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Cardiovascular Research 1999 43(1):67-76; doi:10.1016/S0008-6363(99)00053-X
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Copyright © 1999, European Society of Cardiology

Regional expression of phospholamban in the human heart

Peter Bokníka,*, Claus Unkela, Uwe Kirchhefera, Ulrich Kleideitera, Oliver Klein-Wielea, Jörg Knappa, Bettina Lincka, Hartmut Lüssa, Frank Ulrich Müllera, Wilhelm Schmitza, Ute Vahlensiecka, Norbert Zimmermannb, Larry R. Jonesc and Joachim Neumanna

aInstitut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität, Domagkstraße 12, D-48129 Münster, Germany
bKlinik für Thorax- und Kardiovaskuläre Chirurgie, Heinrich Heine-Universität, Moorenstraße 5, D-40001 Düsseldorf, Germany
cKrannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

* Corresponding author. Tel.: +49-251-835-5503; fax: +49-251-835-5501.

Received 30 July 1998; accepted 11 January 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Background: Several independent lines of evidence indicate that phospholamban (PLB) expression correlates positively with depression of force of contraction and duration of contraction in isolated cardiac preparations of several animal species. Here, we studied whether PLB levels correlate with attenuation of contractility and enhancement of contractile time parameters in different parts of the human heart. Methods: Force of contraction was measured in isolated electrically driven atrial and ventricular preparations from human hearts. Ca2+-uptake by human atrial and ventricular homogenates was assayed at different ionized Ca2+-concentrations. Protein expression of PLB and the sarcoplasmic Ca2+-ATPase (SERCA) was measured in homogenates by quantitative immunoblotting using specific antibodies. PLB mRNA expression was quantified in human cardiac preparations by Northern blot analysis. Results: The duration of contraction in isolated preparations of human right ventricle (RV) was double that found in right atrial preparations (RA) (620±25 ms versus 308±15 ms). In RA, PLB expression was reduced by 44% at the protein level and by 34% at the mRNA level compared to RV. In contrast, the SERCA protein content was increased by 104% in RA compared to RV. Ca2+-uptake at low ionized Ca2+-concentration, where the inhibiting effect of PLB is maximal, amounted to 1.39±0.28 nmol Ca2+/mg protein in RA and to 0.62±0.09 nmol Ca2+/mg protein in RV (n=6 both). Conclusions: It is suggested that duration of contraction is shorter in human atrium versus ventricle due to the combined effect of decreased PLB levels (which inhibits SERCA function) and increased SERCA levels. The lower relative ratio of PLB to SERCA leads to less inhibition of SERCA and increased Ca2+-uptake which enhances relaxation and contraction in human atrium.

KEYWORDS Atrial function; Contractile function; Gene expression; SR (function); Ventricular function


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Ca2+ is a prominent regulator of cardiac contractility. Many interventions that increase force of contraction in the heart, such as sympathetic innervation and pharmacological interventions increase force of contraction by increasing intracellular Ca2+. For example, digitalis, phosphodiesterase inhibitors and catecholamines elevate force of contraction by increasing intracellular Ca2+-levels. Elevated Ca2+ binds to troponin C of the myofilaments to initiate the generation of force. β-Adrenoceptor agonists elevate Ca2+ by stimulating cAMP generation, by subsequent activation of protein kinase A (PKA) and phosphorylation of regulatory proteins. The predominant protein phosphorylated in the sarcoplasmic reticulum (SR) by PKA is phospholamban (PLB) the major function of which is to regulate the activity of the SR Ca2+-pump (SERCA). In metabolically [32P]-labeled isolated guinea-pig hearts β-adrenergic stimulation phosphorylates PLB and at the same time increases the Ca2+-uptake into the SR [1]. PLB in the dephosphorylated state is an inhibitor of the Ca2+-pump; phosphorylation of PLB by PKA or Ca2+/calmodulin dependent protein kinase reverses the inhibition, increasing the rate of Ca2+-transport into the SR, and thus augmenting the rate of cardiac relaxation as well as contraction [2,3].

