Muscle Ca2 Research Articles

Triclosan Impairs Hippocampal Synaptic Plasticity and Spatial Memory in Male Rats.

Triclosan, a widely used industrial and household agent, is present as an antiseptic ingredient in numerous products of everyday use, such as toothpaste, cosmetics, kitchenware, and toys. Previous studies have shown that human brain and animal tissues contain triclosan, which has been found also as a contaminant of water and soil. Triclosan disrupts heart and skeletal muscle Ca2+ signaling, damages liver function, alters gut microbiota, causes colonic inflammation, and promotes apoptosis in cultured neocortical neurons and neural stem cells. Information, however, on the possible effects of triclosan on the function of the hippocampus, a key brain region for spatial learning and memory, is lacking. Here, we report that triclosan addition at low concentrations to hippocampal slices from male rats inhibited long-term potentiation but did not affect basal synaptic transmission or paired-pulse facilitation and modified the content or phosphorylation levels of synaptic plasticity-related proteins. Additionally, incubation of primary hippocampal cultures with triclosan prevented both the dendritic spine remodeling induced by brain-derived neurotrophic factor and the emergence of spontaneous oscillatory Ca2+ signals. Furthermore, intra-hippocampal injection of triclosan significantly disrupted rat navigation in the Oasis maze spatial memory task, an indication that triclosan impairs hippocampus-dependent spatial memory performance. Based on these combined results, we conclude that triclosan exerts highly damaging effects on hippocampal neuronal function in vitro and impairs spatial memory processes in vivo.

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.

Dysfunction of muscle contraction with impaired intracellular Ca2+ handling in skeletal muscle and the effect of exercise training in male db/db mice.

Type 2 diabetes is characterized by reduced contractile force production and increased fatigability of skeletal muscle. While the maintenance of Ca2+ homeostasis during muscle contraction is a requisite for optimal contractile function, the mechanisms underlying muscle contractile dysfunction in type 2 diabetes are unclear. Here, we investigated skeletal muscle contractile force and Ca2+ flux during contraction and pharmacological stimulation in type 2 diabetic model mice ( db/db mice). Furthermore, we investigated the effect of treadmill exercise training on muscle contractile function. In male db/db mice, muscle contractile force and peak Ca2+ levels were both lower during tetanic stimulation of the fast-twitch muscles, while Ca2+ accumulation was higher after stimulation compared with control mice. While 6 wk of exercise training did not improve glucose tolerance, exercise did improve muscle contractile dysfunction, peak Ca2+ levels, and Ca2+ accumulation following stimulation in male db/db mice. These data suggest that dysfunctional Ca2+ flux may contribute to skeletal muscle contractile dysfunction in type 2 diabetes and that exercise training may be a promising therapeutic approach for dysfunctional skeletal muscle contraction. NEW & NOTEWORTHY The purpose of this study was to examine muscle contractile function and Ca2+ regulation as well as the effect of exercise training in skeletal muscle in obese diabetic mice ( db/db). We observed impairment of muscle contractile force and Ca2+ regulation in a male type 2 diabetic animal model. These dysfunctions in muscle were improved by 6 wk of exercise training.

Growth performance, nutrient digestibility, carcass traits, body composition, and meat quality of goat fed Chinese jujube (Ziziphus Jujuba Mill) fruit as a replacement for maize in diet

