Role of Intracellular Calcium in Vitamin D‐Treated L6 Myoblast Differentiation and Fiber Type Specification

Abstract

Emerging evidence suggests that vitamin D plays a role in skeletal muscle differentiation and fiber type‐specific gene expression. It has been demonstrated that vitamin D receptor (VDR) and/or intracellular calcium are involved in this process. The present study was conducted to examine the role of intracellular calcium in vitamin D‐mediated L6 myoblast differentiation. An intracellular calcium chelator, 1,2‐Bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA), was treated to the cells and incubated for 24 hrs prior to differentiation. Then, 100 pM 1,25‐dihydroxyvitamin D3 was treated to the cells for 8 days of differentiation. A total of 4 treatments (a 2×2 factorial design) used in this study were: CON, BAPTA, 1,25‐dihydroxyvitamin D3 (VitD), BAPTA+1,25‐dihydroxyvitamin D3 (BAPTA‐D). Ca2+‐chelating effect of BAPTA was confirmed after BAPTA incubation by using fluorescent Ca2+ indicator, Indo‐1 AM. On day 8 after differentiation, mRNA levels of myogenic regulatory factors (MRFs) and skeletal muscle fiber type‐specific markers were analyzed by qPCR. In VitD treatment, mRNA levels of two MRFs, MyoD and Myogenin, were suppressed in comparison with those of CON (0.8 and 0.6 folds, respectively, p<0.05). In contrast, slow fiber type‐specific markers, including PGC‐1α, ATP2a2 and Tnni1, were upregulated (1.8, 1.7, and 2.9 folds, respectively, p<0.05), while Tnni2, a fast fiber type‐specific gene, was downregulated (0.49 folds, p<0.05) in VitD treatment compared to those of CON. In BAPTA treatment, mRNA levels of Myogenin and Mrf4 were decreased when compared to those of CON (0.75 and 0.42 folds, respectively, p<0.05). However, it was shown that slow fiber type‐specific gene mRNA levels (PGC‐1α, ATP2a2, and Tnni1) were not affected by intracellular calcium level. In BAPTA‐D treatment, decreased gene expression levels of Myogenin and Mrf4 were similar to those of BAPTA treatment, while MyoD was at further lower level compared to that of VitD treatment (0.5 folds, p<0.05). In addition, Tnni1, which was increased in VitD treatment, was not increased in BAPTA‐D treatment. This results may indicate that 1,25‐dihydroxyvitamin D3 modulated Tnni1 mRNA expression via intracellular calcium level. At gene expression level, the inhibitory effect in VitD treatment on MyoD gene expression was amplified in BAPTA‐D treatment. However, the enhancing effect in VitD treatment on Tnni1 mRNA level was abolished in BAPTA‐D treatment. Therefore, intracellular calcium level partially mediates the effects of 1,25‐dihydroxyvitamin D3 on gene expression during L6 myoblast differentiation.