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Constitutive ₂-adrenergic signalling enhances sarcoplasmic reticulum Ca²⁺ cycling to augment contraction in mouse heart

Ying-Ying Zhou, Long-Sheng Song, Edward G. Lakatta, Rui-Ping Xiao and Heping Cheng

MS 9779 Received 25 June 1999; accepted after revision 9 September 1999.

	ABSTRACT

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1. Transgenic overexpression of the ₂-adrenergic receptor (₂AR) in mouse heart augments baseline cardiac function in a ligand-independent manner, due to the presence of spontaneously active ₂AR (₂AR*). This study aims to elucidate the mechanism of ₂AR*-mediated modulation of cardiac excitation-contraction (EC) coupling.

2. Confocal imaging was used to analyse Ca²⁺ sparks and spatially resolve Ca²⁺ transients in single ventricular myocytes from transgenic (TG4) and non-transgenic (NTG) littermates. Whole-cell voltage- and current-clamp techniques were used to record L-type Ca²⁺ currents (I_Ca) and action potentials, respectively.

3. In the absence of any ₂AR ligand, TG4 myocytes had greater contraction amplitudes, larger Ca²⁺ transients and faster relaxation times than did NTG cells.

4. The action potentials of TG4 and NTG myocytes were similar, except for a prolonged end-stage repolarization in TG4 cells; the I_Ca density and kinetics were nearly identical. The relationship between peak Ca²⁺ and contraction, which reflects myofilament Ca²⁺ sensitivity, was similar.

5. In TG4 cells, the frequency of Ca²⁺ sparks (spontaneous or evoked at -40 mV) was 2-7 times greater, despite the absence of change in the resting Ca²⁺, sarcoplasmic reticulum (SR) Ca²⁺ content, and I_Ca. Individual sparks were brighter, broader and lasted longer, leading to a 2·3-fold greater signal mass. Thus, changes in both spark frequency and size underlie the greater Ca²⁺ transient in TG4 cells.

6. The inverse agonist ICI 118,551 (ICI, 5 × 10^-7 M), which blocks spontaneous ₂AR activation, reversed the aforementioned ₂AR* effects on cardiac EC coupling without affecting the sarcolemmal I_Ca. However, ICI failed to detect significant constitutive ₂AR activity in NTG cells.

7. We conclude that ₂AR*-mediated signalling enhances SR release channel activity and Ca²⁺-induced Ca²⁺ release in TG4 cardiac myocytes, and that ₂AR* enhances EC coupling by reinforcing SR Ca²⁺ cycling (release and reuptake), but bypassing the sarcolemmal I_Ca.

	INTRODUCTION

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Excitation-Ca²⁺ release coupling is a pivotal event in cardiac function. During membrane depolarization, sarcolemmal Ca²⁺ influx through the voltage-operated L-type Ca²⁺ channels triggers the sarcoplasmic reticulum (SR) to release Ca²⁺ via the Ca²⁺-induced Ca²⁺ release (CICR) mechanism (Fabiato, 1983). The resultant cytosolic Ca²⁺ transient activates myofilament proteins and initiates contraction. Cardiac relaxation ensues when the elevated cytoplasmic Ca²⁺ is resequestered by the SR Ca²⁺ pump, or, to a lesser extent, extruded via the sarcolemmal Na⁺-Ca²⁺ exchanger. In order to meet the body's variable circulatory demands, cardiac excitation-contraction (EC) coupling is constantly modulated by an array of physiological mechanisms. Driven by sympathetic neurotransmitters and adrenal hormones, -adrenergic receptor (AR) signalling interacts with virtually all constituents of the EC coupling cascade, including the L-type Ca²⁺ channel current (I_Ca) (Tsien et al. 1986; Xiao & Lakatta, 1993), SR Ca²⁺ release channels/ryanodine receptors (RyRs) (Yoshida et al. 1992; Valdivia et al. 1995), phospholamban (PLB) (Lindemann et al. 1983; Kuschel et al. 1999a), and contractile myofilaments (Rapundalo et al. 1989; Kuschel et al. 1999b). Additionally, -adrenergic stimulation may modulate other sarcolemmal ionic currents, such as K⁺ currents (Scamps, 1996), and electrogenic transporters, such as Na⁺-Ca²⁺ exchanger (in non-mammalian species) (Shuba et al. 1998), to alter the action potential configuration, and subsequently, the Ca²⁺ fluxes during a cardiac cycle.

Although both ₁AR and ₂AR stimulation enhance cardiac contractility, recent studies have revealed several features unique to ₂AR subtype signal transduction. Specifically, while ₁AR couples exclusively to stimulatory G protein (G_s), agonist-occupied ₂AR in several species dually couples to G_s and inhibitory G protein (G_i), generating cross-talk between the two signalling pathways through a single receptor (Xiao et al. 1995, 1999; Kuschel et al. 1999a). As a result, ₂AR signalling is uncoupled from phosphorylation of cytoplasmic and SR proteins (in rat and dog), and induces a less potent relaxant effect (in rat) (Xiao et al. 1994; Altschuld et al. 1995; Kuschel et al. 1999a,b). The contractile response to ₂AR signalling depends largely, if not exclusively, on an augmentation of I_Ca (Xiao & Lakatta, 1993; Zhou et al. 1997). In an extreme case, potent ₂AR-G_i signalling in the mouse heart fully negates the concurrent ₂AR-G_s signalling in most mouse ventricular myocytes, such that a contractile response is unmasked only if the G_i function is inhibited (Xiao et al. 1999).

