Muscle Excitation-contraction Coupling Research Articles

High Resolution Structure of the Membrane Embedded Skeletal Muscle Ryanodine Receptor

The type 1 ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum (SR) is required for skeletal muscle excitation-contraction coupling and is the largest known ion channel, comprised of four 565 kDa protomers. Cryogenic electron microscopy (cryoEM) studies of the RyR have primarily used detergent to solubilize the channel; in the present study, we have used cryoEM to solve high-resolution structures of the channel in liposomes using a gel-filtration approach with on-column detergent removal to form liposomes and incorporate the channel simultaneously, improving the incorporation rate by >20-fold compared to a dialysis-based approach. This allowed us to resolve the structure of the channel in the closed and open states at 3.36 and 3.98 A, respectively. This method offers validation for detergent-based structures of the RyR and offers a method for utilizing an electrochemical gradient mimicking the SR, where Ca2+ concentrations are millimolar in the lumen and nanomolar in the cytosol.

Ryanodine Receptor Type 2: A Molecular Target for Dichlorodiphenyltrichloroethane- and Dichlorodiphenyldichloroethylene-Mediated Cardiotoxicity.

DDT/DDE interactions with RyR2 alter cardiomyocyte Ca2+ dynamics that may contribute to adverse cardiovascular outcomes associated with exposures.

Multiple Sequence Variants in STAC3 Affect Interactions with CaV1.1 and Excitation-Contraction Coupling

STAC3 is a soluble protein essential for skeletal muscle excitation-contraction (EC) coupling. Through its tandem SH3 domains, it interacts with the cytosolic II-III loop of the skeletal muscle voltage-gated calcium channel. STAC3 is the target for a mutation (W284S) that causes Native American myopathy, but multiple other sequence variants have been reported. Here, we report a crystal structure of the human STAC3 tandem SH3 domains. We analyzed the effect of five disease-associated variants, spread over both SH3 domains, on their ability to bind to the CaV1.1 II-III loop and on muscle EC coupling. In addition to W284S, we find the F295L and K329N variants to affect both binding and EC coupling. The ability of the K329N variant, located in the second SH3 domain, to affect the interaction highlights the importance of both SH3 domains in association with CaV1.1. Our results suggest that multiple STAC3 variants may cause myopathy.

Multi-species transcriptomics reveals evolutionary diversity in the mechanisms regulating shrimp tail muscle excitation-contraction coupling.

The natural flight response in shrimp is powered by rapid contractions of the abdominal muscle fibres to propel themselves backwards away from perceived danger. This muscle contraction is dependent on repetitive depolarization of muscle plasma membrane, triggering tightly spaced cytoplasmic [Ca2+] transients and rapidly rising tetanic force responses. To achieve such high amplitude and high frequency of Ca2+ transients requires a high abundance of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) to rapidly clear cytoplasmic Ca2+ between each transient and an efficient Ca2+ release system consisting of the Ryanodine Receptor (RyR), and voltage gated Ca2+ channels (CaVs). With the aim to expand our knowledge of muscle gene function and identify orthologous genes regulating muscle excitation-contraction (EC) coupling, this study assembled nine Penaeid shrimp muscle transcriptomes. On average, the nine transcriptomes contained 27,000 contigs, with an annotation rate of 36% and a BUSCO completeness of 70%. Despite maintaining their function, the crustacean RyR and CaV proteins showed evidence of significant diversification from mammalian orthologs, while SERCA remained more conserved. Several key components of protein interaction were conserved, while others showed distinct crustacean specific evolutionary adaptations. Lastly, this study revealed approximately 1,000 orthologous genes involved in muscle specific processes present across all nine species.

Neuronal Junctophilin 3 Can Replace Muscle Junctophilin 2 in Voltage-induced Calcium Release

