Ryanodine receptor allosteric coupling and the dynamics of calcium sparks

Puffs and sparks are localized intracellular Ca²⁺ elevations that arise from the cooperative activity of Ca²⁺-regulated inositol 1,4,5-trisphosphate receptors and ryanodine receptors clustered at Ca²⁺ release sites on the surface of the endoplasmic reticulum or the sarcoplasmic reticulum. While the synchronous gating of Ca²⁺-regulated Ca²⁺ channels can be mediated entirely though the buffered diffusion of intracellular Ca²⁺, interprotein allosteric interactions also contribute to the dynamics of ryanodine receptor (RyR) gating and Ca²⁺ sparks. In this article, Markov chain models of Ca²⁺ release sites are used to investigate how the statistics of Ca²⁺ spark generation and termination are related to the coupling of RyRs via local [Ca²⁺] changes and allosteric interactions. Allosteric interactions are included in a manner that promotes the synchronous gating of channels by stabilizing neighboring closed-closed and/or open-open channel pairs. When the strength of Ca²⁺-mediated channel coupling is systematically varied (e.g., by changing the Ca²⁺ buffer concentration), simulations that include synchronizing allosteric interactions often exhibit more robust Ca²⁺ sparks; however, for some Ca²⁺ coupling strengths the sparks are less robust. We find no evidence that the distribution of spark durations can be used to distinguish between allosteric interactions that stabilize closed channel pairs, open channel pairs, or both in a balanced fashion. On the other hand, the changes in spark duration, interspark interval, and frequency observed when allosteric interactions that stabilize closed channel pairs are gradually removed from simulations are qualitatively different than the changes observed when open or both closed and open channel pairs are stabilized. Thus, our simulations clarify how changes in spark statistics due to pharmacological washout of the accessory proteins mediating allosteric coupling may indicate the type of synchronizing allosteric interactions exhibited by physically coupled RyRs. We also investigate the validity of a mean-field reduction applicable to the dynamics of a ryanodine receptor cluster coupled via local [Ca²⁺] and allosteric interactions. In addition to facilitating parameter studies of the effect of allosteric coupling on spark statistics, the derivation of the mean-field model establishes the correct functional form for cooperativity factors representing the coupled gating of RyRs. This mean-field formulation is well suited for use in computationally efficient whole cell simulations of excitation-contraction coupling.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Polymorphism of Ca2+ Sparks Evoked from In-Focus Ca2+ Release Units in Cardiac Myocytes

Ca²⁺ sparks are the elementary release events in many types of cells. Here we present a morphometric analysis of Ca²⁺ sparks (i.e., amplitude and kinetic parameters) using an approach that minimizes the confounding factor of the detection of out-of-focus events. By activation and visualization of Ca²⁺ sparks from Ca²⁺ release units under loose-seal patch-clamp conditions, we found that the amplitude and rising rate of in-focus sparks exhibited a broad modal distribution, whereas spark rise time and spatial width appeared to be stereotyped. Spark morphometrics were constant irrespective of the latency of spark production and the time-dependent L-type Ca²⁺ channel activation. Polymorphism of Ca²⁺ sparks in terms of variable amplitude and rising rate was evident for events from the same release units, and intra- and interrelease unit variability contributed equally to the overall variability. The rising rate, a reporter of the underlying Ca²⁺ release flux, displayed a strong positive correlation with spark amplitude, but a negative correlation with spark rise time, an index of Ca²⁺ release duration. On the basis of Ca²⁺ spark morphometrics measured here, we suggested a model in which cohorts of variable number of ryanodine receptors are activated in the genesis of Ca²⁺ sparks, and the ensuing negative feedback overrides the regenerative Ca²⁺-induced Ca²⁺ release to extinguish the ongoing Ca²⁺ spark.     

