Major Muscle Proteins Research Articles

Cardiac manifestations of human ACTA2 variants recapitulated in a zebrafish model

The ACTA2 gene encodes actin α2, a major smooth muscle protein in vascular smooth muscle cells. Missense variants in the ACTA2 gene can cause inherited thoracic aortic diseases with characteristic symptoms, such as dysfunction of smooth muscle cells in the lungs, brain vessels, intestines, pupils, bladder, or heart. We identified a heterozygous missense variant of Gly148Arg (G148R) in a patient with a thoracic aortic aneurysm, dissection, and left ventricular non-compaction. We used zebrafish as an in vivo model to investigate whether or not the variants might cause functional or histopathological abnormalities in the heart. Following the fertilization of one-cell stage embryos, we injected in vitro synthesized ACTA2 mRNA of wild-type, novel variant G148R, or the previously known pathogenic variant Arg179His (R179H). The embryos were maintained and raised for 72 h post-fertilization for a heart analysis. Shortening fractions of heart were significantly reduced in both pathogenic variants. A histopathological evaluation showed that the myocardial wall of ACTA2 pathogenic variants was thinner than that of the wild type, and the total cell number within the myocardium was markedly decreased in all zebrafish with pathogenic variants mRNAs. Proliferating cell numbers were also significantly decreased in the endothelial and myocardial regions of zebrafish with ACTA2 variants compared to the wild type. These results demonstrate the effects of ACTA2 G148R and R179H on the development of left ventricle non-compaction and cardiac morphological abnormalities. Our study highlights the previously unknown significance of the ACTA2 gene in several aspects of cardiovascular development.

265 Interactive Effects of Dietary Isoleucine and Valine Ratios to Lysine in Response to Varying Leucine to Lysine Ratios on Pectoralis Major Muscle Protein Expression in Commercial Broilers

Abstract Optimizing the dietary concentrations and ratios of branched-chain amino acids (BCAA) is necessary to optimize broiler chicken growth performance and carcass meat yield. A central composite design (CCD) study was conducted to understand the impact of dietary BCAA concentrations and ratios on Pectoralis major (PM) muscle protein expression involved in mTOR and related protein synthesis pathways as well as and investigate the mechanisms behind how BCAA ratios affect broiler growth and muscle protein accretion. A total of 2,592 d-old Ross 344 × 708 male broilers were randomly placed in 144 floor pens (n = 18 birds per pen). Before the initiation of the experimental finisher phase, all pens were standardized to contain 15 birds per pen. Each pen received 1 of 15 dietary treatments in the 23 CCD with 6 center points from 20 to 35 d of age, varying in digestible ratios of valine:lysine (Val:Lys; 64 to 87), leucine:lysine (Leu:Lys; 110 to 185), and isoleucine:lysine (Ile:Lys; 52 to 75). On d 35, 1 bird per pen was randomly selected for PM protein extraction and proteomic analysis via data independent acquisition protein sequencing with a timsTOF Pro 2 LC/MS/MS and Spectronaut 15 software. Ribosomal protein S6 kinase (RPS6KA3), eukaryotic elongation factor 2 (EEF2), eukaryotic translation initiation factor 4B (EIF4B), eukaryotic translation initiation factor 4E (EIF4E), CAP-Glycine domain containing linker protein 1 (CLIP1), mitogen-activated protein kinase 3 (MAPK3), Protein kinase domain-containing protein (MAP2K1), Dual-specificity mitogen-activated protein kinase kinase 2 (MAP2K2), paxillin (PXN), and regulator complex protein (LAMTOR3) were identified, quantified, and analyzed as a CCD using the RSREG procedure of SAS ver. 9.4 with significance set at P ≤ 0.10. The surface response models for RPS6KA3, EEF2, EIF4B, EIF4E, MAPK3, MAP2K1, MAP2K2, CLIP1, PXN, and LAMTOR3 were not significant (P ≥ 0.926; R2 values ≤ 0.1). No linear (P ≥ 0.5223), quadratic (P ≥ 0.0.634) or cross product (P ≥ 0.6238) regression effects were observed for any of the proteins analyzed. A minimum stationary point was observed for RPS6KA3, EEF2, MAPK3, and LAMTOR3. However, optimal values for the other proteins could not be obtained due to stationary saddle points. Overall, the results in this study indicate that varying concentrations of dietary BCAA may not impact expression of proteins related to broiler skeletal muscle growth on d 35 following the 15-d treatment period, despite the influence on live performance. This is a limitation of this study and suggests that if the mTOR pathway was involved it was not still differentially up- or down-regulated at this time point. Further investigation and new studies with multiple time points will be required to better understand the mechanisms behind how BCAA impact broiler growth and muscle development.

