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J. Biol. Chem., Vol. 275, Issue 50, 39193-39199, December 15, 2000

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Functional Embryonic Cardiomyocytes after Disruption of the L-type _1C (Ca_v1.2) Calcium Channel Gene in the Mouse^*

Claudia Seisenberger, Verena Specht, Andrea Welling, Josef Platzer^§, Alexander Pfeifer, Susanne Kühbandner, Jörg Striessnig^§, Norbert Klugbauer, Robert Feil, and Franz Hofmann^¶

From the  Institut für Pharmakologie und Toxikologie, TU München, Biedersteiner Strasse 29, D-80802 München, Germany and ^§ Institut für Biochemische Pharmakologie, Universität Innsbruck, Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria

Received for publication, July 20, 2000, and in revised form, August 28, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The L-type _1C (Ca_v1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca_v1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca_v1.2/) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/), and homozygous (/) Ca_v1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 µM nisoldipine. In (+/+), (+/), and (/) cardiomyocytes, an L-type Ba²⁺ inward current (I_Ba) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of 80 mV, nisoldipine blocked I_Ba of day 12.5 p.c. (+/+) and (+/) cells with two IC₅₀ values of 0.1 and 1 µM. Inhibition of I_Ba of (/) cardiomyocytes was monophasic with an IC₅₀ of 1 µM. The low affinity I_Ba was also present in cardiomyocytes of homozygous _1D (Ca_v1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Calcium channels play an important role in the function of different tissues. The calcium entry via high voltage-activated (HVA)¹ calcium channels leads to excitation-contraction coupling in the heart, tension development in smooth muscle, neurotransmitter release in brain, and endocrine secretion in gland tissues. In the cardiovascular system, voltage-activated calcium channels are essential for the generation of normal cardiac rhythm, for induction of rhythm propagation through the atrioventricular node, and for the contraction of the atrial and ventricular muscle (1). In diseased myocardium, calcium channels can contribute to abnormal impulse generation and cardiac arrhythmias (2).

Calcium channels are hetero-oligomeric complexes of up to four subunits as follows: ₁, , ₂, and  subunit. The ₁ subunit contains the voltage sensor, the selectivity filter, the ion-conducting pore, and the binding sites for all known calcium channel blockers. The other subunits are auxiliary subunits, which modulate the channel function. Calcium channels can be further modulated by a variety of hormones, protein kinases, and protein phosphatases (3-5).

High voltage-activated calcium channels have been classified as L-type and non-L-type channels. L-type channels are encoded by four distinct genes, namely Ca_v1.1 to Ca_v1.4 (6), that give rise to numerous splice variants. Mammalian L-type channels have a similar ion selectivity and inactivation kinetics and are affected by dihydropyridines at similar concentrations. The expression pattern and the electrophysiology of L-type calcium channels have been studied extensively in pre- and postnatal heart cells of the mouse (7-10). The major L-type channel expressed in the cardiac and smooth muscle is the Ca_v1.2 (11, 12). In addition, the expression of the Ca_v1.3 gene was reported (13-15). However, the exact function of these channels often remained unclear. To analyze the functional relevance of the L-type Ca_v1.2 calcium channel for various tissues, two mouse lines were generated in which the Ca_v1.2 gene was disrupted at different exons. Both mouse lines had an identical phenotype. The homozygous (/) embryos died before day 14.5 p.c. Surprisingly, cardiomyocytes of 12.5-day-old p.c. embryos beat spontaneously using an unidentified L-type like calcium current.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vector Construction (Mouse Line A)-- Murine calcium channel Ca_v1.2 genomic DNA was obtained from a 129SV-P1 library (Genome Systems, St. Louis, MO) by screening with two primers amplifying 204 bp of the second exon of the Ca_v1.2 gene. Two exons were identified in the P1 clone coding for the second and third exon of mouse Ca_v1.2 gene (16, 17). The third exon encoding part of the domain I of Ca_v1.2 was used for the construction of the targeting vector since alternative splicing has not been described for this exon. The key features of the targeting vector are shown in Fig. 1A. A neomycin resistance cassette (Neo) was placed into the MunI site of the third exon in the reverse direction of transcription. Additionally, a herpes simplex virus type I-thymidine kinase cassette (HSV-TK) was inserted 5'-terminal of the homologous region. Analysis of the genotype of the offsprings and proof for the correct insertion of the Neo were performed with primer pair NeoPA (5'-GCC TGC TCT TTA CTG AAG GCT CT-3') and VS3 (5'-ACC ATT TGA AAT CAT TAT TTT ACT-3') that amplify a 400-bp fragment of the mutated RNA and primer pair CSI (5'-ACG CCC AGC TCA TGC CAA CAT-3') and mun3 (5'-TAA GGC CAC ACA ATT GGC AA-3') that amplify a 354-bp fragment of the Ca_v1.2 exons 2 and 3 (Fig. 1C).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Targeted disruption of the calcium channel Cav1.2 gene (mouse line A). A, a partial restriction map of the Cav1.2 (1C) wild type locus (Aa), targeting vector (Ab), and targeted locus (Ac). Exons 2 and 3 are indicated as solid boxes and introns as solid lines. The Neo cassette was inserted into the MunI site of the third exon in opposite orientation. 5' to the Neo cassette, three stop codons were inserted in the three different reading frames. Double arrowhead lines in Aa and Ac represent the expected DNA fragments after SphI digest and hybridization with the EV500 probe (striped box in Ac). S, SphI; M, MunI; B, BamHI; A, AspI; C, ClaI. B, identification of Cav1.2 (+/+, wild type), (+/, heterozygous), and (/, homozygous) embryos by Southern blot analysis of SphI-digested DNA. Genomic DNA was derived from a single litter of 12.5 p.c. embryos from mating of heterozygous Cav1.2 (+/) mice. Hybridization with the EV500 probe yielded signals of 6.5 (+/+) and 4.5 kb (/). C, RT-PCR of RNA isolated from 12.5 p.c. embryos. PCR strategy to identify (Ca) wild type (WT) and (Cb) mutated (/) RNA. Cc, the primer pair CSI/mun3 amplified a 354-bp fragment from the wild type locus but did not amplify RNA from the knockout locus. Lanes 1 and 2, the RNA was reverse-transcribed; lane 3 (K), control plasmid containing the Cav1.2 cDNA; lanes 4 and 5, the RNA was not reverse-transcribed. The negative result of lane 5 shows that the amplicon of lane 2 was not derived from genomic DNA. Cd, the primer pair VS3/NeoPA yielded a 400-bp fragment only when the mutated locus is present. Lanes 1 and 2, the RNA was reverse-transcribed; lanes 3 and 4, the RNA was not reverse-transcribed. The negative result of lanes 3 and 4 shows that the amplicons of lanes 1 and 2 were not derived from genomic DNA.

