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Cardiovascular Research 2005 68(3):464-474; doi:10.1016/j.cardiores.2005.06.020
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Copyright © 2005, European Society of Cardiology

Endothelin-1 and isoprenaline co-stimulation causes contractile failure which is partially reversed by MEK inhibition

Felix Münzela, Ulrike Mühlhäusera, Wolfram-Hubertus Zimmermannb, Michael Didiéb, Karin Schneiderbangera, Pia Schuberta, Sven Engmanna, Thomas Eschenhagenb and Oliver Zolka,*

aInstitut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany
bInstitut für Experimentelle und Klinische Pharmakologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany

* Corresponding author. Tel.: +49 9131 8522783; fax: +49 9131 8522773. Email address: Zolk{at}pharmakologie.uni-erlangen.de

Received 1 July 2004; revised 8 June 2005; accepted 17 June 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The mitogen-activated kinase kinases (MEK)-extracellular signal-regulated kinases (ERK) signaling pathway is activated by agonists like catecholamines or endothelin-1 (ET-1) and has been implicated in cardiac pathology, such as the progression from cardiac hypertrophy to failure. The purpose of the present study, performed in an in vitro model of contractile failure, was to evaluate whether MEK inhibition prevents functional deterioration.

Methods and results: Contractile dysfunction was induced in reconstituted rat heart tissue by concomitant treatment with ET-1 (10 nmol/l) and isoprenaline (ISO, 10 nmol/l) for 5 days. While basal force of contraction was unchanged, contractile responsiveness to β-adrenoceptor agonists was markedly impaired (active force declined to 51% of controls) and was associated with decreased lusitropy. Moreover, in ET-1+ISO-treated heart tissues, reprogramming of gene expression was observed with an increased ratio of β-myosin heavy chain (MHC) to {alpha}-MHC mRNA and increased transcript levels of ANF and skeletal/smooth muscle {alpha}-actin isoforms. The MEK inhibitor U0126 (10 µmol/l) almost completely prevented the reduction in β-adrenergic responsiveness and the negative lusitropic effect of ET-1+ISO co-stimulation. In addition, U0126 completely normalized ANF gene expression, but did not affect or only marginally affected expression of MHC and {alpha}-actin isoforms.

Conclusions: These results suggest that interruption of the MEK-ERK signaling pathway with a specific MEK inhibitor prevents, in part, the occurrence of a pathologic phenotype secondary to excessive stimulation with neurohumoral factors. The MEK-ERK pathway seems to be an important but not exclusive regulatory pathway responsible for the development of contractile dysfunction.

KEYWORDS Endothelin-1; Isoprenaline; MAP kinases; ERK1/2; ERK5; Contractile function; Engineered heart tissue; U0126; Cardiac myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Chronic heart failure is a complex syndrome involving the activation of multiple cellular, metabolic, and neurohumoral pathways following the initial myocardial insult. Activated neurohormones, growth factors, cytokines and increased mechanical stress have been implicated as mediators of cardiac hypertrophy and failure. Consequently, the most successful pharmacological strategy thus far has been the blockade of activated neurohumoral vasoconstrictor systems associated with disease progression, specifically the sympathetic nervous system and the renin–angiotensin system.

Despite these advances, chronic heart failure remains a debilitating condition with high mortality. Currently, two major new approaches are being pursued. The first one includes inhibitors of additional neurohormonal vasoconstrictor systems, antagonists of cytokines, and agents that favourably modulate extracellular matrix. A number of novel drugs are currently under clinical evaluation. The second approach, which still awaits proof of concept by preclinical studies, aims at targeting intracellular signaling molecules, such as mitogen activated protein (MAP) kinases, and, thus, differs fundamentally from the traditional concept of blocking specific neurohormone receptors [1]. This novel concept is based on the observation that the MAP kinase family represents an intersection connecting different signaling pathways and thereby modulates diverse processes including cardiac hypertrophy, reexpression of fetal genes such as atrial natriuretic factor, and apoptosis.

