Calcium flux measurements during hypoxia in cultured heart cells

We tested the hypothesis that cultured chick embryo ventricular cells grown in monolayer could be used to study calcium fluxes across the myocardial sarcolemmal membrane during simulated ischemia. A specially adapted anaerobic chamber allowed the exposure of heart cells to profound hypoxia (PO2 less than 1.5 Torr) for prolonged periods of time. Ca2+ flux studies were conducted in this chamber following hypoxia and substrate deprivation to simulate important components of myocardial ischemia. To prove we were measuring cellular rather than interstitial cation contents we conducted 51Cr-EDTA interstitial space marker washout studies and showed that our procedures were sufficient to remove at least 99.7% of the extracellular fluid. The addition of lanthanum (1 mM) to the wash solution reduced nonspecific 45Ca2+ binding to the polystyrene culture plates to less than 0.03% of applied counts, but did not alter 45Ca2+ uptake under normoxic conditions. Following 2 h of hypoxia and substrate deprivation the Ca2+ content of the rapidly exchangeable Ca2+ pool of cultured monocytes increased 282% compared to control values during normoxia. We conclude that cultured chick embryo ventricular cells grown in monolayer are suitable for investigation of cation fluxes during simulated ischemia. Under the conditions studied, lanthanum displaceable Ca2+ did not make a major contribution to Ca2+ influx. The system permitted clear resolution of alterations in Ca2+ flux kinetics under conditions of profound hypoxia.