Disfuncion Muscular Respiratoria

new england
The
journal of medicine
established in 1812 march 27, 2008 vol. 358 no. 13
Rapid Disuse Atrophy of Diaphragm Fibers in Mechanically

Ventilated Humans
Sanford Levine, M.D., Taitan Nguyen, B.S.E., Nyali Taylor, M.D., M.P.H., Michael E. Friscia, M.D.,
Murat T. Budak, M.D., Ph.D., Pamela Rothenberg, B.A., Jianliang Zhu, M.D., Rajeev Sachdeva, M.D.,
Seema Sonnad, Ph.D., Larry R. Kaiser, M.D., Neal A. Rubinstein, M.D., Ph.D., Scott K. Powers, Ph.D., Ed.D.,
and Joseph B. Shrager, M.D.
A BS T R AC T
Background
The combination of complete diaphragm inactivity and mechanical ventilation (for From the Department of Surgery (S.L.,
more than 18 hours) elicits disuse atrophy of myofibers in animals. We hypothe- T.N., N.T., M.E.F., M.T.B., P.R., J.Z., S.S.,
L.R.K., J.B.S.), the Department of Cell and
sized that the same may also occur in the human diaphragm. Developmental Biology (N.A.R.), and the
Pennsylvania Muscle Institute (S.L., M.T.B.,
Methods N.A.R., J.B.S.), University of Pennsylva-
nia; the Gift of Life Donor Program (S.L.);
We obtained biopsy specimens from the costal diaphragms of 14 brain-dead organ Medical Research, Surgical, and Labora-
donors before organ harvest (case subjects) and compared them with intraoperative tory Medicine Services, Department of
biopsy specimens from the diaphragms of 8 patients who were undergoing surgery Veterans Affairs Medical Center (S.L.,
T.N., R.S., J.B.S.) — all in Philadelphia;
for either benign lesions or localized lung cancer (control subjects). Case subjects and the Center for Exercise Science, Uni-
had diaphragmatic inactivity and underwent mechanical ventilation for 18 to 69 versity of Florida, Gainesville (S.K.P.).
hours; among control subjects diaphragmatic inactivity and mechanical ventilation Address reprint requests to Dr. Levine at
1495 Wesleys Run, Gladwyne, PA 19035,
were limited to 2 to 3 hours. We carried out histologic, biochemical, and gene-expres- or at sdlevine@mail.med.upenn.edu.
sion studies on these specimens.
N Engl J Med 2008;358:1327-35.
Copyright © 2008 Massachusetts Medical Society.
Results
As compared with diaphragm-biopsy specimens from controls, specimens from
case subjects showed decreased cross-sectional areas of slow-twitch and fast-twitch
fibers of 57% (P = 0.001) and 53% (P = 0.01), respectively, decreased glutathione
concentration of 23% (P = 0.01), increased active caspase-3 expression of 100%
(P = 0.05), a 200% higher ratio of atrogin-1 messenger RNA (mRNA) transcripts to
MBD4 (a housekeeping gene) (P = 0.002), and a 590% higher ratio of MuRF-1 mRNA
transcripts to MBD4 (P = 0.001).
Conclusions
The combination of 18 to 69 hours of complete diaphragmatic inactivity and me-
chanical ventilation results in marked atrophy of human diaphragm myofibers.
These findings are consistent with increased diaphragmatic proteolysis during in-
activity.
n engl j med 358;13 www.nejm.org march 27, 2008 1327

