L-type Ca2+ channels serve as a sensor of the SR Ca2+ for tuning the efficacy of Ca2+-induced Ca2+ release in rat ventricular myocytes

doi:10.1113/jphysiol.2003.050823

L-type Ca²⁺ channels serve as a sensor of the SR Ca²⁺ for tuning the efficacy of Ca²⁺-induced Ca²⁺ release in rat ventricular myocytes

Hajime Takamatsu, Taku Nagao*, Hidenori Ichijo and Satomi Adachi-Akahane

Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences,University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 and * National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

In cardiac excitation-contraction coupling, Ca²⁺-induced Ca²⁺ release (CICR) from ryanodine receptors (RyRs), triggered by Ca²⁺ entry through the nearby L-type Ca²⁺ channel, induces Ca²⁺-dependent inactivation (CDI) of the Ca²⁺ channel. Aiming at elucidating the physiological role of CDI produced by CICR (CICR-dependent CDI), we investigated the contribution of the CICR-dependent CDI to action potential (AP) waveform and the amount of Ca²⁺-influx through Ca²⁺ channels during AP in rat ventricular myocytes. The elimination of the CICR-dependent CDI, by depletion of the SR Ca²⁺ with thapsigargin, significantly prolonged AP duration (APD). APD changed in parallel with the magnitude of CICR during the recovery of the SR Ca²⁺ content after transient depletion by caffeine. Such CICR-dependent change of APD persisted under the highly Ca²⁺ buffered condition where the Ca²⁺ signalling was restricted to nanoscale domains. Blockers of the Ca²⁺-dependent Cl^- channel or the BK channel did not affect AP waveform. The amount of Ca²⁺-influx through Ca²⁺ channels during the SR-depleted type AP waveform, measured in the SR-depleted myocyte, was increased by 40 % over that during the SR-intact type AP waveform measured in the SR-intact myocyte. The protein kinase A stimulation further enhanced the Ca²⁺-influx during AP under the SR-depleted condition to 70 % of that under the SR-intact condition. These results indicate that the CICR-dependent CDI of L-type Ca²⁺ channels, under control of the privileged cross-signalling between L-type Ca²⁺ channels and RyRs, play important roles for monitoring and tuning the SR Ca²⁺ content via changes of AP waveform and the amount of Ca²⁺-influx during AP in ventricular myocytes.

(Resubmitted 4 July 2003; accepted 30 July 2003 ; first published online 1 August 2003)
Corresponding author S. Adachi-Akahane: Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Email: satomiaa{at}mol.f.u-tokyo.ac.jp

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

In cardiac excitation-contraction coupling (E-C coupling), gating kinetics of L-type Ca²⁺ channels and ryanodine receptors (RyRs) are mutually regulated via advantageous cross-signalling between the two channels in the confined space of the dyad junction that is largely inaccessible to exogenous Ca²⁺ buffers (Sham et al. 1995; Adachi-Akahane et al. 1996).

It is well known that the inactivation of L-type Ca²⁺ channels depends on voltage and cytosolic Ca²⁺ ([Ca²⁺]_i) (Bers, 2001). Recent studies proposed that, in response to Ca²⁺ influx through the L-type Ca²⁺ channel, the Ca²⁺-bound calmodulin (CaM) interacts with the IQ motif located in the carboxyl tail of the pore-forming $alpha$ _1C subunit of L-type Ca²⁺ channels to cause a conformational change of the Ca²⁺ channel leading to Ca²⁺-dependent inactivation (CDI) (Pitt et al. 2001; Erickson et al. 2001). In contrast to recent advances in the clarification of the molecular mechanism underlying CDI of the Ca²⁺ channel, however, its physiological role in cardiac E-C coupling, with respect to its contribution to action potential (AP) waveform still remains to be elucidated. In rat ventricular myocytes, the fast Ca²⁺-dependent inactivation of the L-type Ca²⁺ channel produced by the Ca²⁺-induced Ca²⁺ release (CICR-dependent CDI) occurs during AP. In adult ventricular myocytes especially, the CICR trigged by the nanoscale cross communication between the Ca²⁺ channel and RyR, further accelerates the inactivation rate of Ca²⁺ channels so its time constant becomes less than 10 ms (Hadley & Hume, 1987). The role of CDI on AP waveform or on the amount of Ca²⁺ influx during the fixed AP waveform has been discussed based on experimental and simulation studies (Linz & Meyer, 1998; Winslow et al. 1999; Fanconnier et al. 2003). However, the contribution of the CICR-dependent CDI of the L-type Ca²⁺ channel to AP waveform and the consequent change of the total amount of Ca²⁺ influx during AP has never been directly addressed. In this study, we aimed at elucidating the physiological role of the CICR-dependent CDI of Ca²⁺ channels in the regulation of AP waveform and also the total amount of Ca²⁺ influx during AP in ventricular myocytes. For this purpose, we investigated the impact of the elimination of CICR-dependent CDI of the Ca²⁺ channel on AP waveform and the relationship between AP and Ca²⁺ currents (I_Ca) under the SR-intact and SR-depleted conditions in adult rat ventricular myocytes. Our results indicate that the CICR-dependent CDI of the L-type Ca²⁺ channel manipulates AP duration (APD) and the total Ca²⁺ influx through Ca²⁺ channels during AP. We conclude that the L-type Ca²⁺ channel serves as a sensor of the SR Ca²⁺ content via the CICR-dependent CDI, thus fine tuning the SR Ca²⁺ content at optimum level to ensure the efficacy of CICR in ventricular myocytes.