The evidence in favour of a link between phospholamban and force of contraction is strengthened by genetic experiments from several groups. Overexpression of PLB reduces basal contractility and reduces Ca2+-transients whereas the opposite is noted by PLB ablation [4–6]. Hence, it is a central tenet that PLB is closely linked to contractile force. Moreover, PLB is also tightly linked to time parameters of contraction. Overexpression of PLB prolongs the duration of contraction by both elevating time to peak tension and time of relaxation [4,5]. The link is substantiated by the fact that the absence of PLB in myocardium shortens time to peak tension and time of contraction and enhances Ca2+-uptake into the SR [4,6]. It has also been demonstrated that subsequent changes of SERCA expression can also contribute to these effects [7]. Using a transgenic animal model, we noted that overexpression of PLB in heart was accompanied by reduced expression of SERCA [6].

Another line of evidence for a prominent role of PLB to regulate force of contraction and time parameters is derived from observations in atria and ventricles. In wild type mouse atrium the PLB content is very low in comparison to the ventricle [5,8,9]. Fittingly, the duration of contraction is much shorter in atrium than in ventricle in mouse and SR vesicles from atria exhibit increased rates of Ca2+-transport at low ionized Ca2+-concentration, because the inhibitory effect of PLB on the Ca2+-pump is absent. It has not yet been reported in humans, whether similar differences in atrial versus ventricular contractility are present and whether these potential differences can be correlated with different levels of PLB and SERCA.

Hence, we addressed the question of whether PLB expression is altered in different chambers of the human heart and whether these differences parallel those in Ca2+-transport and contractile time parameters. For comparison, we also measured SERCA expression and the ratio of PLB and SERCA. We report here that PLB and SERCA expression is regionally regulated in the human heart.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
Procedures for obtaining human tissue conforms with the principles outlined in the Declaration of Helsinki. The study is in accordance with the guidelines from the local ethics’ committee and patients gave written informed consent. Nonfailing hearts were obtained from prospective organ donors whose hearts could not be used owing to surgical reasons or blood group incompatibility. On inspection these hearts appeared to have normal ventricles. Aortic and pulmonary valves were excised from nonfailing hearts and used for valve replacement.

Failing hearts were obtained from patients undergoing orthotopic heart transplantation due to end-stage heart failure (NYHA IV) resulting from idiopathic dilated cardiomyopathy (IDC) or ischemic cardiomyopathy (ICM). Care was taken not to take scarred, fibrotic or adipose tissue, endocardium or large vessels. Medical treatment for the patients consisted of nitrates, cardiac glycosides, diuretics and angiotensin-converting enzyme inhibitors. The tissue samples for mRNA and protein quantification were frozen in liquid nitrogen immediately after cardiectomy in the operating room. For contractile experiments, atrial and ventricular tissue was placed in ice-cold gassed bathing solution (composition, see below) and delivered to the laboratory within 10 min.

2.1 Contraction experiments
Contraction experiments were performed as described previously [10]. In brief, trabeculae carneae were isolated from right atria and right ventricles (diameter less than 1 mm, length 5–8 mm) which were dissected in gassed bathing solution. The bathing solution contained (in mM): NaCl 119.8, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaH2PO4 0.42, NaHCO3 22.6, Na2EDTA (ethylenediaminotetraacetic acid) 0.05, ascorbic acid 0.28, glucose 5.05, continuously gassed with 95% O2 and 5% CO2 and maintained at 35°C (pH 7.4). Preparations were attached by threads to a force transducer and suspended individually in 10 ml glass tissue chambers for recording isometric contractions. Isometric force of contraction was measured after preloading each muscle to optimal length. Resting force (about 5 mN) was kept constant throughout the experiments. Preparations were electrically driven by field stimulation at 0.5 Hz with rectangular pulses of 5 ms duration, the voltage was about 10–20% greater than threshold. All preparations were equilibrated in bathing solution until complete stabilization (60–90 min). During this period the bathing solution was changed every 15 min. The time course of isometric contraction was calculated using twitches recorded at high chart speed.

