Optical imaging of cardiomyocyte membrane voltage in the Langendorff-perfused mouse heart with cellular and subcellular resolution

Guixue Bu, Purdue University

Abstract

Heart disease has been the leading cause of death in the United States for more than one century. More specifically, sudden death from cardiac arrhythmias accounts for the majority of all cardiac deaths. It is therefore of significant importance to understand the arrhythmogenic mechanisms for developing new treatment strategies. The measurement of transmembrane voltage changes with cellular and subcellular resolution from the intact heart to study the mechanisms linking abnormal cellular/subcellular events to cardiac arrhythmias would be a significant technological advancement. Fluorescence-based techniques have been used extensively to assess electrical behavior of individual cardiomyocytes in culture with microsecond resolution, but approaches to monitor transmembrane voltage changes in the intact heart with similar spatiotemporal resolution have not been reported previously. Accordingly, this thesis developed a technique to monitor cardiomyocyte transmembrane voltage changes in the electromechanically dissociated, Langendorff-perfused mouse heart on a cellular and subcellular scale using laser scanning confocal microscopy in conjunction with the voltage sensitive fluorescent dyes ANNINE-6 and ANNINE-6plus. Action potential induced fluorescence transients, ΔF/F0(t), were recorded from peripheral sarcolemmal segments, intercalated disks, and transverse tubules in this preparation using repetitive unidirectional line scan at a rate of 1042 lines per second. Signal processing algorithms were also developed to resolve and to improve signal-to-noise ratio (SNR) of the fluorescence transients. Peak fractional changes in fluorescence intensity averaged -21.6±2.5% (347 recordings from 24 hearts loaded with ANNINE-6 or ANNINE-6plus). Signal processing increased the SNR of the fluorescence transient by a factor close to [special characters omitted] where N is the number of action potentials in the recording. Fluorescence transients were validated using microelectrode recorded transmembrane action potentials from the hearts under the same experimental conditions. This study provides a novel and unique experimental technique for integrative cardiac electrophysiology research. Direct measurement of transmembrane voltage changes in selected segments of the cardiomyocyte sarcolemma (t-tubule, intercalated disks, peripheral membrane) that are not accessible by the conventional microelectrode technique will facilitate future research on mechanisms of cardiac excitation-contraction coupling and arrhythmogenesis. This technique also bodes well for assessment of heterocellular coupling in the intact heart.

Degree

Ph.D.

Advisors

Berbari, Purdue University.

Subject Area

Cellular biology|Medical imaging|Biophysics

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