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Cardiovascular Research 1999 44(2):381-389; doi:10.1016/S0008-6363(99)00216-3
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Use-dependent facilitation and depression of L-type Ca2+ current in guinea-pig ventricular myocytes: modulation by Ca2+ and isoprenaline

Susan E. Bates1 and Alison M. Gurney*

Department of Physiology and Pharmacology, University of Strathclyde, Strathclyde Institute for Biomedical Sciences, 27 Taylor Street, Glasgow G4 0NR, UK

* Corresponding author. Tel.: +44-141-548-4119; fax: +44-141-552-2562 a.m.gurney{at}strath.ac.uk

Received 8 April 1999; accepted 16 June 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: An increase in stimulation frequency can facilitate or depress cardiac Ca2+ current (ICa). The aim was to examine the Ca2+ dependence of these effects, to determine if facilitation is sustained, and to elucidate the mechanism by which isoprenaline modulates facilitation. Methods: We examined the effects of increasing the stimulation frequency for 1 min, from 0.05 to 1 Hz, on ICa recorded from guinea-pig ventricular myocytes, using the whole-cell, voltage-clamp technique. Results: 1 Hz stimulation caused a facilitation of ICa that peaked in 5 s and was followed by depression towards the basal level. Metabolic inhibitors or replacement of extracellular Ca2+ with Ba2+ abolished facilitation without affecting depression, implying that they are independent processes and that facilitation required ATP and Ca2+. Subtraction of the depression observed in either condition, from the response to 1 Hz stimulation recorded under control conditions, revealed that ICa facilitation was well maintained during 1 Hz stimulation. Increased intracellular Ca2+ buffering reduced both phases of the response. Furthermore, varying the extracellular Ca2+ concentration ([Ca2+]o) revealed a Ca2+-dependent enhancement of depression and a bell-shaped dependence of facilitation on [Ca2+]o. Facilitation increased with [Ca2+]o up to 1 mM, then declined at higher concentrations due to partial masking by the overlapping depression. Isoprenaline produced concentration-dependent inhibition of facilitation and enhancement of depression when pipettes contained 2 mM EGTA, but not BAPTA. For an equivalent increase in ICa amplitude, the effects of isoprenaline and elevated [Ca2+]o on the response to 1 Hz stimulation were quantitatively the same. Conclusions: Facilitation is sustained during increased activity, but appears transient due to overlapping depression. Both responses are promoted by increased submembrane [Ca2+]. Isoprenaline appears to modulate facilitation and depression as a consequence of increased Ca2+ influx, rather than cAMP-dependent phosphorylation. The apparent block of facilitation by isoprenaline may result from masking by the enhanced depression.

KEYWORDS Calcium; Ca-channel; Membrane currents; Myocytes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
In mammalian cardiac muscle, frequency-dependent modulation of the L-type calcium current (ICa) influences the action potential duration, Ca2+ influx and contractile force [1–3]. Depending on the experimental conditions, a sudden increase in the frequency of current activation causes an increase (facilitation) and/or a decrease (depression) of ICa peak amplitude, as reviewed in Ref. [4]. It is generally agreed that facilitation requires Ca2+ influx [3,5–7]. Consistent with this, increasing the intracellular [Ca2+] has been shown to produce Ca channel facilitation [8–10]. An early study of the frequency-dependent modulation of ICa in guinea-pig ventricular myocytes concluded that Ca2+ entry is also a determinant of negative current staircases [5]. More recent studies conclude that they depend on membrane voltage, but not Ca2+ [11–13]. Proposed mechanisms include incomplete recovery from voltage-dependent inactivation between pulses [11] or depolarization-induced, ultra-slow inactivation that accumulates during each pulse [12].

