Myosin Heavy Chain Content Research Articles

Effect of thermal and defatting treatments on the composition, protein profile and structure of house cricket (Acheta domesticus) protein extracts

This study investigated the impact of blanching (100 °C, 40 s), defatting method (maceration, Soxhlet) and solvent polarity (hexane, ethanol) on the profile, structure and solubility of house cricket protein extracts. Blanching and Soxhlet using ethanol impacted the protein profile, with a lower content of myosin heavy chain and a higher abundance of low molecular weight proteins (<25 kDa). Moreover, ethanol induced aggregation of non-blanched cricket proteins, with a 13–72% reduction in protein recovery yield. The protein secondary structure of non-blanched extracts was also affected by ethanol with 18% more β-sheets. Furthermore, blanching resulted in a lower protein surface hydrophobicity by a factor of 3 to 7, with no impact of solvent polarity. Finally, the solubility of protein extracts remained >75%, regardless of the blanching and defatting methods. These findings, combined with the evaluation of techno-functional properties, could be used for the development of cricket-based protein ingredients for food formulations.

Fluorescent characterization of differentiated myotubes using flow cytometry.

Flow cytometry is routinely used in the assessment of skeletal muscle progenitor cell (myoblast) populations. However, a full gating strategy, inclusive of difficult to interpret forward and side scatter data, which documents cytometric analysis of differentiated myoblasts (myotubes) has not been reported. Beyond changes in size and shape, there are substantial metabolic and protein changes in myotubes allowing for their potential identification within heterogenous cell suspensions. To establish the utility of flow cytometry for determination of myoblasts and myotubes, C2C12 murine cell populations were assessed for cell morphology and metabolic reprogramming. Laser scatter, both forward (FSC; size) and side (SSC; granularity), measured cell morphology, while mitochondrial mass, reactive oxygen species (ROS) generation and DNA content were quantified using the fluorescent probes, MitoTracker green, CM-H2DCFDA and Vybrant DyeCycle, respectively. Immunophenotyping for myosin heavy chain (MyHC) was utilized to confirm myotube differentiation. Cellular viability was determined using Annexin V/propidium iodide dual labelling. Fluorescent microscopy was employed to visualize fluorescence and morphology. Myotube and myoblast populations were resolvable through non-intuitive interpretation of laser scatter-based morphology assessment and mitochondrial mass and activity assessment. Myotubes appeared to have similar sizes to the myoblasts based on laser scatter but exhibited greater mitochondrial mass (159%, p < 0.0001), ROS production (303%, p < 0.0001), DNA content (18%, p < 0.001) and expression of MyHC (147%, p < 0.001) compared to myoblasts. Myotube sub-populations contained a larger viable cluster of cells which were unable to be fractionated from myoblast populations and a smaller population cluster which likely contains apoptotic bodies. Imaging of differentiated myoblasts that had transited through the flow cytometer revealed the presence of intact, 'rolled-up' myotubes, which would alter laser scatter properties and potential transit through the laser beam. Our results indicate that myotubes can be analyzed successfully using flow cytometry. Increased mitochondrial mass, ROS and DNA content are key features that correlate with MyHC expression but due to myotubes 'rolling up' during flow cytometric analysis, laser scatter determination of size is not positively correlated; a phenomenon observed with some size determination particles and related to surface properties of said particles. We also note a greater heterogeneity of myotubes compared to myoblasts as evidenced by the 2 distinct sub-populations. We suggest that acoustic focussing may prove effective in identifying myotube sub populations compared to traditional hydrodynamic focussing.

Cathepsin S activity controls chronic stress-induced muscle atrophy and dysfunction in mice

