ATP as a first messanger controlling excitation-transcription coupling of skeletal muscle

Inactivity and microgravity cause both significant muscle atrophy and fibre type switching. Muscle mass and fibre type composition is under the control of signalling pathways that only recently are being defined. The overall process governing muscle growth and fibre type determination is defined excitation-transcription coupling. Mass reduction (atrophy) is associated to the prevailing of catabolism over anabolism and, in particular, to the activation of the ubiquitin-proteasome system. Muscle growth is primarily mediated by the PI3K-PKB/Akt system through the recruitment of protein kinase mTOR and NFATc2, a downstream effector of calcineurin. In addition, muscle growth is also the result of PI3K-PKB repressing activity over the transcription factors of the Foxo family, which activators of ubiquitin ligase atrogin-1 and MuRF1 activities. As a consequence, the atrophic program is initiated by the removal of the inhibitory action of the Akt system over Foxo. Thus, it is emerging a scenario where a wide cross talk of diverse signalling pathways appears to govern muscle growth or reduction. However, it is still unclear the major determinant initiating these signalling pathways. What is certain is that the “first messenger” is related to the electrical activity and, in part, to trophic factors released from the nerve. It is predictable that the initiating events occur at cell membrane, however, the molecular element(s) involved in the process are still unknown. It is also certain that the lack of such messenger(s) represents the primary event leading to mass reduction. The main objective of our project is to investigate the upstream processes governing the overall excitation-transcription coupling of skeletal muscle and disclose the nature of the first messenger that mediates the transcriptional activity of skeletal muscle electrical stimulation. The proposed project is also willing to introduce a novel approach to study the signal transduction pathways involved in muscle atrophy. In fact, besides animal experimental models that mimic microgravity, we want to utilize adult muscle fibres isolated and cultivated in vitro. In our laboratory we have established culture conditions that permit to cultivate in vitro isolated muscle fibres for as long as three weeks. The isolation procedure, by removing the nerve, eliminates any contractile activity of cultivated fibres and starts the atrophic program and the cognate signalling pathways. Since atrophy in cultured muscle fibres progresses very slowly, this protocol appears to be an ideal tool to scrutinize in details signalling pathways that activate atrophy. Importantly, we have demonstrated recently that cultivated muscle fibres release ATP in the extracellular medium each time they are electrically stimulated, a condition that mimic contractile activity. This finding makes the nucleotide a good candidate as a first messenger promoting excitation-transcription coupling. In fact, extracellular ATP signalling is a well known modulator of various physiological responses, including cell growth, platelet activation, neurotransmission, immune responses, cell migration, proliferation and apoptosis. Consistent with our hypothesis is the fact that skeletal muscle expresses diverse cell membrane ATP receptors, whose function is still undefined. Importantly, we have recently demonstrated that the nucleotide exerts an autocrine action in potentiating muscle contraction, an effect that is abolished if soluble ATP-hydrolyzing enzymes are added to the contracting muscle or in the presence of specific inhibitors of ATP receptors. We propose that under microgravity and, more in general, under conditions of reduced muscle use (bed rest, cachexia, and aging) the decreased contractile activity is strictly correlated with the drop of ATP release and of the cognate signalling functions.