Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in rapid diaphragmatic atrophy and contractile dysfunction, collectively termed ventilator-induced diaphragm dysfunction (VIDD). Recent evidence reveals that endurance exercise training, performed prior to MV, protects the diaphragm against VIDD. While the mechanism(s) responsible for this exercise-induced protection against VIDD remain unknown, increased diaphragm antioxidant expression may be required. To investigate the role that increased antioxidants play in this protection, we tested the hypothesis that elevated levels of the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) is required to achieve exercise-induced protection against VIDD. Cause and effect was investigated in two ways. First, we prevented the exercise-induced increase in diaphragmatic SOD2 via delivery of an antisense oligonucleotide targeted against SOD2 post-exercise. Second, using transgene overexpression of SOD2, we determined the effects of increased SOD2 in the diaphragm independent of exercise training. Results from these experiments revealed that prevention of the exercise-induced increases in diaphragmatic SOD2 results in a loss of exercise-mediated protection against MV-induced diaphragm atrophy and a partial loss of protection against MV-induced diaphragmatic contractile dysfunction. In contrast, transgenic overexpression of SOD2 in the diaphragm, independent of exercise, did not protect against MV-induced diaphragmatic atrophy and provided only partial protection against MV-induced diaphragmatic contractile dysfunction. Collectively, these results demonstrate that increased diaphragmatic levels of SOD2 are essential to achieve the full benefit of exercise-induced protection against VIDD.

Mechanical ventilation (MV) is a life-saving instrument used to provide ventilatory support for critically ill patients and patients undergoing surgery. Unfortunately, an unintended consequence of prolonged MV is the development of inspiratory weakness due to both diaphragmatic atrophy and contractile dysfunction; this syndrome is labeled ventilator-induced diaphragm dysfunction (VIDD). VIDD is clinically important because diaphragmatic weakness is an important contributor to problems in weaning patients from MV. Investigations into the pathogenesis of VIDD reveal that oxidative stress is essential for the rapid development of VIDD as redox disturbances in diaphragm fibers promote accelerated proteolysis. Currently, no standard treatment exists to prevent VIDD and, therefore, developing a strategy to avert VIDD is vital. Guided by evidence indicating that activation of the classical axis of the renin-angiotensin system (RAS) in diaphragm fibers promotes oxidative stress and VIDD, we hypothesized that activation of the nonclassical RAS signaling pathway via angiotensin 1-7 (Ang1-7) will protect against VIDD. Using an established animal model of prolonged MV, our results disclose that infusion of Ang1-7 protects the diaphragm against MV-induced contractile dysfunction and fiber atrophy in both fast and slow muscle fibers. Further, Ang1-7 shielded diaphragm fibers against MV-induced mitochondrial damage, oxidative stress, and protease activation. Collectively, these results reveal that treatment with Ang1-7 protects against VIDD, in part, due to diminishing oxidative stress and protease activation. These important findings provide robust evidence that Ang1-7 has the therapeutic potential to protect against VIDD by preventing MV-induced contractile dysfunction and atrophy of both slow and fast muscle fibers.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, 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 diaphragmatic 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 spectrometry, 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 soluble 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 exercise 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.

Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.

Oxidative damage to the diaphragm as a result of cervical spinal cord injury (SCI) promotes muscle atrophy and weakness. Respiratory insufficiency is the leading cause of morbidity and mortality in cervical spinal cord injury (SCI) patients, emphasizing the need for strategies to maintain diaphragm function. Hyperbaric oxygen (HBO) increases the amount of oxygen dissolved into the blood, elevating the delivery of oxygen to skeletal muscle and reactive oxygen species (ROS) generation. It is proposed that enhanced ROS production due to HBO treatment stimulates adaptations to diaphragm oxidative capacity, resulting in overall reductions in oxidative stress and inflammation. Therefore, we tested the hypothesis that exposure to HBO therapy acutely following SCI would reduce oxidative damage to the diaphragm muscle, preserving muscle fiber size and contractility. Our results demonstrated that lateral contusion injury at C3/4 results in a significant reduction in diaphragm muscle-specific force production and fiber cross-sectional area, which was associated with augmented mitochondrial hydrogen peroxide emission and a reduced mitochondrial respiratory control ratio. In contrast, rats that underwent SCI followed by HBO exposure consisting of 1 h of 100% oxygen at 3 atmospheres absolute (ATA) delivered for 10 consecutive days demonstrated an improvement in diaphragm-specific force production, and an attenuation of fiber atrophy, mitochondrial dysfunction and ROS production. These beneficial adaptations in the diaphragm were related to HBO-induced increases in antioxidant capacity and a reduction in atrogene expression. These findings suggest that HBO therapy may be an effective adjunctive therapy to promote respiratory health following cervical SCI.

