Ratiometric fluorescence imaging and marker-free motion tracking of Langendorff perfused beating rabbit hearts

Abstract

Optical mapping is a fluorescence based imaging technique used extensively in cardiac research to study the electrophysiological properties of isolated hearts kept at physiological conditions. The major limitation of optical mapping studies are the distortions of electrophysiological signals due to the contractile motion of the hearts. To reduce electrophysiological signal distortions due to the contractile motion artifacts, the mechanical motion has been suppressed in optical mapping experiments using electromechanical uncouplers such as Blebbistatin. Recent studies used marker-free numerical motion tracking and motion stabilization techniques to record electrophysiological parameters in the absence of electromechanical uncouplers and showed that contractile motion of the cardiac tissue is no longer a limitation in optical mapping studies. However, despite these developments, accurate measurements of quantities such as action potential duration (APD) and cardiac restitution are still challenging due to the residual motion artifacts present in the electrophysiological signals even after numerical motion tracking. A combination of marker-free motion tracking and ratiometric optical mapping technique is used in this thesis to minimize motion-related artifacts from contracting hearts. This combined experimental and numerical technique reduced motion artifacts significantly and hence, the combination is used to precisely measure APD, cardiac restitution and ventricular fibrillation frequencies from Langendorff perfused contracting and deforming rabbit hearts. A systematic comparison of these electrophysiological parameters in contracting and Blebbistatin-uncoupled conditions showed, on average, 27 ± 5% (N=5 hearts) shortening of APD in contracting hearts as compared to Blebbistatin-uncoupled hearts. Ventricular fibrillation frequency significantly increased in contracting hearts (13 ± 3.5 Hz) in comparison with Blebbistatin-uncoupled hearts (8 ± 1.5 Hz).