METHODS

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Abstract
Introduction
Methods
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Isolation of rat ventricular myocytes

The experiments were carried out according to the Guidelines of Animal Experiments in the University of Tokyo. Rat ventricular myocytes were isolated according to the previously described method (Adachi-Akahane et al. 1996). Sprague-Dawley rats (male, 5- to 6- weeks old) were deeply anaesthetized with sodium pentobarbital (50 mg kg^-1, I.P.), the hearts were quickly excised and perfused in a Langendorff apparatus first with Ca²⁺-free Tyrode solution containing (mM): NaCl 137, KCl 5.4, MgCl₂ 1, Hepes 10, glucose 10, pH 7.4, at 37 °C. Then they were perfused with Ca²⁺-free Tyrode solution containing collagenase (collagenase S-1, Nitta Gelatin Inc.) and protease (type XIV, Sigma), and finally with Tyrode solution containing 0.2 mM Ca²⁺. The myocytes were then dispersed in 0.2 mM Ca²⁺-Tyrode solution and stored at room temperature.

Electrophysiological recordings

Electrophysiological recordings were performed in the whole cell patch-clamp configuration with Axopatch 200B via an A/D converter Digidata 1200 (Axon Instruments, Inc.). The resistance of the electrode pipette was 1 to 3 M $Omega$ when filled with the internal solution. The series resistances were less than three times as large as the original pipette resistances, which were electronically compensated. Signals were sampled at 5-10 kHz, filtered at 2-5 kHz, digitized, and stored. External solutions were quickly exchanged via a concentration-clamp device (Cleemann & Morad, 1991). All measurements were performed at room temperature.

Action potential and K⁺ currents were recorded with a pipette solution containing (mM): 130 KCl, 5 sodium phosphocreatine, 5 Mg-ATP, 0.1 K₄-BAPTA, 10 Hepes, pH 7.3. In some experiments as shown in Fig. 3 and Fig. 6, APs were recorded with the pipette solution containing (mM): 30 KCl, 100 potassium glutamate, 5 sodium phosphocreatine, 5 Mg-ATP, 0.1 K₄-BAPTA, 10 Hepes, pH 7.3. Normal Tyrode solution containing (mM): NaCl 137, KCl 5.4, MgCl₂ 1, CaCl₂ 2, Hepes 10, glucose 10, pH 7.4, was used as the extracellular solution. In experiments with a highly Ca²⁺ buffered condition (Fig. 6), the pipette solution contained K₄-BAPTA (1 or 2 mM) and [Ca²⁺]_i was buffered to 100 nM. APs were evoked by brief depolarizing current pulses (1 ms, 150 % threshold). APD was assessed as the time from the overshoot to the indicated percentage of repolarization.

Calcium currents through L-type Ca²⁺ channels (I_Ca) and the transient outward K⁺ currents were recorded under AP-clamp conditions. The AP waveforms that most closely matched the mean APD were used for the AP-clamp experiments (Figs 3Ba, 7A, and 8A). I_Ca was recorded with the pipette solution containing (mM): 120 caesium methanesulfonate, 20 TEACl, 10 Hepes, 5 sodium phosphocreatine, 0.2 Na-GTP, 5 Mg-ATP, 0.1 K₄-BAPTA (or 2 K₄-BAPTA in Fig. 5Bb), pH 7.3; under the blockade of a Na⁺ current and inward-rectifier K⁺ current with tetrodotoxin (10 µM) and Ba²⁺ (0.2 mM), respectively. I_Ca was estimated as the difference between the current in the absence and presence of Cd²⁺ (0.2 mM). This Cd²⁺-sensitive current was integrated to estimate the total Ca²⁺ influx through the L-type Ca²⁺ channels ( int I_Ca dt). The transient outward K⁺ current was measured as the 4-aminopyridine (4-AP, 1 mM)-sensitive current component (I_K-4AP).

Measurement of intracellular Ca²⁺ transients

Intracellular Ca²⁺ transients and membrane potentials were simultaneously recorded in rat ventricular myocytes with fura-2 (100 µM), introduced into myocytes through the patch pipettes, according to the method previously described (Adachi-Akahane et al. 1996). Fura-2 fluorescence was exited at 340 and 410 nm, and measured at 510 nm. The free-Ca²⁺ concentration was ratiometrically calculated.

Statistics

All data are presented as the mean ± S.E.M. with n given as the number of cells. Statistical analysis was performed with Student's t test. P < 0.05 was accepted as being statistically significant.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

APD was prolonged by the depletion of the SR Ca²⁺

Since I_Ca are responsible for the plateau phase of APs, we investigated the role of the CICR-dependent CDI of Ca²⁺ channels in shaping AP waveform. For this purpose, we first examined the effect of the depletion of SR Ca²⁺ by treatment with thapsigargin (1 µM) on AP waveform (Fig. 1). Resting membrane potential (E_rest) and the peak amplitude of overshoot (E_peak) were unchanged by the depletion of the SR Ca²⁺ (E_rest and E_peak before vs. after treatment with thapsigargin, -73.2 ± 0.7 mV and 52.9 ± 2.2 mV vs. -74.7 ± 0.6 mV and 53.5 ± 1.8 mV, respectively, P n.s.). Surprisingly APD, particularly the plateau phase, was gradually prolonged as the SR Ca²⁺ content was depleted (Fig. 1A). Eventually, APD₅₀ of SR-depleted cells was prolonged by twofold as compared with that of SR-intact cells (Fig. 1B).

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Figure 1. Effect of elimination of Ca²⁺-dependent inactivation by depletion of the SR Ca²⁺ on action potential waveform
A, time course of the change of action potential (AP) waveform by treatment with thapsigargin (1 µM). Typical recordings of APs electrically evoked at a frequency of 0.033 Hz in a ventricular myocyte are superimposed. B, summary of AP duration (APD) recorded before ( circle ) and after ( filled circle ) the depletion of the SR Ca²⁺ by the blockade of SR Ca²⁺-ATPase with thapsigargin. APs were evoked at a frequency of 0.2 Hz (n = 7). Values are represented as the mean ± S.E.M. * P < 0.05, ** P < 0.01 vs. SR intact.

