The Danger Signal Extracellular ATP Is Involved in the Immunomediated Damage of α-Sarcoglycan-Deficient Muscular Dystrophy.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

The administration of high-mobility group box 1 fragment prevents deterioration of cardiac performance by enhancement of bone marrow mesenchymal stem cell homing in the delta-sarcoglycan-deficient hamster.

OBJECTIVES: We hypothesized that systemic administration of high-mobility group box 1 fragment attenuates the progression of myocardial fibrosis and cardiac dysfunction in a hamster model of dilated cardiomyopathy by recruiting bone marrow mesenchymal stem cells thus causing enhancement of a self-regeneration system. METHODS: Twenty-week-old J2N-k hamsters, which are δ-sarcoglycan-deficient, were treated with systemic injection of high-mobility group box 1 fragment (HMGB1, n = 15) or phosphate buffered saline (control, n = 11). Echocardiography for left ventricular function, cardiac histology, and molecular biology were analyzed. The life-prolonging effect was assessed separately using the HMGB1 and control groups, in addition to a monthly HMGB1 group which received monthly systemic injections of high-mobility group box 1 fragment, 3 times (HMGB1, n = 11, control, n = 9, monthly HMGB1, n = 9). RESULTS: The HMGB1 group showed improved left ventricular ejection fraction, reduced myocardial fibrosis, and increased capillary density. The number of platelet-derived growth factor receptor-alpha and CD106 positive mesenchymal stem cells detected in the myocardium was significantly increased, and intra-myocardial expression of tumor necrosis factor α stimulating gene 6, hepatic growth factor, and vascular endothelial growth factor were significantly upregulated after high-mobility group box 1 fragment administration. Improved survival was observed in the monthly HMGB1 group compared with the control group. CONCLUSIONS: Systemic high-mobility group box 1 fragment administration attenuates the progression of left ventricular remodeling in a hamster model of dilated cardiomyopathy by enhanced homing of bone marrow mesenchymal stem cells into damaged myocardium, suggesting that high-mobility group box 1 fragment could be a new treatment for dilated cardiomyopathy.

Xanthine oxidase is hyper-active in Duchenne muscular dystrophy.

Generation of superoxide by xanthine oxidase can be stimulated under ischemic and aberrant calcium homeostasis. Because patients and mice with Duchenne muscular dystrophy (DMD) suffer from ischemia and excessive calcium influx, we tested the hypothesis that xanthine oxidase activity is elevated and contributes to disease pathology. Xanthine oxidase activity was measured by urinary isoxanthopterin in DMD patients at rest and in response to exercise. Urinary isoxanthopterin/creatinine was elevated compared to age-matched controls and Becker muscular dystrophy (BMD) patients. Concentrations were also increased after a six minute walk test in ambulatory patients. We also measured urinary isoxanthopterin in wildtype mice and a number of dystrophic mouse models; the DMD mouse model (mdx), mdx mice overexpressing a variety of transgenic miniaturized and chimeric skeletal muscle-specific dystrophins and utrophin and the ß-sarcoglycan deficient (Scgb-/-) mouse which represents type 2E human limb-girdle muscular dystrophy. Mdx and Scgb-/-mice had greater urinary isoxanthopterin/creatinine than wildtype mice while mdx mice expressing dystrophin or utrophin linking the extracellular matrix to the actin cytoskeleton were not different than wildtype. We also measured higher levels of urinary ortho-tyrosine in humans and mice deficient for dystrophin to confirm elevated oxidative stress. Surprisingly, mdx had lower xanthine oxidase protein levels and higher mRNA in gastrocnemius muscle compared to wildtype mice, however, the enzymatic activity of skeletal muscle xanthine oxidase was elevated above wildtype and a transgenic rescued mdx mouse (DysΔMTB-mdx). Downhill treadmill running also caused significant increases in mdx urinary isoxanthopterin that was prevented with the xanthine oxidase inhibitor allopurinol. Similarly, in vitro eccentric contraction-induced force drop of mdx muscle was attenuated by the allopurinol metabolite, oxypurinol. Together, our data suggests hyper-activity of xanthine oxidase in DMD, identifies xanthine oxidase activity as a contributing factor in eccentric contraction-induced force drop of dystrophin-deficient skeletal muscle and highlights the potential of isoxanthopterin as a noninvasive biomarker in DMD.

