Analysis of cellular calcium fluxes in cardiac muscle to understand calcium homeostasis in the heart

Central to controlling intracellular calcium concentration ([Ca(2+)](i)) are a number of Ca(2+) transporters and channels with the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) being of particular note in the heart. This review concentrates on the regulation of [Ca(2+)](i) in cardiac muscle and the homeostatic mechanisms employed to ensure that the heart can operate under steady-state conditions on a beat by beat basis. To this end we discuss the relative importance of various sources and sinks of Ca(2+) responsible for initiating contraction and relaxation in cardiac myocytes and how these can be manipulated to regulate the Ca(2+) content of the major Ca(2+) store, the sarcoplasmic reticulum (SR). We will present a simple feedback system detailing how such control can be achieved and highlight how small perturbations to the steady-state operation of the feedback loop can be both beneficial physiologically and underlie changes in systolic Ca(2+) in ageing and heart disease. In addition to manipulating the amplitude of the normal systolic Ca(2+) transient, the tight regulation of SR Ca(2+) content is also required to prevent the abnormal, spontaneous or diastolic release of Ca(2+) from the SR. Such diastolic events are a major factor contributing to the genesis of cardiac arrhythmias in disease situations and in recently identified familial mutations in the SR Ca(2+) release channel (ryanodine receptor, RyR). How such diastolic release arises and potential mechanisms for controlling this will be discussed.     

Ginsenoside Re suppresses electromechanical alternans in cat and human cardiomyocytes

Ginseng botanicals are increasingly used as complementary or alternative medicines for a variety of cardiovascular diseases, yet little is known about their cellular actions in cardiac muscle. Electromechanical alternans (EMA) is a proarrhythmic cardiac abnormality that results from disturbances of intracellular Ca(2+) homeostasis. This study sought to determine whether a purified ginsenoside extract of ginseng, Re, exerts effects to suppress EMA and to gain insight into its mechanism of action. Alternans was induced by electrically pacing cardiomyocytes at room temperature. Re (> or = 10 nM) reversibly suppressed EMA recorded from cat ventricular and atrial myocytes and Langendorff-perfused cat hearts. In cat ventricular myocytes, Re reversibly suppressed intracellular Ca(2+) concentration ([Ca(2+)](i)) transient alternans. Re exerted no significant effects on baseline action potential configuration or sarcolemmal L-type Ca(2+) current (I(Ca,L)), Na(+) current, or total K(+) conductance. In human atrial myocytes, Re suppressed mechanical alternans and exerted no effect on I(Ca,L). In cat ventricular myocytes, Re increased [Ca(2+)](i) transient amplitude and decreased sarcoplasmic reticulum (SR) Ca(2+) content, resulting in an increase in fractional SR Ca(2+) release. In SR microsomes isolated from cat ventricles, Re had no effect on SR Ca(2+) uptake. Re increased the open probability of ryanodine receptors (RyRs), i.e., SR Ca(2+)-release channels, isolated from cat ventricles and incorporated into planar lipid bilayers. We concluded that ginsenoside Re suppresses EMA in cat atrial and ventricular myocytes, cat ventricular muscle, and human atrial myocytes. The effects of Re are not mediated via actions on sarcolemmal ion channels or action potential configuration. Re acts via a subcellular mechanism to enhance the opening of RyRs and thereby overcome the impaired SR Ca(2+) release underlying EMA.     

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.     

Expression and functional characterization of the cardiac muscle ryanodine receptor Ca(2+) release channel in Chinese hamster ovary cells.

