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(Circulation Research. 2001;88:794.)
© 2001 American Heart Association, Inc.

Cellular Biology

ß-Adrenergic Stimulation Synchronizes Intracellular Ca²⁺ Release During Excitation-Contraction Coupling in Cardiac Myocytes

Long-Sheng Song, Shi-Qiang Wang, Rui-Ping Xiao, Harold Spurgeon, Edward G. Lakatta, Heping Cheng

From the Laboratory of Cardiovascular Sciences (L.-S.S., S.-Q.W., R.-P.X., H.S., E.G.L., H.C.), National Institute on Aging, National Institutes of Health, Baltimore, Md; and National Laboratory of Biomembrane and Membrane Biotechnology (H.C.), College of Life Sciences, Peking University, Beijing, China.

Correspondence to Heping Cheng, PhD, Laboratory of Cardiovascular Sciences, National Institute on Aging, NIH, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail chengp{at}grc.nia.nih.gov

	Abstract

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Abstract—To elucidate microscopic mechanisms underlying the modulation of cardiac excitation-contraction (EC) coupling by ß-adrenergic receptor (ß-AR) stimulation, we examined local Ca²⁺ release function, ie, Ca²⁺ spikes at individual transverse tubule–sarcoplasmic reticulum (T-SR) junctions, using confocal microscopy and our recently developed technique for release flux measurement. ß-AR stimulation by norepinephrine plus an ₁-adrenergic blocker, prazosin, increased the amplitude of SR Ca²⁺ release flux (J_SR), its running integral (J_SR), and L-type Ca²⁺ channel current (I_Ca), and it shifted their bell-shaped voltage dependence leftward by 10 mV, with the relative effects ranking I_Ca> J_SR>J_SR. Confocal imaging revealed that the bell-shaped voltage dependence of SR Ca²⁺ release is attributable to a graded recruitment of T-SR junctions as well as to changes in Ca²⁺ spike amplitudes. ß-AR stimulation increased the fractional T-SR junctions that fired Ca²⁺ spikes and augmented Ca²⁺ spike amplitudes, without altering the SR Ca²⁺ load, suggesting that more release units were activated synchronously among and within T-SR junctions. Moreover, ß-AR stimulation decreased the latency and temporal dispersion of Ca²⁺ spike occurrence at a given voltage, delivering most of the Ca²⁺ at the onset of depolarization rather than spreading it out throughout depolarization. Because the synchrony of Ca²⁺ spikes affects Ca²⁺ delivery per unit of time to contractile myofilaments, and because the myofilaments display a steep Ca²⁺ dependence, our data suggest that synchronization of SR Ca²⁺ release represents a heretofore unappreciated mechanism of ß-AR modulation of cardiac inotropy.

Key Words: excitation-contraction coupling • ß-adrenergic receptor • L-type Ca²⁺ channel current • ryanodine receptors • heart cells

	Introduction

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During cardiac excitation-contraction (EC) coupling, depolarization activates the voltage-gated L-type Ca²⁺ channels in the sarcolemmal membrane encompassing the transverse (T) tubules. The ensuing Ca²⁺ influx triggers Ca²⁺ release from ryanodine receptors (RyRs) in the junctional sarcoplasmic reticulum (SR) via Ca²⁺-induced Ca²⁺ release (CICR).¹ It is now widely accepted that Ca²⁺ sparks originating from a single RyR or a cluster of RyRs constitute elementary functional units of the SR Ca²⁺ release,² and that spatial and temporal summation of discrete Ca²⁺ sparks gives rise to the global Ca²⁺ transient,^{3 4} which in turn, activates contractile proteins to generate a contraction. Increasing evidence also suggests that Ca²⁺ sparks are under the exquisite local control of single L-type Ca²⁺ channels.^{3 4 5} Thus, the microscopic properties of the RyR in response to L-type channel Ca²⁺ currents are important determinants of the efficiency of EC coupling.

