Fast Skeletal Muscle Troponin Research Articles

Investigating the effects of low-salt processing on the umami peptides of dry-cured ham using peptidomics techniques

This study investigated the effect of low-salt processing on the umami peptide profile of dry-cured hams. Peptidomics data showed 633 umami peptides in the low- and full-salt groups. Among them, 36.2% and 26.5% of shared umami peptides in the low-salt group were significantly down- and up-regulated in relative abundance. Multivariate statistical analysis showed 1011 significantly different umami peptides (SDUPs) in the low- and full-salt groups. Creatine kinase M-type (CKM) and fast skeletal muscle troponin T (TnTf) were the main precursor proteins of these SDUPs. At the end of processing, the relative expression of CKM was lower in the low-salt group than in the full-salt group (P < 0.05), but there was no significant difference in TnTf. More dipeptidyl peptidase cleavage sites were observed in CKM and TnTf proteins in the low-salt group.

Tirasemtiv enhances submaximal muscle tension in an Acta1:p.Asp286Gly mouse model of nemaline myopathy.

Nemaline myopathies are the most common form of congenital myopathies. Variants in ACTA1 (NEM3) comprise 15-25% of all nemaline myopathy cases. Patients harboring variants in ACTA1 present with a heterogeneous disease course characterized by stable or progressive muscle weakness and, in severe cases, respiratory failure and death. To date, no specific treatments are available. Since NEM3 is an actin-based thin filament disease, we tested the ability of tirasemtiv, a fast skeletal muscle troponin activator, to improve skeletal muscle function in a mouse model of NEM3, harboring the patient-based p.Asp286Gly variant in Acta1. Acute and long-term tirasemtiv treatment significantly increased muscle contractile capacity at submaximal stimulation frequencies in both fast-twitch extensor digitorum longus and gastrocnemius muscle, and intermediate-twitch diaphragm muscle in vitro and in vivo. Additionally, long-term tirasemtiv treatment in NEM3 mice resulted in a decreased respiratory rate with preserved minute volume, suggesting more efficient respiration. Altogether, our data support the therapeutic potential of fast skeletal muscle troponin activators in alleviating skeletal muscle weakness in a mouse model of NEM3 caused by the Acta1:p.Asp286Gly variant.

Intermolecular And Dynamic Investigation of The Mechanism of Action of Reldesemtiv on Fast Skeletal Muscle Troponin Complex Toward the Treatment of Impaired Muscle Function

Muscle weakness as a secondary feature of attenuated neuronal input often leads to disability and sometimes death in patients with neurogenic neuromuscular diseases. These impaired muscle function has been observed in several diseases including amyotrophic lateral sclerosis, Charcot–Marie–Tooth, spinal muscular atrophy and Myasthenia gravis. This has spurred the search for small molecules which could activate fast skeletal muscle troponin complex as a means to increase muscle strength. Discovered small molecules have however been punctuated by off-target and side effects leading to the development of the second-generation small molecule, Reldesemtiv. In this study, we investigated the impact of Reldesemtiv binding to the fast skeletal troponin complex and the molecular determinants that condition the therapeutic prowess of Redesemtiv through computational techniques. It was revealed that Reldesemtiv binding possibly potentiates troponin C compacting characterized by reduced exposure to solvent molecules which could favor the slow release of calcium ions and the resultant sensitization of the subunit to calcium. These conformational changes were underscored by conventional and carbon hydrogen bonds, pi-alkyl, pi-sulfur and halogen interactions between Reldesemtiv the binding site residues. Arg113 (−3.96 kcal/mol), Met116 (−2.23 kcal/mol), Val114 (−1.28 kcal/mol) and Met121 (−0.63 kcal/mol) of the switch region of the inhibitory subunit were among the residues that contributed the most to the total free binding energy of Reldesemtiv highlighting their importance. These findings present useful insights which could lay the foundation for the development of fast skeletal muscle small molecule activators with high specificity and potency.

