Dihydropyridine Receptor Research Articles

Calmodulin-Binding Proteins in Muscle: A Minireview on Nuclear Receptor Interacting Protein, Neurogranin, and Growth-Associated Protein 43.

Calmodulin (CaM) is an important Ca2+-sensing protein with numerous downstream targets that are either CaM-dependant or CaM-regulated. In muscle, CaM-dependent proteins, which are critical regulators of dynamic Ca2+ handling and contractility, include calcineurin (CaN), CaM-dependant kinase II (CaMKII), ryanodine receptor (RyR), and dihydropyridine receptor (DHPR). CaM-regulated targets include genes associated with oxidative metabolism, muscle plasticity, and repair. Despite its importance in muscle, the regulation of CaM—particularly its availability to bind to and activate downstream targets—is an emerging area of research. In this minireview, we discuss recent studies revealing the importance of small IQ motif proteins that bind to CaM to either facilitate (nuclear receptor interacting protein; NRIP) its activation of downstream targets, or sequester (neurogranin, Ng; and growth-associated protein 43, GAP43) CaM away from their downstream targets. Specifically, we discuss recent studies that have begun uncovering the physiological roles of NRIP, Ng, and GAP43 in skeletal and cardiac muscle, thereby highlighting the importance of endogenously expressed CaM-binding proteins and their regulation of CaM in muscle.

PharmGKB summary: very important pharmacogene information for CACNA1S.

The CACNA1S gene encodes the α1s subunit of the dihydropyridine receptor (DHPR), a voltage-gated calcium channel and voltage sensor for Ca2+ release in skeletal muscle. Variants in the CACNA1S gene have been linked to the pharmacogenetic disorder known as malignant hyperthermia susceptibility (MHS) and hypokalemic periodic paralysis (hypoPP). Two variants in CACNA1S are verified by the European Malignant Hyperthermia Group (EMHG) to be associated with MHS [1]. Although the occurrence of MHS during anesthesia is relatively rare, the genetic prevalence of MHS-causative mutations is estimated to be between 1 in 400 [2] to 1 in 2000–3000 [3]. The American College of Medical Genetics (ACMG) “Guidelines for Reporting Incidental Findings in Clinical Exome and Genome Sequencing” includes “known pathogenic” and “likely pathogenic” variants in CACNA1S related to MHS in its list of genetic variants to report as incidental findings [4, 5]. In addition, 9 CACNA1S variants are linked to hypoPP [6]. Finally, several CACNA1S variants and polymorphisms are proposed to be associated with thyrotoxic periodic paralysis (TPP) [7], hyperCKemia [8] and statin-associated myopathy [9]. Thus, the CACNA1S gene is associated with an eclectic array of muscle disorders with clinical manifestations ranging from subclinical myopathies, episodic paralysis, and life-threatening response to anesthesia.

Excitation-contraction coupling in skeletal muscle: recent progress and unanswered questions.

Excitation-contraction coupling (ECC) is a physiological process that links excitation of muscles by the nervous system to their mechanical contraction. In skeletal muscle, ECC is initiated with an action potential, generated by the somatic nervous system, which causes a depolarisation of the muscle fibre membrane (sarcolemma). This leads to a rapid change in the transmembrane potential, which is detected by the voltage-gated Ca2+ channel dihydropyridine receptor (DHPR) embedded in the sarcolemma. DHPR transmits the contractile signal to another Ca2+ channel, ryanodine receptor (RyR1), embedded in the membrane of the sarcoplasmic reticulum (SR), which releases a large amount of Ca2+ ions from the SR that initiate muscle contraction. Despite the fundamental role of ECC in skeletal muscle function of all vertebrate species, the molecular mechanism underpinning the communication between the two key proteins involved in the process (DHPR and RyR1) is still largely unknown. The goal of this work is to review the recent progress in our understanding of ECC in skeletal muscle from the point of view of the structure and interactions of proteins involved in the process, and to highlight the unanswered questions in the field.