Mechanical ventilation (MV) is a life-saving measure for patients in respiratory failure. However, prolonged MV results in significant diaphragm atrophy and contractile dysfunction, a condition referred to as ventilator-induced diaphragm dysfunction (VIDD). While there are currently no clinically approved countermeasures to prevent VIDD, increased expression of heat shock protein 72 (HSP72) has been demonstrated to attenuate inactivity-induced muscle wasting. HSP72 elicits cytoprotection via inhibition of NF-κB and FoxO transcriptional activity, which contribute to VIDD. In addition, exercise-induced prevention of VIDD is characterized by an increase in the concentration of HSP72 in the diaphragm. Therefore, we tested the hypothesis that increased HSP72 expression is required for the exercise-induced prevention of VIDD. We also determined whether increasing the abundance of HSP72 in the diaphragm, independent of exercise, is sufficient to prevent VIDD.

Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. However, prolonged MV causes the rapid development of diaphragm muscle atrophy, and diaphragmatic weakness may contribute to difficult weaning from MV. Therefore, developing a therapeutic countermeasure to protect against MV-induced diaphragmatic atrophy is important. MV-induced diaphragm atrophy is due, at least in part, to increased production of reactive oxygen species (ROS) from diaphragm mitochondria and the activation of key muscle proteases (i.e., calpain and caspase-3). In this regard, leakage of calcium through the ryanodine receptor (RyR1) in diaphragm muscle fibers during MV could result in increased mitochondrial ROS emission, protease activation, and diaphragm atrophy. Therefore, these experiments tested the hypothesis that a pharmacological blockade of the RyR1 in diaphragm fibers with azumolene (AZ) would prevent MV-induced increases in mitochondrial ROS production, protease activation, and diaphragmatic atrophy. Adult female Sprague-Dawley rats underwent 12 hours of full-support MV while receiving either AZ or vehicle. At the end of the experiment, mitochondrial ROS emission, protease activation, and fiber cross-sectional area were determined in diaphragm muscle fibers. Decreases in muscle force production following MV indicate that the diaphragm took up a sufficient quantity of AZ to block calcium release through the RyR1. However, our findings reveal that AZ treatment did not prevent the MV-induced increase in mitochondrial ROS emission or protease activation in the diaphragm. Importantly, AZ treatment did not prevent MV-induced diaphragm fiber atrophy. Thus, pharmacological inhibition of the RyR1 in diaphragm muscle fibers is not sufficient to prevent MV-induced diaphragm atrophy.

Mechanical ventilation (MV) is a life-saving intervention in patients in respiratory failure. Unfortunately, prolonged MV results in the rapid development of diaphragm atrophy and weakness. MV-induced diaphragmatic weakness is significant because inspiratory muscle dysfunction is a risk factor for problematic weaning from MV. Therefore, developing a clinical intervention to prevent MV-induced diaphragm atrophy is important. In this regard, MV-induced diaphragmatic atrophy occurs due to both increased proteolysis and decreased protein synthesis. While efforts to impede MV-induced increased proteolysis in the diaphragm are well-documented, only one study has investigated methods of preserving diaphragmatic protein synthesis during prolonged MV. Therefore, we evaluated the efficacy of two therapeutic interventions that, conceptually, have the potential to sustain protein synthesis in the rat diaphragm during prolonged MV. Specifically, these experiments were designed to: 1) determine if partial-support MV will protect against the decrease in diaphragmatic protein synthesis that occurs during prolonged full-support MV; and 2) establish if treatment with a mitochondrial-targeted antioxidant will maintain diaphragm protein synthesis during full-support MV. Compared to spontaneously breathing animals, full support MV resulted in a significant decline in diaphragmatic protein synthesis during 12 hours of MV. In contrast, diaphragm protein synthesis rates were maintained during partial support MV at levels comparable to spontaneous breathing animals. Further, treatment of animals with a mitochondrial-targeted antioxidant prevented oxidative stress during full support MV and maintained diaphragm protein synthesis at the level of spontaneous breathing animals. We conclude that treatment with mitochondrial-targeted antioxidants or the use of partial-support MV are potential strategies to preserve diaphragm protein synthesis during prolonged MV.

