RyR Clusters Research Articles

Calcium Spark Termination: Ryanodine Receptor Unitary Flux Dependent Mechanism

Spontaneous sparks seem to terminate at a fixed free sarcoplasmic recticulum Ca concentration ([Ca]SR) indicating that the SR luminal Ca level is a key factor in terminating Ca sparks. In principle, such luminal Ca control could be achieved by different mechanisms. One is that luminal Ca may alter RyR2 gating by acting at intra-SR sites. Another is that, as luminal Ca falls, RyR2 unitary release flux (iCa) may become insufficient to support continued inter-RyR2 Ca-induced Ca release within a RyR2 cluster (a cytosolic process). To date, it has been virtually impossible to experimentally distinguish these possibilities in cells. We have overcome this obstacle by devising a means to manipulate iCa independently of [Ca]SR. This was accomplished by exploiting RyR2 permeation properties. Briefly, sparks and [Ca]SR were simultaneously recorded in permeabilized rat myocytes. Unitary RyR2 iCa in the tested cellular solutions was defined using single RyR2 measurements in bilayers as well as a well-established RyR permeation model. Preliminary data reveal that reducing RyR2 iCa (at a relatively constant [Ca]SR) dramatically decreases spark frequency. We believe this method is the first to experimentally delineate the contribution of RyR2 iCa flux in the SR luminal Ca control of sparks in cardiomyocytes.

Impaired Local Calcium Signaling in Primary Cultured Adult Rat Ventricular Myocytes

Although cultured adult cardiac myocytes have been adopted in studying protein functions in combination with cell-level genetic modifications, cellular alterations by culturing itself need to be clarified to understand real function of the protein genetically altered. We systematically compared contractile properties, calcium ion (Ca2+) signaling, transverse (t)-tubules, ryanodine receptor distributions between freshly isolated and two-days cultured adult rat ventricular myocytes. Density of t-tubules was remarkably decreased by culture. In cultured myocytes, cell shortenings were attenuated by ∼60% and relaxation was slowed. Consistently, magnitudes of action potential-induced Ca2+ transients were decreased to ∼50% and decays of the Ca2+ transients were retarded by culture. In cultured cells, density of L-type Ca2+ current was reduced to ∼40% and its inactivation was retarded. The latter is consistent with smaller Ca2+ transients in cultured group. However, sarcoplasmic reticulum Ca2+ contents were not different between two groups. To know the mechanism for smaller Ca2+ transient in cultured cells we examined Ca2+ sparks in these two groups of cells. The frequency of spontaneous Ca2+ sparks was significantly decreased by culturing. The amplitude, duration, and time-to-peak of individual Ca2+ sparks were not different between the two groups. Mean spark width was two-fold larger in cultured cells compared with freshly isolated cells. Quantitative analysis of immunofluorescence revealed shortening of longitudinal spacing between RyR2 clusters, and less dense and disorganized distributions of RyR2 clusters in cultured cells, which may be related to lower frequency of sparks in these cells. These results provide evidence on significant difference in Ca2+ sparks as well as excitation-contraction coupling in primary cultured adult ventricular myocytes. (This work was supported by National Research Foundation of Korea grants funded by the Ministry of Education, Science and Technolgy (2010-0000070).)

Tetracaine is a Potent Inhibitor of SR Ca Leak in Ventricular Cardiac Myocytes

Tetracaine is commonly used to inhibit RyR-dependent SR Ca leak, however ability to do so in intact cardiac myocytes has not been fully characterized. In single RyR channel bilayer a significant portion of leak would not be inhibited even by low millimolar concentrations of tetracaine. In this study we investigated the effectiveness of tetracaine on RYR-dependent SR Ca leak in ventricular myocytes. Experiments were performed in isolated rabbit myocytes loaded with fluo-4. Myocytes were perfused with 2mM Ca NT with 200µM caffeine in order to increase basal SR Ca leak in the presence of varying concentrations of tetracaine (1, 10, 30, 100µM). Myocytes were then rapidly perfused with 0 Na, 0 Ca NT with 1mM tetracaine following washout with same solution but with varying amounts of tetracaine. SR Ca leak was quantified as the shift of Ca from the cytosol to the SR upon blockage of the RyR with high tetracaine. The EC50 for tetracaine is significantly reduced in intact myocytes compared to single RyR channel. Among the potential explanations for this effect is the possibility that Ca flux regulation makes the RyR clusters in the SR junction more sensitive to blockage of release.

