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1 Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710; 2 Medical Biotechnology Center, University of Maryland Biotechnology Institute, and 3 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201; 4 Institute of Neurobiology, University of Puerto Rico, San Juan, Puerto Rico 00901; and 5 Departamento de Bioquimica e Imunologia, Laboratorio de Membranas Excitaveis, Universidade Federal de Minas Gerais, Minas Gerais, Brazil
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Heart failure and dilated cardiomyopathy develop in mice that
lack the muscle LIM protein (MLP) gene (MLP
/). The
character and extent of the heart failure that occurs in MLP/ mice were investigated using echocardiography and
in vivo pressure-volume (P-V) loop measurements. P-V loop data were
obtained with a new method for mice (sonomicrometry) using two pairs of
orthogonal piezoelectric crystals implanted in the endocardial wall.
Sonomicrometry revealed right-shifted P-V loops in MLP/
mice, depressed systolic contractility, and additional evidence of
heart failure. Cellular changes in MLP/ mice were
examined in isolated single cells using patch-clamp and confocal
Ca2+ concentration ([Ca2+]) imaging
techniques. This cellular investigation revealed unchanged Ca2+ currents and Ca2+ spark characteristics
but decreased intracellular [Ca2+] transients and
contractile responses and a defect in excitation-contraction coupling.
Normal cellular and whole heart function was restored in
MLP/ mice that express a cardiac-targeted transgene,
which blocks the function of 
-adrenergic receptor (-AR) kinase-1
(ARK1). These data suggest that, despite the persistent stimulus to
develop heart failure in MLP/ mice (i.e., loss of the
structural protein MLP), downregulation and desensitization of the
-ARs may play a pivotal role in the pathogenesis. Furthermore, this
work suggests that the inhibition of ARK1 action may prove an
effective therapy for heart failure.
contractility; -adrenergic receptor; excitation-contraction
coupling; calcium signaling; transgenic; -adrenergic receptor
kinase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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HEART FAILURE IS
a clinical syndrome in which molecular and cellular changes in cardiac
myocytes lead to inadequate myocardial function. Much attention has
been focused on the role played by the -adrenergic receptor (-AR)
signaling pathway in heart failure after the surprising discovery that
-AR blockers, known to have negative inotropic action, provide
benefit to patients in heart failure (4-6). Indeed,
it has been well established that -ARs are downregulated and/or
desensitized during heart failure (3). Characteristic changes in
-ARs include uncoupling of both 1- and
2-AR subtypes, threefold increase in activity of the
-adrenergic receptor kinase (ARK1), and increased levels of
Gi (13, 40). Exploration of the mechanism of
action of -AR modulation in the context of heart failure requires
cellular and molecular examination of cardiac myocytes from animals
whose myocardial function is well defined.
A useful genetically defined murine model of heart failure was recently
described (1). In this model, the muscle LIM protein (MLP)
is knocked out to produce the MLP/ animal. Two genetic
interventions have been described that suggest that alterations in
either Ca2+ transport (29, 38) or -AR
function (31) play a central role in MLP/
heart failure. We investigated the nature and extent of heart failure
associated with the MLP/ mouse, and that rescued by the
ARK1 inhibitor, by examining the intrinsic myocardial contractile
state in vivo using a pressure-volume (P-V) analysis and intracellular
Ca2+ concentration ([Ca2+]i)
signaling using patch-clamp methods and confocal Ca2+
imaging. We present here data showing that MLP/ animals
have a defect in excitation-contraction (EC) coupling that contributes
to their contractile dysfunction and that this pathology is
significantly more severe than the loss of the MLP per se.
Moreover, -AR dysfunction appears to be a necessary component because restoration of -AR signaling in these animals is sufficient to prevent the abnormalities of in vivo contractile dysfunction, of
poor cellular contraction, of weak Ca2+ signaling, and of
defective EC coupling.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental Animals
Adult MLP/ and MLP//ARK1
inhibitor (ARKct) littermate mice of either sex, age 5 to 7 mo, were
used in this study. The mice were generated by mating transgenic mice
with cardiac-targeted overexpression of the ARKct (24)
with mice homozygous for the deletion of the MLP gene
(MLP/) (1) to create
MLP//ARKct mice (31). Nonlittermate
wild-type mice of either 129Sv/B6 or CD-1 strain were used as controls.