The New England Journal of Medicine
Downloaded from nejm.org on July 30, 2015. For personal use only. No other uses without permission.
Copyright © 2008 Massachusetts Medical Society. All rights reserved.
The n e w e ng l a n d j o u r na l of m e dic i n e
M
echanical ventilation is a criti- gen and stored at −80°C until used. Specimens
cal component of modern intensive care from case subjects were obtained before circula-
medicine, but the process of discontin- tory arrest or removal of any organs, and speci-
uing mechanical ventilation can be difficult.1,2 mens from control subjects were obtained during
Laboratory studies have shown that the combina- the surgery for their lung lesions. In addition, to
tion of diaphragmatic inactivity and mechanical determine whether our hypothesis regarding at-
ventilation for prolonged periods (more than 18 rophy was limited to the diaphragm or primary
hours) is associated with atrophy of myofibers in respiratory muscles, we obtained specimens of
the rat diaphragm.3-5 We hypothesized that simi- the pectoralis major muscle at the level of the
lar changes occur in the human diaphragm and third interspace in six subjects from each group.
that disuse atrophy of human diaphragm myofi- (These subjects were the only ones for whom ap-
bers could be a major contributor to the weaning propriate consent was obtained for these biop-
problems that occur in some of our patients. sies.) To avoid surgical trauma to this superficial
We evaluated the diaphragms of brain-dead muscle, these specimens were obtained immedi-
organ donors, who show respiratory-muscle in- ately after the skin incision and processed in the
activity and undergo mechanical ventilation for same manner as the diaphragm specimens.
prolonged periods, to determine whether disuse
atrophy of the diaphragm occurs in ventilated Measurements
humans. We compared intraoperative biopsy We carried out histologic, biochemical, and gene-
specimens obtained from the costal diaphragms expression measurements on diaphragm speci-
of 14 brain-dead organ donors before harvest mens. Only histologic data were obtained from
(case subjects) and compared them with intraop- the pectoralis specimens.
erative biopsy specimens obtained from the dia- We measured fiber-type proportions, fiber-
phragms of 8 patients who were undergoing type cross-sectional areas, and area fractions to
surgery for either benign lesions or stage 1 lung characterize fiber atrophy in diaphragm-biopsy
cancer (control subjects). Case subjects with specimens. We also measured the concentrations
diaphragmatic inactivity underwent mechanical of glutathione, active caspase-3, and procaspase-3.
ventilation for 18 to 69 hours, whereas in control Glutathione-concentration analyses were used to
subjects, the combination of diaphragm inac- assess the presence of oxidative stress6; we used
tivity and mechanical ventilation was limited to active caspase-3 and procaspase-3 as indicators
2 to 3 hours. of caspase activity. Active caspase is known to
dissociate proteins from the myofibrillar lattice,7
Me thods which is a critical step in muscle proteolysis.
We quantitatively assessed the number of mes-
Subjects senger RNA (mRNA) transcripts for atrogin-1 and
Our protocol for case subjects was approved by MuRF-1 relative to MBD4, a housekeeping gene,
the Gift of Life Donor Program (http://www. using real-time reverse-transcriptase polymerase
donors1.org), and our protocol for control sub- chain reaction.8 Atrogin-1 and MuRF-1 are ubiq-
jects was approved by the University of Pennsyl- uitin ligases that are key components of the
vania institutional review board; the protocols ubiquitin–proteasome pathway for proteolysis.7,9
appear in the Supplementary Appendix, available Histologic studies were carried out using pre-
with the full text of this article at www.nejm.org. viously described immunohistologic methods,10
All biopsy specimens were obtained with appro- and a minimum of 400 fibers were studied in
priate written informed consent. each specimen. Biochemical and gene-expression
studies were performed in triplicate on each
Biopsies specimen. Glutathione was measured using an
Full-thickness biopsy specimens (about 20 to enzyme-recycling assay kit (glutathione assay kit,
24 mm by 6 to 8 mm in size) were obtained from Cayman Chemicals). Caspase measurements were
the same region of the right anterior costal dia- conducted using sodium dodecyl sulfate–poly-
phragm in all case and control subjects, frozen acrylamide-gel electrophoresis, followed by im-
in isopentane after 3 to 5 minutes for length munoblotting with monoclonal antibodies specif
equilibration, and then transferred to liquid nitro- ic for the 32- and 17-kD fragments. We used the
1328 n engl j med 358;13 www.nejm.org march 27, 2008

