From the Department of Pharmacology and Cell
Biophysics, and
Division of Cardiology, University of
Cincinnati, Cincinnati, Ohio 45267, the § Division of
Xenobiotics, Metabolism and Disposition, National Institute of Health
Sciences, Tokyo 158, Japan, and the ** Department of Anatomy, Case
Western Reserve University, Cleveland, Ohio 44106
Received for publication, August 1, 2000, and in revised form, December 11, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cardiac-specific overexpression of murine
cardiac calsequestrin results in depressed cardiac contractile
parameters, low Ca2+-induced Ca2+ release
from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic
mice. To test the hypothesis that inhibition of phospholamban activity
may rescue some of these phenotypic alterations, the calsequestrin
overexpressing mice were cross-bred with phospholamban-knockout mice.
Phospholamban ablation in calsequestrin overexpressing mice led to
reversal of the depressed cardiac contractile parameters in
Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca2+ storage, assessed by
caffeine-induced Na+-Ca2+ exchanger currents.
The inactivation time of the L-type Ca2+ current
(ICa), which has an inverse correlation with
Ca2+-induced SR Ca2+ release, and the relation
between the peak current density and half-inactivation time were also
normalized, indicating a restoration in the ability of
ICa to trigger SR Ca2+ release. The
prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels
of atrial natriuretic factor and Hypertrophy of ventricular myocardium is postulated to be an
adaptive response to relative increases in external workload, induced
by endocrine, paracrine, autocrine, and mechanical factors or decreased
myocardial contractility (1). The increase in heart mass has been
implicated to normalize cardiac function by decreasing wall stress.
However, a sustained imbalance between workload and muscle
contractility may lead to progressive thinning of the left ventricular
wall and chamber dilation associated with decompensated hypertrophy and
heart failure (2, 3). Studies in human and animal models have shown
that cardiac hypertrophy is associated with impaired sarcoplasmic
reticulum (SR)1
Ca2+ modulation, leading to aberrant cardiac contraction
and relaxation (4-8). Although several Ca2+-related
signaling molecules, such as calcineurin, Ca2+-calmodulin
kinase, and Ca2+-dependent protein kinase C
have been suggested to play key roles in myocardial hypertrophic
responses (9-12), it is not clear yet whether the abnormal SR
Ca2+ handling per se contributes to the
generation, maintenance, and characteristics of cardiac hypertrophy
in vivo. Furthermore, it is unknown whether reversal of the
SR Ca2+ handling defects can mediate functional benefits in
myocardial hypertrophy.
The SR plays a key role in the regulation of Ca2+
homeostasis and contractility in cardiac muscle. During relaxation,
Ca2+ is transported from the cytosol into the lumen of the
SR by a Ca2+-ATPase. Subsequently, in response to
Ca2+ influx through L-type Ca2+ channels, the
Ca2+ loaded in the SR is released through the ryanodine
receptors for the initiation of muscle contraction. The activity of the SR Ca2+-ATPase is regulated by phospholamban, a 52-amino
acid phosphoprotein (13). Decreases in the levels of phospholamban or
increases in its phosphorylation status result in an increase in the
apparent Ca2+ affinity of the SR Ca2+-ATPase,
and augment cardiac contractile parameters. The mechanisms underlying
these regulatory effects of phospholamban have been suggested to
reflect facilitation of SR Ca2+ transport, Ca2+
loading, and Ca2+ release (13-19). These studies also
indicate that the SR Ca2+ load is one of the major
determinants of myocardial contractility (18, 19). The SR
Ca2+ load and subsequent Ca2+ release are
regulated by calsequestrin, a luminal SR protein, with high capacity
and low affinity for Ca2+ (20). Calsequestrin has been
reported to form a stable complex with the ryanodine receptor, junctin,
and triadin (21). Recent transgenic approaches have shown that
increased expression of calsequestrin in the heart was associated with
increased SR Ca2+ storage capacity, but this SR
Ca2+ was not available for release during
excitation-contraction coupling, leading to depressed Ca2+
transients and contractile parameters (22, 23). The depressed cardiac
function was associated with re-expression of a fetal gene program and
hypertrophy (23) or failure (24). To better define the role of SR
Ca2+ handling defects in the hypertrophic response, the
current study employed a genetic approach to improve SR function
through phospholamban inhibition in the calsequestrin overexpressing
hearts (23). Ablation of phospholamban restored the depressed
contractile parameters and reversed the compensatory alterations in SR
Ca2+ handling protein levels. Furthermore, induction of
atrial natriuretic factor (ANF) and Experimental Animals--
Phospholamban knockout 129SvJ/CF-1
mice (15) were mated with transgenic FVB/N mice overexpressing murine
cardiac calsequestrin specifically in cardiac muscle (line number 418)
(23). F1 heterozygous phospholamban offsprings carrying the
calsequestrin transgene were inbred with their littermates without the
transgene to obtain F2 pups with three different genotypes:
wild-type (WT), calsequestrin overexpressing (CSQOE), and
calsequestrin overexpressing with phospholamban ablation
(CSQOE/PLBKO). To identify the genotypes, polymerase chain reaction analysis of tail genomic DNA was carried out
as described previously (15, 23). The cardiac phenotype of the WT mice
with mixed genetic background was similar to that in the FVB/N genetic
background (23). The handling and maintenance of animals in this study
was approved by the ethics committee of the University of Cincinnati.
Eight to 15-week-old mice of either gender were used for the following
studies unless otherwise indicated.
Morphological Analyses--
Immunocytochemistry of cardiac
ventricles were performed as described previously (23). Ventricular
myocyte cross-sectional areas were obtained, as described previously
(25).
In Vivo and ex Vivo Left Ventricular Function--
Left
ventricular contractile parameters were determined in closed-chest
anesthetized mice using a 1.4-French scale Millar MIKRO-TIP catheter,
as previously described (25). Contractile parameters of isolated hearts
were also determined in Langendorff mode at 37 °C with a constant
perfusion pressure of 50 mm Hg, as described previously (23).
