Junction Of Muscle Research Articles

Discovery of Protein Degradation Machinery at the Desmosome Reveals Novel Triggers of the Desmosomal Disease, Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic‐based cardiac disease which is characterized by right/bi‐ventricular dysfunction, fibro‐fatty replacement of the myocardium and ventricular arrhythmias, leading to sudden death in young people and athletes. ARVC is termed a “disease of the desmosome” (mechanical cell‐cell junction) as 40% of ARVC patients harbor mutations and/or loss of desmosomal components. However, limited information exists on how cardiac desmosomal protein loss/degradation triggers ARVC. Through a yeast‐two‐hybrid screen in an adult human heart cDNA library, we uncovered a new interaction between the desmosomal protein, desmoplakin (DSP, N terminus) and a protein degradation machinery component, CSN6 (COP9 signalosome subunit 6). Traditionally, CSN proteins are thought to prevent proteins from being degraded; however, a role for CSN6 in the heart is undefined. We show that CSN6 localizes to desmosomal junctions in cardiac muscle and structurally complex with desmoplakin, suggesting that the novel hypothesis that CSN6 may protect the cardiac desmosome from degradation and loss/destablization this pathway may lead to ARVC. Using two novel cardiac‐specific CSN6 knockout (CSN6‐KO) mice, we further show that CSN6 loss accelerates desmosome destruction and triggers ARVC, as CSN6‐KO hearts exhibit selective loss of desmosomal proteins and recapitulate the key cardiomyopathy features and sudden death associated with ARVC, similar to classic mouse model of ARVC (DSP‐cKO). CSN6‐KO and DSP‐cKO hearts also harbor underlying hyper‐accumulation of protein degradation machinery specifically at cell‐cell junctions. Restoration of CSN function by the protein degradation inhibitor (MLN4924) could rescue the loss of desmosomal protein expression associated with CSN6 loss in vitro, further highlighting that the cardiac desmosome is regulated by protein degradation. CSN6 pathways are also relevant to human ARVC as a N‐terminus desmoplakin mutation (R315C) identified in a human ARVC patient was sufficient to abrogate DSP‐CSN6 binding and result in loss of CSN6 localization at the cell‐cell junction in cardiac biopsies. DSP R315C knock‐in mice also exhibited cardiac CSN6 reduction and early electrical defects found in ARVC. We altogether identify CSN6 as a novel “brake” to prevent desmosomal protein degradation, and reveal that disruption of this complex is an important “accelerator” of sudden death and ARVC.Support or Funding InformationAmerican Heart Association Postdoctoral Fellowship 17POST33670205This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 112: Vegf-receptor Inhibitor Impedes Macrophage Function At The End Target Muscle After Nerve Injury

PURPOSE: Recovery in peripheral nerve injuries is limited by the critical time point beyond which muscle denervation is irreversible. A major component of functional recovery is muscle reinnervation, which depends on terminal Schwann cells (tSCs) at the neuromuscular junction (NMJ). The hallmark feature of the tSC response to injury is cytoplasmic process elongation that guides axon growth. In response to injury, macrophages secrete Vascular Endothelial Growth Factor (VEGF) which may induce angiogenesis to support tSC process extension. We hypothesize that VEGF inhibition impedes tSC function at the NMJ, which in turn will impede reinnervation of the end target muscle. METHODS: Sciatic nerve transection with immediate repair was performed in C57BL/6 mice. To evaluate whether VEGF inhibition impedes end target muscle reinnervation, Cabozantinib, a VEGF Receptor-2 (VEGFR-2) inhibitor, was administered to mice via oral gavage for seven days following nerve injury to suppress VEGF secretion. For the control group, saline was given for the same duration. Mice were sacrificed on 14, 21, 28 and 42 days after injury (n = 3 per time point), and the leg muscles were harvested. Immunohistochemical stains for NF200 (axon marker), S100 (Schwann cell marker), VEGF, CD68 (macrophage marker), and bungarotoxin (BTX, motor endplate marker) were used on extensor digitorum longus and tibialis anterior muscle sections. Functional innervation was determined by two methods: 1) immunohistochemical staining with NF-200 for axonal reinnervation into motor endplates marked by BTX in whole muscle mounts and 2) electrophysiological stimulation of the sciatic nerve to test tibialis anterior CMAP amplitude. RESULTS (PRELIMINARY): After nerve injury, extracellular and intracellular VEGF expression increased significantly at postoperative day (POD) 3 compared to non-injured muscle, and then peaked at POD 12. At two weeks (POD14) after injury, motor end plates were 41.2% fully innervated and 37.1% partially innervated in the control group, while the Cabozantinib group had only 7.5% fully innervated and 4.3% partially innervated motor end plates. VEGF expression at POD 14 was greatly different between the two groups (11.48 percent pixels vs 3.49 percent pixels, p <0.001). By POD 21, however, 97% of the motor end plates in the Control group and 93.2% in the Cabozantinib group had reached full innervation. Axonal sprouting between motor end plates was more prevalent in the control group (46.9% vs 18.5%). VEGF expression decreased in both groups by POD 21 and POD 28, while CD68 positive cells co-stained with VEGF remained significantly higher in the control group at POD 28. Electrophysiological stimulation at 10 Hz showed significant differences at POD 21 (t test, mean difference 2.151 mV, p = 0.047) but no differences at POD 42 (t test -0.822, p = 421). CONCLUSIONS: Drug induced VEGF inhibition had a marked but temporary effect on the functional recovery of denervated muscle after nerve injury. Although the differences between the two experimental groups became nonsignificant over time, the effects seen at the neuromuscular junction of the target muscles highlighted the unrecognized role of VEGF in macrophages on the functional recovery after nerve injury.