Diaphr agm Disuse and Mechanical Ventilation
relative standard curve method to compute the areas of slow-twitch and fast-twitch fibers were
gene-expression level in each of our diaphragm 2025±745 and 1871±589 µm2, respectively, where-
samples.8 Complete details for all methods are as in control specimens these cross-sectional
available in the Supplementary Appendix. areas were 4725±1547 and 3949±1805 µm2, re
spectively. Therefore, in case specimens, the cross-
Statistics sectional area of slow-twitch fibers decreased
Means (±SD) and medians are presented for all 57% (P = 0.001) as compared with control values,
continuous data. Demographic, histologic, bio- and the cross-sectional area of fast-twitch fibers
chemical, and gene-expression variables were decreased 53% (P = 0.01) (Fig. 2A). Case and con-
compared between case and control groups using trol specimens did not differ with respect to the
t tests for normally distributed continuous data, numerical proportions or area fractions of slow-
Mann–Whitney tests for non-normally distribut- twitch and fast-twitch fibers (Fig. 2B and 2C).
ed data, and Fisher’s exact tests for categorical In addition, in an age-matched subgroup of five
variables.11,12 case and five control subjects, the cross-sectional
areas of both slow-twitch and fast-twitch fibers
R e sult s did not differ statistically from those of the full
groups; in this subgroup, slow-twitch and fast-
Characterization of Experimental Cohort twitch fibers in case specimens exhibited mean
Demographic information, reason for inclusion decreases in cross-sectional areas of 39% (P = 0.004)
in the study, and medical history for case and and 41% (P = 0.02), respectively, as compared with
control subjects are summarized in Table 1; ven- controls.
tilator settings, measurements of arterial blood
gases, and vital signs are summarized in Table 2. Biochemistry
Clinical data for each of the case subjects are Total glutathione concentration in diaphragm-
presented in the Supplementary Appendix in Ta- biopsy specimens from case subjects was
ble S1; Tables S2 and S3 contain histologic data 1.03±0.17 mM, whereas that in control specimens
for case and control subjects; and Table S4 con- was 1.35±0.21 mM; therefore, case specimens ex-
tains usual laboratory measurements. Case sub- hibited a decrease of 23% (P = 0.01) from that
jects were younger than control subjects (mean noted in controls (Fig. 3A). Figures 3B and 3C
age, 35±16 years vs. 57±18; P = 0.008). The two show immunoblots and a quantitative compari-
groups did not differ with respect to proportions son of case and control specimens with respect
of men and women or to body-mass index. After to the expression of the active 17-kD caspase-3
brain death, case subjects’ diaphragms were in- fragment and the 32-kD inactive procaspase frag-
active for 18 to 69 hours, whereas inactivity in ment. Figure 3C shows that active caspase-3 in
control subjects’ diaphragms was limited to 2 to case specimens had a value of 1.52±1.15 optical-
3 hours; the mean inactivity time for case sub- density units, whereas that in controls had a
jects’ diaphragms was appreciably greater — more value of 0.66±0.45 optical-density unit; therefore,
than 10 times that of control subjects’ diaphragms the diaphragm-biopsy specimens from case sub-
(2.4±0.5 vs. 34±16, P<0.001). jects showed an increase of 154% above controls
(P = 0.05). In addition, Figure 3C shows that pro-
Analysis of Diaphragm-Biopsy Specimens caspase in case specimens measured 0.72±0.40
Histology optical-density unit, whereas that in control speci-
A comparison of Figures 1A and 1B indicates mens was 1.13±0.51 optical-density units; this
that the fibers in the diaphragm-biopsy specimens higher value of procaspase in the control speci-
from case subjects were appreciably smaller than mens approached but did not reach statistical
those from control subjects. Figures 1C, 1D, 1E, significance (P = 0.07).
and 1F show that both slow-twitch and fast-twitch
fibers in the case specimens were affected by at- Gene Expression
rophy. Importantly, all panels in Figure 1 indicate Expression of MBD4 was used to normalize the
that fiber atrophy in case specimens was not ac- number of transcripts of atrogin-1 and MuRF-1
companied by an inflammatory-cell infiltrate. (the two ubiquitin ligases of interest), since its
In case specimens, the mean cross-sectional expression in the diaphragm-biopsy specimens

Table 1. Summary of Demographic Characteristics, Reason for Surgery, and Medical History for Control and Case Subjects.*
Subject Reason for Surgery or Cause

No. Age (yr) Sex BMI of Brain Death Relevant Medical History
Control subjects
1 79 M 31 Stage 1A adenocarcinoma of the lung Prostate carcinoma, nonsmoker, farmer
2 64 M 36 Stage 1A adenocarcinoma of the lung Peripheral arterial disease, rheumatoid arthritis, hyper-
tension, coronary artery disease, smoked 90 pack/yr
3 55 F 23 Stage 1A benign fatty tumor Hypercholesterolemia, osteoarthritis, smoked 10 pack/yr
4 76 M 27 Stage 1A adenocarcinoma of the lung Coronary artery disease with history of myocardial in-
farction, macular degeneration, prostate carcinoma
(radiation therapy, 1999), coronary-artery bypass
graft (1988), pipe smoker (quit 30 yr ago)
5 58 F 30 Stage 1 carcinoid tumor Hypercholesterolemia, primary hyperparathyroidism,
kidney stones, smoked 40 pack/yr
6 25 F 27 Ganglioneuroma Gallstones, nonsmoker
7 54 F 23 Ganglioneuroma Glaucoma, seasonal allergies, nonsmoker
8 41 F 26 Hamartoma Herniated lumbar disks, dysfunctional uterine bleed-
ing, smoked 24 pack/yr (quit 2 yr ago)
Case subjects
1 18 F 24 Motor vehicle accident None
2 21 F 29 Drug overdose Drug abuse
3 18 M 25 Gunshot wound to head None
4 19 M 24 Respiratory arrest secondary to seizure Seizure disorder with implanted pacemaker
5 49 M 24 Motor vehicle accident Hypertension, peptic ulcer disease, depression, hypo-
gonadism, smoker
6 33 F 44 Drug overdose Drug and ethyl alcohol abuse, metronidazole and
ceftriaxone for vaginitis
7 25 F 21 Motor vehicle accident Pregnant
8 50 M 21 Stroke Hypertension, ethyl alcohol abuse, smoked 30 pack/yr
9 23 M 20 Motor vehicle accident Hypertension, ethyl alcohol abuse, marijuana abuse
10 53 F 45 Stroke Hypertension, type 2 diabetes mellitus, gastresopha-
geal reflux disease, atrial fibrillation (new onset)
11 45 M 32 Stroke Hypertension, ethyl alcohol abuse, drug abuse
12 26 M 28 Cardiac arrest Seizure disorder
13 56 F 26 Stroke Smoked 80 pack/yr
14 58 F 36 Stroke Hypertension, chronic obstructive pulmonary disease,
hypothyroidism, schizoaffective disorder, bipolar
disorder, smoked 25 pack/yr, obesity, oral cortico-
steroid prescription
* All control subjects had normal values for spirometry. BMI denotes body-mass index (defined as the weight in kilograms divided by the
square of the height in meters).
from case and control subjects did not differ (data mens contained 885±294 ANCU. Therefore, the
not shown). Control specimens contained 72±19 case specimens showed 3.0 times as much ex-
arbitrary normalized copy units (ANCU) of atro- pression of atrogin-1 mRNA transcripts (P = 0.002)
gin-1, whereas case specimens contained 216±67 and 6.9 times as much expression of MuRF-1
ANCU. In addition, control specimens contained mRNA transcripts (P = 0.001) as control specimens
128±51 ANCU of MuRF-1, whereas case speci- (Fig. 4).

