Skeletal Muscle Protein Turnover Research Articles

Post-natal muscle growth and protein turnover: a narrative review of current understanding.

A model explaining the dietary-protein-driven post-natal skeletal muscle growth and protein turnover in the rat is updated, and the mechanisms involved are described, in this narrative review. Dietary protein controls both bone length and muscle growth, which are interrelated through mechanotransduction mechanisms with muscle growth induced both from stretching subsequent to bone length growth and from internal work against gravity. This induces satellite cell activation, myogenesis and remodelling of the extracellular matrix, establishing a growth capacity for myofibre length and cross-sectional area. Protein deposition within this capacity is enabled by adequate dietary protein and other key nutrients. After briefly reviewing the experimental animal origins of the growth model, key concepts and processes important for growth are reviewed. These include the growth in number and size of the myonuclear domain, satellite cell activity during post-natal development and the autocrine/paracrine action of IGF-1. Regulatory and signalling pathways reviewed include developmental mechanotransduction, signalling through the insulin/IGF-1-PI3K-Akt and the Ras-MAPK pathways in the myofibre and during mechanotransduction of satellite cells. Likely pathways activated by maximal-intensity muscle contractions are highlighted and the regulation of the capacity for protein synthesis in terms of ribosome assembly and the translational regulation of 5-TOPmRNA classes by mTORC1 and LARP1 are discussed. Evidence for and potential mechanisms by which volume limitation of muscle growth can occur which would limit protein deposition within the myofibre are reviewed. An understanding of how muscle growth is achieved allows better nutritional management of its growth in health and disease.

Skeletal muscle protein turnover responses to parenteral nutrition in patients with alcoholic liver cirrhosis and sarcopenia.

Alcoholic liver cirrhosis (ALC) is accompanied by sarcopenia. The aim of this study was to investigate the acute effects of balanced parenteral nutrition (PN) on skeletal muscle protein turnover in ALC. Eight male patients with ALC and seven age- and sex-matched healthy controls were studied for 3 h of fasting followed by 3 h of intravenous PN (SmofKabiven 1,206 mL: amino acid = 38 g, carbohydrates = 85 g, and fat = 34 g) 4 mL/kg/h. We measured leg blood flow and sampled paired femoral arteriovenous concentrations and quadriceps muscle biopsies while providing a primed continuous infusion of [ring-2d5]-phenylalanine to quantify muscle protein synthesis and breakdown. Patients with ALC exhibited shorter 6-min walking distance (ALC: 487 ± 38 vs. controls: 722 ± 14 m, P < 0.05), lower hand-grip strength (ALC: 34 ± 2 vs. controls: 52 ± 2 kg, P < 0.05), and computed tomography (CT)-verified leg muscle loss (ALC: 5,922 ± 246 vs. controls: 8,110 ± 345 mm2, P < 0.05). Net leg muscle phenylalanine uptake changed from negative (muscle loss) during fasting to positive (muscle gain) in response to PN (ALC: -0.18 ± +0.01 vs. 0.24 ± 0.03 µmol/kg muscle·min-1; P < 0.001 and controls: -0.15 ± 0.01 vs. 0.09 ± 0.01 µmol/kg muscle·min-1; P < 0.001) but with higher net muscle phenylalanine uptake in ALC than controls (P < 0.001). Insulin concentrations were substantially higher in patients with ALC during PN. Our results suggest a higher net muscle phenylalanine uptake during a single infusion of PN in stable patients with ALC with sarcopenia compared with healthy controls.NEW & NOTEWORTHY Muscle protein turnover responses to parenteral nutritional (PN) supplementation have not previously been studied in stable alcoholic liver cirrhosis (ALC). We applied stable isotope tracers of amino acids to directly quantify net muscle protein turnover responses to PN in sarcopenic males with ALC and healthy controls. We found a higher net muscle protein gain in ALC during PN, thereby providing the physiological rationale for future clinical trials of PN as a potential countermeasure to sarcopenia.

Alterations in skeletal muscle abundance of protein turnover, stress, and antioxidant proteins during the periparturient period in dairy cows fed ethyl-cellulose rumen-protected methionine