Vector Construction (Mouse Line B)-- A second P1 plasmid was obtained from Genome Systems containing a different part of the murine Ca_v1.2 gene. Restriction map analysis and sequencing of this P1 plasmid revealed that exons 13-16 encoding part of repeat II of Ca_v1.2 were present (16, 17). The key features of the targeting vector are shown in Fig. 2A. The vector contains a Neo and HSV-TK cassette and three 34-bp loxP sequences. Two of the loxP sites flank the Neo/HSV-TK cassette, which was inserted into the intron between exons 13 and 14. A third loxP site was placed into the intron between exons 15 and 16. To confirm the Cre-mediated deletion of the exons 14 and 15, RT-PCR was performed using the primer pair VS11 (5'-CTG GAA TTC CTT GAG CAA CCT TGT-3') and VS16 (5'-AAT TTC CAC AGA TGA AGA GG- ATG-3') to amplify exons 14 and 15 (329 bp) in (+/+), (+/), and (/) cells and the primer pair VS9 (5-ACA CAG CCA ATA AAG CCC TCC TG-3') and VS18 (5'-GGC CAG CTT CTT CCT CTC CTT-3') to amplify the sequence between exons 13 and 16 in the (/) mouse (341 bp).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Targeted disruption of the calcium channel Cav1.2 gene using the Cre/loxP system (mouse line B). Aa, a partial restriction map of the Cav1.2 wild type allele. Exons are indicated as solid boxes. The location of the DNA fragment used as the 5'-hybridization probe in B is shown. Ab, the targeting vector contains a Neo/HSV-TK cassette and three loxP sequences (gray triangles numbered I-III). Two loxP sites flank the Neo/HSV-TK cassette, which is located in the intron upstream of exon 14. The third loxP site is located in the intron between exons 15 and 16. Ac, the targeted locus after homologous recombination. Ad, knockout locus after Cre-mediated excision of exons 14 and 15 and the Neo/HSV-TK cassette. The double arrowhead line in Aa and Ad shows the DNA fragment obtained after digestion with BamHI. A, Acc65I; B, BamHI; C, ClaI; EI, EcoRI; H, HindIII; SI, SstI. B, Southern blot analysis. DNA isolated from embryos at day 12.5 p.c. was digested with BamHI and then hybridized with the 5' probe yielding a 9- (+/+) and 17-kb (/) fragment. C, RT-PCR of RNA isolated from 12.5 p.c. embryos. Ca, primer pair VS11/VS16 amplifies a 329-bp fragment (arrow) of exons 14 and 15 in the wild type (+/+) and heterozygous (+/) but not in knockout (/) embryos. Cb, primer pair VS9/VS18 amplifies a 341-bp fragment (arrow) of exons 13-16 in RNA from homozygous knockout embryos (/). Left part, schematic drawing of spliced RNA. Right part, gels of PCR products; M, marker. D, the amplicon obtained by primer pair VS9/VS18 was sequenced and aligned with the murine cDNA sequence of Cav1.2 (16). Only part of the relevant sequence is shown. 1st line, exons and exon borders (|). 2nd line (+/+), sequence of murine cDNA; only part of the sequence from exons 14 and 15 is shown (//, interruption of sequence). 3rd line (/), sequence of cDNA amplified from the RNA of a knockout embryo. Lowercase italic letters, sequence from the intron upstream of exon 16. *, stop codon caused by the frameshift.

