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. Author manuscript; available in PMC: 2010 Feb 17.
Published in final edited form as: Circulation. 2009 Feb 2;119(6):788–796. doi: 10.1161/CIRCULATIONAHA.108.817379

Intracellular Calcium Dynamics and the Acceleration of Sinus Rhythm by β-Adrenergic Stimulation

Boyoung Joung 1, Liang Tang 1, Mitsunori Maruyama 1, Seongwook Han 1, Zhenhui Chen 1, Marcelle Stucky 1, Larry R Jones 1, Michael C Fishbein 2, James N Weiss 3, Peng-Sheng Chen 1, Shien-Fong Lin 1
PMCID: PMC2735196  NIHMSID: NIHMS126503  PMID: 19188501

Abstract

Background

Recent evidence indicates that membrane voltage and Ca2+ clocks jointly regulate sinoatrial node (SAN) automaticity. Here we test the hypothesis that sinus rate acceleration by β-adrenergic stimulation involves synergistic interactions between these clock mechanisms.

Methods and Results

We simultaneously mapped intracellular calcium (Cai) and membrane potential (Vm) in 25 isolated canine right atrium (RA), using previously described criteria of the timing of late diastolic Cai elevation (LDCAE) relative to the action potential (AP) upstroke to detect the Ca2+ clock. Before isoproterenol, the earliest pacemaking site occurred in the inferior SAN, and LDCAE was observed in only 4/25 preparations. Isoproterenol (1 μmol/L) increased sinus rate and shifted pacemaking site to superior SAN, concomitant with the appearance of LDCAE preceding the AP upstroke by 98 ± 31 ms. Caffeine had similar effects, while SR Ca2+ depletion with ryanodine and thapsigargin prevented isoproterenol-induced LDCAE and blunted sinus rate acceleration. Cai transient relaxation time during ISO was shorter in superior SAN (124 ± 34 ms) than inferior SAN (138 ± 24 ms, p = 0.01) or RA (164 ± 33 ms, p = 0.001), and was associated with a lower SR Ca2+ ATPase pump to phospholamban protein ratio in SAN than in RA. If current blockade with ZD 7288 modestly blunted, but did not prevent LDCAE or sinus rate acceleration by isoproterenol.

Conclusions

Acceleration of the Ca2+ clock in the superior SAN plays an important role in sinus acceleration during β-adrenergic stimulation, interacting synergistically with the voltage clock to increase sinus rate.

Keywords: calcium; sinoatrial node; sarcoplasmic reticulum; nervous system, sympathetic

Spontaneous diastolic depolarization (DD) of sinoatrial node (SAN) cells periodically initiates action potentials (APs) to set the rhythm of the heart. The mechanism of spontaneous DD has traditionally been attributed to a “voltage clock” mechanism, mediated by voltage-sensitive membrane currents, such as the hyperpolarization-activated pacemaker current (If) regulated by cAMP.1, 2 However, recent studies implicate a complementary “Ca2+ clock” mechanism mediated by Ca2+ release from the sarcoplasmic reticulum (SR) causing DD via activation of Na/Ca exchanger current (INCX), which coordinately regulates sinus rate along with the voltage clock.3-11 Since the intact SAN is a heterogeneous structure that includes multiple different cell types interacting with each other, the relative importance of the voltage and Ca2+ clocks for pacemaking in different regions of the SAN, and in response to neurohumeral stimuli such as β-agonists, may be different. Indeed, activation maps in intact canine right atrium (RA) showed that SAN impulse origin is multicentric,12 and sympathetic stimulation predictably results in a cranial (superior) shift of the pacemaking site in human and dogs.13 Based on evidence from isolated SAN myocytes showing that late diastolic Ca elevation (LDCAE) relative to the AP upstroke is a key signature of pacemaking by the Ca2+ clock,3-11 we examined whether this criterion could provide insight into the relative importance of the Ca2+ and voltage clock mechanisms to pacemaking in intact SAN tissue. To test this hypothesis, we performed dual optical mapping of transmembrane potential (Vm) and intracellular calcium (Cai) in intact canine SAN and RA under control conditions, during isoproterenol (ISO) infusion and in response to other pharmacologic interventions.

