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Rationale: 3′,5′-cAMP is a ubiquitous second messenger which, upon β-AR (β-adrenergic receptor) stimulation, acts in microdomains to regulate cardiac excitation-contraction coupling by activating phosphorylation of calcium handling proteins. One crucial microdomain is in vicinity of the cardiac RyR2 (ryanodine receptor type 2) which is associated with arrhythmogenic diastolic calcium leak from the sarcoplasmic reticulum often occurring in heart failure. Objective: We sought to establish a real-time live-cell imaging approach capable of directly visualizing cAMP in the vicinity of mouse and human RyR2 and to analyze its pathological changes in failing cardiomyocytes under β-AR stimulation. Methods and Results: We generated a novel targeted fluorescent biosensor Epac1 (exchange protein directly activated by cAMP 1)-JNC (junctin) for RyR2-associated cAMP and expressed it in transgenic mouse hearts as well in human ventricular myocytes using adenoviral gene transfer. In healthy cardiomyocytes, β 1 -AR but not β 2 -AR stimulation strongly increased local RyR2-associated cAMP levels. However, already in cardiac hypertrophy induced by aortic banding, there was a marked subcellular redistribution of PDEs (phosphodiesterases) 2, 3, and 4, which included a dramatic loss of the local pool of PDE4. This was also accompanied by measurable β 2 -AR/AMP signals in the vicinity of RyR2 in failing mouse and human myocytes, increased β 2 -AR–dependent RyR2 phosphorylation, sarcoplasmic reticulum calcium leak, and arrhythmia susceptibility. Conclusions: Our new imaging approach could visualize cAMP levels in the direct vicinity of cardiac RyR2. Unexpectedly, in mouse and human failing myocytes, it could uncover functionally relevant local arrhythmogenic β 2 -AR/cAMP signals which might be an interesting antiarrhythmic target for heart failure.

Pulmonary hypertension is a complex disorder characterized by pulmonary vascular remodeling and right ventricular hypertrophy, leading to right heart failure. The mechanisms underlying this process are not well understood. We hypothesize that the structural remodeling occurring in the cardiomyocytes of the right ventricle affects the cytosolic Ca 2+ handling leading to arrhythmias. After 12 days of monocrotaline-induced pulmonary hypertension in rats, epicardial mapping showed electrical remodeling in both ventricles. In myocytes isolated from the hypertensive rats, a combination of high-speed camera and confocal line-scan documented a prolongation of Ca 2+ transients along with a higher local Ca 2+ -release activity. These Ca 2+ transients were less synchronous than in controls, likely due to disorganized transverse-axial tubular system. In fact, following pulmonary hypertension, hypertrophied right ventricular myocytes showed significantly reduced number of transverse tubules and increased number of axial tubules; however, Stimulation Emission Depletion microscopy demonstrated that the colocalization of L-type Ca 2+ channels and RyR2 (ryanodine receptor 2) remained unchanged. Finally, Stimulation Emission Depletion microscopy and super-resolution scanning patch-clamp analysis uncovered a decrease in the density of active L-type Ca 2+ channels in right ventricular myocytes with an elevated open probability of the T-tubule anchored channels. This may represent a general mechanism of how nanoscale structural changes at the early stage of pulmonary hypertension impact on the development of the end stage failing phenotype in the right ventricle.

Right ventricular (RV) failure is a major cause of death in patients with pulmonary arterial hypertension, and the mechanism of RV failure remains unclear. While the malfunction of RyR2 (ryanodine receptor type 2) on sarcoplasmic reticulum (SR) and aberrant Ca 2+ cycling in cardiomyocytes have been recognized in some cardiovascular diseases, their roles in RV failure secondary to pulmonary arterial hypertension require further investigation. In a monocrotaline-induced rat model of pulmonary arterial hypertension, the RV remodeling process was divided into normal, compensated, and decompensated stages according to the hemodynamic and morphological parameters. In both compensated and decompensated stages, significant diastolic SR Ca 2+ leakage was detected along with reduced intracellular Ca 2+ transient amplitude and SR Ca 2+ contents in RV myocytes. RyR2 protein levels decreased progressively during the process, and the thiol oxidation proportions of RyR2 were higher in compensated and decompensated stages than in normal stage. Inhibition of RyR2 oxidation by dithiothreitol or repairing RyR2 directly by dantrolene could restore Ca 2+ homeostasis in RV myocytes. Daily intraperitoneal injection of dantrolene delayed decompensation progression and significantly improved the survival rate of pulmonary hypertension rats in decompensated stage (79.3% versus 55.9%; P =0.026). Our findings suggest that diastolic SR Ca 2+ leakage via oxidized RyR2 facilitates the development of RV failure. Dantrolene can inhibit diastolic SR Ca 2+ leakage in RV cardiomyocytes, delay right cardiac dysfunction, and improve the survival of rats with pulmonary arterial hypertension.

Hypoxia during pregnancy profoundly affects uterine vascular adaptation and increases the risk of pregnancy complications, including preeclampsia and fetal intrauterine growth restriction. We recently demonstrated that increases in Ca 2+ sparks and spontaneous transient outward currents (STOCs) played an essential role in pregnancy-induced uterine vascular adaptation. In the present study, we hypothesize that gestational hypoxia suppresses Ca 2+ sparks/STOCs coupling leading to increased uterine vascular tone via enhanced endoplasmic reticulum (ER)/oxidative stress. Uterine arteries were obtained from nonpregnant and near-term pregnant sheep residing in low altitude or acclimatizing to high-altitude (3801 m) hypoxia for ≈110 days. High-altitude hypoxia suppressed pregnancy-induced upregulation of RyR1 and RyR2 (ryanodine receptor 1 and 2) protein abundance, Ca 2+ sparks, and STOCs in uterine arteries. Inhibition of Ca 2+ sparks/STOCs with the RyR inhibitor ryanodine significantly increased pressure-dependent myogenic tone in uterine arteries from low-altitude normoxic pregnant animals but not those from high-altitude hypoxic pregnant animals. Gestational hypoxia significantly increased ER/oxidative stress in uterine arteries. Of importance, the hypoxia-mediated suppression of Ca 2+ sparks/STOCs and increase in myogenic tone in uterine arteries of pregnant animals were reversed by inhibiting ER/oxidative stress. Of great interest, the impaired sex hormonal regulation of STOCs in high-altitude animals was annulled by scavenging reactive oxygen species but not by inhibiting ER stress. Together, the findings reveal the differential mechanisms of ER and oxidative stresses in suppressing Ca 2+ sparks/STOCs and increasing myogenic tone of uterine arteries in hypoxia during gestation, providing new insights into the understanding of pregnancy complications associated with hypoxia.

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary heart disease characterized by fatty infiltration, life-threatening arrhythmias, and increased risk of sudden cardiac death. The guideline for management of ARVC in patients is to improve quality of life by reducing arrhythmic symptoms and to prevent sudden cardiac death. However, the mechanism underlying ARVC-associated cardiac arrhythmias remains poorly understood. Methods: Using protein mass spectrometry analyses, we identified that integrin β1 is downregulated in ARVC hearts without changes to Ca 2+ -handling proteins. As adult cardiomyocytes express only the β1D isoform, we generated a cardiac specific β1D knockout mouse model and performed functional imaging and biochemical analyses to determine the consequences of integrin β1D loss on function in the heart in vivo and in vitro. Results: Integrin β1D deficiency and RyR2 Ser-2030 hyperphosphorylation were detected by Western blotting in left ventricular tissues from patients with ARVC but not in patients with ischemic or hypertrophic cardiomyopathy. Using lipid bilayer patch clamp single channel recordings, we found that purified integrin β1D protein could stabilize RyR2 function by decreasing RyR2 open probability, mean open time, and increasing mean close time. Also, β1D knockout mice exhibited normal cardiac function and morphology but presented with catecholamine-sensitive polymorphic ventricular tachycardia, consistent with increased RyR2 Ser-2030 phosphorylation and aberrant Ca 2+ handling in β1D knockout cardiomyocytes. Mechanistically, we revealed that loss of DSP (desmoplakin) induces integrin β1D deficiency in ARVC mediated through an ERK1/2 (extracellular signal–regulated kinase 1 and 2)–fibronectin–ubiquitin/lysosome pathway. Conclusions: Our data suggest that integrin β1D deficiency represents a novel mechanism underlying the increased risk of ventricular arrhythmias in patients with ARVC.

