Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes.

The importance of Na-Ca exchange as a trigger for sarcoplasmic reticulum (SR) Ca release remains controversial. Therefore, we measured whole-cell Ca currents (ICa), Na-Ca exchange currents (INaCa), cellular contractions, and intracellular Ca transients in adult rabbit cardiac myocytes. We found that changing pipette Na concentration markedly affected the relationship between cell shortening (or Ca transients) and voltage, but did not affect the Ca current-voltage relationship. We then inhibited Na-Ca exchange and varied SR content (by changing the number of conditioning pulses before each test pulse). Regardless of SR Ca content, the relationship between contraction and voltage was bell-shaped in the absence of Na-Ca exchange. Next, we rapidly and completely blocked ICa by applying nifedipine to cells. Cellular shortening was variably reduced in the presence of nifedipine. The component of shortening blocked by nifedipine had a bell-shaped relationship with voltage, whereas the "nifedipine-insensitive" component of contraction increased with voltage. With the SR disabled (ryanodine and thapsigargin pretreatment), ICa could initiate late-peaking contractions that were approximately 70% of control amplitude. In contrast, nifedipine-insensitive contractions could not be elicited in the presence of ryanodine and thapsigargin. Finally, we recorded reverse Na-Ca exchange currents that were activated by membrane depolarization. The estimated sarcolemmal Ca flux occurring by Na-Ca exchange (during voltage clamp steps to +30 mV) was approximately 10-fold less than that occurring by ICa. Therefore, Na-Ca exchange alone is unlikely to raise cytosolic Ca concentration enough to directly activate the myofilaments. We conclude that reverse Na-Ca exchange can trigger SR Ca release. Because of the sigmoidal relationship between the open probability of the SR Ca release channel and pCa, the effects of ICa and INaCa may not sum in a linear fashion. Rather, the two triggers may act synergistically in the modulation of SR release.     

Calcium alternans in a couplon network model of ventricular myocytes: role of sarcoplasmic reticulum load

Intracellular calcium (Ca) alternans in cardiac myocytes have been shown in many experimental studies, and the mechanisms remain incompletely understood. We recently developed a "3R theory" that links Ca sparks to whole cell Ca alternans through three critical properties: randomness of Ca sparks; recruitment of a Ca spark by neighboring Ca sparks; and refractoriness of Ca release units. In this study, we used computer simulation of a physiologically detailed mathematical model of a ventricular myocyte couplon network to study how sarcoplasmic reticulum (SR) Ca load and other physiological parameters, such as ryanodine receptor sensitivity, SR uptake rate, Na-Ca exchange strength, and Ca buffer levels affect Ca alternans in the context of 3R theory. We developed a method to calculate the parameters used in the 3R theory (i.e., the primary spark rate and the recruitment rate) from the physiologically detailed Ca cycling model and paced the model periodically to elicit Ca alternans. We show that alternans only occurs for an intermediate range of the SR Ca load, and the underlying mechanism can be explained via its effects on the 3Rs. Furthermore, we show that altering the physiological parameters not only directly changes the 3Rs but also alters the SR Ca load, having an indirect effect on the 3Rs as well. Therefore, our present study links the SR Ca load and other physiological parameters to whole cell Ca alternans through the framework of the 3R theory, providing a general mechanistic understanding of Ca alternans in ventricular myocytes.     

An integrative model of the cardiac ventricular myocyte incorporating local control of Ca2+ release

The local control theory of excitation-contraction (EC) coupling in cardiac muscle asserts that L-type Ca(2+) current tightly controls Ca(2+) release from the sarcoplasmic reticulum (SR) via local interaction of closely apposed L-type Ca(2+) channels (LCCs) and ryanodine receptors (RyRs). These local interactions give rise to smoothly graded Ca(2+)-induced Ca(2+) release (CICR), which exhibits high gain. In this study we present a biophysically detailed model of the normal canine ventricular myocyte that conforms to local control theory. The model formulation incorporates details of microscopic EC coupling properties in the form of Ca(2+) release units (CaRUs) in which individual sarcolemmal LCCs interact in a stochastic manner with nearby RyRs in localized regions where junctional SR membrane and transverse-tubular membrane are in close proximity. The CaRUs are embedded within and interact with the global systems of the myocyte describing ionic and membrane pump/exchanger currents, SR Ca(2+) uptake, and time-varying cytosolic ion concentrations to form a model of the cardiac action potential (AP). The model can reproduce both the detailed properties of EC coupling, such as variable gain and graded SR Ca(2+) release, and whole-cell phenomena, such as modulation of AP duration by SR Ca(2+) release. Simulations indicate that the local control paradigm predicts stable APs when the L-type Ca(2+) current is adjusted in accord with the balance between voltage- and Ca(2+)-dependent inactivation processes as measured experimentally, a scenario where common pool models become unstable. The local control myocyte model provides a means for studying the interrelationship between microscopic and macroscopic behaviors in a manner that would not be possible in experiments.     

Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration

Our aim was to measure the influence of sarcoplasmic reticulum (SR) calcium content ([Ca](SRT)) and free SR [Ca] ([Ca](SR)) on the fraction of SR calcium released during voltage clamp steps in isolated rabbit ventricular myocytes. [Ca](SRT), as measured by caffeine application, was progressively increased by conditioning pulses. Sodium was absent in both the intracellular and in the extracellular solutions to block sodium/calcium exchange. Total cytosolic calcium flux during the transient was inferred from I(Ca), [Ca](SRT), [Ca](i), and cellular buffering characteristics. Fluxes via the calcium current (I(Ca)), the SR calcium pump, and passive leak from the SR were evaluated to determine SR calcium release flux (J(rel)). Excitation-contraction (EC) coupling was characterized with respect to both gain (integral J(rel)/integral I(Ca)) and fractional SR calcium release. Both parameters were virtually zero for a small, but measurable [Ca](SRT). Gain and fractional SR calcium release increased steeply and nonlinearly with both [Ca](SRT) and [Ca](SR). We conclude that potentiation of EC coupling can be correlated with both [Ca](SRT) and [Ca](SR). While fractional SR calcium release was not linearly dependent upon [Ca](SR), intra-SR calcium may play a crucial role in regulating the SR calcium release process.     

The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes

We have investigated the effects on spontaneous SR Ca release of modulating the sarcoplasmic reticulum ryanodine receptor (RyR) with low (<0.5 mM) concentrations of caffeine. Experiments were performed on isolated rat ventricular myocytes. Intracellular Ca concentration was measured with Indo-1 or Fluo-3 in voltage-clamped cells. Spontaneous Ca release was produced by elevating external Ca to 5 mM. Caffeine application increased the frequency of spontaneous release. Both the magnitude of the spontaneous Ca transients and the integral of the resulting Na-Ca exchange current were decreased by caffeine. The combination of increased frequency of spontaneous release and decreased Ca efflux per event meant that the Ca efflux per unit time was unaffected by low concentrations of caffeine. The SR Ca content was reduced by caffeine. The extra Ca efflux calculated from the Na-Ca exchange current integrals occurring during the initial burst of spontaneous activity on application of caffeine accounted for this reduction of SR Ca content. In contrast to these maintained effects on spontaneous release, caffeine had only transient effects on stimulated Ca release produced by depolarizing pulses. We conclude that stimulation of the RyR results in spontaneous release at SR Ca contents lower than those at which release would normally occur. Therefore, the balance between normal and spontaneous Ca release can be shifted by modulation of the RyR. This will have important consequences for arrhythmogenesis due to spontaneous Ca release.     

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.     

Regulation of cardiac sarcoplasmic reticulum Ca release by luminal [Ca] and altered gating assessed with a mathematical model

Cardiac excitation-contraction coupling is initialized by the release of Ca from the sarcoplasmic reticulum (SR) in response to a sudden increase in local cytosolic [Ca] ([Ca]i) within the junctional cleft. We have tested the hypothesis that functional ryanodine receptor (RyR) regulation plays a major role in the regulation of myocyte Ca. A mathematical model with unique characteristics was used to simulate Ca homeostasis. Specifically, the model was designed to accurately represent the SR [Ca]-dependence of release from a variety of experimentally produced data sets. The simulated data for altered RyR Ca sensitivity demonstrated a regulatory feedback loop that resulted in the same release at lower [Ca]SR. This suggests that the primary role of myocyte RyR regulation may be to decrease SR [Ca] without decreasing the size of the [Ca]i transient. The model results suggest that this action moderates the increased SR [Ca] observed with adrenergic stimulation and may keep the [Ca]SR below the threshold for delayed afterdepolarizations and arrhythmia. However, increased Ca affinity of the RyR increased the probability of delayed afterdepolarizations when heart failure was simulated. We conclude that RyR regulation may play a role in preventing arrhythmias in healthy myocytes but that the same regulation may have the opposite effect in chronic heart failure.     