Ca 2+ Leak and Ca 2+ Sparks in Mammalian Heart: Insights from a Computational Model

Calcium (Ca2+) signaling in muscle, neuronal, and non-excitable cells has benefited significantly from advances in biological tools and imaging technology, however, the molecular interactions of nanoscopic molecules, structures and compartments has been challenging to study under physiological conditions. Here, we exploit novel computational modeling techniques to examine real-time molecular and cellular physiology in cardiac ventricular myocytes. The model focuses on local and cell-wide Ca2+ signaling phenomena related to calcium induced calcium release from intracellular calcium channels, ryanodine receptors (RyR2s), located on the sarcoplasmic reticulum (SR) membrane. This work is informed by the latest molecular investigations and recent characterizations of channels, transporters, and buffers located in mammalian heart. We have created a detailed, whole-cell model of Ca2+ signaling using a realistic number of calcium release units (CRU) each containing a cluster of stochastically gating RyR2s. During systole the opening of these RyR2s is triggered by Ca2+ entry via voltage gated L-type Ca2+ channels. The synchronized opening of the RyR2 cluster leads to localized elevations of [Ca2+]i known as Ca2+ sparks. During diastole Ca2+ sparks are still observed and are attributed to the finite opening rate of the RyR2. RyR2s are also believed to display unsynchronized or non-spark openings where only a few channels in the CRU open without triggering the remainder of the RyR2 cluster. This non-spark Ca2+ release would be below current experimental detection thresholds and therefore “invisible.” These spark and non-spark openings of RyR2s constitute a molecular basis for Ca2+ leak from the SR. The computational model suggests that a significant fraction of SR Ca2+ leak is due to RyR2s openings that fail to trigger a “visible” Ca2+ spark. Additionally, the fraction of non-spark or “invisible” SR Ca2+ leak increases as SR Ca2+ content declines.

Differential sensitivity of Ca2+wave and Ca2+spark events to ruthenium red in isolated permeabilised rabbit cardiomyocytes

Spontaneous Ca²(+) waves in cardiac muscle cells are thought to arise from the sequential firing of local Ca²(+) sparks via a fire-diffuse-fire mechanism. This study compares the ability of the ryanodine receptor (RyR) blocker ruthenium red (RuR) to inhibit these two types of Ca²(+) release in permeabilised rabbit ventricular cardiomyocytes. Perfusing with 600 nm Ca²(+) (50 μm EGTA) caused regular spontaneous Ca²(+) waves that were imaged with the fluorescence from Fluo-5F using a laser-scanning confocal microscope. Addition of 4 μm RuR caused complete inhibition of Ca²(+) waves in 50% of cardiomyocytes by 2 min and in 100% by 4 min. Separate experiments used 350 μm EGTA (600 nm Ca²(+)) to limit Ca²(+) diffusion but allow the underlying Ca(2+) sparks to be imaged. The time course of RuR-induced inhibition did not match that of waves. After 2 min of RuR, none of the characteristics of the Ca²(+) sparks were altered, and after 4 min Ca²(+) spark frequency was reduced ∼40%; no sparks could be detected after 10 min. Measurements of Ca(2+) within the SR lumen using Fluo-5N showed an increase in intra-SR Ca²(+) during the initial 2-4 min of perfusion with RuR in both wave and spark conditions. Computational modelling suggests that the sensitivity of Ca²(+) waves to RuR block depends on the number of RyRs per cluster. Therefore inhibition of Ca²(+) waves without affecting Ca²(+) sparks may be explained by block of small, non-spark producing clusters of RyRs that are important to the process of Ca²(+) wave propagation.

