CLINICAL PERSPECTIVE

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(Circulation. 2005;112:1936-1944.)
© 2005 American Heart Association, Inc.

Arrhythmia/Electrophysiology

Functional Roles of Ca_v1.3(_1D) Calcium Channels in Atria

Insights Gained From Gene-Targeted Null Mutant Mice

Zhao Zhang, MD, PhD; Yuxia He, MD; Dipika Tuteja, PhD; Danyan Xu, MD; Valeriy Timofeyev, PhD; Qian Zhang, MD; Kathryn A. Glatter, MD; Yanfang Xu, MD, PhD; Hee-Sup Shin, PhD; Reginald Low, MD; Nipavan Chiamvimonvat, MD

From the Division of Cardiovascular Medicine (Z.Z., X.H., D.T., D.X., V.T., Q.Z., K.A.G., Y.X., R.L., N.C.), Department of Internal Medicine, University of California, Davis; the Department of Veterans Affairs (N.C.), Northern California Health Care System, Mather, Calif; the Center for Calcium and Learning (H.-S.S.), Division of Life Sciences, Korea Institute of Science and Technology, Seoul, Korea; and the Department of Physiology (Z.Z.), Henan Medical University, Zhingzhou, China.

Correspondence to Nipavan Chiamvimonvat, Division of Cardiovascular Medicine, University of California, Davis, One Shields Ave, GBSF 6315, Davis, CA 95616. E-mail nchiamvimonvat{at}ucdavis.edu

Received January 31, 2005; revision received June 15, 2005; accepted June 27, 2005.

	Abstract

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Background— Previous data suggest that L-type Ca²⁺ channels containing the Ca_v1.3(_1D) subunit are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Ca_v1.2(_1C) subunit are found in the brain, vascular smooth muscle, and cardiac tissue. However, our previous report as well as others have shown that Ca_v1.3 Ca²⁺ channel–deficient mice (Ca_v1.3^–/–) demonstrate sinus bradycardia with a prolonged PR interval. In the present study, we extended our study to examine the role of the Ca_v1.3(_1D) Ca²⁺ channel in the atria of Ca_v1.3^–/– mice.

Methods and Results— We obtained new evidence to demonstrate that there is significant expression of Ca_v1.3 Ca²⁺ channels predominantly in the atria compared with ventricular tissues. Whole-cell L-type Ca²⁺ currents (I_Ca,L) recorded from single, isolated atrial myocytes from Ca_v1.3^–/– mice showed a significant depolarizing shift in voltage-dependent activation. In contrast, there were no significant differences in the I_Ca,L recorded from ventricular myocytes from wild-type and null mutant mice. We previously documented the hyperpolarizing shift in the voltage-dependent activation of Ca_v1.3 compared with Ca_v1.2 Ca²⁺ channel subunits in a heterologous expression system. The lack of Ca_v1.3 Ca²⁺ channels in null mutant mice would result in a depolarizing shift in the voltage-dependent activation of I_Ca,L in atrial myocytes. In addition, the Ca_v1.3-null mutant mice showed evidence of atrial arrhythmias, with inducible atrial flutter and fibrillation. We further confirmed the isoform-specific differential expression of Ca_v1.3 versus Ca_v1.2 by in situ hybridization and immunofluorescence confocal microscopy.

Conclusions— Using gene-targeted deletion of the Ca_v1.3 Ca²⁺ channel, we established the differential distribution of Ca_v1.3 Ca²⁺ channels in atrial myocytes compared with ventricles. Our data represent the first report demonstrating important functional roles for Ca_v1.3 Ca²⁺ channel in atrial tissues.

Key Words: arrhythmias • ion channels • atrial fibrillation • calciumatrium

	Introduction

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Voltage-gated Ca²⁺ channels are heteromultimeric complexes of a pore-forming, transmembrane-spanning ₁-subunit, a disulfide-linked complex of ₂- and -subunits, and an intracellular ß- and -subunit.^1,2 The ₁-subunit is the largest and incorporates the conduction pore, the voltage sensor, gating apparatus, and the known sites of channel regulation by second messengers, drugs, and toxins. Mammalian ₁-subunits of voltage-gated Ca²⁺ channels are encoded by at least 10 distinct genes.³ Previous data suggest that L-type Ca²⁺ channels (LTCCs) containing the Ca_v1.3(_1D) subunit (D-LTCC) are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Ca_v1.2(_1C) subunit (C-LTCCs) are found in the brain, vascular smooth muscle, and cardiac tissue. Recently, we and others, have shown that the Ca_v1.3 Ca²⁺ channel is highly expressed in cardiac pacemaking tissue and plays an important role in the spontaneous diastolic depolarization and frequency of beating in sinoatrial (SA) node cells.^4–6 Specifically, using a mouse model of gene-targeted deletion of Ca_v1.3 Ca²⁺ channel, we established a role for the Ca_v1.3 Ca²⁺ channel in the generation of spontaneous action potential in SA node cells.⁴ The Ca_v1.3-null mutant mouse shows evidence of profound SA and atrioventricular (AV) node dysfunction. We observed that D-LTCCs show a low activation threshold compared with that of C-LTCCs. The hyperpolarizing shift in the activation threshold of the Ca_v1.3 Ca²⁺ channel can be directly documented in isolated SA node cells as well as in a nonexcitable expression system, wherein the Ca_v1.3 subunit can be expressed and studied alone.⁴ This gating property of D-LTCCs contributes importantly to the generation of spontaneous action potential and pacemaking activities within SA node cells.