Activation of MAP kinases has been demonstrated in transgenic models of heart failure [2] as well as in man [3]. For example, samples obtained from patients with end-stage heart failure due to dilated cardiomyopathy revealed a marked increase in extracellular signal-regulated kinases 1/2 (ERK1/2) activity, while the activation of p38 kinase was decreased [4]. By comparison, levels of activated ERK1/2 were unchanged in heart samples from patients with heart failure secondary to ischemic heart disease and levels of p38 activation were significantly increased [3,5]. Flesch et al. showed that mechanical unloading of the heart through a left ventricular assist device (LVAD) lead to significant reductions in the activity of ERK1/2, whereas p38 activity levels were significantly increased after LVAD support [6].

Recent findings obtained in transgenic animals suggest that activation of ERK signaling molecules is sufficient to generate a hypertrophic response, and under certain conditions it induces concomitant cardiac dysfunction. Transgenic mice in which ERK1/2 were activated by cardiac overexpression of either Ras or MAP kinase kinase 1 (MEK1) developed massive cardiac hypertrophy [7,8]. Transgenic overexpression of constitutively active Ras was associated with abnormal left ventricular diastolic function [7]. MEK1 transgenic mice, however, exhibited concentric cardiac hypertrophy without signs of cardiomyopathy or increased lethality [8]. In contrast, cardiac specific expression of constitutively active MEK5, the activating MAPKK for ERK5, resulted in eccentric cardiac hypertrophy that rapidly progressed to dilated cardiomyopathy and sudden death [9].

The studies performed so far demonstrate that MAP kinases are differentially regulated during heart disease and highlight the possibility that pharmacological modulation may restore the balance among the MAP kinase branches. Thus, the present study addressed the question whether inhibition of the MAP kinase kinases (MEK)-ERK pathway, which has been linked to cardiac hypertrophy and contractile dysfunction, benefits or further exacerbates the heart failure phenotype, i.e. improves or deteriorates contractile function. We investigated reconstituted rat heart tissue (engineered heart tissue, EHT) in which contractile failure was induced by β-adrenoceptor agonist and endothelin-1 (ET-1) co-stimulation. This in vitro model mimics co-activation of several neurohumoral pathways in heart failure under well defined conditions. We thought, therefore, that this in vitro model will be suitable to assess general implications of MEK-ERK inhibition for contractile performance of heart tissue. In detail, we tested the drug U0126, which specifically prevents the activation of MEK1 and MEK5 (and hence the activation of ERK1/2 and ERK5) for its effects on twitch tension, twitch kinetics, and expression of hypertrophy-related genes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Engineered heart tissue
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Engineered heart tissue was prepared as described in detail before [10,11]. Cardiac cells isolated from hearts of 1–3 days old Wistar rats (2.5 x 106/EHT) were mixed with collagen type I, Matrigel, Dulbecco's modified Eagles medium (DMEM), 10% horse serum, 2% chick embryo extract, 100 U/ml penicillin, and 100 µg/ml streptomycin. The resulting reconstitution mix was pipetted into circular casting molds and incubated for 45 min to let the reconstitution of mixture gel. Medium (DMEM, 10% horse serum, 2% chick embryo extract, 100 U/ml penicillin, 100 µg/ml streptomycin) was added. After 6 days in culture, ring-shaped EHTs were transferred to a holder, where EHTs were exposed to continuous unidirectional static stretch secondary to collagen elasticity and further collagen condensation. Culture medium was changed 12 h after EHT casting and then every other day while the culture was performed in casting molds. After transfer to the holder, the culture medium was changed every day. EHTs were treated with 10 nmol/l ET-1 and 10 nmol/l ISO for another 5 days. To investigate the effect of MAP kinase kinase (MEK) inhibition, EHTs were exposed to ET-1+ISO in the presence or absence of U0126, a selective inhibitor of MEK-dependent intracellular ERK phosphorylation. U0126 was added at the same time when ET-1+ISO was added. The concentration of U0126 (10 µmol/l) was chosen from knowledge of its inhibitory potency [12] and its ability to inhibit the activation of ERK1/2 and ERK5 in neonatal cardiomyocytes.

2.2 Force measurement
EHTs were transferred into gassed organ baths (37 °C, modified Tyrode's solution with 0.2 mmol/l Ca2+). After 30 min equilibration without pacing, EHTs were electrically stimulated with rectangular pulses (2 Hz, 5 ms, 100–120 mA). Preload was adjusted to Lmax, i.e. the length where EHTs developed maximal active force. Inotropic and lusitropic responses to cumulative concentrations of Ca2+ (0.2–2.8 mmol/l) and isoprenaline (0.1–1000 nmol/l) were recorded. Twitch tension, contraction duration (T1: time from 50% to peak force development), and relaxation duration (T2: time to 50% relaxation) were evaluated by BMON software (Jäckel, Hanau, Germany). Maximum twitch tension achieved with cumulative Ca2+ concentrations was used to correct for differences in the number of cardiomyocytes reconstituted in individual EHTs.

