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Am J Physiol Heart Circ Physiol 292: H2898-H2905, 2007. First published February 9, 2007; doi:10.1152/ajpheart.00546.2006
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Losartan prevents stretch-induced electrical remodeling in cultured atrial neonatal myocytes

Erol Saygili,1 Obaida R. Rana,1 Esra Saygili,1 Hannes Reuter,1 Konrad Frank,1 Robert H. G. Schwinger,2 Jochen Müller-Ehmsen,1 and Carsten Zobel1

1Laboratory of Muscle Research and Molecular Cardiology, Department of Internal Medicine III, University of Cologne, Cologne, and 2Medical Clinic II, Klinikum Weiden, Weiden, Germany

Submitted 27 May 2006 ; accepted in final form 5 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Atrial fibrillation (AF) is the most frequent arrhythmia found in clinical practice. In recent studies, a decrease in the development or recurrence of AF was found in hypertensive patients treated with angiotensin-converting enzyme inhibitors or angiotensin receptor-blocking agents. Hypertension is related to an increased wall tension in the atria, resulting in increased stretch of the individual myocyte, which is one of the major stimuli for the remodeling process. In the present study, we used a model of cultured atrial neonatal rat cardiomyocytes under conditions of stretch to provide insight into the mechanisms of the preventive effect of the angiotensin receptor-blocking agent losartan against AF on a molecular level. Stretch significantly increased protein-to-DNA ratio and atrial natriuretic factor mRNA expression, indicating hypertrophy. Expression of genes encoding for the inward rectifier K+ current (IK1), Kir2.1, and Kir2.3, as well as the gene encoding for the ultrarapid delayed rectifier K+ current (IKur), Kv1.5, was significantly increased. In contrast, mRNA expression of Kv4.2 was significantly reduced in stretched myocytes. Alterations of gene expression correlated with the corresponding current densities: IK1 and IKur densities were significantly increased in stretched myocytes, whereas transient outward K+ current (Ito) density was reduced. These alterations resulted in a significant abbreviation of the action potential duration. Losartan (1 µM) prevented stretch-induced increases in the protein-to-DNA ratio and atrial natriuretic peptide mRNA expression in stretched myocytes. Concomitantly, losartan attenuated stretch-induced alterations in IK1, IKur, and Ito density and gene expression. This prevented the stretch-induced abbreviation of action potential duration. Prevention of stretch-induced electrical remodeling might contribute to the clinical effects of losartan against AF.

atrial fibrillation; mechanical stretch; hypertrophy; ion channels

ATRIAL FIBRILLATION (AF) is the most frequent arrhythmia found in clinical practice. In patients with arterial hypertension, the risk of developing AF is increased as much as 42% (25). Considerable interest in the role of the renin-angiotensin-aldosterone system (RAAS) in the development of AF has been generated by recent reports suggesting a decrease in the development or recurrence of AF in patients with hypertension treated with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor-blocking agents (ARBs) (14, 20, 21, 30, 42). The preventive effect of RAAS inhibition is not only related to the reduction in blood pressure, since the LIFE (losartan vs. atenolol) study (42) and a study by Fogari et al. (14) (losartan vs. amlodipine) demonstrated the superiority of losartan, despite similar reductions in blood pressure, in terms of prevention or recurrence of AF. Therefore, specific mechanisms related to activation of the RAAS are likely to be involved in the development of AF.

Electrical remodeling resulting in an abbreviation of the action potential (AP) duration (APD) has been described in various animal models (33, 45) and in humans (40). This might be one important mechanism contributing to the self-perpetuation of AF (45), since it encourages the initiation and maintenance of multiple reentrant wavelets in a limited mass of atrial tissue (32). It has been suggested that alterations in different ionic currents are responsible for the reduction of APD, depending on the species and the model studied. However, a consistent finding seems to be downregulation of the transient outward K+ current (Ito) and upregulation of the inward rectifier K+ current (IK1) (5, 40, 46). In addition, a modeling study recently suggested that upregulation of IK1 plays an important role in the shortening of the human AP in AF (52).

Patients with hypertension show structural and electrical remodeling of the atria comparable to the alterations observed in patients with AF (3). The increased wall tension associated with hypertension is considered essential for the process of cardiac remodeling (13).

In the present study, we used an in vitro stretch model for hypertensive load employing rat atrial cardiomyocytes to provide insight into the mechanisms resulting in the preventive effect of losartan against AF on a molecular level. We were able to show that stretch induces hypertrophy and electrical remodeling, which could be prevented by the ARB losartan.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and mechanical stretch. Neonatal rat atrial myocytes were isolated and cultured as described previously (54). Cells were grown in DMEM-Ham's F-12 supplemented with 10% horse serum and 5% fetal bovine serum. After 24 h, the serum-containing medium was removed, and the cells were washed and maintained in serum-free DMEM-Ham's F-12. The stretch device, which was based on the apparatus described by Lee et al. (28), was applied as described previously for ventricular neonatal myocytes (27, 35). Cells were plated onto silicone membranes coated with collagen type I (Bioflex) at a density of 1.5 x 105 cells per well. The device applies plane equibiaxial stretch by pulling the elastic membranes over a rigid polyvinylchloride disk. The device was calibrated to allow for a standardized equibiaxial stretch of 13%.

