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.

Non-technical summary Chronic mechanical loading (CML) of skeletal muscle induces growth and this effect can be blocked by the drug rapamycin. Rapamycin is considered to be a highly specific inhibitor of the mammalian target of rapamycin (mTOR), and thus, many have concluded that mTOR plays a key role in CML-induced growth. However, direct evidence that mTOR confers the CML-induced activation of growth promoting events such as hypertrophy, hyperplasia and ribosome biogenesis is lacking. This study addressed that gap in knowledge by using a specialized line of transgenic mice. Surprisingly, the results indicate that only a few of the growth promoting events induced by CML are fully dependent on mTOR signalling (e.g. hypertrophy). These results advance our understanding of the molecular mechanisms that regulate skeletal muscle mass and should help future studies aimed at identifying targets for therapies that can prevent the loss of muscle mass during conditions such as bedrest, immobilization, and ageing.Abstract Chronic mechanical loading (CML) of skeletal muscle induces compensatory growth and the drug rapamycin has been reported to block this effect. Since rapamycin is considered to be a highly specific inhibitor of the mammalian target of rapamycin (mTOR), many have concluded that mTOR plays a key role in CML-induced growth regulatory events. However, rapamycin can exert mTOR-independent actions and systemic administration of rapamycin will inhibit mTOR signalling in all cells throughout the body. Thus, it is not clear if the growth inhibitory effects of rapamycin are actually due to the inhibition of mTOR signalling, and more specifically, the inhibition of mTOR signalling in skeletal muscle cells. To address this issue, transgenic mice with muscle specific expression of various rapamycin-resistant mutants of mTOR were employed. These mice enabled us to demonstrate that mTOR, within skeletal muscle cells, is the rapamycin-sensitive element that confers CML-induced hypertrophy, and mTOR kinase activity is necessary for this event. Surprisingly, CML also induced hyperplasia, but this occurred through a rapamycin-insensitive mechanism. Furthermore, CML was found to induce an increase in FoxO1 expression and PKB phosphorylation through a mechanism that was at least partially regulated by an mTOR kinase-dependent mechanism. Finally, CML stimulated ribosomal RNA accumulation and rapamycin partially inhibited this effect; however, the effect of rapamycin was exerted through a mechanism that was independent of mTOR in skeletal muscle cells. Overall, these results demonstrate that CML activates several growth regulatory events, but only a few (e.g. hypertrophy) are fully dependent on mTOR signalling within the skeletal muscle cells.

Reactive oxygen species (ROS) have been linked with both depressed Na + ,K + -pump activity and skeletal muscle fatigue. This study investigated N -acetylcysteine (NAC) effects on muscle Na + ,K + -pump activity and potassium (K + ) regulation during prolonged, submaximal endurance exercise. Eight well-trained subjects participated in a double-blind, randomised, crossover design, receiving either NAC or saline (CON) intravenous infusion at 125 mg kgfor 15 min, then 25 mg kg −1 h −1 for 20 min prior to and throughout exercise. Subjects cycled for 45 min at 71%V O 2 peak , then continued at 92%V O 2 peak until fatigue. Vastus lateralis muscle biopsies were taken before exercise, at 45 min and fatigue and analysed for maximal in vitro Na + ,K + -pump activity (K + -stimulated 3-O-methyfluorescein phosphatase; 3-O-MFPase). Arterialized venous blood was sampled throughout exercise and analysed for plasma K + and other electrolytes. Time to fatigue at 92%V O 2 peak was reproducible in preliminary trials (C.V. 5.6 ± 0.6%) and was prolonged with NAC by 23.8 ± 8.3% (NAC 6.3 ± 0.5 versus CON 5.2 ± 0.6 min, P < 0.05). Maximal 3-O-MFPase activity decreased from rest by 21.6 ± 2.8% at 45 min and by 23.9 ± 2.3% at fatigue (P < 0.05). NAC attenuated the percentage decline in maximal 3-O-MFPase activity (%Δactivity) at 45 min (P < 0.05) but not at fatigue. When expressed relative to work done, the %Δactivity-to-work ratio was attenuated by NAC at 45 min and fatigue (P < 0.005). The rise in plasma [K + ] during exercise and the Δ[K + ]-to-work ratio at fatigue were attenuated by NAC (P < 0.05). These results confirm that the antioxidant NAC attenuates muscle fatigue, in part via improved K + regulation, and point to a role for ROS in muscle fatigue.

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It is well known that an increase in mechanical loading can induce skeletal muscle hypertrophy, and a long standing model in the field indicates that mechanical loads induce hypertrophy via a mechanism that requires signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Specifically, it has been widely proposed that mechanical loads activate signaling through mTORC1 and that this, in turn, promotes an increase in the rate of protein synthesis and the subsequent hypertrophic response. However, this model is based on a number of important assumptions that have not been rigorously tested. In this study, we created skeletal muscle specific and inducible raptor knockout mice to eliminate signaling by mTORC1, and with these mice we were able to directly demonstrate that mechanical stimuli can activate signaling by mTORC1, and that mTORC1 is necessary for mechanical load‐induced hypertrophy. Surprisingly, however, we also obtained multiple lines of evidence that indicate that mTORC1 is not required for a mechanical load‐induced increase in the rate of protein synthesis. This observation highlights an important shortcoming in our understanding of how mechanical loads induce hypertrophy and illustrates that additional mTORC1‐independent mechanisms play a critical role in this process.—You, J.‐S., McNally, R. M., Jacobs, B. L., Privett, R. E., Gundermann, D. M., Lin, K.‐H., Steinert, N. D., Goodman, C. A., Hornberger, T. A. The role of raptor in the mechanical load‐induced regulation of mTOR signaling, protein synthesis, and skeletal muscle hypertrophy. FASEB J. 33, 4021–4034 (2019). http://www.fasebj.org