Diaphragm remodeling and compensatory respiratory

mechanics in a canine model of Duchenne muscular

dystrophy
A. F. Mead, M. Petrov, A. S. Malik, M. A. Mitchell, M. K. Childers, J. R. Bogan,
G. Seidner, J. N. Kornegay and H. H. Stedman
J Appl Physiol 116:807-815, 2014. First published 9 January 2014; doi:10.1152/japplphysiol.00833.2013

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Diaphragm remodeling and compensatory respiratory mechanics in a canine
model of Duchenne muscular dystrophy
A. F. Mead,1 M. Petrov,1 A. S. Malik,1 M. A. Mitchell,1 M. K. Childers,2 J. R. Bogan,3 G. Seidner,1
J. N. Kornegay,3 and H. H. Stedman1
1
Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 2Institute for
Regenerative Medicine, School of Medicine, Wake Forest University, Winston-Salem, North Carolina; and 3Department of
Pathology and Laboratory Medicine, School of Medicine, University of North Carolina, Chapel Hill, North Carolina
Submitted 16 July 2013; accepted in final form 1 January 2014
Mead AF, Petrov M, Malik AS, Mitchell MA, Childers MK,
Bogan JR, Seidner G, Kornegay JN, Stedman HH. Diaphragm re-
modeling and compensatory respiratory mechanics in a canine model of
Duchenne muscular dystrophy. J Appl Physiol 116: 807– 815, 2014. First
published January 9, 2014; doi:10.1152/japplphysiol.00833.2013.—Ven-
tilatory insufficiency remains the leading cause of death and late stage
morbidity in Duchenne muscular dystrophy (DMD). To address crit-
ical gaps in our knowledge of the pathobiology of respiratory func-
tional decline, we used an integrative approach to study respiratory
mechanics in a translational model of DMD. In studies of individual
dogs with the Golden Retriever muscular dystrophy (GRMD) muta-
tion, we found evidence of rapidly progressive loss of ventilatory
capacity in association with dramatic morphometric remodeling of the
diaphragm. Within the first year of life, the mechanics of breathing at
rest, and especially during pharmacological stimulation of respiratory
control pathways in the carotid bodies, shift such that the primary role
of the diaphragm becomes the passive elastic storage of energy
transferred from abdominal wall muscles, thereby permitting the
expiratory musculature to share in the generation of inspiratory
pressure and flow. In the diaphragm, this physiological shift is
associated with the loss of sarcomeres in series (⬃60%) and an
increase in muscle stiffness (⬃900%) compared with those of the
nondystrophic diaphragm, as studied during perfusion ex vivo. In
addition to providing much needed endpoint measures for assessing
the efficacy of therapeutics, we expect these findings to be a starting
point for a more precise understanding of respiratory failure in DMD.
respiratory mechanics; dystrophin; animal models; diaphragm; mus-
cular dystrophy
DUCHENNE MUSCULAR DYSTROPHY (DMD) is a severe, childhood-
onset muscle wasting disease with an approximate incidence of
1 in 3,500 boys caused by mutations in the X-linked dystrophin
gene (28). As with less common childhood-onset muscular
dystrophies, progressive weakness in muscles of locomotion
dominates the clinical course to loss of ambulation, but the
leading cause of late-stage morbidity and mortality is progres-
sive failure of the respiratory pump (4, 21, 25). Patient-oriented
studies have defined the rate of decline in standardized indices
from pulmonary function tests (4, 21, 27), and studies in mdx
mice have provided mechanistic information about some of the
molecular and cellular correlates of muscle degeneration (9,
42). However, a reliable model of the effect of dystrophin’s
absence on respiratory mechanics and loss of ventilatory ca-
pacity has been elusive. Whereas the diaphragm of the mdx
Address for reprint requests and other correspondence: H. H. Stedman,
Dept. of Surgery, Perelman School of Medicine, Univ. of Pennsylvania, 422
Curie Blvd. Rm. 709a, Philadelphia, PA 19104 (e-mail: hstedman@mail.
med.upenn.edu).
http://www.jappl.org 8750-7587/14 Copyright © 2014 the American Physiological Society
J Appl Physiol 116: 807–815, 2014.
First published January 9, 2014; doi:10.1152/japplphysiol.00833.2013.
mouse is its most severely affected muscle, studies of respira-
tory mechanics have been limited to whole-body plethysmog-
raphy, limiting the extent to which the contribution of different
respiratory muscle groups can be determined (29, 30). There-
fore, there is a compelling need for a model of respiratory
mechanics in DMD to improve our understanding of the
disease process in man, and to provide a platform for thera-
peutic development. Golden Retriever muscular dystrophy
(GRMD) is an important translational model of DMD that
exhibits the major hallmarks of the human disease on a greatly
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accelerated time scale (33, 47), but its potential as a model of
respiratory pathobiology in DMD has not been fully exploited.
To address these needs, we applied both novel and classical
experimental approaches to assess the age-related loss of ven-
tilatory capacity in GRMD and the accompanying changes in
respiratory mechanics. In DMD, exertional increase in meta-
bolic rate is limited by global weakness of the muscles of
locomotion, rendering the progressive decline of ventilatory
capacity early in DMD clinically silent (4). Consequently,
assessment of respiratory mechanics at higher ventilatory rates