This study was conducted to evaluate the effect of Chinese jujube meal replacement for maize in ration on the growth performance, digestibility coefficient, carcass traits, body composition and meat quality in Jinlan cashmere goats. Thirty approximately 14-months-old male Jinlan cashmere goats with similar body weight (25.00 ± 1.53 kg) were randomly assigned to five groups in a completely randomized design. Five levels of jujube meal were formulated in the ration, in which maize was replaced by different levels of jujube meal 0 (C), 50 (J5), 100 (J10), 150 (J15), 200 (J20) g/kg dietary dry matter (DM). The experimental period lasted 50 days: 10 days for adaption and 40 days for measurements. The results showed that goats in J15 had higher dry matter intake (DMI) and average daily gain (ADG) compared with C group, which had a lower feed convention ratio (FCR, g DMI/g ADG) than that in C group (P < 0.05). The increase of jujube replacement in the ration enhanced the digestibility coefficients of DM, organic matter (OM) and crude protein (CP) in a linear, quadratic and cubic manner (P < 0.05), and acid detergent fiber (ADF) in a quadratic and cubic manner (P < 0.05). Dressing percentage and meat weight were higher in J15 than those of C group (P < 0.05). Animals fed the jujube diets had a higher rumen ratio than those in C group (P < 0.05). Warner-bratzler shear force (WBSF) and pH in longissimus dorsi (LD) declined in quadratic and cubic manner with supplementation of jujube meal (P < 0.05). There was no effect of jujube meal on moisture and ash contents of LD muscle (P > 0.05). Ether extract (EE) content in LD muscle was significantly lower, whereas protein content was significantly higher in treatment groups than in C group (P < 0.05). Substitution of jujube meal for maize stimulated the accumulation of Ca, K, Mg Na and Fe in LD muscle of J15 group in comparison with that in C group. The results of the non-essential amino acids (NEEA) composition of LD muscle were similar for all groups except histidine. Diet in J15 and J20 stimulated the accumulation of valine, isoleucine, leucine, C18:0 and C18:2n-6 cis (P < 0.05) in LD muscle. In conclusion, our study suggests that jujube meal, at concentration up to 150 g/kg dietary DM, is considered as an optimal alternative feed resource for maize as it promotes growth performance, improves meat quality and composition.

Loss of S100A1 expression leads to Ca2+ release potentiation in mutant mice with disrupted CaM and S100A1 binding to CaMBD2 of RyR1.

Calmodulin (CaM) and S100A1 fine‐tune skeletal muscle Ca2+ release via opposite modulation of the ryanodine receptor type 1 (RyR1). Binding to and modulation of RyR1 by CaM and S100A1 occurs predominantly at the region ranging from amino acid residue 3614‐3640 of RyR1 (here referred to as CaMBD2). Using synthetic peptides, it has been shown that CaM binds to two additional regions within the RyR1, specifically residues 1975‐1999 and 4295‐4325 (CaMBD1 and CaMBD3, respectively). Because S100A1 typically binds to similar motifs as CaM, we hypothesized that S100A1 could also bind to CaMBD1 and CaMBD3. Our goals were: (1) to establish whether S100A1 binds to synthetic peptides containing CaMBD1 and CaMBD3 using isothermal calorimetry (ITC), and (2) to identify whether S100A1 and CaM modulate RyR1 Ca2+ release activation via sites other than CaMBD2 in RyR1 in its native cellular context. We developed the mouse model (RyR1D‐S100A1KO), which expresses point mutation RyR1‐L3625D (RyR1D) that disrupts the modulation of RyR1 by CaM and S100A1 at CaMBD2 and also lacks S100A1 (S100A1KO). ITC assays revealed that S100A1 binds with different affinities to CaMBD1 and CaMBD3. Using high‐speed Ca2+ imaging and a model for Ca2+ binding and transport, we show that the RyR1D‐S100A1KO muscle fibers exhibit a modest but significant increase in myoplasmic Ca2+ transients and enhanced Ca2+ release flux following field stimulation when compared to fibers from RyR1D mice, which were used as controls to eliminate any effect of binding at CaMBD2, but with preserved S100A1 expression. Our results suggest that S100A1, similar to CaM, binds to CaMBD1 and CaMBD3 within the RyR1, but that CaMBD2 appears to be the primary site of RyR1 regulation by CaM and S100A1.

Importance of breed aptitude (beef or dairy) in determining trace element concentrations in bovine muscles

The aim of this study was to determine the concentrations of various trace elements in muscles with different oxidative/glycolytic profiles (cardiac [CA]; diaphragm [DI], as oxidative; trapezius [TR], as intermediate oxidative/glycolytic; and semimembranosus [SM], as glycolytic muscle) of ten dairy-aptitude (Holstein-Friesian, HF), ten beef-aptitude (Galician Blonde, GB) and ten cross-breed (GBxHF) calves. The type of muscle was a highly significant factor in relation to the concentrations of all elements, whereas breed was only significant for Fe, Mn and Zn in the SM muscle. The concentrations of the main trace elements (Cu, Fe, Se and Zn) were significantly lower in GB and GBxHF than in HF, that were mainly associated with differences in the oxidative/glycolytic profile, probably due to the muscular hypertrophy characteristic of heavily muscled breeds. The pattern of distribution was similar in all breeds, with significantly higher concentrations in the CA muscle, followed by the DI; trace element concentrations in the SM and TR muscles were very similar.

