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During exercise or stress, the sympathetic system stimulates cardiac contractility via β‐adrenergic receptor (β‐AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation–contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca2+/calmodulin kinase type‐2‐specific site. To examine the contribution of these phosphosites to Ca2+ signalling, we generated double knock‐in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2‐S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca2+ imaging and electrophysiology experiments. Isoprenaline was used to stimulate β‐ARs. Measurements of Ca2+ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca2+ releases in DKI myocytes, both in control conditions and during β‐AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca2+ inside the sarcoplasmic reticulum, suggesting higher Ca2+ sensitivity of the RyRs. The refractoriness of Ca2+ spark triggering depends on the Ca2+ sensitivity of the RyR2. We found that RyR2‐S2808A/S2814A channels were more Ca2+ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2‐Ser2808 and RyR2‐S2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β‐AR‐mediated sensitization of RyRs. Key points: Phosphorylation of cardiac sarcoplasmic reticulum Ca2+‐release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function.Ablation of both the RyR2‐Ser2808 and RyR2‐Ser2814 sites increases the propensity for pro‐arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs.The intra‐sarcoplasmic reticulum Ca2+ threshold for spontaneous Ca2+ wave generation is lower in RyR2‐double‐knock‐in cells.The RyR2 from double‐knock‐in cells exhibits increased Ca2+ sensitivity.Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel.Phosphorylation of Ser2030 might be sufficient for a β‐adrenergic response. [ABSTRACT FROM AUTHOR]

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, and oral squamous cell carcinoma (OSCC) is one of the most common types. There is strong evidence that ryanodine receptor 2 (RYR2) plays an important role in different types of cancer according to previous studies. Its expression is associated with survival in patients with HNSCC, but it is unknown whether altered RYR2 expression contributes to tumorigenesis. Therefore, we examined how RYR2 polymorphisms affect OSCC susceptibility and clinicopathological characteristics. Five single nucleotide polymorphisms (SNPs) of RYR2, rs12594, rs16835904, rs2779359, rs3765097, and rs3820216, were analyzed in 562 cases of OSCC and 332 healthy controls using real-time PCR. We demonstrated that RYR2 SNP rs12594 was significantly different between the case and control groups, but this difference was not significant after adjusting for personal habits. In contrast, we found that different genotypes of SNP rs2779359 were significantly associated with the characteristics of clinical stage and tumor size in OSCC patients, according to the odds ratios and the adjusted odds ratios; specifically, patients with the T genotype had 1.477-fold (95% CI, 1.043 to 2.091; p = 0.028) and 1.533-fold (95% CI, 1.087–2.162; p = 0.015) increases in clinical stage and tumor size, respectively, compared with patients with the C allele. The results of our study, in which RYR2 SNPs associated with OSCC progression and development were examined for the first time, suggest that clinicopathological characteristics may alter OSCC susceptibility. Finally, RYR2 SNP rs2779359 not only plays a role in both the prognosis and diagnosis of oral cancer but is also likely an important predictive factor for recurrence, response to treatment, and medication toxicity. [ABSTRACT FROM AUTHOR]

Abstract Aims Hyperactivity of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII‐induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII‐dependent RyR2 phosphorylation for CaMKII‐induced heart failure development in vivo. Methods and results We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2‐S2814A knock‐in mice that are resistant to CaMKII‐dependent RyR2 phosphorylation. Ca2+‐spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65 ± 0.73 mF/F0 vs. 1.88 ± 0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+‐leak induction in the CaMKIIδC TG, both regarding Ca2+‐spark size and frequency, demonstrating that the CaMKIIδC‐induced SR Ca2+ leak entirely depends on the CaMKII‐specific RyR2‐S2814 phosphorylation. Yet, the RyR2‐S2814A mutation did not affect the massive contractile dysfunction (ejection fraction = 12.17 ± 2.05% vs. 45.15 ± 3.46% in WT), cardiac hypertrophy (heart weight/tibia length = 24.84 ± 3.00 vs. 9.81 ± 0.50 mg/mm in WT), or severe premature mortality (median survival of 12 weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. Conclusions S2814 phosphoresistance of RyR2 prevents the CaMKII‐dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms—independent of SR Ca2+ leak—are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure.

