Necdin enhances muscle reconstitution of dystrophic muscle by vessel-associated progenitors, by promoting cell survival and myogenic differentiation

Improving stem cell therapy is a major goal for the treatment of muscle diseases, where physiological muscle regeneration is progressively exhausted. Vessel-associated stem cells, such as mesoangioblasts (MABs), appear to be the most promising cell type for the cell therapy for muscular dystrophies and have been shown to significantly contribute to restoration of muscle structure and function in different muscular dystrophy models. Here, we report that melanoma antigen-encoding gene (MAGE) protein necdin enhances muscle differentiation and regeneration by MABs. When necdin is constitutively overexpressed, it accelerates their differentiation and fusion in vitro and it increases their efficacy in reconstituting regenerating myofibres in the α-sarcoglycan dystrophic mouse. Moreover, necdin enhances survival when MABs are exposed to cytotoxic stimuli that mimic the inflammatory dystrophic environment. Taken together, these data demonstrate that overexpression of necdin may be a crucial tool to boost therapeutic applications of MABs in dystrophic muscle.     

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.     

PDGF-receptor concentration is elevated in regenerative muscle fibers in dystrophin-deficient muscle.

Dystrophin-deficient muscle undergoes sudden, postnatal onset of muscle necrosis that is either progressive, as in Duchenne muscular dystrophy, or successfully arrested and followed by regeneration, as in most muscles of mdx mice. The mechanisms regulating regeneration in mdx muscle are unknown, although the possibility that there is renewed expression of genes regulating embryonic muscle cell proliferation and differentiation may provide testable hypotheses. Here, we examine the possibility that necrotic and regenerating mdx muscles exhibit renewed or increased expression of PDGF-receptors. PDGF-binding to receptors on muscle has been shown previously to be associated with myogenic cell proliferation and delay of muscle differentiation. We find that PDGF-receptors are present in 4-week-old mdx mice in muscles that undergo brief, reversible necrosis (hindlimb muscles) or progressive necrosis (diaphragm), as well as in 4-week-old control mouse muscles. Immunoblots indicate that the concentrations of PDGF-receptors in 4-week-old dystrophic (necrotic) and control muscles are similar. Prenecrotic, dystrophic fibers and control fibers possess some cell surface labeling of fibers treated with anti-PDGF-receptor and viewed by indirect immunofluorescence. Necrotic fibers in dystrophic muscle show cytoplasmic labeling for PDGF-receptors and labeling of perinuclear regions at the muscle cell surface. Adult dystrophic muscle displays higher concentrations of PDGF-receptor in both regenerated muscle (hindlimb) and progressively necrotic muscle (diaphragm) than found in controls. Anti-PDGF-receptor labeling of regenerated, dystrophic muscle is observed primarily in granules surrounding central nuclei or surrounding nuclei located at the surface of regenerated fibers. No labeling of perinuclear regions of control muscle or prenecrotic fibers was observed. Myonuclei fractionated from adult mdx hindlimb muscles contained no PDGF-receptor, indicating that PDGF-receptor-positive structures are not tightly associated with nuclei or within nuclei. L6 myoblasts show PDGF-receptor distributed diffusely on the cell surface. Stimulation of L6 myoblasts with 10 ng/ml of PDGF-BB causes receptor internalization and concentration in granules at perinuclear regions. Thus, PDGF stimulation of myoblasts causes a redistribution of PDGF-receptors to resemble receptor localization observed during muscle regeneration. These findings implicate PDGF-mediated mechanisms in regeneration of dystrophic muscle.     

