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Abstract

Introduction: Cardiac remodelling is controlled by complex systems, including activation of the renin-angiotensin system (RAS) and signalling through MAP kinases and Ca2+-activated calcineurin. Whether Ang II, which increases [Ca2+]i and stimulates MAP kinases, mediates myocardial effects through calcineurin-dependent pathways remain unclear. We investigated effects of two calcineurin inhibitors, cyclosporine A (CsA) and tacrolimus (FK506) (10-10-10-6 mol/L, 20 mins) on activation of MAP kinases and on growth, pro-fibrotic and pro-inflammatory responses in Ang II-stimulated rat cardiac fibroblasts.

Methods and Results: Ang II increased phosphorylation of ERK1/2 and p38MAPK (1.5-1.8-fold, p<0.05) without effect on JNK. FK506, but not CsA, attenuated Ang II-stimulated MAP kinase activation. Molecular indices of cell growth (proliferating cell nuclear antigen (PCNA)), fibrosis (fibronectin, pro-collagen) and inflammation (iNOS), were upregulated by Ang II (12 hrs). FK506 and CsA inhibited PCNA effects. Ang II-induced pro-fibrotic and pro-inflammatory responses were inhibited by CsA. Ang II receptors, AT1R and AT2R, were not influenced by calcineurin inhibitors. Our data indicate differential calcineurin inhibitor sensitivity of MAP kinases and cellular responses in Ang II-stimulated fibroblasts. p38MAP kinase and ERK1/2 are regulated in a FK506-sensitive manner, whereas fibrosis and inflammation are CsA-sensitive. Cell proliferation is inhibited by both FKC506 and CsA. These are post-receptor phenomena, since AT1R and AT2R status was unaltered by treatment.

Conclusions: Our findings identify an important role for calcineurin in MAP kinase/growth/pro-fibrotic/pro-inflammatory signalling by Ang II in cardiac fibroblasts. Although both FK506 and CsA inhibit calcineurin, they exert differential effects on molecular and cellular responses. Such differences may contribute to variable clinical responses of these agents.

Keywords

heart immunomodulators MAP kinase signal transduction

Introduction

In response to pathological stress and injury, cardiac tissue undergoes hypertrophy, inflammation, fibrosis and apoptosis. Fundamental to these processes is the activation of mitogen-activated protein (MAP) kinases, which directly modify transcriptional regulatory factors leading to altered cardiac gene expression [Dengler and Pober, 2000; Barry et al. 2008]. To date at least six major mammalian MAP kinase subfamilies have been identified, of which extracellular signal-regulated protein kinase (ERK1/2), c-Jun aminoterminal kinase (JNK) and p38MAP kinase (p38MAPK) are the best characterized. ERK1/2 and p38MAPK are major growth and profibrotic signalling kinases, whereas JNK and p38MAPK influence cell survival, apoptosis, differentiation and inflammation [Clerk et al. 2007; Wang, 2007]. Increased activation of MAP kinases has been demonstrated in cardiac remodelling [Zhen et al. 2007; Lorenz et al. 2009]. Of the many factors influencing MAP kinases in the heart is activation of the renin–angiotensin system. Increased angiotensin II (Ang II) signalling has been demonstrated in cardiac hypertrophy, cardiac failure, hypertensive heart disease and aortic aneurysms [De Mello, 2011; Kurdi and Booz, 2011].

In addition to MAP kinases, the Ca²⁺-calmodulin-activated phosphatase, calcineurin (phosphatase 2B) and its downstream transcriptional effector, nuclear factor of activated T-cells (NFAT), have been implicated as transducers of cardiac hypertrophy and remodelling [Zhang 2002; Rao, 2009]. Interestingly, some of the signalling components of the calcineurin pathway found in T cells are also present in cardiac cells and have been implicated in the hypertrophic response, possibly through interaction with MAP kinases and Ang II [Rana et al. 2009; Tan et al. 2008; Ikeda et al. 2006].

