Download Progression from hypertrophic to dilated cardiomyopathy in mice

Am J Physiol Heart Circ Physiol
280: H151–H159, 2001.
Progression from hypertrophic to dilated cardiomyopathy
in mice that express a mutant myosin transgene
KALEV FREEMAN,1 CYNTHIA COLON-RIVERA,1 M. CHARLOTTE OLSSON,2
RUSSELL L. MOORE,2 HOWARD D. WEINBERGER,3 INGRID L. GRUPP,4
KAREN L. VIKSTROM,5 GUIDO IACCARINO,6 WALTER J. KOCH,6
AND LESLIE A. LEINWAND1
1
Department of Molecular Cellular and Developmental Biology and 2Department of Kinesiology and
Applied Physiology, University of Colorado, Boulder 80309-0347; 3Division of Cardiology, University
of Colorado Health Sciences Center, Denver, Colorado 80262; 4Department of Pharmacology,
University of Cincinnati Medical Center, Cincinnati, Ohio 45267; 5Department of Pharmacology,
State University of New York Upstate Medical University, Syracuse, New York 13210; and
6
Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710
Received 30 May 2000; accepted in final form 11 August 2000
Freeman, Kalev, Cynthia Colon-Rivera, M. Charlotte
Olsson, Russell L. Moore, Howard D. Weinberger, Ingrid L. Grupp, Karen L. Vikstrom, Guido Iaccarino,
Walter J. Koch, and Leslie A. Leinwand. Progression
from hypertrophic to dilated cardiomyopathy in mice that
express a mutant myosin transgene. Am J Physiol Heart Circ
Physiol 280: H151–H159, 2001.—A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of
a cardiac ␣-myosin transgene including the R403Q mutation
and a deletion of a segment of the actin-binding domain.
HCM mice show early histopathology and hypertrophy, with
progressive hypertrophy in females and ventricular dilation
in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM,
we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and
embryonic gene expression in 8- to 11-mo-old male transgenic
animals. Significantly impaired exercise tolerance and both
systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also
decreased, and lusitropic responsiveness to the ␤-adrenergic
agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled ␤-adrenergic receptor kinase 1
(␤-ARK1) were increased by more than twofold over controls,
and total ␤-ARK1 activity was also significantly elevated.
Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have
undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be
part of a pathological continuum rather than independent
diseases.
of the heart muscle that
are associated with cardiac dysfunction. Dilated cardiomyopathy (DCM), the most common form, is char-
acterized by ventricular chamber dilation with normal
or decreased wall thickness and impaired systolic function, which often manifests as heart failure (33). Hypertrophic cardiomyopathy (HCM) is characterized by
abnormal cardiac hypertrophy, fibrosis, and myofibrillar disarray (33). Systolic function is typically normal
or enhanced, but cardiac relaxation is impaired due to
the thickened, fibrotic ventricular walls. Patients can
remain asymptotic for many years, or they can display
symptoms and consequences of outflow tract obstruction, diastolic dysfunction, or atrial fibrillation, including sudden cardiac death (39).
It has been estimated that 20% of idiopathic DCM
and 70% of HCM is familial (26, 33), and recent studies
have elucidated the genetic basis of some of these
cases. Eight different disease genes have been linked to
familial HCM (2, 28). Significantly, all of the eight
genes encode distinct molecular components of the
cardiac sarcomere, the fundamental force-generating
unit of heart muscle fibers. Linkage analysis in families with idiopathic DCM has been less informative, in
part because of the later onset of disease, but recently
a mutation in cardiac actin, a component of the sarcomeric thin filament, was linked to DCM (32). Mutations in cardiac actin have also been associated with
familial HCM (28). In hamsters, both HCM and DCM
are caused by mutations in the same gene, ␦-sarcoglycan, which encodes a protein of the dystrophin-associated glycoprotein complex (34). In addition, cases in
which HCM has progressed to DCM have been reported (18, 42); this decompensation occurs in ⬃10–
15% of patients (37, 38). An important question raised
by these findings is whether HCM and DCM are inherently independent diseases or whether these diseases
are part of the same pathological spectrum (2).
Address for reprint requests and other correspondence: L. A.
