Muscle & Nerve

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is a genetic disorder characterized by progressive weakening of the muscles. While the two types of FSHD (FSHD1 and FSHD2) have distinct genetic causes, they share similarities in their clinical presentations. Both result in muscle weakness, particularly in the face, shoulders, and upper arms. Genetic testing is essential for accurately diagnosing the specific type of FSHD and guiding treatment and management. MethodWe acquired bulk and single-cell gene expression data for FSHD2 from the NIH portal website. Our analysis involved an extensive array of differentially expressed genes, and pathway and gene ontology analysis. Using statistical tests, we identified the top up- and down-regulated genes, and the pathways and gene ontology terms characterizing those genes that exhibited substantial changes across both bulk and single-cell transcriptomes. ResultsThe top 10 up-regulated genes identified in the bulk gene expression analysis represent a diverse range of biological functions, but all are associated with FSHD. In contrast to the bulk down-regulated genes, the single-cell top 10 down-regulated genes are primarily linked to muscle-related functions. These genes, such as ACTC1, ACTA1, MYL11, MYH3, MYL6B, MYBPH, TPM2, MYL2, MYL1, and TNNI1 are integral to muscle contraction and skeletal muscle function. Moreover, all the top 10 single-cell down-regulated pathways are implicated in the pathogenesis of muscle dystrophy. Finally, the top 10 down-regulated gene ontology terms are all relevant to the pathogenesis of muscular dystrophy. ConclusionsThis study unequivocally demonstrates that single-cell transcriptomics surpasses bulk transcriptomics in elucidating the genes, pathways, biological processes, molecular functions, and cellular components associated with FSHD2. While bulk transcriptomics offers a broader perspective on gene expression, single-cell transcriptomics shines in its capacity to unveil cell-specific gene regulation, especially in the realm of muscle-related functions.

ObjectiveDegeneration of motor endplates (MEPs) in denervated muscle is thought to be a key factor limiting functional regeneration after peripheral nerve injury (PNI) in humans. However, there is currently no paradigm to determine MEP status in denervated human muscle to estimate likelihood of reinnervation success. Here, we present a quantitative analysis of MEP status in biopsies of denervated muscles taken during nerve repair surgery and ensuing functional recovery. MethodsThis is a retrospective single-surgeon cohort study of patients (n=22) with upper extremity PNI confirmed with electromyography (EMG), treated with nerve transfers. Muscle biopsies were obtained intra-operatively from 10 patients for MEP morphometric analysis. Age at time of surgery ranged from 22-77 years and time from injury to surgery ranged from 2.5 -163 months. Shoulder range of motion (ROM) and Medical Research Council (MRC) scores were recorded pre-op and at final follow-up. ResultsSurviving MEPs were observed in biopsies of denervated muscles from all patients, even those greater than six months from injury. Average postoperative ROM improvement (assessed between 6-9 months post-surgery) was: forward flexion 84.3 {+/-} 51.8{degrees}, abduction 62.5 {+/-} 47.9{degrees}, and external rotation 25.3 {+/-} 28.0{degrees}. InterpretationWhile it is believed that MEP degeneration 6 months post-injury prevents reinnervation, this data details MEP persistence beyond this timepoint along with significant functional recovery after nerve surgery. Accordingly, persistence of MEPs in denervated muscles may predict the extent of functional recovery from nerve repair surgery.

BackgroundDuchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disease caused by mutations in the DMD gene, leading to the absence or dysfunction of dystrophin. While cardiac and skeletal muscles are both affected, tissue-specific differences in disease manifestation and dystrophin regulation remain poorly understood. MethodsTo investigate these differences, we established a human induced pluripotent stem cell (hiPSC) model of DMD from peripheral blood mononuclear cells (PBMC) of a patient carrying a splice-site mutation in intron 68 (c.9975-1G>T). An isogenic control line was generated via CRISPR/Cas9 correction. Both repaired and DMD hiPSCs were differentiated into cardiomyocytes (hiPSC-CMs) and skeletal muscle cells (hiPSC-SMs). Transcript and protein analyses were performed, along with functional assessment using microelectrode array. ResultsTranscript analysis revealed an in-frame deletion of two amino acids (Tyr3325 and Arg3326) due to skipping of the first six nucleotides of exon 69. Despite this, near full-length Dp427 was detected by western blot, along with expression of Dp116 in hiPSC-CMs. Dystrophin levels were preserved in DMD hiPSC-CMs but markedly reduced in hiPSC-SMs, suggesting tissue-specific regulation. Functional analysis showed altered {beta}-adrenergic responsiveness in DMD hiPSC-CMs, with increased beating frequency and accelerated repolarization upon isoproterenol stimulation. ConclusionsOur study identifies a splice-site mutation that preserves high level of dystrophin expression in cardiac but reduced in skeletal muscle and reveals Dp116 expression in cardiomyocytes. These findings highlight the importance of tissue context in DMD and demonstrate the power of hiPSC-based systems for dissecting mutation-specific effects.

BackgroundDuchenne muscular dystrophy (DMD) is a lethal pediatric degenerative muscle disease for which there is no cure. Robust preclinical models that recapitulate major clinical features of DMD are required to investigate efficacy of potential DMD therapeutics. Rat models of DMD have emerged as promising small animal models to accomplish this; however, there have been no comprehensive studies investigating the functional skeletal muscle decrements associated with the modeling of DMD in rats. MethodsCRISPR/Cas9 gene editing was used to generate a dystrophin-deficient Sprague-Dawley muscular dystrophy rat (MDR). Biochemical and immunofluorescent analyses were performed to confirm loss of dystrophin in striated muscles of this rat model. In situ and ex vivo muscle function was assessed in wild-type (WT) and MDR muscles at 3, 6, and 12 months of age, followed by histopathological analyses. ResultsMDR muscle tissues exhibited loss of full-length dystrophin and reduced content of other dystrophin glycoprotein complex members. MDR extensor digitorum longus (EDL) muscles and diaphragms displayed pronounced and progressive muscle weakness beginning at 3 months of age, compared to WT littermates. EDLs also exhibit susceptibility to eccentric contraction-induced damage. Functional deficits in soleus muscles were less severe and were associated with a right shift in force-frequency relationship and a muscle fiber-type shift. MDR muscles display progressive histopathology including degenerative lesions, fibrosis, regenerative foci, and modest adipose deposition. ConclusionsMDR is a preclinical model of DMD that exhibits many translational features of the human disease, including a large dynamic range of muscle decrements, that has high utility for the evaluation of potential therapeutics for DMD.

The health of the infralesional lower motor neuron (LMN) has received little attention in individuals with traumatic cervical spinal cord injuries (SCI). Infralesional LMN health is clinically relevant in the context of nerve transfer surgery to restore critical upper limb functions, as those demonstrating LMN damage below the neurological level of injury may experience irreversible sequelae of denervation (e.g., atrophy, fibrosis) without timely intervention. In this two-centre retrospective cohort study, we examined the health of the infralesional LMN in individuals with traumatic cervical SCI, using data derived from the clinical electrodiagnostic examination performed early after SCI. We assessed 66 limbs in 42 individuals with traumatic cervical SCI (40 males, mean age = 43.6{+/-}17.2, mean duration from injury = 3.3{+/-}1.5 months, 25 with motor complete injuries). Analysis was stratified by injury level as 1) C4 and above, 2) C5 and 3) C6-7. EMG performed on representative muscles from C5-6, C6-7, C7-8 and C8-T1, were included in analysis. LMN abnormality was dichotomized as present (abnormal spontaneous activity) or absent. Data were pooled for the most caudal infralesional segment (C8-T1). Overall, a high frequency of denervation potentials was seen in all infralesional segments for all injury levels. The pooled frequency of denervation potentials at C8-T1 was 74.6% of limbs tested. There was also evidence of denervation potentials at the rostral border of the neurological level of injury, as high as 64.3% of C5-6 muscles for C5 injuries. These data support a high prevalence of infralesional LMN abnormality following SCI, which has implications to candidacy, timing of the intervention, donor nerve options and motor prognosis following SCI.

