In silico simulations reveal that RYR distribution affects the dynamics of calcium release in cardiac myocytes

The dyads of cardiac myocytes contain ryanodine receptors (RYRs) that generate calcium sparks upon activation. To test how geometric factors of RYR distribution contribute to the formation of calcium sparks, which cannot be addressed experimentally, we performed in silico simulations on a large set of models of calcium release sites (CRSs). Our models covered the observed range of RYR number, density, and spatial arrangement. The calcium release function of CRSs was modeled by RYR openings, with an open probability dependent on concentrations of free Ca²⁺ and Mg²⁺ ions, in a rapidly buffered system, with a constant open RYR calcium current. We found that simulations of spontaneous sparks by repeatedly opening one of the RYRs in a CRS produced three different types of calcium release events (CREs) in any of the models. Transformation of simulated CREs into fluorescence signals yielded calcium sparks with characteristics close to the observed ones. CRE occurrence varied broadly with the spatial distribution of RYRs in the CRS but did not consistently correlate with RYR number, surface density, or calcium current. However, it correlated with RYR coupling strength, defined as the weighted product of RYR vicinity and calcium current, so that CRE characteristics of all models followed the same state-response function. This finding revealed the synergy between structure and function of CRSs in shaping dyad function. Lastly, rearrangements of RYRs simulating hypothetical experiments on splitting and compaction of a dyad revealed an increased propensity to generate spontaneous sparks and an overall increase in calcium release in smaller and more compact dyads, thus underlying the importance and physiological role of RYR arrangement in cardiac myocytes.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

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.     

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.     

Modulation of the Frequency of Spontaneous Sarcoplasmic Reticulum Ca2+ Release Events (Ca2+ Sparks) by Myoplasmic [Mg2+] in Frog Skeletal Muscle

The modulation by internal free [Mg²⁺] of spontaneous calcium release events (Ca²⁺ “sparks”) from the sarcoplasmic reticulum (SR) was studied in depolarized notched frog skeletal muscle fibers using a laser scanning confocal microscope in line-scan mode (x vs. t). Over the range of [Mg²⁺] from 0.13 to 1.86 mM, decreasing the [Mg²⁺] induced an increase in the frequency of calcium release events in proportion to [Mg²⁺]^−1.6. The change of event frequency was not due to changes in [Mg-ATP] or [ATP]. Analysis of individual SR calcium release event properties showed that the variation in event frequency induced by the change of [Mg²⁺] was not accompanied by any changes in the spatiotemporal spread (i.e., spatial half width or temporal half duration) of Ca²⁺ sparks. The increase in event frequency also had no effect on the distribution of event amplitudes. Finally, the rise time of calcium sparks was independent of the [Mg²⁺], indicating that the open time of the SR channel or channels underlying spontaneous calcium release events was not altered by [Mg²⁺] over the range tested. These results suggest that in resting skeletal fibers, [Mg²⁺] modulates the SR calcium release channel opening frequency by modifying the average closed time of the channel without altering the open time. A kinetic reaction scheme consistent with our results and those of bilayer and SR vesicle experiments indicates that physiological levels of resting Mg²⁺ may inhibit channel opening by occupying the site for calcium activation of the SR calcium release channel.     

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.     

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.     

Luminal Mg2+, a key factor controlling RYR2-mediated Ca2+ release: cytoplasmic and luminal regulation modeled in a tetrameric channel

In cardiac muscle, intracellular Ca²⁺ and Mg²⁺ are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca²⁺] in the SR ([Ca²⁺]_L) stimulates the Ca²⁺ release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg²⁺, which has not been regarded as an important regulator of Ca²⁺ release.     

