Cardiovascular Research

Cardiovascular Research 2003 57(3):670-680; doi:10.1016/S0008-6363(02)00731-9
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Ca²⁺ current-mediated regulation of action potential by pacing rate in rat ventricular myocytes

Jérémy Fauconnier^a,1, Stéphane Bedut^b,1, Jean-Yves Le Guennec^c, Dominique Babuty^b and Sylvain Richard^a,*

^aINSERM U-390, Physiopathologie Cardiovasculaire, CHU Arnaud de Villeneuve, 34295 Montpellier Cedex 5, France
^bCNRS UMR 6542, Faculté des Sciences, Parc de Grandmont, 37200 Tours, France
^cINSERM Emi-U 0211 N2C, Faculté de Médecine, 2 Boulevard Tonnellé, 37032 Tours, France

srichard{at}montp.inserm.fr

^* Corresponding author. Tel.: +33-4-6741-5244; fax: +33-4-6741-5242.

Received 6 June 2002; accepted 14 October 2002

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: Pacing rate regulates the duration of the cardiac action potential (AP). It also regulates the decay kinetics of the L-type Ca²⁺ current (I_Ca-L) which occurs via modulation of Ca²⁺-dependent inactivation. We investigated whether and how this latter process contributes to frequency-dependent (FD) changes in the AP waveform in rat ventricular cells. Methods: We recorded APs using a microelectrode technique in rat papillary muscles, and using the whole-cell current patch-clamp technique in single rat ventricular cells. Results: The AP duration (APD) was increased by high rates encompassing the physiological range (0.1–5.7 Hz) in both papillary muscles and single cells. This prolongation was accompanied by concomitant depolarisation (7 mV at 5.7 Hz) of the membrane potential (MP) in papillary muscles. Equivalent artificial depolarisation of the MP enhanced the FD prolongation in single cells. The FD prolongation was enhanced in presence of the K⁺ current blocker 4-aminopyridine (5 mmol/l), and decreased in absence of extracellular Ca²⁺. It was antagonised by Ca²⁺ channel blockers (Co²⁺, nifedipine, nitrendipine) and decreased by use of high EGTA (10 vs. 0.5 mmol/l EGTA) or BAPTA (20 mmol/l) in the patch-pipette. It was prevented by ryanodine or thapsigargin, two drugs that reduce or abolish SR-Ca²⁺ function. Conclusion: I_Ca-L contributes to the FD modulation of the AP, which occurs following a sudden change in cardiac frequency in rat ventricular cells. This highly dynamic physiological process is related to SR-Ca²⁺ release and occurs through beat-to-beat adaptation of Ca²⁺-dependent inactivation of I_Ca-L.

KEYWORDS Ca channel; Calcium (cellular); Heart rate (variability); Membrane potential; SR (function)

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
In most mammalian species, including human, elevation of the cardiac beating rate increases steady-state twitch force of the ventricle, which is referred to as the Bowditch phenomenon or the positive force-frequency relationship [1,2]. This short-term regulation of the excitation–contraction (E–C) coupling appears to be an important determinant of cardiac contractility [1–3]. Contractile force is governed by the amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) [4]. This release is triggered predominantly by transmembrane Ca²⁺ entry through L-type Ca²⁺ channels (I_Ca-L) during the action potential (AP) [4]. I_Ca-L is also electrogenic and contributes to maintain the AP plateau [3,4]. Amplitude or kinetic variations in the net Ca²⁺ influx via I_Ca-L have potential effects on the shape and duration of the AP, and also on contraction [5,6].