The binding interactions that maintain excitation-contraction coupling junctions in skeletal muscle.

Calcium for contraction of skeletal muscles is released via tetrameric ryanodine receptor (RYR1) channels of the sarcoplasmic reticulum (SR), which are assembled in ordered arrays called couplons at junctions where the SR abuts T tubules or plasmalemma. Voltage-gated Ca2+ (CaV1.1) channels, found in tubules or plasmalemma, form symmetric complexes called CaV tetrads that associate with and activate underlying RYR tetramers during membrane depolarization by conveying a conformational change. Intriguingly, CaV tetrads regularly skip every other RYR tetramer within the array; therefore, the RYRs underlying tetrads (named V), but not the voltage sensor-lacking (C) RYRs, should be activated by depolarization. Here we hypothesize that the checkerboard association is maintained solely by reversible binary interactions between CaVs and RYRs and test this hypothesis using a quantitative model of the energies that govern CaV1.1-RYR1 binding, which are assumed to depend on number and location of bound CaVs. A Monte Carlo simulation generates large statistical samples and distributions of state variables that can be compared with quantitative features in freeze-fracture images of couplons from various sources. This analysis reveals two necessary model features: (1) the energy of a tetramer must have wells at low and high occupation by CaVs, so that CaVs positively cooperate in binding RYR (an allosteric effect), and (2) a large energy penalty results when two CaVs bind simultaneously to adjacent RYR protomers in adjacent tetramers (a steric clash). Under the hypothesis, V and C channels will eventually reverse roles. Role reversal justifies the presence of sensor-lacking C channels, as a structural and functional reserve for control of muscle contraction.

The Binding Interactions that Maintain the Ec Coupling Junctions in Skeletal Muscle

The Binding Interactions that Maintain the Ec Coupling Junctions in Skeletal Muscle

Histologic characteristics of the subscapularis tendon from muscle to bone: reference to subscapularis lesions

Although subscapularis tendon lesions seem to differ from those of the supraspinatus tendon, the features they have in common suggest that the subscapularis tendon may also have 2 distinct layers. Our aim was therefore to characterize the histologic structure of the subscapularis tendon from its humeral insertion point to the musculotendinous junction. A histologic study was performed on 10 autopsy samples. The subscapularis tendon was extracted in one piece from the musculotendinous junction to the humeral insertion point and was prepared using standard (hematoxylin-eosin-saffron) staining. Histologic analysis revealed 2 fibrous layers, distinguishable by the orientation of the collagen bundles. The deep layer was thinner and composed of parallel longitudinal collagen fibers inserting onto the lesser tuberosity flush with the cartilage. The superficial layer was thicker and composed of interdigitated collagen bundles inserting onto the lesser and the greater tuberosity after splitting into 2 bands, 1 lining the floor of the bicipital groove, and the other extending over the long head of the biceps tendon across the groove. Each layer formed an independent musculotendinous junction in the subscapularis muscle. The subscapularis tendon is composed of 2 distinct fibrous layers, just like the supraspinatus tendon, but arranged differently. The superficial layer of the subscapularis tendon passes across the bicipital groove and forms a fibrous ring around the long head of the biceps tendon that stabilizes the latter in the bicipital groove. These results explain some of the specific features of subscapularis tears described in the literature, namely, delamination and biceps subluxation.

