Spatial characteristics of sarcoplasmic reticulum Ca²⁺ release events triggered by L-type Ca²⁺ current and Na⁺ current in guinea-pig cardiac myocytes

Abstract

Ca²⁺ signals in cardiac muscle cells are composed of spatially limited elementary events termed Ca²⁺ sparks. Several studies have also indicated that Ca²⁺ signals smaller than Ca²⁺ sparks can be elicited. These signals have been termed Ca²⁺ quarks and were proposed to result from the opening of a single Ca²⁺ release channel of the sarcoplasmic reticulum. We used laser-scanning confocal microscopy to examine the subcellular properties of Na⁺ current (I_Na)- and L-type Ca²⁺ current (I_Ca,L)-induced Ca²⁺ transients in voltage-clamped ventricular myocytes isolated from guinea-pigs. Both currents, I_Na and I_Ca,L, evoked substantial, global Ca²⁺ transients. To examine the spatiotemporal properties of such Ca²⁺ signals, we performed power spectral analysis of these Ca²⁺ transients and found that both lacked spatial frequency components characteristic for Ca²⁺ sparks. The application of 10 μm verapamil to partially block L-type Ca²⁺ current reduced the corresponding Ca²⁺ transients down to individual Ca²⁺ sparks. In contrast, I_Na-induced Ca²⁺ responses were still spatially homogeneous and lacked Ca²⁺ sparks even for small current amplitudes. By using high resistance patch pipettes (> 4 MΩ) to exaggerate the loss of voltage control during I_Na, Ca²⁺ sparks appeared superimposed on a homogeneous Ca²⁺ release component and were exclusively triggered during the flow of I_Na. In the presence of 10 μm ryanodine both I_Ca,L and I_Na elicited small, residual Ca²⁺ transients that were spatially homogeneous but displayed distinctively different temporal profiles. We conclude that I_Na is indeed able to cause Ca²⁺ release in guinea-pig ventricular myocytes. In contrast to I_Ca,L-induced Ca²⁺ transients, which are built up from the recruitment of individual Ca²⁺ sparks, the I_Na-evoked cellular responses were always homogeneous, indicating that their underlying elementary Ca²⁺ release event is distinct from the Ca²⁺ spark. Thus, I_Na-induced Ca²⁺ transients are composed of smaller Ca²⁺ signals, most likely Ca²⁺ quarks.

Cardiac excitation-contraction coupling (E-C coupling) links the electrical excitation of the cell membrane, the action potential, to the mechanical activity of the contractile machinery (Callewaert, 1992; Bers, 2001). An important step during this signal transduction is a transient rise in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). This Ca²⁺ transient is composed of both, Ca²⁺ influx across the cell membrane and Ca²⁺ release from intracellular stores, the sarcoplasmic reticulum (SR). The precise contribution of each mechanism is dependent on the species (Bers, 2001) and on SR Ca²⁺ load (Han et al. 1994; Spencer & Berlin, 1995; Santana et al. 1997). Ca²⁺ entry through voltage-activated Ca²⁺ channels in the plasma membrane is believed to trigger the release of Ca²⁺ from the SR by a mechanism referred to as Ca²⁺-induced Ca²⁺ release (CICR; for reviews see Callewaert, 1992; Niggli, 1999). The relaxation of cardiac myocytes is mediated by various Ca²⁺ transport processes. The two most important mechanisms responsible for the removal of ${\text{Ca}}_{\text{i}}^{2+}$ are the SR Ca²⁺ pump and the Na⁺-Ca²⁺ exchanger in the plasma membrane (for a recent review see Egger & Niggli, 1999; Lipp & Bootman, 1999). The plasma membrane Ca²⁺ pump removes a minor component, although the exact quantitative contributions again vary between different species (Bers, 2001).

The Na⁺-Ca²⁺ exchange is a process controlled by the concentration gradients of both Na⁺ and Ca²⁺ as well as the membrane voltage across the plasma membrane. This enables the exchanger to mediate Ca²⁺ extrusion in its ‘forward mode’ (i.e. Na⁺ influx and Ca²⁺ efflux), but Ca²⁺ can also enter the cell during its ‘reverse mode’ of operation (Na⁺ efflux and Ca²⁺ influx; e.g. Horackova & Vassort, 1979; Crespo et al. 1990). While it was known that slow and tonic Ca²⁺ entry can occur via Na⁺-Ca²⁺ exchange, LeBlanc & Hume (1990) provided experimental evidence for the ability of the Na⁺-Ca²⁺ exchange to trigger fast SR Ca²⁺ release during E-C coupling. Their observation also implied the existence of a subsarcolemmal space with restricted diffusion, referred to as the ‘fuzzy space’, to which Ca²⁺ and Na⁺ channels as well as the Na⁺-Ca²⁺ exchanger have preferential access (Lederer et al. 1990a, b) and from which ions would only slowly diffuse into the cytoplasm. Activation of the fast I_Na is believed to lead to a short-lived accumulation of Na⁺ in the ‘fuzzy’ space (model calculation revealed concentrations of up to 30 mm; Lederer et al. 1990b; see also Wendt-Gallitelli et al. 1993), which in turn would be sufficient to cause reverse-mode Na⁺-Ca²⁺ exchange. It was postulated that this Ca²⁺ entry might be able to trigger SR Ca²⁺ release. Although evidence supporting such a sodium current (I_Na)-induced Ca²⁺ release has subsequently been reported by others (e.g. Lipp & Niggli, 1994), this notion did not remain unquestioned. Instead, various experimental studies could not find I_Na-induced Ca²⁺ release under similar experimental conditions and proposed other mechanisms causing this phenomenon (see for example Sham et al. 1992; Bouchard et al. 1993; Sipido et al. 1995; Evans & Cannell, 1997).

