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Possible Functional Linkage Between the Cardiac Dihydropyridine and Ryanodine Receptor: Acceleration of Rest Decay by Bay K 8644
J Mol Cell Cardiol 28, 79–93 (1996)
Possible Functional Linkage Between the Cardiac Dihydropyridine and Ryanodine Receptor: Acceleration of Rest Decay by Bay K 8644 E. McCall, L. V. Hryshko, V. M. Stiffel, D. M. Christensen and D. M. Bers Department of Physiology, Loyola University Medical Center, 2160 South First Avenue, Maywood, Illinois 60153, USA (Received 8 June 1995, accepted in revised form 12 July 1995)
E. MC, L. V. H, V. M. S, D. M. C D. M. B. Possible Functional Linkage Between the Cardiac Dihydropyridine and Ryanodine Receptor: Acceleration of Rest Decay by Bay K 8644. Journal of Molecular and Cellular Cardiology (1996) 28, 79–93. The effect of the dihydropyridine -Type Ca chanel agonist Bay K 8644 on post-rest contractions in ferret ventricular muscle and isolated myocytes was investigated. Bay K 8644 was shown to abolish rest potentiation and greatly accelerate rest decay. The post-rest contraction suppressed by Bay K 8644 was accompanied by action potentials of large amplitude and longer duration, but voltage-clamp measurements showed that this suppression was not due to a supra-optimal ICa trigger. Caffeineinduced contractures and rapid cooling contractures demonstrated an accelerated rest-dependent decline in sarcoplasmic reticulum (SR) Ca content in the presence of Bay K 8644, which was present even with Ca-free superfusion during rest. Thus, the Bay K 8644-induced decline of SR Ca during rest was independent of extracellular Ca or ICa. To explore whether the binding of Bay K 8644 to the dihydropyridine receptor could alter the SR Ca release channel/ryanodine receptor in a more direct way, ryanodine binding was measured in the absence and presence of Bay K 8644. Ryanodine binding to isolated ferret ventricular myocytes was increased by Bay K 8644 under conditions where sarcolemmal-SR junctions might be expected to be intact, but not after physical disruption. These results are consistent with a working hypothesis where Bay K 8644 may bind to the dihydropyridine receptor and this may lead to physical changes in the linkage between the dihydropridine receptor and a subset of ryanodine receptors, thereby increasing the opening of the SR Ca release channel during rest (and accelerating resting Ca loss). 1996 Academic Press Limited K W: Cardiac muscle, Sarcolemmal Ca channel, Sarcoplasmic reticulum, Dihydropyridine receptor, Ryanodine receptor, Bay K 8644.
Introduction
(+) enantiomer behaves as a partial Ca channel antagonist (Sanguinetti et al. 1986; Kass, 1987). Bay K 8644 is now widely used as a specific and diagnostic activator of -Type Ca channels. When mammalian ventricular muscle is stimulated after a period of rest, the contraction amplitude at the first post-rest contraction can either be increased or decreased. This is commonly referred
Voltage clamp studies of Ca current (ICa) have demonstrated that the dihydropyridine (DHP) compound Bay K 8644 is a selective -Type Ca channel agonist (Hess et al., 1985). The (−) enantiomer and the racemic mixture (+−) of Bay K 8644 exhibit this Ca channel agonist effect, while the
Please address all correspondence to: Donald M. Bers, Ph.D. Department of Physiology, Loyola University Medical School, 2160 South First Ave., Maywood, Il 60153, USA.
0022–2828/96/010079+15 $12.00/0
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to as rest potentiation or rest decay, respectively (Bers, 1991). In some species (e.g. rabbit and guinea-pig ventricle), only rest decay is normally observed and this is thought to be due to the gradual depletion of sarcoplasmic reticulum (SR) Ca during rest with subsequent extrusion from the cell by Na/Ca exchange (Bers et al, 1989, 1993). In rat ventricle, rest potentiation is more prominent and can persist for many minutes. This rest potentiation may be due to either a rest-dependent increase in SR Ca content or slow recovery of the excitation– contraction coupling mechanism (Bers, 1989; Bers et al., 1993). Some species (e.g. ferret and canine ventricle) exhibit a combination, with rest potentiation for short rest periods, followed by rest decay for longer rests (Hryshko et al., 1989a; Bers et al., 1983). An unexpected action of Bay K 8644 has previously been observed in canine ventricular muscle (Hryshko et al., 1989a,b; Saha et al., 1989; Bouchard et al., 1989). It was found that Bay K 8644 abolished rest potentiation and greatly accelerated rest decay in canine ventricle. This action was seen with both the (−) enantiomer and racemic Bay K 8644, but not with the (+) enantiomer (i.e. just as with the increase in ICa). Their results suggested that the acceleration of rest decay seen with Bay K 8644 in dog ventricle was due to a more rapid loss of SR Ca. Here we report similar effects of Bay K 8644 in ferret ventricular muscle. The aims of the present study were to evaluate the fundamental mechanism by which Bay K 8644 is able to accelerate rest decay. The results are consistent with a hypothesis whereby Bay K 8644 binds to the DHP receptor and transmits a signal (independent of ICa) to a subset of SR Ca release channels to increase channel opening and SR Ca decline during rest. Some of this work has previously appeared in abstract form (Hryshko et al., 1990; McCall and Bers, 1994).
Methods Muscle isolation procedure The isolation procedures used were similar to those described previously (Hryshko et al., 1989c). Briefly, hearts were excised from adult male ferrets (Marshall Farms, North Rose, NY, USA) under sodium pentobarbital anaesthesia (140 mg/kg i.p.), the right ventricle was opened and suitable isolated trabeculae and papillary muscles (0.1–1 mm in diameter) were removed and the ends were tied
with fine suture. They were then suspended in a 0.15 ml muscle chamber, with one end attached to a post and the other to a piezoresistive force transducer (AE 875, Aksjeselskapet Micro-Elektronikk, Horten, Norway).
Measurement of post-rest contraction The preparations were superfused, at flow rates of 15 ml/min, with normal Tyrode (NT) solution containing (m): 140 NaCl, 6 KCl, 2 CaCl2, 1 MgCl2, 2 CaCl2, 10 glucose, 5 HEPES, pH 7.4 at 37°C. The regular stimulation frequency was 0.5 Hz, induced by field stimulation using platinum electrodes during the equilibration period (>45 min) and between rests. Force developed by the preparation during steady-state (SS) stimulation and upon resumption of regular stimulation after a 10 to 300 s rest period (post-rest, PR) was measured. After obtaining results with NT, the perfusate was changed to NT plus 1 l Bay K 8644, and the experimental protocol was repeated. The effects of Bay K 8644 at 1 l were similar to those at 100 nM and were progressively weaker at lower concentrations. Quantitative dose-response curves were not conducted, but most experiments were done with 0·1 or 1 l Bay K 8644 to produce nearly maximal effects.
Assessment of SR Ca load: rapid cooling contractures (RCCs) The theory and practical applications of RCCs have been considered in earlier studies (Kurihara and Sakai, 1985; Bers et al., 1987, 1989). Briefly, rapid cooling of cardiac muscle to temperatures lower than 5°C results in Ca release from the SR, such that the amplitude of the resultant RCC is indicative of the SR Ca content at the moment of cooling. In this series of experiments, RCCs were evoked by rapidly lowering superfusate temperature to 0°C. This was accomplished by having NT flowing through perfusion lines jacketed with either water (at 37°C) or a 3:1 water:propylene glycol mixture (−2°C), with the specific solution perfusing the chamber controlled by means of solenoid pinch valves. At a flow rate of 15 ml/min switching to the cooled perfusate reduced muscle surface temperature to below 5°C in <0.5 s, and the chamber temperature was kept at >0°C during constant perfusion (for >30 s). The protocol used was similar to that described above for the assessment of PR
Acceleration of Rest Decay by Bay K 8644
contractions except that RCCs were initiated at the end of the rest intervals rather than twitches.
Action potential measurements Action potentials (APs) were measured in ferret isolated trabeculae and papillary muscles perfused with NT, in the presence and absence of 1 l Bay K 8644, at 37°C, using conventional glass microelectrodes filled with 3 KCl (typically 15–20 MX resistance). The stimulation and rest periods were as described above, with SS and PR APs and contractions measured.
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allowed to equilibrate at 37°C for >20 min prior to experimental protocols. The protocol used was similar to that described for the whole muscle experiments (0·5 Hz and 30–90 s rest periods). However, the extracellular solution was sometimes changed during the rest period to a 0Ca/0Na solution to prevent Ca influx (and efflux via Na/Ca exchange). In this 0Ca/0Na solution Na was replaced with Li and 1 m EGTA replaced the Ca. For control runs [Ca]o in the control solution was increased to 3 m, whereas after equilibration with 100 n Bay K 8644 [Ca]o was reduced to 1 m. This was done so that the amplitude of contractions was similar in the presence and absence of Bay K 8644.
Myocyte isolation procedure Assessment of SR Ca load: caffeine contractures (CafC) Isolation of ventricular myocytes was carried out as previously described (Hryshko et al., 1989c; Bassani et al., 1994). Hearts were excised from adult male ferrets (1–1.3 kg, Marshall Farms, North Rose, NY, USA.) under sodium pentobarbital anesthesia (140 mg/kg i.p.), then mounted in a Langendorff perfusion apparatus and perfused with nominally Ca-free, HEPES-buffered Tyrode solution for 5 min at 37°C and a flow rate of 20 ml/min. The heart was then perfused with the same solution containing 1 mg/ml collagenase (Type B, Boehringer Mannheim) and 0·16 mg/ml pronase (Boehringer Mannheim) until it became flaccid (10–30 min), after which the digested tissue was separated and filtered. The resultant cell suspension was rinsed several times and the Ca concentration gradually increased to 2 m. Prior to experimental use the myocytes were plated onto Plexiglas superfusion chambers, with the glass bottoms of the chambers treated with laminin (Gibco, Grand Island, NY, USA) to increase cell adhesion.
The rapid application of a caffeine-containing solution induces a contracture in myocytes, and the amplitude can be used as an index of SR Ca content (Smith et al., 1988). Caffeine (10 m) was added to the 0Ca/0Na solution (see above) as a powder, at 37°C. The caffeine-containing solution was introduced into the chamber via a quick-switching device, similar to that described previously (Bassani et al., 1994). The stimulation and perfusion protocols used were similar to those described above, except that a rapid switch to caffeine was applied at the end of the rest intervals rather than an electrical stimulus. Caffeine application was continued for approximately 6 s, by which time the caffeine contracture had begun to decline. The tip of the quick-switching device was then washed, to remove any residual caffeine solution, and the cell perfused with either the 3 m Ca or 100 nM Bay K 8644 with 1 m Ca Tyrode solution for about 2 min prior to resumption of stimulation.
Measurement of cell shortening and rest decay
ICa measurement
Myocyte shortening was measured as previously described (Bassani et al., 1994). Myocytes were superfused with Tyrode solution at 37°C and a flow rate of 6 ml/min. Field stimulation at 0·5 Hz was induced via platinum electrodes and cell shortening at both ends was measured using a video-edge detection system (Crescent Electronics, Sandy, UT, USA) connected to a strip chart recorder, for immediate display of contraction records, and stored using a video recorder for off-line analysis. The basic normal Tyrode’s solution (NT) used in these experiments was as described above and cells were
ICa was measured by the whole-cell voltage-clamp technique using an Axopatch 1C patch clamp amplifier (Axon Instruments, Foster City, CA, USA) (see Hryshko and Bers, 1990, for further details) in ferret cardiac myocytes isolated as described above. Patch electrodes were used with resistances of 0·5–1·5 MX (glass type TW150–6, World Precision Instruments, Sarasota, FL, USA) and containing: (m) 140 CsCl, 10 EGTA and 5 Mg-ATP, pH 7.1 at 37°C or 30°C. Myocytes were superfused with: (m) 140 Tetraethylammonium (TEA) Chloride, 6 CsCl, 2 CaCl2, 1 MgCl2, 10 glucose, 5 HEPES, pH
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7.4. These solutions were chosen to eliminate contaminating Na and K currents (i.e. no Na inside or out, and Cs and TEA replacing K). Cells were held at −90 mV and then pulsed to 0 mV for 100 ms at 0.5 Hz. On achieving a SS level of ICa the cells were then held at −90 mV during the rest intervals (30–300 s), after which 0.5 Hz depolarizing pulses to 0 mV were resumed.
and linear regression for Scatchard plots (using GraphPad InPlot, GraphPad Software Inc.). Since we are measuring ryanodine binding in physiological, not optimal equilibrium binding conditions, Kd and Bmax values must be interpreted cautiously. Thus, we focus on differences in ryanodine binding rather than inferred pharmacological parameters which are condition dependent (Lai et al., 1989). Protein concentration was measured by the Lowry method.
