RyR2 Channels Research Articles

MDFI regulates fast-to-slow muscle fiber type transformation via the calcium signaling pathway

Muscle fiber is the basic unit of skeletal muscle with strong self-adaptability, and its type is closely related to meat quality. Myod family inhibitor (Mdfi) has the function of regulating myogenic regulatory factors during cell differentiation, but how Mdfi regulates muscle fiber type transformation in myoblasts is still unclear. In the present study, we constructed overexpressing and interfering with Mdfi C2C12 cell models by lipofection. The immunofluorescence, quantitative real-time PCR (qPCR), and western blot results show that the elevated MDFI promoted mitochondrial biogenesis, aerobic metabolism and the calcium level by activating CaMKK2 and AMPK phosphorylation and then stimulated the conversion of C2C12 cells from fast glycolytic to slow oxidative type. In addition, after inhibiting IP3R and RYR channels, the higher MDFI reversed the blockage of calcium release from the endoplasmic reticulum by calcium channel receptor inhibitors and increased intracellular calcium levels. Therefore, we propose that the higher MDFI promotes muscle fiber types conversion through the calcium signaling pathway. These findings further broaden our understanding of the regulatory mechanism of MDFI in muscle fiber type transformation. Furthermore, our results suggest potential therapeutic targets for skeletal muscle and metabolic-related diseases.

Orai, RyR, and IP3R channels cooperatively regulate calcium signaling in brain mid-capillary pericytes

Pericytes are multifunctional cells of the vasculature that are vital to brain homeostasis, yet many of their fundamental physiological properties, such as Ca2+ signaling pathways, remain unexplored. We performed pharmacological and ion substitution experiments to investigate the mechanisms underlying pericyte Ca2+ signaling in acute cortical brain slices of PDGFRβ-Cre::GCaMP6f mice. We report that mid-capillary pericyte Ca2+ signalling differs from ensheathing type pericytes in that it is largely independent of L- and T-type voltage-gated calcium channels. Instead, Ca2+ signals in mid-capillary pericytes were inhibited by multiple Orai channel blockers, which also inhibited Ca2+ entry triggered by endoplasmic reticulum (ER) store depletion. An investigation into store release pathways indicated that Ca2+ transients in mid-capillary pericytes occur through a combination of IP3R and RyR activation, and that Orai store-operated calcium entry (SOCE) is required to sustain and amplify intracellular Ca2+ increases evoked by the GqGPCR agonist endothelin-1. These results suggest that Ca2+ influx via Orai channels reciprocally regulates IP3R and RyR release pathways in the ER, which together generate spontaneous Ca2+ transients and amplify Gq-coupled Ca2+ elevations in mid-capillary pericytes. Thus, SOCE is a major regulator of pericyte Ca2+ and a target for manipulating their function in health and disease.

