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Vol. 286, Issue 1, 531-538, July 1998
Institut für Pharmakologie und Toxikologie der
Westfälischen Wilhelms-Universität, Domagkstra
e 12, D-48149 Münster, Germany (B.L., P.B., U.H., U.K., J.K., F.U.M.,
W.S., A.S., U.V., J.N.);
Institut für Pathologie der
Westfälischen Wilhelms-Universität, Domagkstrae 17, D-48149 Münster, Germany (H.A.B.);
Abteilung Allgemeine
Pharmakologie, Universitäts-Krankenhaus Eppendorf,
Martinistrae 52, D-20246 Hamburg, Germany (T.E., E.J., S.L., H.S);
and
Department of Medicine and Krannert Institute of Cardiology,
Indiana University School of Medicine, Indianapolis, Indiana (L.R.J.)
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We studied the influence of prolonged administration of the beta adrenoceptor agonist isoproterenol on contractile parameters and expression of sarcoplasmic reticulum (SR) Ca++-ATPase and phospholamban, genes important for Ca++ uptake into the SR. Isoproterenol (Iso), 0.9% NaCl (Ctr), propranolol (Prop) or Iso plus Prop were administered to rats by subcutaneous infusion with osmotic minipumps for 1, 2, 3, 4, 8, 13 and 26 days, respectively. The positive inotropic effect of Iso was impaired in rats pretreated with Iso in vivo. Iso pretreatment shortened time to peak tension (TPT) by 28%, time of relaxation (RT) by 27% and total contraction time (TCT) by 27% compared with the appropriate controls (day 2). The shortening of time-dependent contractile indices started after 1 day of Iso pretreatment, reached a maximum after 2 days and remained reduced for 4 days. Longer treatment by Iso failed to affect time parameters, whereas the positive inotropic effect of Iso added to the isolated muscles persisted. The shortened contractile time parameters were accompanied by diminished mRNA and protein expression of phospholamban (PLB) and SR-Ca++-ATPase (SERCA). The mRNA levels for PLB and SERCA were maximally reduced by 31 ± 1.3% and 41 ± 1.4% in the Iso-pretreated group (2 days) respectively. The reduced mRNA levels were accompanied by reduced levels of the corresponding proteins. It is concluded that altered levels of PLB and SERCA probably account for the noted changes in contractile time parameters in the mammalian heart.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cardiac
hypertrophy is an important step leading to heart failure in patients.
Heart failure is accompanied by prolonged relaxation, which is deemed
detrimental to cardiac performance. Relaxation is mediated by the
removal of Ca++ from the sarcoplasma through the
combined action of Na+/Ca++
exchanger in the sarcolemma and SERCA 2a. The physiological inhibitor of SERCA is PLB, an intrinsic protein of the SR. PLB lowers the affinity of SERCA for Ca++ at low
Ca++ concentrations (Odermatt et al.,
1996
). Indeed, overexpression of PLB prolongs the duration of
contraction in the heart of transgenic mice (Kadambi et al.,
1996) and ablation of the PLB gene shortens contraction duration (Luo
et al., 1994). To elucidate a possible causal relationship
between hypertrophy and altered relaxation several animal models have
been used. For instance, pressure overload or thyroid hormone induced
hypertrophy, changed relaxation and altered expression of SR proteins
(Lompre et al., 1989; Nagai et al., 1989; De la
Bastie et al., 1990; Kiss et al., 1994; Matsui et al., 1995).
We focused on changes in cardiac function after stimulation of
beta adrenoceptors in an in vivo model (Mende
et al., 1992). Chronic beta adrenoceptor
stimulation leads to cardiac hypertrophy in this model. This is
accompanied by biochemical and mechanical alterations. The density of
beta-1 and beta-2 adrenoceptors was reduced, the
expression of the alpha subunit of inhibitory G-proteins was
increased and the positive inotropic effect of beta
adrenoceptor stimulation was attenuated (Chang et al., 1982;
Eschenhagen et al., 1992). These alterations were in part
reversible by beta adrenoceptor blockade (Eschenhagen
et al., 1992). One could hypothesize that this model might
mimic important alterations that occur in progressive human heart
failure, which is characterized by hypertrophy and subsequent decline
in cardiac function leading ultimately to terminal heart failure
accompanied by increasing adrenergic stimulation. There is strong
negative correlation between catecholamine levels and life expectancy.
