The Role And Mechanism Of Mitochondrial Inner Membrane Protein HIGD1A In Pathological Cardiac Hypertrophy

Background: Pathological cardiac hypertrophy is an independent risk factor for the development of heart failure and other cardiovascular diseases,which is also an adaptive cardiac response caused by a variety of factors.Persistent pressure overload can promote maladaptive cardiac remodelling,which often develops into heart failure.Mitochondria is the powerhouse of heart.mitochondrial dysfunction is associated with the development of cardiovascular disease.However,the relationship between mitochondrial dysfunction and pathological cardiac hypertrophy have not been elucidated.Therefore,modulation of mitochondrial function may be a potential treatment for cardiac hypertrophy.Hypoxiainducible gene structural domain protein 1A(HIGD1A),which is a mitochondrial inner membrane protein,is specifically expressed in stressful conditions,such as hypoxia or hypoglycaemia.It is reported that HIGD1 A promote cell survival through increasing the efficiency of the electron transport chain(ETC)and improving mitochondrial function.HIGD1 A also play a regulatory role in diseases,including hyperlipidaemia,non-alcoholic fatty liver,and cancer,However,whether HIGD1 A affects the development of pathological cardiac hypertrophy is unknown.In our previous study,we established myocardial hypertrophy in mice using thoracic aortic constriction,and found that the expression of HIGD1 A was downregulated in the myocardial tissue after TAC surgery 8 weeks,which indicated that HIGD1 A may participate in the regulation of myocardial hypertrophy.Therefore,to clarify the role and mechanism of HIGD1 A is in the occurrence and development of cardiac hypertrophy,we will systematically study this scientific problem in vivo and in vitro,to find new targets for the prevention and treatment of cardiac hypertrophy.Objectives: 1.To determine the role of HIGD1 A in pathological myocardial hypertrophy;2.To elucidate whether HIGD1 A alleviates pathological myocardial hypertrophy by regulating mitochondrial function and oxidative stress.Methods:1.Experimental Animals:(1)Experimental groups: male C57 BL/6 mice(6-8 weeks,20-22 mg)were divided into 8 groups randomly(n=6): Sham group,TAC 1w,2w,4w,6w,8w group,TAC 8w + saline group(TAC 8w + NS),TAC 8w+simvastatin group(TAC 8w+SIM).Establishment of a mouse model of myocardial hypertrophy using transverse aortic arch constriction(TAC)surgery,the Sham group not with transverse aortic arch constriction.(2)A transthoracic echocardiography system(Visual Sonics,Vevo 2100)was used to evaluate cardiac function.(3)Hematoxylin-eosin staining and Masson’s trichrome staining.(4)ANP,β-MHC m RNA and protein measurements to reflect myocardial hypertrophy.(5)Western blot and RT-q PCR to detect the expression level of HIGD1 A.2.Cellular experiment:(1)Experimental groups: H9c2 cells were cultured and treated with angiotensin II(Ang Ⅱ1 μmol/L)to induce hypertrophy model,which were divided into the following 7 groups: control group(control),Ang Ⅱ 24 h group(Ang Ⅱ 24h),Ang Ⅱ 48 h group(Ang Ⅱ 48h),over-expressed HIGD1 A group(ad HIGD1A),silent HIGD1 A group(sh HIGD1A),Ang Ⅱ+ overexpressed HIGD1 A group(Ang Ⅱ+ad HIGD1A)and Ang Ⅱ+ silent HIGD1 A group(Ang Ⅱ+sh HIGD1A).(2)Plasmid transfection to overexpress and silence HIGD1 A.(3)Determination of mitochondrial membrane potential by JC-1 staining.(4)Determination of ROS content in cells using DCFH-DA fluorescence probe method.Results: 1.HIGD1 A expression was significantly reduced in cardiac hypertrophy mice 1.1 Heart mass(HW),lung weight(LW),heart weight/body weight ratio(HW/BW),lung weight to body weight ratio(LW/BW)and heart mass/tibial length ratio(HW/TL)were overly higher in C57BL/6J mice in TAC group compared to Sham.1.2 Compared to Sham group mice,left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD)began to increase and shortening fraction(FS)and ejection fraction(EF)began to decrease after 4w of TAC.1.3 HE staining results showed that myocardial fibres in the sham group of mice were normally aligned,whereas myocardial fibres in the TAC4 w and 8w groups were broken and lost their normal aligned structure.Masson results showed that myocardial fibrosis was aggravated and myocardial collagen fibres were significantly increased in the TAC group of C57BL/6J mice compared to the Sham group.1.4 RT-q PCR and Western blot detected myocardial hypertrophy markers ANP and β-MHC,and the results showed that ANP and β-MHC were significantly increased after TAC4 w.1.5 RT-q PCR,Western blot detected the expression of HIGD1 A after TAC surgery,and the results showed that HIGD1 A was significantly down-regulated after TAC4w(p<0.05).2.Expression of HIGD1 A in Ang II-induced cardiomyocyte hypertrophy 2.1 RT-q PCR for HIGD1 A m RNA content revealed that Ang II induced a decrease in HIGD1 A expression at 24 h and 48 h.2.2 Plasmid constructs overexpressed and silenced HIGD1A-specific cardiomyocytes.2.3 The results of the surface area of cardiomyocytes measured by the ghost pen cyclic peptide assay and the results of ANP and β-MHC measured by RT-q PCR showed that overexpression of HIGD1 A could reverse Ang II-induced cardiac hypertrophy(p < 0.01).2.4 The results of cardiomyocyte surface area by ghost pencil cyclic peptide assay and ANP and β-MHC by RT-q PCR showed that silencing of HIGD1 A reversed Ang II-induced myocardial hypertrophy(p < 0.01).2.5 HIGD1 A was found to affect cardiomyocyte hypertrophy levels by altering mitochondrial membrane potential and ROS levels as detected by JC-1,ROS kit 3.Up-regulation of HIGD1 A expression is involved in the anti-hypertrophic effect of simvastatin.We screened simvastatin by bioinformatics and pharmacological analysis and showed that HIGD1 A expression was elevated in the TAC8w+SIM group compared to the sham group,(p < 0.01).Conclusions: 1.The mitochondrial endosomal protein HIGD1 A was significantly down-regulated in cardiac hypertrophy mice and Ang Ⅱ-induced cardiac hypertrophic cells.2.In cardiomyocytes,overexpression of HIGD1 A retarded Ang II-induced cardiomyocyte hypertrophy,whereas silencing of HIGD1 A expression exacerbated Ang II-induced cardiomyocyte hypertrophy;3.Up-regulation of HIGD1 A expression is involved in the anti-hypertrophic effect of simvastatin 4.This study identifies the role of HIGD1 A in myocardial hypertrophy for the first time,and provides novel evidence and goal for the prevention and treatment of myocardial hypertrophy and heart failure by affecting mitochondrial function.