Sykes Benjamin G, Van Steyn Peter M, Vignali Jonathan D, Winalski John, Lozier Julie, Bell Wade E, Turner James E
Department of Biology, Center for Molecular, Cellular, and Biological Chemistry, Virginia Military Institute, Lexington, VA 24450, USA.
Brain Sci. 2016 Oct 26;6(4):51. doi: 10.3390/brainsci6040051.
It has been known that both estrogen (E2) and nitric oxide (NO) are critical for proper cardiovascular system (CVS) function. It has also been demonstrated that E2 acts as an upstream effector in the nitric oxide (NO) pathway. Results from this study indicate that the use of a nitric oxide synthase (NOS) inhibitor (NOSI) which targets specifically neuronal NOS (nNOS or NOS1), proadifen hydrochloride, caused a significant depression of fish heart rates (HR) accompanied by increased arrhythmic behavior. However, none of these phenotypes were evident with either the inhibition of endothelial NOS (eNOS) or inducible NOS (iNOS) isoforms. These cardiac arrhythmias could also be mimicked by inhibition of E2 synthesis with the aromatase inhibitor (AI), 4-OH-A, in a manner similar to that of nNOSI. In both scenarios, by using an NO donor (DETA-NO) in either NO + nNOSI or E2 + AI co-treatments, fish could be significantly rescued from decreased HR and increased arrhythmias. However, the addition of an NOS inhibitor (L-NAME) to the E2 + AI co-treatment fish prevented the rescue of low heart rates and arrhythmias, which strongly implicates the NO pathway as a downstream E2 targeted molecule for the maintenance of healthy cardiomyocyte contractile conditions in the developing zebrafish. Cardiac arrhythmias could be mimicked by the S-nitrosylation pathway inhibitor DTT (1,4-dithiothreitol) but not by ODQ (1H-[1-3]oxadiazolo[4,3-a]quinoxalin-1-one), the inhibitor of the NO receptor molecule sGC in the cGMP-dependent pathway. In both the nNOSI and AI-induced arrhythmic conditions, 100% of the fish expressed the phenotype, but could be rapidly rescued with maximum survival by a washout with dantrolene, a ryanodine Ca channel receptor blocker, compared to the time it took for rescue using a control salt solution. In addition, of the three NOS isoforms, eNOS was the one most implicated in the maintenance of an intact developing fish vascular system. In conclusion, results from this study have shown that nNOS is the prominent isoform that is responsible, in part, for maintaining normal heart rates and prevention of arrhythmias in the developing zebrafish heart failure model. These phenomena are related to the upstream stimulatory regulation by E2. On the other hand, eNOS has a minimal effect and iNOS has little to no influence on this phenomenon. Data also suggests that nNOS acts on the zebrafish cardiomyocytes through the S-nitrosylation pathway to influence the SR ryanidine Ca channels in the excitation-coupling phenomena. In contrast, eNOS is the prominent isoform that influences blood vessel development in this model.
已知雌激素（E2）和一氧化氮（NO）对心血管系统（CVS）的正常功能都至关重要。也已证明E2在一氧化氮（NO）途径中作为上游效应物起作用。本研究结果表明，使用特异性靶向神经元型一氧化氮合酶（nNOS或NOS1）的一氧化氮合酶抑制剂（NOSI）盐酸普罗地芬，会导致鱼心率（HR）显著降低，并伴有心律失常行为增加。然而，抑制内皮型一氧化氮合酶（eNOS）或诱导型一氧化氮合酶（iNOS）亚型均未出现这些表型。用芳香化酶抑制剂（AI）4-羟基雄烯二酮抑制E2合成，也能以类似于nNOSI的方式模拟这些心律失常。在这两种情况下，通过在NO + nNOSI或E2 + AI联合处理中使用NO供体（DETA-NO），鱼的心率降低和心律失常增加的情况可得到显著缓解。然而，在E2 + AI联合处理的鱼中添加一氧化氮合酶抑制剂（L-NAME）可阻止低心率和心律失常的缓解，这强烈表明NO途径是E2的下游靶向分子，用于维持斑马鱼发育中心肌细胞的健康收缩状态。心律失常可被S-亚硝基化途径抑制剂二硫苏糖醇（DTT，1,4-二硫苏糖醇）模拟，但不能被cGMP依赖性途径中NO受体分子可溶性鸟苷酸环化酶（sGC）的抑制剂ODQ（1H-[1,3]恶二唑并[4,3-a]喹喔啉-1-酮）模拟。在nNOSI和AI诱导的心律失常情况下，100%的鱼表现出该表型，但与使用对照盐溶液进行挽救所需的时间相比，用兰尼碱钙通道受体阻滞剂丹曲林冲洗可使其迅速得到最大程度的挽救并存活。此外，在三种一氧化氮合酶亚型中，eNOS最与维持完整的发育中鱼血管系统有关。总之，本研究结果表明，nNOS是在斑马鱼发育性心力衰竭模型中部分负责维持正常心率和预防心律失常的主要亚型。这些现象与E2的上游刺激调节有关。另一方面，eNOS的影响最小，iNOS对此现象几乎没有影响。数据还表明，nNOS通过S-亚硝基化途径作用于斑马鱼心肌细胞，以影响兴奋-偶联现象中的肌浆网兰尼碱钙通道。相比之下，eNOS是在该模型中影响血管发育的主要亚型。
