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Vitamin D and its receptor in skeletal muscle are associated with muscle disease manifestation, lipid metabolism and physical fitness of patients with myositis

Arthritis Research & Therapy volume 27, Article number: 48 (2025) Cite this article

Abstract

Background

Low levels of vitamin D have been associated with several autoimmune diseases. A growing body of evidence supports the association of vitamin D with skeletal muscle damage, regeneration, and energy and lipid metabolism. The aim was to analyse vitamin D and its receptor (VDR) in the muscle tissue of patients with idiopathic inflammatory myopathies (IIM) and to relate them to clinical parameters and muscle lipid and energy metabolism.

Methods

Forty-six patients with IIM and 67 healthy controls (HC) were included in the study. 27 IIM patients participated in a 24-week exercise intervention. Muscle biopsies were obtained from 7 IIM patients before/after training, 13 non-exercising IIM controls, and 21 HC. Circulating concentrations of 25(OH)D and 1,25(OH)D were measured. Gene expression of VDR and CYP27B1, the enzyme converting 25(OH)D to hormonally active 1,25(OH)D, was determined by qPCR in muscle tissue and primary muscle cells. Lipid oxidative metabolism was assessed in muscle tissue (mRNA, qPCR) and primary muscle cells (radioactive assays).

Results

Lower levels of active 1,25(OH)D were observed in IIM patients compared with HC (mean ± SD: 125.0 ± 45.4 vs. 164.7 ± 49.2 pmol/L; p < 0.0001). 25(OH)D was associated with CRP (r = -0.316, p = 0.037), MITAX (r = -0.311, p = 0.040) and HAQ (r = -0.390, p = 0.009) in IIM. After 24 weeks of training, active 1,25(OH)D was associated with MMT8 (r = 0.866, p < 0.0001), FI-2 (r = 0.608, p = 0.013) and HAQ (r = -0.537, p = 0.032). Gene expression of both VDR and CYP27B1 in primary muscle cells decreased after training (p = 0.031 and p = 0.078, respectively). Associations of VDR mRNA in muscle tissue with MMT-8 (IIM: r = -0.559, p = 0.013), serum CK (HC: r = 0.484, p = 0.031), myoglobin (IIM: r = 0.510, p = 0.026) and myostatin (IIM: r = -0.519, p = 0.023) were observed. The expression of VDR in differentiated muscle cells correlated negatively with the complete oxidation of palmitic acid (r = -0.532, p = 0.028). Muscle mRNA of carnitine palmitoyl transferase 1 (CPT1) (downregulated in IIM, p = 0.001) correlated positively with serum 1,25(OH) vitamin D (r = 0.410, p = 0.042).

Conclusion

Reduced biologically active vitamin D in circulation suggests its impaired metabolism in IIM. Serum vitamin D levels and gene expression of its receptor and activating enzyme in muscle tissue were modified by regular exercise and associated with disease manifestations, physical fitness, and muscle lipid metabolism of IIM patients.

Introduction

Muscle weakness and fatigue, elevated serum muscle enzymes, mononuclear cell infiltration, and myofiber degeneration/fibrosis represent clinical and histological features common to all subtypes of idiopathic inflammatory myopathies (IIM) [1]. Based on the particular combination of key features, the IIMs can be divided into dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM). Recently, other subsets of IIM have been defined such as clinically amyopathic dermatomyositis (CADM), antisynthetase syndrome (ASyS), and immune-mediated necrotizing myopathy (IMNM) [2]. After initial immunosuppressive treatment, inflammatory symptoms are suppressed in most patients, but only a few recover previous muscle function and their physical limitations persist [3]. Therefore, in addition to inflammation, an important role of non-immune mechanisms, such as endoplasmic reticulum stress [4], hypoxia [5, 6], autophagy [7], and metabolic dysfunction [8] has been suggested.

Recent studies have associated vitamin D deficiency with several autoimmune diseases such as rheumatoid arthritis [9], multiple sclerosis [10], systemic lupus erythematosus [11], and IIM [12, 13]. In IIM, vitamin D levels were correlated with circulating muscle enzymes, the presence of anti-Jo-1 and anti-Mi-2 antibodies, and the absolute number of total T and Treg cells [13]. Patients with IIM with extremely low vitamin D levels at the time of diagnosis also frequently had a heliotrope rash, and gastrointestinal and hepatic involvement [13]. Interestingly, VDR agonists with their emerging anti-inflammatory properties have been suggested as candidates for the future treatment of IIM [14].

Vitamin D is partially produced in the skin after UVB-mediated conversion of 7-dehydrocholesterol, and obtained from food (cholecalciferol). In the liver, cholecalciferol is hydroxylated into prohormone 25-hydroxyvitamin D (25(OH)D; calcidiol). Since serum 25(OH)D best reflects vitamin D production and absorption and has a relatively long half-life, its concentrations are most commonly used as an indicator of vitamin D status [15]. In the kidneys and several other tissues [16], 25(OH)D is converted by CYP27B1 (25-hydroxyvitamin D 1-alpha-hydroxylase) into biologically active metabolite 1,25-dihydroxy vitamin D (1,25(OH)D; calcitriol) [17]. Calcitriol activates the nuclear vitamin D receptor (VDR), a member of the ligand-induced transcription factor family. After binding of 1,25(OH)D to VDR, a complex with the retinoid X receptor (RXR) is formed and binds to specific DNA sequences for transcriptional modulation of target genes involved in calcium/phosphate homeostasis, cellular proliferation/differentiation, immune and mitochondrial function [18]. The 1,25(OH)D-VDR complex also shows rapid, non-genomic effects on intracellular signalling and calcium flux [19].

