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Am J Physiol Heart Circ Physiol 290: H1165-H1171, 2006. First published October 21, 2005; doi:10.1152/ajpheart.00821.2005
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Calcium receptor is functionally expressed in rat neonatal ventricular cardiomyocytes

Jacob Tfelt-Hansen,1,3,4 Jakob Lerche Hansen,2,3 Sanela Smajilovic,1,3 Ernest F. Terwilliger,5 Stig Haunso,1,3 and Soren P. Sheikh2,3

1Laboratory of Molecular Cardiology and 2Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Copenhagen University Hospital, Rigshospitalet, Copenhagen; 3Copenhagen Heart Arrhythmia Research Center, Copenhagen, Denmark; 4Division of Endocrinology, Diabetes and Hypertension, Department of Medicine and Membrane Biology Program, Brigham and Women's Hospital and Harvard Medical School, Boston; and 5Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Institutes of Medicine, Boston, Massachusetts

Submitted 3 August 2005 ; accepted in final form 18 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Both intra- and extracellular calcium play multiple roles in the physiology and pathophysiology of cardiomyocytes, especially in stimulus-contraction coupling. The intracellular calcium level is closely controlled through the concerted actions of calcium channels, exchangers, and pumps; however, the expression and function(s) of the so-called calcium-sensing receptor (CaR) in the heart remain less well characterized. The CaR is a seven-transmembrane receptor, which, in response to noncovalent binding of extracellular calcium, activates intracellular effectors, including G proteins and extracellular signal-regulated kinases (ERK1/2). We have shown that cultured neonatal cardiomyocytes express the CaR messenger RNA and the CaR protein. Furthermore, increasing concentrations of extracellular calcium and a type II CaR activator "calcimimetic" caused inositol phosphate (IP) accumulation, downregulated tritiated thymidine incorporation, and supported ERK1/2 phosphorylation, suggesting that the CaR protein is functionally active. Interestingly, the calcimimetic induced a more rapid ERK1/2 phosphorylation than calcium and left-shifted the IP concentration-response curve for extracellular calcium, supporting the hypothesis that CaR is functionally expressed in cardiac myocytes. This notion was underscored by studies using a virus containing a dominant-negative CaR construct, because this protein blunted the calcium-induced IP response. In conclusion, we have shown that the CaR is functionally expressed in neonatal ventricular cardiomyocytes and that the receptor activates second messenger pathways, including IP and ERK, and decreases DNA synthesis. A specific calcium-sensing receptor on cardiac myocytes could play a role in regulating cardiac development, function, and homeostasis.

calcium-sensing receptor; DNA synthesis; extracellular signal-regulated kinase 1/2; inositol phosphate; G protein-coupled receptor; seven-transmembrane receptor

THE PIVOTAL ROLE OF CALCIUM in the normal physiology and pathophysiology of the cardiomyocyte is well known (41). Both intra- and extracellular calcium play multiple roles in cardiac myocytes, especially in stimulus-contraction coupling. In normal physiology, calcium acts via rapid changes in intracellular calcium as a result of changes in the activities of calcium channels, exchangers, and pumps; i.e., calcium is a so-called second messenger. However, it is conceivable that calcium also may act as a first messenger through an extracellular signaling mechanism. The calcium receptor (CaR) senses the extracellular calcium level and can tell the cell the exact level of calcium outside the cell. The change in calcium levels will be transformed into activation of intracellular signaling pathways that will induce downstream effects on the cell.