The mechanisms underlying the Ca2+-dependent facilitation of cardiac ICa are not clear. Modulation of facilitation by the β-adrenergic agonist, isoprenaline [5,7,14–16] and intracellular cAMP [13,15], along with a requirement for intracellular ATP [8], suggested that phosphorylation is involved. It was subsequently proposed to involve phosphorylation via Ca2+ and calmodulin-dependent protein kinase II (CamKII), because the frequency-dependent facilitation measured during short trains of pulses was suppressed by CamKII inhibitors [17,18]. Such a mechanism was demonstrated for the facilitation of smooth muscle ICa [19]. On the other hand, intracellular Ca2+ has been shown to induce an ATP-dependent, but phosphorylation-independent facilitation of ICa in cardiac myocytes [8,10]. It is possible that the apparent effects of CamKII inhibitors resulted from modulation of an overlapping depression, because facilitation and depression are activated simultaneously by an increase in stimulation frequency. During maintained stimulation, ICa amplitude usually declines after the facilitation to the baseline or beyond [2,3,5,6]. Since the experiments with kinase inhibitors investigated only the first few records of ICa following increased activity, an effect on ICa depression might not have been seen.

This study addresses the Ca2+-dependence of the activity-dependent facilitation and depression of ICa in guinea-pig ventricular myocytes, as well as interactions between these processes. The stimulation frequency was raised from 0.05 to 1 Hz for 1 min periods, during which the peak amplitude of ICa was seen to increase and then decline towards the basal level. This response is shown to reflect the simultaneous activation of independent processes leading to facilitation and depression. Moreover, changes in the amplitude of depression had marked effects on the apparent facilitation. Increased depression may explain the apparent inhibitory effect of isoprenaline on facilitation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell dissociation
Experiments were performed on ventricular myocytes isolated from male, Dunkin Hartley guinea-pigs (250–400 g). The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animals were killed by cervical dislocation and hearts removed into ice-cold physiological salt solution (PSS). The cell isolation method is described in Ref. [20]. Briefly, hearts were retrogradely perfused for 5 min at 36°C with a nominally Ca2+-free solution containing (in mM) NaCl 120, KCl 5.4, MgSO4 5, Na-pyruvate 5, glucose 20, taurine 20, HEPES (4-[2-hydroxyethyl] piperazine-1-ethanesulphonic acid) 10, pH 6.96, then for 2 min with 4 U/ml protease (Sigma, type XXIV) added. Solution containing collagenase (Worthington Class 2, ~0.3 mg/ml) and hyaluronidase (Sigma, 0.6 mg/ml) was then perfused for 5–10 min before separating and finely chopping the ventricles in ‘KB’ solution (see Ref. [21]). The tissue was strained through gauze, the cell suspension centrifuged briefly at low speed and resuspended in ‘KB’ solution.

2.2 Electrophysiology
Cells were transferred to a 0.5 ml recording chamber and superfused continuously with PSS at room temperature (~22–24°C). PSS contained (in mM) NaCl 120, KCl 2.5, CaCl2 (or BaCl2) 1, MgCl2 2.5, NaHCO3 15, NaH2PO4 1, HEPES 5, glucose 10 and was continuously bubbled with 95% O2/5% CO2 (pH 7.4). This was sometimes replaced with HEPES-buffered solution containing (in mM) NaCl 150, KCl 2.5, MgCl2 2.5, HEPES 10 and varying [CaCl2], pH adjusted to 7.4. The whole-cell configuration of the patch-clamp technique was used to voltage clamp cells at –70 or –80 mV. Recording pipettes pulled from borosilicate capillaries (Clark Electromedical) had resistances of 2–4 M{Omega} when filled with internal solution, which usually contained (in mM) CsCl 130, MgCl2 1, HEPES 15, EGTA 2, ATP-Na2 2 (pH 7.2). After forming the whole-cell configuration, the superfusion was changed to a K+-free solution, prepared by replacing the KCl in PSS with equimolar NaCl. This suppressed the inwardly rectifying K+ current, which was often substantial with Cs+-filled pipettes. At least 10 min was allowed for dialysis of the cell before experiments began.

