Oxygen concentration modulates the differentiation of muscle stem cells toward myogenic and adipogenic fates

The physiological oxygen concentration of many tissues is far lower than that in which cells are typically cultured in vitro and this may inadvertently influence the proliferation and differentiation potential of many cell types. Muscle derived stem cells, known as satellite cells are responsible for the maintenance and repair of muscle tissue post-natally and in vivo would be exposed to oxygen concentrations of ～2-5%. Relatively few studies describe the function of these cells in large animal models and here we investigate the influence oxygen concentration has on modulating porcine muscle derived stem cell fate. We compared cells derived from two metabolically distinct muscles, the diaphragm and the hind limb semi-membranosus (SM) muscle. The two sub-populations responded differently to culture at atmospheric (～20%) and physiological (～5%) oxygen concentration. While myogenesis was enhanced in both populations at low oxygen, noticeably diaphragm derived cells exhibited greater myotube formation, than those from SM. The trans-differentiation of cells derived from these two sources was similarly affected, with considerable differences seen in adipogenic and neuronal tendencies. In addition to the effect of oxygen on cell phenotype, the expression of key signalling proteins varied between the two sub-populations during early time-points of induced differentiation, suggesting altered regulation of muscle specific stem cells under these conditions. While differences in muscle stem cell potential requires further investigation, the culture of cells in physiological oxygen concentration appears as fundamental to recreating the micro-environmental niche as routinely used factors such as cytokines, substrata and matrices.     

Characterization of myosin isoforms in satellite cell cultures from adult rat diaphragm, soleus and tibialis anterior muscles

Satellite cells were isolated by enzymatic dissociation and Percoll gradient centrifugation from adult rat diaphragm, soleus, and tibialis anterior muscles with fairly reproducible yields. Diaphragm and soleus muscle yielded approximately five times more satellite cells than tibialis anterior muscle. According to light microscopic criteria, no morphological differences existed between the satellite cell cultures of different origin. Contrary to the donor muscles, myotubes from the 10-day-cultured satellite cells contained a uniform myosin heavy chain (MHC) pattern with predominance of an immunochemically identified embryonic heavy chain. The three types of cultures displayed a typical embryonic light chain (LC) pattern with LC1emb, LC1f, LC2f, and traces of LC3f. The isomyosin pattern was characterized by four embryonic isomyosins, eM1-eM4, with similar distributions in the three cultures. In summary, these myosin analyses provide no evidence for the existence of satellite cell diversity among three rat muscles of different fiber-type composition, at least not under the applied in vitro conditions.     

Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin

Extraocular muscles (EOM) represent a unique muscle group that controls eye movements and originates from head mesoderm, while the more typically studied body and limb muscles are somite-derived. Aiming to investigate myogenic progenitors (satellite cells) in EOM versus limb and diaphragm of adult mice, we have been using flow cytometry in combination with myogenic-specific Cre-loxP lineage marking for cell isolation. While analyzing cells from the EOM of mice that harbor Myf5^Cre-driven GFP expression, we identified in addition to the expected GFP⁺ myogenic cells (presumably satellite cells), a second dominant GFP⁺ population distinguished as being Sca1⁺, non-myogenic, and exhibiting a fibro/adipogenic potential. This unexpected population was not only unique to EOM compared to the other muscles but also specific to the Myf5^Cre-driven reporter when compared to the MyoD^Cre driver. Histological studies of periocular tissue preparations demonstrated the presence of Myf5^Cre-driven GFP⁺ cells in connective tissue locations adjacent to the muscle masses, including cells in the vasculature wall. These vasculature-associated GFP⁺ cells were further identified as mural cells based on the presence of the specific XLacZ4 transgene. Unlike the EOM satellite cells that originate from a Pax3-negative lineage, these non-myogenic Myf5^Cre-driven GFP⁺ cells appear to be related to cells of a Pax3-expressing origin, presumably derived from the neural crest. In all, our lineage tracing based on multiple reporter lines has demonstrated that regardless of common ancestral expression of Myf5, there is a clear distinction between periocular myogenic and non-myogenic cell lineages according to their mutually exclusive antecedence of MyoD and Pax3 gene activity.     

