A Comparative Assessment of Fluo Ca 2+ Indicators in Rat Ventricular Myocytes

Abstract

Improvements in the fluo series of fluorescent Ca2+ indictors routinely used to measure cytosolic free Ca2+ concentration ([Ca2+]i) have increased signal-to-noise ratio, enabling more quantitative [Ca2+] measurements. The improved fluo derivatives show increased cellular loading efficiency, reduced pH sensitivity, and excitation maxima that better match the wavelengths of common lasers. Nevertheless, the extent to which these indicators interfere with native intracellular Ca2+ homeostasis has not been systematically characterized. Here, we have examined three different fluo derivatives (fluo-2, fluo-3 and fluo-4) in freshly isolated rat ventricular myocytes. Cells were loaded with a fluo indicator either by incubation with the acetoxymethyl (AM) ester or by introducing the K+ salt of the indicator through a whole-cell pipette (injection-loaded). Fluorescence changes in cardiomyocytes were measured using confocal microscopy during field simulation or current injection. Three significant differences were identified among the three indicators and two loading methods. 1. Ca2+ kinetics in AM-loaded cells were slower than in injection-loaded cells; thus the decay of the cardiac [Ca2+]i transient appeared to be slower. In AM-loaded cells, fluo-3 reported the fastest response while fluo-2 and fluo-4 gave comparable, slower responses. When injection-loaded as the K+ salt, all three indicators reported comparable responses that were faster than any of the AM-loaded indicators. 2. When AM-loaded, all three indicators showed apparent spatial inhomogeneities in cellular fluorescence, in contrast to injection-loaded cells. Thus diverse cellular structures appear brighter in AM-loaded cells than in injection-loaded cells. 3. For each indicator, the calibrated signals (F/Fo) in the AM-loaded cells were higher than in injection-loaded cells. We conclude that injection-loaded cells produce more accurate [Ca2+]i measurements and spatially resolved signals. If AM loading is needed, the use of fluo-3 would appear to provide more accurate calibrated signals.