Markers Of Protein Degradation Research Articles

High-Intensity Interval Training Mitigates Sarcopenia and Suppresses the Myoblast Senescence Regulator EEF1E1.

The optimal exercise regimen for alleviating sarcopenia remains uncertain. This study aimed to investigate the efficacy of high-intensity interval training (HIIT) over moderate-intensity continuous training (MICT) in ameliorating sarcopenia. We conducted a randomized crossover trial to evaluate plasma proteomic reactions to acute HIIT (four 4-min high-intensity intervals at 70% maximal capacity alternating with 4 min at 30%) versus MICT (constant 50% maximal capacity) in inactive adults. We explored the relationship between a HIIT-specific protein relative to MICT, identified via comparative proteomic analysis, eukaryotic translation elongation factor 1 epsilon 1 (EEF1E1) and sarcopenia in a paired case-control study of elderly individuals (aged over 65). Young (3 months old) and aged (20 months old) mice were randomized to sedentary, HIIT and MICT groups (five sessions/week for 4 weeks; n = 8 for each group). Measurements included skeletal muscle index, hand grip strength, expression of atrophic markers Atrogin1 and MuRF1 and differentiation markers MyoD, myogenin and MyHC-II via western blotting. We examined the impact of EEF1E1 siRNA and recombinant protein on D-galactose-induced myoblast senescence, measuring senescence-associated β-galactosidase and markers like p21 and p53. The crossover trial, including 10 sedentary adults (32 years old, IQR 31-32) demonstrated significant alterations in the abundance of 21 plasma proteins after HIIT compared with MICT. In the paired case-control study of 84 older adults (84 years old, IQR 69-81; 52% female), EEF1E1 was significantly increased in those with sarcopenia compared to those without (14.68 [95%CI, 2.02-27.34] pg/mL, p = 0.03) and was associated with skeletal muscle index (R2 = 0.51, p < 0.001) and hand grip strength (R2 = 0.54, p < 0.001). In the preclinical study, aged mice exhibited higher EEF1E1 mRNA and protein levels in skeletal muscle compared to young mice, accompanied by a lower muscle mass and strength, increased cellular senescence and protein degradation markers and reduced muscle differentiation efficiency (all p < 0.05). HIIT reduced EEF1E1 expression and mitigated age-related muscle decline and atrophy in aged mice more effectively than MICT. Notably, EEF1E1 downregulation via siRNA significantly counteracted D-galactose-induced myoblast senescence as evidenced by reduced markers of muscle protein degradation and improved muscle differentiation efficiency (all p < 0.05). Conversely, treatments that increased EEF1E1 levels accelerated the senescence process (p < 0.05). Further exploration indicated that the decrease in EEF1E1 was associated with increased SIRT1 level and enhanced autophagy. This study highlights the potential of HIIT as a promising approach to prevent and treat sarcopenia while also highlighting EEF1E1 as a potential intervention target.

Skeletal muscle myosin heavy chain fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We examined how resistance exercise (RE), cycling exercise and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation. The 1boutRE study involved younger men (n=8; 5±2years of RE experience) performing a lower body RE bout with vastus lateralis (VL) biopsies being obtained prior to and acutely following exercise. With the 10weekRT study, VL biopsies were obtained in 36 younger adults before and 24h after their first/naïve RE bout. Participants also engaged in 10weeks of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined in an acute cycling study (n=7) and a study involving 2weeks of leg immobilization (n=20). In the 1boutRE study, fragmentation of all MyHC isoforms (MyHCTotal) increased 3h post-RE (∼200%, P=0.018) and returned to pre-exercise levels by 6h post-RE. Interestingly, a greater magnitude increase in MyHC type IIa versus I isoform fragmentation occurred 3h post-RE (8.6±6.3-fold vs. 2.1±0.7-fold, P=0.018). In 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, P<0.001); however, the last RE bout response was attenuated compared to the first bout (P=0.045). Although cycling exercise did not alter MyHCTotal fragmentation, ∼8% VL atrophy with 2weeks of leg immobilization increased MyHCTotal fragmentation (∼108%, P<0.001). Mechanistic C2C12 myotube experiments indicated that MyHCTotal fragmentation is likely due to calpain proteases. In summary, RE and disuse atrophy increase MyHC protein fragmentation. Research into how ageing and disease-associated muscle atrophy affect these outcomes is needed. HIGHLIGHTS: What is the central question of this study? How different exercise stressors and disuse affect skeletal muscle myosin heavy chain fragmentation. What is the main finding and its importance? This investigation is the first to demonstrate that resistance exercise and disuse atrophy lead to skeletal muscle myosin heavy chain protein fragmentation in humans. Mechanistic in vitro experiments provide additional evidence that MyHC fragmentation occurs through calpain proteases.