Aims: Hyperactivity of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) has emerged as a central cause of pathologic remodelling in heart failure. It has been suggested that CaMKII‐induced hyperphosphorylation of the ryanodine receptor 2 (RyR2) and consequently increased diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) is a crucial mechanism by which increased CaMKII activity leads to contractile dysfunction. We aim to evaluate the relevance of CaMKII‐dependent RyR2 phosphorylation for CaMKII‐induced heart failure development in vivo. Methods and results: We crossbred CaMKIIδC overexpressing [transgenic (TG)] mice with RyR2‐S2814A knock‐in mice that are resistant to CaMKII‐dependent RyR2 phosphorylation. Ca2+‐spark measurements on isolated ventricular myocytes confirmed the severe diastolic SR Ca2+ leak previously reported in CaMKIIδC TG [4.65 ± 0.73 mF/F0 vs. 1.88 ± 0.30 mF/F0 in wild type (WT)]. Crossing in the S2814A mutation completely prevented SR Ca2+‐leak induction in the CaMKIIδC TG, both regarding Ca2+‐spark size and frequency, demonstrating that the CaMKIIδC‐induced SR Ca2+ leak entirely depends on the CaMKII‐specific RyR2‐S2814 phosphorylation. Yet, the RyR2‐S2814A mutation did not affect the massive contractile dysfunction (ejection fraction = 12.17 ± 2.05% vs. 45.15 ± 3.46% in WT), cardiac hypertrophy (heart weight/tibia length = 24.84 ± 3.00 vs. 9.81 ± 0.50 mg/mm in WT), or severe premature mortality (median survival of 12 weeks) associated with cardiac CaMKIIδC overexpression. In the face of a prevented SR Ca2+ leak, the phosphorylation status of other critical CaMKII downstream targets that can drive heart failure, including transcriptional regulator histone deacetylase 4, as well as markers of pathological gene expression including Xirp2, Il6, and Col1a1, was equally increased in hearts from CaMKIIδC TG on a RyR WT and S2814A background. Conclusions: S2814 phosphoresistance of RyR2 prevents the CaMKII‐dependent SR Ca2+ leak induction but does not prevent the cardiomyopathic phenotype caused by enhanced CaMKIIδC activity. Our data indicate that additional mechanisms—independent of SR Ca2+ leak—are critical for the maladaptive effects of chronically increased CaMKIIδC activity with respect to heart failure. [ABSTRACT FROM AUTHOR]

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 β-adrenergic receptor (βAR) 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 βAR 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that simply reducing β‐amyloid (Aβ) 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 Aβ‐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. [ABSTRACT FROM AUTHOR]

Creation of disease models utilizing hiPSCs in combination with CRISPR/Cas9 gene editing enable mechanistic insights into differential pharmacological responses. This allows translation of efficacy and safety findings from a healthy to a diseased state and provides a means to predict clinical outcome sooner during drug discovery. Calcium handling disturbances including reduced expression levels of the type 2 ryanodine receptor (RYR2) are linked to cardiac dysfunction; here we have created a RYR2 deficient human cardiomyocyte model that mimics some aspects of heart failure. RYR2 deficient cardiomyocytes show differential pharmacological responses to L-type channel calcium inhibitors. Phenotypic and proteomic characterization reveal novel molecular insights with altered expression of structural proteins including CSRP3, SLMAP, and metabolic changes including upregulation of the pentose phosphate pathway and increased sensitivity to redox alterations. This genetically engineered in vitro cardiovascular model of RYR2 deficiency supports the study of pharmacological responses in the context of calcium handling and metabolic dysfunction enabling translation of drug responses from healthy to perturbed cellular states.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, and oral squamous cell carcinoma (OSCC) is one of the most common types. There is strong evidence that ryanodine receptor 2 (RYR2) plays an important role in different types of cancer according to previous studies. Its expression is associated with survival in patients with HNSCC, but it is unknown whether altered RYR2 expression contributes to tumorigenesis. Therefore, we examined how RYR2 polymorphisms affect OSCC susceptibility and clinicopathological characteristics. Five single nucleotide polymorphisms (SNPs) of RYR2, rs12594, rs16835904, rs2779359, rs3765097, and rs3820216, were analyzed in 562 cases of OSCC and 332 healthy controls using real-time PCR. We demonstrated that RYR2 SNP rs12594 was significantly different between the case and control groups, but this difference was not significant after adjusting for personal habits. In contrast, we found that different genotypes of SNP rs2779359 were significantly associated with the characteristics of clinical stage and tumor size in OSCC patients, according to the odds ratios and the adjusted odds ratios; specifically, patients with the T genotype had 1.477-fold (95% CI, 1.043 to 2.091; p = 0.028) and 1.533-fold (95% CI, 1.087–2.162; p = 0.015) increases in clinical stage and tumor size, respectively, compared with patients with the C allele. The results of our study, in which RYR2 SNPs associated with OSCC progression and development were examined for the first time, suggest that clinicopathological characteristics may alter OSCC susceptibility. Finally, RYR2 SNP rs2779359 not only plays a role in both the prognosis and diagnosis of oral cancer but is also likely an important predictive factor for recurrence, response to treatment, and medication toxicity.

