β-Adrenergic Stimulation Modulates Ryanodine Receptor Ca2+ Release During Diastolic Depolarization to Accelerate Pacemaker Activity in Rabbit Sinoatrial Nodal Cells
It has long been recognized that activation of sympathetic β-adrenoceptors (β-ARs) increases the spontaneous beating rate of sinoatrial nodal cells (SANCs); however, the specific links between stimulation of β-ARs and the resultant increase in firing rate remain an enigma. In the present study, we show that the positive chronotropic effect of β-AR stimulation is critically dependent on localized subsarcolemmal ryanodine receptor (RyR) Ca2+ releases during diastolic depolarization (CRDD). Specifically, isoproterenol (ISO; 0.1 μmol/L) induces a 3-fold increase in the number of CRDDs per cycle; a shift to higher CRDD amplitudes (from 2.00±0.04 to 2.17±0.03 F/F0; P<0.05 [F and F0 refer to peak and minimal fluorescence]); and an increase in spatial width (from 3.80±0.44 to 5.45±0.47 μm; P<0.05). The net effect results in an augmentation of the amplitude of the local preaction potential subsarcolemmal Ca2+ transient that, in turn, accelerates the diastolic depolarization rate, leading to an increase in SANC firing rate. When RyRs are disabled by ryanodine, β-AR stimulation fails to amplify subsarcolemmal Ca2+ releases, fails to augment the diastolic depolarization rate, and fails to increase the SANC firing rate, despite preserved β-AR stimulation-induced augmentation of L-type Ca2+ current amplitude. Thus, the RyR Ca2+ release acts as a switchboard to link β-AR stimulation to an increase in SANC firing rate: recruitment of additional localized CRDDs and partial synchronization of their occurrence by β-AR stimulation lead to an increase in the heart rate.
Arobust fight or flight response, largely mediated via abrupt sympathetic nervous input to the heart to increase its beating rate, is an essential component of the vertebrate survival instinct. This β-adrenoceptor (β-AR)–induced increase of the sinoatrial nodal cell (SANC) beating rate had initially been attributed exclusively to modulation of ionic channels located within the surface membrane.1,2⇓ Given the more recently recognized critical role of intracellular Ca2+ signaling,3–8⇓⇓⇓⇓⇓ an important issue arises as to whether the increase in SANC beating rate effected by β-AR stimulation also involves a β-AR effect on intracellular Ca2+ dynamics. In this regard, evidence has been presented to suggest that altered Ca2+ flux via ryanodine receptors (RyRs) is involved in the β-AR chronotropic effect in frog9 and guinea pig10 SANCs. However, the specific mechanisms of RyR involvement in β-AR stimulation-induced increase in the SANC firing rate are unknown. In the present study, we show in single, isolated rabbit sinoatrial (SA) node cells that β-AR stimulation increases the number of RyR-generated CRDDs. This increased likelihood for CRDD occurrence functionally results in their partial synchronization, producing a localized subsarcolemmal Ca2+ transient that spreads by Ca2+-induced Ca2+ release, activating Na+-Ca2+ exchange current to accelerate the diastolic depolarization,8 and thus increases the SANC firing rate. Inhibition of RyRs blocks the chronotropic effect of β-AR stimulation, although this maneuver does not prevent the β-AR stimulation-induced increase in L-type Ca2+ current (ICa,L). Thus, modulation of localized CRDD characteristics during pacemaker depolarization is a novel mechanism involved in β-AR modulation of cardiac chronotropy.
Materials and Methods
SA Node Cell Preparations and Electrophysiological Recordings
Single SA node cells were isolated, using protocols approved by our institution’s Animal Care and Use Committee, from rabbit (Harlan Industries, Indianapolis, Ind) hearts as previously described.11 We studied spindle-shaped SA node cells that exhibited spontaneous contractions in normal Tyrode solution containing 1.8 mmol/L Ca2+. Perforated or ruptured patch-clamp techniques were used to record spontaneous action potentials or currents, respectively, with Axopatch-1D patch-clamp amplifier (Axon Instruments). The bath temperature was maintained at 35±0.5°C. For perforated patch-clamp experiments, β-escin12 (50 μmol/L, Sigma) was added to the pipette solution.
For ICa,L recordings, depolarizing voltage-clamp pulses (300 ms) were applied from a holding potential of −50 mV; 30 μmol/L tetrodotoxin (TTX) and 4 mmol/L 4-aminopyridine (4-AP) were added to block interfering currents. To minimize interference from rundown, ICa,L was measured every 11 seconds (step to −10 mV), and the effect of isoproterenol (ISO) was expressed as maximal ICa,L amplitude in the presence of ISO normalized to amplitude immediately before ISO addition. For recordings of diastolic depolarization current, a voltage ramp protocol was used from a holding potential of −60 to −40 mV.
