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.

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.

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.

Discovery and Structure-Activity Relationship of a Ryanodine Receptor 2 Inhibitor.

Ryanodine receptor 2 (RyR2) is a large Ca2+-release channel in the sarcoplasmic reticulum (SR) of cardiac muscle cells. It serves to release Ca2+ from the SR into the cytosol to initiate muscle contraction. RyR2 overactivation is associated with arrhythmogenic cardiac disease, but few specific inhibitors have been reported so far. Here, we identified an RyR2-selective inhibitor 1 from the chemical compound library and synthesized it from glycolic acid. Synthesis of various derivatives to investigate the structure-activity relationship of each substructure afforded another two RyR2-selective inhibitors 6 and 7, among which 6 was the most potent. Notably, compound 6 also inhibited Ca2+ release in cells expressing the RyR2 mutants R2474S, R4497C and K4750Q, which are associated with cardiac arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT). This inhibitor is expected to be a useful tool for research on the structure and dynamics of RyR2, as well as a lead compound for the development of drug candidates to treat RyR2-related cardiac disease.

Generation and Accumulation of Various Advanced Glycation End-Products in Cardiomyocytes May Induce Cardiovascular Disease.

Cardiomyocyte dysfunction and cardiovascular diseases (CVDs) can be classified as ischemic or non-ischemic. We consider the induction of cardiac tissue dysfunction by intracellular advanced glycation end-products (AGEs) in cardiomyocytes as a novel type of non-ischemic CVD. Various types of AGEs can be generated from saccharides (glucose and fructose) and their intermediate/non-enzymatic reaction byproducts. Recently, certain types of AGEs (Nε-carboxymethyl-lycine [CML], 2-ammnonio-6-[4-(hydroxymetyl)-3-oxidopyridinium-1-yl]-hexanoate-lysine [4-hydroxymethyl-OP-lysine, hydroxymethyl-OP-lysine], and Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine [MG-H1]) were identified and quantified in the ryanodine receptor 2 (RyR2) and F-actin-tropomyosin filament in the cardiomyocytes of mice or patients with diabetes and/or heart failure. Under these conditions, the excessive leakage of Ca2+ from glycated RyR2 and reduced contractile force from glycated F-actin-tropomyosin filaments induce cardiomyocyte dysfunction. CVDs are included in lifestyle-related diseases (LSRDs), which ancient people recognized and prevented using traditional medicines (e.g., Kampo medicines). Various natural compounds, such as quercetin, curcumin, and epigallocatechin-3-gallate, in these drugs can inhibit the generation of intracellular AGEs through mechanisms such as the carbonyl trap effect and glyoxalase 1 activation, potentially preventing CVDs caused by intracellular AGEs, such as CML, hydroxymethyl-OP, and MG-H1. These investigations showed that bioactive herbal extracts obtained from traditional medicine treatments may contain compounds that prevent CVDs.

Stabilizing cardiac ryanodine receptor with dantrolene treatment prevents left ventricular remodeling in pressure-overloaded heart failure mice.

Dantrolene (DAN) directly binds to cardiac ryanodine receptor 2 (RyR2) through Leu601-Cys620 in the N-terminal domain and subsequently inhibits diastolic Ca2+ leakage through RyR2. We previously reported that therapy using RyR2 V3599K mutation, which inhibits diastolic Ca2+ leakage by enhancing calmodulin (CaM) binding ability to RyR2, prevents left ventricular (LV) remodeling in transverse aortic constriction (TAC) heart failure. Here, we examined whether chronic administration of DAN prevents LV remodeling in TAC heart failure via the same mechanism as genetic therapy. A pressure-overloaded hypertrophy mouse model was developed using TAC. Wild-type (WT) mice were divided into three groups: sham-operated mice (Sham group), TAC mice (TAC group), and TAC mice treated with DAN (TAC-DAN group, 20 mg/kg/day, i.p.). They were then followed up for 8 weeks. The survival rate was higher in the TAC-DAN group (83%) than in the TAC group (49%), and serial echocardiography studies and pathological tissue analysis showed that LV remodeling was significantly prevented in the TAC-DAN group compared to the TAC group. An increase in the diastolic Ca2+ spark frequency and a decrease in the binding affinity of CaM to RyR2 were observed at 8 weeks in the TAC group but not in the TAC-DAN group. Stabilization of RyR2 with DAN prevented LV remodeling and improved survival after TAC by enhancing CaM binding to RyR2 and inhibiting RyR2-mediated diastolic Ca2+ leakage.

Novel mutation in N-terminal fragment of ryanodine receptor 2 causing catecholaminergic polymorphic ventricular tachycardia.

