Spontaneous Ca Release Research Articles

SparkMaster 2: A New Software for Automatic Analysis of Calcium Spark Data.

Calcium (Ca) sparks are elementary units of subcellular Ca release in cardiomyocytes and other cells. Accordingly, Ca spark imaging is an essential tool for understanding the physiology and pathophysiology of Ca handling and is used to identify new drugs targeting Ca-related cellular dysfunction (eg, cardiac arrhythmias). The large volumes of imaging data produced during such experiments require accurate and high-throughput analysis. We developed a new software tool SparkMaster 2 (SM2) for the analysis of Ca sparks imaged by confocal line-scan microscopy, combining high accuracy, flexibility, and user-friendliness. SM2 is distributed as a stand-alone application requiring no installation. It can be controlled using a simple-to-use graphical user interface, or using Python scripting. SM2 is shown to have the following strengths: (1) high accuracy at identifying Ca release events, clearly outperforming previous highly successful software SparkMaster; (2) multiple types of Ca release events can be identified using SM2: Ca sparks, waves, miniwaves, and long sparks; (3) SM2 can accurately split and analyze individual sparks within spark clusters, a capability not handled adequately by prior tools. We demonstrate the practical utility of SM2 in two case studies, investigating how Ca levels affect spontaneous Ca release, and how large-scale release events may promote release refractoriness. SM2 is also useful in atrial and smooth muscle myocytes, across different imaging conditions. SparkMaster 2 is a new, much-improved user-friendly software for accurate high-throughput analysis of line-scan Ca spark imaging data. It is free, easy to use, and provides valuable built-in features to facilitate visualization, analysis, and interpretation of Ca spark data. It should enhance the quality and throughput of Ca spark and wave analysis across cell types, particularly in the study of arrhythmogenic Ca release events in cardiomyocytes.

Pkd1 is necessary for full cardiac AngII-induced hypertrophy but not for full acute AngII-induced calcium handling changes.

The renin-angiotensin system (RAS) regulates blood pressure, extracellular fluid and electrolyte balance, cardiac inotropy, and vascular resistance. In the heart, angiotensin II (AngII) can induce hypertrophy directly by activating Protein Kinase D (PKD), a signaling kinase known to contribute to cardiac remodeling. Little is known, however, about the acute and chronic functional effects of AngII-induced PKD activation on calcium (Ca) handling. To investigate this, we used hearts from cardiac-specific PKD1 knockout (cKO) mice and their WT littermates. We measured Ca transients (CaT) and Ca spark (CaSp) frequencies in cardiomyocytes at 0.5 and 1 Hz pacing under acute and chronic AngII stimulation (100 nM in vitro and 3 mg/kg/day in osmotic minipump, respectively). Sarcoplasmic reticulum (SR) Ca load was assessed by rapid caffeine-induced Ca release. At baseline and after acute AngII treatment, CaT amplitude and tau decay, CaSp frequency and SR Ca load were similar in WT vs. PKD1 cKO. This was also the case for chronic exposure to AngII except that the tau decay was significantly slower in PKD1 cKO myocytes. In agreement with previous reports, echocardiographic measurements revealed that PKD1 cKO mice were also more resistant to chronic AngII-induced hypertrophy (LVPW increase 24.69 ± 2.74% vs. 54.73 ± 7.73 %) and ejection fraction reduction (4.08 ± 1.5% vs. 12.67 ± 0.52%). We conclude that despite PKD being necessary for the remodeling changes induced by chronic AngII, it does not alter the amplitude and kinetics of CaT and spontaneous Ca release, except for the slowed decay kinetics in the PKD1 cKO after chronic AngII treatment and decreased acute SR Ca load.

