Calcium Sensing Receptor Promotes Cardiac Fibroblast Proliferation and Extracellular Matrix Secretion

Cardiac fibroblasts are the main non-myocyte cell type in the heart. Under physiological circumstances, cardiac fibroblasts play an important role in maintaining homeostasis of the extracellular matrix, in addition to producing growth factors, cytokines and matrix metalloproteinases (MMPs) [1]. However, cardiac fibroblasts promote myocardial scarring and remodeling in injured hearts in ischemic, hypertensive and hypertrophic heart diseases. Excessive proliferation and migration of cardiac fibroblasts result in fibrosis [2,3]. Myocardial fibrosis can alter the mechanical properties of the hearts, impairing cardiac function and promoting heart failure [4]. Some evidence has shown that the cytosolic calcium ion ([Ca²⁺]_i) acts as a second messenger to regulate versatile cell functions including excitation-condition coupling, gene transcription, cell growth, differentiation and apoptosis. However, little information implies that the Ca²⁺ signaling pathway is involved in cardiac fibroblast proliferation and extracellular matrix (ECM) production [5].

Calcium sensing receptor (CaR), a G protein coupled receptor, activates the phospholipase C (PLC) signaling pathway, resulting in an increase in inositol phosphate (IP) accumulation and Ca²⁺ mobilization [6]. Our previous work demonstrated that CaR induced cardiomyocyte apoptosis through the mitochondrial and endo (sarco) plasmic reticulum apoptotic pathway in the failing heart. Moreover, our results showed obvious myocardial fibrosis following administration of a CaR activator [7]. However, at present, no evidence had shown that CaR was expressed in cardiac fibroblasts.

In this study, our results demonstrate that CaR is present in rat cardiac fibroblasts. We identified both the protein levels and the functional regulation of CaR in cardiac fibroblasts.

Isoproterenol (ISO), calindol, calhex231, sodium tauroursodeoxy cholate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for CaR, MMP-2, MMP-3, MMP-9, TIMP-3, TIMP-4, α-SMA and GAPDH were from Santa Cruz Biotechnology. A secondary antibody was AP-IgG (Promega). Polyvinylidene difluoride (PVDF) membranes were from Whatman (now part of GE Healthcare Life Sciences, Buckinghamshire, UK).

Neonatal rat cardiac fibroblasts were prepared from 2- to 3-day-old neonatal Wistar rats (Animal Research Institute of Harbin Medical University, China). The rats were anesthetized and sacrificed by immersion into 75% (v/v) alcohol. The ventricles were removed and washed three times in D-Hanks balanced salt solution (mmol/L: 0.4 KCl, 0.06 KH₂PO₄, 8.0 NaCl, 0.35 NaHCO₃, and 0.06 Na₂HPO₄ 7H₂O, pH 7.2) at 4 °C, then minced and incubated with 0.25 % (w/v) trypsin for 10 min at 37°C. To terminate the digestion, an equal volume of cold Dulbecco's modified Eagle's medium (DMEM) containing 10 % (v/v) newborn calf serum was added. The supernatant was discarded. Cells were incubated with fresh 0.25 % trypsin for 15 min at 37 °C, and the supernatant was collected. The latter digestion step was repeated four times. Cells in the supernatant were then isolated by centrifugation for 13 min at 2400 g/min at room temperature. Cells were re-suspended in DMEM containing 20 % (v/v) newborn calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin, and then were cultured as monolayers at a density of 5× 10⁴ cells/cm² at 37 °C in a humidified atmosphere containing 5 % (v/v) CO₂. After 1 h incubation in flasks at 37 °C, a humidified atmosphere with 5 % CO₂, the unattached cells were discarded and the attached cells were continuously cultured. The medium was changed two times per week.

Three days after being seeded, the cardiac fibroblasts were divided randomly into four groups: (1) control group: no treatment; (2) angiotensin (Ang) II group: pre-treated with 100 nmol/L AngII followed by treatment with 2.5 mM CaCl₂ for 24 h; (3) Calindol+ angiotensin (Ang) II group: pre-incubated with 100 nmol/L AngII followed by treatment with 3 µM calindol for 24 h; (4) Calhex231+ angiotensin (Ang) II group: pre-incubated with 100 nmol/L AngII followed by treatment with 2 µM calhex231 for 24 h.

