Determining Resistance to Protein Turnover in Aged Skeletal Muscle Collagen Using a Novel Stable Isotope Timecourse Approach
Authors: Lawrence Marcus M. , Abbott Claire , Peelor Frederick F. , Lopes Erika Barboza Prado , Griffin Timothy M. , Miller Benjamin F. 
Published: April 2020

BACKGROUND

Aged skeletal muscle presents with an accumulation of extracellular matrix (ECM) proteins, including collagen, that are stiffer and fibrotic (i.e., unresolvable and resistant to turnover) which alters the cellular microenvironment leading to reduced muscle function. A limitation of current assessments of protein turnover using a single timepoint is that they do not account for how much of a protein pool is actively turning over. In addition, defining the proportion of the protein pool actively turning over also informs on how much of the same pool is fibrotic. Thus, determining aging muscle changes in both collagen protein synthesis and the collagen protein pool actively turning over is vital for a more comprehensive understanding of fibrosis in aged skeletal muscle ECM.

PURPOSE

To test the hypothesis that with age the fraction of collagen proteins actively turning over would decrease, indicating an increase in fibrotic collagen, and that the fraction of the protein pool turning over would be inverse to the amount of collagen cross‐linking and overall protein oxidative damage.

METHODS

Young adult (6 mo.) and old (23 mo.) female C57BL/6 mice were labeled with the stable isotope deuterium oxide (D2O) for 4, 15, 30, 45, or 60 days (n=3–5/timepoint). Gastrocnemius muscles were analyzed for collagen protein synthesis (k 1/day) and the plateau (P) value for fraction new as a novel indicator of the protein pool capable of turning over (with 1.0 equal to 100% of the total pool turning over). Collagen cross‐linking was assessed using a pepsin digestion collagen solubility and hydroxyproline assay and Western Blotting for lysyl oxidase (LOX, catalyzes collagen cross‐links), while muscle protein carbonylation was assessed as a marker of oxidative damage.

RESULTS

Collagen protein synthesis and collagen plateau fraction new were significantly (p < 0.05) lower in old compared to young (0.011±0.002 vs 0.027±0.003 k 1/day and 0.141±0.015 vs 0.280±0.051 P, respectively). The lower collagen turnover in old compared to young muscle was paralleled by higher total LOX expression (20183±600 vs 16333±435 arbitrary density units, respectively), lower pepsin soluble collagen (15.1±1.28 vs 37.4±5.96 ng/mg muscle, respectively), higher pepsin insoluble (285±17.5 vs 201±16.4 ng/mg muscle, respectively) and total collagen (303±19.8 vs 237±18.4 ng/mg muscle, respectively), and unchanged protein carbonylation (0.083±0.002 vs 0.083±0.008 arbitrary density units, respectively).

CONCLUSIONS

The pool of collagen proteins actively turning over was significantly reduced in aged compared to young muscle, indicating that a greater proportion of the collagen proteins in aged muscle were fibrotic. The reduced proportion of collagen turning over was associated with increased markers of collagen cross‐linking, but not overall protein oxidative damage. Further research is warranted to identify which individual ECM proteins become fibrotic with aging to develop therapeutics to rescue muscle function.

Support or Funding Information

This research was supported by NIA Training Grant T32AG052363.