Positive End-Expiratory Pressure Ventilation Induces Longitudinal Atrophy in Diaphragm Fibers

AJRCCM Articles in Press. Published on 26-March-2018 as 10.1164/rccm.
201709-1917OC
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POSITIVE END-EXPIRATORY PRESSURE VENTILATION INDUCES

LONGITUDINAL ATROPHY IN DIAPHRAGM FIBERS
Johan Lindqvist, PhD7*; Marloes van den Berg, MD1*; Robbert van der Pijl1,7; Pleuni E.
Hooijman, PhD1; Albertus Beishuizen, MD, PhD5; Judith Elshof, B.Sc3; Monique de
Waard, PhD3; Armand Girbes, MD, PhD3; Angelique Spoelstra-de Man, MD, PhD3;
Zhong-Hua Shi, MD, PhD6; Charissa van den Brom, PhD2; Sylvia Bogaards1; Shengyi
Shen, PhD7; Joshua Strom, PhD7; Henk Granzier, PhD7; Jeroen Kole1; René J.P.
Musters, PhD1; Marinus A. Paul, MD, PhD4; Leo M.A. Heunks, MD, PhD3; and Coen
A.C. Ottenheijm, PhD1,7
1
Dept of Physiology, 2Dept of Anesthesiology; 3Dept of Intensive Care; 4Dept of
Cardiothoracic Surgery, VU University Medical Center, Amsterdam, the Netherlands;
5
Dept of Intensive Care, Medisch Spectrum Twente, Enschede, the Netherlands; 6Dept
of Critical Care Medicine, Beijing Tiantan Hospital, Capital Medical University, Beijing,
PR China; 7Cellular and Molecular Medicine, University of Arizona, Tucson, USA
*: both authors contributed equally
Corresponding Author:
Coen A.C. Ottenheijm, PhD, Cellular and Molecular Medicine, University of Arizona,
Tucson, USA; Phone: 520-626-4198; E-mail: coeno@email.arizona.edu
Author’s contributions:
Study design: JL, AB, MAP, LMAH, CACO. Study coordination: JL, MB, LMAH, CACO.
Patient inclusion: AB, MCW, MAP, AG. Biopsy obtaining: MAP. Data collection and
analysis: JL, MB, PEH, SS, JS, CB, ZHS, RP, JE (biopsy handling, contractility
experiments, animal experiments), MB, PEH, SB, JK, RM (biopsy handling, contractility-
, STED experiments), Data interpretation: JL, MB, PEH, MAP, LMAH, AB, CACO.
Drafting the article: JL, MB, LMAH, CACO. Figures: MB, PEH. Critical revision and final
Copyright © 2018 by the American Thoracic Society

AJRCCM Articles in Press. Published on 26-March-2018 as 10.1164/rccm.201709-1917OC
Page 2 of 55
approval of the draft to be published: JL, MB, PEH, AB, MCW, AG, JE, ASM, MAP,
ZHS, CB, SS, HG, JS, RP, JK, RJPM, SB, LMAH, CACO.
Conflicts of interest: None of the authors has a financial relationship with a

commercial entity that has an interest in the subject of this manuscript.
Sources of support: Coen A. C. Ottenheijm was supported by National Heart, Lung,

and Blood Institute (NHLBI) Grant HL-121500; H. Granzier was supported by
R01AR053897.
Running head: Longitudinal atrophy of the diaphragm in ICU
Descriptor number: 4.08 Mechanical Ventilation: Physiology & Pathophysiology
Total word count: 3500
This article has an online data supplement, which is accessible from this issue's table of
content online at www.atsjournals.org

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AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject: Diaphragm weakness is highly prevalent in
critically ill patients and contributes to weaning failure. The mechanisms underlying
diaphragm weakness include reduced fiber thickness (i.e. cross-sectional atrophy) and
impaired contractile function of the remaining fibers. Disuse is a recognized risk factor
for critical illness associated diaphragm weakness. However, the effects of positive end-
expiratory pressure (PEEP) are unknown.
What This Study Adds to the Field:
This study demonstrates that in critically ill patients, mechanical ventilation with PEEP
results in reduced diaphragm fiber length due to a loss of contractile units in series, i.e.
longitudinal atrophy. Consequently, diaphragm fibers are at a mechanical disadvantage
when PEEP is released. In experimental models we have identified specific molecular
pathways, including titin-based mechanosensing, that are involved in the development
of longitudinal atrophy.

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ABSTRACT
RATIONALE: Diaphragm weakness in critically ill patients prolongs ventilator
dependency and duration of hospital stay, and increases mortality and health care
costs. The mechanisms underlying diaphragm weakness include cross-sectional fiber
atrophy and contractile protein dysfunction, but whether additional mechanisms are at
play is unknown. OBJECTIVES: To test the hypothesis that mechanical ventilation with
positive end-expiratory pressure (PEEP) induces longitudinal atrophy by displacing the
diaphragm in caudal direction and reducing the length of fibers. METHODS: We studied
structure and function of diaphragm fibers of mechanically ventilated critically ill
patients, and mechanically ventilated rats with normal and increased titin compliance.
MEASUREMENTS AND MAIN RESULTS: (1) PEEP causes a caudal movement of the
diaphragm, both in critically ill patients and in rats, and this caudal movement reduces
fiber length; (2) diaphragm fibers of 18h mechanically ventilated rats (PEEP: 2.5
cmH2O) adapt to the reduced length by absorbing serially-linked sarcomeres, the
smallest contractile units in muscle (i.e. longitudinal atrophy); (3) increasing the
compliance of titin molecules reduces longitudinal atrophy. CONCLUSION: Mechanical
ventilation with PEEP results in longitudinal atrophy of diaphragm fibers, a response
which is modulated by the elasticity of the giant sarcomeric protein titin. We postulate
that longitudinal atrophy, in concert with the aforementioned cross-sectional atrophy,
hampers spontaneous breathing trials in critically ill patients: during these efforts end-
expiratory lung volume is reduced, and the shortened diaphragm fibers are stretched to
excessive sarcomere lengths. At these lengths, muscle fibers generate less force and
diaphragm weakness ensues.

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INTRODUCTION
Critically ill patients admitted to the intensive care unit rapidly develop diaphragm
muscle weakness1-6. Diaphragm weakness contributes to weaning failure and is
associated with increased mortality rates6-9. For establishing strategies to prevent or
treat diaphragm weakness, a fundamental understanding of the underlying cellular
mechanisms is crucial. Studies in mechanically ventilated brain dead organ donors
suggest that reduced cross-sectional area of diaphragm fibers caused by activation of
proteolytic pathways plays an important role10-12. Using biopsy specimens from critically
ill patients, we recently established that the contractility of individual diaphragm muscle
fibers is severely impaired13-15, partly accounted for by a reduced cross-sectional area
of fibers13. A mechanism that has been largely neglected is the effect of mechanical
ventilation on diaphragm fiber length reduction, from here on referred to as longitudinal
fiber atrophy (as opposed to the aforementioned cross-sectional fiber atrophy).
Typically, critically ill patients are ventilated with positive end-expiratory pressure
(PEEP) to limit alveolar collapse. However, PEEP-induced increase of end-expiratory
lung volume may flatten the shape of the diaphragm dome resulting in structural
modifications in the diaphragm fibers.
We hypothesized that: (1) the PEEP-induced increase in end-expiratory lung volume
flattens the diaphragm dome, and forces the muscle fibers to act at a shorter length
during the respiratory cycle; (2) the shorter fiber length causes structural adaptations in
muscle fibers, either by loss of the number of sarcomeres (the smallest contractile units)
in series to maintain optimal overlap of the myosin-based thick and actin-based thin
filaments or by reducing the length of the thick and/or thin filaments; (3) diaphragm fiber
length adaptations are modulated by titin: a giant protein that spans the length of the

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half sarcomere, forming a contiguous filament along the myofibril, and functions as a
molecular spring that develops tension when sarcomere length changes. This layout of
titin makes it ideally suited to sense changes in diaphragm length during mechanical
ventilation with PEEP16.
To test these hypotheses, we studied muscle fiber structure and contractility in
diaphragm biopsies of mechanically ventilated critically ill patients, and in mechanically
ventilated rats with and without a mutation in the titin splice factor Rbm20 that results in
a more compliant titin molecule17.
METHODS
Additional details are in the online data supplement.
Patients
Ultrasound: Ultrasound was performed in critically ill patients (n=15, Table 1) with
clinical indication for analysis of diaphragm function. M-mode ultrasound images were
acquired while PEEP was acutely reduced with either five or ten cmH2O within one
breath cycle. From the M-mode images obtained during subsequent breaths (Fig.1A),
the caudal-cranial displacement of the diaphragm dome was calculated.
Biopsies: Diaphragm biopsies were obtained from mechanically ventilated critically ill
patients undergoing abdominal or thoracic surgery for clinical indication (critically ill
group; n=12, Table 3). As a control group, diaphragm muscle biopsies were obtained
from patients undergoing tumor removal of a suspected early-stage lung malignancy
(n=12, Table 2). Exclusion criteria are in the online data supplement. The protocol was

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approved by the Medical Ethics Committee of the VU University Medical Center. Written
informed consent was obtained from each patient and/or legal representative.
Biopsy handling, contractility of single fibers, and microscopy: As described in the online
data supplement13;15;18-21;18;22-26.
Rats
Long-term mechanical ventilation and contractility of intact diaphragm strips: Rats
(n=12) were mechanically ventilated for eighteen hours. For details, see online data
supplement13;27. To test the effect of titin-based mechanosensing on diaphragm
remodeling, we utilized rats harboring a mutation in Rbm20 causing expression of a
giant titin isoform17;28. For details see online data supplement29;30-32.
Acute effect of PEEP on diaphragm position: In a subset of ventilated rats, ultrasound
was applied to visualize the effect of PEEP on diaphragm length. For details, see online
data supplement
Statistical analyses
For details, see online supplement. Differences between groups were considered
significant if P<0.05.
RESULTS
Patient characteristics
Patient characteristics are shown in Tables 1-4. Pulmonary function data of control
subjects are described in Table E1 (online data supplement).

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Acute effect of mechanical ventilation with PEEP on diaphragm position and fiber
length in critically ill patients and rats.
To establish the acute effect of PEEP on the position of the diaphragm, we used
ultrasound to determine diaphragm displacement in critically ill patients caused by a
decrease in PEEP (∆PEEP) of 5 cmH2O or 10 cmH2O. Figure 1A (left and middle
panels) show typical B- and M-mode ultrasound images in which the cranial
displacement of the diaphragm during ∆PEEP is visualized. Acute release of 5 cmH2O
PEEP caused a cranial diaphragm displacement in all five patients, with an average
displacement of 0.40±0.10 cm (Fig.1A, right panel). Acute ∆PEEP of 10 cmH2O caused
a cranial diaphragm displacement in all ten patients, with an average displacement of
0.89±0.17 cm (Fig.1A, right panel).
Investigating whether mechanical ventilation with PEEP, and the concomitant caudal
movement of the diaphragm shortens diaphragm fibers requires the isolation of whole-
length (central tendon to rib cage) muscle fibers. This is impossible to establish in
patients as biopsies do not contain whole-length fibers, but is feasible in rats. Thus, we
first determined the effect of mechanical ventilation with PEEP (2, 3 and 5 cmH2O) on
diaphragm length in rats using ultrasonography. Figure 1B shows a typical B- (left) and
M-mode (middle) ultrasound image, in which the acute effect of 3 cmH2O PEEP is
visualized in M-mode. Similar as in patients, PEEP caused a caudal movement of the
diaphragm (0.24mm/cmH2O, Fig.1B, right).

