rapidly progressive diaphragmatic weakness and injury during mechanical ventilation in humans

Rapidly Progressive Diaphragmatic Weakness and Injuryduring Mechanical Ventilation in Humans

Samir Jaber1,2,6, Basil J. Petrof3, Boris Jung1,2, Gerald Chanques1,2, Jean-Philippe Berthet4, Christophe Rabuel5,Hassan Bouyabrine6, Patricia Courouble1,2, Christelle Koechlin-Ramonatxo7, Mustapha Sebbane1,2, Thomas Similowski8,Valerie Scheuermann9, Alexandre Mebazaa5, Xavier Capdevila1,2, Dominique Mornet2, Jacques Mercier2,10,Alain Lacampagne9, Alexandre Philips2, and Stefan Matecki2,10

1Department of Anesthesiology and Critical Care (DAR B), and 6Liver Transplant Unit, Saint-Eloi University Hospital, Montpellier, France; 2Equipesoutenue par la Region et l’Institut National de la Sante et de la Recherche Medicale (INSERM) 25, Universite Montpellier, Montpellier, France;3Meakins-Christie Laboratories and Respiratory Division, McGill University Health Centre Research Institute, Montreal, Quebec, Canada;4Department of Vascular and Thoracic Surgery, Hospital A de Villeneuve, Montpellier, France; 5Department of Anesthesiology and Critical Care

Medicine, Hopital Lariboisiere, Universite Paris Diderot, Equipe INSERM U 942, Paris, France; 7INRA, Universite Montpellier I, Montpellier, France;8Service de Pneumologie et Reanimation Medicale, Groupe Hospitalier Pitie-Salpetriere, Assistance Publique-Hopitaux de Paris, and ER10 Universite

Paris 6, Paris, France; 9Equipe INSERM U 637, Physiologie Cardio Vasculaire, Montpellier, France; and 10Department of Clinical Physiology, Arnaud

de Villeneuve University Hospital, Montpellier, France

Rationale: Diaphragmatic function is a major determinant of theability to successfully wean patients from mechanical ventilation(MV). Paradoxically, MV itself results in a rapid loss of diaphragmaticstrength in animals. However, very little is known about the timecourse or mechanistic basis for such a phenomenon in humans.Objectives: To determine in a prospective fashion the time course fordevelopment of diaphragmatic weakness during MV; and the re-lationship between MV duration and diaphragmatic injury or atro-phy, and the status of candidate cellular pathways implicated inthese phenomena.Methods: Airway occlusion pressure (TwPtr) generated by the di-aphragmduringphrenicnervestimulationwasmeasured inshort-term(0.5 h; n 5 6) and long-term (.5 d; n 5 6) MV groups. Diaphragmaticbiopsies obtained during thoracic surgery (MV for 2–3 h; n 5 10) andfrombrain-deadorgandonors(MVfor24–249h;n5 15)wereanalyzedforultrastructural injury, atrophy,andexpressionofproteolysis-relatedproteins (ubiquitin, nuclear factor-kB, and calpains).Measurements and Main Results: TwPtr decreased progressively duringMV, with a mean reduction of 32 6 6% after 6 days. Longer periods ofMV were associated with significantly greater ultrastructural fiberinjury (26.2 6 4.8 vs. 4.7 6 0.6% area), decreased cross-sectional areaof muscle fibers (1,904 6 220 vs. 3,100 6 329 mm2), an increase ofubiquitinated proteins (119%), higher expression of p65 nuclearfactor-kB (177%), and greater levels of the calcium-activated pro-teasescalpain-1, -2,and-3(1104%, 1432%,and1266%,respectively)in the diaphragm.Conclusions:Diaphragmaticweakness, injury,andatrophyoccur rapidlyin critically ill patients during MV, and are significantly correlated withthe duration of ventilator support.

