1α,25-Dihydroxyvitamin D3 Regulates Mitochondrial Oxygen Consumption and Dynamics in Human Skeletal Muscle Cells*

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.

Vitamin D 3 , through the activity of its active metabolite, 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 D 3 ), 2 is essential for normal calcium and phosphorus balance and the maintenance of skeletal health (1)(2)(3)(4). Vitamin D 3 and its metabolites also have important clinical effects on muscle function, the biochemical basis of which is not well understood. Skeletal muscle weakness and myopathy are prominent in humans with vitamin D deficiency, who rapidly respond to treatment with vitamin D 3 (5,6). Sarcopenia is common in chronic and end stage kidney disease (7,8), where concentrations of 1␣,25(OH) 2 D 3 are low. The administration of vitamin D 3 has been reported to improve muscle strength in humans (9). Reduced serum 1␣,25(OH) 2 D 3 concentrations have been linked to falls in humans (10,11), and clinical trials have demonstrated salutary effects of ␣calcidol on fall prevention (12). Improvement in muscle fiber size has been documented in elderly subjects treated with 1␣,25(OH) 2 D 3 (13).
Previous reports have shown that in avian and rodent isolated skeletal muscle cells and cultured myoblast cell lines, vitamin D 3 metabolites, such as 25-hydroxyvitamin D 3 (25(OH)D 3 ) and 1␣,25(OH) 2 D 3 , influence cellular calcium and phosphorus uptake, cellular growth, differentiation, and the expression of a limited number of genes (14 -19). Many reports suggest that the vitamin D receptor (VDR) is expressed in skeletal muscle (20 -24), and VDR deletion in mice results in alterations in muscle function and strength (25,26). Treatment of vitamin D-deficient humans with cholecalciferol improves muscle phosphocreatine recovery after exercise (27), suggesting that vitamin D 3 or its metabolites alter skeletal muscle oxidative capacity.
To assess the mechanism of action of the active metabolite of vitamin D 3 , 1␣,25(OH) 2 D 3 , 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 following treatment of cells with 1␣,25(OH) 2 D 3 . The dependence of the observed changes upon the presence of the VDR was assessed. Our results show extensive 1␣,25(OH) 2 D 3 -mediated changes in skeletal muscle mitochondrial OCR, dynamics, pyruvate dehydrogenase phosphorylation, and expression of nuclear genes encoding mitochondrial proteins. These changes suggest important effects on skeletal muscle that are likely to alter skeletal muscle performance.
Muscle Biopsies and Mitochondrial Isolation-Muscle biopsies were performed, and mitochondria were isolated as described previously (28,29).
Immunohistochemistry/Microscopy-Skeletal muscle cells were grown on collagen-treated glass plates (MatTek Corp.) and were fixed and treated with VDR antibody as described earlier (32). Skeletal muscle sections (American MasterTech Scientific) were stained with VDR antibody or preadsorbed VDR antibody, as described (32).
Construction of VDR pEGFP Expression Vectors and Transfection into Human Skeletal Muscle Cells-Human VDR cDNA was amplified by PCR with primers with a KpnI site in the 5Ј primer and BamHI site in the 3Ј primer. The product was ligated into pEGFP-C1 or pEGFP-N1 (Clontech). HSkMCs were transfected using Lipofectamine 2000 (Life Technologies).
Knockdown of VDR with Silencing RNA-VDR in skeletal muscle cells was knocked down using two Silencer Select siRNAs to the human VDR (siRNA ID s14777 and s14779, Life Technologies). Silencer Select negative control 1 non-specific siRNA was used in matching cells in equimolar amounts. Lipofectamine (Life Technologies) siRNA complex in Opti-MEM medium was used for transfection. RNA was isolated as detailed below and reverse-transcribed using Superscript III and an oligo(dT) or random hexamer primer, and percentage knockdown of the VDR RNA was determined following quantitative PCR relative to the RPL13A reference gene.