2.3 Gene expression analysis by real-time RT-PCR
Total RNA (2 µg) isolated from EHTs according to standard protocols was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase and cDNA was subsequently amplified with the TaqMan system (Prism 7700, PE Biosystems) as described previously [11]. The level of calsequestrin mRNA in each sample was used to normalize for the variability in RNA quantity or differences in the efficiency of the RT-reaction. Calsequestrin gene expression was not significantly affected by any intervention and therefore could be used as a housekeeping gene.

2.4 Histology
Immunofluorescent staining and confocal microscopy of whole-mount EHTs were performed essentially as described [11]. A monoclonal antibody against sarcomeric actinin (Sigma, 1:800) and a Cy3-conjugated secondary antibody against mouse IgG (Sigma, 1:1000) were used. Nonstriated fiber length was measured (in micrometer) from the end of the cell to the first striation. Fig. 5B illustrates the definition of myofibril diameters.


Figure 5
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  		Fig. 5 Contractile properties of EHTs treated with U0126 in the presence or absence of ET-1+ISO. Concentration-dependent effects of isoprenaline on twitch tension and twitch kinetics were determined in control EHTs (n=20), EHTs treated with ET-1+ISO (n=15), U0126 (n=15) or ET-1+ISO+U0126 (n=15). (A) Force of contraction (FOC) and change in force of contraction from baseline ({Delta}FOC); (B) clinotropic (time to peak tension, T1) and lusitropic effects (time to relaxation, T2). *p<0.05 vs. control, two-way ANOVA; #p<0.05 vs. ET-1+ISO+U0126, two-way ANOVA.

 
2.5 Binding assay
Binding assays were performed to determine the density of β-adrenoceptors (Bmax) in control EHTs or EHTs treated with ISO, ET-1, ISO+ET-1, U0126, or ISO+ET-1 in the presence of U0126, respectively. After treatment, each individual EHT was cut into halves for measurement of total and nonspecific binding. EHTs were washed twice with binding buffer (MEM, 20 mmol/l HEPES, pH 7.4), followed by incubation with the β1/2 adrenoceptor ligand 3H-CGP-12177 (2 nmol/l, Perkin Elmer) in binding buffer for 2 h at 4 °C. After three rapid washes with ice-cold ligand-free binding buffer, 1 mol/l NaOH containing 0.01% SDS was added, and dissociated radioligand was quantified by liquid scintillation counting. Nonspecific binding was determined in the presence of 3 µmol/l propranolol.

2.6 Western blotting
Immunoblots were performed as described earlier [11]. Blots were probed with antibodies against calsequestrin (Affinity Bioreagents, 1:2000), sarcomeric {alpha}-actin (Sigma, 1:5000), or Thr202/Tyr204 phosphorylated ERK1/2 (Cell Signaling, 1:2000). Phospho-ERK1/2 blots were stripped in 100 mmol/l mercaptoethanol, 2% SDS, 62.5 mmol/l Tris–HCl pH 6.7 at 50 °C for 30 min and reprobed for total ERK protein with an ERK1/2 antibody (Cell Signaling, 1:2000).

2.7 Immunoprecipitation and Western blot analysis of ERK5
After stimulation, cardiomyocytes or EHTs were immediately lysed in lysis buffer (50 mmol/l Tris–HCl, pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Triton X-100, 1 mmol/l PMSF, 1 mmol/l NaF, 1 mmol/l sodium orthovanadate, 1 µg/ml aprotinin). The cleared lysates were incubated with 3 µl of anti-ERK5 rabbit antiserum (Upstate) and 25 µl protein A-sepharose for 12 h at 4 °C. After 4 washes with lysis buffer, collected immune complexes were eluted with 20 µl of 2 x Laemmliés SDS loading buffer, separated on an 8% SDS polyacrylamide gel, electrotransferred to a nitrocellulose membrane, and processed for immunoblotting using the ERK5 polyclonal antiserum (Upstate, 1:2000).

