The mechanisms of skeletal muscle atrophy in response to transient knockdown of the vitamin D receptor in vivo

Abstract

Key points: Reduced vitamin D receptor (VDR) expression prompts skeletal muscle atrophy. Atrophy occurs through catabolic processes, namely the induction of autophagy, while anabolism remains unchanged. In response to VDR-knockdown mitochondrial function and related gene-set expression is impaired. In vitro VDR knockdown induces myogenic dysregulation occurring through impaired differentiation. These results highlight the autonomous role the VDR has within skeletal muscle mass regulation.

Abstract: Vitamin D deficiency is estimated to affect ∼40% of the world's population and has been associated with impaired muscle maintenance. Vitamin D exerts its actions through the vitamin D receptor (VDR), the expression of which was recently confirmed in skeletal muscle, and its down-regulation is linked to reduced muscle mass and functional decline. To identify potential mechanisms underlying muscle atrophy, we studied the impact of VDR knockdown (KD) on mature skeletal muscle in vivo, and myogenic regulation in vitro in C2C12 cells. Male Wistar rats underwent in vivo electrotransfer (IVE) to knock down the VDR in hind-limb tibialis anterior (TA) muscle for 10 days. Comprehensive metabolic and physiological analysis was undertaken to define the influence loss of the VDR on muscle fibre composition, protein synthesis, anabolic and catabolic signalling, mitochondrial phenotype and gene expression. Finally, in vitro lentiviral transfection was used to induce sustained VDR-KD in C2C12 cells to analyse myogenic regulation. Muscle VDR-KD elicited atrophy through a reduction in total protein content, resulting in lower myofibre area. Activation of autophagic processes was observed, with no effect upon muscle protein synthesis or anabolic signalling. Furthermore, RNA-sequencing analysis identified systematic down-regulation of multiple mitochondrial respiration-related protein and genesets. Finally, in vitro VDR-knockdown impaired myogenesis (cell cycling, differentiation and myotube formation). Together, these data indicate a fundamental regulatory role of the VDR in the regulation of myogenesis and muscle mass, whereby it acts to maintain muscle mitochondrial function and limit autophagy.

Keywords: atrophy; metabolism; skeletal muscle; vitamin D.

Figure 1. In vivo experimental design and grouping

A, schematic design of in vivo experiments. B, confirmation of contralateral VDR‐KD by qRT‐PCR (N = 7). C, representative western blot and quantification of VDR‐KD (N = 7). Scale bars represent 200 μm. Data are individual values with mean ± SD. Data were analysed using paired t‐tests. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2. In vivo VDR‐KD results in muscle fibre atrophy

A, representative images of muscle fibres stained for dystrophin (green), DAPI (blue) and VDR (red). Scale bars represent 200 μm. B, all fibre CSA analysis; C, Type IIa; and D, IIb fibre CSA distribution. Three random fields of view were measured per section in both L and R TA muscles in each animal (N = 7), with CSA measured for all intact fibres. E, alkaline soluble protein measures; F, RNA; and G, DNA quantification per mg dried muscle (n = 7). Data are individual values with mean ± SD. Data were analysed using paired t‐tests. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3. In vivo VDR‐KD increases autophagy‐related pathways

A, measurement of mixed lysate MPS rates by D₂O incorporation (N = 7). B, quantification and representative western blots of phosphorylated and total protein anabolic signalling intermediates (n = 7). C, qRT‐PCR measurement of proteolysis‐related gene expression. D, quantification and representative western blots of autophagy‐related protein expression (n = 7). Data are individual values with mean ± SD. ^* P < 0.05, ^** P < 0.01 between the indicated groups. Data were analysed using paired t‐tests. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4. VDR‐KD upregulates autophagy‐related gene‐sets whilst downregulating mitochondrial metabolic processes

A, volcano plot of P < 0.05 statistically significant up‐/downregulated genes. B, top five upregulated and downregulated gene‐sets from the Molecular signatures database for VDR‐OE muscles (n = 7). See also Supplemental file 1. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5. In vivo VDR‐KD reduces electron transport chain‐related gene expression

A, RNA‐seq pathway analysis of Rattus norvegicus electron transport chain gene expression. Log fold changes are shown as a gradient from red (upregulated) to blue (downregulated). B, quantification and representative western blot analysis of individual mitochondrial electron transport chain complex protein expression (n = 7). Data are individual values with mean ± SD. P values are for comparisons between the indicated groups. Data were analysed using paired t‐tests. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6. In vitro VDR‐KD impairs myoblast cell cycle regulation and proliferation

A, qRT‐PCR analysis of VDR‐KD by shRNA (n = 5). B, representative western blot and quantification showing VDR‐KD (n = 6). C, cell cycle proportions of proliferating myoblasts (n = 7). D, total cell populations (n = 4). E, BrdU incorporation within proliferating myoblasts (n = 10). Data are individual values with mean ± SD, ^* P < 0.05. Data were analysed using t‐tests. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7. In vitro VDR‐KD impairs myogenesis and differentiation

A, representative images of myotubes 5 days after differentiation, stained with phalloidin (red) and DAPI (blue). B, quantification of myotube population (n = 4). C, myotube diameter quantification (n = 4). D, quantification of the number of myonuclei per myotube (n = 4). E, total alkaline soluble protein, DNA and RNA content throughout differentiation (n = 6). F, western blot analysis of myosin IIx expression throughout differentiation (n = 6). G, myofibrillar protein synthesis rates 5 days after differentiation (n = 6). Scale bars represent 100 μm. For all grouped plots non‐pairwise comparisons were made using two‐sided t‐tests. For time‐course measurements, ANOVA with multiple comparison by Tukey analysis were used. Data are individual values with mean ± SD. ^* P < 0.05, ^**** P < 0.0001 between the groups indicated. ^†† P < 0.01, ^††† P < 0.001 between VDR‐KD and shRNA controls at that time point; ^§ P < 0.05, ^§§ P < 0.01 versus the initial time point. [Color figure can be viewed at wileyonlinelibrary.com]