Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca2+ release from Ryanodine Receptors (RyRs). We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca2+ sparks (Ca2+ release units, CRUs) also became less solid. An increased fraction of small CRUs in HF was linked to augmented ‘silent’ Ca2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca2+ leak and slows Ca2+ release kinetics, leading to weakened contraction in this disease.

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na+/Ca2+ exchange (NCX) and Na+/K+-ATPase (NKA). While the relevance of Ca2+ homeostasis in cardiac function has been extensively investigated, the role of Na+ regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na+ content have multiple effects on the heart by influencing intracellular Ca2+ and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na+ homeostasis. Among the proteins that accomplish this task are the Na+/Ca2+ exchanger (NCX) and the Na+/K+ pump (NKA). By transporting three Na+ ions into the cytoplasm in exchange for one Ca2+ moved out, NCX is one of the main Na+ influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na+ ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na+ and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na+ homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na+/Ca2+ exchanger (NCX1) and Na+/K+ pump and the controversies that still persist in the field.

interaction between perturbed Na + fluxes and CaMKII, and suggest that pharmacological inhibition of intracellular Na + loading can contribute to normalizing Ca 2+ and membrane potential dynamics in heart failure.

Key points• We have developed a detailed computational model of a cardiac Ca 2+ spark based on a three dimensional reconstruction of electron tomograms.• Our model predicts near total junctional Ca 2+ depletion after the spark, while regional Ca 2+ reserve is preserved. The local Ca 2+ gradient inferred by these findings reconciles previous model predictions with experimental measurements.• Differences in local distribution of calsequestrin have a profound impact on spark termination time, as reported by Fluo5, solely based on its Ca 2+ buffering capacity.• The SERCA pump can prolong spark release time by pumping Ca 2+ back into the junctional SR during the spark. Abstract Triggered release of Ca2+ from an individual sarcoplasmic reticulum (SR) Ca 2+ release unit (CRU) is the fundamental event of cardiac excitation-contraction coupling, and spontaneous release events (sparks) are the major contributor to diastolic Ca 2+ leak in cardiomyocytes. Previous model studies have predicted that the duration and magnitude of the spark is determined by the local CRU geometry, as well as the localization and density of Ca 2+ handling proteins. We have created a detailed computational model of a CRU, and developed novel tools to generate the computational geometry from electron tomographic images. Ca 2+ diffusion was modelled within the SR and the cytosol to examine the effects of localization and density of the Na + /Ca 2+ exchanger, sarco/endoplasmic reticulum Ca 2+ -ATPase 2 (SERCA), and calsequestrin on spark dynamics. We reconcile previous model predictions of approximately 90% local Ca 2+ depletion in junctional SR, with experimental reports of about 40%. This analysis supports the hypothesis that dye kinetics and optical averaging effects can have a significant impact on measures of spark dynamics. Our model also predicts that distributing calsequestrin within non-junctional Z-disc SR compartments, in addition to the junctional compartment, prolongs spark release time as reported by Fluo5. By pumping Ca 2+ back into the SR during a release, SERCA is able to prolong a Ca 2+ spark, and this may contribute to SERCA-dependent changes in Ca 2+ wave speed. Finally, we show that including the Na + /Ca 2+ exchanger inside the dyadic cleft does not alter local [Ca 2+ ] during a spark.

Key points Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non‐dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z‐axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. Abstract Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z‐axis and obscured detection by 2D techniques. Non‐overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single ‘rogue’ RyR. Internal CRUs were more tightly packed along z‐lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t‐tubules, which enabled quantification of dyadic and non‐dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