Keywords: diaphragm disuse; atrophy; calpain; weaning; ventilator-

induced diaphragmatic dysfunction

Difficulties in weaning patients from mechanical ventilation(MV) account for a large proportion of time spent in the

intensive care unit (ICU), and thus have a major impact onthe use of health care resources (1). Diaphragmatic function isa major determinant of the ability to successfully wean patientsfrom MV (2). Recently, concern has been raised that MV mayitself have harmful effects on the diaphragm (2). In animals (3–9),diaphragmatic inactivity associated with MV leads to muscle fiberatrophy in the diaphragm and a reduction in its force-generatingcapacity, a condition referred to as ‘‘ventilator-induced diaphrag-matic dysfunction’’ (VIDD) (2, 10). Recently, Levine and co-workers (11) reported that prolonged diaphragmatic inactivityinduced by MV in brain-dead organ donors is associated withpreferential fiber atrophy and an increase in markers of proteolysis(E3 ubiquitin ligases and caspase-3) within the diaphragm, thussupporting the existence of VIDD in these patients.

Importantly, the impact of such changes on diaphragmaticcontractile function, and the rapidity with which diaphragmaticatrophy develops during MV in humans, remains unknown.Accordingly, in the present study, our first hypothesis was thatMV would be associated with time-dependent reductions indiaphragmatic force-generating capacity and muscle fiber size.In addition, animal studies suggest that diaphragmatic weaknessduring MV is caused not only by atrophy, but also by thepresence of muscle fiber injury (8, 12). However, the existenceof such an injury phenomenon in mechanically ventilatedhumans has not been established. Therefore, our secondhypothesis was that histologic signs of fiber injury would befound in the diaphragms of mechanically ventilated individuals,

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

There is strong evidence from animal models that mechan-ical ventilation causes atrophy and impaired contractility ofthe diaphragm. However, little is known regarding the timecourse and mechanisms underlying this phenomenon inhumans.

What This Study Adds to the Field

This study demonstrates the rapid onset of diaphragmaticweakness and atrophy in mechanically ventilated humans.In addition, we show that mechanical ventilation is associ-ated with structural injury to diaphragm muscle fibers andup-regulation of the calpain proteolytic system.

(Received in original form April 29, 2010; accepted in final form September 2, 2010)

Supported by the Program Hospitalier de Recherche Clinique (PHRC) 2005 of the

French Ministry of Health and from Association Francxaise contre les Myopathies

(AFM) (#12,815), and the Fonds de la recherche en sante du Quebec.

Correspondence and requests for reprints should be addressed to Stefan Matecki,

M.D., Ph.D., Department of Clinical Physiology, Arnaud de Villeneuve University

Hospital, CHU de Montpellier, France. E-Mail: [email protected]

This article has an online supplement, which is accessible from this issue’s table of

contents at www.atsjournals.org

Am J Respir Crit Care Med Vol 183. pp 364–371, 2011

Originally Published in Press as DOI: 10.1164/rccm.201004-0670OC on September 2, 2010

Internet address: www.atsjournals.org

and that the magnitude of this injury would also be signifi-cantly correlated with the duration of MV. Finally, we soughtto expand on prior work implicating E3 ubiquitin ligases inVIDD (11) and to additionally examine previously unex-plored cellular pathways of muscle atrophy and injury in thehuman diaphragm during MV. Hence, our last hypothesis wasthat prolonged MV would be associated with increased ubiquiti-nation of diaphragmatic proteins, and an up-regulated expressionof nuclear factor-kB (NF-kB) and calpains, which have all beenlinked to various pathologies causing skeletal muscle atrophy orinjury (13–17).

To test these hypotheses, we took advantage of severaldifferent clinical scenarios, which provided naturally occurringmodels of MV applied for variable periods of time in humansubjects. Our specific objectives in this study of mechanicallyventilated patients were as follows: to evaluate the time courseand extent of adverse changes in diaphragmatic force productionassociated with long-term MV (defined as .24 h); and toexamine the relationship between the duration of MV and thedevelopment of structural injury or atrophy within diaphragmaticmuscle fibers, together with the status of the previously men-tioned cellular pathways hypothesized to be associated with thesephenomena. Some of the results of this study have been pre-viously reported in the form of an abstract (18).

METHODS

Additional details are provided in the online supplement.

Study Subjects

The study was conducted in accordance with the World MedicalAssociation guidelines for research in humans, and approved by theinstitutional ethics board of the Montpellier University Hospital (pro-tocol NCT00786526). All subjects or their surrogates provided writteninformed consent to participate in the study.

