Normal Control Muscles Research Articles

Effect of khat leaves extract (Catha edulis) on the excitability of frog gastrocnemius.

This study was held on the isolated excitable tissue of frog's gastrocnemius muscle. Experiments were down by determining the dose dependent of leaves of khat extract. Work down in accordance with personal presupposition since reported that khat have psycho stimulant effects accordingly we thought khat might have effects on systemically biology that mediated by chticol amines. Results showed the effect of khat leaves extract on excitability of muscle contraction that was easily attested when the electrical stimuli were applied either through the nerve or directly to the muscle. Whereas khat treatment showed a significant increase in the contraction period of the gastrocnemius muscle and both of the relaxation periods and half relaxation time were shortened. Also it was found that pretreatment by khat leaves extract caused changes in the tension output, increased the height of effect intensity with prolonged the contraction period, while the latent period was decreased and reduced slightly the half relaxation period of the twitch comparison with the normal control muscle. These experimental results indicated that khat had highly stimulant effects on muscle, whereas caused liberation of thermoenzymatic and chemical energy during contraction which converted into mechanical energy, which manifested its self as work.

Isometric training improves fatigue resistance in dystrophin-deficient muscle

Eccentric contractions, in which the muscle is stretched during contraction, cause substantially greater damage than isometric (ISO) contractions, in which the length of the muscle does not change during contraction. Here, we tested the hypothesis that ISO training improves fatigue resistance in skeletal muscle from dystrophin-deficient mdx52 mice (15-22 wk old). ISO training (100 Hz stimulation frequency, 0.25-s contractions every 0.5 s, 6 sets of 60 contractions) was performed on the left plantar flexor muscles in vivo with supramaximal electrical stimulation every other day for 4 wk. Compared with the normal control muscle, resistance to fatigue was reduced in the nontrained muscle from mdx52 mice, which was accompanied by a reduction in citrate synthase activity and the LC3BII/I ratio and an increase in the phosphorylation levels of Akt Ser473 and the expression levels of p62. ISO training restored these alterations and markedly increased in vivo fatigue resistance and PGC-1α expression in mdx52 muscles. Moreover, an increased number of Evans Blue dye-positive fibers was significantly reduced by ISO training in mdx52 muscles. In contrast, ISO training did not restore a reduction in the amount of SH3 and cysteine-rich domain 3 in mdx muscles. Thus, our data suggest that mitochondrial function is impaired in dystrophin-deficient muscles, which is likely to be induced by the defective autophagy due to persistent activation of Akt. ISO training inhibits the aberrant activation of Akt presumably by up-regulating the PGC-1α expression, which results in improved mitochondrial function and thus fatigue resistance in dystrophin-deficient muscles.

Ultrasonographic and Histological Correlation after Experimental Reconstruction of a Volumetric Muscle Loss Injury with Adipose Tissue.

Different types of scaffolds are used to reconstruct muscle volume loss injuries. In this experimental study, we correlated ultrasound observations with histological findings in a muscle volume loss injury reconstructed with autologous adipose tissue. The outcome is compared with decellularized and porous matrix implants. Autologous adipose tissue, decellularized matrix, and a porous collagen matrix were implanted in volumetric muscle loss (VML) injuries generated on the anterior tibial muscles of Wistar rats. Sixty days after implantation, ultrasound findings were compared with histological and histomorphometric analysis. The muscles with an autologous adipose tissue implant exhibited an ultrasound pattern that was quite similar to that of the regenerative control muscles. From a histological point of view, the defects had been occupied by newly formed muscle tissue with certain structural abnormalities that would explain the differences between the ultrasound patterns of the normal control muscles and the regenerated ones. While the decellularized muscle matrix implant resulted in fibrosis and an inflammatory response, the porous collagen matrix implant was replaced by regenerative muscle fibers with neurogenic atrophy and fibrosis. In both cases, the ultrasound images reflected echogenic, echotextural, and vascular changes compatible with the histological findings of failed muscle regeneration. The ultrasound analysis confirmed the histological findings observed in the VML injuries reconstructed by autologous adipose tissue implantation. Ultrasound can be a useful tool for evaluating the structure of muscles reconstructed through tissue engineering.

Increased taurine in pre-weaned juvenile mdx mice greatly reduces the acute onset of myofibre necrosis and dystropathology and prevents inflammation.