Cell type mapping of inflammatory muscle diseases highlights selective myofiber vulnerability in inclusion body myositis

Inclusion body myositis (IBM) is the most prevalent inflammatory muscle disease in older adults with no effective therapy available. In contrast to other inflammatory myopathies such as subacute, immune-mediated necrotizing myopathy (IMNM), IBM follows a chronic disease course with both inflammatory and degenerative features of pathology. Moreover, causal factors and molecular drivers of IBM progression are largely unknown. Therefore, we paired single-nucleus RNA sequencing with spatial transcriptomics from patient muscle biopsies to map cell-type-specific drivers underlying IBM pathogenesis compared with IMNM muscles and noninflammatory skeletal muscle samples. In IBM muscles, we observed a selective loss of type 2 myonuclei paralleled by increased levels of cytotoxic T and conventional type 1 dendritic cells. IBM myofibers were characterized by either upregulation of cell stress markers featuring GADD45A and NORAD or protein degradation markers including RNF7 associated with p62 aggregates. GADD45A upregulation was preferentially seen in type 2A myofibers associated with severe tissue inflammation. We also noted IBM-specific upregulation of ACHE encoding acetylcholinesterase, which can be regulated by NORAD activity and result in functional denervation of myofibers. Our results provide promising insights into possible mechanisms of myofiber degeneration in IBM and suggest a selective type 2 fiber vulnerability linked to genomic stress and denervation pathways.

Skeletal muscle myosin heavy chain protein fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We sought to examine how resistance exercise (RE), cycling exercise, and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation in humans. In the first study (1boutRE), younger adult men (n=8; 5±2 years of RE experience) performed a lower body RE bout with vastus lateralis (VL) biopsies obtained immediately before, 3-, and 6-hours post-exercise. In the second study (10weekRT), VL biopsies were obtained in untrained younger adults (n=36, 18 men and 18 women) before and 24 hours (24h) after their first/naïve RE bout. These participants also engaged in 10 weeks (24 sessions) of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined from a third acute cycling study (n=7) and a fourth study involving two weeks of leg immobilization (n=20, 15 men and 5 women) to determine how MyHC fragmentation was affected. In the 1boutRE study, the fragmentation of all MyHC isoforms (MyHCTotal) increased 3 hours post-RE (~ +200%, p=0.018) and returned to pre-exercise levels by 6 hours post-RE. Immunoprecipitation of MyHCTotal revealed ubiquitination levels remained unaffected at the 3- and 6-hour post-RE time points. Interestingly, a greater increase in magnitude for MyHC type IIa versus I isoform fragmentation occurred 3-hours post-RE (8.6±6.3-fold versus 2.1±0.7-fold, p=0.018). In all 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, respectively; p<0.001); however, the last RE bout response was attenuated compared to the first bout (p=0.045). The first/naïve bout response was significantly elevated in females only (p<0.001), albeit females also demonstrated a last bout attenuation response (p=0.002). Although an acute cycling bout did not alter MyHCTotal fragmentation, ~8% VL atrophy with two weeks of leg immobilization led to robust MyHCTotal fragmentation (+108%, p<0.001), and no sex-based differences were observed. In summary, RE and disuse atrophy increase MyHC protein fragmentation. A dampened response with 10 weeks of resistance training, and more refined responses in well-trained men, suggest this is an adaptive process. Given the null polyubiquitination IP findings, more research is needed to determine how MyHC fragments are processed. Moreover, further research is needed to determine how aging and disease-associated muscle atrophy affect these outcomes, and whether MyHC fragmentation is a viable surrogate for muscle protein turnover rates.

Moringa oleifera improves skeletal muscle metabolism and running performance in mice

Moringa oleifera (M. oleifera) is a subtropical plant routinely used to treat inflammation and chronic disease. However, the mechanism of action of M. oleifera has not been fully elucidated. Thus, this study aims to evaluate its role as a novel ergogenic aid to improve exercise performance by driving signaling pathways implicated in mitochondrial biogenesis and oxidative metabolism in skeletal muscle tissue. To accomplish this, adult male C57BL/6 mice were treated with 1.0 g of M. oleifera (N = 20) per day or vehicle control (N = 20) for five weeks. Following three weeks of supplementation, half of each group (RUN) was given access to running wheels every night for two weeks (Remaining half = SED). After treatment protocols were complete, the gastrocnemius muscles were excised and assayed for markers of mitochondrial biogenesis, angiogenesis, endurance capacity, and capillary density using immunohistochemistry and RT-PCR. Our results showed a significant increase in average distance run in the M. oleifera + RUN compared to SED mice. In addition, key metabolic markers such as PGC-1α, PPARγ, SDHB, SUCLG1, VEGF, PGAM-2, PGK1, and MYLPF were increased in the M. oleifera treated groups compared to vehicle + SED. Moreover, M. oleifera also increased CSA and decreased markers of protein degradation. This data suggests that M. oleifera has the potential to be an ergogenic aid that enhances energy metabolism in adult skeletal muscle by increasing the expression of key metabolic markers.

Cornflower Extract and Its Active Components Alleviate Dexamethasone-Induced Muscle Wasting by Targeting Cannabinoid Receptors and Modulating Gut Microbiota.

Sarcopenia, a decline in muscle mass and strength, can be triggered by aging or medications like glucocorticoids. This study investigated cornflower (Centaurea cyanus) water extract (CC) as a potential protective agent against DEX-induced muscle wasting in vitro and in vivo. CC and its isolated compounds mitigated oxidative stress, promoted myofiber growth, and boosted ATP production in C2C12 myotubes. Mechanistically, CC reduced protein degradation markers, increased mitochondrial content, and activated protein synthesis signaling. Docking analysis suggested cannabinoid receptors (CB) 1 and 2 as potential targets of CC compounds. Specifically, graveobioside A from CC inhibited CB1 and upregulated CB2, subsequently stimulating protein synthesis and suppressing degradation. In vivo, CC treatment attenuated DEX-induced muscle wasting, as evidenced by enhanced grip strength, exercise performance, and modulation of muscle gene expression related to differentiation, protein turnover, and exercise performance. Moreover, CC enriched gut microbial diversity, and the abundance of Clostridium sensu stricto 1 positively correlated with muscle mass. These findings suggest a multifaceted mode of action for CC: (1) direct modulation of the muscle cannabinoid receptor system favoring anabolic processes and (2) indirect modulation of muscle health through the gut microbiome. Overall, CC presents a promising therapeutic strategy for preventing and treating muscle atrophy.