In the present report, we present new evidence that demonstrates that there is significant expression of Ca_v1.3 Ca²⁺ channels in atrial but not in ventricular tissue. Specifically, using in situ hybridization and immunocytochemistry, we show that there is robust expression of Ca_v1.3 Ca²⁺ channels in mouse atrial but not ventricular tissue. Because both isoforms have similar pharmacological properties, it is difficult to isolate one current from the other with conventional electrophysiology. The Ca_v1.3-null mutant mouse model provides a unique opportunity to directly determine the contribution of D- versus C-LTCCs in atrial versus ventricular tissues. Here, using in vitro and in vivo electrophysiological recordings, we document for the first time the functional roles of the Ca_v1.3 Ca²⁺ channel in mouse atrial myocytes.

	Methods

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Animals and Protocols
The present investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication 85-23, revised 1985) and was performed in accordance with the guidelines of the Animal Care and Use Committee of the University of California, Davis. Generation of Ca_v1.3-null mutant mice has been previously described.^4,7

Electrophysiological Recordings
Single atrial and ventricular myocytes were isolated from Ca_v1.3^–/–, Ca_v1.3^+/–, and wild-type (WT, Ca_v1.3^+/+) littermates on a C57BL/6J background, as previously described.⁸ Whole-cell I_Ca was recorded at room temperature with patch-clamp techniques.^9,10 The external solution contained (in mmol/L) N-methyl glucamine (NMG) 140, CsCl 5, MgCl₂ 0.5, CaCl₂ 2, 4-amino pyridine 2, glucose 10, and HEPES 10, and the internal solution contained NMG 135, tetraethylammonium chloride 20, disodium ATP 4, EGTA 1, and HEPES 10. All chemicals were purchased from Sigma Chemical unless stated otherwise. Cell capacitance was calculated by integrating the area under a curve of an uncompensated capacitative transient elicited by a 20-mV hyperpolarizing pulse from a holding potential of –40 mV. Whole-cell current records were filtered at 2 kHz and sampled at 10 kHz. Liquid junction potentials were measured as previously described,¹¹ and all data were corrected for liquid junction potentials. Curve fitting and data analysis were performed with Origin software (MicroCal Inc).

Reverse Transcription–Polymerase Chain Reaction
Total RNA was prepared from the atria and ventricles of wild-type (WT) C57BL/6J mice with TRIzol Reagent (Invitrogen). cDNA was synthesized from total RNA samples by oligo(dT)-primed reverse transcription (RT) (Superscript II RNase H-reverse transcriptase, Invitrogen). cDNA was then subjected to polymerase chain reaction (PCR) amplification with HotStarTaq DNA polymerase (Qiagen). Primers used in the PCR were designed from mutually unique regions of Ca_v1.2 and Ca_v1.3 channels as follows: (1) for Ca_v1.3, 5'-ATGAACCTTCCGACATTTTC-3' (forward) and 5'-GTGCTCATAGTCTGGGCGGC-3' (reverse), according to the published sequence of mouse Ca_v1.3 (accession No. NM_028981) and (2) for Ca_v1.2, 5'-ATGGTCAATGAAAACACGA-3' (forward) and 5'- ACTGACGGTAGAGATGGTTG-3' (reverse), according to the published sequence of mouse Ca_v1.2 (accession No. NM_009781). The absence of genomic contamination in the RNA samples was confirmed by RT-negative controls for each experiment.