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Next, we determined whether the caudal displacement shortens diaphragm fibers by
perfusing rats with fixative immediately after the start of PEEP ventilation (2.5 cmH2O)
and during unassisted breathing (control). Arrest of diaphragm movement was complete
within ten seconds after start of fixation. At arrest of diaphragm movement, the phase of
the respiratory cycle was not possible to determine, but based on ultrasound
observations in mice (unpublished data) we anticipate that arrest occurred at end-
expiration. The in vivo fixated diaphragm was excised from the rats and whole length
(central tendon to rib cage) fibers were isolated from the mid-costal region (note that
diaphragm fibers were isolated from the same location in each rat; Fig.1C). The length
of these fibers was significantly shorter in rats receiving PEEP ventilation (MV vs.
control: 18.0±0.5 vs. 19.9±0.4 mm; Fig.1D), which was reflected by a reduced
sarcomere length in the fibers (MV vs. control: 2.64±0.06 vs. 2.85±0.07 µm, Fig.1E).
Thus, PEEP ventilation in rats results in a caudal displacement of the diaphragm, with a
concomitant reduction of fiber and sarcomere length.
Long-term effects of mechanical ventilation with PEEP on diaphragm fiber length
in critically ill patients and rats.
Length adaptations in diaphragm fibers of critically ill patients.
The average duration of mechanical ventilation in the critically ill patients prior to biopsy
was 133±38 hours (range: 17-435 hours; Tables 3 and 4), and the average PEEP in the
critically ill patients was 9.3±1.0 cmH2O (range: 6±0.7-12±1.7 cmH20; Table 4). To study
whether PEEP and the concomitant shortening of the diaphragm induced length
adaptations (i.e. structural changes in diaphragm fibers), we first applied a functional

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10
assay to determine thick and thin filament length in diaphragm fibers. Permeabilized
individual muscle fibers were maximally activated at incremental sarcomere lengths and
the generated force was determined. Force at incremental sarcomere lengths was fitted
using a 2nd order polynomial19, which yielded three parameters that describe the force-
sarcomere length relation: (1) the sarcomere length at which maximum force is
generated (SLopt), (2) the sarcomere length at which 50% of maximum force is
generated (SL50) and (3) the sarcomere length at which the fit crosses the X-axis, thus
no force is generated (SLmax, Fig 2A). Figure 2B shows that the average force-
sarcomere length relation (with force normalized to its maximum) of the critically ill
patients overlaps with that of the control patients. Figures 2C-E show that SLopt, SL50
and SLmax are comparable between critically ill and control patients (critically ill vs.
control, SLopt: 2.66±0.12 vs. 2.68±0.12 µm; SL50: 3.68±0.11 vs. 3.70±0.10 µm; SLmax:
4.10±0.11 vs. 4.12±0.12 µm). Thus, the sarcomere length-dependence of force is
unaffected in muscle fibers of critically ill patients, suggesting that the length of the thin
and thick filaments is not shortened.
Next, we directly measured the length of the thin and thick filaments. Thin and thick
filament length were measured by stimulated emission depletion (STED)
superresolution microscopy. An example of phalloidin-stained thin filaments is shown in
Fig.3A. Average thin filament length was comparable between fibers of critically ill and
control patients (critically ill vs. control: 1.28±0.03 vs. 1.26±0.05 µm). An example of
myosin heavy chain-stained thick filaments is shown in Fig.3B. Average thick filament
length was comparable between fibers of critically ill and control patients (critically ill vs.
control: 1.79±0.04 vs. 1.86±0.03 µm). Thus, microscopy analyses confirm that thin and

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thick filament lengths are not shorter in diaphragm fibers of critically ill patients
compared to those of control patients. Determining the number of sarcomeres in series
in the diaphragm of patients is not feasible (this would require biopsies spanning the
length from central tendon to rib cage), and therefore we performed these studies in rats
that were mechanically ventilated for 18 hours
Length adaptations in diaphragm fibers of 18h mechanically ventilated rats.
Rats were mechanically ventilated for 18 hours with a positive end-expiratory pressure
of 2.5 cmH2O. First, we validated the model and determined whether MV with PEEP
had an effect on the contractile properties of the diaphragm. Mid-costal diaphragm strips
(isolated from the same location as for the determination of sarcomere length, Fig.1C)
were isolated and electrically activated (schematic, Fig.4A). Similar to previous studies
33-36
, maximal tension was decreased after 18h of MV (MV vs. control: 176±12 vs.
278±11 mN/mm2; Fig.4B). Importantly, the diaphragm muscle strip length for maximal
force production (optimal length, MLopt, Fig.4A) was significantly reduced, by 12%, in
ventilated rats (MV vs. control: 2.05±0.36 vs. 2.33±0.02 cm; Fig.4C). Thus, in rats, 18
hours MV with PEEP causes contractile weakness with the maximal force generated at
a shorter muscle length.
Next, we determined whether the reduction of MLopt was a reflection of a change in thick
and/or thin filament length. Individual, permeabilized muscle fibers were maximally
activated at incremental sarcomere lengths. Fig.5A (top) shows that the average force-
sarcomere length relation of fibers of the MV-rats overlaps with that of fibers of control

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rats; SLopt (Fig.5A, bottom), SL50, and SLmax were comparable between MV and control
rats (MV vs. control, SLopt: 2.89±0.41 vs. 2.81±0.13 µm; SL50: 3.69±0.04 vs. 3.63±0.03
µm; SLmax: 4.01±0.05 vs. 3.97±0.04 µm). To confirm that the length of the thin and thick
filaments is not shorter in diaphragm fibers of the rats that received 18 hours MV, we
measured thin and thick filament length by STED superresolution microscopy.
Examples of phalloidin-stained thin filaments and myosin heavy chain-stained thick
filaments are shown in Figure 5B&C. Average thin filament length (MV vs control:
1.27±0.02 vs. 1.29±0.03 µm, Fig.5B), and average thick filament length (MV vs. control:
1.75±0.02 vs. 1.74±0.03 µm, Fig.5C) were comparable between fibers of MV and
control rats. Thus, similar to the findings in critically ill patients, the length of the thin and
thick filaments is not reduced in diaphragm fibers of rats that received 18 hours of MV
with PEEP.
To determine whether the reduced MLopt in MV rats was caused by a reduction in the
number of sarcomeres in series, diaphragm fibers were isolated with the excised fibers
spanning the whole distance from the central tendon to the ribs (Fig.1C, illustrated by
line X). The length of these fibers was divided by the length of the sarcomeres, as
determined by α-actinin stainings (Fig.5D), to render the number of sarcomeres in
series. Importantly, the number of sarcomeres in series was reduced by 12% in rats
receiving MV compared to control animals (MV vs. control: 5488±128 vs. 6250±170
sarcomeres; Fig.5E). Thus, 18 hours of mechanical ventilation with PEEP causes
longitudinal atrophy of diaphragm muscle fibers, due to absorption of sarcomeres in
series.

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13
Effect of compliant titin on diaphragm fiber length remodeling during MV.
We tested the hypothesis that low titin stiffness, by preconditioning the diaphragm to
reduced titin-based mechanosensing, blunts the longitudinal atrophy response during
diaphragm shortening caused by mechanical ventilation with PEEP. We studied rats
with a mutation in the titin splicing factor Rbm2017;28, resulting in a more compliant titin
molecule (the sarcomeric location of titin is shown in Fig.6A). As shown in Fig.6B,
diaphragm fibers of Rbm20-deficient rats express a titin isoform that is larger than in
fibers of wildtype (wt) rats. By co-electrophoresing diaphragm samples of wt or Rbm20-
deficient rats with titin isoforms of known sizes (see methods) we estimated that the
difference in titin isoform size is ~110 kDa. To verify that this larger isoform results in
lower titin-based passive tension, individual diaphragm fibers were passively stretched:
Fig.6C shows that, as expected, fibers of Rbm20-deficient rats generate lower passive
tension than fibers from wildtype rats. Subsequently, these rats were mechanically
ventilated with 2.5 cmH20 PEEP for 18 hours. Mid-costal diaphragm strips were isolated
and electrically activated. Maximal tension was reduced after 18 hours MV (298±13 vs.
189±9 mN/mm2, con vs. MV, respectively), but this decrease was less pronounced in
rats with the compliant titin isoform (241±7 vs. 215±7 mN/mm2, con vs. MV respectively;
tension decrease after MV, wt vs. Rbm20-def: 37±3% vs. 11±3%; p<0.001; Fig.6D).
Furthermore, the MLopt of diaphragm strips from wt rats was reduced after MV
(2.39±0.02 vs. 2.03±0.07 cm, con vs. MV, respectively), and this reduction was smaller
and not significant in Rbm20-deficient rats that had received mechanical ventilation
(2.24±0.06 vs. 2.22±0.10 cm, con vs. MV, respectively; MLopt decrease after MV, wt vs.

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Rbm20-def: 15±3% vs. 1±4%; Fig.6E). Finally, the number of sarcomeres in series was
not significantly reduced by 18h of MV in the Rbm20-deficient rats (7155±173 vs.
7005±317, con vs. MV, respectively; p=0.69; Fig.6F), whereas this reduction was
significant in wt rats (6255±170 vs. 5488±117, con vs. MV, respectively; sarcomere
number decrease after MV, wt vs. Rbm20-def: 12±2% vs. 2±4%).
Thus, these results suggest that titin is involved in diaphragm fiber length remodeling
during mechanical ventilation with PEEP.
DISCUSSION
Our findings show that (1) mechanical ventilation with PEEP causes a caudal
movement of the diaphragm dome, both in critically ill patients and in rats. Additional
studies in rats reveal that this caudal movement of the diaphragm reduces fiber and
sarcomere length; (2) diaphragm fibers adapt to the reduced length by absorbing
sarcomeres in series, with no changes in thick or thin filament length in both rats and
critically ill patients; (3) titin’s elastic properties modulate this length adaptation.
Longitudinal atrophy of diaphragm fibers.
PEEP is applied in nearly all mechanically ventilated critically ill patients to mitigate
alveolar collapse, thereby improving oxygenation and respiratory mechanics. However,
application of PEEP is associated with adverse effects in some patients due to alveolar
overdistention and hemodynamic compromise. The present study provides strong
evidence that PEEP may have adverse effects on diaphragm function as well. In
particular, PEEP causes a caudal movement of the diaphragm dome, with a shortening

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15
of the zone of apposition (Fig.1). This shortening leads to structural adaptions in muscle
fibers, as indicated by the reduced length at which diaphragm fibers generate maximal
force after 18h of mechanical ventilation (Fig.4C). Such reduction had been suggested
in previous studies36, but remained controversial37. This controversy is at least partly
due to difficulties in the exact determination of optimal muscle length: determination of
where muscle fibers end and the tendon starts with the naked eye is prone to error.
Therefore, to obtain conclusive evidence for fiber shortening during mechanical
ventilation with PEEP, we used microscopy analyses to measure the number of
sarcomeres in series in whole-length diaphragm fibers in rats and found this number to
be decreased (Fig.5). The magnitude of the decrease completely accounts for the
reduction of optimal length (both 12%; Figs. 4C and 5E). Thus, the present study is the
first to show that mechanical ventilation with PEEP not only causes cross-sectional fiber
atrophy, but also longitudinal fiber atrophy, i.e. reduced length of fibers. Although
adaptations might occur more rapidly in rats than in humans, we speculate that the
rapid time course of longitudinal atrophy in the diaphragm fibers of rats suggests that
this mechanism contributes to the rapid development, within 24 hours9, of in vivo
diaphragm weakness in critically ill patients. In these studies9, PdiTw was determined at
the end of expiration and at zero PEEP, thus with the length of the diaphragm fibers on
the descending limb of the force-length relation.
Disuse of skeletal muscle causes reduction of the length of the myosin-based thick
filaments38, but such reduction was not observed in diaphragm fibers of mechanically
ventilated critically ill patients or in ventilated rats. To rule out that length changes were
too small to detect, we determined thick and thin filament length by STED