has been challenging. In a canine model, we used doxapram to
exploit the pharmacodynamics of direct channel activation in
the sensory afferents controlling respiratory drive, thereby
dissociating respiratory mechanics from metabolic loading.
This allowed us to test hypotheses related to changes in the
mechanics of tidal breathing in the setting of elevated respira-
tory drive in relatively young dogs with the GRMD mutation
and with no overt clinical signs of ventilatory insufficiency.
In DMD, the diaphragm is affected earlier and more severely
than other skeletal muscles. This is possibly due to heavy use
as the primary driver of ventilation at rest. We used esophageal
and gastric balloon manometry to test the hypothesis that
doxapram stimulation would uncover deficits in transdiaphrag-
matic pressure (Pdi) caused by loss of diaphragm contractile
capacity in GRMD during tidal breathing.
The two-compartment chest wall model of respiration first
established by Konno and Mead allows for the relative contri-
bution of opposing inspiratory muscles to be estimated nonin-
vasively by comparing volume changes in the abdomen (VAB)
and rib cage (VRC) approximated by respiratory inductance
plethysmography (RIP) (11, 31). Because positive change in
VAB during inspiration is caused largely by the shortening and
caudal motion of the diaphragm, the diaphragm’s contribution
to inspiration can be estimated by measuring ⌬VAB expressed
as a percentage of total tidal volume (Vt) during normal
breathing. In DMD, clinical studies suggest that the dia-
phragm’s contribution to inspiration at rest progressively di-
minishes during the first two decades of life, with ⌬VAB
807
808 Respiratory Dysfunction in the Dystrophin-Deficient Dog
reduced from ⬃70% to ⬃30% by the onset of nocturnal
oxygen desaturation (37, 44). In very advanced DMD, severe
diaphragm weakness sometimes results in abdominal paradox:
cranial diaphragm motion during inspiration as negative chest
wall pressure developed by the intercostal and auxiliary mus-
cles overwhelms the diaphragm (11, 23, 39). We hypothesized
that a similar shift in VRC/VAB partitioning would be observed
in GRMD at rest, that loss of abdominal volumetric contribu-
tion would limit increases in Vt under stimulated conditions,
and that stimulated metabolic loading would uncover abdom-
inal paradox absent at rest in dogs with no clinical signs of
ventilatory insufficiency.
Simultaneous recording of compartmental volumes and
pressures can be used to assess the contribution of nondia-
phragm inspiratory and expiratory muscles. We predicted that
during periods of stimulated respiratory drive, dogs with the
GRMD mutation would rely more heavily on inspiratory and
expiratory intercostal and abdominal muscles than normal dogs
to achieve similar rates of ventilation.
Dystrophin-deficient muscles undergo progressive changes
in passive as well as active properties (49), and radiographic
and NMR studies have reported thickening of the GRMD
diaphragm (7, 51). We tested the hypothesis that changes to the
morphometry of the GRMD diaphragm itself, in particular the
presence of additional interstitial collagen resulting from fibro-
sis, would affect the muscle’s passive mechanical properties,
potentially impacting overall respiratory mechanics at rest and
under load. To test this, we developed and utilized a novel
system for oxygenated perfusion of the isolated hemidia-
phragm.
MATERIALS AND METHODS
Animals. This study included 34 dogs with the GRMD mutation; 4
dystrophin-expressing GRMD-carrier dogs; and 17 unaffected, nor-
mal dogs, either from the GRMD breeding colony or mongrel, ranging
from 4 to 18 mo of age. Seven dogs with the GRMD mutation and five
normal control dogs were included in doxapram studies under anes-
thesia at the time of euthanasia. All animals used in this study were
cared for and used humanely in accordance with the requirements
specified by the Institutional Animal Care and Use Committees at the
University of Pennsylvania, the University of North Carolina-Chapel
Hill, and Wake Forest University, and in accordance with the Guide
for the Care and Use of Laboratory Animals (revised 1996; National
Research Council, Washington, DC). RIP studies of awake dogs were
conducted at the Perelman School of Medicine, University of Penn-
sylvania, University of North Carolina-Chapel Hill, and Wake Forest
University. All doxapram studies were conducted at the University of
Pennsylvania. Although all dogs were a part of other research proj-
ects, no intervention was expected to affect parameters assessed in the
present study.
Respiratory inductance plethysmography. RIP traces were gener-
ated by inductance bands embedded in the Lifeshirt jacketed telemetry
system (Vivometrics) and analyzed using Vivologic software (Vivo-
metrics). Shirts from a range of sizes were fitted to dogs with the
primary aim being proper location of rib cage (RC) and abdominal
(AB) inductance bands. The RC band was located at the level of the
fourth rib, and the AB band just caudal to the rib cage. Measurements
of awake and anesthetized dogs were made in the laterally recumbent
posture. The standard method of calibrating RIP traces involves
performing an isovolume maneuver to establish the relative contribu-
tion of RC and AB signals to Vt (31). Because this method is
inapplicable to dogs, we used the quantitative diagnostic calibration