Predicting Local SR Ca2+ Dynamics during Ca2+ 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 (DCa,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), DCa,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, DCa,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.

Ryanodine Receptors Outside of Couplons are Involved in Excitation-Contraction Coupling in Rabbit Ventricular Myocytes

Current theories of excitation-contraction coupling (ECC) in ventricular myocytes assert that L-type Ca channels interact with clusters of ryanodine-sensitive Ca release channels (RyRs) within couplons. We hypothesized that RyR clusters exist also outside of couplons and contribute to ECC. We investigated this hypothesis by three-dimensional imaging of RyR clusters and sarcolemma of isolated myocytes lying flat (horizontal) and on end (vertical). We deconvolved the image stacks, created reconstructions of cell segments, and identified RyR cluster types. RyR clusters remote to sarcolemma were assumed to be outside of couplons. Similar studies were performed on intact ventricular tissue. Furthermore, we imaged evoked Ca transients and sarcolemma of horizontal cells labeled with fluo-4 and di-8-anepps. Image sequences were acquired using rapid two-dimensional scanning (Zeiss LSM5Live, rates up to 300HZ). The image sequences were corrected for bleaching and cross-talk. In horizontal and vertical isolated cells, RyR clusters appeared to be arranged in sheets in the vicinity of Z-disks. Some RyR clusters were associated with sarcolemma, in particular transverse tubules, and are presumably part of couplons. However, most RyR clusters were not. Examination of cells in intact tissue revealed a smaller number of RyR clusters not associated with sarcolemma than in isolated cells. The density of transverse tubules was higher than in isolated cells. This loss of transverse tubules might be caused by the isolation procedure. Analysis of the rapid image sequences indicated that both types of RyR clusters were activated during an action potential. However, the RyR clusters not associated with sarcolemma were activated with delays of up to 10ms. In conclusion, we demonstrated that RyR clusters outside of couplons are involved in ECC. We suggest that activation of RyR clusters outside of couplons occurs by a common pool mechanism.

Occurrence of Spontaneous Sparks in Ventricular Myocytes From Junctional and Non-junctional Ryr Clusters

In isolated rabbit ventricular myocytes, we found a significant number of ryanodine receptor (RyR) clusters that are not associated with the sarcolemma (non-junctional RyRs). The contribution of non-junctional RyR clusters to calcium transients is unclear. Here, we investigated if these non-junctional RyRs are able to produce spontaneous local calcium release events (sparks), and compared the probability of non-junctional versus junctional sparks. We imaged spontaneous sparks in cells loaded with fluo-4 and bathed in Tyrode solution with dextran (Molecular Weight: 10 kDa) linked to Texas Red dye. We evoked spontaneous sparks using field stimulation in the presence of 1 μM isoproterenol and 4 mM calcium. After 5 stimuli applied with a frequency of 0.5 Hz, we simultaneously imaged the sarcolemma and spontaneous sparks using a line scan confocal microscope (Biorad MRC-1024). Furthermore, a 3D image stack of the Texas Red associated signal was acquired to identify the sarcolemma including the transverse tubular system. We classified sparks as non-junctional if their distance to the sarcolemma is larger than 1 μm. All other sparks were assumed to be junctional. In measurements on 12 isolated cells, 38 sparks (51%) were identified as non-junctional, 36 (49%) as junctional. Our measurements clearly demonstrate that non-junctional RyR clusters are able to release calcium and produce spontaneous sparks. We expect that our approach for distinguishing between non-junctional and junctional sparks underestimates the number of non-junctional sparks. If this is true, the probabilities of the types of sparks are similar to probabilities of the two types of RyR clusters identified in related immunolabeling and microscopic studies. This would suggests that spark generation probability of RyR clusters does not depend on their type.

