Optical mapping of human embryonic stem cell-derived cardiomyocyte graft electrical activity in injured hearts

Abstract

Background Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac regeneration, but the successful development of hESC-CM-based therapies requires improved tools to investigate their electrical behavior in recipient hearts. While optical voltage mapping is a powerful technique for studying myocardial electrical activity ex vivo, we have previously shown that intra-cardiac hESC-CM grafts are not labeled by conventional voltage-sensitive fluorescent dyes. We hypothesized that the water-soluble voltage-sensitive dye di-2-ANEPEQ would label engrafted hESC-CMs and thereby facilitate characterization of graft electrical function and integration. Methods We developed and validated a novel optical voltage mapping strategy based on the simultaneous imaging of the calcium-sensitive fluorescent protein GCaMP3, a graft-autonomous reporter of graft activation, and optical action potentials (oAPs) derived from di-2-ANEPEQ, which labels both graft and host myocardium. Cardiomyocytes from three different GCaMP3+ hESC lines (H7, RUES2, or ESI-17) were transplanted into guinea pig models of subacute and chronic infarction, followed by optical mapping at 2 weeks post-transplantation. Results Use of a water-soluble voltage-sensitive dye revealed pro-arrhythmic properties of GCaMP3+ hESC-CM grafts from all three lines including slow conduction velocity, incomplete host-graft coupling, and spatially heterogeneous patterns of activation that varied beat-to-beat. GCaMP3+ hESC-CMs from the RUES2 and ESI-17 lines both showed prolonged oAP durations both in vitro and in vivo. Although hESC-CMs partially remuscularize the injured hearts, histological evaluation revealed immature graft structure and impaired gap junction expression at this early timepoint. Conclusion Simultaneous imaging of GCaMP3 and di-2-ANEPEQ allowed us to acquire the first unambiguously graft-derived oAPs from hESC-CM-engrafted hearts and yielded critical insights into their arrhythmogenic potential and line-to-line variation.

In Vitro Matured Human Pluripotent Stem Cell–Derived Cardiomyocytes Form Grafts With Enhanced Structure and Function in Injured Hearts

“…To detect host electric activity, recipient hearts were loaded with RH237, a voltage-sensitive dye that preferentially labels host myocardium. 5,15 Graft- (ASAP1) and host- (RH237) derived oAPs were then acquired under both spontaneous and paced conditions (Figure 4A–4H and Video S1). Although grafts formed with TCP hPSC-CMs were nearly always uncoupled from the host, grafts formed with PDMS hPSC-CMs were far more likely to show 1:1 host-graft coupling, with PDMS day 40 grafts showing near-perfect host-graft electromechanical integration (Figure 4I).…”

“…All animal procedures were approved by the Animal Care Committee of the University Health Network. In brief, 650- to 750-g male Hartley guinea pigs (Charles River) underwent cardiac cryoinjury by thoracotomy, 5,14,15 followed 10 days later by repeat thoracotomy and direct intramyocardial injection of 1×10 8 ASAP1 + PDMS- or TCP-generated hPSC-CMs or vehicle. To prevent rejection, all animals were treated with an immunosuppression regimen including cyclosporine A and methylprednisolone starting 2 days before cell transplantation and continuing until euthanasia.…”

“…We next took advantage of this system to compare in vivo outcomes after the transplantation of hPSC-CMs at 4 distinct stages of in vitro maturation (ie, PDMS versus TCP hPSC-CMs harvested at either day 20 or 40). For these experiments, we used the previously reported guinea pig MI model 5,10,15 and the sequence of experimental procedures depicted in Figure 2A. In brief, adult guinea pigs underwent cardiac cryoinjury by thoracotomy, followed 10 days later by direct intracardiac injection of 1×10 8 PDMS versus TCP hPSC-CMs (n=15 and 13, respectively).…”

KairoSight: Open-Source Software for the Analysis of Cardiac Optical Data Collected From Multiple Species

“…To date, cardiac optical mapping, also known as optocardiography (Boukens and Efimov, 2014;George and Efimov, 2019;Swift et al, 2019), has been used to investigate changes in electrical activity, spiral wave formation (Jalife, 2003), excitation-contraction coupling (Christoph et al, 2017), sarcoplasmic reticulum-specific calcium cycling (Wang et al, 2014), metabolic status (Mercader et al, 2012;Wengrowski et al, 2014;Kuzmiak-Glancy et al, 2015;Jaimes et al, 2016a;Garrott et al, 2017), the efficacy of ablation therapies (Swift et al, 2014), stem-cell engraftment (Costa et al, 2012;Shiba et al, 2012;Filice et al, 2020), cardiotoxicity (Jaimes et al, 2019b), and the pathobiology of heart failure (Spragg and Kass, 2006; Abbreviations: APD 80 , action potential duration at 80% repolarization; AUF, arbitrary units of fluorescence; Ca 2+ , intracellular calcium; CaD 80 , calcium transient duration at 80% reuptake; dF/F0, change in fluorescence over baseline; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocytes; PCL, pacing cycle length; SNR, signal-to-noise ratio; V m , transmembrane voltage. Ng et al, 2014;Zasadny et al, 2020).…”

Correction to: Optical mapping of human embryonic stem cell-derived cardiomyocyte graft electrical activity in injured hearts

“…The original article [ 1 ] contained a typo in co-author, Rocco Romagnuolo’s name which has since been corrected.…”