Muscle Endplate Research Articles

The effect of increasing motor end-plate innervation on smile activation in acute and early facial palsy.

While it has been over half a century since primary cross-facial nerve grafting was first described for facial reanimation, the outcome of this procedure, remains inconsistent and provide lesser smile excursion when compared to the likes of the masseteric nerve. However, the latter itself has limitations in terms of the lack of spontaneity and resting tone. While combinations have been attempted more proximally, we ask the question as to whether more distal nerve transfers with vascularized nerve grafts are a better option. In a retrospective review of clinical practice at our institute, 16 consecutive patients had single, double, and finally triple distal nerve transfers, close to the target facial muscle to reinnervate the motor endplates directly, over a 6-year period (2018-23). All patients had the onset of facial palsy within 18 months. Statistical analysis of the comparison between three sub-cohorts was performed using student's t-test and one-way ANOVA, respectively. Qualitatively, masseteric neurotization of a single facial nerve branch translated into smile improvement in 50% of cases, as opposed to all cases of double- and triple-neurotization of the smile muscles. In terms of upper lip elevation, single neurotization showed improvement in 25% of cases, double-neurotization in 40% of cases and triple-neurotization in 100% of cases. Upper lip elevation was also significantly better in those who had a vascularized cross-facial nerve graft (Student's t-test <0.05). In summary, increasing neural input to the motor endplates of smile muscles can significantly improve smile activation, in acute flaccid facial palsies.

Advances in Understanding and Managing Myasthenia Gravis: Current Trends and Future Directions.

Myasthenia gravis (MG) is an autoimmune illness characterized by autoantibodies against the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), and an increasing number of extra postsynaptic proteins. Pathogenic autoantibodies reduce the number of functional AChRs in the neuromuscular junction's (NMJ)muscle end plate. The cause of the autoimmune response is unknown, but thymic abnormalities and immune regulatory deficiencies are significant. The disease's incidence is likely influenced by genetic predisposition, with sex hormones and exercise playing a role. MGcan affect any age, race, or gender and can be caused by any stressor, with infections being the most frequent cause. Treatment focuses on airway support and the triggering incident. MG is a rare autoimmune disease causing fatigue-inducing weakness in the axial, respiratory, leg, and bulbar muscles. Initially affecting the eyes, most MGpatients experience at least one worsening symptom during their illness. The disease is mainly caused by antibodies against the AChR, dependence on the immune system within cells, and engagement of the complement system. The complement system plays a significant role in MG, and complement inhibition can both prevent the onset and slow its development. Ocular MG affects around 15% of people, with most patients having blocking antibodies against the cholinergic receptor. There may be correlations between thymoma and other autoimmune conditions, especially thyroid illness. Treatment and management for MGinvolve removing autoantibodies from circulation or blocking effector mechanisms using techniques such as complement inhibition, plasmapheresis, and B-cell elimination.

Differences in neuromuscular junctions between intrinsic muscles of the forepaw and biceps muscles in rats.

Motor endplates of the interossei muscles become destabilized, whereas those of the biceps muscles remain stable in a rat model of obstetric brachial plexus palsy. However, it is unclear whether the morphology of the motor endplates of the interossei muscles is different from that of the biceps muscles in normal rat. We hypothesized that the motor endplates in the interossei muscles have specific characteristics different from those in the biceps muscles. The motor endplates were labeled with α-bungarotoxin and synaptophysin. The cross-sectional areas of the muscle fibers, the morphologies of the motor endplates, and the absolute and normalized areas (corrected by muscle fiber diameter) of the motor endplates of the interossei muscles and the biceps muscles were compared in rats at 1, 3, and 5 weeks after birth. The cross-sectional area of the interossei muscles and biceps muscle fibers were found to have increased gradually at 1, 3, and 5 weeks, but that of the biceps muscles was larger than that of the interossei muscles. The motor endplates of the interossei muscles and the biceps muscles gradually develop from crescent to pretzel shape after birth, and those of the interossei muscles have a smaller area. At 1, 3, and 5 weeks postnatally, the area of postnatal normalized motor endplates of the interossei muscles was much smaller than that of the biceps muscles. A better understanding of the morphological differences of the motor endplates between the interossei muscles and the biceps muscles may help to understand their physiological and pathological changes.

