Satellite cell dysfunction contributes to the progressive muscle atrophy in myotonic dystrophy type 1.

AIMS: Myotonic dystrophy type 1 (DM1), one of the most common forms of inherited neuromuscular disorders in the adult, is characterized by progressive muscle weakness and wasting leading to distal muscle atrophy whereas proximal muscles of the same patients are spared during the early phase of the disease. In this report, the role of satellite cell dysfunction in the progressive muscular atrophy has been investigated. METHODS: Biopsies were obtained from distal and proximal muscles of the same DM1 patients. Histological and immunohistological analyses were carried out and the past regenerative history of the muscle was evaluated. Satellite cell number was quantified in vivo and proliferative capacity was determined in vitro. RESULTS: The size of the CTG expansion was positively correlated with the severity of the symptoms and the degree of muscle histopathology. Marked atrophy associated with typical DM1 features was observed in distal muscles of severely affected patients whereas proximal muscles were relatively spared. The number of satellite cells was significantly increased (twofold) in the distal muscles whereas very little regeneration was observed as confirmed by telomere analyses and developmental MyHC staining (0.3-3%). The satellite cells isolated from the DM1 distal muscles had a reduced proliferative capacity (36%) and stopped growing prematurely with telomeres longer than control cells (8.4 vs. 7.1 kb), indicating that the behaviour of these precursor cells was modified. CONCLUSIONS: Our results indicate that alterations in the basic functions of the satellite cells progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.

ZASP: a new Z-band alternatively spliced PDZ-motif protein.

PDZ motifs are modular protein-protein interaction domains, consisting of 80-120 amino acid residues, whose function appears to be the direction of intracellular proteins to multiprotein complexes. In skeletal muscle, there are a few known PDZ-domain proteins, which include neuronal nitric oxide synthase and syntrophin, both of which are components of the dystrophin complex, and actinin-associated LIM protein, which binds to the spectrin-like repeats of alpha-actinin-2. Here, we report the identification and characterization of a new skeletal muscle protein containing a PDZ domain that binds to the COOH-terminal region of alpha-actinin-2. This novel 31-kD protein is specifically expressed in heart and skeletal muscle. Using antibodies produced to a fragment of the protein, we can show its location in the sarcomere at the level of the Z-band by immunoelectron microscopy. At least two proteins, 32 kD and 78 kD, can be detected by Western blot analysis of both heart and skeletal muscle, suggesting the existence of alternative forms of the protein. In fact, several forms were found that appear to be the result of alternative splicing. The transcript coding for this Z-band alternatively spliced PDZ motif (ZASP) protein maps on chromosome 10q22.3-10q23.2, near the locus for infantile-onset spinocerebellar ataxia.

Heat shock treatment increases engraftment of transplanted human myoblasts into immunodeficient mice.

Myoblast transfer therapy (MTT) is a strategy that has been proposed to treat some striated muscle pathologies. However, the first therapeutic trials using this technique were unsuccessful due to the limited migration and early cell death of the injected myoblasts. Various strategies have been considered to increase myoblast survival in the host muscle after MTT. Overexpression of heat shock proteins (HSPs) in mouse myoblasts has been shown to improve cell resistance against apoptosis in vitro and in vivo. Our objective was to determine whether heat shock (HS) treatment increased the survival of human myoblasts leading to better participation of the injected cells in muscle regeneration. For this study, HS-treated human myoblasts were injected into the tibialis anterior (TA) muscles of immunodeficient RAG-/- gammaC-/- mice. TA muscles were excised at 24 hour and at 1 month after injection. Our results showed that HS treatment increased the expression of the hsp70 protein and protected the cells from apoptosis in vitro. HS treatment dramatically increased the number of human fibers present at 1 month after injection when compared with nontreated cells. Interestingly, HS treatment decreased apoptosis at 24 hour after human myoblast injection, but no differences were observed concerning proliferation, suggesting that the increased fiber formation among the HS-treated group was probably due to decreased cell death. These data suggested that HS treatment might be used in the clinical context to improve the success of MTT.

