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Am J Physiol Heart Circ Physiol 308: H303–H315, 2015.
First published December 5, 2014; doi:10.1152/ajpheart.00485.2014.
Early right ventricular fibrosis and reduction in biventricular cardiac reserve
in the dystrophin-deficient mdx heart
Tatyana A. Meyers and DeWayne Townsend
Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, Minnesota
Submitted 9 July 2014; accepted in final form 1 December 2014
right ventricle; dystrophic cardiomyopathy; dystrophin; Duchenne
muscular dystrophy
DUCHENNE MUSCULAR DYSTROPHY is an X-linked disease characterized by progressive skeletal and cardiac muscle degeneration (14, 21), resulting from the loss of the protein dystrophin
(29). Dystrophin is a large multidomain cytoskeletal protein
that has many cellular functions, including forming a link
between the actin cytoskeleton and the sarcolemmal membrane
of the striated muscle cell (24, 25). From this subsarcolemmal
location it has been demonstrated that dystrophin provides
mechanical support to the membrane (47, 73), contributes to
the transmission of force from the sarcomeres to the extracellular matrix (52), and serves as a signaling scaffold (8, 27).
Clinically, DMD presents initially with progressive skeletal
muscle weakness during the first decade of life. The initial
stages of the disease are primarily the result of degeneration of
skeletal muscles which are, over time, replaced with a mixture
Address for reprint requests and other correspondence: D. Townsend, Dept.
of Integrative Biology and Physiology, Univ. of Minnesota, 2231 6th St. SE,
Minneapolis, MN 55455 (e-mail: [email protected]).
http://www.ajpheart.org
of adipose tissue and fibrosis such that most patients are
nonambulatory by their teenage years (11). As the disease
progresses, the muscles of respiration are increasingly affected
resulting in significant respiratory insufficiency (4, 31, 42).
This respiratory failure is a leading cause of death in DMD and
therapies that improve ventilation have resulted in significant
increases in life expectancy for DMD patients (22, 23).
Around the time that respiratory dysfunction becomes apparent, so too does cardiac function begin to falter. Careful
examination of cardiac function reveals that subclinical dysfunction is often present even earlier in the disease process (33,
54). As in the skeletal muscle, the cardiac manifestation of
DMD is characterized by a relentless downward trajectory (15,
53). This decline in cardiac function can be slowed using
steroids and standard heart failure treatments (40, 56), but there
remains no effective specific therapy for DMD, and inevitably
these patients die of respiratory and/or cardiac complications.
Our understanding of the molecular pathogenesis of DMD
has been greatly assisted by the dystrophin-deficient mdx
mouse (10). This mouse has a relatively mild phenotype, with
subclinical myopathic disease evident at baseline. However, it
is clear that the mdx mouse is susceptible to muscle injury as
either stress (18, 20, 34, 47, 73) or age (16, 37, 51, 68) result
in significant myopathic changes becoming evident. In some
sense the mdx mouse provides insights into the earlier aspects
of the disease, offering the opportunity to understand the
mechanisms underlying the initiating aspects of the dystrophic
disease process. Of all the skeletal muscles in the mdx mouse,
the diaphragm shows the most significant levels of fibrosis and
degeneration (61). This pathology translates into a progressive
reduction in respiratory function in the mdx mouse, with
dysfunction present as early as 3 mo of age (30, 32, 44). This
chronic respiratory insufficiency of the mdx mouse makes this
model particularly valuable for examining how respiratory
dysfunction interacts with the dystrophic heart.
Alveolar hypoxia secondary to hypoventilation by weakened respiratory muscles presents a unique hemodynamic
challenge to the dystrophic heart. Specifically, decreases in
alveolar oxygen levels induce constriction of the pulmonary
arterial tree. This increase in pulmonary resistance places an
additional afterload upon the right ventricle and decreases the
preload filling the left ventricle. Evidence of increased pulmonary vascular resistance has been observed in mdx mice where
studies using cardiac magnetic resonance imaging show right
ventricular dilation (17, 74). These changes are consistent with
an increase in right ventricular afterload. Other studies using
catheter-based hemodynamic approaches have shown a reduction in left ventricular preload, also consistent with an increase
in pulmonary vascular resistance decreasing the loading of the
left ventricle (66). Interestingly, transgenic replacement of
dystrophin in skeletal muscle, including the diaphragm, cor-
0363-6135/15 Copyright © 2015 the American Physiological Society
H303
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Meyers TA, Townsend D. Early right ventricular fibrosis and reduction in biventricular cardiac reserve in the dystrophin-deficient mdx heart.
Am J Physiol Heart Circ Physiol 308: H303–H315, 2015. First published
December 5, 2014; doi:10.1152/ajpheart.00485.2014.—Duchenne muscular dystrophy (DMD) is a progressive disease of striated muscle
deterioration. Respiratory and cardiac muscle dysfunction are particularly clinically relevant because they result in the leading causes of
death in DMD patients. Despite the clinical and physiological significance of these systems, little has been done to understand the
cardiorespiratory interaction in DMD. We show here that prior to the
onset of global cardiac dysfunction, dystrophin-deficient mdx mice
have increased cardiac fibrosis with the right ventricle being particularly affected. Using a novel biventricular cardiac catheterization
technique coupled with cardiac stress testing, we demonstrate that
both the right and left ventricles have significant reductions in both
systolic and diastolic function in response to dobutamine. Unstimulated cardiac function is relatively normal except for a significant
reduction in the ventricular pressure transient duration compared with
controls. These biventricular analyses also reveal the absence of a
dobutamine-induced increase in isovolumic relaxation in the right
ventricle of control hearts. Simultaneous assessment of biventricular
pressure demonstrates a dobutamine-dependent enhancement of coupling between the ventricles in control mice, which is absent in mdx
mice. Furthermore, studies probing the passive-extension properties
of the left ventricle demonstrate that the mdx heart is significantly
more compliant compared with age-matched C57BL/10 hearts, which
have an age-dependent stiffening that is completely absent from
dystrophic hearts. These new results indicate that right ventricular
fibrosis is an early indicator of the development of dystrophic cardiomyopathy, suggesting a mechanism by which respiratory insufficiency
may accelerate the development of heart failure in DMD.
H304
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
METHODS
Animals. Initial studies defining the procedures and measures of the
biventricular recordings were performed in young 3- to 4-mo-old
C57BL/6J mice obtained directly from Jackson Labs (Bar Harbor,
ME). Both mdx (C57BL/10ScSn-Dmdmdx) and the background control (C57BL/10 SnJ) mice were obtained from a colony maintained at
the University of Minnesota. The genetics of these colonies were
maintained by purchasing new breeders from Jackson Labs every four
generations. Subanalysis of previously published hemodynamic data
demonstrated that there was no significant difference between male
and female mdx mice in the 4- to 7-mo-old age range, furthermore no
significant sex differences were observed in the 9- to 10-mo-old
cohort in this study, although very old female mdx mice do display
decreased cardiac function (7). The lack of evidence for sex-dependent hemodynamic changes in our target age group led us to use both
sexes in these studies. All studies were reviewed and approved by the
University of Minnesota’s Institutional Animal Care and Use Committee.
Cardiac catheterization. Biventicular catheterization was developed from the techniques required for left ventricular catheterization
described elsewhere (64, 73). Briefly, animals were anesthetized with
2% isoflurane and ventilated with 4 cmH2O positive end-expiratory
pressure (PEEP). The apex of the heart was exposed through a
thoracotomy and removal of the pericardium. A 1.2-Fr pressure
transducer (Transonic, Ithaca, NY) was inserted into the right ventricle through an apical stab incision made with a 27-gauge needle. The
course of this incision included a small part of the septum (see Fig. 3)
to provide sufficient myocardial tissue to prevent leakage through the
right ventricular wall. This was particularly important for smaller
mice with right ventricular walls too thin to seal around the catheter.
Next, the 1.2-Fr pressure-volume catheter (Transonic) was inserted
through a second stab incision into the left ventricular cavity. Several
parameters used to calculate the volume were obtained in preliminary
pilot studies including blood resistivity (1.2 ohm·m) and epislon:
sigma ratio (800,000). Left ventricular catheter placement was optimized to obtain low-amplitude oscillations of the degree parameter,
indicating that the catheter was centered in the ventricular chamber.
