Termination of cardiac Ca2+ sparks: role of intra-SR [Ca2+], release flux, and intra-SR Ca2+ diffusion

Ca(2+) release from cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) is regulated by dyadic cleft [Ca(2+)] and intra-SR free [Ca(2+)] ([Ca(2+)](SR)). Robust SR Ca(2+) release termination is important for stable excitation-contraction coupling, and partial [Ca(2+)](SR) depletion may contribute to release termination. Here, we investigated the regulation of SR Ca(2+) release termination of spontaneous local SR Ca(2+) release events (Ca(2+) sparks) by [Ca(2+)](SR), release flux, and intra-SR Ca(2+) diffusion. We simultaneously measured Ca(2+) sparks and Ca(2+) blinks (localized elementary [Ca(2+)](SR) depletions) in permeabilized ventricular cardiomyocytes over a wide range of SR Ca(2+) loads and release fluxes. Sparks terminated via a [Ca(2+)](SR)-dependent mechanism at a fixed [Ca(2+)](SR) depletion threshold independent of the initial [Ca(2+)](SR) and release flux. Ca(2+) blink recovery depended mainly on intra-SR Ca(2+) diffusion rather than SR Ca(2+) uptake. Therefore, the large variation in Ca(2+) blink recovery rates at different release sites occurred because of differences in the degree of release site interconnection within the SR network. When SR release flux was greatly reduced, long-lasting release events occurred from well-connected junctions. These junctions could sustain release because local SR Ca(2+) release and [Ca(2+)](SR) refilling reached a balance, preventing [Ca(2+)](SR) from depleting to the termination threshold. Prolonged release events eventually terminated at a steady [Ca(2+)](SR), indicative of a slower, [Ca(2+)](SR)-independent termination mechanism. These results demonstrate that there is high variability in local SR connectivity but that SR Ca(2+) release terminates at a fixed [Ca(2+)](SR) termination threshold. Thus, reliable SR Ca(2+) release termination depends on tight RyR regulation by [Ca(2+)](SR).     

Sarcoplasmic reticulum Ca2+ refilling controls recovery from Ca2+-induced Ca2+ release refractoriness in heart muscle

In cardiac muscle Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is initiated by Ca2+ influx via L-type Ca2+ channels. At present, the mechanisms underlying termination of SR Ca2+ release, which are required to ensure stable excitation-contraction coupling cycles, are not precisely known. However, the same mechanism leading to refractoriness of SR Ca2+ release could also be responsible for the termination of CICR. To examine the refractoriness of SR Ca2+ release, we analyzed Na+-Ca2+ exchange currents reflecting cytosolic Ca2+ signals induced by UV-laser flash-photolysis of caged Ca2+. Pairs of UV flashes were applied at various intervals to examine the time course of recovery from CICR refractoriness. In cardiomyocytes isolated from guinea-pigs and mice, beta-adrenergic stimulation with isoproterenol-accelerated recovery from refractoriness by approximately 2-fold. Application of cyclopiazonic acid at moderate concentrations (<10 micromol/L) slowed down recovery from refractoriness in a dose-dependent manner. Compared with cells from wild-type littermates, those from phospholamban knockout (PLB-KO) mice exhibited almost 5-fold accelerated recovery from refractoriness. Our results suggest that SR Ca2+ refilling mediated by the SR Ca2+-pump corresponds to the rate-limiting step for recovery from CICR refractoriness. Thus, the Ca2+ sensitivity of CICR appears to be regulated by SR Ca2+ content, possibly resulting from a change in the steady-state Ca2+ sensitivity and in the gating kinetics of the SR Ca2+ release channels (ryanodine receptors). During Ca2+ release, the concomitant reduction in Ca2+ sensitivity of the ryanodine receptors might also underlie Ca2+ spark termination by deactivation.     

Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease

The cardiac ryanodine receptor (RyR2) is the sarcoplasmic reticulum (SR) Ca(2+) release channel which is responsible for generation of the cytosolic Ca(2+) transient required for activation of cardiac contraction. RyR2 functional activity is governed by changes in [Ca(2+)] on both the cytosolic and luminal phase of the RyR2 channel. Activation of RyR2 by cytosolic Ca(2+) results in Ca(2+)-induced Ca(2+) release (CICR) from the SR. The decline in luminal [Ca(2+)] following release contributes to termination of CICR and Ca(2+) signalling refractoriness through the process of luminal Ca(2+)-dependent deactivation of RyR2s. The control of RyR2s by luminal Ca(2+) involves coordinated interaction of the channel with several SR proteins, including the Ca(2+)-binding protein calsequestrin (CASQ2), and the integral proteins triadin 1 (TRD) and junctin (JCN). CASQ2 in addition to serving as a Ca(2+) storage site and a luminal Ca(2+) buffer modulates RyR2 function more directly as a putative luminal Ca(2+) sensor. TRD and JCN, stimulatory by themselves, mediate the interactions between CASQ2 and RyR2. Acquired and genetic defects in proteins of this junctional Ca(2+) signalling complex lead to disease states such as cardiac arrhythmia and heart failure by impairing luminal Ca(2+) regulation of RyR2.     

Intra-sarcoplasmic reticulum free [Ca2+] and buffering in arrhythmogenic failing rabbit heart

Smaller Ca2+ transients and systolic dysfunction in heart failure (HF) can be largely explained by reduced total sarcoplasmic reticulum (SR) Ca2+ content ([Ca]SRT). However, it is unknown whether low [Ca]SRT is manifest as reduced: (1) intra-SR free [Ca2+] ([Ca2+]SR), (2) intra-SR Ca2+ buffering, or (3) SR volume (as percentage of cell volume). Here we assess these possibilities in a well-characterized rabbit model of nonischemic HF. In HF versus control myocytes, diastolic [Ca2+]SR is similar at 0.1-Hz stimulation, but the increase in both [Ca2+]SR and [Ca]SRT as frequency increases to 1 Hz is blunted in HF. Direct measurement of intra-SR Ca2+ buffering (by simultaneous [Ca2+]SR and [Ca]SRT measurement) showed no change in HF. Diastolic [Ca]SRT changes paralleled [Ca2+]SR, suggesting that SR volume is not appreciably altered in HF. Thus, reduced [Ca]SRT in HF is associated with comparably reduced [Ca2+]SR. Fractional [Ca2+]SR depletion increased progressively with stimulation frequency in control but was blunted in HF (consistent with the blunted force-frequency relationship in HF). By studying a range of [Ca2+]SR, analysis showed that for a given [Ca]SR, fractional SR Ca2+ release was actually higher in HF. For both control and HF myocytes, SR Ca2+ release terminated when [Ca2+]SR dropped to 0.3 to 0.5 mmol/L during systole, consistent with a role for declining [Ca2+]SR in the dynamic shutoff of SR Ca2+ release. We conclude that low total SR Ca2+ content in HF, and reduced SR Ca2+ release, is attributable to reduced [Ca2+]SR, not to alterations in SR volume or Ca2+ buffering capacity.     

Abnormal intrastore calcium signaling in chronic heart failure

Diminished Ca release from the sarcoplasmic reticulum (SR) is an important contributor to the impaired contractility of the failing heart. Despite extensive effort, the underlying causes of abnormal SR Ca release in heart failure (HF) remain unknown. We used a combination of simultaneous imaging of cytosolic and SR intraluminal [Ca] in isolated cardiomyocytes and recordings from single-ryanodine receptor (RyR) channels reconstituted into lipid bilayers to investigate alterations in intracellular Ca handling in an experimental model of chronic HF. We found that diastolic free [Ca] inside the SR was dramatically reduced because of a Ca leak across the SR membrane, mediated by spontaneous local release events (Ca sparks), in HF myocytes. Additionally, the magnitudes of intrastore Ca depletion signals during global and focal Ca release events were blunted, and [Ca]SR recovery was slowed after global but not focal Ca release in HF myocytes. At the single-RyR level, the sensitivity of RyRs to activation by luminal Ca was greatly enhanced, providing a molecular mechanism for the maintained potentiation of Ca sparks (and increased Ca leak) at reduced intra-SR [Ca] in HF. This work shows that the diminished SR Ca release characteristic of failing myocardium could be explained by increased sensitivity of RyRs to luminal Ca, leading to enhanced spark-mediated SR Ca leak and reduced intra-SR [Ca].     

