Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle

Abstract

Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral "rejuvenating" factors that can restore regenerative function. Here, we demonstrate that the circulating protein growth differentiation factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction.

Figure 1. Rejuvenation of muscle stem cells by heterochronic parabiosis

(A). Frequency of clone-sorted satellite cells from isochronic (Iso) or heterochronic (Het) mice forming colonies after 5 days in culture. All colonies showed characteristic morphology of muscle lineage cells. (B) DNA damage in freshly sorted satellite cells assessed by single cell gel electrophoresis under alkaline conditions. Damage was quantified using a visual scoring metric (25) (key at top) and represented by color-coding: no damage (green), moderate damage (orange), maximal damage (red). (C) Representative images (confocal z-stacks) of freshly sorted satellite cells stained with DAPI (blue) and anti-pH2AX (green); data are quantified in (D). All graphs represent mean ± SD, with p-values, calculated by Mann-Whitney analysis. “n=” indicates number of mice used for each analysis. Scale bar = 10µm.

Figure 2. Rejuvenation of muscle stem cells by rGDF11 supplementation

(A, B). Frequency (A) and myogenic colony formation (B) of satellite cells from vehicle- or rGDF11-treated mice. (C). Quantification of DNA damage assays using freshly sorted satellite cells from vehicle- or rGDF11-treated mice, scored as in Fig. 1B. (D, E). H&E staining (D) and frequency distribution of myofiber size (E) in regenerating TA muscles 7 days after cryoinjury in vehicle- or rGDF11-treated young and aged mice. Scale bar = 100µm. (F). Representative images of transverse cryosections of TA muscles 2 weeks after transplantation. (G) Quantification of transplant data as maximal number of GFP⁺ myofibers found in each engrafted muscle. Graphs represent mean ± SD (in A–C, G). p-values were calculated by Mann-Whitney analysis (A–C), Wilcoxon Exact analysis (E), or Student’s t-test (G). “n=” indicates number of mice used for each analysis.

Figure 3. Improved muscle physiology and physical function after rGDF11 supplementation

(A). Electron micrographs of transverse sections of TA muscle from vehicle- or rGDF11-treated aged mice (representative of n=4 per group). Arrows indicate swollen mitochondria. (B, C). Western blot of PGC-1α (B) and LC3 forms I and II (C) in TA muscle extracts from cardiotoxin-injured or uninjured vehicle- or rGDF11-treated aged mice. Three animals are shown for each experimental group. Densitometric quantification of Western data are provided below each blot, normalized to GAPDH (B) or Actin (C). (D, E). Scatter plots of exercise endurance (D, maximum treadmill runtime in a 90 minute window) or forelimb grip-strength (E) of vehicle- or rGDF11-treated aged mice. Grip-strength is plotted as maximum force (Newton, N) exerted in triplicate trials. Red line represents the maximum grip-strength of 33-39 week-old young male mice. Data are presented for individual mice (black symbols) overlaid with mean ± SD (orange lines). p-values were calculated by Mann-Whitney analysis. “n=” indicates number of mice used for each analysis.