β-Adrenergic Stimulation Synchronizes Intracellular Ca2+ Release During Excitation-Contraction Coupling in Cardiac Myocytes
Abstract—To elucidate microscopic mechanisms underlying the modulation of cardiac excitation-contraction (EC) coupling by β-adrenergic receptor (β-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. β-AR stimulation by norepinephrine plus an α1-adrenergic blocker, prazosin, increased the amplitude of SR Ca2+ release flux (JSR), its running integral (∫JSR), and L-type Ca2+ channel current (ICa), and it shifted their bell-shaped voltage dependence leftward by ≈10 mV, with the relative effects ranking ICa> JSR>∫JSR. 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. β-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, β-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 β-AR modulation of cardiac inotropy.
- excitation-contraction coupling
- β-adrenergic receptor
- L-type Ca2+ channel current
- ryanodine receptors
- heart cells
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.
β-Adrenergic receptor (β-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 β-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 level14 15 16 17 18 or on individual EC coupling components, such as L-type channels6 7 8 and Ca2+ sparks,17 19 20 the exact microscopic mechanisms underlying β-AR–mediated modulation of EC coupling are still not well understood. For instance, although it has been well established that β-AR stimulation increases the functional availability of L-type Ca2+ channels6 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 β-AR–mediated effect on spatial-temporal properties of SR Ca2+ release and on the relationship between the triggering L-type Ca2+ channel current (ICa) and the SR Ca2+ release flux (JSR). Using Ca2+ spikes and spatially averaged SR Ca2+ release fluxes (JSR) as direct readouts of EC coupling, we have demonstrated microscopic mechanisms of β-AR modulation of excitation-induced Ca2+ release in cardiac myocytes. Specifically, we found that β-AR stimulation enhances the JSR 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
Confocal Ca2+ Imaging and Simultaneous Recording of ICa
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 ICa 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. ICa 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 ICa,22 and that the JSR determined by this method21 is comparable with that derived by mathematical models from conventional Ca2+ transients.23
NE (10–6 mol/L) plus a selective α1-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 β-AR stimulation in quiescent cells or electric field–stimulated (1.0 Hz) cells.
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.
Measurements of Global and Single T-SR Junctional Ca2+ Release Function
In rat ventricular myocytes, the trigger Ca2+ signal, ICa, was elicited by membrane depolarization under whole-cell voltage-clamp conditions, and the evoked Ca2+ release flux from the SR, JSR, 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 JSR and a minor component that represents the running integral of JSR (∫JSR)21 (Figure 1B⇓). For simplicity, a small contribution caused by the ICa 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 ΔF/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 β-AR stimulation (see below).
β-AR Stimulation Enhances SR Ca2+ Release Flux
Figure 2A⇓ illustrates typical simultaneous recordings of spatially averaged JSR, its running integral ∫JSR, and the corresponding trigger ICa. Unlike conventional Ca2+ transients that last typically for ≈200 ms, the JSR is rather brief (≈30 ms at 0 mV). After β-AR stimulation by NE in the presence of the α1-adrenergic blocker, prazosin, both ICa and JSR are markedly enhanced (Figure 2A⇓). Figures 2B⇓ through 2D plot the average voltage dependence of peak JSR, ∫JSR, and ICa in the presence or absence of NE. Under control conditions, the peak JSR and ∫JSR exhibit a bell-shaped voltage dependence, as does the trigger ICa. In the presence of the β-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 β-AR stimulation, with a prominent effect on peak ICa, a modest effect on JSR, and a rather small effect on ∫JSR (Figure 2⇓). For example, at 0 mV, NE increased ICa, JSR, and ∫JSR by 138%, 67%, and 25%, respectively.
β-Adrenergic Modulation of Frequency and Intensity of Ca2+ Spikes
To elucidate microscopic mechanisms underlying β-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 β-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.
Based on this perspective of excitation-induced Ca2+ release, we hypothesized that β-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 β-AR stimulation, the curve for fraction of active T-SR junctions (ε) is shifted upward and exhibits a plateau with ε ≈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 β-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 β-AR stimulation.
β-AR Stimulation Reduces Latency and Temporal Dispersion of the Occurrence of Ca2+ Spikes
Next, we determined whether β-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 (Lmd), which is inversely related to the temporal togetherness of Ca2+ spikes, are shown in Figure 4D⇓. Both display a curved L-shaped voltage dependence. β-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, β-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.
