Articles in PresS. Am J Physiol Regul Integr Comp Physiol (February 10, 2016). doi:10.1152/ajpregu.00550.2015
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Janus Kinase Inhibition Prevents Cancer- and Myocardial Infarction-Mediated
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Diaphragm Muscle Weakness in Mice
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Ira J. Smith1, Brandon Roberts2, Adam Beharry2, Guillermo L. Godinez1, Donald G. Payan ,
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Todd M. Kinsella1, Andrew R. Judge2, and Leonardo F. Ferreira3
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Rigel Pharmaceuticals, South San Francisco, California, USA; 2Department of Physical Therapy,
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University of Florida, Gainesville, Florida, USA; 3Department of Applied Physiology and
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Kinesiology, University of Florida, Gainesville, Florida, USA
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Correspondence:
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Todd M. Kinsella, Ph.D.
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Rigel Pharmaceuticals, Inc
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South San Francisco, CA 94080
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Email:
[email protected]
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Office Phone: 650.624.1225
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RUNNING TITLE: JAK inhibition prevents cancer- and MI-mediated diaphragm weakness
Copyright © 2016 by the American Physiological Society.
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Abstract
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Respiratory dysfunction is prevalent in critically ill patients, and can lead to adverse
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clinical outcomes including respiratory failure and increased mortality. Respiratory muscles,
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which normally sustain respiration through inspiratory muscle contractions, become weakened
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during critical illness, and recent studies suggest that respiratory muscle weakness is related to
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systemic inflammation. Here we investigate the pathophysiological role of the inflammatory
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JAK1/3 signaling pathway in diaphragm weakness in two distinct experimental models of critical
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illness. In the first experiment, mice received subcutaneous injections of PBS or C26 cancer cells,
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and were fed chow formulated with or without the JAK1/3 inhibitor R548 for twenty-six days.
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Diaphragm specific force was significantly reduced in tumor bearing mice receiving standard
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chow, however treatment with the JAK1/3 inhibitor completely prevented diaphragm weakness.
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Diaphragm cross-sectional area was diminished by approximately 25% in tumor bearing mice,
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but was similar to healthy mice in tumor-bearing animals treated with R548. In the second study,
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mice received sham surgery or coronary artery ligation leading to myocardial infarction (MI),
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and were treated with R548 or vehicle one-hour post-surgery, and once daily for three days.
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Diaphragm specific force was comparable between sham-surgery/vehicle, sham-surgery/R548
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and MI/R548 groups, but significantly decreased in the MI/vehicle group. Markers of oxidative
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damage and activated caspase-3, mechanisms previously identified to reduce muscle contractility,
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were not elevated in diaphragm extracts. These experiments implicate JAK1/3 signaling in
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cancer- and MI-mediated diaphragm weakness in mice, and provide a compelling case for further
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investigation.
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Introduction
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Respiratory dysfunction is a common and serious problem in critically ill patients.
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Approximately one-third of critically ill patients develop respiratory failure, and the mortality
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rate of patients with respiratory failure is twice the rate of patients without respiratory failure
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(16). Inadequate ventilatory patterns and expulsive behavior limit gas exchange and airway
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clearance, causing pulmonary complications such as pneumonia. While the etiology is poorly
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understood, and likely complex and multifactorial, recent studies suggest that respiratory
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dysfunction in critically ill patients is due, at least in part, to diaphragm weakness. Diaphragm
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weakness compromises ventilatory and non-ventilatory behavior (6, 7), which can contribute to
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increased morbidity and mortality in patients.
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To begin to address this important problem, here we investigate diaphragm muscle
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weakness in two distinct and highly relevant experimental models of critical illness, cancer-
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cachexia and acute phase myocardial infarction. Though fundamentally different in their
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pathogenesis, one notable commonality is elevated systemic inflammation, which is reported in
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critically ill human patients (2, 4), as well as preclinical models (9, 13). Experimental models
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characterized by systemic inflammation were selected for the current investigation because we
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recently found that inhibition of the inflammatory Janus Kinase 1/3 (JAK 1/3) signaling pathway
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prevented diaphragm muscle dysfunction in a different preclinical model, mechanical ventilation
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(12, 14). The goal of the current investigation was to determine whether this same pathway
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contributes to cancer cachexia- and acute MI-mediated diaphragm muscle weakness in mice.
