(Circulation. 1995;92:3220-3228.)
© 1995 American Heart Association, Inc.
Articles |
Unchanged Protein Levels of SERCA II and Phospholamban but Reduced Ca2+ Uptake and Ca2+-ATPase Activity of Cardiac Sarcoplasmic Reticulum From Dilated Cardiomyopathy Patients Compared With Patients With Nonfailing Hearts
From the Universität zu Köln, Medizinische Klinik III, Joseph-Stelzmannstr 9, D-50924 Köln, and the Max Delbrück Zentrum für Molekulare Medizin, Robert-Rössle Str 10, D-13125 Berlin-Buch (P.K., E.-G.K.), Germany.
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Abstract |
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Background The aim of the present study was to investigate whether Ca2+ uptake into the sarcoplasmic reticulum (SR) is altered in failing human myocardium resulting from dilated cardiomyopathy.
Methods and Results Ca2+-ATPase (SERCA II) activity and Ca2+-dependent 45Ca2+ uptake (oxalate supported, steady state) in isolated vesicles from the SR (VSR) and in crude membrane preparations (CSR) (free Ca2+, 0.01 to 100 µmol/L) from nonfailing (donor hearts, n=13) and terminally failing (heart transplants, dilated cardiomyopathy, n=17) human myocardium were studied. In the same hearts, protein levels (Western blot analysis) and mRNA levels (Northern blot analysis) of SERCA II and phospholamban were measured. Increasing concentrations of Ca2+ were followed by an increased Ca2+-ATPase activity and Ca2+ uptake. Ca2+ uptake activity and Ca2+-ATPase activity in CSR preparations from failing myocardium were significantly reduced compared with nonfailing hearts (Ca2+-ATPase, 163±8 and 125±7 nmol ATP/mg protein per minute for nonfailing tissue and failing tissue in New York Heart Association [NYHA] class IV, respectively; Ca2+ uptake, 7.1±0.8 and 3.5±0.3 nmol/mg protein per minute in CSR from nonfailing and NYHA class IV hearts, respectively; P<.05). In contrast, no significant difference was measured in VSR. In the same preparations (CSR and VSR), both SERCA II and phospholamban levels (Western blot technique with monoclonal antibodies) were unchanged in failing compared with nonfailing tissue. mRNA expression relative to GAPDH mRNA for SERCA IIa and for phospholamban was significantly reduced in failing human myocardium (P<.05).
Conclusions These findings provide evidence that in failing human myocardium caused by dilated cardiomyopathy, protein levels of SERCA II and phospholamban are unchanged even though mRNA levels for SERCA II and phospholamban and the SERCA II function are reduced compared with nonfailing myocardium.
Key Words: calcium • contractility • heart failure • sarcoplasmic reticulum
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Introduction |
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Elevation of the extracellular concentration of Ca2+ has been reported by various groups to stimulate the force of contraction in failing and nonfailing human tissue to the same degree.1 2 3 4 Thus, the contractile apparatus in terminally failing human myocardium was suggested to maximally increase force development even in failing hearts. However, inotropic stimulation by cAMP-dependent and cAMP-independent mechanisms results in an altered diastolic relaxation in papillary muscle strip preparations from patients with DCM.5 During inotropic stimulation, abnormalities in diastolic rather than in systolic contraction become evident. Furthermore, several investigators demonstrated an altered force-frequency relation in failing human myocardium.5 6 7 8 In these studies, force of contraction increased only in nonfailing tissue but not in diseased human hearts. Similar findings have been reported from in vivo measurements after rapid atrial pacing in patients with heart failure.9 These alterations in contraction coupling have been suggested to be due to an altered [Ca2+]i handling10 11 12 ; ie, the positive force-frequency relation has been related to changes in Ca2+ content of SR.12 Consistently, differences in [Ca2+]i handling have been reported in isolated cardiac myocytes from patients with DCM compared with control cells.13 Similar findings have been reported after simultaneous measurement of force of contraction and [Ca2+]i signal by use of the chemiluminescent aequorin in intact muscle preparations.1 2 7 If in failing human myocardium a reduced Ca2+ loading of the SR is present, then diastolic relaxation and the frequency-dependent increase in force development will be altered. A reduction of the SR Ca2+ uptake may lead to a slower diastolic Ca2+ decay and a reduced SR Ca2+ content, which in turn would cause less available Ca2+ content to be released during the depolarization of the next beat. Measurements of Ca2+ uptake by SR in VSR prepared from nonfailing human hearts and from hearts with DCM revealed similar values.14 However, these findings contrast measurements in right ventricular biopsies from humans.15 One problem in the interpretation of these data is that they are carried out in different origins of human tissue and under different preparation procedures (CSR versus VSR).14 15 In addition, the SERCA II mRNA in the myocardium from patients with heart failure has been reported to be reduced compared with nonfailing control myocardium.16 17 18 The authors of these studies16 17 18 concluded that the reduced mRNA levels play an important role in alterations of Ca2+ movements and myocardial relaxation reported in human heart failure.14 However, there may be a discrepancy between mRNA levels and protein expression or function. Therefore, it is still a matter of debate whether [Ca2+]i uptake into the SR and SERCA II activity are altered in human DCM, possibly because of a reduced amount of SERCA II expression. Consequently, the present study was aimed at investigating Ca2+ uptake and SERCA II activity in CSR and VSR to overcome possible influences of the isolation procedure. In the same preparations, protein expression of SERCA II and phospholamban was measured with monoclonal antibodies. Northern blot analyses were performed to compare protein expression and the amount of encoding mRNA for SERCA II and phospholamban. To characterize the human tissue studied, the force-frequency relation was performed in left ventricular papillary muscle strips from the same hearts.
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Human Myocardium
Electrically Driven Human Papillary Muscle Strips
Myocardium from terminally failing human hearts (left ventricular myocardium) was obtained from patients after cardiectomy during cardiac transplantation (10 men, 7 women; age, 44.9±5.2 years; range, 17 to 62 years; left ventricular end-diastolic volume, 270±24 mL; ejection fraction, 27±4%). The preoperative diagnosis was DCM in all patients. All patients had been classified as being in New York Heart Association class IV. The pretreatment of the patients consisted of diuretics, nitrates, and angiotensin-converting enzyme inhibitors. None of the patients had received Ca2+ channel antagonists within 7 days of surgery or ß-adrenoceptor agonists with 48 hours of surgery. Drugs used for general anesthesia were flunitrazepam, fentanyl, and pancuronium bromide with isoflurane. Cardiac surgery was performed on cardiopulmonary bypass with cardioplegic arrest during hypothermia. Patients gave written informed consent before surgery. Nonfailing human myocardium was obtained from 13 donors (10 men, 3 women; age, 32±11 years; normal ejection fraction by echocardiographic examination; absence of cardiovascular medication in patient history; no inotropic support with isoprenaline or noradrenaline; patients were on dopamine) who were brain dead as a result of traumatic injury. These nonfailing hearts could not be used for transplantation for technical reasons (eg, death of the recipient, logistic problems, suspected chest trauma seen after explantation, and fever shortly before explantation). To identify these hearts as controls, the clinical situation before death was considered. Data showing that the heart was useful as a donor were given to the attending cardiologist and the cardiac surgeon explanting the heart. There was no evidence of left ventricular dysfunction by echocardiography. The number of ß-adrenoceptors also was measured. It has been demonstrated that the number of ß-adrenoceptors is decreased in failing human myocardium. Also, the maximal increase in force of contraction after stimulation with isoprenaline has been examined. Histological examination was performed to identify myocardial disease. The failing human myocardium exerted typical histomorphological alterations as described for DCM. The cardioplegic solution used was a modified Bretschneider solution containing (in mmol/L) NaCl 15, KCl 10, MgCl2 4, histidine HCl 180, tryptophan 2, mannitol 30, and potassium dihydrogen oxoglutarate 1.
