ABSTRACT
In vivo testing of glucocorticoid steroids in dystrophic mice offers important insights in benefits and risks of those drugs in the pathological context of muscular dystrophy. Frequency of dosing changes the spectrum of glucocorticoid effects on muscle and metabolic homeostasis. Here, we describe a combination of non-invasive and invasive methods to quantitatively discriminate the specific effects of intermittent (once-weekly) versus mainstay (once-daily) regimens on muscle fibrosis, muscle function, and metabolic homeostasis in murine models of Duchenne and limb-girdle muscular dystrophies.
Subject(s)
Muscular Dystrophies, Limb-Girdle , Muscular Dystrophy, Duchenne , Mice , Animals , Glucocorticoids/pharmacology , Glucocorticoids/metabolism , Disease Models, Animal , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/pathology , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophies, Limb-Girdle/pathologyABSTRACT
Limb-girdle muscular dystrophy (MD) type 2B (LGMD2B) and Duchenne MD (DMD) are caused by mutations to the Dysferlin and Dystrophin genes, respectively. We have recently demonstrated in typically mild dysferlin- and dystrophin-deficient mouse models that increased plasma cholesterol levels severely exacerbate muscle wasting, and that DMD patients display primary dyslipidemia characterized by elevated plasma cholesterol and triglycerides. Herein, we investigate lipoprotein abnormalities in LGMD2B and if statin therapy protects dysferlin-deficient mice (Dysf) from muscle damage. Herein, lipoproteins and liver enzymes from LGMD2B patients and dysferlin-null (Dysf) mice were analyzed. Simvastatin, which exhibits anti-muscle wasting effects in mouse models of DMD and corrects aberrant expression of key markers of lipid metabolism and endogenous cholesterol synthesis, was tested in Dysf mice. Muscle damage and fibrosis were assessed by immunohistochemistry and cholesterol signalling pathways via Western blot. LGMD2B patients show reduced serum high-density lipoprotein cholesterol (HDL-C) levels compared to healthy controls and exhibit a greater prevalence of abnormal total cholesterol (CHOL)/HDL-C ratios despite an absence of liver dysfunction. While Dysf mice presented with reduced CHOL and associated HDL-C and LDL-C-associated fractions, simvastatin treatment did not prevent muscle wasting in quadriceps and triceps muscle groups or correct aberrant low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) protein expression. LGMD2B patients present with reduced serum concentrations of HDL-C, a major metabolic comorbidity, and as a result, statin therapy is unlikely to prevent muscle wasting in this population. We propose that like DMD, LGMD2B should be considered as a new type of genetic dyslipidemia.
Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors , Muscular Dystrophies, Limb-Girdle , Mice , Animals , Dysferlin/genetics , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Dystrophin , Cholesterol, HDL , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Atrophy , Simvastatin/pharmacology , Simvastatin/therapeutic useABSTRACT
Muscle repair in dysferlinopathies is defective. Although macrophage (Mø)-rich infiltrates are prominent in damaged skeletal muscles of patients with dysferlinopathy, the contribution of the immune system to the disease pathology remains to be fully explored. Numbers of both pro-inflammatory M1 Mø and effector T cells are increased in muscle of dysferlin-deficient BlAJ mice. In addition, symptomatic BlAJ mice have increased muscle production of immunoproteasome. In vitro analyses using bone marrow-derived Mø of BlAJ mice show that immunoproteasome inhibition results in C3aR1 and C5aR1 downregulation and upregulation of M2-associated signaling. Administration of immunoproteasome inhibitor ONX-0914 to BlAJ mice rescues muscle function by reducing muscle infiltrates and fibro-adipogenesis. These findings reveal an important role of immunoproteasome in the progression of muscular dystrophy in BlAJ mouse and suggest that inhibition of immunoproteasome may produce therapeutic benefit in dysferlinopathy.
