STIM1-Ca2+ signaling modulates automaticity of the mouse sinoatrial node.

Cardiac pacemaking is governed by specialized cardiomyocytes located in the sinoatrial node (SAN). SAN cells (SANCs) integrate voltage-gated currents from channels on the membrane surface (membrane clock) with rhythmic Ca(2+) release from internal Ca(2+) stores (Ca(2+) clock) to adjust heart rate to meet hemodynamic demand. Here, we report that stromal interaction molecule 1 (STIM1) and Orai1 channels, key components of store-operated Ca(2+) entry, are selectively expressed in SANCs. Cardiac-specific deletion of STIM1 in mice resulted in depletion of sarcoplasmic reticulum (SR) Ca(2+) stores of SANCs and led to SAN dysfunction, as was evident by a reduction in heart rate, sinus arrest, and an exaggerated autonomic response to cholinergic signaling. Moreover, STIM1 influenced SAN function by regulating ionic fluxes in SANCs, including activation of a store-operated Ca(2+) current, a reduction in L-type Ca(2+) current, and enhancing the activities of Na(+)/Ca(2+) exchanger. In conclusion, these studies reveal that STIM1 is a multifunctional regulator of Ca(2+) dynamics in SANCs that links SR Ca(2+) store content with electrical events occurring in the plasma membrane, thereby contributing to automaticity of the SAN.

Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells.

BACKGROUND AND OBJECTIVES: The creation of cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) has spawned broad excitement borne out of the prospects to diagnose and treat cardiovascular diseases based on personalized medicine. A common feature of hiPS-CMs is their spontaneous contractions but the mechanism(s) remain uncertain. METHODS: Intrinsic activity was investigated by the voltage-clamp technique, optical mapping of action potentials (APs) and intracellular Ca(2+) (Cai) transients (CaiT) at subcellular-resolution and pharmacological interventions. RESULTS: The frequency of spontaneous CaiT (sCaiT) in monolayers of hiPS-CMs was not altered by ivabradine, an inhibitor of the pacemaker current, If despite high levels of HCN transcripts (1-4). HiPS-CMs had negligible If and IK1 (inwardly-rectifying K(+)-current) and a minimum diastolic potential of -59.1±3.3mV (n=18). APs upstrokes were preceded by a depolarizing-foot coincident with a rise of Cai. Subcellular Cai wavelets varied in amplitude, propagated and died-off; larger Cai-waves triggered cellular sCaTs and APs. SCaiTs increased in frequency with [Ca(2+)]out (0.05-to-1.8mM), isoproterenol (1µM) or caffeine (100µM) (n≥5, p<0.05). HiPS-CMs became quiescent with ryanodine receptor stabilizers (K201=2µM); tetracaine; Na-Ca exchange (NCX) inhibition (SEA0400=2µM); higher [K(+)]out (5→8mM), and thiol-reducing agents but could still be electrically stimulated to elicit CaiTs. Cell-cell coupling of hiPS-CM in monolayers was evident from connexin-43 expression and CaiT propagation. SCaiTs from an ensemble of dispersed hiPS-CMs were out-of-phase but became synchronous through the outgrowth of inter-connecting microtubules. CONCLUSIONS: Automaticity in hiPS-CMs originates from a Ca(2+)-clock mechanism involving Ca(2+) cycling across the sarcoplasmic reticulum linked to NCX to trigger APs.

Bradycardia alters Ca(2+) dynamics enhancing dispersion of repolarization and arrhythmia risk.

