BACKGROUND Mechanical ventilation (MV) is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged MV is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during MV, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of MV-induced reactive oxygen species (ROS) production in the diaphragm is important. OBJECTIVE These experiments tested the hypothesis that elevated mitochondrial ROS emission is required for MV-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm. DESIGN Cause and effect was determined by preventing MV-induced mitochondrial ROS emission in the diaphragm of rats using a novel mitochondrial-targeted antioxidant (SS-31). MEASUREMENTS AND MAIN RESULTS Compared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against MV-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against MV-induced myofiber atrophy and contractile dysfunction. CONCLUSIONS These results reveal that prevention of MV-induced increases in diaphragmatic mitochondrial ROS emission protects the diaphragm MV-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of ROS production in the diaphragm during prolonged MV. These results could lead to the development of a therapeutic intervention to impede MV-induced diaphragmatic weakness.

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Combined chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) has enabled genome-wide epigenetic profiling of numerous cell lines and tissue types. A major limitation of ChIP-seq, however, is the large number of cells required to generate high-quality data sets, precluding the study of rare cell populations. Here, we present an ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) and sequencing method to generate genome-wide histone mark profiles with high resolution from as few as 10 3 cells. We demonstrate that ULI-NChIP-seq generates high-quality maps of covalent histone marks from 10 3 to 10 6 embryonic stem cells. Subsequently, we show that ULI-NChIP-seq H3K27me3 profiles generated from E13.5 primordial germ cells isolated from single male and female embryos show high similarity to recent data sets generated using 50-180 Â more material. Finally, we identify sexually dimorphic H3K27me3 enrichment at specific genic promoters, thereby illustrating the utility of this method for generating high-quality and -complexity libraries from rare cell populations.

Mechanical ventilation (MV) is a life-saving intervention used in patients with acute respiratory failure. Unfortunately, prolonged MV results in diaphragmatic weakness, which is an important contributor to the failure to wean patients from MV. Our laboratory has previously shown that reactive oxygen species (ROS) play a critical role in mediating diaphragmatic weakness after MV. However, the pathways responsible for MV-induced diaphragmatic ROS production remain unknown. These experiments tested the hypothesis that prolonged MV results in an increase in mitochondrial ROS release, mitochondrial oxidative damage, and mitochondrial dysfunction. To test this hypothesis, adult (3-4 months of age) female Sprague-Dawley rats were assigned to either a control or a 12-h MV group. After treatment, diaphragms were removed and mitochondria were isolated for subsequent respiratory and biochemical measurements. Compared to control, prolonged MV resulted in a lower respiratory control ratio in diaphragmatic mitochondria. Furthermore, diaphragmatic mitochondria from MV animals released higher rates of ROS in both State 3 and State 4 respiration. Prolonged MV was also associated with diaphragmatic mitochondrial oxidative damage as indicated by increased lipid peroxidation and protein oxidation. Finally, our data also reveal that the activities of the electron transport chain complexes II, III, and IV are depressed in mitochondria isolated from diaphragms of MV animals. In conclusion, these results are consistent with the concept that diaphragmatic inactivity promotes an increase in mitochondrial ROS emission, mitochondrial oxidative damage, and mitochondrial respiratory dysfunction. KeywordsMitochondria; Superoxide; Antioxidants; Free radicals Mechanical ventilation (MV) is a life-saving measure used to maintain alveolar ventilation in patients incapable of doing so on their own (e.g., respiratory failure, coma, or spinal cord injury). Unfortunately, prolonged MV reduces the activity of the principal muscle of inspiration (i.e., diaphragm) and results in diaphragmatic wasting and contractile dysfunction [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Indeed, MV results in a rapid onset of diaphragmatic atrophy that is accompanied by oxidative stress [1,15,16]. Although extended periods of disuse also lead to locomotor skeletal muscle atrophy [13,[17][18][19][20][21][22][23][24][25][26], a unique characteristic of MV-induced diaphragmatic atrophy is the rapidity of the atrophic response [25,26]. Although the molecular steps that regulate MVinduced diaphragm atrophy remain unclear, growing evidence indicates that redox disturbances in diaphragmatic fibers play a key signaling role in this process. In this regard, our laboratory was the first to report that prolonged MV results in both protein oxidation and lipid peroxidation in the diaphragm [13,15]. Specifically, diaphragm unloading via MV is associated with a rapid onset of diaphragmatic oxidative stress that develops within 3-6 h after the initiation of MV [15]. Impor...

Prolonged mechanical ventilation (MV) results in diaphragmatic weakness due to fiber atrophy and contractile dysfunction. Recent work reveals that activation of the proteases calpain and caspase-3 is required for MV-induced diaphragmatic atrophy and contractile dysfunction. However, the mechanism(s) responsible for activation of these proteases remains unknown. To address this issue, we tested the hypothesis that oxidative stress is essential for the activation of calpain and caspase-3 in the diaphragm during MV. Cause-and-effect was established by prevention of MV-induced diaphragmatic oxidative stress using the antioxidant Trolox. Treatment of animals with Trolox prevented MV-induced protein oxidation and lipid peroxidation in the diaphragm. Importantly, the Trolox-mediated protection from MV-induced oxidative stress prevented the activation of calpain and caspase-3 in the diaphragm during MV. Furthermore, the avoidance of MV-induced oxidative stress not only averted the activation of these proteases but also rescued the diaphragm from MV-induced diaphragmatic myofiber atrophy and contractile dysfunction. Collectively, these findings support the prediction that oxidative stress is required for MV-induced activation of calpain and caspase-3 in the diaphragm and are consistent with the concept that antioxidant therapy can retard MV-induced diaphragmatic weakness.