Myogenic, genomic and non‐genomic influences of the vitamin D axis in skeletal muscle

Despite vitamin D‐deficiency clinically presenting with myopathy, muscle weakness and atrophy, the mechanisms by which vitamin D exerts its homeostatic effects upon skeletal muscle remain to be fully established. Recent studies have shown that the receptor by which 1α,25‐dihydroxyvitamin D3 (1,25[OH]2D3) exerts its biological actions (ie, the vitamin D receptor, VDR) elicits both genomic and non‐genomic effects upon skeletal muscle. The controversy surrounding skeletal muscle VDR mRNA/protein expression in post‐natal muscle has been allayed by myriad recent studies, while dynamic expression of VDR throughout myogenesis, and association of higher VDR levels during muscle regeneration/immature muscle cells, suggests a role in myogenesis and perhaps an enrichment of VDR in satellite cells. Accordingly, in vitro studies have demonstrated 1,25(OH)2D3 is anti‐proliferative in myoblasts, yet pro‐differentiation in latter stages of myogenesis. These effects involve modulation of gene expression (VDR as a transcriptional co‐activator controls ~3% of the genome) and post‐genomic intracellular signalling for example, via c‐Src and alterations to intramuscular calcium homeostasis and proteostasis. The aim of this review is to consider the biomolecular role for the vitamin D/VDR axis in myogenesis, while also exploring global evidence for genomic and non‐genomic mechanisms of action for 1,25(OH)2D3/VDR.


| INTRODUCTION
Vitamin D is a fat-soluble hormone that exists in two main forms.
Cholecalciferol (vitamin D 3 ), the primary source of vitamin D, is obtained predominantly via subcutaneous ultraviolet B (UVB; 290-320 nm) irradiation of 7-dehydrocholesterol (7-DHC) into vitamin D, although it can also be attained through dietary consumption of animal products such as oily fish, egg yolks and red meat. 1 Comparatively, ergocalciferol (vitamin D 2 ) is the product of UV irradiation of ergosterol, a yeast sterol present in some plants and fungi, and has commonly been prescribed as a supplement. 2 Approximately, 1 billion people worldwide are considered to be either vitamin D deficient or insufficient. 1 This pandemic of hypovitaminosis D is regarded as an important global public health issue due to vitamin D's role in human physiology, for example, the immune system, and its deficiency being implicated in a number of chronic diseases. 5 Vitamin D-deficiency is classically recognized as being associated with bone diseases, such as rickets and osteoporosis. 6 However, vitamin D has effects on tissues outside the skeleton that may be crucial for a wide range of other chronic health issues, with one such area being skeletal muscle. Expression of both the vitamin D receptor (VDR) 7,8 and CYP27B1 has been observed in the skeletal muscle of humans and rodents. 9,10 As a member of the nuclear receptor super-family, the VDR is localized to the nucleus where it binds DNA vitamin D response elements (VDREs), thereby acting as a transcription factor. 11 Through observational studies, it is now evident that low vitamin D is associated with reduced muscle mass (sarcopenia) and increased risk of falls in the elderly. 12 As individuals age, the expression of VDR decreases which, paired with a higher prevalence of deficiency within the older population, suggests involvement of the vitamin D system in musculoskeletal ageing. 8 Indeed, vitamin D deficiency is associated with reduced muscle mass and weakness, myalgia and an increased risk of sarcopenia, regardless of activity levels. 13,14 Moreover, low vitamin D has also been implicated in muscle dysfunction in chronic obstructive pulmonary disease (COPD), cancer and diabetes. [15][16][17] Finally, numerous randomized controlled trials (RCTs) have suggested an important physiological role of the hormone within muscle, demonstrating increases in muscle function following vitamin D 3 supplementation. [18][19][20] While the mechanisms by which vitamin D and the VDR exert its actions upon skeletal muscle remain to be fully established, here we review the current state of this area.

| VDR EXPRESSION AND SKELETAL MUSCLE
Early studies reported a positive association between VDR content of classical tissue targets of vitamin D, for example, bone and intestine, and the level of biological activity as a response to treatment with vitamin D 3 . 21 It has since been demonstrated, that upon treatment with 1,25(OH) 2 D 3, VDR expression is also upregulated in skeletal muscle cells. 16,22,23 However, the confirmed expression of VDR in skeletal muscle has been subject to controversy. Initial animal studies used immunohistochemistry as a means for detection, but selectivity of the widely used anti-VDR antibody 9A7 was questionable in crossreacting with unrelated proteins on western blots. 24 More recent data, confirming the presence of VDR in both human and murine skeletal muscle cells, have been generated through studies using multiple VDR antibodies. 22,25,26 However, in fully differentiated murine and human muscle, VDR levels have been detected at extremely low levels or not at all, whereas VDR expression is higher in cell-cycling myoblasts than differentiated cells. 22,27,28 One potential explanation for these observations is that VDR levels are dynamic in nature, being highest during immaturity followed by an age-related decline. This hypothesis is supported by the fact that VDR is displayed to be upregulated in regenerating muscle of mice that have undergone muscular injuries or exercise. 10,29,30 This raises an important question-is VDR expression enriched in satellite cells? In postnatal muscle fibres, this population of muscle stem cells represents approximately 30% to 35% of the total muscle cell population, before declining to 5% in healthy adult muscle. 31 As satellite cells normally exist in a quiescent state, unless stimulated to differentiate into myocytes by exercise or muscular damage, this could explain the difficulties of detecting VDR in adult muscle. 32 Nonetheless, DNA-bound VDR may also represent a challenge for detection using standard extraction techniques. Next, we review in vitro and in vivo evidence linking vitamin D/VDR to muscle biology.

