Rapidly Progressive Diaphragmatic Weakness and ... - ATS Journals

Rapidly Progressive Diaphragmatic Weakness and Injury during Mechanical Ventilation in Humans Samir Jaber1,2,6, Basil J. Petrof 3, Boris Jung1,2, Ge´rald Chanques1,2, Jean-Philippe Berthet 4, Christophe Rabuel5, Hassan Bouyabrine6, Patricia Courouble1,2, Christelle Koechlin-Ramonatxo7, Mustapha Sebbane1,2, Thomas Similowski8, Vale´rie 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 Re´gion et l’Institut National de la Sante´ et de la Recherche Me´dicale (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 ˆ pital Lariboisie`re, Universite´ Paris Diderot, Equipe INSERM U 942, Paris, France; 7INRA, Universite´ Montpellier I, Montpellier, France; Medicine, Ho 8 ˆ pitaux de Paris, and ER10 Universite´ Service de Pneumologie et Re´animation Me´dicale, Groupe Hospitalier Pitie´-Salpeˆtrie`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 2–3 h; n 5 10) and from brain-dead organ donors (MV for 24–249 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 Que´bec. 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

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 (3–9), 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 (13–17). 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 1–2 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 2–3 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 24–36 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

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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 (13–17).

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 3–4 days of MV. By the end of the evaluation period at 5–6 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 3A–3D 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 4A–4F). 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.


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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

M

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 (13–17). 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 5–6 days. In addition, the fact that ‘‘baseline’’ TwPtr values (obtained at a mean of 12.5 6 7.5 h


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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. (C–F) 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.

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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 (13–17). 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 Ce´cile Sanchez for their technical assistance and interpretation of data concerning ultrastructural evaluation.

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