Table 2. Summary of Ventilator Settings, Arterial Blood Gas Measurements, and Vital Signs for Control and Case Subjects.*
Control Subjects Case Subjects

Measurement (N = 8) (N = 14) P Value
Ventilator settings and related measurements
Tidal volume (ml/kg of body weight) 7.5±1.3 8.0±2.0 0.47
Ventilation frequency (breaths/min) 11±1.7 14±3.0 0.02
PEEP (cm H2O) 0.0±0.0 6.0±1.0 <0.001
FiO2 (%) — 52±0.11 —
SaO2 (%) 99±2.0 — —
PaO2 (mm H2O) — 147±88 —
PetCO2 (mm Hg) 31±3.8 — —
PaCO2 (mm Hg) — 34±6.0 —
Arterial pH (units) — 7.39±0.05 —
PaO2/FiO2† — 412±167 —
Vital signs
Systolic pressure (mm Hg) 115±10 125±20 0.20
Diastolic pressure (mm Hg) 62±6.0 70±10 0.08
Heart rate (beats/min) 72±9.0 105±18 <0.001
Body temperature (°C) 35.7±0.3 36.4±1.1 0.06
* Plus–minus values are means ±SD. FiO2 denotes fractional concentration of inspired oxygen, PaCO2 arterial carbon di-
oxide pressure, PaO2 arterial oxygen pressure, PEEP positive end-expiratory pressure, PetCO2 end-tidal carbon dioxide
pressure, and SaO2 arterial oxygen saturation.
† These measurements were made at an FiO2 of 1.0.
Histology of Pectoralis Major–Biopsy the diaphragm. Muscle inactivity is known to ef-

Specimens fect oxidative stress and increase cytosolic calci-
The cross-sectional areas of slow-twitch and fast- um concentration, perturbations known to elicit
twitch fibers in the biopsy specimens from the increases in the activity of proteases (e.g., cas-
pectoralis major in case subjects were 3084±796 pases) that cause increased dissociation of the
and 2933±1343 µm2, respectively, whereas the myofibrillar lattice, the critical initial step in
cross-sectional areas of these fiber-types in the con proteolysis.7,16 In case subjects, the decrease in
trol specimens were 3325±1256 and 3418±1281 diaphragmatic glutathione concentration is con-
µm2, respectively. These data show that the pec- sistent with oxidative stress,6,17 and the increases
toralis fibers from case and control subjects did in active caspase-3 suggest an increased rate of
not differ with respect to cross-sectional area of protein release from the myofibrillar lattice.16
any fiber type (see Fig. S1 in the Supplementary After release from the lattice, the major route
Appendix). Likewise, these case and control speci- of proteolysis for muscle proteins is the ubiqui-
mens did not differ with respect to the numerical tin–proteasome pathway, which consists of the
proportion or area fractions of slow-twitch and following sequential steps: activation of the
fast-twitch fibers (Table S7 in the Supplementary small protein cofactor ubiquitin (76 amino acid
Appendix). residues), formation of activated ubiquitin-chain
moieties catalyzed by specific ubiquitin-conju-
Dis cus sion gase enzymes (i.e., E2 enzymes), attachment of
ubiquitin chains to specific proteins by ubiqui-
In our case subjects, the combination of 18 to 69 tin-ligase enzymes (i.e., E3 enzymes such as
hours of diaphragmatic inactivity and mechani- atrogin-1 and MuRF-1), and recognition of specif
cal ventilation was associated with marked atro- ic ubiquitin-protein chains by the 26S proteasome,
phy of both slow-twitch and fast-twitch fibers of followed by release of ubiquitin residues and deg-

