Influence of sex and fiber type on the satellite cell pool in human skeletal muscle.

The repair, remodeling, and regeneration of myofibers are dependent on satellite cells (SCs), although, the distribution of SCs in different fiber types of human muscle remains inconclusive. There is also a paucity of research comparing muscle fiber characteristics in a sex-specific manner. Therefore, the aim of this study was to investigate fiber type-specific SC content in men and women. Muscle biopsies from vastus lateralis were collected from 64 young (mean age 27 ± 5), moderately trained men (n = 34) and women (n = 30). SCs were identified by Pax7-staining together with immunofluorescent analyses of fiber type composition, fiber size, and myonuclei content. In a mixed population, comparable number of SCs was associated to type I and type II fibers (0.07 ± 0.02 vs 0.07 ± 0.02 SCs per fiber, respectively). However, unlike men, women displayed a fiber type-specific distribution, with SC content being lower in type II than type I fibers (P = .041). Sex-based differences were found specifically for type II fibers, where women displayed lower SC content compared to men (P < .001). In addition, positive correlations (r-values between 0.36-0.56) were found between SC content and type I and type II fiber size in men (P = .03 and P < .01, respectively), whereas similar relationships could not be detected in women. Sex-based differences were also noted for fiber type composition and fiber size, but not for myonuclei content. We hereby provide evidence for sex-based differences present at the myocellular level, which may have important implications when studying exercise- and training-induced myogenic responses in skeletal muscle.

Effects of Simulated Microgravity on Muscle Stem Cells Activity.

BACKGROUND/AIMS: The study of the effects of simulated microgravity on primary cultures of human satellite cells represents a reliable model for identifying the biomolecular processes involved in mechanic load-related muscle mass loss. Therefore, this study aims to investigate the role of myostatin and Bone Morphogenetic Protein-2 in human satellite cells response to simulated microgravity condition. METHODS: In order to identify the main molecules involved in the phenomena of degeneration/regeneration of muscle tissue related to the alteration of mechanic load, we performed a morphological and immunohistochemical study on 27 muscle biopsies taken from control, osteoporotic and osteoarthritic patients, underwent hip arthroplasty. For each patient, we set up primary satellite cell cultures subjected to normogravity and simulated microgravity (110h) regimens. Cellular functionality has been studied through a morphological evaluation performed by optical microscopy, and an ultrastructural evaluation carried out by transmission electron microscopy. Furthermore, we evaluated the expression of Bone Morphogenetic Protein-2 and myostatin through immunocytochemical reactions. RESULTS: Our results showed that in the very early phases of simulated microgravity condition the satellite cells are more active than those subjected to the normogravity regime, as demonstrated by both the increase in the number of myotubes and the significant increase in the expression of Bone Morphogenetic Protein-2 in all experimental groups. However, with prolongated exposure to simulated microgravity regime (>72h), satellite cells and new formed myotubes underwent to cell death. It is important to note that, in early phases, simulated microgravity can stimulate the formation of new myotubes from satellite cells derived by osteoporotic patients. Furthermore, we observed that simulated microgravity can induce changes in myostatin expression levels by group-dependent variations. CONCLUSION: The results obtained allowed us to hypothesize a possible molecular mechanism of response to simulated microgravity, confirming the importance of Bone Morphogenetic Protein-2 and myostatin in the physio-pathogenesis of muscle tissue. In addition, these data can lay the foundation for new therapeutic approached in the prevention/cure of osteoporosis and sarcopenia.

No effect of the endurance training status on senescence despite reduced inflammation in skeletal muscle of older individuals.

