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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 7, 2004 as doi:10.1096/fj.03-1158fje. |
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,1
,2
* Department of Animal and Human Biology, INFM University of Turin, Italy;
Institutes of Neurophysiology and
Anatomy, University of Cologne, Germany; and
Institute of Physiology I, University of Bonn, Germany
2Correspondence: Institute of Physiology I, University of Bonn, Argelanderstr. 2a, D-53115 Bonn. E-mail: bernd.fleischmann{at}uni-bonn.de
SPECIFIC AIMS
Nitric oxide (NO) is thought to play an important role as a signaling molecule in embryonic and adult cardiomyocytes, but its putative role in muscarinic modulation of ICa is still unclear.
The aim of this work was to analyze the muscarinic modulation of the L-type Ca2+ current (ICa) during embryonic development. This study was motivated by our earlier findings in embryonic stem (ES) cell-derived pacemaker and atrial-like cardiomyocytes, where the involvement of NO in muscarinic signaling was proposed.
PRINCIPAL FINDINGS
1. Muscarinic receptor activation depresses basal ICa in early- but not late-stage embryonic ventricular cardiomyocytes
Analysis of the muscarinic response in wild-type (wt) ventricular cardiomyocytes showed a carbachol (CCh, 1 µM) -induced depression of basal peak ICa in early-stage (E10.5–E12.5) embryonic cardiomyocytes. Conversely, in late-stage (E16.5–E18.5) ventricular cardiomyocytes, ICa did not show a muscarinic response without ß-adrenergic prestimulation (Fig. 1
). Thus, early embryonic ventricular cardiomyocytes display prominent depression of basal ICa in the presence of CCh whereas late-stage cardiomyocytes were found to be reminiscent of terminally differentiated cells.
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2. Muscarinic agonists evoke NO production
To verify whether the generation of NO played a role in the CCh effect in early-stage cells, we performed single cell imaging experiments using the NO-sensitive fluorescence dye DAF-FM DA. We could show that CCh led to a sustained increase of DAF fluorescence, indicating NO generation. The involvement of NO was further substantiated when the NOS inhibitor L-NG-monomethyl-L-arginine (L-NMMA, 1 mM) blocked the CCh action and coapplication of excess L-arginine (400 µM) in the presence of L-NMMA restored the inhibitory effect of CCh on ICa.
3. Pertussis toxin, soluble guanylyl-cyclase (sGC), PDE inhibitors, and the catalytic subunit of PKA block the CCh effect on ICa in early-stage ventricular cardiomyocytes
To analyze the signaling pathway underlying the muscarinic depression of basal ICa in early cardiomyocytes, we first inhibited Gi/o proteins by preincubating the cells with pertussis toxin (PTX, 1 µM) for 12 h. In all cells pretreated with PTX, CCh no longer decreased basal ICa. The signaling pathway involved was further analyzed with ODQ (100 µM), a selective inhibitor of the sGC. ODQ enhanced basal ICa density whereas CCh had no effect. Accordingly, application of 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1, 15 µM), a direct activator of the sGC, caused prominent depression of ICa. We next investigated whether inhibition of PDE activity blocked the CCh effect onto ICa. The current was not reduced in the presence of IBMX and CCh; because of the obvious involvement of cGMP and PDEs in the CCh-mediated inhibition of ICa, we investigated next whether, similar to the ES cell system, a cGMP-dependent PDE isoform was involved in this signaling cascade. We used erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA, 30 µM), a selective inhibitor of PDE2. EHNA enhanced basal ICa and led to a complete block of the CCh effect on ICa. To determine whether the NO-mediated activation of the PDE2 worked through lowering of cAMP-PKA levels, we applied via patch pipette the catalytic subunit of PKA (200 µ/mL). This resulted in maximal stimulation of ICa density by 144 ± 50.4% in all cells tested. Thus, early ventricular cardiomyocytes display a different feature of muscarinic regulation: CCh-induced generation of NO via Gi/o proteins, activation of sGC-cGMP, and the cGMP-dependent PDE2 are critical determinants leading to reduced PKA activity and the ensuing decline of ICa density. These data further prove that the CCh-mediated effect onto ICa occurs via sGC, cGMP, and cGMP-dependent PDE2 and not by direct modulation of AC. Our functional data further demonstrate that in early embryonic cardiomyocytes there is a constitutive activity of sGC and AC.