We recently showed that skeletal-muscle, voltage-induced calcium release could be recapitulated in tsA201 cells by expression of 5 muscle junctional proteins: CaV1.1, CaVβ1a, Stac3, RyR1 and Junctophilin-2 (JP2). In tsA201 cells, JP2 induced junctions at which CaV1.1, CaVβ1a, Stac3 accumulated on the PM side, and RyR1 accumulated on the ER side. To determine whether isoform-specific properties of JP2 are required for voltage-gated Ca2+ release, we have now replaced JP2 with the neuronal Junctophilin-3 (JP3). We found that JP3 is more effective at recruiting RyR1 to junctions than JP2, either when the JP's are expressed alone or when CaV1.1, CaVβ1a and Stac3 are also present, which increased RyR1 recruitment for both junctophilins. Strikingly, a truncated, cytoplasmically diffusible RyR1 (RyR11:4300), lacking the ER transmembrane domain, strongly co-localizes with JP3 alone, but very poorly with JP2 alone. CaV1.1, CaVβ1a and Stac3 co-expression confers on JP2 the ability to recruit RyR11:4300 and further increases the recruitment of RyR11:4300 by JP3. We next tested whether JP3 could not only recruit the other junctional proteins but functionally replace JP2. Freeze-fracture electron microscopy of cells expressing JP3 and the other four proteins revealed the presence of CaV1.1 arranged in tetrads, indicating mechanical links between CaV1.1 and RyR1. Moreover, depolarization elicited Ca2+ transients not requiring extracellular Ca2+ entry (as in skeletal muscle). However, the transients for JP3 were slightly smaller and slower than those for JP2. Our results indicate that the major role of JP2 in striated muscle excitation-contraction coupling is to form and stabilize ER-PM junctions and to recruit the necessary proteins to these junctions. Voltage-dependent Ca2+ release does not require the presence of a muscle Junctophilin, because such release still occurs, albeit less efficiently, with the neuronal JP3. Supported by NIH-AR070298.

Effect of Dimethyl Sulfoxide on Excitation-Contraction Coupling in Chicken Slow Skeletal Muscle

Effect of Dimethyl Sulfoxide on Excitation-Contraction Coupling in Chicken Slow Skeletal Muscle

Interactions of Dichlorodiphenyltrichloroethane (DDT) and Dichlorodiphenyldichloroethylene (DDE) With Skeletal Muscle Ryanodine Receptor Type 1.

Dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiphenyldichloroethylene (DDE) are ubiquitous in the environment and detected in tissues of living organisms. Although DDT owes its insecticidal activity to impeding closure of voltage-gated sodium channels, it mediates toxicity in mammals by acting as an endocrine disruptor (ED). Numerous studies demonstrate DDT/DDE to be EDs, but studies examining muscle-specific effects mediated by nonhormonal receptors in mammals are lacking. Therefore, we investigated whether o,p'-DDT, p,p'-DDT, o,p'-DDE, and p,p'-DDE (DDx, collectively) alter the function of ryanodine receptor type 1 (RyR1), a protein critical for skeletal muscle excitation-contraction coupling and muscle health. DDx (0.01-10 µM) elicited concentration-dependent increases in [3H]ryanodine ([3H]Ry) binding to RyR1 with o,p'-DDE showing highest potency and efficacy. DDx also showed sex differences in [3H]Ry-binding efficacy toward RyR1, where [3H]Ry-binding in female muscle preparations was greater than male counterparts. Measurements of Ca2+ transport across sarcoplasmic reticulum (SR) membrane vesicles further confirmed DDx can selectively engage with RyR1 to cause Ca2+ efflux from SR stores. DDx also disrupts RyR1-signaling in HEK293T cells stably expressing RyR1 (HEK-RyR1). Pretreatment with DDx (0.1-10 µM) for 100 s, 12 h, or 24 h significantly sensitized Ca2+-efflux triggered by RyR agonist caffeine in a concentration-dependent manner. o,p'-DDE (24 h; 1 µM) significantly increased Ca2+-transient amplitude from electrically stimulated mouse myotubes compared with control and displayed abnormal fatigability. In conclusion, our study demonstrates DDx can directly interact and modulate RyR1 conformation, thereby altering SR Ca2+-dynamics and sensitize RyR1-expressing cells to RyR1 activators, which may ultimately contribute to long-term impairments in muscle health.

Interaction of the Homer1 EVH1 domain and skeletal muscle ryanodine receptor

The skeletal muscle ryanodine receptor (RyR1) proteins are intracellular calcium (Ca2+) release channels on the membrane of the sarcoplasmic reticulum (SR) and required for skeletal muscle excitation-contraction coupling. Homer (Vesl) is a family of scaffolding proteins that modulate target proteins including RyRs (ryanodine receptors), mGluRs (group 1 metabotropic glutamate receptors) and IP3Rs (inositol-1,4,5-trisphosphate receptors) through a conserved EVH1 (Ena/VASP homology 1) domain. Here, we examined the interaction between Homer1 EVH1 domain and RyR1 by co-immunoprecipitation, continuous sucrose density-gradient centrifugation, and bio-layer interferometry binding assay at different Ca2+ concentrations. Our results show that there exists a high-affinity binding between the Homer1 EVH1 domain and RyR1, especially at 1 mM of Ca2+. Based on our data and the known structures of Homer1 EVH1 domain and RyR1, we found two consensus proline-rich sequences in the structure of RyR1, PPHHF and FLPPP, and proposed two corresponding binding models to show mechanisms of recognition different from those used by other proline-rich motifs. The side proline residues of two proline-rich motifs from RyR1 are away from the hydrophobic surface of Homer1 EVH1, rather than buried in this hydrophobic surface. Our results provide evidence that Homer1 regulates RyR1 by direct interaction.

