Prostaglandin F2alpha promotes muscle cell survival and growth through upregulation of the inhibitor of apoptosis protein BRUCE.

During skeletal muscle growth and regeneration, the majority of differentiating myoblasts undergoes cell-cell fusion to form multinucleated myofibers, whereas a proportion of myoblasts undergoes apoptosis. The treatment of myoblasts with prostaglandin F2alpha (PGF2alpha) during myogenesis in vitro leads to the formation of large myotubes, but the mechanism by which PGF2alpha promotes myotube growth has not been investigated. Here, we demonstrate that PGF2alpha reduces cell death during myogenesis in vitro and in vivo. In addition, we show that PGF2alpha increases expression of the inhibitor of apoptosis protein (IAP) BRUCE through a pathway dependent on the nuclear factor of activated T cell 2 transcription factor. Importantly, PGF2alpha-mediated reduction in muscle cell death is dependent on BRUCE, and overexpression of BRUCE is sufficient to promote muscle cell survival and growth. These results establish a previously unrecognized link between NFAT signaling and regulation of IAP expression and are the first to identify a signaling pathway that increases BRUCE expression. In addition, our results provide evidence that increasing the pool of muscle cells available for fusion by inhibiting cell death enhances myotube growth.

A muscle precursor cell-dependent pathway contributes to muscle growth after atrophy.

Slow-twitch skeletal muscle atrophies greatly in response to unloading conditions. The cellular mechanisms that contribute to the restoration of muscle mass after atrophy are largely unknown. Here, we show that atrophy of the mouse soleus is associated with a 36% decrease in myonuclear number after 2 wk of hindlimb suspension. Myonuclear number is restored to control values during the 2-wk recovery period in which muscle mass returns to normal, suggesting that muscle precursor cells proliferate and fuse with myofibers. Inhibition of muscle precursor cell proliferation by local gamma-irradiation of the hindlimb completely prevents this increase in myonuclear number. Muscle growth occurs normally during the first week in irradiated muscles, but growth during the second week is inhibited, leading to a 50% attenuation in the restoration of muscle mass. Thus early muscle growth occurs independently of an increase in myonuclear number, whereas later growth requires proliferating muscle precursor cells leading to myonuclear accretion. These results suggest that increasing the proliferative capacity of muscle precursor cells may enhance restoration of muscle mass after atrophy.

Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway.

The nuclear factor of activated T cells (NFAT) family of transcription factors regulates the development and differentiation of several tissue types. Here, we examine the role of NFATC2 in skeletal muscle by analyzing adult NFATC2(-/)- mice. These mice exhibit reduced muscle size due to a decrease in myofiber cross-sectional area, suggesting that growth is blunted. Muscle growth was examined during regeneration after injury, wherein NFATC2-null myofibers form normally but display impaired growth. The growth defect is intrinsic to muscle cells, since the lack of NFATC2 in primary muscle cultures results in reduced cell size and myonuclear number in myotubes. Retroviral-mediated expression of NFATC2 in the mutant cells rescues this cellular phenotype. Myonuclear number is similarly decreased in NFATC2(-/)- mice. Taken together, these results implicate a novel role for NFATC2 in skeletal muscle growth. We demonstrate that during growth of multinucleated muscle cells, myoblasts initially fuse to form myotubes with a limited number of nuclei and that subsequent nuclear addition and increases in myotube size are controlled by a molecular pathway regulated by NFATC2.

Altered primary myogenesis in NFATC3(-/-) mice leads to decreased muscle size in the adult.

Signal transduction pathways involving calcineurin and its downstream effector NFAT have been implicated in regulating myogenesis. Several isoforms of NFAT exist that may differentially contribute to regulating skeletal muscle physiology. The purpose of this study was to determine the role of the NFATC3 isoform in skeletal muscle development. Adult mice lacking NFATC3 have reduced muscle mass compared to control mice. The smaller size of the muscles is not due to atrophy or blunted myofiber growth, but rather to a reduced number of myofibers. This reduction in myofiber number is not limited to a specific fiber type nor are the proportions of fiber types altered. The lower fiber number found in the adult NFATC3(-/-) mice is a consequence of impaired muscle development during embryogenesis. Immunohistochemical studies of E15 EDL muscles indicate that the total number of primary myofibers is decreased in NFATC3(-/-) embryos. At E17.5 no further decrease in primary myofiber number occurs; the size and organization of the myofibers are unaltered, and secondary myogenesis proceeds normally, suggesting a role for NFATC3 during early events in primary myogenesis. These results suggest a heretofore unknown role for the transcription factor NFAT in early skeletal muscle development.

