The Effect Of FAT10 On Cardiac Hypertrophy By Regulating Calcium/calmodulin-dependent Protein Kinase ? Delta And Its Mechanisms

Background:Sudden cardiac death?SCD?is one of the major causes of human death.Generally,lethal ventricular arrhythmia is the direct cause of onset,and cardiac hypertrophy can be considered as one of the risk factors of SCD.According to the etiology,cardiac hypertrophy can be divided into idiopathic cardiac hypertrophy?Hypertrophic cardiomyopathy,HCM?and secondary cardiac hypertrophy.Hypertrophic cardiomyopathy is usually caused by a series of specific gene mutations,however secondary cardiac hypertrophy is often induced by many common disease stimuli,including:long-standing hypertension;ischemia associated with coronary artery disease;valvular insufficiency and stenosis;myocarditis due to an infectious agent;congenital malformations,diabetic cardiomyopathy,etc.Calcium homeostasis in cardiomyocyte is fundamental to the normal systolic function and electrophysiological activity.The calcium release channel of sarcoplasmic reticulum?ryanodine receptor 2,RyR2?plays an essential role in Ca²⁺release and maintaining intracellular Ca²⁺homeostasis.RyR2 can be phosphorylated by CaMK2D,one of the calmodulin kinase II family members,which is an important mechanism for regulating channel function.Increased RyR2 Ca²⁺release mediated by CaMK2D hyper-activity is one of the important mechanisms for intracellular calcium overload of cardiac hypertrophy and ventricular arrhythmia.Ubiquitin-like?UBL?proteins,with structural similarities to ubiquitin,even more recently,more and more attention has been drawn to identify the role of UBL in the cardiovascular diseases.FAT10,a member of UBL family,is recently discovered as a main functional molecule of mediating protein degradation by the proteasome,which plays an important role in the regulation of apoptosis and cell cycle.Objective:The studies have found that intracellular Ca²⁺cycle abnormalities play a key role in cardiac hypertrophy,however,the regulatory mechanisms are still unclear.Our team first found FAT10 was expressed in heart tissue and had an anti-apoptotic effect against myocardium ischemia,however,the role of FAT10 in the development of cardiac hypertrophy was unknown.The objective of this study is to investigate whether FAT10 is involved in cardiac hypertrophy by regulating CaMK2D-RyR2function,and to explore the underlying molecular mechanisms,which provide a theoretical basis of cardiac hypertrophy biomarkers screening and drug intervention targets finding.Methods:1.Confirm FAT10 was expressed in heart tissue:The total RNA and protein were extracted from rat,mouse,and human heart tissue.And FAT10 expression was detected by RT-PCR and Western Blot.2.Construction and verification of the cardiac hypertrophy model:The angiotensin II-induced cardiomyocyte hypertrophic model was established with the neonatal rat cardiomyocytes in vitro.HE staining of the heart tissue slices and heart weight/body weight ratio was used to confirm the hypertrophic phenotype in25-week-old spontaneously hypertensive rats?SHR?.The mRNA expression level of hypertrophic markers-Acta1,Myh7 and BNP in the heart of SHR was detected by qRT-PCR.3.Detection of the expression level of FAT10 in caridac hypertrophic models:the expression level of FAT10 was detected by qRT-PCR and Western Blot in SHR and AngII induced hypertrophic cardiomyocytes.Inadditon,GEO?Gene Expression Omnibus?database profiles were used to analyze the expression level of FAT10 in other studies of cardiac hypertrophy.4.To observe the effect of FAT10 on cardiac hypertrophy:The FAT10overexpression and interference adenovirus were used as the gain-of-functional and loss-of-functional approaches to study the effects of FAT10 in angiotensin II induced hypertrophic cardiomyocytes.The influence of FAT10 on the cell surface of hypertrophic cardiomyocytes was detected by F-actin staining.Then the expression level of ANF,BNP,MYH7 and ACTA1 was also studied by these two approaches.5.To screen the putative substrates of FAT10:The target proteins were screened by tandem mass spectrometry and the protein cluster analysis was performed using the bioinformatics approach.Then the endogenous interaction of FAT10 and CaMK2Dwasconfirmedbyco-immunoprecipitation?co-IP?and immunofluorescence.6.To study the regulatory effect of FAT10 on