The prolonged APD was shortened in parallel with the restoration of the SR Ca²⁺

We next examined whether the prolonged APD by depletion of the SR Ca²⁺ was restored by the refilling of the SR Ca²⁺ (Fig. 2). The SR Ca²⁺ was transiently depleted by the exposure to caffeine (5 mM) and then was loaded up by stimulation of the I_Ca after the removal of caffeine. In the presence of caffeine, Ca²⁺ transient was small and APD was as long as that recorded after treatment with thapsigargin (Fig. 2A). After the removal of caffeine, as expected, APD was gradually shortened to the basal length in parallel with the restoration of the magnitude of Ca²⁺ transients during the refilling of Ca²⁺ in the SR as the myocyte was stimulated (Fig. 2B). These results suggest that the CICR-dependent CDI of Ca²⁺ channels may abbreviate the plateau phase of AP.

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Figure 2. The relationship between APD and Ca²⁺ transient
A, time course of the change of AP waveform during the reloading phase of SR Ca²⁺ after wash out of caffeine. Typical recordings of AP (upper) and Ca²⁺ transients (lower) electrically evoked at a frequency of 0.2 Hz in a ventricular myocyte before and on exposure to caffeine, and during the reloading phase of the SR Ca²⁺ by stimulation are shown. B, time course of APD₅₀ ( circle ), representing time from peak to 50 % repolarization of AP, and peak magnitude of Ca²⁺ transients ( filled circle ) measured before and during the depletion of the SR Ca²⁺ by caffeine, and during the reloading phase of the SR Ca²⁺ after wash out of caffeine.

The contribution of other ionic currents to the prolongation of AP

Other ionic currents sensitive to CICR could also contribute to the prolongation of APD by the depletion of the SR Ca²⁺. Alteration of Ca²⁺-sensitive current components such as Cl^- or K⁺ channels, if any, is likely to prolong APD. We checked the possible involvement of Ca²⁺-sensitive Cl^- channel to AP waveform. Application of niflumic acid at 50 µM had no effect on the AP waveform (Fig. 3Aa). Another Cl^- channel blocker, diisothiocyanatestilbene-2,2'-disulfonic acid (DIDS) at 200 µM had no effect on AP waveform either (data not shown). Iberiotoxin (0.1 µM), a selective blocker of BK channels, had no effect on the AP waveform (Fig. 3Ab). In the presence of these blockers, AP continued to be prolonged by the elimination of the CICR by thapsigargin (data not shown). These results confirm that neither Ca²⁺-sensitive Cl^- channels nor BK channels are involved in AP waveform in rat ventricular myocytes.

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Figure 3. Effect of the elimination of CICR on other Ca²⁺ -activated ionic currents
A, typical traces of APs before (black line) and after (grey line) the treatments with niflumic acid (NFA, 50 µM; a) or iberiotoxin (IbTX, 0.1 µM; b). NFA and IbTX had no effects on AP waveform (APD₅₀ : control 16.4 ± 2.4 ms vs. NFA 15.8 ± 1.7 ms, n = 5; control 16.3 ± 8.2 ms vs. IbTX 17.1 ± 8.6 ms, n = 3). B, SR-intact type AP waveform used for voltage clamp (a) and typical I_K-4AP traces, assessed as the 4-AP-sensitive component of membrane currents under AP-clamp condition, recorded in ventricular myocytes in the absence (b) and the presence (c) of Cd²⁺ (0.2 mM) in order to block the Ca²⁺ influx and CICR. The currents recorded before (black) and after (grey) the depletion of the SR Ca²⁺ by caffeine are superimposed.

Transient outward K⁺ current components such as Kv channels could be involved in the CICR-dependent APD shortening, since Ca²⁺-dependent modulation of Kv4 channel by Kv channel-interacting proteins (KChIPs) and neuronal calcium sensor protein-1 have been suggested (An et al. 2000; Deschênes et al. 2002; Guo et al. 2002). Therefore, we examined the dependence of the 4-AP-sensitive K⁺ current (I_K-4AP) on CICR. The depletion of the SR Ca²⁺ by thapsigargin or caffeine (5 mM) slightly attenuated I_K-4AP (Fig. 3Bb) that was recorded with AP clamp (Fig. 3Ba). This 4-AP sensitive CICR-dependent outward current component was abolished when the Ca²⁺ channel was blocked by Cd²⁺ (0.2 mM, Fig. 3Bc) or when K⁺ channels were blocked by Cs⁺ and TEA⁺ from the intracellular side (data not shown). These results suggest that the CICR-dependent 4-AP-sensitive K⁺ currents (I_K-4AP,CICR) may exist in rat ventricular myocytes. In order to exclude the possible contribution of I_K-4AP,CICR to the CICR-dependent prolongation of APD, we examined the effects of the depletion of SR Ca²⁺ on AP waveform under the blockade of I_K-4AP,CICR by 4-AP at 1 mM (Fig. 4). Overall APD was prolonged to threefold after the treatment with 4-AP. Under this condition, the depletion of the SR Ca²⁺ by thapsigargin further prolonged APD by another twofold. Especially, the plateau phase of AP was markedly prolonged. These results indicate that the CICR-dependent change of APD is not explained by the Ca²⁺-dependent Cl^- channels or K⁺ channels.

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Figure 4. AP prolongation by the depletion of the SR Ca²⁺ under the blockade of transient K⁺ currents by 4-AP
A, AP recorded from a ventricular myocyte at a stimulation frequency of 0.2 Hz at baseline (black line) and after the application of 1 mM 4-AP (dotted line) or 4-AP + 1 µM thapsigargin (grey line) are superimposed. B, summary of APD recorded at a frequency of 0.2 Hz (n = 4). Values are represented as the mean ± S.E.M. * P < 0.05 vs. SR intact + 4-AP.

