Heat Stress Protects Against Mechanical Ventilation-Induced Diaphragmatic Atrophy

doi:10.1152/japplphysiol.00170.2014 117:518-524, 2014. First published 24 July 2014;J Appl PhysiolK. Powers and Hisashi NaitoNoriko Ichinoseki-Sekine, Toshinori Yoshihara, Ryo Kakigi, Takao Sugiura, Scottventilation-induced diaphragmatic atrophyHeat stress protects against mechanical

You might find this additional info useful...

46 articles, 20 of which can be accessed free at:This article cites /content/117/5/518.full.html#ref-list-1

including high resolution figures, can be found at:Updated information and services /content/117/5/518.full.html

can be found at:Journal of Applied Physiologyabout Additional material and information http://www.the-aps.org/publications/jappl

This information is current as of February 23, 2015.

ISSN: 0363-6143, ESSN: 1522-1563. Visit our website at http://www.the-aps.org/.Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2014 by the American Physiological Society.those papers emphasizing adaptive and integrative mechanisms. It is published 12 times a year (monthly) by the American

publishes original papers that deal with diverse areas of research in applied physiology, especiallyJournal of Applied Physiology

on February 23, 2015

Dow

nloaded from on F

ebruary 23, 2015D

ownloaded from

http://www.the-aps.org/publications/jappl

Heat stress protects against mechanical ventilation-induced diaphragmaticatrophy

Noriko Ichinoseki-Sekine,1 Toshinori Yoshihara,1 Ryo Kakigi,2 Takao Sugiura,3 Scott K. Powers,4

and Hisashi Naito1

1School of Health and Sports Science, Juntendo University, Inzai, Chiba, Japan; 2School of Medicine, Juntendo University,Bunkyo-ku, Tokyo, Japan; 3Faculty of Education, Yamaguchi University, Yamaguchi, Yamaguchi, Japan; and 4Department ofApplied Physiology and Kinesiology, University of Florida, Gainesville, Florida

Submitted 20 February 2014; accepted in final form 17 July 2014

Ichinoseki-Sekine N, Yoshihara T, Kakigi R, Sugiura T, PowersSK, Naito H. Heat stress protects against mechanical ventilation-induceddiaphragmatic atrophy. J Appl Physiol 117: 518–524, 2014. First pub-lished July 24, 2014; doi:10.1152/japplphysiol.00170.2014.—Mechani-cal ventilation (MV) is a life-saving intervention in patients who areincapable of maintaining adequate pulmonary gas exchange due torespiratory failure or other disorders. However, prolonged MV is asso-ciated with the development of respiratory muscle weakness. We hypoth-esized that a single exposure to whole body heat stress would increasediaphragm expression of heat shock protein 72 (HSP72) and that thistreatment would protect against MV-induced diaphragmatic atrophy.Adult male Wistar rats (n � 38) were randomly assigned to one of fourgroups: an acutely anesthetized control group (CON) with no MV; 12-hcontrolled MV group (CMV); 1-h whole body heat stress (HS); or 1-hwhole body heat stress 24 h prior to 12-h controlled MV (HSMV).Compared with CON animals, diaphragmatic HSP72 expression in-creased significantly in the HS and HSMV groups (P � 0.05). ProlongedMV resulted in significant atrophy of type I, type IIa, and type IIx fibersin the costal diaphragm (P � 0.05). Whole body heat stress attenuatedthis effect. In contrast, heat stress did not protect against MV-induceddiaphragm contractile dysfunction. The mechanisms responsible for thisheat stress-induced protection remain unclear but may be linked toincreased expression of HSP72 in the diaphragm.

heat shock protein; proteolysis; antioxidants; respiratory muscleweakness

MECHANICAL VENTILATION (MV) is a life-saving intervention inpatients who are incapable of maintaining adequate pulmonarygas exchange due to respiratory failure or other disorders (14).Unfortunately, prolonged MV is associated with the develop-ment of respiratory muscle weakness, specifically weakness ofthe diaphragm (9, 19). MV-induced respiratory muscle weak-ness is significant because it may contribute to difficulty inweaning from the ventilator (8), which in turn is associatedwith increased patient mortality and morbidity (7). Therefore,it is important to develop clinical strategies to protect patientsagainst MV-induced diaphragmatic weakness.

It has been established that whole body heat stress per-formed 24 h prior to locomotor muscle inactivity prevents orattenuates disuse locomotor muscle atrophy by inducing 72-kDa heat shock protein (HSP72). HSP72 is a highly conservedstress protein that can be induced in skeletal muscle fibers byboth environmental (e.g., heat stress) and intracellular stresses(e.g., disturbed pH) (4, 28), eccentric exercise (24, 25), andoxidative stress (47). Although the precise mechanisms under-

lying the protective effect of increased HSP72 expressionagainst disuse muscle atrophy are unclear, it is thought thatHSP72 prevents proteolysis by inhibiting key atrophy signalingpathways (36). Further, increased HSP72 expression may alsoprotect against muscle atrophy by assisting in the refolding ofoxidized proteins, thus protecting these proteins from proteol-ysis (20, 26). Regardless of the mechanisms responsible forHSP72-mediated protection against limb muscle atrophy, in-creased muscle levels of HSP72 clearly show a protectiveeffect against inactivity-induced atrophy of locomotor skeletalmuscles.

While exposure of animals to heat stress has been shown tobe protective against disuse locomotor muscle atrophy (26, 35),it is unclear whether whole body heat stress can protect thediaphragm against MV-induced atrophy. Therefore, the presentstudy was performed to determine whether whole body heatstress performed 24 h prior to prolonged MV could protect thediaphragm against MV-induced atrophy. We hypothesized thata single exposure to whole body heat stress would increase theexpression of HSP72 in the diaphragm, and that this treatmentwould protect against MV-induced diaphragmatic atrophy.