(-)-Epicatechin inhibits development of dilated cardiomyopathy in δ sarcoglycan null mouse.

BACKGROUND AND AIMS: Several studies propose that (-)-epicatechin, a flavonol present in high concentration in the cocoa, has cardioprotective effects. This study aimed to evaluate the impact of (-)-epicatechin on the development of dilated cardiomyopathy in a δ sarcoglycan null mouse model. METHODS AND RESULTS: δ Sarcoglycan null mice were treated for 15 days with (-)-epicatechin. Histological and morphometric analysis of the hearts treated mutant mice showed significant reduction of the vasoconstrictions in the coronary arteries as well as fewer areas with fibrosis and a reduction in the loss of the ventricular wall. On the contrary, it was observed a thickening of this region. By Western blot analysis, it was shown, and increment in the phosphorylation level of eNOS and PI3K/AKT/mTOR/p70S6K proteins in the heart of the (-)-epicatechin treated animals. On the other hand, we observed a significantly decreased level of the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) heart failure markers. CONCLUSION: All the results indicate that (-)-epicatechin has the potential to prevent the development of dilated cardiomyopathy of genetic origin and encourages the use of this flavonol as a pharmacological therapy for dilated cardiomyopathy and heart failure diseases.

Oxidative stress preconditioning of mouse perivascular myogenic progenitors selects a subpopulation of cells with a distinct survival advantage in vitro and in vivo.

Cell engraftment, survival and integration during transplantation procedures represent the crux of cell-based therapies. Thus, there have been many studies focused on improving cell viability upon implantation. We used severe oxidative stress to select for a mouse mesoangioblast subpopulation in vitro and found that this subpopulation retained self-renewal and myogenic differentiation capacities while notably enhancing cell survival, proliferation and migration relative to unselected cells. Additionally, this subpopulation of cells presented different resistance and recovery properties upon oxidative stress treatment, demonstrating select advantages over parental mesoangioblasts in our experimental analysis. Specifically, the cells were resistant to oxidative environments, demonstrating survival, continuous self-renewal and improved migration capability. The primary outcome of the selected cells was determined in in vivo experiments in which immunocompromised dystrophic mice were injected intramuscularly in the tibialis anterior with selected or non-selected mesoangioblasts. Resistant mesoangioblasts exhibited markedly enhanced survival and integration into the host skeletal muscle, accounting for a more than 70% increase in engraftment compared with that of the unselected mesoangioblast cell population and leading to remarkable muscle recovery. Thus, the positive effects of sorting on mesoangioblast cell behaviour in vitro and in vivo suggest that a selection step involving oxidative stress preconditioning may provide a novel methodology to select for resistant cells for use in regenerative tissue applications to prevent high mortality rates upon transplantation.

ß-Sarcoglycan Deficiency Reduces Atherosclerotic Plaque Development in ApoE-Null Mice.

BACKGROUND: Smooth muscle cells are important for atherosclerotic plaque stability. Their proper ability to communicate with the extracellular matrix is crucial for maintaining the correct tissue integrity. In this study, we have investigated the role of ß-sarcoglycan within the matrix-binding dystrophin-glycoprotein complex in the development of atherosclerosis. RESULTS: Atherosclerotic plaque development was significantly reduced in ApoE-deficient mice lacking ß-sarcoglycan, and their plaques contained an increase in differentiated smooth muscle cells. ApoE-deficient mice lacking ß-sarcoglycan showed a reduction in ovarian adipose tissue and adipocyte size, while the total weight of the animals was not significantly different. Western blot analysis of adipose tissues showed a decreased activation of protein kinase B, while that of AMP-activated kinase was increased in mice lacking ß-sarcoglycan. Analysis of plasma in ß-sarcoglycan-deficient mice revealed reduced levels of leptin, adiponectin, insulin, cholesterol, and triglycerides but increased levels of IL-6, IL-17, and TNF-α. CONCLUSIONS: Our results indicate that the dystrophin-glycoprotein complex and ß-sarcoglycan can affect the atherosclerotic process. Furthermore, the results show the effects of ß-sarcoglycan deficiency on adipose tissue and lipid metabolism, which may also have contributed to the atherosclerotic plaque reduction.