Immediately after excision, the muscle strips were placed in ice-cold preaerated Tyrode's solution (for composition, see below) and delivered to the laboratory within 10 minutes. From each native myocardial tissue sample, papillary muscle strips were prepared (1 to 2 mm wide and 8 to 10 mm long), with muscle fibers running parallel to the length of the strips. Connective tissue if visible was carefully trimmed away, and areas of dense necrosis or fibrosis were avoided. The muscles were suspended in an organ bath (75 mL) maintained at 37°C containing a modified Tyrode's solution of the following composition (in mmol/L): NaCl 119.8, KCl 5.4, MgCl2 1.05, CaCl2 1.8, NaHCO3 22.6, NaH2PO4 0.42, glucose 5.05, ascorbic acid 0.28, and Na2-EDTA 0.05. The bathing solution was aerated continuously with 95% O2 and 5% CO2. The muscles were stimulated by two platinum electrodes with field stimulation from a Grass S 88 stimulator (frequency, 1 Hz; duration, 5 milliseconds; intensity, 10% to 20% above threshold). Preparations were allowed to equilibrate for at least 90 minutes, with the bathing solution changed after 45 minutes. Isometric force of contraction was measured with an inductive force transducer (W. Fleck and FMI) attached to a Hellige Helco scripter or Gould recorder. Concentration-dependent mechanical effects, eg, force of contraction, were obtained. After complete mechanical stabilization, the force-frequency relation was studied starting with a rate of 0.5 Hz.
Preparation of the SR
The SR was prepared according to the
methods of Meissner and
Henderson19 and Sitsapesan and Williams.20
The preparation was made at 4°C; myocardial tissue was chilled in
ice-cold homogenization buffer of the following
composition (in mmol/L): sucrose 300, PMSF 1, and PIPES 20, pH 7.4).
Connective tissue was carefully trimmed away, and myocardial tissue was
homogenized with a motor-driven
homogenizer (Braun). The homogenate was
spun at 8000 rpm (Beckman JA 20) for 20 minutes. The pellet was
discarded. The supernatant was filtered through four layers of gauze
and centrifuged at 35 000 rpm for 60 minutes (Sorvall A 641).
The pellet was resuspended in a 10% sucrose buffer containing (in
mmol/L) KCl 400, MgCl2 0.5, CaCl 0.5, EGTA 0.5, and PIPES
25, pH 7.0. This was the CSR. The total yield of protein recovered per
gram of wet weight of myocardial tissue was 1.32±0.29 and
1.58±0.38
mg/g in nonfailing and failing myocardium, respectively.
The dilution was then loaded on a sucrose gradient.
Centrifugation was carried out for 2 hours in a
swinging bucket rotor at 21 000 rpm (Beckman SW 40). The fraction in
the interface between 20% and 30% sucrose was collected and
resuspended in 400 mmol/L KCl. This fraction was centrifuged at
35 000 rpm for 60 minutes (Sorvall A 641). The preparations were
stored at -80°C in a buffer containing (in mmol/L) sucrose 400,
HEPES 5, and Tris 5, pH 7.2. Protein content was measured by the method
of Lowry et al.21
Measurement of Ca2+-ATPase Activity
The
reaction was carried out according to the method of
Chu et al,22 which is based on coupled enzyme reactions.
(1) ATP
ADP+Pi. This reaction was catalyzed by the SR
Ca2+-ATPase. (2)
ADP+PhosphoenolpyruvateATP+Pyruvate. The reaction was
catalyzed by
the pyruvate kinase. (3)
Pyruvate+NADHLactate+NAD+.
The reaction was catalyzed by the lactate dehydrogenase.
The oxidation of NADH was monitored continuously by the decreased absorbance at 340 nm with a spectrophotometer (Beckman DU 640). The reaction was carried out in 1 mL at 37°C. The SR preparations (final concentration, 50 µg/mL) were suspended in the reaction mixture of the following composition (in mmol/L): MOPS 21, NaN3 4.9, EGTA 0.06, KCl 100, MgCl2 3, phosphoenolpyruvate 1, and NADH 0.2, and pyruvate–lactate dehydrogenase kinase LDR enzyme mixture 8.4/12 U. CaCl2 was added to the reaction mixture to yield the desired free Ca2+ concentrations calculated according to the method of Fabiato.23 The reaction was started with ATP (1 mmol/L) and was constant over at least 5 minutes. The basal activity was measured in the absence of Ca2+ and in the presence of EGTA (4 mmol/L) simultaneously. All experiments were carried out in triplicate. The activity of the Ca2+-ATPase was given in nanomoles of ATP per milligram of protein per minute. The action of the specific Ca2+-ATPase inhibitor CPA also was studied.
Ca2+ Uptake Into SR
The assays were performed
in a total volume of 200 µL
incubation buffer consisting of (in mmol/L) ATP 5,
creatine-phosphate 6, NaN3 10, KCl 100, EGTA 0.2,
imidazol 18, MgCl2 5, and K+-oxalate 10, pH
6.9.19 24 The medium was supplemented by different
concentrations of EGTA and CaCl2. The reaction was started
with 45Ca2+. The incubation was performed at
37°C for 1 to 30 minutes. The reaction was terminated by rapid vacuum
filtration through Whatman GF/F filters. The filters were immediately
washed with 6 mL ice-cold buffer (120 mmol/L KCl; 2 mmol/L EGTA).
Radioactivity was determined in a ß-counter (Pharmacia LKB).
Nonspecific uptake was determined in the absence of oxalate. All
experiments were carried out in triplicate. According to Movsesian et
al,14 the 45Ca2+
uptake rate was calculated by least-squares linear regression
analysis (r>.98 in all cases). To assess the
contribution of SERCA II, the inhibitory actions of CPA
(Sigma Chemical Co), an indole tetramic acid metabolite of
Aspergillus and Penicillium, and thapsigargin
were studied in a concentration-dependent manner (1 to 10
µmol/L).25 26 Furthermore, the effect of the
antibiotic
A23187 (Sigma), a calcium ionophore that forms lipophilic complexes
with divalent cations, on Ca2+ uptake was examined. A23187
has been reported to inhibit oxalate-supported Ca2+
uptake.27
Immunoblotting
For determination of SERCA II and
phospholamban in failing and
nonfailing human myocardium, immunoblotting
techniques were performed as described previously with slight
modifications.28 SERCA II and phospholamban were
determined in CSR and VSR from failing and nonfailing
myocardium. To identify SERCA II and phospholamban,
specific commercially available antibodies were
used.29 30
Equal amounts of membrane proteins (10 µg) were separated by
SDS-PAGE–urea (4 mol/L urea, 7.5% acrylamide, 0.1%
SDS)31 on a Biorad Mini Protean electrophoresis
apparatus. After electrophoretic separation, proteins were
electrotransfered from the SDS-PAGE onto
polyvinylidenfluoride membrane sheets (Immobilon, SERVA).