Moreover, we have shown that after chronic beta adrenoceptor
stimulation, the relaxation of papillary muscles was enhanced and the
expression of PLB and SERCA on protein levels was reduced (Stein
et al., 1996). This was in part compensated by reduced phosphorylation of PLB (Stein et al., 1996). Moreover, the
expression of mRNA for SERCA was reversibly altered in this model
(Boluyt et al., 1995). However, the time course of changes
in contractile parameters and the expression of SR genes that might
underlie these changes have not been demonstrated. It is unknown
whether chronic beta adrenoceptor stimulation leads to
transient or persistent shortening of cardiac relaxation and whether
these alterations are blunted by beta adrenoceptor
antagonists. If a causal relationship exists, it is expected that
altered relaxation is preceded by altered expression of mRNA and
protein levels for PLB and/or SERCA. In addition, it has not been
studied whether Ca++ uptake is actually altered
in this model.
Thus, we addressed the following questions. Is the enhanced relaxation in hypertrophy due to chronic beta adrenoceptor stimulation transient or persistent? Is it preceded or followed by a alterations in SERCA and/or PLB expression? Are alterations in SERCA and PLB expression accompanied by changes in Ca++ uptake? What is the temporal relationship between mRNA and protein levels of PLB and SERCA?
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals.
Male Wistar rats (270-300 g) were treated with
subcutaneous infusions by osmotic minipumps (Alzet osmotic pump type
ML2; Alza, Palo Alto, CA) as described before (Eschenhagen et
al., 1992; Mende et al., 1992). Minipumps were
implanted subcutaneously in the neck of the animals under short-term
ether anesthesia. Mean rate of infusion was 5 µl/hr. Rats were
treated 1, 2, 3, 4, 8, 13 or 26 days with either 0.9% NaCl as control,
(±)-Iso HCl alone (2.4 mg/kg/day dissolved in 0.002 M HCl;
Boehringer-Ingelheim, Ingelheim, Germany), (±)-Prop HCl alone (29.7 mg/kg/day; Sigma Chemical, St. Louis, MO) or a combination (Iso and
Prop). The animals had free access to food and tap water. For the 26 days' treatment, minipumps were removed after 13 days under short-term ether anesthesia, and a new freshly filled pump was implanted. Because
drug delivery was stable for 14 days, pumps were reimplanted into
another rat when the total time of drug delivery was <13 days. The
rats were killed by a blow to the neck and bleeding from the carotid
arteries. Hearts were rapidly removed and exsanguinated in ice-cold
0.9% NaCl. Papillary muscles were used for contraction experiments,
and ventricles were frozen immediately in liquid nitrogen for further
analysis.
Contraction experiments.
The experiments were performed on
electrically driven (frequency, 1 Hz; duration, 5 msec; intensity, 20%
greater than threshold) papillary muscles from the left ventricles of
the pretreated rats (Eschenhagen et al., 1992; Mende
et al., 1992). The preparations were isolated, mounted and
suspended individually in glass tissue chambers for recording isometric
contractions. The bathing solution (10 ml) was a modified Tyrode's
solution containing (mM) NaCl 119.8, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaH2PO4 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28 and glucose 5.0. It was continuously gassed with
95% O2 and 5% CO2 and
maintained at 35°C. Force of contraction was measured with an
inductive force transducer. Concentration-response curves were obtained
cumulatively and expressed as increases in force of contraction (mN).
Immunohistochemical determinations.
