Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H.

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of ßPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and ß-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.

Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation.

Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the fundamental role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel rac3b/rfng/sgca regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by chromosomal rearrangement that generated new cis-regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying hindbrain boundary-specific cis-regulatory elements. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.

Altered functional differentiation of mesoangioblasts in a genetic myopathy.

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 ß-sarcoglycan-null mice (ßSG(-/-)), a murine model of genetic myopathy with early myocardial involvement. Although complementation with ßSG gene was inconsequential, knock-in of miRNA669a (missing in ßSG(-/-) cMabs) partially rescued the mutation-induced molecular phenotype. Here, we undertook a detailed evaluation of functional differentiation of ßSG(-/-) 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 Ca(2+)-handling properties of ßSG(-/-) cMabs were closer to C2C12 cells than to CM ones. Nevertheless, subtler aspects, including action potential contour, Ca(2+)-spark properties and RyR isoform expression, distinguished ßSG(-/-) 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 ßSG(-/-) 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 ßSG deletion, may prevent functional differentiation of cMabs towards the skeletal muscle phenotype.

Preliminary study on sarcoglycan sub-complex in rat cerebral and cerebellar cortex.

The sarcoglycan sub-complex is a protein system which plays a key role in sarcolemma stabilization during muscle activity. Although numerous studies have been conducted on this system, there are few data about its localization in non-muscular tissues. On this basis we carried out an indirect immunofluorescence study on normal rat cerebral and cerebellar cortex. In particular, we carried out single localization reactions to analyze if these proteins are present in brain and double localization reactions between sarcoglycans and either SMI-32 or GFAP to verify if they are expressed both in neurons and glial cells. We found that all tested sarcoglycans are present both in cerebral and cerebellar cortex and that they are expressed both in neurons and glial cells. The typical staining pattern of all sarcoglycans is represented by "spot-like" fluorescence, with spots of 0.5-2 microm average diameter laid out mainly around the soma of the cells. The main difference about sarcoglycans expression between cerebral and cerebellar cortex is that in the cerebellar cortex the sarcoglycans positivity is detectable only in an area which is likely to correspond to Purkinje cells layer. The presence of sarcoglycans in cerebral and cerebellar cortex and their disposition mainly around the soma of the cells suggest a role of these proteins in cellular signalling and in regulating postsynaptic receptor assembly mainly in axo-somatic synapses.

Cell-matrix interactions in muscle disease.

The extracellular matrix (ECM) provides a solid scaffold and signals to cells through ECM receptors. The cell-matrix interactions are crucial for normal biological processes and when disrupted they may lead to pathological processes. In particular, the biological importance of ECM-cell membrane-cytoskeleton interactions in skeletal muscle is accentuated by the number of inherited muscle diseases caused by mutations in proteins conferring these interactions. In this review we introduce laminins, collagens, dystroglycan, integrins, dystrophin and sarcoglycans. Mutations in corresponding genes cause various forms of muscular dystrophy. The muscle disorders are presented as well as advances toward the development of treatment.

Age-related changes in dystrophin-glycoprotein complex and in utrophin are not correlated with intrinsic laryngeal muscles protection in mdx mice.

In this study we investigate whether dystrophic intrinsic laryngeal muscles (ILM) from aged mdx mice show alterations in dystrophin-glycoprotein complex (DGC) components.Immunofluorescence and immunoblotting analyses of beta-sarcoglycan, beta-dystroglycan, and utrophin showed that aged ILM had a similar pattern of changes in aged affected muscles (diaphragm and limb), suggesting that aging leads to changes in utrophin and DGC proteins in dystrophic ILM that cannot be correlated with their protection from dystrophic change.

Sarcoglycan[s] are not muscle-specific: hypothetical roles.

The sarcoglycan complex is a multimember transmembrane complex interacting with other proteins to provide a mechano-signaling connection from the cytoskeleton to the extracellular matrix in myofibers. This complex plays a key role at the membrane and is crucial in maintaining sarcolemma viability in muscle fibers. Recent observations have demonstrated that in the lung this glycoprotein is associated with both alveoli and bronchioles, and that the urogenital and digestive tracts are epsilon-sarcoglycan positive. Further addressing this issue, in this work we extend our previous studies to better verify whether the sarcoglycan complex also exists in epithelial tissue. All our observations showed staining for all sarcoglycans to be a normal pattern in all tested epithelial cells. We hypothesize a key role for sarcoglycans in bidirectional signaling between cells and extracellular matrix, and an important role in the regulation of inhibitory synapses and of blood brain barrier.

A novel mutation of the epsilon-sarcoglycan gene in a Chinese family with myoclonus-dystonia syndrome.