ß-Adrenergic receptor (ß-AR) stimulation by the sympathetic neurotransmitter norepinephrine (NE) and the adrenal hormone epinephrine plays a pivotal role in modulation of cardiac function in response to stress or exercise. Major Ca²⁺ cycling proteins involved in EC coupling, including L-type Ca²⁺ channels,^{6 7 8} RyRs,^{9 10} and the SR Ca²⁺-ATPase regulator phospholamban,^{11 12 13} are all known target proteins of ß-AR–adenylyl cyclase-cAMP–protein kinase A (PKA) signaling pathway. The resultant phosphorylation of these proteins increases the intracellular Ca²⁺ transient and contraction amplitudes, and it accelerates their kinetics. Despite extensive studies at the whole-cell level^{14 15 16 17 18} or on individual EC coupling components, such as L-type channels^{6 7 8} and Ca²⁺ sparks,^{17 19 20} the exact microscopic mechanisms underlying ß-AR–mediated modulation of EC coupling are still not well understood. For instance, although it has been well established that ß-AR stimulation increases the functional availability of L-type Ca²⁺ channels^{6 7} and alters the gating pattern of the channel,^{7 8} little is known about how these affect the coupling between the L-type Ca²⁺ channels and RyRs.

By using a high-affinity Ca²⁺ indicator, oregon green 488 BAPTA-5N (OG-5N), in conjunction with a slow Ca²⁺ buffer, EGTA, we have directly visualized local Ca²⁺ release at the single transverse tubule–sarcoplasmic reticulum (T-SR) junction level, ie, Ca²⁺ spikes, which may consist of one or a few Ca²⁺ sparks.²¹ In the present study, we intended to determine the ß-AR–mediated effect on spatial-temporal properties of SR Ca²⁺ release and on the relationship between the triggering L-type Ca²⁺ channel current (I_Ca) and the SR Ca²⁺ release flux (J_SR). Using Ca²⁺ spikes and spatially averaged SR Ca²⁺ release fluxes (J_SR) as direct readouts of EC coupling, we have demonstrated microscopic mechanisms of ß-AR modulation of excitation-induced Ca²⁺ release in cardiac myocytes. Specifically, we found that ß-AR stimulation enhances the J_SR by recruiting more functional T-SR junctions, by enhancing Ca²⁺ spike amplitude, and perhaps most importantly, by synchronizing the occurrence of Ca²⁺ spikes after excitation.

	Materials and Methods

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Confocal Ca²⁺ Imaging and Simultaneous Recording of I_Ca
Cardiac ventricular myocytes were isolated from adult Sprague-Dawley rats by using a standard enzymatic technique.²¹ Confocal Ca²⁺ imaging microscopy and simultaneous recording of whole-cell I_Ca were achieved by using the techniques previously reported.²¹ Patch pipette–filling solution containing 4 mmol/L EGTA was made by mixing the following two solutions (3:2): an EGTA-free solution containing (in mmol/L) CsCl 120, MgCl₂ 1.5, MgATP 5, NaCl 10, tetraethylammonium chloride 10, and HEPES 20 (pH 7.2 adjusted with CsOH); and a high-EGTA solution containing (in mmol/L): CsCl 100, MgCl₂ 1.5, MgATP 5, NaCl 10, tetraethylammonium chloride 10, EGTA 10, CaCl₂ 5, and HEPES 20 (pH 7.2 adjusted with CsOH). The Ca²⁺ indicator OG-5N hexapotassium salt (Molecular Probes) (1 mmol/L) was directly dissolved in the pipette solution. Tetrodotoxin (20 µmol/L) was included in the bath solution to avoid contamination of the sodium current. I_Ca and SR Ca²⁺ release were activated by 200-ms depolarizing pulses from a holding potential of -60 mV to test potentials at 10- or 15-second intervals.

Optical Measurement of SR Ca²⁺ Release Function
Global and localized SR Ca²⁺ release function were measured by using the OG-5N/EGTA method.²¹ Briefly, a slow, high-affinity, and nonfluorescent Ca²⁺ buffer, EGTA, was used to minimize the resident time of Ca²⁺ released into the cytosol, and a low-affinity, fast Ca²⁺ indicator, OG-5N, was chosen to optimize the detection of localized high [Ca²⁺] in release site microdomains. Because of its slow kinetics for Ca²⁺ association, EGTA at the concentration used is not expected to disturb, to a significant extent, Ca²⁺ signaling between L-type channels and RyRs in the T-SR junctions. This, in fact, has been evidenced by the observations that intracellular dialysis of up to 10 mmol/L EGTA has little effect on the release-dependent inactivation of I_Ca,²² and that the J_SR determined by this method²¹ is comparable with that derived by mathematical models from conventional Ca²⁺ transients.²³