Measurement of protein-to-DNA ratio. Cells were lysed after 48 h of stretch in lysis buffer containing 2.5 mg/ml SDS and 5% saline-sodium citrate. The amount of protein in the lysates was measured with the Bradford method using the DC Protein Assay (Bio-Rad). DNA concentration in the lysis buffer was determined using Hoechst 33258 dye (bis-benzimide).

Quantitative real-time RT-PCR. Total RNA was extracted from atrial cardiomyocytes using the RNeasy Mini Kit (catalog no. 74104, Qiagen) according to the manufacturer's instructions. A total of 1 µg of RNA was reverse transcribed using random hexamers from a first-strand cDNA synthesis kit (catalog no. K1622, Fermentas). Real-time PCR was performed in 96-well plates on a sequence detection system (Prism 7700, ABI). Two-step RT-PCR was performed using dilutions of first-strand cDNA with a final concentration of 1x Assays-On-Demand and 1x TaqMan Universal PCR Master Mix, No AmpErase UNG (catalog no. P/N 4324018, ABI). The final reaction volume was 50 µl. Each sample was analyzed in duplicate. The thermal cycler conditions were 95°C for 10 min and 40 cycles of 15 s at 95°C (denaturation) and 1 min at 60°C (annealing/extension). A validation experiment was performed for each primer/probe set. The comparative cycle threshold ({Delta}{Delta}CT) method of data analysis was used for relative quantification. PCR primers and fluorogenic probes for all the target genes and the endogenous control were purchased as Assays-On-Demand (Applied Biosystems, Foster City, CA). The assay identification numbers for the endogenous control and target genes were as follows: Rn00560865_m1 (beta2-microglobulin), Rn00561661_m1 (Nppa), Rn00581941_m1 (Kv4.2), Rn00821554_m1 (Kir2.3), Rn01464023_m1 (Kir2.2), Rn00568808_s1 (Kir2.1), and Rn00564245_s1 (Kv1.5).

Electrophysiological measurements in neonatal rat atrial myocytes. For electrophysiological measurements, the silicone membrane was cut into small pieces, which were placed in the recording chamber. Current densities were recorded using the whole cell patch-clamp technique with a Heka EPC10 amplifier. Micropipettes were pulled from thin-walled borosilicate glass using a Flaming-Brown micropipette puller (model P-87, Sutter Instruments). The pipette tip was heat polished with a heating filament. When filled with intracellular solutions, tip resistances were typically 2.5–3 M{Omega}. Leakage compensation was not used. After membrane rupture, the cell capacitance was estimated by integration of the capacity currents and compensation of cell capacitance and pipette series resistance. Only seals in the gigaohm range were taken for measurements. All experiments were performed at room temperature (21°C). The extracellular solution for measurement of K+ currents (Ito and IK1) contained (mmol/l) 140 NaCl, 4 KCl, 1 MgCl2, 10 HEPES, 2 CaCl2, 10 glucose, and 0.5 CdCl2, with pH adjusted to 7.4 with 1 M NaOH. The intracellular solution contained (mmol/l) 90 potassium aspartate, 20 KCl, 10 HEPES, 1 MgCl2, 5 Na2ATP, and 5 EGTA, with pH adjusted to 7.3 with 2 M KOH. APs were recorded at room temperature in the extracellular solution (see above) without CdCl2. The pipette solution for the AP studies contained (in mmol/l) 140 KCl, 1 MgCl2, 5 HEPES, and 5 Na2ATP (pH 7.2).

For measurement of IK1, myocytes were held at a membrane potential of –40 mV, and voltage steps from –130 to –10 mV in 10-mV increments were applied for 500 ms with an interval of 1 s. Ito was measured from a holding potential of –80 mV, and a 20-ms prepulse to –40 mV was used to inactivate the Na+ currents. Voltage steps from –20 to +60 mV in 10-mV increments were applied for 500 ms with an interval of 1 s. Ultrarapid delayed rectifier K+ current (IKur) was estimated from the current level at the end of the 500-ms pulse. APs were elicited by a brief (2-ms) suprathreshold pulse applied at a frequency of 2 Hz.

Statistical analysis. Values are means ± SE. Comparisons were made by Student's t-test for two groups and by 1-way ANOVA followed by least significant difference post hoc test for multiple groups. P < 0.05 was considered statistically significant. For measurements of protein-to-DNA ratio and atrial natriuretic peptide (ANP) mRNA, n represents the number of myocyte preparations, with each condition studied in triplicate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mechanical stretch induces hypertrophy. We initially determined whether our in vitro stretch model resulted in hypertrophy, which was measured as alterations of the protein-to-DNA ratio and ANP gene expression after 48 h of stretch. Because contractile activity can have major effects on cell growth and hypertrophy signaling pathways (22), it is conceivable that stretch- or losartan-induced alterations of the spontaneous beating frequency would influence hypertrophy. However, no differences in spontaneous contraction rates were observed between the different groups. Stretched myocytes were noticeably larger than nonstretched control myocytes (Fig. 1). Stretch induced a significant increase in the protein-to-DNA ratio, as well as the relative abundance of ANP mRNA, suggesting hypertrophic growth of the cardiomyocytes (Fig. 2). Blockade of the angiotensin receptor with 1 µM losartan (18, 31) prevented the increase in protein-to-DNA ratio, as well as in ANP mRNA expression, in a significant manner (Fig. 2; P < 0.05). Cell capacitance as an indicator of cell size showed a trend toward a significant increase in stretched compared with nonstretched myocytes [16.41 ± 0.82 pF for control (n = 12) and 18.57 ± 1.05 pF for stretched (n = 11) myocytes], supporting the notion of stretch-induced hypertrophy in our model. Losartan significantly reduced average cell capacitance in stretched myocytes (13.51 ± 1.06 pF, n = 10, P < 0.05).