Assessment of Mitochondrial Volume and Fragmentation-To observe dynamic changes in mitochondrial structure, cultured hSkMCs (treated with either vehicle or 1␣,25(OH) 2 D 3 for 48 h) were plated on 8-well glass bottom plates (LabTek) and incubated with 100 nM MitoTracker Deep Red (Life Technologies; excitation 638 nm/emission 700 nm) for 15 min at room temperature. Following thorough rinsing with dye-free solution, cells were imaged using a Nikon A1R confocal system (Nikon Instruments Inc.). Images were acquired at 2048 ϫ 2048 pixels and 12-bit depth using a Plan-Apo ϫ60/1.4 numerical aperture oil objective. Images were corrected for background intensity variations using ImageJ software (National Institutes of Health).
For mitochondrial volume measurement, we performed optical sectioning with a 0.5-m step. For each cell, mitochondria volume was determined using Nikon Elements software.
Mitochondrial morphometric analysis was performed using procedures developed by Koopman et al. (33)(34)(35). To assess the extent of filamentous versus fragmented morphometry, the form factor (an index of mitochondrial branching) and aspect ratio (an index of mitochondrial branch length) were calculated for each cell using a custom-written MATLAB (The Math-Works)-based program (36,37). A decrease in aspect ratio and/or form factor indicates mitochondrial fragmentation (33)(34)(35)(36)(37).
Digital PCR and Quantitative PCR-Human mRNAs (VDR, CYP24A1, MSTN, PDK4) were quantitated by real-time digital PCR with the QuantStudio three-dimensional digital PCR system using FAM-labeled TaqMan assays. RPL13A was used as a reference gene. Data were analyzed on-line using Quant-Studio 3D AnalysisSuite TM Cloud Software. qPCR for determination of VDR following siRNA knockdown was carried out using a Roche LightCycler 480 qPCR apparatus (Roche Applied Science) with SYBR Green master mix, Universal RT mix (Roche Applied Science), and an intron-spanning qPCR primer pair for the VDR (Roche Applied Science).
Assessment of Mitochondrial Protein Expression Using Western Blotting with Specific Antibodies-We assessed changes in mitochondrial protein expression using antibodies or antibody mixtures (all from Abcam unless otherwise noted) directed against the following mitochondrial proteins or complexes: VDAC1 or porin (ab15895), pyruvate dehydrogenase Western blot antibody mixture (ab110416), pyruvate dehydrogenase E1-␣ subunit (phosphorylation at position 293) (ab177461), pyruvate dehydrogenase phosphatase 2 (ab133982), pyruvate dehydrogenase kinase 4 (ab71240), total OXPHOS human Western blot mixture (ab110411), mitofusin 1 (ab57602), mitofusin 2 (ab56889), OPA1 (ab42364), Drp-1 (ab56788), and Fis1 (sc-98900, Santa Cruz Biotechnology, Inc.). Human skeletal muscle cells were plated in T175 flasks and treated with vehicle or 1␣,25(OH) 2 D 3 (10 Ϫ8 M) for 48 h. Homogenates of cells were prepared in extraction buffer (ab193970). Cellular protein was quantitated . 15 g of cellular protein was treated with SDSloading buffer containing 20 mM dithiothreitol and loaded on 10% bis-tris polyacrylamide gels. Proteins were separated by electrophoresis and transferred onto PVDF membranes. The membranes were probed with the appropriate primary antibodies at concentrations recommended by the manufacturer. Peroxidase-labeled secondary antibodies were used to generate a chemiluminescent signal that was detected on x-ray film. The intensity of the bands was quantitated using ImageJ software. Equivalence of mitochondrial protein loading was assured by assessing porin intensity.
Measurement of Pyruvate Dehydrogenase in Cell Homogenates-hSkMCs were treated with vehicle (n ϭ 5) or 1␣,25(OH) 2 D 3 , 10 Ϫ8 M (n ϭ 6), for 48 h. Pyruvate dehydrogenase (PDH) activity was measured in 96-well plates with a pyruvate dehydrogenase activity colorimetric assay (BioVision, Milpitas, CA). The increase in absorbance at 450 nm with time was measured. Values for PDH activity were obtained using a standard curve of increasing NADH concentrations.
