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

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. Support or Funding Information The authors' work was supported by the Health and Medical Research Fund (04151816, Hong Kong) and General Research Fund (17117217M, Research Grants Council of Hong Kong). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .