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Sarcoglycanopathies, also known as limb girdle muscular dystrophy 3-6, are rare muscular dystrophies characterized, although heterogeneous, by high disability, with patients often wheelchair-bound by late adolescence and frequently developing respiratory and cardiac problems. These diseases are currently incurable, emphasizing the importance of effective treatment strategies and the necessity of animal models for drug screening and therapeutic verification. Using the CRISPR/Cas9 genome editing technique, we generated and characterized δ-sarcoglycan and β-sarcoglycan knockout zebrafish lines, which presented a progressive disease phenotype that worsened from a mild larval stage to distinct myopathic features in adulthood. By subjecting the knockout larvae to a viscous swimming medium, we were able to anticipate disease onset. The δ-SG knockout line was further exploited to demonstrate that a δ-SG missense mutant is a substrate for endoplasmic reticulum-associated degradation (ERAD), indicating premature degradation due to protein folding defects. In conclusion, our study underscores the utility of zebrafish in modeling sarcoglycanopathies through either gene knockout or future knock-in techniques. These novel zebrafish lines will not only enhance our understanding of the disease's pathogenic mechanisms, but will also serve as powerful tools for phenotype-based drug screening, ultimately contributing to the development of a cure for sarcoglycanopathies. [ABSTRACT FROM AUTHOR]

Sarcoglycanopathies, also known as limb girdle muscular dystrophy 3-6, are rare muscular dystrophies characterized, although heterogeneous, by high disability, with patients often wheelchair-bound by late adolescence and frequently developing respiratory and cardiac problems. These diseases are currently incurable, emphasizing the importance of effective treatment strategies and the necessity of animal models for drug screening and therapeutic verification. Using the CRISPR/Cas9 genome editing technique, we generated and characterized δ-sarcoglycan and β-sarcoglycan knockout zebrafish lines, which presented a progressive disease phenotype that worsened from a mild larval stage to distinct myopathic features in adulthood. By subjecting the knockout larvae to a viscous swimming medium, we were able to anticipate disease onset. The δ-SG knockout line was further exploited to demonstrate that a δ-SG missense mutant is a substrate for endoplasmic reticulum-associated degradation (ERAD), indicating premature degradation due to protein folding defects. In conclusion, our study underscores the utility of zebrafish in modeling sarcoglycanopathies through either gene knockout or future knock-in techniques. These novel zebrafish lines will not only enhance our understanding of the disease’s pathogenic mechanisms, but will also serve as powerful tools for phenotype-based drug screening, ultimately contributing to the development of a cure for sarcoglycanopathies.

Introduction: Limb-girdle muscular dystrophy type 2E (LGMD2E) is caused by mutations in the β-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscles. β-Sarcoglycan-deficient (Sgcb-null) mice develop severe muscular dystrophy and cardiomyopathy with focal areas of necrosis.Methods: In this study we performed morphological (histological and cellular characterization) and functional (isometric tetanic force and fatigue) analyses in dystrophic mice. Comparison studies were carried out in 1-month-old (clinical onset of the disease) and 7-month-old control mice (C57Bl/6J, Rag2/γc-null) and immunocompetent and immunodeficient dystrophic mice (Sgcb-null and Sgcb/Rag2/γc-null, respectively).Results: We found that the lack of an immunological system resulted in an increase of calcification in striated muscles without impairing extensor digitorum longus muscle performance. Sgcb/Rag2/γc-null muscles showed a significant reduction of alkaline phosphate-positive mesoangioblasts.Discussion: The immunological system counteracts skeletal muscle degeneration in the murine model of LGMD2E. Muscle Nerve, 2018. [ABSTRACT FROM AUTHOR]

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 β-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 β-sarcoglycan, and their plaques contained an increase in differentiated smooth muscle cells. ApoE-deficient mice lacking β-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 β-sarcoglycan. Analysis of plasma in β-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 β-sarcoglycan can affect the atherosclerotic process. Furthermore, the results show the effects of β-sarcoglycan deficiency on adipose tissue and lipid metabolism, which may also have contributed to the atherosclerotic plaque reduction. [ABSTRACT FROM AUTHOR]