Frozen cross sections (5 µm) of each heart were mounted on Silan-coated glass slides and fixed
in 4°C cold acetone for 90 sec. For histological examination, the
slides were stained with hematoxylin and eosin; for immunohistological
examination, the slides were incubated with the monoclonal antibody A1
against PLB (Drago and Colyer, 1994) in a dilution of 1:200 in 0.6%
bovine serum albumin, followed by a rabbit anti-mouse bridging antibody (1:30 in phosphate-buffered solution; 30 min at room temperature; Dako
Diagnostika, Hamburg, Germany) and a polyclonal mouse APAAP complex
(1:100 in RPMI; 60 min at room temperature; Dianova, Hamburg, Germany).
The enzyme reaction was developed for 25 min at room temperature in a
freshly prepared new fuchsin solution containing naphthol-bisphosphate
and levamisole. Finally, the sections were counterstained with
hematoxylin and mounted with Kayser's glycerin gelatin. All slides
were stained in one preparation. Omission of the primary antibody
served as negative control.
Ca++ uptake measurement.
Frozen
hearts were homogenized in 250 mM sucrose, 10 µM cantharidin and 30 mM histidine (pH 7.0). Cantharidin was added to inhibit protein
phosphatases. Cantharidin (10 µM) inhibits completely type 1 and type
2A phosphatases (Neumann et al., 1995).
Ca++ uptake in homogenates was measured by the
microfiltration technique (Martonosi and Feretos, 1964). The reaction
buffer contained 50 mM MOPS ([N-morpholino]propanesulfonic acid; pH
7.0), 3 mM MgCl2, 100 mM KCl, 5 mM
NaN3, 10 mM potassium oxalate, 0.5 mM EGTA, 10 µM cantharidin and different CaCl2
concentrations to give pCa values of 7.49. Free calcium concentrations
were calculated by the method of Bers (1994).
Ca++ uptake was measured by preincubation of
homogenates with antibody for 20 min on ice. Ca++
uptake could be stimulated by preincubation of homogenates with an
anti-PLB antibody (2D12) for 20 min on ice. Ca++
uptake was initiated by the addition of 3 mM ATP and then performed at
37°C. Aliquots of 100 µl were filtered at various time points on
0.22-µm filters (GS type, Millipore) and washed twice with 5 ml of
150 mM NaCl. Quantification of
45Ca++ was measured by
scintillation counting.
Total RNA preparation.
Total RNA was isolated from
ventricular tissue (frozen immediately after death) according to the
protocol of Chomczynski and Sacchi (1987). RNA concentration was
determined by absorbance at 260 nm in triplicate. RNA was denatured
with 42% formamide and 5.8% formaldehyde at 95°C for 2 min and
size-fractionated by electrophoresis in 1% agarose gels containing 0.5 µg/ml ethidium bromide (Fluka Chemie, Buchs, Switzerland). RNA was
transferred to Hybond N nylon membranes overnight (Amersham,
Braunschweig, Germany) by Northern blot capillary transfer using 20×
SSC (3 M NaCl and 0.3 M sodium citrate, pH 7.0) as the transfer medium. Transfer was controlled on an ultraviolet transilluminator.
cDNA probes.
The plasmids (pBS) with cDNA inserts for rat
SERCA 2a and rat PLB were kindly provided from Dr. K. R. Boheler
(Lompré et al., 1989). Plasmid (pGEM-2) with cDNA insert
for rat Gs
was a kind gift from Dr.
R. R. Reed. The plasmids were transformed into Escherichia
coli, and positive clones were picked and grown in rich medium.
Plasmids were obtained by large-scale preparation. Inserts were
isolated by digestion with EcoRI for SERCA 2a, PLB and
Gsalpha. The cDNA inserts were
purified from 1% agarose gels. Sizes were ~2000 bp for SERCA 2a,
~1100 bp for PLB and ~1100 bp for
Gsalpha. The cDNA probes were labeled
(Mega-Prime kit; Amersham Buchler, Braunschweig, Germany) with
[32P]dCTP (3.000 Ci/mmol, New England
Nuclear-Dupont, Bad Homburg, Germany) to a specific activity of 3.5 to
8.0 × 108 dpm/µg. Unbound radioactivity
was separated by gel filtration with Sephadex G-50 DNA grade (Pharmacia
Fine Chemicals, Uppsala, Sweden).
