Skeletal Muscle Acetylcholine Receptor Research Articles

Recombinant human neuregulin-1 alleviates immobilization-induced neuromuscular dysfunction via neuregulin-1/ErbB signaling pathway in rat

Immobilization-induced Neuromuscular Dysfunction (NMD) increases morbidity and mortality of patients in Intensive Care Units. However, the underlying mechanism of NMD remain poorly elucidated which limited the development of therapeutic method for NMD. Here we developed an immobilization rat model and tested the hypothesis that decreased expression of NRG-1, abnormal expression and distribution of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle caused by immobilization can lead to NMD. To investigate the role of NRG-1/ErbB pathway on immobilization-induced NMD, exogenous recombinant human neuregulin-1 (rhNRG-1) was used to increase the expression of NRG-1 in skeletal muscle during immobilization. It was observed rhNRG-1 significantly alleviated the muscle loss and enhanced the expression of ε-nAChR, while diminished the expression of γ- and α7-nAChR and NMD. Interestingly, ErbB inhibitor PD158780 blocked the protective effects of rhNRG-1. Collectively, the results of present study suggested that rhNRG-1 attenuated immobilization-induced muscle loss and NMD, suppressed γ- and α7-nAChR production, enhanced ε-nAChR synthesis via activating NRG-1/ErbB pathway. Taken together, our findings provide novel insights into NMD contribution, suggesting that the rhNRG-1 is a promising therapy to protect against immobilization-induced myopathy.

Nicotinic acetylcholine receptor in skeletal muscle

This article is a systematic review carried out in order to recognize the importance of the nicotinic acetylcholine receptor in skeletal muscle and the relationship between this and the development of human characteristics such as the development of strength. Strength is one of the fundamental motor skills of athletic performance, in training there are neuromuscular adaptations and metabolic alterations that improve performance during competition, physical strength training, improves the performance of athletes during the competitive period, as well Also the plasticity of the skeletal muscle tissue, thus allowing the manifestation of different adaptations to the functional demands of each athlete linked to processes of a mechanical, metabolic and electrophysiological character specific to any sports modality.

Human skeletal muscle acetylcholine receptor gene expression in elderly males performing heavy resistance exercise.

Muscle fiber denervation is a major contributor to the decline in muscle mass and function during aging. Heavy resistance exercise is an effective tool for increasing muscle mass and strength, but whether it can rescue denervated muscle fibers remains unclear. Therefore, the purpose of this study was to investigate the potential of heavy resistance exercise to modify indices of denervation in healthy elderly individuals. Thirty-eight healthy elderly men (72 ± 5 yr) underwent 16 wk of heavy resistance exercise, whereas 20 healthy elderly men (72 ± 6 yr) served as nonexercising sedentary controls. Muscle biopsies were obtained pre and post training, and midway at 8 wk. Biopsies were analyzed by immunofluorescence for the prevalence of myofibers expressing embryonic myosin [embryonic myosin heavy chain (MyHCe)], neonatal myosin [neonatal myosin heavy chain (MyHCn)], nestin, and neural cell adhesion molecule (NCAM), and by RT-qPCR for gene expression levels of acetylcholine receptor (AChR) subunits, MyHCn, MyHCe, p16, and Ki67. In addition to increases in strength and type II fiber hypertrophy, heavy resistance exercise training led to a decrease in AChR α1 and ε subunit messenger RNA (mRNA; at 8 wk). Changes in gene expression levels of the α1 and ε AChR subunits with 8 wk of heavy resistance exercise supports the role of this type of exercise in targeting stability of the neuromuscular junction. The number of fibers positive for NCAM, nestin, and MyHCn was not affected, suggesting that a longer timeframe is needed for adaptations to manifest at the protein level.

Alleviating Sepsis-Induced Neuromuscular Dysfunction Linked With Acetylcholine Receptors by Agrin

BackgroundAbnormal expression and distribution of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle caused by sepsis can lead to neuromuscular dysfunction. Here, we asked whether neural agrin regulates nAChRs to ameliorate muscle function, which could be associated with the agrin/muscle-specific kinase pathway. MethodsRats were subjected to cecal ligation and puncture (CLP) group, sham group, or control group to observe the alteration caused by sepsis. To verify the effect of improving function, rats were injected with agrin or normal saline intramuscularly after CLP. Electromyogram was used to measure neuromuscular function. Cytokines levels of serum and the expression of related proteins and mRNA were tested after treatment. ResultsCompared with the rats in control or sham group, CLP-treated rats showed an acute inflammatory status and a reduction of neuromuscular dysfunction in tibialis anterior muscle, which was associated with abnormal expression in agrin/muscle-specific kinase pathway and increased expression of γ- and α7-nAChR. Exogenous agrin alleviated neuromuscular dysfunction and decreased the expression of γ- and α7-nAChR through agrin-related signaling pathway. ConclusionsThe decreased expression of agrin may lead to skeletal muscle dysfunction. Early enhancement of intramuscular agrin levels after sepsis may be a potential strategy for the treatment of sepsis-induced muscle dysfunction.

