Long-term monitoring of stimulated lower leg skeletal muscle forces compared with voluntary contractions in myopathy patients - A five-year follow-up report on 5 adults.

OBJECTIVE: Long-term assessments of lower leg muscle forces in ambulant patients with distal myopathies. METHODS AND MATERIALS: Over a five-year period, we measured involuntary, nerve-stimulated, isometric torques of the ankle dorsiflexors in a group of ambulant patients with myopathies and compared results with voluntary Manual Muscle Tests (MMT). RESULTS: From ten recruited patients, five could finish the five-year protocol. Twenty-seven force measurements sessions (one per year; 1,5 hours duration each) were performed. These patients exhibited low, stable torques or increased minimally (0.2 Newtonmeter, versus 0.1 Nm, ns; 0.7 vs. 1.0, ns; 3.4 vs. 3.5, ns; 0.2 vs. 0.1, ns; 0.8 vs. 1.5, P 0.0004 initial values vs. 5-year values, [norm: 3.9-5.7 Nm]). A 6th patient, eliciting low torque values (0.1 Nm) early passed away. Contraction times inversely correlated with MMT. MMT provided similar overall force abilities. CONCLUSIONS: Long-term monitoring of lower leg muscle forces in ambulant patients is limited by the patient's health status. In a small group of patients, stimulated lower leg forces did not worsen over many years relative to their diagnosed myopathies. Tracking involuntary forces, could be a useful monitoring providing phenotypic information, in addition to MMT. Future devices should be small and be simply self-applying, designed for subjects' domestic use and web-based data transfer. CLINICALTRIALS: gov NCT00735384.

Cellular and molecular alterations to muscles and neuromuscular synapses in a mouse model of MEGF10-related myopathy.

Loss-of-function mutations in MEGF10 lead to a rare and understudied neuromuscular disorder known as MEGF10-related myopathy. There are no treatments for the progressive respiratory distress, motor impairment, and structural abnormalities in muscles caused by the loss of MEGF10 function. In this study, we deployed cellular and molecular assays to obtain additional insights about MEGF10-related myopathy in juvenile, young adult, and middle-aged Megf10 knockout (KO) mice. We found fewer muscle fibers in juvenile and adult Megf10 KO mice, supporting published studies that MEGF10 regulates myogenesis by affecting satellite cell differentiation. Interestingly, muscle fibers do not exhibit morphological hallmarks of atrophy in either young adult or middle-aged Megf10 KO mice. We next examined the neuromuscular junction (NMJ), in which MEGF10 has been shown to concentrate postnatally, using light and electron microscopy. We found early and progressive degenerative features at the NMJs of Megf10 KO mice that include increased postsynaptic fragmentation and presynaptic regions not apposed by postsynaptic nicotinic acetylcholine receptors. We also found perisynaptic Schwann cells intruding into the NMJ synaptic cleft. These findings strongly suggest that the NMJ is a site of postnatal pathology in MEGF10-related myopathy. In support of these cellular observations, RNA-seq analysis revealed genes and pathways associated with myogenesis, skeletal muscle health, and NMJ stability dysregulated in Megf10 KO mice compared to wild-type mice. Altogether, these data provide new and valuable cellular and molecular insights into MEGF10-related myopathy.

Type selective ablation of postnatal slow and fast fatigue-resistant motor neurons in mice induces late onset kinetic and postural tremor following fiber-type transition and myopathy.