Exposure to chronic psychological stress (CPS) is an intractable risk factor for inflammatory and metabolic diseases. Lysosomal cysteinyl cathepsins play an important role in human pathobiology. Given that cathepsin S (CTSS) is upregulated in the stressed vascular and adipose tissues, we investigated whether CTSS participates in chronic stress-induced skeletal muscle mass loss and dysfunction, with a special focus on muscle protein metabolic imbalance and apoptosis. Eight-week-old male wildtype (CTSS+/+) and CTSS-knockout (CTSS−/−) mice were randomly assigned to non-stress and variable-stress groups. CTSS+/+ stressed mice showed significant losses of muscle mass, dysfunction, and fiber area, plus significant mitochondrial damage. In this setting, stressed muscle in CTSS+/+ mice presented harmful alterations in the levels of insulin receptor substrate 2 protein content (IRS-2), phospho-phosphatidylinositol 3-kinase, phospho-protein kinase B, and phospho-mammalian target of rapamycin, forkhead box-1, muscle RING-finger protein-1 protein, mitochondrial biogenesis-related peroxisome proliferator-activated receptor-γ coactivator-α, and apoptosis-related B-cell lymphoma 2 and cleaved caspase-3; these alterations were prevented by CTSS deletion. Pharmacological CTSS inhibition mimics its genetic deficiency-mediated muscle benefits. In C2C12 cells, CTSS silencing prevented stressed serum- and oxidative stress-induced IRS-2 protein reduction, loss of the myotube myosin heavy chain content, and apoptosis accompanied by a rectification of investigated molecular harmful changes; these changes were accelerated by CTSS overexpression. These findings demonstrated that CTSS plays a role in IRS-2-related protein anabolism and catabolism and cell apoptosis in stress-induced muscle wasting, suggesting a novel therapeutic strategy for the control of chronic stress-related muscle disease in mice under our experimental conditions by regulating CTSS activity.

Impacts of myosin heavy chain phenotypes on recovery of leg extension force after ACL-reconstructed knee

It is well known that myosin heavy chain (MyHC) phenotypes impact skeletal muscle function. Anterior cruciate ligament (ACL) tear, a common sport injury, induces skeletal muscle atrophy around the injured knee. However, the interrelationship between MyHCs and the functional recovery of ACL reconstruction surgery (ACL-R) remains unclear. The purpose of this study was to investigate the interrelationship between MyHC phenotypes and the functional recovery of leg extension force in patients with ACL-R. Under a clinical setting, 27 patients with ACL-R participated in this study. ACL-R in each patient was carried out using a semitendinous (ST) tendon autograft. During ACL-R, a small muscle sample was dissected from the ST tendon of each patient. MyHC phenotypes in the muscle sample were evaluated using SDS-PAGE. The leg extension force was evaluated 1 day before and then 3, 6, and 12 months after ACL-R. Based on the dominant (>40%) MyHC isoforms in the ST muscle, patients were classified into 4 groups as follows: Groups I, IIa, IId/x, and Even (E: all MyHC isoforms less than 40%). There is a positive correlation between the relative content of MyHC II (IIa + IId/x) and the leg extension force before ACL-R. Functional recovery of knee in subjects with ACL-R was facilitated in patients having the dominant MyHC IIa. This study suggests that the analysis of MyHC phenotypes in ST muscle dissected during ACL-R is a clinically useful index for the functional assessment of the knee in subjects with ACL injury.

Revisiting specific force loss in human permeabilised single skeletal muscle fibres obtained from older individuals.

Specific force (SF) has been shown to be reduced in some but not all studies of human ageing using chemically skinned single muscle fibres. This may be due in part to the health status/physical activity levels of different older cohorts, but also from methodological differences in studying skinned fibres. The aim of the present study was to compare SF in fibres from older hip fracture patients (HFP), healthy master cyclists (MC) and healthy non-trained young adults (YA) using two different activating solutions. Quadriceps muscle samples and 316 fibres were obtained from HFPs (74.6±4 years, n=5), MCs (74.8±1, n=5) and YA (25.5±2, n=6). Fibres were activated (pCa 4.5, 15oC) in solutions containing either 60 mM TES or 20 mM imidazole. SF was determined by normalising force to fibre CSA assuming either an elliptical or circular shape and to fibre myosin heavy chain content. Activation in TES resulted in significantly higher MHC-I SF in all groups and YA MHC-IIA fibres, irrespective of normalisation method. Whilst there were no differences in SF between the participant groups, the ratio of SF between the TES and imidazole solutions was lower in HFPs compared to YAs (MHC-I p<0.05; MHC-IIA p=0.055). Activating solution composition, as opposed to donor characteristics, had a more notable effect on single fibre SF. However, this two-solution approach revealed an age-related difference in sensitivity in HFPs which was not shown in MCs. This suggests further novel approaches may be required to probe age/activity-related differences in muscle contractile quality.

Effects of ultrasound on the oxidation and structures of the myofibrillar protein in the presence or absence of soybean oil.