Impact of ciguatoxins on human cardiac and neuronal physiology

Ciguatera (CG) is a syndrome induced by Ciguatoxins (CTXs), natural phycotoxins among the most harmful we know. They are produced by benthic dinoflagellates of the Gambierdiscus and Fukuyoa. CG is the result of food poisoning caused by the consumption of contaminated fish meat along the food chain, from herbivorous fish to higher carnivorous fish. It is the most common non-bacterial seafood poisoning in the world with an increasing 50,000 to 100,000 new cases per year although only 2 to 10% of them are actually reported. CG is manifested by multiple undefined symptoms (gastrointestinal, cardiovascular, neurosensory, skin and inflammatory) in the form of polymorphic syndrome. CTXs are known to modify the cellular membrane permeability. Several fundamental studies have highlighted a common action of CTXs by modulating the ion channels (Na+, K+, Ca2+) essential to excitable cells such as cardiomyocytes (CM) and neurons (NR). Our main objective is to understand and characterize the impact of CTXs on human cardiac and neuronal physiology. We hypothesized that CTXs can affect the intracellular calcium handling and therefore the cardiac excitation-contraction coupling (ECC) and neuronal functional and molecular properties. We differentiated human induced pluripotent stem cell-derived ventricular-like cardiomyocytes (hiPSC-CMs) and neurons (hiPSC-NRs), we used 2 types of purified CTXs (CTX1B and CTX3C) and 2 types of hiPSC healthy control (HC). We investigated the effects of CTXs on cytosolic calcium, cells morphology and CM contractile properties. To determine if CTXs modulate the RyR2 channel activity, we performed single-channel measurements using the planar lipid bilayer technique. We observed that CTXs lead to intracellular calcium leaks and increase in cardiac and neuronal basal calcium level. They cause increased activity of the RyR2 channels as reflected by higher opening frequency Fo (Fo of 244 ± 112 events/s for HC vs. 2279 ± 836 events/s for HC + CTX1B, P < 0.01). Exposure of CTXs also cause reduced contractions of the hiPSC-derived cardiac sheets and cardiac hypertrophy as evidenced by an increase in cell area (1997 ± 119 μm2 for HC vs. 2852 ± 475 μm2 for HC + CTX1B, P < 0.01). Our preliminary data suggest that CTXs lead to RyR2 aberrant kinetic properties. CTXs also induce reduced beat rate and hiPSC-CM hypertrophy.

FKBP12 binds to the cardiac ryanodine receptor with negative cooperativity: implications for heart muscle physiology in health and disease.

Cardiac ryanodine receptors (RyR2) release the Ca2+ from intracellular stores that is essential for cardiac myocyte contraction. The ion channel opening is tightly regulated by intracellular factors, including the FK506 binding proteins, FKBP12 and FKBP12.6. The impact of these proteins on RyR2 activity and cardiac contraction is debated, with often apparently contradictory experimental results, particularly for FKBP12. The isoform that regulates RyR2 has generally been considered to be FKBP12.6, despite the fact that FKBP12 is the major isoform associated with RyR2 in some species and is bound in similar proportions to FKBP12.6 in others, including sheep and humans. Here, we show time- and concentration-dependent effects of adding FKBP12 to RyR2 channels that were partly depleted of FKBP12/12.6 during isolation. The added FKBP12 displaced most remaining endogenous FKBP12/12.6. The results suggest that FKBP12 activates RyR2 with high affinity and inhibits RyR2 with lower affinity, consistent with a model of negative cooperativity in FKBP12 binding to each of the four subunits in the RyR tetramer. The easy dissociation of some FKBP12/12.6 could dynamically alter RyR2 activity in response to changes in in vivo regulatory factors, indicating a significant role for FKBP12/12.6 in Ca2+ signalling and cardiac function in healthy and diseased hearts. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

The selective RyR2 inhibitor ent-verticilide suppresses atrial fibrillation susceptibility caused by Pitx2 deficiency

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/−) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/−) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent-verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca2+ sparks and spontaneous Ca2+ release events increased in permeabilized and intact atrial myocytes from Pitx2+/− mice. Atrial burst pacing consistently increased the incidence and duration of AF in Pitx2+/− mice. The RyR2 inhibitor ent-verticilide significantly reduced the frequency of spontaneous Ca2+ release in intact atrial myocytes and attenuated AF susceptibility with reduced AF incidence and duration. Our data demonstrate that RyR2 hyperactivity enhances SR Ca2+ leak and AF inducibility in Pitx2+/− mice via abnormal Ca2+ handling. Therapeutic targeting of hyperactive RyR2 in AF using ent-verticilide may be a viable mechanism-based approach to treat atrial arrhythmias caused by Pitx2 deficiency.

Long-term potentiation and spatial memory training stimulate the hippocampal expression of RyR2 calcium release channels.