The CaR belongs to family C II of the superfamily of seven-transmembrane (7TM) receptors, also termed G protein-coupled receptors (5). The human CaR is 1,078 amino acid residues long and, like other 7TM receptors, has three structural domains: 1) an unusually large extracellular domain, characteristic of the family C 7TM receptors; 2) a transmembrane domain; and 3) an intracellular domain, which is the hydrophilic COOH terminus of the protein. A wide range of intracellular signaling pathways of the CaR have been identified, including phospholipase C (PLC), which stimulates inositol phosphate (IP) production, and mitogen-activated protein kinases (28). Although the major ligand of the CaR is extracellular calcium, it is a promiscuous receptor that recognizes many ligands (28). CaR agonists are divided into type I, which are direct agonists, and type II, which work as allosteric modulators; i.e., they require the presence of calcium to activate the receptor. The best described CaR function is its regulation of the secretion of parathyroid hormone (4), a key calcium-regulating hormone in the calcium homeostatic system. Type II CaR agonists, termed calcimimetics, have been introduced in the treatment of uremic hyperparathyroidism (24). The calcimimetics bind to the transmembrane domain of the CaR and increase its sensitivity to the calcium ion (24). AMG 073 is currently the drug of choice for clinical use because of pharmacokinetic considerations. Interestingly, Holstein et al. (14) reported that calcimimetic-induced extracellular signal-related kinase (ERK1/2) activation was slower and more sustained in human embryonic kidney (HEK-293) cells stably transfected with the CaR (HEK CaR) compared with stimulation by calcium. The CaR has been shown to be expressed in adult rat cardiomyocytes (35). The authors found that challenging the cells with type I CaR agonist induced the production of IP, suggesting that the CaR is linked to the PLC pathway. Furthermore, a recent study (36) elegantly showed that the CaR also is present in the endothelial cells of mesentery and coronary arteries and that stimulation of the receptor induced hyperpolarization of the vascular smooth muscle cells. This study indicated that CaR is present in the heart. The function of the CaR in heart in normo- and pathophysiology remains to be clarified.

It is thought that neonatal cardiomyocytes are terminally differentiated, although this view has recently been challenged (6, 7). However, DNA synthesis is observed in neonatal cardiomyocytes undergoing hypertrophy (7), perhaps because hypertrophy of different cells involves a partial progression through the cell cycle (3). Other G protein-coupled receptors such as angiotensin receptor type 1 and adrenergic receptors are known to induce hypertrophy in this primary cell culture (6).

We hypothesized that the CaR also is functionally expressed in rat neonatal cardiomyocytes and that it regulates DNA synthesis. Thus the aim of this study was to characterize the CaR in myocardial cells, to identify its downstream intracellular signaling pathways, and, finally, to investigate whether the CaR plays a role in regulating DNA synthesis of cardiomyocytes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Polyclonal antisera against phosphorylated and nonphosphorylated ERK1/2 were purchased from New England Biolabs (Beverly, MA). The enhanced chemiluminescence kit Supersignal was purchased from Pierce (Rockford, IL), and other reagents were obtained from Sigma Chemical (St. Louis, MO).

Neonatal ventricular myocyte culture. Neonatal ventricular cardiomyocytes were prepared from 1- to 5-day-old neonatal Wistar rats (University of Copenhagen, Copenhagen, Denmark) by modification of a previously described method (27). All protocols were in accordance with institutional guidelines and approved by the Danish Animal Experimentation Inspectorate under the Ministry of Justice. Cells were plated at a density of 5 x104 cells/cm2 in minimal essential medium (MEM) supplemented with 1% L-glutamine, 0.1 mM bromodeoxyuridine, 0.15 mM vitamin B12, 1 µg/ml insulin, and 6,250 U/ml penicillin. Cell culture plates were precoated with 8% FCS for 5 h at 37°C.

Immunofluorescence. Cells cultured on coverslips for 3 days were fixed with 4% formaldehyde. To detect CaR-positive cells, we performed immunohistochemical studies using a monoclonal anti-CaR LRG antibody (peptide sequence used to raise this antibody was LRGHEESGDRFSNSSTAF) and a polyclonal anti-CaR FF7 antibody (peptide sequence used to raise this antibody was HNGFAKEFWEETFNC) as previously described (20). Actin was stained with 0.4 µg/ml tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin (Sigma). Alexa Fluor 488 conjugated to goat anti-mouse secondary antibody was used for the LRG antibody, and Alexa Fluor 546 conjugated to goat anti-rabbit secondary antibody was employed for the FF7 antibody (Alexa-conjugated antibodies were both from Molecular Probes, Eugene, OR). Fluorescence images were collected with a Zeiss LSM 510 Meta confocal microscope (Jena, Germany) at the Harvard Center for Neurodegeneration and Repair (Boston, MA).