Bioactive Peptides in Meat and Meat Products

A number of bioactive peptides with health benefits have been reported to be generated in meat through the proteolysis phenomena taking place during postmortem aging or further processing. Bioactive peptides consist of short sequences, less than 20 amino acid residues, that are inactive while in the parent protein. These sequences may be activated when they are released from the parent protein by peptidases. Such peptides are released through the hydrolysis of major muscle proteins by muscle endopeptidases, like calpains and cathepsins, and resulting protein fragments and polypeptides are further hydrolyzed by muscle exopeptidases, mainly, tri- and di-peptidylpeptidases, aminopeptidases, and carboxypeptidases. As a result, a variety of peptides with different sequences and lengths and large amounts of free amino acids are generated. The highest amounts of bioactive peptides, and their major health benefits, have been reported in dry-cured ham as a result of the intense proteolysis experienced during the long processing time under mild conditions that favor the action of muscle peptidases. The generated peptides must be bioaccesible and bioavailable to exert their physiological benefit.This means that they must be resistant to gastrointestinal digestion and be absorbed through the intestinal barrier. Some bioactive peptides may be generated through hydrolysis during gastrointestinal digestion. This review is focused on the generation of bioactive peptides in meat and processed meats, on the most relevant bioactivities exerted by such peptides(such as inhibition of angiotensin I–converting enzyme and dipeptidyl peptidase IV as well as antioxidant and anti-inflammatory activity), and on their reported benefits to consumers’health.

The influence of KCl concentration on the gelation of myofibrillar protein giant squid (Dosidicus gigas) due to molecular conformation change.

Protein gelation process is of importance in food industry. The objective of this study is to investigate the influence of salt concentration variation, which induced protein conformation change, on protein's intermolecular interactions and its gelation process. Paramyosin has been separated and purified from myofibrillar protein extracted from giant squid. Then Giant squid's paramyosin molecular mass and intermolecular interactions were quantified by means of light scattering techniques. Finally, the micro-rheology study via diffusing wave spectroscopy (DWS) technique revealed that this conformation change dramatically affected myofibrillar protein gelation process. The obtained apparent molecular weight (ca 2 × 105 g/mol) suggested that protein molecules existed as dimers, while the second virial coefficient A2 significantly reduced from -3.98456 × 10-5 to -5.07575 × 10-4 ml mol/g2 when KCl concentrated from 0.15 to 1 mol/L. Light scattering data also suggest that paramyosin dimers are stiff, with a persistence length of 120 nm, almost the length of a molecule and independent of salt concentration. Mean-square displacement (MSD) of tracer particles at 5 temperatures with 4 salt concentrations displayed that this conformation change had dramatic effect. Therefore, G' and G" were remarkably altered with at least one order of magnitude difference owing to this event occurrence. Paramyosin conformation change due to KCl concentrated enhances attractive interactions with apparent molecular mass increase, which resulted in majority paramyosin molecules (> 99%) in dimeric form and promoted aggregates formation. DWS technique revealed that the conformation change dramatic affected this process characterized by the correlation functions, MSD, and G' and G". This study brings forward data on understanding the effect of a major salt supplement, KCl, on the chemical physics of a major muscle protein.

