High content imaging of relative ATP levels for mitochondrial toxicity prediction in human induced pluripotent stem cell derived cardiomyocytes.

Abstract

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are increasingly being evaluated in assays aimed at better understanding potential cardiotoxic liability of newly developed therapeutic compounds. Disruption of mitochondria has been implicated in the mechanism of drug-induced cardiotoxicity of some compounds. Therefore, we have developed a high content imaging assay for the investigation of mitochondrial toxicity in hiPSC-CMs using ATP-Red, a fluorescent dye capable of detecting subcellular localization of relative ATP levels in living cells. We demonstrated time-dependent decreases in ATP-Red signal over 6h treatment with known mitochondrial toxicants antimycin (0.03, 0.1µM) or oligomycin (3, 10µM). Concentration-dependent decreases in ATP-Red signal with antimycin (0.001-0.3µM) and oligomycin (0.003-1µM) were rescued by restoring glycolysis through glucose supplementation. Decreased ATP levels were also identified in a selection of clinically available drugs with reported association with mitochondrial toxicity but absent in compounds with no known association with mitochondrial dysfunction. ATP measurements using the ATP-Red imaging assay were consistent with orthogonal measurements of whole cell ATP levels in lysed hiPSC-CMs following compound treatment. Similar findings were also obtained with measurement of mitochondrial membrane potential, except amiodarone which had no change despite decreased ATP levels. The developed high throughput imaging assay, assessing subcellular ATP dynamics, could provide mechanistic insights for tested compounds.