The study design included four groups of subjects (Figure 1) asfollows: (1) functional evaluation short-term group, patients anesthe-tized and supported with MV for 1–2 hours during digestive systemendoscopic procedures; (2) functional evaluation long-term group,critically ill patients admitted to the ICU who required MV for atleast 5 days; (3) histobiochemical evaluation short-term group, patientsanesthetized and supported with MV for 2–3 hours during thoracicsurgery for localized (Stage 1A) lung cancers; and (4) histobiochemicalevaluation long-term group, patients with brain death destined fororgan donation, who had received MV for at least 24 hours beforeorgan harvest. All subjects were required to have undergone MV via anendotracheal tube in fully controlled mode (i.e., without significantspontaneous breathing efforts during the MV period).

Functional Evaluation by Magnetic Stimulation

of the Phrenic Nerves

Diaphragmatic function was assessed by measuring the change inendotracheal tube pressure induced by application of bilateral mag-netic twitch stimulation of the phrenic nerves during airway occlusion(TwPtr). TwPtr values were obtained at the end of the endoscopicprocedure in the short-term MV group and every 24–36 hours in thelong-term MV group.

Histobiochemical Evaluation of Biopsy Specimens

Diaphragm biopsies (z1 cm3) were obtained from the zone ofapposition of the costal diaphragm at the midaxillary line. In thelong-term MV group, the biopsies were obtained before circulatoryarrest and removal of other organs. Each biopsy was partitioned andtissue blocks were prepared as required for analysis of the parameterslisted next.

Histologic signs of injury and atrophy. Tissue blocks were preparedfor transmission electron microscopy (Hitachi H7100, Tokyo, Japan)using standard methods. As described in previous studies (8, 12, 19),disrupted sarcomeres were used as an index of respiratory muscle

injury. For evaluation of muscle fiber atrophy, transverse frozensections were stained with hematoxylin and eosin, and with antibodiesdirected against type I (slow) and type II (fast) isoforms of myosinheavy chain to determine fiber types. Computer images captured fromrandomly selected microscopic fields were then analyzed to determinethe mean percent area of fiber injury, fiber cross-sectional area, andfiber type proportions (11).

Biochemical markers of injury and atrophy. Total ubiquitinated pro-teins (20) and NF-kB p65 subunit (21) expression were quantified byimmunoblotting. Immunoblotting was also used to evaluate expression ofcalpain isoforms (calpain-1, -2, and -3) known to be involved in thedisassembly of myofilaments from their native state, a process that has beenimplicated in both atrophy and structural injury to muscle fibers (13–17).

Statistical Analysis

Data are presented as mean values 6 standard deviation. We used ttests for normally distributed continuous data, Mann-Whitney tests fornonnormally distributed continuous data, Friedman analysis of vari-ance, and Spearman correlation coefficient. A repeated measuresanalysis of variance was used to evaluate time-dependent effects ofMV on diaphragmatic contractile function. Statistical significance wasdefined as P less than or equal to 0.05.

RESULTS

Patient and Ventilation Characteristics

The experimental cohorts included in the functional andhistobiochemical components of the study are described inTables 1 and 2, respectively; Table 3 shows ventilator settings,gas exchange parameters, and vital signs in all patient groups.The mean duration of MV in the long-term group greatlyexceeded that in the short-term group for both functional andhistobiochemical study patients (P , 0.0001). In patients whounderwent functional evaluation, no significant differences werepresent between the short- and long-term MV groups withrespect to age, sex, or anthropometric characteristics. In addi-tion, there were no significant differences in age or anthropo-metrics when comparing the two short-term MV groups shownin Tables 1 and 2. In the histobiochemical study, patients in theshort-term MV group were significantly older than in the long-term MV group (P 5 0.045) and also contained a higherproportion of males, which is in keeping with the knowndemographics of lung cancer. However, there were no signifi-cant differences in age or anthropometric characteristics be-tween the two long-term MV groups shown in Tables 1 and 2.