Size Matters: Ryanodine Receptor Cluster Size Heterogeneity Potentiates Calcium Waves

Ryanodine receptors (RyRs) mediate calcium (Ca)-induced Ca release and intracellular Ca homeostasis. In a cardiac myocyte, RyRs group into clusters of variable size from a few to several hundred RyRs, creating a spatially nonuniform intracellular distribution. It is unclear how heterogeneity of RyR cluster size alters spontaneous sarcoplasmic reticulum (SR) Ca releases (Ca sparks) and arrhythmogenic Ca waves. Here, we tested the impact of heterogeneous RyR cluster size on the initiation of Ca waves. Experimentally, we measured RyR cluster sizes at Ca spark sites in rat ventricular myocytes and further tested functional impacts using a physiologically detailed computational model with spatial and stochastic intracellular Ca dynamics. We found that the spark frequency and amplitude increase nonlinearly with the size of RyR clusters. Larger RyR clusters have lower SR Ca release threshold for local Ca spark initiation and exhibit steeper SR Ca release versus SR Ca load relationship. However, larger RyR clusters tend to lower SR Ca load because of the higher Ca leak rate. Conversely, smaller clusters have a higher threshold and a lower leak, which tends to increase SR Ca load. At the myocyte level, homogeneously large or small RyR clusters limit Ca waves (because of low load for large clusters but low excitability for small clusters). Mixtures of large and small RyR clusters potentiates Ca waves because the enhanced SR Ca load driven by smaller clusters enables Ca wave initiation and propagation from larger RyR clusters. Our study suggests that a spatially heterogeneous distribution of RyR cluster size under pathological conditions may potentiate Ca waves and thus afterdepolarizations and triggered arrhythmias.     

Reverse mode of the sarcoplasmic reticulum calcium pump and load-dependent cytosolic calcium decline in voltage-clamped cardiac ventricular myocytes

We have characterized [Ca](i) decline in voltage-clamped rabbit ventricular myocytes with progressive increases in sarcoplasmic reticulum (SR) calcium load. "Backflux" through the SR calcium pump is a critical feature which allows realistically small values for SR calcium leak fluxes to be used. Total cytosolic calcium was calculated from the latter part of [Ca](i) decline using rate constants for cellular calcium buffers. Intra-SR calcium buffering characteristics were also deduced. We found that the net SR calcium pump flux and rate of [Ca](i) decline decreased as the SR free [Ca] rose, with pump parameters held constant. We have therefore characterized for the first time in intact myocytes both forward and reverse SR calcium pump kinetics as well as intra-SR calcium buffering and SR calcium leak. We conclude that the reverse flux through the SR calcium pump is an important factor in comprehensive understanding of dynamic SR calcium fluxes.     

Sodium-calcium exchange affects local calcium signal decay and the rate of exocytotic secretion in single chromaffin cells

The effects of Na+ deprivation on local calcium signal decay and the rate of exocytotic secretion were measured in single bovine chromaffin cells to determine whether Na-Ca exchange influences the local cytosolic Ca2+ signal for neurohormone release. Na+ replacement with N-methylglucamine caused a marked slowing of the decay of the local Ca2+ signal near points of its initiation, as measured by high-resolution fluorescent Ca2+ imaging in the confocal laser scanning microscope. Na+ replacement also resulted in a doubling of the rate and magnitude of exocytotic secretion measured in single cells by high-resolution microamperometry. Release rates provide an independent measure of local active zone Ca2+. Five repetitive stimulations of the same cell in Na+-free, but not in Na+-containing, medium resulted in a progressively increasing rate of catecholamine release, suggesting an increasing level of active zone Ca2+ and a role of Na-Ca exchange activity in Ca2+ clearance between stimulations. As secretory activity and its triggering Ca2+ signals are known to be co-localized in active zones along the plasma membrane, the results suggest that Na-Ca exchange may influence the decay of the local Ca2+ signal for exocytotic secretion. This would be consistent with a contribution to local Ca2+ clearance by a novel mechanism utilizing the insertion of secretory vesicle Na-Ca exchangers into the plasma membrane during exocytosis.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.     