In Situ Hybridization
Ca_v1.2- and Ca_v1.3-specific cDNA fragments were subcloned into a TA cloning vector (Invitrogen). All clones were sequenced. The sense and antisense riboprobes were synthesized in the presence of UTP-digoxigenin label with use of a DIG RNA labeling kit (Roche). Mouse hearts procured from 8- to 10-week-old WT mice were dissected and perfused first with Rnase-free phosphate-buffered saline and later with 4% paraformaldehyde (made in phosphate-buffered saline). Ca_v1.3^–/– mouse hearts were also used as negative controls for the Ca_v1.3 probe. Perfused hearts were fixed overnight at 4°C in 4% paraformaldehyde. Infiltration of hearts was done with a mixture of 10% and 30% sucrose in ratios of 2:1, 1:1, and 1:2 at room temperature for 30 minutes (each) with gentle rotation. Hearts were transferred to 30% sucrose and allowed to settle at 4°C. Hearts were then transferred to degassed OCT medium (Tissue-Tek) and maintained at 4°C overnight with rotation. Embedding was done in OCT medium, and the samples were frozen on a dry ice/ethanol bath. Cryosectioning was completed, and sections were laid on gelatin-coated slides (Fisher Scientific). After air-drying, in situ hybridization was performed with the anti-sense as well as the corresponding sense cRNA probes on adjacent sections. After hybridization and washes, the sections were subjected to immunologic detection with anti-digoxigenin Fab fragments conjugated to alkaline phosphatase by using a DIG nucleic acid detection kit (Roche). The signals were developed with nitro blue tetrazolium and bromochloroindolyl phosphate (Roche) added in alkaline phosphatase buffer in the presence of levamisole (Sigma) to inhibit endogenous alkaline phosphatase. The specimens were inspected for development of purple precipitate by bright-field microscopy (Carl Zeiss Vision). Digitized images were obtained with AxioVision 4 (Zeiss).

Immunofluorescence Confocal Microscopy
Immunofluorescence labeling was performed as described previously.¹² The following primary antibodies were used: (1) anti-Ca_v1.3 (Santa Cruz Biotechnology, Inc), a polyclonal antibody raised in goat against a purified peptide corresponding to amino acid residues 859 to 875 of rat Ca_v1.3 (accession No. P27732)¹³ and (2) anti-Ca_v1.2 (Alomone Labs), a polyclonal antibody raised in rabbit against a glutathione-S-transferase fusion protein with residues 1 to 46 of rabbit Ca_v1.2 (accession No. P15381).¹⁴ The cells were treated with anti-Ca_v1.3 or anti-Ca_v1.2 antibodies (1:200 dilution for 1 hour). Immunofluorescence labeling for confocal microscopy was performed by treatment with Texas red–conjugated goat anti-rabbit antibody or rabbit anti-goat antibody (Calbiochem, 1:500 dilution). Immunofluorescence-labeled samples were examined with a Pascal Zeiss confocal laser scanning microscope. Control experiments performed by incubation with secondary antibody only did not show positive staining under the same experimental conditions. Identical settings were used for all specimens.

Transient Transfection of Ca_v1.2 and Ca_v1.3 Ca²⁺ Channels in HEK Cells
HEK 293 cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 2 mmol/L L-glutamine, and 1% penicillin/streptomycin (Invitrogen) and kept at 37°C in a 5% CO₂ incubator. Cells were transiently transfected with the calcium phosphate precipitation procedure (Invitrogen) as described previously.⁴ Channel subunits to be studied were subcloned into pGW1H, an expression vector with a cytomegalovirus promoter (British Biotechnology). Cells were transiently transfected with 7.5 µg of plasmid containing the Ca_v1.3 Ca²⁺ channel (a gift from Dr S. Seino, Kobe University, Kobe, Japan) or the Ca_v1.2 Ca²⁺ channel and coexpressed with 5 µg of plasmid containing the gene that encodes a ß_1A-subunit (derived from skeletal muscle).

In Vivo Electrophysiological Studies in Mice
In vivo electrophysiological studies were performed as previously described.¹⁵ Standard pacing protocols were used to determine the electrophysiological parameters, including sinus node recovery time; atrial, AV nodal, and ventricular refractory periods; and AV nodal conduction properties. Each animal underwent an identical pacing and programmed stimulation protocol. The Q-T interval was determined manually by placing cursors on the beginning of the QRS and the end of the T wave. The rate-corrected QT interval was calculated with a modified Bazett’s formula as reported by Mitchell et al,¹⁶ whereby the RR interval was first expressed as a unitless ratio (RR in ms/100 ms). The rate-corrected QT interval was defined as QT interval (in ms)/(RR/100)^1/2.

To induce atrial and ventricular tachycardia and fibrillation, programmed extrastimulation techniques and burst pacing were used. Programmed right atrial and right ventricular double and triple extrastimulation techniques were performed at a 100-ms drive cycle length, down to a minimum coupling interval of 10 ms. Right atrial and right ventricular burst pacing was performed as eight 50-ms and four 30-ms cycle-length train episodes repeated several times, up to a maximum 1-minute time limit of total stimulation. For comparison of inducibility, programmed extrastimulation techniques and stimulation duration of atrial and ventricular burst pacing were the same in all mice. Sustained atrial or ventricular arrhythmias were defined as atrial arrhythmias lasting >30 seconds. Reproducibility was defined as >1 episode of induced atrial or ventricular tachycardia.