P.235Clinico-pathological characterisation of CACNA1S related congenital myopathy in children

Mutations in the CACNA1S gene were recently identified as a new cause of congenital myopathy (CM). To date only a few cases have been described, with limited phenotypic data. We report the clinical, pathological and radiological phenotype in five children with genetically confirmed CACNA1S-CM. →Patients were referred to our national highly specialised service for CM at The Dubowitz Neuromuscular Centre, UK. We conducted retrospective case note analysis. Two patients had recessive inheritance and three autosomal dominant. →Four of five patients (age range 7-18 years) had severe neonatal presentation with foetal akinesia, weakness, hypotonia, contractures, feeding difficulties and respiratory compromise. The 5th patient presented with motor delay in infancy. All five patients had proximal predominant, bulbar and facial weakness but showed spontaneous improvement over time. Ophthalmoplegia was noted in three. Two patients remain non-ambulant. Two developed scoliosis. Two are on nocturnal non-invasive ventilation initiated at age of 5 months and 7 years. All apart from one required nasogastric/gastrostomy feeding up until 2-5 years of age, but all were able to feed orally from age 5 years. One patient had clear history of periodic weakness well responding to Acetazolamide. Two children reported fatigue and limited endurance but no abnormalities of neuromuscular transmission were detected. All muscle biopsies showed fibre size variation and marked slow fibre predominance. Additional findings included mild central nucleation, few central cores and focally accentuated pseudo-dystrophic changes. Three out of the five patients had abnormal lower limb muscle MRI but with no specific muscle pattern. →CACNA1S related myopathy is a recently described form of congenital myopathy and should be considered in cases of undiagnosed dominant and recessive congenital myopathies with phenotypical features presented above.

Triclosan Affects Ca2+ Regulatory Module and Musculature Development in Skeletal Myocyte during Early Life Stages of Zebrafish (Danio rerio).

Advanced technologies for toxicity tests are designed to identify biomarkers with superior predictive power or end points of the complex web of biological pathways. However, the data obtained need to be fully characterized for dose-response, physiological systems, and relevance to a system or (sub) population before biological interpretation and decision making. In this study, the toxicity of triclosan (TCS) on zebrafish was selected as a case study to correlate the observed morphological effects with existing data and identify the critical events by receptor activity sensitivity analysis. Triclosan exhibited weak acute toxicity against zebrafish and significantly affected the development of trunk muscles at 0.52, 1.04, and 1.73 μM. Through receptor-mediated screening, we found that the adverse effects of TCS induce Ryanodine receptor 1 (RyR1) activity and distort Ca2+ signaling. The trunk skeletal muscle abnormalities occurred only when the dihydropyridine receptor (DHPR) was blocked, demonstrating that TCS mainly influenced the Ca2+ regulatory module associated with signaling between DHPRs and RyR1; DHPRs mainly regulated the orthograde and retrograde signaling in skeletal muscles. This unexpected result could integrate the mode of action of TCS and provide insight for high-throughput screening and toxicity prediction using zebrafish.

Dihydropyridine Receptor Congenital Myopathy In A Consangineous Turkish Family.

Dihydropyridine receptor congenital myopathy is a recently described congenital myopathy caused by dominant or recessive mutations in the CACNA1S gene. To date, only 11 cases from 7 families were described in a single report. Here, we describe a consanguineous family with three affected children, presenting congenital hypotonia, contractures, ophthalmoplegia and respiratory insufficiency, with a novel homozygous mutation in the CACNA1S gene. They also showed cognitive delay, pes equinovarus deformity and neurogenic changes that have not been associated with this myopathy in the previous reports. This report expands the phenotypic spectrum of dihydropyridine receptor congenital myopathy and underscores the importance of whole exome sequencing in early onset neuromuscular disorders.

From synaptic input to muscle contraction: arm muscle cells of Octopus vulgaris show unique neuromuscular junction and excitation-contraction coupling properties.