Effects of High-Volume Versus High-Load Resistance Training on Skeletal Muscle Growth and Molecular Adaptations.

We evaluated the effects of higher-load (HL) versus (lower-load) higher-volume (HV) resistance training on skeletal muscle hypertrophy, strength, and muscle-level molecular adaptations. Trained men (n = 15, age: 23 ± 3 years; training experience: 7 ± 3 years) performed unilateral lower-body training for 6 weeks (3× weekly), where single legs were randomly assigned to HV and HL paradigms. Vastus lateralis (VL) biopsies were obtained prior to study initiation (PRE) as well as 3 days (POST) and 10 days following the last training bout (POSTPR). Body composition and strength tests were performed at each testing session, and biochemical assays were performed on muscle tissue after study completion. Two-way within-subject repeated measures ANOVAs were performed on most dependent variables, and tracer data were compared using dependent samples t-tests. A significant interaction existed for VL muscle cross-sectional area (assessed via magnetic resonance imaging; interaction p = 0.046), where HV increased this metric from PRE to POST (+3.2%, p = 0.018) whereas HL training did not (−0.1%, p = 0.475). Additionally, HL increased leg extensor strength more so than HV training (interaction p = 0.032; HV < HL at POST and POSTPR, p < 0.025 for each). Six-week integrated non-myofibrillar protein synthesis (iNon-MyoPS) rates were also higher in the HV versus HL condition, while no difference between conditions existed for iMyoPS rates. No interactions existed for other strength, VL morphology variables, or the relative abundances of major muscle proteins. Compared to HL training, 6 weeks of HV training in previously trained men optimizes VL hypertrophy in lieu of enhanced iNon-MyoPS rates, and this warrants future research.

Adaptation of the two-dimensional electrophoresis method for canned meat

Studies of the qualitative indicators of canned meat in accordance with regulatory documents are carried out on average samples of specimens, but when studying by proteomic methods, such sampling does not allow high-quality separation of protein components due to the high fat content in the product. When two-dimensional electrophoresis was carried out on an average sample, fragments of the main muscle and connective tissue proteins of beef were found in small quantities, but the electrophoretogram was not very informative. A significantly better separation was achieved after removing the fat fraction from the product. When studying broth from canned meat, the largest amount of intensely coloured high-molecular-weight protein fractions with a mass of more than 50 kDa was revealed. The electrophoretogram of the meat pieces showed a wide range of proteins across the entire molecular weight range of the polyacrylamide gel, including major muscle proteins. The study of broth together with meat pieces but after fat removal is optimal for the primary screening of the protein component of canned meat.

Effects of dietary energy levels on Pectoralis major mixed muscle protein turnover and body composition in two broiler lines housed in different grow-out environments.

This study determined the Pectoralis (P) major mixed muscle protein turnover (PT) in two meat broiler lines, Line A and Line B, during the finishing grow-out feeding period (21-42days) as affected by the dietary metabolizable energy (ME) levels and ambient temperatures. Experimental finishing diets consisted of 80, 90, 100, 110 and 120% ME of recommended nutrient guidelines for energy level. Fractional synthesis rates (FSR) or fractional degradation rates (FDR) were measured in P.major at day 36 and 42. Protein and fat mass gain were measured, and respective energy retention efficiencies as protein and fat (EREp and EREf) were determined. Metabolic heat production (HP) was also reported. Experimental feeding studies were conducted in cool season (24hr mean: 69.91˚F and 63.98% RH) and in hot season (24hr mean: 77.55˚F and 86.04% RH). Results showed that FSR or FDR values were not affected by dietary ME levels at day 36, whereas reduced FSR (p<.05) were observed at day 42 fed diets with reduced ME levels (≤100% ME) which could have resulted from greater maintenance energy requirement of maturing broilers at that age. Broilers fed reduced ME diets (≤100% ME) maintained protein mass (equivalent to broilers fed ≥100%-120% ME) by reduced FDR and increased feed intake. Grow-out ambient temperature did not affect FSR or FDR values across ME levels. Line B retained higher protein mass, lower fat mass and greater HP compared to Line A. This was followed by higher feed intake in Line B. Further, Line B exhibited higher EREp and lower EREf across dietary ME levels. In summary, PT homeostasis and body composition changes in broiler lines studied seemed to be regulated by the birds' intent to normalize energy intake as per physiological need by controlling feed intake.

