Our study aimed to characterize sarcopenia in C57BL/6J mice using a clinically relevant definition and age range and investigate the underlying mechanisms leading to sarcopenia. Aged male C57BL/6J mice (23-32 months) were classified as non-sarcopenic (no deficit), probable sarcopenic (1 deficit), and sarcopenic (2-3 deficits) based on assessments of grip strength, muscle mass, and treadmill running time and using 2 standard deviations below the mean of the young (4-9 months) as cut-off points. A 9-22% prevalence of sarcopenia was identified in 23-26-month-old mice. Age-related declines in muscle function were more severe than in muscle mass and outcomes of all three assessments were positively correlated in aged mice. As sarcopenia progressed, there were decreases in specific force as well as fiber size and number of IIB skeletal muscle fibers. Mitochondrial biogenesis, oxidative capacity, and AMPK-autophagy signaling decreased significantly while no increase in atrogenes was detected. Our study is the first to characterize sarcopenia in C57BL/6J mice using a clinically relevant definition and highlights the different trajectories of age-related declines in muscle mass and function. These critical insights into the molecular changes associated with sarcopenia progression will facilitate future development of therapeutic interventions.

Large chunks of mouse quadricep muscle tissue (30 mg to 100 mg) were first homogenized in 5% sodium dodecyl sulfate (SDS)/50 mM triethylammonium bicarbonate (TEAB) lysis buffer (Sigma) with protease and phosphatase inhibitors in a Bullet Blender Storm (Next Advance, Inc) with 0.5 mm zirconium oxide beads at 4°C. Buffer volume was added to 5X tissue weight and bead volume was 3X tissue weight. The homogenate was then further lysed with a probe sonicator (Fisher Scientific) for 3 cycles of setting #3 for 2 minutes on ice in between lysis steps. Protein concentration was measured using the Pierce BCA assay (Thermo Scientific). Homogenate of 50 μg was added to a process control of 800 ng of yeast enolase protein (Sigma) and then reduced with 20 mM DTT and alkylated with 40 mM IAA. Lysates were prepared for S-trap column (Protifi) binding by the addition of 1.2% phosphoric acid and 350 μl of binding buffer (90% Methanol, 100 mM TEAB). The acidified lysate was bound to the column incrementally, followed by 3 wash steps with binding buffer to remove SDS and 3 wash steps with 50:50 methanol:chloroform to remove lipids and a final wash step with binding buffer. Trypsin (1:10) in 50mM TEAB was added to the S-trap column for digestion at 47°C for one hour. Peptides were eluted with increasing hydrophobicity with a first elution of 50 mM TEAB, followed by elutions with 0.1% trifluoroacetic acid (TFA) and then 50% acetonitrile in water. Elutions were pooled, speed vacuumed and resuspended in 0.1% TFA. Peptides were eluted from a PepSep C18 15 cm column with 150 μm inner diameter and 1.9 μm particle size (Bruker), combined with a PepMap C18 5 mm Neo Trap Cartridge with 300 μm inner diameter (Thermo Scientific), and a Fossil Sharp Singularity LOTUS 5 cm hydrophobic coated nESI emitter with 20 μm inner diameter and 363um outer diameter (Fossil Ion Technology). A 50°C heated source (CorSolutions) was used to electrospray two μg of each digested sample with 300 femtomole of Pierce Retention Time Calibrant (PRTC) onto a Thermo Scientific Vanquish Neo UHPLC system coupled with a Thermo Orbitrap Eclipse Tribrid Mass Spectrometer with the application of a distal 3 kV spray voltage. The PRTC was used to assess the system suitability of the column and instruments before and during analysis. We analyze system suitability runs prior to any sample analysis and then after every six to eight sample runs another system suitability run is analyzed. Buffer A is 0.1% formic acid in water and buffer B is 0.1% formic acid in 80% acetonitrile. The 25-minute system suitability gradient consists of a 4 to 6% B in 0.7 minutes, 6 to 6.5% B in 0.3 minutes, 6.5 to 40% B in 20 minutes, 40 to 55% B in 0.5 minutes, followed by a wash of 99% B for 3.5 minutes at a flow rate of 1.3 uL/min. The 60-minute sample LC gradient consists of a 4 to 6% B for 0.7 minutes, 6 to 6.5% B in 0.3 minutes, 6.5 to 40% B in 55.7 minutes, 40 to 55% B in 0.5 minutes, followed by a wash of 99% B for 2.8 minutes at a flow rate of 0.8 to 1.3 uL/min. For the system suitability analysis, a cycle of one 30,000 resolution full-scan mass spectrum (400-810 m/z) with AGC target of 4e5 and maximum injection time of 50 millisecond (ms) followed by a data-independent acquisition (DIA) MS/MS spectra on a loop count of 20 using an inclusion list of +2 precursor charges at 15,000 resolution, AGC target of 5e4, 22 ms maximum injection time, 30% normalized collision energy with a 2 m/z isolation window. For the sample digest, first a chromatogram library of 6 independent injections is analyzed from a pool of all samples within a batch. For each injection a cycle of one 30,000 resolution full-scan mass spectrum with AGC target of 4e5 and maximum injection time of 50 ms and a mass range of 110 m/z (395-505 m/z, 495-605 m/z, 595-705 m/z, 695-805 m/z, 795-905 m/z, 895-1005 m/z) followed by a DIA MS/MS spectra on a loop count of 25 using an inclusion list of +3 precursor charges at 30,000 resolution, AGC target of 5e5, 54 ms maximum injection time, 27% normalized collision energy with a 4 m/z overlapping isolation window. The chromatogram library data is used to quantify proteins from individual sample runs. These individual runs consist of a cycle of one 30,000 resolution full-scan mass spectrum with a mass range of 350-1005 m/z with AGC target of 4e5 and maximum injection time of 50 ms followed by a DIA MS/MS spectra on a loop count of 75 using an inclusion list of +3 precursor charges at 30,000 resolution, AGC target of 4e5, 54 ms maximum injection time, 27% normalized collision energy with an overlapping 12 m/z isolation window. Application of the mass spectrometer and LC solvent gradients are controlled by the ThermoFisher Xcalibur data system.

Batch design was randomly balanced based on the 4 condition ratios. The condition groups were: Young (6-month old) GHSR-1a WT, n=6 (Young WT), Old (30-month old) adult GHSR-1a WT, n=6 (Old WT), Young (6-month old) GHSR-1a KO C57BL/6J mice, n=6 (Young KO), and Old (30-month old) adult GHSR-1a KO C57BL/6J mice, n=6 (Old KO). The samples were divided into 2 batches of 12 individual samples and 2 pooled references for a total of 14 samples per batch. A pool of all 24 samples was used to create a reference pool to be used as a common reference, which was homogenized, aliquoted, frozen, and used to compare between the two batches. 4 conditions, 6 mice each for 24 samples