Introduction: Neuronal Ca2+ signals generated through the activation of Ca2+-induced Ca2+ release in response to activity-generated Ca2+ influx play a significant role in hippocampal synaptic plasticity, spatial learning, and memory. We and others have previously reported that diverse stimulation protocols, or different memory-inducing procedures, enhance the expression of endoplasmic reticulum-resident Ca2+ release channels in rat primary hippocampal neuronal cells or hippocampal tissue. Methods and Results: Here, we report that induction of long-term potentiation (LTP) by Theta burst stimulation protocols of the CA3-CA1 hippocampal synapse increased the mRNA and protein levels of type-2 Ryanodine Receptor (RyR2) Ca2+ release channels in rat hippocampal slices. Suppression of RyR channel activity (1 h preincubation with 20 μM ryanodine) abolished both LTP induction and the enhanced expression of these channels; it also promoted an increase in the surface expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR1 and GluR2 and caused a moderate but significant reduction of dendritic spine density. In addition, training rats in the Morris water maze induced memory consolidation, which lasted for several days after the end of the training period, accompanied by an increase in the mRNA levels and the protein content of the RyR2 channel isoform. Discussion: We confirm in this work that LTP induction by TBS protocols requires functional RyR channels. We propose that the increments in the protein content of RyR2 Ca2+ release channels, induced by LTP or spatial memory training, play a significant role in hippocampal synaptic plasticity and spatial memory consolidation.

Molecular Aspects Implicated in Dantrolene Selectivity with Respect to Ryanodine Receptor Isoforms

Dantrolene is an intra-cellularly acting skeletal muscle relaxant used for the treatment of the rare genetic disorder, malignant hyperthermia (MH). In most cases, MH susceptibility is caused by dysfunction of the skeletal ryanodine receptor (RyR1) harboring one of nearly 230 single-point MH mutations. The therapeutic effect of dantrolene is the result of a direct inhibitory action on the RyR1 channel, thus suppressing aberrant Ca2+ release from the sarcoplasmic reticulum. Despite the almost identical dantrolene-binding sequence exits in all three mammalian RyR isoforms, dantrolene appears to be an isoform-selective inhibitor. Whereas RyR1 and RyR3 channels are competent to bind dantrolene, the RyR2 channel, predominantly expressed in the heart, is unresponsive. However, a large body of evidence suggests that the RyR2 channel becomes sensitive to dantrolene-mediated inhibition under certain pathological conditions. Although a consistent picture of the dantrolene effect emerges from in vivo studies, in vitro results are often contradictory. Hence, our goal in this perspective is to provide the best possible clues to the molecular mechanism of dantrolene's action on RyR isoforms by identifying and discussing potential sources of conflicting results, mainly coming from cell-free experiments. Moreover, we propose that, specifically in the case of the RyR2 channel, its phosphorylation could be implicated in acquiring the channel responsiveness to dantrolene inhibition, interpreting functional findings in the structural context.

Dynamic calcium-calmodulin-dependent stabilization of leaky RyR2 in live cardiac myocytes.

Calmodulin (CaM) is a major regulator of cardiac ryanodine receptors (RyR2). CaM binding shifts RyR2 channel conformation to a state that suppresses diastolic SR Ca leak that can become pro arrhythmic as commonly observed in cardiac diseases such as heart failure (HF) or diabetic cardiomyopathy. In these diseases, CaM affinity for RyR2 is reduced, promoting a leaky pathological RyR2 structural state that is arrhythmogenic. Conversely, increased CaM binding can suppress RyR2-mediated Ca leak and promote a conformational shift to the stable physiological non-leaky state. Still, there are unknowns regarding the dynamic interplay between CaM-RyR2 during each heartbeat and in pathogenic RyR states in live cardiac myocytes. We hypothesize that high [Ca] elevations reached during Ca transients in a healthy beating myocyte would increase CaM-RyR binding throughout the cardiac cycle, promoting RyR stability. Further, the lower CaM-RyR2 affinity in disease, exacerbated by reduced Ca transient amplitude, resulting in lower CaM-RyR2 binding, destabilized RyR2s and enhance arrhythmogenic diastolic SR Ca leak. We used permeabilized cardiac myocytes from WT and HF mouse models as a controlled approach to measuring both the [Ca]i and [CaM] dependence of CaM-RyR2 binding via FRET between donor-labeled FKBP12.6 and acceptor-labeled CaM. Moreover, we simultaneously recorded [Ca]i transients within myocytes, enabling measurement of dynamic CaM-RyR2 saturation during periodic Ca waves that emulate paced Ca transients. We find that CaM-RyR2 FRET is reduced in the leaky pathological state and associated with alterations in Ca leak event frequencies. We infer that the healthy higher CaM-RyR2 affinity favors more complete saturation of RyR2 with CaM throughout the cardiac cycle, versus pathological states. These data suggest that a modest fraction of CaM-free RyR2s with pathological diastolic conformation might suffice to elicit proarrhythmic SR Ca leak in cardiac disease.

