Muscle Fiber Repair Research Articles

Sprouty1 Regulates Reversible Quiescence of a Self-Renewing Adult Muscle Stem Cell Pool during Regeneration

SummarySatellite cells are a heterogeneous population of skeletal muscle specific stem cells capable of self-renewal and differentiation after transplantation. Whether quiescent satellite cells can self-renew and contribute to muscle fiber repair in their endogenous environment in normal regenerating muscle has remained unknown. The transcription factor Pax7 is expressed in satellite cells and is critical for establishing the adult satellite cell pool. Using a temporally-inducible genetic lineage tracing approach (Pax7-CreERtm; R26R-lacZ) to fate-map adult satellite cells, we show that in response to injury quiescent adult Pax7+ cells enter the cell cycle; a subpopulation return to quiescence to fully replenish the satellite cell compartment and the others contribute to de novo muscle fiber formation. We demonstrate that Sprouty1 (Spry1), an inhibitor of receptor tyrosine kinase signaling, is robustly expressed in quiescent Pax7+ satellite cells in uninjured adult muscle, down-regulated in proliferating myogenic cells in injured muscles, and re-induced as Pax7+ cells return to quiescence in regenerated muscles. We show through deletion of Spry1 specifically in cycling adult Pax7+ satellite cells, that Spry1 is required for the return to quiescence and homeostasis of the self-renewing Pax7+ satellite cell pool during repair. Satellite cells unable to return to quiescence succumb to apoptosis leading to a diminished self-renewing Pax7-derived satellite cell pool. Our results define a novel role for Spry1 in adult stem cell biology and tissue repair.

Long-Term Survival of Transplanted Stem Cells in Immunocompetent Mice with Muscular Dystrophy

Satellite cells refer to resident stem cells in muscle that are activated in response to damage or disease for the regeneration and repair of muscle fibers. The use of stem cell transplantation to treat muscular diseases has been limited by impaired donor cell survival attributed to rejection and an unavailable stem cell niche. We isolated a population of adult muscle mononuclear cells (AMMCs) from normal, strain-matched muscle and transplanted these cells into delta-sarcoglycan-null dystrophic mice. Distinct from other transplant studies, the recipient mice were immunocompetent with an intact endogenous satellite cell pool. We found that AMMCs were 35 times more efficient at restoring sarcoglycan compared with cultured myoblasts. Unlike cultured myoblasts, AMMC-derived muscle fibers expressed sarcoglycan protein throughout their entire length, consistent with enhanced migratory ability. We examined the capacity of single injections of AMMCs to provide long-term benefit for muscular dystrophy and found persistent regeneration after 6 months, consistent with augmentation of the endogenous stem cell pool. Interestingly, AMMCs were more effectively engrafted into aged dystrophic mice for the regeneration of large clusters of sarcoglycan-positive muscle fibers, which were protected from damage, suggesting that the stem cell niche in older muscle remains permissive.

All muscle satellite cells are equal, but are some more equal than others?

Skeletal muscle is an accessible adult stem-cell model in which differentiated myofibres are maintained and repaired by a self-renewing stem-cell compartment. These resident stem cells, which are known as satellite cells, lie on the surface of the muscle fibre, between the plasmalemma and overlying basal lamina. Although they are normally mitotically quiescent in adult muscle, satellite cells can be activated when needed to generate myoblasts, which eventually differentiate to provide new myonuclei for the homeostasis, hypertrophy and repair of muscle fibres, or fuse together to form new myofibres for regeneration. Satellite cells also self-renew in order to maintain a viable stem-cell pool that is able to respond to repeated demand. The study of the control of self-renewal has led to the idea that the satellite-cell pool might be heterogeneous: that is it might contain both self-renewing satellite ;stem' cells and myogenic precursors with limited replicative potential in the same anatomical location. The regulatory circuits that control satellite-cell self-renewal are beginning to be deciphered, with Pax7, and Notch and Wnt signalling being clearly implicated. This Commentary seeks to integrate these interesting new findings into the wider context of satellite-cell biology, and to highlight some of the many outstanding questions.