Assessment of Mitochondrial and Nuclear DNA-Total DNA was prepared from hSkMCs grown in 6-well plates and treated with vehicle (ethanol, n ϭ 9) or 10 Ϫ8 M 1␣,25(OH) 2 D 3 (n ϭ 9) for 48 h. Confluent cells were lifted using 0.25% trypsin-EDTA, pelleted at 300 ϫ g in a microcentrifuge, and then resuspended in 200 l of Dulbecco's phosphate-buffered saline. DNA was prepared using QIAamp DNA Mini Kit spin columns. DNA was treated with RNase, and purified DNA was eluted from columns into 200 l of water. Purified DNA was used to measure the human mitochondrial genes ND1 and ND6 and nuclear genes BECN1 and NEB using a NovaQUANT TM human mitochondrial to nuclear DNA ratio kit (EMD Millipore). Quantitative PCR was performed on 2 ng of DNA using specific PCR primers and a Roche LightCycler 480 qPCR apparatus with SYBR Green master I mix (Roche Applied Science). The ratios of mitochondrial genes ND1 and ND6 to nuclear genes BECN1 and NEB in cells treated with 10 Ϫ8 M 1␣,25(OH) 2 D 3 or vehicle (ethanol) were determined using crossing points and a standard curve generated with 0.02-20 ng of human DNA.
Preparation of Libraries-mRNA-seq libraries were prepared as described previously (38). RNA libraries were prepared with a TruSeq RNA Sample Prep Kit version 2 (Illumina). Reverse transcription and adaptor ligation steps were performed manually. Poly(A) mRNA was purified from total RNA using oligo(dT) magnetic beads. RNA was treated with RNase-free DNase during preparation of RNAs using the Nucleospin RNA/ Protein kit (Clontech). Purified mRNA was fragmented at 95°C for 8 min, eluted from the beads, and primed for first strand cDNA synthesis. The RNA fragments were then copied into first strand cDNA using SuperScript III reverse transcriptase and random primers (Invitrogen). Second strand cDNA synthesis was performed using DNA polymerase I and RNase H. The double-stranded cDNA was purified using a single AMPure XP bead (Agencourt) clean-up step. The cDNA ends were repaired and phosphorylated using Klenow, T4 polymerase, and T4 polynucleotide kinase followed by AMPure XP bead clean-up. The blunt-ended cDNAs were modified to include a single 3Ј-adenylate (A) residue using Klenow exoϪ (3Ј-5Ј exo minus). Paired end DNA adaptors (Illumina) with a single "T" base overhang at the 3Ј-end were immediately ligated to the "A-tailed" cDNA population. Unique indexes, included in the standard TruSeq Kits (12-Set A and 12-Set B) were incorporated at the adaptor ligation step for multiplex sample loading on the flow cells. miRNA-seq libraries from total RNA samples were synthesized with a NEBNext multiplex small RNA kit (New England Biolabs) as described previously (39). Adaptors were ligated to the 3Ј-ends of the small non-coding RNAs present in 500 ng of total RNA. A complementary primer was annealed to the 3Јadaptor sequences, followed by ligation of a 5Ј RNA adaptor. A cDNA library was created by reverse transcriptase (Superscript III, Invitrogen) treatment of adaptor-ligated and annealed small RNAs. The library was enriched by 15 cycles of PCR employing a common 5Ј primer and a 3Ј primer containing one of eight index primers (3Ј-adaptor complement; index sequences equivalent to Illumina TruSeq Small RNA sequences). The PCR products were purified. The libraries were assessed for miRNA products by Agilent Bioanalyzer DNA 1000 (Santa Clara, CA) analysis. The 130 -160-bp region was quantitated to determine equimolar amounts of vehicle and 1␣,25(OH) 2 D 3 -treated sample libraries to pool. The pooled small RNA libraries were fractionated to extract an miRNA-enriched sample via 3% Pippin Prep (Sage Science) gel cassettes. Pooled miRNA fractions were assessed by a second Agilent DNA 1000 assay. A predominant peak at 140 -150 bp indicated that the miRNA modification and size selection steps were performed as expected. Libraries were loaded onto single end flow cells at concentrations of 8 -10 pM to generate cluster densities of 700,000/mm 2 following Illumina's standard protocol using the Illumina cBot cluster kit version 3. The flow cells were sequenced as two reads: 51 cycles using the small RNA sequencing primer (read 1) and an index read to demultiplex the samples. Libraries were sequenced on an Illumina HiSeq 2000 using TruSeq SBS sequencing kit version 3 and SCS version 1.4.8 data collection software. Base calling was performed using Illumina's RTA version 1.12.4.2.
