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Journal of Physiology (2001), 533.3, pp. 837-848
© Copyright 2001 The Physiological Society

Modulation of the Ca²⁺-induced Ca²⁺ release cascade by -adrenergic stimulation in rat ventricular myocytes

Serge Viatchenko-Karpinski and Sandor Györke

	ABSTRACT

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1. To define the sub-cellular mechanisms of modulation of cardiac excitation-contraction (E-C) coupling by the -adrenergic pathway, we carried out confocal Ca²⁺ imaging in conjunction with recordings of inward Ca²⁺ current in fluo-3-loaded patch-clamped rat ventricular myocytes.
2. Isoproterenol (isoprenaline; ISO) increased the amplitude of the inward Ca²⁺ current and the globally measured intracellular Ca²⁺ transients. The gain of calcium-induced calcium release (CICR) was increased at all membrane potentials but especially at positive membrane potentials (> +30 mV). ISO dramatically broadened the bell-shaped voltage dependence of intracellular Ca²⁺ transients by shifting the descending portion of the curve to very high membrane potentials.
3. The number of local release events (solitary sparks and conglomerates of overlapping sparks) induced by depolarizing steps to +30 mV was increased significantly by ISO. This potentiation of events was due to increased trigger calcium current (I_Ca) as well the enhanced ability of I_Ca to induce release. The amplitude and duration of solitary sparks were increased in the presence of ISO. In addition, ISO dramatically increased the proportion and the size ('mass') of clustered events.
4. Exclusion of Na⁺ from the intra- and extracellular solutions prevented ISO from enhancing the ability of I_Ca to trigger sparks.
5. We conclude that -adrenergic stimulation enhances the gain of the CICR cascade by increasing the fidelity of dihydropyridine receptor (DHPR)-ryanodine receptor (RyR) coupling and by promoting cross-activation of RyRs in neighbouring release sites. Reverse Na⁺-Ca²⁺ exchange (NCX) appears to play a role in the -adrenergic enhancement of CICR by effectively contributing to the Ca²⁺ trigger.

	INTRODUCTION

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During cardiac E-C coupling Ca²⁺ entry through voltage-gated Ca²⁺ channels in the sarcolemma locally controls the activity of Ca²⁺-sensitive Ca²⁺ release channels located in the adjacent junctional sarcoplasmic reticulum (SR) (Cheng et al. 1996; Bers & Perez-Reyes, 1999; Wier & Balke, 1999; Niggli, 1999). The release of Ca²⁺ arising from activation of functional clusters of RyRs has been visualized as Ca²⁺ sparks by confocal imaging in cells loaded with fluo-3 (Cheng et al. 1993). These microscopic release events appear to be due to the concerted opening of 10-30 RyRs (Bridge et al. 1999; Lukyanenko et al. 2000a). They amplify the initial trigger signal and sum to produce an elevation of cell-averaged myoplasmic [Ca²⁺], leading to activation of contractile proteins. It has been suggested that CICR can be triggered by Ca²⁺ entry through other pathways besides I_Ca, such as T-type Ca²⁺ channels (Sipido, 1998) and NCX (Leblanc & Hume, 1990; Lipp & Niggli, 1994; Kohomoto et al. 1994). In addition, it has been proposed that a voltage-dependent process, similar to that known to operate in skeletal muscle, controls Ca²⁺ release in cardiac myocytes (Ferrier & Howlett, 1995; Howlett et al. 1998). However, the conditions of operation and the physiological relevance of these additional trigger mechanisms remain uncertain.

Cardiac E-C coupling is subject to regulation by various physiological and pharmacological agents. One of the most physiologically important regulatory influences affecting cardiac contractile function is stimulation through the -adrenergic pathway. -Adrenergic agonists increase the magnitude of Ca²⁺ release, resulting in increased contractile activation of cardiac muscle. The biochemical signalling mechanisms underlying -adrenergic stimulation involve cAMP-dependent phosphorylation by protein kinase A (PKA) of certain target proteins including DHPRs, RyRs and phospholamban (e.g. Bers, 1991). Studies in which I_Ca and global Ca²⁺ transients were measured have suggested that the increase in SR Ca²⁺ release is caused by either increased I_Ca liberating more Ca²⁺ or an increase in the size of the SR Ca²⁺ pool enabling the same trigger I_Ca to release more Ca²⁺ (Callewaert et al. 1988; Hussain & Orchard, 1997). However, the precise sub-cellular features of these stimulatory effects remain to be elucidated. -Adrenergic stimulation of the E-C coupling process could occur at any level of the CICR cascade: (1) increasing the number of Ca²⁺ entry points, thereby triggering more release sites; (2) enhancing the fidelity of the DHPR-RyR interactions; (3) augmenting cross-activation of neighbouring release sites. In addition, any of the proposed additional trigger mechanisms, such as reverse NCX, could contribute to the potentiation of Ca²⁺ release. To better understand the principles of regulation of E-C coupling, we performed confocal Ca²⁺ imaging in conjunction with recordings of I_Ca in patch-clamped cardiac myocytes loaded with fluo-3. Preliminary results from this study were presented at the 44th Biophysical Society Meeting (Viatchenko-Karpinski & Györke, 2000).

