Lack of vitamin D signalling shifts skeletal muscles towards oxidative metabolism.

BACKGROUND: Mice lacking vitamin D receptor (VDR) exhibit a glycogen storage disorder, disrupting carbohydrate utilization in muscle. Here, we asked if the defective carbohydrate metabolism alters the fat utilization by the skeletal muscles of vdr-/- mice. METHODS: To check the effect of high-fat-containing diets on muscle mass and metabolism of vdr-/- mice, we subjected them to two different milk fat-based diets (milk fat diet with 60% of energy from milk fat and milk-based diet [MBD] with 37% of energy from milk fat) and lard-based high-fat diet (HFD) containing 60% of energy from lard fat. Skeletal muscles and pancreas from these mice were analysed using RNA sequencing, quantitative reverse transcription polymerase chain reaction and western blot to understand the changes in signalling and metabolic pathways. Microscopic analyses of cryosections stained with haematoxylin and eosin, BODIPY, succinate dehydrogenase and periodic acid-Schiff reagent were performed to understand changes in morphology and metabolism of muscle fibres and pancreatic islets. RESULTS: Transcriptomic analyses showed that the skeletal muscles of vdr-/- mice exhibit upregulation of the fatty acid oxidation pathways, suggesting a shift towards increased lipid utilization even in a carbohydrate-enriched regular chow diet (chow). Two different milk fat-enriched diets restored body weight (12.01 ± 0.33 g in chow vs. 17.99 ± 0.62 g in MBD) and muscle weights (38.58 ± 3.84 mg in chow vs. 110.72 ± 1.96 mg in MBD for gastrocnemius [GAS]) of vdr-/- mice. Muscle ATP levels (0.56 ± 0.18 µmol in chow vs. 1.48 ± 0.08 µmol in MBD) and protein synthesis (0.25 ± 0.04 A.U. in chow vs. 2.02 ± 0.06 A.U. in MBD) were upregulated by MBD. However, despite increasing muscle energy levels, HFD failed to restore the muscle mass and cross-sectional area to that of wild-type (WT) mice (104.95 ± 2.6 mg for WT mice on chow vs. 77.26 ± 1.7 mg for vdr-/- mice on HFD for GAS). Moreover, HFD disrupted glucose homeostasis in vdr-/- mice, while MBD restored it. We further analysed insulin response and pancreatic insulin levels of these mice to show that HFD led to reduced insulin levels in pancreatic beta cells of vdr-/- mice (mean intensity of 1.5 × 10-8 for WT mice on chow vs. 4.3 × 10-9 for vdr-/- mice on HFD). At the same time, MBD restored glucose-stimulated pancreatic insulin response (mean intensity of 9.2 × 10-9 ). CONCLUSIONS: Skeletal muscles of vdr-/- mice are predisposed to utilize fatty acids as their primary energy source to circumvent their defective carbohydrate utilization. Thus, HFDs could restore energy levels in the skeletal muscles of vdr-/- mice. This study reveals that when mice are subjected to a lard-based HFD, VDR signalling is essential for maintaining insulin levels in pancreatic islets. Our data show a critical role of VDR in muscle metabolic flexibility and pancreatic insulin response.

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage.

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the reliability and robustness of the source. We describe here a stepwise protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue (uMSCs), their immunophenotyping, and the propagation of such cultures over several passages. In this procedure, the viability of the uMSCs is high because there is no enzymatic digestion. Further, the removal of blood vessels, including the umbilical cord arteries and the vein, ensures that there is no contamination of cells of endothelial origin. Using flow cytometry, uMSCs upon isolation are CD45-CD34-, indicating an absence of cells from the hematopoietic lineage. Importantly, they express key surface markers, CD105, CD90, and CD73. Upon establishment of cultures, this paper describes an efficient method to induce differentiation in these uMSCs into the skeletal muscle lineage. A detailed analysis of myogenic progression in differentiated uMSCs reveals that uMSCs express Pax7, a marker for myogenic progenitors in the initial stages of differentiation, followed by the expression of MyoD and Myf5, and, finally, a terminal differentiation marker, myosin heavy chain (MyHC).

Systemic ablation of vitamin D receptor leads to skeletal muscle glycogen storage disorder in mice.

