Optical multisite monitoring of cell excitation phenomena in isolated cardiomyocytes

Abstract

An especially designed setup which consists of an inverted fluorescence microscope, an argon ion laser and a photodiode array system permits membrane potential monitoring in isolated guinea-pig ventricular cardiomyocytes, stained with the voltage-sensitive dye di-4-ANEPPS, which responds linearly with relative fluorescence changes (delta F/F) approximately -8% per 100 mV. About a dozen measuring spots covering a single cell were simultaneously monitored with a spatial and temporal resolution of 15 microns and about 20 microseconds, respectively. In general, the rising phases of the action potentials within a single cell were highly synchronized (i.e. all upstroke velocities peaked within about 20 microseconds); however, in one cell (out of 25 examined) significant (P < 0.05) time lags exceeding the signal-dependent time resolution were also found. Experiments, simultaneously performed with our optical system and a widely used patch-clamp setup, revealed a slowed and delayed response of the clamp amplifier depending on the cell access resistance. Optical monitoring during whole-cell voltage-clamping demonstrated the influence of graduated series resistance compensation. When field stimulation was used, our results clearly demonstrated the spatially dependent polarization of the cell membrane during the stimulus, as well as a highly synchronized upstroke development. Slight differences in the maximum upstroke velocities within a single cell were also found and were basically in agreement with mathematical models.

Null Mutation of Connexin43 Causes Slow Propagation of Ventricular Activation in the Late Stages of Mouse Embryonic Development

“…Highresolution optical mapping of voltage-dependent changes in cardiac tissue [21][22][23] has previously been used in the adult mouse, 24,25 the embryonic chick, 26,27 and other rodent hearts, 26 as well as cellular preparations. 28,29 This study uses optical mapping to explore the role of Cx43 in cardiac impulse propagation during the embryonic development and the early postnatal life in the mouse.…”

Mechanisms of defibrillation

“…When V m in the depolarized section of the cell reaches the activation threshold, a sufficient number of ion channels are opened to initiate an action potential, which then spreads into the entire cell. These predictions about the distribution of shock-induced V m changes and activation pattern were confirmed on a qualitative level in measurements from isolated cardiac myocytes using fluorescent V m -sensitive dyes and optical mapping techniques (11)(12)(13).…”

Artifactual contractions triggered by field stimulation of cardiomyocytes

“…The two methods of stimulation are fundamentally different. A current injection triggers a uniform depolarization, whereas an electric field will expose each part of the membrane to different potentials during stimulation (8,22,31,41,47,48). We observed that contraction characteristics depend on the stimulation method used.…”

“…What can the reason be for this? When a cell is stimulated by a uniform electric field and aligned parallel to it, the two ends are nonuniformly (and oppositely) polarized, and different regions of the cell experience very different transmembrane voltages (8,22,31,33,41,47,48). Spatial differences have also been observed in the intracellular Ca 2ϩ ([Ca 2ϩ ] i ) transient (7,32).…”

“…Alternating polarity of the pulse will result in different patterns of depolarization of a cell (32,41,47,48), and Tung and Borderies (41) predicted mathematically that the time for the membrane to reach the threshold potential for triggering an action potential depended on the total length of the pulse. However, they also predicted that the excitability would be independent of the waveform (mono-or biphasic).…”