mRNA-seq Data Analysis-mRNA-seq data were processed by the Mayo Bioinformatics Core Facility to identify genes with differential expressions between vehicle-and 1␣,25(OH) 2 D 3 -treated groups. The processing of the mRNA data was performed using MAP-RSeq (40) workflow (version 1.2.1.3). MAP-RSeq consists of the following steps: alignment, quality control, obtaining genomic features per sample, and finally summarizing the data across samples. The pipeline provides detailed quality control data across genes using the RSeQC (41) software (version 2.3.2). Paired end reads are aligned by TopHat (42) (version 2.0.6) against the hg19 genome build using the bowtie1aligner (43). Gene counts were generated using HTSeq (44) software (version 0.5.3p9), and the gene annotation files were obtained from Illumina. Differential expression analysis was performed with edgeR version 2.6.2 (45) to identify genes with altered expression by 1␣,25(OH) 2 D 3 treatment. A cut-off for false discovery rate-adjusted p value was set at 0.01 to determine the genes with significant expression change between conditions. miRNA-seq Data Analysis-miRNA-seq data were processed with a microRNA deep sequencing analysis workflow called CAP-miRSeq version 1.1 (46). This workflow provides comprehensive analysis of miRNA sequencing data, including read preprocessing, alignment, mature/precursor/novel miRNA quantification, and variant detection in the miRNA coding region. In particular, CAP-miRSeq utilizes miRDeep2 (47) to classify and quantify the expressions of known and novel miRNAs, based on the annotations from miRBase version 19 (48). Normalization and differential analysis were performed FIGURE 1. The vitamin D receptor is expressed in human muscle and muscle cells. A, localization of VDR in sections of normal human skeletal muscle using a polyclonal VDR antiserum (magnification, ϫ100; inset magnification, ϫ400). B, localization of VDR in sections of normal human skeletal muscle using a polyclonal VDR antiserum, preadsorbed with VDR (magnification, ϫ100; inset magnification, ϫ400). C, localization of VDR in primary cultures of human skeletal muscle cells using a polyclonal VDR antiserum (magnification, ϫ100; inset magnification, ϫ630). D, localization of VDR in primary cultures of human skeletal muscle cells using a polyclonal VDR antiserum preadsorbed with VDR (magnification, ϫ100; inset magnification, ϫ630). E, Western blot of homogenates of normal human skeletal muscle tissue obtained by biopsy using a polyclonal VDR antibody. F, Western blot of homogenates of primary cultures of human skeletal muscle cells using a polyclonal VDR antibody. R, recombinant VDR.
miRNA Target Identification-A two-step approach was used to identify potential target genes for the miRNAs differentially expressed between 1␣,25(OH) 2 D 3 treatment and control. First, predicted targets were collected from TargetScan version 6.2 (49) for each miRNA. Second, the correlations between differentially expressed miRNAs and mRNAs were evaluated with the Spearman correlation coefficient and its associated statistical significance. The miRNA-mRNA pairs with statistically significant correlation (p Յ 0.05) were used to identify target genes. Finally, the common target genes identified by both steps were treated as high confidence targets for each miRNA.
Pathway Analysis-DAVID (version 6.7) (50, 51) was used to perform pathway enrichment analysis for differentially expressed genes or miRNA-targeted genes. DAVID provides pathways from databases, including the Kyoto Encyclopedia of Genes and Genomes (52)(53)(54), Panther (55), BioCarta, and Reactome (56). A cut-off p value was set at 0.01 to determine the significantly enriched pathways. The results were further confirmed with Ingenuity Pathway Analysis (Ingenuity Systems).