Generation of Gene-targeted Mouse Lines-- Sixty µg of each targeting vector were linearized with NotI and electroporated into 1 × 10⁷ R1 embryonic stem (ES) cells obtained from Samuel Lunenfeld Research Institute, Toronto, Canada. G418/ganciclovir- (mouse line A) and G418 (mouse line B)-resistant clones were screened by Southern blot analysis. Analysis of 311 G418-resistant clones revealed two clones (77 and 94 in mouse line B) that carried the floxed Neo/HSV-TK cassette and the third loxP site at the correct genomic region. 1 × 10⁷ ES cells of clone 77 were electroporated with 6 µg of a Cre-expressing plasmid. Cells were plated at different dilutions and were selected with ganciclovir. Ganciclovir-resistant clones in which the Neo/HSV-TK cassette and exons 14 and 15 had been excised were identified by Southern analysis. ES cells carrying the disrupted allele (line A) or the Ca_v1.2 gene with the deleted exons 14 and 15 (line B) were microinjected into blastocysts from C57BL/6 mice to generate chimeric mice. Chimeric males were crossed with C57BL/6 females. Germ line transmission of the mutated Ca_v1.2 genes was verified by PCR analysis and Southern hybridization using tail DNA. The generation of the Ca_v1.3(/) mice has been described recently (18).

RNA Isolation and First Strand cDNA Synthesis-- Total RNA was isolated from 12.5-day-old mouse embryos using TRIZOL LS Reagent (Life Technologies, Inc.). For the first strand synthesis, 4 µg of total RNA were used according to the manufacturer's instructions. Primer pairs for the detection of the other calcium channels were as follows: D4-1 (5'-CGT GGT GAA CTC CTC GCC T-3') and D4-2 (5'-AAA AGG TGA TGG AGA TTC TAT T-3') to amplify a 312-bp fragment of Ca_v1.3; ISHSK1 (5'-CGC GGA TCC ATC TAC TTT GTC ACC CTC ATT CT -3') and ISHSK2 (5'-TAG GGT ACC ATG ATT TTG TTC AAG CCT TCG AT-3') to amplify a 348-bp fragment of Ca_v1.1; and a1F1 (5'-TAG GGA TCC GGC CCC GTG ATG ATG AC-3') and a1F2 (5'-CGC GGT ACC GTA CCA GGT CCC CAT CCA-3') to amplify a 525-bp fragment of Ca_v1.4.

Dissection and Cell Culturing of Murine Embryonic Cardiomyocytes-- Individual embryos were obtained after breeding of heterozygous Ca_v1.2 (+/) mice at day 9.5 p.c. or later. Cardiac myocytes were isolated as described (8) at day 12.5 p.c. or later. Myocytes were plated on plastic coverslips and cultured in Dulbecco's modified Eagle's medium (8) supplemented with 10% fetal calf serum and 5% penicillin/streptomycin (stock 10 mg/ml). Pharmacological tests were done in normal Tyrode's solution containing (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 glucose, 5 HEPES, pH 7.4. Genotyping of embryos was done by PCR.

Electrophysiology-- Whole-cell currents were recorded at room temperature 18-48 h after plating using fire-polished electrodes with resistances of 2-3.5 M. Pipettes were filled with (in mM) 60 CsCl, 50 aspartic acid, 68 CsOH, 1 MgCl₂, 5 K-ATP, 1 CaCl₂, 10 HEPES, 11 EGTA, pH 7.4. Extracellular solution for sealing and recording of sodium current (I_Na) was (in mM) 130 NaCl, 4.8 KCl, 5 BaCl₂, 1 MgCl₂, 5 glucose, 5 HEPES, pH 7.4. To isolate barium currents (I_Ba), the bath solution was changed to (in mM) 130 N-methyl-D-glucamine, 4.8 CsCl, 5 BaCl₂, 5 glucose, 5 HEPES, pH 7.4. The holding potential (HP) was 80 mV. Trains of test pulses were to 40 mV for I_Na or to 0 mV for I_Ba of L-type calcium channel applied once every 10 s for 40 ms. Data were collected and stored at an EPC-9 computer under control of Pulse software (HEKA electronics). Total cell membrane capacitance was determined by compensation mechanisms of the EPC9 computer and used as a measurement of membrane area. (+/+), (+/), and (/) cardiomyocytes had similar capacities of 30 ± 2.6 (n = 60), 26 ± 2.3 (n = 58), and 26 ± 2 (n = 53) pF, respectively. Inactivation curves were fitted by a Boltzmann relation as follows: I/I_max = (1  A)/(1 + exp((V  V_0.5)/k) + A, where I is the current, I_max is the maximal current at the beginning of the experiment, V is the potential, V_0.5 is the midpoint of the curve, k is the slope factor, and A is the non-inactivating part. Ba²⁺/Ca²⁺ selectivity of the current was determined by a 100-ms pulse from 80 mV (HP) to 0 mV for (+/+) and (+/) and to 10 mV for (/) cardiomyocytes at 0.2 Hz. Five mM Ba²⁺ was exchanged for 5 mM Ca²⁺ in the bath solution. In some experiments the sequence was reversed.