Figure 1
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  		Fig. 1. Images of stretched and nonstretched (control) cells. Stretched myocytes are larger than nonstretched cells.

 

Figure 2
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  		Fig. 2. Protein-to-DNA ratio and relative abundance of atrial natriuretic peptide (ANP) mRNA in losartan-treated and untreated atrial myocytes stretched for 48 h. Stretch significantly increased protein-to-DNA ratio (12 preparations with 3 samples each for control and stretch) and significantly increased relative abundance of ANP mRNA (3 preparations with 3 samples each for control and stretch) compared with nonstretched (control) cells. Losartan (1 µM) prevented stretch-induced increase of protein-to-DNA ratio (5 preparations with 3 samples each), as well as increase in ANP expression (3 preparations with 3 samples each) in a significant manner. Losartan was without effect in nonstretched myocytes. *P < 0.05 vs. control. **P < 0.05 vs. stretched.

 
Expression of genes encoding for Ito, IKur, and IK1. Relative gene expression was assessed with quantitative real-time PCR (see MATERIALS AND METHODS). The main gene encoding for Ito under physiological conditions is Kv4.2 (6). Figure 3 shows an ~50% reduction of Kv4.2 gene expression (P < 0.05) after 48 h of mechanical stretch. Losartan significantly increased Kv4.2 mRNA in nonstretched control cells (P < 0.05). The stretch-induced reduction in Kv4.2 gene expression could be fully prevented by losartan (P < 0.05).


Figure 3
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  		Fig. 3. Kv4.2, Kv1.5, Kir2.3, and Kir2.1 gene expression in losartan-treated and untreated atrial myocytes stretched for 48 h. Stretch significantly decreased Kv4.2 gene expression, significantly increased Kv1.5 gene expression, significantly increased Kir2.3 gene expression, and slightly, but significantly, increased Kir2.1 gene expression compared with nonstretched control cells. Losartan (1 µM) prevented stretch-induced decrease of Kv4.2 (P < 0.05) and attenuated stretch-induced increase of Kv1.5 (P < 0.05), as well as stretch-induced increase of Kir2.3. For each condition, data were obtained from 4 preparations with 3 samples each. Data were evaluated by quantitative real-time PCR. *P < 0.05 vs. control. **P < 0.05 vs. stretched.

 
Expression of Kv1.5, the gene encoding for IKur, was significantly increased (~2-fold) in stretched atrial myocytes (Fig. 3). In control myocytes, losartan was without effect on Kv1.5 expression levels. In stretched myocytes, losartan significantly attenuated the increase in Kv1.5 expression.

The genes Kir2.1, Kir2.2, and Kir2.3 encode for IK1. We were able to detect Kir2.1 and Kir2.3, but not Kir2.2, mRNA in our samples. Stretch slightly, but significantly, increased Kir2.1 gene expression (Fig. 3; P ≤ 0.05). Losartan did not influence Kir2.1 mRNA in nonstretched or stretched myocytes. In contrast to the minor changes in Kir2.1 gene expression, Kir2.3 gene expression was drastically increased (~2.8-fold) by stretch (Fig. 3; P ≤ 0.05). Losartan was without effect on Kir2.3 mRNA in nonstretched control cells but partially prevented the increase in Kir2.3 mRNA expression in stretched cells (Fig. 3; P ≤ 0.05).

Densities of Ito, IK1, and IKur. Representative recordings of Ito from isolated atrial myocytes cultured on silicone membranes are shown in Fig. 4A. Mean density-voltage relations of Ito from control cells, stretched cells, and losartan-treated stretched and nonstretched control cells are depicted in Fig. 4B. The significant stretch-induced reduction in Ito density (P < 0.05) correlated with the gene expression data. This reduction was more than compensated in the presence of 1 µM losartan (P < 0.05). Consistent with the mRNA data, application of losartan to nonstretched control cells was associated with a significant increase in Ito density.


Figure 4
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  		Fig. 4. A: typical recordings of transient outward K+ current (Ito) in isolated neonatal rat atrial myocytes. Currents were obtained by application of voltage steps from –20 to +60 mV for 500 ms. B: current-voltage (I-V) relations in control cells (6 cells from 6 preparations), stretched cells (5 cells from 3 preparations), stretched cells treated with 1 µM losartan (5 cells from 4 preparations), and control cells treated with 1 µM losartan (7 cells from 6 preparations). Stretch significantly decreased Ito density (stretch vs. control from +40 to +60 mV). Losartan (1 µM) completely prevented stretch-induced decrease of Ito. *P < 0.05 vs. control. **P < 0.05 vs. stretched. ***P < 0.05 vs. stretched + 1 µM losartan. {dagger}P < 0.05 vs. control.

 
The sustained current at the end of a 500-ms pulse was taken as a measure of IKur, since it is the main component of this current when the L-type Ca2+ current (ICa2+) is blocked (2, 19). IKur densities were significantly increased in stretched compared with control myocytes (Fig. 5). The losartan-induced attenuation of the increase in IKur density corresponded with the mRNA data. Losartan was without effect on current density in control myocytes.