Fast Skeletal Muscle Troponin and Tropomyosin as a Dietary Source of Antidiabetic and Antihypertensive Bioactive Peptides: An In Silico Study

Background : The nutraceutical properties of food hydrolysates rely on multiple biochemical interactions involving the modulation of enzymes and cellular receptors. Numerous bioactive peptides released from troponin and tropomyosin digestion have been identified. Their characterization has mostly been performed by hydrolysis catalyzed by proteases unrelated to the human digestive system. Objective: This study aimed to determine the bioactive profile of beef, pork, and chicken meat by analyzing the frequency and pharmacokinetics of biopeptides released from troponin and tropomyosin. Methods: In silico digestion and biopeptide release frequency were studied by three parameters; bioactive fragments release frequency (AE), frequency percentage (W), and mean occurrence (AS), all stated on the BIOPEP-UWM platform. Further on, hydrolysis end-products were screened based on gastrointestinal-absorption probability and pharmacokinetic profiling performed on SwissADME, SwissTargetPrediction, and ADME/Tlab bioinformatics web tools. Statistical analyses were performed using a one-way ANOVA test. Results: Dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibiting biopeptides exhibited the highest release frequency. Moreover, W and AS parameters showed no significant difference (p&gt;0.05) between the myofibrillar isoforms assessed. Seven biopeptides were classified as highly absorbable and reported optimal drug-likeness compliance. Although biopeptides hold good pharmacokinetic properties, the therapeutic potency of biopeptides showed to be lower than those of DPP-IV and ACE-inhibiting drugs. Conclusions: Troponin and tropomyosin are rich dietary sources of bioactive peptides, mainly DPP-IV and ACE inhibitors. Digestion end-products are mainly dipeptides with optimal pharmacokinetic and drug-like properties, suggesting a potential therapeutic application in hypertensive and hyperglycemic disorders.

Sarcomere dynamics revealed by a myofilament integrated FRET-based biosensor in live skeletal muscle fibers

The sarcomere is the functional unit of skeletal muscle, essential for proper contraction. Numerous acquired and inherited myopathies impact sarcomere function causing clinically significant disease. Mechanistic investigations of sarcomere activation have been challenging to undertake in the context of intact, live skeletal muscle fibers during real time physiological twitch contractions. Here, a skeletal muscle specific, intramolecular FRET-based biosensor was designed and engineered into fast skeletal muscle troponin C (TnC) to investigate the dynamics of sarcomere activation. In transgenic animals, the TnC biosensor incorporated into the skeletal muscle fiber sarcomeres by stoichiometric replacement of endogenous TnC and did not alter normal skeletal muscle contractile form or function. In intact single adult skeletal muscle fibers, real time twitch contractile data showed the TnC biosensor transient preceding the peak amplitude of contraction. Importantly, under physiological temperatures, inactivation of the TnC biosensor transient decayed significantly more slowly than the Ca2+ transient and contraction. The uncoupling of the TnC biosensor transient from the Ca2+ transient indicates the biosensor is not functioning as a Ca2+ transient reporter, but rather reports dynamic sarcomere activation/ inactivation that, in turn, is due to the ensemble effects of multiple activating ligands within the myofilaments. Together, these findings provide the foundation for implementing this new biosensor in future physiological studies investigating the mechanism of activation of the skeletal muscle sarcomere in health and disease.

P.124 EDG-5506 targets fast skeletal myosin and reduces muscle damage biomarkers in a phase 1 trial in Becker muscular dystrophy (BMD)

To assess safety, pharmacokinetics (PK), pharmacodynamics and biomarkers of muscle damage in healthy adults and adults with BMD. Fast (Type II) muscle fibers are affected early and disproportionately in dystrophinopathies. EDG-5506 targets fast skeletal muscle myosin, and in animal models of Duchenne muscular dystrophy (DMD), decreased muscle damage biomarkers and fibrosis, while increasing muscle strength and habitual activity. Ascending EDG-5506 doses (5-40 mg daily) were administered orally for 14 days in healthy adults who underwent assessments of safety, plasma and muscle PK, and biomarkers. Seven ambulatory adults with BMD were enrolled to assess safety, PK and effects of EDG-5506 or placebo in the setting of dystrophic muscle after 14 days. EDG-5506 was well-tolerated at all doses in healthy adults. Most common adverse events were somnolence and dizziness, which were mild and transient. Plasma and muscle exposures exceeded exposures that had demonstrated positive effects in animal models of DMD. In healthy adults and in participants with BMD, muscle concentrations substantially exceeded those in plasma, consistent with high affinity of EDG-5506 for fast fiber myosin. Muscle strength was maintained in all cohorts. In BMD participants, EDG-5506 was well-tolerated and significantly reduced creatine kinase, myoglobin, and fast skeletal muscle troponin I (TNNI2). In a plasma proteomic panel (SOMAscan), proteins associated with muscle damage and muscle metabolism were elevated in participants with BMD when compared to healthy controls. This panel of proteins decreased significantly with EDG-5506, particularly for those proteins that become elevated in response to exercise. EDG-5506 demonstrated favorable safety and PK profiles at dose levels that resulted in robust reductions in muscle damage biomarkers in adults with BMD. By targeting fast skeletal muscle myosin to protect dystrophic muscle, EDG-5506 has potential to be a novel disease-modifying approach in DMD and BMD regardless of mutation type. Phase 2 studies in BMD and DMD are planned.

