The Role Of CaMK? In Ischemia-induced Cardiac Angiogenesis

Background Ischemic heart disease(IHD)is the primary cause of death in cardiovascular diseases.Although the prognosis has been significantly improved by percutaneous coronary intervention,there are still considerable patients who are not suitable for the intervention.Coronary angiogenesis is a compensatory coronary collateral vessel growth which significantly improves blood supply in the ischemic area,reduces the infarct size and mortality in patients with myocardial infarction.Considerable studies have been conducted to investigate the mechanisms of coronary angiogenesis in order to develop better therapeutic strategies for IHD.However,the underlying mechanism remains largely unclear.CaMKII is a kind of serine/threonine protein kinase,which is regulated by Ca2+.CaMKII is excessively activated in ischemic heart disease,that mediates the inflammation and ischemia/reperfusion damage.However,whether it participates in the coronary angiogenesis is still unknown.Here,we investigated the role of CaMKII activation in ischemia-induced cardiac angiogenesis to figure out the underlying mechanism of angiogenesis.Methods 1)6 to 8-week-old male C57BL/6 mice at weight of 20-22g were used to build the repetitive transient ischemia murine model.ECG and echocardiography were used to check the model.HE staining was carried out to observe the pathological change of heart.?-SMA,CD31 and vWF immunohistochemical staining were conducted to detect the coronary angiogenesis.2)The mice were divided into sham,ischemia,ischemia+KN93,ischemia+KN92 group.Echocardiography was used to evaluate the cardiac function,RT-qPCR and Western blot were conducted to detect the expression of CaMKII and VEGF,immunofluorescence staining was used to detect the expression of CD31 and Ki67.3)We isolated the primary cardiac microvascular endothelial cells(CMECs)to establish the hypoxia endothelial model.After the intervention of KN93/KN92,we detected the proliferation and migration of endothelium by MTT and transwell experiment.Also,the protein expression of CaMK?,p-CaMK? and VEGF were tested by Western blot.Results 1)ST-segment elevation in leads ? and aVL were observed during ischemia.Cardiac function was unchanged during the whole ischemic process.a-SMA,vWF and CD31 were increased remarkably detected by immunofluorescence staining indicating that we established a repetitive transient ischemia murine model.2)Angiogenesis was induced by ischemia and suppressed by the intervention of CaMKII inhibitor KN93.RT-qPCR and Western blot analyses showed that myocardial ischemia induced an increasing expression and phosphorylation of CaMK?.Moreover,VEGF expression was increased in the ischemic model and blunted by KN93.3)KN93 suppressed the proliferation and migration of cardiac endothelial cells in hypoxic condition,in which the expression of p-CaMK? and VEGF were remarkably increased.Conclusions 1)Increasing expression and phosphorylation level of CaMKII were detected in the ischemia-induced coronary angiogenesis model.2)Experiments in vitro and in vivo indicated that CaMK? triggered the expression of VEGF and thus played a role in ischemia-induced coronary angiogenesis.3)Ischemia increased CaMKII expression in myocardium but not in the endothelial cells.