Objective This study is to evaluate the effects of the Adenoviral βARKct (Adv. βARKct) myocardial gene transfection following cardioplegic arrest on cardiopulmonary bypass (CPB) in a swine model. Methods Swine models of cardioplegic arrest on CPB were established after 5 days of myocardial injection of Adv. βARKct or Adv. luciferase. The pigs were randomized into Adv. βARKct, Control, and Sham groups. Invasive hemodynamics, cardiac function, biomarkers, and tissue morphology were assessed. Results Baseline data were similar among these groups. Hemodynamics and cardiac function showed a deteriorating trend throughout 6 h after weaning in βARKct and Control groups. Compared with Control group, Adv. βARKct treatment significantly elevated global and regional ventricular function (cardiac output, dp/dtmax, Ejection fraction, peak systolic longitudinal strain, and peak systolic strain rate) and altered hemodynamics (cardiac cycle efficiency and systemic vascular resistance). Moreover, inotropic score in βARKct group was gradually decreased to 5.0 ± 1.1, compared with Control group (6.2 ± 0.9), at 6 h after weaning. Biomarkers in βARKct group were significantly better than in Control group. Meanwhile, βARKct treatment reduced the histopathologic injuries, rescued β1-AR, SERCA2a, and RyR2 levels, and decreased the GRK2 levels in myocardial cells. Conclusion Adv.βARKct inhibits GRK2 and ameliorates myocardial injuries following cardioplegic arrest on CPB, via stabilizing β1-AR, reducing mitochondrial damages and restoring sarcoplasmic reticulum Ca-handling protein expression.

A central point in heart research is to understand the mechanisms behind Ca2+ handling and to find alternatives to correct defective Ca2+ cycling associated with heart failure and arrhythmias. In cardiac myocytes, entry of Ca2+ occurs through L-type Ca2+ channels and mediates the opening of RyR2 (ryanodine receptor 2) channels allowing systolic movement of Ca2+ from the sarcoplasmic reticulum (SR) to the cytosol. In addition, SR Ca2+ release occurs randomly due to RyR2 opening in response to a combination of factors that favor (luminal and cytosolic Ca2+, ATP, posttranslational modifications; gain-of-function mutations or drugs) or preclude it (Mg2+, FKBPs, loss-of-function mutations or drugs). Due to its dependence on luminal SR Ca2+, the concept of SOICR (store overload-induced SR Ca2+ release) has emerged, and the capability of these events to generate delayed afterdepolarizations and arrhythmias is widely accepted. Thus, understanding the mechanisms that regulate SR Ca2+ release and its capacity to promote SOICR is of critical importance. Fil: Gonano, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina Fil: Vila Petroff, Martin Gerarde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina

Cardiomyocyte (CM) maturation is an essential step in heart development that prepares the organ for robust and sustainable contractions. However, very little is known about the contribution of CM maturation to cardiac pathogenesis, partly due to the paucity of basic knowledge about maturation. For example, although calcium plays well-established roles in cardiac stress responses, whether normal calcium oscillations are required for CM maturation has not been examined. To address this question, we created mice with mosaic inactivation of ryanodine receptor 2 (RYR2), the central channel that mediates calcium release from sarcoplasmic/endoplasmic reticulum (SR/ER), in CMs. RYR2 was depleted in a small fraction of neonatal CMs by adeno-associated virus (AAV)-mediated Cas9-based somatic mutagenesis (CASAAV). The majority of CMs intentionally retained RYR2 expression, which circumvented cardiac dysfunction and death that would result from widespread calcium transient ablation. In RYR2-depleted CMs, sarcomere expansion, transverse-tubule formation, mitochondrial metabolism and maturational hypertrophy were impaired. Strikingly, these maturation defects were associated with unfolded protein response, with the activation of three major ER stress effectors: spliced Xbp1 (XBP1s), ATF6 N-terminus (ATF6N), and ATF4. Tauroursodeoxycholic acid, an ER stress-relieving drug, partially rescued the maturation defects in RYR2-depleted CMs. Over expression (OE) of XBP1s, ATF6N or ATF4 each impaired CM maturation, with phenotypic severity ranked as ATF4>XBP1s>ATF6N. ATF4 OE in neonatal CMs triggered both ultrastructural and transcriptomic changes highly correlated with RYR2 depletion phenotypes. Interestingly, ATF4 OE altered distinct transcriptomic programs in neonatal vs. adult CMs and caused ultrastructural defects only at the neonatal stage. No terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was detected upon RYR2 depletion or ATF4 OE, excluding the contribution of apoptosis to maturation defects. Collectively, these data indicate that RYR2-mediated calcium handling prevents an adverse and stage-specific impact of ER stress on CM maturation, which lead to CM dysfunction.

A 13-year-old girl had a witnessed loss of consciousness after a scuffle with another student at school and was found in ventricular fibrillation at the time of arrival of emergency medical services personnel. The patient was successfully defibrillated in the field and was transported to the emergency department as a presumed "traumatic arrest". The patient's initial electrocardiogram was remarkable for a prolonged QT interval, and it was discovered that multiple family members had died of cardiac events as young adults. Genetic testing subsequently revealed a mutation in the RYR2 gene, which is implicated in catecholaminergic polymorphic ventricular tachycardia.

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

Rationale: Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM, including lack of T-tubules, immature excitation–contraction coupling, and inefficient Ca-induced Ca release remain major limitations. Objective: Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote T-tubule development and mature excitation–contraction coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). Methods and Results: hiPSC-CM were generated using a standard chemical differentiation method supplemented with T3 (triiodothyronine) and/or Dex (dexamethasone) during days 16 to 30 followed by single-cell culture for 5 days on Matrigel mattress. hiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive T-tubule network. Notably, Matrigel mattress was necessary for T-tubule formation. Compared with adult human ventricular cardiomyocytes, T-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of excitation–contraction coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks and promoted RyR2 (ryanodine receptor) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex-treated cells. Conclusions: Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process, which when coupled with further maturation on Matrigel mattress, is sufficient for T-tubule development, enhanced Ca-induced Ca release, and more ventricular-like excitation–contraction coupling. This new hormone maturation method could advance the use of hiPSC-CM for disease modeling and cell-based therapy.

Rationale: Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Previous studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. Objective: To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate transverse tubules (T-tubules), a hallmark of mature CMs. Methods and Results: We developed CRISPR/Cas9/AAV9-based somatic mutagenesis, a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin-T promoter–driven Cre to Rosa Cas9GFP/Cas9GFP neonatal mice. When directed against junctophilin-2 ( Jph2 ), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both adeno-associated virus–transduced and nontransduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure and that JPH2 protects T-tubules in this context. We then used CRISPR/Cas9/AAV9-based somatic mutagenesis to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified RYR2 (Ryanodine Receptor-2) as a novel, cell-autonomously required T-tubule maturation factor. Conclusions: CRISPR/Cas9/AAV9-based somatic mutagenesis is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.

Article, see p 1595 Arrhythmogenic cardiomyopathy (AC) is a genetic disorder characterized by high risk of life-threatening ventricular arrhythmias, sudden cardiac death, and progressive heart failure. Currently, there is evidence that AC includes a spectrum of cardiomyopathy phenotypes, ranging from the classical form of arrhythmogenic right ventricular cardiomyopathy (ARVC) to more recently identified forms of arrhythmogenic left ventricular cardiomyopathy.1,2 ARVC is considered a disease of the desmosome, because in the majority of cases, it is caused by mutations in genes encoding proteins of the cardiac desmosomes.3 However, mutations in nondesmosomal genes have also been found in ARVC, such as the genes encoding the cardiac ryanodine receptor 2 ( RYR2 ), the transforming growth factor β-3 ( TGFB3 ), the nuclear transmembrane protein 43 ( TMEM43 ), and desmin ( DES ). Mutations in LMNA , encoding the nuclear envelope protein lamin A/C, known to cause dilated cardiomyopathy with an arrhythmic phenotype, was reported to cause also an ARVC phenotype,3 even though the association between LMNA and ARVC is still questioned. In the left dominant form, which clinically presents as an arrhythmogenic dilated cardiomyopathy, the same desmosomal genes causing ARVC can be found,1 as well as LMNA and the more recently identified filamin C gene ( FLNC ), encoding an actin …

