Human cardiac organoids highlight cardiotoxicity of the tire rubber antioxidant 6PPD.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), a tire rubber antioxidant and environmental contaminant, poses potential human cardiovascular health risks. However, these remain poorly understood. In this study, we established human induced pluripotent stem-cell-derived cardiac organoids (hiPSC-COs) to evaluate 6PPD cardiotoxicity. The organoids exhibited mature cardiac characteristics, containing multiple cell types, including cardiomyocytes, smooth muscle cells, and cardiac fibroblasts. There was also robust expression of cardiac structural and electrophysiological markers. 6PPD exposure induced concentration-dependent cytotoxicity and apoptosis, with increased cleaved caspase-3 and Bax expression with concurrent BCL2 suppression. Sub-cytotoxic concentrations disrupted cardiac electrophysiology within 60 min, causing tachycardia and shortened field potential duration that persisted at 24 h. Transcriptomic profiling showed dose-dependent molecular responses. 10 μM 6PPD activated DNA damage responses while suppressing cardiac contractile genes. Meanwhile, 100 μM triggered endoplasmic reticulum stress, autophagy, and apoptosis alongside disrupted calcium homeostasis and cardiac development programs. 6PPD induces multifaceted cardiotoxicity with functional impairment occurring below cytotoxic thresholds, highlighting the potential cardiovascular risks from environmental exposure and establishing hiPSC-COs as a valuable platform for environmental cardiotoxicant assessment.