Background We have previously shown that non-equilibrium Na+ current (INa) reactivation drives isoproterenol-induced phase-3 early afterdepolarizations (EADs) in mouse ventricular myocytes. In these cells, EAD initiation occurs secondary to potentiated sarcoplasmic reticulum Ca2+ release and enhanced Na+/Ca2+ exchange (NCX). This can be abolished by tetrodotoxin-blockade of INa, but not ranolazine, which selectively inhibits ventricular late INa. Aim Since repolarization of human atrial myocytes is similar to mouse ventricular myocytes in that it is relatively rapid and potently modulated by Ca2+, we investigated whether similar mechanisms can evoke EADs in human atrium. Indeed, phase-3 EADs have been shown to re-initiate atrial fibrillation (AF) during autonomic stimulation, which is a well-recognized initiator of AF. Methods We integrated a Markov model of INa gating in our human atrial myocyte model. To simulate experimental results, we rapidly paced this cell model at 10 Hz in the presence of 0.1 µM acetylcholine and 1 µM isoproterenol, and assessed EAD occurrence upon return to sinus rhythm (1 Hz). Results Cellular Ca2+ loading during fast pacing results in a transient period of hypercontractility after return to sinus rhythm. Here, fast repolarization and enhanced NCX facilitate INa reactivation via the canonical gating mode (i.e., not late INa burst mode), which drives EAD initiation. Simulating ranolazine administration reduces atrial peak INa and leads to faster repolarization, during which INa fails to reactivate and EADs are prevented. Conclusions Non-equilibrium INa reactivation can critically contribute to arrhythmias, specifically in human atrial myocytes. Ranolazine might be beneficial in this context by blocking peak (not late) atrial INa.

Genome-wide association studies have implicated many ion channels in schizophrenia pathophysiology. Although the functions of these channels are relatively well characterized by single-cell studies, the contributions of common variation in these channels to neurophysiological biomarkers and symptoms of schizophrenia remain elusive. Here, using computational modeling, we show that a common biomarker of schizophrenia, namely, an increase in delta-oscillation power, may be a direct consequence of altered expression or kinetics of voltage-gated ion channels or calcium transporters. Our model of a circuit of layer V pyramidal cells highlights multiple types of schizophrenia-related variants that contribute to altered dynamics in the delta-frequency band. Moreover, our model predicts that the same membrane mechanisms that increase the layer V pyramidal cell network gain and response to delta-frequency oscillations may also cause a deficit in a single-cell correlate of the prepulse inhibition, which is a behavioral biomarker highly associated with schizophrenia.

Background Early-afterdepolarizations (EADs) are triggers of cardiac arrhythmia driven by L-type Ca2+ current (ICaL) reactivation or sarcoplasmic reticulum (SR) Ca2+ release and Na+/Ca2+ exchange. In large mammals the positive action potential (AP) plateau promotes ICaL reactivation, and the current paradigm holds that cardiac EAD dynamics are dominated by interaction between ICaL and the repolarizing K+ currents. However, EADs are also frequent in the rapidly repolarizing mouse AP, which should not readily permit ICaL reactivation. This suggests that murine EADs exhibit unique dynamics, which are key for interpreting arrhythmia mechanisms in this ubiquitous model organism. We investigated these dynamics in myocytes from arrhythmia-susceptible CaMKIIδC-overexpressing mice (Tg), and via computational simulations. Methods and Results In Tg myocytes, β-adrenergic challenge slowed late repolarization, potentiated SR Ca2+ release, and initiated EADs below the ICaL activation range (−47±0.7 mV). These EADs were abolished by caffeine and tetrodotoxin (but not Ranolazine), suggesting that SR Ca2+ release and Na+ current (INa), but not late INa, are required for EAD initiation. Simulations suggest that potentiated SR Ca2+ release and Na+/Ca2+ exchange triangulate late AP repolarization, which permits non-equilibrium reactivation of INa, and thereby drives the EAD upstroke. AP clamp experiments suggest that lidocaine eliminates virtually all inward current elicited by EADs, and that this effect occurs at concentrations (40-60 μM) for which lidocaine remains specific for inactivated Na+ channels. This strongly suggests that previously inactive channels are recruited during the EAD upstroke, and that non-equilibrium INa dynamics underlie murine EADs. Conclusions Non-equilibrium reactivation of INa drives murine EADs.