(QDC) technique first described by Sackner et al. for use in humans,
J Appl Physiol • doi:10.1152/japplphysiol.00833.2013 • www.jappl.org
• Mead AF et al.
and subsequently applied to dogs (41, 42, 45). QDC compares
standard deviations of raw RC and AB signals collected over ⬃5 min
of regular, quiet breathing to calculate a proportionality constant, K,
which relates the relative contributions of those signals to Vt. Subse-
quently, Vt as measured by spirometry over the same period, is used
to calculate the scaling factor, M, which scales the proportionally
calibrated sum of RC and AB signals to real Vt. Our calibration
procedures differed slightly between dogs studied during wakeful
breathing and those included in the anesthetized doxapram study. In
awake dogs, when we were interested in the proportional contribution
of AB and RC compartments, we calculated K alone without spirom-
etry. These animals were fitted with the Lifeshirt as described above,
placed in lateral recumbency, and allowed to breath quietly for ⬃5
min. QDC calibration was performed on RC and AB traces from this
period. For the doxapram study, dogs were intubated and anesthetized
(2% isoflurane), and placed in lateral recumbency before performing
the QDC calibration as described above. In addition, we calculated the
scaling factor, M, to establish approximate, RIP-derived values for Vt,
VAB, and VRC by using spirometry data from a Datex Omeida
anesthesia machine, which integrates signals from syringe-calibrated
flow sensors to determine Vt.
We measured respiratory phase angle from RIP tracing as an index
of abdominal paradox. Phase angle is a measure of the degree to
which RC and AB movements are synchronized during tidal breath-
ing. A phase angle of zero indicates fully synchronous motion,
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whereas 180° indicates a state in which RC and AB are exactly
asynchronous. Values from 1° to 180° reflect AB lagging behind RC
by a proportional amount of the breathing cycle. Vivologic software
calculates phase angle on a breath-by-breath basis as described (3).
We averaged breath-by-breath phase angle measurements over ap-
proximately 30-s periods of regular quiet breathing or, in the case of
doxapram, 30 s beginning at peak Vt following doxapram adminis-
tration.
Pressures. Gastric pressure (Pgas) and esophageal pressure (Pes)
were measured as described (40) using balloons constructed from an
intraaortic balloon pump coronary perfusion support system (Fi-
deltity; Datascope). These polyethylene balloons have the advantage
of being mounted on long, flexible cannulae. The balloon material is
inelastic, and therefore does not contribute to Pgas or Pes as a result of
any inherent recoil. The balloons were connected to calibrated
TSD104A pressure transducers (Biopac Systems), the output digitized
at 500 Hz with an MP150c data acquisition system (Biopac Systems),
and analyzed using AcqKnowledge software (Biopac Systems). Inter-
breath pressures were set to zero during restful breathing. Transdia-
phragmatic pressure (Pdi) was calculated by subtracting Pes from Pgas.
Anesthesia and doxapram studies. Dogs were induced with dex-
medetomidine hydrochloride (375 ␮g/m2) (Pfizer Animal Health)
before being masked down with isoflurane (Baxter Healthcare), intu-
bated, and maintained at 2% isoflurane continuously throughout the
study in a laterally recumbent position. Gastric and esophageal bal-
loon catheters were placed, and the Lifeshirt jacket was fitted to the
dog as described above. Doxapram hydrochloride (Baxter Healthcare)
bolus was administered via the saphenous vein at 1 mg/kg, followed
by a second dose of 2 mg/kg 5 min later.
Histology. The length of the costal muscular diaphragm was mea-
sured in vivo at three standardized places through an incision in the
abdominal wall at necropsy. To determine sarcomere length at these
dimensions, strips were clamped with two-headed biopsy forceps,
excised, and fixed in 10% formalin. Diaphragm thickness was mea-
sured once at this point. Fixed tissue blocks were sectioned longitu-
dinally, stained with hematoxylin and eosin (Fisher Scientific) as
described (6), and imaged at 20⫻. Sarcomere length was determined
by measuring the length of 10 sarcomeres by eyepiece micrometer and
dividing by 10. In addition, diaphragm, external and internal inter-
costal, abdominal wall, and cranial tibialis muscle blocks were ex-
cised and frozen in liquid nitrogen-cooled isopentane. These were
later thawed and fixed in 10% formalin for 24 h prior to paraffin
 Respiratory Dysfunction in the Dystrophin-Deficient Dog • Mead AF et al. 809
strip was fixed to the arm of a calibrated 305C-LR muscle lever
(Aurora Scientific), and the rib was solidly fixed in place. Unipolar
electrodes (Biopac Systems) connected to a model S88 stimulator
(Grass Technologies) were used to elicit twitch and tetanus contrac-
tions to establish viability and L0 as described (41). Sets of five
lissajous loops were performed at 0.5, 1, 2, and 5 Hz. Length and force
were sampled at 500 Hz using an MP150 data acquisition system
(Biopac Systems) and analyzed in AcqKnowledge software (Biopac
Systems).
Statistics. All data in figures are presented as means ⫾ SD.
Significance between groups was tested with paired or unpaired
Student’s t-test as appropriate; P ⱕ 0.05 is considered significant and
is denoted with an asterisk.