	METHODS

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Single ventricular myocytes were obtained from Sprague-Dawley rat hearts by enzymatic dissociation (Györke et al. 1997). Adult rats (200-300 g) were killed with an overdose of sodium pentobarbitone in accordance with the guidelines of the TTUHSC Animal Care and Use Committee. Whole-cell patch-clamp recordings of membrane currents were made using an Axopatch 200B amplifier (Axon Instruments, USA). Pipettes (1-3 M) were filled with a solution that contained (mM): 120 caesium aspartate, 20 CsCl, 3 Na₂ATP or 3 Cs₂ATP, 3.5 MgCl₂ and 5 Hepes, with 50 µM fluo-3, pH 7.3. We used three different external solutions. Standard Tyrode solution contained (mM): 140 NaCl, 5.4 KCl, 1 CaCl₂, 0.5 MgCl₂, 10 Hepes and 5.6 glucose, pH 7.3. Low-Ca²⁺ solution contained (mM): 140 NaCl, 5.4 KCl, 0.2 CaCl₂, 2 MgCl₂, 10 Hepes and 5.6 glucose, pH 7.3. Low-Ca²⁺ Na⁺-free solution contained (mM): 140 LiCl, 5.4 KCl, 0.2 CaCl₂, 2 MgCl₂, 10 Hepes and 5.6 glucose, pH 7.3. Voltage pulses were applied from a holding potential of -50 mV at 1 min intervals. Current signals were sampled at 5 kHz and filtered at 2 kHz. To inhibit the voltage-dependent Na⁺ current the external solutions contained 20 µM TTX. To isolate currents through the Ca²⁺ channels we used the Ca²⁺ channel blocker Cd²⁺. At the end of each series of measurements 0.5 mM Cd²⁺ was introduced into the bathing solution to inhibit the Ca²⁺ currents. The currents flowing through Ca²⁺ channels were defined by subtracting the Cd²⁺-insensitive current from the total current recorded in the absence of Cd²⁺ under otherwise identical conditions. We used ISO and TTX from Sigma (USA) and diltiazem from ICN Biomedicals Inc. (USA). Ca²⁺ imaging was performed using a BioRad 1024 laser scanning confocal system (LSM-GB200) equipped with an Olympus 60, 1.4 NA objective. Fluo-3 was excited by light at 488 nm (25 mW argon laser, intensity attenuated to 1-3 %), and fluorescence was measured at wavelengths of > 515 nm. All experiments were performed at 20-23 °C. Local fluorescence events were detected and quantified by using an automated computer program kindly provided by Dr Heping Cheng (NIA, NIH, Baltimore, MD, USA). Using a thresholding algorithm, all events were qualified as either solitary sparks or conglomerates of overlapping sparks (Zhou et al. 1999). [Ca²⁺] was calculated from fluo-3 fluorescence using an equation and calibration parameters described previously (Cheng et al. 1993). The size of the events was described as the signal mass, defined as the integral of the change in normalized fluorescence (F/F, where F = F - F₀, F is recorded fluorescence intensity and F₀ is background fluorescence) over space - time (x,t) in the linescan image (M = F/F(x,t)dxdt, Zhou et al. 1999). Image processing and analysis were performed by using NIH Image (NIH, Bethesda, MD, USA) and IDL software (Research Systems Inc., Boulder, CO, USA).