Expression of mdr1 is required for efficient long term regeneration of dystrophic muscle

The mouse mdr1a and mdr1b genes are expressed in skeletal muscle, though their precise role in muscle is unknown. Dystrophic muscle is characterized by repeated cycles of degeneration and regeneration. To explore the role of the mdr1 genes during muscle regeneration, we have created a triple knockout mouse lacking the mdr1a, mdr1b, and the dystrophin genes. The resulting ReX mice developed normally and were fertile. However, as adults, ReX had a higher proportion of degenerating muscle fibers and greater long-term loss of muscle mass than mdx. ReX muscles were also characterized by a reduced proportion of muscle side population (mSP) cells, of myogenic cells, and a reduced capacity for muscle regeneration. We found too that mSP cells derived from dystrophic muscle are more myogenic than those from normal muscle. Thus, in dystrophic muscle, the mdr1 gene plays an important role in the preservation of the mSP and of the myogenic regenerative potential. Moreover, our results suggest a hitherto unappreciated role of mdr1 in precursor cells of regenerating tissue; they therefore provide an important clue to the physiological significance of mdr1 expression in stem cells.     

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.     

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.     

A role for cell sex in stem cell-mediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency

We have shown that muscle-derived stem cells (MDSCs) transplanted into dystrophic (mdx) mice efficiently regenerate skeletal muscle. However, MDSC populations exhibit heterogeneity in marker profiles and variability in regeneration abilities. We show here that cell sex is a variable that considerably influences MDSCs' regeneration abilities. We found that the female MDSCs (F-MDSCs) regenerated skeletal muscle more efficiently. Despite using additional isolation techniques and cell cloning, we could not obtain a male subfraction with a regeneration capacity similar to that of their female counterparts. Rather than being directly hormonal or caused by host immune response, this difference in MDSCs' regeneration potential may arise from innate sex-related differences in the cells' stress responses. In comparison with F-MDSCs, male MDSCs have increased differentiation after exposure to oxidative stress induced by hydrogen peroxide, which may lead to in vivo donor cell depletion, and a proliferative advantage for F-MDSCs that eventually increases muscle regeneration. These findings should persuade researchers to report cell sex, which is a largely unexplored variable, and consider the implications of relying on cells of one sex.     

Increased expression of deltaCaMKII isoforms in skeletal muscle regeneration: Implications in dystrophic muscle disease

The expression of delta isoforms of calcium-calmodulin/dependent protein kinase II (CaMKII) has been reported in mammalian skeletal muscle; however, their functions in this tissue are largely unknown. This study was conducted to determine if deltaCaMKII expression was altered during regeneration of skeletal muscle fibers in two distinct models. In the first model, necrosis and regeneration were induced in quadriceps of normal mice by intramuscular administration of 50% glycerol. Immunostaining and confocal microscopy revealed that deltaCaMKII expression was clearly enhanced in fibers showing centralized nuclei. The second model was the mdx mouse, which undergoes enhanced muscle necrosis and regeneration due to a mutation in the dystrophin gene. sern blot analysis of hind leg extracts from 4 to 6 week old mdx mice revealed that deltaCaMKII content was decreased when compared to age-matched control mice. This loss in delta kinase content was seen in myofibrillar and membrane fractions and was in contrast to unchanged deltaCaMKII levels in cardiac and brain extracts from dystrophic mice. Confocal microscopy of mdx quadriceps and tibialis muscle showed that deltaCaMKII expression was uniformly decreased in most fibers from dystrophic mice; however, enhanced kinase expression was observed in regenerating muscle fibers. These data support a fundamental role for deltaCaMKII in the regeneration process of muscle fibers in normal and mdx skeletal muscle and may have important implications in the reparative process following muscle death.     

Fibro-adipogenic progenitors in skeletal muscle homeostasis,regeneration and diseases

Skeletal muscle possesses a remarkable regenerative capacity that relies on the activity of muscle stem cells, also known as satellite cells. The presence of non-myogenic cells also plays a key role in the coordination of skeletal muscle regeneration. Particularly, fibro-adipogenic progenitors (FAPs) emerged as master regulators of muscle stem cell function and skeletal muscle regeneration. This population of muscle resident mesenchymal stromal cells has been initially characterized based on its bi-potent ability to differentiate into fibroblasts or adipocytes. New technologies such as single-cell RNAseq revealed the cellular heterogeneity of FAPs and their complex regulatory network during muscle regeneration. In acute injury, FAPs rapidly enter the cell cycle and secrete trophic factors that support the myogenic activity of muscle stem cells. Conversely, deregulation of FAP cell activity is associated with the accumulation of fibrofatty tissue in pathological conditions such as muscular dystrophies and ageing. Considering their central role in skeletal muscle pathophysiology, the regulatory mechanisms of FAPs and their cellular and molecular crosstalk with muscle stem cells are highly investigated in the field. In this review, we summarize the current knowledge on FAP cell characteristics, heterogeneity and the cellular crosstalk during skeletal muscle homeostasis and regeneration. We further describe their role in muscular disorders, as well as different therapeutic strategies targeting these cells to restore muscle regeneration.     

