Background: There is paucity of data indicating the role of cytokines including TNFa in the development of disuse muscle atrophy, despite the growing interest to this problem emerging in the recent years. The aim of the present study was to test the hypothesis that TNFa/ TNFR1 may be involved in the development of disuse muscle atrophy caused by unloading through aSMase/ ceramide/ ROS mechanism.
Methods: The experiments were performed on male Wistar rats (180 – 230g) subjected to 4 or 14 days of hindlimb suspension (HS) and treated with clomipramine (HS+Clom) or vehicle. The following parameters were studied: TNFR1, aSMase, nSMase and Nox2 proteins and ceramide in detergent-resistant membrane (DRM) fraction isolated from soleus muscle homogenates, pro-oxidant/anti-oxidant and pro-apoptotic/anti-apoptotic activities, immune fluorescence intensity and distribution of ceramide, Nox2, Nox4 and caveolin-3 on longitudinal and transverse muscle sections. The relative muscle mass, cross-sectional area (CSA) and Feret’s diameter (FD) of muscle fibers were used to confirm muscle atrophy. Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post hoc test, or Cruskall-Wallis and Mann-Whitney U test.
Results: Disuse caused an increase in membrane TNFR1, aSMase, ceramide abundance in DRM, up-regulation of pro-oxidant and pro-apoptotic capacities (increased Nox2, Nox4, TBA-active products, Bax/Bcl-2 ratio, elevated activity of caspase-3/7 and -6). The most of alterations were maximal on the 4 th day of unloading. The inhibitor of aSMase clomipramine attenuated ceramide accumulation, decreased pro-oxidant and pro-apoptotic activities and diminished muscle atrophy induced by unloading. It has been shown that in suspended for 14 days rats the loss in relative muscle mass, CSA and FD averaged -35%, -65% and -49%, respectively, whereas in clomipramine treated rats it was -25%, -45% and -25%, in comparison with the control values. Clomipramine also mitigated the inhibition of the mTORC1/p70S6 kinase inhibition caused by 14-day HS.
Conclusions: The obtained results indicate the involvement of aSMase/ ceramide pathway in the development of disuse muscle atrophy. This effect may be triggered by TNFR1 and realized through enhanced prooxidant NADPH oxidase activity and pro-apoptotic signaling.