Abstract
AimThe objective of this study is to determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion.Methods and resultsBy using immunoassaying and western blot analyses, we found that β1 and β2-adrenergic receptors (AdR) were expressed in C2C12 cells. The differentiated satellite cells exhibited an increased expression of β2-AdR, as compared with the proliferating cells. Continuous exposure of isoprenaline (ISO), a β-AdR agonist, delayed C2C12 cell differentiation, and myoblast fusion in time- and dose-dependent manner. ISO also increased short myotube numbers while decreasing long myotube numbers, consistent with the greater reduction in MyHC1, MyHC2a, and MyHC2x expression. Moreover, continuous exposure of ISO gradually decreased the ratio of PKA RI/RII, and PKA RI activator efficiently reversed the ISO effect on C2C12 cell differentiation and myoblast fusion while PKA inhibitor H-89 deteriorated the effects. Continuous single-dose ISO increased β1-AdR expression in C2C12 cells. More importantly, the cells showed enhanced phospho-ERK1/2 levels, resulting in increasing phospho-β2-AdR levels while decreasing β2-AdR levels, and the specific effects could be abolished by ERK1/2 inhibitor. Furthermore, continuous exposure of ISO induced FOXO1 nuclear translocation and increased the levels of FOXO1 in nuclear extracts while reducing pAKT, p-p38MAPK, and pFOXO1 levels. Conversely, blockade of ERK1/2 signaling partially abrogated ISO effects on AKT, p38MAPK, and FOXO1signaling, which partially restored C2C12 cell differentiation and myoblast fusion, leading to an increase in the numbers of medium myotube along with the increased expression of MyHC1 and MyHC2a.ConclusionContinuous exposure of ISO impedes satellite cell differentiation and myoblast fusion, at least in part, through PKA-ERK1/2-FOXO1 signaling pathways, which were associated with the reduced β2-AdR and increased β1-AdR levels.
Highlights
Muscular dystrophy (MD) is a destructive neuromuscular disease characterized by progressive muscle weakness, muscle atrophy, and cardiac dysfunction [1]
Continuous stimulation with ISO inhibited C2C12 cell differentiation and myotube formation To explore the potential role of ISO in C2C12 cell differentiation, myotube formation following C2C12 cell differentiation with 2% horse serum (HS)-Dulbecco’s modified Eagle’s medium (DMEM) was firstly observed under a microscope, and the differentiated C2C12 cells showed myotube formation characterized by multinucleated myoblast fusion in a time-dependent manner
Less reduction of Myosin heavy chain (MyHC) protein level was detected by western blot in ISO-transiently treated cells compared to the ISO-continuously treated cells (Additional file 1: Figure S1D-F). These results suggested that the inhibition to C2C12 cell differentiation and myotube formation by continuous stimulation of ISO was stronger than the transient stimulation
Summary
Muscular dystrophy (MD) is a destructive neuromuscular disease characterized by progressive muscle weakness, muscle atrophy, and cardiac dysfunction [1]. An autonomic imbalance has been observed in which sympathetic activity is increased while coupled with diminished parasympathetic activity, contributing to the development of dilated cardiomyopathy (DCM), ventricular arrhythmias, and sudden cardiac death in patients with Duchenne and Becker MD [1, 3]. Most studies have focused on the impact of autonomic dysfunction on cardiac performance and morphology in MD with little attention to its possible role in the development of skeletal myopathy. It has been speculated that autonomic imbalance inhibits the anabolism of skeletal muscle, attributing to the reduced expression and sensitivity of β2-AdR in skeletal muscle [8]. These results suggest that β-AdR is involved in MD
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