An increase in the production of endothelin-1 (ET-1) by cells of the heart has been implicated in the generation of arrhythmias in chronic heart failure and following acute myocardial infarction (Duru et al. 2001). Here we attempt to demonstrate that mobilisation of inositol 1, 4, 5-triphosphate (IP₃) is sufficient to generate spontaneous extra Ca²⁺ transients (ECTs) in isolated rat ventricular myocytes (IRVMs) (rats were humanely killed by CO₂ inhalation and cervical dislocation). This has been done using IP₃BM (a membrane-permeable ester of IP₃, the ester is cleaved by endogenous esterases leaving free IP₃ in the cytosol) and the non-competitive IP₃ receptor inhibitor 2-amino-ethoxydiphenyl borate (2APB) (Maruyama et al. 1997).

The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in indo-1-loaded IRVMs using dual emission ratiometric microscopy. Cells were paced throughout the 25 min long experiments (0.33 Hz) and the F₄₀₅/F₄₉₀ ratio was recorded for 30 s with 60 s intermissions.

Control recordings (n = 17), using Hepes buffer solution induced no change to the amplitude of electrically evoked Ca²⁺ signals for 25 min of continuous pacing (amplitude 114 ± 0.59 % of control at 15 min). During that period there was a slight but significant increase in the occurrence of ECTs (0.59 ± 0.59); the application of 2 µM 2APB did not change that behaviour (amplitude 122 ± 8.7 %, number of ECTs 0 ± 0; n = 8) indicating that this substance does not affect excitation-contraction coupling per se.

Administration of 100 nM ET-1 (n = 11) caused a robust increase in the amplitude of electrically induced Ca²⁺ signals (202 ± 34.6 %) and also promoted the generation of ECTs (9.7 ± 2). The application of 2 µM 2APB did not significantly alter the amplitude response (240 ± 67.7 %) but specifically diminished the occurrence of ECTs to control levels (1.125 ± 0.64). These data suggested an involvement of IP₃Rs in the ET-1 response.

Stimulation of IRVMs with IP₃BM (10 µM) increased the amplitude of evoked Ca²⁺ signals (154 ± 15.2% n = 5) and the occurrence of ECTs (8.6 ± 5.6). Application of 2 µM 2APB together with IP₃BM suppressed both the amplitude increase and generation ECTs (88 ± 22.7 % and 0 ± 0; n = 3 respectively). All figures are averages ± S.E.M. (all signifances were determined by Student’s independent, two-tailed t test).

From these data we conclude that IP₃R are functionally present in ventricular myocytes. Thus the ET-1-induced inotropic response and the generation of ECTs is (at least partially) due to the mobilisation of IP₃ in rat ventricular myocytes.

This research was funded by the BBSRC.