Rapidly Progressive Diaphragmatic Weakness and Injury

during Mechanical Ventilation in Humans

Samir Jaber1,2,6, Basil J. Petrof 3, Boris Jung1,2, Gerald Chanques1,2, Jean-Philippe Berthet 4, 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
1

Department of Anesthesiology and Critical Care (DAR B), and 6Liver Transplant Unit, Saint-Eloi University Hospital, Montpellier, France; 2Equipe
soutenue par la Region et lInstitut National de la Sante et de la Recherche Medicale (INSERM) 25, Universite Montpellier, Montpellier, France;
3
Meakins-Christie Laboratories and Respiratory Division, McGill University Health Centre Research Institute, Montreal, Quebec, Canada;
4
Department of Vascular and Thoracic Surgery, Hospital A de Villeneuve, Montpellier, France; 5Department of Anesthesiology and Critical Care
pital Lariboisie`re, Universite Paris Diderot, Equipe INSERM U 942, Paris, France; 7INRA, Universite Montpellier I, Montpellier, France;
Medicine, Ho
8
pitaux de Paris, and ER10 Universite
Service de Pneumologie et Reanimation Medicale, Groupe Hospitalier Pitie-Salpetrie`re, Assistance Publique-Ho
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 the

ability to successfully wean patients from mechanical ventilation
(MV). Paradoxically, MV itself results in a rapid loss of diaphragmatic
strength in animals. However, very little is known about the time
course or mechanistic basis for such a phenomenon in humans.
Objectives: To determine in a prospective fashion the time course for
development of diaphragmatic weakness during MV; and the relationship between MV duration and diaphragmatic injury or atrophy, and the status of candidate cellular pathways implicated in
these phenomena.
Methods: Airway occlusion pressure (TwPtr) generated by the diaphragm during phrenic nerve stimulation was measured in short-term
(0.5 h; n 5 6) and long-term (.5 d; n 5 6) MV groups. Diaphragmatic
biopsies obtained during thoracic surgery (MV for 23 h; n 5 10) and
from brain-dead organ donors (MV for 24249 h; n 5 15) were analyzed
for ultrastructural injury, atrophy, and expression of proteolysis-related
proteins (ubiquitin, nuclear factor-kB, and calpains).
Measurements and Main Results: TwPtr decreased progressively during
MV, with a mean reduction of 32 6 6% after 6 days. Longer periods of
MV were associated with significantly greater ultrastructural fiber
injury (26.2 6 4.8 vs. 4.7 6 0.6% area), decreased cross-sectional area
of muscle fibers (1,904 6 220 vs. 3,100 6 329 mm2), an increase of
ubiquitinated proteins (119%), higher expression of p65 nuclear
factor-kB (177%), and greater levels of the calcium-activated proteases calpain-1, -2, and -3 (1104%, 1432%, and 1266%, respectively)
in the diaphragm.
Conclusions: Diaphragmatic weakness, injury, and atrophy occur rapidly
in critically ill patients during MV, and are significantly correlated with
the duration of ventilator support.
Keywords: diaphragm disuse; atrophy; calpain; weaning; ventilatorinduced diaphragmatic dysfunction

Difficulties in weaning patients from mechanical ventilation

(MV) account for a large proportion of time spent in the

(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: s-matecki@chu-montpellier.fr
This article has an online supplement, which is accessible from this issues table of
contents at www.atsjournals.org
Am J Respir Crit Care Med Vol 183. pp 364371, 2011
Originally Published in Press as DOI: 10.1164/rccm.201004-0670OC on September 2, 2010
Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject

There is strong evidence from animal models that mechanical ventilation causes atrophy and impaired contractility of
the diaphragm. However, little is known regarding the time
course and mechanisms underlying this phenomenon in
humans.
What This Study Adds to the Field

This study demonstrates the rapid onset of diaphragmatic

weakness and atrophy in mechanically ventilated humans.
In addition, we show that mechanical ventilation is associated with structural injury to diaphragm muscle fibers and
up-regulation of the calpain proteolytic system.