Fig. 1. A: cross-sectional study of diaphragmatic contribution to breathing RESULTS

(⌬VAB) at rest in 22 dogs with the GRMD mutation (x), 3 dystrophin-
expressing carrier dogs (⌬), and 12 normal dogs (o) shows a loss of diaphragm
function in GRMD. B: decline in ⌬VAB at rest in seven individual dogs with
the GRMD mutation measured at two time points.
embedding and sectioning at 7 ␮m. Sections were stained with
hematoxylin and eosin or picrosirius red (Sigma-Aldrich) and imaged
at 20⫻ in brightfield. Images were analyzed for collagen cross-
sectional area using Image Pro 7. Image files were converted from the
Diaphragms in animals with the GRMD mutation are dys-
functional during quiet breathing. We used RIP to measure
⌬VAB in 22 dogs with the GRMD mutation, 12 normal dogs,
and 3 dogs that were dystrophin-expressing GRMD carriers
that were laterally recumbent, during periods of wakeful, quiet
breathing. Dogs with the GRMD mutation showed reduced
⌬VAB compared with normal and carrier dogs (20.2 ⫾ 15.8%
vs. 54.2 ⫾ 16.3%; P ⬍ 0.01) indicating reduced diaphragm

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RGB (red, green, blue) color model to the YIQ (in-phase/quadrature)
color model. Channel Y (luminance) was extracted from the YIQ
image. The extracted Y channel is an 8-bit, gray-scale image. Image
segmentation was histogram-based; double-threshold intensity ranges
were set as follows: contractile apparatus, 0 –140; collagen, 140 –205.
Data were exported to Microsoft Excel for further analysis. Investi-
gators were blinded to genotype and muscle origin for each slide, and
collagen cross-sectional area (CSA) was quantified for each by aver-
aging three 500-␮m2, nonoverlapping images.
Ex vivo diaphragm mechanics. A section of costal hemidiaphragm
was removed at the time of euthanasia and rapidly cooled on ice. The
internal thoracic artery was cannulated, and the muscle was perfused
with perfusate warmed to 37°C. Perfusate was a modified Krebs-
Henseleit buffer containing, in mmol/l: CaCl2, 1.5; glucose, 5.5; KCl,
4.7; MgSO4, 1.66; NaCl, 118.0; NaH2PO4, 1.18; NaHCO3, 24.88; and
Na-pyruvate, 2.0 in H2O, to which was added 20% heparinized whole
blood. The perfusate was oxygenated and warmed by means of an
Apex cardiopulmonary bypass membrane oxygenator (Cobe Cardio-
vascular) fed with a mixture of 95% O2 and 5% CO2. The oxygen-
ator’s integral heat exchanger was warmed by a water bath (Haake A
28F/SC 100; Thermo Fisher) to maintain perfusate at 37°C. After
warming for 10 min, a strip roughly 1 cm wide was cut in the
muscular diaphragm parallel to the direction of shortening and left
attached to the rib at the costal margin. The central-tendon end of the
range of motion and contribution to inspired volume. The
difference was larger in older dogs (Fig. 1A). Seven dogs with
the GRMD mutation from this group were studied at two time
points, and all showed an age-related decline in ⌬VAB (Fig.
1B) at rest. Although normal dogs were not measured at
multiple time points, no normal (or carrier) dog over 1 year of
age showed a ⌬VAB of less than 60%.
Pdi in dogs with the GRMD mutation is reduced at rest and
during direct stimulation of respiratory drive. To address
whether doxapram would uncover deficits in Pdi caused by loss
of diaphragm contractile capacity in GRMD we used balloon
manometry to trace Pdi throughout the hyperventilatory re-
sponse to doxapram bolus injection. In anesthetized and instru-
mented dogs with the GRMD mutation (n ⫽ 7) and normal
(n ⫽ 5) dogs, we administered doxapram intravenously to
temporarily stimulate an abrupt increase in ventilation while
monitoring respiratory performance. Table 1 shows ages,
weights, and basic respiratory parameters of dogs included in
the doxapram study. End-inspiratory Pdi in dogs with the
GRMD mutation was lower than in normal dogs at rest (5.45 ⫾
2.39 mmHg vs. 7.8 ⫾ 1.11 mmHg; P ⬍ 0.05) and at the peak

Table 1. Major ventilatory parameters of 7 dogs with the GRMD mutation and 5 normal control dogs included in the
doxapram study
Ventilation, Ventilation, Respiratory rate, Respiratory rate,
Animal Dystrophin, Age, Weight, ml·kg⫺1·min⫺1 ml·kg⫺1·min⫺1 Tidal volume, Tidal volume, ml/kg breaths/min breaths/min
ID ⫾ months kg (baseline) (1 mg/kg doxapram) ml/kg (baseline) (1 mg/kg doxapram) (baseline) (1 mg/kg doxapram)

g892 ⫺ 10 16.6 17.5 246.5 3.5 12.1 5 25

g092 ⫺ 13 13 142 375.3 10.9 22.1 13 17
g124 ⫺ 6 9.8 58.2 189.8 5.8 9.7 10 20
g547 ⫺ 17 17.3 57.5 372.3 8.2 18.6 7 23
g033 ⫺ 16 19.3 116.5 408 9 17.3 13 25
g566 ⫺ 16 13.8 38.8 333.8 7.8 19.6 5 17
g890 ⫺ 17 20 90.8 308.1 5.7 10.6 16 29
n290 ⫹ 7 21.5 109.8 304.4 11 23.3 10 16
n559 ⫹ 7 20.3 83.1 463.3 13.8 31.3 6 15
n530 ⫹ 9 23 82.8 346.4 10.3 22.6 8 16
n256 ⫹ 13 27.8 135.1 330.9 11.3 21.6 12 19
n016 ⫹ 7 20 115 576.3 10.5 24 10 25

