Hemodynamic Melody of Postnatal Cardiac and Pulmonary Development in Children with Congenital Heart Diseases.

Hemodynamics is the eternal theme of the circulatory system. Abnormal hemodynamics and cardiac and pulmonary development intertwine to form the most important features of children with congenital heart diseases (CHDs), thus determining these children's long-term quality of life. Here, we review the varieties of hemodynamic abnormalities that exist in children with CHDs, the recently developed neonatal rodent models of CHDs, and the inspirations these models have brought us in the areas of cardiomyocyte proliferation and maturation, as well as in alveolar development. Furthermore, current limitations, future directions, and clinical decision making based on these inspirations are highlighted. Understanding how CHD-associated hemodynamic scenarios shape postnatal heart and lung development may provide a novel path to improving the long-term quality of life of children with CHDs, transplantation of stem cell-derived cardiomyocytes, and cardiac regeneration.

Corrigendum: Identification of ACKR4 as an immune checkpoint in pulmonary arterial hypertension.

[This corrects the article DOI: 10.3389/fimmu.2023.1153573.].

A surgical mouse model of neonatal right ventricular outflow tract obstruction by pulmonary artery banding.

BACKGROUD: Diseased animal models play an extremely important role in preclinical research. Lacking the corresponding animal models, many basic research studies cannot be carried out, and the conclusions obtained are incomplete or even incorrect. Right ventricular (RV) outflow tract (RVOT) obstruction leads to RV pressure overload (PO) and reduced pulmonary blood flow (RPF), which are 2 of the most important pathophysiological characteristics in pediatric cardiovascular diseases and seriously affect the survival rate and long-term quality of life of many children. Due to the lack of a neonatal mouse model for RVOT obstruction, it is largely unknown how RV PO and RPF regulate postnatal RV and pulmonary development. The aim of this study was to construct a neonatal RVOT obstruction mouse model. METHODS AND RESULTS: Here, we first introduced a neonatal mouse model of RVOT obstruction by pulmonary artery banding (PAB) on postnatal day 1. PAB induced neonatal RVOT obstruction, RV PO, and RPF. Neonatal RV PO induced cardiomyocyte proliferation, and neonatal RPF induced pulmonary dysplasia, the 2 features that are not observed in adult RVOT obstruction. As a result, PAB neonates exhibited overall developmental dysplasia, a sign similar to that of children with RVOT obstruction. CONCLUSIONS: Because many pediatric cardiovascular diseases are associated with RV PO and RPF, the introduction of a neonatal mouse model of RVOT obstruction may greatly enhance our understanding of these diseases and eventually improve or save the lives of many children.

Volume overload impedes the maturation of sarcomeres and T-tubules in the right atria: a potential cause of atrial arrhythmia following delayed atrial septal defect closure.

Introduction: Adult patients with atrial septal defects (ASD), the most common form of adult congenital heart disease, often die of arrhythmias, and the immaturity of cardiomyocytes contributes significantly to arrhythmias. ASD typically induces a left-to-right shunt, which then leads to the right atrium (RA) volume overload (VO). Whether or not VO contributes to RA cardiomyocyte immaturity and thereby causes arrhythmias in adult patients with ASD remains unclear. Methods: Here, we developed the first neonatal RA VO mouse model by creating a fistula between the inferior vena cava and abdominal aorta on postnatal day 7. RA VO was confirmed by increases in the mean flow velocity, mean pressure gradient, and velocity time integral across the tricuspid valve, and an increase in the RA diameter and RA area middle section. Results: We found that VO decreased the regularity and length of sarcomeres, and decreased the T-element density, regularity, and index of integrity of T-tubules in RA cardiomyocytes, suggesting that the two most important maturation hallmarks (sarcomere and T-tubules) of RA cardiomyocytes were impaired by VO. Accordingly, the calcium handling capacity of cardiomyocytes from postnatal day 21 (P21) RA was decreased by VO. VO caused a significant elongation of the PR interval. The expression of connexin 43 (Cx43) was decreased in RA VO. Moreover, gene ontology (GO) analysis of the downregulated genes in RA demonstrated that there was an abundance of enriched terms associated with sarcomeres and T-tubules exposed to VO. The results were further verified by qRT-PCR. Conclusions: In conclusion, the first neonatal RA VO mouse model was developed; furthermore, using this neonatal RA VO mouse model, we revealed that VO impeded RA sarcomere and T-tubule maturation, which may be the underlying causes of atrial arrhythmias in adult patients with ASD.