It has long been accepted that vitamin D plays a critical role in the regulation of Ca2+ and phosphate homeostasis and is therefore critical for bone function. However, the discovery of a VDR in skeletal muscle cells provided evidence of the important role of this hormone also in skeletal muscle function and metabolism [20]. Serum concentrations of 25(OH)D are associated with muscle strength and muscle mass, and the incidence of injury across a broad range of age groups [21,22,23]. Many studies show that muscle weakness and low physical performance associated with vitamin D deficiency can be prevented or restored by adequate vitamin D supplementation [24, 25]. In vitro experiments in human skeletal muscle myoblasts showed a positive effect of vitamin D treatment on myogenesis and mitochondrial oxidative capacity [26]. However, the effect of additional factors such as age, medical condition, physical exercise, or protein consumption needs to be considered in the interpretation of the results [27,28,29].

Limited contractile activity, muscle disuse, and glucocorticoid therapy promote lipid accumulation, lipotoxicity, inflammation, and metabolic dysfunction that contribute to muscle atrophy and functional deterioration in IIM [30,31,32]. In our previous work, we showed the reduced capacity of skeletal muscle cells derived from patients with IIM to adapt to increased lipid load, which was linked to reduced mitochondrial function and fat oxidative metabolism [33]. Others showed that muscle cells exposed to saturated fatty acid in vitro displayed excessive fat accumulation and cellular damage. This effect was reversed by 1,25(OH)D treatment, in parallel with increased ATP production and expression of genes related to mitochondrial biogenesis and lipid oxidative metabolism, such as PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1-α), CPT1 (carnitine palmitoyl transferase 1) and PPARα (peroxisome proliferator-activated receptor-α) [34]. VDR silencing reduced mitochondrial oxidative phosphorylation in C2C12 muscle cells [35]. These results suggest that reduced vitamin D levels could contribute to reduced oxidative capacity and deteriorated lipid metabolism in the skeletal muscle of patients with IIM.

In the present study, the potential role of vitamin D in disease manifestations related to skeletal muscle function and metabolism was investigated in patients with idiopathic inflammatory myopathies. Apart from 25(OH)D, serum levels of biologically active 1,25(OH)D, a vitamin D metabolite that skeletal muscle cells are exposed to in vivo, were analysed and compared to those of healthy individuals. The association of vitamin D status with clinical and physical activity parameters was assessed. Gene expression of vitamin D receptor and 25(OH)D activating enzyme CYP27B1 in muscle tissue and primary muscle cells was evaluated, together with their relation to muscle function parameters and lipid metabolism.

Methods

Study design and population

A total of 46 IIM patients with established disease and 67 healthy controls (HC) matched by age, gender, and body mass index were recruited. Of these, 27 patients with IIM attended 24-week physiotherapy program (personal training, PT) focused on activities of daily living and resistance and stability training for muscle strength and endurance. 19 IIM patients (mostly living outside of Prague) represented a non-exercising control patients group (CG). The intervention included physical exercise for 1 hour per session twice a week performed under the supervision of a physiotherapist and for the remaining five days a week an individual home exercise for 30 min per session according to an instruction manual (a detailed description of the physical therapy program and all outcome measures is available in [36], intervention trial registration number ISRCTN35925199). Peripheral blood collection and clinical examination were performed before and after the training intervention. All IIM patients were recruited from the inpatient and outpatient wards of the Institute of Rheumatology in Prague. Patients with IIM were diagnosed according to EULAR/ACR criteria [37] and ENMC criteria for IMNM [38] and were divided into 3 groups – PM, which also contained patients with antisynthetase syndrome, DM, and IMNM. Patients with inclusion body myositis were excluded. All study participants were ≥18 years of age, and each of them signed an informed consent form. The study design and informed consent form were approved by the local ethics committee at the Institute of Rheumatology in Prague.

Clinical disease activity was evaluated by the disease activity core set measures proposed by International Myositis Assessment & Clinical Studies Group (IMACS): myositis disease activity assessment (MYOACT), patient and physician global activity using visual analogue scales (VAS), myositis intention-to-treat activity index (MITAX), and the myositis damage index (MDI) [39]. Physical functioning was evaluated by MMT-8 (manual muscle test scores of individual eight muscle groups), Functional index-2 (FI-2), Health assessment questionnaire (HAQ), and Medical outcomes study 36-item short form health survey (SF-36: the physical component score (PCS) and the mental component score (MCS)) [40]. Anthropometric parameters and body composition were assessed by Dual energy X-ray absorptiometry (DEXA) (iDXA Lunar). Laboratory analyses of biochemical parameters for clinical practice (serum C-reactive protein (CRP) and markers of muscle impairment creatine kinase (CK), lactate dehydrogenase (LD), and myoglobin, insulin, lipid profile), and both vitamin D isoforms were performed in an accredited laboratory of Clinical Biochemistry and Hematology of the Institute of Rheumatology using Beckman Coulter AU 680 (Beckman Coulter, USA), Roche cobas e601 (Roche, Switzerland) and Liaison XL (Diasorin, Italy) analytical systems.

