Characterization of the dystrophin-associated protein complex by mass spectrometry.

The dystrophin-associated protein complex (DAPC) is a highly organized multiprotein complex that plays a pivotal role in muscle fiber structure integrity and cell signaling. The complex is composed of three distinct interacting subgroups, intracellular peripheral proteins, transmembrane glycoproteins, and extracellular glycoproteins subcomplexes. Dystrophin protein nucleates the DAPC and is important for connecting the intracellular actin cytoskeletal filaments to the sarcolemma glycoprotein complex that is connected to the extracellular matrix via laminin, thus stabilizing the sarcolemma during muscle fiber contraction and relaxation. Genetic mutations that lead to lack of expression or altered expression of any of the DAPC proteins are associated with different types of muscle diseases. Hence characterization of this complex in healthy and dystrophic muscle might bring insights into its role in muscle pathogenesis. This review highlights the role of mass spectrometry in characterizing the DAPC interactome as well as post-translational glycan modifications of some of its components such as α-dystroglycan. Detection and quantification of dystrophin using targeted mass spectrometry are also discussed in the context of healthy versus dystrophic skeletal muscle.

Analysis on sarcoglycans expression as markers of septic cardiomyopathy in sepsis-related death.

The post-mortem assessment of sepsis-related death can be carry out by many methods recently suggested as microbiological and biochemical investigations. In these cases, the cause of death is a multiple organ dysfunction due to a dysregulated inflammatory response occurring after the failure of infection control process. It was highlighted also that the heart can be a target organ in sepsis which determines the so-called septic cardiomyopathy characterized by myocardial depression. Several mechanisms to explain the pathophysiology of septic cardiomyopathy were suggested, but very few studies about the structural alterations of cardiac cells responsible for myocardial depression were carried out. The aim of this study was to evaluate whether sarcoglycans (SG) were involved in septic cardiac damage analyzing their expression in sepsis-related deaths and, particularly, if these proteins can be used as markers of septic myocardial dysfunction. Cases of septic-related death confirmed by clinical and autopsy records were investigated and compared to a control group of traumatic deaths. Indirect immunofluorescence analysis was performed to analyze α-SG, ß-SG, δ-SG, ζ-SG, ε-SG, and γ-SG. Decrease of fluorescence staining pattern for all tested sarcoglycans was observed in the septic-related deaths compared to normal fluorescence staining pattern of control group. These results provide new findings about the myocytes structural alterations due to sepsis and suggest that these proteins could be used in forensic assessment of septic cardiomyopathy.

Role of major and brain-specific Sgce isoforms in the pathogenesis of myoclonus-dystonia syndrome.

Loss-of-function mutations in SGCE, which encodes ε-sarcoglycan (ε-SG), cause myoclonus-dystonia syndrome (OMIM159900, DYT11). A "major" ε-SG protein derived from CCDS5637.1 (NM_003919.2) and a "brain-specific" protein, that includes sequence derived from alternative exon 11b (CCDS47642.1, NM_001099400.1), are reportedly localized in post- and pre-synaptic membrane fractions, respectively. Moreover, deficiency of the "brain-specific" isoform and other isoforms derived from exon 11b may be central to the pathogenesis of DYT11. However, no animal model supports this hypothesis. Gene-trapped ES cells (CMHD-GT_148G1-3, intron 9 of NM_011360) were used to generate a novel Sgce mouse model (C57BL/6J background) with markedly reduced expression of isoforms derived from exons 3' to exon 9 of NM_011360. Among those brain regions analyzed in adult (2month-old) wild-type (WT) mice, cerebellum showed the highest relative expression of isoforms incorporating exon 11b. Homozygotes (SgceGt(148G1)Cmhd/Gt(148G1)Cmhd or SgceGt/Gt) and paternal heterozygotes (Sgcem+/pGt, m-maternal, p-paternal) showed 60 to 70% reductions in expression of total Sgce. Although expression of the major (NM_011360) and brain-specific (NM_001130189) isoforms was markedly reduced, expression of short isoforms was preserved and relatively small amounts of chimeric ε-SG/ß-galactosidase fusion protein was produced by the Sgce gene-trap locus. Immunoaffinity purification followed by mass spectrometry assessments of Sgcem+/pGt mouse brain using pan- or brain-specific ε-SG antibodies revealed significant reductions of ε-SG and other interacting sarcoglycans. Genome-wide gene-expression data using RNA derived from adult Sgcem+/pGt mouse cerebellum showed that the top up-regulated genes were involved in cell cycle, cellular development, cell death and survival, while the top down-regulated genes were associated with protein synthesis, cellular development, and cell death and survival. In comparison to WT littermates, Sgcem+/pGt mice exhibited "tiptoe" gait and stimulus-induced appendicular posturing between Postnatal Days 14 to 16. Abnormalities noted in older Sgcem+/pGt mice included reduced body weight, altered gait dynamics, and reduced open-field activity. Overt spontaneous or stimulus-sensitive myoclonus was not apparent on the C57BL/6J background or mixed C57BL/6J-BALB/c and C57BL/6J-129S2 backgrounds. Our data confirm that mouse Sgce is a maternally imprinted gene and suggests that short Sgce isoforms may compensate, in part, for deficiency of major and brain-specific Sgce isoforms.