Ligand binding studies The effect of Bay K 8644 on ryanodine binding was investigated using isolated ferret myocytes. For ligand binding studies, 3H-ryanodine (62.5 lCi/ lol) and 3H-PN200-110 (90.9 lCi/lol) purchased from New England Nuclear were used. Ryanodine binding was carried out essentially as previously described (Lai et al., 1989; Bers and Stiffel, 1993) at physiological ionic strength. Except where noted, the incubation medium contained 140 m KCl, 20 m Tris, 1 m EGTA, 0.1–3 m free [Ca], 1–200 n 3H-ryanodine at pH 7.4 with protease inhibitor cocktail (final concentrations: 75 n aprotinin, 0.23 l PMSF, 0.83 m benzamidine, 1 m iodoacetimide, 1.1 l leupeptin and 0.7 l pepstatin A). Samples were preincubated in the presence and absence of 0.1–1 l Bay K 8644 for 30 min prior to addition of ryanodine with similar results between concentrations (1 l was used for most subsequent experiments to obtain maximal effects). Cold ryanodine (17 l was used to displace 3H-ryanodine and allow measurement of specific ryanodine binding. Incubations were at 37°C for 90 min, which was found to be optimal for ryanodine binding. Longer times would be required for true equilibrium, but long incubations at 37°C resulted in a gradual decrease in apparent ryanodine binding (presumably due to degradation). The reaction was terminated by 3×3 ml washes of distilled water with a Brandel Cell Harvester through Whatman GF/B filters. The dihydropyridine PN200-110 was used to measure DHPR using a buffer consisting of a final concentration of 25 m Tris, 10 m HEPES-Na, 1 m EDTA, and 1.1 m MgCl2, 0.1–15 nM 3HPN200−100, pH 7.4. Nifedipine (16·6 l) was used to allow measurement of nifedipine-sensitive PN200-110 binding. All tubes were incubated at room temperature in the dark for 90 min. The reaction was terminated by 2×3 ml washes with ice cold 10 m Na-HEPES, pH 7·4 through GF/B filters with a Brandel Cell Harvester. Curve fitting of pooled binding results was by a least squares method for simple Michaelis binding
Reagents Unless otherwise stated experimental reagents used were of analytical grade and supplied by Sigma (St. Louis, MO, USA). (+−) Bay K 8644 was obtained from Miles Pharmaceuticals (West Haven, CT, USA). A stock solution was made up in ethanol and stored at −20°C. Aliquots of this solution were added to the perfusate immediately prior to use. In functional studies ethanol never exceeded 0.1% in the perfusate.
Results Acceleration of rest decay by Bay K 8644 The characteristic effect of Bay K 8644 on PR contraction in ferret cardiac muscle is illustrated in Figure 1. The upper panel shows SS and PR force development in a ferret papillary muscle in the absence and presence of Bay K 8644. Under control conditions the first PR contraction after a 1 min rest in this preparation showed post-rest potentiation, i.e. it had a greater amplitude (84% increase) than that elicited by regular stimulation. The subsequent contractions were initially smaller than the SS contractions, then gradually increased to the SS level (not shown). In contrast, the first PR contraction after a 5-min rest was smaller than the SS level (50% decrease), resumably reflecting Ca loss from the SR, with subsequent contractions progressively increasing back to SS level. The addition of 1 l Bay K 8644 increased SS force development 2.8-fold in this preparation. The positive inotropic effect of both the (−)-enantiomer and the racemic mixture of Bay K 8644 has been shown in a number of studies, and is thought to reflect an increase in -Type ICa (Tiaho et al., 1990). The surprising effect was that Bay K 8644 also alters the PR response pattern of the preparation, with no rest potentiation after the 1-min rest in
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Acceleration of Rest Decay by Bay K 8644 (a) Control
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Figure 1 The effect of Bay K 8644 exposure on the contractile response of isolated ferret papillary muscle. (a) Original experimental tracings of force developed by a ferret papillary muscle at 37°C in response to regular stimulation at 0.5 Hz and after 1- and 5-min rest periods, in the absence (left) and presence (right) of 1 l Bay K 8644. Note the scale change with Bay K 8644. (b) Pooled results for experiments as in (a) in the absence and presence of 1 l Bay K 8644. Data shown are mean±... (n=4–5, except at 15 s where n=3 and no ...). Differences are significant (P<0.5) for all points beyond 4 s.
Bay K 8644. The PR contraction after a 5-min rest was significantly smaller than both the SS level in Bay K 8644 (93% decrease) and the 5-min PR response under control conditions (even in absolute terms, despite the larger SS twitch after Bay K 8644). This was a consistent effect. Pooled results from experiments like this with additional rest intervals are shown in Figure 1b. Under control conditions increasing the rest interval led to a biphasic pattern of PR force development, with increases in PR twitch force observed for rest intervals up to 1 min and progressive decline thereafter (similar to other control results in this preparation, Wier and Yue, 1986;
McCall and Orchard, 1991). The addition of Bay K 8644 completely eliminated rest potentiation, and depressed PR force at all rest intervals. This effect of Bay K 8644 has previously been shown in canine ventricular muscle (Hryshko et al., 1989a), but was not readily apparent in preliminary experiments in rabbit, rat or guinea-pig ventricle, or in ferret preparations at room temperature (not shown).
Electrophysiological measurements One possibility to explain the Bay K 8644 accelerated rest decay is that Bay K 8644 could induce
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Figure 2 The effect of Bay K 8644 on action potentials. Control membrane potential and force recordings before and after a 5-min rest at 37°C before (left) and after equilibration with 1 l Bay K 8644 (right).
an unexpected effect on the action potential at the PR contraction (i.e. decreased amplitude or duration). This does not seem to be the case as illustrated by the action potential recordings shown in Figure 2, which are typical of the results obtained in three muscles. The PR action potential exhibited an increased duration and amplitude in both the absence and presence of Bay K 8644. While the amount of change differed from muscle to muscle, both rest and Bay K 8644 always increased action potential duration. The pattern of SS and PR force developed by the preparation and the effect of Bay K 8644 in Figure 2 was similar to that in Figure 1. Since Bay K 8644 is expected to increase inward ICa the increase in action potential duration and SS contraction is not surprising. However, the strong suppression of the PR contraction is still apparent, even though the AP characteristics would be consistent with greater Ca influx. These results indicate that, under these conditions, rest decay of force occurs even though Ca influx into the cell is expected to be increased. In an elegant series of experiments demonstrating Cainduced Ca-release (CICR) in mechanically ‘‘skinned’’ cardiac cells Fabiato (1985) showed a
biphasic effect of ‘‘trigger’’ [Ca] on SR Ca release. SR Ca release increased with increasing [Ca] trigger levels (up to 1–4 l). At higher levels of trigger [Ca] (e.g. 10 l) SR Ca release appeared to be effectively inactivated. To test the possibility that Bay K 8644 leads to supraoptimal triggering for SR Ca release during PR contractions, ICa was measured in the absence and presence of Bay K 8644 in isolated ferret ventricular myocytes. Representative results are shown in Figure 3. The intrinsic effect of rest on ICa amplitude in ferret ventricular myocytes from physiological holding potentials is to decrease the ICa amplitude as shown in the control traces in Figure 3 (see also Hryshko and Bers, 1990; Bers et al., 1993). Addition of 1 l Bay K 8644 increased control ICa amplitude twoto three-fold, but the ICa amplitude was still lower at the PR pulse than at SS. This suggests that the PR trigger Ca would be lower than the SS trigger (in the absence and presence of Bay K 8644). Therefore, it is unlikely that the small PR contraction could be attributed to supra-optimal Ca triggering with Bay K 8644. The PR ICa amplitude with Bay K 8644 is also much larger than the
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Figure 3 The effect of Bay K 8644 on ICa. ICa elicited under SS conditions and after a 5-min rest interval from a holding potential of −90 mV to a test potential of 0 mV. Data are shown for control conditions (left) and after equilibration with 1 l Bay K 8644. This cell was studied at 30°C and is representative of 10 cells studied at either 30 or 37°C.
control, indicating that a decrease in PR ICa trigger with Bay K 8644 is also not the reason for the small PR twitch with the DHP. Rapid cooling contractures and SR Ca content The SR is the main source of activating Ca in ferret ventricular muscle and is important in PR contractile behavior (Bers, 1991). As alterations in Ca influx do not appear to adequately account for the observed PR effects of Bay K 8644, we tested the possibility that Bay K 8644 affects SR function. This hypothesis was tested by studying SS and PR RCCs before and after exposure to Bay K 8644. The results are shown in Figure 4. The 2 s RCC represents the degree of SR Ca loading under SS conditions. Under control conditions the Ca content of the SR decreased monotonically as the rest interval lengthened, as indicated by a reduction in the amplitude of the RCCs. The transient increase in twitch force (rest potentiation in Fig. 1) despite a monotonic decline in SR Ca in ferret ventricular myocytes is explained by a relatively slow change in recovery of the E-C coupling mechanism during rest (Bers et al., 1993). Figure 4b shows that the gradual decline in SR Ca content based on RCCs in control (t1/2 >200 s) was greatly accelerated after equilibration with Bay K 8644 (t1/2 <30 s). This accelerated decline in SR Ca content during rest could explain the acceleration of rest decay of twitches observed with
Bay K 8644, as hypothesized by Hryshko et al. (1989b) for canine ventricular muscle. That is, Bay K 8644 promotes diastolic SR Ca loss, which may underlie the acceleration of rest decay induced by Bay K 8644. During rest at −90 mV it is not expected that there is any significant activity of Type Ca channels. This, then, raises the possibility that Bay K 8644 might exert its effects on resting SR Ca fluxes via a physical link between the DHP receptor and the Ca release channel, independent of ICa. Thus, the effect of Bay K 8644 on ryanodine binding was studied in ferret ventricular myocytes to determine if Bay K 8644 exhibited effects consistent with a promotion of SR Ca channel opening.
Ryanodine binding in cardiac myocytes There is substantial evidence for a physical linkage between the sarcolemmal DHP receptor and the SR ryanodine receptor in skeletal muscle (Brandt et al., 1990; Kim et al., 1990). This linkage seems to be central in E-C coupling even if additional proteins are involved (Rı´os and Pizarro´, 1988). Furthermore, ryanodine binding in both cardiac and skeletal ryanodine receptor is known to be sensitive to a variety of agents which alter channel open probability or SR Ca release. That is, concentrations of adenine nucleotides and Ca which are optimal for channel opening also stimulate ryanodine binding.
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(a) Control
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Figure 4 The effect of Bay K 8644 on SR Ca loading in isolated ferret papillary muscle. Original experimental tracings of SS force at 0.5 Hz and RCCs after 2 s (SS RCC), 1- and 5-min rest periods, in the absence (left) and presence (right) of 1 l Bay K 8644. Arrow indicates where rapid cooling was applied in the trace where no RCC was observed. Pooled results showing force developed during RCCs in ferret ventricular muscle over a range of rest intervals, in the absence and presence of 1 l Bay K 8644 (Bay K). Data are shown as mean±... (n=3–5).