The aim of the present study was to determine if the training status decreases inflammation, slows down senescence, and preserves telomere health in skeletal muscle in older compared with younger subjects, with a specific focus on satellite cells. Analyses were conducted on skeletal muscle and cultured satellite cells from vastus lateralis biopsies (n = 34) of male volunteers divided into four groups: young sedentary (YS), young trained cyclists (YT), old sedentary (OS), and old trained cyclists (OT). The senescence state and inflammatory profile were evaluated by telomere dysfunction-induced foci (TIF) quantification, senescence-associated ß-galactosidase (SA-ß-Gal) staining, and quantitative (q)RT-PCR. Independently of the endurance training status, TIF levels (+35%, P < 0.001) and the percentage of SA-ß-Gal-positive cells (+30%, P < 0.05) were higher in cultured satellite cells of older compared with younger subjects. p16 (4- to 5-fold) and p21 (2-fold) mRNA levels in skeletal muscle were higher with age but unchanged by the training status. Aging induced higher CD68 mRNA levels in human skeletal muscle (+102%, P = 0.009). Independently of age, both trained groups had lower IL-8 mRNA levels (-70%, P = 0.011) and tended to have lower TNF-α mRNA levels (-40%, P = 0.10) compared with the sedentary subjects. All together, we found that the endurance training status did not slow down senescence in skeletal muscle and satellite cells in older compared with younger subjects despite reduced inflammation in skeletal muscle. These findings highlight that the link between senescence and inflammation can be disrupted in skeletal muscle.

Satellite Cells in Skeletal Muscle of the Hibernating Dormouse, a Natural Model of Quiescence and Re-Activation: Focus on the Cell Nucleus.

Satellite cells (SCs) participate in skeletal muscle plasticity/regeneration. Activation of SCs implies that nuclear changes underpin a new functional status. In hibernating mammals, periods of reduced metabolic activity alternate with arousals and resumption of bodily functions, thereby leading to repeated cell deactivation and reactivation. In hibernation, muscle fibers are preserved despite long periods of immobilization. The structural and functional characteristics of SC nuclei during hibernation have not been investigated yet. Using ultrastructural and immunocytochemical analysis, we found that the SCs of the hibernating edible dormouse, Glis glis, did not show apoptosis or necrosis. Moreover, their nuclei were typical of quiescent cells, showing similar amounts and distributions of heterochromatin, pre-mRNA transcription and processing factors, as well as paired box protein 7 (Pax7) and the myogenic differentiation transcription factor D (MyoD), as in euthermia. However, the finding of accumulated perichromatin granules (i.e., sites of storage/transport of spliced pre-mRNA) in SC nuclei of hibernating dormice suggested slowing down of the nucleus-to-cytoplasm transport. We conclude that during hibernation, SC nuclei maintain similar transcription and splicing activity as in euthermia, indicating an unmodified status during immobilization and hypometabolism. Skeletal muscle preservation during hibernation is presumably not due to SC activation, but rather to the maintenance of some functional activity in myofibers that is able to counteract muscle wasting.

Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers from Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cell.

Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7+ myogenic progenitors on their surface between the myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are the primary myogenic stem cells in adult muscle. This chapter describes our laboratory protocols for isolating, culturing, and immunostaining intact myofibers from mouse skeletal muscle as a means for studying satellite cell dynamics. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are plated in dishes coated with PureCol collagen (formerly known as Vitrogen) and maintained in a mitogen-poor medium (± supplemental growth factors). Employing such conditions, satellite cells remain at the surface of the parent myofiber while synchronously undergoing a limited number of proliferative cycles and rapidly differentiate. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. These EDL myofibers are routinely plated individually as adherent myofibers in wells coated with Matrigel and maintained in a mitogen-rich medium, conditions in which satellite cells migrate away from the parent myofiber, proliferate extensively, and generate numerous differentiating progeny. Alternatively, these EDL myofibers can be plated as non-adherent myofibers in uncoated wells and maintained in a mitogen-poor medium (± supplemental growth factors), conditions that retain satellite cell progeny at the myofiber niche similar to the FDB myofiber cultures. However, the adherent myofiber format is our preferred choice for monitoring satellite cells in freshly isolated (Time 0) myofibers. We conclude this chapter by promoting the Nestin-GFP transgenic mouse as an efficient tool for direct analysis of satellite cells in isolated myofibers. While satellite cells have been often detected by their expression of the Pax7 protein or the Myf5nLacZ knockin reporter (approaches that are also detailed herein), the Nestin-GFP reporter distinctively permits quantification of satellite cells in live myofibers, which enables linking initial Time 0 numbers and subsequent performance upon culturing. We additionally point out to the implementation of the Nestin-GFP transgene for monitoring other selective cell lineages as illustrated by GFP expression in capillaries, endothelial tubes and neuronal cells. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular, can also be isolated and analyzed using protocols described herein. Collectively, this chapter provides essential tools for studying satellite cells in their native position and their interplay with the parent myofiber.

Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish.

Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4-5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite-like cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse.

Bone Morphogenetic Proteins and myostatin pathways: key mediator of human sarcopenia.

BACKGROUND: Sarcopenia, osteoporosis and osteoarthritis are the most frequent musculoskeletal disorders affecting older people. The main aim of this study was to test the hypothesis that the balance between BMPs and myostatin pathways regulates the age-related muscle degeneration in OP and OA patients. To this end, we investigated the relationship among the expression of BMP-2/4-7, myostatin and phosphorylated Smads1-5-8 and the muscle quality, evaluated in term of fibers atrophy and satellite cells activity. METHODS: In this retrospective study, we collected 123 biopsies of vastus lateralis: 48 biopsies from patients who underwent hip arthroplasty for subcapital fractures of the femur (OP), 55 biopsies from patients who underwent hip arthroplasty for osteoarthritis (OA) and 20 biopsies from patients who underwent hip arthroplasty for high-energy hip fractures (CTRL). Muscle biopsies were fixed in 4% paraformaldehyde and paraffin embedded. Serial sections were used for morphometrical and immunohistochemical analysis (BMP/2/4-7, myostatin, Smads1-5-8, Pax7 and myogenin). In addition, 1 mm3 of muscle tissue of each patient was embedded in epon for ultrastructural study. RESULTS: Morphometric data indicated an increase of the number of atrophic fibers in OP patients compare to OA. In line with these data, we found an high regenerative potential in muscle tissues of OA patients due to the significant amount of both Pax7 and myogenin positive satellite cells detected in OA group. In addition, our data showed the decrease of BMP2/4 and -7 expression in OP patients compared to both OA group and CTRL. Conversely, OP patients were characterized by high levels of myostatin expression. A different expression profile was also found for phosphorylated Smad1-5-8 between OP and OA patients. In particular, OP patients showed a low number of positive phosphorylated Smad1-5-8 nuclei. CONCLUSION: The identification of molecular pathways involved in the pathogenesis of sarcopenia open new prospective for the development of drugs able to prevent/treat the muscle impairment that occur in elderly. Results here reported, highlighting the role of BMPs and myostatin pathways in physio-pathogenesis of human sarcopenia, allow us to propose human recombinant BMP-2/7 and anti-myostatin antibodies as a possible therapeutic option for the sarcopenia.

Early ultrastructural events of skeletal muscle damage following cardiotoxin-induced injury and glycerol-induced injury.

In this study, we investigated the early changes of skeletal muscle damage in response to injuries induced by cardiotoxin (CTX) and glycerol by using both light microscopy and transmission electron microscopy. Normal, non-dystrophic, adult male mice were used in this study. Tibialis anterior (TA) muscles were injected either with CTX or glycerol. Samples were collected at intervals starting from 1h up to 4days after injury. Injured muscles were subjected to both histological and ultrastructural analyses. CTX-induced injury caused mitochondrial accumulation and swelling followed by lysis, while glycerol-induced injury caused accumulation of vesicles with focal disruption of the basal lamina, indicating that the injuries have different mechanisms of damage to myofibers. Moreover, inflammatory cells, including neutrophils and macrophages, were recruited earlier and in larger numbers after CTX-induced injury than after glycerol-induced injury. On the other hand, satellite cells (SCs) activation started at 6h after both injuries, as indicated by an increase in both the length and cytoplasmic-to-nuclear ratio. However, there were significantly longer SCs with a higher cytoplasmic-to-nuclear ratio in the CTX-injured muscles than in the glycerol-injured muscles at day 4. In conclusion, our results demonstrated a difference between CTX and glycerol in their damage to myofibers; CTX damages myofiber mitochondria, while glycerol damages the myofiber cell membrane and alters osmosis. In addition, CTX-induced injury caused earlier and more extensive inflammatory infiltration than did glycerol-induced injury. This study is the first study to shed light on the early events following skeletal muscle injury induced by CTX and glycerol.

