Calcium release channel RyR2 regulates insulin release and glucose homeostasis.
ABSTRACT: The type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic reticulum (ER) of several types of cells, including cardiomyocytes and pancreatic ? cells. In cardiomyocytes, RyR2-dependent Ca2+ release is critical for excitation-contraction coupling; however, a functional role for RyR2 in ? cell insulin secretion and diabetes mellitus remains controversial. Here, we took advantage of rare RyR2 mutations that were identified in patients with a genetic form of exercise-induced sudden death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). As these mutations result in a "leaky" RyR2 channel, we exploited them to assess RyR2 channel function in ? cell dynamics. We discovered that CPVT patients with mutant leaky RyR2 present with glucose intolerance, which was heretofore unappreciated. In mice, transgenic expression of CPVT-associated RyR2 resulted in impaired glucose homeostasis, and an in-depth evaluation of pancreatic islets and ? cells from these animals revealed intracellular Ca2+ leak via oxidized and nitrosylated RyR2 channels, activated ER stress response, mitochondrial dysfunction, and decreased fuel-stimulated insulin release. Additionally, we verified the effects of the pharmacological inhibition of intracellular Ca2+ leak in CPVT-associated RyR2-expressing mice, in human islets from diabetic patients, and in an established murine model of type 2 diabetes mellitus. Taken together, our data indicate that RyR2 channels play a crucial role in the regulation of insulin secretion and glucose homeostasis.
Project description:Atrial fibrillation (AF) is the most common cardiac arrhythmia, however the mechanism(s) causing AF remain poorly understood and therapy is suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca2+) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle.In the present study, we sought to determine whether intracellular diastolic SR Ca2+ leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF.We generated 3 knock-in mice with mutations introduced into RyR2 that result in leaky channels and cause exercise induced polymorphic ventricular tachycardia in humans [catecholaminergic polymorphic ventricular tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse models harboring RyR2 mutations to explore the role of diastolic SR Ca2+ leak in AF. AF was stimulated with an intra-esophageal burst pacing protocol in the 3 CPVT mouse models (RyR2-R2474S+/-, 70%; RyR2-N2386I+/-, 60%; RyR2-L433P+/-, 35.71%) but not in wild-type (WT) mice (P<0.05). Consistent with these in vivo results, there was a significant diastolic SR Ca2+ leak in atrial myocytes isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit that stabilizes the closed state of RyR2 and prevents a Ca2+ leak through the channel. Atrial RyR2 from RyR2-R2474S+/- mice were oxidized, and the RyR2 macromolecular complex was depleted of calstabin2. The Rycal drug S107 stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation induced dissociation of calstabin2 from the channel. S107 reduced the diastolic SR Ca2+ leak in atrial myocytes and decreased burst pacing-induced AF in vivo. S107 did not reduce the increased prevalence of burst pacing-induced AF in calstabin2-deficient mice, confirming that calstabin2 is required for the mechanism of action of the drug.The present study demonstrates that RyR2-mediated diastolic SR Ca2+ leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca2+ leak through RyR2 and pacing-induced AF associated with CPVT mutations.
Project description:The Ca2+ release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exercise-induced sudden cardiac death.
Project description:BACKGROUND:Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disorder caused by mutations in the cardiac ryanodine receptor RyR2 that increase diastolic calcium cation (Ca2+) leak from the sarcoplasmic reticulum (SR). Calmodulin (CaM) dissociation from RyR2 has been associated with diastolic Ca2+ leak in heart failure. OBJECTIVE:Determine whether the tetracaine-derivative compound EL20 inhibits abnormal Ca2+ release from RyR2 in a CPVT model and investigate the underlying mechanism of inhibition. METHODS:Spontaneous Ca2+ sparks in cardiomyocytes and inducible ventricular tachycardia were assessed in a CPVT mouse model, which is heterozygous for the R176Q mutation in RyR2 (R176Q/+ mice) in the presence of EL20 or vehicle. Single-channel studies using sheep cardiac SR or purified RyR2 reconstituted into proteoliposomes with and without exogenous CaM were used to assess mechanisms of inhibition. RESULTS:EL20 potently inhibits abnormal Ca2+ release in R176Q/+ myocytes (half-maximal inhibitory concentration = 35.4 nM) and diminishes arrhythmia in R176Q/+ mice. EL20 inhibition of single-channel activity of purified RyR2 occurs in a similar range as seen in R176Q/+ myocytes (half-maximal inhibitory concentration = 8.2 nM). Inhibition of single-channel activity for cardiac SR or purified RyR2 supplemented with 100-nM or 1-?M CaM shows a 200- to 1000-fold reduction in potency. CONCLUSION:This work provides a potential therapeutic mechanism for the development of antiarrhythmic compounds that inhibit leaky RyR2 resulting from CaM dissociation, which is often associated with failing hearts. Our data also suggest that CaM dissociation may contribute to the pathogenesis of arrhythmias with the CPVT-linked R176Q mutation.
Project description:Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor (RyR2). The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under ?-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.
Project description:Glucose-stimulated insulin secretion (GSIS) from pancreatic ?-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]). Glucose uptake into ?-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in ?-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated ?-cells. Conventional PCR assays and immunostaining confirmed that ?-cells express RyR2, the cardiac RyR isoform. Extended incubation of ?-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated ?-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose) to ?-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.