The electrotransfer was controlled by staining the
polyvinylidenfluoride membranes with Ponceau red (Sigma).
For the immunoreaction, a monoclonal mouse anti-phospholamban
antibody (IgG1) (Biomol, Germany; immunogen, canine phospholamban
purified from canine cardiac SR) and a monoclonal mouse anti–SERCA
II-ATPase antibody (IgG1) (Dianova; immunogen, canine SERCA purified
from canine cardiac SR) were used. For detection, a second antibody, a
peroxidase-conjugated mouse IgG (Sigma), and the enhanced
chemoluminescence assay (Amersham) were used. After exposure to
x-ray film (ORWO), the bands were quantified by densitometry (PDI
Imaging System).
Northern Blot Analysis
Total RNA from frozen left ventricular
tissue
samples was prepared according to the protocol of Chomczynski and
Sacchi.32 Typically, between 50 and 100 µg total RNA was
obtained from 150 mg tissue. The amount of RNA was determined by UV
absorption. The optical density ratio of 260:280 nm was 1.8:2.0 in all
cases. Then 15 µg total RNA was separated in a 6% formaldehyde/1.2%
agarose gel, blotted on nylon membranes (Schleicher und Schuell) by
overnight capillary blotting, and fixed by UV irradiation. After
fixation, the blots were prehybridized in 50% formamide solution (5x
SSC, 5x Denhardt's solution, 50% formamide, 1% SDS, 50 mmol/L
sodium phosphate, pH 6.8, 10% dextran sulfate, and 100 g/mL salmon
sperm DNA). Hybridization was performed in 50% formamide solution at
42°C for at least 16 hours. The membrane was successively hybridized
with a 2-kb SERCA II cDNA fragment (BamHI-BamHI,
kindly provided by D.H. MacLennan, Toronto, Canada) and a
0.3-kb phospholamban cDNA fragment (HindIII-Not
I, kindly provided by Prof Dr W. Schmitz, Münster, Germany) that
was generated by polymerase chain reaction amplification with specific
primer chosen from the human phospholamban sequence published by Fujii
et al.33 The fragments had been cut from the plasmid
vector with the appropriate restriction enzymes, separated from the
vector DNA on a 1% low-melt agarose gel, and labeled with
[
-32P]dCTP (Amersham Buchler Ltd) by use of the
Multiprime DNA Labeling Kit (Amersham Buchler Ltd). The concentration
of the labeled probe in the hybridization solution was
1x106 cpm/mL. After overnight hybridization with
the SERCA II or phospholamban cDNA at 42°C, the membrane was
successively washed twice in 2x SSC/0.1% SDS at room temperature for
15 minutes and twice in 0.2x SSC/0.1% SDS at 68°C for 45 minutes.
Standardization was performed by hybridization of the same membrane
with a 40-base single-stranded synthetic
oligonucleotide probe for GAPDH (Dianova).
Hybridization conditions were the same as described above. Stringency
washes were performed with 2x SSC/0.1% SDS at room temperature for 30
minutes with 2x SSC/0.1% SDS at 65°C and twice for 5 minutes with
2x SSC/0.1% SDS. Membranes were exposed to Kodak films (Kodak
X-OMAT). Signals were quantified by densitometric analysis with
the Image Quant Densitometric System (Molecular Dynamics). The size (in
kilobases) of the detected mRNA was calculated by the 18S (1.8 kb) and
28S (4.6 kb) ribosomal RNA migration from the gel wells.
Binding Experiments
The ouabain binding assays in CSR were
performed in a total
volume of 250 µL incubation buffer. The incubation was carried out at
37°C for 180 minutes. These conditions allowed complete equilibration
of the receptors with the radioligand. The reaction was
terminated by rapid vacuum filtration through Whatman GF/C filters and
were immediately washed three times with 6 mL ice-cold incubation
buffer. All experiments were performed in triplicate. Filters were
dried at 90°C and placed in 10 mL scintillation fluid (Quickszint
501, Zinsser Analytics), and radioactivity was determined in a liquid
scintillation counter. 3H-ouabain binding was performed
according to the method of Schwinger et al.3
Materials
45CaCl and 3H-ouabain were
purchased
from Amersham-Buchler. Antibodies were a monoclonal (mouse) anti–SERCA
II-ATPase-IgG1 antibody (Dianova)29 and
anti–phospholamban (mouse)-IgG1 (Biomol).30 All other
chemicals were of analytic grade or the best grade commercially
available. Only deionized and double-distilled water was used
throughout.
Statistical Analysis
The data shown are mean±SEM. For
comparison within one group,
the paired t test was applied. Otherwise, statistical
significance was analyzed with Student's t test for
unpaired observations or by ANOVA. A value of P<.05 was
considered significant. Statistical evaluation was performed by the
Institut für Medizinische Informationsverarbeitung, Biometrie und
Epidemiologie of the University of Munich, Germany.
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Force-Frequency Relation in Failing and Nonfailing Human Myocardium
The force-frequency relation in failing human myocardium resulting from DCM and in nonfailing tissue was examined to functionally characterize the human tissue used. After an increase in frequency of stimulation in only human nonfailing myocardium, the force of contraction increased but remained the same or even decreased in human failing myocardium. After an increase in frequency of stimulation, the force of contraction increased significantly in nonfailing tissue, from 2.6±0.2 to 4.2±0.2 mN at 0.5 and 2.0 Hz, respectively. The corresponding values for failing myocardium were 2.8±0.3 and 2.4±0.3 mN at 0.5 and 2.0 Hz, respectively. Therefore, the myocardium used exerted the force-frequency relation as typically described in previous studies involving nonfailing and terminally failing (DCM) human myocardium.6 7 8
Ca2+-ATPase Measurement in Human SR
SERCA II
activity was measured by an optical assay in CSR and VSR
from failing and nonfailing human myocardium. The specific
Ca2+-ATPase inhibitor CPA depressed
Ca2+-ATPase activity concentration-dependently in
preparations from failing and nonfailing tissue. This holds true for
CSR and VSR. Fig 1
shows the concentration-dependent
effect of CPA on Ca2+-ATPase activity for human failing and
nonfailing myocardium. After an increase in free
Ca2+ (up to 35.5 µmol/L), the activity of SERCA II
increased in failing and nonfailing tissue. The maximal
Ca2+-ATPase activity was recorded at 35.5 µmol/L free
Ca2+ in the assay for both failing and nonfailing
myocardium (Fig 2). The
Ca2+-ATPase activity in VSR from failing (175±11 nmol
ATP/mg SR per minute) and nonfailing (169±14 nmol ATP/mg SR per
minute) human tissue was not significantly different. In contrast, in
CSR Ca2+-ATPase activity was significantly reduced in
failing human myocardium compared with control tissue (Fig 2).
The
corresponding values for CSR were 125±7 and 163±8 nmol/mg
protein per
minute, respectively (P<.05; Fig 3).
Measurement of Ca2+-ATPase activity relative
to the density of Na+/K+-ATPase also was
significantly reduced in failing compared with nonfailing
myocardium (11.7±0.6 and 18.7±0.7 nmol ATP/min per 1 pmol
ouabain binding site, failing versus nonfailing tissue, respectively).