Short-term ursolic acid promotes skeletal muscle rejuvenation through enhancing of SIRT1 expression and satellite cells proliferation.

Ursolic acid (UA) is a triterpenoid compound, which exerts its influences on the skeletal muscles. However, the mechanisms underlying these effects are still unclear. In this study, muscle satellite cells were isolated and purified by high-throughput pre-plating method (∼>60%) from 10 days old mice skeletal muscles. Evaluation of paired-box 7 (Pax7) expressions then confirmed the purification. Treatment of the cells with UA showed that UA up-regulated SIRT1 (∼35 folds) and overexpressed PGC-1α (∼175 folds) gene significantly. Moreover, the number of muscle satellite cells, which accompanied by initiation of neomyogenesis in the animal skeletal muscles, was increased (∼3.4 times). We also evaluated UA-mediated changes in the cellular energy status in the skeletal muscles. The results revealed that in the UA-treated mice, ATP and ADP contents in the various skeletal muscle tissue types, including: Gastrocnemius (Gas), Tibialis Anterior (Tib) and Gluteus Maximus (Glu) have been significantly decreased (P≤0.001); 2.2, 3.2, 2 times for ATP, and 9.6, 35.7, 11.6 times for ADP, respectively; however to compensate this process mitochondrial biogenesis occurred (12.33%±1.5 times). Furthermore, a rise in ATP/ADP ratio was observed 2.5, 4.5, 2.05 times for Gas, Tib and Glu muscles, respectively (P≤0.001). Alternatively, UA enhanced the expression of myoglobin (∼2 folds) in concert with remodeling of glycolytic muscle fibers to mainly fast IIA (∼30%) and slow-twitch (∼4%) types as well. Finally, our study indicated that UA indirectly mimicked beneficial effects of short-term calorie restriction and exercise (fast-oxidative) by directing the skeletal muscle composition toward oxidative metabolism.

Effects of Dexamethasone on Satellite Cells and Tissue Engineered Skeletal Muscle Units.

Tissue engineered skeletal muscle has potential for application as a graft source for repairing soft tissue injuries, a model for testing pharmaceuticals, and a biomechanical actuator system for soft robots. However, engineered muscle to date has not produced forces comparable to native muscle, limiting its potential for repair and for use as an in vitro model for pharmaceutical testing. In this study, we examined the trophic effects of dexamethasone (DEX), a glucocorticoid that stimulates myoblast differentiation and fusion into myotubes, on our tissue engineered three-dimensional skeletal muscle units (SMUs). Using our established SMU fabrication protocol, muscle isolates were cultured with three experimental DEX concentrations (5, 10, and 25 nM) and compared to untreated controls. Following seeding onto a laminin-coated Sylgard substrate, the administration of DEX was initiated on day 0 or day 6 in growth medium or on day 9 after the switch to differentiation medium and was sustained until the completion of SMU fabrication. During this process, total cell proliferation was measured with a BrdU assay, and myogenesis and structural advancement of muscle cells were observed through immunostaining for MyoD, myogenin, desmin, and α-actinin. After SMU formation, isometric tetanic force production was measured to quantify function. The histological and functional assessment of the SMU showed that the administration of 10 nM DEX beginning on either day 0 or day 6 yielded optimal SMUs. These optimized SMUs exhibited formation of advanced sarcomeric structure and significant increases in myotube diameter and myotube fusion index, compared with untreated controls. Additionally, the optimized SMUs matured functionally, as indicated by a fivefold rise in force production. In conclusion, we have demonstrated that the addition of DEX to our process of engineering skeletal muscle tissue improves myogenesis, advances muscle structure, and increases force production in the resulting SMUs.

Reciprocal interaction between TRAF6 and notch signaling regulates adult myofiber regeneration upon injury.