intensive care unit (ICU), and thus have a major impact on

the use of health care resources (1). Diaphragmatic function is
a major determinant of the ability to successfully wean patients
from MV (2). Recently, concern has been raised that MV may
itself have harmful effects on the diaphragm (2). In animals (39),
diaphragmatic inactivity associated with MV leads to muscle fiber
atrophy in the diaphragm and a reduction in its force-generating
capacity, a condition referred to as ventilator-induced diaphragmatic dysfunction (VIDD) (2, 10). Recently, Levine and coworkers (11) reported that prolonged diaphragmatic inactivity
induced by MV in brain-dead organ donors is associated with
preferential fiber atrophy and an increase in markers of proteolysis
(E3 ubiquitin ligases and caspase-3) within the diaphragm, thus
supporting the existence of VIDD in these patients.
Importantly, the impact of such changes on diaphragmatic
contractile function, and the rapidity with which diaphragmatic
atrophy develops during MV in humans, remains unknown.
Accordingly, in the present study, our first hypothesis was that
MV would be associated with time-dependent reductions in
diaphragmatic force-generating capacity and muscle fiber size.
In addition, animal studies suggest that diaphragmatic weakness
during MV is caused not only by atrophy, but also by the
presence of muscle fiber injury (8, 12). However, the existence
of such an injury phenomenon in mechanically ventilated
humans has not been established. Therefore, our second
hypothesis was that histologic signs of fiber injury would be
found in the diaphragms of mechanically ventilated individuals,

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

and that the magnitude of this injury would also be significantly correlated with the duration of MV. Finally, we sought
to expand on prior work implicating E3 ubiquitin ligases in
VIDD (11) and to additionally examine previously unexplored cellular pathways of muscle atrophy and injury in the
human diaphragm during MV. Hence, our last hypothesis was
that prolonged MV would be associated with increased ubiquitination of diaphragmatic proteins, and an up-regulated expression
of nuclear factor-kB (NF-kB) and calpains, which have all been
linked to various pathologies causing skeletal muscle atrophy or
injury (1317).
To test these hypotheses, we took advantage of several
different clinical scenarios, which provided naturally occurring
models of MV applied for variable periods of time in human
subjects. Our specific objectives in this study of mechanically
ventilated patients were as follows: to evaluate the time course
and extent of adverse changes in diaphragmatic force production
associated with long-term MV (defined as .24 h); and to
examine the relationship between the duration of MV and the
development of structural injury or atrophy within diaphragmatic
muscle fibers, together with the status of the previously mentioned cellular pathways hypothesized to be associated with these
phenomena. Some of the results of this study have been previously 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 Medical
Association guidelines for research in humans, and approved by the
institutional ethics board of the Montpellier University Hospital (protocol NCT00786526). All subjects or their surrogates provided written
informed consent to participate in the study.
The study design included four groups of subjects (Figure 1) as
follows: (1) functional evaluation short-term group, patients anesthetized and supported with MV for 12 hours during digestive system
endoscopic procedures; (2) functional evaluation long-term group,
critically ill patients admitted to the ICU who required MV for at
least 5 days; (3) histobiochemical evaluation short-term group, patients
anesthetized and supported with MV for 23 hours during thoracic
surgery for localized (Stage 1A) lung cancers; and (4) histobiochemical
evaluation long-term group, patients with brain death destined for
organ donation, who had received MV for at least 24 hours before
organ harvest. All subjects were required to have undergone MV via an
endotracheal tube in fully controlled mode (i.e., without significant
spontaneous breathing efforts during the MV period).

Functional Evaluation by Magnetic Stimulation

of the Phrenic Nerves
Diaphragmatic function was assessed by measuring the change in
endotracheal tube pressure induced by application of bilateral magnetic twitch stimulation of the phrenic nerves during airway occlusion
(TwPtr). TwPtr values were obtained at the end of the endoscopic
procedure in the short-term MV group and every 2436 hours in the
long-term MV group.

