Forecasting individual exercise sweat losses from forecast air temperature and energy expenditure.

Introduction: Recent success in predicting individual sweat losses from air temperature and energy expenditure measurements suggests a potential for forecasting individual sweat losses for future combinations of environment and exercise. The purpose of this study is to determine the plausibility of accurately forecasting exercise sweat losses from meteorological air temperature forecasts and individual running energy expenditure forecasts. The potential impact on plasma sodium is also estimated when setting drinking rates equal to forecast sweat losses. Materials and methods: Individual exercise sweat losses (equated to water needs) and energy expended while running were measured in 33 participants along with air temperature and compared with forecasts of the same. Forecast inputs were used in a web app to forecast exercise sweat losses for comparison with observed values. The bias between forecast and observed exercise sweat losses was used to calculate the potential drinking impact on plasma sodium. Results: The concordance correlation coefficient between forecast and observed values was 0.95, 0.96, and 0.91 for air temperature, energy expenditure, and exercise sweat losses, respectively, indicating excellent agreement and no significant differences observed via t-test. Perfect matching of water intake to sweat losses would lower plasma sodium concentrations from 140 to 138 mmol/L; calculations using the 95% limits of agreement for bias showed that drinking according to forecast exercise sweat losses would alter plasma sodium concentrations from 140 to between 136 and 141 mmol/L. Conclusions: The outcomes support the strong potential for accurately forecasting exercise sweat losses from commonly available meteorological air temperature forecasts and energy expenditure from forecast running distance.

Predicted sweat rates for group water planning in sport: accuracy and application.

This study tested the accuracy of a novel, limited-availability web application (H2Q™) for predicting sweat rates in a variety of sports using estimates of energy expenditure and air temperature only. The application of predictions for group water planning was investigated for soccer match play. Fourteen open literature studies were identified where group sweat rates were reported (n = 20 group means comprising 230 individual observations from 179 athletes) with fidelity. Sports represented included: walking, cycling, swimming, and soccer match play. The accuracy of H2Q™ sweat rates was tested by comparing to measured group sweat rates using the concordance correlation coefficient (CCC) with 95% confidence interval [CI]. The relative absolute error (RAE) with 95% [CI] was also assessed, whereby the mean absolute error was expressed relative to an acceptance limit of 0.250 L/h. The CCC was 0.98 [0.95, 0.99] and the RAE was 0.449 [0.279, 0.620], indicating that the prediction error was on average 0.112 L/h. The RAE was < 1.0 for 19/20 observations (95%). Drink volumes modeled as a proxy for sweat losses during soccer match play prevented dehydration (< 1% loss of body mass). The H2Q™ web application demonstrated high group sweat prediction accuracy for the variety of sports activities tested. Water planning for soccer match play suggests the feasibility of easily and accurately predicting sweat rates to plan group water needs and promote optimal hydration in training and/or competition.

Comparative Efficacy of Angiotensin II Type 1 Receptor Blockers Against Ventilator-Induced Diaphragm Dysfunction in Rats.

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.

The accurate prediction of sweat rate from energy expenditure and air temperature: a proof-of-concept study.

This proof-of-concept study used a web application to predict runner sweat losses using only energy expenditure and air temperature. A field study (FS) of n = 37 runners was completed with n = 40 sweat loss observations measured over 1 h (sweat rate, SR). Predictions were also compared with 10 open literature (OL) studies in which individual runner SR was reported (n = 82; 109 observations). Three prediction accuracy metrics were used: for FS, the mean absolute error (MAE) and concordance correlation coefficient (CCC) were calculated to include a 95% confidence interval [CI]; for OL, the percentage concordance (PC) was examined against calculation of accumulated under- and over-drinking potential. The MAE for FS runners was 0.141 kg [0.105, 0.177], which was less than estimated scale weighing error on 85% of occasions. The CCC was 0.88 [0.82, 0.93]. The PC for OL was 96% for avoidance of both under- and over-drinking and 93% overall. All accuracy metrics and their CIs were below acceptable error tolerance. Input errors of ±10% and ±1 °C for energy expenditure and air temperature dropped the PC to between 84% and 90%. This study demonstrates the feasibility of accurately predicting SR from energy expenditure and air temperature alone. Novelty Results demonstrate that accurate runner SR prediction is possible with knowledge of only energy expenditure and air temperature. SR prediction error was smaller than scale weighing error in 85% of observations. Accurate runner SR prediction could help mitigate the common risks of over- and under-drinking.

