Ca Leak Research Articles

The oral Ca/calmodulin-dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure.

AimsExcessive activation of Ca/calmodulin‐dependent kinase II (CaMKII) is of critical importance in heart failure (HF) and atrial fibrillation. Unfortunately, lack of selectivity, specificity, and bioavailability have slowed down development of inhibitors for clinical use. We investigated a novel CaMKIIδ/CaMKIIɣ‐selective, ATP‐competitive, orally available CaMKII inhibitor (RA608) on right atrial biopsies of 119 patients undergoing heart surgery. Furthermore, we evaluated its oral efficacy to prevent deterioration of HF in mice after transverse aortic constriction (TAC).Methods and resultsIn human atrial cardiomyocytes and trabeculae, respectively, RA608 significantly reduced sarcoplasmic reticulum Ca leak, reduced diastolic tension, and increased sarcoplasmic reticulum Ca content. Patch‐clamp recordings confirmed the safety of RA608 in human cardiomyocytes. C57BL6/J mice were subjected to TAC, and left ventricular function was monitored by echocardiography. Two weeks after TAC, RA608 was administered by oral gavage for 7 days. Oral RA608 treatment prevented deterioration of ejection fraction. At 3 weeks after TAC, ejection fraction was 46.1 ± 3.7% (RA608) vs. 34.9 ± 2.6% (vehicle), n = 9 vs. n = 12, P < 0.05, ANOVA, which correlated with significantly less CaMKII autophosphorylation at threonine 287. Moreover, a single oral dose significantly reduced inducibility of atrial and ventricular arrhythmias in CaMKIIδ transgenic mice 4 h after administration. Atrial fibrillation was induced in 6/6 mice for vehicle vs. 1/7 for RA608, P < 0.05, 'n − 1' χ 2 test. Ventricular tachycardia was induced in 6/7 for vehicle vs. 2/7 for RA608, P < 0.05, 'n − 1' χ 2 test.ConclusionsRA608 is the first orally administrable CaMKII inhibitor with potent efficacy in human myocytes. Moreover, oral administration potently inhibits arrhythmogenesis and attenuates HF development in mice in vivo.

Protocol Development for Expressing Functional Ryanodine Receptors in HEK293-6E Suspension Cells

We have developed a protocol for expressing ryanodine receptors (RyR), specifically the rabbit skeletal RyR isoform (rbRyR1) and human cardiac RyR isoform (hRyR2), that respond to known RyR modulators, with the goal of developing molecular tools for FRET-based structural readouts of RyRs. Both RyR1 and RyR2 are composed of four 565 kDa subunits and are the primary Ca2+ channels in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle, respectively. Triggered SR Ca2+ release is required for muscle contraction, but RyR dysregulation can result in SR Ca leak during rest, which is linked to several myopathies, diabetes, and neurological disorders.

Increased SR Calcium Leak is Promoted by O-GlcNAcylation of CaMKII in Diabetes and Hyperglycemia

Abnormal calcium (Ca) leak via cardiac ryanodine receptor 2 (RyR2) is enhanced by high extracellular [glucose] via O-GlcNAcylation of CaMKII and may be important in diabetic cardiomyopathy (PMID: 24077098). Indeed, CaMKII O-GlcNAcylation and activity are elevated in humans and animal diabetic models. Here, we tested whether CaMKIIδ and the site identified as an O-GlcNAcylation target (Ser280) are required for promoting SR Ca leak. We measured Ca sparks, Ca transients, and sarcoplasmic reticulum (SR) Ca content in isolated mouse ventricular cardiomyocytes.

Exendin-4 Reduces Ventricular Arrhythmia Activity and Calcium Sparks-Mediated Sarcoplasmic Reticulum Ca Leak in Rats with Heart Failure.

