The Role Of Transient Receptor Potential Ion Channel Protein 7 In Myocardial Hypertrophy And Underlying Mechanisms

SectionⅠThe role of transient receptor potential ion channelprotein 7 in pathological myocardial hypertrophyBackground and purpose:Myocardial hypertrophy is one of the main causes of increased morbidity and mortality of cardiovascular diseases,and is closely related to the development of a variety of cardiovascular events.A persistent state of myocardial hypertrophy will lead to systolic dysfunction,decompensated dilated cardiomyopathy,cardiac fibrosis,heart failure and even sudden death.Transient receptor potential ion channel protein 7（Trpm7）is increasingly studied in cardiovascular diseases,playing a very important role in regulating cell cycle,magnesium ion and calcium ion balance,while its role in myocardial hypertrophy still remains unclear.Objective:In this study,the effect of Trpm7 on the pathological process of myocardial hypertrophy in the mouse model of myocardial hypertrophy constructed by abdominal aortic coarctation（AAC）and in the model of myocardial hypertrophy induced by phenylephrine（PE）induced in H9C2 rat cardiomyocytes was discussed,It provides a new therapeutic target for clinical treatment.Methods:This study is divided into in vivo part and in vitro part.In vivo experiments were conducted on healthy adult SPF-level male B129 mice.A model of myocardial Trpm7-specific knockout(Trpm7^MYKO)was established based on Myh6-Cre-Lox P gene recombination technology.A mouse model of myocardial hypertrophy was constructed by AAC,and the mice were randomly divided into four groups:（1）Trpm7^fl/flsham surgery group(Sham+Trpm7^fl/fl);（2）Trpm7^MYKOsham surgery group(Sham+Trpm7^MYKO);（3）Trpm7^fl/flabdominal aortic coarctation group(AAC+Trpm7^fl/fl);（4）Trpm7^MYKOabdominal aortic coarctation group(AAC+Trpm7^MYKO),8 animals in each group.After 4 weeks of AAC,small animal echocardimeter was used to detect mouse heart Left ventricular ejection fraction（LVFE）,fraction shortening（FS）,left ventricular internal diameter at end-diastole（LVIDd）and interventricular septal thickness at diastole（IVSd）.Mouse heart tissue was taken and ratio of heart weight to body weigh（HW/BW）,ratio of heart weight to tibial length（HW/TL）,left ventricular weight were measured.H&E and Masson staining detected the morphology of heart disease in mice.Trpm7&WGA immunofluorescence double staining to assess Trpm7 expression and cardiomyocyte size changes in mouse heart tissues.q RT-PCR and Western Blot detected Trpm7,ANP,BNP,β-MHC m RNA and protein expression levels in the heart tissues of each group of mice.In vitro experiments took rat cardiomyocytes（H9C2 cells）with good growth status,The hypertrophic model of H9C2 cardiomyocytes was established by PE stimulation.CCK-8 detected the effect of different concentrations of PE on cardiomyocytes viability,and obtained the optimal concentration of PE-induced H9C2 cardiomyocyte hypertrophic model.Transfection of lentivirus sh-Trpm7-RNAi into H9C2 cells interfered with Trpm7 expression.Cardiomyocytes were randomly divided into 4 groups:（1）sh-NC group:PBS treatment was added to cardiomyocytes transfected by lentiviral negative control;（2）sh-Trpm7 group:PBS treatment was added to lentiviral sh-Trpm7-RNAi transfected cardiomyocytes;（3）PE+sh-NC group:treatment with PE was added to cardiomyocytes transfected with lentiviral negative control;（4）PE+sh-Trpm7 group:Treatment with PE was added to lentiviral sh-Trpm7-RNAi transfected cardiomyocytes.Western Blot and q RT-PCR detected the expression levels of Trpm7,ANP,BNP,andβ-MHC in cardiomyocytes in each treatment group.Cell