Analysis of Genes That Encode Mitochondrial Proteins-We specifically examined the differential expression of genes that encode mitochondrial proteins. Mitochondrial proteins were identified based on a compendium from MitoCarta (57). 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) 2 D 3 -or vehicle-treated cells.
Statistical Methods-Statistical differences between samples were analyzed using Student's two-tailed t test, assuming equal variance. A p value of Ͻ0.05 was regarded as statistically significant.
Data Sharing-All of the sequencing data that were analyzed in this report have been deposited in the Gene Expression Omnibus (GSE70934).

The Vitamin D Receptor Is Expressed in Human Muscle Cells and Skeletal Muscle
Homogenates-We confirmed the presence of the VDR in human muscle tissue and hSkMCs by immunohistochemistry. VDR was detected in cellular cytoplasm but not in mitochondria (Fig. 1, A and C). Immunostaining was reduced when VDR preadsorbed antibody was used (Figs. 1, B and D). Western blot analysis with a VDR antibody demonstrated bands of the appropriate mobility (M r ϳ48,000) that co-migrated with recombinant VDR in homogenates of human skeletal muscle biopsies and hSkMC (Fig. 1, E and F). The absence of localization of the VDR in mitochondria was corroborated in cells transfected with a VDR-eGFP expression plasmid, where no mitochondrial localization of fluorescence was noted. We confirmed the presence of the VDR mRNA in muscle cells by isolating mRNA, synthesizing cognate cDNA, and sequencing the VDR mRNA.
The Increase in Mitochondrial Oxygen Consumption Rate by 1␣,25(OH) 2 D 3 Is VDR-dependent-We treated hSkMCs with either a VDR-specific antisense silencing RNA (siRNA) or a scrambled/control siRNA. Following treatment of cells with VDR siRNA, cellular VDR mRNA decreased by Ͼ80%; treatment of cells with control siRNA did change VDR mRNA. Human skeletal muscle cells with reduced VDR expression had decreased basal OCR (p ϭ 0.002), maximal respiration (p ϭ 0.003), coupled OCR (p Ͻ 0.001), and respiratory reserve OCR (p ϭ 0.02) following 1␣,25(OH) 2 D 3 treatment compared with cells with normal VDR expression. The direct addition of 1␣,25(OH) 2 D 3 to isolated mitochondria failed to increase OCR, suggesting that the effects of 1␣,25(OH) 2 D 3 on OCR are dependent on extramitochondrial biochemical events.
C-1 and C-25 Hydroxyl Groups Are Required for Vitamin D 3 Analogs to Increase OCR-Both C-1 and C-25 hydroxyl groups are required for optimal binding of vitamin D 3 analogs to the VDR (58). To determine the influence of C-1 or C-25 hydroxyl groups or both in vitamin D analogs or metabolites on OCR, we treated hSkMCs with 10 Ϫ8 M vitamin D 3 , 25(OH)D 3 , 24R,25(OH) 2 D 3 , or 1␣(OH)D 3 for 48 h, following which OCR was determined. Treatment of cells with vitamin D 3 (Fig. 3, top left) and 25(OH)D 3 (Fig. 3, top right) decreased maximal OCR; treatment with 24R,25(OH) 2 D 3 did not change maximal OCR (Fig. 3, bottom left), and treatment with 1␣(OH)D 3 , a 1␣-hydroxylated vitamin D analog, showed a small but statistically insignificant change in OCR (Fig. 3, bottom right). The data demonstrate that only 1␣,25(OH) 2 D 3 that has both C-1 and C-25 hydroxyl groups and that binds to the VDR with high affinity has a significant effect on OCR in skeletal muscle cells.
1␣,25(OH) 2 D 3 Decreases the Expression of Phosphorylated Pyruvate Dehydrogenase and Pyruvate Dehydrogenase Kinase 4 -The activity of the PDH complex, located in the outer mitochondrial matrix, regulates the formation of acetyl-CoA from pyruvate and is altered by the phosphorylation status of its E1

1␣,25(OH) 2 D 3 Does Not Significantly Alter the Expression of Proteins in the Mitochondrial Respiratory Chain-Treatment
of hSkMCs with 1␣,25(OH) 2 D 3 did not significantly change proteins in complexes I-V in the mitochondrial respiratory chain (Table 3).