2.8 Statistical analysis
All summary values are expressed as mean ± SEM. Two-way analysis of variance (ANOVA) was used for statistical analysis of concentration–response curves. All other comparisons were made by ANOVA followed by post hoc analysis with the Bonferroni correction for multiple comparisons. All statistical analyses were done with the GraphPad software, and a value of p<0.05 was considered to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Contractile properties of EHTs treated with ET-1, ISO or ET-1+ISO
Force of contraction (FOC) and twitch kinetics (T1, T2) were measured after 5 days of intervention. Fig. 1 summarises the effects of ET-1 (10 nmol/l), ISO (10 nmol/l) and combined ET-1 and ISO treatment on isometric force and twitch kinetics in EHTs. ET-1 treatment alone caused a significant increase in basal FOC (0.57 ± 0.05 vs. 0.33 ± 0.03 mN) and markedly diminished the inotropic response to ISO ({Delta}FOC; 0.16 ± 0.04 vs. 0.41 ± 0.03 mN; two-way ANOVA p<0.05). In contrast, ISO treatment did not change basal FOC (0.38 ± 0.05 mN) and only slightly decreased {Delta}FOC (0.31 ± 0.06 mN, nonsignificant vs. controls). Co-stimulation with ET-1 and ISO induced a contractile phenotype which was different from that provoked by separate ET-1 or ISO stimulation. Basal FOC did not differ from controls (0.35 ± 0.04 mN), but {Delta}FOC was markedly decreased (0.21 ± 0.03 mN; two-way ANOVA p<0.05). ET-1, ISO, as well as ET-1 and ISO co-treatment significantly prolonged T1 and T2 by ~50% and ~40%, respectively (Fig. 1B).


Figure 1
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  		Fig. 1 Contractile properties of EHTs treated with ET-1, ISO or ET-1+ISO. Concentration-dependent effects of isoprenaline on twitch tension and twitch kinetics were determined in control EHTs (n=20), EHTs treated with ET-1 (n=6), ISO (n=6) or ET-1+ISO (n=15). (A) Force of contraction (FOC) and change in force of contraction from baseline ({Delta}FOC); (B) clinotropic (time to peak tension, T1) and lusitropic effects (time to relaxation, T2). *p<0.05 vs. control, two-way ANOVA; #p<0.05 vs. ET-1+ISO, two-way ANOVA.

 
3.2 Effects on β-adrenoceptor density
As shown in Fig. 2 ISO treatment of EHTs significantly downregulated β-adrenoceptor density by 40%. ET-1 slightly increased β-adenoceptor density which was significantly reduced by concomitant treatment with ISO. Compared to control EHTs, total number of β-adrenoceptors was not significantly changed after treatment with ET-1+ISO, U0126, or ET-1+ISO in the presence of U0126.


Figure 2
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  		Fig. 2 Treatment effects on the β-adrenoceptor density in EHTs. β-adrenoceptor density was determined in EHTs after treatment with ISO, ET-1, U0126, ET-1+ISO, and ET-1+ISO in the presence of U0126. *p<0.05.

 
3.3 Phosphorylation of ERK
Figs. 3 and 4Go show the effects of ET-1+ISO and U0126 on phosphorylation of ERK in isolated neonatal cardiomyocytes (A) and in EHTs (B). Cultured myocytes or EHTs were treated with ET-1+ISO for 15 min, and cell lysates were analysed by quantitative immunoblotting using antibodies against ERK5 or phosphospecific ERK1/2. In the case of ERK5 precipitation was necessary for adequate immunodetection of the protein. ET-1+ISO-stimulation caused a reduced electrophoretic mobility (phosphorylation shift, Fig. 3) of ERK5, indicative of ERK5 activation. In cardiomyocytes, ET-1+ISO increased the amount of phosphorylated ERK5 by 69 ± 12% (n=7, p<0.05). The stimulated phosphorylation of ERK5 was effectively blocked by pretreatment with the MEK inhibitor U0126. The representative blot shown in Fig. 3B demonstrates that ERK5 is similarly activated in EHTs and that this activation is blocked by U0126. In parallel, ET-1+ISO treatment significantly increased the activity of ERK1/2 in EHTs by 98 ± 15% (n=5, p<0.001) which was completely blocked by U0126 (Fig. 4). U0126 by itself diminished basal ERK1/2-phosphorylation by 71 ± 11% (n=5, p<0.01) compared to untreated controls. In the presence of U0126 ET-1+ISO augmented ERK1/2 phosphorylation by 236 ± 49% compared to U0126 treated controls (n=5, p<0.01).


