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.

A mouse model of inherited choline kinase ß-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia.

The CHKB gene encodes choline kinase ß, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb-/- mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb+/- mice. Unlike wildtype mice, 60% of the Chkb+/- and all Chkb-/- mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb+/- and Chkb-/- hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb-/- hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb+/- and Chkb-/- mice.

A long-read RNA-seq approach to identify novel transcripts of very large genes.

RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap: 5 kb), large (Neb: 22 kb), and very large (Ttn: 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type-specific differential expression of these novel transcripts. The improved transcript identification and quantification shown by our approach removes previous impediments to studies aimed at quantitative differential expression of ultralong transcripts.

A novel estrogen receptor 1: sphingomyelin phosphodiesterase acid-like 3B pathway mediates rituximab response in myositis patients.

OBJECTIVES: The B-cell depleting biologic, rituximab, is used to treat refractory autoimmune myositis. However, the beneficial effects of rituximab appear to outweigh the known contribution of B cells in myositis. We aimed to elucidate how myositis patients respond differently to rituximab and possible alternative mechanisms of action. METHODS: Here we have: (i) comprehensively investigated concurrent mRNA and microRNA expression in muscle biopsies taken at baseline and 16 weeks post treatment in 10 patients who were part of the rituximab in myositis (RIM) trial; and (ii) investigated the beneficial effect of rituximab on myositis muscle cells. RESULTS: Our analyses identified an increased number of changes in gene expression in biopsies from patients who had a clinical response to rituximab (n = 5) compared with non-responders (n = 5). The two groups had completely different changes in microRNA and mRNA expression following rituximab therapy, with the exception of one mRNA, BHMT2. Networks of mRNA and microRNA with opposite direction of expression changes highlighted ESR1 as upregulated in responders. We confirmed ESR1 upregulation upon rituximab treatment of immortalized myotubes and primary human dermatomyositis muscle cells in vitro, demonstrating a direct effect of rituximab on muscle cells. Notably, despite showing a response to rituximab, human dermatomyositis primary muscle cells did not express the rituximab target, CD20. However, these cells expressed a possible alternative target of rituximab, sphingomyelinase-like phosphodiesterase 3 b (SMPDL3B). CONCLUSION: In addition to B-cell depletion, rituximab may be beneficial in myositis due to increased ESR1 signalling mediated by rituximab binding to SMPDL3B on skeletal muscle cells.

Disruption of a key ligand-H-bond network drives dissociative properties in vamorolone for Duchenne muscular dystrophy treatment.

Duchenne muscular dystrophy is a genetic disorder that shows chronic and progressive damage to skeletal and cardiac muscle leading to premature death. Antiinflammatory corticosteroids targeting the glucocorticoid receptor (GR) are the current standard of care but drive adverse side effects such as deleterious bone loss. Through subtle modification to a steroidal backbone, a recently developed drug, vamorolone, appears to preserve beneficial efficacy but with significantly reduced side effects. We use combined structural, biophysical, and biochemical approaches to show that loss of a receptor-ligand hydrogen bond drives these remarkable therapeutic effects. Moreover, vamorolone uniformly weakens coactivator associations but not corepressor associations, implicating partial agonism as the main driver of its dissociative properties. Additionally, we identify a critical and evolutionarily conserved intramolecular network connecting the ligand to the coregulator binding surface. Interruption of this allosteric network by vamorolone selectively reduces GR-driven transactivation while leaving transrepression intact. Our results establish a mechanistic understanding of how vamorolone reduces side effects, guiding the future design of partial agonists as selective GR modulators with an improved therapeutic index.