The muscular-hydrostat configuration of octopus arms allows high manoeuvrability together with the efficient motor performance necessary for its multitasking abilities. To control this flexible and hyper-redundant system the octopus has evolved unique strategies at the various levels of its brain-to-body organization. We focus here on the arm neuromuscular junction (NMJ) and excitation-contraction (E-C) properties of the arm muscle cells. We show that muscle cells are cholinergically innervated at single eye-shaped locations where acetylcholine receptors (AChR) are concentrated, resembling the vertebrate neuromuscular endplates. Na+ and K+ contribute nearly equally to the ACh-activated synaptic current mediating membrane depolarization, thereby activating voltage-dependent L-type Ca2+ channels. We show that cell contraction can be mediated directly by the inward Ca2+ current and also indirectly by calcium-induced calcium release (CICR) from internal stores. Indeed, caffeine-induced cell contraction and immunohistochemical staining revealed the presence and close association of dihydropyridine (DHPR) and ryanodine (RyR) receptor complexes, which probably mediate the CICR. We suggest that the dynamics of octopus arm contraction can be controlled in two ways; motoneurons with large synaptic inputs activate vigorous contraction via activation of the two routs of Ca2+ induced contraction, while motoneurons with lower-amplitude inputs may regulate a graded contraction through frequency-dependent summation of EPSP trains that recruit the CICR. Our results thus suggest that these motoneuronal pools are likely to be involved in the activation of different E-C coupling modes, thus enabling a dynamics of muscles activation appropriate for various tasks such as stiffening versus motion generation.

Estrone exposure interacts with temperature to alter predator evasion performance and systemic mRNA abundances

Environmental estrogens from anthropogenic activities are ubiquitous in aquatic ecosystems. Ambient temperature in these systems also fluctuates in daily, seasonal, and long-term rhythms. While both factors have been studied extensively, their interaction on aquatic life is critical to understand. The objective of this study was, therefore, to examine how behavior and gene expression are impacted by estrogenic exposure across a range of environmental temperatures. Larval fathead minnows (Pimephales promelas) were exposed to estrone (E1) at two concentrations (nominal 625 and 1250 ng/L) or to an ethanol solvent control, at one of four temperatures (15, 18, 21 and 24 °C) from fertilization to 21 days post-hatch. Exposed larvae were assessed for alterations in predator evasion performance and mRNA abundances of two genes for calcium channel receptors found in muscles - dihydropyridine receptor (dhpr) and ryanodine receptor 1, and the gonadal genes anti-Müllerian hormone, cytochrome P450 gonadal aromatase (cyp19a), doublesex and mab-3 related transcription factor 1 (dmrt1) and estrogen receptor 1 (esr1). Larval escape angle, escape latency, as well as systemic esr1 and cyp19a mRNA abundances were altered by an interaction between E1 concentration and temperature. E1-exposed larval exhibited reduced escape performance across all tested temperatures, whereas decreased systemic dhpr mRNA abundance was observed only at 18 °C. E1-exposure reduced systemic mRNA abundances of amh, cyp19a, dhpr, and ryr1, while temperature significantly reduced systemic cyp19a and dhpr mRNA abundances. E1-exposure and temperature significant enhanced systemic mRNA abundances of esr1 and cyp19a, respectively. These complex results illustrate the importance of considering how abiotic factors may moderate the effects of contaminant exposure during the sensitive larval developmental stage, as temperature modulates effects of estrogenic exposure on animal performance and mRNA abundances.

Nanoscale reorganization of sarcoplasmic reticulum in pressure-overload cardiac hypertrophy visualized by dSTORM

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca2+-ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.

Blocking skeletal muscle DHPRs/Ryr1 prevents neuromuscular synapse loss in mutant mice deficient in type III Neuregulin 1 (CRD-Nrg1).