Contractile Protein Expression and Phosphorylation and Contractility of Gastric Smooth Muscles from Obese Patients and Patients with Obesity and Diabetes.

Ingested food is received, mixed, and ground into chyme by distinct gastric motility patterns. Diabetes impairs gastric muscle function, but the mechanisms underlying diabetes-induced gastric muscle dysfunction are unknown. Here, we compared the expression and phosphorylation of Ca2+ sensitization and contractile proteins in human gastric muscles from obese nondiabetic and diabetic patients. We also compared the spontaneous phasic contractions and the contractile responses evoked by electrical field stimulation of cholinergic motor neurons. Fundus and antrum muscles were obtained from sleeve gastrectomies and were used in in vitro myobath contractile studies and for capillary electrophoresis and immunodetection of γ-actin, CPI-17, pT38-CPI-17, MYPT1, pT853-MYPT1, pT696-MYPT1, myosin light chain (MYL9), pS19-MYL9, myosin light chain kinase (MYLK), protein phosphatase-1δ (PP1δ), and Rho-associated kinase (ROCK2). In diabetic fundus muscles, MYLK, ROCK2, and PP1δ expression was unchanged; MYPT1 and CPI-17 expression was decreased; and the pT853/MYPT1 and pT38/CPI-17 ratios, but not the pT696/MYPT1 ratio, were increased. Although MYL9 expression was increased, the pS19/MYL9 ratio was unchanged in diabetic fundus muscles. In diabetic antrum muscles, MYLK and MYL9 expression was unchanged, but ROCK2, CPI-17, and PP1δ expression was decreased. The pT38/CPI-17 ratio was unchanged, while the pS19/MYL9, pT853/MYPT1, and pT696/MYPT1 ratios were decreased, consistent with the reduced ROCK2 expression. The frequencies of spontaneous phasic contractions from nondiabetic and diabetic gastric fundus and antrum muscles did not significantly differ from each other, regardless of age, sex, or diabetic status. The fold increases in the contractions of diabetic fundus and antrum muscles in response to increased frequencies of electrical field stimulation were significantly lower compared to nondiabetic fundus and antrum muscles. The altered contractile responses and the protein expression and phosphorylation in gastric muscles of obese patients with diabetes illustrate the importance of understanding how smooth muscle Ca2+ sensitization mechanisms contribute to gastric motility.

Evaluation of mutant muscle Ca2+ channel properties using two different expression systems.

Allard appraises recent studies investigating the pathological mechanism of hypokalemic periodic paralysis mutations.

Inositol trisphosphate receptor-mediated Ca2+ signalling stimulates mitochondrial function and gene expression in core myopathy patients.

Core myopathies are a group of childhood muscle disorders caused by mutations of the ryanodine receptor (RyR1), the Ca2+ release channel of the sarcoplasmic reticulum. These mutations have previously been associated with elevated inositol trisphosphate receptor (IP3R) levels in skeletal muscle myotubes derived from patients. However, the functional relevance and the relationship of IP3R mediated Ca2+ signalling with the pathophysiology of the disease is unclear. It has also been suggested that mitochondrial dysfunction underlies the development of central and diffuse multi-mini-cores, devoid of mitochondrial activity, which is a key pathological consequence of RyR1 mutations. Here we used muscle biopsies of central core and multi-minicore disease patients with RyR1 mutations, as well as cellular and in vivo mouse models of the disease to characterize global cellular and mitochondrial Ca2+ signalling, mitochondrial function and gene expression associated with the disease. We show that RyR1 mutations that lead to the depletion of the channel are associated with increased IP3-mediated nuclear and mitochondrial Ca2+ signals and increased mitochondrial activity. Moreover, western blot and microarray analysis indicated enhanced mitochondrial biogenesis at the transcriptional and protein levels and was reflected in increased mitochondrial DNA content. The phenotype was recapitulated by RYR1 silencing in mouse cellular myotube models. Altogether, these data indicate that remodelling of skeletal muscle Ca2+ signalling following loss of functional RyR1 mediates bioenergetic adaptation.

When muscle Ca2+ channels carry monovalent cations through gating pores: insights into the pathophysiology of type 1 hypokalaemic periodic paralysis.

