The Role Of FoxP3 In Cardiac Hypertrophy And Transcription Process Of Myh7 With The Effect Of Triptolide

Background and objective:Almost all pathological changes in cardiovascular systems will lead to cardiac hypertrophy.Although early-stage cardiac hypertrophy has certain compensatory functions for the heart,long-term cardiac hypertrophy eventually leads to impaired cardiac function and even progresses to heart failure,resulting in death.Currently,there are limited treatment options for cardiac hypertrophy in clinical practice,and their efficacy is often unsatisfactory due to the complex pathological mechanism of cardiac hypertrophy.Despite extensive research on the mechanisms of cardiac hypertrophy,the underlying mechanisms are not yet clear.Transcription factors play an unparalleled role in the molecular biology mechanisms of cardiac hypertrophy,as they can regulate gene expression.Our previous studies showed that triptolide(Tp),a kind of traditional Chinese medicine,could up-regulate forkhead box transcriptional factor P3(FoxP3)expression in cardiomyocytes to exert a protective effect in the early stages of cardiac hypertrophy by inhibiting the autophagy of cardiomyocytes.However,the direct role of cardiomyocytic FoxP3 in cardiac hypertrophy is still unclear.In cardiac hypertrophy,fetal genes such as atrial natriuretic peptide(Anp),brain natriuretic peptide(Bnp),and myosin heavy chain 7(Myh7)are re-activated and re-expressed,called fetal gene reprogramming which is a common feature of pathological hypertrophic cardiomyocytes,and the re-expression of fetal genes often damages the function of cardiomyocytes.Therefore,intervening in fetal gene reprogramming may be an effective strategy for treating cardiac hypertrophy.In fetal gene reprogramming,nuclear factor of activated T-cells(NFAT)is a core regulatory factor.However,due to the weak DNA binding capability of NFATc3,the combination of other transcription factors is necessary for the gene regulatory function of NFATc3.Inspired by the interaction between FoxP3 and NFAT in CD4~+T cells downregulating interleukin 17(IL 17)expression,we hypothesized that FoxP3 could reduce the downstream fetal gene transcription driven by NFAT and inhibit fetal gene reprogramming by binding to NFAT,thereby affecting cardiac hypertrophy.To verify the above hypothesis,this study aims to explore the molecular biological mechanism of FoxP3-mediated regulation of fetal genes in cardiomyocytes and investigate the intervention effect of triptolide to further understand the pathogenesis of cardiac hypertrophy and lay the foundation for exploring new preventive and therapeutic strategies.Methods:1.Using isoproterenol(ISO)to cause hypertrophic response of neonatal rat ventricular myocyte(NRVM)and H9c2 cardiomyocyte.These cells were stained with an actin-tracker,and the cell area was measured to evaluate the quality of the hypertrophic model and the therapeutic effect of Tp.The expression levels of fetal genes(Anp,Bnp,and Myh7)and Foxp3 were assessed by real-time PCR and western blot.Moreover,the cytoplasmic and nuclear proteins were isolated to determine the spatial expression characteristics of FoxP3in hypertrophic cardiomyocytes,along with the effect of Tp on FoxP3 nuclear translocation.2.By transfecting si RNA or plasmid,the expression of FoxP3 in NRVM was knocked down or overexpressed.Cardiac hypertrophy model was constructed by ISO,and Tp was used to treat cardiac hypertrophy.Cell skeleton staining was used to measure cell area to determine the effect of modulated expression of FoxP3 on cardiac hypertrophy and therapeutic effects of Tp.Real-time PCR was used to determine whether the modulated expression of FoxP3 affected the expression of fetal genes(Anp,Bnp and Myh7)hypertrophy-related genes(Cox2),and NFATc3.3.To induce an increase in FoxP3 expression in the hearts of mice,an adenovirus-associated virus expressing FoxP3(AAV-FoxP3)was injected through tail vein.Meanwhile,using a mini-osmotic pump containing to build a cardiac hypertrophy model by continuously and stably releasing ISO.Tp was used to treat cardiac hypertrophy.Immunohistochemistry,Western blot and immunofluorescence techniques were used to confirm whether AAV-FoxP3 successfully induced an increase in FoxP3 expression in the hearts of mice and cardiomyocytes in vitro.Hematoxylin-eosin(HE)staining,Masson staining,wheat germ agglutinin(WGA)staining,and ultrasound inspection were used to determine the effects of increased cardiomyocytic FoxP3 expression on cardiac hypertrophy and investigate the effect of Tp.4.Immunofluorescence was used to determine whether there was an interaction between NFATc3 and FoxP3 in the nuclei of cardiac cells.Subsequently,the total protein and nuclear protein were extracted from cardiac cells.Using immunoprecipitation(IP)to confirm whether NFATc3 and FoxP3 interact with each other,as well as changes in their interaction under hypertrophy and the effect of Tp.5.Using chromatin immunoprecipitation(Ch IP)technology to confirm the target genes regulated by FoxP3 and the impact of cardiac hypertrophy on this regulatory effect.Electrophoretic mobility shift assay was used to identify the characteristic binding sequences of FoxP3 on the target genes.Probe enzyme-linked immunosorbent assay(ELISA)was used to determine the binding ability of FoxP3 and the modulating effect of Tp.Results:1.ISO successfully induced cardiac hypertrophy in cardiomyocytes,which was characterized by an increase in cell surface area and upregulation of fetal gene expression.In hypertrophic cardiomyocytes,the expression and nuclear translocation of FoxP3 were significantly downregulated.Tp effectively restored the expression and nuclear translocation of FoxP3,which might be associated with the therapeutic effects of Tp on cardiac hypertrophy.2.Knockdown of FoxP3 in cardiomyocytes increased their sensitivity to pathological stimuli,resulting in an increase in cell size and a significant upregulation of ANP,BNP,MYH7,COX2,and NFATc3 expression.Conversely,overexpression of FoxP3 in cardiomyocytes significantly inhibited ISO-induced cardiac hypertrophy,leading to a decrease in cell size and a significant reduction in ANP,BNP,MYH7,COX2,and NFATc3expression.3.Upregulation of FoxP3 expression in myocardial tissue inhibited ISO-induced cardiac hypertrophy and MYH7 expression.Myocardial tissue inflammation cell infiltration,myocardial fibrosis,and cardiac function were all improved by the amplification of FoxP3.Tp treatment restored FoxP3 expression in hypertrophic hearts and effectively alleviated cardiac hypertrophy.4.In the nuclei of hypertrophic cardiomyocytes,the interaction between FoxP3 and NFATc3 was weakened,while Tp treatment up-regulated this interaction.5.FoxP3 in cardiomyocytes could bind to characteristic sequences upstream of the Myh7 gene to regulate transcription of Myh7,a NFATc3-driven gene.In hypertrophic cardiomyocytes,the binding of FoxP3 to this characteristic sequence is weakened,while Tp treatment restored the binding of FoxP3 to the Myh7 promoter.Conclusion:1.Expression and nuclear translocation of FoxP3 were both reduced in hypertrophic cardiomyocytes.2.Downregulation of FoxP3 expression exacerbates isoproterenol-induced hypertrophy of cardiomyocytes,while upregulation of FoxP3 expression effectively alleviates myocardial hypertrophy.3.Cardiomyocytic FoxP3 inhibited the transcription of fetal gene Myh7 by interacting with NFATc3,thereby ameliorated cardiac hypertrophy.4.Triptolide could upregulate FoxP3 expression,and promote its nuclear translocation,thereby resulting in the amelioration of cardiac hypertrophy in vitro and in vivo.