Identification of MicroRNAs linked to regulators of muscle protein synthesis and regeneration in young and old skeletal muscle

Over the course of ageing there is a natural and progressive loss of skeletal muscle mass. The onset and progression of age-related muscle wasting is associated with an attenuated activation of Akt-mTOR signalling and muscle protein synthesis in response to anabolic stimuli such as resistance exercise. MicroRNAs (miRNAs) are novel and important post-transcriptional regulators of numerous cellular processes. The role of miRNAs in the regulation of muscle protein synthesis following resistance exercise is poorly understood. This study investigated the changes in skeletal muscle miRNA expression following an acute bout of resistance exercise in young and old subjects with a focus on the miRNA species predicted to target Akt-mTOR signalling.

The role of microRNAs in regulating muscle protein synthesis

 The focus of Evelyn’s PhD was to improve the current body of knowledge in the area of age-related muscle wasting. Evelyn identified two new molecules that are potentially important in the maintenance of skeletal muscle mass. These regulators may one day serve as therapeutic targets for age-related muscle wasting.

Erythropoietin does not enhance skeletal muscle protein synthesis following exercise in young and older adults

PURPOSE: Erythropoietin (EPO) is a renal cytokine that is primarily involved in hematopoiesis while also playing a role in non-hematopoietic tissues expressing the EPO-receptor (EPOR). The EPOR is present in human skeletal muscle. In mouse skeletal muscle, EPO stimulation can activate the AKT serine/threonine kinase 1 (AKT) signaling pathway, the main positive regulator of muscle protein synthesis. We hypothesized that a single intravenous EPO injection combined with acute resistance exercise would have a synergistic effect on skeletal muscle protein synthesis via activation of the AKT pathway.

METHODS: Ten young (24.2 ± 0.9 years) and 10 older (66.6 ± 1.1 years) healthy subjects received a primed, constant infusion of [ring-13C(6)] L-phenylalanine and a single injection of 10,000 IU epoetin-beta or placebo in a double-blind randomized, cross-over design. 2 h after the injection, the subjects completed an acute bout of leg extension resistance exercise to stimulate skeletal muscle protein synthesis.

RESULTS: Significant interaction effects in the phosphorylation levels of the members of the AKT signaling pathway indicated a differential activation of protein synthesis signaling in older subjects when compared to young subjects. However, EPO offered no synergistic effect on vastus lateralis mixed muscle protein synthesis rate in young or older subjects.

CONCLUSIONS: Despite its ability to activate the AKT pathway in skeletal muscle, an acute EPO injection had no additive or synergistic effect on the exercise-induced activation of muscle protein synthesis or muscle protein synthesis signaling pathways.

Protecting skeletal muscle with protein and amino acid during periods of disuse

Habitual sedentary behavior increases risk of chronic disease, hospitalization and poor quality of life. Short-term bed rest or disuse accelerates the loss of muscle mass, function, and glucose tolerance. Optimizing nutritional practices and protein intake may reduce the consequences of disuse by preserving metabolic homeostasis and muscle mass and function. Most modes of physical inactivity have the potential to negatively impact the health of older adults more than their younger counterparts. Mechanistically, mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis are negatively affected by disuse. This contributes to reduced muscle quality and is accompanied by impaired glucose regulation. Simply encouraging increased protein and/or energy consumption is a well-intentioned, but often impractical strategy to protect muscle health. Emerging evidence suggests that leucine supplemented meals may partially and temporarily protect skeletal muscle during disuse by preserving anabolism and mitigating reductions in mass, function and metabolic homeostasis.

Growth of Australian shortfin eel (Anguilla Australis) elvers given different dietary protein and lipid levels

The Australian shortfin eel, Anguilla australis is a potential candidate for intensive aquaculture. The present study was undertaken to evaluate the growth of elvers (5.4 g ± 0.1 initial weight) fed with diets of varying protein and lipid content, and to assess the potential of using soya-bean meal as a dietary ingredient. A 10 week experiment was conducted at 24 (±1.0) °C by rearing fish, in 60 L conical fibre glass tanks using a closed recirculation system. Diets having protein concentrations of 40 or 50% (by dry weight) were tested at three lipid levels (15, 20, 25%); diets being designated P₄₀L₁₅, P₄₀L₂₀, P₄₀L₂₅, P₅₀L₁₅, etc. All these diets contained 5% soya-bean meal. In addition P₅₀L₂₀ diets were formulated to contain 10 and 20% soya-bean meal in the diet (Diets S1 & S2). Shortfin eel grew best on the P₅₀L₁₅ diet, with an average specific growth rate of 2.26%. Food conservation ratio (FCR) and Protein efficiency ratio (PER) ranged from 1.21 (P₅₀L₁₅) to 2.12 (P₄₀L₂₅), and 0.92 (P₅₀L₂₅) to 1.65 (P₅₀L₁₅), respectively. Based on all criteria the best growth performance of shortfin eel was on the P₅₀L₁₅ diet, followed by P₄₀L₂₀ and P₄₀L₁₅. At both protein levels fish reared on diets with 25% lipid performed poorly. The performance of shortfin eel was not affected by the amount of soya-bean meal in the diet, up to a maximum of 20% dietary inclusion. No significant differences in muscle protein were evident in shortfin eel reared on different dietary treatments, nor was the lipid content of muscle related to dietary lipid level.