Ultrasound is widely used as a novel non-thermal processing technique to improve protein properties. In recent decades, applying ultrasound-assisted emulsification (UAE) to produce protein-stabilized emulsion has attracted people's attention. Instead of applying ultrasound to treat a single protein solution, UAE treatment refers to the use of sonication to a mixture of protein and oil. The purpose of this study was to compare the different effects of ultrasound treatment on the properties of the myofibrillar protein (MP) in the presence or absence of soybean oil. A suitable sonication power was selected based on the change in emulsion properties. The 300W sonication power was selected due to its most effectively decreased emulsion droplet size and increased absolute zeta potential. Sonication more significantly increased the protein carbonyl content and disulfide bonds of the MP-soybean oil sample than MP sample. Due to the existence of oil, ultrasound could unfold more protein molecules illustrated by a lower α-helix content and intrinsic fluorescence intensity, and a higher surface hydrophobicity. LC-MS/MS results illustrated that sonication enhanced the myosin heavy chain and actin content at the soybean oil interface as well as accelerated the myosin light chain to separate from myosin in the MP-soybean oil system. In summary, ultrasound treatment could lead to a higher level of protein oxidation and more protein molecule exposure in the MP with the presence of oil system than in the oil-free MP system. This article is protected by copyright. All rights reserved.

Human and African ape myosin heavy chain content and the evolution of hominin skeletal muscle

Humans are unique among terrestrial mammals in our manner of walking and running, reflecting 7 to 8 Ma of musculoskeletal evolution since diverging with the genus Pan. One component of this is a shift in our skeletal muscle biology towards a predominance of myosin heavy chain (MyHC) I isoforms (i.e. slow fibers) across our pelvis and lower limbs, which distinguishes us from chimpanzees. Here, new MyHC data from 35 pelvis and hind limb muscles of a Western gorilla (Gorilla gorilla) are presented. These data are combined with a similar chimpanzee dataset to assess the MyHC I content of humans in comparison to African apes (chimpanzees and gorillas) and other terrestrial mammals. The responsiveness of human skeletal muscle to behavioral interventions is also compared to the human-African ape differential. Humans are distinct from African apes and among a small group of terrestrial mammals whose pelvis and lower limb muscle is slow fiber dominant, on average. Behavioral interventions, including immobilization, bed rest, spaceflight and exercise, can induce modest decreases and increases in human MyHC I content (i.e. -9.3% to 2.3%, n = 2033 subjects), but these shifts are much smaller than the mean human-African ape differential (i.e. 31%). Taken together, these results indicate muscle fiber content is likely an evolvable trait under selection in the hominin lineage. As such, we highlight potential targets of selection in the genome (e.g. regions that regulate MyHC content) that may play an important role in hominin skeletal muscle evolution.

The dynamic change in the degradation and in vitro digestive properties of porcine myofibrillar protein during freezing storage

The myofibrillar protein (MP) degradation and in vitro digestive properties of porcine longissimus during freezing at −8, −18, −25 and − 40 °C for 1, 3, 6, 9 and 12 months were investigated. As the freezing temperature and duration of frozen storage increased, the amino nitrogen and TCA (trichloroacetic acid)-soluble peptides of the samples were significantly increased, while the total sulfhydryl content and band intensity of myosin heavy chain, actin, troponin T, tropomyosin were significantly decreased (P < 0.05). At higher freezing storage temperatures and durations, the particle size of MP samples and the green fluorescent spots detected using a laser particle size analyzer and confocal laser scanning microscopy became large. After 12 months of freezing, the digestibility and the degree of hydrolysis of the trypsin digestion solution of the samples frozen at −8 °C were significantly decreased by 15.02 % and 14.28 %, respectively, when compared to fresh samples, whereas, the mean surface diameter (d3,2) and mean volume diameter (d4,3) were significantly increased by 14.97 % and 21.53 %, respectively. Therefore, frozen storage induced protein degradation and impaired the ability of digestion in the pork proteins. This phenomenon was more evident as the samples were frozen at high temperatures over a long storage period.