Functional Evaluation of the Diaphragm During MV

In absolute terms, the mean baseline value of TwPtr in long-term MV patients was significantly lower than in the short-termMV group (16.5 6 5.2 vs. 20.1 6 2.5 cm H2O; P 5 0.03).Furthermore, TwPtr decreased progressively over time relativeto its initial baseline value in the long-term MV group (Figure2), with a statistically significant reduction after 3–4 days of MV.By the end of the evaluation period at 5–6 days of MV, TwPtr inthe long-term MV group was reduced by approximately 32%compared with its initial value (P , 0.01), and by about 50%relative to the value obtained in the short-term MV patients(P , 0.001). Static compliance of the respiratory systemaveraged 38 6 12 ml/cm H2O on the day of the first TwPtrmeasurement in the long-term MV group, and did not changesignificantly during the evaluation period.

Histobiochemical Evaluation of the Diaphragm During MV

Figures 3A–3D show representative longitudinal electron mi-croscopic images of diaphragms from the short- and long-termMV groups. In short-term MV patients, the ultrastructure of thediaphragm appeared normal. In comparison, diaphragms from

Jaber, Petrof, Jung, et al.: Mechanical Ventilation and Human Diaphragmatic Injury 365

the long-term MV group exhibited a significant increase in theprevalence of ultrastructural abnormalities (P 5 0.001), con-sisting of disruption of the normal myofibrillar organizationwith enlarged spaces containing disorganized sarcomeric mate-rial (Figure 3E). Furthermore, there was a significant positivecorrelation (r2 5 0.8; P , 0.001) between the magnitude ofdiaphragmatic injury and the duration of MV (Figure 3F). With

the exception of one patient, all subjects in the long-term MVgroup demonstrated a level of injury that exceeded the highestvalue obtained in the short-term MV group.

Figure 4 shows representative histologic images used toevaluate diaphragm muscle fiber size and fiber type proportions(Figures 4A–4F). There was no significant alteration in theproportions of type I (slow-twitch) and type II (fast-twitch)

Figure 1. Schematic illustration of experimental design.

NF-kB 5 nuclear factor-kB.

TABLE 1. FUNCTIONAL EVALUATION: PATIENT CHARACTERISTICS AND DURATION OF MECHANICAL VENTILATION

Subjects (n) Age (yr)

Sex

(M/F)

Weight

(kg)

Height

(cm)

BMI

(kg/m2)

Reason for MV

or Intensive Care

Unit Admission Relevant Medical History

Duration of

MV (h)

Short-term MV group

1 62 F 59 156 24 Digestive endoscopy Alcoholism, cirrhosis 0.5

2 42 M 70 168 24 Digestive endoscopy Gastrointestinal bleeding, alcoholism 0.5

3 34 M 87 183 26 Digestive endoscopy Alcoholism, cirrhosis 0.5

4 24 M 65 159 26 Digestive endoscopy Hepatitis B 0.5

5 43 F 76 167 27 Digestive endoscopy Gastrointestinal bleeding, Crohn disease 0.5

6 55 M 82 175 28 Digestive endoscopy Alcoholism, cirrhosis 0.5

Mean 6 SD 43 6 14 4/2 73 6 11 168 6 10 26 6 2 0.5

Long-term MV group

1 46 M 65 165 23 Facial trauma None 140

2 41 F 74 159 29 Digestive hemorrhage Laryngeal carcinoma 175

3 34 F 61 163 22 Stroke None 120

4 57 M 80 180 24 Polytrauma Alcoholism, cirrhosis 160

5 68 M 75 156 31 Stroke Parkinson disease 135

6 42 M 61 164 23 Facial trauma Bipolar disorder 150

Mean 6 SD 48 6 12 4/2 69 6 8 164 6 9 25 6 4 146 6 19*

Definition of abbreviations: BMI 5 body mass index (defined as the weight in kilograms divided by the square of the height in meters); MV 5 mechanical ventilation.

* P , 0.01.

