The clustering of ryanodine receptors (RyR2) into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling. Using immunolabeling and confocal microscopy, we have analyzed the distribution of RyR2 clusters in rat and ventricular atrial myocytes. The resolution of the three-dimensional structure was improved by a novel transverse sectioning method as well as digital deconvolution. In contrast to earlier reports, the mean RyR2 cluster transverse spacing was measured 1.05 microm in ventricular myocytes and estimated 0.97 microm in atrial myocytes. Intercalated RyR2 clusters were found interspersed between the Z-disks on the cell periphery but absent in the interior, forming double rows flanking the local Z-disks on the surface. The longitudinal spacing between the adjacent rows of RyR2 clusters on the Z-disks was measured to have a mean value of 1.87 microm in ventricular and 1.69 microm in atrial myocytes. The measured RyR2 cluster distribution is compatible with models of Ca2+ wave generation. The size of the typical RyR2 cluster was close to 250 nm, and this suggests that approximately 100 RyR2s might be present in a cluster. The importance of cluster size and three-dimensional spacing for current E-C coupling models is discussed.

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.

Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca2+ dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca2+ transient, which enhanced contractility to counter mechanical load, but also caused spontaneous Ca2+ sparks during diastole that could be arrhythmogenic. The increases in the Ca2+ transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2+ in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2+ sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca2+-calmodulin–dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca2+ sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca2+ sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2+ sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress–induced Ca2+ dysregulation, arrhythmias, and cardiomyopathy.

Spontaneously generated calcium (Ca2+) waves can trigger arrhythmias in ventricular and atrial myocytes. Yet, Ca2+ waves also serve the physiological function of mediating global Ca2+ increase and muscle contraction in atrial myocytes. We examine the factors that influence Ca2+ wave initiation by mathematical modeling and large-scale computational (supercomputer) simulations. An important finding is the existence of a strong coupling between the ryanodine receptor distribution and Ca2+ dynamics. Even modest changes in the ryanodine receptor spacing profoundly affect the probability of Ca2+ wave initiation. As a consequence of this finding, we suggest that there is information flow from the contractile system to the Ca2+ control system and this dynamical interplay could contribute to the increased incidence of arrhythmias during heart failure.

Cardiac atrial cells lack a regular system of transverse tubules like that in cardiac ventricular cells. Nevertheless, many atrial cells do possess an irregular internal transverse‐axial tubular system (TATS). To investigate the possible role of the TATS in excitation‐contraction coupling in atrial myocytes, we visualized the TATS (labelled with the fluorescent indicator, Di‐8‐ANEPPS) simultaneously with Ca2+ transients and/or Ca2+ sparks (fluo‐4). In confocal transverse linescan images of field‐stimulated cells, whole‐cell Ca2+ transients had two morphologies: ‘U‐shaped’ transients and irregular or ‘W‐shaped’ transients with a varying number of points of origin of the Ca2+ transient. About half (54 %, n=289 cells, 13 animals) of the cells had a TATS. Cells with TATS had a larger mean diameter (13.2 ± 2.8 μm) than cells without TATS (11.7 ± 2.0 μm) and were more common in the left atrium (n= 206 cells; left atrium: 76 with TATS, 30 without TATS; right atrium: 42 with TATS, 58 without TATS). Simultaneous measurement of Ca2+ sparks and sarcolemmal structures showed that cells without TATS had U‐shaped transients that started at the cell periphery, and cells with TATS had W‐shaped transients that began simultaneously at the cell periphery and the TATS. Most (82 out of 102 from 31 cells) ‘spontaneous’ (non‐depolarized) Ca2+ sparks occurred within 1 μm of a sarcolemmal structure (cell periphery or TATS), and 33 % occurred within 1 pixel (0.125 μm). We conclude that the presence of a sarcolemmal membrane either at the cell periphery or in the TATS in close apposition to the sarcoplasmic reticulum is required for the initiation of an evoked Ca2+ transient and for spontaneous Ca2+ sparks.