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Cardiovascular Research 1999 43(3):675-684; doi:10.1016/S0008-6363(99)00160-1
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Copyright © 1999, European Society of Cardiology

Nitric oxide synthase expression and role during cardiomyogenesis

W Bloch1,a, B.K Fleischmannb, D.E Lorkec, C Andressena, B Hopsb, J Heschelerb and K Addicksa,*

aInstitute of Anatomy I, University of Cologne, Cologne, Germany
bInstitute of Neurophysiology, University of Cologne, Cologne, Germany
cDepartment of Neuroanatomy, University of Hamburg, Hamburg, Germany

* Corresponding author. Tel: +49-221-478-5202; fax: +49-221-478-6711 Addicks.Anatomie{at}uni-koeln.de

Received 29 December 1998; accepted 27 April 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of the present study was the investigation of the expression of NOS during cardiomyogenesis and its functional role. Design: The qualitative and quantitative expression of NOS isoforms during different stages of cardiac development was evaluated using immunocytochemistry and dot blots, respectively. The functional relevance of NOS expression during cardiomyogenesis was investigated using the in vitro ES cell-differentiation model and selective pharmacological agents. Results: On day 7.5 of embryonic development (E7.5) none of the NOS isoforms were expressed in the embryo, whereas the inducible (iNOS), as well as the endothelial (eNOS) isoforms were detected in the extraembryonic parts. In contrast, starting from E9.5 rat and murine embryos displayed prominent iNOS and eNOS expression. This was correlated with high expression of soluble guanylylcyclase (sGC) as well as high cyclic GMP (cGMP) content. During further development after E14.5 both, iNOS as well as eNOS, started to be downregulated and shortly prior to birth reduced staining for eNOS was found, whereas iNOS was hardly detectable. We further investigated whether NO plays a role for cardiomyogenesis, using in vitro ES cell-derived cardiomyocytes differentiating within embryoid bodies (EBs). The NOS expression pattern in these cells paralleled the one detected in vivo. We demonstrate that continuous incubation of EBs with the NOS inhibitors L-NMMA (2–10 mM) or L-NA (2–10 mM) for 4 to 9 days after plating resulted in a pronounced differentiation arrest of cardiomyocytes, whereas this effect could be reversed by coapplication of the NO-donor spermine-NONOate (10 µM). Conclusions: Both, iNOS and eNOS isoforms are prominently expressed during early stages of cardiomyogenesis. Around E14.5 NOS expression starts to decline. Moreover, the NO-generation is required for cardiomyogenesis since NOS inhibitors prevent the maturation of terminally differentiated cardiomyocytes using the ES cell system.

KEYWORDS Developmental biology; Gene expression; Nitric oxide; Signal transduction; Cell culture


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
NO is a universal signaling molecule, whose physiological role beyond a vasodilator and retrograde neurotransmitter has been evidenced for many biological processes over recent years [1]. So far three distinct NOS isoforms have been identified [2]. For the synthesis of NO preferentially involved in intracellular signaling the neuronal NOS (nNOS) and eNOS isoforms have been implied [3], whereas iNOS has been suggested to play a role in inflammatory processes [4] as well as tumor-growth [5]. iNOS is usually triggered by endotoxins and cytokines [4], whereas eNOS is activated by an increase in the intracellular free Ca2+ concentration [1]. The iNOS and the eNOS isoforms have been detected in the diseased heart muscle [6–8] and their expression has been discussed controversially as a possible cause for the negative inotropy found in such disorders as cardiomyopathies [9–11].

Since pathologically transformed cells can recapitulate the early embryonic phenotype we have investigated in our previous work the establishment of intracellular signaling cascades involved in the regulation of the cardiac L-type Ca2+ channel (ICa) using embryonic stem (ES) cell-derived cardiomyocytes during different stages of development [12]. We found, that during early embryonic development, muscarinic signaling was entirely modulated through eNOS mediated generation of NO, whereas in late embryonic development, muscarinic receptor activation depressed prestimulated ICa independent of NO generation [12]. This was accompanied by distinct changes in the expression of the eNOS and iNOS isoform.