2.2 Ca2+-uptake measurement
Frozen tissue was homogenized three times for 30 s at 4°C in buffer containing (in mmol/l) sucrose 250, histidine 10, NaF 10 with a Polytron PT-10 (Kinematica, Luzern, Switzerland). 100 µl aliquots of homogenate protein were incubated with 25 µl of anti PLB monoclonal antibody 2D12 [11] or antibody control buffer for 20 min on ice. To initiate Ca2+-uptake, 100 µl of pretreated homogenate sample was added to 500 µl of Ca2+-uptake medium containing 50 mmol/l MOPS (pH 7.0), 3 mmol/l MgCl2, 100 mmol/l KCl, 10 mmol/l oxalate, 0.5 mmol/l EGTA, 5 mmol/l NaN3, and 3 mmol/l ATP at 37°C. For time course assays of Ca2+-uptake, 0.05 mmol/l with tracer [45Ca2+] was included giving an ionized Ca2+-concentration of 30 nmol/l. [45Ca2+] accumulated at different times was determined by filtration [8]. Free ionized Ca2+-concentration was calculated according to Bers [12]. Ca2+-uptake by atrial and ventricular homogenates was linear in range from 3 min to 23 min (data not shown). To measure the Ca2+-dependence of [45Ca2+]-transport, the EGTA concentration was set at 0.5 mmol/l in the presence of different concentrations of [45Ca2+] to give the desired ionized Ca2+-concentration (pCa values from 8.25 to 6.75) and Ca2+-uptake was determined by filtration at 8 min of incubation.

2.3 Preparation of homogenates for protein quantification
Frozen tissue was homogenized in 100 µl of 10 mM NaHCO3 by use of microdismembrator (B. Braun Melsungen, Melsungen, Germany), then 200 µl of 20% of SDS (sodium dodecyl sulphate) was added and protein was extracted at 25°C for 30 min. The samples were pelleted at 14 000xg at 4°C for 20 min and the protein concentration in the supernatant was assayed according to Lowry [13].

2.4 Gel electrophoresis and western blotting
SDS extracts made as described above were thawed and SDS buffer according to Laemmli [14] was added. The samples were heat-treated for 10 min at 30°C. Thirty µg (unless stated otherwise) of homogenate sample protein were loaded per lane. These amounts were in the linear range for PLB (cf. Fig. 3) and for SERCA2a (Fig. 4) for ventricular as well as atrial homogenates.


Figure 3
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  		Fig. 3 Immunological quantification of phospholamban (PLB) in human right atrium (RA) and right ventricle (RV). Autoradiogram (A), protein dependency of immunological signals (B). Human atrial and ventricular homogenates were prepared as described in Methods, subjected to gel electrophoresis and transferred to nitrocellulose. Nitrocellulose strips were treated with an antibody against PLB and subsequently with [125I]-labeled anti-mouse IgG. Radioactive bands were excised from nitrocellulose sheet and bound radioactivity was quantitated by gamma-counting.

 

Figure 4
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  		Fig. 4 Immunological quantification of sarcoplasmic Ca2+-ATPase (SERCA) in human right atrium (RA) and right ventricle (RV). Autoradiogram (A), protein dependency of immunological signals (B). Human atrial and ventricular homogenates were prepared as described in Methods, subjected to gel electrophoresis and transferred to nitrocellulose. Nitrocellulose strips were treated with an antibody against SERCA2a and subsequently with [125I]-labeled protein A. Radioactive bands were excised from nitrocellulose sheet and bound radioactivity was quantitated by gamma-counting.

 
Gels were run according to Movsesian [15] using 8% polyacrylamide separating gels. Electrophoresis was initially run at 40 mA per gel for 30 min and then the current was increased to 60 mA. Proteins were electrophoretically transferred to nitrocellulose membranes (TA 85, 0.45 µM pore size, Schleicher & Schuell, Dassel, Germany) in 50 mM sodium phosphate buffer (pH 7.4) 180 min at 1.5 A at 4°C as reported [16]. Then membranes were blocked in Tris buffered saline containing 2.0% bovine serum albumin (BSA) for 30 min and incubated overnight at 4°C with antibodies directed against PLB (monoclonal A1, PhosphoProtein Research, Bardsey, England [17]), SERCA (2A7-A1, [18]) and calsequestrin (CSQ, [19]), PLB-antibody was detected using [125I]-labeled goat-anti-mouse IgG (ICN Biomedicals, Eschwege, Germany), SERCA- and CSQ-antibody using [125I]-labeled protein A (ICN Biomedicals, Eschwege, Germany). Radioactive bands identified by autoradiography were excised from nitrocellulose sheet and bound radioactivity was quantitated by gamma-counting (Cobra II, Canberra-Packard, Dreieich, Germany). Background counts, which were less than 15% of total counts for each band, were substracted from all measurements.

2.5 Total RNA preparation
In order to extract total RNA a modification of the method described by Chomczynski and Sacchi [20] was employed. Frozen tissue was homogenized using a microdismembrator (B. Braun Melsungen, Melsungen, Germany) in TriStar ReagentTM (AGS, Heidelberg, Germany) containing guanidinium thiocyanate and phenol. Total RNA was extracted according to the manufacturer’s instruction.

