The Mechanisms Of Sorting Nexin 17 In Doxorubicin Induced Myocardial Injury

Objective:To study whether SNX17 is involved in the regulation of doxorubicin induced myocardial injury and the related mechanisms.Methods:1.The isolated and purified primary neonatal rat ventricular myocytes(NRVMs)were used to construct cell model of doxorubicin induced myocardial injury.Quantitative real-time PCR and Western Blot were applied to detect the changes in m RNA and protein expression of SNX17 to clarify the connection between SNX17 and doxorubicin myocardial injury.2.Cardiomyocytes were transfected with SNX17 si RNA and induced by doxorubicin,Western Blot and flow cytometry were used to detect the effect of SNX17 deficiency on doxorubicin myocardial injury..3.SNX17 heterozygous knockout rats and their littermates were injected with doxorubicin through tail vein to construct animal model of doxorubicin myocardial injury.TUNEL staining,sirius scarlet staining and echocardiography were used to detect the effect of SNX17 deficiency on doxorubicin induced myocardial injury.Meanwhile,the survival rates of rats were counted.4.Immunoprecipitation was carried out using SNX17 antibody,and immunoprecipitation-mass spectrometry was applied to find the potential downstream target of SNX17.The expression of LMOD2 was detected by Western Blot in doxorubicin-induced cardiomyocyte damage model.Both Myc-SNX17 and HA-LMOD2 plasmids were transfected into the HEK293 cells,and immunofluorescence staining was used to observe the localization of SNX17 and LMOD2.Western Blot was used to verify the effect of SNX17 changes on LMOD2 after interference and overexpression of SNX17 in doxorubicin myocardial injury model.SNX17 heterozygous knockout and control rats were treated with doxorubicin,and Western Blot was conducted to verify the changes of LMOD2 before and after treatment to determine the regulatory roles of SNX17 on LMOD2.5.The changes in myofilament structure of cardiomyocytes after intervention of SNX17 were observed by immunofluorescence staining.The NRVMs were transfected with SNX17 si RNA and overexpression plasmids respectively,and MEA was applied to detect changes in myocardial contractility after SNX17 intervention.Meanwhile,NRVMs were interfered with SNX17 si RNA and transfected with LMOD2 overexpression plasmid to observe whether overexpression of LMOD2 can compensate for the decrease in myocardial contractility caused by the down-regulation of SNX17.6.Doxorubicin-induced NRVMs were transfected with SNX17 si RNA and LMOD2 overexpression plasmid to observe whether LMOD2 overexpression can inhibit the deterioration of doxorubicin myocardial damage caused by SNX17 deficiency by Western Blot.7.The plasmids with different domains of SNX17 were constructed and co-transfected with LMOD2 plasmid in HEK293 cells.Immunofluorescence staining and immunoprecipitation were used to identify the domain of SNX17 that interacts with LMOD2..8.NRVMs were transfected with SNX17 si RNA and lysosomal inhibitors,the way SNX17 regulates LMOD2 were determined by Western Blot and verified by immunofluorescence staining.Results:1.The m RNA and protein expression levels of SNX17 were down-regulated in the doxorubicin induced cardiomyocyte injury model.2.At the cellular level,the interference of SNX17 aggravated doxorubicin induced cardiomyocyte injury.At the animal level,the deficiency of SNX17 increases the mortality and aggravates the myocardial imjury caused by doxorubicin.3.LMOD2 is a potential target gene regulated by SNX17 through analysis of immunoprecipitation-mass spectrometry results.LMOD2 is down-regulated in the doxorubicin-induced myocardial injury model.SNX17 and LMOD2 interact and co-localize.The interference of SNX17 inhibits the expression of LMOD2,and overexpression of SNX17 can compensates for the decrease of LMOD2 caused by doxorubicin..4.Interference with SNX17 cause structural disorder of myofilament in cardiomyocytes and lead to decrease in myocardial contractility,while overexpression of LMOD2 can inhibit the decrease in myocardial contractility caused by down-regulation of SNX17.5.LMOD2 overexpression can inhibit the deterioration of doxorubicin-induced myocardial injury caused by SNX17 deficiency6.SNX17 binds to LMOD2 through its C-terminal domain,and SNX17 deficiency leads to increased degradation of LMOD2 via the lysosomal pathway,and lysosomal inhibitor can inhibit the degradation of LMOD2 in lysosome.Conclusion:1.In doxorubicin myocardial injury model,the expression of SNX17 was down-regulated,and the deficiency of SNX17 aggravates doxorubicin myocardial injury.2.In doxorubicin myocardial injury model,the deficiency of SNX17 aggravates the downregulation of LMOD2.3.The deficiency of SNX17 leads to the disorder of myofilament structure and the decrease of cardiomyocyte contractility by down-regulating LMOD2.4.SNX17 interacts with LMOD2 through its C-terminal domain,SNX17 deficiency leads to the increased degradation of LMOD2 in the lysosome.