Causative variants linked with limb girdle muscular dystrophy in an Iranian population: 6 novel variants.

BACKGROUND: Limb-girdle muscular dystrophy (LGMD) is a non-syndromic muscular dystrophy caused by variations in the genes involved in muscle structure, function and repair. The heterogeneity in the severity, progression, age of onset, and causative genes makes next-generation sequencing (NGS) a necessary approach for the proper diagnosis of LGMD. METHODS: In this article, 26 Iranian patients with LGMD criteria were diagnosed with disease variants in the genes encoding calpain3, dysferlin, sarcoglycans and Laminin α-2. Patients were referred to the hospital with variable distribution of muscle wasting and progressive weakness in the body. The symptoms along with biochemical and EMG tests were suggestive of LGMD; thus the genomic DNA of patients were investigated by whole-exome sequencing including flanking intronic regions. The target genes were explored for the disease-causing variants. Moreover, the consequence of the amino acid alterations on proteins' secondary structure and function was investigated for a better understanding of the pathogenicity of variants. Variants were sorted based on the genomic region, type and clinical significance. RESULTS: In a comprehensive investigation of previous clinical records, 6 variations were determined as novel, including c.1354-2 A > T and c.3169_3172dupCGGC in DYSF, c.568 G > T in SGCD, c.7243 C > T, c.8662_8663 insT and c. 4397G > C in LAMA2. Some of the detected variants were located in functional domains and/or near to the post-translational modification sites, altering or removing highly conserved regions of amino acid sequence.

Novel γ-sarcoglycan interactors in murine muscle membranes.

BACKGROUND: The sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma. The SC coordinates changes in phosphorylation and Ca++-flux during mechanical deformation, and these processes are disrupted with loss-of-function mutations in gamma-sarcoglycan (Sgcg) that cause Limb girdle muscular dystrophy 2C/R5. METHODS: To gain insight into how the SC mediates mechano-signaling in muscle, we utilized LC-MS/MS proteomics of SC-associated proteins in immunoprecipitates from enriched sarcolemmal fractions. Criteria for inclusion were co-immunoprecipitation with anti-Sgcg from C57BL/6 control muscle and under-representation in parallel experiments with Sgcg-null muscle and with non-specific IgG. Validation of interaction was performed in co-expression experiments in human RH30 rhabdomyosarcoma cells. RESULTS: We identified 19 candidates as direct or indirect interactors for Sgcg, including the other 3 SC proteins. Novel potential interactors included protein-phosphatase-1-catalytic-subunit-beta (Ppp1cb, PP1b) and Na+-K+-Cl--co-transporter NKCC1 (SLC12A2). NKCC1 co-localized with Sgcg after co-expression in human RH30 rhabdomyosarcoma cells, and its cytosolic domains depleted Sgcg from cell lysates upon immunoprecipitation and co-localized with Sgcg after detergent permeabilization. NKCC1 localized in proximity to the dystrophin complex at costameres in vivo. Bumetanide inhibition of NKCC1 cotransporter activity in isolated muscles reduced SC-dependent, strain-induced increases in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In silico analysis suggests that candidate SC interactors may cross-talk with survival signaling pathways, including p53, estrogen receptor, and TRIM25. CONCLUSIONS: Results support that NKCC1 is a new SC-associated signaling protein. Moreover, the identities of other candidate SC interactors suggest ways by which the SC and NKCC1, along with other Sgcg interactors such as the membrane-cytoskeleton linker archvillin, may regulate kinase- and Ca++-mediated survival signaling in skeletal muscle.

