Noncanonical WNT Activation in Human Right Ventricular Heart Failure

Abstract

Background: No medical therapies exist to treat right ventricular (RV) remodeling and RV failure (RVF), in large part because molecular pathways that are specifically activated in pathologic human RV remodeling remain poorly defined. Murine models have suggested involvement of Wnt signaling, but this has not been well-defined in human RVF. Methods: Using a candidate gene approach, we sought to identify genes specifically expressed in human pathologic RV remodeling by assessing the expression of 28 WNT-related genes in the RVs of three groups: explanted nonfailing donors (NF, n = 29), explanted dilated and ischemic cardiomyopathy, obtained at the time of cardiac transplantation, either with preserved RV function (pRV, n = 78) or with RVF (n = 35). Results: We identified the noncanonical WNT receptor ROR2 as transcriptionally strongly upregulated in RVF compared to pRV and NF (Benjamini-Hochberg adjusted P < 0.05). ROR2 protein expression correlated linearly to mRNA expression (R ² = 0.41, P = 8.1 × 10^-18) among all RVs, and to higher right atrial to pulmonary capillary wedge ratio in RVF (R ² = 0.40, P = 3.0 × 10^-5). Utilizing Masson's trichrome and ROR2 immunohistochemistry, we identified preferential ROR2 protein expression in fibrotic regions by both cardiomyocytes and noncardiomyocytes. We compared RVF with high and low ROR2 expression, and found that high ROR2 expression was associated with increased expression of the WNT5A/ROR2/Ca²⁺ responsive protease calpain-μ, cleavage of its target FLNA, and FLNA phosphorylation, another marker of activation downstream of ROR2. ROR2 protein expression as a continuous variable, correlated strongly to expression of calpain-μ (R ² = 0.25), total FLNA (R ² = 0.67), calpain cleaved FLNA (R ² = 0.32) and FLNA phosphorylation (R ² = 0.62, P < 0.05 for all). Conclusion: We demonstrate robust reactivation of a fetal WNT gene program, specifically its noncanonical arm, in human RVF characterized by activation of ROR2/calpain mediated cytoskeleton protein cleavage.

Keywords: Ror2; Wnt; calpain; heart failure; remodeling; right ventricle (RV); right ventricular (RV) failure.

Figure 1

Relative mRNA expression of WNT-related genes with differential transcription between pRV and RVF as noted in (A–D). WNT-related gene expression was normalized to average of GAPDH and TBP for each patient and then to the median NF expression for each gene. Genes were initially filtered for statistically significant differential expression between NF/pRV/RVF for DCM and ICM separately using Kruskal–Wallis (see Supplementary Table 4) and then were assessed for differential pRV/RVF expression using Mann–Whitney U (NF n = 29, DCM-pRV n = 47, DCM-RVF n = 26, ICM-pRV n = 31, ICM-RVF n = 9) (see also Table 2). Benjamini-Hochberg corrected P < 0.05 was used for significance.

Figure 2

ROR2 protein expression increases in RVF and correlates with hemodynamics. (A) Dot plot demonstrating ROR2 protein expression (NF n = 29, pRV n = 78, RVF n = 35) normalized to GAPDH. To allow interblot comparison for 143 samples, two samples from the original blot (Supplementary Figure 1) were loaded alongside remaining samples on subsequent blots. Finally, expression was normalized to the median NF expression and plotted using a logarithmic base-10 scale to facilitate visual interpretation. Differential protein expression was assessed by Kruskal–Wallis comparing NF/pRV/RVF and by Mann–Whitney U comparing pRV/RVF. (B) Linear regression comparing ROR2 protein expression to RA:PCWP in RVF (n = 35).

Figure 3

Preferential ROR2 protein expression in fibrotic areas. Representative pRV (A,D) and RVF (B,C,E,F) with low and high ROR2 expression by western blot, respectively. In these serial trichrome stained (A,C) and ROR2 immunohistochemistry (D,F) sections obtained at 10X using light microscopy, we found a pattern of preferential ROR2 expression in regions of greater fibrosis by both cardiomyocytes and noncardiomyocytes.

Figure 4

ROR2 expression correlates with increases in calpain expression and calpain-mediated cleavage. (A) Western blots comparing expression of downstream targets between a subset of high (n = 6) and low (n = 6) ROR2 expressing RVF patients. (B) Logarithmic regressions comparing ROR2 expression to full length, calpain cleaved, and phosphorylated FLNA (n = 12 total). Y-axis is normalized to the maximal expression of each target protein among these 12 patient samples.

Figure 5

Proposed RVF model consisting of reactivation of a ROR2 fetal gene program with increasing RVF severity, which results in increased calpain expression and activity leading to calpain-mediated cleavage of cytoskeleton structural proteins including FLNA.