Introduction

Osteomalacia and rickets are classical disorders of bone and mineral homeostasis. Defective skeletal mineralisation due to deficiency of vitamin D and substrates necessary for hydroxyapatite deposition, namely calcium and phosphate, are predominant mechanisms. However, effects of vitamin D on muscle are less well recognised. In the clinic, muscle weakness, pain and hypotonia are observed in subjects with severe vitamin D deficiency. Under the microscope, changes in muscle fibre size with preferential atrophy of type 2 (i.e. fast twitch) muscle fibres are seen and on electromyogram (EMG), reduced motor unit action potentials have long been reported in patients with vitamin D deficiency [1, 2]. In fact, in his seminal report on rickets in 1645, Daniel Whistler gave equal credence to the muscle and bone defects seen in children with this condition (i.e. he described both their “flabby, toneless muscles and flexible, waxy bones”) [3].

Vitamin D deficiency is generally defined as a 25-OH-vitamin D less than 50 nmol/l (~ 20 ng/ml) [4], but seasonal variations, ethno-specific differences, body composition and alterations in the vitamin D-binding globulin amongst individuals may impact on “normal” levels. Optimal levels of vitamin D for musculoskeletal function may be lower than this threshold, and levels > 20 nmol/l (~ 8 ng/ml) are needed for calcium homeostasis.

How does vitamin D exert such critical effects on skeletal muscle? Attempts to answer this seemingly straightforward question have met challenges and controversy over the decades. The answer begins with the vitamin D receptor (VDR), a cognate nuclear receptor to which the active hormone, 1,25(OH)2Vitamin D (calcitriol) binds to exert both genomic and non-genomic effects in cells. The VDR is the modus operandi by which vitamin D exerts its diverse effects in physiology, from a central role in calcium and mineral homeostasis to non-classical effects on cell division, tissue pleiotropy, fibrosis and immune modulation [5]. The VDR is widely expressed, both at classical sites of vitamin D activity including intestine, parathyroid and bone, and non-classical sites such as immune cells, the mammary gland and brain [6]. However, its presence in skeletal muscle has been hotly contested for years. This special issue article will focus on morphologic and functional roles of vitamin D in muscle, from strength to contraction to development and ageing and will characterise the controversy of VDR’s expression in skeletal muscle, central to our understanding of vitamin D’s effects on this tissue.

Vitamin D Receptor and Muscle

We have learnt a great deal about the VDR since its discovery in 1974 by Brumbaugh and Haussler [7]. Key insights in its biological activity, its protein and structural composition examined by xray crystallography and conformational changes induced by binding to its ligand have come to light [8]. It exerts its effects on the genome by binding to its ligand 1,25(OH)2D together with Retinoid X-receptor (RXR) to form a transcription factor complex (1,25D-VDR-RXR). However, VDR also functions as an orphan nuclear receptor, that is, by direct binding to DNA without its ligand. VDR’s ligand-bound and orphan nuclear effects differ, the former possibly being responsible for its classical effects in skeletal and mineral homeostasis and the latter being associated with its less classical effects, such as the maintenance of healthy hair follicles. Hence, mice with whole-body VDR knockout display alopecia (i.e. hair loss), an effect that is not seen in subjects with ligand deficiency (i.e. low levels of vitamin D) [5]. Classical effects of ligand deficiency (i.e. vitamin D and 1,25(OH)2D deficiency) include reduced intestinal absorption of calcium and phosphate, reduced substrate for hydroxyapatite formation and subsequent osteomalacia (impaired mineralization of bone).

VDR is widely expressed in nature, in vertebrates and invertebrates, fish, avian species and mammals, and its expression spans a wide range of tissues and organs. However, its presence in skeletal muscle has been unclear for some time. Confirmation of VDR’s expression in skeletal muscle is essential in determining whether vitamin D’s effects are direct or indirect effects in this tissue. A number of issues have confounded the clear demonstration of this nuclear receptor in muscle. These include the heterogeneous, multi-cellular nature of skeletal muscle which comprises muscle fibres (i.e. a multinucleated, contractile syncytia of mycoytes) in addition to extracellular matrix, fibroblasts and satellite cells; time-dependent changes in VDR expression during the process of muscle development and; technical, experimental factors, such as the low specificity of VDR antibodies [9].