The animals in this study were handled according to approved protocols
and animal welfare regulations of the authors' Institutional Review Boards.
Transthoracic Echocardiography
Two-dimensional (2D) guided M-mode echocardiography was performed using an HDI 5000 echocardiograph (ATL, Bothell, WA) as previously described (11, 31). Mice were first studied in the conscious state using gentle manual restraint after a period of acclimation. A soft plastic collar was fashioned to prevent the animals from biting the probe. After acquisition of satisfactory echocardiograms, the same mice were restudied under anesthesia (Avertin 2.5%, 14 µl/g ip).P-V Loops
Several days after echocardiography, mice were reanesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg) and were connected to a rodent ventilator after endotracheal intubation. After bilateral vagotomy, the chest was opened and the pericardium was dissected to expose the heart. A 7-0 suture ligature was placed around the transverse aorta to manipulate loading conditions (see Fig. 2A). A 1.4-Fr (0.46 mm) high-fidelity micromanometer catheter (Millar Instruments, Houston, TX) was inserted into the right carotid and advanced retrograde into the left ventricle (LV). A polyethylene (PE)-50 catheter was inserted into the external jugular vein for drug infusion. Two pairs of miniature omnidirectional piezoelectric crystals (0.7 mm; Sonometrics, London, ON, Canada) were implanted in the endocardium of the LV by inserting the crystals through the epicardial layer into the chamber and then withdrawing until there was resistance. Short-axis dimension was measured with a pair of crystals implanted in the anterior-posterior orientation. Long-axis dimension was measured with a crystal implanted in the apex and one crystal attached to the base of the heart at the level of the aortic valve using cyanoacrylate adhesive (Vetbond, 3M Animal Care Products, St. Paul, MN). The space and time resolution of the sonomicrometry system are 0.015 mm and 0.001 s, respectively.After a brief period of stabilization, simultaneous LV pressure
and LV dimensions were recorded at baseline and during increases in
afterload generated by gently pulling on the suture to transiently constrict the transverse aorta. The ventilator was stopped during data
acquisition to eliminate effects of positive ventilation. After return
of LV pressure to baseline values, the contractile state was increased
with a dobutamine infusion (2 µg · kg1 · min1). After
steady state was reached (~5 min), recordings of variably loaded
pressure-dimension measurements were made as above. All data was
recorded digitally at 2,000 Hz and stored on a computer for off-line
analysis. At the end of the experiment, animals were killed and proper
positioning of the crystals was documented by direct inspection.
Data Analysis
The digitized data were analyzed using a computer algorithm. Baseline and dobutamine hemodynamic values were obtained by averaging 10 beats recorded during the steady-state periods. Parameters measured were LV systolic and end-diastolic pressure, maximal (LV dP/dtmax) and minimal first derivative of LV pressure, and heart rate. The end-systolic point of each P-V loop was determined by an automated iterative linear regression technique as previously described (21, 30). LV volume was calculated as a modified general ellipsoid using the equation V = (
/6) · Dla · (Dap)2
where Dla is the apex to base long-axis LV
dimension and Dap is the anterior to posterior
short-axis LV dimension. The end-systolic points were then fitted to a
parabolic curvilinear model, Pes = a(Ves 
V0)2 + b(Ves V0), where
Pes is end-systolic pressure, Ves is
end-systolic volume, V0 is the volume axis intercept,
b is the local slope at V0, and a is
the curvilinearity coefficient (21). If the 95%
confidence interval of a did not include zero, the
end-systolic P-V relation (ESPVR) was considered curvilinear (see Table
3).
Cell Isolation and Electrophysiology
Adult animals (wild type, n = 5; MLP/, n = 6; and
MLP//ARKct, n = 7) were killed by
intraperitoneal injection of pentobarbital sodium (100 mg/kg). Single
mouse ventricular myocytes were isolated (37) and stored
at room temperature (22-25°C) in Dulbecco's modified Eagle's
medium (JRH Biosciences, Lanexa, KS) until being used. An Axopatch-200A
amplifier (Axon Instruments) was used to patch clamp the cells (whole
cell configuration) and measure membrane currents. Patch pipettes of
nominal resistance of 0.5-3 M
were used and were filled with an
internal solution of (in mM) 130 CsCl, 20 tetraethylammonium chloride,
5 Mg-ATP, 10 HEPES, and 0.05 fluo 3-K5; pH 7.2 (with NaOH).
Some cells were preloaded with fluo 3 by 30-min exposure to 1.5 µM
fluo 3-AM at room temperature.