2.6 cDNA probes
The cDNA probes for phospholamban and human atrial natriuretic factor (ANF) were constructed by reverse transcription-polymerase chain reaction (RT-PCR). First strand cDNA was reverse transcribed from 1 µg of total human ventricle mRNA in 10 µl of 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6.0 mM MgCl2, 1.0 mM each dNTP (Pharmacia, Uppsala, Sweden), 5.0 mM DL-dithiothreitol, 50 µg/ml bovine serum albumin, 10 units of human placental RNAse inhibitor (AGS, Heidelberg, Germany) and 30 units of TrueScriptTM reverse transcriptase (AGS, Heidelberg, Germany) at 41°C for 60 min. Primers based on the published cDNA sequences for human PLB [21] and human ANF [22] were employed to generate specific probes.

Primers for PLB:
Forward Reverse
5'-GGCAAGCTGCAGATCTAGAGG-3' 5'-GATGATCACAGCTGCCAAGGC-3'

Primers for ANF:
Forward Reverse
5'-CTCCACCACCACCGTGAGCTT-3' 5'-TCTGTCCATGGTGCTGAAGTT-3'

All PCR reactions were carried out in a total volume of 50 µl containing 20 mM Tris–HCl (pH 8.55 at 25°C), 16 mM (NH4)2SO4, 200 µM each dNTP, 1.5 mM MgCl2 and 1.5 units Taq DNA polymerase (AGS, Heidelberg, Germany). Each reaction was subjected to 30 cycles of denaturation (1 min at 94°C), annealing (2 min) and extension (2 min at 72°C). All PCR reactions were performed in a thermal cycler (Omnigene TR3 CM220, MWG-Biotech, Ebersberg, Germany). The size of PCR-products was compared to DNA size markers (MBI Fermentas, Vilnius, Latvia). Single bands of the expected size were obtained. PCR-products were cut out from agarose gels and purified by dialysis [23]. In order to confirm the identity of PCR-products cycle sequencing using AmplitaqTM-FS DNA polymerase (Applied Biosystems, Weiterstadt, Germany) and a ABI PRISM-310 automated sequencer (Applied Biosystems, Weiterstadt, Germany) was performed. Purified probes were labeled with [32P]-{alpha}-dCTP (NEN DuPont, Bad Homburg, Germany) by random priming (Megaprime-kit, Amersham Buchler, Braunschweig, Germany) and unbound radioactivity was separated by gel filtration with SephadexTM G-50 DNA grade (Pharmacia, Uppsala, Sweden).

2.7 Northern blotting and hybridization
Twenty µg (unless otherwise stated) of total RNA were separated on 1% denaturing agarose gels and transferred to HybondTM N nylon membranes (Amersham Buchler, Braunschweig, Germany) by capillary transfer in 20xSSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0). Transfer was controlled on an ultraviolet transilluminator.

Membranes were prehybridized in a solution containing 50% deionized formamide, 5xDenhardt solution, 0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA, 0.2 mg/ml tRNA from yeast and 0.1% SDS at pH 7.4. Hybridization was performed overnight at 42°C in the same buffer containing labeled probes (specific activity 3.5–8.0x108 dpm/µg). The membranes were washed twice in 2xSSC, 0.1% SDS at room temperature, followed by 15 min washing in 2xSSC, 0.1% SDS at 65°C. The blots were washed three times to a final stringency of 0.2xSSC, 0.1% SDS at 65°C and exposed to PhosphorImagerTM-screens. Bound radioactivity was visualized and quantified in a PhosphorImagerTM using ImageQuantTM software (version 3.30, Molecular Dynamics, Krefeld, Germany). The blots were hybridized subsequently against PLB and ANF. For further hybridization, the membranes were stripped by boiling 0.1% SDS. In order to normalize the amount of RNA bound to membranes, all blots were also hybridized against 18S ribosomal RNA as described [24].

2.8 Data analysis
Data shown are means±SEM. Statistical significance was estimated with Student’s t-test for paired or unpaired observations. In addition, one way analysis of variance was followed by Bonferroni’s test as appropriate. A p-value <0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
Fig. 1 depicts original tracings of single contractions of human cardiac preparations at high time resolution. It is obvious that time parameters like time to peak tension, time of relaxation and total time of contraction are markedly longer in isolated electrically driven preparations from human ventricle compared to atrium. Several experiments confirmed these dramatic regional differences and, as summarized in Table 1, total time of contraction is doubled in right ventricle compared to right atrium.