High pacing rates abbreviate the AP duration (APD) in various mammalian species. This is often associated with increased Ca²⁺ transient and contraction, suggesting that Ca²⁺-dependent mechanisms play a role [3,5,7–9]. However, a quite different effect is observed in rat ventricular cells. The APD is increased, whereas contraction is usually decreased [10–12]. In parallel, I_Ca-L undergoes frequency-dependent (FD) facilitation that occurs through changes in current amplitude and kinetics, and results into a slowing of current inactivation promoting increased Ca²⁺ entry. This phenomenon, which exists in several mammalian species including human, is particularly robust and consistent in rat ventricular cells [13–17]. The present study was designed to test the controversial hypothesis that, at rates of stimulation encompassing the physiological range, facilitation of I_Ca-L plays a significant role in the FD prolongation of the AP in rat ventricular cells [11,18,19]. Experiments were performed both on single cells and on papillary muscles in order to achieve more physiological conditions.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 Animals
The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH, No. 85-23, revised 1996) and European directives (96/609/EEC). Briefly, 6–10-week-old Wistar Kyoto rats (Janvier, Le Genest, St. Isle, France) were heparinized (0.2 ml, Heparin Gibco^® 1000 UI/ml) and anaesthetized with sodium pentobarbital (100 mg/kg, Sanofi Santé, France) by intraperitoneal injection.

2.2 Single cells
Ventricular myocytes were isolated as described [20]. The heart was perfused at 35 °C with a Ca²⁺-free Hepes-buffered solution (5 min) containing (in mmol/l): NaCl (117), KCl (5.7), NaHCO₃ (4.4), KH₂PO₄ (1.5), MgCl₂ (1.7), Hepes (21), glucose (11.7), taurine (20); pH adjusted at 7.2 with NaOH. Then, collagenase (1.35 mg/ml; Worthington type IV, 247 UI/mg) was used for 35 min. Successive steps were made to increase Ca²⁺ progressively and to eliminate dead cells before storage in a solution containing 1 mmol/l CaCl₂ and 1% BSA, and maintained at 36 °C.

2.3 APs and I_Ca-L in single cells
APs were recorded in rod-shaped Ca²⁺-tolerant myocytes at 22–24 °C by use of the whole-cell patch-clamp technique (Axopatch 200A, Axon instrument, Burlingham, CA, USA). Pipettes had resistance of 2–3 M when filled with the recording solution containing (mmol/l): KCl (130), Hepes (25), ATP(Mg) (3), GTP(Na) (0.4), EGTA (10 or 0.5) or BAPTA (20) as specified in the legends of figures; pH adjusted to 7.2 with KOH; 290–310 mOsm. The myocytes were superfused with a Tyrode's solution containing (in mmol/l): NaCl (135), MgCl₂ (1), KCl (4), glucose (11), Hepes (2), CaCl₂ (1.8); pH adjusted to 7.4 with NaOH. In Ca²⁺-free solutions, Mg²⁺ replaced Ca²⁺. APs were elicited by 0.2 ms current injection of supra-threshold intensity. Acquisition rate was 10 kHz. Signals were filtered at 5 kHz (lowpass Bessel Filter). Cells were stimulated routinely at 0.1 Hz until stabilisation of APs (3–5 min). This frequency was taken as the reference since no change occurred at rates lower than 0.2 Hz. For testing at a given frequency, stimulation was started after a rest period of at least 10 s. Between each trial, the frequency was returned to 0.1 Hz. In one set of experiments, I_Ca-L was recorded in voltage clamp conditions optimised to eliminate contaminating inward Na⁺ and outward K⁺ currents [13–17]. Bath solutions contained (mmol/l): TEACl (140), CaCl₂ (1.8), MgCl₂ (2), Hepes (10), glucose (10); pH adjusted to 7.4 with TEAOH; 290–310 mOsm. The pipette solution contained (mmol/l): CsCl (140), Hepes (10), ATP(Mg) (3), GTP(Mg) (0.4), EGTA (10); pH adjusted to 7.2 with CsOH; 290–310 mOsm.

2.4 Measurements of contraction in single cells
Only myocytes showing clear sarcomeric patterning were selected. Cells were field-stimulated by platinum electrodes at 0.2 and 1.0 Hz. Sarcomere length (SL) was determined by using a fast Fourier transform algorithm on an acquired video image of the cell as described before [21]. Due to technical limitations, it was not possible to measure accurately SL at 3.3 Hz. The sampling frequency of the video image acquisition was 50 Hz.

