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J Appl Physiol 99: 313-322, 2005; doi:10.1152/japplphysiol.00609.2004
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Expressional reprogramming of survival pathways in rat cardiocytes by neuregulin-1{beta}

Marie-Noëlle Giraud,1,* Martin Flück,2,* Christian Zuppinger,1 and Thomas M. Suter1

1Swiss Cardiovascular Center, and 2Department of Anatomy, University of Bern, Bern, Switzerland

Submitted 14 June 2004 ; accepted in final form 7 March 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Neuregulin/ErbB2-induced kinase signaling provides essential survival and protection clues for functional integrity of the adult heart and skeletal muscle. To define the regulatory pathways involved in neuregulin-dependent muscle cell survival, we set out to map the largely unknown transcript targets of this growth/differentiation factor in cardiocytes. Freshly isolated adult primary rat cardiocytes were treated for 24 h with recombinant human neuregulin-1{beta} (NRG-1{beta}, 30 ng/ml). Transcript level alterations in NRG-1{beta}-treated and control cardiocytes (n = 6) were identified with Atlas Rat Toxicology 1.2 cDNA arrays (BD Clontech) and established permutation L1 regression analysis. Selected transcriptional adjustments were confirmed by RT-PCR and Western blotting. Involvement of MAPK pathways was verified with the inhibitor PD-98059. Application of the single dose of NRG-1{beta} to quiescent cardiocytes induced expressional reprogramming of distinct cellular processes. This response included a prominent 50–100% increase in transcripts of multiple redox systems. It also involved a comparable mRNA augmentation of protein synthetic and folding factors together with augmented message for the trigger of cardiac hypertrophy, cyclin D1 (CCND1). First evidence for a role of neuregulin in promotion of mitochondrial turnover, voltage-gated ion channel expression, and the suppression of fatty acid transporter mRNAs was revealed. Subsequent analysis confirmed a corresponding upregulation of redox factor proteins thioredoxin and the thioredoxin reductase 1, GSTP-1, and CCND1 and demonstrated downregulation of the related transcripts by PD-98059 in neuregulin-stimulated cultures. These MAPK-dependent expressional adjustments point to novel oxidative defense and hypertrophy pathways being involved in the longer lasting protective function of neuregulin in the heart.

heart; transcriptome; cardioprotection; redox; microarray

GROWTH AND DIFFERENTIATION factors exert important control on the phenotype of muscular tissues through altering gene expression and protein synthesis (9, 14, 15, 23, 39, 61). Accumulating evidence implies that growth and differentiation factor signaling take part in the adaptive response of cardiac and skeletal muscle to external stressors (23, 42–44).

Neuregulins (NRG) are a family of soluble/transmembrane polypeptide ligands of ErbB tyrosine kinase receptors, which play an essential role in the transmission of growth and differentiation signals in the developing and adult heart (2, 13, 21). Specifically the rapid activation of MAPK and AKT kinase pathways via soluble NRG-1{beta} isoform has been implied in control of development, growth, and repair of cardiocytes (1, 2, 21, 31, 46, 60). The importance of NRG-1 for heart integrity beyond embryonic stages was highlighted by the recent investigations showing that genetic alterations blocking the production of the NRG-1 receptor, ErbB2, cause dilated cardiomyopathy (38). In addition, cancer patients treated with Trastuzumab, a humanized monoclonal antibody targeting the proto-oncogene ErbB2, were found to have increased risk for severe left ventricular dysfunction and heart failure (reviewed in Refs. 13, 21, 46). Conversely, recombinant NRG-1{beta} isoforms protect cardiocytes from apoptosis and the damage induced by antineoplastic drug therapy (20, 45, 46, 60). Similarly, NRG-1 and NRG-2 isoforms are involved in myogenesis and the nerve-dependent prevention of muscle fiber apoptosis (17, 55, 61). NRG also induces enhanced glucose uptake in muscle cells (51). Importantly, evidence for a physiological regulation of NRG/ErbB signaling in cardiocytes and skeletal muscle was recently provided (31, 32). For instance, phosphorylation of the ErbB receptors and proteolytic processing of transmembrane NRG forms in rat hindlimb muscles are increased after a single bout of electric stimulation or treadmill running (32). Furthermore, NRG protected both quiescent and paced adult rat ventricular cardiocytes against {beta}-adrenergic-induced apoptosis (31). These observations strongly support that NRG-1 provides important survival and protection clues for cardiac and skeletal muscle.

