Ryanodine Receptor Phosphorylation Research Articles

Ca2+/calmodulin-dependent kinase IIδC-induced chronic heart failure does not depend on sarcoplasmic reticulum Ca2+ leak.

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

KUS121, a VCP modulator, has an ameliorating effect on acute and chronic heart failure without calcium loading via maintenance of intracellular ATP levels

AimsAs heart failure (HF) progresses, ATP levels in myocardial cells decrease, and myocardial contractility also decreases. Inotropic drugs improve myocardial contractility but increase ATP consumption, leading to poor prognosis. Kyoto University Substance 121 (KUS121) is known to selectively inhibit the ATPase activity of valosin-containing protein, maintain cellular ATP levels, and manifest cytoprotective effects in several pathological conditions. The aim of this study is to determine the therapeutic effect of KUS121 on HF models. Methods and resultsCultured cell, mouse, and canine models of HF were used to examine the therapeutic effects of KUS121. The mechanism of action of KUS121 was also examined. Administration of KUS121 to a transverse aortic constriction (TAC)-induced mouse model of HF rapidly improved the left ventricular ejection fraction and improved the creatine phosphate/ATP ratio. In a canine model of high frequency-paced HF, administration of KUS121 also improved left ventricular contractility and decreased left ventricular end-diastolic pressure without increasing the heart rate. Long-term administration of KUS121 to a TAC-induced mouse model of HF suppressed cardiac hypertrophy and fibrosis. In H9C2 cells, KUS121 reduced ER stress. Finally, in experiments using primary cultured cardiomyocytes, KUS121 improved contractility and diastolic capacity without changing peak Ca2+ levels or contraction time. These effects were not accompanied by an increase in cyclic adenosine monophosphate or phosphorylation of phospholamban and ryanodine receptors. ConclusionsKUS121 ameliorated HF by a mechanism totally different from that of conventional catecholamines. We propose that KUS121 is a promising new option for the treatment of HF.

5-Methoxytryptophan alleviates atrial structural remodeling in ibrutinib-associated atrial fibrillation

BackgroundIbrutinib is an effective and well-tolerated treatment for B-cell lymphomas but is associated with an increased risk of atrial fibrillation (AF) by altering the structure of the atrium. 5-Methoxytryptophan (5-MTP) inhibits inflammatory and fibrotic processes. This study aimed to determine the effects and mechanisms of 5-MTP on the underlying mechanisms of AF caused by ibrutinib. MethodsThe effect of 5-MTP on ibrutinib-related AF was investigated in male Sprague Dawley rats using echocardiographic, electrophysiological, immunofluorescent, Masson staining, and molecular analyses. RusultsThe ibrutinib+5-MTP group showed (1) a lower incidence and shorter duration of AF and accelerated atrial conduction; (2) a decreased left atrial mass and left atrial diameter; (3) decreased myocardial fibrosis in the left atrium; (4) lower atrial inflammation; (5) increased sarcoplasmic reticulum Ca2+-ATPase 2a protein expression, decreased phosphorylation of phospholamban at Thr17, and decreased sodium/calcium exchanger 1 protein expression and phosphorylation of ryanodine receptor 2 at S2814; and (6) decreased phosphorylation of CaMKII expression. 5-MTP treatment markedly activated the PI3K-Akt signaling. Inhibiting PI3K-Akt signaling significantly reversed the protective effect of 5-MTP on ibrutinib-related AF. ConclusionsThese findings suggest that 5-MTP administration decreases the vulnerability of ibrutinib-related AF mainly caused by ameliorated maladaptive left atrial remodeling and dysregulation of calcium handling proteins. Mechanistically, 5-MTP treatment markedly enhanced the activation of cardiac PI3K-Akt signaling.

Electrical and Structural Insights into Right Ventricular Outflow Tract Arrhythmogenesis.

The right ventricular outflow tract (RVOT) is the major origin of ventricular arrhythmias, including premature ventricular contractions, idiopathic ventricular arrhythmias, Brugada syndrome, torsade de pointes, long QT syndrome, and arrhythmogenic right ventricular cardiomyopathy. The RVOT has distinct developmental origins and cellular characteristics and a complex myocardial architecture with high shear wall stress, which may lead to its high vulnerability to arrhythmogenesis. RVOT myocytes are vulnerable to intracellular sodium and calcium overload due to calcium handling protein modulation, enhanced CaMKII activity, ryanodine receptor phosphorylation, and a higher cAMP level activated by predisposing factors or pathological conditions. A reduction in Cx43 and Scn5a expression may lead to electrical uncoupling in RVOT. The purpose of this review is to update the current understanding of the cellular and molecular mechanisms of RVOT arrhythmogenesis.