Peptidomics insights into the interplay between the pre-digestion effect of mixed starters and the digestive pattern of sausage proteins

This work investigated the influence of mixed starters (Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201) on the digestive pattern of dry fermented sausage proteins. The addition of starters resulted in a higher degree of proteolysis and looser protein conformation as evidenced by the decreased carbonyl content and electrophoresis bands intensity of myosin heavy chain (MHC) and actin, increased sulfhydryl content and surface hydrophobicity, and transformation of protein secondary structure from α-helix to β-sheet and β-turn. These changes induced by the starters acted as a “pre-digestion” step and improved the digestive pattern of sausage protein. The most remarkable improvement in gastric (33.98%–49.46%) and whole gastrointestinal digestibility (87.41%–94.57%) was observed on sausages of day 21. Comparative peptidomics analysis during digestion revealed that the digested peptides of the inoculated sample were more widely distributed throughout the 3D structure of the sausage proteins, namely, myoglobin, MHC, and creatine kinase M-type. More peptides were degraded in the rod region (780–1933) of MHC. The region was possibly expanded by starters. New bioactive peptides, including MNVKHWPWMK, FFL, and LLF, were released from the MHC and actin altered by the starters. This work highlighted the application of mixed starters as a potential tool to enhance the nutrition of fermented sausage.

Effect of Heating on Protein Denaturation, Water State, Microstructure, and Textural Properties of Antarctic Krill (Euphausia superba) Meat

Antarctic krill (Euphausia superba) is an important source of biomass and high-quality protein. However, heat treatment negatively impacts the quality of Antarctic krill and compromises its utilization in the food industry. This study aimed to investigate the mechanisms underlying changes in Antarctic krill meat characteristics and physicochemical properties treated at different temperatures and holding times. Our findings indicated that hardness and cooking loss of Antarctic krill meat increased dramatically at higher temperatures and holding times. The low-field nuclear magnetic resonance (LF-NMR) analysis revealed that the loss of immobilized water increased, whereas the SDS-PAGE analysis showed that the content of myosin heavy chain decreased significantly and that protein degradation occurred. Fourier transform infrared spectroscopy (FT-IR) and intrinsic fluorescence spectra suggested that α-helix motifs were transformed into β-sheets and that more hydrophobic groups were exposed. The scanning electron microscopy (SEM) results showed that Antarctic krill meat formed corrugated folded regions after heat treatment without forming a three-dimensional water-entrapped structure, which led to significant water loss, resulting in rapid deterioration of Antarctic krill meat. These results provide the basis for a deeper understanding of the processing characteristics of Antarctic krill meat.

Identification of enhancers responsible for the coordinated expression of myosin heavy chain isoforms in skeletal muscle

BackgroundSkeletal muscles consist of fibers of differing contractility and metabolic properties, which are primarily determined by the content of myosin heavy chain (MYH) isoforms (MYH7, MYH2, MYH1, and MYH4). The regulation of Myh genes transcription depends on three-dimensional chromatin conformation interaction, but the mechanistic details remain to be determined.ResultsIn this study, we characterized the interaction profiles of Myh genes using 4C-seq (circular chromosome conformation capture coupled to high-throughput sequencing). The interaction profile of Myh genes changed between fast quadriceps and slow soleus muscles. Combining chromatin immunoprecipitation-sequencing (ChIP-seq) and transposase accessible chromatin with high-throughput sequencing (ATAC-seq), we found that a 38 kb intergenic region interacting simultaneously with fast Myh genes promoters controlled the coordinated expression of fast Myh genes. We also identified four active enhancers of Myh7, and revealed that binding of MYOG and MYOD increased the activity of Myh7 enhancers.ConclusionsThis study provides new insight into the chromatin interactions that regulate Myh genes expression.

Cathepsin K activity controls cachexia-induced muscle atrophy via the modulation of IRS1 ubiquitination.

