Timothy Etheridge

Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling

BACKGROUND We previously showed that human muscle protein synthesis (MPS) increased during infusion of amino acids (AAs) and peaked at ≈120 min before returning to baseline rates, despite elevated plasma AA concentrations. OBJECTIVE We tested whether a protein meal elicited a similar response and whether signaling responses that regulate messenger RNA translation matched MPS changes. DESIGN Eight postabsorptive healthy men (≈21 y of age) were studied during 8.5 h of primed continuous infusion of [1,2-¹³C₂]leucine with intermittent quadriceps biopsies for determination of MPS and anabolic signaling. After 2.5 h, subjects consumed 48 g whey protein. RESULTS At 45-90 min after oral protein bolus, mean (± SEM) myofibrillar protein synthesis increased from 0.03 ± 0.003% to 0.10 ± 0.01%/h; thereafter, myofibrillar protein synthesis returned to baseline rates even though plasma essential AA (EAA) concentrations remained elevated (+130% at 120 min, +80% at 180 min). The activity of protein kinase B (PKB) and phosphorylation of eukaryotic initiation factor 4G preceded the rise of MPS and increases in phosphorylation of ribosomal protein kinase S6 (S6K1), and 4E-binding protein 1 (4EBP1) was superimposable with MPS responses until 90 min. However, although MPS decreased thereafter, all signals, with the exception of PKB activity (which mirrored insulin responses), remained elevated, which echoed the slowly declining plasma EAA profile. The phosphorylation of eukaryotic initiation factor 2α increased only at 180 min. Thus, discordance existed between MPS and the mammalian target of rapamycin complex 1 (mTORC1) and signaling (ie, S6K1 and 4EBP1 phosphorylation). CONCLUSIONS We confirm our previous findings that MPS responses to AAs are transient, even with oral protein bolus. However, changes in MPS only reflect elevated mTORC1 signaling during the upswing in MPS.

Effects of leucine and its metabolite β-hydroxy-β-methylbutyrate on human skeletal muscle protein metabolism

•  The branched‐chain amino acid (BCAA) leucine acts as both a ‘trigger’ for the initiation of protein synthesis, and as a substrate for newly synthesized protein. •  As a BCAA, leucine can be metabolized within skeletal muscle, leaving open the possibility that leucine metabolites might possess anabolic properties. •  One metabolite in particular, β‐hydroxy‐β‐methylbutyrate (HMB), has shown positive effects on lean body mass and strength following exercise, and in disease‐related muscle wasting, yet its impact on acute human muscle protein turnover is undefined. •  We report here that HMB stimulates muscle protein synthesis to a similar extent to leucine. HMB was also found to decrease muscle protein breakdown. •  Our observation that HMB enhances muscle protein anabolism may partly (or wholly) underlie its pre‐defined anabolic/anti‐catabolic supplemental efficacy in humans.

Effects of hypoxia on muscle protein synthesis and anabolic signaling at rest and in response to acute resistance exercise.

Chronic reductions in tissue O(2) tension (hypoxia) are associated with muscle atrophy and blunted hypertrophic responses to resistance exercise (RE) training. However, the effect of hypoxia on muscle protein synthesis (MPS) at rest and after RE is unknown. In a crossover study, seven healthy men (21.4 ± 0.7 yr) performed unilateral leg RE (6 × 8 repetitions at 70% 1-repetition maximum) under normoxic (20.9% inspired O(2)) and normobaric hypoxic (12% inspired O(2) for 3.5 h) postabsorptive conditions. Immediately after RE the rested leg was biopsied, and a primed continuous infusion of [1,2-(13)C(2)]leucine was maintained for 2.5 h before final biopsies from both legs to measure tracer incorporation and signaling responses (i.e., ribosomal S6 kinase 1). After 3.5 h of hypoxia, MPS was not different from normoxia in the rested leg (normoxia 0.033 ± 0.016 vs. hypoxia 0.043 ± 0.016%/h). MPS increased significantly from baseline 2.5 h after RE in normoxia (0.033 ± 0.016 vs. 0.104 ± 0.038%/h) but not hypoxia (0.043 ± 0.016 vs. 0.060 ± 0.063%/h). A significant linear relationship existed between MPS 2.5 h after RE in hypoxia and mean arterial blood O(2) saturation during hypoxia (r(2) = 0.49, P = 0.04). Phosphorylation of p70S6K(Thr389) remained unchanged in hypoxia at rest but increased after RE in both normoxia and hypoxia (2.6 ± 1.2-fold and 3.4 ± 1.1-fold, respectively). Concentrations of the hypoxia-responsive mTOR inhibitor regulated in development and DNA damage-1 were unaltered by hypoxia or RE. We conclude that normobaric hypoxia does not reduce MPS over 3.5 h at rest but blunts the increased MPS response to acute RE to a degree dependent on extant SpO(2).

A single protein meal increases recovery of muscle function following an acute eccentric exercise bout.