Effect of meat temperature on moisture loss, water properties, and protein profiles of broiler pectoralis major with the woody breast condition

The objective of this study was to investigate the influence of meat temperature on moisture loss, muscle water properties, and protein profiles in broiler pectoralis major with the severe woody breast (WB) condition. Broiler breast samples were collected from a commercial plant and sorted into normal, WB, and pale, soft, and exudative (PSE). Temperature treatments included 23°C, 40°C, 53°C, 57°C, 68°C, and 90°C based on denaturation of major muscle protein types during heating. Moisture loss was estimated with weight changes, water properties were measured with time-domain nuclear magnetic resonance measurements, and protein profiles were determined with SDS electrophoresis gel. There were no differences in moisture loss between 3 groups at meat temperature 23°C, 40°C, and 57°C. Moisture loss of WB samples was greater than normal and PSE at either 68°C or 90°C; however, it was the least at 53°C. Only close changing trend was noted between the intramyofibrillar water (T21) reduction and moisture loss. The extramyofibrillar (T22) water content and reduction in WB meat during heating were consistent greater, and electrophoretic profiles differed among 3 muscle conditions. Data suggest that greater reductions in intramyofibrillar and extramyofibrillar water are responsible for the increased moisture loss in WB meat at higher temperature.

Sturgeon protein-derived peptides exert anti-inflammatory effects in LPS-stimulated RAW264.7 macrophages via the MAPK pathway

• KIWHHTF, VHYAGTVDY and HLDDALRGQE are novel anti-inflammatory peptides derived from sturgeon muscle. • Three novel peptides suppress the release of NO、IL-1β, and IL-6. • Three novel peptides increase the SOD activity in the LPS-induced RAW264.7 cells. • Three novel peptides can down-regulate the MAPK pathway. Previous studies have demonstrated that polypeptides extracted from fish protein possess potential anti-inflammatory effects, but few studies have reported the underlying anti-inflammatory mechanism of sturgeon peptides. Hence, the goal of this study was to explore the peptide sequences with potential anti-inflammatory activity derived from sturgeon muscle protein in the lipopolysaccharide (LPS)-induced RAW264.7 inflammatory model. Fourteen novel peptides were identified in major muscle proteins by mass spectrometry, and three synthetic peptides – KIWHHTF, VHYAGTVDY, and HLDDALRGQE - decreased the release of inflammatory mediators and inflammatory cytokines (NO, IL-6, and IL-1β), while increasing SOD activity significantly in the LPS-induced RAW264.7 inflammatory model. Western blotting analysis revealed that these peptides suppressed the phosphorylation levels of MAPKs, indicating that these peptides exerted anti-inflammatory effects by inhibiting the MAPK pathway. The novel peptides potentially have application prospects as functional food ingredients.

Bioactive peptides generated in the processing of dry-cured ham

Peptides and free amino acids are naturally generated in dry-cured ham as a consequence of proteolysis phenomenon exerted by muscle peptidases. The generation of bioactive peptides in different types of dry-cured ham produced in Spain, Italy and China is reviewed in this manuscript. Major muscle proteins are extensively hydrolysed firstly by endogenous endo-peptidases followed by the successive action of exo-peptidases, mainly, tri- and di-peptidylpeptidases, aminopeptidases and carboxypeptidases. Such proteolysis is very intense and consists of the generation of large amounts of free amino acids and a good number of peptides with different sequences and lengths, some of them exerting relevant bioactivities like angiotensin converting enzyme inhibitory activity, antioxidant activity, di-peptidylpeptidase IV inhibitory activity among other and in vivo antihypertensive, hypoglycemic or anti-inflammatory activity. This manuscript reviews the recent findings showing that dry-cured ham constitutes a good source of natural bioactive peptides that have potential benefit for human health.