Leinwand, Dept. of Molecular, Cellular, and Developmental Biology,
Univ. of Colorado, Campus Box 347, Boulder, CO 80309-0347
(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
myosin heavy chain; cardiac decompensation; exercise
intolerance; ␤-adrenergic receptor kinase 1
CARDIOMYOPATHIES ARE DISEASES
http://www.ajpheart.org
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
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DEVELOPMENT OF DILATED CARDIOMYOPATHY IN HCM MICE
Valuable insight into the pathogenesis of familial
HCM has been gained from studies of the ␤-myosin
heavy chain (␤-MyHC) gene. Over 50 mutations at this
disease locus have been linked to HCM, and it has been
estimated that ␤-MyHC mutations account for onethird of the familial HCM cases (39). Nearly all of the
␤-MyHC mutations associated with HCM lie in the
“head” region of the heavy chain (2), which includes
both the ATPase and actin-binding regions critical for
generating muscle force. These mutations are predominantly missense mutations or short deletions that do
not disrupt the genetic reading frame, but they instead
appear to produce full-length mutated myosin molecules that become incorporated into the sarcomere (40).
The advent of mouse models for HCM demonstrated
that mutant MyHCs could disrupt the cardiac muscle
apparatus, causing a stress on the heart, which results
in HCM (10, 45). We engineered a mutant ␣-MyHC
transgene (the normal murine myocardium is almost
exclusively ␣-MyHC) that includes a well-characterized R403Q missense mutation associated with markedly reduced survival in humans (45). An additional
deletion of amino acids 468–527 in the actin-binding
domain bridged by nine nonmyosin amino acids allowed the mutant protein to be distinguished electrophoretically from endogenous mouse MyHC. Targeted
expression of this transgene in the mouse heart was
achieved by using a rat ␣-MyHC promoter, and 10–
12% of total MyHC in purified myofibrils from transgene-positive animals was the mutant protein (45). At
3 mo of age, the hearts of transgene-positive animals
display hypertrophy of both right and left ventricles,
with a pattern of myocyte hypertrophy, myocellular
disarray, interstitial fibrosis, and small vessel coronary disease that closely replicates the human histopathology (45). Areas of severe myocyte damage, examined at the electron microscope level, contained
degenerating myofibrils and collagen deposits (45). A
gender difference was also observed in these animals.
Cardiac hypertrophy increases with age in female animals, but older male animals have dilated left ventricular (LV) chambers, suggesting a more severe phenotype resembling DCM.
The purpose of this study was to determine whether
DCM is part of the phenotypic presentation of this
transgenic mouse model of HCM. We hypothesized
that older male animals would demonstrate not only
morphological indications of DCM but also functional
and biochemical defects, including systolic dysfunction
and adrenergic desensitization. Exercise testing, echocardiography, and isolated heart studies determined
cardiac function. Because adrenergic signal abnormalities are well established in DCM, we assessed the
sensitivity of isolated hearts to the adrenergic agonist
isoproterenol. In addition, we measured adrenergic receptor density, adenylyl cyclase activity, and the levels
and activity of G protein-coupled receptor kinases
(GRK) in heart extracts. We also assessed the expression of genetic markers of cardiac hypertrophy in the
HCM animals.
METHODS
Experimental animals. Mice heterozygous for the mutant
myosin transgene (45) were backcrossed with C57/Bl6 mice
to generate experimental animals (HCM) and nontransgenic
(NTG) littermate controls. The transgene coding region consists of a rat ␣-MyHC cDNA containing a G1445A point
mutation, resulting in Arg403Gln, and a deletion of amino
acids 468–527 bridged by the addition of nine nonmyosin
amino acids: SerSerLeuProHisLeuLysLeu. Male offspring
were genotyped by PCR and allowed to reach 8–11 mo of age
under identical conditions, when some of the mice were
selected for noninvasive echocardiography and exercise testing. Separate groups of age-matched male mice were euthanized for isolated heart experiments, histology, pharmacology, or RNA extraction. All of the animals were handled
according to approved protocols of the University of Colorado.
Treadmill exercise. The mice were exercised on a custombuilt eight-lane treadmill with an infrared detection system
similar to that previously described (9). The mice were acclimated to the treadmill at a 7° incline with one 15-min
low-speed (5–7 m/min) session without the shock grid and
two 15-min sessions with the shock grid (5–7 m/min and 20
m/min). The mice were exercised once daily at 20 m/min for
60 min over a 2-wk period. If a mouse became exhausted
during exercise, it was removed from the apparatus. Exercise
tolerance was measured by counting the average number of
infrared beam breaks per minute for each animal over all
exercise sessions. To determine exercise endurance, the animals were exercised once at a high speed (27 m/min) for 60
min, and the time at which each animal became exhausted
was recorded.