Histobiochemical Evaluation of Biopsy Specimens

Diaphragm biopsies (z1 cm3) were obtained from the zone of
apposition of the costal diaphragm at the midaxillary line. In the
long-term MV group, the biopsies were obtained before circulatory
arrest and removal of other organs. Each biopsy was partitioned and
tissue blocks were prepared as required for analysis of the parameters
listed next.
Histologic signs of injury and atrophy. Tissue blocks were prepared
for 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

365

injury. For evaluation of muscle fiber atrophy, transverse frozen

sections were stained with hematoxylin and eosin, and with antibodies
directed against type I (slow) and type II (fast) isoforms of myosin
heavy chain to determine fiber types. Computer images captured from
randomly selected microscopic fields were then analyzed to determine
the mean percent area of fiber injury, fiber cross-sectional area, and
fiber type proportions (11).
Biochemical markers of injury and atrophy. Total ubiquitinated proteins (20) and NF-kB p65 subunit (21) expression were quantified by
immunoblotting. Immunoblotting was also used to evaluate expression of
calpain isoforms (calpain-1, -2, and -3) known to be involved in the
disassembly of myofilaments from their native state, a process that has been
implicated in both atrophy and structural injury to muscle fibers (1317).

Statistical Analysis
Data are presented as mean values 6 standard deviation. We used t
tests for normally distributed continuous data, Mann-Whitney tests for
nonnormally distributed continuous data, Friedman analysis of variance, and Spearman correlation coefficient. A repeated measures
analysis of variance was used to evaluate time-dependent effects of
MV on diaphragmatic contractile function. Statistical significance was
defined as P less than or equal to 0.05.

RESULTS
Patient and Ventilation Characteristics

The experimental cohorts included in the functional and

histobiochemical components of the study are described in
Tables 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 greatly
exceeded that in the short-term group for both functional and
histobiochemical study patients (P , 0.0001). In patients who
underwent functional evaluation, no significant differences were
present between the short- and long-term MV groups with
respect to age, sex, or anthropometric characteristics. In addition, there were no significant differences in age or anthropometrics when comparing the two short-term MV groups shown
in Tables 1 and 2. In the histobiochemical study, patients in the
short-term MV group were significantly older than in the longterm MV group (P 5 0.045) and also contained a higher
proportion of males, which is in keeping with the known
demographics of lung cancer. However, there were no significant differences in age or anthropometric characteristics between 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 longterm MV patients was significantly lower than in the short-term
MV group (16.5 6 5.2 vs. 20.1 6 2.5 cm H2O; P 5 0.03).
Furthermore, TwPtr decreased progressively over time relative
to its initial baseline value in the long-term MV group (Figure
2), with a statistically significant reduction after 34 days of MV.
By the end of the evaluation period at 56 days of MV, TwPtr in
the 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 system
averaged 38 6 12 ml/cm H2O on the day of the first TwPtr
measurement in the long-term MV group, and did not change
significantly during the evaluation period.
Histobiochemical Evaluation of the Diaphragm During MV

Figures 3A3D show representative longitudinal electron microscopic images of diaphragms from the short- and long-term
MV groups. In short-term MV patients, the ultrastructure of the
diaphragm appeared normal. In comparison, diaphragms from

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Figure 1. Schematic illustration of experimental design.

NF-kB 5 nuclear factor-kB.

the long-term MV group exhibited a significant increase in the

prevalence of ultrastructural abnormalities (P 5 0.001), consisting of disruption of the normal myofibrillar organization
with enlarged spaces containing disorganized sarcomeric material (Figure 3E). Furthermore, there was a significant positive
correlation (r2 5 0.8; P , 0.001) between the magnitude of
diaphragmatic injury and the duration of MV (Figure 3F). With

the exception of one patient, all subjects in the long-term MV

group demonstrated a level of injury that exceeded the highest
value obtained in the short-term MV group.
Figure 4 shows representative histologic images used to
evaluate diaphragm muscle fiber size and fiber type proportions
(Figures 4A4F). There was no significant alteration in the
proportions of type I (slow-twitch) and type II (fast-twitch)

