Different Roles And Mechanisms Of BAG3 And MALAT1 In Cardiac Physiological Hypertrophy And Cardiac Pathological Hypertrophy

Background:Heart failure caused by a variety of heart diseases,is the leading cause of death worldwide.Stimulated heart undergoes physiological hypertrophy or pathological hypertrophy（also known as physiological remodeling or pathological remodeling）.Physiological hypertrophy characterized by adaptive growth of heart,unchanged or mildly enhanced systolic and diastolic function but not developed to cardiac dysfunction.There is no fibrosis and upregulation of fetal genes such as ANP,ACTC and Myh7 occur in physiological hypertrophy.While the expression of SERCA2a is increased.On the contrary,pathological hypertrophy characterized by maladaptive growth,fibrosis and activation of fetal genes such as ANP,ACTC and Myh7.Although a compensatory response may occur in the short term,heart function will deteriorate and eventually lead to heart failure.Physiological hypertrophy and pathological hypertrophy often occur simultaneously in the early stages of many heart diseases.Therefore,studying the mediating factors and mechanisms of both beneficial and harmful hypertrophy is helpful to protect against heart function deterioration and heart failure caused by many kinds of heart diseases.BAG3 is a protein highly expressed in heart,skeletal muscles and some tumors and regulates a number of biological processes including development,apoptosis,autophagy,and tumor development.A growing number of studies have shown that BAG3also plays important roles in heart,which can promote the degradation of misfolded proteins and damaged organelles,inhibit apoptosis of cardiomyocytes,maintain the structural integrity of z-disc of cardiomyocytes,regulate myocardial contractility and promote the transcription of filaments.In addition,in patients with advanced heart failure,the amount of BAG3 in myocardium decreased while the amount in blood increased.MALAT1 is a lncRNA highly expressed in heart and many tumors,which plays regulatory roles in tumor developing through various mechanisms.MALAT1 can promote angiogenesis in ischemic skeletal muscle and activate variety pathways,including serum response factor（SRF）and ERK/MAPK which have important effects on the pathophysiology of myocardium.However,it is not known how BAG3 and MALAT1are regulated and what their functions are during physiological hypertrophy and pathological remodeling.Objective:To investigate the regulation,function,and underlying mechanism of BAG3and MALAT1 in physiological hypertrophy and pathological remodeling.Methods:1,The first part:We generated TMX-induced heart-specific BAG3heterozygous and homozygous knockout mice.Then we constructed mouse models of cardiac physiological hypertrophy and cardiac pathological hypertrophy by using swimming training for tree weeks or PE osmotic minipumps implanting for two weeks respectively.Insulin growth factor 1（IGF1）and phenylephrine（PE）treated Neonatal rat ventricular cardiomyocytes（NRVC）were used to study functions and mechanisms in vitro.Adenoviruses containing flag-tagged full-length BAG3 or BAG3 specific shRNA were used to overexpress or knockdown BAG3 in NRVC.Echocardiography was used to detect the changes of heart function and morphology in mice.Immunohistochemical H&E staining was used to detect the changes in myocardial cross-sectional area.Masson staining was used to detect the fibrosis in myocardium.Western blot was used to test the different changes of two hypertrophies and pathways.Immunofluorescence was used to detect the changes in cell area.2,The second part:IGF1 or PE treated Human adult ventricular cardiomyocytes（AC16 cell line）were used to study functions and mechanisms of MALAT1 in two different hypertrophies.Lipofectamine 3000 reagent was used to transfect MALAT1 specific siRNA,miR-26a mimics or miR-26a inhibitor into AC16 cells.Real-time quantitative PCR was used to detect the expression levels of various RNAs.Western blot was used to test the different changes of two hypertrophies.Luciferase activity assay was used to verify the interaction between RNAs.Western blot,immunofluorescence and immunohistochemical images were processed by Photoshop2020.Gray values of Western blots were measured by image J（Ver 1.8.0）software.Immunofluorescence and immunohistochemical cell area were measured by Image Pro Plus software.Relative expression quantity in Real-time quantitative PCR was calculated by 2^-ΔΔCtΔΔCt formula.Bar charts were made by Graphpad Prism（Ver 7.0.4）software.All statistical analyses were performed by SPSS（IBM）software,and the results were presented as mean±standard error.The statistical methods were one-way ANOVA（followed by Bonferroni or Dunnet’s post-hoc tests as appropriate）and independent sample t-test analysis.P<0.05 was considered statistically significant.Results:1,The first part:We found that BAG3 was upregulated in both physiological hypertrophy and pathological hypertrophy in vivo and in vitro.Knockdown of BAG3 in NRVC（and heterozygous-KO BAG3 in heart）blunted IGF1（and swimming training）induced cardiac physiological hypertrophy.Overexpression of BAG3 promoted IGF1induced physiological hypertrophy in NRVC.On the contrary,BAG3 knocking-down in NRVC（and BAG3 heterozygous-KO in heart）aggravated PE induced cardiac pathological hypertrophy.Overexpression of BAG3 inhibited PE induced pathological hypertrophy in NRVC.In addition,BAG3 overexpression promoted physiological hypertrophy by activating Akt-mTOR pathway and could be reversed by PI3K or mTOR inhibitors（PF-04691502 or rapamycin）.BAG3 knocking-down aggravated pathological hypertrophy through activating calcineurin–NFATc2 pathway and could be reversed by calcineurin inhibitor（Cyclosporin A）.2,The second part:We found that MALAT1 was upregulated in both IGF1 induced physiological hypertrophy and PE induced pathological hypertrophy.Knockdown of MALAT1 in AC16 blunted PE induced pathological hypertrophy but had no effect on IGF1 induced physiological hypertrophy.We found that MALAT1 specifically binds to miR-26a and observed a reciprocal negative regulatory relationship between these factors.Knockdown of MALAT1repressed GATA4 expression and could be rescued by miR-26a inhibitor.The inhibition of pathological hypertrophy caused by MALAT1 knocking down could be reversed by miR-26a inhibitor.Conclusion:BAG3 and MALAT1 are upregulated in both physiological and pathological cardiac hypertrophy.BAG3 promotes cardiac physiological hypertrophy through activating Akt-mTOR pathway.BAG3 inhibits cardiac pathological hypertrophy through inhibiting calcineurin–NFAT_c22 pathway.MALAT1 promotes cardiac pathological hypertrophy through binding and inhibiting miR-26a.