Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.     

Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes

The possible contribution of Na(+)-Ca(2+) exchange to the triggering of Ca(2+) release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the "trigger flux" of Ca(2+) crossing the cell membrane in rabbit ventricular myocytes with Ca(2+) release disabled pharmacologically. Under conditions that promote Ca(2+) entry via Na(+)-Ca(2+) exchange, internal [Na(+)] (10 mM), and positive membrane potential, the Ca(2+) trigger flux (measured using a fluorescent Ca(2+) indicator) was much greater than the Ca(2+) flux through the L-type Ca(2+) channel, indicating a significant contribution from Na(+)-Ca(2+) exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca(2+) channels was assessed by whole-cell patch-clamp recordings of Ca(2+) current and complementary experiments in which internal [Na(+)] was reduced. However, Ca(2+) entry via Na(+)-Ca(2+) exchange measured in the absence of L-type Ca(2+) current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca(2+) channels increase [Ca(2+)] near Na(+)-Ca(2+) exchanger molecules and activate this protein. These results help to resolve seemingly contradictory results obtained previously and have implications for our understanding of the triggering of Ca(2+) release in heart cells under various conditions.     

Modulation of Ca2+ channel-gated Ca2+ release by W-7 in cardiac myocytes.

Cardiac muscle excitation-contraction coupling is controlled by the Ca(2+)-induced Ca2+ release mechanism. The present study examines the effects of a calmodulin antagonist W-7 on Ca2+ current (ICa)-induced Ca2+ release in whole cell-clamped rat ventricular myocytes. Exposure of cells to W-7 suppressed ICa, but the intracellular Ca(2+)-transients showed a lesser degree of reduction, suggesting possible enhancement of Ca(2+)-induced Ca2+ release. The effects of W-7 on the efficacy of Ca2+ release were most prominent at negative potentials. At test potentials of -30 mV, 20 microM W-7 almost completely blocked ICa, but significant Ca(2+)-transients remained, thus causing a four to six-fold increase in the efficacy of Ca(2+)-induced Ca2+ release. The depolarization-dependent Ca(2+)-transients were eliminated in absence of extracellular Ca2+, blocked by Cd2+, and were absent when the sarcoplasmic reticulum was depleted of Ca2+, implicating dependency on Ca(2+)-signaling between the L-type channel and the ryanodine receptor. W-7 mediated increase in the efficacy of Ca(2+)-induced Ca2+ release was eliminated when myocytes were dialyzed with the internal solution containing gluathione (5 mM), suggesting the possible role of cellular redox state in the regulation of Ca2+ release by the calmodulin antagonist.     

SR Ca2+ refilling upon depletion and SR Ca2+ uptake rates during development in rabbit ventricular myocytes

While it has been reported that a sparse sarcoplasmic reticulum (SR) and a low SR Ca(2+) pump density exist at birth, we and others have recently shown that significant amounts of Ca(2+) are stored in the neonatal rabbit heart SR. Here we try to determine developmental changes in SR Ca(2+) loading mechanisms and Ca(2+) pump efficacy in rabbit ventricular myocytes. SR Ca(2+) loading (load(SR)) and k(0.5) (Ca(2+) concentration at half-maximal SR Ca(2+) uptake) were higher and lower, respectively, in younger age groups. Inhibition of the L-type calcium current (I(Ca)) with 15 microM nifedipine dramatically reduced load(SR) in older but not in younger age groups. In contrast, subsequent inhibition of the Na(+)/Ca(2+) exchanger (NCX) with 10 microM KB-R7943 strongly reduced load(SR) in the younger but not the older age groups. Accordingly, the time integral of the inward NCX current (tail I(NCX)) elicited on repolarization was highly sensitive to nifedipine in the older groups and sensitive to KB-R7943 in the younger groups. Interestingly, slow SR loading took place in the presence of both nifedipine and KB-R7943 in all age groups, although it was less prominent in the older groups. We conclude that the SR loading capacity at the earliest postnatal stages is at least as large as that of adult myocytes. However, reverse-mode NCX plays a prominent role in SR Ca(2+) loading at early postnatal stages while I(Ca) is the main source of SR Ca(2+) loading at late postnatal and adult stages.     

