Background-Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias.Although abnormal Ca 2ϩ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca 2ϩ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca 2ϩ /calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results-Mice in which the S2814 Ca 2ϩ /calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca 2ϩ release events, resulting in reduced sarcoplasmic reticulum Ca 2ϩ load on confocal microscopy. These Ca 2ϩ 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 2ϩ /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 2ϩ /calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca 2ϩ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure. (Circulation. 2010;122:2669-2679.)Key Words: arrhythmia Ⅲ calcium Ⅲ calcium-calmodulin-dependent protein kinase type 2 Ⅲ heart failure Ⅲ ryanodine receptor calcium release channel Ⅲ sarcoplasmic reticulum C ongestive heart failure (HF) is a leading cause of mortality and morbidity worldwide. Approximately 50% of HF patients die of sudden cardiac death (SCD) attributed to ventricular arrhythmias (Ͼ300 000 in the United States annually). 1,2 A large fraction of these arrhythmias are thought to be initiated by focal triggered mechanisms, such as spontaneous diastolic Ca 2ϩ release from cardiac myocyte ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR), which activates an arrhythmogenic depolarizing inward Na ϩ /Ca 2ϩ exchange (NCX) current. 3,4 Indeed, in HF there is enhanced diastolic SR Ca 2ϩ release and other changes in electrophysiological substrate that greatly enhance the propensity for triggered arrhythmias. Likewise, patients with inherited RyR2 point mutations exhibit catecholaminergic polymorphic ventricular tachycardia, a known cause of SCD with sensitivity to adrenergic conditions such as exercise or stress. 5,6 HF is a chronic hyperadrenergic state, and a prominent theory suggested that ␤-adrenergic activation of protein kinase A (PKA) destabilized ...

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 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, N98S) or LQTS (D96V, D130G, 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 (100nM) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared to wild-type (WT) 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:WT-CaM activated Ca waves, demonstrating functional dominance. By contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2 binding affinity (D130G, 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.

Key Words: FKBP12.6 Ⅲ FKBP12 Ⅲ Ca sparks Ⅲ binding properties Ⅲ rapamycin Ⅲ RyR2 C ardiac ryanodine receptors (RyR2) are sarcoplasmic reticulum (SR) Ca release channels, crucial in excitation-contraction coupling. 1 Dysfunctional RyR2, exhibiting enhanced Ca leak has been implicated in arrhythmogenesis and heart failure (HF). 1 Homotetrameric RyR2s have a transmembrane channel domain, and regulatory and scaffolding cytoplasmic domain.In heart, FK506 binding protein (FKBP) isoforms FKBP12 and FKBP12.6 are coexpressed and can bind RyR2 at a stoichiometry of 4 FKBP per RyR tetramer. 2,3 FKBP12 binds RyR2 with much lower affinity, but is much higher in concentration in heart than FKBP12.6. 4 Nevertheless, FKBP12 and 12.6 share 85% sequence homology and similar 3D structures. 5 Human and rat FKBP12 differ in only 3 residues, and human and rat FKBP12.6 are identical. This makes study of human FKBP function in rat myocytes reasonable.Effects of FKBPs on RyR2 activity in myocytes are controversial. Some groups reported that dissociation of FKBP12.6 from RyR2 by immunosuppressants (rapamycin or FK506) activated RyR2 channels and induced subconductance states. 6 -8 RyR2 point mutations associated with cardiac sudden death may also exhibit altered FKBP12.6 interaction, 9 and FK506 can alter resting Ca 2ϩ spark frequency (CaSpF) and SR Ca 2ϩ content. 7,8,10 FKBP12.6 overexpression also increased SR load and enhanced contraction. 11 However, others reported that FKBP12.6 removal had no effect on RyR2 activity, 3,12 and failed to observe RyR2 subconductance states in channels from FKBP12.6 knockout mice. 13,14 Some groups suggest that FKBP12 affects RyR2 differently from FKBP12.6. [15][16][17]20 Therefore, FKBP12/12.6 binding and RyR2 effects remain unclear, especially in the cardiomyocyte environment (as studied here).Altered FKBP-RyR2 interaction is a prominent hypothesis explaining increased SR Ca leak in HF via RyR2 hyperphos- Here we characterize in the myocyte environment the interaction between FKBP12/12.6 and RyR2, its functional consequences, and modulation by PKA-dependent phosphorylation. Using fluorescent FKBP12/12.6 (F-FKBP), we simultaneously assessed physical association-dissociation of FKBP12/12.6 with RyR2, and RyR2 activity (via Ca sparks) in permeabilized ventricular myocytes. We found that: (1) both FKBP12.6 and FKBP12 bound to RyR2 (K d Ϸ1 nmol/L and 200 nmol/L, respectively); (2) FKBP12.6 but not FKBP12 inhibited CaSpF; (3) the binding properties of FKBP12.6/12-RyR2 were not changed by PKA-dependent phosphorylation of RyR2; and (4) endogenous FKBP12 was Ϸ1 mol/L cytosol and FKBP12.6 Ϸ100 nmol/L cytosol. MethodsRat and mouse ventricular myocytes were isolated and permeabilized as previously described (see the Online Data Supplement, available at http://circres.ahajournals.org). 23 F-FKBP12.6 and F-FKBP12 were characterized using circular dichroism spectroscopy and ligandbinding studies (Online Figure I). 24 Consistent with previous reports, 25 F-FKBP constructs have the same secondary structure, RyR ...