	RESULTS

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The effects of ISO on the relationship between I_Ca and intracellular Ca²⁺ transients

Figure 1 shows inward Ca²⁺ currents and linescan-averaged Ca²⁺ transients induced by depolarization to different test potentials in the range -30 to +60 mV before and after exposure of the cell to 500 nM ISO. In accordance with previous studies, ISO increased the magnitude of I_Ca and Ca²⁺ transients and also accelerated the rate of decay of the Ca²⁺ transients. This series of experiments is summarized in Fig. 2A, which plots the voltage dependence of I_Ca and Ca²⁺ transients. Under control conditions the Ca²⁺ transient showed a typical, for cardiac muscle, bi-phasic dependence on the membrane potential, similar to I_Ca. This indicates that the release process is tightly graded by I_Ca. In the presence of ISO the amplitude of the Ca²⁺ transients was no longer graded with changes in I_Ca. Ca²⁺ transients reached high values at low membrane potentials (small I_Ca) and did not show a decrease at positive membrane potentials. We also analysed the effects of ISO on the gain of E-C coupling. E-C coupling gain is defined as the ratio of the amount of Ca²⁺ released during a Ca²⁺ transient to the amount of the trigger Ca²⁺ (Stern, 1992). We calculated the E-C coupling gain as the ratio of F/F to the amplitude of I_Ca (Wier et al. 1994; duBell et al. 1996). Figure 2B shows the voltage dependence of gain at voltages between -30 and +30 mV normalized to averaged gain at 0 mV. We limited our estimates of gain to membrane potentials of +30 mV because at higher membrane potentials measurements of I_Ca become highly unreliable due to concomitant outward currents. Under control conditions the gain gradually decreased between -30 and +10 mV and showed a slight increase between +10 and +30 mV. ISO enhanced the gain in this range of membrane potentials. The increase was disproportionately high at positive membrane potentials resulting in essentially U-shaped voltage dependence. At higher membrane potentials we were unable to quantify the gain because Ca²⁺ entry could not be measured. It is evident, however, that the release gain at these potentials should be very high considering that Ca²⁺ release remained close to maximum while the influx of Ca²⁺ would be expected to decrease to very low levels.

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Figure 1. The effect of -adrenergic stimulation on intracellular Ca2+ transients and ICa Representative recordings of F/F0 changes and whole-cell currents from a myocyte during depolarizing steps to different membrane potentials before (A) and after (B) exposure of the cell to 500 nM ISO. The holding potential was -50 mV, test voltage step duration was 150 ms. The extracellular solution contained 1 mM Ca2+ and 140 mM Na+.

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Figure 2. Effects of bath application of ISO on voltage dependence of Ca2+ release, ICa and gain of CICR in the presence of Na+ in the pipette and bathing solution A, voltage dependence of peak fluorescence changes (upper panel) and ICa (lower panel) in voltage-clamped cardiomyocytes before () and after exposure to ISO (500 nM, ). Peak F/F0 and calcium current amplitude values were normalized to their respective values at 0 mV. B, relative gain of CICR (ratio of peak F/F0 to the peak amplitude of ICa) in control () and -adrenergically stimulated () myocytes. Data are presented as means ± S.E.M. of 12 experiments in different cells.

Effects of depolarization to highly positive membrane potentials

To further investigate the effects of ISO, we performed measurements of Ca²⁺ transients during test pulses in the range +90 to +200 mV. Figure 3 illustrates linescan-averaged Ca²⁺ transients recorded at these highly positive membrane potentials. Under control conditions no Ca²⁺ transients were observed during depolarizing steps above +120 mV. Ca²⁺ transients occurred only upon repolarization. These Ca²⁺ signals were apparently triggered by the I_Ca tail currents. In the presence of ISO, pulses to +120 mV still triggered Ca²⁺ transients with a magnitude close to maximal. However, depolarization to +150 mV completely abolished the Ca²⁺ transients during the voltage step. The voltage dependence of Ca²⁺ transient amplitude is plotted in Fig. 3B. In addition to the data recorded with our standard Tyrode solutions (1 mM Ca²⁺, circles), the figure also includes data acquired using bathing solutions with reduced [Ca²⁺] (0.2 mM, triangles). At both Ca²⁺ concentrations ISO dramatically shifted the descending phase of voltage dependency of release towards more positive membrane potentials. The extent of the shift, however, was clearly less in the presence of reduced bathing [Ca²⁺]. This difference in the voltage dependence of release is likely to reflect the lowered equilibrium potential for Ca²⁺ (E_Ca) at reduced extracellular [Ca²⁺]. These results indicate that potentiation of release by ISO does not involve a voltage-dependent mechanism but rather relies on Ca²⁺ entry from the extracellular milieu. It also rules out the possibility that release at high membrane potentials is triggered directly by NCX. Indeed, Ca²⁺ influx through NCX would be expected to increase at higher membrane potentials.