Case (N=14) Control (N=8)

Case Control
A B A
6000
Fiber Cross-Sectional Area (µm2)

Fiber Size 5000
4000
50 µm 50 µm
3000
P=0.001
P=0.01
C D 2000
Slow Myosin 1000

Heavy Chain
0
50 µm 50 µm Slow-Twitch Fibers Fast-Twitch Fibers
E F B
100
Fast Myosin
Heavy Chain
80
Fiber Proportion (%)

50 µm 50 µm
60 P=0.14
P=0.11
Figure 1. Comparison of Representative Case and Control Diaphragm-Biopsy
AUTHOR: Levine RETAKE 1st 40
Specimens with ICMRespect to Fiber Size.
FIGURE: 1 of 4 2nd
The slow-twitch REGand
F fast-twitch fibers in the case specimens (Panels A, C,
3rd
20
and E) are smaller
CASE than those in the control diaphragmsRevised (Panels B, D, and F).
Panels A and BEMail Line 4-C
(hematoxylin and eosin) show that neitherSIZE inflammatory
ARTIST: ts H/T H/T specimens.
infiltrate nor necrosis
Enon is present in case or control 22p3 The sections 0
Combo Slow-Twitch Fibers Fast-Twitch Fibers
in Panels C and D were preincubated with NOQ7.5.4D antibody,10,13 which
AUTHOR, PLEASE NOTE:
is specific for the Figure
slow myosin heavy chain, whereas sections in Panels E
has been redrawn and type has10,14been reset. C
and F were preincubated withPlease the MY-32 antibody,
check carefully. which reacts with all
100
fast myosin heavy chains. In addition, in each section, all fibers are outlined
10,15 In each of the sections, fibers react-
by an antibody
JOB:reactive
35813 to laminin. ISSUE: 03-27-08
ing with the antibody appear orange-red, whereas fibers not reacting with 80
Fiber Area Fraction (%)
the antibody appear black. In Panels C, D, E, and F, a representative slow-

twitch fiber is indicated by an open circle and a fast-twitch fiber by an open 60 P=0.56
square. P=0.51
40
radation of proteins to small peptides (8 to 11
amino acid residues) by the 20S catalytic core of 20
the proteasome.18 In conditions characterized by

0
degradation of muscle protein, there is an up- Slow-Twitch Fibers Fast-Twitch Fibers
regulation of mRNAs coding for atrogin-1 and
MuRF-118,19; therefore, the marked increases in Figure 2. Group Comparisons of Case and Control
AUTHOR:Specimens
Diaphragm-Biopsy
ICM
Levine with Respect RETAKE
to 1st
these transcripts noted in the diaphragm-biopsy 2nd
REG F FIGURE:
Histologic Features.2 of 4
specimens from case subjects are consistent with BothCASE
the slow-twitch fibers and the fast-twitch
3rd
fibers in
Revised
increased proteolysis. the EMail
case specimens Line
are appreciably 4-C
smaller than those
SIZE
ARTIST: ts
Since our data are histologic or biochemical, in the control specimens (Panel
Enon
H/T A). However,
Combo
H/T 16p6
the case
we can only speculate on the functional signifi- and control specimens do not differ with respect to
cance of our findings. One report re-emphasized20 proportions
Figureof slow-
has beenand fast-twitch
redrawn and type fibers (Panel
has been reset. B),
nor do they differ with respect
Please check to area fractions of slow-
carefully.
the idea that weaning patients from ventilators and fast-twitch fibers (Panel C) (i.e., the proportion of
is closely linked to diaphragm force generation diaphragm-fiber
JOB: 35813 cross-sectional area accounted for by
ISSUE: 03-27-08
(usually assessed clinically as transdiaphragmatic slow- or fast-twitch fiber types).
pressure). The question then becomes how mea-

A 1200 Case (N=6) Control (N=6)

1.6
P=0.01
Ratio of mRNA Transcripts

900
Total Glutathione (mM)
Relative to MBD4
1.2 P=0.01
600
0.8
300 P=0.002
0.4
0
0 Atrogin-1:MBD4 MuRF-1:MBD4
Case Control
(N=11) (N=5) Figure 4. Group Comparisons of Case and Control
Diaphragm-Biopsy
ICM
AUTHOR:Specimens
Levine with Respect to mRNA
RETAKE 1st
B Expression for Atrogin-1
FIGURE: 4 of 4 and MuRF-1 Normalized 2nd
REG F
3rd
Molecular to aCASE
Housekeeping Gene. Revised
Weight Control Case
specimens have aboutLine
CaseEMail 4-C the ratio
three times of
SIZE
37 ARTIST: ts H/T H/T mRNA
Procaspase-3 atrogin-1
Enon to MBD4 (a housekeeping gene) 16p6tran-
Combo
scripts and about seven times the ratio of MuRF-1 to
25 AUTHOR,as
MBD4 mRNA transcripts PLEASE NOTE:with control
compared
Figure has been redrawn and type has been reset.
20 Active caspase-3 specimens. Please check carefully.
JOB: 35813 ISSUE: 03-27-08