Paraffin-embedded tissue sections (0.2 µm) were deparaffinized with xylenes and rehydrated with graded washes with ethanol followed by phosphate-buffered saline (PBS). Then, each slice was treated with 30 µl 3 % H₂O₂ (reagent A), incubated at room temperature for 20 min and washed twice with PBS. Then, 30 µl goat serum (reagent B) was added, followed by incubation at room temperature for 20 min and two washes with PBS. Each slice was incubated in 30 µl primary antibody (mouse anti-rat CaR monoclonal antibody, 1:200 dilution) and placed in a wet box at 4°C overnight. After washing with PBS, the slices were incubated in 30 µl biotinylated polyclonal secondary antibody (reagent C) at room temperature for 30 min, followed by washing with PBS. The diaminobenzidine (DAB) method was used for color development, followed by washing with tap water. Slices were then counterstained with hematoxylin, incubated ammonia, dehydrated with gradient ethanol, transparentized with xylenes and finally sealed with neutral gum. Cells with brown staining particles in their cytoplasm and nucleus were denoted positive under a light microscope.

Protein isolation and analysis were performed as described previously [8]. Briefly, whole-cell lysates were collected in lysis buffer (62.5 mmol/L of Tris-HCl, 2 mmol/L of EDTA, 2.3% SDS and 10% glycerol [pH 6.8]), with a protease inhibitor mixture), and total protein content was determined by the bicinchoninic acid method (Pierce). Equal amounts of protein samples were separated by SDS-PAGE and transferred to nitrocellulose using standard techniques.

Cardiac fibroblasts were loaded with 1 µmol/L Flou-4/AM at 37°C for 30 minutes in HEPES buffered saline (in mmol/L: 130 NaCl, 5 KCl, 10 glucose, 1 MgCl₂,1.0 CaCl₂, and 25 HEPES [pH 7.4]), and groups of 5 to 8 cells were monitored using an inverted Olympus IX-70 microscope. Spectrofluorometric measurements were collected using the Delta Scan System spectrofluorometer (Photon Technology), where the field was excited at 488 nm, with emission at 530 nm [9].

Cardiac fibroblasts (1x10⁵ cells per well) were seeded in 6-well plates and treated with or without different reagents for 48 hours. Thereafter, MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (5 mg/mL) was added to each well, and plates were incubated at 37°C for 4-6 hours. The medium was then replaced with 150 µL dimethyl sulfoxide (DMSO) and agitated for 10 minutes. Absorbance was measured at 490 nm using a microplate reader.

Cardiac fibroblasts (1 x10⁵ per well) were seeded in 6-well plates and treated with or without different reagents for a designated period. The cells were then digested by 0.25% trypsin for 6-8 minutes at 37°C. The cell number was counted using a hemocytometer.

Rat cardiac fibroblasts were treated with calcium, calindol and calhex231, and then subjected to the in vitro scratch assay as described previously [10,11]. Images were captured at 0, 24 and 48 hours after treatment using phase-contrast microscopy.

Male Wistar rats (200-250 g) were obtained from the Experimental Animal Center of Harbin Medical University (Harbin, People's Republic of China). All animal experimental protocols complied with the "Guide for the Care and Use of Laboratory Animals" published by the Chinese National Institutes of Health. The study was approved by the Institutional Animal Research Committee of Harbin Medical University. All animals were housed at the animal care facility of Harbin Medical University at 25°C with 12/12 h light/dark cycles and were allowed free access to normal rat chow and water throughout the study period. Rats were randomly assigned to different treatment groups.

β-Adrenergic stimulation can be induced by a high dose of isoproterenol (ISO), injected subcutaneously as reported before [12]. Sixty rats were randomly divided into four groups. In group 1 (n= 10), the rats were subcutaneously injected with saline for 4 days as a control. In group 2 (ISO, n= 15), the rats were subcutaneously injected with ISO (170 mg/kg/d in saline) for 4 days to induce cardiac hypertrophy. In group 3 (ISO-A, n= 18), the rats were subcutaneously injected with calindol (10 µmol/kg/d in saline) for 4 weeks after administration of ISO for 4 days. Calindol is a specific activator of CaR. In group 4 (ISO-I, n= 17), the rats were subcutaneously injected with calhex231 (10 µmol/kg/d in saline) for 4 weeks after administration of ISO for 4 days. Calhex231 is a specific inhibitor of CaR.

