Nataliya Kibiryeva

Cardiopulmonary bypass (CPB) is required during most cardiac surgeries. CBP drives systemic inflammation and multiorgan dysfunction that is especially severe in neonatal patients. Limited understanding of molecular mechanisms underlying CPB-associated inflammation presents a significant barrier to improve clinical outcomes. To better understand these clinical issues, we performed mRNA sequencing on total circulating leukocytes from neonatal patients undergoing CPB. Our data identify myeloid cells, particularly monocytes, as the major cell type driving transcriptional responses to CPB. Furthermore, IL-8 and TNF-α were inflammatory cytokines robustly upregulated in leukocytes from both patients and piglets exposed to CPB. To delineate the molecular mechanism, we exposed THP-1 human monocytic cells to CPB-like conditions, including artificial surfaces, high shear stress, and cooling/rewarming. Shear stress was found to drive cytokine upregulation via calcium-dependent signaling pathways. We also observed that a subpopulation of THP-1 cells died via TNF-α–mediated necroptosis, which we hypothesize contributes to post-CPB inflammation. Our study identifies a shear stress–modulated molecular mechanism that drives systemic inflammation in pediatric CPB patients. These are also the first data to our knowledge to demonstrate that shear stress causes necroptosis. Finally, we observe that calcium and TNF-α signaling are potentially novel targets to ameliorate post-CPB inflammation.

The heart is the first major organ to develop during embryogenesis and must receive proper spatiotemporal signaling for proper development. Failure of proper signaling between the first and second heart fields at twenty days gestation contributes to the generation of a congenital heart defect. The most common cyanotic congenital heart defect is tetralogy of Fallot (TOF) which requires surgical intervention in the first year of life. In right ventricular tissue of infants born with TOF, the levels of scaRNA1 are reduced and mRNA splicing is dysregulated. In this study, we investigate a method of quantifying pseudouridylation levels in relation to scaRNA1 levels in spliceosomal RNA U2 in three different groups of samples: right ventricular (RV) tissue of infants born with TOF versus RV tissue from normally developing infants, scaRNA1 knockdown in primary normal cardiomyocytes derived from normally developing infants, and scaRNA1 overexpression in primary cells derived from RV tissue from infants born with TOF. We hypothesize that the amount of pseudouridylation is dependent on scaRNA1 level, compromising spliceosomal function and therefore, contributing to the generation of a congenital heart defect. Our results revealed a statistically significant decrease of pseudouridylation levels in the right ventricular tissue of infants born with TOF compared to the controls. Knocking down the scaRNA1 levels in normal primary cardiomyocytes resulted in a statistically significant decrease of pseudouridylation. Finally, an overexpression of scaRNA1 in TOF primary cells resulted in an increase in pseudouridylation levels, but it did not achieve statistical significance. Our previous research provided an association between scaRNA levels, alternative splicing, and development. Here, we demonstrate that pseudouridylation levels in spliceosomal RNA U2 is dependent on the expression level of scaRNA1. Although further investigation is needed, we believe that scaRNA expression regulates biochemical modifications to spliceosomal RNAs, adjusting the fidelity of the spliceosome, allowing for controlled alternative splicing of mRNA that is important in embryonic development. If validated, this is an underappreciated mechanism that is critical for regulating proper embryonic development.

Congenital heart disease (CHD) is a leading cause of death in children <1 year of age. Despite intense effort in the last 10 years, most CHDs (~70%) still have an unknown etiology. Conotruncal based defects, such as Tetralogy of Fallot (TOF), a common complex of devastating heart defects, typically requires surgical intervention in the first year of life. We reported that the noncoding transcriptome in myocardial tissue from children with TOF is characterized by significant variation in levels of expression of noncoding RNAs, and more specifically, a significant reduction in 12 small cajal body-associated RNAs (scaRNAs) in the right ventricle. scaRNAs are essential for the biochemical modification and maturation of small nuclear RNAs (spliceosomal RNAs), which in turn are critical components of the spliceosome. This is particularly important because we also documented that splicing of mRNAs that are critical for heart development was dysregulated in the heart tissue of infants with TOF. Furthermore, we went on to show, using the zebrafish model, that altering the expression of these same scaRNAs led to faulty mRNA processing and heart defects in the developing embryo. This review will examine how scaRNAs may influence spliceosome fidelity in exon retention during heart development and thus contribute to regulation of heart development.

The importance of microRNAs for maintaining stability in the developing vertebrate heart has recently become apparent. In addition, there is a growing appreciation for the significance of microRNAs in developmental pathology, including the formation of congenital heart defects. We examined the expression of microRNAs in right ventricular (RV) myocardium from infants with idiopathic tetralogy of Fallot (TOF, without a 22q11.2 deletion), and found 61 microRNAs to be significantly changed in expression in myocardium from children with TOF compared to normally developing comparison subjects (O’Brien et al. 2012). Predicted targets of microRNAs with altered expression were enriched for gene networks that regulate cardiac development. We previously derived a list of 229 genes known to be critical to heart development, and found 44 had significantly changed expression in TOF myocardium relative to normally developing myocardium. These 44 genes had significant negative correlations with 33 microRNAs, each of which also had significantly changed expression. Here, we focus on miR-421, as it is significantly upregulated in RV tissue from infants with TOF; is predicted to interact with multiple members of cardiovascular regulatory pathways; and has been shown to regulate cell proliferation. We knocked down, and over expressed miR-421 in primary cells derived from the RV of infants with TOF, and infants with normally developing hearts, respectively. We found a significant inverse correlation between the expression of miR-421 and SOX4, a key regulator of the Notch pathway, which has been shown to be important for the cardiac outflow track. These findings suggest that the dysregulation of miR-421 warrants further investigation as a potential contributor to tetralogy of Fallot.

Congenital heart defects (CHD) are the most common cause of death in children under the age of 1. Tetralogy of Fallot (TOF) is a severe CHD that results from developmental defects in the conotruncal outflow tract. Recently, a tissue-specific gene expression template (GET) was derived from microarray data that accurately characterized multiple normal human tissues. We used the GET to examine spatial, temporal, and a pathological condition (TOF) within a single organ, the heart. The GET, as previously defined, generally identified temporal and spatial differences in the cardiac tissue. Differences in the stoichiometry of the GET reflected the severe developmental disturbance associated with TOF. Our analysis suggests that the homoeostatic equilibrium assessed by the GET at the inter-organ level is generally maintained at the intra-organ level as well.

Tetralogy of Fallot (TOF) is the most commonly observed conotruncal congenital heart defect. Treatment of these patients has evolved dramatically in the last few decades, yet a genetic explanation is lacking for the failure of cardiac development for the majority of children with TOF. Our goal was to perform genome wide analyses and characterize expression patterns in cardiovascular tissue (right ventricle, pulmonary valve and pulmonary artery) obtained at the time of reconstructive surgery from 19 children with tetralogy of Fallot.