Biological variation of arginine vasopressin.

PURPOSE: There is growing interest in the measurement of plasma levels of arginine vasopressin (AVP) for the assessment of mild dehydration. However, the principles of biological variation have not been applied to the study of AVP and understanding biological variation of AVP may provide insights regarding measurement thresholds. The purpose of this investigation was to determine the biological variation of AVP in healthy euhydrated individuals to understand the potential for establishing both static and/or change thresholds of importance. METHODS: We studied 29 healthy volunteers (24 men and 5 women) while controlling for hydration and pre-analytical factors. All subjects completed between 2-8 trials where biological variation was determined using widely published methods. We determined the intraindividual, interindividual, and analytical coefficients of variation (CVI, CVG, and CVA, respectively) and subsequently the index of individuality and heterogeneity (II and IH, respectively). RESULTS: AVP did not reach the IH threshold required to be considered useful in the dynamic assessment of physiological deviations from normal. AVP levels approached the II threshold required to be considered useful in the static assessment of physiological deviations from normal. CONCLUSIONS: This analysis demonstrates that AVP assessment is unlikely to yield useful information about hydration status.

Osmolality of Commercially Available Oral Rehydration Solutions: Impact of Brand, Storage Time, and Temperature.

Oral rehydration solutions (ORS) are specifically formulated with an osmolality to optimize fluid absorption. However, it is unclear how many ORS products comply with current World Health Organization (WHO) osmolality guidelines and the osmotic shelf-life stability is not known. Therefore, the purpose of this investigation was to examine the within and between ORS product osmolality variation in both pre-mixed and reconstituted powders. Additionally, the osmotic stability was examined over time. The osmolality of five different pre-mixed solutions and six powdered ORS products were measured. Pre-mixed solutions were stored at room temperatures and elevated temperatures (31 °C) for two months to examine osmotic shelf stability. Results demonstrated that only one pre-mixed ORS product was in compliance with the current guidelines both before and after the prolonged storage. Five of the six powdered ORS products were in compliance with minimal inter-packet variation observed within the given formulations. This investigation demonstrates that many commercially available pre-mixed ORS products do not currently adhere to the WHO recommended osmolality guidelines. Additionally, due to the presence of particular sugars and possibly other ingredients, the shelf-life stability of osmolality for certain ORS products may be questioned. These findings should be carefully considered in the design of future ORS products.

Effects of exercise preconditioning and HSP72 on diaphragm muscle function during mechanical ventilation.

BACKGROUND: 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. METHODS: Cause and effect was determined by inhibiting the endurance exercise-induced increase in HSP72 in the diaphragm of exercise trained animals exposed to prolonged MV via administration of an antisense oligonucleotide targeting HSP72. Additional experiments were performed to determine if increasing HSP72 in the diaphragm via genetic (rAAV-HSP72) or pharmacological (BGP-15) overexpression is sufficient to prevent VIDD. RESULTS: Our results demonstrate that the exercise-induced increase in HSP72 protein abundance is required for the protective effects of exercise against VIDD. Moreover, both rAAV-HSP72 and BGP-15-induced overexpression of HSP72 were sufficient to prevent VIDD. In addition, modification of HSP72 in the diaphragm is inversely related to the expression of NF-κB and FoxO target genes. CONCLUSIONS: HSP72 overexpression in the diaphragm is an effective intervention to prevent MV-induced oxidative stress and the transcriptional activity of NF-κB and FoxO. Therefore, overexpression of HSP72 in the diaphragm is a potential therapeutic target to protect against VIDD.

Importance of sample volume to the measurement and interpretation of plasma osmolality.