In this study we have investigated the expression pattern of NOS isoforms and of the target signaling components sGC and cGMP [13] during rat/mouse embryonic cardiomyogenesis. In order to identify a possible role of NO in cardiogenesis we have further monitored cardiac myofibrillogenesis in the presence and absence of selective NOS inhibitors using ES cell-derived cardiomyocytes [14].

We demonstrate, that the iNOS as well as eNOS isoforms are expressed in early embryonic cardiomyocytes whereas they are downregulated starting from E14.5. Moreover, we show that in the presence of NOS inhibitors the number of differentiated cardiomyocytes is significantly reduced.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Murine and rat embryos: animal breeding and histology
Crl:(WI)BR rats from the Wistar stock were bred and kept under defined environmental conditions (21°C; relative humidity 50%; 12 h light–dark cycle) at the laboratory animal facilities of the University Hospital Hamburg. They were fed a standard diet (Altromin 1314, Altromin, Germany), and water was given ad libidum. For mating, one male rat was placed with three females for 2 h; fertilization was assumed to have occurred when a positive vaginal smear was found (gestational day 0). On each day between gestational day 8 (E8) and 15, at least two pregnant females were killed by cervical dislocation and, after laparotomy, the uterus was opened along the antimesometrial side. Then the fetuses including fetal membranes were dissected and placed for further preparation into Petri dishes containing Hank’s salt solution. After removal of both, the covering decidua and the fetal membrane under a dissecting microscope, the embryos (32 altogether) were fixed by immersion fixation in buffered paraformaldhyde (4% in 0.1 M phosphate buffer, pH 7.4). Mouse embryos of the C57 BL/CJ stock were essentially obtained in the same way: one male was mated with three females overnight (12 h) and the appearance of a vaginal plug was marked as day E0.5 of gestation. After preparation as described above, the murine embryos (E7.5–E18.5 of age, daily intervals, 50 embryos altogether) were immersion fixed in buffered 4% paraformaldehyde, as well. Both rat and murine embryos were kept in the fixative for 2 h (E7.5–E12.5) or overnight (E13.5 and older), dehydrated in ascending series of ethanol, and after three steps of methylbenzoate embedded in Paraplast+(Shendon).

2.2 Immunocytochemistry in murine embryos
For immunohistochemistry the paraffin slices of murine embryos were prepared as described before [15]. Immunocytochemistry was performed using the same primary and secondary antibodies as described for the ES cell-derived cardiomyocytes. Content of cGMP was evaluated using a cGMP rabbit antibody (Quartett, Hamburg, Germany) at a dilution of 1:600. As 4% paraformaldehyde has been shown to result in optimal fixation of cGMP on the cell’s protein matrix [16], this solution was used for immunohistochemical detection of the water soluble cGMP.

2.3 Dot blotting
Murine hearts were dissected from E9.5 (21 embryos), E14.5 (14 embryos), E19.5 (10 embryos) old embryos as well as from adult mice (three animals). Protein fractions were prepared by homogenization in hypotonic buffer (10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM phenyl-methyl-sulfonyl-fluoride (PMSF), 1 mM iodoacetamide). Cell debris and the nuclear fraction were removed by centrifugation at 1000 g for 30 min. The protein concentrations were determined by Lowry densitometry and balanced by dilution to 5.1 mg/ml in Laemmli buffer [17]. After dotting 20 µl samples onto a PVDF membrane (Immobilon, Bedford, USA), immunological detection of the iNOS and eNOS isoforms was carried out as described for immunocytochemistry except for the use of alkaline phosphatase histochemical detection enzyme. The incubation medium consisted of 0.25 M Tris, 0.26 mM Naphtol AS-MX phosphate (Sigma, Deisenhofen, Germany), and 1.5 mM Fast Blue salt (Gurr, UK), adjusted to pH 8.6. The specificity of the antibodies has been showed in earlier Western blot experiments [15]. For densitometry, grey value determination was performed using the Optimas software (Seattle, USA). For the quantitative comparison of protein concentration at the different time points of differentiation the grey value at the E9.5 stage was taken as 100%.

