Induced degeneration and regeneration in aged muscle reduce tubular aggregates but not muscle function.

Introduction: Tubular aggregates (TA) are skeletal muscle structures that arise from the progressive accumulation of sarcoplasmic reticulum proteins. Cytoplasmic aggregates in muscle fibers have already been observed in mice and humans, mainly during aging and muscle disease processes. However, the effects of muscle regeneration on TA formation have not yet been reported. This study aimed to investigate the relationship between degeneration/regeneration and TA in aged murine models. We investigated the presence and quantity of TA in old males from two murine models with intense muscle degeneration and regeneration. Methods: One murine lineage was a Dmdmdx model of Duchenne muscular dystrophy (n = 6). In the other model, muscle damage was induced by electroporation in C57BL/6J wild-type mice, and analyzed after 5, 15, and 30 days post-electroporation (dpe; n = 15). Regeneration was evaluated based on the quantity of developmental myosin heavy chain (dMyHC)-positive fibers. Results: The frequency of fibers containing TA was higher in aged C57BL/6J (26 ± 8.3%) than in old dystrophic Dmdmdx mice (2.4 ± 2%). Comparing the data from induced degeneration/regeneration in normal mice revealed a reduced proportion of TA-containing fibers after 5 and 30 dpe. Normal aged muscle was able to regenerate and form dMyHC+ fibers, mainly at 5 dpe (0.1 ± 0.1 vs. 16.5 ± 2.6%). However, there was no difference in force or resistance between normal and 30 dpe animals, except for the measurements by the Actimeter device, which showed the worst parameters in the second group. Discussion: Our results suggest that TA also forms in the Dmdmdx muscle but in smaller amounts. The intense degeneration and regeneration of the old dystrophic model resulted in the generation of new muscle fibers with a lower quantity of TA. Data from electroporated wild-type mice support the idea that muscle regeneration leads to a reduction in the amount of TA. We suggest that TA accumulates in muscle fibers throughout physiological aging and that regeneration leads to the formation of new fibers without these structures. In addition, these new fibers do not confer functional benefits to the muscle.

Tetraspanin CD82 Associates with Trafficking Vesicle in Muscle Cells and Binds to Dysferlin and Myoferlin.

Tetraspanins organize protein complexes at the cell membrane and are responsible for assembling diverse binding partners in changing cellular states. Tetraspanin CD82 is a useful cell surface marker for prospective isolation of human myogenic progenitors and its expression is decreased in Duchenne muscular dystrophy (DMD) cell lines. The function of CD82 in skeletal muscle remains elusive, partly because the binding partners of this tetraspanin in muscle cells have not been identified. CD82-associated proteins are sought to be identified in human myotubes via mass spectrometry proteomics, which identifies dysferlin and myoferlin as CD82-binding partners. In human dysferlinopathy (Limb girdle muscular dystrophy R2, LGMDR2) myogenic cell lines, expression of CD82 protein is near absent in two of four patient samples. In the cell lines where CD82 protein levels are unaffected, increased expression of the ≈72 kDa mini-dysferlin product is identified using an antibody recognizing the dysferlin C-terminus. These data demonstrate that CD82 binds dysferlin/myoferlin in differentiating muscle cells and its expression can be affected by loss of dysferlin in human myogenic cells.

Rhabdomyosarcoma Associated with Core Myopathy/Malignant Hyperthermia: Combined Effect of Germline Variants in RYR1 and ASPSCR1 May Play a Role.

Rhabdomyosarcomas have been described in association with thyroid disease, dermatomyositis, Duchenne muscular dystrophy, and in muscular dystrophy models but not in patients with ryanodine receptor-1 gene (RYR1) pathogenic variants. We described here an 18-year-old male who reported a cervical nodule. Magnetic resonance images revealed a mass in the ethmoidal sinus corresponding to rhabdomyosarcoma. As his father died from malignant hyperthermia (MH), an in vitro contracture test was conducted and was positive for MH susceptibility. Muscle histopathological analysis in the biopsy showed the presence of cores. Molecular analysis using NGS sequencing identified germline variants in the RYR1 and ASPSCR1 (alveolar soft part sarcoma) genes. This report expands the spectrum of diseases associated with rhabdomyosarcomas and a possible differential diagnosis of soft tissue tumors in patients with RYR1 variants.

