Spontaneous Sarcoplasmic Reticulum Research Articles

The ryanodine receptor channel as a molecular motif in atrial fibrillation: pathophysiological and therapeutic implications

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with substantial morbidity and mortality. It causes profound changes in sarcoplasmic reticulum (SR) Ca(2+) homeostasis, including ryanodine receptor channel dysfunction and diastolic SR Ca(2+) leak, which might contribute to both decreased contractile function and increased propensity to atrial arrhythmias. In this review, we will focus on the molecular basis of ryanodine receptor channel dysfunction and enhanced diastolic SR Ca(2+) leak in AF. The potential relevance of increased incidence of spontaneous SR Ca(2+) release for both AF induction and/or maintenance and the development of novel mechanism-based therapeutic approaches will be discussed.

Delayed Afterdepolarization in Intact Canine Sinoatrial Node as a Novel Mechanism for Atrial Arrhythmia

Recent evidence indicates that spontaneous sarcoplasmic reticulum Ca release and Na-Ca exchanger current activation contribute to the sinoatrial node (SAN) automaticity. These findings suggest that SAN activity may share mechanisms that underlie both automaticity and triggered activity. The aim of this study is to test the hypothesis that spontaneous, nonvoltage gated, intracellular Ca (Ca(i)) elevation may induce delayed afterdepolarization (DAD) in intact SAN during isoproterenol infusion. We simultaneously mapped Ca(i) and membrane potential in 31 isolated Langendorff-perfused canine right atriums (RA). Isoproterenol increased heart rate and late diastolic Ca(i) elevation (LDCAE) of the superior SAN, leading to consistent SAN automaticity in all 31 RAs. However, DAD-like diastolic depolarizations (DD) were transiently observed in 4 RAs during isoproterenol infusion. These DAD-like DDs were preceded by LDCAE, but did not trigger a full action potential. The LDCAE preceding DAD-like DDs had smaller amplitude (0.41 ± 0.08 AU vs 0.48 ± 0.07 AU, P = 0.001) and less steep slopes (3.7 ± 1.3 AU/s vs 4.8 ± 1.4 AU/s, P = 0.001) than that of sinus beats. The coupling interval of DAD-like DDs was longer than that of the preceding normal beats (407 ± 48 ms vs 371 ± 44 ms, P = 0.002). The isoproterenol-induced LDCAE of superior SAN induced a full action potential in most cases. However, if the LDCAE was too small to trigger an action potential, then it induces only DAD-like DD. The failure of DAD-like DD to consistently trigger a sinus beat is a novel mechanism of atrial arrhythmogenesis.

Calcium Dynamics and the Mechanisms of Atrioventricular Junctional Rhythm

The purpose of this study was to test the hypothesis that rhythmic spontaneous sarcoplasmic reticulum calcium (Ca) release (the "Ca clock") plays an important role in atrioventricular junction (AVJ) automaticity. The AVJ is a primary backup pacemaker to the sinoatrial node. The mechanisms of acceleration of AVJ intrinsic rate during sympathetic stimulation are unclear. We simultaneously mapped transmembrane potential and intracellular Ca in Langendorff-perfused canine AVJ preparations that did not contain sinoatrial node (n = 10). Baseline AVJ rate was 37.5 +/- 4.0 beats/min. The wavefront from leading pacemaker site propagated first through the slow pathway, then the fast pathway and atria. There was no late diastolic Ca elevation (LDCAE) at baseline. Isoproterenol up to 3 micromol/l increased heart rate to 100 +/- 6.8 beats/min, concomitant with the appearance of LDCAE that preceded the phase 0 of action potential by 97.3 +/- 35.2 ms and preceded the onset of late diastolic depolarization by 23.5 +/- 3.5 ms. Caffeine also produced LDCAE and AVJ acceleration. The maximal slope of LDCAE and diastolic depolarization always colocalized with the leading pacemaker sites. Ryanodine markedly slowed the rate of spontaneous AVJ rhythm. Isoproterenol did not induce LDCAE in the presence of ryanodine. The I(f) blocker ZD 7288 did not prevent LDCAE or AVJ acceleration induced by isoproterenol (n = 2). Isoproterenol and caffeine induced LDCAE and accelerated intrinsic AVJ rhythm. Consistent colocalization of the maximum LDCAE and the leading pacemaker sites indicates that the Ca clock is important to the intrinsic AVJ rate acceleration during sympathetic stimulation.