MATERIALS AND METHODS

Animals. All experimental protocols were approved by the institu-tional ethics review committee of Juntendo University (H24-03), andthe experiments were performed according to the American Physio-logical Society “Guiding Principles in the Care and Use of Animals.”

Adult male Wistar rats (3 mo old) were randomly assigned to oneof four groups: 1) an acutely anesthetized control group (CON, n � 9)with no MV; 2) a 12-h controlled MV group (CMV, n � 10); 3) 1-hwhole body heat stress group (HS, n � 10); or 4) 1-h whole body heatstress 24 h prior to 12-h controlled MV (HSMV, n � 9). All rats weremaintained under a 12:12-h light-dark cycle with standard chow andwater ad libitum for at least 1 wk before use.

Experimental design. Control animals were acutely anesthetizedusing pentobarbital sodium (ip injection of 60 mg/kg body wt) and thediaphragm was quickly removed. Animals in the HS and HSMVgroups were exposed to 1-h whole body heat stress using a heatchamber. To ensure the same time intervals between heat stress anddissection with HSMV rats, rats from the HS group were anesthetizedand the diaphragm was removed 36 h after completion of heat stress.Immediately following the 12-h controlled MV, CMV and HSMVanimals were killed and diaphragm was removed. The time intervalbetween completion of heat stress and initiation of controlled MV inthe HSMV group was 24 h. After the entire diaphragm was removed,the costal diaphragm was divided into several segments for subse-quent analysis.

Application of heat stress. Animals in HS and HSMV groups wereexposed to heat stress (40–41°C, 60 min) in a heat chamber(TVG321AA; Advantec, Tokyo, Japan) without anesthesia (26, 41).

Address for reprint requests and other correspondence: N. Ichinoseki-Sekine, School of Health and Sports Science, Juntendo Univ., 1-1 Hira-gagakuendai, Inzai, Chiba 270-1695, Japan (e-mail: [email protected]).

J Appl Physiol 117: 518–524, 2014.First published July 24, 2014; doi:10.1152/japplphysiol.00170.2014.

8750-7587/14 Copyright © 2014 the American Physiological Society http://www.jappl.org518


Dow

nloaded from

mailto:[email protected]

This heat stress increased the colonic temperature of the animals to�40°C following 30 min of heat exposure (LT8A; Gram, Saitama,Japan). Specifically, following 60 min of heat stress, colonic temper-ature increased to 41.1–41.4°C, suggesting that colonic temperaturewas maintained above 40°C for 30 min.

Mechanical ventilation. All surgical procedures were performedusing aseptic technique. Animals from the CMV and HSMV groupswere anesthetized by intraperitoneal injection of pentobarbital sodium(60 mg/kg body wt), tracheostomized, and mechanically ventilated(tidal volume � 0.6 ml/100 g body wt, 80 breaths/min, humidifiedambient air) with a volume-cycled ventilator (SAR-1000; CWE,Ardmore, PA) for 12 h. ECG and heart rate were monitored via alead-II ECG using needle electrodes placed subcutaneously. Thecarotid artery was cannulated to permit the continuous measurementof arterial blood pressure and periodic blood sampling (every 4 h) foranalysis of arterial blood gas (cobas b 221; Roche Diagnostics, Tokyo,Japan). Arterial PO2, PCO2, and pH were maintained within theirrespective normal ranges by adjustment of tidal volume. A venouscatheter was placed in the jugular vein for continuous infusion ofpentobarbital sodium (10 mg·kg�1·h�1). Body (i.e., colonic) temper-ature was maintained at 36.5–37.5°C with a water-circulating warm-ing pad. Continuous care during MV included expressing the bladder,lubricating the eyes, rotating the animal, and passive limb movement.Animals also received intramuscular injections of glycopyrrolate(0.04 mg/kg) prior to MV every 2 h during MV to reduce airwaysecretions.

Histological measures. Serial cross-sections from frozen costaldiaphragm muscle samples were cut at a thickness of 10 �m using amicrotome (CM3050S; Leica, Wetzlar, Germany) at �20°C. Immu-nohistochemical staining was performed to identify muscle fibertypes. Unfixed cryosections were rinsed in 0.01 M phosphate-bufferedsaline (PBS) and incubated in 0.3% Triton X-100/0.01 M PBS beforeblocking with 3% bovine serum albumin (BSA) in 0.01 M PBS atroom temperature. Primary antibodies used for fiber typing were:M8421 (type I, 1:500; Sigma, St. Louis, MO), M4276 (type II, 1:500;Sigma), SC-71 (type IIa, 1:400), and BF-35 (all fibers except type IIx,1:200). All antibodies were diluted in 3% BSA/0.01 M PBS andapplied to sections overnight at 4°C. After rinsing with 0.01 M PBS,Alexa Fluor 488 goat anti-mouse IgG antibody (A11003; InvitrogenJapan, Tokyo, Japan) was applied as a secondary antibody (1:500).Hematoxylin and eosin staining was also performed for measurementof cross-sectional area (CSA). Sections were viewed under a fluores-cence microscope (DM5000B; Leica) with a 10� objective andcaptured using a digital camera (IM 500; Leica). Diaphragm musclefiber CSA was determined using Scion Image (NIH, Bethesda, MD).