Subject(s)
Muscle, Skeletal , Muscular Dystrophies, Limb-Girdle , Mice , Animals , Dysferlin/genetics , Muscle, Skeletal/pathology , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/pathology , Immunity, InnateABSTRACT
Galectin-1 is a small (14.5 kDa) multifunctional protein with cell-cell and cell-ECM adhesion due to interactions with the carbohydrate recognition domain (CRD). In two types of muscular dystrophies, this lectin protein has shown therapeutic properties, including positive regulation of skeletal muscle differentiation and regeneration. Both Duchenne and limb-girdle muscular dystrophy 2B (LGMD2B) are subtypes of muscular dystrophies characterized by deficient membrane repair, muscle weakness, and eventual loss of ambulation. This chapter explains confocal techniques such as laser injury, calcium imaging, and galectin-1 localization to examine the effects of galectin-1 on membrane repair in injured LGMD2B models.
Subject(s)
Galectin 1 , Muscular Dystrophies, Limb-Girdle , Sarcolemma , Galectin 1/metabolism , Galectin 1/pharmacology , Galectin 1/therapeutic use , Humans , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Dystrophies, Limb-Girdle/drug therapy , Sarcolemma/drug effects , Sarcolemma/physiologyABSTRACT
BACKGROUND: Glucocorticoid steroids are standard of care in Duchenne Muscular Dystrophy (DMD) to slow disease course. Use of glucocorticoids in other muscular dystrophies, including Becker (BMD) and Limb Girdle (LGMD), has been less explored. Recently, preclinical studies conducted in DMD and LGMD mouse models showed once-weekly prednisone was associated with improved muscle performance without activation of muscle atrophy genes. OBJECTIVE: To determine safety and tolerability of once-weekly prednisone in patients with LGMD and BMD. METHODS: We conducted an open label, exploratory single center study of of once-weekly prednisone at 0.75-1âmg/Kg in LGMD (nâ=â19) and BMD (nâ=â1) (mean age 35, range 18-60). The LGMD participants represented multiple different LGMD subtypes, and the study included ambulatory and non-ambulatory participants. Participants were assessed at baseline and 24 weeks for vital signs, blood biomarkers, and for patient-reported side effects. As secondary endpoints, functional muscle testing and body composition were measured. RESULTS: Over the 24-week study, there were no significant changes in blood pressure, HgbA1C, or lipid profiles. We observed a reduction in serum creatine kinase over the study interval. Whole body DEXA scanning suggested a possible increase in lean mass and a reduction in adiposity. Functional measures suggested trends in improved muscle performance. CONCLUSIONS: In this single center, open label pilot study, once-weekly prednisone was safe and well tolerated. Additional investigation of once-weekly prednisone in a larger cohort and for a longer period of time is warranted.
Subject(s)
Muscular Dystrophies, Limb-Girdle , Muscular Dystrophy, Duchenne , Prednisone , Drug Administration Schedule , Humans , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophy, Duchenne/drug therapy , Pilot Projects , Prednisone/administration & dosage , Prednisone/adverse effectsABSTRACT
Limb-girdle muscular dystrophy D2 (LGMDD2) is an ultrarare autosomal dominant myopathy caused by mutation of the normal stop codon of the TNPO3 nuclear importin. The mutant protein carries a 15 amino acid C-terminal extension associated with pathogenicity. Here we report the first animal model of the disease by expressing the human mutant TNPO3 gene in Drosophila musculature or motor neurons and concomitantly silencing the endogenous expression of the fly protein ortholog. A similar genotype expressing wildtype TNPO3 served as a control. Phenotypes characterization revealed that mutant TNPO3 expression targeted at muscles or motor neurons caused LGMDD2-like phenotypes such as muscle degeneration and atrophy, and reduced locomotor ability. Notably, LGMDD2 mutation increase TNPO3 at the transcript and protein level in the Drosophila model Upregulated muscle autophagy observed in LGMDD2 patients was also confirmed in the fly model, in which the anti-autophagic drug chloroquine was able to rescue histologic and functional phenotypes. Overall, we provide a proof of concept of autophagy as a target to treat disease phenotypes and propose a neurogenic component to explain mutant TNPO3 pathogenicity in diseased muscles.