Abstract P1094: Sexual Dimorphism In Histone Deacetylases Activity Regulates Ventricular Calcium Handling Properties

Introduction: There is a sexual dimorphism in cardiac excitation contraction coupling (ECC) with female ventricular myocytes (F VM ) exhibiting an attenuated basal calcium concentration ([Ca] i ), Ca transient (CaT) amplitude, and a prolonged CaT decay constant (τ). Increased expression levels of the sodium-calcium exchanger (NCX) have been described in F VM however, the role NCX expression plays in sexual dimorphism of physiological and pathophysiological Ca handling properties, and how NCX expression is regulated in a sex specific manner remains to be determined. Here we tested the hypothesis, that a sexual dimorphism in HDAC activity regulates NCX expression and NCXs differential contribution to the CaT adds to the sexual dimorphism in ventricular ECC. Method: [Ca] i was quantified in field stimulated, freshly isolated ventricular myocytes (VMs) from male (M VM ) and female (F VM ) FVB/N mice loaded with the Ca sensitive fluorescent dye Fluo-4/AM. Changes in NCX expression were quantified by qPCR and western blotting. Results: Under basal conditions F VM exhibited an attenuated CaT amplitude (ΔF/F 0 ; M VM :1.8±0.08, F VM :1.4±0.06, p=0.038) and prolonged CaT decay constant (τ: M VM :0.37±0.01ms, F VM :0.46±0.02ms, p<0.01). The caffeine induced Ca transient (CaT caf ) amplitude was comparable in F VM and M VM , but the CaT caf decay constant (τ caf: M VM : 3.34±0.22s ; F VM :4.81±0.36s) was longer in F VM although increased NCX expression was determined on the protein and mRNA level. Consistent with increased NCX expression, F VM showed an attenuated increase in basal [Ca] i (F/F 0 ; M= 60%, F=44%, p=0.0033) and τ (M=0.37±0.01s, F=0.46±0.02s, p<0.0001) when SERCA activity was attenuated (CPA: 10μM). Also during conditions favoring NCX reverse mode ([Na] o =93, 46 mM) F VM exhibited a larger increase in CaT amplitude (ΔF/F 0 ; M=81%, F=133%, p<0.01) and an increased propensity for spontaneous Ca release events. Inhibition of HDAC class II (LMK: 5mg/kg/day) did not change Ca handling properties in F VM but slowed τ (M LMK : 0.44±0.03ms) and τ caf in M VM (M LMK =5.1±0.33s) making them comparable to F VM . Conclusion: The sexual dimorphism in ventricular ECC is mediated by attenuated HDAC activity in F VM s and facilitates NCX Ca overload.

The Purkinje-myocardial junction is the anatomic origin of ventricular arrhythmia in CPVT.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome caused by gene mutations that render RYR2 Ca release channels hyperactive, provoking spontaneous Ca release and delayed afterdepolarizations (DADs). What remains unknown is the cellular source of ventricular arrhythmia triggered by DADs: Purkinje cells in the conduction system or ventricular cardiomyocytes in the working myocardium. To answer this question, we used a genetic approach in mice to knock out cardiac calsequestrin either in Purkinje cells or in ventricular cardiomyocytes. Total loss of calsequestrin in the heart causes a severe CPVT phenotype in mice and humans. We found that loss of calsequestrin only in ventricular myocytes produced a full-blown CPVT phenotype, whereas mice with loss of calsequestrin only in Purkinje cells were comparable to WT mice. Subendocardial chemical ablation or restoration of calsequestrin expression in subendocardial cardiomyocytes neighboring Purkinje cells was sufficient to protect against catecholamine-induced arrhythmias. In silico modeling demonstrated that DADs in ventricular myocardium can trigger full action potentials in the Purkinje fiber, but not vice versa. Hence, ectopic beats in CPVT are likely generated at the Purkinje–myocardial junction via a heretofore unrecognized tissue mechanism, whereby DADs in the ventricular myocardium trigger full action potentials in adjacent Purkinje cells.

Abstract 15420: Resolving a Controversy: Inhibition of Cardiac Ryanodine Receptors is the Principal Mechanism of Antiarrhythmic Action of Flecainide in Catecholaminergic Polymorphic Ventricular Tachycardia

Rationale: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive cardiac ryanodine receptor (RyR2) calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro , reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide’s efficacy in CPVT. Objective: To determine whether RyR2 block independently contributes to flecainide’s efficacy for suppressing spontaneous sarcoplasmic reticulum (SR) Ca release and for preventing ventricular tachycardia in vivo . Methods and Results: We synthesized N -methyl flecainide analogues (QX-FL and NM-FL) and showed that N -methylation reduces flecainide’s inhibitory potency on RyR2 channels but not on cardiac sodium channels. Antiarrhythmic efficacy was tested utilizing a calsequestrin knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes — lacking intact sarcolemma and devoid of sodium channel contribution — flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin (TTX) to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous SR Ca release, while QX-FL and NM-FL did not. In vivo , flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas NM-FL did not, despite comparable sodium channel block. Conclusions: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone was not enough to prevent arrhythmias. Hence, RyR2 inhibition by flecainide is critical for its mechanism of antiarrhythmic action.

