In cardiac muscle, Ca 2 -induced Ca 2 release (CICR) from the

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1 Cellular Biology Transmission of Information From Cardiac Dihydropyridine Receptor to Ryanodine Receptor Evidence From BayK 8644 Effects on Resting Ca 2 Sparks Hideki Katoh, Klaus Schlotthauer, Donald M. Bers Abstract Coupling between L-type Ca 2 channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs) plays a pivotal role in excitation-contraction (E-C) coupling in cardiac myocytes, and Ca 2 influx is generally accepted as the trigger of sarcoplasmic reticulum (SR) Ca 2 release. The L-type Ca 2 channel agonist BayK 8644 (BayK) has also been reported to alter RyR gating via a functional linkage between DHPR and RyR, independent of Ca 2 influx. Here, the effect of rapid BayK application on resting RyR gating in intact ferret ventricular myocytes was measured as Ca 2 spark frequency (CaSpF) by confocal microscopy and fluo 3. BayK increased resting CaSpF by % within 10 seconds in Ca 2 -free solution, and depolarization had no additional effect. The effect of BayK on CaSpF was dose-dependent, but even 50 nmol/l BayK induced a rapid % increase in CaSpF. Nifedipine (5 mol/l) had no effect by itself on CaSpF, but it abolished the BayK effect (presumably by competitive inhibition at the DHPR). The nondihydropyridine Ca 2 channel agonist FPL (1 mol/l) did not alter CaSpF (despite rapid and potent enhancement of Ca 2 current, I Ca ). In striking contrast to the very rapid and depolarization-independent effect of BayK on CaSpF, BayK increased I Ca only slowly ( 18 seconds), and the effect was greatly accelerated by depolarization. We conclude that in ferret ventricular myocytes, BayK effects on I Ca and CaSpF both require drug binding to the DHPR, but postreceptor pathways may diverge in transmission to the gating of the L-type Ca 2 channel and RyR. (Circ Res. 2000;87: ) Key Words: Ca 2 channel sarcoplasmic reticulum excitation-contraction coupling confocal microscopy FPL In cardiac muscle, Ca 2 -induced Ca 2 release (CICR) from the sarcoplasmic reticulum (SR) is pivotal in excitationcontraction (E-C) coupling. 1,2 In skeletal muscle, depolarization causes SR Ca 2 release, and the dihydropyridine receptor (DHPR) is thought to be the membrane potential sensor that transmits a direct intermolecular signal to the ryanodine receptor (RyR), causing SR Ca 2 release. 3,4 In both muscle types, the DHPR and RyR are in relatively close physical proximity, but exactly how they communicate is unclear. Spatially localized [Ca 2 ] i elevations (Ca 2 sparks) at the sarcomere level can be detected by laser scanning confocal microscopy, allowing direct visualization of SR Ca 2 release events in cardiac myocytes. 5 9 In cardiac muscle E-C coupling, it is generally thought that Ca 2 current (I Ca ) via single L-type Ca 2 channels (DHPRs) goes into a restricted space, triggering local SR Ca 2 release via RyRs. 1,2 Ca 2 sparks evoked by I Ca are believed to summate spatially and temporally, giving rise to the normal whole-cell twitch Ca 2 transient. 6 8 Possible alterations in E-C coupling in hypertrophic and failing rat heart emphasize the importance of understanding the basis of cardiac DHPR-RyR interactions. Evidence suggests that the intracellular loop between domains II and III of the skeletal muscle DHPR is important in transmitting a gating signal to the skeletal RyR. 3 Peptides from this II-III loop can also alter ryanodine binding and RyR gating in skeletal RyR. 13,14 The analogous cardiac II-III loop peptides also alter cardiac RyR gating in lipid bilayers and intact myocytes. 15 This raises the possibility of a physical and/or functional link between cardiac DHPR and RyR. BayK 8644 (BayK) is a dihydropyridine L-type Ca 2 channel agonist 16 that can indirectly modulate resting RyR gating (ie, via DHPR-RyR interaction). 17,18 BayK converts postrest potentiation to postrest decay in canine and ferret ventricular myocytes secondary to a rapid loss of SR Ca 2 during rest The loss of SR Ca 2 at rest was found to be due to a dramatic increase in Ca 2 spark frequency (CaSpF) that occurred even in the complete absence of extracellular Ca 2 and could be competitively blocked by nifedipine. BayK had no effect on single isolated RyR channel gating in lipid bilayers. 18 BayK also increases ryanodine binding in intact ventricular myocytes, but this effect was abolished after homogenization. 17 Thus, an intact physical DHPR-RyR linkage may be needed for the effect of BayK on SR Ca 2 release. Our working hypothesis is that BayK binds to DHPR and that this signal is transmitted to the RyR (independent of Ca 2 entry), Received March 27, 2000; accepted May 24, From the Department of Physiology, Loyola University Chicago, Maywood, Ill. Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Medical School, 2160 S First Ave, Maywood, IL dbers@lumc.edu 2000 American Heart Association, Inc. Circulation Research is available at 106