A mathematical model of cardiocyte Ca(2+) dynamics with a novel representation of sarcoplasmic reticular Ca(2+) control

Cardiac contraction and relaxation dynamics result from a set of simultaneously interacting Ca(2+) regulatory mechanisms. In this study, cardiocyte Ca(2+) dynamics were modeled using a set of six differential equations that were based on theories, equations, and parameters described in previous studies. Among the unique features of the model was the inclusion of bidirectional modulatory interplay between the sarcoplasmic reticular Ca(2+) release channel (SRRC) and calsequestrin (CSQ) in the SR lumen, where CSQ acted as a dynamic rather than simple Ca(2+) buffer, and acted as a Ca(2+) sensor in the SR lumen as well. The inclusion of this control mechanism was central in overcoming a number of assumptions that would otherwise have to be made about SRRC kinetics, SR Ca(2+) release rates, and SR Ca(2+) release termination when the SR lumen is assumed to act as a simple, buffered Ca(2+) sink. The model was sufficient to reproduce a graded Ca(2+)-induced Ca(2+) release (CICR) response, CICR with high gain, and a system with reasonable stability. As constructed, the model successfully replicated the results of several previously published experiments that dealt with the Ca(2+) dependence of the SRRC (, J. Gen. Physiol. 85:247-289), the refractoriness of the SRRC (, Am. J. Physiol. 270:C148-C159), the SR Ca(2+) load dependence of SR Ca(2+) release (, Am. J. Physiol. 268:C1313-C1329;, J. Biol. Chem. 267:20850-20856), SR Ca(2+) leak (, J. Physiol. (Lond.). 474:463-471;, Biophys. J. 68:2015-2022), SR Ca(2+) load regulation by leak and uptake (, J. Gen. Physiol. 111:491-504), the effect of Ca(2+) trigger duration on SR Ca(2+) release (, Am. J. Physiol. 258:C944-C954), the apparent relationship that exists between sarcoplasmic and sarcoplasmic reticular calcium concentrations (, Biophys. J. 73:1524-1531), and a variety of contraction frequency-dependent alterations in sarcoplasmic [Ca(2+)] dynamics that are normally observed in the laboratory, including rest potentiation, a negative frequency-[Ca(2+)] relationship, and extrasystolic potentiation. Furthermore, under the condition of a simulated Ca(2+) overload, an alternans-like state was produced. In summary, the current model of cardiocyte Ca(2+) dynamics provides an integrated theoretical framework of fundamental cellular Ca(2+) regulatory processes that is sufficient to predict a broad array of observable experimental outcomes.     

Sarcoplasmic Reticulum Structure and Functional Properties that Promote Long-Lasting Calcium Sparks

Calcium (Ca) sparks are the fundamental sarcoplasmic reticulum (SR) Ca release events in cardiac myocytes, and they have a typical duration of 20–40 ms. However, when a fraction of ryanodine receptors (RyRs) are blocked by tetracaine or ruthenium red, Ca sparks lasting hundreds of milliseconds have been observed experimentally. The fundamental mechanism underlying these extremely prolonged Ca sparks is not understood. In this study, we use a physiologically detailed mathematical model of subcellular Ca cycling to examine how Ca spark duration is influenced by the number of functional RyRs in a junctional cluster (which is reduced by tetracaine or ruthenium red) and other SR Ca handling properties. One RyR cluster contains a few to several hundred RyRs, and we use a four-state Markov RyR gating model. Each RyR opens stochastically and is regulated by cytosolic and luminal Ca. We varied the number of functional RyRs in the single cluster, diffusion within the SR network, diffusion between network and junctional SR, cytosolic Ca diffusion, SERCA uptake activity, and RyR open probability. For long-lasting Ca release events, opening events within the cluster must occur continuously because the typical open time of the RyR is only a few milliseconds. We found the following: 1) if the number of RyRs is too small, it is difficult to maintain consecutive openings and stochastic attrition terminates the release; 2) if the number of RyRs is too large, the depletion of Ca from the junctional SR terminates the release; and 3) very long release events require relatively small-sized RyR clusters (reducing flux as seen experimentally with tetracaine) and sufficiently rapid intra-SR Ca diffusion, such that local junctional intra-SR [Ca] can be maintained by intra-SR diffusion and overall SR Ca reuptake.     