Electrophysiology of Isolated Cardiomyocytes--
Left
ventricular myocytes were isolated from mice and whole cell currents
were recorded using patch clamp techniques as described previously (17,
26, 27). Membrane capacitance was measured using voltage ramps of 0.8 V/s from a holding potential of Immunoblotting and Dot-blot Analysis--
SDS-polyacrylamide
electrophoresis and quantitative immunoblotting of cardiac ventricular
homogenates were performed as described previously (23). Dot-blot
analysis of total RNA from cardiac ventricles was performed as
described previously (25).
Statistics--
Data are presented as mean ± S.E.
Comparisons across groups were evaluated using analysis of variance
(ANOVA). When the p value was less than 0.05, Fisher's
least significant difference (LSD) procedure was employed to
discriminate which means differed from the others.
Calsequestrin Overexpressing Mice Deficient in
Phospholamban--
To determine whether enhanced SR Ca2+
transport activity may modify the time course of cardiac dysfunction
and hypertrophy, we generated transgenic mice, which overexpress
calsequestrin and are deficient in phospholamban. To achieve this,
CSQOE mice, which exhibit depressed function and
hypertrophy (23), were mated with phospholamban knockout mice, which
demonstrated enhanced SR Ca2+ transport and hyperdynamic
function (15). F2 littermates with WT, CSQOE,
and CSQOE/PLBKO genotypes were characterized in
parallel to eliminate any potential effects of the genetic background. There were no phenotypic alterations at the gross morphological level
between the three models up to 7 months of age. Furthermore, ablation
of phospholamban did not alter the levels of calsequestrin overexpression (Fig. 1A),
which was increased by 18 ± 4 (n = 8) in
CSQOE and 19 ± 4-fold (n = 8) in
CSQOE/PLBKO ventricles, compared with WT
littermates. Phospholamban was undetectable in
CSQOE/PLBKO, whereas it was increased by 37%
in CSQOE, compared with WTs, consistent with previous
findings (23).
Subcellular Distribution of Calsequestrin--
To assess the
effects of phospholamban ablation on the subcellular localization of
overexpressed calsequestrin, immunostaining and confocal microscopy
were performed. Cryostat sections from both CSQOE and
CSQOE/PLBKO transgenic hearts exhibited similar striated staining patterns as WTs (Fig. 1B). The increased
staining in CSQOE and CSQOE/PLBKO
tissue was consistent with the immunoblotting data. Further examination
of sections from the CSQOE and
CSQOE/PLBKO littermates revealed that the
staining patterns of transverse striations were predominant. No obvious
morphological deterioration, such as abnormal striations, fibrosis, and
necrosis, was observed in these sections.
Rescue of Cardiac Contractile Parameters--
An important
question is whether the depressed cardiac contractile parameters
associated with calsequestrin overexpression may be restored in
CSQOE/PLBKO mice. Thus, cardiac catheterization was performed in 2-month-old intact anesthetized animals and their hemodynamic function was evaluated. The intrinsic heart rate was not
different between groups. However, the maximal rates of left ventricular pressure development (+dP/dt) and decline (
These studies were extended to the organ level, and isolated mouse
hearts from 2-month-old littermates were perfused in parallel, using
the Langendorff mode (Fig. 2). Hearts
from CSQOE mice exhibited depressed +dP/dt values, similar
to previous observations in the CSQOE mouse of the FVB/N
genetic background (23). However, the rates of left ventricular
relaxation, which were attenuated in the original model (23), did not
reveal any statistical differences from their WT littermates (FVB/N + 129SvJ/CF-1). This apparent discrepancy may be due to the differences
in the genetic background (FVB/N versus FVB/N+129SvJ/CF-1),
which is known to play a major role in determining the cardiac
phenotype of mice (30, 31). Interestingly, the contractile parameters
in CSQOE hearts did not deteriorate upon aging to 7 months,
in contrast to the progressive deterioration of cardiac function and
morbidity of a transgenic mouse model with 10-fold overexpression of
dog cardiac calsequestrin (24, 32). Upon ablation of phospholamban,
both +dP/dt and
Another important question is whether Rescue of Ca2+ Handling Defects in Calsequestrin
Overexpressing Myocytes--
Stable Ca2+ tolerant
cardiomyocytes could be achieved with voltage clamping and manipulating
the intracellular ionic environment (17, 26, 27). Since
caffeine-induced SR Ca2+ release fully saturates the
fluorescent dye signal in calsequestrin overexpressing myocytes (35),
the INCX current was used to estimate the SR
Ca2+ content. When INCX was measured
in the absence of caffeine by replacing external Na+ with
Li+, the average values of outward
INCX current densities were 0.99 ± 0.06 pA/pF (n = 31), 0.90 ± 0.06 pA/pF
(n = 32), 0.91 ± 0.06 pA/pF (n = 13), in WT, CSQOE, and CSQOE/PLBKO,
respectively. This suggests that the functional activity of
Na+-Ca2+ exchanger was not significantly
altered in CSQOE or CSQOE/PLBKO, compared with WT myocytes. In subsequent studies, caffeine (5 mM) was added to the external solution in the presence of
Na+ to examine whether phospholamban ablation increased the
SR Ca2+ content in myocytes overexpressing calsequestrin.
Rapid application of caffeine elicited inward
INCX currents by sarcolemmal
Na+-Ca2+ exchange (Fig.
3A). The peak current density
of caffeine-induced INCX currents was increased
in CSQOE myocytes, indicating high SR Ca2+
storage, as previously described (23). Phospholamban ablation resulted
in further increases in the SR Ca2+ storage, as evidenced
by the higher caffeine-induced INCX current density of CSQOE/PLBKO, compared with the
CSQOE myocytes (Fig. 3B).
