Mechanism Study Of CKIP-1 Regulating Myocardial Ischemia Reperfusion Injury

Acute myocardial infarction (AMI) due to coronary artery occlusion results in acute myocardial ischemia and cardiomyocytes death. Reperfusion therapy is the most effective treatment to limit the infarct size and salvage the dying cardiomyocytes. However, reperfusion will induce secondary damage to the ischemia myocardium reffered to as reperfusion injury, which compromises the benefits of blood flow restoration. Cell death due to necrosis, apoptosis are responsible for the cardiomyocytes loss in myocardial infarction. Apoptosis has been recognized as a highly programmed process. Recently, data suggest that part of necrosis is also regulated in some instances called "necroptosis", which dependant on receptor-interacting protein kinase 3 (RIPK3). Necrotic cell death can also be driven by some key molecular pathways in a regulated manner.CKIP-1 is abundantly expressed in tissue and organs and is involved in regulating cytoskeleton actin, cell proliferation, differentiation, and apoptosis. Previous study showed that CKIP-1-knockout mice displayed spontaneous heart hypertrophy with aging and high sensitivity to pressure overload-induced cardiac hypertrophy, which indicated CKIP-1 is probably a protective factor in the adult heart. The role of CKIP-1 in myocardial I/R inury is still not known.This study established I/R model with CKIP-1 knockout (KO) mice and CKIP-1 transgene (TG) mice to test whether CKIP-1 is involved in the pathogenesis of mouse myocardial I/R injury and investigated the mechanisms.Methods and Results:(1) The hearts of CKIP-1 KO and littermates subjected or not to I/R injury was performed TTC/evens blue stain. The CKIP-1-deficient hearts exhibited significantly increase of myocardial infarction size and circulating plasma myocardial damage biomarker cTnT level, compared with WT hearts. Apoptosis induced by I/R in CKIP-1-deficient hearts, evaluated by TUNEL, showed no significant difference with WT hearts. The results indicate that CKIP-1 regulates necrosis other than apoptosis. To explore the effect of CKIP-1 on myocardial fibrosis and function following I/R 1 month, masson trichrome stain and echocardiography was performed. In comparison with WT mice, the ejection fraction and fractional shortening of CKIP-1-defeicient mice were decreased, the LVIDs and LVIDd of which were increased. Masson trichrome staining revealed increased fibrosis in the myocardial infarction border zone of CKIP-1-defeicient mice, compared with WT mice.(2) Since CKIP-1-knockout mice showed spontaneous heart hypertrophy with aging. Wether the myocardial damage following I/R was due to high sensitivity to ischemia because of cardiac hypertrophy or other mechanisms? Wether CKIP-1 is a protective factor of cardiovascular system? Wether overexpression of CKIP-1 could protect cardiomyocytes against of I/R injury? To answer scientific questions above, we repeated the experiments of part (1) with CKIP-1 TG mice to confirm the data produced by CKIP-1 KO mice. Consistant with the results of CKIP-1 KO mice, overexpression of CKIP-1 decreased the infarction area and cTnT level in comparison with WT mice.1 month after I/R, compared with WT mice, the ejection fraction and fractional shortening of CKIP-1 TG mice were increased, the LVIDs and LVIDd of which were decreased, masson trichrome staining revealed fibrosis in the myocardial infarction border zone of CKIP-1 TG mice was decreased.(3) The outcomes above indicate that CKIP-1 regulates necrosis other than apoptosis induced by myocardial I/R injury. FACS was performed to analyze cell death stained by annexin V/PI after treatment by TNFa+z-VAD for 5h. Compared with WT, overexpression of CKIP-1 decreased cell necrosis induced by T/z. QPCR analysis showed TNFa gene upregulation activated by T/z treatment was suppressed by CKIP-1 overexpression. Immunoprecipitation identified that CKIP-1 interacts with TRAF2, which is a regulator of TNFa synthesis. Knockdown of TRAF2 by siRNA in CKIP-1 overexpressed L929 cell lines blocked the inhibition of TNFa.Conclusion:Our results demonstrate that CKIP-1 is implicated in the regulation of I/R-induced cardiac injury and dysfunction via suppressing the expression of TNFa. The present study has identified critical roles of CKIP-1 in regulating cardiomyocyt necroptosis.