Exercise running and tetracycline as means to enhance skeletal muscle stem cell performance after external fixation

Prolonged limb immobilization, which is often the outcome of injury and illness, results in the atrophy of skeletal muscles. The basis of muscle atrophy needs to be better understood in order to allow development of effective countermeasures. The present study focused on determining whether skeletal muscle stem cells, satellite cells, are directly affected by long-term immobilization as well as on investigating the potential of pharmacological and physiological avenues to counterbalance atrophy-induced muscle deterioration. We used external fixation (EF), as a clinically relevant model, to gain insights into the relationships between muscle degenerative and regenerative conditions to the myogenic properties and abundance of bona fide satellite cells. Rats were treated with tetracycline (Tet) through the EF period, or exercise trained on a treadmill for 2 weeks after the cessation of the atrophic stimulus. EF induced muscle mass loss; declined expression of the muscle specific regulatory factors (MRFs) Myf5, MyoD, myogenin, and also of satellite cell numbers and myogenic differentiation aptitude. Tet enhanced the expression of MRFs, but did not prevent the decline of the satellite cell pool. After exercise running, however, muscle mass, satellite cell numbers (enumerated through the entire length of myofibers), and myogenic differentiation aptitude (determined by the lineal identity of clonal cultures of satellite cells) were re-gained to levels prior to EF. Together, our results point to Tet and exercise running as promising and relevant approaches for enhancing muscle recovery after atrophy.     

The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts

Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.     

Transient neonatal denervation alters the proliferative capacity of myosatellite cells in dystrophic (129ReJdy/dy) muscle.

It has been previously shown that transiently denervated, neonatal dystrophic muscle fails to undergo the degeneration-regeneration cycle characteristic of murine dystrophy (Moschella and Ontell, 1987). Thus, the myosatellite cells (myogenic stem cells) in these muscles have been spared the mitotic challenge to which dystrophic myosatellite cells are normally subjected early in the time course of the disease. By in vitro evaluation of the proliferative capacity of myosatellite cells derived from extensor digitorum longus (EDL) muscles of 100-day-old genetically normal (+/+) and genetically dystrophic [dy/dy (129ReJdy/dy)] mice and from muscles of age-matched mice that had been neonatally denervated (by sciaticotomy) and allowed to reinnervate, it has been possible to directly determine whether the cessation of spontaneous regeneration in older dy/dy muscles in vivo, is due to an innate defect in the proliferative capacity of the myosatellite cells or exhaustion of the myosatellite cells' mitotic activity during the regenerative phase of the disease. This study demonstrates that transient neonatal denervation of dystrophic muscle (Den.dy/dy) increases the number of muscle colony-forming cells (MCFs) per milligram of wet weight muscle tissue, increases the plating efficiency, and significantly increases the in vitro mitotic activity of dystrophic myosatellite cells toward normal values. The increased mitotic capability of myosatellite cells derived from Den.dy/dy muscle as compared to unoperated dy/dy muscle suggests that there is no innate defect in the proliferative capacity of the myosatellite cells of dy/dy muscles and that the cessation of spontaneous regeneration in the dy/dy muscles is related to the exhaustion of their myosatellite cells' mitotic capability.     

Reduced satellite cell numbers and myogenic capacity in aging can be alleviated by endurance exercise

BACKGROUND: Muscle regeneration depends on satellite cells, myogenic stem cells that reside on the myofiber surface. Reduced numbers and/or decreased myogenic aptitude of these cells may impede proper maintenance and contribute to the age-associated decline in muscle mass and repair capacity. Endurance exercise was shown to improve muscle performance; however, the direct impact on satellite cells in aging was not yet thoroughly determined. Here, we focused on characterizing the effect of moderate-intensity endurance exercise on satellite cell, as possible means to attenuate adverse effects of aging. Young and old rats of both genders underwent 13 weeks of treadmill-running or remained sedentary. METHODOLOGY: Gastrocnemius muscles were assessed for the effect of age, gender and exercise on satellite-cell numbers and myogenic capacity. Satellite cells were identified in freshly isolated myofibers based on Pax7 immunostaining (i.e., ex-vivo). The capacity of individual myofiber-associated cells to produce myogenic progeny was determined in clonal assays (in-vitro). We show an age-associated decrease in satellite-cell numbers and in the percent of myogenic clones in old sedentary rats. Upon exercise, there was an increase in myofibers that contain higher numbers of satellite cells in both young and old rats, and an increase in the percent of myogenic clones derived from old rats. Changes at the satellite cell level in old rats were accompanied with positive effects on the lean-to-fat Gast muscle composition and on spontaneous locomotion levels. The significance of these data is that they suggest that the endurance exercise-mediated boost in both satellite numbers and myogenic properties may improve myofiber maintenance in aging.     

Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle

The deteriorating in vivo environment is thought to play a major role in reduced stem cell function with age. The capacity of stem cells to support tissue maintenance depends not only on their response to cues from the surrounding niche, but also on their abundance. Here, we investigate satellite cell (myogenic stem cell) pool size and its potential to participate in muscle maintenance through old age. The numbers and performance of mouse satellite cells have been analyzed using molecular markers that exclusively characterize quiescent satellite cells and their progeny as they transit through proliferation, differentiation and generation of reserve cells. The study establishes that abundance of resident satellite cells declines with age in myofibers from both fast- and slow-twitch muscles. Nevertheless, the inherent myogenic potential of satellite cells does not diminish with age. Furthermore, the aging satellite cell niche retains the capacity to support effective myogenesis upon enrichment of the mitogenic milieu with FGF. Altogether, satellite cell abundance, but not myogenic potential, deteriorates with age. This study suggests that the population of satellite cells that participate in myofiber maintenance during routine muscle utilization is not fully replenished throughout life.     

Changes in proliferation,differentiation, fibroblast growth factor 2 responsiveness and expression of syndecan‐4 and glypican‐1 with turkey satellite cell age

Myogenic satellite cells are heterogeneous multipotential stem cells that are required for muscle repair, maintenance, and growth. The membrane‐associated heparan sulfate proteoglycans syndecan‐4 and glypican‐1 differentially regulate satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) signal transduction, and expression of the myogenic regulatory factors MyoD and myogenin. The objective of the current study was to determine the effect of age on syndecan‐4 and glypican‐1 satellite cell populations, proliferation, differentiation, FGF2 responsiveness, and expression of syndecan‐4, glypican‐1, MyoD, and myogenin using satellite cells isolated from the pectoralis major muscle of 1‐day‐old, 7‐week‐old and 16‐week‐old turkeys. Proliferation was significantly reduced in the 16‐week‐old satellite cells, while differentiation was decreased in the 7‐week‐old and the 16‐week‐old cells beginning at 48 h of differentiation. Fibroblast growth factor 2 responsiveness was highest in the 1‐day‐old and 7‐week‐old cells during proliferation; during differentiation there was an age‐dependent response to FGF2. Syndecan‐4 and glypican‐1 satellite cell populations decreased with age, but syndecan‐4 and glypican‐1 were differentially expressed with age during proliferation and differentiation. MyoD and myogenin mRNA expression was significantly decreased in 16‐week‐old cells compared to the 1‐day‐old and 7‐week‐old cells. MyoD and myogenin protein expression was higher during proliferation in the 16‐week‐old cells and decreased with differentiation. These data demonstrate an age‐dependent effect on syndecan‐4 and glypican‐1 satellite cell subpopulations, which may be associated with age‐related changes in proliferation, differentiation, FGF2 responsiveness, and the expression of the myogenic regulatory factors MyoD and myogenin.     

‘From Death,Lead Me to Immortality’ – Mantra of Ageing Skeletal Muscle

Skeletal muscle is a post-mitotic tissue maintained by repair and regeneration through a population of stem cell-like satellite cells. Following muscle injury, satellite cell proliferation is mediated by local signals ensuring sufficient progeny for tissue repair. Age–related changes in satellite cells as well as to the local and systemic environment potentially impact on the capacity of satellite cells to generate sufficient progeny in an ageing organism resulting in diminished regeneration. ‘Rejuvenation’ of satellite cell progeny and regenerative capacity by environmental stimuli effectors suggest that a subset of age-dependent satellite cell changes may be reversible. Epigenetic regulation of satellite stem cells that include DNA methylation and histone modifications which regulate gene expression are potential mechanisms for such reversible changes and have been shown to control organismal longevity. The area of health and ageing that is likely to benefit soonest from advances in the biology of adult stem cells is the emerging field of regenerative medicine. Further studies are needed to elucidate the mechanisms by which epigenetic modifications regulate satellite stem cell function and will require an increased understanding of stem-cell biology, the environment of the aged tissue and the interaction between the two.     