Calcineurin is the common cellular target for the immunosuppressive drugs cyclosporine A (CsA) and tacrolimus (FK506), which block cytokine-induced immune responses [Ikeda et al. 2006; Braun et al. 1995; Bierer et al. 1993; McCormack and Keating, 2006]. These immunosuppressive agents produce similar effects on signal transduction pathways in T lymphocytes. In other cells, however, including cardiac cells, they may not interact with the same target and their effects on signalling appear to differ. Whereas CsA binds to cyclophilin, FK506 binds to FK506-binding protein. This may contribute to some of the different metabolic profiles that these two agents exhibit, such as hyperglycaemia and hypertension [Dengler and Pober, 2000; Braun et al. 1995; Bierer et al. 1993; McCormack and Keating, 2006; Seibert et al. 2011]. Novel calcineurin-independent mechanisms and targets of action for CsA and FK506 have been proposed, including transforming growth factor β (TGFβ)-activated kinase (TAK1), sarcoplasmic reticulum Ca²⁺-release channel (SERCA), L-type Ca²⁺ channels, ryanodine receptors and MAP kinases [Santana et al. 2002; Smaili et al. 2001; Akool et al. 2008; Molkentin, 2000].

In the present study we questioned whether Ang II influences myocardial MAP kinases and cellular responses (growth, fibrosis and inflammation) through calcineurin. In particular we investigated the effects of CsA and FK506 on the activation of MAP kinases, and on the expression of proliferating cell nuclear antigen (PCNA), fibronectin, procollagen 1 and inducible nitric oxide synthase (iNOS), signalling molecules important in cell growth, fibrosis and inflammation. We studied cardiac fibroblasts, which are critically involved in cardiac remodelling in response to stress [Porter and Turner, 2009].

Methods

The study was approved by the Animal Ethics Committee of Ottawa Hospital Research Institute, University of Ottawa, and carried out according to the recommendations of the Canadian Council for Animal Care.

Cell culture and drug protocols

Studies were performed in cultured cardiac fibroblasts (passages 2–7) derived from adult Wistar Kyoto (WKY) rats as we previously described [Touyz et al. 1996a; Touyz et al. 1996b]. Cardiac fibroblasts were studied because they contribute to cardiac hypertrophy and remodelling [Porter and Turner, 2009]. Serum-deprived cells were treated with ionomycin (10⁻⁴ mol/l, 5 min), a Ca²⁺ ionophore that stimulates calcineurin signalling (as a positive control), or Ang II (10⁻⁹ mol/l, 5 min to 24 h) in the absence and presence of CsA (10⁻¹⁰–10⁻⁶ mol/l) or FK506 (10⁻¹⁰–10⁻⁶ mol/l) (20 min preincubation).

Measurement of intracellular free Ca²⁺ concentration in response to ionomycin

[Ca²⁺]_i was measured using the fluorescent probe fura 2AM (Molecular Probes, Life Technologies, Grand Island, NY, USA) as we previously described [Touyz et al. 1996a]. On the day of the study, the culture medium was replaced with warmed modified Hanks buffered saline containing (mmol/l): 137 NaCl, 4.2 NaHCO₃, 3 Na₂HPO₄, 5.4 KCl, 0.4 KH₂PO₄, 1.3 CaCl₂, 0.5 MgCl₂, 10 glucose, 0.8 MgSO₄ and 5 HEPES (pH 7.4). Cardiac fibroblasts were loaded with fura-2-acetoxymethyl ester (fura 2AM) (4 µmol/l) dissolved in dimethyl sulphoxide containing 0.02% pluronic F-127 (Molecular Probes), and incubated for 30 min at 37°C in a humidified incubator. Under these loading conditions, the ratiometric fluorescence cell images are homogeneous, indicating no significant compartmentalization of the dye. After 30 min, the loaded cells were washed and used after a stabilization period of 5–10 min. All washing procedures and experiments were performed at room temperature, thereby minimizing compartmentalization and cell extrusion of the dye. The coverslip containing cells was placed in a stainless steel chamber and mounted on the stage of an inverted microscope (×10 objective), and a Stallion High Speed Digital Microscopy Workstation imaging system (Slidebook; Zeiss, Maple Grove, MN, USA) was used. Fura 2AM-loaded cells were exposed to an excitation wavelength of 340 and 380 nm while monitoring the emission signal at 510 nm. [Ca²⁺]_i responses were measured in cells exposed to ionomycin (10⁻⁷ mol/l).