Predictive factors of the contracture test for diagnosing malignant hyperthermia in a Brazilian population sample: a retrospective observational study

Abstract Introduction Malignant Hyperthermia (MH) is a pharmacogenetic, hereditary and autosomal dominant syndrome triggered by halogenates/succinylcholine. The In Vitro Contracture Test (IVCT) is the gold standard diagnostic test for MH, and it evaluates abnormal skeletal muscle reactions of susceptible individuals (earlier/greater contracture) when exposed to caffeine/halothane. MH susceptibility episodes and IVCT seem to be related to individual features. Objective To assess variables that correlate with IVCT in Brazilian patients referred for MH investigation due to a history of personal/family MH. Methods We examined IVCTs of 80 patients investigated for MH between 2004‒2019. We recorded clinical data (age, sex, presence of muscle weakness or myopathy with muscle biopsy showing cores, genetic evaluation, IVCT result) and IVCT features (initial and final maximum contraction, caffeine/halothane concentration triggering contracture of 0.2g, contracture at caffeine concentration of 2 and 32 mmoL and at 2% halothane, and contraction after 100 Hz stimulation). Results Mean age of the sample was 35±13.3 years, and most of the subjects were female (n=43 or 54%) and MH susceptible (60%). Of the 20 subjects undergoing genetic investigation, 65% showed variants in RYR1/CACNA1S genes. We found no difference between the positive and negative IVCT groups regarding age, sex, number of probands, presence of muscle weakness or myopathy with muscle biopsy showing cores. Regression analysis revealed that the best predictors of positive IVCT were male sex (+12%), absence of muscle weakness (+20%), and personal MH background (+17%). Conclusions Positive IVCT results have been correlated to male probands, in accordance with early publications. Furthermore, normal muscle strength has been confirmed as a significant predictor of positive IVCT while investigating suspected MH cases.

Predictive factors of the contracture test for diagnosing malignant hyperthermia in a Brazilian population sample: a retrospective observational study.

INTRODUCTION: Malignant Hyperthermia (MH) is a pharmacogenetic, hereditary and autosomal dominant syndrome triggered by halogenates/succinylcholine. The In Vitro Contracture Test (IVCT) is the gold standard diagnostic test for MH, and it evaluates abnormal skeletal muscle reactions of susceptible individuals (earlier/greater contracture) when exposed to caffeine/halothane. MH susceptibility episodes and IVCT seem to be related to individual features. OBJECTIVE: To assess variables that correlate with IVCT in Brazilian patients referred for MH investigation due to a history of personal/family MH. METHODS: We examined IVCTs of 80 patients investigated for MH between 2004‒2019. We recorded clinical data (age, sex, presence of muscle weakness or myopathy with muscle biopsy showing cores, genetic evaluation, IVCT result) and IVCT features (initial and final maximum contraction, caffeine/halothane concentration triggering contracture of 0.2g, contracture at caffeine concentration of 2 and 32 mmoL and at 2% halothane, and contraction after 100 Hz stimulation). RESULTS: Mean age of the sample was 35±13.3 years, and most of the subjects were female (n=43 or 54%) and MH susceptible (60%). Of the 20 subjects undergoing genetic investigation, 65% showed variants in RYR1/CACNA1S genes. We found no difference between the positive and negative IVCT groups regarding age, sex, number of probands, presence of muscle weakness or myopathy with muscle biopsy showing cores. Regression analysis revealed that the best predictors of positive IVCT were male sex (+12%), absence of muscle weakness (+20%), and personal MH background (+17%). CONCLUSIONS: Positive IVCT results have been correlated to male probands, in accordance with early publications. Furthermore, normal muscle strength has been confirmed as a significant predictor of positive IVCT while investigating suspected MH cases.

Nemaline Myopathy in Brazilian Patients: Molecular and Clinical Characterization.

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the "typical" form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation.

Central Core Disease: Facial Weakness Differentiating Biallelic from Monoallelic Forms.

