Behaviour of human myoblasts in vitro: role of ageing and inflammatory cytokines

Ageing is associated with a loss of muscle mass, a processes known as “sarcopenia”. It has been postulated that one of the reasons underlying this muscle loss is a decreased ability to repair itself in response to damage. Repair of muscle is facilitated by a specific population of progenitor adult stem cells known as satellite cells in situ and myoblasts ex vivo. The work in this thesis has used a cell culture approach to study the behaviour of human myoblasts and compare the inherent effects of age (by studying myoblasts taken from young and old people) and using an in vitro model of ageing, (i.e. proliferative senescence), and investigating the contribution of environmental factors by culturing the cells in human serum from young and old participants and treating them with recombinant cytokines.<br/>Several parameters were studied, but the main focus was on ability of myoblasts to proliferate and differentiate. Markers of proliferating muscle cells were desmin, NCAM and Ki67, and markers of differentiating muscle cells were myogenin and myosin heavy chain. Additional parameters observed were DNA damage and analysis of specific cytokines in the cell secretome. No differences in any parameter measured were found between cells of young and old people. However senescent myoblasts differed significantly in all parameters from early passage cells. Differentiation was studied over a seven day period. There was a delay of two-four days in the onset of markers expression between senescent and early passage cells, as well as a decrease in the expression levels of myogenin (50 ± 3% in young, 49 ± 3% in old and 6 ± 1% in senescent) after three days of differentiation and myosin heavy chain (MHC; 71 ± 2% in young, 70 ± 1.4% in old and 15 ± 1% in senescent) after five days of differentiation. In addition, increased DNA damage (7 ± 1% in young, 8 ± 1% in old and 90 ± 4% in senescent), increased TGF-β secretion (111 ± 13pg/ml in young, 115 ± 19 pg/ml in old and 268 ± 11pg/ml in senescent), and decreased myotube area (151574 ± 22968μm2 in young, 132531 ± 25106μm2 in old and 47765 ± 763μm2 in senescent) were observed in senescent cells compared to early passage cells.<br/>For influence of environmental factors, freshly isolated cells were cultured in human sera or medium with or without cytokines. No differences were observed in the myoblasts cultured in sera from young and elderly individuals (Ki67 expression was 85 ± 2% in young and 84 ± 2% in old, and desmin expression was 81 ± 2% in young and 83 ± 3% in old at three days after isolation). There was, however, a significant decrease in desmin and myogenin expression when cells were exposed to TGF-β1 (1 ng/ml), TNF-α (1 ng/ml) or IL-1β (1 ng/ml). Committed myoblasts were also cultured in the presence or absence of TGF-β1, TNF-α, or IL-1β. Myogenin but not desmin expression was significantly inhibited in the presence of cytokines.<br/>These findings support the observation of many studies in vivo in both human and animal models which show that satellite cells from old muscle can contribute to muscle repair and regeneration. This suggests the satellite cells per se may not be critically involved in the mechanism responsible for sarcopenia. However, their sensitivity to inflammatory cytokines suggests that if these were present in vivo they would affect the myogenic behaviour of the cells.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

King's College London

Year dc:date.issued

2012

Author dc:creator

• Alsharidah, Mansour

Advisors dc:contributor.advisor

• Harridge, Stephen David Royston
• Velloso, Cristiana Paranhos

Language dc:language

eng

Identifier

oai:kclpure.kcl.ac.uk:studenttheses/f544ecce-946d-40f1-91a4-fd750aacee11

OAI identifier oai:identifier

oai:kclpure.kcl.ac.uk:studenttheses/f544ecce-946d-40f1-91a4-fd750aacee11