Celecoxib ameliorates diabetic sarcopenia by inhibiting inflammation, stress response, mitochondrial dysfunction, and subsequent activation of the protein degradation systems.

Aim: Diabetic sarcopenia leads to disability and seriously affects the quality of life. Currently, there are no effective therapeutic strategies for diabetic sarcopenia. Our previous studies have shown that inflammation plays a critical role in skeletal muscle atrophy. Interestingly, the connection between chronic inflammation and diabetic complications has been revealed. However, the effects of non-steroidal anti-inflammatory drug celecoxib on diabetic sarcopenia remains unclear. Materials and Methods: The streptozotocin (streptozotocin)-induced diabetic sarcopenia model was established. Rotarod test and grip strength test were used to assess skeletal muscle function. Hematoxylin and eosin and immunofluorescence staining were performed to evaluate inflammatory infiltration and the morphology of motor endplates in skeletal muscles. Succinate dehydrogenase (SDH) staining was used to determine the number of succinate dehydrogenase-positive muscle fibers. Dihydroethidium staining was performed to assess the levels of reactive oxygen species (ROS). Western blot was used to measure the levels of proteins involved in inflammation, oxidative stress, endoplasmic reticulum stress, ubiquitination, and autophagic-lysosomal pathway. Transmission electron microscopy was used to evaluate mitophagy. Results: Celecoxib significantly ameliorated skeletal muscle atrophy, improving skeletal muscle function and preserving motor endplates in diabetic mice. Celecoxib also decreased infiltration of inflammatory cell, reduced the levels of IL-6 and TNF-α, and suppressed the activation of NF-κB, Stat3, and NLRP3 inflammasome pathways in diabetic skeletal muscles. Celecoxib decreased reactive oxygen species levels, downregulated the levels of Nox2 and Nox4, upregulated the levels of GPX1 and Nrf2, and further suppressed endoplasmic reticulum stress by inhibiting the activation of the Perk-EIF-2α-ATF4-Chop in diabetic skeletal muscles. Celecoxib also inhibited the levels of Foxo3a, Fbx32 and MuRF1 in the ubiquitin-proteasome system, as well as the levels of BNIP3, Beclin1, ATG7, and LC3Ⅱ in the autophagic-lysosomal system, and celecoxib protected mitochondria and promoted mitochondrial biogenesis by elevating the levels of SIRT1 and PGC1-α, increased the number of SDH-positive fibers in diabetic skeletal muscles. Conclusion: Celecoxib improved diabetic sarcopenia by inhibiting inflammation, oxidative stress, endoplasmic reticulum stress, and protecting mitochondria, and subsequently suppressing proteolytic systems. Our study provides evidences for the molecular mechanism and treatment of diabetic sarcopenia, and broaden the way for the new use of celecoxib in diabetic sarcopenia.

Immune mediated myasthenia gravis in children, current concepts and new treatments: A narrative review article.