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.

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. • Diastolic Ca2+ leakage and CaM dissociation from RyR2 are crucial causes for TAC-induced heart failure. • Dantrolene stabilizes the tetrameric structure, stops Ca2+ leakage and CaM dissociation. • Chronic administration of dantrolene inhibited LV remodeling and improved prognosis in TAC model. [ABSTRACT FROM AUTHOR]

Abstract RYR2 mutation is clinically frequent in non‐small cell lung cancer (NSCLC) with its function being elusive. We downloaded lung squamous cell carcinoma and lung adenocarcinoma samples from the TCGA database, split the samples into RYR2 mutant group (n = 337) and RYR2 wild group (n = 634), and established Kaplan‐Meier curves. The results showed that RYR2 mutant group lived longer than the wild group (p = 0.027). Weighted gene co‐expression network analysis (WGCNA) of differentially expressed genes (DEGs) yielded prognosis‐related genes. Five mRNAs and 10 lncRNAs were selected to build survival prognostic models with other clinical features. The AUCs of 2 models are 0.622 and 0.565 for predicting survival at 3 years. Among these genes, the AUCs of DKK1 and GS1‐115G20.1 expression levels were 0.607 and 0.560, respectively, which predicted the 3‐year survival rate of NSCLC sufferers. GSEA identified an association of high DKK1 expression with TP53, MTOR, and VEGF expression. Several target miRNAs interacting with GS1‐115G20.1 were observed to show the relationship with the phenotype, treatment, and survival of NSCLC. NSCLC patients with RYR2 mutation may obtain better prognosis by down‐regulating DKK1 and up‐regulating GS1‐115G20.1.

RationaleAtrial 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.ObjectiveTo 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 resultsWe 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.ConclusionsWe 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.

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. [ABSTRACT FROM AUTHOR]

During exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation-contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca 2+ /calmodulin kinase type-2-specific site. To examine the contribution of these phosphosites to Ca 2+ signalling, we generated double knock-in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2-S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca 2+  imaging and electrophysiology experiments. Isoprenaline was used to stimulate β-ARs. Measurements of Ca 2+ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca 2+ releases in DKI myocytes, both in control conditions and during β-AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca 2+ inside the sarcoplasmic reticulum, suggesting higher Ca 2+ sensitivity of the RyRs. The refractoriness of Ca 2+ spark triggering depends on the Ca 2+ sensitivity of the RyR2. We found that RyR2-S2808A/S2814A channels were more Ca 2+ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2-Ser2808 and RyR2-S2814 sites increases the propensity for pro-arrhythmic spontaneous Ca 2+ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for β-AR-mediated sensitization of RyRs. KEY POINTS: Phosphorylation of cardiac sarcoplasmic reticulum Ca 2+ -release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function. Ablation of both the RyR2-Ser2808 and RyR2-Ser2814 sites increases the propensity for pro-arrhythmic spontaneous Ca 2+ releases, as previously suggested for hyperphosphorylated RyRs. The intra-sarcoplasmic reticulum Ca 2+ threshold for spontaneous Ca 2+ wave generation is lower in RyR2-double-knock-in cells. The RyR2 from double-knock-in cells exhibits increased Ca 2+ sensitivity. Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel. Phosphorylation of Ser2030 might be sufficient for a β-adrenergic response., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

