# High-Resolution Optical Measurement of Cardiac Restitution, Contraction and Fibrillation Dynamics in Beating versus Blebbistatin-uncoupled Isolated Rabbit Hearts

### Abstract

Optical mapping is a high-resolution fluorescence imaging technique, that uses voltage- or calcium-sensitive dyes to visualize electrical excitation waves on the heart surface. However, optical mapping is very susceptible to the motion of cardiac tissue, which results in so-called motion artifacts in the fluorescence signal. To avoid motion artifacts, contractions of the heart muscle are typically suppressed using pharmacological excitation-contraction uncoupling agents, such as Blebbistatin. The use of pharmacological agents, however, may influence cardiac electrophysiology. Recently, it has been shown that numerical motion tracking can significantly reduce motion-related artifacts in optical mapping, enabling the simultaneous optical measurement of cardiac electrophysiology and mechanics. Here, we combine ratiometric optical mapping with numerical motion tracking to further enhance the robustness and accuracy of these measurements. We evaluate the method’s performance by imaging and comparing cardiac restitution and ventricular fibrillation (VF) dynamics in contracting, non-working versus Blebbistatin-arrested Langendorff-perfused rabbit hearts ($Ntextequals10$). We found action potential durations (APD) to be, on average, $25 ± 5 textpercent$ shorter in contracting hearts compared to hearts uncoupled with Blebbistatin. The relative shortening of the APD was found to be larger at higher frequencies. VF was found to be significantly accelerated in contracting hearts, i.e., $9 ± 2 Hz$ with Blebbistatin and $15 ± 4 Hz$ without Blebbistatin ($Ntextequals10$ hearts), and maintained a broader frequency spectrum. In contracting hearts, the average number of phase singularities was $N_textbraceleftPStextbraceright textequals 11 ± 4$ compared to $N_textbraceleftPStextbraceright textequals 6 ± 3$ with Blebbistatin during VF on the anterior left ventricular surface. VF inducibility was reduced with Blebbistatin. We found the effect of Blebbistatin to be concentration-dependent and reversible by washout. Aside from the electrophysiological characterization, we also measured and analyzed cardiac motion. Our findings may have implications for the interpretation of optical mapping data, and highlight that physiological conditions, such as oxygenation and metabolic demand, must be carefully considered in ex vivo imaging experiments.

Publication
Frontiers in Physiology 11: 464