| Background and Objective:Pathological myocardial hypertrophy,characterized by embryonic gene activation,increased protein synthesis and increased cardiomyocyte size,is an important risk factor for heart failure.Neurohumoral activation(such as angiotensin II [Ang Ⅱ])is a key factor leading to pathological myocardial hypertrophy.Although a large number of studies have shown that multiple signaling pathways are involved in Ang Ⅱ-induced myocardial hypertrophy,the specific mechanism is still not completely clear.CSN5,also known as Jab1,was originally identified as a c-Jun co-activator involved in the regulation of signal transduction,gene transcription,and protein stability.In recent years,there has been increasing evidence that CSN5 is involved in a variety of cellular processes,thereby controlling the occurrence and development of various diseases,including cardiovascular disease.However,the role and mechanism of CSN5 in Ang Ⅱ-induced myocardial hypertrophy are not clear.In this study,we first constructed a mouse model of Ang Ⅱ-induced myocardial hypertrophy and a myocardial mast cell model,and detected the expression changes of CSN5 in heart tissues and cells.Secondly,we verified the effect of CSN5 on myocardial hypertrophy through in vitro cell experiments.Finally,we explored the specific mechanism of CSN5 in regulating Ang Ⅱ-induced myocardial hypertrophy.These results will provide new insights into the biological functions and potential mechanisms of CSN5 in pathological myocardial cell hypertrophy and new ideas for the treatment of pathological myocardial cell hypertrophy.Methods:1.In the Ang Ⅱ-induced myocardial hypertrophy mouse model,the expression of hypertrophic marker genes atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and β-myosin heavy chain(β-MHC)as well as WGA staining were detected by qRT-PCR assay to verify the success of model establishment.After the model was successfully established,the expression changes of CSN5 m RNA and protein in myocardial tissue were detected by qRT-PCR and Western blot assay,and the cellular localization of CSN5 expression in myocardial tissue was detected by tissue immunofluorescence assay.2.After primary neonatal rat cardiomyocytes(NRCM)were isolated,they were treated with Ang Ⅱ to construct an in vitro cardiomyocyte hypertrophy model.qRT-PCR and Western blot experiments were performed to detect the expression changes of CSN5 m RNA and protein in hypertrophic cardiomyocytes,and the subcellular distribution of CSN5 expression was detected by cellular immunofluores-cence experiment.3.The primary cardiomyocytes of neonatal rats infected with adenoviruses Ad-CSN5 and Ad-sh CSN5 were used to up-regulate or down-regulate the expression of CSN5 and to add Ang Ⅱ-treated cells.The expression changes of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and β-myosin heavy chain(β-MHC),the hypertrophic marker genes,were detected by qRT-PCR experiment,and the cell size changes were observed by cellular immunofluorescence.4.The primary cardiomyocytes of neonatal rats infected with adenoviruses Ad-CSN5 and Ad-sh CSN5 were used to up-regulate or down-regulate the expression of CSN5 and detect the expression changes of AMPK-ACC signaling pathway related proteins and CSN5 proteins by Western blot experiment.5.The primary cardiomyocytes of neonatal rats were infected by adenovirus Ad-CSN5,and at the same time,the AMPK pathway was inhibited by adenovirus Ad-sh AMPK or AMPK pathway inhibitor CC,and Ang Ⅱ was added to treat the cells.The expression changes of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and β-myosin heavy chain(β-MHC),the hypertrophic marker genes,were detected by qRT-PCR,and the cell size changes were observed by cellular immunofluorescence.6.The primary cardiomyocytes of neonatal rats were infected with adenoviruses Ad-CSN5 m RNA Ad-sh CSN5 to up-regulate or down-regulate CSN5.The m RNA and protein expression levels of LKB1 and CSN5 were detected by qRT-PCR and Western blot experiments.7.The primary cardiomyocytes of neonatal rats were infected with adenovirus Ad-sh CSN5,and Cycloheximide,CHX)was added to inhibit the synthesis of intracellular protein.The degradation rate of LKB1 protein was detected by Western blot experiment.8.Protein immunoprecipitation experiment was performed to determine whether the endogenous expression in primary myocardial cells and the exogenous CSN5 introduced from 293 T cells could directly bind to LKB1 protein;9.The primary cardiomyocytes of neonatal rats infected with adenoviruses Ad-CSN5 and Ad-sh CSN5 were used to up-regulate or down-regulate the expression of CSN5,and Western blot experiment was conducted to detect the changes in the level of polyubiquitination at k48 of LKB1 protein.10.The primary cardiomyocytes of neonatal rats infected with adenoviruses Ad-CSN5 and Ad-sh CSN5 were used to up-regulate or down-regulate the expression of CSN5,while adenoviruses Ad-sh LKB1 and Ad-LKB1 were used to down-regulate or up-regulate the expression of LKB1.The expression changes of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and β-myosin heavy chain(β-MHC),the hypertrophic marker genes,were detected by qRT-PCR experiments,and the cell size changes were observed by cellular immunofluorescence.Results:1.The