RESUMO
Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.
Assuntos
Exercício Físico/fisiologia , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico/genética , Quinases Proteína-Quinases Ativadas por AMP , Agonistas Adrenérgicos beta/metabolismo , Animais , Aripiprazol/metabolismo , Aripiprazol/farmacologia , Cálcio/agonistas , Cálcio/metabolismo , Interações Medicamentosas , Humanos , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Espectrometria de Massas , Camundongos , Fosfoproteínas/química , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sistemas de Translocação de Proteínas/genética , Sistemas de Translocação de Proteínas/metabolismo , Proteoma/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Estresse Fisiológico/fisiologia , Tapsigargina/metabolismo , Tapsigargina/farmacologiaRESUMO
We have previously developed a high-throughput bioengineered human cardiac organoid (hCO) platform, which provides functional contractile tissue with biological properties similar to native heart tissue, including mature, cell-cycle-arrested cardiomyocytes. In this study, we perform functional screening of 105 small molecules with pro-regenerative potential. Our findings reveal surprising discordance between our hCO system and traditional 2D assays. In addition, functional analyses uncovered detrimental effects of many hit compounds. Two pro-proliferative small molecules without detrimental impacts on cardiac function were identified. High-throughput proteomics in hCO revealed synergistic activation of the mevalonate pathway and a cell-cycle network by the pro-proliferative compounds. Cell-cycle reentry in hCO and in vivo required the mevalonate pathway as inhibition of the mevalonate pathway with a statin attenuated pro-proliferative effects. This study highlights the utility of human cardiac organoids for pro-regenerative drug development, including identification of underlying biological mechanisms and minimization of adverse side effects.