Isoproterenol Increases the Fraction of Spark-Dependent RyR-Mediated Leak in Ventricular Myocytes

Recent research suggests that the diastolic ryanodine-receptor-mediated release of Ca²⁺ (J_leak) from the sarcoplasmic reticulum of ventricular myocytes occurs in spark and nonspark forms. Further information about the role(s) of these release manifestations is scarce, however. This study addresses whether the fraction of spark-mediated J_leak increases due to β-adrenergic stimulation. Confocal microscopy was used to simultaneously image Ca²⁺ sparks and quantify J_leak in intact rabbit myocytes, either in the absence or in the presence of 125 nM isoproterenol. It was found that isoproterenol treatment shifts the spark-frequency-J_leak relationship toward an increased sensitivity to a [Ca²⁺] trigger. In agreement, a small but significant increase in spark width was found for cells with matched baseline [Ca²⁺] and total SR [Ca²⁺]. The reconstruction of release fluxes, when applied to the average sparks from those selected cells, yielded a wider release source in the isoproterenol event, indicating the recruitment of peripheral ryanodine receptors. Overall, the results presented here indicate that β-adrenergic stimulation increases the spark-dependent fraction of J_leak. Working together, the increased Ca²⁺ sensitivity and the greater spark width found during isoproterenol treatment may increase the probability of Ca²⁺ wave generation.     

Intracellular Ca alternans: coordinated regulation by sarcoplasmic reticulum release, uptake, and leak

Beat-to-beat alternation in the cardiac intracellular Ca (Ca_i) transient can drive action potential (AP) duration alternans, creating a highly arrhythmogenic substrate. Although a steep dependence of fractional sarcoplasmic reticulum (SR) Ca release on SR Ca load has been shown experimentally to promote Ca_i alternans, theoretical studies predict that other factors are also important. Here we present an iterated map analysis of the coordinated effects of SR Ca release, uptake, and leak on the onset of Ca_i alternans. Predictions were compared to numerical simulations using a physiologically realistic AP model as well as to AP clamp experiments in isolated patch-clamped rabbit ventricular myocytes exposed to 1), the Ca channel agonist BayK8644 (100 nM) to increase SR Ca load and release fraction, 2), overexpression of an adenoviral SERCA2a construct to increase SR Ca uptake, and 3), low-dose FK506 (20 μM) or ryanodine (1 μM) to increase SR Ca leak. Our findings show that SR Ca release, uptake, and leak all have independent direct effects that promote (release and leak) or suppress (uptake) Ca_i alternans. However, since each factor affects the other by altering SR Ca load, the net balance of their direct and indirect effects determines whether they promote or suppress alternans. Thus, BayK8644 promotes, whereas Ad-SERCA2a overexpression, ryanodine, and FK506 suppress, Ca_i alternans under AP clamp conditions.     

Dynamic regulation of sarcoplasmic reticulum Ca(2+) content and release by luminal Ca(2+)-sensitive leak in rat ventricular myocytes.

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.     

How Does Stochastic Ryanodine Receptor-Mediated Ca Leak Fail to Initiate a Ca Spark?

Spontaneous calcium (Ca) sparks are initiated by single ryanodine receptor (RyR) opening. Once one RyR channel opens, it elevates local [Ca] in the cleft space ([Ca]_Cleft), which opens other RyR channels in the same Ca release unit (CaRU) via Ca-induced Ca-release. Experiments by Zima et al. (J. Physiol. 588:4743–4757, 2010) demonstrate that spontaneous Ca sparks occur only when intrasarcoplasmic-reticulum (SR) [Ca] ([Ca]_SR) is above a threshold level, but that RyR-mediated SR Ca leak exists without Ca sparks well below this threshold [Ca]_SR. We examine here how single RyR opening at lower [Ca]_SR can fail to recruit Ca sparks at a CaRU, while still contributing to SR Ca leak. We assess this using a physiologically detailed mathematical model of junctional SR Ca release in which RyR gating is regulated by [Ca]_SR and [Ca]_Cleft. We find that several factors contribute to the failure of Ca sparks as [Ca]_SR declines: 1), lower [Ca]_SR reduces driving force and thus limits local [Ca]_Cleft achieved and the rate of rise during RyR opening; 2), low [Ca]_SR limits RyR open time (τ_O), which further reduces local [Ca]_Cleft attained; 3), low τ_O and fast [Ca]_Cleft dissipation after RyR closure shorten the opportunity for neighboring RyR activation; 4), at low [Ca]_SR, the RyR exhibits reduced [Ca]_Cleft sensitivity. We conclude that all of these factors conspire to reduce the probability of Ca sparks as [Ca]_SR declines, despite continued RyR-mediated Ca leak. In addition, these same factors explain the much lower efficacy of L-type Ca channel opening to trigger local SR Ca release at low [Ca]_SR during excitation-contraction coupling. Conversely, all of these factors are fundamentally important for increasing the propensity for pro-arrhythmic Ca sparks and waves in cardiac myocytes at high [Ca]_SR.     