The intracellular mechanisms mediating the protective effects of NRG-1 in cardiac muscle are not well understood. The limited number of investigations in embryonic and neonatal cardiocytes indicate that the NRG-1{beta} isoform significantly reduces anthracycline-induced myofibrillar disarray (22, 45) and may induce a program toward hypertrophy in rat cardiocytes (2, 21, 60). The few known NRG-1-dependent expressional alterations in heart muscle cells include markers of the cardiac conduction system (18), {alpha}-skeletal actin and the hypertrophic agent atrial natriuretic factor (2, 40, 60). These few known adaptations, however, incompletely explain the survival effect of NRG on cardiac cells.

The protective effect of NRG against oxidative stress has been demonstrated in neuronal cell types: NRG causes the induction of defense mechanisms by attenuating free radical release (11) and a significant protective effect from H2O2-induced death (12). Last, microarray analysis of tumorigenic epithelial cells has indicated that expression of several other genes involved in signal transduction and transcription are affected in the first hour of NRG-1 treatment (53). These latter aspects indicate that gene expressional adjustments may underlie the longer lasting cardioprotective effect of NRG-1 (61).

The aim of this study was to map gene expressional adaptations governed by the major NRG isoform, NRG-1{beta}, in cardiocytes to gain insight into the pathways involved in NRG-mediated cardioprotection. Using microarray technology, we tested the hypothesis that one dose of recombinant NRG-1{beta} would cause distinct transcript-level alterations of different gene ontologies (i.e., functional categories) in adult primary rat ventricular cardiocytes, which would match NRG-induced functional improvements. Particularly, it was hypothesized that the expressional adaptations would persist until 24 h and match the reported enhancement of cell survival, hypertrophy, carbohydrate metabolism, and altered electric conduction in (heart) muscle cells. Particularly, we speculated that gene expression profiling would expose the unidentified molecular nature of cytoprotective adaptations to NRG-1 related to oxidative stress defense. Finally, transcript level adaptations were suspected to depend on MAPK signaling and to provoke corresponding codirectional adjustments of protein levels.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell culture.   All experiments were carried out according to the Swiss Animal Protection Law and with the permission of The State Veterinary Office, Bern, Switzerland. Adult (200–250 g) male Wistar rats from an in-house breeding facility were killed by pentobarbital sodium injection (Abbott Laboratories, Chicago, IL). According to previously published methods (29), the heart was rapidly removed from anesthetized rats and immediately mounted on a temperature-controlled (37°C) modified Langendorff system for coronary retrograde perfusion. After being perfused with Tyrode solution (in mM: 137 NaCl, 5.4 KCl, 1.2 CaCl2, 0.5 MgCl2, 10 HEPES, and 10 glucose, pH 7.4) and Ca2+-free Tyrode, the heart was digested with collagenase type II (300 U/mg, 0.07%, Worthington) and type XIV protease (0.02%, Sigma Buchs, Switzerland) and rinsed with Krebs buffer solution (in mM: 85 KOH, 30 KCl, 30 KH2PO4, 3 MgSO4, 0.5 EGTA, 10 HEPES, 50 L-glutamic acid, 20 taurine, and 10 glucose, pH 7.4). The left ventricle was dissociated to release cells from the tissue into Krebs buffer solution and filtered. Cells were seeded on four laminin-coated plates (10-cm diameter) and cultured overnight in serum-free ACCT-medium [M-199 supplemental 1% 100 U/ml penicillin-streptomycin (GIBCO-BRL, Basel, Switzerland) and 20 mM creatine (Sigma)]. After medium change, two plates were treated with recombinant human NRG-1{beta} (30 ng/ml, Neomarkers, P. H. Stehelin & Cie, Basel, Switzerland) and two were left untreated (control group). After 24 h, the plates were subjected to RNA isolation, and lysates from the two plates for each treatment were pooled to control for the fluctuating nature of gene expression. Six independent series of paired experiments were performed for microarray experiments. Microscopic inspection of cell morphology revealed that the primary cultures obtained under these conditions consisted of >95% myocytes.

Inhibition experiments with PD-98059 (Calbiochem, VWR International, Lucerne, Switzerland) were carried out with the same paired design in four independent experiments. PD-98059 (50 µM end concentration) or equally diluted DMSO solvent was added for 24 h to overnight serum-free cultured primary rat cardiocytes concomitantly with/or without NRG-1{beta}.

RNA isolation.   Total RNA was isolated with RNeasy kit (Qiagen, Basel, Switzerland) as described by Wittwer et al. (58) but with minor modifications. Briefly, cultures were washed with PBS and cells were scraped into 42°C equilibrated RLT buffer. Total RNA was quantified with Ribogreen assay against a RNA standard (Molecular Probes), and the integrity was verified after separation of the samples on formaldehyde-agarose gels.

