Adaptation of rat fast-twitch muscle to endurance activity is underpinned by changes to protein degradation as well as protein synthesis.

Muscle adaptations to exercise are underpinned by alterations to the abundance of individual proteins, which may occur through a change either to the synthesis or degradation of each protein. We used deuterium oxide (2 H2 O) labeling and chronic low-frequency stimulation (CLFS) in vivo to investigate the synthesis, abundance, and degradation of individual proteins during exercise-induced muscle adaptation. Independent groups of rats received CLFS (10 Hz, 24 h/d) and 2 H2 O for 0, 10, 20, or 30 days. The extensor digitorum longus (EDL) was isolated from stimulated (Stim) and contralateral non-stimulated (Ctrl) legs. Proteomic analysis encompassed 38 myofibrillar and 46 soluble proteins and the rates of change in abundance, synthesis, and degradation were reported in absolute (ng/d) units. Overall, synthesis and degradation made equal contributions to the adaptation of the proteome, including instances where a decrease in protein-specific degradation primarily accounted for the increase in abundance of the protein

Global proteome changes in the rat diaphragm induced by endurance exercise training

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfor- tunately, prolonged MV results in the rapid development of diaphragmatic atrophy and weakness. Importantly, endurance exercise training results in a diaphragmatic phenotype that is protected against ventilator-induced diaphragmatic atrophy and weakness. The mechanisms responsible for this exercise-induced protection against ventilator-induced dia- phragmatic atrophy remain unknown. Therefore, to investigate exercise-induced changes in diaphragm muscle proteins, we compared the diaphragmatic proteome from sedentary and exercise-trained rats. Specifically, using label-free liquid chromatography-mass spectrome- try, we performed a proteomics analysis of both soluble proteins and mitochondrial proteins isolated from diaphragm muscle. The total number of diaphragm proteins profiled in the sol- uble protein fraction and mitochondrial protein fraction were 813 and 732, respectively. Endurance exercise training significantly (P<0.05, FDR <10%) altered the abundance of 70 proteins in the soluble diaphragm proteome and 25 proteins of the mitochondrial proteome. In particular, key cytoprotective proteins that increased in relative abundance following exer- cise training included mitochondrial fission process 1 (Mtfp1; MTP18), 3-mercaptopyruvate sulfurtransferase (3MPST), microsomal glutathione S-transferase 3 (Mgst3; GST-III), and heat shock protein 70 kDa protein 1A/1B (HSP70). While these proteins are known to be cytoprotective in several cell types, the cyto-protective roles of these proteins have yet to be fully elucidated in diaphragm muscle fibers. Based upon these important findings, future experiments can now determine which of these diaphragmatic proteins are sufficient and/or required to promote exercise-induced protection against inactivity-induced muscle atrophy

ANGIOTENSIN II TYPE 1 RECEPTOR CONTRIBUTES TO VENTILATOR-INDUCED DIAPHRAGM DYSFUNCTION

S. E. Hall1, A. J. Smuder2, M. P. Wiggs2, A. B. Morton2, K. J. Sollanek2, S. K. Powers2 1 Boise State University, Boise, ID & 2 University of Florida, Gainesville, FL Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. Unfortunately, prolonged MV results in diaphragmatic atrophy and contractile dysfunction (collectively termed ventilator-induced diaphragm dysfunction, VIDD). The development of VIDD is important because diaphragm weakness can contribute to problems in weaning patients from MV. Currently, no standard treatment exists to prevent VIDD and understanding the causation of VIDD is vital for the development of a therapeutic intervention. Recent evidence reveals that increased plasma levels of Angiotensin II (Ang II) promotes oxidative stress and fiber atrophy in limb skeletal muscles. Our preliminary experiments suggest that Ang II type 1 receptor (AT1R) activation contributes to VIDD, however, MV-induced AT1R activation in the diaphragm is independent of circulating Ang II levels. Indeed, the AT1R activation could be the result of mechanical stretch of the receptor during the passive length change of the diaphragm during MV. PURPOSE: Test the hypothesis that MV-induced stretch activation of AT1R in the diaphragm is required for the development VIDD. METHODS: Two structurally different AT1R antagonists were administered during 12 hours of MV. Olmesartan has an ideal chemical structure to inhibit stretch-induced AT1R activation whereas irbesartan does not prevent stretch-induced AT1R activation. Both drugs are effective at blocking Ang II binding to AT1R. Following MV, animals were sacrificed and measures of VIDD were performed. RESULTS: Treatment of animals with Olmesartan protected against VIDD whereas irbesartan did not protect against VIDD, as determined by contractile force and cross sectional area. Diaphragm contractile force at 160 Hz (in addition to other frequencies) was significantly improved in the MV-Olmesartan group (22.8 N/cm2) compared to the MV group (21.2 N/cm2), p\u3c.05. In addition, the decrease in cross sectional area seen with MV was attenuated by only olmesartan across all fiber types; type I (905.3 μm2 vs. 1205.4 μm2), type IIa (991.0 μm2 vs. 1351.0 μm2), and type IIb/x (2336.9 μm2 vs. 2891.0 μm2), p\u3c.05. CONCLUSION: These results suggest that stretch activation of AT1R is essential for the development of VIDD. Importantly, these experiments provide evidence that the FDA approved drug olmesartan may have clinical benefits in the protection against VIDD in humans

Crosstalk between autophagy and oxidative stress regulates proteolysis in the diaphragm during mechanical ventilation.

Mechanical ventilation (MV) results in the rapid development of ventilator-induced diaphragm dysfunction (VIDD). While the mechanisms responsible for VIDD are not fully understood, recent data reveal that prolonged MV activates autophagy in the diaphragm, which may occur as a result of increased cellular reactive oxygen species (ROS) production. Therefore, we tested the hypothesis that (1) accelerated autophagy is a key contributor to VIDD; and that (2) oxidative stress is required to increase the expression of autophagy genes in the diaphragm. Our findings reveal that targeted inhibition of autophagy in the rat diaphragm prevented MV-induced muscle atrophy and contractile dysfunction. Attenuation of VIDD in these animals occurred as a result of increased diaphragm concentration of the antioxidant catalase and reduced mitochondrial ROS emission, which corresponded to reductions in the activity of calpain and caspase-3. To determine if increased ROS production is required for the upregulation of autophagy biomarkers in the diaphragm, rats that were administered the mitochondrial-targeted peptide SS-31 during MV. Results from this study demonstrated that mitochondrial ROS production in the diaphragm during MV is required for the increased expression of key autophagy genes (i.e. LC3, Atg7, Atg12, Beclin1 and p62), as well as for increased activity of cathepsin L. Together, these data reveal that autophagy is required for VIDD, and that autophagy inhibition reduces MV-induced diaphragm ROS production and prevents a positive feedback loop whereby increased autophagy is stimulated by oxidative stress, resulting in further increases in ROS and autophagy