Rates of Synthesis of Individual Proteins in Muscles of Old Mice: A Heavy Water Approach.

Abstract

Age‐associated loss of muscle mass and function is a major factor in the development of frailty, but the mechanisms responsible for this loss are unknown. Unquestionably, muscle atrophy is the consequence of changes in the balance between protein synthesis and breakdown. However, the nature and extent of these changes in old age is unclear, with some studies reporting reduced basal muscle protein synthetic rate in the aged compared with younger people and others suggesting no difference. Whole organism heavy water ([2H2]O) dynamic labelling of proteins, followed by LC‐MSMS, provides a significant improvement in analysis of protein turnover, allowing the rates of synthesis and degradation of both slow and fast turnover muscle proteins to be accurately modelled. The aim of this study was to use a [2H2]O labelling approach to determine the synthesis rates of individual proteins in the gastrocnemius muscles of adult and old mice.Ten adult and ten old male mice were primed with an intraperitoneal bolus injection of saline, prepared with 99% [2H2]O and subsequently, provided with 8% (v/v) [2H2]O in the drinking water, for up to 60 days. The sampling interval was distributed to obtain sufficient data points to cover both high and low turnover proteins. Muscle proteins were extracted and digested using standard procedures for analysis on a high‐resolution mass spectrometer (Thermo Q‐Exactive) and the raw data files were processed to allow protein identification using Crux‐tide ( McIlwain et al, 2014). The software RIAna (: https://github.com/Molecular‐Proteomics/riana) was then used to track time dependent incorporation of [2H] into tryptic peptides, yielding synthesis rates of proteins to be determined.Muscle mass was significantly reduced in the old mice and preliminary data demonstrated little change in the rate of synthesis of a large proportion of identified muscle proteins between adult and old mice. Several individual proteins showed a marked decrease in protein synthesis rates in muscles of old compared with adult mice, particularly structural proteins. Other proteins, particularly redox‐responsive proteins demonstrated an increased synthesis in muscles of old mice. Proteomic analysis of unlabelled muscle protein pools and RNASeq data will be integrated with the turnover data to further interpret these findings.This study was supported by the University of Liverpool, MRC and NIA (AG051442).