Forward mode Na⁺-Ca²⁺ exchanger (NCX) activity, which induces inward currents leading to depolarization, is probably the cause of the prolongation of APD, because it is the major pathway for extruding Ca²⁺ under the blockade of SR Ca²⁺-ATPase. We examined the influence of the forward mode NCX activity on AP waveform by eliminating the forward mode NCX activity by replacing the extracellular Na⁺ by Li⁺ (Fig. 5). In SR-intact cells, the replacement of Na⁺ by Li⁺ eliminated the low plateau phase of AP at around -40 mV, which resulted in the significant shortening of APD₉₀ (Fig. 5Aa). On the other hand, the blockade of forward mode NCX activity abbreviated overall APD in SR-depleted cells (Fig. 5Ab). This abbreviation of APD may have resulted from CDI of the Ca²⁺ channel due to the build up of subsarcolemmal [Ca²⁺]_i by Ca²⁺ influx through the Ca²⁺ channel under the condition where both NCX and SR Ca²⁺ ATPase were blocked. To test this possibility, we recorded I_Ca in SR-depleted cells under the inhibition of forward mode NCX activity. As shown in Fig. 5Ba, the inhibition of the forward mode NCX activity significantly suppressed I_Ca amplitude in weakly Ca²⁺ buffered condition (BAPTA 0.1 mM). In contrast, when [Ca²⁺]_i was highly buffered by dialysis with BAPTA (2 mM), the reduction of I_Ca was much smaller than in the weakly Ca²⁺-buffered conditions (Fig. 5Bb). These results indicate that the block of forward mode NCX activity, in addition to the block of SR Ca²⁺ ATPase activity, results in the local build-up of [Ca²⁺]_i around the Ca²⁺ channel to produce CDI of I_Ca and the reduction of APD. Therefore the CICR-dependent CDI of the L-type Ca²⁺ channels, but not the forward mode NCX activity, appears to be responsible for the CICR-dependent change of APD.

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Figure 5. Contribution of Na⁺-Ca²⁺ exchange currents to the prolongation of APD by the depletion of the SR Ca²⁺
A, summary of APD recorded in SR-intact (a) and SR-depleted (b) ventricular myocytes at a frequency of 0.2 Hz under conditions where forward mode Na⁺-Ca²⁺ exchanger (NCX) activity was functional (Na⁺) or blunted (Na⁺ replaced by Li⁺; n = 7). Insets, typical AP traces recorded in a single ventricular myocyte with and without forward mode NCX activity (black and grey lines, respectively). B, effect of the inhibition of forward mode NCX activity on I_Ca recorded under voltage clamp with the SR-depleted type AP waveform (top). I_Ca was recorded as Cd²⁺-sensitive component in the SR-depleted cells under the conditions where forward mode NCX activity was intact (black line) or inhibited by the replacement of Na⁺ by Li⁺ (grey line). The averaged current traces are shown. The amplitude of I_Ca was sensitive to the blockade of NCX in the presence of low Ca²⁺ buffer (BAPTA 0.1 mM, n = 7; a), but was significantly less sensitive in the presence of high Ca²⁺ buffer (BAPTA 2 mM, n = 3; b). * P < 0.05 vs. SR intact.

CICR-dependence of APD persisted under the strong buffering of cytosolic Ca²⁺

In order to examine whether the CICR-dependent change of APD was caused by the privileged cross signalling between L-type Ca²⁺ channels and RyRs, we tested the effect of SR Ca²⁺ depletion on AP waveform under the strong buffering of cytosolic Ca²⁺, which allowed us to limit the diffusion distance of free Ca²⁺ within 30 nm (Adachi-Akahane et al. 1996). As shown in Fig. 6, APD was markedly prolonged by the elimination of CICR after treatment with thapsigargin. These results indicate that the AP waveform is regulated by the CICR-dependent CDI as a result of the individual local communication between Ca²⁺ channels and proximate RyRs.

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Figure 6. Effect of the elimination of CICR on APD under the high buffer condition
AP were recorded from a ventricular myocyte at a stimulation frequency of 0.2 Hz under high Ca²⁺ buffer conditions sufficient to abolish contraction and Ca²⁺-activated ionic currents ([Ca²⁺]_i was buffered to 100 nM with BAPTA (1 or 2 mM) and Ca²⁺). A, typical traces of AP before (black line) and after (grey line) the depletion of the SR Ca²⁺ by thapsigargin were superimposed. B, summary of APD recorded at a frequency of 0.2 Hz (n = 4). Values are represented as the mean ± S.E.M. * P < 0.05, ** P < 0.01 vs. SR intact.

Changes in APD by CICR-dependent CDI of Ca²⁺ channels influenced the total amount of Ca²⁺ influx through L-type Ca²⁺ channels

We explored the relationship between AP and I_Ca by measuring I_Ca elicited by voltage clamp with AP waveform to clarify the influence of the prolongation of APD by the elimination of CICR-dependent CDI on the total amount of Ca²⁺ influx through Ca²⁺ channels (Fig. 7). When I_Ca was evoked in SR-depleted cells with the SR-depleted type AP waveform, the duration of I_Ca was longer but the peak amplitude of I_Ca was smaller compared with those recorded with the SR-intact type AP waveform in SR-intact cells (Fig. 7A bottom). Summarized data show that, in SR-depleted cells, the peak amplitude of I_Ca was smaller by 20 % compared with that recorded in SR-intact cells (Fig. 7C). However, current-voltage relationships showed that the slope in the high voltage range, representing the maximal channel conductance during AP, was unchanged between the SR-intact and the SR-depleted groups (Fig. 7B). These results indicate that the Ca²⁺ channel conductance by itself was not altered by the prolongation of APD. Therefore, the reduction of I_Ca peak amplitude appears to be due to the significant reduction of the electrochemical driving force for Ca²⁺ at the plateau phase of the prolonged AP. Nevertheless, interestingly, the total amount of Ca²⁺ influx through Ca²⁺ channels in the SR-depleted cells was increased by 40 % compared with that of SR-intact cells (Fig. 7C). These results demonstrate that the CICR-dependent CDI limits the total Ca²⁺ influx through Ca²⁺ channels during AP by shortening APD.

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Figure 7. Effect of AP prolongation on the amount of Ca²⁺ influx through L-type Ca²⁺ channels
A, AP-clamp waveforms (top) and averaged I_Ca traces recorded under AP-clamp conditions (bottom). B, the relationship between membrane potential and I_Ca during AP clamp (top panel). Bottom panel shows the I_Ca-voltage relationships normalized at the peak amplitude. C, summary of peak amplitude of I_Ca and the total amount of Ca²⁺ influx ( int I_Cadt; n = 7) recorded with the SR-intact AP in the SR-intact ventricular myocytes ( square ) and with the SR-depleted AP in the SR-depleted myocytes ( filled square ). Values are represented as the mean ± S.E.M. *P < 0.05 vs. SR intact.

