In differentiating prefusion myoblasts connexin43 gap junction coupling is upregulated before myoblast alignment then reduced in post-mitotic cells

Abstract

Previously we have shown that during in vivo muscle regeneration differentiating rat primary myoblasts transiently upregulate connexin43 (Cx43) gap junctions and leave cell cycle synchronously. Here, we studied the temporal regulation of Cx expression in relation to functional dye coupling in allogenic primary myoblast cultures using western blotting, immuno-confocal microscopy and dye transfer assays. As in vivo, Cx43 was the only Cx isotype out of Cx26, 32, 37, 40, 43 and 45 found in cultured rat myoblasts by immunostaining. Cultured myoblasts showed similar temporal regulation of Cx43 expression and phenotypic maturation to those regenerating in vivo. Cx43 protein was progressively upregulated in prefusion myoblasts, first by the cytoplasmic assembly in sparse myoblast meshworks and then in cell membrane particles in aligned cells. Dye injection using either Lucifer Yellow alone, Cascade Blue with a non-junction permeant FITC-dextran revealed an extensive gap junction coupling between the sparse interacting myoblasts and a reduced communication between the aligned, but still prefused cells. The aligned myoblasts, uniformly upregulate p21(waf1/cip1) and p27(kip1) cell cycle control proteins. Taken together, in prefusion myoblasts less membrane-bound Cx43 was found to mediate substantially more efficient dye coupling in the growing cell fraction than those in the aligned post-mitotic myoblasts. These and our in vivo results in early muscle differentiation are consistent with the role of Cx43 gap junctions in synchronizing cell cycle control of myoblasts to make them competent for a coordinated syncytial fusion.

Lithium Chloride Regulates Connexin43 in Skeletal Myoblasts In Vitro: Possible Involvement in Wnt/β-Catenin Signaling

“…Recently, Gorbe and his colleagues argue that Cx43 expression in regenerating muscle is correlated with myoblast proliferation and fusion into myotubes. Therefore, these reports highlight that gap junction coupling plays a critical role in myotube formation (Gorbe et al 2005(Gorbe et al , 2006(Gorbe et al , 2007.…”

MicroRNAs and Cell Cycle Regulation

“…1,9 The proliferation and developmental phenotypes associated with microRNA expression in C2C12 can be attributed in part to the repression of specific target genes that affect muscle cell differentiation. Targets of miR-206 and/or miR-1 include Pola1 (the largest subunit of DNA polymerase a, 9 Gkb1 (connexin-43), a gap junction protein that regulates myoblast proliferation and is repressed during myoblast fusion in vitro, [10][11][12][13] and Hdac4 (a repressor of muscle gene expression). 1 Targets of miR-133 include Srf (serum response factor), a regulator of muscle cell differentiation and proliferation, 1 and Ptbp2 a splicing factor that may control alternative splicing of developmentally regulated transcripts during myoblast-myotube transition.…”

18α-Glycyrrhetinic Acid Induces Phenotypic Changes of Skeletal Muscle Cells to Enter Adipogenesis

“…Especially, the role of gap-junctional communication has been extensively studied in myogenesis. Of the so far identified connexins, connexin43 (Cx43) is the major connexin expressed in myoblasts [17,18,[20][21][22]. Upregulation of Cx43 expression in myoblasts during skeletal muscle regeneration has been reported [17], and inhibition of gap-junctional communication in myoblasts in vitro either by chemical inhibitors [18] or by inhibiting Cx43 expression [22] has been shown to inhibit myotube formation.…”