Myofiber-specific inhibition of TGFß signaling protects skeletal muscle from injury and dystrophic disease in mice.

Muscular dystrophy (MD) is a disease characterized by skeletal muscle necrosis and the progressive accumulation of fibrotic tissue. While transforming growth factor (TGF)-ß has emerged as central effector of MD and fibrotic disease, the cell types in diseased muscle that underlie TGFß-dependent pathology have not been segregated. Here, we generated transgenic mice with myofiber-specific inhibition of TGFß signaling owing to expression of a TGFß type II receptor dominant-negative (dnTGFßRII) truncation mutant. Expression of dnTGFßRII in myofibers mitigated the dystrophic phenotype observed in δ-sarcoglycan-null (Sgcd(-/-)) mice through a mechanism involving reduced myofiber membrane fragility. The dnTGFßRII transgene also reduced muscle injury and improved muscle regeneration after cardiotoxin injury, as well as increased satellite cell numbers and activity. An unbiased global expression analysis revealed a number of potential mechanisms for dnTGFßRII-mediated protection, one of which was induction of the antioxidant protein metallothionein (Mt). Indeed, TGFß directly inhibited Mt gene expression in vitro, the dnTGFßRII transgene conferred protection against reactive oxygen species accumulation in dystrophic muscle and treatment with Mt mimetics protected skeletal muscle upon injury in vivo and improved the membrane stability of dystrophic myofibers. Hence, our results show that the myofibers are central mediators of the deleterious effects associated with TGFß signaling in MD.

Sarcolemmal alpha and gamma sarcoglycan protein deficiencies in Turkish siblings with a novel missense mutation in the alpha sarcoglycan gene.

BACKGROUND: The sarcoglycan alpha gene, also known as the adhalin gene, is located on chromosome 17q21; mutations in this gene are associated with limb-girdle muscular dystrophy type 2D. We describe two Turkish siblings with findings consistent with limb-girdle muscular dystrophy type 2D. The evaluation excluded a dystrophinopathy, which is the most common form of muscular dystrophy. PATIENTS: Both siblings had very high levels of creatinine phosphokinase and negative molecular tests for deletions and duplications of the dystrophin gene. The older boy presented at 8 years of age with an inability to climb steps and an abnormal gait. His younger brother was 5 years old and had similar symptoms. The muscle biopsy evaluation was performed only in the older brother. RESULTS: The muscle biopsy showed dystrophic features as well as a deficiency in the expression of two different glycoproteins: the alpha sarcoglycan and the gamma sarcoglycan. Sarcolemmal expressions of dystrophin and other sarcoglycans (beta and delta) were diffusely present. DNA analysis demonstrated the presence of previously unknown homozygous mutations [c.226 C > T (p.L76 F)] in exon 3 in the sarcoglycan alpha genes of both siblings. Similar heterozygous point mutations at the same locus were found in both parents, but the genes of beta, delta, and gamma sarcoglycan were normal in the remaining family members. CONCLUSIONS: We describe two siblings with limb-girdle muscular dystrophy type 2D with a novel missense mutation. These patients illustrate that the differential diagnosis of muscular dystrophies is impossible with clinical findings alone. Therefore, a muscle biopsy and DNA analysis remain essential methods for diagnosis of muscle diseases.

Targeting endothelial junctional adhesion molecule-A/ EPAC/ Rap-1 axis as a novel strategy to increase stem cell engraftment in dystrophic muscles.