Calcium oscillations and T-wave lability precede ventricular arrhythmias in acquired long QT type 2

Alternans of intracellular Ca(2+) (Ca(i)) underlies T-wave alternans, a predictor of cardiac arrhythmias. A related phenomenon, T-wave lability (TWL), precedes torsades de pointes (TdP) in patients and animal models with impaired repolarization. However, the role of Ca(i) in TWL remains unexplored. This study investigated the role of Ca(i) dynamics on TWL in a noncryoablated rabbit model of long QT syndrome type 2 (LQT2) using simultaneous measurements of Ca(i) transient (CaT), action potentials (APs), and electrocardiogram (ECG) during paced rhythms and focused on events that precede ventricular ectopy. APs and CaTs were mapped optically from paced Langendorff female rabbit hearts (n = 8) at 1.2-s cycle length, after atrioventricular node ablation. Hearts were perfused with normal Tyrode solution, then with dofetilide (0.5 μM), and reduced [K(+)] (2 mM) and [Mg(2+)] (0.5 mM) to elicit LQT2. Lability of ECG, voltage, and Ca(i) signals were evaluated during regular paced rhythm, before and after dofetilide perfusion. In LQT2, lability of Ca(i), voltage, and ECG signals increased during paced rhythm, before the appearance of early afterdepolarizations (EADs). LQT2 resulted in AP prolongation and multiple (1 to 3) additional Ca(i) upstrokes, whereas APs remained monophasic. When EADs appeared, Ca(i) rose before voltage upstrokes at the origins of propagating EADs. Interventions (i.e., ryanodine and thapsigargin, n = 3 or low [Ca](o) and nifedipine, n = 4) that suppressed Ca(i) oscillations also abolished EADs. In LQT2, Ca(i) oscillations (Ca(i)O) precede EADs by minutes, indicating that they result from spontaneous sarcoplasmic reticulum Ca(2+) release rather than spontaneous I(Ca,L) reactivation. Ca(i)O likely produce oscillations of Na/Ca exchange current, I(NCX). Depolarizing I(NCX) during the AP plateau contributes to the generation of EADs by reactivating Ca(2+) channels that have recovered from inactivation. TWL reflects CaTs and APs lability that occur before EADs and TdP.

Predicting Local SR Ca2+ Dynamics during Ca2+ Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca2+) wave propagation, and 2), speed of Ca2+ diffusion within the SR. Ca2+ waves are initiated when a spontaneous local SR Ca2+ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca2+ diffusion constant in the SR (DCa,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca2+] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca2+] during Ca2+ waves. Simulations have investigated how dynamic local changes in SR [Ca2+] are influenced by 1), DCa,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca2+ pumps; 4), SR Ca2+ pump dependence on cytosolic [Ca2+]; and 5), the rate of transfer between network and junctional SR. Of these factors, DCa,SR is the primary determinant of how release from one RyR cluster alters SR [Ca2+] in nearby regions. Specifically, our results show that local increases in SR [Ca2+] ahead of the wave can potentially facilitate Ca2+ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca2+] are possible during SR Ca2+release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca2+]SR.

The human CASQ2 mutation K206N is associated with hyperglycosylation and altered cellular calcium handling

Mutations in the human cardiac calsequestrin gene (CASQ2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT-2). This inherited disorder is characterized by life-threatening arrhythmias induced by physical and emotional stress in young patients. Here we identified a novel heterozygous missense mutation (K206N) in the CASQ2 gene in a symptomatic family in which one member died of cardiac arrest. The functional properties of CSQK206N were investigated in comparison to the wild-type form of CASQ2 (CSQWT) by expression in eukaryotic cell lines and neonatal mouse myocytes. The mutation created an additional N-glycosylation site resulting in a higher molecular weight form of the recombinant protein on immunoblots. The mutation reduced the Ca2+ binding capacity of the protein and exhibited an altered aggregation state. Consistently, CSQK206N-expressing myocytes exhibited an impaired response to caffeine administration, suggesting a lower Ca2+ load of the sarcoplasmic reticulum (SR). The interaction of the mutated CSQ with triadin and the protein levels of the ryanodine receptor were unchanged but the maximal specific [3H]ryanodine binding was increased in CSQK206N-expressing myocytes, suggesting a higher opening state of the SR Ca2+ release channel. Myocytes with expression of CSQK206N showed a higher rate of spontaneous SR Ca2+ releases under basal conditions and after β-adrenergic stimulation. We conclude that CSQK206N caused a reduced Ca2+ binding leading to an abnormal regulation of intracellular Ca2+ in myocytes. This may then contribute to the increased propensity to trigger spontaneous Ca2+ transients in CSQK206N-expressing myocytes.

