Quadriceps Biopsy Research Articles

Super-relaxed myosins contribute to respiratory muscle hibernation in mechanically ventilated patients.

Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients (N = 54) to those of non-ICU patients undergoing thoracic surgery (N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.

Skeletal muscle vulnerability in a child with Pitt-Hopkins syndrome

BackgroundTCF4 acts as a transcription factor that binds to the immunoglobulin enhancer Mu-E5/KE5 motif. Dominant variants in TCF4 are associated with the manifestation of Pitt-Hopkins syndrome, a rare disease characterized by severe mental retardation, certain features of facial dysmorphism and, in many cases, with abnormalities in respiratory rhythm (episodes of paroxysmal tachypnea and hyperventilation, followed by apnea and cyanosis). Frequently, patients also develop epilepsy, microcephaly, and postnatal short stature. Although TCF4 is expressed in skeletal muscle and TCF4 seems to play a role in myogenesis as demonstrated in mice, potential myopathological findings taking place upon the presence of dominant TCF4 variants are thus far not described in human skeletal muscle.MethodTo address the pathological effect of a novel deletion affecting exons 15 and 16 of TCF4 on skeletal muscle, histological and immunofluorescence studies were carried out on a quadriceps biopsy in addition to targeted transcript studies and global proteomic profiling.ResultsWe report on muscle biopsy findings from a Pitt-Hopkins patient with a novel heterozygous deletion spanning exon 15 and 16 presenting with neuromuscular symptoms. Microscopic characterization of the muscle biopsy revealed moderate fiber type I predominance, imbalance in the proportion of fibroblasts co-expressing Vimentin and CD90, and indicate activation of the complement cascade in TCF4-mutant muscle. Protein dysregulations were unraveled by proteomic profiling. Transcript studies confirmed a mitochondrial vulnerability in muscle and confirmed reduced TCF4 expression.ConclusionOur combined findings, for the first time, unveil myopathological changes as phenotypical association of Pitt-Hopkins syndrome and thus expand the current clinical knowledge of the disease as well as support data obtained on skeletal muscle of a mouse model.

Amyloid Myopathy as Initial Manifestation of ATTRwt: A Rare Phenomenon (P9-8.007)

<h3>Objective:</h3> To describe a rare presentation of amyloid myopathy. <h3>Background:</h3> Wild-type transthyretin amyloidosis (ATTRwt) is an age-related progressive disease with limited treatment options. Cardiomyopathy and peripheral neuropathy are well-recognized manifestations of transthyretin (ATTRwt) amyloidosis but myopathy is rarely the initial presentation. <h3>Design/Methods:</h3> NA <h3>Results:</h3> An 80-year-old man presented with slowly progressive bilateral lower limb weakness for over 5 years making him bedridden for one year. He denied any oculo-bulbar, sensory, or autonomic symptoms and reported no family history of neuromuscular disease. Physical examination revealed severe symmetric weakness of hip flexors and quadriceps and mild weakness of proximal arms bilaterally. Laboratory workup showed normal CK and no myositis antibodies were detected. Screen for monoclonal protein was negative. EMG performed was consistent with an irritable chronic myopathic process predominantly affecting the proximal leg muscles, along with a mild length-dependent peripheral neuropathy. Right quadriceps biopsy revealed fairly diffuse atrophic changes associated with focal amyloid deposition within adipose tissue and the blood vessel wall without any inflammation. Mass spectrometry suggested ATTR type amyloid and TTR genetic testing was negative confirming diagnosis of ATTRwt amyloid myopathy. A cardiac PYP scan showed diffusely increased myocardial uptake of the radiotracer. Patient was started on Tafamidis by cardiologist to slow disease progression. <h3>Conclusions:</h3> Neurological manifestations of ATTRwt are being increasingly recognized but myopathy is rarely reported as an initial presentation. The clinical presentation of myopathy can mimic other relatively common neuromuscular disorders and hence a high index of suspicion and through workup is needed to confirm diagnosis. Patients can have concomitant cardiomyopathy, which is potentially treatable, and therefore increased awareness is required. Supportive therapy remains the mainstay of therapy for neurological involvement, but future studies can pave the way for targeted treatments. <b>Disclosure:</b> Dr. Bovino has nothing to disclose. Dr. Young has nothing to disclose. Peter Pytel has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for Colianni&amp;Colianni. Peter Pytel has received publishing royalties from a publication relating to health care. Dr. Nadkarni has nothing to disclose. Dr. Paul has nothing to disclose.