Biallelic pathogenic variants in the gene encoding nebulin (NEB) are a known cause of congenital myopathy. We present two individuals with congenital myopathy and compound heterozygous variants (NM_001271208.2: c.2079C>A; p.(Cys693Ter) and c.21522+3A>G) in NEB. Transcriptomic sequencing on patient muscle revealed that the extended splice variant c.21522+3A>G causes exon 144 skipping. Nebulin isoforms containing exon 144 are known to be mutually exclusive with isoforms containing exon 143, and these isoforms are differentially expressed during development and in adult skeletal muscles. Patients MRIs were compared to the known pattern of relative abundance of these two isoforms in muscle. We propose that the pattern of muscle involvement in these patients better fits the distribution of exon 144-containing isoforms in muscle than with previously published MRI findings in NEB-related disease due to other variants. To our knowledge this is the first report hypothesizing disease pathogenesis through the alteration of isoform distributions in muscle.

BackgroundAmyotrophic lateral sclerosis (ALS), the most predominant form of Motor Neuron Disease (MND), is a progressive and fatal neurodegenerative condition that spreads throughout the neuromotor system by afflicting upper and lower motor neurons. Lower motor neurons project from the central nervous system and innervate muscle fibres at motor endplates, which degrade over the course of the disease leading to muscle weakness. The direction of neurodegeration from or to the point of neuromuscular junctions and the role of muscle itself in pathogenesis has continued to be a topic of debate in ALS research. MethodsTo assess the variation in gene expression between affected and nonaffected muscle tissue that might lead to this local degeneration of motor units, we generated RNA-seq skeletal muscle transcriptomes from 28 MND cases and 18 healthy controls and conducted differential expression analyses on gene-level counts, as well as an isoform switching analysis on isoform-level counts. ResultsWe identified 52 differentially-expressed genes (Benjamini-Hochberg-adjusted p < 0.05) within this comparison, including 38 protein coding, 9 long non-coding RNA, and 5 pseudogenes. Of protein-coding genes, 31 were upregulated in cases including with notable genes including the collagenic COL25A1 (p = 3.1 x 10-10), SAA1 which is released in response to tissue injury (p = 3.6 x 10-5) as well as others of the SAA family, and the actin-encoding ACTC1 (p = 2.3 x 10-5). Additionally, we identified 17 genes which exhibited a functional isoform switch with likely functional consequences between cases and controls. ConclusionsOur analyses provide evidence of increased tissue generation in MND cases, which likely serve to compensate for the degeneration of motor units and skeletal muscle.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary neuromuscular disorder. The Russian FSHD Patient Registry was established in 2019 following the development of a PCR-based method for genetic confirmation of the diagnosis. Results: The registry included 470 participants (51% male). Genetic confirmation was obtained for 76% (n=356), the remainder were included based on clinical and anamnestic data. Clinical assessment forms and patient-reported questionnaires were analyzed for 310 and 142 patients, respectively. D4Z4 repeat unit (RU) distribution showed patterns consistent with European cohorts, with a predominance of patients with 3 RUs. A moderate inverse correlation was found between RUs number and clinical severity scales. Periscapular weakness was the most common onset manifestation (46.8%), followed by facial weakness (31.6%) which was often unnoticed by patients. The mean age in the Russian cohort was 37.8 years (range 0-97), indicating a younger cohort compared to international data. A delta-adjusted cluster analysis (n=215) identified three distinct trajectories: a classic phenotype with onset before age 14 and early involvement of various muscle groups (n=177), and two clusters characterized by either facial or periscapular onset with slow progression. Conclusion: The Russian FSHD registry provides a comprehensive characterization of a large national cohort, revealing a predominance of patients with 3 D4Z4 repeats and a younger demographic profile compared to international data. Cluster analysis identified three heterogeneous disease trajectories, offering a framework for improved patient stratification.

Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. Here, we have introduced through CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy, into a Duchenne patients cell line. Dystrophin expression was restored in edited myoblasts and the myogenic defects were ameliorated. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology. SUMMARY STATEMENTWe restored dystrophin expression in a DMD culture by replicating the exact deletion in exons 45-55 harboured by mild patients, testing this therapeutic approach, and creating a new cell model.