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16
superresolution microscopy. STED superresolution microscopy creates non-diffraction-
limited images by the selective deactivation of fluorophores, minimizing the area of
illumination at the focal point, and thus enhancing the achievable resolution39.
Accordingly, thick filament measurements in the present study had a precision of ~50nm
and thin filament measurements of ~90nm, a precision required for detection of small,
yet relevant differences in length. We anticipate that the duration of disuse in the rats
(18h) may have been too short to cause filament length changes in the diaphragm.
Furthermore, although the patients were on mechanical ventilation much longer than the
rats, diaphragm activity was not completely absent in the majority of critically ill patients,
but varied depending on the ventilation mode, respiratory drive, level of sedation, and
the use of neuromuscular blocking agents (Table 4). It is likely that even these low
levels of diaphragm activity are sufficient to attenuate changes in filament length.
Role for the giant muscle protein titin?
Our studies into the mechanisms that underlie the longitudinal atrophy of diaphragm
fibers during mechanical ventilation with PEEP suggest a role for the giant elastic
protein titin. Titin is the largest protein known to date and spans the entire length of the
sarcomere from Z-disk to M-band (Fig.6A). The central I-band region of titin is
extensible and functions as a spring that generates passive tension when the
sarcomere is stretched. Titin filaments overlap in both the Z-disk and M-band, forming a
contiguous filament along the myofibril. This layout of titin within the muscle’s
sarcomere makes it ideally suited to sense length changes. Indeed, titin links
mechanical stress sensing to trophic signaling pathways via several titin-binding

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17
proteins16. We hypothesized that low titin stiffness – by preconditioning the diaphragm
to reduced mechanosensing – blunts the longitudinal atrophy response during
diaphragm shortening caused by mechanical ventilation with PEEP. To test this
hypothesis we took advantage of Rbm20-deficient rats that have reduced titin-based
stiffness17;28. Long-term mechanical ventilation of mice is technically challenging, and
the availability of this mutant rat model provided us with the unique opportunity to
overcome this limitation. The rat model carries a homozygous mutation in the titin splice
factor Rbm20, leading to the expression of a diaphragm titin isoform that is ~110 kDa
larger than in wt rats, with a concomitant reduction in titin-based passive stiffness of
diaphragm fibers (Fig.6C: at end-expiratory sarcomere length (~2.85 µm, Fig.1E)
passive tension is ~30% lower in Rbm20-deficent rats). The data support our
hypothesis: after 18h of mechanical ventilation with PEEP the development of
diaphragm weakness is attenuated in the Rbm20-deficient rats (Fig.6D), and
importantly, the optimal length for force generation and the number of sarcomeres in
series in diaphragm fibers of ventilated Rbm20-deficient rats are not significantly
different from non-ventilated Rbm20-deficient rats (Fig.6E&F). These findings support
the concept in which titin-based mechanosensing modulates the length adaptation of
diaphragm fibers during mechanical ventilation with PEEP. Future studies should
unravel the titin-based signaling pathways that underlie this effect.
Study limitations.
First, for the measurement of diaphragm displacement during PEEP variation
ultrasound was used. In each patient and rat, the ultrasound probe was placed at the

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same position and approximately the same region of the diaphragm was imaged.
However, as 2D images were obtained, we cannot rule out that the magnitude of
displacement was different in other regions of the diaphragm.
Second, we did not include a group of rats which were ventilated with zero PEEP, and
therefore we cannot rule out that the observed longitudinal atrophy of diaphragm fibers
is the result of MV per se, whether or not PEEP is applied. However, ventilation with
zero PEEP might cause atelectasis and an (systemic) inflammatory response40;41, which
in turn might impair diaphragm fiber structure and function, thereby confounding the
results. Furthermore, it is highly unlikely that ventilation with zero PEEP induces
longitudinal atrophy: this type of atrophy, i.e. sarcomere in series absorption, only
occurs when muscles are put at shorter (sarcomere) length42. Our ultrasound data show
a strong correlation between PEEP and caudal diaphragm movement (Fig.1B), a
movement that was associated with sarcomere shortening (Fig.1E). The rats in the
present study were on CMV and it is unknown whether complete inactivity of the
diaphragm affects fiber length, or aggravates the effects of PEEP. However, in rats with
joint immobilization, so that muscle length was fixed at normal operational length,
denervation (i.e., lack of muscle activity) does not induce sarcomere absorption,
whereas denervation of the same muscle but fixed at a shorter length does promote
longitudinal fiber atrophy43. Taken together, this supports a concept in which PEEP-
induced diaphragm shortening is the main factor contributing to longitudinal atrophy of
diaphragm fibers during mechanical ventilation.
Third, we postulate that the force–length relation of the diaphragm is affected by
sarcomere absorption, but this postulation assumes that the mechanical characteristics

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19
of the central tendon and of the tissues that connect the diaphragm muscle to the
costae is unaffected. In theory, lengthening of the central tendon would allow the shorter
fibers to operate at the plateau region of the force-length relation, thereby mitigating the
effects of longitudinal fiber atrophy. However, long-term shortening of lower limb muscle
is associated with a decrease in tendon length, an adaptation that would aggravate (not
mitigate) the effects of longitudinal fiber atrophy (for review see44).
Clinical implications.
The findings of the current study point towards a novel mechanism for failing a
spontaneous breathing trial in patients weaning from mechanical ventilation. The acute
reduction or withdrawal of PEEP decreases end-expiratory lung volume and thereby
stretches the adapted, short diaphragm fibers to long sarcomere lengths. This will force
the muscle fibers to operate on the disadvantageous descending limb of the force-
length relation, where overlap of the thick and thin filaments is sub-optimal, and thus
contribute to diaphragm weakness (for schematic, see Fig.7). This longitudinal atrophy
of fibers adds to the contractile weakness caused by ultrastructural damage and cross-
sectional fiber atrophy33;45. The magnitude of this contribution depends on the level of
applied PEEP: assuming that in rats before the application of MV with PEEP the
sarcomere length in the diaphragm is ~2.85 µm at end-expiration (Fig.1E) and that 12%
of sarcomeres is absorbed during 18h MV with 2.5 cmH2O PEEP (Fig.5E), after removal
of MV with PEEP at similar diaphragm fiber length the sarcomere length would be ~3.24
µm (2.85/0.88) at end-expiration, thus already at the descending limb of the force-length
relation (Fig.5A). Considering that 2.5 cmH2O PEEP in rats is estimated to correspond

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to 5-6 cmH2O PEEP in humans (estimation based on respiratory system compliance of
64 ml/cmH2O and TLC of 5167 ml for humans46 versus 0.51 ml/cmH2O and TLC of
16.7ml for rats47;48, the nearly 2-fold higher average PEEP in our cohort of patients
(Table 4) may have significant detrimental effect on the operating sarcomere length in
the diaphragm (note that PEEP levels in patients varied, but that the average lowest
PEEP level, i.e. PEEPmin in Table 4, was 6.0±0.7 cmH2O, a level comparable to that in
the ventilated rats). We postulate that this longitudinal diaphragm atrophy contributes to
difficulties with weaning patients from mechanical ventilation, especially in patients that
have respiratory disease or pre-existing diaphragm weakness. Our findings suggest that
slow reduction in PEEP is advisable, to allow for the reversal of longitudinal atrophy.
Clearly, this reasoning should be tested in future studies.

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21
REFERENCE LIST
1. Watson, A. C., P. D. Hughes, H. M. Louise, N. Hart, R. J. Ware, J. Wendon, M.
Green, and J. Moxham. 2001. Measurement of twitch transdiaphragmatic,
esophageal, and endotracheal tube pressure with bilateral anterolateral magnetic
phrenic nerve stimulation in patients in the intensive care unit. Crit Care Med.
29:1325-1331.
2. Hermans, G., A. Agten, D. Testelmans, M. DeCramer, and G. Gayan-Ramirez.
2010. Increased duration of mechanical ventilation is associated with decreased
diaphragmatic force: a prospective observational study. Crit Care 14:R127.
3. Jaber, S., B. J. Petrof, B. Jung, G. Chanques, J. P. Berthet, C. Rabuel, H.
Bouyabrine, P. Courouble, C. Koechlin-Ramonatxo, M. Sebbane, et al. 2011.
Rapidly progressive diaphragmatic weakness and injury during mechanical
ventilation in humans. Am.J.Respir.Crit Care Med. 183:364-371.
4. Gottesman, E. and F. D. McCool. 1997. Ultrasound evaluation of the paralyzed
diaphragm. Am.J.Respir.Crit Care Med. 155:1570-1574.
5. Goligher, E. C., E. Fan, M. S. Herridge, A. Murray, S. Vorona, D. Brace, N.
Rittayamai, A. Lanys, G. Tomlinson, J. M. Singh, et al. 2015. Evolution of
Diaphragm Thickness during Mechanical Ventilation. Impact of Inspiratory Effort.
Am.J.Respir.Crit Care Med. 192:1080-1088.
6. Dres, M., B. P. Dube, J. Mayaux, J. Delemazure, D. Reuter, L. Brochard, T.
Similowski, and A. Demoule. 2017. Coexistence and Impact of Limb Muscle and

Page 22 of 55
22
Diaphragm Weakness at Time of Liberation from Mechanical Ventilation in Medical
Intensive Care Unit Patients. Am.J.Respir.Crit Care Med. 195:57-66.
7. Esteban, A., A. Anzueto, F. Frutos, I. Alia, L. Brochard, T. E. Stewart, S. Benito, S.
K. Epstein, C. Apezteguia, P. Nightingale, et al. 2002. Characteristics and
outcomes in adult patients receiving mechanical ventilation: a 28-day international
study. JAMA 287:345-355.
8. Supinski, G. S. and L. A. Callahan. 2013. Diaphragm weakness in mechanically
ventilated critically ill patients. Crit Care 17:R120.
9. Demoule, A., B. Jung, H. Prodanovic, N. Molinari, G. Chanques, C. Coirault, S.
Matecki, A. Duguet, T. Similowski, and S. Jaber. 2013. Diaphragm dysfunction on
admission to the intensive care unit. Prevalence, risk factors, and prognostic
impact-a prospective study. Am.J.Respir.Crit Care Med. 188:213-219.
10. Levine, S., T. Nguyen, N. Taylor, M. E. Friscia, M. T. Budak, P. Rothenberg, J.
Zhu, R. Sachdeva, S. Sonnad, L. R. Kaiser, et al. 2008. Rapid disuse atrophy of
diaphragm fibers in mechanically ventilated humans. N.Engl.J.Med. 358:1327-
1335.
11. Hussain, S. N., M. Mofarrahi, I. Sigala, H. C. Kim, T. Vassilakopoulos, F. Maltais, I.
Bellenis, R. Chaturvedi, S. B. Gottfried, P. Metrakos, et al. 2010. Mechanical
ventilation-induced diaphragm disuse in humans triggers autophagy.

