A Comparative Study of the Toxic Effects of t‐BHP on AC16 and H9c2 Cardiomyocytes

ABSTRACT tert ‐Butyl hydroperoxide (t‐BHP), a common oxidative stress inducer in cellular models, exhibits differential cytotoxic effects on AC16 and H9c2 cardiac cell lines. This study investigated these effects by treating both cell lines with varying t‐BHP concentrations, resulting in a dose‐dependent increase in cell mortality for both. The half‐maximal inhibitory concentration (IC 50 ) was 108.4 μmol/L for H9c2 cells and 419.3 μmol/L for AC16 cells. Under identical t‐BHP exposure, H9c2 cells demonstrated lower survival rates than AC16 cells, suggesting greater t‐BHP tolerance in AC16 cells. While increasing cell mortality in H9c2 cells correlated with a gradual rise in reactive oxygen species (ROS) levels, AC16 cells displayed an “inverted U‐shaped” trend. This implicates ROS as a critical factor in t‐BHP‐induced H9c2 cell mortality, a conclusion applicable to AC16 cells only below 350 μmol/L t‐BHP, above which other non‐ROS mechanisms may accelerate cell mortality in AC16 cells. Seahorse metabolic analysis indicated that t‐BHP exposure significantly reduced basal respiration, adenosine triphosphate (ATP) production, nonmitochondrial respiration, maximal respiration, and respiratory reserve capacity in both cell lines. Metabolically, H9c2 cells depend primarily on mitochondrial oxidative phosphorylation, while AC16 cells favor glycolysis. Moreover, proton leak in H9c2 mitochondria progressively decreased with increasing t‐BHP, whereas AC16 cells exhibited significantly increased mitochondrial proton leak ( p < 0.01) from the initial t‐BHP exposure, independent of concentration. The aforementioned results indicate that, establishing a model of cardiomyocyte death induced by increased oxidative stress with t‐BHP, H9c2 cells robustly simulate this pathological process. However, when selecting AC16 cells, great attention should be given to the t‐BHP concentration.