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Am J Physiol Endocrinol Metab 288: E1214-E1221, 2005. First published January 4, 2005; doi:10.1152/ajpendo.00261.2004
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Chronic cardiotrophin-1 stimulation impairs contractile function in reconstituted heart tissue

Oliver Zolk, Sven Engmann, Felix Münzel, and Rasti Krajcik

Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany

Submitted 17 June 2004 ; accepted in final form 3 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiotrophin-1 (CT-1) is known to promote survival but also to induce an elongated morphology of isolated cardiac myocytes, leading to the hypothesis that CT-1, which is chronically augmented in human heart failure, might induce eccentric cardiac hypertrophy and contractile failure. To address this, we used heart tissues reconstituted from neonatal rat cardiac myocytes (engineered heart tissue, EHT) as multicellular in vitro test systems. CT-1 dose-dependently affected contractile function in EHTs. After treatment with 0.1 nM CT-1 (corresponds to plasma levels in humans) for 10 days, twitch tension significantly decreased to 0.30 ± 0.04 mN (n = 15) vs. 0.45 ± 0.04 mN (n = 16) in controls. Furthermore, positive inotropic effects of cumulative concentrations of Ca2+ and isoprenaline were significantly diminished. Maximum isoprenaline-induced increase in twitch tension amounted to 0.27 ± 0.04 mN (n = 15) vs. 0.47 ± 0.06 mN (n = 16) in controls (P < 0.001). When EHTs were treated for only 5 days, qualitatively similar results were obtained but changes were less pronounced. Immunostaining of whole mount EHT preparations revealed that after CT-1 treatment, the number of nonmyocytes significantly increased by 98% (1 nM, 10 days), and myocytes did not form compact, longitudinally oriented muscle bundles. Interestingly, expression of the Ca2+-handling protein calsequestrin was markedly reduced (69 ± 7% of control) by treatment with CT-1 (0.1 nM, 10 days). In summary, long-term exposure to CT-1 induces contractile dysfunction in EHTs. Structural changes due to impaired differentiation and/or remodeling of heart tissue may play an important role.

engineered heart tissue; signal transducer and activator of transcription 3

CARDIOTROPHIN-1 (CT-1) originally was discovered as a factor that can induce hypertrophy of cardiac myocytes, both in vitro and in vivo (11, 14). Subsequently, CT-1 has been shown to have a wide variety of effects on different cell types, including the ability to induce hypertrophy but also to stimulate the survival of cells (19). The apoptotic death of embryonic or neonatal cardiac myocytes in defined serum-free medium was reduced by treatment with CT-1, which has been attributed to an activation of the types 1 and 2 extracellular signal-regulated kinases (ERK1/2) (11). These findings are consistent with the general observation that, in most cell types and conditions, the ERK cascade seems to have an antiapoptotic effect, and a reduction in its activity is essential for the process of apoptosis to proceed (19).

CT-1 induces a hypertrophic response in cardiac myocytes that is distinct from the hypertrophic response observed after G protein-coupled receptor (GPCR) stimulation (20). Whereas GPCR agonists such as catecholamines, angiotensin II, and endothelin-1 induce a rather uniform increase in size, resulting from addition of myofibrils in parallel (7, 13), CT-1 induces a predominant increase in cell length with the addition of new sarcomeric units in series (20). Recent reports suggest that ERK5 plays a critical role in gp130-mediated serial assembly of sarcomeres in isolated cardiac myocytes. Consistent with these in vitro findings, cardiac-specific expression of activated MEK5 in transgenic mice resulted in eccentric cardiac hypertrophy that progressed to dilated cardiomyopathy and sudden death (9).

In patients with congestive heart failure, plasma concentrations of CT-1 (8, 16, 17) are markedly increased and correspond to disease severity (17). The heart is a prominent source of circulating CT-1 in humans (1), and augmented expression of CT-1 at mRNA and protein levels was found in failing left ventricular myocardium (23). Enhanced CT-1 secretion seems to be an early event that occurs before onset of left ventricular systolic dysfunction and therefore may have an impact on disease progression (15).

Altogether, the studies performed so far demonstrate that 1) CT-1 expression is chronically augmented in human heart failure, 2) CT-1 may play a role in protecting cardiac cells against stressful stimuli, and 3) long-term activation of ERK5, a downstream effector of CT-1/gp130 signaling, induces contractile failure. The latter finding raises the possibility that chronically augmented CT-1, as observed in human heart failure, might promote contractile dysfunction.