Cells were superfused with one of three solutions. Solution 1 contained (in mM) 140 NaCl, 5 KCl, 0.5 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 5.5 glucose, and 5 HEPES. Solution 2 contained (in mM) 140 NaCl, 5 CsCl, 0.5 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 5.5 glucose, and 5 HEPES. Solution 2 was used to measure Ca2+ current (ICa). Solution 3 contained (in mM) 145 CsCl, 0.5 MgCl2, 5.5 glucose, and 5 HEPES. This solution was used to block the Na+/Ca2+ exchanger when the sarcoplasmic reticulum (SR) Ca2+ content was tested by applying 20 mM caffeine. The pH of all extracellular solutions was kept at 7.4 with the temperature at 34-37°C.
Confocal Microscopy and [Ca2+]i Measurements
Confocal microscopy was used to measure [Ca2+]i (7-9) and to investigate cellular function as previously described in rat and mouse heart cells (16, 36, 37). A robust preloading protocol was used to attain similar SR Ca2+ loads (35, 37). We were therefore able to compare Ca2+ signaling in cells from wild-type, MLP/, and MLP//ARKct
animals because they had a constant amount of Ca2+ in the
SR (15). This procedure permits meaningful comparison of
EC coupling gain (35, 37).
Statistical Analysis
Data are expressed as means ± SE. Two-way repeated ANOVA was used to evaluate the hemodynamic measurements, the ESPVR variables under basal conditions and with dobutamine stimulation, and the echocardiographic parameters between anesthetized and conscious states. When appropriate, post hoc analysis was performed with a Newman-Keuls test. Bland-Altman (2) analysis (coefficient of variability) was used to show agreement between the crystal and echocardiographic measurements of LV diastolic dimension. For all analyses, P < 0.05 was considered significant. For the data from single-cell experiments, two-sample comparisons were performed using Student's t-test, and multigroup comparisons were made using a one-way ANOVA and Tukey's test.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Heart Failure in MLP/ Mice
Evidence from echocardiographic studies.
Investigations into the nature and extent of heart failure associated
with the MLP/ mouse are shown in Fig.
1. Echocardiograms performed in mice under both conscious and anesthetized conditions for wild-type (Fig. 1,
left), MLP/ (Fig. 1, middle), and
a genetically crossed hybrid mouse (Fig. 1, right) are
shown. From Fig. 1, it is clear that the MLP/ mice have
hearts with significantly enlarged internal chamber dimensions
[end-diastolic dimension (EDD) and end-systolic dimension (ESD)] and
reduced fractional shortening compared with wild-type mice (see Fig. 1
and Table 1). Such LV dilation and
reduced cardiac function are apparent in both anesthetized and
conscious animals. Although echocardiography is valuable to measure
chamber size and systolic performance in heart failure in vivo, it is
limited in the assessment of the intrinsic contractile state of the
ventricle because of the dependence on afterload (26). To
examine the murine myocardial contractile state in vivo, a new method
was developed for use in mice: sonomicrometry pressure-volume analysis (See Fig. 2A).
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Cardiac function assessed using P-V loop measurements.
With two pairs of endocardial implanted piezoelectric crystals (see
METHODS and Fig. 2A) and a high-fidelity
micromanometer in the LV, in vivo P-V relationships were obtained.
These P-V relationships were obtained at different "loading"
conditions and in the presence and absence of -AR stimulation. P-V
relationships provide a powerful approach to examine contractile
function in vivo (20, 33), and these measurements were
used to investigate in vivo contractile dysfunction in the
MLP/ mice.
-AR agonist dobutamine (2 µg · kg1 · min1, red P-V
loops) slightly increases the LV end-systolic pressure (LVESP) with low
afterload but has an even more dramatic action as afterload is
increased. More importantly, however, there is an increase in the slope
of the relation describing end-systolic pressure and volume as
afterload increases (Figs. 2B and
3A). This pattern of changes
in cardiac function is typical for a normal heart whether it be from a
mouse, dog, pig, or human (19, 20, 22, 33) and is a
fingerprint for the normal myocardial response to increased afterload
in the presence and absence of -AR stimulation.
