385

Journal of Physiology (1992). 453, pp. 385-400 With 6 figures Printed in Great Britain

THE EFFECT OF A CALMODULIN INHIBITOR ON INTRACELLULAR [Ca2"J AND CONTRACTION IN ISOLATED RAT VENTRICULAR MYOCYTES

By J. E. FRAMPTON AND C. H. ORCHARD From the Department of Physiology. University of Leeds, Leeds LS2 9JT

(Received 22 May 1991) SUMMARY

1. The effect of the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7; 1O /Lm) on intracellular [Ca2+] ([Ca2+]i) and [H+], and on contraction, has been studied in myocytes isolated from the ventricles of rat hearts. [Ca2]1i and [H+] were monitored using the fluorescent dyes Fura-2 and 2',7'bis(carboxyethyl)-5.6-carboxyfluorescein (BCECF) respectively. 2. W7 decreased the size of both the Fura-2 fluorescence (a function of [Ca2+]i) transient and twitch, but had no effect on their time course. 3. The decrease in the size of the Fura-2 fluorescence transient in the presence of W7 was accompanied by a decrease in the increase of Fura-2 fluorescence that could be elicited bv releasing Ca2+ from the sarcoplasmic reticulum using 10 mM-caffeine. 4. There was a decrease in the apparent sensitivity of the contractile proteins to Ca2+ in the presence of W7 which may account, in part, for the decrease in the twitch observed in the presence of MW7. 5. Test beats were interpolated at different test intervals after a train of steadystate contractions. Mechanical restitution curves were constructed by plotting the size of the test beat against the test interval. Both the size and the duration of the twitch increased as the test interval was prolonged. W7 slowed this mechanical restitution but had no effect on the changes in the duration of the twitch. 6. Intracellular pH was not altered by W7. 7. These results are discussed in terms of the known actions of calmodulin and W7.

INTRODUCTION

The role of the ubiquitous Ca2+-binding protein calmodulin (Cheung, 1980) in excitation-contraction coupling in cardiac muscle is unclear, although it is thought to affect both the uptake and release of Ca2+ from the sarcoplasmic reticulum (SR): calmodulin produces a Ca2+-dependent increase in the Ca2+ pump activity of isolated SR vesicles (e.g. Katz & Remtulla, 1978; Lopaschuk, Richter & Katz, 1980) which is associated with the phosphorylation of the protein phospholamban in the SR membrane by a Cai± calmodulin-dependent protein kinase (Le Peuch, Haiech & Demaille 1979, Kirchberger & Antonetz 1982; Tada, Inui, Yamada, Kadoma, UIS

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J. E. FRAMPTON AND C. H. ORCHARD

Kuzuya, Abe & Kakiuchi, 1983). Such stimulation of the SR Ca2+-ATPase is thought to increase Ca2+ accumulation by the SR (Fabiato, 1985a). Stimulation of Ca2+ uptake into the SR by the Ca2+-calmodulin-dependent phosphorylation of phospholamban can account for several observations in skinned cardiac cells associated with the withdrawal of calmodulin from the bathing solution: (i) a decrease in the amplitude, and a slowing of the time course, of the Ca2+ transient (Fabiato, 1985a); (ii) a slowing of recovery of the Ca2+ transient following a previous release of Ca2+ from the SR (Fabiato, 1985 b) and (iii) the slowing of the time-course of potentiation of contraction of skinned ventricular myocytes produced by a simulated Ca2+ current. However these data were obtained by using simulated Ca2+ currents to induce Ca2` release from the SR of skinned cardiac cells, and Ca2+-induced Ca2+ release (CICR) may itself be modified by calmodulin (see below). More recently it has been suggested that the rise in intracellular [Ca2+] ([Ca2+]i transient) that induces contraction may also stimulate Ca2+ uptake by the SR via a calmodulin-dependent phosphorylation of phospholamban, but that this uptake decays with time following a [Ca2+]i transient (Schouten, 1990). Thus at short intervals following a [Ca2+]i transient, Ca21 uptake into the SR will be rapid, but as the interval increases, the rate of uptake into the SR will decrease. Such a mechanism can explain the observation that when the interval between contractions is decreased, the rate of relaxation of the twitch is increased (Allen & Kurihara, 1980; Schouten, 1990; Frampton, Orchard & Boyett, 1991) because of an increased rate of Ca2+ accumulation by the SR. It has also been suggested that such a mechanism can explain why the twitch becomes larger as the interval from the previous twitch is increased (i.e. mechanical restitution): when the second twitch occurs following a short interval, most of the Ca2+ released from the SR is rapidly re-accumulated by the stimulated Ca2+ pump, so that little remains free to activate the myofilaments. However after longer intervals the pump is less potentiated, and so more Ca2+ enters the bulk cytoplasmic space to activate the myofilaments (Schouten, 1990). However, the absence of calmodulin modifies, but does not abolish, 'mechanical restitution' in skinned cardiac cells (Fabiato, 1985 b). Thus 'beat-to-beat' control of [Ca2+]i may exist which is mediated via Ca2+-calmodulin-dependent phosphorylation of the SR: an increase in [Ca2+]i resulting in greater activation of the Ca2+ pump of the SR and hence an increase in the rate of removal of Ca2+ from the cytoplasm (Kirchberger & Antonetz, 1982; Movsesian, Nishikawa & Adelstein, 1984). If calmodulin stimulates Ca2+ uptake by the SR, and hence increases the amount of Ca2+ available for release (Fabiato, 1985 a, b) it would be expected that calmodulin inhibitors such as N-(6-aminohexyl)-5-chloro- 1 -naphthalensulphonamide (W7; Hidaka, Yamaki, Naka, Tanaka, Hayashi & Kobayashi, 1980), would decrease Ca2+ uptake and hence decrease Ca2+ release and contraction. However, Kodama, Anno, Satake & Shibate (1990) observed that 100 ,aM-W7 produced a marked increase in the strength of contraction, even though the amplitude of the Ca2+ current in voltageclamped ventricular myocytes was reduced in a dose-dependent manner above 10 /tM. Further, the physiological role of calmodulin-dependent phosphorylation of phospholamban has been questioned by Lindemann & Watanabe (1985), who showed that inotropic agents which produced physiological increases in [Ca2+]i alone (in the absence of an increase in cAMP), did not stimulate phospholamban

