Impairment of IGF-I expression and anabolic signaling following ischemia/reperfusion in skeletal muscle of old mice

Abstract

With the advancement of age, skeletal muscle undergoes a progressive decline in mass, function, and regenerative capacity. Previously, our laboratory has reported an age-reduction in recovery and local induction of IGF-I gene expression with age following tourniquet (TK)-induced skeletal muscle ischemia/reperfusion (I/R). In this study, young (6 mo) and old (24-28 mo) mice were subjected to 2h of TK-induced ischemia of the hindlimb followed by 1, 3, 5, or 7 days of reperfusion. Real time-PCR analysis revealed clear age-related reductions and temporal alterations in the expression of IGF-I and individual IGF-I Ea and Eb splice variants. ELISA verified a reduction of IGF-I peptide with age following 7 day recovery from TK. Western blotting showed that the phosphorylation of Akt, mTOR, and FoxO3, all indicators of anabolic activity, were reduced in the muscles of old mice. These data indicate that an age-related impairment of IGF-I expression and intracellular signaling does exist following injury, and potentially has a role in the impaired recovery of skeletal muscle with age.

Figure 1

Wet masses of gastrocnemius muscles were measured from 6 mo (young) and 24–27 mo (old) C57BL/6 mice following 1, 3, 5, and 7 days of recovery from 2 hour TK-induced I/R. Values are represented as mean ± SEM. Statistical analysis was performed using two-factor ANOVA followed by SNK post-hoc (α = 0.05).

Figure 2

Plantaris muscles from 6 mo (young) and 24–27 mo (old) C57BL/6 mice were paraffin-embedded and stained with hematoxylin & eosin (H&E) following 1, 3, 5, and 7 days of recovery from 2 hour TK-induced I/R. Inset bar represents 100 μm.

Figure 3

Real time-PCR was performed to measure relative expression of IGF-I (A), IGF-I Ea (B), IGF-I Eb (C), and myostatin (D) mRNA in the extensor digitorum longus muscle of 6 mo (young) and 24–27 mo (old) C57BL/6 mice following 1, 3, 5, and 7 days of recovery from 2 hour TK-induced I/R. mRNA values are normalized to 18S rRNA expression and are represented as mean ± SEM relative to Day 1 young control levels. Statistical analysis was performed using two-factor ANOVA followed by SNK post-hoc (α = 0.05).

Figure 4

Whole tissue IGF-I peptide levels were measured by ELISA in the tibialis anterior and extensor digitorum longus muscles of 6 mo (young) and 24–27 mo (old) C57BL/6 mice following 7 days recovery from TK-induced I/R. Values are represented as mean ± SEM. Statistical analysis was performed using two-factor ANOVA followed by SNK post-hoc (α = 0.05).

Figure 5

Relative phosphorylation abundances, total protein contents, and phosphorylation/total protein ratios of Akt (A), mTOR (B), and FoxO3 (C) were measured by Western blotting samples from the gastrocnemius muscle of 6 mo (young) and 24–27 mo (old) C57BL/6 mice following TK-induced I/R. Representative Coomassie-blue stained gel (D) demonstrates consistent loading in all SDS-PAGE experiments. Values are represented as mean ± SEM. Statistical analysis was performed using Student’s t-tests; * p ≤ 0.05.