EXCITATION-CONTRACTION IN ISOLATED CARDIAC CELLS

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We have dissociated cardiac cells from adult rats, rabbits and guinea pigs. These preparations are being used to study the contractile, electrophysiological, and biochemical characteristics in a variety of different conditions. We have developed a system for simultaneous measurement, in single cardiac myocytes, of changes in cytosolic free calcium, cell length and membrane current/voltage, with high time resolution. The system uses the fluorescent probe indo-1 to monitor cytosolic free calcium transient. Indo-1 fluorescence is excited by epi-illumination with 10 microsecond flashes of 350 plus or minus 10 nm light at repetition rates of up to 200 Hz. Indo emission is collected by paired photomultipliers to measure stimultaneously spectral windows of 411 plus or minus 20 nm and 481 plus or minus 25 nM optimizing the trade -off between collected light intensity and cytosolic free calcium sensitivity. The fluorescence emission from each flash is collected by a pair of fast integrator sample- and-hold circuits of custom design under the control of a VAX 11/730 computer which computes the ratio of indo emission at the two wavelengths as a measure of cytosolic free calcium with a time precision of better than 20 micro-seconds. Cells length is measured from the bright-field image of the cell by an optical edge tracking method using a vedio edge detector (or a photodiode array when millisecond time resolution is required). The membrane potential may be monitored simultaneously with patch electodes. Our initial results have shown the widely divergent, calcium-dependent systolic and diastolic properties of intact rat, guinea pig and rabbit cardiac muscle are retained with a high degree of fidelity in the majority of viable single myocytes isolated from the myocardium of these species, and that these myocytes are thus a valid model of studies of calcium-dependent excitation-contraction mechanisms in the heart. Thus, the availability of single myocytes and an apparatus whereby we can simultaneously measure membrane currents, cytosolic calcium and function will permit mechanistic studies of aspects of excitation-contraction coupling that have heretofore not been amenable to direct study. We have already begun some initial experiments of this sort.