Serum biomarkers associated with baseline clinical severity in young steroid-naïve Duchenne muscular dystrophy boys.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin in muscle, and while all patients share the primary gene and biochemical defect, there is considerable patient-patient variability in clinical symptoms. We sought to develop multivariate models of serum protein biomarkers that explained observed variation, using functional outcome measures as proxies for severity. Serum samples from 39 steroid-naïve DMD boys 4 to <7 years enrolled into a clinical trial of vamorolone were studied (NCT02760264). Four assessments of gross motor function were carried out for each participant over a 6-week interval, and their mean was used as response for biomarker models. Weighted correlation network analysis was used for unsupervised clustering of 1305 proteins quantified using SOMAscan® aptamer profiling to define highly representative and connected proteins. Multivariate models of biomarkers were obtained for time to stand performance (strength phenotype; 17 proteins) and 6 min walk performance (endurance phenotype; 17 proteins) including some shared proteins. Identified proteins were tested with associations of mRNA expression with histological severity of muscle from dystrophinopathy patients (n = 28) and normal controls (n = 6). Strong associations predictive of both clinical and histological severity were found for ERBB4 (reductions in both blood and muscle with increasing severity), SOD1 (reductions in muscle and increases in blood with increasing severity) and CNTF (decreased levels in blood and muscle with increasing severity). We show that performance of DMD boys was effectively modeled with serum proteins, proximal strength associated with growth and remodeling pathways and muscle endurance centered on TGFß and fibrosis pathways in muscle.

Sphingosine Phosphate Lyase Is Upregulated in Duchenne Muscular Dystrophy, and Its Inhibition Early in Life Attenuates Inflammation and Dystrophy in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a congenital myopathy caused by mutations in the dystrophin gene. DMD pathology is marked by myositis, muscle fiber degeneration, and eventual muscle replacement by fibrosis and adipose tissue. Satellite cells (SC) are muscle stem cells critical for muscle regeneration. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes SC proliferation, regulates lymphocyte trafficking, and is irreversibly degraded by sphingosine phosphate lyase (SPL). Here, we show that SPL is virtually absent in normal human and murine skeletal muscle but highly expressed in inflammatory infiltrates and degenerating fibers of dystrophic DMD muscle. In mdx mice that model DMD, high SPL expression is correlated with dysregulated S1P metabolism. Perinatal delivery of the SPL inhibitor LX2931 to mdx mice augmented muscle S1P and SC numbers, reduced leukocytes in peripheral blood and skeletal muscle, and attenuated muscle inflammation and degeneration. The effect on SC was also observed in SCID/mdx mice that lack mature T and B lymphocytes. Transcriptional profiling in the skeletal muscles of LX2931-treated vs. control mdx mice demonstrated changes in innate and adaptive immune functions, plasma membrane interactions with the extracellular matrix (ECM), and axon guidance, a known function of SC. Our cumulative findings suggest that by raising muscle S1P and simultaneously disrupting the chemotactic gradient required for lymphocyte egress, SPL inhibition exerts a combination of muscle-intrinsic and systemic effects that are beneficial in the context of muscular dystrophy.

Elevation of fast but not slow troponin I in the circulation of patients with Becker and Duchenne muscular dystrophy.

INTRODUCTION: One of the hallmarks of injured skeletal muscle is the appearance of elevated skeletal muscle proteins in circulation. Human skeletal muscle generally consists of a mosaic of slow (type I) and fast (type IIa, IIx/d) fibers, defined by their myosin isoform expression. Recently, measurement of circulating fiber-type specific isoforms of troponin I has been used as a biomarker to suggest that muscle injury in healthy volunteers (HV) results in the appearance of muscle proteins from fast but not slow fibers. We sought to understand if this is also the case in severe myopathy patients with Becker and Duchenne muscular dystrophy (BMD, DMD). METHODS: An enzyme-linked immunosorbent assay (ELISA) that selectively measures fast and slow skeletal troponin I (TNNI2 and TNNI1) was used to measure a cross-section of patient plasma samples from HV (N = 50), BMD (N = 49), and DMD (N = 132) patients. Creatine kinase (CK) activity was also measured from the same samples for comparison. RESULTS: TNNI2 was elevated in BMD and DMD and correlated with the injury biomarker, CK. In contrast, TNNI1 levels were indistinguishable from levels in HV. There was an inverse relationship between CK and TNNI2 levels and age, but no relationship for TNNI1. DISCUSSION: We define a surprising discrepancy between TNNI1 and TNNI2 in patient plasma that may have implications for the interpretation of elevated muscle protein levels in dystrophinopathies.