Schwann cells are integral components of vertebrate neuromuscular synapses; in their absence, pre-synaptic nerve terminals withdraw from post-synaptic muscles, leading to muscle denervation and synapse loss at the developing neuromuscular junction (NMJ). Here, we report a rescue of muscle denervation and neuromuscular synapses loss in type III Neuregulin 1 mutant mice (CRD-Nrg1−/−), which lack Schwann cells. We found that muscle denervation and neuromuscular synapse loss were prevented in CRD-Nrg1−/−mice when presynaptic activity was blocked by ablating a specific gene, such as Snap25 (synaptosomal-associated 25 kDa protein) or Chat (choline acetyltransferase). Further, these effects were mediated by a pathway that requires postsynaptic acetylcholine receptors (AChRs), because ablating Chrna1 (acetylcholine receptor α1 subunit), which encodes muscle-specific AChRs in CRD-Nrg1−/−mice also rescued muscle denervation. Moreover, genetically ablating muscle dihydropyridine receptor (DHPR) β1 subunit (Cacnb1) or ryanodine receptor 1 (Ryr1) also rescued muscle denervation and neuromuscular synapse loss in CRD-Nrg1−/−mice. Thus, these genetic manipulations follow a pathway–from presynaptic to postsynaptic, and, ultimately to muscle activity mediated by DHPRs and Ryr1. Importantly, electrophysiological analyses reveal robust synaptic activity in the rescued, Schwann-cell deficient NMJs in CRD-Nrg1−/−Cacnb1−/−or CRD-Nrg1−/−Ryr1−/−mutant mice. Thus, a blockade of synaptic activity, although sufficient, is not necessary to preserve NMJs that lack Schwann cells. Instead, a blockade of muscle activity mediated by DHRPs and Ryr1 is both necessary and sufficient for preserving NMJs that lack Schwann cells. These findings suggest that muscle activity mediated by DHPRs/Ryr1 may destabilize developing NMJs and that Schwann cells play crucial roles in counteracting such a destabilizing activity to preserve neuromuscular synapses during development.

Dysferlin Mutants: Defects in Trafficking and Association with Proteins of the Transverse Tubule

Dysferlinopathies are a class of muscular dystrophies caused by a loss of function or expression of dysferlin, a 237 kDa single-pass transmembrane protein expressed in many tissues, including skeletal muscle. Dysferlin has been implicated in sarcolemmal repair and in the regulation of calcium signaling following mechanical stress. Dysferlin binds to several proteins associated with the transverse tubules, including the dihydropyridine receptor (DHPR) and Caveolin 3, to stabilize the triad junction and perhaps to serve as a scaffold for repair machinery. Mutations disrupting these interactions may lead to disruption of Ca signaling after mechanical stress, which in turn leads to fiber loss, inflammation and muscle atrophy associated with muscular dystrophy. Mutations to dysferlin may also to lead to aberrant trafficking, causing accumulation in compartments other than the T-tubules, therefore removing or reducing dysferlin's function. We are studying several point mutations found in dysferlinopathy patients, as well as domain deletion mutants, to determine their effects on trafficking, association with other membrane proteins, and dimerization. Our results to date indicate that mutations or deletions that are thought to be pathogenic are at least partially associated with altered trafficking to the T-tubules and, for some, disruption of binding to Cav3, but not to changes in its ability to dimerize. Supported by the Jain Foundation and NIH RO1 AR064268.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for the Use of Potent Volatile Anesthetic Agents and Succinylcholine in the Context of RYR1 or CACNA1S Genotypes.

The identification in a patient of 1 of the 50 variants in the RYR1 or CACNA1S genes reviewed here should lead to a presumption of malignant hyperthermia susceptibility (MHS). MHS can lead to life-threatening reactions to potent volatile anesthetic agents or succinylcholine. We summarize evidence from the literature supporting this association and provide therapeutic recommendations for the use of these agents in patients with these RYR1 or CACNA1S variants (updates at https://cpicpgx.org/guidelines and www.pharmgkb.org).

Qiliqiangxin attenuates atrial structural remodeling in prolonged pacing-induced atrial fibrillation in rabbits.