TNF-α and IL-1β increase Ca2+ leak from the sarcoplasmic reticulum and susceptibility to arrhythmia in rat ventricular myocytes

Sepsis is associated with ventricular dysfunction and increased incidence of atrial and ventricular arrhythmia however the underlying pro-arrhythmic mechanisms are unknown. Serum levels of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are elevated during sepsis and affect Ca2+ regulation. We investigated whether pro-inflammatory cytokines disrupt cellular Ca2+ cycling leading to reduced contractility, but also increase the probability of pro-arrhythmic spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). Isolated rat ventricular myocytes were exposed to TNF-α (0.05ngml−1) and IL-1β (2ngml−1) for 3 hr and then loaded with fura-2 or fluo-3 to record the intracellular Ca2+ concentration ([Ca2+]i). Cytokine treatment decreased the amplitude of the spatially averaged Ca2+ transient and the associated contraction, induced asynchronous Ca2+ release during electrical stimulation, increased the frequency of localized Ca2+ release events, decreased the SR Ca2+ content and increased the frequency of spontaneous Ca2+ waves at any given cytoplasmic Ca2+. These data suggest that TNF-α and IL-1β increase the SR Ca2+ leak from the SR, which contributes to the depressed Ca2+ transient and contractility. Increased susceptibility to spontaneous SR Ca2+ release may contribute to arrhythmias in sepsis as the resulting Ca2+ extrusion via NCX is electrogenic, leading to cell depolarisation.

Changes in intra-luminal calcium during spontaneous calcium waves following sensitization of ryanodine receptor channels

Cardiac contraction during systole is dependent on action potential-triggered Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) channels. SR Ca2+ release can also occur spontaneously during diastole, which causes a decrease in Ca2+ content within the SR and contributes to arrhythmogenesis. Here, we use measurements of cytosolic Ca2+ and intra-SR Ca2+ ([Ca2+]SR) to examine how RyR sensitization alters spontaneous SR Ca2+ release events in rabbit ventricular myocytes. RyR sensitization with caffeine (250 microM) increased the open probability of single RyR channels, increased the initial frequency and amplitude of local SR Ca2+ release events (Ca2+ sparks), and decreased the [Ca2+]SR level where Ca2+ sparks terminated. In intact myocytes, caffeine applied during rest after steady-state electrical stimulation increased the frequency of spontaneous Ca2+ waves and decreased the [Ca2+]SR level where waves terminated. These effects caused a marked loss of SR Ca2+ content. Therefore, increasing RyR activity has complex effects on cardiac function. Increased RyR activity during systole is beneficial as it increases SR Ca2+ release and contractile strength. However, increased RyR activity during diastole produces spontaneous, arrhythmogenic Ca2+ release events that lower SR Ca2+ content and subsequently decrease contractility.

Arrhythmogenic Foci and the Mechanisms of Atrial Fibrillation

In this issue of the Circulation: Arrhythmia and Electrophysiology , Kurotobi et al1 determined the number of arrhythmogenic foci in atria provocable with isoproterenol infusion in patients with paroxysmal, persistent, and permanent atrial fibrillation (AF). The results show that persistent AF has more foci than paroxysmal AF. However, when AF persists for >12 months, the number of foci were reduced. The authors suggest that the increased number of foci probably is involved in the development of persistent AF. However, because this association is not observed for AF that persists for >12 months, the importance of these foci in AF maintenance remains unclear. Article see p 39 In ambulatory canine models, simultaneous sympathovagal discharges (rather than isolated sympathetic activation) are typical triggers of paroxysmal atrial tachycardia (AT) and AF.2,3⇓ These observations are explained by the late phase-3 early afterdepolarization (EAD), which depends on the coexistence of increased amplitude and duration of intracellular calcium (Cai) transient and the shortened action potential duration (APD).4,5⇓ Vagal stimulation shortens APD and therefore is needed to promote the late phase-3 EAD. In the Kurotobi study, however, arrhythmogenic foci were induced by isoproterenol alone, without the need of vagal stimulation or the use of parasympathomimetic agents. These results are different from that obtained in canine studies. The first possible explanation is that the pulmonary veins (PVs) naturally have shorter APDs with elevated (less negative) membrane potentials.6,7⇓ Rapid activation may further shorten the APD in the PVs.8 These changes are independent of the vagal tone. Jais et al9 reported the effective refractory periods of the PVs were shorter …