Voltage was controlled with an Axopatch 200A or Biologic RK 300 patch-clamp amplifier. Ca2+ currents were activated at 0.05 Hz, by a 150 ms depolarization to 0 mV. Frequency-dependent modulation of ICa was studied by increasing the rate to 1 Hz for 1 min periods. This protocol optimised activity-dependent facilitation of ICa [4] and since ICa was recorded at the Cl– equilibrium potential (0 mV) it would not be influenced by Ca2+-dependent changes in Cl– current. A 75 ms pre-pulse to –40 mV eliminated T-type Ca2+ current and Na+ current; Cs+ in the pipette-filling solution blocked outward K+ currents. Cell capacitance (159±5 pF, n=83) was estimated from the capacitative current generated by small hyperpolarizing voltage steps applied from –80 mV. Uncompensated series resistance was ≤8 M{Omega}. It was routinely compensated. Stimulus protocols were generated and data collected using p-CLAMP (5.5.1) software and a TL-1 interface (Axon Instruments). Current signals were filtered at 3 kHz and digitized at 40 kHz without leak subtraction.

2.3 Data analysis
Data analysis employed p-CLAMP and ORIGIN (MicroCal) software. Data are represented as mean±S.E.M. of n cells. A paired t-test was used to compare two means from one group of cells, the Mann-Whitney nonparametric test compared two cell populations and ANOVA was used for comparison of multiple groups; P<0.05 was considered significant. Exponential functions were fit to data using the following equation:


Formula (1)

where I is the current at time t, t0 is time zero, a1 and a2 are the amplitudes and {tau}1 and {tau}2 the time constants of the individual components and c represents a constant.

2.4 Sources of reagents
The following reagents were used: isoprenaline hydrochloride from Sigma; EGTA (ethyleneglycol-bis-(β-aminoethylether)-N,N,N',N'-tetraacetic acid) and 2,4-dinitrophenol from BDH; BAPTA (1,2-bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid; sodium salt) and cyclopiazonic acid from Calbiochem-Novabiochem; FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) from Aldrich. Isoprenaline was prepared as a 1 mM stock in 1 mM ascorbic acid and 10 mM EDTA (ethylenediamine tetra-acetic acid).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Activity-dependent modulation of ICa
Raising the stimulation frequency from 0.05 to 1 Hz for 1 min, in the presence of 1 mM extracellular CaCl2 and 2 mM internal EGTA, induced a biphasic response (Fig. 1(A)). The peak amplitude of ICa was initially enhanced, reaching a maximum facilitation of 34±3% (n=21; 18 animals) within 5 s. After this, ICa amplitude gradually declined, by 18±1% of the facilitated level after 1 min. Peak ICa amplitude recovered to control levels within 3 min of returning to 0.05 Hz, after which a second 1 Hz train produced a similar response. As previously shown [2,7], facilitation was associated with slowing of ICa inactivation (Fig. 1(A) inset), which was best fit by the sum of two exponential components according to Eq. (1), with the time constants and amplitudes in Table 1. At the peak of facilitation, the amplitude of the slower decaying component (as) had increased, without a change in the amplitude of the fast component (af); the time constants of both the slow ({tau}s) and fast ({tau}f) components were slightly, but significantly, reduced. The overall slowing of inactivation is therefore explained by enhancement of the slower component.


Figure 1
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  		Fig. 1 Biphasic effect of switching from 0.05 to 1 Hz stimulation. (A) Peak ICa amplitude plotted against time after establishing whole-cell recording (WCR). 1 Hz stimulation is indicated by bars. Inset shows voltage protocol and 1st (1), 6th (6) and last (60) current records obtained during the 1 Hz train. (B) Percent change in ICa amplitude plotted against time after switching to 1 Hz stimulation (n=21). Changes were measured relative to the mean of the six records immediately preceding the 1 Hz train. The superimposed curve is a bi-exponential fit to the data; the additional curves show the individual components. One component represents facilitation, with amplitude 40% and time constant 1.6 s. The other represents depression with amplitude –33% and time constant 23 s.

 

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  		Table 1 Influence of 1 Hz stimulation on peak amplitude and decay kinetics of ICa

 
Fig. 1(B) shows mean data from 21 cells (18 animals), in which the stimulation frequency was increased to 1 Hz for 1 min. Peak ICa amplitude is plotted as the percent change relative to basal ICa recorded at 0.05 Hz, basal being the mean of the six records immediately preceding the switch to 1 Hz. The time course of the facilitation and subsequent depression of ICa could be modelled as the sum of two exponential components. The best fit was obtained with a maximum facilitation of 40%, reached with a time constant of 1.6 s, and a maximum depression of –33% reached with a time constant of 23 s.

