Regeneration Of Dystrophic Muscle Research Articles

Bioprinted Notch ligand to function as stem cell niche improves muscle regeneration in dystrophic muscle.

299In Duchenne muscular dystrophy, dystrophic muscle phenotypes are closely associated with the exhaustion of muscle stem cells. Transplantation of muscle stem cells has been widely studied for improving muscle regeneration, but poor cell survival and self-renewal, rapid loss of stemness, and limited dispersion of grafted cells following transplantation have collectively hindered the overall success of this strategy. Optimized mechanisms for maintaining and improving stem cell function are naturally present in the microenvironment of the stem cell niche in healthy muscles. Therefore, one logical strategy toward improving stem cell function and efficiency of stem cell transplantation in diseased muscles would be the establishment of a microenvironment mimicking some key aspects of healthy native stem cell niches. Here, we applied inkjet-based bioprinting technology to engineer a mimicked artificial stem cell niche in dystrophic muscle, comprising stem cell niche regulating factors (Notch activator DLL1) bioprinted onto 3D DermaMatrix construct. The recombinant DLL1 protein, DLL1 (mouse): Fc (human) (rec), was applied here as the Notch activator. Bioprinted DermaMatrix construct was seeded with muscle stem cells in vitro, and increased stem cell maintenance and repressed myogenic differentiation process was observed. DLL1 bioprinted DermaMatrix construct was then engrafted into dystrophic muscle of mdx/scid mice, and the improved cell engraftment and progression of muscle regeneration was observed 10 days after engraftment. Our results demonstrated that bioprinting of Notch activator within 3D construct can be applied to serve as muscle stem cell niche and improve the efficacy of muscle stem cell transplantation in diseased muscle.

Lipin1 plays complementary roles in myofibre stability and regeneration in dystrophic muscles.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by dystrophin mutations, leading to the loss of sarcolemmal integrity, and resulting in progressive myofibre necrosis and impaired muscle function. Our previous studies suggest that lipin1 is important for skeletal muscle regeneration and myofibre integrity. Additionally, we discovered that mRNA expression levels of lipin1 were significantly reduced in skeletal muscle of DMD patients and the mdx mouse model. To understand the role of lipin1 in dystrophic muscle, we generated dystrophin/lipin1 double knockout (DKO) mice, and compared the limb muscle pathology and function of wild-type B10, muscle-specific lipin1 deficient (lipin1Myf5cKO ), mdx and DKO mice. We found that further knockout of lipin1 in dystrophic muscle exhibited a more severe phenotype characterized by increased necroptosis, fibrosis and exacerbated membrane damage in DKO compared to mdx mice. In barium chloride-induced muscle injury, both lipin1Myf5cKO and DKO showed prolonged regeneration at day 14 post-injection, suggesting that lipin1 is critical for muscle regeneration. In situ contractile function assays showed that lipin1 deficiency in dystrophic muscle led to reduced specific force production. Using a cell culture system, we found that lipin1 deficiency led to elevated expression levels of necroptotic markers and medium creatine kinase, which could be a result of sarcolemmal damage. Most importantly, restoration of lipin1 inhibited the elevation of necroptotic markers in differentiated primary lipin1-deficient myoblasts. Overall, our data suggests that lipin1 plays complementary roles in myofibre stability and muscle function in dystrophic muscles, and overexpression of lipin1may serve as a potential therapeutic strategy for dystrophic muscles. KEY POINTS: We identified that lipin1mRNA expression levels are significantly reduced in skeletal muscles of Duchenne muscular dystrophy patients and mdx mice. We found that further depletion of lipin1 in skeletal muscles of mdx mice induces more severe dystrophic phenotypes, including enhanced myofibre sarcolemma damage, muscle necroptosis, inflammation, fibrosis and reduced specific force production. Lipin1 deficiency leads to elevated expression levels of necroptotic markers, whereas restoration of lipin1 inhibits their expression. Our results suggest that lipin1 is functionally complementary to dystrophin in muscle membrane integrity and muscle regeneration.

HDAC inhibitors tune miRNAs in extracellular vesicles of dystrophic muscle-resident mesenchymal cells.