Therefore, there was a statistically significant difference for
Ca2+-ATPase activity in nonfailing and failing human tissue
in CSR only. The potency of Ca2+ to stimulate SERCA II was
identical in CSR from failing and nonfailing human
myocardium as judged by the EC50 values
(nonfailing, 0.56 [0.30 to 1.07] µmol/L; failing, 0.58
[0.39 to
0.85] µmol/L).
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Ca2+ Uptake Into Human SR
Ca2+ uptake in SR prepared from left
ventricular myocardium of nonfailing and
terminally failing hearts was examined. Measurements were performed in
CSR and VSR located between the 20% and 30% sucrose gradient. To
characterize the preparation, the effect of CPA and thapsigargin on SR
Ca2+ uptake was measured. CPA and thapsigargin have been
reported to act as specific inhibitors of the
Ca2+-ATPase.25 26 CPA, thapsigargin,
and
A23187 concentration-dependently reduced oxalate-supported SR
Ca2+ uptake (not shown). At each free Ca2+
concentration used, Ca2+ uptake increased linearly
(r=.98) with time in CSR and VSR in both failing and
nonfailing tissue. Uptake rates were calculated by least-squares
linear regression analysis (r>.98 in all cases) of
six time points according to Movsesian et al.14 Because
Ca2+ uptake also depends on the free Ca2+
concentration used, Ca2+ uptake measurements have been
performed at free Ca2+ concentrations from 0.03 to 50
µmol/L. In both failing and nonfailing myocardial preparations,
Ca2+ uptake increased after an increase in
Ca2+. Ca2+ uptake was significantly reduced in
CSR from failing myocardium compared with control tissue
(Fig 4) at free Ca2+ concentrations >1
µmol/L. However, there was no statistically significant difference
between groups in the concentration range up to or exceeding 1 µmol/L
free Ca2+ in VSR. At a free Ca2+ concentration
of 1 µmol/L, the SR Ca2+ uptakes in VSR from
nonfailing and terminally failing myocardium were 9.7±1.4
and 10.7±1.0 nmol Ca2+/mg SR per minute,
respectively. The corresponding values in CSR were 7.1±0.8 and
3.5±0.3 nmol Ca2+/mg SR per minute
(P<.05), respectively (Fig 4). Therefore, there was
a
significant difference for Ca2+ uptake in CSR but not in
VSR from terminally failing myocardium caused by DCM
compared with nonfailing control tissue. The potency of
Ca2+ to stimulate SR
45Ca2+ uptake was identical in CSR
from failing and nonfailing human myocardium as judged by
the EC50 values (nonfailing, 0.46 [0.35 to 0.60]
µmol/L; failing, 0.42 [0.35 to 0.49] µmol/L).
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SERCA II and Phospholamban Protein Levels
Protein levels of
SERCA II and phospholamban were measured in CSR
and VSR from failing and nonfailing human myocardium with
the immunoblotting technique. Experiments were
performed in identical preparations as used for Ca2+ uptake
and Ca2+-ATPase activity measurements. To identify SERCA II
and phospholamban, specific monoclonal antibodies were used. Fig
5 gives the protein dependency of the
antibody-marked lanes for the measurement of SERCA II and
phospholamban. There was a linear correlation (P<.01)
between optical density and protein content for both phospholamban and
SERCA II. Fig 6 shows representative
Western blots. The antibody-marked lanes were measured by
densitometry; the data are given in Fig 7. There was no
significant difference between nonfailing and terminally failing
myocardium for either SERCA II or phospholamban. When
the optical densities were related to ouabain binding sites of CSR,
there also was no significant difference between failing and
nonfailing myocardium.
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mRNA Expression of SERCA IIa and Phospholamban
The steady
state expression levels of mRNA encoding SERCA IIa and
phospholamban were measured with total RNA from hearts exhibiting
severe heart failure or normal myocardial tissue. Fig 8
gives the Northern blot analysis of the mRNA expression of
SERCA IIa and phospholamban in left ventricular
myocardium from failing and nonfailing human hearts. For
both SERCA II and phospholamban mRNA, the expression was reduced in
failing tissue. Fig 9 gives the amount of mRNA relative
to the expression of GAPDH mRNA. GAPDH mRNA levels were used as an
internal standard for the variations in sample loading and blotting
efficiency of RNA. The expression level of each mRNA was divided by the
GAPDH mRNA value because the GAPDH mRNA level was proportional to the
intensity of 28S and 18S ribosomal RNA on ethidium bromide staining.
The mRNA expressions for SERCA IIa and phospholamban were significantly
reduced in failing compared with nonfailing human
myocardium (see the Table). The densities of
SERCA IIa mRNA relative to GAPDH mRNA (arbitrary unit per arbitrary
unit) in human failing and nonfailing myocardium were
1.77±1.24 (n=9) and 3.87±0.28 (n=9). The
corresponding values for
phospholamban mRNA of the same hearts were 0.99±0.04 and
1.66±0.12,
respectively.
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Discussion |
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In terminally failing human myocardium from patients suffering from DCM, alterations in excitation-contraction coupling have been described by several groups.3 6 7 34 In addition to a blunted response to cAMP-dependent inotropic mechanisms,4 34 35 a lusitropic dysfunction in myocardium from patients with DCM has been reported.3 5 The [Ca2+]i handling has been found to be altered in heart failure.10 Inasmuch as relaxation is suggested to result from sequestration of free Ca2+ from the cytosol into the SR, this uptake mechanism has been suggested to be insufficient to maintain diastolic function. The negative force-frequency relation in failing myocardium also may be due to a reduced SR Ca2+ loading after a reduced SR Ca2+ uptake. Reports of diminished steady state mRNA levels for the Ca2+-ATPase and phospholamban in homogenates of failing human left ventricular myocardium are consistent with this hypothesis.16 17 18 36 37 38 However, mRNA levels, protein synthesis, and enzyme function are not necessarily altered similarly in heart failure. Therefore, the present study aimed at investigating SR Ca2+-uptake and Ca2+-ATPase activity in myocardium from patients with DCM and in control subjects. In addition to these functional studies, the protein levels and the mRNA levels of SERCA II and phospholamban were measured. To ensure that the failing tissue used reveals an altered contraction coupling as typically described for failing tissue, the force-frequency relation was measured. Only in nonfailing myocardium was an increase in frequency of stimulation linked to an increase in force of contraction. In the failing tissue used, the force-frequency relation was negative, as has been described as typical for dilated cardiomyopathic tissue.