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that leads to sudden cardiac death in children and young adults. The most common form of CPVT is caused by autosomal-dominant mutations in the gene encoding cardiac ryanodine receptor type-2 ( RYR2) . Mutations in RYR2 promote excessive calcium (Ca 2+ ) leak from the sarcoplasmic reticulum (SR), triggering potentially lethal arrhythmias. Recently, we demonstrated that the tetracaine derivative EL20 specifically inhibits RYR2, normalizes Ca 2+ handling, and suppresses arrhythmias in a CPVT mouse model. We hypothesized that EL20 will also normalize Ca 2+ handling and arrhythmias in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs). Materials/Methods: Blood samples from a child carrying RYR2 mutation R176Q/+ (RQ) and a mutation-negative relative (WT) were collected, processed into peripheral blood mononuclear cells (pBMCs), and reprogrammed into iPSCs using Sendai virus system. Functional iPSC-CMs were derived using Stemdiff TM kit. Differentiation of CMs was validated using qPCR and immunofluorescent staining. Confocal Ca 2+ imaging was used to monitor RyR2 activity. Axiom Biosystem TM microelectrode arrays (MEA) was used to assess arrhythmogenic action potential (AP) and rates. Results: Successfully differentiated CMs showed increased levels of Ca 2+ -handling markers and decreased levels of stem cell markers. Baseline Ca 2+ imaging revealed a 4-fold increase in calcium spark frequency (CaSpF) in RQ iPSC-CMs (3.08±0.58 a.u., n=12) vs. WT (0.69±0.22 a.u., n=13, p Conclusion: Our results demonstrate that EL20 normalizes Ca 2+ handling and AP beat rate frequency in patient-derived RYR2-R176Q/+ iPSC-CMs with no observed off-target effects, positioning this novel RyR2-inhibitor as a promising therapeutic candidate for treatment of CPVT.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in the cardiac type 2 ryanodine receptor (RyR2). Using knock-in mouse (KI) model (R2474S/+), we previously reported that a single point mutation within the RyR2 sensitizes the channel to agonists, primarily mediated by defective inter-domain interaction within the RyR2 molecule and subsequent dissociation of calmodulin (CaM) from the RyR2. Here, we examined whether CPVT can be genetically rescued by enhancing the binding affinity of CaM to the RyR2. We first determined whether there is a possible amino-acid substitution at the CaM-binding domain in the RyR2 (3584-3603) that can enhancethe binding affinity of CaM, and found that V3599K substitution showed the highest binding affinity of CaM to CaM-binding domain. Hence, we generated a heterozygous KI mouse model (V3599K/+) with a single amino acid substitution in the CaM-binding domain of the RyR2, and crossbred it with the heterozygous CPVT -associated R2474S/+ KI mouse to obtain a double heterozygous R2474S/V3599K KI mouse. The CPVT phenotypes—bidirectional or polymorphic ventricular tachycardia, spontaneous Ca2+ transients, and Ca2+ sparks—were all inhibited in the R2474S/V3599K mice. Thus, enhancement of the CaM binding affinity of the RyR2 is essential to prevent CPVT-associated arrhythmogenesis.

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. A knock-in mouse model that expresses an A165D point mutation was generated using CRISPR/Cas9 technology. Echocardiogram, electrocardiography, and histological analysis were performed on mutant mice. We found that ventricular tachycardia was readily induced by exercise and pharmacological challenges. Functional properties of the mutant RyR2 channel were evaluated in isolated cardiomyocytes. The mutant RyR2 channels enhanced SR Ca 2+ release, triggered delayed afterdepolarization in cardiomyocytes. These recapitulated CPVT phenotypes in both animal models and cardiomyocytes indicated that RyR2 A165D is a CPVT-causing mutation. Furthermore, we pinpointed the 3D localization of the mutation site in the structures determined by cryo-electron microscopy. It revealed that the A165D mutation change the electric charge of the residue, disrupted conformational stability of the RyR2 tetramer structure, and promoted a closed-to-open state transition. Moreover, the location of the A165D mutation is in a loop that harbors several other mutations, and, like A165D, most these mutations result in changes in the electrostatic charge of the side chains of the residues. This demonstrated a common molecular mechanism of CPVT-causing RyR2 mutations. Our data illustrated disease-relevant functional defects and provide a deeper mechanistic understanding of a life-threatening cardiac arrhythmia.

We would like to congratulate Dr Kapplinger et al1 for their successful research article about genetic test interpretations implications in the study of RYR2 (ryanodine receptor 2) in suspected catecholaminergic polymorphic ventricular tachycardia (CPVT) and comment on some important aspects we find of interest. The authors conclude that 13 new exons, added to the 8 previously reported in literature, were found to be spots of pathogenic variants, and they suggest that this information could be used together with clinical workup to mitigate the interpretive ambiguity of …

See Article by Kapplinger and Pundi et al All inherited cardiac channelopathies have their diagnostic hallmark. In the case of catecholaminergic polymorphic ventricular tachycardia (CPVT), this is bidirectional ventricular tachycardia during exercise. If all patients with clinical suspicion of CPVT had this diagnostic feature during an exercise stress test in the setting of a structurally normal heart and normal resting ECG, a CPVT clinical diagnosis could easily be established. Unfortunately, in everyday clinical practice, the next new patient coming to medical attention is unlikely to be a textbook case. And this is where genetics come in. The genetic revolution of the mid-1990s with the first breakthrough discoveries of the genetic substrate underlying inherited arrhythmia syndromes seemed to promise a land where the void created by reduced penetrance and variable expressivity would be filled by a firm genetic diagnosis. Largely, this is indeed what happened and genetics have truly had a major impact on clinical management,1 but from this to a genetic panacea, it is a long way. A crucial element that was missing in the early days of the genetic revolution was human genome variability. Early scientific reports used 50 to 100 controls to cross-check any genetic finding before assigning it pathogenicity. Who would dare say that now, in the era of the 1000 Genomes Project and of the Exome (ExAC) and Genome Aggregation Consortiums (gnomAD)?2 By now, it is in fact well appreciated that rare genetic variation is paradoxically a rather common finding and that this also holds true for the main CPVT-causative gene, the ryanodine receptor 2 ( RYR2 ).3,4 Just as the next new patient is probably not a textbook case, so the next rare or even novel RYR2 variant identified on genetic testing is not a certainly pathogenic variant either. Unfortunately, this …

Background Hypoxia during pregnancy could cause abnormal development and lead to increased risks of vascular diseases in adults. This study determined angiotensin II ( AII )‐mediated vascular dysfunction in offspring middle cerebral arteries ( MCA ). Methods and Results Pregnant rats were subjected to hypoxia. Vascular tension in offspring MCA by AII with or without inhibitors, calcium channel activities, and endoplasmic reticulum calcium stores were tested. Whole‐cell patch clamping was used to investigate voltage‐dependent calcium channel currents. mRNA expression was tested using quantitative real‐time polymerase chain reaction. AII ‐mediated MCA constriction was greater in male offspring exposed to prenatal hypoxia. AT 1 and AT 2 receptors were involved in the altered AII ‐mediated vasoconstriction. Prenatal hypoxia increased baseline activities of L‐type calcium channel currents in MCA smooth muscle cells. However, calcium currents stimulated by AII were not significantly changed, whereas nifedipine inhibited AII ‐mediated vasoconstrictions in the MCA . Activities of IP 3 /ryanodine receptor–operated calcium channels, endoplasmic reticulum calcium stores, and sarcoendoplasmic reticulum membrane Ca 2+ ‐ ATP ase were increased. Prenatal hypoxia also caused dysfunction of vasodilatation via the endothelium NO synthase. The mRNA expressions of AT 1A, AT 1B, AT 2R, Cav1.2α1C, Cav3.2α1H, and ryanodine receptor RyR2 were increased in the prenatal‐hypoxia group. Conclusions Hypoxia in pregnancy could induce dysfunction in both contraction and dilation in the offspring MCA . AII ‐increased constriction in the prenatal‐hypoxia group was not mainly dependent on the L‐type and T‐type calcium channels; it might predominantly rely on the AII receptors, IP 3 /ryanodine receptors, and the endoplasmic reticulum calcium store as well as calcium ATP ase.

