The Role Of E3 Ubiquitin Ligase WWP1 In Simulated Weightlessness-induced Myocardial Remodeling

BackgroundThe myocardium is capable of structural and functional remodeling in response to different workloads.In the weightless environment of spaceflight,the normal loading effect of gravity on the cardiovascular system is lost,which can trigger a series of cardiovascular "aging-like" responses: arteriosclerosis,myocardial atrophy,and cardiac decompensation.Magnetic resonance imaging(MRI)tests of astronauts after space flight showed myocardial atrophy and functional decline,and ground-based human bed rest experiments and animal simulations of weightlessness also found structural remodeling and functional decline of the heart.In conclusion,myocardial remodeling and functional decline due to weightless flight are serious threats to astronaut health.Studies have shown that protein stability regulation and post-translational modifications play an important role in the development of myocardial remodeling.ObjectiveTo find effective ways to cope with cardiovascular dysfunction in the space-specific environment,it is currently of utmost importance to investigate in depth the molecular mechanisms underlying the occurrence of myocardial remodeling induced by weightlessness or simulated weightlessness.Therefore,in this study,we used mouse tail suspension and rhesus monkey bed rest as models of simulated weightlessness effect,combined with a cardiac pressure overload model,to study the changes of E3 ubiquitin ligase WWP1 expression in simulated weightless heart,establish its correlation with myocardial remodeling,investigate the molecular mechanism of myocardial remodeling from the perspective of protein interaction and post-translational modification,provide a theoretical basis for analyzing the mechanism of simulated weightless-induced myocardial remodeling,and provide intervention targets and strategies for combating myocardial remodeling caused by long-term manned space flight.Method1.Rhesus monkey bed rest(BR)model,mouse tail suspension(TS)model,mouse transverse aortic constriction(TAC)model and clinical sample of human heart failure(HF)were used to analyze the protein expression of WWP1 in myocardial remodeling induced by simulated weightlessness and pressure overload by Western blot(WB)and immunohistochemistry.2.We used WWP1 global knockout(KO)mice to construct a mouse tail suspension model and a TAC model to resolve the role of WWP1 in myocardial remodeling by means of echocardiography,histological analysis and real-time quantitative PCR(Q-PCR).3.In cardiomyocytes HL-1 and HEK293 T cell line,mass spectrometry,immunoprecipitation,cell immunofluorescence and GST-Pulldown were used to analyze and screen substrate proteins that directly interact with WWP1;Protein ubiquitination assay,Pulse-chase analysis and protein truncation analysis were used to analyze the regulatory effect and mechanism of WWP1 on substrate proteins;combined with mouse tail suspension,rhesus monkey bed rest,mouse TAC and clinical sample of human heart failure,to validate the regulatory effect and mechanism of WWP1 on substrate protein in vivo.4.In cardiomyocytes HL-1 and HEK293 T cell line,Reporter gene assays,Target gene overexpression and knockdown,protein interaction analysis and adenovirus treatment were used to dissect the WWP1-mediated signaling pathway regulating myocardial remodeling;mouse tail suspension model and mouse TAC model were used to validate the regulatory mechanisms involved in myocardial remodeling by WWP1.5.In mouse TAC model and mouse tail suspension model,myocardium-specific expression of adenoviral sh RNA and natural small molecule compound Indole-3-carbinol(I3C)were used to target WWP1,echocardiography,histological analysis and real-time quantitative PCR were used to explore the therapeutic effects of targeting WWP1 on myocardial remodeling.Results1.WWP1 protein levels were significantly elevated in the rhesus monkey bed rest model,mouse tail suspension model,mouse transverse aortic constriction model and clinical sample of human heart failure,and WWP1 elevation occurred mainly in cardiac myocytes;in addition,WWP1 protein levels were positively correlated with the degree of myocardial remodeling.2.In mouse tail suspension model,WWP1 knockdown alleviated myocardial atrophy,deepened fiber coloration and decreased cardiac function caused by simulated weightlessness;in mouse aortic constriction model,WWP1 knockdown alleviated myocardial hypertrophy,myocardial fibrosis and decreased cardiac function caused by pressure overload.3.In cardiomyocytes HL-1 and HEK293 T cell line,WWP1 co-localizes with DVL2,interacts directly with the DIX domain of DVL2,and increases DVL2 protein stability through K27-linked polyubiquitination;overexpression of WWP1 was able to increase DVL2 protein levels and conversely DVL2 protein levels decreased;in the rhesus monkey bed rest model,mouse tail suspension model,mouse transverse aortic constriction model and clinical sample of human heart failure,WWP1 protein levels were positively correlated with DVL2 protein levels,and there was no significant change in classical Wnt signaling downstream of DVL2.4.WWP1 increases the transcriptional activity of MEF2 C,a key transcription factor for myocardial remodeling,in a dose-dependent manner in cardiomyocytes HL-1 and HEK293 T cell line;mechanistically,WWP1 increases the protein stability of DVL2 by stabilizing it,and DVL2 interacts with CaMKⅡ to increase the activity of the latter,which in turn phosphorylates HDAC4 and translocates it out of the nucleus to derepress the transcriptional activity of MEF2 C.In both cellular and mouse TAC models,knockdown of DVL2 or CaMKⅡ resulted in a loss of the pro-myocardial remodeling effect of WWP1;in addition,DVL2/CaMKⅡ/HDAC4 signaling pathway activity was inhibited after knockdown of WWP1 in a mouse tail suspension simulated weightlessness model.5.In a mouse TAC model,targeting WWP1 using myocardial-specific expression of adenoviral sh RNA and the natural small molecule compound I3 C were both able to alleviate myocardial hypertrophy,myocardial fibrosis and decreased cardiac function caused by pressure overload,respectively.in a mouse tail suspension model,I3 C intraperitoneal injection significantly alleviated the myocardial atrophy and functional decline caused by simulated weightlessness.Conclusion1.WWP1 was able to respond to myocardial remodeling due to pressure load changes and was expressed elevated in both simulated weightlessness and pressure overloadinduced myocardial remodeling.2.WWP1 plays an important role in myocardial remodeling,and knockdown of WWP1 inhibits myocardial remodeling and functional decline induced by simulated weightlessness and pressure overload3.WWP1 is involved in myocardial remodeling regulation through a non-classical Wnt signaling pathway.WWP1 is able to interact directly with DVL2 and increase DVL2 protein stability through K27-linked polyubiquitination,thereby activating the DVL2/CaMKⅡ/HDAC4/MEF2 C signaling pathway involved in myocardial remodeling regulation.4.Myocardial-specific expression of adenovirus and the natural small molecule compound I3 C targeting WWP1 were effective in mitigating myocardial remodeling induced by simulated weightlessness and pressure overload.