End-systolic and end-diastolic parallel conductances were determined
prior to the beginning of the experimental protocol.
Once the animal was instrumented, 6 – 8 ml/kg of 10% albumin was
infused through a jugular catheter over a 10-min period to replace
both insensible and blood volume loss during surgery. Anesthesia was
decreased to 1% isoflurane and the temperature stabilized to 37°C.
Following this initial stabilization period, baseline hemodynamic data
were collected. Hemodynamic data include occlusion of the inferior
vena cava within the thoracic cavity and compression of the abdominal viscera. Stepwise dobutamine doses were given by constant-rate
infusions with a maximal volume of 0.3 ml·kg⫺1·min⫺1 delivered by
a syringe pump (Kent Scientific, Torrington, CT). Analog data were
collected by a Ponemah DAQ16 (DSI, St. Paul, MN).
Imaging studies. Hearts were fixed overnight in 10% formalin
and stored in 70% ethanol prior to paraffin embedding and sectioning.
Short-axis cross-sections of the hearts were sectioned at 4 ␮m and
stained with hematoxylin and eosin and Sirius red/fast green using
standard histochemical procedures. Images were collected on a Nikon
E200 microscope fitted with polarizing filters. Individual images were
merged using an image stitching plug-in in ImageJ (49) to form a
single large high-resolution image of the entire section. These montage images were used for the subsequent quantification. All quantification of the fibrosis was performed in a blinded fashion on images
de-identified using randomly generated numerical file names. For
assessment of the regional distribution of lesions, the montaged heart
images were divided into three domains: the right ventricular free
wall, the left ventricular free wall, and the interventricular septum.
Fibrosis-positive areas of these sections were determined by splitting
the color channels of the bright-field sirius red/fast green images. The
blue channel was thresholded to provide the total number of pixels
within the heart. Fibrotic areas were determined by setting a negative
threshold in the green channel, as the lesions contain very low levels
of green. Two sections from most hearts were analyzed, having been
taken from distinct regions along the long axis of the heart. The raw
quantification results from both sections were combined to produce
the final measure of fibrosis for each heart.
Data analysis and statistics. Hemodynamic data were analyzed by
the Ponehma Physiological suite. Additional analysis of beat-to-beat
interactions was performed by custom Excel (Microsoft, Redman,
WA) macros that allowed for the efficient characterization of changes
associated with various loading conditions. Representative transients
tracings are the average of 15–30 beats, aligned to the peak pressure
and averaged (MatLab, Mathworks, Natick, MA). Statistical analyses
were performed using Prism (GraphPad, La Jolla, CA) and R (version
3.0.2). Student’s t-test was only used for the comparison of two data
sets; all other analyses used a two-way ANOVA with a Tukey post
hoc test to assess the statistical significance of multiple comparisons.
RESULTS
Dystrophic cardiomyopathy is a progressive disease that is
characterized by the accumulation of fibrosis throughout the
myocardium. We examined histological sections of hearts from
9-mo-old mdx and control C57BL/10 mice. In agreement with
previous work, the mdx hearts display significantly more
overall fibrosis than age-matched C57BL/10 controls (Fig. 1).
This increased level of fibrosis is evident in young (4 – 6 mo
old) mdx mice and continues to increase with age. Further
morphological analysis reveals that the right ventricle of the
mdx mouse heart accumulates significantly more fibrosis than
either the septum or left ventricular free wall (Fig. 2). In
contrast, at 9 mo of age, C57BL/10 mice have only small
differences in the levels of fibrosis between regions of the
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rects these changes in both right ventricular geometry (17) and
left ventricular loading (66). Together, these studies and the
natural history of the disease suggest that there are functionally
significant hemodynamic consequences of respiratory dysfunction that contribute to cardiac dysfunction. The consequences
of correcting dystrophic skeletal muscle without the correction
of the heart are not clear. Studies in mice give mixed results,
with some studies suggesting improvements in cardiac function
with the selective correction of skeletal muscle (17), some
showing no effect (69), and others demonstrating increased
myocardial damage (66). Importantly, human patients with
selective disruption of dystrophin expression in the myocardium display a severe cardiac phenotype with earlier onset and
more rapid progress of heart failure compared with DMD
patients (3, 63).
In this study a novel biventricular cardiac catheterization
procedure is used to examine both right and left ventricular
function of the dystrophic mdx heart. The simultaneous recording of both right and left ventricular function permits a detailed
characterization of how the dystrophic disease process affects
the two pumping chambers of the heart. This analysis is
essential for assessing the role that alterations in pulmonary
circulation have on the development of dysfunction in both the
right and left ventricles of the dystrophic heart. These investigations are significant because a detailed understanding of the
hemodynamic consequences of respiratory insufficiency and its
potential to accelerate cardiac dysfunction could impact the
clinical management of DMD patients.
H305
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
A
Left Ventricle
Right Ventricle
B
mdx
10
‡
C57BL/10
% Fibrotic Area
8
6
4
2
0
mdx
Fig. 1. The 9-mo-old mdx hearts have significantly more scarring compared with C57BL/10 hearts. A: representative images of sirius red/fast green staining in
bright-field and cross-polarized light illumination of hearts from 9-mo-old C57BL/10 and mdx mice from both the right and left ventricles. Collagen appears red
in bright-field sections and the birefringence of these lesions further confirms the collagen content of this staining. Note that collagen staining in C57BL/10 hearts
is primarily perivascular with some interstitial staining, while mdx hearts have more intense interstitial staining and scar formation. Bar is 100 ␮m. B: hearts
from mdx mice show significantly higher levels of fibrosis. ‡P ⬍ 0.0001 vs. C57BL/10; data obtained from heart sections from 14 C57BL/10 and 15 mdx mice.
heart: 2.7 ⫾ 0.3%, 1.8 ⫾ 0.2%, and 2.3 ⫾ 0.2% for the right
ventricle, interventricular septum, and left ventricle, respectively. In addition to elevated levels of fibrosis, the rate of
accumulation of right ventricular fibrosis in the dystrophic
heart is nearly 50% greater than that observed in either the left
ventricular free wall or interventricular septum: 0.53 ⫾ 0.13,
0.34 ⫾ 0.06, and 0.37 ⫾ 0.07%/mo for the right ventricle,
interventricular septum, and left ventricle, respectively. The
morphology of the fibrosis consists of areas of replacement
fibrosis and areas with significant increases in interstitial fibrosis. The birefringence signal observed in C57BL/10 sections is
uniformly green to yellow in color in contrast to the orange to
red birefringence associated with fibrosis in the mdx heart
sections. This shift to longer wavelengths of birefringence has
been documented to occur with larger, more tightly packed
collagen bundles (19). This suggests that the fibrosis present in
the dystrophic heart is both more widely distributed and consists of thicker strands of collagen.
To assess global function of the dystrophic heart, both right
and left ventricular function needs to be assessed. To address this need, a murine biventricular catheterization methodology was developed to allow simultaneous measurements of both right and left ventricular function (Fig. 3A).
This approach allows for the detection of alterations in both
ventricular coupling and the contractile function of each ventricle. The left ventricle is assessed using pressure-volume loop
analysis (Fig. 3, B and C). This methodology is well suited for
the left ventricle where the symmetry of its cavity is well
approximated by a cylinder, the geometry assumed by the
single segmented conductance catheter. In contrast, the asymmetrical geometry of the right ventricle makes this methodology more difficult to interpret, so in these studies only a
pressure catheter was used to assess right ventricular function.
An important strength of pressure-volume loop analysis is the
ability to transiently vary the loading conditions on the heart
while collecting pressure and volume data throughout. In these
studies two procedures are used to transiently alter the loading
of the heart. First is the occlusion of the inferior (caudal) vena
cava, a procedure which blocks venous return from the caudal
portion of the mouse. As expected, this decrease in preload is
first evident in the right ventricle, where peak systolic pressure
begins to decline 3– 4 beats before the beginning of the reduction in left ventricular systolic pressure. In young C57BL/6
mice, which were used to validate these procedures, it took this
reduction in preload 3.7 ⫾ 0.6 beats to transit the lungs and
reach the left ventricle. With occlusion of venous return,
systolic pressures of both ventricles drop to ⬇60% of baseline
systolic pressure. The second procedure used to alter loading
on the heart was abdominal compression. In contrast to occlusion of venous return, abdominal compression affects both
ventricles simultaneously. This indicates that this procedure
increases both venous return to the right ventricle, by compression of the mesenteric circulation, and afterload on the left
ventricle, by compression of the abdominal aorta. The effect is
much larger in the left ventricle where systolic pressure increases to 135 ⫾ 3% of baseline compared with 115 ⫾ 2% in
the right ventricle. This likely results from the low compliance
of the abdominal aorta, where small changes in volume result
in large changes in pressure vs. the high compliance of the
abdominal venous compartment.