Subject(s)
Autophagy/drug effects , Chloroquine/pharmacology , Chloroquine/therapeutic use , Disease Models, Animal , Drosophila melanogaster/drug effects , Muscular Atrophy/drug therapy , Muscular Dystrophies, Limb-Girdle/complications , Animals , Animals, Genetically Modified , Autophagy/genetics , Drosophila melanogaster/genetics , Female , Humans , Insect Hormones , Locomotion , Male , Motor Neurons/metabolism , Muscles/metabolism , Muscular Atrophy/complications , Muscular Atrophy/genetics , Muscular Atrophy/pathology , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/pathology , Phenotype , Survival Rate , beta Karyopherins/genetics , beta Karyopherins/metabolismABSTRACT
Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients' muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3ß (GSK-3ß), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.
Subject(s)
Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Muscular Dystrophies, Limb-Girdle/drug therapy , Nerve Tissue Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Adenosine Triphosphate/metabolism , Allosteric Site/drug effects , CD56 Antigen/analysis , Calpain/deficiency , Calpain/genetics , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Glycogen Synthase Kinase 3 beta/chemistry , Humans , Hydrazines/pharmacology , Hydrazines/therapeutic use , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/deficiency , Muscle Proteins/genetics , Muscular Dystrophies, Limb-Girdle/enzymology , Nerve Tissue Proteins/chemistry , Phosphorylation , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/physiology , Quinolones/pharmacology , Quinolones/therapeutic use , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/physiology , Thiadiazoles/pharmacology , Thiadiazoles/therapeutic use , Wnt Signaling Pathway/drug effectsABSTRACT
INTRODUCTION/AIMS: In this study we report the results of a phase Ib/IIa, open-label, multiple ascending-dose trial of domagrozumab, a myostatin inhibitor, in patients with fukutin-related protein (FKRP)-associated limb-girdle muscular dystrophy. METHODS: Nineteen patients were enrolled and assigned to one of three dosing arms (5, 20, or 40 mg/kg every 4 weeks). After 32 weeks of treatment, participants receiving the lowest dose were switched to the highest dose (40 mg/kg) for an additional 32 weeks. An extension study was also conducted. The primary endpoints were safety and tolerability. Secondary endpoints included muscle strength, timed function testing, pulmonary function, lean body mass, pharmacokinetics, and pharmacodynamics. As an exploratory outcome, muscle fat fractions were derived from whole-body magnetic resonance images. RESULTS: Serum concentrations of domagrozumab increased in a dose-dependent manner and modest levels of myostatin inhibition were observed in both serum and muscle tissue. The most frequently occurring adverse events were injuries secondary to falls. There were no significant between-group differences in the strength, functional, or imaging outcomes studied. DISCUSSION: We conclude that, although domagrozumab was safe in patients in limb-girdle muscular dystrophy type 2I/R9, there was no clear evidence supporting its efficacy in improving muscle strength or function.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Muscle Strength/drug effects , Muscular Dystrophies, Limb-Girdle/drug therapy , Adult , Body Composition/drug effects , Female , Humans , Male , Middle Aged , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiopathology , Muscular Dystrophies, Limb-Girdle/physiopathology , Pentosyltransferases/metabolism , Young AdultABSTRACT
Mutations in CAPN3 cause limb girdle muscular dystrophy R1 (LGMDR1, formerly LGMD2A) and lead to progressive and debilitating muscle wasting. Calpain 3 deficiency is associated with impaired CaMKIIß signaling and blunted transcriptional programs that encode the slow-oxidative muscle phenotype. We conducted a high-throughput screen on a target of CaMKII (Myl2) to identify compounds to override this signaling defect; 4 were tested in vivo in the Capn3 knockout (C3KO) model of LGMDR1. The leading compound, AMBMP, showed good exposure and was able to reverse the LGMDR1 phenotype in vivo, including improved oxidative properties, increased slow fiber size, and enhanced exercise performance. AMBMP also activated CaMKIIß signaling, but it did not alter other pathways known to be associated with muscle growth. Thus, AMBMP treatment activates CaMKII and metabolically reprograms skeletal muscle toward a slow muscle phenotype. These proof-of-concept studies lend support for an approach to the development of therapeutics for LGMDR1.