Thus the aim of the present study was to evaluate the net effect of long-term CT-1 stimulation on cardiac contractility. To evaluate myocardial function in vitro, we used reconstituted rat heart tissue (engineered heart tissue, EHT). Single cardiac myocytes suffer from the fact that some basic functional properties vanish when one moves down the hierarchical scale from multicellularity to single cells. We thought, therefore, that EHT, which combines standardization of in vitro test systems with multicellularity, would be suitable to assess general implications of CT-1 for contractile performance. In detail, we tested effects of CT-1 on twitch tension and twitch kinetics in response to calcium and {beta}-agonist stimulation dependent on CT-1 concentrations or the time of exposure to CT-1.


   METHODS
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 METHODS
RESULTS
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Cell culture. All care and treatment of animals were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication 85-23, revised 1985) and were subjected to prior approval by the local animal protection authority. Neonatal rat cardiac myocytes were dissociated from the ventricles of 1- to 3-day-old Wistar rats (Charles River) by serial trypsin-DNase II digestion as described previously (21). Isolated cardiac myocytes were plated on fibronectin/collagen-coated plastic dishes (60-mm diameter, 2 x 106 cells) and maintained for ~24 h in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µM 5-bromo-2'-deoxyuridine. More than 90% of cultured cells were myocytes, as assessed by immunofluorescence staining with an antibody against sarcomeric {alpha}-actinin (Sigma).

Engineered heart tissue. Circular EHT was prepared as described in detail previously (21, 22). Cardiac cells were isolated from hearts of 1- to 3-day-old Wistar rats by fractionated trypsin digestion. Cardiac cells (2.5 x 106 cells per EHT) were mixed with collagen type I, soluble basement membrane extract of the Engelbreth-Holm-Swam tumor (Matrigel; Becton Dickinson), and serum-containing culture medium (DMEM, 10% horse serum, 2% chick embryo extract, 100 U/ml penicillin, and 100 µg/ml streptomycin). The reconstitution mix was pipetted into circular casting molds and incubated for 45 min to let the reconstitution mixture gel. Medium (DMEM, 10% horse serum, 2% chick embryo extract, 100 U/ml penicillin, and 100 µg/ml streptomycin) was added, and EHTs were cultured as described earlier (21). After 7 days in culture, EHTs were transferred to spacers to continue culture under static stretch for an additional 4 days. One day after casting, EHTs were stimulated with recombinant human CT-1 (0.1 and 1 nM; Tebu, Offenbach, Germany) for 10 days, with daily changes of the medium. The recombinant human CT-1 was synthesized in Escherichia coli as a 201-amino acid polypeptide lacking the hydrophobic NH2-terminal secretion signal sequence. Purity was >99% as determined by SDS-PAGE and HPLC analyses. Endotoxin levels were <0.1 ng/µg.

Force measurement. EHTs were transferred into gassed organ baths (37°C, modified Tyrode solution with 0.4 mM Ca2+ gassed with carbogen). After a 30-min equilibration without pacing, EHTs were electrically stimulated with rectangular pulses (2 Hz, 5 ms, 80–100 mA). Preload was adjusted to the length at which EHTs developed maximal active force. Inotropic responses to cumulative concentrations of Ca2+ (0.4–2.8 mM) and isoprenaline (0.1–1,000 nM) were recorded. Twitch tension, resting tension, contraction duration (T1, time from 50% to peak force development), and relaxation duration (T2, time to 50% relaxation) were evaluated using BMON software (Jäckel, Hanau, Germany).

Western blot analysis. Cardiac myocytes were washed with phosphate-buffered saline (PBS) and homogenized in lysis buffer (50 mM HEPES, 100 mM sodium fluoride, 2 mM sodium orthovanadate, 4 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride) in a glass/Teflon homogenizer. Protein concentration was measured with the DC protein assay kit (Bio-Rad). Proteins (30 µg) were separated in 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 5% nonfat dry milk or 5% bovine serum albumin before incubation with a polyclonal antibody against phospho-Tyr705 STAT3 (signal transducer and activator of transcription 3; Cell Signaling), followed by horseradish peroxidase-conjugated anti-rabbit IgG (Sigma). Proteins were visualized using the ECL detection system (Amersham). Membranes were stripped in 100 mM mercaptoethanol, 2% SDS, and 62.5 mM Tris·HCl, pH 6.7, at 50°C for 30 min and then reprobed for total STAT3 protein with a specific antibody (Cell Signaling).

