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

Abstract

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 553±56 nM (P<0.05), implicating a role of elevated intracellular Na⁺ in the anoxia-induced increase in [Ca²⁺]_i. Veratridine (30 μM), 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.