Muscle biopsy and skeletal muscle cell cultures

Muscle biopsies were obtained from 20 chronic IIM patients (7 patients before and after 24-week training, 13 control/nonexercising IIM patients), and 21 healthy controls. Samples of musculus vastus lateralis (100 - 150 mg) were taken by Bergström needle under local anaesthesia, cleaned from blood, snap frozen in liquid nitrogen, and stored at −80°C. Approximately 80 mg of tissue was used to establish a primary muscle cell culture, as previously described [41]. Briefly, satellite cells were isolated and propagated to myoblasts in collagen-coated flasks (culture medium: Dulbecco’s Minimum Essential Medium supplemented with 20% foetal bovine serum and antibiotics), harvested at ~80% confluence and stored in liquid nitrogen. In a subpopulation of patients (before/after 24-week training/healthy controls n=7/7/7), cells were exposed to palmitic fatty acid (100 μM), coupled to a fatty acid-free BSA (in 5:1 molar ratio), or to BSA alone (control cells) for the 3 last days of differentiation. Palmitate treatment represents a chronic metabolic challenge and tests the capacity of muscle cells to handle saturated fatty acid overload [33].

Radiometric fatty acid oxidation (Fox) assay

The assay was described in detail previously and characterizes the metabolic adaptability by measuring a capacity to increase fat oxidation with increasing substrate load [33]. Briefly, differentiated muscle cells (myotubes) were pre-incubated in glucose- and serum-free media, followed by a 3-hour incubation with [1- 14 C]-labeled palmitate (0.5 mCi/ml; ARC, USA), with/without a fatty acid challenge provided by 100μM „cold“/non-labeled palmitate (Sigma, USA), coupled to a fatty acid–free BSA. Following incubation, the medium was transferred into the 48-well custom-made CO2-trapping plate. CO2 released from media by perchloric acid was collected in 1M NaOH. Acidified media were spun twice/4°C, and radioactivity of supernatant containing intermediate Fox metabolites (ASP/Acid-Soluble-Products) was measured (Ecolite/Germany; TriCarb2910TR/Perkin-Elmer/USA). Cells were washed twice (ice-cold-PBS), harvested (0.25ml 0.05%SDS), and used to measure protein (BCA, Thermo-Fisher-Scientific, USA) and to detect neutral lipids content by thin layer chromatography (TLC), as described in [33]. Data were normalized to the protein content of the corresponding well. The impact of IIM and the 24-week training on fatty acid oxidation, lipid accumulation, and mitochondrial OxPHOS proteins was reported elsewhere [33], and in this work, these results were used solely for the correlation analyses.

Enzyme-linked immunosorbent assay (ELISA) and multiplex immunoassay

Myostatin and clusterin concentrations in serum were measured using commercially available ELISA kits (R&D Systems, Minneapolis, USA, and BioVendor, Brno, Czech Republic, respectively) according to the manufacturer’s protocol. The absorbance was detected at 450 nm on a microplate reader (Tecan Sunrise, Salzburg, Austria). Bio-Plex Human Cytokine 27-Plex Panel (Bio-Rad, Hercules, USA) was used to determine cytokine concentrations in serum and analyzed by Bio-Rad Bio-Plex 200 Systems reader (Hercules, USA) according to the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction (qPCR) analysis

The muscle samples were processed by homogenization of the tissue using a bead homogenizer (Retsch, Germany). Total RNA was isolated from 30 – 50 mg of frozen muscle using TRIzol Reagent (Thermo Fisher Scientific, Waltham, USA) or Qiazol (Qiagen, USA). Samples were treated with DNase I (New England BioLabs, France) and quantified with NanoDrop 2000c (Thermo Fisher Scientific, Waltham, USA). cDNA was obtained by reverse transcription with a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, USA) in a thermal cycler (Bio-Rad MyCycler, Hercules, USA). qPCR was carried out by TaqMan Gene Expression Assays (Thermo Fisher Scientific, Waltham, USA) in the QuantStudio 7 Flex qPCR system (Thermo Fisher Scientific, Waltham, USA). qPCR data are expressed as 2(-∆Ct)*1000. After the evaluation of four housekeeping genes using NormFinder software (MOMA, Aarhus, Denmark), the average of ACTB and RPL13 genes was used as an endogenous control in muscle tissue samples. For VDR expression in muscle cell cultures, RPL13 and β−2-microglobulin were used as an internal control.