RT-PCR. One-step RT-PCR (kit from Qiagen, Santa Clarita, CA) was used for determining the presence of CaR transcript(s) with the use of a pair of primers yielding a 331-bp product, as predicted from the rat CaR cDNA (NM_ 016996). Rat kidney CaR sense (bp 3172–3191), 5'-AACACCATTGAGGAAGTCCG-3', and antisense primers (bp 3482–3503), 5'-GAGAAGGTGACCGTACCACTGC-3', were used for the reactions (26). We used the following procedure for RT-PCR (32): in brief, cellular RNA was isolated (11) with the TRI reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. Total RNA (2 µg) was mixed with a master cocktail containing RT-PCR buffer, sense and antisense CaR primers, dNTPs, RNase inhibitor, and an enzyme mixture containing reverse transcriptase (Omniscript and Sensiscript) and HotStart Taq DNA polymerase at the concentrations recommended by the manufacturer (Qiagen) in a final volume of 50 µl (33). The temperature-cycling protocol was as follows: 30 min at 50°C for the RT reaction, followed by denaturation and activation of the HotStart DNA polymerase for 15 min at 95°C, and PCR amplification (30 s at 94°C, 30 s at 58°C, and 1 min at 72°C for 40 cycles). A final extension for 10 min at 72°C was performed after 40 cycles. To eliminate amplification from contaminating genomic DNA, we omitted reverse transcriptase from the RT-PCR as a negative control reaction for each sample. RT-PCR products were fractionated on 1.5% agarose gels. The presence of a 331-bp amplified product was indicative of a positive PCR arising from the presence of a CaR-related sequence within the cDNA.

Western blot analysis for phosphorylated and total ERK1/2. For determination of ERK1/2 phosphorylation and total ERK1/2, a monolayer of neonatal ventricular cardiomyocytes was plated at 1.5 million cells/well on six-well plates. After 72 h in MEM, cells were incubated for 18 h in serum-free, calcium-free Dulbecco's modified Eagle's medium (DMEM) containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl2. This medium was removed and replaced with the same medium supplemented with 6 mM CaCl2 or with 3 µM AMG 073 for the indicated periods and then lysed. Protein concentrations were measured with the Micro BCA protein kit (Pierce). SDS-PAGE and immunoblotting were performed as described previously (31), and the bands were visualized with the ECL system (Amersham Biosciences).

IP assay. Briefly, neonatal ventricular cardiomyocytes were plated in 12-well plates (106 cells/well) and grown as described in Neonatal ventricular myocyte culture in medium containing 5% FCS for 72 h. Next, 16 h before stimulation, the medium was replaced with inositol-free DMEM supplemented with 5% FCS and myo-[2-3H]inositol (1 µCi/ml; Amersham). The accumulation of IPs was measured as described previously (12, 16).

Infecting neonatal ventricular cardiomyocytes with CaR constructs in recombinant adeno-associated virus. High-efficiency gene transfer into neonatal ventricular cardiomyocytes was accomplished using a recombinant adeno-associated virus (rAAV)-based method. The CaR sequence with a naturally occurring dominant negative mutation (R185Q), as well as the same vector containing the cDNA encoding the beta-galactosidase gene, was under the control of a cytomegalovirus immediate-early promoter element and was packaged as previously described (37). The beta-galactosidase served as the control for nonspecific effects of rAAV infection. Cells were seeded (0.2 million cells/well) in 12-well plates in 0.5 ml of growth medium and cultured for 48 h. About 1,000 virus particles/cell were used to infect each well as previously described (30). Cells were washed once with serum-free MEM. Virus particles were then added, and the culture was incubated for 90 min in serum-free medium at 37°C in a cell culture incubator. Equal volumes of MEM containing 20% serum were added to the cells to achieve a final serum concentration of 10%. The cells were then cultured for 48 h, and experiments with calcium were performed as described in IP assay.

DNA synthesis assay. Neonatal ventricular cardiomyocytes were plated at 0.2 million cells/well in 12-well plates in MEM containing 0.1% FBS. The cells were cultured for 48 h, and the medium was changed to serum-free, calcium-free DMEM containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl2. Calcium (0.5–10 mM) or angiotensin (10–7 M) was added alone or with calcimimetic. After 24 h, [3H]thymidine (1 µl/ml; 50 µCi/ml) was added, and the cells were cultured again for 20 h. Twenty hours after application of the [3H]thymidine, the cells were assayed as described previously (29).