387 W77 AND CARDIAC MUSCLE phosphorylation in intact hearts. Thus the evidence that calmodulin is involved in stimulating Ca2" uptake by the SR is not unequivocal. In addition to its effects on Ca2" uptake by the SR, calmodulin also inhibits CICR from isolated canine SR vesicles (Meissner & Henderson, 1987) and has been shown to directly inhibit single cardiac SR Ca2"-release channels in a Ca2"-dependent manner (Smith, Rousseau & Meissner, 1989). This has led to the suggestion that inhibition of normal CICR by calmodulin may compensate for an increase in myoplasmic LCa2J]i during increased (cardiac) muscle activity (Smith et al. 1989). Calmodulin may also have other roles within the cardiac cell: it has been suggested that calmodulin-dependent phosphorylation is involved in the regulation of the Na'-H' antiporter in cultured rat ventricular cells (Weissberg, Little, Cragoe & Bobik, 1989). Such inhibition may alter intracellular pH, and hence contractile function (Orchard & Kentish, 1990). In the present study we have, therefore, examined the effect of the calmodulin inhibitor W7 (Hidaka et al. 1980) on the [Ca2+]i transient, the twitch and the Ca2" content of the SR by using caffeine (cf. Fabiato, 1985b; Smith, Valdeomillos, Eisner & Allen, 1988; Frampton et al. 1991), on mechanical restitution, and on intracellular

pH, using ventricular myocytes isolated from rat hearts and loaded with the ionselective fluorescent dyes Fura-2 (for Ca2") or BCECF; 2',7'-bis (carboxyethyl)-5,6carboxyfluorescein for Hf). METHODS

Isolation of ventricular myocytes and loading with Fura-2 and BCECF The preparation of myocytes has been described previously (Frampton et al. 1991). In brief. adult rats (250-300 g) were deeply anaesthetized with chloroform. The heart was removed and Langendorif perfused at constant flow with physiological salt solution (see below) containing Ca2+ (0 75 mM) at 37 'C. When the preparation was stable, perfusion was switched to a nominally Ca2+free solution (+ 100 /tm-EGTA) for 5 min. The heart was then perfused with salt solution containing collagenase (1 mg/ml; Worthington, type II), protease (01 mg/ml; Sigma, type XIV) and Ca21 (50-100/UM). This solution was recirculated to give a total exposure to enzyme of about 9 min. At the end of the enzyme perfusion, the ventricular tissue was sectioned and shaken in the enzyme-containing solution +2o% bovine serum albumin for 5 min at 37 'C. This mixture was filtered through gauze and the filtrate centrifuged. The supernatant was removed and the cell pellet was resuspended in salt solution containing Ca2" (0 5 mM) and the cells allowed to settle again at room temperature. Isolated myocytes were then incubated in physiological salt solution containing Ca2+ (0 5 mM) and either Fura-2-AM (Fura-2-acetoxymethyl ester form) or BCECF-AM (5 AM) as appropriate, for 12-15 min at room temperature. The cells were then centrifuged, the supernatant removed, and the cells resuspended in control solution (containing 1 mM-Ca2+) and kept at room temperature until they were used.