Muscular dystrophies are severe genetic diseases for which no efficacious therapies exist. Experimental clinical treatments include intra-arterial administration of vessel-associated stem cells, called mesoangioblasts (MABs). However, one of the limitations of this approach is the relatively low number of cells that engraft the diseased tissue, due, at least in part, to the sub-optimal efficiency of extravasation, whose mechanisms for MAB are unknown. Leukocytes emigrate into the inflamed tissues by crossing endothelial cell-to-cell junctions and junctional proteins direct and control leukocyte diapedesis. Here, we identify the endothelial junctional protein JAM-A as a key regulator of MAB extravasation. We show that JAM-A gene inactivation and JAM-A blocking antibodies strongly enhance MAB engraftment in dystrophic muscle. In the absence of JAM-A, the exchange factors EPAC-1 and 2 are down-regulated, which prevents the activation of the small GTPase Rap-1. As a consequence, junction tightening is reduced, allowing MAB diapedesis. Notably, pharmacological inhibition of Rap-1 increases MAB engraftment in dystrophic muscle, which results into a significant improvement of muscle function offering a novel strategy for stem cell-based therapies.

Synthetic prostacyclin agonist, ONO1301, enhances endogenous myocardial repair in a hamster model of dilated cardiomyopathy: a promising regenerative therapy for the failing heart.

OBJECTIVES: Remodeling of the left ventricle (LV) in idiopathic dilated cardiomyopathy (IDCM) is known to be associated with multiple pathologic changes that endogenous factors, such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), protect against. Although a clinically relevant delivery method of these factors has not been established, ONO1301, a synthetic prostacyclin agonist, has been shown to upregulate multiple cardioprotective factors, including HGF and VEGF, in vivo. We thus hypothesized that ONO1301 may reverse LV remodeling in the DCM heart. METHODS: ONO1301 dose-dependently added to the normal human dermal fibroblasts and human coronary artery smooth muscle cells in vitro, to measure the expression of HGF, VEGF, stromal cell-derived factor (SDF)-1, and granulocyte-colony stimulating factor (G-CSF), assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. δ-Sarcoglycan-deficient J2N-k hamsters, which is an established DCM model, were treated by epicardial implantation of an atelocollagen sheet with or without ONO1301 immersion or sham operation. RESULTS: ONO1301 dose-dependently upregulated expression of these 4 factors in vitro. ONO1301 treatment, which induced dominant elevation of ONO1301 levels for 2 weeks, significantly preserved cardiac performance and prolonged survival compared with the other groups. This treatment significantly upregulated expressions of cardioprotective factors and was associated with increased capillaries, attenuated fibrosis, and upregulation of α-sarcoglycan in the DCM heart. CONCLUSIONS: ONO1301 atelocollagen-sheet implantation reorganized cytoskeletal proteins, such as α-sarcoglycan, increased capillaries, reduced fibrosis, and was associated with upregulated expression of multiple cardioprotective factors, leading to preservation of cardiac performance and prolongation of survival in the δ-sarcoglycan-deficient DCM hamster.

Long-term miR-669a therapy alleviates chronic dilated cardiomyopathy in dystrophic mice.

BACKGROUND: Dilated cardiomyopathy (DCM) is a leading cause of chronic morbidity and mortality in muscular dystrophy (MD) patients. Current pharmacological treatments are not yet able to counteract chronic myocardial wastage, thus novel therapies are being intensely explored. MicroRNAs have been implicated as fine regulators of cardiomyopathic progression. Previously, miR-669a downregulation has been linked to the severe DCM progression displayed by Sgcb-null dystrophic mice. However, the impact of long-term overexpression of miR-669a on muscle structure and functionality of the dystrophic heart is yet unknown. METHODS AND RESULTS: Here, we demonstrate that intraventricular delivery of adeno-associated viral (AAV) vectors induces long-term (18 months) miR-669a overexpression and improves survival of Sgcb-null mice. Treated hearts display significant decrease in hypertrophic remodeling, fibrosis, and cardiomyocyte apoptosis. Moreover, miR-669a treatment increases sarcomere organization, reduces ventricular atrial natriuretic peptide (ANP) levels, and ameliorates gene/miRNA profile of DCM markers. Furthermore, long-term miR-669a overexpression significantly reduces adverse remodeling and enhances systolic fractional shortening of the left ventricle in treated dystrophic mice, without significant detrimental consequences on skeletal muscle wastage. CONCLUSIONS: Our findings provide the first evidence of long-term beneficial impact of AAV-mediated miRNA therapy in a transgenic model of severe, chronic MD-associated DCM.

