Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra

The fluorescent Mg(2+) indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, [Mg(2+)](i) averaged 0.842 +/- 0.019 mM. After the cells were loaded with Mg(2+) by exposure to high extracellular [Mg(2+)] ([Mg(2+)](o)), reduction of [Mg(2+)](o) to 1 mM (in the presence of extracellular Na(+)) induced a decrease in [Mg(2+)](i). The rate of decrease in [Mg(2+)](i) was higher at higher [Mg(2+)](i), whereas raising [Mg(2+)](o) slowed the decrease in [Mg(2+)](i) with 50% reduction of the rate at approximately 10 mM [Mg(2+)](o). Because a part of the furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25 microg/ml for 5 min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus approximately 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg(2+)] ([Mg(2+)](c)), whereas the residual 22% likely represented [Mg(2+)] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg(2+)] calibrated from the residual furaptra fluorescence ([Mg(2+)](r)) was 0.6-0.7 mM in bathing solution [Mg(2+)] (i.e., [Mg(2+)](c) of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg(2+)](r) was lower than and virtually insensitive to [Mg(2+)](c). We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg(2+)](c). In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg(2+) buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg(2+) flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na(+), Mg(2+) efflux is markedly activated by [Mg(2+)](c) above the normal basal level (approximately 0.9 mM), with a half-maximal activation of approximately 1.9 mM [Mg(2+)](c). We conclude that [Mg(2+)](c) is tightly regulated by an Mg(2+) efflux that is dependent on extracellular [Na(+)].     

Iminocoumarin-based low affinity fluorescent Ca2+ indicators excited with visible light.

A series of iminocoumarin-based fluorescent Ca2+ indicators were synthesized and the spectral profiles of their free and Ca2+ bound forms were studied. The newly-synthesized compounds incorporate the Ca2+ chelating structure of BAPTA. The chromophore moieties are iminocoumarins substituted at the 3-position with benzothiazolyl, benzoxazolyl and benzimidazolyl groups. These compounds are excited with visible light and their Ca2+ dissociation constants range from 5.4 to 27.5 microM. Fluorescence spectra studies of these probes indicated a clear shift in their excitation wavelength maxima upon Ca2+ binding along with changes in fluorescence intensity that enable the compounds to be used as low Ca2+ affinity, visible excitable probes.     

High and low affinity Ca2+ binding to the sarcoplasmic reticulum: use of a high-affinity fluorescent calcium indicator.

The fluorescent calcium indicator, calcein, has been used as a high-affinity indicator of Ca2+ in the aqueous phase at physiological pH in the study of high-affinity calcium binding to sarcoplasmic reticulum (SR). The binding constant of the indicator at physiological pH is 10(3)-10(4) M-1 and increases with increasing pH. The binding mechanism of the indicator with Ca2+ and Mg2+ is described. Application of calcein as an aqueous indicator of Ca2+ binding to the SR at room temperature has revealed two classes of binding sites: one with high capacity and low affinity (ca. 820 nmol/mg protein, Kd = 1.9 mM), and another with low capacity and higher affinity (ca. 35 nmol/mg protein, Kd = 17.5 micronM). The divalent cation specificity of the low-affinity site is low and Ca2+/Mg2+ specificity of the high-affinity site is high. Quantitative studies of the bindings indicate that the high-affinity site residues in the Ca2+ ATPase (carrier) protein and represents complexation in the active site of the carrier and that the low-affinity site residues in the nonspecific acidic binding proteins. The contribution of Donnan equilibrium effects to the measured binding is shown to be insignificant. Stopped flow kinetic studies of Ca2+ passive binding with calcein and arsenazo III dyes have demonstrated that the binding to high-affinity site is very fast and that the overall binding reaction with the low-affinity site is slow, with a time course of about 4 s. Our analysis has shown that at least part of the low-affinity acidic proteins are within the SR matrix and that Ca2+ can reach them only by transversing the membrane via the Ca2+ carrier (Ca2+ ATPase). A model of the SR is proposed that accounts for several functional properties of the organelle in terms of its known protein composition and topological organization.     