Central Core Disease (CCD) is a genetic neuromuscular disorder characterized by the presence of cores in muscle biopsy. The inheritance has been described as predominantly autosomal dominant (AD), and the disease may present as severe neonatal or mild adult forms. Here we report clinical and molecular data on a large cohort of Brazilian CCD patients, including a retrospective clinical analysis and molecular screening for RYR1 variants using Next-Generation Sequencing (NGS). We analyzed 27 patients from 19 unrelated families: four families (11 patients) with autosomal dominant inheritance (AD), two families (3 patients) with autosomal recessive (AR), and 13 sporadic cases. Biallelic RYR1 variants were found in six families (two AR and four sporadic cases) of the 14 molecularly analyzed families (~43%), suggesting a higher frequency of AR inheritance than expected. None of these cases presented a severe phenotype. Facial weakness was more common in biallelic than in monoallelic patients (p = 0.0043) and might be a marker for AR forms. NGS is highly effective for the identification of RYR1 variants in CCD patients, allowing the discovery of a higher proportion of AR cases with biallelic mutations. These data have important implications for the genetic counseling of the families.

Effects of low-intensity training on the brain and muscle in the congenital muscular dystrophy 1D model.

INTRODUCTION: Congenital Muscular Dystrophy type 1D (MDC1D) is characterized by a hypoglycosylation of α-dystroglycan protein (α-DG), and this may be strongly implicated in increased skeletal muscle tissue degeneration and abnormal brain development, leading to cognitive impairment. However, the pathophysiology of brain involvement is still unclear. Low-intensity exercise training (LIET) is known to contribute to decreased muscle degeneration in animal models of other forms of progressive muscular dystrophies. AIM: The objective of this study was to analyze the effects of LIET on cognitive involvement and oxidative stress in brain tissue and gastrocnemius muscle. METHODS: Male homozygous (Largemyd-/-), heterozygous (Largemyd+/-), and wild-type mice were used. To complete 28 days of life, they were subjected to a low-intensity exercise training (LIET) for 8 weeks. After the last day of training, 24 h were expected when the animals were submitted to inhibitory avoidance and open-field test. The striatum, prefrontal cortex, hippocampus, cortex, and gastrocnemius were collected for evaluation of protein carbonylation, lipid peroxidation, and catalase and superoxide dismutase activity. RESULTS: LIET was observed to reverse the alteration in aversive and habituation memory. Increased protein carbonylation in the striatum, prefrontal cortex, and hippocampus and lipid peroxidation in the prefrontal cortex and hippocampus were also reversed by LIET. In the evaluation of the antioxidant activity, LIET increased catalase activity in the hippocampus and cortex. In the gastrocnemius, LIET decreased the protein carbonylation and lipid peroxidation and increased catalase and superoxide dismutase activity. CONCLUSION: In conclusion, it can be inferred that LIET for 8 weeks was able to reverse the cognitive damage and oxidative stress in brain tissue and gastrocnemius muscle in MDC1D animals.

Spinal cord injury-related thermoregulatory impairment masks a fatal malignant hyperthermia crisis: a case report.

PURPOSE: Malignant hyperthermia (MH) is a hypermetabolic disorder that can occur in genetically susceptible individuals exposed to halogenated anesthetics and succinylcholine. Spinal cord injury (SCI) above the sixth thoracic vertebra is associated with dysfunction of the sympathetic/parasympathetic nervous pathways, including thermoregulatory dysfunction, presenting as hypothermia in cold environments because of vasodilation and heat loss. This effect could mitigate or obscure an MH episode. Here, we describe development of a fatal MH crisis in a patient with SCI. CLINICAL FEATURES: A 27-yr-old male patient with an SCI after fracture of the sixth cervical vertebra was admitted for spinal arthrodesis. Anesthetic medications included remifentanil, propofol, succinylcholine, rocuronium, and isoflurane. After the start of the surgery, muscular contractures resembling myoclonus were noted, which resolved with pancuronium administration. Four hours after the start of anesthesia, the patient presented with hyperthermia, hypercarbia, hypotension, muscle rigidity, arrhythmia, and cardiogenic shock, with metabolic/respiratory acidosis. Malignant hyperthermia was suspected and the treatment was started, but he developed cardiopulmonary arrest and died an hour and a half after the first cardiac arrest. Both parents were investigated and were found to have normal creatine kinase levels and positive in vitro contracture tests. His mother carried a variant in the ryanodine receptor type 1 (RYR1) gene (c.14918C>T), which is associated with MH. CONCLUSION: Spinal cord injury-induced thermoregulatory dysfunction may obscure the early diagnosis of MH and lead to fatal outcome.

