| BackgroundHeart failure(HF)is a refractory and progressive disease,which is considered as an irreversible process characterized by cardiac pump failure and cardiac remodeling.Interstitial fibrosis plays a key role in developing and progressing of HF by causing adverse mechanical and electrical disturbances in the failure hearts.Sirt3 is a highly conserved protein of nicotinamide adenine dinucleotide(NAD)-dependent deacetylase.Sirt3 interacts with mitochondrial fission and fusion,directly acts with key proteins in mitochondrial dynamics,and can provide new targets for the treatment of diseases through these effects.OPA1,the GTPase anchored to the mitochondrial inner membrane(IMM)of mitochondria is an obligatory protein for the IMM fusion and identified as an acetylated protein by proteomic survey which is proved as a direct target of Sirt3.The peptide Szeto-Schiller Compound 31(SS31,elamipretide)interacts specifically with IMM to affect membrane curvature and prevent mitophagy.Mitochondrial dysfunction resulted from the imbalance of mitochondrial fission and fusion,which plays a crucial role in the pathogenesis of HF.Excessive mitochondrial fission and reduced mitochondrial fusion have been demonstrate to lead to the activation of apoptotic cell death during the progression of HF.Therefore,several therapeutic strategies to strengthen mitochondrial fusion or inhibit excessive mitochondrial fission have been reported to preserve the mitochondrial function of the heart and against the process of the heart failureAccordingly,regulating proteins that target mitochondrial dynamics is a potential strategy to prevent stress-induced cardiac damage.In HF,the OPA1 expression is decreased,which aggravates the mitochondrial fragmentation,resulting mitochondrial dysfunction and mitochondrial apoptosis,which contribute to the progression of HF,Increasing evidence indicates that Sirt3 plays a key role in mitochondrial dysfunction,OPA1 expression is potentially regulated by Sirt3.Thus,it is very worthwhile exploring whether SS31 can activate the OPA1-related mitochondrial fusion to alleviate HF injury by activating Sirt3-dependent function.In this study,we investigated the therapeutic effects of SS31 using mouse TAC model and mouse primary myocardial cell and fibroblast injury model induced by angiotensin Ⅱ,especially focusing on mitochondrial fusion via modulating Sirt3/OPA1 expression.Aims1.To investigate the protective effect of SS31 on pressure overload-induced heart failure in mice;2.To investigate whether the protective effect of SS31 on pressure overload-induced heart failure in mice is mediated by mitochondrial fusion;3.To explore whether the regulation of SS31 on mitochondrial fusion is mediated by the Sirt3/OPA1 pathway;Materials and methods1.In vivoThe model of pressure overload-induced heart failure in mice was established with TAC model.In the exploratory studies,The 129 wild-type mice were randomized divided into 3 groups,two of them underwent TAC surgery.one group was given SS31(3mg/kg/d 60d),another group was given saline,and the third group was sham-operated group.In the same way,Sirt3KO mice were randomized divided into 3 groups,two of them underwent TAC surgery.One group was given SS31(3mg/kg/d 60d),another group was given saline,and the third group was sham-operated group.Regular diet feeding continued during the intervention period.Before surgery and at the 8th weekend after surgery,echocardiogram images and data were collected by M-mode,tissue Doppler mode,Pulse Wave Doppler(PWD)echocardiography to evaluate the heart systolic and diastolic functions of the mice.Collagen content was evaluated by Masson trichrome staining with morphology.Collagen Ⅰ,collagen Ⅲ were used to identify the fibrosis level by immunohistochemical.Transdifferentiation of fibroblasts was assessed by immunohistochemical staining of alpha smooth muscle Actin(α-SMA)and transforming growth factor-β(TGF-β).The morphology and size distribution of mitochondria in cardiomyocytes were observed by transmission electron microscopy.Mouse brain natriuretic peptide(BNP)Elisa kit were allowed for the determination of BNP concentrations in mouse serum.2.1n vitroVentricular tissues of neonate mice were collected in asepsis,and fibroblast cells were collected in batches by differential velocity adherent method,and cultured in Dulbecco’s Modified Eagle Medium(DMEM).The cells were routinely cultured in a cell incubator and the growth status were observed daily.The fibroblasts were stimulated with angiotensin Ⅱ to simulate the process of heart failure and cardiac fibrosis.Primary cardiac fibroblasts were spread in cell cultured plate evenly,after cell density grows to 60%-80%,serum-free medium with concentration of SS31(100 nM final concentration)was added to each well to stimulate cells for 48h.After the medium changing and cleaning,angiotensin Ⅱ(Ang Ⅱ)(1μM final concentration)was added to stimulate the cells to concentration for 48h to obtain the cardiac fibroblast cell.The relative protein content was measured by western blot.The degree of transdifferentiation of fibroblasts and the morphology of mitochondria were evaluated by immunofluorescence labeling.3.Statistical analysisAll data are obtained from at least 3 independently-repeated experiments and were processed by GraphPad Prism 8 software package.The quantitative data were expressed as mean ± standard deviation.The t-test was used to evaluate the significance of the differences between 2 groups.Differences with p<0.05 were considered statistically significant.One-way ANOVA was used for the comparison between groups,and p<0.05 was considered statistically significantResults1.In vivoSS31 could effectively alleviate the clinical manifestations of pressure overload-induced heart failure in wild-type mice but could not alleviate the clinical manifestations of heart failure in Sirt3KO mice.SS31 could improve the structure and function of heart myocardium in wild-type mice with heart failure after TAC,but its protective effects on the structure and function of heart myocardium in Sirt3KO mice with heart failure disappeared.SS31 reduced cardiac fibrosis in wild-type heart failure mice,but did not alleviate the degree of myocardial fibrosis in Sirt3KO mice.The results of transmission electron microscopy showed that SS31 treatment could improve the mitochondrial morphology of heart failure in wild-type mice,but the mitochondrial morphology was not improved by SS31 in Sirt3KO mice.SS31 can restore the expression of mitochondrial IMM fusion protein OPA1 in the heart of wild-type mice,but can not restore the expression of OPA1 in Sirt3KO mice.2.In vitroSS31 inhibited the transdifferentiation of myocardial fibroblasts during the process of angiotensin Ⅱ induced transdifferentiation,and promoted the expression of Sirt3 protein in myocardial fibroblasts.SS31 improved mitochondrial fusion of myocardial fibroblasts stimulated by angiotensin Ⅱ in a Sirt3-dependent manner.SS31 could restore the expression of mitochondrial fusion protein OPA1 in wild-type fibroblasts stimulated by angiotensin I,but had no effect on the expression of mitochondrial fusion protein OPA1 in Sirt3KO mouse fibroblasts stimulated by angiotensin Ⅱ.Meanwhile,the expression of mitochondrial fusion protein OPA1 in Sirt3 overexpressed mouse fibroblasts was significantly increased.Conclusions1.SS31 has a protective effect on pressure overload-induced heart failure in mice;2.The protective effect of SS31 on pressure overload-induced heart failure in mice is mediated by mitochondrial fusion;3.The regulation of SS31 on mitochondrial fusion is mediated by Sirt3/OPA1 pathway;... |
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