Exercise, diet and skeletal muscle gene expression

Skeletal muscle, as a consequence of its mass and great capacity for altered metabolism, has a major impact on whole-body metabolic homeostasis and is capable of remarkable adaptation in response to various physiological stimuli, including exercise and dietary intervention. Exercise-induced increases in skeletal muscle mRNA levels of a number of genes have been reported, due to transcriptional activation and/or increased mRNA stability. The cellular adaptations to exercise training appear to be due to the cumulative effects of transient increases in gene transcription after repeated exercise bouts. The relative importance of transcriptional (mRNA synthesis) and translational (mRNA stability or translational efficiency) mechanisms for the training-induced increases in skeletal muscle protein abundance remains to be fully elucidated. Dietary manipulation, and the associated alterations in nutrient availability and hormone levels, can also modify skeletal muscle gene expression, although fewer studies have been reported. A major challenge is to understand how exercise and diet exert their effects on gene and protein expression in skeletal muscle. In relation to exercise, potential stimuli include stretch and muscle tension, the pattern of motor nerve activity and the resultant calcium transients, the energy charge of the cell and substrate availability, oxygen tension and circulating hormones. These are detected by various cellular signaling mechanisms, acting on a range of downstream targets and a wide range of putative transcription factors. A key goal in the years ahead is to identify how alterations at the level of gene expression are coupled to the changes in skeletal muscle phenotype. It is clear that gene expression, although representing a specific site of regulation, is only one step in a complex cascade from the initial stimulus to the final phenotypic adaptation and integrated physiological response.

Cellular adaption to repeated eccentric exercise-induced muscle damage

Eccentrically biased exercise results in skeletal muscle damage and stimulates adaptations in muscle, whereby indexes of damage are attenuated when the exercise is repeated. We hypothesized that changes in ultrastructural damage, inflammatory cell infiltration, and markers of proteolysis in skeletal muscle would come about as a result of repeated eccentric exercise and that gender may affect this adaptive response. Untrained male (n = 8) and female (n = 8) subjects performed two bouts (bout 1 and bout 2), separated by 5.5 wk, of 36 repetitions of unilateral, eccentric leg press and 100 repetitions of unilateral, eccentric knee extension exercises (at 120% of their concentric single repetition maximum), the subjects' contralateral nonexercised leg served as a control (rest). Biopsies were taken from the vastus lateralis from each leg 24 h postexercise. After bout 2, the postexercise force deficit and the rise in serum creatine kinase (CK) activity were attenuated. Women had lower serum CK activity compared with men at all times (P < 0.05), but there were no gender differences in the relative magnitude of the force deficit. Muscle Z-disk streaming, quantified by using light microscopy, was elevated vs. rest only after bout 1 (P < 0.05), with no gender difference. Muscle neutrophil counts were significantly greater in women 24 h after bout 2 vs. rest and bout 1 (P < 0.05) but were unchanged in men. Muscle macrophages were elevated in men and women after bout 1 andbout 2 (P < 0.05). Muscle protein content of the regulatory calpain subunit remained unchanged whereas ubiquitin-conjugated protein content was increased after both bouts (P < 0.05), with a greater increase after bout 2. We conclude that adaptations to eccentric exercise are associated with attenuated serum CK activity and, potentially, an increase in the activity of the ubiquitin proteosome proteolytic pathway.

Muscle p70S6K phosphorylation in response to soy and dairy rich meals in middle aged men with metabolic syndrome: a randomised crossover trial

The mammalian target of rapamycin (mTOR) pathway is the primary regulator of muscle protein synthesis. Metabolic syndrome (MetS) is characterized by central obesity and insulin resistance; little is known about how MetS affects the sensitivity of the mTOR pathway to feeding.

Creatine transporter (SLC6A8) knockout mice display an increased capacity for in vitro creatine biosynthesis in skeletal muscle.

The present study aimed to investigate whether skeletal muscle from whole body creatine transporter (CrT; SLC6A8) knockout mice (CrT(-/y)) actually contained creatine (Cr) and if so, whether this Cr could result from an up regulation of muscle Cr biosynthesis. Gastrocnemius muscle from CrT(-/y) and wild type (CrT(+/y)) mice were analyzed for ATP, Cr, Cr phosphate (CrP), and total Cr (TCr) content. Muscle protein and gene expression of the enzymes responsible for Cr biosynthesis L-arginine:glycine amidotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) were also determined as were the rates of in vitro Cr biosynthesis. CrT(-/y) mice muscle contained measurable (22.3 ± 4.3 mmol.kg(-1) dry mass), but markedly reduced (P < 0.05) TCr levels compared with CrT(+/y) mice (125.0 ± 3.3 mmol.kg(-1) dry mass). AGAT gene and protein expression were higher (~3 fold; P < 0.05) in CrT(-/y) mice muscle, however GAMT gene and protein expression remained unchanged. The in vitro rate of Cr biosynthesis was elevated 1.5 fold (P < 0.05) in CrT(-/y) mice muscle. These data clearly demonstrate that in the absence of CrT protein, skeletal muscle has reduced, but not absent, levels of Cr. This presence of Cr may be at least partly due to an up regulation of muscle Cr biosynthesis as evidenced by an increased AGAT protein expression and in vitro Cr biosynthesis rates in CrT(-/y) mice. Of note, the up regulation of Cr biosynthesis in CrT(-/y) mice muscle was unable to fully restore Cr levels to that found in wild type muscle.