BackgroundRyanodine receptor 2 (RYR2) encodes a component of a calcium channel. RYR2 variants were well-reported to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), but rarely reported in epilepsy cases. Here, we present a novel heterozygous mutation of RYR2 in a child with focal epilepsy.MethodsAt the age of 2 years and 7 months, the patient experienced seizures, such as eye closure, tooth clenching, clonic jerking and hemifacial spasm, as well as abnormal electroencephalogram (EEG). Then, he was analyzed by whole-exome sequencing (WES). The mutations of both the proband and his parents were further confirmed by Sanger sequencing. The pathogenicity of the variant was further assessed by population-based variant frequency screening, evolutionary conservation comparison, and American Association for Medical Genetics and Genomics (ACMG) scoring.ResultsWES sequencing revealed a novel heterozygous truncating mutation [c.12670G > T, p.(Glu4224*), NM_001035.3] in RYR2 gene of the proband. Sanger sequencing confirmed that this mutation was inherited from his mother. This novel variant was predicted to be damaging by different bioinformatics methods. Cardiac investigation showed that the proband had no structural abnormalities, but sinus tachycardia.ConclusionWe proposed that RYR2 is a potential candidate gene for focal epilepsy, and epilepsy patients carried with RYR2 variants should be given more attention, even if they do not show cardiac abnormalities

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. Subclinical cardiotoxicity elicited by low total cumulative doxorubicin (DOX) doses can occur years after treatment in cancer patients. In male B6C3F1 mice, a progressive deterioration in heart function measured by echocardiography was evidenced by a significant time‐dependent decline in systolic function during 24 weeks after treatment with 6‐ or 9‐mg/kg total cumulative DOX doses. A significant dose‐related downregulation of Ryr2 and Serca2 and increased number of apoptotic cardiomyocytes at 24 weeks after treatment may underlie delayed‐onset subclinical cardiac dysfunction. [ABSTRACT FROM AUTHOR]

RYR2 mutation is clinically frequent in non‐small cell lung cancer (NSCLC) with its function being elusive. We downloaded lung squamous cell carcinoma and lung adenocarcinoma samples from the TCGA database, split the samples into RYR2 mutant group (n = 337) and RYR2 wild group (n = 634), and established Kaplan‐Meier curves. The results showed that RYR2 mutant group lived longer than the wild group (p = 0.027). Weighted gene co‐expression network analysis (WGCNA) of differentially expressed genes (DEGs) yielded prognosis‐related genes. Five mRNAs and 10 lncRNAs were selected to build survival prognostic models with other clinical features. The AUCs of 2 models are 0.622 and 0.565 for predicting survival at 3 years. Among these genes, the AUCs of DKK1 and GS1‐115G20.1 expression levels were 0.607 and 0.560, respectively, which predicted the 3‐year survival rate of NSCLC sufferers. GSEA identified an association of high DKK1 expression with TP53, MTOR, and VEGF expression. Several target miRNAs interacting with GS1‐115G20.1 were observed to show the relationship with the phenotype, treatment, and survival of NSCLC. NSCLC patients with RYR2 mutation may obtain better prognosis by down‐regulating DKK1 and up‐regulating GS1‐115G20.1. [ABSTRACT FROM AUTHOR]