Effect of five hours of mixed exercise on urinary nitrogen excretion in healthy moderate-to-well-trained young adults.

Carbohydrates and fats are the primary energy substrates during exercise, but proteins can also contribute. When proteins are degraded in the body, the amino groups are mainly converted to urea and excreted. Therefore, nitrogen excretion has been used as a marker of protein degradation, but a clear conclusion has yet to be reached on the effect of exercise on nitrogen excretion. Thus, we tested whether exercise increases nitrogen excretion. Fifteen young, healthy, moderate-to-well-trained participants (4 females, 11 males, VO2max 54.4 ± 1.7 mL·kg-1·min-1; mean ± SEM) participated in a randomized, balanced cross-over design investigation consisting of 1 day with 5 h of exercise (exercise day, EX) and 1 day with no exercise (control day, CON). The participants recorded their dietary intake the day before from 16:00 and throughout the intervention day. They then repeated these dietary intakes on the second trial day. A standardized lunch was provided on both days. In addition, participants were allowed to consume almost protein-free snacks in EX to ensure the same energy balance during both trial days. Urine was collected throughout the whole testing period, and urinary 3-methylhistidine (3-MH) excretion was measured to examine muscular catabolism. The sweat rate was calculated during the exercise period. The urinary nitrogen and 3-MH excretions did not differ significantly between EX and CON (p = 0.764 and p = 0.953). The sweat rate was 2.55 ± 0.25 L in EX and 0.14 ± 0.15 L in CON (p < 0.001), and by estimating sweat nitrogen excretion, total nitrogen excretion was shown to differ with exercise. Our results showed that 5 hours of mixed exercise did not significantly impact urinary nitrogen and 3-MH excretions in healthy moderate-to-well-trained young adults.

Urea Transporter B Is Produced in the Jk Null Blood Group Yet Fails to Integrate into the Red Cell Membrane Impacting Erythropoiesis and Erythrocytes

The Kidd (Jk) blood group is carried by an integral membrane glycoprotein, urea transporter B (UT-B), which transports urea through the red blood cell (RBC) membrane. The absence of both Jk a and Jk b antigens, designated phenotypically as Jk(a-b-) (Jk null), is very rare with a significantly increased prevalence in certain ethnicities, most abundantly within the Polynesian population where it is found at 0.9% prevalence. Provision of blood for patients requiring this phenotype is challenging. In Australia, there are only 15 active Jk null blood donors. Individuals of the Jk null phenotype have been shown to have suboptimal urine concentrating ability but erythrocytes appear phenotypically normal with shape, size and lifespan within normal reference indices (Lawicki et al., Transfus Med Rev, 2017). Where UT-B integrates in the RBC membrane is unknown although protein 4.1R deficiency is reported to lead to the absence of UT-B (Azouzi et al., Br J Haematol, 2015). Our aim was to identify and characterise the molecular basis for the Jk null phenotype in Australian blood donors and decipher the biological impact of the Jk null phenotype using our ex vivo model of erythropoiesis to help inform clinical transfusion practice. Massively parallel sequencing showed that all blood donors (n=9) were homozygous for JK c.342-1G&amp;gt;A (splice site variant) and c.838G&amp;gt;A polymorphisms resulting in the JK*02N.01/*02N.01 genotype. This genotype predicts a Jk null phenotype, and a truncated UT-B protein caused by the formation of a premature stop codon. The assumption from the genomics data is that the putative truncated UT-B protein would not be translated. To test this hypothesis we examined the RBCs of Jk null individuals and confirmed the lack of UT-B expression on the RBC surface by live flow cytometry and confocal imaging. In control RBCs UT-B is seen in discrete microdomains on the RBC surface by live confocal microscopy. To establish the expression of UT-B during erythropoiesis we cultured CD34+ cells from control and Jk null buffy coats after whole blood donation. Flow cytometry and confocal microscopy of control cells identified that UT-B is expressed intracellularly and at the cell surface in the developing erythroid cells. Notably we identified that the UT-B protein can also be found intracellularly in developing Jk null erythroid cells but is not detectable in the plasma membrane. In Jk null reticulocytes, UT-B colocalises with markers of protein degradation and destruction implying that the truncated UT-B protein is quickly removed by controlled protein degradation pathways. Quantitative proteomics was used to assess the global effect of the Jk null phenotype on the mature RBC membrane proteome. These data confirmed the absence of UT-B but also uncovered that in mature Jk null RBCs, RBC proteins involved in solute transport (aquaporin-1, stomatin) and membrane-cytoskeleton stabilisation (alpha and beta adducins, and dematin) are significantly downregulated. In contrast, actin binding proteins including tropomyosin and myosin IIA were upregulated in Jk null RBCs. The proteomics data also revealed that numerous major plasma proteins including alpha-2-macroglobulin, hemopexin and immunoglobulins are associated exclusively with Jk null RBCs. These findings imply that the lack of functional, membrane embedded UT-B induces various compensatory mechanisms in the Jk null RBCs membrane. In conclusion, we show that a truncated UT-B protein is produced in Jk null erythroid cells but fails to mature into a RBC membrane integrated, functional glycoprotein. Our data also suggest that the absence of a functional UT-B glycoprotein on the RBC surface affects the RBC membrane and cytoskeletal proteome and its interaction with major plasma glycoproteins.