Calcium-dependent inactivation and the dynamics of calcium puffs and sparks

Localized intracellular Ca²⁺ elevations known as puffs and sparks arise from the cooperative activity of inositol 1,4,5-trisphosphate receptor Ca²⁺ channels (IP₃Rs) and ryanodine receptor Ca²⁺ channels (RyRs) clustered at Ca²⁺ release sites on the surface of the endoplasmic reticulum or sarcoplasmic reticulum. When Markov chain models of these intracellular Ca²⁺-regulated Ca²⁺ channels are coupled via a mathematical representation of a Ca²⁺ microdomain, simulated Ca²⁺ release sites may exhibit the phenomenon of “stochastic Ca²⁺ excitability” reminiscent of Ca²⁺ puffs and sparks where channels open and close in a concerted fashion. To clarify the role of Ca²⁺ inactivation of IP₃Rs and RyRs in the dynamics of puffs and sparks, we formulate and analyze Markov chain models of Ca²⁺ release sites composed of 10–40 three-state intracellular Ca²⁺ channels that are inactivated as well as activated by Ca²⁺. We study how the statistics of simulated puffs and sparks depend on the kinetics and dissociation constant of Ca²⁺ inactivation and find that puffs and sparks are often less sensitive to variations in the number of channels at release sites and strength of coupling via local [Ca²⁺] when the average fraction of inactivated channels is significant. Interestingly, we observe that the single channel kinetics of Ca²⁺ inactivation influences the thermodynamic entropy production rate of Markov chain models of puffs and sparks. While excessively fast Ca²⁺ inactivation can preclude puffs and sparks, moderately fast Ca²⁺ inactivation often leads to time-irreversible puffs and sparks whose termination is facilitated by the recruitment of inactivated channels throughout the duration of the puff/spark event. On the other hand, Ca²⁺ inactivation may be an important negative feedback mechanism even when its time constant is much greater than the duration of puffs and sparks. In fact, slow Ca²⁺ inactivation can lead to release sites with a substantial fraction of inactivated channels that exhibit puffs and sparks that are nearly time-reversible and terminate without additional recruitment of inactivated channels.     

Single Ryanodine Receptor Channel Basis of Caffeine's Action on Ca Sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca²⁺ release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca²⁺ sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca²⁺ level on both sides of the channel. Cytosolic Ca²⁺ enhanced RyR2 caffeine affinity, whereas luminal Ca²⁺ essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC₅₀ of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca²⁺ spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca²⁺ sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.     

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca²⁺ release through ryanodine receptors. In the present study we aimed at further understanding how Ca²⁺ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca²⁺ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca²⁺ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca²⁺ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca²⁺ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca²⁺ sparks while the rest take either the form of lower amplitude, longer duration Ca²⁺ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca²⁺ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca²⁺-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.     

Calcium sparks activate calcium-dependent Cl- current in rat corpus cavernosum smooth muscle cells

Spontaneous transient currents, due to activation of Ca²⁺-dependent K⁺ and Cl^– channels, occur in corpus cavernosum smooth muscle cells (CCSMC) of the penis. The Ca²⁺ events responsible for triggering Ca²⁺-dependent Cl^– channels have never been identified in vascular muscle. We used high-speed fluorescence imaging combined with patch-clamp electrophysiology to provide the first characterization of Ca²⁺ events underlying these currents. Freshly isolated rat CCSMC loaded with fluo-4 exhibited localized, spontaneous elevations of intracellular Ca²⁺ (Ca²⁺ sparks) in 57% of cells. There was an average of 6.4 ± 0.5 release sites/cell with a frequency of 0.9 ± 1 Hz/cell and peak amplitude F/F_o of 67 ± 10%. We addressed the controversy of whether these events are mediated by ryanodine or inositol 1,4,5 trisphosphate (IP₃) receptors. Caffeine caused either a global Ca²⁺ rise at high concentrations or an increase in spark frequency at lower concentrations, whereas ryanodine dramatically reduced the amplitude and frequency of sparks. 2-Aminoethoxydiphenyl borate, an inhibitor of IP₃ receptors, had no effect on spark frequency. Combined imaging and electrophysiological recording revealed strong coupling between Ca²⁺ sparks and biphasic transient currents, a relationship never before shown in vascular muscle. Moreover, spark frequency increased on depolarization, an effect abolished with the blockade of Ca²⁺ channels, consistent with Ca²⁺ influx regulating Ca²⁺ release from stores. We establish for the first time that Ca²⁺ sparks occur in CCSMC and arise from Ca²⁺ release through ryanodine receptors. Moreover, the voltage dependence of spark frequency demonstrated here provides novel functional evidence for voltage-dependent Ca²⁺ influx in CCSMC. calcium signaling; potassium and chloride channels; ryanodine receptors     