Activation of RyR2 by class I kinase inhibitors.

Kinase inhibitors are a common treatment for cancer. Class I kinase inhibitors that target the ATP-binding pocket are particularly prevalent. Many of these compounds are cardiotoxic and can cause arrhythmias. Spontaneous release of Ca2+ via cardiac ryanodine receptors (RyR2), through a process termed store overload-induced Ca2+ release (SOICR), is a common mechanism underlying arrhythmia. We explored whether class I kinase inhibitors could modify the activity of RyR2 and trigger SOICR to determine if this contributes to the cardiotoxic nature of these compounds. The impact of class I and II kinase inhibitors on SOICR was studied in HEK293 cells and ventricular myocytes using single-cell Ca2+ imaging. A specific effect on RyR2 was confirmed using single channel recordings. Ventricular myocytes were also used to determine if drug-induced changes in SOICR could be reversed using anti-SOICR agents. Class I kinase inhibitors increased the propensity of SOICR. Single channel recording showed that this was due to a specific effect on RyR2. Class II kinase inhibitors decreased the activity of RyR2 at the single channel level but had little effect on SOICR. The promotion of SOICR mediated by class I kinase inhibitors could be reversed using the anti-SOICR agent VK-II-86. Part of the cardiotoxicity of class I kinase inhibitors can be assigned to their effect on RyR2 and increase in SOICR. Compounds with anti-SOICR activity may represent an improved treatment option for patients.

Generation and Regulation of Spontaneous Contractions in theProstate.

Spontaneous myogenic contractions have been shown to be significantly upregulated in prostate tissue collected from men with Benign Prostatic Hyperplasia (BPH), an extremely common disorder of the ageing male. Although originally thought likely to be involved in 'housekeeping' functions, mixing prostatic secretions to prevent stagnation, these spontaneous myogenic contractions provide a novel opportunity to understand and treat BPH. This treatment potential differs from previous models, which focused exclusively on attenuating nerve-mediated neurogenic contractions. Previous studies in the rodent prostate have provided an insight into the mechanisms underlying the regulation of myogenic contractions. 'Prostatic Interstitial Cells' (PICs) within the prostate appear to generate pacemaker potentials, which arise from the summation of number of spontaneous transient depolarisations triggered by the spontaneous release of Ca2+ from internal stores and the opening of Ca2+-activated Cl- channels. Pacemaker potentials then conduct into neighbouring smooth muscle cells to generate spontaneous slow waves. These slow waves trigger the firing of 'spike-like' action potentials, Ca2+ entry and contraction, which are not attenuated by blockers of neurotransmission. However, these spontaneous prostatic contractions can be modulated by the autonomic nervous system. Here, we discuss the mechanisms underlying rodent and human prostate myogenic contractions and the actions of existing and novel pharmacotherapies for the treatment of BPH. Understanding the generation of human prostatic smooth muscle tone will confirm the mechanism of action of existing drugs, inform the identification and effectiveness of new pharmacotherapies, as well as predict patient outcomes.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

Intercellular uncoupling and Ca2+ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (Vm) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes. We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local Vm and intracellular [Ca] simultaneously. We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized Vm in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed Vm depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models. Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize Vm and trigger action potentials.

Dynamics of sodium current mediated early afterdepolarizations.

Early afterdepolarizations (EADs) have been attributed to two primary mechanisms: 1) recovery from inactivation of the L-type calcium (Ca) channel and/or 2) spontaneous Ca release, which depolarizes the membrane potential through the electrogenic sodium-calcium exchanger (NCX). The sodium (Na) current (INa), especially the late component of the Na current, has been recognized as an important player to set up the conditions for EADs by reducing repolarization reserve and increasing intracellular Na concentration, which leads to Ca overload. However, INa itself has not been considered as a direct initiator of EADs. A recent experimental study by Horvath et al. has shown that the amplitude of the late component of the Na current is as large as potassium (K) and Ca currents (∼1 pA/pF). This result suggests that INa by itself can exceeds the sum of outward currents and depolarize the membrane potential. In this study, we show that INa can also directly initiate EADs. Mathematical analysis reveals a fundamental dynamical origin of EADs arising directly from the Na channel reactivation. This system has three fixed points. The dynamics of the INa mediated EAD oscillation is different from that of the membrane voltage oscillation of the pacemaker cell, which has only one fixed point.

