Doxorubicin-Induced Cardiac Senescence Is Alleviated Following Treatment with Combined Polyphenols and Micronutrients through Enhancement in Mitophagy

Oxidative stress and impaired mitophagy are the hallmarks of cardiomyocyte senescence. Specifically, a decrease in mitophagic flux leads to the accumulation of damaged mitochondria and the development of senescence through increased ROS and other mediators. In this study, we describe the preventive role of A5⁺, a mix of polyphenols and other micronutrients, in doxorubicin (DOXO)-induced senescence of H9C2 cells. Specifically, H9C2 cells exposed to DOXO showed an increase in the protein expression proteins of senescence-associated genes, p21 and p16, and a decrease in the telomere binding factors TRF1 and TRF2, indicative of senescence induction. Nevertheless, A5⁺ pre-treatment attenuated the senescent-like cell phenotype, as evidenced by inhibition of all senescent markers and a decrease in SA-β-gal staining in DOXO-treated H9C2 cells. Importantly, A5⁺ restored the LC3 II/LC3 I ratio, Parkin and BNIP3 expression, therefore rescuing mitophagy, and decreased ROS production. Further, A5⁺ pre-treatment determined a ripolarization of the mitochondrial membrane and improved basal respiration. A5⁺-mediated protective effects might be related to its ability to activate mitochondrial SIRT3 in synergy with other micronutrients, but in contrast with SIRT4 activation. Accordingly, SIRT4 knockdown in H9C2 cells further increased MnSOD activity, enhanced mitophagy, and reduced ROS generation following A5⁺ pre-treatment and DOXO exposure compared to WT cells. Indeed, we demonstrated that A5⁺ protects H9C2 cells from DOXO-induced senescence, establishing a new specific role for A5⁺ in controlling mitochondrial quality control by restoring SIRT3 activity and mitophagy, which provided a molecular basis for the development of therapeutic strategies against cardiomyocyte senescence.