Echocardiography. We performed transthoracic echocardiography with the use of a System Five echocardiography
machine (Vingmed, Horton, Norway) with a 10-MHz phasedarray transducer. Each mouse was injected intraperitoneally
immediately before imaging with successive 0.05- to 0.3-ml
doses of 20 mg/ml tribromoethanol (Avertin) until mild sedation was achieved. The chest was shaved, and the mouse was
positioned on its abdomen on a 1.25-cm-thick acoustic standoff pad. Heart rates were monitored by electrocardiography
during image acquisition. M-mode recordings were acquired
in an M-mode format. To maximize temporal resolution,
images were displayed off-line from the original R␪ sampling
information for measurements by using Echo-Pack software
(Vingmed). Measurements from three cardiac cycles per animal were averaged.
Histology. Hearts were rapidly excised after cervical dislocation and placed in phosphate-buffered saline while still
beating to allow blood to be pumped out of the cardiac
chambers and coronary vessels. The hearts were then placed
in a 10:1 volume of 10% Formalin to tissue for fixation. The
fixed hearts were embedded in paraffin, sectioned, and
stained with Mason’s trichrome according to standard protocols. The first subatrial section from each heart was digitized,
and the internal and external LV areas from each heart were
traced manually and measured with the use of Scion Image
software (Scion, National Institutes of Health).
Isolated heart preparations. For determination of isolated
heart function, ejecting heart preparations were performed
as previously described (13, 14, 31). Hearts with rates under
300 beats/min were paced at that rate (3/5 NTG and 3/7 HCM
hearts). Baseline measurements were taken after the establishment of steady-state conditions, and preload was altered over a range of cardiac work from 200 to 350
mmHg 䡠 ml 䡠 min⫺1 to generate Starling curves for each heart.
Sensitivity to the adrenergic agonist isoproterenol was deter-
DEVELOPMENT OF DILATED CARDIOMYOPATHY IN HCM MICE
mined with additional hearts by using an isovolumic preparation similar to that previously described for the rat heart
(24). Briefly, each heart was excised and retrograde perfusion
was established, and then a highly compliant custom-made
latex balloon was inserted into the left ventricle via the
mitral valve. Balloons were sized and shaped to match the
dimensions of dilated HCM hearts. The balloon was attached
to an airtight catheter filled with distilled water and connected to pressure tubing which housed a 3-Fr transducer
(Millar Instruments, Houston, TX), and the balloon was
inflated to yield an end-diastolic pressure of 5 mmHg. The
hearts were paced at 360 beats/min, and LV pressures were
monitored during steady-state conditions and at 1, 3, and 5
min after exposure to 1 ␮mol/l isoproterenol.
␤-Adrenergic receptor density, adenylyl cyclase activity,
and GRK immunoblotting and activity. The mice were euthanized by cervical dislocation, and the hearts were excised and
placed in phosphate-buffered saline while beating to pump
blood out of the myocardium. Left ventricles were dissected
and frozen at ⫺80°C within 10 min of death. The functional
state of ␤-adrenergic receptors in the HCM and NTG hearts
was determined as described previously (20, 23, 27). Briefly,
we measured total ␤-adrenergic receptor binding on myocardial membranes with the use of the nonselective ␤-adrenergic
ligand [125I]iodocyanopidnolol (20). Membrane adenylyl cyclase activity was determined under basal conditions and in
the presence of isoproterenol or NaF (23, 27). The myocardial
levels of ␤-adrenergic receptor kinase 1 (␤-ARK1) and GRK5
were determined by immunoprecipitation, followed by immunoblotting of detergent-solubilized extracts (20, 23). Total
GRK activity in the myocardial membranes was determined
by using rhodopsin-enriched rod outer segment membranes
as an in vitro substrate, and [␥-32P]ATP incorporation into
rhodopsin was determined (20, 23).
RNA analysis. Total RNA was extracted from the LV
myocardium previously frozen as described above by using
TriZOL reagent (GIBCO-BRL). The expression of ␣-MyHC,
␤-MyHC, ␣-skeletal actin (s-ACT), and atrial natriuretic factor (ANF) mRNA were determined by using a slot blot with
previously described oligonucleotide probes (22, 41). Small
variations in loading were corrected by normalization to
mRNA levels of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).
Statistics. Data are presented as means ⫾ SE. The number
(n) of mice used is indicated. Statistical analysis was performed by Student’s t-test for paired comparisons between
HCM and NTG mice. Starling curves and isoproterenol doseresponse values were tested by ANOVA.
RESULTS
To quantify the extent of ventricular dilation in older
male HCM animals, hearts from age-matched HCM
and NTG mice were dissected and fixed in formaldehyde. The first subatrial section from each heart was
digitized, and internal LV chamber area was directly
measured. The “external LV chamber area” was defined as the area within a closed region circumscribing
the entire left ventricle myocardium, including the
interventricular septum. Both the internal LV chamber area (1.41 ⫾ 0.14 vs. 3.21 ⫾ 0.51 mm2, NTG vs.
