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Cardiovascular Research 2002 54(2):427-437; doi:10.1016/S0008-6363(02)00260-2
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Copyright © 2002, European Society of Cardiology

Analysis of altered gene expression during sustained atrial fibrillation in the goat

Victor L.J.L Thijssena,*,1, Huub M.W van der Veldenb,1, Erwin P van Ankerenb, Jannie Ausmac, Maurits A Allessiec, Marcel Borgersa, Guillaume J.J.M van Eysd and Habo J Jongsmab

aDepartment of Molecular Cell Biology, Cardiovascular Research Institute Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands
bDepartment of Medical Physiology, University Medical Center Utrecht, Utrecht, The Netherlands
cDepartment of Physiology, Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands
dDepartment of Molecular Genetics, Cardiovascular Research Institute Maastricht, Maastricht, The Netherlands

Victor.Thijssen{at}Molcelb.Unimaas.nl

* Corresponding author. Tel.: +31-43-388-1361; fax: +31-43-388-4151

Received 21 September 2001; accepted 22 January 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Atrial fibrillation (AF) is characterised by electrical, gap junctional and structural remodelling. However, the underlying molecular mechanisms of these phenomena are largely unknown. To get more insight into atrial remodelling at the molecular level we have analysed changes in gene expression during sustained AF in the goat. Methods: The differential display technique (DD) was used to identify genes differentially expressed during sustained AF (13.9±5.2 weeks) as compared to sinus rhythm (SR). Dot-blot analysis was performed to confirm the altered gene expression and to establish the changes in expression after 1, 2, 4, 8 and 16 weeks of AF. Immunohistochemistry and western blotting were used to validate the DD approach and to further characterise the changed expression of the β-myosin heavy chain gene at the protein level. Results: Of the approximately 125 fragments that showed changed expression levels during AF, 34 were cloned and sequenced. Twenty-one of these represented known genes involved in cardiomyocyte structure, metabolism, expression regulation, or differentiation status. The changed expression of 70% of the isolated clones could be confirmed by dot-blot analysis. In addition, time course analysis revealed different profiles of expression as well as transient re-expression of genes, e.g. the gene for hypoxia-inducible factor 1{alpha} during the first week of AF. During sustained AF the frequency of cardiomyocytes expressing β myosin heavy chain (βMHC) increased from 21.8±2.1 to 47.9±2.5% (S.E.M.). The overall expression of MHC ({alpha}+β) appeared to be down-regulated during AF. Conclusions: AF is accompanied by changes in expression of proteins involved in cellular structure, metabolism, gene expression regulation and (de-)differentiation. Most alterations in expression confirm or support the hypothesis of cardiomyocyte de-differentiation. Furthermore, the results suggest a role for ischemic stress in the early response of cardiomyocytes to AF, possibly via activation of hypoxia-inducible factor 1{alpha}.

KEYWORDS Arrhythmia (mechanisms); Atrial function; Remodeling; Gene expression; Ischemia; Myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Atrial fibrillation (AF) is characterised by extensive electrical and structural remodelling of the atrium, both at the level of the single myocyte and the atrial tissue as a whole. Electrophysiological remodelling is characterised by shortening of the atrial effective refractory period (AERP), which (in part) is the result of changes in expression of the L-type Ca2+ channel. Several authors have reported, both in animal models [1,2] and in patients [3,4], a down-regulated expression of the L-type Ca2+ channel during AF. In patients with paroxysmal or persistent AF reduced protein levels of L-type Ca2+ channel were shown to correlate with AERP shortening [5]. Besides the L-type Ca2+ channel, the expression of several K+ channel subunits appears to be down-regulated [1–3,6–8].

Since it was shown that time courses of AERP shortening and the increase in the duration of AF episodes did not match, it was concluded that other factors must be important for making the arrhythmia sustained [9]. A candidate is gap junctional remodelling, which was shown to occur both in a goat model of AF and in patients suffering from sustained AF [10–14]. Another factor involved in the stabilisation of AF appears to be structural remodelling [15]. Structural remodelling during AF includes a significant reduction in sarcomere number (myolysis), the accumulation of glycogen, changes in mitochondrial morphology, altered chromatin distribution and last but not least changes in cellular size (hypertrophy), phenomena known from hibernating myocardium in patients with chronic ischemic heart disease [16–19]. In addition, the expression pattern of several (structural) proteins adapts a foetal phenotype indicative of cardiomyocyte de-differentiation; e.g. re-expression of {alpha}-smooth muscle actin and the disappearance of cardiotin [20].

Apart from its effect on the stability of AF, structural remodelling of atrial myocytes is likely to be co-responsible for the temporarily impaired contractile function of the atria after cardioversion from AF [21]. It has been shown that following brief periods of AF, the electrical remodelling and contractile dysfunction are completely reversible within a few days [22]. However, after longer periods of AF, a discrepancy occurs between the normalisation of electrical properties (days to weeks) [23–25] and the recovery of contractile function (weeks to months) [26–29]. Structural remodelling might be important in this context, although the down-regulation and/or altered function of L-type Ca2+ channels seems to be largely responsible [22].

Many aspects of AF-induced remodelling have been studied extensively in the goat model of AF. In fact, the principles of ‘AF begets AF’ and ‘AF-induced de-differentiation’ originate from studies in which this model was used [30]. However, the exact molecular mechanisms underlying various aspects of atrial remodelling still remain unknown. In order to identify factors involved in the electrical and structural remodelling, we studied alterations in gene expression in the fibrillating goat atrium using the method of differential display [31].