Arrhythmia severity in mouse models harboring RyR2 mutations with varied extent of channel activity.

Genetic mutations in the type 2 ryanodine receptor (RyR2) can cause life-threatening arrhythmias, and to date, more than 300 of disease-linked mutations have been reported. Among arrhythmic diseases caused by gain-of-function type RyR2 mutations, catecholaminergic polymorphic ventricular tachycardia (CPVT) is the most common, and its arrhythmia severity ranges from mild to severe depending on mutation sites. In addition, most CPVT-linked RyR mutations are not associated with morphological change in heart, but some RyR2 mutations have reported to cause cardiomyopathy such as dilated cardiomyopathy (DCM) and left ventricular non-compaction (LVNC). The mechanisms underlying the differences in the manifestation of disease phenotypes and the arrhythmia severity are not well understood. We have established a procedure to quantitatively evaluate homozygous mutant RyR2 channel activities using HEK293 cell expression system and investigated the relation to arrhythmia severity in human patients (Kurebayashi at al., J Gen Physiol 2022). Based on the study, we selected multiple lines of mice expressing RyR2 mutations with different degrees of channel activation, and investigated the severity of arrhythmia, the presence or absence of cardiomyopathy, myocardial [3H]ryanodine binding activity, and properties of Ca2+ signals in cardiomyocytes. Our results indicate that arrhythmia severity in RyR2-mutant mice was in good agreement with that predicted from HEK293 cell system.

Kinetics and mapping of calcium-driven calmodulin conformations on ryanodine receptors.

Calmodulin (CaM) is an important Ca2+-sensor for Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during relaxation and contraction in cardiac and skeletal muscle. Although several high-resolution cryo-EM structures have been published recently, the physiologically relevant mechanism by which CaM modulates the SR Ca2+ release channel (ryanodine receptor, RyR) is still not completely understood. Using distances calculated from fluorescence lifetime (FLT)-detected FRET between RyR-bound FKBP to CaM, we have trilaterated the structural states of CaM and mapped Ca2+-driven shifts in the conformation of CaM bound to RyR. As the locations of both the N- and C-lobes can be determined, we found that CaM becomes more compact in contracting high-Ca2+ relative to the relaxing low-Ca2+ and that skeletal and cardiac RyR isoforms show different CaM-RyR conformations and binding. These structural observations correlate with the experimentally determined modulatory action of CaM. By integrating our FLT-FRET system in a stopped-flow experiment, we resolved CaM binding kinetics and millisecond Ca2+-driven structural transitions of CaM-bound to RyR. Distinct binding kinetics of CaM to RyR1 and RyR2 were observed, suggesting different CaM-RyR interactions. Specifically, CaM association to RyR2 is three-fold faster vs. RyR1 at 30 nM and 30 μM. Upon rapid Ca2+ transition from μM to nM, the structural transition is four-fold slower for CaM bound to RyR2 relative to RyR1. The results provide mechanistic insight into CaM's action to transduce Ca2+ signals for RyR channel function during muscle contraction, and furthermore the technology developed holds promise for determining structure-function relationships of other RyR modulators. This work was supported by NIH grants R01HL92097 (RLC, DMB) and R37AG26160 (DDT), and by American Heart Association Fellowship 16POST31010019 (RTR).