Mechanical ventilation (MV) is a clinical tool providing adequate alveolar ventilation in patients that require respiratory support. Although a life-saving intervention for critically ill patients, prolonged MV results in the rapid development of inspiratory muscle weakness due to both diaphragmatic atrophy and contractile dysfunction; collectively known as "ventilator-induced diaphragm dysfunction" (VIDD). VIDD is a severe clinical problem because diaphragmatic weakness is a risk factor for difficulties in weaning patients from MV. Currently, no standard treatment to prevent VIDD exists. Nonetheless, growing evidence reveals that hydrogen sulfide (H2 S) possesses cytoprotective properties capable of protecting skeletal muscles against several hallmarks of VIDD, including oxidative damage, accelerated proteolysis, and mitochondrial damage. Therefore, we used an established animal model of MV to test the hypothesis that treatment with sodium sulfide (H2 S donor) will defend against VIDD. Our results confirm that sodium sulfide was sufficient to protect the diaphragm against both MV-induced fiber atrophy and contractile dysfunction. H2 S prevents MV-induced damage to diaphragmatic mitochondria as evidenced by protection against mitochondrial uncoupling. Moreover, treatment with sodium sulfide prevented the MV-induced activation of the proteases, calpain, and caspase-3 in the diaphragm. Taken together, these results support the hypothesis that treatment with a H2 S donor protects the diaphragm against VIDD. These outcomes provide the first evidence that H2 S has therapeutic potential to protect against MV-induced diaphragm weakness and to reduce difficulties in weaning patients from the ventilator. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Mechanical ventilation (MV) results in diaphragm atrophy and contractile dysfunction, known as ventilator-induced diaphragm dysfunction (VIDD). VIDD is important because diaphragm weakness is a risk factor for problems in weaning patients from MV. Currently, no accepted treatment exists to protect against VIDD. Growing evidence reveals that hydrogen sulfide (H2 S) donors protect skeletal muscle against ischemia-reperfusion-induced injury. Nonetheless, it is unknown if treatment with a H2 S donor can protect against VIDD. WHAT QUESTION DID THIS STUDY ADDRESS? Can treatment with an H2 S donor protect against VIDD? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? This study provides the first evidence that treatment with a H2 S donor protects against VIDD. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? These new findings provide the basis for further exploration of H2 S donors as a therapy to prevent VIDD and reduce the risk of problems in weaning patients from MV.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in the rapid development of inspiratory muscle weakness due to diaphragmatic atrophy and contractile dysfunction (termed ventilator-induced diaphragm dysfunction (VIDD)). Although VIDD is a major risk factor for problems in weaning patients from MV, a standard therapy to prevent VIDD does not exist. However, emerging evidence suggests that pharmacological blockade of angiotensin II type 1 receptors (AT1Rs) protects against VIDD. Nonetheless, the essential characteristics of AT1R blockers (ARBs) required to protect against VIDD remain unclear. To determine the traits of ARBs that are vital for protection against VIDD, we compared the efficacy of two clinically relevant ARBs, irbesartan and olmesartan; these ARBs differ in molecular structure and effects on AT1Rs. Specifically, olmesartan blocks both angiotensin II (AngII) binding and mechanical activation of AT1Rs, whereas irbesartan prevents only AngII binding to AT1Rs. Using a well-established preclinical model of prolonged MV, we tested the hypothesis that compared with irbesartan, olmesartan provides greater protection against VIDD. Our results reveal that irbesartan does not protect against VIDD whereas olmesartan defends against both MV-induced diaphragmatic atrophy and contractile dysfunction. These findings support the hypothesis that olmesartan is superior to irbesartan in protecting against VIDD and are consistent with the concept that blockade of mechanical activation of AT1Rs is a required property of ARBs to shield against VIDD. These important findings provide a foundation for future clinical trials to evaluate ARBs as a therapy to protect against VIDD.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, an unintended consequence of prolonged MV is the rapid development of diaphragmatic atrophy and contractile dysfunction, known as ventilator-induced diaphragm dysfunction (VIDD). Although the mechanism(s) responsible for VIDD are not fully understood, abundant evidence reveals that oxidative stress leading to the activation of the major proteolytic systems (i.e., autophagy, ubiquitin-proteasome, caspase, and calpain) plays a dominant role. Of the proteolytic systems involved in VIDD, calpain has received limited experimental attention due to the longstanding dogma that calpain plays a minor role in inactivity-induced muscle atrophy. Guided by preliminary experiments, we tested the hypothesis that activation of calpains play an essential role in MV-induced oxidative stress and the development of VIDD. This premise was rigorously tested by transgene overexpression of calpastatin, an endogenous inhibitor of calpains. Animals with/without transfection of the calpastatin gene in diaphragm muscle fibers were exposed to 12 h of MV. Results confirmed that overexpression of calpastatin barred MV-induced activation of calpain in diaphragm fibers. Importantly, deterrence of calpain activation protected the diaphragm against MV-induced oxidative stress, fiber atrophy, and contractile dysfunction. Moreover, prevention of calpain activation in the diaphragm forstalled MV-induced mitochondrial dysfunction and prevented MV-induced activation of caspase-3 along with the transcription of muscle specific E3 ligases. Collectively, these results support the hypothesis that calpain activation plays an essential role in the early development of VIDD. Further, these findings provide the first direct evidence that calpain plays an important function in inactivity-induced mitochondrial dysfunction and oxidative stress in skeletal muscle fibers.