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animal model of atrial fibrillation
For the first part of our studies 12 female goats were used (average weight 55 kg). Of these, six were kept in SR while in the others sustained AF was induced as described by Wijffels et al. [9]. In brief, silicon strips containing multiple electrodes were stitched to the epicardial surface of both atria as well as to Bachmann's bundle. After recovery, the electrodes were connected to an external automatic fibrillator programmed to deliver a 1-s burst of stimuli (50 Hz, four times threshold) each time an iso-electrical segment longer than 300 ms was detected. Following this protocol AF becomes sustained (episodes >24 h) in most animals after 1–2 weeks. After at least 2 months of sustained AF samples of the right (RAA) and left atrial appendages (LAA) were immediately frozen in liquid N2 and stored at –80 °C until use.

For the second part of our studies 36 female goats were used (average weight 61 kg) and instrumented as described by Ausma et al. [32]. Six were kept in SR and used as controls. In the other animals an electrode was inserted via the jugular vein in the right atrium and connected to a pacemaker. Sustained (chronic) AF was induced by switching on the pacemaker, producing 2-s bursts (50 Hz, four times threshold) at 1-s intervals. Initially AF was self-terminating within seconds whereas during the following days AF episodes became longer and more stable. The inter-burst period could gradually be prolonged to 30 min while sustained AF was being maintained. Animals were sacrificed after 1, 2, 4, 8 or 16 weeks of AF (n=6) and RAA and LAA samples were prepared and stored as described above.

All animal handling was according to the Dutch Law on Animal Experimentation (WOD) and The European Directive for Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes.

2.1.1 Differential display
From pulverised atrial samples, total RNA was isolated using RNeasy (Qiagen). Chromosomal DNA contaminations were removed during incubation with RNase-free DNase (10 U/µl; Boehringer Mannheim) in the presence of an RNase inhibitor (1 U/µl RNA-guard; Pharmacia). Concentrations were adjusted to 0.2 µg/µl and aliquots were frozen and stored at –80 °C. For reverse transcription 1 µl (0.2 µg) DNA-free total RNA and 1 µl 25 µM anchored primer (T12VA (V is three-fold degenerate for A, C and G), T12VC, T12VG or T12VT; final concentration 2.5 µM) in 8 µl DEPC treated dH2O was heated in a thermal cycler for 5 min at 65 °C and quickly cooled down to 37 °C. Then 10 µl of a mixture of M-MLV Reverse Transcriptase (200 U; GibcoBRL) in 100 mM Tris–HCl, pH 8.3, 150 mM KCl, 6 mM MgCl2, 20 mM DTT, 40 µM dNTPs and 2U/µl RNA-guard was added and incubations were performed for an additional l h at 37 °C, followed by 5 min at 95 °C to heat inactivate the enzyme.

Aliquots of 2 µl were transferred to thin walled 200 µl PCR tubes and used for differential display (DD) analysis. To each aliquot an equal volume of 25 µM of the appropriate anchored primer was added followed by a mixture of 1 unit Taq DNA polymerase (recombinant; GibcoBRL) and 0.5 µM of a specific arbitrary 10-mer primer (26 different ones; Operon) in 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 3 mM MgCl2, 2 µM of each dNTP, 0.02 µCi/µl [{alpha}-32P]–dCTP (all final concentrations). Incubations were performed in a Perkin Elmer GeneAmp PCR System 2400 thermal cycler (Perkin Elmer Applied Biosystems) according to the following program; 1 min at 94 °C was followed by 40 cycles {30 s 94 °C, 90 s 40 °C, 30 s 72 °C} and an extension of 5 min at 72 °C. Stop buffer (10 mM ethylenediaminetetraacetic acid, pH 7.5, 97.5% deionised formamide and 0.3% Bromophenol Blue) was added, samples were heated to 80 °C for 2 min and 3 µl aliquots were electrophoresed on 6% denaturing sequencing gels. Each gel was dried without fixation and exposed to Fuji RX safety film.

Gel strips containing DD PCR fragments were excised, dried and soaked in 100 µl dH2O for 10 min at room temperature, followed by incubation for 15 min in a boiling water bath. Eluted DNA in the presence of a glycogen carrier was ethanol precipitated and re-amplified by Taq DNA polymerase (2 units) in 40 µl of 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 3 mM MgCl2 containing 2.5 µM each of the appropriate anchored primer and specific 10-mer primer and 20 µM of each dNTP. PCR conditions were as described above. Finally, DNAs were cloned into a T/A cloning vector (pGEM-T Easy, Promega) or pBluescript (Stratagene) and at least three clones of each ligation were sequenced using an ABI prism 310 Genetic Analyzer (Perkin Elmer Applied Biosystems).

2.1.2 Slot(dot)-blot analysis
For the screening of the DD DNA products a slot-blot procedure was applied as described by Mou et al. [33]. Of each plasmid DNA 250 ng was denatured for 10 min at 100 °C in 0.4 M NaOH, 10 mM EDTA and transferred onto a pre-cut nylon membrane (Zeta-Probe, Biorad). The DNA in each well was washed with 0.4 M NaOH, 10 mM EDTA and immobilised by UV cross-linking. Each membrane was pre-hybridised for 1 h at 60 °C in 0.5 M Na2HPO4, pH 7.2, 1 mM EDTA, 7% SDS. For probes, 10 µg of pooled (from five goats) total RNA and 2.5 µg oligodT were incubated at 65 °C for 10 min. Reverse transcription was performed for 1.5 h at 37 °C with 600 U M-MLV reverse transcriptase (GibcoBRL) in 50 µl 50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM of dATP, dGTP and dTTP, in the presence of 40 U RNase inhibitor RNasin (Promega) and 50 µCi [{alpha}-32P]dCTP. Following purification over Sephadex G50, probes were denatured for 5 min at 95 °C and added to the pre-hybridisation mix. Following overnight incubation at 60 °C, membranes were washed in phosphate buffer/SDS with increasing stringency and exposed to a phosphorimaging screen (Biorad). For image analysis either Image Quant® (Molecular Dynamics) or Quantity One® version 4.2.1. (Biorad) software was used. For analysis of the time series, 500 ng amplified DNA fragments were transferred onto Hybond N+ membranes (Amersham) following the same procedure. Probe labelling and hybridisation was as described above.