2.4 ES-cell preparation
Murine embryonic stem cells of the line D3 [18] were cultured and differentiated into spontaneously beating cardiomyocytes as previously described [19]. Briefly, cells were grown as spheroidal aggregates (embryoid bodies; EBs) in hanging drops for 2 days, then transferred into suspension for 5 days and finally plated in 24 microwell-plates for different periods (3–4 and 9–12 days, respectively). Single cardiomyocytes were isolated from clusters of spontaneously beating areas by a modified procedure of Isenberg and Klöckner [20]. Beating areas of 15 to 20 EBs were isolated with a sterile microscalpel and collected in low Ca2+-solution containing (in mM): 120 NaCl, 5.4 KCl, 5 MgSO4, 5 Na-pyruvate, 20 glucose, 20 taurine, 10 Hepes (pH 6.9 with NaOH). The tissue was then incubated in enzyme medium (1 mg/ml collagenase B, Boehringer, Mannheim, Germany; 30 µM CaCl2) for 20 min at 37°C. Tissue fragments were transferred to a medium containing (in mM): 85 KCl, 30 K2HPO4, 5 MgSO4, 1 EGTA, 2 Na2ATP, 5 Na-Pyruvate, 5 creatine, 20 taurine, 20 glucose, pH 7.2, in which they were kept at room temperature for 1 h, and then resuspended in DMEM (Gibco, Eggenstein, Germany) complemented with 20% fetal calf serum. Isolated cells were plated on sterile, gelatine-coated glass cover slips and kept in the incubator for 24–48 h. Spontaneously contracting myocytes could be observed within 12 h after cell preparation.

EBs were treated with various concentrations (2–10 mM) of the NOS inhibitors L-NMMA/D-NMMA (Calbiochem, Bad Soden, Germany) or L-NA/D-NA, (Alexis, Gruenberg, Germany) in parallel starting from the first day of plating. Similarly, EBs were coincubated with the NO-donor NO-spermine-NONOate (10 µM, Alexis) and L-NMMA (10 mM). In addition, experiments with the selective SGC inhibitor ODQ (10 µM, Alexis) were performed. For all these experiments untreated control cells were always prepared and analyzed in parallel. The substances used were dissolved in DMEM medium, pH corrected and filtered. Fresh medium containing the substance was replaced every third day. Analysis was performed as described below on most of the EBs 6 to 9 days after plating.

2.5 Immunocytochemistry in ES cell-derived cardiomyocytes
Single cell preparations of 7+4 day- and 7+9 day-old EBs were used for immunocytochemical investigation of NOS isoform distribution and {alpha}-actinin staining. Histochemical estimation of NOS activity was performed by applying the NADPH-diaphorase staining. Single cell preparations were fixed in 4% paraformaldehyde in 0.1 M PBS for 20 min. NADPH-diaphorase staining was applied with a Tris buffer solution (pH 8.0) containing 83 mg nicotinamide adenine dinucleotide phosphate (β-NADPH), 40 mg nitro blue tetrazolium, 125 mg monosodium maleate and 0.1% triton X-100 at 100 ml for 2 h, followed by incubation with a 1:600 dilution of mouse anti-rat {alpha}-actinin antibody (Sigma) for 1 h at 37°C. Peroxidase rabbit IgG kit (Vector Labs., Burlingam, USA) was then used as recommended, with 3,3'-diaminobenzidine as the chromogen. Cell preparations were indirectly immunolabelled with a dilution of 1:600 {alpha}-actinin mouse anti-rat antibody and a rabbit anti-mouse antibody for iNOS, eNOS (Biomol, Hamburg, Germany) or nNOS (Alexis) in a dilution of 1:1000 for 1 h at 37°C, followed by a TRITC-labelled IgG goat anti-rabbit antibody (Sigma) and a biotin-labeled IgG goat anti-mouse antibody (Vector Labs.). Thereafter, cells were treated with extravidin FITC (Sigma). Double immunostaining for eNOS and iNOS was performed with an eNOS rabbit anti-mouse antibody (Biomol) and a 1:1000 dilution of iNOS antibody from mouse (Affinitti, Nottingham, UK), followed by the same treatment as described above.