Microarray experiments.   Total RNA (5 µg) was reverse transcribed with Moloney murine leukemia virus reverse transcriptase using nucleotides from the EZ-kit (Ambion Europe), and radiolabeled targets were hybridized overnight to ATLAS Rat Toxicology 1.2 cDNA arrays (#7860–1, BD Clontech). The nylon arrays were washed (4 times for 60 min in 2% SDS/1% SDS at 68°C, once for 30 min in 0.1% SSC/0.5% SDS at 68°C), rinsed in 2x SSC, and exposed to a phosphoimager. Eight days later, the screen was developed on a Phosphorimager #425E, and the signal was quantified with IMAGE-Quant v. 3.3 (Molecular Dynamics, Sunnyvale, CA) as described by Wittwer et al. (58) but with the modification that the global background method was used for background determination. For repetitive use, the membranes were stripped according to the manufacturer's instruction (Ambion Europe), thereby removing on average >90% of the signal (58). Data sets have been deposited at Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) under sample codes GSM17431 [NCBI GEO] -GSM17435 and GSM17437 [NCBI GEO] -GSM17443, which link to the series GSE1094 [NCBI GEO] .

Microarray data analysis.   Transcript signals were analyzed for an effect of culture condition (NRG-1{beta} vs. control) based on the premise that altered mRNAs show up as outliers in regression analysis as described (35, 58) with the following modifications. Only those transcripts were included in the analysis for which the spot signal was 30% above global background on the majority (>4 of 6) of filters hybridized with targets from one condition (NRG-1{beta} or control) and present on at least two filters hybridized to targets prepared from the cells cultured under the other conditions. These criteria were chosen to include transcripts in the analysis that were expressed in one culture condition (NRG-1{beta} or control) but at low abundance in the other condition. This limitation resulted in 327 transcripts being selected from the 1200 transcripts that were probed for on the array.

Identification of differentially expressed genes.   The signal strength of the 327 cDNAs between the control and NRG-1{beta}-treated cardiocytes was compared using 10log-transformed raw signal data in scatterplots for all combinations of filter pairs from the control vs. NRG-1{beta} treatment. This resulted in 36 scatterplots (6 controls x 6 NRG-1{beta} filters) being analyzed. Then, linear L1 regression analysis was carried out for each scatterplot, and the residues were (= observed – expected value) calculated for each transcript with Statistica software package 6.1 [StatSoft (Europe), Hamburg, Germany]. Residues were exported into Microsoft Excel 2000 and used to test significance of a trend under application of the nonparametric sign test as described (58). The false discovery rate adjustment was used to correct the multiplicity error per hypothesized alteration of a gene ontology at a two-tailed P = 5% (4).

For each transcript, the relative expression ratio was estimated from the respective residue relative to the L1 regression line of NRG-1{beta} vs. control comparisons of the log-transformed expression signals. The residues from all 36 scatterplots were potentiated to the base of 10, and the mean and SE was calculated for each transcript. The a priori {alpha}-level for statistical significance was set to P < 0.05. Confidence intervals of 95% were calculated using Statistica 6.1 (Statsoft). For details on minimal information on gene expression (MIAME; Ref. 6), supplemental data are available at http://jap.physiology.org/cgi/content/full/00609.2004/DC1.

RT-PCR.   Total RNA (600 ng) was reverse transcribed with random hexameres using the OMNIscript kit (Qiagen). The resulting cDNA was subjected to real-time PCR reactions on a Genamp 5700 (Applied Biosystems, Rotkreuz, Switzerland) with gene specific primers (700 nM) using Sybr green PCR Master Mix (Applied Biosystems; see Table 1 for primer sequences). Aliquots corresponding to 6 ng (0.6 ng for 28S) RNA were used for PCR experiments. For PCR experiments with the same primer pair, all samples from the two treatments were run on the same 96-well plate, and two to five replicons for each sample and primer pair were performed. Quantification of the amount of each mRNA relative to the reference 28S ribosomal RNA was done under application of the comparative CT method as described previously (57). 28S-standardized expression values were normalized to the level seen in the respective untreated control (set to 1). Those values that complied with the conditions of an outlier as defined by the Grubbs test (24) were removed for analysis. Significance of a trend in NRG-stimulated cultures was verified with a paired, one-tailed Wilcoxon test based on the hypothesis on a match of RNA-level alteration as detected with microarray and RT-PCR analysis. Similarly, the use of a paired, one-tailed Wilcoxon test was justified given the suspected reduction of individual mRNA levels in the cultures treated with the MAPK inhibitor PD-98059. The a priori {alpha}-level for statistical significance was set to P < 0.05. The use of this nonparametric test was justified, because for all 28S-related mRNA levels, normal distribution could be rejected based on the Kolmogorov-Smirnov/Lillefors test (Statistica 6.1). This was based on the hypothesis of a same directional change as observed in microarray experiments or the speculation on a reduction of RNA levels in PD-98059-treated cultures.


