Vitamin D deficiency down-regulates Notch pathway contributing to skeletal muscle atrophy in old wistar rats

One of the most striking effects of ageing is an involuntary loss of muscle mass known as sarcopenia. The development of sarcopenia appears to be multifactorial and includes anabolic resistance to dietary amino acids, hormonal changes and sedentary lifestyle[1]. The diminished ability of aged muscle to self-repair is also a key factor behind sarcopenia[2‐4]. Muscle loss during ageing may partly depend on the accumulation of repeated episodes of incomplete repair and regeneration throughout the life span following overt injury but also daily small damages that may not be perceived via pain or alteration in function[5]. Muscle repair occurs in 4 interdependent phases: (1) degeneration; (2) inflammation; (3) regeneration, involving satellite cells (SC) that enter the cell cycle and differentiate to form newly multinucleated cells or to repair surviving fibers; (4) remodelling and repair[6]. This process is thus reliant on SC located underneath basal lamina of myofibers[7, 8]. SC pool size shrinks significantly with ageing[9]. Shefer et al.[10] showed that number of SC cells per freshly-isolated mice myofiber declines with age whereas SC differentiation potential remains unchanged[10]. However, the state of SC pool with ageing is controversial because some investigators have demonstrated that although no changes occur in the SC number with ageing, their physiological function, i.e. regenerative potential, was impaired[11‐13].

As recently revealed, the hypothesis of a decreased SC proliferative capacity with age can also be explained by an age-related decrease in Notch pathway activity[14]. Notch is a highly conserved transmembrane receptor whose pathway plays a central role in muscle development and regeneration[15‐17]. Binding of the Notch ligand, e.g. transmembrane protein Delta-1, promotes two proteolytic cleavage events[18]. First, an ADAM (A disintegrin and metalloprotease domain) protease cleaves Notch receptor to generate the transmembrane fragment Notch (^TMNotch)[19]. Second, a γ-secretase complex cleaves ^TMNotch[20], leading to the release of the intracellular domain of Notch receptor (^NicdNotch). ^NicdNotch then translocates to the nucleus where it acts as a transcription factor to promote the transcription of its target genes, such as Hes1 or Hey1[21], which are implicated in the blockade of cell differentiation and the maintenance of cell self-renewal[22‐25]. Mutant mice expressing the Notch inhibitor dnMAML1-gfp in muscle stem cells show smaller muscles and fewer SC[26]. The decline of Notch pathway activity with ageing may in part explain the reduced number of SC able to regenerate muscle cells[27]. Although changes occur in SC cells during aging, environmental factors still play a significant role in muscle regeneration[28].

Observational studies have shown that vitamin D status is positively correlated to muscle strength and function[29]. Vitamin D is derived from the action of ultraviolet (UV) light on skin and from diet[30, 31]. Once produced in skin or absorbed by the gut, vitamin D is carried in blood, mostly by vitamin D-binding protein, to the liver where it undergoes 25-hydroxylation to form calcifediol (25(OH) D), the major circulating metabolite of vitamin D[32]. Vitamin D input is largely reflected by blood 25(OH) D concentrations, and blood 25(OH) D is widely used as a measure of vitamin D status. 25(OH) D undergoes a last hydroxylation step by 1-α-hydroxylase enzyme (CYP27B1), expressed in kidney and many other tissues, to form the active hormone 1,25-dihydroxyvitamin D (1,25(OH)₂D or calcitriol)[33]. Vitamin D plays a role in numerous physiological processes through both genomic and non-genomic effects[33, 34]. Note that the various actions of vitamin D can be dependent on or independent of its binding on the nuclear receptor VDR (vitamin D receptor), as shown by previous studies in myotubes or osteoblasts[35, 36].