The present investigation conforms with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
The expression of the various NOS isoforms was investigated during early and late embryonic development using rat and mouse embryos of different developmental stages. In order to monitor the pattern of NOS-expression during development in the embryonic heart immunocytochemical analysis was performed for each species on a day by day basis starting from E7.5. At least three embryos were analyzed for each developmental stage. In the present work representative data of either mouse or rat are shown, since the expression pattern of NOS and target signaling molecules were parallel in both species at all developmental stages.

Specific antibody staining against the three known NOS isoforms evidenced in a E7.5 rat embryo, that NOS was absent in the embryonic portion (Fig. 1a and b), whereas the extraembryonic part displayed strong positivity for iNOS (Fig. 1b) and weak immunoreactivity for eNOS (Fig. 1a). Accordingly, at this early stage a high cGMP content was detected exclusively in the extraembryonic parts (Fig. 1d), whereas sGC expression was strong in both, the embryonic and intraembryonic components of the embryo (Fig. 1c). In clear contrast to these findings, embryos starting from E8.5 displayed pronounced iNOS (Fig. 2a) and eNOS (Fig. 3a) expression in the murine heart (Fig. 2–6GoGoGoGo show a murine embryo E9.5). During further embryonic development a clear decrease in iNOS expression was observed starting from E14.5 (Fig. 2b). However, the downregulation was more pronounced in the ventricle as compared to the atrium (Fig. 2b). At this stage, only a minor reduction in eNOS expression was detected in the ventricle (Fig. 3b). The continuous decrease of the NOS expression was further proven by the analysis of later embryonic rat/mouse stages. Figs. 2c and 3cGo depict the expression pattern of the NOS isoforms in an E18.5 murine heart. At this stage iNOS displayed very weak immunoreactivity in atrium as well as ventricle (Fig. 2c). Similarly, eNOS expression was very low in the ventricle, whereas it was present in the atrium (Fig. 3c). In order to investigate the NOS isoform expression pattern at a quantitative level, dot blot experiments using protein extracts of embryonic hearts at different developmental stages were performed. These revealed a clearly decreasing immunoreactivity for both, iNOS and eNOS isoforms from E9.5 to the adult stage. The densitometric analysis of the dot blots showed that iNOS expression decreased to approximately 50% in the atrial and 20% in the ventricular myocardium, whereas in the adult myocardium the content was further reduced to about 10% (Fig. 4). The eNOS content was found to decline to a lesser extent since in the adult mouse a residual concentration of about 65% was still detectable (Fig. 4). During all stages of murine and rat embryonic development the nNOS isoform was not detected. In line with the eNOS immunoreactivity in the heart the correlated signaling components sGC (Fig. 5) and cGMP (Fig. 6) followed the identical expression pattern during embryonic development. Next, we tested the possible functional significance of the NOS expression during early embryonic cardiomyogenesis using the in vitro differentiation system of ES cell-derived cardiomyocytes. The cardiomyocytes identified by {alpha}-actinin costaining, were strongly positive to the histochemical NADPH-diaphorase stain, known to react with the reductase domain of all NOS isoforms (Fig. 7a). In addition, similar to the situation detected in the early embryonic heart of mouse and rat, early developmental stage (EDS, 7+3/4 days) murine ES cell-derived cardiomyocytes, identified by {alpha}-actinin costaining, displayed strong iNOS and eNOS expression (Fig. 7c and d). As expected for the intact NO dependent signaling cascade sGC and cGMP were also detected in these cells. Conversely, in late developmental stage (LDS, 7+9–15 days) ES cell-derived cardiomyocytes iNOS was not detected and eNOS expression was found to be very low (Fig. 7e and f). In agreement with these findings, NADPH staining as well as sGC and cGMP were low in LDS cells. Because ES cell-derived cardiomyocytes displayed an identical pattern of NOS expression as detected in mouse and rat heart and since these cells are considered a valid model for embryonic cardiomyogenesis [21,22], we used this in vitro model to investigate the influence of NOS inhibitors on cardiac development within EBs (five independent experiments on at least eight EBs each and controls). In order to pharmacologically interfere with the early stages of cardiomyogenesis, NOS inhibitors (L-NA, L-NMMA, 2–10 mM) were added to the cell culture at the first day of plating and the culture medium was exchanged every third day. Preferentially plating periods of 4, 6 and 9 days were used for analysis. As demonstrated in Fig. 8, incubation of EBs with the NOS inhibitor L-NA (10 mM) for 9 days prominently influenced cardiomyogenesis, as evidenced by {alpha}-actinin staining of single dissociated EB derived-cardiomyocytes: (i) the overall number of cardiomyocytes was unaltered, (ii) cardiomyocytes with the typical distinct cross striation pattern detected in LDS cells were significantly reduced. The effect was most pronounced at the LDS stage, since at this time point prevalently terminally differentiated cardiomyocytes are detected [21]. The effect of the NOS inhibitors on ES cell-derived cardiomyogenesis was considered specific, because EBs treated with the non-active stereoisomer D-NA or D-NMMA (2–10 mM) resulted in unaltered cardiomyogenesis as compared to control cells. These data were further confirmed by the observation that coincubation of both, L-NMMA (10 mM) and the NO-donor spermine-NONOate (10 µM) reversed the L-NMMA effect (Fig. 8e). The functional involvement of the NO/sGC/cGMP pathway was corroborated by application of the soluble guanylyl cyclase inhibitor ODQ (10 µM) [23], which led to similar changes of the cardiomyogenesis as observed in the presence of NOS inhibitors (Fig. 8f).