Muscle calcium stress cleaves junctophilin1, unleashing a gene regulatory program predicted to correct glucose dysregulation.

Calcium ion movements between cellular stores and the cytosol govern muscle contraction, the most energy-consuming function in mammals, which confers skeletal myofibers a pivotal role in glycemia regulation. Chronic myoplasmic calcium elevation ("calcium stress"), found in malignant hyperthermia-susceptible (MHS) patients and multiple myopathies, has been suggested to underlie the progression from hyperglycemia to insulin resistance. What drives such progression remains elusive. We find that muscle cells derived from MHS patients have increased content of an activated fragment of GSK3β - a specialized kinase that inhibits glycogen synthase, impairing glucose utilization and delineating a path to hyperglycemia. We also find decreased content of junctophilin1, an essential structural protein that colocalizes in the couplon with the voltage-sensing CaV1.1, the calcium channel RyR1 and calpain1, accompanied by an increase in a 44 kDa junctophilin1 fragment (JPh44) that moves into nuclei. We trace these changes to activated proteolysis by calpain1, secondary to increased myoplasmic calcium. We demonstrate that a JPh44-like construct induces transcriptional changes predictive of increased glucose utilization in myoblasts, including less transcription and translation of GSK3β and decreased transcription of proteins that reduce utilization of glucose. These effects reveal a stress-adaptive response, mediated by the novel regulator of transcription JPh44.

Targeting ryanodine receptors to treat human diseases.

This Review provides an update on ryanodine receptors (RyRs) and their role in human diseases of heart, muscle, and brain. Calcium (Ca2+) is a requisite second messenger in all living organisms. From C. elegans to mammals, Ca2+ is necessary for locomotion, bodily functions, and neural activity. However, too much of a good thing can be bad. Intracellular Ca2+ overload can result in loss of function and death. Intracellular Ca2+ release channels evolved to safely provide large, rapid Ca2+ signals without exposure to toxic extracellular Ca2+. RyRs are intracellular Ca2+ release channels present throughout the zoosphere. Over the past 35 years, our knowledge of RyRs has advanced to the level of atomic-resolution structures revealing their role in the mechanisms underlying the pathogenesis of human disorders of heart, muscle, and brain. Stress-induced RyR-mediated intracellular Ca2+ leak in the heart can promote heart failure and cardiac arrhythmias. In skeletal muscle, RyR1 leak contributes to muscle weakness in inherited myopathies, to age-related loss of muscle function and cancer-associated muscle weakness, and to impaired muscle function in muscular dystrophies, including Duchenne. In the brain, leaky RyR channels contribute to cognitive dysfunction in Alzheimer's disease, posttraumatic stress disorder, and Huntington's disease. Novel therapeutics targeting dysfunctional RyRs are showing promise.

InsP3R-RyR Ca2+ channel crosstalk facilitates arrhythmias in the failing human ventricle.

Dysregulated intracellular Ca2+ handling involving altered Ca2+ release from intracellular stores via RyR channels underlies both arrhythmias and reduced function in heart failure (HF). Mechanisms linking RyR dysregulation and disease are not fully established. Studies in animals support a role for InsP3 receptor Ca2+ channels (InsP3R) in pathological alterations in cardiomyocyte Ca2+ handling but whether these findings translate to the divergent physiology of human cardiomyocytes during heart failure is not determined. Using electrophysiological and Ca2+ recordings in human ventricular cardiomyocytes, we uncovered that Ca2+ release via InsP3Rs facilitated Ca2+ release from RyR and induced arrhythmogenic delayed after depolarisations and action potentials. InsP3R-RyR crosstalk was particularly increased in HF at RyR clusters isolated from the T-tubular network. Reduced SERCA activity in HF further facilitated the action of InsP3. Nanoscale imaging revealed co-localisation of InsP3Rs with RyRs in the dyad, which was increased in HF, providing a mechanism for augmented Ca2+ channel crosstalk. Notably, arrhythmogenic activity dependent on InsP3Rs was increased in tissue wedges from failing hearts perfused with AngII to promote InsP3 generation. These data indicate a central role for InsP3R-RyR Ca2+ signalling crosstalk in the pro-arrhythmic action of GPCR agonists elevated in HF and the potential for their therapeutic targeting.