Discovery of Reldesemtiv, a Fast Skeletal Muscle Troponin Activator for the Treatment of Impaired Muscle Function.

The discovery of reldesemtiv, a second-generation fast skeletal muscle troponin activator (FSTA) that increases force production at submaximal stimulation frequencies, is reported. Property-based optimization of high throughput screening hit 1 led to compounds with improved free exposure and in vivo muscle activation potency compared to the first-generation FSTA, tirasemtiv. Reldesemtiv demonstrated increased muscle force generation in a phase 1 clinical trial and is currently being evaluated in clinical trials for the treatment of amyotrophic lateral sclerosis.

Prescription and acceptance of durable medical equipment in FORTITUDE-ALS, a study of reldesemtiv in ALS: post hoc analyses of a randomized, double-blind, placebo-controlled clinical trial

Objective: To evaluate the possible effect of reldesemtiv, a fast skeletal muscle troponin activator, on prescription and acceptance of durable medical equipment (DME) in the FORTITUDE-ALS trial. Methods: Health economic outcome information was collected in FORTITUDE-ALS (NCT03160898); sites recorded if and when DME, specifically manual or power wheelchairs, gastrostomy tubes, noninvasive ventilators, or augmentative language devices, was prescribed by a physician and accepted by the patient (DME-PAP) during the trial. Acceptance was defined as the patient agreeing the item was needed. Cox regression analysis compared time to DME-PAP for each reldesemtiv dose with placebo. Post hoc analyses evaluated all reldesemtiv doses compared with placebo. Results: At least one DME item was prescribed and accepted by 33/114 (28.9%) of placebo patients, 19/112 (17.0%) of patients receiving reldesemtiv 150 mg bid, 24/113 (21.2%) receiving 300 mg bid, and 29/117 (24.8%) receiving 450 mg bid. The proportion of new DME-PAP was significantly lower in patients receiving reldesemtiv 150 mg bid vs placebo (17.0% vs 28.9%, p = 0.032). The hazard ratio versus placebo for accepting at least one DME item for all reldesemtiv doses combined was 0.61 (confidence interval: 0.39, 0.96, p = 0.032). 25% of placebo patients were prescribed and agreed to obtain a DME item by 84 days; this threshold was met for reldesemtiv-treated patients at 120 days. Conclusions: Results suggest ALS patients receiving reldesemtiv may have lower risk of and delayed need for DME related to impaired mobility, breathing, swallowing, or speaking; this delay is consistent with other measures indicating delay in disease progression.

Pathogenic variants in TNNC2 cause congenital myopathy due to an impaired force response to calcium.

Troponin C (TnC) is a critical regulator of skeletal muscle contraction; it binds Ca2+ to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has not yet been implicated in muscle disease. Here, we report 2 families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations suggested that the pathomechanisms by which the variants cause muscle disease include disruption of the binding sites for Ca2+ and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients' biopsies revealed a markedly reduced force response of the sarcomeres to [Ca2+]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, wild-type TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients' myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility.

Acute and chronic tirasemtiv treatment improves in vivo and in vitro muscle performance in actin-based nemaline myopathy mice.