Background: The mdx mouse model for the fatal muscle wasting disease Duchenne Muscular Dystrophy (DMD) shows a very mild pathology once growth has ceased, with low levels of myofibre necrosis in adults. However, from about 3 weeks of post-natal age, muscles of juvenile mdx mice undergo an acute bout of severe necrosis and inflammation: this subsequently decreases and stabilises to lower adult levels by about 6 weeks of age. Prior to the onset of this severe dystropathology, we have shown that mdx mice are deficient in the amino acid taurine (potentially due to weaning), and we propose that this exacerbates myofibre necrosis and inflammation in juvenile mdx mice. Objectives: The purpose of this study was to increase taurine availability to pre-weaned juvenile mdx mice (from 14 days of age), to evaluate the impact on levels of myofibre necrosis and inflammation (at 22 days) during the acute period of severe dystropathology. Results: Untreated 22 day old mdx muscle was not deficient in taurine, with similar levels to normal C57 control muscle. However taurine treatment, which increased the taurine content of young dystrophic muscle (by 40%), greatly reduced myofibre necrosis (by 75%) and prevented significant increases in 3 markers of inflammation. Conclusion: Taurine was very effective at preventing the acute phase of muscle damage that normally results in myofibre necrosis and inflammation in juvenile mdx mice, supporting continued research into the use of taurine as a therapeutic intervention for protecting growing muscles of young DMD boys

Abnormal expression of seven myogenesis-related genes in extraocular muscles of patients with concomitant strabismus

Hyperplasia or hypoplasia of muscles gradually leads to strabismus. Myogenesis-related genes are involved in extraocular muscle development, including myogenic differentiation1 (MYOD1), myogenin (MYOG), retinoblastoma1 (RB1), cyclin-dependent kinase inhibitor1A (P21), cyclin‑dependent kinase inhibitor1C (P57), insulin-like growth factor1 (IGF1) and muscle creatine kinase (MCK). This study evaluated the expression of the above seven myogenesis-related genes by real-time quantitative RT-PCR in 18resected extrocular muscles of patients with concomitant strabismus and 12normal control muscle samples from one presumably healthy male 6h after sudden mortality. We found that although there was a great divergence among the expression levels of 6 myogenesis-related regulatory factors, the relative expression patterns were similar in all the normal muscles, including the synergistic, antagonistic and yoke muscles. However, their expression levels in the 18diseased extraocular muscles were abnormal; the expression levels of all the genes, with the exception of P57, were reduced in most of the diseased muscle tissues. These results imply that the abnormal expression of these myogenesis-related genes may contribute to concomitant strabismus.

Methylation alterations of WT1 and homeobox genes in inflamed muscle biopsy samples from patients with untreated juvenile dermatomyositis suggest self‐renewal capacity

To determine the effect of methylation alteration in inflamed muscles from children with juvenile dermatomyositis (DM) and other idiopathic inflammatory myopathies (IIMs). Magnetic resonance imaging-directed diagnostic muscle biopsies yielded samples from 20 children with juvenile DM, which were used for genome-wide DNA methylation profiling, as were muscle biopsy samples from 4 healthy controls. Bisulfite treatment followed by pyrosequencing confirmed methylation status in juvenile DM and other IIMs. Immunohistochemistry defined localization and expression levels of WT1. Comparison of genome-wide DNA methylation profiling between juvenile DM muscle and normal control muscle revealed 27 genes with a significant methylation difference between the groups. These genes were enriched with transcription factors and/or cell cycle regulators and were unrelated to duration of untreated disease. Six homeobox genes were among them; ALX4, HOXC11, HOXD3, and HOXD4 were hypomethylated, while EMX2 and HOXB1 were hypermethylated. WT1 was significantly hypomethylated in juvenile DM (Δβ = -0.41, P < 0.001). Bisulfite pyrosequencing verification in samples from 56 patients with juvenile DM confirmed the methylation alterations of these genes. Similar methylation alterations were observed in juvenile polymyositis (n = 5) and other IIMs (n = 9). Concordant with the other findings, WT1 protein was increased in juvenile DM muscle, with average positive staining of 11.6%, but was undetectable in normal muscle (P < 0.001). These results suggest that affected muscles of children with juvenile DM and IIMs have the capacity to be repaired, and that homeobox and WT1 genes are epigenetically marked to facilitate this repair process, potentially suggesting new avenues of therapeutic intervention.