We show that extracellular vesicles (EVs) released by mesenchymal cells (i.e., fibro-adipogenic progenitors-FAPs) mediate microRNA (miR) transfer to muscle stem cells (MuSCs) and that exposure of dystrophic FAPs to HDAC inhibitors (HDACis) increases the intra-EV levels of a subset of miRs, which cooperatively target biological processes of therapeutic interest, including regeneration, fibrosis, and inflammation. Increased levels of miR-206 in EVs released by FAPs of muscles from Duchenne muscular dystrophy (DMD) patients or mdx mice exposed to HDACi are associated with enhanced regeneration and decreased fibrosis. Consistently, EVs from HDACi-treated dystrophic FAPs can stimulate MuSC activation and expansion exvivo, and promote regeneration, while inhibiting fibrosis and inflammation of dystrophic muscles, upon intramuscular transplantation in mdx mice, invivo. AntagomiR-mediated blockade of individual miRs reveals a specific requirement of miR-206 for EV-induced expansion of MuSCs and regeneration of dystrophic muscles, and indicates that cooperative activity of HDACi-induced miRs accounts for the net biological effect of these EVs. These data point to pharmacological modulation of EV content as novel strategy for therapeutic interventions in muscular dystrophies.

ZEB1 protects skeletal muscle from damage and is required for its regeneration

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration.

Genetic manipulation of CCN2/CTGF unveils cell-specific ECM-remodeling effects in injured skeletal muscle.

In skeletal muscle, extracellular matrix (ECM) remodeling can either support the complete regeneration of injured muscle or facilitate pathologic fibrosis and muscle degeneration. Muscular dystrophy (MD) is a group of genetic disorders that results in a progressive decline in muscle function and is characterized by the abundant deposition of fibrotic tissue. Unlike acute injury, where ECM remodeling is acute and transient, in MD, remodeling persists until fibrosis obstructs the regenerative efforts of diseased muscles. Thus, understanding how ECM is deposited and organized is critical in the context of muscle repair. Connective tissue growth factor (CTGF or CCN2) is a matricellular protein expressed by multiple cell types in response to tissue injury. Although used as a general marker of fibrosis, the cell type-dependent role of CTGF in dystrophic muscle has not been elucidated. To address this question, a conditional Ctgf myofiber and fibroblast-knockout mouse lines were generated and crossed to a dystrophic background. Only myofiber-selective inhibition of CTGF protected δ-sarcoglycan-null ( Sgcd-/-) mice from the dystrophic phenotype, and it did so by affecting collagen organization in a way that allowed for improvements in dystrophic muscle regeneration and function. To confirm that muscle-specific CTGF functions to mediate collagen organization, we generated mice with transgenic muscle-specific overexpression of CTGF. Again, genetic modulation of CTGF in muscle was not sufficient to drive fibrosis, but altered collagen content and organization after injury. Our results show that the myofibers are critical mediators of the deleterious effects associated with CTGF in MD and acutely injured skeletal muscle.-Petrosino, J. M., Leask, A., Accornero, F. Genetic manipulation of CCN2/CTGF unveils cell-specific ECM-remodeling effects in injured skeletal muscle.

Epigenetic Regulators Modulate Muscle Damage in Duchenne Muscular Dystrophy Model.

Histone acetyl transferases (HATs) and histone deacetylases (HDAC) control transcription during myogenesis. HDACs promote chromatin condensation, inhibiting gene transcription in muscle progenitor cells until myoblast differentiation is triggered and HDACs are released. HATs, namely CBP/p300, activate myogenic regulatory and elongation factors promoting myogenesis. HDAC inhibitors are known to improve regeneration in dystrophic muscles through follistatin upregulation. However, the potential of directly modulating HATs remains unexplored. We tested this possibility in a well-known zebrafish model of Duchenne muscular dystrophy. Interestingly, CBP/p300 transcripts were found downregulated in the absence of Dystrophin. While investigating CBP rescuing potential we observed that dystrophin-null embryos overexpressing CBP actually never show significant muscle damage, even before a first regeneration cycle could occur. We found that the pan-HDAC inhibitor trichostatin A (TSA) also prevents early muscle damage, however the single HAT CBP is as efficient even in low doses. The HAT domain of CBP is required for its full rescuing ability. Importantly, both CBP and TSA prevent early muscle damage without restoring endogenous CBP/p300 neither increasing follistatin transcripts. This suggests a new mechanism of action of epigenetic regulators protecting dystrophin-null muscle fibres from detaching, independent from the known improvement of regeneration upon damage of HDACs inhibitors. This study builds supporting evidence that epigenetic modulators may play a role in determining the severity of muscle dystrophy, controlling the ability to resist muscle damage. Determining the mode of action leading to muscle protection can potentially lead to new treatment options for muscular dystrophies in the future.