| Vitamin D and myogenesis
The majority of studies investigating the vitamin D axis and muscle cell function have adopted in vitro cultures of muscle cells.
Myogenesis, the means by which skeletal muscle is generated, is a multi-step process and can be largely broken down into four stages: appears that this effect occurs simultaneously with induction of the cyclin-dependent kinase inhibitors (CDKIs) p21 and p27, 36 and data from studies using VDR knockdown (VDR-KD) cell lines indicate that these cell-cycle influences are VDR-dependent. 37 Fusion of myoblasts into multinucleated myotubes is a strictly controlled process. The first wave occurs during embryogenesis and is responsible for generating the myotubes of the preliminary muscular system, whereas the secondary wave takes place later in a vertebrate's lifespan. It is this second wave that is responsible for the addition of secondary myotubes/myofibres that increase muscle mass, so the impact of vitamin D on this process is of particular interest. 39 Myotube formation can occur spontaneously in high serum media and is believed to rely primarily on myogenin. In culture, C2C12 myoblasts can be induced to exit the cell cycle and differentiate into myotubes through switching "growth culture" medium (10% foetal bovine serum, FBS) to "differentiation medium" (2% FBS). This method of differentiation induction via serum starvation is primarily driven by IGF-1 and has been used to examine the differences that occur when myoblasts are treated with vitamin D 3 either at the point of differentiation initiation or when they are fully differentiated. 40 Interestingly, 1,25(OH) 2 D 3 appears to stimulate myotube formation when high-sera medium is used for both C2C12 and primary muscle cell cultures, 35,41 yet it has the opposing effect in cases of differentiation via serum starvation. 22,42 Thus, it is possible to suggest that vitamin D elicits differential effects depending on the model of myogenesis used. When C2C12 myoblasts are treated with 100 nM 1,25(OH) 2 D 3 during the early differentiation phases (ie, day 0 and day 4), myogenin and neonatal myosin heavy chain (nMHC) isoforms were down-regulated, but this effect was not present in the fully differentiated group (day 8). 36 In fact, treatment with 1,25(OH) 2 D 3 at day 8 post-differentiation appeared to have an opposing effect as myosin heavy chain type IIa expression was upregulated with an additional protective role against cellular detachment. 36 The inhibitory influence of 1,25(OH) 2 D 3 upon myotube formation has been supported further as C2C12 myoblasts also treated at day 4 differentiation were only able to fuse partially, resulting in abnormally shaped myotubes possessing an increased number of myonuclei. 16 The results from these aforementioned in vitro studies have been translated in vivo through the use of vitamin D deficient and/or VDRnull animals. These animals possess similar phenotypes, namely presenting with smaller muscle fibres, reduced muscle strength, higher fatigue, fibre hyper-nuclearity and abnormal gait. [42][43][44][45] In rats, vitamin D deficiency causes cell-cycle arrest, which, while initially seeming undesirable for muscle function, it is possible that this action of 1,25 (OH) 2 D 3 promotes entry of myoblasts into a quiescent state. 46 Notch signalling is considered a master regulator of myoblast quiescence and, while expression of full-length Notch is unaffected by deficiency, a decrease in the cleaved form of Notch is present, indicating reduced activation of the Notch pathway in vitamin D deficient rats. 46 Phenotypic alterations of the VDR-KO mouse model are not limited to just the muscular system-these animals also display alterations, for example, to cardiovascular and gastrointestinal systems. 49 Due to this confounding systemic impact caused by the absence of the VDR gene, it has become difficult to draw final conclusions from results produced from the total-body VDR-null mice. Tissue-specific VDR-KO animal models have been generated in an attempt to bypass this problem, and work using mice with a myocyte-specific VDR deletion (mVDR null) has demonstrated the importance of VDR in muscle function. 26 Stark differences can be seen between these mice and the whole-body knockout animals, as the mVDR mice were of a normal body size yet their muscle fibres were marginally larger in diameter. 26 Thus, efforts should be focused on clarifying conclusions drawn from whole-body models using this new tissue-specific model, while also yielding novel information regarding the biological activity of vitamin D.   66 As osteocalcin has been shown to influence muscular mitochondrial function, myofibre uptake and catabolism of glucose/fatty acids, and insulin sensitivity, 65,67,68 it is sensible to suggest this genomic relationship as one potential explanation for the aberrant muscle phenotype seen in VDR-KO mice. Similar to the VDR, expression levels of the osteocalcin gene have been shown to decline as individuals age. 69 Lastly, expression of integrinβ3 is also recognized to be under the control of a VDRE, and deficiency of this integrin in myoblasts results in impaired myoblast migration and fusion. 70 The integrins are a major family of trans-membrane receptors that act to provide connections between the extracellular matrix and the cytoskeleton. 71  would provide further data that may clarify the issue, while also elucidating any differences that occur at each stage of the myogenic pathway.