2 Katoh et al BayK 8644 Effects on Ca 2 Sparks and I Ca 107 Figure 1. Rapid BayK application increases resting Ca 2 sparks. Confocal line-scan images (along long cell axis) were obtained after 4, 12, and 28 seconds of rest after 1-Hz stimulation in control (A) and with 500 nmol/l BayK (B). Single line scans were stacked from left to right. Selected [Ca 2 ] i line plots are from marked sites. increasing resting RyR opening and CaSpF. Here, we provide new information about (1) the kinetics of this effect of BayK, (2) whether I Ca activation modulates the BayK effect, (3) whether FPL (FPL, a benzoylpyrrole Ca 2 channel agonist that does not compete at the DHPR) exerts the same effect, and (4) comparative kinetics and depolarization dependence of BayK on resting CaSpF versus I Ca. We find that BayK rapidly increases resting CaSpF and is not mimicked by FPL and that the effect is independent of depolarization or Ca 2 entry. All 4 of these effects are in striking contrast to BayK effects on I Ca, suggesting a divergent transduction pathway. Materials and Methods Ferret ventricular myocytes were prepared as described previously, 20 and experiments were performed at 22 C. Standard Tyrode s solution contained (mmol/l) NaCl 140, KCl 6, MgCl 2 1, HEPES 5, glucose 10, and CaCl 2 2. In the 0Ca-0Na solution, LiCl replaced NaCl, CaCl 2 was omitted, and 10 mmol/l EGTA was added. The ph was adjusted to 7.4 with NaOH or LiOH. Cells were loaded with the Ca 2 indicator fluo 3 by exposure to 20 mol/l fluo 3 acetoxymethyl ester (Molecular Probes) for 20 minutes at 22 C, with 30 minutes allowed for deesterification. Unless they were voltage-clamped, cells were field-stimulated (0.5 Hz) to steady state before cessation and quick solution switch (time constant, 300 ms) to 0Ca-0Na solution for Ca 2 spark measurement. CaSpF was then measured during a 30-second rest period (15 images). After the first 10 seconds of rest in 0Ca-0Na/EGTA, the cell was field-stimulated for 10 pulses (at 1 Hz) to depolarize cells during exposure to the test solution. With Li replacing Na in this solution, action potentials are still readily activated. 21 SR Ca 2 content was evaluated by rapid application of 10 mmol/l caffeine dissolved in 0Ca-0Na solution with 1 mmol/l EGTA to the cell via a quick-switcher. 21 Confocal fluorescence imaging was performed as described 9,18 with a laser scanning confocal microscope (LSM410, Zeiss) coupled to an inverted microscope (Axiovert 100, Zeiss) with an 40 oil-immersion objective (NA 1.3), excitation at a wavelength of 488 nm, and emission at 515 nm. Line scans (512 pixels/line, 0.25 m/pixel) were acquired at 250 lines/s and were processed with IDL software (Research Systems) with [Ca 2 ] i calculated 5,9 with a fluo 3 K d 1.1 mol/l and resting [Ca 2 ] i 150 nmol/l. 22,23 Visually identified Ca 2 sparks were accepted if local [Ca] i change (5 adjacent pixels) exceeded 60 nmol/l with duration at half-amplitude 8 ms. 9,18 Ca 2 sparks counted per line scan image were normalized spatially (per m 3 ) and temporally (per second) as CaSpF (pl 1 s 1 ). Global [Ca 2 ] i transients (depolarizationor caffeine-induced) were derived from average fluorescence intensities along the scanned line. I Ca was recorded by whole-cell ruptured-patch voltage clamp as described 24 with pclamp (Axon Instruments), filtered at 10 khz, and sampled at 1 khz. Patch electrodes had resistances of 1.0 to 1.5 M, with an internal solution composed of (mmol/l) CsCl 125, MgCl 2 1, HEPES 20, EGTA 10, MgATP 10, and GTP 0.3 (ph 7.2). Cells were superfused with Tyrode s solution in which KCl and NaCl were replaced by CsCl and TEA-Cl, respectively, to better isolate I Ca. Action potentials were recorded in current-clamp mode with physiological pipette and bath solutions and higher-resistance electrodes (10 to 20 M ). Figure 2. BayK effects on CaSpF. A, Time course of CaSpF during 30 seconds of rest in control and in the presence of 500 nmol/l BayK. Data are mean SEM, n 11, paired. Tick marks indicate 10 pulses at 1 Hz.

