Tetralogy of Fallot: Hypoxia, the villain of the story?

Tetralogy of Fallot (ToF) is a cyanotic congenital heart disease, composed of four malformations: persistent communication between the right and the left ventricle, pulmonary stenosis, overriding aorta, and right ventricle hypertrophy. The etiology of this disease is not entirely known as yet, but it has been proposed that the pathology has genetic components. During embryonic development, the fetus is exposed to a physiological hypoxia to facilitate the formation of blood vessels and blood cells through de novo processes.


| INTRODUCTION
Tetralogy of Fallot (ToF) is the most common cyanotic congenital heart disease (CHD), which affects the normal blood flow through the heart due to malformations during embryonic development (Apitz et al., 2009).The pathology is composed of four principal malformations: 1. Ventricular septal defect (VSD) that results in the communication of the right ventricle with the left ventricle.2. Pulmonary stenosis or the narrowing of the pulmonary valve and the pulmonary artery.3. Overriding of the aorta, which means that the aorta artery opens for both ventricles and not just for the left ventricle.4. Hypertrophy of the right ventricle (Wise-Faberowski et al., 2019).
The causes of ToF are still not entirely known.However, it has been observed and studied that genetic components in chromosomes play a role in the pathology.These genetic factors interfere with the signaling pathways responsible for the proliferation of cardiac cells, particularly those linked with the interventricular septum, which are the most frequent signaling pathways.These pathways include GATA4, FTL4, and Slit-ROBO.When these pathways become dysregulated, it leads to malformations of the outflow tract of the right ventricle (RV), alterations in angiogenesis, and migration and alignment of the cardiac cells (Manshaei et al., 2020).
The normal fetal oxygen levels range between 20 and 30 mmHg (considered as hypoxic levels in adult tissues).Thus, we can define physiological hypoxia as having oxygen levels between 7 and 20 mmHg and we can establish that levels lower than 7 mmHg of oxygen represent pathological levels of hypoxia (Patterson & Zhang, 2010).
Throughout embryonic development, the fetus is exposed to a hypoxic environment, as the essential elements necessary for oxygen and blood transport, including the placenta, blood vessels, and blood itself, are forming de novo.However, when cells are exposed to lower fetal oxygen levels (Ream et al., 2008), it is essential to highlight that chronic pathological hypoxia can threaten homeostasis, physiological conditions, and critical pathways.At this point, Hypoxia-Inducible Factor (HIF) is activated and initiates transcriptional responses that enable adaptation to the hypoxic environment.These responses involve angiogenic activation and vasoactive reactions, which promote cell survival and elevation of tissue oxygen levels (Taylor & Scholz, 2022).