Impact of high-intensity exercise training (HIIT) versus moderate continuous training (MCT) on skeletal muscle alterations in HFrEF: a substudy of the SMART-EX trial

Abstract Background Skeletal muscle dysfunction contributes to exercise intolerance in chronic heart failure. The randomized multicenter Study of Myocardial Recovery After Exercise Training in Heart Failure (SMART-EX) has shown that high-intensity exercise training (HIIT) but not moderate continuous training (MCT) reduced the left ventricular end diastolic diameter. Both training modalities significantly increased exercise tolerance as determined by cardiopulmonary exercise testing (CPET). However, the underlying molecular alterations within the skeletal muscle with respect to different exercise training modalities are largely unknown. Methods In patients with chronic heart failure with reduced ejection fraction (HFrEF) who participated in the SMART-EX skeletal muscle substudy needle biopsies from the vastus lateralis muscle were performed before and after 12 weeks of supervised HIIT, MCT, or recommendation of regular exercise (RRE) to allow molecular assessment of markers for muscle catabolism, inflammation and energy supply by RT-PCR and western blotting. Echocardiography and CPET were performed in all patients. Changes over time were tested within each group. Results Skeletal muscle biopsies at baseline and follow-up were available in 31 patients (HIIT n=9; MCT n=9; RRE n=13). The majority of patients were male (87%). Median left ventricular ejection fraction was 29%. Median improvement in peak VO2 was 2.0 (IQR -0.4-2.8) in HIIT vs. 1.4 (IQR -0.9-3.4) in MCT vs. 0.0 (IQR -2.9-2.1) ml/min/kg in RRE. This was statistically not significant. HIIT reduced mRNA levels of the E3 ligases of the ubiquitine proteasome system MuRF1 by -72% (p&amp;lt;0.01) and MafBx -45% (p=0.09) with no change in MCT (MuRF1 -31%, p=0.23; MafBx -47%, p=0.21) and RRE (MuRF1 -8%, p=0.20; MafBx -6%, p=0.46). There was no change in mRNA expression of the inflammatory markers interleukin 6 (HIIT 12%; MCT 29%; RRE -35%; n.s.) and TNFα (HIIT -9%; MCT 20%; RRE 27%; n.s.) in either group. The mRNA expression of the mitochondrial F1 subunit of complex V (ATP5F1) significantly increased in HIIT by 14% (p&amp;lt;0.05) with no change in MCT (-3%, p=0.75) and RRE (2%, p=0.76). The mRNA expression of mitochondrial complex I subunits NDUFB8 and NDUFB6 remained unchanged in all groups. Western blot analysis of porin expression, which is a marker of total mitochondrial content, revealed a significant increase in HIIT by 69% (p&amp;lt;0.05) whereas MCT (21%, p=0.18) and RRE (4%, p=0.79) remained unchanged. Conclusion High-intensity interval training in HFrEF for 12 weeks resulted in reduced markers of protein degradation via the ubiquitine proteasome system and increased markers of mitochondrial content and energy supply via mitochondrial complex V whereas moderate continuous exercise training had only minor (non-significant) effects. These molecular changes are probably one reason why exercise capacity improved in patients of the SMART-EX trail.

Partial replacement of soybean meal with microalgae biomass on in vitro ruminal fermentation may reduce ruminal protein degradation

The objective of this study was to evaluate the effects of partially replacing soybean meal (SBM) with algal sources on in vitro ruminal fermentation. Using 6 fermenters in a 3 × 3 replicated Latin square with 3 periods of 10 d each, we tested 3 treatments: a control diet (CRT) with SBM at 17.8% of the diet dry matter (DM); and 50% SBM biomass replacement with either Chlorella pyrenoidosa (CHL); or Spirulina platensis (SPI). The basal diet was formulated to meet the requirements of a 680-kg Holstein dairy cow producing 45 kg/d of milk with 3.5% fat and 3% protein. All diets had a similar nutritional composition (16.0% CP; 34.9% NDF; 31.0% starch, DM basis) and fermenters were provided with 106 g DM/d split into 2 portions. After 7 d of adaptation, samples were collected for 3 d of each period for analyses of ruminal fermentation at 0, 1, 2, 4, 6, and 8 h after morning feeding for evaluation of the ruminal fermentation kinetics. For the evaluation of the daily production of total metabolites and for the evaluation of nutrient degradability, samples from the effluent containers were collected daily. Statistical analysis was performed with the MIXED procedure of SAS with treatment, time, and their interactions considered as fixed effects; day, square, and fermenter were considered as random effects. Orthogonal contrasts (CRT vs. algae; and CHL vs. SPI) were used to depict the treatment effect, and significance was declared when P ≤ 0.05. Fermenters that received algae-based diets had a greater propionate molar concentration and molar proportion when compared with the fermenters fed CRT diets. In addition, those algae-fed fermenters had lower branched short-chain fatty acids (BSCFA) and isoacids (IA), which are biomarkers of ruminal protein degradation, along with lower ammonia (NH3-N) concentration and greater nonammonia nitrogen (NAN). When contrasting with fermenters fed SPI-diets, fermenters fed based CHL-diets had a lower molar concentration of BSCFA and IA, along with lower NH3-N concentration and flow, and greater NAN, bacterial nitrogen flow, and efficiency of nitrogen utilization. Those results indicate that CHL protein may be more resistant to ruminal degradation, which would increase efficiency of nitrogen utilization. In summary, partially replacing SBM with algae biomass, especially with CHL, is a promising strategy to improve the efficiency of nitrogen utilization, due to the fact that fermenters fed CHL-based diets resulted in a reduction in BSCFA and IA, which are markers of protein degradation, and it would improve the efficiency of nitrogen utilization. However, further validation using in vivo models are required.

