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-adrenergic
receptor signaling in hypertrophied myocytes overexpressing
G
q
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Transgenic overexpression of
G
q causes cardiac hypertrophy and depressed contractile
responses to 
-adrenergic receptor agonists. The electrophysiological
basis of the altered myocardial function was examined in left
ventricular myocytes isolated from transgenic (Gq) mice.
Action potential duration was significantly prolonged in
Gq compared with nontransgenic (NTG) myocytes. The densities of inward rectifier K+ currents, transient
outward K+ currents (Ito), and
Na+/Ca2+ exchange currents were reduced in
Gq myocytes. Consistent with functional measurements,
Na+/Ca2+ exchanger gene expression was reduced
in Gq hearts. Kinetics or sensitivity of
Ito to 4-aminopyridine was unchanged, but
4-aminopyridine prolonged the action potential more in
Gq myocytes. Isoproterenol increased L-type
Ca2+ currents (ICa) in both groups,
with a similar EC50, but the maximal response in
Gq myocytes was ~24% of that in NTG myocytes. In NTG
myocytes, the maximal increase of ICa with
isoproterenol or forskolin was similar. In Gq myocytes,
forskolin was more effective and enhanced ICa up
to ~55% of that in NTG myocytes. These results indicate that the
changes in ionic currents and multiple defects in the -adrenergic
receptor/Ca2+ channel signaling pathway contribute to
altered ventricular function in this model of cardiac hypertrophy.
action potential; potassium currents; sodium-calcium exchanger; calcium currents; heart failure; transgenic model
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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MYOCARDIAL HYPERTROPHY is an initial adaptive process to a variety of physiological and pathological conditions associated with increased cardiac work. The hypertrophic response initially normalizes wall stress and maintains ventricular function. However, decompensated congestive heart failure occurs when the adaptive process fails. The molecular mechanisms that trigger cardiac hypertrophy and regulate the progression to heart failure have not been well elucidated (7). Results from recent studies in transgenic mice indicate that stimulation of the signal transduction pathways mediated by heterotrimeric Gq protein is closely related to the induction of cardiac hypertrophy and its progression to heart failure (1, 2, 8, 20, 23).
In ventricular myocytes isolated from failing human hearts, the magnitude of contraction is diminished and relaxation is prolonged. These changes are associated with changes in electrophysiological properties and abnormal intracellular Ca2+ handling (3, 4, 11). Prolongation of the action potential is the most consistently observed electrophysiological abnormality in failing human hearts. Downregulation of the repolarizing K+ currents, the inward rectifier K+ currents (IK1) and the transient outward K+ currents (Ito), has been postulated to underlie the observed action potential prolongation (5). Molecular and biochemical studies in failing human hearts indicate that altered expression of the sarcoplasmic reticulum (SR) Ca2+ regulatory proteins, such as SR Ca2+-ATPase and phospholamban, may contribute to altered intracellular Ca2+ transients (10, 12, 16, 21). Upregulation of the sarcolemmal Na+/Ca2+ exchanger has also been suggested as a compensatory mechanism for decreased SR Ca2+ uptake (9, 22).
Another hallmark of human heart failure is depressed contractile
responsiveness to catecholamines. Multiple changes in the -adrenergic receptor (-AR) signaling pathway, including changes in -AR expression (a selective loss of 1-ARs) and G
protein activity levels [inhibitory G protein (Gi) and/or
stimulatory G protein (Gs)], increased -AR kinase
levels, impaired -AR/adenylyl cyclase coupling, and decreased
adenylyl cyclase activity, occur in the failing human heart
(13). However, most studies in human hearts are limited to
a single time point, usually in hearts with severe hypertrophy or
terminal heart failure, and the relationships between the degree of
ventricular dysfunction and cellular abnormalities are not well characterized.
