Inhibition of COX-2/PGE2/EP4 signaling protects against cell apoptosis during hypoxia/reoxygenation in H9C2 cardiomyocytes

Abstract

Myocardial infarction is a condition associated with significant mortality. Reperfusion therapy restores blood flow, it may also induce lethal tissue injury, known as ischemia-reperfusion injury (IRI) in clinical practice. Thus, exploring ways to control or attenuate IRI is of clinical interest for improving post-ischemic recovery. Cyclooxyenase-2 (COX-2) – which is induced during ischemia - is highly expressed at the site of recent acute myocardial infarction (Heart. 2004;90:440–443). Furthermore, the presence of COX-2 is positively correlated with progressively increased apoptosis which played a central role in the pathophysiology of IRI. However, whether there is causal link or underlying signaling cascade between ischemia-driven COX-2 expression and apoptosis in IRI is unclear. Thus, the present study aimed to investigate the molecular basis of potential cause-effect link between COX-2 expression and enhanced apoptosis during ischemia using rat origin cardiomyocytes (H9C2) subjected to hypoxia/reoxygenation (H/R, 6 hours hypoxia followed by 12 hours reoxygenation). The results showed that H/R significantly increased the protein expression of COX-2 (P<0.05 vs. Control), which was prevented by 2 hours pretreatment with Helenalin (NFkB inhibitor, at 10μM, P<0.05 vs. H/R) but not by pretreatment with SC-205346A [Hypoxia inducible factor alpha (HIF-1α) inhibitor, at 20μM]. This suggests that NFkB but not HIF-1α mediated the expression of COX-2 during H/R in H9C2 cardiomyocytes. Further, under the experimental setting, H/R induced H9C2 cells damage [evidenced by increased lactic acid dehydrogenase (LDH) leakage (marker of cell injury, P<0.05 vs. Control)] was concomitant with increased cleaved caspase-3 expression (marker of cell apoptosis, P<0.05 vs. Control) but no changes in the protein expressions of the anti-apoptotic Bcl-2 and pro-apoptotic Bax, suggesting that H/R induced caspase-3 dependent cell apoptosis which was independent of the intrinsic mitochondrial pathway. Pretreat the cells with NS398 (COX-2 selective inhibitor, at 10μM, 1 hour) or L161,982 [prostaglandin E receptor subtype 4 (EP4) selective antagonist, at 100nM, 1 hour] suppressed the H/R induced increases of LDH leakage and cleaved caspase-3 expression (P<0.05 vs. H/R), indicating that both COX-2 and EP4 mediated cell apoptosis during H/R. Given that prostaglandin E2 (PGE2) is a major metabolite of COX-2 and an endogenous ligand of EP4 receptor, H9C2 cardiomyocytes were exposed to exogenous PGE2 (from 5nM to 50μM, 18 hours) in a concentration dependent manner. As anticipated, PGE2, at a high concentration (50μM), significantly increased cellular injury as evidenced by enhanced LDH leakage (P<0.05 vs. Control). It is concluded that H/R may induce cardiomyocytes apoptosis via NFkB/COX-2/PGE2/EP4 signaling, which may serve as a novel target for anti-ischemic therapy.