A central aim of this study is to assess the role of right
ventricular dysfunction in the pathophysiology of dystrophic
cardiomyopathy. The methodologies described above were
applied to a population of 9-mo-old mdx and C57BL/10 mice.
As seen with the younger C57BL/6, with the occlusion of
venous return, the decline in the peak systolic pressure of the
right ventricle precedes the decline in the left ventricle (Fig.
3E). The time between reductions in right ventricular pressure
and left ventricular pressures is modulated by the capacitance
of the pulmonary circulation, which is controlled by changes in
pulmonary arterial resistance and pulmonary venous compliance. In C57BL/10 mice, but not mdx, dobutamine induces a
significant reduction in the transit time of this pressure wave
through the pulmonary circulation (Fig. 3E), likely through a
reduction in pulmonary venous compliance. Importantly, hemoglobin saturation levels are not different between genotypes
or dobutamine dose (all conditions average ⬎96%); indicating
that hypoxia is not a confounding variable altering pulmonary
circulation in this preparation. In the left ventricle the magnitude of the pressure decline resulting from the reduction of
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C57BL/10
H306
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
A
Right Ventricle
> 15 Months
LV
Se
pt
um
R
V
0
20
10
10
0
0
LV
*
*
LV
10
*
>15 Months
Old
Se
pt
um
20
20
30
9-10 Months
Old
R
V
30
4-6 Months
Old
Se
pt
um
30
R
V
% Sirius Red
B
Fig. 2. A: regional distribution of fibrosis in the dystrophic heart. B: quantification of the percentage of regional fibrosis in the hearts of young adult (4 – 6 mo
old), middle-aged adult (9 –10 mo old), and old (⬎15 mo old) mdx mice revealed continuous increase in overall fibrosis and a consistent distribution of scarring.
The right ventricle has a greater percentage of fibrosis compared with other regions of the heart in all age groups examined. *P ⬍ 0.05; data are derived from
12–15 mice.
venous return is significantly lower in mdx mice with pressure
drops of 30.5 ⫾ 2.0 mmHg compared with a 39.3 ⫾ 1.9 mmHg
drop in wild-type hearts. A similar difference is present in the
right ventricle where pressures drop 11.9 ⫾ 0.5 mmHg in
C57BL/10 compared with 9.5 ⫾ 0.8 mmHg in mdx. Interestingly, abdominal compression results in a significantly greater
increase in left ventricular systolic pressure in mdx mice (25.5 ⫾
1.7 mmHg) compared with C57BL/10 (20.8 ⫾ 1.1 mmHg).
There is no significant difference between the genotypes in the
right ventricular response to abdominal compression.
The 9-mo-old mdx mice used in this study have relatively
normal cardiac function at baseline, but significant deficits are
revealed with stimulation by dobutamine. Two-way ANOVA
revealed significant decreases in left and right ventricular
systolic function in mdx mice compared with C57BL/10 mice
(Fig. 4, A–E). The differences between dystrophic and wildtype hearts are most evident with dobutamine stimulation
where the systolic function of the left ventricle of mdx mice
is significantly attenuated compared with control hearts at
both doses of dobutamine. Despite the increased fibrosis, the
right ventricle has a relatively normal response to dobutamine,
although higher doses are required to observe statistically
significant increases in systolic function. Interestingly, both
strains demonstrate that the load-independent measure of contractility, preload recruitable stroke work (PRSW), is significantly increased by dobutamine at both doses examined here
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9-10 Months
4-6 Months
Left Ventricle
H307
A
B
LV Pressure
(mmHg)
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
150
100
50
C
PV-Catheter
0
20
IVCO
150
Abdominal
Compression
50
0
0
20
40
LV Volume (µl)
100
75
50
30
20
10
0
Time
500 msec
E
Number of Beats
that RV precedes LV
60
100
LV Pressure
RV Pressure
125
40
LV Volume (µl)
4
60
Fig. 3. Left ventricular pressure-volume loops
with simultaneous right ventricular pressure
measurements in the mouse. A: a schematic of
the approach used to assess right and left
ventricular function in aged mdx mice. Representative pressure-volume loops from young
wild-type mice under steady-state conditions
(B) and with loading conditions altered by
occlusion of the inferior vena cava (IVCO;
black) or compression of the abdomen (red)
(C). D: representative tracings of left and
right ventricular pressure during an occlusion
of the inferior vena cava. D, left trace: the
beginning of the decline in right ventricular
pressure is at the left edge of the plot (red
arrow) and the black arrow denotes the beginning of pressure decline in the left ventricle.
This delay allows for the assessment of coupling between the right and left ventricles (D
and E). Changes in ventricular pressures following abdominal compression occur simultaneously in both ventricles (D, right trace:
red and black arrows). *P ⬍ 0.05 from
C57BL/10 baseline data.
C57BL/10
3
2
*
*
mdx
1
0
Dobutamine
(µg/kg/min)
(Fig. 4E). Two-way ANOVA demonstrates that the mdx hearts
have lower PRSW but respond to dobutamine to the same
degree as C57BL/10 hearts. These data indicate that alterations
in the loading of the heart play an important role in the control
of the systolic response to dobutamine in the dystrophic heart.
Evaluation of parameters of global cardiac function also
reveal significant reductions in the cardiac reserve of 9-mo-old
mdx mice. Compared with C57BL/10 mice, mdx mice have
lower left ventricular cardiac output and stroke work, a difference that is increased at both doses of dobutamine used in this
study (Fig. 5, A and B). Interestingly, there is no difference
between the strains in the chronotropic response to dobutamine
(Fig. 5D). Two-way ANOVA demonstrates a significant genotype effect indicating a lower stroke volume in mdx hearts
compared with that of C57BL/10 hearts (Fig. 5C), suggesting
that the significant reductions in cardiac output and stroke work
are driven primarily by reductions in stroke volume. These
increases are driven by the significant dobutamine-induced
reductions in end-systolic volume observed in C57BL/10
hearts, which are not significantly evident in dobutaminestimulated mdx hearts (Fig. 5E). No detectable changes in the
end-diastolic volume in C57BL/10 or mdx hearts are observed
[50.0 ⫾ 2.5 (C57Bl/10) vs. 47.1 ⫾ 1.8 ␮l (mdx) at baseline and
51.9 ⫾ 3.0 vs. 51.1 ⫾ 3.7 ␮l with 15 ␮g·kg⫺1·min⫺1 dobutamine]. While right ventricular volumes are not assessed in
these studies, it is likely that the changes in stroke volume
observed in the left ventricle are mirrored in the right ventricle
as any significant mismatch in cardiac output between the
ventricles would be expected to alter diastolic pressures, which
was not observed in these studies.
Both at baseline and with dobutamine stimulation, the integral of the left ventricular pressure transient is significantly
lower in the mdx heart (Fig. 6). At baseline this reduction is
due to a combination of slightly lower systolic pressures and a
significant reduction in the duration of the pressure transient
(Fig. 6, A and E). For these analyses the pressure transient
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D
Pressure (mmHg)
Pressure-Only
LV Pressure
(mmHg)
0
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
LV Systolic
Pressure (mmHg)
150
*
‡
50
D
*
*
†‡
‡
10000
5000
0
*
30
20
10
6000
*
4000
†
*
2000
Dobutamine
(µg/kg/min)
Dobutamine
(µg/kg/min)
Preload Recruitable
Stroke Work
*
Dobutamine
(µg/kg/min)
20000
E
40
0
0
15000
C57BL/10
mdx
50
0
150
100
*
†
*
†
50
Dobutamine
(µg/kg/min)
0
duration is defined as the period between beginning of the
pressure increase and the minimum dP/dt. Dobutamine stimulation significantly shortens the transient duration in both
C57BL/10 and mdx hearts, such that the pressure transient duration becomes equal between both genotypes. These changes in
pressure transient duration are also observed in the right ventricle of mdx and C57BL/10 hearts (Fig. 6, F–J). This reduction in pressure transient duration is also evident in 4- to
6-mo-old mdx and C57BL/10, with left ventricular pressure
transient durations of 47.6 ⫾ 1.0 and 51.5 ⫾ 1.4 ms in mdx and
C57BL/10, respectively, and right ventricular pressure transient durations of 47.5 ⫾ 1.0 and 52.2 ⫾ 1.3 ms for mdx and
C57BL/10, respectively.
