It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes. Aging led to an increase in oxidation of protein thiols demonstrated by increased S-glutathionylation of cysteine residues on proteins from Old (24 months old at the start of the study) mouse hearts compared to Young (5–6 months old). This shift in the oxidation state of the proteome was almost completely reversed by 8 weeks of treatment with elamipretide. Many of the significant changes that occurred were in proteins involved in mitochondrial or cardiac function. We also found changes in the mouse heart phosphoproteome that were associated with age, some of which were partially restored with elamipretide treatment. Parallel reaction monitoring of a subset of phosphorylation sites revealed that the unmodified peptide reporting for Myot S231 increased with age, but not its phosphorylated form and that both phosphorylated and unphosphorylated forms of the peptide covering cMyBP-C S307 increased, but that elamipretide treatment did not affect these changes. These results suggest that changes to thiol redox state and phosphorylation status are two ways in which age may affect mouse heart function, which can be restored by treatment with elamipretide.

已经证明，elamipretide (SS-31) 可以挽救心脏中与年龄相关的功能缺陷，但其背后的全套机制尚未确定。我们研究了 elamipretide 影响心脏蛋白翻译后修饰的假设。使用鸟枪法蛋白质组学分析小鼠心脏的 S-谷胱甘肽化和磷酸化蛋白质组，以评估衰老对这些翻译后修饰的影响以及线粒体靶向药物 elamipretide 逆转年龄相关变化的能力。与年轻（5-6 个月大）相比，衰老（研究开始时 24 个月大）小鼠心脏蛋白质上半胱氨酸残基的 S-谷胱甘肽化增加表明，衰老导致蛋白质硫醇氧化增加。这种蛋白质组氧化态的转变在 elamipretide 治疗 8 周后几乎完全逆转。发生的许多重大变化都发生在与线粒体或心脏功能有关的蛋白质中。我们还发现了与年龄相关的小鼠心脏磷酸化蛋白质组的变化，其中一些通过 elamipretide 治疗部分恢复。对磷酸化位点子集的平行反应监测显示，Myot S231 的未修饰肽报告随着年龄的增长而增加，但其磷酸化形式不增加，并且覆盖 cMyBP-C S307 的肽的磷酸化和非磷酸化形式均增加，但依拉米肽治疗没有增加影响这些变化。