Current theories predict that, in addition to agonist-induced activation, receptors can undergo spontaneous conformational changes, resulting in ligand-independent activity. Since these spontaneously activated receptors are scarce (see Samama et al. 1993 for constitutively active mutants), their functional manifestation and significance have not been appreciated until recently. In TG4 transgenic mice, which have 200-fold overexpression of wild-type human ₂ARs, the existence of ligand-independent, spontaneously activated ₂AR (₂AR*) has been convincingly documented in the intact animal, in isolated atria, and in single myocytes (Milano et al. 1994; Bond et al. 1995; Du et al. 1996; Xiao et al. 1999). In retrospect, the observation that ₂AR* induces a robust inotropic effect is rather surprising, because our recent report illustrated that ligand-occupied ₂AR is usually quiescent in both TG4 and non-transgenic (NTG) littermates due to the G_i coupling (Xiao et al. 1999). These results indicate that, unlike liganded ₂AR, ₂AR* modulates EC coupling in a distinctly different fashion.

The present study aims to elucidate the cellular mechanism(s) that underlies ₂AR*-mediated contractile responses. We systematically characterized the major components of EC coupling, including action potential, I_Ca, SR Ca²⁺ content, the elementary SR Ca²⁺ release events (Ca²⁺ sparks), Ca²⁺ transients and cell contraction in ventricular myocytes from TG4 and NTG mice. The present findings reveal profound modulatory effects of ₂AR* on both CICR sensitivity and SR Ca²⁺ reuptake, in contrast to the lack of effect on I_Ca. These results suggest a novel mode of cardiac inotropic regulation: an enhanced SR Ca²⁺ cycling mediates the positive inotropic effect, while the sarcolemmal I_Ca and myofilament Ca²⁺ sensitivity remain unchanged.

	METHODS

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Cell isolation

This investigation conforms to NIH guidelines on the care and use of animals. Transgenic mice with ₂AR 200-fold overexpression (TG4) and their non-transgenic littermates (NTG) were generously provided by Drs Walter J. Koch and Robert J. Lefkowitz (Duke University Medical Center, Durham, NC, USA). Single murine cardiac myocytes were isolated from the hearts of 2- to 3-month-old mice using a standard enzymatic technique (Korzick et al. 1997). Briefly, mice were anaesthetized by intraperitoneal injection with 100 mg kg^-1 sodium pentobarbitone. After all reflex activity had ceased, the chest was opened and the heart quickly removed and retrogradely perfused with collagenase B and protease using the Langendorff method. Cells were then shaken loose from the minced heart and suspended in Hepes-buffered solution consisting of (mM): CaCl₂ 1, NaCl 137, KCl 5·4, dextrose 15, MgSO₄ 1·3, NaH₂PO₄ 1·2, Hepes 20; pH 7·4 adjusted with NaOH.

Confocal microscopy

Cells were placed on the stage of a Zeiss LSM-410 inverted confocal microscope (Carl Zeiss, Inc., Germany) and excited by the 488 nm line of an argon laser. In whole-cell patch-clamp experiments, cells were dialysed with 100 µM fluo-3 pentapotassium salt (Molecular Probes) via a patch pipette. In experiments using field electrical stimulation, fluo-3 loading was achieved by a 10 min incubation of the myocytes in Hepes buffer containing 10 µM of the acetoxymethyl ester form fluo-3 AM (Molecular Probes), dissolved in DMSO, followed by a 15 min wash in fresh Hepes buffer (Cheng et al. 1993).

All images were taken in line-scan mode, with the scan line usually oriented along the long axis of the myocyte, avoiding the nucleus or nuclei of the cell. Each image consisted of 512 line-scan images 2·09-4·38 ms apart, and each line consisted of 512 pixels spaced at 0·10-0·25 µm intervals. The microscope was equipped with a Zeiss Plan-Neofluar × 40 oil immersion objective lens (NA, 1·3). The confocal pinhole was set to render spatial resolutions of 1·0 µm (axial) and 0·4 µm (perpendicular to the optical axis). Image processing, data analysis and presentation were performed with IDL software (Research Systems, Boulder, CO, USA).

Spark detection and measurement

To detect and characterize Ca²⁺ sparks objectively, we used a modified version of the automated spark detector recently described (Cheng et al. 1999). The basic steps in the algorithm include: (1) locating tentative spark regions as pixels/regions that are 2 × standard deviation (S.D.) above the mean fluorescence intensity; (2) excising these provisional sparks from the image, and recomputing the statistics of the remaining regions (presumed to have no spark); (3) determining final spark regions by their excessive deviation (3·8 S.D.) from the background. However, some sparks were clustered and overlapped in space and time, usually with a 'seeder' spark at the beginning of the cluster. To measure the rate of occurrence of sparks spontaneously activated at rest, we considered each cluster as a primary event; secondary sparks usually reflect a local regeneration driven by the seeder. Therefore, a primary event was defined as a connected area that exceeded a weak threshold condition (2·0 S.D.), while containing at least one pixel that exceeded a stronger (i.e. 3·8 S.D.) threshold. An event so detected was then compared with a reference image formed by high-pass filtering and thresholding the raw image, to determine spark clustering. Solitary sparks were those whose non-zero pixels in the reference image were all connected in a single islet. Otherwise, it was deemed as a spark cluster. All parametric measurements were performed on solitary Ca²⁺ sparks.