The aim of this study was to investigate the effect of exendin-4 (Ex-4) on ventricular arrhythmias and calcium sparks-mediated calcium leak in a myocardial infarction-heart failure model.We studied the influence of exendin-4 on ventricular arrhythmogenesis in a rat myocardial infarction-heart failure model. In vivo arrhythmia studies (electrocardiogram [ECG] telemetry studies), ex vivo arrhythmia studies calcium sparks tests, and analysis of total and phosphorylated ryanodine receptor (RyR) 2 and CaMK-II were carried out in sham group, myocardial infarction (MI) group, MI + Ex-4 and MI + Ex-4 + Exendin9-39 (Ex9-39) groups.ECG telemetry studies showed an antiarrhythmic effect of exendin-4 with reduction of spontaneous ventricular arrhythmias. Exendin-4 abbreviated the APD90, which was longer in the heart failure model, and increase the APD alternans thresholds. Exendin-4 also reduced the susceptibility to burst pacing-induced arrhythmia ex vivo. Subcellular sarcoplasmic reticulum (SR) calcium leak characteristics were tested in four groups of rat cardiomyocytes. Exendin-4 reduced calcium spark mass, spark frequency, and calcium leak, which may be due to reduced S2814-RyR2 and CaMK-II phosphorylation. Co-administration of exendin 9-39 with exendin-4 partly abolished the above-mentioned effect of exendin-4.These findings suggest that exendin-4 exerts an antiarrhythmic effect through decreasing SR calcium leak in spontaneous and burst pacing-induced ventricular arrhythmias, which may be due to reduced RyR2 phosphorylation and suppressed CaMK-II activity. Exendin-4 may act as a novel antiarrhythmic strategy in heart failure.

P3508A novel mouse model of sleep-disordered breathing is associated with contractile dysfunction and CaMKII overexpression

Abstract Background Patients with sleep-disordered breathing (SDB) develop arrhythmias and contractile dysfunction, but the mechanisms are poorly understood, possibly due to the lack of mouse models that mimic airway obstruction in spontaneously sleeping mice. Interestingly, New Zealand obese mice have been shown to develop airway obstruction with inspiratory flow limitation resulting in apneas, but these mice also develop diabetes. Purpose We developed a novel mouse model of increased airway obstruction in spontaneously sleeping lean mice and investigated the impact on sleep-related apneas and contractile function. Methods and results Male C57BL6 mice at 8–12 weeks of age were subjected to polytetrafluoroethylene (PTFE) injection (100 μl) into the tongue. This resulted in an increased tongue volume and reduced pharyngeal luminal diameter. Conscious mice behave normal and there was no difference in body weight between PTFE injected mice and untreated littermates (control). Whole body plethysmography was used to monitor spontaneous breathing for 8h in a quiet environment. Interestingly, compared to control, mice with PTFE injection showed a significantly increased frequency of apneas (lasting &gt;1s, fig. A, * indicated P&lt;0.05, t-test). Echocardiographic analysis revealed that ejection fraction was significantly reduced in PTFE-treated mice 8 weeks after surgery (vs. control, fig. B). In accordance, the developed force of isolated papillary muscles from hearts of PTFE mice was significantly reduced compared to control (fig. C). Ca/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the development of heart failure. Intriguingly, compared to control, CaMKII expression was significantly increased in ventricular heart homogenates of PTFE-treated mice (fig. D). Moreover, the magnitude of CaMKII overexpression correlated significantly with the frequency of apneas (fig. E). Papillary muscle post-pause contractions can be used as measure of diastolic sarcoplasmic reticulum (SR) Ca leak, which is known to be enhanced by CaMKII. Compared to control, post-pause contraction amplitude was significantly smaller in PTFE-treated mice, indicating an increased SR Ca leak (fig. F). Figure 1 Conclusion PTFE injection in mice results in an increased frequency of spontaneous apneas. PTFE-treated mice develop mild contractile dysfunction, which is accompanied by CaMKII overexpression. This novel mouse model offers great opportunities for investigation of sleep-related breathing disorders. Acknowledgement/Funding This study was supported by the Medical Faculty of the University of Regensburg (ReForM programme).

Structural development of a type-1 ryanodine receptor (RyR1) Ca2+-release channel inhibitor guided by endoplasmic reticulum Ca2+ assay

Type-1 ryanodine receptor (RyR1) is a calcium-release channel localized on sarcoplasmic reticulum (SR) of the skeletal muscle, and mediates muscle contraction by releasing Ca2+ from the SR. Genetic mutations of RyR1 are associated with skeletal muscle diseases such as malignant hyperthermia and central core diseases, in which over-activation of RyR1 causes leakage of Ca2+ from the SR. We recently developed an efficient high-throughput screening system based on the measurement of Ca2+ in endoplasmic reticulum, and used it to identify oxolinic acid (1) as a novel RyR1 channel inhibitor. Here, we designed and synthesized a series of quinolone derivatives based on 1 as a lead compound. Derivatives bearing a long alkyl chain at the nitrogen atom of the quinolone ring and having a suitable substituent at the 7-position of quinolone exhibited potent RyR1 channel-inhibitory activity. Among the synthesized compounds, 14h showed more potent activity than dantrolene, a known RyR1 inhibitor, and exhibited high RyR1 selectivity over RyR2 and RyR3. These compounds may be promising leads for clinically applicable RyR1 channel inhibitors.