Females display relatively preserved muscle quality compared with males during the onset and early stages of C26-induced cancer cachexia.

Cancer cachexia is clinically defined by involuntary weight loss >5% in <6 mo, primarily affecting skeletal muscle. Here, we aimed to identify sex differences in the onset of colorectal cancer cachexia with specific consideration to skeletal muscle contractile and metabolic functions. Eight-weeks old BALB/c mice (69 males, 59 females) received subcutaneous C26 allografts or PBS vehicle. Tumors were developed for 10-, 15-, 20-, or 25 days. Muscles and organs were collected, in vivo muscle contractility, protein synthesis rate, mitochondrial function, and protein turnover markers were assessed. One-way ANOVA within sex and trend analysis between sexes were performed, P < 0.05. Gastrocnemius and tibialis anterior (TA) muscles became atrophic in male mice at 25 days, whereas female mice exhibited no significant differences in muscle weights at endpoints despite presenting hallmarks of cancer cachexia (fat loss, hepatosplenomegaly). We observed lowered muscle contractility and protein synthesis concomitantly to muscle mass decay in males, with higher proteolytic markers in muscles of both sexes. mRNA of Opa1 was lower in TA, whereas Bnip3 was higher in gastrocnemius after 25 days in male mice, with no significant effect in female mice. Our data suggest relative protections to skeletal muscle in females compared with males despite other canonical signs of cancer cachexia and increased protein degradation markers; suggesting we should place onus upon nonmuscle tissues during early stages of cancer cachexia in females. We noted potential protective mechanisms relating to skeletal muscle contractile and mitochondrial functions. Our findings underline possible heterogeneity in onset of cancer cachexia between biological sexes, suggesting the need for sex-specific approaches to treat cancer cachexia.NEW & NOTEWORTHY Our study demonstrates biological-sex differences in phenotypic characteristics of cancer cachexia between male and female mice, whereby females display many common characteristics of cachexia (gonadal fat loss and hepatosplenomegaly), protein synthesis markers alterations, and common catabolic markers in skeletal muscle despite relatively preserved muscle mass in early-stage cachexia compared with males. Mechanisms of cancer cachexia appear to differ between sexes. Data suggest need to place onus of early cancer cachexia detection and treatment on nonmuscle tissues in females.

Endoplasmic reticulum stress and ubiquitin-proteasome system impairment in natural scrapie.

Chronic accumulation of misfolded proteins such as PrPSc can alter the endoplasmic reticulum homeostasis triggering the unfolded protein response (UPR). In this pathogenic event, the molecular chaperones play an important role. Several reports in humans and animals have suggested that neurodegeneration is related to endoplasmic reticulum stress in diseases caused by the accumulation of misfolded proteins. In this study, we investigated the expression of three endoplasmic reticulum stress markers: PERK (protein kinase R-like endoplasmic reticulum kinase), BiP (binding immunoglobulin protein), and PDI (Protein Disulfide Isomerase). In addition, we evaluated the accumulation of ubiquitin as a marker for protein degradation mediated by the proteasome. These proteins were studied in brain tissues of sheep affected by scrapie in clinical and preclinical stages of the disease. Results were compared with those observed in healthy controls. Scrapie-infected sheep showed significant higher levels of PERK, BiP/Grp78 and PDI than healthy animals. As we observed before in models of spontaneous prion disease, PDI was the most altered ER stress marker between scrapie-infected and healthy sheep. Significantly increased intraneuronal and neuropil ubiquitinated deposits were observed in certain brain areas in scrapie-affected animals compared to controls. Our results suggest that the neuropathological and neuroinflammatory phenomena that develop in prion diseases cause endoplasmic reticulum stress in brain cells triggering the UPR. In addition, the significantly higher accumulation of ubiquitin aggregates in scrapie-affected animals suggests an impairment of the ubiquitin-proteasome system in natural scrapie. Therefore, these proteins may contribute as biomarkers and/or therapeutic targets for prion diseases.

Capsaicin alleviates cisplatin-induced muscle loss and atrophy in vitro and in vivo.