Skeletal muscle is a target of vitamin D[34], and several in vitro and in vivo studies have been led to analyze its effects on muscle. In vitro studies show that vitamin D modulates muscle cell proliferation and differentiation[35, 37, 38]. In vivo, vitamin D injections in adult rats promote cell proliferation and consequently the regenerative process in skeletal muscle after a crush injury[39]. Besides its key role in muscle cell proliferation and differentiation, vitamin D also regulates muscle contractile function[40, 41]. Whole together, these data demonstrate the necessity of vitamin D for the maintenance of structural integrity and function of skeletal muscle.

Vitamin D deficiency is common in elderly populations and has been associated with muscle weakness[42]. However, an in vivo study has highlighted that muscle weakness was rather a consequence of hypophosphatemia associated with hypovitaminosis D[43]. Furthermore, VDR expression in human muscle tissue decreases with age[44].

Bone morphogenetic protein 4 (Bmp4) and fibroblast growth factor-2 (Fgf-2) are two controllers known to be involved in the modulation of muscle cell proliferation and differentiation. Bmp4 regulates the transition from proliferation to differentiation[45], and Fgf-2 enhances the number of SC held in a proliferative state without suppressing the transition to the state of differentiation[46]. Interestingly, previous studies have shown that vitamin D modulates Bmp4 and FgF-2 expression[47, 48], and Notch pathway and Bmp4 interact to control proliferation/differentiation transition[49].

Prompted by these previous investigations, we addressed the hypothesis that vitamin D deficiency in old rats reduces the potential of skeletal muscle to regenerate by down-regulating the activity of the Notch signalling pathway, leading to muscle atrophy. After 9 months of vitamin D depletion, we studied the expression of markers implicated in the Notch pathway activity and in the modulation of proliferation in skeletal muscles in 24-month-old rats. We found that 9-month of vitamin D depletion induced a significant vitamin D deficiency in old rats and led to skeletal muscle atrophy, due at least in part to a reduced Notch pathway activity which controls muscle cell proliferation.

Sarcopenia is defined as the involuntary loss of muscle mass and strength with ageing[1]. The diminished ability of aged muscle to self-repair is a key driver of this process[4, 6, 28, 57], whether in situations of overt injury as in small daily damages not perceived via pain or altered contractile function[5]. The regulatory activity of the Notch pathway ―a key factor of muscle development and regeneration[17, 58]― also decreases with age and may contribute to muscle atrophy[12, 14]. Besides these endogenous regulatory pathways, environmental factors also play a key role in muscle regeneration[28]. Vitamin D could be central to maintained muscle mass due to its known effects on skeletal muscle[34]. Vitamin D status is positively correlated to muscle strength/function[34]. Older populations commonly develop vitamin D deficiency, causing muscle weakness[42]. Here, we tested the hypothesis that vitamin D deficiency contributes to the age-related muscle atrophy, due at least in part to a reduced Notch pathway activity. At the end the experimental period, blood 25(OH)D concentration had fallen by 75% in vitamin D-depleted rats. Despite similar food intake levels, average body weight increased in D-depleted rats compared to controls. Furthermore, while body fat mass increased in D-depleted rats, percent lean mass decreased. In accordance with this decreased fat free mass, D-depleted old rats showed a significant reduction in muscle mass, particularly in type II muscle mass. Interestingly, age-related muscle atrophy is characterized by a loss of muscle fibers, notably type II fibers[59, 60]. In this context, our study focused on TA, as it is mainly composed of type II fibers[61]. Our results on muscle mass are consistent with studies showing that vitamin D status is inversely correlated with body fat mass[62, 63] and that muscle is a direct target of vitamin D[34, 64]. Vitamin D potentiates protein synthesis in C2C12 following leucine and insulin treatment and regulates muscle contractile function[40, 41, 53]. Vitamin D is required for normal skeletal muscle development, and it promotes skeletal muscle regeneration following injury in adulthood[39, 65]. Finally, hypovitaminosis D develops with ageing and is linked to muscle weakness which increases the risks of falls[66]. All data from literature show that vitamin D is essential in regulating skeletal muscle structure and function. Interestingly, Schubert et al. have demonstrated that hypophosphatemia is responsible for skeletal muscle weakness in vitamin-D deficient rat[43], demonstrating that vitamin D effects on muscle could also be dependent of other factors. Vitamin D is known to regulate phosphocalcic homeostasis[67]. In our study, vitamin D depletion had no effect on serum phosphorus and calcium levels indicating that muscle atrophy was not a consequence of a phosphocalcic imbalance but was likely due to the vitamin D deficiency.

