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Introduction: We recently identified the fetal noncanonical WNT receptor ROR2 as being re-expressed in human right ventricular failure (RVF), but the myocardial role of ROR2 remains poorly described. Hypothesis: We assessed if ROR2 expression influences cardiomyocyte structure or contributes to RVF pathogenesis. Methods: ROR2 gain- and loss-of-function (ROR2 GOF and ROR2 LOF ) NRVMs were generated using adenoviral-delivered ROR2 cDNA or shRNA and cultured on nanopatterned substrates. NRVM structure was assessed by confocal microscopy (minimum n=3 replicates, >30 cells/condition). NRVM gene expression was characterized by RNAseq. The impact of ROR2 on RV structure and function was assessed using AAV9-mediated cardiac ROR2 delivery, in 4-week old C57BL/6 mice (vs GFP), and echocardiography and hemodynamics (n= 8, 50% male). Results: ROR2 GOF disrupts non-sarcomeric and sarcomeric NRVM structure, exhibiting smaller (500 vs. 711 μm 2 , p = 7.4x10 -23 ) and rounder (aspect ratio 2.1 vs. 3.1, p=1.9x10 -16 ) shape and fragmented sarcomeres (Figure). Whereas, ROR2 LOF NRVMs demonstrate a striking peripheralization of α-actinin and β-catenin. Gene-set enrichment analyses of ROR2 GOF NRVMs reveal upregulation of cytokinesis, mitosis, microtubule regulation, and insulin like growth factor receptor signaling. By 4 weeks after induction of ROR2 GOF in vivo , mice exhibit biventricular dilation and systolic dysfunction (TAPSE 0.85 vs 1.2 mm, p = 2.1x10 -5 ; right ventricular outflow dimension 2.0 vs 1.7 mm, p = 0.001, LV EDV 33.5 vs 28.6 μL, p = 0.05; LV ejection fraction 63% vs 69%, p = 0.015). RV diastolic function is reduced in ROR2 GOF (E:e’ 21.8 vs 18.6, p = 0.004 and RV EDP 2.9 vs 1.0 mmHg, p = 0.06). Conclusions: ROR2 GOF causes biventricular dilation, systolic and diastolic dysfunction in vivo . In vitro, ROR2 GOF NRVMs exhibit an immaturity phenotype with maintenance of proliferation gene program. In contrast, ROR2 LOF NRVMs form a tight monolayer with enhanced cell-cell borders.

Background: Drivers of adaptive and pathologic human right ventricular (RV) remodeling are poorly defined, but there is some evidence that WNT signaling contributes to these processes. Methods: Using a candidate gene approach, we sought to identify genes specifically expressed in RV failure by assessing the expression of 28 WNT-related genes in the RVs of three groups: human explanted nonfailing donor hearts (NF, n = 29), pre-transplant cardiomyopathy hearts with preserved RV function (pRV, n = 78), and pre-transplant cardiomyopathy hearts with RV failure (RVF, n = 35). Results: We identified the noncanonical WNT receptor ROR2 as transcriptionally strongly upregulated in RVF compared to pRV and NF (p < 0.05, adjusted for multiple hypothesis testing using Benjamin-Hochberg). By western blot, ROR2 protein expression correlated to mRNA expression (R 2 = 0.41, p = 8.1 x 10-18) and increased linearly with higher right atrial to pulmonary capillary wedge ratio in RVF (R 2 =0.40, p = 3.0 x 10-5). Using trichrome, immunohistochemistry, and RNAscope, we identified preferential ROR2 mRNA and protein expression in fibrotic regions by both cardiomyocytes and noncardiomyocytes. High ROR2 expression correlated with increased expression of a known ROR2 target, the WNT/Ca2+ responsive protease calpain and its target FLNA (R 2 0.25 and 0.67, respectively, p < 0.05), as well as with increased relative phosphorylation of FLNA (R 2 0.62, p < 0.05), a marker of its activation by ROR2. Finally, mice with a range of RVF severity, generated by pulmonary artery constriction, demonstrated a robust increase in ROR2 and FLNA expression compared to sham-treated animals, and ROR2 and FLNA expression correlated strongly with each other (R 2 0.89, p = 6.9 x 10-5) and with increasing RV weight normalized to body weight (R 2 ~0.8, p < 0.001, both). Conclusion: ROR2 expression in the RV is dramatically increased in humans and mice with severe RVF, and ROR2 expression correlates with worsening RV hemodynamics, calpain expression, FLNA cleavage, and FLNA phosphorylation. Taken together, the data reveal the robust activation of noncanonical WNT signaling in human RVF, and identify ROR2-mediated cytoskeleton protein cleavage as a potential novel target of human pathologic RV remodeling.

Background: A greater understanding of the gene programs that drive adaptive and pathologic right ventricle (RV) remodeling are needed given the poor response of the RV to standard reverse remodeling agents and the absence of RV-specific therapies. Left ventricular failure (LVF) provides an ideal model to study human RV remodeling given the spectrum of secondary RV involvement. Methods: Dilated (DCM) and ischemic cardiomyopathy (ICM) hearts in the Penn Human Heart Tissue Bank were identified as having preserved RV function (pRV) or RVF using preexplant right atrial to wedge pressure ratio. Using RT-PCR and a candidate gene approach, we assessed RV differential expression of 28 WNT -related genes and NPPA/NPPB as positive controls between nonfailing (NF, n= 29), DCM-pRV (n = 47), DCM-RVF (n = 26), ICM-pRV (n = 31), and ICM-RVF (n = 10). Genes which met significance for differential expression between NF, pRV, and RVF for both DCM and ICM using Kruskal-Wallis were further assessed by Mann Whitney U comparing pRV to RVF after combining ICM and DCM groups (Benjamini-Hochberg p < 0.05 for significance). Results: Most (20/28) WNT -related genes were differentially expressed in at least one cardiomyopathy group, but only 13, as well as NPPA, were significant for both DCM and ICM. Only, CREBBP, NFATC2, and ROR2 were differentially expressed between pRV and RVF (Table). Discussion: WNT -related genes show significant DGE in RV remodeling. But, of those tested, only CREBBP, NFATC2, and ROR2 appear to be implicated in the progression from adaptive to pathologic RV remodeling. Additional studies are underway to confirm differential protein expression.