3 108 Circulation Research July 21, 2000 Figure 3. Low-concentration BayK also alters CaSpF. Same as Figure 2 except 50 nmol/l BayK was used (n 6, paired). Tick marks indicate 1-Hz stimulation. Nifedipine (Sigma) and ( )BayK (Calbiochem) were dissolved in ethanol, and FPL (Alexis) was dissolved in DMSO (final ethanol and DMSO concentrations 0.1%). Caffeine was dissolved directly in 0Ca-0Na solution. Results were expressed as mean SEM for the indicated number (n) of myocytes, and a value of P 0.05 was considered significant (Student s t test). Results Rapid BayK Application: CaSpF and SR Ca 2 Content To prevent Ca 2 influx from affecting CaSpF, we removed extracellular Ca 2 and added 10 mmol/l EGTA immediately after the interruption of electrical stimulation. Furthermore, to avoid any change in SR Ca 2 load or [Ca 2 ] i at the beginning of the rest period (for control versus experimental), identical solutions and stimulation protocols preceded the rest. During rest, 0Ca-0Na inhibited Ca 2 extrusion via Na /Ca 2 exchange and minimized SR Ca 2 loss. 20 Under control 0Ca-0Na/EGTA conditions, CaSpF during 30 seconds of rest remained nearly constant (Figures 1A and 2). Thus, CaSpF at resting membrane potential is not altered by complete removal of [Ca 2 ] o. Furthermore, stimulation in 0Ca- 0Na/EGTA did not produce any detectable changes in [Ca 2 ] i. After this control measurement, Tyrode s solution was restored Figure 4. BayK accelerates rest-dependent loss of SR Ca 2. Peak [Ca 2 ] i of caffeine-induced [Ca 2 ] i transient was used as a measure of SR Ca 2 content. Caffeine (10 mmol/l) was applied rapidly in 0Ca-0Na/EGTA solution 1 second after steady-state stimulation (SS) and after 30 seconds of rest in the absence (control, Ctl) and presence of BayK (n 6, paired *P 0.01). Figure 5. Nifedipine inhibits BayK effect on CaSpF but has no effect alone. A, CaSpF during 30 seconds in control and with 500 nmol/l BayK plus 5 mol/l nifedipine (n 5, paired). Inset shows action potentials recorded in control and in 0Ca-0Na with or without 500 nmol/l BayK plus 5 mol/l nifedipine. B, Same as in A, but in the absence and presence of 5 mol/l nifedipine (n 7, paired). In these experiments, [Ca 2 ] o before 0Ca-0Na was 3 mmol/l (rather than 2 mmol/l) to improve detection of possible inhibitory effect of nifedipine on CaSpF (control CaSpF was slightly higher than in Figures 2, 3, and 5A). and the cell stimulated to return to the initial steady state. When the protocol was repeated with 500 nmol/l BayK (Figures 1B and 2), CaSpF increased rapidly by % at maximum in 10 seconds (P 0.01, n 11). Field stimulation after 10 seconds of rest in BayK had no effect, indicating that the BayK effect on CaSpF was voltage-independent, in marked contrast to the effect of BayK on I Ca. 16,25 The lack of increased CaSpF on stimulation could have been due to the BayK effect being maximal already at 10 seconds with 500 nmol/l BayK. To test this possibility, BayK concentration was lowered to 50 nmol/l, which increased CaSpF by only % (P 0.01, n 6; Figure 3) at the maximum point. Depolarization still had no effect on CaSpF with 50 nmol/l BayK. There was no apparent change in time course of BayK effect on CaSpF between 50 and 500 nmol/l BayK. These results suggest that the peak BayK effect on CaSpF was dose-dependent and rapid, but independent of both depolarization and Ca 2 influx. To measure SR Ca 2 content, we applied caffeine (10 mmol/l) either after the last steady-state pulse or after the 30-second rest period ( 500 nmol/l BayK). Figure 4 shows that after 30 seconds in control 0Ca-0Na/EGTA, there was a small (5%) loss of SR Ca 2 content (versus steady state). In the presence of BayK, the SR Ca 2 content in 0Ca-0Na/EGTA was