RNA isolation and dot-blot analysis. Total RNA and DNA were isolated from EHTs with TRIzol reagent (GIBCO-BRL) as recommended. The RNA was resuspended in water, quantitated by optical density at 260 nm, diluted, and denatured, and 1.5 µg were blotted to nylon filters using a dot-blot filtration manifold. After blotting, the filters were baked at 80°C for 15 min, prehybridized, hybridized, and washed as described previously (24). A 400-bp fragment of the rat atrial natriuretic peptide (ANP) cDNA, a 400-bp fragment of the rat calsequestrin cDNA, and a 893-bp fragment of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were used to generate specific probes by random prime labeling. Hybridization signals were quantitated using autoradiography and densitometry.

Confocal microscopy. EHTs were fixed as whole mount samples in 4% paraformaldehyde in PBS, pH 7.4, overnight at 4°C. After a wash in PBS, samples were blocked and permeabilized at 4°C overnight in PBS, pH 7.4, containing 10% fetal calf serum, 1% bovine serum albumin, 0.5% Triton-X 100, and 0.05% thimerosal. After two washes in PBS, samples were incubated with a primary antibody against sarcomeric {alpha}-actinin (1:800; Sigma) at 4°C for 24 h. Costaining of myosin binding protein C (MyBP-C) was performed with a polyclonal antiserum raised against cardiac MyBP-C (1:500). After an overnight wash in PBS, samples were incubated with the Cy3-conjugated secondary antibody against mouse IgG (1:1,000; Sigma) or the Alexa 488-conjugated secondary antibody against rabbit IgG (1:1,000; Molecular Probes) at room temperature for 3 h. After repeated washes, samples were mounted in medium with 4,6-diamidino-2-phenylindole (DAPI) to counterstain nuclei (Vectashield; Vector Laboratories). Confocal imaging was performed with a Zeiss LSM 5 Pascal system using a Zeiss Axiovert microscope. {alpha}-Actinin fluorescence together with DAPI counterstaining of nuclei allowed us to discriminate myocytes and nonmyocytes in whole mount preparations and to count both cell populations separately.

For immunocytochemistry, neonatal rat cardiac myocytes cultured on chamber slides were fixed with 4% paraformaldehyde for 10 min before Triton X-100 treatment (0.1% in Tris-buffered saline), which allowed permeabilization of the cells. Detection of sarcomeric actinin was performed as described for EHTs.

WST-1 cell viability assay. Cell viability of cardiac myocytes following CT-1 incubation was assessed with the reagent WST-1 (Roche, Mannheim, Germany). Cells were cultured at a density of 50,000 cells/well into 96-well microtiter plates using DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µM 5-bromo-2'-deoxyuridine. Medium was changed every 24 h, and CT-1 (0.1 and 1 nM) was added from day 1 to day 11 after plating. Thereafter, 10 µl of WST-1 were applied to each well and incubated for 2 h at 37°C. The optical density was read at 450 nm using a microplate reader (Tecan, Crailsheim, Germany).

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed using Student's t-test to compare two groups and analysis of variance (ANOVA) with post hoc Bonferroni correction for multiple comparisons. A value of P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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CT-1 induces STAT3 phosphorylation in a concentration-dependent manner. First, we determined the effective concentration of CT-1 to be used in our in vitro experiments. One important CT-1 signaling pathway involves activation of Janus kinase tyrosine kinases, which results in tyrosine phosphorylation of the STAT3 transcription factor. Thus we investigated the concentration-dependent effects of CT-1 on STAT3 phosphorylation in neonatal cardiac myocytes. The resulting concentration response curve is shown in Fig. 1. Incubation of cells with increasing concentrations of CT-1 for 5 min augmented STAT3 Tyr705 phosphorylation with a half-maximal effective concentration of 0.25 nM. The earliest effect was observed with 0.1 nM CT-1; the maximum was reached with 1 nM CT-1. Based on these results, subsequent studies in EHTs were performed with 0.1 and 1.0 nM CT-1. Another argument to test 0.1 nM CT-1 was the fact that this concentration corresponds to plasma levels found in humans. Talwar et al. (17) have reported that plasma CT-1 concentration in normal humans is ~10–50 pM and increases to 30–500 pM in patients with left ventricular systolic dysfunction.



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  		Fig. 1. Concentration-dependent effects of cardiotrophin-1 (CT-1) on signal transducer and activator of transcription 3 (STAT3) phosphorylation. Protein extracts from cardiac myocytes treated for 5 min with different concentrations of CT-1 (0.03–3 nM) or vehicle were blotted to detect STAT3 Tyr705 phosphorylation (p-STAT3). Membranes were stripped and reprobed for total STAT3 as a control for protein loading. Concentration-response curve shows results from densitometric analysis of bands (n = 4). *P < 0.05 vs. vehicle control.

