Investigation Of Calpain/Calpastatin Expression In Isolated Heart

With the science and technology advance,surgical level by leaps and bounds,Heart transplantation has become the effective treatment of end-stage heart disease,However,it still faces many problems,such as the serious imbalance between organ demand and the number of available organs,the low utilization rate of organs,the short preservation time of organs,the immune rejection after heart transplantation and the complications caused by long-term immunosuppressive therapy,infection and malignant tumor and many other issues,Therefore,it is of great clinical significance to improve the preservation time of organs,increase the utilization rate of organs,and improve the inflammatory response during cardiac storage to alleviate the imbalance between supply and demand and the incidence of postoperative complications.Calpain is a kind of protease which is activated by calcium ions,Calpain-1 and calpain-2 are two classical/ubiquitous Calpain proteinases,Calpain regulates the biological activity of other proteins by restricting the enzymatic hydrolysis of various enzymes and cytoskeletal protein systems in cells.It plays an important role in cell cycle,contraction,apoptosis,migration,differentiation and signal transduction[1].Cytoskeletal non-erythroid alpha-spectrin is an endogenous calcineurin substrate,Under pathophysiological conditions or during cell necrosis,cellular Calpain cleavage Spectrin produces 150kDa and 145kDa decomposition products(SBDPs)[2],which can be used as a marker of Calpain activation.Calpastatin is an endogenous inhibitory protein that specifically acts on calpain-1 and calpain-2,and Calpastatin can specifically bind to calpain-1 and calpain-2,which can inhibite the enzymatic hydrolysis activity of calpain-1 and calpain-2[3].Calpain/Calpastatin balance is found to play an important role in acute immune rejection,and inhibition of Calpain by overexpression of Calpastatin can delay allograft immune rejection[4].The purpose of this study was to analyze the expression changes of Calpain/Calpastatin in the isolated heart,vivo overexpression of Calpastatin inhibits Calpain activation,which provides a theoretical basis for the study on the protective mechanism of isolated heart.Objective1.To study the expression of Calpain/Calpastatin in isolated heart.2.Establish a high expression method of CalpastatinMethods1.Heart perfusion in vitro model was established,After obtaining the heart,HTK solution was perfused for 2h via the ascending aorta with a microperfusion pump,Then it was placed in HTK cardiac preservation solution at 4? and respectively placed in the solution at Oh,1h,2h,3h and 4h to simulate the state of cold ischemia of isolated heart at 2h,3h,4h,5h and 6h.The protein expressions of calpain-1,calpain-2,Spectrin and Calpastatin were measured by Western blotting.2.The recombinant adenovirus containing Calpastatin gene was amplified.The recombinant adenovirus vector containing Calpastatin gene was injected into the left ventricular cavity.Myocardial tissue was extracted 48 hours later and the expression of Calpastatin was detected by Western Blot.Results1.Calpain-1 protein expression in isolated myocardial tissue was increased at 3h,4h,5h and 6h compared with 2h,and reached the highest level at 5h.Calpain-2 protein expression was increased at 3h,4h,5h and 6h compared with 2h,and reached the highest level at 5h and 6h.The protein expression levels of Spectrin at 3h,4h,5h and 6h in isolated myocardium were all higher than that at 2h,and reached the maximum value at 6h.Calpastatin protein expression was increased at 4h and 6h,but there was no significant difference at 3h and 5h.Calpain/Calpastatin balance is broken,presenting a state of high stress.2.The expression of Calpastatin protein in myocardial tissue was significantly increased 48 hours later after injection of adenovirus vector containing Calpastatin gene.Conclusions(1)HTK solution was used to preserve isolated hearts of mice.Expressions of Calpain-1 and calpain-2 and enzyme activity were increased.(2)injecting adenovirus vector which contains Calpastatin gene into the left ventricle of mice can increase the expression of Calpastatin protein in myocardial tissue,which is expected to play a protective role on the isolated heart.