BackgroundCachexia is a complicated metabolic disorder that is characterize by progressive atrophy of skeletal muscle. Cathepsin K (CTSK) is a widely expressed cysteine protease that has garnered attention because of its enzymatic and non‐enzymatic functions in signalling in various pathological conditions. Here, we examined whether CTSK participates in cancer‐induced skeletal muscle loss and dysfunction, focusing on protein metabolic imbalance.MethodsMale 9‐week‐old wild‐type (CTSK+/+, n = 10) and CTSK‐knockout (CTSK−/−, n = 10) mice were injected subcutaneously with Lewis lung carcinoma cells (LLC; 5 × 105) or saline, respectively. The mice were then subjected to muscle mass and muscle function measurements. HE staining, immunostaining, quantitative polymerase chain reaction, enzyme‐linked immunosorbent assay, and western blotting were used to explore the CTSK expression and IRS1/Akt pathway in the gastrocnemius muscle at various time points. In vitro measurements included CTSK expression, IRS1/Akt pathway‐related target molecule expressions, and the diameter of C2C12 myotubes with or without LLC‐conditioned medium (LCM). An IRS1 ubiquitin assay, and truncation, co‐immunoprecipitation, and co‐localization experiments were also performed.ResultsCTSK+/+ cachectic animals exhibited loss of skeletal muscle mass (muscle weight loss of 15%, n = 10, P < 0.01), muscle dysfunction (grip strength loss > 15%, n = 10, P < 0.01), and fibre area (average area reduction > 30%, n = 5, P < 0.01). Compared with that of non‐cachectic CTSK+/+ mice, the skeletal muscle of cachectic CTSK+/+ mice exhibited greater degradation of insulin receptor substrate 1 (IRS1, P < 0.01). In this setting, cachectic muscles exhibited decreases in the phosphorylation levels of protein kinase B (Akt308, P < 0.01; Akt473, P < 0.05) and anabolic‐related proteins (the mammalian target of rapamycin, P < 0.01) and increased levels of catabolism‐related proteins (muscle RING‐finger protein‐1, P < 0.01; MAFbx1, P < 0.01) in CTSK+/+ mice (n = 3). Although there was no difference in LLC tumour growth (n = 10, P = 0.44), CTSK deletion mitigated the IRS1 degradation, loss of the skeletal muscle mass (n = 10, P < 0.01), and dysfunction (n = 10, P < 0.01). In vitro, CTSK silencing prevented the IRS1 ubiquitination and loss of the myotube myosin heavy chain content (P < 0.01) induced by LCM, and these changes were accelerated by CTSK overexpression even without LCM. Immunoprecipitation showed that CTSK selectively acted on IRS1 in the region of amino acids 268 to 574. The results of co‐transfection of IRS1‐N‐FLAG or IRS1‐C‐FLAG with CTSK suggested that CTSK selectively cleaves IRS1 and causes ubiquitination‐related degradation of IRS1.ConclusionsThese results demonstrate that CTSK plays a novel role in IRS1 ubiquitination in LLC‐induced muscle wasting, and suggest that CTSK could be an effective therapeutic target for cancer‐related cachexia.

Contractility and myosin heavy chain content of skeletal muscle engineered from adult and aged rats

Functional three-dimensional skeletal muscle constructs (myooids) were engineered from myogenic cells harvested from the muscles of adult and aged rats. Myooids formed by self-organization of myogenic cells in the absence of an artificial scaffold and were maintained in culture. Myooid excitability and contractility were measured at days 1, 3, 7, and 24 after myooid formation. Skeletal muscle myosin heavy chain (MHC) content was measured and MHC isoforms were separated on SDS-PAGE gels and quantified. Adult and aged rat myooids had only ~35-60% of the skeletal muscle MHC content of control skeletal muscle from rats, the remaining MHC content consisting of isoforms found in cultured fibroblasts but not in control skeletal muscle. In addition, myooids expressed only developmental isoforms of skeletal muscle MHC, known to generate less specific force than adult isoforms. Myooids from aged rats generated greater normalized force than myooids from adult rats, but only 1-3% of that for adult skeletal muscle controls. Normalizing for skeletal muscle MHC content and isoform expression predicts myooid forces of ~6-18% of control adult skeletal muscles. We hypothesize that the remaining force deficit is due to cellular and sub-cellular disorganization—Myooids lack the density and organization of sarcomeric arrays seen in skeletal muscle.

Early movement restriction deteriorates motor function and soleus muscle physiology