Biochemical measures. Frozen costal diaphragm muscle sampleswere minced, and a portion of the muscle (�80 mg) was homogenizedin 10 volumes of ice-cold homogenization buffer [20 mM HEPES, pH7.4, 60 mM dithiothreitol, 1% (wt/vol) Triton X-100] containingprotease inhibitor cocktail (Complete EDTA-free and PhosSTOP;Roche, Penzberg, Germany). The homogenates were centrifuged at12,000 g for 20 min at 4°C to collect the cytosolic fraction. Thesupernatant protein concentration was determined using a Bio-Rad(Bradford) Protein Assay kit (Bio-Rad, Hercules, CA). The insolublepellets corresponding to the particulate fraction were subsequentlywashed three times in 5 volumes of ice-cold homogenization bufferand centrifuged at 12,000 g for 5 min at 4°C. The pellets were thenresuspended in 10 volumes of lysis buffer consisting of 20 mMHEPES (pH 7.4), 250 mM NaCl, and 1% (wt/vol) sodium dodecylsulfate (SDS), and centrifuged at 17,000 g for 5 min at 4°C. Thesupernatant was saved and the protein concentration of the solubilizedparticulate fraction was determined using a BCA Protein Assay kit(Thermo Scientific, Rockford, IL).

Protein extracts were solubilized in sample buffer [30% glycerol,5% 2-�-mercaptoethanol, 2.3% SDS, 62.5 mM Tris-HCl (pH 6.8),and 0.05% bromophenol blue] at 2 mg/ml and incubated at 95°C for5 min. Proteins were then loaded onto 6.5–12.5% SDS-polyacryl-

amide gels and run at 120–130 V for 60–80 min. Separated proteinswere transferred onto polyvinylidene difluoride (PVDF) membranes(Bio-Rad) using a Bio-Rad mini transblot cell at 100 V for 60 min at4°C in transfer buffer [25 mM Tris-HCl (pH 8.3), 192 mM glycine,and 20% methanol]. After transfer, membranes were stained withPonceau-S and visually inspected to control for equal loading andprotein transfer. The membranes were then blocked for 1 h at roomtemperature in blocking buffer [5% nonfat dry milk in Tween-Tris-buffered saline (T-TBS: 40 mM Tris-HCl, 300 mM NaCl, and 0.1%Tween 20, pH 7.5)]. After three washes with T-TBS, membranes wereincubated with primary antibodies against the following: calpain-1(1:1,000; Cell Signaling, Beverly, MA), cleaved caspase-3 (1:1,000;Cell Signaling), mono- and polyubiquitinated proteins (1:1,000; Enzo,Farmingdale, NY), superoxide dismutase (SOD)-1 (1:5,000; AssayDesigns, Ann Arbor, MI), SOD-2 (1:2,000; Assay Designs), fastmyosin (1:5,000; Sigma), and actin (1:5,000; Sigma). Primary anti-body incubations were carried out in dilution buffer for 2 h at roomtemperature. After several washes in T-TBS, membranes were incu-bated with horseradish peroxidase (HRP)-conjugated anti-rabbit sec-ondary antibodies (Cell Signaling, 1:5,000) in dilution buffer for 1 hat room temperature. After several washes, bands were visualizedusing Immobilon Western Chemiluminescent HRP Substrate (Milli-pore, Billerica, MA), and the signals were recorded with a LightCapture system (ATTO, Tokyo, Japan). Analyses were performedusing CS Analyzer 3.0 software (ATTO).

To determine relative HSP72 levels in the diaphragm, membraneswere incubated with anti-HSP70 alkaline phosphatase conjugate(SPA-810AP, 1:2,000; Stressgen Biotechnologies, BC, Canada) inT-TBS with 5% nonfat dry milk for 1 h at room temperature. Themembranes were subsequently washed in T-TBS and then reactedwith alkaline phosphatase substrate (Bio-Rad) at room temperature.Bands were quantified using computerized densitometry (Scion Im-age, NIH).

To determine the level of protein oxidation in the diaphragm, wemeasured the levels of protein carbonyls in muscle proteins separatedby electrophoresis using an oxidized protein detection kit (Oxyblot,no. S7150; Millipore), which is based on immunochemical detectionof protein carbonyl groups derivatized with 2,4-dinitrophenylhydra-zine (DNPH). The samples were treated with 20 mM DNPH in 10%trifluoroacetic acid or derivatization-control solution (lacking DNPH),and incubated for 15 min. A total of 8 �g of myofibrillar protein fromeach sample was loaded onto 10% SDS-polyacrylamide gels, andSDS-polyacrylamide gel electrophoresis (PAGE) and Western blot-ting were performed according to the manufacturer’s instructions.Proteins were then transferred onto PVDF membranes and stainedwith Ponceau S, which is specific for proteins. The primary antibodywas a rabbit polyclonal antibody raised against dinitrophenyl hydra-zone (DNP) (S7150–4, 1:150; Millipore). An HRP-conjugated sec-ondary antibody was used (S7150–5, 1:300; Millipore). After severalwashes, DNP signals were visualized using Immobilon WesternChemiluminescent HRP Substrate (Millipore), and the signals wererecorded with an ATTO Light Capture system (ATTO). To quantifythe amount of oxidation, the ratio between densitometric values of theOxyblot bands and those stained with Ponceau S was defined as theoxidative index (Oxy/PS).

Measurement of in vitro diaphragmatic contractile properties.Upon the euthanizing of the animal, the entire diaphragm was re-moved and a muscle strip, including the tendinous attachments at thecentral tendon and rib cage, was dissected from the midcostal regionin a dissecting chamber containing Krebs-Henseleit solution main-tained at 25°C and equilibrated with a 95% O2-5% CO2 gas (pH �7.4). The strip was suspended vertically with one end connected to anisometric force transducer (MLT500; AD Instruments Japan, Nagoya,Japan) within a jacketed tissue bath. The force output was recordedusing a computerized data-acquisition system (PowerLab; AD Instru-ments Japan). After a 15-min equilibration period, optimal contractilelength of the in vitro strip was determined using a micrometer while

519Heat Stress Protects Against MV-Induced Diaphragmatic Atrophy • Ichinoseki-Sekine N et al.

