Mechanisms of cardiac ischemia-reperfusion injury and cardioprotection

National Heart, Lung, And Blood Institute

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Abstract

The goal of this project is to 1) understand the role of mitochondria in ischemia-reperfusion injury and cardiioprotection and 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection. To accomplish these goals we study ischemia reperfusion and cardioprotection in a Langendorff perfused heart model. We follow changes in high energy phosphates and ions using nuclear magnetic resonance and fluorescent measurements. We also measure contractility and infarct size using standard methods. We also isolated heart extracts and mitochondria and correlate changes in ischemia-reperfusion injury and cardioprotection with changes in protein levels, protein location and protein post translational modifications. We performed studies to examine the role of nitric oxide in cardioprotection. Nitric oxide has been shown to be an important signaling messenger in ischemic preconditioning (IPC). Accordingly, we investigated whether protein S-nitrosylation occurs in IPC hearts and whether S-nitrosoglutathione (GSNO) elicits similar effects on S-nitrosylation and cardioprotection. Preceding 20 minutes of no-flow ischemia and reperfusion, hearts from C57BL/6J mice were perfused in the Langendorff mode and subjected to the following conditions: (1) control perfusion; (2) IPC; or (3) 0.1 mmol/L GSNO treatment. Compared with control, IPC and GSNO significantly improved postischemic recovery of left ventricular developed pressure and reduced infarct size. IPC and GSNO both significantly increased S-nitrosothiol contents and S-nitrosylation levels of the L-type Ca2+ channel alpha1 subunit in heart membrane fractions. We identified several candidate S-nitrosylated proteins by proteomic analysis following the biotin switch method, including the cardiac sarcoplasmic reticulum Ca2+-ATPase, alpha-ketoglutarate dehydrogenase, and the mitochondrial F1-ATPase alpha1 subunit. The activities of these enzymes were altered in a concentration-dependent manner by GSNO treatment. We further developed a 2D DyLight fluorescence difference gel electrophoresis proteomic method that used DyLight fluors and a modified biotin switch method to identify S-nitrosylated proteins. IPC and GSNO produced a similar pattern of S-nitrosylation modification and cardiac protection against ischemia/reperfusion injury, suggesting that protein S-nitrosylation may play an important cardioprotective role in heart.[unreadable] We also examined mechanisms by which inhibition of glycogen synthase kinase (GSK) mediates cardioprotection. Inhibition of GSK-3 reduces ischemia-reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to determine the molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSKninhibitors prior to ischemia, significantly improved recovery of function. To assess the effect of GSKninhibitors on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSKninhibitors and treated with uncoupler or cyanide, or were made anoxic. We found that GSKninhibition slowed ATP consumption under these conditions, which could be due to inhibition of ATP entry into the mitochondria through VDAC and/or ANT or to inhibition of the F1F0 ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer but not the inner mitochondrial membrane, and we found that GSKninhibitors slow AMP production similar to their effect on ATP consumption. This suggests that GSKninhibitors are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not due to opening of the mitochondrial permeability transition pore. Since GSKnis a kinase, we wanted to determine if protein phosphorylation might be involved. Therefore, we performed western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSK inhibitors. LC/MS analysis revealed one of these proteins to be VDAC2. Taken together, we found that GSK mediated signaling modulates transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest that GSK regulates VDAC and suggest that VDAC may be an important regulatory site in ischemia-reperfusion injury.[unreadable] Most of these studies showing that inhibition of GSK is protective, however, were performed in rats. We therefore did additional studies to determine whether GSK- inhibition mimicked the protective effects of PC in mice. Langendorff murine hearts were treated with a specific GSK-3 inhibitor AR-A014418 (GSK Inhib VIII) for 10 min or subjected to 4 cycles of 5-min ischemia/reperfusion (PC) prior to a 20-min global ischemia and 120-min reperfusion. PC and GSK had improved post-ischemic LVDP recovery compared to their time-matched controls (57.0 2.5 and 51.7 3.8% vs. 33.6 2.4% of their pre-ischemic LVDP). Consistent with improved functional recovery, infarct size in PC and GSK Inhib VIII hearts was decreased relative to control (11.9 1.6 and 19.2 3.5 vs. 34.8 1.9%). Lactate levels were measured in hearts snap-frozen after a 20-min ischemic period and found to be significantly lower in PC and GSK Inhib VIII-treated groups (6.69 0.73 and 8.30 0.29 moles lactate/g wet weight) with respect to control (10.34 0.67 moles lactate/g wet weight). We used a comparative proteomics analysis to look at mitochondrial protein expression/phosphorylation changes that were present only in PC and GSK Inhib VIII-treated, but not in control. Levels of ATP synthase e, cytochrome c oxidase subunit VIb, ATP synthase coupling factor 6, cytochrome c oxidase subunit 5A, and cytochrome b-c1 complex subunit were increased while cytochrome c was decreased in PC and GSK Inhib VIII. These changes were not the result of alterations in protein expression and but post-translational modifications. PC and GSK Inhib VIII hearts had reduced levels of phospho-Tyr cytochrome c as measured with Western blotting. Two-dimensional Blue Native gels revealed that complex V had significantly less Ser/Thr phosphorylation in PC and GSK Inhib VIII-treated hearts. Data also suggest that PC cardioprotection increase the amount of the complex IV in the supercomplex assembly of the electron transport chain. The composition of the superstructure is reported to play a role in electron transport flow, generation of reactive oxygen species and mitochondrial morphology. Thus the ability of PC and GSK to alter post-translational modifications of the electron transport complexes and to alter supercomplex composition will have important implications for mitochondrial function.