Children with low physical activity and interactions with environment experience atypical sensorimotor development and maturation leading to anatomical and functional disorganization of the sensorimotor circuitry and also to enduring altered motor function. Previous data have shown that postnatal movement restriction in rats results in locomotor disturbances, functional disorganization and hyperexcitability of the hind limb representations in the somatosensory and motor cortices, without apparent brain damage. Due to the reciprocal interplay between the nervous system and muscle, it is difficult to determine whether muscle alteration is the cause or the result of the altered sensorimotor behavior (Canu et al., 2019). In the present paper, our objectives were to evaluate the impact of early movement restriction leading to sensorimotor restriction (SMR) during development on the postural soleus muscle and on sensorimotor performance in rats, and to determine whether changes were reversed when typical activity was resumed. Rats were submitted to SMR by hind limb immobilization for 16 h / day from birth to postnatal day 28 (PND28). In situ isometric contractile properties of soleus muscle, fiber cross sectional area (CSA) and myosin heavy chain content (MHC) were studied at PND28 and PND60. In addition, the motor function was evaluated weekly from PND28 to PND60. At PND28, SMR rats presented a severe atrophy of soleus muscle, a decrease in CSA and a force loss. The muscle maturation appeared delayed, with persistence of neonatal forms of MHC. Changes in kinetic properties were moderate or absent. The Hoffmann reflex provided evidence for spinal hyperreflexia and signs of spasticity. Most changes were reversed at PND60, except muscle atrophy. Functional motor tests that require a good limb coordination, i.e. rotarod and locomotion, showed an enduring alteration related to SMR, even after one month of ‘typical’ activity. On the other hand, paw withdrawal test and grip test were poorly affected by SMR whereas spontaneous locomotor activity increased over time. Our results support the idea that proprioceptive feedback is at least as important as the amount of motor activity to promote a typical development of motor function. A better knowledge of the interplay between hypoactivity, muscle properties and central motor commands may offer therapeutic perspectives for children suffering from neurodevelopmental disorders.

Pressing Intervention Promotes the Skeletal Muscle Repair of Traumatic Myofascial Trigger Points in Rats.

ObjectiveTo observe the effect of pressing intervention on the skeletal muscle repair of myofascial trigger points (MTrPs) in rats and explore the mechanism of pressing intervention on the deactivation of trigger points.MethodsThirty SPF rats were randomly divided into blank group, model group and press group, with 10 rats in each group. The MTrPs models were established by blunt striking plus eccentric exercise, and then evaluated. The press group was given a pressing intervention with a self-made device for 14 days, and the rats in the other two groups were fed normally. Soft tissue tension (STT) D0.2 and pressure pain threshold (PPT) were measured before and after intervention. The skeletal muscle tissue at MTrPs was extracted and assessed by hematoxylin–eosin (HE) and Masson staining. The expression of collagen I, collagen III, α- smooth muscle actin (α-SMA), myosin heavy chain (MHC) and fibronectin (FN) were detected by Western Blotting. Enzyme linked immunosorbent assay (ELISA) was used to evaluate the expression of substance P (SP), 5-hydroxytryptamine (5-HT), cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2).Results(1) Compared with the blank group, the PPT and D0.2 reduced (P < 0.05) in the model group; while compared with the model group, the PPT and D0.2 increased (P < 0.05) in the press group. (2) Compared with the blank group, the model group showed obvious spontaneous potentials with higher amplitude and frequency, which were also much higher than those of the press group (P < 0.05). (3) The HE and Masson staining results showed evident fibrosis in the muscle tissue of the model group, with a larger area of collagen fibers relative to that of the press group (P < 0.05). (4) The amount of collagen I, collagen III, FN, α- SMA, SP, 5-HT, COX-2 and PGE2 increased and the content of MHC decreased (P < 0.05) in the model group, as compared to the blank group; while all the substances (P < 0.05), instead of MHC which increased (P < 0.05), in the press group were decreased as compared to the model group.ConclusionPressing intervention on the MTrPs in rats can alleviate chronic inflammation, inhibit fibrosis, promote skeletal muscle repair and relieve pain.

Comparative study on the rheological properties of myofibrillar proteins from different kinds of meat

In this study, the gel properties of myofibrillar proteins (MPs) from four meat sources (fish, beef, sheep, and pork) were compared. Oscillatory rheology measurements including temperature sweep, frequency sweep, and strain sweep were conducted to characterise the small and large deformation rheological properties of the MPs. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy (SEM) were used to evaluate differences in the molecular weight distribution as well as the microstructures in gel among different MPs. Frequency sweep measurements showed that all MP gels were weak gels. MPs extracted from pork exhibited the highest gel strength and most compact gel structure, whereas those from fish exhibited the lowest gel strength and loosest gel structure. In addition, the MP extracted from pork (PSM) had the highest content of myosin heavy chain (MHC) and actin. In conclusion, the MPs extracted from fish source and mammalian sources varied significantly in terms of rheological properties and microstructural characteristics. These results provided useful information for developing mixed gel products with different gel strengths.