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calpain/genetics , Cardiac Myosins/genetics , Muscle Proteins/genetics , Muscular Dystrophies, Limb-Girdle/drug therapy , Myosin Light Chains/genetics , Pyrimidines/pharmacology , Small Molecule Libraries/pharmacology , Acyltransferases/genetics , Acyltransferases/metabolism , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Calpain/deficiency , Cardiac Myosins/metabolism , Cell Line , Creatine Kinase, Mitochondrial Form/genetics , Creatine Kinase, Mitochondrial Form/metabolism , Female , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/deficiency , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/metabolism , Muscular Dystrophies, Limb-Girdle/pathology , Myoblasts/drug effects , Myoblasts/metabolism , Myoblasts/pathology , Myosin Light Chains/metabolism , Oxidative Stress , Phenotype , Physical Conditioning, Animal , Protein Isoforms/genetics , Protein Isoforms/metabolism , Signal TransductionABSTRACT
We identified the known c.1_9del mutation in the PLEC gene in four unrelated females from consanguineous families of Turkish origin. All individuals presented with slowly progressive limb-girdle weakness without any dermatological findings, and dystrophic changes observed in their muscle biopsies. Additionally, the neurological examination revealed ptosis, facial weakness, fatigability, and muscle cramps in all four cases. In two patients, repetitive nerve stimulation showed a borderline decrement and a high jitter was detected in all patients by single-fiber electromyography. Clinical improvement was observed after treatment with pyridostigmine and salbutamol was started. We further characterize the phenotype of patients with limb-girdle muscular dystrophy R17 clinically, by muscle magnetic resonance imaging (MRI) features and by describing a common 3.8 Mb haplotype in three individuals from the same geographical region. In addition, we review the neuromuscular symptoms associated with PLEC mutations and the role of plectin in the neuromuscular junction.
Subject(s)
Muscular Dystrophies, Limb-Girdle/genetics , Mutation , Myasthenic Syndromes, Congenital/genetics , Phenotype , Plectin/genetics , Adolescent , Adult , Child , Exons , Female , Homozygote , Humans , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophies, Limb-Girdle/pathology , Myasthenic Syndromes, Congenital/drug therapy , Myasthenic Syndromes, Congenital/pathologyABSTRACT
Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.
Subject(s)
Gain of Function Mutation , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Muscle Strength/genetics , Muscular Dystrophies, Limb-Girdle/metabolism , Nerve Tissue Proteins/metabolism , Animals , Disease Models, Animal , HSP40 Heat-Shock Proteins/antagonists & inhibitors , HSP40 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/antagonists & inhibitors , HSP70 Heat-Shock Proteins/genetics , HeLa Cells , Humans , Mice , Molecular Chaperones/antagonists & inhibitors , Molecular Chaperones/genetics , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/pathology , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Saccharomyces cerevisiaeABSTRACT
Dysferlinopathies are a non-lethal group of late-onset muscular dystrophies. Here, we evaluated the fusion ability of primary myoblasts from young dysf-/- mice and the muscle histopathology prior to, and during early stages of disease onset. The ability of primary myoblasts of 5-week-old dysf-/- mice to form large myotubes was delayed compared to their wild-type counterparts, as evaluated by scanning electron microscopy. However, their fusion activity, as reflected by the presence of actin filaments connecting several cells, was enhanced by the antifibrotic drug halofuginone. Early dystrophic signs were already apparent in 4-week-old dysf-/- mice; their collagen level was double that in wild-type mice and continued to rise until 5 months of age. Continuous treatment with halofuginone from 4 weeks to 5 months of age reduced muscle fibrosis in a phosphorylated-Smad3 inhibition-related manner. Halofuginone also enhanced myofiber hypertrophy, reduced the percentage of centrally nucleated myofibers, and increased muscle performance. Together, the data suggest an inhibitory effect of halofuginone on the muscle histopathology at very early stages of dysferlinopathy, and enhancement of muscle performance. These results offer new opportunities for early pharmaceutical treatment in dysferlinopathies with favorable outcomes at later stages of life.
