Cardiomyocytic FoxP3 Is Involved In Mitophagy During Cardiac Remodeling And The Regulatory Role Of Triptolide

Objective:Forkhead box/winged helix transcriptional factor P3(FoxP3)is a well-studied transcription factor with various reported functions.FoxP3 is responsible for controlling the expression of a series genes related with inflammation,growth and maintaining T cell activity,thereby participating in some serious diseases such as infections,cancers and cardiovascular diseases.Our previous study reported that FoxP3 was expressed in cardiomyocytes for the first time,and its expression was down-regulated during cardiac remodeling,but whether cardiomyocytic FoxP3 participates in cardiac remodeling remains unclear.To our knowledge,mitochondrial dyshomeostasis is an important intracellular pathological change during cardiac remodeling,leading to mitochondrial damage and mitophagy activation,which is considered a pathogenic factor of cardiac remodeling.Therefore,this study was aimed to unveil the regulatory role of cardiomyocytic FoxP3 during cardiac remodeling from the perspective of mitophagy.An intervention study using triptolide(TP)was also carried out,which was helpful for developing future research of translational medicine.Methods:In this study,isoproterenol(Iso)was used to induce cardiac remodeling in male C57BL/6 mice or male FoxP3DTR C57BL/6 mice in vivo,and angiotensin Ⅱ(AngⅡ)was used to induce cell hypertrophy of rat cardiomyocyte cell line H9c2 or neonatal rat ventricular myocardiocytes(NRVMs)in vitro.Here,TP was used to inhibit cardiac remodeling in vitro and in vivo.Hematoxylin-eosin(HE)staining,fluorescein isothiocyanate(FITC)-conjugated wheat germ agglutinin(WGA)staining,and Masson’s trichrome staining were carried out to observe the histological alterations of cardiac remodeling.Rhodamine-phalloidine staining was used for detecting cardiomyocyte hypertrophy.Immunohistochemistry(IHC),immunoblotting(IB),and immunofluorescence(IF)were used to detect the expression and distribution of proteins in the in vivo experiments.IF and IB were used to detect protein level and distribution,and real-time PCR was used to detect mRNA expression in vitro.For monitoring autophagy,microtubule-associated protein 1 light chain 3(LC3)was employed as an autophagy marker to indicate autophagy activation,autophagy flux,and autophagic vesicle localization using IF and IB assessments.Mitophagy was observed by transmission electron microscopy(TEM)and fluorescencent co-localization.Moreover,the key factors mediating mitophagy activation were identified by IB and fluorescencent co-localization.The interactions between FoxP3 and its targets were detected by fluorescencent colocalization,immunoprecipitation(IP)and chromatin immunoprecipitation(ChIP).Small interference RNA(siRNA)was used to knock down the expressions of FoxP3 and some key autophagy genes,while adeno-associated virus(AAV)was used as a vector to overexpress FoxP3.This study was divided into three parts,and the research approaches were as follows:Part 1:First,the expression and intracellular distribution of FoxP3 were observed in the cardiac remodeling model in vitro and in vivo,and the spatial characteristics of its expression were discussed.Subsequently,the dynamic changes of mRNA expression,protein expression and distribution of FoxP3 were observed to characterize its temporal feature during in vitro cardiac remodeling.Finally,the effects of different concentrations of TP on the cytoplastic and nuclear distribution of FoxP3 were observed in vitro.Taking together,the spatiotemporal expression feature of FoxP3 during cardiac remodeling was characterized.Part 2:First,the spatial distribution and temporal changes of LC3 were observed in vitro and in vivo,to characterize the cell type associated with autophagy and the check pointed time.Then,autophagy type and autophagic flux changes were observed to further characterize autophagy in the in vitro model.Importantly,siRNAs were used to knock down the expression of autophagy-related gene 5(Atg5),PTEN-induced kinase 1(Pink1)and Parkin,to confirm whether these molecules were involved in mitophagy activation during cardiac remodeling.Combining these strategies for observing the autophagic changes in vitro and in vivo,we aimed to unveil the type,pathway,and trigger of autophagy in cardiac remodeling.Part 3:First,the expression patterns of Parkin and its transcription factor,activating transcription factor 4(ATF4),were determined in the in vitro model.Cardiomyocytes with FoxP3 knockdown or overexpression were exposed to AngⅡ,then the changes of Parkin mRNA expression and Parkin-mitophagy were assessed to confirm that cardiomyocytic FoxP3 was responsible for the negatively