Thus we sought to test the hypothesis that ryanodine binding in ventricular myocytes was increased by Bay K 8644 (perhaps secondary to an increased in the probability of resting openings of the SR Ca release channel). Initial studies with isolated ferret ventricular myocytes which were subjected to vigorous homogenization (with a Polytron) and/or subcellular fractionation in attempts to prepare “cardiac diads” failed to display consistent effects of Bay K 8644 on ryanodine binding.
Figure 5 shows the opposite extreme in terms of preparations, i.e. ryanodine binding in intact isolated ventricular myocytes in a normal extracellular solution (except that [Ca] was reduced to 0.5 m to minimize spontaneous activity during the required long incubation). In isolated single cell experiments, the mean resting [Ca]i is not altered appreciably by Bay K 8644 (not shown). Thus, we do not anticipate any difference in [Ca]i during the binding experiments in Figure 5. This figure shows that Bay K 8644 increased ryanodine binding and this effect was significant at all ryanodine concentrations above 10 n. Additionally the 95% confidence bands for the linear regressions of the Scatchard plots (Fig. 5, right) were virtually nonoverlapping. While true equilibrium conditions were not achieved for ryanodine binding experiments, the parallel shift in the Scatchard plot would suggest an increase in apparent Bmax without a change in apparent Kd. Experiments were also performed with isolated cells suspended in an intracellular type solution (140 m KCl, 6 m Na) with [Ca] reduced to 30 l (Fig. 6a). This [Ca] was selected because it is near the optimum for ryanodine binding (see Fig. 7). The aims were to control intracellular [Ca] better, and to create minimal mechanical disturbance. Ryanodine binding is still increased with Bay K 8644, but the difference is smaller than in Figure 5 (and is only significant at 30 and 100 n ryanodine, although the apparent Bmax was still increased by 27% in Bay K 8644). In Figure 6b the same preparations as in Figure 6a were exposed to the same medium, but with the addition of 10 l digitonin to ensure that [Ca] was reasonably controlled at 30 l. The difference between the control and the Bay K 8644 curves is further diminished. Two explanations for the loss of Bay K 8644’s effect with high K buffer and digitonin are that (1) the clear difference in NT (Fig. 5) was secondary to a more optimal [Ca] inside Bay K 8644-treated cells (a difference eleminated after digitonin permeabilization) or (2) either depolarization in high K or disruption of the linkage with digitonin decreases an intrinsically real effect of Bay K 8644 on polarized and intact junctions. Permeabilized myocytes exposed to 30 l Ca may also hypercontract and this could alter mechanical linkages at the membrane. Figure 7 shows the [Ca] dependence of ryanodine binding to digitonin permeabilized myocytes. In this case the free [Ca] in the test tubes during the assay were measured directly with Ca-selective mini-electrodes (see legend). Digitonin permeabilization was required to ensure that the [Ca] measured in the
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Acceleration of Rest Decay by Bay K 8644 Ryanodine binding, intact ventricular myocytes NT, 0.5 mM Ca 20 Bay K Bound/free
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Figure 5 Ryanodine binding to isolated ferret ventricular myocytes. Freshly dissociated myocytes were incubated in a modified normal Tyrode’s solution (NT) with free [Ca] reduced to 0.5 m. Results are from four to five cell preparations in the presence and absence of 1 l Bay K 8644. The curve fits shown in the left panel for control and Bay K 8644 suggest Kd of 39 and 46 n and Bmax of 656 and 904 fmol/mg respectively (from pooled data at each [ryanodine], whereas the average from curve fits to individual preparations gave Kd values of 49±10 and 53±6 n and Bmax values of 743±73 and 963±102 fmol/mg respectively). The Scatchard plots at the right show linear regressions for control and Bay K 8644 with Kd of 36 and 40 n and Bmax of 642 and 854 fmol/mg respectively.
incubation medium was the same as in the cells. The optimal [Ca] was >100 l but varied from 30–300 l in different preparations. The results shown in Figure 8 provide evidence that the Bay K 8644 effect requires some level of mechanical integrity. These cells were homogenized mildly (five passes with a glass-teflon homogenizer). After homogenization there was still an apparent stimulation of ryanodine binding by Bay K 8644. However, in part of the sample also subjected to sonication before assay (to increase disruption), the effect of Bay K 8644 was eliminated. Similar effects were seen in two other preparations treated in a similar manner, and, furthermore, in two other preparations in which aggressive homogenization (2×30 s Polytron bursts) was performed prior to assay. While the effects of Bay K 8644 on ryanodine binding are not dramatic, the observed stimulatory effects of Bay K 8644 on ryanodine binding appear to be sensitive to the mechanical integrity of the cells or junctions, and may be sufficient to account for the observed functional effects (i.e. a reduction in PR contractions). That is, modulation of only a small fraction of ryanodine receptors could be sufficient to produce the physiological effects observed.
Is acceleration of rest decay secondary to increased Ca entry? Another potential explanation for the Bay K 8644induced acceleration not excluded so far is the following. If Bay K 8644 increases Ca entry locally by occasional openings of sarcolemmal Ca channels even at rest, this could activate individual local SR Ca release channels (via Ca-induced Ca-release). The result would be more rapid depletion of SR Ca during rest. This mechanism would, therefore, provide an explanation which does not require any physical communication between the sarcolemma and SR (rather Ca would serve as a second messenger). The final experimental series evaluated this possibility. The strategy was to remove all extracellular Ca during the rest (and include EGTA), such that no diastolic Ca influx would be possible. Then the SR Ca content was tested at the end of different rest periods. In this series we also changed the extracellular [Ca] so that both control and Bay K 8644-treated cells had similar SR Ca loads at the start of the rest period. We found that using 3 m Ca in control conditions and lowering [Ca]o to 1 m when Bay K 8644 was added produced this effect (see Fig. 9a and b).
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(a) Bound ryanodine (fmol/mg)
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Intact ferret myocytes 1000
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Figure 7 The [Ca]-dependence of ryanodine binding to isolated ferret ventricular myocytes in the presence and absence of 1 l Bay K 8644. The free [Ca] in the test tubes during the assay procedure were measured directly with Ca-selective mini-electrodes (Hove-Madsen and Bers, 1992) and was often slightly different from the calculated value. Thus, the free [Ca] varied somewhat in different preparations. The points represent average values for both free [Ca] and the ryanodine binding measured in the samples. The incubation included 140 m KC1, 6 m NaC1, 1 m EGTA, 30 n 3H-ryanodine, 10 m HEPES at pH 7.4.
140 mM K, 30 µ M Ca +10 µ M Digitonin Bay K
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Figure 6 Ryanodine binding to isolated ferret ventricular myocytes in high K solutions containing 140 m KC1, 6 m Na and 30 l Ca at pH 7.4. Results are from four to eight preparations. The same preparations were used in both panels, but in (b) 10 l digitonin was included to permeabilize the sarcolema. The curve fits in the panel (a) for control and Bay K 8644 used Kd of 24 and 26 n and Bmax of 541 and 686 fmol/mg respectively. For panel (b) Kd was 45 and 48 n and Bmax was 680 and 762 fmol/mg respectively.
The SS twitch contraction amplitudes were similar in the absence and presence of Bay K 8644 (5.7±0.5 l for control, 6.2±0.6 l with Bay K 8644, n=18), although there was a clear prolongation of twitch relaxation with Bay K 8644, presumably due to the effects on action potential duration (see Fig. 2). Bay K 8644 accelerated rest decay of twitches in the isolated myocytes in a similar way to multicellular preparations (data not shown, P<0.05; ANOVA followed by Bonferroni t-test for multiple, repeated, pairwise comparisons, n=18). Therefore this accelerative effect on rest decay did not reflect the inotropic state of the preparation. The acceleration of the rest decay of twitches with Bay K 8644 was still observed in the absence of extracellular Ca and Na during the rest (not
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Figure 8 The effect of disruption by sonication on the effect of 1 l Bay K 8644 on ryanodine binding. Isolated ferret ventricular myocytes were lightly homogenized in 140 m KC1, 6 m NaC1, 1 m EGTA, 10 m HEPES at pH 7.4. A portion of this homogenate was further disrupted by 5 min of sonication in a bath sonicator. Free [Ca] in the incubation buffer was 450 l.
shown), evaluated by switching to 0Ca/0Na solution during rest (±Bay K 8644) and returning to the steady-state just prior to the test twitch. However, clear quantitative interpretation of these results are complicated by the longer action potential duration and Ca influx which occurs. Thus,
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Figure 9 The effect of Bay K 8644 on twitch contraction and caffeine contracture amplitudes. (a) Original records of contractions elicited at 0.5 Hz in a representative ferret ventricular myocyte under control conditions (3 m Ca) and in the presence of Bay K (100 n Bay K 8644, 1 m Ca). (b) Original records of CafC elicited in the same cell 2s after cessation of SS stimulation, in the absence and presence of Bay K 8644, Note change in time scale. (c) Original records of post-rest CafC, obtained after the rest periods indicated, obtained from the same cell, during continuous superfusion with control solution with and without Bay K 8644 (left panels), or after superfusion with 0Ca/0Na during the rest period (in the absence and presence of Bay K 8644, right panels).
caffeine-induced contractures were used to assess SR Ca content (Figs 9b and c). In Figure 9b replacing a steady-state twitch with a CafC (2 s interval) produced contractions of very similar amplitude for control (3 m Ca) and 1 m Ca plus 100 nM Bay K 8644 (25.8±3.8 l for control, 28.0±4.3 l with Bay K 8644, n=11). This indicates that the level of SR Ca loading is about the same in both cases. Figure 9c shows post-rest CafC where the cell was in the steadystate solution (left) or in the 0Ca/0Na solution during the rest (right). Bay K 8644 accelerates the rest-dependent decline in SR Ca content in both the presence and absence of extracellular Ca. This can be seen graphically for pooled experimental data in Figure 10. The reduction in CafC amplitude with Bay K 8644 was significant at each interval tested and for both control and 0Ca solution during the rest (P<0.05, n=9–11). Thus, diastolic Ca influx
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Figure 10 The effect of Bay K 8644 on the rest decay of SR Ca content assessed by CafC. In the upper panel cells were rested under control conditions (3 m Ca, filled circles) and in the presence of Bay K 8644 (100 n+1 m Ca, open circles). In the lower panel cells were rested in 0Ca/0Na solution (with m EGTA) in the absence (filled circles) and presence (open circles) of 100 n Bay K 8644. Data shown as mean±..., n=9–11.
is not responsible for the effects of Bay K 8644 on rest decay and SR Ca loss.
Discussion Relation to previous work The acceleration of rest decay induced by Bay K 8644 in ferret ventricular myocytes is similar to that observed in canine ventricle (Hryshko et al., 1989a,b; Saha et al., 1989; Bouchard et al., 1989). The present results indicate that this effect is not due to changes in action potential or ICa associated with either the steady-state or post-rest twitch. Rather, results from RCCs and caffeine-induced contractures provide compelling evidence that this acceleration in rest decay is due to a faster decline in
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SR Ca content during rest (even when the initial SR Ca content was the same). Despite the apparent dissociation from ICa, the acceleration of rest decay may still be due to Bay K 8644 binding to its expected pharmacological target, the -Type Ca channel or dihydropyridine receptor. Although detailed dose-response curves have not been done, the concentration where Bay K 8644 produced this effect are similar to those required for increasing ICa (and are maximal at 100–1000 n). Acceleration of rest decay and increased ICa are both observed with either the (−) enantiomer or racemic Bay K 8644, but not with the (+) enantiomer or other DHP Ca channel antagonists (Sanguinetti et al., 1986; Kass, 1987; Saha et al., 1989). Ryanodine greatly accelerates rest decay of twitches and SR Ca content even after short times of exposure to low concentrations (Bers et al., 1987; Male´cot and Katzung, 1987). Ryanodine (100 n) shortened the half-time for loss of SR Ca from >100 s to <1 s in rabbit ventricle. This is believed to be due to the ability of ryanodine to increase the open probability of SR Ca release channels (Rousseau et al., 1987; Rousseau and Meissner, 1989). The effect of Bay K 8644 is qualitatively similar with respect to rest decay, although the acceleration is markedly smaller (about five-fold rather than 100-fold). In this context there would seem to be three ways in which Bay K 8644 might reduce SR Ca content during rest; (1) a direct effect on the SR (like ryanodine), (2) an enhanced Ca influx during rest (e.g. via Ca channels) or (3) a functional linkage between the dihydropyridine receptor and the SR or ryanodine receptor therein. These are considered below.