Myasthenia gravis (MG) is the most frequent transmission disease in the neuromuscular junction. Juvenile myasthenia gravis (JMG) is an autoimmune antibody-mediated disease of postsynaptic endplate defined as MG presentation in patients before the age of 18 years old. While many clinical features of JMG are identical to the adults, there are some significant differences between them regarding presentation, clinical course, antibody level, and thymus histopathology. In JMG, ocular symptoms are more frequent, the clinical course is comparably benign, and the outcome is better than adult MG. Antibodies attack the muscle endplate proteins in the postsynaptic membrane and interfere with transmission. These antibodies in most patients are against the acetylcholine receptors, but they may also be directed toward muscle-specific kinase, lipoprotein-related protein 4, and agrin. Findings show racial influences and genetic effects on the occurrence of JMG. The essential clinical symptom is fatigable weakness of muscles that can be in the form of isolated ocular type or more disseminated weakness. The diagnosis of JMG is essentially clinical, with fluctuating patterns of weakness and easy fatigability, but a series of diagnostic evaluations can confirm the diagnosis. Precise diagnostic evaluation and distinction from congenital myasthenic syndromes is critical. The treatment plan is conducted according to the clinical course (ocular or generalized), antibody type, and disease severity. The mainstay of treatment includes symptomatic therapy, long-lasting immunosuppressive treatment and treatment of myasthenic crisis. Novel medications are introduced and conducted to the specific pathophysiologic mechanisms of the disease, and they are used primarily in the refractory MG.

Increased Frequency of Giant Miniature End-Plate Potentials at the Neuromuscular Junction in Diabetic Rats.

There is a need for research addressing the functional characteristics of the motor end-plate in diabetes to identify mechanisms contributing to neuromuscular dysfunction. Here, we investigated the effect of diabetes on spontaneous acetylcholine release in the rat neuromuscular junction. We studied two randomized groups of male Wistar rats (n = 7 per group, 350 ± 50 g, 12-16 weeks of age): one with streptozotocin-induced experimental diabetes, and a healthy control group without diabetes. After 8 weeks of monitoring after diabetes induction, rats in both groups were anesthetized with pentobarbital. Then, the diaphragm muscle was dissected for electrophysiological recordings of miniature end-plate potentials (MEPPs) using a single electrode located at the region of the muscle end-plate. All experiments were conducted at environmental temperature (20-22 °C) in rat Ringer solution with constant bubbling carbogen (95% O2, 5% CO2). Compared to healthy controls, in the diaphragm neuromuscular end-plate derived from diabetic rats, the MEPPs were higher in amplitude and frequency, and the proportion of giant MEPPs was elevated (7.09% vs. 1.4% in controls). Our results showed that diabetes affected the acetylcholine MEPP pattern and increased the number of giant potentials compared to healthy controls.

Porcine Acellular Nerve-Derived Hydrogel Improves Outcomes of Direct Muscle Neurotization in Rats.

Background: The ability to reinnervate a muscle in the absence of a viable nerve stump is a challenging clinical scenario. Direct muscle neurotization (DMN) is an approach to overcome this obstacle; however, success depends on the formation of new muscle endplates, a process, which is often limited due to lack of appropriate axonal pathfinding cues. Objective: This study explored the use of a porcine nerve extracellular matrix hydrogel as a neuroinductive interface between nerve and muscle in a rat DMN model. The goal of the study was to establish whether such hydrogel can be used to improve neuromuscular function in this model. Materials and Methods: A common peroneal nerve-to-gastrocnemius model of DMN was developed. Animals were survived for 2 or 8 weeks following DMN with or without the addition of the hydrogel at the site of neurotization. Longitudinal postural thrust, terminal electrophysiology, and muscle weight assessments were performed to qualify and quantify neuromuscular function. Histological assessments were made to qualify the host response at the DMN site, and to quantify neuromuscular junctions (NMJs) and muscle fiber diameter. Results: The hydrogel-treated group showed a 132% increase in postural thrust at 8 weeks compared with that of the DMN alone group. This was accompanied by an 80% increase in the number of NMJs at 2 weeks, and 26% increase in mean muscle fiber diameter at 8 weeks. Conclusions: These results suggest that a nerve-derived hydrogel may improve the neuromuscular outcome following DNM.

Distal anterior interosseous nerve to ulnar nerve motor branch reverse end-to-side transfer in a case of severe ulnar neuropathy.