Statistics
Data are presented as mean±SEM. Comparison among the 3 genotypes was performed with SigmaStat with ANOVA and pairwise multiple comparison procedures (Holm-Sidak method). We analyzed each litter separately and did not find significant outliers from the different litters. Because each litter was too small to allow for statistical analysis, we combined the data from all litters for the final statistical analysis. Comparison of the occurrence of atrial arrhythmias was performed with Fisher’s exact test.

	Results

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Ca_v1.3 Transcripts Are Highly Expressed in Mouse Atria Compared With Ventricles
We directly probed for the existence of the Ca_v1.3 Ca²⁺ channel in mouse cardiac myocytes by RT-PCR. Figure 1A shows representative RT-PCR–amplified products with primers specific for Ca_v1.2 versus Ca_v1.3 Ca²⁺ channels and primers specific for glyceraldehyde 3-phosphate dehydrogenase as a positive control from total RNA from mouse right atria, left atria, right ventricles, left ventricles, septum, and brain. Primers designed from mutually unique regions of Ca_v1.2 and Ca_v1.3 channels in the N-termini are shown in Figure 1B. Whereas Ca_v1.2 transcripts are expressed throughout the different regions in atria and ventricles, Ca_v1.3 transcripts are highly expressed in the atria compared with the ventricles. The signals obtained from the right and left ventricles and interventricular septum were very low compared with the atria.

View larger version (69K): [in this window] [in a new window]   Figure 1. A, Representative agarose gels of RT-PCR–amplified products with the use of primers specific for Cav1.2 vs Cav1.3 Ca2+ channels and primers specific for glyceraldehyde 3-phosphate dehydrogenase as a positive control from total RNA from mouse RA (right atria), LA (left atria), RV (right ventricles), LV (left ventricles), S (septa), and B (brains). –ve refers to a negative control (PCR-amplified product without RT to ensure that there was no genomic contamination of the RNA samples). Lane 1 is the HI-LO DNA markers (Bioscience, Inc). B, Nucleotide sequence alignment (ClustalW) of the N-termini of mouse Cav1.2 and Cav1.3 Ca2+ channels. Highlighted regions refer to the primers used for the PCRs to generate the sense and antisense riboprobes. *Conserved nucleotide between the 2 isoforms. Nucleotide sequence numbers are given on the right. C, Photomicrographs comparing the distribution of Cav1.2 vs Cav1.3 transcripts in mouse atria and ventricles. Sections obtained with the corresponding sense riboprobes are shown to the right as the negative controls. RA and LV refer to right atrium and left ventricle, respectively.

To further confirm that the RT-PCR products generated with primers specific to the Ca_v1.3 Ca²⁺ channel were indeed amplified from cardiac myocytes and not other cell types in the cardiac homogenate (eg, vascular smooth muscle cells), we further generated sense and antisense riboprobes in the presence of UTP-digoxigenin label for in situ hybridization. Figure 1C is a photomicrograph comparing the distribution of Ca_v1.2 versus Ca_v1.3 transcripts in mouse atria and ventricles. Whereas Ca_v1.2 transcripts are present in both the atria and ventricles, Ca_v1.3 transcripts are present mainly in the atria. Sense riboprobes were used as negative controls from consecutive sections (labeled as sense).

Immunodetection of Ca_v1.2 Versus Ca_v1.3 Ca²⁺ Channels in Dissociated Mouse Atrial and Ventricular Myocytes
To further examine the regional distribution of the Ca_v1.2 and Ca_v1.3 Ca²⁺ channels at the protein level, we performed an immunofluorescence confocal microscopy study of single, isolated, mouse atrial and ventricular myocytes. The specificity of the antibodies and the lack of cross reactivity at the dilutions used for the 2 different isoforms of the Ca²⁺ channels were first tested in expressed Ca_v1.2 and Ca_v1.3 Ca²⁺ channels in HEK 293 cells (Figure 2A, 2B, 2D, and 2E) compared with nontransfected cells (Figure 2G and 2H). Figure 2C and 2F represent negative controls treated with secondary antibodies only.

View larger version (41K): [in this window] [in a new window]   Figure 2. Confocal photomicrographs of HEK 293 cells expressing Cav1.2 (A–C) vs Cav1.3 (D–F) Ca2+ channels. Anti-Cav1.2 (A and D) and anti-Cav1.3 (B and E) antibodies were used. Immunofluorescence labeling was done by treatment with secondary antibodies (Texas red–conjugated anti-rabbit antibodies). C and F show samples treated with secondary antibodies only as negative controls. G and H included nontransfected cells treated with anti-Cav1.2 vs anti-Cav1.3, respectively. Corresponding differential interference contrast (DIC) images are shown in the right panels. The scale bar is 20 µm.

