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Aim: The objective of this study is to determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion. Methods and results: By 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. Conclusion: Continuous 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. [ABSTRACT FROM AUTHOR]

Background: Clinically, Charcot-Marie-Tooth disease (CMT)-associated muscle atrophy still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT type 4F (CMT4F) by destroying the myelin sheath form, which may be related to the inhibitory role of Ezrin in the self-association of L-periaxin. However, it is still unknown whether L-periaxin and Ezrin are independently or interactively involved in the process of muscle atrophy by affecting the function of muscle satellite cells. Method: A gastrocnemius muscle atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells were treated with adenovirus-mediated overexpression or knockdown of Ezrin. Then, overexpression of L-periaxin and NFATc1/c2 or knockdown of L-periaxin and NFATc3/c4 mediated by adenovirus vectors were used to confirm their role in Ezrin-mediated myoblast differentiation, myotube formation and gastrocnemius muscle repair in a peroneal nerve injury model. RNA-seq, real-time PCR, immunofluorescence staining and Western blot were used in the above observation. Results: For the first time, instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/fusion in vitro. In vivo transduction of adenovirus vectors carrying Ezrin, but not Periaxin, into the gastrocnemius muscle in a peroneal nerve injury model increased the numbers of muscle myosin heavy chain (MyHC) I and II type myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of overexpressed Ezrin combined with incubation of knockdown L-periaxin within the injured peroneal nerve or injection of knockdown L-periaxin into peroneal nerve-injured gastrocnemius muscle not only increased the number of muscle fibers but also recovered their size to a relatively normal level in vivo. Overexpression of Ezrin promoted myoblast differentiation/fusion, inducing increased MyHC-I+ and MyHC-II + muscle fiber specialization, and the specific effects could be enhanced by the addition of adenovirus vectors for knockdown of L-periaxin by shRNA. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by knockdown of Ezrin by shRNA in vitro but decreased myotube length and size. Mechanistically, overexpressing Ezrin did not alter protein kinase A gamma catalytic subunit (PKA-γ cat), protein kinase A I alpha regulatory subunit (PKA reg Iα) or PKA reg Iβ levels but increased PKA-α cat and PKA reg II α levels, leading to a decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the effects of overexpressing-Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by an increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, overexpressing Ezrin enhanced type I muscle fiber specialization, accompanied by an increase in NFATc2/c3 levels and a decrease in NFATc1 levels. Furthermore, overexpressing NFATc2 or knocking down NFATc3 reversed the inhibitory effects of Ezrin knockdown on myoblast differentiation/fusion. Conclusions: The spatiotemporal pattern of Ezrin/Periaxin expression was involved in the control of myoblast differentiation/fusion, myotube length and size, and myofiber specialization, which was related to the activated PKA-NFAT-MEF2C signaling pathway, providing a novel L-Periaxin/Ezrin joint strategy for the treatment of muscle atrophy induced by nerve injury, especially in CMT4F. [ABSTRACT FROM AUTHOR]

We aimed to explore whether superfluous sympathetic activity affects myoblast differentiation, fusion, and myofiber types using a continuous single-dose isoprenaline exposure model in vitro and to further confirm the role of distinct NFATs in ISO-mediated effects. Compared with delivery of single and interval single, continuous single-dose ISO most obviously diminished myotube size while postponing myoblast differentiation/fusion in a time- and dose-dependent pattern, accompanied by an apparent decrease in nuclear NFATc1/c2 levels and a slight increase in nuclear NFATc3/c4 levels. Overexpression of NFATc1 or NFATc2, particularly NFATc1, markedly abolished the inhibitory effects of ISO on myoblast differentiation/fusion, myotube size and Myh7 expression, which was attributed to a remarkable increase in the nuclear NFATc1/c2 levels and a reduction in the nuclear NFATc4 levels and the associated increase in the numbers of MyoG and MEF2C positive nuclei within more than 3 nuclei myotubes, especially in MEF2C. Moreover, knockdown of NFATc3 by shRNA did not alter the inhibitory effect of ISO on myoblast differentiation/fusion or myotube size but partially recovered the expression of Myh7, which was related to the slightly increased nuclear levels of NFATc1/c2, MyoG and MEF2C. Knockdown of NFATc4 by shRNA prominently increased the number of MyHC +, MyoG or MEF2C + myoblast cells with 1 ~ 2 nuclei, causing fewer numbers and smaller myotube sizes. However, NFATc4 knockdown further deteriorated the effects of ISO on myoblast fusion and myotube size, with more than 5 nuclei and Myh1/2/4 expression, which was associated with a decrease in nuclear NFATc2/c3 levels. Therefore, ISO inhibited myoblast differentiation/fusion and myotube size through the NFAT-MyoG-MEF2C signaling pathway. [ABSTRACT FROM AUTHOR]

Background/Aims: Vagus nerve stimulation (VNS) suppresses arrhythmic activity and minimizes cardiomyocyte injury. However, how VNS affects angiogenesis/arteriogenesis in infarcted hearts, is poorly understood. Methods: Myocardial infarction (MI) was achieved by ligation of the left anterior descending coronary artery (LAD) in rats. 7 days after LAD, stainless-steel wires were looped around the left and right vagal nerve in the neck for vagus nerve stimulation (VNS). The vagal nerve was stimulated with regular pulses of 0.2ms duration at 20 Hz for 10 seconds every minute for 4 hours, and then ACh levels by ELISA in cardiac tissue and serum were evaluated for its release after VNS. Three and 14 days after VNS, Real-time PCR, immunostaining and western blot were respectively used to determine VEGF-A/B expressions and α-SMA- and CD31-postive vessels in VNS-hearts with pretreatment of α7-nAChR blocker mecamylamine (10 mg/kg, ip) or mACh-R blocker atropine (10 mg/kg, ip) for 1 hour. The coronary function and left ventricular performance were analyzed by Langendorff system and hemodynamic parameters in VNS-hearts with pretreatment of VEGF-A/B-knockdown or VEGFR blocker AMG706. Coronary arterial endothelial cells proliferation, migration and tube formation were evaluated for angiogenesis following the stimulation of VNS in coronary arterial smooth muscle cells (VSMCs). Results: VNS has been shown to stimulate VEGF-A and VEGF-B expressions in coronary arterial smooth muscle cells (VSMCs) and endothelial cells (ECs) with an increase of α-SMA- and CD31-postive vessel number in infarcted hearts. The VNS-induced VEGF-A/B expressions and angiogenesis were abolished by m-AChR inhibitor atropine and α7-nAChR blocker mecamylamine in vivo. Interestingly, knockdown of VEGF-A by shRNA mainly reduced VNS-mediated formation of CD31+ microvessels. In contrast, knockdown of VEGF-B powerfully abrogated VNS-induced formation of α-SMA+ vessels. Consistently, VNS-induced VEGF-A showed a greater effect on EC tube formation as compared to VNS-induced VEGF-B. Moreover, VEGF-A promoted EC proliferation and VSMC migration while VEGF-B induced VSMC proliferation and EC migration in vitro. Mechanistically, vagal neurotransmitter acetylcholine stimulated VEGF-A/B expressions through m/nACh-R/PI3K/Akt/Sp1 pathway in EC. Functionally, VNS improved the coronary function and left ventricular performance. However, blockade of VEGF receptor by antagonist AMG706 or knockdown of VEGF-A or VEGF-B by shRNA significantly diminished the beneficial effects of VNS on ventricular performance. Conclusion: VNS promoted angiogenesis/arteriogenesis to repair the infracted heart through the synergistic effects of VEGF-A and VEGF-B. [ABSTRACT FROM AUTHOR]