Cardiac Protein Kinase D1 ablation alters the myocytes β-adrenergic response

β-adrenergic (β-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute β-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte β-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal β-adrenergic responses in Ca2+ handling.

Enzastaurin cardiotoxicity: QT interval prolongation, negative inotropic responses and negative chronotropic action

Several clinical trials observed that enzastaurin prolonged QT interval in cancer patients. However, the mechanism of enzastaurin-induced QT interval prolongation is unclear. Therefore, this study aimed to assess the effect and mechanism of enzastaurin on QT interval and cardiac function. The Langendorff and Ion-Optix MyoCam systems were used to assess the effects of enzastaurin on QT interval, cardiac systolic function and intracellular Ca2+ transient in guinea pig hearts and ventricular myocytes. The effects of enzastaurin on the rapid delayed rectifier (IKr), the slow delayed rectifier K+ current (IKs), transient outward potassium current (Ito), action potentials, Ryanodine Receptor 2 (RyR2) and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) expression and activity in HEK 293 cell system and primary cardiomyocytes were investigated using whole-cell recording technique and western blotting. We found that enzastaurin significantly prolonged QT interval in guinea pig hearts and increased the action potential duration (APD) in guinea pig cardiomyocytes in a dose-dependent manner. Enzastaurin potently inhibited IKr by binding to the human Ether-à-go-go-Related gene (hERG) channel in both open and closed states, and hERG mutant channels, including S636A, S631A, and F656V attenuated the inhibitory effect of enzastaurin. Enzastaurin also moderately decreased IKs. Additionally, enzastaurin also induced negative chronotropic action. Moreover, enzastaurin impaired cardiac systolic function and reduced intracellular Ca2+ transient via inhibition of RyR2 phosphorylation. Taken together, we found that enzastaurin prolongs QT, reduces heart rate and impairs cardiac systolic function. Therefore, we recommend that electrocardiogram (ECG) and cardiac function should be continuously monitored when enzastaurin is administered to cancer patients.

Spatial Distribution of Calcium Sparks Determines Their Ability to Induce Afterdepolarizations in Human Atrial Myocytes

Analysis of the spatio-temporal distribution of calcium sparks showed a preferential increase in sparks near the sarcolemma in atrial myocytes from patients with atrial fibrillation (AF), linked to higher ryanodine receptor (RyR2) phosphorylation at s2808 and lower calsequestrin-2 levels. Mathematical modeling, incorporating modulation of RyR2 gating, showed that only the observed combinations of RyR2 phosphorylation and calsequestrin-2 levels can account for the spatio-temporal distribution of sparks in patients with and without AF. Furthermore, we demonstrate that preferential calcium release near the sarcolemma is key to a higher incidence and amplitude of afterdepolarizations in atrial myocytes from patients with AF.

Nanoscale organization of ryanodine receptor distribution and phosphorylation pattern determines the dynamics of calcium sparks.

Super-resolution imaging techniques have provided a better understanding of the relationship between the nanoscale organization and function of ryanodine receptors (RyRs) in cardiomyocytes. Recent data have indicated that this relationship is disrupted in heart failure (HF), as RyRs are dispersed into smaller and more numerous clusters. However, RyRs are also hyperphosphorylated in this condition, and this is reported to occur preferentially within the cluster centre. Thus, the combined impact of RyR relocalization and sensitization on Ca2+ spark generation in failing cardiomyocytes is likely complex and these observations suggest that both the nanoscale organization of RyRs and the pattern of phosphorylated RyRs within clusters could be critical determinants of Ca2+ spark dynamics. To test this hypothesis, we used computational modeling to quantify the relationships between RyR cluster geometry, phosphorylation patterns, and sarcoplasmic reticulum (SR) Ca2+ release. We found that RyR cluster disruption results in a decrease in spark fidelity and longer sparks with a lower amplitude. Phosphorylation of some RyRs within the cluster can play a compensatory role, recovering healthy spark dynamics. Interestingly, our model predicts that such compensation is critically dependent on the phosphorylation pattern, as phosphorylation localized within the cluster center resulted in longer Ca2+ sparks and higher spark fidelity compared to a uniformly distributed phosphorylation pattern. Our results strongly suggest that both the phosphorylation pattern and nanoscale RyR reorganization are critical determinants of Ca2+ dynamics in HF.

