Recent studies have suggested an involvement of calcineurin in the regulation of muscle mass. It has been shown that calcineurin transduces signals leading to cardiac hypertrophy and that it is required for skeletal muscle differentiation. The present study has been aimed at investigating the modulations of calcineurin expression and activity in C2C12 myotubes exposed to TNFa.
Methods: C2C12 myotubes have been cultured (24h or 48h) in the presence or in the absence of TNFa (100 ng/ml). Calcineurin mRNA expression has been evaluated by semi-quantitative RT-PCR. The DNA-binding of the transcription factor NF-AT has been detected by EMSA, and assumed as an indirect indicator of calcineurin bioactivity. As a control of TNFa action, NF-kB activation has also been evaluated by EMSA. Cleavage of the caspase-3 substrate PARP has been checked by western blot.
Results: Treatment ofC2C12myotubeswithTNFa (100 ng/ml) for 48h induces the appearance of several apoptotic figures. However, there is no evidence of PARP cleavage, suggesting the occurrence of capase-independent cell death. Calcineurin mRNA expression does not significantly differ between controls and TNFa-treated C2C12myotubes, both at 24h and 48h after treatment. The DNA-binding activity ofNF-ATis impaired inmyotubes exposed toTNFa for 48h (C=44329±649, TNFa=26609±775, p=0.039), while no changes can be observed at 24h. C2C12 myotubes typically responde to TNFa by increasing the DNA-binding activity of NF-kB.
Conclusions: These data show that treatment of C2C12 myotubes with TNFa results in modulations of calcineurin activity, not paralleled by changes in mRNA levels, that suggest an involvement of this phosphatase in TNFa signaling. These results lead to hypothesize that the development of strategies aimed at modulating calcineurin activity may be of interest for the treatment of TNFa-related cachexia.