Figure 3A and 3C shows specific labeling with anti-Ca_v1.2 antibody in isolated mouse atrial and ventricular myocytes, respectively. In contrast, only atrial myocytes show specific labeling with the anti-Ca_v1.3 antibody (Figure 3B). Only a low level of staining with the anti-Ca_v1.3 antibody was observed in ventricular myocytes (Figure 3D). We further documented the specificity of the anti-Ca_v1.3 antibody in isolated atrial myocytes from Ca_v1.3^–/– mutant mice (Figure 3E and 3F). Whereas atrial myocytes from Ca_v1.3^–/– mutant mice showed specific labeling with the anti-Ca_v1.2 antibody (Figure 3E), no staining was observed with the anti-Ca_v1.3 antibody (Figure 3F). Figure 3G shows additional images of negative controls with secondary antibody only.

View larger version (48K): [in this window] [in a new window]   Figure 3. Confocal photomicrographs from freshly isolated WT mouse atrial (A and B) and ventricular (C and D) myocytes treated with anti-Cav1.2 (A and C) vs anti-Cav1.3 (B and D) antibodies. Immunofluorescence labeling was done by treatment with secondary antibodies (Texas red–conjugated antibodies). E and F represent mouse atrial myocytes isolated from Cav1.3–/– mutant mice. G, Mouse ventricular myocytes isolated from WT mice treated with secondary antibodies only as negative controls. Corresponding DIC images are shown in the right panels. The scale bar is 20 µm.

Functional Roles of the Ca_v1.3 Ca²⁺ Channel in the Heart Assessed in Ca_v1.3^–/– Mutant Mice
Our data on the regional localization of the Ca_v1.3 Ca²⁺ channel transcript and protein with in situ hybridization and immunofluorescence confocal microscopy are consistent with expression of the Ca_v1.3 Ca²⁺ channel mainly in the atria. However, the functional roles of the differential expression of the Ca_v1.3 Ca²⁺ channel are unknown. Because Ca_v1.2 and Ca_v1.3 Ca²⁺ channels have similar pharmacological properties, we reasoned that Ca_v1.3^–/– mutant mice would be an ideal model to study the functional role of Ca_v1.3 Ca²⁺ channels in the atria. We undertook in vivo electrophysiological studies comparing mutant mice with heterozygous and WT animals. All mutant mice showed evidence of SA and AV nodes dysfunction, as assessed by sinus cycle length, sinus node recovery time, PR interval, and Wenckebach cycle length (see the Table). Furthermore, atrial arrhythmias, mainly atrial fibrillation, were induced in all mutant mice and a small number of heterozygous littermates. In contrast, atrial arrhythmias were induced in none of the WT littermates (P<0.01 comparing WT and mutant animals by Fisher’s exact test). Indeed, previous studies of the same background mouse model have shown that WT mice are not inducible for atrial arrhythmias in the absence of carbachol.¹⁷Figure 4 shows examples of atrial fibrillation and atrial flutter that were induced in a Ca_v1.3^–/–-null mutant mouse. In contrast, ventricular arrhythmias were not induced in either the WT, heterozygous or the homozygous mutant mice. The in vivo electrophysiological parameters are summarized in the Table.

View this table: [in this window] [in a new window]   In Vivo Electrophysiological Studies in Cav1.3–/– and Cav1.3+/– Mice Compared With WT Littermates

View larger version (27K): [in this window] [in a new window]   Figure 4. In vivo electrophysiological studies in Cav1.3-null mutant mice, showing evidence of inducible atrial fibrillation (A) and atrial flutter (B) after atrial extrastimuli. Upper tracings are surface ECG (leads I and II). Lower traces are intracardiac electrograms showing atrial and ventricular electrograms, with induced sustained rapid atrial fibrillation and atrial flutter with relatively slow ventricular response.

