P. Schaffer

Di-4-ANEPPS causes photodynamic damage to isolated cardiomyocytes

Action potential recordings from isolated guinea pig ventricular cells in the whole-cell recording mode were used to study the toxic and photodynamic properties of the voltage-sensitive fluorescent dye di-4-ANEPPS. Staining of the cardiomyocytes with di-4-ANEPPS (30 or 60 μM; 10 min) did not alter the action potential shape. When the stained cells were illuminated (1W/cm2) severe effects on the action potential were observed. There was a prolongation of the action potential duration, occurrence of early afterdepolarizations, reduction of the membrane resting potential and eventually inexcitability. Addition of the antioxidant catalase (100 IU/ml) to the extracellular solution delayed the onset of these effects, suggesting that reactive-oxygen-intermediates take part in di-4-ANEPPS induced photodynamic damage. Since di-4-ANEPPS is a very important tool for optical membrane potential recordings in heart tissue and single cardiomyocytes catalase might be useful in suppressing photodynamic damage during optical potential recordings.

Optical multisite monitoring of cell excitation phenomena in isolated cardiomyocytes

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 (ΔF/F) ≈ −8% per 100 mV. About a dozen measuring spots covering a single cell were simultaneously monitored with a spatial and temporal resolution of 15 μm and about 20 μs, 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 μs); 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.

Optical Monitoring Of Excitation Patterns In Single Cardiomyocytes

We have developed an optical multisite recording system to monitor membrane excitationes in isolated or cultured cells using potential-sensitive fluorescent dyes. The system offers in its current configuration up to 24 out of 100 selectable myuring spots each with a pixel size at as low as 14 x 14 pm separated by 1 pm. Each spot is re resented by one element of a 10 x 10 photodyode array wkich, after individual amplification, is linked to one channel of a 24channel transient recorder (sample rate 200 kHz per channel). Simultaneous optical and electrical measurements on isolated cardiomyocytes of adult guinea pigs allow us to study the electrical behaviour of the cell membrane with subcellular spatial resolution.

Fast optical potential mapping in single cardiomyocytes during field stimulation

We have studied the process of cell excitation which was evoked and influenced by electrical fields. Optical potential mapping with very high spatial and temporal resolution (15 μm and about 30 μm, respectively) was performed in isolated ventricular cells from guinea pig using the fluorescent potential sensitive dye Di-4-Anepps and Laser excitation. Depending on duration and amplitude of the electrical pulses, rising phases were distorted extensively. Action potentials were elicited, sometimes more than a millisecond delayed. A simple model of three coupled Bee-ler-Reuter models was capable to simulate closely the develope-ment of action potentials.

OPTICAL POTENTIAL MAPPING HELPS TO REVEAL DISCRETE-NATURAL-PHENOMENA IN CARDIAC MUSCLE

The cardiac muscle is composed of electrically coupled cells which form a complex, three-dimensional structure. Depending on many physiological parameters (e.g. age), the amount of electrical coupling between the cells varies, which leads to nonuniformities in the propagating wavefronts. Optical potential mapping allows one to monitor all kinds of excitation phenomena in various size scales at many sites simultaneously, implying the whole heart the same as isolated cardiomyocytes. The spatial and temporal resolution requirements depend on the specimen under study and can be increased to about 10 µm and to excitation delays of some µs respectively. An optical mapping system with 256 measuring spots, built in our laboratory, allowed us to clearly demonstrate the discrete and discontinuous nature of propagation in rat cardiac tissues. As a unique application, the optical method allows field stimulation studies which mimic defibrillation-like conditions. Recent results obtained in such experiments did not con...

Optical Multisite Detection Of Membrane Potentials In Single Cardiomyocytes During Voltage Clamp

We have performed voltage clamp experiments in single cardiomyocytes combined with simultaneous optical multisite monitoring of membrane potentials. The fluorescent potential sensitive dye di-4-ANEPPS was used for staining and an argon ion laser for excitation of fluorescence. The percentage change of the fluorescence light intensity was about 5% per lOOmV, and showed a linear relationship (within the limits of noise). Optical signals obtained simultaneously from different measuring spots on a cell sometimes showed timelags of about 100 ps. Normalizing the optical signals to the clamp steps allowed to obtain calibrated membrane potential values within a cell. This promises to be a powerful method to interpret voltage clamp experiments with large and fast currents.