Figure 1
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  		Fig. 1 NOS isoform expression at E7.5 of rat heart development. a: In the extraembryonic tissue slightly positive immunoreactivity for eNOS was detected, whereas the embryonic tissue was negative (* indicates embryonic portion). b: iNOS was also negative in the embryonic part (inset b1) and positive in the extraembryonic part. c: Positive antibody staining for sGC was detected in both, the embryonic (* and c1) and the extraembryonic part. d: Only the extraembryonic tissue showed cGMP content. Bar equals in a–d, 100 µm; in b1 and c1, 70 µm.

 

Figure 2
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  		Fig. 2 iNOS expression in the embryonic murine heart in situ changed during development (A: atrial tissue, V: ventricular tissue). a: E9.5: Atrium (A) and ventricle (V) strongly express iNOS. b: E14.5: iNOS expression is already weaker, but intensities in the atrium and the ventricle are similar. c: E18.5: iNOS immunoreactivity is only present in the atrium, whereas in the ventricle it was very weak or undetectable. Bar equals in a, 50 µm; b and c, 100 µm.

 

Figure 3
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  		Fig. 3 eNOS expression in the embryonic murine heart in situ changed during development (A: atrial tissue, V: ventricular tissue). a: E9.5: Atrium (A) and ventricle (V) strongly expressed eNOS. b: E14.5: eNOS expression remained pronounced in the atrium whereas in the ventricle it started to decrease. c: E18.5: Weak eNOS expression was present in the atrium, whereas in ventricular tissue it was undetectable at this stage. Bar equals in a, 50 µm; b and c, 100 µm.

 

Figure 4
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  		Fig. 4 Dot blots demonstrate, that iNOS and eNOS expression changed during murine heart development (A: atrial tissue, V: ventricular tissue). Original dot blots of heart extracts from murine hearts at different stages of development show the decrease in eNOS expression (upper panel). The lower panel depicts the quantitative evaluation of iNOS (black columns) and eNOS (grey columns) expression using densitometric measurements of the dot blots. iNOS and eNOS intensities determined in E9.5 hearts were taken as reference value (100) for the scaling of the intensities measured at later developmental stages. A strong decrease of iNOS expression during development is noticed, whereas eNOS declines to a lesser degree. Note the different expression level between atrium and ventricle. For control, iNOS and eNOS expression was determined in the adult skeletal muscle. The number of murine hearts used for the dot blots at the various differentiation stages was 21 (E9.5), 14 (E14.5), 10 (E19.5) and 3 (adult animals).

