Effects Of Diet Flavonoid Fisetin On Pathological Cardiac Remodeling And Its Mechanisms

Fisetin is a natural dietary flavonoid,mainly found in various fruits and vegetables such as apples,persimmons,grapes,strawberries,cucumbers,etc.It has powerful anti-inflammatory,anti-oxidant,anti-tumor actions and protective effects against myocardial and cerebral ischemia.In recent years,fisetin has attracted much more attention because of its similar structure to senolytic drug quercetin,which is targeted to eliminate senescent cells.It can be used as a simulated drug for dietary restriction to delay the aging of various organs in rats.Mitochondrial quality control disorders are considered to be an important factor leading to cardiac aging and pathological myocardial remodeling,however,whether fisetin improves pathological myocardial remodeling by regulating mitochondrial quality has not been reported.Objective:To explore the role of dietary flavonoid fisetin in pathological myocardiac remodeling and its related mechanisms,and provide sufficient theoretical basis for fisetin to treat myocardial hypertrophy and fibrosis.Methods:Cardiomyocyte hypertrophy model was induced by isoproterenol（ISO）treatment in vitro.H9c2 cells were divided into blank control group,model group,fisetin group,fisetin+ISO group and fisetin+ISO+Compound C and fisetin+ISO+Ex-527 group.In model group,the H9c2 cells were stimulated with 30μM ISO for 48h;the fisetin group was pretreated with 20μM fisetin 30 min before ISO stimulation.In inhibitor groups,10μM Compound C or Ex-527 was added to the cell culture medium while intervening with fisetin.The control group was treated with a drug-free solvent.CCK-8 kit was used to detect the toxicity of fisetin and ISO on cardiomyocytes and the optimal therapeutic concentration and modeling concentration was selected.The surface area of cardiomyocytes was calculated by FITC phalloidin/DAPI double fluorescent staining,DCFH-DA fluorescent probe was used to measure the level of mitochondrial ROS,and the level of mitochondrial membrane potential in cardiomyocytes was measured by JC-1 kit.Mito Tracker Red was used to observe the morphology and quantity of mitochondria,Annexin V-FITC/PI staining combined with flow cytometry was used to detect the apoptosis of cardiomyocyte.The m RNA levels of cardiomyocyte hypertrophy markers,such as ANP,BNP,β-MHC and mitochondrial copy number were detected by real-time quantitative PCR.Western blot was used to detect the expression of mitochondrial fission/fusion-related proteins,mitochondrial unfolded protein response(UPR^mt)-related proteins,mitochondrial autophagy-related proteins,endoplasmic reticulum stress-related proteins and apoptosis-related proteins.40 male C57/BL6 mice were randomly divided into four groups:saline group,ISO group（20 mg/kg/d）,fisetin control group and fisetin treatment group（25 mg/kg/d,po）,10 mice in each group.ISO was injected subcutaneously once a day for 28 days to induce a model of pathological myocardial remodeling.Another 40 C57/BL6 mice were randomly divided into sham operation group（Sham）,fisetin control group,thoracic aortic constriction（TAC）group,and fisetin treatment group（TAC+fisetin）.On the 28th day after operation or ISO injection,small animal ultrasound imaging system was used to detect the heart function of mice.HE,WGA,Sirius Red or Masson staining and immunohistochemical staining were used to observe the gross pathology,cardiomyocyte hypertrophy and myocardial fibrosis phenotype,tunel staining was used to detect myocardial cell apoptosis,real-time quantitative PCR was used to detect the m RNA levels of ANP,BNP,andβ-MHC,and the changes of related proteins were analyzed by western blot.Results:Fisetin had no significant effect on the viability of cultured H9c2cardiomyocytes within the concentration range of 5～40μM,but when the concentration exceeded 50μM,the cell viability began to decrease significantly（p<0.01）.Compared with the control group,30μM ISO treatment for 48 h significantly induced cardiomyocyte hypertrophy,the m RNA levels of ANP,BNP andβ-MHC,cardiomyocyte surface area and mitochondrial ROS level were significantly increased（P<0.05）,and mitochondrial membrane potential was significantly reduced.The endoplasmic reticulum stress of cardiomyocytes in model group was significantly increased,the mitochondria split in fragments,the copy number was significantly reduced,and the mitochondrial autophagy activity was significantly reduced（P<0.05or P<0.01）.Compared with the model group,fisetin significantly inhibited ISO-induced cardiomyocyte hypertrophy,decreased ANP,BNP,β-MHC m RNA expression levels and cardiomyocyte surface area,mitigated cardiomyocyte apoptosis and mitochondrial ROS content,and increases mitochondrial membrane potential level（P<0.05 or P<0.01）.The copy number of cardiomyocyte mitochondria in fisetin intervention group was increased significantly,the number of fragmented mitochondria decreased,and the number of reticulated mitochondria increased significantly as compared to the model group（P<0.01）.In addition,fisetin significantly reversed the increase in the expression of endoplasmic reticulum stress-related proteins,and elevated the expression of mitochondrial fusion proteins OPA1,Mfn2,mitochondrial UPR-related protein Lon P1 and mitochondrial autophagy proteins Pink1 and Parkin,which are statistically different from the model group significance（P<0.05 or P<0.01）.AMPK inhibitor Compound C and Sirt1 inhibitor Ex-527 significantly antagonized the protective effects of fisetin on ISO-induced myocardial hypertrophy in cultured H9c2 cells in vitro,increased the expression levels of ANP,BNP andβ-MHC m RNA and the surface area of myocardial cells,activated ER stress and inhibited mitochondrial UPR（P<0.05 or P<0.01）.The models of cardiac remodeling in mice were successfully established by ISO subcutaneous injection or TAC surgery for 28 days.Compared with sham operation,the cross-sectional area of myocardial cells,HW/BW,HW/TL and myocardial fibrosis were significantly increased（P<0.05）.Small animal ultrasound results showed that the left ventricular ejection fraction（LVEF%）and left ventricular short axis shortening rate（LVFS%）of mice in model group were significantly decreased（P<0.05）,left ventricular systolic diameters（LVIDs）,left ventricular diastolic diameters（LVIDd）,left ventricular systolic volume（LVOLs）and left ventricular diastolic volume（LVOLd）were elevated significantly（P<0.05）.Compared with model group,the structure and function of heart in fisetin treatment group was significantly improved,LVEF%and LVFS%were significantly increased（P<0.05）,LVIDs,LVIDd,LVOLs,LVOLd,HW/BW values and HW/TL values were obviously reduced（P<0.05）.In addition,fisetin obviously improved the myocardial hypertrophy and fibrosis induced by ISO subcutaneous injection and TAC surgery,inhibited myocardial cell apoptosis,reduced the expression levels of ANP,BNP andβ-MHC m RNA,endoplasmic reticulum stress-related proteins GRP78,CHOP,Calreticulin,mitochondrial fission proteins Drp1 and Fis1 in myocardium,and up-regulated mitochondrial fusion proteins Mfn2,OPA1,mitochondrial autophagy protein Pink1,Parkin and mitochondrial UPR protein Lon P1,compared with the model group,the difference was significant（P<0.05 or P<0.01）.Conclusion:The dietary flavonoid fisetin could significantly reduce the myocardial damage induced by endoplasmic reticulum stress,enhance mitochondrial fusion,mitochondrial autophagy and UPR^mt,and improve pathological myocardial remodeling.Fisetin regulates UPR^mt and endoplasmic reticulum stress through AMPK/Sirt1 signaling pathway,and then controls mitochondrial quality.