The expression level of each clone was normalised to that of at least two out of four different controls; a clone from a goat atrium cDNA library encoding part of the GAPDH housekeeping gene and three cloned DD-DNAs selected on the basis of equal gel display in AF and SR.

2.1.3 Immunohistochemistry
Unfixed cryostat sections (8 µm) were stained essentially as described before [34]. They were incubated at room temperature in phosphate-buffered saline (PBS) containing 0.2% (v/v) Triton X-100 for 1 h, followed by 30 min in 2% (w/v) Bovine Serum Albumin (BSA) in PBS. Sections were washed with PBS and incubated overnight with primary mouse anti-{alpha}-MHC (Alexis Biochemicals, clone F88-12.F8) or mouse anti-β-MHC (169-ID5; gift from Dr. A.F.M. Moorman, AMC) antibodies (1:5 and 1:10 dilution in PBS containing 10% Normal Goat Serum, respectively). Samples were incubated for 2.5 h with secondary antibodies against mouse IgG, conjugated with Fluorescein Isothiocyanate (FITC; Jackson Laboratories) or Texas Red (TR; Jackson Laboratories). Sections were mounted with Vectashield (Vector Laboratories) and examined using a Nikon Optiphot-2 light microscope equipped for epifluorescence. Within each section, percentages of myocytes that did (++ or +) or did not (–) stain for β-MHC were assessed.

2.1.4 Western blotting
Tissue samples were homogenised in RIPA solubilisation buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Nonidet-P40, 1% sodium deoxycholate, 0.1% sodium dodecylsulfate) with protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 1 mM phenantroline, 1 mM benzamidine, 0.5 mM pefabloc, 5 mM sodium bisulfate, 20 µg/ml pepstatin A). They were treated as described [35]. Heparin anti-coagulated blood control samples were immediately mixed with concentrated Laemmli's sample buffer. Of each sample 40 µg of total cellular protein was electrophoresed on 8% SDS–polyacrylamide gels and transferred onto nitrocellulose membranes. Efficiency of transfer was checked by Ponceau Red staining. Membranes were blocked for 1 h at room temperature in 5% Protifar in 0.1% Tween 20 in PBS (T–PBS). They were probed for the presence of {alpha} and β-MHC in an overnight incubation at 4 °C using the antibodies (1:40 and 1:10, respectively) described in the previous section. After the primary antibody incubation, membranes were incubated for 1 h at 4 °C with a horseradish peroxidase labelled secondary antibody (Bio-Rad; 1:7000). Peroxidase activity was detected using ECL detection reagents (Amersham). Film exposure times were 20 s for detection of {alpha}-MHC and 30 min for detection of β-MHC expression. Scans (Microtek ScanMaker 630) were analysed using Image Quant software (Molecular Dynamics).

2.1.5 Statistical analysis
To identify statistically significant changes in expression between AF and SR, a Mann–Whitney rank sum test was used to compare the average normalised values (see Methods) of five independent slot(dot)-blot analyses. Beta-MHC expression in AF was compared to SR using a paired Student's t-test or whenever the data did not have a normal distribution a Mann–Whitney rank sum test. Statistical significance was defined as P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Analysis of differential gene expression between SR and chronic AF
To identify genes involved in AF-induced remodelling of the atrium, we used the DD technique [31] to compare atrial mRNA expression patterns from six goats in sustained AF (induced as described by Wijffels et al. [9]) with those from six goats in SR. For PCR amplification primer combinations of one out of four degenerate anchored oligo(dT) primers with one out of 26 short arbitrary 10-mers (OP-DDRT1-OP-DDRT26) [36] were used. A typical example of expression patterns generated by DD is shown in Fig. 1. About 125 DD bands of amplified gene products, reproducibly displayed differently in AF as compared to SR, were excised from the gels. DNAs from 34 DD fragments, ranging from 90 to 375 bp (average of 227 bp), were cloned and sequenced. They represented 16 (47%) down- and 15 (44%) up-regulated genes, while three (clone 2.2, 3.3, and 4.1) of them did not show any change in expression and were used as controls. Apart from these controls, 21 (62%) clones could be identified based on sequence homology to database entries of known eukaryotic genes. Most clones could be placed into one of four functional categories; ‘structure’ (7/21), ‘metabolism’ (4/21), ‘expression’ (7/21) and ‘embryogenesis/(de-)differentiation’ (3/21). Genes in the first category mainly encoded proteins involved in cardiomyocyte contractility, like {alpha}-cardiac actin ({alpha}-CA), β-myosin heavy chain (β-MHC), titin, and cardiac troponin I (cTnI). In addition, a sarcolemma associated protein (SLAP-2) and a nuclear matrix protein (matrin 3) were identified. Genes involved in metabolic pathways included the VLDL receptor (VLDLr) gene, the myoglobin gene and the ubiquitin hydrolysing enzyme I (UBH1) gene, encoding a de-ubiquitinating enzyme which prevents ubiquitinated substrates from degradation, like ‘fat facets’ in Drosphila [37]. In the category ‘expression’, several genes were identified encoding ribosomal proteins, together with a transcriptional repressor (of c-myc) and a translational activator of EIF2{alpha} (EIF2{alpha} kinase). The remaining known genes (3/21) encoded proteins (H beta 58, LAK-4p, KIAA1610/ER 1) that have been associated with the differentiation status of an eukaryotic cell [38,39]. The ten DD fragments placed in the category ‘unknown’ matched with EST sequences, which means that they represent either unknown genes or known genes whose 3' non-translated sequences are not in the GenBank database.