Extraocular muscles involved in convergence are innervated by an additional set of palisade endings that may differ in their excitability: A human study.

Palisade endings are located at the myotendinous junction of extraocular muscles in most mammals. Irrespective of their unclarified function as motor or sensory nerve endings, a specialized role in convergence is proposed, based on their high number in the medial rectus muscle (MR). Further support comes from a study in monkey demonstrating that only the MR and inferior rectus muscle (IR) contain an additional population of palisade endings that express the calcium-binding protein calretinin (CR) in addition to choline acetyltransferase (ChAT). Here we studied, whether CR-positive palisade endings are present in human as well and confined to extraocular muscles most active during convergence. The systematic analysis of all eye muscles of 17 human specimen revealed that only the MR and IR contain an additional population of CR-positive palisade endings and multiple en-grappe endings, which target non-twitch muscle fibers along their whole length. Approximately 80% of all palisade endings in the MR expressed CR. Furthermore, the intrafusal muscle fibers of some muscle spindles in the MR were innervated by CR-positive annulospiral nerve endings that transmit the signals of muscle length changes to the brain. All extraocular muscles contained few thin CR-positive, but ChAT-negative nerve fibers, possibly representing free sensory or autonomic endings arising from the trigeminal ganglion. As in monkey, in the medial periphery of the human oculomotor nucleus ChAT-positive neurons were found to co-express CR. Therefore these neurons most likely represent the cell bodies of CR-positive palisade endings in the MR. Unlike in monkey, these neurons do not lie within a compact cell group, but are more scattered. In conclusion, the MR and IR in human contain two histochemically different populations of palisade and multiple endings that may contribute to ocular alignment and convergence in a different way.

Isolated rupture of the vastus lateralis tendon: Case report

We report a patient with a history of bariatric surgery who have an isolated rupture of the unilateral vastus lateralis tendon from the adhesion site on the patella. The diagnosis was missed in this case and was placed with magnetic resonance (MR) investigation of the patient during follow-up. A 40-year-old male patient felt pain in his knee after an awkward movement. Initially, because of his bariatric surgery, he had some other complaints, the diagnosis was missed, and the patient had conservative treatment and physical therapy applied. When complaints did not improve, MR was taken, and the vastus lateralis tendon was identified to have ruptured from the patella in isolation. Treated surgically, the patient had no new rupture on check-up performed 1 year later. The patient is fully healed. After the bariatric surgery, some metabolic changes and weakness at the osteotendinous junction of muscles may occur. Isolated vastus lateralis rupture is rarely observed, and diagnosis may be missed. In these patients, surgical treatment is required and other accompanying pathologies of the knee should also be treated at the same time.

Effects of Repeated Eyelid Injections with Botulinum Toxin A on Innervation of Treated Muscles in Patients with Blepharospasm

ABSTRACTPurpose: To assess changes in innervation and muscle morphology after repeated botulinum toxin A injections in subjects with benign essential blepharospasm.Methods: Surgical waste specimens were processed for histologic examination of nerve fibers, neuromuscular junctions, fiber size, and central nucleation and compared to age matched controls and to two subjects with blepharospasm that had not received botulinum toxin A injections.Results: There was a significant increase in amount of nerve fibers and numbers of neuromuscular junctions in the orbicularis oculi muscles from subjects with blepharospasm treated repetitively with botulinum toxin A. In addition there was a significant decrease in mean muscle fiber cross-sectional area and an increase in central nucleation. The specimens from the subjects with only blepharospasm had the same density of nerves but had intermediate levels of neuromuscular junctions.Conclusions: These data suggest that repeated injections of botulinum toxin A has an effect on nerve and neuromuscular junction numbers, which are partly mirrored in orbicularis oculi muscle from subjects with blepharospasm only. These studies suggest the potential for modulating these changes in order to extend the duration of effectiveness of botulinum toxin.