Minocycline Treatment Reduces Mass and Force Output From Fast-Twitch Mouse Muscles and Inhibits Myosin Production in C2C12 Myotubes.

Minocycline, a tetracycline-class of antibiotic, has been tested with mixed effectiveness on neuromuscular disorders such as amyotrophic lateral sclerosis, autoimmune neuritis and muscular dystrophy. The independent effect of minocycline on skeletal muscle force production and signalling remain poorly understood. Our aim here is to investigate the effects of minocycline on muscle mass, force production, myosin heavy chain abundance and protein synthesis. Mice were injected with minocycline (40 mg/kg i.p.) daily for 5 days and sacrificed at day six. Fast-twitch EDL, TA muscles and slow-twitch soleus muscles were dissected out, the TA muscle was snap-frozen and the remaining muscles were attached to force transducer whilst maintained in an organ bath. In C2C12 myotubes, minocycline was applied to the media at a final concentration of 10 μg/mL for 48 h. In minocycline treated mice absolute maximal force was lower in fast-twitch EDL while in slow-twitch soleus there was an increase in the time to peak and relaxation of the twitch. There was no effect of minocycline treatment on the other contractile parameters measured in isolated fast- and slow-twitch muscles. In C2C12 cultured cells, minocycline treatment significantly reduced both myosin heavy chain content and protein synthesis without visible changes to myotube morphology. In the TA muscle there was no significant changes in myosin heavy chain content. These results indicate that high dose minocycline treatment can cause a reduction in maximal isometric force production and mass in fast-twitch EDL and impair protein synthesis during myogenesis in C2C12 cultured cells. These findings have important implications for future studies investigating the efficacy of minocycline treatment in neuromuscular or other muscle-atrophy inducing conditions.

Mitoquinone mesylate (MitoQ) prevents sepsis-induced diaphragm dysfunction.

Sepsis-induced diaphragm dysfunction is a major contributor to respiratory failure in mechanically ventilated patients. There are no pharmacological treatments for this syndrome, but studies suggest that diaphragm weakness is linked to mitochondrial free radical generation. We hypothesized that administration of mitoquinone mesylate (MitoQ), a mitochondrially targeted free radical scavenger, would prevent sepsis-induced diaphragm dysfunction. We compared diaphragm function in 4 groups of male mice: 1) sham-operated controls treated with saline (0.3 mL ip), 2) sham-operated treated with MitoQ (3.5 mg/kg/day given intraperitoneally in saline), 3) cecal ligation puncture (CLP) mice treated with saline, and 4) CLP mice treated with MitoQ. Forty-eight hours after surgery, we assessed diaphragm force generation, myosin heavy chain content, state 3 mitochondrial oxygen consumption (OCR), and aconitase activity. We also determined effects of MitoQ in female mice with CLP sepsis and in mice with endotoxin-induced sepsis. CLP decreased diaphragm specific force generation and MitoQ prevented these decrements (e.g. maximal force averaged 30.2 ± 1.3, 28.0 ± 1.3, 12.8 ± 1.9, and 30.0 ± 1.0 N/cm2 for sham, sham + MitoQ, CLP, and CLP + MitoQ groups, respectively, P < 0.001). CLP also reduced diaphragm mitochondrial OCR and aconitase activity; MitoQ blocked both effects. Similar responses were observed in female mice and in endotoxin-induced sepsis. Moreover, delayed MitoQ treatment (by 6 h) was as effective as immediate treatment. These data indicate that MitoQ prevents sepsis-induced diaphragm dysfunction, preserving force generation. MitoQ may be a useful therapeutic agent to preserve diaphragm function in critically ill patients with sepsis.NEW & NOTEWORTHY This is the first study to show that mitoquinone mesylate (MitoQ), a mitochondrially targeted antioxidant, treats sepsis-induced skeletal muscle dysfunction. This biopharmaceutical agent is without known side effects and is currently being used by healthy individuals and in clinical trials in patients with various diseases. When taken together, our results suggest that MitoQ has the potential to be immediately translated into treatment for sepsis-induced skeletal muscle dysfunction.

Impact of Human Epidermal Growth Factor on Tissue-Engineered Skeletal Muscle Structure and Function.