Sodium-Calcium Exchange Is Essential for Effective Triggering of Calcium Release in Mouse Heart

In cardiac myocytes, excitation-contraction coupling depends upon sarcoplasmic reticular Ca²⁺ release triggered by Ca²⁺ influx through L-type Ca²⁺ channels. Although Na⁺-Ca²⁺ exchange (NCX) is essential for Ca²⁺ extrusion, its participation in the trigger process of excitation-contraction coupling is controversial. To investigate the role of NCX in triggering, we examined Ca²⁺ sparks in ventricular cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice. Myocytes from young NCX KO mice are known to exhibit normal resting cytosolic Ca²⁺ and normal Ca²⁺ transients despite reduced L-type Ca²⁺ current. We loaded myocytes with fluo-3 to image Ca²⁺ sparks using confocal microscopy in line-scan mode. The frequency of spontaneous Ca²⁺ sparks was reduced in KO myocytes compared with WT. However, spark amplitude and width were increased in KO mice. Permeabilizing the myocytes with saponin eliminated differences between spontaneous sparks in WT and KO mice. These results suggest that sarcolemmal processes are responsible for the reduced spark frequency and increased spark width and amplitude in KO mice. When myocytes were loaded with 1 mM fluo-3 and 3 mM EGTA via the patch pipette to buffer diadic cleft Ca²⁺, the number of sparks triggered by action potentials was reduced by 60% in KO cells compared to WT cells, despite similar SR Ca²⁺ content in both cell types. When EGTA was omitted from the pipette solution, the number of sparks triggered in KO and WT myocytes was similar. Although the number of sparks was restored in KO cells, Ca²⁺ release was asynchronous. These results suggest that high subsarcolemmal Ca²⁺ is required to ensure synchronous triggering with short spark latency in the absence of NCX. In WT mice, high subsarcolemmal Ca²⁺ is not required for synchronous triggering, because NCX is capable of priming the diadic cleft with sufficient Ca²⁺ for normal triggering, even when subsarcolemmal Ca²⁺ is lowered by EGTA. Thus, reducing subsarcolemmal Ca²⁺ with EGTA in NCX KO mice reveals the dependence of Ca²⁺ release on NCX.     

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.     

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.     

Sarcoplasmic Reticulum Ca2+ Release in Rat Slow- and Fast-Twitch Muscles

The same isoform of ryanodine receptor (RYR1) is expressed in both fast and slow mammalian skeletal muscles. However, differences in contractile activation and calcium release kinetics in intact and skinned fibers have been reported. In this work, intracellular Ca²⁺ transients were measured in soleus and extensor digitorum longus (EDL) single muscle fibers using mag-fura-2 (K _D for Ca²⁺= 49 μm) as Ca²⁺ fluorescent indicator. Fibers were voltage-clamped at V _h =−90 mV and sarcoplasmic reticulum calcium release was measured at the peak (a) and at the end (b) of 200 msec pulses at +10 mV. Values of a-b and b were assumed to correspond to Ca²⁺-gated and voltage-gated Ca²⁺ release, respectively. Ratios (b/a-b) in soleus and EDL fibers were 0.41 ± 0.05 and 1.01 ± 0.13 (n= 12), respectively. This result suggested that the proportion of dihydropyridine receptor (DHPR)-linked and unlinked RYRs is different in soleus and EDL muscle. The number of DHPR and RYR were determined by measuring high-affinity [³H]PN200-110 and [³H]ryanodine binding in soleus and EDL rat muscle homogenates. The B _max values corresponded to a PN200-110/ryanodine binding ratio of 0.34 ± 0.05 and 0.92 ± 0.11 for soleus and EDL muscles (n= 4–8), respectively. These data suggest that soleus muscle has a larger calcium-gated calcium release component and a larger proportion of DHPR-unlinked RYRs. Received: 31 August 1995/Revised: 25 January 1996     

Comparison of the Effects Exerted by Luminal Ca2+ on the Sensitivity of the Cardiac Ryanodine Receptor to Caffeine and Cytosolic Ca2+