Figure 5
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  		Fig. 5. The sustained part of Ito at the end of a 500-ms pulse was taken as a measure of ultrarapid delayed rectifier K+ current (IKur), since it is the main component of this current when the L-type Ca2+ current (ICa2+) is blocked (2, 19). IKur densities were significantly increased in stretched compared with nonstretched control myocytes. Losartan (1 µM) attenuated increase in IKur density. Losartan was without effect on current density in control myocytes. *P < 0.05 vs. control. **P < 0.05 vs. stretched. ***P < 0.05 vs. stretched + 1 µM losartan.

 
Typical recordings of IK1 elicited by 500-ms voltage steps from –130 to –10 mV from a holding potential of –40 mV are illustrated in Fig. 6A. Averaged current-voltage relations of IK1 are presented in Fig. 6B. Stretch induced a significant increase in IK1 density (~2.5-fold at –130 mV) that could be partially prevented by losartan (P < 0.05). In Fig. 6C, the current-voltage curve from –80 to –20 mV demonstrates a clear trend toward increased outward current densities of IK1 in stretched myocytes.


Figure 6
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  		Fig. 6. A: typical inward rectifier K+ current (IK1) recordings from isolated neonatal rat atrial myocytes. Currents were obtained by application of voltage steps from –130 to –10 mV for 500 ms. B: I-V relations (6 cells from 4 preparations for control and stretched and 5 cells from 3 preparations for stretched + 1 µM losartan). Mechanical stretch significantly increased IK1 density (stretch vs. control from –130 to –90 mV). Losartan (1 µM) prevented stretch-induced increase of IK1 in a significant manner (stretch vs. stretch + 1 µM losartan from –130 to –120 mV). C: I-V curve from –80 to –20 mV. Note clear trend toward increased outward current densities of IK1 in stretched myocytes. *P < 0.05 vs. control. **P < 0.05 vs. stretched. ***P < 0.05 vs. stretched + 1 µM losartan.

 
APs. Figure 7 depicts representative APs from atrial myocytes. Despite the increase in mRNA expression of Kv4.2 mRNA and Ito density induced by losartan in control cells, APDs at 50% and 90% repolarization (APD50 and APD90) were not significantly altered. In contrast, stretch resulted in a significant shortening of the APD50 and APD90 that could be prevented in a significant manner by losartan.


Figure 7
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  		Fig. 7. A: representative action potentials of cultured neonatal atrial myocytes. B: averaged data for control cells, stretched cells, stretched cells treated with 1 µM losartan, and nonstretched control cells treated with 1 µM losartan. Stretch shortened action potential durations (APDs) at 50% and 90% repolarization (APD50 and APD90). Losartan (1 µM) prevented decrease in APD. (control, n = 6; stretched, n = 5; control + losartan; n = 7; stretched + losartan, n = 6.) *P < 0.05 vs. control. **P < 0.05 vs. stretched.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of this study is that the ARB losartan prevents stretch-induced electrical remodeling, resulting in shortening of APD in cultured atrial myocytes. Furthermore, we have shown that alterations of ion channel expression and function in the vitro model resemble alterations of chronic AF.

The angiotensin receptor plays a central role in stretch-induced hypertrophy. The autocrine release of angiotensin II mediates mechanical stretch-induced hypertrophy in vitro (36). Additionally, activation of the angiotensin receptor without involvement of angiotensin II has been reported recently (55). Our demonstration that stretch-induced increases in markers of hypertrophy (protein-to-DNA ratio, ANP mRNA, and cell capacitance) could be prevented by losartan correlates with these observations.

Chronic AF is associated with reduced Ito density (5, 40), which is associated with reduced Kv4.3 mRNA and protein expression (7, 17). In patients with paroxysmal AF, Kv4.3 mRNA is reduced by 29% (7). Similar observations have been made in different animal models. Ito densities were significantly reduced after rapid pacing in dogs (51) and rabbits (4). In rats, short-term (8 h) rapid pacing resulted in diminished Kv4.2 and Kv4.3 mRNA expression (48). Cardiac hypertrophy is associated with reduced mRNA (16), protein (26), and density levels (26) of Ito in ventricular myocytes. In rat atria, Kv4.2, but not Kv4.3, seems to be the molecular correlate of Ito (6). Experiments with spontaneously hypertensive rats suggest that the angiotensin receptor might be directly involved in the regulation of Ito. In this animal model, Ito is inhibited and can be recovered by treatment with the angiotensin receptor-specific antagonist losartan (8). Furthermore, there is evidence that cardiomyocytes, Purkinje fibers, and cardiac fibroblasts produce angiotensin II (12). Therefore, locally produced angiotensin II may interact in a paracrine and/or autocrine manner to regulate functional expression of Ito.

Static stretch induced a pronounced reduction in Kv4.2 mRNA expression (~50%) that corresponded to a significantly reduced Ito density. The reduction in current density could be prevented by losartan. Interestingly, losartan also increased Kv4.2 mRNA in control myocytes and resulted in significantly higher levels of Kv4.2 mRNA and Ito density in stretched than in control myocytes. These findings suggest that the angiotensin receptor also plays an important role in the regulation of Ito under physiological conditions. This observation is supported by the finding that the Ito gradient from endocardial to epicardial ventricular myocytes seems to depend on the activity of the local RAAS (50).