Methods

Langendorff-perfused canine SAN preparation

This study protocol was approved by the Institutional Animal Care and Use Committee of Indiana University School of Medicine and the Methodist Research Institute, and conforms to the guidelines of the American Heart Association. We studied isolated canine RAs in 25 mongrel dogs (22 to 28 kg). The heart was rapidly excised under general anesthesia and the right coronary artery was perfused with 37°C Tyrode's solution equilibrated with 95% O2 and 5% CO2 to maintain a pH of 7.4. The composition of Tyrode's solution was (in mmol/L): 125 NaCl, 4.5 KCl, 0.25 MgCl2, 24 NaHCO3, 1.8 NaH2PO4, 1.8 CaCl2, and 5.5 glucose). The coronary perfusion pressure was regulated between 50 and 60 mmHg. To ensure adequate atrial perfusion, all ventricular coronary branches were tied off. Both ventricles and left atrium were removed. Contractility was inhibited by 10-17 μmol/L of blebbistatin,14 and the motion artifact was negligible even after ISO infusion (online supplement Figure S1). Pseudo-ECG was recorded with widely spaced bipolar RA electrodes using ISO-DAM8A (World precision instruments, FL, USA).

Dual Vm and Cai recordings

The hearts were stained with Rhod-2 AM and RH237 (Molecular Probes) and excited with laser light at 532 nm.15 Fluorescence was collected using 2 cameras (MiCAM Ultima, BrainVision, Tokyo, Japan) at 1 ms/frame and 100×100 pixels with spatial resolution of 0.35×0.35 mm2/pixel. After mapping baseline spontaneous beats, pharmacologic intervention was performed. ISO infusion (1 μmol/L) was used in 11 of 25 dogs, including 5 dogs in which we determined the ISO dose response of sinus rate. In 3 dogs, the ryanodine dose response of heart rate was evaluated. In same dogs, we also determined the ISO dose response of heart rate during ryanodine infusion of 3 μmol/L. In remaining 11 dogs, the pharmacologic interventions were as follows: caffeine (20 mmol/L, 2 ml) given as a bolus injection within 1 second (n=2), caffeine (0.2 and 0.5 mmol/L) continuous infusion (n=2) including a dog with caffeine (20 mmol/L) continuous infusion, ISO (1 μmol/L) to induce LDCAE and then followed by ryanodine (3 μmol/L) (n=2), ryanodine (10 μmol/L) plus thapsigargin (200 nmol/L) without and then with ISO infusion (1 μmol/L) (n=2), and ZD 7288 (3 μmol/L) without and then with ISO infusion (1 μmol/L) (n=3).

Histology and calcium handling protein assay

The tissues were fixed in 4% buffered formalin for one hour, followed by storage in 70% alcohol, processed routinely and stained with hematoxylin and eosin and with trichrome for histopathological studies. We also performed immunostaining of HCN4 with rabbit anti-HCN4 polyclonal antibody (Santa Cruz Inc) at 1:100 dilution. Microscopic examination was performed to confirm the correct localization of SAN.

Superior and inferior parts of SAN, and RA were collected in 4 dogs. SERCA2a and phospholamban (PLB) are detected by the anti-SERCA2a monoclonal antibody, 2A7A1, and the anti-PLB monoclonal antibody, 1F1, respectively.16 The SERCA2a/PLB ratio was evaluated. Antibody-binding protein bands are visualized by 125I-protein A and quantified with a Bio-Rad Personal Fx phosphorimager.

