The Role And Mechanism Of E3 Ubiquitin Ligase RNF5 In Pathological Cardiac Hypertrophy

BackgroundThere are two types of cardiac hypertrophy:physiological and pathological hypertrophy.Physiological hypertrophy is common in athletes and pregnant women,and does not develop into heart failure.However,pathological hypertrophy usually develops into heart failure.Previous studies have showed that hypertension,valvular heart disease,coronary heart disease and hereditary cardiomyopathy are the main causes of pathological hypertrophy.Treatment strategies for heart failure caused by pathological hypertrophy include drug treatments(beta-blockers,angiotensinconverting enzyme inhibitors,angiotensin Ⅱ receptor antagonists,aldosterone receptor inhibitors,ivabradine,levosimendan,etc),and device therapies(cardiac resynchronization therapy,implantable cardioverter defibrillator,etc).Due to these treatment strategies,the symptoms and prognoses of patients with heart failure have been greatly improved.However,the mortality of heart failure remains high.The pathogenesis of pathological hypertrophy is quite complex and still not fully understood.Therefore,it is crucial to clarify the pathogenesis of pathological cardiac hypertrophy to delay and reverse the occurrence of heart failure,as well as find new therapeutic targets.Since cardiomyocytes are terminally differentiated cells,the essence of cardiac hypertrophy is the increase of protein contents in the myocardium.Imbalances in protein quality control lead to disruption of protein homeostasis and are associated with a variety of diseases.Increased cardiac pressure load leads to cardiac hypertrophy with cyclical production,degradation and oxidative damage of proteins in the heart.Previous studies have showed that abundant ubiquitinated proteins existed in the hearts of pathological cardiac hypertrophy and heart failure.Ubiquitination plays an extremely important role in regulating the homeostasis of cardiomyocyte protein metabolism and the normal functions of cardiomyocytes.As one of the main components of the ubiquitin-proteasome system(UPS),ring-finger protein 5(RNF5)is a kind of E3 ubiquitin ligases and involved in a variety of protein substrates degradation.Previous studies have shown that under viral infection conditions,RNF5 can ubiquitinate and degrade interferon stimulator(Stimulator of interferon genes,STING)to reduce the production of interferon I.In pathological cardiac hypertrophy,STING deficiency alleviates the degree of myocardial inflammatory response and fibrosis.However,the current researches regarding RNF5 focus on inflammatory response,innate immune response and malignant tumor.The role of RNF5 in pathological cardiac hypertrophy is unknown and it is unclear that whether RNF5 interacts with STING has an impact on cardiac hypertrophy.Therefore,we aimed to explore the effects of RNF5 on pathological cardiac hypertrophy,and clarify the underlying molecular mechanisms to find a new promising therapeutic target for pathological cardiac hypertrophy.Part 1.The expression of RNF5 is increased in pathological cardiac hypertrophyPurposeWe explored whether RNF5 was differentially expressed in pathological cardiac hypertrophy,by establishing cell and animal models of pathological cardiac hypertrophy.Methods(1)We treated neonatal rat cardiomyocytes(NRCMs)with phenylephrine(PE)to induce cardiomyocyte hypertrophy,and phosphate buffered saline(PBS)-treated NRCMs were used as a control group.(2)The mouse model of pathological cardiac hypertrophy induced by transverse aortic constriction(TAC)was established,and the sham-operated mice were used as the control group.(3)The morphological changes of NRCMs were detected by immunofluorescence.(4)RT-PCR and Western blot were used to detect the mRNA and protein expression of RNF5 and cardiac hypertrophy markers including atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and MYH7 in NRCMs and myocardial tissues of mice,respectively.Results(1)Compared with the PBS control group,in the PE-induced hypertrophy model of NRCMs,immunofluorescence showed that PE stimulation contributed to NRCMs hypertrophy,which indicated the model was successfully established;RT-PCR showed that there was no significant difference in the mRNA expression of Rnf5;Western blot showed the protein expression of RNF5 was significantly increased.(2)Compared with the sham-operated group,RT-PCR showed no significant difference in the mRNA expression of in myocardial tissues in the pathological cardiac hypertrophy mouse model induced by TAC;Western blot showed that the protein expression of RNF5 was significantly increased.ConclusionsThe protein expression of RNF5 is increased in hypertrophic cardiomyocytes and in the cardiac tissues of a mouse model of pathological cardiac hypertrophy.Part 2.Effects of RNF5 on pathological cardiac hypertrophyPurposeBy establishing the RNF5 knockout mouse model,RNF5 knockdown and RNF5 overexpression NRCMs models,the effects of RNF5 intervention on pathological cardiac hypertrophy were explored from in vivo and in vitro experiments,respectively.Methods(1)The TAC-induced cardiac hypertrophy mouse model was established.The mice were randomly divided into 4 groups before surgery:wild type and sham operation group(WT Sham),wild type and TAC group(WT TAC),RNF5 gene knockout and sham surgery group(KO Sham)and RNF5 knockout and TAC group(KO TAC).