Raggi et al. J Transl Med (2020) 18:21 https://doi.org/10.1186/s12967-020-02210-5 Journal of Translational Medicine RESEARCH Open Access Transcriptome analysis defnes myocardium gene signatures in children with ToF and ASD and reveals disease-specifc molecular reprogramming in response to surgery with cardiopulmonary bypass Federica Raggi1†, Davide Cangelosi1†, Pamela Becherini1,3, Fabiola Blengio1,4, Martina Morini1, Massimo Acquaviva1,5, Maria Luisa Belli1,6, Giuseppe Panizzon2, Giuseppe Cervo2, Luigi Varesio1^, Alessandra Eva1† and Maria Carla Bosco1*† Abstract Background: Tetralogy of Fallot (ToF) and Atrial Septal Defects (ASD) are the most common types of congenital heart diseases and a major cause of childhood morbidity and mortality. Cardiopulmonary bypass (CPB) is used during corrective cardiac surgery to support circulation and heart stabilization. However, this procedure triggers systemic infammatory and stress response and consequent increased risk of postoperative complications. The aim of this study was to defne the molecular bases of ToF and ASD pathogenesis and response to CPB and identify new potential biomarkers. Methods: Comparative transcriptome analysis of right atrium specimens collected from 10 ToF and 10 ASD patients was conducted before (Pre-CPB) and after (Post-CPB) corrective surgery. Total RNA isolated from each sample was individually hybridized on Afymetrix HG-U133 Plus Array Strips containing 38,500 unique human genes. Diferences in the gene expression profles and functional enrichment/network analyses were assessed using bioinformatic tools. qRT-PCR analysis was used to validate gene modulation. Results: Pre-CPB samples showed signifcant diferential expression of a total of 72 genes, 28 of which were over- expressed in ToF and 44 in ASD. According to Gene Ontology annotation, the mostly enriched biological processes were represented by matrix organization and cell adhesion in ToF and by muscle development and contractility in ASD specimens. GSEA highlighted the specifc enrichment of hypoxia gene sets in ToF samples, pointing to a role for hypoxia in disease pathogenesis. The post-CPB myocardium exhibited signifcant alterations in the expression profle *Correspondence: [email protected] †Federica Raggi and Davide Cangelosi contributed equally to this work †Alessandra Eva and Maria Carla Bosco share senior authorship ^Luigi Varesio—Deceased. The authors dedicate the manuscript to the memory of Luigi Varesio who prematurely passed away during the course of the study 1 Laboratory of Molecular Biology, IRCSS Istituto Giannina Gaslini, Padiglione 2, L.go G.Gaslini 5, 16147 Genova, Italy Full list of author information is available at the end of the article © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Raggi et al. J Transl Med (2020) 18:21 Page 2 of 20 of genes related to transcription regulation, growth/apoptosis, infammation, adhesion/matrix organization, and oxi- dative stress. Among them, only 70 were common to the two disease groups, whereas 110 and 24 were unique in ToF and ASD, respectively. Multiple functional interactions among diferentially expressed gene products were predicted by network analysis. Interestingly, gene expression changes in ASD samples followed a consensus hypoxia profle. Conclusion: Our results provide a comprehensive view of gene reprogramming in right atrium tissues of ToF and ASD patients before and after CPB, defning specifc molecular pathways underlying disease pathophysiology and myocardium response to CPB. These fndings have potential translational value because they identify new candidate prognostic markers and targets for tailored cardioprotective post-surgical therapies. Keywords: Gene expression profling, Congenital heart disease, Cardiopulmonary bypass, Atrial myocardium, Hypoxia Background surgery at the age of 4/5 years [5, 6]. Surgical closure of Congenital heart diseases (CHDs) are the most frequent atrial septal defects is usually associated with normal life types of birth defects in humans, afecting over 1% of all expectancy [5]. live births worldwide (estimated incidence 8 per 1000), Cardiopulmonary bypass (CPB) with aortic cross- and represent a major cause of morbidity and mortality clamping (AoXC) and hypothermic cardioplegic arrest in children [1]. CHDs can be classifed into three broad (CA) is a commonly used technique in cardiac surgery to categories: cyanotic heart disease, left-sided obstruc- support circulation and heart stabilization and maintain tive defects, and septation defects [2]. Tetralogy of Fal- organ perfusion. It facilitates the repair of cardiac lesions lot (ToF) is the main form of cyanotic CHDs (estimated resulting in the reduction of surgical mortality and incidence 5 per 10,000 live births), characterized by a achievement of complete repair of heart defects also at an conal septum malalignment which leads to aorta right- early age [4, 7]. However, despite the eforts to minimize ward deviation. Tis defect results in a large ventricular organ damage, cardiac surgery with CPB is associated septal malformation and stenosis of the pulmonary valve with postoperative morbidity and multiorgan dysfunc- with consequent pressure and volume overload of the tion syndrome. It is well documented that CPB triggers a right ventricle, adaptive ventricular hypertrophy associ- systemic infammatory response, whose activation in the ated with reduced pulmonary fow, impaired myocardial setting of major surgery and trauma can be exaggerated nutrient and oxygen supply, and fnally heart failure [3]. in some patients, resulting in the inappropriate recruit- ToF patients require primary surgical repair during the ment and hyperactivation of leukocytes (mainly neutro- frst year of life to close the ventricular defect and remove phils and monocyte/macrophages), increased release of the obstruction in order to relieve hypoxemia, eliminate proinfammatory cytokines, excessive stimulation of the the hypertrophic stimulus, and preserve the function of complement and coagulation systems, and endothelial the right ventricle. However, long-term complications dysfunction, eventually leading to unwarranted organ and probability of secondary corrective surgery later damage [8, 9]. In addition, myocardium subjected to CA in life remain important clinical challenges [3, 4]. Atrial undergoes an obligate period of ischemia lasting about Septal Defects (ASD) are the third most common types 1 h. Subsequent reperfusion of ischemic myocardium of CHD (estimated incidence 10 per 10,000 live births). (I/R) causes the release of reactive oxygen species (ROS), Tey are characterized by several defects in the cardiac apoptosis, and necrosis which can further aggravate CPB- terminations of systemic and pulmonary veins and in the induced infammatory and stress response, contributing intratrial septum, which result in the communication of to organ dysfunction and increasing the risk of postop- the heart left and right sides and blood shunt between erative complications and myocardial failure [9–12]. pulmonary and systemic circulations [5]. Only a few ASD Although signifcant advances in the clinical manage- patients present with severe problems during infancy ment of ToF and ASD patients have been made in the and require primary cardiac surgery within the frst year last few decades, much remains to be elucidated regard- of life to prevent the onset of irreversible changes in ing the molecular mechanisms underlying disease patho- the pulmonary vasculature [6]. Most ASD patients are genesis and myocardial response to corrective surgery asymptomatic throughout infancy and childhood but with CPB. In the feld of cardiomyopathy, microarray- may develop complications that increase with age, which based gene expression profling has become an impor- include ventricle dysfunction, atrial arrhythmias, pul- tant approach for the characterization of the molecular monary hypertension, and heart failure. Life expectancy bases of disease pathogenesis, progression, and response is reduced if defects remain untreated, recommending to surgery/therapy, contributing to the identifcation of Raggi et al. J Transl Med (2020) 18:21 Page 3 of 20 novel biomarkers essential for the refnement of patient full-thickness specimens, and was clinically reproducible, diagnostic and prognostic evaluation and the design of as previously reported by Voisine et al. [13]. tailored treatment strategies [10, 13–17]. In this study, we conducted a comparative transcrip- RNA isolation and cRNA synthesis