Optical mapping study of blebbistatin-induced chaotic electrical activities in isolated rat atrium preparations

Abstract

We have studied the spatiotemporal pattern of blebbistatin-induced anomalous electrical activities in isolated rat atrial preparations using the optical mapping of excitation spread. Atrial preparations including the right or left auricle were dissected from adult rat hearts. Each preparation was then stained with a fast merocyanine-rhodanine voltage-sensitive dye (NK2761). Using a multi-element (16 x 16) photodiode array, we assessed the spread of excitation optically by timing the initiation of the action potential-related extrinsic absorption changes. The contraction-related optical signals were suppressed by adding (S)-(-)-blebbistatin (10-100 miocroM) to the bathing solution. Blebbistatin had an effective delay time of about 1.5 h following its application, at which time anomalous electrical activities occurred. These took the form of triggered activities and rhythmical spontaneous excitations. We optically mapped the spatiotemporal patterns of the excitation spread during these anomalous electrical activities. When the triggered activities occurred, the site of ectopic focus, where the triggered action potential first appeared, and the area of excitation spread varied in every event. When the rhythmical spontaneous excitations occurred, the excitation spread from the anomalous pacemaker and, occasionally, their spatial shift was observed. In addition, the combination pattern of the spontaneous excitations and triggered activities was also observed. We suggest that these phenomena are due to the disturbed intracellular calcium dynamics induced by the application of blebbistatin.

Properties of blebbistatin for cardiac optical mapping and other imaging applications

“…We have used blebbistatin in many of our studies [2, 18, 28, 31], and similar to others [16, 17, 24], we found blebbistatin to significantly improve the fidelity of potentiometric recordings. Our imaging setup includes a second camera and a UV light source, and with this, we have observed blebbistatin precipitation within some of our heart preparations.…”

“…Blebbistatin crystals may interrupt normal vascular flow (crystals can be larger than 20 μm) and create regions of underperfused tissue. Notably, occasional arrhythmic events were reported when high blebbistatin concentrations (100 μM) were used in atrial preparations [17]. Secondly, in studies that use GFP or other green/yellow dyes, blebbistatin precipitate could be mistaken for labeled cells.…”

The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart

“…To our knowledge, the present study is the first to demonstrate that blebbistatin causes significant changes in the ventricular electrophysiology, although Kanlop & Sakai (2010) noted spontaneous electrical activity in response to high concentrations of blebbistatin (10–100 μ m ). We did not demonstrate similar spontaneous activity in our experiments; however, the dose required to abolish motion artefacts (5 μ m ) is lower than those reported by Kanlop & Sakai (2010). Nevertheless, these data are worthy of note and are of potential importance.…”

“…The mechanism suggested by Fedorov et al (2007), by which calcium is released into the cytosol, may be a plausible explanation for the APD prolongation we observed, because this increased intracellular calcium would lead to an increase in the calcium transient. The spontaneous electrical excitations observed in the study by Kanlop & Sakai (2010), albeit at higher blebbistatin concentrations (10–100 μ m ), also suggest altered intracellular calcium handling.…”

Low-Cost Optical Mapping Systems for Panoramic Imaging of Complex Arrhythmias and Drug-Action in Translational Heart Models

“…Second, we used blebbistatin as an excitation-contraction uncoupler to minimize motion ‘artifacts’ in our fluorescence signals. Though this is currently standard practice in the optical mapping field, recent studies have shown that this compound can have deleterious effects on cardiac tissue 25 26 . Consequently, it would be desirable to develop a low-cost optical analog to the epicardial electrode sock, designed to record epicardial activity in a beating heart 27 .…”