A system is described that can simultaneously record cytosolic Ca2+ concentration ([Ca2+]i), cell length, and either membrane potential or current in single cardiac myocytes loaded with the fluorescent Ca2+ indicator indo-1. Fluorescence is excited by epi-illumination with 3.8-microsecond flashes of 350 +/- 5 nm light from a xenon arc. Indo-1 fluoresence is measured simultaneously in spectral windows of 391-434 nm and 457-507 nm, and the ratio of indo-1 emission in the two bands is computed as a measure of [Ca2+]i for each flash. With cells loaded with the permeant acetoxymethyl ester of indo-1, quantitation of [Ca2+]i is not precise, owing to subcellular compartmentation of indo-1; however, the instrument would allow full quantitation if indo-1 free acid was introduced by microinjection. Simultaneously, cell length is measured on-line from the bright-field image of the cell. Because fluorescence collection is time gated during the brief flash, and red light (650-750 nm) is used for the bright-field image, cell length and [Ca2+]i measurements are obtained simultaneously without cross talk. Membrane potential or current can be recorded simultaneously with indo-1 fluorescence and cell length via standard patch-clamping techniques.

Anoxic contractile failure in rat heart myocytes is caused by failure of intracellular calcium release due to alteration of the action potential (excitation-contraction ABSTRACTAnoxia of the heart causes failure of contraction before any irreversible injury occurs; the mechanism by which anoxia blocks cardiac excitation-contraction coupling is unknown. Studies in whole muscle are confounded by heterogeneity; however, achieving the low oxygen tensions required to study anoxia in a single myocyte during electrophysiological recording has been a barrier in experimental design. Guided by calculations of oxygen transport, we developed a system to insulate myocytes in an open dish from oxygen by a laminar counterflowing argon column, permitting free access to the cell by microelectrodes while maintaining a P02 <0.02 torr (1 torr = 133 Pa). In the absence of glucose, the amplitude of stimulated contraction of anoxic ventricular myocytes fell to

Under certain conditions, spontaneous release of Ca2+ from the sarcoplasmic reticulum occurs in resting mammalian myocardium. In single rat ventricular myocytes, such spontaneous Ca2+ release appears localized rather than homogeneous. When the increase in cytosolic Ca2+ is present in a single locus within a cell, it causes a small depolarization, which, at the normal resting potential, is subthreshold for generating an action potential. However, when spontaneous Ca2+ release occurs simultaneously at more than a single discrete locus, the resultant sarcolemmal depolarization is augmented to levels that can induce an action potential, even when this depolarization begins at the normal resting membrane potential. Thus, the synchronous occurrence of multifocal localized increases in cytosolic Ca2+ due to spontaneous Ca2+ release from the sarcoplasmic reticulum within ventricular myocytes is a mechanism for "abnormal automaticity."

We used adult rat cardiac myocytes to examine the acute effects of 0.1-5.0% (vol/vol)