Subject(s)
Dysferlin , Muscle, Skeletal/drug effects , Muscular Dystrophies, Limb-Girdle/drug therapy , Piperidines/pharmacology , Protein Synthesis Inhibitors/pharmacology , Quinazolinones/pharmacology , Animals , Disease Models, Animal , Fibrosis/drug therapy , Fibrosis/pathology , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Skeletal/pathology , Muscular Dystrophies, Limb-Girdle/pathology , Muscular Dystrophies, Limb-Girdle/physiopathologyABSTRACT
Mutations in dysferlin are responsible for a group of progressive, recessively inherited muscular dystrophies known as dysferlinopathies. Using recombinant proteins and affinity purification methods combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found that AMP-activated protein kinase (AMPK)γ1 was bound to a region of dysferlin located between the third and fourth C2 domains. Using ex vivo laser injury experiments, we demonstrated that the AMPK complex was vital for the sarcolemmal damage repair of skeletal muscle fibers. Injury-induced AMPK complex accumulation was dependent on the presence of Ca2+, and the rate of accumulation was regulated by dysferlin. Furthermore, it was found that the phosphorylation of AMPKα was essential for plasma membrane repair, and treatment with an AMPK activator rescued the membrane-repair impairment observed in immortalized human myotubes with reduced expression of dysferlin and dysferlin-null mouse fibers. Finally, it was determined that treatment with the AMPK activator metformin improved the muscle phenotype in zebrafish and mouse models of dysferlin deficiency. These findings indicate that the AMPK complex is essential for plasma membrane repair and is a potential therapeutic target for dysferlinopathy.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Dysferlin/chemistry , Dysferlin/metabolism , Metformin/administration & dosage , Muscle, Skeletal/injuries , Muscular Dystrophies, Limb-Girdle/drug therapy , Animals , Cell Line , Disease Models, Animal , Dysferlin/genetics , Humans , Lasers/adverse effects , Metformin/pharmacology , Mice , Muscle, Skeletal/metabolism , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/metabolism , Mutation , Phosphorylation , Protein Domains , Sarcolemma/metabolism , ZebrafishABSTRACT
Dysferlinopathies, which are muscular diseases caused by mutations in the dysferlin gene, remain serious medical problems due to the lack of therapeutic agents. Herein, we report the design, synthesis, and structure-activity relationships of a 2,6-disubstituted 3H-imidazo[4,5-b]pyridine series, which was identified from the phenotypic screening of chemicals that increase the level of dysferlin in myocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Optimization studies with cell-based phenotypic assay led to the identification of a highly potent compound, 19, with dysferlin elevation effects at double-digit nanomolar concentrations. In addition, the molecular target of our chemical series was identified as tubulin, through a tubulin polymerization assay and a competitive binding assay using a photoaffinity labeling probe.