Two pools of IRE1α in cardiac and skeletal muscle cells.

The endoplasmic reticulum (ER) plays a central role in cellular stress responses via mobilization of ER stress coping responses, such as the unfolded protein response (UPR). The inositol-requiring 1α (IRE1α) is an ER stress sensor and component of the UPR. Muscle cells also have a well-developed and highly subspecialized membrane network of smooth ER called the sarcoplasmic reticulum (SR) surrounding myofibrils and specialized for Ca2+ storage, release, and uptake to control muscle excitation-contraction coupling. Here, we describe 2 distinct pools of IRE1α in cardiac and skeletal muscle cells, one localized at the perinuclear ER and the other at the junctional SR. We discovered that, at the junctional SR, calsequestrin binds to the ER luminal domain of IRE1α, inhibiting its dimerization. This novel interaction of IRE1α with calsequestrin, one of the highly abundant Ca2+ handling proteins at the junctional SR, provides new insights into the regulation of stress coping responses in muscle cells.-Wang, Q., Groenendyk, J., Paskevicius, T., Qin, W., Kor, K. C., Liu, Y., Hiess, F., Knollmann, B. C., Chen, S. R. W., Tang, J., Chen, X.-Z., Agellon, L. B., Michalak, M. Two pools of IRE1α in cardiac and skeletal muscle cells.

Neuromuscular evaluation of arm-cycling repeated sprints under hypoxia and/or blood flow restriction.

This study aimed to determine the effects of hypoxia and/or blood flow restriction (BFR) on an arm-cycling repeated sprint ability test (aRSA) and its impact on elbow flexor neuromuscular function. Fourteen volunteers performed an aRSA (10s sprint/20s recovery) to exhaustion in four randomized conditions: normoxia (NOR), normoxia plus BFR (NBFR), hypoxia (FiO2 = 0.13, HYP) and hypoxia plus BFR (HBFR). Maximal voluntary contraction (MVC), resting twitch force (Db10), and electromyographic responses from the elbow flexors [biceps brachii (BB)] to electrical and transcranial magnetic stimulation were obtained to assess neuromuscular function. Main effects of hypoxia, BFR, and interaction were analyzed on delta values from pre- to post-exercise. BFR and hypoxia decreased the number of sprints during aRSA with no significant cumulative effect (NOR 16 ± 8; NBFR 12 ± 4; HYP 10 ± 3 and HBFR 8 ± 3; P < 0.01). MVC decrease from pre- to post-exercise was comparable whatever the condition. M-wave amplitude (- 9.4 ± 1.9% vs. + 0.8 ± 2.0%, P < 0.01) and Db10 force (- 41.8 ± 4.7% vs. - 27.9 ± 4.5%, P < 0.01) were more altered after aRSA with BFR compared to without BFR. The exercise-induced increase in corticospinal excitability was significantly lower in hypoxic vs. normoxic conditions (e.g., BB motor evoked potential at 75% of MVC: - 2.4 ± 4.2% vs. + 16.0 ± 5.9%, respectively, P = 0.03). BFR and hypoxia led to comparable aRSA performance impairments but with distinct fatigue etiology. BFR impaired the muscle excitation-contraction coupling whereas hypoxia predominantly affected corticospinal excitability indicating incapacity of the corticospinal pathway to adapt to fatigue as in normoxia.

Salicylic acid amplifies Carbachol-induced bronchoconstriction in human precision-cut lung slices