2.5 APs in papillary muscles
APs were recorded from right ventricular papillary muscles using a standard microelectrode recording technique (WPI M707, Sarasota, FA, USA) at 37 °C [12]. Muscles were mounted in a bath superfused with Tyrode's solution containing (mmol/l): NaCl (138.6), KCl (5.4), MgCl₂ (1.2), NaH₂PO₄ (0.33), CaCl₂ (1.8), Hepes (10), glucose (11), pH adjusted to 7.4 with NaOH; and gassed with air. In Ca²⁺-free solutions, Mg²⁺ replaced Ca²⁺. Electrodes resistances ranged between 20 and 40 M. They were filled with a 3-mol/l KCl solution. Unstretched preparations were driven at 1.0 Hz for 3–5 min in order to stabilise electrical activity prior to increasing the rates to 2.5, 3.3 and 5.7 Hz. The AP at each rate was recorded after 30 s, corresponding to steady state effect. The zero-potential, adjusted to the ground potential, was calibrated before and after each period. One or more sites were studied in each papillary muscle. The results presented originated from a single impalement.

2.6 Analyses and statistics
Data acquisition and analyses were performed using the Pclamp (version 8.1, Axon instruments) and/or Acquis software (G. Sadoc, CNRS URA 1121 Orsay). The MP, the AP amplitude and the APD at 20% and 50% (APD₅₀) and 90% of repolarisation were measured. For brevity, only measurements of the APD_50, which best reflects the contribution of I_Ca-L are presented. Only maximal variations (either steady-state or transient) have been included in analyses. All averaged or normalised data are presented as mean±S.E.M. The significance between groups of data was assessed using Student's t-test (for paired and unpaired samples as appropriate), and one-way analysis of variance (ANOVA test) when three or more groups were compared, with a Newman–Keuls post-hoc test comparing all pairs of group means. Results were considered significant with P less than 0.05 (*P<0.05, **P<0.01, ***P<0.001).

2.7 Solutions
Ryanodine and thapsigargin (Sigma, St. Quentin Fallavier, France) were prepared as 10-mmol/l stock solution in distilled H₂O and DMSO, respectively. Nifedipine (Sigma) and nitrendipine (Sigma) were prepared as 10 mmol/l stock solutions in ethanol. Stock solutions were diluted to the desired concentration in the Tyrode solution. 4-Aminopyridine (4-AP; 5 mmol/l; Sigma) was added to block the transient outward K⁺ current (I_to) in some experiments. Co²⁺ was used to block I_Ca-L. The control and test solutions were applied as described before [12,17].

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Effect of the rate of stimulation
Fig. 1Aa shows the effect of a stepped increase in the rate of stimulation, from 1.0 to 3.3 Hz and to 5.7 Hz, on the duration of APs recorded in a right papillary muscle at 37 °C. The APD₅₀ increased gradually (Fig. 1Ab). A concomitant depolarisation of the MP occurred (Fig. 1Aa,c). Qualitatively similar results were observed in single ventricular myocytes recorded at 22–24 °C with the patch-clamp technique (Fig. 1Ba), though the range of rates where changes occurred was lower than for the papillary muscles recorded at 37 °C. The APD₅₀ also increased gradually at higher rates (Fig. 1Ba,b). The FD prolongation reached steady state within four to eight stimulations after the beginning of a train (data not shown). Vice versa, the APD₅₀ decreased when the rate of stimulation was lowered (Fig. 1Bc). A small, but significant, depolarisation of the MP occurred (–82.8±0.4 mV at 3.3 Hz vs. –84.2±0.3 mV at 0.1 Hz; n=34, *P<0.05). Then, we investigated whether a moderate depolarisation (<10 mV), induced by artificially changing the MP of single cells, could participate in the FD modulation of the AP in papillary muscles. This manoeuvre induced per se a prolongation of the AP. The APD was longer at the more depolarised MP for a given frequency (0.1 and 3.3 Hz, respectively; Fig. 2A). Moreover, MP depolarisation enhanced significantly the FD prolongation of the APD₅₀ (Fig. 2A,B).