| Genomic action of the vitamin D/VDR axis
Polymorphisms of the VDR gene has been shown to alter the biological activity of the vitamin D-VDR complex. The rs2228570 single nucleotide polymorphism (SNP), more commonly known as FokI, is located at the 5 0 end of the VDR gene, leading to an altered start codon, and a subsequently abnormally sized VDR protein. 73 Comparatively, the rs1544410 (BsmI) and rs731236 (TaqI) SNPs are localized close to the 3 0 un-translated region (3 0 -UTR) and, therefore, have no impact on the structural characteristics of the receptor. 73  Finally, it appears that these genomic influences of vitamin D permit a relationship between the vitamin D/VDR axis and mitochondrial metabolism. Recent supplementation studies have indicated that 1,25 (OH) 2 D 3 results in improved mitochondrial function within skeletal muscle, 79 but the mechanism for this beneficial effect is yet to be fully elucidated. Since mitochondrial translocation of VDR was first described, 80 two key questions remain unanswered: (a) What is the general role of vitamin D in mitochondria, and (b) how does this relationship specifically translate into skeletal muscle? Potential explanations may be that 1,25(OH) 2 D 3 can directly influence energy production (eg, ATP synthesis) by eliciting its genomic effects on mechanisms such as oxidative phosphorylation or the tricarboxylic acid (TCA) cycle. 81 This theory has been explored by several research groups, all of which have demonstrated upregulation of expression levels of genes associated with the TCA cycle and electron transport chain (ETC). 53,82 In addition, studies using VDR-KD cell lines have shown that the rate of ATP synthesis derived from oxidative phosphorylation is reduced by 20% vs a shRNA scrambled control, 83 and that this absence of VDR is also associated with increased production of reactive oxygen species (ROS). 84 While evidence does exist to indicate a relationship between mitochondria and the vitamin D system it is clear that further investigations are required for concise conclusions to be made.

| Signal transduction pathway (non-genomic) regulation of muscle by the vitamin D/VDR axis
Muscle proteostasis is tightly regulated by an intrinsic balance between the rates of muscle protein synthesis (MPS; anabolism) and breakdown (MPB; catabolism). If the net rate of MPS exceeds that of MPB, then the net muscle protein balance (NPB) is considered to be positive, and growth of muscle fibres is achieved; alternatively, muscle atrophy occurs when the NPB is negative due to rate of MPB being higher than MPS. 85,86 Of the various proteolytic systems existing in muscle, vitamin D has been mainly implicated in one in particular, the ubiquitin-proteasomal pathway (UPP). 87,88 In rats, deficiency of vitamin D resulted in increased levels of MPB, significantly elevated expression of both the ubiquitin-conjugating enzyme, E2, and its ubiquitin conjugates, but no alterations to either calpain or lysosomal enzymes were observed. 87 At the heart of the UPP lies the 26S proteasome, a large complex comprised of two smaller sub-complexes: the 20S core peptidase and the 19S regulatory unit. 89 The proteasomal activity of the 20S subunit was seen to be upregulated in biopsies from the vitamin D-deficient mouse model, thereby indicating an increase in non-lysosomal proteolysis and a place for the vita- c-Src, proto-oncogene tyrosineprotein kinase Src; MAPK, mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin; 4EBP1, eukaryotic translation initiation factor 4E-binding protein; p70S6K, ribosomal protein S6 kinase β-1; Raf, proto-oncogene serine/threonine-protein kinase; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; Ca 2+ , calcium ion; SOCE, store-operated calcium entry; VDCC, voltage-dependent calcium channel; MEK, MAPK kinase; ERK, extracellular signal-regulated kinase. Created with BioRender.com meaning it is possible to hypothesize that 1,25(OH) 2 D 3 -induced translocation of VDR permits activation of c-Src signalling, therefore suggesting that c-Src acts as a gateway to cell signalling for vitamin D.
As c-Src is under positive regulation by vitamin D 3 and is also known to be an upstream regulator of Akt, it is, therefore, possible that the hormone is able to have some element of control over mechanistic target of rapamycin (mTOR) and p70kDa S6 kinase (p70S6K) signalling. The Akt/mTOR signalling pathway is deemed to be one of the principal regulators of muscle mass in skeletal muscle, and myogenesis is seen to be impaired when mTOR is inhibited following muscle injury. 97 Akt is able to positively control MPS via its relationship with mTOR, while also repressing MPB through its inhibitive phosphorylation of members of the FOXO family of transcription factors. 98  is notably reduced in antisense PKC knockdown models, 104