Mfn2 Inhibits Cardiac Hypertrophy By Regulating Mams And Cytoplasmic Ca²⁺

Objective:Pathological cardiac hypertrophy is a compensatory cardiac dilatation caused by various injury-stimulating factors and sustained stress load.Prolonged cardiac hypertrophy is one of the pathological factors of arrhythmia,heart failure,and even sudden death.Currently,there is no effective treatment strategy for cardiac hypertrophy.Mitochondria associated endoplasmic reticulum membranes（MAMs）act as the connecting structure of mitochondria and endoplasmic reticulum,and play a role in intracellular calcium homeostasis,mitochondrial metabolism,and mitochondrial autophagy.Studies have shown that abnormal MAMs are associated with a variety of diseases,but the mechanism of MAMs in cardiac hypertrophy has been less studied.Therefore,this study aims to explore whether MAMs are involved in cardiac hypertrophy and whether this effect inhibited cardiac hypertrophy by mitofusin2（Mfn2）regulation of MAMs and cytoplasmic calcium(Ca²⁺).Methods:In animal experiments,after the Mfn2 knockout mouse model and cardiac hypertrophy mouse model establish by angiotensin II（Ang II）were successfully constructed,the heart function of mice was detected by small animal ultrasound apparatus.Hematoxylin&eosin（H&E）staining was used to detect the heart pathological state of mice.Wheat germ agglutinin（WGA）staining was used to detect cardiomyocytes surface area.Real-time quantitative PCR（RT-q PCR）was used to determine cardiac hypertrophy genes,including Atrial natriuretic peptide（ANP）,brain natriuretic peptide（BNP）andβ-myosin heavy chain（β-MHC）.In the cell experiment,Western Blot and RT-q PCR were used to detect Mfn2 protein and m RNA expression levels after Ang II treatment.TRITC-phalloidin staining and RT-q PCR were used to detect cardiomyocytes surface area and hypertrophy genes expression levels after infection with Mfn2 overexpressed virus and transfection with small interfering RNA（si RNA）.Western Blot was used to detect the MAMs-related protein levels after Ang II treatment.Mito-tracker and ER-tracker were used to detect MAMs formation infected by overexpressing Mfn2.Through 2’,7’-Dichlorodihydrofluorescein diacetate（DCFH-DA）and Flou-4,AM probes were used to detect Reactive oxygen species（ROS）and cytoplasmic Ca²⁺levels after overexpressing Mfn2.Finally,RT-q PCR was used to detect hypertrophy genes expression levels after calmodulin（Ca M）inhibitor treatment.Results:Compared with Mfn2^flox/floxmice,Mfn2 knockout mice significantly showed cardiac hypertrophy and cardiac dysfunction.Compared with Mfn2^flox/floxmice,Mfn2knockout mice with Ang II treatment less significantly showed these.Mfn2 protein and m RNA levels appeared a time-dependent decrease in neonatal rat cardiomyocytes treated with Ang II.TRITC-phalloidin staining and RT-q PCR showed that overexpressing Mfn2inhibited cardiac hypertrophy by decreasing the cell surface area and by downregulating ANP,BNP,andβ-MHC hypertrophy genes expression levels,while knockdowning Mfn2sensitized cells to undergo Ang II-induced cardiac hypertrophy.Western Blot showed that MAMs marker proteins inositol 1,4,5-trisphosphate receptor（IP3R）,FUN14 domain containing protein 1（FUNDC1）and phosphofurin acidic cluster sorting protein 2（PACS-2）appeared a time-dependent decrease in cardiomyocytes after Ang II treatment.Mito-tracker and ER-tracker showed that overexpression of Mfn2 significantly attenuated MAMs decreasing in cardiomyocytes induced by Ang II treatment.DCFH-DA and Flou-4,AM probes showed that overexpressing Mfn2 efficiently attenuated Ca²⁺level increasing in cardiomyocytes induced by Ang II treatment.RT-q PCR results showed that inactivation of Ca M inhibited Ang II and Mfn2 deficiency-induced upregulating hypertrophy genes expression levels.Conclusions:At the animal level,Mfn2 knockout can aggravate Ang II-induced cardiac hypertrophy and cardiac dysfunction.At the cellular level,we explored Mfn2inhibits cardiac hypertrophy by regulating MAMs and cytoplasmic Ca²⁺.Our results reveal the pathological mechanism of Mfn2-mediated cardiac hypertrophy and provide effective prevention and treatment strategies for cardiac hypertrophy and cardiovascular disease caused by persistent cardiac hypertrophy.