Miranda D. Grounds

Towards understanding skeletal muscle regeneration.

Factors which effect proliferation and fusion of muscle precursor cells have been studied extensively in tissue culture, although little is known about these events in vivo. This review assesses the tissue culture derived data with a view to understanding factors which may control the regeneration of mature skeletal muscle in vivo. The following topics are discussed in the light of recent developments in cell and molecular biology: 1) Injury and necrosis of mature skeletal muscle fibres 2) Phagocytosis of myofibre debris 3) Revascularisation of injured muscle 4) Activation and proliferation of muscle precursor cells (mpc) in vivo Identification of mpcs; Satellite cell relationships; Extracellular matrix; Growth factors; Hormones; Replication. 5) Differentiation and fusion of muscle precursor cells in vivo Differentiation; Fusion; Extracellular matrix; Cell surface molecules: Growth factors and prostaglandins 6) Myotubes and innervation.

Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes

SummaryThe activation of mononuclear muscle precursor cells after crush injury to mouse tibialis anterior muscles was monitored in vivo by in situ hybridization with MyoD1 and myogenin probes. These genes are early markers of skeletal muscle differentiation and have been extensively studied in vitro. The role in vivo of these regulatory proteins during myogenesis of mature muscle has not been studied previously. MyoD1 and myogenin mRNA were present in occasional mononuclear cells of uninjured muscle. Increased MyoD1 and myogenin mRNA sequences in mononuclear cells were detected as early as 6 h after injury, peaked between 24 and 48 h, and thereafter declined to pre-injury levels at about 8 days. The mRNAs were detected in mononuclear cells throughout the muscle, with the majority of cells located some distance from the site of crush injury. The presence of MyoD1 and myogenin mRNA at 6 to 48 h indicates that transcription of these genes is occurring at the same time as replication of muscle precursor cells in vivo. At no time were significant levels of mRNA for these genes detected in myotubes. MyoD1 and myogenin provide precise markers for the very early identification and study of mononuclear skeletal muscle precusor cells in muscle regenerating in vivo.

Age-associated Changes in the Response of Skeletal Muscle Cells to Exercise and Regenerationa

ABSTRACT: This paper looks at the effects of aging on the response of skeletal muscle to exercise from the perspective of the behavior of muscle precursor cells (widely termed satellite cells or myoblasts) and regeneration. The paper starts by outlining the ways in which skeletal muscle can respond to damage resulting from exercise or other trauma. The age‐related changes within skeletal muscle tissue and the host environment that may affect the proliferation and fusion of myoblasts in response to injury in old animals are explored. Finally, in vivo and in vitro data concerning the wide range of signaling molecules that stimulate satellite cells and other aspects of regeneration are discussed with respect to aging. Emphasis is placed on the important role of the host environment, inflammatory cells, growth factors and their receptors (particularly for FGF‐2), and the extracellular matrix.

Rapid death of injected myoblasts in myoblast transfer therapy

Myoblast transplantation has been proposed as a potential therapy for Duchenne muscular dystrophy (DMD). A Y‐chromosome‐specific probe was used to track the fate of donor male myoblasts injected into dystrophic muscles of female mdx mice (which are an animal model for DMD). In situ analysis with the Y‐probe showed extremely poor survival of isolated normal male (C57B1/10Sn) donor myoblasts after injection into injured or uninjured muscles of dystrophic (mdx) and normal (C57B1/10Sn) female host mice. A decrease in the numbers of donor (male) myoblasts was seen from 2 days and was marked by 7 days after injection: few or no donor myoblasts were detected in host muscles examined at 3–12 months. There was limited movement of the injected donor myoblasts and fusion into host myofibers was rare. The results of this study strongly suggest that the failure of clinical trials of myoblast transplantation therapy in boys with DMD may have been due to rapid and massive death of the donor myoblasts soon after myoblast injection.

The Role of Stem Cells in Skeletal and Cardiac Muscle Repair

In postnatal muscle, skeletal muscle precursors (myoblasts) can be derived from satellite cells (reserve cells located on the surface of mature myofibers) or from cells lying beyond the myofiber, e.g., interstitial connective tissue or bone marrow. Both of these classes of cells may have stem cell properties. In addition, the heretical idea that post-mitotic myonuclei lying within mature myofibers might be able to re-form myoblasts or stem cells is examined and related to recent observations for similar post-mitotic cardiomyocytes. In adult hearts (which previously were not considered capable of repair), the role of replicating endogenous cardiomyocytes and the recruitment of other (stem) cells into cardiomyocytes for new cardiac muscle formation has recently attracted much attention. The relative contribution of these various sources of precursor cells in postnatal muscles and the factors that may enhance stem cell participation in the formation of new skeletal and cardiac muscle in vivo are the focus of this review. We concluded that, although many endogenous cell types can be converted to skeletal muscle, the contribution of non-myogenic cells to the formation of new postnatal skeletal muscle in vivo appears to be negligible. Whether the recruitment of such cells to the myogenic lineage can be significantly enhanced by specific inducers and the appropriate microenvironment is a current topic of intense interest. However, dermal fibroblasts appear promising as a realistic alternative source of exogenous myoblasts for transplantation purposes. For heart muscle, experiments showing the participation of bone marrow-derived stem cells and endothelial cells in the repair of damaged cardiac muscle are encouraging.