366 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 183 2011

fibers between the short- and long-term MV groups (Figure4G). However, in the long-term MV patients, the mean cross-sectional area of all diaphragm fibers was reduced by 39%

compared with the short-term MV group (Figure 4H). Further-more, there was a significant relationship between reductions indiaphragmatic fiber size and the duration of MV (Figure 4I). In

TABLE 2. HISTOBIOCHEMICAL EVALUATION: PATIENT CHARACTERISTICS AND DURATION OF MECHANICAL VENTILATION

Subjects (n)

Age

(yr)

Sex

(M/F )

Weight

(kg)

Height

(cm)

BMI

(kg/m2)

Reason for Surgery or Cause

of Brain Death

Relevant Medical

History

Duration

of MV (h)

Short-term MV group

1 48 M 70 170 24 Stage 1A adenocarcinoma of the lung Smoker 30 pack-years 2.5

2 44 M 68 178 21 Stage 1A adenocarcinoma of the lung Diabetes, smoker 20 pack-years 2

3

39 M 70 170 24 Stage 1A adenocarcinoma of the lung Transient ischemic accident,

smoker 20 pack-years

3

4 54 M 93 163 35 Stage 1A adenocarcinoma of the lung Obesity, smoker 30 pack-years 2

5 61 M 64 151 28 Stage 1A adenocarcinoma of the lung None 2.5

6 44 M 69 173 23 Stage 1A adenocarcinoma of the lung Smoker 40 pack-years 2

7

55 M 72 182 21 Stage 1A adenocarcinoma of the lung Alcoholism, cirrhosis,


2

8

65 M 102 181 31 Stage 1A adenocarcinoma of the lung Larynx resection for carcinoma,


2

9 65 M 63 172 21 Stage 1A adenocarcinoma of the lung None 3

10 52 F 47 158 19 Stage 1A adenocarcinoma of the lung Smoker 40 pack-years 2

Mean 6 SD 53 6 9 9/1 72 6 15 170 6 10 25 6 5 2.3 6 0.4

Long-term MV group

1 50 M 90 180 28 Gunshot wound to head Smoker 20 pack-years 58

2 35 F 65 171 22 Stroke Smoker 40 pack-years 63

3 18 M 80 180 25 Drug overdose None 48

4 57 F 70 162 27 Stroke None 24

5 41 M 102 195 27 Stroke None 48

6 40 F 98 176 32 Stroke None 60

7

54 M 85 170 29 Stroke Alcoholism, smoker

20 pack-years

48

8 58 M 80 160 31 Stroke Hypertension 72

9 57 F 80 162 30 Motor vehicle accident None 68

10 18 F 62 167 22 Stroke None 48

11 18 F 60 160 23 Motor vehicle accident None 144

12

68 F 70 155 29 Stroke Seizure disorder, smoker

40 pack-years

112

13 19 F 80 168 28 Stroke None 249

14 58 F 65 168 23 Stroke None 90

15 28 M 78 173 26 Cardiac arrest Seizure disorder 81

Mean 6 SD 41 6 17* 6/9 77 6 12 170 6 10 27 6 3 80 6 55†

Definition of abbreviations: BMI 5 body mass index (defined as the weight in kilograms divided by the square of the height in meters); MV 5 mechanical ventilation.

* P , 0.05.† P , 0.01.

TABLE 3. VENTILATOR SETTINGS, GAS EXCHANGE, AND VITAL SIGNS FOR ALL PATIENTS

Functional Evaluation Histobiochemical Evaluation

Short-Term MV (n 5 6) Long-Term MV (n 5 6) Short-Term MV (n 5 10) Long-Term MV (n 5 15)

Ventilator settings and gas

exchange parameters

Tidal volume, ml/kg of

body weight

8.1 6 1.2 7.6 6 1.8 7.2 6 1.8 8.5 6 1.7*

Respiratory rate, breaths/min 12 6 1 24 6 4† 12 6 2 24 6 2†

PEEP, cm H2O 5 6 0 5 6 2 2 6 1‡ 5 6 4*

pH — 7.45 6 0.06 — 7.33 6 0.13‡

PaO2/FiO2, mm Hg — 364 6 72 — 370 6 128

PaCO2, mm Hg — 40 6 7 — 37 6 6

Bicarbonates, mmol/L — 27 6 6 — 20 6 3x

SpO2, % 99 6 1 99 6 1 99 6 1 99 6 1

Vital signs

Systolic pressure, mm Hg 116 6 10 120 6 15 114 6 13 118 6 18

Diastolic pressure, mm Hg 68 6 10 73 6 9 63 6 7 71 6 13

Heart rate, beats/min 84 6 15 95 6 18* 79 6 12 99 6 20*

Body temperature, 8C 36.4 6 0.5 36.3 6 0.5 35.8 6 0.4 36.5 6 0.9

Definition of abbreviations: MV 5 mechanical ventilation; PEEP 5 positive end-expiratory pressure.