One alternative explanation involved a loss of voltage control during the large cardiac I_Na, which can reach amplitudes of more than 10 nA (Makielski et al. 1987). The idea was that during I_Na voltage control is partly lost, which in turn accidentally activates I_Ca,L (L-type Ca²⁺ current). Presumably, the resulting spurious Ca²⁺ entry could trigger SR Ca²⁺ release by CICR and I_Na-induced Ca²⁺ release could then be seen as an experimental artefact.

In the present paper, we examined the subcellular characteristics of SR Ca²⁺ release events induced by I_Ca,L and I_Na with rapid line-scanning confocal microscopy of voltage-clamped guinea-pig ventricular myocytes. We show that both ionic currents recruit distinct and separate modes of SR Ca²⁺ release that can be distinguished based on their ‘Ca²⁺ signalling fingerprint’ and based on their pharmacological profile.

METHODS

Cell isolation and solutions

All experiments were carried out according to the Swiss Animal Protection Law and with the permission of The State Veterinary Office, Bern, Switzerland. Guinea-pig ventricular myocytes were isolated using a standard enzymatic procedure described previously (Lipp & Niggli, 1994). In brief, adult animals were killed by cervical dislocation, the heart rapidly removed and retrogradely perfused on a Langendorff perfusion system at 37 °C. The perfusion solution contained (mm): NaCl 135, KCl 5.4, MgCl₂ 1, NaH₂PO₄ 0.33, Hepes 5, glucose 11, pH 7.3 adjusted with NaOH. After 5 min, collagenase B (Boehringer Mannheim, Rotkreuz, Switzerland) and protease type XIV (Sigma, Buchs, Switzerland) were added to a final concentration of 0.2 and 0.04 mg ml⁻¹, respectively, and the perfusion continued for another 4–6 min. Subsequently, the ventricles were minced and placed in perfusion solution containing 200 μm CaCl₂ on a rocking table to allow for dissociation of the tissue. Cells were taken from the supernatant, transferred into a recording chamber which was mounted on the stage of an inverted microscope (Diaphot TMD, Nikon, Küsnacht, Switzerland). Ca²⁺-resistant cells readily adhered to the coverslip without coating and were constantly superfused (1-2 ml min⁻¹) with cellular solution containing (mm): NaCl, 135, KCl 5, CaCl₂ 2, glucose 10, MgCl₂ 2, Hepes 10 (pH 7.4 adjusted with NaOH). In order to inhibit L-type Ca²⁺ channels, verapamil was added (10 μm) from a stock solution. All experiments were performed at room temperature (20-22 °C). Extracellular solutions were exchanged by a custom-made solenoid-driven fast perfusion system (time for solution change < 500 ms).

Electrophysiological recordings

Single guinea-pig ventricular myocytes were voltage clamped in the whole-cell configuration of the patch-clamp technique. Patch pipettes were pulled from borosilicate glass on a horizontal puller (Zeitz, Augsburg, Germany). Filled with the intracellular solution, the pipettes had a final resistance of 1–2 MΩ. In some experiments we deliberately produced pipettes with a higher resistance of > 4 MΩ. The solutions used for filling the pipettes consisted of (mm): caesium aspartate 120, TEA-Cl 20, Hepes 10, MgATP 5, MgCl₂ 1, fluo-3 0.1, pH 7.2 adjusted with CsOH. Application of voltage pulses and recording of the membrane current was performed with an Axopatch 200 patch clamp amplifier (Axon Instruments, Foster City, CA, USA) controlled by custom-made software running under LabView (National Instruments, Ennetbaden, Switzerland). Residual 50 Hz noise was removed off-line by a custom-made routine based on IgorPro software (WaveMetrics, Lake Oswego, OR, USA).

