INTRODUCTIONMechanical ventilation (MV) is an im-

portant component of modern medicalpractice which allows support of breath-ing in the intensive care unit (ICU) andduring surgery requiring general anes-thesia. Many patients, however, fail ini-tial weaning from the ventilator and

enter the difficult realm of prolongedventilation. Patients who develop thisventilator dependence, though a diversegroup, share the common underlyingproblem of substantial dysfunction of themajor inspiratory muscle, the diaphragm(1–8). The development of ventilator-in-duced diaphragm dysfunction (VIDD)

appears to be a major underlying causeof prolonged ventilator-dependence withits attendant dramatic increase in mor-bidity and mortality (9–14).

The pathogenesis of VIDD includesboth atrophy of diaphragmatic myofibersand loss of diaphragmatic contractilefunction (that is, specific force) unrelatedto atrophy (15–17). In previous studies,MV with diaphragm inactivity has beenshown to elicit significant dysfunctionand/or atrophy of myofibers in the di-aphragm of humans (18–21), rats (22),mice (23–25), rabbits (26) and piglets (27).With regard to the atrophy, several prote-olytic events, such as activation of theubiquitin proteasome system (UPS)(28–30), autophagy (24,25,31) and apo-ptosis (32–35), and upregulation of cal-pain (36), have been demonstrated inMV models. We and others have re-

M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4 | T A N G E T A L . | 5 7 9

The JAK–STAT Pathway Is Critical in Ventilator-InducedDiaphragm Dysfunction

Huibin Tang,1,2 Ira J Smith,3 Sabah NA Hussain,4 Peter Goldberg,4 Myung Lee,1,2 Sista Sugiarto,1,2

Guillermo L Godinez,3 Baljit K Singh,3 Donald G Payan,3 Thomas A Rando,5,6 Todd M Kinsella,3 and Joseph B Shrager1,2

1Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California,United States of America; 2Veterans Administration Palo Alto Healthcare System, Palo Alto, California, United States of America;3Rigel Pharmaceuticals, South San Francisco, California, United States of America; 4Critical Care Division, Royal Victoria Hospital,Montreal, Quebec, Canada; 5Department of Neurology and Neurological Science, Stanford University School of Medicine, Stanford,California, United States of America; and 6Neurology Service, Veterans Administration Palo Alto Healthcare System, Palo Alto,California, United States of America

Mechanical ventilation (MV) is one of the lynchpins of modern intensive-care medicine and is life saving in many critically illpatients. Continuous ventilator support, however, results in ventilation-induced diaphragm dysfunction (VIDD) that likely prolongspatients’ need for MV and thereby leads to major associated complications and avoidable intensive care unit (ICU) deaths. Oxidative stress is a key pathogenic event in the development of VIDD, but its regulation remains largely undefined. We reporthere that the JAK–STAT pathway is activated in MV in the human diaphragm, as evidenced by significantly increased phospho-rylation of JAK and STAT. Blockage of the JAK–STAT pathway by a JAK inhibitor in a rat MV model prevents diaphragm musclecontractile dysfunction (by ~85%, p < 0.01). We further demonstrate that activated STAT3 compromises mitochondrial functionand induces oxidative stress in vivo, and, interestingly, that oxidative stress also activates JAK–STAT. Inhibition of JAK–STAT preventsoxidative stress-induced protein oxidation and polyubiquitination and recovers mitochondrial function in cultured muscle cells.Therefore, in ventilated diaphragm muscle, activation of JAK–STAT is critical in regulating oxidative stress and is thereby centralto the downstream pathogenesis of clinical VIDD. These findings establish the molecular basis for the therapeutic promise ofJAK–STAT inhibitors in ventilated ICU patients.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2014.00049

Address correspondence to Joseph B Shrager, Division of Thoracic Surgery, Department of

Cardiothoracic Surgery, Stanford University School of Medicine, 2nd floor, Falk building, 300

Pasteur Dr., Stanford, CA 94305-5407. Phone: 650-721-2086; Fax: 650-724-6259; E-mail:

shrager@stanford.edu; and Todd Kinsella, Rigel Pharmaceuticals, Inc., 1180 Veterans Blvd.,

South San Francisco, CA 94080. Phone: 650-624-1285; E-mail: tkinsella@rigel.com.

Submitted March 12, 2014; Accepted for publication September 30, 2014; Epub

(www.molmed.org) ahead of print September 30, 2014.

ported that oxidative stress is induced inMV diaphragm (35,37,38) and that the el-evated mitochondrial oxidative stress(MOS) in MV human diaphragm appearsto be a key upstream inducer of theseproteolytic events (35).

In addition to promoting proteinturnover, MOS could impact specificforce as well in at least two ways. MOSgenerates free radicals that may directlyoxidize muscle proteins, altering theirstructure and function, includingchanges to myofilament structure, cross-bridge kinetics and/or a reduction of thecalcium sensitivity of myofilaments(39–42). Secondly, MOS is able to inducea metabolic switch, a reduction in mito-chondrial oxidative phosphorylation andan increase in glycolysis (43–46), whichmay lead to reduced overall energy sup-ply to the muscle. Since reduction ofspecific force occurs prior to the pres-ence of muscle atrophy in the ventilatedhuman diaphragm (20,23), this compo-nent of VIDD may account for the earli-est and perhaps most critical phase ofthe process. Therefore, understandingthe basis of MOS generation in the MVdiaphragm would be likely to identifytargets that could be used to impact themost clinically important phase ofVIDD.

The JAK–STAT pathway, consisting ofJanus kinase (JAK) and signal transducerand activator of transactivation (STAT),is a signaling cascade that can be acti-vated by cytokines, hormones andgrowth factors via ligand-receptor inter-actions. The consequentially activatedJAK kinase can further phosphorylateSTAT proteins, and the latter usuallyform dimers, translocate to the nucleusand transcriptionally activate genes. In aprevious study, we found that STAT3gene expression level was upregulated inventilated human diaphragm and thatthis upregulation was linked to the acti-vation of mitochondrial apoptosis (35). Italso was recently reported that overex-pression of STAT3 can lead to skeletalmuscle atrophy (47). In addition, oxida-tive stress has been shown to activate theJAK–STAT pathway in vitro (48). There-

fore, it is reasonable to speculate thatMV-induced oxidative stress elevatesSTAT3 and thereby contributes to themuscle atrophy component of VIDD.However, whether and how theJAK–STAT pathway contributes to the re-duction in diaphragm muscle specificforce associated with prolonged MV re-mains unknown.

In the current study, we report thatJAK and STAT are significantly phos-phorylated/activated in both humanand rat diaphragms subjected to MV.Blockade of the JAK–STAT pathway inventilated rats dramatically prevents theloss of contractile function in their di-aphragms. Overactivation of JAK–STATinduces oxidative stress in skeletal mus-cle in vivo, with increased protein oxida-tion and reduction in mitochondrialmembrane potential. Interestingly,JAK–STAT also can be activated by ox-idative stress, and inhibition of JAK orSTAT can suppress H2O2-induced oxida-tive stress. Together, these findingsstrongly suggest that the regulation ofoxidative stress via activation ofJAK–STAT is crucial in the developmentof VIDD. This work renders theJAK–STAT pathway an optimal targetfor a drug to prevent VIDD.

