Increased cytochrome C threonine 50 phosphorylation in aging heart as a novel defensive signaling against hypoxia/reoxygenation induced apoptosis.

Previous studies have shown that aging promotes myocardial apoptosis. However, the detailed mechanisms remain unclear. Our recent studies revealed that aging not only activates apoptosis, but also activates some anti-apoptotic factors. By quantitative phosphoproteomics, here we demonstrated that aging increases cytochrome c (Cytc) phosphorylation at threonine 50 (T50), a post-translational modification with unknown functional impact. With point mutation and lentivirus transfection, cardiomyocytes were divided into four groups: empty vector group, WT (wild type), T50E (as a phosphomimic variant), and T50A (non-phosphorylatable). TUNEL staining and flow cytometry were used to determine the apoptosis ratio in different groups after hypoxic/reoxygenated (H/R) treatment. The results showed that T50-phosphorylated Cytc suppressed myocardial apoptosis induced by H/R. Furthermore, Western Blot and ELISA measurements revealed that Cytc T50 phosphorylation inhibited caspase-9 and caspase-3 activity without altering caspase-8, BCL-2, BCL-XL, and Bax expression. In our study, we demonstrated that aging increases phosphorylation Cytc at T50 and this aging-increasing phosphorylation site can suppress H/R-induced apoptosis.