J Appl Physiol • doi:10.1152/japplphysiol.00170.2014 • www.jappl.org


Dow

nloaded from

evoking single twitches. After the muscle strip was set at the optimallength, maximal isometric twitch force at 1 Hz (2 ms) and force-frequency response (500 ms duration, 15–160 Hz) was measured withsupramaximal stimulation at 100 V. Contractions were separated by a3-min recovery period. Force production was normalized againstcalculated muscle CSA: total muscle CSA (in cm2) � muscle mass(in g)/[fiber length (in cm) � 1.056], where 1.056 is the density ofmuscle (in g/cm3) (31, 34).

Statistical analysis. Data are reported as mean values SE. Notethat blood gas measurements and blood pressure measurements wereperformed in two groups (i.e., CMV and HSMV); these data weretherefore analyzed using an unpaired t-test. Otherwise, the Brown-Forsythe test was performed to determine the equality of groupvariances, and then a two-way analysis of variance (ANOVA) orone-way ANOVA was performed where appropriate. Bonferroni’spost hoc tests were used for multiple comparisons. In all analyses,P � 0.05 was established a priori as the benchmark for statisticalsignificance.

RESULTS

Systemic and biological responses to MV. The systemic andbiological responses to MV are shown in Table 1. Blood gasesand arterial blood pressure were maintained within their re-spective normal ranges during 12-h MV and did not differbetween the two MV groups. Heart rate (300–400 beats/min)and body temperature (36.5–37.5°C) were maintained within

their respective normal ranges during the MV period. Oncompletion of MV, no visual abnormality of the lungs orperitoneal cavity was observed.

Diaphragmatic fiber atrophy. Fiber CSA was determined forindividual fiber types in all groups (Fig. 1). Similar to previousstudies, prolonged MV resulted in significant atrophy of type I,type IIa, and type IIx fibers in the costal diaphragm. Note thatprolonged MV resulted in a greater rate of atrophy in type IIfibers compared with type I fibers. This greater rate of fiberatrophy resulted in an increase in the percentage of CSAoccupied by type I fibers (Table 2). No significant difference inCSA area was observed for type I, type IIa, or type IIx fibersbetween the CON and HSMV animals, indicating that heatstress performed prior to MV attenuated MV-induced dia-phragmatic atrophy. Further, no differences existed in musclefiber type among the experimental groups.

HSP72 expression in the diaphragm. Compared with CONanimals, diaphragmatic HSP72 expression increased signifi-cantly in the HS and HSMV groups (Fig. 2). Moreover, HSP72expression levels in the diaphragm were greater in HSMV thanin CMV animals. These results indicate that whole body heatstress as performed in this study significantly increased HSP72expression in diaphragm muscle.

Autolyzed calpain-1, cleaved caspase-3, and ubiquitinatedprotein expression in the diaphragm. Prolonged MV signifi-cantly increased cleaved (activated) caspase-3 expression indiaphragm muscle in the CMV group (Fig. 3). Heat stress

Table 1. Initial body weight, blood gas, and arterial bloodpressure at completion of 12-h of mechanical ventilation

Variable CON HS CMV HSMV

Body weight, g 303.6 3.4 307.5 2.0 304.1 2.9 304.2 2.9PaO2, mmHg NA NA 82.2 2.5 84.7 2.5PaCO2, mmHg NA NA 35.1 1.0 36.1 1.0pH NA NA 7.49 0.01 7.49 0.01Arterial blood

pressure,mmHg NA NA 81.7 14.2 85.1 18.6

Values are means SE. PaO2, arterial PO2; PaCO2, arterial PCO2; NA, notapplicable; CON, control (n � 9); HS, 1-h whole body heat stress (n � 10);CMV, 12-h controlled mechanical ventilation (n � 10); HSMV, 1-h whole-body heat stress 24 h prior to 12-h controlled mechanical ventilation (n � 9).No significant differences were observed among groups for any of the vari-ables.

Fig. 1. Fiber cross-sectional area (CSA) in diaphragm muscle fibers from allfour experimental groups. Values are means SE. *Significantly differentfrom all groups (*P � 0.05; **P � 0.01). CON, control (n � 9); HS, 1-hwhole body heat stress (n � 10); CMV, 12-h controlled mechanical ventilation(n � 10); HSMV, 1-h whole body heat stress 24 h prior to 12-h controlledmechanical ventilation (n � 9).

Table 2. Percentage of total cross-sectional area occupiedby each diaphragmatic fiber type

Group Type I Type IIa Type IIx Type IIb

CON 19.8 0.8 20.8 1.5 51.7 1.5 7.7 2.2HS 19.6 1.0 19.2 1.1 51.7 2.4 9.5 2.3CMV 23.4 1.0#§ 21.7 0.8 52.4 1.5 2.5 0.9§HSMV 22.2 0.8 22.4 1.0 53.6 1.0 1.8 0.3§

Values are means SE, in %. CON, n � 9; HS, n � 10; CMV, n � 10;HSMV, n � 9. #Significantly different from CON (P � 0.05). §Significantlydifferent from HS (P � 0.05).

Fig. 2. Heat shock protein 72 (HSP72) expression in diaphragm muscle. Valuesare means SE. #Significantly different from CON (P � 0.05). †Significantlydifferent from CMV (P � 0.05). CON, n � 9; HS, n � 10; CMV, n � 10;HSMV, n � 9.

520 Heat Stress Protects Against MV-Induced Diaphragmatic Atrophy • Ichinoseki-Sekine N et al.



Dow

nloaded from

decreased MV-induced expression of cleaved caspase-3 by�39%; however, the difference was not significant. In contrast,no significant difference among groups was observed for au-tolyzed calpain-1 or ubiquitinated protein levels.

SOD levels in the diaphragm. No significant difference inSOD-1 or SOD-2 expression was observed among groups(Fig. 4). These results indicated that neither 12-h MV nor heatstress affected the expression levels of SOD-1 and SOD-2.