Nemaline myopathy, a disease of the actin-based thin filament, is one of the most frequent congenital myopathies. To date, no specific therapy is available to treat muscle weakness in nemaline myopathy. We tested the ability of tirasemtiv, a fast skeletal troponin activator that targets the thin filament, to augment muscle force—both in vivo and in vitro—in a nemaline myopathy mouse model with a mutation (H40Y) in Acta1. In Acta1H40Y mice, treatment with tirasemtiv increased the force response of muscles to submaximal stimulation frequencies. This resulted in a reduced energetic cost of force generation, which increases the force production during a fatigue protocol. The inotropic effects of tirasemtiv were present in locomotor muscles and, albeit to a lesser extent, in respiratory muscles, and they persisted during chronic treatment, an important finding as respiratory failure is the main cause of death in patients with congenital myopathy. Finally, translational studies on permeabilized muscle fibers isolated from a biopsy of a patient with the ACTA1H40Y mutation revealed that at physiological Ca2+ concentrations, tirasemtiv increased force generation to values that were close to those generated in muscle fibers of healthy subjects. These findings indicate the therapeutic potential of fast skeletal muscle troponin activators to improve muscle function in nemaline myopathy due to the ACTA1H40Y mutation, and future studies should assess their merit for other forms of nemaline myopathy and for other congenital myopathies.

Biallelic Pathogenic Variants in TNNT3 Associated With Congenital Myopathy.

ObjectivePathogenic variants in TNNT3, the gene encoding fast skeletal muscle troponin T, were first described in autosomal dominant distal arthrogryposis type 2B2. Recently, a homozygous splice site variant, c.681+1G>A, was identified in a patient with nemaline myopathy and distal arthrogryposis. Here, we describe the second individual with congenital myopathy associated with biallelic TNNT3 variants.MethodsClinical exome sequencing data from a patient with molecularly undiagnosed congenital myopathy underwent research reanalysis. Clinical and histopathologic data were collected and compared with the single reported patient with TNNT3-related congenital myopathy.ResultsA homozygous TNNT3 variant, c.481-1G>A, was identified. This variant alters a consensus splice acceptor and is predicted to affect splicing by multiple in silico prediction tools. Both the patient reported here and the previously published patient exhibited limb, bulbar, and respiratory muscle weakness from birth, which improved over time. Other shared features include history of polyhydramnios, hypotonia, scoliosis, and high-arched palate. Distal arthrogryposis and nemaline rods, findings reported in the first patient with TNNT3-related congenital myopathy, were not observed in the patient reported here.ConclusionsThis report provides further evidence for the association of biallelic TNNT3 variants with severe recessive congenital myopathy with or without nemaline rods and distal arthrogryposis. TNNT3 sequencing and copy number analysis should be incorporated into the workup of congenital myopathies.

Reldesemtiv in Patients with Spinal Muscular Atrophy: a Phase 2 Hypothesis-Generating Study

This phase 2, double-blind, placebo-controlled, hypothesis-generating study evaluated the effects of oral reldesemtiv, a fast skeletal muscle troponin activator, in patients with spinal muscular atrophy (SMA). Patients ≥ 12 years of age with type II, III, or IV SMA were randomized into 2 sequential, ascending reldesemtiv dosing cohorts (cohort 1: 150 mg bid or placebo [2:1]; cohort 2: 450 mg bid or placebo [2:1]). The primary objective was to determine potential pharmacodynamic effects of reldesemtiv on 8 outcome measures in SMA, including 6-minute walk distance (6MWD) and maximum expiratory pressure (MEP). Changes from baseline to weeks 4 and 8 were determined. Pharmacokinetics and safety were also evaluated. Patients were randomized to reldesemtiv 150 mg, 450 mg, or placebo (24, 20, and 26, respectively). The change from baseline in 6MWD was greater for reldesemtiv 450 mg than for placebo at weeks 4 and 8 (least squares [LS] mean difference, 35.6 m [p = 0.0037] and 24.9 m [p = 0.058], respectively). Changes from baseline in MEP at week 8 on reldesemtiv 150 and 450 mg were significantly greater than those on placebo (LS mean differences, 11.7 [p = 0.038] and 13.2 cm H2O [p = 0.03], respectively). For 6MWD and MEP, significant changes from placebo were seen in the highest reldesemtiv peak plasma concentration quartile (Cmax > 3.29 μg/mL; LS mean differences, 43.3 m [p = 0.010] and 28.8 cm H2O [p = 0.0002], respectively). Both dose levels of reldesemtiv were well tolerated. Results suggest reldesemtiv may offer clinical benefit and support evaluation in larger SMA patient populations.

LATE BREAKING NEWS ORAL PRESENTATION: LBO 1 Pathogenic variants in TNNC2 cause congenital myopathy due to an impaired force response to calcium

Troponin C (TnC) is a critical regulator of skeletal muscle contraction: it binds Ca2+ to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has never been implicated in muscle disease. Here, we report two families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations indicate that the pathomechanisms by which the variants cause muscle disease include disruption of the bindings sites for Ca2+ and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients’ biopsies revealed a markedly reduced force response of the sarcomeres to [Ca2+]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, healthy TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients’ myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility.