Depressed Protein Synthesis and Anabolic Signaling Potentiate ACL Tear–Resultant Quadriceps Atrophy

Background: Anterior cruciate ligament (ACL) tear (ACLT) leads to protracted quadriceps muscle atrophy. Protein turnover largely dictates muscle size and is highly responsive to injury and loading. Regulation of quadriceps molecular protein synthetic machinery after ACLT has largely been unexplored, limiting development of targeted therapies. Purpose: To define the effect of ACLT on (1) the activation of protein synthetic and catabolic signaling within quadriceps biopsy specimens from human participants and (2) the time course of alterations to protein synthesis and its molecular regulation in a mouse ACL injury model. Study Design: Descriptive laboratory study. Methods: Muscle biopsy specimens were obtained from the ACL-injured and noninjured vastus lateralis of young adult humans after an overnight fast (N = 21; mean ± SD, 19 ± 5 years). Mice had their limbs assigned to ACLT or control, and whole quadriceps were collected 6 hours or 1, 3, or 7 days after injury with puromycin injected before tissue collection for assessment of relative protein synthesis. Muscle fiber size and expression and phosphorylation of protein anabolic and catabolic signaling proteins were assessed at the protein and transcript levels (RNA sequencing). Results: Human quadriceps showed reduced phosphorylation of ribosomal protein S6 (–41%) in the ACL-injured limb (P = .008), in addition to elevated phosphorylation of eukaryotic initiation factor 2α (+98%; P = .006), indicative of depressed protein anabolic signaling in the injured limb. No differences in E3 ubiquitin ligase expression were noted. Protein synthesis was lower at 1 day (P = .01 vs control limb) and 3 days (P = .002 vs control limb) after ACLT in mice. Pathway analyses revealed shared molecular alterations between human and mouse quadriceps after ACLT. Conclusion: (1) Global protein synthesis and anabolic signaling deficits occur in the quadriceps in response to ACL injury, without notable changes in measured markers of muscle protein catabolism. (2) Importantly, these deficits occur before the onset of significant atrophy, underscoring the need for early intervention. Clinical Relevance: These findings suggest that blunted protein anabolism as opposed to increased catabolism likely mediates quadriceps atrophy after ACL injury. Thus, future interventions should aim to restore muscle protein anabolism rapidly after ACLT.

FP.16 The effects of resistance exercise training on mitochondrial myopathy patients

Patients with single, large-scale mitochondrial DNA (mtDNA) deletions often present with mitochondrial myopathy (MM) for which no treatment is available. Exercise intervention induces beneficial effects in mitochondrial myopathy. However, the mechanisms for this are not yet understood. The aim of this study is to investigate genetic, biochemical and molecular changes triggered by 16-week resistance exercise training for the development of exercise mimetic drugs for the cure of MM patients. Quadriceps biopsies were taken from a cohort of single, large-scale mtDNA deletion patients before and after 16-week resistance exercise training. Biopsies were assessed for changes after exercise in oxidative phosphorylation (OXPHOS) staining for Laminin, VDAC1, Ndufb8 and COX1; fibre type distribution, size and contraction force staining for Myh7, Myh2 and Myh1; heteroplasmy using real-time PCR for MT-ND4, MT-CO1, MT-CO3 and MT-CYB genes; differentially expressed (DE) genes and related protein level using RNA-sequencing and imaging mass cytometry, respectively. Myopathy score is a strong predictor of resistance exercise training responsiveness. Compared to healthy controls, all patients present with a significantly lower contraction force of SKM fibres (p=0.003) and differential fibre type distribution with increased type 2a (p<0.04) and decreased fibre I (p<0.0001). After exercise, all cases show an increase in the proportion of fibres with higher level of COX1 (p<0.0012) and increase of Myh7⁺ fibre type size (all patients except P2, p < 0.0001). The majority of patients show a significant increase of mitochondrial mass that positively correlates with the increase of aerobic capability (R=0.81). Furthermore, 13,132 DE genes were identified between pre- and post-exercise cases, of which 40 DE genes were selected with p<0.05 and logarithmic fold change <1.5 or >1.5. Among these, 13 novel exercise-modulated targets were established with an increased protein level localising within the SKM fibres (e.g. Dock11, Edil3, Ikbkg, Gem). Further analysis will assess the protein level of targets with nuclear, perinuclear and extracellular localisation in SKM of MM patients. Resistance exercise training is effective in reducing the OXPHOS defects and induces SKM remodelling in MM patients. The identification of novel exercise-modulated targets may provide insight for the development of exercise mimetic drugs for the cure of mitochondrial myopathy patients.