Abstract 11999: Molecular and Cellular Characterization of RyR2 Mutations Linked to Long-QT Syndrome

Long-QT syndrome (LQTS) is a cardiac disorder characterized by prolonged QT interval and abnormal T waves on the electrocardiogram. Electrical abnormality can lead to severe arrhythmias, syncope and sudden cardiac death. Mutations in the Na + and K + channels or associated proteins account for the majority of cases; however, the genetic causes of about 20% of LQTS cases remain poorly understood. Recent genetic studies revealed novel LQTS-associated variants on the cardiac ryanodine receptor (RyR2), a Ca 2+ channel responsible for Ca 2+ -induced Ca 2+ release and excitation-contraction coupling. Given the important roles of RyR2 in the electrical activity of heart and previous evidence of RyR2 mutations in other inherited arrhythmias, we hypothesized that RyR2 mutations lead to functional alterations of the channels and contribute to dysregulated Ca 2+ release and pathological electrical remodeling. Eleven novel RyR2 mutations were identified in genetic studies of LQTS patients, including S166C, H877P, R1760W, G2094S, R2824W, R2920Q, R3673W, Y4287N, V4298M, P4534S and K4594Q. [3H] ryanodine (RyR2 ligand) binding assays revealed variable effects of the mutations on the opening probability of the RyR2 channel. In cell lines with stable expression of RyR2, Ca 2+ overload activates RyR2 channels and leads to spontaneous Ca 2+ release into the cytoplasm. Compared to WT RyR2, LQTS-associated RyR2 mutations cause an increase in the proportion of cells that exhibit spontaneous Ca 2+ release at various Ca 2+ concentrations, suggesting an increase in luminal Ca 2+ sensitivity of the channels. A transgenic mouse model carrying the R2920Q mutation was used to investigate the cellular and molecular pathways leading to LQTS. Homozygous mice showed prolonged QT interval and QTc at 5 months. Electrophysiological studies of the isolated cardiomyocytes revealed a depolarized resting membrane potential in the homozygous R2920Q cardiomyocytes and prolonged action potential duration in both heterozygous and homozygous R2920Q cardiomyocytes, compared to the WT cells. The availability of a mouse model that recapitulates the cardinal signs of LQTS will allow for deeper mechanistic insight into the role of RyR2 dysfunction as a driver of this arrhythmogenic syndrome.

The Role of Ca2+ Sparks in Force Frequency Relationships in Guinea Pig Ventricular Myocytes.

Calcium sparks are the elementary Ca2+ release events in excitation-contraction coupling that underlie the Ca2+ transient. The frequency-dependent contractile force generated by cardiac myocytes depends upon the characteristics of the Ca2+ transients. A stochastic computational local control model of a guinea pig ventricular cardiomyocyte was developed, to gain insight into mechanisms of force-frequency relationship (FFR). This required the creation of a new three-state RyR2 model that reproduced the adaptive behavior of RyR2, in which the RyR2 channels transition into a different state when exposed to prolonged elevated subspace [Ca2+]. The model simulations agree with previous experimental and modeling studies on interval-force relations. Unlike previous common pool models, this local control model displayed stable action potential trains at 7 Hz. The duration and the amplitude of the [Ca2+]myo transients increase in pacing rates consistent with the experiments. The [Ca2+]myo transient reaches its peak value at 4 Hz and decreases afterward, consistent with experimental force-frequency curves. The model predicts, in agreement with previous modeling studies of Jafri and co-workers, diastolic sarcoplasmic reticulum, [Ca2+]sr, and RyR2 adaptation increase with the increased stimulation frequency, producing rising, rather than falling, amplitude of the myoplasmic [Ca2+] transients. However, the local control model also suggests that the reduction of the L-type Ca2+ current, with an increase in pacing frequency due to Ca2+-dependent inactivation, also plays a role in the negative slope of the FFR. In the simulations, the peak Ca2+ transient in the FFR correlated with the highest numbers of SR Ca2+ sparks: the larger average amplitudes of those sparks, and the longer duration of the Ca2+ sparks.