Page 23 of 55
23
12. Picard, M., B. Jung, F. Liang, I. Azuelos, S. Hussain, P. Goldberg, R. Godin, G.
Danialou, R. Chaturvedi, K. Rygiel, et al. 2012. Mitochondrial dysfunction and lipid
accumulation in the human diaphragm during mechanical ventilation.
13. Hooijman, P. E., A. Beishuizen, C. C. Witt, M. C. de Waard, A. R. Girbes, A. M.
Spoelstra-de Man, H. W. Niessen, E. Manders, H. W. van Hees, C. E. van den
Brom, et al. 2015. Diaphragm muscle fiber weakness and ubiquitin-proteasome
activation in critically ill patients. Am.J.Respir.Crit Care Med. 191:1126-1138.
14. van den Berg, M., P. E. Hooijman, A. Beishuizen, M. C. de Waard, M. A. Paul, K.
J. Hartemink, H. W. H. van Hees, M. W. Lawlor, L. Brocca, R. Bottinelli, et al.
2017. Diaphragm Atrophy and Weakness in the Absence of Mitochondrial
Dysfunction in the Critically Ill. Am.J.Respir.Crit Care Med. 196:1544-1558.
15. Hooijman, P. E., A. Beishuizen, M. C. de Waard, F. S. de Man, J. W. Vermeijden,
P. Steenvoorde, R. A. Bouwman, W. Lommen, H. W. van Hees, L. M. Heunks, et
al. 2014. Diaphragm fiber strength is reduced in critically ill patients and restored
by a troponin activator. Am.J.Respir.Crit Care Med. 189:863-865.
16. Ottenheijm, C. A., H. W. van Hees, L. M. Heunks, and H. Granzier. 2011. Titin-
based mechanosensing and signaling: role in diaphragm atrophy during
unloading? Am.J.Physiol Lung Cell Mol.Physiol 300:L161-L166.

Page 24 of 55
24
17. Guo, W., S. Schafer, M. L. Greaser, M. H. Radke, M. Liss, T. Govindarajan, H.
Maatz, H. Schulz, S. Li, A. M. Parrish, et al. 2012. RBM20, a gene for hereditary
cardiomyopathy, regulates titin splicing. Nat.Med. 18:766-773.
18. Ottenheijm, C. A., P. Hooijman, E. T. DeChene, G. J. Stienen, A. H. Beggs, and H.
Granzier. 2010. Altered myofilament function depresses force generation in
patients with nebulin-based nemaline myopathy (NEM2). J.Struct.Biol. 170:334-
343.
19. Ottenheijm, C. A., C. Hidalgo, K. Rost, M. Gotthardt, and H. Granzier. 2009.
Altered contractility of skeletal muscle in mice deficient in titin's M-band region.
J.Mol.Biol. 393:10-26.
20. de Man, F. S., H. W. van Hees, M. L. Handoko, H. W. Niessen, I. Schalij, M.
Humbert, P. Dorfmuller, O. Mercier, H. J. Bogaard, P. E. Postmus, et al. 2011.
Diaphragm muscle fiber weakness in pulmonary hypertension. Am.J.Respir.Crit
Care Med. 183:1411-1418.
21. Manders, E., G. Ruiter, H. J. Bogaard, G. J. Stienen, A. Vonk-Noordegraaf, F. S.
de Man, and C. A. Ottenheijm. 2015. Quadriceps muscle fibre dysfunction in
patients with pulmonary arterial hypertension. Eur.Respir.J. 45:1737-1740.
22. Lawlor, M. W., C. A. Ottenheijm, V. L. Lehtokari, K. Cho, K. Pelin, C. Wallgren-
Pettersson, H. Granzier, and A. H. Beggs. 2011. Novel mutations in NEB cause
abnormal nebulin expression and markedly impaired muscle force generation in
severe nemaline myopathy. Skelet.Muscle 1:23.

Page 25 of 55
25
23. Ottenheijm, C. A., M. W. Lawlor, G. J. Stienen, H. Granzier, and A. H. Beggs.
2011. Changes in cross-bridge cycling underlie muscle weakness in patients with
tropomyosin 3-based myopathy. Hum.Mol.Genet. 20:2015-2025.
24. Ottenheijm, C. A., C. C. Witt, G. J. Stienen, S. Labeit, A. H. Beggs, and H.
Granzier. 2009. Thin filament length dysregulation contributes to muscle weakness
in nemaline myopathy patients with nebulin deficiency. Hum.Mol.Genet. 18:2359-
2369.
25. Ottenheijm, C. A., D. Buck, J. M. de Winter, C. Ferrara, N. Piroddi, C. Tesi, J. R.
Jasper, F. I. Malik, H. Meng, G. J. Stienen, et al. 2013. Deleting exon 55 from the
nebulin gene induces severe muscle weakness in a mouse model for nemaline
myopathy. Brain 136:1718-1731.
26. Winter, J. M., B. Joureau, E. J. Lee, B. Kiss, M. Yuen, V. A. Gupta, C. T. Pappas,
C. C. Gregorio, G. J. Stienen, S. Edvardson, et al. 2016. Mutation-specific effects
on thin filament length in thin filament myopathy. Ann.Neurol. 79:959-969.
27. Manders, E., F. S. de Man, M. L. Handoko, N. Westerhof, H. W. van Hees, G. J.
Stienen, A. Vonk-Noordegraaf, and C. A. Ottenheijm. 2012. Diaphragm weakness
in pulmonary arterial hypertension: role of sarcomeric dysfunction. Am.J.Physiol
Lung Cell Mol.Physiol 303:L1070-L1078.
28. Greaser, M. L., C. M. Warren, K. Esbona, W. Guo, Y. Duan, A. M. Parrish, P. R.
Krzesinski, H. S. Norman, S. Dunning, D. P. Fitzsimons, et al. 2008. Mutation that

Page 26 of 55
26
dramatically alters rat titin isoform expression and cardiomyocyte passive tension.
J.Mol.Cell Cardiol. 44:983-991.
29. Warren, C. M., P. R. Krzesinski, and M. L. Greaser. 2003. Vertical agarose gel
electrophoresis and electroblotting of high-molecular-weight proteins.
Electrophoresis 24:1695-1702.
30. Granzier, H. L. and T. C. Irving. 1995. Passive tension in cardiac muscle:
contribution of collagen, titin, microtubules, and intermediate filaments. Biophys.J.
68:1027-1044.
31. Ottenheijm, C. A., A. M. Knottnerus, D. Buck, X. Luo, K. Greer, A. Hoying, S.
Labeit, and H. Granzier. 2009. Tuning passive mechanics through differential
splicing of titin during skeletal muscle development. Biophys.J. 97:2277-2286.
32. van Hees, H. W., W. J. Schellekens, G. L. ndrade Acuna, M. Linkels, T. Hafmans,
C. A. Ottenheijm, H. L. Granzier, G. J. Scheffer, J. G. van der Hoeven, P. N.
Dekhuijzen, et al. 2012. Titin and diaphragm dysfunction in mechanically
ventilated rats. Intensive Care Med. 38:702-709.
33. Shanely, R. A., M. A. Zergeroglu, S. L. Lennon, T. Sugiura, T. Yimlamai, D. Enns,
A. Belcastro, and S. K. Powers. 2002. Mechanical ventilation-induced
diaphragmatic atrophy is associated with oxidative injury and increased proteolytic
activity. Am.J.Respir.Crit Care Med. 166:1369-1374.

Page 27 of 55
27
34. Le, B. G., N. Viires, J. Boczkowski, N. Seta, D. Pavlovic, and M. Aubier. 1994.
Effects of mechanical ventilation on diaphragmatic contractile properties in rats.
35. Sassoon, C. S., V. J. Caiozzo, A. Manka, and G. C. Sieck. 2002. Altered
diaphragm contractile properties with controlled mechanical ventilation.
J.Appl.Physiol 92:2585-2595.
36. Yang, L., J. Luo, J. Bourdon, M. C. Lin, S. B. Gottfried, and B. J. Petrof. 2002.
Controlled mechanical ventilation leads to remodeling of the rat diaphragm.
37. Sassoon, C. S., E. Zhu, L. Fang, G. C. Sieck, and S. K. Powers. 2014. Positive
end-expiratory airway pressure does not aggravate ventilator-induced
diaphragmatic dysfunction in rabbits. Crit Care 18:494.
38. Udaka, J., S. Ohmori, T. Terui, I. Ohtsuki, S. Ishiwata, S. Kurihara, and N. Fukuda.
2008. Disuse-induced preferential loss of the giant protein titin depresses muscle
performance via abnormal sarcomeric organization. J.Gen.Physiol 131:33-41.
39. Dyba, M., S. Jakobs, and S. W. Hell. 2003. Immunofluorescence stimulated
emission depletion microscopy. Nat.Biotechnol. 21:1303-1304.
40. Vreugdenhil, H. A., C. J. Heijnen, F. B. Plotz, J. Zijlstra, N. J. Jansen, J. J.
Haitsma, B. Lachmann, and A. J. van Vught. 2004. Mechanical ventilation of
healthy rats suppresses peripheral immune function. Eur.Respir.J. 23:122-128.

Page 28 of 55
28
41. Chu, E. K., T. Whitehead, and A. S. Slutsky. 2004. Effects of cyclic opening and
closing at low- and high-volume ventilation on bronchoalveolar lavage cytokines.
Crit Care Med. 32:168-174.
42. Williams, P. E. and G. Goldspink. 1973. The effect of immobilization on the
longitudinal growth of striated muscle fibres. J.Anat. 116:45-55.
43. Goldspink, G., C. Tabary, J. C. Tabary, C. Tardieu, and G. Tardieu. 1974. Effect of
denervation on the adaptation of sarcomere number and muscle extensibility to the
functional length of the muscle. J.Physiol 236:733-742.
44. Harvey, L. A. and R. D. Herbert. 2002. Muscle stretching for treatment and
prevention of contracture in people with spinal cord injury. Spinal Cord. 40:1-9.
45. Bernard, N., S. Matecki, G. Py, S. Lopez, J. Mercier, and X. Capdevila. 2003.
Effects of prolonged mechanical ventilation on respiratory muscle ultrastructure
and mitochondrial respiration in rabbits. Intensive Care Med. 29:111-118.
46. Henderson, W. R., L. Chen, M. B. P. Amato, and L. Bochard. 2017. Fifty years of
research in ARDS. Respiratory mechanics in acute respiratory distress syndrome.
47. Rubini, A., D. El-Mazloum, F. Morra, and G. Bosco. 2013. The effect of body
cooling on respiratory system mechanics and hysteresis in rats.
Respir.Physio.Neurobiol. 189:52-58.