Data Analysis

“Sinus rate” was defined as the rate generated by sinus node activations confirmed with optical mapping. On the other hand, we used the term “heart rate” to describe the activation rate determined by pseudo-ECG tracings only. The Cai and Vm traces were normalized to their respective peak-to-peak amplitude for comparison of timing and morphology. The mean surface area of SAN was measured by the summation of pixel areas showing DD in Vm tracing (Figure 1A and B). The slopes of LDCAE (Figures 2 & 3) and DD during ISO infusion were measured from the onsets of LDCAE and DD (red arrows, Figure 3C) to peak levels of LDCAE and DD, respectively (broken arrows, Figure 3C). The onsets of LDCAE and DD were defined by the time of the transition between negative to positive values in dCai/dt and dVm/dt curves (arrows, Figure 3E). Linear regression was used to determine the correlation between sinus rate and the LDCAE or DD slopes. The “90% Cai relaxation time” was measured from the maximum systolic Cai to 90% reduction of Cai. Student's t-tests were used to compare means between two groups. Analyses of variance with Fisher's least significant difference post hoc test were used to compare the means among 3 and 4 groups. Data were presented as mean ± SEM. A P-value of < 0.05 was considered statistically significant.

Figure 1.

Figure 1

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Identification of SAN. A, Intact canine RA preparation. The white shaded area is the SAN. The numbers show the Vm recording sites in B. B, Vm (blue) recording of a spontaneous sinus beat. Vm was recorded in SAN (sites 1-4), inferior (site 5), posterior (sites 6-10), and anterior (sites 11-15) to SAN. Vm of SAN only shows typical phase 4 DD (arrows). C, Masson's trichrome staining of SAN and adjacent RA. The general outline of the SAN is indicated with the black arrows. RAA, RA appendage; IVC, inferior vena cava; SVC, superior vena cava; ST, sulcus terminalis; A, anterior; P, posterior; S, superior; I, inferior.

Figure 2.

Figure 2

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Activation pattern of SAN and surrounding RA during baseline spontaneous sinus beat. A, Isochronal map. The number on each isochronal line indicates the time of activation (ms), with the earliest activation as time zero. The white shaded area is the SAN. B, The Vm (blue) and Cai (red) recordings from the superior (a), middle (b), inferior (c) SAN and RA (d) presented in A. Arrows point to LDCAE. C, Magnified view of Cai and Vm tracings of inferior SAN. Note that LDCAE (arrow) occurred prior to phase 0 of AP (0 ms), and much earlier than the p wave on ECG. D, Cai and Vm ratio maps at times from −40 ms before to 20 ms after phase 0 AP of C.

Figure 3.

Figure 3

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Activation pattern of SAN and surrounding RA during ISO infusion of 0.3 μmol/L. A, Isochronal map of Vm. The number on the each isochronal line indicates time (ms). White shaded area is the SAN. B, The Vm (blue) and Cai (red) recordings from the superior (a), middle (b), inferior (c) SA nodes and RA (d) presented in A. C, Magnified view of Cai and Vm tracings of superior SAN. Note the robust LDCAE (solid arrow) before phase 0 of AP (0 ms), which in turn was much earlier than onset of p wave on ECG. D, The Vm and Cai ratio maps at times from −60 ms before to 180 ms after phase 0 AP of C. The LDCAE (broken arrows in frame −40 and −20 ms) was followed by the Cai sinkhole during early diastole (solid arrow in frame 180 ms). E, (a) Cai and dCai/dt. (b) Vm and dVm/dt. The onset of LDCAE and DD were marked with arrows.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Results

Identification of SAN

The baseline sinus rate was 95 ± 18 bpm (range 81-134 bpm). Because SAN is subepicardial in dogs,17 we mapped the epicardial side of the tissue (Figure 1A). The APs in the SAN (sites 1-4) exhibited spontaneous phase 4 DD (arrows in Figure 1B), whereas the RA (sites 5-15) did not. Sites with DD were typically located posterior to the sulcus terminalis and along the SAN artery (dashed line in Figure 1A). The mean surface area of SAN was 41.2 ± 7.7 mm2 (indicated by white colored area in Figure 1A). The correct identification of the SAN was confirmed by histological studies (Figure 1C) and by immunostaining of anti-HCN4 (online supplement Figure S2).