A.3 A novel recessive TNNT1 congenital core-rod myopathy in French Canadians

Background: Mutations in the slow skeletal muscle troponin T (TNNT1) gene cause a congenital nemaline myopathy resulting in death from respiratory insufficiency in early infancy. We report on four French Canadians with a novel congenital TNNT1 myopathy. Methods: Patients underwent lower extremity and paraspinal MRI, quadriceps biopsy and genetic testing. TNNT1 expression in muscle was assessed by quantitative PCR and immunoblotting. Wild type or mutated TNNT1 mRNAs were co-injected with morpholinos in a zebrafish knockdown model to assess for rescue of the morphant phenotype. Results: Four patients shared a novel missense homozygous mutation in TNNT1. They developed from childhood slowly progressive limb-girdle weakness with spinal rigidity and contractures. They suffered from restrictive lung disease and recurrent episodes of rhabdomyolysis. Older patients remained ambulatory into their sixties. Lower extremity MRI showed symmetrical myopathic changes. Paraspinal MRI showed diffuse fibro-fatty involution. Biopsies showed multi-minicores. Nemaline rods were seen in half the patients. TNNT1 mRNA expression was similar in controls and patients, while levels of TNNT1 protein were reduced in patients. Wild type TNNT1 mRNA rescued the zebrafish morphants but mutant transcripts failed to do so. Conclusions: This study expands the spectrum of TNNT1-related myopathy to include a milder clinical phenotype caused by a functionally-confirmed novel mutation.

Anterior Cruciate Ligament Reconstruction Upregulates Vitamin D Activating Pathways within the Quadriceps

Anterior cruciate ligament (ACL) tears are prevalent sport‐related injuries that result in protracted quadriceps atrophy and weakness despite surgical reconstruction and physical therapy. Vitamin D and its receptor are integral for skeletal muscle development and homeostasis. In addition, vitamin D status is positively associated with muscle size and strength. If the vitamin D receptor and related enzymes are upregulated with ACL injury and reconstruction, then overall vitamin D status may be an important effector of recovery. Concentrations of vitamin D needed to support optimal recovery may differ from typical clinical cut points used to diagnose vitamin D deficiency, which could have powerful therapeutic implications following ACL injury and reconstruction. This is especially important given recent research showing over 50% of athletes have vitamin D inadequacy. We hypothesized that subjects with circulating vitamin D sufficient to suppress parathyroid hormone secretion (≥ 40 ng/mL) would be better protected against quadriceps atrophy at the conclusion of rehabilitation. Muscle biopsies (vastus lateralis) were obtained from ACL‐injured and Healthy limbs of 16 participants (6M,10F; 19±5yr, body mass index: 25.1±3.7kg/m2) prior to surgical reconstruction, 1 week following reconstruction (Recon), and following the completion of post‐surgical rehabilitation (6 months following surgery, Post‐Rehab). RNA was isolated from all quadriceps biopsies, followed by library construction, sequencing, and bioinformatics analysis. Transcript abundance of vitamin D activating pathway regulators (vitamin D receptor [VDR], and Cytochrome P450 2R1 [CYP2R1] and Family 27 Subfamily B Member 1 [CYP27B1]) was assessed following analysis using DESeq2 and false discovery rate (FDR) adjustment of p‐values. Additionally, 25‐hydroxy vitamin D (25(OH)D) was assessed in serum samples obtained at the time of reconstruction surgery. Quadriceps fiber cross‐sectional area was measured via laminin immunohistochemistry. Recon (1 week post‐surgery) muscle showed increased transcript abundance of the vitamin D receptor, VDR(elevated 7.8 fold, FDR&lt;0.05), and vitamin D‐activating pathway regulators CYP2R1 (elevated 1.5 fold, FDR&lt;0.05) and CYP27B1 (elevated 2.6 fold, FDR&lt;0.05) when compared to the Healthy limb. Following the completion of rehabilitation at the 6‐month follow‐up, participants with 25(OH)D above 40 ng/mL showed 6.4% atrophy of quadriceps fiber size compared to 22.8% atrophy in participants with 25(OH)D below 40 ng/mL (p=0.06). These findings provide evidence for surgically induced elevations in the vitamin D activating pathway within skeletal muscle. Upregulation of the vitamin D activating pathway may support quadriceps mitochondrial health and satellite cell activity and self‐renewal capacity, both of which are compromised following ACL reconstruction. In addition, elevated vitamin D concentrations may protect against protracted quadriceps atrophy. Future research should investigate if manipulation of vitamin D concentrations can enhance quadriceps functional recovery following ACL reconstruction.

