Simultaneous measurement of calcium transients and motion in cultured heart cells

Abstract

The fluorescent Ca2+ probe indo-1 is a new intracellular Ca2+ concentration [( Ca2+]i) indicator that may be suitable for measurement of [Ca2+]i transients in intact heart cells. We exposed spontaneously contracting cultured chick embryo ventricular cells (37 degrees C) to the membrane-permeable indo-1-acetoxymethyl ester (indo-1 AM). Indo-1 loading was associated with a decrease in the amplitude of contraction measured with a video motion detector, but contractility returned to control levels during a subsequent 30-min wash. Analysis of emission spectra of dye obtained by digitonin permeabilization of cells loaded in indo-1 AM showed that the active intracellular dye was not pure indo-1 but probably includes partially deesterified molecules. With the use of an inverted X40 objective epifluorescence system, washed cells containing indo-1 were excited at 360 nm, and fluorescence intensity was measured at 410 nm (increases with increasing [Ca2+]) and 480 nm (decreases with increasing [Ca2+]). Calibration of the [Ca2+]i signals, reflected by the ratio of 410 to 480 nm fluorescence, was achieved by use of ethylen-glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-Ca2+ buffered solutions containing the nonfluorescent Ca2+ ionophore Bromo-A23187. Average end-diastolic and peak-systolic [Ca2+]i were 328 +/- 32 and 813 +/- 72 nM (means +/- SE, n = 8). The onset of the [Ca2+]i transient preceded motion by 27 +/- 5 ms (means +/- SE, n = 4), but generally resembled the motion signals in contour. These findings indicate that indo-1 may be used to detect [Ca2+]i transients in isolated ventricular cells without causing significant alterations in mechanical performance.