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Figure 3. Effects of bath application of ISO on intracellular Ca2+ transients and ICa at depolarizing pulses to high membrane potentials in the presence of Na+ in the pipette and bathing solution A, representative traces of F/F0 changes in a myocyte during depolarizing steps to the indicated membrane potentials before (control) and after exposure of the cell to 500 nM ISO. The holding potential was -50 mV. The extracellular solution contained 1 mM Ca2+ and 140 mM Na+. B, voltage dependence of F/F (normalized to peak F/F at 0 mV in control) in control (filled symbols) and -adrenergically stimulated (open symbols) myocytes in the membrane potential range +90 to +200 mV. Circles, 1 mM Ca2+; triangles, 0.2 mM Ca2+ in bath solution. Data are presented as means ± S.E.M. of 9 experiments in different cells.

The effects of ISO on the relationship between I_Ca and Ca²⁺ sparks

In cardiac myocytes, global Ca²⁺ transients result from a summation of elementary release events (sparks) (Cheng et al. 1993). To gain insight into the mechanism of up-regulation of E-C gain by ISO we carried out confocal imaging of Ca²⁺ sparks in conjunction with simultaneous measurements of inward Ca²⁺ currents in whole-cell patch-clamped myocytes. The experiments were performed in the presence of either diltiazem (0.2 µM) or lowered extracellular Ca²⁺ (0.2 mM). These manoeuvres decreased the number of Ca²⁺ sparks triggered by depolarization, allowing us to quantify spark properties during depolarization.

Figure 4 shows representative images of Ca²⁺ sparks and I_Ca measured during 150 ms depolarizing pulses to +30 mV before and after addition of 500 nM ISO to the bath solution. Under control conditions, depolarization induced occasional Ca²⁺ sparks (Fig. 4A). ISO caused a dramatic increase in the number of evoked events (Fig. 4B). Importantly, many events in the presence of ISO appeared to present not single isolated sparks but also conglomerates of sparks caused by sequential activation of adjacent release sites. These changes in release events were accompanied by a substantial increase in the magnitude of I_Ca.

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Figure 4. Effects of ISO on Ca2+ sparks and ICa in the presence of Na+ in the pipette and bathing solutions Representative recordings of whole-cell currents and confocal linescan images of Ca2+ sparks during depolarizing steps to +30 mV before (A) and after (B) addition of ISO (500 nM) to the bathing solution. The holding potential was -50 mV. The extracellular solution contained 0.2 mM Ca2+ and 140 mM Na+. C, ISO caused an increase in the frequency (f) of event production (number of events per 150 ms depolarizing stimulation time per 100 µm space) by about 2.5-fold, and increased the mass of events by about 2-fold and mass of release events (M = F/F(x,t)dxdt), peak amplitude of ICa and efficiency of ICa in triggering release events (f/ICadt) during the depolarizing pulses to +30 mV. Data are presented as means ± S.E.M. of 5 experiments in different cells. *P < 0.05 vs. control (Student's paired t test).

The effects of ISO on Ca²⁺ sparks and I_Ca are summarized in Fig. 4C and Table 1. Data from five different experiments were pooled. ISO increased the probability of event production (individual sparks and conglomerates of sparks) by about 2.5-fold and increased the mass of events by about 2-fold. The increase in the overall spatio-temporal size of events was due to an increase in the magnitude and duration of individual sparks, as well as to an increased ability of individual sparks to ignite neighbouring release sites, resulting in conglomerates of events. The amplitude of I_Ca was increased by about 30 % (Fig. 4C). These data were also used to assess the effects of ISO on the ability of I_Ca to trigger Ca²⁺ release. The relationship between I_Ca and the probability of evoking Ca²⁺ sparks characterizes the fidelity or efficiency of coupling between Ca²⁺ entry through DHPRs and activation of RyRs. To assess the relationship between I_Ca and Ca²⁺ spark production, we divided the frequency of events by the integral of I_Ca during the depolarizing step (Santana et al. 1996). As shown in Fig. 4C, ISO enhanced the efficiency of I_Ca to trigger release by about 60 %.

Effects of removal of extracellular and intracellular Na⁺

Recent studies suggest that NCX can influence E-C coupling by modulating the gain of CICR (Litwin et al. 1998; Goldhaber et al. 1999). To explore the role of NCX in ISO-induced potentiation of Ca²⁺ release we carried out experiments using Na⁺-free pipette and bath solutions. Removal of Na⁺ from the extra- and intracellular environment of the cell is expected to halt NCX. Figure 5A illustrates representative traces of Ca²⁺ transients recorded at different membrane potentials, while Fig. 5B shows the voltage dependence of release based on results from seven cells. In the absence of Na⁺, ISO was no longer able to induce release at highly positive membrane potentials. For comparison, in Na⁺-containing solutions, depolarizing steps up to +120 mV triggered Ca²⁺ release under otherwise similar experimental conditions (Fig. 3B, open triangles).