Nonuniform response of the field stimulated cardiomyocyte: a study by optical micromapping

Optical potential mapping with very high temporal (S20 p) and subcellular spatial (15 pm) resolution allows detailed investigation of excitation phenomena in isolated cardiomyocytes. Using the fluorescent potential sensitive dye di-4-ANEPPS and laser excitation we have investigated the rising phases of action potentials from isolated guinea pig ventricular cells. When field stimulation was performed, different areas of the cell showed significantly different upstroke velocities and often significantly related time lags depending upon the local polarization produced during the stimulus pulse. This complex nonuniform behaviour is a challenge for mathematical modelling.

Optical micromapping reveals potential distributions on cardiomyocytes during field stimulation

Cells which are exposed to electrical fields show a spatial dependent distribution of the membrane potential: parts closer to the anode are hyperpolarized and parts closer to the cathode are depolarized. High resolution optical potential mapping allows, after a normalization procedure based on the common action potential amplitude, a detailed description of this distribution. Our first results, obtained from rod-shaped single ventricular cardiomyocytes showed, that the membrane potential distribution smoothly followed the applied homogenous electrical field. There was no hint for a cell shape dependent nonuniform potential distribution, which, if present, would be restricted to very small areas significantly below our spatial resolution of 15 /spl mu/m, or which would only lead to small potential changes hidden within the signal noise.

Die Messung von Aktionspotentialen und Mustern der Erregungsausbreitung mittels optischer Methoden

Zusammenfassung Neben der Verwendung von Elektroden zur Erfassung von Aktionspotentialen und der Erregungsausbreitung in elektrisch aktiven Zellen und Geweben hat sich die optische Membranspannungsmessung als zusatzliche wertvolle Methode etabliert. Sie beruht auf der Eigenschaft spezieller Farbstoffe, die, wenn sie an die Zellmembran gebunden sind, ihre optischen Eigenschaften in linearer Abhangigkeit von der Membranspannung andern. Wir haben zwei eigene Meβplatze, einen zur Untersuchung von Herzteilpraparaten und einen fur isolierte Herzmuskelzellen, aufgebaut und das elektrische Verhalten dieser Praparate (von Ratte und Meerschweinchen) untersucht. Simultane optische und elektrische Messungen von Aktionspotentialen an isolierten Herzmuskelzellen zeigten das uberlegene ortliche und zeitliche Auflosungsvermogen der optischen Methode. Mittels optischen Mappings konnten wir direkt Inhomogenitaten der Erregungsausbreitung in Papillarmuskeln zeigen; auch der storende Einfluβ von Flecainid (ein Antiarrhythmikum) auf die Ausbreitung war nachweisbar. Optische Vielpunktmessungen an der isolierten Zelle zeigten im allgemeinen hochst synchrone Anstiegsflanken der Aktionspotentiale innerhalb der Zelle, als Ausnahme aber auch signifikante Zeitunterschiede. Dieselbe Methode zeigte auch (als einzig mogliche) die komplexen elektrischen Muster in Zellen und Geweben unter Feldstimulation.

Evaluation of noisy single shot signals in cardiac optical potential mapping

The authors have developed a novel data evaluation method which allows them to gain statistically relevant information even from noisy single shot records. The method may be applied to all signals which consist not only of a part of interest (yielding the relevant information) but also of a part which contains only noise. It is based on two assumptions: (i) the noise remains the same throughout a record and (ii) the influence of noise on a noisy signal is the same as on a signal without noise. The authors have applied this method to signals which were obtained via optical potential monitoring using a potential sensitive dye in single cardiomyocytes. Calculating time lags of excitation in cardiomyocyte yielded confidence intervals which allowed the authors to quantify the reliability of the data.<<ETX>>