A logical effect of the reduction in body vitamin D status is that the expression of its receptor VDR was, as expected, down-regulated in the skeletal muscle of old D-depleted rats. Ceglia et al.[68] had previously shown that vitamin D₃ supplementation increases VDR expression and fiber size in skeletal muscle of elderly women[68]. Furthermore, vitamin D, and specifically its active form 1,25(OH)₂D, auto-regulates the expression of the VDR gene through intronic and upstream enhancers[53, 69]. Taken together, these results highlight that vitamin D depletion for 9 months provoked a vitamin D deficiency in old rats and subsequently generated morphological and molecular changes related to hypovitaminosis D.

In order to understand why muscle mass is reduced in D-depleted rats, we ran a PCR array to study the expression of genes related to autocrine signalling, apoptosis, metabolism, anabolism regulation, myogenesis, notch pathway, cell proliferation and cell structure and function. Except for the autocrine signalling, the expression of at least one gene from each listed clusters was down regulated in the vitamin D depleted group. These results highlighted that vitamin D deficiency displays a large variety of metabolic and functional changes in skeletal muscle. Thus, the effects of vitamin D depletion on these pathways need to be further investigated in skeletal muscle. In our study, we choose to focus on cell proliferation and Notch pathway. Previous studies have demonstrated that Notch signalling, muscle cell proliferation and vitamin D status are impaired in older people, and that vitamin D modulates muscle cell proliferation and stimulates regeneration. We found that the expressions of key genes related to the regulation of cell proliferation, particularly Bmp-4 and Fgf-2, were down-regulated with vitamin D depletion. The BMP proteins are known to be involved in myogenesis[70, 71]. In a murine C2C12 myoblast cell line, Terada et al.[45] found that Bmp-4 regulates myoblast proliferation[45]. On Fgf-2, studies using cultures of muscle cells or fibers in which SC were maintained in their in situ position, i.e. under the fiber basement membrane, have established that Fgf-2 enhances proliferative rate or the number of SC[46, 72]. Interestingly, previous studies have shown that 1,25-dihydroxyvitamin D modulates Bmp-4 and FgF-2 expression[47, 48].

As Bmp4 and Fgf-2 regulates muscle cell proliferation, we aimed to confirm that proliferation state where diminished in TA muscle of D-depleted rats. PCNA protein expression was decreased in vitamin D-depleted old rats compared to controls. PCNA reflects the proliferative activity, particularly in regenerating skeletal muscle[73‐75]. The reduction in the muscle proliferative capacity of depleted old rats is consistent with a previous study showing that vitamin D stimulates muscle cell proliferation in rat skeletal muscle[39]. However, in vitro studies have established that 1,25-dihydroxyvitamin D treatment of a C2C12 muscle cell line inhibited cell proliferation[38, 76], whereas others have concluded that this hormone stimulates muscle proliferation[35, 37]. These works demonstrate that i) in vitro, the ability of vitamin D to modulate cell proliferation depends on cell culture conditions, and that ii) in vivo models make it possible to account for intrinsic and extrinsic factors, both of which can influence cell proliferation[27, 77, 78].