Hybridization procedure.
Hybond N nylon membranes were
prehybridized for 2 to 6 hr at 42°C in a prehybridization solution
containing 50% formamide, 5× Denhardt's solution (1 mg/ml Ficoll,
polyvinylpyrrolidone and bovine serum albumin), 0.9 M NaCl, 0.06 M
NaH2PO4, 0.006 M EDTA, 0.1% SDS and 400 µg/ml tRNA from yeast. The
32P-labeled probes were added to the
prehybridization solution in a concentration of 0.5 to 1 × 106 dpm/ml. Hybridization of the membrane was
performed at 42°C for 16 to 20 hr. The membranes were washed twice in
2× SSC, 0.1% SDS at room temperature followed by 15 min washing in
2× SSC, 0.1% SDS at 65°C. The blots were washed three times to a
final stringency in 0.2× SSC, 0.1% SDS at 65°C. Wet blots were
sealed in plastic wrap and exposed using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA) for 24 hr, followed by a subsequent exposure
to X-ray films (X-OMAT AR; Kodak) for 2 to 5 days at 
80°C by using
intensifier screens. For further hybridizations with other radiolabeled
probes, the blot membrane was washed with boiling in 0.1% SDS. After a control exposure in the PhosphorImager to assess loss of label, membranes were then used for subsequent hybridizations.
Quantification of specific mRNA.
Hybridization intensity of
autoradiographic signals on Northern blots were measured quantitatively
by two-dimensional densitometry (ImageQuant; Molecular Dynamics). Then,
20 µg total RNA was used for quantitative analysis. Northern blots
were hybridized subsequently against SERCA 2a, PLB and
Gsalpha as described above. Gsalpha was used to correct the amount of
total RNA bound to the membrane (Eschenhagen et al., 1992).
Furthermore, an external standard (microscales; Amersham) was used to
normalize methodical variation. Total RNA of several rat hearts was
isolated, and 20 µg was used for each Northern blot as an internal
standard. The final result was obtained by normalizing to
Gsalpha, external standard and internal
standard.
Preparation of homogenate.
Next, 50 mg of powdered heart
tissue were homogenized at 4°C three times for 30 sec each with a
Polytron PT-1O (Kinematica, Luzern, Switzerland) in 300 µl of 10 mM
NaHCO3. Then, 600 µl of 20% SDS was added
(Movsesian et al., 1994; Linck et al., 1996). Mixtures were incubated at 25°C for 20 min before centrifugation. Supernatants were collected and assayed for protein, according to the
method of Lowry (1951) after trichloroacetic acid precipitation.
SDS-PAGE and autoradiography.
SDS extracts made as described
above were thawed, and additional SDS buffer made according to Laemmli
(1970) was added. Samples were heat-treated for 10 min at 95°C to
convert the high-molecular-weight form of PLB into the
low-molecular-weight form. Next, 50 µg of homogenate sample protein
were loaded per lane. These amounts were in the linear range for PLB. A
similar linearity was obtained for SERCA in homogenates (data not
shown) as has been published before (Linck et al., 1996).
using 10%
acrylamide separating gels. After gel electrophoresis, separated
proteins were transferred to nitrocellulose membranes as described
(Neumann et al., 1991). Nitrocellulose sheets were incubated
with monoclonal antibody A1 raised against PLB (Drago and Colyer, 1994)
and monoclonal antibody AB 465 to SERCA (MacDougall et al.,
1991). Protein binding antibodies were visualized using [125I]protein A. Radioactive bands identified
by autoradiography were excised from the nitrocellulose sheet, and
bound radioactivity was quantified by 
counting. Background counts,
which were <15% of total counts for each band, were subtracted from
all measurements.