Reverse end-to-side (RETS) distal transfer is gaining popularity in cases of proximal nerve damage with the nerve in continuity, allowing the nerve to potentially retain its ability to regenerate and recover. While preserving the original axon pool, RETS could provide an additional pool of motor axons and/or possibly "babysit" the muscle endplates and distal denervated nerve Schwann cells until reinnervation from the original pool occurs. The authors present a video demonstrating anterior subcutaneous transposition of the ulnar nerve at the elbow coupled with a distal anterior interosseous nerve to ulnar nerve RETS in a case of severe posttraumatic ulnar neuropathy at the elbow. The video can be found here: https://stream.cadmore.media/r10.3171/2022.9.FOCVID2282.

The Clinical Outcome in AChR-Positive Generalized Myasthenia Gravis: A Retrospective Observational Study.

Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction, and in over 80% of cases, antibodies are identified against the nicotinic type of acetylcholine receptor (AChR) on the muscle endplate. Despite the availability of various treatment options, patients with MG experience relapses and remission during the course of the disease. Aims and Objective: To understand the clinical profile, predictors of outcomes in acetyl choline receptor (AChR) antibody positive generalized MG. This is a retrospective, single-centre, observational study of 108 patients with AChR positive generalized MG. We collected data on clinical and demographical profiles, treatments received, and treatment responses from those who fulfilled inclusion criteria over a mean follow up period of 33.75 ±7.30 months. Clinical outcomes were studied in terms of the type of remission and crisis or disease exacerbations patients had, considering different variables and treatment received. We found the commonest initial symptoms were ocular or oculo-bulbar, which progressed to generalized MG in the first year of disease onset. 36 (33.3%) patients experienced a crisis requiring mechanical ventilation within a mean period of 9.4 ±4.77 months from the disease onset. Multivariate regression analysis showed late-onset MG (age of onset between 50-70 years) and treatment with rituximab were better correlated with remission, (odd ratio of 4.7; 95 % CI ,1.12 -12.6; P value < 0.05 and odd ratio of 4.56; 95 % CI ,1.2 -10.04; P value < 0.05) respectively. While treatment with Mycofenolate Mofetile (MMF) was associated with a higher number of relapses (odd ratio of 1.8; 95 % CI ,0.08 -0.96; P value < 0.05). Treatment with Rituximab showed a higher rate of remission as compared to treatment refractory (TR) on conventional immunosuppressant therapy (IST). Out of 35(32 %) thymoma patients, 21 patients underwent thymectomy and these patients showed significantly greater rate of remission as compared both thymoma patients who denied thymectomy as a treatment option (N = 10 ;55.60 % vs N = 4; 23.50%). In this study of AChR antibody positive generalized MG patients, we found that nearly one-third of them experienced myasthenic crisis despite receiving the best medical care. Rituximab appeared to be effective in the treatment of refractory MG and those who failed thymectomy. Thymectomy was associated with better outcomes in patients, both with or without a thymoma.

A novel phenotype of AChR-deficiency syndrome with predominant facial and distal weakness resulting from the inclusion of an evolutionary alternatively-spliced exon in CHRNA1

Primary acetylcholine receptor deficiency is the most common subtype of congenital myasthenic syndrome, resulting in reduced amount of acetylcholine receptors expressed at the muscle endplate and impaired neuromuscular transmission. AChR deficiency is caused mainly by pathogenic variants in the ε-subunit of the acetylcholine receptor encoded by CHRNE, although pathogenic variants in other subunits are also seen. We report the clinical and molecular features of 13 patients from nine unrelated kinships with acetylcholine receptor deficiency harbouring the CHRNA1 variant NM_001039523.3:c.257G>A (p.Arg86His) in homozygosity or compound heterozygosity. This variant results in the inclusion of an alternatively-spliced evolutionary exon (P3A) that causes expression of a non-functional acetylcholine receptor α-subunit. We compare the clinical findings of this group to the other cases of acetylcholine receptor deficiency within our cohort. We report differences in phenotype, highlighting a predominant pattern of facial and distal weakness in adulthood, predominantly in the upper limbs, which is unusual for acetylcholine receptor deficiency syndromes, and more in keeping with slow-channel syndrome or distal myopathy. Finally, we stress the importance of including alternative exons in variant analysis to increase the probability of achieving a molecular diagnosis.

