Kinetics of intracellular Ca2+ concentration changes and cell contraction of electrically stimulated cardiomyocytes as analysed by automated digital-imaging microscopy

Abstract

Enzymatically disaggregated, electrically stimulated cardiomyocytes from adult rats were examined by television-mediated vital microscopy for intracellular Ca2+ concentration and contractile activity. Using an inverted microscope in the epifluorescence mode, the Ca2+ signal was imaged with a low-light-level CCD camera and traced by means of the intracellular concentration of the fluorescent complex of Ca2+ with its indicator Fluo-3. Using the transmitted-light mode, cardiomyocytes that were not loaded were imaged with a conventional CCD camera with automatic gain control and traced by length measurements. Optical images of at least 40 cardiomyocytes per batch of cells from one heart were recorded in up to 20 microscopic fields of observation on videotape within 20 min. They were consecutively analysed by a personal computer installed with an image analysis card at a time-resolution of 20 ms, employing a discrete convolution operation, filtering and threshold setting for fluorescence measurements, and contour description and vectorial analysis for length measurements. Frames of fluorescent images were corrected for the halo effect caused by the increase in the Ca(2+)-dependent fluorescence signal after electrical stimulation. The cell contraction had to be measured in the transmission mode without Fluo-3 due to the inhibition caused by the intracellular Fluo-3. The following coefficients of variation (V) were determined: Vfluorescence < 0.033 and Vtransmission < 0.003 for the precision of measurement, and Vfluorescence < 0.05 and Vtransmission < 0.04 for the reproducibility. The system was validated with isoprenaline and ouabain as agents to modify the Ca(2+)-signal and the contraction. The response of cardiomyocytes of various rats to electrical stimulation, with respect to amplitude and its time point, had a V < 0.08 for both the Ca(2+)-signal and the contraction.