In this regard, a transgenic mouse model, overexpressing the
-subunit of Gq in the heart, which reproduces many
biochemical and hemodynamic changes seen in human heart disease, may
provide an informative model system to investigate the cellular
mechanisms of cardiac hypertrophy and failure (8,
23). For example, at higher levels of Gq
overexpression (5-fold over endogenous levels), frank cardiac
decompensation occurs, with development of biventricular failure,
pulmonary congestion, and death between 11 and 14 wk of age
(8). Twofold overexpression of Gq shows no
detectable effects on cardiac gene expression, function, or hypertrophy, suggesting that a threshold level of Gq
expression is necessary to transduce these effects. By contrast,
relatively modest (~4-fold) overexpression of Gq
(Gq-25) results in moderate cardiac hypertrophy and
increased hypertrophy-associated gene expression. Echocardiography and
in vivo cardiac hemodynamic studies revealed significantly impaired
intrinsic contractility and responses to -AR agonists. Nevertheless,
Gq-25 mice exhibit relatively compensated left
ventricular function. These mice have a normal life span and do not
develop overt heart failure. Thus Gq-25 mice provide an
intermediate-phase cardiac hypertrophy model, neither fully compensated
nor decompensated, that has been designated previously as
"compromised" heart (23).
Recently, we reported that left ventricular myocytes isolated from
transgenic Gq-25 (Gq) mice exhibit
prolonged contractions and Ca2+ transients
associated with reductions in Ca2+ uptake rates and the
apparent affinity of SR Ca2+-ATPase for Ca2+
(30). There was no change in L-type Ca2+
current (ICa) density. If impaired
Ca2+ uptake by the SR were the only cellular abnormality,
then significantly decreased amplitude of contractions and
Ca2+ transients should be observed in Gq
myocytes. However, we found no such differences, suggesting that other
Ca2+ regulatory mechanisms may be involved in the abnormal
Ca2+ signaling observed in Gq myocytes.
The most consistent electrophysiological change in a variety of
experimental models as well as in human heart failure is action potential prolongation. Prolongation of the action potential may have a
profound effect on cellular excitation-contraction coupling. Because a
normal action potential is the result of an orderly sequence of changes
in the permeability of the membrane inward and outward ionic currents,
the delayed repolarization can occur secondary to an increase in the
inward currents, a decrease in the outward currents, and/or more
complex changes generated by the sarcolemmal
Na+/Ca2+ exchanger. Accordingly, we examined
electrophysiological parameters such as action potentials,
K+ channel currents, and Na+/Ca2+
exchange currents in left ventricular myocytes isolated from Gq and nontransgenic (NTG) control mice. In addition,
because the cardiac Ca2+ channel is a well-characterized
physiological effector of -AR signal transduction, we examined the
effects of isoproterenol (Iso) to identify cellular mechanisms related
to -AR dysfunction.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Generation of transgenic mice.
Transgenic (Gq) mice with a nearly fourfold increase of
the murine -subunit of Gq in the heart
(Gq-25) were generated as previously described
(8). The mice overexpressing moderate levels of
Gq do not exhibit cardiomyocyte replacement or apoptotic heart failure at the age (12-15 wk) used in the present studies (1).
Preparation of myocytes.
Cardiac myocytes from 14 Gq mice and 14 control NTG
littermates were used for electrophysiolgical measurements. Single left ventricular myocytes were isolated from the hearts of NTG and Gq mice by a method described previously
(19). Briefly, the heart was perfused with
Ca2+-free Tyrode solution containing collagenase (type II,
0.5 mg/ml; Worthington) and BSA (1 mg/ml) for 10-20 min by the
Langendorff method at 37°C. At the end of the perfusion period, the
heart was removed, myocytes were prepared from the apical two-thirds of
the left ventricle, and these myocytes were passed through 200-µm
nylon mesh. In some experiments, atrial myocytes were prepared (18) and used to test the ability of pertussis toxin (PTX)
to block muscarinic receptor-mediated activation of K+
channel currents. The isolated cells were stored in
low-Cl
, high-K+ medium, and all experiments
were performed at room temperature (20-22°C).
q myocytes (30).
Cell capacitance was 129.0 ± 2.7 pF (n = 120) in
NTG and 146.1 ± 3.9 pF (n = 131) in
Gq myocytes (P < 0.01).
Electrophysiology.
Whole cell currents were recorded using patch-clamp techniques, as
previously described (19). Patch electrodes had 1.5- to
2.5-M
tip resistance for whole cell current recordings and 10- to
15-M tip resistance for action potential measurements. Membrane
capacitance was measured using voltage ramps of 0.8 V/s from a holding
potential of 
50 mV. The experimental chamber (0.2 ml) was placed on a
microscope stage, and external solution changes were made rapidly using
a modified Y-tube technique (29).