Ca2+ Release Events in Cardiac Myocytes Up Close: Insights from Fast Confocal Imaging

The spatio-temporal properties of Ca²⁺ transients during excitation-contraction coupling and elementary Ca²⁺ release events (Ca²⁺ sparks) were studied in atrial and ventricular myocytes with ultra-fast confocal microscopy using a Zeiss LSM 5 LIVE system that allows sampling rates of up to 60 kHz. Ca²⁺ sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR) release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca²⁺ sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca²⁺ spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca²⁺]_i. 2-D imaging of Ca²⁺ transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca²⁺ entry through surface membrane Ca²⁺ channels and subsequent activation of Ca²⁺-induced Ca²⁺ release. With a latency of 2.5 ms after application of an electrical stimulus, Ca²⁺ entry could be detected that was followed by SR Ca²⁺ release after an additional 3 ms delay. Maximum Ca²⁺ release was observed 4 ms after the beginning of release. The timing of Ca²⁺ entry and release was confirmed by simultaneous [Ca²⁺]_i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca²⁺ release events that fused into a peripheral ring of elevated [Ca²⁺]_i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca²⁺ release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca²⁺ transient. In summary, ultra-fast confocal imaging allows investigation of Ca²⁺ signals with a time resolution similar to patch clamp technique, however in a less invasive fashion.     

Ca2+ release from the sarcoplasmic reticulum activated by the low affinity Ca2+ chelator TPEN in ventricular myocytes

The Ca2+ content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca2+ release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca2+ within the SR with the membrane-permeant low affinity Ca2+ chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca2+ content and SR Ca2+ depletion can influence Ca2+ release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca2+ release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca2+ releases increased in frequency and developed into cell-wide Ca2+ waves. SR Ca2+ load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40microTPEN did not significantly inhibit the SR-Ca2+-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca2+ chelator in intracellular Ca2+ stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca2+ leak from the SR leading to its Ca2+ depletion. Lowering of SR Ca2+ content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.     

Modification of sarcoplasmic reticulum (SR) Ca2+ release by FK506 induces defective excitation-contraction coupling only when SR Ca2+ recycling is disturbed

This study examined whether the effects of FK506-binding protein dissociation from sarcoplasmic reticulum (SR) Ca(2+) release channels on excitation-contraction (EC) coupling changed when SR Ca(2+) reuptake and (or) the trans-sarcolemmal Ca(2+) extrusion were altered. The steady-state twitch Ca(2+) transient (CaT), cell shortening, post-rest caffeine-induced CaT, and Ca(2+) sparks were measured in rat ventricular myocytes using laser-scanning confocal microscopy. In the normal condition, 50 micromol FK506/L significantly increased steady-state CaT, cell shortening, and post-rest caffeine-induced CaT. When the cells were solely perfused with thapsigargin, FK506 did not reduce any of the states, but when low [Ca(2+)](0) (0.1 mmol/L) was perfused additionally, FK506 reduced CaT and cell shortening, and accelerated the reduction of post-rest caffeine-induced CaT. FK506 significantly increased Ca(2+) spark frequency in the normal condition, whereas it mainly prolonged duration of individual Ca(2+) sparks under the combination of thapsigargin and low [Ca(2+)](0) perfusion. Modification of SR Ca(2+) release by FK506 impaired EC coupling only when released Ca(2+) could not be taken back into the SR and was readily extruded to the extracellular space. Our findings could partly explain the controversy regarding the contribution of FK506-binding protein dissociation to defective EC coupling.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sar-coplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of-10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

Rate of diastolic Ca release from the sarcoplasmic reticulum of intact rabbit and rat ventricular myocytes.