P.187 SelN expression in activated satellite cells following muscle injury

SELENON-related congenital myopathy is characterized by proximal weakness starting in infancy, early respiratory insufficiency, and early development of severe scoliosis. While changes in the <i>SELENON</i> gene, which encodes the protein SelN, are known to cause this disease the mechanisms through which loss of SelN lead to myopathy are not well understood. Previous studies suggest that SelN may have multiple roles in muscle, including regulating development of Type II muscle fibers, modulating excitation-contraction coupling through interactions with RYR1 and other muscle calcium channels, and possibly supporting satellite cell activation and proliferation following muscle injury. One particular challenge to understanding the role of SelN in skeletal muscle has been the inability to directly visualize SelN expression within muscle fibers and supporting cells due to a lack of robust antibodies for immunohistochemistry. Studies of mRNA expression and Western blot analysis of protein expression suggest significant post-transcriptional regulation of protein expression with an overall pattern of high expression in developing muscle and other developing tissues and low-level ubitquitous expression in mature tissues but evaluation of SelN expression in more limited sub-populations of cells has not been possible. Experiments in mouse suggest that loss of SelN expression results in decreased satellite cell proliferation following muscle injury. Here, I use a newly developed zebrafish model with mNeonGreen-tagged SelN to directly visualize SelN expression in satellite cells following muscle injury and show that SelN expression increases in activated satellite cells following mechanical muscle injury. This provides support SelN playing a role in satellite cell activation and proliferation during muscle repair following injury.

P.08 Phase 1 open-label trial of Rycal S48168 (ARM210) for RYR1-related myopathies

Pathogenic variants in the <i>RYR1</i> gene, encoding the skeletal muscle ryanodine receptor (RyR1), are the most common cause of non-dystrophic neuromuscular disorders. Pathogenic <i>RYR1</i> variants exhibit downstream consequences, including aberrant sarcoplasmic reticulum (SR) Ca²⁺ leak, hypersensitivity to RyR1 agonists, diminished SR Ca²⁺ release, and/or decreased RyR1 protein expression. Decreased RyR1-calstabin1 association, due to <i>RYR1</i> variants and/or post-translational modifications of the RyR1 channel, exacerbates SR Ca²⁺ leak with detrimental downstream effects on muscle function. Rycal compounds have been shown to restore RyR1-calstabin1 association, stabilize the <i>RyR1</i> closed state, and mitigate aberrant Ca²⁺ leak in <i>RYR1</i>-related myopathy (<i>RYR1</i>-RM) muscle biopsies. We conducted a phase 1 trial of S48168 (ARM210), a Rycal compound, in ambulatory adults with <i>RYR1</i>-RM (NCT04141670). The primary endpoint was safety and tolerability over a one-month dosing period. Pharmacokinetic (PK) studies, muscle biopsies, motor/muscle function tests, and a patient-reported fatigue questionnaire were also included. Seven eligible individuals were enrolled and received either the low dose (120 mg, n=3) or high dose (200 mg, n=4) of S48168 (ARM210) daily. Three treatment emergent adverse effects grade 2 or above were reported, all unrelated to S48168 (ARM210) and there were no serious adverse events. S48168 (ARM210) showed a dose-dependent PK profile and C_max remained within pre-defined safety margins. At one month, self-reported fatigue scores decreased and shoulder abduction strength trended higher in the high dose group, while handgrip strength increased in both dose groups, though these exploratory comparisons were descriptive. Other preliminary efficacy results were mixed. Given the favorable safety profile and efficacy trends, further clinical development of S48168 (ARM210) is warranted for <i>RYR1</i>-RM.

Holiday Heart Syndrome, Atrial Fibrillation, and RyR2 Antagonist.

The Holiday Heart Syndrome (HHS) refers to binge alcohol-associated cardiac arrhythmia of which atrial fibrillation (AF) being the most occurred arrhythmia type. Excessive alcohol consumption is a serious public health and economic issue in the US and globally. Despite a tremendous education effort, binge drinking among the population of young and older adults continues to rise. During this current COVID-19 pandemic, social isolation has significantly increased alcohol consumption nationwide. In this issue, Greco et al. reported a straightforward study on the role of RyR2 dysfunction in binge alcohol-evoked AF using an HHS rat model, and evaluated the anti-AF potential of dantrolene, an RyR channel blocker. However, further investigations including off-target effects of dantrolene and therapeutic potential of other RyR2 blockers in AF are needed.

Statin activation of skeletal ryanodine receptors (RyR1) is a class effect but separable from HMG-CoA reductase inhibition.

Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolaemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. Using a range of structurally diverse statin analogues, we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property. Compounds were screened for RyR1 activity utilising [3 H]ryanodine binding. Mechanistic insight into RyR1 activity was studied by incorporating RyR1 channels from sheep, mouse or rabbit skeletal muscle into bilayers. All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic Ca2+ . We synthesised the one essential structural moiety that all statins must possess for HMG-CoA reductase inhibition, the R-3,5-dihydroxypentanoic acid unit, and it did not activate RyR1. We also identified five analogues retaining potent HMG-CoA reductase inhibition that inhibited RyR1 and four that lacked the ability to modulate RyR1. That cerivastatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin regulation of RyR1 and HMG-CoA reductase are separable effects will allow the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds we have identified, allowing development of effective cardioprotective statins with improved patient tolerance.

How does flecainide impact RyR2 channel function?

Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ release. It has been introduced as a clinical antiarrhythmic agent for catecholaminergic polymorphic ventricular tachycardia (CPVT), a condition most commonly associated with gain-of-function RyR2 mutations. Current debate concerns both cellular mechanisms of its antiarrhythmic action and molecular mechanisms of its RyR2 actions. At the cellular level, it targets NaV1.5, RyR2, Na+/Ca2+ exchange (NCX), and additional proteins involved in excitation-contraction (EC) coupling and potentially contribute to the CPVT phenotype. This Viewpoint primarily addresses the various direct molecular actions of flecainide on isolated RyR2 channels in artificial lipid bilayers. Such studies demonstrate different, multifarious, flecainide binding sites on RyR2, with voltage-dependent binding in the channel pore or voltage-independent binding at distant peripheral sites. In contrast to its single NaV1.5 pore binding site, flecainide may bind to at least four separate inhibitory sites on RyR2 and one activation site. None of these binding sites have been specifically located in the linear RyR2 sequence or high-resolution structure. Furthermore, it is not clear which of the inhibitory sites contribute to flecainide's reduction of spontaneous Ca2+ release in cellular studies. A confounding observation is that flecainide binding to voltage-dependent inhibition sites reduces cation fluxes in a direction opposite to physiological Ca2+ flow from SR lumen to cytosol. This may suggest that, rather than directly blocking Ca2+ efflux, flecainide can reduce Ca2+ efflux by blocking counter currents through the pore which otherwise limit SR membrane potential change during systolic Ca2+ efflux. In summary, the antiarrhythmic effects of flecainide in CPVT seem to involve multiple components of EC coupling and multiple actions on RyR2. Their clarification may identify novel specific drug targets and facilitate flecainide's clinical utilization in CPVT.