Immunoblotting

Muscle cell protein lysates (40 μg) containing Complete protease inhibitor cocktail (Roche, Germany) were mixed with a loading dye (240mM Tris-HCl pH 6.8, 40% glycerol, 8% SDS, 5% β-mercaptoethanol, and 0.4% bromophenol blue), incubated (96°C for 5 min), separated on the 10%-SDS-PAGE and transferred to PVDF membrane (Millipore, USA). After blocking (Odyssey blocking buffer, LI-COR, USA), membranes were incubated overnight with an OxPHOS human antibody cocktail (Abcam/UK,1:250). Appropriate IRDye 680RD or 800CW antibodies (LI-COR, USA, 1:10000) were used to visualize protein content (LI-COR, USA). Protein ladder 10–180 kDa (Thermo-Fisher-Scientific, USA) was used. The relative content of individual OxPHOS protein complexes was calculated as % of the sum of protein signals for all five complexes (%OxPHOS).

Statistical analysis

Student’s t-test or Mann-Whitney U test was used for the comparison of two groups of subjects. One-way ANOVA or the Kruskal-Wallis test and corresponding posthoc analyses were conducted for comparison of data obtained from three or more groups of subjects. The associations of serum and gene expression levels with clinical and laboratory markers were assessed by Pearson’s product-moment correlation coefficient or Spearman’s rank correlation coefficient, as applicable. Correlations were adjusted for body mass index, glucocorticoid dose equivalent to prednisone, and vitamin D supplementation using the partial correlation technique [42]. The analyses and the graphs were performed using GraphPad Prism version 9 (GraphPad Software , Boston, USA) and IBM SPSS version 22 (IBM SPSS, NY, USA).

Results

Characteristics of the study population

The characteristics of all IIM patients and HC enrolled in the study are summarized in Table 1. From the total of 46 IIM patients recruited in the study, 27 participated in a 24-week personal training (PT) and 19 patients represented a control patients group (CG). 7 patients from the PT group underwent a skeletal muscle biopsy before and after the training period along with an additional 13 non-training patients (CG) and 21 healthy controls who had a single muscle biopsy. Full clinical data for each patient subgroup according to the biological material available for subsequent analyses are shown in Supplementary Table S1.

Table 1 Characteristics of the study population
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The levels of biologically active 1,25(OH)D are significantly decreased in myositis patients

Serum levels of 25(OH)D and 1,25(OH)D were analyzed in 46 IIM patients and 67 age-/sex-matched healthy controls. Decreased levels of active 1,25(OH)D were observed in IIM patients compared to HC (125.0±45.4 vs. 164.7±49.2 pmol/l; p<0.0001). No difference was found for 25(OH)D (Fig. 1). There was no significant difference in either 25(OH)D or 1,25(OH)D serum levels between myositis subgroups PM, DM, and IMNM (Suppl. Fig. 1). The 24-week training program did not alter serum 25(OH)D or 1,25(OH)D levels (Suppl. Fig. 2). No season-related difference in 25(OH)D or 1,25(OH)D serum levels in either IIM patients or healthy controls was observed (Suppl. Fig. 3). Also, no difference in 25(OH)D or 1,25(OH)D serum levels was observed between patients positive and negative for the 3 most abundant autoantibodies – antinuclear (ANA), anti-Jo-1 and anti-Ro-52 (data not shown).

Fig. 1
figure 1

Vitamin D levels in myositis patients (IIM) and healthy controls (HC). An unpaired t-test was used to compare the means between groups. Columns represent mean, whiskers represent SD

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Serum 25(OH)D and 1,25(OH)D levels are associated with clinical parameters and physical activity

The correlation analyses of circulating 25(OH)D and 1,25(OH)D were performed in a cohort of IIM patients before (N=46) and after (N=27) the training intervention. Since a significant association of 25(OH)D serum levels with BMI (r=−0.424, p=0.004), borderline association with daily GC dose (r=−0.273, p=0.074) and an association of 1,25(OH)D with a vitamin D supplementation daily dose (r=0.342, p=0.025) were observed, another set of correlation analyses adjusted for these three parameters was performed. At the baseline, significant associations of 25(OH)D with inflammatory markers (CRP, TNF, IL-8), disease activity (MITAX), overall function (HAQ), and physical activity (HAP, SF-36 PSC) were observed (Table 2). The associations remained significant even after correction for BMI, GC, and vitamin D supplementation daily dose. In the intervention group after 24 weeks of training, a positive association of active 1,25(OH)D with muscle strength (MMT-8) and endurance (FI-2) and overall function (HAQ), independent of BMI, GC, and vitamin D supplementation was observed. In the group of non-exercising patients, only borderline association of 1,25(OH)D with FI-2 (r=0.435, p=0.062) and MMT-8 (r=0.435, p=0.062) was observed, and the significance was lost after correction for BMI + GC dose + vitamin D supplementation dose. The correlation analysis of training-induced changes (Δ) in studied parameters did not show statistically significant associations (Suppl. Table 2).