 
CT-1 induces sustained activation of STAT3. Additional experiments were performed to determine the time course of STAT3 activation. As Fig. 2 shows, CT-1 (0.3 nM) produced a biphasic increase in STAT3 Tyr705 phosphorylation. A rapid and transient increase leveled off after 90 min and a sustained phase lasted for at least 6 days.



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  		Fig. 2. Time course of CT-1-induced activation of STAT3. Protein extracts from cardiac myocytes treated for various times with 0.3 nM CT-1 or vehicle control (Ctr) were blotted to detect STAT3 Tyr705 phosphorylation (p-STAT3). Membranes were stripped and reprobed for total STAT3 as a control for protein loading. Data from densitometric analysis of bands are shown. Values are expressed as percentages of the corresponding control (n = 4). *P < 0.05 vs. Ctr.

 
CT-1 impairs contractile function of EHTs. Figure 3A shows the time course of the experiments performed in rat EHTs. After treatment with 0.1 nM CT-1 for 10 days, basal twitch tension was significantly decreased to 0.30 ± 0.04 mN (n = 15) vs. 0.45 ± 0.04 mN (n = 16, P = 0.034) in untreated controls, as shown in Fig. 3, B (representative twitch recordings) and C. Furthermore, the positive inotropic responses to cumulative concentrations of Ca2+ and isoprenaline were significantly diminished. Maximum isoprenaline-induced increase in twitch tension from basal force (change in force of contraction, {Delta}FOC) amounted to 0.27 ± 0.04 mN (n = 15) in the CT-1-treated group compared with 0.47 ± 0.06 mN (n = 16) in controls (P < 0.001). Apparent affinity values for the effect of isoprenaline did not differ between ET-1-treated and control EHTs (9.0 ± 0.10 vs. 8.9 ± 0.06, not significant). Only minor effects on twitch kinetics were observed (Fig. 3D). Time of contraction (T1) was slightly prolonged, whereas time of relaxation (T2) remained unchanged. Treatment with 1 nM CT-1 totally abolished the contractile response to Ca2+ and isoprenaline. When the treatment period was reduced to 5 days, CT-1 (1 nM) significantly decreased both basal force and {Delta}FOC (Fig. 4). However, effects were less pronounced compared with the 10-day stimulation. CT-1 administered at a concentration of 0.1 nM for 5 days did not significantly change contractility in EHTs.



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  		Fig. 3. Effects of CT-1 treatment on contractile function of engineered heart tissues (EHTs). A: schematic representation of the time course of the experiments. B: representative twitch recordings. EHTs were treated for 10 days with 0.1 nM (n = 15), 1 nM CT-1 (n = 15), or vehicle (Ctr, n = 16), and isometric force of contraction (FOC) in response to increasing preload and increasing concentrations of extracellular Ca2+ or isoprenaline was determined (C). D: contraction kinetics of EHTs treated with 0.1 nM CT-1 (n = 15) compared with control EHTs (Ctr, n = 15). For increasing concentrations of isoprenaline, the time of contraction (T1) and the time of relaxation (T2) have been determined. *P < 0.05 vs. Ctr.

 


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  		Fig. 4. Effects of CT-1 treatment for 5 days on contractile function of EHTs. EHTs were treated for 5 days with 0.1 nM (n = 6), 1 nM CT-1 (n = 6), or vehicle (Ctr, n = 6), and isometric FOC in response to increasing concentrations of extracellular Ca2+ or isoprenaline was determined.

 
Morphological changes of cardiac myocytes induced by CT-1. As illustrated in Fig. 5A, in serum-deprived cells treatment with CT-1 (1 nM) for 2 days stimulated the synthesis and longitudinal assembly of myofilaments, as assessed by {alpha}-actinin immunofluorescence, and increased cell surface area to 298 ± 36% of controls (n = 10, P < 0.001). These findings confirmed the well-known hypertrophic effect of CT-1 on isolated cardiac myocytes. In contrast, CT-1 induced no significant additional changes in the overall morphology when the cardiac myocytes were treated in the presence of 15% serum.