Functional evaluation group data in the table were obtained at the time of the last twitch airway occlusion pressure measurement, whereas histobiochemical

evaluation group data were obtained at the time of diaphragmatic biopsy.

* , 0.05 and † P , 0.01 for comparisons between the short- and long-term MV groups within the functional and histobiochemical components of the study.‡ P , 0.01 and x P , 0.05 for comparisons between the two long-term MV groups and the two short-term MV groups across the functional and histobiochemical

components of the study.


the long-term MV patients who had been ventilated for at least72 hours, the values for diaphragmatic fiber size were all lowerthan the lowest value observed in the short-term MV group.Interestingly, although both injury and atrophy were separatelycorrelated with the duration of MV, the absolute levels ofdiaphragmatic injury and atrophy were not significantly corre-lated with one another in individual patients of the long-termMV group (P 5 0.39).

We next determined whether long-term MV was associatedwith increased ubiquitination of muscle proteins in the di-aphragm. As shown in Figure 5A, several proteins demon-strated greater ubiquitination in the long-term MV group, andtotal protein ubiquitination quantified from the entire lane ofantiubiquitin immunoblots was significantly increased (119%;P 5 0.04) in the long-term MV group. In addition, the level ofp65 Nf-kB, which has also been linked to skeletal muscleatrophy and injury, was greater (177%; P 5 0.02) in thediaphragms of long-term MV patients (Figure 5B). Finally, wealso quantified calpains, which are calcium-dependent proteasesinvolved in myofilament cleavage and the degradation ofcytoskeletal proteins. As indicated in Figure 6, immunoblottingrevealed significant increases for all three calpain isoforms(calpain-1 1104%, P 5 0.0014; calpain-2 1432%, P 5 0.0009;and calpain-3 1266%, P 5 0.001) in the diaphragms of long-term MV patients compared with the short-term MV group.

DISCUSSION

The principal findings of this investigation are that in criticallyill patients undergoing long-term controlled MV, there aremultiple deleterious changes in the human diaphragm, consist-ing of (1) decreased force-generating capacity, (2) muscle fiberinjury, (3) muscle atrophy, and (4) increased expression ofubiquitinated proteins, Nf-kB, and calpain isoforms, all of whichhave been previously implicated in different aspects of skeletalmuscle injury and atrophy responses (13–17).

Before discussing these results in detail, certain limitationsof this study are addressed. First, we were unable to performphrenic nerve stimulation studies on the brain-dead organdonor patients because of ethical and logistical considerations.With respect to the latter, brain-dead organ donors patientswere managed at different hospital centers within our univer-sity health care network, and only one of these locations hadthe equipment needed to perform magnetic stimulation of the

phrenic nerves. For obvious reasons it is also not possible toobtain diaphragmatic biopsies from critically ill patients admittedto the ICU. Therefore, it was necessary to perform the functionaland histobiochemical components of this study in separatepatient populations. Nonetheless, the long-term MV cohorts inthe functional and histobiochemical groups were well-matchedfor most characteristics. Second, although we eliminated pa-tients who were clinically unstable or suffering from otherconditions known to alter respiratory muscle function in ourstudy, we cannot exclude the possibility that factors other thanMV per se were involved in the functional and histobiochem-ical alterations found in the long-term MV groups (see later).Third, the short-term MV patients in the histobiochemicalstudy were older than in the long-term group and consistedof patients with underlying Stage 1A lung cancers. However,these factors would, if anything, be expected to favor muscleinjury and atrophy in the short-term MV group (22), and arethus unlikely to have affected the main findings.

In two previous studies, significant reductions in TwPtr havebeen reported in mechanically ventilated patients (23, 24),obtained at a single point and without any systematic relation-ship to the duration of MV. Importantly, the serial measure-ments of TwPtr performed from the first day of MV in our studyrevealed a decline in TwPtr values after the onset of MV thatwas extremely rapid, with a mean reduction to approximatelytwo-thirds of its baseline value after 5–6 days. In addition, the factthat ‘‘baseline’’ TwPtr values (obtained at a mean of 12.5 6 7.5 h

0

Figure 2. Relationship between duration of mechanical ventilation

(MV) and diaphragmatic function. Maximal twitch airway occlusion

pressure (TwPtr) generated by magnetic stimulation of the phrenicnerves at different time points in short-term MV (open bar; n 5 6) and

long-term MV (solid bars; n 5 6) groups. H 5 number of hours of MV;

D 5 number of days of MV.