Table 2 The heat map of 25(OH)D and 1,25(OH)D vitamin D associations with clinical parameters in myositis patients before and after the exercise intervention
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Vitamin D receptor and vitamin D activating and deactivating enzymes (CYP27B1 and CYP24A1) gene expression in muscle decreases after training and is associated with muscle function parameters

We did not observe a statistically significant difference in gene expression of VDR and CYP27B1 in muscle tissue and primary muscle cells in IIM compared to HC. However, after the 24-week training, gene expression of both VDR and CYP27B1 in primary muscle cells decreased significantly (Fig. 2). Associations of VDR gene expression with myoglobin (Fig. 3A IIM, Fig. 3E HC), MMT-8 (Fig. 3D IIM), myostatin (Fig. 3B IIM, Fig. 3F HC), CK (HC: r=0.484, p=0.031), and lipid profile (Fig. 3G, H HC) were observed in muscle tissue. Lower CYP27B1 gene expression in the muscle was also associated with higher MMT-8 (Fig. 4B IIM), clusterin (Fig. 4C IIM), TGFβ mRNA (Fig. 4D IIM), lower myoglobin (Fig. 4F HC), and lower VDR (Fig. 4A IIM, Fig. 4E HC). Moreover, lower CYP27B1 correlated with better metabolic parameters in IIM patients, such as higher lean body mass (r=−0.651, p=0.0026), basal metabolic rate (r=−0.630, p=0.0038), and lower 120-min blood glucose levels in oral glucose tolerance test (oGTT) (r=0.532, p=0.023).

Fig. 2
figure 2

Vitamin D receptor (VDR), CYP27B1, and CYP24A1 gene expression in muscle tissue and in primary myotubes derived from myositis patients before and after training intervention (pre- and post-PT) and healthy controls (HC). Mann–Whitney (HC vs pre-PT IIM) and Wilcoxon (pre- vs post-PT IIM) tests were used to compare the means between groups. Columns represent mean, whiskers represent SD

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Fig. 3
figure 3

Vitamin D receptor (VDR) gene expression in muscle tissue correlates with serum myoglobin (A, E), serum myostatin (B, F), vitamin D levels (C), muscle strength (D), and serum lipids (G, H) in myositis patients and healthy controls (baseline levels). The bivariate relationships between variables were assessed using the Spearman correlation coefficient. MMT-8, manual muscle testing of 8 muscles, TG—triglycerides, HDL, high-density lipoprotein

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Fig. 4
figure 4

Vitamin D 1A hydroxylase (CYP27B1) gene expression in muscle tissue correlates with VDR (A, E), muscle strength assessed by MMT-8 (B), and indicators of muscle damage/dysfunction (C, D, F) in myositis patients and healthy controls (baseline levels). The bivariate relationships between variables were assessed using the Spearman correlation coefficient. MMT-8, manual muscle testing of 8 muscles

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Vitamin D receptor and circulating vitamin D levels are related to muscle oxidative capacity and lipid metabolism in cultured muscle cells

There was a decline of VDR mRNA expression related to 3-day palmitate challenge in primary myotubes, irrespective of the disease or training state of the IIM patients (Fig. 5A, B).

Fig. 5
figure 5

Vitamin D receptor expression in primary myotubes exposed to a 3-day treatment with saturated fatty acid. Myotubes were treated with 100 µM palmitic fatty acid coupled to BSA (3-day palmitate) or to BSA alone (control cells). Data are presented as means ± SEM, n = 21 per group. A Comparison between IIM before PT- white; IIM after PT- light grey; healthy controls—dark gray, n = 7 per group. Paired t-test was used to asses the effect of stimulation in each group of subjects. B global effect of palmitate independent from IIM or training effect, n = 21. Unpaired t-test was used to compare the means between groups. PT – personal training

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The expression of VDR in differentiated muscle cells correlated negatively with the complete oxidation of saturated palmitic fatty acid (CO2 production) (Fig. 6A), total palmitate disposal (oxidative + non-oxidative palmitate disposal) (Fig. 6B), non-oxidative palmitate disposal (Fig. 6C), and relative content of complex V (% of the sum of all five OxPHOS complexes) (Fig. 6F). Moreover, the expression of VDR correlated positively with the relative content of complex I and complex IV (Fig. 6D, E).

Fig. 6
figure 6

Vitamin D receptor expression in primary myotubes correlates with characteristics of lipid metabolism in vitro. Lipid metabolism was assessed by radioactive assays, using saturated14C-labeled palmitic fatty acid, and normalized to protein content. VDR, Vitamin D receptor; Fox, fatty acid oxidation. Individual complexes of mitochondrial oxidative phosphorylation (OxPHOS) are presented as % from the total signal, obtained as a sum of signals of all 5 protein complexes (immunoblotting). The bivariate relationships between variables were assessed using the Pearson correlation coefficient. Points colour scheme: white – HC, light grey—IIM before PT, dark grey – IIM after PT PT – personal training

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The expression of genes important for the mitochondrial oxidative capacity, carnitine palmitoyl transferase (CPT1, the rate-limiting enzyme of mitochondrial fatty acid oxidation) (Fig. 7A) and frataxin (the nuclear-encoded mitochondrial protein), was downregulated in the skeletal muscle of IIM patients (n=12, p=0.035). Muscle mRNA levels of CPT1 correlated positively with serum 1,25(OH) vitamin D levels (Fig. 7B) and negatively with levels of 25(OH) vitamin D (Fig. 7C). mRNA expression of MYH7, the marker of oxidative muscle fibers, did not differ between IIM patients and controls (p>0.1, data not shown). Due to the limited amount of biological material obtained, the impact of training intervention on the expression of these genes could not be determined.