Regulation of acetylcholine receptor gene expression in human myasthenia gravis muscles. Evidences for a compensatory mechanism triggered by receptor loss.

Myasthenia gravis (MG) is a neuromuscular disorder mediated by antibodies directed against the acetylcholine receptor (nAChR) resulting in a functional nAChR loss. To analyze the molecular mechanisms involved at the muscular target site, we studied the expression of nAChR subunits in muscle biopsy specimens from MG patients. By using quantitative PCR with an internal standard for each subunit, we found that the levels of beta-, delta-, and epsilon-subunit mRNA coding for the adult nAChR were increased in severely affected MG patients, matching our previous data on the alpha-subunit. Messenger levels were highly variable in MG patients but not in controls, pointing to individual factors involved in the regulation of nAChR genes. The fetal subunit (gamma-chain) transcripts were almost undetectable in the extrajunctional region of MG muscle, suggesting that gene regulation in MG differs from that in the denervation model, in which nAChR gamma-subunit mRNA is reexpressed. Nicotinic AChR loss mediated by monoclonal anti-nAChR antibodies in both the TE671 muscle cell line and cultured normal human myotubes induces a similar increase in beta- alphand delta-subunit mRNA levels, suggesting the existence of a new muscular signaling pathway system coupled to nAChR internalization and independent of muscle electrical activity. These data demonstrate the existence of a compensatory mechanism regulating the expression of the genes coding for the adult nAChR in patients with MG.

Impact on oxidative phosphorylation of immortalization with the telomerase gene.

Skin fibroblasts are essential tools for biochemical, genetic and physiopathological investigations of mitochondrial diseases. Their immortalization has been previously performed to overcome the limited number of divisions of these primary cells but it has never been systematically evaluated with respect to efficacy and impact on the oxidative phosphorylation (OXPHOS) characteristics of the cells. We successfully immortalized with the human telomerase gene 15 human fibroblasts populations, 4 derived from controls and 11 from patients with diverse respiratory chain defects. Immortalization induced significant but mild modification of the OXPHOS characteristics of the cells with lower rates of oxygen consumption and ATP synthesis associated with their loose coupling. However, it never significantly altered the type and severity of any genetic OXPHOS defect present prior to immortalization. Furthermore, it did not significantly modify the cells' dependence on glucose and sensitivity to galactose thus showing that immortalized cells could be screened by their nutritional requirement. Immortalized skin fibroblasts with significant OXPHOS defect provide reliable tools for the diagnosis and research of the genetic cause of mitochondrial defects. They also represent precious material to investigate the cellular responses to these defects, even though these should afterwards be verified in unmodified primary cells.

Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

PABPN1 gene therapy for oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.

Myoblast transfer therapy: is there any light at the end of the tunnel?

Myoblast transfer therapy (MTT) was proposed in the 70's as a potential treatment for muscular dystrophies, based upon the early results obtained in mdx mice: dystrophin expression was restored in this model by intramuscular injections of normal myoblasts. These results were quickly followed by clinical trials for patients suffering from Duchenne Muscular Dystrophy (DMD) in the early 90's, based mainly upon intramuscular injections of allogenic myoblasts. The clinical benefits obtained from these trials were minimal, if any, and research programs concentrated then on the various pitfalls that hampered these clinical trials, leading to numerous failures. Several causes for these failures were identified in mouse models, including a massive cell death of myoblasts following their injection, adverse events involving the immune system and requiring immunosuppression and the adverse events linked to it, as well as a poor dispersion of the injected cells following their injection. It should be noted that these studies were conducted in mouse models, not taking into account the fundamental differences between mice and men. One of these differences concerns the regulation of proliferation, which is strictly limited by proliferative senescence in humans. Although this list is certainly not exhaustive, new therapeutic venues were then explored, such as the use of stem cells with myogenic potential, which have been described in various populations, including bone marrow, circulating blood or muscle itself. These stem cells presented the main advantage to be available and not exhausted by the numerous cycles of degeneration/regeneration which characterize muscle dystrophies. However, the different stem candidates have shown their limits in terms of efficiency to participate to the regeneration of the host. Another issue was raised by clinical trials involving the injection of autologous myoblasts in infacted hearts, which showed that limited targets could be aimed with autologous myoblasts, as long as enough spared muscle was available. This resulted in a clinical trial for the pharyngeal muscles of patients suffering from Oculo-Pharyngeal Muscular Dystrophy (OPMD). The results of this trial will not be available before 2 years, and a similar procedure is being studied for Fascio-Scapulo-Humeral muscular Dystrophy (FSHD). Concerning muscular dystrophies which leave very few muscles spared, such as DMD, other solutions must be found, which could include exon-skipping for the eligible patients, or even cell therapy using stem cells if some cell candidates with enough efficiency can be found. Recent results concerning mesoangioblasts or circulating AC133+ cells raise some reasonable hope, but still need further confirmations, since we have learned from the past to be cautious concerning a transfer of results from mice to humans.

