Angiotensin II Increases Expression of α1C Subunit of L-Type Calcium Channel Through a Reactive Oxygen Species and cAMP Response Element–Binding Protein–Dependent Pathway in HL-1 Myocytes
Angiotensin II (Ang II) is involved in the pathogenesis of atrial fibrillation (AF). L-type calcium channel (LCC) expression is altered in AF remodeling. We investigated whether Ang II modulates LCC current through transcriptional regulation, by using murine atrial HL-1 cells, which have a spontaneous calcium transient, and an in vivo rat model. Ang II increased LCC α1C subunit mRNA and protein levels and LCC current density, which resulted in an augmented calcium transient in atrial myocytes. An ≈2-kb promoter region of LCC α1C subunit gene was cloned to the pGL3 luciferase vector. Ang II significantly increased promoter activity in a concentration- and time-dependent manner. Truncation and mutational analysis of the LCC α1C subunit gene promoter showed that cAMP response element (CRE) (−1853 to −1845) was an important cis element in Ang II-induced LCC α1C subunit gene expression. Transfection of dominant-negative CRE binding protein (CREB) (pCMV-CREBS133A) abolished the Ang II effect. Ang II (1 μmol/L, 2 hours) induced serine 133 phosphorylation of CREB and binding of CREB to CRE and increased LCC α1C subunit gene promoter activity through a protein kinase C/NADPH oxidase/reactive oxygen species pathway, which was blocked by the Ang II type 1 receptor blocker losartan and the antioxidant simvastatin. In the rat model, Ang II infusion increased LCC α1C subunit expression and serine 133 phosphorylation of CREB, which were attenuated by oral losartan and simvastatin. In summary, Ang II induced LCC α1C subunit expression via a protein kinase C–, reactive oxygen species–, and CREB-dependent pathway and was blocked by losartan and simvastatin.
- angiotensin II
- dihydropyridine receptor α 1C subunit
- transcriptional regulation
- signal transduction
- cAMP response element–binding protein
Atrial fibrillation (AF) is the most common sustained arrhythmia in clinical practice. Recent reports suggest that AF is associated with activation of the local renin–angiotensin system in the atria of humans1 and in a dog model of AF.2 This indicates that angiotensin II (Ang II) may be involved in the pathogenesis of AF.2–4 Moreover, blockade of the renin–angiotensin system has been shown to be an effective treatment of AF.5
Ang II increases spontaneous calcium sparks6,7 and L-type calcium channel (LCC) current (ICaL) in cardiomyocytes.7,8 However, downregulation of ICaL9 and channel expression10,11 are observed in AF. Whether Ang II increases or decreases ICaL channel subunit expression in atrial myocytes and the detailed signaling mechanisms are unknown. Accordingly, in the present study, we explored the chronic effect of Ang II on the transcriptional regulation of LCC channel subunits in atrial myocytes. We used a murine atrial cell line HL-1, which is the only available atrial myocyte cell line that continuously divides and maintains a differentiated cardiac phenotype with spontaneous depolarization.10 We also used an in vivo rat model of Ang II infusion to verify the results obtained in the cellular study. We first found that Ang II increased LCC channel α1C subunit expression and augmented ICaL and calcium transient amplitude in HL-1 atrial myocytes. The detailed signaling mechanisms by which Ang II regulates the expression of LCC α1C subunits were also studied.
Materials and Methods
Culture and Transfection of HL-1 Cardiomyocytes
HL-1 myocytes were cultured in the Claycomb medium. Transient transfection of the HL-1 myocytes was performed using Lipofectamine 2000 reagent. The efficiency of transfection based on the green fluorescent protein fluorescence was between 60% and 80%. Normalized luciferase activities were measured using the Dual Luciferase Reporter Assay System. All of the experiments were conducted in a standardized fashion when the cells were in the confluent phase. Further detailed methods are provided in the online data supplement at http://circres.ahajournals.org.