Skeletal muscle is a postmitotic tissue that repairs and regenerates through activation of a population of stem-cell-like satellite cells. However, signaling mechanisms governing adult skeletal muscle regeneration remain less understood. In the present study, we have investigated the role of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein involved in receptor-mediated activation of multiple signaling pathways in regeneration of adult myofibers. Skeletal muscle-specific depletion of TRAF6 in mice (TRAF6(mko)) improved regeneration of myofibers upon injury with a concomitant increase in the number of satellite cells and activation of the Notch signaling pathway. Ex vivo cultures of TRAF6(mko) myofiber explants demonstrated an increase in the proliferative capacity of myofiber-associated satellite cells accompanied by an upregulation of Notch ligands. Deletion of TRAF6 also inhibited the activity of transcription factor NF-κB and the expression of inflammatory cytokines and augmented the M2c macrophage phenotype in injured muscle tissues. Collectively, our study demonstrates that specific inhibition of TRAF6 improves satellite cell activation and skeletal muscle regeneration through upregulation of Notch signaling and reducing the inflammatory repertoire.

Role of syndecan-4 side chains in turkey satellite cell apoptosis and focal adhesion formation.

S4 (syndecan-4) is a cell membrane heparan sulfate proteoglycan that functions in muscle growth and development. It is composed of a central core protein and two types of side chains: GAGs (glycosaminoglycans) and N-glycosylated (N-linked glycosylated) chains. The N-glycosylated chains and GAG chains are required for S4 to regulate turkey myogenic satellite cell proliferation. The objective of the current study was to determine whether the S4 side chains regulate cell proliferation through muscle cell focal adhesion formation and apoptosis. S4 mutants with only one or without any N-glycosylated chains attached to the core protein with or without GAG chains were generated to study the function of N-glycosylated chains and the interaction between N-glycosylated chains and GAG chains. The wild-type S4 and all of the S4 side chain mutants were transfected into turkey myogenic satellite cells. Cell apoptosis and focal adhesion formation were measured, and PKCα (protein kinase Cα) cell membrane localization was investigated. S4 increased FAK (focal adhesion kinase) activity and the deletion of the side chains decreased this effect. S4 and the S4 mutants had no effect on ß1-integrin expression, but increased the cell membrane localization of ß1-integrin and PKCα. Furthermore, cell apoptosis and vinculin containing focal adhesions were not affected by S4 and its mutants. The results suggest that S4 and its side chains play important roles in regulating FAK activity, and PKCα and ß1-integrin cell membrane localization, but not cell apoptosis and vinculin-containing focal adhesion formation.

Nitric oxide synthase inhibition delays low-frequency stimulation-induced satellite cell activation in rat fast-twitch muscle.

This study examined the effect of nitric oxide synthase (NOS) inhibition via N(ω)-nitro-l-arginine methyl ester (l-NAME) administration on low-frequency stimulation-induced satellite cell (SC) activation in rat skeletal muscle. l-NAME only delayed stimulation-induced increases in SC activity. Also, stimulation-induced increases in hepatocyte growth factor (HGF) mRNA and protein expression were only abrogated at the mRNA level in l-NAME-treated animals. Therefore, early stimulation-induced SC activation appears to be NOS-dependent, while continued activation may involve NOS-independent HGF translational control mechanisms.

Adult skeletal muscle stem cell migration is mediated by a blebbing/amoeboid mechanism.

Adult skeletal muscle possesses a resident stem cell population called satellite cells, which are responsible for tissue repair following damage. Satellite cell migration is crucial in promoting rapid tissue regeneration, but it is a poorly understood process. Furthermore, the mechanisms facilitating satellite cell movement have yet to be elucidated. This study investigates the process of satellite cell migration, revealing that they undergo two distinct phases of movement, first under the basal lamina and then rapidly increasing their velocity when on the myofiber surface. Most significantly, we show that satellite cells move using a highly dynamic blebbing or amoeboid-based mechanism and not via lamellipodia-mediated propulsion. We show that nitric oxide and noncanonical Wnt signaling pathways are necessary for regulating the formation of blebs and the migration of satellite cells. In summary, we propose that the formation of blebs and their necessity for satellite cell migration has significant implications in the future development of therapeutic regimes aimed at promoting skeletal muscle regeneration.

