Cardiovascular Research

Cardiovascular Research 1999 42(2):443-454; doi:10.1016/S0008-6363(99)00045-0
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Gene expression of proteins influencing the calcium homeostasis in patients with persistent and paroxysmal atrial fibrillation

Bianca J.J.M. Brundel^a,b, Isabelle C. Van Gelder^a,*, Robert H. Henning^b, Anton E. Tuinenburg^a, Leo E. Deelman^b, Robert G. Tieleman^a, Jan G. Grandjean^c, Wiek H. Van Gilst^b and Harry J.G.M. Crijns^a

^aDepartment of Cardiology, Thoraxcenter, University Hospital Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands
^bDepartment Clinical Pharmacology, Thoraxcenter, University Hospital Groningen, Groningen, The Netherlands
^cDepartment of Thoracic Surgery, Thoraxcenter, University Hospital Groningen, Groningen, The Netherlands

i.c.van.gelder{at}thorax.azg.nl

^* Corresponding author. Tel.: +31-50-3612355; fax: +31-50-3614391

Received 16 December 1998; accepted 25 January 1999

	Abstract

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

 
Objective: Persistent atrial fibrillation (AF) results in an impairment of atrial function. In order to elucidate the mechanism behind this phenomenon, we investigated the gene expression of proteins influencing calcium handling. Methods: Right atrial appendages were obtained from eight patients with paroxysmal AF, ten with persistent AF (>8 months) and 18 matched controls in sinus rhythm. All controls underwent coronary artery bypass grafting, whereas most AF patients underwent Cox’s MAZE surgery (n=12). All patients had a normal left ventricular function. Total RNA was isolated and reversely transcribed into cDNA. In a semi-quantitative polymerase chain reaction the cDNA of interest and of glyceraldehyde-3-phosphate dehydrogenase were coamplified and separated by ethidium bromide-stained gel electrophoresis. Slot blot analysis was performed to study protein expression. Results: L-type calcium channel ₁ and sarcoplasmic reticulum Ca²⁺-ATPase mRNA (–57%, p=0.01 and –28%, p=0.04, respectively) and protein contents (–43%, p=0.02 and –28%, p=0.04, respectively) were reduced in patients with persistent AF compared to the controls. mRNA contents of phospholamban, ryanodine receptor type 2 and sodium/calcium exchanger were comparable. No changes were observed in patients with paroxysmal AF. Conclusions: Alterations in gene expression of proteins involved in the calcium homeostasis occur only in patients with long-term persistent AF. In the absence of underlying heart disease, the changes are rather secondary than primary to AF.

KEYWORDS Atrial fibrillation; Ca²⁺-handling proteins; Gene expression

	1. Introduction

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

 
Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting millions of people worldwide and its incidence increases with age [1]. Clinical observations showed that immediately after restoration of sinus rhythm, atrial contractile function is severely impaired or even absent [2,3]. The contractile dysfunction is reversible after restoration of sinus rhythm, its time course being related to the previous duration of AF [2]. Restoration does not seem to be complete in all patients presumably related to the extent of damage occurring during AF [4,5].

In an experimental pig model atrial contractile dysfunction was observed after cessation of pacing induced AF of a duration of only 1–30 min, indicating that contractile remodeling, like electrical remodeling in the goat [6], is an early process [7]. There are strong indications that abnormalities in calcium handling, in response to tachycardia induced intracellular calcium overload [8–10], play a pivotal role in the induction of atrial contractile dysfunction [5,7,11–14]. The proteins and ion channels involved in the adaptation processes during AF have not been clarified yet. Most likely, identification of the signaling pathways and their target genes may lead to new therapeutic options for the treatment of AF. Therefore, we investigated alterations in mRNA and protein expression of proteins involved in calcium handling of right atrial appendages (RAA) of patients with paroxysmal and persistent AF undergoing cardiac surgery. To overcome the problem whether changes were caused by AF itself, or by the concomitant underlying heart disease, we selected AF patients with a normal left ventricular function and matched them for age, sex and left ventricular function with patients in sinus rhythm who underwent coronary artery bypass surgery.