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48. Bolle, J., G. Eder, S. Takenaka, K. Ganquly, S. Karrasch, C. Zeller, M. Neuner, W.
G. Kreyling, A. Tsuda, and H. Schulz. 2008. Postnatal lung function in the
developing rat. J.Appl.Physiol 104:1167-1176.
49. Schellekens, W. J., H. W. van Hees, M. Kox, M. Linkels, G. L. Acuna, P. N.
Dekhuijzen, G. J. Scheffer, J. G. van der Hoeven, and L. M. Heunks. 2014.
Hypercapnia attenuates ventilator-induced diaphragm atrophy and modulates
dysfunction. Crit Care 18:R28.
FIGURE LEGENDS
Figure 1. A) Ultrasonographic view of the diaphragm in a critically ill patient in the
region of the liver dome, with B-mode image in (left) and M-mode image in (middle).
The M-mode image shows the acute effect of a 10 cmH2O PEEP change on the
position of the diaphragm, measured at end-expiration (indicated by dashed line); note
that in this patient PEEP was decreased from 12 to 2 cmH2O, resulting in a cranial
diaphragm displacement. Right: Cranial diaphragm displacement in critically ill patients
caused by a 5 cmH2O PEEP reduction (n=5) or by a 10 cmH2O PEEP reduction (n=10);
each data point is the average of three measurements per patient. (B) Ultrasonographic
view of the diaphragm in rat during mechanical ventilation in the region of the liver
dome, with B-mode image left and corresponding M-mode image in the middle. The M-
mode image shows that an increase of PEEP with 3 cmH2O causes an acute caudal
movement of the diaphragm, measured at end-expiration (dashed line). Right panel
shows the effect of 2, 3, and 5 cmH2O ∆PEEP on caudal diaphragm displacement; each
data point is the average of five rats. C) image of an excised rat diaphragm, illustrating

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the mid-costal position (X) where a strip was isolated for measurement of diaphragm
fiber and sarcomere length in in vivo-fixated rats. D) Diaphragm fiber length in in vivo-
fixated PEEP-ventilated (2.5 cmH2O) rats and non-ventilated rats; each data point
represents one rat. E) Diaphragm sarcomere length in in vivo-fixated PEEP-ventilated
(2.5 cmH2O) rats and non-ventilated rats; each data point represents one rat.
Figure 2. A) Schematic showing the force-sarcomere length relation of a diaphragm
fiber of a control patient; SLopt: sarcomere length at which maximal force is generated;
SL50: sarcomere length at which 50% of maximal force is generated; SLmax: sarcomere
length at which no force is generated. Right panel shows schematics of a sarcomere
with the corresponding lengths. B) Force-sarcomere length relation of diaphragm fibers
of twelve critically ill and twelve control patients; note that both relations overlap. Force
is presented as percentage of maximal force. SLopt (C), SL50 (D), and SLmax (E) are
comparable between critically ill and control patients. (Note that (1) SLopt, SL50 and
SLmax were not different between slow- and fast-twitch fibers, data not shown, and
therefore these data are pooled; and (2) in line with previous work 13-15 the maximal
force generating capacity was lower in both slow- and fast-twitch fibers of critically ill
patients, data not shown). Note that each data point represents the average of ~ten
fibers per subject.
Figure 3. A) Deconvolved stimulated emission depletion (STED) superresolution
microscopy images of sarcomeres in a diaphragm fiber of a control and a critically ill
patient, labeled with AlexaFluor-conjugated phalloidin to visualize the thin filaments.

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Intensity measurements were used to determine thin filament length, which was
comparable between critically ill and control patients. (B) Deconvolved STED
superresolution microscopy images of sarcomeres in a diaphragm fiber of a control and
a critically ill patient, labeled with myosin heavy chain antibodies to visualize the thick
filaments. Intensity measurements were used to determine thick filament length, which
was comparable between critically ill and control patients. Note that each data point
represents the average of 50-100 sarcomeres per subject.
Figure 4. In vitro contractility of electrically-stimulated diaphragm strips of 18h
mechanically ventilated rats. A) Schematic showing a full-length diaphragm strip in the
experimental setup. The strips were isolated from the mid-costal region, similar to
location ‘X’ in figure 2B. MLopt: the strip length at which maximal force is generated. B)
The maximal tension (force normalized to the cross sectional are of the strip) was
significantly lower in rats that were mechanically ventilated for 18h. C) MLopt was
significantly shorter in the rats that were mechanically ventilated for 18h. Note that each
data point represents one rat.
Figure 5. A) Top: Force-sarcomere length relation of diaphragm fibers of 18h
mechanically ventilated rats and control rats; note that both relations overlap. Force is
presented as percentage of maximal force; each data point is the average of five rats.
Bottom: SLopt is comparable between mechanically ventilated and control rats. Note that
~90% of fibers were fast-twitch and therefore both fiber types were pooled, and that in
line with previous work32;49 the maximal force generating capacity was lower in fibers of

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mechanically ventilated rats, data not shown. B) Deconvolved stimulated emission
depletion (STED) superresolution microscopy images of sarcomeres in a diaphragm
fiber of a control and an 18h mechanically ventilated rat, labeled with AlexaFluor-
conjugated phalloidin to visualize the thin filaments. Intensity measurements were used
to determine thin filament length, which was comparable between 18h mechanically
ventilated and control rats. Each data point represents one rat. C) Deconvolved STED
superresolution microscopy images of sarcomeres in a diaphragm fiber of a control and
an 18h mechanically ventilated rat, labeled with myosin heavy chain antibodies to
visualize the thick filaments. Intensity measurements were used to determine thick
filament length, which was comparable between 18h mechanically ventilated and
control rats. Each data point represents one rat. D) Typical α-actinin staining in a rat
diaphragm fiber to visualize the z-discs in order to determine sarcomere length; E) The
number of sarcomeres in series is significantly lower in 18h mechanically ventilated rats
compared to control rats. Each data point represents the average of one rat; per rat
2000-3000 sarcomeres were measured.
Figure 6. A) Schematic of the layout of the giant protein titin in the muscle sarcomere.
B) bottom: 1% SDS-agarose gel illustrating the slower mobility of the larger titin isoform
in the Rbm20-deficient rats. T1 is full length titin, T2 is a titin degradation product. Top:
magnification of T1 titin, note the lower titin mobility in Rbm20-deficient rats. C) Passive
tension-sarcomere length relation in diaphragm fibers. Note that the larger titin isoform
in Rbm20-deficient rats results in lower passive tension (each data point represents the
average of six rats). D) 18h of mechanical ventilation causes significant contractile

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weakness of intact diaphragm strips in control rats.. The development of this weakness
is blunted in 18h mechanically ventilated Rbm20-deficient rats. E) MLopt is reduced in
18h mechanically ventilated control rats, but this reduction is absent in Rbm20-deficient
rats. F) The number of sarcomeres in series is not significantly reduced in 18h
mechanically ventilated Rbm20-deficient rats compared to non-ventilated Rbm20-
deficient rats. Each data point represents one rat; per rat 2000-3000 sarcomeres were
measured.
Figure 7. Schematic illustrating the development of longitudinal atrophy in diaphragm
fibers during mechanical ventilation with PEEP. A) muscle length in a spontaneous
breathing rat at end-expiration. For simplicity only three sarcomeres are drawn. B) the
acute effect of PEEP on sarcomere length. C) Long-term PEEP causes sarcomere
absorption to restore sarcomere length. D) During weaning, and normalization of end-
expiratory pressure, the diaphragm fibers are stretched to lengths beyond optimal
sarcomere length. The schematic shows a length at which thick-thin filament overlap is
absent and no force can be generated by the muscle fiber.

Page 34 of 55
Table 1. General characteristics of critically ill patients on whom ultrasound was performed
Subject # Age Sex BMI Relevant medical history Reason of admission PEEP induced Delta PEEP at Support Tidal volume
(yr) (M/F) (kg/m²) to ICU excursion (cm) PEEP baseline pressure (ml)
(cmH2O) (cmH2O) (cmH2O)
1 63 F 19.1 COPD, pancreatitis Pneumonia 0.204 5 5 18 371
2 67 F 29.4 AVNRT, polymyalgia Septic arthritis 0.500 5 5 20 344

rheumatica, adnectomy,
lumpectomy
3 32 F 37.5 Tetraplegia Sepsis, APL 0.147 5 14 12 483
4 57 M 23.4 None OHCA 0.630 5 10 14 478
5 62 M 30.6 Diabetes mellitus Type II, Necrotizing fasciitis 0.534 5 10 4 595

CVA
6 70 M 25.2 COPD OHCA 1.232 10 10 8 499
7 61 M 26.3 Diabetes mellitus Type II S. aureus septic arthritis 1.073 10 10 12 583
8 67 F 27.3 Myelofibrosis, Hypertension ARDS 0.374 10 10 4 552
9 65 F 32.7 None Multiple trauma 0.314 10 12 12 488

(including thorax)
10 89 M 24.1 Benign prostatic Neurotrauma (including 0.803 10 10 6 435

hyperplasia; Paroxysmal multiple rib fracture)
atrial fibrillation
11 70 F 21.6 Multiple myeloma, DVT Pneumonia 0.380 10 12 23 332
12 77 M 27.9 CABG, right upper IHCA during 0.612 10 10 10 385

lobectomy due to elective CAG
adenocarcinoma
13 56 M 21.6 None Intracranial hemorrhage 2.210 10 10 14 404
14 48 M 24.7 LBBB OHCA 0.879 10 10 8 470
15 70 F 29.4 Diabetes mellitus Type II Peritonitis 0.740 10 10 10 374
Definition of abbreviations: yr = year; BMI = body mass index; PEEP = positive end-expiratory pressure; COPD: chronic obstructive pulmonary disease; ARDS = acute respiratory distress syndrome;
OHCA = out of hospital cardiac arrest; DVT = deep vein thrombosis; CABG = coronary artery bypass graft; IHCA = in hospital cardiac arrest; CAG = coronary angiography; AVNRT = AV nodal
reentrant tachycardia; APL = acute promyeloid leukemia; DM = diabetes mellitus; CVA = cerebrovascular accident; LBBB = left bundle branch block

Page 35 of 55
Table 2. Characteristics of control patients

Control Age Sex BMI (kg/m²) Relevant medical history TNM classification MV (h)
# (yr) (M/F)
1 52 M 25 T2DM, HT, COPD GOLD II, ex-smoker pT3N0M0 1.5
2 58 F 28 None pT1aN0M0 2
3 56 M 21 COPD GOLD II, ex-smoker pT4N2M0 2
4 60 F 26 HT pT2aN0R0 0.75
5 59 F 28 Chronic bronchitis, smoker benign, cyst 0.75
6 64 F 26 COPD GOLD II, smoker pT1bN0M0 1.25
7 64 M 31 HT, MI, T2DM, smoker benign, bronchiectasis and inflammation 1
8 55 F 27 Allergic asthma, recurrent pneumonia, smoker carcinoid T1bN0 1
9 40 M 24 None carcinoid pT1aN0R0 1.5
10 50 M 24 COPD GOLD I, smoker mucinous adeno-carcinoma T1aN0M0 1.25
11 58 F 23 Chronic bronchitis, COPD GOLD I, ex-smoker adenocarcinoma pT0N0M0 2.5
12 69 M 33 HT, ex-smoker adenocarcinoma pT3N1M0 1.5
Definitions of abbreviations: yr = year; BMI = body mass index; MV (h) = hours of mechanical ventilation; T2DM = type 2 diabetes mellitus; HT = hypertension; COPD = chronic obstructive pulmonary
disease (GOLD classification); MI = myocardial infarction.

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Table 3. Characteristics of critically ill patients from whom biopsies were obtained
Crit. ill Age Sex BMI (kg/m²) Relevant medical history Reason of admission Surgery where biopsy MV Septic Died APACHE II
# (yr) (M/F) to ICU was obtained (h) ICU
1 47 F 22 None Severe trauma Re-laparotomy: gauze 40 N N 35
removal
2 25 F 33 None Severe trauma Re-relaparotomy: closure 219 Y N 45
abdomen
3 66 M 29 HT Abdominal sepsis due Re-laparotomy: rinsing 28 Y N 11
to perforated abdominal cavity
appendicitis
4 69 M 22 None Esophageal rupture Thoracotomy: esophageal 17 Y N 27
(Boerhaave syndrome) repair
5 51 M 29 Hypothyreoidism Severe trauma 4th re-laparotomy: closure 85 N N 30
abdomen
6 72 M 20 T2DM, colon carcinoma, Abdominal sepsis due Re-laparotomy: 20 Y Y 19
chronic bronchitis, COPD to duodenal perforation cholecystectomy
GOLD II
7 74 M 25 HT, AFib Respiratory failure due Re-laparotomy: evacuation 435 Y Y 19
to bilateral pneumonia empyema
8 22 F 20 None Severe trauma 2nd re-relaparotomy: 203 N N 31
closure abdomen
9 55 F 21 Lower leg amputation, right Abdominal sepsis due Re-laparotomy: closure 69 Y N 19
kidney infarction, splenic to perforations by abdomen
infarction intestinal ischemia
10 75 M 22 HT, chronic coronary artery Postoperative care 4th re-laparotomy: 290 Y Y 22
disease, chronic kidney after thoraco- Hartmann procedure
disease, smoker abdominal aortic
aneurysm repair
11 54 M 26 None Postoperative care 4th re-laparotomy: 50 N N 40
after ruptured sigmoidresection with
abdominal aortic placement colostomy
aneurysm repair
12 66 F 21 colitis Severe trauma with 4th re-laparotomy: rinsing 134 Y N 24
intestinal damage abdominal cavity
Definition of abbreviations: yr = year; BMI = body mass index; ICU = intensive care unit; MV (h) = hours of mechanical ventilation; APACHE = acute physiology and chronic health evaluation; HT =
hypertension; T2DM = type 2 diabetes mellitus; COPD = chronic obstructive pulmonary disease (GOLD classification); HT= hypertension; AF= atrial fibrillation.