Bradycardia prolongs action potential (AP) durations (APD adaptation), enhances dispersion of repolarization (DOR), and promotes tachyarrhythmias. Yet, the mechanisms responsible for enhanced DOR and tachyarrhythmias remain largely unexplored. Ca(2+) transients and APs were measured optically from Langendorff rabbit hearts at high (150 × 150 µm(2)) or low (1.5 × 1.5 cm(2)) magnification while pacing at a physiological (120 beats/min) or a slow heart rate (SHR = 50 beats/min). Western blots and pharmacological interventions were used to elucidate the regional effects of bradycardia. As a result, bradycardia (SHR 50 beats/min) increased APDs gradually (time constant τf→s = 48 ± 9.2 s) and caused a secondary Ca(2+) release (SCR) from the sarcoplasmic reticulum during AP plateaus, occurring at the base on average of 184.4 ± 9.7 ms after the Ca(2+) transient upstroke. In subcellular imaging, SCRs were temporally synchronous and spatially homogeneous within myocytes. In diastole, SHR elicited variable asynchronous sarcoplasmic reticulum Ca(2+) release events leading to subcellular Ca(2+) waves, detectable only at high magnification. SCR was regionally heterogeneous, correlated with APD prolongation (P < 0.01, n = 5), enhanced DOR (r = 0.9277 ± 0.03, n = 7), and was gradually reversed by pacing at 120 beats/min along with APD shortening (P < 0.05, n = 5). A stabilizer of leaky ryanodine receptors (RyR2), 3-(4-benzylcyclohexyl)-1-(7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)propan-1-one (K201; 1 µM), suppressed SCR and reduced APD at the base, thereby reducing DOR (P < 0.02, n = 5). Ventricular ectopy induced by bradycardia (n = 5/15) was suppressed by K201. Western blot analysis revealed spatial differences of voltage-gated L-type Ca(2+) channel protein (Cav1.2α), Na(+)-Ca(2+) exchange (NCX1), voltage-gated Na(+) channel (Nav1.5), and rabbit ether-a-go-go-related (rERG) protein [but not RyR2 or sarcoplasmic reticulum Ca(2+) ATPase 2a] that correlate with the SCR distribution and explain the molecular basis for SCR heterogeneities. In conclusion, acute bradycardia elicits synchronized subcellular SCRs of sufficient magnitude to overcome the source-sink mismatch and to promote afterdepolarizations.

The link between abnormal calcium handling and electrical instability in acquired long QT syndrome--Does calcium precipitate arrhythmic storms?

Release of Ca(2+) ions from sarcoplasmic reticulum (SR) into myocyte cytoplasm and their binding to troponin C is the final signal form myocardial contraction. Synchronous contraction of ventricular myocytes is necessary for efficient cardiac pumping function. This requires both shuttling of Ca(2+) between SR and cytoplasm in individual myocytes, and organ-level synchronization of this process by means of electrical coupling among ventricular myocytes. Abnormal Ca(2+) release from SR causes arrhythmias in the setting of CPVT (catecholaminergic polymorphic ventricular tachycardia) and digoxin toxicity. Recent optical mapping data indicate that abnormal Ca(2+) handling causes arrhythmias in models of both repolarization impairment and profound bradycardia. The mechanisms involve dynamic spatial heterogeneity of myocardial Ca(2+) handling preceding arrhythmia onset, cell-synchronous systolic secondary Ca(2+) elevation (SSCE), as well as more complex abnormalities of intracellular Ca(2+) handling detected by subcellular optical mapping in Langendorff-perfused hearts. The regional heterogeneities in Ca(2+) handling cause action potential (AP) heterogeneities through sodium-calcium exchange (NCX) activation and eventually overwhelm electrical coupling of the tissue. Divergent Ca(2+) dynamics among different myocardial regions leads to temporal instability of AP duration and - on the patient level - in T wave lability. Although T-wave alternans has been linked to cardiac arrhythmias, non-alternans lability is observed in pre-clinical models of the long QT syndrome (LQTS) and CPVT, and in LQTS patients. Analysis of T wave lability may provide a real-time window on the abnormal Ca(2+) dynamics causing specific arrhythmias such as Torsade de Pointes (TdP).

Synchronous systolic subcellular Ca2+-elevations underlie ventricular arrhythmia in drug-induced long QT type 2.