MicroRNA regulatory networks associated with abnormal muscle repair in survivors of critical illness.

BackgroundIntensive care unit (ICU)‐acquired weakness is characterized by muscle atrophy and impaired contractility that may persist after ICU discharge. Dysregulated muscle repair and regeneration gene co‐expression networks are present in critical illness survivors with persistent muscle wasting and weakness. We aimed to identify microRNAs (miRs) regulating the gene networks and determine their role in the self‐renewal of muscle in ICU survivors.MethodsMuscle whole‐transcriptome expression was assessed with microarrays in banked quadriceps biopsies obtained at 7 days and 6 months post‐ICU discharge from critically ill patients (n = 15) in the RECOVER programme and healthy individuals (n = 8). We conducted an integrated miR–messenger RNA analysis to identify miR/gene pairs associated with muscle recovery post‐critical illness and evaluated their impact on myoblast proliferation and differentiation in human AB1167 and murine C2C12 cell lines in vitro. Select target genes were validated with quantitative PCR.ResultsTwenty‐two miRs were predicted to regulate the Day 7 post‐ICU muscle transcriptome vs. controls. Thirty per cent of all differentially expressed genes shared a 3'UTR regulatory sequence for miR‐424‐3p/5p, which was 10‐fold down‐regulated in patients (P < 0.001) and correlated with quadriceps size (R = 0.86, P < 0.001), strength (R = 0.75, P = 0.007), and physical function (Functional Independence Measures motor subscore, R = 0.92, P < 0.001) suggesting its potential role as a master regulator of early recovery of muscle mass and strength following ICU discharge. Network analysis demonstrated enrichment for cellular respiration and muscle fate commitment/development related genes. At 6 months post‐ICU discharge, a 14‐miR expression signature, including miRs‐490‐3p and ‐744‐5p, identified patients with muscle mass recovery vs. those with sustained atrophy. Constitutive overexpression of the novel miR‐490‐3p significantly inhibited AB1167 and C2C12 myoblast proliferation (cell count AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 7926 ± 4060 vs. 14 159 ± 3515 respectively, P = 0.006; proportion Ki67‐positive nuclei AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 0.38 ± 0.07 vs. 0.54 ± 0.06 respectively, P < 0.001; proliferating cell nuclear antigen expression AB1167 miR‐490‐3p mimic or scrambled‐miR transfected myoblasts 11.48 ± 1.97 vs. 16.75 ± 1.19 respectively, P = 0.040). Constitutive overexpression of miR‐744‐5p, a known regulator of myogenesis, significantly inhibited AB1167 and C2C12 myoblast differentiation (fusion index AB1167 miR‐744‐5p mimic or scrambled‐miR transfected myoblasts 8.31 ± 7.00% vs. 40.29 ± 9.37% respectively, P < 0.001; myosin heavy chain expression miR‐744‐5p mimic or scrambled‐miR transfected myoblasts 0.92 ± 0.39 vs. 13.53 ± 5.5 respectively, P = 0.01).ConclusionsCombined functional transcriptomics identified 36 miRs including miRs‐424‐3p/5p, ‐490‐3p, and ‐744‐5p as potential regulators of gene networks associated with recovery of muscle mass and strength following critical illness. MiR‐490‐3p is identified as a novel regulator of myogenesis.

