To understand the relevance of the work published by Zhao et al in this journal, it may be helpful to first describe some of the mechanistic and structural characteristics of cardiac excitation–contraction coupling. It is now well established through the extensive work of Fabiato that in heart a small rise of Ca2þ in the vicinity of the sarcoplasmic reticulum (SR) induces a much larger Ca2þ release from this intracellular store, e.g. This is known as Ca2þ-induced Ca2þ release (CICR). It is also known that large SR release channels or ryanodine receptors (RyR) in the terminal cisternae of the SR can be gated by rises in Ca2þproduce by L-type Ca2þChannel (LCC) current. Thus LCCs provide the trigger which induces Ca2þ release from the SR via RyRs in functioning heart cells. This provides the molecular basis of CICR. It was originally assumed that Ca2þ release sites were distributed in a ‘common pool’ into which both triggering and released Ca2þwere discharged. However, this immediately introduced a difficulty. It had been known from some time that in heart both contraction and Ca2þ release can be graded. In fact, Ca2þ release and LCC current magnitude both display a bell-shaped relationship suggesting that Ca2þ release can be graded with trigger size. It was not clear how graded responses could be obtained in a common pool since one would expect that Ca2þ release through one RyR would induce release from adjacent RyRs producing an all or none response (Figure 1A). To deal with this difficulty, Michael Stern proposed a ‘cluster bomb’ model (Figure 1B) in which release units were arranged in clusters separated one from another by a distance which could limit common pool regeneration by reducing the likelihoods of adjacent clusters activation. In this model, which is largely accepted today, a single LCC could supply Ca2þacross a Ca2þ synapse that would activate RyRs in the cluster and lead to a regenerative activation of release from the entire cluster. It is now understood that a cluster of Ca2þ transporters, including LCCS, in the sarcolemma are separated from a cluster of RyR in the terminal cisternae of the SR by a junctional regions of 10–15 nm which forms a Ca2þ synapse. This entire arrangement, referred to as a couplon, forms a Ca2þ release unit (CRU). It is now possible to detect local release events or Ca2þ sparks which emanate from these geometrically isolated CRUs that sum to produce a Ca2þ transient. In this model, individual CRUs can be recruited stochastically as a function of voltage thus producing graded Ca2þ release. This stochastic recruitment of CRUs is possible because they are controlled locally. However, local control is not necessarily constrained to one CRU in all parts of the cell. Moreover, it is known that under conditions of extreme SR loading large spontaneous releases of Ca2þoccur independent of LCC activation. Under these circumstances, CRUs behave as if they are in a common pool. It has been suggested that additional controls exist within CRUs to limit the sensitivity of the RyRs to triggering Ca2þ so that activation between adjacent CRUs and common pool activation of all CRUs is less likely to occur providing an additional level of local control. It has been proposed for example that the 12.6-kDa-FK506 binding protein (FKP12.6) reduces the sensitivity of RyRs to Ca2þ stabilizing the CICR system and preventing, or at least limiting, common pool regeneration of multiple CRUs (Figure 1C). However, this has proved controversial. In this edition of Cardiovascular Research, Zhao et al. report elegant studies that strongly suggest that indeed FKBP12.6 can reduce the sensitivity of RyRs to Ca2þby modifying RyR open probability (PRyR). Presumably this effect is mediated by FKBP12.6 binding with recently studied structural domains present in RyR2. This effectively stabilizes the system against undesirable Ca2þ release produced by common pool regeneration during for example b-adrenergic stimulation. These authors used a combination of patch clamping and confocal imaging to detect both ‘sparklets’ produced by unitary Ca2þcurrents and local release events or sparks in both WT and FKBP12.6 KO mice. This allowed them to establish the temporal relationship between isolated triggers (sparklets) and sparks in situ. Near threshold depolarization, which activated a small fraction of the LCC pool, produced a stochastic activation of sparks by what was very likely a single LCC opening. Spark frequency was found to be higher in the FKBP12.6 KO than in WT mice. In addition, the delay between trigger and sparks was shortened. These observations, among others, led the authors to conclude that FKBP12.6 stabilizes RyRs by desensitizing them to Ca2þ. An important finding that helps to resolve controversy surrounding this subject is that neither FKBP12.6 KO nor b-adrenergic stimulation acting independently

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.

