Effects of substrate-free anoxia and veratridine on intracellular calcium concentration in isolated rat ventricular cardiomyocytes.

Cytosolic free Ca2+ concentration ([Ca2+]i) was measured in freshly isolated rat ventricular cardiomyocytes during substrate-free anoxia. Cardiomyocytes were loaded with fura-2 and incubated in an anoxic chamber in which a pO2 equal to 0 mmHg was realized by inclusion of Oxyrase. [Ca2+]i was measured in individual cells using digital imaging fluorescence microscopy. During anoxia, the shape of cardiomyocytes changed from a relaxed-elongated form into a rigor configuration within 15 min after the onset of anoxia. After the cells had developed the rigor state, a delayed rise in [Ca2+]i reached a stable maximal level within 45 min. The mean values for the pre-anoxic and maximal anoxic [Ca2]i were 52 +/- 3 nM (N = 42) and 2115 +/- 59 nM (N = 45), respectively. The purported Na+ overload blocker R 56865, significantly reduced maximal anoxic [Ca2+]i to 533 +/- 56 nM (P < 0.05), implicating a role of elevated intracellular Na+ in the anoxia-induced increase in [Ca2+]i. Veratridine (30 microM), with induces Na+ overload, increased [Ca2+]i to 787 +/- 39 nM. The compound R 56865 reduced veratridine-induced increases in [Ca2+]i to 152 +/- 38 nM. Upon reperfusion, after 45 min of anoxia, two distinct responses were observed. Most often, [Ca2+]i decreased upon reperfusion without a change in morphology or viability, while in the minority of cases, [Ca2+]i increased further followed by hypercontraction and loss of cell viability. The mean value for [Ca2+]i 10 min after reperfusion of the former group, was 752 +/- 46 nM (N = 38).(ABSTRACT TRUNCATED AT 250 WORDS)