In hSkMCs in which VDR expression was inhibited by anti-VDR siRNA (Ͼ80% inhibition observed), only 15 mRNAs changed following treatment of cells with 1␣,25(OH) 2 D 3 , compared with 1947 mRNAs altered following treatment of cells with 1␣,25(OH) 2 D 3 when the VDR expression is normal. The mRNAs whose expression changed are identical to those altered by 1␣,25(OH) 2 D 3 in cells expressing normal amounts of the VDR, but the changes are quantitatively reduced. Knockdown of the VDR in cells resulted in changes in gene expression that were in the opposite direction of those observed following 1␣,25(OH) 2 D 3 treatment of VDRexpressing cells (supplemental Table 2).

Discussion
The salient findings of our report are that 1␣,25(OH) 2 D 3 has important effects on mitochondrial physiology, morphology, and expression of key mitochondrial proteins. Mitochondrial OCR increases in skeletal muscle cells treated with 1␣,25 (OH) 2 D 3 . In particular, respiration coupled to the generation of ATP is increased, which suggests that the hormone increases energy production in muscle. The increase in mitochondrial OCR is specific for 1␣,25(OH) 2 D 3 that has both C-1 and C-25 hydroxyl groups and does not occur with other vitamin D 3 ana-logs that lack either one of both C-1 and C-25 hydroxyls, such as 25(OH)D 3 , 25R,25(OH) 2 D 3 , 1␣-(OH)D 3 , and vitamin D 3 . The findings are consistent with the high binding affinity of 1␣,25(OH) 2 D 3 for the VDR relative to the lower affinities of the other tested analogs (58).
Several mechanisms might account for the increases in mitochondrial OCR. First, increases in mitochondrial volume fraction and branching consistent with mitochondrial fusion and biogenesis (69 -72) occur following treatment of muscle cells with 1␣,25(OH) 2 D 3 . Indeed, there are appropriate increases and decreases in mediators of mitochondrial fusion (OPA1) and fission (Fis1 and Drp1) that are consistent with the observed changes in mitochondrial morphology (59, 60, 62).

1␣,25(OH) 2 D 3 and Skeletal Muscle
Increases in the mRNA for mediators of increased mitochondrial biogenesis, such as MYC, MAPK13, and EPAS1, are likely to play a role in increasing the numbers of mitochondrial. An increase in mitochondrial volume could account for the increase in mitochondrial OCR (59 -62, 73). Second, we observe decreases in the amount of inactive, phosphorylated pyruvate dehydrogenase following treatment of skeletal muscle cells with 1␣,25(OH) 2 D 3 . The alterations in the phosphorylation state of PDH are associated with a concomitant decrease in the expression of the PDH kinase, PDK4. There is a trend toward an increase in the expression of the PDH phosphatase, PDP2. The changes in protein expression are supported by changes in mRNA expression for PDK4 and PDP2. Treatment of cells with 1␣,25(OH) 2 D 3 was associated with a trend to an increase in PDH activity (p ϭ 0.09). The small increase in PDH complex (PDC) activity could potentially increase the amount of acetyl-CoA entering the tricarboxylic acid cycle and thus might account for an increase in mitochondrial OCR. Insulin is known to decrease PDK4 expression, whereas glucocorticoids have the opposite effect (63, 67, 74 -77). In aggregate, the data

1␣,25(OH) 2 D 3 and Skeletal Muscle
suggest that 1␣,25(OH) 2 D 3 might regulate carbohydrate and fatty acid metabolism in muscle cells. Confirmation of such effects in vivo would be of great interest. The change in expression of ϳ2000 nuclear mRNAs, 83 of which encode proteins known to localize in mitochondria following treatment of skeletal muscle cells with 1␣,25(OH) 2 D 3 , is of interest. In addition to the quantitatively large up-regulation of the mitochondrial CYP24A1, mRNAs for other mitochondrial proteins that play a role in carbohydrate and fatty acid metabolism were noted to be either induced or repressed. Of note, a significantly down-regulated mRNA, PDK4, encodes the pyruvate dehydrogenase kinase, isoenzyme 4, a serine/threonine kinase that phosphorylates the pyruvate dehydrogenase subunits PDHA1 and PDHA2, and regulates metabolite flux through the tricarboxylic acid cycle (63, 67, 76 -78). Conversely, the mRNA for PDP2, which encodes pyruvate dehydrogenase phosphatase catalytic subunit 2, is significantly increased. PDP2 catalyzes the dephosphorylation and concomitant reactivation of the ␣ subunit of the E1 component of the pyruvate dehydrogenase complex (78 -80). The changes in the expression of mRNAs for PDP2 and PDK4 and documented alterations in the expression of the encoded proteins would function together to increase PDC activity.