Role of TLR4 signaling on Porphyromonas gingivalis LPS-induced cardiac dysfunction in mice.

Oral infections, particularly periodontitis, are a well-established risk factor for cardiovascular diseases, although the molecular mechanisms involved remain elusive. The aims of the present study were to investigate the effects of lipopolysaccharide derived from Porphyromonas gingivalis (PG-LPS) on cardiac function in mice, and to elucidate the underlying mechanisms. Mice (C57BL/6) were injected with PG-LPS (0.8 mg/kg/day) with or without an inhibitor of Toll-like receptor 4 (TLR4) signaling (TAK-242, 0.8 mg/kg/day) for 4 weeks. Left ventricular ejection function was significantly decreased at 1 week (from 67 ± 0.5 to 58 ± 1.2%) and remained low at 4 weeks (57 ± 1.0%). The number of apoptotic myocytes was increased (approximately 7.4-fold), the area of fibrosis was increased (approximately 3.3-fold) and the number of 8-hydroxydeoxyguanosine-positive myocytes, a sensitive indicator of oxidative DNA damage, was increased (approximately 7.6-fold) at 4 weeks in the heart of PG-LPS treated mice. However, levels of various serum pro-inflammatory cytokines in PG-LPS-treated mice were similar to those in control mice. The impairment of cardiac function in PG-LPS-treated mice appears to involve activation of TLR4-NADPH oxidase (NOX) 4 signaling, leading to abundant production of reactive oxygen species and Ca2+ leakage from sarcoplastic reticulumn induced by calmodulin kinase II (CaMKII)-mediated phosphorylation of phospholamban (at Thr-17) and ryanodine receptor 2 (at Ser-2448). Pharmacological inhibition of TLR4 with TAK-242 attenuated the changes in cardiac function in PG-LPS-treated mice. Our results indicate that TLR4-NOX4 signaling may be a new therapeutic target for treatment of cardiovascular diseases in patients with periodontitis.

Phosphorylation of cardiac ryanodine receptor (RyR2): a structure-function study of phosphomimetic and phospho-null triple mutants

The activity of ryanodine receptor type 2 (RyR2), a key player in cardiac contractility, is modulated by various post-translational modifications. Phosphorylation at multiple sites mediated through a dynamic balance between various site-specific kinases and phosphatases was proposed as an important regulator, but the subject remains contentious. Ser2808 is thought to be phosphorylated by protein kinase A (PKA) and Ca2+/calmodulin dependent protein kinase II (CaMKII), while Ser2814 and Ser2030 are phosphorylated mainly by CaMKII and PKA, respectively.

Nanoscale organization of ryanodine receptor distribution and phosphorylation pattern determines the dynamics of calcium sparks

Superresolution imaging techniques have provided a better understanding of the relationship between nanoscale organization and function of the ryanodine receptors (RyRs) and, consequently, the impact of organization on calcium spark properties in heart failure (HF). While hyperphosphorylation of RyRs is generally observed during HF, the impact of RyR phosphorylation on calcium (Ca2+) sparks remains incompletely understood because several experimental studies show seemingly contradictory results.

Interplay between mineral bone disorder and cardiac damage in acute kidney injury: from Ca2+ mishandling and preventive role of Klotho in mice to its potential mortality prediction in human

Biomarkers of mineral bone disorders (MBD) including phosphorus, fibroblast growth factor (FGF)-23 and Klotho are strongly altered in patients with acute kidney injury (AKI) who have high cardiac outcomes and mortality rates. However, the crosslink between MBD and cardiac damage after an AKI episode still remains unclear. We tested MBD and cardiac biomarkers in an experimental AKI model after 24 or 72 hours of folic acid injection and we analyzed structural cardiac remodeling, intracellular calcium (Ca2+) dynamics in cardiomyocytes and cardiac rhythm. AKI mice presented high levels of FGF-23, phosphorus and cardiac troponin T and exhibited a cardiac hypertrophy phenotype accompanied by an increase in systolic Ca2+ release 24 hours after AKI. Ca2+ transients and contractile dysfunction were reduced 72 hours after AKI while diastolic sarcoplasmic reticulum Ca2+ leak, pro-arrhythmogenic Ca2+ events and ventricular arrhythmias were increased. These cardiac events were linked to the activation of the calcium/calmodulin-dependent kinase II pathway through the increased phosphorylation of ryanodine receptors and phospholamban specific sites after AKI. Cardiac hypertrophy and the altered intracellular Ca2+ dynamics were prevented in transgenic mice overexpressing Klotho after AKI induction. In a translational retrospective longitudinal clinical study, we determined that combining FGF-23 and phosphorus with cardiac troponin T levels achieved a better prediction of mortality in AKI patients at hospital admission. Thus, monitoring MBD and cardiac damage biomarkers could be crucial to prevent mortality in AKI patients. In this setting, Klotho might be considered as a new cardioprotective therapeutic tool to prevent deleterious cardiac events in AKI conditions.

