Vitamin D3 Exerts Beneficial Effects on C2C12 Myotubes through Activation of the Vitamin D Receptor (VDR)/Sirtuins (SIRT)1/3 Axis

Abstract

The onset of sarcopenia is associated with a decline in vitamin D receptor (VDR) expression, wherein reduced VDR levels contribute to muscle atrophy, while heightened expression promotes muscle hypertrophy. Like VDR, the age-related decline in protein deacetylase sirtuin (SIRT) expression is linked to the development of sarcopenia and age-related muscle dysfunction. This study aimed to investigate whether the VDR agonist 1,25-dihydroxyvitamin D3 (1,25VD3) exerts beneficial effects on muscles through interactions with sirtuins and, if so, the underlying molecular mechanisms. Treatment of 1,25VD3 in differentiating C2C12 myotubes substantially elevated VDR, SIRT1, and SIRT3 expression, enhancing their differentiation. Furthermore, 1,25VD3 significantly enhanced the expression of key myogenic markers, including myosin heavy chain (MyHC) proteins, MyoD, and MyoG, and increased the phosphorylation of AMPK and AKT. Conversely, VDR knockdown resulted in myotube atrophy and reduced SIRT1 and SIRT3 levels. In a muscle-wasting model triggered by IFN-γ/TNF-α in C2C12 myotubes, diminished VDR, SIRT1, and SIRT3 levels led to skeletal muscle atrophy and apoptosis. 1,25VD3 downregulated the increased expression of muscle atrophy-associated proteins, including FoxO3a, MAFbx, and MuRF1 in an IFN-γ/TNF-α induced atrophy model. Importantly, IFN-γ/TNF-α significantly reduced the mtDNA copy number in the C2C12 myotube, whereas the presence of 1,25VD3 effectively prevented this decrease. These results support that 1,25VD3 could serve as a potential preventive or therapeutic agent against age-related muscle atrophy by enhancing the VDR/SIRT1/SIRT3 axis.

Keywords: mitochondrial biogenesis; muscle atrophy; oxidative phosphorylation (OXPHOS); sarcopenia; sirtuins; vitamin D; vitamin D receptor (VDR).

Figure 1

Effects of 1,25VD3 on VDR, SIRT1, SIRT3 expression, myogenic differentiation, and AMPK/AKT activation. C2C12 myotubes were differentiated for four days and treated with various concentrations of 1,25VD3 (0 nM, 1 nM, 10 nM, 100 nM, 200 nM) for 24 h. (A) Representative Western blotting images of VDR, SIRT1, SIRT3, MyHC proteins (MyHC I and II), myogenic markers (MyoD and MyoG), p-AMPK, AMPK, p-AKT, and AKT. (B) qPCR analysis of the VDR, SIRT1, and SIRT3 mRNA levels. (C) qPCR analysis of MyHC I, IIa, IIx and IIb mRNA levels. The symbols in (B,C) serve as points to represent values. Different shapes are used to visually distinguish the values for each group. The data are representative of three independent experiments and are presented as the mean ± SEM. One-way ANOVA was used for statistical analysis between the negative control and 1,25VD3-treated groups. Statistical significance is represented as * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared with 0 nM (control group), and † p < 0.05, †† p < 0.01, and ††† p < 0.001 when compared to 1 nM group. See Supplementary Figure S1 for myotube diameter measurements and Western blot quantifications.

Figure 2

Effects of transient VDR knockdown in C2C12 myotubes on VDR, SIRT1, SIRT3, and MyHC expression. C2C12 myotubes were differentiated by serum depletion and transfected with VDR DsiRNA on the third day of differentiation for 48 h. (A) Representative Western blotting images of VDR, SIRT1, SIRT3, and MyHC (MyHC I and II). (B) Western blot quantification of VDR, SIRT1, and SIRT3 expression. (C) qPCR analysis of the VDR, SIRT1, and SIRT3 mRNA levels. (D) Western blot quantification of MyHC I and MyHC II expressions. The symbols in (B–D) serve as points to represent values. Different shapes are used to visually distinguish the values for each group. The data are representative of three independent experiments Mann–Whitney U test was used for statistical analysis between the negative control (NC) and VDR-KD (VDR knockdown) groups. Statistical significance is represented by ** p < 0.01, and *** p < 0.001.

Figure 3

Effects of IFN-γ/TNF-α-induced VDR, SIRT1, and SIRT3 downregulation on myotube atrophy and apoptosis. After four days of differentiation, C2C12 myotubes were exposed to various concentrations of IFN-γ/TNF-α, including control (PBS) and 20 ng/mL IFN-γ combined with 50, 100, or 150 ng/mL TNF-α for 24 h. (A) Representative Western blot images of VDR, SIRT1, SIRT3, MyHC (MyHC I and II), atrophy markers (FoxO3a, MuRF1, and MAFbx), and apoptosis markers (cleaved caspase-3, cleaved PARP, and Bax). (B) Quantification of atrophy markers (pFoxO3a/FoxO3a, MAFbx, and MuRF1). (C) Quantification of apoptosis-related proteins (cleaved caspase-3, cleaved PARP, and Bax). The expression of phosphorylated FoxO3a (p-FoxO3a) was normalized to that of total FoxO3a, whereas the expression of other proteins was normalized to that of α-tubulin. The symbols in (B,C) serve as points to represent values. Different shapes are used to visually distinguish the values for each group. The data are representative of three independent experiments. One-way ANOVA was used for statistical analysis between the negative control and IFN-γ/TNF-α treated groups. Statistical significance is represented as * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to the control group, and † p < 0.05 when compared to the 20 ng/mL IFN-γ + 50 ng/mL TNF-α group. Refer to Supplementary Figure S2 for Western blot quantification of VDR, SIRT1, SIRT3, MyHC I, and MyHC II.