Abnormalities of NBR1, a novel autophagy-associated protein, in muscle fibers of sporadic inclusion-body myositis

Intra-muscle fiber accumulation of ubiquitinated protein aggregates containing several conformationally modified proteins, including amyloid-β and phosphorylated tau, is characteristic of the pathologic phenotype of sporadic inclusion-body myositis (s-IBM), the most common progressive degenerative myopathy of older persons. Abnormalities of protein-degradation, involving both the 26S proteasome and autophagic-lysosomal pathways, were previously demonstrated in s-IBM muscle. NBR1 is a ubiquitin-binding scaffold protein importantly participating in autophagic degradation of ubiquitinated proteins. Whereas abnormalities of p62, a ubiquitin-binding protein, were previously described in s-IBM, abnormalities of NBR1 have not been reported in s-IBM. We have now identified in s-IBM muscle biopsies that NBR1, by: (a) immunohistochemistry, was strongly accumulated within s-IBM muscle-fiber aggregates, where it closely co-localized with p62, ubiquitin, and phosphorylated tau; (b) immunoblots, was increased threefold (p < 0.001); and (c) immunoprecipitation, was associated with p62 and LC3. By real-time PCR, NBR1 mRNA was increased twofold (p < 0.01). None of the various disease- and normal-control muscle biopsies had any NBR1 abnormality. In cultured human muscle fibers, NBR1 also physically associated with both p62 and LC3, and experimental inhibition of either the 26S proteasome or the lysosomal activity resulted in NBR1 increase. Our demonstration of NBR1 abnormalities in s-IBM provides further evidence that altered protein degradation pathways may be critically involved in the s-IBM pathogenesis. Accordingly, attempts to unblock defective protein degradation might be a therapeutic strategy for s-IBM patients.

Novel mutations in NEB cause abnormal nebulin expression and markedly impaired muscle force generation in severe nemaline myopathy

BackgroundNemaline myopathy (NM) is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods) in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB), which is thought to account for roughly 50% of cases.ResultsWe describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost.ConclusionsOur findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease.

Impaired Autophagy in Sporadic Inclusion-Body Myositis and in Endoplasmic Reticulum Stress-Provoked Cultured Human Muscle Fibers

The hallmark pathologies of sporadic inclusion-body myositis (s-IBM) muscle fibers are autophagic vacuoles and accumulation of ubiquitin-positive multiprotein aggregates that contain amyloid-beta or phosphorylated tau in a beta-pleated sheet amyloid configuration. Endoplasmic reticulum stress (ERS) and 26S proteasome inhibition, also associated with s-IBM, putatively aggrandize the accumulation of misfolded proteins. However, autophagosomal-lysosomal pathway formation and function, indicated by autophagosome maturation, have not been previously analyzed in this system. Here we studied the autophagosomal-lysosomal pathway using 14 s-IBM and 30 disease control and normal control muscle biopsy samples and our cultured human muscle fibers in a microenvironment modified to resemble aspects of s-IBM pathology. We report for the first time that in s-IBM, lysosomal enzyme activities of cathepsin D and B were decreased 60% (P < 0.01) and 40% (P < 0.05), respectively. We also detected two indicators of increased autophagosome maturation, the presence of LC3-II and decreased mammalian target of rapamycin-mediated phosphorylation of p70S6 kinase. Moreover, in cultured human muscle fibers, ERS induction significantly decreased activities of cathepsins D and B, increased levels of LC3-II, decreased phosphorylation of p70S6 kinase, and decreased expression of VMA21, a chaperone for assembly of lysosomal V-ATPase. We conclude that in s-IBM muscle, decreased lysosomal proteolytic activity might enhance accumulation of misfolded proteins, despite increased maturation of autophagosomes, and that ERS is a possible cause of s-IBM-impaired lysosomal function. Thus, unblocking protein degradation in s-IBM muscle fibers may be a desirable therapeutic strategy.