Levels of oxidized proteins in the diaphragm. Levels ofoxidized proteins in the diaphragm increased significantly inresponse to prolonged MV (Fig. 5). Although heat stresstended to protect the diaphragm against MV-induced proteinoxidation, the effect was not statistically significant.

Actin and myosin protein content. Actin content in the costaldiaphragm decreased significantly as a result of prolonged MV,but this loss of actin protein was significantly attenuated byheat stress (Fig. 6). No change in myosin content was observedamong groups.

Diaphragm contractile properties. To examine whether heatstress can protect against diaphragmatic contractile dysfunc-tion, we measured both in vitro maximal isometric twitch force

and the force-frequency responses in excised strips of costaldiaphragm muscle. Compared with controls, 12 h of controlledMV resulted in a significant reduction in diaphragm muscleforce production at all stimulation frequencies above 15 Hz(Fig. 7). Note that heat stress did not rescue the diaphragmfrom MV-induced contractile dysfunction.

DISCUSSION

Overview of principal findings. Application of heat stress didnot protect against MV-induced diaphragmatic contractile dys-function. A detailed discussion of these and other major find-ings follows.

Heat stress protects against MV-induced atrophy. In agree-ment with previous studies in limb skeletal muscle suggestingthat heat stress can protect against muscle atrophy (11, 26, 35),our results indicated that whole body heat stress has a protec-tive effect against MV-induced diaphragmatic atrophy. Impor-tantly, this heat stress-induced protection against ventilator-induced atrophy was observed in all diaphragm muscle fibertypes. Note that the magnitude of MV-induced diaphragmatic

Fig. 3. Autolyzed calpain-1, cleaved caspase-3,and ubiquitinated protein expression. Valuesare means SE. #Significantly different fromCON (P � 0.05). §Significantly different fromHS (P � 0.05). CON, n � 9; HS, n � 10;CMV, n � 10; HSMV, n � 9.

Fig. 4. Superoxide dismutase (SOD) expres-sion. Values are means SE. CON, n � 9;HS, n � 10; CMV, n � 10; HSMV, n � 9.




Dow

nloaded from

atrophy observed in the present study was similar to thosedescribed in previous reports (10, 17, 22, 30, 38).

HSP72 level in type I fibers typically exceeds that in type IIfibers (13, 18). Nonetheless, although heat stress increasedHSP72 levels in the diaphragm, it did not alter the relativepercentages of type I and II fibers in this key inspiratorymuscle. Regarding HSP72 in skeletal muscle, previous studieshave postulated that heat stress-induced expression of HSP72in skeletal muscle is responsible for the protective effect ofwhole body heat stress against disuse locomotor muscle atro-phy (26, 35). In this regard, exposing animals to brief periodsof whole body heat stress has been shown to significantlyelevate HSP levels in rat tissue (5), and the magnitude of thisresponse depends on muscle fiber type. For example, Oishi etal. (28, 29) demonstrated that heat stress results in smallincreases in the expression of HSP72 in the soleus muscle,which has a high percentage of type I fibers, whereas heatstress promotes a much larger increase in HSP72 expression inthe plantaris muscle, which is composed primarily of type IImuscle fibers. Note that compared with a previous study (29)the magnitude of heat stress-induction of HSP72 in the dia-phragm was lower than that of fast muscle fibers in locomotorskeletal muscles; however, it was similar to the magnitude ofinduced HSP72 expression in the diaphragm following a shortperiod of endurance exercise training (38). Importantly, endur-ance exercise training prior to MV has been shown to have apreventive effect against MV-induced diaphragmatic atrophy(38). Therefore, it is possible that the protective effect of heat

stress against MV-induced diaphragmatic atrophy is directlylinked to the increase in HSP72 levels in the diaphragm.

With regard to the mechanisms underlying the protectiveeffect of HSP72, it has been established that HSP72 can protectagainst calcium- and mitochondrial-related damage in musclemyotubes (21). In addition, HSP72 is known to inhibit apo-ptosis through caspase-dependent and -independent mecha-nisms (33). Previous work has shown that HSP72 attenuatesthe expression of the activated form of caspase-3, suggestingthat HSP72 plays a role in protection against apoptosis (44). Inthe present study, the expression of cleaved caspase-3 in-creased significantly in the diaphragm following 12 h of MV(Fig. 3). In contrast, prior heat stress successfully protected thediaphragm against MV-induced cleaved caspase-3 expressionin the diaphragm (Fig. 3). This finding is important becausepharmacological inhibition of caspase-3 activation in the dia-phragm can attenuate MV-induced diaphragmatic atrophy (22,27). Therefore, prior heat stress may protect the diaphragmagainst MV-induced atrophy, at least in part, by preventingcaspase-3 activation. With regard to the role of protease acti-vation in MV-induced diaphragm atrophy, previous studiesindicated that prolonged MV activates numerous proteolyticsystems in the diaphragm, including calpain and caspase-3

Fig. 5. Protein oxidation as determined by normalization of protein carbonyllevels against Ponceau S-stained protein expression. Values are means SE.#Significantly different from CON (P � 0.05). CON, n � 9; HS, n � 10;CMV, n � 10; HSMV, n � 9.

Fig. 6. Actin and myosin protein content.Values are means SE. #Significantly dif-ferent from CON (P � 0.05). CON, n � 9;HS, n � 10; n � 10; HSMV, n � 9.

Fig. 7. Effect of 12-h controlled mechanical ventilation on diaphragmaticmuscle force-frequency responses in vitro. Values are means SE. #Signif-icantly different from CON (P � 0.05). §Significantly different from HS (P �0.05). CON, n � 9; HS, n � 10; CMV, n � 10; HSMV, n � 9.