Time-course of changes in the myonuclear domain during denervation in young-adult and old rat gastrocnemius muscle.

If myonuclear loss initiates muscle wasting, it should precede the loss of muscle mass. As aging affects muscle plasticity, the time-course of muscle atrophy during disuse may differ between young and old animals. To investigate this, gastrocnemius muscles of 5- and 25-month-old rats were exposed to 1, 2, or 4 weeks of denervation, whereas the contralateral gastrocnemius muscles served as controls. Muscle fibers of each type responded similarly to 4 weeks of denervation. For both ages most of the atrophy (36%; P < 0.001) occurred in the first 2 weeks. In young-adult muscles, the myonuclear number remained constant, but in old muscles it decreased to below control level after 4 weeks of denervation (P < 0.05). Despite this differential response, myonuclear domain size decreased similarly at both ages (P < 0.001). In both young-adult and old rats, denervation-induced atrophy was not preceded by a loss of myonuclei.

In situ real-time imaging of the satellite cells in rat intact and injured soleus muscles using quantum dots.

The recruitment of satellite cells, which are located between the basement membrane and the plasma membrane in myofibers, is required for myofiber repair after muscle injury or disease. In particular, satellite cell migration has been focused on as a satellite cell response to muscle injury because satellite cell motility has been revealed in cell culture. On the other hand, in situ, it is poorly understood how satellite cell migration is involved in muscle regeneration after injury because in situ it has been technically very difficult to visualize living satellite cells localized within skeletal muscle. In the present study, using quantum dots conjugated to anti-M-cadherin antibody, we attempted the visualization of satellite cells in both intact and injured skeletal muscle of rat in situ. As a result, the present study is the first to demonstrate in situ real-time imaging of satellite cells localized within the skeletal muscle. Moreover, it was indicated that satellite cell migration toward an injured site was induced in injured muscle while spatiotemporal change in satellite cells did not occur in intact muscle. Thus, it was suggested that the satellite cell migration may play important roles in the regulation of muscle regeneration after injury. Moreover, the new method used in the present study will be a useful tool to develop satellite cell-based therapies for muscle injury or disease.

Steroid therapy is associated with decreased numbers of dendritic cells and fibroblasts, and increased numbers of satellite cells, in the dystrophic skeletal muscle.

BACKGROUND: The possible therapeutic benefits of using steroids to enhance muscle strength and slow disease progression in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) has been examined previously. In this investigation, it was hypothesised that steroid therapy is associated with morphological changes in the dystrophic muscle. OBJECTIVES AND METHODS: To test this hypothesis, two muscle biopsies were obtained (one biopsy before treatment, and the second 6 months following prednisone therapy) from 24 patients with dystrophies (18 DMD, 6 BMD). The participants were categorised into: control (6 specimens, normal muscle), untreated and treated groups. The muscle was evaluated for ultrastructural changes using transmission electron microscopy (TEM). RESULTS: In the untreated group, the muscle fibres were degenerated and of variable sizes. The myofibrils were thin with either complete loss of bands and/or abnormal banding patterns. The Z-lines were irregularly spaced and loosely registered. The mitochondria of the myofibrils were small, few, spherical and irregularly distributed. Numerous dendritic cells (DCs) with euchromatic nuclei, and multiple and long dendrites, were seen among the myofibrils. The collagen fibres among the muscle fibres (endomysium) were numerous and large. The satellite cells had euchromatic nuclei with clumps of heterochromatin. In the treated group, the muscle fibres had a relatively uniform size with occasional fibres showing partial degeneration. The myofibrils had a relatively similar diameter comparable to that of normal muscle .The degenerated areas were small in size with occasional foci showing loss of banding pattern, and abnormal short bands with thick and hazy Z-lines. The mitochondria of the myofibrils were numerous, spherical, small in size and regularly arranged between the myofibrils. Few DCs, with heterochromatic nuclei, and few and short dendrites appeared between the myofibrils. The collagen fibres between the muscle fibres (endomysium) were numerous and large. As compared with the treated group, there was a statistically significant increase (p<0.05) in the numbers of DCs (0.7+/-0.2 vs 1.6+/-0.3) and fibroblasts (1.9+/-0.2 vs 2.9 +/-0.3) in the untreated group. Alternatively, there was a statistically significant decrease (p<0.05) in the numbers of satellite cells (1.2+/-0.2 vs 0.6+/-0.1). CONCLUSION: The ability of steroids to induce ultrastructural features of improvement supports the notion that they have beneficial therapeutic role. The clinical ramifications of these observations mandate further studies.