The sympathetic nervous system regulates skeletal muscle motor innervation and acetylcholine receptor stability.

Symptoms of autonomic failure are frequently the presentation of advanced age and neurodegenerative diseases that impair adaptation to common physiologic stressors. The aim of this work was to examine the interaction between the sympathetic and motor nervous system, the involvement of the sympathetic nervous system (SNS) in neuromuscular junction (NMJ) presynaptic motor function, the stability of postsynaptic molecular organization, and the skeletal muscle composition and function. Since muscle weakness is a symptom of diseases characterized by autonomic dysfunction, we studied the impact of regional sympathetic ablation on muscle motor innervation by using transcriptome analysis, retrograde tracing of the sympathetic outflow to the skeletal muscle, confocal and electron microscopy, NMJ transmission by electrophysiological methods, protein analysis, and state of the art microsurgical techniques, in C57BL6, MuRF1KO and Thy-1 mice. We found that the SNS regulates motor nerve synaptic vesicle release, skeletal muscle transcriptome, muscle force generated by motor nerve activity, axonal neurofilament phosphorylation, myelin thickness, and myofibre subtype composition and CSA. The SNS also modulates the levels of postsynaptic membrane acetylcholine receptor by regulating the Gαi2 -Hdac4-Myogenin-MuRF1pathway, which is prevented by the overexpression of the guanine nucleotide-binding protein Gαi2 (Q205L), a constitutively active mutant G protein subunit. The SNS regulates NMJ transmission, maintains optimal Gαi2 expression, and prevents any increase in Hdac4, myogenin, MuRF1, and miR-206. SNS ablation leads to upregulation of MuRF1, muscle atrophy, and downregulation of postsynaptic AChR. Our findings are relevant to clinical conditions characterized by progressive decline of sympathetic innervation, such as neurodegenerative diseases and aging.

Natural polymeric 3-alkylpyridinium salt affects vertebrate skeletal muscle contractility by preferentially blocking neuromuscular transmission

The effects of natural polymeric alkylpyridinium salt (nPoly-3-APS), a potent acetylcholinesterase inhibitor isolated from the marine sponge Reniera sarai, were studied on isolated mouse phrenic nerve-hemidiaphragm muscle preparations using electrophysiological approaches. nPoly-3-APS inhibited nerve-evoked isometric muscle twitch and tetanic contraction in a concentration-dependent manner (IC50=29.4μM and 18.5μM, respectively) and produced a 30–44% decrease of directly muscle-elicited twitch and tetanus amplitudes at 54.4μM. Additionally, nPoly-3-APS (9.1–27.2μM) markedly decreased the amplitude of miniature endplate potentials, while their frequency was only affected at the highest concentration used. Endplate potentials were also inhibited by nPoly-3-APS in a concentration-dependent manner (IC50=20.1μM), without significant change in the resting membrane potential of muscle fibers (up to 54.4μM). In conclusion, our results show, for the first time, that nPoly-3-APS preferentially blocks the neuromuscular transmission, in vitro, by a non-depolarizing mechanism. This strongly suggests that the in vivo toxicity of nPoly-3-APS mainly occurs through an antagonist action of the compound on nicotinic acetylcholine receptors of skeletal muscles.

53. Palmitoylethanolamide may have a role in enhancing pulmonary function and muscular strength in ALS patients

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease.

Acetylcholine receptors from human muscle as pharmacological targets for ALS therapy

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. By microtransplanting muscle membranes from selected ALS patients into Xenopus oocytes, we show that PEA reduces the desensitization of acetylcholine-evoked currents after repetitive neurotransmitter application (i.e., rundown). The same effect was observed using muscle samples from denervated (non-ALS) control patients. The expression of human recombinant α1β1γδ (γ-AChRs) and α1β1εδ AChRs (ε-AChRs) in Xenopus oocytes revealed that PEA selectively affected the rundown of ACh currents in ε-AChRs. A clear up-regulation of the α1 subunit in muscle from ALS patients compared with that from non-ALS patients was found by quantitative PCR, but no differential expression was found for other subunits. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease.