Protein levels of SERCA II and phospholamban, as detected with highly specific antibodies, were unchanged in VSR and CSR preparations from failing human myocardial tissue compared with controls. Consistent with the results reported here, Movsesian and coworkers14 39 40 found the immunodetectable levels of the SERCA II protein, phospholamban, and calsequestrin unchanged in the left ventricular myocardium from heart failure patients (DCM) compared with healthy control subjects. In contrast to the findings at the protein level, Mercadier and coworkers16 reported a decrease in the mRNA content of SERCA II in patients undergoing cardiac transplantation for idiopathic DCM (n=6), coronary artery disease (n=4), and diverse etiologies (n=31). These findings are in line with the present study. Because mRNA levels for SERCA IIa and phospholamban were decreased in failing myocardium, it is not unreasonable to speculate that the expression of these two genes is coordinately regulated. Several groups reported a decreased mRNA level for SERCA II in failing human myocardium.17 18 37 This decrease correlates with results from animal models of heart failure. In rat ventricular hypertrophy, the decrease in SERCA II mRNA content parallels a decrease in Ca2+-ATPase protein concentration, which suggests a decrease in Ca2+-ATPase pump density.41 42 However, the situation in animals is not necessarily transferable to the situation in humans.43 Furthermore, protein levels may be regulated independent of the encoding mRNA levels. Large differences in the ratio of mRNA levels to protein content for both the SERCA I and SERCA II isoforms of the Ca2+-transporting ATPase have been observed.44 45 Therefore, steady state mRNA levels cannot be assumed to be a certain predictor of protein content or even protein function. The differences may be related to mRNA processing, mRNA translation, posttranslational modification, and rates of protein synthesis and protein degradation. In hearts from patients with end-stage heart failure, mRNA levels for the SR Ca2+ release channel, Ca2+-ATPase, and phospholamban were altered.17 36 However, the function of the SR Ca2+ release channel was not different in DCM compared with nonfailing myocardium as judged by measurements of caffeine-induced Ca2+ release in skinned fiber preparations.46 Because both SERCA II and phospholamban protein levels determined with monoclonal antibodies were unchanged in failing myocardium compared with nonfailing tissue, mRNA levels of SERCA II or phospholamban do not correlate with the protein expression or function in humans.
Ca2+ uptake is regulated by several factors. Phospholamban phosphorylation has been reported to stimulate Ca2+ uptake by SR. Because both the phosphorylation state and the amount of phospholamban determine the activity of SERCA II, Ca2+ uptake–regulating proteins have to be studied functionally. The Ca2+-dependent Ca2+-uptake into the SR and the Ca2+-ATPase activity were significantly reduced in CSR from terminally failing myocardium compared with nonfailing control tissue. In contrast, no significant differences were found between groups in VSR. Movsesian and coworkers14 40 47 consistently found no significant differences between normal hearts and excised failing hearts with respect to Vmax of SR Ca2+ uptake in VSR (homogenization and differential sedimentation). Movsesian et al47 used monoclonal antibodies, which mimic the effects of cAMP-dependent phospholamban phosphorylation, to study the capacity of Ca2+ uptake in human cardiac SR. They observed similar cAMP-dependent phosphorylation and Ca2+ uptake in nonfailing and failing myocardium. These findings in VSR are in agreement with the data presented here for VSR preparations. In contrast to the findings in isolated vesicles of left ventricular myocardium,47 in homogenates (CSR) of the left12 and right15 ventricular myocardium (crude membranes from human biopsy specimens) from patients with DCM, SR Ca2+ uptake12 and Ca2+-ATPase activity were reduced. The differences between CSR and VSR reported here are therefore in line with previously published data. One explanation may be the isolation procedure itself. During the gradient centrifugation, proteins responsible for the regulation of the Ca2+-ATPase or the amount of those regulatory units relative to the ATPase may change. This may be one reason why SR Ca2+ uptake in VSR increased only marginally compared with CSR. Furthermore, when membrane or subcellular preparations are investigated, one could potentially lose or enrich one component. To overcome this problem, CSR and VSR have been studied simultaneously to characterize SR function of the same hearts.
Protein levels of SERCA II and phospholamban do not correlate with functional studies in vitro, indicating a reduced maximal ATPase-activity and a reduced Ca2+ uptake into the SR in failing tissue compared with control tissue. Ca2+ uptake mechanisms and regulation of the force of contraction may be affected by factors, either extrinsic or intrinsic to the SR, that are not present in VSR but can be studied in CSR homogenates, the intact muscle preparation, or isolated myocytes. Therefore, the Ca2+-ATPase activity and the Ca2+ uptake into the SR in CSR from nonfailing human myocardium were significantly higher compared with failing tissue resulting from DCM. A decrease in Ca2+ uptake in the human failing heart has been also deducted from measurements of [Ca2+]i movements10 by use of intact preparations. Beuckelmann and coworkers48 observed in isolated myocytes from patients with severe heart failure a reduced rate of Ca2+ uptake into the SR. This finding may indicate that regulation of SR Ca2+ uptake is altered in DCM. Therefore, it is not unreasonable to speculate that in the intact heart differences in regulation of Ca2+ uptake into the SR and in Ca2+ release from the SR cause the alteration in [Ca2+]i handling observed by various investigators.49 In these studies, [Ca2+]i handling was examined with isolated myocytes,13 48 intact papillary muscle strip preparations,10 or CSR homogenates.
In summary, the present study provides evidence that the protein levels of SERCA II and phospholamban are not significant different in myocardium from patients with DCM compared with control tissue. However, the Ca2+-ATPase activity and the rate of Ca2+ uptake in CSR preparations from failing hearts were significantly reduced compared with control tissue. One possible explanation for the alterations in [Ca2+]i handling evident in DCM may be an altered regulation of the Ca2+ uptake into the SR and the Ca2+ release from the SR, ie, an altered regulation of the phosphorylation status. To elucidate the subcellular mechanisms for the altered [Ca2+]i handling, further studies focusing on intracellular regulatory mechanisms are required.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Experimental work was supported by the Deutsche Forschungsgemeinschaft (Drs Schwinger and Böhm) and the Zentrum für Molekulare Medizin Köln. This work contains data from the doctoral thesis of U. Bavendiek (University of Cologne, in preparation). Our special thanks go to Prof Dr B. Reichart, Department of Cardiac Surgery, University of Munich, and Prof Dr E.R. DeVivie, Department of Cardiac Surgery, University of Cologne, and their colleagues for providing the myocardial tissue. We thank Heidrun Villena, Britta Baulig, and Susanne Hoischen for their excellent assistance.
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Footnotes |
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Reprint requests to Drmed Robert H.G. Schwinger, Universität zu Köln, Medizinische Klinik III, Joseph-Stelzmannstr 9, D-50924 Köln, Germany.
Received October 4, 1994; revision received June 28, 1995; accepted July 20, 1995.