BackgroundAsthma exacerbations evoke emergency room visits, progressive loss of lung function and increased mortality. Environmental and industrial toxicants exacerbate asthma, although the underlying mechanisms are unknown. We assessed whether 3 distinct toxicants, salicylic acid (SA), toluene diisocyanate (TDI), and 1-chloro-2,4-dinitrobenzene (DNCB) induced airway hyperresponsiveness (AHR) through modulating excitation-contraction coupling in human airway smooth muscle (HASM) cells. The toxicants include a non-sensitizing irritant (SA), respiratory sensitizer (TDI) and dermal sensitizer (DNCB), respectively. We hypothesized that these toxicants induce AHR by modulating excitation-contraction (EC) coupling in airway smooth muscle (ASM) cells.MethodsCarbachol-induced bronchoconstriction was measured in precision-cut human lung slices (hPCLS) following exposure to SA, TDI, DNCB or vehicle. Culture supernatants of hPCLS were screened for mediator release. In HASM cells treated with the toxicants, surrogate readouts of EC coupling were measured by phosphorylated myosin light chain (pMLC) and agonist-induced Ca2+ mobilization ([Ca2+]i). In addition, Nrf-2-dependent antioxidant response was determined by NAD(P) H quinone oxidoreductase 1 (NQO1) expression in HASM cells.ResultsIn hPCLS, SA, but not TDI or DNCB, potentiated carbachol-induced bronchoconstriction. The toxicants had little effect on release of inflammatory mediators, including IL-6, IL-8 and eotaxin from hPCLS. In HASM cells, TDI amplified carbachol-induced MLC phosphorylation. The toxicants also had little effect on agonist-induced [Ca2+]i.ConclusionSA, a non-sensitizing irritant, amplifies agonist-induced bronchoconstriction in hPCLS via mechanisms independent of inflammation and Ca2+ homeostasis in HASM cells. The sensitizers TDI and DNCB, had little effect on bronchoconstriction or inflammatory mediator release in hPCLS.ImplicationsOur findings suggest that non-sensitizing irritant salicylic acid may evoke AHR and exacerbate symptoms in susceptible individuals or in those with underlying lung disease.

Pomolic acid reduces contractility and modulates excitation-contraction coupling in rat cardiomyocytes

Pomolic acid (PA) isolated from Licania pittieri has hypotensive effects in rats, inhibits human platelet aggregation and elicits endothelium-dependent relaxation in rat aortic rings. The present study was designed to investigate the effects of PA on cardiomyocytes. Trabeculae and enzymatically isolated cardiomyocytes from rats were used to evaluate the concentration-dependent effects of PA on cardiac muscle tension and excitation-contraction coupling (ECC) by recording Ca2+ transients reported with Fluo-3 and Fura-2, as well as L-type Ca2+ currents (LTCC). PA reduced the contractile force in rat cardiac trabeculae with an EC50 = 14.3 ± 2.4 μM. PA also reduced the amplitude of Ca2+ transients in a concentration-dependent manner, with an EC50 = 10.5 ± 1.3 μM, without reducing sarcoplasmic reticulum (SR) Ca2+ loading. PA decreased the half width of the Ca2+ transient by 31.7 ± 3.3% and increased the decay time and decay time constant (τ) by 7.6 ± 2.7% and 75.6 ± 3.7%, respectively, which was associated with increased phospholamban (PLN) phosphorylation. PA also reversibly reduced the macroscopic LTCC in the cardiomyocyte membrane, but did not demonstrate any effects on skeletal muscle ECC. In conclusion, PA reduces LTCC, Ca2+ transients and cardiomyocyte force, which along with its vasorelaxant effects explain its hypotensive properties. Increased PLN phosphorylation protected the SR from Ca2+ depletion. Considering the effects of PA on platelet aggregation and the cardiovascular system, we propose it as a new potential, multitarget cardiovascular agent with a demonstrated safety profile.

Correcting the R165K substitution in the first voltage-sensor of CaV1.1 right-shifts the voltage-dependence of skeletal muscle calcium channel activation

ABSTRACTThe voltage-gated calcium channel CaV1.1a primarily functions as voltage-sensor in skeletal muscle excitation-contraction (EC) coupling. In embryonic muscle the splice variant CaV1.1e, which lacks exon 29, additionally function as a genuine L-type calcium channel. Because previous work in most laboratories used a CaV1.1 expression plasmid containing a single amino acid substitution (R165K) of a critical gating charge in the first voltage-sensing domain (VSD), we corrected this substitution and analyzed its effects on the gating properties of the L-type calcium currents in dysgenic myotubes. Reverting K165 to R right-shifted the voltage-dependence of activation by ~12 mV in both CaV1.1 splice variants without changing their current amplitudes or kinetics. This demonstrates the exquisite sensitivity of the voltage-sensor function to changes in the specific amino acid side chains independent of their charge. Our results further indicate the cooperativity of VSDs I and IV in determining the voltage-sensitivity of CaV1.1 channel gating.

