Cardiovascular Research

Cardiovascular Research 1997 36(2):216-222; doi:10.1016/S0008-6363(97)00180-6
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Effects of -adrenergic stimulation on the sarcolemmal Na⁺/Ca²⁺-exchanger in adult rat ventricular cardiocytes

Hans Reinecke^a,*, Roland Vetter^b and Helmut Drexler^a

^aUniversität Freiburg, Innere Medizin III, Kardiologie und Angiologie, Freiburg, Germany
^bMax-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin-Buch, Germany

^* Corresponding author. Present address: University of Washington, Department of Pathology, Box 357470, Room E520 HSB, Seattle, WA 98195-7335, Tel. (+1-206) 6168684; Fax (+1-206) 5433644; E-mail: hreineck@u.washington.edu

Received 17 February 1997; accepted 6 June 1997

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The cardiac sarcolemmal Na⁺/Ca²⁺-exchanger (NCX) plays an important role in the maintenance of the myocardial Ca²⁺ homeostasis which is altered in cardiac hypertrophy and failure. The aim of the present study was to investigate whether -adrenergic stimulation known to induce cardiac hypertrophy might be involved in the regulation of the sarcolemmal NCX. Methods: Adult rat ventricular cardiocytes (ARC) were isolated from male Sprague–Dawley rats. Phenylephrine, an -adrenergic agonist, was used as hypertrophic agent. NCX expression was measured by competitive RT-PCR and Western blot analysis. Results: -Adrenergic stimulation of ARC with 10 µM phenylephrine for 24 h resulted in a significant increase of the NCX mRNA (2.5-fold) and the NCX protein level (1.8-fold). The changes on the expression level were blocked by the ₁-adrenoceptor antagonist prazosin. Conclusions: The data demonstrate that the NCX expression level is up-regulated by the activation of the -adrenergic signal transduction pathway. The increased NCX mRNA level induced by -adrenergic stimulation appeared to be translated into an increased NCX protein level.

KEYWORDS Na⁺/Ca²⁺-exchanger; Rat; Cardiocytes; -adrenoceptor; Expression

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The pathophysiology of heart failure is closely associated with neuroendocrine changes [1]. These changes involve the activation of the adrenergic system leading to increased plasma concentrations of catecholamines and, thereby, serving as a compensatory mechanism for the failing circulation. However, overshooting activation is likely to have negative long-term effects [2]. Therefore, it is worth to investigate whether disturbances of the myocardial Ca²⁺ homeostasis, which occur in experimental cardiac hypertrophy as well as in human heart failure [3], are affected by catecholamines.

The cardiac sarcolemmal Na⁺/Ca²⁺-exchanger (NCX) plays an important role in the control of Ca²⁺ fluxes across the sarcolemma of the cardiomyocyte and, therefore, in the control and maintenance of the myocardial Ca²⁺ homeostasis [4, 5]. Previous studies have presented evidence for an increase of the NCX gene expression and/or activity in animal models of pressure-overload induced hypertrophy [6, 7]. Studies from our laboratory have shown that the NCX gene expression and the functional activity of the antiporter are increased in patients with severe heart failure [8, 9]. It was hypothesised that the increased NCX expression and function in failing hearts might, in part, compensate for depressed sarcoplasmic reticulum (SR) Ca²⁺-ATPase function, implying that an increased NCX expression in the failing heart might limit intracellular Ca²⁺ overload during diastole and improve relaxation [8, 9].

Several experimental studies have shown that cardiac hypertrophy could be initiated and maintained by chronic infusion of adrenergic agonists (for a review, see [10]). In vitro studies using neonatal cardiocytes have clearly demonstrated load-independent hypertrophic effects of ₁-adrenergic agonists. The growth effect was characterised by increased rates of protein synthesis, myocyte surface area, protein content, total transcriptional activity [10], and induction of protooncogene gene expression [11, 12]. The hypertrophic response to ₁-adrenergic stimuli also involved the selective up-regulation of the early developmental contractile protein isogenes, skeletal -actin, and β-myosin heavy chain genes [13–15]. Increased protein synthesis due to ₁-adrenoceptor stimulation was also shown for adult rat cardiocytes [16]. However, regarding a transcriptional activation by ₁-adrenergic agonists the data are inconsistent [16–18]. ₁-Adrenergic activation is known to activate protein kinase C [19], and to generate increased levels of inositol trisphosphates, which in turn induce the release of Ca²⁺ from intracellular stores [20, 21]. Since those effects were blocked by the ₁-specific antagonist prazosin they are likely to be mediated by ₁-adrenoceptor subtype. In addition, there is no evidence for an ₂-adrenoceptor subtype in rat cardiac myocytes [22, 23].