Subject(s)
Imidazoles/chemistry , Muscular Dystrophies, Limb-Girdle/drug therapy , Pyridines/chemistry , Tubulin Modulators/therapeutic use , Binding Sites , Cell Differentiation , Cell Proliferation/drug effects , Drug Design , Dysferlin/metabolism , Hep G2 Cells , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Molecular Docking Simulation , Muscular Dystrophies, Limb-Girdle/pathology , MyoD Protein/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Protein Structure, Tertiary , Pyridines/pharmacology , Pyridines/therapeutic use , Structure-Activity Relationship , Tubulin/chemistry , Tubulin/metabolism , Tubulin Modulators/chemistry , Tubulin Modulators/pharmacologyABSTRACT
There is no cure or beneficial management option for Limb-Girdle muscular dystrophy (MD) type 2B (LGMD2B). Losartan, a blood pressure (BP) lowering angiotensin II (AngII) receptor type 1 (ATR1) blocker (ARB) with unique anti-transforming growth factor-ß (TGF-ß) properties, can protect muscles in various types of MD such as Duchenne MD, suggesting a potential benefit for LGMD2B patients. Herein, we show in a mild, dysferlin-null mouse model of LGMD2B that losartan increased quadriceps muscle fibrosis (142%; P<0.0001). In a severe, atherogenic diet-fed model of LGMD2B recently described by our group, losartan further exacerbated dysferlin-null mouse muscle wasting in quadriceps and triceps brachii, two muscles typically affected by LGMD2B, by 40% and 51%, respectively (P<0.05). Lower TGF-ß signalling was not observed with losartan, therefore plasma levels of atherogenic lipids known to aggravate LGMD2B severity were investigated. We report that losartan increased both plasma triglycerides and cholesterol concentrations in dysferlin-null mice. Other protective properties of losartan, such as increased nitric oxide release and BP lowering, were also reduced in the absence of dysferlin expression. Our data suggest that LGMD2B patients may show some resistance to the primary BP-lowering effects of losartan along with accelerated muscle wasting and dyslipidemia. Hence, we urge caution on the use of ARBs in this population as their ATR1 pathway may be dysfunctional.
Subject(s)
Angiotensin II Type 1 Receptor Blockers/therapeutic use , Blood Pressure/drug effects , Losartan/therapeutic use , Muscle, Skeletal/drug effects , Muscular Dystrophies, Limb-Girdle/drug therapy , Animals , Cholesterol/blood , Creatine Kinase/blood , Disease Models, Animal , Fluorescent Antibody Technique , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/pathology , Muscular Dystrophies, Limb-Girdle/pathology , Triglycerides/bloodABSTRACT
Limb-girdle myasthenia with tubular aggregates, a subtype of congenital myasthenic syndrome, is an extremely rare autosomal recessive genetic disease characterized by prominent limb-girdle weakness and good response to acetylcholinesterase inhibitor therapy. Herein, we reported two novel mutations of GFPT1 gene in a Chinese pedigree. Two siblings presented with fatigue, weakness of limb-girdle and decrement of the muscle action potential with repetitive nerve stimulation. Thus, myasthenia gravis was initially suspected, but anti-AChR antibodies were negative. Two novel missense mutations (p.Lys154Asn and p.Asn363Ser) in GFPT1 were identified through genetic testing conducted on 167 well-established genes associated with muscular diseases by targeted high throughput sequencing. Both mutations have not been recorded in the dsSNP database, Exome Aggregation Consortium database and 1000 Genomes Project database. The mutation sites were co-segregated with the phenotype and conserved between the different species. The mutations were not found in the 200 unrelated normal controls. Muscle biopsies revealed tubular aggregates, in accordance with previous reports with GFPT1 mutations. Subsequently, dramatic improvement in strength occurred following anti-cholinesterase therapy. Our study will be helpful for the diagnosis and treatment for Limb-girdle myasthenia with tubular aggregates.