Calcium Current Measurement
Transmembrane currents were measured using a patch–clamp amplifier (Dagan 8900, Dagan Corporation, Minneapolis, Minn) by the whole-cell recording technique. Total calcium currents including L-type (ICaL) and T-type calcium currents (ICaT) were obtained by a family of depolarization steps to +60 mV from the holding potential at −80 mV. ICaL was obtained by a family of depolarization steps to +70 mV from the holding potential at −50 mV and was measured as the nifedipine-sensitive current (3 μmol/L). ICaT was measured as subtracting total calcium currents by currents with the holding potential at −50 mV. Further detailed methods are provided in the online data supplement.
Fluo-3 Dye Staining and Confocal Laser Scanning Microscopy for Recordings of Intracellular Calcium
Spontaneous calcium transient was monitored in HL-1 cells using the fluorescent dye Fluo 3-acetoxymethyl ester and an inverted CLS microscope (LSM 510, Carl Zeiss, Jena, Germany). For fluorescence excitation, the 488-nm band of an argon laser was used. Emission was recorded using a long-pass LP 515 filter set. Line-scan images were acquired every 1.5 ms. The calcium level was reported as F/F0, where F0 is the resting or diastolic Fluo-3 fluorescence. The sarcoplasmic reticulum (SR) calcium load was measured by rapid caffeine application (50 mmol/L). Further detailed methods are provided in the online data supplement.
Construction of LCC α1C Subunit Promoter–Luciferase Fusion Plasmids and cAMP-Response Element Site Mutagenesis
A 1951-bp promoter fragment of rat LCC α1C subunit gene was cloned by PCR using forward and reverse primers designed according to the published rat dihydropyridine receptor gene sequence (GenBank accession no. AF221551). The ≈1.9-kb PCR product was subcloned into the pGL3Basic vector at BglII and HindIII sites (P1). Using a similar strategy, 4 deletion constructs (P2 to P5) were generated. All constructs were confirmed by DNA sequencing. The putative cAMP-response element (CRE) between nucleotide positions −1853 and −1845 of LCC α1C subunit gene promoter (CATACGTAA) was changed to CAGATCTAA by inserting a restriction enzyme sequence (BglII). Further detailed methods are provided in the online data supplement.
RNA Extraction, Quantitative Real-Time RT-PCR, and RNA Stability Assays
Total RNA was isolated and reverse-transcribed. The single-stranded cDNA was amplified (ABI-Prism 7900, Applied Biosystems, Foster City, Calif), using SYBR Green dye. For mouse/rat LCC α1C, α2D, and β subunits, the primers were designed according to the published gene sequences (CACNA1C, CACNA2D1-D3, and CACNB1-B3, respectively). GAPDH mRNA was used as the internal control. For RNA stability assay, actinomycin D (5 μg/mL) was added alone or simultaneously with Ang II (10−6 mol/L), and then the cells were harvested after 0, 2, 6, 18, and 24 hours for RNA extraction and quantification. The primer sequences and other detailed methods are provided in the online data supplement.
Protein Extracts, Western Blot Analyses, and Electrophoretic Mobility Shift Assays
Preparations for cytosolic and nuclear extracts, Western blot analyses, and nonisotopic electrophoretic mobility shift assays were as previously described.12
Assay of Intracellular Reactive Oxygen Species and Superoxide
Intracellular reactive oxygen species (ROS) production was measured using the ROS sensitive fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCF-DA) and confocal laser scanning microscopy as described above for the monitoring of intracellular calcium. The fold change from the baseline fluorescence level was calculated (F/F0). Intracellular superoxide production was measured by lucigenin-amplified chemiluminescence in the cell lysates. Detailed methods are provided in the online data supplement.