Neuromuscular junction morphology, fiber-type proportions, and satellite-cell proliferation rates are altered in MyoD(-/-) mice.

Gene compensation by members of the myogenic regulatory factor (MRF) family has been proposed to explain the apparent normal adult phenotype of MyoD(-/-) mice. Nerve and field stimulation were used to investigate contraction properties of muscle from MyoD(-/-) mice, and molecular approaches were used to investigate satellite-cell behavior. We demonstrate that MyoD deletion results in major alterations in the organization of the neuromuscular junction, which have a dramatic influence on the physiological contractile properties of skeletal muscle. Second, we show that the lineage progression of satellite cells (especially initial proliferation) in the absence of MyoD is abnormal and linked to perturbations in the nuclear localization of beta-catenin, a key readout of canonical Wnt signaling. These results show that MyoD has unique functions in both developing and adult skeletal muscle that are not carried out by other members of the MRF family.

Human satellite progenitor cells for use in myofascial repair: isolation and characterization.

Current use of prosthetic meshes and implants for myofascial reconstruction has been associated with infectious complications, long-term failure, and dissatisfying cosmetic results. Our laboratory has developed a small animal model for ventral hernia repair, which uses progenitor cells isolated from a skeletal muscle biopsy. In the model, progenitor cells are expanded in vitro, seeded onto a nonimmunogenic, novel aligned scaffold of bovine collagen and placed into the defect as a living adjuvant to the innate repair mechanism. The purpose of the current investigation is to examine the feasibility of translating our current model to humans. As a necessary first step we present our study on the efficacy of isolating satellite cells from 9 human donor biopsies. We were able to successfully translate our progenitor cell isolation and culture protocols to a human model with some modifications. Specifically, we have isolated human satellite muscle cells, expanded them in culture, and manipulated these cells to differentiate into myotubes in vitro. Immunohistochemical analysis allowed the characterization of distinct progenitor cell cycle stages and quantification of approximate cell number. Furthermore, isolated cells were tracked via cytoplasmic nanocrystal labeling and observed using confocal microscopy.

Satellite cell characterization from aging human muscle.

OBJECTIVES: Satellite cells (SCs) are skeletal muscle progenitor cells located between the basal lamina and the sarcolemma of muscle fibers. They are responsible for muscle growth and repair. In humans, aging results in the depletion of the SC population and in its proliferative activity, but not in its function. It has not yet been determined whether under conditions of massive muscle fiber death in vivo, the regenerative potential of SCs is totally or partially compromised in old muscle. No studies have yet tested whether advanced age is a factor that restrains the response of SCs to muscle denervation in humans; this is also due to difficulties in the isolation and in the culture of SCs from a small human surgery fragment. The aim of this study was to study in depth muscle regeneration analysing the SC ability of SCs to proliferate and differentiate in aging human patients. METHODS: In order to study in more detail the molecular mechanism, the proliferative and differentiative ability of aging SCs, we isolated SCs from aging human muscle biopsies and analysed their morphology by transmission electron microscopy and immunocytochemical analysis (antibodies against desmin, N-CAM and M-cadherin) and their capacity to grow and to expand in vitro. Moreover, in order to evaluate gene expression of myogenic regulatory factors Myf5, MyoD and myogenin (Myf4), RT-PCR was performed. RESULTS AND DISCUSSION: SCs isolated from aging human muscle biopsies and plated into favorable proliferation and differentiation conditions were able to proceed through the myogenic program and actively form myotubes, although taking longer than the young control sample. The RT-PCR analysis together with the ultrastructural SC features showed that the myogenic potential seemed to be compromised during the aging human muscle proliferation in vitro.