Dow

nloaded from

(22, 37). Emerging evidence suggests that MV-induceddiaphragmatic atrophy is dependent on the activation ofboth calpain and caspase-3 via a cross-talk activation mech-anism (27). In contrast to these reports, Supinski et al. (40)concluded that calpain and caspase-3 are activated in par-allel and remain independently active in septic animalsexposed to prolonged MV.

It was reported recently that nuclear factor of activated Tcells (NFAT) proteins function in cellular adaptations to envi-ronmental changes (15, 16, 45). Indeed, NFAT-regulated geneexpression is activated in response to cellular heat stress (12).NFAT activity is regulated by calcineurin and heat shocktranscription factor 1 (HSF1) (12). Further, HSF1 also regu-lates the expression of HSPs. It follows that NFAT may alsocontribute to the protective effect of heat stress against MV-induced diaphragmatic atrophy. However, further studies arerequired to determine if NFAT activation contributes to heatstress-induced protection against diaphragmatic atrophy.

In the present study, calpain-1 expression increased in thediaphragm during prolonged MV, but the change did not reachstatistical significance. Similarly, the levels of ubiquitinatedproteins tended to increase in the diaphragm during MV, butthese increases were not statistically significant. Nonetheless,these results do not preclude a potential role for the ubiquitin-proteasome pathway in diaphragmatic atrophy because the 20Sprotease can degrade nonubiquitinated proteins (43).

Heat stress does not protect against MV-induced decreasesin diaphragmatic specific force production. Although severalstudies have investigated the effects of heat stress on locomotormuscle atrophy, there has been no investigation to date regard-ing the impact of heat stress on muscle contractile dysfunctioninduced by prolonged disuse. In this first report on this impor-tant issue, we found that 1 h of heat stress 24 h prior to MVdoes not confer protection against MV-induced diaphragmcontractile dysfunction.

It has been reported that heat stress increases the antioxidantcapacity of skeletal muscles (35). Oxidants decrease calciumrelease and calcium sensitivity of the contractile apparatus (1,23) and impair sarcomeric protein function (3). Indeed, admin-istration of antioxidants prevents endotoxin-induced dia-phragm dysfunction (39). In this study, oxyprotein levelsincreased significantly after prolonged MV, as expected. Con-trary to our expectations, however, heat stress did not attenuateoxyprotein levels. Moreover, heat stress did not increaseSOD-1 or -2 levels in this study. While it has been establishedthat oxidative stress plays a necessary role in the developmentof ventilator-induced diaphragmatic dysfunction, whole bodyheat stress apparently does not elevate diaphragmatic oxidantsto a level that provides protection. Yamashita et al. (46)reported that peak SOD values were observed after 48 h ofwhole body heat stress at 41°C. Thus it is possible that the timeinterval between heat stress and MV in the present study wasnot sufficient to increase antioxidant capacity.

Finally, prolonged MV is known to result in a progressiveimpairment of diaphragm specific force production at all stim-ulation frequencies (2, 31). Further, prolonged MV also resultsin diaphragm fiber atrophy (32). Therefore, the combination ofMV-induced diaphragm fiber atrophy and the MV-inducedreduction in diaphragm specific force production act in concertto decrease total force generation in the diaphragm. Althoughheat stress performed before MV was not successful in pro-

tecting the diaphragm against MV-induced reductions in dia-phragmatic specific force production, it was effective in pro-tecting against MV-induced diaphragmatic atrophy. This isimportant because several lines of evidence suggest that de-pressed inspiratory muscle strength contributes to difficultweaning [6, 42; see Powers et al. (32) for more details on theinfluence of prolonged MV on diaphragmatic contractile dys-function and the impact of impaired inspiratory muscle func-tion on weaning difficulties].

Conclusions. In summary, 1 h exposure of animals to wholebody heat stress 24 h prior to prolonged MV does not protectagainst MV-induced diaphragm contractile dysfunction butdoes provide protection against MV-induced diaphragmaticatrophy. The mechanisms underlying this heat stress-inducedprotection remain unclear but may be linked to increasedexpression of HSP72 and subsequent protection againstcaspase-3 activation in the diaphragm. Regardless of the mech-anisms responsible for this protective effect, the discovery thatheat stress provides protection against MV-induced diaphragmatrophy is a new and important finding that could have signif-icant clinical implications in the prevention of MV-induceddiaphragmatic weakness.

GRANTS

This work was supported by Japan Society for the Promotion of Science (JSPS)KAKENHI Grant No. 23700781 (N. Ichinoseki-Sekine) and JSPS ResearchFellowships for Young Scientists Grant No. 11J11122 (T. Yoshihara).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: N.I.-S., T.Y., and H.N. conception and design ofresearch; N.I.-S., T.Y., and R.K. performed experiments; N.I.-S. and T.Y.analyzed data; N.I.-S. and T.Y. interpreted results of experiments; N.I.-S.prepared figures; N.I.-S. drafted manuscript; N.I.-S., T.S., S.K.P., and H.N.edited and revised manuscript; N.I.-S., T.Y., R.K., T.S., S.K.P., and H.N.approved final version of manuscript.

REFERENCES

1. Andrade FH, Reid MB, Allen DG, Westerblad H. Effect of hydrogenperoxide and dithiothreitol on contractile function of single skeletalmuscle fibres from the mouse. J Physiol 509: 565–575, 1998.

2. Betters JL, Criswell DS, Shanely RA, Van Gammeren D, Falk D,Deruisseau KC, Deering M, Yimlamai T, Powers SK. Trolox attenuatesmechanical ventilation-induced diaphragmatic dysfunction and proteoly-sis. Am J Respir Crit Care Med 170: 1179–1184, 2004.

3. Callahan LA, She ZW, Nosek TM. Superoxide, hydroxyl radical, andhydrogen peroxide effects on single-diaphragm fiber contractile apparatus.J Appl Physiol (1985) 90: 45–54, 2001.