100 and 0 mV) from a holding potential of 80 mV. The
extent of activation was quantified by the peak current measured at +40
mV. The prepulse inactivation curves were normalized to the maximum
current fitted with a Boltzmann equation:
I/Imax = 1/{1 + exp[(Vm V0.5)/k]}, where I is
current, Imax is maximum current,
Vm is membrane potential,
V0.5 is midpotential, and k is the
slope factor.
Ca2+ currents were recorded using an external solution
containing (mM) 2 CaCl2, 1 MgCl2, 135 tetraethylammonium chloride, 5 4-aminopyridine (4-AP), 10 glucose, and
10 HEPES (pH 7.3). To determine responses to -AR stimulation,
myocytes were dialyzed with the pipette solution containing (mM) 100 cesium aspartate, 20 CsCl, 1 MgCl2, 2 MgATP, 0.5 GTP, 10 BAPTA, and 5 HEPES (pH 7.3). These solutions isolated
ICa from other membrane currents such as
Na+ and K+ channel currents and also
Ca2+ flux through the Na+/Ca2+
exchanger. As we previously showed, -AR regulation of the
Ca2+ channel can be reliably measured under these
experimental conditions (25). The voltage dependence of
activation was determined using an interactive nonlinear regression
fitting procedure to the Boltzmann equation: Gmax = 1/{1 + exp[(V0.5 Vm)/k]}, where Gmax is
maximum conductance.
For measurements of the Na+/Ca2+ exchanger
currents, the external solution contained (mM) 150 NaCl, 2 CsCl, 2 MgCl2, 1 CaCl2, 0.001 nifedipine, 0.02 ouabain,
and 5 HEPES (pH 7.3). The pipette solution contained (mM) 20 NaOH, 110 CsOH, 50 aspartic acid, 1 MgCl2, 2 MgATP, 42 EGTA, and 5 HEPES (pH 7.4). The concentration of free internal Ca2+ was
adjusted to 67 nM by addition of CaCl2 (15).
To activate Na+/Ca2+ exchange currents, the
cells were held at 40 mV, and the external solution was rapidly
switched to one in which equimolar LiCl was substituted for NaCl
(15, 17). Exposure of the myocytes to Na+-free solution produced outward Na+
extrusion through the Na+/Ca2+ exchanger.
RNase protection assay.
Total RNA was isolated from the hearts of five Gq mice
and five NTG littermates by use of the ULTRASPEC-II RNA Isolation System (Biotecx Laboratories, Houston, TX). The MAXIscript In Vitro
Transcription Kit (Ambion, Austin, TX) was used to synthesize radiolabeled cRNA probes from linearized DNA riboprobe templates. The
mouse Na+/Ca2+ exchanger (NCX-1) riboprobe
template was prepared by subcloning an RT-PCR-generated cDNA fragment
(spanning nucleotides 31 to +118 bp relative to the AUG translational
start codon) into pBluescript SK(+). Gene expression levels were
determined from total RNA samples by use of the RNase Protection Assay
(RPA) Kit (Ambion). The RPA gel was dried and then exposed to BIOMAX-MR
X-ray film (Kodak, New Haven, CT). Overall NCX-1 levels were determined
by 1) exposing the RPA gel to a Kodak phosphor screen,
2) scanning signal levels with a PhosphorImager
(Molecular Dynamics, Wayzata, MN), and 3) analyzing the data
with NIH Image Analysis software.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Action potential.
Action potentials (Fig. 1) were recorded
in control NTG and Gq left ventricular myocytes in
Tyrode solution with physiological pipette solution (see
MATERIALS AND METHODS). NTG myocytes displayed a brief
action potential with a rapid initial phase of repolarization without a
discernible plateau phase. The shape and duration of the action
potential recorded in NTG myocytes were similar to those observed in
adult mouse ventricular myocytes reported previously (27,
31). In contrast, the action potential recorded in
Gq myocytes showed a relatively small initial notch
followed by a clear plateau phase. Action potential duration quantified
at 50 and 70% repolarization (APD50 and APD70)
was significantly prolonged in Gq myocytes (Table
1). There was no significant difference in the resting membrane potentials between the two groups.
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q myocytes.
K+ channel currents.