G-CSF treatment can attenuate dexamethasone-induced reduction in C2C12 myotube protein synthesis

Granulocyte-colony stimulating factor (G-CSF) has been demonstrated to enhance skeletal muscle recovery following injury and increases muscle function in the context of neuromuscular disease in rodent models. However, understanding of the underlying mechanisms used by G-CSF to mediate these functions remains poor. G-CSF acts on responsive cells through binding to a specific membrane spanning receptor, G-CSFR. Recently identified, the G-CSFR is expressed in myoblasts, myotubes and mature skeletal muscle tissue. Therefore, elucidating the actions of G-CSF in skeletal muscle represents an important prerequisite to consider G-CSF as a therapeutic agent to treat skeletal muscle. Here we show for the first time that treatment with moderate doses (4 and 40ng/ml) of G-CSF attenuates the effects of dexamethasone in reducing protein synthesis in C2C12 myotubes. However, a higher dose (100ng/ml) of G-CSF exacerbates the dexamethasone-induced reduction in protein synthesis. In contrast, G-CSF had no effect on basal or dexamethasone-induced protein degradation, nor did G-CSF influence the phosphorylation of Akt, STAT3, Erk1/2, Src, Lyn and Erk5 in C2C12 myotubes. In conclusion, physiologically relevant doses of G-CSF may attenuate reduced skeletal muscle protein synthesis during catabolic conditions, thereby improving recovery.

Soy protein ingestion results in less prolonged p70S6 kinase phosphorylation compared to whey protein after resistance exercise in older men

BACKGROUND: The phosphorylation of p70S6 Kinase (p70S6K) is an important step in the initiation of protein translation. p70S6K phosphorylation is enhanced with graded intakes of whey protein after resistance exercise. Soy protein ingestion results in lower muscle protein synthesis after exercise compared with whey; however, the underlying mechanisms responsible for this difference have not been reported. FINDINGS: 13 older men (60-75) completed an acute bout of lower body resistance exercise and ingested 30 g of soy protein or carbohydrate. Muscle biopsies were obtained in the rested and fasted state and 2 and 4 hours post exercise. Phosphorylation status of p70S6K was measured with western blot. Results were compared with previously reported data from the ingestion of 30 g of whey protein or placebo. p70S6K phosphorylation was increased 2, but not 4 hours post exercise with soy protein ingestion. p70S6K phosphorylation was not increased post exercise with carbohydrate ingestion. CONCLUSIONS: Ingesting 30 g of either whey or soy protein resulted in equivalent p70S6K phosphorylation at 2 hours post exercise, however, unlike whey, soy protein failed to promote prolonged phosphorylation of p70S6K to 4 hours post-exercise.

Protein requirements and recommendations for older people: a review

Declines in skeletal muscle mass and strength are major contributors to increased mortality, morbidity and reduced quality of life in older people. Recommended Dietary Allowances/Intakes have failed to adequately consider the protein requirements of the elderly with respect to function. The aim of this paper was to review definitions of optimal protein status and the evidence base for optimal dietary protein. Current recommended protein intakes for older people do not account for the compensatory loss of muscle mass that occurs on lower protein intakes. Older people have lower rates of protein synthesis and whole-body proteolysis in response to an anabolic stimulus (food or resistance exercise). Recommendations for the level of adequate dietary intake of protein for older people should be informed by evidence derived from functional outcomes. Randomized controlled trials report a clear benefit of increased dietary protein on lean mass gain and leg strength, particularly when combined with resistance exercise. There is good consistent evidence (level III-2 to IV) that consumption of 1.0 to 1.3 g/kg/day dietary protein combined with twice-weekly progressive resistance exercise reduces age-related muscle mass loss. Older people appear to require 1.0 to 1.3 g/kg/day dietary protein to optimize physical function, particularly whilst undertaking resistance exercise recommendations.

Effects of aging, exercise, and disease on force transfer in skeletal muscle

The loss of muscle strength and increased injury rate in aging skeletal muscle has previously been attributed to loss of muscle protein (cross-sectional area) and/or decreased neural activation. However, it is becoming clear that force transfer within and between fibers plays a significant role in this process as well. Force transfer involves a secondary matrix of proteins that align and transmit the force produced by the thick and thin filaments along muscle fibers and out to the extracellular matrix. These specialized networks of cytoskeletal proteins aid in passing force through the muscle and also serve to protect individual fibers from injury. This review discusses the cytoskeleton proteins that have been identified as playing a role in muscle force transmission, both longitudinally and laterally, and where possible highlights how disease, aging, and exercise influence the expression and function of these proteins.