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) disease progression is associated with muscle inflammation, although its role in FSHD muscle pathology is unknown. MethodsWe have developed a novel humanized mouse strain, NSG-SGM3-W41, that supports the co- engraftment of human hematopoietic stem cells (HSCs) and muscle myoblasts as an experimental model to investigate the role of innate immunity in FSHD muscle pathology. ResultsThe NSG-SGM3-W41 mouse supports the selective expansion of human innate immune cell lineages following engraftment of human HSCs and the co-engraftment and differentiation of patient-derived FSHD or control muscle myoblasts. Immunohistological and NanoString RNA expression assays establish that muscle xenografts from three FSHD subjects were immunogenic compared to those from unaffected first-degree relatives. FSHD muscle xenografts preferentially accumulated human macrophages and B cells and expressed early complement genes of the classical and alternative pathways including complement factor C3 protein, which is a mediator of early complement function through opsonization to mark damaged cells for macrophage engulfment. FSHD muscle xenografts also underwent immune donor dependent muscle turnover as assayed by human spectrin {beta}1 immunostaining of muscle fibers and by NanoString RNA expression assays of muscle differentiation genes. ConclusionsThe NSG-SGM3-W41 mouse provides an experimental model to investigate the role of innate immunity and complement in FSHD muscle pathology and to develop FSHD therapeutics targeting DUX4 and the innate immunity inflammatory responses.

Background and ObjectivesSporadic inclusion body myositis (IBM) is the most common acquired myopathy in individuals over age 50. The disorder is slowly progressive and while many therapies have been investigated, response has generally been poor. Clinical heterogeneity may influence treatment responsiveness; however, data regarding heterogeneity in IBM is limited and often conflicting. We aim to identify clinically distinct subgroups within a large IBM cohort, as well as prognostic factors for disease progression. MethodsClinical, histologic, radiologic, and electrophysiologic data were analyzed for all patients with IBM and other forms of myositis enrolled in a longitudinal cohort from The Johns Hopkins Myositis Center from 2003-2018. Univariate, multivariate, and graphical analyses were used to identify prognostic factors in IBM patients. ResultsAmong the 335 IBM patients meeting inclusion criteria, 64% were male with an average age of disease onset of 58.7 years and a delay to diagnosis of 5.2 years. Initial misdiagnosis (52%) and immunosuppressant treatment (42%) were common. Less than half (43%) of muscle biopsies demonstrated all three pathologic hallmarks: endomysial inflammation, mononuclear cell invasion, and rimmed vacuoles. Black patients had significantly weaker arm abductors, hip flexors, and knee flexors compared to non-Black patients but were less likely to develop dysphagia. Female patients had stronger finger flexors and knee extensors compared to their male counterparts but were more likely to develop dysphagia. A significant number (20%) of patients had an age of onset less than 50 years. This group of younger patients was weaker at their first visit; however, this may be accounted for by a longer disease duration at first visit. DiscussionAlthough IBM has long been considered a disorder predominately of older, White men, female, and non-White patients comprise a significant proportion of the IBM population. Our study demonstrates that female and Black patients have distinct clinical phenotypes within the overarching IBM clinical phenotype.