Matching Participants for Triceps Surae Muscle Strength and Tendon Stiffness Does Not Eliminate Age-Related Differences in Mechanical Power Output During Jumping.

Reductions in muscular power output and performance during multi-joint motor tasks with aging have often been associated with muscle weakness. This study aimed to examine if matching younger and middle-aged adults for triceps surae (TS) muscle strength and tendon stiffness eliminates age-related differences in muscular power production during drop jump. The maximal ankle plantar flexion moment and gastrocnemius medialis tendon stiffness of 29 middle-aged (40–67 years) and 26 younger (18–30 years) healthy physically active male adults were assessed during isometric voluntary ankle plantar flexion contractions using simultaneous dynamometry and ultrasonography. The elongation of the tendon during the loading phase was assessed by digitizing the myotendinous junction of the gastrocnemius medialis muscle. Eight younger (23 ± 3 years) and eight middle-aged (54 ± 7 years) adults from the larger subject pool were matched for TS muscle strength and tendon stiffness (plantar flexion moment young: 3.1 ± 0.4 Nm/kg; middle-aged: 3.2 ± 0.5 Nm/kg; tendon stiffness: 553 ± 97 vs. 572 ± 100 N/mm) and then performed series of drop jumps from different box heights (13, 23, 33, and 39 cm) onto a force plate (sampling frequency 1000 Hz). The matched young and middle-aged adults showed similar drop jump heights for all conditions (from lowest to highest box height: 18.0 ± 3.7 vs. 19.7 ± 4.8 cm; 22.6 ± 4.2 vs. 22.9 ± 4.9 cm; 24.8 ± 3.8 vs. 23.5 ± 4.9 cm; 25.2 ± 6.2 vs. 22.7 ± 5.0 cm). However, middle-aged adults showed longer ground contact times (on average 36%), lower vertical ground reaction forces (36%) and hence lower average mechanical power (from lowest to highest box height: 2266 ± 563 vs. 1498 ± 545 W; 3563 ± 774 vs. 2222 ± 320 W; 4360 ± 658 vs. 2475 ± 528 W; 5008 ± 919 vs. 3034 ± 435 W) independent of box height. Further, leg stiffness was lower (48%) in middle-aged compared to younger adults for all jumping conditions and we found significant correlations between average mechanical power and leg stiffness (0.70 ≤ r ≤ 0.83; p < 0.01). Thus, while jumping performance appears to be unaffected when leg extensor muscle strength and tendon stiffness are maintained, the reduced muscular power output during lower limb multi-joint tasks seen with aging may be due to age-related changes in motor task execution strategy rather than due to muscle weakness.

A Review on Nerve Conduction Studies

Electrophysiological measurements are indispensable tool for investigating the functional integrity of peripheral nerves in both clinical and laboratory environments. Nerve conduction study is a property of both the axons and myelin sheath. The nerve conduction velocity is highly dependent on rapid signal propagation enabled by myelination. The compound motor action potential (CMAP) which correlates with the number of functional axons is an indicator for axonal damage when significantly reduced. Because nerve conduction velocity (NCV) is sensitive to both myelin sheath and axonal changes, it is an excellent measure of the function of the peripheral nervous system. Nerve conduction studies may be diagnostically helpful in patients suspected of having almost any peripheral nervous system disorder including disorders of nerve roots, peripheral nerves, muscle and neuromuscular junction. Cranial nerves and spinal cord function may also be assessed. Here, we mentioned the components and properties of nerve conduction study.

Social isolation alters ultrasonic vocalizations but not thyroarytenoid neuromuscular junctions in old rats.