Fig. 7
figure 7

Carnitin palmitoyl transferase 1, CPT1, the rate-limiting enzyme in mitochondrial fatty acid oxidation is decreased in IIM skeletal muscle (A), and correlates with vitamin D levels (B, C). An unpaired t-test (A) and Pearson correlation coefficient (B, C) were used to assess the variables relationships. Points colour scheme: white – HC, light grey—IIM before PT, dark grey – IIM after PT. PT – personal training

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Circulating 1,25(OH) vitamin D levels correlated positively with in vitro parameters of lipid metabolism in human primary myotubes, including total fatty acid disposal (r=0.656, p=0.0012), the relative proportion of lipid oxidative metabolism (expressed as a percentage of total fatty acid disposal, r=0.573, p=0.0067), and incomplete fatty acid oxidation (the production of intermediate metabolites of Fox; r=0.587, p=0.0051).

Discussion

Active vitamin D is decreased in IIM and correlates with clinical parameters and physical activity

Our study on vitamin D in idiopathic inflammatory myopathies shows decreased levels of biologically active 1,25(OH)D in IIM patients compared to healthy individuals, even though the levels of commonly measured 25(OH)D were comparable with controls. Vitamin D deficiency in IIM has been reported by two studies so far [12, 13], however, only 25(OH)D levels were assessed. We have not found a significant difference in vitamin D levels between the myositis subgroups similar to Azali et al. [12]. Yu et al. [13] showed the correlation of vitamin D levels with muscle enzymes, the presence of anti-Jo-1 and anti-Mi-2 antibodies, and the absolute numbers of total T and Treg cells suggesting a role of vitamin D in the immunological pathogenesis of IIM. In the presented study, higher 25(OH)D was significantly associated with lower inflammation (CRP, TNF, IL-8), lower disease activity (MITAX), better overall function (HAQ), and physical activity parameters (HAP, SF-36 PCS) in IIM patients despite the 25(OH)D levels comparable to healthy controls. These associations highlight the importance of vitamin D from the clinical perspective. Moreover, the correlations were independent of BMI, glucocorticoid treatment, and vitamin D supplementation dose. The correlation analyses were adjusted for these parameters, as increased BMI and steroid use have been associated with 25(OH)D deficiency [43,44,45]. An inverse correlation between 25(OH) vitamin D and CRP in a large patient cohort has been reported and this effect was more pronounced in patients with inflammatory diseases compared to patients with non-inflammatory diseases [46]. The protolerogenic activity of vitamin D relies on its capability to control the maturation, differentiation, and activation of immune cells through VDR that is either constitutively present or inducible in the majority of the immune cells [47,48,49].

Implementing physical activity and exercise in the treatment of IIM has been shown to reduce impairment and improve patients’ quality of life [50]. Specifically, the improvement of muscle strength and endurance [36], aerobic and mitochondrial capacity [31], and reversed metabolic dysfunction [33, 51] represent the contribution of exercise to both systemic and intrinsic muscle adaptations in IIM patients. Even though vitamin D status depends largely on sunlight exposure and dietary or supplementary intake, recent studies have found that endurance and resistance exercise can also influence circulating levels of 25(OH)D in various study populations [52]. However, the intensity of exercise, the initial vitamin D deficiency, BMI, and seasonal factors may play a role in the observed inconsistent effect of exercise on 25(OH)D levels [52]. In our study, a 24-week training intervention (a tailored program focused on activities of daily living, resistance, and stability training provided indoors) did not affect either 25(OH)D or 1,25(OH)D serum levels in IIM patients. However, higher levels of 1,25(OH)D correlated with higher muscle strength and endurance parameters (MMT-8 and FI-2, respectively), and the overall function evaluated by HAQ. These findings suggest a possible connection of 1,25(OH)D with physical fitness improvement. For instance, in a study by Marantes et al. [53] no consistent association between 25(OH)D and measurements of muscle mass or strength was found. However, low 1,25(OH) (2) D was associated with low skeletal mass and low knee extension moment.

VDR and CYP gene expression in skeletal muscle is associated with muscle fitness indicators

The gene expression of VDR and 25(OH)D metabolizing enzymes (CYP27B1 and CYP24A1) in primary muscle cells derived from IIM patients’ muscles significantly decreased after 24-week training to levels comparable with healthy controls. As the muscle strength and endurance of patients participating in the training intervention increased [36] without changes in vitamin D levels, we may suggest that the local metabolism of vitamin D in muscle tissue of IIM patients reflects the changes in overall physical fitness and a lower requirement for vitamin D. This may be supported by the correlation of lower VDR mRNA with higher 25(OH)D levels, higher muscle strength measured by MMT-8, lower release of myoglobin into circulation, and higher serum myostatin. The latter has been shown to correlate with the improvement in muscle inflammation in IIM [54, 55]. Lower expression of vitamin D activating enzyme 25-hydroxyvitamin D 1-alpha-hydroxylase encoded by CYP27B1 gene in muscle tissue correlated with lower expression of VDR, suggesting a mutual regulation that could be expected based on their function. Similarly to VDR, lower expression of CYP27B1 correlated with higher muscle strength. Moreover, lower CYP27B1 mRNA was associated with lower TGFβ mRNA in muscle tissue and lower clusterin levels in serum. We have previously shown upregulated clusterin in the serum and muscle of IIM patients with higher disease activity [56]. TGFβ is considered a negative regulator of adult skeletal muscle [57] through reduction of myoblast fusion [58] and contribution to muscle fibrosis [59].

