Abstract
Muscle weakness and myopathy are observed in vitamin D deficiency and chronic renal failure, where concentrations of the active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), are low. To evaluate the mechanism of action of 1α,25(OH)2D3 in skeletal muscle, we examined mitochondrial oxygen consumption, dynamics, and biogenesis and changes in expression of nuclear genes encoding mitochondrial proteins in human skeletal muscle cells following treatment with 1α,25(OH)2D3. The mitochondrial oxygen consumption rate (OCR) increased in 1α,25(OH)2D3-treated cells. Vitamin D3 metabolites lacking a 1α-hydroxyl group (vitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) decreased or failed to increase OCR. 1α-Hydroxyvitamin D3 did not increase OCR. In 1α,25(OH)2D3-treated cells, mitochondrial volume and branching and expression of the pro-fusion protein OPA1 (optic atrophy 1) increased, whereas expression of the pro-fission proteins Fis1 (fission 1) and Drp1 (dynamin 1-like) decreased. Phosphorylated pyruvate dehydrogenase (PDH) (Ser-293) and PDH kinase 4 (PDK4) decreased in 1α,25(OH)2D3-treated cells. There was a trend to increased PDH activity in 1α,25(OH)2D3-treated cells (p = 0.09). 83 nuclear mRNAs encoding mitochondrial proteins were changed following 1α,25(OH)2D3 treatment; notably, PDK4 mRNA decreased, and PDP2 mRNA increased. MYC, MAPK13, and EPAS1 mRNAs, which encode proteins that regulate mitochondrial biogenesis, were increased following 1α,25(OH)2D3 treatment. Vitamin D receptor-dependent changes in the expression of 1947 mRNAs encoding proteins involved in muscle contraction, focal adhesion, integrin, JAK/STAT, MAPK, growth factor, and p53 signaling pathways were observed following 1α,25(OH)2D3 treatment. Five micro-RNAs were induced or repressed by 1α,25(OH)2D3. 1α,25(OH)2D3 regulates mitochondrial function, dynamics, and enzyme function, which are likely to influence muscle strength.
Highlights
OPA1 increased, whereas expression of the pro-fission proteins Fis1 and Drp1 decreased
To assess the mechanism of action of the active metabolite of vitamin D3, 1␣,25(OH)2D3, in human skeletal muscle cells, we examined changes in mitochondrial oxygen consumption (OCR), mitochondrial dynamics, mitochondrial OXPHOS proteins, pyruvate dehydrogenase phosphorylation, and nuclear gene expression using whole transcriptome shotgun sequencing (WTSS, RNA-seq) of messenger RNAs and micro-RNAs
Western blot analysis with a vitamin D receptor (VDR) antibody demonstrated bands of the appropriate mobility (Mr ϳ48,000) that co-migrated with recombinant VDR in homogenates of human skeletal muscle biopsies and human skeletal muscle cells (hSkMCs) (Fig. 1, E and F)
Summary
General—All human studies were by approved by the institutional review board of the Mayo Clinic. 25-Hydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, 1␣-hydroxyvitamin D3, and l␣,25-dihydroxyvitamin D3 were gifts from Dr Milan Uskokovic (Hoffman-La Roche, Nutley, NJ). The ratios of mitochondrial genes ND1 and ND6 to nuclear genes BECN1 and NEB in cells treated with 10Ϫ8 M 1␣,25(OH)2D3 or vehicle (ethanol) were determined using crossing points and a standard curve generated with 0.02–20 ng of human DNA. The adaptor-modified DNA fragments were enriched by 12 cycles of PCR using primers included in the Illumina Sample Prep Kit. The concentration and size distribution of the libraries were determined on an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA). Libraries were loaded onto single end flow cells at concentrations of 8 –10 pM to generate cluster densities of 700,000/mm following Illumina’s standard protocol using the Illumina cBot cluster kit version 3. The collection of 1158 nuclear and mitochondrial DNA genes encoding proteins with strong support of mitochondrial localization was searched for differentially expressed genes from 1␣,25(OH)2D3- or vehicle-treated cells. Data Sharing—All of the sequencing data that were analyzed in this report have been deposited in the Gene Expression Omnibus (GSE70934)
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