Developing an approach for evaluating the cardiotoxic potential of botanicals

Figures

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  FIGURE 1. Adapted from the cardiovascular failure modes described in Berridge et al. (2024). Endpoints currently prioritized by the Botanical Safety Consortium’s cardiotoxicity working group are highlighted in green. Figure created in BioRender.

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  FIGURE 2. Overview of the model system and experimental setups used to determine the suitability of these assays for botanicals. Microelectrode Array (MEA) recordings can be used to assess cardiac-modulatory potential of botanical extracts including changes in several functional parameters, like beat rate, field potential duration, and spike amplitude. Mitochondrial function assessment is used to assess mitochondrial dysfunction in cardiomyocytes that could contribute to impaired ATP production, increased oxidative stress, and cell death. The optical mapping of transient calcium measurements can provide insights into calcium-handling abnormalities associated with arrhythmogenesis, contractile dysfunction, and other cardiac pathologies. The contractile force assay provides key insights into potential effects on the contractile properties of heart cells. Figure created in BioRender.

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  FIGURE 3. Microelectrode Array (MEA) recordings rely on 48-well or 96-well plates that have 8–16 electrodes on the bottom of each well, on top of which cardiomyocytes can be cultured (A). Cardiac function is recorded as an electrical signal with distinct features, such as the depolarization peak (arrow), repolarization (arrowhead), and beat period (horizontal arrow) that can change between baseline (black) and exposure (red) (B). The cardiac-modulatory potential of botanical extracts can be assessed by comparing activity before (i.e., baseline recording) and after acute exposure (C). Figure created in BioRender.

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  FIGURE 4. Schematic diagram showing how optical mapping is conducted. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are plated onto well plates and exposed to a botanical extract or a solvent control for a period of time before a voltage or calcium-sensitive dye is added. The activation of the dye by the depolarizing hiPSC-CMs is then measured and used to measure action potentials and intracellular calcium transients to capture key electrophysiological parameters. Figure created in BioRender.

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  FIGURE 5. Schematic diagram showing how mitochondrial dysfunction is measured in the seahorse assay. Beating human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are plated and then exposed to a botanical extract or vehicle control. After exposure, oxygen consumption rates are measured to assess potential mitochondrial dysfunction.

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  FIGURE 6. A schematic illustrating the direct measurement of contractile force. Human-induced pluripotent stem cell-derived cardiomyocytes (A) cardiomyocytes stained with a combined phalloidin/DAPI (4′,6-diamidino-2-phenylindole) for visualizing F-actin and nuclei, respectively are plated on freely swinging polydimethylsiloxane membranes (B), which move when the plated cardiomyocytes contract. A baseline recording is taken (C) before the same cells are exposed to a botanical extract or a vehicle control. Then another recording is taken and compared to the baseline to evaluate changes in the contractile force of the cardiomyocytes (C). EAC, early after contraction.