Figure 3
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  		Fig. 3 Analysis of ERK5 phosphorylation by Western blot mobility shift. Cardiomyocytes (A) or EHTs (B) were stimulated for 15 min with ET-1 (10 nmol/l)+isoprenaline (ISO, 10 nmol/l) in the presence or absence of U0126 (10 µmol/l). For control, cells were treated with U0126 alone. Cell lysates were prepared and ERK5 immunoprecipitated, followed by Western blot analysis. Four EHTs per group were pooled for immunoprecipitation and Western blot analysis. Phosphorylated and unphosphorylated forms of ERK5 are indicated. The ratio of phosphorylated ERK5 to total ERK5 in cardiomyocytes was quantified by densitometry. *p<0.05 vs. control; #p<0.05 vs. ET-1+ISO.

 

Figure 4
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  		Fig. 4 Phosphorylation of ERK1/2. Cardiomyocytes (A) or EHTs (B) were stimulated for 15 min with ET-1 (10 nmol/l)+isoprenaline (ISO, 10 nM) in the presence or absence of U0126 (10 µmol/l). For control, cells were treated with U0126 alone. Cell lysates were prepared and ERK1/2 phosphorylation was measured by Western blot analysis with a phospho-specific ERK antibody (upper). No difference in the amount of ERK1/2 protein was observed (lower). The ratio of phosphorylated ERK1/2 to total ERK1/2 in EHTs was quantified by densitometry. *p<0.05 vs. control; #p<0.05 vs. ET-1.

 
3.4 Effects of MEK inhibition on contractile function in EHTs treated with ET-1+ISO
Fig. 5 summarises the effects of the MEK inhibitor U0126 (10 µmol/l) on contractile performance. U0126 alone had no significant effects on {Delta}FOC and twitch kinetics, but induced a parallel downward shift of the concentration–response curve for the inotropic effects of ISO (Fig. 5A). Compared to control EHTs, basal and maximun FOC were decreased by about 0.1 mN (two-way ANOVA p<0.05 for group effect). In ET-1+ISO-stimulated EHTs, co-incubation with U0126 partially normalised twitch kinetics (Fig. 5B) and almost completely normalised the inotropic response to ISO ({Delta}FOC 0.35 ± 0.03 vs. 0.21 ± 0.03 mN in the ET-1+ISO group and 0.41 ± 0.03 in controls).

3.5 Morphology of cardiac myocytes
Myocardial hypertrophy is characterised by increases in cell size and sarcomerogenesis over and above that which would be predicted for a given stage of development. In cultured cardiomyocytes cell surface area is generally used as an estimate of cellular hypertrophy. Because of its three dimensional nature estimation of cell surface area is difficult in EHTs. Instead, we determined the diameter of myofibril bundles as a morphological correlate of cellular hypertrophy. To identify cardiomyocytes and to depict their contractile apparatus immunofluorescence staining of whole-mount EHT preparations was performed with an anti-{alpha}-actinin antibody. Compared to control EHTs, stimulation with ET-1+ISO significantly increased the diameter of myofibril bundles (13.1 ± 0.4 vs. 10.2 ± 0.5 µm, n=30, p<0.05) and the length of nonstriated premyofibrils (23.2 ± 1.7 vs. 4.8 ± 0.9 µm, n=30, p<0.05). These alterations were prevented by treatment with U0126 (Fig. 6).


Figure 6
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  		Fig. 6 Immunohistochemistry. Immunofluorescent staining of {alpha}-actinin was performed in EHTs treated with ET-1+ISO in the presence or absence of U0126 for 5 days or in control EHTs. (A) Representative sections from 4 different EHTs per group were investigated. Photomicrographs were taken of randomly chosen fields at x 400. Nonstriated portions of actinin-positive filaments (arrow) were mainly observed in ET-1+ISO treated EHTs. (B) Diameter of myofibril bundles and (C) length of nonstriated cables. The schematic representation in (B) illustrates the definition of myofibril diameters.