Mechanisms of Calcium Leak from Cardiac Sarcoplasmic Reticulum Revealed by Statistical Mechanics

Heart muscle contraction is normally activated by a synchronized Ca release from sarcoplasmic reticulum (SR), a major intracellular Ca store. However, under abnormal conditions, Ca leaks from the SR, decreasing heart contraction amplitude and increasing risk of life-threatening arrhythmia. The mechanisms and regimes of SR operation generating the abnormal Ca leak remain unclear. Here, we employed both numerical and analytical modeling to get mechanistic insights into the emergent Ca leak phenomenon. Our numerical simulations using a detailed realistic model of the Ca release unit reveal sharp transitions resulting in Ca leak. The emergence of leak is closely mapped mathematically to the Ising model from statistical mechanics. The system steady-state behavior is determined by two aggregate parameters: the analogs of magnetic field (h) and the inverse temperature (β) in the Ising model, for which we have explicit formulas in terms of SR [Ca] and release channel opening and closing rates. The classification of leak regimes takes the shape of a phase β-h diagram, with the regime boundaries occurring at h = 0 and a critical value of β (β∗) that we estimate using a classical Ising model and mean field theory. Our theory predicts that a synchronized Ca leak will occur when h > 0 and β >β∗, and a disordered leak occurs when β <β∗ and h is not too negative. The disorder leak is distinguished from synchronized leak (in long-lasting sparks) by larger Peierls contour lengths, an output parameter reflecting degree of disorder. Thus, in addition to our detailed numerical model approach, we also offer an instantaneous computational tool using analytical formulas of the Ising model for respective ryanodine receptor parameters and SR Ca load that describe and classify phase transitions and leak emergence.

Abnormal Calcium Leak from Cardiac Sarcoplasmic Reticulum: New Insights Offered by Statistical Physics

Heart muscle contraction is activated by a synchronized systolic Ca release from sarcoplasmic reticulum (SR) via Ca sparks. In disease, Ca sparks fail to terminate, causing a diastolic Ca leak that decreases contraction amplitude and increases the risk of arrhythmia. The mechanisms and treatment of the abnormal Ca leak remain unclear. We have recently shown that spark termination emerges as collective behavior (synchronized closings) of Ca release channels (RyRs) that is identical to synchronization of spin orientation in ferromagnets described by a phase transition in Ising model. We employed the Ising approach to investigate and classify mechanisms of spark termination failure and Ca leak. Methods and Results: The key parameters determining whether spark termination succeeds or fails are SR Ca and RyR opening/closing rates. They define analogues of magnetic field h and the inverse temperature (β) in Ising model. Sparks terminate via a phase transition known as “h polarity reversal” and the leak emerges when h fails to change its sign. This happens when the SR depletes insufficiently and RyR openings remained partially synchronized via Ca-induced-Ca-release, generating long-lasting sparks. Leak can also occur via β, known as Onsager's “order-disorder” transition. This happens at low SR Ca, reducing RyR current and RyRs interactions, resulting in independent RyR openings. The disorder leak is distinguished from synchronized leak by larger Peierls contour lengths reflecting degree of disorder. Abnormal leak results from either a probability imbalance during synchronized firing of RyRs or from disordered RyR firing. Each leak type requires different and balanced treatment to shift RyR operation towards normal spark termination.