PKC-{epsilon} seems to be a possible candidate for intracellular signaling pathway involved in the angiotensin receptor-dependent regulation of Kv4.2. Activation of PKC-{epsilon} has been demonstrated in stretched myocytes through the effect of angiotensin II receptor stimulation by an autocrine/paracrine release of angiotensin II (36, 49) or a direct activation of these receptors without angiotensin II release (55). Furthermore, regulation of Kv4.2 expression and current density by this pathway has been elegantly demonstrated in an experimental setting of diabetes, as well as hypothyroidism (37). These findings suggest that this pathway might also be relevant in stretch-dependent regulation of Kv4.2 expression.

IKur has been found to be unchanged (5) or reduced (40) in chronic human AF. However, in a rat rapid pacing model, a transient increase of Kv1.5 mRNA and protein expression was observed in atrial tissue (48). Therefore, one might speculate that an increase in IKur density in the early phase of electrical remodeling might be followed by a reduction in current density in chronic AF.

IK1 plays an important role in the late repolarization phase of the AP and maintenance of the resting membrane potential. The genes Kir2.1, Kir2.2, and Kir2.3 encode for IK1 in cardiomyocytes, resulting in heterotetrameric channels (53). The increase in IK1 in atrial myocytes of patients with chronic AF (5, 10, 15, 46) corresponds to an increased expression of Kir2.1, Kir2.2, and Kir2.3 (10, 15). Interestingly, patients with valvular heart disease without AF show increased expression of Kir2.x genes (15). An important role for increased IK1 density in the development of AF is also suggested by the observations that mice with overexpression of Kir2.1 (29) and humans with a gain-of-function mutation of this channel develop AF (47). Animal models of cardiac hypertrophy show reduced (41) or unchanged (34) IK1 densities in ventricular myocytes.

Kir2.1, Kir2.2, and Kir2.3 are expressed in human atrial tissue (15, 43). Kir2.1, but not Kir2.2 or Kir2.3, is expressed in guinea pig atrial tissue, whereas Kir2.1 and Kir2.3 are expressed in sheep atria (9). We were able to detect Kir2.1 and Kir2.3, but not Kir2.2, mRNA in the rat neonatal atrial myocytes. To our knowledge, no data are available regarding expression of Kir2.x genes in rat atrial tissue.

Stretch induced a modest, but significant, increase in Kir2.1 mRNA expression, whereas Kir2.3 mRNA was increased ~2.8-fold, corresponding to a significant increase in IK1 density. Losartan significantly attenuated the stretch-induced increase in Kir2.3 mRNA, as well as IK1 density. Kir2.1 mRNA expression was unaffected in stretched or control myocytes. Similar observations have been made in human atrial samples from patients with valvular (aortic and mitral) heart disease without AF, where mRNA levels of Kir2.3 were significantly upregulated, whereas Kir2.1 expression was unchanged (15). Kir2.3 mRNA is 12-fold more abundant in atrial than in ventricular tissue, where Kir2.3 expression is very low. Since IK1 density is unchanged (34) or reduced (1) in diseased ventricular myocardium, one might speculate that the increase of IK1 in stretched cardiomyocytes or atrial tissue exposed to volume overload is related to the more pronounced role of Kir2.3 in atrium than in ventricle. These findings might imply that, especially, Kir2.3 gene expression and, thereby, IK1 density are positively regulated by stretch involving stimulation of the angiotensin receptor.

Recently, there has been evidence that IK1 is involved in the mechanism of ventricular fibrillation maintenance by electrical isolation effects, and pharmacological blockade of this current can terminate reentry and ventricular fibrillation in guinea pigs (44). This has been attributed to an essential role of IK1 in rotor stability and rotation frequency (24). The notion of a periodic activity of a small number of isolated rotors has also been suggested to be important in AF (23). Increased IK1 density might therefore contribute to the occurrence of AF.

The finding of shortened APDs in stretched atrial myocytes corresponds with observations in chronic AF in humans (11, 39). The results are also consistent with observations from a rat rapid pacing model (48). Although the shortening of APD90 can be readily explained by the increase in IK1 density, the reduction of APD50 seems to be in contrast to the reduction of Ito density, since it is the main current responsible for early repolarization in rat. However, alterations of other currents responsible for the shape and duration of the AP might be responsible for this observation. For example, upregulation of atrial Kv1.5 mRNA has been attributed to a decrease in atrial APD in the presence of reduced Kv4.2 mRNA levels in a rat pacing model (48). Furthermore, artificial overexpression of Kv1.5 in cultured rat myocytes resulted in a significant shortening of APD50 and APD90 (38), supporting the notion that the increase in IKur density might contribute to the reduction in APD50 observed in our model.

In summary, our observations suggest that stretch leads to electrical remodeling, resulting in shortening of the AP in atrial myocytes, which might predispose it to the development of AF. The ARB losartan prevents the remodeling process and, thereby, might contribute to the clinically observed protection against AF.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Hans und Gertie Fischer-Stiftung.


   ACKNOWLEDGMENTS
 
This work contains data from the doctoral thesis of E. Saygili (University of Cologne).