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  		Table 1. Primer sequences

 
Microarray data interpretation.   Detected transcripts were grouped into functional categories based on the information deposited in the ExPASy data bank (http://www.expasy.org/cgi-bin/sprot-search-ful). This grouping is not ultimate.

Protein analysis.   Isolation, Western blotting, and immunodetection was basically carried out as described (16). In brief, proteins were extracted with 50 µl per 10-cm dish of modified RIPA buffer (1% NP-40, 0.25% deoxycholate, 50 mM Tris·HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 mM NaF, 1 mM PMSF, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 2 µg/ml pepstatin, 1 µg/ml aprotinin; all reagents were received from Sigma Chemie). Soluble proteins were isolated in the supernatant of a centrifugation step (5 min, 10,000 g, 4°C). Protein concentration was estimated with bicinchoninic acid (B9643, Sigma) against BSA standard and adjusted to 1 mg/ml with SDS-PAGE loading buffer (50 mM Tris·HCl, pH 6.8, 10% glycerol, 2% SDS, 5% {beta}-mercaptoethanol, 0.1% bromphenol blue). Denatured protein (15 µg; 5 min at 95°C) was loaded per lane on reducing 10% or 15% SDS-PAGE gels and wet transferred via Western blotting onto nylon membranes (Protran BA85, Schleicher & Schuell, Dassel, Germany). Protein samples from paired experiments (control vs. NRG-1{beta}) were run simultaneously on the same gel. Two gels were run to separate all the samples. Immunodetection was carried out after initial blocking in 2.5% milk-1% BSA in TTBS (20 mM Tris·base, pH 7.5, 150 mM NaCl, 0.05% Tween 20) and incubation with diluted specific antibodies. The primary antibodies were directed against thioredoxin (TXN; 1:1,000, polyclonal rabbit, anti-mouse thioredoxin, Redox Bioscience, Kyoto, Japan), thioredoxin reductase 1 (TXNRD1; 1:500, polyclonal rabbit, sc-20147, Santa Cruz, LabForce, Nunningen, Switzerland), GSTP1 (1:500, polyclonal rabbit, #354212, Calbiochem, VWR International, Lucerne, Switzerland), CCND1 (1:500, monoclonal, #CC12, Oncogene Research Products, San Diego, CA), and EEF2 (1:500, polyclonal rabbit, sc-25634, LabForce, Santa Cruz, CA). After being washed in TTBS, a 1:2,000 dilution of the respective horseradish peroxidase-conjugated (HRP) secondary antibody, i.e., goat HRP-anti-rabbit antibody #55676 from Cappel (ICN, Eschwege, Germany) or goat HRP-anti-mouse (antibody #2304, Sigma), was added. The specificity of the respective antibodies has been established before and was verified with running positive control rat soleus muscle or rat/human rat tumor cell lines. Signal detection was carried out with SuperSignal WestFemto (Pierce, Perbio Science, Lausanne, Switzerland) and recorded on film (Kodak BiomaxMR, Sigma). Integration of the respective band intensity vs. background was carried out as described (16). To combine the data from the two immunoblots per protein detection, background-corrected signal values were normalized to the mean of signals from the controls on the respective immunoblot. Subsequently, the hypothesis of a protein level increase of EEF2, CCND1, TXN, TXNRD1, or GSTP1 in codirectional correspondence to the identified mRNA-level change was verified with the appropriate one-tailed statistical test. For all proteins, except CCND1, a paired, one-tailed t-test was applied (Statistica 6.1). For CCND1 a paired, one-tailed Wilcoxon test was justified because the hypothesis of a normal distribution was rejected based on the Kolmogorov-Smirnov/Lillefors test (Statistica 6.1). The a priori {alpha}-level for statistical significance was set to P < 0.05. Confidence intervals of 95% were calculated using Statistica 6.1 (Statsoft). For data presentation as means ± SE of normalized signals, the values were imported into Microsoft Excel 2000 and the created graph was exported into Microsoft Powerpoint 2002 for final figure assembly.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell culture.   Freshly isolated cardiocytes from adult rats were cultured overnight in serum-free medium and stimulated for 24 h with NRG-1{beta} or mock (control group). Total RNA (13.5 ± 2.8 and 13.9 ± 1.9 µg) was isolated in each series from the NRG-1{beta}-treated and control cultures. There was no significant difference in RNA amount in rat adult cardiocytes between the two treatments. Furthermore, the amount of 28S ribosomal RNA and protein in cardiocytes was not altered by NRG-1{beta} treatment (data not shown).

