Mechanisms of exercise-induced mitophagy: a new avenue to drug discovery
| Grant number: | 17/20730-4 |
| Support type: | Scholarships in Brazil - Scientific Initiation |
| Effective date (Start): | January 01, 2018 |
| Effective date (End): | December 31, 2018 |
| Field of knowledge: | Biological Sciences - Physiology |
| Principal Investigator: | Luiz Henrique Marchesi Bozi |
| Grantee: | Gabriel Santos Arini |
| Home Institution: | Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil |
Abstract Physical inactivity is considered one of the main factors for the increase in the incidence of chronic-degenerative diseases. The benefits of physical exercise are directly linked to the improvement of mitochondrial bioenergetic input. Recently, we demonstrated that the optimization of cardiac mitochondrial metabolism in physical exercise is associated to the degree of mitochondrial plasticity, characterized by changes in the size, number and location of mitochondria in the affected musculature. These events are controlled mainly by mitochondrial fusion, fission and removal (mitophagy) processes. However, it is still unclear which of these processes is the limiting factor in the mitochondrial bioenergetic adaptations resulting from physical exercise. In this view, in the present proposal we will test the hypothesis that the aerobic physical exercise stimulates the mitochondrial fission, facilitating the removal of the mitochondria less effectives and, subsequently, the fusion of the remaining mitochondria. This process will result in the selection/production of mitochondria more efficient in meeting the high-energy demand of the exercise. This kind of research is incipient and difficult to approach since the existing experimental models make it impossible to perform an in vivo cross-sectional analysis of the effect of physical exercise on mitochondrial dynamics and mitophagy. Recently, we established a model of physical exercise in swimming for the nematode Caenorhabditis elegans. We chose this experimental model because this animal has important characteristics: easy genetic manipulation, high homology with the human genome, being transparent (allowing analysis of integrated physiology in real time) and, more importantly, enable the performance of large-scale functional genomic analyzes using siRNA libraries. For this, Caenorhabditis elegans exhibiting dual labeling for mitochondria and autophagy in muscle cells will undergo a protocol of physical swimming exercise. At the time of 0.5h, 1h, 2h, 4h after exercise and 24h after a 4h exercise session, we will evaluate the mitochondrial morphology and autophagy by confocal microscopy. Next, the animals will be incubated prior to physical exercise with chloroquine (20mM/3h) to test the involvement of autophagy in the possible removal of fragmented mitochondria. To characterize the mitophagic process in the exercise, we will use animals that present, in addition to the marked mitochondria, mitophagic proteins PINK1 or PDR1 fused to fluorophores. We will also evaluate if the mitochondrial fission process and consequent selective removal by mitophagy is associated with the loss of mitochondrial membrane potential in physical exercise. For this, animals with cell muscle mitochondria labeled with GFP will be previously incubated with MitoTracker (100nM/20min), a fluorescent probe that accumulates in mitochondria given its membrane potential. In the aforementioned evaluation times, we will determine the physical exercise effects on membrane potential, evaluating the fluorescence ratio of Mitotraker and GFP acquired by confocal microscopy. Finally, we will determine if the mitophagic process is necessary for the adaptative response to physical exercise. For that, mutant animals for PARKIN and PINK1 proteins will undergo a swimming exercise session of with a minimum duration to activate the mitophagic process. Then, we will evaluate if a single exercise session will be sufficient to protect the nematodes against thermal and oxidative stress. This proposal is achievable and feasible, once all the tests are standardized. Our preliminary results are promising as they point to mitochondrial fission throughout the exercise. We count on the collaboration of Profs. Alexander van der Bliek (UCLA), Eyleen O'Rourke (U. Virginia), Marcelo Mori (Unicamp) and Julio Ferreira (ICB - USP). (AU) | |