HCM, respectively, P ⬍ 0.005) and the ratio of the
internal to the external LV chamber area (9.27 ⫾ 0.81
vs. 18.77 ⫾ 1.99, P ⬍ 0.01) were significantly increased
in the HCM hearts (Fig. 1). Foci of myofibrillar disar-
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Fig. 1. Measurement of left ventricular (LV) chamber dimensions in
hypertrophic cardiomyopathy (HCM) and nontransgenic (NTG) littermate control hearts. Representative sections from HCM and NTG
hearts. Average LV chamber area in NTG (n ⫽ 8) and HCM (n ⫽ 7)
heart sections. *P ⬍ 0.01, t-test.
ray and fibrosis were observed in HCM hearts, as
described in detail previously (45).
Exercise intolerance is one of the hallmarks of DCM.
HCM is linked to sudden cardiac death in young athletes. Furthermore, exercise intolerance has been
found in cases of symptomatic HCM with diastolic
dysfunction alone as well as in cases that have progressed to dilation and systolic dysfunction (39). We
assessed exercise tolerance in the HCM mice by using
a custom-built treadmill equipped with infrared beams
above the shock stimuli at the rear of the treadmill
belt. We first measured the ability of mice to keep pace
with a treadmill belt moving at 20 m/min on a 7°
incline. Compared with NTG, the HCM mice were
exercise intolerant, as indicated by a significantly
higher number of beam breaks per minute during the
exercise test (Fig. 2). When the mice ran at a higher
speed (27 m/min) for 60 min, HCM mice showed significantly depressed exercise endurance compared
with NTG controls (Fig. 2). Whereas all of the six
control mice completed 60 min of exercise at this speed,
only one of eight HCM mice was able to complete the
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DEVELOPMENT OF DILATED CARDIOMYOPATHY IN HCM MICE
Fig. 2. Treadmill exercise tolerance of HCM (n ⫽ 8) and NTG (n ⫽ 6)
mice. Top: beam breaks per min during 1 h of exercise at 20 m/min.
Averaged results from 12 exercise sessions; *P ⬍ 0.05, t-test. Bottom:
time to failure in 1-h exercise at 27 m/min. Averaged results from 1
exercise session.
entire exercise session. One HCM mouse died after 34
min of exercise, six failed from exhaustion between 35
and 52 min, and only one was able to complete the full
60-min session (Fig. 2).
To assess in vivo hemodynamics, we studied HCM
and NTG mice by transthoracic echocardiography at
comparable near-physiological heart rates (560 ⫾ 10
beats/min). Systolic function, measured as percent
fractional shortening, was significantly decreased in
the HCM mice (Fig. 3). Because of the high murine
heart rate, standard Doppler parameters for assessment of diastolic function were not measurable. Therefore, we chose to assess diastolic function by evaluating
the rate of relaxation of the posterior wall of the left
ventricle, measured directly from the digitized M-mode
images as the slope of a line tangent to the posterior
wall during diastole. The posterior wall relaxation
slope of the HCM mice was significantly depressed,
suggesting impaired in vivo relaxation (Fig. 3). The
internal diameter of the left ventricle in diastole was
modestly increased in the HCM animals as measured
from M-mode images, but the difference from NTG
hearts was not significant.
To assess myocardial function directly in the absence
of neurohumoral or peripheral cardiovascular effects,
we conducted isolated ejecting heart experiments. A
small number of transgenic and control hearts were
excised and cannulated via the pulmonary veins and
aorta to establish an isolated system in which the
hearts performed measurable preload-dependent pressure work against a tightly controlled afterload. Under
identical load conditions, both contractility and relax-
ation, measured by the first derivative of pressure
development over time (⫹dP/dt) and first derivative of
pressure relaxation over time (⫺dP/dt), respectively,
were decreased, and time to peak pressure (TPP) and
half-time to relaxation (RT1⁄2) were increased in the
HCM mice (Table 1). The maximal systolic pressure
developed by the HCM hearts was also diminished
(Table 1). These changes were modest, ranging from 9
to 16%, but were statistically significant as indicated.
Despite an overall reduction in both ⫹dP/dt and
⫺dP/dt over a range of cardiac work obtained by varying the preload, HCM hearts were able to increase both
⫹dP/dt and ⫺dP/dt to the same relative extent as
controls, suggesting that the Starling response is preserved in these hearts (Fig. 4).