Stress synchronizes calcium release and promotes SR calcium leak

Stress synchronizes calcium release and promotes SR calcium leak

Synergistic Dual Automaticity in Sinoatrial Node Cell and Tissue Models

The mechanism of sinoatrial node (SAN) automaticity is traditionally attributed to membrane ion currents. Recent evidence indicates spontaneous sarcoplasmic reticulum (SR) Ca(2+) cycling also plays an important role. A computer simulation on SAN cell and 1D tissue model was performed. In the SAN cells, SR Ca(2+) cycling broadly modulated the sinus rate from 1.74 Hz to 3.87 Hz. Shortening of the junctional SR refilling time and increase of SR Ca(2+) release were responsible for sinus rate acceleration. However, under the fast SR Ca(2+) cycling, decreased L-type Ca(2+) current (I(CaL)) resulted in irregular firing. When Ca(2+) cycling was suppressed, I(f) and I(CaT) both acted to stabilize the pacemaker rhythm, but I(CaT) had less effect than I(f). At the 1D level, the electrical coupling between neighboring cells had little effect on the earliest pacemaker location. The leading pacemaking site always colocalized with the site with the highest SR Ca(2+) cycling rate, but shifted to the site with less inhibited I(CaL). The rate of SR Ca(2+) cycling can effectively and broadly modulate the sinus rate. I(f), I(CaL) and I(CaT) play integral roles to guarantee SAN cell rhythmic firing. The leading pacemaker site is determined by intracellular Ca(2+) dynamics and membrane currents, indicating the synergistic dual automaticity not only exists in single SAN cells, but also at the tissue level.

Enhanced Beta-Adrenergic Response, Spontaneous Calcium Release, and Arrhythmogenic Propensity in Mice with a Phosphomimetic Mutation of a PKA Site (S2030D) in the Cardiac Ryanodine Receptor

The phosphorylation of the cardiac calcium release channel/ryanodine receptor (RyR2) by protein kinase A (PKA) has been extensively investigated, but its functional consequences remain poorly understood and controversial. We have previously shown that S2030 is a major PKA site in RyR2 responding to beta-adrenergic stimulation, and that phosphomimetic mutation of this PKA site (S2030D) enhances luminal calcium activation of single RyR2 channels and increases the propensity for spontaneous calcium release in HEK293 cells during store calcium overload. To further investigate the physiological and pathophysiological significance of PKA phosphorylation of S2030, we generated a knock-in mouse model harboring the phosphomimetic S2030D mutation in RyR2. We found that S2030D mutant mice displayed higher isoproterenol-induced heart rate increase than wt mice. ECG recordings revealed that S2030D mutant mice were more susceptible to bidirectional ventricular tachycardia induced by the injection of epinephrine plus caffeine as compared to wt mice. To determine the impact of the S2030D mutation on sarcoplasmic reticulum (SR) calcium handling, ventricular myocytes isolated from wt and mutant mice were loaded with fluo-4 and field-stimulated. Calcium imaging showed that S2030D mutant cells exhibited enhanced spontaneous calcium release activity in the presence of isoproterenol. These observations indicate that the S2030D mutation in RyR2 enhances the beta-adrenergic response, and suggest that excessive PKA phosphorylation of S2030 in RyR2 may increase the propensity for stress-induced spontaneous SR calcium release and ventricular arrhythmias (supported by CIHR).