Figure 2 illustrates typical outward
currents recorded in NTG (A) and Gq
(B) myocytes. In both groups, depolarization positive to
30 mV activated outward currents, which then decayed slowly to a
sustained outward current at the end of a 300-ms voltage step. Details
of electrophysiological characteristics of the outward currents in
adult mouse ventricular myocytes that exhibit a sum of fast and slow
components have been described elsewhere (31). In the
present study, we refer to the total K+ current components
simply as Ito. Myocytes isolated from
Gq hearts showed a smaller current amplitude than those
from NTG hearts.
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f) and slow
(s) time constants and the relative amplitude
[Af/(Af + As)] in Gq myocytes were
comparable to those in NTG myocytes. The f,
s, and
Af/(Af + As) at +60 mV were 13.8 ± 1.1 ms,
99.1 ± 8.2 ms, and 0.48 ± 0.02 for NTG (n = 10) and 15.3 ± 1.5 ms, 102.5 ± 8.8 ms, and 0.46 ± 0.02 for Gq (n = 21) myocytes,
respectively. The current-voltage (I-V) relationships of
peak and sustained current amplitudes, normalized relative to cell
capacitance (pA/pF), are plotted in Fig. 2C. The average
current densities of peak and sustained components were significantly
decreased in Gq myocytes. At +60 mV, the peak and
sustained currents were 25.4 ± 1.1 and 13.0 ± 0.8 pA/pF
(n = 52) for NTG and 17.8 ± 1.4 and 10.0 ± 0.8 pA/pF (n = 46) for Gq myocytes. The
voltage dependence of Ito inactivation was
similar between the two groups (Fig. 2D). The mean values of
V0.5 and k were 41.9 ± 0.9 mV
and 6.4 ± 1.1 mV for NTG myocytes (n = 10) and
44.9 ± 2.2 mV and 8.0 ± 1.8 mV for Gq
myocytes (n = 7), respectively.
Peak and sustained currents were blocked by 4-AP (Fig.
3, A and B).
Cumulative concentration-response relationships revealed that
IC50 was similar between the two groups (Fig.
3C).
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40 mV to test potentials between
50 and 100 mV. Figure 4 shows
representative IK1 recorded from NTG
(A) and Gq (B) myocytes. The
current was blocked by external application of 0.5 mM Ba2+
(5). Gq myocytes showed a significant
reduction of the current density (Fig. 4C). The mean current
density at 100 mV was 12.1 ± 0.9 and 7.2 ± 0.9 pA/pF
for NTG (n = 22) and Gq
(n = 32) myocytes, respectively.
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Na+/Ca2+ exchange currents.
The Na+/Ca2+ exchanger is the principal
mechanism for Ca2+ extrusion from the cell. However, it has
been proposed that inward Na+ flux in exchange for
Ca2+ efflux via the Na+/Ca2+
exchanger can contribute to depolarizing current during the action potential plateau (6). Therefore,
Na+/Ca2+ exchange currents were compared
between NTG and Gq myocytes. To measure
Na+/Ca2+ exchange activity, the cells were held
at 40 mV, and the external solution was rapidly switched from
Na+-containing solution to Na+-free solution in
which LiCl was substituted for NaCl. Figure 5 shows typical examples of
Na+/Ca2+ exchange currents recorded from NTG
(A) and Gq myocytes
(B). When the external Na+ was changed to
Li+, the membrane current shifted to an outward direction
in both groups; however, peak current amplitude was significantly
smaller in Gq myocytes. Under our experimental
conditions, the average Na+/Ca2+ exchange
current density in NTG and Gq myocytes was 0.76 ± 0.1 (n = 15) and 0.38 ± 0.04 pA/pF
(n = 22), respectively (Fig. 5C).
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Na+/Ca2+ exchanger gene expression.
To determine whether the functional changes were associated with
alterations in the Na+/Ca2+ exchanger gene
expression, we used an RPA to quantify NCX-1 mRNA levels (Fig.
6). Total RNA from the hearts of
Gq mice and NTG littermates was hybridized with a
riboprobe specific for the mouse NCX-1 transcript. The hybridization
reactions were subjected to RNase treatment, electrophoresed, and
visualized by autoradiography (Fig. 6A). Overall NCX-1
expression levels were determined by exposing the RPA gel to a phosphor
plate, scanning the plate with a PhosphorImager, and analyzing the
results with NIH Image Analysis software. As summarized in Fig.
