Continuous exposure of isoprenaline inhibits myoblast differentiation and fusion through PKA/ERK1/2-FOXO1 signaling pathway

Abstract

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. Electronic supplementary material The online version of this article (10.1186/s13287-019-1160-x) contains supplementary material, which is available to authorized users.

Ezrin regulated myoblast differentiation/fusion and muscle fiber specialization through PKA-NFAT-MyoD/MEF2csignalling pathway

“…However, our results did not show anyapparentchanges in expressions of Myf-5 and MyoD in overexpressingEzrin in myoblast cells, but showed increased numbers of MyoGandMEF2C-positive myoblast cells and myotubes. Actually, MyoG and MEF2C play an essential role in the initiation and later stage of myoblast differentiation, respectively 26 .Recent reports have also associated PKA RI and RII to myoblast differentiation and myotube formation 11,12 . Of interest, we found that overexpressingEzrin markedly increased PKA RI levels, leading to an increased PKA RI/RII ratio, accompanied by an acceleration of myoblast differentiation and fusion.…”

“…Previous results have shown that PKA and PKAreg I/II ratio play crucial roles in controlling myoblast differentiation and fusion 11,12 . To con rm if Ezrin's role in myoblast differentiation and fusion was involved in PKA signalling pathway, we used western blot to detect PKA signaling (Fig.…”

“…PKA activity is found to have effects on myoblast differentiation and fusion 11,12 . Combining with the above results that the overexpression or knockdown of Ezrinaffected myoblast differentiation and fusion by altering PKA activity, we treated C2C12 cells with PKA inhibitor (H-89).…”

“…Traditionally, the activated protein kinase A(PKA) has been linked to the unique phenomenon of myoblast differentiation/fusion and myotube formation, ascribing to the alteration in PKA regulatory subunit I (PKA RI) under normal differentiation condition 11 . Our previous study has shown that it abated the ratio of PKA RI/RII in myoblast cells, resulting in the postponement of myoblast differentiation and fusion 12 . Further evidence shows that ERM proteins act as PKA-anchoring proteins and sequester PKA close to its target proteins for their effective phosphorylation and functional regulation 13 .…”

S100B is required for maintaining an intermediate state with double-positive Sca-1+ progenitor and vascular smooth muscle cells during neointimal formation

“…To confirm the dynamic process of Sca-1+ progenitor cell migration from the adventitia to intima during the process of neointimal formation, the artery sections from different time points were incubated with Sca-1 (ab4336, Abcam) antibody followed by fluorescent dye-conjugated secondary antibody (Jackson ImmunoResearch) and counterstained with DAPI (Sigma) [28].…”