Drug Delivery
NE (10^–6 mol/L) plus a selective ₁-adrenergic blocker, prazosin (10^–6 mol/L), or an L-type Ca²⁺ channel agonist, FPL64176 (FPL, 10^–5 mol/L), was locally delivered through a glass pipette positioned near the cell. In some experiments, caffeine (20 mmol/L, 1 second) was rapidly applied onto the cells by pressure-ejection through a pipette. In a subset of experiments, cells were loaded with fluo-3 AM (5 µmol/L for 15 minutes); the caffeine-releasable SR Ca²⁺ content was then assessed before and 4 minutes after ß-AR stimulation in quiescent cells or electric field–stimulated (1.0 Hz) cells.

Statistics
Data were reported as mean±SEM. Student’s t test or a paired t test was applied, when appropriate, to determine statistical differences. Differences between voltage-dependent curves were assessed by using ANOVA for repeated measures. A P value less than 0.05 was considered statistically significant.

	Results

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Measurements of Global and Single T-SR Junctional Ca²⁺ Release Function
In rat ventricular myocytes, the trigger Ca²⁺ signal, I_Ca, was elicited by membrane depolarization under whole-cell voltage-clamp conditions, and the evoked Ca²⁺ release flux from the SR, J_SR, was measured simultaneously with confocal microscopy by using the OG-5N/EGTA method²¹ (Figure 1A). The spatially averaged OG-5N fluorescence signal consists of two separable components: a spiky transient that is proportional to J_SR and a minor component that represents the running integral of J_SR (J_SR)²¹ (Figure 1B). For simplicity, a small contribution caused by the I_Ca influx (6% of the peak Ca²⁺ release from the SR²¹ ) was neglected in the data presentation. Linescan confocal imaging visualized discrete SR Ca²⁺ release events (Ca²⁺ spikes) at the Z line/T tubule regions that were identified by the OG-5N–stained bands separated by 1.8 µm (Figure 1A). It is noteworthy that the voltage pulse did not always elicit a Ca²⁺ spike from a T-SR junction. At T-SR junctions that fired one or more Ca²⁺ spikes (local F/F₀>0.2), the visualization of Ca²⁺ release flux in space and time makes it possible to measure the latency and peak amplitude of Ca²⁺ spikes (Figure 1C) and to determine how these are affected by ß-AR stimulation (see below).

View larger version (42K): [in this window] [in a new window]   Figure 1. Measurement of local and global SR Ca2+ release function. A, Representative image of OG-5N/EGTA fluorescence acquired at -20 mV in a rat ventricular myocyte. Left inset, Contrast-enhanced OG-5N staining at rest; bright bands correspond to Z line/T tubule regions. Discrete Ca2+ spikes ignited by membrane depolarization are localized to the center of Z line/T tubule regions (white lines). B, Spatially averaged OG-5N signal. The smooth line refers to the running integral of JSR (JSR); its steady-state level (FSS/F0) reflects the total amount of released Ca2+. C, SR Ca2+ release function at the single T-SR junction level. Vertical dashed lines mark the beginning and end of the voltage pulse. A T-SR junction is considered to be active if its OG-5N signal exceeds a threshold set at F/F0>0.2 (top horizontal dotted lines). Open circles mark the initiation of Ca2+ release at individual T-SR junctions.