6C, NCX-1 mRNA levels were reduced by 40% in
Gq hearts compared with NTG hearts. These changes could not be accounted for by changes in loading conditions (Fig.
6B).
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Contribution of Ito to action potential prolongation.
In an earlier study, we found that the time course of
ICa inactivation was significantly slower in
Gq than in NTG myocytes, although
ICa density was not altered (30).
It is therefore possible that slower ICa
inactivation may also contribute to action potential prolongation in
Gq myocytes. The possible contribution of this effect to
action potential prolongation was tested with BAPTA (10 mM) used as an
intracellular Ca2+ buffer to minimize
Ca2+-dependent ICa inactivation
(19, 24, 25). In NTG myocytes, action potential duration was significantly longer in the presence of BAPTA than without an intracellular Ca2+ buffer. The
APD50 and APD70 were 27.8 ± 1.4 and
37.7 ± 2.1 ms (n = 11), respectively.
q myocytes. To address
this question, we measured APD50 and APD70 in
NTG myocytes after application of 4-AP (100 µM) to reduce
Ito by ~40% (Fig. 3C). Although
4-AP prolonged action potential duration in NTG myocytes, action
potential duration was still significantly shorter than in
Gq myocytes: APD50 before and after 4-AP was
13.5 ± 1.0 and 18.5 ± 2.0 ms, and APD70 before
and after 4-AP was 16.7 ± 1.9 and 24.8 ± 3.3 ms
(n = 7), respectively.
Furthermore, when action potentials were compared in the presence of a
higher concentration of 4-AP (2 mM), action potential duration became
significantly longer in Gq than in NTG myocytes (Fig.
7). These measures of APD50
and APD70 are summarized in Table
2. Taken together, these data indicate
that a decreased Ito expression may contribute
to the action potential prolongation observed in Gq
myocytes. However, additional changes, including a slower
ICa inactivation, may also participate in the
overall changes in action potential duration.
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-AR signaling: effects of Iso and forskolin on ICa.
To minimize Ca2+-dependent inactivation and subsequent
negative -AR regulation of the Ca2+ channels, myocytes
were dialyzed with BAPTA (24, 25). As in our
previous study (30), the peak ICa
densities in NTG and Gq myocytes were
similar: 13.4 ± 0.7 (n = 36) and 12.3 ± 0.8 pA/pF (n = 32), respectively. When
ICa inactivation was measured in myocytes
dialyzed with EGTA (30), the time to half-decay was
significantly slower in Gq myocytes (30.9 ± 1.6 ms, n = 30) than in NTG myocytes (18.5 ± 1.8 ms,
n = 29). However, with BAPTA, the mean times to
half-decay were similar: 44.2 ± 2.4 (n = 10) and
44.4 ± 3.4 ms (n = 8) for NTG and
Gq myocytes, respectively.
q myocytes.
In these experiments, Iso increased peak ICa by
160% of basal value in NTG myocytes (Fig. 8A) but only by
15% in Gq myocytes (Fig. 8B). A negative shift of the I-V relationships was also observed in both
groups. When data were fitted to a Boltzmann relationship (see
MATERIALS AND METHODS), the shift in the I-V
relationship was 13.1 ± 1.0 and 4.8 ± 0.7 mV for NTG
(n = 15) and Gq myocytes
(n = 18), respectively. Figure
9A summarizes the cumulative
dose-response effects of Iso on peak ICa.
Although EC50 was similar in both groups (28.9 and 27.4 nM
for NTG and Gq, respectively), Iso was significantly
less effective in Gq myocytes. The increase of peak
ICa by Iso (1 µM) was 124 ± 13% for NTG
and 30 ± 5% for Gq myocytes.
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q myocytes
to Iso could be due to changes at several levels in the -AR
signaling cascade (13). To test the possibility that loss
of response to Iso was caused by reduced adenylyl cyclase activity, we
examined the effects of an adenylyl cyclase activator, forskolin. In
NTG myocytes, the maximal responses of ICa to
forskolin (5 µM) and Iso (1 µM) were similar. Furthermore,
forskolin did not lead to a further increase of
ICa after maximum ICa
induced by Iso. In contrast, we found that subsequent forskolin
application further enhanced ICa in
Gq myocytes. However, the combined effect was still
significantly less than the effect of Iso in NTG myocytes. Thus the
attenuated responsiveness of the Gq myocytes to Iso
occurs not only at the receptor level but also at or below the adenylyl
cyclase level. To test this hypothesis, we compared the effects of
forskolin (5 µM) on ICa in NTG and
Gq myocytes. As shown in Fig. 9B, forskolin increased ICa by 125 ± 21% in NTG
myocytes (n = 17), whereas the stimulatory effect was
significantly less in Gq myocytes (67 ± 6%,
n = 25).