The Role of Cysteine 3602 in RyR2 Regulation by Calmodulin and Oxidative Stress

In the skeletal ryanodine receptor (RyR1), cysteine 3635 (C3635) is considered to be an important residue responsible for the redox regulation of the channel. It has been shown that oxidation of C3635 causes intersubunit cross-linking and calmodulin (CaM) dissociation from RyR1. These structural modifications can lead to channel activation. While the role of RyR1 C3635 has been extensively studied, the functional significance of the corresponding cysteine 3602 (C3602) in the cardiac RyR (RyR2) remains unclear. To characterize the role of C3602 in RyR2 response to oxidative stress, we co-expressed either wild type (WT) GFP-RyR2 or the single mutant C3602A GFP-RyR2, together with the ER-targeted Ca sensor R-CEPIA1er and SERCA2a, in HEK293 cells. Cells transfected with either WT- or C3602A-RyR2 produced Ca waves that were sensitive to oxidative stress induced by diamide. Diamide equally increased the endoplasmic reticulum (ER) Ca leak in the cells expressing WT- and the C3602A-RyR2 mutant. Both WT-RyR2 and the mutant RyR2 expressed in HEK293 cells demonstrated the same level of disulfide cross-linking upon diamide treatment. Moreover, the nitric oxide donor NOC-12 had similar effect on ER Ca leak via WT and the mutant RyR2. Given the localization of C3602 within the CaM binding site in RyR2, we assessed the effect of C3602A mutation on the degree of CaM dissociation during oxidative stress. The C3602A mutation did not significantly affect CaM binding to RyR2 during oxidative stress, and the degree of this binding was comparable to the one observed in WT RyR2. Taken together, these results suggest that C3602 does not play a key role in RyR2 response to oxidative stress. Despite ∼70% homology between RyR1 and RyR2, these channels appear to exhibit important structural and functional differences, particularly with respect to redox regulation.

Size Matters: Ryanodine Receptor Cluster Size Heterogeneity Potentiates Calcium Waves

Ryanodine receptors (RyRs) mediate calcium (Ca)-induced Ca release and intracellular Ca homeostasis. In a cardiac myocyte, RyRs group into clusters of variable size from a few to several hundred RyRs, creating a spatially nonuniform intracellular distribution. It is unclear how heterogeneity of RyR cluster size alters spontaneous sarcoplasmic reticulum (SR) Ca releases (Ca sparks) and arrhythmogenic Ca waves. Here, we tested the impact of heterogeneous RyR cluster size on the initiation of Ca waves. Experimentally, we measured RyR cluster sizes at Ca spark sites in rat ventricular myocytes and further tested functional impacts using a physiologically detailed computational model with spatial and stochastic intracellular Ca dynamics. We found that the spark frequency and amplitude increase nonlinearly with the size of RyR clusters. Larger RyR clusters have lower SR Ca release threshold for local Ca spark initiation and exhibit steeper SR Ca release versus SR Ca load relationship. However, larger RyR clusters tend to lower SR Ca load because of the higher Ca leak rate. Conversely, smaller clusters have a higher threshold and a lower leak, which tends to increase SR Ca load. At the myocyte level, homogeneously large or small RyR clusters limit Ca waves (because of low load for large clusters but low excitability for small clusters). Mixtures of large and small RyR clusters potentiates Ca waves because the enhanced SR Ca load driven by smaller clusters enables Ca wave initiation and propagation from larger RyR clusters. Our study suggests that a spatially heterogeneous distribution of RyR cluster size under pathological conditions may potentiate Ca waves and thus afterdepolarizations and triggered arrhythmias.

Redox Dependent Modifications of Ryanodine Receptor: Basic Mechanisms and Implications in Heart Diseases.

Heart contraction vitally depends on tightly controlled intracellular Ca regulation. Because contraction is mainly driven by Ca released from the sarcoplasmic reticulum (SR), this organelle plays a particularly important role in Ca regulation. The type two ryanodine receptor (RyR2) is the major SR Ca release channel in ventricular myocytes. Several cardiac pathologies, including myocardial infarction and heart failure, are associated with increased RyR2 activity and diastolic SR Ca leak. It has been suggested that the increased RyR2 activity plays an important role in arrhythmias and contractile dysfunction. Several studies have linked increased SR Ca leak during myocardial infarction and heart failure to the activation of RyR2 in response to oxidative stress. This activation might include direct oxidation of RyR2 as well as indirect activation via phosphorylation or altered interactions with regulatory proteins. Out of ninety cysteine residues per RyR2 subunit, twenty one were reported to be in reduced state that could be potential targets for redox modifications that include S-nitrosylation, S-glutathionylation, and disulfide cross-linking. Despite its clinical significance, molecular mechanisms of RyR dysfunction during oxidative stress are not fully understood. Herein we review the most recent insights into redox-dependent modulation of RyR2 during oxidative stress and heart diseases.

