RyR2 Serine-2030 PKA Site Governs Ca2+ Release Termination and Ca2+ Alternans.

BACKGROUND: PKA (protein kinase A)-mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. METHODS: We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in human embryonic kidney 293 cells. Here we determined the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation and carried out confocal Ca2+ imaging. RESULTS: We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM-wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM-wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. Moreover, we showed that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM-wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide and the CaMKII inhibitor N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide individually and together, respectively. S2030L abolished the impact of CaM-wild-type, CaM-M, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide-sensitive component, but not the N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide-sensitive component, of isoproterenol.

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

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

Modulation of cardiac ryanodine receptor 2 by calmodulin.

The high-conductance intracellular calcium (Ca2+) channel RyR2 is essential for the coupling of excitation and contraction in cardiac muscle. Among various modulators, calmodulin (CaM) regulates RyR2 in a Ca2+-dependent manner. Here we reveal the regulatory mechanism by which porcine RyR2 is modulated by human CaM through the structural determination of RyR2 under eight conditions. Apo-CaM and Ca2+-CaM bind to distinct but overlapping sites in an elongated cleft formed by the handle, helical and central domains. The shift in CaM-binding sites on RyR2 is controlled by Ca2+ binding to CaM, rather than to RyR2. Ca2+-CaM induces rotations and intradomain shifts of individual central domains, resulting in pore closure of the PCB95 and Ca2+-activated channel. By contrast, the pore of the ATP, caffeine and Ca2+-activated channel remains open in the presence of Ca2+-CaM, which suggests that Ca2+-CaM is one of the many competing modulators of RyR2 gating.

A Gating Mutation in Ryanodine Receptor Type 2 Rescues Phenotypes of Alzheimer's Disease Mouse Models by Upregulating Neuronal Autophagy.

It is well established that ryanodine receptors (RyanRs) are overactive in Alzheimer's disease (AD), and it has been suggested that inhibition of RyanR is potentially beneficial for AD treatment. In the present study, we explored a potential connection between basal RyanR activity and autophagy in neurons. Autophagy plays an important role in clearing damaged organelles and long-lived protein aggregates, and autophagy dysregulation occurs in both AD patients and AD animal models. Autophagy is known to be regulated by intracellular calcium (Ca2+) signals, and our results indicated that basal RyanR2 activity in hippocampal neurons inhibited autophagy through activation of calcineurin and the resulting inhibition of the AMPK (AMP-activated protein kinase)-ULK1 (unc-51-like autophagy-activating kinase 1) pathway. Thus, we hypothesized that increased basal RyanR2 activity in AD may lead to the inhibition of neuronal autophagy and accumulation of ß-amyloid. To test this hypothesis, we took advantage of the RyanR2-E4872Q knock-in mouse model (EQ) in which basal RyanR2 activity is reduced because of shortened channel open time. We discovered that crossing EQ mice with the APPKI and APPPS1 mouse models of AD (both males and females) rescued amyloid accumulation and LTP impairment in these mice. Our results revealed that reduced basal activity of RyanR2-EQ channels disinhibited the autophagic pathway and led to increased amyloid clearance in these models. These data indicated a potential pathogenic outcome of RyanR2 overactivation in AD and also provided additional targets for therapeutic intervention in AD. Basal activity of ryanodine receptors controls neuronal autophagy and contributes to development of the AD phenotype.SIGNIFICANCE STATEMENT It is well established that neuronal autophagy is impaired in Alzheimer's disease (AD). Our results suggest that supranormal calcium (Ca2+) release from endoplasmic reticulum contributes to the inhibition of autophagy in AD and that reduction in basal activity of type 2 ryanodine receptors disinhibits the neuronal autophagic pathway and leads to increased amyloid clearance in AD models. Our findings directly link neuronal Ca2+ dysregulation with autophagy dysfunction in AD and point to additional targets for therapeutic intervention.

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.