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Efficacy and safety of vamorolone in Duchenne muscular dystrophy: An 18-month interim analysis of a non-randomized open-label extension study.

BACKGROUND: Treatment with corticosteroids is recommended for Duchenne muscular dystrophy (DMD) patients to slow the progression of weakness. However, chronic corticosteroid treatment causes significant morbidities. Vamorolone is a first-in-class anti-inflammatory investigational drug that has shown evidence of efficacy in DMD after 24 weeks of treatment at 2.0 or 6.0 mg/kg/day. Here, open-label efficacy and safety experience of vamorolone was evaluated over a period of 18 months in trial participants with DMD. METHODS AND FINDINGS: A multicenter, open-label, 24-week trial (VBP15-003) with a 24-month long-term extension (VBP15-LTE) was conducted by the Cooperative International Neuromuscular Research Group (CINRG) and evaluated drug-related effects of vamorolone on motor outcomes and corticosteroid-associated safety concerns. The study was carried out in Canada, US, UK, Australia, Sweden, and Israel, from 2016 to 2019. This report covers the initial 24-week trial and the first 12 months of the VBP15-LTE trial (total treatment period 18 months). DMD trial participants (males, 4 to <7 years at entry) treated with 2.0 or 6.0 mg/kg/day vamorolone for the full 18-month period (n = 23) showed clinical improvement of all motor outcomes from baseline to month 18 (time to stand velocity, p = 0.012 [95% CI 0.010, 0.068 event/second]; run/walk 10 meters velocity, p < 0.001 [95% CI 0.220, 0.491 meters/second]; climb 4 stairs velocity, p = 0.001 [95% CI 0.034, 0.105 event/second]; 6-minute walk test, p = 0.001 [95% CI 31.14, 93.38 meters]; North Star Ambulatory Assessment, p < 0.001 [95% CI 2.702, 6.662 points]). Outcomes in vamorolone-treated DMD patients (n = 46) were compared to group-matched participants in the CINRG Duchenne Natural History Study (corticosteroid-naïve, n = 19; corticosteroid-treated, n = 68) over a similar 18-month period. Time to stand was not significantly different between vamorolone-treated and corticosteroid-naïve participants (p = 0.088; least squares [LS] mean 0.042 [95% CI -0.007, 0.091]), but vamorolone-treated participants showed significant improvement compared to group-matched corticosteroid-naïve participants for run/walk 10 meters velocity (p = 0.003; LS mean 0.286 [95% CI 0.104, 0.469]) and climb 4 stairs velocity (p = 0.027; LS mean 0.059 [95% CI 0.007, 0.111]). The vamorolone-related improvements were similar in magnitude to corticosteroid-related improvements. Corticosteroid-treated participants showed stunting of growth, whereas vamorolone-treated trial participants did not (p < 0.001; LS mean 15.86 [95% CI 8.51, 23.22]). Physician-reported incidences of adverse events (AEs) for Cushingoid appearance, hirsutism, weight gain, and behavior change were less for vamorolone than published incidences for prednisone and deflazacort. Key limitations to the study were the open-label design, and use of external comparators. CONCLUSIONS: We observed that vamorolone treatment was associated with improvements in some motor outcomes as compared with corticosteroid-naïve individuals over an 18-month treatment period. We found that fewer physician-reported AEs occurred with vamorolone than have been reported for treatment with prednisone and deflazacort, and that vamorolone treatment did not cause the stunting of growth seen with these corticosteroids. This Phase IIa study provides Class III evidence to support benefit of motor function in young boys with DMD treated with vamorolone 2.0 to 6.0 mg/kg/day, with a favorable safety profile. A Phase III RCT is underway to further investigate safety and efficacy. TRIAL REGISTRATION: Clinical trials were registered at www.clinicaltrials.gov, and the links to each trial are as follows (as provided in manuscript text): VBP15-002 [NCT02760264] VBP15-003 [NCT02760277] VBP15-LTE [NCT03038399].