Measurement of Cytosolic pH Simultaneouly with Isometric Tension in Canine Trabeculae

Summary

Dye leakage is a major problem for experiments used to measure cytosolic pH that last over several hours. The ratio of epifluorescence (FI₅₀₀/FI₄₄₀, 540 nm emission) of the pH indicator 2′,7′-bis(carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) changes with time when it is loaded in the tissue. This change is enhanced by experimental protocols with large changes in pH and is probably due to dye leakage from the cells. Measurements of cytosolic pH (pH_i) which are performed long before the calibrations, are hence rendered unreliable. We describe a calibration technique for measuring pH_i during long experimental protocols performed on canine right ventricular trabeculae loaded with BCECF. The equation was modified to calculate pH_i according to the intracellular concentration of dye present during each measurement. This concentration-based correction compensates for dye leakage during long experiments. Fluorescence was measured simultaneously with isometric twitch tension. With this technique we show that prolonged acidification (45 minutes) in the presence or absence of bicarbonate in the buffer medium did not produce significant irreversible damage to the ventricular trabeculae as judged by developed isometric contractile tension, which was measured simultaneously.