3.2 Selective block of facilitation by metabolic inhibitors or substitution of Ca2+ with Ba2+
The effect of metabolic inhibition on the response to 1 Hz stimulation was investigated by superfusing cells with 200 µM dinitrophenol (n=3) or 5 µM FCCP (n=2). Both drugs inhibit mitochondrial function and had similar effects, reducing ICa at 0.05 Hz and abolishing facilitation, but not depression (Fig. 2(A)). After washing, both basal ICa and the response to 1 Hz stimulation (not shown) recovered fully. Fig. 2(B), lefthand panel, shows mean responses to 1 Hz stimulation before and during metabolic block. After block there was no evidence of facilitation, only a gradual depression of ICa, which was fit by a single exponential with time constant 24±8 s and maximum amplitude –34±8% (n=4, 3 animals). These values compare with depression under control conditions, implying that metabolic inhibition had little effect on the activity-induced depression. The righthand panel of Fig. 2(B) shows the mean result of subtracting responses in the presence of metabolic inhibitors from control responses. Facilitation revealed in this way was fit by a single exponential with amplitude 43±4% and time constant 2.7±0.7 s. These values closely resemble the amplitude and time constant of facilitation estimated from bi-exponential fits to the control responses, according to Fig. 1(B).


Figure 2
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  		Fig. 2 Separation of facilitation from depression. (A) Peak ICa amplitude plotted against time of whole-cell recording. 1 Hz trains are indicated by bars. Dinitrophenol (DNP, 200 µM) was applied as indicated. (B) Lefthand panel, percent change in peak ICa amplitude plotted against time of 1 Hz stimulation, under control conditions (bullet) and after superfusing with 200 µM DNP or 5 µM FCCP ({circ} data combined, n=4). The superimposed curve is an exponential fit to the mean response after metabolic inhibition, with amplitude 30%, time constant 14 s. (B) Righthand panel, component sensitive to metabolic inhibition, obtained by subtracting the inhibited responses in the lefthand panel from controls. The superimposed curve is an exponential fit with amplitude 39%, time constant 2.1 s. (C) Lefthand panel, percent change in ICa amplitude plotted against time of 1 Hz stimulation, in 1 mM CaCl2 (bullet) or BaCl2 ({circ}). Inset shows Ca2+ and Ba2+ currents immediately before switching to 1 Hz. (C) Righthand panel, the Ca2+-dependent component, obtained by subtracting each point in the response measured in BaCl2 from that in CaCl2.

 
The facilitation and depression of ICa could also be separated by replacing extracellular Ca2+ with equimolar Ba2+, as illustrated in Fig. 2(C). The records of ICa and IBa inset in the lefthand panel were obtained from a cell immediately before increasing the stimulation frequency and show the expected enhancement of current amplitude and slowing of inactivation by Ba2+. Ba2+ abolished facilitation in five cells from two animals without obviously affecting depression. Upon reperfusing with the Ca2+-PSS, ICa returned to control within 2 min, although the response to 1 Hz stimulation recovered fully in only one cell. In the presence of Ba2+, 1 Hz stimulation was maintained for only 30 s, because cells generally failed to survive if it was more prolonged. This period was too short to allow exponential fits, but it is clear from Fig. 2(C) that depression followed a similar time course whether Ca2+ or Ba2+ carried the current. The righthand panel of Fig. 2(C) shows the Ca2+-dependent component of the response, obtained by subtracting current amplitudes recorded in the presence of BaCl2 from those in the presence of CaCl2. It reveals a Ca2+-dependent facilitation that peaked in ~5 s and appeared to be sustained thereafter.