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References |
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R. Vlasblom, A. Muller, R. J.P Musters, M. J Zuidwijk, C. van Hardeveld, W. J Paulus, and W. S Simonides Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes Cardiovasc Res, August 15, 2004; 63(3): 537 - 544. [Abstract] [Full Text] [PDF]
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A. M Janczewski, M. Zahid, B. H Lemster, C. S Frye, G. Gibson, Y. Higuchi, E. G Kranias, A. M Feldman, and C. F McTiernan Phospholamban gene ablation improves calcium transients but not cardiac function in a heart failure model Cardiovasc Res, June 1, 2004; 62(3): 468 - 480. [Abstract] [Full Text] [PDF]
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Y. Ke, L. Wang, W. G. Pyle, P. P. de Tombe, and R. J. Solaro Intracellular Localization and Functional Effects of P21-Activated Kinase-1 (Pak1) in Cardiac Myocytes Circ. Res., February 6, 2004; 94(2): 194 - 200. [Abstract] [Full Text] [PDF]
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 T. Zhang, S. Miyamoto, and J. H. Brown Cardiomyocyte Calcium and Calcium/Calmodulin-dependent Protein Kinase II: Friends or Foes? Recent Prog. Horm. Res., January 1, 2004; 59(1): 141 - 168. [Abstract] [Full Text]
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D. W. Rodenbaugh, H. L. Collins, D. G. Nowacek, and S. E. DiCarlo Increased susceptibility to ventricular arrhythmias is associated with changes in Ca2+ regulatory proteins in paraplegic rats Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2605 - H2613. [Abstract] [Full Text] [PDF]
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O. J. Muller, M. Lange, H. Rattunde, H.-P. Lorenzen, M. Muller, N. Frey, C. Bittner, W. Simonides, H. A. Katus, and W.-M. Franz Transgenic rat hearts overexpressing SERCA2a show improved contractility under baseline conditions and pressure overload Cardiovasc Res, August 1, 2003; 59(2): 380 - 389. [Abstract] [Full Text] [PDF]
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D. J. Lips, L. J. deWindt, D. J.W. van Kraaij, and P. A. Doevendans Molecular determinants of myocardial hypertrophy and failure: alternative pathways for beneficial and maladaptive hypertrophy Eur. Heart J., May 2, 2003; 24(10): 883 - 896. [Abstract] [Full Text] [PDF]
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 R. H. G. Schwinger and K. F. Frank Calcium and the Failing Heart: Phospholamban, Good Guy or Bad Guy? Sci. Signal., April 29, 2003; 2003(180): pe15 - pe15. [Abstract] [Full Text] [PDF]
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W. Schillinger, J. W Fiolet, K. Schlotthauer, and G. Hasenfuss Relevance of Na+-Ca2+ exchange in heart failure Cardiovasc Res, March 15, 2003; 57(4): 921 - 933. [Full Text] [PDF]
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J. Weisser-Thomas, V. Piacentino III, J. P Gaughan, K. Margulies, and S. R Houser Calcium entry via Na/Ca exchange during the action potential directly contributes to contraction of failing human ventricular myocytes Cardiovasc Res, March 15, 2003; 57(4): 974 - 985. [Abstract] [Full Text] [PDF]
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W Schillinger, A Ohler, S Emami, F Muller, C Christians, P.M.L Janssen, H Kogler, N Teucher, B Pieske, T Seidler, et al. The functional effect of adenoviral Na+/Ca2+ exchanger overexpression in rabbit myocytes depends on the activity of the Na+/K+-ATPase Cardiovasc Res, March 15, 2003; 57(4): 996 - 1003. [Abstract] [Full Text] [PDF]
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 F. del Monte and R. J Hajjar Targeting calcium cycling proteins in heart failure through gene transfer J. Physiol., January 1, 2003; 546(1): 49 - 61. [Abstract] [Full Text] [PDF]
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K. F Frank, B. Bolck, E. Erdmann, and R. H.G Schwinger Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation Cardiovasc Res, January 1, 2003; 57(1): 20 - 27. [Abstract] [Full Text] [PDF]
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M. U Braun, P. LaRosee, S. Schon, M. M Borst, and R. H Strasser Differential regulation of cardiac protein kinase C isozyme expression after aortic banding in rat Cardiovasc Res, October 1, 2002; 56(1): 52 - 63. [Abstract] [Full Text] [PDF]
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 G. Plenz, H. Eschert, M. Erren, T. Wichter, M. Bohm, M. Flesch, H. H. Scheld, and M. C. Deng The interleukin-6/interleukin-6-receptorsystem is activated in donor hearts J. Am. Coll. Cardiol., May 1, 2002; 39(9): 1508 - 1512. [Abstract] [Full Text] [PDF]
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J. Kneller, H. Sun, N. Leblanc, and S. Nattel Remodeling of Ca2+-handling by atrial tachycardia: evidence for a role in loss of rate-adaptation Cardiovasc Res, May 1, 2002; 54(2): 416 - 426. [Abstract] [Full Text] [PDF]
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 C. Communal, M. Sumandea, P. de Tombe, J. Narula, R. J. Solaro, and R. J. Hajjar Functional consequences of caspase activation in cardiac myocytes PNAS, April 18, 2002; (2002) 92022999. [Abstract] [Full Text] [PDF]
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N. R. Alpert, L. A. Mulieri, and D. Warshaw The failing human heart Cardiovasc Res, April 1, 2002; 54(1): 1 - 10. [Full Text] [PDF]
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F. del Monte, S. E. Harding, G. W. Dec, J. K. Gwathmey, and R. J. Hajjar Targeting Phospholamban by Gene Transfer in Human Heart Failure Circulation, February 26, 2002; 105(8): 904 - 907. [Abstract] [Full Text] [PDF]
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J. B Sande, I. Sjaastad, I. B Hoen, J. Bokenes, T. Tonnessen, E. Holt, P. K Lunde, and G. Christensen Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity Cardiovasc Res, February 1, 2002; 53(2): 382 - 391. [Abstract] [Full Text] [PDF]
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F. del Monte, E. Williams, D. Lebeche, U. Schmidt, A. Rosenzweig, J. K. Gwathmey, E. D. Lewandowski, and R. J. Hajjar Improvement in Survival and Cardiac Metabolism After Gene Transfer of Sarcoplasmic Reticulum Ca2+-ATPase in a Rat Model of Heart Failure Circulation, September 18, 2001; 104(12): 1424 - 1429. [Abstract] [Full Text] [PDF]
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N. Satoh, T. Sato, M. Shimada, K. Yamada, and Y. Kitada Lusitropic Effect of MCC-135 Is Associated with Improvement of Sarcoplasmic Reticulum Function in Ventricular Muscles of Rats with Diabetic Cardiomyopathy J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 1161 - 1166. [Abstract] [Full Text] [PDF]
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H. Kubo, K. B. Margulies, V. Piacentino III, J. P. Gaughan, and S. R. Houser Patients With End-Stage Congestive Heart Failure Treated With {beta}-Adrenergic Receptor Antagonists Have Improved Ventricular Myocyte Calcium Regulatory Protein Abundance Circulation, August 28, 2001; 104(9): 1012 - 1018. [Abstract] [Full Text] [PDF]
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G. Munch, B. Bolck, A. Sugaru, K. Brixius, W. Bloch, and R. H.G. Schwinger Increased Expression of Isoform 1 of the Sarcoplasmic Reticulum Ca2+-Release Channel in Failing Human Heart Circulation, June 5, 2001; 103(22): 2739 - 2744. [Abstract] [Full Text] [PDF]