Dyssynchronous heart failure (DHF) is associated with structural and functional remodeling in cardiomyocytes from subcellular to whole organ level. Several studies have demonstrated remodeling of Ca2+ signaling and excitation-contraction coupling. This study aimed at quantification of the subcellular spatial distribution of diastolic local Ca2+ release events (sparks) in cardiomyocytes and their remodeling in DHF. Adult canines were used as control and DHF models. DHF was induced by right ventricular tachypacing at 200 beats per minute. Left ventricular cardiomyocytes were isolated via enzymatic digestion, loaded with Fluo-4 and Di-8-ANEPPS, and imaged with rapid scanning confocal microscopy (Zeiss LSM 5 Duo). Acquisition of 2D image sequences was initiated 460 ms after stimulation of the myocytes. Images were acquired at a xy resolution of 0.1 µm with a field of view of 102.4 µm (x) x 25.6 µm (y). Image sequences comprised 100 frames at 9.3 ms per frame. Image analysis involved cell segmentation and spark detection. We compared spark density in regions 0-10 µm and 10-40 µm from the longitudinal cell end. Our analyses yielded a similar diastolic spark density in control and DHF cells (0.0119 ± 0.0023 vs 0.0155 ± 0.0027 sparks/µm2/s, respectively). In control cells spark density was homogeneously distributed. In contrast, DHF cells exhibited a decreased spark density within 0-10 µm versus 10-40 µm from the longitudinal cell end (0.0101 ± 0.0039 vs 0.0227 ± 0.0047 sparks/µm2/s, respectively). DHF is associated with a spatially heterogeneous remodeling of spark density. This finding provides a foundation for understanding basic mechanisms of arrhythmogenesis in heart failure cells. The heterogeneity of spark density may result from heterogeneous structural remodeling, for instance, remodeling of ryanodine receptor clusters and the transverse tubular system.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuronal Na channel blockade suppresses arrhythmogenic diastolic Ca release

Background— Cardiac resynchronization therapy (CRT) is a major advance for treatment of patients with dyssynchronous heart failure (DHF). However, our understanding of DHF-associated remodeling of subcellular structure and function and their restoration after CRT remains incomplete. Methods and Results— We investigated subcellular heterogeneity of remodeling of structures and proteins associated with excitation–contraction coupling in cardiomyocytes in DHF and after CRT. Three-dimensional confocal microscopy revealed subcellular heterogeneity of ryanodine receptor (RyR) density and the transverse tubular system (t-system) in a canine model of DHF. RyR density at the ends of lateral left ventricular cardiomyocytes was higher than that in cell centers, whereas the t-system was depleted at cell ends. In anterior left ventricular cardiomyocytes, however, we found a similar degree of heterogeneous RyR remodeling, despite preserved t-system. Synchronous heart failure was associated with marginal heterogeneity of RyR density. We used rapid scanning confocal microscopy to investigate effects of heterogeneous structural remodeling on calcium signaling. In DHF, diastolic Ca 2+ spark density was smaller at cell ends versus centers. After CRT, subcellular heterogeneity of structures and function was reduced. Conclusions— RyR density exhibits remarkable subcellular heterogeneity in DHF. RyR remodeling occurred in lateral and anterior cardiomyocytes, but remodeling of t-system was confined to lateral myocytes. These findings indicate that different mechanisms underlie remodeling of RyRs and t-system. Furthermore, we suggest that ventricular dyssynchrony exacerbates subcellular remodeling in heart failure. CRT efficiently reduced subcellular heterogeneity. These results will help to explain remodeling of excitation–contraction coupling in disease and restoration after CRT.