To gain further insights into the mechanisms underlying the rescue
effects of phospholamban ablation, whole cell
ICa measurements were performed in the three
models (Fig. 4A). The
current-voltage relations of CSQOE and
CSQOE/PLBKO were similar to WT (data not shown). However, the current density of peak L-type Ca2+
current (ICa) at 2 mM external
Ca2+ concentration was significantly reduced in
CSQOE myocytes, consistent with previous observations in
the FVB/N genetic background (23). Phospholamban ablation resulted in
significant increases in the current density of peak
ICa, although these values were lower than WT
(Fig. 4B). Furthermore, the inactivation kinetics of
ICa, which was significantly slower in
CSQOE myocytes, was restored upon phospholamban ablation
(Fig. 4B).
We have previously shown that ICa inactivation
in mouse myocytes correlates with the local increase in
Ca2+ released from the SR, which promotes
Ca2+-dependent inactivation. Thus, the
inactivation kinetics of ICa can be used to
evaluate the efficiency of ICa-induced SR
Ca2+ release (17, 26). To elucidate the functional coupling
of L-type Ca2+ channels and the SR Ca2+
release, the correlations between densities and inactivation time
courses of ICa were examined by varying the
external Ca2+ concentration (1-20 mM). As
shown in Fig. 4C, the relation between the peak current
density and the half-inactivation time was shifted upward in
CSQOE, reflecting an inability of
ICa to trigger SR Ca2+ release. In
contrast, CSQOE/PLBKO and WT myocytes exhibited
similar relations between the maximal ICa
density and the half-inactivation time, suggesting that phospholamban
ablation restored the ability of ICa to trigger
SR Ca2+ release.
To examine the stimulatory effects of Rescue of Action Potential Prolongation in Calsequestrin
Overexpressing Myocytes--
A major electrophysiological abnormality,
observed in a variety of experimental models of myocardial disease, as
well as human heart failure, is action potential prolongation. Thus, we
examined the characteristics of action potentials in isolated
ventricular myocytes from the three groups. WT myocytes displayed a
brief action potential with a rapid initial phase of repolarization without a discernible plateau phase (Fig.
6), similar to previous observations in
adult mouse ventricular myocytes (27). However, the action potential
duration, quantified at 50% repolarization (APD50), was
significantly longer in CSQOE (35.7 ± 5.7 ms,
n = 5), compared with WT (17.0 ± 1.4 ms,
n = 22) myocytes. Interestingly, the
CSQOE/PLBKO myocytes displayed
APD50 values (19.8 ± 1.7 ms, n = 11)
similar to WTs. There was no significant difference in the resting
membrane potentials among the three groups (WT, SR-associated Proteins--
Calsequestrin overexpression was
previously shown to result in altered expression of several SR
Ca2+ cycling proteins (23). Thus, quantitative
immunoblotting was utilized to determine whether phospholamban ablation
might restore these changes. The protein levels of the SR
Ca2+-ATPase were increased in parallel with up-regulation
of phospholamban in CSQOE hearts, but ablation of
phospholamban prevented the compensatory up-regulation of the SR
Ca2+-ATPase (Table I). The
ryanodine receptor levels, which were decreased in CSQOE
hearts, were further reduced in CSQOE/PLBKO hearts, similar to previous observations in phospholamban-deficient mice (16). Furthermore, the levels of GRP78 (78-kDa glucose-regulated protein, also known as BiP) and calreticulin, which are
Ca2+-binding proteins localized in the lumen of the
endoplasmic reticulum (ER) or the SR, were up-regulated by 3.4- and
2.0-fold in CSQOE, respectively. These proteins are known
to be molecular chaperones and to play a role in the storage of the
exchanging pool of ER Ca2+ in cultured cells (36, 37). They
are also known to be up-regulated in response to accumulation of mis-
or unfolded ER proteins by ER stress, such as perturbation of
Ca2+ homeostasis (38, 39). Phospholamban ablation abolished
the increases in GRP78 and calreticulin, which may indicate a relative reduction in ER/SR stress in CSQOE/PLBKO
versus CSQOE.
Cardiac Hypertrophy--
One of the characteristics of hypertrophy
is the increase in heart muscle mass, associated with reactivation of a
fetal gene program, to compensate for increases in workload. To assess
whether improvement of cardiac function in
CSQOE/PLBKO mice is accompanied by rescue of
the hypertrophic response, ventricular and atrial weights were
evaluated. In 2-3-month-old CSQOE mice, the wet
ventricular/body weight ratio was increased approximately by 35%,
compared with WT (Fig. 7), in agreement
with previous observations in the FVB/N genetic background (23).
Examination of gross cardiac morphology revealed enlargement of the
left atrium (data not shown) and the atrial/body weight ratio was
increased by 38%. These increases in weight ratios were not
progressive at least up to 7 months of age. Ablation of phospholamban
restored the atrial/body weight ratio to WT levels, but it did not
reverse the increases in the ventricular/body weight ratio. Two-way
ANOVA indicated that the wet tissue/body weight ratios of the
CSQOE/PLBKO were not dependent on age.
Furthermore, these alterations did not reflect changes in body weights,
which were similar among the three genotypes with the same age.
Ventricular myocyte size was also assessed by cell capacitance of
patch-clamped isolated cells. CSQOE myocytes were
significantly larger than WTs, but phospholamban ablation restored cell
capacitance to WT levels (WT, 140 ± 3 pF; CSQOE,
167 ± 4* pF; CSQOE/PLBKO, 142 ± 3# pF, n = 76-107, *, p < 0.05 versus WT, #, p < 0.05 versus CSQOE). Furthermore, examination of
cross-sectional areas of ventricular myocytes indicated significant
increases upon calsequestrin overexpression and reversal upon
phospholamban ablation (WT, 319 ± 27 µm2;
CSQOE, 409 ± 20* µm2;
CSQOE/PLBKO, 354 ± 12#
µm2, n = 6, *p < 0.05 versus WT; #, p < 0.05 versus
CSQOE).