3.3 Effect of intracellular Ca2+ buffering and extracellular [Ca2+]
Fig. 3(A) shows the effect of intracellular Ca2+ buffering on the response to 1 Hz stimulation. When EGTA in the pipette was increased from 2 to 15 mM, or EGTA was replaced with 2 mM BAPTA, ICa responded in a qualitatively similar way; a transient facilitation was followed by depression. As before, facilitation was associated with slowing of ICa inactivation due to enhancement of as (Table 1). 1 Hz stimulation induced a significant increase in as and in the ratio, as/af, with BAPTA present. It also increased as in all four cells (three animals) studied with 15 mM EGTA, although the mean increase did not reach statistical significance. The peak facilitation amounted to 23±1% with 15 mM EGTA in the pipette and 13±1% (n=12, 9 animals) with 2 mM BAPTA. In both cases, facilitation was significantly (P<0.05) reduced compared with 2 mM EGTA. Facilitation measured at 2 mM BAPTA was also significantly (P<0.01) smaller than with 15 mM EGTA. The depression of ICa after 1 min at 1 Hz amounted to –10±4% of the facilitated level with 15 mM EGTA and –13±1% with 2 mM BAPTA. In comparison with 2 mM EGTA, these levels of depression were significantly reduced (15 mM EGTA, P<0.05; BAPTA, P<0.001).


Figure 3
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  		Fig. 3 Influence of intracellular Ca2+ buffering and [Ca2+]o. (A) Percent change in ICa amplitude plotted against time of 1 Hz stimulation when pipettes contained 2 mM EGTA (bullet; n=21), 15 mM EGTA ({blacksquare}; n=4) or 2 mM BAPTA ({blacksquare}; n=12). (B) Percent change in ICa amplitude plotted against time of 1 Hz stimulation when [CaCl2]o was 0.1 mM ({circ}), 0.5 mM ({bigtriangleup}) or 2 mM (bullet). (C) Maximum ICa facilitation plotted against [CaCl2]o. (D) ICa depression after 1 min at 1 Hz, measured relative to the facilitated current and plotted against [CaCl2]o. The curve shows the best fit of Eq. 2, with KD=2.0 mM, Dmax=–55% and Dmin=–5%. N=3, except at 1 mM (n=21) and 4 mM (n=4) CaCl2.

 
Substituting 2 mM BAPTA for EGTA in the pipette increased ICa by ~50% and slowed its inactivation due to a preferential increase in as with a reduction in {tau}s (see Table 1). These effects are similar to the changes in ICa kinetics observed during facilitation at 1 Hz. BAPTA may therefore inhibit the response to 1 Hz stimulation because it enhances and slows ICa, thereby limiting the potential for an additional increase. However, the effect of 15 mM EGTA was not associated with a change in ICa amplitude or kinetics (Table 1), suggesting that Ca2+ buffering was an important factor.

Both facilitation and depression were influenced by the extracellular CaCl2 concentration ([CaCl2]o). For [CaCl2]o above 1 mM we employed a HEPES-buffered bath solution, which had no effect on the response to 1 Hz stimulation at 1 mM CaCl2. Fig. 3(B) shows mean responses to 1 Hz stimulation with 2 mM EGTA in the pipette, in the presence of varying [CaCl2]o. Peak facilitation increased with increasing [CaCl2]o between 0.1 and 1 mM CaCl2, but fell to around 20% above 2 mM (Fig. 3(C)). The maximal facilitation was also reached earlier in the train as the [CaCl2]o was raised, and was less well maintained due to enhanced depression (Fig. 3(B)). As shown in Fig. 3(D), the amplitude of depression (D), measured relative to the maximally facilitated current, increased with increasing [CaCl2]o according to the following hyperbolic function:


Formula (2)

where c represents [CaCl2], Dmax and Dmin are the maximum and minimum amplitudes of depression reached at saturating or zero [CaCl2]o, respectively, and KD is the apparent dissociation constant for Ca2+ binding. The best fit to the data indicated Dmin=–5%, which along with the effects of extracellular Ba2+ implies that a minimal depression persists in the absence of Ca2+.