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/) are dramatically different from those
observed in wild-type mice. The Ves and Ved of
the hearts are dramatically increased (7-fold and 4-fold,
respectively), and fractional shortening is significantly reduced from
47-55% in control animals to 26-29% in MLP/
animals (P < 0.001). The responses to increases in
afterload are less steep in MLP/ animals (Fig.
3A, Table 3) compared with
wild-type hearts, and there is little change in the slope of the ESPVR
in response to the -AR agonist dobutamine (Figs. 2C and
3A). Together, these data indicate that MLP/
mice are in functional heart failure, a finding consistent with clinical observation of these animals. These data also support the
large body of published work indicating that the -AR signaling system is poorly responsive or unresponsive in many models of heart
failure (3). It is also worth noting that although LV dP/dtmax is mildly reduced in the
MLP/ mice compared with wild-type mice, it does not
have the sensitivity to detect the marked abnormalities in intrinsic
contractility as shown by the P-V loop analysis (12, 27).
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EC Coupling Defect in MLP/ Mice
Cellular function.
The cellular cause of the contractile dysfunction in
MLP/ mice is unknown. However, Arber et al.
(1) suggested that the absence of the MLP, a structural
protein in striated muscle, might be responsible. Although this
hypothesis seems reasonable, Ca2+ signaling defects in
heart failure have been detected in other models of heart failure even
when alternative reasonable causes were found (16, 41).
Consequently, we decided to test the possibility that Ca2+
signaling may be altered and may contribute to the heart failure phenotype in MLP/ mice. We examined membrane currents,
[Ca2+]i, and cellular shortening and
relaxation in patch-clamped heart cells (whole cell mode). Figure
5A shows the
voltage protocol, the [Ca2+]i measurement as
fractional fluorescence (F/Fo), the shortening record, and
the membrane current density (pA/pF) in a single control heart cell. It
is clear from the data that the cardiomyocytes from
MLP/ mice produce significantly smaller
[Ca2+]i changes and reduced contractions for
similar Ca2+ currents (see Fig. 5B). These
differences are demonstrated more clearly when the voltage dependence
for these data is shown in Fig. 6. There
is also a clear reduction in the rate of cellular shortening shown in
Fig. 5. Figure 6D shows the voltage dependence decrease in
shortening and relaxation in the cardiac myocytes taken from
MLP/ animals.
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20, 0, and +20 mV), as shown in Fig.
7. There is a clear hysteresis in the
relationship, as illustrated in Fig. 7A, middle.
The arrows along the dashed line reveal the trajectory of the plot. The
hysteresis reflects the rapid release of Ca2+ by the EC
coupling mechanism (Ca2+-induced Ca2+
release, CICR) and the relatively slower responsiveness of the contractile machinery. Hysteresis in wild-type mouse heart cells is
expected because of the known kinetics of contraction. In
MLP/ animals, the heart cells show a characteristic
flattened hysteresis loop. This can be seen best if one compares Fig.
7A, left, with Fig. 7B,
middle. These examples have a similar elevation of
[Ca2+]i, but the extent of the
shortening is considerably decreased (~5%) in the
MLP/ animals compared with controls (~10%). The data
in Fig. 7B show the consistent pattern of reduced
contractile responsiveness to [Ca2+]i
changes. The reduced extent of contraction leads to the flattened appearance of each of the trajectories shown in Fig. 7B.
Together, the data suggest that, in addition to the decreased
shortening due to the reduction in the
[Ca2+]i transients (as shown in Fig.
6C), there is a further reduction in contractility due to an
additional factor that has not yet been defined. In
MLP/ heart failure, the additional factor may be
alterations in troponin or other contractile proteins that have been
observed in heart failure (28, 39) or may be directly
related to the chronic loss of MLP.
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/ animals. This reinforces the
data shown in Fig. 7 but does not answer the question of considerable
interest. Why is the [Ca2+]i transient
reduced in the cells from the MLP/ mice?