A calcineurin- and NFAT-dependent pathway regulates Myf5 gene expression in skeletal muscle reserve cells.

Myf5 is a member of the muscle regulatory factor family of transcription factors and plays an important role in the determination, development, and differentiation of skeletal muscle. However, factors that regulate the expression and activity of Myf5 itself are not well understood. Recently, a role for the calcium-dependent phosphatase calcineurin was suggested in three distinct pathways in skeletal muscle: differentiation, hypertrophy, and fiber-type determination. We propose that one downstream target of calcineurin and the calcineurin substrate NFAT in skeletal muscle is regulation of Myf5 gene expression. For these studies, we used myotube cultures that contain both multinucleated myotubes and quiescent, mononucleated cells termed 'reserve' cells, which share many characteristics with satellite cells. Treatment of such myotube cultures with the calcium ionophore ionomycin results in an approximately 4-fold increase in Myf5 mRNA levels, but similar effects are not observed in proliferating myoblast cultures indicating that Myf5 is regulated by different pathways in different cell populations. The increase in Myf5 mRNA levels in myotube cultures requires the activity of calcineurin and NFAT, and can be specifically enhanced by overexpressing the NFATc isoform. We used immunohistochemical analyses and fractionation of the cell populations to demonstrate that the calcium regulated expression of Myf5 occurs in the mononucleated reserve cells. We conclude that Myf5 gene expression is regulated by a calcineurin- and NFAT-dependent pathway in the reserve cell population of myotube cultures. These results may provide important insights into the molecular mechanisms responsible for satellite cell activation and/or the renewal of the satellite cell pool following activation and proliferation.

Sexually dimorphic expression of myosin heavy chains in the adult mouse masseter.

Little is known regarding the role of androgenic hormones in the maintenance of myosin heavy chain (MHC) composition of rodent masticatory muscles. Because the masseter is the principal jaw closer in rodents, we felt it was important to characterize the influence of androgenic hormones on the MHC composition of the masseter. To determine the extent of sexual dimorphism in the phenotype of masseter muscle fibers of adult (10-mo-old) C57 mice, we stained tissue sections with antibodies specific to type IIa and IIb MHC isoforms. Females contain twice as many fibers containing the IIa MHC as males, and males contain twice as many fibers containing the IIb MHC as females. There is a modest amount of regionalization of MHC phenotypes in the mouse masseter. The rostral portions of the masseter are composed mostly of type IIa fibers, whereas the midsuperficial and caudal regions contain mostly type IIb fibers. Using immunoblots, we showed that castration results in an increase in the expression of type IIa MHC fibers in males. Ovariectomy has no effect on the fiber type composition in females. We conclude that testosterone plays a role in the maintenance of MHC expression in the adult male mouse masseter.

Calcineurin activity is required for the initiation of skeletal muscle differentiation.

Differentiation of skeletal muscle myoblasts follows an ordered sequence of events: commitment, cell cycle withdrawal, phenotypic differentiation, and finally cell fusion to form multinucleated myotubes. The molecular signaling pathways that regulate the progression are not well understood. Here we investigate the potential role of calcium and the calcium-dependent phosphatase calcineurin in myogenesis. Commitment, phenotypic differentiation, and cell fusion are identified as distinct calcium-regulated steps, based on the extracellular calcium concentration required for the expression of morphological and biochemical markers specific to each of these stages. Furthermore, differentiation is inhibited at the commitment stage by either treatment with the calcineurin inhibitor cyclosporine A (CSA) or expression of CAIN, a physiological inhibitor of calcineurin. Retroviral-mediated gene transfer of a constitutively active form of calcineurin is able to induce myogenesis only in the presence of extracellular calcium, suggesting that multiple calcium-dependent pathways are required for differentiation. The mechanism by which calcineurin initiates differentiation includes transcriptional activation of myogenin, but does not require the participation of NFAT. We conclude that commitment of skeletal muscle cells to differentiation is calcium and calcineurin-dependent, but NFAT-independent.