Through a wide range of techniques, including scintillation autoradiography, binding studies [10, 11], Reverse transcription polymerase chain reaction (RT-PCR) [12] and immunohistochemistry [13], VDR has been demonstrated in cultured myocytes of avian, murine and human origin. Table 1 summarises studies examining VDR’s presence in skeletal muscle. Thirty years ago, in one of the first descriptions of VDR in human muscle cells, Costa and colleagues reported specific binding of tritiated 1,25-(OH)2D3 with a protein component, presumably the VDR, with dose-dependent increases in the expression of CYP24A1, the classic VDR target gene [11]. VDR mRNA has been detected in muscle, both in cultured muscle cells and whole muscle tissue, but at much lower (4000-fold difference) levels compared to classical sites of VDR expression such as the duodenum [14]. Because RT-PCR is an exquisitely sensitive method that uses amplification of mRNA levels to produce a signal, the relevance of such low transcript levels of VDR in muscle that are found by this method remains unclear.

Table 1 Studies on VDR Expression in Skeletal Muscle
Full size table

Contradictory reports on the detection of VDR protein by western blot and immunohistochemistry have been published [15,16,17]. Using a specific VDR antibody (D6-Ab), VDR protein was either detected at low levels [14] or not at all [17, 18] in skeletal muscle from mice and adult humans. In contrast to RT-PCR which is exquisitely sensitive, protein detection methods may not be sufficiently sensitive to detect extremely low levels of VDR in muscle at baseline. However, priming VDR by treatment with its ligand may lift levels above a critical threshold of detection. To support this, VDR protein was detected in the quadriceps of older human subjects following a course of oral vitamin D supplementation [19, 20].

Taking a novel approach to the question of whether VDR is present in muscle, Lee and colleagues explored whole-body expression patterns of VDR in a mouse model in which native VDR was replaced with a VDR transgene expressing luciferase, using a bacterial artificial chromosome (BAC)) [21]. Luciferase refers to a group of enzymes that produce bioluminescence. The BAC-VDR protein was not detected in adult muscle using western blot, immunohistochemistry or luciferase studies, but by comparison was expressed at classical sites of vitamin D action. Reasons for this lack of VDR expression in muscle may be methodological. Owing to the relatively low baseline levels of VDR in muscle, a finite amount of BAC-VDR in this model may be preferentially expressed in classical VDR sites.

A number of in vitro studies have characterised the activity of VDR in cultured myoblasts. Rapid translocation of VDR from nucleus to the cytoplasm was demonstrated following treatment of myoblasts with the ligand 1,25(OH)2D3 [16, 22], a process dependent on microtubular transport. VDR interacted with a range of intracellular signalling pathways, the tyrosine phosphorylation cascade, [23], and membrane-scaffolding proteins such as caveolin-1 [16]. Following prolonged treatment of myoblasts with 1,25(OH)2D3, VDR translocated back to the nucleus to carry out its role in regulating transcription [24]. Hence, depending on its location within the cell and exposure times to its ligand, VDR participated in both rapid and genomic actions in these cultured myoblasts.

VDR levels within muscle may change over time, particularly across the various stages of muscle development. Studies employing tissue culture report substantially greater levels of VDR in immature muscle cells, myoblasts and muscle precursor cells, as opposed to fully differentiated myotubes and whole muscle fibres [14, 18]. Newborn mice, whose muscles are actively involved in the process of secondary myogenesis (a process that occurs in utero in humans) express significantly higher levels of muscle VDR than 3-week old mice and adult mice [14]. However, following muscle injury in adult mice, the process of post-natal muscle repair is activated, essentially a recapitulation of embryonic myogenesis, and a significant increase in muscle VDR is seen [25, 26]. Therefore, higher VDR expression levels in primordial muscle cells and newborn mice, together with muscle fibres undergoing repair support a pleiotropic role of VDR in muscle and potential effects in muscle development and repair. It is conceivable that VDR in adult muscle may sequester within satellite cells and hence, escape detection on assays that examine whole muscle tissue.

In summary, the controversy of VDR’s presence in skeletal muscle has stemmed from wide differences in muscle models used to answer this question, non-specificity of VDR antibodies, and protein detection methods that may be insufficiently sensitive to detect low levels of VDR protein in this tissue. Low baseline levels do not preclude a biological role for VDR in this tissue. Indeed, transcription factors are known to exert genomic effects even at low levels of expression, dependent on their binding affinity to DNA [27]. In addition, VDR may sequester within a specific cell population, such as satellite cells, and thus escape detection methods employing whole muscle (in which satellite cells comprise a minority, i.e. ~ 5% cells in adult muscle) [28]. To date, evidence indicates that VDR is indeed expressed in muscle but at very low baseline levels in adults. VDR in muscle predominates in precursor cells and in developing and regenerating muscle fibres, and hence its activity in this tissue appears related to muscle development and pleiotropy.