Skeletal muscle turnover helps support the physiological needs of dairy cows during the transition into lactation. We evaluated effects of feeding ethyl-cellulose rumen-protected methionine (RPM) during the periparturient period on abundance of proteins associated with transport AA and glucose, protein turnover, metabolism, and antioxidant pathways in skeletal muscle. Sixty multiparous Holstein cows were used in a block design and assigned to a control or RPM diet from -28 to 60 d in milk. The RPM was fed at a rate of 0.09% or 0.10% of dry matter intake (DMI) during the prepartal and postpartal periods to achieve a target Lys:Met ratio in the metabolizable protein of ∼2.8:1. Muscle biopsies from the hind leg of 10 clinically healthy cows per diet collected at -21, 1, and 21 d relative to calving were used for western blotting of 38 target proteins. Statistical analysis was performed using the PROC MIXED statement of SAS version 9.4 (SAS Institute Inc.) with cow as random effect, whereas diet, time, and diet × time were the fixed effects. Diet × time tended to affect prepartum DMI, with RPM cows consuming 15.2 kg/d and controls 14.6 kg/d. However, diet had no effect on postpartum DMI (17.2 and 17.1 ± 0.4 kg/d for control and RPM, respectively). Milk yield during the first 30 d in milk was also not affected by diet (38.1 and 37.5 ± 1.9 kg/d for control and RPM, respectively). Diet or time did not affect the abundance of several AA transporters or the insulin-induced glucose transporter (SLC2A4). Among evaluated proteins, feeding RPM led to lower overall abundance of proteins associated with protein synthesis (phosphorylated EEF2, phosphorylated RPS6KB1), mTOR activation (RRAGA), proteasome degradation (UBA1), cellular stress responses (HSP70, phosphorylated MAPK3, phosphorylated EIF2A, ERK1/2), antioxidant response (GPX3), and de novo synthesis of phospholipids (PEMT). Regardless of diet, there was an increase in the abundance of the active form of the master regulator of protein synthesis phosphorylated MTOR and the growth-factor-induced serine/threonine kinase phosphorylated AKT1 and PIK3C3, whereas the abundance of a negative regulator of translation (phosphorylated EEF2K) decreased over time. Compared with d 1 after calving and regardless of diet, the abundance of proteins associated with endoplasmic reticulum stress (XBP1 spliced), cell growth and survival (phosphorylated MAPK3), inflammation (transcription factor p65), antioxidant responses (KEAP1), and circadian regulation (CLOCK, PER2) of oxidative metabolism was upregulated at d 21 relative to parturition. These responses coupled with the upregulation of transporters for Lys, Arg, and His (SLC7A1) and glutamate/aspartate (SLC1A3) over time were suggestive of dynamic adaptations in cellular functions. Overall, management approaches that could take advantage of this physiological plasticity may help cows make a smoother transition into lactation.

Mutant and curli-producing E. coli enhance the disease phenotype in a hSOD1-G93A mouse model of ALS

The gut microbiome is a potential non-genetic contributing factor for Amyotrophic Lateral Sclerosis. Differences in gut microbial communities have been detected between ALS subjects and healthy controls, including an increase in Escherichia coli in ALS subjects. E. coli and other gram-negative bacteria produce curli proteins, which are functional bacterial amyloids. We examined whether long-term curli overexposure in the gut can exacerbate the development and progression of ALS. We utilized the slow-developing hSOD1-G93A mouse model of ALS with their C57BL/6J WT littermate controls, including males and females, with a total of 91 animals. These mice were on a normal chow diet and fed curli-producing or curli-nonproducing (mutant) E. coli in applesauce (vehicle) 3 times/week, from 1 through 7 months of age. Male hSOD1 mice demonstrated gradual slowing in running speed month 4 onwards, while females exhibited no signs of locomotive impairment even at 7 months of age. Around the same time, male hSOD1 mice showed a gradual increase in frequency of peripheral CD19+ B cells. Among the male hSOD1 group, chronic gut exposure to curli-producing E. coli led to significant shifts in α- and β-diversities. Curli-exposed males showed suppression of immune responses in circulation, but an increase in markers of inflammation, autophagy and protein turnover in skeletal muscle. Some of these markers were also changed in mutant E. coli-exposed mice, including astrogliosis in the brainstem and demyelination in the lumbar spinal cord. Overall, chronic overexposure to a commensal bacteria like E. coli led to distant organ pathology in our model, without the presence of a leaky gut at 6 months. Mechanisms underlying gut-distant organ communication are of tremendous interest to all disciplines.

Comparison of the Effect of a Protein-Free and Restricted High Protein—Low Carbohydrate Diet on Ventricular Myosin ATPase Activity and Isomyosin Profile in Young Rats: Evidence That Protein-Depleted Animals Are Euthyroid

