PHRENIC NERVE STIMULATION PROTECTS AGAINST MECHANICALVENTILATION-INDUCED DIAPHRAGM DYSFUNCTION IN RATSMEIRONG YANG, PhD, HAITAO WANG, MD, GUANGWEI HAN, PhD, LIANHUA CHEN, PhD, LINA HUANG, PhD,

JIHONG JIANG, PhD, and SHITONG LI, MD

Department of Anesthesiology, First People’s Hospital, School of Medicine, Shanghai Jiaotong University,100 Hai Ning Road, Shanghai 200080, China

Accepted 13 March 2013

ABSTRACT: Introduction: We investigated a novel applicationof phrenic nerve stimulation (PNS) in diaphragm dysfunctioninduced by mechanical ventilation (MV). Methods: Twenty-oneSprague-Dawley rats were assigned randomly to 3 groups:spontaneous breathing, 18-h controlled MV, and 18-h controlledMV with PNS. Upon completion of the experimental protocol,diaphragm contractility, gene expression of insulin-like growthfactor-1 (IGF-1) and ubiquitin ligases, and serum IGF-1 levelswere analyzed. Results: Compared with the spontaneouslybreathing rats, impaired diaphragm contractile function, includ-ing force-related properties and force-frequency responses,were pronounced with MV. Furthermore, MV suppressed IGF-1and induced muscle ring finger 1 mRNA expression in the dia-phragm. In contrast, PNS counteracted MV-induced geneexpression changes in the diaphragm and restored diaphragmfunction. Conclusions: PNS exerted a protective effect againstMV-induced diaphragm dysfunction by counteracting alteredexpression of IGF-1 and ubiquitin ligase in the diaphragm.

Muscle Nerve 48: 958–962, 2013

Mechanical ventilation (MV) is an important life-supporting technique used for intensive carepatients with respiratory failure. However, absenceof diaphragm electrical activity and contractile dys-function induced by MV are major factors respon-sible for ventilator weaning difficulties.1,2

Therefore, there is a need for protective strategiesagainst the deleterious effects of MV on thediaphragm.

The cellular mechanisms responsible for MV-induced diaphragmatic dysfunction have not beenelucidated fully. Recent discoveries have suggestedthat diaphragm disuse caused by MV triggers alter-ations in signaling that regulates myofibrillar pro-tein synthesis and proteolysis.3,4 Among these,insulin-like growth factor-1 (IGF-1) has been impli-cated to play a critical role in the regulation ofmuscle growth and function.5 In addition topromoting myofibrillar protein synthesis, IGF-1inhibits ubiquitin-dependent protein degradation

through the phosphatidylinositol 3-kinase (PI3K)/AKT/FOXO pathway.6,7 Increasing evidence sug-gests that muscle contractile activity may be neces-sary for locally produced IGF-1 in skeletal musclethrough mechanical signal transduction.8 Hence,the prevention of MV-induced diaphragm inactivitymay be a key to preserving diaphragm function.

The use of phrenic nerve stimulation (PNS) torestore breathing in quadriplegic patients9,10 sug-gests that PNS could be used as a compensatorytechnique during mechanical ventilation to main-tain diaphragm function. Recent studies haveshown that PNS may play a valuable therapeuticrole in patients with acute respiratory insufficiencyor neuromuscular disease.11,12 The purpose of thisstudy was to determine whether PNS could protectagainst MV-induced diaphragm dysfunction bycounteracting altered expressions of IGF-1 andubiquitin-protein ligase in the diaphragm.

MATERIALS AND METHODS

Animals. All experiments were approved by theAnimal Care and Use Committee of the ShanghaiJiaotong University School of Medicine (Shanghai,PR China). A total of 21 male adult Sprague–Daw-ley rats (9 weeks old) were included in the experi-ment. All animals were housed individually undera 12-h:12-h light–dark cycle at ambient tempera-ture (23–25�C) and were allowed free access to astandard laboratory diet and water. The rats wereassigned randomly to 3 groups, with 7 rats in eachgroup: spontaneous breathing (SB); 18-h con-trolled MV; and 18-h controlled MV with PNS(MS).