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  		Fig. 5. Effects of CT-1 (0.1 nM, 48 h) on morphology of cardiac myocytes. A: serum-deprived (w/o) or serum-stimulated neonatal rat cardiac myocytes (CM) were treated with vehicle (Ctr) or CT-1 (0.1 nM) for 48 h. Thereafter, cells were fixed and immunostained with an antisarcomeric {alpha}-actinin primary antibody and a Cy3-labeled secondary antibody. B: whole mount preparations of EHTs treated with vehicle (Ctr), 0.1 nM CT-1, or 1 nM CT-1 were stained with the antisarcomeric {alpha}-actinin antibody. Control EHTs display longitudinally oriented bundles of cardiac myocytes. In CT-1-treated EHTs, longitudinal growth of cardiac myocytes was disturbed. Nonstriated portions of {alpha}-actinin-positive filaments (arrows) were mainly observed in CT-1-treated EHTs. C: costaining of sarcomeric {alpha}-actinin (localized in the Z-band) and cardiac myosin binding protein C (MyBP-C; localized in doublets in the C-zone of the A-band of the sarcomere) in cardiac myocytes from EHTs treated with vehicle or 0.1 nM CT-1 for 10 days.

 
The net effect on contractility of cardiac myocytes reconstituted in EHTs may depend on their ability to maintain survival during the cultivation period and the degree of differentiation. Therefore, it became of interest to determine whether CT-1 affects the number and the phenotype of cardiac myocytes reconstituted in EHTs. As shown in Fig. 5B, we found marked differences in the morphology of cardiac myocytes in controls or EHTs treated with CT-1 (0.1 nM) for 10 days. In vehicle-treated controls, intensively interconnected cell bundles consisting of cardiac myocytes were found throughout the EHT. As demonstrated before, histological features of myocytes in these cell bundles resemble those of myocytes within adult native differentiated myocardium (22). In contrast, after treatment with CT-1 (0.1 nM) for 10 days, cardiac myocytes formed a loose network and displayed less intense {alpha}-actinin staining. Condensed muscle bundles were not observed. Moreover, nonstriated premyofibrils were mainly observed in CT-1 (1 nM)-treated EHTs. Investigation of the striated portions with confocal microscopy revealed no significant difference in the morphology of the sarcomeres, as assessed using immunofluorescent costaining of the Z-band protein {alpha}-actinin and the A-band protein MyBP-C (Fig. 5C).

Effects of CT-1 on cell count and gene expression in EHTs. To investigate whether depressed contractile function following CT-1 treatment was caused by a loss of cardiac myocytes that is compensated by an increased number of nonmyocytes, we selectively stained cardiac myocytes with a monoclonal antibody against sarcomeric {alpha}-actinin in whole mount preparations of EHTs. As shown in Fig. 6A, 1 nM CT-1 provoked an increase in the total number of cells by ~37%, which was due mainly to an increase in the number of nonmyocytes (by 98%). In parallel, the RNA and DNA content increased, whereas the RNA/DNA ratio remained constant (Fig. 6B).



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  		Fig. 6. A: total cell count of cardiac myocytes and nonmyocytes in EHTs exposed to 0.1 or 1 nM CT-1 for 10 days (n = 4) relative to vehicle-treated controls (Ctr, n = 4). *P < 0.05 vs. Ctr. B: total RNA content and the RNA/DNA ratio in EHTs treated with vehicle (Ctr) or 0.1 or 1 nM CT-1 for 10 days (n = 8). *P < 0.05 vs. Ctr. C: effect of CT-1 treatment for 10 days on the viability of cultured neonatal cardiac myocytes (n = 10). Colorimetric quantification of WST-1 cleavage to formazan corresponding to the mitochondrial metabolic activity was used for measurement of cell viability.

 
Viability of isolated cardiac myocytes after 10-day incubation with CT-1 (0.1 nM and 1 nM) was assessed using WST-1 colorimetry assay. These experiments showed no significant difference in the mitochondrial metabolic activity, a measure of cell viability, of control and CT-1-treated cardiac myocytes (Fig. 6C). Thus it seems unlikely that CT-1 at concentrations <1 nM may exert direct toxic effects on cardiac myocytes.

To investigate whether altered cellular morphology in EHTs corresponds to changes in gene expression, we determined mRNA concentrations of calsequestrin, a myocyte-specific Ca2+-handling protein, and ANP, a marker protein of cardiac myocyte hypertrophy that is known to be regulated by CT-1 in monolayer cell culture systems (5, 18, 20), using dot-blot analysis. As shown in Fig. 7, mRNA concentrations of the housekeeping gene GAPDH were not significantly affected by CT-1 treatment. However, CT-1 (0.1 nM) significantly depressed calsequestrin mRNA in EHTs by 31.3 ± 6.9%, whereas ANP mRNA expression was stimulated to 125.7 ± 7.3% of controls (P < 0.05).