Figure 1
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  		Fig. 1 Differential display expression patterns. Typical examples of DD expression patterns obtained by using two different anchored primers in combination with two 10-mer primers. Gel patterns from two goats in SR and three goats in sustained AF were compared. The arrow indicates a DD band, representing a gene whose expression is up-regulated in AF.

 
3.2 Confirmation of altered gene expression
Since one of the criticisms of the DD technique is the relatively high number of false positives, reported to range from 5 to 50% [40], it is essential to confirm differential gene expression by a hybridisation-based assay such as Northern blotting. In order to screen large numbers of DD-products simultaneously without the need for large amounts of RNA, we used a reverse Northern-like procedure, essentially as described by Mou et al. [33]. A total of five independent experiments were performed to determine the ratios of normalised signals between AF and SR. It was assessed whether each ratio was statistically higher or lower than 1, indicating up- or down-regulation of gene expression (Fig. 2). The four controls showed a maximal deviation of 10% in AF as compared to SR, which suggests that they are stable expressed. The DD data for 20 (65%) of the remaining 31 clones could be confirmed by dot-blot analyses and ratios for 15 of these were statistically significant (P<0.05). The dot-blot data from ten clones did not corroborate those from the DD analyses and showed either no change (1) or an opposite change (9) in expression. Although statistically significant, overall changes in the levels of expression for most genes were not very large; up-regulation ranged from +22 to +107% and down-regulation from –7 to –38%.


Figure 2
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  		Fig. 2 Gene expression analysis of sequences differentially displayed during sustained atrial fibrillation. {blacktriangleup}=up-regulated; {blacktriangledown}=down-regulated; {diamondsuit}=unchanged; ND is not determined; *P<0.050.

 
3.3 Time course of altered gene expression
Changes in AF-induced gene expression levels were studied in a time course series by comparing dot-blot data after 1, 2, 4, 8 and 16 weeks of AF with those in SR. We also investigated a number of genes that were likely to undergo changes in expression during AF, i.e. the genes for {alpha}-smooth muscle actin ({alpha}-SMA), slow skeletal troponin I (ssTnI), and hypoxia-inducible factor 1{alpha} (HIFl{alpha}). The results are listed in Fig. 3. After 1 week of AF, five out of 13 genes investigated showed a similar change in expression as observed during sustained AF (average 12.7 weeks). At 4 weeks, this number had increased to eight out of 13 and at 16 weeks, the expression level of most of the clones (12 out of 13) was similar or comparable to that measured before. Several genes were down-regulated throughout the time course (titin, UBH1, {alpha}-SMA, EIF{alpha}2 kinase), whereas others changed progressively (β-MHC, cTnI, ssTnI, VLDLr) or displayed transient alterations (HIF1{alpha}, {alpha}-CA). In addition, genes that had not changed their overall expression in sustained AF as compared to SR did show changes during the time course. For example, the gene represented by DD-DNA 7 appeared to follow an expression pattern (0–8 weeks) similar to HIF1{alpha}, while the gene represented by DD-DNA 2 showed a transient decreased expression early (1 week) in AF.


Figure 3
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  		Fig. 3 Time course of altered gene expression during atrial fibrillation. * P<0.050; {blacktriangleup}=up-regulated; {blacktriangledown}=down-regulated; {diamondsuit}=unchanged; NA=not applicable.

 
3.4 Changes in myosin heavy chain isoform expression
Since the results from the DD and dot-blot analyses with respect to the β-MHC mRNA expression were rather contradictory, we investigated the protein expression of {alpha}-MHC and β-MHC by immunohistochemistry and western blotting. Fig. 4A shows immunolabelling patterns of representative sections of the atrial appendage from a goat in SR incubated with antibodies against {alpha}-MHC or β-MHC. Most cardiomyocytes (<90%) stained positive for {alpha}-MHC while only a subpopulation (21.8+2.1% (S.E.M.)) expressed the β-isoform of MHC (+ and ++). Within this subpopulation a small fraction of cardiomyocytes (3.2+/–0.7%) expressed β-MHC at high levels (++). During sustained AF the percentage of cardiomyocytes expressing {alpha}-MHC seemed unaffected, while the percentage of those expressing β-MHC (+ and ++) had clearly increased (47.9+/–2.5% (S.E.M.)). The percentage of cardiomyocytes expressing β-MHC at high levels (++) had increased even more (18.0+/–2.5% (S.E.M.)). The observed AF-induced increase in the relative numbers of β-MHC expressing cells is in support of the DD-data. The relative levels of expressed {alpha}- and β-MHC protein were assessed by western blot analysis (Fig. 4B). Contrary to what has been shown for chicken atrium [41], both antibodies reacted with several bands in the goat atrium extracts. We focused on bands with the same electrophoretic mobility as the 200 kD marker, representing rabbit skeletal muscle myosin. Competitor studies could not be performed since specific peptides were not available. Consequently, we used adult mouse heart and goat blood extracts for controls. Cross-reactivity of the {alpha}-(human)MHC antibody to mouse cardiac myosin in a western blot was stated by the supplier. Indeed a prominent 200 kD band showed up, as can be seen in the positive (+) control lane. There was no reaction with the goat blood sample (control –). The β-MHC antibody was (almost) negative with the mouse cardiac extract, which is in accordance with the negligible expression of β-MHC in adult mouse heart [42]. There was no specific reaction with goat blood. However, a non-specific band with a higher molecular weight became visible, possibly as a result of the almost 100 times longer exposure time needed for ECL detection of β-MHC (30 min) as compared to {alpha}-MHC (20 s). Fig. 4B shows the goat atrial myosin expression in two representative experiments. The quantitative analysis of the data from four experiments revealed a significantly lower expression of {alpha}-MHC in AF as compared to SR and a tendency of up-regulated β-MHC expression (not statistically significant).