The increase in ventricular/body weight ratio of the CSQOE
mice was associated with increased expression of a fetal gene program (Fig. 8), including ANF, Defects in SR Ca2+ uptake and release are common
features of hypertrophied and failing animal and human myocardia,
although the mechanisms underlying the etiology of these defects have
not been clear. Furthermore, the contribution of impaired SR
Ca2+ cycling to the onset and progression of the
hypertrophic response is unknown. To address this question, the current
study utilized the calsequestrin overexpressing model with cardiac
hypertrophy (23) and introduced phospholamban-deficient alleles to
improve the SR Ca2+ transport properties. Phospholamban
ablation did not alter the levels or the localization of the
overexpressed calsequestrin, which was associated with the terminal
cisternae at the z-lines, similar to endogenous calsequestrin. However,
the contractile parameters were restored to wild-type levels in
vivo and they were super-rescued in Langendorff-perfused hearts,
upon ablation of phospholamban. The observed differences in the degree
of cardiac function enhancement between the intact animal and the
isolated organ levels are presumably due to differences in external
loading conditions. This functional improvement was at least partially due to enhanced SR Ca2+ storage in phospholamban-deficient
hearts, as estimated by the caffeine-induced
INCX. Calsequestrin overexpression has been
previously shown to increase the SR Ca2+ content but this
Ca2+ pool was not accessible for release (22, 23). However,
upon phospholamban ablation, the additional Ca2+ load,
accompanied by the enhanced SR Ca2+ uptake, is likely to
increase the intraluminal free SR Ca2+ concentration, which
is associated with higher amounts of Ca2+ released (19). In
addition, the density of ICa, which was
decreased in calsequestrin overexpressing myocytes, was partially
restored in CSQOE/PLBKO cells, leading to
higher activation of the Ca2+-induced SR Ca2+
release despite the reduced levels of the ryanodine receptor, elicited
by phospholamban ablation (16). Moreover, the inactivation kinetics of
ICa, which are regulated by the Ca2+
influx as well as SR Ca2+ release (17, 26) were rescued and
the ICa density-inactivation relation of
CSQOE/PLBKO was similar to the WTs. These
results suggest that phospholamban ablation restored the
impaired SR Ca2+ release properties or defective
excitation-contraction coupling of calsequestrin overexpressing
cardiomyocytes. Furthermore, prolongation of the action potential,
observed in CSQOE myocytes, which may reflect alterations
in the transient outward K+ currents
(Ito) and possible induction of arrhythmias
(27), was restored upon phospholamban ablation.
Cardiac-specific overexpression of dog cardiac calsequestrin has been
suggested to attenuate The increases in the expression levels of: (a) calreticulin,
which has also been reported to increase in pressure overload hypertrophy (42) and may contribute to increased ER/SR Ca2+
buffering; (b) GRP78, which is another
Ca2+-binding molecular chaperone in the ER/SR; and
(c) SR Ca2+-ATPase, which constituted an
important compensatory response for SR function in CSQOE
hearts, were reversed by phospholamban deficiency, indicating a
reduction in ER stress or restored ER/SR Ca2+ homeostasis
in CSQOE/PLBKO hearts. However, further
examinations are needed to reveal the mechanisms by which calsequestrin
buffers SR luminal Ca2+, and the effects of phospholamban
ablation on free luminal Ca2+ and ryanodine receptor gating properties.
In the calsequestrin overexpressing heart as well as a variety of other
experimental models, cardiac hypertrophy was associated with increased
expression of fetal genes (23, 43-45). Interestingly, the
up-regulation of Phospholamban deficiency also resulted in significant reduction of
atrial mass in the calsequestrin overexpressing mice. The reduction of
atrial mass suggested that stress on the atria was attenuated,
presumably due to the enhanced ventricular function. Enlargement of
left atrial size in a hypertrophic heart is postulated as a predictor
of atrial fibrillation and thromboembolism leading to stroke, which is
associated with increased risk of cardiovascular mortality (46-49).
Therefore, inhibition of phospholamban function may not only improve
ventricular performance but also prevent atrial-induced morbidity.
Recently, phospholamban ablation was shown to rescue the depressed
function and heart failure phenotype in an MLP-deficient mouse model,
which displays many phenotypic features of human dilated cardiomyopathy
(50). Cardiac-specific overexpression of the -skeletal actin mRNA as well as
ventricular myocyte size. These results indicate that attenuation of
phospholamban function may prevent or overcome functional and
remodeling defects in hypertrophied hearts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-skeletal actin was normalized,
and myocyte hypertrophy was rescued in the calsequestrin overexpressing hearts.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
50 mV. Whole cell
Na+-Ca2+ exchanger currents
(INCX) were recorded by the method Kimura et al. (28). To activate Na+-Ca2+
exchanger, the cells were held at
40 mV and the external solution was
rapidly switched to one in which equimolar LiCl was substituted for
NaCl. The Ca2+ content of the SR was evaluated in
voltage-clamped myocytes by the transient inward
INCX currents evoked by a rapid application of
caffeine (5 mM) to release the SR Ca2+ (29).
Whole cell L-type Ca2+ currents
(ICa) were recorded by applying depolarization
pulses every 10 s from a holding potential of
50 mV (26, 27). To obtain relations between peak current density and inactivation kinetics, ICa was measured in a series of
external Ca2+ concentrations (1.0-20 mM).
Action potentials were recorded, as previously described (27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (72K):
[in a new window]
Fig. 1.
Quantitative immunoblotting and
immunolocalization studies confirming calsequestrin overexpression in
the phospholamban knockout background. A, a dilution
series of a control homogenate from pooled wild-type hearts was used to
generate a standard line for quantitation of calsequestrin and
phospholamban ventricular protein levels in WT, CSQOE, and
CSQOE/PLBKO littermate mice. Calsequestrin
levels were ~20-fold above WT in CSQOE and
CSQOE/PLBKO. Phospholamban was increased in
CSQOE (37%), but was absent in
CSQOE/PLBKO. B, longitudinal
sections of ventricular tissue. Images were obtained using identical
brightness and contrast. Clearly calsequestrin-specific staining was
much brighter in CSQOE and
CSQOE/PLBKO. There were no obvious differences
in staining patterns in any of the sections, suggesting that the
overexpression of calsequestrin or the absence of phospholamban does
not affect the distribution of calsequestrin. Bar, 8 µm.
dP/dt), which
were significantly attenuated in the CSQOE mice (6,677 ± 199 and 6,244 ± 546 mm Hg/s, respectively), exhibited values
that were similar between CSQOE/PLBKO
(10,807 ± 906 and 10,552 ± 942 mm Hg/s, respectively) and
WT mice (10,333 ± 1,054 and 12,483 ± 1,363 mm Hg/s,
respectively), indicating restoration of cardiac function in
vivo.
dP/dt values of calsequestrin overexpressing hearts
were significantly increased to levels even higher than those in the WT
littermates. Similar effects were observed in older (7 month) mice,
suggesting the functional benefit of phospholamban ablation over the
long term. Furthermore, there was no difference in the intrinsic heart rate across the three groups with different ages.