Telomere length as a tool to monitor satellite cell amplification for cell-mediated gene therapy.

Cell-mediated gene therapy requires an in vitro amplification of modified cells prior to their injection into target tissue. Since the proliferative capacity of normal human cells is limited, we have tested a method to follow in vitro the proliferative potential of human satellite cells. Our results show that telomere length can be used to predict the proliferative potential of human satellite cells. In this short communication, the telomere shortening and the limited replicative potential are discussed in the context of the possible use of human satellite cells for gene transfer and why cell-mediated gene therapy has been less successful in humans than in mice.

Replicative potential and telomere length in human skeletal muscle: implications for satellite cell-mediated gene therapy.

In this study, we have evaluated the ability of human satellite cells isolated from subjects aged from 5 days to 86 years to proliferate in culture. Cells were cultivated until they became senescent. The number of cell divisions was calculated by counting the number of cells plated in culture compared to the number of cells removed following proliferation. Telomere length, which is known to decrease during each round of cell division, has been used to analyze the in vitro replicative capacity and in vivo replicative history of human satellite cells at isolation. The rate of telomere shortening in myonuclei of these muscle biopsies was also examined. Our results show that both proliferative capacity and telomere length of satellite cells decreases with age during the first two decades but that the myonuclei of human skeletal muscle are remarkably stable because telomere length in these myonuclei remains constant from birth to 86 years. The lack of shortening of mean terminal restriction fragments (TRF) in vivo confirms that skeletal muscle is a stable tissue with little nuclear turnover and therefore an ideal target for cell-mediated gene therapy. Moreover, our results show that it is important to consider donor age as a limiting factor to obtain an optimal number of cells.

Human muscle precursor cell regeneration in the mouse host is enhanced by growth factors.

The aim of this study was to optimize human muscle formation in vivo from implanted human muscle precursor cells. We transplanted donor muscle precursor cells (MPCs) prepared from postnatal or fetal human muscle into immunodeficient host mice and showed that irradiation of host muscle significantly enhanced muscle formation by donor cells. The amount of donor muscle formed in cryodamaged host muscle was increased by exposure of donor cells to growth factors before their implantation into injured host muscle. Insulin-like growth factor type I (IGF-I) significantly increased the amount of muscle formed by postnatal human muscle cells, but not by fetal human MPCs. However, treatment of fetal muscle cells with IGF-I, in combination with basic fibroblast growth factor and plasmin, significantly increased the amount of donor muscle formed. In vivo, human MPCs formed mosaic human-mouse muscle fibers, in which each human myonucleus was associated with a zone of human sarcolemmal protein spectrin.

Extended amplification in vitro and replicative senescence: key factors implicated in the success of human myoblast transplantation.