Patients suffering from type 1 hypokalaemic periodic paralysis (HypoPP1) experience attacks of muscle paralysis associated with hypokalaemia. The disease arises from missense mutations in the gene encoding the α1subunit of the dihydropyridine receptor (DHPR), a protein complex anchored in the tubular membrane of skeletal muscle fibres which controls the release of Ca2+ from sarcoplasmic reticulum and also functions as a Ca2+ channel. The vast majority of mutations consist of the replacement of one of the outer arginines in S4 segments of the α1subunit by neutral residues. Early studies have shown that muscle fibres from HypoPP1 patients are abnormally depolarized at rest in low K+ to the point of inducing muscle inexcitability. The relationship between HypoPP1 mutations and depolarization has long remained unknown. More recent investigations conducted in the closely structurally related voltage-gated Na+ and K+ channels have shown that comparable S4 arginine substitutions gave rise to elevated inward currents at negative potentials called gating pore currents. Experiments performed in muscle fibres from different models revealed such an inward resting current through HypoPP1 mutated Ca2+ channels. In mouse fibres transfected with HypoPP1 mutated channels, the elevated resting current was found to carry H+ for the R1239H arginine-to-histidine mutation in a S4 segment and Na+ for the V876E HypoPP1 mutation, which has the peculiarity of not being located in S4 segments. Muscle paralysis probably results from the presence of a gating pore current associated with hypokalaemia for both mutations, possibly aggravated by external acidosis for the R1239H mutation.

Epigenetic responses to exertional heat stroke in mice: a potential link to long term Ca2+ dysregulation in skeletal muscle

Exposure to extreme environments can induce a long‐term epigenetic memory of stress that is stored as altered DNA‐methylation and/or histone posttranslational modifications. These ultimately affect gene and protein expression as well as cellular functions. Exertional heat stroke (EHS) is a life‐threatening stress, but whether it induces epigenetic changes is unknown. Previous studies have suggested long‐term complications of EHS that could be mediated by epigenetics, including greater vulnerability to disease, heat stress susceptibility and disordered muscle Ca2+ signaling; the latter characterized as a malignant‐hyperthermia‐type muscle phenotype, based on the in vitro contracture test (IVCT).PURPOSETo determine whether a single exposure to EHS using a preclinical mouse model induces significant changes in DNA methylation and altered skeletal muscle Ca2+ regulation.METHODSMice were either subjected to a standardized EHS protocol using a forced running wheel at 37.5°C or a matched exercise control trial (22.5 °C), N=4/group. The EHS mice achieved peak core temps of ~42.2 °C, accompanied by transient loss of consciousness. Mice were euthanized after 4 or 30 days. At day 4, cells of a monocyte lineage were isolated from bone marrow, and DNA was extracted and tested using bisulfite DNA sequencing. At 30 days, soleus muscles were excised (N=5/group) and tested for a Ca2+ response to caffeine and halothane (IVCT test).RESULTS&gt;3,000 genes from monocytes were significantly hyper‐ or hypomethylated in EHS mice compared to matched controls. Some genes of physiological interest included Ryr1&amp;2, Cacna1s&amp;c (DHPR isoforms), Orai1 (store operated calcium channel), Camk2d, Pde4d (cAMP signaling), Atp2a1/2, Sod2, Nox4, Nos1&amp;2 isoforms, Hsp72, a number of K+ channels, and isoforms of Mir669a‐1 (inhibitors of skeletal muscle myogenesis). The IVCT displayed an abnormal response in solei from EHS mice (3/5) vs. control (0/5) (p=0.031; adjusted Wald Chi‐Square test). Two mice showed an abnormal response to caffeine; one displayed abnormal responses to both caffeine and halothane.CONCLUSIONSThe data demonstrates that EHS induces striking alterations in the epigenome at 4 days. Most biological and behavioral responses return to normal by this time. DNA methylation profiles from accessible blood cell populations may prove to be a useful biomarker for severity of EHS exposures. The IVCT findings mirror the incidence in military EHS populations. Coupled with the epigenetic changes observed in Ca2+‐related genes, studying skeletal muscle Ca2+ regulation may prove a promising target for future discovery.Support or Funding InformationBK and Betty Stevens Endowment and DOD Grant W81XWH‐15‐2‐0038. Disclaimers: Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calsequestrin, a new modulator of unfolded protein response in skeletal and cardiac muscle