Plasma catecholamine and Iso levels were determined using
high-performance liquid chromatography with electrochemical detection. Blood was collected from the carotid arteries and centrifuged at
14,000 × g for 10 min at 4°C. The supernatant was
stored at 80°C until use. Then, 200 µl of serum and 100 µl of
10 pg/µl dihydroxybenzylamine as internal standard were transferred
into sample cartridges (Recipe, Munich, Germany) containing 1 ml of 2 M
Tris/2% EDTA buffer (pH 8.7) and 20 mg of alumina. The cartridges were
shaken upside down for 10 min for adsorption of the catecholamines onto
the alumina. After that, the buffer was removed, and the residue was
washed three times, using 1 ml of a 0.2% Tris buffer, pH 8.6. For
desorption of catecholamines from the alumina, 125 µl of glacial
acetic acid was added, and after vigorous shaking, the supernatant
(containing the catecholamines) was centrifuged into the sample vial.
Nest, 40 µl was injected into the high-performance liquid
chromatography system, which consisted of an LC Workstation Class LC10
(Shimadzu, Kyoto, Japan) with an SIL-10A autoinjector, an LC-10AT
liquid chromatograph and a Waters model 410 electrochemical detector
(Waters Millipore, Eschborn, Germany) with a glassy carbon working
electrode set at a potential of 0.7 V vs. Ag/AgCl and software provided by the manufacturer. Chromatographic separation was
performed on a reversed-phase C18 column (18 µm, 150 mm × 4.6 mm I.D.; Recipe) at ambient temperature. The mobile phase consisted of
50 mM citric acid, 27 mM sodium dihydrogen phosphate buffer, 0.27 mM
sodium EDTA and 1.5 mM sodium 1-octane-sulfonate (Sigma, Munich,
Germany) dissolved in distilled water/methanol (90:10% v/v). Flow rate
was 1 ml/min.
Data analysis. Data shown are mean ± S.E.M. Statistical significance was estimated with Student's t test for unpaired observations. P < 0.05 was considered significant. In addition, one-way analysis of variance was performed as indicated.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Iso plasma levels amounted to 4.49 ± 1.55 ng/ml (2 days Iso
alone, n = 9), 3.70 ± 1.16 (2 days Iso plus Prop,
n = 9), 2.77 ± 0.66 (8 days Iso alone,
n = 11) and 3.55 ± 0.92 (26 days Iso alone,
n = 11). This indicates the appropriate function of
osmotic minipumps. Moreover, Iso led to cardiac hypertrophy starting at day 1 and persisted throughout the whole period of investigation (fig.
1) as described previously (Mende
et al., 1992; Eschenhagen et al., 1992;
Müller et al., 1994).
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As reported (Mende et al., 1992), the positive inotropic
effect of Iso was persistently attenuated in the Iso-pretreated rats in
comparison with NaCl pretreated animals (day 1 to day 26, data not
shown). Additionally applied Prop abolished the effect of Iso
pretreatment on heart weight and hypertropy (table
1). The first new finding of the present
work is the transient alteration in time parameters. TCT (fig.
2A), TPT (fig. 2B) and RT (fig. 2C) were
shortened after Iso pretreatment for 1, 2, 3 and 4 days in comparison
with NaCl-pretreated animals. Time parameters were maximally reduced
after 2 days of Iso pretreatment with a reduction of TCT from 136 ± 1.7 to 99 ± 3.4 msec (27%), of TPT from 57 ± 1.2 to
41 ± 1.1 msec (28%) and of RT from 79 ± 1.5 to 58 ± 2.8 msec (27%). In contrast to the persistently blunted inotropy, the
effect on time parameter was thus transient. The Iso-induced reduction
of contraction time parameters was blocked in the presence of Prop,
demonstrating beta adrenergic-induced effects (day 2, table
1).