Microarray analysis.   Three hundred twenty-seven genes were detected by microarray analysis. Comparison of the expression profiles from these 327 transcripts between NRG-1{beta}-treated and untreated primary cardiocyte cultures showed a high inter- and intragroup correlation (mean r2 = 0.71 for control vs. control, 0.72 for NRG-1{beta} vs. NRG-1{beta} and 0.74 for control vs. NRG-1{beta}). Statistical analysis with a multiplicity error corrected P of 5% identified differential expression of 130 of these 327 detected transcripts in cardiocytes upon 24 h treatment with NRG-1{beta}. Transcripts were compiled into gene ontologies, which are reported separately below.

Redox regulation.   The hypothesis for an mRNA-level increase of gene ontologies related to oxidative stress defense was confirmed for 11 factors (see Fig. 1). Eleven factors [catalase (CAT), Cu-Zn-dismutase (SOD1), TXN, TXNRD1, PDIA1, PDIA4, PDIA3, GSTP1, GPX1, GCLC, TST] involved in redox regulation were upregulated at the transcript level. The mRNA concentration of one redox factor only, i.e., HO-1, was reduced.



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  		Fig. 1. Neuregulin (NRG)-1{beta} induced adaptations of transcripts for factors of redox regulation. Data are means ± SE of the estimated signal ratio NRG-1{beta} over control (CTL) for each transcript. Black and gray filled bars indicate significantly up- or downregulated transcripts (sign test, P = 5%). Open bars indicate other detected but nonsignificant altered transcripts. To the left, the gene name, GenBank identifier, and the 95% confidence interval (brackets) are given for each transcript.

 
Protein turnover.   The suspected transcript level enhancement of genes involved in hypertrophy, i.e., protein synthesis, was evident for five factors. From the other RNAs involved in protein turnover, the expression of four chaperones was increased. On the contrary, the mRNA levels of factors involved in intracellular proteolytic degradation were differentially affected with the increase of CPD and CPE on NRG-1{beta} treatment being the most striking observation (Fig. 2).



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  		Fig. 2. Changes in mRNA levels of functional categories involved in protein turnover after a 24-h treatment of adult rat primary cardiocytes with NRG-1{beta}. For details see legend to Fig. 1.

 
Energy metabolism.   mRNA levels of several genes involved in energy metabolism were affected. On NRG-1{beta} treatment, the suspected mRNA concentration changes of carbohydrate metabolism in primary cardiocytes was confirmed for several factors. Transcript levels of three factors involved in carbohydrate metabolism were significantly increased (Fig. 3). Additionally, the mRNA levels of factors involved in different aspects of mitochondrial metabolism were prominently affected: two factors each involved in {beta}-oxidation and the import of amino acids into the mitochondrial citrate cycle. Likewise, five factors of mitochondrial oxidative phosphorylation were induced after NRG-1{beta} treatment. In contrast, the mRNA levels of three main factors of fatty acid transport were reduced (Fig. 3).



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  		Fig. 3. NRG-1{beta}-provoked changes in mRNAs for components of energy metabolism. For details see legend to Fig. 1.

 
Cell regulation.   Multiple transcript levels of the 143 detected factors involved in regulatory aspects of cell cycle, gene expression, and DNA repair were affected. Foremost, the transcript level of the cell cycle regulator cyclin D1 (CCND1) was increased, and two factors involved in apoptosis (HRK, PDCD2) were reduced on NRG treatment of cardiocytes (Fig. 4). The mRNA level of 11 factors involved in gene transcription and DNA repair was differentially affected.



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  		Fig. 4. mRNA alterations of cell regulatory factors upon NRG-1{beta} treatment. For details see legend to Fig. 1.

 
Among the 10 measured factors involved in maintenance of ion gradients, the supposed increases in mRNA levels of the four voltage-gated ion channels reached significance (Fig. 4).

Cytoskeletal organization.   From the measured factors involved in the structural framework and trafficking, five were induced on the transcript level and nine were reduced (data not shown).

Data verification.   A comparison of microarray and RT-PCR on the same RNA samples was performed on a subset of differentially expressed targets (Table 2). RT-PCR confirmed the trend of alterations and the statistical significance observed with microarray analysis.


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  		Table 2. Verification of mRNA-level changes by RT-PCR

 
Protein expression.   Immunblotting experiments demonstrated an increase of redox factors TXN, TXNR1, GSTP1, and CCND1 per total protein after 24 or 48 h of NRG-1{beta} stimulation, as suspected from the upregulation of the corresponding mRNA. This change corresponded to the observed upregulation of the respective transcripts (Fig. 5). In contrast EEF2 protein levels were not altered.