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Materials and Methods
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University of Florida and Rigel Pharmaceuticals Institutional Animal Care and Use
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Committees approved animal experiments conducted at respective institutions. C26 cancer
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studies were conducted as described elsewhere (10), and MI was performed by ligation of the left
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anterior descending coronary artery as previously described (1). Total and cleaved caspase-3 and
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actin were assessed via Western blot, as explained previously (12). Statistical analysis was
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performed using one-way or two-way ANOVA, as appropriate, followed by Tukey’s multiple
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comparisons test. Two-tailed P values of < 0.05 were considered significant. Data are reported as
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mean ± SEM.
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Results and Discussion
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In the first set of experiments, male CD2F1 mice received subcutaneous injections with
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either PBS or C26 colon carcinoma cells, and were further sub-divided into groups receiving
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either the JAK 1/3 inhibitor R548 or vehicle. R548 was formulated in chow (0.3g R548/kg
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chow), and provided to the animals upon PBS or C26 cell delivery, and throughout the study.
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The R548 dose was based on pharmacokinetic studies demonstrating that 0.3g R548/kg chow
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maintained blood exposure levels above the levels associated with recovery of diaphragm muscle
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specific force during mechanical ventilation (12, 14). Chow intake was similar between the
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experimental groups throughout the study, and animals were euthanized on the 26th day of the
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experiment. As previously reported (10), diaphragm specific force was significantly reduced in
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tumor-bearing mice (P < 0.05; Figure 1). Treatment with the JAK 1/3 inhibitor prevented cancer-
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mediated contractile dysfunction (Figure 1), as well as diaphragm muscle wasting which was
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assessed by muscle cross-sectional area (data not shown). Similarly, R548 blocked
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approximately 80% of tibialis anterior (TA) and plantaris limb muscle atrophy in tumor-bearing
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mice (P < 0.05; data not shown), but did not prevent gastrocnemius or soleus wasting (data not
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shown). JAK 1/3 inhibition also significantly reduced mRNA levels of the STAT3 down-stream
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transcriptional target Socs3, and the atrophy-related genes atrogin-1 and MuRF1 in TA muscle of
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cachectic mice (Figure 2; P < 0.05). Importantly, JAK 1/3 inhibition was well tolerated in tumor-
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bearing mice and did not exacerbate tumor growth or whole body cachexia. These data identify
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JAK 1/3 signaling as a key mediator of diaphragm muscle weakness during cancer-cachexia in
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mice, and establish inhibition of JAK 1/3 signaling as a promising therapeutic strategy worthy of
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additional investigation.
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In the second series of experiments, male C57Bl/6 mice were randomly assigned to one
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of four groups: 1) sham-surgery/vehicle treatment; 2) sham-surgery/R548 treatment; 3)
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myocardial infarction/vehicle (MI/vehicle); and 4) MI/R548. MI was initiated by ligation of the
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left anterior descending coronary artery, and the JAK 1/3 inhibitor was delivered one-hour post-
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MI (75mg/kg BW, subcutaneous injection), and continued once daily for three days.
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Pharmacokinetic studies were conducted to determine the subcutaneous dose required to
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maintain plasma levels of the active drug above the efficacious level identified during
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mechanical ventilation (12, 14). Seventy-two hours post-MI, diaphragm muscle specific force
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was diminished by nearly 20% (P < 0.05), and therapeutic administration of R548 completely
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prevented this loss (Figure 3). Measurements of cardiac pathology, such as mean left ventricular
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weight and epicardium infarct area, were unchanged with JAK 1/3 inhibition (data not shown),
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arguing that improved diaphragm function was likely related to intrinsic factors within
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diaphragm muscle. Inflammation-mediated reactive oxygen species (ROS) and oxidative stress
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may reduce muscle specific force after acute MI (3); therefore we next determined whether MI
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increased cellular markers of oxidative damage. However, protein carbonyl and 4-HNE levels,
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markers of oxidative stress assessed in whole muscle lysates, were not significantly elevated in
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diaphragm muscle from MI mice (data not shown). We also evaluated caspase-3 activation, since
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previous studies suggest that this apoptotic protease contributes to muscle wasting and weakness
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(5, 11), and because we previously showed that JAK 1/3 inhibition during mechanical ventilation
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prevented activation of caspase-3 (12). Western blot analysis revealed no significant differences
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in the protein levels of total or cleaved (activated) caspase-3 (Figure 4). Additionally, there were
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no significant differences in the amount of partially degraded (cleaved) actin, an established
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caspase-3 proteolytic substrate (Figure 4). Together these data suggest that caspase-3 was
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unlikely to have contributed to diaphragm weakness at the time point evaluated here. While
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markers of oxidative damage in whole cell lysates were unchanged in the current study,
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oxidative modifications to specific contractile proteins following MI, as was recently reported
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(3), may have contributed to diminished diaphragm force. Indeed, we previously reported that
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inhibition of JAK/STAT3 signaling significantly reduced mitochondrial dysfunction and
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oxidative damage in diaphragm muscle during mechanical ventilation (12). This study
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demonstrates for the first time prevention of diaphragm weakness following acute myocardial
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infarction using a clinically relevant drug treatment regimen. Additionally, our findings identify
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the important role of JAK 1/3 signaling in MI-induced diaphragm weakness in mice.
