Inhibition of cAMP signaling prevents congenital heart defects counteracting oxidative stress in Pde2A deficient embryos

ABSTRACT Background Phosphodiesterases (PDEs) are the enzymes that hydrolyze cyclic nucleotides (cAMP and cGMP) playing a key role in the homeostasis of these two second 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 defects. 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. Methods Transcriptome analysis of Pde2A -/- embryonic heart was performed by RNA sequencing and the most altered genes were also analyzed by quantitative real time PCR. In vivo treatment with drugs acting on cAMP signaling (Metoprolol and H89) and oxidative stress (N-Acetyl-Cysteine, NAC) were carried out on pregnant Pde2A +/- female. Histological, biochemical, and molecular analyses were then performed on embryonic hearts. Results 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. Partial rescue of cardiac defects was observed by using the antioxidant NAC, indicating oxidative stress like one of the molecular mechanisms underpinning the CHDs. Conclusions We identified specific biological processes, molecules and cell signaling that can be targeted by selected drugs with consequent beneficial effects for cAMP-dependent CHDs. Novelty and Significance What is Known? Congenital Heart Defects are the most frequent heart birth defects including septal defects, hypertrabeculation and non-compacted myocardium. Pde2A hydrolyses the cAMP and cGMP second messengers. Pde2A -deficient mice are embryonic lethal and show cAMP-dependent Congenital Heart Defects. What New Information Does This Article Contribute? We identified several novel pathways altered in hearts of Pde2A -/- embryos. We demonstrated that drugs lowering cAMP levels rescued specific CHDs in Pde2A -/- embryos. We discovered that antioxidants are beneficial for CHDs in Pde2A -/- embryos. The significance of this work relay in molecular discoveries and pharmacological approaches to treat CHDs by using a mouse model that recapitulate the major congenital heart defects. Among the pathways involved in specific defects associated with CHDs, the transcriptome analysis revealed an impairment of genes of the immune system, cardiomyocyte development and contractility, angiogenesis, control of gene transcription and oxidative stress in Pde2A -/- hearts. The scientific community will have open access to the RNA-seq data that can be utilized to further understand the congenital cardiac pathology and clarify the molecular implication in selected defects such as septal and ventricular wall defects. Up to date CHDs, when possible and if identified in time, are mostly treated trough surgery. The identification of drugs blunting the cAMP signaling response or reducing oxidative stress pathways will be useful for setting therapeutic approaches to alleviate CHDs.