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

Subjects (n)
Short-term MV group
1
2
3
4
5
6
Mean 6 SD
Long-term MV group
1
2
3
4
5
6
Mean 6 SD

Reason for MV
or Intensive Care
Unit Admission

Age (yr)

Sex
(M/F)

Weight
(kg)

Height
(cm)

BMI
(kg/m2)

62
42
34
24
43
55
43 6 14

F
M
M
M
F
M
4/2

59
70
87
65
76
82
73 6 11

156
168
183
159
167
175
168 6 10

24
24
26
26
27
28
26 6 2

Digestive
Digestive
Digestive
Digestive
Digestive
Digestive

46
41
34
57
68
42
48 6 12

M
F
F
M
M
M
4/2

65
74
61
80
75
61
69 6 8

165
159
163
180
156
164
164 6 9

23
29
22
24
31
23
25 6 4

Facial trauma
Digestive hemorrhage
Stroke
Polytrauma
Stroke
Facial trauma

endoscopy
endoscopy
endoscopy
endoscopy
endoscopy
endoscopy

Relevant Medical History

Alcoholism, cirrhosis
Gastrointestinal bleeding, alcoholism
Alcoholism, cirrhosis
Hepatitis B
Gastrointestinal bleeding, Crohn disease
Alcoholism, cirrhosis

None
Laryngeal carcinoma
None
Alcoholism, cirrhosis
Parkinson disease
Bipolar disorder

Duration of
MV (h)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
140
175
120
160
135
150
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.

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

367

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

Subjects (n)
Short-term MV group
1
2
3
4
5
6

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)

48
44
39

M
M
M

70
68
70

170
178
170

24
21
24

Stage 1A adenocarcinoma of the lung

Stage 1A adenocarcinoma of the lung
Stage 1A adenocarcinoma of the lung

2.5
2
3

54
61
44
55

M
M
M
M

93
64
69
72

163
151
173
182

35
28
23
21

Stage
Stage
Stage
Stage

65

102

181

31

Stage 1A adenocarcinoma of the lung

65
52
53 6 9

M
F
9/1

63
47
72 6 15

172
158
170 6 10

21
19
25 6 5

Stage 1A adenocarcinoma of the lung

Stage 1A adenocarcinoma of the lung

Smoker 30 pack-years
Diabetes, smoker 20 pack-years
Transient ischemic accident,
smoker 20 pack-years
Obesity, smoker 30 pack-years
None
Smoker 40 pack-years
Alcoholism, cirrhosis,
smoker 80 pack-years
Larynx resection for carcinoma,
smoker 35 pack-years
None
Smoker 40 pack-years

50
35
18
57
41
40
54

M
F
M
F
M
F
M

90
65
80
70
102
98
85

180
171
180
162
195
176
170

28
22
25
27
27
32
29

Gunshot wound to head

Stroke
Drug overdose
Stroke
Stroke
Stroke
Stroke

58
57
18
18
68

M
F
F
F
F

80
80
62
60
70

160
162
167
160
155

31
30
22
23
29

Stroke
Motor vehicle accident
Stroke
Motor vehicle accident
Stroke

19
58
28
41 6 17*

F
F
M
6/9

80
65
78
77 6 12

168
168
173
170 6 10

28
23
26
27 6 3

1A
1A
1A
1A

adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma

of
of
of
of

the
the
the
the

lung
lung
lung
lung

7
8
9
10
Mean 6 SD
Long-term MV group
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean 6 SD

Smoker 20 pack-years
Smoker 40 pack-years
None
None
None
None
Alcoholism, smoker
20 pack-years
Hypertension
None
None
None
Seizure disorder, smoker
40 pack-years
None
None
Seizure disorder

Stroke
Stroke
Cardiac arrest

2
2.5
2
2
2
3
2
2.3 6 0.4
58
63
48
24
48
60
48
72
68
48
144
112
249
90
81
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.