Figure 3. Relationship between duration of mechanical ventilation (MV)

and diaphragmatic injury. Representative electron microscopy images of

longitudinal ultrathin sections obtained from the diaphragms of the

short-term MV (A, C) and long-term MV (B, D) groups are shown, at bothlow (A, B) and high (C, D) magnitude amplification. Note the disorga-

nization of sarcomeric structure, which is only present in the long-term

MV group images. (E) Quantitative analysis of the prevalence of these

findings in the two groups. (F ) Significant correlation between the degreeof diaphragmatic injury and the duration of MV (horizontal dashed line

indicates highest value of injury measured in short-term MV group).


after initiation of MV) were also reduced suggests two possi-bilities. The first is that even relatively short periods of con-trolled MV can induce adverse effects on diaphragmaticfunction (i.e., VIDD) in humans. In this regard, animal studieshave shown that VIDD occurs after only 12 hours in rats (6)and 1 day in rabbits (8). Another possibility is that indepen-dently of MV, critical illness causes diaphragmatic weakness. Infact, both explanations may be operative, and it is reasonable tospeculate that VIDD could be accelerated by additional factorsassociated with underlying critical illness, such as increasedsystemic inflammation (25).

Animal studies have found that MV-induced decreases indiaphragmatic force-generating capacity cannot be ascribed toatrophy alone, because the force loss is persistent even aftercorrecting for reductions in muscle cross-sectional area (2).Under these conditions, histologic evidence of myofibrillar

disarray has also been significantly correlated with abnormalcontractile function of the diaphragm (8). Our study is the firstto demonstrate this phenomenon in the human diaphragmduring MV, and a significant correlation between the magnitudeof diaphragmatic injury and the duration of MV. We also foundsignificant muscle fiber atrophy in the diaphragms of long-termMV patients, which is consistent with the recent findings ofLevine and coworkers (11) in a similar patient population.These authors also reported that mRNA transcript levels forE3 ubiquitin ligase enzymes were increased. Here we addition-ally demonstrate a greater level of protein ubiquitination inthe diaphragms of patients undergoing long-term MV. Further-more, in our study we observed that the degree of diaphrag-matic atrophy was directly proportional to the length of MV.

Interestingly, we did not find a significant correlation betweenthe levels of diaphragmatic injury and atrophy present in individual

Figure 4. Relationship be-

tween duration of mechanicalventilation (MV) and diaphrag-

matic atrophy. Representative

images of transverse frozen sec-

tions obtained from the dia-phragms of the short-term MV

(A, C, E ) and long-term MV (B,

D, F) groups are shown. The

diaphragm sections are stainedwith hematoxylin and eosin (A,

B) or with antibodies directed

against slow (C, D) or fast (E, F )

isoforms of myosin heavychain. (C–F) Serial sections, in-

dividual representative slow-

and fast-twitch fibers are markedby an open square and circle,

respectively. (G, H) Quantitative

analyses of diaphragm fiber size

(mean cross-sectional area) andfiber-type proportions in the

two patient groups, respectively. (I) The significant correlation between the degree of diaphragmatic atrophy and the duration of MV (horizontal dashedline indicates lowest value of fiber size measured in short-term MV group).

Figure 5. Total ubiquitinated proteins and nuclear

factor-kB expression in diaphragms of short- and long-term mechanical ventilation (MV) groups. Representa-

tive immunoblots and group mean quantification of

protein levels measured in diaphragm tissues obtained

from the short- and long-term MV groups for totalubiquitinated proteins (A) and nuclear factor-kB (B).