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/ mice, experiments were done under
conditions that produce similar SR loading (16, 35, 37,
41). This control is important because changes in SR
Ca2+ load alone can account for changes in EC coupling gain
(35, 37). Under such conditions, there are at least two
clear ways by which the [Ca2+]i transient can
be reduced. First, the elementary units of SR Ca2+ release,
the Ca2+ sparks, can be altered. They could be smaller or
shorter in duration, for example. Figure 8B shows typical
Ca2+ sparks from cells taken from control and
MLP/ animals. The Ca2+ sparks are found to
be indistinguishable, as shown in the sample records and the
signal-averaged Ca2+ sparks. Second, EC coupling gain could
be altered (16, 34, 37, 41). The EC coupling gain function
indicates how well ICa activates SR
Ca2+ release. At negative potentials (i.e., negative to
20 mV), the single-channel current is largest, and the opening of a
single L-type Ca2+ channel can activate an elementary SR
Ca2+-release unit (9), a Ca2+
spark (9, 34). Thus it is at these negative potentials
where EC coupling gain reflects the efficacy of the coupling between Ca2+ influx through L-type Ca2+ channels and
the triggered response known as CICR (9, 34). Thus we note
at negative potentials in Fig. 8C that there is a significant reduction in EC coupling gain in MLP/
myocytes compared with control myocytes. At more positive potentials (0 and +20 mV), the single-channel current of L-type Ca2+
channels is reduced, and the opening of a single L-type
Ca2+ channel is not sufficient to activate a
Ca2+ spark. Thus, as test depolarizations reach more
positive potentials, local [Ca2+]i becomes
relatively less important and global (i.e., cell wide) [Ca2+]i becomes more important. For that
reason, EC coupling gain declines and differences in EC coupling gain
between wild-type and MLP/ cells vanish (see Ref.
34). It is important to remember that in this analysis, SR
Ca2+ load is being artificially kept constant to permit
comparison of EC coupling gain in cells from the different mouse types.
The reduction of EC coupling gain in MLP/ heart cells
indicates that the efficacy of activation of Ca2+ sparks by
Ca2+ influx through the L-type Ca2+ channel is
reduced in MLP/ heart cells (16). This
important observation provides us with the primary evidence that there
is a defect in EC coupling in MLP/ myocardial cells.
ARKct Transgene Expressed in MLP/ Mice
In vivo function.
We examined the effect of overexpression of the ARKct transgene on
cardiac function. The expression of this transgene largely prevents the
downregulation and desensitization of the -AR by inhibiting the
action of ARK1 to phosphorylate the receptor (31). Figure 1, right, shows that cardiac function measured by
echocardiography has largely returned to normal, a finding similar in
principle to our earlier report (31) but now refined by
improved anesthesia and echocardiography techniques. Most importantly,
however, the P-V analysis shown in Fig. 2D is remarkable. It
shows that the most dramatic change in the
MLP//ARKct animals is the reduction in end-diastolic
and end-systolic cardiac volumes, which have become near normal. Just
as important is the increased steepness of the ESPVR with increasing
afterload, which approaches that observed in wild-type animals.
Furthermore, the increased slope in the ESPVR after the application of
the -AR agonist dobutamine seen in wild-type animals is restored in
the MLP//ARKct animals. This functional restoration
of heart function in the MLP//ARKct animals is also
clearly shown in Fig. 3. These data are also supported by the LV/body
weight data showing normalization of cardiac mass in the
MLP//ARKct mice (wild type 3.28 ± 0.03 mg/g,
MLP/ 5.13 ± 0.21 mg/g,
MLP//ARKct 3.55 ± 0.33 mg/g;
P < 0.005 MLP/ vs. either wild type or
MLP//ARKct).
Cellular function in cells from MLP//ARKct
animals.
The decrease in [Ca2+]i transient observed in
cells from the hearts of the MLP/ mice is profound.
There is, however, a remarkable change in the [Ca2+]i transients of the heart cells in the
MLP//ARKct animals, as shown in Fig. 5C.
Similarly, the cellular contractions are restored to the control levels
as are rates of shortening and relaxation. These alterations in
[Ca2+]i and cell shortening are observed
widely over the voltage range 40 to +50 mV, as shown in Fig. 6, B and
C. Importantly, the Ca2+ current density (pA/pF)
is identical for cells from control, MLP/, and
MLP//ARKct animals. The dependence of cell
shortening as a function of [Ca2+]i for the
cells from MLP//ARKct animals, as shown in Fig.
7C, is very close to that of the wild-type animals. Compare,
for example, the similarity of the shape in the sample records from
wild-type and MLP//ARKct cells at 20 mV [Fig. 7,
A and C, left]. The plot of peak cell
shortening vs. [Ca2+]i shown in Fig.
