Effects of 1,25(OH)2 D3 and 25(OH)D3 on C2C12 Myoblast Proliferation, Differentiation, and Myotube Hypertrophy

Abstract

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2 D by 1α-hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2 D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . We show that myoblasts not only responded to 1,25(OH)2 D3 , but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2 D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2 D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α-hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2 D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2 D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2 D3 . J. Cell. Physiol. 231: 2517-2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Figure 1

Both 25(OH)D3 and 1,25(OH)2D3 attenuated C2C12 myoblast proliferation. Micrographs of C2C12 myoblasts cultured for 4 days in growth medium (A), in growth medium supplemented with 1000 nmol/L 25(OH)D3 (B), or 100 nmol/L 1,25(OH)2D3 (C). Myoblast proliferation was quantified at day 1 and 4 (D). Scale bar indicates 100 µm. Data were analyzed using a two‐way ANOVA followed by Bonferroni's post hoc comparisons test. Values are mean ± SEM (n = 20). ***P < 0.001; ^### P < 0.001 (^# between time period, * between vitamin D3 concentrations).

Figure 2

Both 25(OH)D3 and 1,25(OH)2D3 increased CYP24 and VDR mRNA levels and reduced myogenin mRNA levels in myoblasts. Myoblasts were cultured for 4 days in growth medium supplemented with 1000 nmol/L 25(OH)D3, 100 nmol/L 1,25(OH)2D3, or without any supplements. After 1 and 4 days, mRNA levels of CYP27B1 (A), CYP24 (B), VDR (C), ki67 (D), MyoD (E), and myogenin (F) were determined. Data were analyzed using a two‐way ANOVA followed by Bonferroni's post hoc comparisons test. Values are mean ± SEM (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001; ^# P < 0.05, ^### P < 0.01, ^### P < 0.001 (^# between time period, * between vitamin D3 concentrations).

Figure 3

Effects of 25(OH)D3 and 1,25(OH)2D3 on myotube diameter. Micrographs of C2C12 myoblasts cultured for 3 days in differentiation medium (A), in differentiation medium supplemented with 400 nmol/L 25(OH)D3 (B), or 100 nmol/L 1,25(OH)2D3 (C). After 3 days of culture, myotube diameter (µm) (D) and myotubes/mm² (E) were determined. Scale bar indicates 100 µm. Data were analyzed using a one‐way ANOVA followed by Bonferroni's post hoc test. Values are mean ± SEM (n = 8). *P < 0.05.

Figure 4

Both 25(OH)D3 and 1,25(OH)2D3 increased CYP24 and VDR mRNA levels in differentiating C2C12 cells. C2C12 cells were cultured for 3 days in differentiation medium supplemented with 400 nmol/L 25(OH)D3, 100 nmol/L 1,25(OH)2D3, or without any supplements. After 1 and 3 days of culture, mRNA levels of CYP27B1 (A), CYP24 (B), VDR (C), MyoD (D), myogenin (E), and PGC1α (F) were determined. Data were analyzed using a two‐way ANOVA followed by Bonferroni's post hoc comparisons test. Values are mean ± SEM (n = 6). *P < 0.05, ***P < 0.001; ^## P < 0.01, ^### P < 0.001 (^# between time period, * between vitamin D3 concentrations).

Figure 5

Effects of 25(OH)D3 and 1,25(OH)2D3 on mRNA levels of myosin heavy chain. C2C12 cells were cultured for 3 days in differentiation medium supplemented with 400 nmol/L 25(OH)D3, 100 nmol/L 1,25(OH)2D3, or without any supplements. After 1 and 3 days of culture, mRNA levels of MHC‐I (A), MHC‐IIA (B), MHC‐IIX (C), MHC‐IIB (D), and MHC embryonic (E) were determined. Data were analyzed using a three‐way ANOVA. Values are mean ± SEM (n = 6). ^### P < 0.001 (^# between time period).

Figure 6

Both 25(OH)D3 and 1,25(OH)2D3 did not affect levels of p‐Akt, total Akt, p‐S6, and total S6 in differentiating C2C12 cells. C2C12 cells were cultured for 3 days in differentiation medium supplemented with 400 nmol/L 25(OH)D3, 100 nmol/L 1,25(OH)2D3, or without any supplements. After 1 and 3 days of culture, levels of p‐Akt (A), p‐S6 (B), total Akt (C), total S6 (D) the ratio of p‐Akt/total Akt (E), and p‐S6/total S6 (F) were determined. Data were analyzed using a two‐way ANOVA followed by Bonferroni's post hoc comparisons test. Values are mean ± SEM (n = 4). ^# P < 0.05, ^## P < 0.01 (^# between time period, * between vitamin D3 concentrations).