RéSUMé: OBJECTIF: L'hyperthermie maligne est un trouble hypermétabolique qui peut survenir chez les personnes génétiquement susceptibles exposées à des anesthésiques volatils et à la succinylcholine. Les lésions médullaires situées au-dessus de la sixième vertèbre thoracique sont associées à un dysfonctionnement des voies nerveuses sympathiques / parasympathiques, y compris un trouble de la thermorégulation, et se présentent sous forme d'hypothermie dans des environnements froids en raison de la vasodilatation et de la perte de chaleur. Cet effet pourrait atténuer ou occulter un épisode d'hyperthermie maligne. Nous décrivons ici l'apparition d'une crise mortelle d'hyperthermie maligne chez un patient atteint de lésion médullaire. CARACTéRISTIQUES CLINIQUES: Un patient de 27 ans atteint d'une lésion médullaire après une fracture de la sixième vertèbre cervicale a été admis pour une arthrodèse rachidienne. Les médicaments anesthésiques comprenaient du rémifentanil, du propofol, de la succinylcholine, du rocuronium et de l'isoflurane. Après le début de la chirurgie, des contractures musculaires ressemblant à une myoclonie ont été notées, lesquelles se sont résolues avec l'administration de pancuronium. Quatre heures après l'induction d'anesthésie, le patient a présenté une hyperthermie, une hypercarbie, une hypotension, une rigidité musculaire, une arythmie et un choc cardiogénique, avec acidose métabolique / respiratoire. Une hyperthermie maligne a été suspectée et le traitement a été amorcé, mais le patient a subi un arrêt cardiorespiratoire et est décédé une heure et demie après le premier arrêt cardiaque. Les deux parents ont passés des tests et se sont avérés avoir des taux normaux de créatine kinase et des tests de contracture in vitro positifs. La mère du patient était porteuse d'un variant du gène récepteur de ryanodine de type 1 (RYR1) (c.14918C>T), lequel est associé à l'hyperthermie maligne. CONCLUSION: Un trouble de la thermorégulation induit par une lésion médullaire peut masquer un diagnostic précoce d'hyperthermie maligne et entraîner une issue fatale.

A Novel SPEG mutation causing congenital myopathy with fiber size disproportion and dilated cardiomyopathy with heart transplantation.

Congenital myopathies are a heterogeneous group of conditions diagnosed based on the clinical presentation, muscle histopathology and genetic defects. Recessive mutations in the SPEG gene have been described in recent years and are primarily associated with centronuclear myopathy with cardiomyopathy. In this report, we describe two Brazilian siblings, aged 13 and 6 years, with a novel homozygous mutation (c.8872 C>T:p.Arg2958Ter) in the SPEG gene leading to a congenital myopathy. In the older sibling, the muscle biopsy showed fiber size disproportion. The mean diameter of type 2 fibers (119 µm) was significantly higher than type 1 (57 µm) (P < 0,001) with a 72% prevalence of type 1 fibers. The patient also had progressive cardiomyopathy treated with heart transplantation. The present report expands the muscle histopathological findings related to mutations in the SPEG gene, including fiber size disproportion without central nuclei. Additionally, this report describes the first case of heart transplantation in a patient with SPEG mutations.

Sarcoglycanopathies: an update.

Sarcoglycanopathies are the most severe forms of autosomal recessive limb-girdle muscular dystrophies (LGMDs), constituting about 10-25% of LGMDs. The clinical phenotype is variable, but onset is usually in the first decade of life. Patients present muscle hypertrophy, elevated CK, variable muscle weaknesses, and progressive loss of ambulation. Four subtypes are known: LGMDR3, LGMDR4, LGMDR5 and LGMDR6, caused, respectively, by mutations in the SGCA, SGCB,SGCG and SGCD genes. Their four coded proteins, α-SG, ß-SG, λ-SG and δ-SG are part of the dystrophin-glycoprotein complex (DGC) present in muscle sarcolemma, which acts as a linker between the cytoskeleton of the muscle fiber and the extracellular matrix, providing mechanical support to the sarcolemma during myofiber contraction. Many different mutations have already been identified in all the sarcoglycan genes, with a predominance of some mutations in different populations. The diagnosis is currently based on the molecular screening for these mutations. Therapeutic approaches include the strategy of gene replacement mediated by a vector derived from adeno-associated virus (AAV). Pre-clinical studies have shown detectable levels of SG proteins in the muscle, and some improvement in the phenotype, in animal models. Therapeutic trials in humans are ongoing.

