Reduced muscle function in mice lacking Vitamin D Receptors in muscles

Created November 2019

Mice with myocyte deletion of vitamin D receptor have sarcopenia and impaired muscle function

Journal of Cachexia, Sarcopenia and Muscle https://doi.org/10.1002/jcsm.12460

Christian M. Girgis Kuan Minn Cha Benjamin So Michael Tsang Jennifer Chen Peter J. Houweling Aaron Schindeler Rebecca Stokes …

Items in both categories Vitamin D Receptor and Sports (a proxie for muscle) are listed here: {category} --- The risk of 44 diseases at least double with poor Vitamin D Receptor as of Oct 2019 Omega-3, Magnesium, Zinc, non-daily Vitamin D dosing, curcumin, intense exercise, etc     Note: The founder of VitaminDWiki uses 10 of the 12 known VDR activators

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Background

It has long been recognized that vitamin D deficiency is associated with muscle weakness and falls. Vitamin D receptor (VDR) is present at very low levels in normal muscle. Whether vitamin D plays a direct role in muscle function is unknown and is a subject of hot debate. Myocyte‐specific deletion of VDR would provide a strategy to answer this question.

Methods

Myocyte‐specific vitamin D receptor (mVDR) null mice were generated by crossing human skeletal actin‐Cre mice with floxed VDR mice. The effects of gene deletion on the muscle phenotype were studied in terms of body tissue composition, muscle tissue histology, and gene expression by real‐time PCR.

Results

Unlike whole‐body VDR knockout mice, mVDR mice showed a normal body size. The mVDR showed a distinct muscle phenotype featuring

  • reduced proportional lean mass (70% vs. 78% of lean mass),

  • reduced voluntary wheel‐running distance (22% decrease, P = 0.009),

  • reduced average running speed, and reduced grip strength (7–16% reduction depending on age at testing).

With their decreased voluntary exercise, and decreased lean mass, mVDR have

  • increased proportional fat mass at 20% compared with 13%.

Surprisingly, their muscle fibres showed slightly increased diameter, as well as the presence of angular fibres and central nuclei suggesting ongoing remodelling. There were, however, no clear changes in fibre type and there was no increase in muscle fibrosis. VDR is a transcriptional regulator, and changes in the expression of candidate genes was examined in RNA extracted from skeletal muscle. Alterations were seen in myogenic gene expression, and there was decreased expression of cell cycle genes cyclin D1, D2, and D3 and cyclin‐dependent kinases Cdk‐2 and Cdk‐4. Expression of calcium handling genes sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCA) Serca2b and Serca3 was decreased and Calbindin mRNA was lower in mVDR muscle.

Conclusions

This study demonstrates that vitamin D signalling is needed for myocyte function. Despite the low level of VDR protein normally found muscle, deleting myocyte VDR had important effects on muscle size and strength. Maintenance of normal vitamin D signalling is a useful strategy to prevent loss of muscle function and size.

Conclusions from PDF

Loss of muscle mass and function, most commonly associated with age, is called sarcopenia. It results in greater risks of falls, fracture, disability, and death.25-28 Vitamin D deficiency is associated with sarcopenia and lower muscle mass in most studies in older people.29-36 However, a direct role in muscle has not been able to be shown in people.

The results presented in this study confirm that Vdr is expressed in myocytes in mice. We now demonstrate that myocyte VDR plays a significant role; deletion affects muscle function and gene expression. mVDR mice have significantly decreased grip strength, which persists to at least 4 months of age. It would be interesting to age these mice and determine whether they develop more severe changes with middle and older age . As well as decreased lean mass, mVDR mice had a significant increase in fat mass. There was a significant decrease in voluntary exercise in the mice, and a more than 20% decrease in voluntary exercise would be consistent with the increase in fat mass seen.

In people with colorectal cancer, physical activity measured by accelerometer correlated with serum vitamin D levels.37 This association was also seen in the NHANES study.38 While increased exercise is likely in many people to be associated with increased sun exposure, it is interesting to hypothesize that lower vitamin D may associate, as in mVDR mice, with lower voluntary exercise.

