Rationale: The widely used chemotherapeutic agent Doxorubicin (Dox) induces cardiotoxicity leading to dilated cardiomyopathy and heart failure. This cardiotoxicity has been related to ROS generation, DNA intercalation, bioenergetic distress and cell death. However, alternative mechanisms are emerging, focusing on signaling pathways. Objective: We investigated the role of Exchange Protein directly Activated by cAMP (EPAC), key factor in cAMP signaling, in Dox-induced cardiotoxicity. Methods and Results: Dox was administrated in vivo (10 +/- 2 mg/kg, i.v.; with analysis at 2, 6 and 15 weeks post injection) in WT and EPAC1 KO C57BL6 mice. Cardiac function was analyzed by echocardiography and intracellular Ca2+ homeostasis by confocal microscopy in isolated ventricular cardiomyocytes. 15 weeks post-injections, Dox-treated WT mice, developed a dilated cardiomyopathy with decreased ejection fraction, increased telediastolic volume and impaired Ca2+ homeostasis, which were totally prevented in the EPAC1 KO mice. The underlying mechanisms were investigated in neonatal and adult rat cardiac myocytes under Dox treatment (1-10 uM). Flow cytometry, Western blot, BRET sensor assay, and RT-qPCR analysis showed that Dox induced DNA damage and cardiomyocyte cell death with apoptotic features rather than necrosis, including Ca2+-CaMKKβ-dependent opening of the Mitochondrial Permeability Transition Pore, dissipation of the Mitochondrial membrane potential, caspase activation, cell size reduction, and DNA fragmentation. Dox also led to an increase in both cAMP concentration and EPAC1 protein level and activity. The pharmacological inhibition of EPAC1 (CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations including DNA damage, Mitochondrial membrane potential, apoptosis, mitochondrial biogenesis, dynamic, and fission/fusion balance, and respiratory chain activity, suggesting a crucial role of EPAC1 in these processes. Importantly, while preserving cardiomyocyte integrity, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Conclusion: Thus, EPAC1 inhibition could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity.
Fisetin inhibits growth, induces G2/M arrest and apoptosis of human epidermoid carcinoma A431 cells: role of mitochondrial membrane potential disruption and consequent caspases activation