regulation of Parkin-mitophagy.The changes in cardiac remodeling and mitophagy of FoxP3-deficient mice were also evaluated,to finally confirm that FoxP3 was involved in the negative regulation of Parkin-mitophagy in cardiac remodeling.Subsequently,the possibility that FoxP3 interfered with Parkin transcription by directly acting on its promoter was discussed,by detecting the binding capacity of FoxP3 to the Parkin promoter in the in vitro model.Then the possibility of direct binding of FoxP3 to nuclear ATF4 was also further explored herein.By combining the results of mitophagy observations and two mechanism studies,the regulatory role of FoxP3 on Parkin-mitophagy during cardiac remodeling and the detailed mechanisms were elucidated.Results:Part 1.The expression and nuclear translocation of cardiomyocytic FoxP3 were down-regulated during cardiac remodeling.Iso induced significant left ventricle remodeling of mice comprised of cardiomyocyte hypertrophy,myocardial fiber disruption,interstitial fibrosis,and inflammatory cell infiltration.Meanwhile,the expression of cardiomyocytic FoxP3 was markedly downregulated in the remodeled left ventricle.AngⅡ induced remarkable cell hypertrophy of cardiomyocytes.Consistently,mRNA expression,protein expression and nuclear translocation of FoxP3 were also significantly downregulated from the early stage of cardiomyocytes exposured to AngⅡ.Interestingly,TP could restore or even upregulate the expression and nuclear translocation of cardiomyocytic FoxP3 in both in vitro and in vivo models.Part 2.Mitophagy in cardiac remodeling was activated through Parkin.In mice model of cardiac remodeling,autophagy occurred mainly in cardiomyocytes and accumulated continuously from the early stage of cardiac remodeling.In vitro,we found that cardiomyocytic autophagy also accumulated from the early stage of cardiac remodeling,and that was a consequence of excessive activation but not degradation disorder of autophagy flux.Not surprisingly,TP showed strong inhibition on myocardial autophagy activation.The main type of autophagy in cardiac remodeling was Atg5-dependent excessively activation of mitophagy,which was activated through the Pink1-Parkin pathway.Parkin was identified as the key autophagy adaptor that controlled the excessive activation of mitophagy during cardiac remodeling,as well as an effector for TP,because TP inhibited cardiac mitophagy by decreasing Parkin expression.Part 3.FoxP3 negatively regulated Parkin-mitophagy in cardiac remodeling by interfering with ATF4-mediated Parkin transcription.In vitro,FoxP3 knockdown resulted in the up-regulation of Parkin in cardiomyocytes,leading to further up-regulation of Parkin-mitophagy in cardiomyocytes treated with AngⅡ;the inhibition of Parkin-mitophagy by the TP treatment was also lost completely.In contrast,FoxP3 overexpression resulted in the down-regulation of Parkin in cardiomyocytes,thereby leading to decreased Parkin-mitophagy in cardiomyocytes;moreover,the inhibition of Parkin-mitophagy by TP was also lost.In vivo,FoxP3 deficiency also resulted in excessive activation of cardiac mitophagy and further deteriorative cardiac remodeling.We also confirmed that FoxP3 was critical for the inhibition of cardiac remodeling and mitophagy by TP.Together,these data confirmed that FoxP3 could negatively regulate Parkin expression,thereby inhibit cardiac mitophagy and remodeling.There were two machanisms involved in the negative regulation of Parkin mRNA expression by FoxP3.First,FoxP3 could directly bind to an element located downstream of the ATF4-binding element within the Parkin promoter to interfere ATF4-mediated transcription of Parkin.Second,FoxP3 might bind nuclear ATF4 to indirectly inhibit its activity on Parkin transcription.Collectively,FoxP3 negatively regulated Parkin-mitophagy in cardiac remodeling by interfering ATF4-mediated Parkin transcription;TP inhibited Parkin expression by restoring FoxP3 expression and nuclear translocation,thereby inhibiting mitophagy and cardiac remodeling.Conclusions:The expression and nuclear translocation of cardiomyocytic FoxP3 were downregulated during cardiac remodeling,leading to rapid upregulation of Parkin and excessive activation of mitophagy.Mechanistically,FoxP3 directly interacted with an element located downstream of the ATF4-binding element involved in the promoter of Parkin and binded nuclear ATF4 to decrease ATF4-related Parkin mRNA expression in cardiac remodeling.In addition,TP could ameliorate cardiac remodeling by inhibiting cardiac Parkin-mitophagy via restoring the expression and nuclear translocation of cardiomyocytic FoxP3.Therefore,TP was expected to be used as a novel anti-cardiac remodeling drug by targeting cardiomyocytic FoxP3.