Along with these changes in contraction, the dystrophic
heart displays significant differences in the dobutamine-induced lusitropic function. In control animals dobutamine
causes significant acceleration of isometric relaxation in the
left ventricle (Fig. 7, A, C, and E); however, dobutamine has no
effect on the isometric relaxation of the mdx heart. The
minimum dP/dt in the right ventricle is significantly increased
with dobutamine treatment in C57BL/10 mice and, to a lesser
degree, in mdx mice (Fig. 7B). In contrast, tau, the time
constant of the exponential curve characterizing the decline of
the right ventricular pressure transient, is not altered by dobutamine (Fig. 7, D and F). Minimum dP/dt displays significant
load dependence (Table 1), which may explain the discrepancy
in the right ventricle, favoring tau as a more load-independent
measure of relaxation.
There is a significant difference in the end-diastolic pressure-volume relationship between 9-mo-old C57BL/10 and
mdx mice. Alterations in left ventricular loading provide the
opportunity to examine the passive compliance of the left
ventricle. In the mouse, over the volumes examined in these
studies, this relationship is well approximated by a linear
model (r2 values of 0.83 ⫾ 0.01 for C57BL/10 and 0.83 ⫾ 0.02
for mdx). Surprisingly, these studies demonstrate that older
mdx mice have increased compliance compared with C57BL/
10, despite increased levels of fibrosis (Fig. 8, B and C).
Similar studies performed in 4- to 6-mo-old animals reveal an
age-dependent decrease in compliance in C57BL/10 (young
0.31 ⫾ 0.03 vs. old 0.40 ⫾ 0.04 mmHg/␮l) that is absent in
mdx hearts (young 0.28 ⫾ 0.04 vs. old 0.26 ⫾ 0.03 mmHg/␮l).
DISCUSSION
Real-time biventricular hemodynamics by direct catheterization in mice, as shown here, provides a unique physiological
window into the emerging cardiomyopathy of the dystrophindeficient heart in vivo. We demonstrate that right ventricular
fibrosis precedes left ventricular fibrosis and significant sys-
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LV dP/dtMax
(mmHg/sec)
C
‡
100
Dobutamine
(µg/kg/min)
Fig. 4. Evidence of reduced left and right ventricular systolic function in old mdx mice. A
dobutamine stress test was performed to assess
cardiac reserve of both right and left ventricles of
C57BL/10 and mdx hearts. These tests reveal
significantly reduced cardiac reserve of both right
and left ventricular systolic pressures (A and B)
and maximum dP/dt (C and D) in mdx hearts
compared with C57BL/10. E: dobutamine significantly increased the preload recruitable stroke
work, a load-independent measure of contractility,
in both mdx and C57BL/10 hearts. ‡P ⬍ 0.05,
significant difference between genotypes. *P ⬍
0.05, difference from baseline C57BL/10. †P ⬍
0.05, difference from baseline mdx.
B
RV Systolic
Pressure (mmHg)
A
RV dP/dtMax
(mmHg/sec)
H308
*
20000
†‡
*
15000
10000
5000
0
3000
1000
10
0
*
600
†
500
Dobutamine
(µg/kg/min)
40
Dobutamine
(µg/kg/min)
0
400
0
10
†
Heart Rate (bpm)
*
*
20
20
†‡
‡
700
40
30
*
2000
50
30
*
4000
Dobutamine
(µg/kg/min)
Dobutamine
(µg/kg/min)
End Systolic
Volume (µl)
5000
*
*
80
60
*
*
Fig. 5. Significant reductions in cardiac reserve of
aged mdx hearts. In a variety of measures of global
cardiac function the 9-mo-old mdx show limited
response to dobutamine stimulation. In C57BL/10
mice there are significant changes in cardiac output
(A), stroke work (B), stroke volume (C), heart rate
(D), end-systolic volume (E), and ejection fraction
(F). The hearts of mdx mice show either no change
or only with the highest dose of dobutamine. Specifically, mdx hearts have significantly lower cardiac output and stroke work following dobutamine
stimulation. *P ⬍ 0.05, difference from baseline
C57BL/10. †P ⬍ 0.05, difference from baseline
mdx. ‡P ⬍ 0.05, difference from C57BL/10 at that
dose.
†
40
20
0
Dobutamine
(µg/kg/min)
tolic and diastolic dysfunction is present in both ventricles,
including apparent increased organ-level compliance. These
findings are evidence that the well-known respiratory dysfunction in DMD results in right ventricular damage. Systolic
dysfunction is evident in both ventricles of the mdx heart;
however, despite the increased fibrosis, the right ventricular
function is slightly better compared with the left ventricle. Our
new findings suggest that right ventricular damage precedes the
onset of significant cardiac complications. This observation
places additional focus on the cardiorespiratory interaction in
muscular dystrophy and may have implications for the clinical
management of DMD patients.
In the present study we demonstrate that the right ventricle
is particularly severely affected throughout the life of the mdx
mouse with a larger percentage of fibrosis present in all age
groups examined (Fig. 2). The mechanism underlying the
increased right ventricular fibrosis is not clear; the size and
shapes of the lesions suggest the fibrotic replacement of damaged cardiac myocytes. This is consistent with a model where
the respiratory dysfunction of the mdx mouse (30, 32, 44)
results in hypoventilation and increased constriction of the
pulmonary vessels. This increase in afterload on the right
ventricle has the potential to increase membrane damage and
myocyte loss within this ventricle, as has been documented in
the left ventricle where increases in afterload result in substantial increases in myocyte damage (18, 34). These results
support the hypothesis that respiratory dysfunction in the
mdx mouse contributes to the progression of dystrophic cardiac disease; however, the possibility that the right ventricle is
somehow more susceptible to injury or fibrosis cannot be
definitively ruled out. The presence of early right ventricular
lesions in DMD patients is difficult to assess. Lesions within
the left ventricle are readily assessed using late gadolinium
enhancement (41, 50). This method is well suited for picking
up relatively large regions of fibrosis, which are present where
there is significant muscle mass. The right ventricle, with its
small mass and potentially smaller lesion sizes may be underrepresented in these studies. It is important to note that there
remains little difference in hemodynamic function between the
right and left ventricles of the mdx mouse; similar results are
seen in DMD patients (6, 58). The failure to observe interventricular hemodynamic differences may result from the tight
coupling of the ventricles, in that dysfunction of the right
ventricle will lead to dysfunction of the left ventricle. It should
also be noted that we have not documented right ventricular
volumes. It is possible that right ventricular dilation may be
present, although stroke volume and pressures remain intact.
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Stroke Volume (µl)
Dobutamine
(µg/kg/min)
H309
C57BL/10
mdx
Ejection Fraction (%)
Cardiac Output
(µl/min)
25000
Stroke Work (mmHg•µl)
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
H310
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
A
B
LV Pressure
(mmHg)
Baseline
100
100
100
50
50
50
0
0
50
100
0
50
1
5
0
G
20
20
10
10
0
0
mdx
1.0
0.5
0.0
0
Despite increased fibrosis and the decreased compliance of
isolated dystrophic myocytes (65, 67, 73), the mdx hearts in
this study display significantly greater left ventricular compliance compared with C57BL/10 hearts (Fig. 8). Furthermore,
our data indicate that the passive extension of the left ventricle
is subject to dynamic regulation by ␤-adrenergic stimulation,
with infusion of dobutamine significantly increasing the compliance of the left ventricle in both C57BL/10 and mdx hearts.