4. Chung J, Nguyen AK, Henstridge DC, Holmes AG, Chan MH, MesaJL, Lancaster GI, Southgate RJ, Bruce CR, Duffy SJ, Horvath I,Mestril R, Watt MJ, Hooper PL, Kingwell BA, Vigh L, Hevener A,Febbraio MA. HSP72 protects against obesity-induced insulin resistance.Proc Natl Acad Sci USA 105: 1739–1744, 2008.

5. Currie RW, White FP. Characterization of the synthesis and accumula-tion of a 71-kilodalton protein induced in rat tissues after hyperthermia.Can J Biochem Cell Biol 61: 438–446, 1983.

6. Eskandar N, Apostolakos MJ. Weaning from mechanical ventilation.Crit Care Clin 23: 263–274, 2007.

7. Esteban A, Alia I, Ibanez J, Benito S, Tobin MJ. Modes of mechanicalventilation and weaning. A national survey of Spanish hospitals TheSpanish Lung Failure Collaborative Group. Chest 106: 1188–1193, 1994.

8. Esteban A, Frutos F, Tobin MJ, Alia I, Solsona JF, Valverdu I, FernandezR, de la Cal MA, Benito S, Tomas R, Garriedo D, Magias S, Blanco J. Acomparison of four methods of weaning patients from mechanical ventilation.Spanish Lung Failure Collaborative Group. N Engl J Med 332: 345–350, 1995.




Dow

nloaded from

9. Gayan-Ramirez G, de Paepe K, Cadot P, Decramer M. Detrimentaleffects of short-term mechanical ventilation on diaphragm function andIGF-I mRNA in rats. Intensive Care Med 29: 825–833, 2003.

10. Gayan-Ramirez G, Testelmans D, Maes K, Racz GZ, Cadot P, ZadorE, Wuytack F, Decramer M. Intermittent spontaneous breathing protectsthe rat diaphragm from mechanical ventilation effects. Crit Care Med 33:2804–2809, 2005.

11. Goto K, Kojima A, Kobayashi T, Uehara K, Morioka S, Naito T,Akema T, Sugiura T, Ohira Y, Yoshioka T. Heat stress as a counter-measure for prevention of muscle atrophy in microgravity environment.Jpn J Aerospace Environ Med 42: 51–59, 2005.

12. Hayashida N, Fujimoto M, Tan K, Prakasam R, Shinkawa T, Li L,Ichikawa H, Takii R, Nakai A. Heat shock factor 1 ameliorates proteo-toxicity in cooperation with the transcription factor NFAT. EMBO J 29:3459–3469, 2010.

13. Hernando R, Manso R. Muscle fibre stress in response to exercise:synthesis, accumulation and isoform transitions of 70-kDa heat-shockproteins. Eur J Biochem 243: 460–467, 1997.

14. Hess D, Kacmarek R. Essentials of Mechanical Ventilation (3rd ed.).New York: McGraw-Hill Medical, 2014.

15. Hogan PG, Chen L, Nardone J, Rao A. Transcriptional regulation bycalcium, calcineurin, and NFAT. Genes Dev 17: 2205–2232, 2003.

16. Horsley V, Pavlath GK. NFAT: ubiquitous regulator of cell differentia-tion and adaptation. J Cell Biol 156: 771–774, 2002.

17. Hudson MB, Smuder AJ, Nelson WB, Bruells CS, Levine S, PowersSK. Both high level pressure support ventilation and controlled mechan-ical ventilation induce diaphragm dysfunction and atrophy. Crit Care Med40: 1254–1260, 2012.

18. Larkins NT, Murphy RM, Lamb GD. Absolute amounts and diffusibil-ity of HSP72, HSP25, and alphaB-crystallin in fast- and slow-twitchskeletal muscle fibers of rat. Am J Physiol Cell Physiol 302: C228–C239,2012.

19. Levine S, Nguyen T, Taylor N, Friscia ME, Budak MT, Rothenberg P,Zhu J, Sachdeva R, Sonnad S, Kaiser LR, Rubinstein NA, Powers SK,Shrager JB. Rapid disuse atrophy of diaphragm fibers in mechanicallyventilated humans. N Engl J Med 358: 1327–1335, 2008.

20. Locke M, Tanguay RM, Klabunde RE, Ianuzzo CD. Enhanced post-ischemic myocardial recovery following exercise induction of HSP 72. AmJ Physiol Heart Circ Physiol 269: H320–H325, 1995.

21. Maglara AA, Vasilaki A, Jackson MJ, McArdle A. Damage to devel-oping mouse skeletal muscle myotubes in culture: protective effect of heatshock proteins. J Physiol 548: 837–846, 2003.

22. McClung JM, Kavazis AN, DeRuisseau KC, Falk DJ, Deering MA,Lee Y, Sugiura T, Powers SK. Caspase-3 regulation of diaphragmmyonuclear domain during mechanical ventilation-induced atrophy. Am JRespir Crit Care Med 175: 150–159, 2007.

23. Moopanar TR, Allen DG. Reactive oxygen species reduce myofibrillarCa2 sensitivity in fatiguing mouse skeletal muscle at 37°C. J Physiol564: 189–199, 2005.

24. Morton JP, Kayani AC, McArdle A, Drust B. The exercise-inducedstress response of skeletal muscle, with specific emphasis on humans.Sports Med 39: 643–662, 2009.

25. Morton JP, MacLaren DP, Cable NT, Bongers T, Griffiths RD,Campbell IT, Evans L, Kayani A, McArdle A, Drust B. Time courseand differential responses of the major heat shock protein families inhuman skeletal muscle following acute nondamaging treadmill exercise. JAppl Physiol 101: 176–182, 2006.