View larger version (32K):
[in a new window]
Fig. 2.
Effects of phospholamban ablation on
contractile parameters of isolated hearts overexpressing
calsequestrin. A, Langendorff-perfused hearts from 2- and 7-month-old mice demonstrated depressed maximal rate of contraction
(+dP/dt) in calsequestrin overexpressing
(CSQOE) mice with no changes in the maximal rate of
relaxation ( dP/dt) and the intrinsic heart rate
(HR), compared with wild-types (WT). Preparations from
calsequestrin overexpressing littermates with phospholamban deficiency
(CSQOE/PLBKO) indicated significant increases
in +dP/dt and
dP/dt without alterations in HR, compared with WT and
CSQOE. B, isolated hearts from 4-month-old mice
were perfused with increasing concentrations of isoproterenol
(Iso). In CSQOE hearts, 30-300 nM
Iso elicited pulsus alternans. Therefore, a 10-s average during
an apparent peak response was used as a representative value (points in
parentheses). Values are mean ± S.E.
(n = 3-5). *, p < 0.05 versus WT; #, p < 0.05 versus
CSQOE (Fisher's LSD after two-way ANOVA).
-adrenergic agonists can
stimulate the contractile parameters of the
CSQOE/PLBKO hearts, as phospholamban is
postulated to be a major player in
-adrenergic responses (13-15).
Thus, we examined the effects of isoproterenol stimulation in
Langendorff-perfused hearts (Fig. 2B). The positive chronotropic effects of isoproterenol were similar among WT,
CSQOE, and CSQOE/PLBKO groups.
-Agonist stimulation also increased the +dP/dt and
dP/dt values in
all three groups, including the hyperdynamic CSQOE/PLBKO (33). However, administration of
isoproterenol (30-300 nM) was associated with induction of
pulsus alternans in CSQOE hearts, presumably due to the
positive chronotropic effect (34). The
CSQOE/PLBKO littermates did not display such
mechanical abnormalities, and the maximally stimulated ±dP/dt
parameters as well as their EC50 values for isoproterenol
stimulation were similar between WT and
CSQOE/PLBKO.
View larger version (13K):
[in a new window]
Fig. 3.
Comparison of the
Na+-Ca2+ exchanger currents
(INCX) activated by a rapid application of
caffeine in ventricular myocytes. A, representative
whole cell INCX currents evoked by caffeine (5 mM) in ventricular cardiomyocytes. Myocytes were
voltage-clamped at a holding potential of 40 mV. B, the
peak caffeine-induced INCX currents normalized
to the cell capacitance to give current densities (CD). WT,
wild-type; CSQOE, calsequestrin-overexpressing,
CSQOE/PLBKO,
calsequestrin-overexpressing and phospholamban-deficient. Values are
expressed as mean ± S.E. (n = 17-19); *,
p < 0.05 versus WT; #, p < 0.05 versus CSQOE (Fisher's LSD after one-way
ANOVA).
View larger version (17K):
[in a new window]
Fig. 4.
Properties of L-type
Ca2+ currents (ICa)
in transgenic mouse ventricular myocytes. A,
representative whole cell ICa currents recorded
in ventricular cardiomyocytes. Currents were elicited from a holding
potential of 50 to +10 mV. B, peak current densities
(CD) and half-inactivation times (t1/2)
of ICa. C, ICa
CD versus ICa t1/2
relations in mouse ventricular myocytes obtained using various
concentrations of extracellular Ca2+ (1-20
mM). Values are expressed as mean ± S.E.
(n = 17-78); *, p < 0.05 versus WT; #, p < 0.05 versus
CSQOE (Fisher's LSD after one-way ANOVA).
-adrenergic agonists on
ICa, 5 mM EGTA in the pipette
solution was replaced with 10 mM BAPTA, a more rapid
Ca2+ chelator. This replacement negates the enhanced
ICa inactivation induced by SR Ca2+
release (17, 26, 27). The peak ICa densities in
WT, CSQOE, and CSQOE/PLBKO myocytes
were 12.5 ± 0.5 pA/pF (n = 51), 6.0 ± 0.3 pA/pF (n = 38), and 11.6 ± 1.1 pA/pF
(n = 43), respectively. Potentiation of
ICa was then examined in the presence of various concentrations of isoproterenol, and in the absence or presence of
isobutylmethylxanthine (IBMX: 100 µM), a
phosphodiesterase inhibitor (Fig.
5A). Perfusion with
isoproterenol increased the current amplitude in all groups and the
EC50 values of peak ICa were similar
among them (Fig. 5B). Although the relative increase of
current amplitude was more prominent in CSQOE cells, their ICa density was significantly smaller in the
presence of isoproterenol (1 µM), compared with WT or
CSQOE/PLBKO cells. Additional application of
IBMX enhanced isoproterenol-promoted ICa in all
groups, although the current density in CSQOE myocytes
remained smaller, compared with WT or
CSQOE/PLBKO myocytes (Fig. 5C).
Therefore, overexpression of murine calsequestrin or subsequent
ablation of phospholamban did not lead to low responsiveness to
-adrenergic stimulation and/or high phosphodiesterase activity.
View larger version (32K):
[in a new window]
Fig. 5.
Effects of isoproterenol
(Iso) and isobutylmethylxanthine (IBMX) on
ICa in transgenic mouse ventricular
myocytes. A, typical L-type Ca2+ currents
(ICa) recorded in ventricular cardiomyocytes.