LATE BREAKING NEWS E-POSTER PRESENTATION: LBP 8 A canine model for understanding the pharmacokinetic/pharmacodynamic relationship of muscle-targeted therapies

Skeletal muscle-targeted therapies that increase contractility hold promise for the treatment of neuromuscular diseases. As a compound class, fast skeletal muscle troponin activators (FSTAs) directly increase muscle calcium (Ca²+) sensitivity and amplify the muscle force response to subtetanic neural stimulation. Disease-modifying therapies like systemic gene transfer have previously been shown to improve muscle function in a canine model of myotubular myopathy. The purpose of the current study was to evaluate the expanded utility of a canine skeletal muscle model to understand the pharmacokinetic/pharmacodynamic (PK/PD) relationship of the novel FSTA, CK-3762601.

Effects of electroacupuncture combined with passive stretch exercise on disused muscle atrophy and expression of skeletal troponin in mice

To observe the efficacy of electroacupuncture (EA) plus passive stretch exercise in the treatment of disused atrophy of gastrocnemius and soleus muscles in mice. Fifty C57BL/6 mice were randomly and equally divided into 5 groups: blank control, model, passive stretch exercise (exercise), EA and EA+exercise groups. The muscular atrophy model was established by fixing the gastrocnemius and soleus muscles with plaster immobilization (by putting the right leg into a plastic vial and then twining the vial with medical plaster bandage from the ankle upwards to the thigh and groin to maintain the knee-joint flexion and ankle joint plantar flexion for 7 days). EA (2 Hz/100 Hz, 1 mA)was applied to bilateral "Zusanli"(ST36) for 10 min, once a day for 4 weeks. For mice with the passive exercise, the plastic vial was removed first, followed by pulling out the hindleg to seize the toes to stretch them until the right hindleg is fully extended, then, pushed the leg towards the body. The procedures were repeated once again and again for 10 min. The exercise was conducted once daily, for 4 weeks. The cross-sectional area of fast and slow muscle fibers of the soleus and gastrocnemius was measured under electronic microscope after ATPase histochemical stain and the expression of slow skeletal muscle troponin (TNNI1) and fast skeletal muscle troponin (TNNI2) in the soleus and gastrocnemius was detected by Western blot. Compared with the blank control group, the cross-sectional areas of the fast and slow muscle fibers of the soleus and gastrocnemius muscles were significantly decreased in the model group (P<0.05, P<0.01). Following the interventions, the cross-sectional areas of the fast and slow muscle fibers of soleus muscle in the EA+exercise group, and those of the fast and slow muscle fibers of the gastrocnemius muscle in the EA and EA+exercise groups, and the expression levels of TNNI1 and TNNI2 proteins in the gastrocnemius muscle of the EA+exercise group were significantly increased in comparison with the model group (P<0.05, P<0.01). EA combined with passive stretch exercise can promote the recovery of the soleus and gastrocnemius muscles in disused muscle atrophy mice, which may be related to its effect in up-regulating the expression of TNNI1 and TNNI2 proteins.

Fast skeletal muscle troponin activator CK-2066260 mitigates skeletal muscle weakness independently of the underlying cause.