Numbers of muscle nuclei and myosatellite cell nuclei in red and white axial muscle during growth of the carp (Cyprinus carpio)

We determined the percentages of muscle fibie nuclei and satellite nuclei over a growth range of carp ( Cyprinus carpio ), as the increase in the number of muscle fibre nuclei is an important aspect of the increase in muscle mass, and myosatellite cells are believed to be the source of new muscle fibre nuclei. In white as well as in red axial muscle the percentage of the nuclei present in muscle that are muscle nuclei (muscle fibre nuclei+myosatellite nuclei) remained constant during growth (54 and 32% respectively). The difference in the percentage of non-muscle nuclei between white and red axial muscle is mainly caused by the higher content of endothelial nuclei in red axial muscle.
In white axial muscle the DNA/protein ratio (nucleus/sarcoplasm ratio) decreased between 3 and 15 cm S.l. In red axial muscle we found a continuous decrease in DNA/protein ratio over the entire investigated size range (3–50 cm s.l.). This may be related to a longer occurrence of hyperplasia in red than in white axial muscle.
In both fibre types the percentage of muscle nuclei being myosatellite nuclei decreased with increasing length, In white axial muscle it decreased from about 5% in carp of 5 cm s.l. to less than 1% in carp of 20 cm S.L.; for red muscle these values were 11 and 3% respectively.
For white axial muscle we calculated that, especially in larger fish, the myosatellite ceils alone cannot account for the increase in the number of muscle fibre nuclei during growth. The percentage of proliferating nuclei in muscle tissue, measured by the uptake of 5-bromo-2'-deoxy-uridine, is high enough to account for the total increase in nuclei. So indirect evidence is available that another cell type present in the muscle tissue may also be involved in the formation of additional muscle fibre nuclei.     

Impaired Skeletal Muscle Regeneration in the Absence of Fibrosis during Hibernation in 13-Lined Ground Squirrels

Skeletal muscle atrophy can occur as a consequence of immobilization and/or starvation in the majority of vertebrates studied. In contrast, hibernating mammals are protected against the loss of muscle mass despite long periods of inactivity and lack of food intake. Resident muscle-specific stem cells (satellite cells) are known to be activated by muscle injury and their activation contributes to the regeneration of muscle, but whether satellite cells play a role in hibernation is unknown. In the hibernating 13-lined ground squirrel we show that muscles ablated of satellite cells were still protected against atrophy, demonstrating that satellite cells are not involved in the maintenance of skeletal muscle during hibernation. Additionally, hibernating skeletal muscle showed extremely slow regeneration in response to injury, due to repression of satellite cell activation and myoblast differentiation caused by a fine-tuned interplay of p21, myostatin, MAPK, and Wnt signaling pathways. Interestingly, despite long periods of inflammation and lack of efficient regeneration, injured skeletal muscle from hibernating animals did not develop fibrosis and was capable of complete recovery when animals emerged naturally from hibernation. We propose that hibernating squirrels represent a new model system that permits evaluation of impaired skeletal muscle remodeling in the absence of formation of tissue fibrosis.     

Spontaneous regeneration of older dystrophic muscle does not reflect its regenerative capacity

