Regulation of myosin heavy chain expression during rat skeletal muscle development in vitro

Abstract

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and approximately 10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN-nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.

Figure 1

Protein expression of MHC isoforms in primary cultures of rat myotubes. Myoblasts were isolated from embryonic day 21 rats and cultured for 12–13 d, during which time they fused into multinucleated myotubes. They were fixed and immunostained as described in MATERIALS AND METHODS. (A) Typical view of myotubes stained for slow MHC, showing nonuniform staining and the presence of cross-striations. (B) Myotubes stained for embryonic MHC. Myotubes exhibited well organized cross-striations and often had peripheral nuclei. (C) Myotubes stained for fast/neonatal MHC illustrating the onset of sarcomeric organization and variations in staining intensity. (D) Culture treated with 1.5 μM TTX and stained for embryonic MHC. Myotubes were broader, had a paucity of cross-striations, and more central nuclei. Bar, 20 μm.

Figure 2

Protein levels of slow and fast MHC in myotube cultures. (A) Slow MHC protein after administration of TTX, CSA, and/or transfection with CN*. (B) Slow MHC protein after transfection with CN* and/or VIVIT. (C) Fast MHC protein after administration of TTX, CSA, or transfection with CN* or VIVIT. Myoblasts were cultured for 12–13 d, then myofibrillar proteins were isolated and subjected to Western blotting as described in MATERIALS AND METHODS. Samples were blotted for slow or fast MHC, stripped, and then reblotted for total MHC. Values for slow or fast MHC were taken as a percentage of total MHC and then normalized to controls. Inset, blots from a single experiment shown in the same order as the data bars. Values are the mean ± SE of three to six independent experiments. *Significantly different from control, p < 0.05. Brackets, the two indicated values are significantly different, p < 0.05.

Figure 3

Protein expression of slow MHC in myotube cultures. Cells were cultured as described in Figure 1 and immunostained with an antibody against slow MHC. (A and B) Culture transfected with CN*, showing widespread, intense punctate staining, central nuclei, and a scarcity of cross-striations. (C) Culture administered CSA. No staining above background was detected. (D) Culture transfected with CN* and administered CSA. No staining above background was detected. (E) Culture transfected with CN* and administered tetrodotoxin. Staining was decreased compared with transfection with CN* only. (F) Culture transfected with CN* and VIVIT. Staining was absent, or the intensity was greatly decreased compared with transfection with CN* only, except in limited regions of a few myotubes. Bars, 20 μm (bar in B refers only to that panel).

Figure 4

Protein expression of fast/neonatal MHC in myotube cultures. Cells were cultured as described in Figure 1 and immunostained with an antibody that crosses to both fast and neonatal MHC. (A and B) Culture treated with CSA showing intensely stained myotubes with well aligned sarcomeres and peripheral nuclei. (C) Culture transfected with calcineurin (CN*) demonstrating a typical pattern of organized cross-striations and peripheral nuclei. Note: The field in C is the same field as that of Figure 3A. Myotubes that were positive for fast/neonatal MHC were negative for slow MHC and vice versa. Bars, 20 μm (bar in B refers only to that panel).