MRI grading of spinal stenosis is not associated with the severity of low back pain in patients with lumbar spinal stenosis

BackgroundAlthough lumbar spinal stenosis (LSS) often coexists with other degenerative conditions, few studies have fully assessed possible contributing factors for low back pain (LBP) in patients with LSS. The purpose of this study was to identify factors associated with the severity of LBP in patients with LSS.MethodsThe patients with neurogenic claudication caused by LSS, which was confirmed by magnetic resonance imaging (MRI) were included in this cross-sectional study. Data included ratings of LBP, buttock and leg pain, and numbness on a numerical rating scale (NRS), 36-item Short-Form Survey (SF-36) scores, muscle mass measured by bioelectrical impedance analysis, and radiographic measurements including lumbopelvic alignment and slippage. The severity of LSS, endplate defects, Modic endplate changes, intervertebral disc degeneration, and facet joint osteoarthritis were evaluated on MRI. Spearman correlation and multivariate linear regression analyses were used to examine the factors associated with the severity of LBP (NRS score).ResultsA total of 293 patients (135 male and 158 female, average age 72.6 years) were analyzed. LBP was moderately correlated with buttock and leg pain, and buttock and leg numbness. Significant but weak correlations were observed between LBP and body mass index, appendicular and trunk muscle mass, all domains of SF-36, pelvic tilt, total number of endplate defects and Modic endplate changes, and summary score of disc degeneration grading, but not severity or number of spinal stenoses. In the multivariate regression analysis, age, female sex, trunk muscle mass, diabetes, NRS buttock and leg pain, NRS buttock and leg numbness, SF-36 vitality, pelvic tilt, and total number of endplate defects were associated with the severity of LBP.ConclusionsTrunk muscle mass, lumbopelvic alignment, and endplate defects, but not severity of stenosis are partly associated with severity of LBP, but buttock and leg pain and buttock and leg numbness have strongest relationships with LBP in patients with LSS.

Outcome of thymectomy in myasthenia gravis patients at Neurological Institute of Thailand

Background &amp; Objective: Myasthenia gravis is the most common neuromuscular junction disorders and is caused by autoantibodies against acetylcholine receptors at the postsynaptic muscle endplate. Although thymectomy is current standard treatment but the benefit in long-term outcomes and in non- thymomatous generalized MG patients with negative AChR antibody are still unclear. The objective is to determine the treatment outcomes of thymectomy in non-thymomatous generalized MG patients with or without AChR antibody and identify features related to remission. Methods: Retrospective chart review of the patients diagnosed with non-thymomatous generalized MG from January 1st, 2014 to December 31th, 2018. Patients were classified into two groups: patients with thymectomy (thymectomy group) and patients with no thymectomy (medication group). Results: Seventy-six patients were included. All baseline clinical features, investigations and treatment response were compared. At 6 and 12 months follow up, most patients in both groups had a good response to treatment. At 24 months follow up, ratio of patients in thymectomy group with complete stable remission is higher than in medication group (25% vs. 7.5%) and all patients in this group had positive acetylcholine receptor antibody. The patients in thymectomy group required lower dosage of prednisone, azathioprine and cholinesterase inhibitors than in medication group. Conclusion: Thymectomy was associated with a high probability of achieving remission, reduced cholinesterase inhibitors and immunosuppressive drugs when compared to medication group. Therefore, thymectomy should be strongly considered for non-thymomatous generalized MG patient with positive AChR antibody; and may be considered in non-thymomatous generalized MG patients with negative AChR antibody.