Skeletal muscle tissue engineering technologies have the potential to treat volumetric muscle loss (VML) by growing exogenous muscle tissue. However, there has been limited success in engineering human cell-sourced skeletal muscle with structure and function comparable to native adult human muscle. The use of growth factors at optimal concentrations and delivery times is critical in enhancing the in vitro myogenesis of satellite cells used in engineered skeletal muscle. The mitogenic protein human epidermal growth factor (hEGF) is of particular interest because it enhances satellite cell proliferation and sarcomeric structure formation in myogenic cell cultures. In this study, we used our scaffold-free tissue-engineered skeletal muscle units (SMUs) to examine the effects of hEGF on the structure and function of human cell-sourced engineered skeletal muscle. During our established SMU fabrication process, human muscle cell isolates were exposed to media treated with 7.5 nM hEGF at three different time spans during the 21-day cell culture period: 0 to 6 days postseeding (hEGF-treated Muscle Growth Media [MGM] Only), 7 to 21 days postseeding (hEGF-treated Muscle Differentiation Media (MDM) Only), and 0 to 21 days postseeding (hEGF-treated MGM+MDM). Control cell cultures were fed standard MGM and MDM (no hEGF treatment). During the fabrication process, light microscopy was used to examine proliferation and differentiation of myogenic cells in the monolayer. After SMU formation, the three-dimensional constructs underwent tetanic force production measurements to evaluate contractile function and immunohistochemical staining to examine SMU structure. Results indicated that hEGF administration impacted myogenesis, by increasing myotube diameter in hEGF-treated MGM only and hEGF-treated MDM-only cell cultures, and by increasing myotube density in hEGF-treated MGM+MDM cultures. The exposure of myogenic cells to hEGF during any time period of the fabrication process led to a significant increase in SMU myosin heavy-chain content. SMUs exposed to hEGF-treated MDM and hEGF-treated MGM+MDM exhibited greater cross-sectional areas and more organized sarcomeric structure. Furthermore, hEGF-treated MGM+MDM SMUs displayed significantly enhanced contractile function compared with controls, indicating advanced functional maturation. In conclusion, hEGF supplementation in human primary myogenic cell cultures advances tissue-engineered skeletal muscle structural and functional characteristics. Impact statement Our research suggests that human epidermal growth factor (hEGF) serves as a critical growth factor in enhancing in vitro skeletal muscle cell proliferation and differentiation during myogenesis and advances human skeletal muscle engineered tissues toward a more native adult skeletal muscle phenotype. Understanding the impact of hEGF on engineered skeletal muscle function and structure is valuable in determining the optimal culture conditions for the development of tissue engineering-based therapies for volumetric muscle loss.

Стабильный уровень гигантских белков саркомерного цитоскелета в поперечнополосатых мышцах сони-полчка Glis glis при гибернации

The changes in the content of giant proteins of the sarcomeric cytoskeleton titin (3000-3700 kDa) and nebulin (770 kDa) in skeletal muscles (m. soleus, m. gastrocnemius), titin in the myocardium (left ventricle), as well as membrane cytoskeletal protein dystrophin (427 kDa) in m. soleus and m. extensor digitorum longus of edible dormouse Glis glis during hibernation have been studied. The animals were of two experimental groups: “Summer activity” and “Hypothermia”. It was found that the development of atrophic changes in the skeletal muscles of the animal during hibernation is accompanied by a decrease in the content of dystrophin. In particular, the intensity of fluorescence on cross-sections of skeletal muscles stained with primary antibodies to dystrophin and secondary antibodies conjugated to the Alexa488 fluorochrome decreased 2.7 times (p < 0.05) and 2.0 times (p < 0.05) in m. soleus and m. EDL, respectively, in animals of the “Hypothermia” group. SDS electrophoresis of proteins in agarose-strengthened macroporous 2.2%-polyacrylamide gel revealed a slight decrease (by 15%, p ≤ 0.01) in the titin content in relation to the content of myosin heavy chains in m. gastrocnemius of animals of the “Hypothermia” group. Titin content in m. soleus and cardiac muscle, as well as the nebulin content in the studied skeletal muscles did not decrease during hibernation. These results are consistent with our earlier data for other hibernates: long-tailed ground squirrel, brown and Himalayan black bears. It can be assumed that in the process of evolution in hibernating animals, molecular mechanisms were developed that are responsible for maintaining a stable level of giant proteins of the sarcomeric cytoskeleton during hibernation.