In a Chinese myoclonus-dystonia syndrome (MDS) family presented with a phenotype including a typical MDS, cervical dystonia, and writer's cramp, genetic analyses revealed a novel 662 + 1insG heterozygous mutation in exon 5 in the epsilon-sarcoglycan (SGCE) gene, leading to a frameshift with a down stream stop codon. Low SGCE mRNA levels were detected in the mutation carriers by real-time PCR, suggesting that the nonsense mutation might interfere with the stability of SGCE mRNA. This is the first report on Chinese with a SGCE mutation leading to MDS. Our data support the fact that same mutation of SGCE gene can lead to a varied phenotype, even in the same family.

Reduced life span with heart and muscle dysfunction in Drosophila sarcoglycan mutants.

In humans, genetically diverse forms of muscular dystrophy are associated with a disrupted sarcoglycan complex. The sarcoglycan complex resides at the muscle plasma membrane where it associates with dystrophin. There are six known sarcoglycan proteins in mammals whereas there are only three in Drosophila melanogaster. Using imprecise P element excision, we generated three different alleles at the Drosophila delta-sarcoglycan locus. Each of these deletions encompassed progressively larger regions of the delta-sarcoglycan gene. Line 840 contained a large deletion of the delta-sarcoglycan gene, and this line displayed progressive impairment in locomotive ability, reduced heart tube function and a shortened life span. In line 840, deletion of the Drosophila delta-sarcoglycan gene produced disrupted flight muscles with shortened sarcomeres and disorganized M lines. Unlike mammalian muscle where degeneration is coupled with ongoing regeneration, no evidence for regeneration was seen in this Drosophila sarcoglycan mutant. In contrast, line 28 was characterized with a much smaller deletion that affected only a portion of the cytoplasmic region of the delta-sarcoglycan protein and left intact the transmembrane and extracellular domains. Line 28 had a very mild phenotype with near normal life span, intact cardiac function and normal locomotive activity. Together, these data demonstrate the essential nature of the transmembrane and extracellular domains of Drosophila delta-sarcoglycan for normal muscle structure and function.

Mutation of delta-sarcoglycan is associated with Ca(2+) -dependent vascular remodeling in the Syrian hamster.

We examined whether mutation of the delta-sarcoglycan gene, which causes dilated cardiomyopathy, also alters the vascular smooth muscle cell (VSMC) phenotype and arterial function in the Syrian hamster CHF 147. Thoracic aorta media thickness showed marked variability in diseased hamsters with zones of atrophy and hypertrophied segments. CHF-147 VSMCs displayed a proliferating/"synthetic" phenotype characterized by the absence of the smooth muscle myosin heavy chain SM2, dystrophin, and Ca(2+)-handling proteins, and the presence of cyclin D1. In freshly isolated VSMCs from CHF 147 hamsters, voltage-independent basal Ca(2+) channels showed enhanced activity similar to that in proliferating wild-type (WT) cells. The transcription factor NFAT (nuclear factor of activated T cells) was spontaneously active in freshly isolated CHF 147 VSMCs, as in proliferating VSMCs from WT hamsters. Mibefradil inhibited B-type channels, NFAT activity, and VSMC proliferation. CHF 147 hamsters had abundant apoptotic cells distributed in patches along the aorta, and clusters of inactive mitochondria were observed in 25% of isolated CHF 147 cells, whereas no such clusters were seen in WT cells. In conclusion, mutation of the delta-sarcoglycan gene increases plasma membrane permeability to Ca(2+), activates the Ca(2+)-regulated transcription factor NFAT, and leads to spontaneous mitochondrial aggregation, causing abnormal VSMC proliferation and apoptosis.

Sarcoglycanopathies: a clinico-pathological study.

BACKGROUND: Limb girdle muscular dystrophy (LGMD) is a heterogeneous group of disorders characterized by limb girdle weakness. There are no clear clinical features that distinguish various types of LGMD. MATERIALS AND METHODS: We studied 26 patients with chronic progressive weakness in limb girdle distribution without early facial involvement with muscle biopsies suggestive of dystrophy/myopathy and positive for dystrophin antibodies. Immunohistochemistry studies of muscle biopsies were done on all patients to classify different types of sarcoglycanopathies. RESULTS: The mean age of presentation was in the third decade. There were 14 male and 12 female patients. The common pattern of inheritance was autosomal recessive, seen in 53.8%. The more frequent type of LGMD was sarcoglycanopathy (SGP) (53.8%). Amongst the SGPs, alpha-SGP (26.9%) was the most common followed by beta-SGP (15.3%), gamma-SGP (3.8%) and delta-SGP (7.6%). Calf hypertrophy was noted in 53.5% of LGMD and 57.1% of SGPs, extensor digitorum brevis hypertrophy in 42% of LGMD and 35.7% of SGPs, winging of scapula in 39.2% of the LGMD group and 35.7% of the SGPs, valley sign in 28.5% of the LGMD group and 21.4% of the SGPs. Hip abductor sign was positive in 71.4% of LGMD and 64.2% of SGPs. Differential weakness of knee flexors was more common in SGP (57.1%). The mean creatine phosphokinase (CK) value was 2519IU/L and was elevated in 92.8% patients. Muscle biopsy showed a dystrophic pattern in 75% of LGMD and a myopathic pattern in the remaining. Symptomatic cardiac involvement was seen in one patient. ECG changes were seen in 44% of LGMD patients and 50% of the SGP. The common changes noted were T wave inversion in V1, V2 (16%), left ventricular hypertrophy LVH (12%) and right bundle branch block (RBBB) in 12% of the LGMD group. CONCLUSION: Sarcoglycanopathy is a more frequent form of LGMD whereas alpha type is the most common among the SGP. The four types of SGP do not differ in the pattern of muscle involvement. A relatively earlier onset, selective weakness of knee flexors and a very high CK may help differentiate SGP from other forms of LGMD. Immunohistochemistry is very useful in classifying the different types of LGMD prior to genetic analysis.

Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy.

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-delta, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin (mdx) or sarcoglycan-delta (sgcd-/-) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd-/-, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd-/- arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd-/- arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.

The sarcoglycan complex in Schwann cells and its role in myelin stability.

Sarcoglycans are originally identified in muscle for their involvement in limb-girdle muscular dystrophies. They form a multi-meric complex (alpha-, beta-, gamma-, delta-sarcoglycan) that associates with dystrophin, dystroglycan and other proteins to constitute the larger dystrophin-glycoprotein complex at the muscle membrane. Three sarcoglycan subunits (epsilon-, beta-, delta-sarcoglycan) were previously identified in Schwann cells and shown to associate with dystroglycan and a Schwann cell-specific dystrophin isoform (Dp116) at the outermost Schwann cell membrane. Currently, little is known about the exact composition and function of the sarcoglycan complex in the peripheral nervous system. In this study, we showed that the Schwann cell sarcoglycan complex consists of epsilon-, beta-, delta-sarcoglycan and the newly identified zeta-sarcoglycan subunit. The expression of sarcoglycans precedes the onset of myelination and is induced by neurons. In sarcoglycan-deficient BIO14.6 hamsters, loss of the Schwann cell sarcoglycan complex reduces the steady state levels of alpha-dystroglycan and Dp116. Ultrastructural analysis of sciatic nerves from the mutant animals revealed altered myelin sheaths and disorganized Schmidt-Lanterman incisures indicative of myelin instability. The disruption in myelin structure increased in severity with age. Nerve conduction studies also showed subtle electrophysiological abnormalities in the BIO14.6 hamsters consistent with reduced myelin stability. Together, these findings suggest an important role of sarcoglycans in the stability of peripheral nerve myelin.

Consequences of disrupting the dystrophin-sarcoglycan complex in cardiac and skeletal myopathy.

Mutations that disrupt the dystrophin glycoprotein complex lead to plasma membrane instability of cardiomyocytes and skeletal muscle myofibers. Instability of the plasma membrane leads to degeneration largely due to activation of a necrotic process in these disorders. In response to ongoing degeneration, skeletal muscle exhibits robust regeneration while in cardiac muscle regeneration is not obvious. The dystrophin complex is concentrated along the plasma membrane in costameric structures that correspond to the Z bands of sarcomeres, thus positioning the dystrophin complex to transmit force between the sarcomere and the plasma membrane to the extracellular matrix. Although it is apparent that this position is important for perpendicular force transmission, it is clear that the dystrophin complex also fulfills signaling roles. Nitric oxide synthase and stress-induced signaling cascades are activated to participate in protection but may also contribute to pathology.

Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy.

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (alpha-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.

Delta-sarcoglycan is necessary for early heart and muscle development in zebrafish.

Delta-sarcoglycan, one member of the sarcoglycan complex, is a very conservative muscle-specific protein exclusively expressed in the skeletal and cardiac muscles of vertebrates. Mutations in sarcoglycans are known to be involved in limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy (DCM) in humans. To address the role of delta-sarcoglycan gene in zebrafish development, we have studied expression pattern of delta-sarcoglycan in zebrafish embryos and examined the role of delta-sarcoglycan in zebrafish embryonic development by morpholino. Strong expression of delta-sarcoglycan was observed in various muscles including those of the segment, heart, eye, jaw, pectoral fin, branchial arches, and swim bladder in zebrafish embryo. Delta-sarcoglycan was also expressed in midbrain and retina. Knockdown of delta-sarcoglycan resulted in severe abnormality in both the cardiac and skeletal muscles. Some severe ones displayed serious morphological abnormality such as hypoplastic head, linear heart, very weak heartbeats, and runtish trunk, all dead within 5 dpf. Whole-mount in situ hybridization analysis showed that adaxial cells and muscle pioneers were affected in delta-sarcoglycan knockdown embryos. In addition, absence of delta-sarcoglycan protein severely delayed the cardiac development and influenced the differentiation of cardiac muscle, and the cardiac left-right asymmetry was dramatically changed in morpholino-treated embryos. These data together suggest that delta-sarcoglycan plays an important role in early heart and muscle development.