Western blotting

Expression of MAP kinases, ERK1/2, p38MAP kinase and JNK (total and phosphorylated) was assessed by immunoblotting as we described [Montezano et al. 2008]. Molecular indices of cell proliferation (PCNA), inflammation (iNOS) and fibrosis (fibronectin, procollagen 1) and of the Ang II receptors AT₁ and AT₂ were also assessed by Western blotting. Proteins were extracted from cells, separated by electrophoresis on a 10% polyacrylamide gel, and transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked with 5% skim milk in Tris-buffered saline solution with 0.1% Tween (pH 7.4) for 1 h at 24°C. Membranes were then incubated with phosphospecific antibodies (1:1000) overnight at 4°C. Phosphospecific antibodies were as follows: anti-p38MAPK (Thr¹⁸⁰/Tyr¹⁸²), anti-SAPK (stress-activated protein kinase)/JNK (Thr¹⁸³/Tyr¹⁸⁵) and anti-ERK 1/2 (Thr²⁰²/Tyr²⁰⁴) (Cell Signaling, Danvers, MA, USA). The corresponding nonphosphospecific antibodies (1:2000) were also used. Antibodies to PCNA (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (1:5000, Sigma, St Louis, MO, USA), procollagen 1 (1:1000; Santa Cruz Biotechnology), iNOS (1:500; Santa Cruz Biotechnology), AT₁R (1:1000; Santa Cruz Biotechnology) and AT₂R (1:500; Santa Cruz Biotechnology) were used to probe growth and profibrotic pathways and Ang II receptor status. After incubation with secondary antibodies (1:2000 to 1:5000), signals were revealed with chemiluminescence, visualized by autoradiography and quantified densitometrically using ImageQuant. Results were normalized by the total protein or β-actin (1:10000; Sigma) (housekeeping protein) and expressed as percentage of vehicle used in the experimental protocols.

Results

Effects of ionomycin on [Ca²⁺]_i responses and MAP kinases in cardiac fibroblasts treated with calcineurin inhibitors

As a positive control to demonstrate that cultured cardiac fibroblasts possess a functional Ca²⁺-regulated calcineurin pathway, we examined the effects of ionomycin on [Ca²⁺]_i and on MAP kinase phosphorylation in cells pretreated with FK506 or CsA. As demonstrated in Figure 1a, ionomycin induced a rapid increase in [Ca²⁺]_i. This response was transient as levels returned to basal concentrations within 5 min of stimulation.

Figure 1.

Cardiac fibroblasts possess functionally active Ca²⁺/calcineurin/MAP kinase pathways. (a) Ionomycin increases intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in cardiac fibroblasts. Representative line graphs demonstrate [Ca²⁺]_i responses to ionomycin (10⁻⁴ mol/l) in 10 different cells. Cardiac fibroblasts were loaded with fura 2AM (4 µmol/l) and fluorescence was measured by fluorescence digital imaging. (b, c) Effects of FK506 and cyclosporine A (CsA) on extracellular signal-regulated protein kinase (ERK1/2) phosphorylation (b) and p38MAP kinase (p38MAPK) (c) in cardiac fibroblasts stimulated or not with ionomycin (Iono; 10⁻⁴ mol/litre). Cells were pretreated with FK506 or CsA for 20 min before ionomycin addition (5 min). Upper panels show representative Western blots. Bar graphs in lower panels show mean and SEM from four to six experiments. Results are presented as the ratio of phosphorylated to total ERK1/2 or p38MAPK. **p < 0.01 versus other groups.

In addition to increasing [Ca²⁺]_i, ionomycin induced significant increases in phosphorylation of ERK1/2 and p38MAPK (p < 0.01). Pretreatment of cells with FK506 and CsA abrogated the ionomycin-induced activation of ERK1/2 and p38MAPK (p < 0 1) (Figure 1b and c). These data confirm a functional Ca²⁺-calcineurin–MAPK pathway in our cell model.

Effects of FKC506 and CsA on Ang II-stimulated phosphorylation of MAP kinases

Ang II induced a rapid increase (5 min) in phosphorylation of p38MAP kinase (Figure 2) and ERK1/2 (Figure 3). Ang II did not significantly alter JNK activation (data not shown). Ang II-induced phosphorylation of p38MAP kinase was inhibited by FK506 (10⁻⁸–10⁻⁶ mol/l) (p < 0.05), but not by CsA. FK506 (10⁻⁸ mol/l) significantly reduced ERK1/2 activation by Ang II. CsA had no effect on ERK1/2 phosphorylation. JNK was not influenced by FK506 or CsA (data not shown).

Figure 2.

