Brigitta Szabó, Georgios Kosmidis, Elena Matsa
Cardiotoxicity occurring as a result of acute or chronic exposure to medications is one of the most common adverse clinical side-effects, placing a significant burden on both pharmaceutical companies and healthcare providers.
Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes represent a novel alternative to traditional models used in the field of safety pharmacology and drug development to assess toxicity. Using these cells, significant progress has been made towards validating cellular electrophysiology assays for predicting the clinical potential of arrhythmias. However, for several other clinically relevant safety parameters, there is still a need for standardized methods.
To maintain their physiological function, cardiomyocytes need to possess an abundant network of mitochondria and require a constant supply of respiratory substrates to meet their high energy demand. Disruption in the ATP-generating pathways (oxidative phosphorylation and glycolysis) or the state of mitochondrial network would significantly impact on cardiac health. Therefore, monitoring drug-induced dysfunction in these processes can be a significant contributor to evaluating cardiotoxicity.
Methods
Since the number, function and morphology of mitochondria are critical markers of iPSC-derived cardiomyocytes maturity and function, we first studied culture-conditions enabling these fetal-like cells to expand their oxidative capacity towards an adult-like state by promoting a shift from glycolysis to fatty acid oxidation. iPSC-derived cardiomyocytes, manufactured using Ncardia’s proprietary protocols, were cultured according to manufacturer’s instructions. Oxygen consumption rate (OCR) indicative of oxidative phosphorylation and extracellular acidification rate (ECAR), a marker of glycolysis, were measured using Agilent Ph-Xtra and MitoXpress kits. For signal detection a BMG plate reader in Dual-read Ratiometric Time Resolved Fluorescent mode was utilized. Mitochondria were stained with MitoTracker Red CMX Ros for morphological assessment. High-magnification images (40x) were acquired using an ImageXpress Micro Confocal microscope and image analysis performed in MetaXpress software version 6.6.
Results
First, OCR and ECAR as well as mitochondrial fluorescence signal readouts were used to facilitate selection of iPSC-CM culture medium compositions that resulted in mitochondrial function and morphology changes indicative of maturation. OCR and ECAR assays were standardized using a small set of validation compounds consisting of known inhibitors of the electron transport chain and glycolysis. Subsequently, changes induced by two known cardiotoxic compounds (Doxorubicin and BMS-986094) that are closely associated with mitochondrial dysfunction were studied.
Conclusions
Our results show the diverse applicability of standardized metabolic assays to monitor mitochondrial function in iPSC-CMs. This set of assays can aid in gaining a deeper understanding of metabolic dysfunction as a mechanism for drug-induced cardiotoxicity.
Acknowledgments: This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 858070.
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