Experimental Procedures. The experimental setupwas performed according to previously describedmethods.13 Rats were anesthetized by intraperito-neal injection of pentobarbital (60 mg/kg bodyweight), and tracheotomy was performed. The leftlateral tail vein and carotid artery were cannulatedfor infusion of pentobarbital sodium (10 mg/kg/h)and heparin (2.5 U/mL/h), respectively. Subse-quently, a surgical incision was made at the neck,and 2 silver needle electrodes were implantedbilaterally near the emergence segment of thephrenic nerve. Body fluid homeostasis was main-tained by means of administration of electrolyte

Abbreviations: IGF-1, insulin-like growth factor-1; L0, optimal length;MAFbx, muscle atrophy F-box; MS, mechanical ventilation with phrenicnerve stimulation treatment; MuRF 1, muscle ring finger 1; MV, mechanicalventilation; PaO2, partial pressures of O2; PaCO2, partial pressures ofCO2; PI3K, phosphatidylinositol 3-kinase; PNS, phrenic nerve stimulation;RT-PCR, reverse-transcription polymerase chain reaction; SB, spontane-ous breathingKey words: diaphragm; electric stimulation therapy; mechanical ventila-tion; muscle weakness; phrenic nerveCorrespondence to: S. Li; e-mail: lstsh2010@hotmail.com

VC 2013 Wiley Periodicals, Inc.Published online 20 March 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.23850

958 Phrenic Stimulation Therapy MUSCLE & NERVE December 2013

solution (2.0 mL/kg/h). Body temperature wasmaintained at 37–37.5�C (6 0.5�C) using a heatinglamp. Arterial blood pressure was measured con-stantly, and blood gases were monitored at 6-hintervals throughout the experiment.

Rats from the MV and MS groups were venti-lated using a volume-driven small-animal ventilator(V8S, Alcott Biotech, Shanghai, China) for 18 hwith a tidal volume of 1 mL/100 g body weightand a respiratory rate of 80 breaths/min. Continu-ous care during MV included bladder expression,removal of airway mucus, eye lubrication, animalrotation, and passive limb movement. Care wasmaintained throughout the experimental period athourly intervals. Rats from the SB group were anes-thetized throughout the experiment and treatedsimilarly to the MV group, except for mechanicalventilation, to identify the effects of MV. Phrenicnerve electrostimulation was performed in each ratfrom the MS group. Electrical stimulation outputintensity gradually increased (0.1 mA increments)until visible diaphragm contraction was observed atthe costal margin. Once output intensity was deter-mined, the stimulator was set to deliver 0.2-ms rec-tangular impulses at 20 Hz for 10 s, followed by a20-s recovery period. During the experiment, phre-nic electrical stimulation was applied once perhour for a period of 10 min each.

Upon completion of the experimental proto-col, all animals were euthanized by intraperitonealinjection of sodium pentobarbital (100 mg/kg).Blood samples were collected rapidly from theheart for radioimmunoassay analysis of IGF-1, andcostal diaphragm segments were removed to mea-sure in vitro contractile properties. A section of theremaining costal diaphragm was frozen rapidly inliquid nitrogen and stored at 280�C for real-timereverse transcription polymerase chain reaction(RT-PCR).

In Vitro Diaphragm Contractile Measurements. Thediaphragm muscle was excised rapidly from themid-costal region, and a diaphragm muscle strip(�5 mm wide), with intact fibers inserted at theribs and central tendon, was used for isometriccontractile measurements, as described previ-ously.14 Briefly, the diaphragm muscle strip wassuspended vertically in a 37�C tissue bath contain-ing Krebs solution: 137 mM NaCl, 4 mM KCl, 1mM KH2PO4, 2 mM CaCl2, 1 mM MgCl2, 12 mMNaHCO3, and 6.5 mM glucose. The solution wasaerated continuously with a gas mixture of 95%O2/5% CO2, and a pH of 7.40 was maintained.The rib-end of the muscle was tied to a rigid sup-porter, and the central muscle tendon was con-nected to an isometric force transducer mountedon a micrometer. Two silver stimulating electrodes

were placed parallel to the muscle strip. Afterequilibration for 15 min, isometric contractionswere recorded at the optimal muscle length (L0)at which maximal isometric tetanic force wasobserved. Stimuli were applied using a rectangularpulse duration of 0.2 ms and a train duration of250 ms, respectively. To ensure supramaximal stim-ulation, the strips were stimulated at 20% abovevoltage to obtain maximal forces. The signal wasamplified and recorded using a data acquisitionsystem (MPA 2000; Alcott Biotech, Shanghai,China). Once maximal stimulus intensity and L0

