Exosome dynamics and changes in a selection of their microRNA cargo in response to an acute bout of eccentrically-biased exercise in healthy human volunteers

Lovett, Jason (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Background: Extracellular vesicles (EVs) are nanoscale (30 to 1000 nm diameter) mediators of intercellular information transfer, shuttling diverse cargos including functional nucleic acids, proteins and lipids. EVs are released by all cell types, are stable in circulation and are thus capable of transferring information between distant tissues. It has been established that circulating EV profiles adapt to differing physiological and disease states. However, a paucity of information on this type of communication arising from skeletal muscle is apparent, especially in the context of exercise. This is despite multiple opinion reviews highlighting the importance of EVs and their cargo (particularly microRNA) as biomarkers of muscle myopathies/dystrophies/damage and even as ameliorators of chronic diseases associated with a sedentary lifestyle. Aims: To determine whether circulating EVs, within the exosome size range, display size, number and cargo alterations in response to exercise-induced muscle damage (EIMD) from an acute bout of eccentrically-biased exercise. Specific cargo selected for analysis included: miR-1, 133a, 133b, 206 and 31. Methods: Blood samples were drawn from 9 human volunteers at baseline (BL), 2 and 24 hours after acute, sequential plyometric jumping and downhill running bouts. Leg muscle pain was assessed on a scale from 1 to 10. Serum creatine kinase (CK) activity was quantified spectrophotometrically. Plasma exosomes were isolated using size exclusion columns and visualised with transmission electron microscopy (TEM). Quantitative and qualitative information on exosome protein was determined by micro-bicinchoninic acid assays and Coomassie-stained gel visualisation respectively. Exosome sizes and numbers were quantified by nanoparticle tracking analysis (NTA). Selected microRNA (miR) cargo was reverse transcribed, and quantified using qPCR with normalisation to an exogenous control (cel-miR-39). Results: Perceived muscle pain and serum CK were elevated post-exercise, providing indirect evidence for EIMD. A simplified protocol for fast exosome visualisation using TEM was achieved, and revealed an abundance of exosomes of varying sizes. A concomitant abundance of exosomes was found using the more precise NTA technique (mean = 9 x 1010 exosomes/ml plasma). Mean exosome diameters were 127 ± 15 nm across all timepoints. No change in exosome size or number was seen over time. Similarly, no change in exosome total protein concentration was apparent between timepoints. Exosome enrichment of the skeletal muscle-specific miR-206 was detected in all but one participant. Neither miR-206, nor ubiquitous myomiRs-1, -133a and -133b changed across timepoints. However, exosome miR-31 decreased from BL to 24 hr post-exercise (p < 0.05). Grouping of participants according to increases (responders) or decreases (non-responders) in exosome number from BL to 24 hr post-exercise revealed that exosome miR-133b decreased between BL and 24 hr post-exercise (p < 0.05) in non-responders (n = 6), with no characteristic change present in responders. Conclusion: A profuse number of exosomes is present in human plasma. NTA analysis revealed that size exclusion was an effective exosome isolation method. Following EIMD the number or sizes of circulating exosomes did not change. Rather, exosome cargo profiles changed. Decreased exosome miR-31 following EIMD suggests the specificity of the response, with differences in response noted between responders and non-responders.

AFRIKAANSE OPSOMMING:Agtergrond: Ekstrasellulêrevesikels (EVs) dien as vervoermiddels van biologiese vrag en groottes is op nanoskaalvlak (30 to 1000 nm deursnee). EVs word vrygestel deur alle seltipes en is stabiel in sirkulasie, wat hul gepas maak om informasie intersellulêr te vervoer in die vorm van funksionele nukleïensure, proteïene en lipiede. Dit is voorheen vasgestel dat sirkulerende EV profiele aanpas volgens veranderinge in gesonde of ongesonde fisiologiese toestande. Desnieteenstaande bestaan daar ‘n gebrek aan inligting vir skeletspier geassosieerde EVs, veral in die konteks van oefening. Ongeag hiervan het verskeie opinie resensies die gebruik van EVs en hul vrag (veral mikroRNS) as biomerkers vir spier miopatie/distrofie/beserings beklemtoon, asook hul potensiaal as genesingsmediators vir die kroniese siektes van ‘n onaktiewe lewenswyse. Doelwitte: Om te bepaal of ‘n verandering in die grootte, getal en vrag van sirkulerende EVs, binne die eksosoom grootte beperking, veroorsak word deur oefening-geïnduseerde spierskade (OGSS), teweeggebring deur ‘n eksentries-bevooroordeelde oefeningsessie. Spesifieke vragte geselekteer vir ontleding sluit in: miR-1, 133a, 133b, 206 en 31. Metodes: Bloedmonsters van 9 menslike vrywilligers was geneem by basislyn (BL), 2 en 24 ure na akute, opeenvolgende pleometriese spronge en afdraand hardloopsessies. Beenspier pyn was geasesseer (skaal van 1 tot 10). Serum kreatien kinase (KK) aktiwiteit was spektrofotometries gekwantifiseer. Plasma eksosome was geïsoleer deur gebruik te maak van grootte uitsluitings kolomme en gevisualiseer dmv transmissie elektron mikroskopie (TEM). Kwantitatiewe en kwalitatiewe informasie vir eksosoom proteïene was bepaal deur mikro-bisinkoninieksuur toetse en Coomassie-kleuring van kladde onderskeidelik. Eksosoom groottes en hoeveelhede was gekwantifiseer dmv nanopartikel opsporings analise (NOA). Die geselekteerde mikroRNS (miR) was agteruit getranskribeer en gekwantifiseer met kPCR. En data is genormaliseer deur eksogene kontrole (cel-miR-39). Resultate: Waargenome spierpyn en serum KK was verhoog na oefening en dui op indirekte wyse na OGSS. ‘n Vereenvoudigde protokol vir spoedige EV visualisering dmv TEM was opgestel, wat ‘n oorvloed van EVs van verkeie groottes ontbloot het. ‘n Gepaardgaande oorvloed van eksosome was gevind met gebruik van die akurate NOA tegniek (gemiddeld 9 x1010 eksosome/ml plasma). Gemene eksosoom deursnee was 127 ±15 nm vir alle tydintervalle. Geen verandering in eksosoom grootte profiel of getal was met die verloop van tyd waargeneem nie, asook nie in totale eksosoom proteïen konsentrasie nie. Eksosoom vermeerdering van die skeletspier-geassosieerde miR-206 was gevind vir 8 van die 9 vrywilligers. Die hoeveelheid miR-206 asook alomteenwoordige myomiRs-1, 133a en 133b het onveranderd gebly tussen tydintervalle. ‘n Afname in eksosoom miR-31 was wel waargeneem vanaf BL tot 24 uur na-oefening (p<0.05). Groepering van deelnemers volgens toenames (reageerders) of afnames (nie-reageerders) in eksosoom hoeveelhede vanaf BL tot 24 uur na-oefening het aan die lig gebring dat eksosoom miR-133b verminder het tussen tydintervalle (p<0.05) slegs in nie-reageerders (n=6). Afsluiting: ‘n Oorvloed van eksosome is teenwoordig in menslike plasma. NOA analise beaam dat grootte uitsluiting ‘n effektiewe eksosoom ontginningsmetode was. Na afloop van OGSS het die profiel van die eksosoom vrag gedeeltelik verander, alhoewel die getal en grootte van sirkulerende eksosome onveranderd gebly het. ‘n Vermindering slegs in eksosoom miR-31 na OGSS beklemtoom spesifisiteit. Verskillende reaksies is tussen reageerders en nie-reageerders waargeneem.

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