Figure 4

Effects of 1,25VD3 treatment on myotube diameter and cell viability in IFN-γ/TNF-α-treated C2C12 myotubes. Differentiated C2C12 myotubes were treated with PBS, 20 ng/mL IFN-γ + 100 ng/mL TNF-α, or 20 ng/mL IFN-γ + 100 ng/mL TNF-α in combination with 1 nM or 10 nM 1,25VD3 for 24 h. (A) Representative phase-contrast images (100×) of C2C12 myotubes after 24 h of treatment with IFN-γ/TNF-α with or without 1,25VD3 (scale bar = 200 μm). Red arrows show the changes in the myotube diameter between the groups. (B) Measurement of the myotube diameter. (C) Cell viability assay (MTT). The data are representative of three independent experiments. The symbols in (B,C) serve as points to represent values. Different shapes are used to visually distinguish the values for each group. One-way ANOVA was used for statistical analysis between the negative control and 1,25VD3-treated groups. Statistical significance is represented as *** p < 0.001 when compared to the control group, ††† p < 0.001 when compared to the IFN-γ/TNF-α group, and ### p < 0.001 when compared to all other groups.

Figure 5

Effects of 1,25VD3 on the rescue of C2C12 myotubes from apoptosis and atrophy by modulation of the downstream targets of VDR, SIRT1, and SIRT3. (A) Representative Western blotting images of VDR, SIRT1, SIRT3, MyHC (MyHC I and II), MyoD, MyoG, p-AMPK, AMPK, p-AKT, and AKT expression levels. (B) qPCR analysis of VDR, SIRT1, and SIRT3 mRNA levels; (C) Western blot analysis of p-AMPK/AMPK and p-AKT/AKT. The symbols in (B,C) serve as points to represent values. Different shapes are used to visually distinguish the values for each group. The data are representative of three independent experiments. One-way ANOVA was used for statistical analysis and statistical significance is represented as * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to the control group, † p < 0.05, †† p < 0.01, and ††† p < 0.001 when compared to the IFN-γ/TNF-α group, and ‡ p < 0.05 when compared to IFN-γ/TNF-α + 1,25VD3 (1nM) group. Refer to Supplementary Figure S3 for Western blot quantification of VDR, SIRT1, SIRT3, MyHC I, MyHC II, MyoD, and MyoG.

Figure 6

Effects of 1,25VD3 on the rescue of C2C12 myotubes from apoptosis and atrophy. (A) Western blot analysis of atrophy markers (pFoxO3a/FoxO3a) and apoptosis markers (cleaved PARP, cleaved caspase 3, and Bax). (B) Western blotting quantification of phosphorylated FoxO3a normalized to that of FoxO3a (C). qPCR analysis of atrophy-related genes (FoxO3a3a, MuRF1, and MAFbx). (D) Quantification of apoptosis-related proteins (cleaved caspase-3, cleaved PARP, and Bax). The symbols in (B–D) serve as points to represent values. Different shapes are used to visually distinguish the values for each group. The data are representative of three independent experiments. One-way ANOVA was used for statistical analysis. Statistical significance is represented as * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to the control group, † p < 0.05 and †† p < 0.01 when compared to the IFN-γ/TNF-α group, ### p < 0.001 when compared to all other groups, and ‡ p < 0.05 when compared to IFN-γ/TNF-α + 1,25VD3 (1nM) group.

Figure 7

Effects of 1,25VD3 on the augmentation of oxidative phosphorylation capacity in myotubes treated with IFN-γ/TNF-α. (A) qPCR analysis of relative mitochondrial DNA normalized to nuclear DNA. (B) qPCR analysis of mitochondrial oxidative phosphorylation-related mRNA expression (NDUFB8, SDHB, MTCOI, UQCR2, and ATP5A). The symbols in (A,B) serve as points to represent values. Different shapes are used to visually distinguish the values for each group. The data are representative of three independent experiments. One-way ANOVA was used for statistical analysis, and statistical significance is represented as * p < 0.05 and ** p < 0.01 when compared to the control group, and † p < 0.05, †† p < 0.01, and ††† p < 0.001 when compared to the IFN-γ/TNF-α group.

Figure 8

Schematic diagram by which Vitamin D and the VDR/SIRT1/SIRT3 axis might promote hypertrophy and inhibit apoptosis in skeletal muscle cells. Ac, Acetyl group; P, Phosphate group, C, Cleaved-PARP.