Young rats fed a protein-deficient diet have elevated total triiodothyronine (T3) levels in spite of decreased skeletal muscle protein turnover. Interpretation of the thyroid status of these animals is complicated by increased T3 binding by serum proteins. Free T3 levels ranging from normal to low and decreased resting oxygen consumption have been reported. To investigate the thyroid status of animals fed a protein-free diet, ventricular myosin ATPase activity and isomyosin profile have been used as an index of thyroid hormone activity. The effect of the protein-free diet has been compared to a restricted high protein-low carbohydrate diet, which causes clear evidence of decreased thyroid hormone effect. After 28 d, calcium-activated myosin ATPase activity was 1.50 ± 0.05 µmol P1/(mg protein · min) in animals fed the basal diet, 1.16 ± 0.03 µmol P1/ (mg protein · min) in animals fed the restricted high protein-low carbohydrate diet and 1.48 ± 0.04 µmol P1/(mg protein · min) in animals fed the protein-free diet. In addition, a shift in isomyosin content with the appearance of V2 and V3 isomyosins occurred with the restricted high protein-low carbohydrate diet but not the protein-free diet. The failure of the protein-depleted rats to decrease myosin ATPase activity and alter isomyosin content suggests that they are euthyroid.

Glycine Regulates Protein Turnover by Activating Protein Kinase B/Mammalian Target of Rapamycin and by Inhibiting MuRF1 and Atrogin-1 Gene Expression in C2C12 Myoblasts

Background: The regulation of protein turnover in skeletal muscle is essential for the maintenance of integrity, growth, and function of this tissue. We recently reported that glycine enhances skeletal muscle growth in young pigs. However, the underlying mechanisms remain unknown.Objective: This study was conducted with a mouse myoblast cell line, C2C12, to test the hypothesis that glycine activates protein kinase B/mammalian target of rapamycin (Akt/mTOR), as well as inhibits 5′-adenosine monophosphate-activated protein kinase (AMPK) and the expression of genes for proteolysis.Methods: C2C12 myoblasts were cultured with 0, 0.25 (physiologic concentration in mouse plasma), 0.5, or 1.0 mmol glycine/L. Cell proliferation, activation of mammalian target of rapamycin complex 1 (mTORC1), AMPK signaling, mRNA levels of atrogin-1 and muscle-specific ring finger protein 1 (MuRF1), and protein synthesis and degradation were measured in the absence or presence of an Akt inhibitor, LY294002, or an AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR).Results: Compared with control cells, 0.25–1.0 mmol glycine/L enhanced cell growth (by 12–15%) after 24 h (P < 0.05). Glycine treatment led to increased DNA replication (by 70–80%) while enhancing mTORC1 activation by upregulating Akt and inhibiting AMPK signaling (P < 0.05). Accordingly, glycine exposure increased (P < 0.05) the rate of protein synthesis (by 20–80%) and inhibited (P < 0.05) the rate of protein degradation (by 15–30%) in a concentration-dependent manner in C2C12 cells. These observations were validated by the use of an Akt inhibitor, LY294002, or an AMPK activator, AICAR. Moreover, glycine addition resulted in decreased mRNA levels for atrogin-1 and MuRF1 (by 20–40% and 30–50%, respectively; P < 0.05). The repressing effect of glycine on the expression of MuRF1, instead of atrogin-1, was abolished by LY294002 (P < 0.05).Conclusions: These findings indicate that glycine plays a previously unrecognized role in enhancing protein synthesis and inhibiting protein degradation in C2C12 cells. Glycine regulates protein turnover by activating mTORC1 and by inhibiting the expression of genes for proteolysis. Our results indicate that glycine is a functional amino acid that improves muscle cell growth.

Dietary protein and muscle wasting in chronic kidney disease: new insights.

Muscle wasting is an important health problem in chronic kidney disease (CKD) patients. Protein restriction in the diet can be one of the main causes of muscle wasting in this population. In this review, we aimed to investigate the relationship between dietary protein intake and muscle wasting in CKD patients according to recent literature. The one of the main mechanisms responsible for the muscle wasting is the disturbances in skeletal muscle protein turnover. Muscle wasting primarily occurs when the rates of muscle protein breakdown exceed the muscle protein synthesis. Dietary protein intake represents an important role by causing a potent anabolic stimulus resulting a positive muscle protein balance. Compared to studies made in healthy populations, there are very limited studies in the literature about the relationship between dietary protein intake and muscle wasting in the CKD population. Majority of the studies showed that a more liberal protein intake is beneficial for muscle wasting in especially advanced CKD and hemodialysis population. Although evaluating muscle wasting in CKD patients, the amount of protein in the diet of patients should also be reviewed. Although excessive protein intake has some negative consequences on this patient group, a more liberated dietary protein intake should be taken into account in this patient group with muscle wasting and especially in dialysis patients.

Investigating effects of sodium beta-hydroxybutyrate on metabolism in placebo-controlled, bilaterally infused human leg with focus on skeletal muscle protein dynamics.