Effects of T-2 toxin on the muscle proteins of shrimp (Litopenaeus vannamei)-a proteomics study.

T-2 toxin (T-2) is a potent mycotoxin and a common contaminant of aquatic animal feed, posing a serious risk to health and aquatic animals. We investigated the effect of T-2 on shrimp muscle proteins using proteomics and conventional biochemical methods. Shrimp were fed a diet containing T-2 at 0-12.2mg kg-1 for 20 days, and changes to the muscle protein composition, ATPase activities, and the sulfhydryl (SH) content and hydrophobicity of actomyosin (AM) were determined. A proteomics study of the proteins was conducted with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional (2D) electrophoresis, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS). Exposure to T-2 markedly affected the muscle protein composition of shrimp in a concentration-responsive manner that displayed a diphasic effect. At a low T-2 concentration (1.2mg kg-1 ), the levels of three major muscle proteins (myofibrillar, sarcoplasmic, and stroma) increased but at higher concentrations they declined progressively. T-2 exposure also led to a breakdown of muscle proteins as evidenced by increases in alkali-soluble protein and the surface hydrophobicity (SoANS) of AM. Thirty differentially expressed proteins were detected, 12 of which showed a concentration-response relationship with T-2 exposure. Among them, 11 homologous proteins were identified by mass spectrometry (MS), with several being key enzymes in energy metabolism. This study demonstrated that T-2 exposure at medium to high concentrations could significantly affect the protein composition and quality of shrimp muscle, and potentially some of its key metabolisms. One of the arginine kinases (spot 27) was particularly responsive to T-2 and could potentially be used as a biomarker protein for T-2 intoxication by shrimp. © 2019 Society of Chemical Industry.

The effect of sexual maturity and egg production on skeletal muscle (pectoralis major and gastrocnemius) protein turnover in broiler breeder pure lines

A study was conducted to evaluate the effect of sexual maturity on pectoralis major and gastrocnemius muscle protein turnover in broiler breeder pure lines. Protein turnover in skeletal muscle tissue was determined in 4 broiler breeder pure lines (Line A, Line B, Line C and Line D) at 22, 27, 33, 37, 44, and 50 wk of age. A completely randomized design with a factorial arrangement 4 × 6 (4 lines and 6 time periods (ages)) was used. There were 5 replicates per line/time and each hen represented a replicate. Five hens/line at each age were given an intravenous flooding-dose of 15 N-phenylalanine (150 mM, 40 atom percent excess (APE) at a dose of 10 mL/kg. After 10 min, birds were euthanized using CO2 asphyxiation and the breast and leg muscle excised and snap frozen in liquid nitrogen for protein turnover analysis. Excreta was collected from each breeder for 3-methyl histidine (3-MH) analysis. There was a significant age effect for the breast muscle fractional synthesis rate (FSR), but no main effects (age and line) for leg muscle FSR. The FSR in breast muscle tissue decreased in hens from wk 22 (first egg) to wk 33 (peak egg production). There was a significant age effect on fractional breakdown rate (FBR) in breast and leg muscle. The FBR in breast muscle increased in hens from wk 22 to wk 33 and remained high through wk 37. Breast muscle FBR significantly decreased in hens from wk 37 to wk 50. The FBR in leg muscle tissue increased in hens from wk 33 to wk 37 and then decreased at wk 50. No line effect was seen for FSR or FBR. There is a large increase in skeletal muscle FBR during the transition for the pullet to sexual maturity with increases in skeletal muscle FBR in the breast and leg muscle through peak egg production. Protein turnover in skeletal muscle tissue is believed to be a source of nutrients for egg production.