BACKGROUND: Vitamin D deficiency leads to pathologies of multiple organ systems including skeletal muscle. Patients with severe vitamin D deficiency exhibit muscle weakness and are susceptible to frequent falls. Mice lacking a functional vitamin D receptor (VDR) develop severe skeletal muscle atrophy immediately after weaning. But the root cause of myopathies when vitamin D signalling is impaired is unknown. Because vitamin D deficiency leads to metabolic changes as well, we hypothesized that the skeletal muscle atrophy in mice lacking VDR may have a metabolic origin. METHODS: We analysed wild-type (WT) mice as well as vitamin D receptor null (vdr-/-) mice for skeletal muscle proteostasis, energy metabolism, systemic glucose homeostasis, and muscle glycogen levels. Dysregulation of signalling pathways as well as the glycogen synthesis and utilization machinery were also analysed using western blots. qRT-PCR assays were performed to understand changes in mRNA levels. RESULTS: Skeletal muscles of vdr-/- exhibited higher expression levels of muscle-specific E3 ubiquitin ligases and showed increased protein ubiquitination, suggesting up-regulation of protein degradation. Foxo1 transcription factor was activated in vdr-/- while Foxo3 factor was unaffected. Fasting protein synthesis as well as mTORC1 pathways were severely down-regulated in vdr-/- mice. Skeletal muscle ATP levels were low in vdr-/- (0.58 ± 0.18 µmol/mL vs. 1.6 ± 0.0.14 µmol/mL, P = 0.006), leading to increased AMPK activity. Muscle energy deprivation was not caused by decreased mitochondrial activity as we found the respiratory complex II activity in vdr-/- muscles to be higher compared with WT (0.29 ± 0.007 mU/µL vs. 0.16 ± 0.005 mU/µL). vdr-/- mice had lower fasting blood glucose levels (95 ± 14.5 mg/dL vs. 148.6 ± 6.1 mg/dL, P = 0.0017) while they exhibited hyperlactataemia (7.42 ± 0.31 nmol/µL vs. 4.95 ± 0.44 nmol/µL, P = 0.0032), suggesting systemic energy deficiency in these mice. Insulin levels in these mice were significantly lower in response to intraperitoneal glucose injection (0.69 ± 0.08 pg/mL vs. 1.11 ± 0.09 pg/mL, P = 0.024). Skeletal muscles of these mice exhibit glycogen storage disorder characterized by increased glycogen accumulation. The glycogen storage disorder in vdr-/- muscles is driven by increased glycogen synthase activity and decreased glycogen phosphorylase activity. Increased glycogenin expression supports higher levels of glycogen synthesis in these muscles. CONCLUSIONS: The results presented show that lack of vitamin D signalling leads to a glycogen storage defect in the skeletal muscles, which leads to muscle energy deprivation. The inability of vdr-/- skeletal muscles to use glycogen leads to systemic defects in glucose homeostasis, which in turn leads to proteostasis defects in skeletal muscles and atrophy.

Umbilical cord tissue is a robust source for mesenchymal stem cells with enhanced myogenic differentiation potential compared to cord blood.

Differentiation of mesenchymal stem cells (MSCs) derived from two different sources of fetal tissues such as umbilical cord blood (UCB) and tissue (UCT) into skeletal muscle have remained underexplored. Here, we present a comparative analysis of UCB and UCT MSCs, in terms of surface markers, proliferation and senescence marker expression. We find that CD45-CD34- MSCs obtained from UCT and UCB of term births display differences in the combinatorial expression of key MSC markers CD105 and CD90. Importantly, UCT MSCs display greater yield, higher purity, shorter culture time, and lower rates of senescence in culture compared to UCB MSCs. Using a robust myogenic differentiation protocol, we show that UCT MSCs differentiate more robustly into muscle than UCB MSCs by transcriptomic sequencing and specific myogenic markers. Functional assays reveal that CD90, and not CD105 expression promotes myogenic differentiation in MSCs and could explain the enhanced myogenic potential of UCT MSCs. These results suggest that in comparison to large volumes of UCB that are routinely used to obtain MSCs and with limited success, UCT is a more reliable, robust, and convenient source of MSCs to derive cells of the myogenic lineage for both therapeutic purposes and increasing our understanding of developmental processes.

Inhibition of Stat3 signaling ameliorates atrophy of the soleus muscles in mice lacking the vitamin D receptor.

BACKGROUND: Although skeletal muscle wasting has long been observed as a clinical outcome of impaired vitamin D signaling, precise molecular mechanisms that mediate the loss of muscle mass in the absence of vitamin D signaling are less clear. To determine the molecular consequences of vitamin D signaling, we analyzed the role of signal transducer and activator of transcription 3 (Stat3) signaling, a known contributor to various muscle wasting pathologies, in skeletal muscles. METHODS: We isolated soleus (slow) and tibialis anterior (fast) muscles from mice lacking the vitamin D receptor (VDR-/-) and used western blot analysis, quantitative RTPCR, and pharmacological intervention to analyze muscle atrophy in VDR-/- mice. RESULTS: We found that slow and fast subsets of muscles of the VDR-/- mice displayed elevated levels of phosphorylated Stat3 accompanied by an increase in Myostatin expression and signaling. Consequently, we observed reduced activity of mammalian target of rapamycin (mTOR) signaling components, ribosomal S6 kinase (p70S6K) and ribosomal S6 protein (rpS6), that regulate protein synthesis and cell size, respectively. Concomitantly, we observed an increase in atrophy regulators and a block in autophagic gene expression. An examination of the upstream regulation of Stat3 levels in VDR-/- muscles revealed an increase in IL-6 protein expression in the soleus, but not in the tibialis anterior muscles. To investigate the involvement of satellite cells (SCs) in atrophy in VDR-/- mice, we found that there was no significant deficit in SC numbers in VDR-/- muscles compared to the wild type. Unlike its expression within VDR-/- fibers, Myostatin levels in VDR-/- SCs from bulk muscles were similar to those of wild type. However, VDR-/- SCs induced to differentiate in culture displayed increased p-Stat3 signaling and Myostatin expression. Finally, VDR-/- mice injected with a Stat3 inhibitor displayed reduced Myostatin expression and function and restored active p70S6K and rpS6 levels, resulting in an amelioration of loss of muscle mass in the soleus muscles. CONCLUSIONS: The loss of muscle mass in slow muscles in the absence of vitamin D signaling is due to elevated levels of phosphorylated Stat3 that leads to an increase in Myostatin signaling, which in turn decreases protein synthesis and fiber size through the phosphorylation of p70S6K and rpS6, respectively.