Rationale Calmodulin (CaM) associates with cardiac ryanodine receptors (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure (HF), cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the in situ binding properties for CaM-RyR2 are unknown. Objective We sought to measure the in situ binding affinity and kinetics for CaM-RyR2 in normal and HF ventricular myocytes, estimate the percentage of Z-line localized CaM that is RyR2-bound and test cellular function of defective CaM-RyR2 interaction. Methods & Results Using FRET (fluorescence resonance energy transfer) in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets, and measured CaMRyR2 binding affinity in situ (Kd =10-20 nM). Using RyR2ADA/+ knock-in (KI) mice, in which half of the CaM-RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca2+ waves production and stress induced ventricular arrhythmia in RyR2ADA/+ mice. In a post myocardial infarction (MI) rat HF model, we detected a decrease in the CaMRyR2 binding affinity (Kd ≈ 51nM, ~3 fold increase) and unaltered FKBP12.6-RyR2 binding affinity (Kd ≈ 0.8nM). Conclusions CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits SR Ca2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In HF, RyR2 shows decreased CaM affinity, but unaltered FKBP12.6 affinity.

Neurotransmitter release depends critically on close spatial coupling of Ca(2+) entry to synaptic vesicles at the nerve terminal; however, the molecular substrates determining their physical proximity are unknown. Using the calyx of Held synapse, where "microdomain" coupling predominates at immature stages and developmentally switches to "nanodomain" coupling, we demonstrate that deletion of the filamentous protein Septin 5 imparts immature synapses with striking morphological and functional features reminiscent of mature synapses. This includes synaptic vesicles tightly localized to active zones, resistance to the slow Ca(2+) buffer EGTA and a reduced number of Ca(2+) channels required to trigger single fusion events. Disrupting Septin 5 organization acutely transforms microdomain to nanodomain coupling and potentiates quantal output in immature wild-type terminals. These observations suggest that Septin 5 is a core molecular substrate that differentiates distinct release modalities at the central synapse.

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Oxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in heart failure (HF) and arrhythmias. Altered RyR2 domain-domain interaction (domain unzipping) and calmodulin (CaM) binding affinity are allosterically coupled indices of RyR2 conformation. In HF RyR2 exhibits reduced CaM binding, increased domain unzipping and greater SR Ca leak, and dantrolene can reverse these changes. However, effects of oxidative stress on RyR2 conformation and leak in myocytes are poorly understood. We used fluorescent CaM, FKBP12.6, and domain-peptide biosensor (F-DPc10) to measure, directly in cardiac myocytes, (1) RyR2 activation by hydrogen peroxide (H2O2)-induced oxidation, (2) RyR2 conformation change caused by oxidation, (3) CaM-RyR2 and FK506-binding protein (FKBP12.6)-RyR2 interaction upon oxidation, and (4) whether dantrolene affects 1–3. H2O2 was used to mimic oxidative stress. H2O2 significantly increased the frequency of Ca2+ sparks and spontaneous Ca2+ waves, and dantrolene almost completely blocked these effects. H2O2 pretreatment significantly reduced CaM-RyR2 binding, but had no effect on FKBP12.6-RyR2 binding. Dantrolene restored CaM-RyR2 binding but had no effect on intracellular and RyR2 oxidation levels. H2O2 also accelerated F-DPc10-RyR2 association while dantrolene slowed it. Thus, H2O2 causes conformational changes (sensed by CaM and DPc10 binding) associated with Ca leak, and dantrolene reverses these RyR2 effects. In conclusion, in cardiomyocytes, H2O2 treatment markedly reduces the CaM-RyR2 affinity, has no effect on FKBP12.6-RyR2 affinity, and causes domain unzipping. Dantrolene can correct domain unzipping, restore CaM-RyR2 affinity, and quiet pathological RyR2 channel gating. F-DPc10 and CaM are useful biosensors of a pathophysiological RyR2 state.

BackgroundRyanodine receptors (RyR) mediate sarcoplasmic reticulum calcium (Ca2+) release and influence myocyte Ca2+ homeostasis and arrhythmias. In cardiac myocytes, RyRs are found in clusters of various sizes and shapes, and RyR cluster size may critically influence normal and arrhythmogenic Ca2+ spark and wave formation. However, the actual RyR cluster sizes at specific Ca2+ spark sites have never been measured in the physiological setting.Methods and ResultsHere we measured RyR cluster size and Ca2+ sparks simultaneously to assess how RyR cluster size influences Ca2+ sparks and sarcoplasmic reticulum Ca2+ leak. For small RyR cluster sizes (<50), Ca2+ spark frequency is very low but then increases dramatically at larger cluster sizes. In contrast, Ca2+ spark amplitude is nearly maximal even at relatively small RyR cluster size (≈10) and changes little at larger cluster size. These properties agreed with computational simulations of RyR gating within clusters.ConclusionsOur study explains how this combination of properties may limit arrhythmogenic Ca2+ sparks and wave propagation (at many junctions) while preserving the efficacy and spatial synchronization of Ca2+‐induced Ca2+‐release during normal excitation‐contraction coupling. However, variations in RyR cluster size among individual junctions and RyR sensitivity could exacerbate heterogeneity of local sarcoplasmic reticulum Ca2+ release and arrhythmogenesis under pathological conditions.