Effects of stress caused by iron excess on mitophagy and apoptosis of murine myoblast C2C12 cells

Abstract

Objective To investigate the effects of stress caused by iron excess on biologic activity of murine myoblast C2C12 cells and related mechanism. Methods Murine preosteoblast myoblast C2C12 cells were incubated in a medium supplemented with different concentrations (50, 100, 200 μmol/L) of ferric ammonium citrate (FAC) under induction of horse serum. The proliferation were assessed by cell counting kit (CCK-8). The apoptotic hallmarks were detected by flow cytometer. Intracellular reactive oxygen species (ROS) was measured using dichlorodihydrofluorescein diacetate (DCFH-DA). The quantity of intracellular mitochondria was measured using mitochondrial assay kit. The expression of autophagy proteins microtubule-associated protein 1 light 3Ⅰ (LC3Ⅰ), LC3Ⅱ, bcl-2/adenovirus E1B 19-kilodalton interacting protein3 (BNIP3), and bcl-2/adenovirus E1B 19-kilodalton interacting protein3-like (BNIP3L) were detected by Western blotting. The expression of cytochrome C in the cytoplasm and mitochondria were detected by Western blotting respectively. The cells were treated with 2.5 mmol/L antioxidant N-acetyl cysteine (NAC) and FAC, the proliferation and apoptosis indexes of the cells were tested again. Results Proliferation of C2C12 cells was significantly inhibited by FAC in dose dependent manner (P<0.01). The apoptotic rate, level of ROS, and number of mitochondria were increased by FAC in dose dependent manner (P<0.01). The protein expression of LC3Ⅱ/LC3Ⅰ, BNIP3, and BNIP3L were increased by FAC in dose dependent manner (P<0.01). The protein expression of Cyt C cytoplasm/mitochondria increased by FAC in dose dependent manner (P<0.01). The proliferation indices in FAC+ NAC, FAC groups were 1.46±0.05 and 0.63±0.04 respectively, and apoptotic rate were 4.72±0.57 and 15.78±0.57 respectively. The statistically significant differrence (P<0.01) suggested that addition of NAC partly reversed the decrease of proliferation and the increase of apoptosis in C2C12 cells induced by FAC. Conclusion The stress caused by iron excess can significantly inhibit biologic activity of skeletal muscle cells, and the mechanism behind inhibition may involve activation of autophagy and apoptosis of mitochondria induced by oxidative stress. Key words: C2C12 cell; Iron accumulation; Sarcopenia; Mitochondrion; Autophagy; Apoptosis