Antibacterial activity of dialkyl-aginate biosurfactant cream againts Staphylococcus aureus an Pseudomonas aerugynosa

Dialkyl-alginate biosurfactant is an amphifilik rhamnolipid biosurfactant that has the potential to be developed into an antibacterial agent. The purpose of this study was to prove that the biosurfactant of dialkyl alginate both before and after creams has antibacterial activity especially against Staphylococcus aureus and Pseudomonas aeruginosa . Test of antibacterial activity of biosurfactant dialkyl alginate at concentrations of 5%, 10% and 20% to Staphylococcus aureus and Pseudomonas aeruginosa using Hammond, et al (2011) modified quantitative method. Enbatic® 1% is used as a positive control and sterile aquadest as a negative control. Determination of antibacterial activity of dialkyl alginate biosurfactant followed by analysis of leakage of protein and nucleic acids using UV-vis Spectrophotometry and leakage of Ca 2 + and K + metal ions using Atomic Absorption Spetrophotometry (AAS). The most active concentration was formulated into cream and then performed physical evaluation (organoleptic, pH, adhesion, spreading and viscosity) and tested antibacterial activity using the well method. The test results showed that the biosurfactants of dialkyl-alginate before and after the cream was treated as antibacterial activity. The concentration of 10% was the most active concentration having activity which did not differ significantly to positive control with p value of 0,05

3145Empagliflozin potently reduces sarcoplasmic Ca leak and increases Ca transient amplitude of human failing ventricular cardiomyocytes

3145Empagliflozin potently reduces sarcoplasmic Ca leak and increases Ca transient amplitude of human failing ventricular cardiomyocytes

The novel CaMKII inhibitor GS-680 reduces diastolic SR Ca leak and prevents CaMKII-dependent pro-arrhythmic activity

RationaleCa/calmodulin-dependent protein kinase II (CaMKII) was shown to increase diastolic sarcoplasmic reticulum (SR) Ca leak, which can result in delayed afterdepolarizations and triggered arrhythmias. Since increased CaMKII expression and activity has been mechanistically linked to arrhythmias in human heart failure (HF) and atrial fibrillation (AF), specific strategies aimed at CaMKII inhibition may have therapeutic potential. ObjectiveWe tested the antiarrhythmic and inotropic effects of a novel selective and ATP-competitive CaMKII inhibitor (GS-680). Methods and resultsTrabeculae were isolated from right atrial appendage biopsies of patients undergoing cardiac surgery. Premature atrial contractions (PACs) were induced by stimulation with isoproterenol (ISO, 100 nM) at increased [Ca]o (3.5 mM). Interestingly, compared to vehicle, PACs were significantly inhibited by exposure to GS-680 (at 100 and 300 nM). GS-680 also significantly decreased early and delayed afterdepolarizations in isolated human atrial myocytes. Moreover, GS-680 (at 100 or 300 nM) significantly inhibited diastolic SR Ca leak, measured as frequency of spontaneous SR Ca release events (Ca sparks) in isolated human atrial myocytes (Fluo-4 loaded) similar to the well-established peptide CaMKII inhibitor AIP. In accordance, GS-680 significantly reduced CaMKII autophosphorylation (Western blot) but enhanced developed tension after 10 or 30 s pause of electrical stimulation (post-rest behavior). Surprisingly, we found a strong negative inotropic effect of GS-680 in atrial trabeculae at 1 Hz stimulation rate, which was not observed at 4 Hz and abolished by beta-adrenergic stimulation. In contrast, GS-680 did not impair systolic force of isolated ventricular trabeculae from explanted hearts of heart transplant recipients at 1 Hz, blunted the negative force-frequency relationship (1–3 Hz) and significantly increased the Ca transient amplitude. ConclusionThe novel ATP-competitive and selective CaMKII inhibitor GS-680 inhibits pro-arrhythmic activity in human atrium and improves contractility in failing human ventricle, which may have therapeutic implications.