Arrhythmogenic Delayed Afterdepolarizations Are Promoted by Severe Hypothermia But Not Therapeutic Hypothermia.

Severe hypothermia (SH) is known to be arrhythmogenic, but the effect of therapeutic hypothermia (TH) on arrhythmias is unclear. It is hypothesized that susceptibility to Ca-mediated arrhythmia triggers would be increased only by SH.Methods and Results:Spontaneous Ca release (SCR) and resultant delayed afterdepolarizations (DADs) were evaluated by optical mapping in canine wedge preparations during normothermia (N, 36℃), TH (32℃) or SH (28℃; n=8 each). The slope (amplitude/rise time) of multicellular SCR (mSCR) events, a determinant of triggered activity, was suppressed in TH (24.4±3.4%/s vs. N: 41.5±6.0%/s), but significantly higher in SH (96.3±8.1%/s) producing higher amplitude DADs in SH (35.7±1.6%) and smaller in TH (5.3±1.0% vs. N: 10.0±1.1%, all P<0.05). Triggered activity was only observed in SH. In isolated myocytes, sarcoplasmic reticulum (SR) Ca release kinetics slowed in a temperature-dependent manner, prolonging Ca transient rise time [33±3 (N) vs. 50±6 (TH) vs. 88±12 ms (SH), P<0.05], which can explain the decreased mSCR slope and DAD amplitude in TH. Although the SR Ca content was similar in TH and SH, Ca spark frequency was markedly increased only in SH, suggesting that increased ryanodine receptor open probability could explain the increased triggered activity during SH. Temperature dependence of Ca release can explain susceptibility to Ca-mediated arrhythmia triggers in SH. This may therefore explain the increased risk of lethal arrhythmia in SH, but not during TH.

Mitochondrial Permeability Transition Pore Opening Promotes Calcium Alternans and Waves in Ventricular Myocytes

The interactions between mitochondria and sarcoplasmic reticulum (SR) calcium (Ca) release form a positive feedback loop, which plays important roles in cardiac excitation-contraction coupling and arrhythmogenesis. We developed a spatially-distributed model for rabbit ventricular myocytes, which consists of a three-dimensional network of coupled mitochondria and Ca release units (CRUs). The L-type Ca channels, the ryanodine receptors, and the mitochondrial permeability transition pores (mPTPs) were simulated using random Markov transitions. We then carried out computer simulations using the detailed ventricular cell model to investigate the effects of mitochondrial depolarization on intracellular Ca cycling dynamics. We show that opening of the mPTPs that cause depolarization of a small portion of the mitochondria promotes Ca alternans and Ca waves due to Ca extrusion from mitochondria elevating SR Ca and enhancing CRU coupling. In experiments of isolated rabbit and mouse ventricular myocytes, we applied FCCP to cause mitochondrial depolarization (measured by TMRM) and mPTP opening (measured by calcein loss from the matrix). For rabbit myocytes at pacing cycle length (PCL) 400 ms, no Ca alternans was observed under control conditions, but Ca alternans appeared 2 minutes after FCCP treatment (50-100 nM) coincident with matrix calcein loss indicating mPTP opening. At the same PCL, spontaneous Ca release was observed 2 minutes after FCCP treatment. For mouse myocytes at PCL 200 ms, no Ca alternans was observed under control condition, but Ca alternans appeared 2 minutes after FCCP treatment. The FCCP-mediated Ca alternans was prevented by the mPTP inhibitor cyclosporine A (CsA). FCCP treatment also sped up Ca oscillations which slowed after CsA. These results show that mitochondrial depolarization and mPTP opening can result in Ca alternans and waves which are known to be arrhythmogenic, mechanistically linking metabolic stress to cardiac arrhythmogenesis.

