The Role And Mechanism Of FAT10 In Cardiac Hypertrophy Induced By Transverse Aortic Constriction

Background:Cardiac hypertrophy results in irreversible structural changes in the heart.Recent studies have found that Ca2+-activated calcium/calmodulin-dependent protein kinase II?CaMKII?protein and FAT10 may be involved in the development of cardiac hypertrophy,but the relevant mechanism is unknown.Objective:In previous studies,we have found that FAT10 is expressed in the myocardium and has anti-ischemic and apoptotic effects.Therefore,our cardiac hypertrophy model was established by transverse aortic constriction?TAC?on the basis of heart-specific FAT10 knock-out mice,which is applied to explore the role of FAT10and CaMKII in the development of cardiac hypertrophy,and provide a theoretical basis for appropriate drug intervention targets.Methods:1.Heart-specific conditional Fat10 knock-out mice were provided from Nanjing Biomedical Research Institute of Nanjing University,based on the homologous recombination technology with tamoxifen-inducible Myh6-MerCreMer system[(Fat10^flox/flox;myh6-Cre^+/-),cKO];2.TAC model construction and verification:We use several experimental technologies to verify the TAC model,such as the echocardiography for small animals,H&E staining,Sirius red staining,WGA-FITC staining,RT-PCR and western blot;3.Mechanism of TAC model:The technology of western blot used to explore the interaction between the FAT10 and TAC-induced cardiac hypertrophy.Result:1.Survival analysis:All mice in the sham group survived;Ctrl+TAC group died on the fifth day after TAC,and the 30-day survival rate was 75%;cKO+TAC group died on the first postoperative day,and the 30-day survival rate was 25%.2.FAT10 expression:In mouse heart tissue,the expression of Fat10 in the knock-out group was significantly lower than in the non-knockout group,but there was no significant change in other tissues.3.Construction of the TAC model:The result of echocardiography showed whether FAT10 was knocked out or not,the interventricular septum?IVS?,left ventricular posterior wall thickness?LVPW?and left ventricular mass?LV mass?were increased after TAC in diastole or systole,and this was particularly obvious in the knock-out group.By calculating the relative quality of the heart,we observed the same results whether FAT10 was knocked out or not,the relative weight of the heart was increased after TAC,and it was even more obvious in the knock-out group.4.Verification of the TAC model:The results of H&E,Sirius red and WGA-FICT staining showed whether FAT10 was knocked out or not,the thickness of ventricular wall was increased,the arrangement of the myocardial fibers was disordered,the fibrosis of the interstitium was slightly,the proliferation of collagen fibers was abnormal,and the volume of myocardial cell was increased after TAC,these changes were also evident in the knock-out group.The RT-PCR and western blot results showed whether FAT10 was knocked out or not,the myocardial hypertrophy-associated indicators ANF and MYH7 are both increased after TAC,and it was also more obvious in the knock-out group.5.Molecular mechanism:In non-knockout group,the FAT10 expression was decreased after TAC,but no FAT10 expression was detected in the knock-out group.In addition,we found whether FAT10 was knocked out or not,the CaMK2D protein was increased after TAC,and the phosphorylation level of RYR2-Ser2814 showed the same trend as that of CaMK2D.Conclusion:1.The knock-out group are prone to cardiac hypertrophy when pressure overload increases2.It was revealed for the first time that decreased expression of FAT10 resulted in hyperphosphorylation of RYR2-Ser2814 by CaMKII,which may cause intracellular calcium leakage during diastole,induce calcium overload,and eventually induce myocardial hypertrophy.