Ontogeny of Ca2+-induced Ca2+ release in rabbit ventricular myocytes

It is commonly accepted that L-type Ca(2+) channel-mediated Ca(2+)-induced Ca(2+) release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the adult mammalian heart and that there is no appreciable CICR in neonates. However, we have observed that cell contraction in the neonatal heart was significantly decreased after sarcoplasmic reticulum (SR) Ca(2+) depletion with caffeine. Therefore, the present study investigated the developmental changes of CICR in rabbit ventricular myocytes at 3, 10, 20, and 56 days of age. We found that the inhibitory effect of the L-type Ca(2+) current (I(Ca)) inhibitor nifedipine (Nif; 15 microM) caused an increasingly larger reduction of Ca(2+) transients on depolarization in older age groups [from approximately 15% in 3-day-old (3d) myocytes to approximately 90% in 56-day-old (56d) myocytes]. The remaining Ca(2+) transient in the presence of Nif in younger age groups was eliminated by the inhibition of Na(+)/Ca(2+) exchanger (NCX) with the subsequent addition of 10 microM KB-R7943 (KB-R). Furthermore, Ca(2+) transients were significantly reduced in magnitude after the depletion of SR Ca(2+) with caffeine in all age groups, although the effect was significantly greater in the older age groups (from approximately 40% in 3d myocytes up to approximately 70% in 56d myocytes). This SR Ca(2+)-sensitive Ca(2+) transient in the earliest developmental stage was insensitive to Nif but was sensitive to the subsequent addition of KB-R, indicating the presence of NCX-mediated CICR that decreased significantly with age (from approximately 37% in 3d myocytes to approximately 0.5% in 56d myocytes). In contrast, the I(Ca)-mediated CICR increased significantly with age (from approximately 10% in 3d myocytes to approximately 70% in 56d myocytes). The CICR gain as estimated by the integral of the CICR Ca(2+) transient divided by the integral of its Ca(2+) transient trigger was smaller when mediated by NCX ( approximately 1.0 for 3d myocytes) than when mediated by I(Ca) ( approximately 3.0 for 56d myocytes). We conclude that the lower-efficiency NCX-mediated CICR is a predominant mode of CICR in the earliest developmental stages that gradually decreases as the more efficient L-type Ca(2+) channel-mediated CICR increases in prominence with ontogeny.     

Modulation of the sarcolemmal L-type current by alteration in SR Ca2+ release

Modulation of the L-type current by sarcoplasmicreticulum (SR) Ca²⁺ release hasbeen examined in patch-clamped mouse myotubes. Inhibition of SRCa²⁺ release by inclusion ofryanodine in the internal solution shifted the half-activating voltage(V_0.5) of theL-type current from 1.1 ± 2.1 to 7.7 ± 1.7 mV. Rutheniumred in the internal solution shiftedV_0.5 from 5.4 ± 1.9 to 3.2 ± 4.1 mV. Chelation of myoplasmic Ca²⁺ with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid perfusion shiftedV_0.5 from 4.4 ± 1.7 to 3.5 ± 3.3 mV and increased the peak current.Extracellular caffeine (1 mM), which should enhance SRCa²⁺ release, significantlydecreased the peak Ca²⁺ current.In low (0.1 mM) internal EGTA, myotube contraction was abolished byinternal perfusion with ryanodine or ruthenium red, whereas addition ofcaffeine to the extracellular solution lowered the contractilethreshold, indicating that these modulators of SRCa²⁺ release had the expectedeffects on contraction. Therefore, SR Ca²⁺ release appears to modulatethe sarcolemmal L-type current, suggesting a retrograde communicationfrom the SR to the sarcolemmal L-type channels inexcitation-contraction coupling.

     

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload. APD90 was similarly abbreviated in myocytes exposed to the Na+/Ca2+ exchange inhibitor KB-R7943 (5 microM) or after inhibition of SR Ca2+ release with ryanodine (20 microM). In the absence of extracellular Na+, spontaneous SR Ca2+ releases caused minimal changes in resting membrane potential. After the myocytes were returned to Na+-containing solution, the potentiated intracellular Ca2+ concentration ([Ca2+]i) transients dramatically prolonged APD90 and [Ca2+]i oscillations caused delayed and early afterdepolarizations (DADs and EADs). Laser-flash photolysis of caged Ca2+ mimicked the effects of spontaneous [Ca2+]i oscillations, confirming that APD prolongation, DADs, and EADs could be ascribed to intracellular Ca2+ release. These results suggest that Na+/Ca2+ exchange is a major physiological determinant of APD and that INaCa activation by spontaneous SR Ca2+ release/oscillations, depending on the timing, can account for both DADs and EADs during SR Ca2+ overload.     