	2. Methods

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

 
2.1 Patients
The day before surgery, one investigator (AET) assessed the clinical characteristics of the patients. The patients’ history and previous electrocardiograms were used to establish type and duration of AF. In addition, the patients were asked for medication use and exercise tolerance (according to the New York Heart Association classification). Echocardiography data were obtained within 3 months before surgery. RAAs were obtained from eight patients with paroxysmal AF and from ten with persistent AF without valvular heart disease and a normal left ventricular function. The AF patients were matched for age, sex, left ventricular function, and as far as possible for medication with 18 clinically stable patients in sinus rhythm undergoing coronary artery bypass surgery. The Institutional Review Board approved the study, and all patients gave written informed consent. Immediately after excision, the RAAs were snap-frozen in liquid nitrogen and stored at –85°C.

2.2 RNA isolation and cDNA synthesis
Total RNA was isolated from RAAs using the method of acid guanidinium thiocyanate/phenol/chloroform extraction followed by a RNeasy kit for RNA minipreps from tissues (Qiagen). The amount of RNA was evaluated by absorption at 260 nm, using a GeneQuant II (Pharmacia Biotechnology, The Netherlands). The ratio of absorption (260–280 nm) of all preparations was between 1.8 and 2.0. First strand cDNA was synthesized by incubation of 1 µg of total RNA, reverse transcription 10x buffer and 200 ng of random hexamers with 200 Units of Moloney Murine Leukemia Virus Reverse Transcriptase, 1 mM of each dNTP and 1 Unit of RNase inhibitor (Promega, The Netherlands) in 20 µl. The synthesis reaction lasted 10 min at 20°C, 20 min at 42°C, 5 min at 99°C, and 5 min at 4°C, respectively. All the products were checked on contaminating DNA (data not shown).

2.3 Semi-quantitative PCR analyses
Since a linear relationship between the amount of input template and amplification product exists within the exponential range of amplification, a semi-quantitative polymerase chain reaction (PCR) was developed [15]. The cDNA of interest and the cDNA of the ubiquitously expressed housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were coamplified in a single PCR reaction. Primers were designed for sarcoplasmic reticulum calcium ATPase (SR Ca²⁺-ATPase), phospholamban (PLB), L-type calcium channel ₁ subunit (L-type Ca²⁺), sodium/calcium exchanger (Na⁺/Ca ²⁺ exchanger), Ryanodine receptor type 2 (RyR2) and the housekeeping enzyme GAPDH (Table 1). Eurogentec (Belgium) synthesized the oligonucleotides. For the semi-quantitative PCR co-amplification of 1 µl of cDNA mixture, 0.5 unit of Taq polymerase (Eurogentec, Belgium) was added to 17.5 nM dNTPs, 10x PCR buffer provided with Taq polymerase, 2.5 mM MgCl₂, 40 pmol of sense and antisense primer for the gene of interest, 40 pmol of sense and antisense GAPDH primer and water to bring the final volume to 50 µl. All reaction mixtures were overlaid with 50 µl of mineral oil (Sigma, The Netherlands). After 3 min denaturation at 94°C, n cycles (Table 1) of amplification were performed, each for 1 min at 94°C, 1 min at 56°C and 1 min at 72°C, using the thermocycler Perkin Elmer 480 (The Netherlands). After the last cycle the 72°C elongation step was extended to 5 min. The PCR products were separated on 1–2.5% agarose gels by gel-electrophoresis and stained with ethidium bromide. The densities of the PCR products were quantified by densitometry (Aldus PhotoStyler 2.0, Grafic Workshop and ImageQuant Version 3.3). Linearity for the PCR reactions was established by making a correlation between the number of cycles and the density of the ratio gene of interest/GAPDH (data not shown).

View this table: [in this window] [in a new window]   Table 1 Sequence for the primers

 
During the PCR for L-type Ca²⁺ the three isoforms of this channel were amplified. The primers were designed to amplify the IVS1 upto IVS5 region of the 1 subunit. The PCR fragment contained the IVS3B adult isoform, the IVS3B deleted D1 isoform and the IVS3A fetal isoform, which could be identified as products of 472, 442, and 355 bp, respectively, after digestion with DdeI (Promega, The Netherlands).

