Nitric oxide synthase expression and role during cardiomyogenesis.
OBJECTIVE
The aim of the present study was the investigation of the expression of NOS during cardiomyogenesis and its functional role.


METHODS
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 microM).


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.
Bloch
W
W
Institute of Anatomy I, University of Cologne, Germany.
Fleischmann
B K
BK
Lorke
D E
DE
Andressen
C
C
Hops
B
B
Hescheler
J
J
Addicks
K
K
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005865:Gestational Age; D006321:Heart / Q000196:embryology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019335:Nitroarginine / Q000494:pharmacology; D009589:Nitrogen Oxides; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology; D013234:Stem Cells / Q000187:drug effects / Q000201:enzymology*; D019323:omega-N-Methylarginine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; C091861:spermine nitric oxide complex; D019335:Nitroarginine; D019323:omega-N-Methylarginine; D013096:Spermine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat; C496315:Nos3 protein, mouse; C496316:Nos3 protein, rat