 

Figure 5
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  		Fig. 5 sGC expression in the embryonic murine heart in situ changed during development (A: atrial tissue, V: ventricular tissue). a: E9.5: Atrium (A) and ventricle (V) were strongly immunoreactive for sGC. b: E14.5: Weaker sGC expression was observed, the immunostaining intensity in atrium and ventricle were similar. c: E18.5: sGC expression was only present in the atrium (arrows), whereas in the ventricle very weak or no sGC expression was detected. Bar equals in a, 50 µm; b and c, 100 µm.

 

Figure 6
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  		Fig. 6 cGMP expression in the embryonic murine heart in situ changed during development (A: atrial tissue, V: ventricular tissue). a: E9.5: Atrium (A) and ventricle (V) expressed high amounts of cGMP. b: E14.5: cGMP content decreased in the ventricle, whereas it remained unchanged in the atrium. c: E18.5: cGMP content was only present in the atrium, whereas in ventricular tissue very weak or no cGMP content was seen. Bar equals in a, 50 µm; b and c 100 µm.

 

Figure 7
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  		Fig. 7 Expression of NADPH-diaphorase and eNOS at different developmental stages in EBs and isolated EB-derived cardiomyocytes. Cardiomyocytes were identified by {alpha}-actinin co-staining. a: Early stage EB (7+4 days) showed strong NADPH-diaphorase activity; {alpha}-actinin positive cells are detected, but striated organization is still absent. b: Late stage EB with very weak NADPH-diaphorase activity, organized striations were recognizable. c: In a representative EDS cell, poorly organized striations were visible. d: In the same cell as displayed in c, prominent eNOS expression was seen. e: In a typical LDS cell sarcomeric structures were clearly visible. f: Only weak expression of eNOS was detected. Bar equals in a–d, 10µm; in e and f, 20 µm.

 

Figure 8
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  		Fig. 8 Myofibrillogenesis of EB (7+9 days) derived cardiomyocytes was depressed upon incubation with the NOS inhibitor L-NA (10 mM). Cross-striated myofibrils are detected by {alpha}-actinin staining. a: A representative terminally differentiated EB derived cardiomyocyte displayed prominent sarcomeric structure, e.g. cross striation (Z-stripes) and elongated cell shape. b: Typically, no distinct myofibrils and cross-striations were observed in EB derived-cardiomyocytes incubated with L-NA (10 mM). Only diffuse spots of Z-stripe material ({alpha}-actinin positive) were present in the cardiomyocyte-anlage of these EBs in line with a defective or incomplete myofibrillogenesis. c: PAP-{alpha}-actinin antibody staining demonstrates intact cells with intact myofibrillogenesis in the control cells. d: In L-NA treated EB-derived cardiomyocytes, only incomplete myofibrillar staining was observable at the cell surface. e: Upon coincubation of L-NMMA (10 mM) with the NO-donor spermine-NONOate (10 µM, L-NMMA/S) well differentiated cardiomyocytes were observed. f: Similar as detected in the L-NMMA treated cardiomyocytes, application of ODQ (10 µM) resulted in a pronounced disturbance of the sarcomeric organization. Bar equals in a–d, 20 µm; in e and f, 10 µm.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We demonstrate that iNOS and eNOS isoforms are expressed early during mouse/rat embryonic heart development and that starting from E14.5 they are strongly downregulated. Indeed, prior to birth only weak expression of eNOS can be detected, whereas iNOS expression is hardly detectable. These findings were confirmed also at a quantitative level by the dot blot technique. We noticed, however, that higher expression of eNOS at later stages of embryonic development and at the adult stage compared to our immunocytochemical data were observed. This appears to be related to the different technical approach, since using immunocytochemistry the antibody recognizes exclusively the active form of eNOS whereas upon cell homogenization the inactive form of eNOS is also recognized (W. Bloch, unpublished results). While prominent NOS expression at the murine blastocyst stage has been reported before [24], the herewith described pattern of NOS expression during cardiomyogenesis was so far unknown. In addition, parallel with eNOS immunoreactivity during embryonic development, drastic changes in sGC expression and cGMP content are observed. These data further support the relevance of our findings in the in vitro ES cell-system, where identical NOS-expression at different stages of cardiomyogenesis was detected. It should be noted that these prominent changes were found to be associated with a switch in the signaling cascades involved in the regulation of ICa [12]. In addition to the classic way of action of NO through intracellular signaling cascades via activation of sGC and cGMP, NO-mediated gene regulation by direct interaction with DNA binding domains has been proposed [25]. In the present study, however, the high sGC, cGMP levels as well as the qualitatively disturbed cardiomyogensis by the sGC inhibitor ODQ imply NO action through these regulatory pathways [1].