A potential mechanism underlying this observation is alterations in titin, an important determinant of sarcomere passive
properties (28). In hearts with dilated cardiomyopathy there is
a change in titin isoforms that increases the compliance of the
heart muscle, even in the presence of significant levels of
fibrosis (13, 39). Also PKA phosphorylation of titin has been
shown to be associated with small increases in sarcomere
compliance (72). However, previous studies in the more severely affected golden retriever model of muscular dystrophy
failed to observe any differences in the passive properties
between dystrophic and wild-type membrane permeabilized
myocytes (65), suggesting that titin compliance is not altered in
5
100
0
0
50
Time (msec)
1.5
Dobutamine
(µg/kg/min)
50
J
15
15
C57BL/10
mdx
40
10
C57BL/10
†
*
Dobutamine
15 µg/kg/min
50
20
RV Pressure
Transient
Width (msec)
RVP Integral
(mmHg•sec)
H
30
2.0
†
5
30
0
*
0
40
100
100
20
30
50
50
40
Dobutamine
5 µg/kg/min
50
‡
40
0
0
Time (msec)
60
Dobutamine
(µg/kg/min)
Time (msec)
I
E
0
15
Baseline
50
0
100
Time (msec)
60
‡
*
†
†
*
40
20
0
Dobutamine
(µg/kg/min)
0
5
15
this model. Other studies have demonstrated the decrease in
compliance of the mdx ventricle (1). In this elegant study,
Langendorff perfused hearts were used to assess the passive
properties of the left ventricle in mdx and C57BL/10. These
changes in passive properties were independent of myocyte
contractile function or extracellular Ca2⫹. The studies reported
here further support this observation in the intact heart. The
increased compliance of the dystrophic heart is only observed
in models where the extracellular matrix is present, supporting
a hypothesis that the interface between myocytes and the
extracellular matrix may be weakened without dystrophin.
Recent data demonstrate that ␣-dystroglycan glycosylation is
altered in the dystrophic heart (38, 59) offering a possible
mechanism underlying this weakened interaction with the
extracellular matrix. Additional studies will be required to
further understand the molecular basis underlying the regulation of the passive properties of the dystrophic heart.
In both the right and left ventricle under baseline conditions
the duration of the pressure transient was significantly truncated in the mdx heart relative to C57Bl/10 (Fig. 6). Interest-
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LVP Integral
(mmHg•sec)
‡
2
0
RV Pressure
(mmHg)
‡
3
Dobutamine
(µg/kg/min)
F
‡
4
mdx
LV Pressure
Transient
Width (msec)
C57BL/10
5
100
C57BL/10
mdx
Time (msec)
Time (msec)
D
Dobutamine
15 µg/kg/min
150
150
0
Fig. 6. Reduced pressure integral and shortened
transient width in dystrophic hearts. Both the left
(A–E) and right (F–J) ventricular pressure transients have reduced durations at baseline. Representative transients are shown for baseline (A and
F), 5 ␮g·kg⫺1·min⫺1 dobutamine (B and G), and 15
␮g·kg⫺1·min⫺1 dobutamine (C and H). ‡P ⬍ 0.05,
difference from C57BL/10 at that dose. *P ⬍ 0.05,
difference from baseline C57BL/10. †P ⬍ 0.05, difference from baseline mdx.
C
Dobutamine
5 µg/kg/min
150
H311
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
C57BL/10
mdx
A
B
*
*
15000
‡
‡
4000
RV dP/dtMin
(mmHg/sec)
LV dP/dtMin
(mmHg/sec)
20000
10000
5000
C
2000
1000
Dobutamine
(µg/kg/min)
D
*
4
2
10
5
0
Dobutamine
(µg/kg/min)
Dobutamine
(µg/kg/min)
C57BL/10
100
100
80
Baseline
Dob. 5 µg/kg
Dob. 15 µg/kg
80
60
60
40
40
20
20
0
0
0
10 20 30
F
mdx
%Peak Systolic (RV)
%Peak Systolic (LV)
E
Fig. 7. Evidence of disrupted relaxation in the
mdx heart. The C57BL/10 left ventricle shows
robust increases in relaxation in response to dobutamine documented as increased rate of relaxation (A) and isovolumic relaxation time constant, tau (C and E). In both of these measures of
relaxation the mdx heart is not affected by dobutamine. The right ventricle demonstrates a
dobutamine-induced increase in the rate of relaxation (B), but there is no dobutamine-induced
decrease in tau (D and F). *P ⬍ 0.05, difference
from baseline C57BL/10 data. ‡P ⬍ 0.05, difference from C57BL/10 at that dose. †P ⬍ 0.05,
difference from baseline mdx.
15
RV Tau (msec)
*
6
0
0
C57BL/10
100
80
80
60
60
40
40
20
20
0 10 20 30
0
0
10 20 30
ingly, a similar shortening of the ejection period has been
observed using tissue Doppler echocardiography in DMD patients (58). This shortened pressure transient is also present in
younger mdx mice, suggesting that it is not related to the
progression of dystrophic cardiac disease. The duration of the
pressure transient is determined by the timing of the repolarization of the ventricular myocytes. The repolarization of the
cardiac myocyte is driven by the combination of inactivation of
the L-type Ca2⫹ channel and the activation of a variety of
potassium currents (45). The mechanism by which this repolarization occurs more rapidly in the mdx heart is unknown. A
possible explanation is directly associated with the loss of
dystrophin; it is well documented that mdx cardiac myocytes
have increases in cytoplasmic calcium (71, 73). This calcium
mdx
100
0
0
10 20 30
originates from the extracellular solution and is likely to be
associated with contraction whether it enters through microtears (65, 73), stretch-activated channels (57, 71), and/or
increases in the L-type Ca2⫹ channel current (35). This increased Ca2⫹ influx is localized to the membrane compartment
and would be ideally positioned to increase the Ca2⫹-dependent inactivation of the L-type Ca2⫹ channel current. The
closing of the L-type Ca2⫹ channel results in the cessation of
Ca2⫹-induced Ca2⫹ release and is followed shortly by declines
in intracellular Ca2⫹ and force generation. ␤-Adrenergic receptor stimulation has been shown to invoke a similar Ca2⫹dependent acceleration of inactivation of the L-type channel
(26), which is consistent with the observations in the present
study.
Table 1. Data summarizing the load dependence of measures of diastolic function
C57BL/10
dP/dtmin-EDV slope
Tau-EDV slope
mdx
Baseline
Dobutamine
(5 ␮g·kg⫺1·min⫺1)
Dobutamine
(15 ␮g·kg⫺1·min⫺1)
Baseline
Dobutamine
(5 ␮g·kg⫺1·min⫺1)
Dobutamine
(15 ␮g·kg⫺1·min⫺1)
97.0 ⫾ 6.6*
⫺0.013 ⫾ 0.013
152.0 ⫾ 13.6*
⫺0.005 ⫾ 0.005
224.6 ⫾ 23.2*
0.00006 ⫾ 0.00004
107.2 ⫾ 6.8*
⫺0.0002 ⫾ 0.0002
133.3 ⫾ 9.03*
⫺0.00003 ⫾ 0.0002
149.7 ⫾ 12.2*
⫺0.00003 ⫾ 0.0001
Values are mean ⫾ SE; n ⫽ 17–19. EDV, end-diastolic volume. Data derived from changes in both preload and afterload in the left ventricle. *Significantly
different from zero.
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LV Tau (msec)
8
†
3000
0
Dobutamine
(µg/kg/min)
*
*
H312
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
LVEDP-EDV Slope
(mmHg/µl)
A
0.4
0.3
C57BL/10
LV Pressure (mmHg)
mdx
LV Pressure (mmHg)
C
*
0.1
0
Dobutamine
(5 µg/kg/min)
Baseline
Dobutamine
(15 µg/kg/min)
15
15
15
10
10
10
5
5
5
0
0
0
-5
LV Volume (µl)
-5
LV Volume (µl)
-5
15
15
15
10
10
10
5
5
5
0
0
0
-5
LV Volume (µl)
The reduced cardiac function of the mdx heart is clearly
revealed in the presence of dobutamine where deficiencies in
both systolic and diastolic function become apparent. The
reduced systolic cardiac reserve is roughly the same between
the right and left ventricles, with both being reduced relative to
C57BL/10 hearts (Fig. 4). Other studies have shown that 9- to
12-mo-old and 22-mo-old mdx mice have significantly decreased levels of Ser16-phosphorylated phospholamban, an
important subcellular target of ␤-adrenergic signaling (36, 71).