In summary, ₁-adrenergic agonists were shown to exert potent hypertrophic effects on cardiocytes. The goal of the present study was to investigate whether ₁-adrenergic stimulation might be involved in the regulation of the sarcolemmal NCX expression in isolated adult rat cardiocytes.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Preparation of adult rat ventricular cardiocytes (ARC)
Ca²⁺-tolerant ARC were isolated according to Powell [24]with modifications of Kammermeier and Rose [25]. Briefly, hearts were rapidly removed from adult male Sprague–Dawley rats (SAVO, Kisslegg, Germany) weighing 380 to 440 g following pentobarbital anesthesia (80 mg/kg), washed in Ca²⁺-free perfusion buffer (PB) (128.2 mM NaCl, 4 mM KCl, 0.19 mM NaH₂PO₄, 1.01 mM Na₂HPO₄, 1.39 mM MgSO₄, 10 mM HEPES, 5.5 mM D-glucose, 2 mM pyruvate, 12.5 µg/ml gentamycin, pH 7.4). Hearts were retrogradely perfused with PB, followed by perfusion with enzyme solution containing 40 U collagenase A and 0.1% hyaluronidase (Boehringer Mannheim). After removing the atria and great vessels, the ventricular tissue was finely minced and incubated in PB plus 2% albumin. CaCl₂ was added stepwise to the cell suspension to achieve a final concentration of 1 mM CaCl₂. The cell suspension was filtered through a 300 µm mesh, and ARC were collected by centrifugation (25xg, 2 min). To reduce nonmyocyte contamination, the resulting pellet was resuspended (PB, 2% albumin, 1 mM CaCl₂), carefully overlaid onto a 6% albumin solution (in PB, 1 mM CaCl₂) and centrifuged. The cell pellet was resuspended in serum-free M-199 (Sigma) supplemented with L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), albumin (0.2%), penicillin (100 U/ml) and streptomycin (100 µg/ml). The cells were plated onto laminin-coated (1 µg/cm²) 60 mm cell culture dishes (Falcon) at a density of 2x10⁵/dish and incubated for 2 h (5% CO₂-incubator). The use of laboratory animals for scientific purposes was approved by the ethical committee of the University of Freiburg and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.2 Stimulation of ARC
For -adrenergic stimulation the medium was replaced by medium supplemented with PE (10 µM), PE (10 µM)+PRAZ (20 µM) or PRAZ (20 µM). The influence of general trophic effects was tested in control experiments (n = 5) by the addition insulin (0.1 µM) to the culture medium. In blocking experiments, the antagonist was given 30 min prior to the agonist. The density of ARC after the medium change was 1 to 1.5x10³ ARC/cm² with about 90% rod-shaped cells. PE and PRAZ were purchased from Sigma, dissolved and stored according to the supplier's instructions. 24 h experiments were performed without medium change. When ARC were incubated for 72 h, the medium was changed every 24 h.

2.3 RNA preparation
ARC were washed twice with phosphate-buffered saline (PBS) and total cellular RNA was isolated according to the method of Chomczynski and Sacchi [26]and quantitated in triplicate by absorbance at 260 nm. The integrity of the RNA was checked by agarose gel electrophoresis prior to PCR analysis.

2.4 Competitive RT-PCR
The determination of the original number of NCX transcripts was performed by competitive RT-PCR in the presence of a defined concentration of a shortened NCX competitor RNA (NCX) which served as an internal standard. The protocol was previously described in detail for the quantitation of the angiotensin I-converting enzyme [27]. Equal amounts of total RNA (150 ng) were mixed with increasing quantities of NCX molecules (50 to 1.25x10⁵) in 1x RT buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂) completed with 0.5 mM dNTPs, 250 pmol of random hexanucleotide primers, 10 mM dithiothreitol, RNase inhibitor (20 U/100 ng total RNA; Amersham Buchler, Braunschweig, Germany) and MMLV reverse transcriptase (10 units/100 ng total RNA; Life Technologies, Eggenstein, Germany). The RT reactions (25 µl) were performed by incubation at 42°C for 60 minutes.