Data analysis. All data were analyzed using GraphPad Prism. For the IP measurements, all experimental values were compared with the described controls using a two-tailed, unpaired Student's t-test. Data for DNA synthesis were analyzed using a two-way ANOVA followed by a Tukey-Kramer multiple comparison post hoc test. Data are presented as means ± SE. A P value <0.05 was considered to represent a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CaR mRNA and protein are present in neonatal ventricular cardiomyocytes. The existence of the CaR in cardiomyocytes could have a substantial impact on our view of the mechanisms underlying the effects that extracellular calcium has on the heart. We therefore investigated whether the mRNA of the CaR was expressed in neonatal ventricular cardiomyocytes. Using one-step RT-PCR with previously published primers (26), we showed the amplification of a 331-bp product (Fig. 1A); sequencing of this cDNA product revealed it to be >99% homologous to the cDNA for the rat CaR. To document CaR protein expression, we performed immunostaining on neonatal ventricular cardiomyocytes with two different anti-CaR antibodies. The two antibodies, both targeting the extracellular domain of the CaR, bound to the neonatal ventricular cardiomyocytes as visualized by confocal microscopy (Fig. 1, B and C). Immunostaining was observed both on the cell surface and intracellularly. Only faint staining occurred when the primary antibodies were omitted.


Figure 1
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  		Fig. 1. Calcium-sensing receptor (CaR) is expressed in neonatal ventricular cardiomyocytes at mRNA and protein levels. A: RT-PCR revealed a product arising from CaR transcripts in neonatal ventricular cardiomyocytes (NCM). RT-PCR was performed on RNA extracted from NCM (lanes 4 and 5) or rat kidney (lanes 2 and 3), as described in MATERIALS AND METHODS, by using a primer pair specific for the sequence of the rat kidney CaR. A 331-bp amplified fragment (lanes 2 and 4) is indicative of product arising from authentic CaR-derived transcript(s) in NCM and rat kidney, respectively. No such product was apparent when the reverse transcriptase was omitted from the RT reaction (lanes 3 and 5). Lane 1 is a DNA ladder. B and C: determination of specificity of immunoreactivity of NCM with anti-CaR antibodies. Cells were incubated with polyclonal anti-CaR FF7 antibody (B) or monoclonal anti-CaR LRG antibody (C) against the CaR, respectively, with Alexa Fluor 488 conjugated to goat anti-mouse secondary antibody used for the LRG antibody and Alexa Fluor 546 conjugated to goat anti-rabbit secondary antibody employed for the FF7 antibody. 1 AB, primary antibody; 2 AB, secondary antibody.

 
CaR promotes production of IP. The CaR is linked to the PLC/IP pathway in most cells (28). We therefore investigated whether stimulation with calcium would induce the accumulation of IPs in neonatal ventricular cardiomyocytes. In a concentration-dependent manner, 3, 6, and 10 mM extracellular calcium (Formula) increased IP accumulation by 16 ± 2, 31 ± 2, and 51 ± 2% (means ± SE), respectively, compared with neonatal ventricular cardiomyocytes treated with 0.5 mM Formula(P < 0.005; Fig. 2A). Because the action of calcium may not be specific for the CaR, we investigated whether a selective allosteric activator of the receptor, AMG 073 (24), which is currently used in the treatment of uremic hyperparathyroidism, would potentiate the calcium response. As shown in Fig. 2B, the addition of 3 µM AMG 073 increased the IP accumulation by 33 ± 2% at 3 mM Formula and by 50 ± 2% at 6 mM Formula, compared with 16 ± 2% at 3 mM Formula alone and 31 ± 2% at 6 mM Formula alone (P ≤ 0.05). The effect of the calcimimetic, therefore, was to shift the concentration-response curve for calcium to the left, effectively sensitizing the CaR to Formula. Thus these results strongly support the CaR as a mediator of the Formula-induced IP accumulation.