Current Treatment, Emerging Translational Therapies, and New Therapeutic Targets for Autoimmune Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease associated with the production of autoantibodies against 1) the skeletal muscle acetylcholine receptor; 2) muscle-specific kinase, a receptor tyrosine kinase critical for the maintenance of neuromuscular synapses; 3) low-density lipoprotein receptor-related protein 4, an important molecular binding partner for muscle-specific kinase; and 4) other muscle endplate proteins. In addition to the profile of autoantibodies, MG may be classified according the location of the affected muscles (ocular vs generalized), the age of symptom onset, and the nature of thymic pathology. Immunopathologic events leading to the production of autoantibodies differ in the various disease subtypes. Advances in our knowledge of the immunopathogenesis of the subtypes of MG will allow for directed utilization of the ever-growing repertoire of therapeutic agents that target distinct nodes in the immune pathway relevant to the initiation and maintenance of autoimmune disease. In this review, we examine the pathogenesis of MG subtypes, current treatment options, and emerging new treatments and therapeutic targets.

IL-10 is critical in the regulatory function of CD1dhiCD5+ B cells in Experimental Autoimmune Myasthenia Gravis (THER2P.964)

Abstract Autoimmune Myasthenia Gravis (MG) is a T cell-dependent, B cell-mediated autoimmune disorder in which autoantibodies is produced targeting the skeletal muscle acetylcholine receptor (AChR). IL-10-competent subset within CD1dhiCD5+ B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. In our previous study, adoptive transfer of CD1dhiCD5+ B cells expanded in vivo by GM-CSF prevented and suppressed Experimental Autoimmune Myasthenia Gravis (EAMG), which was associated with downregulation of mature dendritic cell marker and expansion of regulatory T cell resulting in the suppression of acetylcholine receptor (AChR)-specific T cell and B cell response. To further examine the role of IL-10 in the regulatory function of B10 cells in EAMG, we induced EAMG in both wild type and IL-10 knockout mice, and also performed the adoptive transfer IL-10 deficient CD1dhiCD5+ cells to EAMG mice. We found that IL-10 knockout mice showed more severe EAMG disease with more Th1 cytokine production and less Foxp3 expression compared with wild type mice. Adoptive transfer of CD1dhiCD5+ B cells from IL-10 knockout mice to EAMG mice could not prevent and suppress disease. Our observations further confirmed that IL-10 derived from B10 cells played a critical role in maintaining and restoring immune homeostasis in EAMG mice. Administration IL-10 competent CD1dhiCD5+ B cells may represent an effective strategy in the treatment of human myasthenia gravis.

Non-competitive Inhibition of Nicotinic Acetylcholine Receptors by Ladybird Beetle Alkaloids.

Ladybird beetles (Family Coccinellidae) secrete an alkaloid rich venom from their leg joints that protects them from predators. Coccinellines, the major venom constituents, are alkaloids composed of three fused piperidine rings that share a common nitrogen atom. Although many coccinellines have been isolated and chemically characterized, their pharmacological properties are essentially unknown. Using radioligand binding and functional assays we investigated the actions of several coccinellines on skeletal muscle and α7 nicotinic acetylcholine receptors (nAChRs). The alkaloids were shown to displace the specific binding of tritiated piperidyl-N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([(3)H]-TCP), which has been shown to bind deep within the ion channel of the electric fish (Torpedo) muscle nAChR. The stereoisomers precoccinelline and hippodamine (whose nitrogens are predicted to be ionized at physiological pH) and their respective analogs N-methyl-precoccinelline and N-methyl-hippodamine (whose quaternary nitrogens are permanently charged) displayed similar IC50s for inhibition of [(3)H]-TCP binding. However, the corresponding precoccinelline and hippodamine N-oxides, coccinelline and convergine (which have an electronegative oxygen bonded to an electropositive nitrogen) displayed significantly higher binding IC50s. Finally, exochomine, a dimeric coccinelline containing the hippodamine structure, displayed the highest IC50 (lowest affinity) for displacing specific [(3)H]-TCP binding. The presence of a desensitizing concentration (10(-3) M) of carbachol (CCh) had little or no effect on the affinity of the Torpedo nAChR for the three coccinellines tested. High concentrations of the coccinellid alkaloids did not affect binding of [(3)H]-cytisine to Torpedo receptor ACh binding sites. Inhibition of the alpha7 nAChR with pre-equilibrated precoccinelline was insurmountable with respect to ACh concentration. We conclude that the coccinellines bind to one or more allosteric sites rather than to the ACh binding sites, and inhibit nAChR responses to ACh through a non-competitive mechanism. Future chemical and pharmacological investigations of other ladybird beetle alkaloids are likely to reveal other interesting alkaloids affecting ligand-gated receptors.