The currents were elicited by voltage-clamp steps from a holding
potential of 50 to 0 mV. The currents after the application of
isoproterenol (Iso: 1 µM) and subsequent
addition of IBMX (100 µM) were superimposed.
B, concentration-dependent effects of Iso on
ICa in WT, CSQOE, and
CSQOE/PLBKO myocytes. The peak current
amplitude was normalized to myocyte size (pA/pF) and plotted against
Iso concentrations. C, summarized data of the effects of Iso
(1 µM) plus IBMX (100 µM) on
ICa in WT, CSQOE, and
CSQOE/PLBKO myocytes. Values are mean ± S.E. (n = 18-51); *, p < 0.05 versus WT (Fisher's LSD after one-way ANOVA).
69.2 ± 0.4 mV,
n = 22; CSQOE,
70.2 ± 0.6 mV,
n = 5; CSQOE/PLBKO,
70.0 ± 0.6 mV, n = 10).
View larger version (11K):
[in a new window]
Fig. 6.
Effects of phospholamban ablation on action
potentials in calsequestrin overexpressing myocytes. Membrane
potential was recorded in the current clamp mode with the patch
electrode filled with a K+-rich internal solution. The
external solution was normal Tyrode solution. Myocytes were stimulated
at 0.2 Hz through the patch pipette.
Relative levels of SR/ER-associated proteins in cardiac ventricles
View larger version (13K):
[in a new window]
Fig. 7.
Cardiac weights in transgenic mice.
Blotted weights of cardiac ventricles and atria from 2-3- and
7-month-old WT, CSQOE, and
CSQOE/PLBKO mice were normalized to the
respective body weights. There was no difference in body weight across
the groups with similar ages. Values represent the mean ± S.E.
(n = 13-21 (2-3-month-old) or 4-6 (7 months old));
*, p < 0.05 versus WT; #, p < 0.05 versus CSQOE (Fisher's LSD after
two-way ANOVA).
-skeletal
actin, and
-myosin heavy chain. Phospholamban deficiency reversed
the increases in the mRNA levels of ANF and
-skeletal actin in
the CSQOE/PLBKO but did not influence the
expression of
-myosin heavy chain levels. These findings suggest
that ventricular myocyte hypertrophy induced by calsequestrin
overexpression is rescued upon phospholamban ablation.
View larger version (30K):
[in a new window]
Fig. 8.
Attenuation of the hypertrophy gene program
in calsequestrin overexpressing myocardium by phospholamban
ablation. A, mRNA dot-blot analysis demonstrated
increased transcript levels of -myosin heavy chain
(
-MHC), ANF, and skeletal
-actin
(SK-actin), but no significant changes in the levels of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in
ventricles of calsequestrin overexpressing (CSQOE) mice,
compared with wild-type (WT) littermates. Note that the mRNA
levels of ANF and SK-actin in calsequestrin overexpressing littermates
with phospholamban ablation (CSQOE/PLBKO) were
comparable to the WT. B, quantitative grouped analysis of
mRNA expression. Values are the mean ± S.E.
(n = 3). *, p < 0.05 versus
WT; #, p < 0.05 versus CSQOE
(Fisher's LSD after one-way ANOVA).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic responses and enhance myocyte
phosphodiesterase activities (24, 32). However, our results indicated
that responsiveness of ICa to isoproterenol and/or IBMX was not decreased in cardiomyocytes overexpressing murine
cardiac calsequestrin (CSQOE), as well as the
CSQOE/PLBKOs, suggesting that the smaller basal
current density observed in CSQOE myocytes is not directly
mediated by alterations in
-adrenoreceptor signaling pathway or
phosphodiesterase activity, but rather by an indirect effect associated
with cardiac hypertrophy. The reasons for these apparent discrepancies
between the dog and mouse cardiac calsequestrin overexpressing models
may be due to differences in their genetic backgrounds (23, 30-32, 40,
41).
-skeletal actin and ANF mRNA was reversed by
phospholamban ablation. The physiological and pathological roles of
increases in
-skeletal actin transcripts in cardiac hypertrophy are
currently unclear. However, the reduction of ANF mRNA levels by
phospholamban ablation may reflect the recovery of cardiomyocytes from
a pathological state, since ANF is known to be a sensitive indicator of
cardiac pathogenesis rather than the degree of hypertrophy (44). The
increases in the slow
-myosin heavy chain transcript, although
relatively small (~2-fold), were not normalized and were expected to
contribute to attenuation of contraction rates. However, phospholamban
ablation and the restored SR function appeared to overcome the effects
of increased
-myosin heavy chain expression, and the cardiac
contractile parameters in CSQOE/PLBKO hearts
were at least as high as the WTs. Consistent with the reversal of ANF
and
- skeletal actin increased expression, the cell capacitance and
cross-sectional area of myocytes were restored to normal levels upon
phospholamban ablation. Remarkably, these alterations did not reflect
similar observations for the ventricular/body weight ratio, which
remained high in CSQOE/PLBKO mice, suggesting
an increase in the number of myocytes in these hearts. Taken together,
these findings suggest that ablation of phospholamban or concomitant
improvement of SR Ca2+ handling may shift the mode of
ventricular myocyte growth from hypertrophy to hyperplasia, possibly by
reducing trophic factors for the adaptive response to Ca2+
handling defects or by increasing cellular mitotic factors.
-adrenergic receptor
kinase inhibitor was also reported to improve cardiac function in the
same model of cardiomyopathy (51). Since phospholamban is the major
substrate in the cardiac
-adrenergic pathway, it is interesting to
propose that at least a part of the beneficial effects by the latter
genetic manipulation may be mediated by the phosphorylation level of
phospholamban. The rescue effects of phospholamban ablation were
maintained over the long-term without increasing the intrinsic heart
rate in both the calsequestrin overexpressing and MLP-deficient models
(50). However, ablation of phospholamban may not be beneficial for all hypertrophic phenotypes, especially when impaired Ca2+
cycling is not associated with the end point (52, 53). Furthermore, it
remains to be determined whether inhibition of phospholamban activity
in a tissue-specific and inducible manner may still rescue cardiac
function and remodeling, when applied subsequent to the onset of
hypertrophy and failure.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL26057, HL52318, P40RR12358, and HL64018 (to E. G. K.), GM54169 and HL61476 (to A. Y.), the American Heart Association, Ohio Valley Affiliate (to A. Y.), and a Ministry of Health and Welfare (Japan) grant (to Y. S.).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.