In voltage-clamped TG4 cells held at -40 mV, evoked sparks were too numerous to be counted by the automated spark detector. In this case, we estimated the spark number by the 'signal mass'. The signal mass is defined as the integral of the change in the normalized fluorescence (F/F₀ = F/F₀ - 1, where F₀ refers to the background fluorescence intensity) over space-time (xt) in the line-scan image. Specifically, we first calculated the total signal mass (M):

	RESULTS

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₂AR*-mediated positive inotropic and lusitropic effects

In the absence of any agonist, the amplitude of unloaded shortening of ventricular myocytes was greater in TG4 cells (4·97 ± 0·30 % of resting cell length, 56 cells from 18 hearts) than in NTG cells (3·02 ± 0·28 % of resting cell length, 39 cells from 15 hearts, P < 0·01) (Fig. 1A and B). This enhanced baseline contractility is ₂AR* dependent, because it was completely reversed by incubation of these cells with ICI (Fig. 1B), an inverse ₂AR agonist (also known as a negative agonist, it preferentially binds to the inactive conformation R of the receptor, shifting the equilibrium between R and R* towards R) (Bond et al. 1995). These observations are in good agreement with previous reports (Milano et al. 1994; Bond et al. 1995; Du et al. 1996; Xiao et al. 1999) and substantiate the idea that ligand-free ₂ARs possess constitutive activity, which is amplified in TG4 due to the vast overexpression of ₂ARs.

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Figure 1. Transgenic overexpression of 2AR induces positive inotropic and lusitropic effects in the absence of 2AR agonist A, typical traces of cell shortening in ventricular myocytes isolated from non-transgenic (NTG) and transgenic (TG4) mouse hearts. The resting cell lengths were 107·8 and 126·5 µm for NTG and TG4 cells, respectively. B, mean contraction amplitudes of NTG and TG4 cells, and TG4 cells treated with ICI 118,551 (TG4-ICI). The mean resting cell lengths were 127·9 ± 3·9 µm (NTG, n = 39) and 139·1 ± 3·7 µm (TG4, n = 56). C-E, rise time (tpeak, C), half-relaxation time (t50, D) and 90 % relaxation time (t90, E) for NTG, TG4 and TG4-ICI cells. Data were obtained from 39 cells of 15 NTG hearts, 56 cells of 18 TG4 hearts, and 23 TG4-ICI cells of 9 TG4 hearts, under steady-state stimulation at 0·5 Hz. Data are presented as means ± S.E.M. *P < 0·01 vs. NTG and TG4-ICI groups, P < 0·05 vs. NTG group.

In intact TG4 mice, the decline of left ventricle pressure (-dP/dt) is faster than in NTG mice, and this relaxant effect is accompanied by an approximately 50 % (Rockman et al. 1996) reduction in the abundance of PLB, which in its dephosphorylated state, inhibits the SR Ca²⁺ ATPase (Lindemann et al. 1983). Similarly, we found that in single TG4 myocytes, contractile relaxation was also accelerated (Fig. 1A). On average, the half-relaxation time (t₅₀) and the 90 % relaxation time (t₉₀) were reduced by 38 and 36 %, respectively (Fig. 1D and E), whereas no significant difference was observed in the rise time (t_peak) (Fig. 1C). For comparison, NTG myocytes have a similar maximal 30-40 % reduction in t₅₀ after ₁AR stimulation (Korzick et al. 1997). In TG4 cells pretreated with ICI, t₅₀ and t₉₀ were largely restored, although t₅₀ was still significantly briefer than that in NTG (Fig. 1D and E). These results suggest that the enhanced myocardial relaxation is attributable not only to the downregulation of PLB as proposed previously (Rockman et al. 1996), but also to acute ₂AR* signalling, presumably by protein kinase A (PKA)-dependent PLB phosphorylation. The fact that ₂AR* induces a maximal relaxation effect is somewhat surprising, because ligand-activated ₂AR signalling is dissociated from phosphorylation of PLB (Xiao et al. 1994; Altschuld et al. 1995; Kuschel et al. 1999a), and in some species, manifests only a minor relaxant effect (Xiao & Lakatta, 1993; Xiao et al. 1994; Kuschel et al. 1999a).