In Vivo Rat Model of Continuous Ang II Infusion
Ang II (2 mg/kg per day) was infused for 6 hours, 3 days, and 7 days into Wistar rats (weight 300±20 g) with a subcutaneously implanted osmotic minipump (3 days and 7 days) (Alzet Co) or a single intraperitoneal injection (6 hours). In addition to a normal diet, the rats also received pure water (vehicle), losartan (10 mg/kg per day), or simvastatin (5 mg/kg per day) during Ang II infusion. After these treatments, the hearts were rapidly excised and perfused with the Langendorff method. After perfusion for 5 minutes, both atria were excised for subsequent studies.
The experimental protocol conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and was approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine.
Data are presented as means±SE and were analyzed using Student’s t test for independent samples. Probability values of <0.05 were considered statistically significant.
Effect of Ang II on the Expression of LCC α1C Subunit in Atrial Myocytes
Ang II increased the level of LCC α1C subunit mRNA in a time- and concentration-dependent manner (Figure 1A). Ang II also increased the level of LCC α1C subunit protein (Figure 1B). Increased mRNA level may be attributable to increased transcription or increased RNA stability. After inhibition of general transcription by actinomycin D (5 μmol/L), Ang II did not affect the RNA stability of LCC α1C subunit (Figure 1C). Therefore, the increase in mRNA may be through a transcriptional mechanism. Ang II did not increase the expression of LCC β and α2D subunits (Figure 1D).
Effect of Sustained Ang II Stimulation on ICaL and Calcium Transient of Atrial Myocytes
HL-1 atrial cells exhibited a typical nifedipine-sensitive ICaL (Figure 2A), the density of which was significantly increased under Ang II stimulation (10−6 mol/L, 24 hours) (peak inward, −18.2±2.6 pA/pF for Ang II treatment [n=6] and −9.2±2.1 pA/pF for control [n=6]; P<0.001) (Figure 2B). Losartan (10−6 mol/L, 24 hours) attenuated the effect of Ang II on ICaL density (peak inward, −11.1±2.3 pA/pF [n=6]; P<0.001 versus Ang II treatment). Losartan did not affect basal ICaL density (Figure I in the online data supplement). The ICaT density was not significantly increased in response to Ang II stimulation (supplemental Figure I).
Increased ICaL may increase calcium release and the amplitude of the calcium transient. Ang II stimulation (10−6 mol/L; 24 hours) augmented transients in atrial myocytes, which were attenuated by the Ang II type 1 receptor blocker losartan (Figure 2C). Losartan itself did not affect the amplitude of calcium transient (Figure 2C). The mean peak F/F0 was significantly higher in Ang II–treated cells than control cells (Figure 2D).
Although an increase of ICaL may increase calcium release and the amplitude of the calcium transient, the SR is also an important determinant of the calcium transient. The SR calcium load was measured by rapid caffeine application (50 mmol/L). Ang II did not significantly affect the SR calcium load in HL-1 myocytes (supplemental Figure III).
Transcriptional Regulation of LCC α1C Subunit Gene by Ang II Dependent on CRE and CRE-Binding Protein
We cloned the promoter of LCC α1C subunit gene to study the mechanism by which Ang II regulates LCC α1C subunit gene expression. Several putative consensus sequences for transcriptional factor binding sites were identified in this region using the computer program TFSEARCH version 1.3 (Figure 3A). Ang II increased the luciferase activity of the 1951-bp promoter–reporter construct of the LCC α1C subunit gene (Figure 3B) in a time- and concentration-dependent manner. The changes of luciferase activity were similar to those of mRNA (Figure 1).
In the promoter deletion constructs (P1 to P5), Ang II–increased transcriptional activity was only evident when the promoter contained an ≈1-kb segment from the −1071 to −1954 (P1) (Figure 3C), which contains a putative CRE (−1853 to −1845). Mutation of this CRE (Figure 3C) and transient transfection of the dominant-negative CRE-binding protein (CREB) that contains a serine-to-alanine mutation corresponding to amino acid 133 (S133A) (Figure 3D) abolished the Ang II effect of increased transcriptional activity, suggesting that the Ang II effect depends on CREB and CRE.