Identification of ACKR4 as an immune checkpoint in pulmonary arterial hypertension.

Objective: Inflammation is recognized as a contributor in the development of pulmonary arterial hypertension (PAH), and the recruitment and functional capacity of immune cells are well-orchestrated by chemokines and their receptors. This study is aimed at identification of critical chemokines in the progression of PAH via transcriptomic analysis. Methods: Differentially expressed genes (DEGs) from lungs of PAH patients were achieved compared to controls based on Gene Expression Omnibus (GEO) database. Gene set enrichment analysis (GSEA) was applied for functional annotation and pathway enrichement. The abundance of immune cells was estimated by the xCell algorithm. Weighted correlation network analysis (WGCNA) was used to construct a gene expression network, based on which a diagnostic model was generated to determine its accuracy to distinguish PAH from control subjects. Target genes were then validated in lung of hypoxia-induce pulmonary hypertension (PH) mouse model. Results: ACKR4 (atypical chemokine receptor 4) was downregulated in PAH lung tissues in multiple datasets. PAH relevant biological functions and pathways were enriched in patients with low-ACKR4 level according to GSEA enrichment analysis. Immuno-infiltration analysis revealed a negative correlation of activated dendritic cells, Th1 and macrophage infiltration with ACKR4 expression. Three gene modules were associated with PAH via WGCNA analysis, and a model for PAH diagnosis was generated using CXCL12, COL18A1 and TSHZ2, all of which correlated with ACKR4. The ACKR4 expression was also downregulated in lung tissues of our experimental PH mice compared to that of controls. Conclusions: The reduction of ACKR4 in lung tissues of human PAH based on transcriptomic data is consistent with the alteration observed in our rodent PH. The correlation with immune cell infiltration and functional annotation indicated that ACKR4 might serve as a protective immune checkpoint for PAH.

A neonatal rat model of pulmonary vein stenosis.

OBJECTIVES: Pulmonary vein stenosis (PVS), one of the most challenging clinical problems in congenital heart disease, leads to secondary pulmonary arterial hypertension (PAH) and right ventricular (RV) hypertrophy. Due to the lack of a rodent model, the mechanisms underlying PVS and its associated secondary effects are largely unknown, and treatments are minimally successful. This study developed a neonatal rat PVS model with the aim of increasing our understanding of the mechanisms and developing possible treatments for PVS. METHODS: PVS was created at postnatal day 1 (P1) by banding pulmonary veins that receive blood from the right anterior and mid lobes. The condition was confirmed using echocardiography, computed tomography (CT), gross anatomic examination, hematoxylin and eosin (H&E) staining, fibrosis staining, and immunofluorescence. Lung and RV remodeling under the condition of PVS were evaluated using H&E staining, fibrosis staining, and immunofluorescence. RESULTS: At P21, echocardiography revealed a change in wave form and a decrease in pulmonary artery acceleration time-indicators of PAH-at the transpulmonary valve site in the PVS group. CT at P21 showed a decrease in pulmonary vein diameter in the PVS group. At P30 in the PVS group, gross anatomic examination showed pulmonary congestion, H&E staining showed wall thickening and lumen narrowing in the upstream pulmonary veins, and immunofluorescence showed an increase in the smooth muscle layers in the upstream pulmonary veins. In addition, at P30 in the PVS group, lung remodeling was evidenced by hyperemia, thickening of pulmonary small vessel walls and smooth muscle layers, and reduction of the number of alveoli. RV remodeling was evidenced by an increase in RV free wall thickness. CONCLUSIONS: A neonatal rat model of PVS was successfully established, showing secondary lung and RV remodeling. This model may serve as a useful platform for understanding the mechanisms and treatments for PVS.

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model.