ß-AR Stimulation Enhances SR Ca²⁺ Release Flux
Figure 2A illustrates typical simultaneous recordings of spatially averaged J_SR, its running integral J_SR, and the corresponding trigger I_Ca. Unlike conventional Ca²⁺ transients that last typically for 200 ms, the J_SR is rather brief (30 ms at 0 mV). After ß-AR stimulation by NE in the presence of the ₁-adrenergic blocker, prazosin, both I_Ca and J_SR are markedly enhanced (Figure 2A). Figures 2B through 2D plot the average voltage dependence of peak J_SR, J_SR, and I_Ca in the presence or absence of NE. Under control conditions, the peak J_SR and J_SR exhibit a bell-shaped voltage dependence, as does the trigger I_Ca. In the presence of the ß-AR agonist, all three curves are shifted upward and leftward by 10 mV, whereas the gross bell-shaped voltage dependence is retained. Interestingly, the three parameters are not equally affected by ß-AR stimulation, with a prominent effect on peak I_Ca, a modest effect on J_SR, and a rather small effect on J_SR (Figure 2). For example, at 0 mV, NE increased I_Ca, J_SR, and J_SR by 138%, 67%, and 25%, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 2. Effects of ß-AR stimulation on global SR Ca2+ release flux. A, ß-AR–mediated effects on spatially averaged JSR, its running integral JSR, and the corresponding trigger ICa in a representative myocyte. The cell was depolarized from a holding potential of -60 mV to between -40 and 40 mV in the absence (left) or presence (right) of ß-AR stimulation by NE (10-6 mol/L) plus an 1-adrenergic blocker prazosin (10–6 mol/L). B, C, and D show, respectively, average effects of ß-AR stimulation on JSR, JSR, and ICa. The rank order for the ß-AR–stimulated fold-increment is ICa>JSR>JSR. n=9 cells in the control group (), n=7 cells in the NE+Pra group (•). P<0.005 for ß-AR effects on ICa and JSR, P<0.05 for JSR.

ß-Adrenergic Modulation of Frequency and Intensity of Ca²⁺ Spikes
To elucidate microscopic mechanisms underlying ß-AR–mediated modulation of cardiac SR Ca²⁺ release, we examined the spatial and temporal recruitment of Ca²⁺ spikes as well as properties of individual Ca²⁺ spikes in response to ß-AR stimulation. As shown in Figure 3A, Ca²⁺ spikes are discernible at all test voltages, even when the SR release is at its maximum (eg, at 0 mV). Figures 3B and 3C illustrate, respectively, a profound bell-shaped voltage dependence for the likelihood of Ca²⁺ spike occurrence at a given T tubule, and a more shallow voltage dependence for the amplitude of Ca²⁺ spikes under control conditions. Hence, the characteristic bell-shaped voltage dependence of Ca²⁺ transients in cardiac myocytes is mostly attributable to a smoothly graded recruitment of T-SR junctions and, to a lesser extent, to changes in local Ca²⁺ release flux at individual T-SR release sites. The graded nature of Ca²⁺ spike amplitude, however, does suggest that a single T-SR junction is capable of firing a varying number of Ca²⁺ sparks. In this regard, Figure 3A shows that multiple Ca²⁺ spikes can be activated in tandem from a given T-SR site (marked by the arrows) during a 200-ms depolarizing pulse.

View larger version (70K): [in this window] [in a new window]   Figure 3. ß-AR stimulation modulates microscopic properties of Ca2+ spikes. A, Confocal images show that, after the perfusion of NE+Pra (4 minutes), more Ca2+ spikes are activated, Ca2+ spikes are ignited in a more synchronous fashion, and Ca2+ spikes become brighter. B, The fraction of active T-SR junctions () as a function of membrane voltage and its response to ß-AR stimulation. n=9 cells (total of 331 T-SR junctions ) in the control group, and n=7 cells (257 junctions ) in the NE+Pra group. C, Average ß-AR effect on Ca2+ spike amplitude as a function of membrane voltage.

Based on this perspective of excitation-induced Ca²⁺ release, we hypothesized that ß-AR–mediated enhancement of macroscopic SR Ca²⁺ release flux might be accounted for by the increased likelihood of Ca²⁺ spike occurrence, the intensity of individual Ca²⁺ spikes, or both. Figure 3B shows that, after ß-AR stimulation, the curve for fraction of active T-SR junctions () is shifted upward and exhibits a plateau with 95% between -30 and 0 mV. Figure 3C shows that the voltage dependence of the Ca²⁺ spike amplitude is also shifted upward and leftward by ß-AR stimulation. Nevertheless, the duration of Ca²⁺ spikes at 50% amplitude (t₅₀), which reflects local Ca²⁺ release time, was unaltered by NE treatment (at 0 mV, t₅₀ =18.05±0.79 ms, n=9 cells, versus 18.57±0.66 ms, n=7 cells, in the absence or presence of NE, respectively). Thus, both variations in fractional T-SR activation (synchronization among junctions) and spike amplitudes (synchronization of release units within a junction) contribute to the voltage-dependent gradation of SR Ca²⁺ release in the presence of ß-AR stimulation.