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Figure 5. Effects of bath application of ISO on intracellular Ca2+ transients and ICa induced by depolarizing pulses to high membrane potentials in the absence of Na+ in the pipette and bathing solutions A, representative traces of F/F0 changes in a myocyte during depolarizing steps to the indicated membrane potentials before and after exposure of the cell to 500 nM ISO. The holding potential was -50 mV. The extracellular solution contained 0.2 mM Ca2+ and 140 mM Na+. B, voltage dependence of F/F0 (normalized to peak F/F0 at 0 mV in control) in control () and -adrenergically stimulated () myocytes in the membrane potential range 0 to +200 mV. Data are presented as means ± S.E.M. of 4 experiments in different cells.

As illustrated in Fig. 6 and Table 1, in the absence of extra- and intracellular Na⁺, ISO caused a much smaller increase in the frequency of triggered events than with Na⁺ present. The differential increases in the frequency of events were 2.0 ± 0.6 vs. 6.2 ± 1.4 events (150 ms)^-1 (100 µm)^-1 in Na⁺-free and Na⁺-containing solutions, respectively (P < 0.05). The ability of I_Ca to trigger sparks increased only slightly (not significantly) in contrast with the results obtained in the presence of external Na⁺. The overall size of events increased by about 2-fold, similar to the results obtained in the presence of Na⁺. Taken together, these results indicate that NCX plays a role in enhancing the efficiency of DHPR-RyR communication upon -adrenergic stimulation.

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Figure 6. Effects of ISO on Ca2+ sparks and ICa in Na+-free bath and pipette solutions Representative recordings of whole-cell currents and confocal linescan images of Ca2+ sparks during depolarizing steps to +30 mV before (A) and after (B) addition of 500 nM ISO to the bathing solution. The holding potential was -50 mV. The extracellular solution contained 0.2 mM Ca2+ and 140 mM Li+. C, effects of ISO on the frequency (f) of event production (number of events per 150 ms depolarizing stimulation time per 100 µm space) and mass of release events (M = F/F(x,t)dxdt), peak amplitude of ICa and efficiency of ICa in triggering release events (f/ICadt) during the depolarizing pulses to +30 mV. Data are presented as means ± S.E.M. of 5 experiments in different cells. *P < 0.05 vs. control (Student's paired t test).

Effects on SR Ca²⁺ load

The observed acceleration of the decay of Ca²⁺ transients upon addition of ISO (Fig. 1) suggests that Ca²⁺ uptake is enhanced under our experimental conditions. This is consistent with the well-known ability of the -adrenergic pathway to stimulate the SR Ca²⁺-ATPase via phosphorylation of phospholamban (e.g. Luo et al. 1994). Enhanced SR Ca²⁺ uptake could result in increased SR Ca²⁺ content, which in turn would be expected to enhance the functional activity of the Ca²⁺ release mechanism (Bassani et al. 1995; Lukyanenko et al. 1996). To investigate the effect of ISO on SR Ca²⁺ content under our experimental conditions we carried out measurements of Ca²⁺ transients induced by application of 10 mM caffeine. The magnitudes of the caffeine-induced Ca²⁺ transients measured before and after addition of ISO (500 nM) were similar in the presence of 1 or 0.2 mM Ca²⁺ in the bathing solution (Fig. 7A and B). Similarly, ISO had no significant effect on the caffeine-induced transients recorded in our Na⁺-free solutions (Fig. 7C). Thus it appears that ISO has no significant impact on SR Ca²⁺ loading under our experimental conditions. Therefore it is unlikely that the results observed with ISO are due to changes in the SR Ca²⁺ content. We attribute the lack of net gain in SR Ca²⁺ content to the increased leak through RyRs upon stimulation with ISO (see Discussion).

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Figure 7. Estimation of the SR Ca2+ content before and after application of ISO Left-hand panel, representative traces of caffeine-induced Ca2+ transients measured before (left) and after (right) exposure of the cells to 500 nM ISO. Right-hand panel, corresponding mean amplitudes of the signal. The extracellular solution contained 1 mM Ca2+ and 140 mM Na+ (A), 0.2 mM Ca2+ and 140 mM Na+ (B) or 0.2 mM Ca2+ and 140 mM Li+ (C). The Ca2+ transients were induced by rapid application of 10 mM caffeine to the whole bath. The holding potential was -50 mV.