Spontaneous activity in the microvasculature of visceral organs: role of pericytes and voltage-dependent Ca(2+) channels.

The microvasculature plays a primary role in the interchange of substances between tissues and the circulation. In visceral organs that undergo considerable distension upon filling, the microvasculature appears to display intrinsic contractile properties to maintain their flow. Submucosal venules in the bladder or gastrointestinal tract generate rhythmic spontaneous phasic constrictions and associated Ca(2+) transients. These events are initiated within either venular pericytes or smooth muscle cells (SMCs) arising from spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) and the opening of Ca(2+) -activated chloride channels (CaCCs) that trigger Ca(2+) influx through L-type voltage-dependent Ca(2+) channels (VDCCs). L-type VDCCs also play a critical role in maintaining synchrony within the contractile mural cells. In the stomach myenteric layer, spontaneous Ca(2+) transients originating in capillary pericytes appear to spread to their neighbouring arteriolar SMCs. Capillary Ca(2+) transients primarily rely on SR Ca(2+) release, but also require Ca(2+) influx through T-type VDCCs for their synchrony. The opening of T-type VDCCs also contribute to the propagation of Ca(2+) transients into SMCs. In visceral microvasculature, pericytes act as either spontaneously active contractile machinery of the venules or as pacemaker cells generating synchronous Ca(2+) transients that drive spontaneous contractions in upstream arterioles. Thus pericytes play different roles in different vascular beds in a manner that may well depend on the selective expression of T-type and L-type Ca(2+) channels.

Spontaneous inward currents reflecting oscillatory activation of Na+/Ca2+ exchangers in human embryonic stem cell-derived cardiomyocytes

Na(+)/Ca(2+) exchanger current (INCX) triggered by spontaneous Ca(2+) release from sarcoplasmic reticulum (SR) has been suggested as one of the cardiac pacemaker mechanisms ("Ca(2+) clock model"). In human embryonic stem cell-derived cardiomyocytes (hESC-CMs) showing spontaneous action potentials (APs), we found that substantial population (35 %) showed regular oscillation of inward currents (SICs) in nystatin-perforated voltage clamp between -40 and 40 mV (-80 ± 10.6 pA, at -20 mV). SICs were similarly observed between nodal, atrial, and ventricular hESC-CMs. Oscillations of [Ca(2+)]i synchronized with SICs were observed under voltage clamp. SICs were eliminated by lowering [Ca(2+)]e, L-type Ca(2+) channel (VOCCL) blocker (nifedipine, 10 µM), ryanodine receptor (RyR) agonist (caffeine, 10 mM), or NCX inhibitor (1 µM SN-6 and 10 µM KB-R7943). Plasma membrane expression of NCX1 was confirmed using immunofluorescence confocal microcopy. Both caffeine and SN-6 slowed the pacemaker potential but did not abolish the AP generation. The inhibitors of funny current (3 µM ivabradine) or voltage-gated K(+) channel currents (1 µM E4031 and 10 µM chromanol-293B) also did not abolish but slowed the pacemaker potential. In a computational model of cardiac pacemaker by Maltsev and Lakatta (2009), after modifying the spatial distribution of RyR, VOCCL, and NCX by using our multiparameter adjust algorithm, we could successfully reproduce spontaneous SR Ca(2+) release and SICs under voltage clamp. It was proposed that, under the membrane depolarization activating VOCCL, oscillatory Ca(2+) releases via RyR induce sharp increases in subsarcolemmal [Ca(2+)]i and inward INCX (SICs). Since the hESC-CMs without SICs still showed spontaneous APs, the putative "Ca(2+) clock" would provide a redundant pacemaker or augmenting mechanism in hESC-CMs.