Telomere shortening is associated to TRF1 and PARP1 overexpression in Duchenne muscular dystrophy

Telomere shortening is thought to contribute to premature senescence of satellite cells in Duchenne muscular dystrophy (DMD) muscle. Telomeric repeat binding factor-1 (TRF1) and poly (ADP-ribose) polymerase-1 (PARP1) are proteins known to modulate telomerase reverse transcriptase (TERT) activity, which controls telomere elongation. Here we show that an age-dependent telomere shortening occurs in DMD muscles and is associated to overexpression of mRNA and protein levels of TRF1 and PARP1. TERT expression and activity are detectable in normal control muscles and they slightly increase in DMD. This is the first demonstration of TRF1 and PARP1 overexpression in DMD muscles. They can be directly involved in replicative senescence of satellite cells and/or in the pathogenetic cascade through a cross-talk with oxidative stress and inflammatory response. Modulation of these events by TRF1 or PARP1 inhibition might represent a novel strategy for treatment of DMD and other muscular dystrophies.

Microbial Targeting of 99mTc-Labeled Recombinant Human β-Defensin-3 in an Animal Model of Infection: A Feasibility Pilot Study

Human beta-defensin-3 (HBD-3) is an antimicrobial peptide with bactericidal effects on many gram-positive and gram-negative bacteria and some yeast species and, if radiolabeled, might be used to distinguish bacterial infection from sterile inflammation. The goals of the present study were to develop methods for radiolabeling HBD-3 with (99m)Tc and to perform preliminary investigations on (99m)Tc-labeled HBD-3 as a means to evaluate induced infection in an animal model. To this purpose, Staphylococcus aureus-induced infection was used to evaluate the capability of (99m)Tc-HBD-3 to distinguish infection from aseptic inflammation in rats. Twenty to 40 microg of recombinant HBD-3 were labeled with (99m)Tc(+) hexa-coordinated with 3 molecules of CO and H(2)O and separated by a column from free (99m)Tc. (99m)Tc-HBD-3 was added to cultures of a bacterial suspension of S. aureus and Escherichia coli to evaluate in vitro antibacterial activity. A bacterial suspension of S. aureus and a carrageenan solution were used to induce infection and sterile inflammation, respectively, in opposite thighs of 9 adult rats. Three separate experiments were performed on groups of 3 rats each. The animals received different doses of (99m)Tc-HBD-3 injected through a cannula into the jugular vein. After sacrifice of the animals, tissue samples were obtained from sites of infection, inflammation, and control muscle (left foreleg) at 1, 3, and 5 h after (99m)Tc-HBD-3 administration. Tissue samples were weighed and then counted in a well-counter. Simultaneously, 1 mL of a standard solution of (99m)Tc-HBD-3 corresponding to each administered dose was counted. (99m)Tc-HBD-3 retained antibacterial activity. Radioactivity in tissue samples from the infected sites was significantly higher than that in samples of either induced inflammation or normal control muscle (ratio, approximately 3:1) at 3 and 5 h after injection, whereas similar radioactivity counts were observed for tissue samples from aseptic inflammation sites and normal control muscle. In this investigation, (99m)Tc-HBD-3 retained antibacterial activity and successfully distinguished infection from aseptic inflammation in adult rats.

Reduced expression of sarcospan in muscles of Fukuyama congenital muscular dystrophy.

Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy (FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated alpha-dystroglycan, together with reduced expression of beta-dystroglycan and alpha-, beta-, gamma-, delta-sarcoglycans. Although the expression of glycosylated alpha-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of alpha-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P>0.1 by the two-tailed t test). This study implied either O-linked glycosylation defects of alpha-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated alpha-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces.

Activated Satellite Cells in Medial Rectus Muscles of Patients with Strabismus

The goal of this study was to determine whether the medial rectus muscles of patients with a history of medial rectus underaction or overaction show alterations in the process of satellite cell activation when compared with normal age-matched control muscles. Medial rectus muscles were obtained with consent from adult patients undergoing surgical resection due to medial rectus underaction or overaction and were prepared for histologic examination by fixation and paraffin embedding. Control muscles were obtained from cornea donor eyes of adults who had no history of strabismus or neuromuscular disease. Cross sections were obtained and stained immunohistochemically for the presence of activated satellite cells, as identified by MyoD immunoreactivity, and the presence of the total satellite cell population, as identified by Pax7 immunoreactivity. The percentages of MyoD- and Pax7-positive satellite cells per 100 myofibers in cross section were calculated. As predicted from results in the literature, MyoD-positive satellite cells, indicative of activation, were present in both the control and resected muscles. In the underacting medial rectus muscles, the percentages of MyoD- and Pax7-positive satellite cells, based on the number of myofibers, were approximately twofold higher than the percentages in the control muscles. In the overacting medial rectus muscles, the percentage of MyoD-positive satellite cells was twofold less than in the control muscles, whereas the percentage of Pax7-positive satellite cells significantly increased compared with that in the control specimens. The presence of an increased number of activated satellite cells in the resected underacting medial rectus muscles and the decreased numbers of activated satellite cells in the overacting muscles was unexpected. The upregulation in the number of MyoD-positive satellite cells in underacting muscles suggests that there is potential for successful upregulation of size in these muscles, as the cellular machinery for muscle repair and regeneration, the satellite cells, is retained and active in patients with medial rectus underaction. The decreased number of activated satellite cells in overacting MR muscle suggests that factors as yet unknown in these overacting muscles are able to affect the number of satellite cells and/or their responsiveness compared with normal age-matched control muscles. These hypotheses are currently being tested.

