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While many ion channels and transporters involved in cardiac cellular physiology have been identified and described, the relative importance of each in determining emergent cellular behaviors remains unclear. Here we address this issue with a novel approach that combines population-based mathematical modeling with experimental tests to systematically quantify the relative contributions of different ion channels and transporters to the amplitude of the cellular Ca(2+) transient. Sensitivity analysis of a mathematical model of the rat ventricular cardiomyocyte quantified the response of cell behaviors to changes in the level of each ion channel and transporter, and experimental tests of these predictions were performed to validate or invalidate the predictions. The model analysis found that partial inhibition of the transient outward current in rat ventricular epicardial myocytes was predicted to have a greater impact on Ca(2+) transient amplitude than either: (1) inhibition of the same current in endocardial myocytes, or (2) comparable inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). Experimental tests confirmed the model predictions qualitatively but showed some quantitative disagreement. This guided us to recalibrate the model by adjusting the relative importance of several Ca(2+) fluxes, thereby improving the consistency with experimental data and producing a more predictive model. Analysis of human cardiomyocyte models suggests that the relative importance of outward currents to Ca(2+) transporters is generalizable to human atrial cardiomyocytes, but not ventricular cardiomyocytes. Overall, our novel approach of combining population-based mathematical modeling with experimental tests has yielded new insight into the relative importance of different determinants of cell behavior., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Numerous studies have employed mathematical modeling to quantitatively understand release of Ca(2+) from the sarcoplasmic reticulum (SR) in the heart. Models have been used to investigate physiologically important phenomena such as triggering of SR Ca(2+) release by Ca(2+) entry across the cell membrane and spontaneous leak of Ca(2+) from the SR in quiescent heart cells. In this review we summarize studies that have modeled myocardial Ca(2+) at different spatial scales: the sub-cellular level, the cellular level, and the multicellular level. We discuss each category of models from the standpoint of parameter sensitivity analysis, a common simulation procedure that can generate quantitative, comprehensive predictions about how changes in conditions influence model output. We propose that this is a useful perspective for conceptualizing models, in part because a sensitivity analysis requires the investigator to define the relevant parameters and model outputs. This procedure therefore helps to illustrate the capabilities and limitations of each model. We further suggest that in future studies, sensitivity analyses will aid in simplifying complex models and in suggesting experiments to differentiate between competing models built with different assumptions. We conclude with a discussion of unresolved questions that are likely to be addressed over the next several years., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Evidence obtained in recent years indicates that, in cardiac myocytes, release of Ca(2+) from the sarcoplasmic reticulum (SR) is regulated by changes in the concentration of Ca(2+) within the SR. In this review, we summarize recent advances in our understanding of this regulatory role, with a particular emphasis on dynamic and local changes in SR [Ca(2+)]. We focus on five important questions that are to some extent unresolved and controversial. These questions concern: (1) the importance of SR [Ca(2+)] depletion in the termination of Ca(2+) release; (2) the quantitative extent of depletion during local release events such as Ca(2+) sparks; (3) the influence of SR [Ca(2+)] refilling on release refractoriness and the propensity for pathological Ca(2+) release; (4) dynamic changes in SR [Ca(2+)] during propagating Ca(2+) waves; and (5) the speed of Ca(2+) diffusion within the SR. With each issue, we discuss data supporting alternative viewpoints, and we identify fundamental questions that are being actively investigated. We conclude with a discussion of experimental and computational advances that will help to resolve controversies. This article is part of a special issue entitled "Local Signaling in Myocytes.", (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We present what we believe to be a new mathematical model of Ca(2+) leak from the sarcoplasmic reticulum (SR) in the heart. To our knowledge, it is the first to incorporate a realistic number of Ca(2+)-release units, each containing a cluster of stochastically gating Ca(2+) channels (RyRs), whose biophysical properties (e.g., Ca(2+) sensitivity and allosteric interactions) are informed by the latest molecular investigations. This realistic model allows for the detailed characterization of RyR Ca(2+)-release properties, and shows how this balances reuptake by the SR Ca(2+) pump. Simulations reveal that SR Ca(2+) leak consists of brief but frequent single RyR openings (~3000 cell(-1) s(-1)) that are likely to be experimentally undetectable, and are, therefore, "invisible". We also observe that these single RyR openings can recruit additional RyRs to open, due to elevated local (Ca(2+)), and occasionally lead to the generation of Ca(2+) sparks (~130 cell(-1) s(-1)). Furthermore, this physiological formulation of "invisible" leak allows for the removal of the ad hoc, non-RyR mediated Ca(2+) leak terms present in prior models. Finally, our model shows how Ca(2+) sparks can be robustly triggered and terminated under both normal and pathological conditions. Together, these discoveries profoundly influence how we interpret and understand diverse experimental and clinical results from both normal and diseased hearts., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Aims: In heart cells, the mechanisms underlying refractoriness of the elementary units of sarcoplasmic reticulum (SR) Ca(2+) release, Ca(2+) sparks, remain unclear. We investigated local recovery of SR Ca(2+) release using experimental measurements and mathematical modelling., Methods and Results: Repeated Ca(2+) sparks were induced from individual clusters of ryanodine receptors (RyRs) in quiescent rat ventricular myocytes, and we examined how changes in RyR gating influenced the time-dependent recovery of Ca(2+) spark amplitude and triggering probability. Repeated Ca(2+) sparks from individual sites were analysed in the presence of 50 nM ryanodine with: (i) no additional agents (control); (ii) 50 µM caffeine to sensitize RyRs; (iii) 50 µM tetracaine to inhibit RyRs; or (iv) 100 nM isoproterenol to activate β-adrenergic receptors. Sensitization and inhibition of RyR clusters shortened and lengthened, respectively, the median interval between consecutive Ca(2+) sparks (caffeine 239 ms; control 280 ms; tetracaine 453 ms). Recovery of Ca(2+) spark amplitude, however, was exponential with a time constant of ∼100 ms in all cases. Isoproterenol both accelerated the recovery of Ca(2+) spark amplitude (τ = 58 ms) and shortened the median interval between Ca(2+) sparks (192 ms). The results were recapitulated by a mathematical model in which SR [Ca(2+)] depletion terminates Ca(2+) sparks, but not by an alternative model based on limited depletion and Ca(2+)-dependent inactivation of RyRs., Conclusion: Together, the results strongly suggest that: (i) local SR refilling controls Ca(2+) spark amplitude recovery; (ii) Ca(2+) spark triggering depends on both refilling and RyR sensitivity; and (iii) β-adrenergic stimulation influences both processes.

