Effect of cAMP signaling activation during heart development in Pde2A deficient embryos.

Background: Phosphodiesterases (PDEs) are the enzymes that hydrolyze cyclic nucleotides (cAMP and cGMP) playing a key role in the homeostasis of these two secondary messengers. PDE2A is a dual-specific PDE that breaks down both cAMP and cGMP and can be activated by cGMP. It appears peculiar that the Pde2A-deficient (Pde2A-/-) mouse model is embryonically lethal, likely due to a strongly reduced size of liver and to a severe anemia. In addition, the heart of Pde2A-/- embryos shows ventricular and atrial septum defects, hypertrabeculation, heart dilatation and non-compaction defect. We recently highlighted a direct relationship between Pde2A impairment, consequent increase of cAMP and the onset of mouse congenital heart defects (CHDs), however the molecular mechanisms underlining the heart defects remain unknown. We found a significant modulation of more than 500 genes affecting biological processes involved in the immune system, cardiomyocyte development and contractility, angiogenesis, control of gene transcription and oxidative stress in hearts from Pde2A-/- embryos. Metoprolol and H89 administration were able to prevent heart dilatation and hypertabeculation in Pde2A-/- embryos. Metoprolol was also able to partially impede heart septum defect and oxidative stress at tissue and molecular levels. By using the antioxidant NAC partial rescue of cardiac defects was observed straightening the oxidative stress like one of the critical molecular mechanisms underpinning the CHDs associated to cAMP unbalance. Transcriptome analysis of Pde2A-/- embryonic heart was performed by RNA sequencing in 5 knockout and 6 wild-type, the most altered genes were also analyzed by quantitative real time PCR研究背景：磷酸二酯酶（Phosphodiesterases, PDEs）是一类可水解环核苷酸（cyclic nucleotides，包括cAMP与cGMP）的酶类，在这两种第二信使的稳态维持中发挥关键调控作用。PDE2A属于双特异性磷酸二酯酶，能够同时降解cAMP与cGMP，且可被cGMP激活。值得注意的是，Pde2A基因敲除（Pde2A-/-）小鼠模型呈现胚胎致死表型，推测其潜在原因为肝脏体积显著缩小以及重度贫血。此外，Pde2A-/-胚胎的心脏可见心室与心房间隔缺损、肌小梁过度增生、心脏扩张以及心肌非致密化缺陷。本团队近期揭示了Pde2A功能受损、伴随cAMP水平升高，与小鼠先天性心脏病（congenital heart defects, CHDs）发生之间的直接关联，但心脏缺陷背后的分子机制仍未阐明。我们在Pde2A-/-胚胎的心脏组织中发现，超过500个基因存在显著表达调控异常，这些基因涉及免疫系统、心肌细胞发育与收缩功能、血管生成、基因转录调控以及氧化应激等多种生物学过程。给予美托洛尔（Metoprolol）与H89处理，可阻止Pde2A-/-胚胎出现心脏扩张与肌小梁过度增生表型；美托洛尔还可在组织与分子层面部分改善心脏间隔缺损与氧化应激状态。通过使用抗氧化剂N-乙酰半胱氨酸（NAC）进行干预，可观察到心脏缺陷得到部分挽救，这证实氧化应激是介导cAMP失衡相关先天性心脏病的关键分子机制之一。本研究通过RNA测序技术，对5例Pde2A基因敲除胚胎心脏样本与6例野生型对照胚胎心脏样本开展转录组分析，并通过实时定量PCR（quantitative real time PCR）对差异表达最为显著的基因进行了验证分析。