A junctional cAMP compartment regulates rapid Ca2+ signaling in atrial myocytes

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following β-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3′,5′-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas β-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas β-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Abstract 12954: A Genotype-Phenotype Link Between Cardiac Monoamine Oxidase Activity and Arrhythmogenic Potential Using a Translational Approach

Introduction: Monoamine oxidase (MAO) has emerged as an intriguing drug target for heart disease because of the toxicity of the oxidative catechol metabolites produced. Our group has recently shown that a high rate of MAO activity in atrial myocardium is associated with a greater incidence of postoperative atrial fibrillation (POAF) following coronary artery bypass graft surgery (CABG). Hypothesis: Cardiac MAO deficiency lowers arrhythmogenic potential. Methods and Results: In a cohort of age-matched CABG patients (N=288, 26% female, 20% African-American), we performed DNA-sequencing analysis of maoA,B while assessing maximal MAO-A and -B enzyme activity with norepinephrine (NE) in fresh atrial tissue from a subset (N=110) of these patients. Patients homozygous for maoA synonymous variant rs1800464, and maoB intron variant rs5905512, had lower NE-supported MAO activity ( Figure 1 , 0 = ref, 1=het, 2=homoz) and lower incidence of POAF (OR 0.6 ± 0.1 and 0.9 ± 0.02, respectively). Mice with cardiomyocyte-specific MAO-A deficiency (cMAOA -/- ) had 2-fold lower rates of epinephrine/caffeine-induced ventricular tachycardia (VT) compared with wild-type ( Figure 2 , *P<0.05). Moreover, cardiomyocytes from cMAOA -/- mice had increased fractional shortening, faster relaxation and shorter Ca 2+ transient duration (P<0.05). Alterations in Ca 2+ handling in the cMAOA -/- mice were accompanied by decreased epinephrine-induced phosphorylation of CamKII and ryanodine receptor (P<0.05). Conclusions: Collectively, these findings suggest that decreasing MAO activity lowers arrhythmogenic potential in the heart, and that cardiac MAO may be a therapeutic target for POAF.

Structural basis for diamide modulation of ryanodine receptor

Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, we solved the cryo-electron microscopy (cryo-EM) structure of RYR in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo-voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. We also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, we solved the crystal structures of several RYR domains from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, we found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. Our structures provide a foundation for developing novel pesticides to overcome the resistance crisis.

Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

Influence of sex on intracellular calcium homoeostasis in patients with atrial fibrillation

AimsAtrial fibrillation (AF) has been associated with intracellular calcium disturbances in human atrial myocytes, but little is known about the potential influence of sex and we here aimed to address this issue.Methods and resultsAlterations in calcium regulatory mechanisms were assessed in human atrial myocytes from patients without AF or with long-standing persistent or permanent AF. Patch-clamp measurements revealed that L-type calcium current (ICa) density was significantly smaller in males with than without AF (−1.15 ± 0.37 vs. −2.06 ± 0.29 pA/pF) but not in females with AF (−1.88 ± 0.40 vs. −2.21 ± 0.0.30 pA/pF). In contrast, transient inward currents (ITi) were more frequent in females with than without AF (1.92 ± 0.36 vs. 1.10 ± 0.19 events/min) but not in males with AF. Moreover, confocal calcium imaging showed that females with AF had more calcium spark sites than those without AF (9.8 ± 1.8 vs. 2.2 ± 1.9 sites/µm2) and sparks were wider (3.0 ± 0.3 vs. 2.2 ± 0.3 µm) and lasted longer (79 ± 6 vs. 55 ± 8 ms), favouring their fusion into calcium waves that triggers ITIs and afterdepolarizations. This was linked to higher ryanodine receptor phosphorylation at s2808 in women with AF, and inhibition of adenosine A2A or beta-adrenergic receptors that modulate s2808 phosphorylation was able to reduce the higher incidence of ITI in women with AF.ConclusionPerturbations of the calcium homoeostasis in AF is sex-dependent, concurring with increased spontaneous SR calcium release-induced electrical activity in women but not in men, and with diminished ICa density in men only.