Ca2+-handling in heart failure--a review focusing on Ca2+ sparks

[Ca2+]i-transients have been shown to be altered in isolated ventricular myocytes from terminally failing human myocardium. It has been demonstrated that one reason for this alteration is a reduction in the Ca2+ content of the sarcoplasmic reticulum (SR). Further investigations were done to investigate, whether there may be an additional defect of the Ca2+-release mechanisms from the SR. These release mechanisms were investigated through the recording of Ca2+ sparks in single human myocytes. In cardiac myocytes, Ca2+ sparks are elementary units of Ca2+ release, which occur spontaneously, or which are triggered by Ca2+ influx through L-type Ca2+-channels (Ca2+-induced Ca2+ release). Ca2+ sparks have been investigated in various animal models of cardiac hypertrophy and cardiac failure and results were conflicting. Discrepancies may be explained by different species and also by the mechanisms underlying hypertrophy and heart failure. This review summarizes our current knowledge on Ca2+ sparks in heart failure.     

Ca2+ channels, 'quantized' Ca2+ release, and differentiation of myocytes in the cardiovascular system

The application of confocal microscopy to cardiac and skeletal muscle has resulted in the observation of transient, spatially localized elevations in [Ca2+]i, termed 'Ca2+ sparks'. Ca2+ sparks are thought to represent 'elementary' Ca2+ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum. In cardiac muscle, Ca2+ sparks appear to be key elements of excitation-contraction coupling, in which the global [Ca2+]i transient is thought to involve the recruitment of Ca2+ sparks, each of which is controlled locally by single coassociated L-type Ca2+ channels. Recently, Ca2+ sparks have been detected in smooth muscle cells of arteries. In this review, we analyse the complex relationship of Ca2+ influx and Ca2+ release with local, subcellular Ca2+ microdomains in light of recent studies on Ca2+ sparks in cardiovascular cells. We performed a comparative analysis of 'elementary' Ca2+ release units in mouse, rat and human arterial smooth muscle cells, using measurements of Ca2+ sparks and plasmalemmal K(Ca) currents activated by Ca2+ sparks (STOCs). Furthermore, the appearance of Ca2+ sparks during ontogeny of arterial smooth muscle is explored. Using intact pressurized arteries, we have investigated whether RyRs causing Ca2+ sparks (but not smaller 'quantized' Ca2+ release events, e.g. hypothetical 'Ca2+ quarks') function as key signals that, through membrane potential and global cytoplasmic [Ca2+], oppose arterial myogenic tone and influence vasorelaxation. We believe that voltage-dependent Ca2+ channels and local RyR-related Ca2+ signals are important in differentiation, proliferation, and gene expression. Our findings suggest that 'elementary' Ca2+ release units may represent novel potent therapeutic targets for regulating function of intact arterial smooth muscle tissue.     

Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes

Ryanodine receptor (RyR) phosphorylation by protein kinase A (PKA) may be important in modulating resting sarcoplasmic reticulum (SR) Ca2+ release, especially in heart failure. However, clear cellular data on PKA-dependent modulation of cardiac RyRs is limited because of difficulty in distinguishing between PKA effects on RyR, phospholamban (PLB), and Ca2+ current. To clarify this, we measured resting Ca2+ sparks in streptolysin-O permeabilized ventricular myocytes from wild-type (WT) and PLB knockout (PLB-KO) mice and transgenic mice expressing only double-mutant PLB (PLB-DM) that lacks the regulatory phosphorylation sites (S16A/T17A). In WT myocytes, cAMP dramatically increased Ca2+ spark frequency (CaSpF) by 2- and 3-fold when [Ca2+] was clamped at 50 and 10 nmol/L (and the SR Ca2+ content also rose by 40% and 50%). However, in PLB-KO and PLB-DM, neither CaSpF nor SR Ca2+ load was changed by the addition of 10 micromol/L cAMP (even with phosphatase inhibition). PKA activation also increased Ca2+ spark amplitude, duration, and width in WT, but not in PLB-KO or PLB-DM. RyR phosphorylation was confirmed by measurements of 32P incorporation on immunoprecipitated RyR. In intact resting myocytes, PKA activation increased CaSpF 2.8-fold in WT, but not in PLB-KO, confirming results in permeabilized myocytes. We conclude that the PKA-dependent increase in myocyte CaSpF and size is entirely attributable to PLB phosphorylation and consequent enhanced SR Ca2+ load. PKA does not seem to have any appreciable effect on resting RyR function in these ventricular myocytes. Moreover, the data provide compelling evidence that elevated intra-SR [Ca2+] increases RyR gating independent of cytosolic [Ca2+] (which was clamped).     

