Simultaneous Widefield Voltage and Dye-Free Optical Mapping Quantifies Electromechanical Waves in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Coupled electromechanical waves define a heart’s function in health and diseases. Optical mapping of electrical waves using fluorescent labels offers mechanistic insights into cardiac conduction abnormalities. Dye-free/label-free mapping of mechanical waves presents an attractive non-invasive alternative. In this study, we developed a simultaneous widefield voltage and interferometric dye-free optical imaging methodology that was used as follows: (1) to validate dye-free optical mapping for quantification of cardiac wave properties in human iPSC-cardiomyocytes (CMs); (2) to demonstrate low-cost optical mapping of electromechanical waves in hiPSC-CMs using recent near-infrared (NIR) voltage sensors and orders of magnitude cheaper miniature industrial CMOS cameras; (3) to uncover previously underexplored frequency- and space-varying parameters of cardiac electromechanical waves in hiPSC-CMs. We find similarity in the frequency-dependent responses of electrical (NIR fluorescence-imaged) and mechanical (dye-free-imaged) waves, with the latter being more sensitive to faster rates and showing steeper restitution and earlier appearance of wavefront tortuosity. During regular pacing, the dye-free-imaged conduction velocity and electrical wave velocity are correlated; both modalities are sensitive to pharmacological uncoupling and dependent on gap-junctional protein (connexins) determinants of wave propagation. We uncover the strong frequency dependence of the electromechanical delay (EMD) locally and globally in hiPSC-CMs on a rigid substrate. The presented framework and results offer new means to track the functional responses of hiPSC-CMs inexpensively and non-invasively for counteracting heart disease and aiding cardiotoxicity testing and drug development.

Paradoxical effects of Zim3, a CRISPRi effector, on human iPSC-cardiomyocyte electrophysiology

“…cardiac ion channels: KCNJ2, encoding for the Kir2.1 protein -inward rectifier K+ channel, contributing to the maintenance of healthy negative resting membrane potential; KCNH2, encoding for the main repolarizing K+ channel in cardiomyocytes, the rapid delayed rectifier that controls action potential duration, APD; and GJA1, encoding for Cx43 -the main gap-junctional protein in ventricular cardiomyocytes; for Cx43 this was also confirmed at the protein level, Figure 1. Using all-optical electrophysiology [6][7][8][9] , Figure 1a, we pursued functional studies, Figure 1c-e, which indeed corroborated physiologically relevant functional effects, expected for upregulated KCNH2 (APD shortening in paced conditions), Figure 1c, f; upregulated KCNJ2 (reduced or eliminated spontaneous firing), Figure 1d, f; and upregulated GJA1 (conduction velocity increase), Figure 1e-h. In combination, these electrophysiological changes are perceived as desirable and a signature of mature iPSC-CMs with anti-arrhythmic properties 10 .…”

CRISPRi Gene Modulation and All-Optical Electrophysiology in Post-Differentiated Human iPSC-Cardiomyocytes

“…When combined with CRISPR gene modulation, high-throughput platforms for all-optical electrophysiology [42][43][44][45][46] , enabled by optogenetics 47 , can open the door to human functional genomics studies in the cardiac field. Using 96-well plate-reader all-optical platform, developed in our lab as derivative of recent work 48,49 , Figure 7a, we obtained synchronous, multi-well measurements of KCNH2 knockdown allowing us to compare the Dox-inducible dCas9-KRAB, siRNA, and a recent more efficient CRISPRi system, dCas9-KRAB-Zim3 20 , using the Zinc Finger Imprinter 3 gene as part of the effector complex. We developed an adenoviral vector to more uniformly deliver the dCas9-KRAB-Zim3 construct.…”

High-throughput optical sensing of peri-cellular oxygen in cardiac cells: system characterization, calibration, and testing

“…This feature enabled multiparametric optical high-throughput measurements from the other half of each well. Such measurements include cellular action potentials, intracellular calcium transients, and contractility ( Klimas et al, 2020 ; Heinson et al, 2023 ; Liu et al, 2023 ). Laser cutting of the sensors was quick and reproducible ( Figure 1E ), where more than 96 half-moon sensors could be cut in less than 10 min.…”

“…Combination of the high-throughput monitoring of peri-cellular oxygen with microfluidics-based solutions of controlling oxygenation ( Wei et al, 2020 ; Yao et al, 2021 ) can yield feedback-controlled growth environment for cardiac tissue engineering. Coupling label-free measurements of peri-cellular oxygen with label-free measurements of cardiac electromechanical waves ( Liu et al, 2023 ) will also provide valuable insights into the interplay between cellular activity and oxygenation state.…”