Increased Ca2+ leak and spatiotemporal coherence of Ca2+ release in cardiomyocytes during β‐adrenergic stimulation

beta-Adrenergic receptor (beta-AR) stimulation of cardiac muscle has been proposed to enhance Ca(2+) release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs). However, the anticipated increase in RyR Ca(2+) sensitivity has proven difficult to study in intact cardiomyocytes, due to accompanying alterations in SR Ca(2+) content, inward Ca(2+) current (I(Ca)) and diastolic cytosolic Ca(2+) concentration ([Ca(2+)](i)). Here, we studied whole-cell Ca(2+) release and spontaneous Ca(2+) leak (Ca(2+) sparks) in guinea-pig ventricular myocytes with confocal Ca(2+) imaging before and during beta-AR stimulation by isoproterenol (Iso), but under otherwise nearly identical experimental conditions. The extent of SR Ca(2+) loading was controlled under whole-cell voltage-clamp conditions. UV flash-induced uncaging of Ca(2+) from DM-nitrophen was employed as an invariant trigger for whole-cell Ca(2+) release. At matched SR Ca(2+) content, we found that Iso enhanced the spatiotemporal coherence of whole-cell Ca(2+) release, evident from spatially intercorrelated release and accelerated release kinetics that resulted in moderately (20%) increased release amplitude. This may arise from higher RyR Ca(2+) sensitivity, and was also reflected in spontaneous SR Ca(2+) leak. At comparable SR Ca(2+) content and cytosolic [Ca(2+)](i), we observed an approximately 4-fold increase in Ca(2+) spark frequency in Iso that also appeared in quiescent cells within 2 min without increased SR Ca(2+) content. This was likely to have been mediated by Ca(2+)/calmodulin-dependent protein kinase (CaMKII), rather than cAMP dependent protein kinase (PKA). We conclude that Iso increases the propensity of RyRs to open, both in response to rapid elevations of [Ca(2+)](i) and at diastolic [Ca(2+)](i). While this could be beneficial in enhancing and synchronizing systolic whole-cell SR Ca(2+) release, the same behaviour could also be proarrhythmogenic during diastole.

Inhibition of RyR2-S2814 Phosphorylation Prevents Heart Failure by Reducing SR Ca Leak

Abnormal regulation of RyR2 by Ca2+/calmodulin-dependent protein kinase 2 (CaMKII) has been suggested as a cause of sarcoplasmic reticulum (SR) Ca2+ leakage and contractile dysfunction in heart failure. We hypothesized that CaMKII phosphorylation of RyR2 is crucial for heart failure development. We generated RyR2 knockin mice in which CaMKII phosphorylation site S2814 was mutated to alanine (S2814A) to prevent phosphorylation. Cardiac function and dimensions monitored by echocardiography were similar in S2814A and WT mice up to 12 months of age. WT (n=13) and S2814A mice (n=9) were subjected to transverse aortic constriction (TAC) to induce pressure overload. At 8 weeks after TAC, S2814A mice displayed a similar hypertrophic response and decrease in ejection fraction (EF) as WT mice. At 16 weeks after TAC, however, EF was significantly lower in WT (32.5 +/- 3.4%) compared to S2814A mice (43.0 +/- 2.9%) suggesting inhibition of heart failure development in the latter. This rescue effect was further verified by a lower lung-weight-to-tibia-length ratio and a decrease in expression levels of the cardiac stress genes ANF and BNP in S2814A mice compared to WT mice at 16 weeks after TAC. Ca2+ imaging in cardiomyocytes, isolated from S2814A and WT mice at 16 weeks after sham or TAC surgery, demonstrated an decreased incidence of spontaneous SR Ca2+ release (SCR) events in S2814A (29.7% of myocytes) compared to WT (61.3% of myocytes; P<0.01). Whereas CaMKII inhibitor KN93 reduced the incidence of SCR events in WT to 36.1% (P<0.01 vs. WT TAC), KN93 did not have an effect of SCR in S2814A myocytes (30.0%; P=NS vs. S2814A TAC). Together, our results demonstrate that blocking CaMKII phosphorylation of RyR2 prevents SR Ca2+ leak, and inhibits or delays the progression to congestive heart failure in S2814A mice.

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca2+ going local?

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca²⁺ going local?