Differential myogenicity of satellite cells isolated from extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro

Following muscle damage, fast- and slow-contracting fibers regenerate, owing to the activation of their satellite cells. In rats, crush-induced regeneration of extensor digitorum longus (EDL, a fast muscle) and soleus (a slow muscle) present different characteristics, suggesting that intrinsic differences exist among their satellite cells. An in vitro comparative study of the proliferation and differentiation capacities of satellite cells isolated from these muscles is presented there. We observed several differences between soleus and EDL satellite cell cultures plated at high density on gelatin-coated dishes. Soleus satellite cells proliferated more actively and fused into myotubes less efficiently than EDL cells. The rate of muscular creatine kinase enzyme appeared slightly lower in soleus than in EDL cultures at day 11 after plating, when many myotubes were formed, although the levels of muscular creatine kinase mRNA were similar in both cultures. In addition, soleus cultures expressed higher levels of MyoD and myogenin mRNA and of MyoD protein than EDL satellite cell cultures at day 12. A clonal analysis was also carried out on both cell populations in order to determine if distinct lineage features could be detected among satellite cells derived from EDL and soleus muscles. When plated on gelatin at clonal density, cells from both muscles yielded clones within 2 weeks, which stemmed from 3–15 mitotic cycles and were classified into three classes according to their sizes. Myotubes resulting from spontaneous fusion of cells from the progeny of one single cell were seen regardless of the clone size in the standard culture medium we used. The proportion of clones showing myotubes in each class depended on the muscle origin of the cells and was greater in EDL- than in soleus-cell cultures. In addition, soleus cells were shown to improve their differentiation capacity upon changes in the culture condition. Indeed, the proportions of clones showing myotubes, or of cells fusing into myotubes in clones, were increased by treatments with a myotube-conditioned medium, with phorbol ester, and by growth on extra-cellular matrix components (Matrigel). These results, showing differences among satellite cells from fast and slow muscles, might be of importance to muscle repair after trauma and in pathological situations.     

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.     

Angiotensin II infusion induces marked diaphragmatic skeletal muscle atrophy

Advanced congestive heart failure (CHF) and chronic kidney disease (CKD) are characterized by increased angiotensin II (Ang II) levels and are often accompanied by significant skeletal muscle wasting that negatively impacts mortality and morbidity. Both CHF and CKD patients have respiratory muscle dysfunction, however the potential effects of Ang II on respiratory muscles are unknown. We investigated the effects of Ang II on diaphragm muscle in FVB mice. Ang II induced significant diaphragm muscle wasting (18.7±1.6% decrease in weight at one week) and reduction in fiber cross-sectional area. Expression of the E3 ubiquitin ligases atrogin-1 and muscle ring finger-1 (MuRF-1) and of the pro-apoptotic factor BAX was increased after 24 h of Ang II infusion (4.4±0.3 fold, 3.1±0.5 fold and 1.6±0.2 fold, respectively, compared to sham infused control) suggesting increased muscle protein degradation and apoptosis. In Ang II infused animals, there was significant regeneration of injured diaphragm muscles at 7 days as indicated by an increase in the number of myofibers with centralized nuclei and high expression of embryonic myosin heavy chain (E-MyHC, 11.2±3.3 fold increase) and of the satellite cell marker M-cadherin (59.2±22.2% increase). Furthermore, there was an increase in expression of insulin-like growth factor-1 (IGF-1, 1.8±0.3 fold increase) in Ang II infused diaphragm, suggesting the involvement of IGF-1 in diaphragm muscle regeneration. Bone-marrow transplantation experiments indicated that although there was recruitment of bone-marrow derived cells to the injured diaphragm in Ang II infused mice (267.0±74.6% increase), those cells did not express markers of muscle stem cells or regenerating myofibers. In conclusion, Ang II causes marked diaphragm muscle wasting, which may be important for the pathophysiology of respiratory muscle dysfunction and cachexia in conditions such as CHF and CKD.     

Muscle satellite cells are a functionally heterogeneous population in both somite-derived and branchiomeric muscles

Skeletal muscles of body and limb are derived from somites, but most head muscles originate from cranial mesoderm. The resident stem cells of muscle are satellite cells, which have the same embryonic origin as the muscle in which they reside. Here, we analysed satellite cells with a different ontology, comparing those of the extensor digitorum longus (EDL) of the limb with satellite cells from the masseter of the head. Satellite cell-derived myoblasts from MAS and EDL muscles had distinct gene expression profiles and masseter cells usually proliferated more and differentiated later than those from EDL. When transplanted, however, masseter-derived satellite cells regenerated limb muscles as efficiently as those from EDL. Clonal analysis showed that functional properties differed markedly between satellite cells: ranging from clones that proliferated extensively and gave rise to both differentiated and self-renewed progeny, to others that divided minimally before differentiating completely. Generally, masseter-derived clones were larger and took longer to differentiate than those from EDL. This distribution in cell properties was preserved in both EDL-derived and masseter-derived satellite cells from old mice, although clones were generally less proliferative. Satellite cells, therefore, are a functionally heterogeneous population, with many occupants of the niche exhibiting stem cell characteristics in both somite-derived and branchiomeric muscles.     

Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD

Satellite cells are tissue-specific stem cells responsible for skeletal muscle growth and regeneration. Although satellite cells were identified almost 50 years ago, the identity of progenitor populations from which they derive remains controversial. We developed MyoD^iCre knockin mice, and used Cre/lox lineage analysis to determine whether satellite cell progenitors express MyoD, a marker of myogenic commitment. Recombination status of satellite cells was determined by confocal microscopy of isolated muscle fibers and by electron microscopic observation of muscle tissue fixed immediately following isolation, using R26R-EYFP and R26R (β-gal) reporter mice, respectively. We show that essentially all adult satellite cells associated with limb and body wall musculature, as well as the diaphragm and extraocular muscles, originate from MyoD+ progenitors. Neonatal satellite cells were Cre-recombined, but only a small minority exhibited ongoing Cre expression, indicating that most satellite cells had expressed MyoD prenatally. We also show that satellite cell development in MyoD-null mice is not due to functional compensation by MyoD non-expressing lineages. The results suggest that satellite cells are derived from committed myogenic progenitors, irrespective of the anatomical location, embryological origin, or physiological properties of associated musculature.     

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Inflammatory cells and apoptosis in respiratory and limb muscles of patients with COPD

Discrepancies exist regarding the involvement of cellular inflammation and apoptosis in the muscle dysfunction of chronic obstructive pulmonary disease (COPD) patients with preserved body composition. We explored whether levels of inflammatory cells and apoptosis were increased in both respiratory and limb muscles of COPD patients without nutritional abnormalities. In the vastus lateralis, external intercostals, and diaphragms of severe and moderate COPD patients with normal body composition, and in healthy subjects, intramuscular leukocytes and macrophage levels were determined (immunohistochemistry). Muscle structure was also evaluated. In the diaphragm and vastus lateralis of severe and moderate COPD patients and controls, apoptotic nuclei were explored using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, electron microscopy, and caspase-3 expression. In COPD patients compared with controls, diaphragm and intercostal levels of inflammatory cells were extremely low and not significantly different. However, in the vastus lateralis of the severe patients, inflammatory cell counts, although also very low, were significantly greater. In those patients, TUNEL-positive nuclei levels were also significantly greater in diaphragms and vastus lateralis. A significant inverse relationship was found between quadriceps TUNEL-positive nuclei levels and muscle force. Ultrastructural apoptotic nuclei revealed no differences in respiratory or limb muscles between COPD patients and controls. Muscle caspase-3 expression did not differ between patients and controls. In severe COPD patients with preserved body composition, while increased apoptotic nuclei seems to be a contributor to their muscle dysfunction, cellular inflammation does not. The increased numbers of TUNEL-positive nuclei in their muscles suggest that they may also be exposed to a continuous repair/remodeling process.     

Satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice

To determine whether muscle satellite cells and utrophin are correlated with the degree of damage in mdx skeletal muscles, we measured the area of the degenerative region as an indicator of myofiber degeneration in the masseter, gastrocnemius, soleus, and diaphragm muscles of mdx mice. Furthermore, we analyzed the expression levels of the paired box homeotic gene 7 (pax7), m-cadherin (the makers of muscle satellite cells), and utrophin mRNA. We also investigated the immunolocalization of m-cadherin and utrophin proteins in the muscles of normal C57BL/10J (B10) and mdx mice. The expression level of pax7 mRNA and the percentage of m-cadherin-positive cells among the total number of cell nuclei in the muscle tissues in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there was no significant correlation between muscle damage and expression level for pax7 mRNA (R = –0.140), nor was there a correlation between muscle damage and the percentage of satellite cells among the total number of cell nuclei (R = –0.411) in the mdx mice. The expression level of utrophin mRNA and the intensity of immunostaining for utrophin in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there also was not a significant correlation between muscle damage and expression level of utrophin mRNA (R = 0.231) in the mdx mice, although upregulated utrophin was incorporated into the sarcolemma. These results suggest that satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice. dystrophy; pax7; m-cadherin; dystrophin-related proteins     

Autophagy-Associated Atrophy and Metabolic Remodeling of the Mouse Diaphragm after Short-Term Intermittent Hypoxia

Background

Short-term intermittent hypoxia (IH) is common in patients with acute respiratory disorders. Although prolonged exposure to hypoxia induces atrophy and increased fatigability of skeletal muscle, the response to short-term IH is less well known. We hypothesized that the diaphragm and limb muscles would adapt differently to short-term IH given that hypoxia stimulates ventilation and triggers a superimposed exercise stimulus in the diaphragm.