A.4 A Novel Recessive TNNT1 Congenital Core-Rod Myopathy in French Canadians

Background: Mutations in the slow skeletal muscle troponin T (TNNT1) gene cause a congenital nemaline myopathy resulting in death from respiratory insufficiency in early infancy. We report on four French Canadians with a novel congenital TNNT1 myopathy. Methods: Patients underwent lower extremity and paraspinal MRI, quadriceps biopsy and genetic testing. TNNT1 expression in muscle was assessed by quantitative PCR and immunoblotting. Wild type or mutated TNNT1 mRNAs were co-injected with morpholinos in a zebrafish knockdown model to assess for rescue of the morphant phenotype. Results: Four patients shared a novel missense homozygous mutation in TNNT1. They developed from childhood slowly progressive limb-girdle weakness with spinal rigidity and contractures. They suffered from restrictive lung disease and recurrent episodes of rhabdomyolysis. Older patients remained ambulatory into their sixties. Lower extremity MRI showed symmetrical myopathic changes. Paraspinal MRI showed diffuse fibro-fatty involution. Biopsies showed multi-minicores. Nemaline rods were seen in half the patients. TNNT1 mRNA expression was similar in controls and patients, while levels of TNNT1 protein were reduced in patients. Wild type TNNT1 mRNA rescued the zebrafish morphants but mutant transcripts failed to do so. Conclusions: This study expands the spectrum of TNNT1-related myopathy to include a milder clinical phenotype caused by a functionally-confirmed novel mutation.

Skeletal muscle regeneration in facioscapulohumeral muscular dystrophy is correlated with pathological severity.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant myopathy characterized by slowly progressive skeletal muscle weakness and wasting. While a regenerative response is often provoked in many muscular dystrophies, little is known about whether a regenerative response is regularly elicited in FSHD muscle, prompting this study. For comparison, we also examined the similarly slowly progressing myotonic dystrophy type 2 (DM2). To first investigate regeneration at the transcriptomic level, we used the 200 human gene Hallmark Myogenesis list. This myogenesis biomarker was elevated in FSHD and control healthy myotubes compared to their myoblast counterparts, so is higher in myogenic differentiation. The myogenesis biomarker was also elevated in muscle biopsies from most independent FSHD, DM2 or Duchenne muscular dystrophy (DMD) studies compared to control biopsies, and on meta-analysis for each condition. In addition, the myogenesis biomarker was a robust binary discriminator of FSHD, DM2 and DMD from controls. We also analysed muscle regeneration at the protein level by immunolabelling muscle biopsies for developmental myosin heavy chain. Such immunolabelling revealed one or more regenerating myofibres in 76% of FSHD muscle biopsies from quadriceps and 91% from tibialis anterior. The mean proportion of regenerating myofibres per quadriceps biopsy was 0.48%, significantly less than 1.72% in the tibialis anterior. All DM2 muscle biopsies contained regenerating myofibres, with a mean of 1.24% per biopsy. Muscle regeneration in FSHD was correlated with the pathological hallmarks of fibre size variation, central nucleation, fibrosis and necrosis/regeneration/inflammation. In summary, the regenerative response in FSHD muscle biopsies correlates with the severity of pathology.

P.236Myofibres with subsarcolemmal rims and/or central aggregates of mitochondria (SRCAM) are prevalent in congenital titinopathies

With increasing use of massive parallel sequencing over recent years, congenital titinopathies due to recessively inherited truncating and/or splice-site pathogenic variants in TTN (which encodes titin) are emerging as a common cause of congenital myopathy (CM). The clinical phenotype is broad. Muscle biopsies show structural abnormalities including cores, central nuclei (CN) and fibre size disproportion. We report a novel structural abnormality prevalent in muscle biopsies from a cohort of patients with clinical and/or pathological features (cores and/or central nuclei and slow fibre predominance) of CM. Cases were classified genetically as "definite" (2 predicted pathogenic TTN variants), "probable" (1 predicted pathogenic and 1 missense variant) and ''possible'' (1 predicted pathogenic or 2 missense variants) titinopathy. The index case, a male with recessive splice-site TTN variants in trans, presented at birth, with profound hypotonia, respiratory weakness and cardiomegaly. CK was normal. EMG was myopathic. Quadriceps biopsy at 2 months showed mild CN and ill-defined cores. A novel observation concerned a striking sub-population of fibres, often smaller, with circumferential rims with/without central aggregates of oxidative staining. These were designated as myofibres with subsarcolemmal rims and/or central aggregates of mitochondria (SRCAM fibres). We subsequently identified SRCAM fibres in 35.7% of genetically definite or probable/possible TTN-CM cases (10/28 total; 4/9 definite, 2/3 probable and 4/16 possible). In stark contrast, SRCAM fibres were present in only 3.6% (2/56) of cases with pathogenic variants in other core/CN-CM genes (RYR1, SEPN1, MYH7, MYH2, DNM2, MTM1). They were most frequent in neonates. Analysis of additional cases and ultrastructural characterisation is ongoing. We recommend that patients with a clinicopathological picture compatible with CM and presence of SRCAM fibres in their muscle biopsy should be screened for TTN mutations.