Purification and identification of anti-inflammatory peptides from spent hen muscle proteins hydrolysate

Spent hens have low market value and incur significant costs for disposal. This study explored the use of spent hens as a source of bioactive peptides. Spent hen hydrolysates prepared by Protease M or Protex 50FP exhibited interleukin (IL)-6 inhibitory activity in endotoxin-activated macrophage-like U937 cells (p < .05). The potential peptides from Protex 50FP hydrolysate were further fractionated using a combination of ultrafiltration, solid-phase extraction and high-performance liquid chromatography; 17 novel peptides encoded in major muscle proteins were identified by mass spectrometry analysis, of which 7 were chemically synthesized and assayed for the IL-6 inhibitory activity. At a concentration of 100 µg/mL, peptide FLWGKSY induced a 79% reduction of IL-6 production in endotoxin-activated macrophage-like U937 cells, which is comparable to results reported from other food sources. Our results indicate that spent hens have potential to be a source of bioactive peptides for anti-inflammatory applications.

Transcriptome analysis of IFM-specific actin and myosin nulls in Drosophila melanogaster unravels lesion-specific expression blueprints across muscle mutations

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres. Mutations in many genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue. However, the pathways underlying muscle degeneration, and manifestation of myopathy phenotypes are not well understood. In this study, we explored transcriptional alterations that ensue from the absence of the two major muscle proteins – myosin and actin - using the Drosophila indirect flight muscles. Our aim was to understand how the muscle tissue responds as a whole to the absence of either of the major scaffold proteins, whether the responses are generic to the tissue; or unique to the thick versus thin filament systems. Our results indicated that muscles respond by altering gene transcriptional levels in multiple systems active in muscle remodelling, protein degradation and heat shock responses. However, there were some responses that were filament-specific signatures of muscle degeneration, like immune responses, metabolic alterations and alterations in expression of muscle structural genes and mitochondrial ribosomal genes. These general and filament-specific changes in gene expression may be of relevance to human myopathies.

Solubilisation of myosin in a solution of low ionic strength l-histidine: Significance of the imidazole ring

Myosin, a major muscle protein, can be solubilised in a low ionic strength solution containing l-histidine (His). To elucidate which chemical constituents in His are responsible for this solubilisation, we investigated the effects of 5mM His, imidazole (Imi), l-α-alanine (Ala), 1-methyl-l-histidine (M-his) and l-carnosine (Car) on particle properties of myosin suspensions and conformational characteristics of soluble myosin at low ionic strength (1mM KCl, pH 7.5). His, Imi and Car, each containing an imidazole ring, were able to induce a myosin suspension, which had small particle size species and high absolute zeta potential, thus increasing the solubility of myosin. His, Imi and Car affected the tertiary structure and decreased the α-helix content of soluble myosin. Therefore, the imidazole ring of His appeared to be the significant chemical constituent in solubilising myosin at low ionic strength solution, presumably by affecting its secondary structure.

Subnanometer Structure of the Actin/Myosin/Tropomyosin Complex

Actins and myosins play a central role in life as they form the molecular motors that transform chemical energy into movement. Without them, we would be confined to a very static existence.To fully understand how the interplay between actin and myosin leads to the conversion of the energy stored in ATP into mechanical energy, one needs structural information. Therefore, we have set out to determine the complex structure of the three major muscle proteins (actin, myosin and tropomyosin) using cryo electron microscopy and iterative helical real-space reconstruction. Using state of the art instruments and reconstruction algorithms we have determined a sub-nanometer resolution electron density map of the complex. With the help of electron-density guided flexible fitting we have subsequently refined the structure to obtain a quasi-atomic resolution structure of the full complex which enabled us, for the first time, to visualize interactions between actin and myosin that have been postulated by computational methods beforehand. In addition, we could also identify a novel interaction between myosin and tropomyosin.