ALPHA AND BETA BUNGAROTOXIN BINDING PROTEINS IN HUMAN CADAVER MUSCLE

The nicotinic acetylcholine receptors (nAChR) in skeletal muscle, which are targets of the autoimmune response that cause myasthenia gravis (MG). MG is an autoimmune disease caused by anti-acetylcholine (anti-AChR) antibodies, resulting in functional loss of AChR at the neuromuscular junction. Specific binding of radio-iodinated α and β Bgtx was carried out in membranes (rapid filtration method) and triton extract (ammonium sulfate precipitation method) of fresh muscle. The specific binding of α Bgtx to membranes and triton extract of fresh muscle was found to be 19.8 ± 0.2 and 10.2 ± 0.12 fmoles/mg tissue, respectively whereas the same for β-Bgtx was found to be 10.2 ± 0.15 and 7.2 ± 0.12 fmoles/mg tissue, respectively. It was observed that there was no significant decline in specific binding to membranes and triton extract of muscle with increase in post-mortem time from 5 to 24 h. By indirect ELISA with MG pool sera, in addition to age the immunoreactivity varies from one cadaver to another. It was observed that β bungarotoxin binding proteins in muscle shows high immunoreactivity as compared to α Bungarotoxin binding protein.

Selective inhibition of caspases in skeletal muscle reverses the apoptotic synaptic degeneration in slow-channel myasthenic syndrome

Slow-channel syndrome (SCS) is a congenital myasthenic disorder caused by point mutations in subunits of skeletal muscle acetylcholine receptor leading to Ca(2+) overload and degeneration of the postsynaptic membrane, nuclei and mitochondria of the neuromuscular junction (NMJ). In both SCS muscle biopsies and transgenic mouse models for SCS (mSCS), the endplate regions are shrunken, and there is evidence of DNA damage in the subsynaptic region. Activated caspase-9, -3 and -7 are intensely co-localized at the NMJ, and the Ca(2+)-activated protease, calpain, and the atypical cyclin-dependent kinase (Cdk5) are overactivated in mSCS. Thus, the true mediator(s) of the disease process is not clear. Here, we demonstrate that selective inhibition of effector caspases, caspase-3 and -7, or initiator caspase, caspase-9, in limb muscle in vivo by localized expression of recombinant inhibitor proteins dramatically decreases subsynaptic DNA damage, increases endplate area and improves ultrastructural abnormalities in SCS transgenic mice. Calpain and Cdk5 are not affected by this treatment. On the other hand, inhibition of Cdk5 by expression of a dominant-negative form of Cdk5 has no effect on the degeneration. Together with previous studies, these results indicate that focal activation of caspase activity at the NMJ is the principal pathological process responsible for the synaptic apoptosis in SCS. Thus, treatments that reduce muscle caspase activity are likely to be of benefit for SCS patients.

Improved methodology to obtain large quantities of correctly folded recombinant N-terminal extracellular domain of the human muscle acetylcholine receptor for inducing experimental autoimmune myasthenia gravis in rats.

IntroductionHuman myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular system. Experimental autoimmune myasthenia gravis (EAMG) is a well-established animal model for MG that can be induced by active immunization with the Torpedo californica-derived acetylcholine receptor (AChR). Due to the expensive cost of purifying AChR from Torpedo californica, the development of an easier and more economical way of inducing EAMG remains critically needed.Material and methodsFull-length cDNA of the human skeletal muscle AChR α1 subunit was obtained from TE671 cells. The DNA fragment encoding the extracellular domain (ECD) was then amplified by polymerase chain reaction (PCR) and inserted into pET-16b. The reconstructed plasmid was transformed into the host strain BL21(DE3)pLysS, which was derived from Escherichia coli. Isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce the expression of the N-terminal ECD. The produced protein was purified with immobilized Ni2+ affinity chromatography and refolded by dialysis.ResultsThe recombinant protein was efficiently refolded to soluble active protein, which was verified by ELISA. After immunization with the recombinant ECD, all rats acquired clinical signs of EAMG. The titer of AChR antibodies in the serum was significantly higher in the EAMG group than in the control group, indicating successful induction of EAMG.ConclusionsWe describe an improved procedure for refolding recombinant ECD of human muscle AChR. This improvement allows for the generation of large quantities of correctly folded recombinant ECD of human muscle AChR, which provides for an easier and more economical way of inducing the animal model of MG.

