Autophagic flux induced by exercise-like response in muscle cells

• Grant number: 17/03387-4
• Support type: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): May 01, 2017
• Effective date (End): April 30, 2018
• Field of knowledge: Health Sciences - Physical Education
• Principal researcher: Patricia Chakur Brum
• Grantee: Gabriel Carvalho Botelho
• Home Institution: Escola de Educação Física e Esporte (EEFE). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Improved skeletal muscle mitochondrial function is considered a major player for many aerobic exercise (AE)-induced health benefits. In addition to the increased number of mitochondria (biogenesis), AE-induced mitochondrial adaptations also relies on mitochondrial quality control mechanisms such as fission, fusion and removal of damaged/aged mitochondria via autophagy (mitophagy). On this matter, our laboratory has been exploring new mechanisms involved in AE-induced mitophagy. Coupling bioinformatics tools to the analysis of skeletal muscle mitochondrial proteome of mice submitted to a single bout of AE, we have identified proteins that might interact with LC3 (autophagic marker) and induce mitophagy. Validation of these candidates requires strategies to stimulate autophagic flux similarly to AE in cultured muscle cells. Thus, our aim is to investigate autophagic flux in muscle cells treated with compounds that simulate molecular/biochemical effects of AE in vitro. For this, C2C12 myoblasts stably expressing mCherry-GFP-LC3 will be treated with AICAR (AMPK inducer), Caffeine (mobilization of intracellular calcium) or Clenbuterol (²-adrenergic agonist). Using a fluorescence microscope coupled to a CO2 incubator, we will measure autophagic flux in live cells by the differential colocalization of mCherry and GFP. This analysis will be performed every 2 hours, up to a maximum of 12 hours after treatments. Despite of being effective in inducing in vitro responses similar to AE, these compounds do so by different molecular pathways. Hence, identifying the temporal window of action for these compounds on the autophagic flux will aid in understanding the mechanisms involved in the AE-induced autophagy. Finally, the results herein obtained will enable further experiments aimed to test candidates potentially involved in AE-induced mitophagy. (AU)