Systemic administration of beta‐hydroxybutyrate (BHB) decreases whole‐body protein oxidation and muscle protein breakdown in humans. We aimed to determine any direct effect of BHB on skeletal muscle protein turnover when administered locally in the femoral artery. Paired design with each subject being investigated on one single occasion with one leg being infused with BHB and the opposing leg acting as a control. We studied 10 healthy male volunteers once with bilateral femoral vein and artery catheters. One artery was perfused with saline (Placebo) and one with sodium‐BHB. Labelled phenylalanine and palmitate were used to assess local leg fluxes. Femoral vein concentrations of BHB were significantly higher in the intervention leg (3.4 (3.2, 3.6) mM) compared with the placebo‐controlled leg (1.9 (1.8, 2.1) mM) with a peak difference of 1.4 (1.1, 1.7) mM, p < 0.0005. Net loss of phenylalanine for BHB vs Placebo −6.7(−10.8, −2.7) nmol/min vs −8.7(−13.8, −3.7) nmol/min, p = 0.52. Palmitate flux and arterio‐venous difference of glucose did not differ between legs. Under these experimental conditions, we failed to observe the direct effects of BHB on skeletal muscle protein turnover. This may relate to a combination of high concentrations of BHB (close to 2 mM) imposed systemically by spillover leading to high BHB concentrations in the saline‐infused leg and a lack of major differences in concentration gradients between the two sides—implying that observations were made on the upper part of the dose–response curve for BHB and the relatively small number of subjects studied.

O104 The effects of elective abdominal surgery on protein turnover: a meta-analysis of stable isotope techniques to investigate postoperative catabolism

Abstract Introduction Elective surgery induces skeletal muscle wasting driven by an imbalance between muscle protein synthesis and muscle protein breakdown. From examination of diverse stable isotope tracer techniques, the dynamic processes driving this imbalance are unclear. This meta-analysis aimed to investigate the mechanistic driver(s) of postoperative catabolism through stable isotope assessment of protein turnover before and after surgery. Methods Meta-analysis was performed of randomized controlled trials and cohort studies in patients undergoing elective abdominal surgery that contained measurements of whole-body or skeletal muscle protein turnover via stable isotope tracer methodology pre- and postoperatively. Postoperative changes in protein synthesis and breakdown were assessed through subgroup analysis of tracer methodology. Results Surgery elicited no overall change in protein synthesis [SMD -0.47, 95%CI: -1.32, 0.39]. However, subgroup analysis revealed significant suppressions via direct-incorporation methodology [SMD -1.53, 95%CI: -2.89, -0.17] within skeletal muscle distal to the site of trauma. Changes of this nature were not present among arterial-venous [SMD 0.61, 95%CI: -1.48, 2.70] or end product [SMD -0.09, 95%CI: -0.81, 0.64] whole-body measures. Surgery resulted in no overall change in protein breakdown [SMD 0.63, 95%CI: -0.06, 1.32]. Yet, separation by tracer methodology illustrated significant increases in urinary end-product measurements (urea/ammonia) [SMD 0.70, 95%CI: 0.38, 1.02] that were not present among arterial-venous measures [SMD 0.67, 95%CI: -1.05, 2.38]. Conclusion Elective surgery elicits suppressions in skeletal muscle protein synthesis that are not reflected on a whole-body level. Lack of uniform changes across whole-body tracer techniques are likely due to contribution from tissues other than skeletal muscle. Take-home message Further research investigating postoperative catabolism should employ tracer methodologies specific to the metabolic pool of interest.

(Dys)regulation of Protein Metabolism in Skeletal Muscle of Humans With Obesity

Studies investigating the proteome of skeletal muscle present clear evidence that protein metabolism is altered in muscle of humans with obesity. Moreover, muscle quality (i.e., strength per unit of muscle mass) appears lower in humans with obesity. However, relevant evidence to date describing the protein turnover, a process that determines content and quality of protein, in muscle of humans with obesity is quite inconsistent. This is due, at least in part, to heterogeneity in protein turnover in skeletal muscle of humans with obesity. Although not always evident at the mixed-muscle protein level, the rate of synthesis is generally lower in myofibrillar and mitochondrial proteins in muscle of humans with obesity. Moreover, alterations in the synthesis of protein in muscle of humans with obesity are manifested more readily under conditions that stimulate protein synthesis in muscle, including the fed state, increased plasma amino acid availability to muscle, and exercise. Current evidence supports various biological mechanisms explaining impairments in protein synthesis in muscle of humans with obesity, but this evidence is rather limited and needs to be reproduced under more defined experimental conditions. Expanding our current knowledge with direct measurements of protein breakdown in muscle, and more importantly of protein turnover on a protein by protein basis, will enhance our understanding of how obesity modifies the proteome (content and quality) in muscle of humans with obesity.