	DISCUSSION

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E-C coupling in cardiac muscle is composed of Ca²⁺ signalling processes at three levels of a CICR cascade: (1) coupling between individual sarcolemmal DHPRs and RyRs in the apposing junctional SR, (2) interaction of neighbouring RyRs present in junctional SR forming a release unit, and (3) interaction between adjacent release units. In the present study, we used confocal Ca²⁺ imaging in conjunction with I_Ca recordings to examine how these different steps are involved in the modulation of E-C coupling by the positive ionotrope ISO. Our results show that -adrenergic enhancement of E-C coupling is caused by an overall increase in the number of Ca²⁺ entry points as well as by an increase in the gain of the CICR cascade. The increase in gain was due to a greater efficiency of DHPR-RyR coupling and an increased tendency of local release events to ignite neighbouring release sites. The increase in efficiency of I_Ca to trigger release events depended on the presence of extra- and intracellular Na⁺, suggesting that NCX plays an important role in -adrenergic potentiation of E-C coupling.

Modulation of DHPR-RyR interactions

Exposure of the cells to ISO resulted in about a 2.5-fold increase in the efficiency of I_Ca in triggering Ca²⁺ sparks at +30 mV. These results indicate that under our control conditions in the absence of ISO, not more than half of the DHPR openings produce activation of RyRs. Thus, our results do not support the notion that DHPR-RyR coupling operates in a saturated mode (Cannell et al. 1994). Rather, they suggest that the fidelity of DHPR-RyR coupling is sub-optimal, thereby providing reserve capacity for up-regulation during ionotropic interventions. Recently, a similar conclusion was reached based on the increased ability of I_Ca to produce Ca²⁺ sparks in transgenic mice with enhanced constitutive 2-adrenergic receptor activity (Zhou et al. 1999). Our conclusion is also consistent with studies of the kinetics of reconstituted RyRs (Zahradnikova et al. 1999). Those studies demonstrated that brief Ca²⁺ stimuli, which mimic Ca²⁺ signals associated with the openings of single DHPR channels, activate RyRs only a fraction of the time and that the probability of activation can be enhanced by increasing the amplitude and duration of the trigger signal.

In general, the increased efficiency of I_Ca in triggering Ca²⁺ sparks should be due to changes in the parameters of the trigger signal and/or changes in the responsiveness of the release mechanism during -adrenergic stimulation. cAMP-mediated signalling has been shown to increase the frequency and lifetime of DHPR channel openings (McDonald et al. 1994). In addition, phosphorylation of RyRs by PKA enhances the activity of this channel as shown previously both in vitro and in vivo (Valdivia et al. 1995; Zhou et al. 1999). Surprisingly, the ISO-induced enhancement in the ability of I_Ca to activate Ca²⁺ sparks required the presence of Na⁺ in the intra- and extracellular millieu (Fig. 4 and Fig. 5). These results suggest that changes in the properties of DHPRs and RyRs by themselves are not sufficient to account for all of the enhancement of DHPR-RyR coupling caused by ISO. A functional NCX is also required for the enhancement of DHPR-RyR coupling by ISO.

Modulation of the number of Ca²⁺ entry points

The potentiation of Ca²⁺ current through cardiac L-type Ca²⁺ channels has been ascribed to either an increase in the probability of opening (Sperelakis et al. 1994) or an increase in the mean open time of the channel due to a shift to a higher activity gating mode (Yue et al. 1990; Kleppisch et al. 1994). An increase in DHPR mean open time would be expected to increase the probability of spark activation regardless of whether NCX is operating or not. In the absence of a functional NCX, the inability of I_Ca to trigger any of the extra sparks observed with an operational NCX suggests that under our experimental conditions -adrenergic stimulation acts primarily by recruiting more DHPR channels rather than by increasing the lifetime of their openings.

Modulation of RyR-RyR interactions

Stimulation by ISO resulted in a significant increase in the overall size and intensity (mass) of discrete Ca²⁺ release events triggered by I_Ca. This increase was due to the greater magnitude of individual sparks as well as to the enhanced ability of primary sparks to activate adjacent release sites resulting in conglomerates of sparks. The changes in magnitude of Ca²⁺ sparks were accompanied by no significant changes in the SR Ca²⁺ loading, as indexed by caffeine-induced Ca²⁺ transients. Therefore, the increase in the magnitude of individual sparks was more likely to be due to increased functional activity of phosphorylated RyRs rather than to an increased amount of releasable Ca²⁺. The enhanced cross- activation of release units could be ascribed to the increased size of individual sparks and the increased sensitivity of RyRs to the Ca²⁺ trigger.