MATERIALS AND METHODS

Human SamplesThe human samples were collected as

described in detail previously at the Uni-versity of Pennsylvania, Philadelphia,USA (30) and the Royal Victoria Hospi-tal, Montreal, Quebec, Canada (31). Con-trol diaphragm muscle biopsies weretaken from patients 69.4 ± 4.7 years old(range 63 to 83 years old) with bodymass index (BMI) 24.1 ± 0.52 kg/m2

(range 23.3 to 26.6 kg/m2). The ventila-tion time for the controls was 1.21 ± 0.1 h(range 1 to 1.5 h). The MV diaphragmsamples were taken from patients 58.2 ±4.6 years old (range 41 to 75 years), meanBMI 27.1 ± 1.77 kg/m2 (range 23 to31 kg/m2). The average MV time forthese patients was 49 ± 8.6 h (range 12 to74 h). Nonrespiratory control muscles

from these control and MV patients weretaken from the quadriceps muscle, as de-scribed previously (31,35).

Mechanical Ventilation of Rats andMeasurement of DiaphragmContractile Function

Animal protocols were approved bythe Institutional Animal Care and UseCommittees of Rigel Pharmaceuticals,Inc. All animal experiments were carriedout according to recommendations inGuide for the Care and Use of LaboratoryAnimals (eighth edition) (49). All surgicalprocedures were performed using asep-tic techniques. Animals (Sprague Dawleyrats, 270 ± 10g) were anesthetized to asurgical plane of anesthesia with isoflu-rane (2% to 4%) and a tracheotomy wasperformed. Rats were maintained on MVwith isoflurane for 18 h using a volume-driven small-animal ventilator (CWE,Ardmore, PA, USA). Tidal volume wasset at 0.7 mL/100 g body weight, respira-tory rate was 80/minute. A carotid arterycatheter was utilized to monitor bloodpressure and to collect arterial bloodsamples. JAK inhibitor or control vehiclewere delivered continuously through ajugular vein cannula. Heart rate wasmonitored throughout the study usingECG needle electrodes, and body tem-perature was maintained at 37°C by arectal temperature probe connected to aHomeothermic Blanket System. Bodyfluid homeostasis was maintained viasubcutaneous administration of1.7 mL/kg body weight/2.5 h saline. Toreduce airway secretions, glycopyrrolate(0.04 mg/kg) was administered subcuta-neously every 2.5 h. After 18 h continu-ous MV, the rats were euthanized and di-aphragms were collected and either usedimmediately for contractile functionstudies or snap frozen in liquid nitrogenfor biochemical assays stored at –80°C.

Diaphragm contractile function wasdetermined using diaphragm stripsmaintained ex vivo, as described previ-ously (38). Briefly, upon completion ofthe study, the entire diaphragm was re-moved and transferred to a dissectingdish containing Krebs-Hensleit physio-

5 8 0 | T A N G E T A L . | M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4

J A K – S T A T R E G U L A T E S D I A P H R A G M C O N T R A C T I L I T Y

logical solution aerated with 95% O2 to5% CO2 gas. A muscle strip was dis-sected from the midcostal region of thediaphragm, which included a portion ofthe central tendon and ribcage. The stripwas mounted vertically using serratedjaw tissue clamps, with one end fixed toan isometric force transducer on a tissueorgan bath system 750 (DMT, Ann Arbor,MI, USA), and immersed in the same so-lution. After a 15-min equilibration, di-aphragm strips were stimulated with aS88X Pulse Stimulator (Grass Technolo-gies, Warwick, RI, USA), and platinumwire electrodes (Radnotti, Monrovia, CA,USA). Contractile measurements weretaken at the optimal muscle length (Lo),the length at which maximal force is ob-tained. Lo was determined by stimulat-ing the muscle at a supramaximal volt-age, and systematically adjusting themuscle length. The force–frequency rela-tionship was assessed by stimulatingeach strip supramaximally with 8-Vpulses, with a train duration of 500 ms,at 15–160 Hz. Contractions were sepa-rated by a two-minute recovery period.Diaphragm force production was nor-malized as specific force based on mus-cle cross-sectional area (CSA). Total mus-cle CSA at right angles to the long axiswas determined by the following calcu-lation: total muscle CSA (mm2) = [muscle mass/ (fiber length × 1.056)],where 1.056 is the density of muscle (in g/cm3). Fiber length was expressed incentimeters measured at Lo.

Reagents and PlasmidsA pan-JAK inhibitor 1 (JAK I) and

STAT3 inhibitor (stattic) were purchasedfrom EMD Millipore (Billerica, MA,USA). The JAK inhibitors, R545 andR548, were generated at Rigel Pharma-ceuticals. Both are potent JAK inhibitors,with an inhibitory effect over JAK1/3 at0.03 nmol/L and JAK2 at 1.1 nmol/L(Supplementary Table S1). H2O2 was pur-chased from Sigma-Aldrich (St. Louis,MO, USA). Lipofectamine 2000 was pur-chased from Invitrogen/Life Technolo-gies/Thermo Fisher Scientific (Waltham,MA, USA). pcDNA3.1–STAT3C was con-

structed in lab by digestion of an EF1-STAT3C-ubc-GFP construct (AddgeneInc., Cambridge, MA, USA) with Hind3and then ligated into pcDNA3.1 vector atthe Hind3 site. The final construct wassequenced for validation. Prk5-JAK2V617F was a gift from LawrenceArgestinger (University of Michigan)with the permission of James Ihle (SaintJude Children’s Research Hospital). pcs2-GFP and pcs2-bGal plasmids were fromDavid Turner and Daniel Goldman (Uni-versity of Michigan).

Cell Culture and TransfectionPlasmids were transfected into

HEK293 cells with lipofectamine 2000.Three days later, ATP content or lu-ciferase assays were performed. The co-transfected pcs2-β-Gal was used for nor-malization via β-Gal assay according tostandard procedures.

Gene electroporation was performed asdescribed previously (44). Briefly, 20 μg ofcontrol plasmid (pcDNA3 vector) or 20 μgof pcDNA3-STAT3C plasmid was mixedwith 2 μg pcs2-GFP plasmid, respectively,and injected into mouse tibialis anterior(TA) muscles, immediately followed by 8pulses of electrical shocks at 140V/cm of60-ms duration with an interval of100 ms, delivered by ECM830 (HarvardApparatus, Holliston, MA, USA) squarewave electroporation system. After 10 d,the TA muscles were collected, and GFP+fibers (~25% of the muscle) were isolatedwith fine forceps under the fluorescentdissecting scope. The high ratio (10 to 1)of the plasmids containing the gene of in-terest to the plasmid containing the GFPreporter ensures that nearly all GFP+fibers express the gene of interest. Totalprotein was extracted from these GFP+fibers by RIPA buffer and then subjectedto Western blot analysis.

JC1 Mitochondrial MembranePotential Assay

Mitochondrial membrane potentialassay was performed with the JC-1reagent (Cayman Chemical Company,Ann Arbor, MI, USA). The JC-1 reagentwas diluted 1:10 in JC-1 assay buffer to

prepare the JC-1 staining solution. Tenmicroliter of staining solution wasadded to the 100 μL of culture media,incubating at 37°C for 30 min. The platewas centrifuged (400g, 5 min) at roomtemperature, and the supernatant wasremoved. After being washed threetimes with an assay buffer, cells werethen analyzed with a SpectraMax M2eMulti-Mode Microplate Reader (Molec-ular Devices LLC, Sunnyvale, CA, USA)for live cells (excitation/emission at560/595 nm) and dead cells (excitation/emission at 485/535 nm).