Figure 4
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  		Fig. 4 Expression of {alpha}- and β-MHC protein in goat atrial myocytes. (A). Immunolabelling of successive transversely cut thin sections of atrial appendage from a goat in AF with antibodies against {alpha}- or β-MHC. Matching phase contrast images are given underneath each panel. Longitudinally sectioned myocytes were double stained with antibodies against β-MHC and desmin. Bar=100 µm. The histogram shows the data of an immunohistochemical evaluation of thin sections from six goats in SR and six goats in sustained AF. Frequencies are given of atrial myocytes that did not stain (–), stained positive (+) for β-MHC or expressed it at very high levels (++). (B). Western blot analysis of the {alpha}- and β-MHC protein levels in SR and sustained AF (16 weeks) from two representative experiments. Positions of the 200 kD rabbit skeletal muscle myosin marker are indicated. For controls, adult mouse cardiac (+) and goat blood (–) extracts were used. Band intensities were measured and average data from four experiments are given in the bar plot.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
AF is accompanied by electrical [9,43], gap junctional [10,11] and structural remodelling [17,19] of the atria. In this study we have used the goat model of AF to obtain more insight into the underlying molecular processes. To identify genes involved in atrial remodelling, we have compared the expression profiles in SR with those during AF by means of the differential display (DD) technique. DD was used because at present no goat-specific microarrays are available. In addition, DD allows the direct identification of both up-regulated and down-regulated genes between multiple samples within one experiment [31]. We have isolated a number of genes that were either up- or down-regulated during sustained (chronic) AF. For approximately 60% of these genes differential expression could be confirmed by dot-blot analysis. Less than 10% of the DD-signals turned out to be false positive, which is within the margins reported for the technique. In those situations, in which the dot-blot data did not corroborate the DD results, heterogeneity between tissue samples is the likely cause. Since relatively large tissue samples from five goats had to be pooled for the preparation of probes, relative numbers of remodelled cardiomyocytes in these mixed samples might be significantly different from those in the much smaller individual goat samples used for DD analysis. This might account for relatively mild overall changes in gene expression. However, locally these (focal) changes might be very pronounced and promote arrhythmogenic activity that could affect the atrial tissue as a whole.

Characteristic of AF-induced structural remodelling of cardiomyocytes is that several structural proteins adopt a foetal-like expression [20]. The expression of several genes that were detected in our DD analyses and placed in the category ‘structure’ appears to be in line with this phenomenon. The observed down-regulation of the mRNA expression of titin is in agreement with observations at the protein level [20]. Down-regulation of cTnI and {alpha}-CA mRNA also correlates with the observed reduction in sarcomeres. It has been suggested that the 3' non-translated regions of both genes are involved in inhibition of proliferation and promotion of differentiation [44], which suggests that down-regulation of their expression might favour cellular de-differentiation. Yet, re-expression of the embryonically expressed ssTnI and {alpha}-SMA [45–48] could not be detected. The absence of an increase in {alpha}-SMA mRNA is in contrast with the observed increase in {alpha}-SMA protein in the cardiomyocytes during AF [20]. However, since cTnI and {alpha}-CA are abundantly expressed and highly homologous to their embryonically expressed counterparts, the inconsistency is likely due to cross-reactivity of their mRNAs with the ssTnI and {alpha}-SMA DNA on the filter, which would obscure any AF-induced effect in mRNA expression of the latter two. In addition, it has been reported that the expression of {alpha}-SMA is predominantly regulated at a post-transcriptional level [49].

Cross-reactivity of {alpha}-MHC mRNA with β-MHC DNA (homology in humans 95% [50]) on the filters is possibly also responsible for the observed inconsistency between DD and dot-blot data. The relative proportions of the two isoforms of myosin heavy chain in mammalian heart are affected by a wide variety of pathological and physiological stimuli [51]. Cardiomyocytes that predominantly express the faster (higher ATPase activity) {alpha}-MHC can generate more force than those expressing the slower β-isoform [52]. Human ventricular myocardium contains 93% β-MHC and 7% {alpha}-MHC, whereas {alpha}-MHC mRNA makes up 30% of total MHC mRNA [53]. During heart failure {alpha}-MHC decreases to a level 15-fold less for mRNA, while protein could hardly be detected anymore. Adult bovine atrium consists of 80–90% {alpha}-MHC and 10–20% β-MHC [54]. Also in adult human left atria {alpha}-MHC comprises about 90% of total MHC, whereas this relative amount dropped to around 50% in individuals with dilated or ischemic cardiomyopathy [55]. Concomitant changes at the mRNA level are not known. An increase in the relative amount of β-MHC of the left atrium during the development of heart failure has been suggested to be beneficial by increasing the economy of contraction [56]. Consequently, an AF-induced increase in β-MHC expression in combination with a reduction in {alpha}-MHC expression would be in support of an observed reduction in atrial contractility. Since a down-regulation of MHC expression could already be detected early (1 week) in the time course, it might be partly responsible for the developing atrial dysfunction.

