Increased Adipocyte Area in Injured Muscle With Aging and Impaired Remodeling in Female Mice

Abstract

We demonstrated that young male and female mice similarly regenerated injured skeletal muscle; however, female mice transiently increased adipocyte area within regenerated muscle in a sex hormone-dependent manner. We extended these observations to investigate the effect of aging and sex on sarcopenia and muscle regeneration. Cardiotoxin injury to the tibialis anterior muscle of young, middle, and old-aged C57Bl/6J male and female mice was used to measure regenerated myofiber cross-sectional area (CSA), adipocyte area, residual necrosis, and inflammatory cell recruitment. Baseline (uninjured) myofiber CSA was decreased in old mice of both sexes compared to young and middle-aged mice. Regenerated CSA was similar in male mice in all age groups until baseline CSA was attained but decreased in middle and old age female mice compared to young females. Furthermore, adipocyte area within regenerated muscle was transiently increased in young females compared to young males and these sex-dependent increases persisted in middle and old age female mice and were associated with increased Pparg Young female mice had more pro-inflammatory monocytes/macrophages in regenerating muscle than young male mice and increased Sca-1(+)CD45(-)cells. In conclusion, sex and age influence pro-inflammatory cell recruitment, muscle regeneration, and adipocyte area following skeletal muscle injury.

Keywords: Monocyte/macrophage; Muscle regeneration; Sarcopenia.

Figure 1.

Effect of sex and age on body weight, anterior compartment (AC) weight, and myofiber cross-sectional area (CSA) in the tibialis anterior muscle. Baseline (noninjured) male and female mice of different ages included young (4–6 months), middle-aged (12–15 months), old (25–30 months), and for males only, very old (32–33 months) animals. (A) body weight, (B) AC weight, and (C) CSA of tibialis anterior muscle fibers. Data presented as mean ± SE; n = 7–12 mice/sex/age group; #p ≤ .01 male vs. female at the corresponding age group, ⁺ p ≤ .02 compared to middle-aged females; ^{^} p ≤ .008 compared to old age group for each sex; §p ≤ .005 compared to very old male mice.

Figure 2.

Effect of sex and age on residual myonecrosis at Day 7 following cardiotoxin injury. (A–D) Histological detail of residual necrosis within regenerated tibialis anterior muscle obtained at 7 days postinjury in young and old, male and female mice. Note regenerated myocytes with centrally located nuclei and infiltrates of mononuclear cells surrounding residual necrotic myofibers (asterisks). Arrows indicate adipocytes. (E) The extent of injury and residual necrosis in the tibialis anterior muscle was determined by histomorphometry in specimen collected from male and female mice at 3 different ages: young (4–6 months), middle (12–15 months), and old (25–30 months). Data presented as mean ± SE; n = 7–12 mice/sex/age group; ⁺ p < .001 compared to young female mice.

Figure 3.

Effect of sex and age on the progressive growth of regenerated myofibers and the intermuscular adipocyte area. The CSA of myocytes and area (%) of adipocytes in the tibialis anterior muscle of male and female mice of different ages included young (4–6 months), middle (12–15 months), and old (25–30 months) obtained at baseline (no injury, Day 0) and at various time (days) after injury. Myofiber CSA in (A) young, (B) middle, and (C) old age male and female mice. Adipocyte area (%) within injured/regenerated muscle in (D) young, (E) middle, and (F) old age male and female mice. Data presented as mean ± SE; n = 6–13 mice/sex/age group/time point; *p ≤ .04 compared to baseline for each sex; ^# p ≤ .05 male vs female at the corresponding time point.

Figure 4.

