Doctoral Degrees (Physiological Sciences)
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Browsing Doctoral Degrees (Physiological Sciences) by Author "Lovett, Jason Andrew Charles"
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- ItemInvestigating the diversity of extracellular vesicle microRNA in response to exercise-induced muscle micro-damage: comparison of targeted plasma microRNA to targeted and in-depth analysis of extracellular vesicle microRNA(Stellenbosch : Stellenbosch University, 2020-12) Lovett, Jason Andrew Charles; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background: Skeletal muscle (SkM) damage occurs routinely in sport and is present in acquired and inherited myopathies. Damage is confirmed by invasive muscle biopsy. Molecular events that occur during SkM injury and repair, including localized immune cell involvement, may be reflected in circulation. A detailed understanding is essential to the development of diagnosis and intervention strategies. Extracellular vesicles (EVs) are nanoscale (30 to 1000 nm) mediators of intercellular communication. EVs are released in abundance by all cell types, contain cargo including microRNA, and are stable in circulation. Circulating EV profiles adapt to differing physiological and pathological states. EVs may therefore serve as non-invasive biomarkers of SkM injury and regenerative processes. Aims: To determine the biomarker potential of microRNAs in plasma, large EVs and small EVs in regard to skeletal muscle damage induced by unaccustomed eccentrically biased exercise. Methods: Two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by 9 healthy male volunteers. Blood samples were taken at baseline, 2 and 24 hr post-exercise. Serum creatine kinase (CK) and perceived muscle pain (PMP) served as indirect markers of muscle damage. Canonical myomiRs, SkM- important miRs and immune-important miRs were analysed in 3 avenues of biomarker: plasma, large EVs and small EVs. Large EVs were isolated using a 10 000 G centrifugation step, whilst the subsequent supernatant was used for plasma analysis and qEV size exclusion isolation of small EVs. Small and large EV-enriched isolates were visualized using STEM, and size and numbers were quantified using NTA. Based on NTA results the highest particle fractions (7- 10) from qEV columns were pooled for RNA analysis. qPCR analysis of plasma, large EV and small EV miRs was done with normalization to an exogenous control. Small RNA sequencing was done on 3 randomly selected participants’ small EV samples from baseline and 24 hr. Results: PMP and CK increased post-exercise. Small EVs were confirmed using gel electrophoresis, NTA and STEM. Large EVs were confirmed using NTA and STEM. Both small and large EVs were visualised using STEM. No change in small or large EVs size or number was seen post-exercise. Plasma miR-1, 133a and 206 increased in abundance at 2 hr, with miR-1 and 206 returning to BL levels at 24 hr. Conversely, plasma miR-208b & 499 also increased at 2 hr, but showed a trend toward a sustained increase at 24 hr. miR-31, a SkM-important miR, decreased in small EVs at 24 hr when compared to BL and 2 hr. No change in immune-important miRs was found in any biomarker source. Lastly, small RNA sequencing revealed an increase in 18 miRs in small EV at 24 hr post-exercise, with a concomitant decrease in 10 miRs. Conclusion: Both small EV and plasma miRs changed in response to muscle-damaging exercise. Each exhibited distinct miR changes, and timing of changes as shown by qPCR. miR-31, for example, decreased only in small EVs post-exercise, whereas most myomiRs increased only in plasma. RNA sequencing revealed more small EV miR changes. However, these were not corroborated with qPCR.