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J Am Coll Cardiol, 2003; 41:858-869, doi:10.1016/S0735-1097(02)02922-4 © 2003 by the American College of Cardiology Foundation |
* Department of Cardiology, University of Tübingen, Tübingen, Germany
Aventis Pharma Deutschland GmbH, DG Cardiovascular Diseases, Frankfurt, Germany
Max-Delbrück Center for Molecular Medicine, Berlin, Germany
Manuscript received April 9, 2002; revised manuscript received August 10, 2002, accepted September 6, 2002.
* Reprint requests and correspondence: Dr. Ralph F. Bosch, Dept. of Cardiology, University of Tuebingen, Otfried-Mueller-Str. 10, D-72076 Tuebingen, Germany.
ralph.bosch{at}uni-tuebingen.de
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Abstract |
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BACKGROUND: Long-term atrial fibrillation reduces L-type Ca2+ (ICa,L) and transient outward K+ (Ito) currents by transcriptional downregulation of the underlying ionic channels. However, electrical remodeling starts early after the onset of rapid atrial rates. The time course of ion current and channel modulation in these early phases of remodeling is currently unknown.
METHODS: Rapid (600 beats/min) right atrial pacing was performed in rabbits. Animals were divided into five groups with pacing durations between 0 and 96 h. Ionic currents were measured by patch clamp techniques; messenger ribonucleic acid (mRNA) and protein expression were measured by reverse transcription-polymerase chain reaction and Western blot, respectively.
RESULTS: L-type calcium current started to be reduced (by 47%) after 12 h of RAP and continued to decline as pacing continued. Current changes were preceded or paralleled by decreased mRNA expression of the Ca2+ channel ß subunits CaB2a, CaB2b, and CaB3, whereas significant reductions in the 
1 subunit mRNA and protein expression began 24 h after pacing onset. Transient outward potassium current densities were not altered within the first 12 h, but after 24 h, currents were reduced by 48%. Longer pacing periods did not further decrease Ito. Current changes were paralleled by reduced Kv4.3 mRNA expression. Kv4.2, Kv1.4, and the auxiliary subunit KChIP2 were not affected.
CONCLUSIONS: L-type calcium current and Ito are reduced in early phases of electrical remodeling. A major mechanism appears to be transcriptional downregulation of underlying ion channels, which partially preceded ion current changes.
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subunits is a major mechanism for the decreased densities of both currents in chronic electrical remodeling (10–14). In contrast to the well-defined alterations of cellular and molecular electrophysiology underlying chronic AF, the initial changes and their time course is currently unknown, although they might be interesting targets for therapeutic interventions. It is well known from in vivo studies that atrial repolarization starts to shorten after very brief periods of AF or RAP (15,16). These changes are associated with an increased inducibility and duration of AF episodes. The only data available on the molecular level showed a transient increase in Kv1.5 mRNA and protein and a reduced mRNA expression of Kv4.2 and Kv4.3 without changes in protein levels after short periods of RAP in rats (17).
The purpose of the present study was to characterize early changes in electrical remodeling on an ionic current level and to correlate them with alterations in ion channel expression.
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Methods |
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Cell isolation and solutions. Myocytes were isolated by enzymatic dissociation, as described previously (18). Bath solutions contained (in mmol/l) NaCl 136, KCl 5.4, CaCl2 2.0, MgCl2 1.0, NaH2PO4 0.33, HEPES 5, and Glucose 10 to record Ito; CholineCl 136, CsCl 5.6, CaCl2 2.0, MgCl2 1.0, NaH2PO4 0.33, HEPES 5, and Glucose 10 to record ICa,L. Pipette solutions contained KCl 20, K-aspartate 110, MgCl 1.0, HEPES 10, EGTA 5, Mg2ATP 5, GTP 0.1, and phosphocreatine 5 to record Ito; CsCl 20, Cs aspartate 110, HEPES 10, EGTA 10, MgCl 1.0, Mg2ATP 5, GTP 0.1, and phosphocreatine 5 to record ICa,L. Bath temperature was 36 ± 1°C. To record Ito, ICa,L and IKr were blocked with 1 µmol/l nisoldipine (Sigma, Deisenhofen, Germany) and 1 µmol/l dofetilide (Pfizer, Karlsruhe, Germany), respectively.
Voltage-clamp technique.
Currents were recorded using the whole-cell configuration of the patch clamp technique. Pipettes with resistances from 2 to 5 M
when filled with pipette solution were connected to a patch clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, California). Membrane capacitance averaged 77.6 ± 6 pF in P0 (n = 34), 85.2 ± 8.8 pF in P6 (n = 23), 69.5 ± 4.3 pF in P12 (n = 36), 67.8 ± 4.5 pF in P24 (n = 33) and 77.3 ± 5.7 pF in P96 (n = 36, p = NS). Before compensation, Rs averaged 9.88 ± 0.66 M. Corresponding values for Rs after compensation were 4.76 ± 0.30 M. Recordings for measurement of current densities were obtained 5 min after cell membrane rupture, followed by the other protocols for the determination of the biophysical properties.
Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).
Specimen were immediately frozen in liquid nitrogen and stored at –80°C. Specific oligonucleotide primer pairs were designed using the following GenBank accession numbers: rabbit Kv1.4 (AJ291314
[GenBank]
), Kv4.2 (AJ291315
[GenBank]
), Kv4.3 (AF198445
[GenBank]
), rabbit L-type Ca2+ channel 1 (X15539
[GenBank]
), 2 (M21948
[GenBank]
), CaB2a (X64297
[GenBank]
), CaB2b (X64298
[GenBank]
), CaB3 (X64300
[GenBank]
), and rabbit housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as standard (L23961
[GenBank]
). Rabbit KChIP2 was cloned and sequenced in its full length. Specific primer pairs and product lengths for each cDNA are shown in Table 1. One-step RT-PCR was performed using the One Step RT-PCR kit (Qiagen, Hilden, Germany) after determining optimal cycle numbers and annealing temperatures. The thermal cycling program was as follows: 50°C for 30 min (reverse transcription), followed by 95°C for 15 min as activation step for PCR, n cycles of amplification (Table 1), each for 30 s at 94°C, 30 s at the specific annealing temperature (Table 1), and 1 min at 72°C. After the last cycle, the 72°C elongation step was extended to 10 min. Electrophoresis of the amplified products was performed on 2% agarose gels containing ethidium bromide. Gels were photographed with a gel imaging system (Intas, Göttingen, Germany), and the density of each band was analyzed with Gel-Pro Analyzer (Media Cybernetics, Silver Spring, Maryland).
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1 antibodies were raised against the synthetic peptide (KQQLEEDLKGYLDWITQAE) corresponding to the /ß interaction domain within the cytoplasmic linker between repeat I and II of rabbit cardiac 1 subunit (20). The 190kD size form of the 1 subunit is the truncated form lacking a large portion of the C-terminus. It has consistently been the most abundant form isolated from cardiac preparations (21). The antibody was affinity purified on the respective antigen column. Anti-G3PDH was commercially available (Biotrend, Cologne, Germany). The membranes were incubated with primary antibodies prepared in TBS containing 1% BSA and 0.04% NaN3 for 90 min. After washing, the membranes were incubated for 1 h with HRP conjugated anti-rabbit IgG (DAKO, Hamburg, Germany) and anti-mouse IgG (Sigma) in block solution. The protein bands were visualized by ECL reagents and Hyperfilm ECL (Amersham Biosciences). The films were densitometrically evaluated using Kodak 1D Image Analysis Software (Kodak, Stuttgart, Germany).
Data analysis.
Data are expressed as mean ± SEM. Statistical comparisons between groups were performed by one-way analysis of variance. A two-tailed p value <0.05 was considered statistically significant. Comparisons between multiple groups were performed with a Bonferroni corrected t test for pairwise group comparisons. Nonlinear regression fitting was performed in pCLAMP 6.0 software (Axon Instruments) using the Chebyshev method to fit the bi-exponential inactivation (
fast and slow: fast and slow inactivation time constants). In Sigma Plot software (SPSS, Chicago, Illinois), the Marquardt-Levenberg algorithm was used for curve fitting to find the steady state half activation and inactivation voltages (Vh,act and Vh,inact) as well as the slope factors kact and kinact with a Boltzman function. The same algorithm was used to determine the time constant of recovery from inactivation recov using a mono-exponential function.
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Results |
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1 subunit (22) and the auxiliary subunits 2 and CaB1-CaB3 (23,24). The effects of RAP on the expression of these subunits are illustrated in Figure 3. A significant reduction (–24%) of the 1 subunit mRNA expression was first seen after 24 h of RAP (p < 0.05 vs. P0), whereas earlier groups were not different from sham-operated animals. After 96 h a further reduction in 1-mRNA expression was observed, mean expression being 48% of baseline (p < 0.01 vs. P0). The protein expression of the 1 subunit was also decreased if RAP persisted for 12 h or more (Fig. 3C), the relative expression averaged 82%, 84%, and 63% of controls after 12, 24, and 96 h of RAP, respectively (p < 0.05 vs. P0 for all). In contrast, mRNA expression of the 2 subunit was identical in all groups (Fig. 3C). The mRNA expression of all L-type Ca2+ channel ß subunits was also decreased by RAP (Fig. 3D and 3E). CaB2b showed a fast decline, with a 23% reduction after 6 h (P6, p < 0.01 vs. P0) that continued up to 96 h (P96, 34% reduction, p < 0.01 vs. P0). The expression of CaB2a was reduced by 45% after 12 h and continued to decrease up to 96 h (–54%, p < 0.01 vs. P0 for both). CaB3 mRNA expression started to be reduced at 12 h (20% reduction, p < 0.05 vs. P0) of RAP and reached a plateau thereafter.