Age-related muscle atrophy of the laryngeal muscles contributes to presbyphonia. Remodeling of the neuromuscular junction is one aspect underlying age-related muscle atrophy. Although muscle disuse has been shown to exacerbate age-related neuromuscular changes in the limb muscles, it is unknown if reduced vocal use has a similar effect in the laryngeal muscles. The objective of this study was to examine the use of social isolation as a novel method to reduce vocal use in old rats-and the impact of that reduced vocal use on ultrasonic vocalization acoustics and neuromuscular junction morphology in the thyroarytenoid muscle. Animal group comparison. Old F344/BN rats (31 months of age) were socially isolated (n = 8) or communally housed (n = 8) for 8 weeks. Effect of housing condition on ultrasonic vocalization acoustics was assessed by calculating the changes in vocalization fundamental frequency and amplitude from baseline to 8 weeks. Neuromuscular junction morphology was measured in the lateral and medial portions of the thyroarytenoid muscle at the conclusion of the experiment. Vocalization amplitude decreased by a mean of -4.4 dB (standard deviation [SD], 4.49) after social isolation, whereas amplitude increased by a mean of 5.7 dB (SD, 5.07) in the communally housed rats (P = 0.002). There was no significant difference in the change in fundamental frequency between groups. Furthermore, there were no group differences in any measure of neuromuscular junction morphology. These results suggest that neuromuscular junctions in the thyroarytenoid muscle of old rats are unaffected by 8 weeks of social isolation, despite functional changes in vocalizations. NA Laryngoscope, 129:E9-E14, 2019.

Effects of neuromuscular presynaptic muscarinic M1 receptor blockade on rocuronium-induced neuromuscular blockade in immobilized tibialis anterior muscles.

SummaryThis in vivo study tested the hypothesis that the modulation of acetylcholine (ACh) release by the M1 muscarinic receptor (mAChR) in the neuromuscular junction of disused muscles may affect the tensions of the muscles during the neuromuscular monitoring of a rocuronium‐induced neuromuscular block and compared the results with those obtained from normal muscles. A total of 20 C57BL/6 (wild‐type) and 10 α7 knock out (α7KO) mice were used in this experiment. As a pre‐experimental procedure, knee and ankle joints of right hind limbs were fixed by needle pinning at the 90° flexed position. After 2 weeks, the main experiment was performed. Both tendons of the tibialis anterior (TA) muscles were obtained, and the muscle tensions were recorded while the dose‐responses of rocuronium were measured three times in the same mouse by the serial administration of pirenzepine (0, 0.001 and 0.01 μg/g). Weight losses were observed after 2 weeks of immobilization in both groups, and a decrease in the mass of TA muscles at the immobilized side was observed compared to those of the contralateral nonimmobilized side. Tension depression of the TA muscles at immobilized side of the α7KO group was faster than those of the wild‐type group, but these differences decreased after the administration of pirenzepine. The tension depressions were similar regardless of the pirenzepine doses at the same side in the group. Tension depression may become more rapid in the α7 AChR‐expressed disused muscles by the decreased release of ACh release upon neuronal firing by the blockade of facilitatory M1 mAChR

Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in Drosophila.

We use the myotendinous junction of Drosophila flight muscles to explore why many integrin associated proteins (IAPs) are needed and how their function is coordinated. These muscles revealed new functions for IAPs not required for viability: Focal Adhesion Kinase (FAK), RSU1, tensin and vinculin. Genetic interactions demonstrated a balance between positive and negative activities, with vinculin and tensin positively regulating adhesion, while FAK inhibits elevation of integrin activity by tensin, and RSU1 keeps PINCH activity in check. The molecular composition of myofibril termini resolves into 4 distinct layers, one of which is built by a mechanotransduction cascade: vinculin facilitates mechanical opening of filamin, which works with the Arp2/3 activator WASH to build an actin-rich layer positioned between integrins and the first sarcomere. Thus, integration of IAP activity is needed to build the complex architecture of the myotendinous junction, linking the membrane anchor to the sarcomere.