These studies are consistent with the presence of chronic
adrenergic receptor stimulation in these older mdx mice, which
may, in part, explain the reduced cardiac reserve. The retention
of the chronotropic response to dobutamine indicates that the
pacemaking cells in the sinoatrial node retain their responsiveness to ␤-adrenergic receptor activation, indicating that ␤-adrenergic signaling remains partially intact (Fig. 5).
There are some interesting differences in diastolic function
between the right and left ventricle of wild-type hearts (Fig. 7).
In the left ventricle of C57BL/10, dobutamine stimulation
results in large increases in the maximal rate of relaxation and
a marked shortening of tau, the time constant of isovolumetric
relaxation. This is consistent with the well-described lusitropic
function of ␤-adrenergic receptor stimulation in cardiac myocytes. These responses are absent from the mdx left ventricle.
In the right ventricle both C57BL/10 and mdx hearts have
-5
LV Volume (µl)
-5
LV Volume (µl)
LV Volume (µl)
significant increases in the minimum dP/dt induced by dobutamine; however, dobutamine does not affect tau in either
genotype. This apparent contradiction in relaxation properties
likely stems from the differential load dependence of the
minimum dP/dt documented by this study (Table 1) and others
(60, 70). In contrast, tau appears independent of changes in
loading conditions (Table 1) (60, 70). In the left ventricle, the
minimum dP/dt occurs shortly after the closure of the mitral
valve and is driven, in part, by the filling of the coronary
arteries. The rate at which the coronary arteries fill is dependent largely on the pressure within the aorta; thus the minimum
dP/dt is highly dependent upon left ventricular afterload (9). It
is not clear if the dependence on load seen in the left ventricle
is applicable to the right ventricle; however the filling of
the coronary vessels would be expected to have similar effects
on the pressure change within the right ventricle. These data
indicate that tau is a purer, load-independent measure of
myocardial isometric relaxation. The failure of the right ventricle to accelerate its relaxation in the presence of dobutamine
observed here and elsewhere (62) highlights the differences in
the relaxation properties of these two chambers. The sarcoplasmic/endoplasmic reticulum Ca2⫹-ATPase (SERCA) and phospholamban are present at roughly the same levels in both
ventricles (5, 55), although the Ca2⫹ reuptake is slower in right
ventricular myocytes (55). The slower kinetics of Ca2⫹ uptake
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B
‡
0.2
Dobutamine
(µg/kg/min)
Fig. 8. Increased passive compliance of the mdx
ventricle. At baseline the end-diastolic pressurevolume relationship indicates that the mdx left
ventricle is significantly more compliant than that
of C57BL/10. In contrast to C57BL/10, in the mdx
heart these passive properties are not altered by
dobutamine treatment (A). Representative pressure-volume loops (gray) with end-diastolic pressure-volume relationship (red) for C57BL/10 (B)
and mdx (C) hearts. *P ⬍ 0.05, difference from
baseline C57BL/10 data. ‡P ⬍ 0.05, difference
from C57BL/10.
C57BL/10
mdx
0.5
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
GRANTS
This study was supported by National Heart, Lung, and Blood Institute
Grants K08-HL-102066 and R01-HL-114832.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: T.A.M. and D.T. performed experiments; T.A.M. and
D.T. analyzed data; T.A.M. and D.T. interpreted results of experiments;
T.A.M. and D.T. prepared figures; T.A.M. and D.T. edited and revised
manuscript; T.A.M. and D.T. approved final version of manuscript; D.T.
conception and design of research; D.T. drafted manuscript.
REFERENCES
1. Barnabei MS, Metzger JM. Ex vivo stretch reveals altered mechanical
properties of isolated dystrophin-deficient hearts. PLoS ONE 7: e32880,
2012.
2. Bergofsky EH. Humoral control of the pulmonary circulation. Annu Rev
Physiol 42: 221–233, 1980.
3. Berko BA, Swift M. X-linked dilated cardiomyopathy. N Engl J Med 316:
1186 –1191, 1987.
4. Birnkrant DJ, Ashwath ML, Noritz GH, Merrill MC, Shah TA,
Crowe CA, Bahler RC. Cardiac and pulmonary function variability in
Duchenne/Becker muscular dystrophy: an initial report. J Child Neurol 25:
1110 –1115, 2010.
5. Bokník P, Unkel C, Kirchhefer U, Kleideiter U, Klein-Wiele O, Knapp J,
Linck B, Lüss H, Müller FU, Schmitz W, Vahlensieck U, Zimmermann
N, Jones LR, Neumann J. Regional expression of phospholamban in the
human heart. Cardiovasc Res 43: 67–76, 1999.
6. Bosser G, Lucron H, Lethor JP, Burger G, Beltramo F, Marie PY,
Marçon F. Evidence of early impairments in both right and left ventricular inotropic reserves in children with Duchenne’s muscular dystrophy.
Am J Cardiol 93: 724 –727, 2004.
7. Bostick B, Yue Y, Duan D. Gender influences cardiac function in the mdx
model of Duchenne cardiomyopathy. Muscle Nerve 42: 600 –603, 2010.
8. Brenman JE, Chao DS, Xia H, Aldape K, Bredt DS. Nitric oxide
synthase complexed with dystrophin and absent from skeletal muscle
sarcolemma in Duchenne muscular dystrophy. Cell 82: 743–752, 1995.
9. Brutsaert DL, Sys SU. Relaxation and diastole of the heart. Physiol Rev
69: 1228 –1315, 1989.
10. Bulfield G, Siller WG, Wight PA, Moore KJ. X chromosome-linked
muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA 81:
1189 –1192, 1984.
11. Bushby K, Finkel R, Birnkrant DJ, Case LE, Clemens PR, Cripe L,
Kaul A, Kinnett K, McDonald C, Pandya S, Poysky J, Shapiro F,
Tomezsko J, Constantin C, DMD Care Considerations Working
Group. Diagnosis and management of Duchenne muscular dystrophy. 2.
Implementation of multidisciplinary care. Lancet Neurol 9: 177–189,
2010.
12. Chamberlain JS, Metzger J, Reyes M, Townsend D, Faulkner JA.
Dystrophin-deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma. FASEB J 21: 2195–2204,
2007.
13. Chaturvedi RR, Herron T, Simmons R, Shore D, Kumar P, Sethia B,
Chua F, Vassiliadis E, Kentish JC. Passive stiffness of myocardium
from congenital heart disease and implications for diastole. Circulation
121: 979 –988, 2010.
14. Clarke JL, Gowers WR. On a case of pseudo-hypertrophic muscular
paralysis. Med Chir Trans 57: 247–260.5, 1874.
15. Connuck DM, Sleeper LA, Colan SD, Cox GF, Towbin JA, Lowe AM,
Wilkinson JD, Orav EJ, Cuniberti L, Salbert BA, Lipshultz SE.
Characteristics and outcomes of cardiomyopathy in children with Duchenne or Becker muscular dystrophy: a comparative study from the Pediatric Cardiomyopathy Registry. Am Heart J 155: 998 –1005, 2008.
16. Coulton GR, Curtin NA, Morgan JE, Partridge TA. The mdx mouse
skeletal muscle myopathy. II. Contractile properties. Neuropathol Appl
Neurobiol 14: 299 –314, 1988.
17. Crisp A, Yin H, Goyenvalle A, Betts C, Moulton HM, Seow Y, Babbs
A, Merritt T, Saleh AF, Gait MJ, Stuckey DJ, Clarke K, Davies KE,
Wood MJA. Diaphragm rescue alone prevents heart dysfunction in
dystrophic mice. Hum Mol Genet 20: 413–421, 2011.
18. Danialou G, Comtois AS, Dudley R, Karpati G, Vincent G, Rosiers
Des C, Petrof BJ. Dystrophin-deficient cardiomyocytes are abnormally
vulnerable to mechanical stress-induced contractile failure and injury.
FASEB J 15: 1655–1657, 2001.
19. Dayan D, Hiss Y, Hirshberg A, Bubis JJ, Wolman M. Are the
polarization colors of picrosirius red-stained collagen determined only by
the diameter of the fibers? Histochemistry 93: 27–29, 1989.