Apart from structural remodelling, AF is accompanied by electrical and gap junctional remodelling and many authors including ourselves have described changes in the expression or distribution of channel proteins that might underlie these processes [1,2,5,15]. In the present study no changes in the levels of mRNAs that encode ion or gap junction channel proteins have been identified. The reason might be the low abundance of many of these mRNAs and the fact that DD is a technique focussed on the 3' untranslated regions of mRNAs. These regions are highly variable between species, which implies that some of the unidentified DD sequences might actually represent 3' UTRs of goat channel mRNAs. Since not all the fragments isolated following DD analysis have been characterised (only 34 out of 125), it is not unlikely that channel mRNAs might be represented among the remaining 90 sequences.

We did find changes in expression of several genes involved in cellular metabolic pathways. Altered expression of proteins like myoglobin and the VLDLr are indicative of changes in energy utilisation. Down-regulation of the VLDLr has been shown to occur in hypertrophic rat hearts [57]. A shift in the metabolic balance as a result of AF had already been suggested by Ausma et al. [58]. They concluded that a temporary metabolic imbalance was not the result of ischemic injury [58]. However, the transient increase in the expression of HIF1{alpha}, as was observed in the present study, indicates that a certain amount of ischemic stress during the onset of AF cannot be ruled out. It has been reported that HIF1{alpha} up-regulates the glucose transporter 1 and several glycolytic enzymes [59,60]. This, in combination with the observed down-regulation of the VLDLr, is indicative of a switch from fatty acid metabolism to glucose metabolism, subsequently leading to increased glycogen storage, either due to excessive influx of glucose or to a decreasing energy demand as a result of impaired contractile activity.

Apart from known genes, we isolated a number of DD fragments without homology to any of the genes that are currently present in the GenBank database. Some of these might represent genes whose products play an important role in early response mechanisms, for they show an expression pattern similar to that of HIF1{alpha} (clones 2 and 7, down- and up-regulated in the first week of AF, respectively). We are currently trying to isolate and characterise full-length cDNAs of these unknown mRNA species.

In conclusion, we have used DD in our search for changes in gene expression due to AF in the goat. That this approach is feasible was proven by the identification of genes whose protein products are known to be involved in AF-induced structural remodelling. In addition, we found genes that might play an important role in this process, genes potentially involved in ‘AF induced de-differentiation’, as well as genes whose expression is in favour of a short period of ischemic stress during the onset of AF.

Time for primary review 25 days.


   Acknowledgements
 
The authors like to thank Dr. A.F.M. Moorman (Academic Medical Centre, Amsterdam) for providing them with the monoclonal antibody (169-ID5) against myosin heavy chain beta, and M. van Zijverden and A.J.C.G.M. Hellemons for their technical assistance. Part of this study was supported by grants from the Dutch Heart Foundation (NHS M93.002 to H.M.W.v.d.V.; NHS 96.155 to J.A.) and the Dutch organisation for Scientific Research (NWO 900-516-318; M.A.A.).