Animals on Earth need to hold postures and execute a series of movements under gravity and atmospheric pressure. VAChT-Cre is a transgenic Cre driver mouse line that expresses Cre recombinase selectively in motor neurons of S-type (slow-twitch fatigue-resistant) and FR-type (fast-twitch fatigue-resistant). Sequential motor unit recruitment is a fundamental principle for fine and smooth locomotion; smaller-diameter motor neurons (S-type, FR-type) first contract low-intensity oxidative type I and type IIa muscle fibers, and thereafter larger-diameter motor neurons (FInt-type, FF-type) are recruited to contract high-intensity glycolytic type IIx and type IIb muscle fibers. To selectively eliminate S- and FR-type motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which the cytotoxic diphtheria toxin A fragment (DTA) was expressed in Cre-expressing neurons. The VAChT-Cre;NSE-DTA mice were born normally but progressively manifested various characteristics, including body weight loss, kyphosis, kinetic and postural tremor, and muscular atrophy. The progressive kinetic and postural tremor was remarkable from around 20 weeks of age and aggravated. Muscular atrophy was apparent in slow muscles, but not in fast muscles. The increase in motor unit number estimation was detected by electromyography, reflecting compensatory re-innervation by remaining FInt- and FF-type motor neurons to the orphaned slow muscle fibers. The muscle fibers gradually manifested fast/slow hybrid phenotypes, and the remaining FInt-and FF-type motor neurons gradually disappeared. These results suggest selective ablation of S- and FR-type motor neurons induces progressive muscle fiber-type transition, exhaustion of remaining FInt- and FF-type motor neurons, and late-onset kinetic and postural tremor in mice.

Role of microenvironment on muscle stem cell function in health, adaptation, and disease.

The role of the cellular microenvironment has recently gained attention in the context of muscle health, adaption, and disease. Emerging evidence supports major roles for the extracellular matrix (ECM) in regeneration and the dynamic regulation of the satellite cell niche. Satellite cells normally reside in a quiescent state in healthy muscle, but upon muscle injury, they activate, proliferate, and fuse to the damaged fibers to restore muscle function and architecture. This chapter reviews the composition and mechanical properties of skeletal muscle ECM and the role of these factors in contributing to the satellite cell niche that impact muscle regeneration. In addition, the chapter details the effects of satellite cell-matrix interactions and provides evidence that there is bidirectional regulation affecting both the cellular and extracellular microenvironment within skeletal muscle. Lastly, emerging methods to investigate satellite cell-matrix interactions will be presented.

Muscle Properties, Gross Motor Performance, and Quality of Life in Children With Sickle Cell Disease.

PURPOSE: To explore muscle properties, gross motor performance, and quality of life (QoL) in children with sickle cell disease (SCD) compared with controls and to assess relationships among these outcomes. METHODS: A cross-sectional study of 24 children assessed muscle properties including: knee extension strength by dynamometry; vastus lateralis (VL) and rectus femoris (RF) muscle thickness by ultrasonography; and VL and RF neuromuscular activation (rate of muscle activation [RoA]) by electromyography (EMG). Gross motor performance and QoL were assessed by standardized tests and questionnaires. RESULTS: Children with SCD had impaired knee extension strength, VL EMG RoA, gross motor performance, and QoL compared with children without SCD. Relationships among muscle properties, gross motor performance, and QoL were identified. CONCLUSIONS: These findings indicate that comprehensive muscle properties, gross motor performance, and QoL assessments should be considered to support and develop individualized physical therapy plans for children with SCD.

Identification and Evaluation of Traditional Chinese Medicine Natural Compounds as Potential Myostatin Inhibitors: An In Silico Approach.

Myostatin (MSTN), a negative regulator of muscle mass, is reported to be increased in conditions linked with muscle atrophy, sarcopenia, and other muscle-related diseases. Most pharmacologic approaches that treat muscle disorders are ineffective, emphasizing the emergence of MSTN inhibition. In this study, we used computational screening to uncover natural small bioactive inhibitors from the Traditional Chinese Medicine database (~38,000 compounds) for the MSTN protein. Potential ligands were screened, based on binding affinity (150), physicochemical (53) and ADMET properties (17). We found two hits (ZINC85592908 and ZINC85511481) with high binding affinity and specificity, and their binding patterns with MSTN protein. In addition, molecular dynamic simulations were run on each complex to better understand the interaction mechanism of MSTN with the control (curcumin) and the hit compounds (ZINC85592908 and ZINC85511481). We determined that the hits bind to the active pocket site (Helix region) and trigger conformational changes in the MSTN protein. Since the stability of the ZINC85592908 compound was greater than the MSTN control, we believe that ZINC85592908 has therapeutic potential against the MSTN protein and may hinder downstream singling by inhibiting the MSTN protein and increasing myogenesis in the skeletal muscle tissues.