Stimulation protocols

To ensure similar SR Ca²⁺ loading between cells, we employed a pre-pulse protocol. Ten voltage steps (250 ms in duration, at 0.5 Hz) from a holding potential of −50 to +10 mV were applied before stepping to the appropriate holding potential of −50 mV (for eliciting I_Ca,L) or −90 mV (for eliciting I_Na). For experiments in which I_Ca,L was blocked by verapamil, the pre-pulse protocol comprised voltage steps (250 ms in duration, at 0.5 Hz) from −50 to +50 mV and thus used reverse-mode Na⁺-Ca²⁺ exchange to load the SR.

Calcium measurements

The spatiotemporal properties of I_Na- and I_Ca,L-induced Ca²⁺ transients were examined using a laser-scanning confocal microscope (BioRad MRC 1000, Glattbrugg, Switzerland). Due to the limited temporal resolution of the confocal microscope in its image mode, we performed line-scanning at a rate of 500 lines s⁻¹ as shown in Fig. 1. For this, a single line along the longitudinal cell axis was chosen and repetitively scanned. Data analysis was performed off-line on a Macintosh PowerPC 8100 computer (Apple, Walisellen, Switzerland) running a modified version of NIH-image (NIH, Bethesda, USA). Changes of Ca²⁺_i were calculated using the self-ratio method as described earlier (Lipp & Niggli, 1994) assuming a K_d value for fluo-3 of 480 nm (Molecular Probes, Oregon, USA).

Figure 1.

Power spectra of line-scan images (montage)

Traces show from top to bottom: voltage protocol, current recording, time course of average Ca²⁺ concentration, a confocal image of a fluo-3-loaded guinea-pig ventricular myocyte. The black line indicates the position of a single scanned line (longitudinal) to record fluorescence vs. time, shown in the right panel. A voltage-clamp depolarisation from −80 to −50 mV activated I_Na and a homogeneous Ca²⁺ influx signal. Power spectra in the spatial domain and the average power (average P_norm) over time (spatial low-frequency components averaged between > 0 and 0.13 lines μm⁻¹). Spatial uniformity of the Ca²⁺ release is confirmed in the power spectra. For this purpose, the raw line-scan images were Fourier transformed in the spatial domain (i.e. in the vertical direction) line by line and the calculated power spectra of all lines were arranged in an identical order to the original line-scan image. For normalisation the power of each frequency component (P_i) was divided by the zero-frequency component (P₀) to obtain P_norm (see Lipp & Niggli, 1996).

Digital image processing (see also Fig. 1)

Figure 2.

I_Ca,L- and I_Na-induced SR Ca²⁺ release

A-D, from top to bottom: voltage protocols for activating L-type Ca²⁺ currents (A and B) and sodium currents (C and D), the resulting I_Ca,L and I_Na, the averaged power spectra in the spatial domain of the line-scan images and the Ca²⁺ signals as 3D-surface plots of the line-scan images. B and D, I_Ca,L and I_Na, respectively, and the corresponding CICR signal observed in the same cardiac myocyte as in A and C after application of 10 μm verapamil for 2 min. B, although I_Ca,L was reduced by approximately 95 %, inhomogeneous SR Ca²⁺ release was still present, characterised by low-frequency components of the power spectra. D, the I_Na-triggered SR Ca²⁺ release signal was largely homogeneous and verapamil insensitive. The power spectra exhibit no localised Ca²⁺ release events (Ca²⁺ sparks) in the low frequency range. E, comparison of the low-frequency components of the power spectra of I_Ca,L-induced SR Ca²⁺ signals in the presence of verapamil (B, Ca²⁺ sparks, n = 21) vs. the power spectra of homogeneous SR Ca²⁺ signals induced by I_Ca,L (A, n = 15) and I_Na-induced Ca²⁺ transients (D, n = 11) (mean ± s.e.m., **P < 0.05).

For the power spectral analysis of the line-scan images, the IDL software package was used (Research Systems, Boulder, CO, USA). Initially, we calculated the Fourier transform (in space) of each individual line of the line-scan image as a two-dimensional array, whereby the order of the resulting line-data was preserved. Inhomogeneous distribution of fluo-3 fluorescence along the line was compensated for by normalising each line in the power spectrum image by the zero-order component (P₀) (Cheng et al. 1993). We have shown recently that Ca²⁺ sparks can readily be identified in line-scan images by their characteristic spectral component in the lower spatial frequency range (Lipp & Niggli, 1996; for details see also Cannell et al. 1994). For the present paper, we averaged the low frequency components (between > 0 and < 0.13 lines μm⁻¹) and expressed them as normalised power ratio vs. time. In control line-scan images, we found that these low frequency components were a sufficiently sensitive indication for the presence of Ca²⁺ sparks in the raw data (not shown). The myocytes were scanned longitudinally; however, the sarcomeric periodicity was not apparent in the line-scan images and in the power spectral analysis. In this paper, we speak of homogeneous Ca²⁺ signals in the absence of any periodicity in the line-scan image. Nevertheless, noticeable non-periodic inhomogenieties were apparent in almost all line-scan recordings (e.g. Fig. 2A). The values were expressed as means ± s.e.m. Student's paired t tests were used to test for significance. Significance is given as P < 0.05 or < 0.1, as indicated in the figure legends.