Apparatus This has been described previously (Frampton et al. 1991). In brief, ventricular myocytes were allowed to settle on the glass-coverslip bottom of a chamber mounted on the stage of a Nikon Diaphot inverted microscope, enclosed within a darkened Faraday cage. Solutions were pumped to the chamber at approximately 3 ml/min. Two input lines were controlled by electrically operated solenoid valves, which enabled a rapid solution changeover (within 4 s). Experiments were carried out at room temperature (24-27 TC). The cells were field stimulated via two platinum electrodes on either side of the bath. To measure cell length (and hence contraction), cells were illuminated from above with red (> 610 nm) light. This created an image of the cell which was collected by the objective lens and directed to the side port of the microscope, where it was separated from the

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dye fluorescence by a 580 nm dichroic mirror. The cell image was then focused onto a linear 1024element photodiode array (EG & G. Reticon, Wokingham. Berkshire). From the output of the array, the length of the cell was measured electronically as described in detail by Boyett, Moore, Jewell, Montgomery, Kirby & Orchard. 1988). Measurement of Fura-2 and BCECF fluorescence Fura-2-loaded myocytes were alternately (approximately every 2-4 ms) excited with ultra violet light of 340 and 380 nm wavelengths as described previously (Frampton et al. 1991). Excitation light from the filters was transmitted to the microscope where a 430 nm dichroic mirror beneath the microscope nosepiece reflected the excitation light to the cell under study via a 40 x oil immersion FLUOR objective lens (numerical aperture 1-3; Nikon UK Ltd. Telford. Shropshire). The resulting Fura-2 fluorescence was collected by the objective lens and transmitted to the side port of the microscope where it passed through a variable rectangular diaphragm (Nikon, UK Ltd) which was arranged so that it only outlined the cell under study, thus ensuring fluorescence from neighbouring cells was not measured. The fluorescence was reflected by a 580 nm dichroic mirror to a photomultiplier tube (Thorn EMI 9844B, Ruislip, Middlesex) via a 510 nm emission filter (bandwidth 20 nm), which ensured only fluorescence at about 510 nm was detected. The output of the photomultiplier tube (the fluorescence signal) was electronically correlated with the excitation filter in the lightpath. The ratio (emission at 510 nm during excitation at 340 nm/emission at 510 nm during excitation at 380 nm, a function of [Ca2"]) was determined using a custom-built analogue divider circuit and was displayed, with the 340 and 380 nm signals, on a chart recorder (Gould Electronics Ltd, Ilford, Essex) and stored on magnetic tape (Racal 7DS FM recorder) for later off-line analysis. Fluorescence signals were filtered by low-pass filters with a cut-off frequency of 100 Hz unless otherwise stated. Records of Fura-2 fluorescence and cell length (unfiltered) were normally averaged (a = 16 sweeps) and analysed using a Tandon computer fitted with a Data Translation DT2805 A/D board and running 'Vacuum' data acquisition software, sampling each channel at 1 kHz. To obtain beat-by-beat plots of peak Fura-2 fluorescence and twitch shortening, a Cambridge Electronic Design (CED) 1401 A/D interface was used, driven by a Tandon 386 computer. Fluorescence from BCECF-loaded myocytes was excited and collected in a similar manner as that from Fura-2-loaded myocytes, but excitation wavelengths were 440 and 490 nm, and fluorescence was monitored at 540 nm. The ratio (emission at 540 nm during excitation at 440 nm/emission at 540 nm during excitation at 490 nm) was used to obtain a qualitative estimate of intracellular pH (pHi). The BCECF fluorescence ratio was filtered using a low-pass filter (time constant, T = 2-7 s). The problems associated with the use of Fura-2 to monitor [Ca2+]i and the calibration of fluorescence ratio into values of [Ca2+]i have been discussed previously (e.g. Frampton et al. 1991). However, in the present study we are primarily interested in qualitative changes of [Ca2+]i that occur in the presence of calmodulin inhibitors. Similarly. in the experiments in which pH1 was measured, the BCECF fluorescence ratio was not calibrated. since this should not affect the qualitative interpretation of our results.