Diastolic Intracellular Calcium-Membrane Voltage Coupling Gain and Postshock Arrhythmias

Recurrent ventricular arrhythmias after initial successful defibrillation are associated with poor clinical outcome. We tested the hypothesis that postshock arrhythmias occur because of spontaneous sarcoplasmic reticulum Ca release, delayed afterdepolarization (DAD), and triggered activity (TA) from tissues with high sensitivity of resting membrane voltage (V(m)) to elevated intracellular calcium (Ca(i)) (high diastolic Ca(i)-voltage coupling gains). We simultaneously mapped Ca(i) and V(m) on epicardial (n=14) or endocardial (n=14) surfaces of Langendorff-perfused rabbit ventricles. Spontaneous Ca(i) elevation (SCaE) was noted after defibrillation in 32% of ventricular tachycardia/ventricular fibrillation at baseline and in 81% during isoproterenol infusion (0.01 to 1 micromol/L). SCaE was reproducibly induced by rapid ventricular pacing and inhibited by 3 mumol/L of ryanodine. The SCaE amplitude and slope increased with increasing pacing rate, duration, and dose of isoproterenol. We found TAs originating from 6 of 14 endocardial surfaces but none from epicardial surfaces, despite similar amplitudes and slopes of SCaEs between epicardial and endocardial surfaces. This was because DADs were larger on endocardial surfaces as a result of higher diastolic Ca(i)-voltage coupling gain, compared to those of epicardial surfaces. Purkinje-like potentials preceded TAs in all hearts studied (n=7). I(K1) suppression with CsCl (5 mmol/L, n=3), BaCl(2) (3 micromol/L, n=3), and low extracellular potassium (1 mmol/L, n=2) enhanced diastolic Ca(i)-voltage coupling gain and enabled epicardium to also generate TAs. Higher diastolic Ca(i)-voltage coupling gain is essential for genesis of TAs and may underlie postshock arrhythmias arising from Purkinje fibers. I(K)(1) is a major factor that determines the diastolic Ca(i)-voltage coupling gain.

Induction of atrial ectopic beats with calcium release inhibition: Local hierarchy of automaticity in the right atrium

Recent evidence indicates that spontaneous sarcoplasmic reticulum (SR) calcium (Ca) release underlies the mechanism of sinoatrial node (SAN) acceleration during beta-stimulation, indicating the importance of the Ca clock in SAN automaticity. Whether or not the same mechanism applies to atrial ectopic pacemakers (AEPs) remains unclear. The purpose of this study was to assess the mechanism of AEP. We simultaneously mapped intracellular calcium (Ca(i)) and membrane potential in 12 isolated canine right atria. The late diastolic Ca(i) elevation (LDCAE) was used to detect the Ca clock activity. Pharmacological interventions with isoproterenol (ISO), ryanodine, and ZD7288, a blocker of the I(f) membrane current, were performed. Ryanodine, which inhibits SR Ca release, reduced LDCAE in SAN, resulting in an inferior shift of the pacemaking site. Cycle length increased significantly in a dose-dependent way. In the presence of 3 to 10 mumol/l of ryanodine, ISO infusion consistently induces AEPs from the lower crista terminalis. All ectopic beats continuing over 30 seconds were located at the lower crista terminalis. These AEPs were resistant to ryanodine treatment even at high doses. Subsequent blockade of I(f) inhibited the AEP and resulted in profound bradycardia. Spontaneous SR Ca release underlies ISO-induced increase of superior SAN activity. As compared with SAN, the AEP is less dependent on the Ca clock and more dependent on the membrane clock for its automaticity. AEPs outside the SAN can effectively serve as backup pacemakers when the Ca clock functionality is reduced.

Mechanisms of sinoatrial node dysfunction in a canine model of pacing-induced atrial fibrillation

The mechanism of sinoatrial node (SAN) dysfunction in atrial fibrillation (AF) is unclear. The purpose of this study was to test the hypothesis that defective spontaneous sarcoplasmic reticulum (SR) Ca(2+) release (Ca(2+) clock) is in part responsible for SAN dysfunction in AF. Arrhythmic events and SAN function were evaluated in pacing-induced AF dogs (n = 7) and in normal dogs (n = 19) with simultaneous intracellular calcium (Ca(i)) and membrane potential recording. AF dogs had frequent sinus pauses during Holter monitoring. Isolated right atrium (RA) from AF dogs showed slower heart rate (P = .001), longer SAN recovery time (P = .001), and longer sinoatrial conduction time (P = .003) than normal. In normal RAs, isoproterenol 0.3 and 1 mumol/L increased heart rate by 96% and 105%, respectively. In contrast, in RAs from AF dogs, isoproterenol increased heart rate by only 60% and 72%, respectively. Isoproterenol induced late diastolic Ca(i) elevation (LDCAE) at superior SAN in all 19 normal RAs but in only 3 of 7 AF RAs (P = .002). In AF RAs without LDCAE (n = 4), heart rate increased by the acceleration of ectopic foci. Caffeine (20 mmol/L) injection increased heart rate with LDCAE in all 6 normal RAs but did not result in LDCAE in any of the 5 AF RAs (P = .002). Type 2 ryanodine receptor (RyR2) in the superior SAN of AF dogs was decreased to 33% of normal (P = .02). SAN dysfunction in AF is associated with Ca(2+) clock malfunction, characterized by unresponsiveness to isoproterenol and caffeine and down-regulation of RyR2 in SAN.

Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure

Transgenic (TG) Ca/calmodulin-dependent protein kinase II (CaMKII)delta(C) mice have heart failure and isoproterenol (ISO)-inducible arrhythmias. We hypothesized that CaMKII contributes to arrhythmias and underlying cellular events and that inhibition of CaMKII reduces cardiac arrhythmogenesis in vitro and in vivo. Under baseline conditions, isolated cardiac myocytes from TG mice showed an increased incidence of early afterdepolarizations compared with wild-type myocytes (P<0.05). CaMKII inhibition (AIP) completely abolished these afterdepolarizations in TG cells (P<0.05). Increasing intracellular Ca stores using ISO (10(-8) M) induced a larger amount of delayed afterdepolarizations and spontaneous action potentials in TG compared with wild-type cells (P<0.05). This seems to be due to an increased sarcoplasmic reticulum (SR) Ca leak because diastolic [Ca](i) rose clearly on ISO in TG but not in wild-type cells (+20+/-5% versus +3+/-4% at 10(-6) M ISO, P<0.05). In parallel, SR Ca leak assessed by spontaneous SR Ca release events showed an increased Ca spark frequency (3.9+/-0.5 versus 2.0+/-0.4 sparks per 100 microm(-1).s(-1), P<0.05). However, CaMKII inhibition (either pharmacologically using KN-93 or genetically using an isoform-specific CaMKIIdelta-knockout mouse model) significantly reduced SR Ca spark frequency, although this rather increased SR Ca content. In parallel, ISO increased the incidence of early (54% versus 4%, P<0.05) and late (86% versus 43%, P<0.05) nonstimulated events in TG versus wild-type myocytes, but CaMKII inhibition (KN-93 and KO) reduced these proarrhythmogenic events (P<0.05). In addition, CaMKII inhibition in TG mice (KN-93) clearly reduced ISO-induced arrhythmias in vivo (P<0.05). We conclude that CaMKII contributes to cardiac arrhythmogenesis in TG CaMKIIdelta(C) mice having heart failure and suggest the increased SR Ca leak as an important mechanism. Moreover, CaMKII inhibition reduces cardiac arrhythmias in vitro and in vivo and may therefore indicate a potential role for future antiarrhythmic therapies warranting further studies.

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca 2+, competing with Ca 2+ transporters like the sarcoplasmic reticulum (SR) Ca 2+-ATPase. Rapid mitochondrial [Ca 2+] transients have been reported to be synchronized with normal cytosolic [Ca 2+] i transients. However, most intra-mitochondrial free [Ca 2+] ([Ca 2+] mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca 2+] mito in single rat myocytes using the ratiometric Ca 2+ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca 2+] mito dependence on [Ca 2+] i (with 5 mM EGTA) was sigmoid with [Ca 2+] mito < [Ca 2+] i for [Ca 2+] i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca 2+] i (± 15 mM Na +) cyclical spontaneous SR Ca 2+ release occurred (5–15/min). Changes in [Ca 2+] mito during individual [Ca 2+] i transients were small (∼ 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca 2+ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca 2+] mito and spontaneous Ca 2+ transients (confirming that SR Ca 2+ releases caused the [Ca 2+] mito rise). Confocal imaging of local [Ca 2+] mito (using rhod-2) showed that [Ca 2+] mito rose rapidly with a delay after SR Ca 2+ release (with amplitude up to 10 nM), but declined much more slowly than [Ca 2+] i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca 2+ uptake for larger [Ca 2+] mito transients was ∼ 0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca 2+ buffering), consistent with prior indirect estimates from [Ca 2+] i measurements, and corresponds to ∼ 1% of the SR Ca 2+ uptake during a normal Ca 2+ transient. Thus small phasic [Ca 2+] mito transients and gradually integrating [Ca 2+] mito signals occur during repeating [Ca 2+] i transients.