results of qRT-PCR indicated that the expression levels of hypertrophic marker genes(ANP,BNP,and β-MHC)in the heart tissue of the Ang Ⅱ-induced myocardial hypertrophy mouse model were significantly increased,and the volume of myocardial cells stained by WGA was increased,indicating that the myocardial hypertrophy mouse model was successfully established.The results of qRT-PCR and Western blot experiments indicated that the expression levels of CSN5 m RNA and protein in the heart tissue of the successfully established myocardial hypertrophy mouse model were significantly increased,and the increased CSN5 expression was found in the cytoplasm of myocardial cells by tissue immunofluorescence experiments.2.The results of qRT-PCR and Western blot experiments showed that the expression levels of CSN5 m RNA and protein in the Ang Ⅱ-induced primary cardiomyocyte hypertrophy model of neonatal rats were significantly increased.The cell immunofluorescence experiment showed that the increased CSN5 expression was mainly limited to the cardiomyocytes in cardiac tissue.3.The results of qRT-PCR and cellular immunofluorescence experiments showed that the overexpression of CSN5 in primary neonatal rat cardiomyocytes could inhibit the increase in the expression of Ang Ⅱ-induced hypertrophic marker genes(ANP,BNP,β-MHC)in cardiomyocytes and the hypertrophy of cardiomyocytes.However,interfering with the expression of CSN5 in primary neonatal rat cardiomyocytes could further promote the increase in the expression of Ang Ⅱ-induced hypertrophic marker genes(ANP,BNP,β-MHC)in cardiomyocytes and the hypertrophy of cardiomyocytes.4.Western blot results showed that after interfering with the expression of CSN5 in primary neonatal rat cardiomyocytes,the threonine(Thr)phosphorylation at locus172 of AMPK protein and serine(Ser7)phosphorylation at locus 9 of ACC protein were decreased and their total protein expressions remained unchanged.However,after overexpression of CSN5 in primary neonatal rat cardiomyocytes,the threonine(Thr)phosphorylation at locus 172 of AMPK protein and serine(Ser7)phosphorylation at locus 9 of ACC protein were increased and their total protein expressions remained unchanged.5.Results of qRT-PCR and cellular immunofluorescence experiments showed that overexpression of CSN5 in primary cardiomyocytes of neonatal rats while interfering with the expression of AMPK or the addition of AMPK pathway inhibitor CC could significantly block the expression of CSN5 over-expression in Ang Ⅱ-induced cardiomyocyte hypertrophic marker genes(ANP,BNP,β-MHC)and the inhibitory effect on cardiomyocyte hypertrophy.6.The results of qRT-PCR and Western blot experiments showed that there was no significant difference in the expression of LKB1 m RNA after overexpression of CSN5 in primary neonatal rat cardiomyocytes,but the protein expression was significantly increased.Similarly,after interfering with the expression of CSN5,there was no significant difference in the expression of LKB1 m RNA but the protein expression was significantly decreased in primary neonatal rat cardiomyocytes.7.Western blot results showed that the degradation rate of LKB1 protein was significantly increased after the addition of CHX while interfering with the expression of CSN5 in primary cardiomyocytes of neonatal rats.8.The results of protein immunoprecipitation experiment showed that the CSN5 and LKB1 proteins expressed endogenous and introduced exogenously could be directly combined;9.Western blot results show that the overexpression of CSN5 in primary neonatal rat cardiomyocytes results in a significant decrease in the polyubiquitination at k48 of LKB1 protein,while the overexpression of CSN5 in primary neonatal rat cardiomyocytes results in a significant increase in the polyubiquitination at k48 of LKB1 protein.10.Results of qRT-PCR and cellular immunofluorescence experiments showed that overexpression of CSN5 in primary cardiomyocytes of neonatal rats while interfering with the expression of LKB1 could significantly block the inhibition of overexpression of CSN5 on Ang Ⅱ-induced increase in the expression of myocardial cell hypertrophic marker genes(ANP,BNP,β-MHC)and myocardial cell hypertrophy.However,co-expression of LKB1 while interfering with CSN5 expression in primary cardiomyocytes of neonatal rats significantly blocked the promotion of Ang Ⅱ-induced increase in the expression of hypertrophic marker genes(ANP,BNP,β-MHC)in cardiomyocytes and cardiomyocyte hypertrophy by interfering with CSN5 expression.Conclusion:In this study,we found that the expression of CSN5 was increased in the heart of Ang Ⅱ-induced myocardial hypertrophy mouse model and in the primary cardiomyocytes of Ang Ⅱ-treated neonatal rats.Overexpression of CSN5 in primary cardiomyocytes of neonatal rats significantly inhibited Ang Ⅱ-induced myocardial hypertrophy,while interference with CSN5 expression exhibited an opposite phenotype.Further mechanism studies have shown that CSN5 maintains AMPK activity in myocardial cells by stabilizing the expression of LKB1,thereby reducing Ang Ⅱ-induced myocardial hypertrophy.In summary,the results of these studies suggest that strategies based on CSN5/LKB1 axis activation provide new ideas for the treatment of hypertrophic cardiomyopathy. |