Ang II Phosphorylates CREB at Serine 133 Through a Protein Kinase C- and ROS-Dependent Mechanism and Is Inhibited by Losartan and Simvastatin
Ang II phosphorylated CREB at serine 133 in a time- and concentration-dependent manner (Figure 4A). Ang II–mediated CREB serine 133 phosphorylation was attenuated by the protein kinase C (PKC) inhibitor chelerythrine (3 μmol/L), the ROS scavenger N-acetyl cysteine (NAC) (10 mmol/L), and the NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI) (10 μmol/L) but not by the extracellular signal-regulated kinase 1/2 (Erk1/Erk2) inhibitor PD98059 (10 μmol/L), the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 μmol/L), or the p38 inhibitor SB 203580 (1 μmol/L) (Figure 4B). To access the role of PKC in the regulation of CRE/CREB, different classes of PKC inhibitors were also examined (GO-6976, LY-333531, εV1 translocation inhibitor, and rottlerin for PKC-α, -β, -ε, and -δ inhibitors, respectively). PKC-α, -ε, and -δ are the major isoforms expressed in adult cardiomyocytes.13 We found that only PKC-ε blocker attenuated Ang II–induced CREB serine 133 phosphorylation (Figure 4C and 4D).
Ang II–mediated CREB serine 133 phosphorylation was not attenuated by the PKA blocker 8-bromoadenosine-3′,5′-cAMP (8-Br-Rp-cAMP) (100 μmol/L) and was therefore PKA-independent. Ang II–mediated CREB serine 133 phosphorylation was also attenuated by losartan (1 μmol/L) and by simvastatin (1 μmol/L), which is also known to be an NADPH oxidase inhibitor (Figure 4E).14
The PKC activator 4-phorbol myristate 13-acetate (PMA) (10 μmol/L) recapitulated the Ang II–mediated Ser133 phosphorylation of CREB, which was also attenuated by NAC, DPI, and simvastatin (Figure 4F), suggesting that PKC was the upstream signal for ROS production.
Ang II Increases Intracellular ROS and Superoxide Levels via a PKC- and NADPH Oxidase–Dependent Pathway and Is Inhibited by Losartan and Simvastatin
Ang II increased intracellular ROS and superoxide levels, as shown by monitoring the fluorescence intensity of the ROS sensitive dye DCF-DA and lucigenin-amplified chemiluminescence, respectively (Figure 5). Ang II–induced generation of ROS and superoxide was inhibited by losartan, the PKC inhibitor chelerythrine, the ROS scavenger NAC, the NADPH inhibitor DPI, and simvastatin (Figure 5B and 5C), suggesting the important role of NADPH oxidase in the generation of ROS and superoxide in atrial myocytes.
Ang II Increases CREB DNA Binding
Using an electrophoretic mobility shift assay, CREB binding to the consensus CRE sequence was assayed in cells treated with Ang II for 2 hours. As shown in Figure 6, Ang II treatment caused an increase in binding to the consensus CRE-binding sequence (lane 2), which was attenuated by chelerythrine (lane 6), NAC (lane 8), and DPI (lane 9) but not by PD98059 (lane 3), SP600125 (lane 4), SB 203580 (lane 5), or 8-Br-Rp-cAMP (lane 7). Competition with 100-fold molar excess of nonlabeled CRE oligodeoxynucleotide (lane 10) demonstrated the specificity of the protein–DNA complex. The addition of CREB antibody to the binding reaction resulted in 1 super-shift band, further demonstrating the specificity of the CREB-DNA binding (lane 11).
Ang II–Mediated Increase in LCC α1C Subunit Gene Transcription Is Ang II Type 1 Receptor–, PKC-, and ROS-Dependent
The Ang II–mediated increase in LCC α1C subunit gene promoter activity (P1) was attenuated by losartan and simvastatin (Figure 7A), the PKC inhibitor chelerythrine, and the ROS inhibitors NAC and DPI (Figure 7B). The changes in mRNA were similar to those in the promoter activity (data not shown).