4. Identification of the basal active NOS isoform in early embryonic cardiomyocytes: up-regulation of NOS1 activity in NOS2 (–/–) cardiomyocytes
To unequivocally prove NO as the key molecule involved in muscarinic signaling in embryonic ventricular cells, we investigated early- and late-stage cardiomyocytes harvested from NOS2 (–/–) and NOS3 (–/–) mice. Constitutive NOS activity was detected as application of L-NMMA led to stimulation of ICa. Our earlier evidence clearly pointed to the NOS2 isoform underlying this constitutive activity, and therefore we focused on NOS2 (–/–) cardiomyocytes. Surprisingly, the stimulatory effect of L-NMMA on basal ICa was similar in NOS2 (–/–) and wt embryonic cardiomyocytes. To understand the underlying mechanism, we first determined with immunocytochemistry NOS isoform expression in slices from adult hearts, where cardiomyocytes can easily be identified. We detected a clear up-regulation of the anti-NOS1 staining in the NOS2 (–/–) cardiomyocytes compared with wt and NOS3 (–/–) samples. Therefore, the functional relevance of the apparent up-regulation of NOS1 was investigated in embryonic NOS2 (–/–) cardiomyocytes. Trifluoromethylphenylimidazole (100 µM), a selective inhibitor of the NOS1 isoform, led to a significant stimulation of ICa. Thus, in the absence of NOS2, which is highly expressed in early-stage embryonic hearts, basal activity of NO is preserved by compensatory up-regulation of the NOS1.
5. The muscarinic response in early-stage ventricular cardiomyocytes is dependent on NOS3-mediated NO-generation
To determine whether NO and which isoform was involved in the muscarinic response in early-stage cells, the CCh effect on ICa was tested in NOS2 (–/–) cardiomyocytes. We found that muscarinic regulation was preserved (Fig. 2
), indicating that this NOS2 isoform is not involved in the CCh-mediated response. Moreover, as found in wt cells, L-NMMA abolished the CCh-mediated inhibition of ICa. We determined the CCh effect in the early ventricular cardiomyocytes harvested from NOS3 (–/–) mice. As depicted in Fig. 2
, all early-stage ventricular cells tested lacked the CCh-response. Thus, the muscarinic depression of ICa in early ventricular cardiomyocytes occurs via the NOS3-mediated generation of NO.
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6. Muscarinic signaling is intact in NOS3 (–/–) late-stage embryonic cardiomyocytes
We investigated CCh action on ICa in late-stage ventricular cardiomyocytes of wt and NOS3 (–/–) cardiomyocytes. The CCh-mediated inhibition of ICa after prestimulation with ISO is present in wt and NOS3 (–/–) mice. Furthermore, the percentage of inhibition of CCh on ISO-stimulated ICa was almost identical in wt and NOS3 (–/–) cells. These data unequivocally demonstrate that NO is not involved in muscarinic regulation in late-stage embryonic ventricular cardiomyocytes. Thus, a switch in cellular signaling in cardiomyocytes occurs during embryonic development.
CONCLUSION AND SIGNIFICANCE
We have investigated the establishment of the hormonal regulation in ventricular cardiomyocytes at the early embryonic stage. As a sensitive readout we have used the regulation of ICa to identify the signaling cascades.
Our main finding is that muscarinic modulation is mediated via generation of NO in the early embryonic ventricle. This is supported by imaging data proving the CCh-evoked generation of NO and by pharmacological inhibition of the sGC (ODQ) and the cGMP-dependent PDE2 (EHNA), resulting in blockade of the CCh action on ICa. The use of NOS2 and NOS3 (–/–) mice demonstrates that the muscarinic generation of NO occurred through selective activation of the NOS3.
Studies with NO as a signaling molecule are difficult: it is a fast and self-limiting molecule, and levels of NO are low. Interpretation is further complicated because other NO-related (as well as unrelated radicals such as peroxynitrite or anion superoxide) have similar biological effects and cannot be pharmacologically separated. We used ventricular cardiomyocytes harvested from NOS2 (–/–) and NOS3 (–/–) embryos to clarify the role of NO for muscarinic signaling during embryonic development. In early-stage NOS2 (–/–) mice, muscarinic signaling was unaltered, but absent in early-stage NOS3 (–/–) mice. Thus, in early embryonic ventricular cardiomyocytes, muscarinic signaling acts through NOS3-mediated NO generation. Conversely, at the late embryonic stage, muscarinic depression of ß-adrenergic prestimulated ICa was normal in NOS3 (–/–) cardiomyocytes, proving that at this stage NO was not involved and that a switch in the signaling cascades had occurred. The lack of NO involvement in muscarinic signaling at the late embryonic stage supports recent studies investigating the muscarinic regulation of ICa in adult murine cardiomyocytes of NOS3 (–/–) mice. Our results clearly favor lack of NO involvement in muscarinic signaling in the adult heart: in murine- and ES cell-derived cardiomyocytes, the switch to NO-independent muscarinic signaling occurs at the late embryonic stage.
We provide evidence that in ventricular cells of early embryonic stage, basal ICa is depressed by muscarinic agonists through generation of NO via NOS3. At later stages, NOS2 and NOS3 expression are both down-regulated and muscarinic signaling acts via direct inhibition of AC.
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FOOTNOTES
1 Authors contributed equally to the manuscript. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1158fje; doi: 10.1096/fj.03-1158fje
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