Spontaneous release of Ca²⁺ from the sarcoplasmic reticulum (SR) of mammalian cardiac muscle occurs in response to increased SR Ca²⁺ load. Spontaneous Ca²⁺ release takes the form of a localised release that propagates throughout the myocyte in the form of a Ca²⁺ wave. Previous measurements have shown that progressive increases in SR Ca²⁺ load increases the frequency of spontaneous Ca²⁺ release, but there is some uncertainty as to whether the characteristics of the Ca²⁺ wave amplitude remain constant. Hearts were removed from terminally anaesthetised rabbits (100 mg kg^-1 Euthutal), and myocytes were isolated from the left ventricle by perfusion with collagenase solution. Cells were permeabilised by brief exposure to β-escin (0.1 mg ml^-1) and subsequently bathed in a mock intracellular solution of the following composition (mM): 0.05 EGTA, 4.6 MgATP, 25 Hepes, 10 phosphocreatine, 10 NaCl, 0.9 Mg²⁺, 100 KCl and 0.01 fluo-3 (or fluo-5F); pH 7.0 (20-22 °C). Spontaneous Ca²⁺ release within the myocyte was measured using a BioRad Radiance 2000 confocal microscope. Linescan (every 2 ms) images allowed high temporal resolution of Ca²⁺ wave amplitude and velocity. Individual cells were exposed to an extracellular [Ca²⁺] ranging from 40 nM to approximately 3.5 µM (see Fig. 1).In general, sustained periods (> 150 s) of regular Ca²⁺ waves occurred at an extracellular [Ca²⁺] of ~250 nM (frequency 0.13 waves s^-1). Peak [Ca²⁺] reached during a wave was approximately 2 µM, increasing to approximately 3.5 µM at high extracellular [Ca²⁺]. Minimum [Ca²⁺] between waves was approximately 120 nM, increasing to nearly 300 nM at high extracellular [Ca²⁺]. The averaged intracellular [Ca²⁺] taken over 10 s periods were equal to the extracellular [Ca²⁺], indicating complete equilibration of the Ca²⁺ across the permeabilised cell membrane. Increased extra- and intracellular [Ca²⁺] caused a marked increase in frequency of Ca²⁺ waves, reaching maximum values of approximately 1.0 wave s^-1 at extracellular [Ca²⁺] of approximately 3-4 µM. At these high [Ca²⁺], in many cases Ca²⁺ waves were not maintained throughout the 150 s period, Ca²⁺ waves diminished and were replaced with a sustained increase in intracellular [Ca²⁺]. This study indicates that spontaneous release from cardiac SR maintains a low minimum intracellular [Ca²⁺] in the face of increasing average intracellular [Ca²⁺]. Increasing the intracellular [Ca²⁺] leads to a marked increase in the frequency of spontaneous waves rather than a change in Ca²⁺ wave amplitude. This supports the conclusions of an earlier study by Eisner et al. (1998).Research was supported by EPSRC and SHERT.

Figure 1. Plot of extracellular [Ca2+] (ordinate) vs. wave maximum [Ca2+], wave minimum [Ca2+] and mean [Ca2+] (abscissa). Each set of three measurements was made from individual cells.

Eisner, D.A., Trafford, A.W., DÆaz, M.E., Overend, C.L. & O’Neill, S.C. (1998). Cardiovasc Res. 38, 589-604.