The effects of elective abdominal surgery on protein turnover: A meta-analysis of stable isotope techniques to investigate postoperative catabolism

SummaryBackground & aimsElective surgery induces skeletal muscle wasting driven by an imbalance between muscle protein synthesis and breakdown. From examination of diverse stable isotope tracer techniques, the dynamic processes driving this imbalance are unclear. This meta-analysis aimed to elucidate the mechanistic driver(s) of postoperative protein catabolism through stable isotope assessment of protein turnover before and after abdominal surgery.MethodsMeta-analysis was performed of randomized controlled trials and cohort studies in patients undergoing elective abdominal surgery that contained measurements of whole-body or skeletal muscle protein turnover using stable isotope tracer methodologies pre- and postoperatively. Postoperative changes in protein synthesis and breakdown were assessed through subgroup analysis of tracer methodology and perioperative care.ResultsSurgery elicited no overall change in protein synthesis [standardized mean difference (SMD) −0.47, 95% confidence interval (CI): −1.32, 0.39, p = 0.25]. However, subgroup analysis revealed significant suppressions via direct-incorporation methodology [SMD -1.53, 95%CI: −2.89, −0.17, p = 0.03] within skeletal muscle. Changes of this nature were not present among arterio-venous [SMD 0.61, 95%CI: −1.48, 2.70, p = 0.58] or end-product [SMD -0.09, 95%CI: −0.81, 0.64, p = 0.82] whole-body measures. Surgery resulted in no overall change in protein breakdown [SMD 0.63, 95%CI: −0.06, 1.32, p = 0.07]. Yet, separation by tracer methodology illustrated significant increases in urinary end-products (urea/ammonia) [SMD 0.70, 95%CI: 0.38, 1.02, p < 0.001] that were not present among arterio-venous measures [SMD 0.67, 95%CI: −1.05, 2.38, p = 0.45].ConclusionsElective abdominal surgery elicits suppressions in skeletal muscle protein synthesis that are not reflected on a whole-body level. Lack of uniform changes across whole-body tracer techniques are likely due to contribution from tissues other than skeletal muscle.

Multi-targeted therapy of cancer by omega-3 fatty acids-an update

Low in dietary ω3 polyunsaturated fatty acid (PUFA) consumption has been associated with increased incidence of cancers. Many basic and clinical studies have been conducted over the last several decades. We previously reviewed multi-targeted therapy of cancer by omega-3 fatty acids in 2008, and since hundreds of new clinical trials are being conducted to validate the effectiveness of ω3 PUFA in cancer therapy. Because of the availability of such large amount of clinical trial data, in this update we summarize clinical data, sort out trials that show promising results, and discuss potential mechanism(s) responsible for the clinical outcomes. It appears that ω3 PUFA mainly affects cancer-associated symptoms, namely cachexia, inflammation, neuropathy, post operative complications and quality of life. Mechanisms responsible for these effects are possible regulation of skeletal muscle protein turnover, inflammatory response and neuron cell survive by ω3 PUFA.

Muscle wasting in cancer: opportunities and challenges for exercise in clinical cancer trials

BackgroundLow muscle in cancer is associated with an increase in treatment-related toxicities and is a predictor of cancer-related and all-cause mortality. The mechanisms of cancer-related muscle loss are multifactorial, including anorexia, hypogonadism, anaemia, inflammation, malnutrition, and aberrations in skeletal muscle protein turnover and metabolism.MethodsIn this narrative review, we summarise relevant literature to (i) review the factors influencing skeletal muscle mass regulation, (ii) provide an overview of how cancer/treatments negatively impact these, (iii) review factors beyond muscle signalling that can impact the ability to participate in and respond to an exercise intervention to counteract muscle loss in cancer, and (iv) provide perspectives on critical areas of future research.ResultsDespite the well-known benefits of exercise, there remains a paucity of clinical evidence supporting the impact of exercise in cancer-related muscle loss. There are numerous challenges to reversing muscle loss with exercise in clinical cancer settings, ranging from the impact of cancer/treatments on the molecular regulation of muscle mass, to clinical challenges in responsiveness to an exercise intervention. For example, tumour-related/treatment-related factors (e.g. nausea, pain, anaemia, and neutropenia), presence of comorbidities (e.g. diabetes, arthritis, and chronic obstructive pulmonary disease), injuries, disease progression and bone metastases, concomitant medications (e.g., metformin), can negatively affect an individual’s ability to exercise safely and limit subsequent adaptation.ConclusionsThis review identifies numerous gaps and oppportunities in the area of low muscle and muscle loss in cancer. Collaborative efforts between preclinical and clinical researchers are imperative to both understanding the mechanisms of atrophy, and develop appropriate therapeutic interventions.

