Lipid raft disruption is an early event during skeletal muscle unloading. Ceramide (Cer) serves as a signaling lipid that can contribute to lipid raft disturbance and muscle atrophy. Using biochemical and fluorescent approaches, the distribution of Cer and related molecules in the rat soleus muscle subjected to 12 h of hindlimb suspension (HS) was studied. HS led to upregulation of TNFα receptor 1 (TNFR1), Cer-producing enzymes, and acid and neutral sphingomyelinase (SMase) in detergent-resistant membranes (lipid rafts), which was accompanied by an increase in Cer and a decrease in sphingomyelin in this membrane fraction. Fluorescent labeling indicated increased Cer in the sarcoplasm as well as the junctional (synaptic) and extrajunctional compartments of the suspended muscles. Also, a loss of membrane asymmetry (a hallmark of membrane disturbance) was induced by HS. Pretreatment with clomipramine, a functional inhibitor of acid SMase, counteracted HS-mediated changes in the Cer/sphingomyelin ratio and acid SMase abundance as well as suppressed Cer accumulation in the intracellular membranes of junctional and extrajunctional regions. However, the elevation of plasma membrane Cer and disturbance of the membrane asymmetry were suppressed only in the junctional compartment. We suggest that acute HS leads to TNFR1 and SMase upregulation in the lipid raft fraction and deposition of Cer throughout the sarcolemma and intracellularly. Clomipramine-mediated downregulation of acid SMase can suppress Cer accumulation in all compartments, excluding the extrajunctional plasma membrane.

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Muscle disuse and denervation leads to muscle atrophy, but underlying mechanisms can be different. Previously, we have found ceramide (Cer) accumulation and lipid raft disruption after acute hindlimb suspension (HS), a model of muscle disuse. Herein, using biochemical and fluorescent approaches the influence of unilateral denervation itself and in combination with short-term HS on membrane-related parameters of rat soleus muscle was studied. Denervation increased immunoexpression of sphingomyelinase and Cer in plasmalemmal regions, but decreased Cer content in the raft fraction and enhanced lipid raft integrity. Preliminary denervation suppressed (1) HS-induced Cer accumulation in plasmalemmal regions, shown for both nonraft and raft-fractions; (2) HS-mediated decrease in lipid raft integrity. Similar to denervation, inhibition of the sciatic nerve afferents with capsaicin itself increased Cer plasmalemmal immunoexpression, but attenuated the membrane-related effects of HS. Finally, both denervation and capsaicin treatment increased immunoexpression of proapoptotic protein Bax and inhibited HS-driven increase in antiapoptotic protein Bcl-2. Thus, denervation can increase lipid raft formation and attenuate HS-induced alterations probably due to decrease of Cer levels in the raft fraction. The effects of denervation could be at least partially caused by the loss of afferentation. The study points to the importance of motor and afferent inputs in control of Cer distribution and thereby stability of lipid rafts in the junctional and extrajunctional membranes of the muscle.

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.