ATP Content MeasurementATP content in cells was measured

with a Biovision ATP assay kit accordingto the supplier’s instructions. Briefly, cellswere lysed in 200 μL ATP assay bufferprovided by the manufacturer (BioVision,San Diego, CA, USA). The samples werethen centrifuged at 20,183g for 15 min at4° to pellet insoluble materials. Super-natants were collected into a fresh set oftubes for the assay. Fifty microliter of thereaction mix was added to 50 μL of lysateto start the ATP reaction. The optical den-sity (OD) 570 nm was measured at 10 to20 min intervals and the concentrationswere calculated using the standards pro-vided by the manufacturer. The ATP con-centrations were then normalized to totalprotein concentrations.

Immunostaining and Western BlottingCultured C2C12 muscle cells on slides

were fixed with 2% PFA for 30 min, andthe immunostaining was performed bystandard procedures. Anti-STAT3 anti-body was purchased from Cell SignalingTechnology (Danvers, MA, USA); andAlexa555-conjugated anti-rabbit second-ary antibody and Alexa488-WGA werepurchased from Invitrogen/Life Technologies/Thermo Fisher Scientific.Mounted cells were then imaged by con-focal microscopy (Zeiss, Jena, Germany).

Protein expression levels were de-tected by Western blot analysis followingstandard procedures. Primary antibodies,anti-DNP (dinitrophenol) and 4-HNE(4-hydroxy-2-nonenal), were purchased

R E S E A R C H A R T I C L E

M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4 | T A N G E T A L . | 5 8 1

from Abcam (Cambridge, England); pri-mary antibody anti-nitrotyrosine waspurchased from EMD Millipore. The restof the antibodies used in this study werepurchased from Cell Signaling Technol-ogy. The phosphorylation sites specifi-cally recognized by these antibodies arepJAK1-tyr1022/1023, pJAK2-tyr1007/1008, pJAK3-tyr980/981,pSTAT5-tyr694 and pSTAT3-tyr705.

Gene Profiling, Quantitative PCRGene profiling was performed as de-

scribed (35). mRNA expression levelswere detected by real-time PCR by stan-dard procedures. The primers used arelisted in Supplementary Table S2.

Statistical AnalysesQuantitation of gray density was per-

formed with ImageJ software (NationalInstitutes of Health, Bethesda, MD, USA;http://imagej.nih.gov/ij). One-way anal-ysis of variance (ANOVA) was used todetermine the significant changes whenthere were more than three groups forcomparison, followed by Tukey post hoctest. Student t test was used to evaluatethe significance while comparing twogroups in this study. A level of p < 0.05,indicated by and asterisk (*) in figures,was considered significant.

RESULTS

The JAK–STAT Signaling Pathway IsActivated in MV Human Diaphragm

To understand the molecular pathogen-esis of VIDD, we began by analyzing geneprofiling data from control (MV <2 h) andventilated (MV >18 h) human diaphragmsamples. With the DAVID bioinformatictool, we found that at least 15 signalingpathways are significantly (P < 0.05) al-tered in ventilated human diaphragm, in-cluding those linked to previous observa-tions in ventilated diaphragm, such as thetoll-like receptor (24), apoptosis (35), gly-colysis (44), calcium (33) and adipocy-tokine signaling pathways (50). However,several novel pathways not previously re-ported, including JAK–STAT, Wnt,MAPK, TGF-β and the insulin/mTOR sig-

naling pathway, also were identified (Fig-ure 1A and Supplementary Table S3). TheJAK–STAT signaling pathway is amongthe most significantly (p < 0.0001) alteredKEGG pathways (see Figure 1A and Sup-plementary Table S3), as evidenced by increased expression of both upstream inducer genes, such as IL6 and IL24, andthe downstream target gene SOCS3 (Fig-ure 1B). We validated the increased ex-pression of these genes by quantitativePCR (Figure 1C).

Since the increased expression of IL6and IL24 can lead to activation of theJAK–STAT signaling pathway by induc-ing the phosphorylation of JAK and STATproteins, we examined the phosphoryla-tion levels of JAK and STAT. We find thatthe phosphorylated forms of JAK1(pJAK1-Y1022/1023), JAK2 (pJAK2-Y1007), JAK3 (pJAK3-Y980/981), STAT3(pSTAT3-Y705) and STAT5 (pSTAT5-Y694) all are significantly upregulated inhuman ventilated diaphragm (Figure 1D),ranging from two-fold to 3.5-fold. Sincephosphorylated STAT3 forms dimers andtranslocates into nuclei to regulate geneexpression, we examined the subcellulardistribution of STAT3 in MV diaphragmby immunostaining. These results indi-cate that STAT3 protein is indeed en-riched in diaphragmatic myonuclei fol-lowing MV (Figure 1E).

The activation of STAT appears to bediaphragm-specific, since it is not acti-vated in the quadriceps muscles of theventilated patients (Figure 1F). This indi-cates that the activation of STAT3 in di-aphragm muscle is unlikely to be occur-ring through systemic factors.

Blockade of the JAK–STAT Pathwaywith a JAK Inhibitor SubstantiallyPrevents Diaphragm MuscleContractile Dysfunction in a Rat MVModel

To investigate the functional signifi-cance of the activated JAK–STAT signal-ing pathway in MV diaphragm, we em-ployed a JAK inhibitor to block theactivation of JAK–STAT in a rat MVmodel. MV was performed on adult SDrats for 18 h and the JAK inhibitor (R545)

was delivered by continuous intravenousinfusion. As in MV human diaphragm,the phosphorylation level of STAT3(Y705) is significantly induced in MV ratdiaphragm (Figure 2A). Delivery of theJAK inhibitor completely blocks the in-crease in STAT3 phosphorylation (seeFigure 2A).

Although diaphragm muscle fiber sizeis in fact reduced in this model of MV ratdiaphragm (51), the specific force (that is,the maximum contractile force normal-ized to fiber size measured with stimu-lated diaphragm strips), is even moredramatically reduced (down to ~50% ofcontrol). Diaphragm strips also appearsomewhat more fatigable following MV,although the major difference betweencontrol and MV diaphragm strips isclearly the difference in specific force,which remains significant up to the4-min point during a fatiguing protocol.JAK inhibitor treatment prevents the lossof maximal diaphragm force-generatingcapacity (median rescue of ~85% relativeto untreated MV rat diaphragm at100 Hz; p = 0.0028) (Figures 2B–D).

These data indicate that activation ofJAK–STAT is necessary for the reductionin specific force, and thereby the devel-opment of VIDD.

Enhanced Activity of JAK–STATInduces Oxidative Stress In Vitro andIn Vivo

Although the above data indicate thatJAK–STAT might be critical in the devel-opment of VIDD, it remains unclear fromthis data which pathogenic changes thissignaling pathway support that lead toVIDD. Since oxidative stress is known tobe activated in both rat and human MVdiaphragm (35,37,38) and is a key patho-logical event in directing the evolution ofVIDD (35), we hypothesized that acti-vated JAK–STAT may reduce the specificforce in diaphragm muscle by inducingoxidative stress in this disease process.