Evidence for Ca(2+) activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex

We have used tryptic digestion to determine whether Ca(2+) can regulate cardiac ryanodine receptor (RyR) channel gating from within the lumen of the sarcoplasmic reticulum (SR) or whether Ca(2+) must first flow through the channel and act via cytosolically located binding sites. Cardiac RyRs were incorporated into bilayers, and trypsin was applied to the luminal side of the bilayer. We found that before exposure to luminal trypsin, the open probability of RyR was increased by raising the luminal [Ca(2+)] from 10 micromol/L to 1 mmol/L, whereas after luminal trypsin exposure, increasing the luminal [Ca(2+)] reduced the open probability. The modification in the response of RyRs to luminal Ca(2+) was not observed with heat-inactivated trypsin, indicating that digestion of luminal sites on the RyR channel complex was responsible. Our results provide strong evidence for the presence of luminally located Ca(2+) activation and inhibition sites and indicate that trypsin digestion leads to selective damage to luminal Ca(2+) activation sites without affecting luminal Ca(2+) inactivation sites. We suggest that changes in luminal [Ca(2+)] will be able to regulate RyR channel gating from within the SR lumen, therefore providing a second Ca(2+)-regulatory effect on RyR channel gating in addition to that of cytosolic Ca(2+). This luminal Ca(2+)-regulatory mechanism is likely to be an important contributing factor in the potentiation of SR Ca(2+) release that is observed in cardiac cells in response to increases in intra-SR [Ca(2+)].     

Transgenic CaMKIIdeltaC overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release

Ca2+/calmodulin-dependent protein kinase II (CaMKII) delta is the predominant cardiac isoform, and the deltaC splice variant is cytoplasmic. We overexpressed CaMKIIdeltaC in mouse heart and observed dilated heart failure and altered myocyte Ca2+ regulation in 3-month-old CaMKIIdeltaC transgenic mice (TG) versus wild-type littermates (WT). Heart/body weight ratio and cardiomyocyte size were increased about 2-fold in TG versus WT. At 1 Hz, twitch shortening, [Ca2+]i transient amplitude, and diastolic [Ca2+]i were all reduced by approximately 50% in TG versus WT. This is explained by >50% reduction in SR Ca2+ content in TG versus WT. Peak Ca2+ current (ICa) was slightly increased, and action potential duration was prolonged in TG versus WT. Despite lower SR Ca2+ load and diastolic [Ca2+]i, fractional SR Ca2+ release was increased and resting spontaneous SR Ca2+ release events (Ca2+ sparks) were doubled in frequency in TG versus WT (with prolonged width and duration, but lower amplitude). Enhanced Ca2+ spark frequency was also seen in TG at 4 weeks (before heart failure onset). Acute CaMKII inhibition normalized Ca2+ spark frequency and ICa, consistent with direct CaMKII activation of ryanodine receptors (and ICa) in TG. The rate of [Ca2+]i decline during caffeine exposure was faster in TG, indicating enhanced Na+-Ca2+ exchange function (consistent with protein expression measurements). Enhanced diastolic SR Ca2+ leak (via sparks), reduced SR Ca2+-ATPase expression, and increased Na+-Ca2+ exchanger explain the reduced diastolic [Ca2+]i and SR Ca2+ content in TG. We conclude that CaMKIIdeltaC overexpression causes acute modulation of excitation-contraction coupling, which contributes to heart failure.     

PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity

During physical exercise and stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2, at Ser2808. Hyperphosphorylation of RyR2-S2808 has been proposed as a mechanism contributing to arrhythmogenesis and heart failure. However, the role of RyR2 phosphorylation during β-adrenergic stimulation remains controversial. We examined the contribution of RyR2-S2808 phosphorylation to altered excitation-contraction coupling and Ca(2+) signaling using an experimental approach at the interface of molecular and cellular levels and a transgenic mouse with ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Experimentally challenging the communication between L-type Ca(2+) channels and RyR2 led to a spatiotemporal de-synchronization of RyR2 openings, as visualized using confocal Ca(2+) imaging. β-Adrenergic stimulation re-synchronized RyR2s, but less efficiently in RyR2-S2808A than in control cardiomyocytes, as indicated by comprehensive analysis of RyR2 activation. In addition, spontaneous Ca(2+) waves in RyR2-S2808A myocytes showed significantly slowed propagation and complete absence of acceleration during β-adrenergic stress, unlike wild type cells. Single channel recordings revealed an attenuation of luminal Ca(2+) sensitivity in RyR2-S2808A channels upon addition of PKA. This suggests that phosphorylation of RyR2-S2808 may be involved in RyR2 modulation by luminal (intra-SR) Ca(2+) ([Ca(2+)](SR)). We show here by three independent experimental approaches that PKA-dependent RyR2-S2808 phosphorylation plays significant functional roles at the subcellular level, namely, Ca(2+) release synchronization, Ca(2+) wave propagation and functional adaptation of RyR2 to variable [Ca(2+)](SR). These results indicate a direct mechanistic link between RyR2 phosphorylation and SR luminal Ca(2+) sensing.     

Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca(2+)-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2(R33Q) in adult rat myocytes led to an increase in excitation-contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca(2+)-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia.     

Ca2+/Calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes

Previous studies in transgenic mice and with isolated ryanodine receptors (RyR) have indicated that Ca2+-calmodulin-dependent protein kinase II (CaMKII) can phosphorylate RyR and activate local diastolic sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks) and RyR channel opening. Here we use relatively controlled physiological conditions in saponin-permeabilized wild type (WT) and phospholamban knockout (PLB-KO) mouse ventricular myocytes to test whether exogenous preactivated CaMKII or endogenous CaMKII can enhance resting Ca2+ sparks. PLB-KO mice were used to preclude ancillary effects of CaMKII mediated by phospholamban phosphorylation. In both WT and PLB-KO myocytes, Ca2+ spark frequency was increased by both preactivated exogenous CaMKII and endogenous CaMKII. This effect was abolished by CaMKII inhibitor peptides. In contrast, protein kinase A catalytic subunit also enhanced Ca2+ spark frequency in WT, but had no effect in PLB-KO. Both endogenous and exogenous CaMKII increased SR Ca2+ content in WT (presumably via PLB phosphorylation), but not in PLB-KO. Exogenous calmodulin decreased Ca2+ spark frequency in both WT and PLB-KO (K0.5 approximately 100 nmol/L). Endogenous CaMKII (at 500 nmol/L [Ca2+]) phosphorylated RyR as completely in <4 minutes as the maximum achieved by preactivated exogenous CaMKII. After CaMKII activation Ca2+ sparks were longer in duration, and more frequent propagating SR Ca2+ release events were observed. We conclude that CaMKII-dependent phosphorylation of RyR by endogenous associated CaMKII (but not PKA-dependent phosphorylation) increases resting SR Ca2+ release or leak. Moreover, this may explain the enhanced SR diastolic Ca2+ leak and certain triggered arrhythmias seen in heart failure.     

Potentiation of Ca(2+) release by cADP-ribose in the heart is mediated by enhanced SR Ca(2+) uptake into the sarcoplasmic reticulum

cADP-Ribose (cADPR) is a novel endogenous messenger that is believed to mobilize Ca(2+) from ryanodine-sensitive Ca(2+) stores. Despite intense research, the precise mechanism of action of cADPR remains uncertain, and experimental findings are contradictory. To elucidate the mechanism of cADPR action, we performed confocal Ca(2+) imaging in saponin-permeabilized rat ventricular myocytes. Exposure of the cells to cADPR resulted in a slow (>2 minutes) and steady increase in the frequency of Ca(2+) sparks. These effects on local release events were accompanied by a significant increase in sarcoplasmic reticulum (SR) Ca(2+) content. In comparison, sensitization of ryanodine receptors (RyRs) by caffeine, a true RyR agonist, caused a rapid (<1 second) and transient potentiation of Ca(2+) sparks followed by a decrease in SR Ca(2+) content. When the increase in the SR load was prevented by partial inhibition of the SR Ca(2+) with thapsigargin, cADPR failed to produce any increase in sparking activity. cADPR had no significant impact on activity of single cardiac RyRs incorporated into lipid bilayers. However, it caused a significant increase in the rate of Ca(2+) uptake by cardiac SR microsomes. Our results suggest that the primary target of cADPR is the SR Ca(2+) uptake mechanism. Potentiation of Ca(2+) release by cADPR is mediated by increased accumulation of Ca(2+) in the SR and subsequent luminal Ca(2+)-dependent activation of RyRs.     