Enhanced Ca²⁺ transients in TG4 myocytes

In principle, the greater contractility of TG4 myocytes could arise from an enhanced excitation-Ca²⁺ release coupling that gives rise to a greater Ca²⁺ transient, or from an enhanced myofilament sensitivity to Ca²⁺, as is the case with -adrenergic stimulation (Gambassi et al. 1998). To discriminate between these possibilities, we examined Ca²⁺ transients and contraction in cells loaded with the Ca²⁺ indicator fluo-3. As shown in Fig. 2B, the ₂AR*-mediated contractile effect was accompanied by an increase in the amplitude of the Ca²⁺ transients (indexed by peak F/F₀ of fluo-3 signal). The maximal rising rate of F/F₀, which roughly reflects the peak SR release flux (Sipido & Wier, 1991), was also enhanced in TG4. The background fluo-3 signal (F₀) was, however, very similar in TG4 (24·9 ± 0·86, n = 38 cells) and NTG cells (25·6 ± 0·64, n = 24), suggesting similar diastolic Ca²⁺ concentrations in the two groups. In fluo-3-loaded cells, contractile relaxation was considerably slowed (compare Fig. 2 with Fig. 1), probably due to the Ca²⁺ buffering effect of the indicator. Nevertheless, the pattern remained unchanged: TG4 cells relengthened more rapidly (t₅₀, 256 ± 13 ms; n = 15) than NTG cells (t₅₀, 312 ± 19 ms; n = 9) (P < 0·05). A similar result was observed for the Ca²⁺ transient decay in these cells (t₅₀, 146 ± 12 and 239 ± 27 ms in TG4 and NTG, respectively; P < 0·05).

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Figure 2. Cell contraction and Ca2+ transients in cardiac myocytes loaded with fluo-3 A, representative traces of Ca2+ transients (upper trace), represented by fluo-3 fluorescence intensity, and cell contraction (lower trace) after stimulation of a TG4 mouse myocyte after a 1 min rest. Note the negative staircase for both contraction and Ca2+ transients. B, peak amplitudes of cell contraction (left), Ca2+ transient (middle), and the maximal rising rate of the normalized fluorescence, d(F/F0)/dt (right), under steady-state conditions. NTG, n = 9 cells; TG4, n = 15. *P < 0·05 vs. NTG group. C, peak contraction amplitude (expressed as a percentage of resting cell length) plotted as a function of the peak amplitude of the Ca2+ transient (F/F0). Data were obtained from 12 NTG cells and 18 TG4 cells during the negative staircase following stimulation from rest. Slopes of the regression lines are 3·38 ± 0·18 (continuous line) and 3·23 ± 0·38 (dashed line) for TG4 and NTG, respectively.

To determine the possible effect of ₂AR* on myofilaments, we plotted the contraction amplitude as a function of the peak Ca²⁺ transient during the negative staircase that followed stimulation of cells from rest (Fig. 2A and C). Linear regression revealed that the slope for TG4 (3·38 ± 0·18) was not significantly different from that for NTG (3·23 ± 0·38) (Fig. 2C), suggesting that the myofilament-Ca²⁺ interaction is probably not altered in TG4 myocytes. Thus, ₂AR* signalling must interact with the EC coupling cascade at steps upstream of the Ca²⁺ transient.

Action potential and I_Ca

To understand Ca²⁺ handling in TG4 cells, we explored possibilities in two categories: the mechanisms affecting sarcolemmal Ca²⁺ cycling and those related to SR Ca²⁺ cycling. In addition to contributing directly to cytosolic Ca²⁺, I_Ca dictates SR Ca²⁺ release via the CICR mechanism (Fabiato, 1983), and affects the SR Ca²⁺ load in a more complex manner (Eisner et al. 1998). Unexpectedly, our recent study showed that the L-type channel, the key effector of agonist-elicited ₂AR signalling, is untouched by ₂AR* (Zhou et al. 1999). In agreement, we found that the I_Ca amplitudes were nearly identical in TG4 and NTG (806 ± 82·5 pA, n = 14 and 764 ± 63·0 pA, n = 13, respectively, at 0 mV in the presence of EGTA). The I_Ca kinetics were not significantly different (TG4 vs. NTG: ₁, 15·09 ± 1·12 vs. 17·74 ± 1·67 ms; ₂, 56·98 ± 2·98 vs. 61·68 ± 4·94 ms) (see also Zhou et al. 1999). While favouring the interpretation that ₂AR* signalling bypasses the I_Ca to augment cardiac contractility, these data do not exclude the involvement of I_Ca in the field-stimulated cells, because the trigger I_Ca may vary if the action potential configuration differs. Therefore, we recorded action potentials in TG4 and NTG cells (Fig. 3). The mean parameters in Table 1 show that TG4 and NTG cells have similar resting membrane potentials (V_rest), action potential (AP) amplitude, and time to 50 % repolarization (t₅₀). The time to 90 % repolarization (t₉₀) was prolonged in TG4 cells. None of these parameters, nor the entire trace of the action potentials was sensitive to ICI. Since the I_Ca should be deactivated at the end-stage repolarization, and since the prolonged t₉₀ in TG4 cells was unaltered by ICI, the prolongation of t₉₀, which might reflect long-term electrophysiological remodelling, has little relevance to altered EC coupling in TG4 cells. The exact cause, however, was not further explored in the present study.

Table 1. Mean data for action potential parameters from NTG and TG4 cells, and TG4 cells in the presence of ICI (TG4-ICI)

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Figure 3. Effect of ICI 118,551 on properties of the action potential in TG4 myocytes Representative traces were recorded by whole-cell current clamping a TG4 cardiomyocyte before (control) and 10 min after exposure to ICI (5 × 10-7 M).

