increases Production of Oxidized Cardiolipin in Aged Heart HPLC/MS 1496 m/z RIC

  16. Heart Mitochondrial Cardiolipin Oxidation during Ischemia in the Aged Rat • Problem • Mitochondria in aging heart • Mitochondrial Cardiolipin – ischemia in aging heart • Summary

  17. Conclusions • Oxidative damage to cardiolipin occurs in the ischemic-reperfused 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.

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