The Functional Significance of Cardiac SERCA Dimerization

It has been shown that the sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) can form homodimers. However, the functional significance of SERCA dimerization remains unclear. FRET measurements in HEK293 cells transfected with fluorescently labeled cardiac SERCA2a revealed that SERCA forms homodimers in a dose-dependent manner. In parallel experiments, properties of endoplasmic reticulum (ER) Ca uptake were studied at different levels of SERCA2a expression. Intra-ER [Ca] ([Ca]ER) was measured in HEK293 cells transfected with SERCA2a (tagged with cerulean), cardiac ryanodine receptor (RyR2 tagged with GFP) and the ER-targeted Ca sensor Cepia-1er. The plasma membrane was permeabilized with saponin to control cytosolic milieu. Activation of RyR2 with caffeine was used to completely deplete [Ca]ER. Once caffeine was removed, RyR2 inhibitors were applied to measure the rate of [Ca]ER recovery. At the end of each experiment, RyR2-independent Ca leak was measured during SERCA inhibition with thapsigargin. For each individual cell, ER Ca uptake and leak as a function of [Ca]ER were analyzed to determine maximum ER Ca uptake rate and maximum ER Ca load. SERCA2a expression level was estimated from the cerulean fluorescence. This analysis revealed that ER Ca uptake increased as a function of [SERCA], with a particularly steep increase at high SERCA expression level. We also found that maximum ER Ca load (or SERCA thermodynamic limit) increased with an increase of SERCA expression. Partial inhibition of SERCA with thapsigargin reduced not only ER Ca uptake rate but also maximum ER Ca load. These results suggest that SERCA dimerization can facilitate ER Ca uptake and improve the efficacy of coupling between ATP hydrolysis and Ca transport. Malfunction of this mechanism (due to SERCA downregulation) can contribute to reduced SR Ca uptake in the failing heart.

Cellular mechanisms of metabolic syndrome-related atrial decompensation in a rat model of HFpEF

Heart failure (HF) with preserved ejection fraction (HFpEF) is present in about 50% of HF patients. Atrial remodeling is common in HFpEF and associated with increased mortality. We postulate that atrial remodeling is associated with atrial dysfunction in vivo related to alterations in cardiomyocyte Calcium (Ca) signaling and remodeling. We examined atrial function in vivo and Ca transients (CaT) (Fluo4-AM, field stim) in atrial cardiomyocytes of ZSF-1 rats without (Ln; lean hypertensive) and with metabolic syndrome (Ob; obese, hypertensive, diabetic) and HFpEF. ResultsAt 21weeks Ln showed an increased left ventricular (LV) mass and left ventricular end-diastolic pressure (LVEDP), but unchanged left atrial (LA) size and preserved atrial ejection fraction vs. wild-type (WT). CaT amplitude in atrial cardiomyocytes was increased in Ln (2.9±0.2 vs. 2.3±0.2F/F0 in WT; n=22 cells/group; p<0.05). Studying subcellular Ca release in more detail, we found that local central cytosolic CaT amplitude was increased, while subsarcolemmal CaT amplitudes remained unchanged. Moreover, Sarcoplasmic reticulum (SR) Ca content (caffeine) was preserved while Ca spark frequency and tetracaine-dependent SR Ca leak were significantly increased in Ln. Ob mice developed a HFpEF phenotype in vivo, LA area was significantly increased and atrial in vivo function was impaired, despite increased atrial CaT amplitudes in vitro (2.8±0.2; p<0.05 vs. WT). Ob cells showed alterations of the tubular network possibly contributing to the observed phenotype. CaT kinetics as well as SR Ca in Ob were not significantly different from WT, but SR Ca leak remained increased. Angiotensin II (Ang II) reduced in vitro cytosolic CaT amplitudes and let to active nuclear Ca release in Ob but not in Ln or WT. SummaryIn hypertensive ZSF-1 rats, a possibly compensatory increase of cytosolic CaT amplitude and increased SR Ca leak precede atrial remodeling and HFpEF. Atrial remodeling in ZSF-1 HFpEF is associated with an altered tubular network in-vitro and atrial contractile dysfunction in vivo, indicating insufficient compensation. Atrial cardiomyocyte dysfunction in vitro is induced by the addition of angiotensin II.

P21-2 - Protein Kinase a Activation Reduces Calmodulin-Ryanodine Affinity in R176Q/+ and R2474S/+ Knock-in Mouse

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited stress-induced arrhythmogenic disease caused by mutations in cardiac ryanodine receptor (RyR2). These mutations cluster in 3 regions of the RyR2 mutable regions (N-terminal, central, and channel pore). Diastolic calcium (Ca) leak via RyR2 can cause lethal arrhythmia such as CPVT. To further understand the pathophysiology of RyR2 channel pore mutation on calmodulin (CaM)-RyR2 affinity in 3 distinct CPVT mouse models. We tested the hypothesis that RyR2 channel pore mutation increases Ca leak by reduced CaM affinity in distinct knock-in (KI) mouse models with CPVT-associated RyR2 mutation (R176Q/+, R2474S/+, and R4496C/+). Ventricular cardiomyocytes were isolated from hearts of 3 KI and wild-type (WT) mice. Fluorescence resonance energy transfer between donor-F-FKBP (FK506-binding protein 12.6) and acceptor-F-CaM was measured to assess CaM-RyR2 binding in saponin-permeabilized myocytes by using confocal microscopy. Under resting conditions (without cAMP), CaM-RyR2 affinity was unaltered in WT and all KI. Under stress conditions (with cAMP), CaM-RyR2 affinity was unchanged in R4496C/+ KI and WT, but reduced in R176Q/+ and R2474S/+. These findings not only provide novel understanding of the effects of RyR2 channel pore mutation and CaM-RyR2 binding in CPVT, but also serve to advance the knowledge of potential therapeutic targets for this life-threatening arrhythmia.