Near-membrane iminocoumarin-based low affinity fluorescent Ca(2+) indicators

Two new potential near-membrane iminocoumarin-based fluorescent Ca(2+) indicators were synthesized and the spectral profiles of their free and Ca(2+) bound forms were studied. The probes incorporate in their BAPTA-related structures, the 3-(benzimidazolyl)iminocoumarin or the 3-(benzothiazolyl)iminocoumarin moiety, substituted at the imino nitrogen with an n-dodecyl lipophilic chain. The compounds are excited with visible light and have Ca(2+) dissociation constant values of 5.50 and 4.49 microM, respectively, the highest reported to date in the literature. Fluorescence spectra studies indicated a clear shift in their excitation wavelength maxima upon Ca(2+) binding along with changes in fluorescence intensity that enable the compounds to be used as ratiometric near-membrane, low Ca(2+) affinity probes.     

心肌胞内钙瞬变的记录方法——快速二维扫描法与线扫描法

目的:采用共聚焦显微镜快速二维扫描方式和线扫描方式记录心肌胞内钙瞬变,并分析其优缺点。方法:标本为急性分离的SD大鼠心肌单细胞,胞内钙信号由钙指示剂fluo4-AM标记,其变化由共聚显微镜(LSM510META系统)记录。钙瞬变由局部场刺激诱发,刺激器和共聚焦成像系统之间通过触发连接同步工作。结果:快速二维扫描方式可在二维平面上反映全细胞范围内钙瞬变的动态过程,空间信息较全面;特别地,当心肌细胞由于药物或病理状态的改变而出现胞内钙稳态失衡时,快速二维扫描的结果更有利于了解胞内钙变化;其结果可制成动画,真实而直观地再现心肌细胞胞内钙瞬变的动态过程。线扫描方式的时间分辨率较高,也有一定的空间分辨率,可反映钙瞬变的时空特征,并可分析细胞收缩的情况。二种扫描方式所得的结果在实质上是一致的,但各有其侧重点和优缺点,在反映心肌细胞功能状态方面具有互补作用。结论:两种扫描方式所得的结果综合起来更有利于对胞内钙信号变化的特征和意义进行正确解读。     

Extracellular ATP induces Ca2+ transients in cardiac myocytes which are potentiated by norepinephrine

Isolated rat ventricular cardiac myocytes loaded with the fluorescent calcium indicator fura2 showed significant changes in intracellular calcium concentrations upon exposure to greater than 1 microM ATP (EC50 = 7.4 +/- 1.3 microM, n = 4, SE), suggesting that extracellular ATP may have an important influence on myocardial contractility. The response was found to be highly ATP specific and required extracellular calcium. Furthermore, 30 s pretreatment of the cells with 0.2-1 microM norepinephrine decreased the concentration of ATP required for the Ca2+ transient, shifting the EC50 for ATP to 1.7 +/- 0.1 microM (n = 3, SE). beta-Propranolol (a beta 1-receptor antagonist) prevented potentiation, whereas phentolamine (an alpha 1-receptor antagonist) did not, indicating that regulation is through the beta 1-adrenergic receptor. ATP and norepinephrine released locally from sympathetic neurons may act in concert through the ATP and beta 1-adrenergic receptors to regulate myocardial calcium homeostasis.     

Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator

A new fluorescent intracellular pH indicator is described ("quene 1") which is related to the tetracarboxylate Ca2+ indicator based on the quinoline fluorophor ("quin 2"). Quene 1 has excitation and emission maxima at 390 and 530 nm, respectively, and shows a 30-fold increase in fluorescence between pH 5 and 9 with a pK alpha of 7.3. The fluorescence is insensitive to Ca2+ and Mg2+ at free concentrations up to 10(-4) M and to the proportions of Na+ and K+ at total concentrations of Na+ and K+ from 100 to 200 mM. The indicator is loaded into thymocytes using the tetraacetoxymethyl ester derivative which is hydrolyzed in the cells to give the tetracarboxylate anion. Intracellular pH can be measured at intracellular quene 1 concentrations of approximately 0.1 mM and quene 1 does not perturb glycolysis or the ATP level in resting cells at concentrations up to 0.8 mM. The intracellular pH of mouse thymocytes indicated by quene 1 is 7.15 +/- 0.04 and it is insensitive to the concentration of Ca2+ or Mg2+ in the extracellular medium. The intracellular pH decreased when the pH of the medium was lowered by addition of HCl, but was insensitive to NaOH at extracellular pH values up to 8.0. Rapid transient changes in intracellular pH are induced by NH4Cl, NaCO2CH3, or HCO3-/CO2. The thymocytes showed no early changes (within 30 min) in intracellular pH in response to mitogenic concentrations of lectins or 4 beta-phorbol-12-myristate-13-acetate.     

Fluorescence signals from the Mg2+/Ca2+ indicator furaptra in frog skeletal muscle fibers.

The fluorescent Mg2+/Ca2+ indicator, furaptra, was injected into single frog skeletal muscle fibers, and the indicator's fluorescence signals were measured and analyzed with particular interest in the free Mg2+ concentration ([Mg2+]) in resting muscle. Based on the fluorescence excitation spectrum of furaptra, the calibrated myoplasmic [Mg2+] level averaged 0.54 mM, if the value of dissociation constant (KD) for Mg2+ obtained in vitro (5.5 mM) was used. However, if the indicator reacts with Mg2+ with a two-fold larger KD in myoplasm, as previously suggested for the furaptra-Ca2+ reaction (M. Konishi, S. Hollingworth, A.B. Harkins, S.M. Baylor. 1991. J. Gen. Physiol. 97:271-301), the calculated [Mg2+] would average 1.1 mM. Thus, the value 1.1 mM probably represents the best estimate from furaptra of [Mg2+] in resting muscle fibers. Extracellular perfusion of muscle fibers with high Mg2+ concentration solution or low Na+ concentration solution did not cause any detectable changes in the [Mg2+]-related furaptra fluorescence within 4 min. The results suggest that the myoplasmic [Mg2+] is highly regulated near the resting level of 1 mM, and that changes only occur with a very slow time course.     

Unloaded shortening increases peak of Ca2+ transients but accelerates their decay in rat single cardiac myocytes

It is of paramount importance to investigate the relation between the time-dependent change in intracellular Ca2+ concentration ([Ca2+]i) (Ca2+ transients) and the mechanical activity of isolated single myocytes to understand the regulatory mechanisms of heart function. However, because of technical difficulties in performing mechanical measurements with single myocytes, the simultaneous recording of Ca2+ transients and mechanical activity has mainly been performed with multicellular cardiac preparations that give conflicting results concerning Ca2+ transients during isometric twitches and during twitches with unloaded shortening. In the present study, we coupled intracellular Ca2+ measurement optics with a force measurement system using carbon fibers to examine the relation between Ca2+ transients and the mechanical activity of rat single ventricular myocytes over a wide range of load. To minimize the possible load dependence of sarcoplasmic reticulum Ca2+ loading, contraction mode was switched at every twitch from unloaded shortening to isometric contraction. During a twitch with unloaded shortening, the Ca2+ transients exhibited a higher peak and a higher rate of decay than transients during an isometric twitch. Similarly, when we changed the contraction mode in every pair of twitches, Ca2+ transients were dependent only on the mode of contraction. Mechanical uncoupling with 2,3-butanedione monoxime abolished this dependence on the mode of contraction. Our results suggest that Ca2+ transients reflect the affinity of troponin C for Ca2+, which is influenced by the change in strain on the thin filament but not by the length change per se.     