Duplicate samples of PCR reaction were performed as described [27]. Denaturing, annealing, and extension reactions proceeded 30 times at 94°C for 1 min, 60°C for 2 min, and 72°C for 3 min. As a negative control, no amplification product occurred if reverse transcriptase or total RNA was omitted in the first-strand cDNA reaction. The NCX PCR products were found to be of the expected size as shown by gel electrophoresis. The amplification products were separated by agarose gel electrophoresis (1.5%), stained with ethidium bromide and photographed under UV transillumination. The negative film was used to evaluate the band densities (Personal Densitometer, Molecular Dynamics, Krefeld, Germany). To correct for differences in size of the target (861 bp) and competitor (778 bp) PCR products, the band densities of the respective competitor PCR products were multiplied by the specific factor 1.1.

2.5 Selection and synthesis of the PCR primers
Sense and antisense primer oligonucleotides were selected from the rat NCX cDNA sequence [28]which is identical to the guinea pig [29]cDNA sequence in the region of the selected primers (sense: 1981–2001; 5'-AATGAGCTTGGTGGCTTCACA-3' and antisense: 2827–2844; 5'-CCGCCGATACAGCAGCAC-3').

2.6 Construction and in vitro transcription of the competitor templates
For construction of internal standard competitor RNA a fragment of 83 bp was released by digestion of the NCX cDNA with HpaI and BclI. The shortened NCX cDNA fragment was filled with the Klenov fragment and religated. For in vitro transcription, the shortened NCX cDNA clone was linearised with the restriction enzyme XhoI. Then, 1 µg of the digested NCX cDNA template was transcribed into RNA by using a T3/T7-RNA polymerase in vitro transcription kit (Stratagene, Heidelberg, Germany). Subsequently, the DNA template was removed by DNase digestion. The competitor RNA template was purified by phenol extraction and quantitated by absorption at 260 nm.

2.7 Northern blot analysis
Northern blot analysis for the NCX was performed as described previously [8, 33]. For the detection of the NCX mRNA, 20 µg total RNA were separated by agarose gel electrophoresis, transferred to a nitrocellulose membrane and hybridised with a specific 1.5 kb guinea pig NCX cDNA fragment (generous gift from Dr. K.D. Philipson) and a 1.2 kb chicken glyceraldehyde dehydrogenase (GAPDH) fragment. The blot was washed as described [33]and exposed to X-ray film (Kodak Inc.) using intensifying screens (Siemens).

2.8 Western blot analysis
After incubation with hormones, cells were washed with PBS, scraped off into HEPES buffer (20 mM HEPES, 4 mM EGTA, 1 mM dithiothreitol; pH 7.4) completed with protease inhibitors (0.1 mM leupeptin, 0.3 mM PMSF). Preparation of particular protein fractions (i.e. crude membrane protein), SDS–polyacrylamide gel electrophoresis, and the immunodetection of the NCX protein was performed as previously described in detail [8, 9]. 60 µg of particular fraction protein were loaded (7.5% running gel) and subsequently transferred to Hybond-nitrocellulose (Amersham). Equal loading was checked by reversible staining of the membrane with Ponceau S. The immunoreaction was performed as described [8, 9]. The polyclonal rabbit--dog NCX antiserum was purchased from Swiss Antibodies Swant Ltd. (Bellinzona, Switzerland). Quantification of the immunoreactive bands was performed by using a Personal Densitometer and the ImageQuant software (Molecular Dynamics).

2.9 Statistical analysis
All data are given in mean±SEM. Differences between groups were evaluated by analysis of variance (ANOVA) followed by Student-Newman-Keuls test. Statistical significance was accepted at the level of p<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Northern blot analysis was used to evaluate whether ₁-adrenergic stimulation induced changes on the NCX expression level. Fig. 1 shows that PE-stimulation for 24 h induced a marked increase of the NCX hybridisation signal. The effect of PE appeared to be blocked by preincubation of ARC with the ₁-specific adrenoceptor antagonist PRAZ (Fig. 1). The blocking effect of PRAZ indicates a specific ₁-adrenoceptor mediated response to PE. Prazosin alone did not increase the expression of the NCX (Fig. 1).

View larger version (91K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Northern blot analysis of the NCX mRNA in ARC treated for 24 h with phenylephrine (PE), phenylephrine+prazosin (PE+PRAZ), and prazosin alone (PRAZ). Control=C.

 
3.1 Competitive RT-PCR analysis
The original number of NCX transcripts was determined by competitive RT-PCR analysis using the same source of mRNA as above and a shortened fragment (NCX) of the original NCX cDNA as an internal standard (Fig. 2). PE-stimulation induced a significant increase of NCX transcripts after 24 h (n = 11; 18.84±3.29x10⁵/60 ng total RNA) and 72 h (n = 5; 16.66±3.42x10⁵/60 ng total RNA), respectively, as compared to controls (24 h: 7.59±1.92x10⁵ and 72 h: 5.31±1.07x10⁵/60 ng total RNA; both p<0.05 vs. control) (Fig. 3). The effect of PE was completely blocked by preincubation of ARC with PRAZ and subsequent incubation with PE (24 h: 8.41±2.62x10⁵ and 72 h: 4.08±0.92x10⁵/60 ng total RNA; both p<0.05 vs. PE and NS vs. control) (Fig. 3). No changes of the NCX mRNA level were found, when ARC were incubated with PRAZ alone (data not shown).