Figure 1
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  		Fig. 1 Time course of isometric contraction in isolated electrically driven (0.5 Hz) preparations from human right atrium and right ventricle. Contraction experiments were performed as described in Methods and the time course of isometric contraction was recorded at high chart speed. TPT, time to peak tension; TR, time of relaxation. RA, right atrium; RV, right ventricle.

 

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  		Table 1 Time parameters of isometric contraction in isolated electrically driven preparations from human right atrium and right ventricle. Contraction experiments were performed as described in Methods and the time course of isometric contraction was recorded at high chart speed

 
Since the sarcoplasmic reticulum plays a crucial role in maintaining Ca2+-homeostasis, we compared Ca2+-uptake by SR vesicles in human atrial and ventricular homogenates. Time course studies confirmed the linearity of our assay system in range from 3 min to 23 min (data not shown). Ca2+-dependence of Ca2+-uptake in the absence and in the presence of PLB monoclonal antibody 2D12 was determined at 8 min incubation. The PLB monoclonal antibody reverses the inhibition of the Ca2+-pump by PLB at low ionized Ca2+-concentration, giving similar effects as PLB phosphorylation [25]. At low ionized Ca2+-concentration (30 nmol/l free Ca2+, 8 min incubation), a concentration at which the inhibitory effect of PLB on SERCA is most pronounced [11,25,26], Ca2+-uptake was markedly higher in human right atrial homogenates. Addition of PLB antibody reversed the PLB-inhibition and stimulated Ca2+-uptake to about the same maximal level in atrial and ventricular homogenates (Fig. 2A). Fig. 2B plots the fold stimulation of Ca2+-uptake by PLB antibody at different ionized Ca2+-concentrations. At low ionized Ca2+-concentrations stimulation of Ca2+-uptake by PLB antibody was much higher in ventricular homogenates compared to atrial homogenates due to greater depression of Ca2+-transport at low ionized Ca2+-concentration. In contrast, no difference in Ca2+-transport rates was observed at high ionized Ca2+-concentrations, concentrations at which the extent of antibody stimulation did not differ between atrial and ventricular homogenates.


Figure 2
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  		Fig. 2 Ca2+-uptake by right atrial (RA) and right ventricular (RV) homogenates in the absence (Control) or in the presence of PLB monoclonal antibody (2D12) at 30 nmol/l of ionized Ca2+ (2A), fold-stimulation of Ca2+-uptake by the PLB monoclonal antibody 2D12 determined at different ionized Ca2+-concentrations (2B). Homogenates were preincubated with PLB monoclonal antibody or control buffer for 20 min on ice, ionized Ca2+-concentration was set using 0.05 mmol/l EGTA and different concentrations CaCl2. Ca2+-uptake was assayed by filtration at 8 min of incubation. *p<0.05 versus RV, {dagger}p<0.05 versus Control.

 
Next, we addressed whether these regional differences in contractility and Ca2+-uptake were correlated with altered expression of PLB and/or SERCA in the tissues. To verify the linearity of protein detection we performed western blots in pooled homogenates from five different human hearts. Fig. 3 shows the results of quantitative immunoblotting of PLB in both atrium and ventricle. The original autoradiogram (Fig. 3A) and the results from several experiments (Fig. 3B) show that the detection of PLB is linear over a wide range of protein loaded per lane on the gel. The results demonstrate that the PLB content in human right atrium is lower compared to human right ventricle (cf. also Fig. 5A). In contrast, quantitative immunoblotting revealed that the content of SERCA2a is higher in human right atrium compared to right ventricle (Figs. 4 and 5BGo).


Figure 5
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  		Fig. 5 Protein expression of phospholamban (PLB, A) and sarcoplasmic Ca2+-ATPase (SERCA, B) in different chambers of human heart. Human atrial and ventricular homogenates were prepared as described in Methods, subjected to gel electrophoresis and transferred to nitrocellulose. For PLB-quantification, nitrocellulose strips were treated with an antibody against PLB and [125I]-labeled anti-mouse IgG. SERCA-expression was assayed using an antibody against SERCA2a and [125I]-protein A. Radioactive bands were excised from nitrocellulose sheet and bound radioactivity was quantitated by gamma-counting. The number in column indicates number of hearts. LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium. *p<0.05 versus RV, {dagger}p<0.05 versus LV, respectively as estimated by one way analysis of variance followed by Bonferroni’s test.

