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Cardiovascular Research 2004 63(3):545-552; doi:10.1016/j.cardiores.2004.04.015
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Copyright © 2004, European Society of Cardiology

Heme oxygenase-1 inhibition of MAP kinases, calcineurin/NFAT signaling, and hypertrophy in cardiac myocytes

Jörn Tongersa, Beate Fiedlera, Danny Königa, Tibor Kempfa, Gunnar Kleina, Jörg Heinekea, Theresia Kraftb, Stepan Gambaryanc, Suzanne M Lohmannc, Helmut Drexlera and Kai C Wollert*,a

aDepartment of Cardiology and Angiology, Hannover Medical School, 30625 Hannover, Germany
bDepartment of Molecular and Cellular Physiology, Hannover Medical School, 30625 Hannover, Germany
cInstitute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, 97080 Würzburg, Germany

* Corresponding author. Abt. Kardiologie und Angiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany. Tel.: +49-511-532-4055; fax: +49-511-532-5412. Email address: wollert.kai{at}mh-hannover.de

Received 9 January 2004; revised 19 April 2004; accepted 19 April 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Heme oxygenases (HO) are the rate-limiting enzymes in heme degradation, catalyzing the breakdown of heme to equimolar quantities of biliverdin (BV), carbon monoxide (CO), and ferrous iron. The inducible HO isoform, HO-1, confers protection against ischemia/reperfusion (I/R)-injury in the heart. We hypothesized that HO-1 and its catalytic by-products constitute an antihypertrophic signaling module in cardiac myocytes. Methods and results: The G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) (30 nmol/l) stimulated a robust hypertrophic response in cardiac myocytes isolated from 1- to 3-day-old Sprague–Dawley rats, with increases in cell surface area (planimetry), sarcomere assembly (confocal laser scanning microscopy), and prepro-atrial natriuretic peptide (ANP) mRNA expression. Adenoviral overexpression of HO-1, but not β-galactosidase, significantly inhibited ET-1 induced cardiac myocyte hypertrophy. The antihypertrophic effects of HO-1 were mimicked by BV (10 µmol/l) and the CO-releasing molecule [Ru(CO)3Cl2]2 (10 µmol/l), strongly suggesting a critical involvement of BV and CO in the antihypertrophic effects of HO-1. Both BV and CO suppressed extracellular signal-regulated kinases (ERK1/ERK2) and p38 mitogen-activated protein kinase (MAPK) activation by ET-1 stimulation. Moreover, BV and CO inhibited the prohypertrophic calcineurin/NFAT pathway. This inhibition occurred upstream from calcineurin because BV and CO inhibited NFAT activation in response to ET-1 stimulation but not in response to adenoviral expression of a constitutively active calcineurin mutant. Upstream-inhibition of the calcineurin/NFAT pathway by CO occurred independent from cGMP and cGMP-dependent protein kinase type I (PKG I). Conclusions: Heme oxygenase-1 and its catalytic by-products, BV and CO, constitute a novel antihypertrophic signaling pathway in cardiac myocytes. Biliverdin and CO inhibition of MAPKs and calcineurin/NFAT signaling provides a mechanistic framework how heme degradation products may promote their antihypertrophic effects.

KEYWORDS Hypertrophy; Second messengers; Signal transduction; MAP kinase; Calcium (cellular)