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

fibers between the short- and long-term MV groups (Figure

4G). However, in the long-term MV patients, the mean crosssectional area of all diaphragm fibers was reduced by 39%

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

Ventilator settings and gas

exchange parameters
Tidal volume, ml/kg of
body weight
Respiratory rate, breaths/min
PEEP, cm H2O
pH
PaO2/FiO2, mm Hg
PaCO2, mm Hg
Bicarbonates, mmol/L
SpO2, %
Vital signs
Systolic pressure, mm Hg
Diastolic pressure, mm Hg
Heart rate, beats/min
Body temperature, 8C

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)

8.1 6 1.2

7.6 6 1.8

7.2 6 1.8

8.5 6 1.7*

12 6 1
560

99 6 1
116
68
84
36.4

6
6
6
6

10
10
15
0.5

24
5
7.45
364
40
27
99

6
6
6
6
6
6
6

4
2
0.06
72
7
6
1

120
73
95
36.3

6
6
6
6

15
9
18*
0.5

12 6 2
261

99 6 1

114
63
79
35.8

6
6
6
6

13
7
12
0.4

24
5
7.33
370
37
20
99

6
6
6
6
6
6
6

2
4*
0.13
128
6
3x
1

118
71
99
36.5

6
6
6
6

18
13
20*
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.

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Figure 2. Relationship between duration of mechanical ventilation

(MV) and diaphragmatic function. Maximal twitch airway occlusion
pressure ( TwPtr) generated by magnetic stimulation of the phrenic
nerves 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.

the long-term MV patients who had been ventilated for at least

72 hours, the values for diaphragmatic fiber size were all lower
than the lowest value observed in the short-term MV group.
Interestingly, although both injury and atrophy were separately
correlated with the duration of MV, the absolute levels of
diaphragmatic injury and atrophy were not significantly correlated with one another in individual patients of the long-term
MV group (P 5 0.39).
We next determined whether long-term MV was associated
with increased ubiquitination of muscle proteins in the diaphragm. As shown in Figure 5A, several proteins demonstrated greater ubiquitination in the long-term MV group, and
total protein ubiquitination quantified from the entire lane of
antiubiquitin immunoblots was significantly increased (119%;
P 5 0.04) in the long-term MV group. In addition, the level of
p65 Nf-kB, which has also been linked to skeletal muscle
atrophy and injury, was greater (177%; P 5 0.02) in the
diaphragms of long-term MV patients (Figure 5B). Finally, we
also quantified calpains, which are calcium-dependent proteases
involved in myofilament cleavage and the degradation of
cytoskeletal proteins. As indicated in Figure 6, immunoblotting
revealed 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 longterm MV patients compared with the short-term MV group.

DISCUSSION
The principal findings of this investigation are that in critically
ill patients undergoing long-term controlled MV, there are
multiple deleterious changes in the human diaphragm, consisting of (1) decreased force-generating capacity, (2) muscle fiber
injury, (3) muscle atrophy, and (4) increased expression of
ubiquitinated proteins, Nf-kB, and calpain isoforms, all of which
have been previously implicated in different aspects of skeletal
muscle injury and atrophy responses (1317).
Before discussing these results in detail, certain limitations
of this study are addressed. First, we were unable to perform
phrenic nerve stimulation studies on the brain-dead organ
donor patients because of ethical and logistical considerations.
With respect to the latter, brain-dead organ donors patients
were managed at different hospital centers within our university health care network, and only one of these locations had
the equipment needed to perform magnetic stimulation of the

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 both
low (A, B) and high (C, D) magnitude amplification. Note the disorganization 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 degree
of diaphragmatic injury and the duration of MV (horizontal dashed line
indicates highest value of injury measured in short-term MV group).