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Zobel, Laboratory of Muscle Research and Molecular Cardiology, Dept. III of Internal Medicine, Univ. of Cologne, Kerpenerstr. 62, 50924 Cologne, Germany (e-mail: carsten.zobel{at}uni-koeln.de)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aimond F, Alvarez JL, Rauzier JM, Lorente P, Vassort G. Ionic basis of ventricular arrhythmias in remodeled rat heart during long-term myocardial infarction. Cardiovasc Res 42: 402–415, 1999.[Abstract/Free Full Text]
  2. Bachmann A, Gutcher I, Kopp K, Brendel J, Bosch RF, Busch AE, Gogelein H. Characterization of a novel Kv1.5 channel blocker in Xenopus oocytes, CHO cells, human and rat cardiomyocytes. Naunyn Schmiedebergs Arch Pharmacol 364: 472–478, 2001.[CrossRef][Web of Science][Medline]
  3. Barbier P, Alioto G, Guazzi MD. Left atrial function and ventricular filling in hypertensive patients with paroxysmal atrial fibrillation. J Am Coll Cardiol 24: 165–170, 1994.[Abstract]
  4. Bosch RF, Scherer CR, Rub N, Wohrl S, Steinmeyer K, Haase H, Busch AE, Seipel L, Kuhlkamp V. Molecular mechanisms of early electrical remodeling: transcriptional downregulation of ion channel subunits reduces ICa,L and Ito in rapid atrial pacing in rabbits. J Am Coll Cardiol 41: 858–869, 2003.[Abstract/Free Full Text]
  5. Bosch RF, Zeng X, Grammer JB, Popovic K, Mewis C, Kuhlkamp V. Ionic mechanisms of electrical remodeling in human atrial fibrillation. Cardiovasc Res 44: 121–131, 1999.[Abstract/Free Full Text]
  6. Bou-Abboud E, Nerbonne JM. Molecular correlates of the calcium-independent, depolarization-activated K+ currents in rat atrial myocytes. J Physiol 517: 407–420, 1999.[Abstract/Free Full Text]
  7. Brundel BJ, Van Gelder IC, Henning RH, Tuinenburg AE, Wietses M, Grandjean JG, Wilde AA, van Gilst WH, Crijns HJ. 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 37: 926–932, 2001.[Abstract/Free Full Text]
  8. Cerbai E, Crucitti A, Sartiani L, De Paoli P, Pino R, Rodriguez ML, Gensini G, Mugelli A. Long-term treatment of spontaneously hypertensive rats with losartan and electrophysiological remodeling of cardiac myocytes. Cardiovasc Res 45: 388–396, 2000.[Abstract/Free Full Text]
  9. Dhamoon AS, Pandit SV, Sarmast F, Parisian KR, Guha P, Li Y, Bagwe S, Taffet SM, Anumonwo JM. Unique Kir2x properties determine regional and species differences in the cardiac inward rectifier K+ current. Circ Res 94: 1332–1339, 2004.[Abstract/Free Full Text]
  10. Dobrev D, Graf E, Wettwer E, Himmel HM, Hala O, Doerfel C, Christ T, Schuler S, Ravens U. Molecular basis of downregulation of G-protein-coupled inward rectifying K+ current (IK,ACh) in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced IK,ACh and muscarinic receptor-mediated shortening of action potentials. Circulation 104: 2551–2557, 2001.[Abstract/Free Full Text]
  11. Dobrev D, Ravens U. Remodeling of cardiomyocyte ion channels in human atrial fibrillation. Basic Res Cardiol 98: 137–148, 2003.[Web of Science][Medline]
  12. Dostal DE, Rothblum KN, Conrad KM, Cooper GR, Baker KM. Detection of angiotensin I and II in cultured rat cardiac myocytes and fibroblasts. Am J Physiol Cell Physiol 263: C851–C863, 1992.[Abstract/Free Full Text]
  13. Epstein ND, Davis JS. Sensing stretch is fundamental. Cell 112: 147–150, 2003.[CrossRef][Web of Science][Medline]
  14. Fogari R, Mugellini A, Destro M, Corradi L, Zoppi A, Fogari E, Rinaldi A. Losartan and prevention of atrial fibrillation recurrence in hypertensive patients. J Cardiovasc Pharmacol 47: 46–50, 2006.[CrossRef][Web of Science][Medline]
  15. Gaborit N, Steenman M, Lamirault G, Le Meur N, Le Bouter S, Lande G, Leger J, Charpentier F, Christ T, Dobrev D, Escande D, Nattel S, Demolombe S. Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation. Circulation 112: 471–481, 2005.[Abstract/Free Full Text]
  16. Gidh-Jain M, Huang B, Jain P, el-Sherif N. Differential expression of voltage-gated K+ channel genes in left ventricular remodeled myocardium after experimental myocardial infarction. Circ Res 79: 669–675, 1996.[Abstract/Free Full Text]
  17. Grammer JB, Bosch RF, Kuhlkamp V, Seipel L. Molecular remodeling of Kv4.3 potassium channels in human atrial fibrillation. J Cardiovasc Electrophysiol 11: 626–633, 2000.[Web of Science][Medline]
  18. Guo W, Kamiya K, Kada K, Kodama I, Toyama J. Regulation of cardiac Kv1.5 K+ channel expression by cardiac fibroblasts and mechanical load in cultured newborn rat ventricular myocytes. J Mol Cell Cardiol 30: 157–166, 1998.[CrossRef][Web of Science][Medline]