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 FD modulation of the AP and of the MP. (A) Steady state APs recorded at 1.0, 3.3 and 5.7 Hz in papillary muscles: (a) original traces in the same preparation; (b) average percentage increases (±S.E.M.) in the APD50 at 3.3 and 5.7 Hz, with 1.0 Hz taken as the reference; (c) average percentage FD changes in the MP. (B) Steady state APs recorded at 0.1, 1.0 and 3.3 Hz in single ventricular cells (10 mmol/l EGTA in the pipette): (a) original traces in the same myocyte; (b) average percentage increases (±S.E.M.) in the APD50 at 0.1, 1.0 and 3.3 Hz, with 0.1 Hz taken as the reference; (c) steady state effect after a decrease from 3.3 to 0.1 Hz on an AP recorded in a same myocyte (0.5 mmol/l EGTA). Statistical significance between pairs of data is indicated by an asterisk (ANOVA, ***P<0.001; see Section 2).

 

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Influence of the MP on the FD prolongation of APs in single cells (10 mmol/l EGTA). (A) Steady-state APs recorded at 0.1 Hz and 3.3 Hz from three different diastolic MPs, artificially controlled using current injection, in the same cell. (B) Average increases (±S.E.M.) in the APD50 (t, ms) when increasing the rate from 0.1 Hz (reference) to 3.3 Hz. Statistical significance between pairs is indicated by an asterisk (ANOVA, *P<0.05).

 
3.2 Effect in the presence of 4-AP
Two kinetically distinct voltage-dependent K⁺ currents have been identified in rat ventricular cells: (i) a 4-AP-sensitive transient current, I_to, activating and inactivating rapidly, and (ii) a delayed rectifier, I_K, which activates too slowly to be involved in the process described here [22]. To study the participation of I_to in the FD modulation of the APD, we recorded APs in presence of 5 mmol/l 4-AP to totally block I_to. The 4-AP slowed the fast repolarizing phase of the AP and prolonged the plateau markedly, which could be inhibited by the inorganic Ca²⁺ channel blocker Co²⁺ (2 mmol/l) (Fig. 3A). In the presence of 4-AP, a graded increase in frequency between 0.1 and 3.3 Hz produced a graded and large prolongation of the AP (Fig. 3B,C). This effect reached a maximum within two to five stimuli and was followed by a partial recovery after 10–30 stimuli, which occurred mainly at rates higher than 1 Hz (data not shown). Examination of I_to in voltage-clamp conditions revealed the existence of a use-dependent unblock of the effect of 4-AP on I_to, but this occurred only for frequencies higher than 1.0 Hz (data not shown) [23]. Furthermore, FD prolongation was observed following changes from 0.1 Hz to rates as low as 0.3 Hz or even 0.2 Hz that had no effect in control conditions (data not shown), whereas no difference was observed between 1.0 and 3.3 Hz (Fig. 3C). The FD prolongation was abolished by Co²⁺ suggesting involvement of Ca²⁺ influx (Fig. 3D).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of 4-AP in single cells (0.5 mmol/l EGTA). (A) Effect of 4-AP (5 mmol/l) and 4-AP+Co2+ (2 mmol/l) at low rate (0.1 Hz). (B) Original traces of steady state APs recorded at 0.1, 0.33, 1.0, and 3.3 Hz in the presence of 5 mmol/l 4-AP in the same cell. (C) Average percentage increases (±S.E.M.) in the APD50 at various rates, with 0.1 Hz taken as the reference. Statistical significance between pairs is indicated by an asterisk (ANOVA, *P<0.05). (D) Suppression of the FD prolongation by 2 mmol/l Co2+ in the presence of 5 mmol/l 4-AP. The cell was the same as in (B).

