A Novel Stable Isotope Approach Demonstrates Surprising Degree of Age-Related Decline in Skeletal Muscle Collagen Proteostasis

Abstract

Age-related deterioration in turnover of collagen proteins accelerates extracellular matrix fibrosis and hinders adaptation to external stimuli. This project sought to understand factors that increase skeletal muscle fibrosis with age by studying what we term the dynamic protein pool. We hypothesized that the dynamic protein pool size of muscle collagen decreases with age, thus indicating a decrease in proteostatic maintenance (ie, ability to maintain proteostasis), and that failure to account for these changes impacts the interpretation of tracer-measured synthesis rates. We used deuterium oxide (D₂O) labeling for up to 60 days in adult (6 months) and old (23 months) mice. The dynamic protein pool in adult skeletal muscle was 65% in tibialis anterior (TA), but only 28% in gastrocnemius (Gastroc). In aged muscle, the dynamic protein pool was further decreased to only 35% and 14% for TA and Gastroc, respectively. We showed that this loss in dynamic pool size was associated with increases in markers of fibrosis and decreased proteostatic maintenance. We demonstrate that aged muscle has higher rates of collagen protein synthesis and lower rates of collagen protein breakdown, which causes collagen accumulation. We further demonstrated that the normal assumption of complete protein renewal and the standard practice of taking a single sample with isotope labeling have profound impacts on interpretation of the genesis of fibrosis. Strategies to maintain muscle function with aging should focus on the dynamic protein pool with attention to methodological strategies to assess those changes.

Keywords: aging; collagen; extracellular matrix; fibrosis; incomplete product renewal; muscle; stable isotope.

Graphical Abstract

Figure 1.

Overview of Experimental Design. Arrows indicate days when mice were euthanized. Dashed lines indicate the period of D₂O labeling. All labeling periods started with an i.p. injection of 99% D₂O.

Figure 2.

Demonstration of How the Period of Labeling Impacts Fractional Synthesis Rate (FSR) in Myofibrillar and Collagen Fractions of Gastrocnemius (A, B) and TA (C, D) Muscles. Calculation of FSR (%/day) using the precursor–product calculation at each individual time point. There was a significant effect of time indicating that as the period of labeling increased, the FSR became slower. The progressive decrease in FSR is caused by the decreased contribution of faster synthesized proteins (that fully turn over early) and the increased contribution of slower synthesized proteins. Filled boxes = adult mice; open boxes = old mice. N = 3–5 mice/time point (16–23 mice/age). Values expressed as mean ± SEM. Significance was determined by a two-way (age × time) analysis of variance.

Figure 3.

Time-Course Approach to Demonstrate Incomplete Renewal. Rise to plateau of fraction new of Gastroc myofibrillar (A) and collagen (C) proteins, and TA myofibrillar (F) and collagen (H) proteins. Calculated plateau values (p) for Gastroc myofibrillar (B) and collagen (D), and TA myofibrillar (G) and collagen (I) protein pools when it is assumed that p was ≤1.0. Representation of the dynamic protein pool size of the collagen in Gastroc (E) and TA (J), as determined from the p. Filled circles = adult mice; open squares = old mice. N = 3–5 mice/time point (18–20 mice/age). Values expressed as mean ± SEM. Significance was determined by an unpaired t-test.

Figure 4.

Secondary Measurements to Confirm Changes in Collagen Proteostasis, Including LOX Content (A, C), AGEs (B, D), and Protein Carbonylation (E). Protein solubility was performed in Gastroc followed by measurement of the collagen content of pepsin soluble fraction (F), pepsin insoluble fraction (G), total collagen content (H) by OHP assay, and collagen FSR of the soluble and insoluble muscle (I). TA collagen content (J), the ratio of collagen packing (K), and representative images of using polarized-light imaging picrosirius red staining (L). N = 5–12 muscles age group. Values expressed as mean ± SEM. Significance was determined by an unpaired t-test.

Figure 5.

Demonstration That Not Accounting for Differences in the Dynamic Protein Pool Impacts Qualitative and Quantitative Outcomes. In panels(A) (Gastroc) and (B) (TA), we show outcomes when it is assumed that the pool undergoes complete renewal (p = 1.0) or noncomplete renewal (p ≤ 1.0) of myofibrillar and collagen protein pools. Each figure shows p on the left axis and synthesis (k) on the right axis. In panel (C), we demonstrate how the commonly used approach of calculating FSR from a single time point of 15 or 60 days also changes the interpretation of the data. For panels (A) and (B), N = 3–5 mice/time point (18–20 mice/age). For panel (C), N = 3–5 mice/age group. Values expressed as mean ± SEM. Significance was determined by an unpaired t-test.