Figure 2A shows the isochronal map during spontaneous sinus rhythm. Activation in the Vm map was earliest in the anteroinferior region of the SAN (site c). Conduction velocity within SAN was slow, taking 21 ms for the impulse to travel from inferior SAN (dark blue line) to superior SAN (red line). The conduction velocity to the posterior sulcus terminalis was slower than to the anterior sulcus, as indicated by the crowded isochronal lines in the superior vena cava (SVC) direction. The upstrokes of Cai and Vm fluorescence were nearly simultaneous (Figures 2B & 2C), with the AP upstroke preceding the Cai transient upstroke by an average of 2.7 ± 2.0 ms. In comparison, during RA pacing, the AP upstroke preceded Cai transient upstroke by 11.6 ± 4.7 ms in the same area. Small amplitude LDCAEs were observed at inferior SAN in 4 out of 25 preparations during baseline recording (arrows in Figure 2B-D). In the remaining 21 preparations, no LDCAE was observed at baseline. Under basal conditions, the leading pacemaker sites were located in the inferior and middle SANs in 18 and 7 preparations, respectively.

The Effects of β-adrenergic stimulation

Robust LDCAE in SAN during ISO Infusion

ISO infusion resulted in an upward (cranial) shift of the earliest activation site to superior SAN (site a, Figure 3A), coincident with the appearance of robust LDCAEs (arrows in Figure 3B) in this region in all 11 preparations. The conduction time from superior SAN (site a) to inferior SAN (site c) was 10 ms. LDCAEs in the superior SAN preceded the phase 0 AP upstroke by 98.4 ± 31.0 ms (from red arrow to 0 ms in Figure 3C), and were associated with an acceleration of the rate of DD. In contrast, optical traces from the inferior SAN (site c) and RA (site d) showed no LDCAEs, with the Cai transient upstroke always occurring after the AP upstroke. The patterns of activation between −60 ms and 180 ms of Figure 3C is shown in Figure 3D. The figure shows that the superior SAN had the earliest elevation in Cai fluorescence (broken arrow, −40 ms, Figure 3D). This site also showed the most rapid recovery during late diastole (arrow, 180 ms, Figure 3D). Because the recovery of Cai fluorescence at superior SAN was relatively early, the optical map at 180 ms showed an area of low Cai fluorescence surrounded by areas of high Cai fluorescence (Cai sinkhole).

The Cai and Vm Characteristics Around the Leading Pacemaker Site

During ISO infusion, LDCAE and DD slopes were steepest at the leading pacemaker site (asterisks in Figure 4A) and progressively decreased towards the periphery of SAN. The LDCAE and DD were absent >3 mm away from the leading pacemaker site in the anterior and posterior directions (Figure 4B and D), but were present up to 5-6 mm inferior to the leading pacemaker site (Figure 4C and D). The LDCAE peak amplitude (arrow, Figure 4B) occurred before the AP upstroke. LDCAE onset was always the earliest at the leading pacemaker site and progressively delayed towards the periphery of SAN (online supplement Figure S3A). The slope of LDCAE (Figure 4D) was always higher at the leading pacemaker site than other SAN sites.

Figure 4.

Figure 4

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The spatial changes of Cai and Vm around the leading pacemaker site (*) in SAN. A, The Cai and Vm ratio maps showing recording site. B, The changes of Cai and Vm tracings along the anterior (A)-posterior (P) direction. C, The changes of Cai and Vm tracings along the superior (S)-inferior (I) direction. D, The spatial change of LDCAE and DD slopes.

Progressive Superior Shift of Both LDCAE and Leading Pacemaker Sites

There was progressive and concomitant upward shift of both the leading pacemaker site and peak LDCAE during ISO infusion (Figure 5A). At 95 bpm, for example, the sites 4 and 5 had most prominent LDCAEs (asterisks). When the sinus rate increased to 173 bpm, however, the site 2 had the most prominent LDCAE. These upward LDCAE shifts were observed in all hearts studied during ISO infusion, and always co-localized with the leading pacemaker site.

Figure 5.