Cardiac functions were assessed using an echocardiography system (GEVIVID7 10S, USA). Echocardiograms were performed on self-breathing rats under anesthesia (intraperitoneal injection of 10% chloral hydrate at 0.3 ml/100 g body weight). Left ventricular (LV) parameters measured include inlet ventricular septal defects (IVSd) and left ventricular ejection fraction (LVEF). All parameters represent the means of five consecutive cardiac cycles [13].

Hearts from different groups, fixed in 10% formalin, were processed and paraffin embedded. Heart sections (4 mm) were then stained with Masson's trichrome reagent [14]. The percent of fibrosis (blue staining) to total tissue was analyzed and calculated by the NIS-ELEMENTS quantitative automatic program (Nikon, Tokyo, Japan), with the average value representing at least 8 images per heart, analyzed in double-blind fashion. Serial, transverse cryosections (7 mm thick) of hearts were cut with the CM1950 Frigocut (Leica, Wetzlar, Germany) at -20°C and were stored at -80°C.

Data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) was used to determine statistical significance between groups and also the two-tailed Student's t-test. A probability value of p<0.05 was taken to indicate statistical significance.

To determine whether CaR is expressed in cardiac fibroblasts, CaR expression was detected by immunofluorescence and immunohistochemical staining. In neonatal cardiac fibroblasts, deep yellow immunostaining or green fluorescence was present throughout all cardiac fibroblasts indicating the expression of CaR at the protein level in these cells. A lack of specific staining was demonstrated in the control group, in the absence of anti-CaR antibody (Fig. 1a and b).

The expression of CaR protein was also examined in neonatal cardiac fibroblasts by western blotting. CaR proteins with a relative molecular mass between 120-140 kDa were detected in neonatal cardiac fibroblasts (Fig. 1c). Furthermore, preadsorption of anti-CaR antibody with standard CaR antigen eliminated the 130 kDa band. Together, these results indicate that CaR is expressed in neonatal cardiac fibroblasts.

To confirm that CaR functionally regulates intracellular calcium concentration in neonatal cardiac fibroblasts, cells were treated with 2.5 mM CaCl₂, NiCl₂ (1 mM), an inhibitor of the Na⁺-Ca²⁺ exchanger, and CdCl₂ (200 µM), an inhibitor of the L-type calcium channel, in the bath solution. These treatments induced a time-dependent increase in intracellular calcium. Similarly, administration of 10 µM calindol evoked an increase in intracellular calcium concentration in more than 95 % of neonatal cardiac fibroblasts in a given observation field. After pretreatment with calhex231, a specific inhibitor of CaR, intracellular calcium concentration was not significantly increased compared to administration of calindol alone.

Neonatal cardiac fibroblasts were pretreated for 10 min with 10 µM TG (thapsigargin), which inhibits refilling of the IP₃-sensitive calcium release pools. Subsequently, cells were treated with 2.5 mM CaCl₂ and 10 µM calindol, which failed to elicit any increase of intracellular calcium concentration. Preincubation of neonatal cardiac fibroblasts for 10 min with U73122, a phosphatidylinositol-specific PLC blocker, abolished the increase in intracellular calcium induced by 10 µM calindol or 2.5 mM CaCl₂. Pretreatment with 2-APB, an IP₃ receptor-specific inhibitor, for 10 min eliminated the effect of 10 µM calindol and 2.5 mM CaCl₂ on the induction of intracellular calcium release (Fig. 2). These results demonstrate that the activation of CaR resulted in intracellular calcium increase in neonatal cardiac fibroblasts through stimulation of the PLC-IP₃ pathway.