Effects of phospholemman downregulation on contractility and [Ca(2+)]i transients in adult rat cardiac myocytes

Phospholemman (PLM) expression was increased in rat hearts after myocardial infarction (MI). Overexpression of PLM in normal adult rat cardiac myocytes altered contractile function and cytosolic Ca(2+) concentration ([Ca(2+)](i)) homeostasis in a manner similar to that observed in post-MI myocytes. In this study, we tested whether PLM downregulation in normal adult rat myocytes resulted in contractility and [Ca(2+)](i) transient changes opposite to those observed in post-MI myocytes. Compared with control myocytes infected with adenovirus (Adv) expressing green fluorescent protein (GFP) alone, myocytes infected with Adv expressing both GFP and rat antisense PLM (rASPLM) had 23% less PLM protein (P < 0.012) at 3 days, but no differences were found in sarcoplasmic reticulum (SR) Ca(2+)-ATPase, Na(+)/Ca(2+) exchanger (NCX1), Na(+)-K(+)-ATPase, and calsequestrin levels. SR Ca(2+) uptake and whole cell capacitance were not affected by rASPLM treatment. Relaxation from caffeine-induced contracture was faster, and NCX1 current amplitudes were higher in rASPLM myocytes, indicating that PLM downregulation enhanced NCX1 activity. In native rat cardiac myocytes, coimmunoprecipitation experiments indicated an association of PLM with NCX1. At 0.6 mM [Ca(2+)](o), rASPLM myocytes had significantly (P < 0.003) lower contraction and [Ca(2+)](i) transient amplitudes than control GFP myocytes. At 5 mM [Ca(2+)](o), both contraction and [Ca(2+)](i) transient amplitudes were higher in rASPLM myocytes. This pattern of contractile and [Ca(2+)](i) transient behavior in rASPLM myocytes was opposite to that observed in post-MI rat myocytes. We conclude that downregulation of PLM in normal rat cardiac myocytes enhanced NCX1 function and affected [Ca(2+)](i) transient and contraction amplitudes. We suggest that PLM downregulation offers a potential therapeutic strategy for ameliorating contractile abnormalities in MI myocytes.     

Photophysics of the fluorescent Ca2+ indicator Fura-2.

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.     

Computer-aided measurement of cell shortening and calcium transients in adult cardiac myocytes

The contractile cycle of the cardiac myocyte is essentially controlled by the concentration of intracellular calcium ([Ca2+]i). Measurement of [Ca2+]i using Ca2+-dependent fluorescence and simultaneous monitoring of cell dynamics enable characterization of a variety of substances interacting with ion channels and contractile proteins. In this report we describe a novel method featuring up to 480 frames/s for monitoring rapid changes in cellular calcium and cell length, in which every individual cycle allows effective evaluation of major cell parameters. Computers aid in determination of time to peak (in ms), time to 50% decrease (ms), diastolic Ca2+ (relative fluorescence units, rfu), systolic Ca2+ (rfu), Ca2+ transients (rfu), DeltaCa2+/Delta(t) rise (rfu/s), and DeltaCa2+/Delta(t) fall (rfu/s). Contractile parameters are as follows: maximum cell length (microm), minimum cell length (microm), absolute cell shortening (microm), peak DeltaL/Delta(t) shortening (microm/s), and peak DeltaL/Delta(t) relaxation (microm/s). In summary, we succeeded in demonstrating that this system is a unique and valuable tool that allows simultaneous and accurate assessment of contractile parameters and of calcium movements of isolated adult cardiac myocytes.     

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.     