Evaluation of Muscle Mass and Stiffness with Limb Ultrasound in COVID-19 Survivors.

BACKGROUND: acute illnesses, like COVID-19, can act as a catabolic stimulus on muscles. So far, no study has evaluated muscle mass and quality through limb ultrasound in post-COVID-19 patients. METHODS: cross sectional observational study, including patients seen one month after hospital discharge for SARS-CoV-2 pneumonia. The patients underwent a multidimensional evaluation. Moreover, we performed dominant medial gastrocnemius ultrasound (US) to characterize their muscle mass and quality. RESULTS: two hundred fifty-nine individuals (median age 67, 59.8% males) were included in the study. COVID-19 survivors with reduced muscle strength had a lower muscle US thickness (1.6 versus 1.73 cm, p =0.02) and a higher muscle stiffness (87 versus 76.3, p = 0.004) compared to patients with normal muscle strength. Also, patients with reduced Short Physical Performance Battery (SPPB) scores had a lower muscle US thickness (1.3 versus 1.71 cm, p = 0.01) and a higher muscle stiffness (104.9 versus 81.07, p = 0.04) compared to individuals with normal SPPB scores. The finding of increased muscle stiffness was also confirmed in patients with a pathological value (≥ 4) at the sarcopenia screening tool SARC-F (103.0 versus 79.55, p < 0.001). Muscle stiffness emerged as a significant predictor of probable sarcopenia (adjusted OR 1.02, 95% C.I. 1.002 - 1.04, p = 0.03). The optimal ultrasound cut-offs for probable sarcopenia were 1.51 cm for muscle thickness (p= 0.017) and 73.95 for muscle stiffness (p = 0.004). DISCUSSION: we described muscle ultrasound characteristics in post COVID-19 patients. Muscle ultrasound could be an innovative tool to assess muscle mass and quality in this population. Our preliminary findings need to be confirmed by future studies comparing muscle ultrasound with already validated techniques for measuring muscle mass and quality.

Protocol for accelerated skeletal muscle regeneration and hypertrophic muscle formation in mice.

The skeletal muscle system is the major organ associated with movement of the body. Myogenesis and regeneration induced post-injury contribute to muscle formation and maintenance. Here, we provide detailed protocol for the accelerated repair of injured skeletal muscles and generation of hypertrophic muscle fibers. This protocol includes cardiotoxin induced muscle injury and also describes isolation of satellite cells from skeletal muscle tissues of mice. This protocol can be used to study the mechanisms associated with accelerated muscle repair and hypertrophy. For complete details on the use and execution of this protocol, please refer to Ray et al. (2021).

Association between Levocarnitine Treatment and the Change in Knee Extensor Strength in Patients Undergoing Hemodialysis: A Post-Hoc Analysis of the Osaka Dialysis Complication Study (ODCS).

Carnitine deficiency is prevalent in patients undergoing hemodialysis, and it could result in lowered muscle strength. So far, the effect of treatment with levocarnitine on lower limb muscle strength has not been well described. This observational study examined the association between treatment with levocarnitine with the change in knee extensor strength (KES) in hemodialysis patients. Eligible patients were selected from the participants enrolled in a prospective cohort study for whom muscle strength was measured annually. We identified 104 eligible patients for this analysis. During the one-year period between 2014 to 2015, 67 patients were treated with intravenous levocarnitine (1000 mg per shot, thrice weekly), whereas 37 patients were not. The change in KES was significantly higher (p = 0.01) in the carnitine group [0.02 (0.01-0.04) kgf/kg] as compared to the non-carnitine group [-0.02 (-0.04 to 0.01) kgf/kg]. Multivariable-adjusted regression analysis showed the positive association between the change in KES and the treatment with levocarnitine remained significant after adjustment for the baseline KES and other potential confounders. Thus, treatment with intravenous levocarnitine was independently and positively associated with the change in KES among hemodialysis patients. Further clinical trials are needed to provide more solid evidence.