Regeneration capacity of skeletal muscle

The organotypic stem cell of skeletal muscle has previously been known as satellite cell. They allow muscle fiber growth during ontogenesis, enable fiber hypertrophy and are responsible for the very efficient repair of muscle fibers. This efficient apparatus is to some degree counterbalanced by an enormous use of the satellite cell pool: fiber atrophy probably is accompanied by loss of myonuclei such that every reversal of atrophy is bound to use new myonuclei i.e. satellite cells. How often in life does this occur? Hard to say. Moreover, the potent repair capacity is challenged by an unexpected vulnerability of skeletal muscle fibers: Passive stretching of contracted muscles may cause multiple "microdamage," disruption of contractile elements or tiny areas of true necrosis (focal necrosis). How often does this happen? Well, for many of us at least once per year when we go up and down mountains during vacation time, followed by sour muscles. Others may decide to change his/her (locomotor) behaviour by severe onset of jogging; it may happen that they suffer kidney failure on Monday due to muscle microdamage and the transfer of myoproteins into the serum over weekend. Also 20 minutes of stepping up and down something like a chair will do: There is a remarkable increase in kreatin kinase and other muscle derived proteins which lasts for days and is bound to reflect some muscle damage. How about sportsmen and worker who repeatedly use their muscles in such a way? We don't have answers yet to most of these questions, but considerable amount of information has been collected over the last years both in animal and--less--in human. What is common in all cases of growth and repair is the proliferation of the satellite cells and their consequent incorporation and fusion with the parent fiber. This way focal damage is repaired often without visible reminders. We would run out of satellite cells were they not stem cells: After division one daughter remains a satellite cell while the other is free to divide. Divide how often? Important for the human cells since the cell ages and proliferates slower and slower till it stops to divide at all, at least in culture. The same is true for the new satellite cell. This we know from recent experiments in which human biopsies derived myogenic cells were grown in vitro and in vivo (by implanting them into skeletal muscles of immunoincompetent mice): Growth correlates negatively with age of the donor. Between age 2 and some 70 years, about two divisions are performed by each satellite cell in human vastus lateralis and biceps brachii muscle in 10 years in the average. Most important for the older among us: at age 76 there are still some 13 divisions left before complete exhaustion. However, there are diseases like Duchenne Muscular Dystrophy (DMD) in which muscle fibers lack a structural protein with the effect of enhanced vulnerability to mechanical stress. There the enhanced use of the satellite cell pool makes the remaining growth capacity in an 8-years-old child as low as otherwise found at age 80. Some time ago, implantation of genetically intact myoblasts obtained from healthy relatives has been proposed as a treatment of DMD. Every logic would have predicted that some local implantation of whatever numbers of cells was bound to fail rescue the complete masculature or at least the muscles for breathing. The human as guinea pig? Now, even years later, we still collect the basic information on growth of human myoblasts and start thinking of ways for systemic application and quantitatively relevant incorporation of the myogenic stem cell or other--possibly pluripotent--stem cells derived from bone marrow.

Muscle stem cells.

Since its discovery four decades ago, the satellite cell of skeletal muscle has been implicated as the major source of myogenic cells involved in growth and repair of muscle fibres. This review not only looks at the role of the satellite cell in these processes but discusses how cells derived from other sources and tissues have recently been implicated in muscle formation and regeneration. Muscle itself also yields cells that contribute to other cell lineages although it is currently debated as to whether these cells originate within muscle or have migrated there from other tissues. The reality of using cells from muscle or other tissues to repair diseased muscle fibres is also addressed.