RESULTS

Properties of I_Ca,L- but not I_Na-induced Ca²⁺ release were altered by verapamil

In voltage-clamped guinea-pig ventricular myocytes, stepping the membrane voltage from −50 to +5 mV or from −90 to −50 mV elicited I_Ca,L- or I_Na-induced Ca²⁺ release, respectively, that both caused a substantial increase in [Ca²⁺]_i (Fig. 2A and C). In Fig. 2 as well as the following figures, the [Ca²⁺]_i calculated from the line-scan images was coded in the height and the colour of 3D-surface representations in order to allow for a rapid assessment of the spatiotemporal properties of the subcellular Ca²⁺ transients. While the I_Ca,L-induced responses showed a rising phase lasting several milliseconds (Fig. 2A), the signals elicited by I_Na were characterised by a very brief upstroke (Fig. 2C). However, both transients appeared to be spatially uniform, i.e. Ca²⁺ release occurred evenly along the recorded line. In order to substantiate this qualitative observation, we quantified the subcellular properties of the Ca²⁺ signals by calculating power spectra of the line scans, as described earlier (Lipp & Niggli, 1996). Particular attention was paid to the characteristic low spatial frequencies indicative of the appearance of detectable Ca²⁺ sparks (Cannell et al. 1994). For simplification, we averaged those spatial frequencies that were characteristic for the existence of Ca²⁺ sparks (between 0 and 0.13 lines μm⁻¹). The time course for the normalised power ratio present in this range of spatial frequencies is shown above the surface plot in this and all subsequent figures. The lack of abrupt time-dependent changes in this plot indicates the absence of identifiable Ca²⁺ sparks during both I_Ca,L- and I_Na-induced Ca²⁺ transients. Figure 2B and D shows current and Ca²⁺ recordings from the same cardiac myocyte after application of 10 μm verapamil for 2 min. Although the Ca²⁺ current was reduced by more than 95 % in B, SR Ca²⁺ release could still be elicited. In comparison to the control situation (Fig. 2A), the Ca²⁺ signal observed in the presence of verapamil was no longer homogeneous, instead it seemed to comprise a limited number of distinct Ca²⁺ sparks. This observation was substantiated by the power spectral analysis. During the voltage step a transient rise in the low-frequency components could be observed, suggesting the presence of Ca²⁺ sparks. In contrast to the recordings obtained during the activation of the I_Ca,L, the I_Na-induced Ca²⁺ signal was largely unaffected by verapamil (Fig. 2D). Despite a marginal decrease in the signal amplitude, the Ca²⁺ transient was still homogeneous, which was confirmed in the power spectral analysis by the absence of low frequency components. The results are summarised in Fig. 2E, where the power spectra of Ca²⁺ sparks (Fig. 2B) are compared with the power spectra of homogeneous SR Ca²⁺ signals induced by I_Ca,L (Fig. 2A) and I_Na (Fig. 2D). There are several possible explanations for the slight decrease in the amplitude of the Ca²⁺ transient in the presence of verapamil. First, verapamil could have inhibited a contaminating I_Ca,L-induced Ca²⁺ signal present under control conditions. Second, Ca²⁺ loading of the SR might have been slightly lower than under control conditions, since Ca²⁺ entry through L-type Ca²⁺ channels was blocked during the pre-pulse protocol, although we largely relied on the loading of the SR by reverse-mode Na⁺-Ca²⁺ exchange under those conditions (see Methods section). Third, verapamil has also been reported to slightly block I_Na (Bustamante, 1985) and the reverse mode of the Na⁺-Ca²⁺ exchanger (Egger & Niggli, 1999).

From these experiments, we concluded that partial inhibition of I_Ca,L could reduce the Ca²⁺ release signals from a homogeneous signal to a signal consisting of a few Ca²⁺ sparks, while the spatial properties of the I_Na-induced transients remained unaffected by verapamil. Since Ca²⁺ sparks cannot be resolved if their number is too large, the possibility exists that I_Na recruited too many Ca²⁺ sparks and thus prevented their identification as individual signalling events. Alternatively and more interestingly, I_Na could have triggered Ca²⁺ release from the SR with a different spatial signature (i.e. homogeneous SR Ca²⁺ release vs. Ca²⁺ sparks).

