Excessive intracellular labile iron levels exacerbate reactive oxygen species (ROS) production through the Fenton reaction, leading to lipid peroxidation and ferroptotic cell death. Ferroptosis is commonly induced experimentally using chemical inhibitors such as RSL3 (a GPX4 inhibitor) or erastin (an inhibitor of the cystine-glutamate exchanger, Xc-) or by cysteine deprivation. However, these methods often fail to replicate the physiological complexity of ferroptosis and are associated with off-target effects. This study establishes a physiologically relevant model of ferroptosis in cardiomyocytes using ferric acetate (FAC) and tert-butyl hydroperoxide (TBH) to simulate iron overload and ROS generation. The combined application of FAC and TBH induced ferroptotic cell death, characterized by increased cytoplasmic Fe2+ levels, elevated lipid peroxidation, and a 2.5-fold rise in cell death, while FAC or TBH alone had minimal effects. Ferroptosis was confirmed by the complete prevention of cell death using ferrostatin-1 (a lipid peroxidation inhibitor) and ML351 (a 15-lipoxygenase inhibitor). Notably, this model bypasses the limitations of traditional synthetic inducers, such as off-target effects and inefficient mimicry of physiological conditions. Additionally, lipid peroxidation levels induced by the FAC-TBH combination were significantly higher than those induced by RSL3, further validating the relevance of this approach. These findings underscore the critical interplay between iron and ROS in ferroptotic cell death and highlight the utility of this model in advancing our understanding of ferroptosis mechanisms. This physiologically relevant system provides a robust platform for investigating therapeutic interventions targeting iron-induced oxidative stress and ferroptosis, particularly in conditions characterized by pathological iron accumulation, such as cardiomyopathies and ischemia-reperfusion injury. By focusing on the intrinsic drivers of ferroptosis, this work lays the groundwork for developing targeted treatments to mitigate ferroptosis-associated cellular damage.