Young dystrophic (dy) murine muscle is capable of "spontaneous" regeneration (i.e., regeneration in the absence of external trauma); however, by the time the mice are 8 weeks old, this regeneration ceases. It has been suggested that the cessation of regeneration in dystrophic muscle may be due to exhaustion of the mitotic capability of myosatellite cells during the early stages of the disease. To test this hypothesis, orthotopic transplantation of bupivacaine treated, whole extensor digitorum longus muscles has been performed on 14 to 16-week-old 129 ReJ/++ and 129 ReJ/dydy mice. The grafted dystrophic muscle is able to produce and maintain for 100 days post-transplantation 356 +/- 22 myofibers, a number similar to that found in age-matched dystrophic muscle. The ability of old dystrophic muscle to regenerate subsequent to extreme trauma indicates that the cessation of "spontaneous" regeneration is due to factor(s) other than the exhaustion of mitotic capability of myosatellite cells. Moreover, there is no significant difference in myosatellite cell frequencies between grafted normal and dystrophic muscles (100 days post-transplantation). Myosatellite cell frequencies in grafted muscles are similar to those in age-matched, untraumatized muscles. While grafting of young dystrophic muscle modifies the phenotypic expression of histopathological changes usually associated with murine dystrophy, grafts of older dystrophic muscle show extensive connective-tissue infiltration and significantly fewer myofibers than do grafts of age-matched normal muscle. As early as 14 days post-transplantation, it is possible to distinguish between grafts of old, normal and dystrophic muscles. It is suggested that the connective tissue stroma, present in the dystrophic muscle at the time of transplantation, may survive the grafting procedure.     

Transient neonatal denervation alters the proliferative capacity of myosatellite cells in dystrophic (129ReJdy/dy) muscle

It has been previously shown that transiently denervated, neonatal dystrophic muscle fails to undergo the degeneration–regeneration cycle characteristic of murine dystrophy (Moschella and Ontell, 1987). Thus, the myosatellite cells (myogenic stem cells) in these muscles have been spared the mitotic challenge to which dystrophic myosatellite cells are normally subjected early in the time course of the disease. By in vitro evaluation of the proliferative capacity of myosatellite cells derived from extensor digitorum longus (EDL) muscles of 100-day-old genetically normal (+/+) and genetically dystrophic [dy/dy (129ReJ^dy/dy)] mice and from muscles of age-matched mice that had been neonatally denervated (by sciaticotomy) and allowed to reinnervate, it has been possible to directly determine whether the cessation of spontaneous regeneration in older dy/dy muscles in vivo, is due to an innate defect in the proliferative capacity of the myosatellite cells or exhaustion of the myosatellite cells' mitotic activity during the regenerative phase of the disease. This study demonstrates that transient neonatal denervation of dystrophic muscle (Den.dy/dy) increases the number of muscle colony-forming cells (MCFs) permilligram of wet weight muscle tissue, increases the plating efficiency, and significantly increases the in vitro mitotic activity of dystrophic myosatellite cells toward normal values. The increased mitotic capability of myosatellite cells derived from Den.dy/dy muscle as compared to unoperated dy/dy muscle suggests that there is no innate defect in the proliferative capacity of the myosatellite cells of dy/dy muscles and that the cessation of spontaneous regeneration in the dy/dy muscles is related to the exhaustion of their myosatellite cells' mitotic capability. © 1992 John Wiley & Sons, Inc.     

Most apoptotic cells in mdx diaphragm muscle contain accumulated lipofuscin

An age-related pigment, lipofuscin (LF), which accumulates in postmitotic, long-lived cells, is formed by the oxidative degradation of cellular macromolecules by oxygen-derived free radicals. In the present study we show that LF is accumulated in some myofibres, myosatellite cells and interstitial cells in the diaphragm muscles of the X chromosome-linked muscular dystrophic (mdx) mice at the age of 10 weeks when repetitive cycles of de- and regeneration of myofibres occur. In contrast, LF is virtually absent in diaphragm muscles of age-matched C57BL/10 (C57) normal control mice. Therefore, mdx muscle is more susceptible to oxidative stress than normal muscle. We hypothesise that gene-regulated cell death (apoptosis) occurs in dystrophic muscle cells that accumulate LF as a consequence of either oxidative stress or injury. We found that 74-79% of apoptotic myosatellite cells, interstitial cells and myofibres in mdx diaphragm contain accumulated or dotted LF granules, but only 12-20% of non-apoptotic cells contain LF. Apoptotic cells are very rare in the diaphragm of age-matched C57 control mice. This suggests that the regeneration of mdx diaphragm muscle initiated from myosatellite cells is impaired by their apoptosis as the result of either oxidative stress or a product of oxidative injury.     