Axial stretch enhances sarcoplasmic reticulum Ca2+ leak and cellular Ca2+ reuptake in guinea pig ventricular myocytes: experiments and models

Cardiac cellular calcium (Ca2+) handling is the well-investigated mediator of excitation-contraction coupling, the process that translates cardiac electrical activation into mechanical events. The reverse--effects of mechanical stimulation on cardiomyocyte Ca2+ handling--are much less well understood, in particular during the inter-beat period, called 'diastole'. We have investigated the effects of diastolic length changes, applied axially using a pair of carbon fibres attached to opposite ends of Guinea pig isolated ventricular myocytes, on the availability of Ca2+ in the main cellular stores (the sarcoplasmic reticulum; SR), by studying the rest-decay of SR Ca2+ content [Ca2+]SR, and the reloading of the SR after prior depletion of Ca2+ from the cell. Cells were loaded with Fura-2 AM (an indicator of the cytosolic 'free' Ca2+ concentration, [Ca2+]i), and pre-conditioned by field-stimulation (2 Hz) at 37 degrees C, while [Ca2+]i transients and sarcomere length (SL) were recorded simultaneously. After reaching a steady state in the behaviour of observed parameters, stimulation was interrupted for between 5 and 60s, while cells were either held at resting length, or stretched (controlled to cause a 10% increase in SL, to aid inter-individual comparison). Thereafter, each cell was returned to its original resting length, followed by swift administration of 10mM of caffeine (in Na+/Ca2+-free solution), which causes the release of Ca2+ from the SR (caffeine), but largely prevents extrusion of Ca2+ from the cytosol to the cell exterior (Na+/Ca2+-free solution). By comparing the [Ca2+]i in cells exposed/not exposed to diastolic stretch of different duration, we assessed the rest-decay dynamics of [Ca2+]SR. To assess SR reloading after initial Ca2+ depletion, the same stretch protocol was implemented after prior emptying of the cell by application of 10mM of caffeine in normal Tyrode solution (which causes Ca2+ to be released from the SR and extruded from the cell via the Na+/Ca2+ exchanger; NCX). Axial stretch enhanced the rate of both rest-decay and reloading of [Ca2+]SR. Application of 40 microM streptomycin, a blocker of stretch-activated ion channels, did not affect the stretch-induced increase in SR reloading. This behaviour was reproduced in a computer simulation study, using a modified version of the 2006 Iribe-Kohl-Noble model of single cardiac myocyte Ca2+ handling, suggesting that stretch increases both Ca2+ leak from the SR and Ca2+ influx via the sarcolemma. This may have important implications for the mobilisation of Ca2+ in stretched cells, and could contribute to the regional 'matching' of individual cardiomyocyte contractility to dynamic, and regionally varying, changes in mechanical loads, such as diastolic pre-load, of cardiac tissue.     

Role of the Na(+)-Ca(2+) exchanger as an alternative trigger of CICR in mammalian cardiac myocytes

Ca(2+) influx through the L-type Ca(2+) channels is the primary pathway for triggering the Ca(2+) release from the sarcoplasmic reticulum (SR). However, several observations have shown that Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger current (I(Na-Ca)) could also trigger the Ca(2+) release. The aim of the present study was to quantitate the role of this alternative pathway of Ca(2+) influx using a mathematical model. In our model 20% of the fast sodium channels and the Na(+)-Ca(2+) exchanger molecules are located in the restricted subspace between the sarcolemma and the SR where triggering of the calcium-induced calcium release (CICR) takes place. After determining the strengths of the alternative triggers with simulated voltage-clamps in varied membrane voltages and resting [Na](i) values, we studied the CICR in simulated action potentials, where fast sodium channel current contributes [Na](i) of the subspace. In low initial [Na](i) the Ca(2+) influx via the L-type Ca(2+) channels is the major trigger for Ca(2+) release from the SR, and the Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger cannot trigger the CICR. However, depending on the initial [Na](i), the contribution of the Ca(2+) entry via the exchanger may account for 25% (at [Na](i) = 10 mM) to nearly 100% ([Na](i) = 30 mM) of the trigger Ca(2+). The shift of the main trigger from L-type calcium channels to the exchanger reduced the delay between the action potential upstroke and the intracellular calcium transient. This may contribute to the function of the myocyte in physiological situations where [Na](i) is elevated. These main results remain the same when using different estimates for the most crucial parameters in the modeling or different models for the exchanger.     