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac hypertrophy has been viewed as a compensatory mechanism of the heart that helps to maintain cardiac output during pathological states with sustained increases in hemodynamic load. However, hypertrophy often heralds decompensation, transition to heart failure, and sudden death [1]. To understand the molecular mechanisms of this ultimately maladaptive response, much investigation has focussed on the signaling pathways controlling hypertrophy at the level of the single cardiac myocyte. In this regard, growth factors signaling through G protein-coupled receptors (e.g. endothelin-1, norepinephrine, or angiotensin II) have emerged as potent mediators of cardiac myocyte hypertrophy. Downstream from G protein-coupled receptors (GPCRs), a complex network of protein kinases, phospholipid kinases, and protein phosphatases transmits the hypertrophic signal to the cell nucleus [2]. For example, all three branches of the mitogen-activated protein kinase (MAPK) pathway, i.e. extracellular signal-regulated kinases (ERK1 and ERK2), p38 MAPK, and c-Jun N-terminal kinases (JNKs), are activated in response to GPCR stimulation in cardiac myocytes [3,4]. Apart from MAPKs, the protein phosphatase calcineurin (PP2B) constitutes another important prohypertrophic pathway in cardiac myocytes [5,6]. Upon activation by Ca2+/calmodulin, calcineurin dephosphorylates cytoplasmic latent NFAT transcription factors resulting in their nuclear translocation [7]. Adding yet another level of complexity, recent studies have identified counter-regulatory pathways in cardiac myocytes that promote antihypertrophic effects by targeting specific prohypertrophic pathways [8,9]. In this context, nitric oxide (NO), signaling via cGMP and cGMP-dependent protein kinase type I (PKG I) has been shown to suppress GPCR-induced cardiac myocyte hypertrophy by interfering with the calcineurin/NFAT pathway [10–13].

Heme oxygenases (HO) are the rate-limiting enzymes in heme degradation, catalyzing the breakdown of heme to equimolar quantities of carbon monoxide (CO), biliverdin, and ferrous iron. Three HO isoforms have been identified. HO-1 is expressed in many tissues, including the heart, its expression being induced by stressful and inflammatory stimuli; HO-2 is constitutively expressed, primarily in the brain and testes (reviewed in Refs. [14,15]). HO-3 is a weak heme catalyst that is found in several organs including the heart; its role is still poorly understood [16]. Carbon monoxide mediates antiinflammatory, antiapoptotic, and vasodilatory effects some of which are mediated via cGMP-dependent mechanisms [17–19]. On the other hand, biliverdin (BV) and bilirubin (BR), which is rapidly generated by BV reductase, have been identified as cytoprotective, antioxidant moieties [20,21]. In the heart, HO-1 confers protection against ischemia/reperfusion (I/R)-injury [22–26]. However, a role for HO-1 in regulating cardiac myocyte hypertrophy has not been established. Considering that antioxidants [27–29], and cGMP-elevating agents [10–13] promote antihypertrophic effects in cardiomyocytes, we explored the possibility that HO-1 and its catalytic by-products, CO and BV/BR, constitute an antihypertrophic signaling module in cardiac myocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Materials
Endothelin-1 (ET-1), BR, and dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma, BV from ICN Biomedicals, the CO-releasing molecule [Ru(CO)3Cl2]2 from Aldrich, the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) from Calbiochem, FeSO4 from Applichem, and the PKG-selective cGMP analog 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) from Biolog.

2.2. Cell culture
This investigation conforms with the Guide for the Care and Use of Laboratory Animals, as published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996). Ventricular cardiomyocytes were isolated from 1- to 3-day-old Sprague–Dawley rats, as described [11]. Cardiac myocytes were plated at a density of 40,000 cells per cm2 (30% to 40% confluence) in gelatin-coated culture dishes (Nunc) in Dulbecco's modified Eagle's medium (DMEM)/medium 199 (4:1), supplemented with 10% horse serum, 5% fetal calf serum, glutamine, and antibiotics. The next morning, cells were switched to DMEM/medium 199 supplemented only with glutamine and antibiotics (maintenance medium). Endothelin-1 stimulation was used as a model system for GPCR-induced cardiac myocyte hypertrophy, as previously described [30,31].

