Spontaneous Sarcoplasmic Reticulum Ca Research Articles

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Suppression of Spontaneous CA2+ Release by Cardioprotective Drugs

Spontaneous sarcoplasmic reticulum (SR) Ca2+ release in the form of Ca2+ waves occurs in cardiac cells under conditions of SR Ca2+ overload. This store-overload induced Ca2+ release (SOICR) is a well-known cause of delayed afterdepolarization and triggered arrhythmias. Hence, inhibiting SOICR may represent a promising therapeutic strategy for Ca2+-triggered arrhythmias. Indeed, we have recently shown that carvedilol, one of the most effective beta-blockers for preventing ventricular tachyarrhythmias and sudden death in heart failure, possesses a novel anti-SOICR activity. To identify more SOICR inhibitors, we searched the DrugBank database (http://www.drugbank.ca) for chemicals with structures similar to carvedilol. We selected a number of hit compounds and assessed their impact on SOICR in HEK293 cells expressing a SOICR-promoting cardiac ryanodine receptor (RyR2) mutation (R4496C). We found that curcumin and resveratrol (natural phenols) and bevantolol (a beta blocker and a Ca2+ channel blocker) suppress SOICR in HEK293 cells with IC50s of 6.7±0.3, 48.4±3.6, 64.7±11.3μM, respectively. On the other hand, gliclazide, diphenhydramine, crocin, dexrazoxane, and trazodone displayed little or no SOICR inhibition. We also tested several other known cardioprotective drugs. We found that docosahexaenoic acid (DHA), eicosapentaenoic (EPA), arachidonic acid (AA), anandamide, and a novel derivative of anandamide (CS-X-95) inhibit SOICR in HEK293 cells with IC50s of 11.2±4.2, 5.1±0.5, 5.5±0.9, 14.7±5.1, and 11.5±0.7 μM, respectively, whereas ranolazine and S107 have no significant impact on SOICR. These observations indicate that many cardioprotective drugs exhibit anti-SOICR activity, and that SOICR inhibition may contribute, in part, to their cardioprotective effect. (Supported by NIH)

Cardiac ryanodine receptor activation by high Ca2+ store load is reversed in a reducing cytoplasmic redox environment

ABSTRACTHere, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca2+]. Basal leak from the sarcoplasmic reticulum is required for normal Ca2+ handling, but excess diastolic Ca2+ leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca2+ release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca2+] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca2+] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH∶GSSG to a normal reduced cytoplasmic redox potential (−220 mV). Conversely, with 20 µM cytoplasmic 4,4′-DTDP or buffering of the redox potential to an oxidising value (−180 mV), RyR2 activity increased with increasing luminal [Ca2+]. The luminal redox potential was constant at −180 mV in each case. These responses to luminal [Ca2+] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg2+). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm.

Sarcoplasmic reticulum Ca²⁺ release is both necessary and sufficient for SK channel activation in ventricular myocytes.

SK channels are upregulated in human patients and animal models of heart failure (HF). However, their activation mechanism and function in ventricular myocytes remain poorly understood. We aim to test the hypotheses that activation of SK channels in ventricular myocytes requires Ca(2+) release from sarcoplasmic reticulum (SR) and that SK currents contribute to reducing triggered activity. SK2 channels were overexpressed in adult rat ventricular myocytes using adenovirus gene transfer. Simultaneous patch clamp and confocal Ca(2+) imaging experiments in SK2-overexpressing cells demonstrated that depolarizations resulted in Ca(2+)-dependent outward currents sensitive to SK inhibitor apamin. SR Ca(2+) release induced by rapid application of 10 mM caffeine evoked repolarizing SK currents, whereas complete depletion of SR Ca(2+) content eliminated SK currents in response to depolarizations, despite intact Ca(2+) influx through L-type Ca(2+) channels. Furthermore, voltage-clamp experiments showed that SK channels can be activated by global spontaneous SR Ca(2+) release events Ca(2+) waves (SCWs). Current-clamp experiments revealed that SK overexpression reduces the amplitude of delayed afterdepolarizations (DADs) resulting from SCWs and shortens action potential duration. Immunolocalization studies showed that overexpressed SK channels are distributed both at external sarcolemmal membranes and along the Z-lines, resembling the distribution of endogenous SK channels. In summary, SR Ca(2+) release is both necessary and sufficient for the activation of SK channels in rat ventricular myocytes. SK currents contribute to repolarization during action potentials and attenuate DADs driven by SCWs. Thus SK upregulation in HF may have an anti-arrhythmic effect by reducing triggered activity.