   Notes
 
1 Both authors contributed equally to this manuscript. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Yue L, Melnyk P, Gaspo R, Wang Z, Nattel S. Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation. Circ Res (1999) 84:776–784.[Abstract/Free Full Text]
  2. van der Velden H.M.W, van der Zee L, Wijffels M.C, et al. Atrial fibrillation in the goat induces changes in monophasic action potential and mRNA expression of ion channels involved in repolarization. J Cardiovasc Electrophysiol (2000) 11:1262–1269.[CrossRef][ISI][Medline]
  3. Van Gelder I.C, Brundel B.J, Henning R.H, et al. Alterations in gene expression of proteins involved in the calcium handling in patients with atrial fibrillation. J Cardiovasc Electrophysiol (1999) 10:552–560.[ISI][Medline]
  4. Lai L.P, Su M.J, Lin J.L, et al. Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca(2+)-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban: an insight into the mechanism of atrial electrical remodeling. J Am Coll Cardiol (1999) 33:1231–1237.[Abstract/Free Full Text]
  5. Brundel B.J, Van Gelder I.C, Henning R.H, et al. Ion channel remodeling is related to intraoperative atrial effective refractory periods in patients with paroxysmal and persistent atrial fibrillation. Circulation (2001) 103:684–690.[Abstract/Free Full Text]
  6. Van Wagoner D.R, Pond A.L, McCarthy P.M, Trimmer J.S, Nerbonne J.M. Outward K+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ Res (1997) 80:772–781.[Abstract/Free Full Text]
  7. Lai L.P, Su M.J, Lin J.L, et al. Changes in the mRNA levels of delayed rectifier potassium channels in human atrial fibrillation. Cardiology (1999) 92:248–255.[CrossRef][ISI][Medline]
  8. Brundel B.J.J.M, Van Gelder I.C, Henning R.H, et al. Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation: differential regulation of protein and mRNA levels for K+ channels. J Am Coll Cardiol (2001) 37:926–932.[Abstract/Free Full Text]
  9. Wijffels M.C, Kirchhof C.J, Dorland R, Allessie M.A. Atrial fibrillation begets atrial fibrillation. A study in awake chronically instrumented goats. Circulation (1995) 92:1954–1968.[Abstract/Free Full Text]
  10. van der Velden H.M, van Kempen M.J, Wijffels M.C, et al. Altered pattern of connexin40 distribution in persistent atrial fibrillation in the goat. J Cardiovasc Electrophysiol (1998) 9:596–607.[ISI][Medline]
  11. van der Velden H.M, Jongsma H.J. Progress in clinical pacing 2000. Santini M, ed. (2000) Rome: Tipolitografia Fernando Begliomini. 190–197.
  12. van der Velden H.M, van Kempen M.J, Groenewegen W.A, Jongsma H.J. Comparative analysis of the distribution of the gap junction proteins connexins 40 and 43 in chronic atrial fibrillation (AF) in patients and a goat model. Circulation (1997) 96:I-237. (Abstract).
  13. Kangaratnam P, Severs N.J, Peters N.S. The relationship between conduction, activation pattern and quantity of immunoreactive connexin in chronic human atrial fibrillation. Eur Heart J (2000) 21:240. (Abstract).
  14. Dupont E, Ko Y, Rothery S, et al. The Gap-junctional protein connexin40 is elevated in patients susceptible to postoperative atrial fibrillation. Circulation (2001) 103:842–849.[Abstract/Free Full Text]
  15. van der Velden H.M, Ausma J, Rook M.B, et al. Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat. Cardiovasc Res (2000) 46:476–486.[Abstract/Free Full Text]
  16. Borgers M, Thoné F, Wouters L, Ausma J, Shivalkar B, Flameng W. Structural correlates of regional myocardial dysfunction in patients with critical coronary artery stenosis: chronic hibernation? Cardiovasc Pathol (1993) 2:237–245.[Medline]
  17. Ausma J, Wijffels M, Thoné F, Wouters L, Allessie M, Borgers M. Structural changes of atrial myocardium due to sustained atrial fibrillation in the goat. Circulation (1997) 96:3157–3163.[Abstract/Free Full Text]
  18. Thijssen V.L.J.L, Ausma J, Liu G.S, Allessie M.A, van Eys G.J, Borgers M. Structural changes of atrial myocardium during chronic atrial fibrillation. Cardiovasc Pathol (2000) 9:17–28.[CrossRef][ISI][Medline]
  19. Thijssen V, Ausma J, Borgers M. Structural remodelling during chronic atrial fibrillation: act of programmed cell survival. Cardiovasc Res (2001) 52:14–20.[Abstract/Free Full Text]
  20. Ausma J, Wijffels M, van Eys G, et al. Dedifferentiation of atrial cardiomyocytes as a result of chronic atrial fibrillation. Am J Pathol (1997) 151:985–997.[Abstract]
  21. Schotten U, Ausma J, Stellbrink C, et al. Cellular mechanisms of depressed atrial contractility in patients with chronic atrial fibrillation. Circulation (2001) 103:691–698.[Abstract/Free Full Text]
  22. Schotten U, Allessie M. Electrical and contractile remodeling during atrial fibrillation go hand-in-hand. PACE (2001) 24:572. (Abstract).
  23. Yu W.C, Lee S.H, Tai C.T, et al. Reversal of atrial electrical remodeling following cardioversion of long-standing atrial fibrillation in man. Cardiovasc Res (1999) 42:470–476.[Abstract/Free Full Text]
  24. Manios E.G, Kanoupakis E.M, Chlouverakis G.I, Kaleboubas M.D, Mavrakis H.E, Vardas P.E. Changes in atrial electrical properties following cardioversion of chronic atrial fibrillation: relation with recurrence. Cardiovasc Res (2000) 47:244–253.[Abstract/Free Full Text]
  25. Elvan A, Wylie K, Zipes D.P. Pacing-induced chronic atrial fibrillation impairs sinus node function in dogs. Electrophysiological remodeling. Circulation (1996) 94:2953–2960.[Abstract/Free Full Text]
  26. Manning W.J, Silverman D.I, Katz S.E, et al. Impaired left atrial mechanical function after cardioversion: relation to the duration of atrial fibrillation. J Am Coll Cardiol (1994) 23:1535–1540.[Abstract]
  27. Daoud E.G, Marcovitz P, Knight B.P, et al. Short-term effect of atrial fibrillation on atrial contractile function in humans. Circulation (1999) 99:3024–3027.[Abstract/Free Full Text]
  28. Everett T.H, Li H, Mangrum J.M, et al. Electrical, morphological, and ultrastructural remodeling and reverse remodeling in a canine model of chronic atrial fibrillation. Circulation (2000) 102:1454–1460.[Abstract/Free Full Text]
  29. Shapiro E.P, Effron M.B, Lima S, Ouyang P, Siu C.O, Bush D. Transient atrial dysfunction after conversion of chronic atrial fibrillation to sinus rhythm. Am J Cardiol (1988) 62:1202–1207.[CrossRef][ISI][Medline]