1␣,25(OH) 2 D 3 and Skeletal Muscle
growth factor, and p53 signaling. The most profoundly up-regulated mRNA following 1␣,25(OH) 2 D 3 treatment of muscle cells is the CYP24A1 (Ͼ25,000-fold increase) that encodes the cytochrome P450 for the 1␣,25(OH) 2 D 3 /25(OH)D 3 -24hydroxylase, which catalyzes the transformation of 1␣,25 (OH) 2 D 3 and 25(OH)D 3 to less active metabolites 1␣,24,25 (OH) 3 D 3 and 24R,25(OH) 2 D 3 . Of note, the mRNA for the calcium-binding protein, parvalbumin, that regulates muscle relaxation is significantly induced by 1␣,25(OH) 2 D 3 . In addition, the expression of mRNAs encoding several growth factors related to the insulin-like growth factor family and nerve growth factor are up-regulated following treatment of cells with 1␣,25(OH) 2 D 3 . Pathway analysis indicates the regulation of several factors involved in muscle cellular signaling, apoptosis, and growth. The massive change in mRNA expression following treatment of cells with 1␣,25(OH) 2 D 3 is consistent with our earlier findings showing similar large scale, 1␣,25(OH) 2 D 3 -mediated changes in mRNA and miRNA expression in Danio rerio embryos (38,39). The effects of 1␣,25(OH) 2 D 3 on mitochondrial OCR may help to explain the observation that reduced serum 1␣,25 (OH) 2 D 3 concentrations in humans have been linked to falls through reduced muscle strength (10,11) and that clinical trials have demonstrated salutary effects of 1␣(OH)D 3 , a 1␣-hydroxylated vitamin D analog that is rapidly metabolized to 1␣,25(OH) 2 D 3 in vivo, on fall prevention (12). The data also suggest that vitamin D 3 and 25(OH)D 3 will not be useful in the treatment of muscle weakness unless they are metabolized to 1␣,25(OH) 2 D 3 , a circumstance that is operative in the context of vitamin D deficiency, where high parathyroid hormone levels drive the rapid metabolism of 25(OH)D 3 to 1␣,25(OH) 2 D 3 . Our findings of increased OCR following treatment of skeletal muscle cells with 1␣,25(OH) 2 D 3 are consistent with the report of Sinha et al. (27), which showed that treatment of vitamin D-deficient humans with cholecalciferol improves muscle phosphocreatine recovery after exercise, suggesting an effect of 1␣,25(OH) 2 D 3 on the formation of high energy phosphorylated intermediates and mitochondrial function. The increase in the numbers of mitochondria could also account for the increase in cellular OCR.
In conclusion, 1␣,25(OH) 2 D 3 alters mitochondrial OCR, mitochondrial biogenesis, and PDC activity, demonstrating unique effects of the sterol hormone on muscle biochemistry. In addition, there is a profound change in the expression of several hundred nuclear mRNAs, several of which encode mitochondrial proteins and proteins involved in cell signaling and cell growth. The observed effects could explain the myopathy of vitamin D deficiency that is seen in patients with impaired intake of vitamin D and the myopathy of chronic renal failure, where the production of 1␣,25(OH) 2 D 3 is impaired.