Subclinical Abnormalities in Sarcoplasmic Reticulum Ca2+ Release Promote Eccentric Myocardial Remodeling and Pump Failure Death in Response to Pressure Overload

This study sought to explore whether subclinical alterations of sarcoplasmic reticulum (SR) Ca(2+) release through cardiac ryanodine receptors (RyR2) aggravate cardiac remodeling in mice carrying a human RyR2(R4496C+/-) gain-of-function mutation in response to pressure overload. RyR2 dysfunction causes increased diastolic SR Ca(2+) release associated with arrhythmias and contractile dysfunction in inherited and acquired cardiac diseases, such as catecholaminergic polymorphic ventricular tachycardia and heart failure (HF). Functional and structural properties of wild-type and catecholaminergic polymorphic ventricular tachycardia-associated RyR2(R4496C+/-) hearts were characterized under conditions of pressure overload induced by transverse aortic constriction (TAC). Wild-type and RyR2(R4496C+/-) hearts had comparable structural and functional properties at baseline. After TAC, RyR2(R4496C+/-) hearts responded with eccentric hypertrophy, substantial fibrosis, ventricular dilation, and reduced fractional shortening, ultimately resulting in overt HF. RyR2(R4496C+/-)-TAC cardiomyocytes showed increased incidence of spontaneous SR Ca(2+) release events, reduced Ca(2+) transient peak amplitude, and SR Ca(2+) content as well as reduced SR Ca(2+)-ATPase 2a and increased Na(+)/Ca(2+)-exchanger protein expression. HF phenotype in RyR2(R4496C+/-)-TAC mice was associated with increased mortality due to pump failure but not tachyarrhythmic events. RyR2-stabilizer K201 markedly reduced Ca(2+) spark frequency in RyR2(R4496C+/-)-TAC cardiomyocytes. Mini-osmotic pump infusion of K201 prevented deleterious remodeling and improved survival in RyR2(R4496C+/-)-TAC mice. The combination of subclinical congenital alteration of SR Ca(2+) release and pressure overload promoted eccentric remodeling and HF death in RyR2(R4496C+/-) mice, and pharmacological RyR2 stabilization prevented this deleterious interaction. These findings suggest potential clinical relevance for patients with acquired or inherited gain-of-function of RyR2-mediated SR Ca(2+) release.

Cellular and Molecular Mechanisms of Atrial Arrhythmogenesis in Patients With Paroxysmal Atrial Fibrillation

Electrical, structural, and Ca2+ -handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca2+ -release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca2+ -release events in paroxysmal AF (pAF) are unknown. Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10-20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca2+ current, and Na+ /Ca2+ -exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca2+ leak and incidence of delayed after-depolarizations in pAF. Ca2+ -transient amplitude and sarcoplasmic reticulum Ca2+ load (caffeine-induced Ca2+ -transient amplitude, integrated Na+/Ca2+ -exchange current) were larger in pAF. Ca2+ -transient decay was faster in pAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca2+ leak and sarcoplasmic reticulum Ca2+ -release events, causing delayed after-depolarizations/triggered activity in pAF. Increased diastolic sarcoplasmic reticulum Ca2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Cellular mechanisms of ventricular arrhythmias in a mouse model of Timothy syndrome (long QT syndrome 8)

