We analyzed the effects of aging on protein abundance and acetylation, as well as the ability of the mitochondrial-targeted drugs SS-31 and NMN to reverse age-related changes, in mouse hearts. Both drugs had a modest effect on restoring the abundance and acetylation of proteins that are lost with age but only NMN reduced acetylation in a subset of proteins which increased acetylation with age. Primarily, these age-related increases in protein acetylation were in mitochondrial pathways such as mitochondrial dysfunction, oxidative phosphorylation, and TCA cycle signaling. We further assessed how these age-related changes correlated with diastolic function (Ea/Aa) and systolic function (fractional shortening under higher workload) measurements from echocardiography. These results show that diastolic and systolic function in old age correlate inversely with the abundance of a specific subset of proteins. This could indicate that the heart proteome may adapt to preserve diastolic or systolic function with age but cannot preserve both. This adds further evidence to our previous report that SS-31 provides benefit to diastolic function and NMN to systolic function and that the greatest benefit may be when both are administered together, as the combination provides a mechanism to improve both aspects of heart function simultaneously.

All mass spectrometry was performed on an Q Exactive HF-X (Thermo Fisher Scientific, Waltham, MA). Samples were loaded from the autosampler onto a 150-μm Kasil fritted trap packed with ReproSil-Pur 120 Å C18-AQ 3 micron beads (Dr. Maisch High Performance LC, Ammerbuch-Entringen, Germany) to a bed length of 2 cm at a flow rate of 2 µl/min for 4 minutes. After loading and desalting, the trap was brought in-line with a pulled fused-silica capillary tip (75-μm ID) packed with 30 cm of the same chromatography material mounted in a nanospray source with the column and trap heated to 50oC. Peptides were eluted off the trap and column using a Thermo Easy-nLC 1200 HPLC using flow rate of 250 nl/min and a gradient of 0-32% acetonitrile in 0.1% formic acid over 90 minutes, followed by 32 – 60% gradient over 5 minutes, and a subsequent column wash at 80% acetonitrile. The mass spectrometer was operated using electrospray ionization (2 kV) with the heated transfer tube at 275oC in data independent acquisition (DIA) mode covering a precursor range of m/z 400 to 1000. The quadrupole isolation width was set to m/z 8, MS/MS resolution was 15,000 at m/z 200, the automatic gain control target was set to 1e6, and the maximum fill time was set to 22 msec. For every other cycle of DIA covering the entire precursor range, the isolation windows were offset by m/z 4 in order to obtain overlapping DIA windows that could be deconvolved, resulting in an effective isolation width of m/z 4. The normalized collision energy was set at 28% with a default charge state of 2. Each cycle of DIA was preceded by an MS acquisition from m/z 400 to 1000 at a resolution of 30,000 with an AGC target of 3e6 covering m/z 400-1000. Chromatogram libraries were made from the two pooled samples (pre- and post-enrichment) in a similar manner except that the quadrupole isolation width was m/z 4 with alternating overlap to give an effective isolation width of m/z 2. Data dependent acquisition (DDA) data was also acquired on the two pools using resolution of 60,000 in MS1 with 3e6 as the AGC target. For MS2, the quadrupole isolation width was set to 1.6, the resolution 15,000, the AGC target 22 msec, and gas phase fractionation was used covering the precursor ranges of m/z 400-600, 600-900, and 900-1600.

Powdered heart tissue was homogenized by probe sonication in ice-cold extraction buffer (0.1% RapiGest surfactant in 100 mM Tris-HCl pH 8.0) with 10 µM trichostatin A and 10 mM nicotinamide in order to preserve the acetylation state of proteins. Protein was digested using trypsin and samples were spun down at 12000 x g to remove insoluble materials. Peptides were purified using MCX columns following the manufacturer’s instructions (Waters). Samples were dried using a CentriVap Concentrator (LABCONCO, Kansas City, MO) and reconstituted in 0.1% formic acid with 16.67 fmol/µl PRTC. Samples were diluted and solubilized in 180 µl of HPLC loading buffer (0.1% trifluoracetic acid and 2% acetonitrile) by vortexing for 10 minutes at 37oC to give an estimated final concentration of 0.11 µg/µl. The solubilized samples were centrifuged at 10,000 x g for 5 minutes to pellet any particulates that might cause HPLC clogging, and then a portion of each supernatant was placed into autosampler vials. Injection volumes were 3 µl. Samples were run in blocks containing single replicates of each condition, where the order within each block was randomized.700 µg of peptides from each sample were taken prior to drying and subjected to a pulldown for the enrichment of acetylated peptides using the Acetyl-Lysine PTMScan Kit following the manufacturer’s instructions (Cell Signaling Technologies, Danvers, MA). Following this pulldown, peptides were run under the same conditions described for standard DIA proteomics as described above, with the following changes.