RNF207 Exacerbates Pathological Cardiac Hyper-trophy Via Post-Translational Modification Of TAB1

Objective: Pathological cardiac hypertrophy refers to the compensatory hypertrophy of cardiomyocytes when continuous pathological factors stimulate the heart and eventually cause heart failure due to decompensation.Ubiquitination modification is widely involved in biological processes such as protein degradation and cell signaling pathways in eukaryotic organisms.Also,it plays an essential role in developing pathological cardiac hypertrophy.Ring finger protein 207 is an E3 ubiquitin ligase mainly expressed in the heart,but its specific function is still unclear.This study aims to explore the relationship between RNF207 and pathological cardiac hypertrophy and clarify the particular function and mechanism of RNF207.Methods:1.To construct pathological cardiac hypertrophy models in vivo and in vitro,transverse aortic constriction(TAC)was conducted in mice,and neonatal rat cardiomyocytes(NRCMs)were stimulated by phenylephrine(PE),respectively.The expression pattern of RNF207 was studied by RT-PCR and western blot in the two models.2.In vivo,adeno-associated virus type 9(AAV)was used as the vector to realize the overexpression and knockdown of RNF207 in mice.Six weeks after the TAC operation,the myocardial hypertrophy of the RNF207 overexpression/knockdown group and control group was detected.In vitro,adenovirus(Ad)was used as a vector to overexpress and knockdown RNF207.Cardiomyocyte hypertrophy was assessed by ACTN1 staining and RT-PCR after PE stimulation.3.Regarding mechanism,the binding proteins of RNF207 were detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS).After screening out the binding protein most related to pathological cardiac hypertrophy,co-IP and immunofluorescence colocalization verified the combination.On this basis,the effect of RNF207 on the ubiquitination modification of the target protein was explored through ubiquitin binding experiments,and the specific modification types were clarified.Subsequently,the molecules of relevant signaling pathways were detected by western blot to examine the effect of RNF207 on the hypertrophy-related signaling pathway mediated by the target protein.Finally,this study further verified the relationship between RNF207 and target protein in pathological cardiac hypertrophy through rescue experiments in NRCMs.Results:1.RNF207 was mainly expressed in cardiomyocytes.In the models of pathological cardiac hypertrophy,the expression of RNF207 was down-regulated and negatively correlated with the severity of cardiac hypertrophy.2.In vivo,AAV-RNF207 was able to promote pathological cardiac hypertrophy and cardiac fibrosis.On the contrary,AAV-sh-RNF207 possessed a protective effect on the heart.The above conclusions were verified in vitro.3.Regarding mechanism,the results of LC-MS/MS indicated that TAB1 was one of the binding proteins of RNF207.RNF207 bonded to TAB1 through the CHR domain and could increase the K63 polyubiquitination of TAB1.K63 polyubiquitination of TAB1 was increased in pathological cardiac hypertrophy,and the introduction of RNF207 amplified this response.RNF207 enhanced the binding of TAB1 and TAK1,promoted the phosphorylation of TAK1,and then activated the downstream JNK1/2 and P38 pathways.In the rescue experiment,the knockdown of TAB1 could inhibit the hypertrophic effect of RNF207.Conclusion: RNF207 is expressed in cardiomyocytes,and its expression is decreased in pathological cardiac hypertrophy models.RNF207 can aggravate cardiac hypertrophy and cardiac fibrosis.Regarding mechanism,RNF207 selectively activates the MAPK signaling pathway by mediating K63 polyubiquitination of TAB1,thereby promoting the development of pathological cardiac hypertrophy.