On the Adjacency Matrix of RyR2 Cluster Structures

In the heart, electrical stimulation of cardiac myocytes increases the open probability of sarcolemmal voltage-sensitive Ca²⁺ channels and flux of Ca²⁺ into the cells. This increases Ca²⁺ binding to ligand-gated channels known as ryanodine receptors (RyR2). Their openings cause cell-wide release of Ca²⁺, which in turn causes muscle contraction and the generation of the mechanical force required to pump blood. In resting myocytes, RyR2s can also open spontaneously giving rise to spatially-confined Ca²⁺ release events known as “sparks.” RyR2s are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. Our recent work has shown that the spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca²⁺ sparks. We showed that the probability of a Ca²⁺ spark occurring when a single RyR2 in the cluster opens spontaneously can be predicted from the precise spatial arrangements of the RyR2s. Thus, “function” follows from “structure.” This probability is related to the maximum eigenvalue (λ ₁) of the adjacency matrix of the RyR2 cluster lattice. In this work, we develop a theoretical framework for understanding this relationship. We present a stochastic contact network model of the Ca²⁺ spark initiation process. We show that λ ₁ determines a stability threshold for the formation of Ca²⁺ sparks in terms of the RyR2 gating transition rates. We recapitulate these results by applying the model to realistic RyR2 cluster structures informed by super-resolution stimulated emission depletion (STED) microscopy. Eigendecomposition of the linearized mean-field contact network model reveals functional subdomains within RyR2 clusters with distinct sensitivities to Ca²⁺. This work provides novel perspectives on the cardiac Ca²⁺ release process and a general method for inferring the functional properties of transmembrane receptor clusters from their structure.     

Eavesdropping on the social lives of Ca(2+) sparks

Ca²⁺ sparks arise from the stochastic opening of spatially discrete clusters of ryanodine receptors called a Ca²⁺ release unit (CRU). If the RyR clusters were not spatially separated, then Ca²⁺ released from one RyR would immediately diffuse to its neighbor and lead to uncontrolled, runaway Ca²⁺ release throughout the cell. While physical separation provides some isolation from neighbors, CRUs are not incommunicado. When inter-neighbor interactions become large enough, Ca²⁺ waves spontaneously emerge. A more circumscribed interaction shows up in high-speed two-dimensional confocal images as jumping Ca²⁺ sparks that seem to be sequentially activated along the Z-line and across Z-lines. However, since Ca²⁺ sparks are stochastic events how can we tell whether two sparks occurring close together in space and time are causally related or appeared simply by coincidence? Here we develop a mathematical method to disentangle cause and coincidence in a statistical sense. From our analysis we derive three fundamental properties of Ca²⁺ spark generation: 1), the “intrinsic” spark frequency, the spark frequency one would observe if the CRUs were incommunicado; 2), the coupling strength, which measures how strongly one CRU affects another; and 3), the range over which the communication occurs. These parameters allow us to measure the effect RyR regulators have on the intrinsic activity of CRUs and on the coupling between them.     

Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density

Rationale

In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).

Objective

To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.

Methods

Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca²⁺ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca²⁺ release, F>F₅₀ within 20 ms) or their absence (delayed areas). Spontaneous Ca²⁺ release events during diastole, Ca²⁺ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.

Results

In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na⁺/Ca²⁺ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca²⁺ influx via Ca²⁺ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca²⁺ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca²⁺ removal by NCX at the membrane was significantly lower in MI.

Conclusion

TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca²⁺ loss and raise SR Ca²⁺ content, but may promote Ca²⁺ waves.     

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 sarcoplasmic 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.     

Frequency and release flux of calcium sparks in rat cardiac myocytes: a relation to RYR gating

Cytosolic calcium concentration in resting cardiac myocytes locally fluctuates as a result of spontaneous microscopic Ca²⁺ releases or abruptly rises as a result of an external trigger. These processes, observed as calcium sparks, are fundamental for proper function of cardiac muscle. In this study, we analyze how the characteristics of spontaneous and triggered calcium sparks are related to cardiac ryanodine receptor (RYR) gating. We show that the frequency of spontaneous sparks and the probability distribution of calcium release flux quanta of triggered sparks correspond quantitatively to predictions of an allosteric homotetrameric model of RYR gating. This model includes competitive binding of Ca²⁺ and Mg²⁺ ions to the RYR activation sites and allosteric interaction between divalent ion binding and channel opening. It turns out that at rest, RYRs are almost fully occupied by Mg²⁺. Therefore, spontaneous sparks are most frequently evoked by random openings of the highly populated but rarely opening Mg₄RYR and CaMg₃RYR forms, whereas triggered sparks are most frequently evoked by random openings of the less populated but much more readily opening Ca₂Mg₂RYR and Ca₃MgRYR forms. In both the spontaneous and the triggered sparks, only a small fraction of RYRs in the calcium release unit manages to open during the spark because of the limited rate of Mg²⁺ unbinding. This mechanism clarifies the unexpectedly low calcium release flux during elementary release events and unifies the theory of calcium signaling in resting and contracting cardiac myocytes.     

Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes

Abnormalities in cardiomyocyte Ca²⁺ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca²⁺ sparks (brief, localized increased in [Ca²⁺]_i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca²⁺]_i transient amplitude and sarcoplasmic reticulum (SR) Ca²⁺ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca²⁺ sparks were less frequent, whereas the fractional release and the frequency of Ca²⁺ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca²⁺ sparks were more frequent in PMI cells (under conditions of similar SR Ca²⁺ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca²⁺ sparks frequency normalized by the SR Ca²⁺ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca²⁺ spark frequency. Indeed, using an internal “failing solution” with less ATP (as found in HF), we observed a dramatic decrease in Ca²⁺ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca²⁺ load may mask these effects at the Ca²⁺ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca²⁺ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients.     

Control of Sarcoplasmic Reticulum Ca2+ Release by Stochastic RyR Gating within a 3D Model of the Cardiac Dyad and Importance of Induction Decay for CICR Termination

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca²⁺ spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca²⁺, Mg²⁺, and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca²⁺ sparks, Ca²⁺ blinks, and Ca²⁺ spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca²⁺ dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca²⁺ dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca²⁺, as well as the time course of local Ca²⁺ gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca²⁺ spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca²⁺ spark initiation and subsequent Ca²⁺ spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca²⁺ in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca²⁺ spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca²⁺ flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca²⁺ sensitivities, Ca²⁺ spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.     

Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes

During the cardiac action potential, Ca²⁺ entry through dyhidropyridine receptor L-type Ca²⁺ channels (DHPRs) activates ryanodine receptors (RyRs) Ca²⁺-release channels, resulting in massive Ca²⁺ mobilization from the sarcoplasmic reticulum (SR). This global Ca²⁺ release arises from spatiotemporal summation of many localized elementary Ca²⁺-release events, Ca²⁺ sparks. We tested whether DHPRs modulate Ca²⁺sparks in a Ca²⁺ entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca²⁺ sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca²⁺ (100 nM). Under these conditions, Ca²⁺ sparks were detected with a stable frequency of 3–5 sparks·s^–1·100 µm^–1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca²⁺ sparks. The DHPR modulators were also without effect on SR Ca²⁺ load (caffeine-induced Ca²⁺ transients) or sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity (Ca²⁺ loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca²⁺ entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca²⁺-release units in ventricular myocytes is modulated by Ca²⁺ entry-independent conformational changes in neighboring DHPRs. exitation-contraction coupling; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase; dihydropyridine receptor; sarcoplasmic reticulum     

Nanoscale organization of ryanodine receptor distribution and phosphorylation pattern determines the dynamics of calcium sparks

Super-resolution imaging techniques have provided a better understanding of the relationship between the nanoscale organization and function of ryanodine receptors (RyRs) in cardiomyocytes. Recent data have indicated that this relationship is disrupted in heart failure (HF), as RyRs are dispersed into smaller and more numerous clusters. However, RyRs are also hyperphosphorylated in this condition, and this is reported to occur preferentially within the cluster centre. Thus, the combined impact of RyR relocalization and sensitization on Ca²⁺ spark generation in failing cardiomyocytes is likely complex and these observations suggest that both the nanoscale organization of RyRs and the pattern of phosphorylated RyRs within clusters could be critical determinants of Ca²⁺ spark dynamics. To test this hypothesis, we used computational modeling to quantify the relationships between RyR cluster geometry, phosphorylation patterns, and sarcoplasmic reticulum (SR) Ca²⁺ release. We found that RyR cluster disruption results in a decrease in spark fidelity and longer sparks with a lower amplitude. Phosphorylation of some RyRs within the cluster can play a compensatory role, recovering healthy spark dynamics. Interestingly, our model predicts that such compensation is critically dependent on the phosphorylation pattern, as phosphorylation localized within the cluster center resulted in longer Ca²⁺ sparks and higher spark fidelity compared to a uniformly distributed phosphorylation pattern. Our results strongly suggest that both the phosphorylation pattern and nanoscale RyR reorganization are critical determinants of Ca²⁺ dynamics in HF.     

