Role of Sphingosine 1-Phosphate Signalling Axis in Muscle Atrophy Induced by TNFα in C2C12 Myotubes

Abstract

Skeletal muscle atrophy is characterized by a decrease in muscle mass causing reduced agility, increased fatigability and higher risk of bone fractures. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), are strong inducers of skeletal muscle atrophy. The bioactive sphingolipid sphingosine 1-phoshate (S1P) plays an important role in skeletal muscle biology. S1P, generated by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK1/2), exerts most of its actions through its specific receptors, S1P1–5. Here, we provide experimental evidence that TNFα induces atrophy and autophagy in skeletal muscle C2C12 myotubes, modulating the expression of specific markers and both active and passive membrane electrophysiological properties. NMR-metabolomics provided a clear picture of the deep remodelling of skeletal muscle fibre metabolism induced by TNFα challenge. The cytokine is responsible for the modulation of S1P signalling axis, upregulating mRNA levels of S1P2 and S1P3 and downregulating those of SK2. TNFα increases the phosphorylated form of SK1, readout of its activation. Interestingly, pharmacological inhibition of SK1 and specific antagonism of S1P3 prevented the increase in autophagy markers and the changes in the electrophysiological properties of C2C12 myotubes without affecting metabolic remodelling induced by the cytokine, highlighting the involvement of S1P signalling axis on TNFα-induced atrophy in skeletal muscle.

S1P Signalling Axis Is Necessary for Adiponectin-Directed Regulation of Electrophysiological Properties and Oxidative Metabolism in C2C12 Myotubes

“…With regard to Ca 2+ currents, the increase in amplitude evoked by Adn was hampered when S1P biosynthesis was prevented. Notably, the blockade of SK1 altered per se Ca 2+ and K + currents in the absence of Adn as previously reported [ 37 ], supporting the hypothesis that, in control conditions, SK1 activity and the consequent S1P formation may have a role in the modulation of the appropriate ion channel permeability and may help to prevent an excessive Ca 2+ entry through voltage dependent Ca 2+ channels. With regard to SK2 inhibitor treatment alone, this induced the appearance of highly variable currents, but not significantly different to those recorded in control condition, suggesting that the enzyme is not involved per se in the control of ion channel opening.…”

“…S1P indeed plays a crucial role in satellite cell activation [ 27 ], proliferation [ 4 ], myoblast differentiation [ 28 ] and migration [ 56 ]. More importantly, S1P signalling axis is emerged to be under the control of numerous well-known physiological and pathological cues that regulate fundamental skeletal muscle processes such as PDGF [ 53 ], IGF-1 [ 52 ] TGFβ [ 57 , 58 ] and TNFα [ 37 , 54 ].…”

“…Once filled, the pipette resistance was 3–7 MΩ. The apparatus for the electrophysiological measurements consisted of the Axopatch 200 B amplifier, A/D-D/A interfaces Digidata 1200; Pclamp 6 software (Axon Instruments, Foster City, CA, USA) as described previously [ 37 , 38 ]. In the current clamp mode of the 200 B amplifier, we recorded the resting membrane potential (RMP) by using a stimulus I = 0.…”

Antagonizing S1P3 Receptor with Cell-Penetrating Pepducins in Skeletal Muscle Fibrosis

“…Murine C2C12 myoblasts were routinely grown in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C in 5% CO 2 [27]. When requested, cells were incubated with the S1P 3 antagonists KRX-725-II, [Tic] 4 -KRX-725-II and [D-Tic] 4 -KRX-725-II at the indicated concentrations, 1 h before being challenged with 5 ng/mL TGFβ1 for 48 h.…”

A2B Adenosine Receptors and Sphingosine 1-Phosphate Signaling Cross-Talk in Oligodendrogliogenesis

“…Extracellular stimuli including growth factors and cytokines such as epidermal growth factor (EGF), PDGF, vascular endothelial growth factor (VEGF), insulin-like growth factor- 1 (IGF-1), TNFα and TGFβ can induce the activation of SphK1 that translocates from cytosol to the plasma membrane to produce S1P ( Donati et al, 2007 ; Bernacchioni et al, 2012 ; Bernacchioni et al, 2021 ). The bioactive sphingolipid can act in an autocrine or paracrine manner after its release in the extracellular milieu (called “inside-out signaling”), depending on specific and/or unspecific transporters, such as the S1P transporter spinster homolog 2 (SPNS2) ( Hisano et al, 2012 ; Spiegel et al, 2019 ), the major facilitator superfamily transporter 2b (MFSD2B) ( Kobayashi et al, 2018 ) and some, unspecific, ATP-binding cassette (ABC) transporters ( Mitra et al, 2006 ; Figure 2 ).…”