Elamipretide (SS-31) Treatment Attenuates Age-Associated Post-Translational Modifications of Heart Proteins

Abstract

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.

Fast and slow skeletal myosin binding protein-C and aging

“…In the current study investigating cardiac tissue from two C57Bl/6 male age groups (13 months and > 29 months, n = 10), we did observe phosphorylation of cMyBP-C at Ser 282; however, no reduction in the phosphorylated state at this specific residue was noted, even though we report a decrease in abundance of cMyBP-C (noted above). While our results do not report the hypophosphorylation of cMyBP-C at Ser282 as reported in mice comparing ages of 7 months and 18-24 months, the specific phosphorylatable sites in cMyBP-C show varied responses to aging as noted above [45,47]. Given the evidence that implicates phosphorylation as a regulator of cardiac function, future studies focused on both contractility and characterization of the multiple specific sites of phosphorylation will provide valuable information [48].…”

“…At this time, the limited number of studies investigating the phosphorylation status in cMyBP-C with aging report hypophosphorylation cMyBP-C at Ser282 in 28-month-old mice compared to 7-month-old mice (male C57Bl/6, n = 5-6) [19] and in 18-24-month-old mice compared to 2-6-month-old mice (grouped male and female genetically modified cMyBP-C(tWT), n = 5-6) [45]. In contrast, there is a report of no change in phosphorylation cMyBP-C at Ser307 in 24-month-old mice compared to 6-month-old mice [47]. Interestingly, site-specific phosphorylation levels (cMyBP-C Ser273 and Ser282) decreased in a linear manner with increasing age, whereas this correlation was not observed with cMyBP-C 302 phosphorylation in the genetically modified cMyBP-C(tWT) mice [45].…”

Elamipretide effects on the skeletal muscle phosphoproteome in aged female mice

“…This report analyzes these datasets in the context of new phosphoproteomics data from the same muscle allowing direct comparisons between residues identified in separate datasets for the first time. Similar to cardiac muscle [21], these results show little effect of ELAM treatment on abundance proteomics, a modest effect on the phosphoproteome, and a very robust effect on protein S-glutathionylation. In contrast to the changes in S-glutathionylation [13], there are relatively few changes with either age or ELAM treatment to the phosphoproteome.…”

“…Quenching of digestion proceeded with trifluoroacetic acid addition to pH 2.0. Peptide samples were centrifuged and desalted using a 50-mg tC18 SepPak cartridge (Waters Corp, Milford, MA) as previously described [21]. Then, 20-and 500-µg aliquots of eluted peptides were dried by vacuum centrifugation and stored at − 80 °C for proteomic and phosphoproteomic analysis, respectively.…”

“…Dried peptides were resuspended in 80% acetonitrile and 1% trifluoroacetic acid, and enriched for phosphopeptides via immobilized metal affinity chromatography as previously described. LC-MS/MS was performed as previously described [21,22].…”