Figure 5

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Co-localization of LDCAE and the leading pacemaker site. A, Upward shift of the leading pacemaker site with LDCAE during ISO infusion. (a) Cai ratio maps of SAN at each sinus rate. (b) Corresponding Cai tracings from superior (1, 2), middle (3, 4) and inferior (5, 6) SAN. At 95 bpm, the sites 4 and 5 had most prominent LDCAEs (asterisks). As sinus rate gradually increased, the sites of Cai elevation progressively moved upward. At the maximum sinus rate of 173 bpm, the site 2 had the most apparent LDCAE. B, Differential responses of different SAN sites to ISO. (a) The Cai and Vm tracings from inferior, middle, and superior SAN sites at different sinus rates. (b) The LDCAE and DD slopes of superior SAN at different sinus rates.

Differential Responses of Different SAN Sites to ISO Infusion

Figure 5Ba compares the Cai and Vm tracings recorded at inferior, middle, and superior SAN as ISO was increased from 0.01 to 1.0 μmol/L. When sinus rate was less than 100 bpm, the inferior SAN served as the pacemaking site, but did not show clear LDCAE. When the sinus rate progressively increased, LDCAE (asterisks) appeared in the middle and then superior regions as the pacemaking sites progressively moved upwards. The superior SAN served as the leading pacemaker site at sinus rates above 120 bpm. The LDCAE and DD slopes in the superior SAN showed significant positive correlation with the increase of sinus rate (Figure 5Bb). The LDCAE slope at the leading pacemaker site, which was located at different sites within the SAN during increasing doses of ISO infusion, was also well correlated with the increase of sinus rate (online supplement Figure S3B).

Typical Cai Dynamics of the Leading Pacemaker Sites during ISO Infusion

Figure 6A compares the morphologies of Cai tracing at SAN (sites 1-3) and RA (sites 4-5) during ISO infusion. There were no morphological differences of Cai traces between the leading pacemaker sites and other RA sites at baseline before ISO infusion (Figure 6Ab). After ISO infusion, the morphology of Cai trace at the leading pacemaker (site 1) was characterized not only by the earliest onset of LDCAE (*) but also by the fastest Cai reuptake (arrow) as compared with other RA sites. The RA sites 4-5, for example, showed no LDCAE and a slower Cai reuptake. The baseline 90% Cai relaxation time was 279 ± 70 ms, 300 ± 83, and 325 ± 67 ms at superior, inferior SAN and RA, respectively (p = 0.11). After ISO infusion, the 90% Cai relaxation time was shorter at superior SAN (124 ± 34 ms) than at inferior SAN (138 ± 24 ms, p = 0.01) and at other RA sites (164 ± 33 ms, p = 0.001, Figure 6B). The superior and inferior SAN were harvested and analyzed for total expression of SERCA2a and PLB after optical mapping studies. As shown in the immunoblot in Figures 6C, SERCA2a and PLB were present in both SAN and RA. The SERCA2a/PLB ratio of RA (1.81 ± 0.19) was higher than that of superior SAN (1.39 ± 0.16, p=0.006) and the inferior SAN (1.51 ± 0.15, p=0.03, Figure 6D).

Figure 6.

Figure 6

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The differences of Cai physiology between SAN and RA. A, (a) Cai tracing from superior (1), middle (2) and inferior (3) SAN, and from anterior (4) and posterior RA (5). (b) The Cai tracing during baseline (left panels) and ISO infusion of 1 μmol/L (right panels). The earliest activation sites were inferior (3) and superior SAN (1) during baseline and ISO infusion, respectively. Arrow points to a rapid reduction of Cai at the superior SAN, which was followed by rapid onset of LDCAE (asterisk). B, The comparison of 90% Cai relaxation time among superior, inferior SAN and RA during baseline and ISO infusion. C, The Western analyses of SERCA2a (SER) and phospholamban (PLB) at superior SAN (SN-S), inferior SAN (SN-I) and RA. D, The SERCA2a/PLB ratio of all preparations analyzed.