  19. Guo W, Kamiya K, Toyama J. Roles of the voltage-gated K+ channel subunits, Kv1.5 and Kv14, in the developmental changes of K+ currents in cultured neonatal rat ventricular cells. Pflügers Arch 434: 206–208, 1997.[CrossRef][Web of Science][Medline]
  20. Hansson L, Lindholm LH, Ekbom T, Dahlof B, Lanke J, Schersten B, Wester PO, Hedner T, de Faire U. Randomised trial of old and new antihypertensive drugs in elderly patients: cardiovascular mortality and morbidity. The Swedish Trial in Old Patients With Hypertension-2 Study. Lancet 354: 1751–1756, 1999.[CrossRef][Web of Science][Medline]
  21. Hansson L, Lindholm LH, Niskanen L, Lanke J, Hedner T, Niklason A, Luomanmaki K, Dahlof B, de Faire U, Morlin C, Karlberg BE, Wester PO, Bjorck JE. Effect of angiotensin-converting-enzyme inhibition compared with conventional therapy on cardiovascular morbidity and mortality in hypertension: the Captopril Prevention Project (CAPPP) Randomised Trial. Lancet 353: 611–616, 1999.[CrossRef][Web of Science][Medline]
  22. Hines WA, Thorburn J, Thorburn A. Cell density and contraction regulate p38 MAP kinase-dependent responses in neonatal rat cardiac myocytes. Am J Physiol Heart Circ Physiol 277: H331–H341, 1999.[Abstract/Free Full Text]
  23. Jalife J. Rotors and spiral waves in atrial fibrillation. J Cardiovasc Electrophysiol 14: 776–780, 2003.[Web of Science][Medline]
  24. Jalife J, Anumonwo JM, Berenfeld O. Toward an understanding of the molecular mechanisms of ventricular fibrillation. J Interv Card Electrophysiol 9: 119–129, 2003.[CrossRef][Web of Science][Medline]
  25. Kannel WB, Wolf PA, Benjamin EJ, Levy D. Prevalence, incidence, prognosis, and predisposing conditions for atrial fibrillation: population-based estimates. Am J Cardiol 82: 2N–9N, 1998.[CrossRef][Web of Science][Medline]
  26. Kaprielian R, Wickenden AD, Kassiri Z, Parker TG, Liu PP, Backx PH. Relationship between K+ channel down-regulation and [Ca2+]i in rat ventricular myocytes following myocardial infarction. J Physiol 517: 229–245, 1999.[Abstract/Free Full Text]
  27. Komuro I, Kaida T, Shibazaki Y, Kurabayashi M, Katoh Y, Hoh E, Takaku F, Yazaki Y. Stretching cardiac myocytes stimulates protooncogene expression. J Biol Chem 265: 3595–3598, 1990.[Abstract/Free Full Text]
  28. Lee AA, Delhaas T, Waldman LK, MacKenna DA, Villarreal FJ, McCulloch AD. An equibiaxial strain system for cultured cells. Am J Physiol Cell Physiol 271: C1400–C1408, 1996.[Abstract/Free Full Text]
  29. Li J, McLerie M, Lopatin AN. Transgenic upregulation of IK1 in the mouse heart leads to multiple abnormalities of cardiac excitability. Am J Physiol Heart Circ Physiol 287: H2790–H2802, 2004.[Abstract/Free Full Text]
  30. Madrid AH, Bueno MG, Rebollo JM, Marin I, Pena G, Bernal E, Rodriguez A, Cano L, Cano JM, Cabeza P, Moro C. Use of irbesartan to maintain sinus rhythm in patients with long-lasting persistent atrial fibrillation: a prospective and randomized study. Circulation 106: 331–336, 2002.[Abstract/Free Full Text]
  31. Malhotra R, Sadoshima J, Brosius FC 3rd, Izumo S. Mechanical stretch and angiotensin II differentially upregulate the renin-angiotensin system in cardiac myocytes in vitro. Circ Res 85: 137–146, 1999.[Abstract/Free Full Text]
  32. Moe GK. A conceptual model of atrial fibrillation. J Electrocardiol 1: 145–146, 1968.[Medline]
  33. Nattel S. New ideas about atrial fibrillation 50 years on. Nature 415: 219–226, 2002.[CrossRef][Medline]
  34. Rozanski GJ, Xu Z, Zhang K, Patel KP. Altered K+ current of ventricular myocytes in rats with chronic myocardial infarction. Am J Physiol Heart Circ Physiol 274: H259–H265, 1998.[Abstract/Free Full Text]
  35. Sadoshima J, Jahn L, Takahashi T, Kulik TJ, Izumo S. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. An in vitro model of load-induced cardiac hypertrophy. J Biol Chem 267: 10551–10560, 1992.[Abstract/Free Full Text]
  36. Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell 75: 977–984, 1993.[CrossRef][Web of Science][Medline]
  37. Shimoni Y, Liu XF. Role of PKC in autocrine regulation of rat ventricular K+ currents by angiotensin and endothelin. Am J Physiol Heart Circ Physiol 284: H1168–H1181, 2003.[Abstract/Free Full Text]