Zeta-sarcoglycan is a functional homologue of gamma-sarcoglycan in the formation of the sarcoglycan complex.

The sarcoglycans (SGs), transmembrane components of the dystrophin-associated glycoprotein complex, are stable and functional only when they assemble into a tetrameric complex in muscle cells. A defect in any one of the four SG members disrupts the entire SG complex (SGC) and causes limb-girdle muscular dystrophy. zeta-SG has been recently found as a transmembrane protein homologous to gamma-SG and delta-SG. To characterize zeta-SG in complex formation, we co-transfected expression vectors encoding all six SGs (alpha-, beta-, gamma-, delta-, epsilon- and zeta-SG) and dystroglycan into Chinese hamster ovary cells. Immunoprecipitation analysis showed that zeta-SG or gamma-SG formed a SGC with beta-SG and delta-SG plus alpha-SG or epsilon-SG, revealing that zeta-SG can form two types of SGCs (alpha-beta-zeta-delta or epsilon-beta-zeta-delta). This result indicates the functional resemblance of zeta-SG to gamma-SG rather than delta-SG, although phylogenetic analysis suggests that zeta-SG is evolutionally closer to delta-SG than to gamma-SG. Reverse transcription (RT)-PCR showed that the expression pattern of the transcript was almost the reciprocal of that of gamma-SG in various mouse tissues and that the zeta-SG transcript was especially abundant in the brain, suggesting that zeta-SG might play a particular role in the central nervous system.

Identification of functional domains in sarcoglycans essential for their interaction and plasma membrane targeting.

Mutations in sarcoglycans have been reported to cause autosomal-recessive limb-girdle muscular dystrophies. In skeletal and cardiac muscle, sarcoglycans are assembled into a complex on the sarcolemma from four subunits (alpha, beta, gamma, delta). In this report, we present a detailed structural analysis of sarcoglycans using deletion study, limited proteolysis and co-immunoprecipitation. Our results indicate that the extracellular regions of sarcoglycans consist of distinctive functional domains connected by proteinase K-sensitive sites. The N-terminal half domains are required for sarcoglycan interaction. The C-terminal half domains of beta-, gamma- and delta-sarcoglycan consist of a cysteine-rich motif and a previously unrecognized conserved sequence, both of which are essential for plasma membrane localization. Using a heterologous expression system, we demonstrate that missense sarcoglycan mutations affect sarcoglycan complex assembly and/or localization to the cell surface. Our data suggest that the formation of a stable complex is necessary but not sufficient for plasma membrane targeting. Finally, we provide evidence that the beta/delta-sarcoglycan core can associate with the C-terminus of dystrophin. Our results therefore generate important information on the structure of the sarcoglycan complex and the molecular mechanisms underlying the effects of various sarcoglycan mutations in muscular dystrophies.

Ultrastructure of diaphragm from dystrophic alpha-sarcoglycan-null mice.

alpha-Sarcoglycan is a 50 kDa single-pass transmembrane glycoprotein exclusively expressed in striated muscle that, together with beta-, gamma-, and delta-sarcoglycan, forms a sub-complex at the muscle fibre cell membrane. The sarcoglycans are components of the dystrophin-associated glycoprotein (DAG) complex which forms a mechanical link between the intracellular cytoskeleton and extracellular matrix. The DAG complex function is to protect the muscle membrane from the stress of contractile activity and as a structure for the docking of signalling proteins. Genetic defects of DAG components cause muscular dystrophies. A lack or defects of alpha-sarcoglycan causes the severe type 2D limb girdle muscular dystrophy. alpha-Sarcoglycan-null (Sgca-null) mice develop progressive muscular dystrophy similar to the human disorder. This animal model was used in the present work for an ultrastructural study of diaphragm muscle. Diaphragm from Sgca-null mouse presents a clear dystrophic phenotype, with necrosis, regeneration, fibre hypertrophy and splitting, excess of collagen and fatty infiltration. Some abnormalities were also observed, such as centrally located nuclei of abnormal shape, fibres containing inclusion bodies within the contractile structure, and fibres with electron-dense material dispersed over almost the entire cell. Additionally, unusual interstitial cells of uncertain identity were detected within muscle fibres. The abnormal ultrastructure of the diaphragm from Sgca-null mice is discussed.