Craig A. Goodman

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.

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 kg(-1) h(-1) for 15 min, then 25 mg kg(-1) h(-1) for 20 min prior to and throughout exercise. Subjects cycled for 45 min at 71% , then continued at 92% 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% 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 (%Deltaactivity) at 45 min (P < 0.05) but not at fatigue. When expressed relative to work done, the %Deltaactivity-to-work ratio was attenuated by NAC at 45 min and fatigue (P < 0.005). The rise in plasma [K(+)] during exercise and the Delta[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.

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.