BACKGROUND: Small sample volumes may artificially elevate plasma osmolality (Posm) measured by freezing point depression. The purpose of this study was to compare two widely different sample volumes of measured Posm (mmol/kg) to each other, and to calculated osmolarity (mmol/L), across a physiological Posm range (~50 mmol/kg). METHODS: Posm was measured using freezing point depression and osmolarity calculated from measures of sodium, glucose, and blood urea nitrogen. The influence of sample volume was investigated by comparing 20 and 250 µL Posm samples (n = 126 pairs). Thirty-two volunteers were tested multiple times while EUH (n = 115) or DEH (n = 11) by -4.0% body mass. Protinol™ (240, 280, and 320 mmol/kg) and Clinitrol™ (290 mmol/kg) reference solutions were compared similarly (n = 282 pairs). RESULTS: The 20 µL samples of plasma showed a 7 mmol/kg positive bias compared to 250 µL samples and displayed a nearly constant proportional error across the range tested (slope = 0.929). Calculated osmolarity was lower than 20 µL Posm by the same negative bias (-6.9 mmol/kg) but not different from 250 µL Posm (0.1 mmol/kg). The differences between 20 and 250 µL samples of Protinol™ were significantly higher than Clinitrol™. CONCLUSIONS: These results demonstrate that Posm measured by freezing point depression will be ~7 mmol/kg higher when using 20 µL vs 250 µL sample volumes. Approximately half of this effect may be due to plasma proteins. Posm sample volume should be carefully considered when calculating the osmole gap or assessing hydration status.

Validation of a Mobile Application Water Planning Tool for Road Race Event Organizers.

Water planning is an important risk management concern for road race event organizers. PURPOSE: To compare water and cup prediction outputs from a mobile application (app) planning tool against: 1) measured group sweat losses, 2) documented event water and cup usage, and 3) traditional mathematical planning solutions. METHODS: Group mean sweating rates (L·h) from 12 published outdoor running studies were each compared to 12 composite averages using the Road Race Water Planner© (RRWP) app. Estimated water (gallons) and cup (number) needs were also compared with documented usage at a large marathon event and to traditional mathematical solutions. RESULTS: Thirteen group mean sweating rates from 286 runners were compared to composite RRWP estimates. Predicted sweating rate accuracy was 92% for RRWP and ranged from 0% to 69% for traditional mathematical solutions. The 2017 Boston marathon included 27,222 runners on a day averaging 21.5°C. Water and cup usage was 31,740 gallons and 1,036,003 cups, respectively. The RRWP estimates were 33,505 gallons and 1,072,160 cups, respectively. The difference in gallons expressed as liters was 0.236 L per person. For an approximately 4-h marathon, the difference per person as a rate was <60 mL·h. The difference in cups was a 3.5% error. All traditional solutions gave inferior estimates to RRWP due to large errors related to fluctuations in weather, as well as complications related to water station numbers. CONCLUSIONS: The results of the RRWP analysis indicate that it can provide event organizers with a valid, quantitative way to narrow the uncertainties of water planning related to changes in participant numbers, race distance, and weather. "Rule of thumb" alternatives are also discussed.

Neither body mass nor sex influences beverage hydration index outcomes during randomized trial when comparing 3 commercial beverages.

Background: The beverage hydration index (BHI) assesses the hydration potential of any consumable fluid relative to water. The BHI is a relatively new metric, and the impact of body mass, sex, and reproducibility has yet to be investigated. Objectives: To assess the independent impact of body mass and sex on BHI using beverages not previously assessed, including an amino acid-based oral rehydration solution (AA-ORS), a glucose-containing ORS (G-ORS), and a sports drink (SpD), compared with water (control). The reproducibility of the results was examined using statistical modeling (bootstrap analysis). Design: Using a repeated-measures design, 40 euhydrated and fasted subjects (17 male, 23 female; urine specific gravity <1.025) were studied on 4 separate occasions. During each trial, subjects ingested 1 L of each beverage, and urine output was measured immediately postingestion and at 1-h intervals for the next 4 h. The BHI was calculated as a ratio of each individual's cumulative urine output after drinking 1 L of water over their cumulative urine output after drinking each of the test beverages. Results: The calculated mean ± SD BHIs of the beverages were as follows: water (1.0 ± 0.0), AA-ORS (1.15 ± 0.28), G-ORS (1.21 ± 0.28), and SpD (1.09 ± 0.26). The BHI for both AA-ORS and G-ORS was greater than that for water (P < 0.05). Despite overall differences in body mass, neither body mass nor sex independently affected BHI. Based upon statistical modeling, our results demonstrate excellent reproducibility of outcomes and external validity. Conclusions: Our results suggest that the BHI may be used and interpreted with confidence independently of body mass or sex. Furthermore, a novel carbohydrate-free AA-ORS and a traditional commercially available G-ORS were superior to water in optimizing hydration, whereas SpD was not. This trial was registered at clinicaltrials.gov as NCT03262597.