Solutions The composition of physiological salt solution used during the cell isolation procedure was (mm): Na', 130-4; Cl-, 142-4; K+, 5-4; HEPES, 5; glucose, 10; H2PO4, 0 4; Mg2+, 3-5; taruine. 20 creatine, 10; Ca2", 0 75; set to pH 7 2 with NaOH. The solution used during the experiments contained (mM): Na+, 135; K+' 5; Mg2+, 1; HCO32- 20; Cl-, 102; 802-, 1; Ca2+, 1; acetate, 20; glucose, 10; insulin 5 u/l. This solution was equilibrated with 5 o CO2-95 %02 to give a pH of 7-3 or with 15 % C02-85% 02 to give a pH of 6-8 (when required). Caffeine was dissolved in the control solution (above) just before use to give a final concentration of 10 mm. W7 was obtained from Sigma and was used at a concentration of 10 uM in all experiments described in this paper. Neither these concentrations of caffeine and W7, or these changes of pH, had a significant effect on Fura-2 fluorescence in vitro at the excitation and emission wavelengths used in the present study. Statistics All data are expressed as means +S. E.M. of n preparations. Statistical comparisons were made using either a paired t test or Student's t test as appropriate.

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RESULTS

The effect OJ 117' upon Fua 2 fluoresc1ene and twitch contra(tion in isolated rat ceutricular ntyocytesj Figure 1 shows a slow time-base record of cell length and Fura-2 fluorescence from a representative mvocyte before and during exposure to 10 /M-W7. On exposure to W7. there was a gradual decline in both the peak of the Fura-2 fluorescence transient (a function of LCaa21]i) and the amplitude of twitch contraction. In six out of nine mvocytes there was also a small decline in the diastolic fluorescence ratio. It appears likely, therefore, that at least a part of the decrease in the size of the twitch inl the presence of WNT7 is dlue to a decrease in the amount of ("a21 initiating contraction. The changes in Fura-2 fluorescence are shown more clearly in the fast time-base records of Fura-2 fluorescence in Fig. 2A which also shows that despite the changes in the amplitude. 10 /,M-"W7 had no effect on the half-time of relaxation (th) of the Fura-2 fluorescence transient. (The ti of the fluorescence transient in (control conditions was 178 + 6 ms and in the presence of 10,am V-7 was 179 + 11 ms, n = 15; twitch ti (control) was 83+7 ms and after exposure to 10 JtM-W7 was 91+9 ms, n = 8; these changes were not significant.) The effects of W7 on Fura-2 fluorescence and contraction were reversible (not shown), typically within 10 min. The effect of W17 upon the caffeine-induced release of C(a2+ fromt the SR One explanation for the negative inotropic effect of W7 described above may be a reduction in the amount of Caa21 stored within the SR available for release, as a result of inhibition of (la21 calmnodulin-dependent C(a21 transport (see Introduction). Therefore we have used the rapid application of caffeine (it) mm) to assess the effect of XN'i on the (Ca2 (ontent of the SR (cf. Smith et al. 1988: Frampton et al. 1991). The result shown in Fig. 2 is typical of the effect of 10 /IM-W7 upon the caffeine-induced release of Caa2+ from the SR. In this cell exposure to 10 /,tm-WN7 for 20 min resulted in a decrease in systolic Fura-2 fluorescence (top). Stimulation was then stopped and the cell was rapidly superfiised with solution (containing 10 mm-caffeine. In these experiments caffeine normally reached the cell under study within 6-8 s following cessation of stimulation; the response to caffeine appeared stable at intervals between 3 and 30 s under (control conditions, although at shorter intervals the response decreased (not shown). The bottom panel of Fig. 2 shows that the amplitude of the caffeine-induced increase of Fura-2 fluorescence, as a consequence of Caa2+ release froni the SR, was markedly reduced by W7. In six out of seven cells there was a clear decrease in the size of the caffeine-induced increase of Fura-2 fluorescence after exposure to 10 upm-NN17. These results suggest that in the presence of 10 /tmWV7. there is a decrease in the amount of CA2+ that is stored in the SR and is available for release by caffeine (but see Discussion). The effect of Wr7 upon the 'aj)parent' inyofilamnent (Ca21 sensitivity The decrease inl the size of the twit(h induced by W7 usually ap)eare(l more marked than the depressionn of the peak of the Fura-2 fluorescence transient. This suggested that there had been a reduction in myofilament Caa21 sensitivity during

J. E. FRAMPTON AND C. H. ORCHARD

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exposure to W7 which could also, in part, explain the observed negative inotropic effect of W7. To examine the effect of 10 jtM-W7 upon the apparent myofilament Ca2+ sensitivity, the effect of W7 upon the relationship between the size of the twitch and 10 #M-W7

145

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Fig. 1. The effect of W7 on Fura-2 fluorescence and contraction in an isolated ventricular myocyte. Top, slow time-base record of cell length (contraction shown as a downward deflection of the trace). Bottom, simultaneous recording of Fura-2 fluorescence ratio (a function of [Ca2+],). Stimulation frequency 1 Hz.