2.3. Assessment of cardiac myocyte hypertrophy and survival
Cardiomyocyte surface area was determined by planimetry using phase contrast microscopy and a digital image analyzer (Leica Q500 MC) [11]. Sarcomere assembly was analyzed by a sample-blinded observer (T. Kempf) by confocal laser scanning microscopy of cardiac myocytes immunostained for the Z-disc protein {alpha}-actinin (see below). A semiquantitative grading system was applied to determine the [%] of cell area with organized sarcomeres: grade I, <10%; grade II, 10–50%; grade III, 50–90%; grade IV, >90% [31]. Immunostaining was performed as previously described [32], using a murine monoclonal antibody directed against {alpha}-actinin (Sigma, 1:100 dilution), and an FITC-labeled sheep anti-mouse antibody (Sigma, 1:100). Prepro-atrial natriuretic peptide (ANP) mRNA/18S expression was assessed by Northern blot, using a 0.6-kb rat ANP cDNA probe as described [33]. To screen for potential toxic effects of HO-1 and its reaction products in cardiac myocytes, trypan blue (Merck) dye exclusion and MTT assays (Sigma) were performed.

2.4. Immunoblotting
Samples were separated on 12% SDS-polyacrylamide gels. Expression of PKG I was detected by immunoblotting using a polyclonal anti-PKG I antiserum (described in Ref. [34], 1:1500 dilution). Phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at Ser239 was detected by the 16C2 monoclonal antibody, and was used as a sensitive and specific readout for cGMP generation and PKG I activation in cardiac myocytes (antibody described in Refs. [11,35], 1:2000). Protein expression levels and phosphorylation status of distinct MAPK family members were analyzed by using polyclonal antibodies directed against ERK1/2 (p44/42), p38 MAPK, and JNK, and monoclonal antibodies against phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK. All MAPK antibodies were obtained from Cell Signaling and were used at a dilution of 1:1000. All MAPK antibodies were derived from rabbits, except for the antibody directed against phospho-ERK1/2 which was of murine origin. Expression levels of HO-1 were determined by using a polyclonal rabbit anti-HO-1 antibody from Affinity Bioreagents (1:1000). For control purposes, expression levels of {alpha}-actinin were determined by using a murine monoclonal antibody from Sigma (1:1000). The membranes were extensively washed and developed with sheep anti-mouse (1:2000) or donkey anti-rabbit (1:2000) secondary antibodies linked to horseradish peroxidase using enhanced chemiluminescence (secondary antibodies and ECL kit from Amersham).

2.5. Plasmid constructs, transfection, and luciferase assay
A calcium phosphate procedure was used to transfect cardiac myocytes with a luciferase reporter plasmid driven by three NFAT binding sites (p3xNFAT-GL) [36]. Luciferase activity was measured by using a Lumat LB 9501 luminometer (Berthold, Nashua, NH), as described [12].

2.6. Adenoviral infection
Replication-deficient adenoviruses containing the cDNA of human HO-1 (Ad.HO-1) [37] or PKG I (Ad.PKG I) [38] were used to overexpress HO-1 or PKG I in cardiac myocytes. A replication-deficient adenovirus encoding β-galactosidase was used as a negative control (Ad.LacZ). To activate the calcineurin/NFAT signaling pathway in cardiac myocytes independent from Ca2+, cells were infected with a replication-deficient adenovirus encoding a Ca2+-independent, constitutively active mutant form of mouse calcineurin A{alpha} (Ad.Cn) [39]. For adenoviral infection, cells were switched to maintenance medium supplemented with 5% fetal calf serum. Two hours after infection, cells were switched back to maintenance medium, and allowed to produce the virally encoded proteins for 24 h. Viruses were used at a concentration of 1 x 104 viral particles per cell. At this concentration, infection with Ad.HO-1 increases HO-1 expression levels in cardiac myocytes 10-fold (not shown), whereas infection with Ad.PKG I increases PKG I activity levels 12-fold as compared to non-infected cells [11].

