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Circulation. 1995;92:3220-3228

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(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

Robert H.G. Schwinger, MD; Michael Böhm, MD; Ulrich Schmidt, MD; Peter Karczewski, PhD; Udo Bavendiek, MS; Markus Flesch, MD; Ernst-Georg Krause, PhD; Erland Erdmann, MD

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.


*    Abstract
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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


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
down arrowResults
down arrowDiscussion
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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.


*    Methods
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 *Methods
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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+Phosphoenolpyruvate->ATP+Pyruvate. The reaction was catalyzed by the pyruvate kinase. (3) Pyruvate+NADH->Lactate+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 [{alpha}-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.


*    Results
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 up arrowMethods
*Results
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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 1Down 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 2Down). 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 2Down). The corresponding values for CSR were 125±7 and 163±8 nmol/mg protein per minute, respectively (P<.05; Fig 3Down). 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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  		Figure 1. Plot showing the concentration-response curve of the effect of increasing concentrations of CPA (0.01 to 10 µmol/L) on SERCA II activity in CSR from nonfailing and failing human myocardium. Ca2+-ATPase activity at control conditions (CPA, 0 µmol/L) and in the presence of 0.01 µmol/L CPA was significantly higher in nonfailing compared with failing myocardium (P<.05). CPA concentration-dependently inhibited SERCA II activity in human failing and nonfailing myocardium.



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  		Figure 2. Plot showing the concentration-response curve of the effect of increasing free Ca2+ (0.01 to 1000 µmol/L) on SERCA II activity in CSR from terminally failing human myocardium caused by DCM and in nonfailing control myocardium. Elevation of Ca2+ resulted in an increase in Ca2+-ATPase activity in failing and nonfailing myocardium. There was a significantly reduced maximal Ca2+-ATPase activity in CSR from terminally failing human myocardium. *P<.05.



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  		Figure 3. Bar graphs showing maximal SERCA II activity in CSR and VSR from terminally failing human myocardium resulting from DCM and in nonfailing control myocardium at 35 µmol/L free Ca2+. Maximal Ca2+-ATPase activity was significantly reduced in CSR from terminally failing human myocardium. *P<.05.

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 4Down) 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 4Down). 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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  		Figure 4. Bar graphs showing maximal Ca2+-induced Ca2+-uptake in VSR and CSR from terminally failing human myocardium resulting from DCM and in nonfailing control myocardium at 1 µmol/L free Ca2+ concentration. Maximal Ca2+-induced Ca2+ uptake was significantly reduced in CSR from terminally failing human myocardium. *P<.05.

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 5Down 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 6Down shows representative Western blots. The antibody-marked lanes were measured by densitometry; the data are given in Fig 7Down. 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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  		Figure 5. Western blots showing the protein dependency of immunochemical detection of SERCA II (upper level) and phospholamban (PLB) in VSR (5 to 15 µg per lane; A) and CSR (5 to 20 µg per lane; B) from human myocardium. Membranes were separated on SDS-PAGE before electrophoretic transfer to nitrocellulose membranes. There was a linear correlation (P<.01) between optical density and protein content for both phospholamban and SERCA II.



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  		Figure 6. Western blots of original experiment of immunochemical detection of SERCA II and phospholamban (PLB) in CSR (10 µg per lane) from patients with DCM and from nonfailing control myocardium. Membranes were separated on SDS-PAGE before electrophoretic transfer to nitrocellulose membranes. Molecular weight standards are described in "Methods." There was no statistically significant difference between failing and nonfailing tissue.



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  		Figure 7. Bar graphs showing the results of Western blot analysis of immunodetectable SERCA II and phospholamban in VSR and CSR from patients with DCM and from nonfailing control myocardium (NF). There was no statistically significant difference between groups for either SERCA II or phospholamban levels in VSR or CSR.

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 8Down 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 9Down 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 TableDown). 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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  		Figure 8. Northern blot analysis showing SERCA II and phospholamban. Top lane, representative hybridization signals for SERCA II that appeared just below the position of the 28S RNA corresponding to an mRNA size of approximately 4.0 kb. Second lane, representative signals for phospholamban that appeared at a position corresponding to 3.3. kb. The signal for GAPDH mRNA was used as an internal standard (third lane). PLB indicates phospholamban.



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  		Figure 9. Bar graph showing the results of Northern blot analysis for SERCA IIa and phospholamban mRNA/GAPDH mRNA in human failing and nonfailing myocardium. Both phospholamban mRNA and SERCA IIa mRNA levels were significantly reduced in failing human myocardium compared with nonfailing tissue. *P<.05.


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  		Table 1. mRNA Levels of SERCA II and Phospholamban in Myocardium From Patients With DCM and in Nonfailing Myocardium


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
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.


*    Selected Abbreviations and Acronyms
 
CPA = cyclopiazonic acid
CSR = crude membrane SR preparations
DCM = dilated cardiomyopathy
SERCA II = sarcoplasmic reticulum Ca2+-ATPase
SR = sarcoplasmic reticulum
VSR = isolated SR vesicles


*    Acknowledgments
 
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.


*    Footnotes
 
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.


*    References
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*References
 

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