Mutations in skeletal muscle -actin (Acta1) cause a variety of myopathies. In a mouse model of congenital myopathy, heterozygous Acta1 (H40Y) knock-in (Acta1+/Ki) mice exhibit features of human nemaline myopathy, including premature lethality, severe muscle weakness, reduced mobility, and the presence of nemaline rods in muscle fibers. In this study, we investigated the structure and function of the neuromuscular junction (NMJ) in the Acta1+/Ki mice. We found marked impairments in NMJ structure in the mutant mice, including fragmented endplates and nerve terminals, reduced density of acetylcholine receptors (AChRs) on endplate membranes, reduced nerve occupancy at endplates, and increased numbers of muscle fiber subsynaptic nuclei. We compared the NMJs in three different types of muscles - the extensor digitorum longus (EDL, composed of fast-twitch muscle fibers), soleus (Sol, enriched in slow-twitch fibers) and the triangularis sterni muscle (TS, a mixed fiber type muscle). Among these three types of muscles, EDL was affected to the greatest extent, suggesting that fast-twitch fibers may be most susceptible to NMJ fragmentation in Acta1+/Ki nemaline myopathy. Electrophysiological analysis of mutant NMJs showed a reduced quantal size (reduced mEPP amplitude), increased mEPP frequency, and increased quantal content, but normal EPP amplitude compared to wild type (WT) NMJs. The results suggest that affected synapses may have undergone homeostatic compensation to maintain normal levels of neurotransmitter release. In addition, paired-pulse facilitation was reduced and synaptic depression under repetitive nerve stimulation was enhanced, indicating shortterm synaptic plasticity was compromised in the mutant mice. Key pointsO_LIMice heterozygous for an Acta1 (H40Y) knock-in mutation exhibit clinical features of human nemaline myopathy. We report structural and functional alterations of neuromuscular synapses in these mutant mice. The NMJ impairments include endplate fragmentation, reduced endplate nerve occupancy, and increased numbers of subsynaptic nuclei in muscle fibers. C_LIO_LINeuromuscular synaptic transmission was compromised - demonstrating both increased quantal content and changes in short-term synaptic plasticity. C_LIO_LIIncreases in spontaneous neurotransmitter release and quantal content suggest homeostatic compensation of synapses to maintain normal transmitter release in the mutant NMJs. C_LI

Background and ObjectivesTranscutaneous spinal cord stimulation (tSCS) is an emerging treatment for motor recovery following spinal cord injury (SCI). However, the extent of motor recovery with tSCS and the reasons why some individuals with motor-complete SCI respond less effectively, despite having the same injury classification, remain unclear. Here, we demonstrate that lumbosacral tSCS can enable anti-gravity voluntary movement following motor-complete SCI, and identify markers that distinguish responders from non-responders. MethodsTen individuals with chronic motor-complete SCI received 30Hz lumbosacral tSCS for 60 min, 2-5 times per week, for a minimum of 6 weeks. Post-intervention, volitional movement was measured using surface electromyography (EMG) over the quadriceps and tibialis anterior (TA), and knee and ankle joint range of motion. To identify markers of responsiveness, we assessed the integrity of the corticospinal tract (motor evoked potentials; MEPs), ascending sensory pathways (somatosensory evoked potentials; SEPs), spinal cord reflexes (H-reflex), and motor neurons (compound muscle action potential, CMAP), along with muscle morphology using ultrasound echo-intensity. ResultsFive of 10 individuals demonstrated voluntary anti-gravity knee extension and ankle dorsiflexion strength in the presence of tSCS. TA MEPs were observed in one responder only and tibial nerve SEPs were not observed in any participants. All participants showed poor TA muscle morphology. Four responders had a soleus H-reflex (compared to 2/5 non-responders) and a normal amplitude fibular CMAPs (compared to 2/5 non-responders). DiscussionThese results show that tSCS can enable volitional motor activity against gravity in people with motor-complete SCI, but there is variability in responsiveness. Using conventional neurophysiological techniques, we were unable to consistently demonstrate the pathways facilitating voluntary control or the factors differentiating responders versus non-responders, but trends were observed. Spinal cord reflex and peripheral motor nerve integrity may be important for responding to tSCS but may not distinguish responders from non-responders. Additional assessments are needed to develop biomarkers for stratifying motor responders to tSCS.