BPC 157 Therapy Heals Tendon Muscle Junction in Rats

AimAfter tendon was separated from muscle in the right quadriceps muscle in rats, we focused on the tendon‐muscle junction healing and therapy with the stable gastric pentadecapeptide BPC 157. Noteworthy, BPC 157 is an original antiulcer peptide, stable in human gastric juice, used in ulcerative colitis and now multiple sclerosis trials), with particular wound healing ability: healing of transected or crushed muscle, transected or detached tendon, and transected ligament, demonstrated macro/microscopically, biomechanically and functionally (i.e., absent leg contracture), with parenteral and peroral therapy application (Curr Pharm Des. 2017;23(27):4012–4028; J Orthop Res. 2006 May;24(5):1109–17; J Orthop Res. 2006 May;24(5):982–9; J Orthop Res. 2003 Nov;21(6):976–83).MethodsUnder deep rat anesthesia tendon muscle junction of right quadriceps muscle was presented, and without direct transection of tendon muscle junction, the tendon was gently separated from muscle fibres. Medication was BPC 157 (10 ug or 10 ng/kg/day, intraperitoneally or in drinking water (0.16ug or 0.16ng/ml; 12ml/rat/day) while controls received an equivolume of saline (5 ml/kg intraperitoneally) or drinking water only. Macro/microscopical, biomechanical assessment was at 1st, 2nd, 4th, and 6th week after surgery, while functional presentation (walking patterns, leg contracture) was daily assessed.ResultsGross assessment reveals in quadriceps muscle a huge defect between tendon and muscle sustainably progressing throughout the experiment and failed healing. By contrast, in rats underwent tendon muscle junction separation that received BPC 157, the healing was continuously presented. Since very beginning, BPC 157 rats presented defect markedly less and finally grossly reestablished tendon muscle junction (Fig. 1). Also, controls exhibited severe leg contracture, and tottering walk, which were fully counteracted by BPC 157 therapy. These results are completely confirmed by microscopy. Illustratively, at 2nd week controls showed edema of the tendon with moderate lymphocytic infiltrate and formation of granulation tissue, while BPC 157 rats exhibited only minimal lymphocytic infiltrate with no granulation tissue formation in all pentadecapeptide BPC 157 treated animals and with better already reestablished myotendinous junction structures (Fig. 2). Likewise, biomechanic assessment demonstrated only a poor recovery of muscle strength in controls, while a full recovery ensues in BPC 157 treated rats.ConclusionIn addition to the therapy of the injured muscle and tendon, BPC 157 therapy could also specifically benefit the injuries of tendon muscle junction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondrial protein S-nitrosation protects against ischemia reperfusion-induced denervation at neuromuscular junction in skeletal muscle.

Deterioration of neuromuscular junction (NMJ) integrity and function is causal to muscle atrophy and frailty, ultimately hindering quality of life and increasing the risk of death. In particular, NMJ is vulnerable to ischemia reperfusion (IR) injury when blood flow is restricted followed by restoration. However, little is known about the underlying mechanism(s) and hence the lack of effective interventions. New evidence suggests that mitochondrial oxidative stress plays a causal role in IR injury, which can be precluded by enhancing mitochondrial protein S-nitrosation (SNO). To elucidate the role of IR and mitochondrial protein SNO in skeletal muscle, we utilized a clinically relevant model and showed that IR resulted in significant muscle and motor nerve injuries with evidence of elevated muscle creatine kinase in the serum, denervation at NMJ, myofiber degeneration and regeneration, as well as muscle atrophy. Interestingly, we observed that neuromuscular transmission improved prior to muscle recovery, suggesting the importance of the motor nerve in muscle functional recovery. Injection of a mitochondria-targeted S-nitrosation enhancing agent, MitoSNO, into ischemic muscle prior to reperfusion reduced mitochondrial oxidative stress in the motor nerve and NMJ, attenuated denervation at NMJ, and resulted in accelerated functional recovery of the muscle. These findings demonstrate that enhancing mitochondrial protein SNO protects against IR-induced denervation at NMJ in skeletal muscle and accelerates functional regeneration. This could be an efficacious intervention for protecting neuromuscular injury under the condition of IR and other related pathological conditions.

Age-related remodelling of the myotendinous junction in the mouse soleus muscle

The age-related loss of muscle mass and function predominantly affect muscles of the lower limbs and have largely been associated with decline in muscle fibre size and number, although the exact mechanisms underlying these losses are poorly understood. In addition, consistent reports that the loss of muscle strength exceeds that which can be explained by declines in muscle mass has widened the search for causes of sarcopenia to include supporting tissues such as the extracellular matrix and tendons. Although the changes to both muscle and tendon with age are well characterised, little work has focused on the interface between these two tissues, the myotendinous junction (MTJ). Given the crucial role for this structure in force transfer between muscle and tendon, we asked whether the myotendinous junction underwent structural changes with age in lower limb muscle. We used whole muscle to assess gross muscle and tendon morphology, and immunohistochemistry to determine fibre and MTJ profile number in young (6 months), middle aged (18 months) and elderly (24 months) C57BL/6 female mice. MTJ length was quantified using serial cross sections of the soleus muscle. We found an apparent 3.5-fold increase in MTJ profiles per cross section with no increase in fibre number in old mice, and found this to be a result of a doubling in length of the MTJ region with age. This coincided with an increase in proximal tendon length (31%), as well as an increase in collagen deposition between 6 and 24-months of age consistent with an expansion of the fibre termination area. These findings uncover a previously undescribed effect of ageing on the MTJ and open up new lines of investigation into the role of this structure in the age-related loss of muscle function.

