The relationship between intracellular [Ca²⁺] and Ca²⁺ wave characteristics in permeabilised cardiomyocytes from the rabbit

Spontaneous sarcoplasmic reticulum (SR) Ca<sup>2+</sup> release and propagated intracellular Ca<sup>2+</sup> waves are a consequence of cellular Ca<sup>2+</sup> overload in cardiomyocytes. We examined the relationship between average intracellular [Ca<sup>2+</sup>] and Ca<sup>2+</sup> wave characteristics. The amplitude, time course and propagation velocity of Ca<sup>2+</sup> waves were measured using line-scan confocal imaging of beta-escin-permeabilised cardiomyocytes perfused with 10 muM Fluo-3 or Fluo-5F. Spontaneous Ca<sup>2+</sup> waves were evident at cellular [Ca<sup>2+</sup>] > 200 nm. Peak [Ca<sup>2+</sup>] during a wave was 2.0-2.2 muM; the minimum [Ca<sup>2+</sup>] between waves was 120-160 nm; wave frequency was similar to0.1 Hz. Raising mean cellular [Ca<sup>2+</sup>] caused increases in all three parameters, particularly Ca<sup>2+</sup> wave frequency. Increases in the rate of SR Ca<sup>2+</sup> release and Ca<sup>2+</sup> uptake were observed at higher cellular [Ca<sup>2+</sup>], indicating calcium-sensitive regulation of these processes. At extracellular [Ca<sup>2+</sup>] > 2 muM, the mean [Ca<sup>2+</sup>] inside the permeabilised cell did not increase above 2 muM. This extracellular-intracellular Ca<sup>2+</sup> gradient could be maintained for periods of up to 5 min before the cardiomyocyte developed a sustained and irreversible hypercontraction. Inclusion of mitochondrial inhibitors (2 muM carbonyl cyanide m-chlorophenylhydrazone and 2 muM oligomycin) while perfusing with > 2 muM Ca<sup>2+</sup> abolished the extracellular-intracellular Ca<sup>2+</sup> gradient through the generation of Ca<sup>2+</sup> waves with a higher peak [Ca<sup>2+</sup>] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca<sup>2+</sup> uptake acts to delay an increase in [Ca<sup>2+</sup>] 1 by blunting the peak of the Ca<sup>2+</sup> wave