Na⁺ current-induced SR Ca²⁺ release did not recruit Ca²⁺ sparks

To examine these possibilities, we used experimental conditions that were expected to facilitate the activation of a few L-type Ca²⁺ channels and the subsequent activation of Ca²⁺ sparks. In the first series of experiments, we elicited I_Na under conditions of deliberate loss of voltage control. For these experiments, we used patch-clamp electrodes with a slightly higher series resistance and we did not compensate for electrode resistance. Under such conditions, the membrane potential is known to escape towards the reversal potential for Na⁺ during flow of I_Na (Hüser et al. 1996). Figure 3 depicts typical current and Ca²⁺ recordings from such an experiment and shows that the Ca²⁺ signals elicited under those conditions had lost their homogeneity. Similar transients were found in eight other cells studied under similar conditions. The spatial inhomogeneity can be read from the three-dimensional surface plot of the line-scan image and also from the transient surge of low frequencies in the power spectral analysis depicted in Fig. 3A. The Ca²⁺ transient seemed to consist of two superimposed components; the first one was an apparently homogeneous socket of Ca²⁺ and resembled the Na⁺ current-induced Ca²⁺ signal seen in Fig. 2 while the second component resembled Ca²⁺ sparks. These two signal components are indeed compatible with the idea of a simultaneous activation of Na⁺ and Ca²⁺ channels whereby the homogeneous signal is evoked by the Na⁺ current-induced Ca²⁺ release while the Ca²⁺ sparks are triggered by the spurious activation of a few L-type Ca²⁺ channels during the brief loss of voltage control.

Figure 3.

I_Na-induced SR Ca²⁺ release events (Ca²⁺ sparks)

A-C, from top to bottom: voltage protocols for activating sodium currents, the resulting I_Na, the averaged power spectra in the spatial domain and the corresponding Ca²⁺ signals as 3D-surface plots of the line-scan images. A, I_Na measured under conditions of loss of voltage control (patch electrodes with higher series resistance). The resulting inhomogeneous Ca²⁺ transient consisted of two superimposed components; a Ca²⁺ spark component and an apparently homogeneous I_Na-induced component. B, I_Na and the corresponding homogeneous SR Ca²⁺ release signal observed in the same cardiac myocyte after application of 10 μm verapamil for 2 min. In the presence of the L-type Ca²⁺ channel blocker only the Ca²⁺ spark components were suppressed, indicating that spurious activation of L-type Ca²⁺ channels did not contribute to the I_Na-induced SR Ca²⁺ release. C, I_Na-induced Ca²⁺ transients induced by decreasing the depolarisation (voltage step to −65 mV). The resulting Ca²⁺ signal was decreased compared to Fig. 2B. Nevertheless, the spatial properties of the homogeneous Ca²⁺ release transient remained unchanged. Similar results were found in all myocytes tested (n = 8).

We verified this notion by applying verapamil that should, under these conditions, suppress only those Ca²⁺ signals originating from the activation of Ca²⁺ channels but not the homogeneous Ca²⁺ release signal triggered by I_Na and subsequent Na⁺-Ca²⁺ exchange reverse mode. Figure 3B shows the result of a verapamil treatment of the same cell as in Fig. 3A. While the localised Ca²⁺ signals seen in the absence of the L-type Ca²⁺ channel blocker were suppressed, the homogeneous component of the Ca²⁺ signal was maintained. The suppression of inhomogeneities was again confirmed with the power spectral analysis. From these results we concluded that spurious activation of Ca²⁺ channels did not contribute to the homogeneous I_Na-induced SR Ca²⁺ release, but rather was responsible for the few clearly visible Ca²⁺ sparks positioned on top of the homogeneous Ca²⁺ signal.

The second question we addressed in this series of experiments was related to the nature of SR Ca²⁺ release induced by I_Na. We used an approach that was analogous to that applied for the identification of the elementary Ca²⁺ release events underlying I_Ca,L-induced Ca²⁺ release, a reduction of the number of activated channels. In the case of I_Ca,L this can be achieved either by inhibiting Ca²⁺ channels or by depolarisations to the threshold of current activation (Cannell et al. 1994, 1995; Lopez-Lopez et al. 1995). For the I_Na-induced Ca²⁺ release we chose the second protocol by reducing the degree of depolarisation. A representative example of such a small voltage step (here to −65 mV) is shown in Fig. 3C. The traces were obtained from the same cell as in Fig. 3A and B. The resulting Ca²⁺ signal was reduced in amplitude by more than 70 %. Despite this change in the amplitude, the spatial properties of the release transient remained unaltered; it was still homogeneous. This important observation was confirmed for all voltages between −75 (the threshold potential for eliciting I_Na in our experiments) and −50 mV (data not shown). More positive depolarisations were not examined because of the risk of contaminating activation of I_Ca,L. From these findings we concluded that I_Na-induced Ca²⁺ release did not recruit Ca²⁺ sparks, instead Ca²⁺ release events were evoked that resulted in a homogeneous SR Ca²⁺ release signal over the entire voltage range.