Purkinje fibers play an essential role in transmitting electrical impulses through the heart, but they may also serve as triggers for arrhythmias linked to defective intracellular calcium (Ca(2+)) regulation. Although prior studies have extensively characterized spontaneous Ca(2+) release in nondriven Purkinje cells, little attention has been paid to rate-dependent changes in Ca(2+) transients. Therefore we explored the behaviors of Ca(2+) transients at pacing rates ranging from 0.125 to 3 Hz in single canine Purkinje cells loaded with fluo3 and imaged with a confocal microscope. The experiments uncovered the following novel aspects of Ca(2+) regulation in Purkinje cells: 1) the cells exhibit a negative Ca(2+)-frequency relationship (at 2.5 Hz, Ca(2+) transient amplitude was 66 ± 6% smaller than that at 0.125 Hz); 2) sarcoplasmic reticulum (SR) Ca(2+) release occurs as a propagating wave at very low rates but is localized near the cell membrane at higher rates; 3) SR Ca(2+) load declines modestly (10 ± 5%) with an increase in pacing rate from 0.125 Hz to 2.5 Hz; 4) Ca(2+) transients show considerable beat-to-beat variability, with greater variability occurring at higher pacing rates. Analysis of beat-to-beat variability suggests that it can be accounted for by stochastic triggering of local Ca(2+) release events. Consistent with this hypothesis, an increase in triggering probability caused a decrease in the relative variability. These results offer new insight into how Ca(2+) release is normally regulated in Purkinje cells and provide clues regarding how disruptions in this regulation may lead to deleterious consequences such as arrhythmias., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A three dimensional model of calcium dynamics in the rat ventricular myocyte was developed to study the mechanism of calcium homeostasis and pathological calcium dynamics during calcium overload. The model contains 20,000 calcium release units (CRUs) each containing 49 ryanodine receptors. The model simulates calcium sparks with a realistic spontaneous calcium spark rate. It suggests that in addition to the calcium spark-based leak, there is an invisible calcium leak caused by the stochastic opening of a small number of ryanodine receptors in each CRU without triggering a calcium spark. The model also explores the mechanism of calcium wave propagation between release sites under the conditions of calcium overload.

