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 microM 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 MOmega) 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 microM 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.

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).

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).

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).

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).