immunofluorescence assay evaluated the changes in cardiomyocyte surface area in each treatment group.Results:1)The expression of Trpm7 was up-regulated in the heart tissue of AAC-induced hypertrophy mice:the results showed that compared with the Sham group,the AAC group observed that the AAC group observed that the mouse had heart dysfunction,LVEF and FS decreased significantly（P<0.01）,increased left ventricular weight（P<0.001）,myocardial cell arrangement,inconsistent cell size,enlarged cardiomyocytes,and the expression of ANP,BNP andβ-MHC markers associated with myocardial hypertrophy in mouse heart tissue was significantly upregulated（P<0.05）,Trpm7 expression levels were also significantly upregulated（P<0.05）.2)Construction of Trpm7 heart-specific knockout mice:Trpm7 fluorescence staining and q RT-PCR results constructed using Cre-Lox P technology showed that the fluorescence intensity of Trpm7^MYKOgroup was significantly reduced and the expression level of Trpm7 m RNA decreased（P<0.05）compared with the Trpm7^fl/flgroup.3)Trpm7 myocardial specific knockout improves AAC-induced cardiac dysfunction in mice:in the AAC-induced mice myocardial hypertrophy model,significant cardiac dysfunction was observed in the AAC+Trpm7^fl/flgroup,LVIDd and IVSd increased significantly（P<0.05）,LVEF and FS decreased significantly（P<0.01）,and Trpm7 m RNA and protein levels increased（P<0.05）;Cardiac dysfunction was significantly improved in the AAC+Trpm7^MYKOgroup,LVIDd and IVSd decreased（P<0.05）,LVEF and FS increased（P<0.05）,and Trpm7 m RNA and protein levels were significantly reduced（P<0.01）.4)Trpm7 myocardial specific knockout improves AAC-induced hypertrophy:histopathology and mouse physiological parameter detection results showed that the arrangement of cardiomyocytes in the AAC+Trpm7^fl/flgroup was disordered,the cardiomyocyte space was significantly widened and of different sizes,the cell size was inconsistent,interstitial fibrosis appears,the surface area of cardiomyocytes increased（P<0.001）,the weight of HW/BW,HW/TL,and left ventricle was significantly increased（P<0.001）,and the intercellular space of AAC+Trpm7^MYKOgroup became smaller and roughly equal.The arrangement of cardiomyocytes was more neat and normal,the degree of fibrosis was improved,the surface area of cardiomyocytes decreased（P<0.001）,and the weight of HW/BW,HW/TL and left ventricle were significantly reduced（P<0.05）.The results of q RT-PCR showed that the levels of ANP,BNP andβ-MHC m RNA in the AAC+Trpm7^fl/flgroup increased significantly（P<0.05）,and the levels of ANP,BNP andβ-MHC m RNA in the AAC+Trpm7^MYKOgroup decreased significantly（P<0.05）.The results of Western blot showed that the levels of ANP,BNP andβ-MHC proteins in the AAC+Trpm7^fl/flgroup increased significantly（P<0.01）,and the levels of ANP,BNP andβ-MHC proteins in the AAC+Trpm7^MYKOgroup decreased significantly（P<0.05）.5)PE induced H9C2 cardiomyocyte hypertrophy:CCK-8 experimental results showed that compared with the CON group,different concentrations of PE（10μM～200μM）treated rat cardiomyocytes for 48h,100μM PE significantly inhibited cardiomyocyte viability（P<0.05）.Other concentrations have no significant effect on cardiomyocyte viability,therefore,100μM PE was selected for 48h to construct H9C2cardiomyocyte hypertrophy model.Compared with the CON group,cardiomyocytes in the PE group significantly increased their surface area（P<0.05）,the expression levels of ANP,BNP andβ-MHC proteins,and the expression of Trpm7 proteins（P<0.05）were significantly increased.6)The results of q RT-PCR and Western blot transfection of H9C2 cardiomyo-cytes using Trpm7 silencing lentivirus showed