Ca²⁺ released from the sarcoplasmic reticulum (SR) via ryanodine receptor type 2 (RYR2) is the key determinant of cardiac contractility. Although activity of RYR2 channels is primary controlled by Ca²⁺ entry through the plasma membrane, there is growing evidence that Ca²⁺ in the lumen of the SR can also be effectively involved in the regulation of RYR2 channel function. In the present study, we investigated the effect of luminal Ca²⁺ on the response of RYR2 channels reconstituted into a planar lipid membrane to caffeine and Ca²⁺ added to the cytosolic side of the channel. We performed two sets of experiments when the channel was exposed to either luminal Ba²⁺ or Ca²⁺. The given ion served also as a charge carrier. Luminal Ca²⁺ effectively shifted the EC₅₀ for caffeine sensitivity to a lower concentration but did not modify the response of RYR2 channels to cytosolic Ca²⁺. Importantly, luminal Ca²⁺ exerted an effect on channel gating kinetics. Both the open and closed dwell times were considerably prolonged over the whole range (response to caffeine) or the partial range (response to cytosolic Ca²⁺) of open probability. Our results provide strong evidence that an alteration of the gating kinetics is the result of the interaction of luminal Ca²⁺ with the luminally located Ca²⁺ regulatory sites on the RYR2 channel complex.     

Magnesium Inhibition of Ryanodine-Receptor Calcium Channels: Evidence for Two Independent Mechanisms

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca²⁺ and Mg²⁺ concentrations. RyRs from skeletal and cardiac muscle are activated by μm Ca²⁺ and inhibited by mm Ca²⁺ and Mg²⁺. ⁴⁵Ca²⁺ release from skeletal SR vesicles suggests two mechanisms for Mg²⁺-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236–244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg²⁺- and Ca²⁺-dependent gating kinetics confirm that there are two mechanisms for Mg²⁺ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg²⁺ reduces P _o by competing with Ca²⁺ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca²⁺ and Mg²⁺ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg²⁺ effect depend on cytoplasmic [Ca²⁺] in such a way that Mg²⁺ inhibition has the properties of Types-I and II inhibition at low and high [Ca²⁺] respectively. Both mechanisms are equally important when [Ca²⁺] = 10 μm in cardiac RyRs or 1 μm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg²⁺ inhibition, as is often assumed, and we discuss the physiological implications of this finding. Received: 1 January 1996/Revised: 14 November 1996     

Dynamics of calcium sparks and calcium leak in the heart

We present what we believe to be a new mathematical model of Ca²⁺ leak from the sarcoplasmic reticulum (SR) in the heart. To our knowledge, it is the first to incorporate a realistic number of Ca²⁺-release units, each containing a cluster of stochastically gating Ca²⁺ channels (RyRs), whose biophysical properties (e.g., Ca²⁺ sensitivity and allosteric interactions) are informed by the latest molecular investigations. This realistic model allows for the detailed characterization of RyR Ca²⁺-release properties, and shows how this balances reuptake by the SR Ca²⁺ pump. Simulations reveal that SR Ca²⁺ leak consists of brief but frequent single RyR openings (∼3000 cell⁻¹ s⁻¹) that are likely to be experimentally undetectable, and are, therefore, “invisible”. We also observe that these single RyR openings can recruit additional RyRs to open, due to elevated local (Ca²⁺), and occasionally lead to the generation of Ca²⁺ sparks (∼130 cell⁻¹ s⁻¹). Furthermore, this physiological formulation of “invisible” leak allows for the removal of the ad hoc, non-RyR mediated Ca²⁺ leak terms present in prior models. Finally, our model shows how Ca²⁺ sparks can be robustly triggered and terminated under both normal and pathological conditions. Together, these discoveries profoundly influence how we interpret and understand diverse experimental and clinical results from both normal and diseased hearts.     

Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca(2+) sparks in airway myocytes

Short-lived, localized Ca(2+) events mediate Ca(2+) signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca(2+)-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca(2+) signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca(2+) signaling system composed of Ca(2+) sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca(2+)-activated K(+) (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca(2+) sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca(2+) spark, which activates ～15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca(2+)] resulting from Ca(2+) sparks, and the kinetic model of BK channels, we estimate that an average Ca(2+) spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ～600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca(2+) sparks using explicit knowledge of the spatial relationship between RYRs (the Ca(2+) source) and BK channels (the Ca(2+) target).     