TLE4 regulates muscle stem cell quiescence and skeletal muscle differentiation.

Muscle stem (satellite) cells express Pax7, a key transcription factor essential for satellite cell maintenance and adult muscle regeneration. We identify the corepressor transducin-like enhancer of split-4 (TLE4) as a Pax7 interaction partner expressed in quiescent satellite cells under homeostasis. A subset of satellite cells transiently downregulate TLE4 during early time points following muscle injury. We identify these to be activated satellite cells, and that TLE4 downregulation is required for Myf5 activation and myogenic commitment. Our results indicate that TLE4 represses Pax7-mediated Myf5 transcriptional activation by occupying the -111 kb Myf5 enhancer to maintain quiescence. Loss of TLE4 function causes Myf5 upregulation, an increase in satellite cell numbers and altered differentiation dynamics during regeneration. Thus, we have uncovered a novel mechanism to maintain satellite cell quiescence and regulate muscle differentiation mediated by the corepressor TLE4.

Measurement of excitation-contraction coupling time in critical illness myopathy.

OBJECTIVE: This study aimed to develop a simple and reliable technique to assess excitation-contraction (E-C) coupling for early diagnosis of critical illness myopathy (CIM). METHODS: We prospectively performed clinical and electrophysiological examinations on patients admitted to intensive care unit (ICU). In addition to full neurological examinations and routine nerve conduction study, motor related potential (MRP) was recorded using an accelerometer attached to the base of hallux after tibial nerve stimulation, and E-C coupling time (ECCT) was measured from the latency difference between soleus compound muscle action potential (CMAP) and MRP. RESULTS: Of 41 patients evaluated, 25 met the criteria for ICU-acquired weakness, 23 of whom had CIM. The time to the first electrophysiological examination (time to first test) correlated negatively with CMAP and with MRP. Conversely, a positive correlation was observed between the time to first test and ECCT. E-C coupling impairment occurred in most of our patients with CIM by the third day of ICU admission, and prolonged ECCT could be the earliest detectable abnormality. CONCLUSIONS: The ECCT measurement is an easy and reliable technique to detect reduced muscle membrane excitability in the early stage of CIM. SIGNIFICANCE: The ECCT measured by our method using an accelerometer may be a parameter that predicts the development of CIM.

Sepsis-Induced Myopathy and Gut Microbiome Dysbiosis: Mechanistic Links and Therapeutic Targets.

ABSTRACT: Sepsis is currently defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. The skeletal muscle system is among the host organ systems compromised by sepsis. The resulting neuromuscular dysfunction and impaired regenerative capacity defines sepsis-induced myopathy and manifests as atrophy, loss of strength, and hindered regeneration after injury. These outcomes delay recovery from critical illness and confer increased vulnerability to morbidity and mortality. The mechanisms underlying sepsis-induced myopathy, including the potential contribution of peripheral organs, remain largely unexplored. The gut microbiome is an immunological and homeostatic entity that interacts with and controls end-organ function, including the skeletal muscle system. Sepsis induces alterations in the gut microbiota composition, which is globally termed a state of "dysbiosis" for the host compared to baseline microbiota composition. In this review, we critically evaluate existing evidence and potential mechanisms linking sepsis-induced myopathy with gut microbiota dysbiosis.

Genetic defects are common in myopathies with tubular aggregates.

