(G-279) Sonogenetics: Optimization of ex vivo naive rat heart stability during simultaneous ultrasound stimulation and optical mapping

The development of noninvasive techniques for the pacing and defibrillation of cardiac tissue provides an opportunity to improve clinical techniques for arrhythmia management and patient response to cardiac pathologies. Atrial fibrillation (AF), for instance, is a cardiac pathology that has been estimated to impact three to six million Americans, with numbers expected to triple within the next thirty years. Patients with this arrhythmia are at an increased risk of stroke and acute death, suggesting the need for additional precise and effective treatment options. Traditional techniques of cardioversion, ablation, and electronic pacemakers and implantable cardioverter-defibrillators have been associated with higher-than-desired recurrence rates, invasive surgeries, and patient discomfort and intolerance. Optogenetics is a technique that has been investigated as an alternative to electronic pacemakers. It sensitizes the heart to light by transfecting it with light-sensitive ion channels, effectively offering a noninvasive, painless, and low-energy control of cardiac rhythm. While optogenetics is promising, its clinical application is limited by the low degree of light able to penetrate into the myocardium. To overcome this limitation, sonogenetics has become a favorable focus, during which the heart is transfected with ultrasound-sensitive ion channels. This would allow stimulation with ultrasound, which has excellent tissue penetration. The present study focuses on the optimization of an apparatus that will be used to conduct ex vivo sonogenetics studies with optical mapping of Langendorff-perfused rat hearts. Additional considerations of the study account for the development of an experimental chamber design that allows for simultaneous ultrasound stimulation and unimpeded optical mapping.

The hearts of adult wild-type rats (n=7) were explanted and placed in cardioplegic solution for aortic cannulation. A 37±0.5 ⁰C Tyrode solution was used for Langendorff-perfusion and submersion of the hearts. Perfusion occurred at a flow rate corresponding to pressures between 40-70 mmHg. Blebbistatin, an electromechanical uncoupler, was added to the perfusate (5-10μM) to inhibit motion. For optical mapping, the voltage-sensitive fluorophore Cytovolt (di-4-ANBDQBS, Cyto Cybernetics) was added to the perfusate as a bolus (100 nm/g of heart tissue). The heart was illuminated with a Solis 623 nm LED (Thorlabs), and the emitted fluorescence was captured through a long-pass filter (715 nm), allowing for a recording with a sensitive high-speed camera (MiCAM, 1000 Hz, 256x256 pixels). The heart was allowed to beat for two hours following submersion in the experimental chamber. Stability of the preparation was determined by monitoring pressure and perfusion flow rate, electrical stimulation threshold, and the activation sequence following electrical pacing. Action potential graphs for normal sinus rhythms and activation maps for electrical epicardial stimulation were constructed for the ventricles. To determine the stimulation threshold, the stimulation current was slowly increased until consistent pacing was achieved. A slightly submerged focused ultrasound probe (FUS; Doppler Electronic Technologies, 15P10CF40-H), with a focal length of 39 mm, was positioned towards the heart in the experimental chamber to ensure unobstructed optical mapping. The ultrasound varied stimulation parameters of ultrasound intensity (0-10 W/cm2), pulse length (0-10 ms), pulse repetition frequency (0-1kHz), stimulation duration (1-10 s), and stimulation site.

Once optimized, the constructed apparatus housing the explanted rat heart produced approximately two hours of reliable normal sinus rhythm and the heart was able to be electrically stimulated, as confirmed by optical mapping. Figure 1A depicts a successful Langendorff-perfusion and positioning of the FUS probe to ensure an unobstructed perspective for optical mapping. Figure 1B exhibits an action potential graph obtained during documentation of normal sinus rhythm. The action potential can be viewed over a span of 100 ms and presents a peak fluorescence at approximately 400 a.u. Figure 1C denotes an activation map, for the electrical stimulation, with a total activation time of 23 ms. Results of electrical stimulation displayed that the stimulation threshold remained below 1.5 mA, even at the end of the experiment. During the two-hour experimentation period, the heart remained at fairly consistent pressures, with a standard deviation of 3.50 mmHg in the last hour of the trial. By the end of the two hours, it was determined that all stages of the desired control experiment were successful in accomplishing the original goal of apparatus optimization. The apparatus allowed for adequate time for successful completion of the experimental objectives, which were to determine normal sinus rhythm, electrically stimulate the heart to showcase the ability of the heart to be stimulated and paced, and allowed time for an attempt to stimulate the heart via ultrasound waves. Therefore, there is now an opportunity for all required data to be successfully obtained for future sonogenetics studies.

An experimental apparatus that allows for the ultrasound stimulation of Langendorff-perfused rat hearts with simultaneous monitoring of the stability and electrical response, using optical mapping, provides a platform for the evaluation of sonogenetically modified rat hearts. This optimization now provides the opportunity to dramatically increase the reliability and reproducibility of results collected by future sonogenetics studies. Using this platform, sonogenetics can be developed, with the potential to ultimately develop a potent tool for the treatment of arrhythmias. Sonogenetics provides a hopeful opportunity to introduce a new noninvasive cardiac pacing technique into the clinic, further increasing patient outcomes.

Thank you to Dr. Christian Zemlin and Dr. Ralph Damiano, Jr. for allowing your lab to teach the next generation of scientists. Thank you to Grace Nikolaisen and Nick Razos for their daily commitment to laboratory excellence and scientific advancement. Thank you to Jakraphan Yu and Jack Yi for their constant encouragement during this process. Thank you to Dr. Chao Zhou, Dr. Katie Schreiber, and Professor Patricia Widder for the work they dedicated to the excellent Cardiovascular Summer Internship at Washington University in St. Louis. Thank you to the American Heart Association (AHA) for granting GR0023814, which has allowed for this opportunity to occur.