Structural Biochemistry of Muscle Contraction.

Muscles are essential for movement and heart function. Contraction and relaxation of muscles rely on the sliding of two types of filaments-the thin filament and the thick myosin filament. The thin filament is composed mainly of filamentous actin (F-actin), tropomyosin, and troponin. Additionally, several other proteins are involved in the contraction mechanism, and their malfunction can lead to diverse muscle diseases, such as cardiomyopathies. We review recent high-resolution structural data that explain the mechanism of action of muscle proteins at an unprecedented level of molecular detail. We focus on the molecular structures of the components of the thin and thick filaments and highlight the mechanisms underlying force generation through actin-myosin interactions, as well as Ca2+-dependent regulation via the dihydropyridine receptor, the ryanodine receptor, and troponin. We particularly emphasize the impact of cryo-electron microscopy and cryo-electron tomography in leading muscle research into a new era.

Structural insights into the regulation of RyR1 by S100A1.

S100A1, a small homodimeric EF-hand Ca2+-binding protein (~21 kDa), plays an important regulatory role in Ca2+ signaling pathways involved in various biological functions including Ca2+ cycling and contractile performance in skeletal and cardiac myocytes. One key target of the S100A1 interactome is the ryanodine receptor (RyR), a huge homotetrameric Ca2+ release channel (~2.3 MDa) of the sarcoplasmic reticulum. Here, we report cryoelectron microscopy structures of S100A1 bound to RyR1, the skeletal muscle isoform, in absence and presence of Ca2+. Ca2+-free apo-S100A1 binds beneath the bridging solenoid (BSol) and forms contacts with the junctional solenoid and the shell-core linker of RyR1. Upon Ca2+-binding, S100A1 undergoes a conformational change resulting in the exposure of the hydrophobic pocket known to serve as a major interaction site of S100A1. Through interactions of the hydrophobic pocket with RyR1, Ca2+-bound S100A1 intrudes deeper into the RyR1 structure beneath BSol than the apo-form and induces sideways motions of the C-terminal BSol region toward the adjacent RyR1 protomer resulting in tighter interprotomer contacts. Interestingly, the second hydrophobic pocket of the S100A1-dimer is largely exposed at the hydrophilic surface making it prone to interactions with the local environment, suggesting that S100A1 could be involved in forming larger heterocomplexes of RyRs with other protein partners. Since S100A1 interactions stabilizing BSol are implicated in the regulation of RyR-mediated Ca2+ release, the characterization of the S100A1 binding site conserved between RyR isoforms may provide the structural basis for the development of therapeutic strategies regarding treatments of RyR-related disorders.

Voltage-induced calcium release in Caenorhabditis elegans body muscles.

Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in Caenorhabditis elegans body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.

An inherited life-threatening arrhythmia model established by screening randomly mutagenized mice.

Inherited arrhythmia syndromes (IASs) can cause life-threatening arrhythmias and are responsible for a significant proportion of sudden cardiac deaths (SCDs). Despite progress in the development of devices to prevent SCDs, the precise molecular mechanisms that induce detrimental arrhythmias remain to be fully investigated, and more effective therapies are desirable. In the present study, we screened a large-scale randomly mutagenized mouse library by electrocardiography to establish a disease model of IASs and consequently found one pedigree that exhibited spontaneous ventricular arrhythmias (VAs) followed by SCD within 1 y after birth. Genetic analysis successfully revealed a missense mutation (p.I4093V) of the ryanodine receptor 2 gene to be a cause of the arrhythmia. We found an age-related increase in arrhythmia frequency accompanied by cardiomegaly and decreased ventricular contractility in the Ryr2I4093V/+ mice. Ca2+ signaling analysis and a ryanodine binding assay indicated that the mutant ryanodine receptor 2 had a gain-of-function phenotype and enhanced Ca2+ sensitivity. Using this model, we detected the significant suppression of VA following flecainide or dantrolene treatment. Collectively, we established an inherited life-threatening arrhythmia mouse model from an electrocardiogram-based screen of randomly mutagenized mice. The present IAS model may prove feasible for use in investigating the mechanisms of SCD and assessing therapies.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. METHODS: Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. RESULTS: We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. CONCLUSIONS: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

Evolutionary isolation of ryanodine receptor isoform 1 for muscle-based thermogenesis in mammals.