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Systemic inflammation is common among patients with cancer and acute MI, and is also
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reported in experimental models of these conditions. Of the pro-inflammatory cytokines typically
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upregulated in these critical illnesses, preclinical studies link the IL-6/JAK/STAT3 signaling
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pathway to muscle wasting and dysfunction. In this pathway, binding of the IL-6/IL-6 receptor
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complex to the transmembrane glycoprotein 130 (gp130) leads to phosphorylation and activation
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of the intercellular JAKs and their downstream target STAT3 (8). Activated STAT3 translocates
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into the nucleus, and initiates target gene transcription. Recent preclinical studies demonstrate
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the important role of the IL-6/JAK/STAT3 pathway in muscle wasting and weakness in a variety
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of conditions, and further show diverse mechanisms connecting the pathway to muscle atrophy
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and dysfunction. For example, as mentioned above, mechanical ventilation in rats resulted in
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STAT3 accumulation within mitochondria, mitochondrial dysfunction, elevated oxidative
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damage, and diaphragm weakness, which were prevented by JAK 1/3 inhibition (12). In aged
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mice and mice with muscular dystrophy, STAT3 activation blunted satellite cell expansion and
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inhibited muscle repair following injury (15). In an experimental model of chronic kidney
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disease, STAT3 initiated muscle wasting through activation of the transcription factor C/EBPδ,
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and a potent negative regulator of muscle mass, myostatin (17). Similar findings were reported in
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preclinical models of cancer-cachexia (11). Here we expand on previous studies, and report for
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the first time the critical role of JAK 1/3 signaling in diaphragm muscle weakness in animal
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models of cancer-cachexia and acute MI. Improved diaphragm contractility throughout the force
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frequency curve suggests that JAK 1/3 inhibition in these preclinical models may improve low to
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moderate intensity diaphragm function typically utilized at rest and during normal activity, as
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well as maximal force required during high respiratory demand. Further studies are necessary to
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better understand the precise mechanisms of JAK 1/3-mediated improvements in diaphragm
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specific force in these experimental models, as well as the potential impact on respiratory
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function.
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In conclusion, our findings highlight the important role of JAK 1/3 signaling in
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diaphragm muscle dysfunction in both cancer-cachexia and acute MI in mice, and provide a
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promising therapeutic target to preserve diaphragm function in these circumstances. These
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findings may have relevance to respiratory muscle dysfunction in critical illness in humans, and
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warrant further study.
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Acknowledgements
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We recognize Esteban S. Masuda, Vanessa C. Taylor, Rajinder Singh, and Yan Chen for their
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roles discovering and developing the JAK 1/3 inhibitor R548. We also thank Philip Coblentz
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(UF), Nikhil Patel (UF), and Kathy White (Rigel) for excellent technical support with in vivo
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studies. I.J.S., G.L.G., D.G.P., and T.M.K. are employees and/or stockholders of Rigel
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Pharmaceuticals, Inc.
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Figure Legends
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Figure 1. JAK 1/3 inhibition prevents cancer-mediated diaphragm weakness. Specific force-
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frequency relationship in diaphragm muscle strips of control mice or cachectic C26 mice fed
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standard chow or chow containing the JAK 1/3 inhibitor R548. Data represent mean ± SEM, n =
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5-6 per group. * P < 0.05 C26/Veh vs all other groups, ** P < 0.05 C26/Veh vs C26/R548, # P