Automated Reduction of Calcium Release Site Models Via State Aggregation

Realistic simulations of local control of cardiac EC coupling require Ca release unit (CaRU) models generated using Markov chain models of L-type Ca channels and ryanodine receptors (RyRs) as “building blocks.” Because compositionally defined CaRU models result in a combinatorial explosion of release site states, most whole cell simulations to date have utilized ad hoc CaRU models in an effort to maintain computational efficiency (e.g., modeling RyR clusters as a “megachannel”). To overcome this state-space explosion, we have implemented, validated, and benchmarked several methods for automated reduction of mechanistic CaRU models that feature an automated process of state aggregation and evaluation of reduction error through comparison of the jump probability matrices of full and reduced models. When there is separation of time scales in the single channel model (e.g., fast activation and slow inactivation), we perform numerical fast/slow reduction by categorizing rate constants in the single channel model as either fast or slow, aggregating states in the expanded CaRU model that are connected by fast transitions, and calculating transition rates between lumped states using the conditional probability distribution of states within each group. For large problems where the conditional distributions can not be directly calculated from the full model, we employ iterative aggregation/disaggregation to calculate conditional distributions in a memory-efficient fashion. For problems without time scale separation, how states should be aggregated to yield good reductions can not be determined a priori. Consequently, we implemented a genetic algorithm that evolves potential schemes for state aggregation, ultimately yielding simplified CaRU models with low reduction error. We demonstrate that such automated CaRU reduction procedures can be used to accelerate multiscale models of local control of CICR in cardiac myocytes.

Electron-Conformational Model of SR-Based Ca2+ Clock Mode

Most of the calcium that activates cardiac contraction comes from the sarcoplasmic reticulum (SR) from where it is released through the Ryanodine Receptors (RyRs). It is well known that the SR overload results in the release of Ca from the SR in the form of waves driving some cardiac arrhythmias. Recently it has been experimentally documented that the isolated SR is capable to spontaneously and rhythmically release Ca2+ (SR-based Ca2+ clock). This self-sustained intracellular Ca2+ oscillator contributes substantially to the late phase of the diastolic depolarization of cardiac pacemaker cells under normal physiological conditions. Interaction of “a surface membrane oscillator” and “an internal oscillator” with “cycles of Ca2+ uptake and release by the SR” can drive normal cardiac automaticity. To describe the SR-based Ca2+ clock mode we propose a simple, physically-reasonable electron-conformational (EC) model for the RyR and present a theory to describe the RyR lattice dynamics. Each RyR is modelled with a single open and closed electronic state. In addition to the fast electronic degree of freedom, RyR channels are characterized by a slow classical conformational coordinate, which specifies the RyR channel conductance. The RyR gating implies a conformational Langevin dynamics, Ca2+-induced electronic transitions, quantum tunneling and thermal transitions. The cooperativity in the RyR lattice is assumed to be determined by the inter-channel conformational coupling. Model simulations of the of 11x11 RyR cluster revealed different regimes depending on the cis- and trans-Ca concentrations and parameters of EC-model. The SR overload is shown to result in RyR lattice auto-oscillations with spontaneous RyR channel openings and closures. We have studied this Ca2+ clock mode, in particular, its stability, under different model suggestions as regards the RyR conformational potential (diabatic and adiabatic regimes), EC-model parameters.Supported by the RFBR 07-04-96126

Organization of Ryanodine Receptors, Transverse Tubules, and Sodium-Calcium Exchanger in Rat Myocytes

Confocal and total internal reflection fluorescence imaging was used to examine the distribution of caveolin-3, sodium-calcium exchange (NCX) and ryanodine receptors (RyRs) in rat ventricular myocytes. Transverse and longitudinal optical sectioning shows that NCX is distributed widely along the transverse and longitudinal tubular system (t-system). The NCX labeling consisted of both punctate and distributed components, which partially colocalize with RyRs (27%). Surface membrane labeling showed a similar pattern but the fraction of RyR clusters containing NCX label was decreased and no nonpunctate labeling was observed. Sixteen percent of RyRs were not colocalized with the t-system and 1.6% of RyRs were found on longitudinal elements of the t-system. The surface distribution of RyR labeling was not generally consistent with circular patches of RyRs. This suggests that previous estimates for the number of RyRs in a junction (based on circular close-packed arrays) need to be revised. The observed distribution of caveolin-3 labeling was consistent with its exclusion from RyR clusters. Distance maps for all colocalization pairs were calculated to give the distance between centroids of punctate labeling and edges for distributed components. The possible roles for punctate NCX labeling are discussed.

