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Despite the difficulties surrounding the detection of the VDR within skeletal muscle, our understanding on the role of the VDR within skeletal muscle has been enhanced

primarily via the use of in vitro models of myogenesis and the development of VDR- KO mouse models [365-368, 370, 375, 377, 385]. Functionally, VDR-KO mice display reductions in grip strength, impaired swimming performance and increased fatigue indicating impaired skeletal muscle performance [302, 386]. Whole-body impairments in skeletal muscle function are supported by morphological changes within the skeletal muscle of VDR-KO mice. Animals at 3-weeks of age, prior to the onset of the secondary metabolic changes such as hypocalcemia, displayed a shift in muscle fibre diameters as well as a 20% decrease in size [387]. Morphological changes are still observed at both 8- and 12-weeks of age and persisted even with the administration of a rescue diet containing increased mineral content [302, 387]. Interestingly, these changes are observed across multiple muscles suggesting the actions of the VDR are not limited to skeletal muscle of specific fibre types [302, 387]. A number of myogenic regulatory factors including myogenic factor 5 (Myf5),

transcription factor E2-aplha (E2A), myogenic differentiation 1 (MyoD) and myogenin

were all increased [302, 387], whilst the expression of immature forms of MHC were also observed in the small muscle fibres of VDR-KO mice [387]. Myostatin mRNA, a negative regulator of muscle mass, was also increased within the skeletal muscle of VDR-KO mice, possibly explaining the observed reductions in muscle fibre size [302]. Given the ubiquitous expression of the VDR, including within the central nervous and vestibular system, the assessment of the direct effects of the loss of the VDR within skeletal muscle are difficult within this mouse model [388]. Recently, mice with the tissue-specific knockout of the VDR within skeletal muscle have been generated however, limited research has been performed to date utilising this model [385, 389]. Interestingly, these mice develop a number of metabolic defects including increased

serum insulin levels, insulin resistance and glucose intolerance [389]. Whilst the morphological characterisation of these mice was not the primary aim of the study, they did report a slight decrease in muscle fibre size concomitant with increases in forkhead box O1 (FOXO1) at both the gene and protein level [389]. The increased activation of FOXO1 could potentially explain the reductions in fibre size observed in both whole-body and skeletal muscle specific VDR-KO mice given its role in skeletal muscle atrophy [390, 391].

The regeneration of skeletal muscle following an insult or injury is a complex process and recent evidence suggests that VDR activation may be increased during times of regeneration. Interestingly, a significant increase in the activation of both the VDR and CYP27B1 were observed following skeletal muscle injury induced via the injection of barium chloride (BaCL2) or freeze crush [251, 392, 393]. The increase in

VDR expression was localised to the myonuclei of regenerating muscle fibres however, co-localisation was also observed with satellite cells suggesting an increase in activation specifically during the regenerative processes [251, 393]. Similar observations were reported following an acute bout of resistance exercise performed via electrical stimulation in rodent skeletal muscle [394]. Both VDR and CYP27B1 expression increased both immediately and 3 h post electrical stimulation, however endurance exercise failed to stimulate corresponding increases in expression [394]. Similarly, an acute bout of high-intensity treadmill based exercise in rats failed to increase VDR expression alone, although exercise in combination with vitamin D supplementation resulted in an increase in VDR expression [395]. Whilst these data highlight an increase in VDR expression during times of skeletal muscle

regeneration, it could be argued that the means to induce said processes are supraphysiological. Given endurance exercise failed to increase VDR expression alone, significant muscle damage may have to take place to induce VDR activation with skeletal muscle.

Alongside its proposed roles in skeletal muscle regeneration, the VDR has also been studied in the context of skeletal muscle development [251, 377, 387, 396-399]. The VDR appears within 13-days of gestation in rats and resides within the mesoderm, a precursor to the musculoskeletal system [396]. Additionally, components of vitamin D related signalling including the VDR and CYP24A1 are expressed within mesenchymal stem cells [397], whilst the expression of the VDR within skeletal muscle decreases across development in mice [250]. Multiple studies have utilised the myogenic C2C12 skeletal muscle cell line to study the role of vitamin D and its related signalling on myogenesis [251, 377, 387, 398, 399]. The treatment of this cell line with 25(OH)D and 1a,25(OH)2D3 results in a reduction in proliferation [251, 377,

398], an inhibition of myotube formation during serum starvation and an increase in individual myotube size [377]. Alterations in key markers of cell cycle progression including Rb, myc, ATM, and cyclin D1 as well the phosphorylation status of Rb contribute to the vitamin D induced anti-proliferative effects within the C2C12 cell line [377, 398]. Whilst vitamin D treatment resulted in a decrease in overall myotube number, individual myotube size was increased, in concordance with a downregulation of myostatin [377]. In contrast, others have reported that 1a,25(OH)2D3 treatment in C2C12’s stimulates myotube formation when a high

employed to study myogenesis resulted in a different differentiation time course leading to conflicting effects of 1a,25(OH)2D3. Whilst the increased expression of the

VDR is observed following treatments with 25(OH)D and 1a,25(OH)2D3 [377, 398]

these models cannot confirm whether the effects on myogenesis observed are mediated by ligand dependent or independent roles of vitamin D. One study examining myogenesis following siRNA-mediated knock-down of the VDR observed similar effects on proliferation and differentiation [400] suggesting a direct role for the VDR in mediating myogenic signalling. However, given the transient nature and partial deletion often seen with siRNA approaches, improved in vitro models are needed to more clearly elucidate the role of the VDR in the development of skeletal muscle.

As previously discussed, the VDR also possesses non-genomic roles involving transient signalling events [357]. In support of a non-genomic role for the VDR within skeletal muscle, the VDR rapidly translocates (1-10 minutes) from the nucleus to the cytoplasm upon exposure of cultured chick myoblasts to vitamin D [364]. Similar translocation to the plasma membrane was reported in C2C12s, with translocation dependent on intact microtubular transport and caveolae structure [401]. Binding of 1a,25(OH)2D3 with the VDR at the plasma membrane in turn activates c-SRC,

phosphoinositide-3-kinase (PI3K) and inositol triphosphate (IP3) which in turn leads to the release of Ca2+ _{from the sarcoplasmic reticulum [402, 403]. Furthermore, the}

actions of vitamin D signalling have been proposed to result in the translocation of PKCa from the cytosol to the cell membrane [404]. PKCa activates the L-type voltage dependent Ca2+ channel (VDCC) and Ca2+ store-operated entry (SOCE)

channel resulting in an increase in Ca2+ flux within the cell [405]. These translocation events likely govern 1a,25(OH)2D3 induced increases in intracellular calcium flux

within skeletal muscle cell lines [406-408] and within chick skeletal muscle [409] (Figure 1.4). Following longer periods of exposure, the VDR appears to return to the nucleus, possibly to carry out its genomic actions [398].

Figure 1.4. Genomic and non-genomic actions of vitamin D related signalling within skeletal muscle. 1a,25(OH)2D3 enters the cell prior to ligand binding to the VDR. Upon translocation to the nucleus, the VDR binds with RXRa forming a heterodimer protein complex that recruits co-regulatory binding partners and influences genomic transcription. 1a,25(OH)2D3 ligand binding also initiates transient signalling events mediated by the VDR that stimulate intracellular calcium uptake. Adapted from [410].