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

The implication of chromosomal abnormalities in the surgical outcomes of Chinese pediatric patients with congenital heart disease.

Background: Copy number variations (CNVs) have been shown to be overrepresented in children with congenital heart disease (CHD). Genetic evaluation of CHD is currently underperformed in China. We sought to determine the occurrence of CNVs in CNV regions with disease-causing potential among a large cohort of Chinese pediatric CHD patients and investigate whether these CNVs could be the important critical modifiers of surgical intervention. Methods: CNVs screenings were performed in 1,762 Chinese children who underwent at least one cardiac surgery. CNV status at over 200 CNV locus with disease-causing potential was analyzed with a high-throughput ligation-dependent probe amplification (HLPA) assay. Results: We found 378 out of 1,762 samples (21.45%) to have at least one CNV and 2.38% of them were carrying multiple CNVs. The detection rates of ppCNVs (pathogenic and likely pathogenic CNVs) were 9.19% (162/1,762), significantly higher than that of the healthy Han Chinese individuals from The Database of Genomic Variants archive (9.19% vs. 3.63%; P = 0.0012). CHD cases with ppCNVs had a significantly higher proportion of complex surgeries compared to CHD patients with no ppCNVs (62.35% vs. 37.63%, P < 0.001). Duration of cardiopulmonary bypass and aortic cross clamp procedures were significantly longer in CHD cases with ppCNVs (all P < 0.05), while no group differences were identified for complications of surgery and one-month mortality after surgery. The detection rate of ppCNVs in the atrioventricular septal defect (AVSD) subgroup was significantly higher than that in other subgroups (23.10% vs. 9.70%, P = 0.002). Conclusions: CNV burden is an important contributor to Chinese children with CHD. Our study demonstrated the robustness and diagnostic efficiency of HLPA method in the genetic screening of CNVs in CHD patients.

Dynamic changes in P300 enhancers and enhancer-promoter contacts control mouse cardiomyocyte maturation.

Cardiomyocyte differentiation continues throughout murine gestation and into the postnatal period, driven by temporally regulated expression changes in the transcriptome. The mechanisms that regulate these developmental changes remain incompletely defined. Here, we used cardiomyocyte-specific ChIP-seq of the activate enhancer marker P300 to identify 54,920 cardiomyocyte enhancers at seven stages of murine heart development. These data were matched to cardiomyocyte gene expression profiles at the same stages and to Hi-C and H3K27ac HiChIP chromatin conformation data at fetal, neonatal, and adult stages. Regions with dynamic P300 occupancy exhibited developmentally regulated enhancer activity, as measured by massively parallel reporter assays in cardiomyocytes in vivo, and identified key transcription factor-binding motifs. These dynamic enhancers interacted with temporal changes of the 3D genome architecture to specify developmentally regulated cardiomyocyte gene expressions. Our work provides a 3D genome-mediated enhancer activity landscape of murine cardiomyocyte development.

Impaired cell-cell communication and axon guidance because of pulmonary hypoperfusion during postnatal alveolar development.

BACKGROUND: Pulmonary hypoperfusion is common in children with congenital heart diseases (CHDs) or pulmonary hypertension (PH) and causes adult pulmonary dysplasia. Systematic reviews have shown that some children with CHDs or PH have mitigated clinical outcomes with COVID-19. Understanding the effects of pulmonary hypoperfusion on postnatal alveolar development may aid in the development of methods to improve the pulmonary function of children with CHDs or PH and improve their care during the COVID-19 pandemic, which is characterized by cytokine storm and persistent inflammation. METHODS AND RESULTS: We created a neonatal pulmonary hypoperfusion model through pulmonary artery banding (PAB) surgery at postnatal day 1 (P1). Alveolar dysplasia was confirmed by gross and histological examination at P21. Transcriptomic analysis of pulmonary tissues at P7(alveolar stage 2) and P14(alveolar stage 4) revealed that the postnatal alveolar development track had been changed due to pulmonary hypoperfusion. Under the condition of pulmonary hypoperfusion, the cell-cell communication and axon guidance, which both determine the final number of alveoli, were lost; instead, there was hyperactive cell cycle activity. The transcriptomic results were further confirmed by the examination of axon guidance and cell cycle markers. Because axon guidance controls inflammation and immune cell activation, the loss of axon guidance may explain the lack of severe COVID-19 cases among children with CHDs or PH accompanied by pulmonary hypoperfusion. CONCLUSIONS: This study suggested that promoting cell-cell communication or supplementation with guidance molecules may treat pulmonary hypoperfusion-induced alveolar dysplasia, and that COVID-19 is less likely to cause a cytokine storm in children with CHD or PH accompanied by pulmonary hypoperfusion.