Qiliqiangxin (QL) can attenuate myocardial remodeling and improve cardiac function in some cardiac diseases, including heart failure and hypertension. This study was to explore the effects and mechanism of QL on atrial structural remodeling in atrial fibrillation (AF). Twenty-one rabbits were randomly divided into a sham-operation group, pacing group (pacing with 600 beats per minute for 4weeks), and treatment group (2.5g/kg/day). Before pacing, the rabbits received QL-administered p.o. for 1week. We measured atrial electrophysiological parameters in all groups to evaluate AF inducibility and the atrial effective refractory period (AERP). Echocardiography evaluated cardiac function and structure. TUNEL detection, hematoxylin and eosin (HE) staining, and Masson's trichrome staining were performed. Immunohistochemistry and western blotting (WB) were used to detect alterations in calcium channel L-type dihydropyridine receptor α2 subunit (DHPR) and fibrosis-related regulatory factors. AF inducibility was markedly decreased after QL treatment. Furthermore, we found that AERP and DHPR were reduced significantly in pacing rabbits compared with sham rabbits; treatment with QL increased DHPR and AERP compared to the pacing group. The QL group showed significantly decreased mast cell density and improved atrial ejection fraction values compared with the pacing group. Moreover, QL decreased interventricular septum thickness (IVSd) and left ventricular end-diastolic diameter (LVEDD). Compared with the sham group, the levels of TGFβ1 and P-smad2/3 were significantly upregulated in the pacing group. QL reduced TGF-β1 and P-smad2/3 levels and downstream fibrosis-related factors. Our study demonstrated that QL treatment attenuates atrial structural remodeling potentially by inhibiting TGF-β1/P-smad2/3 signaling pathway.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca2+ signaling of differentiated C2C12 myotubes.

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca2+]i transient and reduced sarcoplasmic reticulum (SR) Ca2+ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca2+-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca2+ signaling by downregulating the expression of DHPR and SERCA proteins.

M102 - VARIATION IN VOLTAGE-GATED CALCIUM CHANNEL GENES IS ASSOCIATED WITH ANTIPSYCHOTIC TREATMENT RESPONSE IN A SOUTH AFRICAN FIRST EPISODE SCHIZOPHRENIA COHORT

Background Voltage-gated calcium channels have been implicated in schizophrenia aetiology, however little is known about their involvement in antipsychotic treatment response. This study investigated variants within the calcium channel subunit genes for association with antipsychotic treatment response in a first episode schizophrenia cohort. Methods The patient cohort included 103 unrelated South African FES patients (80% South African Coloured, 12% Xhosa and 8% European descent). Treatment response was assessed using the positive and negative syndrome scale (PANSS) over a period of 12 months, with measurements taken biweekly for the first six weeks, and every three months thereafter. Variants within the calcium channel subunit genes (α1, α2δ, β and γ subunits) were mined from available GWAS data produced using the Infinium OmniExpressExome-8 Kit (Illumina, California, USA). In total 1830 variants were identified within 10 CACNA1 genes, four CACNB genes, four CACNA2D genes and eight CACNG genes. Linear mixed-effects models were used to investigate the effect of genetic variants on change in PANSS scores for each domain (positive, negative, general and total) over the twelve-month period, adjusting for age, gender, proportion ancestry, and baseline PANSS scores. Multiple modes of inheritance were considered and the best-fit model was selected for further analysis. Results Twelve regulatory variants were shown to be significantly associated with treatment outcome. Most notably the CACNA1B rs2229949 CC genotype was associated with improved negative symptomology, where the C-allele was predicted to abolish a miRNA-binding site (has-mir-5002-3p), suggesting a possible mechanism of action through which this variant may have an effect. Discussion These results implicate the calcium channel subunits in antipsychotic treatment response and suggest that increased activation of these channels may be explored to enhance or predict antipsychotic treatment outcome.