SMAC-expression in Denervated Human Skeletal Muscle as a Potential Inhibitor of Coexpressed Inhibitor-of-apoptosis Proteins

In multinucleated skeletal muscle fibers, apoptotic muscle fiber loss is mediated by a consecutive disassembly of single fiber segments. During this period of time, proapoptotic and antiapoptotic factors compete for promotion or inhibition of apoptotic fiber degradation. In 16 patients with a neurogenic muscular atrophy, we studied the immunohistochemical expression of the inhibitor-of-apoptosis proteins (IAP) survivin, cIAP1, and XIAP and also of second mitochondria-derived activator of caspase (SMAC), which is released during apoptosis and binds to IAPs to prevent them from inhibiting caspases. Although normal control muscle fibers show no expression of SMAC and IAPs, there was a distinct sarcoplasmic expression of SMAC (12.0%+/-3.5%), survivin (10.2%+/-4.0%), cIAP1 (9.0%+/-3.1%), and XIAP (11.0%+/-4.6%) in varying numbers of muscle fibers in neurogenic muscular atrophy. Coexpression of SMAC and IAPs varied. Some denervated muscle fibers displayed up-regulation of either SMAC or IAPs. Other groups of atrophic muscle fibers showed coexpression of SMAC and IAPs. All factors were exclusively up-regulated in atrophic muscle fibers. These findings indicate that IAPs may inhibit apoptotic degradation of denervated muscle fibers. However, IAPs are finally insufficient to counterbalance and prevent muscle fiber apoptosis, as up-regulated expression of SMAC can antagonize this antiapoptotic potential and promote apoptotic muscle fiber disassembly and loss. The interplay between IAPs and SMAC may represent a threshold for muscle fiber-degrading caspase activities. If this bears a therapeutic potential in the prevention of loss of denervated muscle fibers, it remains highly speculative.

Altered alpha1-syntrophin expression in myofibers with Duchenne and Fukuyama muscular dystrophies.

Alpha1-syntrophin, a scaffolding adapter and modular protein, is a cytoplasmic component of the dystrophin glycoprotein complex. This study investigated immunohistochemically the expression of alpha1-syntrophin in Duchenne and Fukuyama muscular dystrophies (DMD and FCMD, respectively). Biopsied muscles of five DMD, five FCMD, five normal controls and five disease controls (three myotonic and two facioscapulohumeral dystrophies) were analyzed. Immunoblot analysis showed that anti-alpha1-syntrophin antibody had a decreased reaction in both DMD and FCMD muscle extracts. Biopsied muscle sections and their serial sections were immunostained with rabbit anti-alpha1-syntrophin and rabbit anti-muscle-specific beta-spectrin antibodies, respectively. Immunoreactive patterns of sarcolemma were classified into (i) a continuously positive immunostaining pattern, (ii) a partially positive immunostaining pattern, (iii) a negative immunostaining pattern and (iv) a faint but entire surface positive immunostaining pattern. The group mean percentages of alpha1-syntrophin and beta-spectrin immunonegative myofibers in the DMD group were 39.3% and 10.8%, respectively, while those in the FCMD group were 45.5% and 10.4%, respectively. These values were statistically significant compared with those of disease control and normal control muscles. Thus we found that dystrophin-deficient DMD muscles contained significant numbers of alpha1-syntrophin-positive fibers and significant numbers of alpha1-syntrophin-negative fibers were present in dystrophin-positive muscles of severe muscular dystrophy such as FCMD. Alpha-dystrobrevin immunoreactivity was tested in DMD muscles and appreciable amounts of alpha-dystrobrevin that binds to syntrophin were found in DMD muscle membranes.