Figure 2
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  		Fig. 2. Calcium and calcimimetic (AMG 073) induce inositol phosphate (IP) accumulation. NCM were plated in 12-well plates (106 cells/well) and grown as described in medium containing 5% FCS for 72 h, and then, 16 h before stimulation, the medium was replaced with inositol-free DMEM supplemented with 5% FCS and myo-[2-3H]inositol (1 µCi/ml). The effects of incubation with calcium alone (A) or with AMG 073 (B) on IP accumulation in NCM were determined. Angiotensin II (ANG II; 10–7 M) was added as positive control. Data from 5 individual experiments, each performed in triplicate, represent normalized average values (means ± SE) with reference to the basal condition in the presence of 0.5 mM extracellular calcium Formula). **P < 0.01 compared with 0.5 mM Formula. #P < 0.05 compared with 3 mM Formula in absence of AMG. ^P < 0.05 compared with 6 mM Formula in absence of AMG.

 
Another approach to proving the involvement of the CaR is to infect the neonatal ventricular cardiomyocytes with a dominant negative CaR and compare the IP response to calcium with that of control neonatal ventricular cardiomyocytes infected with the vector expressing beta-galactosidase. As shown in Fig. 3, infecting the neonatal ventricular cardiomyocytes with adeno-associated virus containing the dominant negative CaR [R185Q (1)] significantly inhibited the Formula-induced IP response. At 6 mM Formula, IP accumulation was stimulated by 6 ± 6% in cells infected with dominant negative CaR, which was blunted relative to the response in cells infected with beta-galactosidase (35 ± 9% increase, P ≤ 0.05; Fig. 3). Stimulation of IP accumulation with 10 mM Formula was likewise blunted in the neonatal ventricular cardiomyocytes infected with the dominant negative CaR [e.g., 13 ± 5% increase in IP accumulation compared with a 42 ± 7% increase in neonatal ventricular cardiomyocytes infected with beta-galactosidase (P < 0.05)]. These results further support the notion that the CaR mediates the effects of Formula on IP accumulation in neonatal ventricular cardiomyocytes.


Figure 3
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  		Fig. 3. Dominant negative CaR attenuates calcium-induced increases in IP accumulation. NCM were plated in 12-well plates (106 cells/well) and grown as described in medium containing 5% FCS for 72 h, and then, 16 h before stimulation, the medium was replaced with inositol-free DMEM supplemented with 5% FCS and myo-[2-3H]inositol (1 µCi/ml). NCM in 12-well plates were infected with either the dominant negative (DN) R185Q CaR or the vector expressing the beta-galactosidase protein 48 h before stimulation, as described in MATERIALS AND METHODS. IPs were measured after incubation of the cells with 0.5, 6, or 10 mM Formula in serum-free medium. Data from 5 individual experiments, each performed in triplicate, represent normalized average values (means ± SE) with reference to the basal condition in the presence of 0.5 mM Formula. Subsequently, 1-tailed, paired Student's t-tests were performed. aP < 0.001 compared with 0.5 mM Formula with beta-galactosidase. bP < 0.05 compared with 6 mM Formula with beta-galactosidase. cP < 0.05 compared with 10 mM Formula with beta-galactosidase.

 
CaR stimulation in neonatal ventricular cardiomyocytes induces ERK1/2 activation. ERK1/2 is a component of an important intracellular signaling pathway from the cell surface to the nucleus, which is known to be downstream of the CaR and is a key player in cell cycle regulation. ERK1/2 is activated by phosphorylation of the protein. Previously, we found that the EC50 for CaR-stimulated parathyroid hormone-related protein (PTHrP) secretion in rat H-500 Leydig cancer cells was 3–4 mM calcium (26). In a pilot study investigating the effect of Formula on ERK1/2 phosphorylation, the neonatal ventricular cardiomyocytes showed a concentration-dependent response between 0.5 and 10 mM Formula, with a maximal response at 6 mM Formula and higher. Therefore, we utilized 6 mM Formula to stimulate ERK1/2 in subsequent experiments. Incubating the neonatal ventricular cardiomyocytes with 6 mM Formula for 0–30 min induced the phosphorylation of ERK1/2 with a maximum at 5–10 min. Total ERK was the control for equal loading of protein in the lanes (Fig. 4). To establish whether the effects of calcium on ERK1/2 activation were mediated through the CaR, we used 3 µM AMG 073, the type II CaR activator, in the presence of 0.5 mM Formula. AMG 073 indeed induced ERK1/2 phosphorylation, but surprisingly, the response occurred more rapidly than with calcium alone, with a maximum as early as 2–5 min.