Ca2+ release from the sarcoplasmic reticulum activated by the low affinity Ca2+ chelator TPEN in ventricular myocytes

The Ca2+ content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca2+ release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca2+ within the SR with the membrane-permeant low affinity Ca2+ chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca2+ content and SR Ca2+ depletion can influence Ca2+ release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca2+ release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca2+ releases increased in frequency and developed into cell-wide Ca2+ waves. SR Ca2+ load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40microTPEN did not significantly inhibit the SR-Ca2+-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca2+ chelator in intracellular Ca2+ stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca2+ leak from the SR leading to its Ca2+ depletion. Lowering of SR Ca2+ content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.     

Regulation of sarcoplasmic reticulum Ca2+ release by cytosolic glutathione in rabbit ventricular myocytes

Of the major cellular antioxidant defenses, glutathione (GSH) is particularly important in maintaining the cytosolic redox potential. Whereas the healthy myocardium is maintained at a highly reduced redox state, it has been proposed that oxidation of GSH can affect the dynamics of Ca²⁺-induced Ca²⁺ release. In this study, we used multiple approaches to define the effects of oxidized glutathione (GSSG) on ryanodine receptor (RyR)-mediated Ca²⁺ release in rabbit ventricular myocytes. To investigate the role of GSSG on sarcoplasmic reticulum (SR) Ca²⁺ release induced by the action potential, we used the thiol-specific oxidant diamide to increase intracellular GSSG in intact myocytes. To more directly assess the effect of GSSG on RyR activity, we introduced GSSG within the cytosol of permeabilized myocytes. RyR-mediated Ca²⁺ release from the SR was significantly enhanced in the presence of GSSG. This resulted in decreased steady-state diastolic [Ca²⁺]_SR, increased SR Ca²⁺ fractional release, and increased spark- and non-spark-mediated SR Ca²⁺ leak. Single-channel recordings from RyR’s incorporated into lipid bilayers revealed that GSSG significantly increased RyR activity. Moreover, oxidation of RyR in the form of intersubunit crosslinking was present in intact myocytes treated with diamide and permeabilized myocytes treated with GSSG. Blocking RyR crosslinking with the alkylating agent N-ethylmaleimide prevented depletion of SR Ca²⁺ load induced by diamide. These findings suggest that elevated cytosolic GSSG enhances SR Ca²⁺ leak due to redox-dependent intersubunit RyR crosslinking. This effect can contribute to abnormal SR Ca²⁺ handling during periods of oxidative stress.     

Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

This study examines whether fluid pressure (FP) modulates the L-type Ca²⁺ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (16 dyn/cm²) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca²⁺ current (I_Ca) and cytosolic Ca²⁺ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed I_Ca (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of I_Ca. The level of I_Ca suppression by FP depended on the level and duration of pressure. The Ba²⁺ current through the Ca²⁺ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca²⁺ channel during FP stimulation. The cytosolic Ca²⁺ transients and the basal Ca²⁺ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the I_Ca and on the Ca²⁺ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca²⁺ buffer, eliminated the FP-induced acceleration of I_Ca inactivation and reduced the inhibitory effect of the FP on I_Ca by 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca²⁺ release, eliminated the accelerating effect of FP on the I_Ca inactivation, and they reduced the inhibitory effect of FP on the I_Ca. These results suggest that the fluid pressure indirectly suppresses the Ca²⁺ channel by enhancing the Ca²⁺-induced intracellular Ca²⁺ release in rat ventricular myocytes. L-type Ca²⁺ current; fluid pressure; ventricular myocytes; cytosolic Ca²⁺ transient     

Modulation of local Ca2+ release sites by rapid fluid puffing in rat atrial myocytes

