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.

Foxp3-mediated blockage of ryanodine receptor 2 underlies contact-based suppression by regulatory T cells.

The suppression mechanism of Tregs remains an intensely investigated topic. As our focus has shifted toward a model centered on indirect inhibition of DCs, a universally applicable effector mechanism controlled by the transcription factor forkhead box P3 (Foxp3) expression has not been found. Here, we report that Foxp3 blocked the transcription of ER Ca2+-release channel ryanodine receptor 2 (RyR2). Reduced RyR2 shut down basal Ca2+ oscillation in Tregs, which reduced m-calpain activities that are needed for T cells to disengage from DCs, suggesting a persistent blockage of DC antigen presentation. RyR2 deficiency rendered the CD4+ T cell pool immune suppressive and caused it to behave in the same manner as Foxp3+ Tregs in viral infection, asthma, hypersensitivity, colitis, and tumor development. In the absence of Foxp3, Ryr2-deficient CD4+ T cells rescued the systemic autoimmunity associated with scurfy mice. Therefore, Foxp3-mediated Ca2+ signaling inhibition may be a central effector mechanism of Treg immune suppression.

RyR2 C-terminal truncating variants identified in patients with arrhythmic phenotypes exert a dominant negative effect through formation of wildtype-truncation heteromers.

Gain-of-function missense variants in the cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), whereas RyR2 loss-of-function missense variants cause Ca2+ release deficiency syndrome (CRDS). Recently, truncating variants in RyR2 have also been associated with ventricular arrhythmias (VAs) and sudden cardiac death. However, there are limited insights into the potential clinical relevance and in vitro functional impact of RyR2 truncating variants. We performed genetic screening of patients presenting with syncope, VAs, or unexplained sudden death and in vitro characterization of the expression and function of RyR2 truncating variants in HEK293 cells. We identified two previously unknown RyR2 truncating variants (Y4591Ter and R4663Ter) and one splice site variant predicted to result in a frameshift and premature termination (N4717 + 15Ter). These 3 new RyR2 truncating variants and a recently reported RyR2 truncating variant, R4790Ter, were generated and functionally characterized in vitro. Immunoprecipitation and immunoblotting analyses showed that all 4 RyR2 truncating variants formed heteromers with the RyR2-wildtype (WT) protein. Each of these C-terminal RyR2 truncations was non-functional and suppressed [3H]ryanodine binding to RyR2-WT and RyR2-WT mediated store overload induced spontaneous Ca2+ release activity in HEK293 cells. The expression of these RyR2 truncating variants in HEK293 cells was markedly reduced compared with that of the full-length RyR2 WT protein. Our data indicate that C-terminal RyR2 truncating variants are non-functional and can exert a dominant negative impact on the function of the RyR2 WT protein through formation of heteromeric WT/truncation complex.

RYR2-ryanodinopathies: from calcium overload to calcium deficiency.

The sarcoplasmatic reticulum (SR) cardiac ryanodine receptor/calcium release channel RyR2 is an essential regulator of cardiac excitation-contraction coupling and intracellular calcium homeostasis. Mutations of the RYR2 are the cause of rare, potentially lethal inherited arrhythmia disorders. Catecholaminergic polymorphic ventricular tachycardia (CPVT) was first described more than 20 years ago and is the most common and most extensively studied cardiac ryanodinopathy. Over time, other distinct inherited arrhythmia syndromes have been related to abnormal RyR2 function. In addition to CPVT, there are at least two other distinct RYR2-ryanodinopathies that differ mechanistically and phenotypically from CPVT: RYR2 exon-3 deletion syndrome and the recently identified calcium release deficiency syndrome (CRDS). The pathophysiology of the different cardiac ryanodinopathies is characterized by complex mechanisms resulting in excessive spontaneous SR calcium release or SR calcium release deficiency. While the vast majority of CPVT cases are related to gain-of-function variants of the RyR2 protein, the recently identified CRDS is linked to RyR2 loss-of-function variants. The increasing number of these cardiac 'ryanodinopathies' reflects the complexity of RYR2-related cardiogenetic disorders and represents an ongoing challenge for clinicians. This state-of-the-art review summarizes our contemporary understanding of RYR2-related inherited arrhythmia disorders and provides a systematic and comprehensive description of the distinct cardiac ryanodinopathies discussing clinical aspects and molecular insights. Accurate identification of the underlying type of cardiac ryanodinopathy is essential for the clinical management of affected patients and their families.