Differential regulation of vascular smooth muscle nuclear factor kappa-B by G alpha q-coupled and cytokine receptors.

NF kappaB has been implicated as a downstream effector of G alphaq-coupled receptor signaling, but whether these and cytokine receptors activate NF kappaB similarly remains unclear. Stimulation of rat vascular smooth muscle cell G alphaq-coupled P2Y nucleotide receptors with UTP induces luciferase transcription from a sensitive and specific NF kappaB dependent promoter. However, these responses are only;15% of that to the reference cytokine IL-1 beta. IL-1 beta is a powerful stimulator of I kappaB alpha degradation, RelA nuclear import, and isoform specific NF kappaB enhancer binding in vitro, responses that are not detectable after P2Y receptor stimulation. Expression of two trans -dominant NF kappaB polypeptides suppresses induction of the NF kappaB reporter and also IL-1 beta stimulated monocyte chemoattractant-1 mRNA, which is not induced by UTP. In contrast, UTP induces higher expression of the endogenous COX-2 and IL-6 mRNAs than does IL-1 beta, implying that G alphaq-coupled receptor evokes additional NF kappaB-independent transcription factors in regulating these two genes. P2Y receptors are as effective as the reference growth factor PDGF-BB at inducing CREB, AP-1, SRE and NFAT transcription, which are largely unaffected by IL-1 beta treatment. NF kappaB is less efficiently activated then several other transcriptional effectors of G alphaq-coupled receptor signaling in vascular smooth muscle cells, and is instead preferentially activated by inflammatory cytokines.

Hepatocyte growth factor affects satellite cell activation and differentiation in regenerating skeletal muscle.

Hepatocyte growth factor (HGF) is the only known growth factor that activates quiescent satellite cells in skeletal muscle. We hypothesized that local delivery of HGF may enhance regeneration after trauma by increasing the number of myoblasts available for restoring normal tissue architecture. Injection of HGF into muscle at the time of injury increases myoblast number but does not enhance tissue repair as determined using quantitative histological analyses. Rather, depending on the dose and the timing of HGF administration relative to the injury, regeneration can be inhibited. The greatest inhibitory effect is observed when HGF is administered on the day of injury and continued for 3 days, corresponding to the time when satellite cell activation, proliferation, and early differentiation normally occur. To establish a mechanism for this inhibition, we show that HGF can act directly on primary muscle cells to block differentiation. These results demonstrate that 1) exogenous HGF synergizes with factors in damaged muscle to increase myoblast number, 2) regeneration is not regulated solely by myoblast number, and 3) HGF inhibits muscle differentiation both in vitro and in vivo.

Systemic administration of the NF-kappaB inhibitor curcumin stimulates muscle regeneration after traumatic injury.

Skeletal muscle is often the site of tissue injury due to trauma, disease, developmental defects or surgery. Yet, to date, no effective treatment is available to stimulate the repair of skeletal muscle. We show that the kinetics and extent of muscle regeneration in vivo after trauma are greatly enhanced following systemic administration of curcumin, a pharmacological inhibitor of the transcription factor NF-kappaB. Biochemical and histological analyses indicate an effect of curcumin after only 4 days of daily intraperitoneal injection compared with controls that require >2 wk to restore normal tissue architecture. Curcumin can act directly on cultured muscle precursor cells to stimulate both cell proliferation and differentiation under appropriate conditions. Other pharmacological and genetic inhibitors of NF-kappaB also stimulate muscle differentiation in vitro. Inhibition of NF-kappaB-mediated transcription was confirmed using reporter gene assays. We conclude that NF-kappaB exerts a role in regulating myogenesis and that modulation of NF-kappaB activity within muscle tissue is beneficial for muscle repair. The striking effects of curcumin on myogenesis suggest therapeutic applications for treating muscle injuries.