(2)The body weight,heart weight,lung weight and tibia length of the mice were weighed respectively.(3)The small animal ultrasound was used to assess the cardiac function.(4)H&E and wheat germ agglutinin(WGA)staining were used to evaluate the degree of hypertrophy of cardiomyocytes.(5)The expression of cardiac hypertrophy biomarkers(ANP,BNP and MYH7)were detected by RT-PCR and Western blot,respectively.(6)Cardiac interstitial fibrosis was assessed by PSR staining.The expression of fibrosis markers(Collagen Ⅰ α1,Collagen Ⅲ α1 and CTGF)were detected by RT-PCR and Western blot,respectively.(7)RT-PCR was used to detect the mRNA expression of inflammatory factors including Il-6,Il-1β and Tnf-α.Western blot was used to detect the expression of related proteins in nuclear factor kappa B(NF-κB)signaling pathway.(8)In vitro experiments,NRCMs were infected with short hairpin RNA-containing adenovirus(AdshRNF5)to knock down RNF5,and then treated with PE to establish the cardiac hypertrophy model.The cells were randomly divided into four groups before infection:AdshRNA PBS,AdshRNF5 PBS,AdshRNA PE and AdshRNF5 PE.Additionally,NRCMs were infected by RNF5-overexpressing adenovirus(AdFlag-RNF5),and cells were randomly divided into four groups before infection:AdVector PBS,AdFlag-RNF5 PBS,AdVector PE and AdFlag-RNF5 PE.(9)RT-PCR and Western blot were used to detect the expression of RNF5,ANP,BNP and MYH7 in the above cells,respectively.(10)Immunofluorescence was used to detect the cardiomyocyte size after RNF5 intervention.Results(1)Gross specimens showed that,compared to the WT TAC group,RNF5 knowout significantly increased the cardiac weight and the ratio of heart weight/body weight,lung weight/body weight,and heart weight/tibia length in TAC-induced cardiac hypertrophy model mice.(2)The results of cardiac ultrasound showed that RNF5 knowout significantly significantly increased left ventricular end-diastolic diameter(LVEDd)and left ventricular end-systolic diameter(LVESd),and decreased the ejection fraction(EF)and short-axis shortening rate(FS)in TAC-induced cardiac hypertrophy model mice.(3)The results of H&E and WGA staining showed that RNF5 knowout significantly increased the cross-sectional area ofthe myocardium in mice with TAC-induced cardiac hypertrophy.(4)RT-PCR and Western blot showed that RNF5 knowout significantly increased the expression of ANP,BNP and MYH7 in TAC-induced cardiac hypertrophy model mice.(5)PSR staining showed that RNF5 knowout aggravated myocardial interstitial collagen deposition and perivascular fibrosis in TAC-induced cardiac hypertrophy model mice,and increased the expression of fibrosis markers(Collagen Ⅰ α1,Collagen Ⅲ αl and CTGF).(6)RT-PCR showed that RNF5 knowout increased the mRNA expression of inflammatory factors(Il-6,Il1-β and Tnf-α)in TAC-induced cardiac hypertrophy model mice,and Western blot showed that the protein expression of IKKβ,p-IkBαand p-p65 was increased in the NF-κB related signaling pathway.(7)In vitro experiments,immunofluorescence showed that RNF5 knockdown significantly increased the surface area of PE-induced NRCMs.RT-PCR and Western blot showed that RNF5 knockdown significantly increased the expression of ANP,BNP and MYH7.In contrast,in RNF5-overexpressed NRCMs,immunofluorescence showed that RNF5 overexpression significantly reduced the surface area of PE-induced NRCMs,and RT-PCR and Western blot showed that RNF5 overexpression significantly decreased the expression of ANP,BNP,and MYH7.ConclusionsRNF5 knockout aggravated cardiac hypertrophy in TAC model mice.In the corresponding cellular model,RNF5 knockdown promoted PE-induced hypertrophy of NRCMs,while RNF5 overexpression ameliorated PE-induced hypertrophy of NRCMs.Part 3.The mechanism of RNF5 in pathological cardiac hypertrophyPurposeTo explore the molecular mechanism of RNF5 in pathological cardiac hypertrophy.Methods(1)mRNA-Seq analysis was performed in WT TAC and KO TAC mice to explore the effect of RNF5 deletion on the transcriptome levels of cardiac hypertrophy.(2)Mass spectrometry analysis was used to screen proteins that bind to RNF5.(3)Immunofluorescence was performed to explore the localization of RNF5 and STING proteins in cells.(4)Protein interaction experiments was performed to verify whether RNF5 and STING interacted.(5)Ubiquitination experiments were performed to explore whether RNF5 regulated STING through ubiquitination,as well as ubiquitination types and ubiquitination sites.(6)Rescue experiments further confirmed the role of RNF5 in regulating STING in cardiac hypertrophy.Results(1)mRNA-Seq analysis showed that the expression of genes related to inflammatory response,fibrosis and myocardial function signaling pathways were significantly up-regulated in RNF5-knockout mouse myocardial tissues.(2)Mass spectrometry analysis suggested that STING was a protein bound to RNF5.(3)Immunofluorescence showed that RNF5 and STING co-localized in the cytoplasm.(4)The protein interaction experiments confirmed the direct interaction between RNF5 and STING.(5)The ubiquitination experiments showed that RNF5 ubiquitinated STING through K48-linked polyubiquitin chains leading to its degradation.(6)Rescue experiments confirmed that RNF5 alleviated pathological cardiac hypertrophy through ubiquitination and degradation of STING.ConclusionsRNF5 alleviates pathological cardiac hypertrophy through K48-linked ubiquitination and proteasomal degradation of STING.