AIMS Sudden death resulting from cardiac arrhythmias is the most common consequence of cardiac disease. Certain arrhythmias caused by abnormal impulse formation including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with delayed afterdepolarizations resulting from diastolic Ca2+ release (DCR) from the sarcoplasmic reticulum (SR). Despite high response of CPVT to agents directly affecting Ca2+ cycling, the incidence of refractory cases is still significant. Surprisingly, these patients often respond to treatment with Na+ channel blockers. However, the relationship between Na+ influx and disturbances in Ca2+ handling immediately preceding arrhythmias in CPVT remains poorly understood and is the object of this study. METHODS AND RESULTS We performed optical Ca2+ and membrane potential imaging in ventricular myocytes and intact cardiac muscles as well as surface ECGs on a CPVT mouse model with a mutation in cardiac calsequestrin. We demonstrate that a subpopulation of Na+ channels (neuronal Na+ channels; nNav) colocalize with ryanodine receptor Ca2+ release channels (RyR2). Disruption of the crosstalk between nNav and RyR2 by nNav blockade with riluzole reduced and also desynchronized DCR in isolated cardiomyocytes and in intact cardiac tissue. Such desynchronization of DCR on cellular and tissue level translated into decreased arrhythmias in CPVT mice. CONCLUSIONS Thus, our study offers the first evidence that nNav contribute to arrhythmogenic DCR, thereby providing a conceptual basis for mechanism-based antiarrhythmic therapy.

Sarcomeres are the basic contractile units of cardiac myocytes. Recent studies demonstrated remodeling of sarcomeric proteins in several diseases, including genetic defects and heart failure. Here we investigated remodeling of sarcomeric α-actinin in two models of heart failure, synchronous (SHF) and dyssynchronous heart failure (DHF), as well as a model of cardiac resynchronization therapy (CRT). We applied three-dimensional confocal microscopy and quantitative methods of image analysis to study isolated cells from our animal models. 3D Fourier analysis revealed a decrease of the spatial regularity of the α-actinin distribution in both SHF and DHF versus control cells. The spatial regularity of α-actinin in DHF cells was reduced when compared with SHF cells. The spatial regularity of α-actinin was partially restored after CRT. We found longitudinal depositions of α-actinin in SHF, DHF and CRT cells. These depositions spanned adjacent Z-disks and exhibited a lower density of α-actinin than in the Z-disk. Differences in the occurrence of depositions between the SHF, CRT and DHF models versus control were significant. Also, CRT cells exhibited a higher occurrence of depositions versus SHF, but not DHF cells. Other sarcomeric proteins did not accumulate in the depositions to the same extent as α-actinin. We did not find differences in the expression of α-actinin protein and its encoding gene in our animal models. In summary, our studies indicate that HF is associated with two different types of remodeling of α-actinin and only one of those was reversed after CRT. We suggest that these results can guide us to an understanding of remodeling of structures and function associated with sarcomeres.

Excitation–contraction coupling in cardiomyocytes requires Ca2+ influx through dihydropyridine receptors in the sarcolemma, which gates Ca2+ release through sarcoplasmic ryanodine receptors (RyRs). Ca2+ influx, release and diffusion produce a cytosolic Ca2+ transient. Here, we investigated the relationship between Ca2+ transients and the spatial arrangement of the sarcolemma including the transverse tubular system (t-system). To accomplish this, we studied isolated ventricular myocytes of rabbit, which exhibit a heterogeneously distributed t-system. We developed protocols for fluorescent labeling and triggered two-dimensional confocal microscopic imaging with high spatiotemporal resolution. From sequences of microscopic images, we measured maximal upstroke velocities and onset times of local Ca2+ transients together with their distance from the sarcolemma. Analyses indicate that not only sarcolemmal release sites, but also those that are within 1μm of the sarcolemma actively release Ca2+. Our data also suggest that release does not occur at sites further than 2.5μm from the sarcolemma. The experimental data are in agreement with results from a mathematical model of Ca2+ release and diffusion. Our findings can be explained by a modified local control model, which constrains the region of regenerative activation of non-junctional RyR clusters. We believe that this model will be useful for describing excitation–contraction coupling in cardiac myocytes with a sparse t-system, which includes those from diseased heart tissue as well as atrial myocytes of some species.

Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization based on fluorescent labeling, 3-D scanning confocal microscopy, image processing and reconstruction of tissue micro-structure at sub-micrometer resolution. We applied this methodology to normal rabbit ventricular tissue and tissue from hearts with myocardial infarction. Our analysis revealed that the volume fraction of fibroblasts increased from 4.83±0.42% (mean ± standard deviation) in normal tissue up to 6.51±0.38% in myocardium from infarcted hearts. The myocyte volume fraction decreased from 76.20±9.89% in normal to 73.48±8.02% adjacent to the infarct. Numerical field calculations on 3-D reconstructions of the extracellular space yielded an extracellular longitudinal conductivity of 0.264±0.082 S/m with an anisotropy ratio of 2.095±1.11 in normal tissue. Adjacent to the infarct, the longitudinal conductivity increased up to 0.400±0.051 S/m, but the anisotropy ratio decreased to 1.295±0.09. Our study indicates an increased density of gap junctions proximal to both fibroblasts and myocytes in infarcted versus normal tissue, supporting previous hypotheses of electrical coupling of fibroblasts and myocytes in infarcted hearts. We suggest that the presented methodology provides an important contribution to modeling normal and diseased tissue. Applications of the methodology include the clinical characterization of disease-associated remodeling.

Sodium-calcium exchange (NCX) is the major calcium (Ca) efflux mechanism of ventricular cardiomyocytes. Consequently the exchanger plays a critical role in the regulation of cellular Ca content and hence contractility. Reductions in Ca efflux by the exchanger, such as those produced by elevated intracellular sodium (Na) in response to cardiac glycosides, raise sarcoplasmic reticulum (SR) Ca stores. The result is an increased Ca transient and cardiac contractility. Enhanced Ca efflux activity by the exchanger, for example during heart failure, may reduce diadic cleft Ca and excitation-contraction (EC) coupling gain. This aggravates the impaired contractility associated with SR Ca ATPase dysfunction and reduced SR Ca load in failing heart muscle. Recent data from our laboratories indicate that NCX can also impact the efficiency of EC coupling and contractility independent of SR Ca load through diadic cleft priming with Ca during the upstroke of the action potential. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

This chapter offers brief information on the current knowledge of Na + -Ca 2+ exchange currents. The early studies paved the way for a number of subsequent investigations that have led to molecular cloning and elucidation of the structure of the exchanger molecule itself. This in turn introduced the possibility of studying the relationship between molecular structure and function. The chapter discusses several aspects of Na + -Ca 2+ exchanger, including structure, topology, and distribution, phylogeny, isoforms, energetics, methods and problems associated with the measurement, isolation of Na + -Ca 2+ exchange current, ionic dependencies, regulation of current, current-voltage relationships and voltage dependence of Na + -Ca 2+ exchange current and mechanism, Na + -Ca 2+ exchange currents during the cardiac action potential, and excitation-contraction coupling. Isolation of Na + -Ca 2+ exchange current includes whole-cell patch-clamp studies, and Na + -Ca 2+ exchange current reversal potential. It is mentioned that the generation of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) is an important regulator of Na + -Ca 2+ exchange activity in heart. The ease with which heart cells can be patch-clamped, together with the presence of a vigorous exchange activity, clearly explains the fact that most of our information on exchange current comes from this tissue. Na + -Ca 2+ exchange currents have been measured in other cell types, including the squid giant axon. Although measurements of exchange current are difficult in many tissues, it is now possible to express Na + -Ca 2+ exchangers in frog oocytes. This together with the development of giant excised patches that can be voltage clamped has made studies of the relationship between structure of the exchanger and function much easier. Measurements of both whole-cell currents as well as currents in giant patches have produced evidence in favor of the idea that a consecutive mechanism can explain exchange activity. Most recently, Na + -Ca 2+ exchange currents have been proposed as a trigger for SR Ca 2+ release under physiological conditions. Thus, in the last 30 years, the study of exchange currents has expanded enormously to provide not only insight into the way that the Na + -Ca 2+ exchange controls intracellular Ca 2+ at the whole-cell level, but also into the details of the molecular mechanism of the exchange reaction itself.