Project description:Increased sarcoplasmic reticulum (SR) Ca2+ leak via the cardiac ryanodine receptor/calcium release channel (RyR2) is thought to play a role in heart failure (HF) progression. Inhibition of this leak is an emerging therapeutic strategy. To explore the role of chronic PKA phosphorylation of RyR2 in HF pathogenesis and treatment, we generated a knockin mouse with aspartic acid replacing serine 2808 (mice are referred to herein as RyR2-S2808D+/+ mice). This mutation mimics constitutive PKA hyperphosphorylation of RyR2, which causes depletion of the stabilizing subunit FKBP12.6 (also known as calstabin2), resulting in leaky RyR2. RyR2-S2808D+/+ mice developed age-dependent cardiomyopathy, elevated RyR2 oxidation and nitrosylation, reduced SR Ca2+ store content, and increased diastolic SR Ca2+ leak. After myocardial infarction, RyR2-S2808D+/+ mice exhibited increased mortality compared with WT littermates. Treatment with S107, a 1,4-benzothiazepine derivative that stabilizes RyR2-calstabin2 interactions, inhibited the RyR2-mediated diastolic SR Ca2+ leak and reduced HF progression in WT and RyR2-S2808D+/+ mice. In contrast, ?-adrenergic receptor blockers improved cardiac function in WT but not in RyR2-S2808D+/+ mice.Thus, chronic PKA hyperphosphorylation of RyR2 results in a diastolic leak that causes cardiac dysfunction. Reversing PKA hyperphosphorylation of RyR2 is an important mechanism underlying the therapeutic action of ?-blocker therapy in HF.
Project description:BACKGROUND:Long-term aerobic exercise alters autonomic balance, which may not be favorable in heart rate (HR)-dependent arrhythmic diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT) because of preexisting bradycardia and increased sensitivity to parasympathetic stimulation. OBJECTIVE:The purpose of this study was to determine whether long-term exercise-induced autonomic adaptations modify CPVT susceptibility. METHODS:We determined exercise-induced parasympathetic effects on HR, arrhythmia incidence, and intracellular sarcoplasmic reticulum (SR) Ca2+ leak in atrial (ACM) and ventricular (VCM) cardiomyocytes, in exercised (EX) calsequestrin knockout (CASQ2-/-) mice, a model of CPVT. RESULTS:Although 8-week treadmill running improved exercise capacity in EX CPVT mice, the incidence and duration of ventricular tachycardia also increased. HR variability analyses revealed an increased high-frequency component of the power spectrum and root mean square of successive differences in R-R intervals indicating accentuated vagal antagonism during ?-adrenergic stimulation resulting in negligible HR acceleration. In EX CASQ2-/- VCM, peak amplitude of Ca2+ transient (CaT) increased, whereas SR Ca2+ content decreased. Aberrant Ca2+ sparks occurred at baseline, which was exacerbated with isoproterenol. Notably, although 10 ?M of the cholinergic agonist carbachol prevented isoproterenol-induced Ca2+ waves in ACM, CaT amplitude, SR Ca2+ load, and isoproterenol-induced Ca2+ waves paradoxically increased in VCM. In parallel, ventricular ryanodine receptor (RyR2) protein expression increased, whereas protein kinase A- and calmodulin-dependent protein kinase II-mediated phosphorylation of RyR2 was not significantly altered, which could imply an increased number of "leaky" channels. CONCLUSION:Our novel results suggest that long-term exercise in CASQ2-/- mice increases susceptibility to ventricular arrhythmias by accentuating vagal antagonism during ?-adrenergic challenge, which prevents HR acceleration and exacerbates abnormal RyR2 Ca2+ leak in EX CASQ2-/- VCM.
Project description:Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release (SOICR) in human embryonic kidney 293 (HEK293) cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype (WT) channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.
Project description:Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca(2+)-handling in pancreatic ? cells. Insulin secretion is triggered by elevation in cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt) of ? cells. This elevation in [Ca(2+)]cyt leads to activation of Ca(2+)/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca(2+)-release channel implicated in Ca(2+)-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic ?-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in ? cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca(2+) leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca(2+)]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca(2+) pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.
Project description:Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT-associated RyR2 mutations cause fatal ventricular arrhythmias in young individuals during ?-adrenergic stimulation.This study sought to determine the effects of a novel RyR2-G230C mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the channel to luminal calcium (Ca(2+)).Functional characterizations of recombinant human RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2 mutant channels were generated by site-directed mutagenesis and heterologously expressed in HEK293 cells together with calstabin2. RyR2 channels were measured to examine the regulation of the channels by cytosolic versus luminal sarcoplasmic reticulum Ca(2+). A 50-year-old white man with repeated syncopal episodes after exercise had a cardiac arrest and harbored the mutation RyR2-G230C. cAMP-dependent protein kinase-phosphorylated RyR2-G230C channels exhibited a significantly higher open probability at diastolic Ca(2+) concentrations, associated with a depletion of calstabin2. The luminal Ca(2+) sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like.The RyR2-G230C mutant exhibits similar biophysical defects compared with previously characterized CPVT mutations: decreased binding of the stabilizing subunit calstabin2 and a leftward shift in the Ca(2+) dependence for activation under conditions that simulate exercise, consistent with a "leaky" channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca(2+) activation. Thus, diastolic sarcoplasmic reticulum Ca(2+) leak caused by reduced calstabin2 binding and a leftward shift in the Ca(2+) dependence for activation by diastolic levels of cytosolic Ca(2+) is a common mechanism underlying CPVT.