Atrial myocytes that lack t-tubules appear to have two functionally separate sarcoplasmic Ca2+ stores: a peripheral store associated with plasmalemmal L-type calcium channels and a central store with no apparent proximity to L-type calcium channels. Here we describe a set of calcium sparks and waves that are triggered by puffing of pressurized (200-400 mmH2O) bathing solutions onto resting isolated rat atrial myocytes. Puffing of pressurized (200 mmH2O) solutions, identical to those bathing the myocytes from distances of approximately 150 microm onto the surface of a single myocyte triggered or enhanced spontaneously occurring peripheral sparks by five- to six-fold and central Ca2+ sparks by two- to three-fold, without altering the unitary spark properties. Exposure to higher pressure flows (400 mmH2O) often triggered longitudinally spreading Ca2+ waves. These results suggest that pressurized flows may directly modulate Ca2+ signaling of atrial myocytes by activating the intracellular Ca2+ release sites.     

Differential Ca2+ and Sr2+ regulation of intracellular divalent cations release in ventricular myocytes

The regulation of the Ca2+ -induced Ca2+ release (CICR) from intracellular stores is a critical step in the cardiac cycle. The inherent positive feedback of CICR should make it a self-regenerating process. It is accepted that CICR must be governed by some negative control, but its nature is still debated. We explore here the importance of the Ca2+ released from sarcoplasmic reticulum (SR) on the mechanisms that may control CICR. Specifically, we compared the effect of replacing Ca2+ with Sr2+ on intracellular Ca2+ signaling in intact cardiac myocytes as well as on the function of single ryanodine receptor (RyR) Ca2+ release channels in panar bilayers. In cells, both CICR and Sr2+ -induced Sr2+ release (SISR) were observed. Action potential induced Ca2+ -transients and spontaneous Ca2+ waves were considerably faster than their Sr2+ -mediated counterparts. However, the kinetics of Ca2+ and Sr2+ sparks was similar. At the single RyR channel level, the affinities of Ca2+ and Sr2+ activation were different but the affinities of Ca2+ and Sr2+ inactivation were similar. Fast Ca2+ and Sr2+ stimuli activated RyR channels equally fast but adaptation (a spontaneous slow transition back to steady-state activity levels) was not observed in the Sr2+ case. Together, these results suggest that regulation of the RyR channel by cytosolic Ca2+ is not involved in turning off the Ca2+ spark. In contrast, cytosolic Ca2+ is important in the propagation global Ca2+ release events and in this regard single RyR channel sensitivity to cytosolic Ca2+ activation, not low-affinity cytosolic Ca2+ inactivation, is a key factor. This suggests that the kinetics of local and global RyR-mediated Ca2+ release signals are affected in a distinct way by different divalent cations in cardiac muscle cells.     

Silver ions induce Ca2+ release from the SR in vitro by acting on the Ca2+ release channel and the Ca2+ pump.

Silver nitrate (AgNO3) is a sulfhydryl oxidizing agent that induces a biphasic Ca2+ release from isolated sarcoplasmic reticulum (SR) vesicles by presumably oxidizing critical sulfhydryl groups in the Ca2+ release channel (CRC), causing the channel to open. To further examine the effects of AgNO3 on the CRC and the Ca2+-ATPase, Ca2+ release was measured in muscle homogenates prepared from rat hindlimb muscle using indo 1. Cyclopiazonic acid (CPA) and ruthenium red (RR) were used to inhibit the Ca2+-ATPase and block the CRC, respectively, before inducing Ca2+ release with both AgNO3 and 4-chloro-m-cresol (4-CMC), a releasing agent specific for the CRC. With AgNO3 and CPA, the early rapid rate of release (phase 1) was increased (P < 0.05) by 42% (314 +/- 5 vs. 446 +/- 39 micromol x g protein(-1) x min(-1)), whereas the slower, more prolonged rate of release (phase 2) was decreased (P < 0.05) by 72% (267 +/- 39 vs. 74 +/- 7.7 micromol x g protein(-1) x min(-1)). RR, in combination with AgNO3, had no effect on phase 1 (P > 0.05) (314 +/- 51 vs. 334 +/- 43 micromol x g protein(-1) x min(-1)) and decreased phase 2 (P < 0.05) by 65% (245 +/- 34 vs. 105 +/- 8.2 micromol x g protein(-1) x min(-1)). With 4-CMC, CPA had no effect (P > 0.05) on either phase 1 or 2. With addition of RR, phase 1 was reduced (P < 0.05) by 59% (2,468 +/- 279 vs. 1,004 +/- 87 micromol x g protein(-1) x min(-1)), and RR completely blocked phase 2. Both AgNO3 and 4-CMC fully inhibited Ca2+-ATPase activity measured in homogenates. These findings indicate that AgNO3, but not 4-CMC, induces Ca2+ release by acting on both the CRC and the Ca2+-ATPase.     