3.4 Sarcoplasmic reticulum Ca2+ uptake does not influence activity-dependent modulation of ICa
To test the involvement of SR Ca2+ release in the activity-dependent modulation of ICa, Ca2+ accumulation by the SR was prevented by exposing cells to the Ca-ATPase inhibitor, cyclopiazonic acid (CPA; 30 µM), for at least 10 min prior to and during recording. This depleted the SR so that non-clamped cells failed to exhibit the usual hypercontracture in response to the K+-free bath solution. However, the activity-induced facilitation (37±4%; n=5) and subsequent depression (–15±2%) of ICa were not significantly different from that seen in the absence of the drug. CPA also had no effect on the amplitude of ICa at 0.05 Hz, which was 2.9±0.4 pA/pF (n=5) in its presence. However, CPA accelerated ICa inactivation. This reflected shortening of both {tau}f (P<0.001) and {tau}s (P<0.05), to 7.0±0.5 ms and 84±8 ms (n=5) respectively; the amplitudes of the two components were unchanged, with as/af=1.3±0.8 being similar to control (Table 1).

3.5 Effect of isoprenaline on activity-induced modulation of ICa
When applied during stimulation at 0.05 Hz, isoprenaline enhanced ICa amplitude by a similar amount whether the pipette contained 2 mM EGTA or BAPTA. At a maximal concentration (1 µM), isoprenaline increased ICa 6-fold to 15.1±0.9 pA/pF (n=8, eight animals) with EGTA present and 5-fold to 18±3 pA/pF (n=5, four animals) with BAPTA. Its effects on the response to 1 Hz stimulation were, however, dependent on the intracellular chelator (Fig. 4). With EGTA present, isoprenaline suppressed facilitation in a concentration-dependent manner (Fig. 4(A)); at 1 µM complete block occurred in two of eight cells to leave a mean facilitation of only 3±1%. Additionally, the maximum depression of ICa was enhanced in a concentration-dependent manner (Fig. 4(B)). In contrast, over a wide concentration range, isoprenaline had no significant effect on facilitation (Fig. 4(A)) or depression (Fig. 4(B)) when pipettes contained 2 mM BAPTA. This suggests that the ability of isoprenaline to modulate facilitation and depression is related to the rise in submembrane [Ca2+] brought about by Ca2+flux across the sarcolemma, rather than cAMP-dependent phosphorylation. If this is the case, similar increases in Ca2+ influx, caused by isoprenaline or raised [Ca2+]o, would be expected to have comparable effects. This was the case. The mean ICa density, recorded with 2 mM internal EGTA, was similar in the presence of 2 nM isoprenaline (3.3±0.4 pA/pF, n=7) or 2 mM extracellular CaCl2 (3.4±0.2 pA/pF, n=3). The peak facilitation produced by 1 Hz stimulation was also similar under these conditions (isoprenaline 22±2%; CaCl2 20±3%) as was the depression (isoprenaline –19±2%; CaCl2 –24.7±0.7). Similarly, the mean ICa density recorded in the presence of 5 nM isoprenaline (4±1 pA/pF, n=3) was the same as in 3–5 mM CaCl2 (4±1 pA/pF, n=5) and both conditions produced facilitations (isoprenaline 14±7%; CaCl2 19±4%) and depressions (isoprenaline –24±9%; CaCl2 –27±4%) that were not significantly different.


Figure 4
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  		Fig. 4 Effect of isoprenaline on response to 1 Hz stimulation. (A) ICa facilitation, measured as maximum % increase in ICa after switching to 1 Hz, plotted against isoprenaline concentration. (B) ICa depression plotted against isoprenaline concentration. Depression was measured as maximum % decrease in ICa after switching to 1 Hz, relative to facilitated current. In (A) and (B), pipettes contained 2 mM EGTA ({square}) or 2 mM BAPTA ({circ}). Data in the absence of isoprenaline (EGTA, {blacksquare}; BAPTA, bullet) are also shown. Number of cells are indicated next to symbols in (A).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Increasing the frequency of ICa activation in guinea-pig ventricular myocytes from 0.05 to 1 Hz produced two distinct and opposing effects; a rapidly developing facilitation of peak ICa amplitude and a slower depression. Positive and negative staircases of ICa have been described before. There is strong evidence that facilitation is Ca2+ dependent, but the role of Ca2+ in activity-dependent depression is less clear. Depression has been proposed to result from incomplete recovery from voltage and/or Ca2+-dependent inactivation between stimuli [3,5,22] or from an ultra-slow form of voltage-dependent inactivation [12]. Here we show that both facilitation and depression are influenced by [Ca2+]o and intracellular Ca2+ buffering, but only facilitation requires Ca2+.