P.028 A milder congenital myopathy in the french canadians caused by a novel TNNT1 homozygous missense mutation

Background: Mutations of the slow skeletal muscle troponin-T1 (TNNT1) gene are a rare cause of nemaline myopathy. The phenotype is characterized by severe amyotrophy and contractures. Death from respiratory insufficiency occurs in infancy. We report on four French Canadians with a novel congenital TNNT1-related myopathy. Methods: Patients underwent MRI of leg muscles, quadriceps biopsy and genetic testing. Wild type or mutated human TNNT1 mRNAs were co-injected with morpholinos in a zebrafish knockdown model to assess their relative abilities to rescue the morphant phenotype. Results: Three adults and one child shared a novel missense homozygous pathogenic variant in the TNNT1 gene. They developed from childhood slowly progressive limb-girdle weakness with spinal rigidity and contractures. They suffered from restrictive lung disease and recurrent episodes of infection-triggered rhabdomyolysis, which were relieved by dantrolene in one patient. Older patients remained ambulatory into their sixties. MRI of leg muscles showed symmetrical atrophy and fatty infiltration in a proximal-to-distal gradient. Biopsies showed multi-minicores, while nemaline rods were seen in half the patients. Wild type TNNT1 mRNA rescued the zebrafish morphants but mutant transcripts failed to rescue the morphants. Conclusions: This study expands the spectrum of TNNT1-related myopathy to include a milder clinical phenotype caused by a functionally-confirmed novel missense mutation.

CONGENITAL MUSCULAR DYSTROPHIES: P.334Recessive loss-of-function mutations in ITGA7 cause cardiac arrhythmia with or without structural cardiomyopathy and respiratory muscle weakness

Integrin α7 encoded by ITGA7 is highly expressed in skeletal and cardiac muscle and contributes to sarcolemmal stability by binding to laminin α2. Three unrelated patients were previously reported with ITGA7-linked congenital muscular dystrophy. Here, we report three patients from two unrelated families presenting with adult-onset cardiac arrhythmia and respiratory weakness due to recessive null mutations in ITGA7. Patient I, a 51-year-old male presented with delayed motor milestones, stridor since birth, cardiac arrhythmia (frequent ventricular ectopy, non-sustained ventricular tachycardia and premature conduction disease) requiring ICD at 46 years. Cardiac MRI showed basal septal hypertrophy and fibrosis. Examination showed focal wasting of medial gastrocnemii and respiratory impairment. EMG was myopathic. Two other male siblings also reported cardiac symptoms. Patient IIa, a 61-year-old female presented at 49 years with febrile respiratory distress requiring mechanical ventilation and tracheostomy. She had a history of atrial flutter, left bundle branch block and atrioventricular block requiring a pacemaker. She presented with further episodes of relapsing respiratory insufficiency. Examination showed mild weakness of ankle dorsiflexion (MRC 4+) and vocal cord paresis. EMG was myopathic. Muscle ultrasound showed widespread hyperechogenicity in the limb muscles. Patient IIb, her female sibling aged 55 presented with chronic hypoventilation. She had mild limb weakness. Presently she uses nocturnal non-invasive ventilation. Quadriceps biopsies (PI and PIIa) revealed non-specific myopathic changes. Next generation sequencing revealed a homozygous c.806_818del [p.S269fs] variant (PI) and two canonical splice site variants (PIIa, PIIb), (c.2357+1G>A [r.spl?] and c.2278-1G>A [r.spl?]) in ITGA7. Immunostaining revealed absent sarcolemmal integrin α7 labeling in both biopsies. Evaluation of α7-integrin-null mice showed a mild progressive myopathy with mainly diaphragmatic involvement and similar pathological features. Patients with predominant respiratory weakness and/or cardiac arrhythmias with or without structural cardiomyopathy should be screened for mutations in ITGA7.