Upregulated IL-1β in dysferlin-deficient muscle attenuates regeneration by blunting the response to pro-inflammatory macrophages.

BackgroundLoss-of-function mutations in the dysferlin gene (DYSF) result in a family of muscle disorders known collectively as the dysferlinopathies. Dysferlin-deficient muscle is characterized by inflammatory foci and macrophage infiltration with subsequent decline in muscle function. Whereas macrophages function to remove necrotic tissue in acute injury, their prevalence in chronic myopathy is thought to inhibit resolution of muscle regeneration. Two major classes of macrophages, classical (M1) and alternative (M2a), play distinct roles during the acute injury process. However, their individual roles in chronic myopathy remain unclear and were explored in this study.MethodsTo test the roles of the two macrophage phenotypes on regeneration in dysferlin-deficient muscle, we developed an in vitro co-culture model of macrophages and muscle cells. We assayed the co-cultures using ELISA and cytokine arrays to identify secreted factors and performed transcriptome analysis of molecular networks induced in the myoblasts.ResultsDysferlin-deficient muscle contained an excess of M1 macrophage markers, compared with WT, and regenerated poorly in response to toxin injury. Co-culturing macrophages with muscle cells showed that M1 macrophages inhibit muscle regeneration whereas M2a macrophages promote it, especially in dysferlin-deficient muscle cells. Examination of soluble factors released in the co-cultures and transcriptome analysis implicated two soluble factors in mediating the effects: IL-1β and IL-4, which during acute injury are secreted from M1 and M2a macrophages, respectively. To test the roles of these two factors in dysferlin-deficient muscle, myoblasts were treated with IL-4, which improved muscle differentiation, or IL-1β, which inhibited it. Importantly, blockade of IL-1β signaling significantly improved differentiation of dysferlin-deficient cells.ConclusionsWe propose that the inhibitory effects of M1 macrophages on myogenesis are mediated by IL-1β signals and suppression of the M1-mediated immune response may improve muscle regeneration in dysferlin deficiency. Our studies identify a potential therapeutic approach to promote muscle regeneration in dystrophic muscle.

A novel quantitative morphometry approach to assess regeneration in dystrophic skeletal muscle

Duchenne muscular dystrophy is an inherited degenerative muscle disease with progressive weakness of skeletal and cardiac muscle. Disturbed calcium homeostasis and signalling pathways result in degeneration/regeneration cycles with fibrotic remodelling of muscle tissue, sustained by chronic inflammation. In addition to altered microarchitecture, regeneration in dystrophic muscle fibres is often only classified by centrally located nuclei but correlation of the regeneration process to nuclear volumes, myosin amounts, architecture and functional quality are missing, in particular in old muscles where the regenerative capacity is exhausted. Such information could yield novel regeneration-to-function biomarkers. Here we used second harmonic generation and multi photon fluorescence microscopy in intact single muscle fibres from wild-type, dystrophic mdx and transgenic mdx mice expressing an Δex 17–48 mini-dystrophin to determine the percentage of centronucleated fibres and nucleus-to-myosin volume ratio as a function of age. Based on this ratio we define a ‘biomotoric efficiency’ as an optical measure for fibre maturation, which is close to unity in adult wild-type and mini-dystrophin fibres, but smaller in very young and old mdx mice as a result of ongoing cell maturation (young) and regeneration (aged). With these parameters it is possible to provide a quantitative measure about muscle fibre regeneration.

Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

BackgroundPresently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice.MethodsWe treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles.ResultsShort-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function.ConclusionsThese data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD.

Fibroadipogenic progenitors mediate the ability of HDAC inhibitors to promote regeneration in dystrophic muscles of young, but not old Mdx mice

HDAC inhibitors (HDACi) exert beneficial effects in mdx mice, by promoting endogenous regeneration; however, the cellular determinants of HDACi activity on dystrophic muscles have not been determined. We show that fibroadipogenic progenitors (FAP) influence the regeneration potential of satellite cells during disease progression in mdx mice and mediate HDACi ability to selectively promote regeneration at early stages of disease. FAPs from young mdx mice promote, while FAPs from old mdx mice repress, satellite cell-mediated formation of myotubes. In young mdx mice HDACi inhibited FAP adipogenic potential, while enhancing their ability to promote differentiation of adjacent satellite cells, through upregulation of the soluble factor follistatin. By contrast, FAPs from old mdx mice were resistant to HDACi-mediated inhibition of adipogenesis and constitutively repressed satellite cell-mediated formation of myotubes. We show that transplantation of FAPs from regenerating young muscles restored HDACi ability to increase myofibre size in old mdx mice. These results reveal that FAPs are key cellular determinants of disease progression in mdx mice and mediate a previously unappreciated stage-specific beneficial effect of HDACi in dystrophic muscles.