Predicting Local SR Ca Dynamics during Ca Wave Propagation in Ventricular Myocytes

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 (Ca²⁺) wave propagation, and 2), speed of Ca²⁺ diffusion within the SR. Ca²⁺ waves are initiated when a spontaneous local SR Ca²⁺ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca²⁺ diffusion constant in the SR (D_Ca,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca²⁺] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca²⁺] during Ca²⁺ waves. Simulations have investigated how dynamic local changes in SR [Ca²⁺] are influenced by 1), D_Ca,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca²⁺ pumps; 4), SR Ca²⁺ pump dependence on cytosolic [Ca²⁺]; 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 [Ca²⁺] in nearby regions. Specifically, our results show that local increases in SR [Ca²⁺] ahead of the wave can potentially facilitate Ca²⁺ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca²⁺] are possible during SR Ca²⁺release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca²⁺]_SR.     

Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction

Oxidative stress plays an important role in mediating ventricular remodeling and dysfunction in heart failure (HF), but its mechanism of action has not been fully elucidated. In this study we determined whether a combination of antioxidant vitamins reduced myocyte apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular (SR) Ca2+ ATPase downregulation in HF after myocardial infarction (MI) and whether these effects were associated with amelioration of left ventricular (LV) remodeling and dysfunction. Vitamins (vitamin C 300 mg and vitamin E 300 mg) were administered to rabbits 1 week after MI or sham operation for 11 weeks. The results showed that MI rabbits exhibited cardiac dilation and LV dysfunction measured by fractional shortening and the maximal rate of pressure rise (dP/dt), an index of contractility. These changes were associated with elevation of oxidative stress, decreases of mitochondrial Bcl-2 and cytochrome c proteins, increases of cytosolic Bax and cytochrome c proteins, caspase 9 and caspase 3 activities and myocyte apoptosis, and downregulation of beta-adrenergic receptor sensitivity and SR Ca2+ ATPase. Combined treatment with vitamins C and E diminished oxidative stress, increased mitochondrial Bcl-2 protein, decreased cytosolic Bax, prevented cytochrome c release from mitochondria to cytosol, reduced caspase 9 and caspase 3 activities and myocyte apoptosis, blocked beta-adrenergic receptor desensitization and SR Ca2+ ATPase downregulation, and attenuated LV dilation and dysfunction in HF after MI. The results suggest that antioxidant therapy may be beneficial in HF.     

RyR1/SERCA1 cross-talk regulation of calcium transport in heavy sarcoplasmic reticulum vesicles

We investigated the functional interdependence of sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1 in heavy sarcoplasmic reticulum membranes by synchronous fluorescence determination of extravesicular Ca2+ transients and catalytic activity. Under conditions of dynamic Ca2+ exchange ATPase catalytic activity was well coordinated to ryanodine receptor activation/inactivation states. Ryanodine-induced activation of Ca2+ release channel leaks also produced marked ATPase activation in the absence of measurable increases in bulk free extravesicular Ca2+. This suggested that Ca2+ pumps are highly sensitive to Ca2+ release channel leak status and potently buffer Ca2+ ions exiting cytoplasmic openings of ryanodine receptors. Conversely, ryanodine receptor activation was dependent on Ca2+-ATPase pump activity. Ryanodine receptor activation by cytosolic Ca2+ was (i) inversely proportional to luminal Ca2+ load and (ii) dependent upon the rate of presentation of cytosolic Ca2+. Progressive Ca2+ filling coincided with progressive loss of Ca2+ sequestration rates and at a threshold loading, ryanodine-induced Ca2+ release produced small transient reversals of catalytic activity. These data indicate that attainment of threshold luminal Ca2+ loads coordinates sensitization of Ca2+ release channels with autogenic inhibition of Ca2+ pumping. This suggests that Ca2+-dependent control of Ca2+ release in intact heavy sarcoplasmic reticulum membranes involves a Ca2+-mediated "cross-talk" between sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1.     

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.