4 Katoh et al BayK 8644 Effects on Ca 2 Sparks and I Ca 109 Figure 6. The nondihydropyridine FPL increases I Ca but not resting CaSpF. A, Peak I Ca current-voltage relationship at steady-state FPL exposure (holding E m 80 mv). B and C, FPL effects on I Ca after 10 seconds of perfusion at rest (pulses from 80 to 0 mv for 150 ms at 1 Hz) were fit to a single exponential curve (n 4). D, CaSpF as a function of rest duration in the absence and presence of 1 mol/l FPL (n 6, paired). 16% lower than steady state and significantly lower than control. This is consistent with previous findings on control SR Ca 2 loss in ferret myocytes 20 and also with equilibrium exposure to BayK, 18 in which the faster loss of resting SR Ca 2 content with BayK is due to the higher CaSpF. BayK Effect on RyR Gating Is Mediated via Dihydropyridine Receptor Next, we tested whether the BayK effect could be inhibited by nifedipine competition at the DHPR. Figure 5A illustrates that when 500 nmol/l BayK and 5 mol/l nifedipine were included together in the test solution, the effect of BayK on CaSpF was completely abolished (see also Reference 18). Rapid application of 5 mol/l nifedipine alone did not alter CaSpF (Figure 5B). These results indicate that the BayK effect on RyR is mediated via the DHPR. Figure 5A shows that action potentials were still activated in 0Ca-0Na solution and also when BayK and nifedipine are included (although plateaus are lower than control, because inward I Ca and Na/Ca exchange current are prevented). To further test the hypothesis that the DHPR, rather than altered Ca 2 channel gating, mediates the BayK effect on CaSpF, we used FPL, another potent L-type Ca 2 channel agonist with effects on I Ca similar to those of BayK. In contrast to BayK, FPL does not compete at the dihydropyridine binding sites, indicating that FPL activates I Ca at a site distinct from dihydropyridines. 26,27 Steady-state exposure to 1 mol/l FPL doubled I Ca amplitude at 0 mv (from 8 to 16 A/F) and shifted peak I Ca from 10 to 10 mv (Figure 6A). FPL also altered I Ca activation and inactivation and caused large tail currents even at the first pulse after 10 seconds of exposure to FPL (Figure 6B). These FPL effects on cardiac I Ca confirm previous reports. 26,27 After resting cells had been exposed to FPL for 10 seconds, 10 pulses to 0 mv (1 Hz) further enhanced the FPL effect on I Ca amplitude (Figures 6B and 6C). I Ca amplitude was already 80% of maximum at the first pulse and gradually increased to 99% of maximum (achieved after 1 minute at 1 Hz). Figure 7. Effect of BayK on I Ca.A,I Ca measured before (Ctl) and after exposure to 500 nmol/l BayK for indicated rest durations (voltage steps from 80 to 0 mv for 150 ms at 1 Hz). B, Rest and depolarization dependence of I Ca increase after BayK exposure (n 4 to 6, paired) are fit to single-exponential functions ( 18 seconds for the dotted and 2 to 4 seconds for the solid lines). C, Peak I Ca current-voltage relationship at steady-state BayK exposure (holding E m 80 mv). Figure 6D shows that despite the dramatic changes of I Ca, rapid application of FPL in 0Ca-0Na/EGTA caused no detectable change in CaSpF during the same protocol as used for BayK in Figures 2 and 3. These results indicate that FPL binds to Ca 2 channels during rest but does not alter RyR gating during rest or depolarization. This supports the idea that BayK binding to the DHPR may be the first, essential step for the BayK effect on RyR gating during rest (and FPL cannot mimic BayK). Rapid BayK Effects on I Ca Figure 7A shows I Ca traces before and after exposure to 500 nmol/l BayK for 5, 10, and 20 seconds before depolarizations. The first pulse after 5 seconds of BayK perfusion showed larger I Ca, but I Ca increased progressively during the following 9 pulses. Longer resting exposure to BayK enhanced I Ca amplitude more markedly at the first pulse. Steady-state current-voltage relationships (Figure 7C) show that BayK increased I Ca amplitude at 0 mv from to A/F and