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare cardiac ion channelopathy. This was the first systematic review of published works on the clinical characteristics, outcomes, and genetic findings of patients with CPVT from Hong Kong. PubMed and Embase were searched electronically from their inception until February 2022. The Joanna Briggs Institute Critical Appraisal Checklist was used to critically appraise included studies. Studies written in English describing at least one patient with CPVT were included. Studies describing overlapping CPVT patients from previous reports were excluded. Two studies describing 17 patients with CPVT were included. All included studies were rated of acceptable quality. The largest case series evaluated 16 CPVT patients with a mean presentation age of 11 ± 4 years. Of these, 15 patients (93.8%) were symptomatic at initial presentation. Ten patients presented with both premature ventricular complexes (PVCs) and ventricular tachycardia/ventricular fibrillation (VT/VF), whereas one had PVCs without VT/VF. Among the 14 patients (87.5%) who underwent genetic testing, eight (57.1%) tested positive for the ryanodine receptor 2 (RyR2) gene. Over a mean follow-up duration of 116 ± 36 months, six patients (37.5%) had incident VT/VF. In a 5-year review of autopsy data retrieved from public mortuaries for 289 sudden cardiac death patients, one CPVT patient (0.03%) was identified. All patients with CPVT in Hong Kong presented at or below 19 years old, of whom a majority were initially symptomatic or had incident VT/VF events. Novel genetic variants in the RyR2 gene not reported beyond our locality were identified. [ABSTRACT FROM AUTHOR]

Creation of disease models utilizing hiPSCs in combination with CRISPR/Cas9 gene editing enable mechanistic insights into differential pharmacological responses. This allows translation of efficacy and safety findings from a healthy to a diseased state and provides a means to predict clinical outcome sooner during drug discovery. Calcium handling disturbances including reduced expression levels of the type 2 ryanodine receptor (RYR2) are linked to cardiac dysfunction; here we have created a RYR2 deficient human cardiomyocyte model that mimics some aspects of heart failure. RYR2 deficient cardiomyocytes show differential pharmacological responses to L-type channel calcium inhibitors. Phenotypic and proteomic characterization reveal novel molecular insights with altered expression of structural proteins including CSRP3, SLMAP, and metabolic changes including upregulation of the pentose phosphate pathway and increased sensitivity to redox alterations. This genetically engineered in vitro cardiovascular model of RYR2 deficiency supports the study of pharmacological responses in the context of calcium handling and metabolic dysfunction enabling translation of drug responses from healthy to perturbed cellular states., Competing Interests: LS, AH, DE, A-LC, PG, JG, RH, AP are employees of AstraZeneca and have stock ownership and/or stock options or interests in the company. MK was a master's thesis student at AstraZeneca and is now employed as a PhD student at the University of Gothenburg, Gothenburg Sweden. PG is currently employed by Owkin, Barcelona, Spain., (© 2024 Starnes, Hall, Etal, Cavallo, Grabowski, Gallon, Kha, Hicks and Pointon.)

Objective To investigate the effect of FK506 binding protein 12.6 (FKBP12.6) knockout on detrusor overactivity (DO) in mice and explore the possible mechanism. Methods Mouse models of DO were established by partial bladder outlet obstruction (PBOO) in 30 wild-type mice (WT) and 30 homozygote FKBP12.6 knockout mice (KO group). All the mice were 8-10 weeks old and female. The morphology of the detrusor was observed using HE staining, and void spots and urodynamic test were used to assess the changes in the symptom of frequent urinary and the bladder pressure, respectively. Visceromotor responses (VMRs) and bladder smooth muscle electrical activity in the filling stage were recorded to assess the bladder response to pressure. Spontaneous contraction of the detrusor strips was tested to evaluate the sensitivity of the detrusor to tension, and the expression of ryanodine receptor 2 (RyR2) was detected using Western blotting. Results FKBP12.6 knockout did not cause obvious changes in detrusor morphology, but aggravated the symptom of frequent urination in the mice. Compared with WT mice, the mice in KO group showed a significantly increased micturition frequency (43.500±19.164 vs 22.000±14.717 every 3 h, P < 0.05) with aggravated DO, significantly increased fluctuation frequency of bladder pressure during the urine storage period (13.900±5.430 vs 8.000±3.232 per 30 min, P < 0.05), and significantly increased sensitivity of the detrusors to tension (3.200±0.648 vs 1.200±0.245, P < 0.01). RyR2 expression in the detrusors decreased significantly in FKBP12.6 knockout mice as compared with the WT mice (0.154±0.106 vs 1.000±0.444, P < 0.01). Conclusion FKBP12.6 knockout results in aggravation of DO in mouse models possibly by down-regulation of RyR2 in the detrusors