OBJECTIVE: A group of genes have been reported to be associated with myopathies with tubular aggregates (TAs). Many cases with TAs still lack of genetic clarification. This study aims to explore the genetic background of cases with TAs in order to improve our knowledge of the pathogenesis of these rare pathological structures. METHODS: Thirty-three patients including two family members with biopsy confirmed TAs were collected. Whole-exome sequencing was performed on 31 unrelated index patients and a candidate gene search strategy was conducted. The identified variants were confirmed by Sanger sequencing. The wild-type and the mutant p.Ala11Thr of ALG14 were transfected into human embryonic kidney 293 cells (HEK293), and western blot analysis was performed to quantify protein expression levels. RESULTS: Eleven index cases (33%) were found to have pathogenic variant or likely pathogenic variants in STIM1, ORAI1, PGAM2, SCN4A, CASQ1 and ALG14. Among them, the c.764A>T (p.Glu255Val) in STIM1 and the c.1333G>C (p.Val445Leu) in SCN4A were novel. Western blot analysis showed that the expression of ALG14 protein was severely reduced in the mutant ALG14 HEK293 cells (p.Ala11Thr) compared with wild type. The ALG14 variants might be associated with TAs in patients with complex multisystem disorders. INTERPRETATION: This study expands the phenotypic and genotypic spectrums of myopathies with TAs. Our findings further confirm previous hypothesis that genes related with calcium signalling pathway and N-linked glycosylation pathway are the main genetic causes of myopathies with TAs.

Detection of Left Atrial Myopathy Using Artificial Intelligence-Enabled Electrocardiography.

BACKGROUND: Left atrial (LA) myopathy is common in patients with heart failure and preserved ejection fraction and leads to the development of atrial fibrillation (AF). We investigated whether the likelihood of LA remodeling, LA dysfunction, altered hemodynamics, and risk for incident AF could be identified from a single 12-lead ECG using a novel artificial intelligence (AI)-enabled ECG analysis. METHODS: Patients with heart failure and preserved ejection fraction (n=613) underwent AI-enabled ECG analysis, echocardiography, and cardiac catheterization. Individuals were grouped by AI-enabled ECG probability of contemporaneous AF, taken as an indicator of underlying LA myopathy. RESULTS: Structural heart disease was more severe in patients with higher AI-probability of AF, with more left ventricular hypertrophy, larger LA volumes, and lower LA reservoir and booster strain. Cardiac filling pressures and pulmonary artery pressures were higher in patients with higher AI-probability, while cardiac output reserve was more impaired during exercise. Among patients with sinus rhythm and no prior AF, each 10% increase in AI-probability was associated with a 31% greater risk of developing new-onset AF (hazard ratio, 1.31 [95% CI, 1.20-1.42]; P<0.001). In the population as a whole, each 10% increase in AI-probability was associated with a 12% greater risk of death (hazard ratio, 1.12 [95% CI, 1.08-1.17]; P<0.001) during long-term follow-up, which was no longer significant after adjustments for baseline characteristics. CONCLUSIONS: A novel AI-enabled score derived from a single 12-lead ECG identifies the presence of underlying LA myopathy in patients with heart failure and preserved ejection fraction as evidenced by structural, functional, and hemodynamic abnormalities, as well as long-term risk for incident AF. Further research is required to determine the role of the AI-enabled ECG in the evaluation and care of patients with heart failure and preserved ejection fraction.

Pre Clinical Assessment of AAVrh74.MCK.GNE Viral Vector Therapeutic Potential: Robust Activity Despite Lack of Consistent Animal Model for GNE Myopathy.