The Sgcb-null mouse, with knocked-down β-sarcoglycan, develops severe muscular dystrophy as in type 2E human limb girdle muscular dystrophy. The mdx mouse, lacking dystrophin, is the most used model for Duchenne muscular dystrophy (DMD). Unlike DMD, the mdx mouse has mild clinical features and shows little fibrosis in limb muscles. To characterize ECM protein deposition and the progression of muscle fibrosis, we evaluated protein and transcript levels of collagens I, III and VI, decorin, and TGF-β1, in quadriceps and diaphragm, at 2, 4, 8, 12, 26, and 52 weeks in Sgcb-null mice, and protein levels at 12, 26, and 52 weeks in mdx mice. In Sgcb-null mice, severe morphological disruption was present from 4 weeks in both quadriceps and diaphragm, and included conspicuous deposition of extracellular matrix components. Histopathological features of Sgcb-null mouse muscles were similar to those of age-matched mdx muscles at all ages examined, but, in the Sgcb-null mouse, the extent of connective tissue deposition was generally greater than mdx. Furthermore, in the Sgcb-null mouse, the amount of all three collagen isoforms increased steadily, while, in the mdx, they remained stable. We also found that, at 12 weeks, macrophages were significantly more numerous in mildly inflamed areas of Sgcb-null quadriceps compared to mdx quadriceps (but not in highly inflamed regions), while, in the diaphragm, macrophages did not differ significantly between the two models, in either region. Osteopontin mRNA was also significantly greater at 12 weeks in laser-dissected highly inflamed areas of the Sgcb-null quadriceps compared to the mdx quadriceps. TGF-β1 was present in areas of degeneration-regeneration, but levels were highly variable and in general did not differ significantly between the two models and controls. The roles of the various subtypes of macrophages in muscle repair and fibrosis in the two models require further study. The Sgcb-null mouse, which develops early fibrosis in limb muscles, appears more promising than the mdx mouse for probing pathogenetic mechanisms of muscle fibrosis and for developing anti-fibrotic treatments. Highlights • The Sgcb-null mouse develops severe muscular dystrophy, the mdx mouse does not. • Fibrosis developed earlier in Sgcb-null quadriceps and diaphragm than mdx. • Macrophages were commoner in mildly inflamed parts of Sgcb-null quadriceps than mdx. • The Sgcb-null model appears more useful than mdx for studying fibrotic mechanisms. • The Sgcb-null model also appears more useful for developing anti-fibrotic treatments. [ABSTRACT FROM AUTHOR]

Mutations underlying genetic cardiomyopathies might affect differentiation commitment of resident progenitor cells. Cardiac mesoangioblasts ( cMabs) are multipotent progenitor cells resident in the myocardium. A switch from cardiac to skeletal muscle differentiation has been recently described in cMabs from β-sarcoglycan-null mice (β SG−/−), a murine model of genetic myopathy with early myocardial involvement. Although complementation with β SG gene was inconsequential, knock-in of miRNA669a (missing in β SG−/− cMabs) partially rescued the mutation-induced molecular phenotype. Here, we undertook a detailed evaluation of functional differentiation of β SG−/− cMabs and tested the effects of miRNA669a-induced rescue in vitro. To this end, cMabs were compared with neonatal cardiomyocytes ( CMs) and skeletal muscle C2C12 cells, representative of cardiac and skeletal muscle respectively. Consistent with previous data on molecular patterns, electrophysiological and Ca2+-handling properties of β SG−/− cMabs were closer to C2C12 cells than to CM ones. Nevertheless, subtler aspects, including action potential contour, Ca2+-spark properties and RyR isoform expression, distinguished β SG−/− cMabs from C2C12 cells. Contrary to previous reports, wild-type cMabs failed to show functional differentiation towards either cell type. Knock-in of miRNA669a in β SG−/− cMabs rescued the wild-type functional phenotype, i.e. it completely prevented development of skeletal muscle functional responses. We conclude that miRNA669a expression, ablated by β SG deletion, may prevent functional differentiation of cMabs towards the skeletal muscle phenotype. [ABSTRACT FROM AUTHOR]

Effective therapy to reduce skeletal muscle injury associated with severe or eccentric exercise is needed. The purpose of this study was to determine whether adenosine receptor stimulation can mediate protection from eccentric exercise-induced muscle injury. Downhill treadmill exercise (-15°) was used to induce eccentric exercise-mediated skeletal muscle injury. Experiments were conducted in both normal wild-type (WT) mice and also in β-sarcoglycan knockout dystrophic mice, animals that show an exaggerated muscle damage with the stress of exercise. In the vehicle-treated WT animals, eccentric exercise increased serum creatine kinase (CK) greater than 3-fold to 358.9 ± 62.7 U/l (SE). This increase was totally abolished by stimulation of the A3 receptor. In the dystrophic β-sarcoglycan-null mice, eccentric exercise caused CK levels to reach 55,124 ± 5,558 U/l. A3 receptor stimulation in these animals reduced the CK response by nearly 50%. In the dystrophic mice at rest, 10% of the fibers were found to be damaged, as indicated by Evans blue dye staining. While this percentage was doubled after exercise, A3 receptor stimulation eliminated this increase. Neither the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (0.05 mg/kg) nor the A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (0.07 mg/kg) protected skeletal muscle from eccentric exercise injury in WT or dystrophic mice. The protective effect of adenosine A3 receptor stimulation was absent in mice, in which genes for phospholipase C β2/β3 (PLCβ2/β3) and β-sarcoglycan were deleted. The present study elucidates a new protective role of the A3 receptor and PLCβ2/β3 and points to a potentially effective therapeutic strategy for eccentric exercise-induced skeletal muscle injury. [ABSTRACT FROM AUTHOR]

