Rap1 exacerbates myocardial ischemia/reperfusion injury through enhancing cell apoptosis and inflammatory response

Abstract

Ischemic heart disease caused by partial or complete blockage of coronary arteries is a leading cause for morbidity and mortality worldwide. Repressor activator protein 1 (Rap1), an established telomere-associated protein, is a novel modulator in hypoxia-induced apoptosis and NFκB-mediated inflammatory response, both of which are involved in the pathophysiology of myocardial ischemia/reperfusion injury (I/RI). Thus, the present study aimed to explore whether or not Rap1 mediates cardiac I/RI in cell and animal models and to dissect its molecular mechanism. In a mice cardiac I/RI model (30 min left descending coronary artery ligation followed by 2 hours reperfusion), the protein expression of Rap1 in the heart was significantly increased (P <0.05 vs. Sham group). Deletion of Rap1 in mice significantly attenuated myocardial I/RI, as evidenced by reduced infarction size (Evans blue/TTC staining), reduced circulating levels of troponin I and creatine phosphokinase-MB (cardiac injury markers, P <0.05 vs. Wild-type mice). These changes were associated with reduced apoptosis (increased Bcl2/Bax ratio, reduced cleave caspase-3 level and TUNEL staining) and inflammatory responses [reduced mRNA levels of pro-inflammatory cytokines (IL1β, IL6 and TNFα) and infiltration of F4/80 positive cells (macrophage specific marker)] in the ischemic heart of Rap1 knockout mice (P<0.05 vs. Wild-type mice). In H9C2 cardiomyocytes, hypoxia/reoxygenation (H/R, 6 hours hypoxia followed by 12 hours reoxygenation) significantly increased both mRNA and protein levels of Rap1 (P <0.05 vs. Control). Rap1 knockdown significantly suppressed H/R-induced cell injury [reduced lactic acid dehydrogenase (LDH) leakage and increased cell viability] when compared to mock-transfected H9C2 cardiomyocytes. In addition, Rap1 knockdown significantly suppressed cell apoptosis in response to H/R (increased Bcl2/Bax ratio, reduced cleave caspase-3 level and TUNEL staining) through its ability to increase the phosphorylation/activation of STAT3 at site Ser727, as stattic (selective STAT3 inhibitor) pre-treatment canceled the abovementioned protective effect mediated by Rap1 knockdown. In line with this, Rap1 deficiency in mice also significantly increased the protein levels of p-STAT3 (Ser727) in the ischemic heart (P <0.05 vs. Wild-type mice). Furthermore, in differentiated THP-1 macrophages, Rap1 knockdown significantly suppressed lipopolysaccharide-induced expression of NFκB-mediated pro-inflammatory cytokines (IL1β, IL8 and MCP1). However, Rap1 knockdown did not influence the expression of NFκB-dependent targets in H/R-stimulated H9C2 cells, suggesting that the effect of Rap1 on NFκB-mediated inflammatory response is specific to macrophages. In conclusion, these data indicated that Rap1 can exacerbate myocardial I/RI through enhancing cell apoptosis via inhibiting STAT3 signaling in cardiomyocytes and also augmenting inflammatory response via up-regulation of macrophages infiltration and pro-inflammatory mediators. Thus, Rap1 may represent a novel therapeutic target for myocardial I/RI.