Home > Browse Issues > Vol.39 No.9
Isoprenaline Induced Muscle Atrophy by Inhibiting the Differentiation of C2C12 Cells into Skeletal Muscle Cells
Chen Shaojuan1,2, Xiang Li1,2, Jiang Miao2, Wang Lu2, Zheng Fei2, Zhang Lei2, Yuan Ye2, Tang Junming1,2*
1Department of Physiology, School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China; 2Department of Cardiology, and Institute of Clinical Medicine, Renmin Hospital, Hubei University of Medicine, Shiyan 442000, China
Abstract: This study is to investigate the effect and possible mechanism of isoprenaline (ISO) on C2C12 cells differentiation into skeletal muscle cells and muscle atrophy. Adrenergic receptors expressions in C2C12 cells were detected using immunofluorescence cytochemistry staining， and the cultured C2C12 cells that reached 70% confluence in vitro were used to establish the model of skeletal muscle stem/progenitor cells differentiation under 2% horse serum with high glucose DMEM. And then， using the cells differentiation model to compare the difference of either single or continuous single aderministration， single-dose (only one time) or continuous single-dose (one time each day for 8 days) of ISO with 10–5 mol/L were added into the differentiation medium. Subsequently， the different dosages effects of ISO with 10–8 mol/L， 10–7 mol/L， 10–6 mol/L or 10–5 mol/L on the cells differentiation were performed by using aderministration of continuous single-dose. After that， immunofluorescence cytochemistry staining were used to analyze the expression of myosin heavy chain (MYH) in differentiated cells from C2C12 cells， and the quantitative analysis of the number of nucleus of myotubes of skeletal muscle cells. Western blot was used to detect the expression of p-p38MAPK (phosphorylated p38-mitogen-activated protein kinase)， p-AKT (phosphorylated protein kinase B)， MYH and myogenin (MyoG). C2C12 cells showed the traits of α1-， α2-， β1- and
β2-subtypes of adrenergic receptors expressions. C2C12 cells gradually differentiated into mature skeletal muscle cells during the procedure of differentiation condition， reaching the peak at day 8 of differentiation period. Singledose ISO with 10–5 mol/L slightly inhibited C2C12 cells differentiations into myotubes compared to continuous single-dose ISO. Furthermore， C2C12 cells differentiations into myotubes were gradually inhibited when exposed to continuous single-dose ISO with 10–8 mol/L， 10–7 mol/L， 10–6 mol/L， 10–5 mol/L， showing dose-dependent manner. Furthermore， the numbers of myotubes with the different nuclear fusion numbers were gradually reduced when the differentiating C2C12 cells were exposed to the stimulation of different dosages of continuous singledose ISO. Especially at the 10–5 mol/L continuous single-dose ISO， the potential of differentiation of C2C12 cells into mature skeletal muscle cells were markedly weaken. Simultaneously， the levels of p-p38MAPK and p-AKT， and the expressions of MYH and MyoG as determined by Western blot were dosages-dependently decreased in the differentiating C2C12 cells with aderministration of continuous single-dose ISO. It is suggested that ISO， especially with continuous aderministration， inhibited C2C12 cells differentiation into mature skeletal muscle cells， which was associated with the reduced p-p38MAPK and p-AKT levels， and decreased MyoG expressions. These results provided a new therapeutic target for prolonged sympathetic overactivity-related muscle atrophy.
β2-subtypes of adrenergic receptors expressions. C2C12 cells gradually differentiated into mature skeletal muscle cells during the procedure of differentiation condition， reaching the peak at day 8 of differentiation period. Singledose ISO with 10–5 mol/L slightly inhibited C2C12 cells differentiations into myotubes compared to continuous single-dose ISO. Furthermore， C2C12 cells differentiations into myotubes were gradually inhibited when exposed to continuous single-dose ISO with 10–8 mol/L， 10–7 mol/L， 10–6 mol/L， 10–5 mol/L， showing dose-dependent manner. Furthermore， the numbers of myotubes with the different nuclear fusion numbers were gradually reduced when the differentiating C2C12 cells were exposed to the stimulation of different dosages of continuous singledose ISO. Especially at the 10–5 mol/L continuous single-dose ISO， the potential of differentiation of C2C12 cells into mature skeletal muscle cells were markedly weaken. Simultaneously， the levels of p-p38MAPK and p-AKT， and the expressions of MYH and MyoG as determined by Western blot were dosages-dependently decreased in the differentiating C2C12 cells with aderministration of continuous single-dose ISO. It is suggested that ISO， especially with continuous aderministration， inhibited C2C12 cells differentiation into mature skeletal muscle cells， which was associated with the reduced p-p38MAPK and p-AKT levels， and decreased MyoG expressions. These results provided a new therapeutic target for prolonged sympathetic overactivity-related muscle atrophy.
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