2.7. Statistical analysis
Data are presented as means±S.E.M. Differences between groups were analyzed by one-way ANOVA followed by Student–Newman–Keuls post hoc test. A two-tailed P value of <0.05 was considered to indicate statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Inhibition of cardiac myocyte hypertrophy by heme oxygenase-1 and heme metabolites
Exposure of cardiac myocytes to ET-1 (30 nmol/l) resulted in a robust hypertrophic response as evidenced by significant increases in cell surface area (Fig. 1A), sarcomere assembly (Fig. 1B and C), and prepro-ANP transcript levels (Fig. 1D). ET-1 (30 or 100 nmol/l) did not alter protein expression levels of HO-1 in cardiac myocytes (not shown). Since many inducers of HO-1 (e.g. hypoxia) are non-selective in their function, adenoviral gene transfer was employed to specifically enhance HO-1 expression levels in cardiac myocytes. Adenoviral overexpression of HO-1 significantly blunted cardiac myocyte hypertrophy in ET-1-stimulated cells (Fig. 1A–D). To delineate which HO-1 reaction product(s) may be responsible for this antihypertrophic effect, cardiac myocytes were stimulated with [Ru(CO)3Cl2]2 (10 µmol/l), a CO-releasing molecule that has been used to mimic the actions of heme oxygenase-derived CO [40]. Similar to HO-1 overexpression, [Ru(CO)3Cl2]2 significantly reduced ET-1 induction of all three morphological and molecular markers of cardiac myocyte hypertrophy (Fig. 1A–D). Likewise, stimulation of cardiac myocytes with BV (10 µmol/l) or BR (10 µmol/l) significantly reduced the prohypertrophic effects of ET-1 (Fig. 1A–D). Notably, the HO-1 reaction product ferrous iron (FeSO4, 10 µmol/l) did not promote antihypertrophic effects (reduction of cell size) in ET-1-stimulated cardiomyocytes, and did not affect the antihypertrophic effects of CO, BV, and BR (not shown). [Ru(CO)3Cl2]2, BV, and BR (10 µmol/l each), or adenoviral overexpression of HO-1 did not exert significant effects on cell size, sarcomere assembly, and ANP expression in the absence of ET-1 (not shown). [Ru(CO)3Cl2]2, BV, BR, and FeSO4 (10 µmol/l each), or adenoviral overexpression of HO-1 did not promote cytotoxic effects in cardiac myocytes as revealed by trypan blue dye exclusion and MTT assays performed after 24 h (not shown).


Figure 1
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  		Fig. 1 Inhibition of cardiac myocyte hypertrophy by heme oxygenase-1 and heme metabolites. Cardiac myocytes were stimulated for 24 h with ET-1 (30 nmol/l) in the absence or presence of 10 µmol/l of [Ru(CO)3Cl2]2 (CO), BV, or BR. Where indicated, cells were infected with Ad.HO-1 or Ad.LacZ to overexpress HO-1 or β-galactosidase for 24 h, respectively. Cell surface area was determined by planimetry; >100 cells were analyzed per condition in n=5 experiments (A). Sarcomere assembly was analyzed by confocal laser scanning microscopy of cardiac myocytes immunostained for {alpha}-actinin. Using a semiquantitative grading system, >200 cells from n=3 experiments were analyzed per condition; data are summarized in (B). Typical high power fields are shown in (C). Prepro-ANP mRNA/18S expression levels were assessed by Northern blot. A representative blot and mean data from n=6–8 experiments are shown in (D). *P<0.01, **P<0.001 vs. control. #P<0.05 vs. ET-1 alone.