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Next, we studied whether Ca++ uptake was altered. The Ca++ uptake was time and Ca++ dependent (data not shown). At linear conditions (pCa 7.5), the maximum rate of Ca++ uptake amounted to 0.44 ± 0.10 and 0.98 ± 0.14 (nmol/mg of protein/5 min) in NaCl- and Iso-treated (2 days) ventricles, respectively (fig. 3). This corresponds to an increase by 123% in the Iso-pretreated rats vs. control (P < 0.05, n = 6).
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To test the hypothesis that alterations in SERCA and PLB, proteins
important for Ca++ uptake into the SR, are
responsible for the altered contraction time parameters, we determined
the expression on both mRNA and protein levels. A typical Northern blot
is seen in figure 4. Iso pretreatment
induced a reduction in SERCA mRNA level (figs. 4 and
5A) at days 1 and 2 of Iso pretreatment
by 35% and 41% in comparison to NaCl pretreatment, respectively. The
maximal decrease occurred after 2 days of Iso pretreatment and the
effect vanished after 3 days of treatment and longer. Iso pretreatment
for 2 days decreased SERCA mRNA level from 1.1 ± 0.06 (NaCl)
density units to 0.64 ± 0.05 (Iso) density units (by 41%). When
Iso and Prop were simultaneously applied, SERCA mRNA level amounted to
1.0 ± 0.04 (n = 5). Thus, the reduction of SERCA
mRNA levels were antagonized by simultaneously administered Prop. Prop
alone had no effect on SERCA mRNA levels (table 1). Iso pretreatment
induced a reduction in SERCA protein level from day 1 to day 4 compared with NaCl pretreatment (figs. 4 and 5B). The protein decreased by 19%
after 1 day, by 21% after 2 days, by 19% after 3 days and by 24%
after 4 days of Iso pretreatment. The Iso-induced effect vanished after
treatment of 
8 days. The effect was blocked by simultaneously
administered Prop, whereas Prop alone did not affect protein expression
(table 1).
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SERCA-mediated Ca++ uptake into the SR is
regulated by PLB. Therefore, we determined also the PLB mRNA and
protein expression. The hybridization with the PLB probe revealed two
mRNAs for PLB at 3.3 and 1.3 kb, although both mRNAs encode for the
same protein (fig. 4). The different mRNAs for the same protein could
be caused by distinct polyadenylation sites (Toyofuku and Zak, 1991).
These polyadenylation sites might be important as a protection against endonucleases. Thus, it was conceivable that the PLB mRNA expression were differently regulated between both mRNAs and therefore resulted in
differential alterations. Hence, we quantified both PLB mRNAs either
together or each band separately. However, no differences were apparent
(data not shown) and the sum of both transcripts is plotted in figure
6A. The PLB mRNA levels were reduced in
1- and 2-day Iso-pretreated rats. The mRNA levels decreased from 1.88 ± 0.15 to 1.33 ± 0.15 (29%) density units after 1 day
and from 1.99 ± 0.19 to 1.38 ± 0.06 (31%) density units
after 2 days, respectively. A longer Iso pretreatment of 3 days and
more was not accompanied by altered PLB mRNA expression. Moreover, a
2-day pretreatment with Iso plus Prop abolished the Iso-induced effect (table 1). Furthermore, the corresponding PLB protein levels were
decreased after 1 to 4 days of Iso pretreatment in comparison with NaCl
pretreatment (fig. 6B). The decrease started after 1 day reached a
maximum after 2 days and remained reduced for 4 days. PLB protein level
was maximally reduced from 8839 ± 312 to 5182 ± 367 cpm
(41%) after 2 days of Iso pretreatment in comparison to
NaCl-pretreated rats. The Iso effect on PLB protein levels was
antagonized by simultaneously administered Prop (table 1).
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Furthermore, the tissue distribution of PLB was investigated immunohistochemically (fig. 7). The control hearts (A) showed a strong specific cytoplasmic staining for PLB in cardiomyocytes as well as in smooth muscle cells of the media of coronary arteries, whereas the interstitial cells were negative. This is consistent with the muscle-specific expression of PLB and supports the specificity of the antibody used in the present study. Moreover, the Iso-pretreated rat hearts (B) exhibited a weaker cytoplasmic staining for PLB in cardiomyocytes compared with control (A), whereas the smooth muscle cells of the arteries revealed the same staining intensity regardless of treatment. This suggests that the regulation of PLB expression occurs mainly in cardiomyocytes. Sections from Iso-pretreated hearts displayed interstitial edema.