Moderate intensity continuous training reverses the detrimental effects of ovariectomy on RyR1 phosphorylation in rat skeletal muscle

High 17β-Estradiol (E2) concentrations in isolated ventricular myocytes as well as a lack of ovarian hormones in cardiac muscle of ovariectomized (OVX) rodents has been shown to lead to arrhythmogenic effects by inducing post-translational modifications, including phosphorylation of the sarcoplasmic reticulum (SR) Ca2+ release channel ryanodine receptor-2 (RyR2). The effects of estrogens on the phosphorylation status of the RyR1 in skeletal muscle have not been investigated before. Furthermore, while high intensity exercise has been shown to increase RyR phosphorylation, there is no data on the effects of moderate intensity continuous training (MICT).The aims of the study were to investigate the effects of a 3-day treatment with low (1 nM, moderate (5 nM) and high (10 nM, 100 nM) E2 concentrations on RyR1 mRNA and protein expression and phosphorylation status (pRyRSer2844) in cultured C2C12 myotubes and to study the effects of OVX on RyR1 expression and phosphorylation in rat skeletal muscle in combination with 3 weeks of MICT.Treatment with low, physiological E2 concentrations reduced dihydropyridine receptor (DHPR) and RyR1 mRNA content in C2C12 myotubes compared to untreated control cells, whereas RyR1 protein phosphorylation (pRyRSer2844) was significantly increased after treatment with high, non-physiological E2 concentrations (p ≤ 0.05). RyR1 protein content (p ≤ 0.05) and pRyRSer2844 (p ≤ 0.05) were significantly elevated in skeletal muscle of OVX vs. sham-operated rats. Importantly, pRyRSer2844 levels were similar to sham-operated controls in OVX rats after MICT (OVX vs. OVX + MICT, p ≤ 0.05).Our results indicate, that one of the actions of estrogens is to alter skeletal muscle Ca2+ homeostasis by modulating the expression and phosphorylation of the RyR1 in skeletal muscle. Notably, regular MICT was able to counteract RyR1 phosphorylation in skeletal muscle of OVX rats.

A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.

BackgroundAlthough considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model.ObjectiveWe aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration.MethodsImmortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons.ResultsNeuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and βIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin.ConclusionWe established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy.

Sweroside Alleviated Aconitine-Induced Cardiac Toxicity in H9c2 Cardiomyoblast Cell Line.

Aconitine is the main bioactive ingredient of Aconitum plants, which are well-known botanical herbs in China. Aconitine is also notorious for its high cardiotoxicity, as it can induce life-threatening ventricular arrhythmias. Unfortunately, there are few effective antidotes to aconitine toxicity. This study aimed to evaluate the potent protective effects of the ingredients from V. baillonii on aconitine toxicity on H9c2 cell line. Cell viability was assessed by methylthiazoltetrazolium bromide (MTT). Intracellular Ca2+ concentration alteration and reactive oxygen species (ROS) generation were observed by confocal microscopy and flow cytometry, respectively. Cellular oxidative stress was analyzed by measuring malondialdehyde (MDA) and superoxide dismutase (SOD) levels. Mitochondrial membrane potential (ΔΨ) was determined using JC-1 kit. RT-PCR and Hoechst staining techniques were conducted to determine the levels of autophagy/apoptosis. The mRNA levels of dihydropyridine receptor (DHPR), ryanodine receptors (RyR2) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) were measured by RT-PCR. We screened six components from V. baillonii, among which, sweroside exhibited the strongest protective effects on aconitine-induced cardiac toxicity. Sweroside suppressed the aconitine-induced mRNA expressions of NaV1.5 (encoded by SCN5A), RyR2 and DHPR, and reversed the aconitine-induced decrease in mRNA level of SERCA, thus preventing the aconitine-induced persistent intracellular Ca2+ accumulation and avoiding intracellular Ca2+ overload. We further found that sweroside restabilized the aconitine-disrupted mitochondrial membrane potential (ΔΨ) and reversed the aconitine-induced increase in the mRNA levels of cell autophagy-related factors (Beclin-1, Caspase-3, and LC3- II) in H9c2 cells. In the whole-animal experiments, we observed that sweroside (50 mg/kg) alleviated effectively aconitine-induced arrhythmias by analysis of electrocardiogram (ECG) recording in rats. Our results demonstrate that sweroside may protect cardiomyocytes from aconitine toxicity by maintaining intracellular Ca2+ homeostasis, restabilizing mitochondrial membrane potential (ΔΨ) and avoiding cell autophagy/apoptosis.