 
3.2. Antihypertrophic effects of carbon monoxide are independent of cGMP and PKG I
The antihypertrophic effects of the CO-releasing molecule [Ru(CO)3Cl2]2 are reminiscent of the growth-inhibitory effects of the NO/cGMP/PKG I pathway in cardiac myocytes [11,12]. Similar to NO, CO has been shown to act as a modulator of cGMP levels in a number of cell types [41–43]. To determine whether CO mediates its antihypertrophic effects via cGMP/PKG I, we assessed the phosphorylation status of VASP, a cytoskeleton-associated protein and established PKG I substrate in cardiac myocytes [11]. Stimulation of cardiac myocytes with the NO donor SNAP (250 µmol/l) or the PKG-selective cGMP analog 8-pCPT-cGMP (500 µmol/l) resulted in a rapid phosphorylation of VASP at Ser157 and Ser239 (Fig. 2, left panel). [Ru(CO)3Cl2]2 (10 µmol/l), by contrast, did not promote VASP phosphorylation between 15 min and 48 h (Fig. 2). Prolonged activation by NO has been shown to result in a downregulation of PKG I expression in cardiac myocytes [11]. However, PKG I expression levels remained constant over 48 h in [Ru(CO)3Cl2]2-stimulated cardiac myocytes (Fig. 2, right panel), providing another piece of evidence that CO does not activate PKG I in our system. We have previously reported that adenoviral overexpression of PKG I enhances the antihypertrophic effects of NO and cGMP in cardiac myocytes [11]. In contrast to these observations, adenoviral overexpression of PKG I did not enhance the antihypertrophic effects of [Ru(CO)3Cl2]2 or Ad.HO-1 in cardiac myocytes (not shown).


Figure 2
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  		Fig. 2 Determination of PKG I expression and activation (VASP phosphorylation). Cardiac myocytes were stimulated for various times with [Ru(CO)3Cl2]2 (CO, 10 µmol/l), as indicated. Cells stimulated for 30 min with SNAP (NO, 250 µmol/l) or 8-pCPT-cGMP (cGMP, 500 µmol/l) served as positive controls. Cell homogenates were then prepared and analyzed by immunoblotting (30 µg protein/lane). PKG I expression was analyzed with a polyclonal antibody (top panels). A monoclonal antibody directed against the VASP phosphorylation site containing Ser239 was used to detect VASP phosphorylated only at Ser239 (46 kDa) and VASP phosphorylated at both Ser157 and at Ser239 (50 kDa) [35]. Recombinant PKG I and human platelet VASP phosphorylated on both Ser157 and Ser239 were used as migration markers (standard). Two additional experiments yielded similar results.

 
3.3. Heme metabolites inhibit ERK1/2 and p38 MAPK activation
To explore the molecular mechanisms of the antihypertrophic effects of the heme metabolites CO and BV, we determined whether CO and/or BV inhibit signaling through any of the three MAPK branches. As expected, ET-1 (30 nmol/l) stimulation led to rapid (within 5 min) phosphorylation of ERK1/2 (p44/42), p38 MAPK, and JNK in cardiac myocytes (Fig. 3A–F). Phosphorylation levels of ERK1/2 and JNK returned towards baseline within 30 min, whereas phosphorylation levels of p38 MAPK remained elevated during this observation period. [Ru(CO)3Cl2]2 and BV (10 µmol/l each) exerted no significant effects on peak ERK1/2 phosphorylation levels, but accelerated their return to baseline (Fig. 3A and B). Moreover, [Ru(CO)3Cl2]2 and BV significantly reduced p38 MAPK phosphorylation levels at 5 and 15 min (Fig. 3C and D). By contrast, JNK phosphorylation was not significantly affected by [Ru(CO)3Cl2]2 or BV (Fig. 3E and F). It should be noted that BR mediated virtually identical effects on MAPK activation as compared to BV (not shown).


Figure 3
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  		Fig. 3 Heme metabolites inhibit MAPK activation. Cardiac myocytes were treated with ET-1 (30 nmol/l) in the absence or presence of [Ru(CO)3Cl2]2 (CO, 10 µmol/l) or BV (10 µmol/l). ERK1/2 and phospho-ERK1/2 expression levels (panels A and B), p38 MAPK and phospho-p38 MAPK expression levels (panels C and D), and JNK and phospho-JNK expression levels (panels E and F) were analyzed by immunoblotting (30 µg protein/lane). Representative blots are shown in A, C, and E. Data from n=3–5 experiments are summarized in B, D, and F. *P<0.05, **P<0.01 vs. ET-1 alone.

