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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session D

Poster F6 - Optical Mapping of hiPSC-derived Cardiomyocyte Monolayers for a High Throughput Cardiotoxicity Screening

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Yeonju Kim (she/her/hers)

Undergraduate Student
Rice University
Houston, Texas, United States

Co-Author(s)

AM

Arvydas Matiukas

Director of Neuroscience Microscopy Core
SUNY Upstate Medical University, United States

Primary Investigator(s)

CZ

Christian Zemlin

Associate Professor of Surgery, Division of Cardiothoracic Surgery
Washington University in St. Louis School of Medicine, United States

Introduction: Cardiotoxicity induced by drugs can lead to severe arrhythmias such as ventricular tachycardia or sudden cardiac arrest by altering the cardiac electrophysiology. To ensure the cardiac safety of patients, all drugs undergo cardiotoxicity screening before clinical trials. However, most screening is done in small animal models which do not represent human physiology and are both time-consuming and costly. As an alternative, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a novel in vitro platform for cardiotoxicity assessment and disease modeling due to their physiological relevance and high-throughput capabilities. Optical mapping has also been widely used to observe changes in action potentials or conduction dynamics in hiPSC-CMs. To develop an effective cardiotoxicity screening test, it is crucial to pace hiPSC-CMs at a desired frequency and to understand the relationship between the signal-to-noise ratio (SNR) and optically mapped area. In this study, we designed a customized dish to pace hiPSC-CM monolayers and optimized the signal processing of periodic calcium signals for high-throughput cardiotoxicity screening. We established a protocol for electrically pacing hiPSC-CM monolayers using the electrode-embedded dish. Furthermore, we determined the relationship between the SNR and the binning radius (BR) by spatially filtering the fluorescence signals obtained from optical mapping. The study describes the optical mapping setup and SNR analysis algorithm that contribute to developing a high-throughput in vitro cardiotoxicity screening test.

Materials and

Methods: hiPSC-CM Pacing
We designed a culture chamber for hiPSC-CMs that enables electrical stimulation of the monolayer using graphite electrodes and platinum wires (Figure 1A). Two graphite rods with platinum wires were placed 1.5 cm apart on an autoclavable petri dish with silicone adhesives. hiPSC-CMs (250,000 cells/cm^2), genetically labeled with GFP-calmodulin for intracellular calcium reporting, were seeded between the electrodes. To measure the calcium transients, the platinum wires were connected to the Model 2100 Isolated Pulse Stimulator from A-M Systems (Sequim, Washington). We then applied a monophasic pulse of 3V with a pulse duration of 20 ms. The GFP signal was recorded with a fluorescence microscope for 10 seconds with a frame rate of 96 fps. We also used optical mapping with 100 fps to obtain calcium signals from a hiPSC-CM monolayer that was not stimulated (Figure 1A).

SNR Analysis
To assess the SNR for different well sizes, we used a representative fluorescence movie of stimulated cardiac activity and spatially filtered by binning, with the binning radius ranging from 0 (corresponding to single-pixel well size) to 10 pixels (each well is 21 pixels x 21 pixels wide). For each binned movie, we smoothed the data with a moving average (MA) filter (Figure 1B). A period of a complete action potential was selected to calculate the signal amplitude. The noise signal was defined as the difference between the binned data and the MA-filtered binned data. The noise level was defined as the standard deviation of the noise signal.

Results, Conclusions, and Discussions: We performed hiPSC-CM pacing with a single customized dish and demonstrated that the cells responded to the pacing pulses of set amplitude and duration with frequencies between 1.0 and 1.5 Hz (Figure 1C). For frequencies higher than 1.2 Hz, a pulse duration of 50 ms was utilized instead of 20 ms, as a longer pulse duration facilitated higher frequency stimulation. Pacing at a predefined frequency eliminates the variability in the natural beating frequency and action potential shape, which is crucial for maximizing the sensitivity to action potential changes caused by cardiotoxicity.

We also established that increasing the binning radius improved the SNR of raw fluorescence signals from optical mapping, particularly in regions with uniform illumination (Figure 1D). The SNR of these regions ranged from 8 to 10 for a single pixel (binning radius=0 pixel) with physical dimensions 0.32 mm x 0.32 mm. We calculated the average SNR over 10 pixels x 10 pixels of well-illuminated regions and achieved an SNR of 54.9 with a binning radius of 10 pixels (3.2 mm). Understanding the average SNR of a single dish and its dependence on temporal and spatial filtering allows us to determine the minimum well size for high-throughput screening and filtering parameters required to achieve a desired SNR and detect the changes in action potential.

In this study, we developed an electrical stimulation chamber that successfully paced the hiPSC-CM monolayers and characterized the signal quality of action potentials obtained from optical mapping. Our SNR analysis of GFP signals from a single dish, combined with the electrical stimulation chamber, paves the way for designing an optical mapping setup for high-throughput drug screening for cardiotoxicity. To further improve the current setup, the electrical stimulation procedure for optical mapping including illumination of the specimen and sterilization must be optimized. The work described here improves the understanding of optical mapping with hiPSC-CMs, enabling more efficient and physiologically relevant assessments of drug-induced cardiotoxicity.

Acknowledgements (Optional): We would like to acknowledge the support of the American Heart Association (AHA Award #906519) and the WashU Cardiovascular Summer Research (CardS) program.