systolic Fura-2 fluorescence was compared with the effect of changing bathing [Ca2+] (Ca2+) (cf. McIvor, Orchard & Lakatta, 1988). Figure 3A shows the experimental protocol used. Initially, Ca2+ was decreased from 10 to 0 5 mm which resulted in a decline in cell contraction (top trace) and the peak of the Fura-2 fluorescence transient (lower trace). Following the return to 1 mm-Cao , the cell was exposed to 10 aM-W7. Figure 3B compares the relationship between twitch shortening and the peak of the Fura-2 fluorescence transient during exposure to W7, with that when Ca2+ was decreased, on a beat-by-beat basis throughout the experiment shown in Fig. 3A. It is clear that the relationship is shifted to the right in the presence of 10 4uM-W7, indicating a decrease in apparent myofilament Ca2+ sensitivity. Similar results were obtained in four cells. It appears likely, therefore, that this decrease in sensitivity contributes to the negative inotropic effect of W7. The effect of W7 upon the pattern and time course of early mechanical restitution To test the hypothesis that calmodulin plays a role in mechanical restitution, and the changes in twitch duration that occur at different inter-stimulus intervals (Schouten, 1990; see Introduction), we have studied the effect of the calmodulin inhibitor W7 on mechanical restitution and twitch duration. Figure 4 illustrates the experimental protocol used to test the effect of W7 upon mechanical restitution. The myocytes were stimulated at a frequency of 0 5 Hz. A control mechanical restitution curve was constructed by interpolating test contractions at different test intervals following a train of regular stimuli, and plotting the size of the test contraction against the test interval (Fig. 5A). The cell was then exposed to 10 jM-W7 and

W7 AND CARDIAC MUSCLE

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A

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Fig. 2. Superimposed traces showing the effect of 10 ftm-W7

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transient (A), and the caffeine-induced increase of Fura-2 fluorescence (B) in a single ventricular myocyte. The baselines in control and during exposure to W7 have been set to the same level in order to show more clearly the decrease in the amplitude during exposure to W7. Stimulation frequency before application of caffeine was 1 Hz. See text for further details of methods.

mechanical restitution measurements were repeated after the twitch had decreased to approximately 50 % of the control value. In each experiment, the size of the test contractions were expressed as a percentage of the average contraction amplitude during the regular contractions just before the test interval (since the steady-state twitch continued to decline in W7 during the period of restitution measurements). Figure 5A illustrates typical mechanical restitution curves under control conditions and following exposure to W7, from a representative ventricular myocyte.

H.ORU(0HHAhlR) J. E. FRAMPTON AND C R.

392

These restitution curves were fitted using a double exl)onential in which the first (shortest) time constant, Tl, represented the rate of recovery of (contraction up to the steady-state interval (2 s). In the example in Fig. 5A, T1 increased from 0 33 s during control to 1 46 s during exposure to W7, suggesting that this phase of recovery had A

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Fig. 3. The effect of 0-5mim-C(ao and l() plm-W7 on Fura-2 fluorescence and twitch contraction. A, slow time-base (hart recording of cell length (top) and Fura-2 fluorescence (bottom). Ca 2 was decreased from 1 to 0(5 mM., and WN'7 was added to the superfusate as indicated above the records. Stimulation frequency was 1 Hz. BI plot of the relationship between p)eak systolic fluorescence ratio and tx itch shortening during exposure to 0)5 mMCa 2 (0) and 1i)ujm-W7 (A). Data from experiment shown in Fig. '3A.

been markedly slowed by W7. Similar results have been seen in six cells (means; sr (control) was 062 + 0-1 s which increased to 153 +± )3 s during exposure to Wr7; / = 01015; n = 6).

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In addition. the half-time of relaxation of the test contraction was also measured (cf. Schouten, 1990). Figure 5B shows the effect of 10,aM- W7 on the relationship between test interval and the half-time of relaxation in a representative cell: as the duration of the test interval was increased. so the half-time of relaxation of the test

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8 11 mmn Fig. 4. Protocol used to measure the effect of 10 jUMW-7 upon mechanical restitution in an isolated rat ventricular myocyte. The traces are continuous. The top trace illustrates the construction of the restitution curve under control conditions (1 mnn-Ca"+. 25 0C). The times above the record show the test intervals used at each point. At 'FSD', the gain of the chart recorder was increased (note the change in cell-length scale) before exposure to 10 /IM-W7. The protocol was repeated after cell contraction had declined to approximately 50'0S of the control value. Note that this cell developed mechanical alternans following exposure to 10 gM-NV7. Steady-state stimulation rate = 0(5 Hz.

twitch increased (cf. Schouten, 1990). However, 10 ,mm-XV7 had no significant effect upon the interval dependence of twitch duration (n = 6). Thus although 10,mM-W7 appears to slow the rate of recovery of contraction in isolated rat ventricular myocytes, this effect appears to be independent of any changes in the interval dependence of twitch contraction. It is also worth noting that mechanical alternans (alternation of large and small contractions) occurred in several (eight out of twenty-seven) cells exposed to W7. When recovery was also followed, the alternans disappeared on removal of W7. It has previously been suggested (e.g. Orchard, McCall, Kirby & Boyett, 1991), that such alternans is the consequence of slowed mechanical restitution, due to slowed recycling of Ca2" by the SR. The data shown above supports this idea.