Confocal Imaging of Ca2+ Transients
Cells were placed on the stage of a Zeiss LSM-410 inverted confocal microscope (Carl Zeiss, Inc) and loaded with fluo-3 AM (Molecular Probes). All images were recorded in the line-scan mode (see Figure 4, inset) with the scan line oriented along the long axis of the SANCs close to the sarcolemmal membrane. In this mode, the lines are plotted along the cell every 1.39 to 5 ms, and each line is added to the right of the preceding line to form the line-scan image. A trigger signal and a short flash generated by the voltage-clamp protocol were recorded simultaneously by the electrophysiological and imaging systems to allow synchronization of electrophysiological and Ca2+ measurements. Image processing was performed with IDL software (version 5.2, Research Systems, Boulder, Colo). To identify and measure local CRDDs, a customized software program was used that selected CRDDs on the basis of their statistical deviation from the background noise.13 The amplitude of local CRDDs was expressed as a peak value (F) normalized to minimal fluorescence (F0); spatial diameter of CRDDs was indexed by the FWHM (full width at half-maximum amplitude) and duration, T50, was measured as the time above 50% level of amplitude. The sarcoplasmic reticulum (SR) Ca2+ content was estimated by rapid application of caffeine (20 mmol/L, 1 second) onto the cell by pressure-ejection through a pipette located 100 μm away from the cell.14 To prevent caffeine-induced increase in Na+-Ca2+ exchange current in SANCs,15 caffeine was dissolved in Na+- and Ca2+-free solution (extracellular Na+ was replaced with N-methyl-d-glucamine15).
Solutions and Materials
For action potential recordings, the bath solution had the following composition (in mmol/L): NaCl 140, KCl 5.4, MgCl2 1, HEPES 5, CaCl2 1.8, and glucose 5.5; pH 7.4. Pipette solution for both perforated patch study and diastolic depolarization current recordings contained (in mmol/L) K-gluconate 120, NaCl 5, MgATP 5, HEPES 5, and KCl 20; pH 7.2. For ICa,L recordings, the bath solution contained the following (in mmol/L): NaCl 117, TEA-Cl 20, CsCl 5.4, MgCl2 1, HEPES 5, CaCl2 1.8, and 4-AP 4; pH 7.4. TTX (30 μmol/L) was added to the bath solution before the experiment. The pipette solution contained the following (in mmol/L): NaCl 10, TEA-Cl 20, CsCl 110, EGTA 10, MgATP 5, and HEPES 10; pH 7.2.
Data are presented as mean±SEM. The statistical significance of effects was evaluated by Student’s t test and ANOVA when appropriate. A value of P<0.05 was considered statistically significant.
Results and Discussion
Efficiency of β-AR Stimulation Is Suppressed When RyRs Are Disabled
Table 1 and Figure 1A show that the β-adrenergic agonist ISO significantly increases spontaneous SANC firing rate. This effect is reversible during washout and occurs via β-ARs, as the application of the β-AR antagonist propranolol (2 μmol/L) during ISO stimulation reverses the increase in the firing rate (n=3, data not shown). Table 1 also lists the control parameters of SANC action potentials, which are highly comparable to those reported previously.1,11⇓
To determine whether the mechanism responsible for this β-adrenergic acceleration of SANC firing depends on Ca2+ release from RyRs, we used ryanodine to functionally disable RyRs.16 Consistent with previous results,3–6⇓⇓⇓ ryanodine decreased the firing rate of SANCs in a dose-dependent manner (EC50, 3 μmol/L) and at 30 μmol/L of ryanodine completely abolished spontaneous excitations.8 Changes in SANC action potential parameters induced by ryanodine (see Table 1) are similar to those reported previously for rabbit SANCs.5,6⇓ Application of ISO in the presence of ryanodine (3 μmol/L) induced only a minor increase in the rate of spontaneous firing (Figure 1B) and this small effect was transient (Figure 1C). Compared with control, the ability of ISO (1 μmol/L) to accelerate the diastolic depolarization rate in the presence of ryanodine was decreased by approximately 3-fold (see Table 1). The average concentration response of the relative change in SANC beating rate in response to ISO is illustrated in Figure 2. Ryanodine caused a 2.5-fold decrease in the ability of ISO to increase SANC firing rate and an approximate 6-fold increase in the ISO EC50 (see inset).These results suggest that Ca2+ release via RyRs is an essential feature of β-AR stimulation-induced acceleration of the rabbit SANC firing rate, an observation also made in amphibian pacemaker cells.9 While that study demonstrated that a β-AR stimulation-induced increase in the amplitude of the global Ca2+ transient after the action potential accompanies an increase in the firing rate of pacemaker cells,9 the specific mechanisms of RyR involvement in β-AR stimulation-induced increase in the SANC firing rate have not been demonstrated.