The RyR2 PCR fragment was designed to contain the alternative splicing site, a 24-bp insert between residues 11145 and 11146 with a restriction site for BamHI. The RyR2 isoform with the 24 bp insert could be identified as products of 310 and 326 bp after digestion with BamHI (Promega, The Netherlands).

2.4 Determination of the absolute alterations of mRNA
To validate the semi quantitative PCRs the changes observed in ratios for L-type Ca²⁺ and SR Ca²⁺ ATPase were determined. Increasing amounts of gene of interest were added to a fixed amount of GAPDH. Therefore, known amounts of GAPDH input template (range 10^–2 to 10^–4 ng, 410 bp) were added to a PCR sample mixture of 0.5 unit of Taq polymerase (Eurogentec, Belgium),17.5 nM dNTPs, 10x PCR buffer, 2.5 mM MgCl₂, 40 pmol of sense and antisense GAPDH primer, 0.7 µl of cDNA mixture and water to bring the final volume to 50 µl, and amplified for 25 cycles. Thereafter, a fixed amount of GAPDH input template was used in a PCR with known amounts of the SR Ca²⁺-ATPase input template (range 10^–1.5 ng to 10^–3.5 ng, 657 bp) or L-type Ca²⁺ input template (range 10^–1.5 ng to 10^–3.5 ng, 563 and 530 bp). The ratios SR Ca ²⁺-ATPase input or L-type Ca²⁺ input versus GAPDH input were calculated.

2.5 Protein preparation and slot–blot analysis
Frozen RAAs of five patients in sinus rhythm, five patients with paroxysmal and five with persistent AF were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 mM β-mercaptoethanol, 10 mg/ml PMSF, 5 µl/ml aprotinin, 100 mM sodium orthovanadate, 5 µl/ml benzamidine, 5 µl/ml pepstatine A, 5 µl/ml leupeptine in 1x PBS by use of an ultraturrax (Polytron, The Netherlands) with 10-s intervals. The homogenate was centrifuged at 14 000 rpm for 20 min at 4°C. After centrifugation the supernatant was carefully removed and used for protein concentration measurement. This was done according to the Bradford method (Sigma, The Netherlands) with bovine albumin used as a standard. Samples of 10 µg protein were denaturated by heating to 95°C before spotting on a TBS (10 mM Tris–HCl pH 8.0, 150 mM NaCl) wetted nitrocellulose membrane (Bio-Rad, The Netherlands) by use of a slot blot apparatus (Bio-Rad, The Netherlands). The membrane was washed twice with 200 µl TBS buffer and the transfer was checked by staining the nitrocellulose membrane with Ponceau S solution (Sigma, The Netherlands). Blocking was performed for 20 min in blocking buffer (5% nonfat milk, TBS and 0.1% Tween 20). After three times washing for 5 min in TBS with 0.1% Tween 20 the membranes were incubated for 90 min with primary antibodies: SR Ca ²⁺ ATPase 1:2500, PLB 1:200 (gifts from Dr F. Wuytack, University Leuven, Belgium), GAPDH 1:5000 (Affinity Reagents, USA) or L-type Ca²⁺ anti ₁-subunit (Alomone Labs, Israel). Immunodetection of the primary antibody was performed, after three times washing for 5 min with TBS and 0.1% Tween 20, with peroxidase conjugated secondary antibody anti-rabbit and anti-mouse IgG (Santa Cruz Biotechnology, The Netherlands) for 60 min. The primary and secondary antibodies were diluted in blocking buffer. The blot was washed two times for 5 min in TBS and 0.1% Tween 20 and one time in TBS also for 5 min. Subsequently, the blot was incubated with the ECL-detection reagent (Amersham, The Netherlands) for 1 min, and exposed to a X-OMAT X-ray film (Kodak, The Netherlands) for 15–90 s. The band densities were evaluated by densitometric scanning using a Snap Scan 600 (Agfa, The Netherlands). To test the linearity of the immunodetection system distinct amounts of protein were analyzed. There was a linear relation between protein amounts spotted on the membrane and the immunoreactive signals of L-type Ca²⁺, SR Ca ²⁺-ATPase, PLB and GAPDH (data not shown).