Background— The rapid expansion of genetic testing has led to increased utilization of clinical whole-exome sequencing (WES). Clinicians and genetic researchers are being faced with assessing risk of disease vulnerability from incidentally identified genetic variants which is typified by variants found in genes associated with sudden death-predisposing catecholaminergic polymorphic ventricular tachycardia (CPVT). We sought to determine whether incidentally identified variants in genes associated with CPVT from WES clinical testing represent disease-associated biomarkers. Methods and Results— CPVT-associated genes RYR2 and CASQ2 variants were identified in one of the world’s largest collections of clinical WES referral tests (N=6517, Baylor Miraca Genetics Laboratories) and compared with a control cohort of ostensibly healthy individuals (N=60 706) and a case cohort of CPVT cases (N=155). Within the WES cohort, the rate of rare variants in CPVT-associated genes was 8.8% compared with 6.0% among controls and 60.0% among cases. There was a predominance of variants of undetermined significance (97.7%). After protein topology mapping, WES variants colocalized more frequently to residues with variants found in controls compared with cases. Retrospective clinical evaluation of individuals referred to our institution with WES-positive variants demonstrated no evidence of clinical CPVT in individuals with a low pretest clinical suspicion for CPVT. Conclusions— The prevalence of incidentally identified CPVT-associated variants is ≈9% among WES tests. Variants of undetermined significances in CPVT-associated genes in WES genetic testing, in the absence of clinical suspicion for CPVT, are unlikely to represent markers of CPVT pathogenicity.

Background— Chromogranin A (CgA) levels have previously been found to predict mortality in heart failure (HF), but currently no information is available regarding CgA processing in HF and whether the CgA fragment catestatin (CST) may directly influence cardiomyocyte function. Methods and Results— CgA processing was characterized in postinfarction HF mice and in patients with acute HF, and the functional role of CST was explored in experimental models. Myocardial biopsies from HF, but not sham-operated mice, demonstrated high molecular weight CgA bands. Deglycosylation treatment attenuated high molecular weight bands, induced a mobility shift, and increased shorter CgA fragments. Adjusting for established risk indices and biomarkers, circulating CgA levels were found to be associated with mortality in patients with acute HF, but not in patients with acute exacerbation of chronic obstructive pulmonary disease. Low CgA-to-CST conversion was also associated with increased mortality in acute HF, thus, supporting functional relevance of impaired CgA processing in cardiovascular disease. CST was identified as a direct inhibitor of CaMKIIδ (Ca 2+ /calmodulin-dependent protein kinase IIδ) activity, and CST reduced CaMKIIδ-dependent phosphorylation of phospholamban and the ryanodine receptor 2. In line with CaMKIIδ inhibition, CST reduced Ca 2+ spark and wave frequency, reduced Ca 2+ spark dimensions, increased sarcoplasmic reticulum Ca 2+ content, and augmented the magnitude and kinetics of cardiomyocyte Ca 2+ transients and contractions. Conclusions— CgA-to-CST conversion in HF is impaired because of hyperglycosylation, which is associated with clinical outcomes in acute HF. The mechanism for increased mortality may be dysregulated cardiomyocyte Ca 2+ handling because of reduced CaMKIIδ inhibition.

Background— We hypothesized that S100A1 is regulated during human hypertrophy and heart failure and that it may be implicated in remodeling after left ventricular assist device. S100A1 is decreased in animal and human heart failure, and restoration produces functional recovery in animal models and in failing human myocytes. With the potential for gene therapy, it is important to carefully explore human cardiac S100A1 regulation and its role in remodeling. Methods and Results— We measured S100A1, the sarcoplasmic endoplasmic reticulum Ca 2+ ATPase, phospholamban, and ryanodine receptor proteins, as well as β-adrenergic receptor density in nonfailing, hypertrophied (left ventricular hypertrophy), failing, and failing left ventricular assist device–supported hearts. We determined functional consequences of protein alterations in isolated contracting muscles from the same hearts. S100A1, sarcoplasmic endoplasmic reticulum Ca 2+ ATPase and phospholamban were normal in left ventricular hypertrophy, but decreased in failing hearts, while ryanodine receptor was unchanged in either group. Baseline muscle contraction was not altered in left ventricular hypertrophy or failing hearts. β-Adrenergic receptor and inotropic response were decreased in failing hearts. In failing left ventricular assist device–supported hearts, S100A1 and sarcoplasmic endoplasmic reticulum Ca 2+ ATPase showed no recovery, while phospholamban, β-adrenergic receptor, and the inotropic response fully recovered. Conclusions— S100A1 and sarcoplasmic endoplasmic reticulum Ca 2+ ATPase, both key Ca 2+ -regulatory proteins, are decreased in human heart failure, and these changes are not reversed after left ventricular assist device. The clinical significance of these findings for cardiac recovery remains to be addressed.

This Controversies in Research article discusses the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the Ryanodine Receptor (RyR) at a single serine (RyRS2808) is essential for normal sympathetic regulation of cardiac myocyte contractility and is responsible for the disturbed Ca 2+ regulation that underlies depressed contractility in heart failure. Studies supporting this hypothesis have associated hyperphosphorylation of RyRS2808 and heart failure progression in animals and humans and have shown that a phosphorylation defective RyR mutant mouse (RyRS2808A) does not respond normally to sympathetic agonists and does not exhibit heart failure symptoms after myocardial infarction. Studies to confirm and extend these ideas have failed to support the original data. Experiments from many different laboratories have convincingly shown that PKA-mediated RyRS2808 phosphorylation does not play any significant role in the normal sympathetic regulation of sarcoplasmic reticulum Ca2+ release or cardiac contractility. Hearts and myocytes from RyRS2808A mice have been shown to respond normally to sympathetic agonists, and to increase Ca 2+ influx, Ca 2+ transients, and Ca 2+ efflux. Although the RyR is involved in heart failure–related Ca 2+ disturbances, this results from Ca 2+ -calmodulin kinase II and reactive oxygen species–mediated regulation rather than by RyR2808 phosphorylation. Also, a new study has shown that RyRS2808A mice are not protected from myocardial infarction. Collectively, there is now a clear consensus in the published literature showing that dysregulated RyRs contribute to the altered Ca 2+ regulatory phenotype of the failing heart, but PKA-mediated phosphorylation of RyRS2808 has little or no role in these alterations.

Rationale: Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. Objective: To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. Methods and Results: We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. Conclusions: A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare but particularly disabling disease that manifests as spontaneous transition to VT associated with increased sympathetic activity. In 2 variants of this disease (CPVT1 and CPVT2), the abnormal electric activity of the heart is associated with genetic defects in the coding of either the ryanodine receptor (type 1) or a related sarcoplasmic reticulum (SR) protein, calsequestrin (type 2). For those cases (20%–30%) unresponsive to β-blocker treatment there were few options available to mitigate the risk of VT/VF (ventricular tachycardia/ventricular fibrillation). However, an article published by the Knollman group1 in 2009 indicated that these individuals may respond to the class 1 antiarrhythmic drug flecainide, which has an anesthetic action via inhibition of INa. The article provided evidence that in the case of CPVT, its antiarrhythmic action was not via inhibition of INa, but instead via inhibition of the activity of the cardiac ryanodine receptor (RyR2) and subsequent reduction in the proarrhythmic release of Ca2+ during diastole. This interpretation was supported by further work by the Knollman and his collaborators,2,3 in particular, the demonstration of direct effects of flecainide and the related more potent local anesthetic R-propafenone3 on SR Ca2+ release in permeabilized cardiac muscle preparations. However, the interpretation of this result is controversial. Two independent groups4,5 have failed to reproduce the effects of flecainide on RyR2 activity in either normal ventricular myocardium5 or a mouse model of CPVT1.4 The first group reported effects of flecainide that are entirely consistent with a direct action on the excitability of cardiac muscle via INa,4 whereas the second produced evidence that the antiarrhythmic effect is by depressed Na+ influx through INa and the subsequent effects on cytoplasmic …

Rationale: The recessive form of catecholaminergic polymorphic ventricular tachycardia is caused by mutations in the cardiac calsequestrin-2 gene; this variant of catecholaminergic polymorphic ventricular tachycardia is less well characterized than the autosomal-dominant form caused by mutations in the ryanodine receptor-2 gene. Objective: We characterized the intracellular Ca 2+ homeostasis, electrophysiological properties, and ultrastructural features of the Ca 2+ release units in the homozygous calsequestrin 2-R33Q knock-in mouse model (R33Q) R33Q knock-in mouse model. Methods and Results: We studied isolated R33Q and wild-type ventricular myocytes and observed properties not previously identified in a catecholaminergic polymorphic ventricular tachycardia model. As compared with wild-type cells, R33Q myocytes (1) show spontaneous Ca 2+ waves unable to propagate as cell-wide waves; (2) show smaller Ca 2+ sparks with shortened coupling intervals, suggesting a reduced refractoriness of Ca 2+ release events; (3) have a reduction of the area of membrane contact, of the junctions between junctional sarcoplasmic reticulum and T tubules (couplons), and of junctional sarcoplasmic reticulum volume; (4) have a propensity to develop phase 2 to 4 afterdepolarizations that can elicit triggered beats; and (5) involve viral gene transfer with wild-type cardiac calsequestrin-2 that is able to normalize structural abnormalities and to restore cell-wide calcium wave propagation. Conclusions: Our data show that homozygous cardiac calsequestrin-2–R33Q myocytes develop spontaneous Ca 2+ release events with a broad range of intervals coupled to preceding beats, leading to the formation of early and delayed afterdepolarizations. They also display a major disruption of the Ca 2+ release unit architecture that leads to fragmentation of spontaneous Ca 2+ waves. We propose that these 2 substrates in R33Q myocytes synergize to provide a new arrhythmogenic mechanism for catecholaminergic polymorphic ventricular tachycardia.