Abstract: Sarcoglycans are a multimeric, integral membrane protein complex that is part of the dystrophin glycoprotein complex. Previous findings suggest that the dystrophin glycoprotein complex plays roles not only in maintaining the mechanical structure of the cell membrane but also in signal transduction. To evaluate the functions of sarcoglycans, we here took advantage of Drosophila, which is useful for screening genetic interactions. Morphological aberrancy was observed in the adult compound eyes of Drosophila β-sarcolgycan (dscgβ) knockdown flies. We also detected genetic interactions between dscgβ and Egfr related genes, such as rhomboid-1, rhomboid-3, and mirror. Furthermore two extra cell types with strong expression of Rhomboid were found in the ommatidia of dscgβ knockdown pupal retina. These cells exhibited phosphorylation of ERKA, suggesting that Egfr signaling is activated via Rhomboid. Through these in vivo analyses, we conclude that dscgβ negatively regulates the Egfr signaling pathway. [Copyright &y& Elsevier]

Mutations in the sarcoglycan (SG) genes cause autosomal recessive muscular dystrophies. The absence of each SG complex component in muscle impairs the proper assembly of the entire SG complex, resulting in sarcolemmal damage. We investigated the consequences of β-SG gene mutations in cultured muscle from two β-SG mutated patients, and analysed each individual SG protein expression by cross-sectional immunocytochemistry and Western blot in aneural and innervated myotubes. Patients' muscle biopsy showed total loss of SG complex; however, a limited amount of β-SG was detected in aneural and innervated myotubes, where the protein was localized to the plasma membrane. This paradoxical β-SG expression can be attributable to antibody cross-reaction or to the expression of an unknown SG isoform specific of immature muscle. In our cultured myotubes, the other components of the SG complex were absent, suggesting that β-SG gene mutations result in a defective assembly of the entire SG complex in early stages of muscle development, and that the role of β-SG is crucial for the normal structure and/or function of the SG complex in the sarcolemma. [ABSTRACT FROM AUTHOR]

Muscular dystrophies in dogs and cats represent a heterogeneous group of inherited, sometimes congenital, but infrequently diagnosed, progressive neuromuscular disorders. A correct identification and characterization of canine and feline muscular dystrophies could increase diagnostic and treatment strategies for veterinary neurologists and could identify useful animal models for the study of human dystrophies. However, in dogs and cats, diagnosis of muscular dystrophies is challenging due to a nonspecific clinical phenotype and pathological lesions, thus is most likely underestimated. We performed immunofluorescence and Western blot techniques using a wide panel of antibodies against proteins involved in human dystrophies (dystrophin mid-rod and carboxyterminal domain, α, β, γ, and δ-sarcoglycan, α-dystroglycan, caveolin-3, emerin, merosin, dysferlin, calpain-3, spectrin epitopes), on 9 canine and 3 feline muscle biopsies characterized by myopathic changes. Dystrophin deficiency was detected in 3 dogs and 2 novel canine muscular dystrophies have been identified, characterized by deficiency of caveolin-3 and calpain-3, respectively. In 2 cats, deficiency of β-SG and carboxyterminal domain of dystrophin in all muscle fibers has been detected. Performing immunofluorescence and Western blot analyses with a wider panel of antibodies allowed a correct identification of muscular dystrophies in dogs and cats and provides a direction for subsequent targeted genetic testing. [ABSTRACT FROM AUTHOR]

Limb-girdle muscular dystrophy type 2E (LGMD2E), resulting from mutations in β-sarcoglycan (SGCB), is a progressive dystrophy with deteriorating muscle function, respiratory failure, and cardiomyopathy in 50% or more of LGMD2E patients. SGCB knockout mice share many of the phenotypic deficiencies of LGMD2E patients. To investigate systemic SGCB gene transfer to treat skeletal and cardiac muscle deficits, we designed a self-complementary AAVrh74 vector containing a codon-optimized human SGCB transgene driven by a muscle-specific promoter. We delivered scAAV.MHCK7.hSGCB through the tail vein of SGCB -/- mice to provide a rationale for a clinical trial that would lead to clinically meaningful results. This led to 98.1% transgene expression across all muscles that was accompanied by improvements in histopathology. Serum creatine kinase (CK) levels were reduced following treatment by 85.5%. Diaphragm force production increased by 94.4%, kyphoscoliosis of the spine was significantly reduced by 48.1%, overall ambulation increased by 57%, and vertical rearing increased dramatically by 132% following treatment. Importantly, no adverse effects were seen in muscle of wild-type mice injected systemically with scAAV.hSGCB. In this well-defined model of LGMD2E, we have demonstrated the efficacy and safety of systemic scAAV.hSGCB delivery, and these findings have established a path for clinically beneficial AAV-mediated gene therapy for LGMD2E., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)