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Background and aim: Chronic heart failure (CHF) is the ultimate outcome of most cardiovascular diseases. The apoptosis of cardiomyocyte is one of the most important factors contributing to CHF. Angiotensin II (Ang II) is one of the most important renin-angiotensin system components and plays a critical role in cardiac remodeling via leading to cardiac apoptosis and cardiac fibroblast proliferation. Thus, this study was aimed to investigate the effect of Ang II on cardiomyocyte apoptosis in vitro H9c2 cell line and neonatal rat cardiomyocyte model.
The H9c2 cell line and neonatal rat cardiomyocytes were cultured in serum-free DMEM medium. We observed the effects of Ang II on cardiomyocyte apoptosis at different concentrations (10-4-10-8 M) and incubation times (24-192h). MTT was used to study cell viability, while the induction of apoptosis and the expression of apoptosis-related mRNA and protein were determined by the use of TUNEL, flow cytometry, RT-PCR, or Western blot, respectively.
Treatment with Ang II for 24h reduced the cell viability of H9c2 cell line in a dose-dependent manner compared to control group (P＜0.05). Ang II- treatment enhanced the apoptosis of H9c2 cell line determined by DAPI and TUNEL staining, and flow cytometry, respectively (DAPI: 23.13±3.26 vs 5.63±0.95; TUNEL: 15.44±2.26 vs 5.53±0.78; flow cytometry: 17.70±0.98 vs6.13±0.72, unit: %, P＜0.01). Compared to control group, treatment with Ang II inhibited the mRNA and protein expression of anti-apoptosis Bcl-2 (mRNA expression: 0.85±0.08 vs 1±0.05; protein expression: 0.63±0.08 vs 0.83±0.05, P＜0.05). In contrast, the mRNA and protein expression of pro-apoptosis Caspase-3, Bax and p53 were increased in Ang II-treated group compared to control group (mRNA expression: 1.38±0.08 vs 1±0.05, 1.22±0.12 vs 1±0.01, 1.18±0.06 VS 1±0.03; protein expression: 1.04±0.18 vs 0.47±0.05, 0.82±0.12 vs 0.43±0.04; P＜0.05). In addition, the apoptosis of neonatal rat cardiomyocyte determined by flow cytometry was increased in Ang II treated group compared to control group (7.70±6.98 vs 10.60±3.72, unit: %, P＜0.01).
Our data show that it is easier to induce the apoptosis of cardiomyocyte cultured in serum-free condition by Ang II in a dose-dependent manner. Our studies also demonstrate that the influence of Ang II on apoptosis-related factors is responsible for the mechanisms underlying Ang II- induced apoptosis. The results suggest that inhibition of Ang II could serve as a novel target for treating HF. [This work was supported by a grant from National Natural Science Foundation of China (No. 81373853), * Corresponding Author].