BackgroundMuscle weakness is a common symptom in numerous diseases and a regularly occurring problem associated with ageing. Prolonged low‐frequency force depression (PLFFD) is a form of exercise‐induced skeletal muscle weakness observed after exercise. Three different intramuscular mechanisms underlying PLFFD have been identified: decreased sarcoplasmic reticulum Ca2+ release, decreased myofibrillar Ca2+ sensitivity, and myofibrillar dysfunction. We here used these three forms of PLFFD as models to study the effectiveness of a fast skeletal muscle troponin activator, CK‐2066260, to mitigate muscle weakness.MethodsExperiments were performed on intact single muscle fibres or fibre bundles from mouse flexor digitorum brevis, which were stimulated with electrical current pulses, while force and the free cytosolic [Ca2+] ([Ca2+]i) were measured. PLFFD was induced by three different stimulation protocols: (i) repeated isometric contractions at low intensity (350 ms tetani given every 5 s for 100 contractions); (ii) repeated isometric contractions at high intensity (250 ms tetani given every 0.5 s for 300 contractions); and (iii) repeated eccentric contractions (350 ms tetani with 20% length increase given every 20 s for 10 contractions). The extent and cause of PLFFD were assessed by comparing the force–[Ca2+]i relationship at low (30 Hz) and high (120 Hz) stimulation frequencies before (control) and 30 min after induction of PLFFD, and after an additional 5 min of rest in the presence of CK‐2066260 (10 μM).ResultsProlonged low‐frequency force depression following low‐intensity and high‐intensity fatiguing contractions was predominantly due to decreased sarcoplasmic reticulum Ca2+ release and decreased myofibrillar Ca2+ sensitivity, respectively. CK‐2066260 exposure resulted in marked increases in 30 Hz force from 52 ± 16% to 151 ± 13% and from 6 ± 4% to 98 ± 40% of controls with low‐intensity and high‐intensity contractions, respectively. Following repeated eccentric contractions, PLFFD was mainly due to myofibrillar dysfunction, and it was not fully reversed by CK‐2066260 with 30 Hz force increasing from 48 ± 8% to 76 ± 6% of the control.ConclusionsThe fast skeletal muscle troponin activator CK‐2066260 effectively mitigates muscle weakness, especially when it is caused by impaired activation of the myofibrillar contractile machinery due to either decreased sarcoplasmic reticulum Ca2+ release or reduced myofibrillar Ca2+ sensitivity.

Large increases in plasma fast skeletal muscle troponin I after whole-body eccentric exercises

ObjectivesIt has been reported that plasma fast skeletal muscle troponin I (fsTnI) but not slow skeletal muscle troponin I (ssTnI) increases after a bout of eccentric exercise of the elbow flexors. The present study compared the first and second bouts of whole-body eccentric exercises for changes in plasma fsTnI and ssTnI concentrations. DesignObservational study in an experimental group. MethodsFifteen sedentary men (20–25 y) performed nine eccentric exercises targeting arm, leg and trunk muscles, and repeated them two weeks later. Blood samples were taken before and for five days following each bout, and plasma ssTnI and fsTnl concentrations were measured by enzyme-linked immunosorbent assays. Their changes were compared between bouts and their relationships to plasma CK activity and myoglobin concentrations were analysed. ResultsPlasma fsTnI concentration increased after the first bout and peaked at 4 days post-exercise (2152–40,295 ng/mL), but no significant increases were evident after the second bout. Plasma ssTnI concentration did not change significantly from the baseline (<0.08 ng/mL) after either bout. Peak plasma fsTnI concentration was significantly (p < 0.005) correlated with peak plasma CK activity (peak: 23,238–207,304 IU/L, r = 0.727) and myoglobin concentration (1047–3936 μg/L, r = 0.625) after the first bout. ConclusionsThese results suggest that plasma TnI concentrations are more specific biomarker of muscle damage than plasma CK activity and myoglobin concentration. It seems that the whole-body eccentric exercises induced damage preferentially to fast-twitch muscle fibres, and increases in plasma CK activity and myoglobin concentration after eccentric exercise may reflect fast-twitch muscle fibre damage.

PSV-29 Changes in the PSE and DFD porcine muscle proteome during autolysis

Abstract Despite reducing of pale, soft, exudative (PSE) pork share, its amount is 45% in Russia and 65% in Belarus of the total. Dark, firm, dry (DFD) pork occurrence increases to 20% in Russia and 30% in Belarus. Protein changes in normal (NOR), PSE and (DFD) pork muscle during 5 days ageing were studied. Were found differences in their protein profiles and potential protein markers of such raw materials were identified. Mainly in range 600–1000 Da and up to 450 Da. Analysis of the peptide composition showed two myosin light chain isoforms (MLC) (Mw/pI): MLC-like protein — 21.8/5.00 and phosphorylated MLC f -21.0/4.90, in the sample. One of which is fast, the other is slow MLC isoform. Increase in the number of fragments was noted of such proteins as pyruvatekinase, α-enolase, muscle creatine phosphokinase and GPDH, adenylatekinase, αB-crystallin. A correlation between muscle proteins breakdown (the troponin group/troponin family) and the characteristics of meat was found. In NOR samples, troponin I decomposed quite clearly. The most obvious autolysis marker is the appearance and increase of three additional fragments of fast skeletal muscle troponin T, which differ in molecular weight and pI. According to the tryptic peptides mass spectra, the C- and N-molecule terminal parts peptides are no longer detected sequentially. Moreover, in DFD samples, they are most significantly split into shorter peptides. Appearance of short N-terminal GPDH 21 kDa fragment on the 5th day of autolysis is PSE specific marker. Totally 12, 14 and 18 specific peptides were identified in chilled and 18, 32 and 38 on 5th day of autolysis in PSE, NOR and DFD pork respectively. DFD meat can be considered a source of natural specific low-molecular peptides, specifically to produce meat products with functional properties. (Research was supported by the Russian Science Foundation, project 16-16-10073)