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

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

A Clinical Diagnostic Test for Calcium Release Deficiency Syndrome.

Importance: Sudden death and cardiac arrest frequently occur without explanation, even after a thorough clinical evaluation. Calcium release deficiency syndrome (CRDS), a life-threatening genetic arrhythmia syndrome, is undetectable with standard testing and leads to unexplained cardiac arrest. Objective: To explore the cardiac repolarization response on an electrocardiogram after brief tachycardia and a pause as a clinical diagnostic test for CRDS. Design, Setting, and Participants: An international, multicenter, case-control study including individual cases of CRDS, 3 patient control groups (individuals with suspected supraventricular tachycardia; survivors of unexplained cardiac arrest [UCA]; and individuals with genotype-positive catecholaminergic polymorphic ventricular tachycardia [CPVT]), and genetic mouse models (CRDS, wild type, and CPVT were used to define the cellular mechanism) conducted at 10 centers in 7 countries. Patient tracings were recorded between June 2005 and December 2023, and the analyses were performed from April 2023 to December 2023. Intervention: Brief tachycardia and a subsequent pause (either spontaneous or mediated through cardiac pacing). Main Outcomes and Measures: Change in QT interval and change in T-wave amplitude (defined as the difference between their absolute values on the postpause sinus beat and the last beat prior to tachycardia). Results: Among 10 case patients with CRDS, 45 control patients with suspected supraventricular tachycardia, 10 control patients who experienced UCA, and 3 control patients with genotype-positive CPVT, the median change in T-wave amplitude on the postpause sinus beat (after brief ventricular tachycardia at ≥150 beats/min) was higher in patients with CRDS (P < .001). The smallest change in T-wave amplitude was 0.250 mV for a CRDS case patient compared with the largest change in T-wave amplitude of 0.160 mV for a control patient, indicating 100% discrimination. Although the median change in QT interval was longer in CRDS cases (P = .002), an overlap between the cases and controls was present. The genetic mouse models recapitulated the findings observed in humans and suggested the repolarization response was secondary to a pathologically large systolic release of calcium from the sarcoplasmic reticulum. Conclusions and Relevance: There is a unique repolarization response on an electrocardiogram after provocation with brief tachycardia and a subsequent pause in CRDS cases and mouse models, which is absent from the controls. If these findings are confirmed in larger studies, this easy to perform maneuver may serve as an effective clinical diagnostic test for CRDS and become an important part of the evaluation of cardiac arrest.

JNK2, a Newly-Identified SERCA2 Enhancer, Augments an Arrhythmic [Ca2+]SR Leak-Load Relationship.

RATIONALE: We recently discovered pivotal contributions of stress kinase JNK2 (c-Jun N-terminal kinase isoform 2) in increased risk of atrial fibrillation through enhanced diastolic sarcoplasmic reticulum (SR) calcium (Ca2+) leak via RyR2 (ryanodine receptor isoform 2). However, the role of JNK2 in the function of the SERCA2 (SR Ca2+-ATPase), essential in maintaining SR Ca2+ content cycling during each heartbeat, is completely unknown. OBJECTIVE: To test the hypothesis that JNK2 increases SERCA2 activity SR Ca2+ content and exacerbates an arrhythmic SR Ca2+ content leak-load relationship. METHODS AND RESULTS: We used confocal Ca2+ imaging in myocytes and HEK-RyR2 (ryanodine receptor isoform 2-expressing human embryonic kidney 293 cells) cells, biochemistry, dual Ca2+/voltage optical mapping in intact hearts from alcohol-exposed or aged mice (where JNK2 is activated). We found that JNK2, but not JNK1 (c-Jun N-terminal kinase isoform 1), increased SERCA2 uptake and consequently elevated SR Ca2+ content load. JNK2 also associates with and phosphorylates SERCA2 proteins. JNK2 causally enhances SERCA2-ATPase activity via increased maximal rate, without altering Ca2+ affinity. Unlike the CaMKII (Ca2+/calmodulin-dependent kinase II)-dependent JNK2 action in SR Ca2+ leak, JNK2-driven SERCA2 function was CaMKII independent (not prevented by CaMKII inhibition). With CaMKII blocked, the JNK2-driven SR Ca2+ loading alone did not significantly raise leak. However, with JNK2-CaMKII-driven SR Ca2+ leak present, the JNK2-enhanced SR Ca2+ uptake limited leak-induced reduction in SR Ca2+, normalizing Ca2+ transient amplitude, but at a higher arrhythmogenic SR Ca2+ leak. JNK2-specific inhibition completely normalized SR Ca2+ handling, attenuated arrhythmic Ca2+ activities, and alleviated atrial fibrillation susceptibility in aged and alcohol-exposed myocytes and intact hearts. CONCLUSIONS: We have identified a novel JNK2-induced activation of SERCA2. The dual action of JNK2 in CaMKII-dependent arrhythmic SR Ca2+ leak and a CaMKII-independent uptake exacerbates atrial arrhythmogenicity, while helping to maintain normal levels of Ca2+ transients and heart function. JNK2 modulation may be a novel therapeutic target for atrial fibrillation prevention and treatment.