Having not found relevant changes in the trigger I_Ca and action potentials, we next focused on various aspects of SR Ca²⁺ regulation.

SR Ca²⁺ filling status

The aforementioned results on hastened Ca²⁺ and contractile relaxation suggest that SR Ca²⁺ resequestration is enhanced in TG4 cells. As pointed out by Eisner and his colleagues (Trafford et al. 1998; Eisner et al. 1998), increases in SR Ca²⁺ pump function would induce a delayed but sustained augmentation on the Ca²⁺ transient or contractility, due to the subsequent increase in SR Ca²⁺ filling. SR Ca²⁺ content is known to be an important determinant for the amplitude of elementary Ca²⁺ release events or 'Ca²⁺ sparks' (Cheng et al. 1996; Song et al. 1997; Satoh et al. 1997), the fraction of SR Ca²⁺ released (Spencer & Berlin, 1997; Schlotthauer et al. 1998) and possibly CICR sensitivity (Lukyanenko et al. 1996; Spencer & Berlin, 1997). To determine whether SR Ca²⁺ load is altered in TG4, a high concentration of caffeine (10 mM) was applied to empty the SR Ca²⁺ store rapidly (Fig. 4A). SR Ca²⁺ content, defined as the amplitude of the caffeine-elicited Ca²⁺ transient, was comparable in TG4 and NTG, both at rest and during steady-state field stimulation at 0·5 Hz (Fig. 4B). In both TG4 and NTG groups, the SR Ca²⁺ filling was slightly lower in the paced cells, consistent with another report (Li et al. 1997). The absence of alterations in SR Ca²⁺ content in TG4 myocytes indicates that additional changes in SR Ca²⁺ handling must be involved to sustain the chronic inotropic effect of ₂AR* signalling and at the same time maintain the SR loading against an enhanced SR pump function.

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Figure 4. Measurement of SR Ca2+ content in TG4 and NTG cardiomyocytes A, a caffeine-elicited Ca2+ transient in a TG4 myocyte. SR filling status was determined by emptying the SR via rapid pressure injection (400 ms, bar) of 10 mM caffeine onto the cell. The peak F/F0 of the ensuing Ca2+ transient was used as an index of SR Ca2+ load. B, mean SR Ca2+ load at rest or under steady-state field stimulation (0·5 Hz) in NTG (n = 19) and TG4 (n = 12) cells.

Properties of Ca²⁺ sparks

In cardiac myocytes, global Ca²⁺ transients result from the summation of discrete elementary events, namely Ca²⁺ sparks originating from RyRs in the SR (Cheng et al. 1993). Characterization of Ca²⁺ spark properties may reveal the mechanisms responsible for altered EC coupling. Using confocal microscopy in conjunction with an automated spark detector (Cheng et al. 1999, also see Methods), we examined both spontaneous and evoked Ca²⁺ sparks in TG4 and NTG myocytes (Fig. 5). In resting cells, the rates of occurrence of spontaneous sparks were 7·60 ± 1·58 (n = 16) and 3·74 ± 0·90 sparks s^-1 (100 µm)^-1 (n = 10, P < 0·05) for TG4 and NTG, respectively. Ca²⁺ sparks in TG4 cells sometimes (133 out of 782 events, or 17 %) ignited neighbouring release sites, creating spatially and temporally clustered events (Fig. 5); spark clusters were less frequent in NTG cells (25 out of 280; 9 %). Regardless of ₂AR density, all Ca²⁺ sparks were completely abolished within 5 to 10 min of exposure of the cells to ryanodine (10 µM) plus thapsigargin (5 µM), an SR Ca²⁺ pump inhibitor (data not shown).

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Figure 5. Comparison of properties of unitary Ca2+ sparks in NTG and TG4 myocytes A, representative confocal line-scan images of Ca2+ sparks. As indicated by the black arrows, time runs horizontally, and spatial information is displayed vertically. The white arrow in the right panel image indicates a 'seeder' event which initiates a spark cluster. Such clustered events were excluded from the measurement of unitary spark properties. Each cluster was counted as one primary event when measuring the rate of spontaneous spark occurrence (see Methods). B-E, histograms of spark amplitude (F/F0, B), full width (FWHM, C) and duration (FDHM, D) at half-maximum, and signal mass (E). A total of 280 and 782 sparks were detected from 10 NTG and 16 TG4 cells, respectively. Events with amplitudes larger than 2·5 or durations longer than 130 ms or signal masses greater than 125 µm ms are omitted in the related plots.

Similarly, when membrane potential was held at -40 mV after a ramp from -70 mV, the evoked Ca²⁺ sparks occurred at a 3-fold higher frequency in TG4 (36·4 ± 9·1 sparks (100 ms)^-1 (100 µm)^-1, 12 images from 3 cells; estimated from the signal mass; see Methods) than in NTG cells (12·8 ± 2·1 sparks (100 ms)^-1 (100 µm)^-1, 12 images from 3 cells; P < 0·05 vs. TG4). ICI treatment to inactivate ₂AR* reduced the evoked spark frequency nearly 7-fold to 5·22 ± 1·9 sparks (100 ms)^-1 (100 µm)^-1 (P < 0·01 vs. control) (see Fig. 6). Since I_Ca and the resting Ca²⁺ were similar in TG4 and NTG, these results suggest that CICR sensitivity, operationally defined as the number of sparks activated by a given amount of trigger Ca²⁺, was augmented 2- to 7-fold in TG4 cells.