Congenital heart disease-associated pulmonary dysplasia and its underlying mechanisms.

Clinical observation indicates that exercise capacity, an important determinant of survival in patients with congenital heart disease (CHD), is most decreased in children with reduced pulmonary blood flow (RPF). However, the underlying mechanism remains unclear. Here, we obtained human RPF lung samples from children with tetralogy of Fallot as well as piglet and rat RPF lung samples from animals with pulmonary artery banding surgery. We observed impaired alveolarization and vascularization, the main characteristics of pulmonary dysplasia, in the lungs of RPF infants, piglets, and rats. RPF caused smaller lungs, cyanosis, and body weight loss in neonatal rats and reduced the number of alveolar type 2 cells. RNA sequencing demonstrated that RPF induced the downregulation of metabolism and migration, a key biological process of late alveolar development, and the upregulation of immune response, which was confirmed by flow cytometry and cytokine detection. In addition, the immunosuppressant cyclosporine A rescued pulmonary dysplasia and increased the expression of the Wnt signaling pathway, which is the driver of postnatal lung development. We concluded that RPF results in pulmonary dysplasia, which may account for the reduced exercise capacity of patients with CHD with RPF. The underlying mechanism is associated with immune response activation, and immunosuppressants have a therapeutic effect in CHD-associated pulmonary dysplasia.

Ability of the Right Ventricle to Serve as a Systemic Ventricle in Response to the Volume Overload at the Neonatal Stage.

BACKGROUND: In children with hypoplastic left heart syndrome (HLHS), volume overload (VO) is inevitable, and the right ventricle (RV) pumps blood into the systemic circulation. Understanding the molecular differences and their different responses to VO between the RV and left ventricle (LV) at the neonatal and highly plastic stages may improve the long-term management of children with HLHS. METHODS AND RESULTS: A neonatal rat ventricular VO model was established by the creation of a fistula between the inferior vena cava and the abdominal aorta on postnatal day 1 (P1) and confirmed by echocardiographic and histopathological analyses. Transcriptomic analysis demonstrated that some of the major differences between a normal neonatal RV and LV were associated with the thyroid hormone and insulin signaling pathways. Under the influence of VO, the levels of insulin receptors and thyroid hormone receptors were significantly increased in the LV but decreased in the RV. The transcriptomic analysis also demonstrated that under the influence of VO, the top two common enriched pathways between the RV and LV were the insulin and thyroid hormone signaling pathways, whereas the RV-specific enriched pathways were primarily associated with lipid metabolism and arrhythmogenic right ventricular cardiomyopathy (ARVC); further, the LV-specific enriched pathways were primarily associated with nucleic acid metabolism and microRNAs in cancer. CONCLUSIONS: Insulin and thyroid hormones may play critical roles in the differences between a neonatal RV and LV as well as their common responses to VO. Regarding the isolated responses to VO, the RV favors an ARVC change and the LV favors a reduction in microRNAs in cancer. The current study suggests that insulin, thyroid hormone, and cancer-associated microRNAs are potential therapeutic targets that should be explored by basic science studies to improve the function of the RV to match that of the LV.

Expression and Predictive Functional Profiles of MicroRNAs Data in Vascularized Composite Allotransplantation Acute Rejection.