To test this hypothesis, we first exam-ined if activation of JAK–STAT is able toinduce oxidative stress. We transfected aplasmid with cDNA encoding a constitu-tively active STAT3 (STAT3C) cDNA into

5 8 2 | T A N G E T A L . | M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4

J A K – S T A T R E G U L A T E S D I A P H R A G M C O N T R A C T I L I T Y

R E S E A R C H A R T I C L E

M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4 | T A N G E T A L . | 5 8 3

AGene Experiments

1 2 3 4F.C.

(MV/Con)IL10Ra -3.6CCND1 -7CCND2 -10IL7 -8.8IL20Ra -5.6LEPR -8IL6 2.6IL24 4.3IL6R 5.4CSF3 11.6CISH 2.1PIK3R1 5.4MYC 5.7SOCS3 13.1

B

CD

E

Altered signaling pathways in human MV diaphragm Jak-STAT signaling pathway P < 0.0001Calcium signaling pathway P < 0.0001Wnt signaling pathwayInsulin signaling pathwayToll-like receptor signaling pathway P < 0.0001MAPK signaling pathway P < 0.0001TGF-beta signaling pathway P < 0.0001Cytokine-cytokine receptor interaction P < 0.0001Adipocytokine signaling pathway P < 0.0001Hedgehog signaling pathwayApoptosis

P < 0.001

Phosphatidylinositol signaling systemVEGF signaling pathwayGlycolysis / Gluconeogenesis

P Value

P < 0.0001P < 0.0001

STAT3 WGADAPI Merged

Tang et al., Fig. 1

Con

MV

0123456

IL6 IL6R IL24 IL20Ra IL20Rb SOCS3

ConMV

*

*

*

*

*

F Quadriceps musclesCon MV

pSTAT3

STAT3

Actin

051015

STA

T3+

(% o

f tot

al n

ucle

i)

Con MV

*

mR

NA

leve

ls (M

V/C

on) Con MV

pJAK1pJAK3

pJAK2

JAK2

Actin

pSTAT5

Actin

pSTAT3

STAT3

Actin

00.20.40.60.8

1

* * *

0

1

2

3

pJAK1/Actin

pJAK2/Actin

pJAK3/Actin

pJAK2/JAK2

Con MV Con MV Con MV Con MV

Con MV Con MV Con MV

pSTAT3/Actin

pSTAT5/Actin

pSTAT3/STAT3

**

*

Gra

y de

nsity

(a.u

)G

ray

dens

ity (a

.u)

P < 0.001P < 0.001P < 0.001P < 0.01P < 0.05mTOR signaling pathway

Figure 1. Activated JAK–STAT signaling pathway in MV human diaphragm. (A) Altered signaling pathways in the MV human diaphragm. Alteredgenes (p < 0.05) from microarray analysis of MV versus control diaphragm samples were input into DAVID Bioinformatics analysis. In the pie figure,the percentage indicates the number of genes involved in the specific pathway over the total number of genes involved in all signaling path-ways listed. The p value indicates the significance of the altered signaling pathway. Signal pathways with p < 0.05 are listed. (B) The mean foldchange in MV versus control human diaphragm samples of genes significantly (p < 0.05) altered by MV that are involved in the JAK–STAT signal-ing pathway (n = 4 pairs of samples). The color green in the heat map indicates the reduced expression of genes, while red indicates increasedexpression. Specific fold changes are indicated in the right column. (C) Quantitative PCR was performed to confirm the altered expression ofgenes identified by microarray. Human diaphragm muscle samples (Con n = 6, MV n = 8) were examined, *p < 0.05. (D) Activation of theJAK–STAT signaling pathway. Protein extracts from MV and control human diaphragm muscles were subjected to Western blot analysis (n ≥ 4).The two lower bar graphs show the data quantified, with normalization of phosphorylated protein. *p < 0.05. (E) Nuclear enrichment of STAT3 inMV human diaphragm. Human diaphragm muscles were stained with STAT3 (red), DAPI (blue) or WGA (green) to show STAT3, nuclei and cellmembrane respectively. Arrows indicate the location of nuclei. The lower panel shows the quantitated data of the STAT3- positive nuclei per totalnuclei. n = 4, *p < 0.05. Note that STAT3 is enriched in nuclei in the ventilated but not the control human diaphragms. (F) STAT3 is not activated innonrespiratory muscles during mechanical ventilation. Protein extracts from human quadriceps muscles from control and MV patients, and sub-jected to Western blot analysis. Note that there is no induction of phosphorylated STAT3 in quadriceps muscles under MV. F.C., fold changes; Con,control; IL6R, interleukin-6 receptor; IL20Ra, interleukin-20 receptor alpha subunit; IL20Rb, interleukin-20 receptor beta subunit; a.u., arbitrary unit.

the mouse tibialis anterior (TA) musclevia electroporation. STAT3C is a mutantform of STAT3, which is constitutivelyactive due to the mutation of alanine (A)661 to cysteine (C) and asparagine (N)663 to cysteine (C). After confirming anincreased expression of STAT3C in skele-tal muscle (Figure 3A), we found thatoverexpression of STAT3C results in theinduction of protein expression of Bcl2l11(Bim), a modulator of mitochondrialmembrane potential and a proapoptoticgene. However, the protein levels of sev-eral mitochondrial components, C1(NDUFB8), C2 (SDHB), C3 (UQCRC2)and C5 (ATP5A), do not show significantchange (Figures 3A, B). Overexpressionof STAT3C also leads to increased proteinoxidation, evidenced by increased abun-dance of nitrotyrosine and 4-hydrox-ynonenal (4-HNE) (Figure 3C, D), indi-cating that the increased STAT3 activityin skeletal muscle does induce oxidative

stress. In addition, there is a dramatic in-duction of muscle protein ubiquitinationfollowing overexpression of STAT3C (seeFigures 3C, D). Although increased pro-tein oxidation and ubiquitination oftenare linked to protein degradation andthus contribute to muscle atrophy, it isalso possible that these posttranslationalmodifications affect the contractile func-tion of muscle proteins and thus lead tothe reduction in specific force, prior toprotein degradation. Together, the in-creased protein posttranslational modifi-cations appear to link the proteasome-mediated protein degradation and thereduction in specific force that occurs inVIDD to a common regulatory pathway,that is, the JAK–STAT pathway.

Since constitutively active STAT3 in-creases the expression of Bcl2l11 (Bim)protein, which can alter mitochondrialmembrane potential, activatedJAK–STAT may affect mitochondrial

function. In fact, it has been reported thatoverexpression of STAT3C reduces mito-chondrial membrane potential (52). Herewe demonstrate that overexpression of aconstitutively active JAK2 reduces mito-chondrial membrane potential, shown bythe reduced JC1 index with overexpres-sion (Figure 3E).