Asthma has significant impacts on living quality particularly in children. Long noncoding RNA (lncRNA) MALAT1 plays a crucial role in neonatal respiratory diseases. Meanwhile, MALAT1 knockdown could induce viability and attenuate apoptosis of airway-related cells. However, the role of MALAT1 in neonatal asthma, asthma-related cell, and its possible mechanism is unclear. This study aims to investigate MALAT1 level in asthma and to identify the effects of MALAT1 on bronchial/tracheal smooth muscle cells (B/TSMCs). Newborn asthma modeling rat was constructed by introducing ovalbumin (OVA). MALAT1 levels in tissues or B/TSMCs were determined by RT-qPCR. Exogenous changes of MALAT1, RyR2 or miR-133a in B/TSMCs were fulfilled by cell transfection; cell apoptosis was measured by using Cell Death Detection ELISA kit and Hochest33342; IL-6, TNF-α and IL-1β level was detected by using corresponding ELISA kit; ryanodine receptor 2 (RyR2) mRNA and miR-133a level was determined by RT-qPCR; cleaved caspase-3 (c-caspase-3) and RyR2 expression was detected by Western blot; luciferase reporter assay was performed to confirm the target regulation of miR-133a on RyR2. We found that MALAT1 was significantly upregulated in tracheal tissues of newborn asthma modeling rats. In MALAT1-silenced or -overexpressed B/TSMCs, we found a synchronous change of cell apoptosis, inflammatory factor secretion (IL-6, TNF-α, and IL-1β) or RyR2 level, but a reverse change of miR-133a level with MALAT1. Besides, MALAT1 induced B/TSMCs apoptosis and inflammation increase could be partially reversed when RyR2 was silenced or when miR-133a was overexpressed. The luciferase reporter assay confirmed that RyR2 is a direct target gene of miR-133a in B/TSMCs. Finally, we conclude that MALAT1 knockdown could protect from B/TSMCs injury via regulating miR-133a/RyR2 axis. [ABSTRACT FROM AUTHOR]

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 Ryr2 I4093V/+ signaling analysis and a ryanodine binding assay indicated that the mutant ryanodine receptor 2 had a gain-of-function phenotype and enhanced Ca 2+ signaling analysis and a ryanodine binding assay indicated that the mutant ryanodine receptor 2 had a gain-of-function phenotype and enhanced Ca 2+ 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

In adult cardiomyocytes (CMs), the type 2 ryanodine receptor (RYR2) is an indispensable Ca2+ release channel that ensures the integrity of excitation-contraction coupling, which is fundamental for every heartbeat. However, the role and importance of RYR2 during human embryonic cardiac development are still poorly understood. Here, we generated two human induced pluripotent stem cell (iPSC)-based RYR2 knockout (RYR2–/–) lines using the CRISPR/Cas9 gene editing technology. We found that RYR2–/–-iPSCs could differentiate into CMs with the efficiency similar to control-iPSCs (Ctrl-iPSCs); however, the survival of iPSC-CMs was markedly affected by the lack of functional RYR2. While Ctrl-iPSC-CMs exhibited regular Ca2+ handling, we observed significantly reduced frequency and intense abnormalities of Ca2+ transients in RYR2–/–-iPSC-CMs. Ctrl-iPSC-CMs displayed sensitivity to extracellular Ca2+ ([Ca2+ ]o) and caffeine in a concentration-dependent manner, while RYR2–/–-iPSC-CMs showed inconsistent reactions to [Ca2+ ]o and were insensitive to caffeine, indicating there is no RYR2-mediated Ca2+ release from the sarcoplasmic reticulum (SR). Instead, compensatory mechanism for calcium handling in RYR2–/–-iPSC-CMs is partially mediated by the inositol 1,4,5-trisphosphate receptor (IP3R). Similar to Ctrl-iPSC-CMs, SR Ca2+ refilling in RYR2–/–-iPSC-CMs is mediated by SERCA. Additionally, RYR2–/–-iPSC-CMs showed a decreased beating rate and a reduced peak amplitude of L-type Ca2+ current. These findings demonstrate that RYR2 is not required for CM lineage commitment but is important for CM survival and contractile function. IP3R-mediated Ca2+ release is one of the major compensatory mechanisms for Ca2+ cycling in human CMs with the RYR2 deficiency.

