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.     

Effects of conformational peptide probe DP4 on bidirectional signaling between DHPR and RyR1 calcium channels in voltage-clamped skeletal muscle fibers

In skeletal muscle, excitation-contraction coupling involves the activation of dihydropyridine receptors (DHPR) and type-1 ryanodine receptors (RyR1) to produce depolarization-dependent sarcoplasmic reticulum Ca²⁺ release via orthograde signaling. Another form of DHPR-RyR1 communication is retrograde signaling, in which RyRs modulate the gating of DHPR. DP4 (domain peptide 4), is a peptide corresponding to residues Leu²⁴⁴²-Pro²⁴⁷⁷ of the central domain of the RyR1 that produces RyR1 channel destabilization. Here we explore the effects of DP4 on orthograde excitation-contraction coupling and retrograde RyR1-DHPR signaling in isolated murine muscle fibers. Intracellular dialysis of DP4 increased the peak amplitude of Ca²⁺ release during step depolarizations by 64% without affecting its voltage-dependence or kinetics, and also caused a similar increase in Ca²⁺ release during an action potential waveform. DP4 did not modify either the amplitude or the voltage-dependence of the intramembrane charge movement. However, DP4 augmented DHPR Ca²⁺ current density without affecting its voltage-dependence. Our results demonstrate that the conformational changes induced by DP4 regulate both orthograde E-C coupling and retrograde RyR1-DHPR signaling.     

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

Ca²⁺ spark is the elementary Ca²⁺ signaling event in muscle excitation-contraction coupling. The rise time of Ca²⁺ 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 Ca²⁺ 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.     

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.     

Oxidative Stress and Ca2+ Release Events in Mouse Cardiomyocytes

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca²⁺ signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca²⁺ release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca²⁺ through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca²⁺ signaling in situ, by analyzing Ca²⁺ sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca²⁺ spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca²⁺ release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca²⁺ depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca²⁺ release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca²⁺ release events.     

Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+-release Channel RyR1 by S-Palmitoylation

The ryanodine receptor/Ca²⁺-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca²⁺ release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation. S-Palmitoylation targets 18 Cys within the N-terminal, cytoplasmic region of RyR1, which are clustered in multiple functional domains including those implicated in the activity-governing protein-protein interactions of RyR1 with the L-type Ca²⁺ channel Ca_V1.1, calmodulin, and the FK506-binding protein FKBP12, as well as in “hot spot” regions containing sites of mutations implicated in malignant hyperthermia and central core disease. Eight of these Cys have been identified previously as subject to physiological S-nitrosylation or S-oxidation. Diminishing S-palmitoylation directly suppresses RyR1 activity as well as stimulus-coupled Ca²⁺ release through RyR1. These findings demonstrate functional regulation of RyR1 by a previously unreported post-translational modification and indicate the potential for extensive Cys-based signaling cross-talk. In addition, we identify the sarco/endoplasmic reticular Ca²⁺-ATPase 1A and the α_1S subunit of the L-type Ca²⁺ channel Ca_V1.1 as S-palmitoylated proteins, indicating that S-palmitoylation may regulate all principal governors of Ca²⁺ flux in skeletal muscle that mediates excitation-contraction 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.     

Regulation of the RyR channel gating by Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>

Ryanodine receptors (RyRs) are the Ca²⁺ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca²⁺ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca²⁺. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca²⁺ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca²⁺ at these sites to inhibit RyRs and this plays an important role in modulating their Ca²⁺-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca²⁺ and Mg²⁺ and how these mechanisms control Ca²⁺ release in excitation-contraction coupling and cardiac pacemaking.     

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.     

A Human Ventricular Myocyte Model with a Refined Representation of Excitation-Contraction Coupling