The sarcoplasmic reticulum (SR) of cardiac myocytes loses Ca during rest. In the present study, we estimated the rest-dependent unidirectional Ca efflux from the SR in intact rabbit and rat ventricular myocytes. We determined the time course of depletion of the SR Ca content (assessed as the amount of Ca released by caffeine) after inhibition of the SR Ca-ATPase by thapsigargin. Before rest intervals in Na-containing, Ca-free solution, a 3-min preperfusion with 0Na,0Ca solution was performed to deplete Nai but keep the SR Ca content constant. The decrease in Nai should stimulate Ca efflux via Na/Ca exchange when Nao is reintroduced. Thapsigargin treatment was limited to the last 2 min of preperfusion with 0Na,0Ca solution to minimize SR Ca loss before addition of Na, while attaining complete block of the SR Ca pump. Total SR Ca content was estimated from the [Ca]i transient evoked by caffeine, taking into account passive cellular Ca buffering. The time constants for SR Ca loss after thapsigargin were 385 and 355 s, whereas the pre-rest SR Ca content was estimated to be 106 and 114 microM (mumol/l nonmitochondrial cell volume) in rabbit and rat myocytes, respectively. The unidirectional Ca efflux from the SR was similar in the two cell types (rabbit: 0.27 microM s-1; rat: 0.32 microM s-1). These values are also comparable with that estimated from elementary Ca release events ("Ca sparks," 0.2-0.8 microM s-1). Thus, resting leak of Ca from SR may be primarily via occasional openings of SR Ca release channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal and spatial properties of cellular Ca2+ flux in trout ventricular myocytes

Confocal microscopy was used to investigate the temporal and spatial properties of Ca(2+) transients and Ca(2+) sparks in ventricular myocytes of the rainbow trout (Oncorhynchus mykiss). Confocal imaging confirmed the absence of T tubules and the long ( approximately 160 microm), thin ( approximately 8 microm) morphology of trout myocytes. Line scan imaging of Ca(2+) transients evoked by electrical stimulation in cells loaded with fluo 4 revealed spatial inhomogeneities in the temporal properties of Ca(2+) transients across the width of the myocytes. The Ca(2+) wavefront initiated faster, rose faster, and reached larger peak amplitudes in the periphery of the myocyte compared with the center. These differences were exacerbated by stimulation with the L-type Ca(2+) channel agonist (-)BAY K 8644 or by sarcoplasmic reticulum (SR) inhibition with ryanodine and thapsigargin. Results reveal that the shape of the trout myocyte allows for rapid diffusion of Ca(2+) from the cell periphery to the cell center, with SR Ca(2+) release contributing to the cytosolic Ca(2+) rise in a time-dependent manner. Spontaneous Ca(2+) sparks were exceedingly rare in trout myocytes under control conditions (1 sparking cell from 238 cells examined). This is in marked contrast to the rat where a total of 56 spontaneous Ca(2+) sparks were observed in 9 of 11 myocytes examined. Ca(2+) sparklike events were observed in a very small number of trout myocytes (15 sparks from 9 of 378 cells examined) after stimulation with either (-)BAY K 8644 or high Ca(2+) (6 mM). Reducing temperature to 15 degrees C in intact myocytes or permeabilizing myocytes to adjust intracellular conditions to favor Ca(2+) spark detection was without significant effects. Possible reasons for the rarity of Ca(2+) sparks in a cardiac myocyte with an active SR are discussed.     

SparkMaster: automated calcium spark analysis with ImageJ

Ca sparks are elementary Ca-release events from intracellular Ca stores that are observed in virtually all types of muscle. Typically, Ca sparks are measured in the line-scan mode with confocal laser-scanning microscopes, yielding two-dimensional images (distance vs. time). The manual analysis of these images is time consuming and prone to errors as well as investigator bias. Therefore, we developed SparkMaster, an automated analysis program that allows rapid and reliable spark analysis. The underlying analysis algorithm is adapted from the threshold-based standard method of spark analysis developed by Cheng et al. (Biophys J 76: 606–617, 1999) and is implemented here in the freely available image-processing software ImageJ. SparkMaster offers a graphical user interface through which all analysis parameters and output options are selected. The analysis includes general image parameters (number of detected sparks, spark frequency) and individual spark parameters (amplitude, full width at half-maximum amplitude, full duration at half-maximum amplitude, full width, full duration, time to peak, maximum steepness of spark upstroke, time constant of spark decay). We validated the algorithm using images with synthetic sparks embedded into backgrounds with different signal-to-noise ratios to determine an analysis criteria at which a high sensitivity is combined with a low frequency of false-positive detections. Finally, we applied SparkMaster to analyze experimental data of sparks measured in intact and permeabilized ventricular cardiomyocytes, permeabilized mammalian skeletal muscle, and intact smooth muscle cells. We found that SparkMaster provides a reliable, easy to use, and fast way of analyzing Ca sparks in a wide variety of experimental conditions. myocytes; sarcoplasmic reticulum; confocal microscopy     