TNF/p38α/polycomb signaling to Pax7 locus in satellite cells links inflammation to the epigenetic control of muscle regeneration

How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified an inflammation-activated signaling in muscle stem (satellite) cells, by which the polycomb repressive complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38α kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EZH2, the enzymatic subunit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. TNF-α antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signaling--p38α and EZH2--invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signaling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signaling to Pax7 and satellite cell decision to proliferate or differentiate.     

Tropomyosin 4 defines novel filaments in skeletal muscle associated with muscle remodelling/regeneration in normal and diseased muscle

The organisation of structural proteins in muscle into highly ordered sarcomeres occurs during development, regeneration and focal repair of skeletal muscle fibers. The involvement of cytoskeletal proteins in this process has been documented, with nonmuscle gamma-actin found to play a role in sarcomere assembly during muscle differentiation and also shown to be up-regulated in dystrophic muscles which undergo regeneration and repair [Lloyd et al.,2004; Hanft et al.,2006]. Here, we show that a cytoskeletal tropomyosin (Tm), Tm4, defines actin filaments in two novel compartments in muscle fibers: a Z-line associated cytoskeleton (Z-LAC), similar to a structure we have reported previously [Kee et al.,2004], and longitudinal filaments that are orientated parallel to the sarcomeric apparatus, present during myofiber growth and repair/regeneration. Tm4 is upregulated in paradigms of muscle repair including induced regeneration and focal repair and in muscle diseases with repair/regeneration features, muscular dystrophy and nemaline myopathy. Longitudinal Tm4-defined filaments also are present in diseased muscle. Transition of the Tm4-defined filaments from a longitudinal to a Z-LAC orientation is observed during the course of muscle regeneration. This Tm4-defined cytoskeleton is a marker of growth and repair/regeneration in response to injury, disease state and stress in skeletal muscle.     

Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle.

Phospholipid incorporation of 32P by primary myotube cultures and the tissue activity of sarcolemmal Na+/K(+)-transporting ATPase were studied to determine whether the absence of dystrophin from dystrophic (mdx) muscle would affect membrane lipid synthesis and membrane function. The incorporation of 32P by phospholipid as a ratio with total protein was greater in cultured dystrophic cells compared with control cells. The mdx cells also incorporated more 32P than control cells into phosphatidylethanolamine, which is thought to increase prior to myoblast fusion, and less into phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. There was no difference in total protein content or [3H]leucine or 32P incorporation into the aqueous fraction of dystrophic and control cells, although dystrophic cells incorporated less [35S]methionine into protein than controls. Isolated sarcolemma from mdx skeletal muscle tissue demonstrated a consistently greater specific activity of ouabain-sensitive Na+/K(+)-transporting ATPase than sarcolemmal preparations from control skeletal muscle. These observations suggest that cytoskeletal changes such as dystrophin deficiency may alter the differentiation of membrane composition and function.     