Consequently, an effective hypoxia signaling system is essential in preventing the development of a pathological hypoxic environment during embryonic development, which could ultimately lead to Congenital Heart Disease (CHD).Research has shown that pathological oxygen deprivation during embryonic development can lead to CHD such as Ventricular Septal Defects (VSD), Atrial Septal Defects (ASD), and thinning of the ventricular myocardium, among other conditions (Kenchegowda et al., 2014).Cbp/P300 interacting transactivator with Glu/Asp rich carboxy-terminal Domain 2 (CITED2) is a transcription cofactor located on chromosome 6 (6q23.3), it interacts with multiple other transcription factors and cofactors, playing a critical role in multiple cellular processes, including cell differentiation, proliferation, migration, apoptosis, and autophagy.This cofactor is also a coregulator of the HIF pathway in response to hypoxia since it modulates gene expression making it crucial during the embryonic development of the liver, lungs, heart, neural tube, and eye (An et al., 2020), CITED2 blocks the HIF-1α binding to p300/CBP and represses HIF-1 activity regulating HIF activity in a negative feedback way (Yoon et al., 2011).
Under normoxic conditions, HIF-1α undergoes degradation; however, in a hypoxic environment, HIF-1α stabilizes and translocates to the cell nucleus, in conjunction with CITED2, it orchestrates the expression of genes involved in responding to low oxygen levels, this critical collaboration contributes significantly to the activation of cellular survival mechanisms, thereby ensuring proper development of vital structures such as the heart (Chen et al., 2022).Consequently, disruptions in CITED2 function may create a deleterious environment for cells, potentially leading to malformations like those observed in Tetralogy of Fallot (Li et al., 2012).
The importance of the CITED2 response to hypoxia and its interaction with HIF-1α lies in their pivotal roles in cellular adaptation to low-oxygen environments, CITED2 collaborates with HIF-1α to orchestrate a cautiously tuned transcriptional response (Li et al., 2012).CITED2 acts as a co-regulator, influencing the transcriptional activity of HIF-1α, this interaction enables the activation of genes crucial for adaptive processes, including angiogenesis, erythropoiesis, and glycolytic metabolism (Chen et al., 2022).The synchronized action of CITED2 and HIF-1α ensures that cells can adjust their gene expression to meet the challenges caused by insufficient oxygen, promoting cell survival and tissue homeostasis, dysregulation of this response has been implicated in various pathological conditions, emphasizing the clinical significance of understanding the intricate interplay between CITED2 and HIF-1α in the context of hypoxia (Xu et al., 2007;Yadav et al., 2021).
Therefore, the absence of this gene during embryological development is fatal for the fetus, and disruption or deficiency in CITED2 leads to defects in atrial and ventricular septa as well as outflow tracts and malformations of the neural tube, this absence might stem from a loss of this gene's allele, leading to an overexpression of Vascular Endothelial Growth Factor Receptor type 2 (VEGFR-2), which in turn triggers birth defects such as ToF (Yin et al., 2002).According to studies, CITED2À/À embryos present malfunction in the outflow tract which leads to lethality (Yin et al., 2002).In another study, it is shown that CITED2À/À embryos presented ventricular septum defects (Barbera, 2002) as well as aortic malformations (Bamforth et al., 2001), all these studies contribute to the proposition that this regulator leads to the malformations presented in ToF.CITED2 directly binds to the initial Cysteine-histidine-rich (CH1) region of p300 and CBP with high affinity, competing with HIF-1α.Thus, it interferes with hypoxia-driven transcription, as a result of this competition, CITED2 has the capability to enhance or diminish the function of HIF-1α (Yin et al., 2016).