Cardiac Ca²⁺-induced Ca²⁺ release (CICR) occurs by a regenerative activation of ryanodine receptors (RyRs) within each Ca²⁺-releasing unit, triggered by the activation of L-type Ca²⁺ channels (LCCs). CICR is then terminated, most probably by depletion of Ca²⁺ in the junctional sarcoplasmic reticulum (SR). Hinch et al. previously developed a tightly coupled LCC-RyR mathematical model, known as the Hinch model, that enables simulations to deal with a variety of functional states of whole-cell populations of a Ca²⁺-releasing unit using a personal computer. In this study, we developed a membrane excitation-contraction model of the human ventricular myocyte, which we call the human ventricular cell (HuVEC) model. This model is a hybrid of the most recent HuVEC models and the Hinch model. We modified the Hinch model to reproduce the regenerative activation and termination of CICR. In particular, we removed the inactivated RyR state and separated the single step of RyR activation by LCCs into triggering and regenerative steps. More importantly, we included the experimental measurement of a transient rise in Ca²⁺ concentrations ([Ca²⁺], 10–15 μM) during CICR in the vicinity of Ca²⁺-releasing sites, and thereby calculated the effects of the local Ca²⁺ gradient on CICR as well as membrane excitation. This HuVEC model successfully reconstructed both membrane excitation and key properties of CICR. The time course of CICR evoked by an action potential was accounted for by autonomous changes in an instantaneous equilibrium open probability of couplons. This autonomous time course was driven by a core feedback loop including the pivotal local [Ca²⁺], influenced by a time-dependent decay in the SR Ca²⁺ content during CICR.     

Unique isoform-specific properties of calsequestrin in the heart and skeletal muscle

Calcium signaling in myocytes is dependent on the cardiac ryanodine receptor (RyR2) calcium release channel and the calcium buffering protein in the sarcoplasmic reticulum, cardiac calsequestrin (CSQ2). The overall properties of CSQ2 and its regulation of RyR2 have not been explored in detail or directly compared with skeletal CSQ1 and its regulation of the skeletal RyR1, with physiological ionic strength and Ca²⁺ concentrations. We find that there are major differences between the two isoforms under these physiological conditions. Ca²⁺ binding to CSQ2 is 50% lower than to CSQ1. Only ～30% of CSQ2 is bound to cardiac junctional face membrane (JFM), compared with ～70% of CSQ1 and the ratio of CSQ2 to RyR2 is only 50% of the CSQ1/RyR1 ratio. Chemical crosslinking shows that CSQ2 is mostly monomer/dimer, while CSQ1 is mostly polymerized. In single channel lipid bilayer experiments, CSQ2 monomers and/or dimers increase the open probability of both RyR1 and RyR2 channels, while CSQ1 polymers decrease the activity of RyR1. We speculate that CSQ2 facilitates high rates of Ca²⁺ release through RyR2 during systole, while CSQ1 curtails RyR1 opening in response to a single action potential to maintain Ca²⁺ and allow repeated Ca²⁺ release and graded activation with increased stimulation frequency.     

A calcium-induced calcium release mechanism mediated by calsequestrin

Calcium (Ca²⁺)-induced Ca²⁺ release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca²⁺ with ryanodine receptors (RyRs) and inducing Ca²⁺ release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca²⁺ may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca²⁺ buffer. We investigate how SR Ca²⁺ release via RyR is regulated by Ca²⁺ and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca²⁺ activation and inactivation, and one SR lumenal site for CSQ binding. The open probability (P_o) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca²⁺. Both peak and steady-state P_o effectively increase as SR lumenal Ca²⁺ increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca²⁺ loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca²⁺ release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca²⁺ release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca²⁺ release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes.     

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.     

Ca<Superscript>2+</Superscript>-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca<Superscript>2+</Superscript> Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca²⁺ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca²⁺. We show that hyperphosphorylation by exogenous PKA increases open probability (P _o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca²⁺; lowering [Ca²⁺] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca²⁺-sensitive mode of gating. We additionally found that cytosolic incubation with Mg²⁺/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca²⁺. We show that the endogenous kinase is both Ca²⁺-dependent and sensitive to inhibitors of PKC. Moreover, the Ca²⁺-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca²⁺-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca²⁺ regulation.     

Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca²⁺ concentration ([Ca²⁺]). Glucose uptake into β-cells promotes Ca²⁺ influx and reactive oxygen species (ROS) generation. In other cell types, Ca²⁺ and ROS jointly induce Ca²⁺ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca²⁺ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H₂O₂ in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H₂O₂-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca²⁺] produced by incubation of dissociated β-cells with H₂O₂. Addition of stimulatory glucose or H₂O₂ (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca²⁺ release, induced by the concomitant increases in [Ca²⁺] and ROS produced by stimulatory glucose, is an essential step in GSIS.     

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.     