  38. Tanabe Y, Hatada K, Naito N, Aizawa Y, Chinushi M, Nawa H, Aizawa Y. Over-expression of Kv1.5 in rat cardiomyocytes extremely shortens the duration of the action potential and causes rapid excitation. Biochem Biophys Res Commun 345: 1116–1121, 2006.[CrossRef][Web of Science][Medline]
  39. Van Wagoner DR. Electrophysiological remodeling in human atrial fibrillation. Pacing Clin Electrophysiol 26: 1572–1575, 2003.[CrossRef][Medline]
  40. Van Wagoner DR, Pond AL, McCarthy PM, Trimmer JS, Nerbonne JM. Outward K+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ Res 80: 772–781, 1997.[Abstract/Free Full Text]
  41. Volk T, Nguyen TH, Schultz JH, Faulhaber J, Ehmke H. Regional alterations of repolarizing K+ currents among the left ventricular free wall of rats with ascending aortic stenosis. J Physiol 530: 443–455, 2001.[Abstract/Free Full Text]
  42. Wachtell K, Lehto M, Gerdts E, Olsen MH, Hornestam B, Dahlof B, Ibsen H, Julius S, Kjeldsen SE, Lindholm LH, Nieminen MS, Devereux RB. Angiotensin II receptor blockade reduces new-onset atrial fibrillation and subsequent stroke compared with atenolol: the Losartan Intervention for End Point Reduction in Hypertension (LIFE) Study. J Am Coll Cardiol 45: 712–719, 2005.[Abstract/Free Full Text]
  43. Wang Z, Yue L, White M, Pelletier G, Nattel S. Differential distribution of inward rectifier potassium channel transcripts in human atrium versus ventricle. Circulation 98: 2422–2428, 1998.[Abstract/Free Full Text]
  44. Warren M, Guha PK, Berenfeld O, Zaitsev A, Anumonwo JM, Dhamoon AS, Bagwe S, Taffet SM, Jalife J. Blockade of the inward rectifying potassium current terminates ventricular fibrillation in the guinea pig heart. J Cardiovasc Electrophysiol 14: 621–631, 2003.[CrossRef][Web of Science][Medline]
  45. Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation. A study in awake chronically instrumented goats. Circulation 92: 1954–1968, 1995.[Abstract/Free Full Text]
  46. Workman AJ, Kane KA, Rankin AC. The contribution of ionic currents to changes in refractoriness of human atrial myocytes associated with chronic atrial fibrillation. Cardiovasc Res 52: 226–235, 2001.[Abstract/Free Full Text]
  47. Xia M, Jin Q, Bendahhou S, He Y, Larroque MM, Chen Y, Zhou Q, Yang Y, Liu Y, Liu B, Zhu Q, Zhou Y, Lin J, Liang B, Li L, Dong X, Pan Z, Wang R, Wan H, Qiu W, Xu W, Eurlings P, Barhanin J, Chen Y. A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation. Biochem Biophys Res Commun 332: 1012–1019, 2005.[CrossRef][Web of Science][Medline]
  48. Yamashita T, Murakawa Y, Hayami N, Fukui E, Kasaoka Y, Inoue M, Omata M. Short-term effects of rapid pacing on mRNA level of voltage-dependent K+ channels in rat atrium: electrical remodeling in paroxysmal atrial tachycardia. Circulation 101: 2007–2014, 2000.[Abstract/Free Full Text]
  49. Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Mizuno T, Takano H, Hiroi Y, Ueki K, Tobe K. Mechanical stress activates protein kinase cascade of phosphorylation in neonatal rat cardiac myocytes. J Clin Invest 96: 438–446, 1995.[Web of Science][Medline]
  50. Yu H, Gao J, Wang H, Wymore R, Steinberg S, McKinnon D, Rosen MR, Cohen IS. Effects of the renin-angiotensin system on the current Ito in epicardial and endocardial ventricular myocytes from the canine heart. Circ Res 86: 1062–1068, 2000.[Abstract/Free Full Text]
  51. Yue L, Feng J, Gaspo R, Li GR, Wang Z, Nattel S. Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation. Circ Res 81: 512–525, 1997.[Abstract/Free Full Text]
  52. Zhang H, Garratt CJ, Zhu J, Holden AV. Role of up-regulation of IK1 in action potential shortening associated with atrial fibrillation in humans. Cardiovasc Res 66: 493–502, 2005.[Abstract/Free Full Text]
  53. Zobel C, Cho HC, Nguyen TT, Pekhletski R, Diaz RJ, Wilson GJ, Backx PH. Molecular dissection of the inward rectifier potassium current (IK1) in rabbit cardiomyocytes: evidence for heteromeric co-assembly of Kir2.1 and Kir2.2. J Physiol 550: 365–372, 2003.[Abstract/Free Full Text]
  54. Zobel C, Kassiri Z, Nguyen TT, Meng Y, Backx PH. Prevention of hypertrophy by overexpression of Kv4.2 in cultured neonatal cardiomyocytes. Circulation 106: 2385–2391, 2002.[Abstract/Free Full Text]
  55. Zou Y, Akazawa H, Qin Y, Sano M, Takano H, Minamino T, Makita N, Iwanaga K, Zhu W, Kudoh S, Toko H, Tamura K, Kihara M, Nagai T, Fukamizu A, Umemura S, Iiri T, Fujita T, Komuro I. Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II. Nat Cell Biol 6: 499–506, 2004.[CrossRef][Web of Science][Medline]



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