Since facilitation could be abolished without reducing depression they are independent processes. This has important implications, because a change in the amplitude or kinetics of one response could have pronounced effects on the other. For instance, the bell-shaped relationship between facilitation and [CaCl2]o can be explained by the opposing depression, which became increasingly pronounced as [CaCl2]o was raised. This would mask facilitation and can explain why facilitation peaked earlier at higher [CaCl2]o. This explanation is consistent with earlier findings [7] that facilitation had a similar magnitude at 2, 5 and 20 mM CaCl2 when measured with 20 mM internal EGTA. The greater Ca2+ buffering under these conditions would hinder the effect of high [Ca2+]o on depression. With 2 mM EGTA, depression may partially mask facilitation even at 1 mM CaCl2, giving the impression that facilitation is transient. Contrary to previous assumptions (e.g., Ref. [6]), our results suggest that facilitation is sustained during increased activity. Thus the facilitation blocked by metabolic inhibitors or Ba2+ substitution for extracellular Ca2+ was well maintained throughout the 1 min period at 1 Hz. Furthermore, the control response to 1 Hz stimulation could be modelled as the sum of two exponential processes, the maximum facilitation being reached within a few seconds and maintained throughout the stimulation period.

The speed of intracellular Ca2+ buffering was more important than buffering capacity for inhibiting facilitation and depression, because both were suppressed by equimolar substitution of BAPTA for EGTA, despite their similar Ca2+ affinities [23,24]. In fact, 2mM BAPTA suppressed facilitation more effectively than 15 mM EGTA. As evidenced by the failure of voltage-clamped cells to contract in response to depolarization, both chelators buffered bulk cytoplasmic Ca2+ effectively at 2 mM. BAPTA is more efficient, however, at buffering rapid [Ca2+] transients under the membrane [25,26]. This is demonstrated by the larger and more prolonged ICa observed with BAPTA, but not 15 mM EGTA, reflecting reduced Ca2+-dependent inactivation. Thus, as previously concluded [7,11,15], facilitation results from Ca2+ entering the cell through open Ca channels and binding to a site close to the inner mouth of the channel. This Ca2+ also enhances depression by binding at a site close to the membrane.

Neither facilitation nor depression were influenced by Ca2+ released from the SR, because they were unaffected by SR depletion with CPA. This agrees with previous reports that ryanodine, which blocks SR Ca2+ release, failed to affect frequency-dependent facilitation [7,11], although others found inhibition [2,27]. It is interesting that, despite blocking SR Ca2+ release, CPA accelerated ICa inactivation. This contrasts with the effect of ryanodine, which slows inactivation by removing Ca2+-induced Ca2+ release [28]. The simplest explanation is that the SR Ca2+-ATPase normally contributes to ICa kinetics by removing submembrane Ca2+ as it enters the cell.

4.1 Modulation of facilitation and depression by isoprenaline
Previous studies described various effects of isoprenaline on frequency-dependent facilitation of cardiac ICa. It has been reported to enhance facilitation at low concentrations [6,7,16] and to have either no effect [15] or to enhance [16] or inhibit [5–7] at high concentrations. In this study, isoprenaline caused a concentration-dependent block of facilitation and enhancement of depression, but only when pipettes contained 2 mM EGTA, not BAPTA. Thus, the effect of isoprenaline was seen only when the submembrane [Ca2+] was allowed to rise during increased activity. Furthermore, ICa facilitation and depression were comparable at concentrations of isoprenaline or extracellular CaCl2 giving rise to similar, unstimulated ICa densities. These observations suggest that the effects of isoprenaline were a direct consequence of increased Ca2+ influx. The apparent abolition of facilitation at high isoprenaline concentrations has previously been taken as evidence for the involvement of cAMP-dependent phosphorylation [5–7,9,14–16]. This may be wrong, because the effects of isoprenaline do not seem to depend on cAMP-dependent phosphorylation.