Emerging strategies for cell and gene therapy of the muscular dystrophies

The muscular dystrophies are a heterogeneous group of over 40 disorders that are characterised by muscle weakness and wasting. The most common are Duchenne muscular dystrophy and Becker muscular dystrophy, which result from mutations within the gene encoding dystrophin; myotonic dystrophy type 1, which results from an expanded trinucleotide repeat in the myotonic dystrophy protein kinase gene; and facioscapulohumeral dystrophy, which is associated with contractions in the subtelomeric region of human chromosome 1. Currently the only treatments involve clinical management of symptoms, although several promising experimental strategies are emerging. These include gene therapy using adeno-associated viral, lentiviral and adenoviral vectors and nonviral vectors, such as plasmid DNA. Exon-skipping and cell-based therapies have also shown promise in the effective treatment and regeneration of dystrophic muscle. The availability of numerous animal models for Duchenne muscular dystrophy has enabled extensive testing of a wide range of therapeutic approaches for this type of disorder. Consequently, we focus here on the therapeutic developments for Duchenne muscular dystrophy as a model of the types of approaches being considered for various types of dystrophy. We discuss the advantages and limitations of each therapeutic strategy, as well as prospects and recent successes in the context of future clinical applications.

Perivascular CD45−:Sca-1+:CD34− Cells Are Derived from Bone Marrow Cells and Participate in Dystrophic Skeletal Muscle Regeneration.

Multiple mechanisms may account for bone marrow (BM) cell incorporation into myofibers following muscle damage. Here, we demonstrated that CD45−:Sca-1+:CD34− cells may play a role in the regeneration of mdx4cv skeletal muscles, an animal model for Duchenne muscular dystrophy. To understand the origin of CD45−:Sca-1+:CD34− cells in skeletal muscle, we reconstituted lethally irradiated wild type (wt) or mdx4cv mice with unfractionated BM cells from transgenic mice ubiquitously expressing green fluorescence protein (GFP). 1, 2, and 6 months post-transplantation, we analyzed the skeletal muscle mononuclear cells from the recipients by flow cytometry for GFP, CD45-PerCP, Sca-1-PE, and CD34-APC. To our surprise, we found a small percentage of BM-derived (GFP+) CD45−:Sca-1+:CD34− cells in the skeletal muscles of these GFP+ BM transplant recipients. These BM-derived cells are localized in the perivascular tissue by immunostaining and their frequency increases with time. We were interested in the potential of these cells for clinical application for muscle diseases. Thus, we FACS-sorted CD45−:Sca-1+:CD34− cells from GFP transgenic mouse skeletal muscles and transplanted them in the tibialis anterior (TA) muscle of mdx4cv mice. In ten days we found significant muscle engraftment. In addition, we studied the response of this population upon acute muscle injury. For this, we injected cardiotoxin in the right TA muscle of mdx4cv and wt mice, followed by BrdU administration in drinking water for three days. After 3.5 days, mice were sacrificed and the right and left (control) TA muscles were harvested and muscle CD45−:Sca-1+:CD34− cells were FACS-sorted, fixed and stained for BrdU and Myf-5. In the injured muscle (right TA), more than 70% of these cells were BrdU+ and more than 50% were Myf-5+, compared to baseline levels (close to zero) in the left TA. This indicates that this population can undergo proliferation and myogenic commitment upon muscle injury. To understand how these BM cells migrate to the muscle and once in the muscle how they mobilize, we investigated the in vitro chemotatic response of GFP+ (BM derived) CD45−:Sca-1+ cells isolated from muscles of GFP+ BM transplant recipients. We found that these cells were highly chemoattrated to stroma derived factor, SDF-1, a chemo-attractant for cells expressing CXCR4. We also observed higher frequency of BM-derived CD45−:Sca-1+:CD34− cells in dystrophic muscle than wt muscle, which may be explained by higher expression levels of SDF-1 in dystrophic muscles. In an effort to determine the identity of these cells when ex vivo cultured, we cultured them in several stem cell media, including a low-serum medium containing specific cytokines for the isolation and expansion of multipotent adult progenitor cells (MAPCs). MAPCs can be isolated from skeletal muscle and BM and can differentiate into multiple tissue cells. Strikingly, we found that MAPCs were enriched up to 40 folds by sorting this population from skeletal muscle. The frequency of BM-derived muscle MAPCs also increases with time post-transplantation in dystrophic muscles. These BM-derived muscle MAPCs displayed the typical MAPC immunophenotypes, displayed a normal diploid karyotype and were capable to differentiate into endothelial cells, hepatocytes and neurons.Taken together, our results suggest that dystrophic muscles recruit BM cells that localize in perivascular tissues and can be defined as CD45-:Sca-1+:CD34-. This population when cultured enriches for MAPCs and can participate in muscle regeneration in dystrophic muscles.