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N. Chossat, F. Griscelli, P. Jourdon, D. Logeart, T. Ragot, M. Heimburger, M. Perricaudet, A.-M. Lompre, S. Hatem, and J.-J. Mercadier Adenoviral SERCA1a gene transfer to adult rat ventricular myocytes induces physiological changes in calcium handling Cardiovasc Res, February 1, 2001; 49(2): 288 - 297. [Abstract] [Full Text] [PDF]
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S. E. Litwin, D. Zhang, and J. H. B. Bridge Dyssynchronous Ca2+ Sparks in Myocytes From Infarcted Hearts Circ. Res., November 24, 2000; 87(11): 1040 - 1047. [Abstract] [Full Text] [PDF]
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S. M. Pogwizd Increased Na+-Ca2+ Exchanger in the Failing Heart Circ. Res., October 13, 2000; 87(8): 641 - 643. [Full Text] [PDF]
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P. Trouve, F. Carre, I. Belikova, C. Leclercq, T. Dakhli, L. Soufir, I. Coquard, J. Ramirez-Gil, and D. Charlemagne Na+-K+-ATPase alpha 2-isoform expression in guinea pig hearts during transition from compensation to decompensation Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1972 - H1981. [Abstract] [Full Text] [PDF]
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S. Gupta, A. J.C Prahash, and I. S Anand Myocyte contractile function is intact in the post-infarct remodeled rat heart despite molecular alterations Cardiovasc Res, October 1, 2000; 48(1): 77 - 88. [Abstract] [Full Text] [PDF]
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L. Sen, G. Cui, G. C. Fonarow, and H. Laks Differences in mechanisms of SR dysfunction in ischemic vs. idiopathic dilated cardiomyopathy Am J Physiol Heart Circ Physiol, August 1, 2000; 279(2): H709 - H718. [Abstract] [Full Text] [PDF]
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G. Munch, B. Bolck, K. Brixius, H. Reuter, U. Mehlhorn, W. Bloch, and R. H. G. Schwinger SERCA2a activity correlates with the force-frequency relationship in human myocardium Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1924 - H1932. [Abstract] [Full Text] [PDF]
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T. Netticadan, R. M. Temsah, K. Kawabata, and N. S. Dhalla Sarcoplasmic Reticulum Ca2+/Calmodulin-Dependent Protein Kinase Is Altered in Heart Failure Circ. Res., March 17, 2000; 86(5): 596 - 605. [Abstract] [Full Text] [PDF]
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L. S Maier, C. Schwan, W. Schillinger, K. Minami, U. Schutt, and B. Pieske Gingerol, isoproterenol and ouabain normalize impaired post-rest behavior but not force-frequency relation in failing human myocardium Cardiovasc Res, March 1, 2000; 45(4): 913 - 924. [Abstract] [Full Text] [PDF]
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 C. A. Walker, F. A. Crawford Jr, and F. G. Spinale MYOCYTE CONTRACTILE DYSFUNCTION WITH HYPERTROPHY AND FAILURE: RELEVANCE TO CARDIAC SURGERY J. Thorac. Cardiovasc. Surg., February 1, 2000; 119(2): 388 - 400. [Full Text] [PDF]
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U Ravens and D Dobrev Regulation of sarcoplasmic reticulum Ca2+-ATPase and phospholamban in the failing and nonfailing heart Cardiovasc Res, January 1, 2000; 45(1): 245 - 252. [Full Text] [PDF]
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F. del Monte, S. E. Harding, U. Schmidt, T. Matsui, Z. B. Kang, G. W. Dec, J. K. Gwathmey, A. Rosenzweig, and R. J. Hajjar Restoration of Contractile Function in Isolated Cardiomyocytes From Failing Human Hearts by Gene Transfer of SERCA2a Circulation, December 7, 1999; 100(23): 2308 - 2311. [Abstract] [Full Text] [PDF]
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 X.-Q. Zhang, Y.-C. Ng, R. L. Moore, T. I. Musch, and J. Y. Cheung In situ SR function in postinfarction myocytes J Appl Physiol, December 1, 1999; 87(6): 2143 - 2150. [Abstract] [Full Text] [PDF]
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S. M. Pogwizd, M. Qi, W. Yuan, A. M. Samarel, and D. M. Bers Upregulation of Na+/Ca2+ Exchanger Expression and Function in an Arrhythmogenic Rabbit Model of Heart Failure Circ. Res., November 26, 1999; 85(11): 1009 - 1019. [Abstract] [Full Text] [PDF]
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B. Huang, S. Wang, D. Qin, M. Boutjdir, and N. El-Sherif Diminished Basal Phosphorylation Level of Phospholamban in the Postinfarction Remodeled Rat Ventricle : Role of {beta}-Adrenergic Pathway, Gi Protein, Phosphodiesterase, and Phosphatases Circ. Res., October 29, 1999; 85(9): 848 - 855. [Abstract] [Full Text] [PDF]
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J. A. Fallavollita, S. Jacob, R. F. Young, and J. M. Canty Jr. Regional alterations in SR Ca2+-ATPase, phospholamban, and HSP-70 expression in chronic hibernating myocardium Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1418 - H1428. [Abstract] [Full Text] [PDF]
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U. Schmidt, R. J. Hajjar, C. S. Kim, D. Lebeche, A. A. Doye, and J. K. Gwathmey Human heart failure: cAMP stimulation of SR Ca2+-ATPase activity and phosphorylation level of phospholamban Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H474 - H480. [Abstract] [Full Text] [PDF]
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J. Prestle, S. Dieterich, M. Preuss, U. Bieligk, and G. Hasenfuss Heterogeneous transmural gene expression of calcium-handling proteins and natriuretic peptides in the failing human heart Cardiovasc Res, August 1, 1999; 43(2): 323 - 331. [Abstract] [Full Text] [PDF]
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B. Pieske, L. S. Maier, D. M. Bers, and G. Hasenfuss Ca2+ Handling and Sarcoplasmic Reticulum Ca2+ Content in Isolated Failing and Nonfailing Human Myocardium Circ. Res., July 9, 1999; 85(1): 38 - 46. [Abstract] [Full Text] [PDF]
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G. F. Tomaselli and E. Marban Electrophysiological remodeling in hypertrophy and heart failure Cardiovasc Res, May 1, 1999; 42(2): 270 - 283. [Full Text] [PDF]
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B. J.J.M. Brundel, I. C. Van Gelder, R. H. Henning, A. E. Tuinenburg, L. E. Deelman, R. G. Tieleman, J. G. Grandjean, W. H. Van Gilst, and H. J.G.M. Crijns Gene expression of proteins influencing the calcium homeostasis in patients with persistent and paroxysmal atrial fibrillation Cardiovasc Res, May 1, 1999; 42(2): 443 - 454. [Abstract] [Full Text] [PDF]
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U. Kirchhefer, W. Schmitz, H. Scholz, and J. Neumann Activity of cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human hearts Cardiovasc Res, April 1, 1999; 42(1): 254 - 261. [Abstract] [Full Text] [PDF]
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B. O'Rourke, D. A. Kass, G. F. Tomaselli, S. Kaab, R. Tunin, and E. Marban Mechanisms of Altered Excitation-Contraction Coupling in Canine Tachycardia-Induced Heart Failure, I : Experimental Studies Circ. Res., March 19, 1999; 84(5): 562 - 570. [Abstract] [Full Text] [PDF]
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K. Dipla, J. A. Mattiello, K. B. Margulies, V. Jeevanandam, and S. R. Houser The Sarcoplasmic Reticulum and the Na+/Ca2+ Exchanger Both Contribute to the Ca2+ Transient of Failing Human Ventricular Myocytes Circ. Res., March 5, 1999; 84(4): 435 - 444. [Abstract] [Full Text] [PDF]
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G. Hasenfuss, W. Schillinger, S. E. Lehnart, M. Preuss, B. Pieske, L. S. Maier, J. Prestle, K. Minami, and H. Just Relationship Between Na+-Ca2+–Exchanger Protein Levels and Diastolic Function of Failing Human Myocardium Circulation, February 9, 1999; 99(5): 641 - 648. [Abstract] [Full Text] [PDF]