Ca²⁺ release did not reflect the spatial properties of I_Na and I_Ca,L

We were left with the question of why I_Na and I_Ca,L could recruit different elementary Ca²⁺ release events. Or in other words, does the I_Na- and I_Ca,L-induced Ca²⁺ release reflect the spatial properties of the Ca²⁺ influx signals or does the discrepancy arise from the recruitment of different populations of RyRs? In order to examine this, we analysed the subcellular characteristics of I_Na- and I_Ca,L-induced Ca²⁺ influx signals. Figure 4A and B depicts results from a guinea-pig ventricular myocyte in which SR Ca²⁺ release had been inhibited by preincubation of the cell in 10 μm ryanodine for 20 min, a procedure that completely eliminates Ca²⁺ release via RyRs (Bers, 2001). Depolarisations from holding potentials of −50 and −90 mV elicited I_Ca,L and I_Na, respectively. The Ca²⁺ influx signal carried by I_Ca,L had a slow rising and decay phase, as has been described earlier (Cannell et al. 1994). These Ca²⁺ influx signals were homogeneous over the entire voltage range tested (-30 to +30 mV, data not shown). We could not assess the subcellular properties of the Ca²⁺ signals for smaller or larger depolarisations since the resulting Ca²⁺ transients were too small to allow for a reliable power spectral analysis. Membrane depolarisations from a holding potential of −90 to −50 mV elicited I_Na and an accompanying Ca²⁺ influx component (Fig. 4B). As shown previously, these I_Na-induced Ca²⁺ influx signals were insensitive to L-type Ca²⁺ channel inhibitors (Lipp & Niggli, 1994) and could therefore be attributed to the activation of I_Na and subsequent Ca²⁺ entry via Na⁺-Ca²⁺ exchange. We verified this in experiments where we substituted Li⁺ for Na⁺, an intervention which resulted in a complete suppression of the Ca²⁺ influx transients during voltage protocols activating Na⁺ channels (Fig. 4C). As for I_Ca,L, we could only test a narrow range of voltages eliciting I_Na, because of the limiting signal-to-noise ratio for the power spectral analysis (tested voltages: between −70 and −50 mV; data not shown). Figure 4D shows the statistical comparison of [Ca²⁺]_i transients induced by large (Fig. 4B) and small I_Na (Fig. 3C) in myocytes with intact and blocked SR Ca²⁺ release function (ryanodine-treated cells). In both cases, I_Na significantly triggered SR Ca²⁺ release. From these experimental results, we conclude that the spatial properties of the Ca²⁺ influx signals induced by the activation of I_Na and I_Ca,L did not reflect the subcellular properties of the elementary Ca²⁺ release signals recruited by each particular current. Therefore, submicroscopic spatial features or the kinetics of the respective Ca²⁺ entry signals into the dyadic cleft may determine the type of subsequent SR Ca²⁺ release event that is activated (i.e. Ca²⁺ sparks or homogeneous release).

Figure 4.

I_Na- and I_Ca,L-induced Ca²⁺ transients in the absence of SR Ca²⁺ release function

A and B, from topSR to bottom: voltage protocols for activating L-type Ca²⁺ currents and sodium currents, respectively, the resulting I_Ca,L and I_Na, the averaged power spectra in the spatial domain and the resulting Ca²⁺ signals as 3D-surface plots of the line-scan images under conditions of blocked SR (myocytes were preincubated in 10 μm ryanodine for 20 min). C, I_Na-induced [Ca²⁺]_i transient most probably mediated by Ca²⁺ entry via Na⁺-Ca²⁺ exchange; substitution of Li⁺ for Na⁺ resulted in a complete suppression of this Ca²⁺ influx signal during I_Na. D, I_Na-induced [Ca²⁺]_i transients: comparison of [Ca²⁺]_i changes induced by large I_Na (voltage steps from −90 to −50 mV) in control (n = 25) and ryanodine-treated myocytes (n = 8) and between [Ca²⁺]_i changes induced by small I_Na (voltage steps from −90 to −65 mV) in control (n = 7) and ryanodine-treated myocytes (n = 5; mean ± s.e.m., **P < 0.001 vs. control large I_Na, *P < 0.05 vs. control small I_Na).

DISCUSSION

During the last few years, the established view of cardiac E-C coupling and Ca²⁺ signalling has been challenged by several unexpected experimental findings. Some of these observations appeared to suggest trigger pathways for CICR other than the well-known activation via L-type Ca²⁺ channels. This includes Ca²⁺ entry by reverse mode Na⁺-Ca²⁺ exchange, particularly following activation of Na⁺ influx via I_Na (LeBlanc & Hume, 1990; Lipp & Niggli, 1994). In some studies, depolarisation and the subsequent changes of the electrochemical gradients for Na⁺ and Ca²⁺ appeared to be sufficient to elicit Na⁺-Ca²⁺ exchange reverse mode (i.e. no Na⁺ influx was required; Levi et al. 1994). A possible contribution of T-type Ca²⁺ current to triggering CICR has also been suggested in several reports (for example see Sipido et al. 1998). More recently, changes of Ca²⁺ signalling after β-adrenergic stimulation have been interpreted to arise from a reduced selectivity of the TTX-sensitive Na⁺ channels with subsequent influx of Ca²⁺ (‘slip-mode conductance’; Santana et al. 1998). Based on other experimental results, β-adrenergic stimulation was also proposed to unmask a direct allosteric coupling between L-type Ca²⁺ channels and RyRs, giving rise to voltage-induced Ca²⁺ release (Hobai et al. 1997; Ferrier et al. 1998; Howlett et al. 1998). None of these additional trigger pathways is fully understood and each has been disputed because of published negative results. A common conclusion of many papers reporting negative results was that the apparent additional trigger pathway might have resulted from an experimental artefact (Sham et al. 1992; Bouchard et al. 1993; Evans & Cannell, 1997; Sipido et al. 1997).