BACKGROUND: Repolarization delay is a common clinical problem, which can promote ventricular arrhythmias. In myocytes, abnormal sarcoplasmic reticulum Ca(2+)-release is proposed as the mechanism that causes early afterdepolarizations, the cellular equivalent of ectopic-activity in drug-induced long-QT syndrome. A crucial missing link is how such a stochastic process can overcome the source-sink mismatch to depolarize sufficient ventricular tissue to initiate arrhythmias. METHODS AND RESULTS: Optical maps of action potentials and Ca(2+)-transients from Langendorff rabbit hearts were measured at low (150×150 µm(2)/pixel) and high (1.5×1.5 µm(2)/pixel) resolution before and during arrhythmias. Drug-induced long QT type 2, elicited with dofetilide inhibition of IKr (the rapid component of rectifying K+ current), produced spontaneous Ca(2+)-elevations during diastole and systole, before the onset of arrhythmias. Diastolic Ca(2+-)waves appeared randomly, propagated within individual myocytes, were out-of-phase with adjacent myocytes, and often died-out. Systolic secondary Ca(2+-)elevations were synchronous within individual myocytes, appeared 188±30 ms after the action potential-upstroke, occurred during high cytosolic Ca(2+) (40%-60% of peak-Ca(2+)-transients), appeared first in small islands (0.5×0.5 mm(2)) that enlarged and spread throughout the epicardium. Synchronous systolic Ca(2+-)elevations preceded voltage-depolarizations (9.2±5 ms; n=5) and produced pronounced Spatial Heterogeneities of Ca(2+)-transient-durations and action potential-durations. Early afterdepolarizations originating from sites with the steepest gradients of membrane-potential propagated and initiated arrhythmias. Interestingly, more complex subcellular Ca(2+)-dynamics (multiple chaotic Ca(2+)-waves) occurred during arrhythmias. K201, a ryanodine receptor stabilizer, eliminated Ca(2+)-elevations and arrhythmias. CONCLUSIONS: The results indicate that systolic and diastolic Ca(2+)-elevations emanate from sarcoplasmic reticulum Ca(2+)-release and systolic Ca(2+)-elevations are synchronous because of high cytosolic and luminal-sarcoplasmic reticulum Ca(2+), which overcomes source-sink mismatch to trigger arrhythmias in intact hearts.

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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Analysis of the potential for HIV-1 Vpr as an anti-cancer agent.

Viral protein R (Vpr) is a 14kD, 96 amino acid accessory protein of the HIV virion that has been demonstrated to have important functions in the viral replication cycle including, among others, the induction of cell cycle arrest and apoptosis in rapidly proliferating cells, which results in immune dysfunction in infected individuals. Several investigators have studied the potential use of the apoptosis inducing and cell cycle arrest effect of Vpr as an anti-tumor therapeutic. In vitro studies have indicated that Vpr is cytotoxic against a large number of different tumor cell types including a number that are p53 independent. Likewise, some in vivo tumor studies using different delivery platforms/methods have indicated an anti-cancer effect mediated by Vpr. Our group has used the aggressive and poorly immunogenic murine melanoma tumor line B16.F10 as a model to deliver, through in vivo electroporation, Vpr expressing plasmids to established tumors and have demonstrated that this treatment regimen can induce growth attenuation and tumor regression in a proportion of the treated mice and appears to be associated with the induction of intratumoral apoptosis. Overall, to date, the data from a number of research groups, including our own, have indicated that Vpr has biological activity against a number of tumors in both in vivo and in vitro models and, as such, may be a potential candidate for testing in human clinical trials. In this report, we summarize the evidence supporting this hypothesis.

Chemical ablation of the Purkinje system causes early termination and activation rate slowing of long-duration ventricular fibrillation in dogs.

Endocardial mapping has suggested that Purkinje fibers may play a role in the maintenance of long-duration ventricular fibrillation (LDVF). To determine the influence of Purkinje fibers on LDVF, we chemically ablated the Purkinje system with Lugol solution and recorded endocardial and transmural activation during LDVF. Dog hearts were isolated and perfused, and the ventricular endocardium was exposed and treated with Lugol solution (n = 6) or normal Tyrode solution as a control (n = 6). The left anterior papillary muscle endocardium was mapped with a 504-electrode (21 x 24) plaque with electrodes spaced 1 mm apart. Transmural activation was recorded with a six-electrode plunge needle on each side of the plaque. Ventricular fibrillation (VF) was induced, and perfusion was halted. LDVF spontaneously terminated sooner in Lugol-ablated hearts than in control hearts (4.9 +/- 1.5 vs. 9.2 +/- 3.2 min, P = 0.01). After termination of VF, both the control and Lugol hearts were typically excitable, but only short episodes of VF could be reinduced. Endocardial activation rates were similar during the first 2 min of LDVF for Lugol-ablated and control hearts but were significantly slower in Lugol hearts by 3 min. In control hearts, the endocardium activated more rapidly than the epicardium after 4 min of LDVF with wave fronts propagating most often from the endocardium to epicardium. No difference in transmural activation rate or wave front direction was observed in Lugol hearts. Ablation of the subendocardium hastens VF spontaneous termination and alters VF activation sequences, suggesting that Purkinje fibers are important in the maintenance of LDVF.