Calcitriol increases MBNL1 expression and alleviates myotonic dystrophy phenotypes in HSALR mouse models

Figures

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  Fig. 1. Calcitriol upregulates MBNL1 expression in C2C12 myoblasts and myotubes. Real-time RT-PCR analysis (a, b) and western blot (c) were performed using undifferentiated or differentiated C2C12 cells. The cells were treated with calcitriol as indicated. Undifferentiated C2C12 cells were examined one day after the treatment. Differentiated C2C12 cells were examined on day 5 of differentiation. Expression levels of Mbnl1 mRNA and MBNL1 protein are normalized to those of Gapdh and GAPDH, respectively, and also to 0 nM-treated cells. Mean and SEM (n = 3 and 6 culture dishes for real-time RT-PCR and western blot, respectively) are indicated. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA followed by Turkey multiple comparison correction

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  Fig. 2. Calcitriol alleviates muscle weakness in HSALR mice. (a) WT and HSALR mice were treated with calcitriol as indicated. Vitamin D level of plasma from each group were analyzed. Mean and SEM (n = 8, 8 and 8 in WT, DM1 0 µg/kg/d and DM1 1 µg/kg/d, respectively) are indicated. (b–d) Calcitriol induced partial recovery from the muscle weakness occurring in the HSALR mice. The time that the mice stay on the rotarod (b), the grip strength (c) and the time that the mice hang on the wire mesh (d) of each group was recorded. Mean and SEM (n = 9, 9 and 8 in WT, DM1 0 µg/kg/d and DM1 1 µg/kg/d, respectively) are indicated. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA followed by Turkey multiple comparison correction (a), and by two-way ANOVA followed by Turkey multiple comparison correction (b–d)

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  Fig. 3. In HSALR mice, calcitriol improves muscle histopathology. (a) Representative H&E staining of TA muscles in different groups of mice as indicated. Arrows point to the central nuclei of muscle fibers. Scale bar = 100 μm. (b) The number of fibers with central nuclei was counted in three groups of mice. The data were analyzed by one-way ANOVA followed by Turkey multiple comparison correction. ***p < 0.001. (c) Frequency distribution of cross-sectional TA muscle fiber area was counted in three groups of mice. The mean and SEM (n = 8, 9 and 8 in WT, DM1 0 µg/kg/d and DM1 1 µg/kg/d, respectively) in each group are indicated

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  Fig. 4. Calcitriol upregulates Mbnl1 expression and corrects aberrant splicing in skeletal muscle of HSALR mice.(a, c) Real-time RT-PCR analysis to estimate the expression of Mbnl1 in TA muscles (a) and soleus (c) of WT and HSALR mice treated with calcitriol as indicated. (b, d) Western blot analysis of MBNL1 in mouse TA muscles (b) and soleus (d). Left panel shows representative blots, and right panel shows the quantitative analysis of signal intensities. (e) RT-PCR analysis to evaluate the splicing of Clcn1 exon 7a, Serca1 exon 22 and Nfix exon 7 in TA muscles of four mice from each group as indicated. (f) The ratio of inclusion of relevant exon was calculated. The mean and SEM in each group are indicated. The data were analyzed by one-way ANOVA followed by Turkey multiple comparison correction. *p < 0.05, **p < 0.01 and ***p < 0.001

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  Fig. 5. Calcitriol activates the promoter of Mbnl1 in myogenic cells. (a) Schematic diagram showing the position of promoter of Mbnl1 gene (pMbnl1). Exons are shown by boxes, introns by thin lines, and pMbnl1 by a black closed square. (b) Schematic diagram of pGL3E vector and pMbnl1-pGL3E vector. In pMbnl1-pGL3E, the pMbnl1 region was cloned into pGL3E upstream of the firefly luciferase gene. (c) Luciferase activity of pGL3E or pMbnl1-pGL3E during myogenic differentiation of C2C12 myoblasts. Cells were treated with different concentrations of calcitriol from differentiation day 0, whole cell lysates were extracted and firefly luciferase activity was evaluated on day 5 after induction of differentiation. Firefly luciferase activity was normalized to the Renilla luciferase activity of co-transfected pRL-CMV, and also to 0 nM treated cells. Mean and SD (n = 5 culture dishes) are indicated. ***p < 0.001, and n.s., not significant by one-way ANOVA followed by Turkey multiple comparison correction