Cisplatin (CP) is a widely used chemotherapeutic drug with subsequent adverse effects on different organs and tissues including skeletal muscle loss and atrophy as the most common clinical symptoms. The molecular mechanism of cisplatin-induced muscle atrophy is not clearly understood. However, recent significant advances indicate that it is related to an imbalance in both the protein status and apoptosis. Capsaicin (CAP) is one of the major ingredients in chilli peppers. It is a valuable pharmacological agent with several therapeutic applications in controlling pain and inflammation with particular therapeutic potential in muscle atrophy. However, the mechanisms underlying its protective effects against cisplatin-induced muscle loss and atrophy remain largely unknown. This study aims to investigate capsaicin's beneficial effects on cisplatin-induced muscle loss and atrophy in vitro and in vivo. The anti-muscle-atrophic effect of capsaicin on cisplatin-induced muscle loss was investigated using in vivo and in vitro studies. By using the pretreatment model, pretreated capsaicin for 24h and treated with cisplatin for 48h, we utilized a C2 C12 myotube formation model where cell viability analysis, immunofluorescence, and protein expression were measured to investigate the effect of capsaicin in hampering cisplatin-induced muscle atrophy. C57BL/6 mice were administered capsaicin (10, 40mg/kg BW) as a pretreatment for 5weeks and cisplatin (3mg/kg BW) for seven consecutively days to assess muscle atrophy in an animal model for protein and oxidative stress examination, and the grip strength was tested to evaluate the muscle strength. Our study results indicated that cisplatin caused lower cell viability and showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in the myotube diameter, repression of Akt, and mTOR protein expression. However, pretreatment with capsaicin could ameliorate cisplatin-induced muscle atrophy by up-regulating the protein synthesis in skeletal muscle as well as down-regulating the markers of protein degradation. Additionally, capsaicin was able to downregulate the protein expression of apoptosis-related markers, activated TRPV1 and autophagy progress modulation and the recovery of lysosome function. In vivo, capsaicin could relieve oxidative stress and cytokine secretion while modulating autophagy-related lysosome fusion, improvinggrip strength, and alleviating cisplatin-induced body weight loss and gastrocnemius atrophy. These findings suggest that capsaicincan restore cisplatin-induced imbalance between protein synthesis and protein degradation pathways and it may have protective effects against cisplatin-induced muscle atrophy.

The impact of senescence on muscle wasting in chronic kidney disease.

Muscle wasting is a common complication of chronic kidney disease (CKD) that is associated with higher mortality. Although the mechanisms of myofibre loss in CKD has been widely studied, the contribution of muscle precursor cell (MPC) senescence remains poorly understood. Senescent MPCs no longer proliferate and can produce proinflammatory factors or cytokines. In this study, we tested the hypothesis that the senescence associated secretory phenotype (SASP) of MPCs contributes to CKD-induced muscle atrophy and weakness. CKD was induced in mice by 5/6th nephrectomy. Kidney function, muscle size, and function were measured, and markers of atrophy, inflammation, and senescence were evaluated using immunohistochemistry, immunoblots, or qPCR. To study the impact of senescence, a senolytics cocktail of dasatinib+quercetin (D&Q) was given orally to mice for 8weeks. To investigate CKD-induced senescence at the cellular level, primary MPCs were incubated with serum from CKD or control subjects. The roles of specific proteins in MPC senescence were studied using adenoviral transduction, siRNA, and plasmid transfection. In the hindlimb muscles of CKD mice, (i) the senescence biomarker SA-β-gal was sharply increased (~30-fold); (ii) the DNA damage response marker γ-H2AX was increased 1.9-fold; and (iii) the senescence pathway markers p21 and p16INK4a were increased 1.99-fold and 2.82-fold, respectively (all values, P<0.05), whereas p53 was unchanged. γ-H2AX, p21, and p16INK4A were negatively correlated at P<0.05 with gastrocnemius weight, suggesting a causal relationship with muscle atrophy. Administration of the senolytics cocktail to CKD mice for 8weeks eliminated the disease-related elevation of p21, p16INK4a , and γ-H2AX, abolished positive SA-β-gal, and depressed the high levels of the SASP cytokines, TNF-α, IL-6, IL-1β, and IFN (all values, P<0.05). Skeletal muscle weight, myofibre cross-sectional area, and grip function were improved in CKD mice receiving D&Q. Markers of protein degradation, inflammation, and MPCs dysfunction were also attenuated by D&Q treatment compared with the vehicle treatment in 5/6th nephrectomy mice (all values, P<0.05). Uraemic serum induced senescence in cultured MPCs. Overexpression of FoxO1a in MPCs increased the number of p21+ senescent cells, and p21 siRNA prevented uraemic serum-induced senescence (P<0.05). Senescent MPCs are likely to contribute to the development of muscle wasting during CKD by producing inflammatory cytokines. Limiting senescence with senolytics ameliorated muscle wasting and improved muscle strength in vivo and restored cultured MPC functions. These results suggest potential new therapeutic targets to improve muscle health and function in CKD.

Predictors of muscle hypertrophy responsiveness to electrically evoked resistance training after spinal cord injury.