Cumulative dose-response curves were recorded using 2-3 different nisoldipine concentrations per cell. The number of experiments was 4-9 for each concentration. The stoichiometries and apparent affinities of nisoldipine were determined by fitting the averaged dose-inhibition points to the Hill equation: I/I_max = 1/(1 + ([nisoldipine]/IC₅₀)^H), where [nisoldipine] is the concentration of nisoldipine, IC₅₀ is the half-blocking concentration, and H is the Hill coefficient, I is the averaged current measured at any concentration of nisoldipine, and I_max is the average current measured in the absence of nisoldipine. To obtain apparent affinities for complex dose-inhibition relations, sums of Hill terms similar in form to that described above were fitted to the data.

Stock solutions were as follows: Bay K 8644 10 mM in ethanol; nisoldipine 20 mM in ethanol; isoproterenol + ascorbic acid 10 mM each in H₂O; tetrodotoxin citrate (TTX) 1 mM in H₂O. When required, stock solutions were freshly diluted to the indicated concentrations with the used extracellular solution. Data are shown as mean ± S.E. with the number of cells in parentheses. Graphics and statistical data analysis using Student's t test were carried out using ORIGIN software (Microcal).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genotype-- Two different mouse lines were generated in which the Ca_v1.2 calcium channel gene was disrupted. In mouse line A, a neomycin resistance cassette was inserted into the third exon of the Ca_v1.2 gene (Fig. 1A) in the opposite orientation. 5' to the Neo cassette stop codons were inserted into each reading frame of Ca_v1.2. The generation of functional channels is highly unlikely because the introduced modification leads to a truncated non-functional protein also in the case of an incorrect splicing, i.e. even if exon three is skipped, because of an early stop codon in exon four. Southern hybridization (Fig. 1B) and RT-PCR (Fig. 1C) with different primer pairs confirmed the germ line transmission of the Ca_v1.2 gene mutation in mouse line A. In mouse line B, exons 14 and 15 were deleted using the Cre/lox recombination system (Fig. 2A). Deletion of exons 14 and 15 destroyed the transmembrane segment IIS5 and the pore in repeat II of the Ca_v1.2 channel. In addition, the Neo/HSV-TK cassette was removed to avoid nonspecific effects produced by the cassette and the products of the cassette. Southern hybridization (Fig. 2B) and RT-PCR (Fig. 2C) with different primer pairs confirmed the germ line transmission of the Ca_v1.2 gene mutation in mouse line B. Deletion of the exons 14 and 15 was verified by sequencing of the RT-PCR product obtained with primer pair VS9 and VS18 (Fig. 2Cb). The primary transcript was spliced from exon 13 to an intron sequence directly upstream of exon 16 (Fig. 2D). The new splicing event caused a frameshift resulting in an early stop codon. In agreement with these results, Western analysis with antibodies against the Ca_v1.2 and the Ca_v1.1 protein yielded no specific bands in (/) embryonic hearts.

Identical results were obtained with both knockout lines. All experiments were at least repeated once in the other knockout line. Heterozygous Ca_v1.2 (+/) mice were indistinguishable from wild type (+/+) mice in shape, development, and behavior. The mating of heterozygous mice led to viable (+/+) and (+/) pups. The genotype and number of newborn mice was (+/+) 363 and (+/) 546 for line A and (+/+) 33 and (+/) 79 for line B. No viable knockout (/) mice were born. Examination of various gestation stages showed that viable embryos were present at day 12.5 p.c. at approximately Mendelian ratio ((+/+) 100, (+/) 171, and (/) 91 in line A and (+/+) 12, (+/) 35, and (/) 22 in line B). No viable (/) embryos were detected at day 14.5 p.c.