LGMD. Identification, description and classification.

The term 'limb girdle muscular dystrophy' (LGMD) was first used in the seminal paper by Walton and Nattrass in 1954, were they identified LGMD as a separate clinical entity In LGMD description it is pointed out that the category of LGMD most likely comprises a heterogeneous group of disorders. After that the clinical entity was discussed but the LMGD nosography reached a permanent classification during two ENMC workshops held in 1995 and 2017, in the last one an operating definition of LGMD was agreed. This last classification included dystrophies with proximal or distal-proximal presentation with evidence at biopsy of fibre degeneration and splitting, high CK, MRI imaging consistent with degenerative changes, fibro-fatty infiltration present in individuals that reached independent walking ability. To be considered in this group at least two unrelated families should be identified. A review is done of the first genetic characterisation of a number of LGMDs during the late twentieth century and a historical summary is given regarding how these conditions were clinically described and identified, the progresses done from identification of genetic loci, to protein and gene discoveries are reported. The LGMD described on which such historical progresses were done are the recessive calpainopathy (LGMD 2A/R1), dysferlinopathy (LGMD 2B/R2), sarcoglycanopathy (LGMD 2C-2F/R3-R6) types and the dominant type due to TPNO3 variants named transportinopathy (LGMD 1F/D2). Because of new diagnostic techniques such as exome and genome sequencing, it is likely that many other subtypes of LGMD might be identified in the future, however the lesson from the past discoveries can be useful for scientists and clinicians.

A new mutation of the SCGA gene is the cause of a late onset mild phenotype limb girdle muscular dystrophy type 2D with axial involvement.

Mutations in the SGCA gene cause limb girdle muscular dystrophy type 2D (LGMD2D). We report a family with three affected siblings with a mild phenotype consisting of late onset glutei and axial muscle weakness produced by a new mutation in the SGCA gene leading to a partial expression of the alpha-sarcoglycan protein. The MRI showed muscle atrophy involving paraspinal, pelvic and thigh muscles and a dystrophic pattern was observed in the muscle biopsy. Exome sequencing revealed a homozygous intronic deletion of SGCA and mRNA analysis showed the presence of three different transcripts. The presence, though in a lower proportion, of wild type transcript leads to a milder presentation of the disease. Although clinical symptoms did not entirely correspond with a sarcoglycanopathy, a compatible muscle MRI drove us to look for changes in the sarcoglycan genes. These cases are an example of how clinical, radiological and pathological data enriches the interpretation of exome analysis.

The Sarcoglycan complex is expressed in the cerebrovascular system and is specifically regulated by astroglial Cx30 channels.

Astrocytes, the most prominent glial cell type in the brain, send specialized processes called endfeet, around blood vessels and express a large molecular repertoire regulating the cerebrovascular system physiology. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction proteins Connexin 43 and 30 (Cx43 and Cx30) allowing in particular for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. In this study, we addressed the specific role of Cx30 at the gliovascular interface. Using an inactivation mouse model for Cx30 (Cx30(Δ/Δ); Δ means deleted allele) we showed that absence of Cx30 does not affect blood-brain barrier (BBB) organization and permeability. However, it results in the cerebrovascular fraction, in a strong upregulation of Sgcg encoding γ-Sarcoglycan (γ-SG), a member of the Dystrophin-associated protein complex (DAPC) connecting cytoskeleton and the extracellular matrix. The same molecular event occurs in Cx30(T5M/T5M) mutated mice, where Cx30 channels are closed, demonstrating that Sgcg regulation relied on Cx30 channel functions. We further characterized the expression of other Sarcoglycan complex (SGC) molecules in the cerebrovascular system and showed the presence of α-, ß-, δ-, γ-, ε- and ζ- SG, as well as Sarcospan. Their expression was however not modified in Cx30(Δ/Δ). These results suggest that a full SGC might be present in the cerebrovascular system, and that expression of one of its member, γ-SG, depends on Cx30 channels. As described in skeletal muscles, the SGC may contribute to membrane stabilization and signal transduction in the cerebrovascular system, which may therefore be regulated by Cx30 channel-mediated functions.