The Importance of SR Function on Pacemaking

Caffeine sensitizes the ryanodine receptor 2 to activation, resulting in increased SR Ca2+ release.10 High concentrations of caffeine will deplete the SR calcium store. Indeed, when a 2 ml caffeine bolus (20 mmol/L) was injected directly into the right coronary artery, LDCAE appeared in the superior SAN and the sinus rate increased by 84% in 2 hearts (online supplement Figure S4A). However, after 10-min continuous infusion, heart rate decreased and LDCAE was abolished. Further ISO infusion did not produced LDCAE (n=1). Continuous caffeine infusion at 0.2 mmol/L and 0.5 mmol/L did not produce LDCAE (n=2, online supplement Figure S5).

If the appearance of LDCAE faithfully tracks stimulation of the Ca2+ clock, then agents which interfere with SR Ca2+ cycling should suppress LDCAE. Figure 7A shows the dose response curve for ryanodine on SAN pacemaking rate (n=3). Because the activation rate changes rapidly during the procedure, it was not possible to document the origin of the atrial activation of every beat. Therefore, we have analyzed the heart rate, rather than sinus rate, based on pseudo-ECG recordings. Low doses of ryanodine (<0.3 μmol/L), which sensitize ryanodine receptors to Ca2+-induced Ca2+ release, resulted in a slight (< 10%) increase in heart rate. Higher doses of ryanodine, which block ryanodine receptors, caused a dose-dependent suppression of sinus node activity. The ISO dose-dependent increase of heart rate was suppressed by ryanodine infusion (Figure 7B). Concomitant with these effects on heart rate, ryanodine pretreatment completely prevented ISO-induced LDCAE (n=3, middle panel of Figure 7C). ISO-induced LDCAE was also abolished with ryanodine (n=2, online supplement Figure S4B).

Figure 7.

Figure 7

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The effect of ryanodine and ZD 7288 on intact canine SAN. A, Dose dependent decrease of heart rate with ryanodine infusion. B, The impaired ISO dose-dependent increase of heart rate by ryanodine infusion of 3 μmol/L. C, The Cai and Vm tracings during 3 μmol/L ryanodine alone infusion (left panels), concomitant 3 μmol/L ryanodine and 1 μmol/L ISO infusion (middle panels), and 10 μmol/L ryanodine, 200 nmol/L thapsigargin and 1 μmol/L ISO infusion (right panels). D, The Cai and Vm tracings during 3 μmol/L ZD 7288 infusion (left panels). ISO infusion of 1 μmol/L produced LDCAE (arrows) at the superior SAN in the presence of ZD 7288 (right panels). The Cai and Vm tracings were recorded from superior (1), middle (2) and inferior (3) SANs. Ryd, ryanodine; Thap, thapsigargin.

To fully suppress SR Ca2+ cycling, we also studied the effects of ryanodine (10 μmol/L) in combination with thapsigargin (200 nmol/L). After 30 min pretreatment, the mean heart rate decreased by 54% from 94 ± 4 bpm to 44 ± 7 bpm. Subsequent ISO infusion (1 μmol/L) increased the mean maximum heart rate to 112 ± 13 bpm (18% over the baseline rate before ryanodine and thapsigargin). The heart rate increase was not associated with LDCAE (n=2, right panel of Figure 7C).

In contrast to SR inhibitors, the If blocker ZD 7288 (3 μmol/L), which decreased the basal sinus rate by 8.3% (n=3), did not prevent ISO from increasing sinus rate by 40%, accompanied by the appearance of LDCAE in the superior SAN (Figure 7D).

Together, these findings provide strong support that in intact SAN tissue, LDCAE faithfully reports Ca2+ clock activity, as previously documented in isolated SAN myocytes.4-10

Effects of SR inhibition and If blockade on Sinus Rate and its Acceleration by ISO

If the voltage and Cai clocks act interdependently and synergistically to support each other in determining SAN rate, then the effects of blocking one clock or the other are likely to be complex and not necessarily very informative. This was the case, as shown in Figure 8 which summarizes the relative changes in heart rate caused by various pharmacologic agents under basal conditions and during ISO. Moreover, ryanodine and ryanodine plus thapsigargin often shifted the pacemaking site to outside of the SAN, so that the source of activation was ectopic beats from outside of the mapped region. Therefore, the heart rate changes might underestimate the sinus node suppression by SR inhibition and overestimate the sinus node responses to ISO. The exact magnitude of SAN suppression could not be determined due to the presence of competing ectopic rhythm.