Calcium signaling in cardiomyocytes is strongly influenced by the topology of the sarcolemma (SL) and the distribution of sarcolemmal proteins, including the L-type calcium channel (LCC) and sodium-calcium exchanger (NCX). Peculiar to mammalian ventricular cardiomyocytes are sarcolemmal invaginations called transverse tubules (TT) exhibiting high densities of LCC clusters that trigger Ca2+ release from the sarcoplasmic reticulum (SR). Prior studies of pharmacologically-disabled SR release in rabbit ventricular myocytes have demonstrated that sub-micrometer resolution details of the SL geometry shape local Ca2+ dynamics and suggest a feedback between cytosolic [Ca2+] and SL ion channel and transporter activity. Here we investigate the hypothesis that the ordered spatial arrangement of TT and coupling between adjacent tubules leads to an organized Ca2+ transient. Moreover, we compare Ca2+ transients arising from clustered or continuously-distributed trigger fluxes for the propensity to produce Ca2+ waves. Our findings are that 1) the arrangement of TTs promotes a faster rise in [Ca2+] transversely relative to the longitudinal direction of cardiomyocytes and 2) clustering of SL transporters along the TT promotes an axially-uniform calcium transient. These results evidence contribution of structural detail at sub-micrometer resolution to excitation-contraction coupling and anomalous Ca2+ dynamics underlying cardiac arrhythmias. Supported by NBCR (NIH grant 2 P41 RR08605), NIH GM31749, NSF MCB-0506593, MCA93S013, Center for Theoretical Biological Physics, Howard Hughes Medical Institute, SDSC, W. M. Keck foundation, Richard A. and Nora Eccles Fund for Cardiovascular Research.

The transverse tubular system of rabbit ventricular myocytes consists of cell membrane invaginations (t-tubules) that are essential for efficient cardiac excitation-contraction coupling. In this study, we investigate how t-tubule micro-anatomy, L-type Ca2+ channel (LCC) clustering, and allosteric activation of Na+/Ca2+ exchanger by L-type Ca2+ current affects intracellular Ca2+ dynamics. Our model includes a realistic 3D geometry of a single t-tubule and its surrounding half-sarcomeres for rabbit ventricular myocytes. The effects of spatially distributed membrane ion-transporters (LCC, Na+/Ca2+ exchanger, sarcolemmal Ca2+ pump, and sarcolemmal Ca2+ leak), and stationary and mobile Ca2+ buffers (troponin C, ATP, calmodulin, and Fluo-3) are also considered. We used a coupled reaction-diffusion system to describe the spatio-temporal concentration profiles of free and buffered intracellular Ca2+. We obtained parameters from voltage-clamp protocols of L-type Ca2+ current and line-scan recordings of Ca2+ concentration profiles in rabbit cells, in which the sarcoplasmic reticulum is disabled. Our model results agree with experimental measurements of global Ca2+ transient in myocytes loaded with 50 μM Fluo-3. We found that local Ca2+ concentrations within the cytosol and sub-sarcolemma, as well as the local trigger fluxes of Ca2+ crossing the cell membrane, are sensitive to details of t-tubule micro-structure and membrane Ca2+ flux distribution. The model additionally predicts that local Ca2+ trigger fluxes are at least threefold to eightfold higher than the whole-cell Ca2+ trigger flux. We found also that the activation of allosteric Ca2+-binding sites on the Na+/Ca2+ exchanger could provide a mechanism for regulating global and local Ca2+ trigger fluxes in vivo. Our studies indicate that improved structural and functional models could improve our understanding of the contributions of L-type and Na+/Ca2+ exchanger fluxes to intracellular Ca2+ dynamics.

In most mammalian cardiomyocytes, the transverse tubular system (t-system) is a major site for electrical signaling and excitation-contraction coupling. The t-system consists of membrane invaginations, which are decorated with various proteins involved in excitation-contraction coupling and mechano-electric feedback. Remodeling of the t-system has been reported for cells in culture and various types of heart disease. In this paper, we provide insights into effects of mechanical strain on the t-system in rabbit left ventricular myocytes. Based on fluorescent labeling, three-dimensional scanning confocal microscopy, and digital image analysis, we studied living and fixed isolated cells in different strain conditions. We extracted geometric features of transverse tubules (t-tubules) and characterized their arrangement with respect to the Z-disk. In addition, we studied the t-system in cells from hearts fixed either at zero left ventricular pressure (slack), at 30 mmHg (volume overload), or during lithium-induced contracture, using transmission electron microscopy. Two-dimensional image analysis was used to extract features of t-tubule cross-sections. Our analyses of confocal microscopic images showed that contracture at the cellular level causes deformation of the t-system, increasing the length and volume of t-tubules, and altering their cross-sections shape. TEM data reconfirmed the presence of mechanically induced changes in t-tubular cross sections. In summary, our studies suggest that passive longitudinal stretching and active contraction of ventricular cardiomyocytes affect the geometry of t-tubules. This confirms that mechanical changes at cellular levels could promote alterations in partial volumes that would support a convection-assisted mode of exchange between the t-system content and extracellular space. (C) 2012 Elsevier Ltd. All rights reserved.