Calcium and Excitation-Contraction Coupling in the Heart.

Cardiac contractility is regulated by changes in intracellular Ca concentration ([Ca2+]i). Normal function requires that [Ca2+]i be sufficiently high in systole and low in diastole. Much of the Ca needed for contraction comes from the sarcoplasmic reticulum and is released by the process of calcium-induced calcium release. The factors that regulate and fine-tune the initiation and termination of release are reviewed. The precise control of intracellular Ca cycling depends on the relationships between the various channels and pumps that are involved. We consider 2 aspects: (1) structural coupling: the transporters are organized within the dyad, linking the transverse tubule and sarcoplasmic reticulum and ensuring close proximity of Ca entry to sites of release. (2) Functional coupling: where the fluxes across all membranes must be balanced such that, in the steady state, Ca influx equals Ca efflux on every beat. The remainder of the review considers specific aspects of Ca signaling, including the role of Ca buffers, mitochondria, Ca leak, and regulation of diastolic [Ca2+]i.

Calcium/Calmodulin Protein Kinase II-Dependent Ryanodine Receptor Phosphorylation Mediates Cardiac Contractile Dysfunction Associated With Sepsis.

Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca loss that mediate altered Ca handling and contractile dysfunction associated with sepsis. Randomized controlled trial. Research laboratorySUBJECTS:: Male wild type and transgenic miceINTERVENTIONS:: We induced sepsis in mice using the colon ascendens stent peritonitis model. Twenty-four hours after colon ascendens stent peritonitis surgery, we observed that wild type mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction, and fractional shortening (ejection fraction %, 54.76 ± 0.67; fractional shortening %, 27.53 ± 0.50) compared with sham controls (ejection fraction %, 73.57 ± 0.20; fractional shortening %, 46.75 ± 0.38). At the cardiac myocyte level, colon ascendens stent peritonitis cells showed reduced cell shortening, Ca transient amplitude and sarcoplasmic reticulum Ca content compared with sham cardiomyocytes. Colon ascendens stent peritonitis hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II activity, which could be prevented by pretreating animals with the antioxidant tempol. Pharmacologic inhibition of calcium and calmodulin-dependent protein kinase II with 2.5 µM of KN93 prevented the decrease in cell shortening, Ca transient amplitude, and sarcoplasmic reticulum Ca content in colon ascendens stent peritonitis myocytes. Contractile function was also preserved in colon ascendens stent peritonitis myocytes isolated from transgenic mice expressing a calcium and calmodulin-dependent protein kinase II inhibitory peptide (AC3-I) and in colon ascendens stent peritonitis myocytes isolated from mutant mice that have the ryanodine receptor 2 calcium and calmodulin-dependent protein kinase II-dependent phosphorylation site (serine 2814) mutated to alanine (S2814A). Furthermore, colon ascendens stent peritonitis S2814A mice showed preserved ejection fraction and fractional shortening (ejection fraction %, 73.06 ± 6.31; fractional shortening %, 42.33 ± 5.70) compared with sham S2814A mice (ejection fraction %, 71.60 ± 4.02; fractional shortening %, 39.63 ± 3.23). Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca content, Ca transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis.

The Role of RyR2 Intersubunit Cross-Linking in SR Ca Mishandling in Failing Heart

We previously reported that oxidation of RyR2 caused formation of disulfide bonds between two subunits within the channel - intersubunit cross-linking (XL). Because intersubunit dynamics within the RyR2 determines the channel gating, XL should play an important role in RyR2 regulation. We found a strong positive correlation between the degree of RyR2 XL and sarcoplasmic reticulum (SR) Ca leak in rabbit ventricular myocytes. When the XL reached the maximum level, further oxidation of RyR2 did not enhance SR Ca leak.