 
3.3 Involvement of Ca²⁺ channels
Fig. 4A illustrates the typical FD facilitation of I_Ca-L observed in absence of other currents in rat ventricular cells (for details, see Refs. [13–17]). This regulation is highly consistent among cells. Since AP FD-prolongation is also observed in nearly all cells, the two regulations were expected to occur in association in same cells. Indeed, when AP type of recording solutions and voltage-clamp conditions (HP of –60 mV) designed to minimise contamination by K⁺ and Na⁺ currents were used, both FD prolongation of AP and FD change in the inward current occurred (Fig. 4B) in all of three cells tested. Although this change was reminiscent of I_Ca-L facilitation, we could not exclude the participation of residual K⁺/Na⁺ currents. It was impossible to depolarise the HP further to eliminate these currents completely because this manipulation also abolishes facilitation of I_Ca-L. Thus, we used pharmacological approaches. First, we found that FD prolongation of the AP was decreased in Ca²⁺-free solutions both in papillary muscles (Fig. 5A) and single cells (Fig. 5B). Although a residual prolongation remained, the decrease was highly significant and consistent (Fig. 5Ab,Bb), indicating that Ca²⁺ entry is important. We next assessed the effect of the Ca²⁺ channel antagonists nifedipine and nitrendipine. These dihydropyridines (DHPs) were chosen because they are devoid of use-dependent block, compared to other Ca²⁺ blockers [24]. In single cells, we applied the DHPs at 2 µmol/l because this concentration blocks approximately 50% of I_Ca-L evoked from a HP of –80 mV and has moderate non-specific effects [25]. We used a higher concentration of DHPs (10 µmol/l) for papillary muscles in order to compensate for the less efficient diffusion of the drug. The FD prolongation of the AP was significantly decreased by DHPs both in papillary muscles (Fig. 6Aa,b) and in single cells (Fig. 6Ba,b), suggesting a contribution of I_Ca-L. In addition, the DHPs prevented the effect of MP depolarisation on the FD modulation of the APD in single cells. Not only was the FD prolongation at a given MP (–90 and –80 mV, respectively) attenuated, but also the enhancement of the FD prolongation by MP depolarisation (Fig. 7A,B), indicating that I_Ca-L is also involved.

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 FD modulation of ICa-L and AP in single cells. (A) Effect of a an increase from 0.1 to 3.3 Hz on ICa-L recorded in absence of intra- and extracellular Na+ and K+ (see Section 2). ICa-L was evoked at –10 mV from a holding potential of –80 mV (EGTA, 10 mmol/l). Note the slowing of decay kinetics of ICa-L. (B) Effect of an increase from 0.1 to 3.3 Hz on both ICa-L and AP recorded in a same cell using AP type solutions (intracellular K+, extracellular Na+: see Section 2). ICa-L was recorded at –10 mV from –60 mV in order to minimise the contribution of contaminating inward INa and outward Ito (EGTA, 0.5 mmol/l). Facilitation of ICa-L can be observed in these conditions [13].

 

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effect of extracellular Ca2+-free solution. (A) Steady state APs recorded at 1.0 and 5.7 Hz in normal and Ca2+-free solutions in papillary muscles: (a) original traces in the same preparation; (b) average percentage increases (±S.E.M.) in the APD50 at 5.7 Hz, with 1.0 Hz taken as the reference. (B) Steady state APs recorded at 0.1 and 3.3 Hz in normal and Ca2+-free solutions in single rat ventricular cells (0.5 mmol/l EGTA): (a) original traces in the same cell, with 0.1 Hz taken as the reference; (b) average percentage increases in the APD50 at 3.3 Hz.

 

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effect of dihydropyridines. (A) Steady state APs recorded at 1.0 and 5.7 Hz in the absence and presence of nifedipine (10 µmol/l) in papillary muscles: (a) original traces in the same preparation; (b) average percentage increases (±S.E.M.) in the APD50 at 5.7 Hz. (B) Steady state APs recorded at 0.1 and 3.3 Hz in the absence and presence of nitrendipine (2 µmol/l) in single myocytes (10 mmol/l EGTA): (a) original traces in the same cell; (b) average percentage increases (±S.E.M.) in the APD50 at 3.3 Hz.

 

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Nitrendipine blocks the enhancement of the FD prolongation of APs by depolarised the MP in single cells (10 mmol/l EGTA). Steady state APs recorded at 0.1 and 3.3 Hz from –90 and –80 mV in the same cell: (A) in control conditions; and (B) in presence of 2 µmol/l nitrendipine. Consistent results were observed in eight (A) and five (B) cells tested, respectively.