Functional Coupling of Ryanodine Receptors to KCa Channels in Smooth Muscle Cells from Rat Cerebral Arteries

The relationship between Ca²⁺ release (“Ca²⁺ sparks”) through ryanodine-sensitive Ca²⁺ release channels in the sarcoplasmic reticulum and K_Ca channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca²⁺ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca²⁺ indicator, fluo-3. Virtually all (96%) detectable Ca²⁺ sparks were associated with the activation of a spontaneous transient outward current (STOC) through K_Ca channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca²⁺ spark. The amplitudes of the Ca²⁺ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca²⁺ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca²⁺ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in K_Ca channel open probability (>10⁴-fold) during a Ca²⁺ spark is consistent with local Ca²⁺ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/F_o) measured during a Ca²⁺ spark (mean 2.04 F/F_o or ∼310 nM Ca²⁺) appears to significantly underestimate the local Ca²⁺ that activates K_Ca channels. These results indicate that the majority of ryanodine receptors that cause Ca²⁺ sparks are functionally coupled to K_Ca channels in the surface membrane, providing direct support for the idea that Ca²⁺ sparks cause STOCs.     

Automatic Detection and Classification of Ca2+ Release Events in Line- and Frame-Scan Images

Analysis of Ca²⁺ signals obtained in various cell types (i.e., cardiomyocytes) is always a tradeoff between acquisition speed and signal/noise ratio of the fluorescence signal. This becomes especially apparent during fast two- or three-dimensional confocal imaging when local intracellular fluorescence signals originating from Ca²⁺ release from intracellular Ca²⁺ stores (e.g., sarcoplasmic reticulum) need to be examined. Mathematical methods have been developed to remedy a high noise level by fitting each pixel with a transient function to “denoise” the image. So far, current available analytical approaches have been impaired by a number of constraints (e.g., inability to fit local, concurrent, and consecutive events) and the limited ability to customize implementation. Here, we suggest a, to our knowledge, novel approach for detailed analysis of subcellular micro-Ca²⁺ events based on pixel-by-pixel denoising of confocal frame- and line-scan images. The algorithm enables spatiotemporally overlapping events (e.g., a Ca²⁺ spark occurring during the decaying phase of a Ca²⁺ wave) to be extracted so that various types of Ca²⁺ events can be detected at a pixel time level of precision. The method allows a nonconstant baseline to be estimated for each pixel, foregoing the need to subtract fluorescence background or apply self-ratio methods before image analysis. Furthermore, by using a clustering algorithm, identified single-pixel events are grouped into “physiologically relevant” Ca²⁺ signaling events spanning multiple pixels (sparks, waves, puffs, transients, etc.), from which spatiotemporal event parameters (e.g., full duration at half maximal amplitude, full width at half maximal amplitude, amplitude, wave speed, rise, and decay times) can be easily extracted. The method was implemented with cross-platform open source software, providing a comprehensive and easy-to-use graphical user interface enabling rapid line-scan images and rapid frame-scan image sequences (up to 150 frames/s) to be analyzed and repetitive Ca²⁺ events (Ca²⁺ sparks and Ca²⁺ puffs) originating from clusters of Ca²⁺ release channels located in the sarcoplasmic reticulum membrane (ryanodine receptors and inositol 1,4,5-trisphosphate receptors) of isolated cardiomyocytes to be examined with a high level of precision.     

Time Course of Individual Ca2+ Sparks in Frog Skeletal Muscle Recorded at High Time Resolution

Discrete Ca²⁺ release events (Ca²⁺ “sparks”) were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 μs per line). Fibers loaded with the Ca²⁺ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at −90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca²⁺ sparks at ∼30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5–2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 ± 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca²⁺ ions are being released by the sarcoplasmic reticulum Ca²⁺ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca²⁺ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.     

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca²⁺) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca²⁺ from the sarcoplasmic reticulum (SR). The Ca²⁺ leak out of the SR is an important process for cellular Ca²⁺ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca²⁺ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca²⁺ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca²⁺ sparks and robust Ca²⁺ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca²⁺ spark and nonspark-based SR Ca²⁺ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca²⁺-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca²⁺ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca²⁺ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca²⁺ release properties due to variations in inter-RyR coupling via local subspace Ca²⁺ concentration ([Ca²⁺]_ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.