The present review focusses on the regulation and interplay of cardiac SR Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCa2/PLN and RyR2. Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners’ proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.

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. 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. 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 FROM AUTHOR]

Ryanodine receptor 2 (RyR2) is a Ca2+ release channel mainly located on the sarcoplasmic reticulum (SR) membrane of heart muscle cells and regulates the concentration of Ca2+ in the cytosol. RyR2 overactivation causes potentially lethal cardiac arrhythmias, but no specific inhibitor is yet available. Herein we developed the first highly potent and selective RyR2 inhibitor, TMDJ-035, containing 3,5-difluoro substituents on the A ring and a 4-fluoro substituent on the B ring, based on a comprehensive structure-activity relationship (SAR) study of tetrazole compound 1. The SAR study also showed that the amide conformation is critical for inhibitory potency. Single-crystal X-ray diffraction analysis and variable-temperature 1H NMR revealed that TMDJ-035 strongly favors cis -amide configuration, while the inactive analogue TMDJ-011 with a secondary amide takes trans -amide configuration. Examination of the selectivity among RyRs indicated that TMDJ-035 displayed high selectivity for RyR2. TMDJ-035 suppressed abnormal Ca2+ waves and transients in isolated cardiomyocytes from RyR2-mutated mice. It appears to be a promising candidate drug for treating cardiac arrhythmias due to RyR2 overactivation, as well as a tool for studying the mechanism and dynamics of RyR2 channel gating. TMDJ-035 with cis -amide configuration could suppress abnormal Ca2+ signals in cardiomyocytes from RyR2-mutated mice. [Display omitted] • The novel RyR2 selective inhibitor 1 was identified. • Structure-activity relationship discovered quite potent RyR2 inhibitor TMDJ-035. • The amide conformation of TMDJ-035 was verified as cis -amide, which is crucial for the activity expression. • TMDJ-035 showed complete selectivity among RyRs. • TMDJ-035 could suppress the abnormal Ca2+ signals in cardiomyocytes from RyR2-mutated mice. [ABSTRACT FROM AUTHOR]

Ryanodine receptor 2 (RyR2) is a large Ca 2+ -release channel in the sarcoplasmic reticulum (SR) of cardiac muscle cells. It serves to release Ca 2+ 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 Ca 2+ 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.

Genetic alterations are vital in the progression of thyroid carcinoma. Ryanodine receptor 2 (RyR2) is reported to serve an important role in several types of human carcinoma. However, the expression and effect of RyR2 in thyroid carcinoma remain unknown. Therefore, the present study analyzed the status of RyR2 in thyroid carcinoma using bioinformatics tools. The mRNA profiles of thyroid carcinoma were downloaded from The Cancer Genome Atlas. RyR2 was distinguished as a differentially expressed gene that has not been reported in thyroid carcinoma. Further analysis indicated that there was selective downregulation of RyR2 expression in thyroid carcinoma tissues compared with that in normal thyroid tissues. Survival analysis showed that RyR2 expression was associated with poorer disease-free survival (DFS) for all patients with thyroid carcinoma. Univariate analysis revealed that a low expression of RyR2 was significantly associated with lymphatic metastasis, extracapsular extension, and the Tumor-Node-Metastasis stage. Cox analysis demonstrated that RyR2 was an independent prognostic factor in thyroid carcinoma for DFS. The biological processes and signaling pathways of RyR2 were reviewed with Gene Set Enrichment Analysis. In conclusion, the present study has revealed that RyR2 is downregulated in thyroid carcinoma, and that low expression of RyR2 is associated with poor prognosis in patients with thyroid carcinoma. RyR2 may therefore serve as a promising tumor suppressor gene in thyroid carcinoma. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) is a universal calcium (Ca2+)‐binding messenger that regulates many vital cellular events. In cardiac muscle, CaM associates with ryanodine receptor 2 (RyR2) and regulates excitation–contraction coupling. Mutations in human genes CALM1, CALM2, and CALM3 have been associated with life‐threatening heart disorders, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia. A novel de novo LQTS‐associated missense CaM mutation (E105A) was recently identified in a 6‐year‐old boy, who experienced an aborted first episode of cardiac arrest. Herein, we report the first molecular characterization of the CaM E105A mutation. Expression of the CaM E105A mutant in zebrafish embryos resulted in cardiac arrhythmia and increased heart rate, suggestive of ventricular tachycardia. In vitro biophysical and biochemical analysis revealed that E105A confers a deleterious effect on protein stability and a reduced Ca2+‐binding affinity due to loss of cooperativity. Finally, the CaM E105A mutation resulted in reduced CaM–RyR2 interaction and defective modulation of ryanodine binding. Our findings suggest that the CaM E105A mutation dysregulates normal cardiac function by a complex mechanism involving alterations in both CaM–Ca2+ and CaM–RyR2 interactions. [ABSTRACT FROM AUTHOR]