The purpose of the study was to identify potential predictors of muscle hypertrophy responsiveness following neuromuscular electrical stimulation resistance training (NMES-RT) in persons with chronic spinal cord injury (SCI). Data for twenty individuals with motor complete SCI who completed twice weekly NMES-RT lasting 12-16weeks as part of their participation in one of two separate clinical trials were pooled and retrospectively analyzed. Magnetic resonance imaging (MRI) was used to measure muscle cross-sectional area (CSA) of the whole thigh and knee extensor muscle before and after NMES-RT. Muscle biopsies and fasting biomarkers were also measured. Following the completion of the respective NMES-RT trials, participants were classified into either high-responders (n = 8; muscle CSA > 20%) or low-responders (n = 12; muscle CSA < 20%) based on whole thigh muscle CSA hypertrophy. Whole thigh muscle and knee extensors CSAs were significantly greater (P < 0.0001) in high-responders (29 ± 7% and 47 ± 15%, respectively) compared to low-responders (12 ± 3% and 19 ± 6%, respectively). There were no differences in total caloric intake or macronutrient intake between groups. Extensor spasticity was lower in the high-responders compared to the low-responders as was the dosage of baclofen. Prior to the intervention, the high-responders had greater body mass compared to the low-responders with SCI (87.8 ± 13.7 vs. 70.4 ± 15.8kg; P = 0.012), body mass index (BMI: 27.6 ± 2.7 vs. 22.9 ± 6.0kg/m2; P = 0.04), as well as greater percentage in whole body and regional fat mass (P < 0.05). Furthermore, high-responders had a 69% greater increase (P = 0.086) in total Akt protein expression than low-responders. High-responders also exhibited reduced circulating IGF-1 with a concomitant increase in IGFBP-3. Exploratory analyses revealed upregulation of mRNAs for muscle hypertrophy markers [IRS-1, Akt, mTOR] and downregulation of protein degradation markers [myostatin, MurF-1, and PDK4] in the high-responders compared to low-responders. The findings indicate that body composition, spasticity, baclofen usage, and multiple signaling pathways (anabolic and catabolic) are involved in the differential muscle hypertrophy response to NMES-RT in persons with chronic SCI.

Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis

Photoinhibitory high light stress in Arabidopsis leads to increases in markers of protein degradation and transcriptional up-regulation of proteases and proteolytic machinery, but proteostasis is largely maintained. We find significant increases in the in vivo degradation rate for specific molecular chaperones, nitrate reductase, glyceraldehyde-3 phosphate dehydrogenase, and phosphoglycerate kinase and other plastid, mitochondrial, peroxisomal, and cytosolic enzymes involved in redox shuttles. Coupled analysis of protein degradation rates, mRNA levels, and protein abundance reveal that 57% of the nuclear-encoded enzymes with higher degradation rates also had high light–induced transcriptional responses to maintain proteostasis. In contrast, plastid-encoded proteins with enhanced degradation rates showed decreased transcript abundances and must maintain protein abundance by other processes. This analysis reveals a light-induced transcriptional program for nuclear-encoded genes, beyond the regulation of the photosystem II (PSII) D1 subunit and the function of PSII, to replace key protein degradation targets in plants and ensure proteostasis under high light stress.

Mechanistic Insights into the Role of TRIM28 in the Regulation of Skeletal Muscle Size

Protein homeostasis plays a critical role in the regulation of skeletal muscle size, and the maintenance of skeletal muscle size contributes significantly to disease prevention and quality of life. Over the past few decades, it has become widely accepted that skeletal muscle size is controlled by the net balance between the rates of protein synthesis and protein degradation. Despite this assertion, the mechanisms that modulate this balance and lead to changes in muscle size remain incompletely defined. Nevertheless, advancements have been made. For instance, a recent study from our lab revealed that the myofiber‐specific loss of a transcriptional intermediary factor named TRIM28 led to a significant reduction in basal myofiber size and attenuated the increase in myofiber size that occurs in response to mechanical overload. Moreover, rigorous follow‐up studies indicate that the TRIM28 knockout‐induced deficits in myofiber size are not driven by a decrease in the rate of protein synthesis, but that this phenotype is instead associated with elevated levels of canonical markers of protein degradation. Together, these observations led to our central hypothesis that TRIM28 confers its effects on myofiber size via the regulation of protein degradation. Thus, in an effort to gain further insight into this possibility, we performed a deep RNA‐sequencing analysis, the results of which led to the identification of Mettl21c and Mettl21e as two genes whose expression were significantly downregulated by the loss of TRIM28. We were intrigued by this discovery because recent reports have implicated Mettl21c and Mettl21e in the regulation of protein degradation, and it has been shown that the loss of these proteins in muscle promotes aberrant protein degradation and reduces muscle size. Combined with our initial observations, it became apparent that Mettl21c and Mettl21e might be important parts of the pathway via which TRIM28 regulates protein degradation to control myofiber size. To test this theory, we generated expression plasmids encoding HA‐Mettl21c and HA‐Mettl21e, and then used electroporation to demonstrate that the expression of Mettl21c and Mettl21e in myofibers is sufficient to induce a robust hypertrophic response in control animals, and that the hypertrophic effect of Mettl21c, but not Mettl21e, is conserved in muscles lacking TRIM28. Collectively, these findings provide evidence that TRIM28 regulates myofiber size via the regulation of protein degradation, and that Mettl21c, but not Mettl21e, might play an important role in the pathway via which TRIM28 confers this effect.

Leucine-Rich Diet Improved Muscle Function in Cachectic Walker 256 Tumour-Bearing Wistar Rats.