Enhancing muscle membrane repair by gene delivery of MG53 ameliorates muscular dystrophy and heart failure in δ-Sarcoglycan-deficient hamsters.

Muscular dystrophies (MDs) are caused by genetic mutations in over 30 different genes, many of which encode for proteins essential for the integrity of muscle cell structure and membrane. Their deficiencies cause the muscle vulnerable to mechanical and biochemical damages, leading to membrane leakage, dystrophic pathology, and eventual loss of muscle cells. Recent studies report that MG53, a muscle-specific TRIM-family protein, plays an essential role in sarcolemmal membrane repair. Here, we show that systemic delivery and muscle-specific overexpression of human MG53 gene by recombinant adeno-associated virus (AAV) vectors enhanced membrane repair, ameliorated pathology, and improved muscle and heart functions in δ-sarcoglycan (δ-SG)-deficient TO-2 hamsters, an animal model of MD and congestive heart failure. In addition, MG53 overexpression increased dysferlin level and facilitated its trafficking to muscle membrane through participation of caveolin-3. MG53 also protected muscle cells by activating cell survival kinases, such as Akt, extracellular signal-regulated kinases (ERK1/2), and glycogen synthase kinase-3ß (GSK-3ß) and inhibiting proapoptotic protein Bax. Our results suggest that enhancing the muscle membrane repair machinery could be a novel therapeutic approach for MD and cardiomyopathy, as demonstrated here in the limb girdle MD (LGMD) 2F model.

Intrinsic cell memory reinforces myogenic commitment of pericyte-derived iPSCs.

Mesoangioblasts (MABs) are a subset of muscle-derived pericytes able to restore dystrophic phenotype in mice and dogs. However, their lifespan is limited and they undergo senescence after 25-30 population doublings. Recently, induced pluripotent stem cells (iPSCs) generated from reprogrammed fibroblasts have been demonstrated to have in vitro and in vivo myogenic potential when sorted for the SM/C-2.6 antigen. Furthermore, chimeric mice from mdx-iPSCs (DYS-HAC) cells showed tissue-specific expression of dystrophin. Nevertheless, myogenic differentiation protocols and the potential of iPSCs generated from different cell sources still present unanswered questions. Here we show that iPSCs generated from prospectively sorted MABs (MAB-iPSCs) are pluripotent as fibroblast-derived iPSCs (f-iPSCs). However, both teratoma formation and genetic cell manipulation assays identify a durable epigenetic memory in MAB-iPSCs, resulting in stronger myogenic commitment. Striated muscle tissue accounts for up to 70% of MAB-iPSC teratomas. Moreover, transfection with Pax3 and Pax7 induces a more robust myogenic differentiation in MAB-iPSCs than in f-iPSCs. A larger amount of CD56(+) progenitors can be sorted from the MAB-iPSCs differentiating pool and, after transplantation into αsg-KO mice, can efficiently participate to skeletal muscle regeneration and restore αsg expression. Our data strongly suggest that iPSCs are a heterogeneous population and, when generated from myogenic adult stem cells, they exhibit a stronger commitment, paving the way for creating custom-made cell protocols for muscular dystrophies.

Bilateral deep brain stimulation of the pallidum for myoclonus-dystonia due to ε-sarcoglycan mutations: a pilot study.

