Immortalized Mouse Cardiac Fibroblasts-SV40

DEHP Promotes Apoptosis of Cardiomyocytes and the Transformation of Cardiac Fibroblasts into Myofibroblasts

Di-(2-ethylhexyl) phthalate (DEHP) has been epidemiologically associated with cardiac fibrosis. However, its underlying molecular mechanism remains elusive. Here, Zhang's team performed network toxicology analyses combined with molecular dynamics simulations and in vitro experiments to understand the cardiotoxic mechanism of DEHP.

Treatment with DEHP decreased cell viability of cardiomyocytes in a dose-dependent manner determined by CCK-8 assays, and thus 50 μg/mL DEHP was used for the following studies (Fig. 1A). Treatment with 50 μg/mL DEHP for 24 h significantly increased TUNEL-positive cardiomyocytes in rat H9c2 and human AC16 cells (Fig. 1D, G) and increased the ratio of BAX to BCL2 as well as cytochrome c expression in these cells (Fig. 1B, C, E, F), which demonstrated that DEHP induced apoptosis in cardiomyocytes. Interestingly, treatment with 50 μg/mL DEHP for 24 h did not induce apoptosis in immortalized mouse cardiac fibroblasts (Fig. 2A, B) but significantly increased fibrosis-associated markers including Tgfb1, Col1a1, and Acta2, markers of myofibroblast transdifferentiation (Fig. 2C). Mechanistically, they postulated that DEHP may exacerbate cardiac dysfunction via SRC, STAT3, and EGFR signaling axis. Western blot analyses revealed that the total protein level of SRC, STAT3, or EGFR were not changed upon DEHP treatment but p-SRC and p-STAT3 protein levels were significantly increased with no change in p-EGFR protein level, which confirmed the selective activation of SRC-STAT3 signaling axis (Fig. 2D, E). They performed CETSA and found that DEHP bound to and thermally stabilized SRC and STAT3 (Fig. 2F, G). Further, saracatinib (5 μM), an SRC/STAT3 phosphorylation inhibitor, rescued DEHP-induced increase in TGF-β and ACTA2 expression (Fig. 2H, I). Taken together, they uncovered that DEHP induced cardiomyocyte apoptosis and promoted cardiac fibrosis through direct activation of SRC-STAT3 signaling.