Atrial local Ca signaling and inositol 1,4,5-trisphosphate receptors

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca²⁺ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca²⁺ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in atrial myocytes, and show our new findings on the role of IP₃R subtype in the regulation of spontaneous focal Ca²⁺ releases in the compartmentalized areas of atrial myocytes. The Ca²⁺ sparks, representing focal Ca²⁺ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca²⁺ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP₃Rs compared with ventricular cells and possess significant levels of type 1 IP₃R (IP₃R1) and type 2 IP₃R (IP₃R2). Over the last decade the roles of atrial IP₃R on the enhancement of Ca²⁺-induced Ca²⁺ release and arrhythmic Ca²⁺ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca²⁺ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP₃R1 in the maintenance of peri-nuclear and non-junctional Ca²⁺ sparks via stimulating a posttranslational organization of RyR clusters.     

Estimation of the sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks

Using a combination of experimental and numerical approaches, we have tested two different approaches to calculating the sarcoplasmic reticulum (SR) Ca2+ release flux, which gives rise to cardiac muscle Ca2+ sparks. By using two-photon excited spot photolysis of DM-Nitrophen, known Ca2+ release flux time courses were generated to provide the first experimental validation of spark flux reconstruction algorithms. These artificial Ca2+ sparks show that it is possible to calculate the SR Ca2+ release waveform with reasonable accuracy, provided the flux equations reasonably reflect the properties of the experimental system. Within cardiac muscle cells, we show that Ca2+ flux reconstruction is complicated by the substantial dye binding to proteins, a factor that has not been adequately addressed in previous flux reconstruction algorithms. Furthermore, our numerical experiments suggest that the calculated time course of release flux inactivation based on conventional flux reconstruction algorithms is likely to be in error. We therefore developed novel algorithms based on an explicit dye binding scheme. When these algorithm were applied to evoked Ca2+ sparks in rat cardiac ventricular myocytes, the reconstructed Ca2+ release waveform peaked in ~5 ms and decayed with a halftime of approximately 5 ms. The peak flux magnitude was 7-12 pA, suggesting that sparks must arise from clusters of >15 ryanodine receptors.     

Mechanisms of Calcium Leak from Cardiac Sarcoplasmic Reticulum Revealed by Statistical Mechanics

Heart muscle contraction is normally activated by a synchronized Ca release from sarcoplasmic reticulum (SR), a major intracellular Ca store. However, under abnormal conditions, Ca leaks from the SR, decreasing heart contraction amplitude and increasing risk of life-threatening arrhythmia. The mechanisms and regimes of SR operation generating the abnormal Ca leak remain unclear. Here, we employed both numerical and analytical modeling to get mechanistic insights into the emergent Ca leak phenomenon. Our numerical simulations using a detailed realistic model of the Ca release unit reveal sharp transitions resulting in Ca leak. The emergence of leak is closely mapped mathematically to the Ising model from statistical mechanics. The system steady-state behavior is determined by two aggregate parameters: the analogs of magnetic field (h) and the inverse temperature (β) in the Ising model, for which we have explicit formulas in terms of SR [Ca] and release channel opening and closing rates. The classification of leak regimes takes the shape of a phase β-h diagram, with the regime boundaries occurring at h = 0 and a critical value of β (β¹) that we estimate using a classical Ising model and mean field theory. Our theory predicts that a synchronized Ca leak will occur when h > 0 and β > β¹, and a disordered leak occurs when β < β¹ and h is not too negative. The disorder leak is distinguished from synchronized leak (in long-lasting sparks) by larger Peierls contour lengths, an output parameter reflecting degree of disorder. Thus, in addition to our detailed numerical model approach, we also offer an instantaneous computational tool using analytical formulas of the Ising model for respective ryanodine receptor parameters and SR Ca load that describe and classify phase transitions and leak emergence.     