Invaginations of the sarcolemma, called 'transverse tubules' (t-tubules), allow for rapid communication of electrical activation deep into the interior of ventricular cardiomyocytes. Given the length and radius of t-tubules, the rate of diffusion alone appears to be insufficient for homeostasis of the t-tubular content, especially during exercise [1]. Previously, we reported in rabbit ventricular myocytes that positive longitudinal strain modulates geometrical features of t-tubules [2] and their mouth-region [3] in a way that could support convective re-distribution of t-tubular content. Here, we test the hypothesis that cell contraction also affects t-tubular volume. Isolated ventricular cardiomyocytes from adult New Zealand white (NZW; n=23) rabbits were imaged using an inverted confocal microscope [2] either at slack length or during negative strain to ∼85%, caused by exposure to superfusate-induced tonic contracture. Image stacks of cell segments were deconvolved and t-tubules segmented. In addition, ventricular tissue from NZW rabbit hearts, fixed either at zero intra-ventricular pressure (n=2) or during contracture (n=2), was studied using transmission electron microscopy (TEM; [3]). T-tubular length and volume were assessed in confocal images, while ellipticity and orientation were explored both in confocal and TEM data. Statistical significance was determined using a two-tailed t-test with p<0.05 considered significant. T-tubular cross-section changed, reducing ellipticity in contractured myocytes (compared to control), while apparent length and total volume of t-tubules increased. This suggests that both passive distension and active contraction may give rise to a convective component of fluid transport, mixing, and exchange of t-tubular content. [1] Shepard, N, HB McDonough. Am J Physiol Heart Circ Physiol. 1998/275:H852-H860 [2] McNary, TG, JH Bridge, FB Sachse. Biophys J. 2011/100:L53-L55. [3] Kohl, P, PJ Cooper, H Holloway. Prog Biophys Mol Biol. 2003/82:221-227.

Summation of microscopic release events from RyRs underlies the upstroke of the calcium transient in mammalian ventricular cells. These release events are initiated when RyRs are gated by transmembrane calcium influx. In rabbit cardiac myocytes the majority of RyR clusters are closely associated with the sarcolemma where they form couplons. However, significant numbers of RyR clusters are non-junctional and do not form couplons. It is unclear if or by what mechanism non-junctional RyRs are activated. We analyzed the activation of both types of RyRs. We studied calcium transients in rabbit isolated ventricular cells that exhibit a sparse t-system with Fluo-4 and high-speed two-dimensional confocal microscopy. We constructed distance maps indicating that some intracellular regions are up to 3.5 μm away from the sarcolemma. Image sequences showed that transients were spatially and temporally inhomogeneous. Transients originated in regions where we detected sarcolemma and spread with a significant delay to areas lacking t-system where non-junctional RyRs are present. We found an increasing relationship between sarcolemmal distances and local activation times with activation times of 13.0±6.6 ms at sarcolemmal sites, and 20.5±5.1 ms at distances 3-3.5 μm. The relationship between sarcolemmal distances and maximal upstroke of the transient was more complex. Maximal upstroke at sarcolemmal sites was 13.2±4.1 %/ms versus 8.8±1.6 ms at 3-3.5 μm. To interpret these results we used a one-dimensional model of calcium diffusion, which explains the spread of upstroke that we see experimentally. We conclude that non-junctional RyRs within 1.5 μm of the sarcolemma are activated. Beyond 1.5 μm the rise in calcium could result from either diffusion or slow activation. In both cases, activation of non-junctional RyRs would occur by a mechanism different than local control, i.e. a modified common pool model.