Adenylosuccinate synthetase 1 (ADSS1) myopathy is an ultra-rare disease characterized by progressive muscle dysfunction. The objective of this investigation was to employ a non-invasive biomarker approach to phenotype (fine-)motor skills, speech production and cognition in adults with ADSS1 myopathy. Five individuals with ADSS1 myopathy and five age-sex-matched healthy controls (HCs) underwent a comprehensive multimodal evaluation. Assessments included, (i) evaluation of motor performance, (ii) speech production and cognitive test batteries, (iii) patient-reported outcomes, (iv) electrical impedance myography (EIM), (v) musculoskeletal magnetic resonance imaging (MRI) and (vi) plasma proteomics. ADSS1 participants vs. HCs demonstrated reduced performance on the 9-Hole Peg and grip strength tests as well as lower self-reported mobility. Speech production analysis revealed asthenia (p=0.02), lower intelligibility (p=0.008), and worse voice quality during the sustained vowel task (p=0.03) in the ADSS1 cohort. Cognitive functioning remained unaffected in patients with ADSS1. On EIM, ADSS1 participants vs. HCs, demonstrated a pattern of higher resistance and lower reactance and phase across upper- and lower-extremity measurements, indicative of poorer muscle health, with large effect sizes (Cliffs 8=0.5-0.9). MRI revealed intramuscular fat infiltration, particularly in posterior compartments of the upper leg (e.g., biceps femoris). Proteomics indicated reduced (p=0.04) Neurotrophin-3 (NTF3; implicated in neuronal development, survival and differentiation) levels in the ADSS1 cohort relative to HCs. Lower NTF3 levels associated with poorer performance on hand-motor tasks as well as higher resistance and lower reactance and phase on EIM. This study highlighted the value of multimodal phenotyping for quantifying disease expression and advancing monitoring strategies in ADSS1 myopathy. Take-home messageThis multimodal investigation demonstrates that integrating electrical impedance myography with quantitative motor, speech, musculoskeletal imaging, and proteomic assessments provides a sensitive and non-invasive research framework for capturing neuromuscular dysfunction and functional disease burden in patients with ADSS1 myopathy, thereby supporting the current biomarker strategy for refined phenotyping and longitudinal disease monitoring in this ultra-rare condition.

IntroductionStructural variants (SVs) of the nebulin gene (NEB), including intragenic duplications, deletions, and copy number variation of the triplicate region, are an established cause of recessively inherited nemaline myopathies and related neuromuscular disorders. Large deletions have been shown to cause dominantly inherited distal myopathies. Here we provide an overview of 35 families with muscle disorders caused by such SVs in NEB. MethodsUsing custom Comparative Genomic Hybridization arrays, exome sequencing, short-read genome sequencing, custom Droplet Digital PCR, or Sanger sequencing, we identified pathogenic SVs in 35 families with NEB-related myopathies. ResultsIn 23 families, recessive intragenic deletions and duplications or pathogenic gains of the triplicate region segregating with the disease in compound heterozygous form, together with a small variant in trans, were identified. In two families the SV was, however, homozygous. Eight families have not been described previously. In 12 families with a distal myopathy phenotype, eight unique, large deletions encompassing 52 to 97 exons in either heterozygous (n = 10) or mosaic (n = 2) state were identified. In the families where inheritance was recessive, no correlation could be made between the types of variants and the severity of the disease. In contrast, all patients with large dominant deletions in NEB had milder, predominantly distal muscle weakness. DiscussionFor the first time, we establish a clear and statistically significant association between large NEB deletions and a form of distal myopathy. In addition, we provide the hitherto largest overview of the spectrum of SVs in NEB.

Surface electrode EMG is an established method for studying biomechanical activity. It has not been well studied in detecting laryngeal biomechanical activity of pre-phonatory onset. Our aims were to compare the sensitivity of surface EMG in identifying pre-phonatory laryngeal activity to needle electrode laryngeal EMG and to compare the pre-phonatory period in patients with adductor laryngeal dystonia (ADLD) with that in controls. ADLD patients (n = 10) undergoing needle LEMG prior to Botox(R) injection and participants with normal voices (n = 6) were recruited. Surface EMG electrodes were placed over the cricoid ring and thyrohyoid membrane. Needle EMG electrodes were inserted into the thyroarytenoid muscle. EMG and auditory output samples were collected during phonation onset. Tracings were de-identified and evaluated. Measurements of time from onset in change of the amplitude and motor unit frequency on the interference pattern to onset of phonation were calculated by two blinded raters. 42 of 71 patient and 40 of 50 control tracings were available for analysis. Correlation for pre-phonatory time between electrode configuration was 0.70 for patients, 0.64 for controls and 0.79 for all the data combined. Interrater correlation was 0.97 for needle and 0.96 for surface electrodes. ADLD patients had a longer pre-phonatory time than control subjects by 169.48ms with surface electrode and 140.23ms with needle electrode (p < 0.001). Surface EMG demonstrates equal reliability as Needle EMG in detecting pre-phonatory activity in controls and subjects. Patients with ADLD have a significantly prolonged pre-phonatory period when compared with controls.