Ca2+ flux through l-type CaV1.2 channels shapes the waveform of the ventricular action potential (AP) and is essential for excitation–contraction (EC) coupling. Timothy syndrome (TS) is a disease caused by a gain-of-function mutation in the CaV1.2 channel (CaV1.2-TS) that decreases inactivation of the channel, which increases Ca2+ influx, prolongs APs, and causes lethal arrhythmias. Although many details of the CaV1.2-TS channels are known, the cellular mechanisms by which they induce arrhythmogenic changes in intracellular Ca2+ remain unclear. We found that expression of CaV1.2-TS channels increased sarcolemmal Ca2+ “leak” in resting TS ventricular myocytes. This resulted in higher diastolic [Ca2+]i in TS ventricular myocytes compared to WT. Accordingly, TS myocytes had higher sarcoplasmic reticulum (SR) Ca2+ load and Ca2+ spark activity, larger amplitude [Ca2+]i transients, and augmented frequency of Ca2+ waves. The large SR Ca2+ release in TS myocytes had a profound effect on the kinetics of CaV1.2 current in these cells, increasing the rate of inactivation to a high, persistent level. This limited the amount of influx during EC coupling in TS myocytes. The relationship between the level of expression of CaV1.2-TS channels and the probability of Ca2+ wave occurrence was non-linear, suggesting that even low levels of these channels were sufficient to induce maximal changes in [Ca2+]i. Depolarization of WT cardiomyocytes with a TS AP waveform increased, but did not equalize [Ca2+]i, compared to depolarization of TS myocytes with the same waveform. We propose that CaV1.2-TS channels increase [Ca2+] in the cytosol and the SR, creating a Ca2+overloaded state that increases the probability of arrhythmogenic spontaneous SR Ca2+ release.

Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

Recent studies have suggested that mitochondria may play important roles in the Ca2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca2+ flux can regulate the generation of Ca2+ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca2+ (Cai 2+) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and CaWs were induced in the presence of high (4 mM) external Ca2+ (Cao 2+). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai 2+ levels even after depletion of SR Ca2+ in the absence of Cao 2+ , suggesting Ca2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m) or Ru360 (a mitochondrial Ca2+ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca2+ release and uptake exquisitely control the local Ca2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.

Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation

SummaryCa2+-Calmodulin dependent protein kinase II (CaMKII) is a regulatory node in heart and brain, and its chronic activation can be pathological. CaMKII activation seen in heart failure can directly induce pathological changes in ion channels, Ca2+ handling and gene transcription.1 Here we discover a novel mechanism linking CaMKII and hyperglycemic signaling in diabetes mellitus, which is a key risk factor for heart2 and neurodegenerative diseases.3,4 Acute hyperglycemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser-279 activates CaMKII autonomously, creating molecular memory even after [Ca2+] declines. O-GlcNAc modified CaMKII is increased in heart and brain from diabetic humans and rats. In cardiomyocytes, increased [glucose] significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum (SR) Ca2+ release events that can contribute to cardiac mechanical dysfunction and arrhythmias.1 These effects were prevented by pharmacological inhibition of O-GlcNAc signaling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were enhanced by increased [glucose] via O-GlcNAc-and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signaling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

The mitochondrial Na+-Ca2+ exchanger, NCLX, regulates automaticity of HL-1 cardiomyocytes

Mitochondrial Ca2+ is known to change dynamically, regulating mitochondrial as well as cellular functions such as energy metabolism and apoptosis. The NCLX gene encodes the mitochondrial Na+-Ca2+ exchanger (NCXmit), a Ca2+ extrusion system in mitochondria. Here we report that the NCLX regulates automaticity of the HL-1 cardiomyocytes. NCLX knockdown using siRNA resulted in the marked prolongation of the cycle length of spontaneous Ca2+ oscillation and action potential generation. The upstrokes of action potential and Ca2+ transient were markedly slower, and sarcoplasmic reticulum (SR) Ca2+ handling were compromised in the NCLX knockdown cells. Analyses using a mathematical model of HL-1 cardiomyocytes demonstrated that blocking NCXmit reduced the SR Ca2+ content to slow spontaneous SR Ca2+ leak, which is a trigger of automaticity. We propose that NCLX is a novel molecule to regulate automaticity of cardiomyocytes via modulating SR Ca2+ handling.

Searching for Sudden Death SNPs in Calcium Handling Genes

Cardiac arrhythmias are a major clinical problem and lead to more than 250 000 sudden cardiac deaths (SCDs) each year in the United States alone.[1][1] Our ability to predict those at risk for sudden death remains poor. The majority of patients who die suddenly are not identified as being at high