The limited success of human myoblast transplantation has been related to immune rejection, poor survival, and limited spread of injected myoblasts after transplantation. An important issue that has received little attention, but is nevertheless of fundamental importance in myoblast transplantation protocols, is the proliferative capacity of human satellite cells. Previous studies from our laboratory have demonstrated that the maximum number of divisions that a population of satellite cells can make decreases with age during the first two decades of life then stabilizes in adulthood. These observations indicate that when satellite cells are used as vectors in myoblast transplantation protocols it is important to consider donor age and the number of divisions that the cells have made prior to transplantation as limiting factors in obtaining an optimal number of donor derived muscle fibers. In this study, myoblasts derived from donors of different ages (newborn, 17 years old, and 71 years old) were isolated and amplified in culture. Their potential to participate in in vivo muscle regeneration in RAG2(-/-)/gamma(c)/C5 triple immunodeficient hosts after implantation was evaluated at 4 and 8 weeks postimplantation. Our results demonstrate that prolonged amplification in culture and the approach to replicative senescence are both important factors that may condition the success of myoblast transplantation protocols.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Reversible immortalization of human myogenic cells by site-specific excision of a retrovirally transferred oncogene.

Myogenic cells have a limited life span in culture, which prevents expansion at clinically relevant levels, and seriously limits any potential use in cell replacement or ex vivo gene therapy. We developed a strategy for reversibly immortalizing human primary myogenic cells, based on retrovirus-mediated integration of a wild-type SV40 large-T antigen (Tag), excisable by means of the Cre-Lox recombination system. Myogenic cells were transduced with a vector (LTTN-LoxP) expressing the SV40 Tag under the control of an LTR modified by the insertion of a LoxP site in the U3 region. Clonal isolates of Tag-positive cells showed modified growth characteristics and a significantly extended life span, while maintaining a full myogenic potential. Transient expression of Cre recombinase, delivered by transfection or adenoviral vector transduction, allowed excision of the entire provirus with up to >90% efficiency. Cultures of Cre-treated (Tag-) or untreated (Tag+) myogenic cells were genetically labeled with a lacZ retroviral vector, and injected into the regenerating muscle of SCID/bg immunodeficient mice. Tag- cells underwent terminal differentiation in vivo, giving rise to clusters of beta-Gal+ hybrid fibers with an efficiency comparable to that of control untransduced cells. Tag+ cells could not be detected after injection. Neither Tag+ nor Tag- cells formed tumor in this xenotransplantation model. Reversible immortalization by Tag therefore allows the expansion of primary myogenic cells in culture without compromising their ability to differentiate in vivo, and could represent a safe method by which to increase the availability of these cells for clinical application.

The chicken gene encoding the alpha isoform of tropomyosin of fast-twitch muscle fibers: organization, expression and identification of the major proteins synthesized.

The chicken gene alpha fTM encoding the alpha-tropomyosin of fast-twitch muscle fibers (alpha fTM) covers 20 kb and consists of 15 exons. From this gene, three types of mature transcripts (1.3 kb, 2 kb and 2.8 kb) are expressed through the use of alternative promoters, alternatively spliced exons and multiple 3' end processing. Northern analysis and S1 mapping have shown that the 1.3-kb transcript (exons 1a, 2b, 3, 4, 5, 6b, 7, 8, 9a-9b) is expressed in fast-twitch skeletal muscles and that 2-kb transcripts are expressed in smooth muscle (exons 1a, 2a, 3, 4, 5, 6b, 7, 8, 9d) and in fibroblasts (exons 1a, 2b, 3, 4, 5, 6a or 6b, 7, 8, 9d). These 2-kb transcripts encode distinct proteins which we have identified by two-dimensional (2D) gel electrophoresis. The 2.8-kb transcript which has not been so far characterized in birds is expressed in brain (exons 1b, 3, 4, 5, 6b, 7, 8, 9c-9d). This transcript has been characterized by a cDNA polymerase chain reaction assay and by S1 nuclease mapping. It produces a major TM isoform of chick brain which we have identified by 2D gels.

Plasticity of human satellite cells.