Absence of the Regulator of G-protein Signaling, RGS4, Predisposes to Atrial Fibrillation and Is Associated with Abnormal Calcium Handling

The description of potential molecular substrates for predisposition to atrial fibrillation (AF) is incomplete, and it is unknown what role regulators of G-protein signaling might play. We address whether the attenuation of RGS4 function may promote AF and the mechanism through which this occurs. For this purpose, we studied a mouse with global genetic deletion of RGS4 (RGS4(-/-)) and the normal littermate controls (RGS4(+/+)). In vivo electrophysiology using atrial burst pacing revealed that mice with global RGS4 deletion developed AF more frequently than control littermates. Isolated atrial cells from RGS4(-/-) mice show an increase in Ca(2+) spark frequency under basal conditions and after the addition of endothelin-1 and abnormal spontaneous Ca(2+) release events after field stimulation. Isolated left atria studied on a multielectrode array revealed modest changes in path length for re-entry but abnormal electrical events after a pacing train in RGS4(-/-) mice. RGS4 deletion results in a predisposition to atrial fibrillation from enhanced activity in the Gαq/11-IP3 pathway, resulting in abnormal Ca(2+) release and corresponding electrical events.

Regulation of calcium clock-mediated pacemaking by inositol-1,4,5-trisphosphate receptors in mouse sinoatrial nodal cells.

Inositol-1,4,5-trisphosphate receptors (IP3 Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3 Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3 Rs slows pacing. In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca(2+) clock is uncoupled from the membrane clock, IP3 R agonists and antagonists modulate the rate of spontaneous Ca(2+) waves, suggesting that IP3 R-mediated Ca(2+) release modulates the Ca(2+) clock. IP3 R modulation also regulates Ca(2+) spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca(2+) clock frequency. Modulation of Ca(2+) clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3 Rs are upregulated. Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing 'funny current' (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene.

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca(2+) release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca(2+) on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [(3)H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G(+/-)) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G(+/-) hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca(2+) release during systole, gradually overloading the sarcoplasmic reticulum with Ca(2+). The resultant Ca(2+) overload then randomly caused bursts of prolonged Ca(2+) release, activating electrogenic Na(+)-Ca(2+) exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.

Calmodulin Potentiates RyR2 Block and Ca Wave Suppression by Flecainide

Flecainide, a dual sodium channel and RyR2 Ca release channel blocker classified as a class Ic antiarrhythmic agent, has been used to suppress ventricular arrhythmias caused by spontaneous Ca release from the sarcoplasmic reticulum in animal models and humans. Since flecainide exhibits much lower antiarrhythmic potency in isolated cells and single RyR2 in vitro than in vivo, whether RyR2 block contributes to its antiarrhythmic mechanism of action has remained controversial. Here we test the hypothesis that loss of Calmodulin (CaM), a physiological binding partner of RyR2 Ca release channels, is responsible for the discrepancy of in vivo and in vitro results.Using single channels isolated from sheep hearts and incorporated into lipid bilayers, we constructed concentration-response curves of flecainide in the presence and absence of physiological CaM concentrations (100nM). Presence of CaM potentiated the action of flecainide on single RyR2 channels, with 3-fold increase in potency (IC50 flecainide= 8.6 µM vs. IC50 flecainide + CaM= 2.8 µM) and a modest increase in Emax. We next tested flecainide in permeabilized ventricular myocytes isolated from calsequestrin knockout mice, an established genetic model of Ca-release triggered ventricular arrhythmia, and from C57Bl6 wild-type mice. Addition of 100 nM CaM doubled both the potency (from 3 µM to 1.4 µM) and efficacy (from 40% to 80% inhibition) of Ca wave suppression by flecainide in calsequestrin KO cells. Moreover, in wild type cells, flecainide had no effect in absence of CaM, whereas in the presence of CaM, flecainide exhibited a modest effect in waves suppression with 25% inhibition of Ca waves and IC50 = 1 µM. Thus, the presence of physiological concentrations of Calmodulin is critical for flecainide's block of RyR2 channels and therefore its antiarrhythmic effect.

Multiscale Consequences of Spontaneous Calcium Release on Cardiac Delayed Afterdepolarizations

