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Introduction:Right ventricular (RV) failure is the leading cause of death in patients with pulmonary arterial hypertension (PAH). Capillary rarefaction has been proposed as the hallmark of RV failure, yet our understanding of the capillary architecture is limited by 2D image analysis. Three-dimensional (3D) confocal deep tissue imaging offers the unparalleled opportunity to characterize the architectural microvascular changes in relationship to cardiomyocytes in pressure-overloaded RV failure.Methods and Results:250 μm heart tissue slices from mice with RV failure, 7-weeks after Pulmonary Artery Banding (PAB) were harvested and the microvascular network was assessed for density, segment length, diameter, orientation, as well as microvasculature-cardiomyocyte contact area in regions with and without interstitial fibrosis using confocal 3D imaging. Human heart tissue from end-stage PAH patients was compared to controls with normal RV function. In PAB mice, 3D imaging of the RV free wall revealed tortuous, shorter, thicker and higher-branched microvascular segments as well as development of fibrosis compared to Sham animals (Fig A). When corrected for fibrosis, the microvascular density was preserved even in advanced stages of RV failure. Physical contact between the microvasculature and cardiomyocytes was preserved in areas without fibrosis, yet significantly impaired in areas with interstitial fibrosis (Fig B), accompanied by an overexpression of β-myosin heavy chain suggesting local ischemia (Fig C). 3D imaging of human RV tissue of end-stage PAH patients revealed architectural microvascular remodeling that varied depending on etiologies and length of disease yet showed preserved microvascular density. (Fig. D).Conclusions:3D deep tissue imaging revealed compensatory changes of the microvascular network to maintain a stable cardiomyocyte coverage in pressure-overloaded RV failure, which was impaired in areas of interstitial fibrosis.

Introduction:Patients who have undergone tetralogy of Fallot (TOF) repair are subject to chronic volume and/or pressure overload, leading to a 40% probability of right ventricular (RV) failure by the third decade of life. We sought to identify a plasma proteome profile of the systemic response to right heart stress to better understand the mechanisms of RV failure and identify novel RV remodeling biomarkers.Methods:Patients with repaired TOF were recruited at the time of standard of care cardiac MRI (CMR) (N=14), and blood samples were collected. Plasma proteomics was assessed using data independent acquisition mass spectrometry. Proteins that were significantly affected between groups were identified and used for pathway enrichment analysis.Results:Patients underwent primary TOF repair at median 2.9m (IQR 0.8, 4.1) and CMR at median 16yrs (IQR 15, 18). Patients were grouped based on median indexed RV end diastolic volume (RVEDVi). Group 1: median RVEDVi 146ml/m2(IQR 139, 164) (N=9); Group 2: median RVEDVi 114ml/m2(IQR 104, 130) (N=5); p<0.001. RV ejection fraction was equal between groups (mean 56% vs. 55%; p=0.68). Forty-one proteins were significantly differentially abundant: 19 enriched in Group 1 and 22 enriched in Group 2 (Fig 1; p<0.05). Adaptive signaling in Group 1 was characterized by upregulation of proteins involved in antioxidant mechanisms (SOD1, TXN, PRDX1&2), calcium handling (S100A6, CALM1), and proteostasis (UBB, HSPA8). Maladaptive signaling was characterized by upregulation of glycolytic pathways (MDH1, PGK1) and heme protein oxidation (ALAD) and downregulation of proteins to counter acute stress (complements, ORM1&2).Conclusion:Patients with repaired TOF and moderate-severe RV dilation have a distinct maladaptive plasma proteomic profile independent of ejection fraction. These data suggest that profiling the circulating plasma proteome may be used to monitor disease progression prior to the onset of clinical heart failure.

Introduction:Single ventricle congenital heart disease such as hypoplastic left heart syndrome (HLHS) with a Fontan circulation accounts for the largest group of children hospitalized with circulation failure, with an in-hospital mortality of 20-50%. We sought to (i) evaluate the mechanisms underlying circulation failure and (ii) identify novel therapeutic targets.Methods:Blood was collected from patients with HLHS s/p Fontan and from controls with normal cardiac anatomy and function (N=5/group). Plasma microvesicles (MV) were isolated and proteomics assessed using data independent acquisition mass spectroscopy. Dysregulated proteins with a fold change >1.5 or < -1.5, p<0.05 were evaluated using Ingenuity pathway analysis.Results:Age of Fontan patients vs. controls was 17.04±2 vs. 15.7±2.5, p=0.4; 20% were male in both groups. 60% of Fontan patients were in NYHA class II/III, 40% in NYHA class IV; 80% had Fontan-associated liver disease. Circulating MVs were released from cardiomyocytes, endothelial cells, and hepatocytes based on cell surface markers. Upregulated proteins (N=27) implicated cell death pathways (Solute carrier family 2, Angiotensinogen, CD14); while downregulated proteins (N=88) implicated impaired cell survival (tyrosine-protein kinase Yes, endothelial growth factors). MVs contained mitochondria and endoplasmic reticulum proteins implicating upregulation in reactive oxygen species signaling (catalase A, S100A8) and downregulation in antioxidant enzymes (GPX1, PRDX 5).Conclusion:Circulating MVs from patients with HLHS s/p Fontan are released from the heart, blood vessels, and liver providing a noninvasive signature of organ remodeling. The MV protein cargo implicates heightened cell death, oxidative damage and impaired cell survival, thereby providing insight into the mechanisms of Fontan associated circulation failure.Figure: Decreased Cell Survival and Oxidative Damage in the Fontan Circulation

An abundance of data has provided insight into the mechanisms underlying the development of left ventricular (LV) hypertrophy and its progression to LV failure. In contrast, there is minimal data on the adaptation of the right ventricle (RV) to pressure and volume overload and the transition to RV failure. This is a critical clinical question, because the RV is uniquely at risk in many patients with repaired or palliated congenital heart disease and in those with pulmonary hypertension. Standard heart failure therapies have failed to improve function or survival in these patients, suggesting a divergence in the molecular mechanisms of RV versus LV failure. Although, on the cellular level, the remodeling responses of the RV and LV to pressure overload are largely similar, there are several key differences: the stressed RV is more susceptible to oxidative stress, has a reduced angiogenic response, and is more likely to activate cell death pathways than the stressed LV. Together, these differences could explain the more rapid progression of the RV to failure versus the LV. This review will highlight known molecular differences between the RV and LV responses to hemodynamic stress, the unique stressors on the RV associated with congenital heart disease, and the need to better understand these molecular mechanisms if we are to develop RV-specific heart failure therapeutics.

Introduction:Right ventricular (RV) function determines the prognosis of pulmonary hypertension. Capillary rarefaction using 2D imaging has been proposed as the hallmark of RV failure, although this paradigm is controversially discussed. By applying 3D deep-tissue imaging, we characterized the adaptive and maladaptive response of the capillaries in the pressure-overloaded RV in hypertrophy and failure.Methods and Results:Mice subjected to pulmonary artery banding (PAB) were harvested after 3 weeks (w) (compensated RV hypertrophy) and 7w post-surgery (decompensated RV failure). Hearts were sectioned into 250 μm and capillaries were stained with isolectin B4. Samples were imaged with confocal microscopy and reconstructed into 3D using Imaris imaging software (Fig. A, n=4). In the RV free wall, mean capillary length decreased, and branching, tortuosity, mean and total capillary diameter increased 3w post-PAB. Mean and total diameter, and total length decreased at 7w resulting in a reduced capillary volume normalized to heart volume at 7w vs 3w, but not significantly reduced vs baseline suggesting that this is not the mechanism of RV failure (Fig. B). However, most strikingly we uncovered by sparsely labeling cardiomyocytes that the capillary-cardiomyocyte contact surface was maintained at w3, yet significantly decreased in areas of interstitial fibrosis in the decompensated RV at 7w compared to baseline and 3w (Fig. C, D).Conclusions:3D deep-tissue imaging uncovered the remarkable adaptive response of the RV microvasculature in the pressure-overloaded RV to maintain an adequate capillary-cardiomyocyte contact by increasing capillary branching, length and diameter. These mechanisms failed in the decompensating RV when interstitial fibrosis impaired the capillary-cardiomyocyte interaction. Our method offers the unprecedented opportunity to study microvascular adaption in the pressure overloaded RV and is readily transferable to human tissue.

Introduction:Patients with repaired tetralogy of Fallot (TOF) are subject to chronic volume and pressure loading leading to a 40% probability of right ventricular (RV) failure by the 3rddecade of life. We sought to identify a non-invasive, circulating signature of the systemic response to right heart stress to better understand the mechanisms of RV failure and to develop novel therapies.Methods:High throughput mass spectroscopy was used to assess circulating plasma proteins in controls with structurally normal hearts (N=14) and in TOF s/p repair (N=74): no residual lesion (N=4), severe right heart volume overload (N=28), severe right heart pressure overload (N=20), and moderate right heart volume and pressure overload (N=22). Ingenuity pathway analysis was used to identify enriched biological processes using proteins with log2 fold change>0.6, and q value <0.05.Results:Mean age in controls was 15±2yrs, 50% male and in TOF s/p repair 17±14yrs, 34% male. The RV was moderately dilated with normal function in those with volume and/or pressure overload. We identified (i) excellent proteomic coverage identifying >1500 proteins/sample; (ii) unique subset of proteins distinguishing control from tetralogy of Fallot with residual defects (Fig 1); (iii) upregulated pathways involved signal transduction (CREBBP), catabolic processes (NT5M), cell death and aging (FUCOA 2-fold); (iv) downregulated pathways involved immune response (IGHV4), complement and coagulation activation (VWF), regulation of calcium handling (S100A 12-fold) and cell-extracellular matrix adhesion.Conclusion:Patients with TOF s/p repair with RV volume and pressure overload have a distinct proteomic profile implicating heightened cell death pathways and impaired calcium handling which may predispose to heart failure and arrhythmias. These data suggest peripheral blood proteins can provide insight into the mechanisms of RV failure and can be used for monitoring disease progression.

Introduction:Chronic right ventricular (RV) pressure and volume overload following RV outflow tract reconstruction in children leads to right heart failure. We show the first noninvasive microRNA (miR) signature of myocardial remodeling in Tetralogy of Fallot, pulmonary atresia and major aortopulmonary collateral arteries (TOF/PA/MAPCAs) and associate with a plasma proteomic signature. Identifying biomarkers to detect the early, reversible stages of heart failure may allow for development of novel therapies to preserve RV function.Methods:Clinical data was collected from patients with TOF/PA/MAPCAs (N=10). miR and protein expression was assessed in the peripheral blood using Agilent Human miR Microarray and high throughput discovery mass spectroscopy (DIA) respectively. GeneSpring and IPA software programs were used to identify miRs and proteins with >2-fold change (ANOVA, corrected p <0.05).Results:Median patient age was 2.5 years (0.02-10.83). Patients were grouped into 4 categories: (i) Neonatal - undergoing primary complete repair (N=2), (ii) Mild RV hypertrophy (N=3), (iii) Moderate RV hypertrophy (N=3), and (iv) Severe RV hypertrophy (N=2), with increasing RV pressure and volume overload in each group. 15 miRs were downregulated (>5-fold) between the neonatal and hypertrophy groups, and were associated with upregulation in their target plasma proteins involved in extracellular matrix receptor interactions and TGF-b signaling (e.g. miR 122-5p, 369-3p). 10 miRs were downregulated only in moderate-severe hypertrophy and were associated with upregulation in their target plasma proteins involved in structural protein biosynthesis, adrenergic signaling, defects in carnitine and Coenzyme A metabolism (e.g. miR 216a, 571). The hypertrophic miR signature reclassified 2 patients with moderate hypertrophy as severe and 1 with severe hypertrophy as moderate.Conclusion:In our discovery cohort of patients with TOF/PA/MAPCAs, we have identified a non-invasive unique signature of RV hypertrophy. Even in the stage of mild RV hypertrophy, fibrosis sets in. With moderate-severe RV hypertrophy, adrenergic signaling is heightened and energy generation is impaired allowing to further risk stratify patients with adverse remodeling.

Introduction:Repaired tetralogy of Fallot (TOF) comprises the largest group of complex, cyanotic congenital heart diseases surviving into adulthood. However, this growing population has a 40% incidence of right ventricular (RV) failure. We sought to identify a non-invasive, blood-based signature of adverse myocardial remodeling to better understand the mechanisms of RV failure.Methods:Demographic, clinical data, and blood samples were collected from adults with repaired TOF (N=20) and whole genome microRNA (miRNA) expression was profiled from the buffy coat using Agilent miRNA microarray platform. Fold change, target gene, and pathway analysis was performed using GeneSpring statistical software.Results:MiRNA expression was profiled in the following groups: 1. normal RV size/function (N=4), 2. mild/moderate RV enlargement (N=11), 3. severe RV enlargement with systolic dysfunction (N=5). 267 miRNAs were downregulated, and 66 were upregulated across the three groups (FC >2.0, FDR corrected p<0.05), demonstrating a progressive change in miRNA expression as RV function decreased. Unbiased hierarchical clustering showed three distinct clusters coinciding with progressive RV enlargement and dysfunction. The most dysregulated miRNAs regulate cell cycle, adherens junction, extracellular matrix (ECM) interaction, and signal transduction pathways including MAPK, PI3K/Akt (Downregulated: miRNA 33a-3p, 122-5p, and 34b-3p; Upregulated: miRNA 431-3p, 196a-3p, and 485-5p). Since many upregulated miRNAs predicted decreased endothelial proliferation, we assessed the effect of plasma from TOF patients with RV dysfunction on endothelial cell function and show a 50% decrease in endothelial cell proliferation and tube formation (p<0.05).Conclusion:Adults with TOF demonstrate a progressive change in circulating whole genome miRNA profiles as RV enlargement and failure develop. The miRNA profile can reclassify patients into greater and lesser severity and can be used in conjunction with imaging to guide the timing of pulmonary valve replacement. The pathways predicted to be dysregulated provide unique insights into the mechanisms of RV failure and can serve as therapeutic targets for future drug development.