Vitamin D and Muscle Development

The VDR makes its first appearance early during embryonic life (i.e. day 13 in rats) and is initially expressed within the mesoderm, the embryonic tissue which gives rise to the musculoskeletal system [29]. Mesenchymal stem cells (MSCs) express VDR in addition to components of the vitamin D –endocrine system including CYP27B1 (1α-hydroxylase) and CYP24A1 (24-hydroxylase) and respond to treatment with the ligand 1,25(OH)2D3, which induces myogenic and osteogenic programs [30].

Experiments on C2C12 cells, an immortalized murine muscle cell line, indicate that vitamin D alters myogenesis in its various stages, including myoblast proliferation, myocyte differentiation and fusion to form myotubes and the determination of myotube size [12, 24, 30,31,32]. 1,25(OH)2D3 exerted effects on post-translational modification in C2C12 myoblasts by altering phosphorylation of Rb (Retinoblastoma protein), JNK (c-Jun N-terminal kinase), Raf-1 (retinoblastoma associated factor 1), cAMP response element binding protein (CREB) and ElK-1 signalling, resulting in an anti-proliferative effect in these cells [33,34,35,36]. Vitamin D modulated C2C12 myotube formation, a complex process in which myocytes fuse to form tubular, contractile syncytia, dependent on well characterised pathways involving IGF-1 and myogenin [12, 24, 32]. Key myogenic regulatory factors includng myogenin, myf5, myoD and IGF were all modulated by vitamin D treatment in these cells. Vitamin D exerted an anabolic effect in increasing C2C12 myotube size by downregulation of myostatin, a TGF-β which negatively regulates muscle mass [24, 32]. These effects of the ligand 1,25(OH)2D3 were directly related to VDR activation and were negated by VDR knockdown [37].

The Vitamin D receptor knockout (VDRKO) mouse model has provided key insights in the biologic activity of the vitamin D and its role in muscle development [5]. In addition to wide-ranging defects including rickets, reduced calcium and phosphate levels, alopecia and immune dysfunction, these mice displayed lighter muscles, global reduction in muscle fibre size and increased myonuclei, changes that persisted despite normalisation of calcium and phosphate by the provision of a rescue diet [12, 38]. This muscle phenotype was seen early in VDRKO mice, initially at 3 weeks of age, preceding the development of phosphate and calcium abnormalities and systemic defects. Increased transcript levels of myostatin (> 2-fold), alteration of myogenic regulatory factors myf5 and myogenin and persistent expression of neonatal myosin heavy chain (MHC) isoforms were seen in the muscle of adult VDRKO mice [12, 38]. However, the VDRKO mouse model is subject to confounding, specifically whole-body changes in these mice may have independent effects on muscle morphology. In addition, attempts to identify vitamin D response elements (VDRE) in the promoter regions of myf5 and myogenin genes have been unsuccessful, suggesting VDR’s effects on key myogenic regulatory factors may be indirect [39, 40].

To bypass potential confounding by systemic effects of the VDR, tissue-specific knockout mice have been generated [41]. The promoter gene used to ablate VDR in the muscle of these mice, MLC1f, is expressed in embryonic life, making this an appropriate model to examine effects on muscle differentiation. Reductions in type II muscle fibre diameter were demonstrated in these mice but the primary focus of this paper was to examine effects on insulin sensitivity and hence, further detail on the muscle morphology of these mice was not presented in detail [41].

Effects of maternal vitamin D on muscle development in offspring have been examined in several species. In humans, maternal vitamin D levels were associated with arm muscle size in offspring as well as grip strength [42, 43]. In rodent studies, offspring of vitamin D-deficient dams demonstrated smaller muscle fibres with effects on protein catabolism and genes involved in muscle differentiation and the cytoskeleton [44, 45]. In pigs, vitamin D supplementation during pregnancy led to increased muscle fibre size and number in offspring, associated with higher transcript levels of myoD, myogenin and reduced myostatin transcript [46]. European sea bass treated with vitamin D after hatching demonstrated increases in muscle fibre size that were dose-dependent and associated with changes in myogenic genes [47]. Maternal vitamin D levels may possibly play an epigenetic role in foetal development, being associated with methylation at 4 sites of the RXR-A (retinoid X receptor alpha) in umbilical cord tissue [48].