Satellite cells were isolated from human quadriceps and masseter muscles and the phenotype of these cells examined in vitro. The expression of the different isoforms of the myosin heavy chains (embryonic, fetal, fast and slow) and light chain isoforms was used to assay myotube diversification. We found that fused cultures of human satellite cells express adult fast and slow MHCs in addition to the embryonic and fetal isoforms. Only the four fast light chains (MLC1emb, MLC1F, MLC2F and MLC3F) were synthesized. No slow MLCs were ever detected in these cultures. In order to determine if the human satellite cells were committed to distinct fast and slow myogenic lineages, a clonal analysis was carried out on both cell populations. All myogenic clones expressed fast and slow MHCs, suggesting that there is no evidence for different fast and slow satellite cell lineages in human skeletal muscle.

A discrepancy resolved: human satellite cells are not preprogrammed to fast and slow lineages.

Satellite cells from chicken and mouse muscle when differentiated in vitro have been shown to display a myosin heavy chain phenotype that corresponds to the fibre from which they originated. Indirect evidence has suggested that this might not be the case for human satellite cells. In the present study we have compared the myosin heavy chain (MHC) profile expressed by differentiated cultures of satellite cells isolated from single fast or slow muscle fibres. The MHC composition of the isolated fibres was determined by sodium dodecyl sulfate glycerol gel electrophoresis and Western blotting. The MHC profile expressed by the differentiated myotubes was identified by immunostaining using specific antibodies. Our results show that all human satellite cells isolated from either fast or slow fibres form myotubes in vitro which co-express both fast and slow MHCs independently of the fibre type from which they originated. These results confirm that human satellite cells, in contrast to those of birds and rodents, are not confined to distinct fast and slow lineages.

Shorter telomeres in dystrophic muscle consistent with extensive regeneration in young children.

Muscular dystrophies are characterised by continuous cycles of degeneration and regeneration resulting in an eventual diminution of the muscle mass and extensive fibrosis. In somatic cells chromosomal telomeres shorten with each round of cell division and telomere length is considered to be a biomarker of the replicative history of the cell. We have previously shown that human myoblasts have a limited proliferative capacity, and that normal skeletal muscle has a very low level of nuclear turnover. However, in patients suffering from muscular dystrophy the satellite cells will be forced to make repeated rounds of cell division, driving the cells towards senescence. In this study we have used the telomere length to quantify the intensity of the muscle cell turnover in biopsies from dystrophic patients of different ages. Our results show that as soon as the first clinical symptoms become apparent the muscle has already undergone extensive regeneration and the rate of telomere loss is 14 times greater than that observed in controls. This confirms that the decline in regenerative capacity is due to the premature senescence of the satellite cells induced by their excessive proliferation during muscle repair.

Skeletal muscle regeneration and the mitotic clock.

Regeneration of muscle fibers following damage requires activation of quiescent satellite cells, their proliferation and finally their differentiation and fusion into multinucleated myotubes, which after maturation will replace the damaged fiber. The regenerative potential of human skeletal muscle will be determined, at least partly, by the proliferative capacity of the satellite cells. In this study, we have measured the proliferative life span of human satellite cells until they reach senescence. These analyses were performed on cell populations isolated from old and young donors as well as from one child suffering from Duchenne muscular dystrophy, where extensive regeneration had occurred. In order to see if there are any age-related changes in the myogenic program we have also compared the program of myogenic differentiation expressed by satellite cells from these subjects at different stages of their proliferative lifespan.

Comparison of the enzyme-linked immunosorbent assay (ELISA) with standard tests used to detect yellow fever virus antibodies.

The enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to yellow fever virus in 110 sera from patients living in an epidemic yellow fever area. The results were then compared with those obtained with the hemagglutination-inhibition (HI), complement-fixation (CF), neutralization (NT), and indirect immunofluorescence (IFA) tests. This ELISA, which used a type-specific antigen, showed the same results as the NT test and was found to be more sensitive and more specific than the HI and CF tests.