2.6 Definitions
2.6.1 Persistent AF
Continuous presence of AF until the moment of cardiac surgery, i.e. at least two consecutive electrocardiograms of AF more than 24 h apart, without intercurrent sinus rhythm. Persistent AF has a non-spontaneously converting character [16,17]. Previously, this type of AF was classified as chronic AF.

2.6.2 Paroxysmal AF
AF typically occurring in episodes of a duration shorter than 24 h (but longer lasting paroxysms are not unusual) with intermittent sinus rhythm. Paroxysmal AF is either converting spontaneously or is terminated with an intravenously administered antiarrhythmic drug [16,17]. It is non-controlled whether paroxysmal AF is present at the moment of cardiac surgery.

2.7 Statistical analysis
All PCRs and SDS-PAGEs were performed twice. The mean values of the ratios were used for statistical analysis. To compare the baseline characteristics between groups for normally distributed variables, mean values and standard deviations are reported. In case of skewed distribution of variables, the median values and ranges are given. Baseline comparison between groups for normally distributed variables was performed by one-way ANOVA for skewed distributed variables by the Wilcoxon two-sample test. The chi-square test with continuity correction or Fisher’s exact test was performed for group comparison for categorical variables when appropriate. To determine which variables influenced mRNA levels of proteins, univariate regression analyses were performed. Only variables with a p value <0.15 were selected for multiple regression analysis. To determine differences in mRNA levels of these proteins between the four groups, a Tukey correction for multiple comparison was performed.

For determination of correlations the Spearman correlation test was used. The Wilcoxon two-sample test was performed for group to group comparison in case of skewed distributed variables or in case of small numbers.

All p-values are two-sided, a p-value <0.05 was considered statistically significant. SAS version 6.12 (Cary, NC) was used for all statistical evaluations.

	3. Results

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

 
3.1 Patients
Included were eight patients with paroxysmal and ten patients with persistent AF. These two groups were compared with two groups of control patients in sinus rhythm, who were matched for sex, age and left ventricular function (Table 2). Six of the eight patients with paroxysmal AF suffered from intractable AF and were scheduled for Cox’s MAZE surgery. The median duration of sinus rhythm before surgery was 1.5 days. The median frequency of paroxysms was once a day (median duration of each paroxysm was 3 h). Three patients with paroxysmal AF had AF at the moment of surgery and harvesting of the RAA. Control RAAs were obtained from clinically stable patients in sinus rhythm who were scheduled for coronary artery bypass surgery. Although the AF groups and their controls in sinus rhythm differed with respect to the underlying heart disease, all had a normal left ventricular function and were in the functional class I or II for exercise tolerance. Also, echocardiographic atrial and left ventricular dimensions were similar among groups.

View this table: [in this window] [in a new window]   Table 2 Baseline characteristics of patients with paroxysmal AF, persistent AF and patients in sinus rhythm at the moment of surgerya

 
3.2 Alterations in mRNA levels in paroxysmal and persistent AF
Changes in mRNA levels of the gene of interest were determined by comparison of gene-of-interest/GAPDH ratios between patients with persistent AF or with paroxysmal AF, and their matched controls in sinus rhythm. The densities of the amplified GAPDH of the four groups of patients were the same for all the genes investigated (data not shown).