Increased Ca2+ Transient Underlies RyR2-Related Left Ventricular Noncompaction.

BACKGROUND: A loss-of-function cardiac ryanodine receptor (RyR2) mutation, I4855M+/-, has recently been linked to a new cardiac disorder termed RyR2 Ca2+ release deficiency syndrome (CRDS) as well as left ventricular noncompaction (LVNC). The mechanism by which RyR2 loss-of-function causes CRDS has been extensively studied, but the mechanism underlying RyR2 loss-of-function-associated LVNC is unknown. Here, we determined the impact of a CRDS-LVNC-associated RyR2-I4855M+/- loss-of-function mutation on cardiac structure and function. METHODS: We generated a mouse model expressing the CRDS-LVNC-associated RyR2-I4855M+/- mutation. Histological analysis, echocardiography, ECG recording, and intact heart Ca2+ imaging were performed to characterize the structural and functional consequences of the RyR2-I4855M+/- mutation. RESULTS: As in humans, RyR2-I4855M+/- mice displayed LVNC characterized by cardiac hypertrabeculation and noncompaction. RyR2-I4855M+/- mice were highly susceptible to electrical stimulation-induced ventricular arrhythmias but protected from stress-induced ventricular arrhythmias. Unexpectedly, the RyR2-I4855M+/- mutation increased the peak Ca2+ transient but did not alter the L-type Ca2+ current, suggesting an increase in Ca2+-induced Ca2+ release gain. The RyR2-I4855M+/- mutation abolished sarcoplasmic reticulum store overload-induced Ca2+ release or Ca2+ leak, elevated sarcoplasmic reticulum Ca2+ load, prolonged Ca2+ transient decay, and elevated end-diastolic Ca2+ level upon rapid pacing. Immunoblotting revealed increased level of phosphorylated CaMKII (Ca2+-calmodulin dependent protein kinases II) but unchanged levels of CaMKII, calcineurin, and other Ca2+ handling proteins in the RyR2-I4855M+/- mutant compared with wild type. CONCLUSIONS: The RyR2-I4855M+/- mutant mice represent the first RyR2-associated LVNC animal model that recapitulates the CRDS-LVNC overlapping phenotype in humans. The RyR2-I4855M+/- mutation increases the peak Ca2+ transient by increasing the Ca2+-induced Ca2+ release gain and the end-diastolic Ca2+ level by prolonging Ca2+ transient decay. Our data suggest that the increased peak-systolic and end-diastolic Ca2+ levels may underlie RyR2-associated LVNC.

A Gain-of-function Mutation in the Gating Domain of ITPR1 Impairs Motor Movement and Increases Thermal and Mechanical Sensitivity.

Inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) is an intracellular Ca2+ release channel important for a number of fundamental cellular functions. Consistent with its critical physiological significance, mutations in ITPR1 are associated with disease. Surprisingly, nearly all the disease-associated ITPR1 mutations characterized to date are loss of function. Despite the paucity of ITPR1 gain-of-function (GOF) mutations, enhanced ITPR1 function as a result of dysregulation by ITPR1 interacting proteins is thought to be associated with ataxia, learning and memory impairments, Alzheimer's disease (AD) progression, and chronic pain. However, direct evidence for the role of ITPR1 GOF in disease is lacking. To determine whether GOF in ITPR1 itself has pathological ramifications, we employed a newly developed mouse model expressing an ITPR1 mutation in the gating domain of the channel, D2594K, that markedly increased the channel's sensitivity to activation by IP3. Behavioral studies showed that the ITPR1-D2594K+/- mutant mice displayed motor deficits and reduced muscle strength. However, the ITPR1-D2594K+/- mutation did not significantly alter hippocampal learning and memory and did not change learning and memory impairments when crossed with the 5xFAD AD model mice. On the other hand, ITPR1-D2594K+/- mice exhibited increased sensitivity to thermal and mechanical stimulation compared to WT. Interestingly, R-carvedilol treatment attenuated the enhanced thermal and mechanical nociception in ITPR1-D2594K+/- mice. Thus, the ITPR1-D2594K+/- mutation in the channel's gating domain has a marked impact on motor movements and pain perception, but little effect on hippocampal learning and memory.

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.

Beta-blocker treatment of patients with atrial fibrillation attenuates spontaneous calcium release-induced electrical activity.

AIMS: Atrial fibrillation (AF) has been associated with excessive spontaneous calcium release, linked to cyclic AMP (cAMP)-dependent phosphorylation of calcium regulatory proteins. Because ß-blockers are expected to attenuate cAMP-dependent signaling, we aimed to examine whether the treatment of patients with ß-blockers affected the incidence of spontaneous calcium release events or transient inward currents (ITI). METHODS: The impact of treatment with commonly used ß-blockers was analyzed in human atrial myocytes from 371 patients using patch-clamp technique, confocal calcium imaging or immunofluorescent labeling. Data were analyzed using multivariate regression analysis taking into account potentially confounding effects of relevant clinical factors RESULTS: The L-type calcium current (ICa) density was diminished significantly in patients with chronic but not paroxysmal AF and the treatment of patients with ß-blockers did not affect ICa density in any group. By contrast, the ITI frequency was elevated in patients with either paroxysmal or chronic AF that did not receive treatment, and ß-blocker treatment reduced the frequency to levels observed in patients without AF. Confocal calcium imaging showed that ß-blocker treatment also reduced the calcium spark frequency in patients with AF to levels observed in those without AF. Furthermore, phosphorylation of the ryanodine receptor (RyR2) at Ser-2808 and phospholamban at Ser-16 was significantly lower in patients with AF that received ß-blockers. CONCLUSION: Together, our findings demonstrate that ß-blocker treatment may be of therapeutic utility to prevent spontaneous calcium release-induced atrial electrical activity; especially in patients with a history of paroxysmal AF displaying preserved ICa density.

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.

R-carvedilol, a potential new therapy for Alzheimer's disease.

For decades, the amyloid cascade hypothesis has been the leading hypothesis in studying Alzheimer's disease (AD) pathology and drug development. However, a growing body of evidence indicates that simply removing amyloid plaques may not significantly affect AD progression. Alternatively, it has been proposed that AD progression is driven by increased neuronal excitability. Consistent with this alternative hypothesis, recent studies showed that pharmacologically limiting ryanodine receptor 2 (RyR2) open time with the R-carvedilol enantiomer prevented and reversed neuronal hyperactivity, memory impairment, and neuron loss in AD mouse models without affecting the accumulation of ß-amyloid (Aß). These data indicate that R-carvedilol could be a potential new therapy for AD.

Impact of R-Carvedilol on ß2-Adrenergic Receptor-Mediated Spontaneous Calcium Release in Human Atrial Myocytes.