 
3.4. Heme metabolites inhibit the calcineurin/NFAT signaling pathway
To determine whether heme metabolites interfere with the prohypertrophic calcineurin/NFAT signaling pathway, cardiac myocytes were transfected with a luciferase reporter plasmid driven by three NFAT consensus binding sites. [Ru(CO)3Cl2]2, BV, and BR (10 µmol/l each), or adenoviral overexpression of HO-1 did not affect basal NFAT transcriptional activity (not shown). As shown in Fig. 4A, ET-1 stimulation for 24 h (30 nmol/l) increased NFAT transcriptional activity, whereas [Ru(CO)3Cl2]2, BV, BR, or adenoviral overexpression of HO-1 significantly reduced ET-1 stimulated NFAT activation. To distinguish whether HO-1, CO, and BV/BR inhibit calcineurin activation of NFAT upstream or downstream of calcineurin, cardiac myocytes were infected with an adenovirus encoding a constitutively active calcineurin mutant. Expression of the calcineurin mutant for 24 h significantly enhanced NFAT transcriptional activity (Fig. 4B). In contrast to NFAT activation in response to ET-1 treatment (Fig. 4A), NFAT activation by the calcineurin mutant was not significantly affected by [Ru(CO)3Cl2]2, BV, BR, or adenoviral overexpression of HO-1 (Fig. 4B), indicating that HO-1, CO, and BV/BR inhibit the calcineurin/NFAT signaling cascade upstream of calcineurin.


Figure 4
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  		Fig. 4 Inhibition of NFAT transcriptional activity by heme metabolites. Cardiac myocytes were transfected with the luciferase reporter plasmid p3xNFAT-GL. NFAT activation was induced by ET-1 (30 nmol/l, panel A) or adenoviral expression of a constitutively active calcineurin mutant (Ad.Cn, panel B), in the absence or presence of 10 µmol/l of [Ru(CO)3Cl2]2 (CO), BV, or BR. Where indicated, cells were infected with Ad.HO-1 or Ad.LacZ to overexpress HO-1 or β-galactosidase, respectively. Luciferase activities were determined 24 h later. Data from n=4–9 experiments are presented. *P<0.01, **P<0.001 vs. control. #P<0.05 vs. ET-1 alone.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This study provides the first evidence that signaling pathways emanating from heme oxygenase-1 promote antihypertrophic effects in cardiac myocytes. Intriguingly, the growth-inhibitory effects of HO-1 were mimicked by CO and BV/BR (but not by ferrous iron), strongly suggesting their critical involvement in the antihypertrophic effects of HO-1. Biliverdin, BR, and CO targeted MAPKs and the calcineurin/NFAT signaling pathway in cardiac myocytes, thus providing a mechanistic framework of how heme degradation products may promote their antihypertrophic effects.

The recent characterization of CO-releasing molecules has greatly facilitated investigations of the versatile actions of CO in a variety of (patho)physiological conditions [40,44]. For example, [Ru(CO)3Cl2]2 has been used as a CO source to achieve sustained vasodilatory effects in isolated rat aortas [40]. In agreement with previous reports that HO-1 and gaseous CO promote vasorelaxation via cGMP-dependent pathways [19,41], the vasodilatory effects of [Ru(CO)3Cl2]2 were shown to be mediated by soluble guanylyl cyclase [40]. However, three lines of evidence indicate that the antihypertrophic effects of CO are mediated via cGMP/PKG I-independent mechanisms. In contrast to a NO donor and a PKG-selective cGMP analog, [Ru(CO)3Cl2]2 did not promote phosphorylation of VASP, a sensitive downstream target of PKG I in cardiac myocytes [11]. In addition, the CO-releasing molecule did not downregulate PKG I expression in cardiac myocytes, an effect typically observed during prolonged activation of the enzyme [11,45]. Finally, in contrast to the antihypertrophic effects of NO and cGMP which are augmented by adenoviral overexpression of PKG I [11], forced expression of PKG I did not enhance the antihypertrophic effects of CO or adenoviral HO-1 expression in our system.