 
3.4 Effects of intracellular Ca²⁺ buffering
To assess the role of intracellular Ca²⁺, we performed patch-clamp recordings using two different concentrations of EGTA in the pipette. At 0.1 Hz, high EGTA increased the APD (Fig. 8Aa,b). On average, the APD₅₀ increased from 10.6±0.8 ms (EGTA 0.5 mmol/l; n=24) to 16.2±1.5 ms (EGTA 10 mmol/l; n=60; ***P<0.001). For comparison, the APD₅₀ was 11.2±0.3 (n=52) in papillary muscles driven at low rate (1.0 Hz). We found that an increase in the stimulation frequency from 0.1 to 3.3 Hz induced a larger prolongation of the APD₅₀ in presence of low EGTA (42±5%; n=7) as compared to high EGTA (20±5%; n=12) (Fig. 8Abc,B). Moreover, FD prolongation was abolished when high BAPTA (20 mM), a faster and more efficient Ca²⁺ chelator than EGTA at the sub-cellular level, was used (Fig. 8Ac,B). These results suggested that the intracellular Ca²⁺ concentration not only influences the APD at low pacing rates but also determines the lengthening of AP at high rates. It should also be noted that high rates induced a transient effect at frequencies <1 Hz when low EGTA (0.5 mmol/l) was used (Fig. 8C). First, the FD prolongation reached a maximum within four to 10 stimuli after starting a train. Then, a partial recovery developed within 10 to 20 stimuli before the AP reached its final waveform. This recovery was absent when external Na⁺ was replaced by Li⁺ (data not shown). The overall FD prolongation was also less in these conditions, suggesting a secondary role of the I_Na–Ca exchanger.

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Effect of intracellular Ca2+ buffer. (A) Steady state APs recorded at 0.1 and 3.3 Hz in single myocytes loaded with 10 mmol/l EGTA (a: high), 0.5 mmol/l EGTA (b: low) or 20 mmol/l BAPTA (c), respectively, in the patch-pipette. (B) Average percentage increases (±S.E.M.) in the APD50 at 3.3 Hz. Statistical significance between pairs is indicated by an asterisk (ANOVA, ***P<0.001). (C) Partial recovery of the FD prolongation of the AP observed over time with 0.5 mmol/l EGTA in the patch pipette.

 
3.5 Effect of ryanodine and thapsigargin
Ryanodine binds to SR-Ca²⁺ release channels (RyRs) and is known to reduce SR Ca²⁺ release and contraction, at micromolar concentration [26,27]. A secondary effect is to prevent the SR-Ca²⁺ release-induced fast inactivation of I_Ca-L [17,27]. We found that ryanodine (1–10 µmol/l) markedly prolonged the AP both in papillary muscles (Fig. 9Aa) and in single cells at low rates (Fig. 9Ba). In addition, ryanodine attenuated the FD prolongation at 5.7 Hz in papillary muscles (Fig. 9Aa,b) and abolished it at 3.3 Hz in single cells (Fig. 9Ba,b). Thapsigargin, known to inhibit SR-Ca²⁺ uptake and to slow the decay kinetics of I_Ca-L [28], had essentially similar effects as ryanodine in single cells. Thapsigargin (1 µmol/l) prolonged dramatically the AP 5 min of extracellular superfusion, and abolished the FD prolongation (n=3; data not shown).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 Effect of ryanodine. (A) Steady state APs recorded at 1.0 and 5.7 Hz in absence and presence of 10 µmol/l ryanodine in papillary muscles: (a) original traces in the same preparation; (b) averaged percentage increases (±S.E.M.) in the APD at 5.7 Hz. (B) steady state APs recorded at 0.1 and 3.3 Hz in absence and presence of 1 µmol/l ryanodine in single myocytes (10 mmol/l EGTA): (a) original traces in the same cell; (b) averaged percentage increases (±S.E.M.) in the APD at 3.3 Hz. Similar effect was observed at 10 µmol/l (data not shown).

 
3.6 FD decrease of contraction in single cells
Fig. 10 shows that an increase in the stimulation rate from 0.2 to 1.0 Hz induced a decrease of the contraction in single cells. This decrease occurred immediately at the first beat following the change and contraction remained stable. Consistent results were obtained in eight cells.