for force production were determined, peak twitchtension, time to peak tension, and half-relaxationtime were determined at L0 from a series of con-tractions induced by single-pulse stimuli. Maximaltetanic tension was produced by a supramaximal,250-ms stimulus train (120 HZ). To measure theforce–frequency response, each strip was stimu-lated with a 250-ms train at 10, 20, 40, 60, 80, 100,and 120 HZ, with at least a 2-min interval betweeneach stimulus train. Finally, fatigability of eachstrip was assessed by 330-ms stimulations repeatedat 25 HZ and applied every second for 5 min. Fol-lowing completion of all measurements, forceswere normalized for muscle cross-sectional areas,which were estimated using the following formula:muscle mass (g)/[L0 (cm) 3 muscle density (g/cm3)], where 1.056 g/cm3 represented muscledensity.

Determination of IGF-1 Serum Levels. Total serumIGF-1 concentrations were determined by radioim-munoassay from blood samples obtained upon ter-mination. Blood samples were centrifuged at 3,000cycles/min at room temperature for 10 min. Se-rum was collected and stored at 220�C. Serum lev-els of IGF-1 were measured using a commercialradioimmunoassay kit (Diagnostic Systems Labora-tories, Webster, Texas) according to the manufac-turer’s instructions. Briefly, 50 lL of the dilutedplasma samples, standards, and controls werepipetted to respective tubes. Next, 100 ll of 125I-IGF-1 reagent was added to each tube. Then, 100ll rat IGF-1 antiserum was pipetted into all tubes(except for nonspecific binding and total counttubes) and incubated overnight at room tempera-ture (�25�C). On the second day, 1 mL of precipi-tating reagent was pipetted into all tubes (exceptfor total count tubes) and incubated for 20 min atroom temperature (�25�C). The tubes were thencentrifuged at 1,500 3 g for 20 min, and theresulting supernatant was removed. Radioactivitywas counted using a gamma counter. Serum IGF-1concentrations were determined by linear regres-sion analysis of the net counts per minute from 6IGF-1 standards.

Phrenic Stimulation Therapy MUSCLE & NERVE December 2013 959

Real-Time RT-PCR Detection. Total RNA was pre-pared from snap-frozen diaphragm samples (50mg) using TRIzol (Invitrogen Life Technologies,Carlsbad, California). RNA was quantified by spec-trophotometry (k 5 260 nm), and RNA concentra-tions were adjusted to 0.25 lg/lL using RNase-freewater. RNA was reverse-transcribed into cDNAusing a PrimeScript RT reagent kit (Takara, Shiga,Japan) according to the manufacturer’s instruc-tions. Following reverse-transcription, cDNA wasamplified using the SYBR Premix Ex TaqTM kit(Takara) and the DNA Engine Option 2 System(Bio-Rad Laboratories, Hercules, California). Real-time RT-PCR primers were designed and produced(Sangon, Shanghai, China) for rat IGF-1, musclering finger 1 (MuRF1), muscle atrophy F-box(MAFbx), and b-actin. The primer sequences wereas follows: rat b-actin (forward: CAC CCG CGAGTA CAA CCT TC; reverse: CCC ATA CCC ACCATC ACA CC); rat MAFbx (forward: TAC AAAGCA TCT TCC AAG GAC A; reverse: AGC AGCTCA CTC TTC TTC TCG T); rat MuRF1 (forward:TCC AGA CCC TCT ACA CAT CCT T; reverse:CCT CTG CAT GAT GTT CAG TTG T); rat IGF-1(forward: GGA GGC TGG AGA TGT ACT GTGCT; reverse: TCC TTT GCA GCT TCC TTT TCTTG). All RT-PCR experiments were performed intriplicate, and RNase-free water was used as a nega-tive control. The amount of target gene was nor-malized to the endogenous control (b-actin), andresults were expressed as relative fold changesusing the 2-DDCtmethod.15

Statistical Analysis. Values given are expressed asmean 6 standard deviation, unless otherwisespecified. Statistical analysis was performed withthe SPSS 17.0 statistical package (SPSS Inc, Chi-cago, Illinois). Data were tested for normalityand equal variance. Comparisons between groupsfor each dependent variable were made usinganalysis of variance with post hoc Student-Newman-Keuls test. Statistical significance was setat P< 0.05.