Homogeneous Ca²⁺ release is distinct from Ca²⁺ sparks

Similar to many other cell types, cardiac Ca²⁺ signalling and E-C coupling is notoriously complex. This partly results from the diversity of possible pathways for Ca²⁺ entry, including several types of voltage-dependent Ca²⁺ channels. In addition, Ca²⁺ transporters that usually remove cytosolic Ca²⁺, such as the Na⁺-Ca²⁺ exchanger, can run in a reverse or Ca²⁺ influx mode. Although some separation between these pathways can be achieved with specific voltage-clamp protocols, the voltage range over which activation or steady-state inactivation occurs shows considerable overlap between the different current components. Moreover, the available pharmacological tools are less than perfect, many show use- or voltage-dependent block or lack specificity. Therefore, experiments aimed at the identification of specific pathways need to be designed and analysed very carefully and the necessary control experiments need to be done.

However, some of the mentioned alternative pathways for Ca²⁺ entry can be ruled out for the present study. The pipette filling solution did not contain cAMP and the cells were not β-adrenergically stimulated. Thus, both voltage-activated Ca²⁺ release and slip-mode influx of Ca²⁺ would not be expected under the experimental conditions used. In addition, for the Ca²⁺ release signals observed here we can exclude T-type Ca²⁺ current as the underlying trigger, since the Ca²⁺ signals were selective for Na⁺ over Li⁺, which is more consistent with the Na⁺-Ca²⁺ exchange as the underlying transporter. Taken together, the homogeneous Ca²⁺ release signals elicited by the activation of I_Na most probably resulted from activation of the Na⁺-Ca²⁺ exchange in the Ca²⁺ influx mode, but, as discussed below, another possible complication had to be considered to further substantiate this conclusion.

The notion that reverse-mode Na⁺-Ca²⁺ exchange is activated during I_Na and is subsequently able to trigger SR Ca²⁺ release, has been challenged in the past (Sham et al. 1992, Bouchard et al. 1993; Sipido et al. 1997). The issue is not yet resolved completely, but species differences in the expression of the Na⁺-Ca²⁺ exchanger have been implied to be important (Sham et al. 1995). As another alternative, it was proposed that a pronounced loss of voltage control and the resulting voltage escape during the large I_Na may induce spurious activation of L-type Ca²⁺ channels, that in turn could have activated SR Ca²⁺ release (Evans & Cannell, 1997). By using a combination of whole-cell voltage-clamp and fluorescent potential-sensitive indicators, we have previously been able to visualise the inevitable voltage escape during I_Na directly (Hüser et al. 1996). Furthermore, we found that in cells that had undergone β-adrenergic stimulation, spurious activation of L-type Ca²⁺ channels during voltage escape was indeed dramatically enhanced (DelPrincipe et al. 2000). We therefore had to account for such a possibility in the current study.

With the recent introduction of sophisticated fluorescence imaging techniques, in particular confocal microscopy, spatially restricted Ca²⁺ signals had been resolved, such as the Ca²⁺ sparks in muscle cells (for a review see Lipp & Niggli, 1996). These local Ca²⁺ signals exhibit well defined and easy to recognise spatial and temporal features (also referred to as ‘Ca²⁺ signalling fingerprint’). In the present study, we took advantage of the confocal imaging technique to discriminate among different triggers and subsequent SR Ca²⁺ release signals.