To study the function and regulation of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel, we expressed the RyR2 proteins in a Chinese hamster ovary (CHO) cell line, and assayed its function by single channel current recording and confocal imaging of intracellular Ca(2+) ([Ca(2+)](i)). The 16-kb cDNA encoding the full-length RyR2 was introduced into CHO cells using lipofectAmine and electroporation methods. Incorporation of microsomal membrane vesicles isolated from these transfected cells into lipid bilayer membrane resulted in single Ca(2+) release channel activities similar to those of the native Ca(2+) release channels from rabbit cardiac muscle SR membranes, both in terms of gating kinetics, conductance, and ryanodine modification. The expressed RyR2 channels were found to exhibit more frequent transitions to subconductance states than the native RyR2 channels and RyR1 expressed in CHO cells. Caffeine, an exogenous activator of RyR, induced release of [Ca(2+)](i) from these cells. Confocal imaging of cells expressing RyR2 did not detect spontaneous or caffeine-induced local Ca(2+) release events (i.e., "Ca(2+) sparks") typically seen in cardiac muscle. Our data show that the RyR2 expressed in CHO cells forms functional Ca(2+) release channels. Furthermore, the lack of localized Ca(2+) release events in these cells suggests that Ca(2+) sparks observed in cardiac muscle may involve cooperative gating of a group of Ca(2+) release channels and/or their interaction with muscle-specific proteins.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.     

Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes

Cardiac alternans is a recognized risk factor for cardiac arrhythmia and sudden cardiac death. At the cellular level, Ca(2+) alternans appears as cytosolic Ca(2+) transients of alternating amplitude at regular beating frequency. Cardiac alternans is a multifactorial process but has been linked to disturbances in intracellular Ca(2+) regulation. In atrial myocytes, we tested the role of voltage-gated Ca(2+) current, sarcoplasmic reticulum (SR) Ca(2+) load, and restitution properties of SR Ca(2+) release for the occurrence of pacing-induced Ca(2+) alternans. Voltage-clamp experiments revealed that peak Ca(2+) current was not affected during alternans, and alternans of end-diastolic SR Ca(2+) load, evaluated by application of caffeine or measured directly with an intra-SR fluorescent Ca(2+) indicator (fluo-5N), were not a requirement for cytosolic Ca(2+) alternans. Restitution properties and kinetics of refractoriness of Ca(2+) release after activation during alternans were evaluated by four different approaches: measurements of 1) the delay (latency) of occurrence of spontaneous global Ca(2+) releases and 2) Ca(2+) spark frequency, both during rest after a large and small alternans Ca(2+) transient; 3) the magnitude of premature action potential-induced Ca(2+) transients after a large and small beat; and 4) the efficacy of a photolytically induced Ca(2+) signal (Ca(2+) uncaging from DM-nitrophen) to trigger additional Ca(2+) release during alternans. The results showed that the latency of global spontaneous Ca(2+) release was prolonged and Ca(2+) spark frequency was decreased after the large Ca(2+) transient during alternans. Furthermore, the restitution curve of the Ca(2+) transient elicited by premature action potentials or by photolysis-induced Ca(2+) release from the SR lagged behind after a large-amplitude transient during alternans compared with the small-amplitude transient. The data demonstrate that beat-to-beat alternation of the time-dependent restitution properties and refractory kinetics of the SR Ca(2+) release mechanism represents a key mechanism underlying cardiac alternans.     

Transmembrane redox sensor of ryanodine receptor complex

Inositol 1,4,5-trisphosphate receptors (IP(3)R) and ryanodine receptors (RyR) mediate the release of endoplasmic and sarcoplasmic reticulum (ER/SR) Ca(2+) stores and regulate Ca(2+) entry through voltage-dependent or ligand-gated channels of the plasma membrane. A prominent property of ER/SR Ca(2+) channels is exquisite sensitivity to sulfhydryl-modifying reagents. A plausible role for sulfhydryl chemistry in physiologic regulation of Ca(2+) release channels and the fidelity of Ca(2+) release from ER/SR is lacking. This study reveals the existence of a transmembrane redox sensor within the RyR1 channel complex that confers tight regulation of channel activity in response to changes in transmembrane redox potential produced by cytoplasmic and luminal glutathione. A transporter selective for glutathione is co-localized with RyR1 within the SR membrane to maintain local redox potential gradients consistent with redox regulation of ER/SR Ca(2+) release. Hyperreactive sulfhydryls previously shown to reside within the RyR1 complex (Liu, G., and Pessah, I. N. (1994) J. Biol. Chem. 269, 33028-33034) are an essential biochemical component of a transmembrane redox sensor. Transmembrane redox sensing may represent a fundamental mechanism by which ER/SR Ca(2+) channels respond to localized changes in transmembrane glutathione redox potential produced by physiologic and pathophysiologic modulators of Ca(2+) release from stores.     