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For thermogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions optimized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.

Structural and functional interactions between the EF hand domain and S2-S3 loop in the type-1 ryanodine receptor ion channel.

Previous cryo-electron micrographs suggested that the skeletal muscle Ca2+ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca2+ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293 cells for [3H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca2+/Mg2+-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca2+-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca2+/Mg2+-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca2+-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca2+/Mg2+-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.

RyR2 Serine-2030 PKA Site Governs Ca2+ Release Termination and Ca2+ Alternans.

BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.

Protein mishandling and impaired lysosomal proteolysis generated through calcium dysregulation in Alzheimer's disease.

Impairments in neural lysosomal- and autophagic-mediated degradation of cellular debris contribute to neuritic dystrophy and synaptic loss. While these are well-characterized features of neurodegenerative disorders such as Alzheimer's disease (AD), the upstream cellular processes driving deficits in pathogenic protein mishandling are less understood. Using a series of fluorescent biosensors and optical imaging in model cells, AD mouse models and human neurons derived from AD patients, we reveal a previously undescribed cellular signaling cascade underlying protein mishandling mediated by intracellular calcium dysregulation, an early component of AD pathogenesis. Increased Ca2+ release via the endoplasmic reticulum (ER)-resident ryanodine receptor (RyR) is associated with reduced expression of the lysosome proton pump vacuolar-ATPase (vATPase) subunits (V1B2 and V0a1), resulting in lysosome deacidification and disrupted proteolytic activity in AD mouse models and human-induced neurons (HiN). As a result of impaired lysosome digestive capacity, mature autophagosomes with hyperphosphorylated tau accumulated in AD murine neurons and AD HiN, exacerbating proteinopathy. Normalizing AD-associated aberrant RyR-Ca2+ signaling with the negative allosteric modulator, dantrolene (Ryanodex), restored vATPase levels, lysosomal acidification and proteolytic activity, and autophagic clearance of intracellular protein aggregates in AD neurons. These results highlight that prior to overt AD histopathology or cognitive deficits, aberrant upstream Ca2+ signaling disrupts lysosomal acidification and contributes to pathological accumulation of intracellular protein aggregates. Importantly, this is demonstrated in animal models of AD, and in human iPSC-derived neurons from AD patients. Furthermore, pharmacological suppression of RyR-Ca2+ release rescued proteolytic function, revealing a target for therapeutic intervention that has demonstrated effects in clinically-relevant assays.

Ca2+ leak through ryanodine receptor 1 regulates thermogenesis in resting skeletal muscle.

Mammals rely on nonshivering thermogenesis (NST) from skeletal muscle so that cold temperatures can be tolerated. NST results from activity of the sarcoplasmic reticulum (SR) Ca2+ pump in skeletal muscle, but the mechanisms that regulate this activity are unknown. Here, we develop a single-fiber assay to investigate the role of Ca2+ leak through ryanodine receptor 1 (RyR1) to generate heat at the SR Ca2+ pump in resting muscle. By inhibiting a subpopulation of RyR1s in a single-fiber preparation via targeted delivery of ryanodine through transverse tubules, we achieve in-preparation isolation of RyR1 Ca2+ leak. This maneuver provided a critical increase in signal-to-noise of the SR-temperature-sensitive dye ER thermoyellow fluorescence signal from the fiber to allow detection of SR temperature changes as either RyR1 or SR Ca2+ pump activity was altered. We found that RyR1 Ca2+ leak raises cytosolic [Ca2+] in the local vicinity of the SR Ca2+ pump to amplify thermogenesis. Furthermore, gene-dose-dependent increases in RyR1 leak in RYR1 mutant mice result in progressive rises in leak-dependent heat, consistent with raised local [Ca2+] at the SR Ca2+ pump via RyR1 Ca2+ leak. We also show that basal RyR Ca2+ leak and the heat generated by the SR Ca2+ pump in the absence of RyR Ca2+ leak is greater in fibers from mice than from toads. The distinct function of RyRs and SR Ca2+ pump in endothermic mammals compared to ectothermic amphibians provides insights into the mechanisms by which mammalian skeletal muscle achieves thermogenesis at rest.

