Inhibition Of COX‐2/PGE2/EP4 Signaling Protects Against Post‐hypoxic Apoptosis 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.Support or Funding InformationThis study was supported by General Research Fund (17124614, and 17123915) from the Research Grants Council of Hong Kong and by National Natural Science Foundation of China (NSFC 81270899).