The effect of W7 upon pHi in isolated rat ventricular myocytes Weissberg et al. (1989) have shown that W7 impairs the ability of cultured rat ventricular myocytes to recover from an ammonium chloride-induced acid load and eventually results in a fall in pHi. Thus the effects of 10 gtm-W7 upon the apparent

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myofilament sensitivity and the slowing of mechanical restitution could be explained, in part, if an intracellular acidosis occurs during exposure to W7 (Orchard, 1987; McCall & Orchard, 1991). Figure 6 shows an experiment designed to investigate whether exposure to 10 aivmW7 resulted in an intracellular acidosis under the conditions of the present study. A A

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Fig. 5. A, the effect of 10 #M-W7 on mechanical restitution in a single rat ventricular myocyte (@, control; A, 10 /LM-W7). Twitch amplitude was expressed as a percentage of previous steady-state value (see text for further discussion). B, the effect of 10 ,UM-W7 on the interval dependence of the half-time of twitch relaxation.

BCECF-loaded myocyte was initially exposed to a respiratory acidosis by increasing CO2 to 15 % (see Methods), which resulted in a marked decrease in twitch contraction and the BCECF fluorescence ratio (a function of pHi). Following recovery, the cell was then exposed to 10 ftM-W7 for 25 min. Although there was a slight decrease in pHi upon initial exposure to 10 /LM-W7, this was small compared to the fall induced by the preceding acidosis. The fluorescence ratio then remained stable whilst contraction continued to decline. Similar results have been seen in five cells. Since exposure to 10 /aM-W7 does not result in a significant intracellular acidosis, it appears unlikely that the effects of W7 upon fluorescence and contraction described above result from an intracellular acidosis.

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DISCUSSION

The main finding of the present study is that the calmodulin antagonist W7 exerts a negative inotropic effect upon isolated rat ventricular myocytes, which is not accompanied by a change in the time course of either the Fura-2 fluorescence

15%

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C02 128 (D.

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5 min Fig. 6. The effect of 10 /M-W7 on the twitch (upper panel) and BCECF fluorescence ratio (a function of intracellular pH, lower panel). The cell was exposed to a respiratory acidosis (15% C02) and 10 #M-W7 as indicated above the records. Note the development of mechanical alternans in this cell during superfusion with 10lM-W7.

transient or the twitch. This negative inotropic effect appears to be accompanied by, and may, therefore, be caused by, a decrease in the Ca2+ content of the SR that can be released by caffeine and a reduction in the apparent myofilament Ca2+ sensitivity. In addition, W7 significantly slowed mechanical restitution, athough there was no effect on the interval dependence of twitch duration. The effect of W7 upon [Ca2+]i and twitch The negative inotropic effect of 10 jM-W7 in the present study contrasts with the observation of Kodama et al. (1990) that 10 ,uM-W7 had virtually no effect on tension recorded in isolated guinea-pig papillary muscle, but produced a small positive inotropic effect on guinea-pig atrial muscle. Our results do agree, however, with those of Lindemann & Watanabe (1985), who showed that perfusion of isolated rat ventricles with the calmodulin antagonist trifluoperazine (10#JM), resulted in a clear decrease in developed tension with no significant effect upon the half-time of relaxation. It may be, therefore, that there is a species difference in the response to such antagonists. The decrease in the size of the Fura-2 fluorescence transient in the presence of W7 may be explained if inhibition of calmodulin by W7 decreases the uptake, and hence release, of Ca2+ by the SR (see Introduction), and so leads to a decrease in the size of the Fura-2 fluorescence transient and twitch. More unexpected, however, was the observation that W7 had no effect on the half-

