The Role of Caspase Inhibitors in Protecting the Myocardium from Ischemia Reperfusion Injury

  • Hajar Mohammed Amor al-Rajaibi

    Student thesis: Doctoral ThesisDoctor of Philosophy

    Abstract

    Introduction
    Rapid restoration of blood flow to ischemic myocardium is essential, however it causes further injury called reperfusion injury. Apoptosis contributes significantly to cardiomyocyte cell death during ischemia reperfusion injury, in which caspase family
    proteases play an essential role as they are the executioners of apoptosis. Caspase inhibitors showed promising cardioprotective results when administered before ischemia or at the start of reperfusion. However, before applying them in pre clinical studies of myocardial ischemia, several investigations needed to be taken to determine their therapeutic window post reperfusion, their effect on functional recovery of myocardium post ischemia, their mechanism of action.

    Methods
    Isolated perfused rat hearts were subjected to 35 min ischemia followed by 2 hr reperfusion where caspase inhibitors [broad spectrum caspase inhibitor (ZVAD, 0.1µM), specific caspase 3 inhibitor (DEVD, 0.07µM)] were added at the start of reperfusion, 15, 30 and 60 min after starting reperfusion at the presence or absence of Wortmannin (WORT, 100nM, PI3-kinase inhibitor). Hearts underwent triphenyl tetrazolium staining for infarct size
    assessment, or were frozen for Western blot analysis. Freshly isolated adult rat ventricular myocytes were subjected to 6 hr hypoxia followed by either 18 hr, where caspase inhibitors
    (ZVAD, 25µM and DEVD, 25µM)] were added at the start of reoxygenation, 15, 30 and 60 min after starting reoxygenation at the presence or absence of Wortmannin (WORT,100nM). Cardiomyocytes were analysed for viability, apoptosis, necrosis and intracellular caspase-3 activity using flow cytometry analysis. Isolated adult rat ventricular papillary muscles were subjected to 35 min hypoxia followed by 100 min reperfusion where caspase
    inhibitors [ZVAD (0.1 µM, 2.5µM) and DEVD (2.5µM)] were added at the start of reperfusion throughout. Power output was measured using work loop technique.

    Results
    Both caspase inhibitors significantly reduced infarcted tissue within the risk zone, apoptotic, necrotic cellular death and intracellular caspase 3 activity level when administered at the start of reperfusion, 15, 30 and 60 min after initiation of reperfusion.
    Work loop studies showed that the broad spectrum caspase inhibitors at high dose of 2.5µM significantly improved post ischemic power out put recovery, however the specific caspase
    3 inhibitor has no significant effect on power output recovery post ischemia. Inhibition of PI3-Akt by WORT at the start of reperfusion abrogated the cardioprotective effect of both caspase inhibitors. However, WORT did not block the cardiprotection of both caspase
    inhibitors when administered at 15 min and 30 min after starting reperfusion. Western blot analysis showed that cytochrome c is released to cytosol during ischemia/reperfusion injury; however both caspase inhibitors failed to inhibit cytochrome c release during reperfusion injury at all time points during reperfusion. Western blot showed a significant increase in phospho-Akt at 5 min and 10 min reperfusion in both caspase inhibitors treated
    groups compared to ischemic control group. However there was no significant increase at 20 min and 120 min reperfusion when compared to ischemic control. WORT blocked the increase in phospho-Akt at 10 min reperfusion.

    Conclusion
    This study is the first to show that administration of caspase inhibitors at various time points post reperfusion can still reduce myocardial injury, which implies that the therapeutic window of caspase inhibitors in myocardial infarction treatment can be
    extended for an hour after starting reperfusion. It also showed that broad inhibition of caspase resulted in a significant functional recovery post ischemia using for the first time work loop technique to measure the power output. This study is also the first to show that cardioprotection by caspase inhibitors at early reperfusion phase is mediated via PI3-Akt cell survival pathway.
    Date of Award2008
    Original languageEnglish
    Awarding Institution
    • Coventry University
    SupervisorHelen Maddock (Supervisor) & Rob James (Supervisor)

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