ß-AR Stimulation Reduces Latency and Temporal Dispersion of the Occurrence of Ca²⁺ Spikes
Next, we determined whether ß-AR stimulation alters the temporal profile of SR Ca²⁺ release among T-SR junctions. The images in Figure 3A indicate that Ca²⁺ spike occurrence is, indeed, substantially more synchronous among T-SR junctions in the presence of NE compared with that in the control cells. To quantitate the synchrony of Ca²⁺ spikes, we measured the latency of Ca²⁺ spike occurrence from the onset of the voltage pulse. The ensembles resulting from a large number of Ca²⁺ spikes at 4 representative voltages (-40, -30, 0, and 20 mV) are shown in Figure 4A. In the absence of NE and at low voltages (eg, -40 or -30 mV), Ca²⁺ spikes were scattered over the entire 200-ms pulse. In contrast, at 0 and 20 mV, the vast majority of Ca²⁺ spikes were concentrated in the first 30 ms. To quantify the temporal pattern of spike occurrence, the mean values of spike latency (L) are plotted against membrane voltage in Figure 4C, and the mean deviations of spike latency (L_md), which is inversely related to the temporal togetherness of Ca²⁺ spikes, are shown in Figure 4D. Both display a curved L-shaped voltage dependence. ß-AR stimulation by NE not only shortened the latency for spike activation (Figure 4C), but it also reduced the temporal dispersion of Ca²⁺ spikes, particularly at the negative voltages (Figure 4D). Thus, in addition to modulating Ca²⁺ spike recruitment and augmenting Ca²⁺ release at a given T-SR junction, ß-AR stimulation appears to front-load the system, delivering most of the Ca²⁺ at the onset of depolarization rather than spreading it out throughout depolarization.

View larger version (39K): [in this window] [in a new window]   Figure 4. ß-AR stimulation synchronizes the occurrence of Ca2+ spikes. A, Histogram of Ca2+ spike latency at different voltages. For a T-SR junction that fires multiple Ca2+ spikes, the latency is measured for the first event only. B, Traces of ICa averaged from the same cells. C and D, Mean latency (L) and its mean deviation (Lmd) of Ca2+ spikes before and after NE. Data are from 9 control cells and 7 cells exposed to NE. *P<0.05, **P<0.01.

The time courses of average I_Ca in the same cells are shown in Figure 4B. It is noteworthy that ß-AR signaling not only increases the amplitude, but it also hastens the decay of I_Ca, at negative potentials in particular (at -30 mV, _fast 10.25 and 8.61 ms; at 0 mV, _fast 11.96 and 9.34 ms, for control and NE, respectively). Because SR Ca²⁺ release is under tight local control by Ca²⁺ entry through L-type Ca²⁺ channels,^{3 4 5} the synchronization of I_Ca explains, at least in part, the ß-AR–mediated synchronization of SR Ca²⁺ release.

Effect of ß-AR Stimulation on SR Ca²⁺ Content
The ß-AR–simulated increase in SR Ca²⁺ release might also reflect an increase in SR Ca²⁺ content. To test this possibility, we examined the possible effect of ß-AR stimulation on the SR Ca²⁺ load. Figure 5A shows that, when the cell membrane potential was held at -60 mV, a pulse of caffeine (20 mmol/L, 1 second) applied by pressure-ejection rapidly empties the SR Ca²⁺ store; the J_SR indexed by the F/F₀ at the steady state is, on average, 2-fold greater than the depolarization-elicited release at 0 mV (Figures 2C and 5B). ß-AR stimulation by NE did not significantly alter the caffeine-releasable Ca²⁺ store under our experimental conditions (Figure 5B).

View larger version (34K): [in this window] [in a new window]   Figure 5. Effect of ß-AR stimulation on SR Ca2+ content. Cells were voltage-clamped at -60 mV, and a caffeine puff (20 mmol/L, 1 second) was delivered by pressure-ejection through a pipette located 100 µm away from the cell. A, Linescan image and line plot showing caffeine-induced OG-5N signal. The smooth line depicts the integral of released Ca2+, JSR. B, Average data on caffeine-induced total SR Ca2+ release before and after ß-AR stimulation. n=5 cells. C, Caffeine-induced SR Ca2+ release (F/F0) in a fluo-3–loaded cell in the absence of EGTA. D, Average data in quiescent (left) and field-stimulated (1.0 Hz, right) cells before and 4 minutes after NE perfusion.