Activation and cellular localization of the cyclosporine A-sensitive transcription factor NF-AT in skeletal muscle cells.

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT-mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT-responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities.

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.

The cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) proteins are expressed in vascular smooth muscle cells. Differential localization of NFAT isoforms and induction of NFAT-mediated transcription by phospholipase C-coupled cell surface receptors.

Expression of the antigen-regulated, cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) is not restricted to lymphoid cells, as thought initially, but the physiological inducers of NFAT-mediated transcription in non-lymphoid cells are unknown. Here, cultured vascular smooth muscle cells (VSMC) are shown to express two isoforms of the NFAT family endogenously, which are localized differentially in cells under resting conditions. Using a retroviral NFAT-specific luciferase reporter, we show that VSMC support previously unrecognized complexities in NFAT-mediated transcription, including evidence for negative regulation by Ca2+ signaling and positive regulation through co-activation of adenylyl cyclase and Ca2+ mobilization. The VSMC mitogen platelet derived growth factor-BB (PDGF-BB) induces NFAT-mediated transcription in VSMC. Thrombin and angiotensin II, which activate Galphaq-coupled receptors, are significantly weaker inducers of NFAT-mediated luciferase expression than is PDGF-BB. However, co-stimulation studies show that Galphaq receptor agonists augment the NFAT-mediated transcriptional response to PDGF-BB. This synergy can be explained in part by augmented intracellular Ca2+ transients elicited by multiple agonist challenges. These data indicate that agonists for phospholipase C-coupled receptors stimulate NFAT-mediated transcription in VSMC differentially, and that NFAT can function to integrate co-activating signals in the extracellular environment.

Myoblast implantation in Duchenne muscular dystrophy: the San Francisco study.

We evaluated myoblast implantation in 10 boys with Duchenne muscular dystrophy (DMD) and absent dystrophin (age 5-10 years) who were implanted with 100 million myoblasts in the anterior tibial muscle of one leg and placebo in the other. Cyclosporine (5 mg/kg/day) was administered for 7 months. Pre- and postimplantation (after 1 and 6 months) muscle biopsies were analyzed. Force generation (tetanic tension and maximum voluntary contraction) was measured monthly in a double-blind design. There was increased force generation in both legs of all boys, probably due to cyclosporine. Using the polymerase chain reaction, evidence of myoblast survival and dystrophin mRNA expression was obtained in 3 patients after 1 month and in 1 patient after 6 months. These studies suggest a salutary effect of cyclosporine upon muscular force generation in Duchenne muscular dystrophy; however, myoblast implantation was not effective in replacing clinically significant amounts of dystrophin in DMD muscle.

Isolation, purification, and growth of human skeletal muscle cells.

Skeletal muscle cells can be used in vitro for the study of myogenests, as well as in VIVO as gene-delivery vehicles for the therapy of muscle and nonmuscle diseases (1-9). These skeletal muscle cells are derived from muscle satellite cells, which he between the basal lamina and the sarcolemma of differentiated muscle fibers (10) Normally quiescent after the period of muscle development and growth during fetal life and the early postnatal period, these cells are induced to proliferate on muscle damage and fuse with existing muscle fibers Satellite cells isolated and grown in vitro are called myoblasts Myoblasts proliferate in mitogen-rich media, but on reaching high cell density followed by exposure to mitogen-poor media, are induced to differentiate and become postmitotic Muscle differentiation is characterized by the fusion of myoblasts to form multinucleated myotubes, which express differentiation-specific proteins. In this chapter, methods are given for the isolation of myoblasts from human muscle tissue using two different techinques flow cytometry (11) and cell cloning (12-14). These methods are applicable to muscle tissue from both fetal and postnatal donors, as well as from normal and diseased individuals.

The fate of myoblasts following transplantation into mature muscle.