Dynamic Interreceptor Coupling Contributes to the Consistent Open Duration of Ryanodine Receptors

Ca 2+ spark is the elementary Ca 2+ signaling event in muscle excitation-contraction coupling. The rise time of Ca 2+ spark is rather stable under different conditions, suggesting consistent open duration of ryanodine receptors (RyRs) in vivo. It has been proposed that the array-based behavior of RyRs plays an important role in shaping Ca 2+ spark dynamics, particularly in controlling the open duration of RyR clusters. Therefore, we investigated the possible role of inter-RyR coupling in stabilization of the open time of arrayed RyRs under several potential perturbations, for instance, array size, inter-RyR coupling noise, and up-regulation or down-regulation of the activity of partial RyRs in the array. We found that RyR arrays with dynamic coupling showed consistent open duration against the perturbations, whereas the RyR array with constant coupling did not. On the other hand, the open probability and amplitude of RyR arrays with dynamic interreceptor coupling were sensitive to the perturbations. These two points were consistent with experimental observations, indicating that the RyR array with dynamic coupling could recapitulate in vivo open properties of RyRs. Our findings support the idea that dynamic coupling is a feasible in vivo working mechanism of RyR arrays.

Loss of Intracellular and Intercellular Synchrony of Calcium Release in Systolic Heart Failure

Normal contraction of cardiac muscle requires a coordinated cellular mechanical response to the electric pacemaker signal that begins in the sinoatrial node and spreads through the conduction system and ultimately across the ventricular myocardium via gap junctions. In ventricular myocytes, depolarization by the action potential causes clusters of L-type calcium channels (LCCs) to open, allowing a small amount of Ca to enter the diadic cleft space between the sarcolemma and the sarcoplasmic reticulum (SR). When the Ca concentration in this space becomes sufficiently high to gate ryanodine receptors (RyRs), they open and release SR Ca into the cytoplasm, so that it can bind to and activate myofilaments, resulting in force development. Each cluster of LCCs on the sarcolemma, its opposing cluster of RyRs across the 10- to 12-nm diameter diadic cleft space, and associated SR regulatory proteins is a couplon.1 According to the local control theory of cardiac excitation-contraction (EC) coupling, couplons are activated independently. Independent activation of couplons depends on the extent of LCC activation, providing a mechanism for regulation of contractile force by stochastic recruitment of couplons.2 In systolic heart failure, numerous cellular defects in EC coupling and Ca regulation have been identified that contribute to contractile dysfunction (and ventricular arrhythmias), although there is a host of other major abnormalities leading to loss of force development, including fibrosis,3 defective energy metabolism,4 pH changes,5 heterogeneous conductance across connexin hemichannels,6 and defects in sarcomeric proteins.7 Thus, it is important to keep in mind that no single defect can explain the contractile dysfunction of systolic failure. Article see p 223 Recent studies of defective EC coupling in heart failure have addressed not only reductions in the extent and kinetics of Ca released in response to the action potential (ie, reduced recruitment of couplons and …

Three‐dimensional high‐resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes

Quantitative understanding of the Ca(2+) handling in cardiac ventricular myocytes requires accurate knowledge of cardiac ultrastructure and protein distribution. We have therefore developed high-resolution imaging and analysis approaches to measure the three-dimensional distribution of immunolabelled proteins with confocal microscopy. Labelling of single rat cardiac myocytes with an antibody to the Z-line marker alpha-actinin revealed a complex architecture of sarcomere misalignment across single cells. Double immunolabelling was used to relate the Z-line structure to the distribution of ryanodine receptors (RyRs, the intracellular Ca(2+) release channels) and the transverse tubular system. Both RyR and transverse tubular system distributions exhibited frequent dislocations from the simple planar geometry generally assumed in existing mathematical models. To investigate potential effects of these irregularities on Ca(2+) dynamics, we determined the three-dimensional distribution of RyR clusters within an extended section of a single rat ventricular myocyte to construct a model of stochastic Ca(2+) dynamics with a measured Ca(2+) release unit (CRU) distribution. Calculations with this model were compared with a second model in which all CRUs were placed on flat planes. The model with a realistic CRU distribution supported Ca(2+) waves that spread axially along the cell at velocities of approximately 50 mum s(-1). By contrast, in the model with planar CRU distribution the axial wave spread was slowed roughly twofold and wave propagation often nearly faltered. These results demonstrate that spatial features of the CRU distribution on multiple length scales may significantly affect intracellular Ca(2+) dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.