Calpain‐dependent α‐fodrin cleavage at the sarcolemma in muscle diseases

To clarify the involvement of calpains in sarcolemmal remodeling, we examined the expression of calpains and their substrate, alpha-fodrin, in various disorders of muscle. Although immunohistological reactions for alpha-fodrin and calpains were weak in normal control muscles, intense immunoreactivity for alpha-fodrin at the sarcolemma and for calpains throughout the cytoplasm were detected in small muscle fibers from patients with inflammatory myositis (IM), rhabdomyolysis (Rhab), and Duchenne muscular dystrophy (DMD). Most of the calpain-alpha-fodrin double-positive muscle fibers in IM and Rhab also expressed the developmental form of myosin heavy chain. The sarcolemma of these small muscle fibers reacted with an antibody that specifically recognizes the 150-kDa fragments of alpha-fodrin (SBDP 150s) cleaved by calpain, but not caspase 3. Western blot analysis confirmed these results. These observations indicate that calpain is activated and reacts with alpha-fodrin as a substrate at the sarcolemma, and plays a key role in modulating sarcolemmal proteins to adapt to the specific conditions in each myopathy.

Peut-on envisager une thérapie cellulaire autologue de la dystrophie musculaire facio-scapulo-humérale ?

Facioscapulohumeral dystrophy (FSHD), one of the most common forms of muscular dystrophy, derives its name from the patients' selective, often asymmetric clinical distribution of muscle weakness. Interestingly, affected and non affected areas can coexist in the same patient for many years. The molecular hallmark is total deletion of the subtelomeric D4Z4 repeat on chromosome 4q. There is no specific treatment. Gene therapy is unlikely to be feasible, as no alterations have been found in the genes located in this subtelomeric region. Muscular dystrophies are characterized by the coexistence of genetically induced muscle degeneration and compensatory muscle regeneration by myoblast proliferation from satellite cells; muscle weakness and atrophy appears when this mechanism is overwhelmed. Cell therapy with autologous myoblasts would, in theory, be a simple way of boosting the regenerative process and of preventing or delaying muscle degeneration. This approach might also avoid the use of toxic immunotherapies. By using a recent very-high-yield cell culture method, we analyzed the proliferation and differentiation of myoblasts obtained from FSHD patients, both ex vivo and in vivo (by intramuscular injection to immunodeficient mice). Myoblasts were obtained by muscle biopsy from five FSHD patients harboring the D4Z4 deletion. We selected the vastus lateralis muscle, which exhibited no clinical, radiological or pathological signs of dystrophy. The growth characteristics of these cells were compared with those of cells from normal control muscles, based on the culture yield, phenotypic characterization with anti-CD56 and anti-desmin antibodies, and the capacity for differentiation (myotube production in vitro and human dystrophin expression one month after injection to Rag2 immunodeficient mice). Patients' cells recovered from 1 g of muscle biopsy specimen resembled control cells in terms of their growth kinetics, culture yield, and capacity to differentiate and produce mature muscle cells. These results indicate that myoblasts taken from unaffected muscle of patients with FSHD warrant testing in a human cell therapy trial.

Tumour necrosis factor-mediated cell death pathways do not contribute to muscle fibre death in dystrophinopathies

There is evidence that apoptotic cell death mechanisms contribute to muscle fibre loss in dystrophinopathies, but little knowledge about the activators of the final degrading caspase cascade in muscle fibre apoptosis. As mitochondria-related activation of this caspase cascade, through e.g. APAF-1, could not be proven in dystrophin-deficient muscle, this study searches for other prospective candidates that may directly trigger apoptotic cell degradation by mitochondria-independent pathways involving the interaction of tumour necrosis factor-alpha (TNF-alpha) and TRAIL with death receptors and subsequent activation of caspase-8. The expression of TNF-alpha, TNF-R1, TRAIL, NF-(kappa)B and caspase-8 were studied in muscle biopsy specimens from 14 patients with a dystrophinopathy [10 Duchenne muscular dystrophies (DMD), 2 Becker MD, and 2 DMD carriers] by immunohistochemistry and Western blotting. In all types of dystrophinopathies, necrotic muscle fibres undergoing myophagocytosis displayed strong expression of TNF-alpha, TNF-R1, and TRAIL, which, however, was attributed to phagocytosing cells and not to the muscle fibres themselves. There was no up-regulation in normal-shaped or atrophic non-necrotic muscle fibres, or in intact muscle fibre segments adjacent to segmental necrosis and myophagocytosis. The expression profiles of caspase-8 and NF-(kappa)B resembled that of normal control muscle. There were likewise no significant differences in the Western blot analyses between normal control and dystrophin-deficient muscle. Based on these findings, a contribution of TNF-alpha or TRAIL-mediated cell death pathways to muscle fibre apoptosis or necrosis in dystrophinopathies could not be confirmed.