To investigate whether neonatal cardiac fibroblast proliferation is regulated by CaR, the cells were starved overnight and pretreated with CaR activators, either CaCl₂ (2.5 mM) or calindol (10 µM), for 1 hour and then incubated with AngII for 48 hours. The results indicated that both CaCl₂ and calindol enhanced cell density, whereas the CaR blocker calhex231 abolished these effects. As shown in Figures 3a and b, CaCl₂ and calindol induced cardiac fibroblast proliferation, as evidenced by cell viability (Fig. 3a) and increased cell number (Fig. 3b).

To address whether the migration of neonatal cardiac fibroblast is induced by CaR, cell migration was studied in a wound-healing assay. Cells in culture were scraped off with a pipette tip, producing a wide acellular area. Cardiac fibroblasts migrating into this acellular area were counted and expressed as the number of migrated cells. Treatment with 2.5 mM CaCl₂ and 10 µM calindol significantly increased the migration of neonatal cardiac fibroblasts after 24 h incubation, while administration of calhex231 reduced this effect (Fig. 3c).

To investigate CaR regulating ECM production, CaR expression was detected. In the calindol and CaCl₂ groups, CaR expression was upregulated compared to control and calhex231-treated group (Fig. 4 a).

MMPs not only play a role in matrix degradation, but are also involved in collagen synthesis regulation. To reveal whether CaR effecting on ECM secretion, MMP-2 and MMP-9 were detected by Western blotting. As expected, 10 µM calindol and 2.5 mM CaCl₂ significantly enhanced the expression of MMP-2 and MMP-9 compared to control and calhex231-treated groups (Fig. 4b, c). TIMP-4 expression was significantly decreased in calindol and CaCl₂ groups, but was increased in calhex231-treated group (Fig. 4d).

α-SMA (alpha-smooth muscle actin) positive fibroblasts were identified as primary cell type responsible for interstitial matrix accumulation in fibrotic diseases. Our results showed that α-SMA expression was increased in the calindol and CaCl₂ groups (Fig. 4e).

To further investigate the in vivo effect of CaR on cardiac fibrosis in cardiac hypertrophy, we established a model of cardiac hypertrophy by injecting rats with a high dose of ISO (170 mg/Kg) for 4 days. Masson's staining analyses showed that collagen deposition was increased in the ISO-4w and ISO-4w-calindol groups, compared with the ISO-4w-calhex231 groups (Fig. 5c).

Hemodynamic parameters were monitored by echocardiography. Compared with the control group, the interventricular septal diastolic thickness (IVSd) was increased in the ISO-4w, ISO-4w-calindol and ISO-4w-calhex231 groups. The left ventricular ejection fraction was decreased in the ISO-4w and ISO-4w-calindol groups after ISO administration for 4 weeks, compared with the control group (Fig. 5a,b).

We also found that the expression of CaR in heart tissues was significantly increased in the ISO-4w and ISO-4w-calindol groups (Fig. 6a). To explore the in vivo effect of CaR on cardiac fibrosis in cardiac hypertrophy, we detected the expression of several molecular indicators of cardiac fibrosis. The expression of both MMP-2, 3 and -9 in the ISO-4w and ISO-4w-calindol groups was increased compared to the ISO-4w-calhex231 and control groups. These results further demonstrate that activation of CaR is involved in the formation of cardiac fibrosis in ISO-induced cardiac hypertrophy (Fig. 6 b‚c‚d).

The expression of CaR in various cells is related to multiple cellular functions, such as cell proliferation, apoptosis and the regulation of systemic calcium homeostasis [15]. For instance, Brown et al. have confirmed that CaR is activated by extracellular calcium in parathyroid cells and is responsible for the release of PTH [16,17,18]. Our previous studies demonstrated that the activation of CaR induced cardiomyocyte apoptosis during ischemia/reperfusion injury and in failing hearts [7,8]. Our present study demonstrates, for the first time, the existence of CaR in rat cardiac fibroblasts. This conclusion is based on several lines of evidence: (a) CaR proteins were identified in neonatal cardiac fibroblasts by Western blot analysis; (b) CaR proteins were shown to be located in neonatal cardiac fibroblasts by immunohistological and immunofluorescence staining; (c) extracellular calcium and calindol, a specific activator of CaR, increased intracellular calcium levels, thereby activating the PLC-IP₃ pathway and inducing the release of calcium from ER; (d) extracellular calcium and calindol promoted neonatal cardiac fibroblast proliferation, migration and ECM secretion through the activation of CaR; and (e) CaR played a role in ISO-induced cardiac fibrosis in rats.

