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

Abstract

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 antiapoptotic 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.

Phosphorylations and Acetylations of Cytochrome c Control Mitochondrial Respiration, Mitochondrial Membrane Potential, Energy, ROS, and Apoptosis

“…Therefore, these phosphorylation sites were also functionally characterized using phosphomimetic replacement strategies, most commonly by glutamate replacement, which mimics the negative charge of the phosphate group. Following Y97 phosphorylation, Y48 phosphorylation was identified in bovine liver [ 41 ], followed by T28 [ 42 ] and T58 [ 43 ] in bovine and rat kidney, respectively, S47 phosphorylation in rat and porcine brain [ 44 ], and, most recently, T58 in rat heart [ 45 ].…”

“…T49 phosphorylation (numbering based on the mature protein, which lacks the start methionine) was reported to increase in aged mouse heart after detection via high-throughput phosphoproteomics [ 45 ]. It was characterized using the AC16 cell line, a human cardiomyocyte cell line, which was transfected using lentiviral constructs containing the sequence for phosphomimetic T49E Cyt c .…”

Prostate Cancer-Specific Lysine 53 Acetylation of Cytochrome c Drives Metabolic Reprogramming and Protects from Apoptosis in Intact Cells

“…Six tissue-specific phosphorylations, a cancer-specific acetylation, and an acetylation that is gained during ischemia in skeletal muscle have been mapped on Cyt c . The phosphorylations are tyrosine 97 and threonine 49 in the normal and aging hearts, respectively [ 9 , 10 , 11 ], tyrosine 48 in the liver [ 12 , 13 ], threonine 28 and threonine 58 in kidney [ 14 , 15 ], and serine 47 in the brain [ 16 , 17 ]. Generally, these modifications were found to be present under basal conditions in their respective tissues and were lost after ischemia, contributing to ischemia-reperfusion injury [ 18 ].…”