C
Case (N=8) Control (N=5)
surements of fiber atrophy in our case subjects
P=0.05
relate to force generation. If the findings in our
2.0
samples occurred throughout the diaphragm, the
Caspase-3 Fragments (OD)
degree of atrophy we observed would predict an

1.5 approximately 55% decrease in transdiaphrag-
matic pressure (i.e., to 45% of control values).
1.0 P=0.07
Therefore, we believe that fiber atrophy of the
magnitude noted in case specimens could have
clinical significance.
0.5
There are limitations to our study. We recog-
nize that the decreases in fiber cross-sectional
0.0 area — noted in the diaphragm-biopsy specimens
32-kD 17-kD Active
Procaspase-3 Caspase-3 from case subjects — can be explained by either
artifactual increases in mean sarcomere length
Figure 3. Group Comparisons of Case and Control Dia- due to improper fixation or actual functional de-
phragm-Biopsy Specimens with Respect to Measure- creases in mean fiber volume. To distinguish
ments AUTHOR: Levine RETAKE
ICMof Glutathione, Active Caspase, and Procaspase.
1st
REG F FIGURE: 3 of 4 2nd between these possibilities, we used both longi-

Total glutathione concentration is lower in case speci- 3rd
mens than in control specimens (Panel A). Representa-
CASE Revised
tudinal and transverse sections of case speci-
tive EMail
immunoblots Line
and two4-C mens and determined a mean sarcomere length
ARTIST:oftstwo caseH/T control specimens
SIZE
H/T
(Panel
EnonB) indicate that these case specimens show
Combo
16p6great of 2.0±0.2 μm for both slow-twitch and fast-
er protein expression of 17-kD active caspase-3 and
twitch fibers. This value is very similar to that
slightly Figure
lower has
protein
beenexpression
redrawn andof 32-kD
type procaspase-3.
has been reset. noted for the diaphragm fibers from control sub-
The group comparisons Pleasein Panel
check C show that the great-
carefully.
er 17-kD active caspase-3 expression in case speci-
jects.21 On the basis of these observations, we
mens is statistically significant. In contrast,
JOB: 35813 the03-27-08
ISSUE: ten- conclude that the decrease in cross-sectional area
dency toward lower 32-kD procaspase-3 expression in of diaphragm fibers from case subjects could be
case specimens is not statistically significant. OD de- attributed entirely to atrophy.
notes optical density. Another limitation is that our case subjects
were younger than the control subjects. Although