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  		Fig. 5. Corresponding protein-level alterations in NRG-1{beta} stimulated cardiocytes. A: examples of each immunodetected protein (top) in 24-h-stimulated cardiocytes and the loading control (bottom) showing a Ponceau S-stained Western blot. Arrows indicate the respective protein band in immunoblots. Molecular mass of detected proteins as estimated by the markers is indicated to the left of each panel. B: mean and SE of normalized protein levels after 24 and 48 h of NRG-1{beta} stimulation of primary rat cardiocytes. n = 5–8. Values in parentheses denote the 95% confidence interval for each protein measure. {ddagger}P < 0.05 of a paired, 1-tailed Wilcoxon test. + and *, P < 0.05, and 0.05 ≤ P < 0.10, respectively, of a paired, 1-tailed t-test.

 
MAPK signaling.   Pharmacological experiments with the MEK inhibitor PD-98059 demonstrated a significant reduction of NRG-stimulated transcript levels of TXN (–44%) and CCND1 (–68%). Additionally, a trend toward a reduction of GSTP1 transcripts (–45%, P = 0.06) was observed. In contrast, PD-98059 did not reduce the level of these transcripts when added in the absence of NRG-1{beta} (data not shown) and did not alter TXNRD1 and EEF2 transcript levels in NRG-1{beta}-stimulated cardiocytes.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
NRGs and their receptors play an essential role during the development and growth of the heart (30, 37) and the maintenance of functional integrity of adult heart (13, 21). In addition, several lines of evidence highlight that NRG-1{beta} acts as a cardioprotective agent in adult rats (8, 20, 31, 41, 60). The importance of identifying the complexity of NRG's regulatory function in the heart is obvious considering the growing interest of new therapeutic opportunities targeting ErbB receptor tyrosine kinase signaling (28, 50, 59), the fundamental role of this pathway in repair and survival of adult cardiomyocytes.

Recent investigations in the role of NRG-1{beta} in adult heart have focused on the identification of activated signaling pathways or activation of single effecter chains (2, 18, 21, 22, 45, 60). The present expressional exploration is particularly helpful in unraveling the regulation of molecular pathways that are engaged in NRG-1{beta}-promoted cell survival. The current data consisting of coincidental transcript level adaptations of distinct gene ontologies obtained by a microarray analysis indicate that the NRG-1{beta} stimulation of cardiocytes results in a discrete pattern of gene expressional alterations. Several of the altered transcripts after 24 h of NRG-1{beta} stimulation represent novel transcriptional targets of NRG-1. The transcript level regulation indicates previously not described alterations in mitochondrial and protein turnover, as well as enhanced ion channel expression in quiescent adult cardiocytes after one dose of NRG-1{beta}. Particularly, corresponding-level adjustments of redox and cell cycle regulator mRNAs and proteins point to novel functional adaptations that may explain the cardioprotective effect of NRG-1{beta} as discussed below.

Statistical rationale.   Several possibilities exist as to which kind of statistical method should be employed to sort out differentially expressed transcripts from microarray data. For the identification of alterations in transcript expression on stimulation of adult rat cardiocytes with NRG-1{beta} from our microarray data, we applied permutation analyses on L1 regression models of scatterplots of log-transformed raw cDNA signals for all array pairs control vs. NRG-1{beta} treatment. Differentially expressed genes were assigned as described with a nonparametric sign test from the difference relative to the regression line and multiplicity error correction for the hypothesized expressional alterations of a gene ontology (57, 58). The justification for the application of regression analysis in our study is given based on the linear relation between expression signals from the experimental conditions and on the basis that the altered mRNAs show up as outliers in these comparisons (35). This regression approach is useful to compare large sets of data from two conditions because it is robust to outliers, does not depend on normal distributions, background correction, and normalization, therefore circumventing this type of bias while developing a high discriminatory power.

The strength of this regression-based approach for the identification of differentially expressed genes for the experimental condition under study is highlighted by the agreement ofidentified mRNA alterations with the expressional trends revealed by alternative RT-PCR technology from 28S-standardized values.

Expressional adaptations.   Following the molecular physiological consequences of observed differential alterations in gene expression for each functional category are discussed. A synopsis of the results is provided in Fig. 6.



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  		Fig. 6. Synopsis of the main findings on expressional reprogramming in primary cardiocytes by NRG-1{beta}. Stippled frames denote gene ontologies, bold names indicate those transcripts where a corresponding protein-level alteration was noted. Arrows and numbers reflect the direction and quantity of altered transcripts within a functional category where levels changed codirectionally.