Skeletal muscle atrophy occurs in several pathological conditions, such as cancer, especially during cancer-induced cachexia. This condition is associated with increased morbidity and poor treatment response, decreased quality of life, and increased mortality in cancer patients. A leucine-rich diet could be used as a coadjutant therapy to prevent muscle atrophy in patients suffering from cancer cachexia. Besides muscle atrophy, muscle function loss is even more important to patient quality of life. Therefore, this study aimed to investigate the potential beneficial effects of leucine supplementation on whole-body functional/movement properties, as well as some markers of muscle breakdown and inflammatory status. Adult Wistar rats were randomly distributed into four experimental groups. Two groups were fed with a control diet (18% protein): Control (C) and Walker 256 tumour-bearing (W), and two other groups were fed with a leucine-rich diet (18% protein + 3% leucine): Leucine Control (L) and Leucine Walker 256 tumour-bearing (LW). A functional analysis (walking, behaviour, and strength tests) was performed before and after tumour inoculation. Cachexia parameters such as body weight loss, muscle and fat mass, pro-inflammatory cytokine profile, and molecular and morphological aspects of skeletal muscle were also determined. As expected, Walker 256 tumour growth led to muscle function decline, cachexia manifestation symptoms, muscle fibre cross-section area reduction, and classical muscle protein degradation pathway activation, with upregulation of FoxO1, MuRF-1, and 20S proteins. On the other hand, despite having no effect on the walking test, inflammation status or muscle oxidative capacity, the leucine-rich diet improved muscle strength and behaviour performance, maintained body weight, fat and muscle mass and decreased some protein degradation markers in Walker 256 tumour-bearing rats. Indeed, a leucine-rich diet alone could not completely revert cachexia but could potentially diminish muscle protein degradation, leading to better muscle functional performance in cancer cachexia.

Deterioration of mitochondrial function in the human intercostal muscles differs among individuals with sarcopenia, obesity, and sarcopenic obesity

Sarcopenic obesity (SO) increases the risk of mortality more than sarcopenia or obesity alone. Sarcopenia weakens the peripheral and respiratory muscles, leading to respiratory complications. It also induces mitochondrial dysfunction in the peripheral muscle; however, whether mitochondrial dysfunction in respiratory muscles differs among individuals with obesity, sarcopenia, and SO remains unknown. We evaluated the deterioration of respiratory muscle strength and mitochondrial function among normal, sarcopenia, obesity, and SO subjects. Twenty-five patients who underwent lung resections were enrolled between April 2017 and January 2021, and their intercostal muscles were harvested. Based on their L3 muscle index and visceral fat area, the patients were divided into four groups (normal, obesity, sarcopenia, and SO). The clinical data, mRNA expression, and protein expressions associated with mitochondrial biogenesis/fusion/fission in the intercostal muscles were compared among the four groups. The respiratory muscle strength was evaluated using peak expiratory flow rate (PEFR). The PEFR values of the four groups were not significantly different. The levels of pAkt/Akt and mTOR (a marker of protein synthesis) were not significantly different among the four groups; however, those in the SO group were substantially lower than those in the sarcopenia or obesity groups. The levels of Atrogen-1 and MuRF1 (a marker of protein degradation) were not significantly different among the four groups; however, those in the SO group were substantially higher than those in the sarcopenia or obesity groups. Expression of PGC1-α (a marker of mitochondrial biogenesis) in the SO group was significantly lower than that in the normal group. MFN1 and MFN2 (marker of mitochondrial fusion) levels were significantly lower in the SO group than those in the normal group. DRP1 (a marker of mitochondrial fission) level in the SO group was substantially lower than that in the normal group. The expression of TNF-α (a pro-inflammatory cytokine) in the SO group was substantially lower than that in the normal group. Our results suggest that the deterioration of protein synthesis and degradation of mitochondrial function in the respiratory muscles was most prominent in the SO before the weakening of the respiratory muscles. The deterioration mechanism may differentially regulate obesity, sarcopenia, and SO.

Chronic social stress alters protein metabolism in juvenile rainbow trout, Oncorhynchus mykiss

When confined in pairs, juvenile rainbow trout (Oncorhynchus mykiss) form dominance hierarchies in which subordinate fish exhibit characteristic physiological changes including reduced growth rates and chronically elevated plasma cortisol concentrations. We hypothesized that alterations in protein metabolism contribute to the reduced growth rate of socially stressed trout, and predicted that subordinate trout would exhibit reduced rates of protein synthesis coupled with increases in protein degradation. Protein metabolism was assessed in dominant and subordinate fish after 4 days of social interaction, and in fish that were separated after 4 days of interaction for a 4 days recovery period, to determine whether effects on protein metabolism recovered when social stress was alleviated. Protein metabolism was assessed in liver and white muscle by measuring the fractional rate of protein synthesis and markers of protein degradation. In the white muscle of subordinate fish, protein synthesis was inhibited and activities of the ubiquitin-proteasome pathway (UPP) and the autophagy lysosomal system (ALS) were elevated. By contrast, the liver of subordinate fish exhibited increased rates of protein synthesis and activation of the ALS. When allowed to recover from chronic social stress for 4 days, differences in protein metabolism observed in white muscle of subordinate fish during the interaction period disappeared. In liver, protein synthesis returned to baseline levels during recovery from social stress, but markers of protein degradation did not. Collectively, these data support the hypothesis that inhibition of muscle protein synthesis coupled with increases in muscle protein breakdown contribute to the reduced growth rates of subordinate rainbow trout.