Possible explanations for effect of Bay K 8644 on resting SR Ca (1) Direct effects of Bay K 8644 on SR It is difficult to prove that Bay K 8644 does not directly affect the SR. However, the lack of effect of Bay K 8644 on ryanodine binding in heavy skeletal SR vesicles, solubilized triads or aggressively homogenized cardiac myocytes does not provide evidence for a direct effect of Bay K 8644 on SR. On the other hand, Bay K 8644 increased ryanodine binding under conditions where the SR-sarcolemmal junctions are likely to be more intact. This would be consistent with an indirect effect of Bay K 8644 on the SR release channel (requiring
intact functional coupling). SR Ca-pump inhibition could, in principle, also explain accelerated rest decay with Bay K 8644. However, there is no evidence for significant depression of SR Ca-ATPase at the concentration of Bay K 8644 used here (Zorzato et al., 1985; L. Hove-Madsen, unpublished observations). Additionally, there is no direct evidence for Bay K 8644 effects on cardiac myofilament Ca sensitivity or on SR Ca release in isolated cardiac or skeletal muscles SR (Thomas et al., 1985; Zorzato et al., 1985). Therefore it seems unlikely that Bay K 8644 affects the SR directly.
(2) Bay K 8644-induced resting Ca influx Another possibility is that exposure to Bay K 8644 leads to diastolic Ca influx. Talo et al. (1990) reported the existence of a steady Ca “window” current when rat ventricular myocytes were subject to sustained depolarization near to, but below the threshold for activation of a twitch (−40 to −20 mV). This “window” current appeared to be sufficient to induce graded SR Ca release, with consequent generation of very small contractions. The well characterized leftward shift in the currentvoltage relationship of cardiac ICa with Bay K 8644 (Sanguinetti et al., 1986) could lead to either the generation or the enhancement of similar “window” currents in ferret myocytes. Even if a window current in the classical sense is not active at resting Em (−80 mV) it is possible that individual -Type Ca channels could open, albeit extremely rarely at rest, but this could be increased by Bay K 8644. These currents may not be readily detectable by conventional electrophysiological techniques, but could induce local SR Ca release during the rest periods, with consequent acceleration of rest decay by Bay K 8644. This could provide an important caveat when considering the results from the whole cell binding studies and rest decay experiments. However, the results in Figures 9 and 10 rule out this possibility since the accelerative effect of Bay K 8644 on rest decay of SR Ca content occurs even in Ca-free, EGTA solution where Ca influx cannot occur. Thus, Ca influx does not mediate the effect of Bay K 8644 on SR Ca content.
(3) Functional linkage from DHP receptor to ryanodine receptor This most intriguing possibility is consistent with the experimental results and cannot be excluded at this stage. In skeletal muscle a voltage-dependent
Acceleration of Rest Decay by Bay K 8644
change in the DHP receptor is thought to induce opening of the SR Ca release channel/ryanodine receptor (see review by Rı´os and Pizarro´, 1988). This coupling may be mechanical as envisaged by Chandler et al. (1976) and could be direct or involve other proteins (e.g. triadin). There may also be Cainduced Ca-release in skeletal muscle, but this may be an amplification system which is not essential for E-C coupling. If Bay K 8644 can bind to the DHP receptor and even partly mimic the effect of depolarization, the chain of mechanical effects could alter the ryanodine receptor and cause increased SR Ca release. Evidence of similar functional linkage is equivocal in cardiac tissue, where Ca-induced Ca-release appears to be essential for SR Ca release and voltage dependent release does not seem to occur (Na¨bauer et al., 1989; Niggli and Lederer, 1990; Bers, 1991). While the effects of Bay K 8644 on ryanodine binding are small (>30% increase in apparent Bmax in Fig. 5), there are two reasons why the modest change in ryanodine binding may be sufficient to explain the clear acceleration of rest decay. First, the ratio of ryanodine:dihydropyridine receptors in ferret ventricular myocytes is 10:1 (Bers and Stiffel, 1993) and each ryanodine receptor is probably a tetramer of RyR. Thus even if every DHPR were directly linked to a ryanodine receptor, the maximum fraction of ryanodine receptors with altered characteristics would be about 10%. Thus the small observed change in ryanodine binding is almost a surprisingly large effect (if a direct RyRDHPR linkage were involved). Second, one only need change a small number of SR Ca release channels (perhaps by a moderate degree) to produce the function effects observed. For example only 6% of ryanodine receptors must open to produce the Ca flux associated with E-C coupling (Bassani and Bers, 1995) and during rest there are only about 10−4 openings/channel/sec (Cheng et al., 1993; Bassani and Bers, 1995). In addition, there is no reason to expect the relation between apparent ryanodine binding and resting SR Ca efflux to be linear. Thus a modest change observed in ryanodine binding could easily be a reflection of the increased rate of SR Ca loss observed during rest. That is, even a small change in the probability of SR Ca channel opening during rest would be enough to accelerate rest decay of SR Ca content. Even a brief exposure to very low concentrations of ryanodine (1–100 pM), where few release channels are likely to be altered, accelerates rest decay markedly in ferret ventricle (Male´cot and Katzung, 1987). In this way the functional consequence of
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Bay K 8644 on SR Ca is rather like a very low ryanodine concentration. Again, the relatively small effects of Bay J 8644 on ryanodine binding could be due to either a small fraction of ryanodine receptors participating in sarcolemmal-SR couplings (consistent with the stoichiometry of DHP: ryanodine receptor) or a small change in Ca release channel properties (or both). In addition, the apparent sensitivity to mechanical disruption of the Bay K 8644 effect on ryanodine binding would be consistent with a mechnical connection transmitting the signal. Thus, while our results are consistent with such a direct functional link, additional work will be required to prove this to be the case.
Species and condition dependence An interesting aspect of this particular effect of Bay K 8644 is that it is readily demonstrable in dog and ferret, but not in rat, rabbit or guinea-pig cardiac preparations. Additionally, it is also more difficult to observe the effect at room temperature compared to 37°C. The species differences could be indicative of morphological and/or functional species differences. It may imply that functional linkage between DHP and ryanodine receptors exists in dog and ferret cardiac tissue but not in the other preparations. There may also be species differences in the properties of the SR release channels or the DHP receptors (e.g. Ca sensitivity, Rousseau and Meissner, 1989; Ashley and Williams, 1990). Another simple explanation could be that the modest accelerative effects of Bay K 8644 on rest decay in species other than ferrets and dogs are masked by the rest decay characteristics of that species. For example the relatively rapid rest decay in control guinea-pig and rabbit ventricle might make it harder to discern. Conversely the very slow rest decay in rat ventricle may be limited by sarcolemmal Na/Ca exchange rather than the rate of SR Ca leak (Bassani and Bers, 1994). Thus, a slightly faster SR Ca leak may not affect rest decay much in rat. In summary, we have shown that the DHP agonist, Bay K 8644, accelerates the rest decay of twitches and SR Ca content in ferret ventricular muscle. The results are most consistent with an underlying mechanism whereby the binding of Bay K 8644 to the DHP receptor alters the state of the ryanodine receptor/SR Ca release channel via a functional coupling between the two receptors.
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Acknowledgements The authors would like to thank Mrs L. Manestar, Ms K. Cha, Mrs M. Robinson and Ms B. Tumulty for their assistance in the preparation of the cardiac myocytes. Thid work was supported by a grant from the USPHS (HL-44583) and E. Mc. was a British-American Research Fellow of the American Heart Association and the British Heart Foundation.
References A RH, W AJ, 1990. Divalent cation activation and inhibition of single calcium release channels from sheep cardiac sarcoplasmic reticulum. J Gen Physiol 95: 981–1005. B RA, B JW, B DM, 1994. Relaxation in ferret ventricular myocytes: unusual interplay among calcium transport systems. J Physiol 476: 295–308. B RA, B DM, 1994. Na-Ca exchange is required for rest-decay but not for rest-potentiation of twitches in rabbit and rat ventricular myocytes. J Mol Cell Cardiol 26: 1335–1347. B RA, B DM, 1995. Rate of diastolic Ca release from the sarcoplasmic reticulum of intact rabbit and rat ventricular myocytes. Biophys J 68: 2015–2022. B DM, 1989. SR Ca loading in cardiac muscle preparations based on rapid cooling contractures. Am J Physiol 256: C109–C120. B DM, 11991. Excitation-Contraction Coupling and Cardiac Contractile Force. Dordrecht, Netherlands, Kluwer Academic Press, 258. B DM, B RA, B JW, B S, H LV, 1993. Paradoxical twitch potentiation after rest in cardiac muscle—increased fractional release of SR calcium. J Mol Cell Cardiol 25: 1047–1057. B DM, B JHB, ML KT, 1987. The mechanism of ryanodine action in cardiac muscle assessed with Ca selectrive microelectrodes and rapid cooling contractures. Can J Physiol Pharmacol 65: 610–618. B DM, B JHB, S KW, 1989. Intracellular Ca transients during rapid cooling contractures in guinea-pig ventricular myocytes. J Physiol 417: 537– 553. B DM, S VM, 1993. Ratio of ryanodine to dihydropyridine receptors in cardiac and skeletal muscle and implications for E-C coupling. Am J Physiol 264: C1587–C1593. B RA, H LV, S JK, B D, 1989. Effects of caffeine and ryanodine on depression of postrest tension development produced by Bay K 8644 in canine ventricular muscle. Br J Pharmacol 97: 1279– 1291. B NR, C AN, W S-R, T JA, 1990. Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads. J Membr Biol 113: 237–251. C WK, R RF, S MF, 1976. Effects of glycerol treatment and maintained depolarization on charge movement in skeletal muscle. J Physiol 254: 285–316. C H, L WJ, C MB, 1993. Calcium
sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262: 740– 744. F A, 1985. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol 85: 247– 290. H P, L JB, T RW, 1985. Different modes of Ca channel gating behavior favored by dihydropyridine Ca agonists and antagonists. Nature 311: 538–544. H-M L, B DM, 1992. Indo-1 binding in permeabilized myocytes alters its spectral and Ca binding properties. Biophys J 63: 89–97. H LV, B DM, 1990. Ca current facilitation during post-rest recovery depends on Ca entry. Am J Physiol 259: H951–H961. H LV, B R, C T, B D, 1989a. Inhibition of rest potentiation in canine ventricular muscle by BAY K 8644: comparison with caffeine. Am J Physiol 257: H399–H406. H LV, K T, B D, 1989b. Possible inhibition of canine ventricular sarcoplasmic reticulum by BAY K 8644. Am J Physiol 257: H407–H414. H LV, S VM, B DM, 1989c. Rapid cooling contractures as an index of SR Ca content in rabbit ventricular myocytes. Am J Physiol 257: H1369–1377. H LV, S VG, B DM, 1990. Bay K 8644 may affect cardiac SR via direct communication between sarcolemmal and SR Ca channel. (Abstract). Biophys J 57: 167a. K RS, 1987. Voltage-dependent modulation of cardiac calcium channel current by optical isomers of Bay K 8644: implications for channel gating. Circ Res 61 [suppl.1] I-1-I-5. K KC, C AH, T JA, B NR, 1990. Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle. Biochemistry 29: 9281– 9289. K S, S T, 1985. Effects of rapid cooling on mechanical and electrical responses in ventricular muscle of guinea pig. J Physiol 361: 361–378. L FA, M M, X L, S HA, M G, 1989. The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum. J Biol Chem 264: 16775–16785. M´ CO, K BG, 1987. Use dependence of ryanodine on postrest contraction in ferret cardiac muscle. Circ Res 60: 560–567. MC E, B DM, 1994. Is the Bay K 8644 induced loss of SR Ca during rest in cardiac muscle due to a small Ca influx at rest? (Abstract). Biophys J 66: A133. MC E, O CH, 1991. Effects of acidosis on interval-force relation and mechanical restitution in ferret papillary muscle. J Physiol 432: 45–63. N¨ M, C G, C L, M M, 1989. Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes. Science 244: 800–803. N E, L WJ, 1990. Voltage-independent calcium release in heart muscle. Science 250: 565–568. Rı´ E, P´ G, 1988. Voltage sensors and calcium channels of excitation-contraction coupling. NIPS 3: 223–227.