Whereas NOS expression and its functional involvement in cardiomyogenesis have so far been unknown, Peunova et al. [26] have reported on a prominent role of NO in a cell culture model of neuronal differentiation. It was shown, that NO in the presence of neuronal growth factor arrests the proliferative phase and induces neuronal differentiation. Accordingly, transient NOS activation could also be detected in embryonic retina [27] as well as embryonic olfactory bulbus [15]. Moreover, investigations in Drosophila melanogaster have confirmed, that NO not only plays a role in neuronal differentiation, but was shown to have an antiproliferative effect on embryonic tissues by regulating the critical equilibrium between cell-proliferation and -differentiation [28].

In the present piece of work we have therefore investigated a possible role of NO in cardiomyogenesis. Our study demonstrates that NOS inhibitors, but not the inactive stereoisomers do indeed strongly alter ES cell-derived cardiomyogenesis. These data are further confirmed by the abolition of the L-NMMA effect in the presence of an NO donor. The clear reduction of differentiated cardiomyocytes in the L-NMMA/L-NA treated EBs points towards the aforementioned inhibition of differentiation. A purely toxic effect can be ruled out due to the normal number of EDS cardiomyocytes in the L-NMMA/L-NA treated EBs and regular cardiomyogenesis in the presence of D-NMMA/D-NA. Furthermore, the number of vessel branches was unaltered in the presence of L-NMMA/L-NA as compared to control EBs, excluding a general, unspecific action of NOS inhibitors.

Future studies should be aimed at in detail analysis of the molecular mechanisms of NO action during cardiomyogenesis. Moreover, due to the known NOS expression pattern in heart muscle disease, it is tempting to speculate that either specific cytokines and/or other noxious stimuli may de-differentiate cardiomyocytes into an early embryonic phenotype.

Time for primary review 36 days.


   Acknowledgements
 
This study was supported by the Cologne Fortune program to W. Bloch and B.K. Fleischmann.