 
Based on the methodological experiments, 30 µg of homogenate protein was used to quantify PLB and SERCA. PLB was subsequently quantified in both tissues as one band at 25 kDa (probably the pentameric form). Data are summarized in Fig. 5. It is obvious that PLB expression (Fig. 5A) is similar in left and right atrium and similar in left ventricle and right ventricle. However, in atria the expression is consistently lower than in ventricles. SERCA expression on the protein level is similar in right ventricle and left ventricle. The expression tended to be somewhat higher in right atria than in left atria. However, the most important finding in this figure is that SERCA expression in left ventricle is lower than in left atria and SERCA expression is lower in right ventricle compared to right atria. In these data results from failing and nonfailing hearts are pooled. This seems to be legitimate as subgroup analysis reveals that PLB protein levels are not different between failing and nonfailing human hearts. Namely protein expression of PLB was 3873±752 cpm in nonfailing (n=7), 3358±521 cpm in idiopathic dilated cardiomyopathy (IDC, n=12) and 2803±521 cpm in ischemic cardiomyopathy (ICM, n=4) in human right ventricular preparations. Similar data were obtained for SERCA on protein level. They amounted to 11296±2266 cpm in nonfailing (n=7), 11435±1489 cpm in IDC (n=12) and 11450±1014 cpm in ICM (n=4) in human right ventricular preparations. This is in agreement with previous findings from our group [15,24]. Therefore, to gain higher statistical power, data from failing and nonfailing preparations were combined for all regions of the hearts. In right atria, PLB expression at the protein level was reduced by 44% compared to right ventricle. In contrast, the SERCA protein content was increased by 104% in right atria compared to right ventricles.

Calsequestrin, a protein located within the lumen of sarcoplasmic reticulum, is also tightly linked to the regulation of Ca2+-homeostasis. We have shown that calsequestrin (CSQ)-overexpression is accompanied by prolonged relaxation [30]. Thus, differences in CSQ-expression might also contribute to the observed differences between atria and ventricles. However, protein expression of CSQ was similar in atria and ventricles and amounted to 8 924±1 461 cpm in human right atria (n=7) and to 10 819±1 144 cpm in human right ventricles (n=12).

For comparison, we also investigated PLB-expression on the mRNA level. A representative experiment is seen in Fig. 6. The first two lanes from the left indicate that equal amounts of total RNA are loaded per lane. This is supported by hybridization of the 18S ribosomal RNA. Moreover, three transcripts of PLB mRNA at 3.3, 1.9 and 0.6 kb were detectable in agreement with our previous report [24]. Finally, total RNA was hybridized with a probe directed against atrial natriuretic factor (ANF). As reported by others before, it is obvious that the ANF expression is much higher in atria compared to ventricle [28,29].


Figure 6
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  		Fig. 6 mRNA-expression of phospholamban (PLB) and atrial natriuretic factor (ANF) in human right ventricle (RV) and right atrium (RA). Total RNA was isolated as described in Methods and 20 µg were separated on 1% denaturing agarose gels and transferred to HybondTM N nylon membranes. The blots were hybridized subsequently with [32P]-labeled probes against PLB, ANF and 18S ribosomal RNA. Bound radioactivity was visualized in a PhosphorImagerTM using ImageQuantTM software. EtBr, ethidium bromide stain.

 
Data on PLB expression at mRNA level (combined for all three transcripts) are summarized in Fig. 7. Qualitatively similar findings were noted as on the protein level. Namely PLB mRNA was similar in right atria versus left atria and in right ventricle versus left ventricle. However, PLB expression at the mRNA level in right atria was 66% of expression in ventricle. The opposite was noted for atrial natriuretic factor (ANF): ANF mRNA expression in right atria did not differ between nonfailing, IDC and ICM hearts and amounted to (in arbitrary units) 127.2±15.0 (n=23). mRNA levels for ANF in ventricles were markedly lower and amounted to (in arbitrary units) 3.3±1.5 in right ventricles from nonfailing hearts (n=7) and 7.7±1.8 in right ventricles from failing hearts (IDC and ICM, n=16), respectively.


