In this study, the principles of surface sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [ ³ H]phenylalanine method when detecting synergist ablation‐induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation‐induced increase in PS when used in vivo (IV‐SUnSET). In addition, IV‐SUnSET detected food deprivation‐induced decreases in PS in the heart, kidney, and skeletal muscles, with similar changes being visualized with an immunohistochemical version of IV‐SUnSET (IV‐IHC‐SUnSET). By combining IV‐IHC‐SUnSET with in vivo transfection, we demonstrate that constitutively active PKB induces a robust increase in skeletal muscle PS. Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB‐independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV‐IHC‐SUnSET exposed the existence of fiber type‐dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.—Goodman, C. A., Mabrey, D. M., Frey, J. W., Miu, M. H., Schmidt, E. K., Pierre, P., Hornberger, T. A. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J. 25, 1028–1039 (2011). www.fasebj.org

MyoD , a master regulator of myogenesis, exhibits a circadian rhythm in its mRNA and protein levels, suggesting a possible role in the daily maintenance of muscle phenotype and function. We report that MyoD is a direct target of the circadian transcriptional activators CLOCK and BMAL1, which bind in a rhythmic manner to the core enhancer of the MyoD promoter. Skeletal muscle of Clock ^Δ19 and Bmal1 ^−/− mutant mice exhibited ∼30% reductions in normalized maximal force. A similar reduction in force was observed at the single-fiber level. Electron microscopy (EM) showed that the myofilament architecture was disrupted in skeletal muscle of Clock ^Δ19 , Bmal1 ^−/− , and MyoD ^−/− mice. The alteration in myofilament organization was associated with decreased expression of actin, myosins, titin, and several MyoD target genes. EM analysis also demonstrated that muscle from both Clock ^Δ19 and Bmal1 ^−/− mice had a 40% reduction in mitochondrial volume. The remaining mitochondria in these mutant mice displayed aberrant morphology and increased uncoupling of respiration. This mitochondrial pathology was not seen in muscle of MyoD ^−/− mice. We suggest that altered expression of both Pgc-1α and Pgc-1β in Clock ^Δ19 and Bmal1 ^−/− mice may underlie this pathology. Taken together, our results demonstrate that disruption of CLOCK or BMAL1 leads to structural and functional alterations at the cellular level in skeletal muscle. The identification of MyoD as a clock-controlled gene provides a mechanism by which the circadian clock may generate a muscle-specific circadian transcriptome in an adaptive role for the daily maintenance of adult skeletal muscle.

In humans, progressive resistance exercise is recognized for its ability to induce skeletal muscle hypertrophy. In an attempt to develop an animal model which mimics human progressive resistance exercise, Sprague-Dawley rats were trained to climb a 1.1-m vertical (80° incline) ladder with weights secured to their tail. The rats were trained once every 3 days for 8 weeks. Each training session consisted of 4-9 (6.02 ± 0.23) climbs requiring 8-12 dynamic movements per climb. Based on performance, the weight carried during each session was progressively increased. Over the course of 8 weeks, the maximal amount of weight the rats could carry increased 287%, p ≤ 0.001. The improved training performance was associated with a 23% absolute increase in the weight of the flexor hallucis longus (FHL), with a concomitant 24% increase in both total and myofibrillar protein, p ≤ 0.001. Peak tetanic tension (Po) of the FHL increased 20%, p ≤ 0.001, while specific tetanic tension (SPo) was not altered. No change in twitch tension (Pt) was observed, which resulted in a 22% decrease in specific twitch tension (SPt) p ≤ 0.01. Despite a decrease in resistance to fatigue, p ≤ 0.05, myosin heavy chain composition, ATP, ADP, creatine, and creatine phosphate concentrations of the FHL were not altered. The results of this study describe an animal model that mimics many of the training parameters and physiological adaptations observed with human progressive resistance exercise. Key words: contractile properties, high-energy phosphates, myosin heavy chain, fatigue resistance

Signaling by the mammalian target of rapamycin (mTOR) has been reported to be necessary for mechanical load-induced growth of skeletal muscle. The mechanisms involved in the mechanical activation of mTOR signaling are not known, but several studies indicate that a unique [phosphotidylinositol-3-kinase (PI3K)- and nutrient-independent] mechanism is involved. In this study, we have demonstrated that a regulatory pathway for mTOR signaling that involves phospholipase D (PLD) and the lipid second messenger phosphatidic acid (PA) plays a critical role in the mechanical activation of mTOR signaling. First, an elevation in PA concentration was sufficient for the activation of mTOR signaling. Second, the isozymes of PLD (PLD1 and PLD2) are localized to the z-band in skeletal muscle (a critical site of mechanical force transmission). Third, mechanical stimulation of skeletal muscle with intermittent passive stretch ex vivo induced PLD activation, PA accumulation, and mTOR signaling. Finally, pharmacological inhibition of PLD blocked the mechanically induced increase in PA and the activation of mTOR signaling. Combined, these results indicate that mechanical stimuli activate mTOR signaling through a PLD-dependent increase in PA. Furthermore, we showed that mTOR signaling was partially resistant to rapamycin in muscles subjected to mechanical stimulation. Because rapamycin and PA compete for binding to the FRB domain on mTOR, these results suggest that mechanical stimuli activate mTOR signaling through an enhanced binding of PA to the FRB domain on mTOR.

Protein synthesis rates commonly are measured using isotopic tracers to quantify the incorporation of a labeled amino acid into muscle proteins. Here we provide evidence supporting our hypothesis that the nonisotopic SUnSET technique is a valid and accurate method for the measurement of in vivo changes in protein synthesis at the whole-muscle and single–muscle fiber levels.