Per- and Polyfluoroalkyl Substances Induce Cardiotoxicity and Alter Protein Profiles of Extracellular Matrix, Metabolism, and Mitochondrial Function in Human Cardiomyocytes

Per- and polyfluoroalkyl substances (PFAS), common environmental contaminants, can cause cardiotoxic effects particularly during fetal development. However, the effect of combined PFAS exposure, which more closely reflects real-world environmental conditions, remains poorly understood. In this study, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to three common PFAS compounds─perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and perfluorodecanoic acid (PFDA)─individually or in combination (20-200 μM; consistent with serum levels reported in occupationally exposed populations). Compared with single compounds, combined PFAS exposure induced synergistic cytotoxicity, significantly reducing hiPSC-CM viability after 5 or 10 days. Sublethal combined exposure for 10 days altered mitochondrial membrane potential and mitochondrial content in a dose-dependent manner and shifted cysteine metabolism, potentially reflecting adaptation to oxidative challenge. After 14 days, combined PFAS increased vimentin, a fibroblast marker, and reduced NKX2.5, α-actinin, and cardiac troponin T, key markers of cardiomyocytes, as detected by immunocytochemistry. Proteomics further showed enrichment of pathways in extracellular matrix organization, cholesterol metabolism, and antioxidant defense, as well as downregulation of mitochondrial proteins. Consistent with changes in protein profiles related to oxidative stress and bioenergetic impairment, exposure of hiPSC-CMs to combined PFAS also increased the level of mitochondrial superoxide, reduced ATP content, and decreased cellular respiration. Together, these data demonstrate that PFAS mixtures drive mitochondrial dysfunction, oxidative stress, metabolic changes, and extracellular matrix remodeling in hiPSC-CMs, underscoring the importance of evaluating PFAS mixtures to better understand cardiac risks from environmental exposure.