Figure 8.

Figure 8

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Spontaneous heart rate of canine intact SAN depends upon both Ca2+-related mechanisms and If current. Bars show a change in heart rate (% from baseline) induced by different pharmacological interventions. The grey bars show the changes during 3 μmol/L ZD 7288, 3 μmol/L ryanodine, and 10 μmol/L ryanodine plus 200 nmol/L thapsigargin infusion without ISO, while the black bars show the changes during 1 μmol/L ISO infusion.

Discussion

Major Findings

The present study supports the importance of spontaneous SR Ca2+ release and the Ca2+ clock to pacemaking in the intact canine SAN. Key findings include the demonstration of a robust LDCAE and its correlation with sinus rate acceleration during β-adrenergic stimulation, the demonstration of LDCAE during caffeine-induced sinus rate acceleration, and the concomitant suppression of ISO-induced LDCAE and sinus rate acceleration by ryanodine and thapsigargin. In addition, sinus rate acceleration during ISO infusion was partially (but not completely) blocked by ZD 7288, a specific If current inhibitor. These findings have reproduced several key observations used to support the Ca2+ clock hypothesis in isolated SAN myocytes and in intact dogs.4-11 In addition, we have demonstrated that ISO infusion resulted in a superior shift of pacemaking site within the SAN, such that the leading pacemaking site always co-localized with the site with most robust LDCAE. Together, these findings support the conclusion that spontaneous SR Ca2+ release acts synergistically with activation of membrane ionic currents such as If, to accelerate the sinus rate in intact canine SAN during β-adrenergic stimulation.

Heterogeneous Cai dynamics in the SAN

Cells in different portions of the SAN exhibit a range of electrophysiological and Ca2+ handling characteristics.18-21 Mapping intact RA to study SAN function has the advantage that differential responses to β-adrenergic stimulation and other interventions can be characterized regionally within the SAN and compared to RA. LDCAEs were observed in only a small percentage of the preparations at baseline. One possible explanation of the latter finding is that the Ca2+ clock lagged behind the voltage clock in regulating DD under basal conditions. Alternatively, because each pixel in an optical map contains information from multiple cells, the effects of spatial averaging might have prevented us from documenting smaller spontaneous SR Ca2+ releases or Ca2+ releases from individual SAN cells. The importance of SR Ca2+ release on baseline heart rate is also supported by the finding that the impact of ryanodine and ryanodine plus thapsigargin on heart rate is much greater than the blockage of If current alone. However, LDCAE occurred in all preparations during ISO infusion, associated with a superior shift of the leading pacemaker site. The superior shift of LDCAE and the pacemaking site was also consistently observed with caffeine infusion. Most importantly, the site of maximum LDCAE slope always co-localized with the leading pacemaking site, suggesting a shift in which the voltage clock now lagged the Ca2+ clock. This observation indicates a strong association between LDCAE and pacemaking during β-adrenergic stimulation, and provides new insights into pacemaker hierarchy in the canine RA.12, 13

Mechanisms of Diastolic Depolarization

Multiple time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells which contribute to DD, including ICa-L, ICa-T, IST and various types of delayed rectifier K currents.22 Many of these membrane currents are known to respond to β adrenergic stimulation. Some of these currents, such as ICa-L, also promote LDCAE and the acceleration of sinus rate by the Ca2+ clock as well as the voltage clock. The interdependence and synergy between the two clocks is evident from the pharmacologic responses (Figure 8). If the two clocks were independent, with the faster clock driving the heart rate under a given condition, then blocking the slower clock should have no effect on SAN rate, whereas blocking the faster clock should decrease the SAN rate to that of the slower clock (which could be either markedly or only slightly slower than the faster clock). However, since both SR inhibitors and If blockade slowed sinus rate under basal conditions, as well as blunted the ISO-induced increase in sinus rate, the hypothesis of independent clocks can be excluded. This is not surprising, given that common ionic mechanisms, such as ICa-L are known to regulate both clocks. However, as the two clocks are interdependent, the relative potency of SR inhibition versus If blockade on slowing sinus rate in figure 8 becomes very difficult to interpret, since interdependency implies that inhibiting the SR will affect the voltage clock as well as the Ca2+ clock, and vice versa. The interpretation is further complicated by the presence of ectopic foci outside the mapped region during sinus node suppression. Thus, the presence or absence of LDCAE appears to be the most reliable indicator of which clock is predominantly driving the sinus rate under any given condition.