Satellite cells deficiency and defective regeneration in dynamin 2-related centronuclear myopathy.

Dynamin 2 (DNM2) is a ubiquitously expressed protein involved in many functions related to trafficking and remodeling of membranes and cytoskeleton dynamics. Mutations in the DNM2 gene cause the autosomal dominant centronuclear myopathy (AD-CNM), characterized mainly by muscle weakness and central nuclei. Several defects have been identified in the KI-Dnm2R465W/+ mouse model of the disease to explain the muscle phenotype, including reduction of the satellite cell pool in muscle, but the functional consequences of this depletion have not been characterized until now. Satellite cells (SC) are the main source for muscle growth and regeneration of mature tissue. Here, we investigated muscle regeneration in the KI-Dnm2R465W/+ mouse model for AD-CNM. We found a reduced number of Pax7-positive SCs, which were also less activated after induced muscle injury. The muscles of the KI-Dnm2R465W/+ mouse regenerated more slowly and less efficiently than wild-type ones, formed fewer new myofibers, and did not recover its normal mass 15 days after injury. Altogether, our data provide evidence that the muscle regeneration is impaired in the KI-Dnm2R465W/+ mouse and contribute with one more layer to the comprehension of the disease, by identifying a new pathomechanism linked to DNM2 mutations which may be involved in the muscle-specific impact occurring in AD-CNM.

Association of Three Different Mutations in the CLCN1 Gene Modulating the Phenotype in a Consanguineous Family with Myotonia Congenita.

Myotonia congenita is a genetic disease caused by mutations in the CLCN1 gene, which encodes for the major chloride skeletal channel ClC-1, involved in the normal repolarization of muscle action potentials and consequent relaxation of the muscle after contraction. Two allelic forms are recognized, depending on the phenotype and the inheritance pattern: the autosomal dominant Thomsen disease with milder symptoms and the autosomal recessive Becker disorder with a severe phenotype. Before the recent advances of molecular testing, the diagnosis and genetic counseling of families was a challenge due to the large number of mutations in the CLCN1 gene, found both in homozygous or in heterozygous state. Here, we studied a consanguineous family in which three members presented a variable phenotype of myotonia, associated to a combination of three different mutations in the CLCN1 gene. A pathogenic splicing site mutation which causes the skipping of exon 17 was present in homozygosis in one very severely affected son. This mutation was present in compound heterozygosis in the consanguineous parents, but interestingly it was associated to a different second variant in the other allele: c.1453 A > G in the mother and c.1842 G > C in the father. Both displayed variable, but less severe phenotypes than their homozygous son. These results highlight the importance of analyzing the combination of different variants in the same gene in particular in families with patients displaying different phenotypes. This approach may improve the diagnosis, prognosis, and genetic counseling of the involved families.

Manifesting carriers of X-linked myotubular myopathy: Genetic modifiers modulating the phenotype.

OBJECTIVE: To analyze the modulation of the phenotype in manifesting carriers of recessive X-linked myotubular myopathy (XLMTM), searching for possible genetic modifiers. METHODS: Twelve Brazilian families with XLMTM were molecularly and clinically evaluated. In 2 families, 4 of 6 and 2 of 5 manifesting female carriers were identified. These females were studied for X chromosome inactivation. In addition, whole-exome sequencing was performed, looking for possible modifier variants. We also determined the penetrance rate among carriers of the mutations responsible for the condition. RESULTS: Mutations in the MTM1 gene were identified in all index patients from the 12 families, being 4 of them novel. In the heterozygotes, X chromosome inactivation was random in 3 of 4 informative manifesting carriers. The disease penetrance rate was estimated to be 30%, compatible with incomplete penetrance. Exome comparative analyses identified variants within a segment of 4.2 Mb on chromosome 19, containing the killer cell immunoglobulin-like receptor cluster of genes that were present in all nonmanifesting carriers and absent in all manifesting carriers. We hypothesized that these killer cell immunoglobulin-like receptor variants may modulate the phenotype, acting as a protective factor in the nonmanifesting carriers. CONCLUSIONS: Affected XLMTM female carriers have been described with a surprisingly high frequency for a recessive X-linked disease, raising the question about the pattern of inheritance or the role of modifier factors acting on the disease phenotype. We demonstrated the possible existence of genetic mechanisms and variants accountable for the clinical manifestation in these women, which can become future targets for therapies.

