Characterization of skeletal muscle atrophy in Smad3-null mice. (A) Frequency distribution of M. tibialis anterior (TA) skeletal muscle fiber CSA between WT and Smad3-null mice. (B, C) Smad3-null primary myoblasts and shSmad3 C2C12 cells were grown under proliferating conditions for a period of 60 h and 72 h, respectively, with proliferation monitored using the methylene blue assay. Error bars represent ± SEM (n = 2). (D) Western blot analysis of Cdk2, cyclin-E and Rb in primary myoblasts (left) and shSmad3 C2C12 cells (right). Ponceau S staining and analysis of tubulin expression were performed to ensure equal loading of samples. The hyperphosphorylated (pRb^PP) and hypophosphorylated (pRb) forms of Rb are indicated. (E) Western blot analysis of Myf5, p21, MyoD, myogenin, MyHC and tubulin expression at 0 h, 24 h, 48 h, 72 h and 96 h of differentiation in WT and Smad3-null primary myotubes. Tubulin expression was analyzed to ensure equal loading of samples. (F) Fusion index of WT and Smad3-null myoblasts at 24 h, 48 h, 72 h and 96 h of differentiation. Error bars represent ± SEM (n = 3). (G) Total average myonuclei number in WT and Smad3-null myotubes at 24 h, 48 h, 72 h and 96 h of differentiation. Error bars represent ± SEM (n = 3). (H-J) Relative mRNA expression levels of caveolin 3, β₁D integrin and SHP-2 in WT and Smad3-null primary myotubes as measured by quantitative real-time PCR. Graphs display fold change ± SEM, normalized to Gapdh (n = 3). For all relevant figure panels, statistical differences are indicated, P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

Myostatin and IGF-1 expression in Smad3-null muscle and primary myotubes. Relative mRNA expression levels of myostatin (A) and Igf-1 (B), as measured by quantitative real-time PCR in quad muscle tissue from WT and Smad3-null mice. Relative mRNA expression levels of myostatin (C) and Igf-1 (D), as measured by quantitative real-time PCR in primary myotube cultures from WT and Smad3-null mice. Graphs display fold change ± SEM, normalized to Gapdh (n = 3). For all figure panels, statistical differences are indicated, P < 0.01 (**) and P < 0.001 (***).

Reduced atrophy in double-knockout (DKO) mice (Smad3^−/−, Mstn^−/−). (A) Quantification of the change in individual skeletal muscle weights between DKO and WT mice. TA denotes M. tibialis anterior. Gas and sol denotes M. gastrocnemius and soleus. Quad denotes M. quadriceps. Error bars represent ± SEM (n = 3). (B) Individual muscle weights from DKO and WT mice expressed as a percentage of total body weight. Error bars represent ± SEM (n = 3). (C) Comparison of the average TA muscle CSA (μm²) between Smad3-null, WT, DKO and myostatin-null mice. Difference between Smad3-null and DKO mice is not significant (P > 0.05). Error bars represent ± SEM (n = 3). (D) Quantification of the total muscle fiber number in TA muscles from Smad3-null, WT, DKO and myostatin-null mice. Error bars represent ± SEM (n = 3). (E) Frequency distribution of individual muscle fiber CSA in TA muscles from Smad3-null, WT and DKO mice, expressed as a percentage of total fiber number. Muscle fiber areas were grouped based on size: < 1 000 μm², 1 000-2 500 μm² and > 2 500 μm². (F) WT, myostatin-null and DKO primary myoblasts were grown under proliferating conditions for a period of 60 h, with proliferation monitored using the methylene blue assay. Error bars represent ± SEM (n = 2). (G) The fusion index of WT, Smad3-null, myostatin-null and DKO myoblasts at 24 h, 48 h, 72 h and 96 h of differentiation. Error bars represent ± SEM (n = 3). (H) Average myonuclei number in WT, Smad3-null, myostatin-null and DKO myotubes at 24 h, 48 h, 72 h and 96 h of differentiation. Error bars represent ± SEM (n = 3). (I) Neutralization of myostatin in WT and Smad3-null primary culture by soluble ActRIIB treatment. WT and Smad3-null primary myoblasts were seeded at a density of 25 000 cells/cm². Following a 12 h attachment period, cells were switched to differentiation media with (0 h + 3 μg/ml soluble ActRIIB) or without soluble ActRIIB (0 h + 0 μg/ml soluble ActRIIB) for 48 h. The MyHC and MyLC expression were measured by western blot analysis. Tubulin levels were measured to ensure equal loading of samples. (J) The expression of MyHC and MyLC in WT and Smad3-null primary culture treated with or without soluble ActRIIB, normalized to tubulin expression. For all relevant figure panels, statistical differences are indicated, P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

Satellite cell (SC) number and self-renewal in Smad3-null mice. Representative images of MyoD and Pax7 immunostaining on tissue-dissociated SCs (A) or SCs migrated from single muscle fibers (C). MyoD⁻/Pax7⁺ (yellow arrowheads), MyoD⁺/Pax7⁻ (red arrows) and MyoD⁺/Pax7⁺ (white asterisks) populations are indicated. Scale bar represents 50 μm. The graph represents the proportions of MyoD⁺/Pax7⁺, MyoD⁻/Pax7⁺ and MyoD⁺/Pax7⁻ cells expressed as a percentage of total cell number in tissue-dissociated SCs (B) or in single muscle fiber cultures (D). (E) The graph represents the number of SCs, per 100 myonuclei (%), in single muscle fibers isolated from Smad3-null and WT mice. (F) The graph represents the number of SCs per single muscle fiber. Error bars represent ± SEM (n = 3). For all relevant figure panels, statistical differences are indicated, P < 0.01 (**) and P < 0.001 (***).

Ubiquitination of intracellular proteins in Smad3-null skeletal muscle. (A) Western blot analysis of MuRF1, ubiquitin, MyHC, Atrogin1, FoxO1, p-FoxO1, Akt, p-Akt (Thr308) and p-Akt (Ser473) expression in quad muscle from WT and Smad3-null mice. Ponceau S staining was performed to ensure equal loading of samples. (B) Quantitative real-time PCR analysis of MuRF1, MyHC I, MyHC IIB and MyHC IIX expression in muscle of WT and Smad3-null mice. Graphs display fold change ± SEM, normalized to Gapdh (n = 3). (C) Treatment of proteasome inhibitor epoxomicin in WT and Smad3-null primary myotubes. Differentiated WT and Smad3-null primary myotubes (48 h) were incubated with epoxomicin (48 h + 100 nM epox) or without epoxomicin (48 h + 0 nM epox) for 10 h before sample collection. The MyHC expression was measured by western blot analysis. Tubulin levels were measured to ensure equal loading of samples. (D) The expression of MyHC in WT and Smad3-null primary myotubes treated with or without epoxomicin, normalized to tubulin expression. For all the relevant figure panels, statistical differences are indicated, P < 0.001 (***).