Publisher Summary This chapter offers brief information on the current knowledge of Na+-Ca2+ exchange currents. The early studies paved the way for a number of subsequent investigations that have led to molecular cloning and elucidation of the structure of the exchanger molecule itself. This in turn introduced the possibility of studying the relationship between molecular structure and function. The chapter discusses several aspects of Na+-Ca2+ exchanger, including structure, topology, and distribution, phylogeny, isoforms, energetics, methods and problems associated with the measurement, isolation of Na+-Ca2+ exchange current, ionic dependencies, regulation of current, current-voltage relationships and voltage dependence of Na+-Ca2+ exchange current and mechanism, Na+-Ca2+ exchange currents during the cardiac action potential, and excitation-contraction coupling. Isolation of Na+-Ca2+ exchange current includes whole-cell patch-clamp studies, and Na+-Ca2+ exchange current reversal potential. It is mentioned that the generation of phosphatidylinositol-4,5-bisphosphate (PIP2) is an important regulator of Na+-Ca2+ exchange activity in heart. The ease with which heart cells can be patch-clamped, together with the presence of a vigorous exchange activity, clearly explains the fact that most of our information on exchange current comes from this tissue. Na+-Ca2+ exchange currents have been measured in other cell types, including the squid giant axon. Although measurements of exchange current are difficult in many tissues, it is now possible to express Na+-Ca2+ exchangers in frog oocytes. This together with the development of giant excised patches that can be voltage clamped has made studies of the relationship between structure of the exchanger and function much easier. Measurements of both whole-cell currents as well as currents in giant patches have produced evidence in favor of the idea that a consecutive mechanism can explain exchange activity. Most recently, Na+-Ca2+ exchange currents have been proposed as a trigger for SR Ca2+ release under physiological conditions. Thus, in the last 30 years, the study of exchange currents has expanded enormously to provide not only insight into the way that the Na+-Ca2+ exchange controls intracellular Ca2+ at the whole-cell level, but also into the details of the molecular mechanism of the exchange reaction itself.

Rationale: Cardiac resynchronization therapy (CRT) is an established treatment for patients with chronic heart failure. However, CRT-associated structural and functional remodeling at cellular and subcellular levels is only partly understood. Objective: To investigate the effects of CRT on subcellular structures and protein distributions associated with excitation-contraction coupling of ventricular cardiomyocytes. Methods and Results: Our studies revealed remodeling of the transverse tubular system (t-system) and the spatial association of ryanodine receptor (RyR) clusters in a canine model of dyssynchronous heart failure (DHF). We did not find this remodeling in a synchronous heart failure model based on atrial tachypacing. Remodeling in DHF ranged from minor alterations in anterior left ventricular myocytes to nearly complete loss of the t-system and dissociation of RyRs from sarcolemmal structures in lateral cells. After CRT, we found a remarkable and almost complete reverse remodeling of these structures despite persistent left ventricular dysfunction. Studies of whole-cell Ca 2+ transients showed that the structural remodeling and restoration were accompanied with remodeling and restoration of Ca 2+ signaling. Conclusions: DHF is associated with regional remodeling of the t-system. Myocytes undergo substantial structural and functional restoration after only 3 weeks of CRT. The finding suggests that t-system status can provide an early marker of the success of this therapy. The results could also guide us to an understanding of the loss and remodeling of proteins associated with the t-system. The steep relationship between free Ca 2+ and contraction suggests that some restoration of Ca 2+ release units will have a disproportionately large effect on contractility.

The transverse tubular system (t-system) is a major site for signaling in mammalian ventricular cardiomyocytes including electrical signaling and excitation-contraction coupling. It consists of membrane invaginations, which are decorated with various proteins including mechanosensitive ion channels. Here, we investigated mechanical modulation of the t-system. By applying fluorescent markers, three-dimensional scanning confocal microscopy, and methods of digital image analysis, we studied isolated ventricular cardiomyocytes under different strains. We demonstrate that strain at the cellular level is transmitted to the t-system, reducing the length and volume of tubules and altering their cross-sectional shape. Our data suggest that a cellular strain of as little as 5% affects the shape of transverse tubules, which has important implications for the function of mechanosensitive ion channels found in them. Furthermore, our study supports a prior hypothesis that strain can cause fluid exchange between the t-system and extracellular space.