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM.

Ryanodine receptors (RyRs) are main regulators of intracellular Ca2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca2+, increasing by ∼20-fold the potency of Ca2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

A Gating Mutation in Ryanodine Receptor Type 2 Rescues Phenotypes of Alzheimer's Disease Mouse Models by Upregulating Neuronal Autophagy.

It is well established that ryanodine receptors (RyanRs) are overactive in Alzheimer's disease (AD), and it has been suggested that inhibition of RyanR is potentially beneficial for AD treatment. In the present study, we explored a potential connection between basal RyanR activity and autophagy in neurons. Autophagy plays an important role in clearing damaged organelles and long-lived protein aggregates, and autophagy dysregulation occurs in both AD patients and AD animal models. Autophagy is known to be regulated by intracellular calcium (Ca2+) signals, and our results indicated that basal RyanR2 activity in hippocampal neurons inhibited autophagy through activation of calcineurin and the resulting inhibition of the AMPK (AMP-activated protein kinase)-ULK1 (unc-51-like autophagy-activating kinase 1) pathway. Thus, we hypothesized that increased basal RyanR2 activity in AD may lead to the inhibition of neuronal autophagy and accumulation of ß-amyloid. To test this hypothesis, we took advantage of the RyanR2-E4872Q knock-in mouse model (EQ) in which basal RyanR2 activity is reduced because of shortened channel open time. We discovered that crossing EQ mice with the APPKI and APPPS1 mouse models of AD (both males and females) rescued amyloid accumulation and LTP impairment in these mice. Our results revealed that reduced basal activity of RyanR2-EQ channels disinhibited the autophagic pathway and led to increased amyloid clearance in these models. These data indicated a potential pathogenic outcome of RyanR2 overactivation in AD and also provided additional targets for therapeutic intervention in AD. Basal activity of ryanodine receptors controls neuronal autophagy and contributes to development of the AD phenotype.SIGNIFICANCE STATEMENT It is well established that neuronal autophagy is impaired in Alzheimer's disease (AD). Our results suggest that supranormal calcium (Ca2+) release from endoplasmic reticulum contributes to the inhibition of autophagy in AD and that reduction in basal activity of type 2 ryanodine receptors disinhibits the neuronal autophagic pathway and leads to increased amyloid clearance in AD models. Our findings directly link neuronal Ca2+ dysregulation with autophagy dysfunction in AD and point to additional targets for therapeutic intervention.

Purinergic Agonists Increase [Ca2+]i in Rat Conjunctival Goblet Cells Through Ryanodine Receptor Type 3 (RyR3).

ATP and BzATP increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First passage cultured rat CGCs were incubated with Fura-2/AM and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M) which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and RT-qPCR were used to investigate RyR presence in rat and human CGCs. ATP stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, PKA inhibitor H89, nifedipine and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i, and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP and BzATP-induced Ca2+i increases originate from the ER, and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in non-excitable cells.

RyR2-dependent modulation of neuronal hyperactivity: A potential therapeutic target for treating Alzheimer's disease.

Increasing evidence suggests that simply reducing ß-amyloid (Aß) plaques may not significantly affect the progression of Alzheimer's disease (AD). There is also increasing evidence indicating that AD progression is driven by a vicious cycle of soluble Aß-induced neuronal hyperactivity. In support of this, it has recently been shown that genetically and pharmacologically limiting ryanodine receptor 2 (RyR2) open time prevents neuronal hyperactivity, memory impairment, dendritic spine loss and neuronal cell death in AD mouse models. By contrast, increased RyR2 open probability (Po) exacerbates the onset of familial AD-associated neuronal dysfunction and induces AD-like defects in the absence of AD-causing gene mutations. Thus, RyR2-dependent modulation of neuronal hyperactivity represents a promising new target for combating AD.

Putative malignant hyperthermia mutation CaV1.1-R174W is insufficient to trigger a fulminant response to halothane or confer heat stress intolerance.

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.

ER stress increases expression of intracellular calcium channel RyR1 to modify Ca2+ homeostasis in pancreatic beta cells.

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+ levels, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestospongin-C inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis.