221 REDUCING SARCOLEMMAL SODIUM CURRENT DECREASES SPONTANEOUS SR CA2+ RELEASE

Aim Ca2+ waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There is some evidence that INa blocking agents can reduce spontaneous sarcoplasmic reticulum (SR) Ca2+ release via effects on the RyR. We tested the hypothesis that direct modulation of INa may also be important in altering SR Ca2+ release. Methods and Results Imaging of spontaneous SR Ca2+ release events in healthy adult rat cardiomyocytes was performed. Variation in frequency of stimulation was used to produce Ca2+ sparks or waves. When SR Ca2+ content was held constant, spark frequency, wave frequency and wave velocity were reduced by a variety of INa blockers (including flecainide, lidocaine, propafenone and TTX). To assess the contribution of INa to spark and wave production voltage clamping was used to activate contraction from holding potentials of -80mV or -40mV. This confirmed that reducing Na+ influx during myocyte stimulation is sufficient to reduce waves and that such agents only cause Ca2+ wave reduction when INa is active. It was found that Na+/Ca2+-exchanger (NCX)-mediated Ca2+ efflux was significantly enhanced by INa blockade and that the effects of INa blockade on wave frequency could be reversed by reducing [Na+]o, suggesting an important downstream role for NCX function in the changes in SR Ca2+ release. Veratridine, an INa activator, increased wave frequency and the effects were abrogated by increasing [Na]o during the wave-detection period. Conclusions Our results show, for the first time, that reducing Na+ influx reduces spontaneous SR Ca2+ release. We have also shown that alterations in [Na+]i modulate wave frequency through alterations in NCX function. A reduction in INa, for example, can increase the [Na+]o:[Na+]i gradient, hence causing more effective Ca2+ efflux at resting membrane potentials. This not only suggests a clear mechanism for the reduction in waves seen with INa blockers, but also emphasises the detrimental effects of high [Na]i seen in disease states such as heart failure. It also allows us to understand an alternative way in which INa blockers exert their antiarrhythmic actions.

Targeted ablation of the histidine-rich Ca2+-binding protein (HRC) gene is associated with abnormal SR Ca2+-cycling and severe pathology under pressure-overload stress

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

Triple mode of action of flecainide in catecholaminergic polymorphic ventricular tachycardia: reply

We thank Steele et al . for their interest and comments regarding our recent study focusing on the effects of INa reduction on spontaneous sarcoplasmic reticulum (SR) Ca2+ release.1 In the context of the authors' previous publications regarding the mechanism of action of flecainide at the cardiac ryanodine receptor (RyR2),2–4 it appears they have interpreted our study as an attempt to infer that flecainide has no effect on RyR2. We would like to clarify this misinterpretation. Our work demonstrates that a reduction in Na+ influx into the cardiomyocyte can, via the enhancement of Ca2+ efflux through the Na+/Ca2+ exchanger, reduce [Ca2+]i in the vicinity of the RyR2 and thus reduce the frequency of spontaneous SR Ca2+ release events. We demonstrate that this is a class effect of INa blockers, and that flecainide causes no reduction in SR Ca2+ leak when INa is eliminated by altering the holding potential via voltage clamp. This mechanism also appears to be relevant in whole-heart models of arrhythmia induced by increased SR Ca2+ release as evidenced by recent data from Radwanski et al .5 Our data should not be taken as a dismissal of the effects of flecainide at RyR2. We merely conclude that, under our experimental conditions, INa blockade confers a greater reduction in spontaneous SR Ca2+ release than effects at the RyR2. Flecainide has been …

β-adrenergic stimulation activates early afterdepolarizations transiently via kinetic mismatch of PKA targets

Sympathetic stimulation regulates cardiac excitation–contraction coupling in hearts but can also trigger ventricular arrhythmias caused by early afterdepolarizations (EADs) in pathological conditions. Isoproterenol (ISO) stimulation can transiently cause EADs which could result from differential kinetics of L-type Ca current (ICaL) vs. delayed rectifier potassium current (IKs) effects, but multiple PKA targets complicate mechanistic analysis. Utilizing a biophysically detailed model integrating Ca and β-adrenergic signaling, we investigate how different phosphorylation kinetics and targets influence β-adrenergic-induced transient EADs. We found that: 1) The faster time course of ICaL vs. IKs increases recapitulates experimentally observed ISO-induced transient EADs (which are due to ICaL reactivation). These EADs disappear at steady state ISO and do not occur during more gradual ISO application. 2) This ICaL vs. IKs kinetic mismatch with ISO can also induce transient EADs due to spontaneous sarcoplasmic reticulum (SR) Ca release and Na/Ca exchange current. The increased ICaL, SR Ca uptake and action potential duration (APD) raise SR Ca to cause spontaneous SR Ca release, but eventual IKs activation and APD shortening abolish these EADs. 3) Phospholemman (PLM) phosphorylation decreases both types of EADs by increasing outward Na/K-ATPase current (INaK) for ICaL-mediated EADs, and reducing intracellular Na and Ca loading for SR Ca-release-mediated EADs. Slowing PLM phosphorylation kinetics abolishes this protective effect. 4) Blocking phospholamban (PLB) phosphorylation has little effect on ICaL-mediated transient EADs, but abolishes SR Ca-release-mediated transient EADs by limiting SR Ca loading. 5) RyR phosphorylation has little effect on either transient EAD type. Our study emphasizes the importance of understanding non-steady state kinetics of several systems in mediating β-adrenergic-induced EADs and arrhythmias. This article is part of a Special Issue entitled "Calcium Signaling in Heart".

