Department of Cardiology, Beijing An Zhen Hospital, Capital Medical University, and Beijing Institute of Heart, Lung, and Blood Vessel Disease, Beijing, China.
Aging (Albany NY). 2022 Jul 25;14(14):5699-5709. doi: 10.18632/aging.204159.
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.
先前的研究表明，衰老会促进心肌细胞凋亡。然而，其详细的机制仍不清楚。我们最近的研究表明，衰老是通过增加细胞色素 c（Cytc）在苏氨酸 50 位的磷酸化（T50）来激活凋亡，而这一翻译后修饰的功能影响尚未可知。通过定量磷酸化蛋白质组学，我们在此证明衰老可增加 Cytc 在苏氨酸 50 位的磷酸化（T50），这是一种功能未知的翻译后修饰。通过点突变和慢病毒转染，将心肌细胞分为四组：空载体组、WT（野生型）、T50E（磷酸模拟突变体）和 T50A（非磷酸化突变体）。TUNEL 染色和流式细胞术用于检测不同组在缺氧/复氧（H/R）处理后的凋亡比例。结果表明，T50 磷酸化 Cytc 抑制了 H/R 诱导的心肌细胞凋亡。此外，Western blot 和 ELISA 测量结果表明，Cytc T50 磷酸化不改变 caspase-8、BCL-2、BCL-XL 和 Bax 的表达，但抑制了 caspase-9 和 caspase-3 的活性。在我们的研究中，我们证明了衰老可增加 Cytc 在 T50 位的磷酸化，而这一衰老诱导的磷酸化位点可抑制 H/R 诱导的凋亡。