Vitamin D and VDR correlate with parameters of muscle oxidative capacity and lipid metabolism in vitro

There is some evidence linking vitamin D, its regulation, and signalling to the functioning of the lipid system in muscle cells. VDR knockdown models have demonstrated the crucial role of vitamin D for mitochondrial oxidative phosphorylation capacity, supporting also its role in muscle regeneration [60]. VDR silencing in murine C2C12 muscle cells reduced ATP production and mitochondrial respiration, without the impact on mitochondrial protein content and markers of mitochondrial fission [35]. Differentiated C2C12 muscle cells exposed to palmitic fatty acid displayed excessive fat accumulation and cellular damage, which was reversed by 1,25(OH) vitamin D treatment [34]. Vitamin D has increased ATP levels and activation of AMPK and SIRT1 as well as gene expression related to mitochondrial biogenesis and lipid oxidative metabolism such as NRF1, PGC-1α or CPT1, PPARα, and others [34]. Sinha et al. [61] demonstrated an increase in the maximal mitochondrial oxidative phosphorylation rate measured by 31P-NMR spectroscopy in vitamin D deficient humans after vitamin D treatment. Similarly, the mitochondrial oxygen consumption rate coupled to the generation of ATP was increased after 1,25(OH) vitamin D treatment in muscle cells in vitro [62]. These observations support the role of vitamin D in modulating / stimulating mitochondrial oxidative phosphorylation.

In this work, VDR mRNA expression was reduced in muscle cells exposed to saturated palmitic fatty acid and correlated negatively with in vitro parameters of lipid metabolism and relative content of complex V, which plays a decisive role in the ATP-producing capacity of mitochondria. The gene expression of the rate-limiting enzyme of mitochondrial fatty acid oxidation CPT1 was downregulated in the skeletal muscle of IIM patients; however, it correlated positively with serum 1,25(OH) vitamin D levels. Moreover, circulating 1,25(OH) vitamin D levels correlated positively with in vitro parameters of lipid metabolism in human primary myotubes in this study.

We also observed higher VDR gene expression in muscle from healthy controls (suggesting a compensatory mechanism for insufficient vitamin D signalling) associated with higher serum TG and lower HDL levels. Low vitamin D levels could result in an increase in triglycerides, total cholesterol, and low-density lipoprotein and a decrease in high-density lipoprotein cholesterol levels [63,64,65]. Interestingly, mice fed with a high-fat diet displayed downregulated vitamin D-related metabolic genes and changes in gene-specific methylation in the liver [66]. In our previous work, we showed that muscle cells from patients with IIM display reduced capacity to adapt to the saturated fatty acid exposure, compared to healthy controls [33]. It is plausible to speculate that a diet higher in saturated fat can deteriorate lipid metabolism also via the negative impact on vitamin D metabolism/signaling in the skeletal muscle of IIM patients. Overall, our data support the existence of a feedback loop linking vitamin D deficiency in patients with IIM to impaired lipid metabolism.

Study limitations

A relative heterogeneity of the study population and a limited availability of biological material resulted in a small sample size in some analyses. Low numbers of individuals per group precluded the realization of the correlation analysis in the individual subgroups. We made an effort to compensate for this limitation by carefully matching the cohort of healthy controls. All patients involved in the study had an established disease and were receiving treatment at the time of sample collection. Even though the analyses were adjusted for therapeutics with the presumed highest impact where possible, the effect of treatment on the presented results cannot be excluded. The adaptive response to training in health and disease could be better discerned if exercise interventions were also performed in healthy control subjects and repeated muscle biopsies obtained from non-exercising IIM patients. Protein levels of VDR and 25(OH)D metabolizing enzymes (CYP27B1 and CYP24A1) would help to specify the suggested response of muscle tissue and primary muscle cells to vitamin D deficiency. We plan to address at least some of these limitations in our future studies.