Only patients with persistent AF showed a significant reduction of the cDNA ratios of L-type Ca²⁺/GAPDH (–57%) and SR Ca²⁺-ATPase/GAPDH (–28%) (Fig. 1A,B). The cDNA ratios of PLB/GAPDH, Na⁺/Ca²⁺ exchanger/GAPDH and RyR2/GAPDH were unchanged compared to the controls in sinus rhythm (Fig. 1C–E). No changes were observed in patients suffering from paroxysmal AF. Table 3 shows that the cDNA ratio L-type Ca²⁺/GAPDH in patients with persistent AF was neither influenced by the underlying heart disease nor by any (calcium handling influencing) drug.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Individual cDNA ratios for L-type Ca2+/GAPDH (A), SR Ca2+ ATPase/GAPDH (B), PLB/GAPDH (C), Na+/Ca2+ exchanger/GAPDH (D) and RyR2/GAPDH (E). Mean values are given for the four different groups±S.E.M. SR-CAF is the sinus rhythm control group for patients with chronic persistent AF, SR-PAF is the sinus rhythm control group for patients with paroxysmal AF. All data are represented in density units/density units. Significance was determined using the Tukey correction for multiple regression analysis for each mean of the four groups.

 

View this table: [in this window] [in a new window]   Table 3 Effects of underlying heart disease and medication on the mRNA levels of l-type Ca2+ channela

 
3.3 Validation of the absolute mRNA contents
To assess the amounts of L-type Ca²⁺ and SR Ca²⁺-ATPase in the cDNA mixtures in different groups, a semi-quantitative PCR was developed with increasing amounts of input cDNA of interest to a standard amount of GAPDH cDNA (0.018 ng).The ratios were determined and plotted in a logarithmic way. This resulted in a straight line demonstrating the validity of the method and enabling estimations of differences of L-type Ca²⁺ and SR Ca²⁺ ATPase in the cDNA mixture (Fig. 2A,B).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Plot showing a significant correlation between the increasing doses of L-type Ca2+ (A) and SR Ca2+ ATPase (B) input template and the ratios of L-type Ca2+/GAPDH and SR Ca2+ ATPase/GAPDH, respectively. Correlation was determined by the Spearman correlation test.

 
3.4 Alterations in proteins levels in paroxysmal and persistent AF
Changes in protein levels were studied for the genes which showed alterations in mRNA ratios (L-type Ca²⁺ and SR Ca²⁺-ATPase) and for PLB. Sufficient RAA tissue to carry out protein isolations were available from five patients with paroxysmal AF, five with persistent AF and five patients in sinus rhythm. The protein levels of gene-of-interest/GAPDH ratios were determined. Fig. 3A shows the specificity of the antibodies used, Fig. 3B the results of the slot–blot analysis. The protein ratio of L-type Ca²⁺/GAPDH and SR Ca²⁺-ATPase/GAPDH were significantly reduced in patients with persistent AF compared to the controls (–43%, p=0.02 and –28%, p=0.04, respectively, Fig. 4A,B). The PLB/GAPDH ratio between the groups was not statistically significant (Fig. 4C). The GAPDH levels were comparable between the different groups (mean values not shown). No alterations were found in the protein levels of patients with paroxysmal AF (Fig. 4). A positive correlation between the mRNA ratios and the protein ratios of L-type Ca²⁺ and SR Ca²⁺-ATPase for patients with persistent AF and sinus rhythm could be demonstrated (Fig. 5).

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) Western blot analysis showing the specificity of the antibodies on human RAA tissue. Anti-L-type calcium channel 1 subunit (200 kDa, lane 1), anti-SR Ca2+ ATPase (105 kDa, lane 2), anti-PLB (6.5 kDa, lane 3), GAPDH (36 kDa, lane 4) and the Marker (lane 5, Bio Rad, The Netherlands). (B) Slot–blot analysis of L-type Ca2+, SR Ca2+ ATPase, PLB and GAPDH of five control patients, five patients with chronic persistent AF (CAF) and five patients with paroxysmal AF (PAF).

 

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Protein ratios of L-type Ca2+/GAPDH (A), SR Ca2+ ATPase/GAPDH (B) and PLB/GAPDH (C) of the individual patients. SR is the sinus rhythm control group. All data are presented as density units/density units. Values are mean±S.E.M. Significant differences are indicated (Wilcoxon two-sample test).