In summary, in vitro studies in muscle cells suggest a role for VDR in muscle proliferation, differentiation and myotube development and size. In vivo studies in mice corroborate this effect by demonstrating reduced muscle mass and smaller fibres in mice with whole-body or muscle-specific VDR ablation. Alterations in myogenic regulatory gene and TGF-β expression have been demonstrated as potential mechanisms for these changes. To confirm direct genomic effects of VDR in muscle, chromatin immunoprecipitation (ChIP) studies are necessary to characterise the VDR cistrome in this tissue (i.e., DNA binding sites).

Vitamin D and Muscle Strength

People with vitamin D deficiency display muscle weakness and higher risk of falls, features that are reversible with vitamin D supplementation [49]. In addition to changes in muscle mass described in the previous section, VDRKO mice displayed reduced grip strength [38] and abnormal swimming with reduced buoyancy and greater fatigue [50]. In open field testing, VDRKO mice displayed shorter steps and abnormal gait and on rotarod testing, they displayed reduced balance with shorter retention times implying abnormal muscle coordination [50, 51]. These defects in muscle function progressed with ageing and a dose-dependent effect of VDR on grip strength was seen [38, 51]. However, no impairment in swimming was demonstrated in 1α-hydroxylase knockout mice [51]. The contrasting phenotypes of VDR and 1α-hydroxylase knockout mice suggest different activities of the vitamin D endocrine system on muscle function. Whilst VDR is important in muscle function, as suggested by the VDRKO model, the active hormone, 1,25(OH)2D is not prerequisite for VDRs actions in this tissue, as suggested by lack of muscle phenotype in 1α-hydroxylase knockout mice. Taken together, this indicates that VDR exerts ligand-independent functions in muscle.

On the other hand, reduced muscle strength has been demonstrated in animal models of vitamin D deficiency [38, 52, 53]. Significant reduction in muscle contraction and impaired recovery in vitamin D-deficient rats and chicks were demonstrated and reversed with vitamin D supplementation [53, 54]. Alterations in expression of components of the sarcomere, including actin and the troponin-tropomyosin complex, provide a mechanism for reduced strength in these studies [55,56,57]. Alternatively, vitamin D may exert its effects on muscle strength via intracellular calcium handling. In vitamin D deficient mice, reduced grip strength was associated with altered expression of mRNAa responsible for calcium-handling and sarco-endoplasmic reticulum calcium transport ATPase (Serca) channels [38]. Reduced calcium concentrations within muscle mitochondria and sarcoplasmic reticulum have been reported in vitamin D deficient animals [54, 58].

A study proposed a primary function for phosphate in vitamin D’s effects on muscle. Rats deficient in vitamin D, phosphorus and calcium underwent muscle strength testing via force transduction on soleus muscle [52]. Phosphate levels displayed the strongest correlation with muscle dysfunction and phosphate repletion reversed defects independent of calcium and vitamin D levels. However, in another study muscle defects in vitamin D deficient mice persisted despite adjusting calcium and phosphate deficiency [38].

Vitamin D effects on the neuromuscular junction were examined in a study of vitamin D deficient rats. In association with defects in muscle balance and coordination, vitamin D deficient rats had increased muscle hypersensitivity and a higher number of nocioceptor axons [59]. In other studies, treatment of WT mice with eldecalcitol, a vitamin D analogue, improved coordination and locomotor performance, with increased expression of IGF1 and myelin in Schwann cells and increased AchR density in neuromuscular junction [60]. Therefore, vitamin D exerts neuronal effects that may further impact on muscle function.

Recent studies have reported a novel ex vivo effect of VDR on muscle function [14, 61]. Muscles of VDRKO were dissected and examined in a controlled environment, unperturbed by systemic changes, and in these studies, VDR modulated the uptake and retention of 25(OH)D3 within muscle fibres [14, 61]. Upon entry into muscle, 25(OH)D3 may be locally converted to 1,25(OH)2D3 and elicit rapid effects on calcium handling thereby altering contraction and muscle strength. Alternatively, muscle may be a storage depot for 25(OH)D3, in which the molecule may be bound to the D-binding protein (DBP), and as required, diffuse back into circulation upon degradation of DBP and its release from actin [61].