Phenotype-- Visual inspection suggested that Ca_v1.2 (+/+), (+/), and (/) embryos developed normally up to day 12.5 p.c. Hearts contracted with the same frequency at day 12.5 p.c. (Fig. 3A). After day 14.5 p.c., the beating frequency of the remaining (+/+) and (+/) embryos increased. Cardiac cells from day 12.5 p.c. (+/+), (+/), and (/) embryos could be cultured for more than a week. During this time, the frequency of spontaneous contractions increased in each genotype from about 30 to around 160 beats/min (Fig. 3B) suggesting that the Ca_v1.2 gene is not necessary for rhythmic activity or is compensated during embryonic development but is required after day 13 p.c. A previous report (9) indicated that the spontaneous contractions of cardiomyocytes from stage II embryoid bodies were caused by oscillations of intracellular [Ca²⁺] and did not require the influx of extracellular Ca²⁺ during each beat. To confirm these results, (+/) and (/) heart cells were superfused with Ca²⁺-free normal Tyrode's solution. Within 6 s, all cardiomyocytes stopped contracting. Addition of 1.8 mM Ca²⁺ restored beating in 2-5 s (n = 7 experiments for each genotype isolated from 3 different embryos). Repeated cycles of Ca²⁺ withdrawal and re-addition stopped and started contractions each time, respectively. Superfusion of (+/) or (/) cardiomyocytes with the dihydropyridine (DHP) nisoldipine (1 µM) stopped beating in 0.5 min (n = 8). The inhibitory effect of nisoldipine was reversed by washout of the compound. These results suggested that the rhythmic activity of day 12.5 p.c. heart cells was not caused by intracellular [Ca²⁺] oscillations but depended on the influx of extracellular Ca²⁺ through a channel with the characteristics of an L-type calcium channel. The nature of this putative channel was analyzed by the patch clamp technique using isolated cardiomyocytes.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Contracting hearts in each genotype. Embryos were collected from both mouse lines. A, cardiac contractions of intact embryos at day 12.5 p.c. and days 14.5-16.5 p.c. after opening the thorax. The three genotypes are as follows: (+/+), filled column; (+/), striped column; (/), open column. Number of cells are given within each column. B, ventricle cells were isolated at day 12.5 p.c. (day 0) and cultured for the indicated days. Values are means from 5 to 10 cells counted each day for each genotype. (+/+), filled circles; (+/), open triangles; (/), open circles.

Electrophysiology of Isolated Cardiomyocytes-- Barium inward currents (I_Ba) were observed in atrial and ventricular primary cultures of day 12.5 p.c. (+/+), (+/), and (/) cardiomyocytes. Voltage-dependent I_Na was present in 97% of the cells with I_Ba. Surprisingly, 98.5% (+/+), 95% (+/), and 81% (/) cardiomyocytes showed calcium channel activity with typical L-type kinetics (Fig. 4). The L-type current of wild type cardiomyocytes activated at 40 mV and reached its maximum between 0 and +10 mV. Due to large outward directed currents, the reversal potential could not be determined reliably. In (+/) and (/) cardiomyocytes, I_Ba activated and peaked at slightly more negative potentials of 50 and 10 mV, respectively. In (+/+), (+/), and (/) cells, I_Ba activated rapidly and inactivated slowly. Steady-state inactivation curves were obtained with 1-s prepulses to potentials between 60 and +30 mV. The V_0.5 values were 19 ± 3.6 mV (n = 8) in (+/+), 24 ± 3.1 mV (n = 8) in (+/), and 30 ± 8.2 mV (n = 5) in (/) cardiomyocytes. The voltage dependence was steeper in (+/+) cells with k = 6.3 ± 0.5 compared with k = 9.4 ± 1.2 and 10.0 ± 4.0 in (+/) and (/) cells. The non-inactivating part was 28 ± 5% in (+/+), 26 ± 5% in (+/), and 36 ± 7% in (/) cells. Although the values were not significantly different from each other, they were in line with the observation that I-V relations of (+/) and (/) cardiomyocytes were shifted slightly to hyperpolarized membrane potentials when compared with that of (+/+) cardiomyocytes (see also Fig. 4). The ion selectivity of the channel of (+/+), (+/), and (/) cardiomyocytes was identical (Fig. 4, D and E). Maximal inward currents with Ca²⁺ as charge carrier were reduced in each genotype, and current inactivation was increased to 90% in the presence of Ca²⁺ (Fig. 4, D and E) suggesting Ca²⁺-dependent inactivation of the channel in each genotype.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Kinetics and pharmacology of cardiac L-type IBa. A and B show results obtained with cardiac myocytes isolated at day 12.5 p.c. from embryos of mouse line A and B, respectively. Left column, current voltage (I-V) relations in the absence () and presence of 1 µM Bay K 8644 () or 1 µM nisoldipine (). The HP was 80 mV. Cells were depolarized to potentials between 80 and +30 mV with 10-mV increments for 100 ms at 0.2 Hz (n = 5 cells per genotype). Center column, genotypes. Right column, representative current traces in the absence (control) and presence of 1 µM Bay K 8644 (Bay K) or 1 µM nisoldipine (nis). Depolarization was from 80 to 0 mV. The horizontal line represents 0 mV (scale bars, 20 ms and 20 pA/pF). C, IBa densities of cardiac myocytes from the three genotypes ((+/+), filled column; (+/), striped column; (/), open column) at day 12.5 p.c. and days 14.5-16.5 p.c. 100-ms pulses were from 80 mV to the peak of the I-V relations. Number of cells are given within each column. Barium- (D) and calcium-dependent (E) inactivation of current in 12.5 p.c. (+/+, filled column), (+/, striped column), and (/, open column) cardiomyocytes. Maximum peak IBa or ICa (Imax) and the sustained IBa or ICa after 100 ms (I100) were determined. r100 represents (Imax  I100)/Imax. Number of cells are given within each column. Inset in D, representative current traces of a (/) cardiomyocyte in the presence of either 5 mM barium or 5 mM calcium. The horizontal line represents 0 mV (scale bars, 20 ms and 100 pA).