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  		Fig. 7. Changes in gene expression. RNA dot blots were prepared with 1.5 µg of RNA per dot and probed with DNA probes specific for the indicated gene. The percent change in induction or repression of gene expression for CT-1 (0.1 nM, 10 days)-treated EHTs (n = 8) relative to controls (Ctr, n = 8) is shown. *P < 0.05 vs. Ctr.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of the present study obtained in rat EHTs were that CT-1 significantly depressed basal force of contraction and the inotropic response to calcium and {beta}-agonist stimulation. Most importantly, these alterations were observed with CT-1 (100 and 1,000 pM) concentrations comparable to CT-1 plasma levels found in patients with heart failure [300–500 pM (17)]. The duration of CT-1 exposure seems to be important, because impairment of contractile function was less pronounced after 5 days compared with 10 days of exposure. However, timing of the exposure also might be crucial. After reconstitution, cardiac cells begin to differentiate to form a contractile heart tissue. Thus it might be important whether the exposure to CT-1 starts immediately after reconstitution (affecting rather immature cells) or 5 days later, after advanced differentiation.

Until now, data on the effects of CT-1 on cardiac contractility were rare. Intravenous injection of CT-1 in conscious rats had no significant effect on left ventricular maximal change in rate of pressure compared with vehicle-treated controls (6). This finding indicates that acute administration of CT-1 does not induce a meaningful change in ventricular contractility. Our data suggest that the long-term action of CT-1, rather than the short-term action, may be important for its modulating effect on contractile function. The present study supports the hypothesis that chronically increased synthesis and release of CT-1, as observed in human heart failure, may further accelerate contractile dysfunction and disease progression. In addition, our findings do not necessarily conflict with previous in vitro or in vivo experiments demonstrating protective effects of CT-1 on cardiac cells. The major argument is that previous studies investigated the stress response, i.e., acute effects. Sheng et al. (12), for example, demonstrated that treatment with CT-1 was able to enhance the survival of neonatal rat cardiac myocytes that were serum deprived. Pretreatment with CT-1 was able to protect cultured neonatal rat cardiac myocytes against subsequent exposure to either elevated temperature (heat shock) or simulated ischemia-hypoxia (14). Altogether, these findings support the view that CT-1 may help cardiac myocytes to compensate acute stress. In the short run, CT-1 obviously helps to preserve contractility; in the long run, however, it may induce contractile dysfunction.

What are the mechanisms inducing contractile failure in EHTs treated with CT-1? CT-1 was not detrimental to cardiac myocytes per se, because the number of myocytes in EHTs that survived the 11-day culture period was unchanged. In addition, direct measurement of cardiac myocyte viability revealed no significant difference between controls and CT-1-treated cells. However, the three-dimensional arrangement of myocytes and the expression of myocyte-specific genes was markedly affected. As expected, CT-1 significantly increased ANP expression in EHTs. This result is in line with previous results obtained from isolated neonatal rat cardiac myocytes. For example, Wollert et al. (20) demonstrated that CT-1 upregulates the ANP gene but does not affect skeletal {alpha}-actin or myosin light chain-2v expression. In addition, in EHTs, CT-1 downregulated expression of calsequestrin by 32%, a protein involved in Ca2+ handling. Moreover, formation of longitudinally oriented bundles of cardiac myocytes was prevented. Changes in both gene expression and arrangement of cardiac myocytes might contribute to ineffective force generation.