Protein kinase A (PKA) stimulation enhances Ca²⁺ signalling in cardiac myocytes through an increase of the Ca²⁺ influx via L-type Ca²⁺ channels and the upregulation of the SR Ca²⁺-uptake activity. Therefore, the enhanced CICR may produce larger CDI, which may increase the contribution of CICR-dependent CDI in the AP waveform and the resulting total amount of Ca²⁺ influx. This possibility was tested by examining the effects of the elimination of CICR on the AP waveform under the stimulation of PKA by cAMP that was intracellularly applied via patch pipette. As shown in Fig. 8A, in the presence of the intracellular cAMP (0.2 mM), the low plateau of AP was markedly prolonged when the SR was intact (Fig. 1). This prolongation appears to be due to the CICR-induced forward mode NCX activity (Fig. 5), which was confirmed by the results that the low plateau component was abolished by the elimination of CICR with thapsigargin (Fig. 8A). Interestingly, the thapsigargin treatment prolonged AP waveform with marked prolongation of the early repolarizing phase at +20 to -20 mV as summarized in Fig. 8B. Similar to that observed in the absence of cAMP (Fig. 7C), under an AP-clamp condition, the peak amplitude of I_Ca was slightly smaller with the SR-depleted AP compared with the SR-intact AP (Fig. 8D). However, the amount of Ca²⁺ influx was significantly larger with the SR-depleted AP than with the SR-intact AP by approximately 70 % (Fig. 8D), which was more pronounced than the results without cAMP (Fig. 7C).

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Figure 8. Effect of the elimination of CICR on APD and Ca²⁺ influx under the stimulation of PKA
AP and I_Ca were recorded with pipette solution containing cAMP (0.2 mM). A, typical traces of AP before (black line) and after (grey line) the depletion of the SR Ca²⁺ by thapsigargin. These AP traces were used as command pulses in the AP-clamp experiments. B, summary of APD recorded at a frequency of 0.2 Hz (n = 6). C, averaged I_Ca traces recorded under AP-clamp conditions. D, summary of peak amplitude of I_Ca and the total Ca²⁺ influx ( int I_Ca dt; n = 5). Values are represented as the mean ± S.E.M. * P < 0.01 vs. SR intact.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

In this study, we demonstrated that the CICR-dependent CDI of voltage-dependent L-type Ca²⁺ channels limits APD and thereby regulates the total amount of Ca²⁺ influx through Ca²⁺ channels during AP in rat ventricular myocytes.

The contribution of Ca²⁺-dependent ion channels other than L-type Ca²⁺ channels to APD in ventricular myocytes

We observed that part of I_K-4AP was sensitive to Cd²⁺ (0.2 mM) and CICR (Fig. 3). Importantly, under the blockade of I_K-4AP by 4-AP (1 mM), the CICR-dependent CDI of Ca²⁺ channel turned out to exert a marked effect on APD (Fig. 3 and Fig. 4). We, however, did not further characterize I_K-4AP,CICR, because its contribution appeared to be minor compared with that of the CICR-dependent CDI of Ca²⁺ channel.

As shown in Fig. 5A, the prolongation of APD by the depletion of SR Ca²⁺ was sensitive to the block of forward mode NCX activity. There are two reasons for such an abbreviation of APD by the blockade of the forward mode NCX activity. (1) NCX actually participates in shaping AP waveform under SR-depleted conditions, or (2) the blockade of NCX attenuates I_Ca as a result of the local build-up of subsarcolemmal [Ca²⁺]_i. As forward mode NCX currents, activated by Ca²⁺ released from the SR by caffeine, has a much smaller conductance compared with other ion currents, forward mode NCX currents in the absence of CICR are implausible as an influence on AP waveform. This could be due to the large background membrane conductance deriving from K⁺ channels and Ca²⁺ channels, even though, in rat ventricular myocytes, forward mode NCX currents contribute to the low plateau phase of AP where membrane conductance is small (Fig. 5A). Under the blockade of SR Ca²⁺ ATPase activity by thapsigargin, the inhibition of NCX suppressed I_Ca and this suppression was attenuated by high Ca²⁺ buffer (Fig. 5B), suggesting that I_Ca was inactivated because of the elevation of local [Ca²⁺]_i by the blockade of the Ca²⁺ extrusion via NCX. Thus, the reduction of APD by the block of forward mode NCX activity, combined with the block of the SR Ca²⁺ATPase activity, was primarily due to the CDI of Ca²⁺ currents by the local build-up of subsarcolemmal [Ca²⁺]_i.

CICR-dependent CDI of L-type Ca²⁺ channels determines APD and the SR Ca²⁺ content

Voltage-clamp experiments with the fixed AP waveform have demonstrated that CICR-dependent inactivation of I_Ca reduced the integrated Ca²⁺ influx during APs by 30 % (Grantham et al. 1996). However, regarding the contribution of CICR to AP waveform, it was reported in rat cardiac myocytes, that ryanodine eliminates the low plateau phase of AP, but does not affect the early plateau phase that is determined by I_Ca (Mitchell et al. 1984a). Since Ca²⁺ channels are inactivated by a Ca²⁺ influx through them, the effect of CDI on AP waveform could have been underestimated. Indeed, it has been reported that, when Sr²⁺ was used as a charge carrier in place of Ca²⁺, APD was markedly prolonged in rat ventricular myocytes (Mitchell et al. 1984b). As are shown in Fig. 1 and Fig. 2, APD of rat ventricular myocytes was strongly influenced by the SR Ca²⁺ content through the CICR-dependent CDI of Ca²⁺ channels. Since the electrochemical driving force for Ca²⁺ is relatively constant in the plateau phase of APs in ventricular myocytes of guinea-pig or ferret, in contrast to rat, the reduction of the peak I_Ca by the prolongation of APD would be much smaller. Thus, in these species, a much larger effect on the SR Ca²⁺ content of the change of APD by the CICR-dependent CDI of Ca²⁺ currents would be expected. In fact, Trafford et al. showed that the transient depletion of the SR Ca²⁺ by exposure to caffeine increased APD in ferret ventricular myocytes (Trafford et al. 1997). In addition, this APD modulation by CDI is supported by the result that the elimination of CDI, by use of the Ca²⁺-insensitive mutant CaM, markedly prolonged APD in guinea-pig ventricular myocytes (Alseikhan et al. 2002).