 

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Relationship between the mRNA and protein expression of L-type Ca2+(A), SR Ca2+ ATPase (B) and PLB (C). () Represents chronic persistent AF patients, () represents SR patients. Correlation was determined by the Spearman correlation test.

 
3.5 Isoforms of L-type calcium channel
To investigate whether dedifferentiation occurred as an adaptation process in patients with AF, the mRNA expression level of the fetal isoform compared to the adult isoforms of L-type Ca²⁺ was determined. The amplified PCR fragment of L-type Ca²⁺ contained the IVS3A (fetal form), the IVS3B (adult form) and the IVS3B deleted D1 form after digestion with DdeI (Fig. 6). No differences in percentages of the adult and fetal isoforms of L-type Ca²⁺ were found in relation to the total quantity of the amplified L-type Ca²⁺ in the different groups (Fig. 7A). The ratio of the IVS3B form was significantly reduced in patients with persistent AF (p=0.01, Fig. 7B). No significant changes were seen in the IVS3B deleted D1 form and the IVS3A form. Any significant alteration in the L-type Ca²⁺ ratios was observed in patients with paroxysmal AF (Fig. 7B–D).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Typical example of an agarose gel. Here, the L-type Ca2+ isoforms (IVS3B, 472 bp; IVS3B deletion, 442 and IVS3A, 355 bp) of four patients with chronic persistent AF and their matched controls in sinus rhythm and patients with paroxysmal AF and their controls are shown.

 

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 (A) Shows the percentage IVS3B, IVS3B deletion and IVS3A in relation to the total L-type Ca2+ protein mRNA expression. No differences in percentage were found between the three isoforms. The cDNA ratios of the IVS3B/GAPDH was significantly lower in patients with persistent AF (B). No alterations were observed between the groups of cDNA ratios of IVS3B deletion/GAPDH (C) and the fetal form IVS3A/GAPDH (D). SR-CAF are the matched control patients in sinus rhythm for chronic persistent AF and SR-PAF for paroxysmal AF. All data are presented as density units/density units. Values are mean±S.E.M. Significant differences are indicated (Wilcoxon two-sample test).

 

	4. Discussion

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

 
4.1 Main findings
We examined five different genes which play an important role in the calcium homeostasis of the myocardial cell. By examining the mRNA and protein expression in patients with paroxysmal AF and persistent AF, and for age, sex and cardiac function matched controls in sinus rhythm we observed two important features. First, the mRNA and protein contents of both SR Ca²⁺-ATPase and L-type Ca²⁺ were significantly reduced in patients with persistent AF. Second, significant changes occurred only in patients with persistent AF but not in those with paroxysmal AF.

4.2 Alterations in gene expression of ion channels and proteins involved in the calcium handling
The alterations observed in the present study of a reduction of the mRNA and protein contents for the L-type Ca²⁺ without changes in L-type Ca²⁺ isoforms in patients with persistent AF, fit in with the described changes in the above mentioned experimental models. Clearly, a lower mRNA and protein expression of L-type Ca²⁺ may underlie reduced L-type Ca²⁺ current densities [11,12,18]. Whereas in the experiments of Yue et al. [11] significant reduction of L-type Ca²⁺ current densities was observed within 6 weeks of rapid continuous atrial pacing, we, however, observed significant alterations only in patients with persistent long-standing (>8 months) AF.

Data on alterations in the sarcoplasmic reticulum proteins influencing calcium handling in RAA tissue of patients with AF are lacking. The observed reduction of the SR Ca²⁺-ATPase mRNA and protein expression in atrial tissue of patients with persistent AF is comparable to data on left ventricular tissue of patients with severe heart failure due to dilated and ischemic cardiomyopathy [19–24]. In our population, both mRNA and protein expression were reduced. In contrast, a discrepancy between adaptation of the SR Ca²⁺-ATPase mRNA, protein and activity levels have been reported in ventricular tissue of patients suffering from severe heart failure [20,21,24–26]. The mRNA and protein expression of PLB, the regulatory protein of SR Ca²⁺-ATPase, was not significantly different between the AF groups and their controls. A reduced gene expression of SR Ca²⁺-ATPase and a not significantly changed expression of PLB yields a reduced ratio of SR Ca²⁺-ATPase to PLB in patients with persistent AF. If we assume that the stoichiometry of PLB to SR Ca²⁺-ATPase determines the level of SR Ca²⁺-ATPase inhibition, this finding may indicate that in the basal low-phosphorylated state, depression of SR calcium uptake is even more pronounced than would be expected from the lower SR Ca²⁺-ATPase mRNA level in patients with persistent AF. This interpretation would be consistent with functional abnormalities observed in the failing human ventricular myocardium [23].