Characteristics of prolonged Ca2+ release events associated with the nuclei in adult cardiac myocytes

Confocal microscopy was used to study the properties of nuclear Ca2+ regulation in adult ventricular myocytes. Prolonged nuclear Ca2+ release (PNCR) events were identified in both intact and permeabilized rat myocytes. PNCR occurred spontaneously and was restricted to localized regions at the ends of the elongated nuclei. Typically, PNCR took the form of a rapid rise in [Ca2+] followed by a maintained plateau. The mean duration of PNCR (1.78+/-0.19 seconds) was markedly greater than the half decay time for cytosolic Ca2+ sparks (31.2+/-0.56 ms) obtained under the same conditions. The PNCR width at half maximum amplitude (5.0+/-0.2 microm) was also significantly greater than that of cytosolic Ca2+ sparks (2.6+/-0.05 microm) obtained under the same conditions. Experiments involving the use of syto-11 to accurately locate the nuclei demonstrated that PNCR originates from the nuclear envelope or a closely associated structure. The spatial spread of PNCR was asymmetrical, with greater diffusion of Ca2+ toward the center of the nucleus than the cytosol. Both PNCR and Ca2+ sparks were abolished by interventions that deplete SR Ca2+ stores or inhibit RYR activation. Experiments on intact, electrically stimulated cells revealed that diffusion of Ca2+ from the ends of the nucleus toward the center is a prominent feature of the nucleoplasmic Ca2+ transient. The possibility that recruitment of Ca2+ release sites involved in PNCR might influence the temporal and spatial characteristics of the nucleoplasmic [Ca2+] transient is considered.     

Imaging microdomain Ca2+ in muscle cells

Ca2+ ions passing through a single or a cluster of Ca2+-permeable channels create microscopic, short-lived Ca2+ gradients that constitute the building blocks of cellular Ca2+ signaling. Over the last decade, imaging microdomain Ca2+ in muscle cells has unveiled the exquisite spatial and temporal architecture of intracellular Ca2+ dynamics and has reshaped our understanding of Ca2+ signaling mechanisms. Major advances include the visualization of "Ca2+ sparks" as the elementary events of Ca2+ release from the sarcoplasmic reticulum (SR), "Ca2+ sparklets" produced by openings of single Ca2+-permeable channels, miniature Ca2+ transients in single mitochondria ("marks"), and SR luminal Ca2+ depletion transients ("scraps"). As a model system, a cardiac myocyte contains a 3-dimensional grid of 104 spark ignition sites, stochastic activation of which summates into global Ca2+ transients. Tracking intermolecular coupling between single L-type Ca2+ channels and Ca2+ sparks has provided direct evidence validating the local control theory of Ca2+-induced Ca2+ release in the heart. In vascular smooth muscle myocytes, Ca2+ can paradoxically signal both vessel constriction (by global Ca2+ transients) and relaxation (by subsurface Ca2+ sparks). These findings shed new light on the origin of Ca2+ signaling efficiency, specificity, and versatility. In addition, microdomain Ca2+ imaging offers a novel modality that complements electrophysiological approaches in characterizing Ca2+ channels in intact cells.     

SR calcium depletion following reversal of the Na+/Ca2+-exchanger in rat ventricular myocytes