phrenic nerves. For obvious reasons it is also not possible to

obtain diaphragmatic biopsies from critically ill patients admitted
to the ICU. Therefore, it was necessary to perform the functional
and histobiochemical components of this study in separate
patient populations. Nonetheless, the long-term MV cohorts in
the functional and histobiochemical groups were well-matched
for most characteristics. Second, although we eliminated patients who were clinically unstable or suffering from other
conditions known to alter respiratory muscle function in our
study, we cannot exclude the possibility that factors other than
MV per se were involved in the functional and histobiochemical alterations found in the long-term MV groups (see later).
Third, the short-term MV patients in the histobiochemical
study were older than in the long-term group and consisted
of patients with underlying Stage 1A lung cancers. However,
these factors would, if anything, be expected to favor muscle
injury and atrophy in the short-term MV group (22), and are
thus unlikely to have affected the main findings.
In two previous studies, significant reductions in TwPtr have
been reported in mechanically ventilated patients (23, 24),
obtained at a single point and without any systematic relationship to the duration of MV. Importantly, the serial measurements of TwPtr performed from the first day of MV in our study
revealed a decline in TwPtr values after the onset of MV that
was extremely rapid, with a mean reduction to approximately
two-thirds of its baseline value after 56 days. In addition, the fact
that baseline TwPtr values (obtained at a mean of 12.5 6 7.5 h

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

369

Figure 4.
Relationship between duration of mechanical
ventilation (MV) and diaphragmatic atrophy. Representative
images of transverse frozen sections obtained from the diaphragms of the short-term MV
(A, C, E ) and long-term MV (B,
D, F) groups are shown. The
diaphragm sections are stained
with hematoxylin and eosin (A,
B) or with antibodies directed
against slow (C, D) or fast (E, F )
isoforms of myosin heavy
chain. (CF) Serial sections, individual representative slowand fast-twitch fibers are marked
by an open square and circle,
respectively. (G, H) Quantitative
analyses of diaphragm fiber size
(mean cross-sectional area) and
fiber-type proportions in the
two patient groups, respectively. (I) The significant correlation between the degree of diaphragmatic atrophy and the duration of MV (horizontal dashed
line indicates lowest value of fiber size measured in short-term MV group).

after initiation of MV) were also reduced suggests two possibilities. The first is that even relatively short periods of controlled MV can induce adverse effects on diaphragmatic
function (i.e., VIDD) in humans. In this regard, animal studies
have shown that VIDD occurs after only 12 hours in rats (6)
and 1 day in rabbits (8). Another possibility is that independently of MV, critical illness causes diaphragmatic weakness. In
fact, both explanations may be operative, and it is reasonable to
speculate that VIDD could be accelerated by additional factors
associated with underlying critical illness, such as increased
systemic inflammation (25).
Animal studies have found that MV-induced decreases in
diaphragmatic force-generating capacity cannot be ascribed to
atrophy alone, because the force loss is persistent even after
correcting for reductions in muscle cross-sectional area (2).
Under these conditions, histologic evidence of myofibrillar

disarray has also been significantly correlated with abnormal

contractile function of the diaphragm (8). Our study is the first
to demonstrate this phenomenon in the human diaphragm
during MV, and a significant correlation between the magnitude
of diaphragmatic injury and the duration of MV. We also found
significant muscle fiber atrophy in the diaphragms of long-term
MV patients, which is consistent with the recent findings of
Levine and coworkers (11) in a similar patient population.
These authors also reported that mRNA transcript levels for
E3 ubiquitin ligase enzymes were increased. Here we additionally demonstrate a greater level of protein ubiquitination in
the diaphragms of patients undergoing long-term MV. Furthermore, in our study we observed that the degree of diaphragmatic atrophy was directly proportional to the length of MV.
Interestingly, we did not find a significant correlation between
the levels of diaphragmatic injury and atrophy present in individual

Figure 5. Total ubiquitinated proteins and nuclear

factor-kB expression in diaphragms of short- and longterm mechanical ventilation (MV) groups. Representative immunoblots and group mean quantification of
protein levels measured in diaphragm tissues obtained
from the short- and long-term MV groups for total
ubiquitinated proteins (A) and nuclear factor-kB (B).
Equal loading was demonstrated by Coomassie blue
staining. NF-kB 5 nuclear factor-kB.