Modification of cardiac RYR2 gating by a peptide from the central domain of the RYR2

The effect of a domain peptide DP_CPVTc from the central region of the RYR2 on ryanodine receptors from rat heart has been examined in planar lipid bilayers. At a zero holding potential and at 8 mmol L^?1 luminal Ca²⁺ concentration, DP_CPVTc induced concentrationdependent activation of the ryanodine receptor that led up to 20-fold increase of P_O at saturating DP_CPVTc concentrations. DP_CPVTc prolonged RyR2 openings and increased RyR2 opening frequency. At all peptide concentrations the channels displayed large variability in open probability, open time and frequency of openings. With increasing peptide concentration, the fraction of high open probability records increased together with their open time. The closed times of neither low- nor high-open probability records depended on peptide concentration. The concentration dependence of all gating parameters had EC₅₀ of 20 μmol L^?1 and a Hill slope of 2. Comparison of the effects of DP_CPVTc with the effects of ATP and cytosolic Ca²⁺ suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DP_CPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.     

The Orai1 inhibitor BTP2 has multiple effects on Ca2+ handling in skeletal muscle

BTP2 is an inhibitor of the Ca²⁺ channel Orai1, which mediates store-operated Ca²⁺ entry (SOCE). Despite having been extensively used in skeletal muscle, the effects of this inhibitor on Ca²⁺ handling in muscle cells have not been described. To address this question, we used intra- and extracellular application of BTP2 in mechanically skinned fibers and developed a localized modulator application approach, which provided in-preparation reference and test fiber sections to enhance detection of the effect of Ca²⁺ handling modulators. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of resting extracellular Ca²⁺ flux. Increasing concentrations of BTP2 caused a shift from inducing accumulation of Ca²⁺ in the t-system due to Orai1 blocking to reducing the resting [Ca²⁺] in the sealed t-system. This effect was not observed in the absence of functional ryanodine receptors (RYRs), suggesting that higher concentrations of BTP2 impair RYR function. Additionally, we found that BTP2 impaired action potential–induced Ca²⁺ release from the sarcoplasmic reticulum during repetitive stimulation without compromising the fiber Ca²⁺ content. BTP2 was found to have an effect on RYR-mediated Ca²⁺ release, suggesting that RYR is the point of BTP2-induced inhibition during cycles of EC coupling. The effects of BTP2 on the RYR Ca²⁺ leak and release were abolished by pre-exposure to saponin, indicating that the effects of BTP2 on the RYR are not direct and require a functional t-system. Our results demonstrate the presence of a SOCE channels–mediated basal Ca²⁺ influx in healthy muscle fibers and indicate that BTP2 has multiple effects on Ca²⁺ handling, including indirect effects on the activity of the RYR.     

Temperature acclimation has no effect on ryanodine receptor expression or subcellular localization in rainbow trout heart

In cardiomyocytes, ryanodine receptors (RYRs) mediate Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum (SR) during excitation–contraction (e–c) coupling. In rainbow trout heart, the relative importance of CICR increases with cold-acclimation. Thus, the aim of this study was to investigate the effect of temperature acclimation (4, 11 and 18°C) on RYR intracellular localization and expression density. We used immunocytochemistry to assess intracellular localization in ventricular myocytes and Western blotting to assess RYR expression in both atrial and ventricular tissue. In ventricular myocytes, RYRs were localized peripherally in transverse bands aligning with sarcomeric m-lines and centrally around mitochondria and the nucleus. Localization did not change with temperature acclimation. RYR expression was also unaffected by temperature acclimation. The localization of RYRs at the m-line is similar to neonatal mammalian cardiomyocytes. We suggest this positioning is indicative of myocytes which rely predominantly on transsarcolemmal Ca²⁺-influx, rather than CICR, during e–c coupling.     

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.