BACKGROUND: This study aimed to investigate the mechanisms of acute rejection for vascularized composite allotransplantation (VCA) using microRNAs (miRNAs) differential expression in a VCA animal model. METHODS: Brown Norway rats were used as transplant donors and Lewis rats as VCA receptors. The changes were divided into different stages before and after transplantation in Lewis rats, and all appearance changes were recorded. Also, histological evaluations were performed on all recipients, and the expression of microRNAs was analyzed when acute immune rejection occurred. Then, we used GO and KEGG Pathway enrichment analyses to predict miRNA targets. Finally, differentially expressed miRNAs were detected by RT-qPCR. RESULTS: Compared to pre-operation, 22 miRNAs were differentially expressed after operations. Among them, nine were upregulated, and 13 were downregulated in skin tissues. The RT-qPCR results revealed that rno-miR-340-5p and rno-miR-21-5p were significantly upregulated and enriched in the PI3K-Akt signaling pathway. Moreover, rno-miR-145-5p and rno-miR-195-5p were significantly downregulated, and most of their target genes were enriched in the Hippo signaling pathway. The histological evaluations showed that, after VCA, the skin tissue presented severe acute rejection. CONCLUSIONS: The miRNAs rno-miR-340-5p, rno-miR-21-5p, rno-miR-145-5p, and rno-miR-195-5p were significantly regulated during VCA acute rejection, when the four miRNAs analyses were done on skin biopsies. These miRNAs might be potential biomarkers for objective, early, and minimally invasive rejection diagnosis.

Potential Roles of miRNAs in Acute Rejection for Vascularized Composite Allotransplantation.

Aim: The development of microsurgery has greatly advanced vascularized composite allotransplantation (VCA). However, like organ transplantation, VCA is also limited by acute rejection, and concerns regarding long-term survival and function of the transplanted graft. Therefore, it is necessary to elucidate the molecular mechanisms underlying acute rejection caused by VCA, in order to improve patient survival. Methods: Firstly, we used Brown Norway rats and Lewis rats to construct animal model of VCA. Regularly record the appearance changes of all subjects. Specimens were collected for histological examination, microRNAs (miRNAs) sequencing and RT-qPCR verification when acute immune rejection occurred. Then, bioinformatics analysis was employed to predict miRNA related molecules and pathway information. Finally, differentially expressed miRNAs were tested and verified. Results: MiRNAs are small non coding RNA transcripts that regulate gene expression at the post-transcriptional level. Studies have shown that miRNAs are involved in immune regulation and several miRNAs have been identified that are potential diagnostic and prognostic biomarkers of acute rejection. In this study, we found that the expression levels of rno-miR-21-5p, rno-miR-340-5p, rno-miR-1-3p and rno-miR-195-5p are significantly associated with acute rejection following VCA. Conclusion: This miRNA signature can potentially an auxiliary diagnostic indicator of rejection, which can help clinicians adjust the immunosuppressive program in time during acute rejection.

GATA4 Regulates Developing Endocardium Through Interaction With ETS1.

BACKGROUND: The pioneer transcription factor (TF) GATA4 (GATA Binding Protein 4) is expressed in multiple cardiovascular lineages and is essential for heart development. GATA4 lineage-specific occupancy in the developing heart underlies its lineage specific activities. Here, we characterized GATA4 chromatin occupancy in cardiomyocyte and endocardial lineages, dissected mechanisms that control lineage specific occupancy, and analyzed GATA4 regulation of endocardial gene expression. METHODS: We mapped GATA4 chromatin occupancy in cardiomyocyte and endocardial cells of embryonic day 12.5 (E12.5) mouse heart using lineage specific, Cre-activated biotinylation of GATA4. Regulation of GATA4 pioneering activity was studied in cell lines stably overexpressing GATA4. GATA4 regulation of endocardial gene expression was analyzed using single cell RNA sequencing and luciferase reporter assays. RESULTS: Cardiomyocyte-selective and endothelial-selective GATA4 occupied genomic regions had features of lineage specific enhancers. Footprints within cardiomyocyte- and endothelial-selective GATA4 regions were enriched for NKX2-5 (NK2 homeobox 5) and ETS1 (ETS Proto-Oncogene 1) motifs, respectively, and both of these TFs interacted with GATA4 in co-immunoprecipitation assays. In stable NIH3T3 cell lines expressing GATA4 with or without NKX2-5 or ETS1, the partner TFs re-directed GATA4 pioneer binding and augmented its ability to open previously inaccessible regions, with ETS1 displaying greater potency as a pioneer partner than NKX2-5. Single-cell RNA sequencing of embryonic hearts with endothelial cell-specific Gata4 inactivation identified Gata4-regulated endocardial genes, which were adjacent to GATA4-bound, endothelial regions enriched for both GATA4 and ETS1 motifs. In reporter assays, GATA4 and ETS1 cooperatively stimulated endothelial cell enhancer activity. CONCLUSIONS: Lineage selective non-pioneer TFs NKX2-5 and ETS1 guide the activity of pioneer TF GATA4 to bind and open chromatin and create active enhancers and mechanistically link ETS1 interaction to GATA4 regulation of endocardial development.