Combined in vivo muscle mass, muscle protein synthesis and muscle protein breakdown measurement: a \u2018Combined Oral Stable Isotope Assessment of Muscle (COSIAM)\u2019 approach

Optimising approaches for measuring skeletal muscle mass and turnover that are widely applicable, minimally invasive and cost effective is crucial in furthering research into sarcopenia and cachexia. Traditional approaches for measurement of muscle protein turnover require infusion of expensive, sterile, isotopically labelled tracers which limits the applicability of these approaches in certain populations (e.g. clinical, frail elderly). To concurrently quantify skeletal muscle mass and muscle protein turnover i.e. muscle protein synthesis (MPS) and muscle protein breakdown (MPB), in elderly human volunteers using stable-isotope labelled tracers i.e. Methyl-[D3]-creatine (D3-Cr), deuterium oxide (D2O), and Methyl-[D3]-3-methylhistidine (D3-3MH), to measure muscle mass, MPS and MPB, respectively. We recruited 10 older males (71 ± 4 y, BMI: 25 ± 4 kg.m2, mean ± SD) into a 4-day study, with DXA and consumption of D2O and D3-Cr tracers on day 1. D3-3MH was consumed on day 3, 24 h prior to returning to the lab. From urine, saliva and blood samples, and a single muscle biopsy (vastus lateralis), we determined muscle mass, MPS and MPB. D3-Cr derived muscle mass was positively correlated to appendicular fat-free mass (AFFM) estimated by DXA (r = 0.69, P = 0.027). Rates of cumulative myofibrillar MPS over 3 days were 0.072%/h (95% CI, 0.064 to 0.081%/h). Whole-body MPB over 6 h was 0.052 (95% CI, 0.038 to 0.067). These rates were similar to previous literature. We demonstrate the potential for D3-Cr to be used alongside D2O and D3-3MH for concurrent measurement of muscle mass, MPS, and MPB using a minimally invasive design, applicable for clinical and frail populations.

Recent advances in measuring and understanding the regulation of exercise-mediated protein degradation in skeletal muscle.

Skeletal muscle protein turnover plays a crucial role in controlling muscle mass and protein quality control, including sarcomeric (structural and contractile) proteins. Protein turnover is a dynamic and continual process of protein synthesis and degradation. The ubiquitin proteasome system (UPS) is a key degradative system for protein degradation and protein quality control in skeletal muscle. UPS-mediated protein quality control is known to be impaired in aging and diseases. Exercise is a well-recognized, nonpharmacological approach to promote muscle protein turnover rates. Over the past decades, we have acquired substantial knowledge of molecular mechanisms of muscle protein synthesis after exercise. However, there have been considerable gaps in the mechanisms of how muscle protein degradation is regulated at the molecular level. The main challenge to understand muscle protein degradation is due in part to the lack of solid stable isotope tracer methodology to measure muscle protein degradation rate. Understanding the mechanisms of UPS with the concomitant measurement of protein degradation rate in skeletal muscle will help identify novel therapeutic strategies to ameliorate impaired protein turnover and protein quality control in aging and diseases. Thus, the goal of this present review was to highlight how recent advances in the field may help improve our understanding of exercise-mediated protein degradation. We discuss 1) the emerging roles of protein phosphorylation and ubiquitylation modifications in regulating proteasome-mediated protein degradation after exercise and 2) methodological advances to measure in vivo myofibrillar protein degradation rate using stable isotope tracer methods.

The Role of GSK-3β in the Regulation of Protein Turnover, Myosin Phenotype, and Oxidative Capacity in Skeletal Muscle under Disuse Conditions.

Skeletal muscles, being one of the most abundant tissues in the body, are involved in many vital processes, such as locomotion, posture maintenance, respiration, glucose homeostasis, etc. Hence, the maintenance of skeletal muscle mass is crucial for overall health, prevention of various diseases, and contributes to an individual’s quality of life. Prolonged muscle inactivity/disuse (due to limb immobilization, mechanical ventilation, bedrest, spaceflight) represents one of the typical causes, leading to the loss of muscle mass and function. This disuse-induced muscle loss primarily results from repressed protein synthesis and increased proteolysis. Further, prolonged disuse results in slow-to-fast fiber-type transition, mitochondrial dysfunction and reduced oxidative capacity. Glycogen synthase kinase 3β (GSK-3β) is a key enzyme standing at the crossroads of various signaling pathways regulating a wide range of cellular processes. This review discusses various important roles of GSK-3β in the regulation of protein turnover, myosin phenotype, and oxidative capacity in skeletal muscles under disuse/unloading conditions and subsequent recovery. According to its vital functions, GSK-3β may represent a perspective therapeutic target in the treatment of muscle wasting induced by chronic disuse, aging, and a number of diseases.