Slow skeletal muscle troponin T, titin and myosin light chain 3 are candidate prognostic biomarkers for Ewing's sarcoma.

Ewing's sarcoma (ES) is a common malignant bone tumor in children and adolescents. Although great efforts have been made to understand the pathogenesis and development of ES, the underlying molecular mechanism remains unclear. The present study aimed to identify new key genes as potential biomarkers for the diagnosis, targeted therapy or prognosis of ES. mRNA expression profile chip data sets GSE17674, GSE17679 and GSE45544 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were screened using the R software limma package, and functional and pathway enrichment analyses were performed using the enrichplot package and GSEA software. The NetworkAnalyst online tool, as well as Cytoscape and its plug-ins cytoHubba and NetworkAnalyzer, were used to construct a protein-protein interaction network (PPI) and conduct module analysis to screen key (hub) genes. LABSO COX regression and overall survival (OS) analysis of the Hub genes were performed. A total of 211 DEGs were obtained by integrating and analyzing the three data sets. The functions and pathways of the DEGs were mainly associated with the regulation of small-molecule metabolic processes, cofactor-binding, amino acid, proteasome and ribosome biosynthesis in eukaryotes, as well as the Rac1, cell cycle and P53 signaling pathways. A total of one important module and 20 hub genes were screened from the PPI network using the Maximum Correlation Criteria algorithm of cytoHubba. LASSO COX regression results revealed that titin (TTN), fast skeletal muscle troponin T, skeletal muscle actin α-actin, nebulin, troponin C type 2 (fast), myosin light-chain 3 (MYL3), slow skeletal muscle troponin T (TNNT1), myosin-binding protein C1 slow-type, tropomyosin 3 and myosin heavy-chain 7 were associated with prognosis in patients with ES. The Kaplan-Meier curves demonstrated that high mRNA expression levels of TNNT1 (P<0.001), TTN (P=0.049), titin-cap (P=0.04), tropomodulin 1 (P=0.011), troponin I2 fast skeletal type (P=0.021) and MYL3 (P=0.017) were associated with poor OS in patients with ES. In conclusion, the DEGs identified in the present study may be key genes in the pathogenesis of ES, three of which, namely TNNT1, TTN and MYL3, may be potential prognostic biomarkers for ES.

Functional Characterization of the Intact Diaphragm in a Nebulin-Based Nemaline Myopathy (NM) Model-Effects of the Fast Skeletal Muscle Troponin Activator tirasemtiv.

Respiratory failure due to diaphragm dysfunction is considered a main cause of death in nemaline myopathy (NM) and we studied both isometric force and isotonic shortening of diaphragm muscle in a mouse model of nebulin-based NM (Neb cKO). A large contractile deficit was found in nebulin-deficient intact muscle that is frequency dependent, with the largest deficits at low–intermediate stimulation frequencies (e.g., a deficit of 72% at a stimulation frequency of 20 Hz). The effect of the fast skeletal muscle troponin activator (FSTA) tirasemtiv on force was examined. Tirasemtiv had a negligible effect at maximal stimulation frequencies, but greatly reduced the force deficit of the diaphragm at sub-maximal stimulation levels with an effect that was largest in Neb cKO diaphragm. As a result, the force deficit of Neb cKO diaphragm fell (from 72% to 29% at 20 Hz). Similar effects were found in in vivo experiments on the nerve-stimulated gastrocnemius muscle complex. Load-clamp experiments on diaphragm muscle showed that tirasemtiv increased the shortening velocity, and reduced the deficit in mechanical power by 33%. Thus, tirasemtiv significantly improves muscle function in a mouse model of nebulin-based nemaline myopathy.