Superfusion of individual cells with the calcium channel agonist Bay K 8644 (1 µM) increased I_Ba and induced a shift of the I-V relation to hyperpolarized potentials in all three genotypes. The calcium channel blocker nisoldipine (1 µM) inhibited I_Ba in each cell line but with less efficiency in (/) cardiomyocytes (Fig. 4). The same results were obtained in mouse line A and B (Fig. 4, A and B). However, the current amplitude differed significantly between the three genotypes (Fig. 4C). I_Ba increased slightly in the (+/) cells after day 14.5 p.c. and was equal to I_Ba of (+/+) cells. The difference in current densities was not due to distinct cell sizes or differences in I_Na. The I_Na amplitudes were similar with 251 ± 19 (n = 57) for (+/+), 219 ± 18 (n = 54) for (+/), and 208 ± 16 (n = 61) pA/pF for (/) cells. This analysis suggested that embryonic cardiac cells from two independent Ca_v1.2 knockout mouse lines expressed a bona fide L-type calcium channel. An alternative explanation for this phenotype was that the observed L-type channel was the so-called slip-mode sodium conductance channel (19). This channel is blocked by TTX with an IC₅₀ of 0.1 µM and allows permeation of calcium in the presence of cAMP kinase or after activation of the -adrenergic receptor. In support, I_Ba was stimulated in each cell line 1.8-2.0-fold (n = 8 to 20 cells) by isoproterenol. A similar adrenergic stimulation of I_Ba has been reported for day 9.5 p.c. mouse embryonic heart cells (10). However, I_Ba was not affected at all by 10 µM TTX, whereas I_Na was blocked reversibly in each cell line (not shown). Therefore, it was concluded that the slip-mode channel did not cause the observed DHP-sensitive I_Ba in Ca_v1.2(/) cardiac cells.

DHP Sensitivity of I_Ba-- The experiments shown in Fig. 4 indicated that I_Ba of the Ca_v1.2(/) cells was less sensitive to nisoldipine than that of the cells with a wild type or heterozygous genotype. Therefore, the extent of channel block was tested by superfusion of the (+/+), (+/), or (/) cells with 1 µM nisoldipine at the HP of 80 mV with trains of test pulses (Fig. 5A). Nisoldipine reduced I_Ba of (+/+) and (+/) cardiomyocytes to 23 ± 3.7% (n = 10) and 27 ± 3.3% (n = 8), respectively. In contrast, I_Ba of (/) cells was reduced only to 65 ± 3.3% (n = 11) of the control. A shift of the HP from 80 to 40 mV reduced I_Ba to zero in all three genotypes. I_Ba recovered to 80% of the previous value in each cell line after reversal of the HP from 40 to 80 mV. The voltage-dependent reversibility of the block indicated that nisoldipine bound preferentially to the inactivated state of the channel, a phenomenon described extensively for the Ca_v1.2 channel (see Refs. 3, 5, and 20 and references cited therein). The affinity of nisoldipine to block I_Ba at a HP of 80 mV was determined from dose-inhibition curves (Fig. 5B) that were fitted to the Hill equation. The calculations yielded a low (0.1 µM) and a high (3 µM) IC₅₀ value for (+/+) and (+/) cells and only a high (1.1 µM) IC₅₀ value for (/) cells (Table I). The inhibition curves were well fitted with a Hill coefficient of 1.0 suggesting that nisoldipine blocked two independent currents in the (+/+) and (+/) cells. Considering the relative variability introduced by the calculation method, we suggest that the high IC₅₀ values were not different between the three genotypes and that the real high IC₅₀ value is close to 1 µM.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Nisoldipine block of IBa from (+/+), (+/), and (/) cardiomyocytes. A, time course and voltage dependence of block. Filled and open symbols represent IBa of a (+/+) and (/) cardiomyocyte under control conditions ( and), in the presence of 1 µM nisoldipine at HP of 80 mV ( and ), and at HP of 40 mV ( and ). Current was determined as current amplitude plus nisoldipine divided by the amplitude in the absence of nisoldipine. Inset, representative current traces (scale bars, 10 ms and 200 pA). B, concentration-inhibition relations. Data points are the mean ± S.E. (n = 4-9 per point). The lines are the fits obtained by the Hill equation. Data for (/) cardiomyocytes were fitted with a one-component Hill equation, whereas the data for (+/+) and (+/) cardiomyocytes, the (+/+) cells in the late stage (day 15.5 p.c.), and the Cav1.3 knockout cells at day 12.5 p.c. were fitted with a two-component Hill equation. , Cav1.2 +/+ cells at day 12.5 p.c.; , Cav1.2 ± cells at day 12.5 p.c.; , Cav1.2 / cells at day 12.5 p.c.; , Cav1.2 +/+ cells at day 15.5 p.c.; , Cav1.3 / cells at day 12.5 p.c.