RYR1 and CACNA1S genetic variants identified with statin-associated muscle symptoms.

To examine the genetic differences between subjects with statin-associated muscle symptoms and statin-tolerant controls. Next-generation sequencing was used to characterize the exomes of 76 subjects with severe statin-associated muscle symptoms and 50 statin-tolerant controls. 12 probably pathogenic variants were found within the RYR1 and CACNA1S genes in 16% of cases with severe statin-induced myopathy representing a fourfold increase over variants found in statin-tolerant controls. Subjects with probably pathogenic RYR1 or CACNA1S variants had plasma CK 5X to more than 400X the upper limit of normal in addition to having muscle symptoms. Genetic variants within the RYR1 and CACNA1S genes are likely to be a major contributor to the susceptibility to statin-associated muscle symptoms.

NEXT GENERATION SEQUENCING AND EXPERIMENTAL MYOLOGY: P.374Identification of gene mutations in patients with primary periodic paralysis using targeted next-generation sequencing

Primary periodic paralysis comprises rare skeletal ion channelopathies characterized by recurrent quadriplegia typically associated with high or low serum potassium levels. We developed a gene panel that includes 10 ion channel-related genes and 245 muscular dystrophy- and myopathy-related genes and used this panel to diagnose 60 patients with primary periodic paralysis and identify the disease-causing or risk-associated gene mutations. The average sequencing depth using this panel was 488.11 ×, and the average coverage was 99.45%. Mutations in 5 genes were discovered in 40 patients (66.67%). Variants in SCN4A, KCNJ2 and CACNA1S genes accounted for 92.5% of the patients with a genetic diagnosis. SCN4A (NM_000334) mutations accounted for the majority in this primary PP cohort (47.5%). We identified 10 SCN4A variants including 5 reported mutations (c.2014C>T p.Arg672Cys, c.2024G>A p.Arg675Gln, c.2111C>T p.Thr704Met, c.4352G>T p.Arg1451Leu, and c.4774A>G p.Met1592Val) and 5 novel mutations (c.107_109del p.Glu36del, c.121C>T p.Arg41Trp, c.718G>A p.Val240Met, c.3868T>C p.Phe1290Leu and c.5293G>A p.Ala1765Thr). Among them, c.2024G>A p.Arg675Gln was identified in 8 patients from 6 families as a hotspot. KCNJ2 is the second most common genetic cause.We identified 9 reported variants (c.199C>T p.Arg67Trp, c.211G>A p.Asp71Asn, c.556C>A p.Pro186Thr, c.566G>T p.Arg189Ser, c.644G>A p.Gly215Asp, c.652C>T p.Arg218Trp, c. 899G>A p.Gly300Asp, c.919A>G p.Met307Val, and c.921G>C p.Met307Ile) in a total of 14 patients. Moreover, we identified 4 likely pathogenic variants (c.614T>A p.Phe205Tyr, c.1583G>A p.Arg528His, c.2965G>A p.Glu989Lys and c.3716G>A p.Arg1239His) in CACNA1S gene (NM_000069). Two RYR1 variants (p.Glu2764Lys and p.Ala4143Val) were identified in this cohort, together with one reported OPA1 variant (p.Ala357Thr) which is previously responsible for optic atrophy related phenotypes. Using targeted NGS, we achieved a diagnostic success rate of 66.67% in a cohort of primary PP patients. We expanded the spectrum of genotypes of primary PP and clinical phenotypes of known myopathy-related genes. We'd like to attribute this diagnostic rate to the inclusive strategy of screening for other causal factors of hypokalaemic muscle weakness and performing accurate clinical examinations and history inquiries prior to interpreting the NGS results.