Purpose: Ryanodine receptor 2 (RYR2) mutation is well-established in the aetiology of an inherited cardiac disorder known as catecholaminergic polymorphic ventricular tachycardia (CPVT). The RYR2 receptor is expressed in cardiomyocytes, and also in the hippocampus. The RYR2 mutation has not been reported as a potential cause of adult-onset genetic generalised epilepsy (GGE).Method: Case report.Results: A 32-year-old right-handed female presented with three unprovoked generalised seizures over twelve years. Electroencephalogram showed epileptiform activity which coincided with normal electrocardiogram recording. Her brother survived a cardiac arrest in his 20's and was diagnosed with CPVT and found to be heterozygous for a novel mutation in the RYR2 gene at chromosome 1q43, c.229 G > A p.(Ala77Thr). The patient inherited the same missense variant, predicted to be damaging by numerous in silico analytic tools. This mutation affects the N-terminal domain of the RYR2 receptor which plays a role in channel activation. However, the patient had repeatedly normal cardiac investigations including normal exercise stress tests.Conclusion: We propose that the RYR2 mutation is a potentially novel neurocardiac calcium channelopathy that may manifest with either CPVT or GGE depending on selective involvement of RYR2 receptors expressed in the heart or in the brain. RYR2 mutant mice have demonstrated spontaneous EEG-positive seizures independent of cardiac arrhythmia. Whole exome sequencing analyses have identified RYR2 as a candidate gene in GGE. This case is a reminder for careful assessment of episodes of transient loss of consciousness in an individual with CPVT, so as to not mistake possible neurogenic seizure for cardiogenic syncope, carrying obvious implications for treatment. [ABSTRACT FROM AUTHOR]

Background/Aims: Spinal cord injury (SCI) is a severe health problem worldwide. Ryanodine receptors (RyRs) are a class of intracellular calcium channels in various excitable tissues such as muscles and nervous tissues. The current study was designed to investigate the possible role of RyR2 upregulation in SCI and to elucidate the possible molecular mechanisms. Methods: Rats were injected with LVshRNAi- RyR2 and then exposed to spinal cord contusion injury. Results: The results showed that knockdown of RyR2 significantly promoted the recovery of structural and functional injury in spinal cord, as evidenced by reduction of lesion volume and increase of Basso, Beattie and Bresnahan (BBB) and combined behavioral score (CBS) scores. Knockdown of RyR2 inhibited the increase of proinflammatory cytokines, including IL-1β and TNFα. Moreover, downregulation of RyR2 increased oxygen consumption rate and decreased the expression of glucose-regulated protein 78 (GRP78), activating transcription factor 3 (ATF3) and ATF6, indicating the improvement of mitochondrial dysfunction and endoplasmic reticulum stress after SCI. Furthermore, silence of RyR2 reduced oxidative stress, as reflected by decrease of TBARS and GSSG content and increase of GSH level. The expression of NADPH oxidase 2 (NOX2), NOX4 and p66shc were increased in SCI rats. Knockdown of RyR2 significantly decreased NOX2 expression, but had no evident effect on NOX4 and p66shc expression. These results indicated NOX2 may be involved in RyR2-induced ROS generation which mediated contusion-induced spinal cord injury. Conclusion: The data provide novel insights into the mechanism of RyR2-mediated injury and the potential therapeutic targets for injury in spinal cord.