Figure 4
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  		Fig. 4. Calcium and calcimimetic (AMG 073) induce ERK activation. NCM were plated in 6-well plates for 72 h and starved in serum-free medium for 18 h. Cells were stimulated in the presence of 6 mM Formula and AMG 073 at the indicated times. In gels for phosphorylated ERK (pERK), the top band is ERK1 and the bottom band is ERK2. Western blotting shows total ERK (tERK) and pERK stimulation by 6 mM Formula with a maximal activation at 5–10 min and by AMG 073 with a maximal activation at 2–5 min. The Western blots are representative of 5 experiments.

 
Calcium induces a biphasic response in DNA synthesis in neonatal ventricular cardiomyocytes. Because activation of the CaR in neonatal ventricular cardiomyocytes leads to induction of ERK1/2 phosphorylation, we investigated a possible role of the CaR in stimulating DNA synthesis. Incubating the neonatal ventricular cardiomyocytes with 0.5–10 mM Formula induced a biphasic response in DNA synthesis, as assessed by [3H]thymidine incorporation. A concentration of 3 mM Formula increased DNA synthesis in neonatal ventricular cardiomyocytes by 30 ± 8% compared with 0.5 mM Formula(P < 0.05; Fig. 5). This stimulation was as potent as that caused by 10–7 M angiotensin II. In contrast, at 6 and 7.5 mM Formula, DNA synthesis was reduced to 65 ± 9 and 61 ± 11%, respectively, compared with that at 0.5 mM Formula(P < 0.01 and P < 0.001, respectively). There was no effect on cell number at any of the calcium concentrations used, as assessed by cell counting (data not shown).


Figure 5
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  		Fig. 5. Calcium downregulates DNA synthesis in NCM. NCM were plated in 12-well plates for 96 h and starved in serum-free media for 24 h. Cells were stimulated in the presence of [3H]thymidine and the indicated concentrations of Formula or 10–7 M ANG II for 18 h. Results are pooled data from 6 independent experiments, each performed in triplicate. *P < 0.05;**P < 0.01; ***P < 0.001 compared with cells treated with 0.5 mM Formula.

 
CaR type II agonist suppresses DNA synthesis in neonatal ventricular cardiomyocytes. We again utilized the type II calcimimetic AMG 073 to assess the role of the CaR in the biphasic actions of calcium on DNA synthesis in neonatal ventricular cardiomyocytes. In the presence of 3 µM AMG 073, DNA synthesis at 0.5, 3, or 6 mM Formula was reduced to 39 ± 4, 61 ± 9, and 12 ± 5%, respectively, compared with that at 0.5 mM Formula(P < 0.01, P < 0.01, and P < 0.001, respectively; Fig. 6). Furthermore, this effect appeared to be concentration dependent, because 3 µM AMG 073 was more potent than 0.3 µM AMG 073 at 0.5 mM Formula.


Figure 6
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  		Fig. 6. Calcimimetic (AMG 073) inhibits DNA synthesis in NCM. NCM were plated in 12-well plates for 96 h and starved in serum-free medium for 24 h. Cells were stimulated in the presence of [3H]thymidine and the indicated concentrations of Formula or with calcimimetic (AMG 073) at 0.3 or 3 µM for 18 h. Results are pooled data from 6 independent experiments, each performed in triplicate. **P < 0.01; ***P < 0.001 compared with cells treated with 0.5 mM Formula.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Calcium is pivotal for the function of the heart. The importance of intracellular calcium has been a major focus of the field of cellular cardiology. Therefore, our finding that the CaR is present on the cell membrane of neonatal ventricular cardiomyocytes supports the hypothesis that calcium is not only an intracellular second messenger in the heart but also a first messenger acting from outside the cardiomyocyte. We established, using one-step RT-PCR, that the CaR is expressed on neonatal ventricular cardiomyocytes at the messenger RNA level. The presence of CaR protein was demonstrated using two antibodies targeting the extracellular domain of the receptor. Although the technique used presently was not meant to establish the exact cellular localization of the CaR, both antibodies revealed immunoreactivity not only on the plasma membrane but also intracellularly in the neonatal ventricular cardiomyocytes, which suggests potentially intriguing roles of the CaR within these cells. In the calvarial osteoblast (8) and the osteoblastic cell line MG-63 (39), the CaR also was found to be present on the cell membrane and intracellularly as assessed using confocal microscopy.