Many issues remain unresolved concerning how local, subcellular Ca(2+) signals interact with bulk cellular concentrations to maintain homeostasis in health and disease. To aid in the interpretation of data obtained in quiescent ventricular myocytes, we present here a minimal whole cell model that accounts for both localized (subcellular) and global (cellular) aspects of Ca(2+) signaling. Using a minimal formulation of the distribution of local [Ca(2+)] associated with a large number of Ca(2+)-release sites, the model simulates both random spontaneous Ca(2+) sparks and the changes in myoplasmic and sarcoplasmic reticulum (SR) [Ca(2+)] that result from the balance between stochastic release and reuptake into the SR. Ca(2+)-release sites are composed of clusters of two-state ryanodine receptors (RyRs) that exhibit activation by local cytosolic [Ca(2+)] but no inactivation or regulation by luminal Ca(2+). Decreasing RyR open probability in the model causes a decrease in aggregate release flux and an increase in SR [Ca(2+)], regardless of whether RyR inhibition is mediated by a decrease in RyR open dwell time or an increase in RyR closed dwell time. The same balance of stochastic release and reuptake can be achieved, however, by either high-frequency/short-duration or low-frequency/long-duration Ca(2+) sparks. The results are well correlated with recent experimental observations using pharmacological RyR inhibitors and clarify those aspects of the release-reuptake balance that are inherent to the coupling between local and global Ca(2+) signals and those aspects that depend on molecular-level details. The model of Ca(2+) sparks and homeostasis presented here can be a useful tool for understanding changes in cardiac Ca(2+ )release resulting from drugs, mutations, or acquired diseases.

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 (Ca(2+)) wave propagation, and 2), speed of Ca(2+) diffusion within the SR. Ca(2+) waves are initiated when a spontaneous local SR Ca(2+) release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca(2+) diffusion constant in the SR (D(Ca,SR)) severely complicates our understanding of whether dynamic local changes in SR [Ca(2+)] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca(2+)] during Ca(2+) waves. Simulations have investigated how dynamic local changes in SR [Ca(2+)] are influenced by 1), D(Ca,SR); 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca(2+) pumps; 4), SR Ca(2+) pump dependence on cytosolic [Ca(2+)]; and 5), the rate of transfer between network and junctional SR. Of these factors, D(Ca,SR) is the primary determinant of how release from one RyR cluster alters SR [Ca(2+)] in nearby regions. Specifically, our results show that local increases in SR [Ca(2+)] ahead of the wave can potentially facilitate Ca(2+) wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca(2+)] are possible during SR Ca(2+)release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca(2+)](SR)., (Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In prior work, we introduced a probability density approach to modeling local control of Ca2+-induced Ca2+ release in cardiac myocytes, where we derived coupled advection-reaction equations for the time-dependent bivariate probability density of subsarcolemmal subspace and junctional sarcoplasmic reticulum (SR) [Ca2+] conditioned on Ca2+ release unit (CaRU) state. When coupled to ordinary differential equations (ODEs) for the bulk myoplasmic and network SR [Ca2+], a realistic but minimal model of cardiac excitation-contraction coupling was produced that avoids the computationally demanding task of resolving spatial aspects of global Ca2+ signaling, while accurately representing heterogeneous local Ca2+ signals in a population of diadic subspaces and junctional SR depletion domains. Here we introduce a computationally efficient method for simulating such whole cell models when the dynamics of subspace [Ca2+] are much faster than those of junctional SR [Ca2+]. The method begins with the derivation of a system of ODEs describing the time-evolution of the moments of the univariate probability density functions for junctional SR [Ca2+] jointly distributed with CaRU state. This open system of ODEs is then closed using an algebraic relationship that expresses the third moment of junctional SR [Ca2+] in terms of the first and second moments. In simulated voltage-clamp protocols using 12-state CaRUs that respond to the dynamics of both subspace and junctional SR [Ca2+], this moment-closure approach to simulating local control of excitation-contraction coupling produces high-gain Ca2+ release that is graded with changes in membrane potential, a phenomenon not exhibited by common pool models. Benchmark simulations indicate that the moment-closure approach is nearly 10,000-times more computationally efficient than corresponding Monte Carlo simulations while leading to nearly identical results. We conclude by applying the moment-closure approach to study the restitution of Ca2+-induced Ca2+ release during simulated two-pulse voltage-clamp protocols.

