Calponin 3 Regulates Myoblast Proliferation and Differentiation Through Actin Cytoskeleton Remodeling and YAP1-Mediated Signaling in Myoblasts

Abstract

An actin-binding protein, known as Calponin 3 (CNN3), modulates the remodeling of the actin cytoskeleton, a fundamental process for the maintenance of skeletal muscle homeostasis. Although the roles of CNN3 in actin remodeling have been established, its biological significance in myoblast differentiation remains largely unknown. This study investigated the functional significance of CNN3 in myogenic differentiation, along with its effects on actin remodeling and mechanosensitive signaling in C2C12 myoblasts. CNN3 knockdown led to a marked increase in filamentous actin, which promoted the nuclear localization of Yes-associated protein 1 (YAP1), a mechanosensitive transcriptional coactivator required for response to the mechanical cues that drive cell proliferation. Subsequently, CNN3 depletion enhanced myoblast proliferation by upregulating the expression of the YAP1 target genes related to cell cycle progression, such as cyclin B1, cyclin D1, and PCNA. According to a flow cytometry analysis, CNN3-deficient cells displayed higher S and G2/M phase fractions, which concurred with elevated proliferation rates. Furthermore, CNN3 knockdown impaired myogenic differentiation, as evidenced by reduced levels of MyoD, MyoG, and MyHC, key markers of myogenic commitment and maturation, and immunocytochemistry showed that myotube formation was diminished in CNN3-suppressed cells, which was supported by lower differentiation and fusion indices. These findings reveal that CNN3 is essential for myogenic differentiation, playing a key role in regulating actin remodeling and cellular localization of YAP1 to orchestrate the proliferation and differentiation in myogenic progenitor cells. This study highlights CNN3 as a critical regulator of skeletal myogenesis and suggests its therapeutic potential as a target for muscle atrophy and related disorders.

Keywords: YAP1; actin cytoskeleton remodeling; calponin; mechanotransduction; myogenic differentiation; proliferation.

Figure 1

Expression of CNN3 in mouse tissues and throughout myoblast differentiation. (A) Immunoblotting was conducted to assess CNN3 expression in C2C12 myoblasts and tissues from 8-week-old mice; α-tubulin was used as a loading control. (B) C2C12 myoblasts were collected and subjected to immunoblotting of MyoD, MyoG, MyHC, and CNN3 at designated differentiation time points; β-actin was used as a loading control. (C) Expression levels were normalized versus β-actin, and relative ratios were calculated, with day 0 set as the baseline for MyoD and CNN3, day 1 for MyoG, and day 2 for MyHC. Data are expressed as means ± SEM (n = 3), with asterisks denoting statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

CNN3 knockdown enhanced F-actin levels and increased nuclear YAP1. C2C12 myoblasts were transfected with 200 nM of either control scRNA or siCNN3 (siCNN3-1 or siCNN3-2). (A) CNN3 expressions were determined 24 h after transfection by immunoblotting. Expression levels were normalized versus β-actin, and relative ratios were calculated using the control scRNA as the baseline (set to one). (B) Cells were stained with FITC-phalloidin (green) to visualize F-actin and Hoechst 33,342 (blue) to label nucleus. Scale bar: 25 μm. Phalloidin fluorescence intensities were quantified using ImageJ program. (C) F-actin levels were analyzed by flow cytometry following staining with FITC-phalloidin. (D) Cytoplasmic and nuclear fractions were subjected to immunoblotting to detect YAP1, pYAP1 (phosphorylated YAP1), and CNN3. α-Tubulin and lamin B2 were used as markers for the cytoplasmic and nuclear fractions, respectively; β-actin was used as a loading control. (E) Expression levels were normalized versus β-actin, and relative ratios were calculated versus scRNA. Data are expressed as means ± SEM (n = 3), with asterisks denoting statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

CNN3 depletion facilitated myoblast proliferation. C2C12 myoblasts were transfected with either control scRNA or siCNN3 and analyzed 24 h post-transfection. (A) Cell proliferation was assessed using EdU incorporation (green) to label replicating cells, and Hoechst 33,342 (blue) was used to counterstain the nucleus. Scale bar: 50 µm. (B) The percentages of EdU-positive cells were determined using ImageJ program. (C) Viable cell numbers were measured using a cell viability assay kit. (D) mRNA levels of proliferation markers (PCNA, Cyclin B1, and Cyclin D1) were assessed by RT-qPCR and normalized versus GAPDH expression. (E,F) Cell cycle analysis was performed using flow cytometry with scatter plots. Data are expressed as means ± SEM (n = 3), with asterisks denoting statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

CNN3 knockdown suppressed expressions of myogenic factors. (A) C2C12 myoblasts were transfected with either control scRNA or siCNN3, allowed to differentiate, and subjected to immunoblotting at designated differentiation time points; β-actin was used as a loading control. Expression of myogenic regulatory factors and CNN3 were evaluated by immunoblotting. (B) Expression intensities of proteins in myoblasts transfected with scRNA (open column) and siCNN3 (blue column) were normalized versus β-actin. Results are expressed as relative ratios compared to scRNA levels on day 0 for CNN3 and MyoD and day 3 for MyoG and MyHC. Data are expressed as means ± SEM (n = 3), with asterisks denoting statistically significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

Depletion of CNN3 disrupted myogenic differentiation. C2C12 myoblasts were transfected with control scRNA or siCNN3 and then allowed to differentiate for 5 days. (A) Representative immunocytochemistry results after staining with MyHC antibody (green) and Hoechst 33,342 (blue). Scale bar: 50 μm. (B) MyHC-positive areas, differentiation and fusion indices, and myotube widths were evaluated as outlined in Section 2.7. Data are expressed as means ± SEM (n = 3), with asterisks denoting statistically significant differences (*** p < 0.001).