Combined transcriptomic, proteomic and synthesis profiling reveal distinct proteostatic signatures for skeletal muscle of adult and old mice

Abstract

Muscle mass is reduced with age and numerous studies have examined the potential role of global changes in proteostasis, through protein synthesis and/or degradation in the overall loss of muscle mass with age, although no consensus has been reached.We hypothesise that changes in expression, synthesis and degradation may be very different for individual proteins and such findings may be overshadowed by examining global changes in protein turnover. In addition, we hypothesize that alterations in the synthesis rates of specific individual proteins in the muscles of old mice when compared with that of adult are likely to impact on the age‐related loss of muscle mass. We used deuterated water labelling to determine the synthesis rates of individual muscle proteins and have integrated these data with RNA Sequencing data and protein abundances to provide a powerful analysis of individual proteins in muscles of adult (6‐8months) and old (26months) C57BL/6J mice by which time ~15% of gastrocnemius muscle had been lost.For protein synthesis analysis, all animals received an initial loading dose of 2H2O via two IP injections of 99% 2H2O in PBS, 4hrs apart. Mice were then given 8% 2H2O in normal drinking water to maintain plasma enrichment of 2H2O at around 4.5% and gastrocnemius muscles were harvested for up to 60 days when mice were culled. Muscle RNA was isolated, RNA libraries generated using poly‐A selection, sequenced using Illumina HiSeq 4000 platform and analysed with DESEQ2 and gene ontology and pathway tools. To determine the effect of age on differentially expressed genes, protein abundance and turnover, we analysed the common list of 185 proteins and cognate transcripts for which we had data on transcript and turnover parameters.Functional annotation of proteins in the unlabelled dataset demonstrated an upregulation of protein degradation pathways with aging, including ubiquitin protein ligase binding and the unfolded protein response suggesting a clear role of protein degradation as a major contributor to the loss of muscle mass with age in mice. The top four KEGG disease pathways enriched when comparing protein abundance in the muscles of adult and old mice were all degenerative diseases caused or exacerbated by protein misfolding and accumulation. For most proteins, synthesis rates were unchanged, and thus, any change in abundance has to be reconciled with a commensurate change in degradation rate. These global data demonstrate that the net negative protein balance within the muscles of old mice is driven by elevated protein degradation rather than reduced translation. However, there were some notable outliers from the analysis of protein synthesis data showing a decrease in the synthesis of several structural proteins and an increase in the synthesis of metabolic or cytoprotective proteins in muscles of old compared with adult mice. The rate of synthesis of a protein was not always associated with transcript level suggesting caution in extrapolating the suggestion that static mRNA levels reflect the rate of protein synthesis or the protein levels in skeletal muscle.