shifted peak I Ca from 10 to 0 mv. BayK altered I Ca activation and inactivation, and these BayK effects on I Ca agree with previous work. 16,27,28 BayK application induced both rest-dependent and depolarization- (or pulse-) dependent effects on I Ca amplitude (Figure 7B). Rest-dependent I Ca activation was slow ( 18 seconds) and incomplete until 1 minute. The depolarizationdependent increase of I Ca was much faster ( 2.5 to 4 seconds). The maximal BayK effect on resting CaSpF was achieved within 10 seconds (Figure 2), whereas at this time the BayK effect on I Ca was only 43%. Furthermore, the BayK effect on CaSpF (RyR gating) was independent of depolarization, but the BayK effect on I Ca was strongly depolarization-dependent. These results revealed marked differences between BayK effects on CaSpF and I Ca.

5 110 Circulation Research July 21, 2000 Discussion In 4 major ways, we extend our original steady-state findings that BayK increases resting SR Ca 2 release. 17,18 We measured (1) onset kinetics of BayK effects on CaSpF and I Ca, (2) depolarization dependence of BayK effects on CaSpF and I Ca, (3) whether the nondihydropyridine Ca 2 channel agonist FPL could mimic BayK, and (4) disparities in kinetics and depolarization dependency of BayK effects on CaSpF and I Ca. BayK effects on CaSpF were maximal within 10 seconds and were unaffected by depolarization, whereas effects on I Ca were slower in onset and highly depolarization-dependent. Both effects of BayK could be blocked by the dihydropyridine nifedipine. However, the nondihydropyridine FPL did not alter resting CaSpF, despite increasing I Ca. Kinetics of BayK Effect on Resting CaSpF BayK was applied in 0Ca-0Na/EGTA solution to rule out possible CICR, which could have been enhanced by BayK at negative membrane potential. To reduce transverse tubule [Ca 2 ] to negligible levels in our conditions requires 1 second. 18,29,30 This does not affect our conclusions here, because resting Ca 2 sparks are not due to Ca 2 influx, CaSpF is unaltered in 0Ca-0Na/EGTA, and all comparisons are at times 2 seconds. 18 Nevertheless, this geometric constraint limits temporal resolution such that we can only claim BayK effects on CaSpF as maximal in 10 seconds (for both 50 and 500 nmol/l BayK). Rampe et al 31 found biphasic BayK association and dissociation rates in canine sarcolemma, with a rapid association rate constant [k on (mol/l) 1 s 1 ]. On the basis of this k on (and k off s 1 ), 31 our 10-second exposure to BayK would result in 99% and 65% saturation with 500 and 50 nmol/l BayK, respectively (in line with our time course of BayK effect on the CaSpF). Ca 2 agonist effects are also less dependent on Ca 2 channel state than is the case for Ca 2 antagonist. 32 BayK Binding to DHPR Is Necessary for BayK Effect on RyR Gating Nifedipine (5 mol/l) inhibited the BayK effect on CaSpF (Figure 5A) but did not alter CaSpF by itself (Figure 5B). This indicates that BayK binding to the DHPR is necessary for altering RyR gating and agrees with steady-state findings. 18 To further test this hypothesis here, we used the Ca 2 channel agonist FPL, which does not compete for binding at the DHPR. 26 Although BayK and FPL produce comparable effects on I Ca (Figures 6 and 7), FPL had no effect at all on resting CaSpF. This has 2 relevant implications: (1) BayK binding to the DHPR is an essential step in altering RyR gating and (2) similar alterations in Ca 2 channel gating properties are not sufficient to mimic the effect of BayK on CaSpF. Although BayK could have direct effects on the RyR, our previous data showed no effect on single-channel RyR current amplitude or open probability in lipid bilayer experiments. 18 Although BayK increased ryanodine binding to intact ferret ventricular myocytes, mechanical disruption of SR-sarcolemmal junctions eliminated the effect. 