Schwann Cell Derived Signals Enhance AChR Clustering in C2C12 Myotubes

The neuromuscular junction (NMJ) is a tripartite structure made up of the pre‐synaptic motor neuron terminal, the post‐synaptic muscle end plate, and the perisynaptic Schwann cells (SC). Degeneration of the NMJ is strongly associated with age‐associated muscle mass and strength declines as well as other neurodegenerative diseases; however, the cellular interactions and signals that initiate the degeneration are poorly understood. In particular, understanding of the role of perisynaptic SCs in NMJ maintenance is scant, whereas the signaling between motor neurons and muscle fibers has been extensively studied. For example, agrin is a motor neuron derived secreted factor that is the primary signal for the induction of acetylcholine receptor (AChR) clustering in myofibers, and treatment of myotubes in culture with agrin has been shown to similarly promote AChR clustering in the absence of neurons. The presence of SCs at motor nerve terminals implicates signaling from these cells in both NMJ formation and maintenance. Thus, the goal was to determine if SC derived signals impact AChR clustering. We hypothesized that SC derived factors would potentiate agrin‐induced aneural AChR clustering in C2C12 myotubes. Differentiated C2C12 myotubes were treated with agrin for either 10 minutes or 4 hours to minimally or maximally induce AChR clustering, respectively. Additional myotubes were treated with agrin for 10 minutes followed by treatment with conditioned media (CM) derived from SW10 Schwann cell line cultures for the remaining 4 hours. Finally, additional myotube cultures were treated for 4 hours with CM alone or normal complete media as controls. The number of clusters and mean cluster area were increased in myotube cultures treated with the combination of a 10‐minute agrin pulse followed by and CM from SW10 cells compared to cultures treated for either 10 minutes or 4 hours with agrin. Neither cluster number nor area were elevated compared with controls in cultures treated with CM alone. These data suggest that SC‐derived signals can potentiate agrin‐induced post‐synaptic AChR clustering in vitro. We are currently investigating the critical SC‐derived signals that potentiate AChR clustering to determine factors that may provide therapeutic benefit in neurodegenerative phenotypes including aging.

Degeneration and Regeneration of Nerve and Muscle end Plates of Athletes after Peripheral Nerve Injury

Degeneration and Regeneration of Nerve and Muscle end Plates of Athletes after Peripheral Nerve Injury

An Approach to Maximize Retrograde Transport Based on the Spatial Distribution of Motor Endplates in Mouse Hindlimb Muscles.

Knowledge regarding the relationship between muscles and the corresponding motor neurons would allow therapeutic genes to transport into specific spinal cord segments. Retrograde tracing technique by targeting the motor endplate (MEP), a highly specialized structure that offers direct access to the spinal motor neurons, has been used to elucidate the connectivity between skeletal muscles and the innervating motor neuron pools. However, current injection strategies mainly based on blind injection or the local MEP region might lead to an underestimation of the motor neuron number due to the uneven distribution of MEP in skeletal muscles. In this work, we proposed a novel intramuscular injection strategy based on the 3D distribution of the MEPs in skeletal muscles, applied the 3D intramuscular injection to the gastrocnemius and tibialis anterior for retrograde tracing of the corresponding motor neurons, and compared this with the existing injection strategy. The intramuscular diffusion of the tracer demonstrated that 3D injection could maximize the retrograde transport by ensuring a greater uptake of the tracer by the MEP region. In combination with optical clearing and imaging, we performed 3D mapping and quantification of the labeled motor neurons and confirmed that 3D injection could label more motor neurons than the current injection method. It is expected that 3D intramuscular injection strategy will help elucidate the connective relationship between muscles and motor neurons faithfully and becomes a promising tool in the development of gene therapy strategies for motor neuron diseases.