CPVT is a rare inherited arrhythmogenic disorder characterized by bidirectional, polymorphic ventricular arrhythmias triggered by catecholamines released during exercise, stress, or sudden emotion in individuals with a normal resting electrocardiogram and structurally normal heart. Mutations in the ryanodine receptor 2 gene are the most common known etiology of this disorder. The c.1195A > G(p.Met399Val) variant in Exon 14 of RyR2 is currently classified as a Variant of Uncertain Significance. We present a case of CPVT caused by this novel disease-causing RyR2 variant and discuss its pathophysiology. The role of SSRIs in treating patients with CPVT unresponsive to mainstream therapies is also highlighted.

Doxorubicin-induced delayed-onset subclinical cardiotoxicity in mice.

Subclinical cardiotoxicity at low total cumulative doxorubicin (DOX) doses can manifest into cardiomyopathy in long-term cancer survivors. However, the underlying mechanisms are poorly understood. In male B6C3F1 mice, assessment of cardiac function by echocardiography was performed at 1, 4, 10, 17, and 24 weeks after exposure to 6, 9, 12, and 24 mg/kg total cumulative DOX doses or saline (SAL) to monitor development of delayed-onset cardiotoxicity. The 6- or 9-mg/kg total cumulative doses resulted in a significant time-dependent decline in systolic function (left ventricular ejection fraction (LVEF) and fractional shortening (FS)) during the 24-week recovery although there was not a significant alteration in % LVEF or % FS at any specific time point during the recovery. A significant decline in systolic function was elicited by the cardiotoxic cumulative DOX dose (24 mg/kg) during the 4- to 24-week period after treatment compared to SAL-treated counterparts. At 24 weeks after DOX treatment, a significant dose-related decrease in the expression of genes and proteins involved in sarcoplasmic reticulum (SR) calcium homeostasis (Ryr2 and Serca2) was associated with a dose-related increase in the transcript level of Casp12 (SR-specific apoptosis) in hearts. These mice also showed enhanced apoptotic activity in hearts indicated by a significant dose-related elevation in the number of apoptotic cardiomyocytes compared to SAL-treated counterparts. These findings collectively suggest that a steady decline in SR calcium handling and apoptosis might be involved in the development of subclinical cardiotoxicity that can evolve into irreversible cardiomyopathy later in life.

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.

Limiting RyR2 open time prevents Alzheimer's disease-related deficits in the 3xTG-AD mouse model.

Increasing evidence suggests that Alzheimer's disease (AD) progression is driven by a vicious cycle of soluble ß-amyloid (Aß)-induced neuronal hyperactivity. Thus, breaking this vicious cycle by suppressing neuronal hyperactivity may represent a logical approach to stopping AD progression. In support of this, we have recently shown that genetically and pharmacologically limiting ryanodine receptor 2 (RyR2) open time prevented neuronal hyperactivity, memory impairment, dendritic spine loss, and neuronal cell death in a rapid, early onset AD mouse model (5xFAD). Here, we assessed the impact of limiting RyR2 open time on AD-related deficits in a relatively late occurring, slow developing AD mouse model (3xTG-AD) that bears more resemblance (compared to 5xFAD) to that of human AD. Using behavioral tests, long-term potentiation recordings, and Golgi and Nissl staining, we found that the RyR2-E4872Q mutation, which markedly shortens the open duration of the RyR2 channel, prevented learning and memory impairment, defective long-term potentiation, dendritic spine loss, and neuronal cell death in the 3xTG-AD mice. Furthermore, pharmacologically shortening the RyR2 open time with R-carvedilol rescued these AD-related deficits in 3xTG mice. Therefore, limiting RyR2 open time may offer a promising, neuronal hyperactivity-targeted anti-AD strategy.

Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes.

KEY POINTS: Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation-contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP-1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in the diseased heart. ABSTRACT: Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+ ). In wild-type myocytes, PP-1 increased Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of RyR2 upon de-phosphorylation by PP-1. Furthermore, we have confirmed the S2808 site as a target for PP-1 and as a potential link between RyR2s modulation and the cellular response.

Loss of Protein Phosphatase 1 Regulatory Subunit PPP1R3A Promotes Atrial Fibrillation.

BACKGROUND: Abnormal calcium (Ca2+) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN. METHODS: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln-knockout mice. Ppp1r3a-knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a-knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca2+ imaging. RESULTS: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF. CONCLUSIONS: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.

Stress Signaling JNK2 Crosstalk With CaMKII Underlies Enhanced Atrial Arrhythmogenesis.