ObjectivesTo screen the entire genome for genes associated with risk for lateral epicondylopathy and improve understanding of underlying biological mechanisms and inform future research aimed at risk stratification and personalized prevention and treatment strategies. MethodsA genome-wide association study was conducted using UK Biobank data. Lateral epicondylopathy cases were identified based on electronic health records from individuals of European ancestry. Logistic regression tested associations between single-nucleotide polymorphisms and disease status, adjusting for sex, age, height, weight and ancestry principal components. Previously-identified candidate genes from the literature were also tested for association with lateral epicondylopathy. ResultsAmong 20,390 cases of lateral epicondylopathy, two loci reached genome-wide significance: one comprising 144 linked SNPs and one single SNP. The first locus, led by rs13127477 (p=7.7x10-12; OR 0.93, 95% CI 0.91 to 0.95), is located near three SIBLING genes (IBSP, MEPE and SPP1) involved in extracellular matrix remodelling at fibrocartilaginous entheses. The risk allele was associated with increased SIBLING gene expression, suggesting that excessive entheseal matrix remodelling contributes to disease susceptibility. The second locus was defined by rs138254824 (p=3.69x10-8; OR 3.42, 95% CI 2.23 to 5.25) near NEDD9 and TMEM170B. Previously reported collagen gene associations were not replicated. ConclusionIn the first genome-wide screen for lateral epicondylopathy, two loci were identified. These loci provide insight regarding the pathophysiology of lateral epicondylopathy and a roadmap for preventing and treating this injury with personalized medicine. Summary BoxO_ST_ABSWhat is already known on this topicC_ST_ABSLateral epicondylopathy is a common and disabling overuse tendon condition, yet its genetic basis has remained poorly characterised, with prior studies limited to small candidate gene analyses. What this study addsThis study provides the first genome-wide association analysis of lateral epicondylopathy, identifying two risk loci on chromosomes 4 and 6 and implicating SIBLING genes (IBSP, MEPE, and SPP1) involved in entheseal extracellular matrix remodelling. How this study might affect research, practice or policyThese findings offer new biological insight into disease susceptibility and challenge previously reported collagen gene associations.

BackgroundIdiopathic inflammatory myopathies (IIMs) are a group of rare acquired muscular diseases. In healthy muscle, myofibers do not express major histocompatibility complex (MHC) class I and II. It was established that MHC-I positive immunostaining, although non-specific, is a marker for IIM diagnosis, while the significance of MHC-II immunostaining remains unclear. The present study investigates the expression of MCH-II in myofibers and capillaries of IIM muscles, taking into account the current IIM classification. Patients & MethodsA historical cohort was designed, including dermatomyositis (DM), inclusion body myositis (IBM), anti-synthetase syndrome (ASyS), immune-mediated necrotizing myopathy (IMNM), or overlap myositis (OM). MHC-II immunostaining was performed on patient muscle sections and was analyzed in a standardized and blind manner. ResultsMuscle sections from biopsies of 72 IIM patients were included: 23 DM, 17 IBM, 12 IMNM, 9 ASyS, and 11 OM. Overall, abnormal MHC-II immunostaining was found in myofibers and/or capillaries in 67 (93%) patients. Myofiber MHC-II immunostaining patterns differed according to the IIM subgroup: the immunostaining was diffuse in IBM (100%), negative in IMNM (75%), perifascicular in ASyS (67%), and either diffuse heterogeneous, clustered, or perifascicular in OM (27%, 27%, and 18%, respectively). MHC-II expression was found in 50% of DM (n=11/22). While all IIM subgroups presented quantitative and qualitative abnormalities of MHC-II immunostaining in capillaries, some subgroups displayed specificities. Most IBM and IMNM muscles presented frequent dilated capillaries (88% and 67%, respectively). DM, ASyS, and OM exhibited high frequencies of capillary lesions, including capillary dropout, leaky capillaries, and dilated capillaries. ConclusionWhile recent expert opinion (EURO-NMD pathology working group) recommended that MHC-II immunostaining of muscle biopsy remains optional, the present work demonstrates that the expression pattern of MHC-II allows to distinguish between several IIM subgroups. Our data argue for the inclusion of MHC-II immunostaining in the routine histological diagnosis for IIMs.