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Figure 6. Simultaneous recordings of ICa, intracellular Ca2+ and cell length in a representative TG4 ventricular myocyte From top to bottom: confocal line-scan image of fluo-3 fluorescence, voltage-clamp protocol, spatially averaged fluo-3 fluorescence transient (F/F0), shortening of cell length and ICa. Data were recorded immediately before (left), or 5 min after (middle) application of the 2AR inverse agonist ICI 118,551, and 5 min after washout of ICI (wash). Note that ICI markedly and reversibly inhibits the contraction and the Ca2+ transient with a profound reduction in Ca2+ spark frequency, but has no effect on the simultaneously recorded ICa. Similar results were obtained from 3 cells.

Figure 5B-E shows histogram distributions of properties of unitary Ca²⁺ sparks. Compared with those in NTG cells, Ca²⁺ sparks in TG4 were enlarged in all dimensions, including amplitude (mean F/F₀, 1·61 vs. 1·43), spatial width (FWHM, 1·81 vs. 1·52 µm), and duration (FDHM, 45·9 vs. 25·8 ms). As a result, the integral of F/F₀ in space-time or signal mass was increased from 15·8 µm ms in NTG to 36·4 µm ms in TG4. Interestingly, a long tail was evident in the F/F₀, FDHM and signal mass plots for TG4, but was absent in NTG plots. This suggests that TG4 cells have a novel population of events: those with F/F₀ > 1·80 (84 events; 13 %), or FDHM > 70 ms (98 events; 15 %), or signal mass > 65 µm ms (100 events; 15 %). These large events account for 50 % of the increases in unitary spark parameters, and suggest that ₂AR* signalling may increase local release flux. These local increases in Ca²⁺ flux could be the result of recruiting more RyRs within the release unit, longer RyR openings, or an increase in local CICR endurance. Thus, increases in both spark size and frequency contribute to the greater Ca²⁺ transients in TG4.

Simultaneous recordings of I_Ca, Ca²⁺ sparks, Ca²⁺ transients and contraction

To monitor simultaneously the main events of EC coupling in the same cell, we employed confocal imaging combined with whole-cell voltage clamping. Representative results from a TG4 cell are shown in Fig. 6, displaying, from top to bottom, line-scan images of intracellular Ca²⁺, membrane potential, spatially averaged Ca²⁺ transients, cell shortening and whole-cell I_Ca. ICI markedly and reversibly suppressed the cell-wide Ca²⁺ transient (F/F₀ from 5·46 ± 0·18 to 2·44 ± 0·1, n = 3, P < 0·05), contraction (from 12·6 ± 2·3 to 2·45 ± 0·68 % of resting length, n = 3, P < 0·01), and Ca²⁺ sparks prior to the transients. Notably, the kinetics of both the Ca²⁺ transient and contraction slowed in the presence of ICI (Fig. 6). In contrast, the amplitude of I_Ca recorded simultaneously in the same cells was unaffected by ICI (from -616 ± 18 to -675 ± 88 pA, n = 3, P > 0·05), although there was a slight slowing in the fast inactivation time constant of I_Ca (₁) (from 12·67 ± 1·24 to 16·14 ± 1·91 ms, n = 3, P < 0·05). The slowing of ₁ by ICI, which reduced SR Ca²⁺ release, is consistent with the Ca²⁺-dependent inactivation of the L-type channel reported previously (Sham, 1997). Control experiments performed in NTG cells using the same protocol revealed only minor effects of ICI on basal contractility, Ca²⁺ transient, I_Ca amplitude, and Ca²⁺ spark frequency at -40 mV (93·8 ± 6·2 %, 91·9 ± 2·2 %, 97·8 ± 1·2 % and 91·4 ± 4·9 %, respectively, n = 3), consistent with previous observations (Xiao et al. 1999; Zhou et al. 1999). Taken together, these data strongly support the notion that ₂AR*-mediated signalling does not regulate I_Ca; instead, it reinforces excitation-Ca²⁺ release coupling and promotes SR Ca²⁺ reuptake.

	DISCUSSION

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A novel cellular mechanism regulating cardiac EC coupling

Ligand-independent, spontaneous activation of ₂AR overexpressed in mouse cardiomyocytes produces positive inotropic and lusitropic effects, even in the absence of any related changes in action potential, I_Ca, SR Ca²⁺ content, or myofilament Ca²⁺ sensitivity. In contrast, Ca²⁺ spark frequency and solitary signal mass are grossly increased, resulting in enhanced SR release and greater global Ca²⁺ transients in TG4 myocytes. The efficiency of SR Ca²⁺ recycling, as reflected by the kinetics of the Ca²⁺ transient and contraction, is enhanced concomitantly in the presence of ₂AR*. Based on these findings, we propose a simple mechanistic model to explain ₂AR*-mediated modulation of cardiac EC coupling (Fig. 7). In this model, ₂AR* reinforces the CICR and the ability of the SR to recycle the released Ca²⁺, while not necessarily affecting the sarcolemmal Ca²⁺ influx and efflux. The resultant Ca²⁺ transient and contraction are both characterized by increased amplitudes, but a compressed relaxation time course. Since ₂AR* activates adenylyl cyclase (Milano et al. 1994) and increases cAMP production (Zhou et al. 1999) in an ICI-sensitive manner, and since the blockade of the PKA activation (by inhibitory cAMP analogues) mimics the effect of ICI to reverse ₂AR*-induced augmentation in cell contraction (Zhou et al. 1999), the ₂AR* signalling is most probably mediated by a cAMP/PKA-dependent protein phosphorylation, as is the case with ligand-elicited AR stimulation.

