Cardiac Specific Deletion Of Sorting Nexin Protein 16(SNX16) Protects Heart From Ang?-Induced Cardiac Hypertrophy In Mice

Background and objective: Cardiac hypertrophy plays a key role in the development of heart failure.Studies have shown that sorting nexin protein 16(SNX16)is closely related to heart failure.GWAS analysis from Ensemble database revealed that a single base mutation of Snx16 gene was detected in heart failure patients.In zebrafish,the level of Snx16 m RNA was significantly increased in the heart failure model.Studies have demonstrated that SNX16 is widely involved in the processes of protein transport,cargo endocytosis,cell signal transduction,etc.However,the effects of SNX16 on the maintenance of cardiac function and its roles in the development of cardiac diseases have not been reported.Our study aims to clarify the role of SNX16 in normal and hypertrophic cardiomyocytes,and to explore its molecular mechanism.Obviously,our study should provide an insight in searching the novel targets for prevention and treatment of cardiac hypertrophy.Methods: 1.Construction of cardiac specific knockout Snx16 mice: CRISPR/Cas9 gene editing technique was used for preparing SNX16f/+ mice.We obtained SNX16f/+ heterozygous mice by inserting lox P sequence at both sides of mouse Snx16 gene exon 2.The mice were then backcrossed with Mlc-2v cre mice to obtain heart specific Snx16 gene knockout mice(SNX16f/f+Cre+).2.Establishment of mouse cardiac hypertrophy model: WT,SNX16f/f-Cre and SNX16f/f+Cre mice at 8-12 weeks were selected and continuously injected with Ang?(2 ?g/kg/min)for 14 days by subcutaneous osmotic pumps to induce cardiac hypertrophy.The mice were analyzed by cardiac morphology,HW/BW ratio,echocardiography and blood pressure.3.Establishment of cardiomyocyte hypertrophy model in vitro and drug stimulation: SNX16 overexpression vector was constructed firstly and then the vector was transfected into neonatal rat ventricular myocytes(NRVM),neonatal mouse ventricular myocytes(NMVM)and rat cardiomyocytes(H9C2).To determine the effect of SNX16 overexpression on cardiac hypertrophy,cardiomyocytes by Ang? stimulation.4.Detection of gene m RNA and protein levels and immunohistochemical analysis: 1)Cardiac cytoskeletal protein was stained by phalloidin or ?-actinin to calculate the surface area of single cardiomyocytes(? 50 cells).The effect of SNX16 knockout or overexpression on Ang? induced cardiomyocyte hypertrophy was analyzed.2)Western bolt and RT-q PCR were used to detect the expression of cardiac hypertrophic markers,such as ANP and BNP,and the key protein levels in EGFR/ERK1/2 signaling pathway of cardiac hypertrophy in the cardiomyocytes with SNX16 deficiency or overexpression.Results: 1.The expression levels of SNX16 were inconsistent in different tissues of 2-month-old C57BL/6 mice,including the heart,liver,brain,spleen,lung,kidney,muscle and fat.The level of SNX16 was relatively low in the heart,lungs and kidneys of mice while was relatively high in the liver,brain,spleen,muscle and fat.2.Snx16 m RNA and protein expression levels were significantly increased in Ang?-induced hypertrophic myocardial cells.Overexpressed SNX16 cardiomyocytes showed some phenotypes of myocardial hypertrophy,such as the enlargement in cell surface area and the increase levels of hypertrophic markers(ANP,BNP).3.Cardiac-specific knockout of Snx16 gene had no significant effect on mouse body weight,cardiac function,cardiac volume and tissue structure.Cardiac Snx16 deficiency significantly inhibited Ang?-induced pathological cardiac hypertrophy,which could significantly relieve the increase of cardiac volume,cardiac weight ratio,cardiomyocyte area,expression of myocardial hypertrophy markers,cardiac function and blood pressure induced by Ang?.4.The EGFR/ERK1/2 signaling pathway is activated in Ang?-induced cardiac hypertrophy.In Snx16 knockout mice,the phosphorylation of EGFR and its downstream key molecule ERK1/2 was significantly inhibited.However,overexpression of SNX16 markedly increased the phosphorylation of EGFR and ERK1/2.5.AZD9291 is an irreversible inhibitor of EGFR,which can effectively inhibit the phosphorylation of EGFR induced by EGF and further to prevent the activation of downstream molecule ERK.ADZ9291 remarkably inhibited the EGFR/ERK1/2 signaling pathway that activated by SNX16 overexpression in cardiomyocytes,thereby reducing cardiac hypertrophy,indicating that SNX16 promoted cardiac hypertrophy through EGFR-dependent signaling pathway.Conclusions: 1.Both in vivo and in vitro experiments have demonstrated that Snx16 m RNA and protein levels was significantly increased in Ang?-induced hypertrophic cardiomyocytes.2.In myocardial cell,SNX16 overexpression caused the enlargement of cardiomyocyte area and the increased expression of ANP and BNP,the markers of cardiac hypertrophy.The mechanism may be related to the activation of EGFR/ERK1/2 signaling pathway by overexpression of SNX16.3.Cardiac-specific deletion of Snx16 gene had no significant effect on the structure and function of heart in mice.Cardiac Snx16 deficiency can significantly reduce myocardial hypertrophy induced by Ang? in mice,and its mechanism is related to the significant inhibition of EGFR/ERK1/2 signaling pathway activation induced by Ang? stimulation.