A hallmark of atrial fibrillation is an excess of spontaneous calcium release events, which can be mimicked by ß1- or ß2-adrenergic stimulation. Because ß1-adrenergic receptor blockers (ß1-blockers) are primarily used in clinical practice, we here examined the impact of ß2-adrenergic stimulation on spontaneous calcium release and assessed whether the R- and S-enantiomers of the non-selective ß- blocker carvedilol could reverse these effects. For this purpose, human atrial myocytes were isolated from patients undergoing cardiovascular surgery and subjected to confocal calcium imaging or immunofluorescent labeling of the ryanodine receptor (RyR2). Interestingly, the ß2-adrenergic agonist fenoterol increased the incidence of calcium sparks and waves to levels observed with the non-specific ß-adrenergic agonist isoproterenol. Moreover, fenoterol increased both the amplitude and duration of the sparks, facilitating their fusion into calcium waves. Subsequent application of the non ß-blocking R-Carvedilol enantiomer reversed these effects of fenoterol in a dose-dependent manner. R-Carvedilol also reversed the fenoterol-induced phosphorylation of the RyR2 at Ser-2808 dose-dependently, and 1 µM of either R- or S-Carvedilol fully reversed the effect of fenoterol. Together, these findings demonstrate that ß2-adrenergic stimulation alone stimulates RyR2 phosphorylation at Ser-2808 and spontaneous calcium release maximally, and points to carvedilol as a tool to attenuate the pathological activation of ß2-receptors.

Subcellular localization of hippocampal ryanodine receptor 2 and its role in neuronal excitability and memory.

Ryanodine receptor 2 (RyR2) is abundantly expressed in the heart and brain. Mutations in RyR2 are associated with both cardiac arrhythmias and intellectual disability. While the mechanisms of RyR2-linked arrhythmias are well characterized, little is known about the mechanism underlying RyR2-associated intellectual disability. Here, we employed a mouse model expressing a green fluorescent protein (GFP)-tagged RyR2 and a specific GFP probe to determine the subcellular localization of RyR2 in hippocampus. GFP-RyR2 was predominantly detected in the soma and dendrites, but not the dendritic spines of CA1 pyramidal neurons or dentate gyrus granular neurons. GFP-RyR2 was also detected within the mossy fibers in the stratum lucidum of CA3, but not in the presynaptic terminals of CA1 neurons. An arrhythmogenic RyR2-R4496C+/- mutation downregulated the A-type K+ current and increased membrane excitability, but had little effect on the afterhyperpolarization current or presynaptic facilitation of CA1 neurons. The RyR2-R4496C+/- mutation also impaired hippocampal long-term potentiation, learning, and memory. These data reveal the precise subcellular distribution of hippocampal RyR2 and its important role in neuronal excitability, learning, and memory.

Increased RyR2 open probability induces neuronal hyperactivity and memory loss with or without Alzheimer's disease-causing gene mutations.

INTRODUCTION: Neuronal hyperactivity is an early neuronal defect commonly observed in familial and sporadic Alzheimer's disease (AD), but the underlying mechanisms are unclear. METHODS: We employed a ryanodine receptor 2 (RyR2) mutant mouse model harboring the R4496C+/- mutation that markedly increases the channel's open probability (Po) to determine the impact of increased RyR2 activity in neuronal function without AD gene mutations. RESULTS: Genetically increasing RyR2 Po induced neuronal hyperactivity in vivo in anesthetized and awake mice. Increased RyR2 Po induced hyperactive behaviors, impaired learning and memory, defective dendritic spines, and neuronal cell death. Increased RyR2 Po exacerbated the onset of neuronal hyperexcitability and learning and memory impairments in 5xFAD mice. DISCUSSION: Increased RyR2 Po exacerbates the onset of familial AD-associated neuronal dysfunction, and induces AD-like defects in the absence of AD-causing gene mutations, suggesting that RyR2-associated neuronal hyperactivity represents a common target for combating AD with or without AD gene mutations.

ß2-adrenergic stimulation potentiates spontaneous calcium release by increasing signal mass and co-activation of ryanodine receptor clusters.