Wang et al. (35) previously reported that rat ventricular adult cardiomyocytes express the CaR at the mRNA and protein levels. Stimulation with calcium as well as with gadolinium and spermine, both type 1 agonists of the CaR, in ventricular adult cardiomyocytes induced intracellular calcium spikes. The effects of calcium could be blocked by the sarco(endo)plasmic reticulum Ca2+-ATPase blocker thapsigargin and the phosphatidylinositol-specific PLC inhibitor U-73122. Stimulation of the ventricular adult cardiomyocytes with calcium and the type 1 CaR agonists gadolinium and spermine also stimulated the accumulation of IPs. Therefore, we next assessed whether the CaR was active in neonatal ventricular cardiomyocytes by investigating the IP production after calcium stimulation. Our results revealed an Formula-induced, concentration-dependent increase in IP levels in neonatal ventricular cardiomyocytes. IP accumulation upon stimulation of the CaR by its ligands was first demonstrated in dispersed parathyroid cells (4). Later, it was shown that in both HEK CaR cells and parathyroid cells, the phospholipases A2, C, and D were activated by the CaR (18). The CaR-mediated PLC activation leading to PI hydrolysis is most likely mediated through G{alpha}q11, because pertussis toxin did not inhibit the calcium-stimulated IP response in HEK CaR cells. CaR-induced accumulation of IP is well documented to induce the release of calcium into the cytosol (5), i.e., calcium as a second messenger, and activation of this pivotal intracellular pathway has been observed in several cell types. Therefore, when studying the CaR, the use of Formula as a ligand is not sufficient to claim the involvement of the receptor, because the effects of Formula may occur through the effect of calcium as a second messenger. We used two approaches to verify that the observed IP accumulation was CaR dependent. First, we utilized a calcimimetic, a newly developed drug targeting the transmembrane domain of the CaR, to prove the CaR's role as a mediator of the effects of Formula. Our results showed that, at 3 and 6 mM Formula, the calcimimetic augmented the effects of calcium on IP accumulation, effectively left-shifting the relationship between Formula and IP accumulation. The IP response to 6 mM Formula with AMG 073 was comparable to that to angiotensin II. Another 7TM receptor, the thrombin receptor, has similar effects on IP3 (10). The fact that the thrombin and angiotensin II, two 7TM receptors coupled to G{alpha}q like the CaR, have a similar range of effects on IP accumulation favors the CaR as the mediator of the effects. The mediatory role of the CaR in calcium-induced IP accumulation in our study was further proven by infecting the neonatal ventricular cardiomyocytes with the dominant negative R185Q CaR with the use of the adeno-associated viral vector and comparing the effects of Formula on IP accumulation with those in cells infected with the control adeno-associated virus expressing beta-galactosidase, a protein approximately the same size as the CaR. Introducing the dominant negative CaR produced a downward and rightward shift in the concentration-response curve for calcium-induced IP accumulation. These results are similar to the effect of this mutant dominant negative CaR on the response of the wild-type CaR to Formula in transiently transfected HEK-293 cells (1). Similar to our results, inhibitory effects also were produced by the same dominant negative CaR in rat H-500 Leydig cancer cells, which likewise express the CaR endogenously, with regard to upregulation of inducible nitric oxide synthase and pituitary tumor-transforming gene (PTTG), a protooncogne, as well as calcium-induced PTHrP release (30–32).