Flecainide reduces Ca2+ spark and wave frequency via inhibition of the sarcolemmal sodium current

AimsCa2+ waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There have been conflicting results regarding whether flecainide reduces Ca2+ waves in isolated cardiomyocytes. We sought to confirm whether flecainide inhibits waves in the intact cardiomyocyte and to elucidate the mechanism.Methods and resultsWe imaged spontaneous sarcoplasmic reticulum (SR) Ca2+ release events in healthy adult rat cardiomyocytes. Variation in stimulation frequency was used to produce Ca2+ sparks or waves. Spark frequency, wave frequency, and wave velocity were reduced by flecainide in the absence of a reduction of SR Ca2+ content. Inhibition of INa via alternative pharmacological agents (tetrodotoxin, propafenone, or lidocaine) produced similar changes. To assess the contribution of INa to spark and wave production, voltage clamping was used to activate contraction from holding potentials of −80 or −40 mV. This confirmed that reducing Na+ influx during myocyte stimulation is sufficient to reduce waves and that flecainide only causes Ca2+ wave reduction when INa is active. It was found that Na+/Ca2+-exchanger (NCX)-mediated Ca2+ efflux was significantly enhanced by flecainide and that the effects of flecainide on wave frequency could be reversed by reducing [Na+]o, suggesting an important downstream role for NCX function.ConclusionFlecainide reduces spark and wave frequency in the intact rat cardiomyocyte at therapeutically relevant concentrations but the mechanism involves INa reduction rather than direct ryanodine receptor (RyR2) inhibition. Reduced INa results in increased Ca2+ efflux via NCX across the sarcolemma, reducing Ca2+ concentration in the vicinity of the RyR2.

Mitochondria-Derived Ros Disturb Ca2+ Cycling and Evoke Abnormal Action Potentials in Guinea-Pig Ventricular Myocytes: A Theoretical Study

Abnormal Ca2+ handling often results in contractile dysfunction and ventricular arrhythmias, the leading causes of sudden cardiac death. However, in spite of extensive efforts, the molecular mechanisms underlying disturbance of Ca2+ cycling are not completely understood, hindering the development of effective antiarrhythmic therapies. Accompanying dysfunctional Ca2+ handling, increased mitochondrial reactive oxygen species (ROS) production is also observed in the diseased heart. Since mitochondria are in close proximity to the redox-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors, RyRs) and the SR Ca2+ ATPase (SERCA), mitochondria-derived ROS could play a crucial role in modulating Ca2+ cycling under pathological conditions. Previous work has shown that mitochondrial depolarization and ROS-induced ROS release significantly enhance spontaneous SR Ca2+ release (Ca2+ spark frequency) in resting myocytes, but the role of mitochondria-derived ROS on Ca2+ dynamics and action potentials in paced cardiac myocytes has not been examined. We hypothesize that the pathological mitochondrial ROS burst forms a ROS microdomain between mitochondria and SR, altering the proximal SR Ca2+ handling channels and consequently leading to disturbed Ca2+ cycling and abnormal electrical activity. To test this hypothesis, we developed a new multiscale myocyte model that incorporates mitochondria and local Ca2+ control, and links RyRs and SERCA to the ROS environment surrounding the SR. The simulations show that the mitochondria-derived ROS burst stimulates RyRs and inhibits SERCA, inducing a cytosolic Ca2+ ([Ca2+]i) transient. This extra [Ca2+]i transient activates the Na+/Ca2+ exchanger and Ca2+-sensitive nonspecific cationic channels, forming a transient inward current (Iti) that evokes early or delayed afterdepolarizations. This study defines the role of mitochondria-derived ROS in Ca2+ overload-mediated ventricular arrhythmias, and underscores the importance of considering mitochondrial targets in designing new antiarrhythmic drugs in the context of sudden cardiac death.