Acceleration of Rest Decay by Bay K 8644 R E, M G, 1989. Single cardiac sarcoplasmic reticulum Ca2+-release channel: Activation by caffeine. Am J Physiol 256: H328–H333. R E, S JS, M G, 1987. Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am J Physiol 253: C364–C368. S JK, H LV, B RA, C T, B D, 1989. Analysis of (−) and (+) isomers of the 1,4dihydropyridine calcium channel agonist Bay K 8644 on postrest potentiation in canine ventricular muscle. Can J Physiol Pharmacol 67: 788–794. S MC, K DS, K RS, 1986. Voltagedependent modulation of Ca channel current in heart cells by Bay K 8644. J Gen Physiol 88: 369–392. S GL, V M, E DA, A DG, 1988. Effects of rapid application of caffeine on the intracellular calcium concentration in ferret papillary muscles. J Gen Physiol 92: 351–368. T A, S MD, S HA, I GI, L EG, 1990. Sustained subthreshold-for-twitch depolarisation in rat single ventricular myocytes causes
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sustained calcium channel activation and sarcoplasmic reticulum calcium release. J Gen Physiol 96: 1085– 1103. T G, GB R, P G, R JC, 1985. The positive inotropic dihydropyridine BAY K 8644 does not affect calcium sensitivity or calcium release of skinned cardiac fibres. Naunyn-Schmied Arch Pharmacol 328: 378–381. T F, R S, L P, N JM, N J, 1990. Cyclic-AMP-dependent phosphorylation modulates the stereospecific activation of cardiac Ca channels by Bay K 8644. Pflu¨gers Arch 417: 58–66. W WG, Y DT, 1986. Intracellular calcium transients underlying the short-term force-interval relationship in ferret ventricular myocardium. J Physiol 376: 507– 530. Z F, V P, S G, M A, 1985. Ca2+ channel agonist BAY K 8644 does not elicit Ca2+ release from skeletal muscle sarcoplasmic reticulum. FEBS Lett 186: 255–258.
Possible Functional Linkage Between the Cardiac Dihydropyridine and Ryanodine Receptor: Acceleration of Rest Decay by Bay K 8644 E. McCall, L. V. Hryshko, V. M. Stiffel, D. M. Christensen and D. M. Bers Department of Physiology, Loyola University Medical Center, 2160 South First Avenue, Maywood, Illinois 60153, USA (Received 8 June 1995, accepted in revised form 12 July 1995)
E. MC, L. V. H, V. M. S, D. M. C D. M. B. Possible Functional Linkage Between the Cardiac Dihydropyridine and Ryanodine Receptor: Acceleration of Rest Decay by Bay K 8644. Journal of Molecular and Cellular Cardiology (1996) 28, 79–93. The effect of the dihydropyridine -Type Ca chanel agonist Bay K 8644 on post-rest contractions in ferret ventricular muscle and isolated myocytes was investigated. Bay K 8644 was shown to abolish rest potentiation and greatly accelerate rest decay. The post-rest contraction suppressed by Bay K 8644 was accompanied by action potentials of large amplitude and longer duration, but voltage-clamp measurements showed that this suppression was not due to a supra-optimal ICa trigger. Caffeineinduced contractures and rapid cooling contractures demonstrated an accelerated rest-dependent decline in sarcoplasmic reticulum (SR) Ca content in the presence of Bay K 8644, which was present even with Ca-free superfusion during rest. Thus, the Bay K 8644-induced decline of SR Ca during rest was independent of extracellular Ca or ICa. To explore whether the binding of Bay K 8644 to the dihydropyridine receptor could alter the SR Ca release channel/ryanodine receptor in a more direct way, ryanodine binding was measured in the absence and presence of Bay K 8644. Ryanodine binding to isolated ferret ventricular myocytes was increased by Bay K 8644 under conditions where sarcolemmal-SR junctions might be expected to be intact, but not after physical disruption. These results are consistent with a working hypothesis where Bay K 8644 may bind to the dihydropyridine receptor and this may lead to physical changes in the linkage between the dihydropridine receptor and a subset of ryanodine receptors, thereby increasing the opening of the SR Ca release channel during rest (and accelerating resting Ca loss). 1996 Academic Press Limited K W: Cardiac muscle, Sarcolemmal Ca channel, Sarcoplasmic reticulum, Dihydropyridine receptor, Ryanodine receptor, Bay K 8644.
Introduction
(+) enantiomer behaves as a partial Ca channel antagonist (Sanguinetti et al. 1986; Kass, 1987). Bay K 8644 is now widely used as a specific and diagnostic activator of -Type Ca channels. When mammalian ventricular muscle is stimulated after a period of rest, the contraction amplitude at the first post-rest contraction can either be increased or decreased. This is commonly referred
Voltage clamp studies of Ca current (ICa) have demonstrated that the dihydropyridine (DHP) compound Bay K 8644 is a selective -Type Ca channel agonist (Hess et al., 1985). The (−) enantiomer and the racemic mixture (+−) of Bay K 8644 exhibit this Ca channel agonist effect, while the
Please address all correspondence to: Donald M. Bers, Ph.D. Department of Physiology, Loyola University Medical School, 2160 South First Ave., Maywood, Il 60153, USA.
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to as rest potentiation or rest decay, respectively (Bers, 1991). In some species (e.g. rabbit and guinea-pig ventricle), only rest decay is normally observed and this is thought to be due to the gradual depletion of sarcoplasmic reticulum (SR) Ca during rest with subsequent extrusion from the cell by Na/Ca exchange (Bers et al, 1989, 1993). In rat ventricle, rest potentiation is more prominent and can persist for many minutes. This rest potentiation may be due to either a rest-dependent increase in SR Ca content or slow recovery of the excitation– contraction coupling mechanism (Bers, 1989; Bers et al., 1993). Some species (e.g. ferret and canine ventricle) exhibit a combination, with rest potentiation for short rest periods, followed by rest decay for longer rests (Hryshko et al., 1989a; Bers et al., 1983). An unexpected action of Bay K 8644 has previously been observed in canine ventricular muscle (Hryshko et al., 1989a,b; Saha et al., 1989; Bouchard et al., 1989). It was found that Bay K 8644 abolished rest potentiation and greatly accelerated rest decay in canine ventricle. This action was seen with both the (−) enantiomer and racemic Bay K 8644, but not with the (+) enantiomer (i.e. just as with the increase in ICa). Their results suggested that the acceleration of rest decay seen with Bay K 8644 in dog ventricle was due to a more rapid loss of SR Ca. Here we report similar effects of Bay K 8644 in ferret ventricular muscle. The aims of the present study were to evaluate the fundamental mechanism by which Bay K 8644 is able to accelerate rest decay. The results are consistent with a hypothesis whereby Bay K 8644 binds to the DHP receptor and transmits a signal (independent of ICa) to a subset of SR Ca release channels to increase channel opening and SR Ca decline during rest. Some of this work has previously appeared in abstract form (Hryshko et al., 1990; McCall and Bers, 1994).
Methods Muscle isolation procedure The isolation procedures used were similar to those described previously (Hryshko et al., 1989c). Briefly, hearts were excised from adult male ferrets (Marshall Farms, North Rose, NY, USA) under sodium pentobarbital anaesthesia (140 mg/kg i.p.), the right ventricle was opened and suitable isolated trabeculae and papillary muscles (0.1–1 mm in diameter) were removed and the ends were tied
with fine suture. They were then suspended in a 0.15 ml muscle chamber, with one end attached to a post and the other to a piezoresistive force transducer (AE 875, Aksjeselskapet Micro-Elektronikk, Horten, Norway).
Measurement of post-rest contraction The preparations were superfused, at flow rates of 15 ml/min, with normal Tyrode (NT) solution containing (m): 140 NaCl, 6 KCl, 2 CaCl2, 1 MgCl2, 2 CaCl2, 10 glucose, 5 HEPES, pH 7.4 at 37°C. The regular stimulation frequency was 0.5 Hz, induced by field stimulation using platinum electrodes during the equilibration period (>45 min) and between rests. Force developed by the preparation during steady-state (SS) stimulation and upon resumption of regular stimulation after a 10 to 300 s rest period (post-rest, PR) was measured. After obtaining results with NT, the perfusate was changed to NT plus 1 l Bay K 8644, and the experimental protocol was repeated. The effects of Bay K 8644 at 1 l were similar to those at 100 nM and were progressively weaker at lower concentrations. Quantitative dose-response curves were not conducted, but most experiments were done with 0·1 or 1 l Bay K 8644 to produce nearly maximal effects.
Assessment of SR Ca load: rapid cooling contractures (RCCs) The theory and practical applications of RCCs have been considered in earlier studies (Kurihara and Sakai, 1985; Bers et al., 1987, 1989). Briefly, rapid cooling of cardiac muscle to temperatures lower than 5°C results in Ca release from the SR, such that the amplitude of the resultant RCC is indicative of the SR Ca content at the moment of cooling. In this series of experiments, RCCs were evoked by rapidly lowering superfusate temperature to 0°C. This was accomplished by having NT flowing through perfusion lines jacketed with either water (at 37°C) or a 3:1 water:propylene glycol mixture (−2°C), with the specific solution perfusing the chamber controlled by means of solenoid pinch valves. At a flow rate of 15 ml/min switching to the cooled perfusate reduced muscle surface temperature to below 5°C in <0.5 s, and the chamber temperature was kept at >0°C during constant perfusion (for >30 s). The protocol used was similar to that described above for the assessment of PR
Acceleration of Rest Decay by Bay K 8644
contractions except that RCCs were initiated at the end of the rest intervals rather than twitches.
Action potential measurements Action potentials (APs) were measured in ferret isolated trabeculae and papillary muscles perfused with NT, in the presence and absence of 1 l Bay K 8644, at 37°C, using conventional glass microelectrodes filled with 3 KCl (typically 15–20 MX resistance). The stimulation and rest periods were as described above, with SS and PR APs and contractions measured.
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allowed to equilibrate at 37°C for >20 min prior to experimental protocols. The protocol used was similar to that described for the whole muscle experiments (0·5 Hz and 30–90 s rest periods). However, the extracellular solution was sometimes changed during the rest period to a 0Ca/0Na solution to prevent Ca influx (and efflux via Na/Ca exchange). In this 0Ca/0Na solution Na was replaced with Li and 1 m EGTA replaced the Ca. For control runs [Ca]o in the control solution was increased to 3 m, whereas after equilibration with 100 n Bay K 8644 [Ca]o was reduced to 1 m. This was done so that the amplitude of contractions was similar in the presence and absence of Bay K 8644.