 
3.6 Gene expression
The molecular program of cardiac hypertrophy is often associated with altered expression of contractile genes or fetal encoded genes in the heart. Accordingly, real-time reverse-transcription polymerase chain reactions were performed to quantify expression of ANF, {alpha}-actin isoforms and myosin heavy chain (MHC) isoforms. Calsequestrin was selected as the internal standard for the target genes, as the levels of calsequestrin transcripts were most consistent and did not significantly differ between control and treatment groups.

In EHTs treated with ET-1+ISO gene expression of ANF and the ratio of β-MHC mRNA/{alpha}-MHC mRNA were markedly increased by 110 ± 27% (p<0.001) and 106 ± 29% (p<0.001), respectively (Fig. 7). Moreover, smooth muscle and skeletal {alpha}-actin transcript levels were significantly augmented while cardiac actin mRNA remained unchanged. U0126 itself significantly decreased ANF mRNA levels by 69 ± 5% (p<0.001) when compared to untreated control EHTs, but did not affect MHC or skeletal/smooth muscle {alpha}-actin isoform gene expression. U0126 almost completely prevented ET-1+ISO-stimulated ANF induction, but did not (β-MHC, skeletal {alpha}-actin) or only moderately (smooth muscle {alpha}-actin) affect induction of fetal isoforms of contractile proteins. Similar to mRNA findings, U0126 did not prevent agonist-stimulated increase in sarcomeric {alpha}-actin at protein level (Fig. 8).


Figure 7
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  		Fig. 7 mRNA expression. mRNA expression of atrial natriuretic factor (ANF), β-myosin heavy chain (MHC), {alpha}-MHC, cardiac {alpha}-actin, skeletal muscle {alpha}-actin, and smooth muscle {alpha}-actin was determined by real-time RT-PCR in EHTs stimulated with ET-1+ISO in the presence (n=10) or absence of U0126 (n=10). Untreated EHTs (Ctr, n=10) or EHTs treated with U0126 alone (n=10) served as controls. Shown are the ratios of {alpha}-MHC/β-MHC, skeletal/cardiac {alpha}-actin and smooth muscle/cardiac {alpha}-actin gene expression. For quantification of ANF transcripts, calsequestrin mRNA was used as an internal standard. *p<0.05 vs. Ctr, #p<0.05 vs. ET-1+ISO.

 

Figure 8
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  		Fig. 8 Sarcomeric {alpha}-actin concentrations. The amount of sarcomeric {alpha}-actin protein (skeletal and cardiac isoforms) was determined by Western blot analysis in EHTs stimulated with ET-1+ISO in the presence (n=10) and absence of U0126 (n=9). Untreated EHTs (Ctr, n=9) and EHTs treated with U0126 alone (n=10) served as the respective controls. Calsequestrin (CSQ) protein expression was not significantly changed by treatment and therefore served as an internal control. *p<0.05 vs. Ctr.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Among a plethora of vasoactive peptides, growth factors and cytokines activated in heart failure, endothelin-1 and catecholamines have been implicated in disease progression and contractile failure [13,14]. To better understand the effects of endothelin-1 (ET-1) and catecholamine stimulation on contractile performance, we employed an in vitro approach using reconstituted rat heart tissue (engineered heart tissue, EHT). Our main novel finding is that combined stimulation with endothelin-1 and the β-adrenoceptor agonist isoprenaline (ISO) for 5 days induced contractile dysfunction in EHTs. Specifically, the inotropic response to β-agonist stimulation was markedly diminished and kinetics of contraction and relaxation were significantly impaired, while basal force was unchanged. The contractile dysfunction induced by ET-1+ISO co-stimulation was prevented in part by pharmacological MEK inhibition.

The finding that long-term treatment of EHTs with 10 nmol/l ET-1 raised baseline developed tension but decreased delta force response to ISO confirms previous observations [11]. As demonstrated before, inhibition of both the protein kinase C (PKC) and the Na+/H+-exchanger (NHE) completely reversed the effects of ET-1 on twitch tension and significantly improved twitch dynamics [11]. The NHE has been demonstrated to alter intracellular pH and calcium concentration, both potentially influencing myocyte contractility [16]. These mechanisms may explain elevated baseline developed tension in ET-1-treated EHTs. Moreover, PKC activation has been associated with abnormally slowed myocardial relaxation due to the downregulation of SERCA2a. Indeed, in EHTs treated with ET-1 we observed decreased SERCA2a transcript and protein levels [11]. Of note, PKC has also been linked to ERK activation, which in turn represents one of the strongest NHE activating kinases [17].