We previously reported that cytosolic calcium transiently increases after reversal of the sarcolemmal Na+/Ca2+-exchanger. Calcium released from sarcoplasmic reticulum (SR) constituted the major part of this cytosolic transient. The aim of this study was to test whether reversal of the Na+/Ca2+-exchanger affects SR calcium content, and whether altered SR calcium content is associated with direct triggering of SR calcium release or calcium release secondary to SR calcium overload. To this purpose we studied the change of SR calcium content after reversal of the Na+/Ca2+-exchanger and the dependence on the magnitude of change of its free energy (delta Gexch) in isolated rat ventricular myocytes. The Na+/Ca2+-exchanger was reversed by abrupt reduction of extracellular sodium ([Na+]o). The magnitude of change of deltaGexch was varied with [Na+]o. Cytosolic free calcium ([Ca2+]i) was measured with indo-1 and SR calcium content was estimated from the increase of [Ca2+]i after rapid cooling (RC). SR function was manipulated either by blockade of the SR Ca2+-ATPase with thapsigargin or by blockade of SR calcium release channels with tetracaine. Reversal of the Na+/Ca2+-exchanger caused a transient increase of [Ca2+]i of about 180 s duration with a time to peak of about 30 s. During the first 30 s rapid small amplitude cytosolic calcium fluctuations were superimposed on this transient. The magnitude of the response of [Ca2+]i to RC, during the course of the cytosolic [Ca2+]i transient, also transiently increased from 174 in control myocytes to 480 nmol/l at the time of the peak value. After correction of [Ca2+]i data for the fraction of mitochondrially compartmentalized indo-1 and mitochondrial calcium, total calcium released from SR after RC was calculated with the use of literature data on cytosolic calcium buffer capacity. Contrary to the measured RC-dependent increase of measured [Ca2+]i, after reversal of the Na+/Ca2+-exchanger, calculated total calcium released from SR transiently decreased. The extent of SR calcium depletion after reversal of the Na+/Ca2+-exchanger increased with the magnitude of change of deltaGexch. Restitution of [Na+]o 30 s after reversal of the Na+/Ca2+-exchanger, greatly accelerated both recovery of [Ca2+]i and SR calcium content. Pretreatment of myocytes with thapsigargin caused almost entire depletion of SR and substantial reduction of the cytosolic transient of [Ca2+]i following reversal of the Na+/Ca2+-exchanger. Application of tetracaine hardly affected SR calcium content, but caused an increase of the SR calcium content following reversal of the Na+/Ca2+-exchanger, while the cytosolic transient increase of [Ca2+]i was substantially reduced. We conclude that reversal of the Na+/Ca2+-exchanger directly triggers SR calcium release and decreases SR calcium content in a deltaGexch dependent manner.     

Ca2+ blinks: rapid nanoscopic store calcium signaling

Luminal Ca(2+) in the endoplasmic and sarcoplasmic reticulum (ER/SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca(2+) entry, Ca(2+)-induced Ca(2+) release, and ER/SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca(2+)], called "Ca(2+) blinks," within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca(2+) release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca(2+),orCa(2+) sparks, but changes of [Ca(2+)] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s(-1), which is approximately 6-fold faster than the recovery of local Ca(2+) release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca(2+) signaling thus broadens our understanding of cardiac store Ca(2+) regulation and function and opens the possibility for local regulation of diverse store-dependent functions.     

Ca2+ scraps: local depletions of free [Ca2+] in cardiac sarcoplasmic reticulum during contractions leave substantial Ca2+ reserve

Free [Ca2+] inside the sarcoplasmic reticulum ([Ca2+]SR) is difficult to measure yet critically important in controlling many cellular systems. In cardiac myocytes, [Ca2+]SR regulates cardiac contractility. We directly measure [Ca2+]SR in intact cardiac myocytes dynamically and quantitatively during beats, with high spatial resolution. Diastolic [Ca2+]SR (1 to 1.5 mmol/L) is only partially depleted (24% to 63%) during contraction. There is little temporal delay in the decline in [Ca2+]SR at release junctions and between junctions, indicating rapid internal diffusion. The incomplete local Ca2+ release shows that the inherently positive feedback of Ca2+-induced Ca2+ release terminates, despite a large residual driving force. These findings place stringent novel constraints on how excitation-contraction coupling works in heart and also reveal a Ca2+ store reserve that could in principle be a therapeutic target to enhance cardiac function in heart failure.     

Pernicious attrition and inter-RyR2 CICR current control in cardiac muscle

In cardiac muscle cells, ryanodine receptor (RyR) mediated Ca^2 + release from the sarcoplasmic reticulum (SR) drives the contractile apparatus. Spontaneous bouts of inter-RyR Ca^2 + induced Ca^2 + release (CICR) generate an elemental unit of SR Ca^2 + release called a spark. Sparks are localized events that terminate soon after they begin. The local control of sparks is not clearly understood. In this article, we review the potential regulatory role that the changing single RyR Ca^2 + current may play. Moreover, we aggregate RyR data into a working scheme of inter-RyR CICR current control of sparks and a potential inter-RyR CICR termination mechanism that we call pernicious attrition. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.