Abnormalities in ␤-adrenergic signaling have been
well documented in heart failure, including DCM, and
recently ␤-adrenergic receptor downregulation, abnormal adrenergic control of the force-frequency relation,
and reduced catecholamine reuptake have been reported in primary HCM (6, 21, 35). To measure the
␤-adrenergic responsiveness of HCM hearts, we used
an isolated isovolumic preparation, in which a balloon
is inserted into the left ventricle and inflated to a
constant diastolic pressure. Isovolumic pressure
changes are recorded as the heart is stimulated to
contract. In this preparation, the effects of adrenergic
activation on coronary perfusion are minimized by retrograde perfusion. Additionally, endogenous pacemaker activity is abolished by crushing the atria, permitting direct assessment of myocardial inotropic and
lusitropic responsiveness to the adrenergic agonist
without chronotropic effects. We measured the isovolumic response of transgenic and NTG hearts to a single
Fig. 3. Echocardiography of HCM (n ⫽ 15) and NTG (n ⫽ 16) mice.
Top: systolic function, % fractional shortening (%FS); *P ⬍ 0.001,
t-test. Bottom: diastolic function, posterior wall relaxation slope
(PWRS); *P ⬍ 0.01, t-test.
DEVELOPMENT OF DILATED CARDIOMYOPATHY IN HCM MICE
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Table 1. Baseline parameters in isolated workperforming heart preparation
NTG
n
Age, mo
Body wt, g
Heart wt (both ventricles), mg
Heart wt/body wt, mg/g
Heart rate, beats/min
Mean aortic pressure
(afterload), mmHg
Left IVP, mmHg
Systolic
Diastolic
End diastolic
Cardiac output (venous return;
preload), ml/min
Aortic output
Coronary flow
Stroke volume, ␮l
Cardiac work (left ventricle),
mmHg 䡠 ml 䡠 min⫺1
⫹dP/dt, mmHg/s
⫺dP/dt, mmHg/s
TPP/unit pressure, ms/mmHg
RT1/2/unit pressure, ms/mmHg
PaO2, mmHg
PvO2, mmHg
MV̇O2, ␮l O2 䡠 min⫺1 䡠 g⫺1
HCM
5
8.4 ⫾ 0.2
30.5 ⫾ 1.0
196 ⫾ 8.2
6.5 ⫾ 0.32
337 ⫾ 11.5
6
8.7 ⫾ 0.1
27.3 ⫾ 0.8
202 ⫾ 3.9
7.4 ⫾ 0.23*
346 ⫾ 11.6
50.3 ⫾ 0.22
50.0 ⫾ 0.24
99.0 ⫾ 2.3
⫺5.0 ⫾ 1.1
9.5 ⫾ 0.8
89.1 ⫾ 1.2†
⫺4.2 ⫾ 1.4
10.1 ⫾ 1.8
5.04 ⫾ 0.02
3.04 ⫾ 0.26
1.97 ⫾ 0.23
15.0 ⫾ 0.5
5.03 ⫾ 0.01
2.65 ⫾ 0.28
2.34 ⫾ 0.28
14.6 ⫾ 0.5
251.8 ⫾ 0.8
3,737 ⫾ 72
⫺3,599 ⫾ 61
0.455 ⫾ 0.007
0.445 ⫾ 0.010
653 ⫾ 5.2
179.6 ⫾ 8.8
153.8 ⫾ 16.5
250.2 ⫾ 0.8
3,399 ⫾ 56†
⫺3,017 ⫾ 171*
0.493 ⫾ 0.005†
0.504 ⫾ 0.017*
656 ⫾ 3.3
251 ⫾ 35.5
149 ⫾ 19.9
Values are means ⫾ SE. n, number of mice for each group; HCM,
mice with hypertrophic cardiomyopathy; IVP, internal venous pressure; NTG, nontransgenic control mice; TPP, time to peak pressure; ⫹dP/dt and ⫺dP/dt, first derivative of pressure development and relaxation, respectively, over time; RT1⁄2, half-time to relaxation; PaO2 and PvO2, partial arterial and venous, respectively, pressures; MV̇O2, myocardial oxygen consumption. *P ⬍ 0.05; †P ⬍ 0.01.
maximal dose of isoproterenol (1 ␮M) at 1-, 3-, and
5-min time points. Systolic responsiveness (⫹dP/dt) of
the HCM hearts to isoproterenol was not impaired
(Fig. 5). However, the lusitropic response, ⫺dP/dt of
the HCM hearts, was significantly diminished (Fig. 5).
The initial relaxation in response to the ␤-agonist was
normal in HCM hearts, but this was followed by an
additional augmentation of relaxation after 1 min in
the NTG but not the HCM hearts.