Relationship of Ryanodine Receptors to the Sarcolemma in Rabbit Ventricular Myocytes

To investigate the spatial relationship between the sarcolemma and ryanodine receptors (RyRs) we dual-labeled cells using Alexa fluor dyes and simultaneously imaged them with confocal microscopy. We deconvolved the images and subjected them to digital processing. We obtained three-dimensional reconstructions from cells in two configurations: lying flat and standing on end. In the flat configuration, RyR clusters appeared to be arranged in sheets near Z-disks. The distance between sheets was ∼2 μm. Although some clusters are closely associated with detectable sarcolemma, the majority of them are not (>70%). With cells standing on end in agar we obtained XY scans orthogonal to the cell long axis. This improved the separation of RyR clusters and the classification based on their relationship to the sarcolemma. These data confirmed our initial finding obtained on cells lying flat, that most of RyRs are non-junctional (see figure). It is unclear whether they contribute to the Ca transient. If they do, the transient would reflect the combined activity of locally controlled couplons with the activation of non-junctional RyRs, which are not locally controlled.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Triadin Deletion Alters Calcium Sparks in Murine Cardiomyocytes

Gene-targeted deletion of the sarcoplasmic reticulum (SR) protein triadin (Trdn-/-) causes 50% reduction in ryanodine receptor (RyR2) Ca2+ release channels and cardiac calsequestrin, and a 50% decrease in the size of t-tubule SR junctions in mouse heart muscle. Here we report on the Ca2+ spark properties of Trdn-/- cardiomyocytes. Isolated ventricular myocytes from Trdn-/- mice (N=5) and wild-type littermates (Trdn+/+, N=8) were loaded with the Ca-sensitive fluorescent indicator Fluo4-AM and Ca2+ sparks were measured in 2mM Ca2+ by confocal microscopy in line scan mode. As illustrated in the figure, triadin deletion caused a dramatic reduction in spark amplitude (ΔF/Fo: Trdn-/- 0.43±0.01, n=893; Trdn+/+ 0.61±0.02, n=745, p<7.27E-22), spark width (FWHM (μm): Trdn-/- 2.64±0.03 n=893, Trdn+/+ 2.90 ± 0.03, n=745, p<1.77E-09) and spark upstroke velocity (Δ(F/Fo)/Δtmax(Δ(F/Fo)/s): Trdn-/- 31.67±0.90, n=891, Trdn+/+ 58.35±1.62, n=741, p<3.49E-48), whereas spark frequency was modestly increased (sparks/100μm/s: Trdn-/- 0.92±0.08, n=255 myocytes, Trdn+/+ 0.71±0.06, n=321 myocytes, p<0.03). The changes in spark properties occurred in absence of significant changes in SR Ca2+ content measured by rapid caffeine application. These data suggest that loss of triadin has a drastic effect on spark properties, possibly by altering the number of RyR2 and/or the RyR2 cluster size.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Beta-Adrenergic Stimulation Does Not Affect Calcium Sparks Refractoriness in Ventricular Myocytes