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The activity of SERCA is regulated by PLB. Thus, not only the total amount of SERCA and PLB levels is important but also the ratio of expression of the two proteins. The ratio at protein level (fig. 8) decreased after 2 and 3 days of Iso pretreatment from 2.52 ± 0.14 to 1.69 ± 0.16 (33%) after 2 days and from 2.56 ± 0.13 to 2.08 ± 0.15 (18%) after 3 days of Iso pretreatment. At mRNA level, the ratio remained unchanged (table 1).
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In the present study, we demonstrate that chronic beta adrenoceptor stimulation in vivo leads to persistent hypertrophy but to transiently enhanced relaxation in the mammalian heart. We studied the altered relaxation in ventricular preparations (fig. 2) but similarly shortened relaxation was also noted in electrically driven left atria (2 days, data not shown). It was important to show that hastened relaxation was accompanied by enhanced Ca++ uptake in this model. This supports the hypothesis that alterations in SERCA and/or PLB may underlie the altered relaxation observed.
Alterations in SERCA and PLB levels in various model systems.
Alterations of SERCA and PLB expression have been noted in postnatal
development, with drug treatment and mechanical intervention. The
expression of only one protein or both was altered in previous publications. In postnatal development, only SERCA was increased but
PLB remained unchanged (for review see Lompre et al., 1994). A discoordinate regulation has been shown for hormonal stimulation. For
instance, thyroid hormone treatment led to a decrease of PLB (mRNA and
protein) and an increase in SERCA (mRNA and protein) expression.
Concomitantly, the Ca++ uptake was increased and
time of relaxation was shortened (Rohrer and Dillmann, 1988; Nagai
et al., 1989; Arai et al., 1991; Kiss et
al., 1994; for extensive review, Lompre et al., 1994).
Drug-induced hypothyroidism increased the ratio of PLB to SERCA (mRNA
and protein level), decreased Ca++ uptake and
prolonged relaxation (Nagai et al., 1989; Kiss et al., 1994). These studies tend to indicate that the ratio of PLB to SERCA is inversely related to Ca++ uptake by
the SR, regardless which intervention and species are studied.
Moreover, we have presented evidence that PLB is dephosphorylated after
chronic beta adrenergic stimulation (Stein et
al., 1996). This would at least in part compensate for the reduced
level of PLB because only unphosphorylated PLB inhibits SERCA activity. Finally, there are several examples for coordinate reduction of PLB and
SERCA levels. Cardiac hypertrophy due to pressure overload (pulmonary
artery banding) was accompanied by reduced levels of PLB (mRNA) and
SERCA (mRNA and protein) and reduced Ca++ uptake
(Matsui et al., 1995). PLB was not measured on protein level, and therefore the ratio of PLB to SERCA was not given. In
another model of pressure hypertrophy (aortic banding leading to
pulmonary congestion), both PLB and SERCA were reduced at the protein
level. The relaxation was impaired, Ca++ uptake
was reduced, and the ratio of PLB to SERCA was diminished (Kiss
et al., 1995). Of note, in a subgroup of animals with
cardiac hypertrophy without heart failure, no changes in PLB or SERCA were noted. Thus, the findings by Kiss et al. (1995)
obtained in a completely different model are in agreement with the
present finding.
Time course studies.
To address the question of whether
chronic beta adrenergic stimulation leads to transient or
persistent alterations in relaxation and PLB and/or SERCA gene
expression, a complete time course of beta adrenergic
stimulation has been studied in the present work. Gene expression in
eukaryotes is usually primarily regulated at the transcriptional level.