Immuno-electron-microscopic localization of basic fibroblast growth factor in the dystrophic mdx mouse masseter muscle

Summary The localization of basic fibroblast growth factor (bFGF)-like immunoreactivity in the masseter muscle of dystrophic mdx mice on postnatal day 28 was investigated by immunoblot analysis and electron microscopy. Crude homogenate of the masseter muscle, when subjected to immunoblotting with a bFGF antiserum, exhibited a main band with the same molecular weight (18 kDa) as bovine bFGF. By electron microscopy, bFGF immunoreactivity was detected in small regenerating myocytes; the smaller cells were the premature myocytes, the most intense staining was the immunoreactivity within the cytoplasm. Putative precursors of the muscle cells with a few myofilaments, which were most intensely labeled with anti-bFGF, contacted each other and possibly developed into multinucleated myocytes through cell fusion. Mature myocytes with densely packed myofilaments and peripherally located nuclei did not exhibit bFGF immunoreactivity; they formed myoneural junctions with motor nerve endings immunoreactive for bFGF. Early differentiating myocytes with intense bFGF-like immunoreactivity did not make contact with immunoreactive nerve terminals. Degenerating large myocytes with a limited number of distorted and/or disrupted myofilaments exhibited electron-dense deposits in the cristae of mitochondria; these deposits were not abolished by immunoadsorption control experiments. Thus, the cell-size-dependent decrease in bFGF immunoreactivity in regenerating but not in degenerating myocytes provides a morphological basis for an autoregulatory role of bFGF in muscle regeneration. This study suggests that neuronal bFGF is not involved in initial muscle regeneration in the dystrophic mdx mouse.     

Contribution of human muscle-derived cells to skeletal muscle regeneration in dystrophic host mice

Background

Stem cell transplantation is a promising potential therapy for muscular dystrophies, but for this purpose, the cells need to be systemically-deliverable, give rise to many muscle fibres and functionally reconstitute the satellite cell niche in the majority of the patient''s skeletal muscles. Human skeletal muscle-derived pericytes have been shown to form muscle fibres after intra-arterial transplantation in dystrophin-deficient host mice. Our aim was to replicate and extend these promising findings.

Methodology/Principal Findings

Isolation and maintenance of human muscle derived cells (mdcs) was performed as published for human pericytes. Mdscs were characterized by immunostaining, flow cytometry and RT-PCR; also, their ability to differentiate into myotubes in vitro and into muscle fibres in vivo was assayed. Despite minor differences between human mdcs and pericytes, mdscs contributed to muscle regeneration after intra-muscular injection in mdx nu/nu mice, the CD56+ sub-population being especially myogenic. However, in contrast to human pericytes delivered intra-arterially in mdx SCID hosts, mdscs did not contribute to muscle regeneration after systemic delivery in mdx nu/nu hosts.

Conclusions/Significance

Our data complement and extend previous findings on human skeletal muscle-derived stem cells, and clearly indicate that further work is necessary to prepare pure cell populations from skeletal muscle that maintain their phenotype in culture and make a robust contribution to skeletal muscle regeneration after systemic delivery in dystrophic mouse models. Small differences in protocols, animal models or outcome measurements may be the reason for differences between our findings and previous data, but nonetheless underline the need for more detailed studies on muscle-derived stem cells and independent replication of results before use of such cells in clinical trials.     

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.     

Effect of nuclear factor κB inhibition on serotype 9 adeno-associated viral (AAV9) minidystrophin gene transfer to the mdx mouse

Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery.     

Regenerative activity of muscle tissue and thymus status in young and old mdx mice with muscle injury and wound xenoplasty

Responses of the skeletal muscle tissue and thymus to muscle injury (complete transection) and wound xenoplasty with the minced muscle tissue of newborn rats (tissue therapy) were studied in mdx mice aged 12–16 and 40–48 weeks. The muscle tissue of mdx mice has genetic defects causing chronic dystrophic processes in it. The muscle tissue of young mdx mice proved to retain a relatively high capacity for regeneration. Under conditions of tissue therapy of the wound, the formation of muscle fibers from muscle cells of the graft and active regeneration of muscle fibers in the recipient mice were observed, and no structural defects were detected in the thymus. The capacity of posttraumatic regeneration in old mdx mice was lower. The xenogenic graft was undergoing resorption, thereby suppressing regeneration of muscle fibers and causing further tissue destruction in the injured muscle. The thymus parenchyma was subject to degenerative changes such as the formation of gaps, hemorrhagic foci, and increased numbers of macrophages and mast cells.