Spontaneous and experimentally evoked [Ca2+]i-transients in cardiac myocytes measured by means of a fast Fura-2 technique

A setup for dual wavelength-excitation fluorescence measurements is introduced which permits a temporal resolution of up to 1 KHz, using the Ca2(+)-sensitive fluorescent dye Fura-2. The system makes use of a novel technical solution for chopping between two excitation wavelengths which does not move any optical components. Two beams, which are alternatively opened or shut by a rotating chopper wheel, are united by a dichroic mirror and are used for low-noise epifluorescence microscopy. The system includes a device for fast changes of extracellular solution that can be used for studying various components of [Ca2+]i-regulation in excitable and non-excitable cells. Sample recordings of spontaneous and experimentally-evoked [Ca2+]i-transients from cardiac myocytes are presented. Cardiac myocytes are a cell species that produces particularly fast [Ca2+]i-transients and therefore, a high temporal resolution is required in order to study physiological and/or pharmacological properties of these transients.     

Differential integration of Ca2+-calmodulin signal in intact ventricular myocytes at low and high affinity Ca2+-calmodulin targets

Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca2+ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin). However, little is known about the dynamics of how CaM targets process the Ca2+ signals to generate appropriate biological responses in the heart. We hypothesized that the different affinities of CaM targets for the Ca2+-bound CaM (Ca2+-CaM) shape their actions through dynamic and tonic interactions in response to the repetitive Ca2+ signals in myocytes. To test our hypothesis, we used two fluorescence resonance energy transfer-based biosensors, BsCaM-45 (Kd = approximately 45 nm) and BsCaM-2 (Kd = approximately 2 nm), to monitor the real time Ca2+-CaM dynamics at low and high affinity CaM targets in paced adult ventricular myocytes. Compared with BsCaM-2, BsCaM-45 tracks the beat-to-beat Ca2+-CaM alterations more closely following the Ca2+ oscillations at each myocyte contraction. When pacing frequency is raised from 0.1 to 1.0 Hz, the higher affinity BsCaM-2 demonstrates significant elevation of diastolic Ca2+-CaM binding compared with the lower affinity BsCaM-45. Biochemically detailed computational models of Ca2+-CaM biosensors in beating cardiac myocytes revealed that the different Ca2+-CaM binding affinities of BsCaM-2 and BsCaM-45 are sufficient to predict their differing kinetics and diastolic integration. Thus, data from both experiments and computational modeling suggest that CaM targets with low versus high Ca2+-CaM affinities (like CaM-dependent kinase versus calcineurin) respond differentially to the same Ca2+ signal (phasic versus integrating), presumably tuned appropriately for their respective and distinct Ca2+ signaling pathways.     

ICPBCZin: a red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions

A new fluorescent Zn²⁺ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn²⁺ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3′,2′:3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn²⁺ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn²⁺ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn²⁺ probe.     

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies

In cardiac myocytes, the activity of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca(2+) release from and Ca(2+) uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37 degrees C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca(2+) release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca(2+) uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca(2+)](i) transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca(2+)](i) transient, indicating that the PLN-mediated feedback on [Ca(2+)](i) removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca(2+) release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca(2+)](i) transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca(2+) uptake is diminished at higher stimulation frequencies within the physiological range.     

Negative control mechanism with features of adaptation controls Ca2+ release in cardiac myocytes.

The central paradox of cardiac excitation-contraction coupling is that Ca(2+)-induced Ca2+ release (CICR), an inherently self-regenerating process, is finely graded by surface membrane Ca2+ current (ICa). By using FPL64176, a novel Ca2+ channel agonist that reduces inactivation of ICa, a rapid negative control mechanism was unmasked at the Ca2+ release level in isolated rat ventricular myocytes. This mechanism terminates CICR independently of the duration of trigger ICa and before the sarcoplasmic reticulum becomes depleted of Ca2+. In its ability to be reactivated by incremental increases in trigger ICa, this mechanism differs from conventional inactivation/desensitization and is similar to the mechanism of increment detection or adaptation described for intracellular Ca2+ release channels. These results indicate that ryanodine receptor adaptation regulates Ca2+ release in cardiac muscle, accounting for or contributing to the graded nature of CICR and, additionally, permitting stores to reload at later times during Ca2+ entry.