1127. Perivascular CD45-:Sca-1+:CD34- Cells Are Derived from Bone Marrow Cells and Participate in Dystrophic Skeletal Muscle Regeneration

Multiple mechanisms may account for bone marrow (BM) cell incorporation into myofibers following muscle damage. Here, we demonstrated that mouse CD45|[minus]|:Sca-1+:CD34|[minus]| cells may play a role in the maturation of skeletal muscles and regeneration of mdx4cv dystrophic skeletal muscles, an animal model for Duchenne muscular dystrophy. To understand the origin of CD45|[minus]|:Sca-1+:CD34|[minus]| cells in mouse skeletal muscle, we reconstituted lethally irradiated wild type or mdx4cv mice with unfractionated BM cells from transgenic mice ubiquitously expressing green fluorescence protein (GFP). 1, 2, and 6 months post-transplantation, we analyzed the skeletal muscle mononuclear cells from the recipients by flow cytometry for GFP, CD45-PerCP, Sca-1-PE, and CD34-APC. To our surprise, we found BM-derived (GFP+) CD45|[minus]|:Sca-1+:CD34|[minus]| cells in the skeletal muscles of these GFP+ BM transplant recipients. We also demonstrated that these BM-derived cells were localized in the perivascular tissue by immuno-staining and that their frequency increased with time. We were interested in the potential clinical application of these cells for muscle diseases. Thus, we sorted CD45|[minus]|:Sca-1+:CD34|[minus]| cells by fluorescence activated cell sorting (FACS) skeletal muscle mononuclear cells and cultured them in several stem cell media (recipes), including a low-serum medium containing specific cytokines for isolating multipotent adult progenitor cells (MAPCs). MAPCs can be isolated from skeletal muscle and BM and differentiate to form myotubes in vitro and in vivo. Strikingly, we found that MAPCs were enriched up to 40 folds by sorting this population from skeletal muscle mononuclear cells. Concomitantly with the increase in frequency of BM-derived muscle CD45|[minus]|:Sca-1+:CD34|[minus]| cells, frequency of BM-derived muscle MAPCs also increased with time post-transplantation in dystrophic muscles. Furthermore, BM-derived muscle MAPCs displayed similar phenotypes of endogenous muscle MAPCs, suggesting a potential mechanism of BM cell migration to dystrophic skeletal muscles. To understand how these BM-derived cells migrate to the muscle and once in the muscle how they mobilize, we investigated the in vitro chemotatic response of GFP+ (BM derived) muscle MAPCs and CD45|[minus]|:Sca-1+ cells isolated from muscles of GFP+ BM transplant recipients. We found that these cells were highly chemoattrated to stroma derived factor, SDF-1, a chemo-attractant for cells expressing CXCR4. We also observed higher frequency of BM-derived CD45|[minus]|:Sca-1+:CD34|[minus]| cells in mdx dystrophic muscle than wild type muscle, which may be explained by higher expression levels of SDF-1 in mdx dystrophic muscles. Taken together, our results suggest that dystrophic muscles recruit BM cells that localize in perivascular tissues and can be defined as CD45|[minus]|:Sca-1+:CD34|[minus]|. This population when cultured enriches for MAPCs and can participate in muscle regeneration in dystrophic muscles.