Measuring Protein Turnover in the Field: Implications for Military Research

Protein turnover reflects the continual synthesis and breakdown of body proteins, and can be measured at a whole-body (i.e. aggregated across all body proteins) or tissue (e.g. skeletal muscle only) level using stable isotope methods. Evaluating protein turnover in free-living environments, such as military training, can help inform protein requirements. We undertook a narrative review of published literature with the aim of reviewing the suitability of, and advancements in, stable isotope methods for measuring protein turnover in field research. The 2 primary approaches for measuring protein turnover are based on precursor- and end-product methods. The precursor method is the gold-standard for measuring acute (over several hours) skeletal muscle protein turnover, whereas the end-product method measures chronic (over several weeks) skeletal muscle protein turnover and provides the opportunity to monitor free-living activities. Both methods require invasive procedures such as the infusion of amino acid tracers and muscle biopsies to assess the uptake of the tracer into tissue. However, the end-product method can also be used to measure acute (over 9-24 h) whole-body protein turnover noninvasively by ingesting 15N-glycine, or equivalent isotope tracers, and collecting urine samples. The end-product method using 15N-glycine is a practical method for measuring whole-body protein turnover in the field over short (24 h) time frames and has been used effectively in recent military field research. Application of this method may improve our understanding of protein kinetics during conditions of high physiological stress in free-living environments such as military training.

Assessing the Dynamic Mitochondrial Fission and Fusion Events in Skeletal Muscle in vivo

Skeletal muscle plays an important role in systemic metabolism and overall health. Within skeletal muscle, there is a decline in mitochondrial function with age. Under normal healthy conditions, mitochondria remodel via two dynamic processes, mitochondrial protein turnover and mitochondrial dynamics, which includes mitochondrial fission and fusion events. While capturing the dynamic nature of mitochondrial protein turnover in skeletal muscle is now possible, assessing mitochondrial fission and fusion relies on snapshots of protein markers from muscle homogenate at distinct timepoints. Recent technical advancements allow for measurement of mitochondrial dynamics in skeletal muscle in vitro; however, rates of mitochondrial dynamics in other tissues are significantly slower in vivo compared to in vitro conditions. Therefore, the purpose of this study was to develop novel imaging methods to assess mitochondrial dynamics in vivo. Our hypothesis was that we could capture and assess in vivo mitochondrial fission/fusion events using a novel imaging approach. Using multiphoton microscopy, we activated a mitochondrial-targeted photo-activated GFP that was electroporated into the tibialis anterior (TA) muscle of 5 adult C57Bl6 mice to assess the rates of mitochondrial fission and fusion events. In vivo mitochondrial fission and fusion rates were about 50 times slower in comparison to previously reported rates in in vitro models. Due to the slower than expected rates of mitochondrial fission/fusion events in skeletal muscle in vivo, we used an additional imaging approach to explore changes mitochondrial morphology over more prolonged periods of time. To do so, we imaged control and denervated TA muscles of 5 adult C57Bl6 mice using an electroporated mitochondrial-targeted GFP (mitochondrial matrix) and TOM20_tdTomato (outer mitochondrial matrix). We determined that two weeks of denervation decreased the cross-sectional area that was co-stained in the denervated limb compared to a control limb, suggesting a decrease in cristae structure. In addition, two weeks of denervation decreased the cross-sectional area of the population of subsarcolemmal mitochondria in the denervated limb compared to a control limb. Together, our data emphasizes the importance of assessing mitochondrial dynamics in vivo. Additionally, changes to mitochondrial morphology in skeletal muscle fibers are regional, which stresses the importance of assessing mitochondrial subpopulations.