CaMK2D:the mRNA and protein expression level of CaMK2D was studied by qRT-PCR and Western Blot.The phosphorylation status of RyR2 on Ser2814 was detected by Western Blot.The regulation effect of FAT10 on CaMK2D was verified with CaMK2D specific inhibitor KN-93 and the change of interaction status of FAT10 with CaMK2D was detected by co-IP in hypertrophic heart of SHR.7.To definite the characteristics of the interaction of FAT10 with CaMK2D:MG132 was used to study whether FAT10 affect proteasome degradation of CaMK2D.The exogenous interaction of FAT10 with CaMK2D was confirmed by co-IP in HEK293 cell line and GST-pulldown in a cell-free reaction system.A recombinant plasmid with double fluorescence of CaMK2D was constructed to verify whether FAT10 changed the molecular conformation of CaMK2D by fluorescence resonance energy transfer method,and protein modification was detected as well.The interaction status of CaMK2D and FAT10 was assessed in the condition of UBA6 and UBE2Z deficiency.8.To determine the binding region of CaMK2D with FAT10:The truncated mutants of CaMK2D were generated by standard molecular cloning methods,and the binding region of CaMK2D with FAT10 was identified by co-IP and immunofluorescence.Results:1.It was the first time to find ubiquitin-like protein FAT10 was expressed in inrodent heart tissues,and the expression was also confirmed in human heart tissue.2.In the AngII induced cardiac hypertrophy model,the mRNA and protein expression level of FAT10 was down-regulated in a dose-dependent manner.In the hypertrophic heart tissue of SHR,the expression of FAT10 was also decreased.The decreased expression of FAT10 was confirmed in two datasets of GEO database involved with cardiac hypertrophy.3.FAT10 overexpression attenuated cardiac hypertrophy induced by angiotensin II,decreased the mRNA expression of ANF and BNP and protein expression of MYH7 and ACTN1.On the contrary,FAT10 deficiency up-regulate the expression level of ANF,BNP,MYH7 and ACTN1 and led to aggravate the hypertrophic phenotype.4.CaMK2D was identified as one of the putative substrate partners of FAT10 by mass spectrometry and the interaction of FAT10 and CaMK2D was the verified by co-IP and immunofluorescence in the cardiomyocytes.Further study revealed that over-expression of FAT10 decreased the protein expression of CaMK2D.5.In the cardiac Hypertrophic model,over-expression of FAT10 significently reduced the phosphorylation of the primary CaMK2D site Ser2814 on RyR2 and the interference of FAT10 had an opposite effect on the phosphorylation of RyR2Ser2814.6.CaMK2D activity inhibition by KN-93 reversed cardiac hypertrophy with FAT10 deficiency by reducing RyR2 phosphorylation.In addition,a weakened interaction of FAT10 with CaMK2D was observed in the SHR heart,which indicated the interaction of FAT10 and CaMK2D played a role in development of cardiac hypertrophy.7.Proteasome inhibition could not eliminate the effect of FAT10 on CaMK2D,indicating FAT10 did not regulate the degradation process of CaMK2D.The direct interaction of FAT10 and CaMK2D was demonstrated by exogenous co-IP and GST-pulldown,which was independent by UBA6 and UBE2Z cascade,as well as C-terminal double glycines motif of FAT10.In addition,the result of fluorescence resonance energy transfer revealed FAT10 might affect molecular conformational change of CaMK2D without alteration of autophosphorylation and oxidized modification of CaMK2D.8.We demonstrated that only catalytic domain,probably within a region comprising residues 1-273,bound to FAT10.Conclusion:1.We first revealed that UBL protein FAT10 was expressed in heart tissue,and the expression levle was down-regulated in cardiac hypertrophy.2.FAT10 could attenuate the phenotype of caridac hypertrophy.3.CaMK2D was one the substrates of FAT10 in the caridac myocytes.4.FAT10 attenuated the cardiac hypertrophy by reducing the expression of CaMK2D and function which decreased the phosphorylation of RyR2.5.FAT10 directly and non-covalent interacted with CaMK2D,which did not affect the degradation of it,and the interaction was independent of E1 and E2 of FAT10.6.FAT10 binded to the catalytic domain of CaMK2D,which affected the molecular conformation without an alteration of post-translational modification of CaMK2D.In conclusion,the above results reveal that FAT10 is an UBL protein that may resiste myocardial hypertrophy.