Cardiac optical mapping uses potentiometric fluorescent dyes to image membrane potential (V_m). An important limitation of conventional optical mapping is that contraction is usually arrested pharmacologically to prevent motion artifacts from obscuring V_m signals. However, these agents may alter electrophysiology, and by abolishing contraction, also prevent optical mapping from being used to study coupling between electrical and mechanical function. Here, we present a method to simultaneously map V_m and epicardial contraction in the beating heart. Isolated perfused swine hearts were stained with di-4-ANEPPS and fiducial markers were glued to the epicardium for motion tracking. The heart was imaged at 750 Hz with a video camera. Fluorescence was excited with cyan or blue LEDs on alternating camera frames, thus providing a 375-Hz effective sampling rate. Marker tracking enabled the pixel(s) imaging any epicardial site within the marked region to be identified in each camera frame. Cyan- and blue-elicited fluorescence have different sensitivities to V_m, but other signal features, primarily motion artifacts, are common. Thus, taking the ratio of fluorescence emitted by a motion-tracked epicardial site in adjacent frames removes artifacts, leaving V_m (excitation ratiometry). Reconstructed V_m signals were validated by comparison to monophasic action potentials and to conventional optical mapping signals. Binocular imaging with additional video cameras enabled marker motion to be tracked in three dimensions. From these data, epicardial deformation during the cardiac cycle was quantified by computing finite strain fields. We show that the method can simultaneously map V_m and strain in a left-sided working heart preparation and can image changes in both electrical and mechanical function 5 min after the induction of regional ischemia. By allowing high-resolution optical mapping in the absence of electromechanical uncoupling agents, the method relieves a long-standing limitation of optical mapping and has potential to enhance new studies in coupled cardiac electromechanics.

Fluorescence optical imaging techniques have revolutionized the field of cardiac electrophysiology and advanced our understanding of complex electrical activities such as arrhythmias. However, traditional monocular optical mapping systems, despite having high spatial resolution, are restricted to a two-dimensional (2D) field of view. Consequently, tracking complex three-dimensional (3D) electrical waves such as during ventricular fibrillation is challenging as the waves rapidly move in and out of the field of view. This problem has been solved by panoramic imaging which uses multiple cameras to measure the electrical activity from the entire epicardial surface. However, the diverse engineering skill set and substantial resource cost required to design and implement this solution have made it largely inaccessible to the biomedical research community at large. To address this barrier to entry, we present an open source toolkit for building panoramic optical mapping systems which includes the 3D printing of perfusion and imaging hardware, as well as software for data processing and analysis. In this paper, we describe the toolkit and demonstrate it on different mammalian hearts: mouse, rat, and rabbit.

BACKGROUND: When human induced pluripotent stem cells (hiPSCs) that CCND2-OE (overexpressed cyclin-D2) were differentiated into cardiomyocytes ( ^CCND2-OE hiPSC-CMs) and administered to the infarcted hearts of immunodeficient mice, the cells proliferated after administration and repopulated >50% of the scar. Here, we knocked out human leukocyte antigen class I and class II expression in ^CCND2-OE hiPSC-CMs ( ^KO/OE hiPSC-CMs) to reduce the cells’ immunogenicity and then assessed the therapeutic efficacy of ^KO/OE hiPSC-CMs for the treatment of myocardial infarction. METHODS: ^KO/OE hiPSC-CM and wild-type hiPSC-CM ( ^WT hiPSC-CM) spheroids were differentiated in shaking flasks, purified, characterized, and intramyocardially injected into pigs after ischemia/reperfusion injury; control animals were injected with basal medium. Cardiac function was evaluated via cardiac magnetic resonance imaging, and cardiomyocyte proliferation was assessed via immunostaining and single-nucleus RNA sequencing. RESULTS: Measurements of cardiac function and scar size were significantly better in pigs treated with ^KO/OE hiPSC-CM spheroids than in animals treated with medium or ^WT hiPSC-CM spheroids. ^KO/OE hiPSC-CMs were detected for just 1 week after administration, but assessments of cell cycle activity and proliferation were significantly higher in the endogenous pig cardiomyocytes of the hearts from the ^KO/OE hiPSC-CM spheroid group than in those from the other 2 groups. Single-nucleus RNA-sequencing analysis identified a cluster of proliferating cardiomyocytes that was significantly more prevalent in the ^KO/OE hiPSC-CM spheroid–treated hearts (3.65%) than in the hearts from the medium (0.89%) or ^WT hiPSC-CM spheroid (1.33%) groups at week 1. YAP (Yes-associated protein) protein levels and nuclear localization were also significantly upregulated in pig cardiomyocytes after treatment with ^KO/OE hiPSC-CM spheroids. Follistatin, which interacts with the HIPPO/YAP pathway, was significantly more abundant in the medium from ^KO/OE hiPSC-CM spheroids than ^WT hiPSC-CM spheroids (30.29±2.39 versus 16.62±0.83 ng/mL, P =0.0056). Treatment with follistatin increased ^WT hiPSC-CM cell counts by 28.3% over 16 days in culture and promoted cardiomyocyte proliferation in the infarcted hearts of adult mice. CONCLUSIONS: ^KO/OE hiPSC-CM spheroids significantly improved cardiac function and reduced infarct size in pig hearts after ischemia/reperfusion injury by secreting follistatin, which upregulated HIPPO/YAP signaling and proliferation in endogenous pig cardiomyocytes.