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

Spontaneous sarcoplasmic reticulum (SR) Ca 2+ release and propagated intracellular Ca 2+ waves are a consequence of cellular Ca 2+ overload in cardiomyocytes. We examined the relationship between average intracellular [Ca 2+ ] and Ca 2+ wave characteristics. The amplitude, time course and propagation velocity of Ca 2+ waves were measured using line-scan confocal imaging of β-escin-permeabilised cardiomyocytes perfused with 10 μM Fluo-3 or Fluo-5F. Spontaneous Ca 2+ waves were evident at cellular [Ca 2+ ] > 200 nM. Peak [Ca 2+ ] during a wave was 2.0–2.2 μM; the minimum [Ca 2+ ] between waves was 120–160 nM; wave frequency was ≈0.1 Hz. Raising mean cellular [Ca 2+ ] caused increases in all three parameters, particularly Ca 2+ wave frequency. Increases in the rate of SR Ca 2+ release and Ca 2+ uptake were observed at higher cellular [Ca 2+ ], indicating calcium-sensitive regulation of these processes. At extracellular [Ca 2+ ] > 2 μM, the mean [Ca 2+ ] inside the permeabilised cell did not increase above 2 μM. This extracellular-intracellular Ca 2+ 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 μM carbonyl cyanide m -chlorophenylhydrazone and 2 μM oligomycin) while perfusing with > 2 μM Ca 2+ abolished the extracellular-intracellular Ca 2+ gradient through the generation of Ca 2+ waves with a higher peak [Ca 2+ ] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca 2+ uptake acts to delay an increase in [Ca 2+ ] by blunting the peak of the Ca 2+ wave.