RATIONALE: Atrial fibrillation (AF) is the most common arrhythmia, and advanced age is an inevitable and predominant AF risk factor. However, the mechanisms that couple aging and AF propensity remain unclear, making targeted therapeutic interventions unattainable. OBJECTIVE: To explore the functional role of an important stress response JNK (c-Jun N-terminal kinase) in sarcoplasmic reticulum Ca2+ handling and consequently Ca2+-mediated atrial arrhythmias. METHODS AND RESULTS: We used a series of cutting-edge electrophysiological and molecular techniques, exploited the power of transgenic mouse models to detail the molecular mechanism, and verified its clinical applicability in parallel studies on donor human hearts. We discovered that significantly increased activity of the stress response kinase JNK2 (JNK isoform 2) in the aged atria is involved in arrhythmic remodeling. The JNK-driven atrial proarrhythmic mechanism is supported by a pathway linking JNK, CaMKII (Ca2+/calmodulin-dependent kinase II), and sarcoplasmic reticulum Ca2+ release RyR2 (ryanodine receptor) channels. JNK2 activates CaMKII, a critical proarrhythmic molecule in cardiac muscle. In turn, activated CaMKII upregulates diastolic sarcoplasmic reticulum Ca2+ leak mediated by RyR2 channels. This leads to aberrant intracellular Ca2+ waves and enhanced AF propensity. In contrast, this mechanism is absent in young atria. In JNK challenged animal models, this is eliminated by JNK2 ablation or CaMKII inhibition. CONCLUSIONS: We have identified JNK2-driven CaMKII activation as a novel mode of kinase crosstalk and a causal factor in atrial arrhythmic remodeling, making JNK2 a compelling new therapeutic target for AF prevention and treatment.

Cardiac-Specific Caveolin-3 Overexpression Prevents Post-Myocardial Infarction Ventricular Arrhythmias by Inhibiting Ryanodine Receptor-2 Hyperphosphorylation.

INTRODUCTION: Ventricular arrhythmia is the most important risk factor for sudden cardiac death (SCD) after acute myocardial infarction (MI) worldwide. However, the molecular mechanisms underlying these arrhythmias are complex and not completely understood. OBJECTIVE: Here, we evaluated whether caveolin-3 (Cav3), the structural protein of caveolae, plays an important role in the therapeutic strategy for ventricular arrhythmias. METHODS: A model of cardiac-specific overexpression of Cav3 was established to evaluate the incidence of ventricular arrhythmias after MI in mice. Ca2+ imaging was employed to detect the propensity of adult murine cardiomyocytes to generate arrhythmias, and immunoprecipitation and immunofluorescence were used to determine the relationship of proteins. Additionally, qRT-PCR and western blotting were used to detect the mRNA and protein expression. RESULTS: We found that cardiac-specific overexpression of Cav3 delivered by a recombinant adeno-associated viral vector reduced the incidence of ventricular arrhythmias and SCD after MI in mice. Ca2+ imaging and western blotting revealed that overexpression of Cav3 reduced diastolic spontaneous Ca2+ waves by inhibiting the hyperphosphorylation of ryanodine receptor-2 (RyR2) at Ser2814, rather than at Ser2808, compared to in rAAV-red fluorescent protein control mice. Furthermore, we demonstrated that Cav3-regulated RYR2 hyperphosphorylation relied on plakophilin-2 in hypoxia-stimulated cultured cardiomyocytes by western blotting, immunoprecipitation, and immunofluorescence in vitro. CONCLUSIONS: Our results suggested a novel role for Cav3 in the prevention of ventricular arrhythmias, thereby identifying a new target for preventing SCD after MI.

Isoproterenol Promotes Rapid Ryanodine Receptor Movement to Bridging Integrator 1 (BIN1)-Organized Dyads.

BACKGROUND: The key pathophysiology of human acquired heart failure is impaired calcium transient, which is initiated at dyads consisting of ryanodine receptors (RyRs) at sarcoplasmic reticulum apposing CaV1.2 channels at t-tubules. Sympathetic tone regulates myocardial calcium transients through ß-adrenergic receptor (ß-AR)-mediated phosphorylation of dyadic proteins. Phosphorylated RyRs (P-RyR) have increased calcium sensitivity and open probability, amplifying calcium transient at a cost of receptor instability. Given that bridging integrator 1 (BIN1) organizes t-tubule microfolds and facilitates CaV1.2 delivery, we explored whether ß-AR-regulated RyRs are also affected by BIN1. METHODS AND RESULTS: Isolated adult mouse hearts or cardiomyocytes were perfused for 5 minutes with the ß-AR agonist isoproterenol (1 µmol/L) or the blockers CGP+ICI (baseline). Using biochemistry and superresolution fluorescent imaging, we identified that BIN1 clusters P-RyR and CaV1.2. Acute ß-AR activation increases coimmunoprecipitation between P-RyR and cardiac spliced BIN1+13+17 (with exons 13 and 17). Isoproterenol redistributes BIN1 to t-tubules, recruiting P-RyRs and improving the calcium transient. In cardiac-specific Bin1 heterozygote mice, isoproterenol fails to concentrate BIN1 to t-tubules, impairing P-RyR recruitment. The resultant accumulation of uncoupled P-RyRs increases the incidence of spontaneous calcium release. In human hearts with end-stage ischemic cardiomyopathy, we find that BIN1 is also 50% reduced, with diminished P-RyR association with BIN1. CONCLUSIONS: On ß-AR activation, reorganization of BIN1-induced microdomains recruits P-RyR into dyads, increasing the calcium transient while preserving electric stability. When BIN1 is reduced as in human acquired heart failure, acute stress impairs microdomain formation, limiting contractility and promoting arrhythmias.

Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits.

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.

Prominent QTc prolongation in a patient with a rare variant in the cardiac ryanodine receptor gene.

We report the case of a 12-year-old female patient with a history of four syncopal episodes related to exercise over 2 years and who showed prominent QTc prolongation on electrocardiogram; therefore, she was clinically diagnosed with long QT syndrome type-1. However, genetic analysis did not identify any LQT-related genes but showed a rare missense variant in the cardiac ryanodine receptor gene. From the results of drug-loading tests, administration of oral propranolol was initiated; thereafter, she experienced no syncopal episodes. This is a case report demonstrating the "overlapping clinical features" of long QT syndrome and catecholaminergic polymorphic ventricular tachycardia.

A novel RYR2 loss-of-function mutation (I4855M) is associated with left ventricular non-compaction and atypical catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channelopathy usually caused by gain-of-function mutations ryanodine receptor type-2 (RyR2). Left ventricular non-compaction (LVNC) is an often genetic cardiomyopathy. A rare LVNC-CPVT overlap syndrome may be caused by exon 3 deletion in RyR2. We sought to characterize the phenotypic spectrum and molecular basis of a novel RyR2 mutation identified in a family with both conditions. METHODS: Several members of an affected family underwent clinical and genetic assessments. A homology model of the RyR2 pore-region was generated to predict the location and potential impact of their RyR2 mutation. Ca2+-release assays were performed to characterize the functional impact of the RyR2 mutant expressed in HEK293 cells. RESULTS: A multigenerational family presented with a history of sudden death and a phenotype of atypical CPVT and LVNC. Genetic testing revealed a RYR2 mutation (I4855M) in two affected individuals. A homology model of the RyR2 pore-region showed that the I4855M mutant reside is located in the highly conserved 'inner vestibule', a water-filled cavity. I4855M may interfere with Ca2+ permeation and affect interactions between RyR2 pore subunits, and is thus predicted in silico to be damaging. Expression and functional studies in HEK293 cells revealed that I4855M inhibited caffeine-induced Ca2+ release and exerted a dominant-negative impact on wild type RyR2. CONCLUSIONS: This study identifies a potentially lethal overlapping syndrome of LVNC and atypical CPVT related to a novel RYR2 variant. Structural and functional studies suggest that this is a loss-of-function mutation, which exerts a dominant-negative effect on wild type RyR2.

Inhibition of the mevalonate pathway ameliorates anoxia-induced down-regulation of FKBP12.6 and intracellular calcium handling dysfunction in H9c2 cells.

Statins have beneficial pleiotropic effects beyond lipid lowering on the cardiovascular system. These cardio-protective effects are mediated through inhibition of the intracellular mevalonate pathway, by decreasing isoprenoid intermediate synthesis and the subsequent post-translational modification of small GTPases, such as Ras, Rho, and Rac. Impaired intracellular calcium handling is considered an important pathophysiologic mechanism responsible for cardiac dysfunction. Our study aimed at investigating the influence of mevalonate pathway, including its downstream small GTPases (Ras, RhoA, and Rac1) on anoxia-mediated alterations of calcium handling in H9c2 cardiomyocytes. Cultured H9c2 cardiomyocytes were exposed to acute anoxia after pretreatment with different drugs that specifically antagonize five key components in the mevalonate pathway, including 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, Rho-kinase, Rac1 and Ras farnesyltransferase. Thereafter, we evaluated the effects of the mevalonate pathway on anoxia-induced cell death, expression of the sarcoplasmic reticulum calcium release channel (ryanodine receptor 2) and its regulator FK506-binding protein 12.6, as well as functional calcium release from intracellular calcium stores. Our experiments confirmed the role of prenylated proteins in regulating cardiomyocyte dysfunction, especially via RhoA- and Ras-related signaling pathways. Furthermore, our data demonstrated that inhibition of the mevalonate pathway could ameliorate anoxia-mediated calcium handling dysfunction with the up-regulated expression of FK506-binding protein 12.6 and consequently provided evidence for FK506-binding protein 12.6 as a "stabilizer" of ryanodine receptor 2.

Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias.

Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1-606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410-437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545-606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C(α) atom movements of up to 8 Šupon channel gating, and predicts the location of the leucine-isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.