Sphingolipids are involved in short-term muscle disuse: effects of the inhibitor of acid sphingomyelinase clomipramine

Our previous data (Bryndina et al., 2014, 2018) evidence the accumulation of sphingolipid backbone ceramide in disused during short-term (6-12 h) or more prolonged period (4-14 days) rat soleus muscle. We also established previously that one of the main mechanisms of ceramide accumulation in unloaded soleus muscle is activation of sphingomyelinase hydrolysis (Bryndina et al., 2017). To further elucidation of the possible effects of ceramide enhancement we performed the experiments in which HS was combined with the pretreatment of rats with the functional inhibitor of acid sphingomyelinase (aSMase) clomipramine. Disuse was simulated by hindlimb suspension (HS) using well known model of Novikov-Iĺin in Morey-Holton modification. By immunofluorescence study (IF), we found that clomipramine partly prevented ceramide increase both in sarcolemma and cytoplasma of muscle fibers. Confocal microscopy demonstrated that this drug also prevented disuse-induced lipid raft disturbance, predominatly in the region of neuromuscular junction (Bryndina et al., 2018) and effectively declined the junctional ceramide level (Petrov et al., 2019). It is known that TNFa can induce heightened sphingomyelinases activity (Schütze et al., 1992; Wiegmann et al., 1994; Schwandner et al., 1998; Wiegmann et al., 1999, Moylan et al., 2014). Therefore, we hypothesized that the possible mechanism of aSMase activation during muscle disuse is an enhancement of TNFa level. We did not find the increase of TNFa (WB, IF) in rat soleus muscle suspended during 12 h. This is consistent with the results of Hirose et al. (2008), who observed an increase in TNFa content only by the 3rd day of HS. But interestingly, TNFa receptors 1 (TNFR1, WB) expression was higher in membrane rafts isolated from muscle by ultracentrifugation in sucrose gradient (Petrov et al., 2019). It suggests that the expression of TNFR1 can precede the growth in TNFa concentration, apparently increasing the sensitivity of the muscle to the action of this cytokine. Clomipramine did not affect TNFa and TNFR1 levels, evidenced the downstream effect of cytokine upon sphingolipids. In order to evaluate the apoptotic / antiapoptotic mechanisms, which can be activated through the TNFa/TNFR1 mechanism, the expression of Bax and Bcl-2 (WB, IF) was performed. It was found that both Bax and Bcl-2 amount was increased after 12-h unloading. Clomipramine administration led to additional increase in Bcl-2 level in soleus muscle of suspended animals, while the level of Bax did not differ significantly from HS. The data obtained indicate that short-term muscle disuse leads to the enhanced TNFR1 expression and aSMase activation followed by ceramide accumulation in muscle fibers. Induced by unloading proapoptotic and antiapoptotic mechanisms may partly depend on sphingolipid signaling.