J Appl Physiol • doi:10.1152/japplphysiol.00833.2013 • www.jappl.org

810 Respiratory Dysfunction in the Dystrophin-Deficient Dog
Fig. 2. A: maximum ventilation achieved in response to intravenous doxapram
administration under anesthesia in seven dogs with the GRMD mutation and
five normal dogs. Although both groups responded equally to the initial
doxapram dose (1 mg/kg), dogs with the GRMD mutation failed to further
increase maximum ventilation after a second, higher dose (2 mg/kg). B and C:
ventilation-Vt and ventilation-RR relationships after doxapram bolus (1 mg/
kg) in seven dogs with the GRMD mutation (x) and five normal (o) dogs.
Points along curves are at 10-s intervals following bolus from lower left to
upper right. Dogs with the GRMD mutation employed higher respiratory rates
earlier to achieve equivalent increases in ventilation. D: respiratory phase angle
was not different between groups before or during maximal doxapram-
mediated ventilation.
effect following 1 mg/kg doxapram (10.44 ⫾ 4.4 mmHg vs.
16.26 ⫾ 2.92 mmHg; P ⬍ 0.05).
Dogs with the GRMD mutation lose volumetric reserve
during direct stimulation of respiratory drive. We were inter-
ested in whether the measured loss of diaphragmatic contribu-
tion to Vt at rest would result in Vt limitation during periods of
respiratory drive. After an initial dose (1 mg/kg) of doxapram,
dogs with the GRMD mutation and normal dogs reached
similar peak rates of ventilation (GRMD, 343.9 ⫾ 90.6
ml·kg⫺1·min⫺1; normal, 403.8 ⫾ 113.9 ml·kg⫺1·min⫺1) and
relative reductions in end-tidal CO2 (GRMD, 26.7 ⫾ 6.3%;
normal, 29.4 ⫾ 6.8%) within 2 min of the doxapram bolus,
before returning to near-baseline rates of ventilation by 5 min
postbolus. A subsequent higher dose (2 mg/kg) further in-
creased ventilation in normal dogs but not in dogs with the
GRMD mutation (Fig. 2A), indicating a loss of ventilatory
capacity in the GRMD animals. In both cases and in both
groups, dogs responded to the onset of doxapram first by
increasing Vt, then respiratory rate in a manner consistent with
graded exercise, as shown by the typical J shape in Fig. 2B.
J Appl Physiol • doi:10.1152/japplphysiol.00833.2013 • www.jappl.org
• Mead AF et al.
However, at equal rates of ventilation, Vt was consistently
lower in the dogs with the GRMD mutation, which compen-
sated with higher respiratory rates (Fig. 2B). Neither anesthesia
nor doxapram had an appreciable effect on ⌬VAB in individual
dogs from either group (data not shown). At rest and at peak
ventilation following doxapram administration (1 mg/kg), we
observed no difference in phase angle between dogs with the
GRMD mutation and normal dogs, indicating an absence of
abdominal paradox (Fig. 2D).
Expiratory muscles compensate for diaphragm dysfunction
in GRMD. DMD is associated with reduced functional residual
capacity (FRC). We were interested in whether loss of volu-
metric reserve observed in GRMD would be accompanied by
abnormal end-inspiratory chest wall volume (EIVcw) and end-
expiratory chest wall volume (EEVcw). Figure 3 shows relative
maximum EIVcw and minimum EEVcw by compartment in
dogs with GRMD and normal dogs before and after a doxa-
pram bolus (1 mg/kg). We found that the increases in Vt that
dogs with the GRMD mutation achieved were almost entirely
the result of lowered EEVcw and not increased EIVcw (Fig. 3A).
In contrast, normal dogs achieved larger increases in Vt pri-
marily by tapping into inspiratory reserve. Furthermore, RIP
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allowed us to differentiate between changes in rib cage and
abdominal contributions to these volumes. The reduction in
EEVcw observed in dogs with the GRMD mutation upon
doxapram administration was primarily caused by lower end-
expiratory VAB rather than VRC, indicating increased cranial
motion of the diaphragm at end-expiration (Fig. 3, B and C).
If abdominal expiratory muscles drive VAB and chest wall
volume below its equilibrium point during expiration, we
would expect to observe a consequent increase in abdominal
pressure (Pgas) (18, 27). Therefore, we performed a detailed
analysis of gastric and esophageal pressures (Pgas and Pes,
respectively) before and after doxapram administration. In-
deed, dogs with the GRMD mutation demonstrated a pattern of
transient increases in Pgas over two time scales following the
initial dose of doxapram (1 mg/kg) (Fig. 4). The first was
repeated each breath at end-expiration, observed as a rightward
Fig. 3. Volumetric reserve. A: chest wall volume (Vcw) changes in response to
doxapram (1 mg/kg) as measured by respiratory inductance plethysmography
in seven dogs with the GRMD mutation (x) and five normal (o) dogs.
Difference between end-inspiratory volume (EIV) (solid lines) and end-
expiratory volume (EEV) (dashed lines) Vcw is equivalent to total tidal volume
(Vt). EEV during quiet breathing (baseline) is set to zero. Doxapram (1 mg/kg)
elicits very different increases in Vt; by means of increased EIVcw in normal
dogs, and decreased EEVcw in dogs with the GRMD mutation. B and C:
doxapram-mediated changes in Vcw by compartment: rib cage (VRC) and
abdomen (VAB). Reduced EEVcw after doxapram in GRMD is primarily due to
cranial displacement of the diaphragm throughout the breathing cycle as shown
by reduction of abdominal end-inspiratory and end-expiratory volumes.
 Respiratory Dysfunction in the Dystrophin-Deficient Dog • Mead AF et al. 811
phragm for potential transplantation (34), we found that the
collateral connections between the internal thoracic and inter-
costal arteries afforded adequate delivery of oxygen to a large
volume of tissue (Fig. 7A) (5). These studies showed that the
specific passive stiffness of the GRMD diaphragm strip was
much higher and invariant at all physiologically relevant cy-
clical shortening rates than in a dystrophin-expressing carrier
diaphragm at muscle lengths above 100% of L0 (Fig. 7B). The
dynamic elastic modulus between 100% and 107% of L0 was
21.4 g/mm2 in dogs with the GRMD mutation vs. 2.4 g/mm2 in
a carrier dog.
DISCUSSION