Myocyte isolation procedure Assessment of SR Ca load: caffeine contractures (CafC) Isolation of ventricular myocytes was carried out as previously described (Hryshko et al., 1989c; Bassani et al., 1994). Hearts were excised from adult male ferrets (1–1.3 kg, Marshall Farms, North Rose, NY, USA.) under sodium pentobarbital anesthesia (140 mg/kg i.p.), then mounted in a Langendorff perfusion apparatus and perfused with nominally Ca-free, HEPES-buffered Tyrode solution for 5 min at 37°C and a flow rate of 20 ml/min. The heart was then perfused with the same solution containing 1 mg/ml collagenase (Type B, Boehringer Mannheim) and 0·16 mg/ml pronase (Boehringer Mannheim) until it became flaccid (10–30 min), after which the digested tissue was separated and filtered. The resultant cell suspension was rinsed several times and the Ca concentration gradually increased to 2 m. Prior to experimental use the myocytes were plated onto Plexiglas superfusion chambers, with the glass bottoms of the chambers treated with laminin (Gibco, Grand Island, NY, USA) to increase cell adhesion.
The rapid application of a caffeine-containing solution induces a contracture in myocytes, and the amplitude can be used as an index of SR Ca content (Smith et al., 1988). Caffeine (10 m) was added to the 0Ca/0Na solution (see above) as a powder, at 37°C. The caffeine-containing solution was introduced into the chamber via a quick-switching device, similar to that described previously (Bassani et al., 1994). The stimulation and perfusion protocols used were similar to those described above, except that a rapid switch to caffeine was applied at the end of the rest intervals rather than an electrical stimulus. Caffeine application was continued for approximately 6 s, by which time the caffeine contracture had begun to decline. The tip of the quick-switching device was then washed, to remove any residual caffeine solution, and the cell perfused with either the 3 m Ca or 100 nM Bay K 8644 with 1 m Ca Tyrode solution for about 2 min prior to resumption of stimulation.
Measurement of cell shortening and rest decay
ICa measurement
Myocyte shortening was measured as previously described (Bassani et al., 1994). Myocytes were superfused with Tyrode solution at 37°C and a flow rate of 6 ml/min. Field stimulation at 0·5 Hz was induced via platinum electrodes and cell shortening at both ends was measured using a video-edge detection system (Crescent Electronics, Sandy, UT, USA) connected to a strip chart recorder, for immediate display of contraction records, and stored using a video recorder for off-line analysis. The basic normal Tyrode’s solution (NT) used in these experiments was as described above and cells were
ICa was measured by the whole-cell voltage-clamp technique using an Axopatch 1C patch clamp amplifier (Axon Instruments, Foster City, CA, USA) (see Hryshko and Bers, 1990, for further details) in ferret cardiac myocytes isolated as described above. Patch electrodes were used with resistances of 0·5–1·5 MX (glass type TW150–6, World Precision Instruments, Sarasota, FL, USA) and containing: (m) 140 CsCl, 10 EGTA and 5 Mg-ATP, pH 7.1 at 37°C or 30°C. Myocytes were superfused with: (m) 140 Tetraethylammonium (TEA) Chloride, 6 CsCl, 2 CaCl2, 1 MgCl2, 10 glucose, 5 HEPES, pH
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7.4. These solutions were chosen to eliminate contaminating Na and K currents (i.e. no Na inside or out, and Cs and TEA replacing K). Cells were held at −90 mV and then pulsed to 0 mV for 100 ms at 0.5 Hz. On achieving a SS level of ICa the cells were then held at −90 mV during the rest intervals (30–300 s), after which 0.5 Hz depolarizing pulses to 0 mV were resumed.
and linear regression for Scatchard plots (using GraphPad InPlot, GraphPad Software Inc.). Since we are measuring ryanodine binding in physiological, not optimal equilibrium binding conditions, Kd and Bmax values must be interpreted cautiously. Thus, we focus on differences in ryanodine binding rather than inferred pharmacological parameters which are condition dependent (Lai et al., 1989). Protein concentration was measured by the Lowry method.
Ligand binding studies The effect of Bay K 8644 on ryanodine binding was investigated using isolated ferret myocytes. For ligand binding studies, 3H-ryanodine (62.5 lCi/ lol) and 3H-PN200-110 (90.9 lCi/lol) purchased from New England Nuclear were used. Ryanodine binding was carried out essentially as previously described (Lai et al., 1989; Bers and Stiffel, 1993) at physiological ionic strength. Except where noted, the incubation medium contained 140 m KCl, 20 m Tris, 1 m EGTA, 0.1–3 m free [Ca], 1–200 n 3H-ryanodine at pH 7.4 with protease inhibitor cocktail (final concentrations: 75 n aprotinin, 0.23 l PMSF, 0.83 m benzamidine, 1 m iodoacetimide, 1.1 l leupeptin and 0.7 l pepstatin A). Samples were preincubated in the presence and absence of 0.1–1 l Bay K 8644 for 30 min prior to addition of ryanodine with similar results between concentrations (1 l was used for most subsequent experiments to obtain maximal effects). Cold ryanodine (17 l was used to displace 3H-ryanodine and allow measurement of specific ryanodine binding. Incubations were at 37°C for 90 min, which was found to be optimal for ryanodine binding. Longer times would be required for true equilibrium, but long incubations at 37°C resulted in a gradual decrease in apparent ryanodine binding (presumably due to degradation). The reaction was terminated by 3×3 ml washes of distilled water with a Brandel Cell Harvester through Whatman GF/B filters. The dihydropyridine PN200-110 was used to measure DHPR using a buffer consisting of a final concentration of 25 m Tris, 10 m HEPES-Na, 1 m EDTA, and 1.1 m MgCl2, 0.1–15 nM 3HPN200−100, pH 7.4. Nifedipine (16·6 l) was used to allow measurement of nifedipine-sensitive PN200-110 binding. All tubes were incubated at room temperature in the dark for 90 min. The reaction was terminated by 2×3 ml washes with ice cold 10 m Na-HEPES, pH 7·4 through GF/B filters with a Brandel Cell Harvester. Curve fitting of pooled binding results was by a least squares method for simple Michaelis binding
Reagents Unless otherwise stated experimental reagents used were of analytical grade and supplied by Sigma (St. Louis, MO, USA). (+−) Bay K 8644 was obtained from Miles Pharmaceuticals (West Haven, CT, USA). A stock solution was made up in ethanol and stored at −20°C. Aliquots of this solution were added to the perfusate immediately prior to use. In functional studies ethanol never exceeded 0.1% in the perfusate.
Results Acceleration of rest decay by Bay K 8644 The characteristic effect of Bay K 8644 on PR contraction in ferret cardiac muscle is illustrated in Figure 1. The upper panel shows SS and PR force development in a ferret papillary muscle in the absence and presence of Bay K 8644. Under control conditions the first PR contraction after a 1 min rest in this preparation showed post-rest potentiation, i.e. it had a greater amplitude (84% increase) than that elicited by regular stimulation. The subsequent contractions were initially smaller than the SS contractions, then gradually increased to the SS level (not shown). In contrast, the first PR contraction after a 5-min rest was smaller than the SS level (50% decrease), resumably reflecting Ca loss from the SR, with subsequent contractions progressively increasing back to SS level. The addition of 1 l Bay K 8644 increased SS force development 2.8-fold in this preparation. The positive inotropic effect of both the (−)-enantiomer and the racemic mixture of Bay K 8644 has been shown in a number of studies, and is thought to reflect an increase in -Type ICa (Tiaho et al., 1990). The surprising effect was that Bay K 8644 also alters the PR response pattern of the preparation, with no rest potentiation after the 1-min rest in
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Acceleration of Rest Decay by Bay K 8644 (a) Control
BAY K 8644
1-min rest
10 mN/mm2
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(b)
Post-rest twitch amplitude
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Figure 1 The effect of Bay K 8644 exposure on the contractile response of isolated ferret papillary muscle. (a) Original experimental tracings of force developed by a ferret papillary muscle at 37°C in response to regular stimulation at 0.5 Hz and after 1- and 5-min rest periods, in the absence (left) and presence (right) of 1 l Bay K 8644. Note the scale change with Bay K 8644. (b) Pooled results for experiments as in (a) in the absence and presence of 1 l Bay K 8644. Data shown are mean±... (n=4–5, except at 15 s where n=3 and no ...). Differences are significant (P<0.5) for all points beyond 4 s.
Bay K 8644. The PR contraction after a 5-min rest was significantly smaller than both the SS level in Bay K 8644 (93% decrease) and the 5-min PR response under control conditions (even in absolute terms, despite the larger SS twitch after Bay K 8644). This was a consistent effect. Pooled results from experiments like this with additional rest intervals are shown in Figure 1b. Under control conditions increasing the rest interval led to a biphasic pattern of PR force development, with increases in PR twitch force observed for rest intervals up to 1 min and progressive decline thereafter (similar to other control results in this preparation, Wier and Yue, 1986;
McCall and Orchard, 1991). The addition of Bay K 8644 completely eliminated rest potentiation, and depressed PR force at all rest intervals. This effect of Bay K 8644 has previously been shown in canine ventricular muscle (Hryshko et al., 1989a), but was not readily apparent in preliminary experiments in rabbit, rat or guinea-pig ventricle, or in ferret preparations at room temperature (not shown).
Electrophysiological measurements One possibility to explain the Bay K 8644 accelerated rest decay is that Bay K 8644 could induce
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SS
SS
PR
50 mV 10 mN/mm2
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Figure 2 The effect of Bay K 8644 on action potentials. Control membrane potential and force recordings before and after a 5-min rest at 37°C before (left) and after equilibration with 1 l Bay K 8644 (right).
an unexpected effect on the action potential at the PR contraction (i.e. decreased amplitude or duration). This does not seem to be the case as illustrated by the action potential recordings shown in Figure 2, which are typical of the results obtained in three muscles. The PR action potential exhibited an increased duration and amplitude in both the absence and presence of Bay K 8644. While the amount of change differed from muscle to muscle, both rest and Bay K 8644 always increased action potential duration. The pattern of SS and PR force developed by the preparation and the effect of Bay K 8644 in Figure 2 was similar to that in Figure 1. Since Bay K 8644 is expected to increase inward ICa the increase in action potential duration and SS contraction is not surprising. However, the strong suppression of the PR contraction is still apparent, even though the AP characteristics would be consistent with greater Ca influx. These results indicate that, under these conditions, rest decay of force occurs even though Ca influx into the cell is expected to be increased. In an elegant series of experiments demonstrating Cainduced Ca-release (CICR) in mechanically ‘‘skinned’’ cardiac cells Fabiato (1985) showed a
biphasic effect of ‘‘trigger’’ [Ca] on SR Ca release. SR Ca release increased with increasing [Ca] trigger levels (up to 1–4 l). At higher levels of trigger [Ca] (e.g. 10 l) SR Ca release appeared to be effectively inactivated. To test the possibility that Bay K 8644 leads to supraoptimal triggering for SR Ca release during PR contractions, ICa was measured in the absence and presence of Bay K 8644 in isolated ferret ventricular myocytes. Representative results are shown in Figure 3. The intrinsic effect of rest on ICa amplitude in ferret ventricular myocytes from physiological holding potentials is to decrease the ICa amplitude as shown in the control traces in Figure 3 (see also Hryshko and Bers, 1990; Bers et al., 1993). Addition of 1 l Bay K 8644 increased control ICa amplitude twoto three-fold, but the ICa amplitude was still lower at the PR pulse than at SS. This suggests that the PR trigger Ca would be lower than the SS trigger (in the absence and presence of Bay K 8644). Therefore, it is unlikely that the small PR contraction could be attributed to supra-optimal Ca triggering with Bay K 8644. The PR ICa amplitude with Bay K 8644 is also much larger than the
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BAY K 8644
0
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PR PR SS
–2
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SS
Figure 3 The effect of Bay K 8644 on ICa. ICa elicited under SS conditions and after a 5-min rest interval from a holding potential of −90 mV to a test potential of 0 mV. Data are shown for control conditions (left) and after equilibration with 1 l Bay K 8644. This cell was studied at 30°C and is representative of 10 cells studied at either 30 or 37°C.