The proximity of the cross communication between L-type Ca²⁺ channels and RyRs were confirmed by the resistance of the CICR-dependent change of AP waveform to the strong buffering of the cytosolic Ca²⁺ (Fig. 6). These results demonstrate that L-type Ca²⁺ channels are the important determinants of APD. We, however, failed to observe the APD modulation by the CICR-dependent CDI in mouse ventricular myocytes (data not shown), indicating that the large K⁺ currents causing the ultra-rapid repolarization and extremely short APD in mouse ventricular myocytes could invalidate the effect of the CICR-dependent CDI on AP waveform.

We observed the additive prolongation of APD by the depletion of the SR Ca²⁺ under the blockade of K⁺ channel current by 4-AP (Fig. 4). It is well known that, in heart failure, in addition to the depression of K⁺ currents, Ca²⁺ transients are generally depressed (Winslow et al. 1999). Therefore, not only the depression of K⁺ currents but also the attenuation of the CICR-dependent CDI could be involved in the prolongation of APD observed in heart failure.

Considering that the prolonged APD, caused by the depletion of the SR Ca²⁺, went back to the original APD in parallel with the recovery of Ca²⁺ transients (Fig. 2) and that SR-depleted type AP waveform induced a larger amount of Ca²⁺ influx through Ca²⁺ channels than the SR-intact type AP waveform (Fig. 7), the CICR-dependent CDI plays an important physiological role in maintaining the SR Ca²⁺ content constant to keep the optimum coupling efficacy of CICR. It is also suggested that the alteration of the balance between Ca²⁺ influx through Ca²⁺ channels and Ca²⁺ efflux via NCX determines the SR Ca²⁺ content (Trafford et al. 1997). The mechanism for maintaining the SR Ca²⁺ content is explained as follows. If the SR has a sufficiently high Ca²⁺ content, the CICR-dependent CDI of Ca²⁺ channels prevents the SR from Ca²⁺ overload by restricting Ca²⁺ influx through Ca²⁺ channels via abbreviation of APD (Bouchard et al. 1995; Sah et al. 2001). Conversely, if the SR Ca²⁺ content is decreased, the CICR-dependent CDI is eliminated and APD is prolonged, thus resulting in an increase of the total amount of Ca²⁺ influx through Ca²⁺ channels during AP (Fig 7). In addition, the reduction of CICR also blunts the Ca²⁺ efflux via NCX (Eisner et al. 2000). The depolarized membrane potential during prolonged APD reduces the forward mode NCX activity, which also helps the load up of the SR Ca²⁺. Consequently, the enhanced total Ca²⁺ influx through Ca²⁺ channels and the reduced Ca²⁺ extrusion via NCX promote the recovery of the SR Ca²⁺ content to the optimum level.

The voltage-dependent inactivation (VDI) component of the L-type Ca²⁺ current is fitted to fast and slow inactivation time constants. The fast component with time constants of 20-50 ms is strongly voltage dependent and its inactivation rate and amplitude becomes larger at voltages positive to 0 mV, while the slow component has time constants of 300-500 ms and is mostly independent of membrane voltages (Mitarai et al. 2000; Findlay, 2002). At the overshoot of AP, the fast VDI component would make a significant contribution to the repolarizing phase. The kinetics of the repolarizing phase determines the driving force for the Ca²⁺ influx and thus determines the peak amplitude of I_Ca as we show in the present study (Fig. 7 and Fig. 8). The fast VDI component would make a more significant contribution to APD in ventricular myocytes having longer APD, such as guinea-pigs and ferrets, than that observed in rat ventricular myocytes in the present study. In cardiac myocytes that do not have a rapidly repolarizing component of AP, such as guinea-pigs, the fast VDI of L-type Ca²⁺ channels may govern the first decaying component of Ca²⁺ currents during AP (Linz & Meyer, 1998). The contribution of the fast VDI component would become larger at more depolarized voltages, while the contribution of the CICR-dependent CDI component becomes less, because the gain of CICR becomes smaller as the voltage approaches the reversal potential for Ca²⁺ (E_Ca). On the other hand, in myocytes with a rapidly repolarizing component of AP, such as rat or mouse, the relative contribution of the fast VDI component may be compromised. The fast VDI component and the deactivation component of the Ca²⁺ currents could overlap during the short APD (Fig. 7). In contrast, in these myocytes, the gain of CICR is higher and the contribution of the CICR-dependent CDI to APD may be larger. The inactivation kinetics of the CICR-dependent CDI matches well with the rising kinetics of Ca²⁺ transients, typically 5-10 ms, as has been shown (Adachi-Akahane et al. 1996). This inactivation component determines the early repolarizing phase of AP and thus the total amount of Ca²⁺ influx during AP (Figs 1, 4 and 6).

It has been reported that, on PKA stimulation, the contribution of the fast VDI component is diminished and the contribution of the slow VDI component becomes dominant, which makes the overall VDI slower and allows CDI to govern the inactivation kinetics (Mitarai et al. 2000; Findlay, 2002). Our data demonstrate that PKA stimulation enhances the contribution of the CICR-dependent CDI to APD and thus to the total amount of Ca²⁺ influx during AP (Fig. 8). This result is explained by the upregulation of CICR and possibly by the enhancement of the relative contribution of the CDI to the inactivation kinetics of Ca²⁺ channels by PKA stimulation as has been recently proposed (Mitarai et al. 2000; Findlay, 2002). Under PKA stimulation, the early repolarizing phase, APD₃₀ at around +10 mV, was significantly prolonged by the elimination of CICR (compare Fig. 8 with Fig. 1), which suggests that PKA stimulation, in addition to the acceleration of the CICR, shifted the relative contribution of the CDI to the inactivation kinetics of the Ca²⁺ channel towards more depolarized voltages.