Whole-Cell I_Ca,L Recorded From Ca_v1.3^–/– Atrial and Ventricular Myocytes Compared With Those From WT Littermates
To further corroborate the findings from the in vivo functional studies described earlier, we directly recorded I_Ca,L from atrial and ventricular myocytes from Ca_v1.3^–/– and compared them with those of heterozygous and WT littermates. Whole-cell I_Ca,L was recorded at a holding potential of –55 mV. Figure 5A shows examples of I_Ca,L current traces elicited at the various step potentials from atrial myocytes isolated from Ca_v1.3^+/+ and Ca_v1.3^+/– littermates compared with Ca_v1.3^–/–. The current-voltage relations are summarized in Figure 5B. Even though there were no significant differences in current density among the 3 different groups of animals, there was a depolarizing shift in the voltage-dependent activation of the current when we compared Ca_v1.3^+/+ to Ca_v1.3^+/– and Ca_v1.3^+/– with Ca_v1.3^–/– mice. We further confirmed this initial impression by generating activation curves from WT, heterozygous, and mutant animals (Figure 5C). I_Ca,L recorded from Ca_v1.3^+/– atrial myocytes showed an 5-mV depolarizing shift at the midpoint of activation compared with WT animals, whereas current from Ca_v1.3^–/– mice showed a further depolarizing shift of 7 mV compared with the heterozygous animals. Figure 5D shows data obtained with a 2-pulse protocol to examine the voltage- and Ca²⁺-dependent inactivation of I_Ca,L in WT, heterozygous, and mutant animals. The curves appear nearly superimposed, with no significant differences in the half-inactivation voltages. In addition, the curves show the typical U-shape configuration for Ca²⁺-dependent inactivation of L-type Ca²⁺ current. Typical traces elicited with the test pulse are shown in the insert. Prepulses more positive than +20 mV elicited a progressively smaller inward current as the command voltages approach the reversal potential, leading to partial recovery of the L-type Ca²⁺ current elicited with the test pulse, owing to a decrease in Ca²⁺-dependent inactivation. There were no significant differences in voltage dependence of the inactivation profile among the 3 groups of animals.

View larger version (23K): [in this window] [in a new window]   Figure 5. A, Example of whole-cell ICa,L recorded from a holding potential of –55 mV elicited at various step potentials (+10, +20, and +30 mV) from atrial myocytes isolated from Cav1.3+/+ and littermates Cav1.3+/– compared with Cav1.3–/– mice. The test potentials used are shown to the left of the current traces. The current-voltage relations are summarized in B (n=10 for each group). C, Voltage-dependent activation curves showing the normalized conductances (g/gmax) from WT, heterozygous, and mutant animals. The solid lines represent fits to the Boltzmann function yielding half-activation voltages (V1/2) of 0.95±0.40, 6.30±0.40, and 13.75±0.38 mV for Cav1.3+/+, Cav1.3+/–, Cav1.3–/–, and respectively (P<0.05 comparing Cav1.3–/– with Cav1.3+/+, Cav1.3–/– with Cav1.3+/–, and Cav1.3+/– with Cav1.3+/+) and slope factors of 6.8, 7.2, and 7.6 mV (P=NS) for Cav1.3+/+, Cav1.3+/–, and Cav1.3–/–, respectively (n=9 for each group). D, Voltage-dependent inactivation according to a 2-pulse protocol to examine the voltage- and Ca2+-dependent inactivation of ICa,L in WT, heterozygous, and mutant animals. Normalized current traces at a test potential of +20 mV, comparing ICa,L recorded from Cav1.3+/+, Cav1.3+/–, and Cav1.3–/– atrial myocytes. Examples of traces obtained from the test pulse are shown in the insert. Prepulses more positive than +20 mV elicited a progressively smaller inward current as the command voltages approach the reversal potential, leading to partial recovery of the LTCC elicited with the test pulse owing to a decrease in the Ca2+-dependent inactivation.

Figure 6 shows the same set of experiments obtained from free-wall left ventricular myocytes, comparing the 3 groups of animals. In contrast with the data obtained from atrial myocytes, there were no significant differences in I_Ca,L recorded from the left ventricular myocytes among the 3 groups of animals. These functional data are consistent with our in situ hybridization and immunofluorescence studies showing that the Ca_v1.3 transcript and protein are present predominantly in atrial tissues.

View larger version (21K): [in this window] [in a new window]   Figure 6. A, Example of whole-cell ICa,L recorded from a holding potential of –55 mV in the same set of experiments as in Figure 5 from free-wall left ventricular myocytes, comparing the 3 groups of animals. In contrast to the data obtained from the atrial myocytes, there were no significant differences in ICa,L recorded from the left ventricular myocytes among the 3 groups of animals. The current-voltage relations are summarized in B (n=10 for each group). C shows the corresponding activation curves and the normalized conductances (g/gmax) from Cav1.3+/+,Cav1.3+/–, andCav1.3–/– ventricular myocytes. The solid lines represent fits to the Boltzmann function yielding half-activation voltages (V1/2) of 9.22±0.42, 9.41±0.60, and 9.5±0.48 mV (P=NS) and slope factors of 5.9±0.38, 6.1±0.54, and 6.4±0.43 mV for Cav1.3+/+, Cav1.3+/–, and Cav1.3–/–, respectively (P=NS, n=9 to 11 for each group). D, Voltage-dependent inactivation according to a 2-pulse protocol to examine the voltage- and Ca2+-dependent inactivation of ICa,L in WT, heterozygous, and mutant animals. Normalized current traces at a test potential of +20 mV, comparing ICa,L recorded from Cav1.3+/+, Cav1.3+/–, andCav1.3–/– ventricular myocytes. Examples of traces obtained from the test pulse are shown in the insert.