BACKGROUND: GNE myopathy is a unique adult onset rare neuromuscular disease caused by recessive mutations in the GNE gene. The pathophysiological mechanism of this disorder is not well understood and to date, there is no available therapy for this debilitating disease. We have previously established proof of concept that AAV based gene therapy can effectively deliver the wild type human GNE into cultured muscle cells from human patients and in mice, using a CMV promoter driven human wild type GNE plasmid delivered through an adeno associated virus (AAV8) based platform. OBJECTIVE: In the present study we have generated a muscle specific GNE construct, driven by the MCK promoter and packaged with the AAVrh74 serotype for efficacy evaluation in an animal model of GNE Myopathy. METHODS: The viral vector was systemically delivered at 2 doses to two age groups of a Gne-/- hGNED207V Tg mouse described as a preclinical model of GNE Myopathy, and treatment was monitored for long term efficacy. RESULTS: In spite of the fact that the full described characteristics of the preclinical model could not be reproduced, the systemic injection of the rAAVrh74.MCK.GNE viral vector resulted in a long term presence and expression of human wt GNE in the murine muscles and in some improvements of their mild phenotype. The Gne-/- hGNED207V Tg mice are smaller from birth, but cannot be differentiated from littermates by muscle function (grip strength and Rotarod) and their muscle histology is normal, even at advanced age. CONCLUSIONS: The rAAVrh74.MCK.GNE vector is a robust tool for the development of GNE Myopathy therapies that supply the intact GNE. However, there is still no reliable animal model to fully assess its efficacy since the previously developed Gne-/- hGNED207V Tg mice do not present disease characteristics.

The role of potassium in muscle membrane dysfunction in end-stage renal disease.

OBJECTIVE: Uremic myopathy is a condition seen in end-stage renal disease (ESRD), characterized by muscle weakness and muscle fatigue, in which the pathophysiology is uncertain. The aim of this study was to assess the role of abnormal serum constituents in ESRD patients by relating them to the excitability properties of the tibialis anterior muscle, at rest and during electrically induced muscle activation, by recording muscle velocity recovery cycles (MVRC) and frequency ramp responses. METHODS: Eighteen ESRD patients undergoing hemodialysis were evaluated by blood sample, MVRC, and frequency ramp (before and near the end of dialysis treatment), quantitative electromyography, and nerve conduction studies. Patients were compared to 24 control subjects. RESULTS: In patients, muscle relative refractory period, early supernormality, late supernormality after 5 conditioning stimuli, and latency of the last of 15 and 30 frequency ramp pulses were strongly associated with potassium levels (p < 0.01), showing depolarization before and normalization in the end of hemodialysis. CONCLUSIONS: In ESRD patients, the muscle membrane is depolarized, mainly due to hyperkalemia. SIGNIFICANCE: Since normal muscle fatigue has been attributed to potassium-induced depolarization, it seems likely that this mechanism is also a major cause of the exaggerated muscle fatigue and weakness in ESRD patients.

Histopathological characteristics of cervical extensor tissue in patients with dropped head syndrome.

BACKGROUND: To date, the histopathologic characteristics of dropped head syndrome (DHS) have not been reported sufficiently. The present study investigates the histopathology of biopsy specimens from the cervical paravertebral region in patients with DHS. METHODS: Histopathological parameters were evaluated in biopsy specimens of the cervical paravertebral soft tissue from 15 patients with DHS. RESULTS: Among the 15 cases of DHS examined, skeletal muscle was identified in 7 cases, all of which showed necrosis, microvessel proliferation and atrophy. The ligament was identified in 12 cases, 8 of which showed degeneration. The lag time between the onset of symptoms and the performance of a biopsy in all 8 cases, which showed degeneration was over 3 months. Microvessel proliferation in the ligament was observed in 1 of the 4 cases, in which the lag time between the onset of symptoms and the performance of a biopsy was less than 3 months (acute or subacute phase), and in 7 of the 8 cases, in which the lag time between the symptoms and the performance of a biopsy was over 3 months (chronic phase). Chronic inflammation in the ligament was identified in 1 of the 12 cases. CONCLUSIONS: The identification of necrosis, microvessel proliferation, and atrophy in the skeletal muscle of patients with DHS and the presence of ligament degeneration and microvessel proliferation in the chronic but not acute or subacute phases may suggest that persistent skeletal muscle damage of the cervical paravertebral region causes subsequent ligament damage in patients with DHS.