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subunits (26,27). The consequences of RAP on the mRNA expression of these subunits are illustrated in Figure 5. Kv4.3 mRNA expression was not affected after 12 h of RAP but decreased significantly after 24 h to reach a plateau. The relative reductions at 24 and 96 h of RAP were 21.4% (p < 0.01 vs. P0) and 16.7% (p < 0.05 vs. P0), respectively. In contrast, the mRNA expression of Kv4.2 was not affected over 96 h of RAP in this model (Fig. 5). Kv1.4 expression was also reduced by RAP; however, changes were highly variable, even within a single group. Overall expression was therefore not significantly different among the different experimental groups. KChIP2 mRNA displayed a reduced expression after 24 and 96 h of RAP that, however, did not reach statistical significance when all groups were compared.
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1 subunit first showed a significant decrease in expression after 12 h of RAP. At 96 h, expression of all but the 2 subunit was decreased, probably contributing to the strong reduction of ICa,L in this group.
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Discussion |
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1 and all ß subunits of the L-type Ca2+ channel. Reductions in mRNA expression of Ca2+ channel ß subunits preceded those of ionic currents and of the 1 subunit, suggesting that a reduced transcription of these auxiliary subunits importantly contributes to reducing the number of functional channels in the cell membrane.
Comparison with previous reports in the literature.
L-type Ca2+ channels
Several studies in patients with chronic AF or in animal models of chronic AF have addressed changes in ionic currents and channels in electrical remodeling. Yue et al. (9) were the first to demonstrate a strong reduction of ICa,L in dogs subjected to RAP between 1 and 42 days. These findings were consistent with those of patients with long-lasting AF (6,8,28). A major mechanism of reduced ICa,L in chronic AF appears to be transcriptional downregulation of the pore-forming 1c subunit (10,11,14).
In the present study, we report the time course of reduction of ICa,L and of the mRNA and protein expression of the underlying channel subunits within the first 96 h of RAP. Before the first changes in current densities were detectable, transcription of the auxiliary channel subunit CaB2b was already strongly depressed. Together with the first detectable reduction in ICa,L, all 3 ß subunit mRNAs, as well as 1 subunit protein expression, were reduced in parallel. Wei et al. (29) have demonstrated that ß subunits are the limiting factor for the expression of L-type Ca2+ channels in the heart. Additionally, ß subunits carry phosphorylation sites that might be important in altering channel function (30). Our group has recently demonstrated in human AF that gene expression of the Ca2+ channel ß1 subunit isoform b (ß1b) is significantly reduced compared with sinus rhythm (31). From these data it appears that the reduced expression of ß subunits is an important mechanism for the reduction of functional channels in the cell membrane.
Kv4.3, Kv4.2, Kv1.4, and KChIP2 (Ito)
A strong reduction of Ito is a consistent finding in patients with chronic AF and in animals with long episodes of rapid atrial stimulation (6,7,9). In the rapid pacing dog model and in humans, a decreased expression of Kv4.3 is suggested to be the mechanism underlying reduced current densities in long-term AF and Kv4.3 expression correlated with Ito current densities (13,14). In our model of short-term RAP, we observed the first reductions in Ito and in Kv4.3 mRNA expression after 24 h; no further reduction was seen thereafter. A recent report by Yamashita et al. (17) described the effects of short episodes of RAP on the expression of different potassium channel subunits in rats. In that model, Kv4.2 and Kv4.3 mRNA expression were decreased after 2 and 4 h, respectively. In both studies Kv1.4 expression was not altered by RAP. The reasons for the different findings are not clear, but they may be due to species differences in the molecular compositions of the channel or to differences in the experimental design. Although our results suggest a transcriptional downregulation of Kv4.3 as a mechanism underlying reduced Ito, these findings need to be interpreted cautiously. First, the reduction in Ito densities was much more pronounced than the decrease in Kv4.3 mRNA expression, indicating that other mechanisms are also involved. Additionally, the molecular basis of Ito in rabbit atrium seems to be different from other species. Fermini et al. (32) have shown that rabbit Ito has a much slower recovery from inactivation than other species, including humans. The same group has recently provided several lines of evidence that, besides Kv4.3, Kv1.4 channels are importantly involved in rabbit atrial Ito, whereas they do not appear to play a role in human Ito (25). Kv1.4 mRNA expression was not significantly reduced by RAP for up to 96 h. However, we cannot exclude the fact that post-transcriptional alterations of Kv1.4 have importantly contributed to the reduced current density in RAP. To our knowledge, no data are available on the effects of AF on the expression of KChIP2 auxiliary potassium channel subunits. Our results provide evidence that, in contrast to the strong changes observed in L-type Ca2+ channel ß subunit expression, KChIP2 is only slightly decreased in the early phases of electrical remodeling and should, therefore, at best play a minor role in the reduction of Ito currents. The effect of long-term AF on KChIP2 expression awaits further study.