Calcium signaling in insulin action on striated muscle

Striated muscles (skeletal and cardiac) are major physiological targets of insulin and this hormone triggers complex signaling pathways regulating cell growth and energy metabolism. Insulin increases glucose uptake into muscle cells by stimulating glucose transporter (GLUT4) translocation from intracellular compartments to the cell surface. The canonical insulin-triggered signaling cascade controlling this process is constituted by well-mapped tyrosine, lipid and serine/threonine phosphorylation reactions. In parallel to these signals, recent findings reveal insulin-dependent Ca²⁺ mobilization in skeletal muscle cells and cardiomyocytes. Specifically, insulin activates the sarco-endoplasmic reticulum (SER) channels that release Ca²⁺ into the cytosol i.e., the Ryanodine Receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP₃R). In skeletal muscle cells, a rapid, insulin-triggered Ca²⁺ release occurs through RyR, that is brought about upon S-glutathionylation of cysteine residues in the channel by reactive oxygen species (ROS) produced by the early activation of the NADPH oxidase (NOX2). In cardiomyocytes insulin induces a fast and transient increase in cytoplasmic [Ca²⁺]_i trough L-type Ca²⁺ channels activation. In both cell types, a relatively slower Ca²⁺ release also occurs through IP₃R activation, and is required for GLUT4 translocation and glucose uptake. The insulin-dependent Ca²⁺ released from IP₃R of skeletal muscle also promotes mitochondrial Ca²⁺ uptake. We review here these actions of insulin on intracellular Ca²⁺ channel activation and their impact on GLUT4 traffic in muscle cells, as well as other implications of insulin-dependent Ca²⁺ release from the SER.     

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).     

R4496C RyR2 mutation impairs atrial and ventricular contractility

Ryanodine receptor (RyR2) is the major Ca²⁺ channel of the cardiac sarcoplasmic reticulum (SR) and plays a crucial role in the generation of myocardial force. Changes in RyR2 gating properties and resulting increases in its open probability (P_o) are associated with Ca²⁺ leakage from the SR and arrhythmias; however, the effects of RyR2 dysfunction on myocardial contractility are unknown. Here, we investigated the possibility that a RyR2 mutation associated with catecholaminergic polymorphic ventricular tachycardia, R4496C, affects the contractile function of atrial and ventricular myocardium. We measured isometric twitch tension in left ventricular and atrial trabeculae from wild-type mice and heterozygous transgenic mice carrying the R4496C RyR2 mutation and found that twitch force was comparable under baseline conditions (30°C, 2 mM [Ca²⁺]_o, 1 Hz). However, the positive inotropic responses to high stimulation frequency, 0.1 µM isoproterenol, and 5 mM [Ca²⁺]_o were decreased in R4496C trabeculae, as was post-rest potentiation. We investigated the mechanisms underlying inotropic insufficiency in R4496C muscles in single ventricular myocytes. Under baseline conditions, the amplitude of the Ca²⁺ transient was normal, despite the reduced SR Ca²⁺ content. Under inotropic challenge, however, R4496C myocytes were unable to boost the amplitude of Ca²⁺ transients because they are incapable of properly increasing the amount of Ca²⁺ stored in the SR because of a larger SR Ca²⁺ leakage. Recovery of force in response to premature stimuli was faster in R4496C myocardium, despite the unchanged rates of recovery of L-type Ca²⁺ channel current (I_Ca-L) and SR Ca²⁺ content in single myocytes. A faster recovery from inactivation of the mutant R4496C channels could explain this behavior. In conclusion, changes in RyR2 channel gating associated with the R4496C mutation could be directly responsible for the alterations in both ventricular and atrial contractility. The increased RyR2 P_o and fractional Ca²⁺ release from the SR induced by the R4496C mutation preserves baseline contractility despite a slight decrease in SR Ca²⁺ content, but cannot compensate for the inability to increase SR Ca²⁺ content during inotropic challenge.     

Ca2+-induced Ca2+ Release in Chinese Hamster Ovary (CHO) Cells Co-expressing Dihydropyridine and Ryanodine Receptors

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca²⁺ release channel (in the membrane of internal Ca²⁺ stores) and the dihydropyridine receptor (DHPR)-Ca²⁺ channel (in the plasma membrane). To ensure expression of functional L-type Ca²⁺ channels, we expressed α₂, β, and γ DHPR subunits and a chimeric DHPR α₁ subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca²⁺ transient (CaT) in the absence of extracellular Ca²⁺ ([Ca²⁺]_o). However, in the presence of [Ca²⁺]_o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca²⁺]_i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca²⁺ influx through Ca²⁺ channels, whereas the second component resulted from Ca²⁺ release through the RyR expressed in the membrane of internal Ca²⁺ stores. However, the onset and the rate of Ca²⁺ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca²⁺ current. These results suggest that the skeletal muscle RyR isoform supports Ca²⁺-induced Ca²⁺ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.