After addition sellectchem of a chemiluminescent substrate chemiluminescence was read using a spectrophotometer. In vivo experiments All animal procedures were approved by the cantonal veterinarian office of Basel. Male Wistar rats of various ages were housed for 2 weeks on a 12 hour light/dark cycle with unrestricted access to food and water. Rats were asphyxiated using CO2 at 6, 18, 21 and 24 months of age, and the gastrocnemius muscle was imme diately dissected, weighed and snap frozen in liquid nitrogen before processing for RNA extraction. Com pared with 6 month old rats, the gastrocnemius weight was unchanged for 18 month old rats and reduced by 41% and 49% in 21 and 24 month old rats, respectively. Statistical analysis Differences between groups were analyzed using one way ANOVA for all experiments.
P 0. 05 was consid ered significant. Results and discussion The transforming growth factor b/SMAD and insulin like growth factor 1/AKT pathways modulate interleukin 1a and tumor necrosis factor Inhibitors,Modulators,Libraries a induced inhibition of human skeletal myoblast differentiation In this study, we were interested in determining the effect of cytokines on Inhibitors,Modulators,Libraries human skeletal myoblast differen tiation. As a first step, it was important to characterize whether HuSKMCs respond to cytokines in a simi lar manner to myoblast lines from other species. We used IL 1a to stimulate IL 1 receptors throughout this study, but very similar effects were seen whenever IL 1b was tested as well. HuSKMCs were differentiated in the absence or presence of IL 1a and TNF a for 5 days after the onset of differentiation, and immunostained with antibodies to MyHC as a marker for differentiation.
Similar to pre vious studies, both IL 1a and TNF a caused a marked reduction in HuSKMC differentiation, seen as a decrease in both myotube number and fusion index, which were decreased by 73% with IL 1a and by 55% witu TNF a. Moreover, CK activity, which normally increases during differentiation, was also Inhibitors,Modulators,Libraries markedly reduced by IL 1a and TNF a. no Inhibitors,Modulators,Libraries increase in CK activity was seen at any concentration tested. We next investigated whether the anti differentiation effect of IL 1a and TNF a on HuSKMCs is perturbed by IGF 1, a positive regulator of myogenesis. Treatment with IGF 1 promoted basal differentiation of HuSKMCs by up to 77%, and partially rescued them Inhibitors,Modulators,Libraries from the inhibitory effects of IL 1a and TNF a, as determined by FI and CK activity.
Because IGF 1 mediated AKT signaling has been shown to block the inhibition of differentiation caused by TGF b family mem bers, we investigated whether the intersection between cytokine signaling and IGF 1 signaling might involve the TGF b pathway. Therefore we sought to deter mine the influence Vorinostat HDAC3 of the TGF b/ALK pathway in IL 1a and TNF a action, using the ALK4/5/7 inhibitor SB431542.