Effects of FK506 and cyclosporine A (CsA) on p38MAP kinase (p38MAPK) phosphorylation in cardiac fibroblasts stimulated or not with angiotensin II (Ang II; 10⁻⁹ mol/litre). Cells were pretreated with FK506 or CsA for 20 min before Ang II addition (5 min). Upper panels show representative Western blots. Bar graphs in lower panels show mean and SEM from four to six experiments. Results are presented as the ratio of phosphorylated to total p38MAPK compared with vehicle (V), taken as 100%. *p < 0.01 versus vehicle; +p < 0.05 versus Ang II.

Figure 3.

Effects of FK506 and cyclosporine A (CsA) on phosphorylation of extracellular signal-regulated protein kinase (ERK1/2) in cardiac fibroblasts stimulated or not with angiotensin II (Ang II; 10⁻⁹ mol/l). Cells were pretreated with FK506 or CsA for 20 min before Ang II addition (5 min). Upper panels show representative Western blots. Bar graphs in lower panels show the mean and SEM from four to six experiments. Results are presented as the ratio of phosphorylated to total ERK1/2 compared with vehicle (V), taken as 100%. *p < 0.01 versus vehicle; +p < 0.05 versus Ang II.

Effects of calcineurin inhibitors on Ang II-stimulated cell growth, inflammation and fibrosis

Expression of PCNA, a molecular marker of cell proliferation, was significantly increased by Ang II after 12 h stimulation. This effect was abrogated by FK506 and CsA (Figure 4). PCNA responses were transient, because at 24 h after stimulation there was no significant change in expression. To support these findings, we evaluated the effects of FK506 on the cell cycle in Ang II-stimulated cells. As shown in Figure 4b, Ang II induced a significant increase in the percentage of cells in the S (synthetic) phase of the cell cycle. This was inhibited by FK506.

Figure 4.

(a) Effects of FK506 and cyclosporine A (CsA) on proliferating cell nuclear antigen (PCNA) expression in cardiac fibroblasts stimulated or not with angiotensin II (Ang II; 10⁻⁹ mol/l). Cells were pretreated with FK506 or CsA for 20 min before Ang II addition (12, 24 h). Upper panels show representative Western blots. Bar graphs in lower panel are the mean and SEM from four to six experiments. Results are presented as the ratio of PCNA to β-actin. *p < 0.05 versus other groups, +p < 0.05 versus Ang II alone. (b) Effects of Ang II (10⁻⁹ mol/l, 1 h) in the absence and presence of FK506 on the S-phase of the cell cycle. *p < 0.05 versus other groups.

Stimulation of cells with Ang II provoked an increase in expression of iNOS, fibronectin and procollagen (Figures 5 and 6). These effects were sustained for up to 24 h. CsA inhibited Ang II-induced proinflammatory and profibrotic responses. In FK506-treated and untreated cells, Ang II-induced expression of iNOS, fibronectin and procollagen 1 was significantly increased compared with vehicle (Figures 5 and 6).

Figure 5.

Effects of FK506 and cyclosporine A (CsA) on expression of inducible nitric oxide synthase (iNOS; proinflammatory molecule) in cardiac fibroblasts stimulated or not with angiotensin II (Ang II; 10⁻⁹ mol/l). Cells were pretreated with FK506 or CsA for 20 min before Ang II addition (12, 24 h). Upper panels show representative Western blots. Bar graphs in lower panel show mean and SEM from four to six experiments. Results are presented as the ratio of iNOS to β-actin compared with vehicle (V), taken as 100%. *p < 0.01 versus vehicle; +p < 0.05 versus Ang II.

Figure 6.

Effects of FK506 and cyclosporine A (CsA) on expression of fibronectin and procollagen 1 (profibrotic responses) in cardiac fibroblasts stimulated or not with angiotensin II (Ang II; 10⁻⁹ mol/l). Cells were pretreated with FK506 or CsA for 20 min before Ang II addition (12, 24 h). Upper panels show representative Western blots. Bar graphs in lower panel show mean and SEM from four to six experiments. Results are presented as the ratio of fibronectin or procollagen 1 to β-actin compared with vehicle (V), taken as 100%. Data are mean and SEM from four to six experiments *p < 0.01 versus vehicle; +p < 0.05 vs Ang II.

AT₁R and AT₂R expression in treated cardiac fibroblasts

Both Ang II receptor types were present in cardiac fibroblasts (Figure 7). Receptor status was not affected by Ang II and treatment with FK506 and CsA did not alter expression of either receptor.