Nebulin Alters Cross-bridge Cycling Kinetics and Increases Thin Filament Activation: A NOVEL MECHANISM FOR INCREASING TENSION AND REDUCING TENSION COST

Nebulin is a giant filamentous F-actin-binding protein ( approximately 800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (-log[Ca(2+)]) and force-ATPase relations, as well as the rate of tension re-development (k(tr)) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons(-1) mm(-1) s(-1)) and reductions in k(tr) (4.7 versus 7.3 s(-1)), calcium sensitivity (pCa(50) 5.74 versus 5.90), and cooperativity of activation (n(H) 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.

Lectin-based proteomic profiling of aged skeletal muscle: Decreased pyruvate kinase isozyme M1 exhibits drastically increased levels of N-glycosylation

Since various neuromuscular diseases are associated with abnormal glycosylation, it was of interest to determine whether this key post-translational modification is also altered in aged skeletal muscle. Lectins represent highly versatile carbohydrate-binding proteins that are routinely employed for the characterization of glycoproteins. Here, we used the lectin wheat germ agglutinin (WGA) for the proteomic profiling of senescent fibers. WGA labeling of the soluble proteome from 3-month- versus 30-month-old rat gastrocnemius muscle, following two-dimensional gel electrophoretic separation, resulted in the identification of 13 distinct protein species. Analysis of WGA binding levels, in conjunction with mass spectrometric fingerprinting, revealed that one isoform of a major metabolic muscle protein exhibited a drastic alteration in the content of sialic acid and N-acetylglucosaminyl sugar residues. Pyruvate kinase isoform M1 with protein accession number gi|16757994|, exhibiting a pI of 6.6 and an apparent molecular mass of 57.8 kDa, showed a six fold increase in N-glycosylation and a three fold decrease in protein expression. In contrast to comparable levels of N-glycosylated proteins in young adult versus senescent muscle, as judged by fluorescein-conjugated WGA labeling of transverse muscle cryosections, staining with antibodies to the M1 isoform of pyruvate kinase showed reduced expression of this cytosolic element. Furthermore, activity assays demonstrated a reduced activity of this glycolytic enzyme in senescent muscle. This agrees with the idea that abnormal post-translational modifications in key metabolic enzymes may be involved in the conversion of aged muscle to slower twitch patterns and a drastic shift to more aerobic-oxidative metabolism.

A Proteomic Assessment of Muscle Contractile Alterations during Unloading and Reloading

Unloading of skeletal muscle causes atrophy and altered contractility. To identify major muscle proteins responding significantly to the altered loading and to elucidate how the contractile alterations reflect potential proteomic modifications, we examined protein expression in the rat soleus muscle during 3-week hindlimb suspension and 2-week reloading. Compared with unsuspended controls, experimental animals had a 0.5- to 0.6-fold decrease in tension during unloading and early reloading, comparable to 0.2- to 0.6-fold decreases in the protein levels of myosin light chain 1 (MLC1), alpha-actin, tropomyosin beta-chain, and troponins T1 and T2. The observed 1.4- to 1.6-fold increase in shortening velocity appears to reflect 1.2- to 9.0-fold increases in the protein levels of fast-type MLC2, glycolytic enzymes, and creatine kinase, and 0.2- to 0.3-fold decreases in slow-type troponins T1 and T2. The levels of three heat shock proteins (p20, alpha crystallin B chain, and HSP90) decreased during unloading but returned to control levels during reloading. These results imply that proteomic responses to unloading change overall myofibrillar integrity and metabolic regulation, resulting in altered contractility.

Thermal properties of titin from porcine and bovine muscles

The thermal properties of titin isolated from porcine and bovine longissimus muscles were investigated by differential scanning calorimetry in the temperature range from 20 to 100 °C. A single peak with average maximum temperatures of 75.6 and 78.4 °C characterized porcine and bovine titin denaturation, respectively. The peaks were much broader than those from the other major muscle proteins. Titin denaturation enthalpy values (1.6–2.6 J/g) were only about half those of whole meat and also lower than those previously determined for myosin, actin, or collagen. The relatively high titin denaturation temperature suggests that it may be partially responsible for meat toughening when muscle tissue is heated above 60 °C.