In the present study, we demonstrated that the CICR-dependent CDI of L-type Ca²⁺ channels manipulates APD and thereby regulates the total Ca²⁺ influx through Ca²⁺ channels during AP in rat ventricular myocytes. We conclude that, in cardiac E-C coupling, L-type Ca²⁺ channels serve as a sensor and a manipulator of the SR Ca²⁺ content on the basis of nanoscale cross communication between the L-type Ca²⁺ channel and RyRs, thus fine tuning the SR Ca²⁺ content for maintaining the efficacy of I_Ca to trigger CICR.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

Adachi-Akahane S, Cleemann L & Morad M (1996). Cross-signaling between L-type Ca²⁺ channels and ryanodine receptors in rat ventricular myocytes. J Gen Physiol 108, 435-454 [Abstract]

Alseikhan BA, Demaria CD, Colecraft HM & Yue DT (2002). Engineered calmodulins reveal the unexpected eminence of Ca²⁺ channel inactivation in controlling heart excitation. Proc Natl Acad Sci U S A 99, 17185-17190 [Abstract/Full Text]

An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS & Rhodes KJ (2000). Modulation of A-type potassium channels by a family of calcium sensors. Nature 403, 553-556 [Medline]

Bers DM, (2001). Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer Academic Press, Dordrecht, Netherlands

Bouchard RA, Clark RB & Giles WR (1995). Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes. Action potential voltage-clamp measurements. Circ Res 76, 790-801 [Abstract/Full Text]

Cleemann L , & Morad M (1991). Role of Ca²⁺ channel in cardiac excitation-contraction coupling in the rat: evidence from Ca²⁺ transients and contraction. J Physiol 432, 283-312 [Abstract]

Desch, ênes I, DiSilvestre D, Juang GJ, Wu RC, An WF & Tomaselli GF (2002). Regulation of Kv4. 3 current by KChIP2 splice variants: a component of native cardiac I_to? Circulation 106, 423-429

Eisner DA, Choi HS, Diaz ME, Okneill SC & Tradfford AW (2000). Integrative analysis of calcium cycling in cardiac muscle. Circ Res 87, 1087-1094 [Abstract/Full Text]

Erickson MG, Alseikhan BA, Peterson BZ & Yue DT (2001). Pre-association of calmodulin with voltage-gated Ca²⁺ channels revealed by FRET in single living cells. Neuron 31, 973-985 [Medline]

Fauconnier J, Bedut S, Guennec JYL, Babuty D & Richard S (2003). Ca²⁺ current-mediated regulation of action potential by pacing rate in rat ventricular myocytes. Cardiovasc Res 57, 670-680 [Medline]

Findlay I, (2002). $beta$ -Adrenergic stimulation modulates Ca²⁺- and voltage-dependent inactivation of L-type Ca²⁺ channel currents in guinea-pig ventricular myocytes. J Physiol 541, 741-751 [Abstract/Full Text]

Grantham CJ , & Cannell MB (1996). Ca²⁺ influx during the cardiac action potential in guinea pig ventricular myocytes. Circ Res 79, 194-200 [Abstract/Full Text]

Guo W, Malin SA, Johns DC, Jeromin A & Nerbonne JM (2002). Modulation of Kv4-encoded K⁺ currents in the mammalian myocardium by neuronal calcium sensor-1. J Biol Chem 277, 26436-26443 [Abstract/Full Text]

Hadley RW , & Hume JR (1987). An intrinsic potential-dependent inactivation mechanism associated with calcium channels in guinea-pig myocytes. J Physiol 389, 205-222 [Abstract]

Linz KW , & Meyer R (1998). Control of L-type calcium current during the action potential of guinea-pig ventricular myocytes. J Physiol 513, 425-442 [Abstract/Full Text]

Mitarai S, Kaibara M, Yano K & Taniyama K (2000). Two distinct inactivation processes related to phosphorylation in cardiac L-type Ca²⁺ channel currents. Am J Physiol Cell Physiol 279, C603-610 [Abstract/Full Text]

Mitchell MR, Powell T, Terrar DA & Twist VW (1984a). The effects of ryanodine, EGTA and low-sodium on action potentials in rat and guinea-pig ventricular myocytes: evidence for two inward currents during the plateau. Br J Pharmacol 81, 543-550 [Abstract]

Mitchell MR, Powell T, Terrar DA & Twist VW (1984b). Strontium, nifedipine and 4-aminopyridine modify the time course of the action potential in cells from rat ventricular muscle. Br J Pharmacol 81, 551-556 [Abstract]

Pitt GS, Zuhlke RD, Hudmon A, Schulman H, Reuter H & Tsien RW (2001). Molecular basis of calmodulin tethering and Ca²⁺-dependent inactivation of L-type Ca²⁺ channels. J Biol Chem 276, 30794-30802 [Abstract/Full Text]

Sah R, Ramirez RJ, Kaprielian R & Backx PH (2001). Alterations in action potential profile enhance excitation-contraction coupling in rat cardiac myocytes. J Physiol 533, 201-214 [Abstract/Full Text]

Sham JS, Cleemann L & Morad M (1995). Functional coupling of Ca²⁺ channels and ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci U S A 92, 121-125 [Abstract]

Trafford AW, Diaz ME, Negretti N & Eisner DA (1997). Enhanced Ca²⁺ current and decreased Ca²⁺ efflux restore sarcoplasmic reticulum Ca²⁺ content after depletion. Circ Res 81, 477-484 [Abstract/Full Text]

Winslow RL, Rice J, Jafri S, Marbán E & O'Rourke B (1999). Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, II: model studies. Circ Res 84, 571-586 [Abstract/Full Text]

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science.