OBJECTIVE: To assess the efficacy of bilateral deep brain stimulation of the internal pallidum in patients with myoclonus-dystonia due to genetically proved ε-sarcoglycan (SGCE-M-D) deficiency. DESIGN: Patients with documented SGCE-M-D undergoing bilateral deep brain stimulation of the internal pallidum were recruited. Standardized assessments of M-D were videorecorded before surgery and 6 to 9 months and 15 to 18 months after surgery, using the movement and disability subscales of the Burke-Fahn-Marsden Dystonia Rating Scale and the Unified Myoclonus Rating Scale. The analysis was based on blinded evaluation of the recordings. SETTING: Movement disorder unit in a university hospital in Paris. PATIENTS: Five consecutive patients with documented SGCE-M-D. MAIN OUTCOME MEASURES: Myoclonus and dystonia scores at follow-up. RESULTS: The median myoclonus score decreased from 76 before surgery (range, 38-116) to 10 at 6 to 9 months after surgery (range, 6-31). The median dystonia score decreased from 30.0 before surgery (range, 18.5-53.0) to 4.5 after surgery (range, 3.5-16.0). Disability was also improved and symptoms remained stable between the postoperative evaluations. No adverse effects occurred. CONCLUSIONS: Bilateral deep brain stimulation of the internal pallidum is safe and highly effective in this homogeneous population of patients with SGCE-M-D. This therapeutic option should therefore be considered for patients with severe, drug-resistant forms of the disorder.

Altered calcium pump and secondary deficiency of gamma-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from delta-sarcoglycan knockout mice.

Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and delta-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of delta-SG isoforms in TT and SR results in a secondary deficiency of gamma-SG and microSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the delta-SG isoforms may stabilize the expression of gamma-SG and microSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle.

Mice lacking dystrophin or alpha sarcoglycan spontaneously develop embryonal rhabdomyosarcoma with cancer-associated p53 mutations and alternatively spliced or mutant Mdm2 transcripts.

Altered expression of proteins in the dystrophin-associated glycoprotein complex results in muscular dystrophy and has more recently been implicated in a number of forms of cancer. Here we show that loss of either of two members of this complex, dystrophin in mdx mice or alpha sarcoglycan in Sgca(-/-) mice, results in the spontaneous development of muscle-derived embryonal rhabdomyosarcoma (RMS) after 1 year of age. Many mdx and Sgca(-/-) tumors showed increased expression of insulin-like growth factor 2, retinoblastoma protein, and phosphorylated Akt and decreased expression of phosphatase and tensin homolog gene, much as is found in a human RMS. Further, all mdx and Sgca(-/-) RMS analyzed had increased expression of p53 and murine double minute (mdm)2 protein and contained missense p53 mutations previously identified in human cancers. The mdx RMS also contained missense mutations in Mdm2 or alternatively spliced Mdm2 transcripts that lacked an exon encoding a portion of the p53-binding domain. No Pax3:Fkhr or Pax7:Fkhr translocation mRNA products were evident in any tumor. Expression of natively glycosylated alpha dystroglycan and alpha sarcoglycan was reduced in mdx RMS, whereas dystrophin expression was absent in almost all human RMS, both for embryonal and alveolar RMS subtypes. These studies show that absence of members of the dystrophin-associated glycoprotein complex constitutes a permissive environment for spontaneous development of embryonal RMS associated with mutation of p53 and mutation or altered splicing of Mdm2.

Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins.

OBJECTIVE: alpha-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [LGMD2D]) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy and persistence of gene expression. METHODS: A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis muscle was conducted. Control sides received saline. A 3-day course of methylprednisolone accompanied gene transfer without further immune suppression. RESULTS: No adverse events were encountered. SGCA gene expression increased 4-5-fold over control sides when examined at 6 weeks (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in the 3-month subject. Adeno-associated virus serotype 1 (AAV1)-neutralizing antibodies were seen as early as 2 weeks. Neither CD4+ nor CD8+ cells were increased over contralateral sides. Scattered foci of inflammation could be found, but showed features of programmed cell death. Enzyme-linked immunospot (ELISpot) showed no interferon-gamma response to alpha-SG or AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 subjects successfully achieved study aims, precluding the need for additional enrollment. INTERPRETATION: The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy. Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design of vascular delivery gene therapy trials.

