Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae <em>Ex Vivo</em>

Abstract

In cardiac muscle, intracellular Ca 2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric deformation. Our understanding of the internal relationships between these events has been limited because we can neither 'see' inside the muscle nor precisely track the spatiotemporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca 2+ transient, and an optical coherence tomograph to capture the tissue's geometric changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 µm by 540 µm) and the length of a typical trabecula (>2000 µm). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying imageprocessing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into

Work-loop contractions reveal that the afterload-dependent time course of cardiac Ca2+ transients is modulated by preload

“…All experiments were conducted on isolated ventricular trabeculae from male Wistar rats in the cardiomyometer ( 20 , 21 ). In this device, trabeculae developed force-length work loops by contracting against a mechanical admittance computed in real time in a field-programmable gate array (FPGA) and imposed by a linear actuator based on a voice coil motor ( 22 ).…”

“…Each dissected trabecula was mounted between two platinum hooks in the cardiomyometer ( 20 ). Voice-coil motors mechanically coupled to each hook set the slack length of the trabecula.…”

“…Steering the measurement arm along a single axis enabled the imaging of muscle cross section, and using a second axis, enabled the imaging of muscle volume. The resultant images were segmented, and the cross-sectional area was calculated by scaling the pixel area by the lateral and depth resolutions of the optical coherence tomography (OCT) ( 20 ). The seven trabeculae had an average cross-sectional area of 0.06 ± 0.04 mm 2 , an average length at L 100 of 2.2 ± 0.5 mm, and a major:minor width ratio of 1.2 ± 0.3 (values expressed as means ± SD).…”

“…It is important to consider the possibility that any changes to the Ca 2+ transient may be a consequence of motion artifacts, as has been considered previously ( 7 ). Here, motion artifacts were investigated in a separate experiment using a gated imaging protocol ( 20 ). The imaging systems of the cardiomyometer are fixed at the center of the measurement chamber and have limited fields of view.…”

“…The upstream and downstream hooks can move in synchrony to achieve such a movement. By moving the trabecula to five different positions within the measurement chamber each 50 μm apart, we were able to perform gated imaging on an actively contracting trabecula ( 20 ). At each position, the trabecula performed work-loop contractions with the same loading conditions (i.e., preload and afterload) while bright-field images (100 fps) and stress and fluorescence data (20 kHz) were captured.…”