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S. Currie and G. L. Smith Enhanced phosphorylation of phospholamban and downregulation of sarco/endoplasmic reticulum Ca2+ ATPase type 2 (SERCA 2) in cardiac sarcoplasmic reticulum from rabbits with heart failure Cardiovasc Res, January 1, 1999; 41(1): 135 - 146. [Abstract] [Full Text] [PDF]
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M. Anger, A.-M. Lompre, O. Vallot, F. Marotte, L. Rappaport, and J.-L. S. MD Cellular Distribution of Ca2+ Pumps and Ca2+ Release Channels in Rat Cardiac Hypertrophy Induced by Aortic Stenosis Circulation, December 1, 1998; 98(22): 2477 - 2486. [Abstract] [Full Text] [PDF]
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Y. Sato, D. G. Ferguson, H. Sako, G. W. Dorn II, V. J. Kadambi, A. Yatani, B. D. Hoit, R. A. Walsh, and E. G. Kranias Cardiac-specific Overexpression of Mouse Cardiac Calsequestrin Is Associated with Depressed Cardiovascular Function and Hypertrophy in Transgenic Mice J. Biol. Chem., October 23, 1998; 273(43): 28470 - 28477. [Abstract] [Full Text] [PDF]
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H. K. B. SIMMERMAN and L. R. JONES Phospholamban: Protein Structure, Mechanism of Action, and Role in Cardiac Function Physiol Rev, October 1, 1998; 78(4): 921 - 947. [Abstract] [Full Text] [PDF]
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F. Schroder, R. Handrock, D. J. Beuckelmann, S. Hirt, R. Hullin, L. Priebe, R. H. G. Schwinger, J. Weil, and S. Herzig Increased Availability and Open Probability of Single L-Type Calcium Channels From Failing Compared With Nonfailing Human Ventricle Circulation, September 8, 1998; 98(10): 969 - 976. [Abstract] [Full Text] [PDF]
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P. A Doevendans, M. J. Daemen, E. D de Muinck, and J. F Smits Cardiovascular phenotyping in mice Cardiovasc Res, July 1, 1998; 39(1): 34 - 49. [Abstract] [Full Text] [PDF]
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O. Zolk, M. Flesch, G. Nickenig, P. Schnabel, and M. Bohm Alteration of intracellular Ca2+-handling and receptor regulation in hypertensive cardiac hypertrophy: insights from Ren2-transgenic rats Cardiovasc Res, July 1, 1998; 39(1): 242 - 256. [Abstract] [Full Text] [PDF]
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L. Priebe and D. J. Beuckelmann Simulation Study of Cellular Electric Properties in Heart Failure Circ. Res., June 15, 1998; 82(11): 1206 - 1223. [Abstract] [Full Text] [PDF]
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G. Hasenfuss Alterations of calcium-regulatory proteins in heart failure Cardiovasc Res, February 1, 1998; 37(2): 279 - 289. [Full Text] [PDF]
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R. M Phillips, P. Narayan, A. M Gomez, K. Dilly, L. R Jones, W.J. Lederer, and R. A Altschuld Sarcoplasmic reticulum in heart failure: central player or bystander? Cardiovasc Res, February 1, 1998; 37(2): 346 - 351. [Full Text] [PDF]
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M. A Movsesian and R. H.G Schwinger Calcium sequestration by the sarcoplasmic reticulum in heart failure Cardiovasc Res, February 1, 1998; 37(2): 352 - 359. [Full Text] [PDF]
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M. Meyer and W. H Dillmann Sarcoplasmic reticulum Ca2+-ATPase overexpression by adenovirus mediated gene transfer and in transgenic mice Cardiovasc Res, February 1, 1998; 37(2): 360 - 366. [Abstract] [Full Text] [PDF]
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P. P de Tombe Altered contractile function in heart failure Cardiovasc Res, February 1, 1998; 37(2): 367 - 380. [Abstract] [Full Text] [PDF]
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R. Handrock, F. Schroder, S. Hirt, A. Haverich, C. Mittmann, and S. Herzig Single-channel properties of L-type calcium channels from failing human ventricle Cardiovasc Res, February 1, 1998; 37(2): 445 - 455. [Abstract] [Full Text] [PDF]
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K. R Sipido, T. Stankovicova, W. Flameng, J. Vanhaecke, and F. Verdonck Frequency dependence of Ca2+ release from the sarcoplasmic reticulum in human ventricular myocytes from end-stage heart failure Cardiovasc Res, February 1, 1998; 37(2): 478 - 488. [Abstract] [Full Text] [PDF]
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A. R.W. Money-Kyrle, C. H. Davies, H. K. Ranu, P. O'Gara, N. Singh Kent, P. A. Poole-Wilson, and S. E. Harding The role of cAMP in the frequency-dependent changes in contraction of guinea-pig cardiomyocytes Cardiovasc Res, February 1, 1998; 37(2): 532 - 540. [Abstract] [Full Text] [PDF]
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C. F. McTiernan, B. H. Lemster, C. Frye, S. Brooks, A. Combes, and A. M. Feldman Interleukin-1ß Inhibits Phospholamban Gene Expression in Cultured Cardiomyocytes Circ. Res., October 19, 1997; 81(4): 493 - 503. [Abstract] [Full Text]
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R. H. G. Schwinger, K. Brixius, U. Bavendiek, S. Hoischen, J. Müller-Ehmsen, B. Bölck, and E. Erdmann Effect of Cyclopiazonic Acid on the Force-Frequency Relationship in Human Nonfailing Myocardium J. Pharmacol. Exp. Ther., October 1, 1997; 283(1): 286 - 292. [Abstract] [Full Text]
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R. J. Hajjar, U. Schmidt, J. X. Kang, T. Matsui, and A. Rosenzweig Adenoviral Gene Transfer of Phospholamban in Isolated Rat Cardiomyocytes : Rescue Effects by Concomitant Gene Transfer of Sarcoplasmic Reticulum Ca2+-ATPase Circ. Res., August 19, 1997; 81(2): 145 - 153. [Abstract] [Full Text]
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K. Brixius, M. Pietsch, S. Hoischen, J. Muller-Ehmsen, and R. H. G. Schwinger Effect of inotropic interventions on contraction and Ca2+ transients in the human heart J Appl Physiol, August 1, 1997; 83(2): 652 - 660. [Abstract] [Full Text] [PDF]
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K. L. Koss and E. G. Kranias Phospholamban: A Prominent Regulator of Myocardial Contractility Circ. Res., December 1, 1996; 79(6): 1059 - 1063. [Full Text]
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M. Flesch, R. H.G. Schwinger, F. Schiffer, K. Frank, M. Sudkamp, F. Kuhn-Regnier, G. Arnold, and M. Bohm Evidence for Functional Relevance of an Enhanced Expression of the Na+-Ca2+ Exchanger in Failing Human Myocardium Circulation, September 1, 1996; 94(5): 992 - 1002. [Abstract] [Full Text]
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Z. Wang, B. Nolan, W. Kutschke, and J. A. Hill Na+-Ca2+ Exchanger Remodeling in Pressure Overload Cardiac Hypertrophy J. Biol. Chem., May 18, 2001; 276(21): 17706 - 17711. [Abstract] [Full Text] [PDF]
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C. Communal, M. Sumandea, P. de Tombe, J. Narula, R. J. Solaro, and R. J. Hajjar Functional consequences of caspase activation in cardiac myocytes PNAS, April 30, 2002; 99(9): 6252 - 6256. [Abstract] [Full Text] [PDF]
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 K. DAVIA, R. J. HAJJAR, C. M. N. TERRACCIANO, N. S. KENT, H. K. RANU, P. O'GARA, A. ROSENZWEIG, and S. E. HARDING Functional alterations in adult rat myocytes after overexpression of phospholamban with use of adenovirus Physiol Genomics, August 31, 1999; 1(2): 41 - 50. [Abstract] [Full Text] [PDF]
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