Investigation of myostatin and relevant regulators during muscle regeneration after an acute bout of eccentric exercise

Conradie, Johannes David (2014-12)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: The aim of this study was to investigate the powerful muscle regulator, myostatin, and its regulators in response to an acute bout of plyometric training. The participants were recruited and screened by characterization by means of isometric force production tests, baseline blood creatine kinase levels and VO2 max results. The selected individuals (n=15) were subjected to a baseline muscle biopsy for comparative purposes. The study made use of plyometric jumping, as source of eccentric exercise, to serve as an exercise intervention after which muscle biopsies (4 hours post and 24 hours post) and blood draw (4 hours post, 24 hours post and 48 hours post) samples were taken. Maximal voluntary isometric contractions of the knee extensors were also measured immediately after the exercise protocol and after 1 week recovery. Creatine kinase (CK) analysis on the serum samples was used to conclude muscle damage. The muscle biopsy samples were used for protein quantification (Western blot) and gene expression assessment (semi-quantitative and real-time PCR). The results showed decreased force production immediately after eccentric exercise (p < 0.05), while returning back to baseline values at 1 week post exercise and CK results showed a significant increases at 4 hours (p<0.05), 24 hours (p<0.001) and 48 hours (p<0.01) after exercise. There were no significant differences in myostatin precursor protein (43 kDa), phosphorylated Smad2,3, Smad7 or activin receptor IIb in response to eccentric exercise. However, the follistatin protein was increased at both 4 hours and 24 hours after exercise (p<0.01). RNA analysis of the extracellular matrix (ECM) protein, decorin, revealed the existence of the splice variants A1 and A2 in human skeletal muscle. The RT-PCR analysis (n=4) of these variants showed no significant difference when comparing pre- to post-exercise. The decorin core protein was also investigated by means of antibody probing and results revealed the need for ABC chondroitinase enzyme treatment before immunoblotting of human skeletal muscle samples. The results concerning knee extensor force reduction and circulating creatine kinase showed the effectiveness of plyometric jumping in producing skeletal muscle damage in the lower limbs of unfit individuals, unaccustomed to eccentric exercise. In conclusion, myostatin, and its associated signalling cascade, are not activated in early muscle regeneration, but follistatin is increased during this phase possibly aiding and initiating the muscle repair process. Future studies: Variants of decorin are expressed in human skeletal muscle, increasing the complexity that should be taken into account in studies concerning the regulation of decorin in a human model. Investigation into myostatin protein at different post-translational levels needs more clarification. Published methods and materials used in different laboratories are not consistent and investigators should attempt to standardise protocols in order to compare results between studies more effectively. Of importance, these results show that the myostatin at protein level report different results compared to mRNA analysis and that more investigation into myostatin regulatory factors, with special reference to follistatin and decorin, is needed in future human models.

AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om die kragtige spiere reguleerder, miostatin, en sy reguleerders in reaksie op 'n akute aanval van pliometriese spronge te ondersoek. Die deelnemers is gewerf en gekeur deur karakterisering deur middel van isometriese krag produksie toetse, basislyn bloed kreatien kinase vlakke en VO2maks resultate. Die geselekteerde individue (N = 15) is onderhewig aan 'n basislyn spierbiopsie vir vergelykende doeleindes. Die studie het gebruik gemaak van pliometriese spronge (essentriese spier aksie) as die oefening intervensie waarna spierbiopsie (4 uur na en 24 uur na) en bloed (4 uur na, 24 uur na en 48 uur na) monsters geneem is. Isometriese kontraksies van die knieverlengers is ook gemeet onmiddellik na die oefening protokol en na 1 week se herstel. Kreatine kinase (KK) ontleding van die serum monsters is gebruik om spierskade aftelei. Die spierbiopsie monsters was gebruik vir proteïen kwantifisering (Western klad) en die assessering van geen uitdrukking (semi-kwantitatiewe en real-time PCR). Die resultate het gewys dat krag produksie afgeneem het onmiddellik na essentriese oefening (p <0.05), terwyl dit terugkeer na die oorspronklike waardes 1 week na oefening en KK resultate toon 'n beduidende toename by 4 uur (p <0,05), 24 uur (p <0,001) en 48 uur (p <0,01) na oefening. Daar was geen betekenisvolle verskille in Miostatien voorloper proteïen (43 kDa), gefosforileerde Smad2,3, Smad7 of Activin reseptoor IIb in reaksie op essentriese oefening. Dit is egter die follistatien proteïen wat verhoog by beide 4 uur en 24 uur na oefening (p <0,01). RNS ontleding van die ekstrasellulêre matriks (ESM) proteïen, decorin, het die bestaan van die splitsing variante A1 en A2 in menslike skeletspier, aan die lig gebring. Die RT-PCR analise (n = 4) van hierdie variante het geen betekenisvolle verskille getoon wanneer voor met na-oefening vergelyk is. Die decorin kern proteïen is ook ondersoek deur middel van teenliggaam afhanklike metodes en resultate het die behoefte aan ABC chondroitinase ensiem behandeling voor immunokladding van menslike skeletspier monsters gesteun. Die resultate aangaande knieverlenger krag vermindering en sirkuleerende kreatien kinase het die doeltreffendheid van pliometriese spronge in die vervaardiging van skeletspier skade in die onderste ledemate van individue ongewoond aan essentriese oefening verseker. Ten slotte, Miostatien, en sy verwante sein kaskade, is nie geaktiveer vroeg in spier herstelling, maar follistatien is tydens hierdie fase verhoog en help moontlik met die aanvang van die spier herstel. Toekomstige studies: variante van decorin word uitgedruk in menslike skeletspier, wat die kompleksiteit aangaande decorin verhoog en dit is iets wat in ag geneem moet word in studies wat handel oor die regulering van decorin in mens modelle. Ondersoek na miostatien proteïen op verskillende na-translasie vlakke moet meer duidelikheid verkry. Gepubliseer metodes en materiaal wat gebruik word in verskillende laboratoriums is nie konsekwent en ondersoekbeamptes moet probeer om protokolle te standaardiseer sodat resultate van studies meer effektief kan vergelyk word. Van belang is, die resultate wys dat miostatien op proteïen vlak verskillende resultate vertoon in vergelyking met boodskapper-RNS ontleding en dat meer ondersoek na miostatien regulerende faktore, met spesiale verwysing na follistatien en decorin, nodig is in toekomstige menslike modelle.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/95947
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