The time courses of average ICa in the same cells are shown in Figure 4B⇑. It is noteworthy that β-AR signaling not only increases the amplitude, but it also hastens the decay of ICa, at negative potentials in particular (at −30 mV, τfast 10.25 and 8.61 ms; at 0 mV, τfast 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 ICa explains, at least in part, the β-AR–mediated synchronization of SR Ca2+ release.
Effect of β-AR Stimulation on SR Ca2+ Content
The β-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 β-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 ∫JSR indexed by the ΔF/F0 at the steady state is, on average, 2-fold greater than the depolarization-elicited release at 0 mV (Figures 2C⇑ and 5B⇓). β-AR stimulation by NE did not significantly alter the caffeine-releasable Ca2+ store under our experimental conditions (Figure 5B⇓).
However, previous studies have shown that β-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 β-adrenergic stimulation.
Reduction of the Gain of EC Coupling by β-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 JSR over the corresponding ICa.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 β-AR stimulation by NE, despite a net increment of SR Ca2+ release. One possible explanation for this unexpected β-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 β-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 β-AR target proteins involved in the EC coupling cascade. Figure 7⇓ shows that FPL rapidly induced a 4.2-fold increment in peak ICa, but only a 0.4-fold enhancement of peak JSR, 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 β-AR–induced reduction in SR gain function.
There are three major findings of the present study. First, using Ca2+ spikes and JSR as the immediate readouts of ICa-elicited SR Ca2+ release, we showed that β-AR stimulation by NE increases the functional availability of T-SR junctions (ε), ie, the fraction of T-SR junctions that fire Ca2+ spikes during a 200-ms depolarization pulse. Second, we demonstrated that β-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 β-AR modulation of EC coupling, ie, synchronization of SR Ca2+ release to the onset of depolarization at a given voltage. Specifically, β-AR stimulation abbreviates the latency of Ca2+ spikes and markedly reduces the temporal dispersion of spike events among T-SR junctions. The β-AR–mediated effects on Ca2+ spikes provide a subcellular perspective for the well documented β-AR modulation of the whole-cell Ca2+ transients14 15 16 17 18 .
β-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 (ε =0.57, Lmd=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 ∫JSR is discharged in a more uniform (ε =0.87) and synchronous (Lmd=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 β-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, β-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 β-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 β-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 β-AR–Mediated Synchronization of SR Ca2+ Release
One possible mechanism for β-AR–mediated synchronization of SR Ca2+ release is related to β-AR modulation of the trigger ICa. At the single-channel level, β-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 ICa amplitude.7 8 At the whole-cell level, these effects translate into an increased ICa amplitude and accelerated activation and inactivation (Figure 4B⇑). Interestingly, the time course of JSR essentially mirrors the waveform of ICa, suggesting that the change in ICa kinetics largely accounts for β-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 β-AR stimulation, increased trigger ICa would enhance the early SR Ca2+ release, which in turn, suppresses the late occurrence of Ca2+ spikes, reducing both L and Lmd of Ca2+ spikes. It is noteworthy that RyR inactivation per se may not be altered by β-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 JSR, is also unaffected by β-AR stimulation by isoproterenol.36
In principle, an increase in CICR sensitivity to ICa 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 β-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
β-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 β-AR stimulation (Figure 6⇑). This observation is in contrast to previous observations that a small ICa 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 JSR directly triggered by ICa and to examine its response to β-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 β-AR stimulation6 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 ICa to trigger SR Ca2+ release (Figure 7⇑). It is also noteworthy that the β-AR–induced reduction in the gain function occurs predominantly at the low voltages (Figure 6⇑), as if a greater unitary current, ICa, makes the local trigger Ca2+ more readily saturated.
It is noteworthy that SR Ca2+ content remained unchanged during β-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 β-AR effects on coupling between L-type channels and RyRs.
In summary, the present study demonstrates that β-AR stimulation in cardiac myocytes synchronizes SR Ca2+ release among T-SR junctions, reducing the latency and the temporal dispersion of Ca2+ spike occurrence. β-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 JSR amplitude. The more synchronous delivery of Ca2+ to myofilaments in the presence of β-AR stimulation likely facilitates the cooperative interaction of Ca2+ to activate contraction. Thus, synchronization of intracellular Ca2+ release constitutes a newly discovered mechanism underlying β-adrenergic modulation of cardiac SR Ca2+ release and EC coupling.
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.).
Original received November 9, 2000; revision received February 23, 2001; accepted March 20, 2001.
- © 2001 American Heart Association, Inc.
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