BackgroundPeripheral nerve trauma denervates skeletal muscle resulting in paralysis and atrophy that is reversible if timely reinnervation occurs, due to its regenerative capacity. If reinnervation is delayed muscles regenerative ability is exhausted and resident fibroadipogenic progenitors (FAPs) differentiate into adipocytes and fibroblasts that replace muscle with non-contractile fibrotic tissue and fat, resulting in physical disability. Prostaglandin E2 (PGE2) inhibits adipogenesis and fibrosis in other tissues. We determined whether PGE2 could inhibit fibro-fatty degradation of long-term denervated muscle. MethodsWe utilized the rat tibial nerve transection model, denervating the gastrocnemius and selected a 5 week post-denervation time point to represent short-term muscle denervation injury (reversible with reinnervation), and 12 weeks to represent sustained, irreversible injury. Gastrocnemius FAPs were isolated via FACS and grown in culture to assess endogenous PGE2 production and the proliferative and differentiation response to exogenous PGE2. We evaluated transcript and protein expression of PGE2 synthesizing enzyme PTGS2, PGE2 degrading enzyme 15-PGDH and markers of proliferation, adipogenesis and fibrogenesis using RT-qPCR, immunofluorescence and SDS-PAGE/Western blotting. Paracrine impact of FAPs produced PGE2 was assessed by treating C2C12 myoblasts with FAPs conditioned media. ResultsTranscript expression of PTGS2 was increased and 15-PGDH decreased (4.37{+/-}2.63 and -3.06{+/-}0.85 fold change respectively, p<0.05) in 5 week, but not 12 week denervated gastrocnemius, consistent with increased PGE2 production in 5 week denervated muscle. Similarly, PTGS2 transcript levels were significantly increased (2.58{+/-}0.33 fold change, p<0.05) and 15-PGDH decreased (-5.24{+/-}3.19 fold change, p<0.05) in FAPs isolated from 5 week, but not 12 week denervated muscle, demonstrating that FAPs are a source of PGE2 in short-term denervated muscle. 16,16-dimethyl PGE2 did not impact naive FAPs in vitro proliferation, but significantly inhibited their differentiation as demonstrated by 88.9%, 82.3% and 94.2% decreases in FAPs expression of adipogenic marker perilipin-1, fibrogenic marker -smooth muscle actin (-SMA) and lipid content respectively, mediated via PGE2 binding to the FAPs EP4 receptor. FAPs isolated from 12 week denervated muscle demonstrated increased adipogenesis and fibrogenesis vs. naive FAPs (perilipin-1 and -SMA 7.93{+/-}2.96 and 2.00{+/-}0.33 fold increase respectively, p<0.05) and remained fully susceptible to PGE2 inhibition of fibro-adipogenic differentiation. Conditioned media from FAPs derived from 5 week, but not 12 week, denervated gastrocnemius stimulated C2C12 myoblast proliferation which was prevented by EP4 blockade. ConclusionsPGE2 is identified as a novel negative regulator of FAPs differentiation in traumatically denervated muscle, suggesting the therapeutic potential of PGE2 to prevent fibro-fatty degradation of long-term denervated muscle awaiting reinnervation.

Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1{Delta}E3/{Delta}E3 mice, two established mouse models for DM1. DM1 muscle showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models displayed increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, expression of synaptic genes was up-regulated in DM1 muscle, which may be linked to the abnormally high activity of histone deacetylase 4 (HDAC4), a known target of CaMKII. Consistently, expression of myosin heavy chains was deregulated as well, leading to a major switch to type IIA fibres in Mbnl1{Delta}E3/{Delta}E3 muscle, and to a lesser extent in HSALR muscle. Interestingly, although HDAC4 was efficiently induced upon nerve injury, synaptic gene up-regulation was abrogated in DM1 muscle, together with a reduced increase in AChR turnover. This suggested that HDAC4-independent mechanisms lead to the defective response to denervation in DM1 muscle. Our study shows that activity-dependent signalling pathways are disturbed in DM1 muscle, which may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.