Heterochronic Plasma Transfer Alters Proteostatic Maintenance in Skeletal Muscle

Studies using heterochronic blood transfer (HBT) and heterochronic plasma transfer (HPT) show that circulating factors in young blood and plasma can rejuvenate the ability of aged skeletal muscle to respond to damage. Additionally, the transfer of old blood and plasma to young mice inhibits skeletal muscle regeneration. These changes in the ability of skeletal muscle to regenerate in response to modifications in the systemic environment are largely mediated by satellite cells. However, the role of satellite cells in maintaining normal muscle function is unclear. Therefore, how the systemic environment influences skeletal muscle in the absence of extreme muscle damage is still unknown. Proteostatic maintenance, which includes protein synthesis and breakdown, is necessary for cellular stability in skeletal muscle. One measure of proteostatic maintenance is protein:DNA ratio (i.e. how much protein is synthesized for cellular maintenance verses cellular proliferation). Here we determined if altering the systemic environment of old mice with young plasma and the systemic environment of young mice with old plasma would change proteostatic maintenance of skeletal muscle. We hypothesized that compared to old mice that receive old plasma (OOp), old mice that underwent HPT from young mice (OYp) would improve proteostatic maintenance. Additionally, we hypothesized that compared to young mice that receive young plasma (YYp), young mice that underwent HPT from old mice (YOp) would decrease proteostatic maintenance. To test this, we implanted young (5-month, n=3 YYp and 5 YOp) and old (24-months, n=5 OOp, and 5 OYp) C57BL/6J mice with a jugular catheter. All mice underwent heterochronic plasma transfer (HPT), receiving 100μl plasma injections, every three days for 24 days. The day before HPT, mice began stable isotope labeling with a bolus injection of 99% deuterium oxide (D2O) followed by 8% D2O-enriched drinking water. After 4 weeks, the quadriceps, gastrocnemius, and tibialis anterior muscles were harvested and analyzed for DNA and protein synthesis to calculate the protein:DNA ratios. The protein:DNA ratio for the mitochondrial fraction trended toward a higher ratio in the tibialis anterior of the OYp versus OOp (11.93 ± 1.161 and 8.928 ± 1.128, respectively: p=0.10). The protein:DNA ratio for the myofibrillar fraction trended toward being lower in the gastrocnemius of the YOp compared to YYp (8.338 ± 0.429 and 6.281 ± 0.737, respectively: p=0.06). These results suggest that manipulating the systemic environment with young or old plasma can change proteostatic maintenance of skeletal muscle so that the muscle reflects the systemic environment. Therefore, strategies to improve the systemic environment, like with exercise, may be a feasible strategy to improve proteostatic maintenance in aged skeletal muscle. Further research is needed to determine what circulating factors alter protein and DNA turnover in skeletal muscle.

PHD3 mediates denervation skeletal muscle atrophy through Nf-κB signal pathway.

Skeletal muscle is the largest organ of the body, the development of skeletal muscle is very important for the health of the animal body. Prolyl hydroxylases (PHDs) are the classical regulator of the hypoxia inducible factor (HIF) signal pathway, many researchers found that PHDs are involved in the muscle fiber type transformation, muscle regeneration, and myocyte differentiation. However, whether PHDs can impact the protein turnover of skeletal muscle is poorly understood. In this study, we constructed denervated muscle atrophy mouse model and found PHD3 was highly expressed in the atrophic muscles and there was a significant correlation between the expression level of PHD3 and skeletal muscle weight which was distinct from PHD1 and PHD2. Then, the similar results were getting from the different weight muscles of normal mice. To further verify the relationship between PHD3 and skeletal muscle protein turnover, we established a PHD3 interference model by injecting PHD3 sgRNA virus into tibialis anterior muscle (TA) muscle of MCK-Cre-cas9 mice and transfecting PHD3 shRNA lentivirus into primary satellite cells. It was found that the Knock-out of PHD3 in vivo led to a significant increase in muscle weight and muscle fiber area (P<.05). Besides, the activity of protein synthesis signal pathway increased significantly, while the protein degradation pathway was inhibited evidently (P<.05). In vitro, the results of 5-ethynyl-2'-deoxyuridine (EdU) and tetramethylrhodamine ethyl ester (TMRE) fluorescence detection showed that PHD3 interference could lead to a decrease in cell proliferation and an increase of cell apoptosis. After the differentiation of satellite cells, the production of puromycin in the interference group was higher than that in the control group, and the content of 3-methylhistidine in the interference group was lower than that in the control group (P<.05) which is consistent with the change of protein turnover signal pathway in the cell. Mechanistically, there is an interaction between PHD3, NF-κB, and IKBα which was detected by immunoprecipitation. With the interfering of PHD3, the expression of the inflammatory signal pathway also significantly decreased (P<.05). These results suggest that PHD3 may affect protein turnover in muscle tissue by mediating inflammatory signal pathway. Finally, we knocked out PHD3 in denervated muscle atrophy mice and LPS-induced myotubes atrophy model. Then, we found that the decrease of PHD3 protein level could alleviate the muscle weight and muscle fiber reduction induced by denervation in mice. Meanwhile, the protein level of the inflammatory signal pathway and the content of 3-methylhistidine in denervated atrophic muscle were also significantly reduced (P<.05). In vitro, PHD3 knock-out could alleviate the decrease of myotube diameter induced by LPS, and the expression of protein synthesis pathway was also significantly increased (P<.05). On the contrary, the expression level of protein degradation and inflammatory signal pathway was significantly decreased (P<.05). Through these series of studies, we found that the increased expression of PHD3 in denervated muscle might be an important regulator in inducing muscle atrophy, and this process is likely to be mediated by the inflammatory NF-κB signal pathway.