Under normal loading conditions, Ca²⁺ sparks remain localized to individual sites (Cheng et al. 1993). Under conditions of Ca²⁺ overload they give rise to regenerative Ca²⁺ waves that propagate through the entire cell via sequential activation of release sites (Cheng et al. 1996; Lukyanenko et al. 1996). Our results suggest a third scenario in which a propagating signal involving a limited number of release sites is part of the normal physiological response of the cell to Ca²⁺ stimuli during -adrenergic stimulation. Theoretical studies have suggested that there is a certain range of values for RyR Ca²⁺ sensitivity and release flux magnitude within which local Ca²⁺ elevations trigger propagating signals that do not become fully self-sustaining (Lukyanenko et al. 1999). This range might be utilized for physiological modulation of release to increase the gain without causing full-scale spontaneous Ca²⁺ release.

The role of SR Ca²⁺ loading

Interestingly, ISO did not cause an increase in the SR Ca²⁺ load under our experimental conditions (Fig. 7). Therefore, changes in SR Ca²⁺ content are not likely to play a significant role in the observed effects of ISO. We attribute this lack of potentiation to increased cycling of Ca²⁺ through the SR. Indeed exposure to ISO led to significant increases in the frequency and magnitude of spontaneous Ca²⁺ sparks (Table 1). Thus it appears that enhanced Ca²⁺ uptake is balanced by increased Ca²⁺ leak through RyRs so that no net gain in the SR Ca²⁺ content is attained. A similar increase in SR Ca²⁺ cycling was reported in transgenic mice over-expressing the 2-adrenergic receptor (Zhou et al. 1999). Cardiac cells from these transgenic animals show profound increases in Ca²⁺ transients and sparks without changes in either I_Ca or SR Ca²⁺ content. It is important to note that several previous studies reported that -adrenergic stimulation increases the SR Ca²⁺ content in cardiac myocytes (Hussain & Orchard, 1997; Piacentino et al. 2000). The apparent discrepancy between these and our results could be due to differences in the experimental conditions. For example, the changes in SR Ca²⁺ content reported in some previous studies could be ascribed to the enhanced SR Ca²⁺ entry through Ca²⁺ channels upon the periodic electrical stimulations used in those studies. On the other hand, in our experiments performed in resting cells the SR Ca²⁺ content should be at steady state with respect to the resting cytosolic [Ca²⁺] and should not be significantly influenced by Ca²⁺ entry. Although our conditions of low stimulation rate might be useful for studying the modulation of E-C coupling at constant SR Ca²⁺ load, we should stress that alterations in SR Ca²⁺ load are likely to be an important part of the response of the cell to -adrenergic stimulation at more physiological stimulation frequencies.

The role of NCX

One of the most intriguing results of the present study is that the ISO-induced enhancement of the efficiency of I_Ca in triggering Ca²⁺ release required the presence of Na⁺ in the intra- and extracellular milieu. We interpret these results as a strong indication that the reverse mode of NCX might be involved in the observed effects of ISO. Several previous studies have suggested that the reverse mode of NCX can activate CICR (Leblanc & Hume, 1990; Lipp & Niggli, 1994; Kohmoto et al. 1994). The possibility of direct activation of release by NCX under our conditions is excluded by the fact that large voltage steps above the E_Ca failed to cause Ca²⁺ release. The thermodynamic E_Ca under our experimental conditions should be near +110 mV (with 1 mM extracellular Ca²⁺) or +90 mM (0.2 mM extracellular Ca²⁺). In our experiments, Ca²⁺ release was completely abolished only at depolarizing steps above +120 mV. This apparent discrepancy could be ascribed to insufficient voltage control during application of large voltage-clamp steps as well as to the highly non-linear nature of CICR in the presence of ISO. It is clear from our data that both I_Ca and an operational NCX are needed for the observed potentiation of release by ISO.

Because removal of Na⁺ had no apparent impact on the frequency of sparks under our baseline conditions (i.e. in the absence of ISO, Table 1), it is likely that the effect of NCX on Ca²⁺ release results from an increase in its functional activity by ISO. In this regard it has been reported that ISO can increase the NCX current in voltage-clamped cardiac myocytes (Perchenet et al. 2000; but see Main et al. 1997). In addition, preliminary results obtained in our laboratory show that ISO enhances the SR Ca²⁺ entry measured fluorometrically during depolarizing steps to high membrane potentials (above E_Ca) in rat ventricular myocytes in which the SR Ca²⁺ release was abolished with ryanodine.