| miRNAs AND HIF-1
MicroRNAs (miRNAs) are RNA molecules that do not code for any protein during gene transcription.They are known as negative regulators of gene expression.This is due to their ability to prevent the synthesis of active proteins by binding to their corresponding mRNA region.Several factors can influence the expression of these molecules, including hypoxia.When oxygen levels drop, the HIF protein is synthesized.It then directly binds with specific miRNAs causing alterations in processes like angiogenesis, cell proliferation, and ischemia-reperfusion (Neudecker et al., 2016).This suggests that miRNA-940, identified in patients with DiGeorge's syndrome and ToF (Liang et al., 2014), might play an important role in the main pathway during the development of ToF when oxygen levels reach the pathological stage.

| HYPOXIA-INDUCIBLE FACTOR (HIF-1) PATHWAY
HIF-1α can be expressed at high levels in most cells, and its transcription is regulated by the HIF1 gene.This transcription is regulated through mechanisms that are both dependent on and independent of oxygen, toward the alpha subunit of the HIF (as shown in Figure 1).This hydroxylation produces ubiquitination by the von Hippel-Lindau protein-recruited E3 complex and proteasomal degradation.This involves the prior activation of second messengers that participate in this process (Taylor & Scholz, 2022).
Regulating mechanisms of HIF.HIF is regulated through both oxygen-dependent and oxygen-independent ways targeting the HIFα subunit.Oxygen-independent mechanisms are activated by the presence of ROS, cytokines, and lipopolysaccharides (LPS), which through second messenger cascades activate the transcription of the HIF1A gene, this transcription can occur at specific levels by the action of microRNA (miRNA) and long non-coding RNAs (lncRNA) mediated by angiotensin II.Oxygen-dependent mechanisms activate under normoxia conditions, where hydroxylation of two distinct HIFα proline residues leads to HIFα poly-ubiquitination and its subsequent degradation, and prevents the recruitment of p300 and CBP, reducing HIF transactivation.An additional regulation uses molecular oxygen to regulate histone methylation and thereby gene expression under hypoxic situations, all of this to promote necessary and correct adjustments to the needs of each cell.
The structure of this factor consists of two alpha subunits and two beta subunits.The expression of the alpha subunits is regulated by oxygen levels and is activated in a hypoxic environment.The beta subunits are constitutively expressed and are known as Aryl Hydrocarbon Nuclear Translocator (ARNT) as they bind to the Aryl Hydrocarbon Receptor (AhR) and thus facilitate its translocation to the nucleus.This interaction helps to regulate the expression of specific genes that enhance cell survival in hypoxic conditions.However, the pivotal aspect of the HIF-1 pathway activation lies in the role of the alpha subunit, as it is responsible for initiating hydroxylation, acetylation, ubiquitination, and phosphorylation reactions.
Activating the phosphatidylinositol-4, 5-bisphosphate-3-kinase (PI3K) regulates protein synthesis, which eventually activates downstream pathways and enhances HIF-1a protein translation.Additionally, certain growth factors that stimulate kinase cascades increase mRNA translation into HIF-1α protein, which is also a key regulator in angiogenesis by inducing the migration of endothelial cells in a hypoxic environment and facilitating the transcription of VEGF to supply oxygen with new vessels to a specific region (Luo et al., 2022;Bruick, 2003).This suggests that many factors that cause cellular stress can trigger the activation and synthesis of HIF-1a and its pathways (Masoud & Li, 2015).Knutson et al., 2021 stated that throughout embryonic development, HIF-1 is detected until the septation of the ventricles occurs.After this event, HIF-1 is no longer detectable, indicating that it is a key regulator during ventricular septation in fetal development.HIF-1 has also been associated with, and identified in, the processes of cell survival and migration, particularly during the development of the cardiac chambers (Knutson et al., 2021).
Although the activation of HIF-1 is crucial for the appropriate development of the heart, it can also be triggered by the external environment to which the mother is exposed, particularly during the developmental stages.These observations show that the impact on cardiac tissue as a result of external factors like the mother's exposure to a hypoxic environment, extends beyond the heart and is also seen in other organs, such as the bone (Bishop & Ratcliffe, 2015).
Furthermore, a study involving groups with pregnancies at higher altitudes has revealed that such groups exhibit a higher prevalence of CHD when compared to groups residing at sea level (Kalisch-Smith et al., 2020).This occurs as a result of lower oxygen pressure at higher altitudes.For example, at sea level (0 feet) oxygen pressure is approximately 21% or 160 mmHg.However, in regions above 8000 feet (Mexico City, for instance, lies at an altitude above 7349 feet), oxygen pressure decreases down to 15% or ±114 mmHg (Sharma & Hashmi, 2023).
The second isoform of the Hypoxia Inducible Factor, HIF-2α, has proved to be a key regulator of cardioprotection by inducing Amphiregulin (AREG) in the cardiac myocytes, which is a ligand of the epidermal growth factor receptor.This has been tested on murine models where the deletion of AREG or its receptor indicates a higher susceptibility to myocardial ischemia.The study showed that under ischemic or hypoxic conditions this signaling pathway is activated and provides cardioprotection.This is because HIF-1α provides protection only under ischemic pre-conditioning situations (Koeppen et al., 2018).In addition to this finding, Dimethyloxalylglycine, an inhibitor of HIF, has proved its ability to stabilize both isoforms, thus providing cardioprotection by triggering this action.