TIME COURSE OF CHANGES IN CAPILLARIZATION IN RAT SKELETAL MUSCLE AFTER STRENUOUS ECCENTRIC EXERCISE

PURPOSE: We examined the time course change of the capillary structure in rat skeletal muscle one, three and seven days after strenuous eccentric exercise. METHODS: The right gastrocnemius muscles of anesthetized male wister rats (n = 20) were subjected to 300 (60 times, 5 sets) controlled eccentric contractions induced by electrical stimulation of surface electrodes. The contralateral gastrocnemius muscle was used as a normal control. All morphometric parameters with respect to capillary and muscle fiber were determined in in situ perfused gastrocnemius muscles in red (Gr; predominant in slow-twitch fiber) and white (Gw; predominant in fast-twitch fiber) portions. RESULTS: Damaged muscle fibers (infiltration of mononuclear cell or a swollen appearance) at days one, three and seven were observed in both Gr (29.3 to 53.9% damaged fibers) and Gw (58.9 to 86.8% damaged fibers) of exercised legs. We could not observe collapsed or obstructe capillaries in any exercised legs in light and electron micrographs of transverse sections. Furthermore, capillary endothelial cells had a normal structure in exercised legs. These results indicate that capillaries were not lost one to seven days after strenuous eccentric exercise. On the first and third day, however, capillary tortuosity and luminal diameter changed in exercised legs. The ratio between minimal and maximal capillary diameter in a transverse section (i.e., capillary tortuosity) significantly differed from control (Gr: 0.75 ± 0.02, Gw: 0.79 ± 0.03) and exercised legs (Gr: 0.65 ± 0.03, Gw: 0.66 ± 0.04) one day after exercise. The mean capillary luminal diameter was significantly increased in exercised legs after one day (Gw: +11.5%) and three days (Gr: + 14.9%, Gw: +25.2%) compared with control legs. These morphological changes in capillaries might be related to muscle fiber repair through the changes in hemodynamics such as erythrocyte and leukocyte flow. CONCLUSION: We conclude that 1) capillary endothelial cell structure was rather stable against strenuous eccentric exercise compared with the muscle fiber structure, and 2) changes in capillary tortuosity and distensibility occur in the degenerated muscle up to three days after the exercise period.

Experimental induction of ring fibers in regenerating skeletal muscle.

Light microscopy and electron microscopy were used to study the formation of ring fibers induced experimentally in regenerating muscle subjected to tenotomy-induced tension deficiency. Anterior tibial rat muscles were injured by intramuscular injection of mepivacain, tenotomized at varying stages of the regenerative process, and analyzed 30 days after sectioning the tendon. The combination of regeneration and tenotomy led to the appearance of ring fibers at different developmental stages. Ring fibers were not observed in regenerating control muscles and were scarce in tenotomized controls. Our results showed that the regenerative phase in which tension deficiency was established had a significant influence on the number of developing ring fibers; the number increased when tenotomy was performed during subsarcolemmic myofibrillogenesis in regenerating fibers. As a consequence, one might hypothesize that tension deficiency during muscle fiber repair plays a critical role in ring fiber formation.

Lymphocyte antigen Leu-19 as a molecular marker of regeneration in human skeletal muscle.

Antigen Leu-19 (Leu19-Ag), a 200- to 220-kDa surface glycoprotein, was originally identified on a subset of human peripheral lymphocytes exhibiting non-major histocompatibility complex-restricted cytotoxicity. Here we report that monoclonal antibody Leu-19 (mAb-Leu19) labels structures in human skeletal muscle: (i) satellite cells, which form the stem cell pool of muscle fiber regeneration, both in normal and diseased muscle; (ii) myotubes and myotube projections in regions of muscle fiber repair; (iii) periodically organized fibrillar structures in areas of regeneration; (iv) the surface of myoblasts and developing myotubes in culture. mAb-Leu19 precipitated a protein of approximately 200 kDa from cultured muscle cells. Our data show that Leu19-Ag is expressed on muscle-specific components of myosegments in repair and thus represents a molecular marker of muscle regeneration. On the basis of this molecular marker and using laser scan microscopy, it is possible to visualize at the light microscopic level hitherto undetectable details of muscle regeneration in routine cryostat sections.