 
Defense against oxidative stress.   The most pronounced finding was the concomitant upregulation of 11 factors involved in redox regulation in 24 h-treated cultures (Fig. 1). Both mRNAs coding for the cytoplasmic TXN and the TXNRD1 were upregulated as well as the protein level. These two radical scavengers are involved in various redox reactions (including deoxynucleotide synthesis) through catalyzing dithiol-disulfide exchange reactions. Similarly, the GSTP1 and GPX1 mRNAs were increased. The encoded proteins regulate the conjugation of hydrophobic electrophiles through exerting control on the oxidation state of glutathione. Additionally, upregulated mRNA levels were noted for three protein disulfide isomerases, PDIA1, PDIA3, and PDIA4. The encoded factors are implicated in the formation of native protein structures via rearrangement of intrachain and interchain disulfide bonds in the endoplasmatic lumen. Further evidence for the supposed modulation of redox regulator expression was provided by the increased mRNA level of the mitochondrial sulfur-containing enzyme TST, as well as CAT and SOD1 after 24 h of NRG-1{beta} addition (see Fig. 6).

The importance of the thioredoxin system in the cellular defense against oxidative stress in cardiovascular diseases has recently gained much attention (49). Interestingly, thioredoxin-mediated reduction of sulfur bonds has been implied to reduce oxidative damage caused by exposure of cardiocytes to the anticancer drug adriamycin (48), and transgenic mouse hearts overexpressing thioredoxin displayed significantly improved postischemic ventricular recovery and reduced myocardial infarct size compared with the corresponding wild-type mouse hearts (56).

Our posteriori demonstration on a corresponding enhancement of protein and mRNA level for TXN, TXNRD1, and GSTP1 (Fig. 5) points to an improvement of cardiac redox regulatory pathways after one dose of NRG-1{beta}. This adjustment relates to, first, increased mRNAs of other factors involved in radical metabolism such as SOD1, CAT, and GPX1 and, second, to altered mRNA level of APEX1. The encoded factors act as a primary defense mechanism against the insult of oxidative stress and DNA repair (10, 26). Taken together, our results imply that NRG-1{beta} induces in cardiocytes expressional reprogramming toward improved defense mechanisms against oxidative stress (Fig. 6).

Protein turnover.   The pattern of increased mRNA levels of five ribosomal protein synthetic factors (EIF4E, EEF2, ETF1, RPL3, and RPL13A) and four chaperones (CANX, HSPA9, HIP, HOP) indicates that expressional adjustments contribute to the reported hypertrophic potential of NRG-1{beta} on cultured adult cardiocytes (Fig. 2; Refs. 2, 60). These anabolic alterations in adult cardiocytes were manifest after a period of 5 days or could be visualized with sensitive labeling techniques beyond 48 h of stimulation. The upregulated EIF4E mRNA level in NRG1{beta}-stimulated cultures relates to the reported association of enhanced levels of this transcript with cardiocyte growth (36). The upregulated chaperone mRNAs also link to the observed expressional induction of oxidative stress defense in cardiocytes and hint for cytoprotection (Figs. 2 and 3). The increased protein level of three verified redox or cell cycle regulators after 24–48 h of NRG-1{beta} stimulation (Fig. 5) supports the notion on enhanced protein synthesis in our study.

It is important to underline that the enhanced abundance of verified protein-level alterations are in line with the doubling of protein synthesis in cultured adult cardiocytes after 1 day of similar NRG-1{beta} stimulation with comparable amounts (2, 60). The relatively small percentage of protein-level increase for redox regulators TXN and TXNRD1 (~20%; Fig. 5) after this duration of stimulation is explained by kinetic limitations with regard to the accumulation rates of newly synthesized proteins. Similar ceiling effects and specific translation effects due to the restrictive serum-free culture conditions possibly relate to the nonaltered protein level for the highly abundant translation factor EEF2 (Fig. 5; Ref. 27). This is supported by our crude measurements of total protein concentration that did not indicate significant difference between control and treated cells after 24 h of NRG treatment. This bears the contention that RNA level alterations are a sensitive measure to probe for cell regulatory adaptations.

Cell regulation.   Intriguingly, 24-h stimulation with NRG-1{beta} induced an increase in cyclin D1 (CCND1) mRNA and protein level in adult primary rat cardiocytes (Fig. 5). This finding is in line with the NRG-1{beta}-induced increase in CCND1 in mammary tumor cell lines (53) and with the well-established implication of cyclin D1 as a central effector of ErbB2/NRG signaling in tumor cells (25).

The experiments with primary rat adult cardiocyte cultures were carried out under serum-free conditions. Proliferation of residual endothelial cells and fibroblasts in NRG-1{beta}-treated cultures is therefore unlikely to have contributed to induced expression of cyclin D1 and EGR1 per se.