The effects of CT-1 on cardiac myocytes 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 (22). This includes the possibility of paracrine signaling between the different cell species. Indeed, recent studies have demonstrated cross talk between the actions of IL-6-related cytokines and growth factors such as endothelin-1 and angiotensin II (2, 10). Moreover, recent work (3) has indicated that CT-1 may participate in cardiac fibrosis post-myocardial infarction. In vitro studies demonstrated that CT-1 treatment induced cardiac fibroblast protein synthesis, proliferation, and migration (3, 18). Furthermore, CT-1 treatment increased mature collagen synthesis (3, 4, 18). The current study shows that CT-1 raises the number of nonmyocytes, most likely cardiac fibroblasts, implicating that "remodeling" also may occur in EHTs treated with CT-1. Second, EHTs already display a hypertrophic phenotype independent from CT-1 treatment, which is provoked by multiple, not well-characterized growth factors added with the serum or liberated from the Matrigel. Thus the present results reflect the modulating action of CT-1 on signaling events induced by multiple growth factors active in EHTs. In contrast, previous experiments in monolayer cell cultures, usually performed in quiescent, serum-deprived cells, were designed to investigate the exclusive action of CT-1 unaffected by other mitogenic factors. However, we believe that this does not correspond to the situation in normal physiology or pathophysiology in vivo, where a plethora of mitogenic factors always are active. In the current study, we have demonstrated that effects of CT-1 on cell size and sarcomere assembly in serum-deprived myocytes were masked in the presence of serum. Whether direct actions of CT-1 on cardiac myocytes is the major cause of contractile failure in EHTs remains to be investigated. Our experiments showed no significant effect of CT-1 on mitochondrial metabolic activity as a measure of cell viability. Thus it seems unlikely that CT-1 at concentrations <1 nM may exert direct toxic effects on cardiac myocytes, inducing cell death. However, CT-1 may directly affect differentiation of cardiac myocytes. Alternatively, indirect mechanisms mediated through stimulation and proliferation of nonmyocytes may play an important role.

In summary, we have demonstrated that chronically augmented CT-1 has detrimental effects on cardiac contractility in EHTs. Although EHTs are well suitable as a test systems, future studies are needed to confirm our results in vivo. Because the neurohumoral milieu, for example, in human heart failure is likely to be rather different from that in our experimental preparations, the in vivo response of ventricular myocytes to CT-1 may be different. Considering these limitations, our study focusing on the contractile effects of CT-1 may help clarify the pathophysiological role of CT-1 in heart disease.


   GRANTS
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 ABSTRACT
 METHODS
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 GRANTS
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This work was supported by Deutsche Forschungsgemeinschaft Grant Zo 123/1-1/2.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Mathias Gautel for the gift of the anti-cardiac MyBP-C antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Zolk, Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany (E-mail: zolk{at}pharmakologie.uni-erlangen.de)