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 10 Immediate decrease in contraction induced by an increase from 0.2 to 1.0 Hz in a single myocyte.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
An increase in the stimulus frequency prolongs the AP at physiological heart rate in rat single ventricular cells and papillary muscles [10–12,18,19]. Several ionic currents, including I_to, are potentially involved [18,19]. The present study demonstrates that I_Ca-L plays a role. This contribution may occur via changes in its decay kinetics in relation with SR-Ca²⁺ release and Ca²⁺-dependent inactivation of Ca²⁺ channels. This regulation probably plays a highly dynamic role in the regulation of APD following sudden changes in cardiac frequency. We also show that FD depolarisation of the MP has a part in the FD prolongation of the AP in papillary muscles. The overall prolongation is likely to reflect the combination of two distinct effects on Ca²⁺ channel activity.

4.1 Modulation of APD by frequency and MP
Adaptation of APD to heart rate varies among species. The APD shortens with stepped increases in frequency in guinea pig and in diseased human cardiomyocytes [5,7–9], whereas a prolongation occurs in rat ventricular cells [10–12,18,19]. Here, we found similar FD prolongation both in rat papillary muscles and rat single ventricular myocytes despite some quantitative differences that may be partly related to differences in intracellular Ca²⁺ buffering. A FD depolarisation also occurred in the papillary muscles, in agreement with previous observation [11]. It is known that increased stimulation frequency causes depolarisation of the MP in thin multicellular preparations, which has been previously related to an increase of the cleft K⁺ concentration [3,7,8]. This depolarisation was, however, very small (<2 mV) in single cells, reflecting possibly better washout or oxygenation of the cells by the bathing solution or, alternatively, temperature difference. Interestingly, we showed here that an artificial moderate depolarisation of the MP (<10 mV) enhances the FD prolongation of the AP in single cells, suggesting that this process contributes to the FD adaptation occurring in papillary muscles.

4.2 Role of I_Ca-L and SR Ca²⁺ release
An increase in the APD is expected to reflect alternatively, or additionally, an increase in an inward current or/and a decrease in an outward current. A decrease of I_to, responsible for the fast repolarisation of the AP in rat ventricular cells, has been proposed [18,19]. Our results, however, showed that the FD prolongation persists largely after blockade of I_to by 4-AP. Suppression by specific Ca²⁺ channel antagonists (DHPs) and by Co²⁺ demonstrated that, in addition to possible effects on I_to, I_Ca-L is involved quite significantly as suggested previously [11]. In addition, both the dependence of the APD on intracellular Ca²⁺ buffering and the antagonistic effect of EGTA and BAPTA on the AP FD-prolongation suggested the involvement of a Ca²⁺-dependent process.

The cardiac I_Ca-L is subject to fast Ca²⁺-dependent inactivation, closely related to a negative feedback created by SR-Ca²⁺ release in nanoscale domains hardly accessible to EGTA [4,29,30]. It can, however, be abolished using high BAPTA [14]. Fast inactivation of I_Ca-L, which is graded with the magnitude of SR-Ca²⁺ release [4,31], is lessened during facilitation of I_Ca-L [17,27,28]. Facilitation of I_Ca-L can be prevented pharmacologically by ryanodine, known to delay current inactivation while suppressing contraction in rat ventricular cells probably because of SR-Ca²⁺ depletion [17,27], and by thapsigargin, a potent and irreversible inhibitor of the SR-Ca²⁺ ATPase preventing SR-Ca²⁺ load [28,32]. Here, we found that both ryanodine and thapsigargin not only prolong the AP but also prevent its adaptation to high rates, as expected from their slowing effect on I_Ca-L decay kinetics. Therefore, we conclude that SR-Ca²⁺ release contributes to control the APD at steady state and is involved in the FD adaptation of the AP in rat ventricular cells. This control occurs via Ca²⁺-dependent inactivation of I_Ca-L as anticipated from mathematical modelling and experimental approaches [33,34]. This mechanism is probably well developed in rat ventricle where high SR Ca²⁺-ATPase activity is known to favour high SR-Ca²⁺ load, and thus high Ca²⁺ release [4]. This interpretation is also consistent with the decrease in contraction that was observed immediately after increasing the pacing rate in single cells (see also Ref. [10,11]), though additional mechanisms may also be involved because changes in APD were slower to reach steady state.