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.

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. 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.

Blockage of the Ryanodine Receptor via Azumolene Does Not Prevent Mechanical Ventilation-Induced Diaphragm Atrophy.

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.

Comparative changes in antioxidant enzymes and oxidative stress in cardiac, fast twitch and slow twitch skeletal muscles following endurance exercise training.

The aim of this study was to evaluate exercise-induced transcriptional and protein responses of heart, soleus (slow oxidative), and plantaris (fast glycolytic) muscle in response to ten days of endurance exercise training. Four-month old female Sprague-Dawley rats were assigned to either a sedentary (SED) or endurance exercise-training (EXE) group (n=8 per group). The heart, plantaris, and soleus were excised and used for biochemical analyses. Our results show that heart and plantaris from EXE animals had higher protein levels of superoxide dismutase 2 (SOD2) compared to SED animals (P<0.05). Also, the protein levels of catalase were higher in plantaris of EXE animals compared to SED animals (P<0.05). No significant differences existed for 4 hydroxynonenal (4HNE) conjugated proteins (index of oxidative damage) in the three tissues between SED and EXE animals. mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) were higher in plantaris of EXE animals compared to SED animals (P<0.05), and mRNA levels of estrogen-related receptor alpha (ERRα) were lower in the heart of EXE animals compared to SED animals. In conclusion, heart and plantaris are responsive to ten days of treadmill training, while greater exercise intensities or durations may be needed to elicit alterations in soleus.

Cervical spinal cord injury exacerbates ventilator-induced diaphragm dysfunction.

Cervical spinal cord injury (SCI) can dramatically impair diaphragm muscle function and often necessitates mechanical ventilation (MV) to maintain adequate pulmonary gas exchange. MV is a life-saving intervention. However, prolonged MV results in atrophy and impaired function of the diaphragm. Since cervical SCI can also trigger diaphragm atrophy, it may create preconditions that exacerbate ventilator-induced diaphragm dysfunction (VIDD). Currently, no drug therapy or clinical standard of care exists to prevent or minimize diaphragm dysfunction following SCI. Therefore, we first tested the hypothesis that initiating MV acutely after cervical SCI will exacerbate VIDD and enhance proteolytic activation in the diaphragm to a greater extent than either condition alone. Rats underwent controlled MV for 12 h following acute (∼24 h) cervical spinal hemisection injury at C2 (SCI). Diaphragm tissue was then harvested for comprehensive functional and molecular analyses. Second, we determined if antioxidant therapy could mitigate MV-induced diaphragm dysfunction after cervical SCI. In these experiments, SCI rats received antioxidant (Trolox, a vitamin E analog) or saline treatment prior to initiating MV. Our results demonstrate that compared with either condition alone, the combination of SCI and MV resulted in increased diaphragm atrophy, contractile dysfunction, and expression of atrophy-related genes, including MuRF1. Importantly, administration of the antioxidant Trolox attenuated proteolytic activation, fiber atrophy, and contractile dysfunction in the diaphragms of SCI + MV animals. These findings provide evidence that cervical SCI greatly exacerbates VIDD, but antioxidant therapy with Trolox can preserve diaphragm contractile function following acute SCI.

Influence of endurance exercise training on antioxidant enzymes, tight junction proteins, and inflammatory markers in the rat ileum.