Cell transplantation has potential benefits for tissue replacement in the the enhancement of tissue regeneration and as cell-mediated gene therapy for systemic diseases. The transplantation of myoblasts into skeletal muscle also allows gene transfer into cells of the host since myoblasts fuse with host fibers thereby forming hybrid myofibers. The success of myoblast transplantation can be determined by a variety of measures, such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression. Each measure is a reflection of the fate of the transplanted cells. In order to compare different measures of transplantation efficacy, we followed the fate of transplanted myoblasts expressing the marker enzyme beta-galactosidase (beta-gal) in two different assays. Two weeks after transplantation, the number of hybrid myofibers was determined histochemically, whereas transgene (beta-gal) expression was measured biochemically. To control for variabilities of transplantation among different animals, we obtained both measurements from each muscle by using alternate cryosections in the two assays. Within each individual muscle, both hybrid fiber number and beta-gal expression were maximal at the site of implantation and diminished in parallel with distance from the site. However, for determining the success of transplantation among groups of muscles, these two measures of efficacy yielded discordant results: the transplants with the highest number of hybrid fibers were not the transplants with the greatest beta-gal activity. Such discrepancies are likely due to regional variations at the transplantation site that arise when cells are introduced into a solid tissue. These results demonstrate the importance of multiple measures of cell fate and transplantation efficacy for studies of cell transplantation and for the application of such studies to cell therapy and cell-mediated gene therapy.

Transient immunosuppressive treatment leads to long-term retention of allogeneic myoblasts in hybrid myofibers.

Normal and genetically engineered skeletal muscle cells (myoblasts) show promise as drug delivery vehicles and as therapeutic agents for treating muscle degeneration in muscular dystrophies. A limitation is the immune response of the host to the transplanted cells. Allogeneic myoblasts are rapidly rejected unless immunosuppressants are administered. However, continuous immunosuppression is associated with significant toxic side effects. Here we test whether immunosuppressive treatment, administered only transiently after allogeneic myoblast transplantation, allows the long-term survival of the transplanted cells in mice. Two immunosuppressive treatments with different modes of action were used: (a) cyclosporine A (CSA); and (b) monoclonal antibodies to intracellular adhesion molecule-1 and leukocyte function-associated molecule-1. The use of myoblasts genetically engineered to express beta-galactosidase allowed quantitation of the survival of allogeneic myoblasts at different times after cessation of the immunosuppressive treatments. Without host immunosuppression, allogeneic myoblasts were rejected from all host strains tested, although the relative time course differed as expected for low and high responder strains. The allogeneic myoblasts initially fused with host myofibers, but these hybrid cells were later destroyed by the massive immunological response of the host. However, transient immunosuppressive treatment prevented the hybrid myofiber destruction and led to their long-term retention. Even four months after the cessation of treatment, the hybrid myofibers persisted and no inflammatory infiltrate was present in the tissue. Such long-term survival indicates that transient immunosuppression may greatly increase the utility of myoblast transplantation as a therapeutic approach to the treatment of muscle and nonmuscle disease.

Specific T cell receptor gene rearrangements at the site of muscle degeneration in Duchenne muscular dystrophy.

Mononuclear cells infiltrate degenerating muscles of Duchenne muscular dystrophy (DMD) patients. Using a quantitative PCR, we first characterized the T cells infiltrating muscle biopsies from six DMD patients. High levels of TCR V beta 2 transcripts were observed in DMD muscle tissue. TCR V beta 2 transcripts from seven DMD patients and five controls were sequenced, and the VDJ junctional region analyzed in 166 clones. One specific amino acid motif, RVSG, was found in the third complementary determining region (CDR3) of TCR V beta 2 chains in samples from five DMD patients, but not in controls. A specific immune reaction at the site of tissue degeneration may play an important role in the pathogenesis of DMD.

Myoblasts in pattern formation and gene therapy.

The tissues of a multicellular animal are composed of diverse cell types arranged in a precisely organized pattern. Features unique to muscle allow an analysis of pattern formation and maintenance in mammals. The progeny of single cells can be taken full cycle from the animal to the culture dish and back to the animal where they fuse into mature myofibers of the host. These features not only facilitate the use of genetically engineered myoblasts in studies of pattern formation, but also in cell-mediated gene therapy: a novel mode of drug delivery for the treatment of muscle and nonmuscle diseases such as hemophilia, cardiac disease and cancer.