Altered in vitro muscle differentiation in X-linked myopathy with excessive autophagy.

X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype.This article has an associated First Person interview with the first author of the paper.

Skeletal Muscle Injury by Electroporation: A Model to Study Degeneration/Regeneration Pathways in Muscle.

Skeletal muscle has a remarkable capacity to regenerate after injuries mainly due to a reservoir of precursor cells named satellite cells (SCs), which are responsible for after-birth growth and response to lesions, either by exercise or disease. Upon injury, the regenerative response includes SCs exit of quiescence, activation, proliferation, and fusion to repair or form new myofibers. This process is accompanied by inflammation, with infiltration of immune cells, primarily macrophages. Every phase of regeneration is highly regulated and orchestrated by many molecules and signaling pathways. The elucidation of players and mechanisms involved in muscle degeneration and regeneration is of extreme importance, especially for therapeutic strategies for muscle diseases.Here we are proposing a model of muscle injury induced by electroporation, which is an efficient method to induce muscle damage in order to follow the steps involved in degeneration and regeneration. Three days after electroporation, the muscle shows prominent signals of degeneration, like areas of necrosis and infiltration of macrophages, followed by regeneration, observed by the presence of centrally nucleated myofibers. After 5 days the regeneration is very active, with small dMyHC positive fibers. Fifteen days later, we observe a general regeneration of the muscle, with fibers with increased diameter after 60 days. This methodology is an easy and simple alternative to induce muscle lesion. It can be employed to study alterations in gene expression and the process of satellite cell recruitment, both in healthy and dystrophic/myopathic animal models for muscular dystrophy.

Isolation and Characterization of Muscle-Derived Stem Cells from Dystrophic Mouse Models.

The study of the population of muscle satellite cells (SC) is important to understand muscle regeneration and its involvement in the different dystrophic processes. We studied two dystrophic mouse models, Largemyd and Lama2dy2j/J, that show an intense and very similar pattern of muscle degeneration, but with differences in the expression of genes involved in the regeneration cascade. They are, therefore, interesting models to study possible differences in the mechanism of activation and action of satellite cells in the dystrophic muscle. The main objectives of this chapter are to describe the isolation and characterization of SC populations, evaluating the presence of myogenic and pluripotent stem cells markers in normal and dystrophic muscles.

Dominant or recessive mutations in the RYR1 gene causing central core myopathy in Brazilian patients.

Central Core Disease (CCD) is an inherited neuromuscular disorder characterized by the presence of cores in muscle biopsy. CCD is caused by mutations in the RYR1 gene. This gene encodes the ryanodine receptor 1, which is an intracellular calcium release channel from the sarcoplasmic reticulum to the cytosol in response to depolarization of the plasma membrane. Mutations in this gene are also associated with susceptibility to Malignant Hyperthermia (MHS). In this study, we evaluated 20 families with clinical and histological characteristics of CCD to identify primary mutations in patients, for diagnosis and genetic counseling of the families. We identified variants in the RYR1 gene in 19/20 families. The molecular pathogenicity was confirmed in 16 of them. Most of these variants (22/23) are missense and unique in the families. Two variants were recurrent in two different families. We identified six families with biallelic mutations, five compound heterozygotes with no consanguinity, and one homozygous, with consanguineous parents, resulting in 30% of cases with possible autosomal recessive inheritance. We identified seven novel variants, four of them classified as pathogenic. In one family, we identified two mutations in exon 102, segregating in cis, suggesting an additive effect of two mutations in the same allele. This work highlights the importance of using Next-Generation Sequencing technology for the molecular diagnosis of genetic diseases when a very large gene is involved, associated to a broad distribution of the mutations along it. These data also influence the prevention through adequate genetic counseling for the families and cautions against malignant hyperthermia susceptibility.