Time course of ion current/channel reduction. We reported a significant reduction of ICa,L and of Ito densities at 12 and 24 h of RAP, respectively. At 96 h of RAP, ICa,L was almost completely absent, and Ito was not further decreased after 24 h of RAP. These changes were paralleled by a decrease in ion channel subunit expression. The time course is much faster than has previously been reported by Yue and co-workers (9,14) in the RAP dog model, in which they saw a progressive decrease of both currents up to 42 days. The exact reasons for the different time courses are not clear; one possible explanation includes differences in the stimulation frequency. We used 600 beats/min, compared with the 400 beats/min used in the study by Yue et al. The higher rate in our model might cause faster changes on current densities and ion channel transcription levels. A second explanation could be interspecies differences in the time course of electrical remodeling, which were described in vivo between goat and dog. Atrial effective refractory period shortening in the goat AF model developed faster than that in the dog RAP model (1,33). Furthermore, electrical remodeling was much slower in the horse model than in the goat or dog model (34). These distinct time courses have been attributed to differences in the coronary reserve or different metabolic demands during AF (34), which, among other effects, also might affect ion channel expression and ion current density differently by mechanisms currently not known.
Potential significance. A shortening and a reduced rate adaptation of atrial refractoriness are consistent findings in AF in humans and in a variety of animal models. Repolarization starts to shorten after a few minutes of AF (15,16), the changes being most prominent within the first 30 min. In the following hours and days, a gradual but less pronounced decline has been described in different experimental models, after which no further shortening was detectable (1,32). Very early changes are most likely due to functional or regulatory modulation of ion channels (35). After these immediate changes, an intermediate response to rapid atrial rate appears to be a transcriptional downregulation of ion channels. We have demonstrated that this occurs within the first hours and closely parallels alterations in ion current densities. The early reductions in ICa,L seem to be particularly functionally important, and decreased ICa,L was shown to be a major mechanism of shortening and impaired rate adaptation of atrial action potentials (9). Within the first days of RAP, these changes become quantitatively comparable to those observed in chronic AF (9–13). These results suggest that transcriptional downregulation of ion channels is a major response to rapid atrial rates even in very early stages, and prevention or early reversal of these changes might be beneficial in breaking the vicious cycle of electrical remodeling.
Study limitations. The transient outward current in rabbits has important differences in biophysical and molecular properties compared with that in humans (25), which might also affect the response of the channel to rapid atrial rates.
Our findings indicate a transcriptional downregulation of ion channel subunits as an important mechanism underlying a reduced ICa,L and Ito in early phases of electrical remodeling. Although we observed a strong correlation between mRNA concentration and channel function, we were not able to show concomitant changes in protein contents for all subunits that were altered on a mRNA level. Other mechanisms, such as alterations of post-transcriptional channel subunit processing or ion channel regulation, might have contributed to changes in ionic currents.
Conclusions.
We have demonstrated for the first time that ICa,L and Ito are decreased as a result of rapid atrial rates in the early phases of electrical remodeling. The underlying mechanism appears to be a transcriptional downregulation of pore-forming () and of auxiliary (ß) subunits that closely paralleled changes in current. These findings provide important insights into pivotal mechanisms of early remodeling, which might be helpful in understanding initial adaptations of the atria to tachyarrhythmias such as AF.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractMethodsResultsDiscussionReferences |
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1c and subunit isoforms. J Cell Biochem. 2000;76:695–703[CrossRef][Medline]
1 subunit (1C-b) by the ß2a subunit: A peptide which inhibits binding of ß to the I-II linker of 1 induces functional uncoupling. Biochem J. 2000;348:657–665[CrossRef][Medline]
1C (Cav1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated 1C subunits. J Biol Chem. 2001;276:21089–21097This article has been cited by other articles:
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F. Holmqvist, M. Stridh, J. E.P. Waktare, L. Sornmo, S. B. Olsson, and C. J. Meurling Atrial fibrillatory rate and sinus rhythm maintenance in patients undergoing cardioversion of persistent atrial fibrillation Eur. Heart J., September 2, 2006; 27(18): 2201 - 2207. [Abstract] [Full Text] [PDF]
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B. J.J.M Brundel, H. H Kampinga, and R. H Henning Calpain inhibition prevents pacing-induced cellular remodeling in a HL-1 myocyte model for atrial fibrillation Cardiovasc Res, June 1, 2004; 62(3): 521 - 528. [Abstract] [Full Text] [PDF]
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