RESULTS

Systemic and Biologic Responses to Treatment. Thebody weight of the rats was similar at baseline andafter 18 h: SB 263 6 7 g and 258 6 9 g, respectively;MV 249 6 7 g and 251 6 7 g, respectively; MS257 6 5 g and 256 6 6 g, respectively. Body weightdid not change significantly between groups, whichsuggested that adequate nutrition and rehydrationwere maintained. Furthermore, rats from all groupsexhibited normal urination and defecation through-out the experimental period. Significant differencesin arterial blood pressure and blood gases were notobserved between groups. Results indicated thatsteady state homeostasis was maintained successfullyduring the 18-h experimental protocol.

Diaphragm Contractile Properties. Peak twitch ten-sion and maximal tetanic tension in the MV groupdecreased significantly by 26% and 19%, respec-tively, compared with the SB rats. However, whenPNS was combined with MV, peak twitch tensionand maximal tetanic tension improved significantlyby 22% and 16%, respectively, compared with MValone. Although MV did not influence time topeak tension or half-relaxation time, the latter wasprolonged in the MV and MS groups comparedwith SB rats (Table 1).

The response of diaphragm strips to increasedstimulus frequencies is shown in Figure 1. Com-pared with SB, MV induced a downward shift inthe force–frequency curve, which suggested signifi-cantly reduced force generation. PNS supplemen-tation during 18 h of MV completely attenuatedthe loss of force production. During the 5-min fa-tigue protocol, a similar pattern of force declinewas exhibited in all groups when tension wasexpressed as a percentage of initial values.

Table 1. In vitro isometric diaphragm contractile properties.

SB MV MS

Peak-twitch tension(mN�mm-2)

8.6 6 1.3 6.4 6 1.0* 7.8 6 0.9†

Maximum tetanictension (mN�mm-2)

17.2 6 2.4 13.9 6 1.8* 16.1 6 1.5†

Time to peak tension (ms) 55 6 8 59 6 17 59 6 8Half-relaxation time (ms) 40 6 16 44 6 16 43 6 11

SB, spontaneous breathing; MV, 18-h controlled mechanical ventilation;MS, MV with phrenic nerve stimulation treatment

*Significant difference vs. SB group (P< 0.05).

†Significant difference vs. MV group (P< 0.05).

FIGURE 1. Force-frequency curves of diaphragm strips. Force-

frequency curves demonstrate that 18-h controlled mechanical

ventilation (MV) reduces diaphragm-specific force production at

different stimulation frequencies compared with spontaneously

breathing (SB). The loss of force generation induced by MV is

completely attenuated by phrenic nerve stimulation supplemen-

tation during MV (MS). *Significant difference versus SB group

(P<0.05), #Significant difference versus MV group (P<0.05).

960 Phrenic Stimulation Therapy MUSCLE & NERVE December 2013

IGF-1 Levels in Serum and Diaphragm. Serum levelsof IGF-1 did not vary between groups (SB,1040 6 235 ng/mL; MV, 1048 6 238 ng/mL; MS,1022 6 283 ng/mL), which suggests that MV or MSdid not affect circulating levels of IGF-1. However,MV significantly reduced diaphragm IGF-1 mRNAexpression by 26% compared with SB rats. In addi-tion, IGF-1 mRNA expression decreased slightly by3.4% in MS diaphragms but was not significantlydifferent from the SB group (Fig. 2A). Thechanges in circulating and diaphragm levels ofIGF-1 indicate that MV or PNS exerted a distincteffect on the local IGF-1 system.

Expression of MuRF 1 and MAFbx mRNA in the

Diaphragm. Expression levels of MuRF1 andMAFbx mRNA in the MV group increased 1.4-fold(P< 0.001) and 0.2-fold, respectively, comparedwith the SB group. During 18 h of MV, bothMuRF1 and MAFbx were induced, but MuRF1increased to a greater extent than MAFbx. Therewere no significant differences in MAFbx expres-sion between the groups. When PNS was supple-mented with MV, MuRF1 and MAFbx expressiondecreased by 52% (P< 0.001) and 15%, respec-tively, compared with MV alone (Fig. 2B and C).

DISCUSSION

These findings demonstrate that PNS improveddiaphragm contractile properties, as evidenced byincreased maximal twitch or tetanic force produc-tion in the diaphragms of PNS-treated mechanical-ventilated rats compared with untreated rats. Inaddition, PNS prevented altered gene expressionof IGF-1, MuRF1, and MAFbx, which suggests anegative protein balance was responsible for the di-aphragm dysfunction induced by prolonged MV.