Page 37 of 55
Table 4. Specification of duration and settings of mechanical ventilation

Crit. CMV AMV O2 NR Total MV MV mode before FiO2 PEEP PEEP min - max RR Vt
ill # (h) (h) (h) (h) (h) obtaining biopsy (%) (cmH2O) (cmH2O) (breaths/min) (mL)
1 40 0 0 0 40 PC (CMV) 47 ± 12 12 ± 0.9 7 - 14 23 ± 2 370 ± 71
2 128 91 0 0 219 PS/CPAP (AMV) 54 ± 20 15 ± 6.7 8 - 26 26 ± 4 392 ± 78
3 2 26 19 0 28 O2-therapy 42 ± 11 5 ± 0.4 3-5 14 ± 4 762 ± 220
4 1 16 0 0 17 PC (AMV)* 57 ± 12 11 ± 1 10 - 12 23 ± 4 515 ± 170
5 85 0 0 0 85 SIMV+ASB (AMV) 44 ± 5 13 ± 2.1 8 - 15 22 ± 2 528 ± 102
6 12 8 1 0 20 CPAP+ASB (AMV) 50 ± 5 5 ± 0.4 5-6 21 ± 1 549 ± 38
7 275 160 152 0 435 SIMV+ASB (CMV) 47 ± 11 11 ± 3 5 - 15 18 ± 5 557 ± 143
8 92 111 0 0 203 PS/CPAP (AMV) 34 ± 8 8 ± 1.0 3 - 10 21 ± 5 337 ± 57
9 29 40 0 0 69 PCV (CMV) 50 ± 10 8 ± 0.4 6-8 17 ± 7 584 ± 157
10 124 166 5 0 290 PCV (CMV) 43 ± 4 7 ±1.4 2-9 24 ± 5 526 ± 113
11 48 2 0 0 50 SIMV+ASB (CMV) 32 ± 2 12 ± 2.1 8 - 16 31 ± 1 494 ± 24
12 130 4 0 0 134 SIMV+ASB (CMV) 41 ± 7 5 ± 0.4 3-7 18 ± 2 400 ± 49
Data are totals or mean±SD. Means and SD of FiO2, PEEP, RR, Vt were calculated from hourly registration from moment of intubation until biopsy. Definition of abbreviations: CMV (h) = hours of
controlled mechanical ventilation; AMV (h) = hours of assisted mechanical ventilation; O2 (h) = hours of received oxygen therapy; NR = non registered (e.g. during transport of the patients or
surgery); Total MV = sum of hours spend on mechanical ventilation from moment of intubation until biopsy; MV = mechanical ventilation; FiO2 (%) = fraction of inspired oxygen in percentages; PEEP
= positive end-expiratory pressure in centimeters of water; RR = respiratory rate in breaths per minute; Vt = tidal volume in ml. MV modes: PC = pressure control; PS/CPAP = pressure
support/continuous positive airway pressure; ASB = assisted spontaneous breathing; SIMV = synchronized intermittent mandatory ventilation; BIPAP = bi-level positive airway pressure; VC = volume
control; MMV = mandatory minute ventilation. Definition of CMV = mechanical ventilation in PC/VC/SIMV/MMV/BIPAP mode, and measured respiratory rate is lower or equal to set respiratory rate.
Definition of AMV = mechanical ventilation in PS/CPAP/ASB/NIV mode, and ventilation in PC/VC/SIMV/MMV/BIPAP mode as the measured respiratory rate is higher than the set respiratory rate. *
Mechanical ventilation of critically ill patient #4 was set on PC ventilation, but by us interpreted as AMV because the measured respiratory rate was higher than the set respiratory rate.

Page 38 of 55
A B-mode M-mode
2.5 PEEP
displacement (cm)
2.0
liver + 1.5
DIA
PEEP: + 1.0
12 cm H20 PEEP:
2 cm H20 0.5
lung
0.0
5 cmH2O 10 cmH2O
B B-mode M-mode
1.5
displacement (mm)
r2=0.9996
p<0.01
vena 1.0
cava
liver +
DIA
+ PEEP:
0.5
PEEP: 3 cm H20
lung 0 cm H20
0.0
0 2 4 6
PEEP (cmH2O)
Sarcomere length (m)

C D 24 * E 3.5
*
Diaph length (mm)
21
3.0
X
18
2.5
vena 15
cava
12 2.0
0 0
Control MV Control MV
FIGURE 1
Page 39 of 55
A thick filament
B
1.00 Z-disk 1.00
Relative force
Relative force
thin filament
SLopt
0.75 0.75
0.50 SL50 0.50
Con
0.25 0.25 Crit. ill
SLopt SL50 SLmax SLmax
0.00 sarcomere length (SL) 0.00
2.0 2.5 3.0 3.5 4.0 2.0 2.5 3.0 3.5 4.0
Sarcomere length (m) Sarcomere length (m)
C 3.0
NS D4.0 NS E 5.0
NS
SLmax (mm)
4.0
SLopt (m)
SL50 (m)
3.0
2.0 3.0
2.0
1.0
2.0
1.0 1.0
0.0 0.0 0.0
Control Crit.Ill Control Crit.Ill Control Crit.Ill
FIGURE 2 Copyright © 2018 by the American Thoracic Society

Page 40 of 55
Control Critically Ill

A
phalloidin
Thin filament length (m)
NS 2.0
1.5 3.0
Z-disc Z-disc Z-disc Z-disc
Intensity (AU)
Intensity (AU)
2.5
1.0 1.5
2.0
0.5 1.5 1.0
2x thin filament 2x thin filament
1.0
0.0 0.5
Control Crit. ill 0 2 4 6 0 2 4 6
Distance (m) Distance (m)
B MHC
Thick filament length (m)
NS
2.0 25 M-line M-line
M-line M-line
Intensity (AU)
Intensity (AU)
1.5 2.0 20
1.0 15
1.5
10 thick filament
thick filament
0.5
1.0 5
0.0
Control Crit. ill 0 2 4 0 2 4 6

Page 41 of 55
A B C
maximal tetanic tension

400 * 2.6 *
force
2.4
300
MLopt (cm)
(mN/mm2)
strip
2.2
diaphragm
MLopt
diaphragm
+ - 200 2.0
1.8
100
1.6
0 0
Control MV Control MV
(18h) (18h)

A B Control
AJRCCM Articles in Press. Published on 26-March-2018 as 10.1164/rccm.201709-1917OC MV
Page 42 of 55
Thin filament length (m)

1.0 phalloidin 1.5
Relative force
0.8 3.0
Z-disc Z-disc Z-disc Z-disc
30 1.0
Intensity (AU)
Intensity (AU)
0.6 2.5
2.0 20
0.4 0.5
Control 1.5
0.2 MV
2x thin filament
10
2x thin filament
1.0
0.0 0 2 4
0.0
0 2 4 6 Control MV
2.0 2.5 3.0 3.5 4.0 Distance (m) Distance (m)
Sarcomere length (m) (18h)
3
C
Thick filament length (m)

MHC 2.0
SLopt (m)
M-line M-line
3.0
2 M-line M-line
3.0 1.5
Intensity (AU)
Intensity (AU)
2.5
2.5 1.0
1 2.0
2.0
1.5
thick filament thick filament 0.5
0 1.5
ControlMV
1.0 0.0
(18h) 0 2 4 6 0 2 4 6 Control MV
(18h)
D a-actinin E 7000 *
# of sarcomeres
5
SL = 2.70 μm 6000
Intensity (AU)
4 5000
4000
3
0
FIGURE 5 0 10 20 30
Con MV
Distance (m)
(18h)
Page 43 of 55
A AJRCCMthick
Articlesfilament
D
in Press. Published on 26-March-2018 as 10.1164/rccm.201709-1917OC
maximal tetanic tension

Z-disc p<0.001 p<0.01
thin filament
M-band
1.0
(relative)
0.5
TITIN 0.0
con MV con MV
wt Rbm20-def
Rbm20
1 2
Rbm20
Rbm20
B E
def
def
def
p<0.01 NS
wt
wt
wt
KO T1
MLopt (relative)
1.0
wt T1
Titin T1 (~3.7MDa)
0.5
T2
Nebulin (~700kDa) 0.0

con MV con MV
wt Rbm20-def
2
MHC (~220kDa)
Number of sarcomeres
p<0.01 NS
C F
in series (relative)
20
Different curves
1.0
Passive tension
p<0.0001
15
(mN/mm2)
10
wt 0.5
5
Rbm20-def
FIGURE 6 0 0.0 con MV
wt
con MV
2.0 2.5
Copyright © 20183.0 3.5 Society
by the American Thoracic
Rbm20-def
Sarcomere length (m)
Page 44 of 55
muscle length
sarcomere length
A Normal, end-expiration
B Acute effect PEEP:

sarcomere length reduction
C Long-term effect PEEP:

sarcomere absorption
thin
D Weaning trial, zero PEEP:
thick
filament
filament
sarcomere length increase
FIGURE 7
Page 45 of 55
ONLINE DATA SUPPLEMENT
METHODS
Patients
Ultrasound:
B- and M-mode ultrasound (Sector array transducer, 5-1MHz; CX 50, Philips Inc., Bothell,
WA, USA) of the diaphragm was performed from subcostal view using the liver as a window
(Figure 1A). Ultrasound was performed in ventilated ICU patients with clinical indication for
analysis of diaphragm function. Ultrasound images were acquired during the acute reduction
in PEEP (Fig.1). Analysis of diaphragm movement was performed on the ultrasound
machine. Informed consent was waived by the Medical Ethics Committee of the VU
University medical center in Amsterdam.
Biopsies:
Diaphragm muscle biopsies were obtained from mechanically ventilated critically ill patients
undergoing abdominal or thoracic surgery for clinical indication (critically ill group, Table 3).
As a control group, diaphragm muscle biopsies were obtained from patients undergoing
tumor removal of a suspected early-stage lung malignancy (Table 2). The following exclusion
criteria were applied for both groups: (1) the presence of COPD GOLD III/IV, chronic heart
failure NYHA III/IV, pulmonary hypertension NYHA III/IV, neuromuscular disease, or chronic
metabolic disease; (2) chronic use of corticosteroids (>7.5 mg/day for at least 3 months); (3)
>10% weight loss within the 6 months prior to surgery for tumor removal or admission to the
intensive care unit; (4) current or recent chemotherapy and/or radiotherapy. Note that control
patients had unaffected total lung capacity (92±11, % predicted). The protocol was approved
by the Medical Ethics Committee of the VU University Medical Center Amsterdam. Patients
were recruited in VU University Medical Center, Netherlands Cancer Institute - Antoni van
Leeuwenhoek Hospital (both Amsterdam), and Medisch Spectrum Twente (Enschede), and
written informed consent was obtained from each patient and/or legal representative.