control, indicating that a decrease in PR ICa trigger with Bay K 8644 is also not the reason for the small PR twitch with the DHP. Rapid cooling contractures and SR Ca content The SR is the main source of activating Ca in ferret ventricular muscle and is important in PR contractile behavior (Bers, 1991). As alterations in Ca influx do not appear to adequately account for the observed PR effects of Bay K 8644, we tested the possibility that Bay K 8644 affects SR function. This hypothesis was tested by studying SS and PR RCCs before and after exposure to Bay K 8644. The results are shown in Figure 4. The 2 s RCC represents the degree of SR Ca loading under SS conditions. Under control conditions the Ca content of the SR decreased monotonically as the rest interval lengthened, as indicated by a reduction in the amplitude of the RCCs. The transient increase in twitch force (rest potentiation in Fig. 1) despite a monotonic decline in SR Ca in ferret ventricular myocytes is explained by a relatively slow change in recovery of the E-C coupling mechanism during rest (Bers et al., 1993). Figure 4b shows that the gradual decline in SR Ca content based on RCCs in control (t1/2 >200 s) was greatly accelerated after equilibration with Bay K 8644 (t1/2 <30 s). This accelerated decline in SR Ca content during rest could explain the acceleration of rest decay of twitches observed with
Bay K 8644, as hypothesized by Hryshko et al. (1989b) for canine ventricular muscle. That is, Bay K 8644 promotes diastolic SR Ca loss, which may underlie the acceleration of rest decay induced by Bay K 8644. During rest at −90 mV it is not expected that there is any significant activity of Type Ca channels. This, then, raises the possibility that Bay K 8644 might exert its effects on resting SR Ca fluxes via a physical link between the DHP receptor and the Ca release channel, independent of ICa. Thus, the effect of Bay K 8644 on ryanodine binding was studied in ferret ventricular myocytes to determine if Bay K 8644 exhibited effects consistent with a promotion of SR Ca channel opening.
Ryanodine binding in cardiac myocytes There is substantial evidence for a physical linkage between the sarcolemmal DHP receptor and the SR ryanodine receptor in skeletal muscle (Brandt et al., 1990; Kim et al., 1990). This linkage seems to be central in E-C coupling even if additional proteins are involved (Rı´os and Pizarro´, 1988). Furthermore, ryanodine binding in both cardiac and skeletal ryanodine receptor is known to be sensitive to a variety of agents which alter channel open probability or SR Ca release. That is, concentrations of adenine nucleotides and Ca which are optimal for channel opening also stimulate ryanodine binding.
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(a) Control
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2-s rest 37°C 0°C 37°C
1-min rest
5-min rest
RCC (% of ss RCC)
(b)
Post-rest RCC amplitude
100 Control 50 Bay K 0
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120 180 Rest interval (s)
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Figure 4 The effect of Bay K 8644 on SR Ca loading in isolated ferret papillary muscle. Original experimental tracings of SS force at 0.5 Hz and RCCs after 2 s (SS RCC), 1- and 5-min rest periods, in the absence (left) and presence (right) of 1 l Bay K 8644. Arrow indicates where rapid cooling was applied in the trace where no RCC was observed. Pooled results showing force developed during RCCs in ferret ventricular muscle over a range of rest intervals, in the absence and presence of 1 l Bay K 8644 (Bay K). Data are shown as mean±... (n=3–5).
Thus we sought to test the hypothesis that ryanodine binding in ventricular myocytes was increased by Bay K 8644 (perhaps secondary to an increased in the probability of resting openings of the SR Ca release channel). Initial studies with isolated ferret ventricular myocytes which were subjected to vigorous homogenization (with a Polytron) and/or subcellular fractionation in attempts to prepare “cardiac diads” failed to display consistent effects of Bay K 8644 on ryanodine binding.
Figure 5 shows the opposite extreme in terms of preparations, i.e. ryanodine binding in intact isolated ventricular myocytes in a normal extracellular solution (except that [Ca] was reduced to 0.5 m to minimize spontaneous activity during the required long incubation). In isolated single cell experiments, the mean resting [Ca]i is not altered appreciably by Bay K 8644 (not shown). Thus, we do not anticipate any difference in [Ca]i during the binding experiments in Figure 5. This figure shows that Bay K 8644 increased ryanodine binding and this effect was significant at all ryanodine concentrations above 10 n. Additionally the 95% confidence bands for the linear regressions of the Scatchard plots (Fig. 5, right) were virtually nonoverlapping. While true equilibrium conditions were not achieved for ryanodine binding experiments, the parallel shift in the Scatchard plot would suggest an increase in apparent Bmax without a change in apparent Kd. Experiments were also performed with isolated cells suspended in an intracellular type solution (140 m KCl, 6 m Na) with [Ca] reduced to 30 l (Fig. 6a). This [Ca] was selected because it is near the optimum for ryanodine binding (see Fig. 7). The aims were to control intracellular [Ca] better, and to create minimal mechanical disturbance. Ryanodine binding is still increased with Bay K 8644, but the difference is smaller than in Figure 5 (and is only significant at 30 and 100 n ryanodine, although the apparent Bmax was still increased by 27% in Bay K 8644). In Figure 6b the same preparations as in Figure 6a were exposed to the same medium, but with the addition of 10 l digitonin to ensure that [Ca] was reasonably controlled at 30 l. The difference between the control and the Bay K 8644 curves is further diminished. Two explanations for the loss of Bay K 8644’s effect with high K buffer and digitonin are that (1) the clear difference in NT (Fig. 5) was secondary to a more optimal [Ca] inside Bay K 8644-treated cells (a difference eleminated after digitonin permeabilization) or (2) either depolarization in high K or disruption of the linkage with digitonin decreases an intrinsically real effect of Bay K 8644 on polarized and intact junctions. Permeabilized myocytes exposed to 30 l Ca may also hypercontract and this could alter mechanical linkages at the membrane. Figure 7 shows the [Ca] dependence of ryanodine binding to digitonin permeabilized myocytes. In this case the free [Ca] in the test tubes during the assay were measured directly with Ca-selective mini-electrodes (see legend). Digitonin permeabilization was required to ensure that the [Ca] measured in the
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Acceleration of Rest Decay by Bay K 8644 Ryanodine binding, intact ventricular myocytes NT, 0.5 mM Ca 20 Bay K Bound/free
Bound ryanodine (fmol/mg)
1000
500 Control
0
50
100 150 [Ryanodine] (nM)
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10
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300 600 Bound ryanodine (fmol/mg)
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Figure 5 Ryanodine binding to isolated ferret ventricular myocytes. Freshly dissociated myocytes were incubated in a modified normal Tyrode’s solution (NT) with free [Ca] reduced to 0.5 m. Results are from four to five cell preparations in the presence and absence of 1 l Bay K 8644. The curve fits shown in the left panel for control and Bay K 8644 suggest Kd of 39 and 46 n and Bmax of 656 and 904 fmol/mg respectively (from pooled data at each [ryanodine], whereas the average from curve fits to individual preparations gave Kd values of 49±10 and 53±6 n and Bmax values of 743±73 and 963±102 fmol/mg respectively). The Scatchard plots at the right show linear regressions for control and Bay K 8644 with Kd of 36 and 40 n and Bmax of 642 and 854 fmol/mg respectively.
incubation medium was the same as in the cells. The optimal [Ca] was >100 l but varied from 30–300 l in different preparations. The results shown in Figure 8 provide evidence that the Bay K 8644 effect requires some level of mechanical integrity. These cells were homogenized mildly (five passes with a glass-teflon homogenizer). After homogenization there was still an apparent stimulation of ryanodine binding by Bay K 8644. However, in part of the sample also subjected to sonication before assay (to increase disruption), the effect of Bay K 8644 was eliminated. Similar effects were seen in two other preparations treated in a similar manner, and, furthermore, in two other preparations in which aggressive homogenization (2×30 s Polytron bursts) was performed prior to assay. While the effects of Bay K 8644 on ryanodine binding are not dramatic, the observed stimulatory effects of Bay K 8644 on ryanodine binding appear to be sensitive to the mechanical integrity of the cells or junctions, and may be sufficient to account for the observed functional effects (i.e. a reduction in PR contractions). That is, modulation of only a small fraction of ryanodine receptors could be sufficient to produce the physiological effects observed.
Is acceleration of rest decay secondary to increased Ca entry? Another potential explanation for the Bay K 8644induced acceleration not excluded so far is the following. If Bay K 8644 increases Ca entry locally by occasional openings of sarcolemmal Ca channels even at rest, this could activate individual local SR Ca release channels (via Ca-induced Ca-release). The result would be more rapid depletion of SR Ca during rest. This mechanism would, therefore, provide an explanation which does not require any physical communication between the sarcolemma and SR (rather Ca would serve as a second messenger). The final experimental series evaluated this possibility. The strategy was to remove all extracellular Ca during the rest (and include EGTA), such that no diastolic Ca influx would be possible. Then the SR Ca content was tested at the end of different rest periods. In this series we also changed the extracellular [Ca] so that both control and Bay K 8644-treated cells had similar SR Ca loads at the start of the rest period. We found that using 3 m Ca in control conditions and lowering [Ca]o to 1 m when Bay K 8644 was added produced this effect (see Fig. 9a and b).
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(a) Bound ryanodine (fmol/mg)
Bound ryanodine (fmol/mg)
Intact ferret myocytes 1000
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30 nM Ryanodine 10 µ M Digitonin
Control
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0.1
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100 [Ca] (µ M)
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Figure 7 The [Ca]-dependence of ryanodine binding to isolated ferret ventricular myocytes in the presence and absence of 1 l Bay K 8644. The free [Ca] in the test tubes during the assay procedure were measured directly with Ca-selective mini-electrodes (Hove-Madsen and Bers, 1992) and was often slightly different from the calculated value. Thus, the free [Ca] varied somewhat in different preparations. The points represent average values for both free [Ca] and the ryanodine binding measured in the samples. The incubation included 140 m KC1, 6 m NaC1, 1 m EGTA, 30 n 3H-ryanodine, 10 m HEPES at pH 7.4.
140 mM K, 30 µ M Ca +10 µ M Digitonin Bay K
500
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Control
50
100 150 [Ryanodine] (nM)
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Figure 6 Ryanodine binding to isolated ferret ventricular myocytes in high K solutions containing 140 m KC1, 6 m Na and 30 l Ca at pH 7.4. Results are from four to eight preparations. The same preparations were used in both panels, but in (b) 10 l digitonin was included to permeabilize the sarcolema. The curve fits in the panel (a) for control and Bay K 8644 used Kd of 24 and 26 n and Bmax of 541 and 686 fmol/mg respectively. For panel (b) Kd was 45 and 48 n and Bmax was 680 and 762 fmol/mg respectively.