Symposium 4: Neuromuscular junction and related disorders SY4.1. Myasthenia gravis and related disorders

Autoimmune myasthenia gravis (MG) results from autoantibodies that bind to the neuromuscular junction (NMJ). Incidence and prevalence vary worldwide; the estimated US prevalence is 20/100,000, or ∼60,000 patients. Women develop MG more often before age 40, men after 50. Drooping eyelids or double vision is the first symptom in 60%; weakness remains limited to periocular muscles in 10–15% – ocular myasthenia. The voice may be nasal or hoarse, especially after prolonged talking, and swallowed liquids may escape through the nose. Weakness typically worsens throughout the day. Spontaneous improvement may occur early on but is rarely permanent. The diagnosis depends on recognizing distinctive patterns of weakness and confirmation by serologic and electrodiagnostic tests. 80–85% of patients have circulating anti-AChR antibodies; repetitive nerve stimulation (RNS) demonstrates abnormal neuromuscular transmission (NMT) in up to 90% of generalized MG and 60% of ocular myasthenia. Increased jitter is found in almost all patients if appropriate muscles are examined. Approximately 10% of patients have antibodies to muscle specific tyrosine kinase (MuSK). Clinical findings resemble non-MuSK MG in some patients; many have predominant weakness in cranio-bulbar muscles with marked atrophy. Others have predominant neck, shoulder and respiratory weakness. With selected treatment most non-MuSK and MuSK-MG patients have only minimal long-term morbidity. Congenital myasthenic syndromes result from genetic defects of muscle endplate molecules involved in NMT; most have autosomal recessive inheritance. The diagnosis is suggested by clinical features and response to cholinesterase inhibitors, or electrodiagnostic findings. Identification of specific genetic or physiologic defects requires genetic studies or specialized morphological and electrophysiological studies. Lambert-Eaton myasthenia results from antibodies to voltage-gated calcium channels (VGCC) on presynaptic nerve terminals, which produces the classic triad of weakness, reduced muscle stretch reflexes and autonomic dysfunction. Approximately 50% have an underlying malignancy, usually small cell lung cancer. The diagnosis is confirmed by a decrementing pattern during low frequency RNS and low muscle responses that increase at least 60% after muscle activation. Antibodies to the VGCC are found in most cases. Treatment is initially directed against any underlying malignancy. Amifampridine produces symptomatic improvement in most patients; immunomodulatory therapy may provide modest benefit in some.

"Time window" effect of Yoda1-evoked Piezo1 channel activity during mouse skeletal muscle differentiation.

AimMechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) and on adult muscle fibres.MethodsImmunostaining, electrophysiological intracellular recordings and Ca2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibres and at the adult neuromuscular junction.ResultsPiezo1 channels were detected by immunostaining in satellite cells (SCs) and myotubes as well as in adult myofibres. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of SCs. Moreover, in myotubes, Yoda1 induced significant [Ca2+] i transients, without detectable [Ca2+] i response in adult myofibres. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve‐evoked or spontaneous synaptic activity or muscle contractions in adult myofibres.ConclusionOur data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration.

Reaction-Diffusion Modeling to Assess what Limits Effective Acetylcholine Fluctuations at High Frequency Cholinergic Electroplaques

In the weakly electric fish Eigenmannia (glass knifefish), high frequency (200-550Hz) electric organ discharge (EOD) is driven by high frequency cholinergic synaptic input onto the electrocytes at their electroplaques. Evolutionarily, this system derives from skeletal muscle endplate synapses. We are interested in what, physically, limits the rapidity with which acetylcholine (ACh) concentrations can oscillate to successfully activate post-synaptic ACh receptors (AChR) and thereby trigger EODs at the oscillating frequency. Assuming periodic release of ACh into a 50nm wide cylindrical synaptic gap, we solve numerically a one dimensional reaction-diffusion model at 200Hz and 500Hz. The model included the diffusion of ACh and its interactions with AChesterase (AChE) and AChRs. For ACh released periodically at the two frequencies, we vary the ratio of ACh input to the AChE concentration. The biochemical/biophysical characteristics of AChE/ACh and AChR/ACh are those typical of neuromuscular junctions. The AChR can be in four states: closed (AR), with one or two bound ACh molecules (AR1 and AR2) and finally open state (AR2open). At 500Hz a higher AChE/ACh ratio is needed to remove ACh from the cleft between consecutive ACh releases. Only a small fraction of the ACh molecules reaches the AChRs, and there are residual amounts of ACh molecules from the preceding release. Previous computational studies showed that the persistently present ACh should not impede high frequency electrocyte firing, provided the cholinergic current is subthreshold for triggering firing. In our simulations, the fast AChE-ACh reactions (not AChR-ACh kinetics) limit the upper frequency for triggering electrocyte action potentials. We suggest, therefore, that carry-over (persistent) activation of AChRs could account for the observed upper limit for EOD frequency in Eigenmannia individuals.

