Investigation of cellular and structural basis of cardiac arrhythmias using two-photon microscopy
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Cardiovascular diseases are often associated with arrhythmias, an electrical dysfunction in which the normal heart rhythm is disturbed. Arrhythmias are multifactorial phenomena, and understanding the mechanisms underlying their initiation and maintenance often requires the investigation of both the electrical properties and the tissue architecture in the intact heart. Two-photon microscopy is an optical sectioning technique used in combination with voltagesensitive fluorophores, which has improved the investigation of electrical conduction deep within the ventricular walls of the intact heart. In this thesis, we employed two-photon microscopy to investigate the cellular and structural basis of cardiac arrhythmias. In the first study, we aimed at optimizing a method for recording transmural electrophysiology with two-photon excitation of voltage sensitive dyes. We compared the performance of two dyes with different voltage sensing mechanism, FluoVolt and di-4-ANEPPS. We observed that, although di-4-ANEPPS tracked action potentials with larger signal-to-noise ratio, FluoVolt delivered action potential signals with higher dynamic range and was more suitable for deep imaging. In the second study, we characterized the electrophysiological properties of the heart in the Scn5a+/- mouse model of Brugada Syndrome, a cardiac conduction disorder causing lethal arrhythmias and sudden cardiac death. We compared the transmural electrophysiology and structural characteristics of the right and left ventricles. We observed that the right ventricle was more prone than the left ventricle to conduction abnormalities. This interventricular difference was mainly explained by the interplay of normal interventricular differences in tissue architecture and the reduction of sodium channel conductivity. In the third study we subjected Scn5a+/- mice to high intensity interval training to assess whether physical activity can reduce the severity of the pro-arrhythmic phenotype of Scn5a+/- mice. We observed that high intensity interval training resulted in a mild but significant improvement in right ventricular conduction, although the mechanism of action remains to be further elucidated. In conclusion, two-photon microscopy allowed the study of transmural conduction that reveal the vulnerability of the RV to conduction block in situations of reduced Na channel activity. Exercise can alleviate these effects not by structural changes but through altered excitability via changes in ion channel expression.