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 NCX mRNA quantification by competitive RT-PCR. (Top) A constant amount of total RNA was mixed with an increasing number of NCX standard molecules, reverse transcribed and amplified by PCR. The PCR products (NCX target: 861 bp, NCX standard: 778 bp) were separated, stained and photographed under UV transillumination. The negative film was used to evaluate the band densities. (Bottom) The ratio of competitor-to-target products was plotted against the known number of competitor molecules on a log scale. At the competition equivalence point (log ratio=0) the original number of target mRNAs corresponds to the initial number of competitor RNA molecules used. M=/HaeIII DNA molecular weight marker.

 

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Bar graphs displaying the results of competitive RT-PCR experiments. The number of NCX transcripts of ARC treated with phenylephrine (PE) for 24 h and 72 h was significantly increased. The effect of PE was blocked by the 1-adrenergic agonist prazosin (PE+PRAZ). Insulin had no effect on the NCX mRNA level (C+Ins vs. C-Ins).

 
To control for general trophic effects (overall increase of protein turn-over), the effect of insulin was tested (n = 5) (Fig. 3 and Fig. 4). Importantly, insulin, which was shown to increase the rates of general protein synthesis in adult rat cardiocytes [16], does not increase the NCX mRNA (Fig. 3) and protein level (Fig. 4). This result suggests, that the observed up-regulation of the NCX is fairly specific and not due to a general anabolic response, i.e. increase of cellular protein. The difference of the NCX mRNA level between control and PE-treated ARC was more pronounced after 72 h of incubation (312% vs. 248% after 24 h compared to the 100% control). However, this effect was rather due to a decrease of NCX transcripts in the controls.

View larger version (70K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (Top) Western blot analysis of the NCX in adult cardiocytes treated for 24 h with prazosin (PRAZ), phenylephrine (PE), phenylephrine+prazosin (PE+PRAZ), or insulin (Ins). Control=C. (Bottom) The amount of immunoreactive NCX protein was significantly increased in ARC treated for 24 h with PE. The PE effect was blocked by prazosin (PE+PRAZ). Insulin treatment had no effect on the NCX expression.

 
3.2 Western blot analysis
Western blot analysis using particular fraction protein of ARC treated with PE for 24 h revealed a specific immunoreactive band at 120 kD as described by Philipson et al. [30](Fig. 4, top). PE-stimulated ARC showed a 1.8-fold increase of the NCX protein level as compared to controls (p<0.05; n = 5). Consistent with the results described above, the increase of the NCX protein was blocked by PRAZ (p<0.05 vs. PE and N.S. vs. control) (Fig. 4, bottom). No significant changes of the NCX protein level were found, when ARC were incubated with insulin or PRAZ (Fig. 4).

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Human heart failure has been shown to be accompanied and promoted by adrenoceptor stimulation and activation of the renin-angiotensin system leading to increased plasma levels of catecholamines [1, 2]. Likewise, it is well established that myocardial Ca²⁺-homeostasis is disturbed in experimental cardiac hypertrophy and in human heart failure [3]. In the present study, we used an adult rat cardiomyocyte cell culture system to investigate the effects of the -adrenergic agonist phenylephrine, representing a well-known promoter of cardiac hypertrophy in vivo and in vitro [10], on the gene expression of the sarcolemmal NCX. The results demonstrate that activation of the ₁-adrenoceptor induced a significant and prolonged up-regulation of the sarcolemmal NCX mRNA and protein level.

Recent studies using experimental models of pressure-overload induced cardiac hypertrophy in different species [6, 7], have presented evidence for an enhanced expression and/or activity of the NCX due to increased mechanical load. In addition, studies from our laboratories have shown that the gene expression [8]as well as the activity [9]of the sarcolemmal NCX is increased in patients with severe heart failure. The hypothesis was raised that an increased NCX expression and activity might, at least in part, compensate for a depressed SR function [8, 9]. However, the benefit of limiting diastolic Ca²⁺ overload might be counteracted by the corresponding influx of Na⁺ leading to membrane depolarization and enhanced arrhythmogenesis. It is well known that increased plasma levels of catecholamines are commonly found in patients with heart failure [1]. Therefore, the present findings might be of interest regarding the situation in man, too.