Our results on the role of NCX in the enhancement of Ca²⁺ release events by ISO are in line with the concept that NCX modulates locally the activity of Ca²⁺ release sites. Bridge and his colleagues (Litwin et al. 1998; Cordeiro et al. 2000) demonstrated that reverse NCX can modulate the gain of CICR in cardiac myocytes under conditions that enhance NCX activity (i.e. elevated intracellular [Na⁺], high membrane potentials). These investigators proposed that when both I_Ca and reverse NCX are operating simultaneously, they might act in synergy to activate Ca²⁺ release. The synergy between the two triggers may arise as a result of the complex, non-linear behaviour of local Ca²⁺ signalling mechanisms, including the steep, sigmoidal dependency of RyR open probability on [Ca²⁺] (Zahradnikova et al. 1999), the binding of Ca²⁺ to saturating buffers in the dyadic cleft (Soeller & Cannell, 1997) and catalytic regulation of NCX by cytosolic Ca²⁺ (Fang et al. 1998). Because of these mechanisms the net Ca²⁺ entry by I_Ca and reverse NCX would not be expected to add in simple linear fashion. Instead the effects of each trigger would tend to amplify the effect of the other. Such non-linear summation of triggers together with the enhanced functional activity of the NCX could potentially explain the combined effects of I_Ca and NCX in the enhancement of E-C coupling by ISO.

Recently, Goldhaber et al. (1999) demonstrated that rapid removal of Na⁺ increases the frequency of Ca²⁺ sparks in patch-clamped cardiac myocytes at a holding potential of -75 mV. This would imply that the forward NCX could also modulate release site activity, in this case by removing Ca²⁺ from the dyadic cleft, thus effectively reducing the threshold for activation of RyRs by Ca²⁺. As mentioned above, our results revealed no differences in the frequency of sparks in Na⁺-containing vs. Na⁺-free solutions in the absence of ISO at steady state. These results, however, do not necessarily contradict the data of Goldhaber et al. (1999). At steady state an initial increase in sparking rate would be expected to reduce the SR Ca²⁺ content, which in turn would cause the sparking activity to decline (Lukyanenko et al. 2000b). This might explain why an increase in sparking rate on removal of Na⁺ was observed in the study by Goldhaber et al. (1999) but not in our study.

Recently it has been proposed that a voltage-dependent mechanism similar to that known to operate in skeletal muscle causes SR Ca²⁺ release in cardiac muscle (Ferrier & Howlett, 1995; Howlett et al. 1998). A key piece of experimental evidence relied upon to support this hypothesis is the occurrence of Ca²⁺ release at strongly positive membrane potentials in the presence of intracellular cAMP. Our results suggest that Ca²⁺ release in this membrane potential range can be ascribed to CICR triggered by a combination of I_Ca and NCX. Our data and the conclusion drawn are in agreement with those communicated in a recent study by Piacentino et al. (2000). These investigators showed that -adrenergic stimulation results in a dramatic broadening of the voltage dependence of contraction in patch-clamped feline ventricular myocytes. This effect could be largely reversed by removal of extra- and intracellular Na⁺ as well as by the inhibitor of the reverse mode of NCX KBR-7943. They concluded that voltage-dependent Ca²⁺ release from the SR in cardiac muscle is explained by CICR.

The role of reverse NCX in cardiac E-C coupling has not been clearly established. Some studies have suggested that NCX can activate release under certain conditions and thereby provide an alternative triggering mechanism to I_Ca (Leblanc & Hume, 1990; Lipp & Niggli, 1994; Kohmoto et al. 1994; Sipido et al. 1997). Other studies, however, have found no evidence of Ca²⁺ release activation by NCX, denying an immediate role for NCX in cardiac E-C coupling (Bouchard et al. 1993; Isenberg & Han, 1994; Lopez-Lopez et al. 1995). Our results help to reconcile these apparently contradictory results and point to a new potential physiological role for the reverse mode of the Na⁺-Ca²⁺ exchanger, this role being modulation of DHPR-RyR coupling during -adrenergic stimulation. The amplitude of the action potential in cardiac muscle reaches +30 to +40 mV. Thus it should be within the range of the effects of NCX on release demonstrated in the present study.

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Acknowledgements

We are grateful to Drs R. Nathan and A. Zahradnikova for critical reading of the manuscript and to Dr J. Bridge for stimulating discussions. This work was supported by the National Institutes of Health (HL 52620, HL 03739).

Corresponding author

S. Györke: Department of Physiology, Texas Tech University HSC, 3601 4th Street, Lubbock, TX 79430, USA.

Email: physg{at}ttuhsc.edu

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