The purpose of this study was to examine the effects of acute protein ingestion on the recovery of muscle function and markers of muscle damage in the 72 h post eccentric-exercise. Nine recreationally active males recorded quadriceps maximum isometric voluntary contraction (MVC), peak 5 s power output (PPO), and perceived muscle soreness. Plasma creatine kinase (CK) and protein carbonyl (PC) content were measured prior to exercise. Delayed-onset muscle soreness (DOMS) was induced by a 30 min downhill run (-10 degrees ) at a target intensity of 75% age-predicted heart rate maximum, immediately followed by ingestion of 100 g protein (containing 40 g essential amino acids; PRO) or placebo (CON) solution. The pre-exercise measures were re-taken in the subsequent 24, 48, and 72 h. CK, PC, and perceived muscle soreness increased significantly following exercise and with each supplement at 24 h. PC and muscle soreness remained elevated at 48 and 72 h (p < 0.05), whereas CK returned to baseline values. No difference between conditions was observed for these measures. Peak MVC significantly declined in CON to -7.9% at 24 h, reaching a nadir of -10% at 48 h (p < 0.05). In the PRO group, MVC remained within pre-exercise values at all time points. PPO followed a similar trend, reaching its nadir of -8.7% at 48 h in CON (p < 0.05), but had recovered in the PRO trial. Ingestion of a single post-exercise protein mixture increases the rate of force and power restoration at 48 h, suggesting potential for protein as an ergogenic aid during the DOMS period.

Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans.

The Effectiveness of RNAi in Caenorhabditis elegans Is Maintained during Spaceflight

Background Overcoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown. Methods Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at −80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions. Results After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions. Conclusions Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.

Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans

Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.

The integrin-adhesome is required to maintain muscle structure, mitochondrial ATP production, and movement forces in Caenorhabditis elegans

The integrin‐adhesome network, which contains >150 proteins, is mechano‐transducing and located at discreet positions along the cell‐cell and cell‐extracellular matrix interface. A small subset of the integrin‐adhesome is known to maintain normal muscle morphology. However, the importance of the entire adhesome for muscle structure and function is unknown. We used RNA interference to knock down 113 putative Caenorhabditis elegans homologs constituting most of the mammalian adhesome and 48 proteins known to localize to attachment sites in C. elegans muscle. In both cases, we found >90% of components were required for normal muscle mitochondrial structure and/or proteostasis vs. empty vector controls. Approximately half of these, mainly proteins that physically interact with each other, were also required for normal sarcomere and/or adhesome structure. Next we confirmed that the dystrophy observed in adhesome mutants associates with impaired maximal mitochondrial ATP production (P < 0.01), as well as reduced probability distribution of muscle movement forces compared with wild‐type animals. Our results show that the integrin‐adhesome network as a whole is required for maintaining both muscle structure and function and extend the current understanding of the full complexities of the functional adhesome in vivo.—Etheridge, T., Rahman, M., Gaffney, C. J., Shaw, D., Shephard, F., Magudia, J., Solomon, D. E., Milne, T., Blawzdziewicz, J., Constantin‐Teodosiu, D., Greenhaff, P. L., Vanapalli, S. A., Szewczyk, N. J. The integrin‐adhesome is required to maintain muscle structure, mitochondrial ATP production, and movement forces in Caenorhabditis elegans. FASEB J. 29, 1235‐1246 (2015). www.fasebj.org

Remote automated multi-generational growth and observation of an animal in low Earth orbit

The ultimate survival of humanity is dependent upon colonization of other planetary bodies. Key challenges to such habitation are (patho)physiologic changes induced by known, and unknown, factors associated with long-duration and distance space exploration. However, we currently lack biological models for detecting and studying these changes. Here, we use a remote automated culture system to successfully grow an animal in low Earth orbit for six months. Our observations, over 12 generations, demonstrate that the multi-cellular soil worm Caenorhabditis elegans develops from egg to adulthood and produces progeny with identical timings in space as on the Earth. Additionally, these animals display normal rates of movement when fully fed, comparable declines in movement when starved, and appropriate growth arrest upon starvation and recovery upon re-feeding. These observations establish C. elegans as a biological model that can be used to detect changes in animal growth, development, reproduction and behaviour in response to environmental conditions during long-duration spaceflight. This experimental system is ready to be incorporated on future, unmanned interplanetary missions and could be used to study cost-effectively the effects of such missions on these biological processes and the efficacy of new life support systems and radiation shielding technologies.

The next phase of life-sciences spaceflight research: Harnessing the power of functional genomics.

Recently we demonstrated that the effectiveness of RNAi interference (RNAi) for inhibiting gene expression is maintained during spaceflight in the worm Caenorhabditis elegans and argued for the biomedical importance of this finding. We also successfully utilized green fluorescent protein (GFP)-tagged proteins to monitor changes in GPF localization during flight. Here we discuss potential applications of RNAi and GFP in spaceflight studies and the ramifications of these experiments for the future of space life-sciences research.