Background— Unexplained cardiac arrest (UCA) may be explained by inherited arrhythmia syndromes. The Cardiac Arrest Survivors With Preserved Ejection Fraction Registry prospectively assessed first-degree relatives of UCA or sudden unexplained death victims to screen for cardiac abnormalities. Methods and Results— Around 398 first-degree family members (186 UCA, 212 sudden unexplained death victims’ relatives; mean age, 44±17 years) underwent extensive cardiac workup, including ECG, signal averaged ECG, exercise testing, cardiac imaging, Holter-monitoring, and selective provocative drug testing with epinephrine or procainamide. Genetic testing was performed when a mutation was identified in the UCA survivor or when the diagnostic workup revealed a phenotype suggestive of a specific inherited arrhythmia syndrome. The diagnostic strength was classified as definite, probable, or possible based on previously published definitions. Cardiac abnormalities were detected in 120 of 398 patients (30.2%) with 67 of 398 having a definite or probable diagnosis (17%), including Long-QT syndrome (13%), catecholaminergic polymorphic ventricular tachycardia (4%), arrhythmogenic right ventricular cardiomyopathy (4%), and Brugada syndrome (3%). The detection yield was similar for family members of UCA and sudden unexplained death victims (31% versus 27%; P =0.59). Genetic testing was performed more often in family members of UCA patients (29% versus 20%; P =0.03). Disease-causing mutations were identified in 20 of 398 relatives (5%). The most common pathogenic mutations were RyR2 (2%), SCN5A (1%), and KNCQ1 (0.8%). Conclusions— Cardiac screening revealed abnormalities in 30% of first-degree relatives of UCA or sudden unexplained death victims, with a clear working diagnosis in 17%. Long-QT, arrhythmogenic right ventricular cardiomyopathy, and catecholaminergic polymorphic ventricular tachycardia were the most common diagnoses. Systematic cascade screening and genetic testing in asymptomatic individuals will lead to preventive lifestyle and medical interventions with potential to prevent sudden cardiac death. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00292032.

Background— Calmodulin (CaM) mutations are associated with severe forms of long QT syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). CaM mutations are found in 13% of genotype-negative long QT syndrome patients, but the prevalence of CaM mutations in genotype-negative CPVT patients is unknown. Here, we identify and characterize CaM mutations in 12 patients with genotype-negative but clinically diagnosed CPVT. Methods and Results— We performed mutational analysis of CALM1 , CALM2 , and CALM3 gene-coding regions, in vitro measurement of CaM-Ca 2+ (Ca)-binding affinity, ryanodine receptor 2–CaM binding, Ca handling, L-type Ca current, and action potential duration. We identified a novel CaM mutation—A103V—in CALM3 in 1 of 12 patients (8%), a female who experienced episodes of exertion-induced syncope since age 10, had normal QT interval, and displayed ventricular ectopy during stress testing consistent with CPVT. A103V modestly lowered CaM Ca-binding affinity (3-fold reduction versus WT-CaM), but did not alter CaM binding to ryanodine receptor 2. In permeabilized cardiomyocytes, A103V-CaM (100 nmol/L) promoted spontaneous Ca wave and spark activity, a cellular phenotype of ryanodine receptor 2 activation. Even a 1:3 mixture of A103V-CaM:WT-CaM activated Ca waves, demonstrating functional dominance. Compared with long QT syndrome D96V-CaM, A103V-CaM had significantly less effects on L-type Ca current inactivation, did not alter action potential duration, and caused delayed afterdepolarizations and triggered beats in intact cardiomyocytes. Conclusions— We discovered a novel CPVT mutation in the CALM3 gene that shares functional characteristics with established CPVT-associated mutations in CALM1 . A small proportion of A103V-CaM is sufficient to evoke arrhythmogenic Ca disturbances via ryanodine receptor 2 dysregulation, which explains the autosomal dominant inheritance.

The first clinical description of long-QT syndrome (LQTS) occurred in 1957 when Drs Anton Jervell and Fred Lange-Nielsen postulated that syncopal/seizure episodes and high propensity for sudden cardiac death (SCD) observed in a subset of children with sensorineural deafness and otherwise unexplained heart rate–corrected QT interval prolongation on ECG stemmed from a novel congenital disorder.1 This was followed by independent descriptions of a syndrome characterized by a cardiac-only phenotype consisting of prolonged QT intervals and an increased risk for syncope, seizures, and SCD in the absence of sensorineural deafness by Drs Cesarino Romano and Owen Ward, in 1963 and 1964, respectively.2,3 During the past 5 decades, insights gleamed from a multitude of clinical, epidemiological, and molecular studies have demonstrated that LQTS is a collection of not only genetically and phenotypically diverse disorders of cardiac repolarization that encompasses the aforementioned predominantly autosomal-dominant, nonsyndromic Romano–Ward syndrome now simply referred to as LQTS4 and autosomal-recessive, multisystem, Jervell–Lange Nielsen syndrome but also exceedingly rare multisystem LQTS subtypes such as Timothy syndrome (TS) characterized by QT prolongation and an increased risk of SCD along with an array of extracardiac manifestations. Although mutations in calcium (Ca2+)-handling proteins, including the CACNA1C -encoded L-type Ca2+ channel (LTCC), RYR2 -encoded ryanodine receptor-2 intracellular calcium release channel (RyR2), and many other auxiliary interacting proteins, are central to the pathogenesis of inherited cardiac arrhythmia syndromes, such as catecholaminergic ventricular tachycardia and Brugada syndrome, until recently, the contribution of dysfunctional Ca2+ handling to the pathogenesis of LQTS was limited to the extremely rare and highly lethal multisystem TS. However, the unexpected recent discoveries of multiple nonsyndromic LQTS-causative mutations in CACNA1C ,5–7 syndromic LQTS-causative mutations in CALM1-3 -encoded Calmodulin8,9 and TRDN -encoded Triadin,10 and common genetic variation at …

Background— Recently, we reported that sarcoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a), the pump responsible for reuptake of cytosolic calcium during diastole, plays a central role in the molecular mechanism of cardiac alternans. Heart failure (HF) is associated with impaired myocardial calcium handling, deficient SERCA2a, and increased susceptibility to cardiac alternans. Therefore, we hypothesized that restoring deficient SERCA2a by gene transfer will significantly reduce arrhythmogenic cardiac alternans in the failing heart. Methods and Results— Adult guinea pigs were divided into 3 groups: control, HF, and HF+AAV9.SERCA2a gene transfer. HF resulted in a decrease in left ventricular fractional shortening compared with controls ( P 2+ release, and increased diastolic Ca 2+ ( P P m alternans and Ca alternans compared with controls ( P P P o secondary to reduction of ryanodine receptor 2–P S2814 ( P P= 0.05). Conclusions— These data show that SERCA2a gene transfer in the failing heart not only improves contractile function but also directly restores electric stability through the amelioration of key arrhythmogenic substrate (ie, cardiac alternans) and triggers (ie, sarcoplasmic reticulum Ca 2+ leak).

Rationale: 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 (Ca 2+ ) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle. Objective: In the present study, we sought to determine whether intracellular diastolic SR Ca 2+ leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF. Methods and Results: 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 Ca 2+ 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 2+ 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 Ca 2+ 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 Ca 2+ 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. Conclusions: The present study demonstrates that RyR2-mediated diastolic SR Ca 2+ leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca 2+ leak through RyR2 and pacing-induced AF associated with CPVT mutations.