Rapid effects of 1,25(OH)2D3 on intramuscular calcium handling have been elucidated by a range of in vitro studies [62,63,64]. 1,25(OH)2D3 resulted in rapid calcium shifts from the sarcoplasmic reticulum to the cytosol, through the action of signal transduction pathway including c-src, phospholipase C gamma (PLC-gamma) and inositol triphosphate (IP3) [65]. Prolonged exposure to 1,25(OH)2D3 led to sustained calcium entry from the extracellular compartment via L-type voltage-dependent calcium-channel (VDCC) and store-operated calcium entry (SOCE) mechanisms. By modulating intracellular calcium, vitamin D may play an indirect role in muscle contraction, plasticity and metabolism, processes which are determined by calcium [66]. To confirm these effects, fura-2 studies in muscle would allow for real-time in vivo assessment of calcium flux in response to vitamin D, and provide a mechanistic basis for effects on contraction and strength.

In summary, muscle weakness and increased fatigue have been reported in vitamin D deficient humans and animals in addition to VDRKO mice. However, there are difficulties in differentiating direct effects of vitamin D deficiency in muscle dysfunction from those of associated phosphate, calcium and parathyroid hormone defects. Cell studies indicate that 1,25(OH)2D regulates muscle cell calcium and phosphate handling in rapid and genomic fashions and uptake of 25(OH)D3 within muscle, positing mechanisms for effects on contraction and strength. Definitive studies are needed to study contractile physiology, endurance and muscle fatigue in mice with distinct alterations of vitamin D signalling, independent of mineral defects.

Vitamin D and Muscle Ageing

Serum vitamin D levels predict the rate of functional decline and age-related atrophy of skeletal muscle in older subjects [67]. Vitamin D deficiency is common in older subjects and levels of VDR decline in muscle with age [15]. Type 2 muscle fibres (i.e. fast twitch) undergo preferential atrophy, predisposing vitamin D deficient subjects to falls [2].

In ageing rodents with vitamin D deficiency, muscle atrophy pathways were upregulated with increased muscle protein catabolism via activation of ubiquitin ligases (MAFBx and MuRF1), TGF-β, FOXO and the ubiquitin-proteosome system were seen [38, 68]. These molecular changes were associated with significant muscle fibre atrophy that was only partly corrected by adjusting calcium levels [68]. Myostatin expression was greater in the muscles of vitamin D deficient rodents and expression of mygenic regulatory factors was also altered [38]. Studies on human myocytes corroborate effects of vitamin D and VDR agonists on pathways known to regulate muscle ageing including ubiquitin ligases, inflammatory markers TNF-alpha and IL6 and PI3K/AKT signalling [20, 69].

Vitamin D may exert indirect effects on muscle ageing by its interaction with other hormones. FGF23 and its co-factor klotho regulate phosphate, vitamin D synthesis and have novel effects on ageing. Mice lacking FGF23 display premature ageing, osteopenia and sarcopenia, and these features were completely reversed by concomitant ablation of CYP27B1 [70]. This suggests that the ligand 1,25(OH)2D may modulate age-related responses to FGF23, possibly through its action on the VDR. Klotho deficient mice display a muscle phenotype remarkably similar to VDRKO mice with weakness, impaired endurance and premature ageing associated with alterations in TGF-β and wnt signalling [71, 72]. It is possible that vitamin D and klotho share inter-connected effects on skeletal muscle morphology and function during ageing on the basis of these similarities.

Vitamin D deficiency results in mitochondrial dysfunction and oxidative stress in muscle cells with reduction in superoxide dismutase (SOD) [73]. Serum Vitamin D levels correlated with lactic acid, creatine kinase and total antioxidant activity (TAC) in elderly subjects following exercise and vitamin D supplementation improved oxidative phosphorylation [74, 75]. In vitro, 1,25(OH)2D3 regulated transcripts of mitochondrial genes in muscle cells with increased mitochondrial volume and oxygen consumption rates [76]. Thus, vitamin D may reduce oxygen free radicals in muscle and alleviate effects of mitochondrial dysfunction, thereby counteracting sarcopenia.

In summary, vitamin D deficiency accelerates muscle ageing with atrophy of muscle fibres and subsequent sarcopenia with a risk of falls and functional decline. Mechanisms elucidated in animal studies include increased muscle protein turnover via activation of ubiquitin-proteosome and oxidative stress. Vitamin D supplementation may reverse these effects and further research is needed on the potential anti-ageing effects of vitamin D on skeletal muscle.

Vitamin D and Muscle Repair

Muscle repair is an intricate process in which satellite cells, unique muscle stem cells, are activated by mitogenic factors and differentiate into myocytes which fuse into muscle fibres, governed by the myogenic regulatory program. Vitamin D may also play a role in this process.