Cellular responses to stress are an integral part of muscle physiology and pathology. To deal with stress, cardiac and skeletal muscle cells utilize various mechanisms, engaging intercellular organelles including the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) [1]. The disruptions of ER functions are often observed in ischemic, hypertrophic and failing hearts [1]. However, the molecular mechanisms governing muscle ER stress regulation in the muscle are still unclear. Disruption of ER/SR hemostasis with physiological and pathological stimuli triggers a coping response, so called unfolded protein response (UPR). One of the UPR signaling pathways involves stress sensor inositol‐requiring enzyme 1α (IRE1α), an integral ER/SR membrane protein kinase and mRNA splicing enzyme. Here, we discovered that calsequestrin, a muscle Ca2+ binding protein localized to junctional SR, interacts with UPR stress sensor IRE1α, and contribute to the modulation of stress responses in the muscle. Both skeletal and cardiac isoforms of calsequestrin bind directly to IRE1α as measured by Microscale Thermophoresis (MST), Surface Plasmon Resonance (SPR), and His‐tag Ni‐NTA‐agarose pull down techniques. Although calsequestrin contains three thioredoxin‐like domains its interaction with IRE1α did not involve cysteine residues in IRE1α. Iodixanol gradient fractionation and immunostaining of skeletal muscle sections or isolated cardiomyocytes showed enrichment of IRE1α protein in junctional SR where it is colocalized with calsequestrin. MST analysis revealed that calsequestrin binding to IRE1α has inhibitory effect on IRE1α by preventing dimerization/oligomerization IRE1α required for X‐box binding protein 1 (XBP1) splicing activity. Consequently, cardiac calsequestrin‐deficient cardiomyocytes have increased activity of IRE1α. We concluded that calsequestrin is a new IRE1α binding partner and the protein plays a role in modulation of stress responses in skeletal and cardiac muscle.Support or Funding InformationCanada Institutes of Health Research (CIHR)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impaired Ca2+ handling in resistance arteries from genetically obese Zucker rats: Role of the PI3K, ERK1/2 and PKC signaling pathways

The impact of obesity on vascular smooth muscle (VSM) Ca2+ handling and vasoconstriction, and its regulation by the phosphatidylinositol 3-kinase (PI3K), mitogen activated protein kinase (MAPK) and protein kinase C (PKC) were assessed in mesenteric arteries (MA) from obese Zucker rats (OZR). Simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and tension were performed in MA from OZR and compared to lean Zucker rats (LZR), and the effects of selective inhibitors of PI3K, ERK-MAPK kinase and PKC were assessed on the functional responses of VSM voltage-dependent L-type Ca2+ channels (CaV1.2). Increases in [Ca2+]i induced by α1-adrenoceptor activation and high K+ depolarization were not different in arteries from LZR and OZR although vasoconstriction was enhanced in OZR. Blockade of the ryanodine receptor (RyR) and of Ca2+ release from the sarcoplasmic reticulum (SR) markedly reduced depolarization-induced Ca2+ responses in arteries from lean but not obese rats, suggesting impaired Ca2+-induced Ca2+ release (CICR) from SR in arteries from OZR. Enhanced Ca2+ influx after treatment with ryanodine was abolished by nifedipine and coupled to up-regulation of CaV1.2 channels in arteries from OZR. Increased activation of ERK-MAPK and up-regulation of PI3Kδ, PKCβ and δ isoforms were associated to larger inhibitory effects of PI3K, MAPK and PKC blockers on VSM L-type channel Ca2+ entry in OZR. Changes in arterial Ca2+ handling in obesity involve SR Ca2+ store dysfunction and enhanced VSM Ca2+ entry through L-type channels, linked to a compensatory up-regulation of CaV1.2 proteins and increased activity of the ERK-MAPK, PI3Kδ and PKCβ and δ, signaling pathways.

Smooth muscle Ca2+ sensitization causes hypercontractility of middle cerebral arteries in mice bearing the familial hemiplegic migraine type 2 associated mutation

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2+/−G301R) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca2+ and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2+/−G301R and WT showed similar contractile responses. The Atp1a2+/−G301R MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca2+ change was smaller than in WT. The enhanced constriction of Atp1a2+/−G301R MCA was associated with increased cSrc activation, stronger sensitization to [Ca2+]i and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2+/−G301R mice had reduced resting blood flow through MCA in comparison with WT mice. FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca2+, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Arsenic, cadmium, and mercury-induced hypertension: mechanisms and epidemiological findings