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


   REFERENCES
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  1. Asai S, Saito Y, Kuwahara K, Mizuno Y, Yoshimura M, Higashikubo C, Tsuji T, Kishimoto I, Harada M, Hamanaka I, Takahashi N, Yasue H, and Nakao K. The heart is a source of circulating cardiotrophin-1 in humans. Biochem Biophys Res Commun 279: 320–323, 2000.[CrossRef][ISI][Medline]
  2. Booz GW, Day JN, Speth R, and Baker KM. Cytokine G-protein signaling crosstalk in cardiomyocytes: attenuation of Jak-STAT activation by endothelin-1. Mol Cell Biochem 240: 39–46, 2002.[CrossRef][ISI][Medline]
  3. Freed DH, Moon MC, Borowiec AM, Jones SC, Zahradka P, and Dixon IM. Cardiotrophin-1: expression in experimental myocardial infarction and potential role in post-MI wound healing. Mol Cell Biochem 254: 247–256, 2003.[CrossRef][ISI][Medline]
  4. Freed DH, Borowiec AM, Angelovska T, and Dixon IM. Induction of protein synthesis in cardiac fibroblasts by cardiotrophin-1: integration of multiple signaling pathways. Cardiovasc Res 60: 365–375, 2003.[Abstract/Free Full Text]
  5. Funamoto M, Fujio Y, Kunisada K, Negoro S, Tone E, Osugi T, Hirota H, Izumi M, Yoshizaki K, Walsh K, Kishimoto T, and Yamauchi-Takihara K. Signal transducer and activator of transcription 3 is required for glycoprotein 130-mediated induction of vascular endothelial growth factor in cardiac myocytes. J Biol Chem 275: 10561–10566, 2000.[Abstract/Free Full Text]
  6. Jin H, Yang R, Ko A, Pennica D, Wood WI, and Paoni NF. Effects of cardiotrophin-1 on haemodynamics and cardiac function in conscious rats. Cytokine 10: 19–25, 1998.[CrossRef][ISI][Medline]
  7. Knowlton KU, Michel MC, Itani M, Shubeita HE, Ishihara K, Brown JH, and Chien KR. The {alpha}1A-adrenergic receptor subtype mediates biochemical, molecular, and morphologic features of cultured myocardial cell hypertrophy. J Biol Chem 268: 15374–15380, 1993.[Abstract/Free Full Text]
  8. Ng LL, O'Brien RJ, Demme B, and Jennings S. Non-competitive immunochemiluminometric assay for cardiotrophin-1 detects elevated plasma levels in human heart failure. Clin Sci (Lond) 102: 411–416, 2002.[Medline]
  9. Nicol RL, Frey N, Pearson G, Cobb M, Richardson J, and Olson EN. Activated MEK5 induces serial assembly of sarcomeres and eccentric cardiac hypertrophy. EMBO J 20: 2757–2767, 2001.[CrossRef][ISI][Medline]
  10. Sano M, Fukuda K, Kodama H, Pan J, Saito M, Matsuzaki J, Takahashi T, Makino S, Kato T, and Ogawa S. Interleukin-6 family of cytokines mediate angiotensin II-induced cardiac hypertrophy in rodent cardiomyocytes. J Biol Chem 275: 29717–29723, 2000.[Abstract/Free Full Text]
  11. Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, and Chien KR. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway. Divergence from downstream CT-1 signals for myocardial cell hypertrophy. J Biol Chem 272: 5783–5791, 1997.[Abstract/Free Full Text]
  12. Sheng Z, Pennica D, Wood WI, and Chien KR. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development 122: 419–428, 1996.[Abstract]
  13. Shubeita HE, McDonough PM, Harris AN, Knowlton KU, Glembotski CC, Brown JH, and Chien KR. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for myocardial cell hypertrophy. J Biol Chem 265: 20555–20556, 1990.[Abstract/Free Full Text]
  14. Stephanou A, Brar B, Heads R, Knight RD, Marber MS, Pennica D, and Latchman DS. Cardiotrophin-1 induces heat shock protein accumulation in cultured cardiac cells and protects them from stressful stimuli. J Mol Cell Cardiol 30: 849–855, 1998.[CrossRef][ISI][Medline]
  15. Talwar S, Squire IB, Davies JE, and Ng LL. The effect of valvular regurgitation on plasma cardiotrophin-1 in patients with normal left ventricular systolic function. Eur J Heart Fail 2: 387–391, 2000.[CrossRef][ISI][Medline]
  16. Talwar S, Downie PF, Squire IB, Barnett DB, Davies JD, and Ng LL. An immunoluminometric assay for cardiotrophin-1: a newly identified cytokine is present in normal human plasma and is increased in heart failure. Biochem Biophys Res Commun 261: 567–571, 1999.[CrossRef][ISI][Medline]
  17. Talwar S, Squire IB, Downie PF, O'Brien RJ, Davies JE, and Ng LL. Elevated circulating cardiotrophin-1 in heart failure: relationship with parameters of left ventricular systolic dysfunction. Clin Sci (Lond) 99: 83–88, 2000.[Medline]
  18. Tsuruda T, Jougasaki M, Boerrigter G, Huntley BK, Chen HH, D'Assoro AB, Lee SC, Larsen AM, Cataliotti A, and Burnett JC Jr. Cardiotrophin-1 stimulation of cardiac fibroblast growth: roles for glycoprotein 130/leukemia inhibitory factor receptor and the endothelin type A receptor. Circ Res 90: 128–134, 2002.[Abstract/Free Full Text]
  19. Wolf I and Seger R. The mitogen-activated protein kinase signaling cascade: from bench to bedside. Isr Med Assoc J 4: 641–647, 2002.[ISI][Medline]
  20. Wollert KC, Taga T, Saito M, Narazaki M, Kishimoto T, Glembotski CC, Vernallis AB, Heath JK, Pennica D, Wood WI, and Chien KR. Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy. Assembly of sarcomeric units in series via gp130/leukemia inhibitory factor receptor-dependent pathways. J Biol Chem 271: 9535–9545, 1996.[Abstract/Free Full Text]
  21. Zimmermann WH, Fink C, Kralisch D, Remmers U, Weil J, and Eschenhagen T. Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnol Bioeng 68: 106–114, 2000.[CrossRef][ISI][Medline]
  22. Zimmermann WH, Schneiderbanger K, Schubert P, Didie M, Münzel F, Heubach JF, Kostin S, Neuhuber WL, and Eschenhagen T. Tissue engineering of a differentiated cardiac muscle construct. Circ Res 90: 223–230, 2002.[Abstract/Free Full Text]
  23. Zolk O, Ng LL, O'Brien RJ, Weyand M, and Eschenhagen T. Augmented expression of cardiotrophin-1 in failing human hearts is accompanied by diminished glycoprotein 130 receptor protein abundance. Circulation 106:1442–1446, 2002.[Abstract/Free Full Text]
  24. Zolk O, Marx M, Jackel E, El-Armouche A, and Eschenhagen T. Beta-adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase. Cardiovasc Res 59: 563–572, 2003.[Abstract/Free Full Text]



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