Conclusions

Lower levels of the biologically active form of vitamin D in circulation suggest an impairment of its metabolism in IIM. Vitamin D serum levels are associated with inflammatory markers, disease activity, muscle function, and metabolic parameters, indicating an important role of vitamin D in disease manifestations and physical fitness of IIM patients. Moreover, we suggest that the positive association of VDR and CYP27B genes in muscle tissue of IIM patients with increased muscle functional decline indicators may represent i) a response of the organism to decreased availability of active 1,25(OH)D, and ii) a mechanism to limit further inflammation- and atrophy-induced muscle damage characteristic for IIM. Lower levels of circulating vitamin D in IIM patients and their associations with impaired lipid metabolism in muscle cells in vitro indirectly indicate that reduced vitamin D may contribute to altered fatty acid metabolism in skeletal muscle, mitochondrial dysfunction, oxidative stress, muscle weakness, and atrophy, which are typical features for patients with IIM.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

IIM:

Idiopathic inflammatory myopathies

HC:

Healthy controls

DM:

Dermatomyositis

PM:

Polymyositis

ASyS:

Antisynthetase syndrome

IMNM:

Immune-mediated necrotizing myopathy

VDR:

Vitamin D receptor

25(OH)D:

25-hydroxy vitamin D

1,25(OH)D:

1,25-dihydroxy vitamin D

CPT1:

Carnitine palmitoyl transferase 1

MYOACT:

Myositis disease activity assessment

MITAX:

Myositis intention-to-treat activity index

MDI:

Myositis damage index

MMT-8:

Manual muscle test scores of individual eight muscle groups

FI-2:

Functional index-2

HAQ:

Health assessment questionnaire

HAP AAS:

Human activity profile adjusted activity score

SF-36:

Medical outcomes study 36-item short form health survey

PCS:

Physical component summary

MCS:

Mental component summary

DEXA:

Dual energy X-ray absorptiometry

CRP:

C-reactive protein

CK:

Creatine kinase

LD:

Lactate dehydrogenase

ELISA:

Enzyme-linked immunosorbent assay

PT:

Personal training

CG:

Control patients group

GC:

Glucocorticoids

DMARDs:

Disease-modifying antirheumatic drugs

SD:

Standard deviation

BMI:

Body mass index

CYP27B1:

25-hydroxyvitamin D 1-alpha-hydroxylase

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Acknowledgements

The authors would like to thank all patients who participated in the study.

Funding

Supported by Ministry of Health of the Czech Republic (NU21-05–00322, 023728), Ministry of Education Youth and Sports of the Czech Republic (SVV 260638), the large research infrastructure project BBMRI.cz (reg. no. LM2023033), and The Ministry of Education, Research, Development and Youth of the Slovak Republic (APVV 20/0466, VEGA-2/0076/22, VEGA-2/0161/24).

Author information

Authors and Affiliations

  1. Institute of Rheumatology, Na Slupi 4, Prague, 12850, Czech Republic

    Lucia Vernerová, Martina Vokurková, Maja Špiritović, Sabína Oreská, Martin Klein, Kateřina Kubínová, Veronika Horváthová, Tereza Kropáčková, László Wenchich, Michal Tomčík & Jiří Vencovský

  2. Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia

    Nikoleta Alchus Laiferová, Michal Nemec, Oksana Mytiai, Jozef Ukropec & Barbara Ukropcová

  3. Department of Health Care Disciplines and Population, Faculty of Biomedical Engineering, Czech Technical University, Prague, Czech Republic

    Maja Špiritović

  4. Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia

    Oksana Mytiai & Barbara Ukropcová

  5. Department of Rheumatology, First Faculty of Medicine, Charles University, Prague, Czech Republic

    Sabína Oreská, Martin Klein, Kateřina Kubínová, Tereza Kropáčková, Michal Tomčík & Jiří Vencovský

  6. Faculty of Science, Charles University, Prague, Czech Republic

    Veronika Horváthová

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  1. Lucia Vernerová
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Contributions

LV and BU were responsible for the study concept and design and drafted the manuscript. JV supervised the study implementation and manuscript preparation. JV, MK, SO, and MT were responsible for patient recruitment and clinical assessment. MS, SO, and MT organized and performed the training intervention. MK and KK performed the muscle biopsies. LV, NAL, MV, OM, MN, VH, and TK conducted the in vitro experiments and laboratory analyses. LW was responsible for analyses of biochemical parameters for clinical practice including both vitamin D isoforms. LV carried out the statistical analysis. LV, JU, BU, and JV are responsible for data interpretation. All authors reviewed the manuscript and gave their final approval of the version to be published.

Corresponding author

Correspondence to Jiří Vencovský.

Ethics declarations

Ethics approval and consent to participate

Ethics approval and consent to participate: all relevant study documentation and amendments were approved by the independent Ethics Committee of the Institute of Rheumatology Prague with reference numbers 5675/2015 and 5688/2015. The study was conducted following the principles outlined in the Declaration of Helsinki, the Guidelines of the International Council for Harmonisation (ICH) on Good Clinical Practise (GCP) Guideline E6 (R2) (EMA/CPMP/ICH/ 135/95) European Union (EU) Directive 95/46/EC, and other applicable regulatory requirements. Patients provided informed written consent before enrolment in the study.

Consent for publication

Not applicable.

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Vernerová, L., Vokurková, M., Laiferová, N.A. et al. Vitamin D and its receptor in skeletal muscle are associated with muscle disease manifestation, lipid metabolism and physical fitness of patients with myositis. Arthritis Res Ther 27, 48 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13075-025-03516-9

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13075-025-03516-9

Keywords

Arthritis Research & Therapy

ISSN: 1478-6362

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