ABSTRACTArsenic (As), cadmium (Cd), and mercury (Hg) are toxic elements widely distributed in the environment. Exposure to these elements was attributed to produce several acute and chronic illnesses including hypertension. The aim of this review is to provide a summary of the most frequently proposed mechanisms underlying hypertension associated with As, Cd, and Hg exposure including: oxidative stress, impaired nitric oxide (NO) signaling, modified vascular response to neurotransmitters and disturbed vascular muscle Ca2+ signaling, renal damage, and interference with the renin–angiotensin system. Due to the complexity of the vascular system, a combination rather than a singular mechanism needs to be considered. In addition, epidemiological findings showing the relationship between various biomarkers of metal exposure and hypertension are described. Given the complex etiology of hypertension, further epidemiological studies evaluating the roles of confounding factors such as age, gender, and life style are still necessary.

Congenital myopathies: disorders of excitation-contraction coupling and muscle contraction.

The congenital myopathies are a group of early-onset, non-dystrophic neuromuscular conditions with characteristic muscle biopsy findings, variable severity and a stable or slowly progressive course. Pronounced weakness in axial and proximal muscle groups is a common feature, and involvement of extraocular, cardiorespiratory and/or distal muscles can implicate specific genetic defects. Central core disease (CCD), multi-minicore disease (MmD), centronuclear myopathy (CNM) and nemaline myopathy were among the first congenital myopathies to be reported, and they still represent the main diagnostic categories. However, these entities seem to belong to a much wider phenotypic spectrum. To date, congenital myopathies have been attributed to mutations in over 20 genes, which encode proteins implicated in skeletal muscle Ca2+ homeostasis, excitation-contraction coupling, thin-thick filament assembly and interactions, and other mechanisms. RYR1 mutations are the most frequent genetic cause, and CCD and MmD are the most common subgroups. Next-generation sequencing has vastly improved mutation detection and has enabled the identification of novel genetic backgrounds. At present, management of congenital myopathies is largely supportive, although new therapeutic approaches are reaching the clinical trial stage.

CD38-cADPR-SERCA Signaling Axis Determines Skeletal Muscle Contractile Force in Response to β-Adrenergic Stimulation

Background/Aims: Cyclic ADP-ribose (cADPR) is a Ca²⁺ -mobilization messenger that acts on ryanodine-sensitive Ca²⁺ channels in the sarcoplasmic reticulum (SR) Ca²⁺ stores. Moreover, it has been proposed that cADPR serves an additional role in activating the sarcoendoplasmic reticulum Ca²⁺ -ATPase (SERCA) pump. The aim of this study was to determine the exact mechanism by which cADPR regulates SR Ca²⁺ stores in physiologically relevant systems. Methods: We analyzed Ca²⁺ signals as well as the production of Ca²⁺ mobilizing messengers in the skeletal muscle cells of mice subjected to intensive exercise or in the SR fractions from skeletal muscle cells after β-adrenergic receptor (β-AR) stimulation. Results: We show that cADPR enhances SERCA activity in skeletal muscle cells in response to β-AR agonists, increasing SR Ca²⁺ uptake. We demonstrate that cADPR is generated by CD38, a cADPR-synthesizing enzyme, increasing muscle Ca²⁺ signals and contractile force during exercise. CD38 is upregulated by the cAMP response element–binding protein (CREB) transcription factor upon β-AR stimuli and exercise. CD38 knockout (KO) mice show defects in their exercise and cADPR synthesis capabilities, lacking a β-AR agonist-induced muscle contraction when compared to wild-type mice. The skeletal muscle of CD38 KO mice exhibits delayed cytosolic Ca²⁺ clearance and reduced SERCA activity upon exercise. Conclusion: These findings provide insight into the physiological adaptive mechanism by which the CD38- cADPR-SERCA signaling axis plays an essential role in muscle contraction under exercise, and define cADPR as an endogenous activator of SERCA in enhancing the SR Ca²⁺ load.

Na leak with gating pore properties in hypokalemic periodic paralysis V876E mutant muscle Ca channel.