Immunoregulatory Cells in Myasthenia Gravis.

Myasthenia gravis (MG) is a T cell-dependent, B-cell mediated autoimmune disease caused by antibodies against the nicotinic acetylcholine receptor or other components of the post-synaptic muscle endplate at the neuromuscular junction. These specific antibodies serve as excellent biomarkers for diagnosis, but do not adequately substitute for clinical evaluations to predict disease severity or treatment response. Several immunoregulatory cell populations are implicated in the pathogenesis of MG. The immunophenotype of these populations has been well-characterized in human peripheral blood. CD4+FoxP3+ regulatory T cells (Tregs) are functionally defective in MG, but there is a lack of consensus on whether they show numerical perturbations. Myeloid-derived suppressor cells (MDSCs) have also been explored in the context of MG. Adoptive transfer of CD4+FoxP3+ Tregs or MDSCs suppresses ongoing experimental autoimmune MG (EAMG), a rodent model of MG, suggesting a protective role of both populations in this disease. An imbalance between follicular Tregs and follicular T helper cells is found in untreated MG patients, correlating with disease manifestations. There is an inverse correlation between the frequency of circulating IL-10–producing B cells and clinical status in MG patients. Taken together, both functional and numerical defects in various populations of immunoregulatory cells in EAMG and human MG have been demonstrated, but how they relate to pathogenesis and whether these cells can serve as biomarkers of disease activity in humans deserve further exploration.

Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species

Better understanding of roles of complement in pathology has fuelled an explosion of interest in complement-targeted therapeutics. The C5-blocking monoclonal antibody (mAb) eculizumab, the first of the new wave of complement blocking drugs, was FDA approved for treatment of Paroxysmal Nocturnal Hemoglobinuria in 2007; its expansion into other diseases has been slow and remains restricted to rare and ultra-rare diseases such as atypical hemolytic uremic syndrome. The success of eculizumab has provoked other Pharma to follow this well-trodden track and made C5 blockade the busiest area of complement drug development. C5 blockade inhibits generation of C5a and C5b, the former an anaphylatoxin, the latter the nidus for formation of the pro-inflammatory membrane attack complex. In order to use anti-complement drugs in common complement-driven diseases, more affordable and equally effective therapeutics are needed. To address this, we explored complement inhibition downstream of C5. Novel blocking mAbs targeting C7 and/or the C5b-7 complex were generated, identified using high throughput functional assays and specificity confirmed by immunochemical assays and surface plasmon resonance (SPR). Selected mAbs were tested in rodents to characterize pharmacokinetics, and therapeutic capacity. Administration of a mouse C7-selective mAb to wildtype mice, or a human C7 specific mAb to C7-deficient mice reconstituted with human C7, completely inhibited serum lytic activity for >48 h. The C5b-7 complex selective mAb 2H2, most active in rat serum, efficiently inhibited serum lytic activity in vivo for over a week from a single low dose (10 mg/kg); this mAb effectively blocked disease and protected muscle endplates from destruction in a rat myasthenia model. Targeting C7 and C7-containing terminal pathway intermediates is an innovative therapeutic approach, allowing lower drug dose and lower product cost, that will facilitate the expansion of complement therapeutics to common diseases.