We also examined the transcriptionalregulation of gene expression by STAT3in cultured cells. Overexpression ofSTAT3C leads to induction of SOCS3 andBcl2l11 (Bim) (Figure 3F). Concomitantly,UCP2, a mitochondrial transporter pro-tein that creates a proton leak across theinner mitochondrial membrane, therebyreducing the efficiency of energy produc-tion by uncoupling oxidative phosphory-lation from ATP synthesis, is induced bySTAT3C (see Figure 3F). On the otherhand, Cox5b, a nuclear-encoded mito-chondrial gene that functions in electrontransport, is significantly downregulatedby STAT3C (see Figure 3F). This profileof gene expression is thus consistentwith the functional decline in mitochon-dria induced by activated STAT3.

Consistent with the STAT3-inducedchanges in gene expression, MV leads tosuppression of the mRNA expression ofNDUFS3 and UQCRC2 in rat diaphragmmuscle, and the pan-JAK inhibitor res-cues the suppression of NDUFS3 (Sup-plementary Figure S1). This clearly indi-cates a role of JAK–STAT in regulatingthe expression of these genes that are rel-evant to mitochondrial function.

JAK–STAT Is Both Activated by H2O2-Induced Oxidative Stress and IsEssential for the Evolution of OxidativeStress

The above work suggests that MV- activated JAK–STAT leads to VIDD inpart by inducing oxidative stress. How-ever, how JAK–STAT is activated in theMV diaphragm remains unclear. We de-veloped an in vitro oxidative stressmodel in cultured muscle cells basedupon H2O2 treatment. We find that H2O2-induced oxidative stress activates theJAK–STAT signaling pathway in musclecells, as evidenced by the increased

5 8 4 | T A N G E T A L . | M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4

J A K – S T A T R E G U L A T E S D I A P H R A G M C O N T R A C T I L I T Y

pSTAT3

STAT3

Con MV MV+R545A

Spe

cific

forc

e (N

/cm

)2

30

20

10

00 25 50 75 100 125 150 175

ConMVMV+R545

B

Spe

cific

forc

e (N

/cm

)2 40

30

20

10

0Con MV MV+R545

p=0.0001p=0.0028

Frequency (Hz)

Spe

cific

forc

e (N

/cm

)2 ConMVMV+R545

25

20

15

10

540

0 5 10Time (min)

C D

Tubulin

Figure 2. JAK inhibition prevents the reduction of muscle-specific force that occurs in me-chanically ventilated rats. (A) Mechanical ventilation induces STAT3 phosphorylation andJAK inhibitor blocks this activation. Control and MV (18 h) rat diaphragm muscles weresubjected to Western blot analysis (n = 4 rats per group). (B–D) Treatment with a JAK in-hibitor (R545) prevents MV-induced contractile dysfunction. The diaphragms from controls(n = 10) and MV rats treated with the JAK inhibitor (n = 8) or vehicle (n = 9) were ana-lyzed: (B) Diaphragm strip ex vivo force–frequency relationship; (C) Specific force genera-tion at 100 Hz; and (D) Fatigue development after 18 h of MV. Con, control.

phosphorylation of JAK2 and STAT3(Figure 4A). Blocking the activation ofJAK–STAT with a JAK inhibitor sup-presses the H2O2-induced STAT3 phos-phorylation/activation (Figure 4B) andconsequently reduces the H2O2-inducednuclear enrichment of STAT3 (Figure 4C).H2O2-induced oxidative stress leads toincreased protein posttranslational modi-fication through carbonylation (anti-DNP)and nitration (anti-nitrotyrosine). This in-duction is reduced by either the JAK in-hibitors (pan-JAK inhibitor I [JAK I] andR545) or the STAT inhibitor stattic (Fig-ures 4D, E; Supplementary Figure S2). Inaddition, H2O2-induced protein ubiquiti-nation also is blocked by either JAK orSTAT inhibitors (see Figures 4D,E, Sup-plementary Figure S2). Mitochondrialmembrane potential and ATP generationare both reduced by H2O2-induced ox-idative stress, and this reduction is atten-uated by pretreatment with either theJAK or STAT inhibitor (Figures 4F, G).

These data demonstrate that H2O2- induced oxidative stress activates theJAK–STAT signaling pathway, and, con-versely, that the JAK–STAT pathway isimportant in the development of H2O2-in-duced oxidative stress. These observa-tions suggest that activation of JAK–STATby ROS might facilitate the developmentof a vicious cycle during MV.

DISCUSSIONProlonged dependence upon MV is an

enormous problem in our hospitals’ in-tensive care units. As the duration of MVrises, so does the incidence of complica-tions such as ventilator-associated pneu-monia (53). The longer the period of fullventilator support, the more severeVIDD becomes (20), creating a down-ward spiral that may become irreversibleand even result in death. An enormouscost in both lives and health system dol-lars is thus incurred by the problem ofVIDD. Prevention of VIDD would belikely to have a major impact upon themorbidity, mortality and costs of inten-sive care medicine.

We report here that the posttransla-tional activation of JAK–STAT in a MV

R E S E A R C H A R T I C L E

M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4 | T A N G E T A L . | 5 8 5

Figure 3. Overexpression of constitutively active STAT3 induces oxidative stress. (A) Constitu-tively active STAT3 (STAT3C) induces the protein expression of Bim (Bcl2l11), but not the com-ponents in electron transportation chain. Twenty microgram of pcDNA3-STAT3C plasmid, to-gether with 2 μg of pcs2GFP plasmid, was electroporated into mouse TA muscle. GFP+ fiberswere isolated 10 d later and total protein extracts were subjected to Western blot analysis (n = 3 pairs of mouse muscles). Left pane shows the efficiency of electroporation. Green fibersare those with the GFP expression. Right panel shows the results of Western blot analysis. C1:NDUFB8, C2: DHB, C3: UQCRC2 and C5: ATP5A. (B) Quantitative data of protein expressionchanges in (A). ImageJ was used for quantitation of gray density on the Western blot (n = 3pairs, *p < 0.05). (C,D) Constitutively active STAT3 (STAT3C) induces protein oxidation andubiquitination in vivo. (C) Samples were prepared as in (A), and Western blot analysis wasperformed. Ponceau S (Pon S) is shown as loading control. (D) Quantitative data of proteinexpression changes in (C). ImageJ was used for quantitation of gray density on the Westernblot (n = 3 pairs, *p < 0.05). (E) Activated JAK2 suppresses mitochondrial membrane potential.Constitutively active JAK2V617F was transfected into HEK293 cells for 3 d, followed by JC-1assay. JC-1 index is the ratio of aggregated JC-1 over monomeric JC-1 (n = 6, *p < 0.05). (F) Activated STAT3 (STAT3C) alters the expression of genes involved in regulating mitochon-drial function. Plasmid expressing constitutively active STAT3C was transfected into HEK293cells for 3 d. Total RNA was collected and quantitative PCR was performed to examine themRNA levels (n = 4 experimental/control pairs, *p < 0.05). Con, control; arb. unit, arbitrary unit.

diaphragm is directly linked to reduc-tions in diaphragm muscle contractility.In both mechanically ventilated humanand rat diaphragms, the JAK–STAT path-way is significantly activated. We showthat activated JAK–STAT results in ox-idative stress and mitochondrial dys-function, and that inhibition ofJAK–STAT prevents this oxidative stressand the reduction in diaphragm musclecontractility. Therefore, a drug that in-hibits JAK–STAT administered early in apatient’s intensive care unit stay, may beable to prevent VIDD (Figure 5).