Report of the 115th ENMC workshop: DM2/PROMM and other myotonic dystrophies: 3rd Workshop, 14–16 February 2003, Naarden, The Netherlands

Myotonic dystrophy (DM1, Steinert’s disease) has been known for about 100 years. Its core features are myotonia, muscle weakness, and cataracts [1]. In 1994 another disorder was published with similar core features but lacking the DM1 mutation [2,3]. The name given to this disorder was proximal myotonic myopathy (PROMM, OMIM *160900). The first ENMC workshop on PROMM in 1997 established the clinical criteria for PROMM [4]. Prior to the second ENMC Workshop on PROMM in 2000, a major breakthrough had been accomplished when, in 1998, Ranum and Day [5] described the mapping of a new genetic locus in a large Minnesota family with myotonic dystrophy-like features on chromosome 3q21. This locus was named DM2. As a result, a new nomenclature emerged and the locus for myotonic dystrophy/Steinert’s disease was renamed DM1 [6]. Subsequent work showed that most PROMM families also mapped to the DM2 locus, whereas another smaller group of PROMM families appeared not to be linked to this locus. One separately described family with marked proximal atrophies, called proximal myotonic dystrophy [7], also mapped to the DM2 locus [8], and the workshop adopted the term myotonic dystrophy type 2 (DM2) for all the progressive myotonic multiorgan disorders linked to the DM2 locus. The clinical course of DM2 appeared to be more favourable compared to DM1. Families with DM2 did not have the severe congenital form of illness that occurs in DM1. Abnormalities in the social and cognitive abilities of adults with DM2 were typically mild or absent, and there was no prominent weakness of the facial and bulbar muscles. In DM2 the manual skills largely remained intact, and hypersomnia and mental retardation were not prominent findings. All these characteristic features of DM2 have not changed since the first ENMC Workshop[4]. They are still valid. In the advent of this 3rd workshop on DM2/PROMM, the definitive milestone of research efforts was achieved in 2001 with the identification of the mutation underlying DM2 by Ranum and Day, in collaboration with Ricker [9]. The mutation is a huge (CCTG)n microsatellite repeat expansion in the first intron of the ZNF9 gene and is thus similar to the (CTG)n repeat causing DM1. The aims with this 3rd ENMC Workshop on DM2/PROMM were:

Platelet-derived growth factor and its receptors are related to the progression of human muscular dystrophy: an immunohistochemical study.

This study has examined the immunological localization of platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF receptor (PDGFR) alpha and beta to clarify their role in the progression of muscular dystrophy. Biopsied frozen muscles from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD) were analysed immunohistochemically using antibodies raised against PDGF-A, PDGF-B, and PDGFR alpha and beta. Muscles from two dystrophic mouse models (dy and mdx mice) were also immunostained with antibodies raised against PDGFR alpha and beta. In normal human control muscle, neuromuscular junctions and vessels were positively stained with antibodies against PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta. In human dystrophic muscles, PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta were strongly immunolocalized in regenerating muscle fibres and infiltrating macrophages. PDGFR alpha was also immunolocalized to the muscle fibre sarcolemma and necrotic fibres. The most significant finding in this study was a remarkable overexpression of PDGFR beta and, to a lesser extent, PDGFR alpha in the endomysium of DMD and CMD muscles. PDGFR was also overexpressed in the interstitium of muscles from dystrophic mice, particularly dy mice. Double immunolabelling revealed that activated interstitial fibroblasts were clearly positive for PDGFR alpha and beta. However, DMD and CMD muscles with advanced fibrosis showed very poor reactivity against PDGF and PDGFR. Those findings were confirmed by immunoblotting with PDGFR beta. These findings indicate that PDGF and its receptors are significantly involved in the active stage of tissue destruction and are associated with the initiation or promotion of muscle fibrosis. They also have roles in muscle fibre regeneration and signalling at neuromuscular junctions in both normal and diseased muscle.