Previous data provide important clues that the differences in biophysical properties of Ca_v1.2 versus Ca_v1.3 Ca²⁺ channels may be directly responsible for the observed findings in the atria.^18,19 I_Ca,L recorded from atrial myocytes isolated from Ca_v1.3^–/– mutant animals were activated at more depolarizing potentials compared with those from Ca_v1.3^+/+ or Ca_v1.3^+/–, which expressed both Ca_v1.2 and Ca_v1.3 Ca²⁺ channels (Figure 5C). Indeed, we have previously documented in a heterologous expression system that there is a significant depolarizing shift in steady-state activation in Ca_v1.2 compared with Ca_v1.3 Ca²⁺ currents, consistent with findings in the Ca_v1.3^–/– mice, which express only the Ca_v1.2 subunit.⁴

	Discussion

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In this study, we directly tested the role of the Ca_v1.3 Ca²⁺ channel in atrial myocytes in Ca_v1.3-null mutant mice. Whole-cell I_Ca,L recordings from atrial myocytes isolated from the null mutant mice showed a depolarizing shift in the voltage-dependent activation compared with WT. In contrast, there were no significant differences in whole-cell I_Ca,L recorded from ventricular myocytes from WT or null mutant mice. Consistent with these findings, we previously documented the hyperpolarizing shift in voltage-dependent activation of Ca_v1.3 compared with Ca_v1.2 Ca²⁺ channel subunits in a heterologous expression system.⁴ The lack of Ca_v1.3 Ca²⁺ channels in the null mutant mice would result in a depolarizing shift in the voltage-dependent activation of I_Ca,L in atrial myocytes. We further confirmed the isoform-specific differential expression of Ca_v1.3 in atrial myocytes by in situ hybridization and immunofluorescence confocal microscopy.

Voltage-Gated Ca²⁺ Channel Subtypes in the Heart
The molecular basis for I_Ca in the heart has previously been investigated. By in situ hybridization, it was found that the most prominently expressed low-voltage activated Ca²⁺ channel in the SA node was Ca_v3.1(_1G), whereas Ca_v3.2(_1H) is present at moderate levels.²⁰ In addition, we and others have previously documented the critical role of Ca_v1.3 in the SA node by using mutant mouse models.^4–6 The dominant high-voltage activated Ca²⁺ channel was Ca_v1.2, whereas only a small amount of Ca_v1.3 mRNA was detected in SA node myocytes of mice.²⁰ The existence the Ca_v1.3 Ca²⁺ channel in different regions of the heart has been further documented in a recent study by Marionneau et al²¹ and is consistent with our findings: Ca_v1.3 was found to be more prominently expressed in the atria compared with the ventricles. However, the functional roles of Ca_v1.3 in the atria or ventricular tissues have never been documented.

Role of Ca_v1.3 Ca²⁺ Channels in Atrial Myocytes
Here, using gene-targeted deletion of the Ca_v1.3 isoform, we were able to document that genetic ablation of the Ca_v1.3 isoform results in the occurrence of atrial arrhythmias. Indeed, this mutant mouse model represents one of the few genetic models of atrial fibrillation. Even though Ca_v1.3 represents only a small amount of the LTCC transcript in the atria, owing to the significant differences in biophysical properties of the Ca_v1.3 isoform, the channel contributes significantly to the overall function in the atria. Our in vivo electrophysiological data as well as patch-clamp recordings are consistent with the notion that Ca_v1.3 LTCCs are expressed and contribute functionally to atrial cardiac myocytes in contrast to ventricular myocytes.

Atrial Fibrillation
Atrial fibrillation is the most common clinical arrhythmia and is associated with a significant increase in morbidity and mortality.²² The underlying mechanisms of atrial fibrillation are very heterogeneous and are often related to underlying heart or pulmonary diseases. However, more recently, several studies have identified mutations in ion channels as possible causes of inherited atrial fibrillation.^23–26 The first gene for an inherited form of atrial fibrillation was identified in a family with autosomal-dominant transmission.²⁴ A mutation was found in the K⁺ channel gene KCNQ1, resulting in a gain-of-function mutation. This is in contrast with the reduction in current density seen with mutations in other residues in this gene causing long QT syndrome type 1. The gain-of-function mutation is consistent with the decrease in action potential duration and effective refractory period, which are thought to be the mechanisms of atrial fibrillation. On the other hand, a number of patients with the mutation also had a prolonged QT interval,²⁴ emphasizing the fact that our understanding of repolarization is incomplete. In addition, recent data also suggest a role for genetic modifiers, or incompletely penetrant disease genes, as mechanisms for the development of atrial fibrillation. Our data showing the development of atrial fibrillation in null mutant mice suggest an important functional role for Ca_v1.3 in the atria. Ablation of the Ca_v1.3 Ca²⁺ channel did not alter the atrial effective refractory period but might nonetheless alter atrial action potential duration or Ca²⁺-activated repolarizing currents. Direct measurements of action potential duration are required to establish this. Additional studies are also needed to further examine the effects of the Ca_v1.3 Ca²⁺ channel on Ca²⁺ transients. Finally, the relevance of this model to human atrial fibrillation remains only speculative at this time.