In HL-1 atrial myocytes, Ang II increased the transcription of α1C subunit of LCC. A CRE in the promoter region of α1C subunit gene, serine 133 phosphorylation of CREB, and the upstream ROS generation were critical for Ang II–induced increased transcription of α1C subunit gene. Losartan and simvastatin inhibited Ang II–induced CREB serine 133 phosphorylation and expression of LCC α1C subunit gene, which was also observed in the native intact atrium from an in vivo rat model of Ang II infusion.
CREB as a Signaling Molecule of Ang II in Atrial Myocytes
The effect of Ang II on the phosphorylation of CREB in cardiomyocytes has been scarcely reported.15 Only 1 study demonstrated that CREB may be involved in long-term cardiac memory, modulating the transcription of genes for potassium channels (Kv1.5 and Kv4.3).15 However, the details of how the upstream signaling pathway activates or inactivates CREB have never been examined. Many reports indicate an important role of CREB to mediate Ang II signaling in various cell types, such as vascular smooth muscle cells16 and cardiac fibroblasts.17 The present study demonstrated that Ang II activated CREB in atrial myocytes, and the study is the first to report that CREB activation is important for the transcriptional regulation of LCC α1C subunit.
Ang II Increases Transmembranous Ionic Current Through a Transcriptional Mechanism
Although several studies have demonstrated that Ang II increases ICaL through directly phosphorylating the channel18–20 in cardiac myocytes, a transcriptional mechanism by which Ang II increases ICaL has never been reported.
The present study demonstrated that Ang II increases the expression of LCC α1C subunit in atrial myocytes. Increased number of available channels increases the transmembranous calcium influx during depolarization and thereby increases intracellular calcium loading,21 which may result in trigger activity and arrhythmia.22
In addition to the present study, other studies have shown that Ang II modulates the transcription of other ionic channels in ventricular myocytes14,23,24 and smooth muscle cells.25 However, these studies, unlike the present study, did not define the upstream detailed signaling pathways.
Ang II may also modulate the level of ion channel subunit mRNA by affecting its mRNA stability26 because Ang II was shown to increase the degradation of Kv4.3 mRNA in ventricular myocytes.26 However, in the present study, Ang II had no effect on the mRNA stability of LCC α1C subunits.
First, the changes in LCC α1C subunit expression and LCC current reported in the present study are the reverse of those observed in dog models of AF9,10 and human AF.11 The relevance of Ang II–mediated LCC α1C subunit expression and LCC current to AF remains unclear. The expression of LCC α1C subunit does not depend solely on Ang II stimulation during AF, and the effect of the complex interaction among rapid depolarization, mechanical stretch, and paracrine Ang II stimulation on LCC α1C subunit gene expression awaits further study.
Second, HL1 cells have only some properties in common with atrial myocytes. For example, the proportions of L- and T-type calcium currents are variable from passage to passage, or even in the same preparation.27 All of the cells have a relatively larger ICaT density compared with that in human atrial myocytes.
We demonstrated that Ang II increased ROS generation through small GTPase Rac1 in atrial myocytes and that simvastatin, which decreases NAPDH oxidase activity and ROS generation through inhibiting Rac1,13 inhibits Ang II–induced ROS generation. ROS have recently been found to play an important role in AF.28 Therefore, our results may implicate the therapeutic effect of statins to prevent Ang II–induced ROS generation in atrial myocytes, although the statin concentrations used in the present study may be high compared with clinically relevant concentrations and are known to have other pleiotropic effects.
Sources of Funding
This work was supported by the National Science Council, Taiwan, Republic of China (grants 95-2314-B-002-087-MY3 and 95-2314-B-002-08); the National Taiwan University Hospital, Taiwan, Republic of China (grants 92N016 and 93N004); and the Institute of Biomedical Sciences, Academia Sinica (grants IBMS-CRC93-T08, IBMS-CRC94-T06, and IBMS-CRC95-T02).
Original received July 28, 2006; revision received April 15, 2007; accepted April 18, 2007.
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