Inflammatory cell recruitment in young male and female mice after injury. Cells isolated from cardiotoxin-injured muscles of the anterior and posterior compartments of the hind limbs representing all the muscle below the knee but not including the foot were analyzed by flow cytometry. (A) total cells, (B) neutrophils (CD11b⁺Ly6G⁺), (C) monocytes (CD11b⁺(CD90/B220/CD49/NK1.1/Ly6G)⁻(F4/80/I-A^b/CD11c)⁻Ly6C^+/−), (D) macrophages (CD11b⁺F4/80⁺), (E and G) monocyte subsets, and (F and H) macrophage subsets. Data presented as mean ± SE, n = 3 mice/sex/time point; ^# p ≤ .008 male vs female at the corresponding time point, †p < .001 Ly6C⁺ monocyte subset male vs female at the corresponding time point, *p ≤ .008 for Ly6C⁺CD206⁻ or Ly6C⁺CD301⁻ macrophage subset male vs female at the corresponding time point.

Figure 5.

Adipocyte area and Sca-1⁺CD45⁻ cells in injured and regenerated tibialis anterior muscle. (A–H) Histological appearance of regenerating muscle. (I and J) Cells isolated from cardiotoxin-injured muscle were analyzed by flow cytometry; isotype controls were used to determine optimal antibody concentrations. (I) Gating strategy for Sca-1⁺CD45⁻ cells; gates (left panel) were set using fluorescence minus one controls. After gating out debris and dead cells based on forward vs side scatter and propidium iodide, respectively, Sca-1⁺CD45⁻ cells from a female at Day 5 (right panel) were identified, (J) Sca-1⁺CD45⁻ cells in young male and female mice. Data represented as mean ± SE, n = 3 mice/sex/time point; ^# p = .05 male vs female at the corresponding time point.

Figure 6.

Inflammatory cell recruitment and Sca-1⁺CD45⁻ cells in young and middle-aged mice 5 days after injury. Cells isolated from cardiotoxin-injured muscles of the anterior and posterior compartments of the hind limbs representing all the muscle below the knee but not including the foot were analyzed by flow cytometry in male and female mice of different ages included young (4–6 months, n = 3/sex) and middle (12 months for males and 19 months for females, n = 4/sex). (A) Total cells, (B) neutrophils (CD11b⁺Ly6G⁺), (C) monocytes (CD11b⁺(CD90/B220/CD49/NK1.1/Ly6G)⁻(F4/80/I-A^b/CD11c)⁻Ly6C^+/−), (D) macrophages (CD11b⁺F4/80⁺), (E) monocyte subsets, (F and G) macrophage subsets, and (H) Sca-1⁺CD45⁻ cells. Data presented as mean ± SE, n = 3–4 mice/sex/age group; †p ≤ .04 compared to young for the corresponding sex, ^# p ≤ .04 male vs female at the corresponding age group, *p = .04 for Ly6C⁺CD206⁻ macrophage subset compared to young for the corresponding sex, §p ≤ .02 for Ly6C⁻CD206⁻ or Ly6C⁻CD301⁻ macrophage subset compared to young for the corresponding sex, ^{^} p = .03 for Ly6C⁻CD301⁻ macrophage subset compared to middle-aged males.

Figure 7.

Effect of sex and age on expression of transcription factors Pparg and Cebpa at baseline, 7, and 21 days after injury. Whole tissue RNA isolated from muscles of the hind limb anterior compartment at baseline (uninjured) or at Day 7 or 21 after cardiotoxin injury were analyzed by quantitative RT-PCR in male and female mice of different ages including young (4–6 months), middle (12–15 months), old (25–30 months). The ranges of raw Ct values for each transcript were: Ubc 20.5 – 26.8, Pparg 22.9 – 29.7 and Cebpa 23.8 – 32.9. (A) Pparg and (B) Cebpa. Data presented as mean 2^-dCt ± SE, n = 4–7 mice/sex/age group; †p ≤ .04 compared to young for the corresponding time point and sex, ^# p ≤ .04 male vs female at the corresponding time point and age group, *p ≤ .04 compared to baseline for the corresponding age group and sex, ¥p ≤ .04 compared to middle-aged for the corresponding time point and sex.