For the ryanodine receptor the amounts of a RyR2 region with or without the 24 bp insert were examined. Alternative transcripts with or without this insertion might provide a means for altering the binding affinity of this putative site for calcium [27]. No differences were found in the expression of RyR2 with or without insert between the groups. This suggests that persistent AF did not induce differences in the calcium binding affinity and changes in fundamental nature of the RyR2. Furthermore, no changes in mRNA and protein expression of RyR2 were observed in patients with AF.

4.3 No dedifferentiation of the L-type calcium channel during AF
Each IVS3 isoform is encoded by a separate but adjacent exon within a single genomic clone and the various isoforms are generated by a developmentally regulated, mutually exclusive exon splicing of the primary transcript [28,29]. No reversion of the adult isoform of L-type Ca²⁺ to its fetal isoform was observed in patients with AF. In contrast, however, Gidh-Jain et al. [30] demonstrated that in patients with left ventricular hypertrophy a significant increase of the mRNA contents of the fetal isoform and reversion of fetal/adult isoform ratio to the fetal phenotype was observed in ventricular tissue. In atrial goat tissue, Ausma et al. [31] showed that during pacing induced AF proteins which are present in embryonic/fetal myocardial cells, e.g. -smooth muscle actin, were reexpressed. A reversion to fetal isoforms of certain ion channels and proteins might be hypothesized to occur in situations comparable to the embryonic situation, e.g. during higher heart rates as is the case during AF. Therefore, a reexpression of fetal proteins might have occurred during AF.

4.4 No changes in gene expression in patients with severe paroxysmal AF
We observed significant alterations in expression of the investigated genes only in patients with long-standing (>8 months) persistent AF. No significant changes were observed in those patients suffering from paroxysmal AF. Importantly, the included paroxysmal AF patients had severe AF with daily episodes of AF. Moreover, three patients had AF at the moment of surgery, i.e. at the moment of harvesting of the right atrial appendage. This may suggest that episodes of sinus rhythm in between episodes of AF protect the myocardial cell from alterations in gene expression.

4.5 Limitations of the study
Drugs and differences in underlying diseases may influence gene expression of proteins and ion channels influencing calcium handling. In this study, to minimize the influence of particular clinical parameters on gene expression, we included only patients with a normal left ventricular function and, when possible, drugs were discontinued before surgery.

The present study does not clarify when the adaptive mechanisms of the atrial myocardial cell, i.e. alterations in gene expression, start. Although our data suggest that this is a late process, no patients with persistent AF with a duration between 1 day and 8 months were included .

Only ten patients with persistent AF and eight patients with paroxysmal AF were included. Therefore, a larger study will be needed to confirm all observations of the present study.

No matched controlled analysis could be performed for determination of protein levels. However, no significant changes in mRNA levels between the control groups were observed. Therefore, in our opinion, a comparison between persistent AF, paroxysmal AF and sinus rhythm patients may to be justified. Clearly, a new study using matched controlled analysis is needed for confirmation of the present protein data.

Time for primary review 27 days.

	Acknowledgements

 
Dr Van Gelder was supported by Grant 94.014 of the Netherlands Heart Foundation, The Hague, The Netherlands. The study was supported by Grant 96.051 of the Netherlands Heart Foundation, The Hague, The Netherlands. We are indebted to Pieter J. De Kam for statistical analysis.

	Notes

 
Presented in part at the Congress of the NASPE (North American Society for Pacing and Electrophysiology), May 6–9 1998, San Diego, California.

	References

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

 

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