We present a probability density approach to modeling localized Ca2+ influx via L-type Ca2+ channels and Ca2+-induced Ca2+ release mediated by clusters of ryanodine receptors during excitation-contraction coupling in cardiac myocytes. Coupled advection-reaction equations are derived relating the time-dependent probability density of subsarcolemmal subspace and junctional sarcoplasmic reticulum [Ca2+] conditioned on "Ca2+ release unit" state. When these equations are solved numerically using a high-resolution finite difference scheme and the resulting probability densities are coupled to ordinary differential equations for the bulk myoplasmic and sarcoplasmic reticulum [Ca2+], a realistic but minimal model of cardiac excitation-contraction coupling is produced. Modeling Ca2+ release unit activity using this probability density approach avoids the computationally demanding task of resolving spatial aspects of global Ca2+ signaling, while accurately representing heterogeneous local Ca2+ signals in a population of diadic subspaces and junctional sarcoplasmic reticulum depletion domains. The probability density approach is validated for a physiologically realistic number of Ca2+ release units and benchmarked for computational efficiency by comparison to traditional Monte Carlo simulations. In simulated voltage-clamp protocols, both the probability density and Monte Carlo approaches to modeling local control of excitation-contraction coupling produce high-gain Ca2+ release that is graded with changes in membrane potential, a phenomenon not exhibited by so-called "common pool" models. However, a probability density calculation can be significantly faster than the corresponding Monte Carlo simulation, especially when cellular parameters are such that diadic subspace [Ca2+] is in quasistatic equilibrium with junctional sarcoplasmic reticulum [Ca2+] and, consequently, univariate rather than multivariate probability densities may be employed.

New information has recently been obtained along two essentially parallel lines of research: investigations into the fundamental mechanisms of Ca(2+)-induced Ca(2+) release (CICR) in heart cells, and analyses of the factors that control the development of unstable rhythms such as repolarization alternans. These lines of research are starting to converge such that we can begin to understand unstable and potentially arrhythmogenic cardiac dynamics in terms of the underlying mechanisms governing not only membrane depolarization and repolarization but also the complex bidirectional interactions between electrical and Ca(2+) signaling in heart cells. In this brief review, we discuss the progress that has recently been made in understanding the factors that control the beat-to-beat regulation of cardiac Ca(2+) release and attempt to place these results within a larger context. In particular, we discuss factors that may contribute to unstable Ca(2+) release and speculate about how instability in CICR may contribute to the development of arrhythmias under pathological conditions.

Ca(2+) efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca(2+) release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca(2+) influx through L-type Ca(2+) channels (dihydropyridine receptors, DHPR), Ca(2+) sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca(2+) together ensure the stability of this positive-feedback system of Ca(2+) amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca(2+)](i) there is little clear evidence to date showing an increase in Ca(2+) spark rate. Indeed, there is some evidence that the SR Ca(2+) content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca(2+) "leak" under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or "rogue" RyRs that may respond differently to local [Ca(2+)](i) in diastole and during the [Ca(2+)](i) transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyper-phosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca(2+) signaling.