In addition to being the first demon-stration of the activation of JAK–STAT inMV human diaphragm tissue, the currentstudy goes beyond a previous publicationconcerning the JAK–STAT pathway inVIDD (51) by elucidating the underlyingmechanism by which JAK–STAT increasesoxidative stress. We observe that the al-tered expression of STAT3- regulatedgenes, such as Bim, UCP and Cox5a, im-pacts mitochondrial function via reducingmembrane potential and/or reducing theefficiency of ATP generation. We alsoshow that the expression of NDUFS3, agene that encodes a core subunit of themitochondrial membrane respiratorychain (complex I), is suppressed by MVbut recovered with JAK inhibitor treat-

5 8 6 | T A N G E T A L . | M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4

J A K – S T A T R E G U L A T E S D I A P H R A G M C O N T R A C T I L I T Y

A

B

Tang et al., Fig. 4

STAT3 DAPI STAT3+DAPI

Con

H2O2

(2 mM, 2h)

H2O2+JAK I(2 mM H2O2

+ 1 µM JAK I)

pJAK2

pSTAT3

STAT3

Actin

- +

JAK2

012345

H2O2

JAK IStattic

- + + +- - + -- - - +M

ito. m

embr

. pot

entia

l(J

C1

inde

x re

d/gr

een) *

**

H2O2 - + + +JAK IStattic- - - +

- - + -

*

**

ATP

leve

ls

0

1

2

C

JAK I (µM)H2O2 - + + + + +

pSTAT3

STAT3

- - 0.2 0.5 1 2E

F

Ub

Actin

Nitro-tyrosine

DNP

D- - + + + + + + + + +

JAK I R545

H2O2

020406080

0

50

100

050

100

150

Gra

y de

nsity

(a.u

., D

NP

) G

ray

dens

ity

(a.u

., N

itro.

)

H2O2- + + +- - + -- - - + R545

JAK I

**

**

* *

G

Gra

y de

nsity

(a.u

., U

b)

*

*

*

*

*

*

H2O2

Figure 4. JAK–STAT is activated by H2O2 and is required for H2O2-induced oxidative stress. (A) H2O2 activates the JAK–STAT pathway by inducing the phosphorylation of JAK2 andSTAT3. C2C12 myotubes were treated with H2O2 (2 mmol/L) for 2 h and total cell lysateswere subjected to Western blot analysis. (B,C) A pan-JAK inhibitor (JAK I) blocks the H2O2-induced phosphorylation (B) and nuclear enrichment (C) of STAT3. (B) C2C12 myotubeswere treated with H2O2 (2 mmol/L) in the presence or absence of JAK I for 2 h, and totalcell lysates were subjected to Western blot analysis. (C) Immunostaining was performed oncultured C2C12 cells. C2C12 myotubes were treated with H2O2 (2 mmol/L) for 2 h in thepresence or absence of JAK I. STAT3 (red) was stained with STAT3 antibody and nucleiwere stained by DAPI (blue), green arrows show the location of nuclei. (D) H2O2-inducedprotein posttranslational modifications are reduced by JAK inhibitors. C2C12 myotubeswere treated with H2O2 (200 μmol/L, 18 h) in the presence or absence of the pan-JAK in-hibitor (JAK I, 1 μmol/L) or R545 (0.5 μmol/L). Protein oxidation and nitration were examinedby antibodies against DNP and nitrotyrosine, and protein ubiquitination was detected byanti-ubiquitin antibody. (E) Quantitation of the modified proteins. The gray density of the in-dicated areas (*) in (D) were quantitated. *p < 0.05. (F,G) The reduction in mitochondrialmembrane potential and ATP production with H2O2 treatment is reversed by JAK and STATinhibitors. C2C12 myotubes were treated with H2O2 (200 μmol/L, 18 h) in the presence orabsence of pan-JAK inhibitor (JAK I, 0.2, 0.5 μmol/L) and STAT inhibitor (stattic; 0.2, 0.5 μmol/L)(n = 6, *p < 0.05). Con, control; Ub, ubiquitin; Nitro., nitrotyrosine; a.u., arbitrary unit.

Increased ROSProtein modification/degradation

Altered energy metabolism

Mechanical Ventilation

JAK-STAT

Mitochondrial dysfunction & oxidative stress

Specific force (early)& muscle mass

VIDD

Drugtarget?

Figure 5. Schematic of the proposed role ofJAK–STAT in the pathogenesis of ventilator-induced diaphragm dysfunction (VIDD).

ment. This transcriptional regulation isconsistent with the fact that the nuclearlocalization of the transcriptional factorSTAT3 increases following phosphoryla-tion at tyrosine 705 (STAT3-Y705) (48). Inaddition to the effect via STAT-regulatedgenes, STAT3 may also directly alter mito-chondrial function by associating withcomponents of the mitochondrial respira-tory chain. This effect is regulated by an-other phosphorylation site on STAT3, theserine 727 (51,54,55).

Oxidative stress may reduce the spe-cific force of skeletal muscle in severalways, which have been previously re-viewed (39–42), including alteration ofmyofilament structure, cross-bridge ki-netics, calcium sensitivity and energysupply. Activated STAT3 has been sug-gested to regulate energy metabolism bysuppressing mitochondrial function incancer cells (52). In the current study, weobserve that STAT3 induces protein oxi-dation and disables mitochondrial func-tion in muscle cells. It is thus possiblethat JAK–STAT affects the specific forceof diaphragm muscle by both posttrans-lational modification (for example, ubiq-uitination, oxidation and nitration) ofproteins, as well as by compromising theenergy supply. Since protein modifica-tions can occur rapidly and directly af-fect muscle contractile function, theymight account for the rapid occurrenceof diaphragm weakness after the institu-tion of MV. The upstream signaling path-ways that regulate oxidative stress inmuscle cells may thus affect muscle con-tractility, and herein we provide the de-tailed evidence that JAK–STAT is an im-portant one of these upstream pathways.

Beyond the functional role of STAT3 inreducing the specific force of diaphragmmuscle in MV, our findings also directlylink STAT3 activation to atrophy of themuscle fibers via increases in proteinubiquitination. Ventilation-induced di-aphragm muscle atrophy is partiallyblocked by JAK inhibition (51), implyingthat JAK–STAT also contributes to VIDDvia regulation of muscle mass. In addi-tion to the 20S proteasome-mediateddegradation of oxidized proteins, STAT3-

induced muscle atrophy, like FoxO- induced protein degradation, occurs alsothrough activation of the ATP-dependent26S ubiquitin-proteasome system (UPS).This observation adds another signalingpathway upstream of muscle atrophythat is additive to the previously re-ported FoxO-induced protein degrada-tion in VIDD, both of which act throughthe activation of UPS. JAK–STAT activa-tion, then, plays a central role underlyingboth of the two changes that constitutethe pathogenesis of VIDD, atrophy andintrinsic dysfunction.