Previous studies in animal models of hypertrophy as well as those in
human heart failure indicate a change in G protein expression levels
(13). To test possible involvement of Gi in
-AR regulation of ICa, we measured effects of
Iso and forskolin in Gq myocytes after
incubation with PTX (2 µg/ml for 4-6 h). The effect of PTX treatment was evaluated by testing the effects of carbachol (10 µM)
on the activation of muscarinic K+ currents in atrial
myocytes. Figure 10A shows,
as expected, carbachol-activated K+ currents in an
untreated atrial myocyte. In contrast, the activation was completely
abolished in cells treated with PTX. Despite such a pretreatment,
Gq myocytes continue to show significantly reduced responses to Iso (Fig. 10B). Maximal Iso (1 µM)-stimulated
increases in ICa in PTX-treated myocytes were
122 ± 17 and 17 ± 8% over baseline in NTG and
Gq myocytes, respectively.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we found that myocytes isolated from
Gq hearts exhibit prolonged action potentials and
decreased densities of the repolarizing K+ currents
(Ito and IK1). These
observations suggest that a common pattern of electrophysiological
changes, associated with human heart failure, occurs in this model of
cardiac hypertrophy. The data also support the hypothesis that enhanced
Ca2+ influx during the prolonged action potential in
Gq myocytes may compensate for the reduced SR
Ca2+ loading to support peak contractions or
Ca2+ transients that are otherwise significantly reduced as
a result of diminished SR function (30). Additionally, our
data indicate that multiple changes, including a decrease in -AR
coupling to adenylyl cyclase and a reduction in the activity of
adenylyl cyclase, contribute to the depressed responses of
ICa to Iso. It is possible that catecholamines
released by tonic sympathetic activation of cardiac nerves or in the
circulation could stimulate -AR, even under basal conditions, and
attenuated responsiveness of ICa to -AR
stimulation in Gq myocytes may contribute to depressed
ventricular dysfunction observed in vivo.
Our experiments demonstrate that action potential duration was
significantly prolonged in Gq myocytes.
APD50 and APD70 were prolonged, and these
changes were associated with reductions in Ito,
IK1, and Na+/Ca2+
exchange current densities. In cardiac myocytes, Ca2+
influx through the L-type Ca2+ channel is the primary
pathway to trigger Ca2+ release from the SR. However,
prolongation of the action potential may result in an enhanced inward
Ca2+ influx and SR Ca2+ loading to support
contractility in Gq myocytes.
The most prominent electrophysiological abnormality found in a variety
of experimental models of heart failure as well as human heart failure
is action potential prolongation. The duration of the cardiac action
potential is controlled by a balance of inward and outward currents. As
in human ventricular myocytes, Ito are present
in adult ventricular myocytes and are responsible for the
repolarization phase of the action potential (27,
31). It is therefore likely that a decrease in
Ito contributes to changes in the action
potential in Gq myocytes. Consistent with this notion,
4-AP prolonged the action potential duration in NTG cells, but 4-AP
prolonged the action potential more in Gq than in NTG myocytes. These results indicate that a change in
Ito alone is not sufficient to account for the
action potential profile observed in Gq myocytes.
Similar results, i.e., that 4-AP prolongs action potential more
dramatically in myocytes from failing hearts than in normal myocytes,
have been reported previously (14).