Background— Catecholaminergic polymorphic ventricular tachycardia is directly linked to mutations in proteins (eg, type 2 ryanodine receptor [RyR2] R4496C ) responsible for intracellular Ca 2+ homeostasis in the heart. However, the mechanism of Ca 2+ release dysfunction underlying catecholaminergic polymorphic ventricular tachycardia has only been investigated in isolated cells but not in the in situ undisrupted myocardium. Methods and Results— We investigated in situ myocyte Ca 2+ dynamics in intact Langendorff-perfused hearts (ex vivo) from wild-type and RyR2 R4496C+/− mice using laser scanning confocal microscopy. We found that myocytes from both wild-type and RyR2 R4496C+/− hearts displayed uniform, synchronized Ca 2+ transients. Ca 2+ transients from beat to beat were comparable in amplitude with identical activation and decay kinetics in wild-type and RyR2 R4496C+/− hearts, suggesting that excitation-contraction coupling between the sarcolemmal Ca 2+ channels and mutated RyR2 R4496C+/− channels remains intact under baseline resting conditions. On adrenergic stimulation, RyR2 R4496C+/− hearts exhibited a high degree of Ca 2+ release variability. The varied pattern of Ca 2+ release was absent in single isolated myocytes, independent of cell cycle length, synchronized among neighboring myocytes, and correlated with catecholaminergic polymorphic ventricular tachycardia. A similar pattern of action potential variability, which was synchronized among neighboring myocytes, was also revealed under adrenergic stress in intact hearts but not in isolated myocytes. Conclusions— Our studies using an in situ confocal imaging approach suggest that mutated RyR2s are functionally normal at rest but display a high degree of Ca 2+ release variability on intense adrenergic stimulation. Ca 2+ release variability is a Ca 2+ release abnormality, resulting from electric defects rather than the failure of the Ca 2+ release response to action potentials in mutated ventricular myocytes. Our data provide important insights into Ca 2+ release and electric dysfunction in an established model of catecholaminergic polymorphic ventricular tachycardia.

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca 2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca 2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca 2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Rationale: Increased activity of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure (HF) progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in HF development and associated diastolic sarcoplasmic reticulum Ca 2+ leak is unclear. Objective: Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with nonischemic and ischemic forms of HF. Methods and Results: Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with nonischemic, but not with ischemic, HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development after transverse aortic constriction compared with wild-type littermates. After transverse aortic constriction, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor. Cardiomyocytes from S2814A mice exhibited significantly lower sarcoplasmic reticulum Ca 2+ leak and improved sarcoplasmic reticulum Ca 2+ loading compared with wild-type mice after transverse aortic constriction. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice after experimental myocardial infarction. Conclusions: Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological sarcoplasmic reticulum Ca 2+ leak and HF development in nonischemic forms of HF such as transverse aortic constriction in mice.

By the time the heart reaches the pathological state clinically recognized as heart failure (HF), it has undergone profound and often irreversible alterations in structure and function at the molecular, cellular, and organ level. Although the etiologies of HF are diverse (hypertension, myocardial infarction, atherosclerosis, valvular insufficiency, mutations in genes encoding sarcomeric proteins, etc), some alterations are commonly found in most forms of HF, and they may account for the maladaptive structural remodeling and systolic dysfunction that characterize this syndrome. At the cellular level, there are well-documented changes in ionic channel density and function (electric remodeling), increased ROS production, mitochondrial dysfunction, imbalanced energy intake and consumption, genetic reprogramming, altered excitation-contraction coupling, and, in general, dysregulation of a multitude of other processes and pathways that are essential for proper cardiac function.1 Combined, this myriad of alterations leads to loss in contractility and ejection fraction, ventricular wall remodeling, increased vascular resistance, and dysregulated fluid homeostasis. In this issue of Circulation Research , Respress et al2 report that preventing phosphorylation of cardiac ryanodine receptors (RyR2) at a single residue, S2814, is sufficient to avert many of these alterations and improve cardiac function in HF. The results presented follow a string of papers that touch on the delicate and controversial subject of ryanodine receptor phosphorylation and HF. They offer a new twist to a contentious story and attempt to reconcile many apparently contradicting results, but key issues remain. Article, see p 1474 ### Calcium Leak in HF Although, as mentioned above, the etiologies of HF are diverse, it appears that suppressing the dysfunction of a select group of biological and molecular signaling pathways may substantially improve or even reverse the cardiac deterioration observed in HF. For example, correcting the characteristically depressed sarcoplasmic reticulum (SR) calcium content of failing cardiomyocytes is a target of HF gene therapy.3 …

Background— Delayed afterdepolarizations (DADs) carried by Na + -Ca 2+ -exchange current (I NCX ) in response to sarcoplasmic reticulum (SR) Ca 2+ leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined. Methods and Results— Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca 2+ ] i (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca 2+ ] i and SR Ca 2+ content (integrated I NCX during caffeine-induced Ca 2+ transient) were unchanged, whereas diastolic SR Ca 2+ leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca 2+ leak. Calmodulin expression (60%), Ca 2+ /calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca 2+ leak, the frequency of spontaneous Ca 2+ release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca 2+ sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca 2+ ] i and I NCX density revealed I NCX upregulation in cAF. Spontaneous Ca 2+ release events accompanied by inward I NCX currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca 2+ ] i (diastolic [Ca 2+ ] i –voltage coupling gain) was higher in cAF compared with control. Conclusions— Enhanced SR Ca 2+ leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I NCX for a given SR Ca 2+ release and increased diastolic [Ca 2+ ] i -voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

In this issue of Circulation Research , Zhang et al1 provide direct and clear evidence that phosphorylation of S2808 in the cardiac ryanodine receptors (RyR2) has no impact on either normal cardiac function, sympathetic stimulation of the heart, or progression of heart failure (HF) after myocardial infarction (MI). They used mice in which the S2808 site was rendered nonphosphorylatable in S2808A knock-in mice, and the results directly contradict results published by the group of A.R. Marks.2–4 This has been a highly controversial area. This new study compels me to assess this pathway in a balanced way, but recognizing that my opinions are influenced by work that my group has done in this area. My goal here is not to microdissect each controversial piece of data in dozens of relevant papers (see recent reviews and tables5,6), but rather to provide my informed opinion about key aspects of this issue in a balanced manner. Let us first consider the model that importantly raised the issue of SR Ca leak as a potential pathophysiological cardiac effect in HF. Article, see p 831 In 2000 the Marks group published an exciting and seminal paper that has fueled intensive research over the past 11 years.7 They proposed that in HF, PKA hyperphosphorylates RyR2-S2808, causing FKBP12.6 dissociation from RyR2, which directly enhances SR Ca leak/depletion and systolic dysfunction in HF (Figure). Tenets later added to this core model were a down-regulation of phosphatases associated with the RyR2 in HF (which would facilitate the PKA-dependent RyR2 hyperphosphorylation) and that this diastolic SR Ca leak could also contribute to triggered arrhythmias. An extensive series of papers by the same group since then have provided highly consistent and compelling support for this working hypothesis, extending the impact of this intriguing PKA/RyR-S2808/FKBP12.6/RyR …

Rationale: Diastolic spontaneous Ca 2+ waves (DCWs) are recognized as important contributors to triggered arrhythmias. DCWs are thought to arise when [Ca 2+ ] in sarcoplasmic reticulum ([Ca 2+ ] SR ) reaches a certain threshold level, which might be reduced in cardiac disease as a consequence of sensitization of ryanodine receptors (RyR2s) to luminal Ca 2+ . Objective: We investigated the mechanisms of DCW generation in myocytes from normal and diseased hearts, using a canine model of post–myocardial infarction ventricular fibrillation (VF). Methods and Results: The frequency of DCWs, recorded during periodic pacing in the presence of a β-adrenergic receptor agonist isoproterenol, was significantly higher in VF myocytes than in normal controls. Rather than occurring immediately on reaching a final [Ca 2+ ] SR , DCWs arose with a distinct time delay after attaining steady [Ca 2+ ] SR in both experimental groups. Although the rate of [Ca 2+ ] SR recovery after the SR Ca 2+ release was similar between the groups, in VF myocytes the latency to DCWs was shorter, and the [Ca 2+ ] SR at DCW initiation was lower. The restitution of depolarization-induced Ca 2+ transients, assessed by a 2-pulse protocol, was significantly faster in VF myocytes than in controls. The VF-related alterations in myocyte Ca 2+ cycling were mimicked by the RyR2 agonist, caffeine. The reducing agent, mercaptopropionylglycine, or the CaMKII inhibitor, KN93, decreased DCW frequency and normalized restitution of Ca 2+ release in VF myocytes. Conclusions: The attainment of a certain threshold [Ca 2+ ] SR is not sufficient for the generation of DCWs. Postrelease Ca 2+ signaling refractoriness critically influences the occurrence of DCWs. Shortened Ca 2+ signaling refractoriness due to RyR2 phosphorylation and oxidation is responsible for the increased rate of DCWs observed in VF myocytes and could provide a substrate for synchronization of arrhythmogenic events at the tissue level in hearts prone to VF.