Although JAK–STAT is shown here toactivate oxidative stress, we also reportthat oxidative stress is able to activateJAK–STAT in muscle cells in vitro. Thecreation of this apparent vicious cyclesuggests that activation of JAK–STAT islikely to be a critical step in the develop-ment of oxidative stress in ventilated di-aphragm muscle, perhaps underlying thesurprisingly rapid evolution of VIDD ascompared with, for example, disuseweakness of limb muscle. It is possiblethat the activation of JAK–STAT by oxida-tive stress occurs via cytokines, such as in-terleukins and VEGF, as these factors areinduced by H2O2 in cultured muscle cellsand in ventilated diaphragm (Figure 1,Supplementary Figure S3). It is not sur-prising that some cytokines/ factors areupregulated in diaphragm by mechanicalventilation (24,51; also see Figures 1A–C).What remains to be elucidated is howthese secretory cytokines/ factors couldfunction locally within diaphragm tissuesubjected to MV without causing sys-temic (for example, nonrespiratory mus-cle) atrophy. It will be worth exploringwhether blocking or neutralizing thesecytokines with competitive compoundsor antibodies would ameliorate VIDD.

Finally, we have demonstrated the ef-fectiveness of a JAK inhibitor, R545, inpreventing MV-induced diaphragmweakness, which indicates the critical roleof the JAK–STAT pathway in the develop-ment of VIDD. R545 is a nominally selec-tive JAK1/3 inhibitor based on an in vitrocell-based assay, but it retains a potent in-hibitory effect on JAK2 (Supplementary

Table S1). When the compound is deliv-ered into a rat by continuous infusion atthe doses used, it would be very difficultto assert that it only inhibits JAK1/3. Inour in vitro oxidative stress model, R545and the pan-JAK inhibitor I (JAK I) ap-pear to work equally well. It remains tobe elucidated in vivo whether or not apan-JAK inhibitor will achieve even betterrecovery of diaphragm muscle dysfunc-tion than the nominally selective JAK 1/3inhibitors. It would be also interesting inthe future to test even more specific JAKinhibitors to investigate their effect.

Our in vivo demonstration of the pre-vention of ventilator-associated di-aphragm weakness by JAK inhibition,based upon the described solid under-standing of the molecular mechanisms ofVIDD, provides strong support for con-tinued development of JAK–STAT– targeted drugs to prevent VIDD in theclinic. Since the reduction in the specificcontractile force of the diaphragm occursat the earliest stage of VIDD, prior to theonset of diaphragm muscle atrophy, it ispossible that JAK–STAT inhibitors wouldhave particular value by impacting thefirst, most clinically important compo-nent of the clinical syndrome.

CONCLUSIONWe have established that the JAK–STAT

signaling pathway is activated in the di-aphragm of rats and humans subjected tomechanical ventilation. This posttransla-tional activation of JAK–STAT leads to areduction of diaphragm contractilitythrough, at least in part, the induction ofmitochondrial oxidative stress. JAK–STATactivation appears to be critical in regulat-ing this oxidative stress and is therebycentral to the downstream pathogenesisof clinical VIDD. This finding establishesthe molecular basis for the therapeuticpromise of JAK–STAT inhibitors in venti-lated ICU patients, and it identifies a clearmechanism of action that would favortaking such a drug to clinical trials.

ACKNOWLEDGMENTSWe are grateful to Lawrence Argetsinger

(University of Michigan) for providing

R E S E A R C H A R T I C L E

M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4 | T A N G E T A L . | 5 8 7

JAK2 plasmids for this study. We alsothank Bianca Kapoor and Isaac Ghansah,and Kun Wang and Yang Gao (StanfordUniversity); and Kelly M McCaugheyand Raniel R Alcantara (Rigel Pharma-ceuticals) for technical assistance. Thiswork is supported by a Veterans Admin-istration (VA) Biomedical LaboratoryR&D Merit Review grant to JB Shrager,and by the grants from the NIH (TR01AG047820 and R37 AG023806) and theVA (Biomedical Laboratory R&D andRehab R&D) Merit Reviews to TA Rando.

DISCLOSUREIJ Smith, GL Godinez, BK Singh,

DG Payan, and TM Kinsella are em-ployees and/or stockholders of RigelPharmaceuticals, Inc.

REFERENCES1. Griffiths RD, Hall JB. (2010) Intensive care unit-

acquired weakness. Crit. Care Med. 38:779–87.2. Knisely AS, Leal SM, Singer DB. (1988) Abnor-

malities of diaphragmatic muscle in neonateswith ventilated lungs. J. Pediatr. 113:1074–7.

3. Laghi F. (2005) Assessment of respiratory outputin mechanically ventilated patients. Respir. CareClin. N. Am. 11:173–99.

4. Liu L, et al. (2012) Neuroventilatory efficiencyand extubation readiness in critically ill patients.Crit. Care. 16:R143.

5. Purro A, et al. (2000) Physiologic determinants ofventilator dependence in long-term mechanicallyventilated patients. Am. J. Respir. Crit. Care Med.161:1115–23.

6. Vallverdú I, et al. (1998) Clinical characteristics,respiratory functional parameters, and outcomeof a two-hour T-piece trial in patients weaningfrom mechanical ventilation. Am. J. Respir. Crit.Care Med. 158:1855–62.

7. Cattapan SE, Laghi F, Tobin MJ. (2003) Can di-aphragmatic contractility be assessed by airwaytwitch pressure in mechanically ventilated pa-tients? Thorax. 58:58–62.

8. Watson AC, et al. (2001) Measurement of twitchtransdiaphragmatic, esophageal, and endotra-cheal tube pressure with bilateral anterolateralmagnetic phrenic nerve stimulation in patients inthe intensive care unit. Crit. Care Med. 29:1325–31.

9. Buscher H, et al. (2005) Assessment of diaphrag-matic function with cervical magnetic stimula-tion in critically ill patients. Anaesth. IntensiveCare 33:483–91.

10. Cader SA, et al. (2010) Inspiratory muscle train-ing improves maximal inspiratory pressure andmay assist weaning in older intubated patients: arandomised trial. J. Physiother. 56:171–7.

11. Carlucci A, et al. (2009) Determinants of weaning

success in patients with prolonged mechanicalventilation. Crit. Care. 13:R97.

12. Laghi F, et al. (2003) Is weaning failure caused bylow-frequency fatigue of the diaphragm? Am. J.Respir. Crit. Care Med. 167:120–7.

13. Martin AD, et al. (2002) Use of inspiratory musclestrength training to facilitate ventilator weaning:a series of 10 consecutive patients. Chest.122:192–6.

14. Martin AD, et al. (2011) Inspiratory musclestrength training improves weaning outcome infailure to wean patients: a randomized trial. CritCare. 15:R84.

15. Hudson MB, et al. (2012) Both high level pressuresupport ventilation and controlled mechanicalventilation induce diaphragm dysfunction andatrophy. Crit. Care Med. 40:1254–60.

16. Powers SK, et al. (2013) Ventilator-induced di-aphragm dysfunction: cause and effect. Am. J.Physiol. Regul. Integr. Comp. Physiol. 305:R464–77.

17. Zhu E, et al. (2005) Early effects of mechanicalventilation on isotonic contractile properties andMAF-box gene expression in the diaphragm. J. Appl. Physiol. (1985). 99:747–56.

18. Grosu HB, et al. (2012) Diaphragm muscle thin-ning in patients who are mechanically ventilated.Chest. 142:1455–60.

19. Hermans G, et al. (2010) Increased duration ofmechanical ventilation is associated with de-creased diaphragmatic force: a prospective obser-vational study. Crit. Care. 14:R127.