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Figure 7. A simplified model of 2AR*-mediated modulation of cardiac EC coupling 2AR* enhances SR Ca2+ release without altering the action potential (AP) and the sarcolemmal Ca2+ fluxes. At the same time, the SR Ca2+ recycling is similarly enhanced, to balance the SR efflux. The resulting Ca2+ transient has a greater amplitude but a compressed time course. The overall Ca2+ extrusion during a complete cardiac cycle is also unchanged, in balance with the sarcolemmal Ca2+ influx. The 2AR inverse agonist ICI 118,551 blocks the effects of 2AR* by stabilizing the receptor in an inactive conformational state.

Eisner and his colleagues (Trafford et al. 1997, 1998; Eisner et al. 1998) demonstrated that targeting RyRs to alter CICR is insufficient to evoke a substantial long-lasting Ca²⁺ or inotropic response. For instance, an increase in SR Ca²⁺ release by low dose caffeine leads only to a brief increase in Ca²⁺ transients because cardiac Ca²⁺ autoregulation can compensate for the primary change by depleting SR Ca²⁺ load. In contrast, a single perturbation of the sarcolemmal Ca²⁺ influx or efflux may permanently alter Ca²⁺ and contractile performances. Theoretical analysis suggested that the sustained effect is linked to an altered SR Ca²⁺ load (Eisner et al. 1998). To this end, our finding in TG4 ventricular myocytes illustrates a previously unidentified scenario: synchronous increases in SR release and reuptake, when acting in concert, can support a marked, chronic positive inotropic effect. Neither an increase in the I_Ca nor an alteration in the SR Ca²⁺ load is obligatory to the enhanced contractility (Fig. 7).

The aforementioned results in this transgenic model suggest that SR Ca²⁺ recycling, independent of sarcolemmal Ca²⁺ fluxes, constitutes an important determinant of cardiac contractility. In this regard, it is noteworthy that alterations of SR Ca²⁺ recycling in TG4 myocytes appear to mirror those in the failing heart, in which a depressed Ca²⁺ transient and contractility are accompanied by a decreased I_Ca to Ca²⁺ spark coupling efficiency (Gómez et al. 1997), reduced SR Ca²⁺ ATPase expression (e.g. Arai et al. 1993), slowed Ca²⁺ transient and contraction (e.g. Gómez et al. 1997), but without evident change in I_Ca density (e.g. Gómez et al. 1997).

₂AR* modulates RyR channel activity in situ

RyR, the SR Ca²⁺ release channel, is phosphorylated by PKA during -adrenergic stimulation (Yoshida et al. 1992; Hohenegger & Suko, 1993). Phosphorylation of RyR increases the probability of channel opening and promotes Ca²⁺ release from the SR (Hain et al. 1995; Lokuta et al. 1995). In agreement with these reports, we found that ₂AR* signalling exerts a profound effect on RyR activity and thereby on CICR sensitivity in situ. In TG4 cells, Ca²⁺ sparks are larger and occur more frequently than those in NTG myocytes, despite the fact that the resting cytosolic Ca²⁺, the SR Ca²⁺ load, and the triggering I_Ca are unaltered. These data suggest that ₂AR*-mediated cAMP/PKA signalling directly modulates the RyR channel molecules to alter their Ca²⁺ sensitivity or release-linked inactivation (Sham et al. 1998). We therefore conclude that RyR, in addition to the L-type channel, PLB and contractile myofilaments, constitutes an important target protein for AR signalling in intact cardiac myocytes. This conclusion is consistent with previous observations that the non-selective AR agonist isoproterenol promotes spontaneous Ca²⁺ sparks in rat ventricular myocytes (Tanaka et al. 1997).

Fidelity of coupling between the L-type channel and RyR

On the molecular level, EC coupling is essentially a tale of two molecules, the L-type channel and RyR, communicating over the nanometre space of the T-tubule-SR junction. L-type channels open and close stochastically, and trigger the associated RyRs in an all-or-none manner to generate Ca²⁺ sparks. The 'digital' coupling fidelity, , is here defined as the ratio of the number of L-type channel openings to the number of Ca²⁺ sparks activated. To accommodate the 7-fold reduction in the frequency of evoked sparks at -40 mV by the ₂AR* blockade, must be far greater than 1:1 under normal conditions. That is, in contrast to the numerical results of Cannell & Soeller (1997), L-type channel opening is not always followed by RyR activation, i.e. only one in several openings triggers an SR release event.