AIMS: It is unknown how ß-adrenergic stimulation affects calcium dynamics in individual RyR2 clusters and leads to the induction of spontaneous calcium waves. To address this, we analysed spontaneous calcium release events in green fluorescent protein (GFP)-tagged RyR2 clusters. METHODS: Cardiomyocytes from mice with GFP-tagged RyR2 or human right atrial tissue were subjected to immunofluorescent labelling or confocal calcium imaging. RESULTS: Spontaneous calcium release from single RyR2 clusters induced 91.4% ± 2.0% of all calcium sparks while 8.0% ± 1.6% were caused by release from two neighbouring clusters. Sparks with two RyR2 clusters had 40% bigger amplitude, were 26% wider, and lasted 35% longer at half maximum. Consequently, the spark mass was larger in two- than one-cluster sparks with a median and interquartile range for the cumulative distribution of 15.7 ± 20.1 vs 7.6 ± 5.7 a.u. (P < .01). ß2-adrenergic stimulation increased RyR2 phosphorylation at s2809 and s2815, tripled the fraction of two- and three-cluster sparks, and significantly increased the spark mass. Interestingly, the amplitude and mass of the calcium released from a RyR2 cluster were proportional to the SR calcium load, but the firing rate was not. The spark mass was also higher in 33 patients with atrial fibrillation than in 36 without (22.9 ± 23.4 a.u. vs 10.7 ± 10.9; P = .015). CONCLUSIONS: Most sparks are caused by activation of a single RyR2 cluster at baseline while ß-adrenergic stimulation doubles the mass and the number of clusters per spark. This mimics the shift in the cumulative spark mass distribution observed in myocytes from patients with atrial fibrillation.

Clinical and Functional Characterization of Ryanodine Receptor 2 Variants Implicated in Calcium-Release Deficiency Syndrome.

Importance: Calcium-release deficiency syndrome (CRDS), which is caused by loss-of-function variants in cardiac ryanodine receptor 2 (RyR2), is an emerging cause of ventricular fibrillation. However, the lack of complex polymorphic/bidirectional ventricular tachyarrhythmias during exercise stress testing (EST) may distinguish it from catecholaminergic polymorphic ventricular tachycardia (CPVT). Recently, in the first clinical series describing the condition, mouse and human studies showed that the long-burst, long-pause, short-coupled ventricular extra stimulus (LBLPS) electrophysiology protocol reliably induced CRDS ventricular arrhythmias. Data from larger populations with CRDS and its associated spectrum of disease are lacking. Objective: To further insight into CRDS through international collaboration. Design, Setting, and Participants: In this multicenter observational cohort study, probands with unexplained life-threatening arrhythmic events and an ultrarare RyR2 variant were identified. Variants were expressed in HEK293 cells and subjected to caffeine stimulation to determine their functional impact. Data were collected from September 1, 2012, to March 6, 2021, and analyzed from August 9, 2015, to March 6, 2021. Main Outcomes and Measures: The functional association of RyR2 variants found in putative cases of CRDS and the associated clinical phenotype(s). Results: Of 10 RyR2 variants found in 10 probands, 6 were loss-of-function, consistent with CRDS (p.E4451del, p.F4499C, p.V4606E, p.R4608Q, p.R4608W, and p.Q2275H) (in 4 [67%] male and 2 [33%] female probands; median age at presentation, 22 [IQR, 8-34] years). In 5 probands with a documented trigger, 3 were catecholamine driven. During EST, 3 probands with CRDS had no arrhythmias, 1 had a monomorphic couplet, and 2 could not undergo EST (deceased). Relatives of the decedents carrying the RyR2 variant did not have EST results consistent with CPVT. After screening 3 families, 13 relatives were diagnosed with CRDS, including 3 with previous arrhythmic events (23%). None had complex ventricular tachyarrhythmias during EST. Among the 19 confirmed cases with CRDS, 10 had at least 1 life-threatening event at presentation and/or during a median follow-up of 7 (IQR, 6-18) years. Two of the 3 device-detected ventricular fibrillation episodes were induced by a spontaneous LBLPS-like sequence. ß-Blockers were used in 16 of 17 surviving patients (94%). Three of 16 individuals who were reportedly adherent to ß-blocker therapy (19%) had breakthrough events. Conclusions and Relevance: The results of this study suggest that calcium-release deficiency syndrome due to RyR2 loss-of-function variants mechanistically and phenotypically differs from CPVT. Ventricular fibrillation may be precipitated by a spontaneous LBLPS-like sequence of ectopy; however, CRDS remains difficult to recognize clinically. These data highlight the need for better diagnostic tools and treatments for this emerging condition.

