Stac3 is required for myotube formation and myogenic differentiation in vertebrate skeletal muscle

Abstract

Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G(1) phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G(1) phase when compared with controls, suggesting a role in the G(1) phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.

FIGURE 1.

Amino acid alignment Stac3 protein sequences from Atlantic salmon, zebrafish, mouse, and human. The protein kinase C domain and SH3 domains are in bold and boxed, respectively, and the predicted phosphorylation sites in salmon and mouse are underlined.

FIGURE 2.

Quantitative PCR analysis of stac3 gene expression in Atlantic salmon. A, mRNA expression profile for Ss stac3α and -β in fish fed a reduced ration for 28 days (day 0) followed by refeeding to satiation for 21 days. B, tissue distribution of Ss stac3α mRNA in gill (G), gut (GT), kidney (K), heart (H), liver (L), eye (E), fast muscle (FM), slow muscle (SM), and skin (S). C, tissue distribution for Ss stac3β as described for B. D, stac3α mRNA levels increase during the maturation of an Atlantic salmon primary myogenic cell culture, and its expression is highly correlated with myog (E) and myoD (F). G, Ss stac3α mRNA levels are decreased in cells starved of amino acid and serum for 72 h (dashed line), when compared with controls (solid line). H, in cells starved for 72 h, Ss stac3α mRNA expression increases in response to amino acid stimulation, but not to Igf-I. Vertical bars represent 0, 3, 6, 12, and 24 h poststimulation.

FIGURE 3.

stac3 and myoD in situ hybridization of zebrafish embryos. stac3 (A, i and ii) and myoD (A, iii) expression during early segmentation. At midsegmentation stac3 (B, i, ii, and iv-vi) is expressed in the adaxial cells, whereas myoD is found throughout the somite (B, iii). stac3 expression is observed in the myotome at late segmentation (C, i–iv), and at the dorsal and ventral extremes of the myotome after the completion of segmentation (D, i–iv). E and F, zebrafish embryos expressing gfp under the control of a fast muscle α actin promoter and in control and stac3 morphant embryos, respectively. Fast muscle fibers in morphant fish (F) have abnormal formation in comparison to controls (E). Antibody specific to slow muscle myosin in stac3 morphant zebrafish embryos showing dysregulated myofibrillar protein assembly in stac3 morphant embryos (H) when compared with controls (G).

FIGURE 4.

Injection of morpholinos in Tg(sMHC-GFP:β-actin-GFPcaax) embryos at 24 h postfertilization. A and B, Mo-control shows normal slow muscle fiber morphology (dose 1.5 ng). A and B, Mo-stac3 shows abnormal slow muscle fiber morphology including loss of slow muscle fibers (white arrowheads) (dose 1.5 ng). Lateral view is shown in A and C. Confocal image of transverse vibratome sections in B and D, insets shows the plane of section cut immediately after the yolk (red line). Expression of ptc1 is reduced in morphant embryos (F) when compared with controls (E). Scale bars represent 100 μm.

FIGURE 5.

stac3 mRNA and protein expression in control and RNAi-treated C2C12 cells. A, stac3 mRNA expression increases during the differentiation of C2C12 cells (dark colored bars), and can be sufficiently knocked down by RNAi (light colored bars). Time points shown are for cells in GM and at 24, 48, 72, and 96 h in DM. B, Myoglobin protein levels (red) in S3RNAi-treated C2C12 cells (lower panels) is decreased in comparison to controls (upper panels) after 72 h in DM. Nuclei were counterstained with DAPI (blue). C, Western blot analysis of Myoglobin protein levels in RNAi-treated (R) and control cells (C) at 72 and 96 h. D, the increased Myog mRNA levels that occur during differentiation of control C2C12 cells (dark bars) is suppressed in S3RNAi-treated cells (light bars). E, there were no differences in myoD mRNA levels between RNAi-treated (light bars) and control cells (dark bars). Scale bars represent 100 μm.

FIGURE 6.

A, Igfbp-5 mRNA levels are lower in S3RNAi-treated cells (light bars) when compared with control cells (dark bars). B, Igf2 mRNA levels are suppressed in S3RNAi-treated cells. C, phosphorylation of Akt (Ser-473) is suppressed in S3RNAi-treated cells (light bars) when compared with controls (dark bars). D, phosphorylation of mTOR (Ser-2448), S6k1 (Thr-389), and S6 ribosomal protein (Ser-235/236) is increased in S3RNAi-treated cells (light bars) when compared with control cells (dark bars). E, exogenously applied Igf2 fails to rescue the S3RNAi-treated cells. Confocal microscopy images showing myosin heavy chain (green) and nuclei (red) of control and RNAi cells stimulated with BSA or 300 ng/ml of Igf2 after growing for 72 h in DM.

FIGURE 7.

A, phospho-Akt (Ser-473), total AKT, phospho-Erk1/2 (Thr-202/Tyr-204), and total Erk1/2 protein levels in RNAi-treated and control cells starved of serum for 24 h and then stimulated with Igf2 (150 ng/ml) for 2 h showing that Igf/Akt signaling is not blocked in S3RNAi-treated cells. B, Western blot analysis of phospho-Erk1/2 (Thr-202/Tyr-204), p38α, phospho-p38α (Thr-180/Tyr-182), MyoD, and phospho-MyoD (Ser-200), in RNAi-treated and control cells. There were no differences for these signaling pathways between S3RNAi-treated and control cells. C, mRNA levels for Stac3 in gfp (control) and STAC3 overexpressing cells. D, Igfbp-5 mRNA levels are increased in Stac3 overexpressing cells, but no differences were found for other markers of differentiation Myog (E) and Ckm (F) in cells overexpressing Stac3, or gfp.

FIGURE 8.

A, Stac3 (black) and MyoD (gray) mRNA levels in cells synchronized at the G₀ phase of the cell cycle and following release of methionine deprivation. Stac3 expression is highest during the G₁ phase of the cell cycle and decreases as cells enter S-phase. B, cell proliferation rates in RNAi-treated and control cells 24 h after transfection and growing in GM. C, cell proliferation rates in RNAi-treated and control cells 24 h after transfection and growing in DM. D, flow cytometry analysis showing the proportion of cells in G₁, S-phase, and G₂ phase of the cell cycle for S3RNAi-treated cells growing in GM (G₁ = 50.82 ± 2.26%, S = 15.52 ± 0.21%, G₂ = 33.65 ± 2.47%). E, flow cytometry analysis showing the proportion of cells in G₁, S-phase, and G₂ phase of the cell cycle for control cells growing in GM (G₁ = 59.65 ± 0.27%, S = 13.79 ± 0.32%, G₂ = 26.55 ± 0.23%). F, flow cytometry analysis of forward linear scatter. S3RNAi-treated cells are smaller (white with black outline, mean = 372.6, half-peak coefficient of variation = 3.3) than control cells (gray, mean = 407.0, half-peak coefficient of variation = 2.0) as indicated by a shift to the left.