26. Naito H, Powers SK, Demirel HA, Sugiura T, Dodd SL, Aoki J. Heatstress attenuates skeletal muscle atrophy in hindlimb-unweighted rats. JAppl Physiol 88: 359–363, 2000.

27. Nelson WB, Smuder AJ, Hudson MB, Talbert EE, Powers SK. Cross-talk between the calpain and caspase-3 proteolytic systems in the dia-phragm during prolonged mechanical ventilation. Crit Care Med 40:1857–1863, 2012.

28. Oishi Y, Taniguchi K, Matsumoto H, Ishihara A, Ohira Y, Roy RR.Differential responses of HSPs to heat stress in slow and fast regions of ratgastrocnemius muscle. Muscle Nerve 28: 587–594, 2003.

29. Oishi Y, Taniguchi K, Matsumoto H, Ishihara A, Ohira Y, Roy RR.Muscle type-specific response of HSP60, HSP72, and HSC73 duringrecovery after elevation of muscle temperature. J Appl Physiol 92: 1097–1103, 2002.

30. Powers SK, Hudson MB, Nelson WB, Talbert EE, Min K, Szeto HH,Kavazis AN, Smuder AJ. Mitochondria-targeted antioxidants protectagainst mechanical ventilation-induced diaphragm weakness. Crit CareMed 39: 1749–1759, 2011.

31. Powers SK, Shanely RA, Coombes JS, Koesterer TJ, McKenzie M,Van Gammeren D, Cicale M, Dodd SL. Mechanical ventilation resultsin progressive contractile dysfunction in the diaphragm. J Appl Physiol 92:1851–1858, 2002.

32. Powers SK, Wiggs MP, Sollanek KJ, Smuder AJ. Ventilator-induceddiaphragm dysfunction: cause and effect. Am J Physiol Regul Integr CompPhysiol 305: R464–R477, 2013.

33. Ravagnan L, Gurbuxani S, Susin SA, Maisse C, Daugas E, ZamzamiN, Mak T, Jaattela M, Penninger JM, Garrido C, Kroemer G.Heat-shock protein 70 antagonizes apoptosis-inducing factor. Nat CellBiol 3: 839–843, 2001.

34. Segal SS, White TP, Faulkner JA. Architecture, composition, andcontractile properties of rat soleus muscle grafts. Am J Physiol CellPhysiol 250: C474–C479, 1986.

35. Selsby JT, Dodd SL. Heat treatment reduces oxidative stress and protectsmuscle mass during immobilization. Am J Physiol Regul Integr CompPhysiol 289: R134–R139, 2005.

36. Senf SM, Dodd SL, McClung JM, Judge AR. Hsp70 overexpressioninhibits NF-kappaB and Foxo3a transcriptional activities and preventsskeletal muscle atrophy. FASEB J 22: 3836–3845, 2008.

37. Shanely RA, Zergeroglu MA, Lennon SL, Sugiura T, Yimlamai T,Enns D, Belcastro A, Powers SK. Mechanical ventilation-induced dia-phragmatic atrophy is associated with oxidative injury and increasedproteolytic activity. Am J Respir Crit Care Med 166: 1369–1374, 2002.

38. Smuder AJ, Min K, Hudson MB, Kavazis AN, Kwon OS, Nelson WB,Powers SK. Endurance exercise attenuates ventilator-induced diaphragmdysfunction. J Appl Physiol 112: 501–510, 2012.

39. Supinski G, Nethery D, DiMarco A. Effect of free radical scavengers onendotoxin-induced respiratory muscle dysfunction. Am Rev Respir Dis148: 1318–1324, 1993.

40. Supinski GS, Wang W, Callahan LA. Caspase and calpain activationboth contribute to sepsis-induced diaphragmatic weakness. J Appl Physiol107: 1389–1396, 2009.

41. Uehara K, Goto K, Kobayashi T, Kojima A, Akema T, Sugiura T,Yamada S, Ohira Y, Yoshioka T, Aoki H. Heat-stress enhances prolif-erative potential in rat soleus muscle. Jpn J Physiol 54: 263–271, 2004.

42. Vassilakopoulos T, Petrof BJ. Ventilator-induced diaphragmatic dys-function. Am J Respir Crit Care Med 169: 336–341, 2004.

43. Vazeille E, Codran A, Claustre A, Averous J, Listrat A, Bechet D,Taillandier D, Dardevet D, Attaix D, Combaret L. The ubiquitin-proteasome and the mitochondria-associated apoptotic pathways are se-quentially downregulated during recovery after immobilization-inducedmuscle atrophy. Am J Physiol Endocrinol Metab 295: E1181–E1190,2008.

44. Xiao R, Ferry AL, Dupont-Versteegden EE. Cell death-resistance ofdifferentiated myotubes is associated with enhanced anti-apoptotic mech-anisms compared to myoblasts. Apoptosis 16: 221–234, 2011.

45. Yamaguchi T, Omori M, Tanaka N, Fukui N. Distinct and additiveeffects of sodium bicarbonate and continuous mild heat stress on fiber typeshift via calcineurin/NFAT pathway in human skeletal myoblasts. Am JPhysiol Cell Physiol 305: C323–C333, 2013.

46. Yamashita N, Hoshida S, Taniguchi N, Kuzuya T, Hori M. Whole-body hyperthermia provides biphasic cardioprotection against ischemia/reperfusion injury in the rat. Circulation 98: 1414–1421, 1998.

47. Zou J, Salminen WF, Roberts SM, Voellmy R. Correlation betweenglutathione oxidation and trimerization of heat shock factor 1, an earlystep in stress induction of the Hsp response. Cell Stress Chaperones 3:130–141, 1998.




Dow

nloaded from

Documents

Heat Stress Protects Against Mechanical Ventilation-Induced Diaphragmatic Atrophy