Apoptosis and mitochondrial bioenergetic dysfunction are hallmarks of cardiotoxicity that drive cardiac damage, particularly following exposure to chemotherapeutic agents such as doxorubicin. Doxorubicin induces pro-apoptotic signaling and disrupts mitochondrial homeostasis and cellular energy metabolism, leading to cardiac injury. Ambrisentan, a selective endothelin A (ETA) receptor antagonist, was investigated in this study for its protective effects against doxorubicin-induced injury in H9c2 cardiomyoblasts, focusing on the p53 and mTOR signaling pathways. Pretreatment with ambrisentan preserved cardiomyocyte viability, attenuated apoptosis, reduced cytosolic and mitochondrial reactive oxygen species accumulation, restored mitochondrial membrane potential, and improved mitochondrial respiration and glycolytic capacity following doxorubicin exposure. Transcriptional and translational analyses showed that these effects were consistent with an induced pro-survival phenotype and improved mitochondrial integrity. Specifically, ambrisentan suppressed apoptotic markers (p-p53/p53, BAX), the mitochondrial fission regulator DNM1, and endothelin components (ET-1, ETA receptor). Concurrently, it restored the anti-apoptotic regulator Bcl-2, key mitochondrial regulators (NRF1, PGC1α, OPA1, ATP5A), and mTOR-associated proteins (p-mTOR/mTOR, Rictor, Raptor), while reducing Beclin-1 expression. Pharmacological modulation further demonstrated that the p53 inhibitor pifithrin-α enhanced the protective effects of ambrisentan; conversely, the p53 activator nutlin-3 reduced them, suggesting a possible role for p53 signaling in mediating these responses. Moreover, inhibition of mTOR signaling with rapamycin attenuated the beneficial effects of ambrisentan on oxidative stress, apoptosis, and mitochondrial dysfunction. Collectively, these findings suggest that ambrisentan protects cardiomyocytes from doxorubicin-mediated injury, with potential involvement of p53- and mTOR-associated signaling in preserving redox homeostasis and cellular bioenergetics.