4.3 Limiting role of MP and I_to
The I_to blocker 4-AP induced large FD prolongation of the AP, suggesting that I_to is a limiting factor, probably because this current shortens the AP and, thereby, limits the recruitment of I_Ca-L. This indirect role of I_to may partially explain the larger FD prolongation correlated with decreased I_to in diabetic rats [18]. Our study also showed that the enhancement of the FD AP-prolongation by depolarisation of the MP, physiologically observed in papillary muscles, reflects the contribution of two distinct effects on I_Ca-L. One effect reflects changes in gating properties, leading to facilitation (slowing of current inactivation). The other is probably related to changes in the number of Ca²⁺ channels activated during the AP. Since there is a reduction in the rate upstroke (dV/dt) and a delay in the repolarisation of the AP, resulting from voltage-dependent inactivation of both I_Na and I_to, more Ca²⁺ channels may be recruited and, thereby, available for FD facilitation.

4.4 Possible role and relevance for pathophysiology
Adaptation of the APD to heart rate is essential for co-ordinated control of electrical activity and Ca²⁺ homeostasis during systolic and diastolic periods. The FD adaptation described in this study occurs very rapidly and at physiological rates. It is expected to occur during sudden variations of the heart rate on a beat-to-beat basis. Its role may be to supply the cell with additional Ca²⁺ ions from the extracellular source in order to refill the SR at high pacing rates. Removal of Ca²⁺ release-induced inactivation of I_Ca-L has indeed been shown to increase Ca²⁺ entry under extreme conditions of SR-Ca²⁺ depletion or during heart failure (HF) [30,35].

Prolongation of the AP is a hallmark feature of hypertrophy and HF, which can disturb electrical heterogeneity and induce arrhythmias leading to sudden death [36–38]. Reduction of I_to is involved, whereas I_Ca-L density is probably unchanged [39,40]. Suppression of I_to by 4-AP or decreased K⁺ channel expression prolongs the AP in post infarction and increases both Ca²⁺ influx and Ca²⁺ transient in rat cardiomyocytes [37,39,41]. Our experiments with 4-AP suggest that one consequence of I_to blunting is to enhance the FD AP-prolongation. This enhancement could be particularly important if the SR-Ca²⁺ release is unaffected or increased [39]. A marked AP prolongation could result in a large, time-dependent positive inotropic effect [41]. Moreover, any depolarisation of the MP, equivalent to that described 8 weeks following left coronary artery ligation in rat (10 mV) [39], might also enhance dramatically the effect of high rates on the APD and on intracellular Ca²⁺ load.

In summary, we have related sudden changes of rat ventricular AP that occur physiologically in a highly dynamic manner during variations in frequency, to changes in Ca²⁺ channel activity. These effects seem to be related to facilitation of I_Ca-L and to SR-Ca²⁺ dependent modulation of Ca²⁺ channel inactivation. We emphasise that this modulation is an obligatory, highly regulated, intermediate in the communication between SR-Ca²⁺ and the APD, which contributes to the electrical control of Ca²⁺ cycling during the diastolic and systolic phases.

Time for primary review 32 days.

	Acknowledgements

 
We thank Drs. G. Vassort, AM. Gomez, AJ. Pappano (Farmington, USA), O. Sejersted (Oslo, Norway), and E. White (Leeds, UK) for interesting discussions and careful reading of the manuscript. This work was supported by a grant from FRM (‘Fondation pour la Recherche Médicale’: INE20001117051) to SR, and by fellowships from ‘Ministère de la Recherche et de la Technologie’ (JF,SB). Part of this work was initiated in the Institute of Human Genetics (Montpellier, France). SR holds CNRS (Centre National de la Recherche Scientifique) researcher position.

	Notes

 
¹ Both authors contributed equally to this work.

	References

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