Figure 7.

Effects of FK506 and cyclosporine A (CsA) on expression of angiotensin II (Ang II) receptors (AT₁R and AT₂R) in cardiac fibroblasts stimulated or not with Ang II (10⁻⁹ mol/l). Cells were pretreated with FK506 or CsA for 20 min before Ang II addition (12, 24 h). Upper panels show representative Western blots. Bar graphs in lower panels are mean and SEM from four to six experiments. Results are presented as the ratio of AT receptor to β-actin compared with vehicle (V), taken as 100%.

Discussion

This study demonstrates that, in cardiac fibroblasts, MAP kinase regulation by Ang II occurs through calcineurin-dependent pathways in a FK506-sensitive manner. Cellular responses are differentially influenced by FK506 and CsA. Whereas FK506 and CsA inhibit Ang II-induced proliferation, as assessed by increased PCNA expression, agonist-stimulated fibrosis and inflammation involves only CsA-dependent processes. These differential cellular responses are postreceptor phenomena, since Ang II receptor status was unaltered by calcineurin inhibitors.

Cardiac hypertrophy is an important clinical predictor of morbidity and mortality. At the cellular level, cardiomyocytes and cardiac fibroblasts exhibit a proinflammatory, fibrogenic and hypertrophic phenotype, induced in large part through activation of MAP kinases [Dengler and Pober, 2000; Barry et al. 2008; Clerk et al. 2007; Wang, 2007]. Once activated by agonists, such as Ang II, MAP kinases phosphorylate intracellular targets that include transcription factors, resulting in the programming of cardiac cell gene expression as part of the stress response [Lunde et al. 2011]. Whereas activation of ERK1/2 and p38MAP kinase mediates hypertrophy, hyperplasia and fibrosis, p38MAP kinase and JNK are involved in inflammation, apoptosis and cell survival. Recent in vivo and in vitro evidence suggests that calcineurin also plays a role in cardiac remodelling, and calcineurin inhibitors have been suggested as potential therapeutic agents in cardiac disease [Kuwahara and Nakao, 2011]. Transgenic mice that overexpress cardiac calcineurin develop severe cardiac hypertrophy and cardiac failure [Dong et al. 2003]. In cultured cardiac cells, FK506 and CsA block Ang II-stimulated cardiomyocyte hypertrophy [Fiedler and Wollert, 2004].

Ang II stimulated activation of cardiac fibroblasts, as demonstrated by increased phosphorylation of ERK1/2 and p38MAP kinase and upregulation of PCNA, fibronectin, procollagen 1 and iNOS. These processes involve calcineurin but sensitivity to calcineurin inhibitors differs. In particular, activation of p38MAP kinase and ERK1/2 is selectively responsive to FK506 whereas molecular events associated with fibrosis and inflammation are more sensitive to CsA. Cell proliferation is responsive to both FK506 and CsA. Calcineurin-mediated regulation of MAP kinases by Ang II is not a generalized phenomenon, because JNK was not significantly activated by Ang II in cardiac fibroblasts.

Differential regulation of MAP kinases by calcineurin has been demonstrated. In activated human basophils, CsA attenuated activation of p38MAP kinase but not of ERK1/2 [Plath et al. 2003]. In injured rat kidneys, CsA decreased the activation of JNK and p38MAPK but not ERK1/2 [Yang et al. 2003]. In osmostressed mammalian embryonic kidney cells, FK506, but not CsA, regulated p38MAPK activation through increased phosphorylation of the eukaryotic initiation factor 2α [Sanchez-Perez et al. 2004]. In osteoblasts, FK506 but not CsA enhanced fibroblast growth factor 2 (FGF-2)-induced vascular endothelial growth factor (VEGF) release [Yamauchi et al. 2009]. Moreover, recent clinical evidence indicates that CsA and FK506 have differential effects on vascular function, the augmentation index, an index of arterial stiffening, being less in patients receiving FK506 than in those receiving CsA [Seibert et al. 2011]. Taken together, these data suggest complex regulation of signalling molecules by calcineurin, processes that may be specific to cell type, agonist or calcineurin inhibitor.