CONGENITAL MYOPATHIES: NEMALINE AND TITINOPATHIES: P.237Congenital fatal cap-rod myopathy due to a de novo autosomal dominant pathogenic ACTA1 variant

Cap disease is a rare structural congenital myopathy (CM) associated with hypotonia, proximal and facial muscle weakness, and frequently scoliosis and respiratory involvement. Mutations in TPM2, TPM3 and ACTA1 have been associated with cap disease, as well as nemaline myopathy. Combined caps and nemaline rods have been reported in the same patient due to a mutation in TPM3. Here, we report the first case of a severe, fatal CM with distinct and separate caps and nemaline rods identified in skeletal muscle biopsies. The patient was born at 37 weeks of gestation, with a history of polyhydramnios, little spontaneous movements at birth, generalised hypotonia, and required immediate ventilatory support. He died 20 days after birth. Ante mortem and post mortem biopsies from the quadriceps, and post mortem biopsies from the biceps and diaphragm showed diffuse fibre hypotrophy without specific structural alterations on light microscopy. Ultrastructural examination of the ante mortem quadriceps biopsy showed several classical cap lesions, and a few of these contained small nemaline rods. The post mortem quadriceps sample showed nemaline rods in several fibres. Both samples showed mitochondrial paracrystalline inclusions in a few interstitial capillary endothelial cells. Ultrastructural findings were key in directing molecular genetic testing. A next generation sequencing panel for 35 congenital myopathy genes identified a de novo ACTA1 c.739G>C p.(Gly247Arg) variant previously reported in the literature in a patient with severe nemaline myopathy, affecting a highly conserved amino acid, and predicted to affect actin function with In Silico analysis. Our case of ACTA1-related cap-rod myopathy is the most severe presentation of a CM with caps or cap-rods described till date. The case further cements the notion of caps and rods being part of the 'nemaline spectrum' and highlights the remarkable heterogeneity of lesions within the same muscle or same group of muscles.

Endo Soc-3-Decrease in AMP Kinase Expression and Activity in Diabetic Patients with Hypogonadism—Increase following Testosterone Supplementation

AMP kinase is a key enzyme involved with energy regulation, the intake of macronutrients, the uptake of glucose and fatty acids by cells and the metabolic combustion of fatty acids. We hypothesized that in patients with hypogonadism associated with diabetes, its expression and activity are diminished since there is a quantitative and qualitative loss of skeletal muscle function. Twenty-two men with HH and T2DM were compared with 20 eugonadal men with T2DM at baseline. From the HH patients, 12 were treated with testosterone 200mg every 2 weeks injected intramuscularly for 24 weeks, during which their plasma concentrations were maintained in the physiological range. Hyperinsulinemic, euglycemic clamps (HEC) were carried out prior to and after testosterone replacement and fat (abdomen) and muscle (quadriceps) biopsies were carried out prior to and after HEC procedure on each occasion. In the hypogonadal state, the expression and the activity (measured as phoso-T172-AMPK-α) of AMP kinase-α was significantly lower in HH patients than eugonadal diabetics both in adipose tissue and skeletal muscle by 37% and 29%, respectively, for expression and by 22.1% and 28%, respectively for activity. Following testosterone, there was no change in expression but there was a significant increase in AMP kinase activity by 69±31% (from 14.9±2.8 to 22.8±2.2U/ml, p&amp;lt;0.05) in the muscle but not in the adipose tissue. At the end of the treatment and following the hyperinsulinemic euglycemic clamp, there was a significant increase in AMP kinase expression by 41±9% and 46±11% in adipose tissue and muscle, respectively, while there was only a modest increase (NS) in activity in both tissues. Clearly, therefore, testosterone is an important modulator of AMP kinase activity. This effect of testosterone may contribute to the increase in glucose uptake and an improvement in insulin sensitivity since AMP kinase also induces an increase in Akt-2 and GLUT-4 expression. Disclosure S. Dhindsa: None. H. Ghanim: None. M. Batra: Speaker's Bureau; Self; Eli Lilly and Company. A. Chaudhuri: Speaker's Bureau; Self; AstraZeneca, Eli Lilly and Company, Boehringer Ingelheim Pharmaceuticals, Inc., Merck &amp; Co., Inc., Novo Nordisk Inc.. S. Abuaysheh: None. P. Dandona: Advisory Panel; Self; AstraZeneca. Consultant; Self; AstraZeneca. Research Support; Self; AstraZeneca.