Page 46 of 55
Biopsy handling:
Diaphragm biopsies were stored in relax/glycerol solution with high concentration protease
inhibitors (Rx/Glyhigh) and initially placed at a roller band for 24 hours at 4°C. Finally,
Rx/Glyhigh was substituted with Rx/Gly with lower concentrations (Rx/Glylow) of protease
inhibitors and stored at -20°C until further use. Solution composition of Rx/Glyhigh and
Rx/Glylow was used as previously1-6 and described below.
Contractile force measurements:
Setup and protocol. Individual muscle fibers were dissected from the biopsies, and were
mounted using aluminum T-clips between a length motor (ASI 403A, Aurora Scientific Inc.,
Ontario, Canada) and a force transducer element (ASI 315C-I, Aurora Scientific Inc., Ontario,
Canada) in a single fiber apparatus (ASI 802D, Aurora Scientific Inc., Ontario, Canada) that
was mounted on the stage of an inverted microscope (Zeiss Axio Observer A1). Sarcomere
length was set using a high speed VSL camera and ASI 900B software (Aurora Scientific
Inc., Ontario, Canada). Mechanical experiments were performed at incremental sarcomere
lengths: 2.1 µm – 2.3 µm – 2.5 µm – 2.8 µm – 3.1 µm – 3.4 µm and 3.7 µm. Fiber width and
diameter were measured at three points along the fiber and the cross-sectional area was
determined assuming an elliptical cross-section. Three different types of bathing solutions
were used during the experimental protocols: a relaxing solution, a pre-activating solution
with low EGTA concentration, and an activating solution (for solution composition, see
below). The temperature of the bathing solutions was kept constant at 20°C using a TEC
controller (ASI 825A, Aurora Scientific Inc. Ontario, Canada). During the experiment, data
were automatically collected by a data acquisition board (sampling rate 10 kHz).
Storage solution. Relax/glycerol solution (Rx/Gly) consisted of 50% (v/v) relaxing solution
and glycerol. In Rx/Glylow the following protease inhibitors were added 1.0 mM DTT, 0.24 mM

Page 47 of 55
PMSF, 0.04 mM leupeptin, 0.01 mM E64. Rx/Glyhigh contained higher concentrations of
leupeptin and E64: 1.0 mM DTT, 0.24 mM PMSF, 0.4 mM leupeptin, 0.1 mM E64.
Solutions for force measurements. All solutions had an ionic strength of 180mM and pH 7.1.
The relaxing solution had a negative logarithm of free calcium concentration (pCa) of 9.0 and
comprised of 5.89 mM Na2ATP, 6.48 mM MgCl2, 40.76 mM Kprop, 100 mM BES, 6.97 mM
EGTA, 14.50 mM CrP and low concentration of freshly added protease inhibitors. Pre-
activating solutions consisted of 5.87 mM Na2ATP, 0.1 mM EGTA, 6.42 mM MgCl, 41.14 mM
Kprop, 100 mM BES, 14.50 mM CrP and 6.9 mM HDTA. Activating solutions consisted of
5.97 mM Na2ATP, 7.0 mM CaEGTA, 6.28 mM MgCl, 40.64 mM Kprop, 100 mM BES and
14.50 mM CrP.
Stimulated emission depletion microscopy:
Individual muscle fibers were dissected from the biopsies and permeabilized with Triton-X.
Immunolabelling was performed as described previously2;7-11. In brief, fibers were stretched
and fixed on a glass slide, and incubated with the following antibodies: Alexa Fluor® 488
conjugated phalloidin (A12379, Invitrogen) to stain the thin filament; mouse anti-myosin
heavy chain (A4.1025 DSHB) to stain all myosin heavy chain isoforms followed by Abberior
STAR 635P (goat anti-mouse, Abberior) as secondary antibody to stain the thick filament.
Images were captured using a Leica TCS SP8 STED 3X superresolution microscope with a
100X 1.4 NA oil objective. To acquire superresolution images, a 592nm CW STED laser was
used for the Alexa488 fluorophore. In addition, a 775nm pulsed laser was used for the
Abberior STAR 635P fluorophore. Images were acquired at Nyquist rate, using Nyquist
Calculator (Scientific Volume Imaging SVI) and subsequently deconvolved using Huygens
Professional (SVI). From the deconvolved images, line scans were derived and analyzed
using ImageJ software (National Institutes of Health). For phalloidin line scans, the half width
at half maximum intensity was used as indication of average thin filament length, for myosin

Page 48 of 55
line scans, the full width at half maximum intensity was assumed to indicate average thick
filament length.
Mechanical ventilation of rats
Long-term mechanical ventilation:
To study the effect of long-term mechanical ventilation on diaphragm structure and function,
rats (n=29; age: 5-6 months; body weight: 300-400g; all males) were sedated with 125 mg/kg
S-ketamine (Ketanest, Pfizer, Netherlands) and 4 mg/kg diazepam (Centrafarm, Netherlands)
intraperitoneally, which was maintained by continuous infusion of 40 mg/kg/h S-ketamine and
1 mg/kg/h diazepam intravenously. Rats were endotracheally intubated with a 16G tube, and
mechanically ventilated (UMV-03, UNO, Zevenaar, Netherlands) with oxygen-enriched air
(40% O2/60% N2), 2.5 cmH2O positive end-expiratory pressure, at ~65 breaths per minute,
with a tidal volume of ~10 mL/kg. The respiratory rate was adjusted to maintain pH and
carbon dioxide within physiological limits (arterial pH and pCO2 after 18h MV: 7.36±0.03 and
40.7±1.8 mmHg, respectively). Rats showed no spontaneous respiratory activity. Body
temperature was maintained (36.5 ± 1.0°C) by using a warm water underbody heating pad.
The right femoral artery was cannulated for blood sampling for blood gas analyses (ABL90,
radiometer, Copenhagen, Denmark) and arterial blood pressure registration. Arterial blood
pressure, ECG, and heart rate were continuously recorded using PowerLab software (Chart
8.0; ADInstruments, Castle Hill, Australia). Then, eighteen rats were sacrificed by cervical
dislocation and the whole diaphagm was immediately excised (duration of mechanical
ventilation ~15 minutes). This group served as the control group. The other eleven rats were
mechanically ventilated for 18 hours, after which the whole diaphragm was excised. From the
excised diaphragms a strip from the costal diaphragm was dissected. Silk suture was
attached to both ends of the strip, and the strip was mounted vertically in a temperature-
controlled tissue bath between a dual-mode lever arm and a fixed hook (1200A Intact Muscle
Test System; Aurora Scientific, Aurora, ON, Canada), as described previously5;12. Muscles
were bathed in continuously oxygenated (95% O2, 5% CO2) mammalian Ringer solution with

Page 49 of 55
pH 7.40 at 30°C, and stimulated directly by using platinum electrodes placed in close
apposition to the muscle. To determine the optimal muscle length (MLopt) for force
generation, the diaphragm strips were stretched from slack length (no passive tension) with
small steps. At each step, maximal tetanic force (150Hz stimulation frequency) was
determined. The length at which force did not increase compared to the force measured at
the previous length was considered MLopt. After completion of the contractility
measurements, length and weight of the muscle preparations were determined. Cross‐
sectional area (in mm2) was calculated by dividing muscle weight (g) by muscle length (cm)
multiplied by specific density (1.056 g/ml) x 100. Force was normalized to muscle cross‐
sectional area (tension, in mN/mm2).
A second strip was dissected from the rat diaphragms and was stored in relax/glycerol with
high concentration protease inhibitors (Rx/Glyhigh) and initially placed on a roller band for 24h
at 4°C. Finally, Rx/Glyhigh was substituted with Rx/Gly with lower concentrations (Rx/Glylow) of
protease inhibitors and stored at -20°C until further use. The protocol for contractility of
individual, permeabilized diaphragm fibers and for STED imaging is similar to that applied on
patient fibers (for details, see above). Note that filament length was determined at 9-12
locations along the entire length of the fibers.
For the determination of the number of sarcomeres in series, a third strip was isolated from
the midcostal region of the diaphragm. This strip was dissected directly adjacent to the one
used for intact strip mechanics, and the dissection location of this strip was identical in each
rat (location demarcated by the inferior phrenic artery). The strip was snap-frozen in liquid
nitrogen and cryosections were cut along the fiber direction. Cryosections were stained with
an α-actinin antibody (Sigma A7811) and sarcomere length was determined at nine locations
along the entire length of the fibers (at each position 20-50 z-discs were included). Total fiber
length was divided by average sarcomere length to determine the number of sarcomeres in
series.

Page 50 of 55
To test the effect of titin-based mechanosensing on diaphragm remodeling during 18h of MV,
we utilized a rat that harbors a homozygous autosomal mutation in the RNA-binding motif-20
13;14
gene (Rbm20) causing expression of a giant titin isoform . The mechanical ventilation
experiment and the diaphragm mechanics studies were identical to those described above.
For passive tension measurements, individual diaphragm fibers were mounted using
aluminum T-clips between a length motor (ASI 403A, Aurora Scientific Inc., Ontario, Canada)
and a force transducer element (ASI 315C-I, Aurora Scientific Inc., Ontario, Canada) in a
single fiber apparatus (ASI 802D, Aurora Scientific Inc., Ontario, Canada) that was mounted
on the stage of an inverted microscope (Zeiss Axio Observer A1). Sarcomere length was set
at slack length and fibers were stretched to preset sarcomere lengths using a high speed
VSL camera and ASI 900B software (Aurora Scientific Inc., Ontario, Canada). Sarcomere
lengths were: 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, and 3.4 µm. Fiber was held at each sarcomere
length for three minutes and passive force was determined at the end of each hold phase.
Fiber width and diameter were measured at three points along the fiber and the cross-
sectional area was determined assuming an elliptical cross-section. For titin mobility studies,
15-17
SDS-agarose electrophoresis was performed as previously described . Briefly, muscle
samples were pulverized to a fine powder and then rapidly solubilized and analyzed by
vertical SDS-agarose electrophoresis. For estimation of molecular mass of titin isoforms, the
samples of interest were co-electrophoresed together with human soleus and left ventricle
samples as these contain titin isoforms of known sizes (human soleus titin T1: 3.7 MDa;
human left ventricle N2B T1: 3.0 MDa and N2BA: 3.3 MDa; Rbm20∆RRM mouse left ventricle
N2BA: 4.0 MDa). Because migration distance in the gel scales with the logarithm of
molecular weight, we could estimate the molecular weight of the proteins based on their
migration distance, as shown previously 15;18.
Acute effect of PEEP on diaphragm position:
In a subset of rats, ten minutes after start of mechanical ventilation ultrasound (Visualsonics
Vevo 200) was applied to visualize the effect of PEEP on diaphragm length. The PEEP level

Page 51 of 55
was set at 0, 2, 3, and 5 cmH20 and at each level the position of the diaphragm was
monitored by M-mode ultrasound using a fixed echo probe (to prevent movement of the echo
probe relative to the diaphragm). Subsequently, PEEP was set at 2.5 cmH2O, and rats were
perfused through the left jugular vein with 8% formaldehyde in PBS. To avoid excess fluid,
the femoral vein was cut. After fixation, the diaphragm was excised and a strip from the mid-
costal region was used to determine fiber and sarcomere length (fiber length measured with
a caliper and sarcomere length using a VSL camera and ASI 900B software (Aurora
Scientific, Canada).
Note that, if feasible, experimentators were ‘blinded’ during data analyses, for example
during analysis of data from the experiments involving microscopy (number of sarcomeres in
series, thin and thick filament lengths) and fiber mechanics (force-sarcomere length relations,
both in rats and humans). Due to the nature of the experiments, those involving intact muscle
mechanics (MLopt, maximal tetanic tension) could not be performed blinded: they needed to
be performed directly after extubating the rats.
Statistical analyses
Normally distributed data are represented as the mean ± standard error of the mean (SEM),
and a Students t-test was used to test for significant differences. When the data was not
normally distributed, a Mann-Whitney-U test was used on the averages per patient. Data
were analyzed by GraphPad Prism version 6.07 (GraphPad Software, USA). Differences
between groups were considered significant if P<0.05.