Evans Blue Dye as an in vivo marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability

Evans Blue Dye (EBD) is widely used to study cellular membrane permeability and has recently been utilised in mdx mice to identify permeable skeletal myofibres that have become damaged as a result of muscular dystrophy. EBD has the potential to be a useful vital stain of myofibre permeability in other models of skeletal muscle injury and membrane‐associated fragility. The parameters for its use for such purposes were optimised in the present study. Of particular interest is the use of EBD to identify the onset of muscle damage. This study compared intravenous vs. intraperitoneal injection; tissue fixation; volume of EBD; time of availability in tissue; and persistence after injection in mdx mice (with endogenous muscle damage) and control mice. Satisfactory labelling of permeable myofibres was seen in frozen sections viewed with fluorescence microscopy when intraperitoneal injection of a 1% EBD solution injected at 1% volume relative to body mass was administered between 16 and 24 h prior to tissue sampling. EBD labelling was then assessed in three mouse models of experimental injury and repair – cut injury, whole muscle grafts, and exercise‐induced muscle damage. These experiments demonstrated that (i) following a cut injury across myofibres, EBD penetrated up to 150 µm from the injury site over a 20‐h period; (ii) EBD was present throughout myofibres of avascular whole muscle graft by one day after transplantation; and (iii) damaged myofibres were detected within 20 min after controlled lengthening–contraction exercise. This simple and inexpensive technique has sensitivity for the detection of increased myofibre permeability and/or sublethal damage that has advantages over other traditional histological techniques at the light microscopy level.

Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy

This review discusses various issues to consider when developing standard operating procedures for pre-clinical studies in the mdx mouse model of Duchenne muscular dystrophy (DMD). The review describes and evaluates a wide range of techniques used to measure parameters of muscle pathology in mdx mice and identifies some basic techniques that might comprise standardised approaches for evaluation. While the central aim is to provide a basis for the development of standardised procedures to evaluate efficacy of a drug or a therapeutic strategy, a further aim is to gain insight into pathophysiological mechanisms in order to identify other therapeutic targets. The desired outcome is to enable easier and more rigorous comparison of pre-clinical data from different laboratories around the world, in order to accelerate identification of the best pre-clinical therapies in the mdx mouse that will fast-track translation into effective clinical treatments for DMD.

Anti-TNFα (Remicade®) therapy protects dystrophic skeletal muscle from necrosis

Necrosis of skeletal muscle fibers in the lethal childhood myopathy Duchenne muscular dystrophy (DMD) results from defects in the cell membrane‐associated protein dystrophin. This study tests the novel hypothesis that the initial sarcolemmal breakdown resulting from dystrophin deficiency is exacerbated by inflammatory cells and that cytokines specifically tumor necrosis factor‐α (TNFα) contribute to muscle necrosis. To block in vivo TNFα bioactivity young dystrophic mdx mice (a model for DMD) were injected weekly from 7 days of age with the anti‐TNFα antibody Remicade® before the onset of muscle necrosis and dystropathology that normally occurs at 21 days postnatally. The extent of inflammation muscle necrosis and myotube formation was measured by histological analysis from 18 to 28 days and muscle damage was also visualized by penetration of Evans blue dye into myofibers. Data from Remicade ‐treated and control mdx mice were compared with mdx/TNFα(−/−) mice that lack TNFα. Pharmacological blockade of TNFα activity with Remicade® clearly delayed and greatly reduced the breakdown of dystrophic muscle in marked contrast to the situation in mdx and mdx/ TNFα(−/−) mice. Remicade had no adverse effect on new muscle formation. Remicade is a highly specific anti‐inflammatory intervention and clinical application to muscular dystrophies is suggested by this marked protective effect against skeletal muscle breakdown.—Grounds M. D. Torrisi J. Anti‐TNFα (Remicade) therapy protects dystrophic skeletal muscle from necrosis.

Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFα function with Etanercept in mdx mice

Necrosis of skeletal muscle fibres in the lethal childhood myopathy Duchenne Muscular Dystrophy results from deficiency of the cell membrane associated protein, dystrophin. We test the hypothesis in dystrophin-deficient mice, that the initial sarcolemmal breakdown resulting from dystrophin deficiency is exacerbated by inflammatory cells, specifically neutrophils, and that cytokines, specifically Tumour Necrosis Factor alpha (TNFalpha), contribute to myofibre necrosis. Antibody depletion of host neutrophils resulted in a delayed and significantly reduced amount of skeletal muscle breakdown in young dystrophic mdx mice. A more striking and prolonged protective effect was seen after pharmacological blockade of TNFalpha bioactivity using Etanercept. The extent of exercise induced myofibre necrosis in adult mdx mice after voluntarily wheel exercise was also reduced after Etanercept administration. These data show a clear role for neutrophils and TNFalpha in necrosis of dystrophic mdx muscle in vivo. Etanercept is a highly specific anti-inflammatory drug, widely used clinically, and potential application to muscular dystrophies is suggested by this reduced breakdown of mdx skeletal muscle.

Molecular and cell biology of skeletal muscle regeneration

When skeletal muscle is damaged, it is repaired by the proliferation of mononuclear muscle precursor cells (mpc) which fuse either with one another to form young multinucleated muscle cells (myotubes) or with the ends of damaged myofibres (Robertson et al., 1990). The success of new muscle formation is related to the size of the injury, as after major trauma and extensive disruption of the external lamina of muscle fibres there is often significant replacement by fibrous and cellular connective tissue. Impaired muscle regeneration and progressive replacement by fat and connective tissue is a feature of myopathies such as Duchenne muscular dystrophy (DMD), although this results from many small discrete lesions constantly recurring over a long period of time rather than from a single large injury. Failed regeneration can be seen in simplistic terms as a failure of muscle precursor replication. In this review we shall concentrate on the biology of muscle precursor cells. For coverage of other aspects of regeneration such as resealing of damaged myofibres, revascularization and reinnervation, see Grounds (1991).