Ca2+-CaM Dependent Inactivation of RyR2 Underlies Ca2+ Alternans in Intact Heart.

RATIONALE: Ca2+ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca2+ alternans remains undefined. Increasing evidence suggests that Ca2+ alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown. OBJECTIVE: To determine the role of CaM (calmodulin) on Ca2+ alternans in intact working mouse hearts. METHODS AND RESULTS: We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type, a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 wild type or mutant mouse hearts. We monitored Ca2+ transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca2+ imaging. We found that CaM-wild type and CaM-M37Q promoted Ca2+ alternans and prolonged Ca2+ transient recovery in intact RyR2 wild type and mutant hearts, whereas CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca2+ current but had no significant impact on sarcoplasmic reticulum Ca2+ content. Furthermore, we developed a novel numerical myocyte model of Ca2+ alternans that incorporates Ca2+-CaM-dependent regulation of RyR2 and the L-type Ca2+ channel. Remarkably, the new model recapitulates the impact on Ca2+ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca2+ elevation as a result of rapid pacing triggers Ca2+-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca2+ release, which, in turn, reduces diastolic cytosolic Ca2+, leading to alternations in diastolic cytosolic Ca2+, RyR2 inactivation, and sarcoplasmic reticulum Ca2+ release (ie, Ca2+ alternans). CONCLUSIONS: Our results demonstrate that inactivation of RyR2 by Ca2+-CaM is a major determinant of Ca2+ alternans, making Ca2+-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.

RYR2-ryanodinopathies: from calcium overload to calcium deficiency.

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

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation.

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+- and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.

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.

The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability.

Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.

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.

Two pools of IRE1α in cardiac and skeletal muscle cells.

The endoplasmic reticulum (ER) plays a central role in cellular stress responses via mobilization of ER stress coping responses, such as the unfolded protein response (UPR). The inositol-requiring 1α (IRE1α) is an ER stress sensor and component of the UPR. Muscle cells also have a well-developed and highly subspecialized membrane network of smooth ER called the sarcoplasmic reticulum (SR) surrounding myofibrils and specialized for Ca2+ storage, release, and uptake to control muscle excitation-contraction coupling. Here, we describe 2 distinct pools of IRE1α in cardiac and skeletal muscle cells, one localized at the perinuclear ER and the other at the junctional SR. We discovered that, at the junctional SR, calsequestrin binds to the ER luminal domain of IRE1α, inhibiting its dimerization. This novel interaction of IRE1α with calsequestrin, one of the highly abundant Ca2+ handling proteins at the junctional SR, provides new insights into the regulation of stress coping responses in muscle cells.-Wang, Q., Groenendyk, J., Paskevicius, T., Qin, W., Kor, K. C., Liu, Y., Hiess, F., Knollmann, B. C., Chen, S. R. W., Tang, J., Chen, X.-Z., Agellon, L. B., Michalak, M. Two pools of IRE1α in cardiac and skeletal muscle cells.