Background— Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca 2+ ) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. Methods and Results— We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca 2+ homeostasis and spontaneous SR Ca 2+ leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca 2+ leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca 2+ load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca 2+ leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. Conclusions— SERCA2a gene therapy stabilizes SR Ca 2+ load, reduces ryanodine receptor phosphorylation and decreases SR Ca 2+ leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease occurring in patients with a structurally normal heart: the disease is characterized by life-threatening arrhythmias elicited by stress and emotion. In 2001, the ryanodine receptor was identified as the gene that is linked to CPVT; shortly thereafter, cardiac calsequestrin was implicated in the recessive form of the same disease. It became clear that abnormalities in intracellular Ca 2+ regulation could profoundly disrupt the electrophysiological properties of the heart. In this article, we discuss the molecular basis of the disease and the pathophysiological mechanisms that are impacting clinical diagnosis and management of affected individuals. As of today, the interaction between basic scientists and clinicians to understand CPVT and identify new therapeutic strategies is one of the most compelling examples of the importance of translational research in cardiology.

Conserved regulation of cardiac calcium uptake by peptides encoded in small open reading frames Magny et al Science . 2013;341:1116–1120. Cardiac contraction requires continuous cycles of calcium release and reuptake between the sarcoplasm and sarcoplasmic reticulum. In vertebrate cardiomyocytes, resequestration of calcium to the sarcoplasmic reticulum is accomplished by the SERCA whose activity is dampened by interaction with the small integral membrane proteins, phospholamban and sarcolipin. In a recent report published in Science , Magny et al identify 2 small peptides in Drosophila encoded in a putative long noncoding RNA that buffers calcium reuptake by sarco/endoplasmic reticulum Ca2+-ATPase 2a in a similar manner to sarco/endoplasmic reticulum Ca2+-ATPase 2a regulation by phospholamban and sarcolipin. These findings demonstrate that regulation of Ca2+-ATPases by small transmembrane peptides is a conserved and ancient strategy. Furthermore, this study highlights the possibility that there may be many undiscovered small peptides encoded within putative long noncoding RNAs that regulate important biological pathways. Regulation of calcium signaling is vitally important to normal heart function, and dysregulation of calcium handling is a common feature of many models of cardiovascular disease and progressive heart failure. The importance of calcium regulation in cardiomyocytes is multifactorial, but stems from its direct role in regulation of the sarcomeric contraction machinery1 as well as roles in gene regulation2 and other processes including cell death.3 During each cycle of contraction and relaxation, calcium is released from the sarcoplasmic reticulum (SR) and binds myofilaments to induce sarcomere shortening. After contraction, the SR calcium pump, SERCA, replenishes the cardiac calcium store by recycling calcium back to the SR from the sarcoplasm (Figure). Clearing calcium from the sarcoplasm allows the sarcomere to relax and the cardiac chambers to refill with blood. Figure. Calcium cycling in cardiomyocytes. Activation of the L-type …

Background Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca 2+ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca 2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca 2+ /calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results Mice in which the S2814 Ca 2+ /calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca 2+ release events, resulting in reduced sarcoplasmic reticulum Ca 2+ load on confocal microscopy. These Ca 2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca 2+ /calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery. Conclusions Our results suggest that Ca 2+ /calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca 2+ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

Purpose of review Emerging evidence highlights the importance of physiological participation of ryanodine receptors (RyR) and Ca2+-induced-Ca2+-release (CICR) from the sarcoplasmic reticulum in Ca2+ signaling and arteriolar contraction in the renal microcirculation. Recent findings Adenosine diphosphate -ribosyl (ADPR) cyclase and its endogenous metabolites cyclic adenosine diphosphate-ribose and nicotinic acid adenine dinucleotide phosphate mobilize intracellular Ca2+ from sarcoplasmic reticulum stores in the renal vasculature via actions on RyR. The ADPR cyclase/cyclic adenosine diphosphate-ribose/RyR/CICR second messenger system mediates significant (>50%) changes in cytosolic Ca2+ concentration ([Ca2+]i) and contractile function of preglomerular arteries/arterioles during angiotensin II and endothelin-1 stimulation of G-protein coupled receptors. These receptors rapidly activate ADPR cyclase via stimulation of superoxide (O2−) production by nicotinamide adenine dinucleotide phosphate oxidases. Basal ADPR cyclase activity and RyR/CICR contribute to [Ca2+]i responses initiated by Ca2+ entry and by inositol trisphosphate receptor-induced sarcoplasmic reticulum Ca2+ release. Acute [Ca2+]i responses in isolated afferent arterioles and renal vasoconstriction in vivo are attenuated by more than 50% by pharmacological inhibition of ADPR cyclase or RyR. Similarly, renal vascular reactivity to angiotensin II, endothelin-1 and norepinephrine is attenuated by approximately 50% in mice lacking CD38, the main mammalian ADPR cyclase. Conclusion RyR and CICR are important regulations of Ca2+ signaling and contractile tone of renal resistance arterioles in healthy kidneys. The role of this novel-signaling pathway in pathophysiological mechanisms awaits investigation.

Background— Congestive heart failure (CHF) is a common cause of atrial fibrillation. Focal sources of unknown mechanism have been described in CHF-related atrial fibrillation. The authors hypothesized that abnormal calcium (Ca 2+ ) handling contributes to the CHF-related atrial arrhythmogenic substrate. Methods and Results— CHF was induced in dogs by ventricular tachypacing (240 bpm �2 weeks). Cellular Ca 2+ -handling properties and expression/phosphorylation status of key Ca 2+ handling and myofilament proteins were assessed in control and CHF atria. CHF decreased cell shortening but increased left atrial diastolic intracellular Ca 2+ concentration ([Ca 2+ ] i ), [Ca 2+ ] i transient amplitude, and sarcoplasmic reticulum (SR) Ca 2+ load (caffeine-induced [Ca 2+ ] i release). SR Ca 2+ overload was associated with spontaneous Ca 2+ transient events and triggered ectopic activity, which was suppressed by the inhibition of SR Ca 2+ release (ryanodine) or Na + /Ca 2+ exchange. Mechanisms underlying abnormal SR Ca 2+ handling were then studied. CHF increased atrial action potential duration and action potential voltage clamp showed that CHF-like action potentials enhance Ca 2+ i loading. CHF increased calmodulin-dependent protein kinase II phosphorylation of phospholamban by 120%, potentially enhancing SR Ca 2+ uptake by reducing phospholamban inhibition of SR Ca 2+ ATPase, but it did not affect phosphorylation of SR Ca 2+ -release channels (RyR2). Total RyR2 and calsequestrin (main SR Ca 2+ -binding protein) expression were significantly reduced, by 65% and 15%, potentially contributing to SR dysfunction. CHF decreased expression of total and protein kinase A–phosphorylated myosin-binding protein C (a key contractile filament regulator) by 27% and 74%, potentially accounting for decreased contractility despite increased Ca 2+ transients. Complex phosphorylation changes were explained by enhanced calmodulin-dependent protein kinase IIδ expression and function and type-1 protein-phosphatase activity but downregulated regulatory protein kinase A subunits. Conclusions— CHF causes profound changes in Ca 2+ -handling and -regulatory proteins that produce atrial fibrillation–promoting atrial cardiomyocyte Ca 2+ -handling abnormalities, arrhythmogenic triggered activity, and contractile dysfunction.