the preferred method for analyzing this type of There are factors affecting fiber atrophy in
data is a matched-pair design,11 we lacked a suf- disuse states that could influence our results. Our
ficient number of subjects to allow matching for data do not elucidate the complex relationships
both age and sex. The results from our previous between conditions present in the diaphragms of
study22 as well as the present data strongly sug- case subjects — inactivity, level of phrenic moto
gest that sex is not a determinant of cross-sec- neuron activity, diaphragm muscle lengths, and
tional area in diaphragm fibers. Therefore, since perhaps additional factors — and the marked
we found no statistically significant differences atrophy of both slow-twitch and fast-twitch fibers
in cross-sectional area between the age-matched in the diaphragms. The orthopedic literature in-
(5 subjects) and full case (14 subjects) groups, we dicates that limb muscles that are used frequent-
reason that the difference in age between case ly show appreciably more inactivity-associated fi-
and control subjects does not account for the at- ber atrophy than muscles used less frequently.26
rophy of diaphragm fibers from case subjects. Because the diaphragm is active in most people
There are other possible causes of atrophy in 24 hours a day, one might expect it to show a
the diaphragm fibers from case subjects. One or greater rate of atrophy than limb muscles that
more of the following conditions may have played are rendered inactive by interventions such as spi
some role in eliciting the atrophy: systemic in- nal cord injury,27 microgravity,28 or bed rest.29
flammatory response syndrome (SIRS) or sep- In summary, our study indicates that the com-
sis,23 barotraum–volutrauma,24 brain death,25 or bination of 18 to 69 hours of diaphragm inactiv-
unmeasured humoral substances. Current con- ity and mechanical ventilation is associated with
cepts suggest that diaphragm atrophy associated marked atrophy of both slow-twitch and fast-
with SIRS, sepsis, or barotrauma–volutrauma twitch fibers in the human diaphragm. Since our
should be associated with an inflammatory-cell observations strongly suggest that increased pro-
infiltrate or increased proinflammatory cyto- teolysis accounts for the fiber atrophy noted in
kines. Neither of these findings, however, was the diaphragm-biopsy specimens from case sub-
evident in the diaphragm fibers from case subjects, we speculate that blocking or attenuating
jects (Fig. 1A, and Table S5 in the Supplementary diaphragm proteolytic pathways in patients on
Appendix). mechanical ventilation might mitigate the wean-
To assess the possibility that noncytokine ing problems that occur in some patients.
humoral substances or other factors associated
Supported by a grant (R01-HL-078834) from the National
with brain death accounted for the diaphragm- Heart, Lung, and Blood Institute (to Dr. Levine) and a grant
fiber atrophy, we compared biopsy specimens from the Department of Veterans Affairs Merit Review Program
from the pectoralis major muscle of six case sub- (to Dr. Shrager).
No potential conflict of interest relevant to this article was
jects and six control subjects. The specimens from reported.
the two groups did not differ with respect to We thank the control subjects and the families of the case
cross-sectional area of either slow-twitch or fast- subjects for providing consent for biopsies, the Gift of Life trans-
plant coordinators (especially Chris Hainsworth and Chris Walsh)
twitch fibers (Fig. S1 in the Supplementary Ap- for presenting our need for research biopsies in a compassionate
pendix). These observations suggest that neither and comprehensible manner to the bereaved families of our case
brain death nor unmeasured humoral factors subjects, and Drs. Greg Lipschick, Gerald Supinski, Michael Reid,
Clara Franzini-Armstrong, Peter McCombs, Clyde F. Barker, Al-
played a role in effecting fiber atrophy in case fred P. Fishman, Peter T. Macklem, Joel D. Cooper, and Sean
subjects’ diaphragms. Levine for their help in completing this work.
References
1. Esteban A, Frutos F, Tobin MJ, et al. oxidative injury and increased proteolytic ren DL, Deering MA, Kavazis AN, Powers
A comparison of four methods of wean- activity. Am J Respir Crit Care Med 2002; SK. Mechanical ventilation promotes re-
ing patients from mechanical ventilation. 166:1369-74. dox status alterations in the diaphragm.
N Engl J Med 1995;332:345-50. 4. Vassilakopoulos T, Petrof BJ. Ventila- J Appl Physiol 2006;101:1017-24.
2. MacIntyre NR, Epstein SK, Carson S, tor-induced diaphragmatic dysfunction. 7. Powers SK, Kavazis AN, DeRuisseau
Scheinhorn D, Christopher K, Muldoon S. Am J Respir Crit Care Med 2004;169:336- KC. Mechanisms of disuse muscle atro-
Management of patients requiring pro- 41. phy: role of oxidative stress. Am J Physiol
longed mechanical ventilation: report of 5. Gayan-Ramirez G, Testelmans D, Maes Regul Integr Comp Physiol 2005;288:
a NAMDRC consensus conference. Chest K, et al. Intermittent spontaneous breath- R337-R344.
2005;128:3937-54. ing protects the rat diaphragm from me- 8. Larionov A, Krause A, Miller W.
3. Shanely RA, Zergeroglu MA, Lennon chanical ventilation effects. Crit Care Med A standard curve based method for rela-
SL, et al. Mechanical ventilation-induced 2005;33:2804-9. tive real time PCR data processing. BMC
diaphragmatic atrophy is associated with 6. Falk DJ, Deruisseau KC, Van Gamme- Bioinformatics 2005;6:62.