Equal loading was demonstrated by Coomassie blue

staining. NF-kB 5 nuclear factor-kB.


long-term MV patients. In animal studies of VIDD, the relation-ship between contractile dysfunction and atrophy is also unclear,and several studies have reported that the two responses canbe dissociated from one another (8, 26, 27). These observationsstrongly suggest that the mechanisms responsible for injuryand atrophy are not identical, although they may be linked.For example, myofilament proteins must first be partiallycleaved and disassembled to be processed and degraded bythe ubiquitin-proteasome system (15). Therefore, one possibil-ity is that the initial disassembly of actomyosin complexes is alsoinvolved in generating injury and early contractile dysfunction.Indeed, this would be consistent with the fact that in our study,diaphragmatic injury seemed to be an earlier phenomenon thanatrophy.

In keeping with the previous hypothesis, we found thatmembers of the calcium-dependent calpain protease systemwere significantly up-regulated in the diaphragms of long-termMV patients. Calpains degrade cytoskeletal proteins in muscle,and are capable of contributing not only to atrophy but also tosarcomeric disassembly and the development of injury re-sponses (13–17). Furthermore, in experimental animals, admin-istration of the calpain inhibitor leupeptin at the onset of MVprevented atrophy and contractile impairment of the diaphragm(28). In animal studies, calpain activation during MV wasrecently reported to be dependent on the presence of increasedoxidative stress (29). Calpains-1 and -2 are ubiquitous and havebeen extensively studied in skeletal muscle (15, 16, 30). Calpain-3 seems to be specific to skeletal muscle, and mutations incalpain-3 are responsible for limb girdle muscular dystrophytype 2a (31). Although its normal physiologic function is stillbeing elucidated, calpain-3 is bound to titin within the sarco-meric apparatus, and is thus ideally located to participate insarcomeric disassembly processes (14). Calpain-3 has also beenreported to play a role in regulating the Nf-kB pathway (32).The transcription factor Nf-kB is triggered by conditionsassociated with skeletal muscle injury and increased oxidativestress (33, 34), and has also been linked to activation of theubiquitin-proteasome system with attendant skeletal muscleatrophy (15, 35, 36). Therefore, taken together with the his-tologic findings of progressive diaphragmatic injury and atrophy

over time, the previously mentioned biochemical changes arelikely to be involved in the loss of diaphragmatic force pro-duction that we observed in long-term MV patients.

Conclusions

In humans, the use of controlled MV is associated with a rapidloss of diaphragmatic force-generating capacity and histobio-chemical signs of diaphragmatic injury and atrophy. We postu-late that these changes could play an important role in thedifficulties encountered in discontinuing ventilatory support inmany critically ill patients.

Author Disclosure: S.M. received sponsored grants from the association francxaisecontre les myopathies for $10,001–$50,000, from projet hospitalier de rechercheclinique for $50,001–$100,000, and from Servier Laboratory for $50,001–$100,000. T.S. served on the advisory board for Nycomed, Boehringer Ingelheim,Astra Zeneca for $1,001–$5,000 each, and Novartis France for $5,001–$10,000;he received lecture fees from Novartis France and Boehringer Ingelheim for$1,001–$5,000 each; and he received a sponsored grant from Maquet FranceS.A. for $10,001–$50,000. S.J. does not have a financial relationship witha commercial entity that has an interest in the subject of this manuscript. B.J.P.does not have a financial relationship with a commercial entity that has aninterest in the subject of this manuscript. B.J. does not have a financial relation-ship with a commercial entity that has an interest in the subject of thismanuscript. G.C. does not have a financial relationship with a commercial entitythat has an interest in the subject of this manuscript. J.-P.B. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. H.B. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. P.C. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. C.K.R. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. M.S. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. V.S. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. A.M. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. X.C. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. D.M. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. A.L. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript. J.M. does not havea financial relationship with a commercial entity that has an interest in the subjectof this manuscript. A.P. does not have a financial relationship with a commercialentity that has an interest in the subject of this manuscript.

Acknowledgment: The authors are grateful to Chantal Cazevieille and CecileSanchez for their technical assistance and interpretation of data concerningultrastructural evaluation.

Figure 6. Expression of calpain isoforms in diaphragms of

short- and long-term mechanical ventilation (MV) groups.Representative immunoblots (A) and group mean quan-

tification of protein levels measured in diaphragm tissues

obtained from the short- and long-term MV groups forcalpain-1 (B), calpain-2 (C ), and calpain-3 (D).


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rapidly progressive diaphragmatic weakness and injury during mechanical ventilation in humans

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