BACKGROUND: This study investigated the effects of endurance exercise training on ileum antioxidant status, as well as tight junction, inflammatory, and nutrient transporter gene expression. METHODS: Sprague-Dawley rats (4 month old) were assigned to sedentary (SED) or endurance exercise-training (EXE) groups (n = 8/group). EXE animals were trained on the treadmill for 10 days at a speed of 30 m/min at 0° incline for 60 min/day. SED and EXE animals were sacrificed (24 h after the final training bout) and the ileum was stored for analyses. RESULTS: The ileum of EXE had higher (p < 0.05) antioxidant protein levels of manganese superoxide dismutase and catalase compared to SED with no change (p > 0.05) in the lipid peroxidation biomarker 4-hydroxynonenal. Ileum mRNA expression of the tight junction gene zonulin increased (p < 0.05) and claudin 1 decreased (p < 0.05) in EXE compared to SED, but occludin and zonula occluden 1 were not different (p > 0.05) between SED and EXE. The ileum mRNA expressions of seven nutrient transporters (SLC5A8, SLC7A6, SLC6A19, SLC7A7, SLC27A2, SLC16A10, and SLC15A1) were not different between the two groups (p > 0.05). EXE had lower ileum TNFα mRNA expression (p < 0.05) compared to SED. No changes (p > 0.05) were found in the other inflammatory mRNAs including NFκB, IFNγ, IL6, CCL2, TLR4, and IL10. In addition, no changes in p-p65:p65 were detected. CONCLUSIONS: These findings suggest that 10 days of endurance exercise training up-regulates key endogenous antioxidant enzymes, decreases select inflammation markers, and alters select markers of tight junction permeability.

AT1 receptor blocker losartan protects against mechanical ventilation-induced diaphragmatic dysfunction.

Mechanical ventilation is a life-saving intervention for patients in respiratory failure. Unfortunately, prolonged ventilator support results in diaphragmatic atrophy and contractile dysfunction leading to diaphragm weakness, which is predicted to contribute to problems in weaning patients from the ventilator. While it is established that ventilator-induced oxidative stress is required for the development of ventilator-induced diaphragm weakness, the signaling pathway(s) that trigger oxidant production remain unknown. However, recent evidence reveals that increased plasma levels of angiotensin II (ANG II) result in oxidative stress and atrophy in limb skeletal muscles. Using a well-established animal model of mechanical ventilation, we tested the hypothesis that increased circulating levels of ANG II are required for both ventilator-induced diaphragmatic oxidative stress and diaphragm weakness. Cause and effect was determined by administering an angiotensin-converting enzyme inhibitor (enalapril) to prevent ventilator-induced increases in plasma ANG II levels, and the ANG II type 1 receptor antagonist (losartan) was provided to prevent the activation of ANG II type 1 receptors. Enalapril prevented the increase in plasma ANG II levels but did not protect against ventilator-induced diaphragmatic oxidative stress or diaphragm weakness. In contrast, losartan attenuated both ventilator-induced oxidative stress and diaphragm weakness. These findings indicate that circulating ANG II is not essential for the development of ventilator-induced diaphragm weakness but that activation of ANG II type 1 receptors appears to be a requirement for ventilator-induced diaphragm weakness. Importantly, these experiments provide the first evidence that the Food and Drug Administration-approved drug losartan may have clinical benefits to protect against ventilator-induced diaphragm weakness in humans.

Inhibition of forkhead boxO-specific transcription prevents mechanical ventilation-induced diaphragm dysfunction.