Depending on the extent of β-adrenergic prestimulation, acute administration of ET-1 had positive or negative inotropic effects in isolated canine trabeculae [15] suggesting a cross-talk between the β-adrenergic system and the ET system. Whether similar interactions take place in a situation of chronic neurohumoral activation characteristic for certain cardiovascular disorders, such as congestive heart failure and ischemic heart disease, is currently unknown. The present study demonstrates that co-stimulation with ET-1 and ISO for 5 days aggravates the contractile failure phenotype induced by ET-1 (diminished delta force response and impaired twitch kinetics) or ISO (impaired twitch kinetics) alone.

Exposure of EHTs to ISO did not show significant desensitisation with regard to force development, although ISO at 10 nM significantly decreased total number of β-adrenoceptors in EHTs by 40%. Moreover, concomitant treatment with U0126 did not change β-adrenoceptor density compared to EHTs treated with ET-1+ISO alone although U0126 partially improved contractile function in EHTs exposed to ET-1+ISO. These apparently discrepant findings are likely explained by the high fraction of spare receptors for β-adrenoceptor-mediated positive inotropic effects in rat cardiomyocytes. Indeed, 50% of maximum contractile response in rat papillary muscles was produced with only 1–3% of beta-adrenoceptor occupancy [18]. Thus, changes in β-adrenoceptor density are unlikely to be directly translated into contractile effects in this model. However, we cannot rule out that changes in postreceptor signaling, i.e. cAMP generation, might explain some of the effects on contractile function, as we did not investigate adenylyl cyclase activity in our experimental setting.

In addition to the functional impairment, ET-1+ISO co-stimulation initiated transcriptional changes which include augmented expression of ANF, β-MHC, skeletal muscle {alpha}-actin, and smooth muscle {alpha}-actin. This response profile of genetic markers is well known from cardiomyocytes treated with ET-1 or β-adrenoceptor agonists alone and characterises the hypertrophic phenotype in these cells [11,19]. Our observations correspond to the current concept which implicates activated neurohormones and growth factors as mediators of cardiac hypertrophy and failure [20]. Most of the alterations described here in EHTs resemble the well known changes observed in human heart failure or are consistent with changes observed in animal models of ventricular dysfunction [21,22].

Besides effects on contractile parameters exposure to ET-1+ISO induced significant changes in cellular morphology with an increase in myofibril diameter. Moreover, a marked increase in premyofibril length was observed, suggesting that ET-1+ISO co-stimulation induced growth of sarcomeric units in parallel and delayed longitudinal differentiation. Of note, ET-1 alone was not able to augment protein synthesis nor increased cardiomyocyte diameter in EHTs although gene expression as part of the hypertrophic growth program was already stimulated [11]. Thus, ET-1 and ISO may have synergistic effects accelerating hypertrophy-associated changes in cellular morphology.

The agonists isoprenaline and endothelin-1 initially utilize dissimilar signaling pathways which finally converge at the level of MEK/ERK [23,24]. While the relevance of MEK/ERK signaling for the hypertrophic response of cardiac myocytes has been studied extensively [25,26], little is known about the effects on contractile performance. Recent transgenic studies by Nicol et al. demonstrate that cardiac-specific overexpression of the activating MAPKK for ERK5, MEK5, induces severe dilated cardiomyopathy characterised by thinning of the ventricular walls and decreased cross-sectional area of individual myocytes [9]. Previously, Bueno et al. showed that MEK1-transgenic animals developed concentric cardiac hypertrophy, which was associated with increased fractional shortening in vivo, as measured by echocardiography [8]. Thus, transgenic studies performed so far produced different results for MEK1 and MEK5 overexpression although both studies indicate that MEK-ERK pathways may play a critical role in the regulation of cardiac function.