Excitation-contraction coupling in the heart depends on the positive feedback process of Ca2+-induced Ca2+ release (CICR). While CICR provides for robust triggering of Ca2+ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca2+ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca2+ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca2+ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca2+ spark sites. Examination of these repetitive Ca2+ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by approximately 80 ms. We conclude that restitution of Ca2+ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca2+ to SR regulatory proteins.

The use of fluorescent dyes over the past two decades has led to a revolution in our understanding of calcium signaling. Given the ubiquitous role of Ca(2+) in signal transduction at the most fundamental levels of molecular, cellular, and organismal biology, it has been challenging to understand how the specificity and versatility of Ca(2+) signaling is accomplished. In excitable cells, the coordination of changing Ca(2+) concentrations at global (cellular) and well-defined subcellular spaces through the course of membrane depolarization can now be conceptualized in the context of disease processes such as cardiac arrhythmogenesis. The spatial and temporal dimensions of Ca(2+) signaling are similarly important in non-excitable cells, such as endothelial and epithelial cells, to regulate multiple signaling pathways that participate in organ homeostasis as well as cellular organization and essential secretory processes.

The elementary event of Ca(2+) release in heart is the Ca(2+) spark. It occurs at a low rate during diastole, activated only by the low cytosolic [Ca(2+)](i). Synchronized activation of many sparks is due to the high local [Ca(2+)](i) in the region surrounding the sarcoplasmic reticulum (SR) Ca(2+) release channels and is responsible for the systolic [Ca(2+)](i) transient. The biophysical basis of this calcium signaling is discussed. Attention is placed on the local organization of the ryanodine receptors (SR Ca(2+) release channels, RyRs) and the other proteins that underlie and modulate excitation-contraction (EC) coupling. A brief review of specific elements that regulate SR Ca(2+) release (including SR lumenal Ca(2+) and coupled gating of RyRs) is presented. Finally integrative calcium signaling in heart is presented in the context of normal heart function and heart failure.

A Ca(2+) spark arises when a cluster of sarcoplasmic reticulum (SR) channels (ryanodine receptors or RyRs) opens to release calcium in a locally regenerative manner. Normally triggered by Ca(2+) influx across the sarcolemmal or transverse tubule membrane neighboring the cluster, the Ca(2+) spark has been shown to be the elementary Ca(2+) signaling event of excitation-contraction coupling in heart muscle. However, the question of how the Ca(2+) spark terminates remains a central, unresolved issue. Here we present a new model, "sticky cluster," of SR Ca(2+) release that simulates Ca(2+) spark behavior and enables robust Ca(2+) spark termination. Two newly documented features of RyR behavior have been incorporated in this otherwise simple model: "coupled gating" and an opening rate that depends on SR lumenal [Ca(2+)]. Using a Monte Carlo method, local Ca(2+)-induced Ca(2+) release from clusters containing between 10 and 100 RyRs is modeled. After release is triggered, Ca(2+) flux from RyRs diffuses into the cytosol and binds to intracellular buffers and the fluorescent Ca(2+) indicator fluo-3 to produce the model Ca(2+) spark. Ca(2+) sparks generated by the sticky cluster model resemble those observed experimentally, and Ca(2+) spark duration and amplitude are largely insensitive to the number of RyRs in a cluster. As expected from heart cell investigation, the spontaneous Ca(2+) spark rate in the model increases with elevated cytosolic or SR lumenal [Ca(2+)]. Furthermore, reduction of RyR coupling leads to prolonged model Ca(2+) sparks just as treatment with FK506 lengthens Ca(2+) sparks in heart cells. This new model of Ca(2+) spark behavior provides a "proof of principle" test of a new hypothesis for Ca(2+) spark termination and reproduces critical features of Ca(2+) sparks observed experimentally.