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J. E. FRAMIPTONS ANAND C H. ORCHARD

time of the decline of the Fura-2 fluorescence transient or the twitch: If calmodulin normally regulates Ca21 uptake by the SR (see Introduction) it might be expected that inhibition of calmodulin by W7 would inhibit uptake, and hence slow the rate of decline of the Fura-2 fluorescence transient, because the rate of decline of the [Ca21]i transient is probably determined largely by the rate at which the SR reaccumulates Ca2+ (Allen & Kurihara, 1980; Wier. 1990). Our observation that W7 did not produce such a slowing has two possible explanations. First, 10 /,M-W7 does not significantly inhibit the rate at which the SR can accumulate Ca2 , possibly because under normal conditions there is no Ca2+-calmodulin regulation of Ca2+ uptake by the SR (Lindemann & Watanabe, 1985). In this case an alternative explanation must be sought for the decrease in the size of the Fura-2 fluorescence transient and the decreased caffeine-induced Ca2+ release observed during the application of W7 (see below). Alternatively the effects of W7 on Ca2+ uptake by the SR may be masked by other effects of W7 on the cell. One possibility is that inhibition of phosphodiesterase by WA7 (Hidaka et al. 1980) leads to an increase of cAMP which would stimulate Ca2+ uptake by the SR, and hence shorten the [Ca2+]i transient. Although possible, it seems unlikely that the effect of calmodulin inhibition (which would lengthen the [Ca2+]i transient) would be exactly offset, in every cell, by a rise in cAMP (which would shorten the [Ca21]i transient). In addition, the concentration of W7 required for 50 % inhibition of phosphodiesterase (67 aM; Hidaka et al. 1980) is much greater than that used in the present study, so that such an effect would be small in the data presented above.

The effect of W7 upon the release of [Ca21]i from the SR by caffeine The rapid application of caffeine was used to assess the effect of exposure to 10 jiMW7 on the Ca2+ load of the SR (cf. Smith et al. 1988; Frampton et al. 1991). It was consistently observed that the amount of Ca21 that could be released from the SR by caffeine was markedly reduced in the presence of W7 (Fig. 2). There are four possible explanations for this result. Firstly, W7 inhibits Ca21 uptake by the SR, so that the SR contains less Ca2+ to be released. However the measurements of time course of the Fura-2 fluorescence transient do not support this idea (above). Secondly, the slowing of mechanical restitution by W7 means that by the time caffeine reaches the cell, less Ca2+ may be ready for release in the presence of W7 than under control conditions (see below). However, caffeine generally reached the cell after a delay of 6-8 s following the last stimulus. This interval should be sufficient for virtual completion of the fast phase of mechanical restitution in both the presence and absence of W7 (Fig. 5). Thus, if caffeine releases Ca2+ from the same pool as the action potential (O'Neill & Eisner, 1990, Frampton et al. 1991), this hypothesis seems unlikely since restitution should be virtually complete in both conditions. Thirdly, W7 (10 /kM) may itself exert a direct inhibitory action upon the Ca2+-release channel at [Ca2+]i values close to those typically reported during diastole (Smith et al. 1989). Thus in the presence of W7, the amplitude of the response to caffeine may be reduced because Ca2+ efflux via the Ca2+-release channel is an integral component of the mechanism by which caffeine induces the release of [Ca2+]i from the SR (e.g. Rousseau & Meissner, 1989; O'Neill & Eisner, 1990). In this case, a decrease in the rate of rise of Fura-2 fluorescence in response to caffeine might be expected. Such a decrease was observed in three out of six cells, suggesting that this might be one contributory

11V7 ANI) CARDLAC MUSCLE9

397

factor. 'hep decrease in the 1)eak of the response may then be explained by the ability of Ca21 sequestration pathways to limit the rise of [(a2+]1i more effectively than when it is released rapidly. Fourthly. the decreasee in the SR Caa2+ content may reflect an inhibition bV 10 /rMV W7 of the size of the slow, C(a2+-loading component of the Ca2+ current, I'a (e.g. Fabiato. 1985c), which will result in less Ca2+ available for uptake by the SR (see below). Consistent with this hypothesis are the observations of Kodama et al. (1990). who reported that W7 reduces the amplitude of the Ca2+ current in isolated voltage-clamped ventricular guinea-pig myocytes in a dosedependent, but non-specific manner (dissociation constant. Kd = 13 8 JtM). Klockner & Isenberg (1987) have also observed a similar non-specific inhibition of 'Ca in isolated guinea-pig mivocytes withl other, structurally unrelated, calmodulin antagonists. Such a decrease in 'Ca would not only decrease the Ca2 -loading component of IC, but would also decrease the trigger for CICR, and hence the amount of ("a2+ released in response to normal stimulation. Either of these latter two mechanisms could. therefore, explain the decrease in both the amplitude of the Fura-2 fluorescence transient, and in the amount of Ca2+ released from the SR bly caffeine.