Improved Cardiac Performance and Decreased Arrhythmia in Hypertrophic Cardiomyopathy With Non-ß-Blocking R-Enantiomer Carvedilol.

BACKGROUND: Hypercontractility and arrhythmia are key pathophysiologic features of hypertrophic cardiomyopathy (HCM), the most common inherited heart disease. ß-Adrenergic receptor antagonists (ß-blockers) are the first-line therapy for HCM. However, ß-blockers commonly selected for this disease are often poorly tolerated in patients, where heart-rate reduction and noncardiac effects can lead to reduced cardiac output and fatigue. Mavacamten, myosin ATPase inhibitor recently approved by the US Food and Drug Administration, has demonstrated the ability to ameliorate hypercontractility without lowering heart rate, but its benefits are so far limited to patients with left ventricular (LV) outflow tract obstruction, and its effect on arrhythmia is unknown. METHODS: We screened 21 ß-blockers for their impact on myocyte contractility and evaluated the antiarrhythmic properties of the most promising drug in a ventricular myocyte arrhythmia model. We then examined its in vivo effect on LV function by hemodynamic pressure-volume loop analysis. The efficacy of the drug was tested in vitro and in vivo compared with current therapeutic options (metoprolol, verapamil, and mavacamten) for HCM in an established mouse model of HCM (Myh6R403Q/+ and induced pluripotent stem cell (iPSC)-derived cardiomyocytes from patients with HCM (MYH7R403Q/+). RESULTS: We identified that carvedilol, a ß-blocker not commonly used in HCM, suppresses contractile function and arrhythmia by inhibiting RyR2 (ryanodine receptor type 2). Unlike metoprolol (a ß1-blocker), carvedilol markedly reduced LV contractility through RyR2 inhibition, while maintaining stroke volume through α1-adrenergic receptor inhibition in vivo. Clinically available carvedilol is a racemic mixture, and the R-enantiomer, devoid of ß-blocking effect, retains the ability to inhibit both α1-receptor and RyR2, thereby suppressing contractile function and arrhythmias without lowering heart rate and cardiac output. In Myh6R403Q/+ mice, R-carvedilol normalized hyperdynamic contraction, suppressed arrhythmia, and increased cardiac output better than metoprolol, verapamil, and mavacamten. The ability of R-carvedilol to suppress contractile function was well retained in MYH7R403Q/+ iPSC-derived cardiomyocytes. CONCLUSIONS: R-enantiomer carvedilol attenuates hyperdynamic contraction, suppresses arrhythmia, and at the same time, improves cardiac output without lowering heart rate by dual blockade of α1-adrenergic receptor and RyR2 in mouse and human models of HCM. This combination of therapeutic effects is unique among current therapeutic options for HCM and may particularly benefit patients without LV outflow tract obstruction.

S100A1's single cysteine is an indispensable redox switch for the protection against diastolic calcium waves in cardiomyocytes.

The EF-hand calcium (Ca2+) sensor protein S100A1 combines inotropic with antiarrhythmic potency in cardiomyocytes (CMs). Oxidative posttranslational modification (ox-PTM) of S100A1's conserved, single-cysteine residue (C85) via reactive nitrogen species (i.e., S-nitrosylation or S-glutathionylation) has been proposed to modulate conformational flexibility of intrinsically disordered sequence fragments and to increase the molecule's affinity toward Ca2+. Considering the unknown biological functional consequence, we aimed to determine the impact of the C85 moiety of S100A1 as a potential redox switch. We first uncovered that S100A1 is endogenously glutathionylated in the adult heart in vivo. To prevent glutathionylation of S100A1, we generated S100A1 variants that were unresponsive to ox-PTMs. Overexpression of wild-type (WT) and C85-deficient S100A1 protein variants in isolated CM demonstrated equal inotropic potency, as shown by equally augmented Ca2+ transient amplitudes under basal conditions and ß-adrenergic receptor (ßAR) stimulation. However, in contrast, ox-PTM defective S100A1 variants failed to protect against arrhythmogenic diastolic sarcoplasmic reticulum (SR) Ca2+ waves and ryanodine receptor 2 (RyR2) hypernitrosylation during ßAR stimulation. Despite diastolic performance failure, C85-deficient S100A1 protein variants exerted similar Ca2+-dependent interaction with the RyR2 than WT-S100A1. Dissecting S100A1's molecular structure-function relationship, our data indicate for the first time that the conserved C85 residue potentially acts as a redox switch that is indispensable for S100A1's antiarrhythmic but not its inotropic potency in CMs. We, therefore, propose a model where C85's ox-PTM determines S100A1's ability to beneficially control diastolic but not systolic RyR2 activity.NEW & NOTEWORTHY S100A1 is an emerging candidate for future gene-therapy treatment of human chronic heart failure. We aimed to study the significance of the conserved single-cysteine 85 (C85) residue in cardiomyocytes. We show that S100A1 is endogenously glutathionylated in the heart and demonstrate that this is dispensable to increase systolic Ca2+ transients, but indispensable for mediating S100A1's protection against sarcoplasmic reticulum (SR) Ca2+ waves, which was dependent on the ryanodine receptor 2 (RyR2) nitrosylation status.