Many issues remain unresolved concerning how local, subcellular Ca2+ signals interact with bulk cellular concentrations to maintain homeostasis in health and disease. To aid in the interpretation of data obtained in quiescent ventricular myocytes, we present here a minimal whole cell model that accounts for both localized (subcellular) and global (cellular) aspects of Ca2+ signaling. Using a minimal formulation of the distribution of local [Ca2+] associated with a large number of Ca2+-release sites, the model simulates both random spontaneous Ca2+ sparks and the changes in myoplasmic and sarcoplasmic reticulum (SR) [Ca2+] that result from the balance between stochastic release and reuptake into the SR. Ca2+-release sites are composed of clusters of two-state ryanodine receptors (RyRs) that exhibit activation by local cytosolic [Ca2+] but no inactivation or regulation by luminal Ca2+. Decreasing RyR open probability in the model causes a decrease in aggregate release flux and an increase in SR [Ca2+], regardless of whether RyR inhibition is mediated by a decrease in RyR open dwell time or an increase in RyR closed dwell time. The same balance of stochastic release and reuptake can be achieved, however, by either high-frequency/short-duration or low-frequency/long-duration Ca2+ sparks. The results are well correlated with recent experimental observations using pharmacological RyR inhibitors and clarify those aspects of the release-reuptake balance that are inherent to the coupling between local and global Ca2+ signals and those aspects that depend on molecular-level details. The model of Ca2+ sparks and homeostasis presented here can be a useful tool for understanding changes in cardiac Ca2+ release resulting from drugs, mutations, or acquired diseases. [ABSTRACT FROM AUTHOR]

Ca2+ sparks in heart muscle are activated on depolarization by the influx of Ca2+ through dihydropyridine receptors in the sarcolemmal (SL) and transverse tubule (TT) membranes. The cardiac action potential is thus able to synchronize the [Ca2+]i transient as Ca2+ release is activated throughout the cell. Increases in the amount of Ca2+ within the sarcoplasmic reticulum (SR) underlie augmented Ca2+ release globally and an increase in the sensitivity of the ryanodine receptors (RyRs) to be triggered by the local [Ca2+]i. In a similar manner, phosphorylation of the RyRs by protein kinase A (PKA) increases the sensitivity of the RyRs to be activated by local [Ca2+]i. Heart failure and other cardiac diseases are associated with changes in SR Ca2+ content, phosphorylation state of the RyRs, [Ca2+]i signaling defects and arrhythmias. Additional changes in transverse tubules and nearby junctional SR may contribute to alterations in local Ca2+ signaling. Here we briefly discuss how TT organization can influence Ca2+ signaling and how changes in SR Ca2+ release triggering can influence excitation-contraction (EC) coupling. High speed imaging methods are used in combination with single cell patch clamp experiments to investigate how abnormal Ca2+ signaling may be regulated in health and disease. Three issues are examined in this presentation: (1) normal Ca2+-induced Ca2+ release and Ca2+ sparks, (2) abnormal SR Ca2+ release in disease, and (3) the triggering and propagation of waves of elevated [Ca2+]i. [ABSTRACT FROM AUTHOR]

Excitation–contraction coupling in the heart depends on the positive feedback process of Ca2+-induced Ca2+ release (CICR). While CICR provides for robust triggering of Ca2+ sparks, the mechanisms underlying their termination remain unknown. At present, it is unclear how a cluster of Ca2+ release channels (ryanodine receptors or RyRs) can be made to turn off when their activity is sustained by the Ca2+ release itself. We use a novel experimental approach to investigate indirectly this issue by exploring restitution of Ca2+ sparks. We exploit the fact that ryanodine can bind, nearly irreversibly, to an RyR subunit (monomer) and increase the open probability of the homotetrameric channel. By applying low concentrations of ryanodine to rat ventricular myocytes, we observe repeated activations of individual Ca2+ spark sites. Examination of these repetitive Ca2+ sparks reveals that spark amplitude recovers with a time constant of 91 ms whereas the sigmoidal recovery of triggering probability lags behind amplitude recovery by∼80 ms. We conclude that restitution of Ca2+ sparks depends on local refilling of SR stores after depletion and may also depend on another time-dependent process such as recovery from inactivation or a slow conformational change after rebinding of Ca2+ to SR regulatory proteins. [ABSTRACT FROM AUTHOR]

Studies calcium permeation in tetrodotoxin (TTX)-sensitive calcium channels in rat cardiac myoctes. Heterologous expression of cardiac sodium channels in HEF-293 cells; Calcium influx via TTX-sensitive calcium channels; Inactivation kinetics of TTX-sensitive calcium channels.