370

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

VOL 183

2011

Figure 6. Expression of calpain isoforms in diaphragms of

short- and long-term mechanical ventilation (MV) groups.
Representative immunoblots (A) and group mean quantification of protein levels measured in diaphragm tissues
obtained from the short- and long-term MV groups for
calpain-1 (B), calpain-2 (C ), and calpain-3 (D).

long-term MV patients. In animal studies of VIDD, the relationship between contractile dysfunction and atrophy is also unclear,
and several studies have reported that the two responses can
be dissociated from one another (8, 26, 27). These observations
strongly suggest that the mechanisms responsible for injury
and atrophy are not identical, although they may be linked.
For example, myofilament proteins must first be partially
cleaved and disassembled to be processed and degraded by
the ubiquitin-proteasome system (15). Therefore, one possibility is that the initial disassembly of actomyosin complexes is also
involved 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 than
atrophy.
In keeping with the previous hypothesis, we found that
members of the calcium-dependent calpain protease system
were significantly up-regulated in the diaphragms of long-term
MV patients. Calpains degrade cytoskeletal proteins in muscle,
and are capable of contributing not only to atrophy but also to
sarcomeric disassembly and the development of injury responses (1317). Furthermore, in experimental animals, administration of the calpain inhibitor leupeptin at the onset of MV
prevented atrophy and contractile impairment of the diaphragm
(28). In animal studies, calpain activation during MV was
recently reported to be dependent on the presence of increased
oxidative stress (29). Calpains-1 and -2 are ubiquitous and have
been extensively studied in skeletal muscle (15, 16, 30). Calpain3 seems to be specific to skeletal muscle, and mutations in
calpain-3 are responsible for limb girdle muscular dystrophy
type 2a (31). Although its normal physiologic function is still
being elucidated, calpain-3 is bound to titin within the sarcomeric apparatus, and is thus ideally located to participate in
sarcomeric disassembly processes (14). Calpain-3 has also been
reported to play a role in regulating the Nf-kB pathway (32).
The transcription factor Nf-kB is triggered by conditions
associated with skeletal muscle injury and increased oxidative
stress (33, 34), and has also been linked to activation of the
ubiquitin-proteasome system with attendant skeletal muscle
atrophy (15, 35, 36). Therefore, taken together with the histologic findings of progressive diaphragmatic injury and atrophy

over time, the previously mentioned biochemical changes are

likely to be involved in the loss of diaphragmatic force production that we observed in long-term MV patients.
Conclusions

In humans, the use of controlled MV is associated with a rapid

loss of diaphragmatic force-generating capacity and histobiochemical signs of diaphragmatic injury and atrophy. We postulate that these changes could play an important role in the
difficulties encountered in discontinuing ventilatory support in
many critically ill patients.
Author Disclosure: S.M. received sponsored grants from the association francxaise
contre les myopathies for $10,001$50,000, from projet hospitalier de recherche
clinique 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 France
S.A. for $10,001$50,000. S.J. does not have a financial relationship with
a 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 an
interest in the subject of this manuscript. B.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this
manuscript. G.C. does not have a financial relationship with a commercial entity
that has an interest in the subject of this manuscript. J.-P.B. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. H.B. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. P.C. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. C.K.R. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. M.S. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. V.S. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. A.M. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. X.C. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. D.M. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. A.L. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript. J.M. does not have
a financial relationship with a commercial entity that has an interest in the subject
of this manuscript. A.P. does not have a financial relationship with a commercial
entity that has an interest in the subject of this manuscript.
Acknowledgment: The authors are grateful to Chantal Cazevieille and Cecile
Sanchez for their technical assistance and interpretation of data concerning
ultrastructural evaluation.

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