20. Jaber S, et al. (2011) Rapidly progressive di-aphragmatic weakness and injury during me-chanical ventilation in humans. Am. J. Respir.Crit. Care Med. 183:364–71.

21. Levine S, et al. (2008) Rapid disuse atrophy of di-aphragm fibers in mechanically ventilated hu-mans. N. Engl. J. Med. 358:1327–35.

22. Powers SK, et al. (2002) Mechanical ventilationresults in progressive contractile dysfunction inthe diaphragm. J. Appl. Physiol. (1985). 92:1851–8.

23. Mrozek S, et al. (2012) Rapid onset of specific di-aphragm weakness in a healthy murine model ofventilator-induced diaphragmatic dysfunction.Anesthesiology. 117:560–7.

24. Schellekens WJ, et al. (2012) Toll-like receptor 4signaling in ventilator-induced diaphragm atro-phy. Anesthesiology. 117:329–38.

25. Tang H, et al. (2013) Diaphragm muscle atrophyin the mouse after long-term mechanical ventila-tion. Muscle Nerve. 48:272–8.

26. Capdevila X, et al. (2003) Effects of controlledmechanical ventilation on respiratory musclecontractile properties in rabbits. Intensive CareMed. 29:103–10.

27. Jung B, et al. (2010) Adaptive support ventilationprevents ventilator-induced diaphragmatic dys-function in piglet: an in vivo and in vitro study.Anesthesiology. 112:1435–43.

28. Agten A, et al. (2012) Bortezomib partially pro-tects the rat diaphragm from ventilator-induceddiaphragm dysfunction. Crit. Care Med.40:2449–55.

29. DeRuisseau KC, et al. (2005) Mechanical ventila-tion induces alterations of the ubiquitin-protea-some pathway in the diaphragm. J. Appl. Physiol.(1985). 98:1314–21.

30. Levine S, et al. (2011) Increased proteolysis,myosin depletion, and atrophic AKT-FOXO sig-naling in human diaphragm disuse. Am. J. Respir.Crit. Care Med. 183:483–90.

31. Hussain SN, et al. (2010) Mechanical ventilation-induced diaphragm disuse in humans triggers au-tophagy. Am. J. Respir. Crit. Care Med. 182:1377–86.

32. McClung JM, et al. (2007) Caspase-3 regulation ofdiaphragm myonuclear domain during mechani-cal ventilation-induced atrophy. Am. J. Respir.Crit. Care Med. 175:150–9.

33. Nelson WB, et al. (2012) Cross-talk between thecalpain and caspase-3 proteolytic systems in thediaphragm during prolonged mechanical venti-lation. Crit. Care Med. 40:1857–63.

34. Welvaart WN, et al. (2011) Diaphragm musclefiber function and structure in humans withhemidiaphragm paralysis. Am. J. Physiol. LungCell Mol. Physiol. 301:L228–35.

35. Tang H, et al. (2011) Intrinsic apoptosis in me-chanically ventilated human diaphragm: linkageto a novel Fos/FoxO1/Stat3-Bim axis. FASEB J.25:2921–36.

36. Maes K, et al. (2007) Leupeptin inhibits ventilator-induced diaphragm dysfunction in rats. Am. J.Respir. Crit. Care Med. 175:1134–8.

37. Shanely RA, et al. (2002) Mechanical ventilation-induced diaphragmatic atrophy is associatedwith oxidative injury and increased proteolyticactivity. Am. J. Respir. Crit. Care Med. 166:1369–74.

38. Whidden MA, et al. (2010) Oxidative stress is re-quired for mechanical ventilation-induced pro-tease activation in the diaphragm. J. Appl. Phys-iol. (1985). 108:1376–82.

39. Powers SK, Jackson MJ. (2008) Exercise-inducedoxidative stress: cellular mechanisms and impacton muscle force production. Physiol. Rev.88:1243–76.

40. Reid MB. (2001) Invited Review: redox modula-tion of skeletal muscle contraction: what weknow and what we don’t. J. Appl. Physiol. (1985).90:724–31.

41. Reid MB. (2008) Free radicals and muscle fatigue:Of ROS, canaries, and the IOC. Free Radic. Biol.Med. 44:169–79.

42. Smith MA, Reid MB. (2006) Redox modulation ofcontractile function in respiratory and limb skele-tal muscle. Respir. Physiol. Neurobiol. 151:229–41.

43. Danshina PV, et al. (2001) Mildly oxidized glycer-aldehyde-3-phosphate dehydrogenase as a possi-ble regulator of glycolysis. IUBMB Life. 51:309–14.

44. Tang H, et al. (2012) Oxidative stress-responsivemicroRNA-320 regulates glycolysis in diverse bi-ological systems. FASEB J. 26:4710–21.

45. Wu SB, Wei YH. (2012) AMPK-mediated increaseof glycolysis as an adaptive response to oxidativestress in human cells: implication of the cell sur-vival in mitochondrial diseases. Biochim. Biophys.Acta. 1822:233–47.

5 8 8 | T A N G E T A L . | M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4

J A K – S T A T R E G U L A T E S D I A P H R A G M C O N T R A C T I L I T Y

46. Kirkinezos IG, Moraes CT. (2001) Reactive oxy-gen species and mitochondrial diseases. Semin.Cell Dev. Biol. 12:449–57.

47. Bonetto A, et al. (2012) JAK/STAT3 pathway inhi-bition blocks skeletal muscle wasting down-stream of IL-6 and in experimental cancercachexia. Am. J. Physiol. Endocrinol. Metab.303:E410–21.

48. Carballo M, et al. (1999) Oxidative stress triggersSTAT3 tyrosine phosphorylation and nucleartranslocation in human lymphocytes. J. Biol.Chem. 274:17580–6.

49. Committee for the Update of the Guide for theCare and Use of Laboratory Animals, Institutefor Laboratory Animal Research, Division onEarth and Life Studies, National Research Coun-cil of the National Academies. (2011) Guide for theCare and Use of Laboratory Animals. 8th edition.Washington (DC): National Academies Press.

50. Picard M, et al. (2012) Mitochondrial dysfunctionand lipid accumulation in the human diaphragmduring mechanical ventilation. Am. J. Respir. Crit.Care Med. 186:1140–9.

51. Smith IJ, et al. (2014) Inhibition of Janus kinasesignaling prevents ventilation-induced di-aphragm dysfunction. FASEB J. 28:2790–803.

52. Demaria M, et al. (2010) A STAT3-mediated meta-bolic switch is involved in tumour transforma-tion and STAT3 addiction. Aging (Albany NY).2:823–42.

53. Kallet RH. (2011) Patient-ventilator interactionduring acute lung injury, and the role of sponta-neous breathing: part 1: respiratory muscle func-tion during critical illness. Respir. Care. 56:181–9.

54. Wegrzyn J, et al. (2009) Function of mitochondrialStat3 in cellular respiration. Science. 323:793–7.

55. Gough DJ, et al. (2009) Mitochondrial STAT3 sup-ports Ras-dependent oncogenic transformation.Science. 324:1713–6.

R E S E A R C H A R T I C L E

M O L M E D 2 0 : 5 7 9 - 5 8 9 , 2 0 1 4 | T A N G E T A L . | 5 8 9