that Trpm7 m RNA and protein expression were significantly reduced（P<0.01）in sh-Trpm7 group compared with sh-NC group.7)Lentiviral silencing Trpm7 significantly reverses elevated Trpm7 expression in mast cells:Trpm7 m RNA and protein expression in sh-NC+PE group was significantly increased（P<0.01）compared with sh-NC group in lentiviral transfection and PE-induced mast cardiomyocytes,and Trpm7 m RNA and protein expression in sh-Trpm7+PE group was significantly reduced（P<0.05）.8)Lentiviral silencing Trpm7 significantly improved PE-induced cardiomyo-cyte hypertrophy:compared with sh-NC group,cardiomyocytes in sh-NC+PE group increased cardiomyocytes and significantly increased surface area（P<0.001）,and compared with sh-NC+PE group,cardiomyocytes decreased and surface area decreased significantly（P<0.01）in sh-NC+PE group.The results of q RT-PCR showed that compared with the sh-NC group,the levels of BNP andβ-MHC m RNA in the sh-NC+PE group increased significantly after 48 hours of PE stimulation（P<0.05）,and compared with the sh-NC+PE group,the levels of BNP andβ-MHC m RNA in the sh-Trpm7+PE group decreased significantly（P<0.05）.Compared with the sh-NC group,the levels of ANP,BNP andβ-MHC proteins in the sh-NC+PE group increased significantly after 48 hours of PE stimulation（P<0.001）,and compared with the sh-NC+PE group,the levels of ANP,BNP andβ-MHC proteins in the sh-Trpm7+PE group decreased significantly（P<0.05）.Conclusions:Trpm7 up-regulated during myocardial hypertrophy,which can promote pathological myocardial hypertrophy.SectionⅡThe molecular mechanism of Trpm7 in pathologicalmyocardial hypertrophyObjective:In this study,the mechanism of JAK2/STAT3 signaling pathway in the promotion of pathologic myocardial hypertrophy by Trpm7 was investigated in mouse myocardial hypertrophy model constructed by AAC and H9C2 rat myocardial hypertrophy model induced by PE.Methods:This study is divided into in vivo part and in vitro part.1)In vivo experiments were performed on mice and grouped as in part I.JAK2and STAT3 phosphorylation levels were detected in myocardial tissue taken from mice 4 weeks after AAC surgery.2)In vitro experiments using PE（100μM）treatment for 48h to construct an H9C2 cardiomyocyte hypertrophy model.They were randomly divided into five groups:control group（CON group）,PE group,PE+Trpm7 lentivirus silencing group（PE+sh-Trpm7 group）,PE+JAK2 inhibitor group（PE+LY2784544 group）,PE+Trpm7lentivirus silencing group+JAK2 activator（PE+sh-Trpm7+RO8191 group）,and after each group was treated accordingly,Western blot detected ANP,BNP,β-MHC and Trpm7 protein expression and JAK2 and STAT3 phosphorylation levels in each group of cells.Results:1)In vivo experiments showed that AAC significantly increased the levels of JAK2 and STAT3 protein phosphorylation in mouse myocardial tissue（P<0.05）.Myocardial specific knockout of Trpm7 significantly inhibited AAC-induced JAK2and STAT3 phosphorylation（P<0.01）.2)In vitro results showed that PE significantly increased the phosphorylation of JAK2 and STAT3 in H9C2 cells（P<0.01）.sh-Trpm7 significantly inhibited PE-induced phosphorylation of JAK2 and STAT3（P<0.01）..3)JAK2 selective inhibitor LY2784544 significantly inhibited the protein levels of cardiac hypertrophy markers ANP,BNP,β-MHC and Trpm7 in PE-induced H9C2cells（P<0.05）.4)sh-Trpm7 significantly inhibited the PE-induced expression of ANP,BNP,β-MHC and Trpm7 proteins,and JAK2 activator RO8191 significantly reversed the inhibitory effect of sh-Trpm7 on the expression of ANP,BNP,β-MHC and Trpm7proteins.Conclusions:Trpm7 promotes pathological myocardial hypertrophy by activating the JAK2/STAT3 signaling pathway.