Phosphoproteomic screening of exercise-like treatments reveals drug interactions regulating myokine secretion

Needham EJ1, Humphrey SJ1, Cooke KC1, Duan X1, Parker BL1 and James DE1,2

  1. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia.
  2. School of Medicine, The University of Sydney, Sydney, NSW, Australia.

Exercise improves health through adaptive metabolic and mechanical remodelling, mediated by a network of kinases in response to homeostatic stress. Secretion of factors from exercising muscle causes systemic benefits. The exercise kinase network provides an approach to understand the regulation of myokine secretion as we predict that activation of a subset of the kinases will trigger secretion pathways. The phosphoproteome of acutely exercised human skeletal muscle provides a blueprint of kinase regulation, however, an in vitro model of exercise is needed to dissect molecular mechanisms. We performed a phosphoproteomic screen of 10 candidate exercise-like treatments in rat L6 myotubes quantifying 20,249 unique Class I phosphopeptides. Of the regulated phosphosites in exercised human skeletal muscle that mapped to the in vitro treatments, 74% were regulated by at least one treatment. Computing in silico combinations predicted that isoproterenol combined with thapsigargin would most closely reproduce the exercise phosphoproteome. The phosphoproteome of the combined treatments had a greater overlap with exercise than the in silico prediction by 16%, highlighting the importance of interactions between stimuli. As exercise involves multiple stimuli, their interactions may be required for myokine secretion. As the combined treatments recapitulated a sizable proportion of exercise signalling, we measured the secretion of exercise factors to test if exercise-related phenotypes were also reproduced. Global mass spectrometry-based secretome analysis of treated L6 myotubes revealed known secreted factors in exercise and novel candidates. The phosphoproteome provides a resource to determine the upstream regulation of myokine secretion.