Cancer cachexia is a syndrome characterized by profound cardiac and diaphragm muscle wasting, which increase the risk of morbidity in cancer patients due to failure of the cardiorespiratory system. In this regard, muscle relies greatly on mitochondria to meet energy requirements for contraction and mitochondrial dysfunction can result in muscle weakness and fatigue. In addition, mitochondria are a major source of reactive oxygen species (ROS) production, which can stimulate increased rates of muscle protein degradation. Therefore, it has been suggested that mitochondrial dysfunction may be an underlying factor that contributes to the pathology of cancer cachexia. To determine if pharmacologically targeting mitochondrial dysfunction via treatment with the mitochondria-targeting peptide SS-31 would prevent cardiorespiratory muscle dysfunction, colon 26 (C26) adenocarcinoma tumor-bearing mice were administered either saline or SS-31 daily (3 mg/kg/day) following inoculation. C26 mice treated with saline demonstrated greater ROS production and mitochondrial uncoupling compared to C26 mice receiving SS-31 in both the heart and diaphragm muscle. In addition, saline-treated C26 mice exhibited a decline in left ventricular function which was significantly rescued in C26 mice treated with SS-31. In the diaphragm, muscle fiber cross-sectional area of C26 mice treated with saline was significantly reduced and force production was impaired compared to C26, SS-31-treated animals. Finally, ventilatory deficits were also attenuated in C26 mice treated with SS-31, compared to saline treatment. These data demonstrate that C26 tumors promote severe cardiac and respiratory myopathy, and that prevention of mitochondrial dysfunction is sufficient to preclude cancer cachexia-induced cardiorespiratory dysfunction.

