Pulmonary Pseudomonas aeruginosa infection induces autophagy and proteasome proteolytic pathways in skeletal muscles: effects of a pressurized whey protein-based diet in mice.

Background: Pulmonary Pseudomonas aeruginosa infection in cystic fibrosis patients is associated with skeletal muscle atrophy. In this study, we investigated the effects of P. aeurginosa infection and a whey protein-rich diet on skeletal muscle proteolytic pathways. Design: An agar bead model of pulmonary P. aeurginosa infection was established in adult C57/Bl6 mice. Protein ubiquitinaiton, lipidation of LC3B protein and expressions of autophagy-related genes and ubiquitin E3 ligases were quantified using immunoblotting and qPCR. The effects of pressure-treated whey protein diet on muscle proteolysis were also evaluated. Results: Pulmonary P. aeurginosa infection reduced diaphragm, tibialis anterior, and soleus muscle weights and increased protein ubiquitination, LC3B protein lipidation, and the expressions of Lc3b, Gabarapl1, Bnip3, Parkin, Atrogin-1, and MuRF1 genes in each muscle. These changes were greater in the tibialis as compared to soleus and diaphragm. Proteolysis indicators increased within one day of infection but were not evident after seven days of infection. A pressurized whey diet attenuated LC3B protein lipidation, expressions of autophagy-related genes (BNIP3), pro-inflammatory cytokines, and protein ubiquitination. Conclusions: We conclude that pulmonary P. aeruginosa infection activates the autophagy, and the proteasome pathways in skeletal muscles and that a pressurized whey protein diet attenuates muscle proteolysis in this model.

Differential Regulation of the Autophagy and Proteasome Pathways in Skeletal Muscles in Sepsis.

OBJECTIVES: Skeletal muscle fiber atrophy develops in response to severe sepsis, but it is unclear as to how the proteolytic pathways that are involved in its development are differentially regulated. We investigated the link between sepsis-induced fiber atrophy and activation of the proteasome and autophagy pathways and whether the degree of activation is more severe and sustained in limb muscles than it is in the diaphragm. DESIGN: Randomized controlled experiment. SETTING: Animal research laboratory. SUBJECTS: Adult male C57/BL6 mice. INTERVENTIONS: Two groups of animals were studied. The sepsis group was subjected to a cecal ligation and perforation technique, whereas the control (sham) group was subjected to abdominal surgery without cecal ligation and perforation. Measurements for both groups were performed 24, 48, and 96 hours after the surgical procedure. MEASUREMENTS AND MAIN RESULTS: Atrophy was quantified in the diaphragm and tibialis anterior by measuring fiber diameter. Autophagy was evaluated using electron microscopic detection of autophagosomes and by measuring LC3B protein lipidation and autophagy-related protein expressions. Proteasomal degradation was quantified by measuring chymotrypsin-like activity of the 26S proteasome and messenger RNA expressions of muscle-specific E3 ligases. Sepsis triggered transient fiber atrophy in the diaphragm that lasted for 24 hours and prolonged atrophy in the tibialis anterior that persisted for 96 hours. The autophagy and proteasome pathways were activated in both muscles at varying intensities over the time course of sepsis. Activation was more pronounced in the tibialis anterior than in the diaphragm. Sepsis inhibited the V-Akt thymoma viral oncogene homolog 1 and complex 1 of the mammalian target of rapamycin pathways and stimulated the AMP-activated protein kinase pathway in both muscles. CONCLUSIONS: Sepsis triggers more severe and sustained muscle fiber atrophy in limb muscles when compared with respiratory muscle. This response is associated with enhanced proteasomal and autophagic proteolytic pathway activities and is triggered by inhibition of the AKT and complex 1 of the mammalian target of rapamycin pathways and activation of the AMPK pathway.