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

Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and propagated intracellular Ca2+ waves are a consequence of cellular Ca2+ overload in cardiomyocytes. We examined the relationship between average intracellular [Ca2+] and Ca2+ wave characteristics. The amplitude, time course and propagation velocity of Ca2+ waves were measured using line-scan confocal imaging of beta-escin-permeabilised cardiomyocytes perfused with 10 muM Fluo-3 or Fluo-5F. Spontaneous Ca2+ waves were evident at cellular [Ca2+] > 200 nm. Peak [Ca2+] during a wave was 2.0-2.2 muM; the minimum [Ca2+] between waves was 120-160 nm; wave frequency was similar to0.1 Hz. Raising mean cellular [Ca2+] caused increases in all three parameters, particularly Ca2+ wave frequency. Increases in the rate of SR Ca2+ release and Ca2+ uptake were observed at higher cellular [Ca2+], indicating calcium-sensitive regulation of these processes. At extracellular [Ca2+] > 2 muM, the mean [Ca2+] inside the permeabilised cell did not increase above 2 muM. This extracellular-intracellular Ca2+ 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 Ca2+ abolished the extracellular-intracellular Ca2+ gradient through the generation of Ca2+ waves with a higher peak [Ca2+] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca2+ uptake acts to delay an increase in [Ca2+] 1 by blunting the peak of the Ca2+ wave.