Type 1 hypokalemic periodic paralysis (HypoPP1) is a poorly understood genetic neuromuscular disease characterized by episodic attacks of paralysis associated with low blood K+ The vast majority of HypoPP1 mutations involve the replacement of an arginine by a neutral residue in one of the S4 segments of the α1 subunit of the skeletal muscle voltage-gated Ca2+ channel, which is thought to generate a pathogenic gating pore current. The V876E HypoPP1 mutation has the peculiarity of being located in the S3 segment of domain III, rather than an S4 segment, raising the question of whether such a mutation induces a gating pore current. Here we successfully transfer cDNAs encoding GFP-tagged human wild-type (WT) and V876E HypoPP1 mutant α1 subunits into mouse muscles by electroporation. The expression profile of these WT and V876E channels shows a regular striated pattern, indicative of their localization in the t-tubule membrane. In addition, L-type Ca2+ current properties are the same in V876E and WT fibers. However, in the presence of an external solution containing low-Cl- and lacking Na+ and K+, V876E fibers display an elevated leak current at negative voltages that is increased by external acidification to a higher extent in V876E fibers, suggesting that the leak current is carried by H+ ions. However, in the presence of Tyrode's solution, the rate of change in intracellular pH produced by external acidification was not significantly different in V876E and WT fibers. Simultaneous measurement of intracellular Na+ and current in response to Na+ readmission in the external solution reveals a rate of Na+ influx associated with an inward current, which are both significantly larger in V876E fibers. These data suggest that the V876E mutation generates a gating pore current that carries strong resting Na+ inward currents in physiological conditions that are likely responsible for the severe HypoPP1 symptoms associated with this mutation.

EC.I.1 - Basıc aspects of excitation-contraction coupling and its relation to clinical disorders

In response to excitation by motor neurons, skeletal muscle fibers develop trains of action potentials that spread along the sarcolemma and in depth along the transverse tubules. Depolarization induces a change in the configuration of a protein complex anchored in the transverse tubular membrane, the dihydropyridine receptor (DHPR), which gets open the ryanodine receptor (RyR) anchored in the membrane of the sarcoplasmic reticulum (SR) in charge of the release of Ca2+ that activates contraction. The succession of events from the action potential to the SR Ca2+ release is referred as the excitation-contraction coupling process. Relaxation of muscle fibers results from both the return of the fiber membrane potential to resting negative values and pumping of cytosolic Ca2+ back into the SR with the help of SR Ca2+-ATPases. The use of the voltage-clamp technique, the measurement of cytosolic or intra-SR Ca2+ changes, together with genetic approaches and recent developments of cryo-electron microscopy have all contributed to a better understanding of the basic molecular and cellular steps involved in the voltage controlled opening of the RyR by the DHPR but also allowed to uncover trans-sarcolemmal Ca2+ influx pathways and accessory proteins that participate in muscle Ca2+ homeostasis. Mutations in the genes encoding proteins involved in intracellular Ca2+ handling induce various muscle disorders such as Brody disease, malignant hyperthermia, central core disease or periodic paralysis with clinical features ranging from abnormal muscle stiffness to muscle weakness, paralysis or degeneration. Deciphering the pathophysiological mechanisms involved in these disorders is critical for the development of therapeutic strategies.

Orosomucoid-like 3 (ORMDL3) upregulates airway smooth muscle proliferation, contraction, and Ca2+ oscillations in asthma

Airway hyperresponsiveness is a major feature of asthma attributed predominantly to an extrinsic immune/inflammatory response increasing airway smooth muscle (ASM) contractility. We investigated whether increased ASM expression of orosomucoid-like 3 (ORMDL3), a gene on chromosome 17q21 highly linked to asthma, induced increased ASM proliferation and contractility invitro and influenced airway contractility and calcium flux in ASM in precision-cut lung slices (PCLSs) from wild-type and hORMDL3Zp3-Cre mice (which express increased levels of human ORMDL3 [hORMDL3]). Levels of ASM proliferation and contraction were assessed in ASM cells transfected with ORMDL3 in vitro. In addition, airway contractility and calcium oscillations were quantitated in ASM cells in PCLSs derived from naive wild-type and naive hORMDL3Zp3-Cre mice, which do not have a blood supply. Increased ASM expression of ORMDL3 invitro resulted in increased ASM proliferation and contractility. PCLSs derived from naive hORMDL3Zp3-Cre mice, which do not have airway inflammation, exhibit increased airway contractility with increased calcium oscillations in ASM cells. Increased ASM ORMDL3 expression increases levels of ASM sarcoplasmic reticulum Ca2+ ATPase 2b (SERCA2b), which increases ASM proliferation and contractility. Overall, these studies provide evidence that an intrinsic increase in ORMDL3 expression in ASM can induce increased ASM proliferation and contractility, which might contribute to increased airway hyperresponsiveness in the absence of airway inflammation in asthmatic patients.