Kifor et al. (19) showed that the CaR in HEK CaR and parathyroid cells activates ERK1/2. CaR activation of ERK1/2 also has been shown in cells expressing the CaR at lower levels, such as the PC-3 human prostate cancer and U87 astrocytoma cell lines and the Leydig cancer H-500 cells (31, 42, 43). In neonatal ventricular cardiomyocytes, we found calcium-induced activation of the ERK1/2 at 5–15 min, similar to the time course for its activation in HEK CaR and parathyroid cells. These kinetics of ERK1/2 activation can vary, however. In H-500 and PC-3 cells, the activation of ERK1/2 is delayed and sustained compared with that in neonatal ventricular cardiomyocytes and parathyroid cells (31, 42). These differences in the duration of ERK1/2 activation may produce different effects on the cell cycle, as observed in Swiss 3T3 fibroblasts (23). An interesting observation in the neonatal ventricular cardiomyocytes was that ERK1/2 activation was more rapid in response to the calcimimetic than with Formula alone. This observation, that Formula and calcimimetics have different effects on ERK1/2 activation, is supported by data from Holstein et al. (14), although these authors saw a delayed response and not a faster response as in our present study.

The CaR has been reported to be a regulator of the cell cycle. Stimulation of the CaR leads to growth arrest in colonic crypt cells, pancreatic carcinoma cells, and keratinocytes, whereas it induces proliferation in astrocytoma cells, osteoblasts, fibroblasts, myeloma, and ovarian surface cells (2, 9, 17, 21, 22, 25, 38, 40). We therefore investigated a possible role for the CaR in DNA synthesis in neonatal ventricular cardiomyocytes. At 3 mM Formula, DNA synthesis was upregulated, whereas at higher levels of Formula, DNA synthesis was downregulated. The effect of 3 mM Formula was not reproduced in the experiments in which AMG 073 was used. The same biphasic phenomenon is seen in chemotaxis when the angiotensin receptor Ia is activated by increasing concentrations of the agonist (15). On the contrary, the calcimimetic inhibited DNA synthesis at all levels of calcium. The difference in the effects of the two CaR ligands may be explained by differences in intracellular signal pathways that they activate, e.g., the duration of ERK1/2 activation. Alternatively, the effects of 3 mM Formula might not be mediated through the CaR.

Interestingly, in utero, the fetus is kept in a hypercalcemic state because of a placental calcium pump (34). This is important, because the cells studied presently are neonatal cardiomyocytes, so the neonatal ventricular cardiomyocytes experienced in utero a constant stimulus of the CaR. This stimulation in utero may be important in the development of the normal myocardium. Changes in Formula in the immediate vicinity of the cell membrane of the cardiomyocyte occur with every heart beat. In an elegant study, it was shown that intracellular signaling events can produce changes in Formula that are detected by the CaR on nearby cells (13). Thus, in theory, the CaR in cardiomyocytes may change its activity with every heart contraction. A recent report (36) showed that the endothelium of the porcine coronary artery expresses the CaR. The effects of the CaR were reported to be in hyperpolarization of vascular myocytes, most likely through a calcium-dependent potassium channel, suggesting that the CaR is important in regulating the tone of the coronary artery and, therefore, blood flow to the heart. Furthermore, it has been shown that the CaR also can regulate a calcium-dependent potassium channel in astrocytoma cells (43). Therefore, we can speculate that the CaR in the cardiomyocyte also regulates the potassium current, and thereby the membrane potential. The CaR may therefore play a role in the electrophysiology of the heart.

In conclusion, we have shown that the CaR, a 7TM receptor, is functionally expressed on the neonatal cardiomyocyte. Stimulating the CaR induced IP accumulation and ERK1/2 activation. DNA synthesis response to calcium was biphasic. Stimulating the CaR with calcimimetics induced a downregulation in DNA synthesis. Therefore, the extracellular calcium through the CaR in the cardiomyocytes should be regarded as a first messenger. Furthermore, the presence of a specific calcium-sensing receptor on cardiac myocytes could play a role in regulating cardiac development, function, and homeostasis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from Copenhagen Heart Arrhythmia Research Center, Statens Sundheds Videnskablige Forskningsråd, and the Danish Heart Association (to J. Tfelt-Hansen).


   ACKNOWLEDGMENTS
 
We thank Katrine Kastberg for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Tfelt-Hansen, Laboratory of Molecular Cardiology, Dept. of Cardiology, Univ. of Copenhagen, 20 Juliane Maries Vej, Section 9312, DK 2100 Copenhagen O, Denmark (e-mail: tfelt{at}dadlnet.dk)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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