Page 52 of 55
Table E1. Pulmonary function data of control patients
Control # FEV1 (L) FEV1 (% pred) FVC (L) FVC (% pred) FEV/FVC
1 2.3 68 3.3 76 0.69
2 2.9 104 3.7 116* 0.77
3 3.1 78 5.9 118 0.52
4 2.9 127 3.4 120* 0.86
5 2.0 79 NR NR 0.75
6 1.9 76 3.1 NR 0.62
7 2.4 92 3.3 86 0.72
8 2.4 102 3.2 115 0.75
9 4.9 104 6.8 116 0.73
10 3.6 84 6.0 110 0.60
11 2.0 73 3.1 95 0.65
12 2.7 86 3.6 87 0.76
Definition of abbreviations: FEV1 (L) = forced expiratory volume; L = liters; % pred = percentage of predicted value of normal;
FVC = forced vital capacity; NR = not registered. * Vital capacity (% pred), because FVC (% pred) was not registered.

Page 53 of 55
Reference List
1. Hooijman, P. E., A. Beishuizen, M. C. de Waard, F. S. de Man, J. W. Vermeijden, P. Steenvoorde,
R. A. Bouwman, W. Lommen, H. W. van Hees, L. M. Heunks, et al. 2014. Diaphragm fiber
strength is reduced in critically ill patients and restored by a troponin activator. Am.J.Respir.Crit
Care Med. 189:863-865.
2. Ottenheijm, C. A., P. Hooijman, E. T. DeChene, G. J. Stienen, A. H. Beggs, and H. Granzier. 2010.
Altered myofilament function depresses force generation in patients with nebulin-based
nemaline myopathy (NEM2). J.Struct.Biol. 170:334-343.
3. Ottenheijm, C. A., C. Hidalgo, K. Rost, M. Gotthardt, and H. Granzier. 2009. Altered contractility
of skeletal muscle in mice deficient in titin's M-band region. J.Mol.Biol. 393:10-26.
4. de Man, F. S., H. W. van Hees, M. L. Handoko, H. W. Niessen, I. Schalij, M. Humbert, P.
Dorfmuller, O. Mercier, H. J. Bogaard, P. E. Postmus, et al. 2011. Diaphragm muscle fiber
weakness in pulmonary hypertension. Am.J.Respir.Crit Care Med. 183:1411-1418.
5. Hooijman, P. E., A. Beishuizen, C. C. Witt, M. C. de Waard, A. R. Girbes, A. M. Spoelstra-de Man,
H. W. Niessen, E. Manders, H. W. van Hees, C. E. van den Brom, et al. 2015. Diaphragm muscle
fiber weakness and ubiquitin-proteasome activation in critically ill patients. Am.J.Respir.Crit
Care Med. 191:1126-1138.
6. Manders, E., G. Ruiter, H. J. Bogaard, G. J. Stienen, A. Vonk-Noordegraaf, F. S. de Man, and C. A.
Ottenheijm. 2015. Quadriceps muscle fibre dysfunction in patients with pulmonary arterial
hypertension. Eur.Respir.J. 45:1737-1740.
7. Lawlor, M. W., C. A. Ottenheijm, V. L. Lehtokari, K. Cho, K. Pelin, C. Wallgren-Pettersson, H.
Granzier, and A. H. Beggs. 2011. Novel mutations in NEB cause abnormal nebulin expression

Page 54 of 55
10
and markedly impaired muscle force generation in severe nemaline myopathy. Skelet.Muscle
1:23.
8. Ottenheijm, C. A., M. W. Lawlor, G. J. Stienen, H. Granzier, and A. H. Beggs. 2011. Changes in
cross-bridge cycling underlie muscle weakness in patients with tropomyosin 3-based
myopathy. Hum.Mol.Genet. 20:2015-2025.
9. Ottenheijm, C. A., C. C. Witt, G. J. Stienen, S. Labeit, A. H. Beggs, and H. Granzier. 2009. Thin
filament length dysregulation contributes to muscle weakness in nemaline myopathy patients
with nebulin deficiency. Hum.Mol.Genet. 18:2359-2369.
10. Ottenheijm, C. A., D. Buck, J. M. de Winter, C. Ferrara, N. Piroddi, C. Tesi, J. R. Jasper, F. I. Malik,
H. Meng, G. J. Stienen, et al. 2013. Deleting exon 55 from the nebulin gene induces severe
muscle weakness in a mouse model for nemaline myopathy. Brain 136:1718-1731.
11. Winter, J. M., B. Joureau, E. J. Lee, B. Kiss, M. Yuen, V. A. Gupta, C. T. Pappas, C. C. Gregorio, G.
J. Stienen, S. Edvardson, et al. 2016. Mutation-specific effects on thin filament length in thin
filament myopathy. Ann.Neurol. 79:959-969.
12. Manders, E., F. S. de Man, M. L. Handoko, N. Westerhof, H. W. van Hees, G. J. Stienen, A. Vonk-
Noordegraaf, and C. A. Ottenheijm. 2012. Diaphragm weakness in pulmonary arterial
hypertension: role of sarcomeric dysfunction. Am.J.Physiol Lung Cell Mol.Physiol 303:L1070-
L1078.
13. Guo, W., S. Schafer, M. L. Greaser, M. H. Radke, M. Liss, T. Govindarajan, H. Maatz, H. Schulz, S.
Li, A. M. Parrish, et al. 2012. RBM20, a gene for hereditary cardiomyopathy, regulates titin
splicing. Nat.Med. 18:766-773.

Page 55 of 55
11
14. Greaser, M. L., C. M. Warren, K. Esbona, W. Guo, Y. Duan, A. M. Parrish, P. R. Krzesinski, H. S.
Norman, S. Dunning, D. P. Fitzsimons, et al. 2008. Mutation that dramatically alters rat titin
isoform expression and cardiomyocyte passive tension. J.Mol.Cell Cardiol. 44:983-991.
15. Warren, C. M., P. R. Krzesinski, and M. L. Greaser. 2003. Vertical agarose gel electrophoresis
and electroblotting of high-molecular-weight proteins. Electrophoresis 24:1695-1702.
16. Ottenheijm, C. A., A. M. Knottnerus, D. Buck, X. Luo, K. Greer, A. Hoying, S. Labeit, and H.
Granzier. 2009. Tuning passive mechanics through differential splicing of titin during skeletal
muscle development. Biophys.J. 97:2277-2286.
17. van Hees, H. W., W. J. Schellekens, G. L. ndrade Acuna, M. Linkels, T. Hafmans, C. A.
Ottenheijm, H. L. Granzier, G. J. Scheffer, J. G. van der Hoeven, P. N. Dekhuijzen, et al. 2012.
Titin and diaphragm dysfunction in mechanically ventilated rats. Intensive Care Med. 38:702-
709.
18. Granzier, H. L. and T. C. Irving. 1995. Passive tension in cardiac muscle: contribution of
collagen, titin, microtubules, and intermediate filaments. Biophys.J. 68:1027-1044.

Positive End-Expiratory Pressure Ventilation Induces Longitudinal Atrophy in Diaphragm Fibers

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Positive End-Expiratory Pressure Ventilation Induces Longitudinal Atrophy in Diaphragm Fibers

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AJRCCM Articles in Press. Published on 26-March-2018 as 10.1164/rccm.

POSITIVE END-EXPIRATORY PRESSURE VENTILATION INDUCES

*: both authors contributed equally

Copyright © 2018 by the American Thoracic Society

Conflicts of interest: None of the authors has a financial relationship with a

Sources of support: Coen A. C. Ottenheijm was supported by National Heart, Lung,

Running head: Longitudinal atrophy of the diaphragm in ICU

Descriptor number: 4.08 Mechanical Ventilation: Physiology & Pathophysiology

Total word count: 3500

Copyright © 2018 by the American Thoracic Society

expiratory pressure (PEEP) are unknown.

What This Study Adds to the Field:

longitudinal atrophy. Consequently, diaphragm fibers are at a mechanical disadvantage

when PEEP is released. In experimental models we have identified specific molecular

pathways, including titin-based mechanosensing, that are involved in the development

Copyright © 2018 by the American Thoracic Society

RATIONALE: Diaphragm weakness in critically ill patients prolongs ventilator

costs. The mechanisms underlying diaphragm weakness include cross-sectional fiber

positive end-expiratory pressure (PEEP) induces longitudinal atrophy by displacing the

structure and function of diaphragm fibers of mechanically ventilated critically ill

cmH2O) adapt to the reduced length by absorbing serially-linked sarcomeres, the

compliance of titin molecules reduces longitudinal atrophy. CONCLUSION: Mechanical

ventilation with PEEP results in longitudinal atrophy of diaphragm fibers, a response

that longitudinal atrophy, in concert with the aforementioned cross-sectional atrophy,

diaphragm weakness ensues.

Copyright © 2018 by the American Thoracic Society

muscle weakness1-6. Diaphragm weakness contributes to weaning failure and is

associated with increased mortality rates6-9. For establishing strategies to prevent or

treat diaphragm weakness, a fundamental understanding of the underlying cellular

mechanisms is crucial. Studies in mechanically ventilated brain dead organ donors

suggest that reduced cross-sectional area of diaphragm fibers caused by activation of

fibers is severely impaired13-15, partly accounted for by a reduced cross-sectional area

ventilation on diaphragm fiber length reduction, from here on referred to as longitudinal

fiber atrophy (as opposed to the aforementioned cross-sectional fiber atrophy).

(PEEP) to limit alveolar collapse. However, PEEP-induced increase of end-expiratory

modifications in the diaphragm fibers.

We hypothesized that: (1) the PEEP-induced increase in end-expiratory lung volume

Copyright © 2018 by the American Thoracic Society

ventilation with PEEP16.

To test these hypotheses, we studied muscle fiber structure and contractility in

diaphragm biopsies of mechanically ventilated critically ill patients, and in mechanically

a more compliant titin molecule17.

Additional details are in the online data supplement.

the caudal-cranial displacement of the diaphragm dome was calculated.

from patients undergoing tumor removal of a suspected early-stage lung malignancy

Copyright © 2018 by the American Thoracic Society

Long-term mechanical ventilation and contractility of intact diaphragm strips: Rats

supplement13;27. To test the effect of titin-based mechanosensing on diaphragm

remodeling, we utilized rats harboring a mutation in Rbm20 causing expression of a

giant titin isoform17;28. For details see online data supplement29;30-32.

Acute effect of PEEP on diaphragm position: In a subset of ventilated rats, ultrasound

subjects are described in Table E1 (online data supplement).

Copyright © 2018 by the American Thoracic Society

length in critically ill patients and rats.

ultrasound to determine diaphragm displacement in critically ill patients caused by a

decrease in PEEP (∆PEEP) of 5 cmH2O or 10 cmH2O. Figure 1A (left and middle

displacement of the diaphragm during ∆PEEP is visualized. Acute release of 5 cmH2O

displacement of 0.40±0.10 cm (Fig.1A, right panel). Acute ∆PEEP of 10 cmH2O caused

a cranial diaphragm displacement in all ten patients, with an average displacement of