20. Dellorusso C, Crawford RW, Chamberlain JS, Brooks SV. Tibialis
anterior muscles in mdx mice are highly susceptible to contractioninduced injury. J Muscle Res Cell Motil 22: 467–475, 2001.
21. Duchenne GB. The pathology of paralysis with muscular degeneration
(paralysie myosclerotique), or paralysis with apparent hypertrophy. Br
Med J 2: 541–542, 1867.
22. Eagle M, Bourke J, Bullock R, Gibson M, Mehta J, Giddings D,
Straub V, Bushby K. Managing Duchenne muscular dystrophy—the
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00485.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 13, 2017
are consistent with the relatively prolonged relaxation of the
right ventricular pressure transient compared with that in the
left ventricle observed in this study. Chamber geometry is
another important difference between the right and left ventricles; it is possible that the increased systolic function induced
by dobutamine is stored in the twisted structure of the contracted left ventricle and its release contributes to the acceleration of the isometric relaxation of the left ventricle. The
geometry and mechanics of right ventricular contraction would
preclude this form of energy storage in the contracted ventricle,
which is then unavailable to contribute to the subsequent
relaxation.
The biventricular recording methods used in this study
provide unique insight into the coupling of the right and left
ventricle through the pulmonary circulation. The transit of a
pressure wave through the pulmonary circulation in the mouse
takes ⬃4 cardiac cycles or ⬃400 ms (Fig. 3). Similar studies
performed in the dog have shown that it takes 2–3 cardiac
cycles, 1.5–2 s, for a similar pressure wave to cross the canine
lung (46). Furthermore, in wild-type C57BL/10 mice this
transit time varies with dobutamine treatment. This is consistent with the presence of ␤-adrenergic receptor-mediated vasoconstriction in both pulmonary arteries and veins (2, 48), as
decreasing the compliance of the pulmonary circulation would
increase the velocity of the pressure wave. Interestingly, this
effect of dobutamine is lost in dystrophic mice, suggesting
abnormalities in the neurohormonal regulation of the mdx
pulmonary circulation.
In summary this study demonstrates the presence of significant fibrosis throughout the mdx heart, with the right ventricle
being particularly affected from an early age. The increased
right ventricular fibrosis may result from increased afterload
following either sympathetic activation or hypoxia-induced
constriction of the pulmonary artery secondary to hypoventilation. The latter of these two is supported by the severely
dystrophic diaphragm in old mdx mice (12) and the already
compromised respiratory function of the young mdx mouse
(30, 32, 43, 44). Together these observations indicate that
respiratory insufficiency occurs in the mdx mouse and is
progressive in nature. This respiratory dysfunction and resulting hypoxia will result in the constriction of the pulmonary
arteries. We propose a model by which this constriction of the
pulmonary vasculature contributes to the increased fibrosis
observed in the right ventricle. This has potential clinical
significance suggesting that earlier initiation of supportive
respiratory therapy could limit right ventricular damage and
delay the onset of cardiomyopathy in DMD patients.
H313
H314
23.
24.
25.
26.
27.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
additive effect of spinal surgery and home nocturnal ventilation in improving survival. Neuromuscul Disord 17: 470 –475, 2007.
Eagle M, Eagle M, Baudouin SV, Baudouin SV, Chandler C, Chandler C,
Giddings DR, Giddings DR, Bullock R, Bullock R, Bushby K, Bushby
K. Survival in Duchenne muscular dystrophy: improvements in life
expectancy since 1967 and the impact of home nocturnal ventilation.
Neuromuscul Disord 12: 926 –929, 2002.
Ervasti JM, Campbell KP. Membrane organization of the dystrophinglycoprotein complex. Cell 66: 1121–1131, 1991.
Ervasti JM, Ohlendieck K, Kahl SD, Gaver MG, Campbell KP.
Deficiency of a glycoprotein component of the dystrophin complex in
dystrophic muscle. Nature 345: 315–319, 1990.
Findlay I. beta-Adrenergic stimulation modulates Ca2⫹- and voltagedependent inactivation of L-type Ca2⫹ channel currents in guinea-pig
ventricular myocytes. J Physiol 541: 741–751, 2002.
Gavillet B, Rougier JS, Domenighetti AA, Behar R, Boixel C, Ruchat
P, Lehr HA, Pedrazzini T, Abriel H. Cardiac sodium channel Nav1.5 is
regulated by a multiprotein complex composed of syntrophins and dystrophin. Circ Res 99: 407–414, 2006.
Granzier H, Labeit S. Cardiac titin: an adjustable multi-functional spring.
J Physiol 541: 335–342, 2002.
Hoffman EP, Brown RH, Kunkel LM. Dystrophin: the protein product
of the Duchenne muscular dystrophy locus. Cell 51: 919 –928, 1987.
Huang P, Cheng G, Lu H, Aronica M, Ransohoff RM, Zhou L.
Impaired respiratory function in mdx and mdx/utrn(⫹/⫺) mice. Muscle
Nerve 43: 263–267, 2011.
Inkley SR, Oldenburg FC, Vignos PJ. Pulmonary function in Duchenne
muscular dystrophy related to stage of disease. Am J Med 56: 297–306, 1974.
Ishizaki M, Suga T, Kimura E, Shiota T, Kawano R, Uchida Y, Uchino K,
Yamashita S, Maeda Y, Uchino M. Mdx respiratory impairment following
fibrosis of the diaphragm. Neuromuscul Disord 18: 342–348, 2008.
James J, Kinnett K, Wang Y, Ittenbach RF, Benson DW, Cripe L.
Electrocardiographic abnormalities in very young Duchenne muscular
dystrophy patients precede the onset of cardiac dysfunction. Neuromuscul
Disord 21: 462–467, 2011.
Kamogawa Y, Biro S, Maeda M, Setoguchi M, Hirakawa T, Yoshida
H, Tei C. Dystrophin-deficient myocardium is vulnerable to pressure
overload in vivo. Cardiovasc Res 50: 509 –515, 2001.
Koenig X, Rubi L, Obermair GJ, Cervenka R, Dang XB, Lukacs P,
Kummer S, Bittner RE, Kubista H, Todt H, Hilber K. Enhanced
currents through L-type calcium channels in cardiomyocytes disturb the
electrophysiology of the dystrophic heart. Am J Physiol Heart Circ
Physiol 306: H564 –H573, 2014.
Lai Y, Zhao J, Yue Y, Wasala NB, Duan D. Partial restoration of cardiac
function with ⌬PDZ nNOS in aged mdx model of Duchenne cardiomyopathy. Hum Mol Genet 23: 3189 –3199, 2014.
Lefaucheur JP, Pastoret C, Sebille A. Phenotype of dystrophinopathy in
old mdx mice. Anat Rec 242: 70 –76, 1995.
Lohan J, Culligan K, Ohlendieck K. Deficiency in cardiac dystrophin
affects the abundance of the ␣-/␤-dystroglycan complex. J Biomed Biotechnol 2005: 28 –36, 2005.
Makarenko I, Opitz CA, Leake MC, Neagoe C, Kulke M, Gwathmey
JK, Del Monte F, Hajjar RJ, Linke WA. Passive stiffness changes
caused by upregulation of compliant titin isoforms in human dilated
cardiomyopathy hearts. Circ Res 95: 708 –716, 2004.
Markham LW, Kinnett K, Wong BL, Woodrow Benson D, Cripe LH.
Corticosteroid treatment retards development of ventricular dysfunction in
Duchenne muscular dystrophy. Neuromuscul Disord 18: 365–370, 2008.
Mazur W, Hor KN, Germann JT, Fleck RJ, Al-Khalidi HR, Wansapura JP, Chung ES, Taylor MD, Jefferies JL, Woodrow Benson D,
Gottliebson WM. Patterns of left ventricular remodeling in patients with
Duchenne muscular dystrophy: a cardiac MRI study of ventricular geometry, global function, and strain. Int J Cardiovasc Imaging 28: 99 –107,
2012.
McCormack WM, Spalter HF. Muscular dystrophy, alveolar hypoventilation, papilledema. JAMA 197: 957–960, 1966.