FKBP12.6 overexpression decreases Ca2+ spark amplitude but enhances [Ca2+]i transient in rat cardiac myocytes

Ryanodine receptors/Ca2+-release channels (RyR2) from the sarcoplasmic reticulum (SR) provide the Ca2+ required for contraction at each cardiac twitch. RyR2 are regulated by a variety of proteins, including the immunophilin FK506 binding protein (FKBP12.6). FKBP12.6 seems to be important for coupled gating of RyR2 and its deficit and alteration may be involved in heart failure. The role of FKBP12.6 on Ca2+ release has not been analyzed directly, but rather it was inferred from the effects of immunophilins, such us FK506 and rapamycin, which, among other effects, dissociates FKBP12.6 from the RyR2. Here, we investigated directly the effects of FKBP12.6 on local (Ca2+ sparks) and global [intracellular Ca2+ concentration ([Ca2+]i) transients] Ca2+ release in single rat cardiac myocytes. The FKBP12.6 gene was transfected in single myocytes using the adenovirus technique with a reporter gene strategy based on green fluorescent protein (GFP) to check out the success of transfections. Control myocytes were transfected with only GFP (Ad-GFP). Rhod-2 was used as the Ca2+ indicator, and cells were viewed with a confocal microscope. We found that overexpression of FKBP12.6 decreases the occurrence, amplitude, duration, and width of spontaneous Ca2+ sparks. FK506 had diametrically opposed effects. However, overexpression of FKBP12.6 increased the [Ca2+]i transient amplitude and accelerated its decay in field-stimulated cells. The associated cell shortening was increased. SR Ca2+ load, estimated by rapid caffeine application, was increased. In conclusion, FKBP12.6 overexpression decreases spontaneous Ca2+ sparks but increases [Ca2+]i transients, in relation with enhanced SR Ca2+ load, therefore improving excitation-contraction coupling.     

IP3 receptor-dependent Ca2+ release modulates excitation-contraction coupling in rabbit ventricular myocytes

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-dependent Ca(2+) signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP(3)R-dependent sarcoplasmic reticulum (SR) Ca(2+) release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP(3)R at levels approximately 3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP(3) (10 microM) produced a transient 21% increase in the frequency of Ca(2+) sparks (P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude (P < 0.05) and a 7% decrease in SR Ca(2+) load (P < 0.05) and was inhibited by IP(3)R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 microM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP(3) production and caused a 38% (P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca(2+) transients. This effect was abolished by the IP(3)R antagonist 2-APB (2 microM) or by using adenoviral expression of an IP(3) affinity trap that buffers cellular IP(3). Together, these data suggest that in rabbit ventricular myocytes IP(3)R-dependent Ca(2+) release has positive inotropic effects on ECC by facilitating Ca(2+) release through ryanodine receptor clusters.     

Propagation of Ca2+ release in cardiac myocytes: role of mitochondria

Factors contributing to "local control" of Ca2+ release in cardiac myocytes are incompletely understood. We induced local release of Ca2+ by regional exposure of mouse atrial and ventricular myocytes to 10mM caffeine for 500 ms using a rapid solution switcher. Propagation of Ca2+ release was imaged by means of a Nipkow confocal microscope, and fluo-3. Under physiologic conditions, a local release of Ca2+ propagated in atrial myocytes, not in ventricular myocytes. Inhibition of SR Ca2+ uptake (500 nM thapsigargin), and of Ca2+ extrusion via Na/Ca exchange (5mM Ni2+), did not result in propagation in ventricular myocytes. The density of mitochondria was greater in ventricular than in atrial myocytes, although the abundance of ryanodine receptors and myofilaments was similar. Partial inhibition of Ca2+ uptake via the mitochondrial Ca2+ uniporter (5 microM Ru360) caused an increase in the [Ca2+]i transient in paced ventricular myocytes, and consistently resulted in propagation of Ca2+ release. This effect of Ru360 did not appear to be due to altered SR Ca2+ content. These data indicate that Ca2+ uptake via the mitochondrial uniporter occurs on a beat-to-beat basis, and may contribute to local control of Ca2+ release. Propagation of Ca2+ release in atrial myocytes may result in part from the relatively low density of mitochondria present.     

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca²⁺-induced Ca²⁺ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca²⁺ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca²⁺ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca²⁺ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca²⁺]_SR, increased SR Ca²⁺ fractional release, and increased spark- and non-spark-mediated SR Ca²⁺ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca²⁺ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca²⁺ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca²⁺ handling during periods of oxidative stress.