9. Reid MB. Response of the ubiquitin- catabolic conditions. J Clin Invest 2004; 24. Belperio JA, Keane MP, Lynch JP III,
proteasome pathway to changes in muscle 113:115-23. Strieter RM. The role of cytokines during
activity. Am J Physiol Regul Integr Comp 17. Lawler JM, Powers SK. Oxidative stress, the pathogenesis of ventilator-associated
Physiol 2005;288:R1423-R1431. antioxidant status, and the contracting dia- and ventilator-induced lung injury. Semin
10. Levine S, Nguyen T, Friscia M, et al. phragm. Can J Appl Physiol 1998;23:23-55. Respir Crit Care Med 2006;27:350-64.
Parasternal intercostal muscle remodeling 18. Mitch WE, Goldberg AL. Mechanisms 25. Amado JA, Lopez-Espadas F, Vazquez-
in severe chronic obstructive pulmonary of muscle wasting: the role of the ubiqui- Barquero A, et al. Blood levels of cytokines
disease. J Appl Physiol 2006;101:1297- tin-proteasome pathway. N Engl J Med in brain-dead patients: relationship with
302. 1996;335:1897-905. circulating hormones and acute-phase re-
11. Dawson B, Trapp R. Basic and clinical 19. Sacheck JM, Hyatt JP, Raffaello A, et al. actants. Metabolism 1995;44:812-6.
biostatistics. 4th ed. New York: McGraw- Rapid disuse and denervation atrophy in- 26. Hudson NJ, Franklin CE. Maintaining
Hill, 2004. volve transcriptional changes similar to muscle mass during extended disuse: aes-
12. Conover W. Practical nonparametric those of muscle wasting during systemic tivating frogs as a model species. J Exp
statistics. New York: John Wiley, 1999. diseases. FASEB J 2007;21:140-55. Biol 2002;205:2297-303.
13. Narusawa M, Fitzsimons RB, Izumo 20. Laghi F, Cattapan SE, Jubran A, et al. 27. Castro MJ, Apple DF Jr, Staron RS,
S, Nadal-Ginard B, Rubinstein NA, Kelly Is weaning failure caused by low-frequen- Campos GE, Dudley GA. Influence of
AM. Slow myosin in developing rat skele- cy fatigue of the diaphragm? Am J Respir complete spinal cord injury on skeletal
tal muscle. J Cell Biol 1987;104:447-59. Crit Care Med 2003;167:120-7. muscle within 6 mo of injury. J Appl Phy
14. Naumann K, Pette D. Effects of 21. Moore AJ, Stubbings A, Swallow EB, siol 1999;86:350-8.
chronic stimulation with different im- et al. Passive properties of the diaphragm 28. Adams GR, Caiozzo VJ, Baldwin KM.
pulse patterns on the expression of myo- in COPD. J Appl Physiol 2006;101:1400-5. Skeletal muscle unweighting: spaceflight
sin isoforms in rat myotube cultures. Dif- 22. Levine S, Nguyen T, Kaiser LR, et al. and ground-based models. J Appl Physiol
ferentiation 1994;55:203-11. Human diaphragm remodeling associat- 2003;95:2185-201.
15. Vater R, Cullen MJ, Harris JB. The ex- ed with chronic obstructive pulmonary 29. Ohira Y, Yoshinaga T, Nonaka I, et al.
pression of vimentin in satellite cells of disease: clinical implications. Am J Respir Histochemical responses of human soleus
regenerating skeletal muscle in vivo. Histo Crit Care Med 2003;168:706-13. muscle fibers to long-term bedrest with
chem J 1994;26:916-28. 23. Bone RC, Balk RA, Cerra FB, et al. Def- or without countermeasures. Jpn J Physiol
16. Du J, Wang X, Miereles C, et al. Acti- initions for sepsis and organ failure and 2000;50:41-7.
vation of caspase-3 is an initial step trig- guidelines for the use of innovative thera- Copyright © 2008 Massachusetts Medical Society.
gering accelerated muscle proteolysis in pies in sepsis. Chest 1992;101:1644-55.
journal editorial fellow

The Journal’s editorial office invites applications for a one-year
research fellowship beginning in July 2009 from individuals at any
stage of training. The editorial fellow will work on Journal projects
and will participate in the day-to-day editorial activities of the Journal
but is expected in addition to have his or her own independent
projects. Please send curriculum vitae and research interests
to the Editor-in-Chief, 10 Shattuck St., Boston, MA 02115
(fax, 617-739-9864), by September 30, 2008.


Disfuncion Muscular Respiratoria

Uploaded by

Copyright:

Available Formats

Disfuncion Muscular Respiratoria

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Disfuncion Muscular Respiratoria

Uploaded by

Copyright:

Available Formats

new england

Rapid Disuse Atrophy of Diaphragm Fibers in Mechanically

n engl j med 358;13 www.nejm.org march 27, 2008 1327

1328 n engl j med 358;13 www.nejm.org march 27, 2008

The New England Journal of Medicine

n engl j med 358;13 www.nejm.org march 27, 2008 1329

Subject Reason for Surgery or Cause

1330 n engl j med 358;13 www.nejm.org march 27, 2008

The New England Journal of Medicine

Control Subjects Case Subjects

Histology of Pectoralis Major–Biopsy the diaphragm. Muscle inactivity is known to ef-

n engl j med 358;13 www.nejm.org march 27, 2008 1331

Case (N=14) Control (N=8)

Fiber Cross-Sectional Area (µm2)

Slow Myosin 1000

Fiber Proportion (%)

the antibody appear black. In Panels C, D, E, and F, a representative slow-

the proteasome.18 In conditions characterized by

1332 n engl j med 358;13 www.nejm.org march 27, 2008

The New England Journal of Medicine

A 1200 Case (N=6) Control (N=6)

Ratio of mRNA Transcripts

JOB: 35813 ISSUE: 03-27-08

degree of atrophy we observed would predict an

REG F FIGURE: 3 of 4 2nd between these possibilities, we used both longi-

n engl j med 358;13 www.nejm.org march 27, 2008 1333

1334 n engl j med 358;13 www.nejm.org march 27, 2008

The New England Journal of Medicine

journal editorial fellow

n engl j med 358;13 www.nejm.org march 27, 2008 1335

You might also like