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Cardiovascular Engineering

Improving Effective Refractory Period Measurements for Non-invasive, Percutaneous Optogenetic Pacing of Mouse Hearts Expressing the ReaChR Opsin

(G-263) Improving Effective Refractory Period Measurements for Non-invasive, Percutaneous Optogenetic Pacing of Mouse Hearts Expressing the ReaChR Opsin

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row G - Poster # 263

Presenting Author(s)

David Honig (he/him/his)

Student Researcher
Washington and Lee University
West Bloomfield, Michigan, United States

Co-Author(s)

Anna Osborn

Student Researcher
Liberty University
Wilmington, Ohio, United States
MA

Marcello Amaral

Visiting Researcher
Washington University in St. Louis, United States

Primary Investigator(s)

CZ

Chao Zhou

Associate Professor
Washington University in St. Louis, United States

Introduction:: Atrial fibrillation, the most common type of irregular heartbeat, affects over 5 million people in the US and is expected to affect over 12 million people by 2030¹. Electronic pacemakers are commonly used to treat arrhythmias but have drawbacks such as invasive surgical procedures, painful electrical shocks, and potential battery failure². An emerging area of research which may alleviate some of these issues is cardiac optogenetics, which uses light-sensitive opsins to control heart rhythms. Cardiac optogenetics has the potential to serve as a pacemaker alternative as it has already been used to pace Drosophila melanogaster, zebrafish, mice, and rat hearts successfully³. Heart rhythm is affected by stimulation frequency and the effective refractory period (ERP), in which no action potentials can be propagated. While electric pacemakers typically utilize a pulse width of 0.5-1 ms⁴, optogenetic pacing utilizes pulse widths that are over ten times wider. Larger pulse widths may affect the accuracy of ERP measurements using the standard method of measure (the longest S1-S2 period which fails to capture⁵) due to partial overlap between pulses and the ERP. Additionally, when pacing at higher stimulation frequencies, the heart rate frequency is half of the stimulation frequency⁶, which reduces pacing efficiency and may represent a possible fatigue effect. Here we measure the ERP of mice hearts expressing the ReaChR excitatory opsin (a red-shifted channelrhodopsin that can induce tachycardia), discuss the limitations of the standard method, and propose an alternative method to measure the ERP in optogenetics to account for wider pulse widths.

Materials and Methods:: Adult mice with the ReaChR gene and the cardiomyocyte Nkx2.5-Cre gene were genetically crossed to produce viable and fertile progeny with ReaChR x Nkx2.5 genotype. Upon maturation, these progeny were anesthetized with isoflurane (1.5 - 2.0%) vaporized in medical air (1.75 L/min) during procedures. The mice were placed on a heating pad in a supine position. The hair on their chest was removed using an electric shaver followed by gel hair remover (Veet) to limit the effect of hair melanin absorption on recorded data. Four electrode needles were inserted subcutaneously into the mices’ limbs and connected to an electrocardiogram (ECG) machine (model ML865, PowerLab 4/25T, ADInstruments Inc., USA). Light stimulation was triggered by the ECG machine to simultaneously record and synchronize data. The ECG signal was recorded using LabChart 7.0 (ADInstruments Inc., USA). Red-light was emitted at 617 nm (LCS-0617-03-11, Mightex Inc., Canada) from a light-emitting diode (LED) perpendicularly onto the mouse’s chest over the heart area (10mm in diameter). Pacing was conducted with a train of 10 S1 pulses at 0.5 Hz above the resting heart rate followed by an S2 pulse ranging from 10 - 33.33 Hz for 15, 20, and 25 ms. Pacing was characterized by an ECG with wider QRS complexes and absent P waves. Three trials were conducted at each S2 pulse frequency and respective pulse width. The ERP was measured with the standard method and an alternative method (the S1-S2 interval minus the S1 mean response time representing the light stimulation to stimuli delay).

Results, Conclusions, and Discussions:: Larger pulse widths were associated with increased capture efficiency as indicated by shorter ERPs recorded when capture was missed. The last ERP of capture at pulse widths of 20 ms (T = 2.80, df = 8, P < 0.023) and 25 ms (T = 3.00, df = 8, P < 0.017) were both significantly shorter than the last ERP of capture at pulse widths of 15 ms. However, there was no significant difference between ERP capture at pulse widths of 20 ms and 25 ms (T = 1.00, df = 8, P = 0.347). These findings are consistent with our hypotheses as a longer pulse width would provide a greater opportunity for capture. Additionally, the alternative method demonstrated a potentially more accurate and consistent measure of the ERP for optogenetic pacing. The alternative method accounted for increases in the delay of stimuli caused by greater pulse widths as demonstrated by ERP values which were not significantly different. In order to test for effects of anesthesia, the order of testing pulse widths was randomized and no significant relationships were found between anesthesia and capture efficiency. Age also did not appear to have any significant effects as pacing was effective between mice of 108 to 400 days old, showing that these transgenic opsins can stay expressed effectively for at least over a year.
Better characterization and understanding of how factors such as pulse width affect pacing and capture efficiency is necessary in order to design more robust pacing models and proceed with successful pacing of more advanced organisms. Further exploration of how long, or if indefinitely, opsins can stay expressed in tissue is an important pursuit to garner evidence towards the effectiveness of optogenetics as a potential alternative to pacemakers. A better understanding is still needed in order to explain the fatigue effect. Altered pacing procedures incorporating a train of S2 stimuli to observe if capturing can resume may help further explain how pacing is affected by the fatigue effect. Additionally, measuring if increased pulse widths and delays of stimuli can act as a potential contributor to the fatigue effect is necessary.

Acknowledgements (Optional): : I would like to express my appreciation to Dr. Chao Zhou and Dr. Marcello Amaral for their instruction and mentorship, the Cardiovascular Summer Research (CardS) Program at Washington University in St. Louis, the American Heart Association, and the Washington University in St. Louis (Start-up fund) for financial support. I would also like to thank other members and associates of the Zhou lab: Abigail Matt, Fei Wang, Elena Gracheva, Hongwu Liang, Stacey Rentchler, and Christian Zemlin.

References (Optional): : (1) Colilla, S.; Crow, A.; Petkun, W.; Singer, D. E.; Simon, T.; Liu, X. Estimates of Current and Future Incidence and Prevalence of Atrial Fibrillation in the U.S. Adult Population. Am. J. Cardiol. 2013, 112 (8), 1142–1147.

(2) Frequently Asked Questions About Pacemakers and Implantable Cardioverter Defibrillators (ICDs).

(3) Gruber, A.; Edri, O.; Gepstein, L. Cardiac Optogenetics: The next Frontier. EP Eur. 2018, 20 (12), 1910–1918.

(4) Cardiac Pacemakers: Function, Troubleshooting, and Management: Part 1 of a 2-Part Series. J. Am. Coll. Cardiol. 2017, 69 (2), 189–210.

(5) Issa, Z. F.; Miller, J. M.; Zipes, D. P. 4 - Electrophysiological Testing: Tools and Techniques. In Clinical Arrhythmology and Electrophysiology (Third Edition); Issa, Z. F., Miller, J. M., Zipes, D. P., Eds.; Elsevier: Philadelphia, 2019; pp 81–124.

(6) Lopez, A. L.; Wang, S.; Larina, I. V. Optogenetic Cardiac Pacing in Cultured Mouse Embryos under Imaging Guidance. J. Biophotonics 2020, 13 (11), e202000223.