Lifelong Exercise Training Modulates Cardiac Mitochondrial Phosphoproteome in Rats

Moderate
physical activity has traditionally been associated with
the improvement of cardiac function and, consequently, with the extension
of life span. Mitochondria play a key role in the adaptation of heart
muscle to exercise-related metabolic demands. In order to disclose
the molecular mechanisms underlying the beneficial effect of lifelong
physical activity in cardiac function, we performed label-free quantitative
mass spectrometry-based proteomics of Sprague–Dawley rat heart
mitochondrial proteome and phosphoproteome. Our data revealed that
54 weeks of moderate treadmill exercise modulates the abundance of
proteins involved in the generation of precursor metabolites and cellular
respiration, suggesting an increase in carbohydrate oxidation-based
metabolism. Moreover, from the 1335 phosphopeptides identified in
this study, 6 phosphosites were exclusively assigned to heart mitochondria
from sedentary rats and 17 to exercised animals, corresponding to
6 and 16 proteins, respectively. Most proteins exhibiting significant
alterations in specific phosphorylation sites were involved in metabolism.
Analysis of the acquired data led to the identification of several
kinases potentially modulated by exercise training, which were selected
for further validation. Indeed, higher protein abundance levels of
RAF and p38 in mitochondria were confirmed to be modulated by sustained
exercise. Our work describes the plasticity of heart mitochondria
in response to long exercise programs manifested by the reprogramming
of phosphoproteome and provides evidence for the kinases involved
in the regulation of metabolic pathways and mitochondrial maintenance.