Interestingly, the effects of myocyte VDR deletion showed some important differences from those seen with whole- body VDR deletion.1 The reduction in size of individual muscles in mVDR mice is significant and of clinically relevant magnitude, but it appears to be less than the effect seen in whole-body VDR-null mice. This suggests that VDR in other cells types may play a role in muscle development in early life and in individual myofibre size and that these may partially negate the atrophic effects of VDR deletion in myocytes. As there was only a 50% decrease in Vdr mRNA in whole muscle, there may also be a contribution of incomplete deletion to the milder phenotype.

Murfl and Mafbx are ubiquitin ligases that regulate proteosomal degradation. In muscle, they are associated with myonuclear apoptosis and reduced fibre size.39-41 MAFbx additionally regulates protein synthesis and muscle regeneration via targeting of regulators including MyoD and eIF3F.41,42 Their expression is increased in whole-body VDR- null mice, in which there is decreased fibre size. There was decreased expression of Murfl in quadriceps and soleus, and significantly decreased expression of Mafbx in two muscles with a trend towards a decrease in the third (P = 0.07). Decreases in Murfl and Mafbx in mVDR mice would support better maintenance of fibre size with inhibition of the ubiquitin-proteosome pathway. Interestingly, these were also decreased in human myotubes treated with vitamin D,43 suggesting the possibility that in mVDR mice, the decrease could be mediated by effects of another cell type with intact vitamin D signalling.

Another potential contributor to the smaller decrease in muscle mass in mVDR mice is Myostatin, which is increased in whole-body VDRKO mice. Myostatin inhibits muscle hypertrophy and hyperplasia, so lower levels permit increased muscle growth.22 In contrast to whole-body VDR-null mice, myostatin was significantly decreased in soleus, and it is interesting to note that there was no decrease in weight of this muscle in mVDR mice.

Because the whole muscles are lighter, but individual fibres are not smaller, it follows that there must be fewer myofibres in mVDR mice , and this was confirmed in quadriceps by counting total fibres at the muscle midpoint for the whole muscle. The decreased expression of cyclin D genes 1-3 and decreased expression of Cdk2 and Cdk4 could also contribute to decreased myofibre number as they would decrease cell cycle progression. Decreased myofibre number and muscle mass would both contribute to decreased strength. Impaired muscle function was shown by two modalities: grip strength and running speed. These techniques test different muscles, and acute vs. more chronic effort; both were significantly affected.

As discussed earlier, calcium handling is important for normal muscle function44 and calcium handling in the whole body is regulated by vitamin D signalling.45,46 We examined expression of intracellular calcium handling genes and found significant changes in expression of Serca2b and 3 in quadriceps. These sarcoplasmic/endoplasmic reticulum calcium ATPase proteins are calcium pumps in the ER and muscle sarcoplasmic reticulum membrane that help to concentrate calcium in the lumen of the ER.47 This permits muscle relaxation after contraction. Calbindin acts both as a calcium buffer and in some tissues as a calcium sensor. Its buffering function would lower the available cytosolic calcium and also help with normal muscle relaxation. It is known to be regulated by vitamin D signalling in other tissues including kidney.48 It is therefore possible that reduced grip strength in these mice was the cause not only of reduced muscle mass but also alterations in contraction-relaxation via alterations in the calcium handling apparatus.

We therefore report significant phenotypic changes in muscle following tissue-specific deletion of VDR. VDR is present in muscle, deletion in myocytes impairs muscle function, muscle size is reduced, but myofibres are slightly larger. The larger myofibres contrast to the findings in whole-body knockout VDR mice. This suggests that as well as the role for myocyte VDR in muscle size and function, vitamin D signalling in other tissues or cell types plays a role in initial muscle development.

The results suggest that maintenance of normal vitamin D signalling is important for preservation of muscle bulk and function. These findings also suggest that therapies targeting VDR could impact skeletal muscle mass and function and may present a novel strategy in addressing or preventing age- related sarcopenia and other disorders of muscle function