Here, we have shown that hypovitaminosis D aggravates the muscle atrophy in old rats as the expressions of key markers modulating muscle cell proliferation are down-regulated. While no experimental injury was induced in our model, discrete episodes of repair and regeneration occur nonetheless due to small daily damages. SC intervene in such situations although their activation is not necessarily detectable[5]. Here we speculate that vitamin D deficiency could result in a poor recruitment of SC into their proliferating state, due at least in part to a down-regulation of Fgf2, making them ready for programmed differentiation, as Bmp-4 is down-regulated. To validate this hypothesis, a model of vitamin D-depleted old rats undergoing muscle acute injury will allow evaluating the effect of vitamin D on SC activation using specific markers as Pax7 and MyoD or Pax7 and PCNA.

The Notch pathway is involved in SC proliferation[14, 79], and PCR arrays showed that the expression of some of the key markers of this pathway were modulated following vitamin D depletion. The mRNA expression of Delta-1, a Notch activator, was lowered in the vitamin D-depleted group whereas the expression of its protein remained unchanged between the two groups. This discrepancy could be due to a post-translational regulation to maintain in-cell Delta levels, as this protein can undergo recycling after its action on the Notch pathway[80]. Here, Notch mRNA expression was unaffected by vitamin D depletion in old rats whereas the expression of the cleaved Notch form (^TMNotch) was reduced, reflecting a drop in activation of the Notch pathway. The down-regulation of ^TMNotch expression in vitamin D-depleted old rats was not the consequence of a change in the expression of Notch pathway activator Delta-1, as previously demonstrated by Conboy et al.[13, 27]. The modulation of ^TMNotch expression in depleted old rats was probably not associated to down-regulation of Notch receptor, as Notch transcript levels remained stable between vitamin D-depleted and control rats. However, Notch receptor, like Delta-1, can undergo recycling after their activation[81]. Therefore, the observed down-regulation of ^TMNotch expression in vitamin D-depleted old rats may be due to a decrease in the proteolytic processing of Notch receptor, involving an ADAM protease and/or the γ-secretase complex[18‐20]. Thus more investigations are needed to evaluate if ADAM or γ-secretase complex could be two targets of vitamin D, providing a new possible explanation of the down-regulation of ^TMNotch expression following vitamin D depletion. The reduced activity of the Notch pathway in vitamin D-depleted rats was further confirmed by the decreased Hes1 mRNA expression in this same D-depleted group. Once the pathway is activated, Notch receptor is cleaved and its intracellular domain acts as a transcription factor to induce Hes1 gene expression. Hence, any up-regulation of Hes1 expression is related to activation of the Notch pathway, and inversely, Hes1 mRNA down-regulation reflects the reduced activity of the Notch signalling pathway[21, 24, 80].

The choice of an animal model in which no overt injury was done provided to us the possibility for studying Notch pathway signalling in the context of repair and regeneration of daily small damages which contributes to age-related muscle atrophy[5, 21]. However, to fully evaluate the effect of vitamin D deficiency on muscle regeneration with aging, future investigations using old and young animal models of regeneration will allow us to investigate the impact of vitamin D on a high regeneration process. The present study has raised new hypotheses. First, since hypovitaminose D affects muscle mass, the severity of muscle damages following acute injury is likely to be increased in vitamin D depleted old rats than in non-depleted animals. Second, it is possible that the efficiency of muscle recovery after injury is slower in vitamin D depleted old rats than in control old or vitamin D-depleted young rats. Third, the rate of satellite cells recruitment in old animals with vitamin D depletion is likely altered. Future investigations should be done in order to answer these questions.

The present work has shown that vitamin D depletion for 9 months efficiently induced vitamin D deficiency in old rats and subsequently generated morphological and molecular changes related to hypovitaminosis D. Vitamin D depletion induces skeletal muscle atrophy in old rats through a reduction in the proliferative ability and in Notch pathway activity in skeletal muscle. The activation of Notch pathway activity is highly implicated in the mechanism of muscle regeneration, vitamin D deficiency could further aggravate the age-related impaired capacity of muscle to regenerate. These findings strongly suggest that vitamin D status needs to be controlled in elderly people to maintain muscle mass.