Aberrant S-nitrosylation mediates calcium-triggered ventricular arrhythmia in the intact heart

Nitric oxide (NO) derived from the activity of neuronal nitric oxide synthase (NOS1) is involved in S-nitrosylation of key sarcoplasmic reticulum (SR) Ca(2+) handling proteins. Deficient S-nitrosylation of the cardiac ryanodine receptor (RyR2) has a variable effect on SR Ca(2+) leak/sparks in isolated myocytes, likely dependent on the underlying physiological state. It remains unknown, however, whether such molecular aberrancies are causally related to arrhythmogenesis in the intact heart. Here we show in the intact heart, reduced NOS1 activity increased Ca(2+)-mediated ventricular arrhythmias only in the setting of elevated myocardial [Ca(2+)](i). These arrhythmias arose from increased spontaneous SR Ca(2+) release, resulting from a combination of decreased RyR2 S-nitrosylation (RyR2-SNO) and increased RyR2 oxidation (RyR-SOx) (i.e., increased reactive oxygen species (ROS) from xanthine oxidoreductase activity) and could be suppressed with xanthine oxidoreductase (XOR) inhibition (i.e., allopurinol) or nitric oxide donors (i.e., S-nitrosoglutathione, GSNO). Surprisingly, we found evidence of NOS1 down-regulation of RyR2 phosphorylation at the Ca(2+)/calmodulin-dependent protein kinase (CaMKII) site (S2814), suggesting molecular cross-talk between nitrosylation and phosphorylation of RyR2. Finally, we show that nitroso-redox imbalance due to decreased NOS1 activity sensitizes RyR2 to a severe arrhythmic phenotype by oxidative stress. Our findings suggest that nitroso-redox imbalance is an important mechanism of ventricular arrhythmias in the intact heart under disease conditions (i.e., elevated [Ca(2+)](i) and oxidative stress), and that therapies restoring nitroso-redox balance in the heart could prevent sudden arrhythmic death.

Crosstalk between Mitochondrial and Sarcoplasmic Reticulum Ca2+ Cycling Modulates Cardiac Pacemaker Cell Automaticity

BackgroundMitochondria dynamically buffer cytosolic Ca2+ in cardiac ventricular cells and this affects the Ca2+ load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca2+ releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na+-Ca2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).ObjectiveTo determine if mitochondrial Ca2+ (Ca2+ m), cytosolic Ca2+ (Ca2+ c)-SR-Ca2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.ResultsInhibition of mitochondrial Ca2+ influx into (Ru360) or Ca2+ efflux from (CGP-37157) decreased [Ca2+]m to 80±8% control or increased [Ca2+]m to 119±7% control, respectively. Concurrent with inhibition of mitochondrial Ca2+ influx or efflux, the SR Ca2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca2+ signal were highly correlated with the change in the SR Ca2+ load (r2 = 0.97). Changes in the spontaneous AP cycle length (Ru360, 111±1% control; CGP-37157, 89±2% control) in response to changes in [Ca2+]m were predicted by concurrent changes in LCR period (r2 = 0.84).ConclusionA change in SANC Ca2+ m flux translates into a change in the AP firing rate by effecting changes in Ca2+ c and SR Ca2+ loading, which affects the characteristics of spontaneous SR Ca2+ release.

A full range of mouse sinoatrial node AP firing rates requires protein kinase A-dependent calcium signaling

Recent perspectives on sinoatrial nodal cell (SANC) ⁎ function indicate that spontaneous sarcoplasmic reticulum (SR) Ca 2+ cycling, i.e. an intracellular “Ca 2+ clock,” driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules (“surface membrane clock”) to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca 2+ signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca 2+ cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca 2+ releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca 2+]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by β-adrenergic receptor (β-AR) stimulation requires normal intracellular Ca 2+ cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca 2+ cycling is disrupted. Thus, AC-cAMP-PKA-Ca 2+ signaling cascade is a major mechanism of normal automaticity in mouse SANC.