Cardiorespiratory dysfunction resulting from doxorubicin (DOX) chemotherapy treatment is a debilitating condition affecting cancer patient outcomes and quality of life. DOX treatment promotes cardiac and respiratory muscle pathology due to enhanced reactive oxygen species (ROS) production, mitochondrial dysfunction and impaired muscle contractility. In contrast, hyperbaric oxygen (HBO) therapy is considered a controlled oxidative stress that can evoke a substantial and sustained increase in muscle antioxidant expression. This HBO-induced increase in antioxidant capacity has the potential to improve cardiac and respiratory (i.e., diaphragm) muscle redox balance, preserving mitochondrial function and preventing muscle dysfunction. Therefore, we determined whether HBO therapy prior to DOX treatment is sufficient to enhance muscle antioxidant expression and preserve muscle redox balance and cardiorespiratory muscle function. To test this, adult female Sprague Dawley rats received HBO therapy (2 or 3 atmospheres absolute (ATA), 100% O2, 1 h/day) for 5 consecutive days prior to acute DOX treatment (20 mg/kg i.p.). Our data demonstrate that 3 ATA HBO elicits a greater antioxidant response compared to 2 ATA HBO. However, these effects did not correspond with beneficial adaptations to cardiac systolic and diastolic function or diaphragm muscle force production in DOX treated rats. These findings suggest that modulating muscle antioxidant expression with HBO therapy is not sufficient to prevent DOX-induced cardiorespiratory dysfunction.

Endurance exercise training promotes a protective phenotype in skeletal muscle known as exercise preconditioning. Exercise preconditioning protects muscle fibers against a variety of threats including inactivity-induced muscle atrophy. The mechanism(s) responsible for exercise preconditioning remain unknown and are explored in these experiments. Specifically, we investigated the impact of endurance exercise training on key components of the renin-angiotensin system (RAS). The RAS was targeted because activation of the classical axis of the RAS pathway via angiotensin II type I receptors (AT1Rs) promotes muscle atrophy whereas activation of the non-classical RAS axis via Mas receptors (MasRs) inhibits the atrophic signaling of the classical RAS pathway. Guided by prior studies, we hypothesized that an exercise-induced decrease in AT1Rs and/or increases in MasRs in skeletal muscle fibers is a potential mechanism responsible for exercise preconditioning. Following endurance exercise training in rats, we examined the abundance of AT1Rs and MasRs in both locomotor and respiratory muscles. Our results indicate that endurance exercise training does not alter the protein abundance of AT1Rs or MasRs in muscle fibers from the diaphragm, plantaris, and soleus muscles compared to sedentary controls (p ​> ​0.05). Furthermore, fluorescent angiotensin II (AngII) binding analyses confirm our results that exercise preconditioning does not alter the protein abundance of AT1Rs in the diaphragm, plantaris, and soleus (p ​> ​0.05). This study confirms that exercise-induced changes in RAS receptors are not a key mechanism that contributes to the beneficial effects of exercise preconditioning in skeletal muscle fibers.

Acute hyperbaric O2 (HBO) therapy after spinal cord injury (SCI) can reduce inflammation and increase neuronal survival. To our knowledge, it is unknown if these benefits of HBO require hyperbaric vs. normobaric hyperoxia. We used a C4 lateralized contusion SCI in adult male and female rats to test the hypothesis that the combination of hyperbaria and 100% O2 (i.e. HBO) more effectively mitigates spinal inflammation and neuronal loss, and enhances respiratory recovery, as compared to normobaric 100% O2. Experimental groups included spinal intact, SCI no O2 therapy, and SCI + 100% O2 delivered at normobaric pressure (1 atmosphere, ATA), or at 2- or 3 ATA. O2 treatments lasted 1-h, commenced within 2-h of SCI, and were repeated for 10 days. The spinal inflammatory response was assessed with transcriptomics (RNAseq) and immunohistochemistry. Gene co-expression network analysis showed that the strong inflammatory response to SCI was dramatically diminished by both hyper- and normobaric O2 therapy. Similarly, both HBO and normobaric O2 treatments reduced the prevalence of immunohistological markers for astrocytes (glial fibrillary acidic protein) and microglia (ionized calcium binding adaptor molecule) in the injured spinal cord. However, HBO treatment also had unique impacts not detected in the normobaric group including upregulation of an anti-inflammatory cytokine (interleukin-4) in the plasma, and larger inspiratory tidal volumes at 10-days (whole body-plethysmography measurements). We conclude that normobaric O2 treatment can reduce the spinal inflammatory response after SCI, but pressured O2 (i.e., HBO) provides further benefit.