In vitro, vitamin D altered muscle cell response to mechanical injury with an increase in muscle cell migration, myotube fusion and expression of tissue regeneration and angiogenic markers [31, 77]. In vivo, muscle damage induced by via freeze-crush or BaCl2-induced mechanisms resulted in significant activation of VDR and CYP27B1 in rodents, specifically within regenerating muscle fibers [26, 78]. Resistance training, inducing lesser degrees of muscle damage, also induced VDR and CYP27B1 in muscles of rodents [25, 79].

Muscle injury induced by freeze-crush injury or high-intensity exercise was ameliorated by vitamin D supplementation in rats, associated with attenuated increase in creatine kinase (CK) and lactate dehydrogenase (LDH) [78, 79]. Functionally, improved recovery in contractile force in the injured muscle was demonstrated. Mechanistically, reduced oxidative stress and inflammation, together with an effect on stress-related proteins (p38 MAPK, ERK1/2, IKK, IkappaB), may explain these beneficial effects [79, 80]. However, there may be a U-shaped effect as excessive doses of 1,25(OH)D3 were not beneficial on muscle regeneration with deleterious effects on satellite cell activity and muscle fibre repair following injury [81].

In human clinical studies, baseline levels of vitamin D correlated inversely with muscle weakness following exercise [82]. Vitamin D altered cytokine levels following exercise, including IL-10, IL-13 and inflammatory mediators TNF-a and IFN-g, suggesting a modulatory effect on inflammation [82, 83]. Levels of VDR protein and IL6 displayed a positive correlation in human muscle, suggesting effects of vitamin D on the inflammatory response to muscle damage [84]. Vitamin D supplementation had a beneficial effect on muscle recovery in adult males in whom muscle injury was induced by repetitive eccentric contractions, i.e. jumping [77, 85].

In summary, vitamin D’s effects on muscle repair are suggested by increases in VDR expression in regenerating muscle tissue. Direct effects on oxidative stress, inflammatory cytokines and satellite cell activity in response to vitamin D have been demonstrated.

Conclusions

This special issue article summarises the current understanding of vitamin D’s effects on skeletal muscle, specifically in development, strength, ageing and repair. While functional effects of vitamin D on muscle, particularly in relation to muscle strength and contraction, appear related to calcium and phosphate levels, pleiotropic effects on muscle development, ageing and repair may be related to direct actions of vitamin D signalling within muscle cells.

At a basic level, vitamin D modulates intramuscular calcium flux via the rapid activation of signalling cascades and second messenger systems [62,63,64]. Genomic responses to vitamin D involve myogenic regulatory factors, TGF-β signalling including myostatin and the ubiquitin-proteosome [24, 32]. Morphologically, muscle mass, fibre size, strength and the regenerative response to muscle damage are altered by vitamin D [26, 38]. Age-related changes in muscle function, protein turnover, oxidative stress and atrophy pathways are postulated mechanisms by which this occurs [38, 68].

The controversial question of VDR’s expression in skeletal muscle has also been discussed. Technical factors giving rise to this controversy, in addition to the heterogeneous, multicellular nature of skeletal muscle, in which individual components may respond differently to vitamin D, have been mentioned. Current evidence indicates that VDR is indeed expressed in muscle, but at low levels that may elude detection. VDR predominates in immature forms of muscle, primordial muscle cells, such as satellite cells, and in developing and regenerating muscle fibres [14, 18]. VDR’s predominant expression in these early muscle cells indicates a primarily pleiotropic role in this tissue. At a functional level, VDR knockout mice display a distinct muscle phenotype, also in support of its presence and activity at this site.

Uncertainties remain. Although gene targets of vitamin D signalling in muscle have been reported, vitamin D response elements (VDRE) within these genes have not been clearly demonstrated and hence, it is unclear if these are direct targets. Chromatin immunoprecipitation (ChIP) studies to characterise the muscle VDR cistrome are needed. Non-genomic effects of vitamin D on calcium flux have been reported in many in vitro studies but the translation of these findings to in vivo muscle physiology is not a foregone conclusion. For confirmation, fura-2 studies in muscle would allow for real-time in vivo assessment of calcium flux in response to vitamin D. A greater understanding of direct effects of VDR on muscle function will come to light with characterisation of tissue-specific models, circumventing systemic effects of vitamin D [41]. Finally, effects of vitamin D on muscle regeneration raise the intriguing possibility that vitamin D modulates satellite cells in their response to damage, and thereby enhances regeneration. Thus, vitamin D exerts diverse effects on skeletal muscle, with a broad functional repertoire in development, pleiotropy and ageing.