However, previous studies have shown that ß-AR stimulation increases SR Ca²⁺ load.¹⁶ This discrepancy might be attributable to the inclusion of EGTA in our experiments. Alternatively, it could be the result of different stimulation/conditioning protocols used, because in the present study cells were electrically stimulated at a low frequency (10- to 15-second intervals), whereas in most previous studies cells were paced at higher frequencies or primed by conditioning pulses. To address these issues, we performed additional experiments to assess SR Ca²⁺ content in fluo-3–loaded cells in the absence of EGTA (Figure 5C). We found that NE increases SR Ca²⁺ in electrically stimulated (1.0 Hz) cells, but it has no significant effect in quiescent cells (Figure 5D). The latter is in agreement with our data obtained in EGTA-loaded, infrequently stimulated cells, and it is consistent with the observation that in saponin-permeabilized ventricular trabeculae the SR Ca²⁺ content is unchanged after isoproterenol stimulation.²⁴ These data thus reconcile our observations with the previous reports.^{16 24} Moreover, the present results suggest that sarcolemmal Ca²⁺ influx elicited by action potentials plays an important role in loading the SR during ß-adrenergic stimulation.

Reduction of the Gain of EC Coupling by ß-AR Signaling
The ability of the SR to amplify the trigger Ca²⁺ influx has been characterized by the gain function of EC coupling, defined as the ratio of peak J_SR over the corresponding I_Ca.²⁵ As shown in Figure 6, the SR gain function decreased monotonically with increasing voltage, in agreement with previous observations.^{5 25} Surprisingly, the gain of EC coupling was significantly reduced, rather than enhanced, after ß-AR stimulation by NE, despite a net increment of SR Ca²⁺ release. One possible explanation for this unexpected ß-AR–induced reduction in the gain of EC coupling may reside in local saturation of the trigger Ca²⁺ signal, caused by the increased L-type channel activity during ß-AR stimulation.^{6 7 8} To determine the possible effect of elevated L-type channel activity on the gain function, we used FPL (10^–5 mol/L), a specific L-type channel agonist, to increase channel-open probability,^{22 26} without directly affecting other ß-AR target proteins involved in the EC coupling cascade. Figure 7 shows that FPL rapidly induced a 4.2-fold increment in peak I_Ca, but only a 0.4-fold enhancement of peak J_SR, resulting in a markedly reduced gain of EC coupling. This supports the notion that local saturation of the trigger Ca²⁺ signal could explain, at least in part, the ß-AR–induced reduction in SR gain function.

View larger version (17K): [in this window] [in a new window]   Figure 6. ß-AR stimulation decreases the gain of EC coupling, the ratio JSR/ICa. The raw data of JSR were converted from the dimensionless F/F0 ratio to absolute Ca2+ flux by using the method by Song et al.21 Under control conditions, the gain monotonically decreases with increasing membrane voltage, in agreement with Wier et al.25 During ß-AR stimulation, the gain is significantly reduced. *P<0.05.

View larger version (19K): [in this window] [in a new window]   Figure 7. FPL differentially augments ICa and JSR. A, Time course of FPL effect on JSR and ICa. FPL (10–5 mol/L) markedly increases ICa by 2.7-fold and the peak Ca2+ release flux, JSR, by 0.33-fold in a single episode of voltage protocol (ie, <15 seconds). At steady state, the ICa and JSR are enhanced by 4.6- and 0.67-fold, respectively. B, Average results (n=7). Note the disparity between the FPL effects on ICa and JSR.

	Discussion

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There are three major findings of the present study. First, using Ca²⁺ spikes and J_SR as the immediate readouts of I_Ca-elicited SR Ca²⁺ release, we showed that ß-AR stimulation by NE increases the functional availability of T-SR junctions (), ie, the fraction of T-SR junctions that fire Ca²⁺ spikes during a 200-ms depolarization pulse. Second, we demonstrated that ß-AR stimulation increases the likelihood of RyRs firing synchronously at a given T-SR junction, augmenting Ca²⁺ spike amplitude without affecting its duration. Third, we identified a heretofore unappreciated mode of ß-AR modulation of EC coupling, ie, synchronization of SR Ca²⁺ release to the onset of depolarization at a given voltage. Specifically, ß-AR stimulation abbreviates the latency of Ca²⁺ spikes and markedly reduces the temporal dispersion of spike events among T-SR junctions. The ß-AR–mediated effects on Ca²⁺ spikes provide a subcellular perspective for the well documented ß-AR modulation of the whole-cell Ca²⁺ transients^{14 15 16 17 18} .

