Background: Physical activity can prevent or delay many obesity-associated diseases. Even though reduction in fat mass clearly is health promoting, beneficial effects of physical activity are also observed regardless of weight change. This suggests additional mechanisms linking physical activity and health status. The recent discovery that contracting muscles secrete peptides (named myokines) has put forward a hypothesis suggesting that working muscles regulate the systemic metabolism through humoral signaling. This may be an important mechanism involved in the beneficial health effects of physical activity. To investigating physical activity on molecular levels, in vitro models of exercise, such as cell cultures of skeletal muscles, is greatly beneficial.
Objectives: This thesis had two major objectives. The first objective was to optimize a protocol for differentiation of human skeletal muscle cells in culture by manipulating experimental conditions, and subsequently evaluate cell morphology and molecular aspects. I aimed to investigate which serum type (HS, FBS, UG) and concentrations that provided highest degree of differentiation, and also to investigate the effect of dexamethasone and insulin. Additionally, I aimed to characterize molecular aspects of cells differentiating in the most promising media. The second objective was to identify and characterize expression of myokines during in vitro myogenic differentiation. I aimed to investigate mRNA expression and secretion of both known and potential myokines.
Results: 1) Romanowsky staining was established and used to calculate a fusion index in order to determine the degree of differentiation in the muscle cell cultures. 2) HS at concentration of 0.5 and 1.0 % showed the best differentiation, but with no statistically significant difference from the other media. 3) Dexamethasone in higher doses inhibited differentiation, whereas increasing concentration of insulin in SFM promotes a tendency towards lower differentiation. 5) mRNA expression of PAX7 was reduced during differentiation of myoblasts in 1.0 and 2.0 % HS, while myogenin, myosin, alpha-actin, PGC1α and GLUT4 were increased. There was no differences between these concentrations of HS, but in SFM and the proliferation medium the gene profile was substantially different. Protein content of myosin and actin increased with differentiation, reaching the highest level on day 7 of differentiation. 6) mRNA expression of the myokines FNDC5, ANGPTL7 and PEDF strongly increased during differentiation, whereas PLAU decreased. Expression of IL-8 showed a minor change, and adiponectin was not detected. Secretion of PLAU and PEDF proteins in to the medium corresponded well with the mRNA expression pattern.
Conclusions: I have found that myoblasts differentiated for 7 days in 1.0 % HS result in well-differentiated muscle cell cultures, exhibiting at best 67 % of all nuclei inside of multinucleated myotubes. All serum-reduced media showed mRNA expression pattern corresponding to the expected changes during differentiation, but it is not possible to draw any conclusions regarding differences between 1.0 and 2.0 % HS. Furthermore, mRNA and protein expression kinetics of myosin and actin reached highest level on day 7, suggesting the cell culture to be most muscle-like at this stage. Finally, I observed distinct mRNA expression dynamics of several myokines during differentiation, and secretion of PLAU and PEDF in to the medium.