  30. Allessie M.A. Atrial electrophysiologic remodeling: another vicious circle? J Cardiovasc Electrophysiol (1998) 9:1378–1393.[ISI][Medline]
  31. Liang P, Pardee A.B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science (1992) 257:967–971.[Abstract/Free Full Text]
  32. Ausma J, Dispersyn G.D, Duimel H, et al. Changes in ultrastructural calcium distribution in goat atria during atrial fibrillation. J Mol Cell Cardiol (2000) 32:355–364.[CrossRef][ISI][Medline]
  33. Mou L, Miller H, Li J, Wang E, Chalifour L. Improvements to the differential display method for gene analysis. Biochem Biophys Res Commun (1994) 199:564–569.[CrossRef][ISI][Medline]
  34. Gros D, Jarry-Guichard T, Ten Velde I, et al. Restricted distribution of connexin40, a gap junctional protein, in mammalian heart. Circ Res (1994) 74:839–851.[Abstract/Free Full Text]
  35. Delorme B, Dahl E, Jarry-Guichard T, et al. Developmental regulation of connexin 40 gene expression in mouse heart correlates with the differentiation of the conduction system. Dev Dyn (1995) 204:358–371.[ISI][Medline]
  36. Bauer D, Muller H, Reich J, et al. Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR). Nucleic Acids Res (1993) 21:4272–4280.[Abstract/Free Full Text]
  37. Chen X, Overstreet E, Wood S.A, Fischer I.A. On the conservation of function of the Drosophila fat facets deubiquitinating enzyme and Fam, its mouse homolog. Dev Genes Evol (2000) 210:603–610.[CrossRef][ISI][Medline]
  38. Paterno G.D, Li Y, Luchman H.A, Ryan P.J, Gillespie L.L. cDNA cloning of a novel, developmentally regulated immediate early gene activated by fibroblast growth factor and encoding a nuclear protein. J Biol Chem (1997) 272:25591–25595.[Abstract/Free Full Text]
  39. Radice G, Lee J.J, Costantini F. H beta 58, an insertional mutation affecting early postimplantation development of the mouse embryo. Development (1991) 111:801–811.[Abstract]
  40. Martin K.J, Pardee A.B. Principles of differential display. Meth Enzymol (1999) 303:234–258.[CrossRef][ISI][Medline]
  41. de Groot I.J, Hardy G.P, Sanders E, Los J.A, Moorman A.F. The conducting tissue in the adult chicken atria. A histological and immunohistochemical analysis. Anat Embryol (1985) 172:239–245.[CrossRef][Medline]
  42. Lyons G.E, Schiaffino S, Sassoon D, Barton P, Buckingham M. Developmental regulation of myosin gene expression in mouse cardiac muscle. J Cell Biol (1990) 111:2427–2436.[Abstract/Free Full Text]
  43. Nattel S, Li D, Yue L. Basic mechanisms of atrial fibrillation-very new insights into very old ideas. Annu Rev Physiol (2000) 62:51–77.[CrossRef][ISI][Medline]
  44. Rastinejad F, Blau H.M. Genetic complementation reveals a novel regulatory role for 3' untranslated regions in growth and differentiation. Cell (1993) 72:903–917.[CrossRef][ISI][Medline]
  45. Bhavsar P.K, Dhoot G.K, Cumming D.V, Butler-Browne G.S, Yacoub M.H, Barton P.J. Developmental expression of troponin I isoforms in fetal human heart. FEBS Lett (1991) 292:5–8.[CrossRef][ISI][Medline]
  46. Ausoni S, De Nardi C, Moretti P, Gorza L, Schiaffino S. Developmental expression of rat cardiac troponin I mRNA. Development (1991) 112:1041–1051.[Abstract]
  47. Ya J, Markman M.W, Wagenaar G.T, Blommaart P.J, Moorman A.F, Lamers W.H. Expression of the smooth-muscle proteins alpha-smooth-muscle actin and calponin, and of the intermediate filament protein desmin are parameters of cardiomyocyte maturation in the prenatal rat heart. Anat Rec (1997) 249:495–505.[CrossRef][Medline]
  48. Woodcock-Mitchell J, Mitchell J.J, Low R.B, et al. Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation (1988) 39:161–166.[CrossRef][ISI][Medline]
  49. Harder B.A, Hefti M.A, Eppenberger H.M, Schaub M.C. Differential protein localization in sarcomeric and nonsarcomeric contractile structures of cultured cardiomyocytes. J Struct Biol (1998) 122:162–175.[CrossRef][ISI][Medline]
  50. Kurabayashi M, Tsuchimochi H, Komuro I, Takaku F, Yazaki Y. Molecular cloning and characterization of human cardiac alpha- and beta-form myosin heavy chain complementary DNA clones. Regulation of expression during development and pressure overload in human atrium. J Clin Invest (1988) 82:524–531.[ISI][Medline]
  51. Wessels A. Spatial distribution of ‘tissue-specific’ antigens in the developing human heart. Amsterdam: University of Amsterdam, 1991. (Thesis).
  52. Pope B, Hoh J.F, Weeds A. The ATPase activities of rat cardiac myosin isoenzymes. FEBS Lett (1980) 118:205–208.[CrossRef][ISI][Medline]
  53. Miyata S, Minobe W, Bristow M.R, Leinwand L.A. Myosin heavy chain isoform expression in the failing and nonfailing human heart. Circ Res (2000) 86:386–390.[Abstract/Free Full Text]
  54. Komuro I, Nomoto K, Sugiyama T, Kurabayashi M, Takaku F, Yazaki Y. Isolation and characterization of myosin heavy chain isozymes of the bovine conduction system. Circ Res (1987) 61:859–865.[Abstract/Free Full Text]
  55. Reiser P.J, Portman M.A, Ning X.H, Schomisch Moravec C. Human cardiac myosin heavy chain isoforms in fetal and failing adult atria and ventricles. Am J Physiol Heart Circ Physiol (2001) 280:H1814–H1820.[Abstract/Free Full Text]
  56. Holubarsch C, Goulette R.P, Litten R.Z, Martin B.I, Mulieri L.A, Alpert N.R. The economy of isometric force development, myosin isoenzyme pattern and myofibrillar ATPase activity in normal and hypothyroid rat myocardium. Circ Res (1985) 56:78–86.[Abstract/Free Full Text]
  57. Masuzaki H, Jingami H, Matsuoka N, et al. Regulation of very-low-density lipoprotein receptor in hypertrophic rat heart. Circ Res (1996) 78:8–14.[Abstract/Free Full Text]
  58. Ausma J, Coumans W, Mast F, et al. High energy phosphates content and mitochondrial enzyme activity during chronic atrial fibrillation. J Mol Cell Card (1999) 31:A62.
  59. Semenza G.L, Roth P.H, Fang H.M, Wang G.L. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem (1994) 269:23757–23763.[Abstract/Free Full Text]
  60. Chen C, Pore N, Behrooz A, Ismail-Beigi F, Maity A. Regulation of glutl mRNA by hypoxia-inducible factor. 1. Interaction between H-ras and hypoxia. J Biol Chem (2001) 276:9519–9525.[Abstract/Free Full Text]

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