H. Windisch

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.

Isoproterenol, norepinephrine and phosphodiesterase inhibitors are blockers of the depressed fast Na+-system in ventricular muscle fibers

It is generally agreed that β-adrenergic transmitters do not influence the maximum rate of rise of action potentials of cardiac fibers with an intact fast Na+-system. Inhibitory effects of epinephrine have only been reported for a Mg-activated fast current [9]. Fibers with a depressed fast Na+-system have been studied under various experimental conditions, some of them mimicking infarct-like environments [1,5]. In some respects the depressed fast sodium-system reacts differently, e.g. recovery from inactivation [3], Ca2+-effects [10], TTX-influences [6] and susceptibility to antiarrhythmic drug influence [4]. We have studied the influence of isoproterenol, norepinephrine and of phosphodiesterase inhibitors on the depressed fast Na+-system in guinea-pig papillary muscles and gained results which are completely different to those in fibers with intact fast Na+-system.

Calcium ion effects on the rising phases of action potentials obtained from guinea-pig papillary muscles at different potassium concentrations.

Abstract In guinea-pig papillary muscles, action potentials, rates of rise ( v max and conduction velocities were measured at various potassium concentrations. An increase in potassium reduced v max in an S-shaped relation to the membrane potential but conduction velocity was influenced differently. At 18 to 20 m m K 0 + , the rising phase often included a remainder of the fast depolarization and v max showed two peaks; the first peak could be abolished by TTX (10 −6 m ), the second peak by D600 (1 μg/ml, 1 Hz). The v max (second peak) of the rising phase of the slow responses as well as overshoot and amplitude was clearly correlated ( r = 0.99) to the log of Ca 0 2+ . The increase in Ca 0 2+ concentration caused an elevation of both overshoot and amplitude of 34.22 mV per decade. In contrast, the v max of the first peak was correlated to the linear Ca 0 2+ concentration. At K 0 + 20 m m increase of Ca 0 2+ led to a marked increase of the first and Na + -dependent peak of v . This v max was increased 4.23 times by a Ca 0 2+ increase from 2.5 to 10 m m . Accordingly, impaired conduction due to 20 m m K 0 + was almost normalized by 10 m m Ca 0 2+ . A Ca 0 2+ -increase (2.5 to 10 m m ) shifted the curve of Na + -inactivation by 4.26 mV in the depolarization direction and reduced v max at normal membrane potential. The results confirm the dependence of the rising phase and the overshoot of slow responses to the log Ca 0 2+ but indicate strong and simultaneous Ca 0 2+ -effects on recovery of the Na + -system which may add to or even dominate the overall changes in v max of slow responses.

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.

The effects of the bradycardia-producing compound alinidine on action potentials and tension development in cardiac fibres.

SummaryAlinidine (ST 567, N-Allyl-Clonidine) exerted concentration-dependent negative chronotropic effects in isolated, spontaneously-beating sinus node cells and Purkinje fibres of guinea pigs and in ventricular strips of chick embryonic myocardium. Reduction of beat frequency by 30% was found after addition of 8.6 μmol/l alinidine in the former. A chronotropic effect was not seen during Ba2+-induced automaticity or triggered activity in guinea-pig papillary muscles and in enzymatically disaggregated cells of embryonic chick myocardium, which lose the β-adrenoceptor responsiveness of the intact embryonic ventricle. In contrast to alinidine, D 600 showed very pronounced and quinidine minor negative chronotropic effects in these latter experiments. Reduction of excitability, rate of rise of the action potential and velocity of repolarization as well as prolongation of the refractory period were seen after applications of very high concentrations of alinidine (285 μmol/l). In electrically-driven atria isometric peak tension was only slightly changed (increased by 85.5 μmol/l, decreased by 285 μmol/l) but it was reduced (to 36.8%) by alinidine (85.5 μmol/l) in papillary muscles. Both in atria and in papillary muscles, the maximum rate of rise of the action potential was unchanged by alinidine up to 85.5 μmol/l and the slight reduction following 285 μmol/l alinidine application was independent of the rate of stimulation. The present findings confirm the selectivity of the bradycardic effects of alinidine which has a main mode of action different to that of membrane stabilizing compounds or inhibitors of the slow inward current.

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.

Quantification of Shock‐Induced Microscopic Virtual Electrodes Assessed by Subcellular Resolution Optical Potential Mapping in Guinea Pig Papillary Muscle

Introduction: The primary objective of this study was the quantitative description of shock‐induced, locally occurring virtual electrodes in natural cardiac tissue.