| Role of HIF during adenosine signaling
When myocardial tissue is exposed to hypoxic conditions, two isoforms of HIF become activated, this leads to an increase in extracellular adenosine levels, a nucleoside present in many cells throughout the body.Adora2a is a receptor for this nucleoside and contains hypoxiaresponse elements that are sensed by HIF1A both during hypoxia and inflammation, as mentioned earlier, AREG is associated with hypoxic processes and is known to promote the survival of adenosine kinases which can decrease during hypoxia, enabling cells to better adapt to inflammatory conditions, and as a result, AREG plays a crucial role in cardioprotection.
Applying these molecular mechanisms to ToF pathogenesis, we find potential alterations in cardiac development under fetal hypoxic conditions and the ensuing responses managed by HIF.Pertinently, a deficiency in Adora2a expression may cause complications in cardiac development, as documented by Ruan et al. in their 2022 study, this crucial finding underscores the critical role of Adora2a and its downstream mechanisms in determining the landscape of cardiac development and, by extension, the pathophysiology of ToF (Ruan et al., 2022).

| CITED2 AND ToF
In animal experiments, mice with homozygous CITED2À/À experienced either embryonic death or a range of cardiac malformations.These findings indicate that the loss of function of CITED2 can lead to cardiac septal malformations or defects.In mouse models, CITED2 knockout embryos experienced in-utero death due to cardiac abnormalities that resembled those observed in children with congenital heart diseases.
Ventricular and atrial septal defects, transposition of the great arteries, and ToF were the predominant heart abnormalities associated with CITED2 variants in humans (Yadav et al., 2021).Depletion of CITED2 results in the loss of differentiation in pluripotent, cardiac, and neural lineages (Wu et al., 2019).In addition, CITED2 regulates the expression of genes critical for many aspects of cardiovascular, kidney, and endoderm development, mesoderm formation, and angiogenesis.
Therefore, defects in or absence of CITED2 in embryos alter the normal development of the heart ring as shown in ToF (Zheng et al., 2021).CITED2 variants were detected in children with different CHD, indicating that CITED2 is the pathogenic gene for human congenital heart malformations.A study conducted in 2022 involving 625 subjects, with 312 having ToF and 293 being healthy individuals, revealed the presence of 9 CITED2 variants in 605 subjects.Interestingly, 6 of these variants were exclusively observed in patients with ToF.These variants significantly affected the transcriptional activity of CITED2 by altering the transcription factor binding site.Normal heart development requires adequate doses of transcription factors such as NKX2-5, TBX5, GATA4, and TBX1.The loss of these factors disrupts cardiac morphogenesis.The loss or malfunction of the CITED2 promoter region leads to the disruption of the transcription factor binding site (Chen et al., 2022).

| CITED2 AND HIF-1α
CITED2 is a negative regulator of HIF-1 because of its competitive binding to the CH1 domain of CBP/p300.Furthermore, HIF-1α has two Transactivation Domains (TADs), namely, N-Terminal TAD (NAD) and C-Terminal TAD (CAD).A study conducted by Yadav et al. (2021), demonstrated that CITED2 can repress either NAD-or CAD-dependent genes during hypoxia.This was indicated by the overexpression of NAD and CAD activities in response to the CITED2 knockdown and their suppression upon CITED2 overexpression.CITED2 represses NAD function by competing for p300 binding.This, alongside CAD, is vital for activation of HIF-1 (Yadav et al., 2021).This competitive binding leads to the inhibition of HIF-1-mediated signaling and a decrease in responsive genes like VEGF.Therefore, in case of a deficiency or disruption in CITED2, HIF will not undergo down-regulation but will instead experience an increase and overexpression during embryonic development.This can lead to an increased expression of VEGF, which, in turn, can cause malfunctions in outflow tract development.
The elevation of mRNA levels of HIF-1 responsive genes in CITED2À/À hearts manifests a conspicuous parallel with the pronounced anomalies observed in VEGF transgenic embryos (Li et al., 2012).These cardiac aberrations observed in CITED 2À/À embryos stem from the disruption of processes critical to crest development and transactivation, additionally, it is imperative to delve into the ramifications of VEGF overexpression (Xu et al., 2007), which will be discussed later in this article.The management of normal embryonic development within the cardiovascular system hinges upon meticulous regulation of VEGF, both in terms of its quantity and the precise timing of its expression, even the slightest deviation from the norm, resulting in an upsurge in VEGF levels, carries dire consequences including risk or lethality, anomalous outflow tracts in the heart, and enlargement of the jugular lymph sac (Yin et al., 2016).