Physiological role for S-nitrosylation of RyR1 in skeletal muscle function and development.

Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca2+ release from the sarcoplasmic reticulum through the ryanodine receptor/Ca2+-release channel RyR1. The RyR1 contains ∼100 Cys thiols of which ∼30 comprise an allosteric network subject to posttranslational modification by S-nitrosylation, S-palmitoylation and S-oxidation. However, the role and function of these modifications is not understood. Although aberrant S-nitrosylation of multiple unidentified sites has been associated with dystrophic diseases, malignant hyperthermia and other myopathic syndromes, S-nitrosylation in physiological situations is reportedly specific to a single (1 of ∼100) Cys in RyR1, Cys3636 in a manner gated by pO2. Using mice expressing a form of RyR1 with a Cys3636→Ala point mutation to prevent S-nitrosylation at this site, we showed that Cys3636 was the principal target of endogenous S-nitrosylation during normal muscle function. The absence of Cys3636 S-nitrosylation suppressed stimulus-evoked Ca2+ release at physiological pO2 (at least in part by altering the regulation of RyR1 by Ca2+/calmodulin), eliminated pO2 coupling, and diminished skeletal myocyte contractility in vitro and measures of muscle strength in vivo. Furthermore, we found that abrogation of Cys3636 S-nitrosylation resulted in a developmental defect reflected in diminished myofiber diameter, altered fiber subtypes, and altered expression of genes implicated in muscle development and atrophy. Thus, our findings establish a physiological role for pO2-coupled S-nitrosylation of RyR1 in skeletal muscle contractility and development and provide foundation for future studies of RyR1 modifications in physiology and disease.

Isolated Cardiac Ryanodine Receptor Function Varies Between Mammals.

Concerted robust opening of cardiac ryanodine receptors' (RyR2) Ca2+ release 1oplasmic reticulum (SR) is fundamental for normal systolic cardiac function. During diastole, infrequent spontaneous RyR2 openings mediate the SR Ca2+ leak that normally constrains SR Ca2+ load. Abnormal large diastolic RyR2-mediated Ca2+ leak events can cause delayed after depolarizations (DADs) and arrhythmias. The RyR2-associated mechanisms underlying these processes are being extensively studied at multiple levels utilizing various model animals. Since there are well-described species-specific differences in cardiac intracellular Ca2+ handing in situ, we tested whether or not single RyR2 function in vitro retains this species specificity. We isolated RyR2-rich heavy SR microsomes from mouse, rat, rabbit, and human ventricular muscle and quantified RyR2 function using identical solutions and methods. The single RyR2 cytosolic Ca2+ sensitivity was similar across these species. However, there were significant species differences in single RyR2 mean open times in both systole and diastole-like solutions. In diastole-like solutions, single rat/mouse RyR2 open probability and frequency of long openings (> 6 ms) were similar, but these values were significantly greater than those of either single rabbit or human RyR2s. We propose these in vitro single RyR2 functional differences across species stem from the species-specific RyR2 regulatory environment present in the source tissue. Our results show the single rabbit RyR2 functional attributes, particularly in diastole-like conditions, replicate those of single human RyR2 best among the species tested.