OBJECTIVES: Mechanical ventilation is a lifesaving measure for patients with respiratory failure. However, prolonged mechanical ventilation results in diaphragm weakness, which contributes to problems in weaning from the ventilator. Therefore, identifying the signaling pathways responsible for mechanical ventilation-induced diaphragm weakness is essential to developing effective countermeasures to combat this important problem. In this regard, the forkhead boxO family of transcription factors is activated in the diaphragm during mechanical ventilation, and forkhead boxO-specific transcription can lead to enhanced proteolysis and muscle protein breakdown. Currently, the role that forkhead boxO activation plays in the development of mechanical ventilation-induced diaphragm weakness remains unknown. DESIGN: This study tested the hypothesis that mechanical ventilation-induced increases in forkhead boxO signaling contribute to ventilator-induced diaphragm weakness. SETTING: University research laboratory. SUBJECTS: Young adult female Sprague-Dawley rats. INTERVENTIONS: Cause and effect was determined by inhibiting the activation of forkhead boxO in the rat diaphragm through the use of a dominant-negative forkhead boxO adeno-associated virus vector delivered directly to the diaphragm. MEASUREMENTS AND MAIN RESULTS: Our results demonstrate that prolonged (12 hr) mechanical ventilation results in a significant decrease in both diaphragm muscle fiber size and diaphragm-specific force production. However, mechanically ventilated animals treated with dominant-negative forkhead boxO showed a significant attenuation of both diaphragm atrophy and contractile dysfunction. In addition, inhibiting forkhead boxO transcription attenuated the mechanical ventilation-induced activation of the ubiquitin-proteasome system, the autophagy/lysosomal system, and caspase-3. CONCLUSIONS: Forkhead boxO is necessary for the activation of key proteolytic systems essential for mechanical ventilation-induced diaphragm atrophy and contractile dysfunction. Collectively, these results suggest that targeting forkhead boxO transcription could be a key therapeutic target to combat ventilator-induced diaphragm dysfunction.

Increased mitochondrial emission of reactive oxygen species and calpain activation are required for doxorubicin-induced cardiac and skeletal muscle myopathy.

Although doxorubicin (DOX) is a highly effective anti-tumour agent used to treat a variety of cancers, DOX administration is associated with significant side effects, including myopathy of both cardiac and skeletal muscles. The mechanisms responsible for DOX-mediated myopathy remain a topic of debate. We tested the hypothesis that both increased mitochondrial reactive oxygen species (ROS) emission and activation of the cysteine protease calpain are required for DOX-induced myopathy in rat cardiac and skeletal muscle. Cause and effect was determined by administering a novel mitochondrial-targeted anti-oxidant to prevent DOX-induced increases in mitochondrial ROS emission, whereas a highly-selective pharmacological inhibitor was exploited to inhibit calpain activity. Our findings reveal that mitochondria are a major site of DOX-mediated ROS production in both cardiac and skeletal muscle fibres and the prevention of DOX-induced increases in mitochondrial ROS emission protects against fibre atrophy and contractile dysfunction in both cardiac and skeletal muscles. Furthermore, our results indicate that DOX-induced increases in mitochondrial ROS emission are required to activate calpain in heart and skeletal muscles and, importantly, calpain activation is a major contributor to DOX-induced myopathy. Taken together, these findings show that increased mitochondrial ROS production and calpain activation are significant contributors to the development of DOX-induced myopathy in both cardiac and skeletal muscle fibres.

Role of intrinsic aerobic capacity and ventilator-induced diaphragm dysfunction.

Prolonged mechanical ventilation (MV) leads to rapid diaphragmatic atrophy and contractile dysfunction, which is collectively termed "ventilator-induced diaphragm dysfunction" (VIDD). Interestingly, endurance exercise training prior to MV has been shown to protect against VIDD. Further, recent evidence reveals that sedentary animals selectively bred to possess a high aerobic capacity possess a similar skeletal muscle phenotype to muscles from endurance trained animals. Therefore, we tested the hypothesis that animals with a high intrinsic aerobic capacity would naturally be afforded protection against VIDD. To this end, animals were selectively bred over 33 generations to create two divergent strains, differing in aerobic capacity: high-capacity runners (HCR) and low-capacity runners (LCR). Both groups of animals were subjected to 12 h of MV and compared with nonventilated control animals within the same strains. As expected, contrasted to LCR animals, the diaphragm muscle from the HCR animals contained higher levels of oxidative enzymes (e.g., citrate synthase) and antioxidant enzymes (e.g., superoxide dismutase and catalase). Nonetheless, compared with nonventilated controls, prolonged MV resulted in significant diaphragmatic atrophy and impaired diaphragm contractile function in both the HCR and LCR animals, and the magnitude of VIDD did not differ between strains. In conclusion, these data demonstrate that possession of a high intrinsic aerobic capacity alone does not afford protection against VIDD. Importantly, these results suggest that endurance exercise training differentially alters the diaphragm phenotype to resist VIDD. Interestingly, levels of heat shock protein 72 did not differ between strains, thus potentially representing an important area of difference between animals with intrinsically high aerobic capacity and exercise-trained animals.