β-Adrenergic Modulation of CRDD Parameters
To define the specific subcellular spatiotemporal characteristics of RyR Ca2+ release and to determine how β-AR stimulation modulates this release to enhance SANC firing, we used confocal microscopy to measure changes in Ca2+ beneath the sarcolemmal membrane. Prior studies in cat atrial pacemaker cells7 and rabbit SANCs8 have noted the occurrence of local subsarcolemmal Ca2+ releases during the latter half of the diastolic depolarization, ie, preceding the action potential upstroke. Figures 3A and 3C demonstrate such CRDDs in rabbit SANCs, most of which occurred during the second half of the diastolic depolarization. The representative image in Figure 3B indicates that β-AR stimulation increases the likelihood for CRDDs to occur, ie, recruits more Ca2+ releases, and increases their brightness. Figure 4 compares the average number of CRDDs during each cycle (Figure 4A), their amplitude (Figure 4B), spatial width (Figure 4C), and duration (Figure 4D) before and after β-AR stimulation. In control conditions, although the mean CRDD duration was the same as in ventricular myocytes, the amplitude and width measured at half-maximum was about twice as large as in ventricular myocytes,17–19⇓⇓ which suggests that each CRDD in SANCs involves more RyR release units than in ventricular myocytes. After β-AR stimulation, there is a 3-fold increase in number of CRDDs per cycle as well as a shift to higher amplitudes and greater width (see Figure 4). There is also a trend for local CRDD duration to increase after ISO, but this is not statistically significant. The net result of the increases in the probability of CRDD occurrence, their increased intensity, and their increased spatial dimensions by β-AR stimulation is a larger subsarcolemmal increase in Ca2+ during pacemaker depolarization as a result of a partial synchronization of local CRDDs. A similar synchronization effect of β-AR stimulation on RyR Ca2+ release has recently been discovered in ventricular myocytes.14 Ryanodine inhibits local CRDDs (Figure 3D) concomitant with the ryanodine-induced decrease in firing rate. Figure 3E demonstrates that in the presence of ryanodine, β-AR stimulation by even a higher ISO concentration (1 μmol/L) is not able to augment CRDD or to increase the SANC firing rate. The combined results in Figure 3 indicate that local Ca2+ transient during pacemaker depolarization occurs via Ca2+ release from RyRs, and that Ca2+ release is amplified, in part at least, by recruitment of additional RyR Ca2+ releases after β-AR stimulation. The resultant amplification has a pivotal role in this β-AR stimulation-induced increase in SANC firing rate.
Effect of β-AR Stimulation on Diastolic Depolarization, Ca2+ Currents, and If
To delineate the ionic mechanisms underlying β-AR–induced Ca2+ release-mediated modulation of SANC pacemaker activity, we simulated the action potential diastolic depolarization by a voltage ramp protocol (Figure 5A, bottom), which followed six preconditioning pulses to ensure steady-state loading of the SR Ca2+. If CRDDs were to activate net inward current, their partial synchronization by β-AR stimulation would augment the slope of the diastolic depolarization; conversely, inhibition of such Ca2+ releases would be expected to decrease net inward current and reduce the diastolic depolarization slope. In control, ISO significantly increased this current (Figure 5B). Ryanodine, per se, reduced average diastolic depolarization current from 0.93±0.13 to 0.48±0.16 pA/pF (n=6, P<0.05). After ryanodine pretreatment, ISO did not produce a significant increase in the diastolic depolarization current (Figure 5B) or in the SANC firing rate (Figure 2). The most likely candidate responsible for this ryanodine-sensitive inward current during the SANC diastolic depolarization is the Na+-Ca2+ exchanger current (INa/Ca), which has been reported to contribute significantly to automaticity of SANCs.1,20,21⇓⇓ That this current was completely abolished after substituting Na+ for Li+ in the bath solution8 provides further evidence that this current is INa/Ca.