¶ Contributed equally to the results of this work.
To whom correspondence should be addressed: Dept. of
Pharmacology and Cell Biophysics, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0575. Tel.: 513-558-2377; Fax: 513-558-2269; E-mail: kraniaeg@email.uc.edu.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M006889200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SR, sarcoplasmic reticulum; CSQOE, calsequestrin overexpressing; CSQOE/PLBKO, calsequestrin overexpressing in phospholamban knockout background; WT, wild-type; INCX, Na+-Ca2+ exchanger current; ICa, L-type Ca2+ current; ANF, atrial natriuretic peptide; ER, endoplasmic reticulum; BAPTA, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid; IBMX, isobutylmethylxanthine; LSD, least significant difference.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Marian, A. J. (2000) Lancet 355, 58-60[CrossRef][Medline] [Order article via Infotrieve] |
2. | Levy, D., Larson, M. G., Vasan, R. S., Kannel, W. B., and Ho, K. K. (1996) J. Am. Med. Assoc. 275, 1557-1562[Abstract] |
3. | Seiler, C., Jenni, R., Vassalli, G., Turina, M., and Hess, O. M. (1995) Br. Heart J. 74, 508-516[Abstract] |
4. | Gwathmey, J. K., Copelas, L., MacKinnon, R., Schoen, F. J., Feldman, M. D., Grossman, W., and Morgan, J. P. (1987) Circ. Res. 61, 70-76[Abstract] |
5. | Gwathmey, J. K., Bentivegna, L. A., Ransil, B. J., Grossman, W., and Morgan, J. P. (1993) Cardiovasc. Res. 27, 199-203[Medline] [Order article via Infotrieve] |
6. |
Wang, J.,
Flemal, K.,
Qiu, Z.,
Ablin, L.,
Grossman, W.,
and Morgan, J. P.
(1994)
Am. J. Physiol.
267,
H918-H924 |
7. | Szymanska, G., Stromer, H., Kim, D. H., Lorell, B. H., and Morgan, J. P. (2000) Pflugers Arch. 439, 339-348[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Bers, D. M.,
Li, L.,
Satoh, H.,
and McCall, E.
(1998)
Ann. N. Y. Acad. Sci.
853,
157-177 |
9. | Molkentin, J. D., Lu, J. R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R., and Olson, E. N. (1998) Cell 93, 215-228[Medline] [Order article via Infotrieve] |
10. | Lim, H. W., and Molkentin, J. D. (1999) Nat. Med. 5, 246-247[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Passier, R.,
Zeng, H.,
Frey, N.,
Naya, F. J.,
Nicol, R. L.,
McKinsey, T. A.,
Overbeek, P.,
Richardson, J. A.,
Grant, S. R.,
and Olson, E. N.
(2000)
J. Clin. Invest.
105,
1395-1406 |
12. |
Bowling, N.,
Walsh, R. A.,
Song, G.,
Estridge, T.,
Sandusky, G. E.,
Fouts, R. L.,
Mintze, K.,
Pickard, T.,
Roden, R.,
Bristow, M. R.,
Sabbah, H. N.,
Mizrahi, J. L.,
Gromo, G.,
King, G. L.,
and Vlahos, C. J.
(1999)
Circulation
99,
384-391 |
13. |
Koss, K. L.,
and Kranias, E. G.
(1996)
Circ. Res.
79,
1059-1063 |
14. | Kiriazis, H., and Kranias, E. G. (2000) Annu. Rev. Physiol. 62, 321-351[CrossRef][Medline] [Order article via Infotrieve] |
15. | Luo, W., Grupp, I. L., Harrer, J., Ponniah, S., Grupp, G., Duffy, J. J., Doetschman, T., and Kranias, E. G. (1994) Circ. Res. 75, 401-409[Abstract] |
16. |
Chu, G.,
Luo, W.,
Slack, J. P.,
Tilgmann, C.,
Sweet, W. E.,
Spindler, M.,
Saupe, K. W.,
Boivin, G. P.,
Moravec, C. S.,
Matlib, M. A.,
Grupp, I. L.,
Ingwall, J. S.,
and Kranias, E. G.
(1996)
Circ. Res.
79,
1064-1076 |
17. |
Masaki, H.,
Sato, Y.,
Luo, W.,
Kranias, E. G.,
and Yatani, A.
(1997)
Am. J. Physiol.
272,
H606-H612 |
18. | Santana, L. F., Kranias, E. G., and Lederer, W. J. (1997) J. Physiol. 503, 21-29[Abstract] |
19. | Li, L., Chu, G., Kranias, E. G., and Bers, D. M. (1998) Am. J. Physiol. 274, H1335-H1347[Medline] [Order article via Infotrieve] |
20. | Yano, K., and Zarain-Herzberg, A. (1994) Mol. Cell. Biochem. 135, 61-70[Medline] [Order article via Infotrieve] |
21. |
Zhang, L.,
Kelley, J.,
Schmeisser, G.,
Kobayashi, Y. M.,
and Jones, L. R.
(1997)
J. Biol. Chem.
272,
23389-23397 |
22. |
Jones, L. R.,
Suzuki, Y. J.,
Wang, W.,
Kobayashi, Y. M.,
Ramesh, V.,
Franzini-Armstrong, C.,
Cleemann, L.,
and Morad, M.
(1998)
J. Clin. Invest.
101,
1385-1393 |
23. |
Sato, Y.,
Ferguson, D. G.,
Sako, H.,
Dorn, G. W., II,
Kadambi, V. J.,
Yatani, A.,
Hoit, B. D.,
Walsh, R. A.,
and Kranias, E. G.
