Dataset Information

Continuous exposure to isoprenaline reduced myotube size by delaying myoblast differentiation and fusion through the NFAT-MEF2C signaling pathway.

ABSTRACT: 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.

SUBMITTER: Yue J

PROVIDER: S-EPMC9829891 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Continuous exposure to isoprenaline reduced myotube size by delaying myoblast differentiation and fusion through the NFAT-MEF2C signaling pathway.

Yue Jing J Xu Wei W Xiang Li L Chen Shao-Juan SJ Li Xin-Yuan XY Yang Qian Q Zhang Ruo-Nan RN Bao Xin X Wang Yan Y Mbadhi MagdaleenaNaemi M Liu Yun Y Yao Lu-Yuan LY Chen Long L Zhao Xiao-Ying XY Hu Chang-Qing CQ Zhang Jing-Xuan JX Zheng Hong-Tao HT Wu Yan Y Chen Shi-You SY Li Shan S Lv Jing J Shi Liu-Liu LL Tang Jun-Ming JM

Scientific reports 20230109 1

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 ...[more]

PMID: 36624121

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Project description: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.

Project description:BackgroundSpinal muscular atrophy is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Recently approved treatments aimed at increasing SMN protein levels have dramatically improved patient survival and have altered the disease landscape. While restoring SMN levels slows motor neuron loss, many patients continue to have smaller muscles and do not achieve normal motor milestones. While timing of treatment is important, it remains unclear why SMN restoration is insufficient to fully restore muscle size and function. We and others have shown that SMN-deficient muscle precursor cells fail to efficiently fuse into myotubes. However, the role of SMN in myoblast fusion is not known.MethodsIn this study, we show that SMN-deficient myoblasts readily fuse with wild-type myoblasts, demonstrating fusion competency. Conditioned media from wild type differentiating myoblasts do not rescue the fusion deficit of SMN-deficient cells, suggesting that compromised fusion may primarily be a result of altered membrane dynamics at the cell surface. Transcriptome profiling of skeletal muscle from SMN-deficient mice revealed altered expression of cell surface fusion molecules. Finally, using cell and mouse models, we investigate if myoblast fusion can be rescued in SMN-deficient myoblast and improve the muscle pathology in SMA mice.ResultsWe found reduced expression of the muscle fusion proteins myomaker (P = 0.0060) and myomixer (P = 0.0051) in the muscle of SMA mice. Suppressing SMN expression in C2C12 myoblast cells reduces expression of myomaker (35% reduction; P < 0.0001) and myomixer, also known as myomerger and minion, (30% reduction; P < 0.0001) and restoring SMN levels only partially restores myomaker and myomixer expression. Ectopic expression of myomixer improves myofibre number (55% increase; P = 0.0006) and motor function (35% decrease in righting time; P = 0.0089) in SMA model mice and enhances motor function (82% decrease in righting time; P < 0.0001) and extends survival (28% increase; P < 0.01) when administered in combination with an antisense oligonucleotide that increases SMN protein levels.ConclusionsHere, we identified reduced expression of muscle fusion proteins as a key factor in the fusion deficits of SMN-deficient myoblasts. This discovery provides a novel target to improve SMA muscle pathology and motor function, which in combination with SMN increasing therapy could enhance clinical outcomes for SMA patients.

Dataset Information

Continuous exposure to isoprenaline reduced myotube size by delaying myoblast differentiation and fusion through the NFAT-MEF2C signaling pathway.

Publications

Continuous exposure to isoprenaline reduced myotube size by delaying myoblast differentiation and fusion through the NFAT-MEF2C signaling pathway.

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