Molecular Changes in Prepubertal Left Ventricular Development Under Experimental Volume Overload.

Background: Left ventricular (LV) volume overload (VO), commonly found in patients with chronic aortic regurgitation (AR), leads to a series of left ventricular (LV) pathological responses and eventually irreversible LV dysfunction. Recently, questions about the applicability of the guideline for the optimal timing of valvular surgery to correct chronic AR have been raised in regard to both adult and pediatric patients. Understanding how VO regulates postnatal LV development may shed light on the best timing of surgical or drug intervention. Methods and Results: Prepubertal LV VO was induced by aortocaval fistula (ACF) on postnatal day 7 (P7) in mice. LV free walls were analyzed on P14 and P21. RNA-sequencing analysis demonstrated that normal (P21_Sham vs.P14_Sham) and VO-influenced (P21_VO vs. P14_VO) LV development shared common terms of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) in the downregulation of cell cycle activities and the upregulation of metabolic and sarcomere maturation. The enriched GO terms associated with cardiac condition were only observed in normal LV development, while the enriched GO terms associated with immune responses were only observed in VO-influenced LV development. These results were further validated by the examination of the markers of cell cycle, maturation, and immune responses. When normal and VO-influenced LVs of P21 were compared, they were different in terms of immune responses, angiogenesis, percentage of Ki67-positive cardiomyocytes, mitochondria number, T-tubule regularity, and sarcomere regularity and length. Conclusions: A prepubertal LV VO mouse model was first established. VO has an important influence on LV maturation and development, especially in cardiac conduction, suggesting the requirement of an early correction of AR in pediatric patients. The underlying mechanism may be associated with the activation of immune responses.

Metabolic maturation during postnatal right ventricular development switches to heart-contraction regulation due to volume overload.

BACKGROUND: Metabolic maturation is one of the primary processes of postnatal cardiomyocyte development. How volume overload (VO), a pathological state of the right ventricle (RV) in children with congenital heart disease (CHD) and patients with heart failure, affects cardiomyocyte metabolic maturation is unclear. METHODS AND RESULTS: A fistula between the abdominal aorta and inferior vena cava on postnatal day 7 (P7) was created in a mouse model to induce a young-aged RV VO. RNA sequencing revealed that the most enriched gene ontology (GO) terms of the upregulated transcriptome had been changed from metabolic maturation to heart contraction by VO. Transmission electron microscopy imaging showed that metabolic maturation marker-mitochondria were converted into the maturation style in the sham group while remaining unchanged in VO group. Calcium imaging showed that the calcium handling ability had slightly increased in the sham group but dramatically increased in the VO group, even with irregular contraction. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the top three enriched KEGG pathways for the upregulated transcriptome during normal RV development were the citrate cycle, cardiac muscle contraction, and protein processing in the endoplasmic reticulum. VO changed those to arrhythmogenic RV cardiomyopathy, dilated cardiomyopathy, and hypertrophic cardiomyopathy. CONCLUSIONS: Metabolic maturation of postnatal RV development was partly interrupted by VO, and the underlining mechanism was associated with the activation of cardiomyopathy pathways.

Volume Overload Initiates an Immune Response in the Right Ventricle at the Neonatal Stage.