The effect of W7 upon the appaz.rent (1(12+ sensitivity of the myoftlaments The result illustrated in Fig. 3 suggests that 10 yuM-W7 may reduce the apparent (Ca2+ sensitivity of the (contractile l)IroteiIls in isolated rat ventricular myocytes. Previously. Kodama et (t. (1990) relorte(1 that 100 ,aM-XV7 significantly reduced the Ca2+ sensitivity of tlhe contractile proteins in canine skinned ventricular muscle. These results are consistent with the report of Silver, Pinto & Dachiw (1986) that W7 inhibits car(liac nyofil)rillar ATPase activity (IC() = 26 /uM). This effect of W7 is probably calmodulin inldependent because Fabiato (1985a) has shown that removal of calmodulin from the medium bathing the myofilaments of a skinned cardiac cell does not affect the (la2+ sensitivity of the contractile proteins. The possibility that \V7 wvas decreasing myofilament Ca2+ sensitivity by producing an intracellular acidosis (e.g. see Orchard & Kentish, 1990) by inhibiting the Na+-H+ exchange mechanismn (Weissberg et al. 1989) seems unlikely because our results with BC(ECF-loaded myocytes suggest that neither the magnitude nor time course of the change of p14j in W7 were appropriate to explain the observed changes in the size of the twitch.

The effect of W17 onl2 mechanical restitation In the )resent stu(ly we ol)serve(1 that the 'fast phase' of mechanical restitution (i.e. the recovery of twitch amplitude at test intervals up to the steady-state value) was significantly slowed in the presence of 10 1M-W7. Since mechanical restitution appears to be predominantly due to Ca2+ recycling by the SR (e.g. McCall & Orchard. 1991) this is consistent with the observation of Fabiato (1985b) that removal of calmodulin slows the re)riming of the Ca2+ release processes in the SR. This suggests that calhnodulinlel)een(lent processes do play a role in determining the pattern of recovery from contraction (Schouten, 1990; see Introduction), although perhaps not by altering the rate of (la2+ uptake by the SR (above). However the data do not support the hypothesis of Schouten (1990; see Introduction) for two reasons. F;.st. in the scheme put forward by Schouten, the twitch becomes larger with

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increasing intervals because of a decrease in Ca2+-calmodulin-dependent activation of the SR Ca2+-ATPase (see Introduction). Thus in the presence of the calmodulin inhibitor W7, the Ca2+-calmodulin activation of the ATPase should be absent throughout, so that the test twitch should be the same size as the preceding twitches, as there should be no transient calmodulin-dependent stimulation of the ATPase. Secondly, there was no concurrent effect on the interval dependence of testinterval twitch duration. Schouten's hypothesis suggests that the rapid twitch observed at short intervals is because of the transient calmodulin-dependent stimulation of the SR Ca2+-ATPase. In this case, W7 should abolish these changes in the time course of the twitch with varying test intervals. However no such effect was observed. Such slowed restitution may, however, underlie the decreased Fura-2 fluorescence transient observed in the presence of W7: slowed Ca2' recycling by the SR will mean that less Ca2+ recycling will occur between stimuli, so that less Ca2+ will be ready for release and so less Ca2+ will be released, and the [Ca2+]i transient will be smaller. The slowed restitution may also explain the mechanical alternans that frequently occurred in the presence of W7: Orchard et al. (1991) suggested that the slowed mechanical restitution observed during respiratory acidosis could explain the appearance of mechanical alternans in isolated ferret ventricular myocytes. Thus the development of mechanical alternans in cells exposed to 10 ,tM-W7 is consistent with the observation that mechanical restitution is slowed by W7. However since W7 induced mechanical alternans in the absence of a change of pHi (e.g. Fig. 6) these results suggest that the slowing of restitution, and subsequent appearance of mechanical alternans, in W7 cannot be attributed to an acidosis. Alternative explanations are that W7 slows mechanical restitution by direct interaction with the SR Ca2+-release channel (Smith et al. 1989; see above) or by inhibiting some unknown calmodulin-dependent process (above).

Summary These data suggest that many of the possible calmodulin-independent effects of W7, can have marked effects on the excitation-contraction coupling pathway in cardiac muscle. However, given that W7 is also a potent calmodulin inhibitor (e.g. Hidaka et al. 1980), the absence of effects of W7 on the time course of the Fura-2 fluorescence transient and the twitch do not support the idea that Ca2+ uptake by the SR is normally regulated by the effects of Ca2+ calmodulin. It remains possible, however, that Ca2+ uptake by the SR is regulated by other Ca2+-dependent mechanisms. We would like to thank Professor David Eisner and Drs Mark Boyett, Stephen O'Neill, Simon Harrison and Godfrey Smith for helpful discussion, and Dr Simon Harrison for useful comments on an earlier version of the manuscript. We thank the Medical Research Council and the Wellcome Trust for financial support. REFERENCES

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