Subject(s)
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Mutation, Missense/genetics , Myopathies, Structural, Congenital/genetics , Action Potentials , Animals , Asian People , Cholinesterase Inhibitors/therapeutic use , Databases, Genetic , Electric Stimulation , Female , Genetic Testing , Humans , Male , Muscle, Skeletal/pathology , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophies, Limb-Girdle/pathology , Myopathies, Structural, Congenital/drug therapy , Myopathies, Structural, Congenital/pathology , Pedigree , Polymorphism, Single Nucleotide , Pyridostigmine Bromide/therapeutic use , Young AdultABSTRACT
Dysferlinopathy is a progressive muscle disorder that includes limb-girdle muscular dystrophy type 2B and Miyoshi myopathy (MM). It is caused by mutations in the dysferlin (DYSF) gene, whose function is to reseal the muscular membrane. Treatment with proteasome inhibitor MG-132 has been shown to increase misfolded dysferlin in fibroblasts, allowing them to recover their membrane resealing function. Here, we developed a screening system based on myocytes from MM patient-derived induced pluripotent stem cells. According to the screening, nocodazole was found to effectively increase the level of dysferlin in cells, which, in turn, enhanced membrane resealing following injury by laser irradiation. Moreover, the increase was due to microtubule disorganization and involved autophagy rather than the proteasome degradation pathway. These findings suggest that increasing the amount of misfolded dysferlin using small molecules could represent an effective future clinical treatment for dysferlinopathy. Stem Cells Translational Medicine 2019;8:1017-1029.
Subject(s)
Drug Evaluation, Preclinical/methods , Induced Pluripotent Stem Cells/transplantation , Muscle Cells/metabolism , Muscular Dystrophies, Limb-Girdle/drug therapy , Adult , Female , Humans , Middle Aged , PhenotypeABSTRACT
Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.
Subject(s)
Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Muscular Dystrophies, Limb-Girdle/drug therapy , Muscular Dystrophies, Limb-Girdle/physiopathology , Muscular Dystrophies/drug therapy , Muscular Dystrophies/physiopathology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Disease Models, Animal , Drug Evaluation, Preclinical , Gene Knockout Techniques , Humans , Locomotion , Movement , Muscle, Skeletal/physiopathology , Muscular Dystrophies/genetics , Muscular Dystrophies, Limb-Girdle/genetics , Mutation , Pentosyltransferases , Phenotype , Proteins , Transcriptome , Walker-Warburg Syndrome , ZebrafishABSTRACT
In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf-/-) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf-/- myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1-43 at the injury site of dysf-/- myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf-/- mice. Halofuginone increased lysosome scattering across the cytosol of dysf-/- primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf-/- versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf-/- myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events.
Subject(s)
Dysferlin/genetics , Muscular Dystrophies, Limb-Girdle/drug therapy , Piperidines/administration & dosage , Quinazolinones/administration & dosage , Synaptotagmins/genetics , Animals , Disease Models, Animal , Humans , Mice , Mice, Knockout , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/pathology , Myoblasts/metabolism , Phosphatidylinositol 3-Kinases/geneticsABSTRACT
The muscular dystrophies are genetically diverse. Shared pathological features among muscular dystrophies include breakdown, or loss of muscle, and accompanying fibrotic replacement. Novel strategies are needed to enhance muscle repair and function and to slow this pathological remodeling. Glucocorticoid steroids, like prednisone, are known to delay loss of ambulation in patients with Duchenne muscular dystrophy but are accompanied by prominent adverse effects. However, less is known about the effects of steroid administration in other types of muscular dystrophies, including limb-girdle muscular dystrophies (LGMDs). LGMD 2B is caused by loss of dysferlin, a membrane repair protein, and LGMD 2C is caused by loss of the dystrophin-associated protein, γ-sarcoglycan. Herein, we assessed the efficacy of steroid dosing on sarcolemmal repair, muscle function, histopathology, and the regenerative capacity of primary muscle cells. We found that in murine models of LGMD 2B and 2C, daily prednisone dosing reduced muscle damage and fibroinflammatory infiltration. However, daily prednisone dosing also correlated with increased muscle adipogenesis and atrophic remodeling. Conversely, intermittent dosing of prednisone, provided once weekly, enhanced muscle repair and did not induce atrophy or adipogenesis, and was associated with improved muscle function. These data indicate that dosing frequency of glucocorticoid steroids affects muscle remodeling in non-Duchenne muscular dystrophies, suggesting a positive outcome associated with intermittent steroid dosing in LGMD 2B and 2C muscle.