Ca²⁺ signals are well studied in cardiac myocytes [19,20]. However, Ca²⁺ signaling pathways are not well understood in neonatal cardiac fibroblasts. Li et al. have revealed that Ca²⁺ activity in human cardiac fibroblasts is mediated by multiple Ca²⁺ signaling pathways, IP₃Rs, SERCAs, NCX, PMCAs and SOCs [21]. CaR, as a G protein coupled receptor, senses changes in extracellular calcium concentration, triggering an increase in intracellular calcium [18]. CaR, in both rat cardiac myocytes and human antral gastrin cells, is sensitive to changes in extracellular calcium concentration [15,22]. Our results in neonatal cardiac fibroblasts are similar to the results described above.

The sarcoplasmic/endoplasmic reticulum, a specialized calcium-storing organelle, is well known to be intimately involved in regulating Ca²⁺ movement within cells. IP₃Rs and RyRs participate in the release of Ca²⁺ from the sarcoplasmic/endoplasmic reticulum [23,24]. Interestingly, Li et al. found that RyRs may not be involved in Ca²⁺ activity in human cardiac fibroblasts. These results demonstrated that intracellular calcium in cardiac fibroblasts are mediated by IP₃Rs [21]. Our results reveal that increased intracellular calcium concentrations were inhibited by U73122 and 2-APB. In addition, the CaR activator calindol induced significantly increased intracellular calcium concentrations. These results demonstrate that CaR is involved in increasing the intracellular Ca²⁺ concentration through the PLC-IP₃ pathway.

Cardiac fibrosis is vital for ventricular remodeling [25]. Cardiac fibroblasts, highly active cells that exhibit increased proliferative, migratory and secretory properties [26,27], represent the largest class of cells residing in the heart. Proliferation of cardiac fibroblasts is the main characteristic of myocardial fibrosis. Although the importance of fibrosis in various cardiac diseases has been well established, the Ca²⁺ signaling mechanisms involved in cardiac fibrosis are not known. Some studies revealed that TRPM7 is located in the membrane of cardiac fibroblasts and mediates higher [Ca²⁺ ]_i oscillation frequencies indicative of higher ECM stiffness, which would result in more efficient ECM remodeling [28,29,30]. Ca²⁺ may play an essential role in cardiac fibroblasts proliferation and differentiation [19]. Our results confirm that activation of CaR could promote migration and elevate the expression levels of MMP-2 and 9 in neonatal cardiac fibroblasts. To further demonstrate the involvement of CaR in cardiac fibrosis, a rat model of ISO-induced cardiac hypertrophy and fibrosis was established, followed by administration of calindol and calhex231. Masson's staining showed that calindol enhanced cardiac fibrosis, whereas calhex231 diminished cardiac fibrosis. The expression of both MMP-2 and -9 was elevated by treatment with calindol.

The CaR-mediated triggering of cardiac fibrosis via Ca²⁺ signaling may serve as a common pathway in the fibrosis cascade. If so, inhibition of Ca²⁺ release from the ER through CaR may attenuate cardiac fibrosis. The contribution of CaR-mediated Ca²⁺ signal in cardiac fibroblasts shown in this study is the first demonstration of an important role of this receptor in heart disease.

In conclusion, our results are the first to report an important role for CaR in Ca²⁺ signaling involved in cardiac fibrosis and that this role is mediated by the PLC-IP3 pathway. Our study opens up a new avenue to explore the potential roles of Ca²⁺ signaling in cardiac fibrosis and could lead to the development of more effective approaches for treating cardiac fibrosis.

The authors declare that they have no conflict of interest.

This study was supported by the National Natural Science Foundation of China (81170289, 81170218, 81370421, 81370330, 81300164, 81170178), the Harbin Medical University grant for excellent younger scientists (to Weihua Zhang) and China Postdoctoral Science Foundation funded project (2012M520766) and Heilongjiang Postdoctoral Fund (No.LBH-Z11084) for Fanghao Lu and Harbin Science and technology bureau Fund (2009RFQXS014) for Lina Wang.