Figure 7
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  		Fig. 7 mRNA expression of phospholamban (PLB) in different chambers of human heart. Total RNA was isolated as described in Methods and 20 µg were separated on 1% denaturing agarose gels and transferred to nylon membranes. The blots were hybridized with [32P]-labeled probes against PLB and subsequently against 18S ribosomal RNA. Bound radioactivity was visualized in a PhosphorImagerTM and quantitated using ImageQuantTM software. Data were normalized to the 18S ribosomal RNA. The number in column indicates number of hearts. LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium. *p<0.05 versus RV, {dagger}p<0.05 versus LV, respectively as estimated by one way analysis of variance followed by Bonferroni’s test.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
The main new finding of the present work is that the PLB content is different between atrium and ventricle in the human heart. PLB expression correlates with regional parameters of Ca2+-transport and duration of contraction. We suggest that the regional differences in time parameters of contraction are causally linked to differences in Ca2+-handling and PLB expression between atria and ventricles.

This causal linkage is based on several independent lines of evidence. For instance, PLB levels can be reduced by chronic β-adrenergic stimulation [31], by L-triiodothyronine application [7] or removed by genetic disruption of the PLB gene [6]. All these interventions shorten time parameters. In contrast, PLB levels can be increased by iodine-deficient diet [7] or genetic overexpression [4,5]. In both latter cases duration of contraction is prolonged. Thus, all these approaches, relying on use of completely different experimental models and methodologies, concur that the level PLB in mammalian heart dramatically regulates myocardial contractility. On the other hand, the expression of cardiac myosin heavy chain (MHC) isoforms is also under hormonal control [32]. Hence, alterations of MHC-pattern could also contribute to the contractile alterations as observed under conditions of hyper- or hypothyroidism.

A distinct regional distribution of PLB has been noted before in mice. In normal mouse atrium versus ventricle, the levels of SERCA2a are equivalent. The PLB content in mouse atrium is substantially decreased, however, and cardiac contraction and relaxation times are shorter than in ventricle [8,9]. Hence, the present data qualitatively agree with results in mice heart. In addition, SERCA protein expression is higher in human atrium than ventricle. Thus, the ratio of PLB/SERCA was three times greater in human ventricle compared to human atrium (0.10±0.02 in human atrium versus 0.35±0.05 in human ventricle). The higher relative ratio of PLB to SERCA in ventricle may reflect a greater portion of SERCA in the inhibited state at low ionized Ca2+-concentration. Consistent with this, we observed that the rate of Ca2+-uptake by ventricular homogenates was reduced at low ionized Ca2+-concentrations compared to atrial homogenates. Furthermore, stimulation of Ca2+-transport by PLB antibody at low ionized Ca2+-concentrations was markedly accentuated in ventricular homogenates [11,26,27]. At high ionized Ca2+-concentrations, there is little inhibition of Ca2+-uptake by PLB. In agreement with this mechanism of SERCA inhibition by PLB [26,27] we found no difference between atrial and ventricular homogenates in antibody stimulation at high ionized Ca2+-concentrations. The rate of Ca2+-transport, however, did not significantly differ between atrium and ventricle although SERCA expression was markedly higher in atrial homogenates. The reason for this discrepancy is not clear. We suggest that higher amounts of Ca2+ are sequestered by atrial sarcoplasmic reticulum due to substantially higher SERCA activity. In contrast, the expression of CSQ did not differ between atrium and ventricle indicating comparable Ca2+-binding capacity. Hence, one could speculate that at high Ca2+-concentrations no further Ca2+ can be taken up into the atrial SR due to saturation of Ca2+-binding capacity. In addition, differences in the expression of another Ca2+-handling proteins such as ryanodine receptor, triadin or junctin [33] may also play a role in this discrepancy.