To further explore ␤-adrenergic function through
biochemical assays, homogenates from HCM and control hearts were assayed for ␤-adrenergic receptor density, adenylyl cyclase activity, and GRK levels and
activity. ␤-Adrenergic receptor density and membrane
adenylyl cyclase activity in HCM hearts, under basal,
sodium fluoride-stimulated, and isoproterenol-stimulated conditions, were not significantly altered compared with NTG hearts (data not shown). Myocardial
␤-ARK1 levels, determined by immunoprecipitation of
cytosolic extracts, were significantly higher in HCM
hearts compared with controls (Fig. 6). Protein levels of
a second GRK found in the heart, GRK5, were not
different in HCM hearts (Fig. 6). ␤-ARK1 exerts its
regulatory activity at the myocardial membrane, so we
also measured total membrane GRK activity in membrane extracts and found a significant increase in HCM
hearts (Fig. 6).
Fig. 4. Response of isolated ejecting hearts to preload altered over a
range of cardiac work from 200 to 350 mmHg 䡠 ml 䡠 min⫺1 (HCM, n ⫽
6, NTG, n ⫽ 4; 1 HCM and 1 NTG heart were excluded because of
irregular heart rates despite pacing). Top: inotropic (pressure contractility over time, ⫹dP/dt) response (P ⬍ 0.05 difference between
groups, ANOVA). Bottom: lusitropic (pressure relaxation over time,
⫺dP/dt) response (P ⬍ 0.05 difference between groups, ANOVA).
Fig. 5. Response of isolated isovolumic hearts to administration of 1
␮M isoproterenol over a 5-min time course (HCM, n ⫽ 5; NTG, n ⫽
5). Top: inotropic (⫹dP/dt) response (P ⫽ no significant difference
between groups, ANOVA). Bottom: lusitropic (⫺dP/dt) response (P ⬍
0.05 difference between groups, ANOVA).
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DEVELOPMENT OF DILATED CARDIOMYOPATHY IN HCM MICE
DISCUSSION
Fig. 6. G protein-coupled receptor kinase (GRK) assessment (HCM,
n ⫽ 8; NTG, n ⫽ 7). Top: protein levels of ␤-adrenergic receptor
kinase 1 (␤-ARK1) and GRK5 in myocardial extracts (*P ⬍ 0.05,
t-test). Bottom: myocardial membrane GRK activity determined by
incorporation of 32P into rhodopsin-enriched rod outer segment membranes (*P ⬍ 0.05, t-test).
It has been previously shown that younger, hypertrophic HCM mice express two genetic markers of
compensatory hypertrophy, ANF and s-ACT (44, 45).
These genes are normally expressed during embryonic
heart development but not in adult myocardium, and
their induction has been well established in cardiac
hypertrophy (4, 36). ␤-MyHC is the predominant isoform in murine myocardium until birth, when ␣-MyHC
is preferentially expressed; reversion to the embryonic
␤-MyHC isoform is also observed in cardiac hypertrophy (see Ref. 30 for a review). We were interested in
assessing the expression of embryonic genes in the
hearts of older dilated male HCM animals. Total RNA
extracted from LV tissue was used for analysis. Slot
blots were hybridized with ␣-MyHC, ␤-MyHC, ANF,
s-ACT, or GAPDH probes, and the signals were normalized to GAPDH levels. Expression of ␤-MyHC,
ANF, and s-ACT was significantly increased in the
HCM ventricles; ␣-MyHC expression was significantly
decreased (Fig. 7). RNAse protection of LV RNA with a
probe that protects fragments of different length from
␣-MyHC, ␤-MyHC, and the mutant MyHC transgene
mRNA confirmed an increased ratio of ␤-MyHC to total
endogenous MyHC in the HCM hearts (data not
shown). These RNA results strongly suggest that an
embryonic gene expression program is still active in
the decompensated hearts of the older transgenic male
animals.
HCM and DCM are clinically recognized as distinct
diseases, although a progression from HCM to ventricular dilation with systolic and diastolic dysfunction has
been observed in a subset of the HCM population (18,
37, 38, 42). These clinical findings suggest that the two
cardiomyopathies may actually be part of the same
pathological spectrum. The data presented here demonstrate that phenotypic progression from HCM to
DCM occurs in mice expressing a mutant myosin
transgene.
The hearts of 8-mo-old male HCM animals are visibly dilated (Fig. 1). Myocardial mass is significantly
increased (Table 1), indicating that eccentric hypertrophy of the heart has resulted in increased LV chamber
dimensions without increased wall thickness (Fig. 1).