The SS twitch contraction amplitudes were similar in the absence and presence of Bay K 8644 (5.7±0.5 l for control, 6.2±0.6 l with Bay K 8644, n=18), although there was a clear prolongation of twitch relaxation with Bay K 8644, presumably due to the effects on action potential duration (see Fig. 2). Bay K 8644 accelerated rest decay of twitches in the isolated myocytes in a similar way to multicellular preparations (data not shown, P<0.05; ANOVA followed by Bonferroni t-test for multiple, repeated, pairwise comparisons, n=18). Therefore this accelerative effect on rest decay did not reflect the inotropic state of the preparation. The acceleration of the rest decay of twitches with Bay K 8644 was still observed in the absence of extracellular Ca and Na during the rest (not
Homogenized ferret myocytes 1500 Bound ryanodine (fmol/mg)
Bound ryanodine (fmol/mg)
1000
Bay K Control
Homog 1000
Sonicated 500 140 mM K 440 µ M Ca 0
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Figure 8 The effect of disruption by sonication on the effect of 1 l Bay K 8644 on ryanodine binding. Isolated ferret ventricular myocytes were lightly homogenized in 140 m KC1, 6 m NaC1, 1 m EGTA, 10 m HEPES at pH 7.4. A portion of this homogenate was further disrupted by 5 min of sonication in a bath sonicator. Free [Ca] in the incubation buffer was 450 l.
shown), evaluated by switching to 0Ca/0Na solution during rest (±Bay K 8644) and returning to the steady-state just prior to the test twitch. However, clear quantitative interpretation of these results are complicated by the longer action potential duration and Ca influx which occurs. Thus,
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90 s rest
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Figure 9 The effect of Bay K 8644 on twitch contraction and caffeine contracture amplitudes. (a) Original records of contractions elicited at 0.5 Hz in a representative ferret ventricular myocyte under control conditions (3 m Ca) and in the presence of Bay K (100 n Bay K 8644, 1 m Ca). (b) Original records of CafC elicited in the same cell 2s after cessation of SS stimulation, in the absence and presence of Bay K 8644, Note change in time scale. (c) Original records of post-rest CafC, obtained after the rest periods indicated, obtained from the same cell, during continuous superfusion with control solution with and without Bay K 8644 (left panels), or after superfusion with 0Ca/0Na during the rest period (in the absence and presence of Bay K 8644, right panels).
caffeine-induced contractures were used to assess SR Ca content (Figs 9b and c). In Figure 9b replacing a steady-state twitch with a CafC (2 s interval) produced contractions of very similar amplitude for control (3 m Ca) and 1 m Ca plus 100 nM Bay K 8644 (25.8±3.8 l for control, 28.0±4.3 l with Bay K 8644, n=11). This indicates that the level of SR Ca loading is about the same in both cases. Figure 9c shows post-rest CafC where the cell was in the steadystate solution (left) or in the 0Ca/0Na solution during the rest (right). Bay K 8644 accelerates the rest-dependent decline in SR Ca content in both the presence and absence of extracellular Ca. This can be seen graphically for pooled experimental data in Figure 10. The reduction in CafC amplitude with Bay K 8644 was significant at each interval tested and for both control and 0Ca solution during the rest (P<0.05, n=9–11). Thus, diastolic Ca influx
% of SS CafC
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Figure 10 The effect of Bay K 8644 on the rest decay of SR Ca content assessed by CafC. In the upper panel cells were rested under control conditions (3 m Ca, filled circles) and in the presence of Bay K 8644 (100 n+1 m Ca, open circles). In the lower panel cells were rested in 0Ca/0Na solution (with m EGTA) in the absence (filled circles) and presence (open circles) of 100 n Bay K 8644. Data shown as mean±..., n=9–11.
is not responsible for the effects of Bay K 8644 on rest decay and SR Ca loss.
Discussion Relation to previous work The acceleration of rest decay induced by Bay K 8644 in ferret ventricular myocytes is similar to that observed in canine ventricle (Hryshko et al., 1989a,b; Saha et al., 1989; Bouchard et al., 1989). The present results indicate that this effect is not due to changes in action potential or ICa associated with either the steady-state or post-rest twitch. Rather, results from RCCs and caffeine-induced contractures provide compelling evidence that this acceleration in rest decay is due to a faster decline in
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SR Ca content during rest (even when the initial SR Ca content was the same). Despite the apparent dissociation from ICa, the acceleration of rest decay may still be due to Bay K 8644 binding to its expected pharmacological target, the -Type Ca channel or dihydropyridine receptor. Although detailed dose-response curves have not been done, the concentration where Bay K 8644 produced this effect are similar to those required for increasing ICa (and are maximal at 100–1000 n). Acceleration of rest decay and increased ICa are both observed with either the (−) enantiomer or racemic Bay K 8644, but not with the (+) enantiomer or other DHP Ca channel antagonists (Sanguinetti et al., 1986; Kass, 1987; Saha et al., 1989). Ryanodine greatly accelerates rest decay of twitches and SR Ca content even after short times of exposure to low concentrations (Bers et al., 1987; Male´cot and Katzung, 1987). Ryanodine (100 n) shortened the half-time for loss of SR Ca from >100 s to <1 s in rabbit ventricle. This is believed to be due to the ability of ryanodine to increase the open probability of SR Ca release channels (Rousseau et al., 1987; Rousseau and Meissner, 1989). The effect of Bay K 8644 is qualitatively similar with respect to rest decay, although the acceleration is markedly smaller (about five-fold rather than 100-fold). In this context there would seem to be three ways in which Bay K 8644 might reduce SR Ca content during rest; (1) a direct effect on the SR (like ryanodine), (2) an enhanced Ca influx during rest (e.g. via Ca channels) or (3) a functional linkage between the dihydropyridine receptor and the SR or ryanodine receptor therein. These are considered below.
Possible explanations for effect of Bay K 8644 on resting SR Ca (1) Direct effects of Bay K 8644 on SR It is difficult to prove that Bay K 8644 does not directly affect the SR. However, the lack of effect of Bay K 8644 on ryanodine binding in heavy skeletal SR vesicles, solubilized triads or aggressively homogenized cardiac myocytes does not provide evidence for a direct effect of Bay K 8644 on SR. On the other hand, Bay K 8644 increased ryanodine binding under conditions where the SR-sarcolemmal junctions are likely to be more intact. This would be consistent with an indirect effect of Bay K 8644 on the SR release channel (requiring
intact functional coupling). SR Ca-pump inhibition could, in principle, also explain accelerated rest decay with Bay K 8644. However, there is no evidence for significant depression of SR Ca-ATPase at the concentration of Bay K 8644 used here (Zorzato et al., 1985; L. Hove-Madsen, unpublished observations). Additionally, there is no direct evidence for Bay K 8644 effects on cardiac myofilament Ca sensitivity or on SR Ca release in isolated cardiac or skeletal muscles SR (Thomas et al., 1985; Zorzato et al., 1985). Therefore it seems unlikely that Bay K 8644 affects the SR directly.
(2) Bay K 8644-induced resting Ca influx Another possibility is that exposure to Bay K 8644 leads to diastolic Ca influx. Talo et al. (1990) reported the existence of a steady Ca “window” current when rat ventricular myocytes were subject to sustained depolarization near to, but below the threshold for activation of a twitch (−40 to −20 mV). This “window” current appeared to be sufficient to induce graded SR Ca release, with consequent generation of very small contractions. The well characterized leftward shift in the currentvoltage relationship of cardiac ICa with Bay K 8644 (Sanguinetti et al., 1986) could lead to either the generation or the enhancement of similar “window” currents in ferret myocytes. Even if a window current in the classical sense is not active at resting Em (−80 mV) it is possible that individual -Type Ca channels could open, albeit extremely rarely at rest, but this could be increased by Bay K 8644. These currents may not be readily detectable by conventional electrophysiological techniques, but could induce local SR Ca release during the rest periods, with consequent acceleration of rest decay by Bay K 8644. This could provide an important caveat when considering the results from the whole cell binding studies and rest decay experiments. However, the results in Figures 9 and 10 rule out this possibility since the accelerative effect of Bay K 8644 on rest decay of SR Ca content occurs even in Ca-free, EGTA solution where Ca influx cannot occur. Thus, Ca influx does not mediate the effect of Bay K 8644 on SR Ca content.
(3) Functional linkage from DHP receptor to ryanodine receptor This most intriguing possibility is consistent with the experimental results and cannot be excluded at this stage. In skeletal muscle a voltage-dependent
Acceleration of Rest Decay by Bay K 8644
change in the DHP receptor is thought to induce opening of the SR Ca release channel/ryanodine receptor (see review by Rı´os and Pizarro´, 1988). This coupling may be mechanical as envisaged by Chandler et al. (1976) and could be direct or involve other proteins (e.g. triadin). There may also be Cainduced Ca-release in skeletal muscle, but this may be an amplification system which is not essential for E-C coupling. If Bay K 8644 can bind to the DHP receptor and even partly mimic the effect of depolarization, the chain of mechanical effects could alter the ryanodine receptor and cause increased SR Ca release. Evidence of similar functional linkage is equivocal in cardiac tissue, where Ca-induced Ca-release appears to be essential for SR Ca release and voltage dependent release does not seem to occur (Na¨bauer et al., 1989; Niggli and Lederer, 1990; Bers, 1991). While the effects of Bay K 8644 on ryanodine binding are small (>30% increase in apparent Bmax in Fig. 5), there are two reasons why the modest change in ryanodine binding may be sufficient to explain the clear acceleration of rest decay. First, the ratio of ryanodine:dihydropyridine receptors in ferret ventricular myocytes is 10:1 (Bers and Stiffel, 1993) and each ryanodine receptor is probably a tetramer of RyR. Thus even if every DHPR were directly linked to a ryanodine receptor, the maximum fraction of ryanodine receptors with altered characteristics would be about 10%. Thus the small observed change in ryanodine binding is almost a surprisingly large effect (if a direct RyRDHPR linkage were involved). Second, one only need change a small number of SR Ca release channels (perhaps by a moderate degree) to produce the function effects observed. For example only 6% of ryanodine receptors must open to produce the Ca flux associated with E-C coupling (Bassani and Bers, 1995) and during rest there are only about 10−4 openings/channel/sec (Cheng et al., 1993; Bassani and Bers, 1995). In addition, there is no reason to expect the relation between apparent ryanodine binding and resting SR Ca efflux to be linear. Thus a modest change observed in ryanodine binding could easily be a reflection of the increased rate of SR Ca loss observed during rest. That is, even a small change in the probability of SR Ca channel opening during rest would be enough to accelerate rest decay of SR Ca content. Even a brief exposure to very low concentrations of ryanodine (1–100 pM), where few release channels are likely to be altered, accelerates rest decay markedly in ferret ventricle (Male´cot and Katzung, 1987). In this way the functional consequence of
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Bay K 8644 on SR Ca is rather like a very low ryanodine concentration. Again, the relatively small effects of Bay J 8644 on ryanodine binding could be due to either a small fraction of ryanodine receptors participating in sarcolemmal-SR couplings (consistent with the stoichiometry of DHP: ryanodine receptor) or a small change in Ca release channel properties (or both). In addition, the apparent sensitivity to mechanical disruption of the Bay K 8644 effect on ryanodine binding would be consistent with a mechnical connection transmitting the signal. Thus, while our results are consistent with such a direct functional link, additional work will be required to prove this to be the case.
Species and condition dependence An interesting aspect of this particular effect of Bay K 8644 is that it is readily demonstrable in dog and ferret, but not in rat, rabbit or guinea-pig cardiac preparations. Additionally, it is also more difficult to observe the effect at room temperature compared to 37°C. The species differences could be indicative of morphological and/or functional species differences. It may imply that functional linkage between DHP and ryanodine receptors exists in dog and ferret cardiac tissue but not in the other preparations. There may also be species differences in the properties of the SR release channels or the DHP receptors (e.g. Ca sensitivity, Rousseau and Meissner, 1989; Ashley and Williams, 1990). Another simple explanation could be that the modest accelerative effects of Bay K 8644 on rest decay in species other than ferrets and dogs are masked by the rest decay characteristics of that species. For example the relatively rapid rest decay in control guinea-pig and rabbit ventricle might make it harder to discern. Conversely the very slow rest decay in rat ventricle may be limited by sarcolemmal Na/Ca exchange rather than the rate of SR Ca leak (Bassani and Bers, 1994). Thus, a slightly faster SR Ca leak may not affect rest decay much in rat. In summary, we have shown that the DHP agonist, Bay K 8644, accelerates the rest decay of twitches and SR Ca content in ferret ventricular muscle. The results are most consistent with an underlying mechanism whereby the binding of Bay K 8644 to the DHP receptor alters the state of the ryanodine receptor/SR Ca release channel via a functional coupling between the two receptors.
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Acknowledgements The authors would like to thank Mrs L. Manestar, Ms K. Cha, Mrs M. Robinson and Ms B. Tumulty for their assistance in the preparation of the cardiac myocytes. Thid work was supported by a grant from the USPHS (HL-44583) and E. Mc. was a British-American Research Fellow of the American Heart Association and the British Heart Foundation.
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