ß-AR–Mediated Synchronization of SR Ca²⁺ Release and Its Physiological Significance
As we reported previously, synchronization of intracellular Ca²⁺ release provides a novel mechanism to enhance cardiac inotropic performance.²¹ The unusually steep Ca²⁺-dependence of cardiac muscle force generation (Hill coefficient 4.87 as in Gao et al²⁷ ) implies that any slight change in Ca²⁺ delivery per unit of time would be amplified in the output of force generation and cell shortening. Because SR release occurs in a discrete manner, spatial synchronization of Ca²⁺ release also plays an important role. In an extreme scenario, it has been shown that solitary Ca²⁺ sparks fail to produce any local movement.²⁸ For multiple Ca²⁺ sparks not overlapping in space and time, they may also be ineffective in initiating local or global cell shortening. Indeed, at -30 mV, when spikes are dyscoordinated in space and time ( =0.57, L_md=37 ms, Figure 4), cell shortening is barely detectable.^{29 30} Unsynchronized Ca²⁺ release at more negative voltages is totally futile in terms of activating cell shortening. At -10 mV, however, when a similar amount of J_SR is discharged in a more uniform ( =0.87) and synchronous (L_md=14 ms) manner (Figure 4), cell contraction amplitude reaches its maximum, as shown in previous studies.^{29 30} Finally, asynchronous SR Ca²⁺ release might create microdomains of refractoriness, hindering the development of a forceful contraction. A slow and inhomogeneous Ca²⁺ release might also be counterbalanced by Ca²⁺ clearance from the cytosol as a result of SR Ca²⁺ resequestration and local Ca²⁺ buffering, limiting the peak Ca²⁺ and peak contraction attained.

The present study demonstrated, for the first time, that ß-AR stimulation provides a physiological mechanism to modulate the degree of synchronization of SR Ca²⁺ release (Figures 3 and 4). By enhancing the synchrony of Ca²⁺ release, ß-AR stimulation may facilitate the highly cooperative action of the released Ca²⁺ on the contractile apparatus, eliciting a greater contraction for a given Ca²⁺ release. Thus, a given level of force development would require less SR Ca²⁺ cycling and its associated energy utilization.

Alterations in the synchrony of SR Ca²⁺ release and its physiological modulation by ß-AR stimulation may have important pathophysiological relevance. In failing hearts of humans and animal models, diminished Ca²⁺ transient and contraction are often characterized by sluggish onset and relaxation.^{19 31 32} In this regard, it has been recently reported that dyssynchronous Ca²⁺ sparks are elicited by action potentials in myocytes from infarcted canine hearts.³³ Furthermore, the ability of ß-AR stimulation to restore the synchrony of SR Ca²⁺ release may also be diminished, as a result of the receptor downregulation and desensitization.³⁴ Thus, the possible contribution of dyssynchronization of intracellular Ca²⁺ release to cardiac contractile dysfunction in the failing heart merits future investigation.

Possible Mechanisms Underlying ß-AR–Mediated Synchronization of SR Ca²⁺ Release
One possible mechanism for ß-AR–mediated synchronization of SR Ca²⁺ release is related to ß-AR modulation of the trigger I_Ca. At the single-channel level, ß-AR modulation increases the functional availability of the L-type channel,⁶ redistributes the channel from mode 0 gating (characterized by infrequent brief openings) toward mode 1 (bursts of brief openings) and mode 2 (very long-lasting openings) gating, without changing I_Ca amplitude.^{7 8} At the whole-cell level, these effects translate into an increased I_Ca amplitude and accelerated activation and inactivation (Figure 4B). Interestingly, the time course of J_SR essentially mirrors the waveform of I_Ca, suggesting that the change in I_Ca kinetics largely accounts for ß-AR–induced synchronization of Ca²⁺ spikes.