17 BayK also had no influence on SR Ca 2 release in skinned guinea pig atrial fibers. 33 Thus, it seems that the effect of BayK on RyR is mediated by the DHPR andaca 2 -independent connection between these receptors. Figure 8. Model for BayK-mediated effects on I Ca and RyR gating. See text for details. BayK Effect on RyR Gating Was Depolarization-Independent The effect of BayK on CaSpF was not influenced by depolarization, in sharp contrast to the effect on I Ca. Indeed, voltage- and use-dependent effects of dihydropyridines (including BayK) on I Ca are classically observed. 25,34 BayK has also been reported to alter gating charge movement attributed to cardiac Ca 2 channels. 35,36 Because the BayK effect on CaSpF was maximal during rest (when no charge movement occurs), it seems unlikely that the BayK effect on CaSpF was mediated by gating charge movement. Because depolarization did not alter CaSpF (or [Ca 2 ] i ), we infer that depolarization per se did not trigger the release of Ca 2 from SR under our experimental conditions. Interestingly, BayK may also cause depolarization-independent SR Ca 2 release in skeletal muscle. 37 Our working hypothesis (Figure 8) is that BayK binding to the DHPR could facilitate protein conformational changes to alter L-type Ca 2 channel gating in a manner that depends on 1 gating cycles (eg, providing access to additional interaction sites). In contrast, the DHPR may transmit a physical signal to the RyR that is independent of I Ca gating or depolarization (and rapid at rest). Our results cannot distinguish whether or not an intermediate protein (R) is involved (eg, sorcin can bind to both DHPR and RyR and alter RyR gating 38,39 ). Conversely, direct DHPR-RyR effects cannot be ruled out, because cardiac DHPR peptides can alter RyR gating in both bilayers and intact voltage-clamped myocytes. 15 Thus, the initial step of BayK binding to the DHPR may be the same for both I Ca and CaSpF, but the functional pathways may diverge between the DHPR and the effector site. In ventricular myocytes, there are 4 to 10 times as many RyRs as there are DHPRs. 40 Thus, even if all DHPRs were coupled in this relatively direct way to RyRs, that would include only 10% to 25% of RyRs. However, activation of 1 RyR may cause sufficiently high local [Ca 2 ] to activate a whole cluster of RyRs via CICR, resulting in a Ca 2 spark. Thus, RyRs coupled to DHPRs may have gating properties different from those of uncoupled RyRs. Niggli 41 suggested such a scenario to explain differential triggering of Ca 2 sparks. It is unclear how the functional DHPR-RyR linkage discussed here might alter E-C coupling. BayK actually depresses E-C coupling, ie, less SR Ca 2 release for a given I Ca and SR Ca 2 load. 28,42 This could be due to altered Ca 2 responsiveness of the RyR. However, this could also be explained by long singlechannel openings induced by BayK and the relatively rapid

6 Katoh et al BayK 8644 Effects on Ca 2 Sparks and I Ca 111 activation of SR Ca 2 release, such that there is wasted I Ca (that does not trigger Ca 2 release). Our data do not indicate any purely voltage-induced Ca 2 release, 43 because no Ca 2 increase accompanied depolarization in 0Ca-0Na solution (in any condition). The BayK-induced increase of resting Ca 2 sparks (and RyR gating) is from an extremely low resting probability (0.0001), 5 whereas the huge CICR during E-C coupling might override this BayK effect. We speculate that the DHPR-RyR interaction responsible for the BayK-induced Ca 2 sparks is weak compared with that in skeletal muscle and that its main physiological importance may be to help colocalize these 2 important Ca 2 channels in heart. Acknowledgments This study was supported by grants from the NIH (HL-30077), the American Heart Association Metropolitan Chicago affiliate, and the Japanese Heart Association. 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