L-type Ca_v1.2 channels have been reported to be blocked by nisoldipine with IC₅₀ values around 10 nM (21) suggesting that day 12.5 p.c. wild type myocytes expressed a relative low affinity L-type Ca_v1.2 channel. This low affinity is apparently a developmental property of the mouse heart, since day 15.5 p.c. wild type myocytes were blocked by nisoldipine with IC₅₀ values of 10 nM and 5.7 µM (Fig. 5B and Table I). The high (10 nM) and intermediate (100 nM) DHP sensitivity was most likely caused by the expression of different splice variants of the Ca_v1.2 gene (21-25). Ca_v1.2 channels that contain the sequence of exon 21 have a lower affinity for DHPs than those containing the alternatively used exon 22 (23, 25). In agreement with the increase in the affinity between day 12.5 and 15.5 p.c., the relative abundance of exon 22 mRNA increased and that of exon 21 decreased in wild type cardiomyocytes between these days. These results confirmed that embryonic cardiomyocytes express L-type Ca_v1.2 channels with different DHP sensitivity in the nanomolar range. In addition to the Ca_v1.2 channel, embryonic cardiomyocytes express a second L-type like calcium channel which is also present in the Ca_v1.2 (/) cells and has a nisoldipine affinity in the µmolar range. The current of (+/+) and (/) cells was blocked by high concentrations of mibefradil with IC₅₀ values of 4.4 and 3.2 µM (n = 3-4 cells for each genotype), respectively. Mibefradil blocks current through the Ca_v3.1 T-type channel (26) and the Ca_v1.2 L-type channel (27) at 0.2 and 4.3 µM, respectively.

Expression of Other L-type Calcium Channels in the Embryonic Heart-- The genes for four L-type calcium channels have been identified as follows: the skeletal muscle Ca_v1.1 (_1S), the cardiac Ca_v1.2 (_1C), the neuro-endocrine Ca_v1.3 (_1D), and the retinal Ca_v1.4 (_1F) channel. RT-PCR amplification of these channels showed that wild type embryonic murine heart expressed the mRNA for Ca_v1.1, Ca_v1.2, and Ca_v1.3. Amplification with L-type channel-specific primers allowed the amplification of the deleted exon 14 and 15 mRNA in (/) cardiomyocytes yielded Ca_v1.1- and Ca_v1.3-specific amplicons in a ratio of 1:10. No specific amplicon was obtained for Ca_V1.4. In addition, Ca_V1.4 mRNA was detected in the adult eye but not the heart of embryos by in situ hybridization with a Ca_V1.4-specific probe (data not shown). The expression of Ca_v1.1 has been reported previously in embryonic heart cell lines (28, 29). However, the possibility that Ca_v1.1 caused the rhythmic activity was rejected, since no specific protein band was detected by Western blot and the fast activation kinetics of the low affinity L-type like channel were not in line with those of a skeletal muscle calcium channel (see Fig. 4). Adult hearts express the Ca_v1.3 channel (13-15). Deletion of the Ca_v1.3 gene caused cardiac arrhythmia (18). Therefore, day 12.5 p.c. Ca_v1.3 (/) heart cells were analyzed. These cells had a regular Ca_v1.2 L-type I_Ba that was blocked half-maximally at 3 nM nisoldipine (Fig. 5B and Table I). However, these cells had still the second I_Ba that was blocked half-maximally at 0.7 µM nisoldipine, which value is very close to the IC₅₀ value of Ca_v1.2 (/) cells. These findings strengthen the hypothesis that the low affinity L-type like I_Ba was caused by a channel not identified so far.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Five conclusions can been drawn from this study as follows. (a) Mouse embryos develop apparently normal in the absence of the Ca_v1.2 gene up to day 12.5 p.c. (b) An intact Ca_v1.2 gene is required for embryo development after day 13 p.c. It is unknown why the Ca_v1.2 channel is needed for development. Other HVA calcium channels (Ca_v1.1, Ca_v1.3, Ca_v1.4, Ca_v2.1, and Ca_v2.3) are not necessary for embryonic and fetal development (18, 30-34). (c) Hearts contract in the absence of an intact Ca_v1.2 gene at day 12.5 p.c. (d) Contraction requires influx of Ca²⁺ through an unidentified L-type like calcium channel. (e) This channel is not the Ca_v1.3 L-type channel. These conclusions are supported by two independent mouse lines in which different exons of the Ca_v1.2 gene were destroyed and a mouse line in which the Ca_v1.3 gene was inactivated.