Because IK1 is responsible for the terminal
phase of repolarization, it is possible that a decrease in
IK1 may also contribute to some of the
prolongation of the terminal phase of the action potential
(14, 28). Many factors affect the
Na+/Ca2+ exchange activity, including membrane
potential and internal and external Na+ and
Ca2+ (6). It is therefore difficult to predict
the extent to which action potential duration is altered by decreasing
Na+/Ca2+ exchange in Gq
myocytes. However, it is unlikely that the reduction of this current
played a significant role in the action potential prolongation found in
Gq myocytes. An alternative explanation for the
prolonged action potential would be a reduced
Ca2+-dependent inactivation. We previously demonstrated
that Gq myocytes exhibit significantly slower
ICa inactivation due to impaired SR function
and/or a defective ICa-induced SR
Ca2+ release process (30). It is therefore
likely that slower ICa inactivation associated
with altered cellular Ca2+ handling contributes to the
action potential prolongation in Gq myocytes. A
modulatory role of increased Ca2+ influx as a result of
reduced Ca2+-dependent inactivation in the plateau phase of
the action potential has been reported in the failing heart by computer
model analysis (28).
It has been reported that Na+/Ca2+ exchange
activity is increased in failing hearts (17,
22). In failing hearts with significantly impaired SR
Ca2+ removal, enhanced Na+/Ca2+
exchange activity during the Ca2+ transients may be an
important compensatory mechanism. However, in our study, which uses a
relatively compensated form of hypertrophy (23), we found
that Na+/Ca2+ exchange current activity
and expression of the NCX-1 gene were reduced. In Gq
hearts the SR Ca2+ uptake rate was reduced by
~30% (30). Therefore, the decrease in NCX-1
expression cannot serve to compensate for defective SR Ca2+
uptake. The cause of this difference is not known. It is possible that
the decrease in NCX-1 may serve to compensate for a different Ca2+ pathway or result from a noncompensatory inhibition of
NCX-1 gene transcript by a Gq-mediated signaling
pathway. Future studies of a direct comparison of
Na+/Ca2+ exchanger function at various stages
of cardiac hypertrophy and failure in the same model would permit
establishment of the functional roles mediated by changes in
Na+/Ca2+ exchanger activity.
Our data show that Iso increased ICa in NTG and
Gq myocytes with similar affinity, but the magnitude of
the response to the drug was significantly reduced in Gq
myocytes. -AR-mediated increases in ICa
depend on the -AR signaling cascade. Because -AR density was
found to be normal in the Gq heart (8), the reduced response to Iso in Gq myocytes suggests
potential changes in -AR and Ca2+ channel coupling. In
NTG myocytes, maximal activation of ICa with Iso
or forskolin was similar, and such activation was not additive. In
contrast, although the relative increase in ICa
with forskolin was still significantly less in Gq than
in NTG myocytes, forskolin was capable of activating
ICa more potently than Iso. Pretreatment of
cells with PTX did not alter the ICa responses to Iso or forskolin. We previously demonstrated that the responses of
ICa to dihydropyridine drugs and a
membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP, were not
altered in Gq myocytes, suggesting that modulation of
the Ca2+ channel by cAMP-dependent protein kinase A
activation is normal (30). Taken together, our data
suggest that impaired -AR-adenylyl cyclase coupling and reduced
adenylyl cyclase activity are involved in the reduced responsiveness of
ICa to Iso. The electrophysiological observations are consistent with biochemical observations that basal
and forskolin-activated adenylyl cyclase activities in
Gq hearts were reduced by ~45%
compared with NTG hearts (26).
In summary, we have studied electrophysiological properties that may
contribute to altered Ca2+ handling and -AR regulation
of ICa in a genetic model of cardiac hypertrophy. The hypertrophy-associated action potential prolongation is a prominent feature of Gq myocytes. Our data
suggest that multiple changes in the -AR signaling pathway that
regulates ICa occur in Gq
myocytes. Because the signal transduction pathway mediated by
Gq is closely associated with the induction of cardiac hypertrophy and the progression of heart failure (1,
2, 8, 20, 23), this
transgenic model may serve as an informative model system for
understanding the cellular mechanisms of heart failure in humans.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. G. W. Dorn for providing the transgenic mice, Dr. R. Millard for helpful comments on the manuscript, and Drs. M. Periasamy and G. J. Babu for guidance during the study. This work was supported by the American Heart Association, Ohio Valley Affiliate, and National Institutes of Health Grants GM-54169 and HL-61476 and Training Grant HL-07382.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Yatani, Dept. of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575 (E-mail: Yatania{at}uc.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. §1734 solely to indicate this fact.
Received 18 October 1999; accepted in final form 10 January 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) and presence of Iso
(
). Bottom: voltage dependence of peak
ICa before and after the application of Iso.