Identification and characterization of a taxon-specific three-finger toxin from the venom of the Green Vinesnake (Oxybelis fulgidus; family Colubridae)

Snake venoms contain a variety of protein and peptide toxins, and the three-finger toxins (3FTxs) are among the best characterized family of venom proteins. The compact nature and highly conserved molecular fold of 3FTxs, together with their abundance in many venoms, has contributed to their utility in structure-function studies. Although many target the nicotinic acetylcholine receptor of vertebrate skeletal muscle, often binding with nanomolar Kds, several non-conventional 3FTxs show pronounced taxon-specific neurotoxic effects. Here we describe the purification and characterization of fulgimotoxin, a monomeric 3FTx from the venom of Oxybelis fulgidus, a neotropical rear-fanged snake. Fulgimotoxin retains the canonical 5 disulfides of the non-conventional 3FTxs and is highly neurotoxic to lizards; however, mice are unaffected, demonstrating that this toxin is taxon-specific in its effects. Analysis of structural features of fulgimotoxin and other colubrid venom 3FTxs indicate the presence of a "colubrid toxin motif" (CYTLY) and a second conserved segment (WAVK) found in Boiga and Oxybelis taxon-specific 3FTxs, both in loop II. Because specific residues in loop II conventional α-neurotoxic 3FTxs are intimately associated with receptor binding, we hypothesize that this loop, with its highly conserved substitutions, confers taxon-specific neurotoxicity. These findings underscore the importance of rear-fanged snake venoms for understanding the evolution of toxin molecules and demonstrate that even among well-characterized toxin families, novel structural and functional motifs may be found.

Regulatory B cells suppress experimental autoimmune myasthenia gravis via production of IL-10 (P5145)

Abstract Autoimmune Myasthenia Gravis (MG) is T cell-dependent, B cell mediated autoimmune disorder in which autoantibodies are produced targeting the skeletal muscle acetylcholine receptor (AChR). Previous studies have shown that regulatory T cells (Tregs) have the capacity to prevent and reverse experimental autoimmune MG (EAMG). In more recent investigations, our studies showed that regulatory B cells (B10 cells) produced high level of IL-10 and suppressed ongoing experimental autoimmune MG (EAMG), resulting in a significant down-modulation of relevant pathogenic anti-AChR autoantibody production and significant preservation of muscle AChR content. In in vitro co-culture studies, B10 cells altered T cell cytokine profiles through increasing IL-10 production and decreasing IFN-r production of CD4+ T cells, but did not directly inhibit T cell proliferation. On the other hand, B10 cells can inhibit B cell proliferation and its autoantibody production in vitro through IL-10 production. Thus, Our studies has provided the significant insights into the nature of the mechanism and effects of regulatory B cells in EAMG. These observations suggest that mobilization of B10 cells in vivo/vitro may represent an effective strategy in the treatment of human MG.

Faster, slower, but never better

The skeletal muscle acetylcholine receptor (AChR) is a beautifully functional molecular complex made up of 5 subunits. Acetylcholine binding initiates pore opening, producing the endplate current (EPC); the resulting action potentials travel over the muscle surface and into the transverse tubules,

Alternatively spliced variants of gamma-subunit of muscle-type acetylcholine receptor in fetal and adult skeletal muscle of mouse.

Gamma-subunit of nicotinic acetylcholine receptor is encoded by chrng gene of mouse. This gene is located on chromosome 1, spans 6.5kb, and contains 12 exons and 11 introns. Previous studies have reported three transcript variants (C1-3) produced by alternative splicing; C1 contains all the 12 reported exons, C2 uses an in-frame alternate splice site in exon-2, and C3 produced by exon-5 skipping. These variants differ in their channel kinetics and opening times. In our study, we report the presence of two new transcript variants (T1 and T2) of chrng expressed in mouse postnatal day 3 and adult skeletal muscles. These transcripts contain novel first coding exon either N1 or N2. N1 is located in the 5' UTR, while N2 is an extended exon-2. 5' extension of exon-2 contains an initiation codon which produces a novel transcript variant. Either of the two exons can splice with the internal exons to produce mature transcripts making different 5' ends of the transcripts. Consequently, the proteins encoded by these two transcripts differ at N-termini. The presence of N2 exon containing transcript was further supported by the availability of EST from the database. These new variants display heterogeneous properties. They differ in the presence of signal peptide, phosphorylation, and acetylation of their amino acid residues of the new N-termini of the gamma subunit.