The Role of CaMKII Overexpression and Oxidation in Atrial Fibrillation-A Simulation Study.

This simulation study aims to investigate how the Calcium/calmodulin-dependent protein kinase II (CaMKII) overexpression and oxidation would influence the cardiac electrophysiological behavior and its arrhythmogenic mechanism in atria. A new-built CaMKII oxidation module and a refitted CaMKII overexpression module were integrated into a mouse atrial cell model for analyzing cardiac electrophysiological variations in action potential (AP) characteristics and intracellular Ca2+ cycling under different conditions. Simulation results showed that CaMKII overexpression significantly increased the phosphorylation level of its downstream target proteins, resulting in prolonged AP and smaller calcium transient amplitude, and impaired the Ca2+ cycling stability. These effects were exacerbated by extra reactive oxygen species, which oxidized CaMKII and led to continuous high CaMKII activation in both systolic and diastolic phases. Intracellular Ca2+ depletion and sustained delayed afterdepolarizations (DADs) were observed under co-existing CaMKII overexpression and oxidation, which could be effectively reversed by clamping the phosphorylation level of ryanodine receptor (RyR). We also found that the stability of RyR release highly depended on a delicate balance between the level of RyR phosphorylation and sarcoplasmic reticulum Ca2+ concentration, which was closely related to the genesis of DADs. We concluded that the CaMKII overexpression and oxidation have a synergistic role in increasing the activity of CaMKII, and the unstable RyR may be the key downstream target in the CaMKII arrhythmogenic mechanism. Our simulation provides detailed mechanistic insights into the arrhythmogenic effect of CaMKII overexpression and oxidation, which suggests CaMKII as a promising target in the therapy of atrial fibrillation.

Abstract 16594: Disruption of Caveolae-Confined Phosphorylation of Ryanodine Receptors Promotes Ca 2+ Handling Abnormalities and Arrhythmogenesis in Mouse Atrial Myocytes

Rationale: Chronic atrial fibrillation has been linked to ectopic triggered activity driven by delayed afterdepolarizations in response to sarcoplasmic reticulum Ca 2+ leak from certain areas in atria. These activities were associated with hyperphosphorylated ryanodine receptors (RyRs), while the underlying mechanisms remain uncertain. Here, we hypothesized that mouse atria have areas with lower transversal-axial tubule system (TATS) organization, where phosphorylation of RyRs is mainly confines by caveolae nanodomains and might be firstly disturbed in disease conditions. Methods and results: In wild type (WT) mouse intact atria stained with RH-237, we found that myocytes located in the intercaval region (ICR, between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum) have a significantly less density of TATS than right atrial appendage (RAA) myocytes: 5.7±0.4% in ICR vs. 13.4±0.9% in RAA, P<0.01. Also an elevated frequency of spontaneous Ca 2+ sparks was observed in myocytes isolated from ICR vs. RAA (12.5±2.6 vs. 1.5±0.3 sparks/μm/s, P<0.01). ICR myocytes isolated from mice with a cardiac-specific knockout of the main structural protein of caveolae, caveolin-3, showed significant increase of Ca 2+ sparks frequency as compared to WT (27.3±4.1 sparks/μm/s, P<0.01). Immunofluorescence staining of RyRs, phosphorylated at protein kinase A/ Ca 2+ /calmodulin-dependent protein kinase-II phosphorylation sites: Ser 2808 -RyR and Ser 2814 -RyR, showed predominant subsarcolemmal localization, with a high co-localization with caveolin-3, in ICR and RAA cells. Myocytes isolated from caveolin-3 knockout mice showed extension of both Ser 2808- RyR and Ser 2808- RyR to whole-cell wide striated pattern in ICR, but not RAA. This redistribution was associated with development of delayed afterdepolarizations in ICR but not RAA cells. Conclusions: Our findings demonstrate that in ICR in mouse atria, caveolae form functional, spatially-confined cAMP nanodomains and control localized RyR phosphorylation. Disruption of caveolae structures, seen in various pathologies, may lead to hyperphosphorylation of RyRs promoting Ca 2+ handling abnormalities and arrhythmogenic triggered activity.