The quasi-parallel lives of satellite cells and atrophying muscle.

Skeletal muscle atrophy or wasting accompanies various chronic illnesses and the aging process, thereby reducing muscle function. One of the most important components contributing to effective muscle repair in postnatal organisms, the satellite cells (SCs), have recently become the focus of several studies examining factors participating in the atrophic process. We critically examine here the experimental evidence linking SC function with muscle loss in connection with various diseases as well as aging, and in the subsequent recovery process. Several recent reports have investigated the changes in SCs in terms of their differentiation and proliferative capacity in response to various atrophic stimuli. In this regard, we review the molecular changes within SCs that contribute to their dysfunctional status in atrophy, with the intention of shedding light on novel potential pharmacological targets to counteract the loss of muscle mass.

A Wnt-TGFß2 axis induces a fibrogenic program in muscle stem cells from dystrophic mice.

We have previously observed that Wnt signaling activates a fibrogenic program in adult muscle stem cells, called satellite cells, during aging. We genetically labeled satellite cells in a mouse model of Duchenne muscular dystrophy to follow their fate during the progression of the disease. We observed that a fraction of satellite cells had a reduced myogenic potential and showed enhanced expression of profibrotic genes compared to age-matched controls. By combining in vitro and in vivo results, we found that expression of transforming growth factor-ß2 (TGFß2) was induced in response to elevated canonical Wnt signaling in dystrophic muscles and that the resulting increase in TGFß activity affected the behavior of satellite cells in an autocrine or paracrine fashion. Indeed, pharmacological inhibition of the TGFß pathway in vivo reduced the fibrogenic characteristics of satellite cells. These studies shed new light on the cellular and molecular mechanisms responsible for stem cell dysfunction in dystrophic muscle and may contribute to the development of more effective and specific therapeutic approaches for the prevention of muscle fibrosis.

FOXO3 promotes quiescence in adult muscle stem cells during the process of self-renewal.

Skeletal muscle stem cells, or "satellite cells" (SCs), are required for the regeneration of damaged muscle tissue. Although SCs self-renew during regeneration, the mechanisms that govern SC re-entry into quiescence remain elusive. We show that FOXO3, a member of the forkhead family of transcription factors, is expressed in quiescent SCs (QSCs). Conditional deletion of Foxo3 in QSCs impairs self-renewal and increases the propensity of SCs to adopt a differentiated fate. Transcriptional analysis of SCs lacking FOXO3 revealed a downregulation of Notch signaling, a key regulator of SC quiescence. Conversely, overexpression of Notch intracellular domain (NICD) rescued the self-renewal deficit of FOXO3-deficient SCs. We show that FOXO3 regulates NOTCH1 and NOTCH3 receptor expression and that decreasing expression of NOTCH1 and NOTCH3 receptors phenocopies the effect of FOXO3 deficiency in SCs. We demonstrate that FOXO3, perhaps by activating Notch signaling, promotes the quiescent state during SC self-renewal in adult muscle regeneration.

Stem cell review series: aging of the skeletal muscle stem cell niche.

Declining stem cell function during aging contributes to impaired tissue function. Muscle-specific stem cells ('satellite cells') are responsible for generating new muscle in response to injury in the adult. However, aged muscle displays a significant reduction in regenerative abilities and an increased susceptibility to age-related pathologies. This review describes components of the satellite cell niche and addresses how age-related changes in these components impinge on satellite cell function. In particular, we review changes in the key niche elements, the myofiber and the basal lamina that are in intimate contact with satellite cells. We address how these elements are influenced by factors secreted by interstitial cells, cells of the immune system, and cells associated with the vasculature, all of which change with age. In addition, we consider more distant sources of influence on the satellite cell niche that change with age, such as neural-mediated trophic factors and electrical activity and systemic factors present in the circulation. A better understanding of the niche elements and their influence on the satellite cell will facilitate the development of therapeutic interventions aimed at improving satellite cell activity and ultimately tissue response to injury in aged individuals.