   Notes
 
1 Authors contributed equally to the manuscript. Back


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

  1. Moncada S., Palmer R.M., Higgs E.A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev (1991) 43:109–142.[Web of Science][Medline]
  2. Forstermann U., Schmidt H.H., Pollock J.S., et al. Isoforms of nitric oxide synthase. Characterization and purification from different cell types. Biochem Pharmacol (1991) 42:1849–1857.[CrossRef][Web of Science][Medline]
  3. Kerwin J.F. Jr., Heller M. The arginine–nitric oxide pathway: a target for new drugs. Med Res Rev (1994) 14:23–74.[CrossRef][Web of Science][Medline]
  4. Xie Q.W., Cho H.J., Calaycay J., et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science (1992) 256:225–228.[Abstract/Free Full Text]
  5. Jenkins D.C., Charles I.G., Thomsen L.L., et al. Roles of nitric oxide in tumor growth. Proc Natl Acad Sci USA (1995) 92:4392–4396.[Abstract/Free Full Text]
  6. Balligand J.L., Kelly R.A., Marsden P.A., Smith T.W., Michel T. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc Natl Acad Sci USA (1993) 90:347–351.[Abstract/Free Full Text]
  7. Balligand J.L., Ungureanu Longrois D., Simmons W.W., et al. Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1 beta and IFN-gamma. Am J Physiol (1995) 268:H1293–1303.[Web of Science][Medline]
  8. de Belder A.J., Radomski M.W., Why H.J., et al. Nitric oxide synthase activities in human myocardium. Lancet (1993) 341:84–85.[CrossRef][Web of Science][Medline]
  9. Hare J.M., Givertz M.M., Craeger M.A., Colucci W.S. Increased sensitivity to nitric oxide synthase inhibition in patients with heart failure: potentiation of beta-adrenergic inotropic responsiveness. Circulation (1998) 97(2):161–166.[Abstract/Free Full Text]
  10. Schulz R., Panas D.L., Catena R., et al. The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha. Br J Pharmacol (1995) 114:27–34.[Web of Science][Medline]
  11. Kilter H., Lenz O., La Rosee K., et al. Evidence against a role of nitric oxide in the indirect negative inotropic-effect of m-cholinoceptor stimulation in human ventricular myocardium. Naunyn Schmiedeberg’s Arch Pharmacol (1995) 352:308–312.[Web of Science][Medline]
  12. Ji G.J., Fleischmann B.K., Bloch W., et al. Regulation of the L-type Ca2+-channel during cardiomyogenesis: Switch from NO to adenylyl-cyclase-mediated inhibition. FASEB J (1999) 13:313–324.[Abstract/Free Full Text]
  13. Murad F., Mittal C.K., Arnold W.P., Katsuki S., Kimura H. Guanylate cyclase: activation by azide, nitro compounds, nitric oxide, and hydroxyl radical and inhibition by hemoglobin and myoglobin. Adv Cyclic Nucleotide Res (1978) 9:145–158.[Medline]
  14. Wobus A.M., Wallukat G., Hescheler J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation (1991) 48:173–182.[CrossRef][Web of Science][Medline]
  15. Arnhold S., Andressen C., Bloch W., Mai J.K., Addicks K. NO synthase-II is transiently expressed in embryonic mouse olfactory receptor neurons. Neurosci Lett (1997) 229:165–168.[CrossRef][Web of Science][Medline]
  16. De Vente J., Steinbusch H.W. On the stimulation of soluble and particulate guanylate cyclase in the rat brain and the involvement of nitric oxide as studied by cGMP immunocytochemistry. Acta Histochem (1992) 92:13–38.[Web of Science][Medline]
  17. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
  18. Doetschman T.C., Eistetter H., Katz M., Schmidt W., Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol (1985) 87:27–45.[Web of Science][Medline]
  19. Maltsev V.A., Wobus A.M., Rohwedel J., Bader M., Hescheler J. Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. Circ Res (1994) 75:233–244.[Abstract/Free Full Text]
  20. Isenberg G., Klockner U. Calcium tolerant ventricular myocytes prepared by preincubation in a ‘KB medium’. Pfluger’s Arch (1982) 395:6–18.[CrossRef][Web of Science][Medline]
  21. Hescheler J., Fleischmann B.K., Lentini S., et al. Embryonic stem cells: a model to study structural and functional properties in cardiomyogenesis. Cardiovasc Res (1997) 36:149–162.[Free Full Text]
  22. Miller-Hance W.C., La Corbiere M., Fuller S.J., et al. In vitro chamber specification during embryonic stem cell cardiogenesis. Expression of the ventricular myosin light chain-2 gene is independent of heart tube formation. J Biol Chem (1993) 268:25244–25252.[Abstract/Free Full Text]
  23. Garthwaite J., Southam E., Boulton C.L., et al. Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Mol Pharmacol (1995) 48:184–188.[Abstract]
  24. Gouge Rc., Marshburn P., Gordon B.E., Nunley W., Huet-Hudson Y.M. Nitric oxide as a regulator of embryonic development. Biol Reprod (1998) 58:875–879.[Abstract/Free Full Text]
  25. Henderson S.A., Lee P.H., Aeberhard E.E., et al. Nitric oxide reduces early growth response-1 gene expression in rat lung macrophages treated with interferon-gamma and lipopolysaccharide. J Biol Chem (1994) 269:25239–25242.[Abstract/Free Full Text]
  26. Peunova N., Enikolopov G. Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells. Nature (1995) 375:68–73.[CrossRef][Medline]
  27. Ientile R., Malecka B., Picciurro V., et al. Nitric oxide synthase in chick embryo retina during development. FEBS Lett (1996) 379:82–84.[CrossRef][Web of Science][Medline]
  28. Kuzin B., Roberts I., Peunova N., Enikolopov G. Nitric oxide regulates cell proliferation during drosophilia development. Cell (1996) 87:639–649.[CrossRef][Web of Science][Medline]

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