8A for cells from the MLP//ARKct animals
has returned toward control (see DISCUSSION). In our
investigation of why the [Ca2+]i transients
returned to normal, we examined Ca2+ sparks and the EC
coupling gain function in the cells from the MLP//ARKct animals. We found that the
Ca2+ sparks in MLP//ARKct myocytes were
similar to those in control cells. Because Ca2+ sparks in
the MLP/ myocytes were also identical to those in
control cells, we learn about the functional similarity of the
SR/T-tubular junction in the myocytes of the three types of mice we
examined. We would conclude, therefore, that there is no significant
difference in the ryanodine receptor cluster organization at
SR/T-tubular junctions. Furthermore, because Ca2+ sparks in
the MLP/ myocytes were identical to those in the
control myocytes, it also shows that there is no defect in SR
Ca2+ uptake in the MLP/ cells. However, as
in other models of heart failure (8, 16, 34, 37, 41),
there is a clear difference in the EC coupling gain function. In the
experiments with MLP//ARKct mice, however, we find
that the EC coupling gain function in cells from the
MLP//ARKct animals has returned to normal.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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To better understand the pathogenesis of heart failure and to
explore novel treatments, we have examined the cellular and molecular
defects that develop with well-defined models of heart failure
(16, 41). Here we study a distinct model of heart failure
that occurs when MLP is "knocked out" (i.e., the
MLP/ mouse) (1, 31). Echocardiographic
abnormalities consistent with dilated cardiomyopathy and heart failure
(1, 29, 31) are known, but intrinsic contractile and
cellular defects have not been investigated until now (see Figs.
2-8). We have identified functional changes during in vivo
measurements of cardiac pressure and volume consistent with an
abnormality in intrinsic contractility. We have also observed an
impaired -AR response and a major defect in EC coupling that we
identified as a decreased sensitivity of the SR Ca2+
release mechanism to triggering Ca2+. There is an
additional defect in Ca2+ signaling consistent with reduced
sensitivity of the contractile proteins to Ca2+. These
findings in the MLP/ animals are interesting because
similar changes have been identified in other animal models of heart
failure that are attributed to unrelated instigating insults [i.e.,
hypertension (16) or viral myocardiopathy
(41)]. The most interesting and provocative finding, however, is that the heart failure pathology in MLP/
animals can be almost completely recovered by the expression of a
single transgene that functionally inhibits the action of ARK1. The
most critical questions raised by these findings are discussed below.
Cellular and Molecular Mechanisms of Heart Failure
MLP/ dilated cardiomyopathy.
Because the MLPs are reported to link actin filaments together at the Z
line, it has been argued that the absence of MLP may contribute to the
development of dilated cardiomyopathy by the removal of its structural
function (10) . The data presented here appear to argue
against that simple conclusion. The reasoning is that there is
virtually full recovery of cardiac contractile function in the
MLP//ARKct animals and yet the MLP remains absent.
Our conclusion is supported by Minamisawa et al. (29), who
found that MLP/ cardiomyopathy appears to be rescued by
knocking out a different protein, phospholamban (PLB).
Initiating insult in MLP/ animals.
The data to date do not permit us to identify which feature of the
MLP/ pathology leads to the dilated cardiomyopathy, and
thus allows the conclusion that some of the structural role of MLP is
important. MLP, however, subserves many functions in addition to its
putative structural role. MLP contributes to muscle development and
serves as a nuclear transcription regulator (10). There
are no compelling data to date to indicate which of these many
functions of MLP is critical to the development of dilated cardiomyopathy.
Why is the MLP/ mouse rescued by cardiac-targeted
expression of ARKct?
We postulate that the MLP/ dilated cardiomyopathy
arises as the result of a pathological spiral that begins with the
yet-unidentified initiating insult. The insult is inadequate to produce
enormous contractile dysfunction on its own but can lead to sufficient dysfunction so that the -AR system is activated in compensatory response. We reason that activation of the -AR system is the primary
means to increase cardiac output when it must be increased transiently. However, tonic activation of the -ARs is not
a normal response and becomes maladaptive because tonic -AR
occupancy by agonist increases -AR desensitization and
downregulation mediated in part by phosphorylation of receptors by
ARK1. This hypothesis, first suggested by us (32), is
supported by our findings presented here, where we show that there is
virtually normal in vivo cardiac function with normalization of -AR
responsiveness in the MLP//ARKct mouse. This can be
better understood in light of the restoration of cellular function in
myocytes from these animals. Presumably, the main benefit of expressing
the ARKct transgene in the hearts of the MLP/ mice
is the protection of -ARs from chronic desensitization and downregulation.
