Loss of REDD1 augments the rate of the overload-induced increase in muscle mass

Abstract

The overload-induced increase in muscle mass is accompanied by protein accretion; however, the initiating events are poorly understood. Regulated in Development and DNA Damage 1 (REDD1), a repressor of the mechanistic target of rapamycin in complex 1 (mTORC1), blunts the elevation in protein synthesis induced by acute muscle contractions. Therefore, this study was designed to determine whether REDD1 alters the rate of the overload-induced increase in muscle mass. Wild-type (WT) and REDD1-null mice underwent unilateral functional overload (OV) of the plantaris, while the contralateral sham leg served as a control. After 3 and 5 days of OV, puromycin incorporation was used as a measurement of protein synthesis. The percent increase in plantaris wet weight and protein content was greater in REDD1-null mice after 3, 5, and 10 days OV. The overload-stimulated rate of protein synthesis in the plantaris was similar between genotypes after 3 days OV, but translational capacity was lower in REDD1-null mice, indicating elevated translational efficiency. This was likely due to elevated absolute mTORC1 signaling [phosphorylation of p70S6K1 (Thr-389) and 4E-BP1 (Ser-65)]. By 5 days of OV, the rate of protein synthesis in REDD1-null mice was lower than WT mice with no difference in absolute mTORC1 signaling. Additionally, markers of autophagy (LC3II/I ratio and p62 protein) were decreased to a greater absolute extent after 3 days OV in REDD1-null mice. These data suggest that loss of REDD1 augments the rate of the OV-induced increase in muscle mass by altering multiple protein balance pathways.

Keywords: autophagy; protein synthesis; resistance exercise; ribosome biogenesis.

Fig. 1.

Loss of REDD1 augments the rate of the overload-induced increase in muscle mass and protein accretion following unilateral synergistic ablation-induced overload. A: percent change in muscle wet weight between the sham control muscle and overloaded muscle after 1, 3, 5, and 10 days. B: percent change in total protein between sham control muscle and the overloaded muscle after 3 and 5 days. *Significant difference between genotypes within the time point. n = 3–8 per group from 1–2 independent experiments. Significance was set at P ≤ 0.05.

Fig. 2.

Loss of REDD1 alters translational capacity independent of a change in ribosome biogenesis. A: percent change in total RNA between the sham control muscle and the overloaded muscle after 1 and 3 days. B: relative abundance of the external transcribed spacer (ETS) of the 45S pre-rRNA was determined by quantitative RT-PCR in the overloaded muscle of both wild-type and REDD1-null mice after 1 and 3 days. C: translational capacity [RNA content per milligram of muscle (ng/mg)] of the overloaded muscle of wild-type and REDD1-null mice after 1 and 3 days of overload. *Significant difference between overloaded muscles of genotypes within a time point. n = 6 or 7 per group. Significance was set at P ≤ 0.05.

Fig. 3.

Loss of REDD1 alters absolute mTORC1 signaling and translational efficiency following 3 days of overload. A: protein synthesis was determined by the SUnSET method. The phosphorylated to total protein ratio of p70S6K1 (Thr-389) (B) and 4E-BP1 (Ser-65) (C) were determined by Western blot analysis. D: REDD1 content was determined by Western blot analysis. E: percent change in indicated measures between the sham and overloaded muscle. F: representative Western blots. Lines connecting bars indicate significant differences. OV, overload; +/+, wild-type mice; −/−, REDD1-null mice; P, phosphorylated protein; T, total protein. n = 7/group from two independent experiments.

Fig. 4.

Loss of REDD1 blunts the overload-induced rate of protein synthesis but does not alter mTORC1 signaling after 5 days of overload. A: protein synthesis was determined by the SUnSET method. The phosphorylated-to-total protein ratio of p70S6K1 (Thr-389) (B) and 4E-BP1 (Ser-65) (C) were determined by Western blot analysis. D: REDD1 content was determined by Western blot analysis. E: percent change in indicated measures between the sham and overloaded muscle. F: representative Western blots. Lines connecting bars indicate significant differences. OV, overload; +/+, wild-type mice; −/−, REDD1-null mice; P, phosphorylated protein; T, total protein. n = 6/8 per group from two independent experiments.

Fig. 5.

Loss of REDD1 leads to a greater absolute suppression of autophagy markers following 3 days of overload. A: The LC3 II-to-LC3 I ratio. B: p62 protein content. C: ratio of phosphorylated ULK1 (Ser-757) to total ULK1. D: total ubiquitin content were determined by Western blot analysis. E: relative p62 mRNA content was determined by quantitative RT-PCR. F: percent change in indicated measures between the sham and overloaded muscle. G: representative Western blots. Lines connecting bars indicate significant difference. OV, overload; +/+, wild-type mice; −/−, REDD1-null mice; P, phosphorylated protein; T, total protein. n = 6/7 per group from two independent experiments.