Mitochondrial fusion dynamics is robust in the heart and depends on calcium oscillations and contractile activity.

Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardiomyocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24-48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2-mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.

The cardiac ryanodine receptor, but not sarcoplasmic reticulum Ca2+-ATPase, is a major determinant of Ca2+ alternans in intact mouse hearts.

Sarcoplasmic reticulum (SR) Ca2+ cycling is governed by the cardiac ryanodine receptor (RyR2) and SR Ca2+-ATPase (SERCA2a). Abnormal SR Ca2+ cycling is thought to be the primary cause of Ca2+ alternans that can elicit ventricular arrhythmias and sudden cardiac arrest. Although alterations in either RyR2 or SERCA2a function are expected to affect SR Ca2+ cycling, whether and to what extent altered RyR2 or SERCA2a function affects Ca2+ alternans is unclear. Here, we employed a gain-of-function RyR2 variant (R4496C) and the phospholamban-knockout (PLB-KO) mouse model to assess the effect of genetically enhanced RyR2 or SERCA2a function on Ca2+ alternans. Confocal Ca2+ imaging revealed that RyR2-R4496C shortened SR Ca2+ release refractoriness and markedly suppressed rapid pacing-induced Ca2+ alternans. Interestingly, despite enhancing RyR2 function, intact RyR2-R4496C hearts exhibited no detectable spontaneous SR Ca2+ release events during pacing. Unlike for RyR2, enhancing SERCA2a function by ablating PLB exerted a relatively minor effect on Ca2+ alternans in intact hearts expressing RyR2 WT or a loss-of-function RyR2 variant, E4872Q, that promotes Ca2+ alternans. Furthermore, partial SERCA2a inhibition with 3 µm 2,5-di-tert-butylhydroquinone (tBHQ) also had little impact on Ca2+ alternans, whereas strong SERCA2a inhibition with 10 µm tBHQ markedly reduced the amplitude of Ca2+ transients and suppressed Ca2+ alternans in intact hearts. Our results demonstrate that enhanced RyR2 function suppresses Ca2+ alternans in the absence of spontaneous Ca2+ release and that RyR2, but not SERCA2a, is a key determinant of Ca2+ alternans in intact working hearts, making RyR2 an important therapeutic target for cardiac alternans.

Dynamic and Irregular Distribution of RyR2 Clusters in the Periphery of Live Ventricular Myocytes.

Cardiac ryanodine receptors (RyR2s) are Ca2+ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca2+ release. The distribution of these Ca2+ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca2+ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca2+ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca2+- and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca2+ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior.

Pathogenic mechanism of a catecholaminergic polymorphic ventricular tachycardia causing-mutation in cardiac calcium release channel RyR2.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition that is characterized by an abnormal heart rhythm in response to physical or emotional stress. The majority CPVT patients carry mutations in the RYR2 gene that encodes the calcium release channel/ryanodine receptor (RyR2) in cardiomyocytes. The pathogenic mechanisms that account for the clinical phenotypes of CPVT are still elusive. We have identified a de novo mutation, A165D, from a CPVT patient. We found that CPVT phenotypes are recapitulated in A165D knock-in mice. The mutant RyR2 channels enhanced sarcoplasmic reticulum Ca2+ release, triggered delayed afterdepolarization in cardiomyocytes. Structural analysis revealed that the A165D mutation is located in a loop that is involved in inter-subunit interactions in the RyR2 tetrameric structure, it disrupted conformational stability of the RyR2, which favored a closed-to-open state transition, resulting in a leaky channel. The loop also harbors several other CPVT mutations, which suggests a common pathogenic molecular mechanism of CPVT-causing mutations. Our data illustrated disease-relevant functional defects and provide a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.

The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor.

A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+ These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction.