Tetralogy of Fallot (TOF) is the most prevalent diagnosis in most follow up cohorts of complex congenital heart disease (CHD) (1, 2). Present day practice of direct early surgical correction significantly reduced mortality and the average life expectancy has risen to well above 60 years of age (3, 4). Unfortunately, the growing cohort of survivors with TOF exhibits a number of late sequelae, e.g., development of heart failure, necessity of re-interventions and arrhythmogenic vulnerability (5).

Heart failure is the most common cause of death in survivors with CHD and its prevention in TOF is one the three major focus areas in research (3, 6). Heart failure in TOF is mostly right ventricular (RV) failure and in many cases associated with chronic pulmonary regurgitation (PR), a common complication following the surgical intervention aimed to relieve pulmonary stenosis (7). The development of the RV failure has been studied extensively in the last decades, albeit mostly in models of acquired RV pressure overload and pulmonary hypertension, a disease phenotype with several major dissimilarities from TOF and chronic PR.

Currently, medical treatment options for RV failure are lacking (8, 9). The most common treatment for patients with PR is pulmonary valve replacement. Timing of pulmonary valve replacement is highly debated, as repeat interventions due to child growth and valve graft degeneration are major limiting factors in its application. Furthermore, re-intervention for pulmonary valve replacement also fails to reduce incidence of RV failure or death, despite improvement of RV volumes and symptoms (10, 11).

Identification of novel targets for treatment of RV failure is necessary, which requires a deeper understanding of the shift from adaptive remodeling to failure. To be able to develop strategies to predict, detect, treat, or prevent heart failure and other late sequelae in TOF, a thorough understanding of the myriad of drivers of disease progression is needed. The drivers of other late sequelae, e.g., malignant arrhythmia, might overlap with those of RV dysfunction but will not be specifically discussed.

This narrative review will focus on what is known of the mechanisms of RV dysfunction in TOF, chronic PR and present knowledge-gaps. We aim to integrate information from functional, cellular and molecular studies with clinical data and explore potential targets for therapy.

Patients with TOF experience a unique sequence of abnormal loading conditions, which affects myocardial adaptation and ultimately cardiac function. Unlike in the normal heart, there is no unloading of the RV at birth when separation of the systemic and pulmonary circulation occurs. Due to right ventricular outflow tract (RVOT) obstruction, the postnatal RV remains subjected to pressure overload, as in the fetal circulation, and the myocardium retains some of its fetal characteristics after birth (12). In addition, most infants with unrepaired TOF develop hypoxemia due to ventricular right-left shunting. This combination of pressure overload and hypoxemia persists and requires corrective surgery to prevent imminent RV failure (13). After corrective surgery, repaired TOF (rTOF) patients experience different degrees of PR inducing volume overload (Figure 1). This volume overload is introduced in an already pressure overloaded RV and will affect RV function differently opposed to introducing isolated PR in an otherwise healthy RV (14). RVOT structure and function may be altered due to patch repair and right bundle branch block may eventually predispose to dyssynchrony. In some cases, residual RVOT obstruction remains present after corrective surgery. Unloading of volume and/or pressure overload can occur after pulmonary valve replacement. For isolated pressure overload, other invasive treatment options may be required. Limited valve graft durability, however, often results in recurrence of PR, RVOT obstruction or a combination of both (15). This might require repetitive pulmonary valve replacements, posing a heavy burden on patients without the guarantee of freedom from adverse events (10, 11).

Thus, this pattern of sequential loading and hypoxemia gives rise to a complex physiology. In understanding this response, the separate mechanisms, their interactions and the time-relationship should be taken into account.

The temporal profile of RV adaptation to RV failure has multiple features, of which the causality is difficult to define. RV dilatation and prolonged QRS duration have been first recognized as hallmarks. Features of (mal)adaptation of the RV may be categorized into three groups: factors affecting ventriculo-arterial interactions (systolic function, PR), factors affecting atrio-ventricular interactions (e.g., impaired relaxation, atrial dysfunction), and factors affecting interventricular interactions (e.g., dyssynchrony, septal shift) (Figure 2).

Studies on histopathology of TOF are scarce. Consequently, much of what is known is derived from historical observations, mostly from patients corrected at what is presently regarded as “later stages”, e.g., more than 6 months of age or at death after uncorrected survival into adulthood (82–86). Hence, results may be affected by survival and selection bias, particularly in the group of patients studied during re-do surgery.

Most commonly described histopathological features are myocyte hypertrophy, fibrosis and altered capillary density. The degree of hypertrophy increases with age, likely initially as an adaptive response until a point of no return is reached with onset of maladaptive features (87–89). In a single-center study comparing biopsies obtained at primary repair between 4 months and 168 months of age, the degree of hypertrophy correlated with RV function, assessed as global strain. The recovery of RV strain after corrective surgery was reduced in the group of patients corrected after 1 year of age, suggesting irreversible remodeling if repair surgery is postponed (89). The hypertrophy is not homogenous; a postmortem study identified larger endocardial cardiomyocytes in the infundibulum as compared with other regions of the RV (90). In biopsies taken at a younger age less cardiomyocyte hypertrophy was found, which might be explained by the shorter exposure to overload (87–89). Alternatively, cardiomyocyte proliferation may be increased, enhancing RV adaptation. After birth, cardiomyocyte proliferation capacity is lost in mammals as a result of several postnatal processes, such as changes in oxygen levels and the transition from placental to enteral nutrition (91). However, recent studies have shown that cardiomyocytes retain the potential to proliferate after birth when under stress (12, 92).

Cardiomyocyte proliferation promotes adaptation to pressure overload better than cellular hypertrophy (92–95). Stimulating proliferation in mature cardiomyocytes or prolonging proliferative capacity after birth is therefore an attractive treatment target to preserve function of the overloaded RV. From cultured cardiomyocytes, isolated from biopsies of TOF patients, Liu et al. reported a high proportion of binucleated cardiomyocytes. This indicated that proliferation was initiated under adverse loading, but arrested in the cytokinesis stage. The authors demonstrated that failure of cytokinesis is mediated by beta-adrenergic signaling and that beta-adrenergic blockade can increase cell division by rescuing cardiomyocytes from cytokinesis failure. Additionally, this was confirmed in a neonatal mouse model of myocardial infarction (96). Although a previous randomized controlled trial (RCT) of beta-blockade in adult rTOF patients could not demonstrate a clinical benefit, a new RCT is being conducted to investigate its effects on cardiomyocyte proliferation in infants before repair surgery (ClinicalTrials.gov Identifier: NCT04713657) (9). Cell cycle activity can also be stimulated by administration of neuregulin-1. In both mature and weaning rats with RV pressure overload, neuregulin-1 attenuated maladaptive remodeling, with improved systolic and diastolic RV function (92, 94, 95). It is important to note that postnatal cardiomyocyte proliferation requires high oxidative energy metabolism (97). If proliferation was to be stimulated in an already metabolically demanding environment, this could exacerbate the energy deficit.

Myocardial fibrosis in rTOF has been associated with the risk of RV dysfunction, ventricular arrhythmia and sudden death (98–100). Recently, CMR studies in TOF have shown that increased late gadolinium enhancement, suggesting increased fibrosis, is a risk factor for late mortality (22). In this study, a composite fibrosis score was used, irrespective of location or type of fibrosis. Interstitial fibrosis, reactive to adverse loading, should be distinguished from replacement fibrosis following scarring, patch material and myocardial ischemia. Interstitial fibrosis has been shown in RV biopsies taken at the time of correction and appears to increase with the age of patients (83, 85, 87–89). Yet, according to RV samples of TOF patients younger than 9 months of age, collagen content is not significantly different from controls (101). These results suggest that neonatal RV pressure overload does not necessarily cause appreciable fibrosis in the first months of life, i.e., collagen production is still part of a beneficial adaptation process. Excessive collagen production being a feature mainly in patients older than 12 months at biopsy in the study of Xie et al. further supports this (89). Additionally, a greater amount of fibrosis was seen in patients with more functioning hypoxia inducible factor 1α (HIF1α) alleles, a mediator of the response to hypoxemia (102). Intriguingly, not all tissue biopsy studies of rTOF patients identified increased fibrosis (49, 87, 103). Assessing fibrosis in rTOF patients late after repair should be done in the context of sequential loading. Irrespective of the duration and degree of PR or residual RVOT obstruction, every rTOF patient was subjected to a period of neonatal pressure overload and underwent corrective surgery with aortic-cross clamping. Prolonged exposure to pressure overload can result in increased reactive fibrosis (48). Prolonged aortic-cross clamping time carries the risk of myocardial injury which will also result in fibrosis, but of the replacement type (104). Unlike in pressure overload, reactive interstitial fibrosis in response to isolated volume overload is much less pronounced or even absent (14, 34, 46, 47, 51, 105). In an older rTOF population with larger RV volumes, Yamamura et al. found that median collagen content of patients undergoing pulmonary valve replacement was 7.1%, which is comparable to the reference value of 7.4% found in healthy RV myocardium (103, 106). A subpopulation in the uppermost quartile of fibrosis in Yamamura's study had a median collagen content of 17.9%. In this small sample, the increased amount of fibrosis was associated with longer cross-clamp time as well as post-operative retention of larger RV end-systolic volumes, RV mass and RA area (103). In a younger, more contemporary managed rTOF population, fibrosis was not significantly different from control tissue samples (49). From these observations, it may be hypothesized that interstitial fibrosis presented in rTOF patients with PR is predominantly the remnant of pre-repair pressure overload and perhaps ischemic remodeling due to hypoxemia and aortic-cross clamping. The association between fibrosis and outcome may suggest a causal relationship, yet, inhibition of reactive fibrosis to either pressure or volume overload did not improve RV dysfunction (107, 108). In addition, interfering with fibrosis in cardiac tissue may have detrimental effects as fibrosis also has a cardioprotective effect (109). In line with this protective function, patients with more functional HIF1α alleles and more fibrosis benefited with less progression of PR, preservation of RV volumes and systolic function and greater freedom from re-interventions (102). These findings suggest that aforementioned adverse outcomes in rTOF patients could be related more to maladaptive fibrosis induced by triggers other than adverse loading. Reactive fibrosis, resulting from pressure and/or volume overload, appears to be merely a marker and not a targetable substrate. Further studies are necessary to identify the nature and optimal balance of adaptive and maladaptive fibrotic remodeling. At present, prevention of fibrosis by early repair and limiting formation of replacement fibrosis, e.g., by optimizing surgical technique and myocardial protection, seem superior to the use of anti-fibrotic treatments.

In addition to hypertrophy and fibrosis, increased capillary density has been demonstrated, both in tissue obtained during initial repair and in human heart specimens (86–88). The increased capillary density measured was attributed to an increased smaller number of vessels, with upregulation of pro-angiogenic factors. These findings indicate angiogenic sprouting to preserve the aerobic metabolic demand, although these vessels have been suggested to be immature and may not be conducting blood (87, 88). In contrast, in many (but not all) experimental models, RV pressure overload is associated with capillary rarefaction and a decrease in angiogenesis has been found to mark the transition from compensated to decompensated RV hypertrophy (110, 111). Based upon these observations, the increased angiogenic response in TOF is different from what is seen in acquired pulmonary hypertension, where cardiomyocyte hypertrophy exceeds the capacity for new capillary formation. Although the vasculature in TOF may not be completely functional, increased angiogenesis seems to be an adaptive feature. A role of hypoxemia and HIF1α in this process is likely, which is supported by the cardioprotective effects of the amount of functioning HIF1α alleles (102, 112). Several studies found increased mRNA expression of angiogenic factors, e.g., vascular endothelial growth factor (VEGF), in TOF patients at the time of repair when compared to healthy and VSD controls (87, 88, 102). However, cyanosis in TOF patients was associated with reduced VEGF expression when compared to those without cyanosis, complicating the interpretation of these angiogenic signals (55). Furthermore, a transcriptomic study demonstrated higher levels of apoptosis associated genes and decreased expression of genes associated with contractility and calcium handling in cyanotic patients (113). In order to preserve adequate oxygen delivery, myocardial vessel formation could be stimulated by targeting angiogenesis. Therapeutic options for promoting angiogenesis in the RV have recently been reviewed elsewhere (114).

In short, the main histopathological findings are cardiomyocyte hypertrophy and increased fibrosis, the severity increasing with older age at repair. Although cardiomyocyte proliferation is temporarily sustained in neonatal models of pressure overload, there is no concrete evidence of increased proliferation in TOF patients. This might suggest that by the time of repair, the adaptive response favors cardiomyocyte hypertrophy rather than proliferation. Late after repair, increased reactive fibrosis due to adverse loading is not necessarily a sign of maladaptation and may not be a suitable target for treatment. A remarkable distinction from pulmonary hypertension and isolated pressure overload is the increased, but potentially incomplete angiogenesis, which requires further investigation.

Identification of pathways involved in RV remodeling is perhaps one of the most important steps in the search for specific treatments. Stimulation of adaptive features and inhibiting maladaptive remodeling might be key in preventing or delaying progression to RV failure. Unfortunately, a comprehensive analysis of patients tissue characteristics is complicated, as tissues may generally only be harvested at initial repair or at pulmonary valve replacement and are compared with different control groups. Intriguingly, there is little overlap in responses described in the studies aiming to elucidate adaptative pathways, as is illustrated in Figure 5, which shows a comparison of the studies investigating differential expression of mRNA between RV tissue samples of TOF patients and healthy controls (88, 115–117). Functional protein association network analysis of all the genes reported in these studies reveals several clusters, the largest encompassing extracellular matrix (ECM) proteins, fibroblasts and angiogenesis (118). Other clusters comprise protein synthesis, mitochondrial metabolism, second messengers, contractile apparatus and cell cycle associated proteins (see Supplementary Figure S1 and Table S1). There is ongoing debate on the contribution of inflammation in the development of heart failure (119). The majority of studies report changes in ECM proteins, which is linked to inflammatory pathways, yet, specific evidence of increased inflammation in the RV of TOF patients is lacking (49, 55, 87, 88, 113, 115–117, 120, 121).

Expression levels of ECM proteins vary between studies in tissue samples taken at the time of repair (54, 121). At the time of pulmonary valve replacement, however, no changes in ECM protein levels were found when comparing to healthy age-matched controls, although mRNA expression of several proteins involved in ECM are increased (49). Similarly, protein and mRNA expression of the ECM proteoglycan lumican was decreased at initial repair, but at the time of pulmonary valve replacement protein levels were unaltered despite increased mRNA expression (49, 121). Interestingly, in an animal model of PR, expression of ECM components also undergo a change in expression, from being initially downregulated to becoming upregulated in the chronic phase (105).

Proteins associated with calcium handling and the contractile machinery of the sarcomere have been found to be affected in uncorrected TOF patients when compared to isolated VSD patients (54). This is also evident from experimental PR models, with myosin heavy chain isoform switch as part of the fetal gene reactivation and altered expression of cardiac muscle actin and Xin actin binding protein for example (105, 122). Whether protein changes in the contractile apparatus are relevant in chronic volume overload late after TOF repair is questioned by the study of Brayson et al. (49). They found that in RV tissue taken at the time of pulmonary valve replacement, myofilament contractility and calcium sensitivity was comparable to non-diseased RV tissue. Enhancement of contractility could be managed by administration of myosin modulators. Omecamtiv mecarbil increases the amount of time in contraction and is investigated extensively in LV failure (123). Studies on its effects in the RV are still scarce however (124, 125). Interestingly, a rat model of chronic aortic regurgitation demonstrated that treatment with omecamtiv mecarbil significantly reduced wall stress in the LV (126). If this also applies to the RV, omecamtiv mecarbil could counteract adverse remodeling in TOF patients with PR. Although controversial, it is important to keep in mind potential side effects of these inotropic treatments. A metabolic burden could be imposed on the myocardium by increasing its oxygen consumption, while reducing myocardial perfusion by decreasing the time spent in diastole (127–130).

Cardiac metabolic inflexibility is the reliance on glycolysis alone for energy production instead of utilizing several different substrates. It has been suggested as a maladaptive phenomenon in a protein analysis study of RV tissue of TOF patients compared to other CHD diagnoses (121). This metabolic shift, with downregulation of beta-oxidation in favor of less efficient glycolysis, is already known from experimental RV pressure overload studies, but has also been observed in a model of chronic PR (105, 131, 132). Accommodating a higher metabolic demand requires optimizing myocardial energetics in the dysfunctional RV to prevent progression to failure. The metabolic shift towards inefficient glycolysis can be (partially) reversed. As an example, dichloroacetate enhances glucose oxidation, thereby restoring mitochondrial function and reducing myocardial apoptosis. Treatment in experimental models of pulmonary hypertension resulted in improved RV systolic function and reverse remodeling (133–135). Similar beneficial effects were observed during treatments to either increase or partially inhibit fatty acid beta oxidation, the latter in order to favor glucose oxidation (136, 137).

A recent study aimed to generate a circRNA-miRNA-mRNA network in TOF. They analyzed three datasets available in GEO databases comparing profiles and identified 29 miRNA, 13 circRNAs and 88 mRNAs (138). Interestingly, a comparison between overlapping miRNA profiles in four other studies yielded two miRNAs that were identified in all 4 studies and 5 miRNAs that were identified in at least 3 of the 4 studies (Table 1) (139–142). MiR-222, also identified in the network by Kan et al. has been associated with exercise-induced myocardial adaptation, atrial fibrillation, is involved in function of L-type calcium channels and is a target of angiogenesis and proliferation via ERBB4 and HIF1α (138, 143–146). MiR-194 has been linked to the p53 pathway and may be involved in pulmonary angiogenesis (147). For disease markers as well as therapeutic targets, these pathways may be worthwhile to explore, albeit that these have not been described in experimental models mimicking TOF.

Generally, experimental studies show that many molecular mechanisms of RV adaptation are similar in pressure and volume overload, albeit that the latter exhibits a milder phenotype with slower disease progression (50, 148). In addition, most clinical studies are too heterogeneous to compare in order to derive a disease-specific molecular signature. Also, the relation between tissue expression patterns and serum biomarkers is difficult to determine. A comprehensive analysis of serum biomarkers associated with adverse outcomes could provide additional insights in adaptation of the overloaded RV, but is beyond the scope of this review (149).

TOF is characterized by a unique sequential loading pattern. Histopathology reflects these different stages of loading, but at present does not provide a targetable substrate for therapy. There is little to no consensus regarding specific adaptation mechanisms due to the wide variation in studies, but it seems likely there is no unifying molecular pathway leading to dysfunction. Knowledge of the distinctive physiology in TOF explains the lack of success of current treatment strategies. Cardiac dysfunction can be described as a combination of impaired ventricular kinetic energy and a disturbed filling pattern. The potential for future strategies is in optimizing RV biomechanics, kinetic energy and treatments to improve myocardial relaxation. In addition to novel drug targets, other promising options include resynchronization therapy, tissue engineered grafts and strategies aimed to strengthen the RV.