Compensatory Changes in Mutant Mice
Because the relative contribution of the Ca_v1.3 Ca²⁺ channel to total I_Ca,L in atrial myocytes is unknown, we directly compared the maximum I_Ca,L density between mutant mice and their WT littermates (Figure 5B). The current was normalized to cell capacity. Cell capacitance of single, isolated, atrial myocytes from the 3 groups of animals was 53.9±2.2, 41.9±2.4, and 52.3±5.0 pF for Ca_v1.3^+/+, Ca_v1.3^+/–, and Ca_v1.3^–/– mice, respectively (n=8, P=NS). There was a 12-mV depolarizating shift in the peak I_Ca,L in the Ca_v1.3^–/– mice compared with their WT littermates; however, the current density was not significantly different between the WT and homozygous mutant animals. This may represent a compensatory change, with upregulation of Ca_v1.2 I_Ca,L in the mutant animals.

In summary, using gene-targeted deletion of the Ca_v1.3 Ca²⁺ channel, we established the important functional roles of Ca_v1.3 Ca²⁺ channels in atrial myocytes in addition to its previously documented role in pacemaking cells. The hyperpolarizing shift in the activation threshold of the Ca_v1.3 Ca²⁺ channel can be directly documented by gene-targeted deletion in the Ca_v1.3 mutant mouse model. Our in vivo electrophysiological data support important roles for Ca_v1.3 in atrial myocytes; the Ca_v1.2 subunit cannot functionally substitute for the Ca_v1.3 subunit. Important phenotypes of atrial fibrillation and atrial flutter were documented in the null mutant mice. Similar to that in neuronal systems, the expression of multiple Ca²⁺ channel subtypes appears to be important in coordinating the different physiological functions in atrial myocytes in addition to cardiac pacemaking cells. Taken together, our data represent the first report on the functional roles for Ca_v1.3 Ca²⁺ channels in atrial cardiomyocytes. Importantly, the differential expression of the 2 different isoforms of the LTCC, with predominant expression of the Ca_v1.3 channel in the atria compared with the ventricles, may offer a unique therapeutic opportunity to directly modify the atrial cells without interfering with ventricular myocytes.

	Acknowledgments

 
This study was supported by NIH/NHLBI grants (RO1, HL67737, and HL75274) and the Nora Ecceles Treadwell Foundation Award (Dr Chiamvimonvat). The authors thank Dr E.N. Yamoah for helpful suggestions and comments and the UC, Davis, Health System Confocal Microscopy Facility.

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CLINICAL PERSPECTIVE

Atrial fibrillation is the most common atrial arrhythmia affecting the American population and is associated with a significant risk of embolism and stroke. The problem is further exacerbated by the fact that treatment strategies have proven largely inadequate. An alteration in ion channel expression (electrical remodeling) has been implicated in the maintenance of the arrhythmias. Importantly, a decrease in Ca²⁺ current density in atrial myocytes of patients with persistent atrial fibrillation has been documented. Previous data suggest that L-type Ca²⁺ channels containing the Ca_v1.3(_1D) subunit are expressed mainly in neurons and neuroendocrine cells, whereas those containing the Ca_v1.2(_1C) subunit are found in the brain, vascular smooth muscle, and cardiac tissue. We and others have shown that Ca_v1.3 Ca²⁺ channel–deficient mice (Ca_v1.3^–/–) demonstrate sinus bradycardia with a prolonged PR interval. This study examined the role of the Ca_v1.3 (_1D) Ca²⁺ channel in the atria of Ca_v1.3^–/– mice. Here, we demonstrate that there is significant expression of Ca_v1.3 Ca²⁺ channels predominantly in atrial compared with ventricular tissues. In addition, Ca_v1.3-null mutant mice have an increased susceptibility to atrial arrhythmias, indicated by inducible atrial flutter and fibrillation. The relevance of this model to human atrial fibrillation is speculative at this time, but the important functional role of the Ca_v1.3 Ca²⁺ channel in atrial tissues in this model warrants additional study to assess its role in humans.

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