Prolonged absence of myostatin reduces sarcopenia

Sarcopenia is a progressive age-related loss of skeletal muscle mass and strength. Parabiotic experiments show that circulating factors positively influence the proliferation and regenerative capacity of satellite cells in aged mice. In addition, we believe that negative regulators of muscle mass also serve to balance the signals that influence satellite cell activation and regeneration capacity with ageing. Myostatin, a negative regulator of pre- and postnatal myogenesis, inhibits satellite cell activation and muscle regeneration postnatally. To investigate the role of myostatin during age-related sarcopenia, we examined muscle mass and regeneration in young and old myostatin-null mice. Young myostatin-null muscle fibers were characterized by massive hypertrophy and hyperplasia and an increase in type IIB fibers, resulting in a more glycolytic muscle. With ageing, wild-type muscle became increasingly oxidative and fiber atrophy was prominent. In contrast no fiber type switching was observed and atrophy was minimal in aged myostatin-null muscle. The effect of ageing on satellite cell numbers appeared minimal, however, satellite cell activation declined significantly in both wild-type and myostatin-null muscles. In young mice, lack of myostatin resulted in increased satellite cell number and activation compared to wild-type, suggesting a greater propensity to undergo myogenesis, a difference maintained in the aged mice. In addition, muscle regeneration of myostatin-null muscle following notexin injury was accelerated and fiber hypertrophy and type were recovered with regeneration, unlike in wild-type muscle. In conclusion, a lack of myostatin appears to reduce age-related sarcopenia and loss of muscle regenerative capacity.     

Satellite cells isolated from aged or dystrophic muscle exhibit a reduced capacity to promote angiogenesis in vitro

Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. In addition to reduced myogenic activity, satellite cells may also lose the ability to communicate with vascular cells for coordination of myogenesis and angiogenesis and restoration of proper muscle function. Objectives of the current study were to determine the angiogenic-promoting capacity of satellite cells from two states characterized by dysfunctional skeletal muscle repair, aging and Duchenne muscular dystrophy. An in vitro culture model composed of satellite cells or their conditioned media and rat adipose tissue microvascular fragments (MVF) was used to examine this relationship. Microvascular fragments cultured in the presence of rat satellite cells from adult muscle donors (9–12 month of age) exhibited greater indices of angiogenesis (endothelial cell sprouting, tubule formation and extensive branching) than MVF co-cultured with satellite cells from aged muscle donors (24 month of age). We sought to determine if the differential degree of angiogenesis we observed in the co-culture setting was due to soluble factors produced by each satellite cell age group. Similar to the co-culture experiment, conditioned media produced by adult satellite cells promoted greater angiogenesis than that of aged satellite cells. Next, we examined differences in angiogenesis-stimulating ability of satellite cells from 12 mo old MDX mice or age-matched wild-type mice. A reduction in angiogenesis activity of media conditioned by satellite cells from dystrophic muscle was observed as compared to healthy muscle. Finally, we found reduced gene expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in both aged and dystrophic satellite cells compared to their adult and normal counterparts, respectively. These results indicate that functional deficits in satellite cell activities during aging and diseased muscle may extend to their ability to communicate with other cells in their environment, in this case cells involved in angiogenesis.     

Myosatellite cells of Cyprinus carpio (Teleostei) in vitro: isolation,recognition and differentiation

Summary We have developed a method for the dissociation and purification of myosatellite cells from white epaxial muscle of carp. The dissociated myosatellite cells were identified by their morphology, their ultrastructure, the formation of multinucleated myotubes containing myofibrils and the immunocytochemical demonstration of desmin. Desmin and 5-bromo-2-deoxyuridine (BrdU) were used to identify terminally differentiated and proliferating myosatellite cells, respectively. The in vitro behavior of myosatellite cells dissociated from carp of 5 cm standard length differed from that described for myosatellite cells of mammals and birds. No substantial proliferation of the myosatellite cells could be observed. Most cells were differentiated (desmin-positive, BrdU-negative) 17 h after plating, regardless of the medium used. This indicates that the investigated white epaxial muscle of carp of 5 cm standard length contains subpopulations of myosatellite cells, arrested at various stages of differentiation.