The increased chamber volume may provide some benefit in terms of a larger stroke volume, but the elevated
systolic and diastolic stress would be detrimental to
cardiac function over the long term.
One of the primary symptoms of DCM is exercise
intolerance. By 8 mo, the HCM mice show a significantly impaired ability to keep pace with a moving
treadmill, and exercise endurance was also significantly decreased (Fig. 2). These results suggest that
cardiac function in the HCM mice is impaired enough
to cause significant physiological consequences. DCM
is also characterized by systolic dysfunction, which
may be accompanied by diastolic abnormalities. In
contrast, HCM patients generally display diastolic dysfunction, with normal or even elevated systolic function (39). Both systolic and diastolic dysfunction were
observed in HCM hearts in vivo by echocardiography.
Isolated ejecting heart studies confirmed that both
systolic and diastolic anomalies are intrinsic to the
HCM myocardium, independent of neurohumoral or
peripheral effects. Although ⫹dP/dt and ⫺dP/dt were
uniformly depressed in HCM hearts over a wide range
of cardiac work, both indexes increased to the same
extent as controls in response to increased load (Fig. 4).
Fig. 7. Gene expression in HCM hearts. ␣-MyHC, ␣-myosin heavy
chain; ␤-MyHC, ␤-myosin heavy chain; s-ACT, ␣-skeletal actin; ANF,
atrial natriuretic factor; LV RNA expression was measured by RNA
slot blot and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. All results are expressed as %NTG, *P ⬍ 0.10;
**P ⬍ 0.05; #P ⬍ 0.01, t-test.
DEVELOPMENT OF DILATED CARDIOMYOPATHY IN HCM MICE
The preserved Starling response in these HCM hearts
is not surprising because even end-stage failing human
hearts have the ability to respond to enhanced preload
with an increase in force development (46). The ability
of the HCM hearts to increase contractility normally,
despite depressed overall contractility, argues against
an intrinsic inability of the HCM hearts to respond to
increased demand. Instead, it is likely that defects in
the regulation of excitation-contraction coupling contribute to the in vivo functional impairment observed
in the HCM mice.
The cardiac ␤-adrenergic signaling system is important for the regulation of excitation-contraction coupling in the heart, and abnormalities in this signaling
system commonly occur with ventricular remodeling
and cardiac decompensation (8). Like hypertrophy, adrenergic activation is part of the compensatory response to cardiac damage, and persistent ␤-adrenergic
stimulation can lead to desensitization of this G protein-coupled receptor system (3, 16). In the isolated
isovolumic heart experiments, the HCM mice exhibited
significantly diminished relaxation in response to the
infusion of the ␤-adrenergic agonist isoproterenol (Fig.
5). This moderate desensitization in lusitropic function
might be expected to have a relatively larger impact on
overall cardiac function because small deficits in myocardial relaxation would in turn affect ventricular filling and result in diminished cardiac output. Desensitization of adrenergic responsiveness is consistent with
the finding of significantly increased levels and activity
of ␤-ARK1 in HCM hearts. ␤-ARK1 acts to uncouple
␤-adrenergic receptors from downstream effectors, including adenylyl cyclase and cardiac contractility (23,
25). In the HCM mice, myocardial protein levels of
␤-ARK1 but not GRK5 were increased. In failing human hearts, ␤-ARK1 mRNA and GRK activity are
elevated approximately two- to threefold (43), but myocardial levels of GRK5 are unaltered (Iaccarino and
Koch, unpublished observations). Similar observations
have been made in other animal models of heart failure, such as cardiomyopathic hamsters (see Ref. 19 for
a review). The increased ␤-ARK1 in the HCM hearts
could lead to physiological adrenergic uncoupling, consistent with the isolated heart results.
Although we observed a diminished lusitropic response to isoproterenol in the HCM animals, no difference in myocardial membrane adenylyl cyclase responsiveness was observed. It is possible that the
sensitivity of the adenylyl cyclase assay was not sufficient to detect the modest change in adrenergic responsiveness observed at the whole organ level. Alternatively, coupling between the ␤-adrenergic receptors
and adenylyl cyclase may be intact in the HCM hearts
despite elevated ␤-ARK1 levels. Considering our observation that the relaxation deficit observed in HCM
hearts after isoproterenol exposure was time dependent (Fig. 5), it is interesting to speculate that the
primary adrenergic abnormality in the HCM hearts
may be a defect in one of the distal components of
relaxation, such as phospholamban or troponin phosphorylation. In a study (15) that used myocardial tis-
H157
sue from human HCM patients, isometric contraction
and relaxation were markedly prolonged, and the calcium transients of the HCM myocardium exhibited two
distinct components, in contrast with controls. Studies
of Ca2⫹ transients in isolated cardiac myocytes from
HCM hearts are in progress.