Methods

We determined the structural, metabolic, and contractile properties of the mouse diaphragm after 4 days of IH (8 hours per day, 30 episodes per hour to a FiO2 nadir=6%), and compared responses in the diaphragm to a commonly studied reference limb muscle, the tibialis anterior. Outcome measures included muscle fiber size, assays of muscle proteolysis (calpain, ubiquitin-proteasome, and autophagy pathways), markers of oxidative stress and mitochondrial function, quantification of intramyocellular lipid and lipid metabolism genes, type I myosin heavy chain (MyHC) expression, and in vitro contractile properties.

Results

After 4 days of IH, the diaphragm alone demonstrated significant atrophy (30% decrease of myofiber size) together with increased LC3B-II protein (2.4-fold) and mRNA markers of the autophagy pathway (LC3B, Gabarapl1, Bnip3), whereas active calpain and E3 ubiquitin ligases (MuRF1, atrogin-1) were unaffected in both muscles. Succinate dehydrogenase activity was significantly reduced by IH in both muscles. However, only the diaphragm exhibited increased intramyocellular lipid droplets (2.5-fold) after IH, along with upregulation of genes linked to activated lipid metabolism. In addition, although the diaphragm showed evidence for acute fatigue immediately following IH, it underwent an adaptive fiber type switch toward slow type I MyHC-expressing fibers, associated with greater intrinsic endurance of the muscle during repetitive stimulation in vitro.

Conclusions

Short-term IH induces preferential atrophy in the mouse diaphragm together with increased autophagy and a rapid compensatory metabolic adaptation associated with enhanced fatigue resistance.     

Regenerative capacity of the dystrophic (mdx) diaphragm after induced injury

Duchenne muscular dystrophy is characterized by myofiber necrosis, muscle replacement by connective tissue, and crippling weakness. Although the mdx mouse also lacks dystrophin, most muscles show little myofiber loss or functional impairment. An exception is the mdx diaphragm, which is phenotypically similar to the human disease. Here we tested the hypothesis that the mdx diaphragm has a defective regenerative response to necrotic injury, which could account for its severe phenotype. Massive necrosis was induced in mdx and wild-type (C57BL10) mouse diaphragms in vivo by topical application of notexin, which destroys mature myofibers while leaving myogenic precursor satellite cells intact. At 4 h after acute exposure to notexin, >90% of diaphragm myofibers in both wild-type and mdx mice demonstrated pathological sarcolemmal leakiness, and there was a complete loss of isometric force-generating capacity. Both groups of mice showed strong expression of embryonic myosin within the diaphragm at 5 days, which was largely extinguished by 20 days after injury. At 60 days postinjury, wild-type diaphragms exhibited a persistent loss ( approximately 25%) of isometric force-generating capacity, associated with a trend toward increased connective tissue infiltration. In contrast, mdx diaphragms achieved complete functional recovery of force generation to noninjured values, and there was no increase in muscle connective tissue over baseline. These data argue against any loss of intrinsic regenerative capacity within the mdx diaphragm, despite characteristic features of major dystrophic pathology being present. Our findings support the concept that significant latent regenerative capacity resides within dystrophic muscles, which could potentially be exploited for therapeutic purposes.     

Androgen receptor expression in satellite cells of the neonatal levator ani of the rat

Androgens are thought to mediate sexual differentiation of spinal nucleus of the bulbocavernosus (SNB) motoneurons via actions on androgen receptors (ARs) within their target muscles bulbocavernosus and levator ani (LA). However, the cells within these muscles which mediate masculinization of the SNB remain undefined. Until recently, myocytes were thought to be the most likely candidate cell type. However, genetic tests of AR function in myocytes have failed to support a sufficient role for these cells in producing masculine SNB morphology, suggesting the involvement of other cell types. To identify other candidate cell types in the LA, we evaluated whether satellite cells or fibroblasts express AR. Fluorescent immunohistochemistry and confocal microscopy were used to evaluate whether satellite cells and fibroblasts express AR in neonatal male and female rats in the LA and an adjacent sexually monomorphic control muscle (CM). We found that a small proportion of satellite cells in the LA express AR and that this proportion is significantly greater in the LA compared to the CM. No sex differences were found between the proportions of satellite cells expressing AR in either muscle. Less colocalization of satellite cells and AR was seen in postnatal day 3 muscle than in postnatal day 1 muscle. In contrast, only negligible amounts of fibroblasts labeled with S100A4 express AR in either the LA or the CM. Together, findings support satellite cells, but not fibroblasts, as a candidate cell type involved in the sexual differentiation of the SNB neuromuscular system. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 448–454, 2013.     

Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche

Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.