Because the diaphragm is only innervated bythe phrenic nerve, PNS enables independent analy-sis of the diaphragm from other respiratorymuscles. This study demonstrates that PNS-inducedcompensatory responses in phrenic nerve activitysufficiently offset the effects of diaphragm unload-ing during 18-h controlled MV. However, patients

often require long-term mechanical ventilation inthe setting of complex, multi-system critical ill-nesses. The major limitation of the study is thatthe therapeutic effect of PNS in a rat model of crit-ical illness was not measured. Further study isneeded to evaluate the therapeutic value of PNS indifferent clinical conditions.

The technique of PNS is a compensatory ther-apy that mimics physiological firing of diaphragmmotor units. Almost all motor units could be cate-gorized broadly into 2 major types with differentphysiological properties, including slow and fastmotor units. Low-frequency (10–20 Hz) tonic stim-ulation mimics the firing pattern of slow motorunits, whereas phasic high-frequency (100–150 Hz)stimulation mimics the firing pattern of fast motorunits.16,17 It has been demonstrated that slow mus-cle fibers are particularly sensitive to decreasedneuromuscular activity compared with fast musclefibers in a variety of muscle disuse conditions.18,19

PNS was delivered at a slow-fiber firing frequencyof 20 Hz in our experiment. However, the clinicalsituation is complex. In addition to MV, factorssuch as sepsis and steroid treatment are associatedwith critical illness myopathy,20 and this affectsboth types of motor units. Over time, with muscledisuse atrophy, both fast and slow muscle fibersare involved. Thus a combination of a high- andlow-frequency stimulation pattern rather than asingle stimulation pattern may be more suited toapplication of PNS in clinical practice.

Evoked-diaphragm activity plays a major role incontrolling gene expression of diaphragm proper-ties. IGF-1 has been identified as the primary medi-ator of activity-induced skeletal muscle adaptationsuch as muscle atrophy and hypertrophy.21,22 Dur-ing MV, the decrease in diaphragmatic IGF-1expression is caused by diaphragm disuse.3 Ourresults further demonstrate that diaphragm activa-tion by means of PNS increases diaphragm IGF-1mRNA expression but serum IGF-1 levels during18-h MV were not affected significantly. Moreover,previous results have shown that increased serum

FIGURE 2. Real-time RT-PCR analysis of IGF-1 (A), MuRF1 (B), and MAFbx (C) mRNA expression in the diaphragm. White bars,

dark gray bars, and light gray bars indicate relative differences between groups of spontaneously breathing (SB), 18-h controlled me-

chanical ventilation (MV), and phrenic nerve stimulation supplementation during MV (MS) after normalization to b-actin expression.

*P<0.05, ** P<0.001 versus SB group; #P<0.05, ##P<0.001 versus MV group.

Phrenic Stimulation Therapy MUSCLE & NERVE December 2013 961

IGF-1 levels as a result of exogenous administra-tion of GH or IGF-1 do not stimulate myofiber hy-pertrophy in the absence of mechanical loading.23

Therefore, the findings suggest that the local IGF-1 diaphragm system plays an important role inmaintaining diaphragm function and acts inde-pendently of any changes in serum IGF-1.

The ubiquitin–proteasome system is an impor-tant proteolytic system involved in the process ofskeletal muscle atrophy in conditions of disuse anddisease.24,25 As key substrate-specific enzymes in theubiquitination process, both MuRF 1 and MAFbxmRNA expression in the diaphragm increased fol-lowing 18-h MV in this study, in accordance withprevious reports.4 However, we found that MuRF 1was more responsive to MV-induced diaphragm dis-use than MAFbx. Importantly, application of PNSduring MV suppressed altered expression of these 2ubiquitin ligases in the diaphragm, which wereinvolved in the negative regulation of protein bal-ance. The degradation of myofibrillar proteins,especially contractile proteins, may be responsiblefor decreased diaphragm force-generating capacityduring prolonged MV. Clinically, this would mani-fest as ventilator dependence. PNS, therefore, mayprovide a new therapeutic alternative for thiscondition.

In summary, our findings indicate that phrenicnerve stimulation protects against MV-induced dia-phragm dysfunction by counteracting alteredexpression of IGF-1 and ubiquitin ligases in the di-aphragm. Our findings provide new informationfor the treatment of MV-induced diaphragm dys-function and could broaden our understanding ofdiaphragmatic protection in clinical practice.

This study was supported by grants from the National NaturalScience Foundation of China (30972859) and Innovation Founda-tion of Shanghai Jiaotong University School of Medicine for PhDGraduates (BXJ 201041).

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