Mechanisms underlying the differential effects of FK506 and CsA may relate, in part, to the fact that these two immunosuppressive agents, both of which target calcineurin, interact with different intracellular molecules. Whereas CsA complexes with cyclophilin, FK506 complexes with FK binding protein 12 [Kajitani et al. 2008; Becker et al. 1993]. These complexes may influence MAP kinases, specifically p38MAP kinase and JNK, independently of calcineurin, as shown in T-cells [Matsuda et al. 2000; Matsuda and Koyasu, 2003]. However whether cyclophilin and FK binding protein 12 have different MAP kinase targets remains unclear. Thus, it seems that CsA and FK506 have two distinct mechanisms of action: one is the inhibition of the protein phosphatase activity of calcineurin, leading to the blockade of the nuclear translocation of NFAT transcription factors and other signalling molecules, and the other is the direct suppression of non-calcineurin-dependent signalling proteins.

Calcineurin signal transduction, through MAP kinases and transcriptional regulators such as myocyte enhancer factor 2 (MEF2), NFAT and GATA4 (transcription factor), play an important role in cardiac hypertrophy, ventricular dilation and heart failure [Balakumar and Jagadeesh, 2010; Barry and Townsend, 2010; Kuwahara and Nakao, 2011]. Accordingly, calcineurin inhibitors, in addition to their immunosuppressive actions, are increasingly being considered as potentially useful drugs in the treatment of cardiac failure [Barry and Townsend, 2010]. The cardioprotective actions of FKC and CsA may relate, at least in part, to antihypertrophic actions [Fiedler and Wollert, 2004]. Although FK506 and CsA both target calcineurin and both agents influence cardiac growth, their clinical profiles differ. For example, FK506 has been associated with lower rates of rejection, fewer drug-specific adverse effects and improved metabolic profiles than CsA [White et al. 2005, 2006; Wang et al. 2008; Ye et al. 2009]. This may be related to the favourable effects of FK506 on vascular stiffness and endothelial function [Seibert et al. 2011; Patel and Kobashigawa, 2007] and to distinct signalling actions by FKC and CsA in cardiac cells, as we show here, and in vascular cells as reported by others [Seibert et al. 2011]. Long-term clinical outcomes in calcineurin inhibitor-treated cardiac failure patients is still unclear [Dandel et al. 2010]. Further investigation is required before these drugs can be used routinely in the management of cardiac disease.

Taken together, our findings suggest that FK506 and CsA have differential effects on stress-related signalling pathways in cardiac fibroblasts, cells that contribute to cardiac hypertrophy and remodelling in pathological conditions. In Ang II-stimulated cells, FK506 attenuates phosphorylation of p38MAP kinase and ERK1/2, whereas CsA inhibits profibrotic and proinflammatory responses. Cell growth is sensitive to both FK506 and CsA. Our data unravel some of the molecular mechanisms whereby calcineurin inhibitors influence cardiac fibroblast function and highlight the fact that although both agents inhibit the calcineurin pathway, they have specific actions on MAP kinase signalling and functional responses. Such differences may relate to specific immunophilin binding proteins and might contribute, at least in part, to the variable clinical responses of these agents.

Footnotes

This study was supported by the Canadian Institute of Health Research (CIHR; grant number 44018) and through an educational grant from Astellas Pharma Canada Inc. RMT is supported through a Canada Research Chair/Canadian Foundation for Innovation award. MW holds the Carolyn and Richard Renaud Research Chair of the Montreal Heart Institute, University of Montreal.

The authors declare no conflicts of interest in preparing this article.

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Angiotensin II signalling and calcineurin in cardiac fibroblasts: differential effects of calcineurin inhibitors FK506 and cyclosporine A

Abstract

Keywords

Introduction

Methods

Cell culture and drug protocols

Measurement of intracellular free Ca2+ concentration in response to ionomycin

Western blotting

Results

Effects of ionomycin on [Ca2+]i responses and MAP kinases in cardiac fibroblasts treated with calcineurin inhibitors

Effects of FKC506 and CsA on Ang II-stimulated phosphorylation of MAP kinases

Effects of calcineurin inhibitors on Ang II-stimulated cell growth, inflammation and fibrosis

AT1R and AT2R expression in treated cardiac fibroblasts

Discussion

Footnotes

References

Measurement of intracellular free Ca²⁺ concentration in response to ionomycin

Effects of ionomycin on [Ca²⁺]_i responses and MAP kinases in cardiac fibroblasts treated with calcineurin inhibitors

AT₁R and AT₂R expression in treated cardiac fibroblasts