It is widely recognized that the positive chronotropic effect of β-AR stimulation in SANCs is accompanied by an increase in the ICa,L amplitude.1 To determine whether the aforementioned effects of ryanodine to reduce the ISO-induced increase in the inward current during the pacemaker depolarization concomitantly affects ICa,L, we measured effects of ISO on ICa,L in the presence and absence of ryanodine.
Ryanodine pretreatment, per se, had no significant effect on the average amplitude of ICa,L (Table 2). Likewise, ryanodine pretreatment had no significant effect on the ISO-induced augmentation of the ICa,L amplitude (Table 2 and Figure 5D). Thus, even when β-AR stimulation substantially increases Ca2+ influx via augmentation of ICa,L amplitude, the net positive chronotropic effect of ISO is suppressed when RyRs are disabled and CRDDs from RyRs are inhibited by ryanodine (Figures 1C, 2, and 3⇑⇑E). In other words, in the absence of intact RyR function, a substantial β-AR stimulation-induced increase of ICa,L in the presence of ryanodine is not sufficient to increase the inward current that underlies SANC diastolic depolarization (Figure 5B) or to increase SANC beating rate. Thus, amplification of local increase of subsarcolemmal Ca2+ during pacemaker depolarization, which accelerates the diastolic depolarization slope via amplification of inward current, is an important link in the cascade of mechanisms that underlies the β-adrenergic modulation of SANC pacemaker activity.
The requirement of intact RyRs for the β-AR stimulation-induced amplification of CRDDs may be attributable to a direct phosphorylation of the RyRs22 or to an effect of β-ARs to increase the SR Ca2+ load.23 To test the latter possibility, we applied a pulse of caffeine (20 mmol/L, 1 second), which rapidly empties the SR Ca2+ store (Figure 6). After 1 minute of ISO superfusion, there was a significant (P<0.05) increase in the SR Ca2+ load by 31% (Figure 6). Thus, the increase in CRDDs by β-AR stimulation could be partly attributable to an increase in RyR release flux due to an increase in the SR Ca2+ load.
The increase in SR Ca2+ load and increased RyR Ca2+ release flux triggered by ICa,L also affected ICa,L decay kinetics. The fast time constant of ICa,L decay, which reflects Ca2+-dependent inactivation,24 was significantly increased after ryanodine pretreatment, whereas the slow time constant remained unchanged (Table 2). This result is similar to that of prior observations of the effect of ryanodine on ICa,L inactivation in rat ventricular myocytes25,26⇓ and human atrial myocytes27 and is consistent with the interpretation that Ca2+ influx through L-type channels and ICa,L-triggered Ca2+ release from SR accelerate time-dependent inactivation of ICa,L. In the present study, the effect of ryanodine to decrease Ca2+ release from the SR in response to an ICa,L of a given amplitude likely reflects a reduction in RyR Ca2+ release flux, a widely recognized effect of ryanodine in ventricular cells.28 That ISO accelerates the fast component of ICa,L decay (Table 2) in spite of the presence of 10 mmol/L EGTA in our pipette solution is consistent with the idea that EGTA is a slow Ca2+ buffer and cannot effectively buffer Ca2+ released from the SR near the mouth of L-type Ca2+ channel.24,29⇓ In the presence of ryanodine, ISO did not affect the kinetics of ICa,L decay (Table 2). This is consistent with the idea that ryanodine reduces RyR Ca2+ release flux in the presence of ISO.