Analysis of total RNA extracted from LV tissue of
HCM animals revealed a pattern of embryonic gene
expression consistent with cardiac hypertrophy (Fig.
7). ␤-MyHC, ANF, and s-ACT were significantly induced, and ␣-MyHC expression was significantly decreased. These findings suggest that the fetal gene
expression program that initially supports compensatory hypertrophy is maintained as the hearts progress
to decompensated, eccentric hypertrophy.
One of the major problems in understanding HCM is
the difficulty in acquiring myocardial specimens from
human patients, and this problem has been an impetus
for the generation of several different mouse models.
Despite different genetic backgrounds, the HCM mice
described here share several phenotypic traits with the
␣-MyHC403/⫹ model of HCM (10). The ␣MyHC403/⫹
mice carry an Arg403-Gln (R403Q) missense mutation
on one allele. In contrast, the HCM mice studied here
have two normal ␣-MyHC alleles but express an additional mutant ␣-MyHC transgene that includes the
same R403Q missense mutation along with a deletion of
amino acids 468–527 bridged by the addition of nine
nonmyosin amino acids (45). Myocardial sections from
both models show the classic histopathology of HCM.
The nature of cardiac hypertrophy is somewhat different between the two models in that the HCM mice
display both LV and right ventricular hypertrophy
(45), whereas in the ␣-MyHC403/⫹ mice only left atrial
weights are increased. Both the ␣-MyHC403/⫹ mice
(17) and the HCM mice are exercise intolerant. Cardiac dysfunction was evident in both HCM and
␣-MyHC403/⫹ mice but with different phenotypic presentations. The ␣-MyHC403/⫹ mice display predominantly diastolic abnormalities, with accelerated systolic kinetics (11). The kinetics of both cardiac
contraction and relaxation are impaired in older HCM
mice. Interestingly, a gender difference is apparent in
both models. Male ␣-MyHC403/⫹ mice more consistently display left atrial enlargement and histopathology than female mice (10). Female HCM mice typically
show a progressive ventricular hypertrophy at 8 mo of
age without the chamber dilation seen in male mice
(45). Gender differences have also been described in
the prevalence and presentation of human cardiac disease, including aortic stenosis (5), idiopathic DCM (7),
and HCM (12). The advent of mouse models that also
display gender differences may help to elucidate the
gender-specific factors that impact the differential response of male and female hearts to similar stresses.
The HCM mice described here are the first mouse
models of a sarcomeric protein mutation to show a
progression from HCM to DCM. The data presented
above demonstrate functional and biochemical defects
in 8- to 11-mo-old male HCM mice consistent with
DCM, with ventricular dilation, systolic and diastolic
H158
DEVELOPMENT OF DILATED CARDIOMYOPATHY IN HCM MICE
dysfunction, exercise intolerance, and adrenergic desensitization. These animals have undergone cardiac
decompensation similar to that of the subgroup of
human HCM patients who progress from primary hypertrophy to symptomatic DCM and systolic dysfunction. The HCM mice do not appear to develop overt
heart failure at this time point, and the extent of the
cardiac dysfunction seen in the HCM mice might be
described as mild or moderate compared with the severe acute abnormalities in other murine models of
heart failure, such as muscle Lin-II, Isl-1, and Mec-3
protein null or calcineurin overexpression (1, 29). However, the progression from HCM to DCM in the HCM
mice occurs over a lifespan period of ⬃30–50%, which
compares favorably with the kinetics of cardiac decompensation in humans. These data support the idea that
HCM and DCM may be part of the same pathological
spectrum. It is likely some cases of human idiopathic
DCM are the result of mutations in sarcomeric proteins
that caused an undiagnosed primary HCM. Identification of a mouse model that recapitulates the transition
from HCM to DCM may ultimately provide valuable
insight not only into HCM but also the more general
phenomenon of cardiac decompensation.
We thank Teresa Bohlmeyer for performing histology, Ole Knudson for performing echocardiograms, Kyle Shotwell for help with
cyclase and binding, and Traci Jackson for help with isolated heart
studies.
This research was supported by National Heart, Lung, and Blood
Institute Grants HL-50560 (to L. A. Leinwand), HL-40306 (to R. L.
Moore), HL-61690 (to W. J. Koch), and HL-22610 (to I. L. Grupp).
9.
10.
11.
12.
13.
14.
15.
16.
17.
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