Evidence for an effect of -adrenoceptor stimulation on the NCX gene expression was recently presented in a study of Menick and co-workers [31], showing that the mRNA level of the NCX is up-regulated by 3 to 5-fold in neonatal feline cardiocytes after only one hour of treatment with 100 µM PE and remained up-regulated for at least four hours. We extend these findings considerably, showing that the effect of adrenergic stimulation on the NCX mRNA level is transferable to the adult cardiocyte maintained in serum-free culture and that this effect is sustained over 3 days. In addition, 24 h after stimulation we observed an increased protein level of the antiporter. It is well established that the gene expression pattern of the neonatal cardiomyocyte differs extensively from the adult phenotype [32]. For example, Ju and colleagues [34]have shown that angiotensin II treatment down-regulated mRNAs encoding Ca²⁺ transport genes (NCX, SR Ca²⁺-ATPase and the SR Ca²⁺-release channel) only in neonatal, but not in adult cardiocytes. The study strongly supports the notion that responses to applied stimuli can be extremely dependent on the developmental stage of cardiocytes. In particular, developmental changes were also shown for the cardiac NCX in the rat heart [33], in which a high expression level of the NCX in the late fetal and early postnatal stage is followed by a steady decline during the development of the adult phenotype resulting in an 8-fold lower expression level compared to postnatal day one. Furthermore, cell cultures of neonatal cardiocytes generally imply the addition of serum, which might provide factors that are not present in the environment of the cardiocyte under physiological conditions. The present study shows that PE treatment for 24 h induced an increase of the NCX mRNA by 2.5-fold and of the protein by 1.8-fold. Regarding the increase of the NCX mRNA level, our result is fairly consistent with the findings of Menick et al. [31], although these data were raised in neonatal cardiocytes under different conditions.

Since -adrenergic stimulation was shown to increase general protein synthesis in vivo and in vitro [10], it has to be considered whether the observed effects of PE on the NCX expression might be unspecific, i.e., reflecting a general increase of cellular protein synthesis. In control experiments, insulin (0.1 µM) was added to the cell culture medium and insulin-treated ARC were incubated for 24 h and 72 h. Insulin is known to be a potent activator of general protein turn-over and, indeed, it has been shown that general protein synthesis in isolated adult rat cardiocytes is characteristically insulin-responsive [16]. Importantly, insulin incubation for 24 h and 72 h had no effect on the NCX gene expression, as measured by competitive RT-PCR (Fig. 3). This result strongly supports the idea of a specific ₁-adrenoceptor mediated response leading to the up-regulation of the NCX. Furthermore, Menick and co-workers [31]also have shown that insulin (0.1 µM) had no effect on the NCX mRNA level in adult cardiocytes.

We also performed Ca²⁺ transport measurements in PE-treated and untreated ARC according to a protocol described elsewhere [9, 33]. However, Ca²⁺ uptake experiments failed for unknown reasons.

Kent and McDermott [35]have shown that passive load applied by step increments of stretch induced an up-regulation of the NCX mRNA. In conjunction with this study, at least two pathways seem to exist for the induction of the NCX up-regulation in the adult rat cardiocyte: load-dependent due to stretch of the cardiomyocyte and load-independent due to -adrenergic stimulation. Load has been shown to alter the Na⁺ and Ca²⁺ gradients across the sarcolemma [36, 37]; -adrenoceptor stimulation results in increased myoplasmic Ca²⁺ concentrations [21]. Evidence that changes of the myoplasmic ionic conditions may affect the NCX gene expression are supported by the fact that veratridine, a Na⁺ and Ca²⁺ influx stimulator in excitable cells, induced an up-regulation of the NCX mRNA level, similar to load-dependent NCX up-regulation [35], and to the -adrenoceptor mediated increase of NCX mRNA and protein, observed in the present study. Since cardiac hypertrophy and heart failure is closely associated with the activation of the adrenergic system and an up-regulation of the cardiac NCX both in animal models as well as in man, future studies will address the pathophysiologic relevance of the present findings in human heart failure.

Time for primary review 25 days.

	Acknowledgements

 
We are grateful to Dr. K.D. Philipson for providing the guinea pig Na⁺/Ca²⁺-exchanger cDNA. The excellent technical assistance of Robert Burzan and Christel Kemsies is greatly appreciated. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Dr 148/6-1, Ve 136/1-3), H.D. is an Established Investigator of the German Research Foundation (DFG, Heisenberg-Stipendium Dr 148/5-2).

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