Overexpression of Galgt2 reduces dystrophic pathology in the skeletal muscles of alpha sarcoglycan-deficient mice.

Recent studies have shown that a number of genes that are not mutated in various forms of muscular dystrophy may serve as surrogates to protect skeletal myofibers from injury. One such gene is Galgt2, which is also called cytotoxic T cell GalNAc transferase in mice. In this study, we show that Galgt2 overexpression reduces the development of dystrophic pathology in the skeletal muscles of mice lacking alpha sarcoglycan (Sgca), a mouse model for limb girdle muscular dystrophy 2D. Galgt2 transgenic Sgca(-/-) mice showed reduced levels of myofiber damage, as evidenced by i) normal levels of serum creatine kinase activity, ii) a lack of Evans blue dye uptake into myofibers, iii) normal levels of mouse locomotor activity, and iv) near normal percentages of myofibers with centrally located nuclei. In addition, the overexpression of Galgt2 in the early postnatal period using an adeno-associated virus gene therapy vector protected Sgca(-/-) myofibers from damage, as observed using histopathology measurements. Galgt2 transgenic Sgca(-/-) mice also had increased levels of glycosylation of alpha dystroglycan with the CT carbohydrate, but showed no up-regulation of beta, gamma, delta, or epsilon sarcoglycan. These data, coupled with results from our previous studies, show that Galgt2 has therapeutic effects in three distinct forms of muscular dystrophy and may, therefore, have a broad spectrum of therapeutic potential for the treatment of various myopathies.

Muscular dystrophy therapy by nonautologous mesenchymal stem cells: muscle regeneration without immunosuppression and inflammation.

BACKGROUND: The use of nonautologous stem cells isolated from healthy donors for stem-cell therapy is an attractive approach, because the stem cells can be culture expanded in advance, thoroughly tested, and formulated into off-the-shelf medicine. However, human leukocyte antigen compatibility and related immunosuppressive protocols can compromise therapeutic efficacy and cause unwanted side effects. METHODS: Mesenchymal stem cells (MSCs) have been postulated to possess unique immune regulatory function. We explored the immunomodulatory property of human and porcine MSCs for the treatment of delta-sarcoglycan-deficient dystrophic hamster muscle without immunosuppression. Circulating and tissue markers of inflammation were analyzed. Muscle regeneration and stem-cell fate were characterized. RESULTS: Total white blood cell counts and leukocyte-distribution profiles were similar among the saline- and MSC-injected dystrophic hamsters 1 month posttreatment. Circulating levels of immunoglobulin A, vascular cell adhesion molecule-1, myeloperoxidase, and major cytokines involved in inflammatory response were not elevated by MSCs, nor were expression of the leukocyte common antigen CD45 and the cytokine transcriptional activator NF-kappaB in the injected muscle. Treated muscles exhibited increased cell-cycle activity and attenuated oxidative stress. Injected MSCs were found to be trapped in the musculature, contribute to both preexisting and new muscle fibers, and mediates capillary formation. CONCLUSIONS: Intramuscular injection of nonautologous MSCs can be safely used for the treatment of dystrophic muscle in immunocompetent hosts without inflaming the host immune system.

[Late-onset sarcoglycanopathy: a cause of repeated muscle cramps after exertion].

A 61-year-old man in generally good health had a 2-year history of repetitive episodes of muscle cramps, weakness, numbness and muscle pain of the bilateral limbs after exerting himself. During each episode, his serum creatine kinase level became elevated then recovered only after sufficient hydration had been provided. Bilateral thigh MRI showed abnormal muscular signals, and the patient was subsequently diagnosed by muscle biopsy, immunohistochemistry, and mini-multiplex western blotting analysis as having solitary sarcoglycanopathy. Late-onset sarcoglycanopathy has rarely been described. In the present patient, his mild clinical condition, subclinical cardiomyopathy and possible involvement of polyneuropathy are unique features.