In this study we combined noninvasive, invasive, ex vivo,

and histological approaches to improve our understanding of
the mechanics of breathing during the progressive loss of
ventilatory capacity in a translational model of DMD. Our data
indicate that respiratory decline in dogs with the GRMD
mutation is accompanied by changes in passive and active
properties of the respiratory pump. In both DMD and GRMD,
widespread muscle disease severely limits the loading of the

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Fig. 4. A and B: representative traces of esophageal vs. gastric pressure
throughout the breathing cycle before and at peak doxapram (1 mg/kg) effect
in a dog with the GRMD mutation and a normal dog. Gray arrows indicate
direction of loop. Gray circles indicate end-expiration. Dogs with the GRMD
mutation showed transient increases in gastric pressure (Pgas) throughout the
breathing cycle consistent with a postexpiratory recoil (PER) breathing strat-
egy. C: representative synchronized traces of pressures and compartmental
volumes in the time domain during two complete breathing cycles before and
at peak doxapram (1 mg/kg) effect in a dog with the GRMD mutation and a
normal dog.

spike in Pes/Pgas loops. The second was a longer-term increase

occurring over several breaths during peak doxapram effect,
observed as a rightward shift of the entire loop. The end-
expiratory Pgas spike emerged only at the higher dose in normal
dogs, and then to a lesser degree than in dogs with the GRMD
mutation, whereas there was no long-term increase in Pgas after
either dose in normal dogs.
Remodeling increases the passive stiffness of the GRMD
diaphragm. We examined the gross morphometry, histopathol-
ogy, and passive viscoelastic properties of the GRMD dia-
phragm. When measured at equal sarcomere lengths, the mus-
cular diaphragms of dogs with the GRMD mutation were
shorter in the direction of contraction compared with normal
dogs (31.7 ⫾ 7.2 mm vs. 75.6 ⫾ 15.6 mm; P ⬍ 0.01) and
thicker in the orthogonal plane (8.06 ⫾ 3.4 mm vs. 2.4 ⫾ 0.4
mm; P ⬍ 0.01). These findings indicate a process of remod-
eling that involves the deletion of sarcomeres rather than Fig. 5. The GRMD diaphragm undergoes a dramatic remodeling that alters
hypercontraction (Fig. 5). Based on histological staining for passive properties of the muscle. A: schematic cranial view of typical dia-
interstitial collagen, the amount of diaphragmatic CSA attrib- phragms from 1-year-old dogs viewed cranially (GRMD, red; normal, blue)
utable to interstitial collagen (46.6% ⫾ 5.6%) was significantly showing reduced muscular length in the direction of shortening and expansion
of central tendon area in GRMD. B: representative photographs of longitudinal
greater than that of other respiratory or locomotory muscles sections of costal diaphragm (location indicated in A) showing reduced muscle
(Fig. 6). To address the physiological correlates of this remod- length and increased thickness in GRMD. White arrowheads indicate central
eling process we developed an isolated, perfused muscle prep- tendon, orange arrowheads indicate insertion on rib. C: plot of muscular
aration to allow direct comparison of passive tension from diaphragm length vs. thickness in six dogs with the GRMD mutation and five
normal dogs. D: sarcomere length at these muscle lengths do not differ
GRMD and dystrophin-expressing carrier diaphragms sub- between dogs with the GRMD mutation and normal dogs, indicating that the
jected to cyclical length changes ex vivo (49). Although the reduced diaphragm length in GRMD results from removal of sarcomeres in
aorta has been used with success to perfuse the canine dia- series.