Cai Sinkhole and the Mechanisms of Pacemaking

Hwang et al15 previously reported that a Cai sinkhole in the postshock period is the site for the first postshock activation that reinitiates ventricular fibrillation. In the present study, we found that a Cai sinkhole is also present at the site of the earliest LDCAE and subsequent onset of the sinus activation. The formation of the Cai sinkhole was facilitated by a rapid decline (short relaxation time) of the Cai fluorescence at the superior SAN during ISO infusion. This finding suggests that Cai reuptake by SR is the fastest in the superior SAN. Because the uptake at superior SAN was the fastest, it resulted in a low Cai region surrounded by high Cai region and the formation of Cai sinkhole during early diastole (Figure 3D). More rapid and complete SR Ca2+ reuptake may ensure that the same site is the earliest to spontaneously release Ca2+ in the next cycle.

Differential SERCA2a/PLB Ratio at SAN and RA

The key protein regulator of Ca uptake is PLB, which inhibits SERCA2a in its dephosphorylated state. β-adrenergic stimulation phosphorylates PLB, reversing its inhibition of SERCA2a and increasing Ca2+ uptake. In canine hearts, the SERCA2a/PLB molar ratio is estimated at about 1:2, implying that at most only half of SERCA2a is inhibited by PLB in the basal state.23, 24 We demonstrated in the present study that there was a significantly lower SERCA2a/PLB ratio at SAN sites than at RA sites, suggesting more PLB molecules are available to regulate SERCA2a molecules in SAN than in RA. ISO infusion phosphorylates PLB and relieves PLB inhibition of SERCA2a, which may account for more robust Ca uptake in SAN than in RA during ISO infusion. However, we did not directly measure the phosphorylation state of PLB before and after ISO infusion to confirm this hypothesis.

Study limitations

An important concern in applying dual optical mapping of Vm and Cai to a thin slowly conducting tissue such as the intact SAN is whether the different dyes are imaging equivalent volumes of tissue, since the dyes have different tissue penetration and scattering properties.25 Since conduction velocity is very slow, this factor could distort the timing of the Ca2+ transient relative to the voltage transient if the respective fluorescence signals were being recorded from nonidentical tissue layers. However, this concern is somewhat mitigated by the same sites which did not exhibit LDCAEs under basal conditions developed LDCAE after ISO, despite identical imaging parameters. Although motion artifact after ISO could potentially create an artifact, as shown in online supplement Figure S1, we documented that motion artifact was negligible in this study. We did not directly measure the SR Ca2+ release. It is therefore possible that some components of LDCAE might have originated from the membrane Ca2+ currents. However, LDCAE were completely suppressed by drugs that inhibited SR function. Finally, pharmacologic agents such as ryanodine, thapsigargin and ZD 7288 are not completely selective, and nonspecific effects cannot be excluded.

Supplementary Material

1

Acknowledgements

We thank Lei Lin, Jian Tan, Juan Song and Stephanie Plummer for their assistance.

Funding Sources

This study was supported in part by NIH Grants P01 HL78931, R01 HL78932, 71140, a Korean Ministry of Information and Communication (MIC) and Institute for Information Technology Advancement (IITA) through research and develop support project (BJ), a Japan Medtronic fellowship (MM), Piansky Family Endowment (MCF), Pauline and Harold Price and Medtronic-Zipes Endowments (PSC) and by an AHA Established Investigator Award (SFL).

Footnotes

Disclosures

None.

References

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