| THE ROLE OF VEGF IN ToF DEVELOPMENT
Angiogenesis is the process by which new blood vessels are created.This process is extremely important during embryonic development to create all the structures of the fetus, as well as to perform many other functions.However, when there are defects in the gene expression or the protein itself, it can cause a range of pathologies in adults.Most importantly, during fetal development, this can manifest as congenital defects such as ToF (Failla, 2018).This angiogenic process is activated by signals that ensure the activation of proteins and growth factors, such as VEGF-A, AGP-2, and FGF.These elements trigger the mechanisms of angiogenesis (Truby & Topkara, 2016).
During embryogenesis, VEGF promotes migration, maturation, and proliferation of mature Endothelial Cells (ECs).Additionally, it supports their branching morphogenesis, including angiogenesis and arterio-venous differentiation.VEGF also helps to improve vascular permeability.Studies indicate that VEGF interacts with another factor, known as the NOTCH pathway, as part of its downstream pathway.This pathway mediates juxtacrine cellular signaling in neuronal, immune, endocrine, and cardiac cells through the activation of VEGFR2.Upon activation, NOTCH translocates to the nucleus where it binds to the DNA and functions as a transcriptional coactivator.This follows the activation of many second messengers (van den Akker et al., 2012).
It has been studied that hypoxia provokes the activation of molecular procedures which in turn activate growth factors, such as VEGF.In response, the formation of new blood vessels ensues.However, if there are any modifications in the growth factor's structure or receptor, angiogenesis might regress (Peters et al., 2013).
A study involving biopsies of myocardial tissue were obtained from patients who underwent primary correction of ToF, those who had pulmonary regurgitation surgery, and valve donors.From these myocardial cells, researchers were able to isolate and extract RNA.Subsequently, these samples underwent analysis through techniques like Northern blot, DNA microarray hybridization, and RT-PCR, as well as Gomori's reticulin staining, immunohistochemistry, and statistical analysis (Peters et al., 2013).
The study found that the expression of angiogenic factors and their receptors, including VEGF, tyrosine kinase receptors flt-1 and flk-1, AGP-1 and 2, and many other factors and proteins, exhibited decreased expression in patients with ToF.This data suggests that VEGF plays an important role in the maturation and development of heart cells during embryogenesis, when the expression of this growth factor or its receptor is at low levels, it can lead to a CHD such as ToF (Peters et al., 2013).

| DISCUSSION
As previously studied, pathological hypoxia occurring during the critical stages of heart development can disrupt cell survival and impede the processes of migration, maturation, and proliferation of the cardiomyocytes and the myocardium.Further investigation is necessary to determine the exact developmental stage at which high hypoxia levels might lead to abnormal interventricular septum development in ToF.As previously stated, CITED2 can be involved in the development of the heart and extravascular structures and its absence can lead to malformations.Studies using murine models have proven that deficiency or disruption of CITED2 can indeed disrupt the process of heart development.
Due to its inability to down-regulate HIF, and coupled with the pre-existing hypoxia, there is a proliferation and over-expression of HIF in response to these conditions.As a result, there is an increased expression of VEGF.This can cause malfunctions in outflow tract development, resulting in a CHD such as ToF.This review takes into consideration the critical role that hypoxia plays in cell migration and survival of cardiac structures.The hyper-or hypofunction of the factors regulating this process during embryogenesis tends to lead to cardiac malformations, including those seen in ToF.It is imperative to consider this while investigating the multifactorial causes of this disease.
The role of HIF-1 expression during fetal development and the consequences of its underexpression warrants further investigation.This could help us understand whether a correlation exists that might lead to the development of ToF, as we have sought to demonstrate in this manuscript.Moreover, it could shed light on the epigenetic aspects of this congenital heart disease.