Background: Pulmonary regurgitation caused by the correction or palliation of pediatric tetralogy of Fallot (TOF) leads to chronic right ventricular (RV) volume overload (VO), which induces adolescent RV dysfunction. A better understanding of the molecular mechanism by which VO initiates neonatal RV remodeling may bring new insights into the post-surgical management of pediatric TOF. Methods and Results: We created a fistula between the abdominal aorta and inferior vena cava on postnatal day 1 (P1) using a rat model to induce neonatal VO. Echocardiography revealed that the velocity and velocity- time-integral of the pulmonary artery (PA) were significantly elevated, and hematoxylin and eosin (H&E) staining showed that the diameter of the RV significantly increased. RNA-seq analysis of the RV on P7 indicated that the top 10 enriched Gene Ontology (GO) terms and the top 20 enriched terms in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were associated with immune responses. Flow-cytometric analysis demonstrated that the number of CD4+and CD8+ immune cells were significantly augmented in the VO group compared with the sham group. Conclusions: A neonatal cardiac VO rat model on P1 was successfully created, providing a platform for studying the molecular biology of neonatal RV under the influence of VO. VO - induces an immune response at the neonatal stage (from P1 to P7), suggesting that immune responses may be an initiating factor for neonatal RV remodeling under the influence of VO and that immunosuppressants may be used to prevent pediatric RV remodeling caused by VO.

Downregulated developmental processes in the postnatal right ventricle under the influence of a volume overload.

The molecular atlas of postnatal mouse ventricular development has been made available and cardiac regeneration is documented to be a downregulated process. The right ventricle (RV) differs from the left ventricle. How volume overload (VO), a common pathologic state in children with congenital heart disease, affects the downregulated processes of the RV is currently unclear. We created a fistula between the abdominal aorta and inferior vena cava on postnatal day 7 (P7) using a mouse model to induce a prepubertal RV VO. RNAseq analysis of RV (from postnatal day 14 to 21) demonstrated that angiogenesis was the most enriched gene ontology (GO) term in both the sham and VO groups. Regulation of the mitotic cell cycle was the second-most enriched GO term in the VO group but it was not in the list of enriched GO terms in the sham group. In addition, the number of Ki67-positive cardiomyocytes increased approximately 20-fold in the VO group compared to the sham group. The intensity of the vascular endothelial cells also changed dramatically over time in both groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the downregulated transcriptome revealed that the peroxisome proliferators-activated receptor (PPAR) signaling pathway was replaced by the cell cycle in the top-20 enriched KEGG terms because of the VO. Angiogenesis was one of the primary downregulated processes in postnatal RV development, and the cell cycle was reactivated under the influence of VO. The mechanism underlying the effects we observed may be associated with the replacement of the PPAR-signaling pathway with the cell-cycle pathway.

Postnatal Right Ventricular Developmental Track Changed by Volume Overload.

Background Current right ventricular (RV) volume overload (VO) is established in adult mice. There are no neonatal mouse VO models and how VO affects postnatal RV development is largely unknown. Methods and Results Neonatal VO was induced by the fistula between abdominal aorta and inferior vena cava on postnatal day 7 and confirmed by abdominal ultrasound, echocardiography, and hematoxylin and eosin staining. The RNA-sequencing results showed that the top 5 most enriched gene ontology terms in normal RV development were energy derivation by oxidation of organic compounds, generation of precursor metabolites and energy, cellular respiration, striated muscle tissue development, and muscle organ development. Under the influence of VO, the top 5 most enriched gene ontology terms were angiogenesis, regulation of cytoskeleton organization, regulation of vasculature development, regulation of mitotic cell cycle, and regulation of the actin filament-based process. The top 3 enriched signaling pathways for the normal RV development were PPAR signaling pathway, citrate cycle (Tricarboxylic acid cycle), and fatty acid degradation. VO changed the signaling pathways to focal adhesion, the PI3K-Akt signaling pathway, and pathways in cancer. The RNA sequencing results were confirmed by the examination of the markers of metabolic and cardiac muscle maturation and the markers of cell cycle and angiogenesis. Conclusions A neonatal mouse VO model was successfully established, and the main processes of postnatal RV development were metabolic and cardiac muscle maturation, and VO changed that to angiogenesis and cell cycle regulation.