Effects Of Oxidative Stress/Ca²⁺/calpain-1 Signaling On Astragaloside IV Attenuates Apoptosis Of Hypertrophied Myocardial Cells Induced By Isoproterenol

ObjectiveTo observe the changes of oxidative stress/Ca²⁺/calpain-1 signaling on apoptosis of hypertrophied myocardial cells induced by isoproterenol and clarify the effect of Astragaloside IV on attenuating apoptosis of hypertrophied cardiomyocytes and the underlying mechanism.MethodsIn in vivo study,fifty SD rats were randomly divided into five groups: control group,ISO(10 mg﹒kg^-1﹒d^-1,i.p.)group,ISO+PRO(40 mg﹒kg^-1﹒d^-1,i.g.)group,ISO+ ASIV(80 mg﹒kg^-1﹒d^-1,i.g.)group and ISO+MDL(3 mg﹒kg^-1﹒d^-1,i.p.)group.Rats received ASIV,PRO or MDL administration for 6 weeks after intraperitoneal injection of ISO for 2 weeks.HE staining was used to measure surface area of cardiomyocytes.HWI and LVWI were used as hypertrophy indicators.TUNEL staining was used to detect apoptotic rate.Electron microscopy was used to observe mitochondrial structure.Fluo-3 AM staining was used to measure cellular calcium concentration.Western blot was used to analyze proteins expression of Bax,Bcl-2,Mito-NOX4 and calpain-1.Elisa was used to determine activities of Mito-SOD,Mito-CAT and calpain.In in vitro study,H9C2 cardiomyocytes were randomly divided into five groups: control group,ISO（10μM）group,ISO+PRO（2μM）group,ISO+ ASIV（100μM）group and ISO+MDL（15μM）group.ASIV,PRO or MDL were added after ISO treatment for 48 h.Rhodaminel-labeled phalloidin staining was used to measure cell surface area.Real-time PCR was used to determine m RNA expression of ANP and BNP.Flow cytometry was used to analyze apoptotic rate.JC-1 staining was used to detect ΔΨ_m.Fluo-3 AM staining was used to measure cellular calcium concentration.Western blot was used to analyze proteins expression of Bax,Bcl-2,Mito-NOX4 and calpain-1.Elisa was used to determine activities of Mito-SOD,Mito-CAT and calpain.ResultsCompared with control group,ISO administration（1）significantly increased cardiomyocyte surface area,HWI and LVWI in rats and up-regulated m RNA expression of ANP and BNP in H9C2 cells.（2）Meanwhile,apoptotic rate was increased in ISO group,as reflected by the increases of TUNEL-positive cells in rats and Annexin V-FITC/PI-positive cells in H9C2 cells respectively.Bax expression in both rats and H9C2 cells was increased while Bcl-2 expression was decreased.（3）Mitochondrial dysfunction and ΔΨ_m dissipation were also found in heart tissue and cell respectively.Furthermore,both in vivo and in vitro study demonstrated that ISO treatment markedly up-regulated Mito-NOX4 and down-regulated activities of Mito-SOD and Mito-CAT.（4）In both isolated adult rats cardiomyocytes and H9C2 cells,cellular calcium concentration was increased after ISO treatment.（5）Calpain activity and calpain-1 protein expression were increased in ISO group.However,in both in vivo and in vitro experiments,（1）ASIV,PRO and MDL treatment groups significantly attenuated cardiomyocyte apoptosis,up-regulated Bax expression and down-regulated Bcl-2 expression.（2）ASIV improved mitochondrial dysfunction,increased ΔΨ_m,decreased Mito-NOX4 expression and up-regulated activities of Mito-SOD and Mito-CAT.（4）ASIV reduced cellular calcium concentration.（5）In addition,ASIV exerted theinhibitory effect as MDL did in suppressing calpain activity and calpain-1expression.ConclusionASIV could significantly attenuate apoptosis of hypertrophied cardiomyocytes induced by ISO and the anti-apoptosis effect was related to inhibiting oxidative stress/Ca²⁺/calpain-1 signaling pathway.