In the current study, the functional relevance of MEK-ERK pathways for contractility of the myocardium has been addressed by inhibitor experiments. The U0126 compound was used, because it is known to inhibit ERK1/2 and ERK5 activation specifically at micromolar concentrations [25,12] without affecting p38 MAPK and JNK in cardiomyocytes [25]. The concentration of U0126 was titrated to completely reverse the effects of ET-1 and ISO co-stimulation on ERK1/2 and ERK5 phosphorylation. U0126 was finally used at a concentration of 10 µmol/l which corresponds to the IC50 for inhibition of MEK1 in vitro (13 µmol/l, [12]). Although U0126 diminished basal force, inotropic responsiveness and contraction kinetics were significantly improved in EHTs chronically exposed to ET-1 and ISO. These findings suggest that contractile dysfunction following ET-1 and ISO stimulation is due, in part, to ERK activation. Altogether, our data provide evidence that MEK inhibition may reverse some facets of the contractile failure phenotype induced by chronic neurohumoral stimulation. However, these beneficial effects are counterbalanced, at least in part, by diminished basal force.

Besides effects on contractile parameters, U0126 completely abolished stimulated expression of ANF mRNA. The latter finding is in line with previous reports suggesting that MEK1/2 and MEK5 cooperate to induce the fetal gene expression program. For example, U0126 effectively inhibited ET-1-induced expression of ANF mRNA in neonatal rat cardiomyocytes [25]. Gillespie-Brown et al. more directly demonstrated that transfection of a dominant negative MEK1 construct attenuated ANF promotor activity. Conversely, specific activation of the ERK cascade by expression of constitutively active MEK1 or MEK5 mutants induced ANF [27,9]. While U0126 completely abolished the increase in ANF transcript expression induced by treatment with ET-1+ISO, only minor effects on mRNA levels of other hypertrophy-related genes, such as β-MHC, skeletal muscle {alpha}-actin, and smooth muscle {alpha}-actin, were observed. Collectively, the current data suggest that the MEK/ERK pathway seems to be an important, but not the exclusive regulatory pathway responsible for hypertrophic and transcriptional responses induced by ET-1+ISO-stimulation. Moreover, our findings suggest that some aspects of the contractile failure phenotype induced by long-term exposure to ET-1+ISO may depend primarily on ERK activation, like the diminished inotropic responsiveness to β-agonist stimulation, whereas other aspects are less dependent on ERK signaling.

The contractile effects of ET-1 and ISO co-stimulation or MEK inhibition in EHTs are not easily explained and several aspects have to be addressed. First, EHTs represent a composite heart muscle construct. Because EHTs were reconstituted from unfractionated heart cells, the different cell types found in the native heart are also found in EHTs [10]. This includes the possibility of paracrine signaling between the different cell species which may affect contractility of the whole muscle construct. Moreover, potential cross-talk between ET-1 and catecholamines might modulate the hypertrophic response [28] and contractile function [18]. Currently we do not know whether ET-1+ISO-induced contractile impairment in EHTs is mediated by direct or indirect mechanisms involving cross-talk or paracrine signaling. Second, recent work has indicated that U0126 reverses stimulated collagen expression in cardiac fibroblasts [29]. Decreased collagen synthesis should change stiffness of the EHT which usually correlates with a decreased resting tension of the heart muscle. However, we did not observe a significant change in the resting tension after U0126 treatment nor after ET-1+ISO co-stimulation (data not shown), and it remains unclear whether significant remodelling occurs which might explain altered contractile performance. Finally, model-specific limitations have to be addressed. EHTs have some advantages over in vitro studies using 2D cultures of fractionated cells. Fractionated single cardiomyocytes suffer from the fact that some basic functional properties vanish when one moves down the hierarchic scale from multicellularity to single cells. EHTs, however, combine standardisation of in vitro test systems with multicellularity. Although histological features of myocytes in EHTs resemble those of myocytes within adult native myocardium [10] one should keep in mind that cardiac cells in EHTs originate from neonatal, i.e. immature hearts.

Considering these potential limitations, our data support the notion that the MEK/ERK signaling pathway might play an important but not an exclusive role in mediating cellular hypertrophy and functional impairment of cardiomyocytes following concomitant stimulation with ET-1 and ISO. Since the contractile failure phenotype was not completely reversed by MEK inhibition, the current results implicate one or more additional classes of MAPK in the mediation of contractile dysfunction. Nevertheless, our study highlights the possibility that pharmacological modulation of the MEK/ERK pathway may improve contractile performance in heart failure. Future animal studies will have to prove this hypothesis.


   Acknowledgements
 
The study was supported by the Deutsche Forschungsgemeinschaft (TE 88/8-2, Zo 123/1-2). The authors thank Nicola Peters for technical assistance.


   Notes
 
Time for primary review 28 days


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

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