Morphological aspects of neuromuscular junctions and gene expression of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle of rats with heart failure

HF is syndrome initiated by a reduction in cardiac function and it is characterized by the activation of compensatory mechanisms. Muscular fatigue and dyspnoea are the more common symptoms in HF; these may be due in part to specific skeletal muscle myopathy characterized by reduced oxidative capacity, a shift from slow fatigue resistant type I to fast less fatigue resistant type II fibers and downregulation of myogenic regulatory factors (MRFs) gene expression that can regulate gene expression of nicotinic acetylcholine receptors (nAChRs). In chronic heart failure, skeletal muscle phenotypic changes could influence the maintenance of the neuromuscular junction morphology and nAChRs gene expression during this syndrome. Two groups of rats were studied: control (CT) and Heart Failure (HF), induced by a single intraperitoneal injection of monocrotaline (MCT). At the end of the experiment, HF was evaluated by clinical signs and animals were sacrificed. Soleus (SOL) muscles were removed and processed for morphological, morphometric and molecular NMJ analyses. Our major finding was an up-regulation in the gene expression of the alpha1 and epsilon subunits of nAChR and a spot pattern of nAChR in SOL skeletal muscle in this acute monocrotaline induced HF. Our results suggest a remodeling of nAChR alpha1 and epsilon subunit during heart failure and may provide valuable information for understanding the skeletal muscle myopathy that occurs during this syndrome.

Effects of sevoflurane and isoflurane on blockade of adult type nicotinic acetylcholine receptors of skeletal muscle of rats by rocuronium

Objective To investigate the effects of sevotlurane and isoflurane on blockade of adult nicotinic acetylcholine receptor (e-nAChR) of skeletal muscle of rats by rocuronium.Methods HEK293 cell line was provided by Molecular Biology Laboratory,Chonqing Medical University and cultured in DMEM liquid culture medium containing 10％ bovine calf serum at 37 ℃in incubator filled with 5％ CO2.e-nAChR was expressed heterologously in HEK293 cell using liposome transfection technology.The whole cell patch clamp technology was used to establish the concentration-effect curves for inhibition of acetylcholine (ACh)-induced current.The 5％ inhibitory concentration values ( IC5 ),25 ％ inhibitory concentration values ( IC25 ) and 50％ inhibitory concentration values (IC50) for sevoflurane,isoflurane and rocuronium were calculated.Firstly the inhibitory effect of rocuronium at a concentration of IC25 was studied in the presence of sevoflurane and isoflurane at concentrations of IC5,IC25 and IC50.Subsequently the inhibitory effect of rocuronium at concentrations of IC5,IC25 and IC50 was studied in the presence of sevoflurane and isoflurane at a concentration of IC25.Inhibition of ACh-induced current amplitude was recorded.Results At the concentrations of IC5,IC25 and IC50 sevoflurane or isoflurane had synergistical inhibitory effect on the current amplitude of e-nAChR with rocuronium at the concentrations of IC5 and IC25,while had addition inhibitory effect on the current amplitude of e-nAChR with rocuronium at the concentration of IC50.The inhibitory effect of sevoflurane at the concentration of IC5 on the current amplitude of e-nAChR with rocuronium at the concentration of IC25 was weaker than that of isoflurane,while the inhibitory effect of sevoflurane at the concentration of IC50 on the current amplitude of e-nAChR with rocuronium at the concentration of IC25 was stronger than that of isoflurane.The inhibitory effect of sevoflurane at the concentration of IC25 on the current amplitude of e-nAChR with rocuronium at the concentration of IC5 was weaker than that of isoflurane.Conclusion Either sevoflurane or isoflurane has synergistical blocking effect on e-nAChR with low concentration of rocuronium,while has addition blocking effect with high concentration of rocuronium.Potentiation of blocking effect of rocuronium on e-nAChR by low concentration of sevoflurane is weaker than that by isoflurane,while the potentiation by sevoflurane at high concentration is stronger than that by isoflurane. Key words: Isoflurane; Androstanols; Receptors, nicotinic; Muscle, skeletal; Sevoflurane