Provocation Testing and Therapeutic Response in a Newly Described Channelopathy: RyR2 Calcium Release Deficiency Syndrome.

BACKGROUND: A novel familial arrhythmia syndrome, cardiac ryanodine receptor (RyR2) calcium release deficiency syndrome (CRDS), has recently been described. We evaluated a large and well characterized family to assess provocation testing, risk factor stratification and response to therapy in CRDS. METHODS: We present a family with multiple unheralded sudden cardiac deaths and aborted cardiac arrests, primarily in children and young adults, with no clear phenotype on standard clinical testing. RESULTS: Genetic analysis, including whole genome sequencing, firmly established that a missense mutation in RYR2, Ala4142Thr, was the underlying cause of disease in the family. Functional study of the variant in a cell model showed RyR2 loss-of-function, indicating that the family was affected by CRDS. EPS (Electrophysiological Study) was undertaken in 9 subjects known to carry the mutation, including a survivor of aborted sudden cardiac death, and the effects of flecainide alone and in combination with metoprolol were tested. There was a clear gradation in inducibility of nonsustained and sustained ventricular arrhythmia between subjects at EPS, with the survivor of aborted sudden cardiac death being the most inducible subject. Administration of flecainide substantially reduced arrhythmia inducibility in this subject and abolished arrhythmia in all others. Finally, the effects of additional metoprolol were tested; it increased inducibility in 4/9 subjects. CONCLUSIONS: The Ala4142Thr mutation of RYR2 causes the novel heritable arrhythmia syndrome CRDS, which is characterized by familial sudden death in the absence of prior symptoms or a recognizable phenotype on ambulatory monitoring or exercise stress testing. We increase the experience of a specific EPS protocol in human subjects and show that it is helpful in establishing the clinical status of gene carriers, with potential utility for risk stratification. Our data provide evidence that flecainide is protective in human subjects with CRDS, consistent with the effect previously shown in a mouse model.

Corrigendum: R-carvedilol, a potential new therapy for Alzheimer's disease.

[This corrects the article DOI: 10.3389/fphar.2022.1062495.].

Using hiPSC-CMs to Examine Mechanisms of Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited cardiac arrhythmia condition, triggered by physical or acute emotional stress, that predominantly expresses early in life. Gain-of-function mutations in the cardiac ryanodine receptor gene (RYR2) account for the majority of CPVT cases, causing substantial disruption of intracellular calcium (Ca2+ ) homeostasis particularly during the periods of ß-adrenergic receptor stimulation. However, the highly variable penetrance, patient outcomes, and drug responses observed in clinical practice remain unexplained, even for patients with well-established founder RyR2 mutations. Therefore, investigation of the electrophysiological consequences of CPVT-causing RyR2 mutations is crucial to better understand the pathophysiology of the disease. The development of strategies for reprogramming human somatic cells to human induced pluripotent stem cells (hiPSCs) has provided a unique opportunity to study inherited arrhythmias, due to the ability of hiPSCs to differentiate down a cardiac lineage. Employment of genome editing enables generation of disease-specific cell lines from healthy and diseased patient-derived hiPSCs, which subsequently can be differentiated into cardiomyocytes. This paper describes the means for establishing an hiPSC-based model of CPVT in order to recapitulate the disease phenotype in vitro and investigate underlying pathophysiological mechanisms. The framework of this approach has the potential to contribute to disease modeling and personalized medicine using hiPSC-derived cardiomyocytes. © 2021 Wiley Periodicals LLC.

Human RyR2 (Ryanodine Receptor 2) Loss-of-Function Mutations: Clinical Phenotypes and In Vitro Characterization.

[Figure: see text].