Our finding that contraction in human atria is much shorter than in ventricle is consistent with previous reports on isolated human electrically driven preparations [34]. However, a caveat is in order. Regional differences in Ca2+-transport and protein expression of SERCA and PLB may not be the only explanation for differences in contractile time parameters between atrium and ventricle. It is well known that crossbridge cycling rate and the velocity of contraction are directly related to the type of the predominant myosin species. Adult human ventricular myosin contains almost exclusively β-myosin heavy chain (β-MHC), whereas {alpha}-myosin heavy chain ({alpha}-MHC) is predominantly expressed in the atrium [35]. Atrial myosin crossbridges having markedly higher ATPase activity differ in their velocity of transition from force generating into non-force generating states from the ventricular myosin. In addition, human ventricles and atria also express different isoforms of myosin essential and regulatory light chains [36]. Morano and coworkers [37] have shown that the expression of atrial myosin essential light chain is directly correlated with maximal shortening velocity of permeabilized human muscle fibers. On the other hand, the function of cardiac contractile proteins is modulated by their phosphorylation state and differences in the activity and/or expression of protein kinases and protein phosphatases can also contribute to regional differences of contractility [38]. In addition, differences between the electrophysiology of atrial and ventricular cardiomyocytes also exist. K+ outward currents are larger and action potential duration is shorter in atrial compared to ventricular preparations in many species including man [39,40]. Similarily, there are differences in the relative abundance of subunits Na+/K+-ATPase and Na+/Ca2+-exchanger between atrium and ventricle [41]. In addition, using two-dimensional gel electrophoresis 40 spots were resolved which differed significantly in intensity between atrium and ventricle [42]. Hence, it is possible that also other regional differences contribute to the longer duration of contraction in human ventricle compared to atrium. Our integrative comparison at various levels (contraction, Ca2+-transport, expression) strongly suggests that the regional differences in the expression of PLB and SERCA represent at least part of the subcellular basis for the different duration of contraction between atrium and ventricle. However, in future work it will be important to compare Ca2+-transients in contracting atrial and ventricular preparations. Moreover, parallel measurement of contractile parameters in preparations from nonfailing human atria and ventricle will be important.

Comparing failing and nonfailing ventricles we found no difference in PLB and SERCA2a expression at protein level. This confirms our previous data [14,25] and is also in agreement with the data from several another groups. This issue and especially SERCA2a protein expression, however, still remains controversial and several groups have reported decreased SERCA2a protein levels in human heart failure (for review see e.g. [25]). Several reasons for this discrepancy are currently the subject of a intensive discussion [25]. On the other hand, discrepancies between alterations in human heart failure and findings from different animal models of heart hypertrophy and failure also exist. Recently, we reported decreased protein expression of PLB and SERCA in a model of isoproterenol-induced heart hypertrophy [31]. We suggest that differences in the cause of heart failure and species differences may explain this discrepancy.

What could be the purpose of the regionally different expression of PLB and SERCA2a and the altered duration of contraction? It is known that the two genes are discoordinately regulated which is conceivably a phylogenetical remnant. Moorman and coworkers [43] have reported that during the rat fetal development the mRNAs encoding PLB and SERCA2a are expressed along the cardiac tube in virtually opposite patterns. SERCA2a mRNA is expressed at highest levels in the upstream part (inflow tract) of the cardiac tube and PLB prevailing in the downstream compartments (outflow tract). These authors suggest that slowly contracting outflow tract functions as a sphincter that substitutes for the semilunar valves to prevent regurgitation of the blood. This mechanism could preserve a unidirectional blood flow in the fetal heart. On the other hand the mechanism may still subserve a reasonal purpose in the mature heart containing cusps. In the cardiac cycle the contraction of the atria precedes that of the ventricles and the time of contraction is shorter. Hence, regional differences in Ca2+-uptake due to different expression of PLB and SERCA might contribute to this functionally meaningful faster contraction of atria in the cardiac circle.

The differential expression of PLB between atria and ventricles may be due to several factors, including tissue-specific expression of trans-acting factors, differential mRNA processing and differential mRNA or protein stability. Usually, the expression of eukaryotic genes is transcriptionally regulated [44]. Hence, the straightforward explanation for the present findings is that PLB expression is transcriptionally regulated and that there is a specific activator (transcription factor) in the ventricle which is less active in atria. Promoter analysis of the murine PLB gene suggests that PLB gene expression may be regulated by the interplay of cis-acting regulatory elements located within the 5'-flanking and intronic regions. Interestingly, the 7-kb upstream region was capable of directing cardiac specific and chamber specific expression in vivo [45].

In summary, we have shown that PLB expression is higher in human ventricle than in atrium and we suggest that this could be the biochemical basis for the differences in Ca2+-handling between the two tissues with resulting longer duration of contration in human ventricle compared to atrium.

Time for primary review 34 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
Supported by the Deutsche Forschungsgemeinschaft and by the Interdisziplinäres Klinisches Forschungszentrum Münster (TP B1, BMBF 01 KS9604). The excellent technical assistance of Cordula Vischedyk is gratefully ackowledged.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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