However, there are a number of exceptions (Kozak, 1991), even for PLB
and SERCA. A physiological intervention (electrically stimulation of
fast twitch skeletal muscle) led to enhanced PLB and SERCA gene
expression (Hu et al., 1995). However, the time courses were
dissimilar. First, the mRNA for PLB increased followed by an enhanced
PLB protein expression. In contrast, the increase in SERCA protein
level preceded the increase in SERCA mRNA level. The authors concluded
that PLB and SERCA expressions were regulated using two levels of
control (Hu et al., 1995). PLB expression was suggested to
be under transcriptional control, whereas SERCA was assumed to be under
translational control. Moreover, it was speculated that PLB stimulates
the translation of the SERCA transcript (Hu et al., 1995).
However, transcription rates were not directly measured by Hu et
al. (1995). Moreover, SERCA mRNA alterations are not always
faithfully followed by SERCA protein levels. After unloading of rat
soleus muscle SERCA mRNA increased, whereas SERCA protein was unchanged
indicating that SERCA can be under translational control (Schulte
et al., 1993). The parallel decrease in mRNA and protein
levels for SERCA and PLB (day 1 and 2) in the present study might
suggest transcriptional control. The persistent decline in protein
levels for SERCA and PLB at times (3 and 4 days) where mRNA for SERCA
has reached control values can have various reasons. Based on work in
skeletal muscle, one might speculate on a nontranscriptional control.
The quotient of PLB to SERCA mRNA stayed constant throughout the time
investigated, indicating a parallel regulation of these levels. The
easiest explanation would be a parallel change in the transcriptional activity of these genes. Promoter analysis will be required to more
rigorously address these questions.
).
Finally, it can been asked whether the changes in PLB are generalized
or occur only in ventricles. Actually, at 2 days of beta
adrenergic stimulation PLB protein level was reduced in left and right
atria (data not shown). This indicates a general underlying mechanism
in atria and ventricles.
In summary, we report here a transient concordant reduction in PLB and
SERCA expression at the protein level. Their altered ratio is suggested
to underlie at least in part the transient shortened time of relaxation
in chronic beta adrenergic stimulation. Thus, chronic
beta adrenoceptor stimulation in vivo is
accompanied by complex transient and persistent alterations in cardiac
function and gene expression.
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Footnotes |
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Accepted for publication March 23, 1998.
Received for publication September 18, 1997.
1 This work was supported by the Deutsche Forschungsgemeinschaft, the Deutsche Herzstiftung, the Deutsche Gesellschaft für Herz- und Kreislaufforschung and the IKF B1.
Send reprint requests to: Dr. Bettina Linck, Institut
für Pharmakologie und Toxikologie der Westfälischen
Wilhelms-Universität, Domagkstrae 12, D-48149 Münster,
Germany.
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Abbreviations |
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Iso, ()-isoproterenol;
TCT, total contraction
time;
TPT, time to peak tension;
RT, time of relaxation;
PLB, phospholamban, Prop, propranolol;
SDS, sodium dodecyl sulfate;
PAGE, polyacrylamide gel electrophoresis, SERCA, sarcoplasmic reticulum
Ca++-ATPase.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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-adrenoceptor-mediated up-regulation of Gi
and Go mRNA level and pertussis toxin-sensitive guanine nucleotide-binding proteins in rat heart.
Mol Pharmacol
42:
773-783[Abstract].- adrenergic stimulation of phospholamban phosphorylation and Ca2+ transport in guinea pig ventricles.
J Biol Chem
260:
13122-13129-adrenergic stimulation.
Circ Res
75:
401-409-adrenergic stimulation leads to biphasic regulation of G1-2 gene transcriptional activity in rat heart.
J Mol Cell Cardiol
26:
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) or Iso (
) (2.4 mg/kg/day). Heart/body weight ratio is the
biventricular wet weight (mg) per body weight (g) at the day of death.
Numbers of animals are shown in brackets. *, Significant differences
vs. control (0). Crosses depict significant differences
vs. NaCl pretreatment.