J Appl Physiol • doi:10.1152/japplphysiol.00833.2013 • www.jappl.org

812 Respiratory Dysfunction in the Dystrophin-Deficient Dog
Fig. 6. A: representative photomicrographs of muscle sections stained with
picrosirius red from one dog with the GRMD mutation and one normal dog.
Scale bar ⫽ 100 ␮m. Collagen (stained red) is increased in GRMD. B: collagen
cross-sectional area (CSA) quantified in muscle sections stained with picro-
sirius red from dogs with the GRMD mutation and normal dogs. The number
of dogs in each group is indicated in white. GRMD diaphragms had signifi-
cantly higher collagen CSA than other GRMD muscles.
respiratory system by way of an exercise-induced increase in
metabolic demand. To circumvent this limitation and to sim-
ulate the effects of moderate exercise, we uncoupled these
processes using the respiratory stimulant doxapram (13, 14).
Normally, increasing demand for ventilation through exer-
cise results in a progressive and sequential recruitment of
additional inspiratory and expiratory contractile reserve that
follows a well characterized pattern. In graded exercise, in-
creased respiratory drive caused by metabolic loading leads
first to the recruitment of additional diaphragmatic motor units,
followed by intercostal and auxiliary muscles to increase Vt,
followed in turn by an increase in respiratory rate (1). Ulti-
mately, at high rates of exercise, expiratory muscles are re-
cruited to shorten breathing cycle time, and to drive EEVcw
below the chest wall’s elastic equilibrium point, thus accessing
expiratory reserve volume and further increasing Vt. Doxa-
pram’s putative mode of action, as well as its independence
from anesthetic concentration, suggested that the complex
neural circuitry underlying the respiratory muscle recruitment
sequence would remain intact (13, 14). Indeed, our observa-
tions show that both normal dogs and those with the GRMD
mutation followed this pattern.
The impairment of ability to increase Vt meant that dogs
with the GRMD mutation required higher respiratory rates and
earlier expiratory muscle recruitment to achieve similar venti-
latory rates as normal dogs after administration of doxapram.
Dogs with the GRMD mutation also failed to further increase
ventilation after a subsequent higher dose. Together, these
findings indicate that ventilatory capacity is severely compro-
mised in dogs with the GRMD mutation by approximately 1
year of age. Furthermore, although a dog with the GRMD
mutation loses ventilatory capacity, a combination of remod-
J Appl Physiol • doi:10.1152/japplphysiol.00833.2013 • www.jappl.org
• Mead AF et al.
eling of the diaphragm and alteration of the timing of accessory
muscle recruitment partially compensate for this loss by en-
abling the expiratory muscles of the abdomen to aid inspira-
tion.
The observation that doxapram reduces EEVAB together
with increased end-expiratory Pgas in GRMD is consistent with
the compensatory adoption of a respiratory muscle recruitment
sequence, termed postexpiratory recoil (PER), which normally
emerges only during heavy exercise or under pathological
conditions such as diaphragm paralysis. Grimby et al. first
showed, in healthy subjects during a period of heavy exercise,
that contraction of abdominal wall muscles at end-expiration
functions not only to increase expiratory flow and Vt, but also
to store elastic and gravitational energy in the diaphragm for
the next breath (26, 19, 35). PER appears to be further aug-
mented in dogs with the GRMD mutation because the entire
range over which the diaphragm moves is shifted cranially
under increased respiratory drive, likely increasing passive
energy storage in the diaphragm’s elastic elements.
Recruitment of the expiratory musculature in GRMD is
further facilitated by a remodeling of the diaphragm, which
affects the passive mechanical properties of the muscle. Al-
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though the GRMD diaphragm becomes weaker, as evidenced
by reduced Pdi, it also becomes shorter, thicker, and severely
fibrotic, with a resulting increase in passive resistance to
distension at three levels. First, series deletion of sarcomeres
alone increases passive stiffness as individual sarcomeres are
stretched further for a given absolute muscle length change.
Second, increased collagen in the extracellular matrix increases
muscle length-specific and CSA-specific stiffness. Finally, the
increase in muscle length-specific stiffness is further multiplied
by the increase in overall CSA of the thickened muscle. It
should be noted that due to animal availability, passive stiff-
ness in the GRMD diaphragm was compared with that from a
heterozygous carrier animal. Although carrier diaphragms ap-
peared grossly normal, and respiratory disease has not been
reported in human or canine carriers, further comparisons to
Fig. 7. Perfusion via the interior thoracic artery enables study of ex vivo
diaphragm mechanics. A: schematic of a region of the costal diaphragm viewed
cranially [see refs. (5, 34)]. A section of costal diaphragm is removed (region
outlined in light blue) and the interior thoracic artery is cannulated (1) and
perfused with a mixture of modified Krebs-Hanselite and heparinized whole
blood at 37°C. A wide strip (⬃1 cm) of costal diaphragm is excised (pink
shaded area) but left attached at the rib (2), whereas the central tendon end is
attached to a muscle ergometer (3). Perfusate is collected and pumped (4)
through a heat exchanger (5) and membrane oxygenator (6) before being
reperfused. B: lissajous loops of specific passive tension vs. length in viable,
perfused ex vivo diaphragm muscle strips from a dog with the GRMD
mutation and a dystrophin-expressing carrier dog showing increased stiffness
at lengths over 100% of L0 in GRMD.
 Respiratory Dysfunction in the Dystrophin-Deficient Dog
normal diaphragms are needed. Regardless, the reliance on
PER combined with the absence of abdominal paradox ob-
served in this study suggests a possible compensatory role for
a stiff diaphragm.
Two clinical examples lend support to this interpretation.
Abdominal paradox resulting from a weak or nonfunctional but
compliant diaphragm is an energetically and volumetrically
unfavorable scenario for gas exchange (53). An example of
severe abdominal paradox in the clinic is acute diaphragmatic
paralysis following iatrogenic phrenic nerve injury during
cardiac surgery. Surgical therapy for refractory cases involves
plication (reduction of the area and excursion of the nonfunc-
tional diaphragm), which serves to eliminate abdominal para-
dox, increase maximum lung volumes, and reduce the work of
breathing for a given Vt (16, 22, 46, 50). In other words, it is
better by these important metrics to have a short and therefore
stiff diaphragm with little or no range of motion, than a
compliant but weak or nonfunctioning diaphragm.
Second, dogs with the GRMD mutation are not a unique
example of compensatory PER in the setting of diaphragm
sarcomere deletion. Hyperinflation of the lungs and increased
FRC in chronic obstructive pulmonary disease leads to a
remodeling of diaphragmatic muscle fibers, whereby sarco-