Multiscale modeling of calcium cycling in cardiac ventricular myocyte: macroscopic consequences of microscopic dyadic function

In cardiac ventricular myocytes, calcium (Ca) release occurs at distinct structures (dyads) along t-tubules, where L-type Ca channels (LCCs) appose sarcoplasmic reticulum (SR) Ca release channels (RyR2s). We developed a model of the cardiac ventricular myocyte that simulates local stochastic Ca release processes. At the local Ca release level, the model reproduces Ca spark properties. At the whole-cell level, the model reproduces the action potential, Ca currents, and Ca transients. Changes in microscopic dyadic properties (e.g., during detubulation in heart failure) affect whole-cell behavior in complex ways, which we investigated by simulating changes in the dyadic volume and number of LCCs/RyR2s in the dyad, and effects of calsequestrin (CSQN) as a Ca buffer (CSQN buffer) or a luminal Ca sensor (CSQN regulator). We obtained the following results: 1), Increased dyadic volume and reduced LCCs/RyR2s decrease excitation-contraction coupling gain and cause asynchrony of SR Ca release, and interdyad coupling partially compensates for the reduced synchrony. 2), Impaired CSQN buffer depresses Ca transients without affecting the synchrony of SR Ca release. 3), When CSQN regulator function is impaired, interdyad coupling augments diastolic Ca release activity to form Ca waves and long-lasting Ca release events.     

RyRCa2+ leak limits cardiac Ca2+ window current overcoming the tonic effect of calmodulinin mice

Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.     

Mice null for calsequestrin 1 exhibit deficits in functional performance and sarcoplasmic reticulum calcium handling

In skeletal muscle, the release of calcium (Ca(2+)) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca(2+) release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca(2+) buffering as well as its potential for modulating RyR1, the L-type Ca(2+) channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca(2+)]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca(2+) content and SR Ca(2+) release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca(2+) release with single action potentials and a collapse of the Ca(2+) release with repetitive trains. Under voltage clamp, SR Ca(2+) release flux and total SR Ca(2+) release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca(2+) release flux appears to be solely due to elimination of the slowly decaying component of SR Ca(2+) release, whereas the rapidly decaying component of SR Ca(2+) release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca(2+)] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca(2+)](free) in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca(2+) buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca(2+) release.     

Organization of Ca2+ release units in excitable smooth muscle of the guinea-pig urinary bladder

Ca(2+) release from internal stores (sarcoplasmic reticulum or SR) in smooth muscles is initiated either via pharmaco-mechanical coupling due to the action of an agonist and involving IP3 receptors, or via excitation-contraction coupling, mostly involving L-type calcium channels in the plasmalemma (DHPRs), and ryanodine receptors (RyRs), or Ca(2+) release channels of the SR. This work focuses attention on the structural basis for the coupling between DHPRs and RyRs in phasic smooth muscle cells of the guinea-pig urinary bladder. Immunolabeling shows that two proteins of the SR: calsequestrin and the RyR, and one protein the plasmalemma, the L-type channel or DHPR, are colocalized with each other within numerous, peripherally located sites located within the caveolar domains. Electron microscopy images from thin sections and freeze-fracture replicas identify feet in small peripherally located SR vesicles containing calsequestrin and distinctive large particles clustered within small membrane areas. Both feet and particle clusters are located within caveolar domains. Correspondence between the location of feet and particle clusters and of RyR- and DHPR-positive foci allows the conclusion that calsequestrin, RyRs, and L-type Ca(2+) channels are associated with peripheral couplings, or Ca(2+) release units, constituting the key machinery involved in excitation-contraction coupling. Structural analogies between smooth and cardiac muscle excitation-contraction coupling complexes suggest a common basic mechanism of action.     

Excitation-contraction coupling in airway smooth muscle

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.     

Physiological and pathological modulation of ryanodine receptor function in cardiac muscle

Calcium release from the sarcoplasmic reticulum (SR) in cardiac muscle occurs through a specialised release channel, the ryanodine receptor, RyR, via the process of Ca-induced Ca release (CICR). The open probability of the RyR is increased by elevation of cytoplasmic Ca concentration ([Ca(2+)](i)). However, in addition to Ca, other modulators affect the RyR open probability. Agents which increase the RyR opening during systole produce a transient increase of systolic [Ca(2+)](i) followed by a return to the initial level due to a compensating decrease of SR Ca content. Increasing RyR opening during diastole decreases SR Ca content and thereby decreases systolic [Ca(2+)](i). We therefore conclude that potentiation of RyR opening will, if anything, decrease systolic [Ca(2+)](i). The effects of specific examples of modulators of the RyR, such as phosphorylation, metabolic changes, heart failure and polyunsaturated fatty acids, are discussed.     

Activation of cardiac ryanodine receptors by the calcium channel agonist FPL-64176

We investigated the possibility that the Ca(2+) channel agonist FPL-64176 (FPL) might also activate the cardiac sarcoplasmic reticulum (SR) Ca(2+) release channel ryanodine receptor (RyR). The effects of FPL were tested on single channel activity of purified and crude vesicular RyR (RyR2) isolated from human and dog hearts using the planar lipid bilayer technique. FPL (100-200 microM) increased single channel open probability (P(o)) when added to the cytoplasmic side of the channel (P(o) = 0.070 +/- 0.021 in control RyR2; 0.378 +/- 0.086 in 150 microM FPL, n = 9, P < 0.01) by prolonging open times and decreasing closed times without changing current magnitude. FPL had no effect on P(o) when added to the trans (luminal) side of the bilayer (P(o) = 0.079 +/- 0.036 in control and 0.103 +/- 0.066 in FPL, n = 4, no significant difference). The bell-shaped [Ca(2+)] dependence of [(3)H]ryanodine binding and of P(o) was altered by FPL, suggesting that the mechanism by which FPL increases channel activity is by an increase in Ca(2+)-induced activation at low [Ca(2+)] (without a change in threshold) and suppression of Ca(2+)-induced inactivation at high [Ca(2+)]. However, the fact that inactivation was restored at elevated [Ca(2+)] suggests a competitive interaction between Ca(2+) and FPL on inactivation. FPL had no effect on RyR skeletal channels (RyR1), where P(o) was 0.039 +/- 0.005 in control versus 0.030 +/- 0.006 in 150 microM FPL (no significant difference). These results suggest that, in addition to its ability to activate the L-type Ca(2+) channels, FPL activates cardiac RyR2 primarily by reducing the Ca(2+) sensitivity of inactivation.     

Defective Mg2+ regulation of RyR1 as a causal factor in malignant hyperthermia

In skeletal muscle, Mg(2+) exerts a dual inhibitory effect on RyR1, by competing with Ca(2+) at the activation site and binding to a low affinity Ca(2+)/Mg(2+) inhibitory site. Pharmacological activators of RyR1 must overcome the inhibitory action of Mg(2+) before Ca(2+) efflux can occur. In normal muscle, where the free [Mg(2+)](i) is approximately 1mM, even prolonged exposure to millimolar levels of volatile anesthetics does not initiate SR Ca(2+) release. However, when the cytosolic [Mg(2+)] is reduced below the physiological range, low levels of volatile anesthetic within the clinically relevant range (1mM) can initiate SR Ca(2+) release, in the form of a propagating Ca(2+) wave. In human muscle fibers from malignant hyperthermia susceptible patients, such Ca(2+) waves occur when 1mM halothane is applied at physiological [Mg(2+)](i). There is increasing evidence to suggest that defective Mg(2+) regulation of RyR1 confers susceptibility to malignant hyperthermia. At the molecular level, interactions between critical RyR1 subdomains may explain the clustering of RyR1 mutations and associated effects on Mg(2+) regulation.     

Oxidative stress-induced leaky sarcoplasmic reticulum underlying acute heart failure in severe burn trauma

Burn trauma causes cardiac dysfunction. However, much of the underlying cellular and molecular mechanisms remain elusive. In the present study, we demonstrate the roles of excessive sarcoplasmic reticulum (SR) Ca(2+) leakage and oxidative stress in burn-associated acute heart failure. In cardiomyocytes from failing rat hearts 12 h after full-thickness cutaneous burn of about 40% of the total body surface area, we found that Ca(2+) transients and contractility were impaired, but the triggering L-type Ca(2+) channel current density was unaltered, giving rise to a significantly reduced gain of excitation-contraction coupling. This deficiency in SR Ca(2+) release was accompanied by a reduction in Ca(2+) content in the SR. Surprisingly, the frequency of spontaneous Ca(2+) sparks was increased by 1.4-fold; Ca(2+) tolerance test (10 mM extracellular Ca(2+)) further showed 2.0- and 1.5-fold more frequent Ca(2+) waves and Ca(2+) sparks, respectively. Myofilament sensitivity to Ca(2+), however, seemed to be unaffected. These results suggest hyperactivity of the ryanodine receptor (RyR) Ca(2+) release channel and a leaky SR in burn. Importantly, pretreatment with antioxidant vitamins C and E seemed to prevent burn-induced RyR hypersensitivity and SR leakage and thereby normalize Ca(2+) transients and contractility. Concomitantly, the in vivo cardiac functions were also more tolerant of traumatic burn. Collectively, our findings suggest that SR leakage due to oxidative stress is likely a major candidate mechanism underlying burn-associated acute heart failure. Antioxidant therapy in burn trauma provides cardioprotection, at least in part, by protecting RyR's from oxidative stress-induced hypersensitivity.     

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.     

Dantrolene prevents arrhythmogenic Ca2+ release in heart failure

In heart failure (HF), arrhythmogenic Ca(2+) release and chronic Ca(2+) depletion of the sarcoplasmic reticulum (SR) arise due to altered function of the ryanodine receptor (RyR) SR Ca(2+)-release channel. Dantrolene, a therapeutic agent used to treat malignant hyperthermia associated with mutations of the skeletal muscle type 1 RyR (RyR1), has recently been suggested to have effects on the cardiac type 2 RyR (RyR2). In this investigation, we tested the hypothesis that dantrolene exerts antiarrhythmic and inotropic effects on HF ventricular myocytes by examining multiple aspects of intracellular Ca(2+) handling. In normal rabbit myocytes, dantrolene (1 μM) had no effect on SR Ca(2+) load, postrest decay of SR Ca(2+) content, the threshold for spontaneous Ca(2+) wave initiation (i.e., the SR Ca(2+) content at which spontaneous waves initiate) and Ca(2+) spark frequency. In cardiomyocytes from failing rabbit hearts, SR Ca(2+) load and the wave initiation threshold were decreased compared with normal myocytes, Ca(2+) spark frequency was increased, and the postrest decay was potentiated. Using a novel approach of measuring cytosolic and intra-SR Ca(2+) concentration (using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR), we showed that treatment of HF cardiomyocytes with dantrolene rescued postrest decay and increased the wave initiation threshold. Additionally, dantrolene decreased Ca(2+) spark frequency while increasing the SR Ca(2+) content in HF myocytes. These data suggest that dantrolene exerts antiarrhythmic effects and preserves inotropy in HF cardiomyocytes by decreasing the incidence of diastolic Ca(2+) sparks, increasing the intra-SR Ca(2+) threshold at which spontaneous Ca(2+) waves occur, and decreasing the loss of Ca(2+) from the SR. Furthermore, the observation that dantrolene reduces arrhythmogenicity while at the same time preserves inotropy suggests that dantrolene is a potentially useful drug in the treatment of arrhythmia associated with HF.