Effects of PGC-1alpha conditional deletion and exercise on mitochondrial biogenesis, autophagy and rev-erb-alpha pathways in mouse skeletal muscle: a proteomics approach

Abstract

A sedentary lifestyle reduces mitochondrial functional integrity and, consequently, an increase in free radicals and reactive species production, leading to the onset of chronic non-communicable diseases. In this sense, the transcription factor PGC-1alfa is a promising molecular target with an essential role in regulating autophagy, mitochondrial content, and oxidative function. However, the mechanisms PGC1 alpha communicates with the circadian cycle remain unknown. The current project will investigate the relationship between PGC-1alpha and Rev-erb-alpha in C2C12 cells (myoblasts/myotubes) in vitro (step 1). After that, we will verify if the effects of physical exercise on mitochondrial biogenesis, oxidative stress, and autophagy pathways in mice's skeletal muscle are dependent on the PGC-1alpha/Rev-erb-alpha axis (step 2). Initially, the manipulation of PGC-1alpha (Knockdown and overexpression) and Rev-erb-alpha (Knockdown, Knockout, and overexpression) will be performed in C2C12 cells, and an evaluation of the protein expression of both molecules. Posteriorly, wild-type mice and conditional knockout (KO) of PGC-1alfa will be used. These groups will be submitted to short-term combined training (3 weeks). The methodologies used in this project will be chromatography and mass spectrometry for proteomics analysis, electronic paramagnetic resonance for reactive oxygen species detection, immunoblotting, histology, immunohistochemistry, immunofluorescence, bioinformatics, mitochondrial respiration, and lysosomal autophagic flow. These functional assessments and morphological, biochemical, and molecular approaches will assist in understanding the complete picture of skeletal muscle adaptation to comprehend the crosstalk between PGC-1alpha and Rev-erb-alpha. (AU)