The analysis of the ‘Ca²⁺ signalling fingerprint’ of Ca²⁺ sparks now allowed us to directly rule out such a possible artefact. Whenever the problem of voltage escape was present in our Ca²⁺ recordings, we could readily identify the evoked Ca²⁺ sparks, even if only a few L-type Ca²⁺ channels were activated. In experiments where we deliberately caused significant loss of voltage control (Fig. 3A), Ca²⁺ sparks appeared as superimposed Ca²⁺ signals on top of the homogeneous, I_Na-induced Ca²⁺ transient. Moreover, these additional local signals were only triggered during the short period of current flow via Na⁺ channels and were sensitive to inhibition by verapamil, an inhibitor of L-type Ca²⁺ channels. Thus, these Ca²⁺ sparks were most likely triggered by spurious activation of L-type Ca²⁺ channels during voltage escape. Taken together, the ‘Ca²⁺ signalling fingerprint’ of the Ca²⁺ sparks allowed us to indirectly detect openings of a few L-type Ca²⁺ channels with a sensitivity far superior to the analysis of the voltage-clamp current traces. Therefore, we are left with the question: what is the nature of the homogeneous Ca²⁺ release signal following I_Na? It clearly arises from SR Ca²⁺ release since it was dramatically reduced after treatment with ryanodine (to eliminate CICR). Since it was spatially homogeneous irrespective of its amplitude, the elementary Ca²⁺ release events underlying this type of SR Ca²⁺ release were most likely smaller than a Ca²⁺ spark. In many respects, these signals resembled those observed following flash-photolytic activation of CICR in the same cell type (Lipp & Niggli, 1996). Based on those homogeneous Ca²⁺ release transients we had proposed the existence of a tiny Ca²⁺ release signal corresponding to the Ca²⁺ release flux via a single RyR, the Ca²⁺ quark. Using two-photon excitation photolysis of caged Ca²⁺, we were later able to elicit Ca²⁺ signals arising from events exhibiting 20 to 40 times smaller release flux than a typical Ca²⁺ spark, possibly corresponding to such Ca²⁺ quarks (Lipp & Niggli, 1998; Niggli, 1999). Similar homogeneous Ca²⁺ release signals were also reported in skeletal muscle after suppression of Ca²⁺ sparks (Shirokova & Rios, 1997), where they were termed ‘small event Ca²⁺ release’. Likewise, when the cardiac muscle isoform of the RyR (RyR2) was heterologously expressed in Chinese hamster ovary (CHO) cells, uniform Ca²⁺ release signals without noticeable Ca²⁺ sparks were recorded (Bhat et al. 1999). Taken together, the homogeneous Ca²⁺ release signals elicited by Na⁺ current most likely arise from the initiation of a large number of unresolved Ca²⁺ quarks.

When comparing the amplification of the Ca²⁺ influx signals by CICR, I_Na-induced signals seem to be more efficient in triggering SR Ca²⁺ release. However, a direct comparison of the trigger efficiency of I_Na and I_Ca,L is very difficult because of the vastly different durations of the two influx signals. While in the present experiments most of the Ca²⁺ influx resulting from I_Na served as a trigger for CICR, only the first few milliseconds of the I_Ca,L are triggering release (Sham et al. 1998). The large remainder of the current only leads to refilling of the SR (Trafford et al. 2001). Thus, when only considering the Ca²⁺ influx during the first few milliseconds of I_Ca,L, the signal amplification and E-C coupling efficiency of this pathway is far superior.

The missing mechanism for RyR inactivation

Contemporary published computer models of Ca²⁺ signalling in the dyadic cleft do not seem to be able to account for the present findings. Generally, these models predict that opening of a single RyR channel results in a rapid spread of the Ca²⁺ signal within the dyadic cleft, reaching almost millimolar concentrations within less than a millisecond (Cannell & Soeller, 1997; Stern et al. 1999). Thus, all RyRs within a cluster of channels would rapidly become activated by CICR and the cluster would exhibit a high degree of positive feedback, essentially resulting in a stereotypical all-or-none response. This model prediction is, however, not only at odds with our observation of Ca²⁺ quarks, but also with more recent estimations of the number of channels participating in a Ca²⁺ spark. By recording single channel currents from L-type Ca²⁺ channels and the resulting Ca²⁺ signals simultaneously, the number of RyRs underlying a Ca²⁺ spark has been estimated to be in the range of four to six (Wang et al. 2001), which is surprising given the fact that freeze-fracture studies seemed to suggest a much larger number of feet per dyad (Franzini-Armstrong et al. 1999). Thus, it appears that the presently prevailing understanding of Ca²⁺ signalling in the dyadic cleft (and the computer models developed based on these ideas) are missing some important features. Most probably, we need to have a better conception for inhibitory mechanisms which may not only govern the degree of activation within a cluster of RyRs, but may also be in charge for the termination of Ca²⁺ release (Sham et al. 1998; DelPrincipe et al. 1999). Unfortunately, the mechanisms linking the individual RyRs within a functional Ca²⁺ release unit are not at all clear. There is indirect evidence indicating that CICR may not be the only message these channels understand. For example, communication and synchronisation by allosteric interaction between the densely packed channel proteins has been proposed (Marx et al. 1998, 2001; Bers & Fill, 1998). In fact, such allosteric mechanisms could not only confer synchronised activation between adjacent channels, but could also communicate the inhibitory signals that seem to be required for both, partial activation of a cluster of RyRs (to account for Ca²⁺ quarks), as well as termination and stability of Ca²⁺ release (Stern et al. 1999).