P.070 Autosomal dominant MARS mutation linked to severe early onset CMT2U

Background: Methionyl-tRNA synthetase (MARS) links methionine to its cognate tRNA required for translation. MARS mutations have been linked to adult-onset CMT2U. Methods: The proband had weakness in her first year of life, sitting at 11 months and walking at 20 months old. At 4 years old she was areflexic with distal &gt; proximal weakness. Nerve conduction studies showed normal median and sural sensory responses with absent common peroneal, low median and tibial motor amplitudes. EMG noted denervation and quadriceps biopsy revealed neurogenic atrophy. Genetic testing for spinal muscular atrophy and sequencing of MNF2, RAB7A, LMNA, MPZ, HSPB1, NEFL, GADP1, TRPV4, HSPB8, GJB1 and PLEK8G5 were negative. She stopped walking at 9 years old and could not raise her arms above her head at 11 years old. Results: Exome sequencing identified MARS: c.1189G&gt;A; p.Ala397Thr. To determine the functional consequences of p.A397T-MARS, yeast complementation assays were performed. Wild type or mutant MARS were cloned into yeast lacking the endogenous MARS ortholog. Wild-type MARS supported robust cellular growth, while the p.A397T-MARS insert did not support cellular growth confirming deleterious effect of this variant. Conclusions: Our patient’s phenotype was similar to children with motor-predominant GARS mutations. Functional data notes this MARS variant to be damaging and predictive of a severe, early-onset phenotype.

Anterior cruciate ligament tear induces a sustained loss of muscle fiber force production.

Patients with anterior cruciate ligament (ACL) tears have persistent quadriceps strength deficits that are thought to be due to altered neurophysiological function. Our goal was to determine the changes in muscle fiber contractility independent of the ability of motor neurons to activate fibers. We obtained quadriceps biopsies of patients undergoing ACL reconstruction, and additional biopsies 1, 2, and 6 months after surgery. Muscles fiber contractility was assessed in vitro, along with whole muscle strength testing. Compared with controls, patients had a 30% reduction in normalized muscle fiber force at the time of surgery. One month later, the force deficit was 41%, and at 6 months the deficit was 23%. Whole muscle strength testing demonstrated similar trends. While neurophysiological dysfunction contributes to whole muscle weakness, there is also a reduction in the force generating capacity of individual muscle cells independent of alpha motor neuron activation. Muscle Nerve, 2018.

PRES leading to the diagnosis of McArdle disease

A 35year-old male developed myalgias after moving furniture and was hospitalized with acute renal failure and rhabdomyolysis requiring hemodialysis. He then had several generalized tonic-clonic seizures. Brain MRI showed findings of posterior reversible encephalopathy syndrome (PRES). Interval history revealed easy fatigability and exercise-induced myalgias in childhood but no preceding history of urine discoloration. Quadriceps biopsy showed absent muscle myophosphorylase reactivity consistent with the diagnosis of McArdle disease. With supportive care he improved and the PRES resolved. This case represents an extreme in the spectrum of complications that can occur in McArdle disease, including downstream central nervous system involvement and highlights the importance of early recognition and aggressive management of rhabdomyolysis.

Using laser capture microdissection to study fiber specific signaling in locomotor muscle in COPD: A pilot study.

Quadriceps dysfunction is important in chronic obstructive pulmonary disease (COPD), with an associated increased proportion of type II fibers. Investigation of protein synthesis and degradation has yielded conflicting results, possibly due to study of whole biopsy samples, whereas signaling may be fiber-specific. Our objective was to develop a method for fiber-specific gene expression analysis. 12 COPD and 6 healthy subjects underwent quadriceps biopsy. Cryosections were immunostained for type II fibers, which were separated using laser capture microdissection (LCM). Whole muscle and different fiber populations were subject to quantitative polymerase chain reaction. Levels of muscle-RING-finger-protein-1 and Atrogin-1 were lower in type II fibers of COPD versus healthy subjects (P = 0.02 and P = 0.03, respectively), but differences were not apparent in whole muscle or type I fibers. We describe a novel method for studying fiber-specific gene expression in optimum cutting temperature compound-embedded muscle specimens. LCM offers a more sensitive way to identify molecular changes in COPD muscle. Muscle Nerve 55: 902-912, 2017.

P.404 - Muscle fibre size revisited: Updated age-stratified normative data in histologically normal/minimal change (HN/MC) paediatric quadriceps biopsies using a high-throughput automated digital script

P.404 - Muscle fibre size revisited: Updated age-stratified normative data in histologically normal/minimal change (HN/MC) paediatric quadriceps biopsies using a high-throughput automated digital script