Search

Transmembrane voltage-sensitive fluorescent dyes are used to study electrical activity in hearts. Green and red fluorescence emissions from di-4-ANEPPS excited with 488 nm light indicate both transmembrane voltage changes and heart movement. We have previously shown that the ratio, green fluorescence divided by red fluorescence, indicates the transmembrane voltage without effects of movement. Here we examine the feasibility of measuring the movement, which is useful for the study of cardiac function, by subtracting this ratiometric signal from the red or green fluorescence signal. The results of this subtraction show tissue movement and its relative changes during cardiac ischemia and perfusion with an electromechanical uncoupling agent. By incorporating the spatial variations in fluorescence intensity from the heart, tissue movement can be qualitatively mapped to examine relative changes, however, with limited ability to quantify absolute displacement. Since these maps are obtained simultaneously with corresponding transmembrane potentials, the method allows study of spatiotemporal cardiac movement patterns and their relationship to the action potential. Index Terms--Electromechanical dissociation, fluorescence, tissue motion, transmembrane voltage-sensitive dye.

Simultaneous mapping of transmembrane voltage (V(m)) and intracellular Ca(2+) concentration (Ca(i)) has been used for studies of normal and abnormal impulse propagation in cardiac tissues. Existing dual mapping systems typically utilize one excitation and two emission bandwidths, requiring two photodetectors with precise pixel registration. In this study we describe a novel, single-detector mapping system that utilizes two excitation and one emission band for the simultaneous recording of action potentials and calcium transients in monolayers of neonatal rat cardiomyocytes. Cells stained with the Ca(2+)-sensitive dye X-Rhod-1 and the voltage-sensitive dye Di-4-ANEPPS were illuminated by a programmable, multicolor LED matrix. Blue and green LED pulses were flashed 180° out of phase at a rate of 488.3 Hz using a custom-built dual bandpass excitation filter that transmitted blue (482 ± 6 nm) and green (577 ± 31 nm) light. A long-pass emission filter (>605 nm) and a 504-channel photodiode array were used to record combined signals from cardiomyocytes. Green excitation yielded Ca(i) transients without significant crosstalk from V(m). Crosstalk present in V(m) signals obtained with blue excitation was removed by subtracting an appropriately scaled version of the Ca(i) transient. This method was applied to study delay between onsets of action potentials and Ca(i) transients in anisotropic cardiac monolayers.

Aims: The present study addresses the controversy regarding the 'primary' role of the subendocardial Purkinje network in triggering torsade de pointes (TdP) ventricular tachyarrhythmia (VAs) in the long QT syndrome (LQTS)., Methods and Results: We investigated the well-established canine anthopleurin-A (AP-A) surrogate model of LQT3 to study the role of the subendocardial Purkinje network in triggering VAs. Three-dimensional activation and repolarization patterns were analysed from unipolar extracellular electrograms utilizing 64 plunge needle electrodes. In 6 dogs, the animals were placed on cardiopulmonary bypass and chemical ablation of the endocardial Purkinje network was obtained using Lugol's solution. Spontaneous VAs consistently developed in response to AP-A infusion and were triggered by a subendocardial focal activity acting on a substrate of spatial three-dimensional dispersion of repolarization. Endocardial ablation was considered successful by the development of complete atrioventricular block in the absence of ventricular escape rhythm. Following endocardial ablation spontaneous VAs were no longer observed. However, an appropriately coupled premature stimulus consistently induced re-entrant VAs., Conclusion: The present study strongly suggests that in the LQTS, focal activity generated in subendocardial Purkinje tissue is the primary, if not the only, trigger for TdP VAs by acting on a substrate of three-dimensional dispersion of myocardial repolarization to induce re-entrant excitation.

Failure of cardiac antiarrhythmic ablation to block action potential conduction produces poor outcomes which lead to repeat procedures. To overcome this, an intraoperative index of the quality of an ablation lesion is needed. We hypothesized that a rise in the translesion stimulus-excitation delay (TED) can indicate a continuous, transmural, linear lesion, and that the TED is related to the path length in the viable tissue around the lesion. Rabbit hearts were isolated, perfused with a warm physiological solution and stained with transmembrane potential-sensitive fluorescent dye. Radiofrequency (RF) ablation was performed on ventricular epicardium with a vacuum-assisted coagulation device to produce either a complete or incomplete lesion. Complete lesions were both transmural and continuous. Incomplete lesions were noncontinuous or nontransmural. The TED was determined with bipolar stimulation at one side of the lesion and either a bipolar electrogram at the other side or optical mapping on both sides. Hearts were then stained with tetrazolium chloride and examined histologically to estimate minimum path lengths of viable tissue from the stimulation site to the recording site. Complete lesions increased the TED by factors of 2.6-3.1 (p < 0.05), whereas incomplete lesions did not significantly increase the TED. Larger minimum path lengths were found for cases that had an increased TED. The TED was quantitatively predictable based on a conduction velocity of 0.38-0.49 m s(-1), which is typical of rabbit hearts. The TED significantly increases when a linear lesion is complete, suggesting that an intraoperative measurement of the TED may help to improve ablation lesions and outcomes. Predictability of the TED based on the viable tissue path suggests that quantitative TEDs for clinical lesions may be anticipated provided that the conduction velocity is considered.

Optical recordings with transmembrane potential (Vm)-sensitive fluorescent dye, or extracellular potential (Ve) recordings are used to map spatiotemporal patterns of cardiac excitation during ventricular fibrillation (VF). While the optical and electrical methods are accepted, there has not been a test of whether they yield equivalent excitation times during VF. Times may differ since previous results indicate optical Vm interrogates deeper than Ve. We tested whether the steepest parts of the downward deflection of the Ve and upward deflection of optical Vm are synchronized during VF. We used simultaneous coepicentral optical and electrical mapping (32 spots, 4 kHz) with translucent indium tin oxide electrodes and a laser scanner on ventricular epicardium. VF was electrically induced in arterially-perfused rabbit hearts stained with di-4-ANEPPS. For both the optical and electrical deflections, maximum magnitudes of the slopes varied over a > 4 fold range, morphologies varied and spatiotemporal distributions were nonuniform. Time differences between the steepest parts of the optical and electrical deflections were typically a few ms. Standard deviations of time differences increased for the deflections that had the smaller slopes, which was only partly due to effects of recording noise as indicated by simulations. For deflections that had slopes ranging from the steepest found at each spot to 1/4 of the steepest, the optical deflections were on average 0.7-1 ms earlier than the Ve deflections. Thus, excitation times during VF measured optically and electrically differ. Considered together with our earlier results indicating that the optical Vm interrogates deeper than Ve, the results suggest that most fibrillatory excitations occur earlier in subsurface tissue than at the heart surface.