Heart development is one of the earliest developmental events, and its pumping action is directly linked to the intensity of development of other organs. Heart contractions mediate the circulation of the nutrients and signalling molecules to the focal points of developing embryos. In the present study, we used in vivo, ex vivo, in vitro, and in silico methods for chick embryo model to characterize and identify molecular targets under the influence of ectopic nitric oxide in reference to cardiogenesis. Spermine NONOate (SpNO) treatment of 10 μM increased the percentage of chick embryos having beating heart at 40th h of incubation by 2.2-fold (p < 0.001). In an ex vivo chick embryo culture, SpNO increased the percentage of embryos having beats by 1.56-fold (p < 0.05) compared with control after 2 h of treatment. Total body weight of SpNO-treated chick embryos at the Hamburger and Hamilton (HH) stage 29 was increased by 1.22-fold (p < 0.005). Cardiac field potential (FP) recordings of chick embryo at HH29 showed 2.5-fold (p < 0.001) increased in the amplitude, 3.2-fold (p < 0.001) increased in frequency of SpNO-treated embryos over that of the control group, whereas FP duration was unaffected. In cultured cardiac progenitors cells (CPCs), SpNO treatment decreased apoptosis and cell death by twofold (p < 0.001) and 1.7-fold (p < 0.001), respectively. Transcriptome analysis of chick embryonic heart isolated from HH15 stage pre-treated with SpNO at HH8 stage showed upregulation of genes involved in heart morphogenesis, heart contraction, cardiac cell development, calcium signalling, structure, and development whereas downregulated genes were enriched under the terms extracellular matrix, wnt pathway, and BMP pathway. The key upstream molecules predicted to be activated were p38 MAPK, MEF2C, TBX5, and GATA4 while KDM5α, DNMT3A, and HNF1α were predicted to be inhibited. This study suggests that the ectopic nitric oxide modulates the onset of cardiac development.

心脏发育是最早的发育事件之一，其泵血作用与其他器官的发育强度直接相关。心脏收缩介导营养物质和信号分子的循环，到达发育中的胚胎的焦点。在本研究中，我们使用体内，离体，体外和计算机模拟的方法对鸡胚模型进行表征和鉴定异位一氧化氮影响下的分子靶标，并以此作为参考。精胺NONOate（SpNO）处理10μM，可在孵化40h时使心脏跳动的鸡胚百分比增加2.2倍（p <0.001）。在离体雏鸡胚胎培养物中，SpNO使具有节律的胚胎百分比增加了1.56倍（p<0.05）与治疗2小时后的对照相比。在汉堡和汉密尔顿（HH）阶段29的SpNO处理的雏鸡的总体重增加了1.22倍（p <0.005）。在HH29处，鸡胚的心脏电势（FP）记录显示，与对照组相比，SpNO处理过的胚胎的振幅增加了2.5倍（p <0.001），而频率增加了3.2倍（p <0.001），而对照组FP持续时间不受影响。在培养的心脏祖细胞（CPC）中，SpNO处理可使凋亡和细胞死亡分别降低两倍（p <0.001）和1.7倍（p<0.001）。在HH8期用SpNO预处理的HH15期分离的雏鸡胚胎心脏的转录组分析显示，参与心脏形态发生，心脏收缩，心脏细胞发育，钙信号传导，结构和发育的基因上调，而下调的基因在细胞外术语下富集基质，wnt途径和BMP途径。预计被激活的关键上游分子是p38 MAPK，MEF2C，TBX5和GATA4，而KDM5α，DNMT3A和HNF1α被抑制。这项研究表明异位一氧化氮调节心脏发育的发作。