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)

Both intact mammalian cardiac muscle and single adult Ca2+-tolerant myocytes, under appropriate experimental conditions, exhibit periodic, spontaneous myofilament oscillations that originate locally within a cell and propagate longitudinally as contractile waves. We have used microscopic imaging techniques to study the effect of electrical stimulation on the oscillation characteristics in single rat and rabbit myocytes. Unstimulated rat cells bathed in Cao of 1-3 mM exhibited these oscillations. During stimulation at rates between 6 and 120 min-1, oscillations did not occur in the interval between stimulated contractions, and following termination of stimulation a transient suppression of the spontaneous oscillation frequency occurred. Conversely, with higher cell Ca2+ loading, achieved by increasing the [Ca2+]o or by addition of isoproterenol or ouabain, stimulation caused de novo oscillations in rabbit cells and increased the spontaneous oscillation frequency in rat cells to levels that resulted in their appearance between stimulated contractions. The tendency for myofilament motion to occur simultaneously at multiple foci was also increased by stimulation at high frequencies, and partial synchronization of these foci resulted in oscillations of an increased amplitude. The modulation of the spontaneous oscillation characteristics in single cells by stimulation may explain, in part, some effects of stimulation on Ca2+-dependent oscillatory phenomena in intact cardiac tissues.

SUMMARY. Spontaneous contractile waves due to spontaneous calcium cycling by the sarcoplasmic reticulum occur in unstimulated bulk rat papillary muscle and single rat cardiac myocytes with intact sarcolemmal function. We used video analytic techniques to quantify the wave characteristics in both bulk muscle and myocytes; laser-light scattering techniques were also employed in muscle. In muscle bathed in physiological concentrations of calcium, the true periodicity of these waves was a fraction of 1 Hz and increased up to several hertz with increases in cell calcium. This was paralleled by an increase in the frequency of scattered laser light intensity fluctuations. In myocytes, a range of spontaneous contractile wave frequencies similar to that which occurred in the muscle was observed; it could be demonstrated that an increase in superfusate calcium concentrations (2-15 mM at 23°Q increases the oscillation frequency but not amplitude. In both myocytes and muscle, low concentrations of caffeine (0.5 DIM) and higher temperature increased the oscillation frequency but diminished their amplitude. However, the scattered light fluctuations did not change with temperature and decreased with caffeine. These results demonstrate that (1) the true frequency of spontaneous sarcoplasmic reticulum oscillations in the unstimulated rat muscle and single myocytes with intact sarcolemmal function is low, i.e., a fraction of a hertz; (2) with cell calcium loading, the oscillation frequency accelerates to those frequencies measured previously in the 'calcium overload" state; (3) while scattered light fluctuations which sample myonlament motion are a sensitive, noninvasive method of detecting the oscillations in bulk muscle, they can be insensitive to the divergent changes in oscillation amplitude and frequency. (Circ Res 57: 844-855, 1985)

This study examines the use of carboxy-seminaphthorhodafluor-1 (C-SNARF-1) as an indicator of cytosolic pH in isolated rat cardiac myocytes. The emission spectrum of C-SNARF-1 when excited at 530 nm contains two well-separated peaks at approximately 590 and 640 nm, corresponding to the acidic and basic forms of the indicator. This spectral feature allows the indicator to be used in the single excitation, dual emission ratio mode. When C-SNARF-1 is loaded into rat cardiac myocytes as the membrane permeant ester derivative, C-SNARF-1/AM, the indicator localizes within the cytosol with virtually no partitioning into the mitochondria. C-SNARF-1 does not load into isolated mitochondria in suspension. There was no evidence for the presence of non-deesterified C-SNARF-1 within the cells. C-SNARF-1 can be calibrated in situ using a technique that abolishes all transsarcolemmal pH gradients. A 0.7-unit shift in the apparent pK (pKapp = pK-log10) between the in vitro calibration and the in situ calibration is consistent with a change in beta (I640 to pH 9/I640 at pH 5) in the cytosolic environment (beta in situ/beta in vitro = 0.21) and not a change in the true pK of the indicator. The contribution of cellular autofluorescence to the total signal can be made negligible. There is no effect of C-SNARF-1 on the contractile properties of rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

A BSTR ACT Previous studies have shown that acidosis increases myoplasmic [Ca 2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca 2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cal, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca ~+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca 2+ transient associated with each contraction and caused the appearance of Cal oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by ~5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemreal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 ~M) abolished all evidence of spontaneous Ca 2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca ~+ release, which can (a) cause spontaneous action potentials, (b) increase resting tension, and (c) negatively affect twitch tension.