Modulation of SR Ca release by luminal Ca and calsequestrin in cardiac myocytes: effects of CASQ2 mutations linked to sudden cardiac death

Cardiac calsequestrin (CASQ2) is an intrasarcoplasmic reticulum (SR) low-affinity Ca-binding protein, with mutations that are associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). To better understand how CASQ2 mutants cause CPVT, we expressed two CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2^DEL) or CASQ2 containing a point mutation (CASQ2^R33Q), in canine ventricular myocytes and assessed their effects on Ca handling. We also measured CASQ2-CASQ2 variant interactions using fluorescence resonance transfer in a heterologous expression system, and evaluated CASQ2 interaction with triadin. We found that expression of CASQ2^DEL or CASQ2^R33Q altered myocyte Ca signaling through two different mechanisms. Overexpressing CASQ2^DEL disrupted the CASQ2 polymerization required for high capacity Ca binding, whereas CASQ2^R33Q compromised the ability of CASQ2 to control ryanodine receptor (RyR2) channel activity. Despite profound differences in SR Ca buffering strengths, local Ca release terminated at the same free luminal [Ca] in control cells, cells overexpressing wild-type CASQ2 and CASQ2^DEL-expressing myocytes, suggesting that a decline in [Ca]_SR is a signal for RyR2 closure. Importantly, disrupting interactions between the RyR2 channel and CASQ2 by expressing CASQ2^R33Q markedly lowered the [Ca]_SR threshold for Ca release termination. We conclude that CASQ2 in the SR determines the magnitude and duration of Ca release from each SR terminal by providing both a local source of releasable Ca and by effects on luminal Ca-dependent RyR2 gating. Furthermore, two CPVT-inducing CASQ2 mutations, which cause mechanistically different defects in CASQ2 and RyR2 function, lead to increased diastolic SR Ca release events and exhibit a similar CPVT disease phenotype.     

IP3 receptor-dependent Ca2+ release modulates excitation-contraction coupling in rabbit ventricular myocytes

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-dependent Ca(2+) signaling exerts positive inotropic, but also arrhythmogenic, effects on excitation-contraction coupling (ECC) in the atrial myocardium. The role of IP(3)R-dependent sarcoplasmic reticulum (SR) Ca(2+) release in ECC in the ventricular myocardium remains controversial. Here we investigated the role of this signaling pathway during ECC in isolated rabbit ventricular myocytes. Immunoblotting of proteins from ventricular myocytes showed expression of both type 2 and type 3 IP(3)R at levels approximately 3.5-fold less than in atrial myocytes. In permeabilized myocytes, direct application of IP(3) (10 microM) produced a transient 21% increase in the frequency of Ca(2+) sparks (P < 0.05). This increase was accompanied by a 13% decrease in spark amplitude (P < 0.05) and a 7% decrease in SR Ca(2+) load (P < 0.05) and was inhibited by IP(3)R antagonists 2-aminoethoxydiphenylborate (2-APB; 20 microM) and heparin (0.5 mg/ml). In intact myocytes endothelin-1 (100 nM) was used to stimulate IP(3) production and caused a 38% (P < 0.05) increase in the amplitude of action potential-induced (0.5 Hz, field stimulation) Ca(2+) transients. This effect was abolished by the IP(3)R antagonist 2-APB (2 microM) or by using adenoviral expression of an IP(3) affinity trap that buffers cellular IP(3). Together, these data suggest that in rabbit ventricular myocytes IP(3)R-dependent Ca(2+) release has positive inotropic effects on ECC by facilitating Ca(2+) release through ryanodine receptor clusters.