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Adachi-Akahane S, Cleemann L & Morad M (1996). Cross-signaling between L-type Ca²⁺ channels and ryanodine receptors in rat ventricular myocytes. J Gen Physiol 108, 435-454	[Abstract]
Alseikhan BA, Demaria CD, Colecraft HM & Yue DT (2002). Engineered calmodulins reveal the unexpected eminence of Ca²⁺ channel inactivation in controlling heart excitation. Proc Natl Acad Sci U S A 99, 17185-17190	[Abstract/Full Text]
An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS & Rhodes KJ (2000). Modulation of A-type potassium channels by a family of calcium sensors. Nature 403, 553-556	[Medline]
Bers DM, (2001). Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer Academic Press, Dordrecht, Netherlands
Bouchard RA, Clark RB & Giles WR (1995). Effects of action potential duration on excitation-contraction coupling in rat ventricular myocytes. Action potential voltage-clamp measurements. Circ Res 76, 790-801	[Abstract/Full Text]
Cleemann L , & Morad M (1991). Role of Ca²⁺ channel in cardiac excitation-contraction coupling in the rat: evidence from Ca²⁺ transients and contraction. J Physiol 432, 283-312	[Abstract]
Desch, ênes I, DiSilvestre D, Juang GJ, Wu RC, An WF & Tomaselli GF (2002). Regulation of Kv4. 3 current by KChIP2 splice variants: a component of native cardiac I_to? Circulation 106, 423-429
Eisner DA, Choi HS, Diaz ME, Okneill SC & Tradfford AW (2000). Integrative analysis of calcium cycling in cardiac muscle. Circ Res 87, 1087-1094	[Abstract/Full Text]
Erickson MG, Alseikhan BA, Peterson BZ & Yue DT (2001). Pre-association of calmodulin with voltage-gated Ca²⁺ channels revealed by FRET in single living cells. Neuron 31, 973-985	[Medline]
Fauconnier J, Bedut S, Guennec JYL, Babuty D & Richard S (2003). Ca²⁺ current-mediated regulation of action potential by pacing rate in rat ventricular myocytes. Cardiovasc Res 57, 670-680	[Medline]
Findlay I, (2002). $beta$ -Adrenergic stimulation modulates Ca²⁺- and voltage-dependent inactivation of L-type Ca²⁺ channel currents in guinea-pig ventricular myocytes. J Physiol 541, 741-751	[Abstract/Full Text]
Grantham CJ , & Cannell MB (1996). Ca²⁺ influx during the cardiac action potential in guinea pig ventricular myocytes. Circ Res 79, 194-200	[Abstract/Full Text]
Guo W, Malin SA, Johns DC, Jeromin A & Nerbonne JM (2002). Modulation of Kv4-encoded K⁺ currents in the mammalian myocardium by neuronal calcium sensor-1. J Biol Chem 277, 26436-26443	[Abstract/Full Text]
Hadley RW , & Hume JR (1987). An intrinsic potential-dependent inactivation mechanism associated with calcium channels in guinea-pig myocytes. J Physiol 389, 205-222	[Abstract]
Linz KW , & Meyer R (1998). Control of L-type calcium current during the action potential of guinea-pig ventricular myocytes. J Physiol 513, 425-442	[Abstract/Full Text]
Mitarai S, Kaibara M, Yano K & Taniyama K (2000). Two distinct inactivation processes related to phosphorylation in cardiac L-type Ca²⁺ channel currents. Am J Physiol Cell Physiol 279, C603-610	[Abstract/Full Text]
Mitchell MR, Powell T, Terrar DA & Twist VW (1984a). The effects of ryanodine, EGTA and low-sodium on action potentials in rat and guinea-pig ventricular myocytes: evidence for two inward currents during the plateau. Br J Pharmacol 81, 543-550	[Abstract]
Mitchell MR, Powell T, Terrar DA & Twist VW (1984b). Strontium, nifedipine and 4-aminopyridine modify the time course of the action potential in cells from rat ventricular muscle. Br J Pharmacol 81, 551-556	[Abstract]
Pitt GS, Zuhlke RD, Hudmon A, Schulman H, Reuter H & Tsien RW (2001). Molecular basis of calmodulin tethering and Ca²⁺-dependent inactivation of L-type Ca²⁺ channels. J Biol Chem 276, 30794-30802	[Abstract/Full Text]
Sah R, Ramirez RJ, Kaprielian R & Backx PH (2001). Alterations in action potential profile enhance excitation-contraction coupling in rat cardiac myocytes. J Physiol 533, 201-214	[Abstract/Full Text]
Sham JS, Cleemann L & Morad M (1995). Functional coupling of Ca²⁺ channels and ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci U S A 92, 121-125	[Abstract]
Trafford AW, Diaz ME, Negretti N & Eisner DA (1997). Enhanced Ca²⁺ current and decreased Ca²⁺ efflux restore sarcoplasmic reticulum Ca²⁺ content after depletion. Circ Res 81, 477-484	[Abstract/Full Text]
Winslow RL, Rice J, Jafri S, Marbán E & O'Rourke B (1999). Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, II: model studies. Circ Res 84, 571-586	[Abstract/Full Text]

				C. Pott, X. Ren, D. X. Tran, M.-J. Yang, S. Henderson, M. C. Jordan, K. P. Roos, A. Garfinkel, K. D. Philipson, and J. I. Goldhaber Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice Am J Physiol Cell Physiol, February 1, 2007; 292(2): C968 - C973. [Abstract] [Full Text] [PDF]

				F. Brette, L. Salle, and C. H. Orchard Quantification of Calcium Entry at the T-Tubules and Surface Membrane in Rat Ventricular Myocytes Biophys. J., January 1, 2006; 90(1): 381 - 389. [Abstract] [Full Text] [PDF]

				J. Fauconnier, A. Lacampagne, J.-M. Rauzier, P. Fontanaud, J.-M. Frapier, O. M. Sejersted, G. Vassort, and S. Richard Frequency-dependent and proarrhythmogenic effects of FK-506 in rat ventricular cells Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H778 - H786. [Abstract] [Full Text] [PDF]