The L-type like channel cannot be an alternatively spliced product of the Ca_v1.2 gene. In mouse line B, exons 14 and 15 were deleted that code for part of the channel pore. Thus, a hypothetical channel would not contain a pore and should, therefore, be non-conducting. Furthermore, the aberrant RNA splicing caused a frameshift that would yield a truncated channel with no pore region. In mouse line A, an unexpected splicing event from exon 2 to exon 4 would lead to a frameshift and stop in exon 4. The L-type like channel is also not coded for by the Ca_v1.3 gene, since it was still present in Ca_v1.3 knockout embryos. The mRNA of the skeletal muscle Ca_v1.1 channel was detected in embryonic mouse cardiac cells. Several lines of evidence suggest that the L-type like channel was not the Ca_v1.1 channel. 1) The activation kinetics of this channel were much faster than those reported for the calcium channel of developing murine skeletal muscle (30). 2) Mice in which the Ca_v1.1 gene is disrupted die at birth but develop normally until birth (30). Therefore, it is very unlikely that the L-type like current was caused by the Ca_v1.1 channel. This current was also not caused by the Ca_v1.4 channel. This channel has been reported to be specifically expressed in the retina (33, 34). In line with this expression pattern, the mRNA of the Ca_v1.4 gene was not detected in the embryonic heart. Its sensitivity against nisoldipine distinguishes the L-type like channel from the brain channels Ca_v2.1, Ca_v2.2, and Ca_v2.3. Furthermore, disruption of the Ca_v2.1 and Ca_v2.3 gene leads to viable pups (31, 32) arguing against an essential role of these channels for cardiac rhythm generation.

The low affinity for nisoldipine would be in line with reports that some T-type calcium currents are blocked at high concentrations by several DHPs (35). Ca_v3.1 and Ca_v3.2 are expressed in the heart (36). However, the Ca²⁺-dependent and slow inactivation of the L-type like channel has not been observed with T-type channels. Furthermore, the expressed Ca_v3.1 channel is inhibited at submicromolar concentrations of mibefradil (37), is not activated by Bay K 8644, and is inhibited marginally by 1 µM (+)-isradipine or 10 µM nifedipine (37). Similarly, amlodipine blocked the expressed Ca_v3.2 channel with an IC₅₀ of 31 µM (38). These considerations strengthen the notion that the L-type like current was caused by an unknown calcium channel.

The preliminary characterization of the new current showed that it has many properties of a classical HVA L-type calcium channel. The current differs from the classical L-type calcium channel by its low affinity for the DHP nisoldipine at negative membrane potentials but resembles Ca_v1.2 channels by the voltage dependence of the block (see Fig. 5A). This property was responsible for the inhibition of cardiac contraction by 1 µM nisoldipine, since at depolarized membrane potentials which are necessary for channel opening the affinity for nisoldipine increased significantly. The affinity of the Ca_v1.2 channel for DHPs is lowered 100-fold by mutation of Tyr-1485, Met-1486, and Ile-1493 in the IVS6 segment (39) and is lost upon mutation of Thr-1061 in the IIIS5 segment (see Refs. 3, 5, and 20). It is possible that the new channel has an altered IVS6 segment but retained other parts of the DHP-binding site.

In conclusion, the present study shows that early cardiac rhythm generation required an unidentified L-type like calcium channel. This channel has many properties of the well characterized Ca_v1.2 channel. Presumably, this similarity has prevented so far its identification in embryonic cells. The channel was also present in fetal hearts and may be present in adult hearts. Its functional significance beyond embryonic development remains to be established and will require the identification of its structure. The Drosophila melanogaster and Caenorhabditis elegans genome contain calcium channel genes of unknown function (40). One may speculate that the L-type like channel is encoded by a similar gene in the mouse.

	ACKNOWLEDGEMENTS

We thank S. Kampf and A. Ebner for excellent technical assistance. The Ca_v1.2 knockout mouse A was provided by C. Seisenberger, and the Ca_v1.2 knockout mouse B was provided by V. Specht; electrophysiology was performed by A. Welling.

	FOOTNOTES

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft, Fond zur Förderung der wissenschaftlichen Forschung Grant P12641-MED, the Östereichische Nationalbank, the VW-Stiftung, and Fond der Chemischen Industrie.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.

^¶ To whom correspondence should be addressed. Tel.: 49-89-4140-3260; Fax: 49-89-4140-3261; E-mail: pharma@ipt.med.tu-muenchen.de.

Published, JBC Papers in Press, September 5, 2000, DOI 10.1074/jbc.M006467200

	ABBREVIATIONS

The abbreviations used are: HVA, high voltage-activated; TTX, tetrodotoxin citrate; RT-PCR, reverse transcriptase-polymerase chain reaction; kb, kilobase pair; p.c., postcoitum; HP, holding potential; DHP, dihydropyridine; bp, base pair; HSV-TK, herpes simplex virus type I-thymidine kinase cassette; pF, picofarad; ES, embryonic stem.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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