It is not clear whether enhanced CRDDs via RyRs during β-AR activation occur spontaneously or are triggered. Spontaneous, localized, diastolic SR Ca2+ release in ventricular myocytes causes spontaneous sarcolemmal diastolic depolarization mainly because of Ca2+ modulation of Na+-Ca2+ exchanger, and when diastolic depolarization amplitude reaches threshold, it elicits a spontaneous action potential.30 The increase in cellular SR Ca2+ load in ventricular myocytes by β-AR stimulation markedly exaggerates this phenomenon. This mechanism of abnormal automaticity in ventricular myocytes may underlie normal automaticity in SANCs. Alternatively, as all CRDDs in SANCs occur during the second half of the diastolic depolarization, it is possible that sarcolemmal ion channel activation during this period evokes CRDDs. A likely candidate of β-AR stimulation-induced augmentation of CRDDs would be L-type Ca2+ channels activated during SANC diastolic depolarization.31
Changes in other ionic currents that underlie SANC diastolic depolarization might also be involved in the triggering of CRDDs and their modulation by β-AR stimulation. It has been observed in latent cat atrial pacemaker cells that a blockade of T-type Ca2+ current with Ni2+ (50 μmol/L) induces a dramatic (230%) prolongation of spontaneous cycle length compared with control.7 To define whether the beating rates and CRDDs in rabbit SANCs in the present study involve T-type Ca2+ current, we exposed SANCs to 50 μmol/L Ni2+. In contrast to the prior observation in cat latent atrial pacemaker cells, Ni2+ induced only a 16% increase in the spontaneous cycle length of rabbit SANCs (from 440±20 to 510±63 ms, n=5). This result is consistent with a previous study in rabbit SANCs in which abolition of T-type Ca2+ current with 40 μmol/L of Ni2+ only slightly (14%) increased the spontaneous cycle length.32 Moreover, in our experiments, 50 μmol/L of Ni2+ did not decrease the number of spontaneous CRDDs per cycle (in control 1.0±0.1 per cycle versus 1.0±0.3 per cycle in Ni2+, n=4). Thus, different perspectives on the role of T-type Ca2+ current in CRDDs, diastolic depolarization, and beating rate are gleaned from experiments in different cell types. That T-type Ca2+ current appears to play a more important role in cat atrial latent pacemaker activity32 than in rabbit SANC primary pacemaker activity might be explained on the basis of a more negative maximum diastolic potential in the former than the latter.32,33⇓
To determine a role of T-type Ca2+ current regulation in β-AR acceleration of SANC beating rate in the present study, we applied 50 μmol/L of Ni2+ after β-AR stimulation. ISO (0.1 μmol/L) increased the beating rate from 108±7 bpm in control to 144±5 bpm with ISO, n=3. Ni2+, applied after ISO, induced only a 10% decrease in the beating rate, to 139±6 bpm, n=3. Thus, under conditions of the present experiments, T-type Ca2+ current likely has, at best, a minor role in β-AR acceleration of rabbit SANC beating rate, a conclusion consistent with that of a prior study32 that failed to demonstrate an effect of ISO on T-type Ca2+ current in rabbit SANCs.
To define the involvement of the hyperpolarization-activated current, If, in β-AR acceleration of SANC beating rate, we applied 2 mmol/L of Cs+34 before and during β-AR stimulation. Cs+ alone induced a 5.2±0.3% (n=5) decrease in beating rate from 205±6 bpm in control to 194±5 bpm with Cs+, consistent with a prior observation in primary pacemaker cells.35 In the presence of ISO (1 μmol/L), Cs+ induced a 7.5±2.6% (n=4) decrease of the SANC beating rate from 225±18 bpm to 208±17 bpm with Cs+. Thus, under the present experimental conditions, the contribution of If to β-AR acceleration of rabbit SANC beating rate did not exceed 10%.
Activation of other ionic currents as a potential mechanism for β-AR stimulation to increase in CRDDs and beating rate in SANCs may be excluded. β-AR–operated chloride current is not involved in the effect of β-AR stimulation, as this current is absent in rabbit SANCs.36,37⇓ Although a small change in the amplitude of deactivation kinetics of the delayed rectifier K+ current38 might theoretically contribute to the positive chronotropic effect of ISO, a significant change in IKr is unlikely because the maximum diastolic potential was not affected by ISO (see Table 1), and the slope of diastolic depolarization was altered only just before the action potential upstroke (Figure 1A).
In summary, the present results show, for the first time, that β-AR stimulation increases the likelihood of occurrence of localized Ca2+ release in the subsarcolemmal space during the pacemaker depolarization and augments its amplitude and spatial dimension, ie, β-AR recruits additional local RyR Ca2+ release and partially synchronizes its occurrence. This spatiotemporal synchronization of CRDDs augments the inward current during the diastolic depolarization, which, in turn, accelerates the diastolic depolarization rate, leading to an increase in SANC firing rate. In the presence of ryanodine, which disables normal RyR function and depletes the SR Ca2+ store, β-AR stimulation fails to amplify local CRDDs, fails to augment the diastolic depolarization, and fails to increase the SANC firing rate (Figures 1 and 2⇑). Thus, the RyR Ca2+ release flux acts as a switchboard that links β-AR stimulation to an increase in SANC firing rate. β-AR modulation of localized subsarcolemmal RyR-generated Ca2+ release characteristics during the diastolic depolarization demonstrated in the present study is a novel mechanism to explain β-AR modulation of cardiac chronotropy.
This work was supported in part by awards from the National Research Council (T.M.V. and K.Yu.B.). The authors are deeply grateful to Drs Heping Cheng and Ira R. Josephson for the comments on the manuscript and to Dr Harold A. Spurgeon and Bruce Ziman for help and technical support.
Original received June 28, 2001; revision received November 6, 2001; accepted November 6, 2001.
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