-AR function is important for the normal
function of the heart. The -AR, like other heptahelical receptors,
not only serves to signal specific G protein-coupled effectors such as
adenylyl cyclase but also signals a host of additional effectors
through diverse intracellular proteins (17). Chronic
downregulation and desensitization of -ARs will blunt all
cAMP-dependent protein kinase-mediated dependent signaling but will
activate other intracellular pathways, such as those linked to cell
growth (e.g., the mitogen-activated protein kinases) (25). We suggest that overexpression of the
ARKct transgene reverses these maladaptations.
MLP//PLB/ rescue of
MLP/.
How effective is the MLP//PLB/ rescue
(see Ref. 29) compared with the
MLP//ARKct rescue shown here? How does this
rescue come about? Although we would like to know the answer to these
questions, there is no way to address them properly at this time
because neither P-V relations nor patch-clamp and confocal cellular
[Ca2+]i signals were studied in the
MLP//PLB/ mouse (29).
/ mice rescued by the PLB knockout did not regain
-AR responsiveness but remained in a highly stimulated state. This
is similar to the result we found with cardiac-targeted expression of
the 2-AR, which we showed to be deleterious in the
MLP/ mouse (31).
It is widely accepted that LV dP/dtmax provides
useful information on relative changes in contractile behavior when all
conditions are controlled in the same animal, (33);
however, it has limited sensitivity when groups of different animals
are compared (12, 27). In this regard, one of the best
measures of cardiac contractile function in vivo is the P-V loop
(23, 33), as we have used here.
P-V loops in the mouse. The analysis of P-V relations with the ESPVR has long been established as a standard method to evaluate cardiac function in larger species. This study in the mouse shows the nonlinear nature of the LV ESPVR (21, 30) and demonstrates how the shift of this relation can detect changes in contractility in the same heart but, most importantly, can identify differences in inotropic state between groups of genetically altered hearts. Recently, it was reported that ESPVRs were obtained by miniaturized conductance micromanometry (14). This study nicely demonstrates the utility of obtaining load-independent measures of ventricular performance to better understand the biology of disease using gene-targeted animals. Conversion to absolute volumes can be achieved using the hypertonic saline dilution method, but this can result in marked hemodynamic changes (42). In our study, we placed two orthogonal pairs of miniature piezoelectric crystals in the endocardial wall of the mouse heart. Because the output of the dimension crystal is a calibrated signal, we were able to obtain internal dimensions in two planes with high accuracy and therefore calculated absolute LV volume throughout the cardiac cycle. An important advantage of this technique is the ability to measure instantaneous wall thickness by placing an additional crystal on the epicardial surface juxtaposed to the endocardial crystal. This will allow for the calculation of wall stress throughout the cardiac cycle and will provide a method for the in-depth analysis of cardiac mechanics in gene-targeted murine hearts. It should be noted that the sonomicrometer technique requires open-chest instrumentation, which is technically demanding and will affect the measurement of basal hemodynamic parameters. For the assessment of murine cardiac function, this technique is complementary to that of closed-chest catheter-based hemodynamic and echocardiography measurements and provides an assessment of intrinsic in vivo contractile function in a load-independent manner.
In summary, a model of dilated cardiomyopathy with heart failure (MLP/) was used to investigate cellular defects in
heart failure and possible new therapies. We have extended our
preliminary result (31) by examining the in vivo and
cellular behavior of the relevant hearts. Importantly, we show that by
expressing the cardiac-targeted ARKct transgene in
MLP/ animals a nearly complete restoration of
contractile function can be achieved. The compelling restoration of
function by transgenic expression of ARKct suggests that modeling
the mechanism of action of the ARKct is an attractive therapeutic approach.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Debbie Colpitts for expert secretarial assistance.
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FOOTNOTES |
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* G. Esposito, L. F. Santana, and K. Dilly contributed equally to this work.
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-61558 (H. A. Rockman) and HL-36974, HL-61602, and HL-25675 (W. J. Lederer). H. A. Rockman is a recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.
Address for reprint requests and other correspondence: H. A. Rockman, Dept. of Medicine, Duke Univ. Medical Center, DUMC 3104, Durham, NC 27710 (E-mail: h.rockman{at}duke.edu).
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.
Received 14 January 2000; accepted in final form 26 June 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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