Cardiac Ca2+ sparks are intracellular Ca2+ release events from clusters of ryanodine receptors (RyR2) in the junctional sarcoplasmic reticulum (jSR). L-type Ca2+ channels (LCC) are located in the nearby apposing sarcolemma (SL) mainly at the transverse tubules. Cellular depolarization permits local Ca2+ influx through LCC that activates RyR2 clusters by Ca2+-induced Ca2+-release (CICR). Ca2+ sparks also occur during diastole due to the finite opening rate of the RyR2s that are sensitive to both cytosolic Ca2+ ([Ca2+]i) and to SR Ca2+ ([Ca2+]SR). There is a significant (at least 50%) depletion of jSR Ca2+ during each Ca2+ spark and this depletion (measured as Ca2+ blinks, Brochet et al. 2005) suggests that refractoriness of Ca2+ sparks is due to the reduction of [Ca2+]SR (Sobie et al. 2006). Additional factors (beyond [Ca2+]i and [Ca2+]SR) have been reported to affect the opening and closing rates of RyR2. Here we examine RyR2 modulation by protein kinase A (PKA) during Beta-adrenergic stimulation. Studying RyR2 refractoriness is complicated because it overlaps with ICa restitution and with the slower SR Ca2+ uptake by SERCA. We assessed RyR2 refractoriness in permeabilized ventricular myocytes from phospholamban-KO mice by studying repeated spontaneous Ca2+ sparks at the same Ca2+ release location in the absence and presence of cAMP (10 μM). We observed under control conditions that Ca2+ spark amplitude restoration (time constant ∼70 ms) was ∼2 fold slower than the reported jSR Ca2+ refilling. RyR2 phosphorylation did not affect Ca2+ sparks amplitude restoration, and the Ca2+ spark frequency distribution peak was slightly diminished, with small increases at longer delays. We conclude that under conditions when neither LCC nor [Ca2+]SR can change to influence Ca2+ sparks rate or Ca2+ sparks refractoriness, RyR2 phosphorylation by PKA activation does not alter RyR2 refractoriness.

Local is as local does: the unitary nature of SR Ca2+ release in cardiac ventricular myocytes

Local is as local does: the unitary nature of SR Ca²⁺ release in cardiac ventricular myocytes

Intracellular Ca 2+ nonlinear wave behaviours in a three dimensional ventricular cell model

Intracellular Ca 2+ activity regulates a wide range of cellular biochemical processes; in muscle cells, it links membrane excitation to contraction. Ca 2+ dynamics includes both synchronous oscillations, and nonlinear wave phenomena, both arising from the superposition of spatially localised stochastic events, such as Ca 2+ sparks. We incorporated individualised cell geometry reconstructed from confocal microscopy with realistic spatial distribution of RyR clusters into the three dimensional ventricular cell model, and reproduced complex spatio-temporal intracellular wave patterns from Ca 2+ sparks. We also introduced a detailed nuclear Ca 2+ handing model to simulate prolonged nuclear Ca 2+ transient, and study the effects of cytosolic-nuclear coupling on intracellular Ca 2+ dynamics. The model provides a computational platform to study intracellular Ca 2+ with the ability to interact with experimental measurements of subcellular structures, and can be modified for other cell types.

New insights into the structure and function of couplons

It is widely accepted that the calcium transient in heart is a summation of many Ca2+ release events or sparks (Cheng et al. 1993). Each local release event is produced by a structure called a couplon (Stern et al. 1997), defined as ‘the functional grouping of RyRs and dihydropyridine receptors, (DHPRs; also known as L-type Ca channels) (and other junctional SR proteins) which may act in concert during EC coupling’ (Franzini-Armstrong et al. 1999). Calcium that enters the couplon through DHPRs triggers Ca2+ release from a cluster of RyRs (ryanodine receptors, sarcoplasmic reticulum Ca2+ release channels), thus producing a local release event. Each couplon obeys local control, which can explain, for example, the gradation of Ca2+ transients with trigger magnitude. The number of recruited couplons determines the transient amplitude. The number of DHPRs found in a couplon is of central importance, but is difficult to investigate experimentally. It seems certain that, at least in rabbits, multiple DHPRs must be present in each couplon because the probability of activation of local Ca2+ release events exceeds the probability that an individual DHPR will open during the action potential (Inoue & Bridge, 2003).