Physiological functions and Ca2+ signal transduction pathway exploring during molting of silkworm, Bombyx mori

Abstract Calcium ions are an important second messagers in the life of organisms, such as in silkworm growth and their developmental processes; these ions can trigger various effects, including the muscle excitation-contraction coupling mechanism, secretion of endocrine and exocrine glands, neuronal activities, midgut nutrients metabolisms, sensory system, enzymatic systems, and acceleration of metabolism activities. This paper reviewed the ecdysis process and mechanisms, where calcium plays an important role in larvae-to-larvae ecdysis and larvae-to-pupae and pupae-to-moth metamorphosis of silkworms; the paper focuses on the advanced achievements in the field of calcium actions during larvae-to-larvae and larvae-to-pupae changes. Calcium ion channels are shown to be the action mechanism of phospholipase C in the G protein cascade of the calcium signal transduction pathway, and the activators and inhibitors of these calcium channels are further discussed.

STAC3 variants cause a congenital myopathy with distinctive dysmorphic features and malignant hyperthermia susceptibility.

SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G>T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.

STAC3 incorporation into skeletal muscle triads occurs independent of the dihydropyridine receptor.

Excitation‐contraction (EC) coupling in skeletal muscles operates through a physical interaction between the dihydropyridine receptor (DHPR), acting as a voltage sensor, and the ryanodine receptor (RyR1), acting as a calcium release channel. Recently, the adaptor protein SH3 and cysteine‐rich containing protein 3 (STAC3) has been identified as a myopathy disease gene and as an additional essential EC coupling component. STAC3 interacts with DHPR sequences including the critical EC coupling domain and has been proposed to function in linking the DHPR and RyR1. However, we and others demonstrated that incorporation of recombinant STAC3 into skeletal muscle triads critically depends only on the DHPR but not the RyR1. On the contrary, here, we provide evidence that endogenous STAC3 incorporates into triads in the absence of the DHPR in myotubes and muscle fibers of dysgenic mice. This finding demonstrates that STAC3 interacts with additional triad proteins and is consistent with its proposed role in directly or indirectly linking the DHPR with the RyR1.

Model discrimination for Ca2+ -dependent regulation of myosin light chain kinase in smooth muscle contraction.

Excitation-contraction coupling in smooth muscle is mediated by the Ca2+ - and calmodulin-dependent regulation of myosin light chain kinase. The precise mechanism of this regulation remains controversial, and several mathematical models have been proposed for the interaction of the three species. These models have previously been analyzed at steady state primarily by numerical simulation of differential equations, for which parameter values must be estimated from data. Here, we use the linear framework for timescale separation to demonstrate that models of this general kind can be solved analytically for an equilibrium steady state, without having to determine parameter values. This analysis leads to parameter-independent methods for discriminating between the models, for which we propose experiments that could be performed with existing methods.

Duplex signaling by CaM and Stac3 enhances CaV1.1 function and provides insights into congenital myopathy.

CaV1.1 is essential for skeletal muscle excitation-contraction coupling. Its functional expression is tuned by numerous regulatory proteins, yet underlying modulatory mechanisms remain ambiguous as CaV1.1 fails to function in heterologous systems. In this study, by dissecting channel trafficking versus gating, we evaluated the requirements for functional CaV1.1 in heterologous systems. Although coexpression of the auxiliary β subunit is sufficient for surface-membrane localization, this baseline trafficking is weak, and channels elicit a diminished open probability. The regulatory proteins calmodulin and stac3 independently enhance channel trafficking and gating via their interaction with the CaV1.1 carboxy terminus. Myopathic stac3 mutations weaken channel binding and diminish trafficking. Our findings demonstrate that multiple regulatory proteins orchestrate CaV1.1 function via duplex mechanisms. Our work also furnishes insights into the pathophysiology of stac3-associated congenital myopathy and reveals novel avenues for pharmacological intervention.

Chapter 38 - Malignant hyperthermia

Malignant hyperthermia (MH) is a form of heat illness caused by increased heat generation exceeding the body's capacity for heat loss. It is classified separately from other forms of heat illness as the latter require assessment of mental function for differential diagnosis. This is not possible with MH which occurs during general anesthesia when mental function cannot be assessed. MH occurs in genetically predisposed individuals exposed to inhalation anesthetics or succinylcholine. The genetic defects identified so far cause perturbation of skeletal muscle excitation-contraction coupling resulting in myoplasmic calcium dysregulation. The most commonly involved gene is RYR1. Increased myoplasmic calcium leads to hypermetabolism and sustained muscle contractile activity with consequent increased oxygen consumption, carbon dioxide production, sympathetic stimulation, muscle rigidity, heat production, rhabdomyolysis, and disseminated intravascular coagulation. Untreated reactions are fatal. In this chapter we summarize clinical features and management and review current understanding of the pathophysiology and molecular genetics of MH.

Structural insights into binding of STAC proteins to voltage-gated calcium channels

Excitation-contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II-III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2.