Myasthenia Gravis – A Review of Current Therapeutic Options

Myasthenia gravis (MG) is an autoimmune disorder that leads to skeletal muscle weakness and fatigue. The autoimmune attack is caused by autoantibodies against the acetylcholine postsynaptic receptors at the neuromuscular junction of skeletal muscles. However, other antigenic targets that are components of the neuromuscular junction have also been implicated in the pathogenesis of MG. The current standard of care is immunosuppressive therapy; however, many existing therapeutic options have not been validated for use in MG in large randomised controlled trials. Furthermore, around 10% of patients with generalised MG are refractory to treatment. The complement system is involved in numerous inflammatory, neurodegenerative and autoimmune diseases, and is a key factor in the pathogenesis of acetylcholine receptor antibody-related MG. Targeting complement and other components involved in the underlying pathogenesis of the disease may provide useful treatment options, particularly for refractory patients.

Anatomical and Functional Changes to the Colonic Neuromuscular Compartment after Experimental Spinal Cord Injury.

A profound reduction in colorectal transit time accompanies spinal cord injury (SCI), yet the colonic alterations after SCI have yet to be understood fully. The loss of descending supraspinal input to lumbosacral neural circuits innervating the colon is recognized as one causal mechanism. Remodeling of the colonic enteric nervous system/smooth muscle junction in response to inflammation, however, is recognized as one factor leading to colonic dysmotility in other pathophysiological models. We investigated the alterations to the neuromuscular junction in rats with experimental high-thoracic (T3) SCI. One day to three weeks post-injury, both injured and age-matched controls underwent in vivo experimentation followed by tissue harvest for histological evaluation. Spontaneous colonic contractions were reduced significantly in the proximal and distal colon of T3-SCI rats. Histological evaluation of proximal and distal colon demonstrated significant reductions of colonic mucosal crypt depth and width. Markers of intestinal inflammation were assayed by qRT-PCR. Specifically, Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) mRNA was acutely elevated after T3-SCI. Smooth muscle thickness and collagen content of the colon were increased significantly in T3-SCI rats. Colonic cross sections immunohistochemically processed for the pan-neuronal marker HuC/D displayed a significant decrease in colonic enteric neuron density that became more pronounced at three weeks after injury. Our data suggest that post-SCI inflammation and remodeling of the enteric neuromuscular compartment accompanies SCI. These morphological changes may provoke the diminished colonic motility that occurs during this same period, possibly through the disruption of intrinsic neuromuscular control of the colon.

Imaging Analysis of the Neuromuscular Junction in Dystrophic Muscle.

Duchenne muscular dystrophy (DMD), caused by the absence of the protein dystrophin, is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but there is little information to determine if damage from contraction-induced injury also occurs at or near the nerve terminal axon. Interestingly, both human patients and the mouse model for DMD (the mdx mouse) present fragmented neuromuscular junction (NMJ) morphology. Studies of mdx mice have revealed presynaptic and postsynaptic abnormalities, nerve terminal discontinuity, as well as increased susceptibility of the NMJ to contraction-induced injury with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyography. Focusing on the NMJ as a contributor to functional deficits in the muscle represents a paradigm shift from the more prevalent myocentric perspectives. Further studies are needed to determine the extent to which the nerve-muscle interaction is disrupted in DMD and the role of the NMJ in the dystrophic progression. This chapter lists the tools needed for nerve terminal and NMJ structural analysis using fluorescence imaging, and provides a step-by-step outline for how to stain, image, and analyze the NMJ in skeletal muscle, with specific attention to mdx muscle.

Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools.

The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChR-Abs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to native-structured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases.