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No Role for the Ryanodine Receptor in Regulating Cardiac Contraction?
Abstract
Cardiac contraction is initiated by Ca(2+) leaving the sarcoplasmic reticulum through the ryanodine receptor (RyR). Although opening of the RyR can be modified by various ligands, these have no maintained effect on contraction. We conclude that modulation of the RyR controls sarcoplasmic reticulum Ca(2+) content rather than cytoplasmic Ca(2+) concentration.
... Thus, there is a dichotomy in their results. It has also been shown that altered RyR Ca 2+ sensitivity can only cause transient (not steady-state) inotropy (Eisner & Trafford, 2000;Lukyanenko et al. 2001). This raises the question of whether OUA-induced alteration of RyR sensitivity could produce sustained inotropy. ...
... It has been demonstrated experimentally and theoretically that simple RyR sensitization (or desensitization) to Ca 2+ , which could increase P open in bilayer studies, can only cause transient inotropic effects in myocytes (Eisner & Trafford, 2000;Lukyanenko et al. 2001;Shannon et al. 2005). Acute RyR sensitization causes an initially larger SR Ca 2+ release, but more Ca 2+ is extruded from the cell (via NCX) and the SR Ca 2+ content decreases. ...
Glycoside-induced cardiac inotropy has traditionally been attributed to direct Na(+)-K(+)-ATPase inhibition, causing increased intracellular [Na(+)] and consequent Ca(2+) gain via the Na(+)-Ca(2+) exchanger (NCX). However, recent studies suggested alternative mechanisms of glycoside-induced inotropy: (1) direct activation of sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors; RyRs); (2) increased Ca(2+) selectivity of Na(+) channels (slip-mode conductance); and (3) other signal transduction pathways. None of these proposed mechanisms requires NCX or an altered [Na(+)] gradient. Here we tested the ability of ouabain (OUA, 3 microm), digoxin (DIG, 20 microm) or acetylstrophanthidin (ACS, 4 microm) to alter Ca(2+) transients in completely Na(+)-free conditions in intact ferret and cat ventricular myocytes. We also tested whether OUA directly activates RyRs in permeabilized cat myocytes (measuring Ca(2+) sparks by confocal microscopy). In intact ferret myocytes (stimulated at 0.2 Hz), DIG and ACS enhanced Ca(2+) transients and cell shortening during twitches, as expected. However, prior depletion of [Na(+)](i) (in Na(+)-free, Ca(2+)-free solution) and in Na(+)-free solution (replaced by Li(+)) the inotropic effects of DIG and ACS were completely prevented. In voltage-clamped cat myocytes, OUA increased Ca(2+) transients by 48 +/- 4% but OUA had no effect in Na(+)-depleted cells (replaced by N-methyl-d-glucamine). In permeabilized cat myocytes, OUA did not change Ca(2+) spark frequency, amplitude or spatial spread (although spark duration was slightly prolonged). We conclude that the acute inotropic effects of DIG, ACS and OUA (and the effects on RyRs) depend on the presence of Na(+) and a functional NCX in ferret and cat myocytes (rather than alternate Na(+)-independent mechanisms).
... 24 This is because, although an increase of L-type Ca current loads the cell and SR with Ca, it also triggers more release from the SR, thereby depleting it and the two effects are balanced. 60 Considering therefore the role of SERCA and the RyR during β-adrenergic stimulation, an effect attributable solely to the RyR is made less likely by the observation (Figure 3A) that increasing RyR po does not itself produce Ca waves in the steady state. It is still, however, necessary to distinguish between these alternatives. ...
Cardiac contraction is activated by an increase of intracellular calcium concentration ([Ca(2+)](i)), most of which comes from the sarcoplasmic reticulum (SR) where it is released, via the ryanodine receptor (RyR), in response to Ca(2+) entering the cell on the L-type Ca(2+) current. This phenomenon is termed Ca(2+)-induced Ca(2+) release (CICR). However, under certain circumstances, the SR can become overloaded with Ca(2+) and once a threshold SR Ca(2+) content is reached Ca(2+) is released spontaneously. Such spontaneous Ca(2+) release from the SR propagates as a Ca(2+) wave by CICR. Some of the Ca(2+) released during a wave is removed from the cell on the electrogenic Na - Ca exchanger resulting in depolarization. This is the cellular mechanism producing delayed afterdepolarizations and is common to those arrhythmias produced by digitalis toxicity and right ventricular outflow tract tachycardia. More recently it has been suggested that arrhythmogenic Ca(2+) waves can also occur if the properties of the RyR are altered, resulting in increase of RyR open probability, for example by phosphorylation. However, in this review experimental evidence will be presented to support the view that such arrhythmias still require a threshold SR Ca(2+) content to be exceeded and that this threshold is decreased by increasing RyR open probability.
... Thus the ability of glycoside to activate RyR2 and stimulate SR Ca 2ϩ release is a result of both direct and indirect actions on the RyR2; the action is direct because it occurs as the result of a highaffinity binding interaction to stimulate single-channel activity and indirect in that it also relies on the ability of NKA inhibition to increase SR Ca 2ϩ load. This mechanism, combining increased SR load (via NKA inhibition) with direct increase in SR release, could explain how the action of glycosides to increase release could contribute to a sustained positive inotropic action without depleting the SR (6). ...
This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.
... If there were an increase in SR gain as a result of s-nitrosylation of the RyR, we would expect the relative magnitude of the slow response to be reduced when the SR was non-functional. Furthermore, Eisner & Trafford (2000) have shown that enhanced RyR activity results in only transient changes in [Ca 2+ ] i and contraction, and for the temporal characteristics of the slow response to be fulfilled, there would have to be a concurrent increase in SR Ca 2+ loading to compensate for the increase in SR Ca 2+ release. This could occur through activation of NHE (and SACs, see below) and stimulation of reverse mode NCX activity. ...
We present the first direct comparison of the major candidates proposed to underlie the slow phase of the force increase seen following myocardial stretch: (i) the Na(+)-H(+) exchanger (NHE) (ii) nitric oxide (NO) and the ryanodine receptor (RyR) and (iii) the stretch-activated channel (SAC) in both single myocytes and multicellular muscle preparations from the rat heart. Ventricular myocytes were stretched by approximately 7% using carbon fibres. Papillary muscles were stretched from 88 to 98% of the length at which maximum tension is generated (L(max)). Inhibition of NHE with HOE 642 (5 microm) significantly reduced (P < 0.05) the magnitude of the slow force response in both muscle and myocytes. Neither inhibition of phosphatidylinositol-3-OH kinase (PtdIns-3-OH kinase) with LY294002 (10 microm) nor NO synthase with L-NAME (1 mm) reduced the slow force response in muscle or myocytes (P > 0.05), and the slow response was still present in the single myocyte when the sarcoplasmic reticulum was rigorously inhibited with 1 microm ryanodine and 1 microm thapsigargin. We saw a significant reduction (P < 0.05) in the slow force response in the presence of the SAC blocker streptomycin in both muscle (80 microm) and myocytes (40 microm). In fura 2-loaded myocytes, HOE 642 and streptomycin, but not L-NAME, ablated the stretch-induced increase in [Ca(2+)](i) transient amplitude. Our data suggest that in the rat, under our experimental conditions, there are two mechanisms that underlie the slow inotropic response to stretch: activation of NHE; and of activation of SACs. Both these mechanisms are intrinsic to the myocyte.
... Additionally, our data show no difference in the time to peak of the Ca 2ϩ transient in nNOS Ϫ/Ϫ and WT myocytes, suggestive of a lack of effect of nNOS-derived NO on the Ca 2ϩ release channel (Sears et al. 2003a). It should also be considered that an isolated effect of NO on the open probability of the RyR may have limited functional significance in regard to the long-term control of contraction, as such changes are thought to result in only transient effects on contraction (Eisner & Trafford 2000). ...
Nitric oxide (NO) has been shown to regulate cardiac function, both in physiological conditions and in disease states. However, several aspects of NO signalling in the myocardium remain poorly understood. It is becoming increasingly apparent that the disparate functions ascribed to NO result from its generation by different isoforms of the NO synthase (NOS) enzyme, the varying subcellular localization and regulation of NOS isoforms and their effector proteins. Some apparently contrasting findings may have arisen from the use of non-isoform-specific inhibitors of NOS, and from the assumption that NO donors may be able to mimic the actions of endogenously produced NO. In recent years an at least partial explanation for some of the disagreements, although by no means all, may be found from studies that have focused on the role of the neuronal NOS (nNOS) isoform. These data have shown a key role for nNOS in the control of basal and adrenergically stimulated cardiac contractility and in the autonomic control of heart rate. Whether or not the role of nNOS carries implications for cardiovascular disease remains an intriguing possibility requiring future study.
... We have argued previously [27,28] that the fact that large changes of Ca current have little effect on the SR content is physiologically important. It means that an abrupt increase of the L-type Ca current will produce an immediate increase of the amplitude of the systolic Ca transient without the delay that would be required if SR Ca had to increase. ...
Most of the calcium that activates contraction in the heart comes from the sarcoplasmic reticulum (SR) and it is therefore essential to control the SR Ca content. SR Ca content reflects the balance between uptake (via the SR Ca-ATPase, SERCA) and release, largely via the ryanodine receptor (RyR). Unwanted changes of SR Ca are prevented because, for example, an increase of SR Ca content increases the amplitude of the systolic Ca transient and this, in turn, results in increased loss of Ca from and decreased Ca entry into the cell thereby restoring cell and SR Ca towards control levels. We discuss the parameters that affect the steady level of SR Ca and how these may change in heart failure. Finally, we discuss disordered Ca regulation with particular emphasis on the condition of alternans where successive heartbeats alternate in amplitude. This behaviour can be explained by excessive feedback gain in the processes controlling SR Ca.
... It is therefore difficult to dissect whether the increase in the velocity of the fast SR Ca 2+ release described here has any significant contribution to the overall increase in intracellular Ca 2+ transient and contractility evoked by βAR stimulation. Experiments from Eisner's group394041 indicated that in conditions where the SR Ca 2+ content is not controlled, factors that alter the sensitivity of RyR2 to trigger Ca 2+ , as is the case of CaMKII-dependent phosphorylation, will have only transitory effects on the amplitude of Ca 2+ transients. This is because the increase in Ca 2+ transient amplitude evoked by the increase in Ca 2+ release due to RyR2 sensitization, decreases Ca 2+ influx via the L-type Ca 2+ channel and increases Ca 2+ efflux via NCX. ...
We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.
... It is likely that chemical modification of RyR2 favors Ca 2+ release in rat cardiac myocytes, since it has been demonstrated that RyR from canine cardiac muscle is activated by S-nitrosylation [32]. An increase in the activity of SERCA and L-type calcium channels is required to increase calcium transients, since the sole increase in RyR's open probability is not enough to keep an increase in calcium transients over time [33]. Interestingly, it has been described in the rabbit heart that the peroxynitrite donor SIN-1 and authentic NO caused Sglutathiolation in SERCA, increasing the activity of the pump [34]. ...
The role of nitric oxide (NO) in cardiac contractility is complex and controversial. Several NO donors have been reported to cause positive or negative inotropism. NO can bind to guanylate cyclase, increasing cGMP production and activating PKG. NO may also directly S-nitrosylate cysteine residues of specific proteins. We used the isolated rat heart preparation to test the hypothesis that the differential inotropic effects depend on the degree of NO production and the signaling recruited. SNAP (S-nitroso-N-acetylpenicillamine), a NO donor, increased contractility at 0.1, 1 and 10 microM. This effect was independent of phospholamban phosphorylation, was not affected by PKA inhibition with H-89 (N-[2((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide), but it was abolished by the radical scavenger Tempol (4-hydroxy-[2,2,4,4]-tetramethyl-piperidine-1-oxyl). However, at 100 microM SNAP reduced contractility, effect reversed to positive inotropism by guanylyl cyclase blockade with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and abolished by PKG inhibition with KT5823, but not affected by Tempol. SNAP increased tissue cGMP at 100 microM, but not at lower concentrations. Consistently, a cGMP analog also reduced cardiac contractility. Finally, SNAP at 1 microM increased the level of S-nitrosylation of various cardiac proteins, including the ryanodine receptor. This study demonstrates the biphasic role for NO in cardiac contractility in a given preparation; furthermore, the differential effect is clearly ascribed to the signaling pathways involved. We conclude that although NO is highly diffusible, its output determines the fate of the messenger: low NO concentrations activate redox processes (S-nitrosylation), increasing contractility; while the cGMP-PKG pathway is activated at high NO concentrations, reducing contractility.
... [23][24][25][26][27][28][29] In addition, the idea that changing the Ca 2+ -mediated open probability of RyR can cause a steady-state increase in the contractility of the normal heart is not consistent with many published studies. [30][31][32] A recent study by Benkusky et al (from a laboratory that is also involved in the present report) used a RyR-S2808A knock-in mouse, in which PKA cannot phosphorylate RyR at Ser2808, to critically test the RyR-Ser2808 phosphorylation hypothesis. 26 The results of this study did not support the idea that Ser2808 is involved in the physiological regulation of cardiac contractility. ...
The sympathetic nervous system is a critical regulator of cardiac function (heart rate and contractility) in health and disease. Sympathetic nervous system agonists bind to adrenergic receptors that are known to activate protein kinase A, which phosphorylates target proteins and enhances cardiac performance. Recently, it has been proposed that protein kinase A-mediated phosphorylation of the cardiac ryanodine receptor (the Ca(2+) release channel of the sarcoplasmic reticulum at a single residue, Ser2808) is a critical component of sympathetic nervous system regulation of cardiac function. This is a highly controversial hypothesis that has not been confirmed by several independent laboratories. The present study used a genetically modified mouse in which Ser2808 was replaced by alanine (S2808A) to prevent phosphorylation at this site. The effects of isoproterenol (a sympathetic agonist) on ventricular performance were compared in wild-type and S2808A hearts, both in vivo and in isolated hearts. Isoproterenol effects on L-type Ca(2+) current (I(CaL)), sarcoplasmic reticulum Ca(2+) release, and excitation-contraction coupling gain were also measured. Our results showed that isoproterenol caused significant increases in cardiac function, both in vivo and in isolated hearts, and there were no differences in these contractile effects in wild-type and S2808A hearts. Isoproterenol increased I(CaL), the amplitude of the Ca(2+) transient and excitation-contraction coupling gain, but, again, there were no significant differences between wild-type and S2808A myocytes. These results show that protein kinase A phosphorylation of ryanodine receptor Ser2808 does not have a major role in sympathetic nervous system regulation of normal cardiac function.
Cardiac contractility is regulated by changes in intracellular Ca concentration ([Ca²⁺]i). Normal function requires that [Ca²⁺]i be sufficiently high in systole and low in diastole. Much of the Ca needed for contraction comes from the sarcoplasmic reticulum and is released by the process of calcium-induced calcium release. The factors that regulate and fine-tune the initiation and termination of release are reviewed. The precise control of intracellular Ca cycling depends on the relationships between the various channels and pumps that are involved. We consider 2 aspects: (1) structural coupling: the transporters are organized within the dyad, linking the transverse tubule and sarcoplasmic reticulum and ensuring close proximity of Ca entry to sites of release. (2) Functional coupling: where the fluxes across all membranes must be balanced such that, in the steady state, Ca influx equals Ca efflux on every beat. The remainder of the review considers specific aspects of Ca signaling, including the role of Ca buffers, mitochondria, Ca leak, and regulation of diastolic [Ca²⁺]i.
Excitation-contraction coupling concerns the processes linking electrical excitation of the surface membrane to contraction. As shown in Fig. 1 A, the action potential produces a systolic increase of calcium that then activates contraction. This chapter will focus on the control of calcium and will largely ignore the contractile protein mechanisms that follow the increase of Ca. These events are described in detail in other chapters. Equally, on grounds of space, we will largely ignore the whole field of pharmacological modulation of e-c coupling. The reader is directed to the following books for recent summaries of the broad field of cardiac electrophysiology and contraction (Bers, 2001;Zipes & Jalife, 2000;Sperelakis et al., 2001). An overview of cellular Ca handling is shown in Fig. 1A.
Synchronized SR calcium (Ca) release is critical to normal cardiac myocyte excitation-contraction coupling, and ideally this release shuts off completely between heartbeats. However, other SR Ca release events are referred to collectively as SR Ca leak (which includes Ca sparks and waves as well as smaller events not detectable as Ca sparks). Much, but not all, of the SR Ca leak occurs via ryanodine receptors and can be exacerbated in pathological states such as heart failure. The extent of SR Ca leak is important because it can (a) reduce SR Ca available for release, causing systolic dysfunction; (b) elevate diastolic [Ca]i, contributing to diastolic dysfunction; (c) cause triggered arrhythmias; and (d) be energetically costly because of extra ATP used to repump Ca. This review addresses quantitative aspects and manifestations of SR Ca leak and its measurement, and how leak is modulated by Ca, associated proteins, and posttranslational modifications in health and disease. Expected final online publication date for the Annual Review of Physiology Volume 76 is February 10, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Diabetes Mellitus is characterised by fasting hyperglycemia and glucose intolerance, due to insulin deficiency, impaired effectiveness
of insulin action or both. There is clear evidence of the negative influence of both type 1 diabetes and type 2 diabetes on
the prevalence, severity and prognosis of cardiovascular disease. Cardiovascular disease represents the commonest cause of
morbidity and mortality within diabetic patients. Human and animal studies have shown that the excess risk of cardiovascular
complications cannot be explained by conventional cardiovascular risk factors alone and therefore, the diabetic state itself
is likely to account for this alteration in cardiac function. The cellular mechanisms associated with contractile dysfunction
and calcium mobilisation will be reviewed with respects to the streptozotocin-induced model of type 1 and type 2 diabetes
mellitus.
The present work investigated the underlying mechanism for the positive inotropic effect of liguzinediol (LZDO) in isolated rat hearts.
Isolated rat heart perfusion, intracellular action potential recording, patch clamp and Ca(2+) imaging were used to measure the isolated rat heart contractility, action potential duration, L-type Ca(2+) current and sarcoplasmic reticulum (SR) Ca(2+) transient in rat cardiomyocyte, respectively.
LZDO (1, 10, and 100μM) significantly enhanced the inotropy of isolated rat hearts, but not heart rates. Nimodipine (1μM, an L-type Ca(2+) channel antagonist), ruthenium red (5μM, a ryanodine receptor inhibitor) and thapsigargin (2μM, an irreversible SR Ca(2+) ATPase inhibitor) completely blocked the positive inotropic effect of LZDO. LZDO significantly enhanced the intracellular Ca(2+) transient in rat cardiomyocyte. However, LZDO (100μM) did not increase L-type Ca(2+) channel current. Moreover, LZDO (100μM) restored the depletion effect of caffeine on Ca(2+) transient. The following compounds also failed to block the positive inotropic effect of LZDO (100μM): β-AR antagonist (propranolol 1μM), phosphodiesterase (PDE) inhibitor (IBMX 5μM), Na(+)-K(+) ATPase inhibitor (ouabain 1μM), α(1)-AR antagonist (prazosin 1μM), dopamine D(1) receptor antagonist (SCH23390 1μM) and Na(+)-Ca(2+) exchange inhibitor (KB-R7943 1μM).
The positive inotropic effect of LZDO in isolated rat hearts was mediated through an elevation of SR Ca(2+) transient, which may act on SR Ca(2+) ATPase. LZDO has a unique biological mechanism that may prove effective in treating heart failure in clinic.
Abnormal behavior of the cardiac ryanodine receptor (RyR2) has been linked to cardiac arrhythmias and heart failure (HF) after myocardial infarction (MI). It has been proposed that protein kinase A (PKA) hyperphosphorylation of the RyR2 at a single residue, Ser-2808, is a critical mediator of RyR dysfunction, depressed cardiac performance, and HF after MI.
We used a mouse model (RyRS2808A) in which PKA hyperphosphorylation of the RyR2 at Ser-2808 is prevented to determine whether loss of PKA phosphorylation at this site averts post MI cardiac pump dysfunction.
MI was induced in wild-type (WT) and S2808A mice. Myocyte and cardiac function were compared in WT and S2808A animals before and after MI. The effects of the PKA activator Isoproterenol (Iso) on L-type Ca(2+) current (I(CaL)), contractions, and [Ca(2+)](I) transients were also measured. Both WT and S2808A mice had depressed pump function after MI, and there were no differences between groups. MI size was also identical in both groups. L type Ca(2+) current, contractions, Ca(2+) transients, and SR Ca(2+) load were also not significantly different in WT versus S2808A myocytes either before or after MI. Iso effects on Ca(2+) current, contraction, Ca(2+) transients, and SR Ca(2+) load were identical in WT and S2808A myocytes before and after MI at both low and high concentrations.
These results strongly support the idea that PKA phosphorylation of RyR-S2808 is irrelevant to the development of cardiac dysfunction after MI, at least in the mice used in this study.
In this article we review the role of the Ryanodine Receptor (RyR) in cardiac inotropy and arrhythmogenesis. Most of the calcium that activates cardiac contraction comes from the sarcoplasmic reticulum (SR) from where it is released through the RyR. The amplitude of the systolic Ca transient depends steeply on the SR Ca content and it is therefore important that SR content be regulated. This regulation occurs via changes of SR Ca content affecting systolic Ca and thence sarcolemmal Ca fluxes. In the steady state, the cardiac myocyte must be in Ca flux balance on each beat and this has implications for understanding even simple inotropic manoeuvres. The main part of the review considers the effects of modulating the RyR on systolic Ca. Potentiation of RyR opening produces an increase of the amplitude of the Ca transient but this effect disappears within a few beats because the increased sarcolemmal efflux of Ca decreases SR Ca content. We conclude that it is therefore unlikely that potentiation of the RyR by phosphorylation plays a dominant role in the actions of positive inotropic agents such as beta-adrenergic stimulation. Some cardiac arrhythmias result from release of Ca from the SR in the form of waves. This is best known to occur when the SR is overloaded with calcium. Mutations in the RyR also produce cardiac arrhythmias attributed to Ca waves due to leaky RyRs and a similar leak has been suggested to contribute to arrhythmias in heart failure. We show that, due to compensatory changes of SR Ca content, simply making the RyR leaky does not produce Ca waves in the steady state and that SR Ca content is critical in determining whether Ca waves occur.
The amount of Ca2+ released from the sarcoplasmic reticulum (SR) is a principal determinant of cardiac contractility. Normally, the SR Ca2+ stores are mobilized through the mechanism of Ca2+-induced Ca2+ release (CICR). In this process, Ca2+ enters the cell through plasmalemmal voltage-dependent Ca2+ channels to activate the Ca2+ release channels in the SR membrane. Consequently, the control of Ca2+ release by cytosolic Ca2+ has traditionally been the main focus of cardiac excitation-contraction (EC) coupling research. Evidence obtained recently suggests that SR Ca release is controlled not only by cytosolic Ca2+, but also by Ca2+ in the lumen of the SR. The presence of a luminal Ca2+ sensor regulating release of SR luminal Ca2+ potentially has profound implications for our understanding of EC coupling and intracellular Ca2+ cycling. Here we review evidence, obtained using in situ and in vitro approaches, in support of such a luminal Ca2+ sensor in cardiac muscle. We also discuss the role of control of Ca2+ release channels by luminal Ca2+ in termination and stabilization of CICR, as well as in shaping the response of cardiac myocytes to various inotropic influences and diseased states such as Ca2+ overload and heart failure.
The human heart proceeds from a relaxed state (diastole) to a fully contracted state (systole) and recovery in 600ms. During this period, Ca(2+) inside the myocardial cell rises from about 10nM to about 100nM and returns to the former. The contractile-relaxation cycle is tightly coupled to the Ca(2+)transient. In the normal physiological state, the autonomic nervous system (ANS) plays a major role in the regulation of cardiac function and important changes occur in diseases of the heart. Sympathetic overdrive is a major determinant of the critical transition from initial compensatory hypertrophy to decompensated failure. Cardiac myocytes from failing hearts are characterized by a number of abnormalities in excitation-contraction coupling, that are a direct consequence of beta-adrenergic signaling defects. Although desensitized in cardiac hypertrophy and failure, the beta-adrenergic signaling pathway retains receptor capacity, a characteristic that is used in therapeutic approaches. There are several putative Ca(2+)-dependent pathways that exert counterbalancing negative regulation over cAMP-dependent positive inotropic effect and may represent potential targets for contractile stimulation. This review is focused on the interactions between sympathetic drive and aspects of calcium signaling in the heart.
The cardiac sarcoplasmic reticulum calcium release channel, commonly referred to as the ryanodine receptor, is a key component in cardiac excitation-contraction coupling, where it is responsible for the release of calcium from the sarcoplasmic reticulum. As our knowledge of the ryanodine receptor has advanced an appreciation that this key E-C coupling component may have a role in the pathogenesis of human cardiac disease has emerged. Heart failure and arrhythmia generation are both pathophysiological states that can result from deranged excitation-contraction coupling. Evidence is now emerging that hyperphosphorylation of the cardiac ryanodine receptor is an important event in chronic heart failure, contributing to impaired contraction and the generation of triggered ventricular arrhythmias. Furthermore the therapeutic benefits of beta blockers in heart failure appear to be partly explained through a reversal of this phenomenon. Two rare inherited arrhythmogenic conditions, which can cause sudden death in children, have also been shown to result from mutations in the cardiac ryanodine receptor. These conditions, catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy (subtype 2), further implicate the ryanodine receptor as a potentially arrhythmogenic substrate and suggest that this channel may offer a new therapeutic target in the treatment of both cardiac arrhythmias and heart failure.
Stretch of the myocardium influences the shape and amplitude of the intracellular Ca(2+)([Ca(2+)](i)) transient. Under isometric conditions stretch immediately increases myofilament Ca(2+) sensitivity, increasing force production and abbreviating the time course of the [Ca(2+)](i) transient (the rapid response). Conversely, muscle shortening can prolong the Ca(2+) transient by decreasing myofilament Ca(2+) sensitivity. During the cardiac cycle, increased ventricular dilation may increase myofilament Ca(2+) sensitivity during diastolic filling and the isovolumic phase of systole, but enhance the decrease in myofilament Ca(2+) sensitivity during the systolic shortening of the ejection phase. If stretch is maintained there is a gradual increase in the amplitude of the Ca(2+) transient and force production, which takes several minutes to develop fully (the slow response). The rapid and slow responses have been reported in whole hearts and single myocytes. Here we review stretch-induced changes in [Ca(2+)](i) and the underlying mechanisms. Myocardial stretch also modifies electrical activity and the opening of stretch-activated channels (SACs) is often used to explain this effect. However, the myocardium has many ionic currents that are regulated by [Ca(2+)](i) and in this review we discuss how stretch-induced changes in [Ca(2+)](i) can influence electrical activity via the modulation of these Ca(2+)-dependent currents. Our recent work in single ventricular myocytes has shown that axial stretch prolongs the action potential. This effect is sensitive to either SAC blockade by streptomycin or the buffering of [Ca(2+)](i) with BAPTA, suggesting that both SACs and [Ca(2+)](i) are important for the full effects of axial stretch on electrical activity to develop.
Cardiac Ca(2+) transients are enhanced by cAMP-dependent protein kinase (PKA). However, PKA-dependent modulation of ryanodine receptor (RyR) function in intact cells is difficult to measure, because PKA simultaneously increases Ca(2+) current (I(Ca)), SR Ca(2+) uptake and SR Ca(2+) loading (which independently increase SR Ca(2+) release). We measured I(Ca) and SR Ca(2+) release +/- 1 microm isoproterenol (ISO; isoprenaline) in voltage-clamped ventricular myocytes of rabbits and transgenic mice (expressing only non-phosphorylatable phospholamban). This mouse model helps control for any effect of ISO-enhanced SR uptake on observed release, but the two species produced essentially identical results. SR Ca(2+) load and I(Ca) were adjusted by conditioning. We thus evaluated PKA effects on SR Ca(2+) release at constant SR Ca(2+) load and I(Ca) trigger (with constant unitary I(Ca)). The amount of SR Ca(2+) release increased as a function of either I(Ca) or SR Ca(2+) load, but ISO did not alter the relationships (measured as gain or fractional release). This was true over a wide range of SR Ca(2+) load and I(Ca). However, the maximal rate of SR Ca(2+) release was approximately 50% faster with ISO (at most loads and I(Ca) levels). We conclude that the isolated effect of PKA on SR Ca(2+) release is an increase in maximal rate of release and faster turn-off of release (such that integrated SR Ca(2+) release is unchanged). The increased amount of SR Ca(2+) release normally seen with ISO depends primarily on increased I(Ca) trigger and SR Ca(2+) load, whereas faster release kinetics may be the main result of RyR phosphorylation.
Myocardial calcium signalling is a vital component of the normal physiological function of the heart. Key amongst the many roles calcium plays is its use as the primary signalling component of excitation-contraction coupling, the intracellular process that links cardiomyocyte depolarisation to contraction. Defective cellular calcium handling, due to abnormalities of the various components which mediate and control excitation-contraction coupling, is widely recognised as a significant patho-physiological event in the contractile dysfunction of the failing heart. In addition, similar defects also appear to be increasingly recognised as mediators of certain forms of cardiac arrhythmias. Such defects include single gene defects in excitation-contraction coupling components that lead to inherited sudden death arrhythmia syndromes. Alternatively, arrhythmogenesis occurring within the context of acquired cardiac disease, in particular heart failure, also appears to be highly dependent on abnormal calcium homeostasis. In this article we review the defects in cardiomyocyte calcium homeostasis that lead to particular pro-arrhythmogenic phenomena and discuss recent insights gained into a variety of inherited and acquired arrhythmia syndromes that appear to involve defective calcium signalling as a central component of their patho-physiology. Potential opportunities for new anti arrhythmic therapeutic strategies based on these recent insights are also discussed.
Cardiac excitation-contraction coupling is initialized by the release of Ca from the sarcoplasmic reticulum (SR) in response to a sudden increase in local cytosolic [Ca] ([Ca]i) within the junctional cleft. We have tested the hypothesis that functional ryanodine receptor (RyR) regulation plays a major role in the regulation of myocyte Ca. A mathematical model with unique characteristics was used to simulate Ca homeostasis. Specifically, the model was designed to accurately represent the SR [Ca]-dependence of release from a variety of experimentally produced data sets. The simulated data for altered RyR Ca sensitivity demonstrated a regulatory feedback loop that resulted in the same release at lower [Ca]SR. This suggests that the primary role of myocyte RyR regulation may be to decrease SR [Ca] without decreasing the size of the [Ca]i transient. The model results suggest that this action moderates the increased SR [Ca] observed with adrenergic stimulation and may keep the [Ca]SR below the threshold for delayed afterdepolarizations and arrhythmia. However, increased Ca affinity of the RyR increased the probability of delayed afterdepolarizations when heart failure was simulated. We conclude that RyR regulation may play a role in preventing arrhythmias in healthy myocytes but that the same regulation may have the opposite effect in chronic heart failure.
This is a concise review of important calcium-transporters on the sarcolemma and organellar membranes of myocardial cells, and their functional roles in cell physiology. It briefly addresses L and T type calcium channels, store-operated calcium channel (SOC), sodium-calcium exchanger (NCX), and the plasma membrane calcium ATPase (PMCA) on the sarcolemma, ryanodine receptor (RyR), IP3 receptor (IP3R) and the sarcoplasmic reticulum (SR) calcium ATPase (SAERCA) on the SR membrane and their contributions to contraction and rhythm-generation. Several agonists and blockers for every transporter that are commonly used in research, and those with therapeutic applications have also been discussed.
The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect.
The major effect of Na/Ca exchange (NCX) on the systolic Ca transient is secondary to its effect on the Ca content of the sarcoplasmic reticulum (SR). SR Ca content is controlled by a mechanism in which an increase of SR Ca produces an increase in the amplitude of the systolic Ca transient. This, in turn, increases Ca efflux on NCX as well as decreasing entry on the L-type current resulting in a decrease of both cell and SR Ca content. This control mechanism also changes the response to other maneuvers that affect excitation-contraction coupling. For example, potentiating the opening of the SR Ca release channel (ryanodine receptor, RyR) with caffeine produces an immediate increase in the amplitude of the systolic Ca transient. However, this increases efflux of Ca from the cell on NCX and then decreases SR Ca content until a new steady state is reached. Changing the activity of NCX (by decreasing external Na) changes the level of SR Ca reached by this mechanism. If the cell and SR are overloaded with Ca then Ca waves appear during diastole. These waves activate the electrogenic NCX and thereby produce arrhythmogenic-delayed afterdepolarizations. A major challenge is how to remove this arrhythmogenic Ca release without compromising the normal systolic release. We have found that application of tetracaine to decrease RyR opening can abolish diastolic release while simultaneously potentiating the systolic release.
Calcium-mediated cross-signaling between the dihydropyridine (DHP) receptor, ryanodine receptor, and Na(+)-Ca2+ exchanger was examined in single rat ventricular myocytes where the diffusion distance of Ca2+ was limited to < 50 nm by dialysis with high concentrations of Ca2+ buffers. Dialysis of the cell with 2 mM Ca(2+)- indicator dye, Fura-2, or 2 mM Fura-2 plus 14 mM EGTA decreased the magnitude of ICa-triggered intracellular Ca2+ transients (Cai-transients) from 500 to 20-100 nM and completely abolished contraction, even though the amount of Ca2+ released from the sarcoplasmic reticulum remained constant (approximately 140 microM). Inactivation kinetics of ICa in highly Ca(2+)-buffered cells was retarded when Ca2+ stores of the sarcoplasmic reticulum (SR) were depleted by caffeine applied 500 ms before activation of ICa, while inactivation was accelerated if caffeine-induced release coincided with the activation of ICa. Quantitative analysis of these data indicate that the rate of inactivation of ICa was linearly related to SR Ca(2+)-release and reduced by > 67% when release was absent. Thapsigargin, abolishing SR release, suppressed the effect of caffeine on the inactivation kinetics of ICa. Caffeine-triggered Ca(2+)-release, in the absence of Ca2+ entry through the Ca2+ channel (using Ba2+ as a charge carrier), caused rapid inactivation of the slowly decaying Ba2+ current. Since Ba2+ does not release Ca2+ but binds to Fura-2, it was possible to calibrate the fluorescence signals in terms of equivalent cation charge. Using this procedure, the amplification factor of ICa-induced Ca2+ release was found to be 17.6 +/- 1.1 (n = 4). The Na(+)-Ca2+ exchange current, activated by caffeine-induced Ca2+ release, was measured consistently in myocytes dialyzed with 0.2 but not with 2 mM Fura-2. Our results quantify Ca2+ signaling in cardiomyocytes and suggest the existence of a Ca2+ microdomain which includes the DHP/ ryanodine receptors complex, but excludes the Na(+)-Ca2+ exchanger. This microdomain appears to be fairly inaccessible to high concentrations of Ca2+ buffers.
This review discusses the mechanism and regulation of Ca release from the cardiac sarcoplasmic reticulum. Ca is released through the Ca release channel or ryanodine receptor (RyR) by the process of calcium-induced Ca release (CICR). The trigger for this release is the L-type Ca current with a small contribution from Ca entry on the Na-Ca exchange. Recent work has shown that CICR is controlled at the level of small, local domains consisting of one or a small number of L-type Ca channels and associated RyRs. Ca efflux from the s.r. in one such unit is seen as a 'spark' and the properties of these sparks produce controlled Ca release from the s.r. A major factor controlling the amount of Ca released from the s.r. and therefore the magnitude of the systolic Ca transient is its Ca content. The Ca content depends on both the properties of the s.r. and the cytoplasmic Ca concentration. Changes of s.r. Ca content and the Ca released affect the sarcolemmal Ca and Na-Ca exchange currents and this acts to control cell Ca loading and the s.r. Ca content. The opening probability of the RyR can be regulated by various physiological mediators as well as pharmacological compounds. However, it is shown that, due to compensatory changes of s.r. Ca, modifiers of the RyR only produce transient effects on systolic Ca. We conclude that, although the RyR can be regulated, of much greater importance to the control of Ca efflux from the s.r. are effects due to changes of s.r. Ca content.
1. The effects of modulating Ca2+-induced Ca2+ release (CICR) in single cardiac myocytes were investigated using low concentrations of caffeine (< 500 microM) in reduced external Ca2+ (0.5 mM). Caffeine produced a transient potentiation of systolic [Ca2+]i (to 800 % of control) which decayed back to control levels. 2. Caffeine decreased the steady-state sarcoplasmic reticulum (SR) Ca2+ content. As the concentration of caffeine was increased, both the potentiation of the systolic Ca2+ transient and the decrease in SR Ca2+ content were increased. At higher concentrations, the potentiating effect decayed more rapidly but the rate of recovery on removal of caffeine was unaffected. 3. A simple model in which caffeine produces a fixed increase in the fraction of SR Ca2+ which is released could account qualitatively but not quantitatively for the above results. 4. The changes in total [Ca2+] during systole were obtained using measurements of the intracellular Ca2+ buffering power. Caffeine initially increased the fractional release of SR Ca2+. This was followed by a decrease to a level greater than that under control conditions. The fraction of systolic Ca2+ which was pumped out of the cell increased abruptly upon caffeine application but then recovered back to control levels. The increase in fractional loss is due to the fact that, as the cytoplasmic buffers become saturated, a given increase in systolic total [Ca2+] produces a larger increase in free [Ca2+] and thence of Ca2+ efflux. 5. These results confirm that modulation of the ryanodine receptor has no maintained effect on systolic Ca2+ and show the interdependence of SR Ca2+ content, cytoplasmic Ca2+ buffering and sarcolemmal Ca2+ fluxes. Such analysis is important for understanding the cellular basis of inotropic interventions in cardiac muscle.
Digital imaging of calcium indicator signals (fura-2 fluorescence) from single cardiac cells has revealed different subcellular patterns of cytoplasmic calcium ion concentration ([Ca2+]i) that are associated with different types of cellular appearance and behavior. In any population of enzymatically isolated rat heart cells, there are mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; spontaneously contracting cells, which have an increased [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous propagating waves of high [Ca2+]i; and cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types. The observed cellular and subcellular heterogeneity of [Ca2+]i in isolated cells indicates that experiments performed on suspensions of cells should be interpreted with caution. The spontaneous [Ca2+]i fluctuations previously observed without spatial resolution in multicellular preparations may actually be inhomogeneous at the subcellular level.
The release of sarcoplasmic reticulum (SR) Ca in cardiac muscle during excitation-contraction coupling is known to be graded by the amount of activating Ca outside the SR (i.e., Ca-induced Ca release). However, little is known about how intra-SR Ca affects the release process. In this study we assessed how the fractional SR Ca release as described by Bassani et al. [Am. J. Physiol. 265 (Cell Physiol. 34): C533-C540, 1993] is affected by alteration of trigger Ca and of SR Ca content. Experiments were done with isolated ferret ventricular myocytes using indo 1 to measure Ca concentration, perforated patch to measure Ca current (ICa), caffeine application to release SR Ca, and thapsigargin to completely block SR Ca uptake. For what we consider a Normal SR Ca load and trigger Ca [action potential at 0.5 Hz with 2 mM extracellular Ca concentration ([Ca]o)], 35 +/- 3% of the SR Ca content was released at a twitch. Changing trigger Ca by altering [Ca]o (to 0.5 and 8 mM) at a test twitch changed this fractional SR Ca release to 10 +/- 2 and 59 +/- 6%, with the same SR Ca load (and peak ICa changed in a parallel manner in separate voltage-clamp experiments). Three different levels of SR Ca load were studied (Low, Normal, and High; by action potential stimulation at different frequencies from 0.05 to 0.8 Hz) using the same standard test trigger Ca (2 mM). Surprisingly, the High-load condition only increased SR Ca content by approximately 4% but appeared to be very close to the limiting SR Ca capacity.(ABSTRACT TRUNCATED AT 250 WORDS)
A nystatin-perforated patch whole cell recording method was used to study the effects of acetylcholine (ACh) on ACh-induced K+ currents in atrial myocytes isolated from cat hearts. The general protocol involved an initial 4-min exposure to ACh (ACh1), followed by a 4-min washout in ACh-free Tyrode solution and then a second 4-min ACh exposure (ACh2). Voltage ramps (40 mV/s) between -130 and +30 mV were used to assess changes in total membrane conductance. ACh2 (10 microM) induced an increase in K+ conductance that was significantly larger than that induced by ACh1 (10 microM) at voltages both negative and positive to the reversal potential. The potentiated current induced by ACh2 reversed at about -80 mV and inwardly rectified at voltages positive to the reversal potential. External Ba2+ (5 mM) or tetraethylammonium (10 mM) abolished all ACh2-induced increases in membrane conductance. The sensitivity to K+ channel blockers, reversal potential, and the rectifying properties indicate that the current potentiated by ACh2 is a K+ current. Atropine (1 microM) blocked all effects of ACh on K+ currents. Potentiation of K+ current by ACh2 required 1) ACh1 concentrations > or = 1 microM, 2) ACh1 duration > or = 2 min, and 3) recovery interval > or = 2 min. We conclude that an initial exposure to ACh potentiates subsequent ACh-induced increases in K+ current. ACh-induced potentiation depends on the concentration and duration of the initial ACh exposure and the recovery interval between consecutive ACh exposures.(ABSTRACT TRUNCATED AT 250 WORDS)
In a variety of vertebrate and invertebrate tissues the ryanodine-sensitive Ca2+ channel is the pathway for Ca2+ release from intracellular stores. The mechanism for activation of the ryanodine receptor-channel complex appears to depend both on the ryanodine receptor isoform and the cell type. In addition, a complex combination of endogenous intracellular compounds regulates channel gating. In this article, Rebecca Sitsapesan, Stephen McGarry and Alan Williams review the mechanisms involved in cyclic ADP-ribose (cADPR)-induced Ca2+ release and discuss the likelihood that cADPR-activated Ca2+ release is mediated by one of the recognized isoforms of the ryanodine receptor-Ca2+ channel complex.
Cardiac hypertrophy and heart failure caused by high blood pressure were studied in single myocytes taken from hypertensive rats (Dahl SS/Jr) and SH-HF rats in heart failure. Confocal microscopy and patch-clamp methods were used to examine excitation-contraction (EC) coupling, and the relation between the plasma membrane calcium current (ICa) and evoked calcium release from the sarcoplasmic reticulum (SR), which was visualized as "calcium sparks." The ability of ICa to trigger calcium release from the SR in both hypertrophied and failing hearts was reduced. Because ICa density and SR calcium-release channels were normal, the defect appears to reside in a change in the relation between SR calcium-release channels and sarcolemmal calcium channels. beta-Adrenergic stimulation largely overcame the defect in hypertrophic but not failing heart cells. Thus, the same defect in EC coupling that develops during hypertrophy may contribute to heart failure when compensatory mechanisms fail.
[Ca2+]i was measured using the fluorescent indicator indo 1 in voltage-clamped ferret and rat ventricular myocytes. The Ca2+ content of the sarcoplasmic reticulum (SR) was estimated from the integral of the Na(+)-Ca2+ exchange current activated by caffeine. Refilling of the SR after caffeine removal was enhanced by stimulation. As the systolic Ca2+ transient recovered, the integral of the L-type Ca2+ current decreased and that of the Na(+)-Ca2+ exchange tail current increased. For the early pulses, the gain of Ca2+ via the Ca2+ current is greater than the loss via the exchanger, and during steady state stimulation, the fluxes are equal. The difference in the integrals gives a measure of the net gain of cell Ca2+ with each pulse. When these are summed, the calculated gain of cell Ca2+ agrees well with the increase of SR Ca2+ produced by stimulation, as measured from the caffeine-evoked currents. There was a nonlinear relationship between SR Ca2+ content and the magnitude of the systolic Ca2+ transient such that at high SR Ca2+ content a given increase of content had a greater effect on the Ca2+ transient than did an increase at low SR content. In conclusion, the effects of systolic Ca2+ on the Ca2+ current and Na(+)-Ca2+ exchange current provide a means to regulate SR Ca2+ content and thence the systolic Ca2+ transient.
Receptor-activated Ca2+ channels (RACCs) play a central role in regulation of the functions of animal cells. Together with voltage-operated Ca2+ channels (VOCCs) and ligand-gated non-selective cation channels, RACCs provide a variety of pathways by which Ca2+ can be delivered to the cytoplasmic space and the endoplasmic reticulum (ER) in order to initiate or maintain specific types of intracellular Ca2+ signal. Store-operated Ca2+ channels (SOCs), which are activated by a decrease in Ca2+ in the ER, are a major subfamily of RACCs. A careful analysis of the available data is required in order to discern the different types of RACCs (differentiated chiefly on the basis of ion selectivity and mechanism of activation) and to properly develop hypotheses for structures and mechanisms of activation. Despite much intensive research, the structures and mechanisms of activation of RACCs are only now beginning to be understood. In considering the physiological functions of the different RACCs, it is useful to consider the specificity for Ca2+ of each type of cation channel and the rate at which Ca2+ flows through a single open channel; the locations of the channels on the plasma membrane (in relation to the ER, cytoskeleton and other intracellular units of structure and function); the Ca2+-responsive enzymes and proteins; and the intracellular buffers and proteins that control the distribution of Ca2+ in the cytoplasmic space. RACCs which are non-selective cation channels can deliver Ca2+ directly to specific regions of the cytoplasmic space, and can also admit Na+, which induces depolarization of the plasma membrane, the opening of VOCCs and the subsequent inflow of Ca2+. SOCs appear to deliver Ca2+ specifically to the ER, thereby maintaining oscillating Ca2+ signals.
It is well established that most of the Ca2+ that activates contraction in mammalian heart is released from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyR) and that the RyR are themselves activated by Ca2+ in the mechanism known as “Ca2+ induced Ca2+ release” (CICR).1 Confocal imaging has made possible the visualization of localized Ca2+ release through RyR, in the form of Ca2+ sparks.2 It appears that Ca2+ sparks are triggered by a local [Ca2+]i,, which is different from the spatial average [Ca2+]i, and which is established first in the region of the RyR by the opening of a single L-type Ca2+ channel.3 4 These phenomena are the basis of the theory of excitation-contraction (E-C) coupling known as “local control,” which was predicted so presciently by Michael D. Stern in 1992.5 Nevertheless, the molecular mechanisms of Ca2+ sparks and the nature of the triggering by Ca2+ entry are still obscure. To complicate matters further, other possible sources of Ca2+ that activate, or “trigger,” this release have been proposed recently, and it has even been suggested that a voltage-sensitive release mechanism, which does not require Ca2+, may exist in cardiac muscle, similar to that in skeletal muscle.6 It is our intention here to review the evidence for local control of E-C coupling in normal heart muscle and to evaluate critically the evidence for additional sources of trigger Ca2+ or mechanisms of SR Ca2+ release. We emphasize, however, that concepts about cardiac Ca2+ sparks, and their possible role in cardiac E-C coupling, do not necessarily extend to Ca2+ sparks that occur in smooth muscle and skeletal muscle. Local Ca2+ release in …