This point may be better appreciated by examining the macro- and microscopic properties of the I_Ca and SR Ca²⁺ release. Let i_L, _L, i_S and _S stand for unitary flux and mean duration of L-type channel opening and elementary SR release, respectively. The ratio of mean unitary fluxes of L-type channel and of Ca²⁺ spark (), which reflects the gain of microscopic coupling if = 1:1, is then given by:

Assuming i_S = 3 pA, _S = 10 ms (Cheng et al. 1993), i_L = 260 fA (at -40 mV, scaling factor of 2 for 10 vs. 1 mM Ca²⁺ as the charge carrier) or 130 fA (at 0 mV), and _L = 0·3 ms (Rose et al. 1992; Sham et al. 1998), the value of would be as low as 1:385 (at -40 mV) or 1:770 at (0 mV). Previous studies on the gain of macroscopic EC coupling (g = ) have established that g 60 at -40 mV and g 15 at 0 mV (Wier et al. 1994; Song et al. 1998). Combined with the microscopic measurements described above, this translates into a value of to the order of 6·4:1 (at -40 mV) to 51:1 (at 0 mV), though the latter might be overestimated because of a significant (1/3) SR depletion at 0 mV (Sham et al. 1998). The higher fidelity (i.e. smaller ratio) at -40 mV associated with the larger unitary current (i_L) at this voltage is consistent with the 'local control' theory (Wier et al. 1994; Santana et al. 1996) and the non-linear dependence of the occurrence of evoked Ca²⁺ sparks on the triggered Ca²⁺ (Santana et al. 1996).

A similar time course for spark generation and L-type Ca²⁺ channel openings has been observed in wild-type rat and guinea-pig ventricular myocytes, under the conditions when SR Ca²⁺ release is small (Cannell et al. 1995; Lopez-Lopez et al. 1995; Santana et al. 1996; Collier et al. 1999) and the release-linked RyR inactivation (Sham et al. 1998) is negligible. Thus, spark probability at a given voltage can be described by a power function of I_Ca with a power of unity, supporting the idea that an individual Ca²⁺ spark is triggered by a single L-type Ca²⁺ channel opening (Cannell et al. 1995; Lopez-Lopez et al. 1995; Santana et al. 1996; Collier et al. 1999). Since the ratio would affect only the coefficient of the power function, the close temporal relationship described above does not contradict our conclusion that the coupling fidelity is far from 1:1 under normal conditions. Rather, it suggests that is essentially time independent during a voltage pulse.

This low digital coupling fidelity indicates that a large reserve capacity resides in the coupling between L-type channels and RyRs. As shown in TG4 mice, facilitation of 'cross-talk' between the L-type channels and RyRs can substantially augment cardiac inotropy, an effect beneficial during stress and excise. However, it should be pointed out that an extremely high CICR sensitivity could be detrimental, because it would create a lot of RyR-RyR 'side-talk' (spark clusters), which would put the system at risk of falling into a regime of instability (generation of Ca²⁺ waves, all-or-none Ca²⁺ transients, etc.).

Intracellular sorting of receptor signalling

The present report provides new evidence for the precision of intracellular sorting of receptor-mediated transmembrane signalling. In adult TG4 myocytes, ₂AR* signalling regulates proteins in the SR (i.e. RyR and PLB) but is diverted from the L-type Ca²⁺ channel in the sarcolemma (Fig. 6 and Zhou et al. 1999). Conversely, ligand-elicited ₂AR signalling, which has no known effector in the cytoplasm, appears to be strictly localized to sarcolemmal microdomains in several mammalian species (Xiao et al. 1994; Altschuld et al. 1995; Zhou et al. 1997; Kuschel et al. 1999a,b). Moreover, in cultured embryonic/neonatal TG4 cells, ₂AR* specifically increases I_Ca, but not cAMP-sensitive K⁺ currents (An et al. 1999). A common thread among these observations is that receptor-mediated signalling is highly compartmentalized and target-protein specific, depending on the conformational state of the receptor or even the developmental status of the cell. This compartmentalization of receptor signalling may help to explain a general enigma in receptor physiology: how do vast numbers of G-protein-coupled receptors converge on a limited portfolio of intracellular messenger molecules (e.g. cAMP, inositol 1,4,5-triphosphate, Ca²⁺), but at the same time, retain a high degree of signalling diversity and specificity? Future studies are required to elucidate the specific mechanisms underlying the precise sorting of receptor signals.

Regarding the interaction between AR and its effector proteins, Muth et al. (1999) recently showed that cardiac specific overexpression of the ₁ subunit of L-type Ca²⁺ channels curtails isoproterenol-induced contractile and I_Ca responses, without altering AR receptor density. Complementary to this finding, ₂AR*, while bolstering contractility, fails to modulate I_Ca (Zhou et al. 1999 and this study), indicating that the usual signalling routed toward the L-type channel is non-existent or disrupted when the receptor is overexpressed. These results suggest a novel bi-directional regulation between the receptor and its effector, which merits further investigation.

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Acknowledgements

The authors wish to thank Drs Walter J. Koch and Robert J. Lefkowitz for their generous supply of the transgenic mice; Dr Harold Spurgeon, Dr Sheng-Jun Zhang and Mr Bruce Ziman for their excellent technical support.

Corresponding author

H. Cheng: Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.

Email: chengp{at}grc.nia.nih.gov

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