Mosqueira M, Baby SM, Lahiri S, Khurana TS. Ventilatory chemosensory drive is blunted in the mdx mouse model of Duchenne muscular
dystrophy (DMD). PLoS ONE 8: e69567, 2013.
Nelson CA, Hunter RB, Quigley LA, Girgenrath S, Weber WD,
McCullough JA, Dinardo CJ, Keefe KA, Ceci L, Clayton NP, McVieWylie A, Cheng SH, Leonard JP, Wentworth BM. Inhibiting TGF-␤
activity improves respiratory function in mdx mice. Am J Pathol 178:
2611–2621, 2011.
45. Nerbonne JM, Kass RS. Molecular physiology of cardiac repolarization.
Physiol Rev 85: 1205–1253, 2005.
46. Olsen CO, Tyson GS, Maier GW, Spratt JA, Davis JW, Rankin JS.
Dynamic ventricular interaction in the conscious dog. Circ Res 52:
85–104, 1983.
47. Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL. Dystrophin protects the sarcolemma from stresses developed during muscle
contraction. Proc Natl Acad Sci USA 90: 3710 –3714, 1993.
48. Porcelli RJ, Bergofsky EH. Adrenergic receptors in pulmonary vasoconstrictor responses to gaseous and humoral agents. J Appl Physiol 34:
483–488, 1973.
49. Preibisch S, Saalfeld S, Tomancak P. Globally optimal stitching of tiled
3D microscopic image acquisitions. Bioinformatics 25: 1463–1465, 2009.
50. Puchalski MD, Williams RV, Askovich B, Sower CT, Hor KH, Su JT,
Pack N, Dibella E, Gottliebson WM. Late gadolinium enhancement:
precursor to cardiomyopathy in Duchenne muscular dystrophy? Int J
Cardiovasc Imaging 25: 57–63, 2009.
51. Quinlan JG, Hahn HS, Wong BL, Lorenz JN, Wenisch AS, Levin LS.
Evolution of the mdx mouse cardiomyopathy: physiological and morphological findings. Neuromuscul Disord 14: 491–496, 2004.
52. Ramaswamy KS, Palmer ML, van der Meulen JH, Renoux A, Kostrominova TY, Michele DE, Faulkner JA. Lateral transmission of force
is impaired in skeletal muscles of dystrophic mice and very old rats. J
Physiol (Lond) 589: 1195–1208, 2011.
53. Romfh A, McNally EM. Cardiac assessment in Duchenne and Becker
muscular dystrophies. Curr Heart Fail Rep 7: 212–218, 2010.
54. Ryan TD, Taylor MD, Mazur W, Cripe LH, Pratt J, King EC, Lao K,
Grenier MA, Jefferies JL, Benson DW, Hor KN. Abnormal circumferential strain is present in young Duchenne muscular dystrophy patients.
Pediatr Cardiol 34: 1159 –1165, 2013.
55. Sathish V, Xu A, Karmazyn M, Sims SM, Narayanan N. Mechanistic
basis of differences in Ca2⫹-handling properties of sarcoplasmic reticulum
in right and left ventricles of normal rat myocardium. Am J Physiol Heart
Circ Physiol 291: H88 –H96, 2006.
56. Schram G, Fournier A, Leduc H, Dahdah N, Therien J, Vanasse M,
Khairy P. All-cause mortality and cardiovascular outcomes with prophylactic steroid therapy in Duchenne muscular dystrophy. J Am Coll Cardiol
61: 948 –954, 2013.
57. Seo K, Rainer PP, Lee DI, Hao S, Bedja D, Birnbaumer L, Cingolani
OH, Kass DA. Hyperactive adverse mechanical stress responses in dystrophic heart are coupled to transient receptor potential canonical 6 and
blocked by cGMP-protein kinase G modulation. Circ Res 114: 823–832,
2014.
58. Shabanian R, Aboozari M, Kiani A, Seifirad S, Zamani G, Nahalimoghaddam A, Kocharian A. Myocardial performance index and atrial
ejection force in patients with Duchenne’s muscular dystrophy. Echocardiography 28: 1088 –1094, 2011.
59. Sharpe KM, Premsukh MD, Townsend D. Alterations of dystrophinassociated glycoproteins in the heart lacking dystrophin or dystrophin and
utrophin. J Muscle Res Cell Motil 34: 395–405, 2013.
60. Starling MR, Montgomery DG, Mancini GB, Walsh RA. Load independence of the rate of isovolumic relaxation in man. Circulation 76:
1274 –1281, 1987.
61. Stedman HH, Sweeney HL, Shrager JB, Maguire HC, Panettieri RA,
Petrof B, Narusawa M, Leferovich JM, Sladky JT, Kelly AM. The mdx
mouse diaphragm reproduces the degenerative changes of Duchenne
muscular dystrophy. Nature 352: 536 –539, 1991.
62. Tabima DM, Hacker TA, Chesler NC. Measuring right ventricular
function in the normal and hypertensive mouse hearts using admittancederived pressure-volume loops. Am J Physiol Heart Circ Physiol 299:
H2069 –H2075, 2010.
63. Towbin JA, Hejtmancik JF, Brink P, Gelb B, Zhu XM, Chamberlain
JS, McCabe ER, Swift M. X-linked dilated cardiomyopathy. Molecular
genetic evidence of linkage to the Duchenne muscular dystrophy (dystrophin) gene at the Xp21 locus. Circulation 87: 1854 –1865, 1993.
64. Townsend D, Blankinship MJ, Allen JM, Gregorevic P, Chamberlain
JS, Metzger JM. Systemic administration of micro-dystrophin restores
cardiac geometry and prevents dobutamine-induced cardiac pump failure.
Mol Ther 15: 1086 –1092, 2007.
65. Townsend D, Turner I, Yasuda S, Martindale J, Davis J, Shillingford
M, Kornegay JN, Metzger JM. Chronic administration of membrane
sealant prevents severe cardiac injury and ventricular dilatation in dystrophic dogs. J Clin Invest 120: 1140 –1150, 2010.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00485.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 13, 2017
28.
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
BIVENTRICULAR DYSFUNCTION IN THE MDX MOUSE
66. Townsend D, Yasuda S, Li S, Chamberlain JS, Metzger JM. Emergent
dilated cardiomyopathy caused by targeted repair of dystrophic skeletal
muscle. Mol Ther 16: 832–835, 2008.
67. Townsend D, Yasuda S, McNally E, Metzger JM. Distinct pathophysiological mechanisms of cardiomyopathy in hearts lacking dystrophin or
the sarcoglycan complex. FASEB J 25: 3106 –3114, 2011.
68. Van Erp C, Loch D, Laws N, Trebbin A, Hoey AJ. Timeline of cardiac
dystrophy in 3–18-month-old MDX mice. Muscle Nerve 42: 504–513, 2010.
69. Wasala NB, Bostick B, Yue Y, Duan D. Exclusive skeletal muscle
correction does not modulate dystrophic heart disease in the aged mdx
model of Duchenne cardiomyopathy. Hum Mol Genet 22: 2634 –2641,
2013.
70. Weiss JL, Frederiksen JW, Weisfeldt ML. Hemodynamic determinants
of the time-course of fall in canine left ventricular pressure. J Clin Invest
58: 751–760, 1976.
H315
71. Williams IA, Allen DG. Intracellular calcium handling in ventricular
myocytes from mdx mice. Am J Physiol Heart Circ Physiol 292: H846 –
H855, 2007.
72. Yamasaki R, Wu Y, McNabb M, Greaser M, Labeit S, Granzier H.
Protein kinase A phosphorylates titin’s cardiac-specific N2B domain and
reduces passive tension in rat cardiac myocytes. Circ Res 90: 1181–1188,
2002.
73. Yasuda S, Townsend D, Michele DE, Favre EG, Day SM, Metzger JM.
Dystrophic heart failure blocked by membrane sealant poloxamer. Nature
436: 1025–1029, 2005.
74. Zhang W, Hove ten M, Schneider JE, Stuckey DJ, Sebag-Montefiore
L, Bia BL, Radda GK, Davies KE, Neubauer S, Clarke K. Abnormal
cardiac morphology, function and energy metabolism in the dystrophic
mdx mouse: an MRI and MRS study. J Mol Cell Cardiol 45: 754 –760,
2008.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 13, 2017
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00485.2014 • www.ajpheart.org