Spontaneous calcium (Ca) release in cardiac myocytes can produce delayed afterdepolarizations (DADs) that promote arrhythmias. In these studies, we combined experimental and computational approaches to investigate quantitatively how Ca release features at subcellular, cellular, and tissue scales influence DADs. Spontaneous Ca release events and corresponding DADs were measured experimentally from isolated rabbit ventricular myocytes exposed to elevated extracellular Ca (2.7 mM) and isoproterenol (0.25 μM), and were compared between lower and higher total intracellular Ca levels following 1 or 5 paced beats at 400 ms intervals, respectively. At the subcellular scale, Ca waves emerged simultaneously from an average of 1.36 (low) and 2.5 (high) sites per Ca release event. Consistent with the criticality-based theory of Ca wave emergence, Ca wave numbers were proportional to sarcoplasmic reticulum (SR) Ca levels that produced INCX with −71.1 pA/pF×ms (low) and −106.0 pA/pF×ms (high) integrated current densities upon rapid exposure to caffeine (10 mM). At the cellular scale, whole-cell calcium transients had peak amplitudes of 0.462 F/Fo (low) and 0.717 F/Fo (high) and full widths at half maximum (FWHMs) of 309.0 ms (low) and 180.0 ms (high). Resultant DADs had peak amplitudes of 1.41 mV (low) and 6.03 mV (high) and FWHMs of 302.3 ms (low) and 140.3 ms (high). Ca wave latencies in single myocytes, which determine Ca release synchrony in cardiac tissue, were 0.996 s (low) and 0.416 s (high). Computer simulation analyses implementing various combinations of the experimentally measured factors suggest that greater numbers of Ca waves, increased SR Ca release, and greater synchrony are associated with larger DAD amplitudes in cardiac tissue. Simulations also suggest that the subcellular number of Ca waves has the largest impact on DAD amplitude in tissue.

Inositol-1,4,5-trisphosphate induced Ca2+ release and excitation-contraction coupling in atrial myocytes from normal and failing hearts.

Impaired calcium (Ca(2+)) signalling is the main contributor to depressed ventricular contractile function and occurrence of arrhythmia in heart failure (HF). Here we report that in atrial cells of a rabbit HF model, Ca(2+) signalling is enhanced and we identified the underlying cellular mechanisms. Enhanced Ca(2+) transients (CaTs) are due to upregulation of inositol-1,4,5-trisphosphate receptor induced Ca(2+) release (IICR) and decreased mitochondrial Ca(2+) sequestration. Enhanced IICR, however, together with an increased activity of the sodium-calcium exchange mechanism, also facilitates spontaneous Ca(2+) release in form of arrhythmogenic Ca(2+) waves and spontaneous action potentials, thus enhancing the arrhythmogenic potential of atrial cells. Our data show that enhanced Ca(2+) signalling in HF provides atrial cells with a mechanism to improve ventricular filling and to maintain cardiac output, but also increases the susceptibility to develop atrial arrhythmias facilitated by spontaneous Ca(2+) release. We studied excitation-contraction coupling (ECC) and inositol-1,4,5-triphosphate (IP3)-dependent Ca(2+) release in normal and heart failure (HF) rabbit atrial cells. Left ventricular HF was induced by combined volume and pressure overload. In HF atrial myocytes diastolic [Ca(2+)]i was increased, action potential (AP)-induced Ca(2+) transients (CaTs) were larger in amplitude, primarily due to enhanced Ca(2+) release from central non-junctional sarcoplasmic reticulum (SR) and centripetal propagation of activation was accelerated, whereas HF ventricular CaTs were depressed. The larger CaTs were due to enhanced IP3 receptor-induced Ca(2+) release (IICR) and reduced mitochondrial Ca(2+) buffering, consistent with a reduced mitochondrial density and Ca(2+) uptake capacity in HF. Elementary IP3 receptor-mediated Ca(2+) release events (Ca(2+) puffs) were more frequent in HF atrial myoctes and were detected more often in central regions of the non-junctional SR compared to normal cells. HF cells had an overall higher frequency of spontaneous Ca(2+) waves and a larger fraction of waves (termed arrhythmogenic Ca(2+) waves) triggered APs and global CaTs. The higher propensity of arrhythmogenic Ca(2+) waves resulted from the combined action of enhanced IICR and increased activity of sarcolemmal Na(+)-Ca(2+) exchange depolarizing the cell membrane. In conclusion, the data support the hypothesis that in atrial myocytes from hearts with left ventricular failure, enhanced CaTs during ECC exert positive inotropic effects on atrial contractility which facilitates ventricular filling and contributes to maintaining cardiac output. However, HF atrial cells were also more susceptible to developing arrhythmogenic Ca(2+) waves which might form the substrate for atrial rhythm disorders frequently encountered in HF.