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Oxidative hotspots on actin promote skeletal muscle weakness in rheumatoid arthritis.

ABSTRACT: Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here we show that oxidative post-translational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde adduct (MDA) modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located to three distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritis mice and RA patients. Moreover, molecular dynamic simulations revealed that these areas, here coined "hotspots", are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and provide novel leads for targeted therapeutic treatment to improve muscle function.

SUBMITTER: Steinz MM

PROVIDER: S-EPMC6538353 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Oxidative hotspots on actin promote skeletal muscle weakness in rheumatoid arthritis.

Steinz Maarten M MM Persson Malin M Aresh Bejan B Olsson Karl K Cheng Arthur J AJ Ahlstrand Emma E Lilja Mats M Lundberg Tommy R TR Rullman Eric E Möller Kristina Ängeby KÄ Sandor Katalin K Ajeganova Sofia S Yamada Takashi T Beard Nicole N Karlsson Björn Cg BC Tavi Pasi P Kenne Ellinor E Svensson Camilla I CI Rassier Dilson E DE Karlsson Roger R Friedman Ran R Gustafsson Thomas T Lanner Johanna T JT

JCI insight 20190328

Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here we show that oxidative post-translational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spect ...[more]

PMID: 30920392

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Project description:MicroRNAs serve an important role in the development of several diseases. Numerous genes regulate the skeletal muscle differentiation of C2C12 myoblasts. The role of miR?760 in rheumatoid arthritis (RA) has not been reported, to the best of our knowledge. Therefore, the aim of the present study was to examine the role of miR?760 in regulating skeletal muscle proliferation in RA. Potential genes functionally involved in the tarsal joint of a collagen?induced RA model were identified using Gene Expression Omnibus. Reverse transcription?quantitative PCR and western blot analyses were performed to determine the mRNA and protein expression levels. The proliferation, cell cycle progression and migration of C2C12 myoblasts were detected using Cell Counting Kit?8, flow cytometry and wound?healing assays, respectively. TargetScan was used to predict the potential target genes of miR?760, and this was verified using a dual?luciferase reporter assay. In the present study, myosin?18b (Myo18b) expression was determined to be downregulated in the RA model. Silencing Myo18b decreased the proliferation, abrogated the cell cycle progression, and reduced the migration and differentiation of C2C12 myoblasts. Expression levels of cyclin?dependent kinase 2, cyclin D1, matrix metalloproteinase (MMP)?2, MMP?9, myogenin and myosin heavy chain 6 were all decreased when Myo18b was silenced. Furthermore, overexpression of Myo18b induced opposing effects on C2C12 myoblasts. It was shown that Myo18b was a target gene of miRNA?760. Overexpression of miR?760 decreased proliferation, cell cycle progression, migration and differentiation in C2C12 myoblasts, and decreased the expression of Myo18b. The opposite results were observed when miR?760 was downregulated. In conclusion, miR?760 inhibited proliferation and differentiation by targeting Myo18b in C2C12 myoblasts. The results of the present study may contribute to understanding the mechanisms underlying RA skeletal muscle proliferation, and miR?760/Myo18b may serve as potential targets for treating patients with RA.

Project description: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.

Project description:Background & aimsMetabolic characterization of a well-defined group of patients could be a powerful tool in revealing metabolic signatures to explain limb muscle weakness in chronic diseases. Studies are currently limited in Chronic Obstructive Pulmonary Disease (COPD) to the identification of differential amino acid concentrations but lack comprehensive analysis of the flux through relevant muscle function related metabolic pathways.MethodsIn 23 stable patients with moderate to very severe COPD and 19 healthy controls, a comprehensive metabolic flux analysis was conducted by administering an intravenous pulse and primed constant infusion of multiple stable tracers of amino acids known to play a role in muscle health. Blood samples were obtained to calculate production (WBP) and interconversion rates, and plasma concentrations of these amino acids. Lower and upper limb muscle strength, muscle mass, lung function, physical activity level, and disease history and characteristics were assessed.ResultsThe COPD group was characterized by lower and upper limb muscle weakness (P < 0.01) despite preserved muscle mass. Higher values were found in COPD for plasma glutamine, WBP of leucine (P < 0.001), 3-methylhistidine (P < 0.01) (marker of enhanced myofibrillar protein breakdown), citrulline (P < 0.05), and arginine to citrulline conversion (P < 0.05) (reflecting enhanced nitric oxide synthesis). Plasma concentration of β-hydroxy β-methylbutyrate (HMB with anticatabolic, anabolic and contractile properties), WBP of glycine (precursor of creatine and glutathione), and transcutaneous O2 saturation explained up to 79% and 65% of the variation in strength of the lower and upper limb muscles, respectively, in COPD.ConclusionsComprehensive metabolic flux analysis revealed a homogenous metabolic signature in stable patients with COPD and a specific metabolic profile in those with skeletal muscle weakness.Clinical trial registryClinicalTrials.gov; No. NCT01787682; URL: www.clinicaltrials.gov.

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Oxidative hotspots on actin promote skeletal muscle weakness in rheumatoid arthritis.

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Oxidative hotspots on actin promote skeletal muscle weakness in rheumatoid arthritis.

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