241. Lentivirus Mediated Dystrophin Expression in mdx Muscles

Top of pageAbstract To study candidate treatments for Duchenne muscular dystrophy, we have generated a series of lentiviral vectors that express various reporter genes and mini-dystrophin cassettes and tested their ability to transduce a variety of cell types in vitro and in vivo. Direct injection of lentiviral vectors into adult skeletal muscle resulted in significantly lower levels of gene expression than were obtained using either AAV or adenoviral vectors. Since an advantage of lentiviral vectors is stable expression of a transgene that has integrated into host genomic DNA, stem cells are considered a good target of lentiviral vectors. During myogenesis, activated-satellite cells or muscle progenitor cells proliferate in the muscle microenvironment. Most of these cells become differentiated and form myofibers, while small numbers of them are stored adjacent to myofibers as mitotically quiescent satellite cells for future muscle regeneration. Skeletal muscle of newborn mice may be a good target for lentiviral vectors, because of relatively high numbers of activated muscle progenitor cells contributing to muscle growth and satellite cell pools. Therefore, we tried targeting satellite cells or muscle progenitor cells using lentivirual vectors expressing dystrophin mini-gene. We demonstrated that relatively higher levels of transuction were obtained with intra-muscular injection into neonatal muscles, and that both muscle fibers and primary cultured satellite cells stably expressed a GFP marker gene. We also demonstrated that lentiviral mediated truncated mini-dystrophin expression in mdx muscles may be useful for correction of pathological changes in dystrophic muscle. These data suggest that myogenic stem cells can be stably transduced with lentiviral vectors and may contribute to stable muscle regeneration in dystrophic muscle by enabling continuous expression of mini-dystrophin, which may have implications for gene therapy of Duchenne muscular dystrophy.

Increased caveolin-3 levels in mdx mouse muscles

The density of skeletal muscle caveolae is increased in Duchenne muscular dystrophy and its genetic homologue, the mdx mouse. This structural change is significant as it may indicate muscle regeneration. We identified in mdx mouse tibialis anterior muscles significantly increased levels of caveolin-3, the chief protein in muscle caveolae, and reduced levels of neuronal nitric oxide synthase, an enzyme regulated by caveolin-3. Similar changes occurred in the corresponding mRNA levels. These data suggest that induction of caveolin-3 occurs and this may at least partly be responsible for increased number of caveolae, altered nNOS-caveolin cycle, and regeneration of dystrophic muscles.

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.

Differential glucocorticoid effects on the fusion of Duchenne/Becker and control muscle cultures: pharmacologic detection of accelerated aging in dystrophic muscle.

We report that the glucocorticoid methylprednisolone (Mepd) enhanced myogenesis in normal primary human muscle cultures, but inhibited myogenesis of most Duchenne/Becker muscle cultures. A decline in the magnitude of myogenic stimulation of Mepd correlated with age in a random group of control patients, including some with neurologic diseases other than Duchenne/Becker dystrophy. A case of Duchenne muscular dystrophy from an exceptionally young patient yielded a muscle culture that was myogenically stimulated by Mepd. These results suggest that continuous cycles of degeneration and regeneration of dystrophic muscle in vivo may result in a change of the glucocorticoid response of the muscle progenitor cells. The glucocorticoid effects suggest caution in the long-term clinical use of these agents for muscle disease such as Duchenne muscular dystrophy.

Progressive Muscular Dystrophy with Particular Reference to Muscle Regeneration

In various progressive muscular dystrophies (PMD), there were a number of fibers with histological and histochemical characteristics of regenerating fibers. With a morphometric analysis in diseased muscles, an identification of type 2C fiber on ATPase stain was the most simple and reliable method for fiber type evaluation. The type 2C fiber comprised 16.5% in Duchenne and 27.5% in Fukuyama type congenital MD, suggesting an active regenerating process taking place in both diseases. Regenerative capacity of muscle fibers after experimental damage to dystrophic chicken and mdx mouse muscles was similar to those seen in nondystrophic control muscles. From the above results, we speculate that a certain environmental factor such as interstitial fibrosis seems to play a major role in delaying regeneration in dystrophic muscles.

Regeneration of dystrophic muscle following multiple injections of bupivacaine.

Regenerated myofibers formed subsequent to orthotopic transplantation of young, dystrophic mouse muscle fail to display the extensive histopathological changes characteristics of murine dystrophy. In order to determine whether this modification of the phenotypic expression of murine dystrophy is unique to the transplantation system or whether it can be found when other extreme trauma induces dystrophic muscle to regenerate, the extensor digitorum longus muscles of 4-6-week-old normal (129 ReJ +/+) and dystrophic (129 ReJ dy/dy) mice were given two series of injections of the myotoxin bupivacaine, spaced 12 hours apart. These injections resulted in necrosis of approximately 90% of the original myofibers. At 100 days after injection, the regenerated normal muscle appeared "healthy," whereas the regenerated dystrophic muscle displayed histopathological changes. It is suggested that the differences in the time course of innervation of the myotubes in the transplantation system as compared with that in the bupivacaine system may be a factor in determining whether regenerated dystrophic myofibers express a dystrophic morphology.

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.