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 pO(2) 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 [Ca-2+](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 553+/-56 nM (P <0.05), implicating a role of elevated intracellular Na+ in the anoxia-induced increase in [Ca2+](i), Veratridine (30 mu M), which 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). The cardiomyocyte cell shape could be followed by monitoring changes in the total fura-2 fluorescence (340+380 nm signal). Within 15 min after the onset of anoxia, the total fluorescence signal increased suddenly, before [Ca2+](i) started to rise, coinciding with the onset of rigor contraction induced by ATP depletion.