Biliverdin, BR, and CO suppressed the activation of p38 MAPK and enhanced the decline in ERK1/2 phosphorylation levels during ET-1 stimulation. Interestingly, JNK phosphorylation was not affected. Reactive oxygen species (ROS) can induce cardiac myocyte hypertrophy by activating multiple signaling cascades, including MAPK pathways (reviewed in Ref. [29]). Therefore, antioxidant effects [20,21] may mediate MAPK inhibition by the HO-1/BV/BR pathway in cardiomyocytes. Indeed, by using dichlorofluorescein (DCF) generation from dichlorofluorescein diacetate (DCFH-DA) to quantitate ROS generation, we could demonstrate that BR (but not CO) significantly reduces DCF production in ET-1 stimulated cardiomyocytes (not shown). Interestingly, CO activates p38 MAPK in macrophages [17], indicating that the effects of CO (inhibition vs. activation) on p38 MAPK may be cell type specific.

The calcineurin/NFAT signaling pathway is a central regulator of the hypertrophic response in cardiac myocytes [5]. HO-1, CO, and BV/BR inhibited calcineurin signaling in ET-1 stimulated cardiomyocytes by targeting the calcineurin/NFAT pathway upstream of calcineurin. Similar to CO, NO targets the calcineurin/NFAT pathway upstream of calcineurin, albeit via a different, cGMP and PKG I-dependent mechanism [12]. It has been proposed that calcineurin activation in cardiac myocytes is triggered by Ca2+ entry via the L-type Ca2+ channel (LTCC) [5]. Along this line, the inhibitory effects of the NO/cGMP/PKG I pathway upstream from calcineurin are mediated by LTCC inhibition [12]. Using the same methodology as in our previous report [12], we did not detect any inhibitory effects of CO on single LTCC gating properties in cardiomyocytes (not shown), confirming that CO and NO use distinct molecular pathways to inhibit calcineurin/NFAT hypertrophy signaling. The inhibitory effects of BV/BR on the calcineurin/NFAT pathway suggest that reactive oxygen species facilitate calcineurin activation in cardiac myocytes. In support of this hypothesis, reactive oxygen species have been reported to cooperate with Ca2+ in the activation of calcineurin in fibroblasts [46].

In conclusion, our study identifies HO-1 and its catalytic by-products, CO and BV/BR, as components of a novel antihypertrophic signaling pathway. Biliverdin, BR, and CO inhibition of ERK1/2, p38 MAPKs, and calcineurin/NFAT signaling provides a mechanistic framework how heme degradation products promote antihypertrophic effects. Our study was performed in neonatal cardiac myocytes which enabled us to dissect downstream mechanisms, but was not designed to assess antihypertrophic effects in vivo. However, it was recently reported that pharmacological induction of cardiac HO-1 expression reduces cardiac hypertrophy in stroke-prone SHR rats by a blood pressure-independent mechanism [47]. Interestingly, cardiac HO-1 expression levels are upregulated in animal models and in patients with heart failure [48,49], suggesting that HO-1 may attenuate hypertrophic growth under these conditions. In the future, studies on the antihypertrophic effects of HO-1 should be extended to investigations with transgenic mice carrying HO-1 gain- or loss-of-function mutations.


   Acknowledgements
 
We gratefully acknowledge Nelli Otto for the expert technical assistance. We thank Drs. X.M. Liu, J.D. Molkentin, and G.R. Crabtree for providing us with Ad.HO-1, Ad.Cn, and p3xNFAT-GL, respectively. This work was supported by grants from the Deutsche Forschungsgemeinschaft to K.C.W. (Wo 552/2-2) and S.M.L. (SFB 355), and an early career grant from Hannover Medical School to J.T. (HiLF Program).


   Notes
 
Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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