Inositol-1,4,5-trisphosphate-dependent Ca(2+) signalling in cat atrial excitation-contraction coupling and arrhythmias

Abstract

Inositol-1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release represents the major Ca(2+) mobilizing pathway responsible for diverse functions in non-excitable cells. In the heart, however, its role is largely unknown or controversial. In intact cat atrial myocytes, endothelin (ET-1) increased basal [Ca(2+)](i) levels, enhanced action potential-evoked [Ca(2+)](i) transients, caused [Ca(2+)](i) transients with alternating amplitudes (Ca(2+) alternans), and facilitated spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) in the form of Ca(2+) sparks and arrhythmogenic Ca(2+) waves. These effects were prevented by the IP(3) receptor (IP(3)R) blocker aminoethoxydiphenyl borate (2-APB), suggesting the involvement of IP(3)-dependent SR Ca(2+) release. In saponin-permeabilized myocytes IP(3) and the more potent IP(3)R agonist adenophostin increased basal [Ca(2+)](i) and the frequency of spontaneous Ca(2+) sparks. In the presence of tetracaine to eliminate Ca(2+) release from ryanodine receptor (RyR) SR Ca(2+) release channels, IP(3) and adenophostin triggered unique elementary, non-propagating IP(3)R-dependent Ca(2+) release events with amplitudes and kinetics that were distinctly different from classical RyR-dependent Ca(2+) sparks. The effects of IP(3) and adenophostin were prevented by heparin and 2-APB. The data suggest that IP(3)-dependent Ca(2+) release increases [Ca(2+)](i) in the vicinity of RyRs and thus facilitates Ca(2+)-induced Ca(2+) release during excitation-contraction coupling. It is concluded that in the adult mammalian atrium IP(3)-dependent Ca(2+) release enhances atrial Ca(2+) signalling and exerts a positive inotropic effect. In addition, by facilitating Ca(2+) release, IP(3) may also be an important component in the development of Ca(2+)-mediated atrial arrhythmias.

Figure 1. Effects of endothelin-1 (ET-1) on Ca²⁺ signalling in intact cat atrial myocytes

Confocal linescan images and spatially averaged [Ca²⁺]_i transients recorded from field-stimulated (0.5 Hz) atrial myocytes. A, ET-1 (100nm) increased diastolic [Ca²⁺]_i and amplitude of electrically evoked [Ca²⁺]_i transients. Spontaneous [Ca²⁺]_i transients and Ca²⁺ waves (marked by the blue triangles in the right panel) occurred between triggered transients in the presence of ET-1. The traces represent [Ca²⁺]_i expressed as normalized changes of fluo-4 fluorescence (F/F₀). B, ET-1 elicited Ca²⁺ alternans. C, confocal linescan images of spontaneous Ca²⁺ sparks (top) and selected subcellular [Ca²⁺]_i traces (F/F₀, averaged over a distance of 1μm marked by the red triangles to the left of the images) under control conditions (left) and after application of ET-1 (right). ET-1 caused a significant increase in Ca²⁺ spark frequency and resting [Ca²⁺]_i. D, the IP₃R inhibitor 2-APB (2μm) prevented the effects of ET-1 on [Ca²⁺]_i.

Figure 2. Effects of IP₃ and adenophostin on [Ca²⁺]_i in saponin-permeabilized atrial and ventricular myocytes

A, top, confocal linescan images (fluo-4 fluorescence images; a.u., arbitrary fluorescence intensity units) under control conditions and 1 and 5min after exposure to 20μm IP₃. Bottom, local subcellular changes of [Ca²⁺]_i (F/F₀ averaged over 1μm indicated by the red triangles to the left of the images). B, effect of adenophostin (5μm; same experimental conditions as in A. C, average data of the effects of IP₃ and adenophostin on basal [Ca²⁺]_i and Ca²⁺ spark frequency. Statistically different at P < 0.01 (*) and P < 0.001 (**). A–C reflect data obtained from atrial myocytes. D, IP₃ had no effect on basal [Ca²⁺]_i and frequency and properties of Ca²⁺ sparks in permeabilized cat ventricular myocytes, suggesting that the IP₃ effects were specific to atrial myocytes. [Ca²⁺]_i was measured with fluo-3 pentapotassium salt (fluo-3 bath concentration was 40μm).

Figure 3. Effect of inhibitors of IP₃ receptors

A, preincubation of permeabilized atrial myocytes with heparin prevented the effect of IP₃ (50μm) on basal [Ca²⁺]_i and Ca²⁺ sparks. B, same experiment as in A but with the IP₃R blocker 2-APB (5μm). C, 2-APB reversed the increase of basal [Ca²⁺]_i and Ca²⁺ spark frequency induced by preceding exposure to IP₃.

Figure 4. Elementary Ca²⁺ release events from IP₃Rs in atrial myocytes

A, confocal linescan images from permeabilized cat atrial myocytes. Top, tetracaine (1mm) abolished spontaneous Ca²⁺ spark activity. In the presence of tetracaine, IP₃ (20μm) caused an increase in basal [Ca²⁺]_i and the occurrence of non-propagating Ca²⁺ release events with significantly different amplitude and kinetics compared to Ca²⁺ sparks. Heparin abolished the elevation of basal [Ca²⁺] and Ca²⁺ release events. Bottom, localized Ca²⁺ release events elicited with adenophostin in the presence of tetracaine. B, left, average linescan images and F/F₀ traces of Ca²⁺ sparks (black), and IP₃- (red) and adenophostin- (blue) mediated Ca²⁺ release events. Right, normalized amplitudes of local [Ca²⁺]_i transients (F/F₀, norm.) and first derivatives (d(F/F₀)/dt). d(F/F₀)/dt serves as a measure of the underlying Ca²⁺ release flux. C, surface plot representation of averaged linescan images of Ca²⁺ sparks and IP₃R-mediated Ca²⁺ release events from B. Bottom, table showing average values of Ca²⁺ release event amplitude (ΔF/F₀), duration (measured at half maximum amplitude), spatial width (full width at half maximum amplitude) and rise time. ‘Control’ indicates RyR-mediated Ca²⁺ sparks recorded in the absence of tetracaine and IP₃R agonists.