meres are deleted in series in such a way that ideal resting
sarcomere length is maintained in the shortened muscle (10).
This allows Pdi to be more effectively generated around a
higher chest wall equilibrium volume and enables compensa-
tory PER breathing (10, 19, 35). Patients with DMD (and dogs
with the GRMD mutation in this study) breathe at a lower than
normal FRC (38), suggesting that the stimulus and mechanism
for diaphragm remodeling are likely different.
There are important caveats in ascribing a compensatory role
to the observed diaphragm remodeling in GRMD. Abdominal
paradox is typically observed in scenarios of diaphragm dys-
function where other respiratory muscles are normal. Because
abdominal paradox requires inspiratory intercostal muscles to
be strong enough to overpower a weak diaphragm and all
respiratory muscles are affected in GRMD, the absence of
abdominal paradox could be a function of insufficient inter-
costal strength. A similar argument could be made against
PER, because clinical examples typically involve normal ab-
dominal muscles. We view changes to the mechanics of breath-
ing in GRMD not so much as compensation for a failing
diaphragm per se, but as a redistribution of the work of
breathing to a larger volume of muscle, thus minimizing peak
force on any individual sarcomere. In this context, the aug-
mentation of diaphragmatic elastance could play an important
role by further enabling the muscles of the abdomen to con-
tribute to inspiration. This interpretation recalls the signifi-
cance of the Gowers’ sign in the clinical assessment of loco-
motive muscle performance in DMD (8, 23). Gower first
observed that when asked to stand from a seated position
without the aid of elevated structures, children with body-wide
neuromuscular disease follow a stereotyped sequence of mo-
tions, the effect of which is to spread the work of standing
among as many muscles as possible. Although the combined
effect of diaphragm remodeling in GRMD appears to be
beneficial, further study is needed to determine processes
underlying the observed changes, and whether any component
of diaphragm stiffness is maladaptive.
J Appl Physiol • doi:10.1152/japplphysiol.00833.2013 • www.jappl.org
• Mead AF et al. 813
There is indirect evidence that a similar process of remod-
eling and compensatory recruitment of the respiratory muscu-
lature takes place in DMD, but there has been no previous
opportunity to integrate the individual components as facili-
tated in this translational model. One study reported sono-
graphically detectable thickening of the muscular diaphragm in
boys with DMD (17), and progressive loss of diaphragm
range-of-motion beginning early in the disease without abdom-
inal paradox has been reported in DMD (30, 35). And, al-
though boys with DMD breathe at a lower FRC, PER has not
been reported. The interpretation of adaptive diaphragm func-
tion put forward here attributes a possible positive functional
role to fibrosis, a process usually considered pathologic. There-
fore, these findings could be relevant to therapies that target
this process. Moreover, the diaphragm has an essential role in
anatomical partition, which might be compromised by necro-
tizing myocytolysis in the absence of fibrosis, leading to a high
incidence of additional morbidity from transdiaphragmatic
herniation of abdominal viscera (7, 12).
The observation that dogs with the GRMD mutation recruit
a broader range of respiratory muscles than normal control
dogs to achieve the same rates of ventilation suggests that the
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lack of a doxapram dose-response in this population reflects a
functional limitation of the respiratory pump. We cannot,
however, rule out the possibility that dystrophin expression in
the central nervous system (CNS) directly affects neuronal
pathways involved in the integration of afferent and efferent
signals responsive to doxapram-receptor binding. Although the
functions of dystrophin and the dystrophin-associated glyco-
protein complex in the CNS are not well understood, cognitive
impairment has been identified in some patients with DMD,
and progress has been made in identifying potential biochem-
ical pathways that might be implicated (2, 15, 52).
Although the phenotype is severe in dystrophin-deficient
dogs, quantifiable biomarkers of disease progression have been
difficult to standardize. The ratio of diaphragmatic length to
thickness reported here is arguably the most extreme morpho-
metric abnormality noted in GRMD to date, and holds promise
as an endpoint measure in translational research. Furthermore,
having established quantifiable changes in respiratory mechan-
ics and capacity in a group of dogs with the GRMD mutation
and with relatively severe disease, we anticipate that longitu-
dinal studies consisting of serial measurements of individual
animals will further enhance the value of this translational
model in view of the inherent phenotypic variability of the
outbred GRMD line. This variability cannot be addressed by
inbreeding without compromising the viability of the overall
colony (32). Noninvasive or minimally invasive biomarkers of
skeletal muscle function in GRMD are currently limited to
analyses of gait, passive range of motion, magnetic resonance
imaging (MRI) analysis of muscle volume and composition,
and of proximal and distal hindlimb muscle force during nerve
electrostimulation (31). Because MRI and electrostimulation
require general anesthesia in dogs, we have recently added
doxapram respiratory studies before anesthesia reversal, with-
out observable detriment to animals undergoing serial studies.
ACKNOWLEDGMENTS
We thank D. Bogan and S. Chakkalakal for excellent technical assistance;
and M. Cereda, D. Cooper, G. Guild, T. Khurana, S. Levine, D. Ren, S.
Singhal, and T. Svitkina for insightful discussions.
814 Respiratory Dysfunction in the Dystrophin-Deficient Dog
Present address of M.K. Childers: Institute for Stem Cell and Regenerative
Medicine, University of Washington, Seattle, WA 98109.
Present address of J.N. Kornegay: Department of Veterinary Integrative
Biosciences, (Mail Stop 4458), College of Veterinary Medicine, Texas A&M
University, College Station, TX 77843– 4458.
GRANTS
Support for this study was provided by National Institutes of Health Grants
NS-042874, NS-052476, and RR-028027; and by the Muscular Dystrophy
Association. A.F.M. and M.P. were supported by National Institute of Arthritis
and Musculoskeletal and Skin Diseases Training Grant T32 AR-053461.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: A.F.M., M.P., A.S.M., M.A.M., G.S., J.N.K., and
H.H.S. conception and design of research; A.F.M., M.P., A.S.M., M.K.C.,
J.R.B., G.S., and H.H.S. performed experiments; A.F.M., G.S., and H.H.S.
analyzed data; A.F.M., M.P., A.S.M., M.A.M., and H.H.S. interpreted results
of experiments; A.F.M. and H.H.S. prepared figures; A.F.M. and H.H.S.
drafted manuscript; A.F.M., M.P., A.S.M., M.A.M., M.K.C., J.R.B., J.N.K.,
and H.H.S. edited and revised manuscript; A.F.M., M.P., A.S.M., M.A.M.,
M.K.C., J.R.B., G.S., J.N.K., and H.H.S. approved final version of manuscript.
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