The control conditions used in the present experiments (2 mM EGTA, 1 mM extracellular CaCl2) proved optimal for recording ICa facilitation, in that facilitation was near maximal while depression was modest. As a consequence, small increases in Ca2+ influx would have little direct effect on facilitation, while enhancing depression sufficiently to partially mask facilitation. This can explain why even the lowest concentration of isoprenaline appeared to inhibit facilitation. Previous studies used different [CaCl2]o and/or intracellular Ca2+ buffers. This would influence the effect of isoprenaline by determining Ca2+ influx and submembrane [Ca2+] both before and during its application. In one study [7], for example, use of 20 mM EGTA would have limited the rise in submembrane [Ca2+] during stimulation, so that a larger Ca2+ influx would be required to produce the same degree of facilitation and depression. The observed enhancement of facilitation at low isoprenaline concentrations could therefore reflect an increase in Ca2+ influx that was just enough to stimulate facilitation without enhancing depression to limiting levels.

4.2 Mechanisms of facilitation and depression
Facilitation was associated with characteristic slowing of ICa inactivation, due to enhancement of the slower inactivating component. This was proposed in rat ventricular cells to reflect a voltage-driven switch from the fast to the slow channel-gating mode, which is regulated by Ca2+ and cAMP-dependent phosphorylation [15]. Since in guinea-pig cells this enhancement occurred without a change in the fast component, it could simply reflect an increased number of slowly inactivating channels. Facilitation was recently proposed to result from Ca2+-dependent activation of CamKII, based on the ability of CamKII inhibitors to block it [17,18]. In contrast, facilitation caused by directly increasing intracellular [Ca2+] was found to involve a phosphorylation-independent, but ATP-dependent mechanism [8,10], which was associated with a similar slowing of ICa inactivation [8]. The inhibitory effect of metabolic inhibitors on activity-induced facilitation is consistent with either mechanism. However, the masking effect of depression argues for caution in the interpretation of data appearing to show modulation of activity-induced facilitation.

Since ICa depression did not require Ca2+, it probably represents the ultra-slow, voltage-dependent inactivation previously described in these cells [12]. The Ca2+-dependent enhancement of depression could reflect direct channel modulation as Ca2+ accumulates under the membrane, either through binding to the channel, changing the internal membrane surface potential or reducing the driving force for Ca2+ entry. Submembrane Ca2+ accumulation could be significant, because during 1 Hz stimulation at [CaCl2]o ≥2 mM, a sizeable inward current appeared at –80 mV (not shown) where Ca2+ is known to activate inward current [29]. Another potential mechanism is Ca2+-induced channel dephosphorylation by calmodulin-dependent phosphatase [30], but this is unlikely to play a major role, because depression was unaffected by ATP depletion with metabolic inhibitors.

4.3 Summary and hypothesis
From the present and previous [8,10] results, we propose that the activity-induced facilitation of ICa is mediated by Ca2+-ATP binding to the Ca2+ channel following Ca2+ entry. A similar mechanism was proposed to explain ATP-dependent, but phosphorylation-independent modulation of ICa by Mg2+ [31]. During increased channel activity, ICa facilitation is counteracted by voltage-dependent but Ca2+-sensitive depression. The relative contributions of facilitation and depression to ICa during increased activity will depend upon factors influencing the trans-sarcolemmal Ca2+ flux or intracellular Ca2+ buffering.

Time for primary review 32 days.


   Acknowledgements
 
We gratefully acknowledge the financial support of the Wellcome Trust, and the help of Ms. S. Davenport and Mr. R. Davies in preparing the cells.


   Notes
 
1 1Present address: Cardiovascular Research, Rayne Institute, St. Thomas's Hospital, Lambeth Palace Road, London SE1 7EH. Back


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

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J Guo and H J Duff
Inactivation of ICa-L is the major determinant of use-dependent facilitation in rat cardiomyocytes
J. Physiol., March 15, 2003; 547(3): 797 - 805.
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