The effect of the 12-kDa isoform of FK-506–binding protein (FKBP)12.0 on cardiac excitation–contraction coupling was studied in adult rabbit ventricular myocytes after transfection with a recombinant adenovirus coding for human FKBP12.0 (Ad-FKBP12.0). Western blots confirmed overexpression (by 2.6±0.4 fold, n=5). FKBP12.0 association with rabbit cardiac ryanodine receptor (RyR2) was not detected by immunoprecipitation. However, glutathione S -transferase pull-down experiments indicated FKBP12.0–RyR2 binding to proteins isolated from human and rabbit but not dog myocardium. Voltage-clamp experiments indicated no effects of FKBP12.0 overexpression on L-type Ca 2+ current ( I Ca,L ) or Ca 2+ efflux rates via the Na + /Ca 2+ exchanger. Ca 2+ transient amplitude was also not significantly different. However, sarcoplasmic reticulum Ca 2+ load was ≈25% higher in myocytes in the Ad-FKBP12.0 group. The reduced ability of I Ca,L to initiate sarcoplasmic reticulum Ca 2+ release was observed over a range of values of sarcoplasmic reticulum Ca 2+ content, indicating that overexpression of FKBP12.0 reduces the sensitivity of RyR2 to Ca 2+ . Ca 2+ spark morphology was measured in β-escin–permeabilized cardiomyocytes. Ca 2+ spark amplitude and duration were significantly increased, whereas frequency was decreased in cells overexpressing FKBP12.0. These changes were accompanied by an increased sarcoplasmic reticulum Ca 2+ content. In summary, the effects of FKBP12.0 overexpression on intact and permeabilized cells were similar to those of tetracaine, a drug known to reduce RyR2 Ca 2+ sensitivity and distinctly different from the effects of overexpression of the FKBP12.6 isomer. In conclusion, FKBP12.0-RyR2 interaction can regulate the gain of excitation–contraction coupling.

INTRODUCTION: Anesthetic preconditioning (APC) with volatile anesthetics improves recovery of contractile function and reduces calcium overload after ischemia/reperfusion (I/R). Mitochondrial and sarcolemmal K ATP channel openings have been implicated in APC-induced cardioprotection. In this study, we investigated the effect of APC on major calcium cycling proteins and its relation to K ATP channels. METHODS: Isolated perfused rat hearts were divided into seven groups: Time control (n = 10), ischemia control (n = 8), APC (n = 8), Mitochondrial K ATP inhibitor 5-hydroxydecanoate (5-HD, 200 μM, n = 8), Sarcolemmal K ATP inhibitor HMR1098 (HMR, 20 μM, n = 8), and APC plus 5-HD or APC plus HMR1098 (n = 8 each). APC was initiated by administering 1.5% isoflurane for 15 min, followed by a 15 min washout before 30 min of myocardial ischemia and 60 min of reperfusion. Ca 2+ -release channels (RyR2), Ca 2+ -adenosine triphosphatase (SERCA2a), phospholamban, plasma membrane Ca 2+ ATPase, and sodium-calcium exchanger in the homogenate were determined by Western blot assay. RESULTS: APC improved contractile recovery (left ventricular developed pressure, +dP/dt, -dP/dt) after I/R, which was blocked by 5-HD and HMR. I/R depressed the density of RyR2, SERCA2a, and phospholamban, with no changes in the density of plasma membrane Ca 2+ ATPase and sodium-calcium exchanger. APC reversed I/R-induced degradation of RyR2 and SERCA2a in the presence or absence of 5HD and HMR. CONCLUSIONS: I/R-induced depression in cardiac performance is associated with a down-regulation of the major sarcoplasmic reticulum Ca 2+ -cycling proteins. Anesthesia preconditioning with isoflurane prevents I/R-related degradation of the RyR2 and SERCA2a in the sarcoplasmic reticulum. However, this effect was independent of its activation of K ATP channels.

See related article, pages 819–829 In response to exercise or other stresses, catecholamines are released into the circulation and within the heart. Catecholaminergic stimulation of β1-adrenergic receptors (β1ARs) in the heart increases heart rate (chronotropy) and contractility (inotropy), resulting in increased cardiac output during acute stress. Concurrent stimulation of β2-ARs dilates blood vessels, which increase blood flow to exercising muscles, thereby matching increased cardiac output to metabolic demands of the organs. The inotropic mechanisms investigated by Valdivia and colleagues1 in the current issue of Circulation Research are essential for the acute stress-dependent increase of cardiac output. Each of the millions of muscle cells in the heart (cardiomyocytes) contribute to myocardial force development. Cardiomyocyte contraction is controlled by intracellular Ca2+ release through a process called excitation-contraction coupling (ECC) that involves the following steps: (1) an action potential (AP) depolarizes the cell membrane; (2) voltage-dependent plasma membrane L-type calcium channels (Cav1.2) opening results in a whole-cell inward Ca2+ current ( I Ca); (3) I Ca activates cardiac ryanodine receptor (RyR2)/Ca2+ release channels located on the junctional sarcoplasmic reticulum (jSR), a process referred to as Ca2+-induced Ca2+ release (CICR); (4) Ca2+ binds to troponin C (TnC) leading to cross-bridge formation between myosin and actin and contraction of the sarcomere. Cardiomyocyte relaxation is signaled by a return of intracellular [Ca2+] to resting levels attributable to the following major mechanisms: (1) Cav1.2 inactivation; (2) RyR2 inactivation; (3) Ca2+ reuptake into the SR by SERCA2a pumps; and (4) Na+/Ca2+ exchange extrusion of Ca2+ to the extracellular space. Under resting conditions, net SR Ca2+ release contributes approximately 66% of Ca2+ necessary for myofilament activation in large mammals including humans, and approximately 90% in rodents.2 Stimulation …

The multifunctional Ca 2+ /calmodulin-dependent protein kinase II δ C (CaMKIIδ C ) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca 2+ release channels in the heart. However, the functional role of CaMKII-dependent phosphorylation of RyR2 is highly controversial. To address this issue, we expressed wild-type, constitutively active, or dominant-negative CaMKIIδ C via adenoviral gene transfer in cultured adult rat ventricular myocytes. CaMKII-mediated phosphorylation of RyR2 was reduced, enhanced, or unaltered by dominant-negative, constitutively active, or wild-type CaMKIIδ C expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing β-galactosidase (β-gal), respectively. In parallel with the phospholamban phosphorylation, the decay kinetics of global Ca 2+ transients was slowed, accelerated, or unchanged, whereas spontaneous Ca 2+ spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKIIδ C groups, respectively. When challenged by high extracellular Ca 2+ , both wild-type and constitutively active CaMKIIδ C protected the cells from store overload-induced Ca 2+ release, manifested by a ≈60% suppression of Ca 2+ waves (at 2 to 20 mmol/L extracellular Ca 2+ ) in spite of an elevated sarcoplasmic reticulum Ca 2+ content, whereas dominant-negative CaMKIIδ C promoted Ca 2+ wave production (at 20 mmol/L Ca 2+ ) with significantly depleted sarcoplasmic reticulum Ca 2+ . Taken together, our data support the notion that CaMKIIδ C negatively regulates RyR2 activity and spontaneous sarcoplasmic reticulum Ca 2+ release, thereby affording a negative feedback that stabilizes local and global Ca 2+ -induced Ca 2+ release in the heart.

Heart failure remains a leading cause of morbidity and mortality worldwide. Although depressed pump function is common, development of effective therapies to stimulate contraction has proven difficult. This is thought to be attributable to their frequent reliance on cAMP stimulation to increase activator Ca 2+ . A potential alternative is nitroxyl (HNO), the 1-electron reduction product of nitric oxide (NO) that improves contraction and relaxation in normal and failing hearts in vivo. The mechanism for myocyte effects remains unknown. Here, we show that this activity results from a direct interaction of HNO with the sarcoplasmic reticulum Ca 2+ pump and the ryanodine receptor 2, leading to increased Ca 2+ uptake and release from the sarcoplasmic reticulum. HNO increases the open probability of isolated ryanodine-sensitive Ca 2+ -release channels and accelerates Ca 2+ reuptake into isolated sarcoplasmic reticulum by stimulating ATP-dependent Ca 2+ transport. Contraction improves with no net rise in diastolic calcium. These changes are not induced by NO, are fully reversible by addition of reducing agents (redox sensitive), and independent of both cAMP/protein kinase A and cGMP/protein kinase G signaling. Rather, the data support HNO/thiolate interactions that enhance the activity of intracellular Ca 2+ cycling proteins. These findings suggest HNO donors are attractive candidates for the pharmacological treatment of heart failure.