In neonatal and adult rat cardiocytes, overexpression of cyclin D1 causes hypertrophic growth (54). The invoked mechanism, however, is not related to its activity required for cell cycle progression (54). Cyclin D1 protein is also involved in left ventricle hypertrophy induced by angiotensin II and pressure overload (7, 19). Our observation of a significant induction of cyclin D1 message and other protein synthesis mediators, in conjunction with the reduced expression of the apoptosis regulatory molecules, CCND1, HRK, RBL2, and PDCD2, is in agreement with the reported induction of a hypertrophic and anti-apoptotic program in cardiac cell cultures treated with NRG-1 (2, 60). Further observations support this hypothesis: we report in this study that NRG-1{beta} treatment induces reprogramming toward enhanced utilization of carbohydrates and amino acids over fatty acids (see below). It is important to underline that cardiac hypertrophy is associated with a suppression of fatty acid oxidation and metabolic reversion toward increased glucose utilization (33).

Transcript level adaptations relating to energy metabolism and electric conductance.   The increased mRNA levels of two glycolytic factors and nine factors involved in central aspects of mitochondrial metabolism (Fig. 3) support a role of NRG-1{beta} in the regulation of energy metabolism in cardiocytes. Such a relation was previously suggested in other cell types (51, 52). The specificity of this response is supported by the concomitant downregulation of mRNAs for major factors of fatty acid metabolism, i.e., FABP3, APO2, and LPL retinoic acid receptor-{alpha} and -{beta} (RXRA and RXRB) (47). Overall, the results provide first evidence for a broad expressional modulation of mitochondrial turnover by NRG-1{beta}.

NRG-1 exerts control on the expression of several neuromuscular junction-associated channels (61). The mRNA level of four voltage-gated plasma membrane ion channels, SCN1B, ATP1B2, ATP2B1, and CLCN3, was increased in the NRG-1{beta}-stimulated cardiocytes (see Fig. 4). These data provide novel evidence for a transcriptional potential of NRG to modulate excitation-contraction coupling in cardiocytes (Fig. 6).

Regulatory events of NRG-modulated gene expression.   NRG-1{beta} caused a rapid (15 min) and sustained activation of AKT and MAPK pathways in adult rat ventricular myocytes and endothelial cells (31, 53). This activation is similar between paced and quiescent cells (31) and is evident before eventual contractile changes are apparent under electric stimulation (34). The downregulation of CCND1, TXN, and GSTP1 mRNA levels by the specific MEK inhibitor PD-98059 (Fig. 6) is in line with the requirement of MAPK signaling for the enhanced CCND1 levels in hypertrophying cardiocytes (54) and the strong relation between GSTP1 and CCND1 mRNAs in certain lymphomas (5). The quiescent adult primary ventricular cardiocytes under study do not develop contractile force. Passive contractions were not observed in quiescent nor NRG-1{beta}-treated cardiocytes (M.-N. Giraud, unpublished observations). We conclude that the identified enhanced cell cycle and redox factor transcript levels are under direct control of the MAPK-dependent signaling pathway via possible effects on transcript synthesis or degradation.

Summarizing, our results indicate that NRG-1{beta} initiated in cardiocytes 1) expressional improvements for salvage pathways involved in redox regulation and cardiac hypertrophy, 2) transcript level reprogramming toward enhanced protein synthesis, and 3) a shift in energy metabolism toward enhanced gene expression of glycolytic factors, mitochondrial components, and ion channels, as well as reduced fatty acid transporters. These expressional adjustments point to the molecular strategy that underlies the cardioprotective effect the NRG growth factor in cultured cells that may come into play in other physiological situations where NRG signaling is implicated.

In conclusion, we report the first large-scale evidence that NRG interacts with quiescent cardiocytes by promoting long-lasting expressional responses of various cellular systems. Our results support the hypothesis of NRG as a survival factor for adult cardiocytes via enhanced levels of redox regulators and cell cycle regulator cyclin D1. They provide as well the first evidence for altered mitochondrial turnover and adjustments of voltage-gated ion channels in cardiocytes by NRG (Fig. 6). The observations hence provide valuable insight on the mode of action of NRG in muscle tissues (30, 61).

This work was supported by Swiss National Science Foundation Grants SCORE-A 32-54985.98 and 32-55136.98 (to T. M. Suter).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Hans Hoppeler for providing access to the equipment for microarray analysis.

Present address of M.-N. Giraud: Cardiovascular Surgery, Inselspital, 3010 Bern, Switzerland.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Flück, Dept. of Anatomy, Univ. of Bern, Baltzerstrasse 2, 3000 Bern 9, Switzerland (E-mail: flueck{at}ana.unibe.ch)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* M.-N. Giraud and M. Flück contributed equally to the work. Back


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