Bloch W, Fleischmann B K, Lorke D E, Andressen C, Hops B, Hescheler J, Addicks K
Institute of Anatomy I, University of Cologne, Germany.
Cardiovasc Res. 1999 Aug 15;43(3):675-84. doi: 10.1016/s0008-6363(99)00160-1.
The aim of the present study was the investigation of the expression of NOS during cardiomyogenesis and its functional role.
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.
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).
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.
本研究旨在探究一氧化氮合酶（NOS）在心肌发生过程中的表达及其功能作用。
分别采用免疫细胞化学和斑点印迹法评估心脏发育不同阶段NOS亚型的定性和定量表达。利用体外胚胎干细胞分化模型和选择性药理试剂研究心肌发生过程中NOS表达的功能相关性。
在胚胎发育第7.5天（E7.5），胚胎中未检测到任何NOS亚型的表达，而在胚胎外部分检测到诱导型（iNOS）和内皮型（eNOS）亚型。相反，从E9.5开始，大鼠和小鼠胚胎显示出显著的iNOS和eNOS表达。这与可溶性鸟苷酸环化酶（sGC）的高表达以及高环磷酸鸟苷（cGMP）含量相关。在E14.5后的进一步发育过程中，iNOS和eNOS均开始下调，在出生前不久发现eNOS染色减少，而iNOS几乎检测不到。我们进一步研究了一氧化氮（NO）是否在心肌发生中起作用，使用体外胚胎干细胞衍生的心肌细胞在胚状体（EBs）中分化。这些细胞中的NOS表达模式与体内检测到的模式相似。我们证明，接种后将EBs与NOS抑制剂L-NMMA（2 - 10 mM）或L-NA（2 - 10 mM）连续孵育4至9天会导致心肌细胞明显的分化停滞，而这种作用可通过共同应用NO供体精胺 - NONOate（10 microM）来逆转。
iNOS和eNOS亚型在心肌发生的早期阶段均显著表达。在E14.5左右，NOS表达开始下降。此外，由于NOS抑制剂使用胚胎干细胞系统可阻止终末分化心肌细胞成熟，因此心肌发生需要NO生成。