The second possible mechanism may be related to a use-dependent inactivation of RyRs in intact cardiac myocytes.³⁵ During a single pulse, unfired RyRs are expected to be exhausted in a time-dependent manner. In response to ß-AR stimulation, increased trigger I_Ca would enhance the early SR Ca²⁺ release, which in turn, suppresses the late occurrence of Ca²⁺ spikes, reducing both L and L_md of Ca²⁺ spikes. It is noteworthy that RyR inactivation per se may not be altered by ß-AR stimulation, because the duration of individual Ca²⁺ spikes (18 ms) remains constant in the absence and presence of NE. The recovery of RyRs from inactivation, assessed by restitution of J_SR, is also unaffected by ß-AR stimulation by isoproterenol.³⁶

In principle, an increase in CICR sensitivity to I_Ca would also promote an early activation of Ca²⁺ spikes and a leftward shift of the spike latency histogram, which would contribute to the synchronization effect. However, it remains controversial whether and how ß-AR– or PKA-dependent phosphorylation of RyRs modulates the gating properties of the Ca²⁺ release channel in vitro and in vivo.^{10 17 20 37}

ß-AR Modulation of the Coupling Efficiency Between L-Type Channels and RyRs
Unexpectedly, we documented a reduction of the gain of EC coupling in response to ß-AR stimulation (Figure 6). This observation is in contrast to previous observations that a small I_Ca elicits a maximal Ca²⁺ transient in isoproterenol-stimulated cells.¹⁶ This discrepancy may be explained by different experimental conditions. In the previous study, a train of conditioning pulses (300 ms, 0 mV) was used to load the SR, resulting in an essentially all-or-none behavior. In the present experimental setting, however, the SR Ca²⁺ load is unaltered by NE, and the inclusion of EGTA makes it possible to dissect the J_SR directly triggered by I_Ca and to examine its response to ß-AR stimulation.

Because cardiac RyRs are under tight local control of single L-type Ca²⁺ channels, a subtle change in microscopic properties of the L-type channel may have profound consequences on the efficiency of EC coupling. The increase in L-type channel availability and the shift of the channel gating to high-activity mode by ß-AR stimulation^{6 7 8} would increase the cumulative L-type channel-open duration in a given T-SR junction, which may cause local saturation of the trigger Ca²⁺ signal. Numerical analysis suggests that normal L-type channel openings (mean open time 0.3 ms³⁸ ) provide a near-optimal trigger, whereas more sustained L-type channel openings are less effective, as they carry excess local Ca²⁺ influx.³⁹ Indeed, increasing L-type channel-open time by FPL markedly reduces the efficiency of I_Ca to trigger SR Ca²⁺ release (Figure 7). It is also noteworthy that the ß-AR–induced reduction in the gain function occurs predominantly at the low voltages (Figure 6), as if a greater unitary current, I_Ca, makes the local trigger Ca²⁺ more readily saturated.

It is noteworthy that SR Ca²⁺ content remained unchanged during ß-AR stimulation in our experiments, and this may obscure SR Ca²⁺ content-mediated changes in the gain of CICR (eg, in paced cells). The CICR between different release units was likely prohibited by the inclusion of millimolar concentrations of exogenous Ca²⁺ buffers. These, in effect, are advantageous with respect to dissecting ß-AR effects on coupling between L-type channels and RyRs.

In summary, the present study demonstrates that ß-AR stimulation in cardiac myocytes synchronizes SR Ca²⁺ release among T-SR junctions, reducing the latency and the temporal dispersion of Ca²⁺ spike occurrence. ß-AR stimulation also promotes synchronous activation of more release units within a given T-SR junction, augmenting Ca²⁺ spike amplitude without altering the SR Ca²⁺ load or spike duration. Both contribute to the increase in J_SR amplitude. The more synchronous delivery of Ca²⁺ to myofilaments in the presence of ß-AR stimulation likely facilitates the cooperative interaction of Ca²⁺ to activate contraction. Thus, synchronization of intracellular Ca²⁺ release constitutes a newly discovered mechanism underlying ß-adrenergic modulation of cardiac SR Ca²⁺ release and EC coupling.

	Acknowledgments

 
This work was supported by National Institutes of Health intramural grants (R.-P.X., E.G.L., and H.C.) and by grants from Major State Basic Research Development Program and National Natural Science Foundation of China (H.C.).

	Footnotes

 
Original received November 9, 2000; revision received February 23, 2001; accepted March 20, 2001.

	References

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