(1998)
J. Biol. Chem.
273,
28470-28477 |
24. |
Cho, M. C.,
Rapacciuolo, A.,
Koch, W. J.,
Kobayashi, Y.,
Jones, L. R.,
and Rockman, H. A.
(1999)
J. Biol. Chem.
274,
22251-22256 |
25. |
D'Angelo, D. D.,
Sakata, Y.,
Lorenz, J. N.,
Boivin, G. P.,
Walsh, R. A.,
Liggett, S. B.,
and Dorn, G. W., II
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
8121-8126 |
26. |
Sako, H.,
Green, S. A.,
Kranias, E. G.,
and Yatani, A.
(1997)
Am. J. Physiol.
273,
C1666-C1672 |
27. | Mitarai, S., Reed, T. D., and Yatani, A. (2000) Am. J. Physiol. 279, H139-H148 |
28. | Kimura, J., Miyamae, S., and Noma, A. (1987) J. Physiol. (Lond.) 384, 199-222[Abstract] |
29. | Eisner, D. A., Trafford, A. W., Diaz, M. E., Overend, C. L., and O'Neill, S. C. (1998) Cardiovasc. Res. 38, 589-604[CrossRef][Medline] [Order article via Infotrieve] |
30. | Alonso, S., Garner, I., Vandekerckhove, J., and Buckingham, M. (1990) J. Mol. Biol. 211, 727-738[Medline] [Order article via Infotrieve] |
31. | Hewett, T. E., Grupp, I. L., Grupp, G., and Robbins, J. (1994) Circ. Res. 74, 740-746[Abstract] |
32. |
Knollmann, B. C.,
Knollmann-Ritschel, B. E.,
Weissman, N. J.,
Jones, L. R.,
and Morad, M.
(2000)
J. Physiol.
525,
483-498 |
33. | Pan, B. S., Hannon, J. D., Wiedmann, R., Potter, J. D., Kranias, E. G., Shen, Y. T., Johnson, R. G., Jr., and Housmans, P. R. (1999) J. Mol. Cell. Cardiol. 31, 159-166[CrossRef][Medline] [Order article via Infotrieve] |
34. | Schmidt, A. G., Kadambi, V. J., Ball, N., Sato, Y., Walsh, R. A., Kranias, E. G., and Hoit, B. D. (2000) J. Mol. Cell. Cardiol. 32, 1735-1744[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Wang, W.,
Cleemann, L.,
Jones, L. R.,
and Morad, M.
(2000)
J. Physiol. (Lond.)
524,
399-414 |
36. |
Lievremont, J. P.,
Rizzuto, R.,
Hendershot, L.,
and Meldolesi, J.
(1997)
J. Biol. Chem.
272,
30873-30879 |
37. |
Liu, N.,
Fine, R. E.,
Simons, E.,
and Johnson, R. J.
(1994)
J. Biol. Chem.
269,
28635-28639 |
38. |
Pahl, H. L.
(1999)
Physiol. Rev.
79,
683-701 |
39. |
Yoshida, H.,
Haze, K.,
Yanagi, H.,
Yura, T.,
and Mori, K.
(1998)
J. Biol. Chem.
273,
33741-33749 |
40. | Nabors, C. E., and Ball, C. R. (1969) Anat. Rec. 164, 153-161[Medline] [Order article via Infotrieve] |
41. | Eaton, G. J., Custer, R. P., Johnson, F. N., and Stabenow, K. T. (1978) Am. J. Pathol. 90, 173-186[Medline] [Order article via Infotrieve] |
42. |
Tsutsui, H.,
Ishibashi, Y.,
Imanaka-Yoshida, K.,
Yamamoto, S.,
Yoshida, T.,
Sugimachi, M.,
Urabe, Y.,
and Takeshita, A.
(1997)
Am. J. Physiol.
272,
H168-H175 |
43. |
Dorn, G. W., II,
Robbins, J.,
Ball, N.,
and Walsh, R. A.
(1994)
Am. J. Physiol.
267,
H400-H405 |
44. |
Vikstrom, K. L.,
Bohlmeyer, T.,
Factor, S. M.,
and Leinwand, L. A.
(1998)
Circ. Res.
82,
773-778 |
45. | Schwartz, K., de la Bastie, D., Bouveret, P., Oliviero, P., Alonso, S., and Buckingham, M. (1986) Circ. Res. 59, 551-555[Abstract] |
46. | Rizzo, V., Maio, F. D., Campbell, S. V., Tallarico, D., Petretto, F., Lorido, A., Bianchi, A., Goubadia, I., and Carmenini, G. (2000) Am. Heart J. 139, 529-536[CrossRef][Medline] [Order article via Infotrieve] |
47. | Fay, W. P., Taliercio, C. P., Ilstrup, D. M., Tajik, A. J., and Gersh, B. J. (1990) J. Am. Coll. Cardiol. 16, 821-826[Medline] [Order article via Infotrieve] |
48. | Thamilarasan, M., and Klein, A. L. (1999) Am. Heart J. 137, 381-383[Medline] [Order article via Infotrieve] |
49. |
Benjamin, E. J.,
D'Agostino, R. B.,
Belanger, A. J.,
Wolf, P. A.,
and Levy, D.
(1995)
Circulation
92,
835-841 |
50. | Minamisawa, S., Hoshijima, M., Chu, G., Ward, C. A., Frank, K., Gu, Y., Martone, M. E., Wang, Y., Ross, J., Jr., Kranias, E. G., Giles, W. R., and Chien, K. R. (1999) Cell 99, 313-322[Medline] [Order article via Infotrieve] |
51. |
Rockman, H. A.,
Chien, K. R.,
Choi, D. J.,
Iaccarino, G.,
Hunter, J. J.,
Ross, J., Jr.,
Lefkowitz, R. J.,
and Koch, W. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7000-7005 |
52. | Delling, U., Sussman, M. A., and Molkentin, J. D. (2000) Nat. Med. 6, 942[CrossRef] |
53. | Chien, K. R. (2000) Nat. Med. 6, 942-943[CrossRef] |