Wnt5a is associated with right ventricular dysfunction and adverse outcome in dilated cardiomyopathy

Abstract

The Wingless (Wnt) pathway has been implicated in the pathogenesis of dilated cardiomyopathy (DCM). To explore the role of Wnt modulators Wnt5a and sFRP3 in DCM patients we analyzed the expression of Wnt5a and sFRP3 in plasma and myocardium of DCM patients and evaluated their effects on NFAT luciferase activity in neonatal mouse cardiomyocytes. Elevated circulating Wnt5a (n = 102) was associated with increased pulmonary artery pressures, decreased right ventricular function and adverse outcome, with a stronger association in more severely affected patients. A higher Wnt5a/sFRP3 ratio (n = 25) was found in the right ventricle vs. the left ventricle and was correlated with NFAT activation as well as pulmonary artery pressures. Wnt5a induced NFAT activation and sFRP3 release in cardiomyocytes in vitro, while sFRP3 antagonized Wnt5a. Wnt5a is associated with right ventricular dysfunction and adverse outcome in DCM patients and may promote the progression of DCM through NFAT signaling.

Figure 1

Wnt5a and sFRP3 associations with adverse outcome in patients with DCM. (A) Circulating Wnt5a, sFRP3 and their ratio (Wnt5a/sFRP3) in DCM patients with adverse outcome (death or HTx) vs survivors at baseline (BL) and 1 year follow-up. (B) Kaplan-Meier curves showing the cumulative incidence of mortality or HTx (n = 13) during the whole study period (median follow-up 3.3 years, 0.2–5.1), according to dichotomized levels of Wnt5a and sFRP3 at enrolment. (C) The relationship between Wnt5a and outcome shown as hazard ratios (HRs) and 95% confidence interval per standard deviation (SD) change with p-values for univariate (UNI), propensity score (PS) adjusted (age, gender, creatinine, NYHA and NT-proBNP) (model 1), and adjusted for RVEF (model 2) Cox hazards models. *p < 0.05, **p < 0.01 vs. survivors. DCM, dilated cardiomyopathy, HTx, heart transplantation; NT-proBNP, N-terminal pro-brain natriuretic peptide; NYHA, New York Heart Association; RVEF, right ventricular ejection fraction; sFRP3, secreted frizzled-related protein 3.

Figure 2

Interactions between Wnt5a and sFRP3 levels and indices of ventricular function. Interaction analysis between Wnt5a, sFRP3, NT-proBNP and RVEF (A) in patients with impaired RV function (i.e. RVEF < 30%, n = 22) and (B) in patients with adverse outcome (n = 13). Interaction analysis was performed including covariate (e.g. Wnt5a), fixed factor (e.g. RVEF with cut-off) and their interaction term. The p-value of the interaction term is shown in blue. HTx, heart transplantation; NT-proBNP, N-terminal pro-brain natriuretic peptide; RVEF, right ventricular ejection fraction; sFRP3, secreted frizzled-related protein 3.

Figure 3

Wnt5a and sFRP3 expression in the myocardium of DCM patients. (A) mRNA and (B) protein levels of Wnt5a, sFRP3 and their ratio (Wnt5a/sFRP3) in the LV and RV myocardial tissue of DCM patients (n = 25). (C) Correlations between Wnt5a/sFRP3 ratio and NT-proBNP (n = 25), LA diameter (n = 20), LVEF (n = 23), PASP (n = 24) and PCWP (n = 24) in the LV and RV of DCM patients. (D) Correlation between Wnt5a/sFRP3 ratio and PASP in the RV and comparison of the Wnt5a/sFRP3 ratio within the LV and RV between DCM patients with pulmonary hypertension (i.e. PASP > 40 mm Hg, n = 11) and without it (i.e. PASP ≤ 40 mm Hg, n = 13). (E) mRNA expression of typical canonical and non-canonical Wnt ligands in the LV and RV of DCM patients obtained in preliminary studies. (F) mRNA expression profiles of other Wnt ligands in the RV of DCM patients (GSE29819) as well as some RNAseq data from the LV (GSE71613) obtained from the GEO repository. DCM, dilated cardiomyopathy; LA, left atrial; LV, left ventricle, LVEF, left ventricular ejection fraction; NT-proBNP, N-terminal pro-brain natriuretic peptide; PASP, pulmonary artery systolic pressure; PCWP, pulmonary capillary wedge pressure; RV, right ventricle; sFRP3, secreted frizzled-related protein 3.

Figure 4

Wnt5a and sFRP3 effects on NFAT signaling. (A) Representative immunoblots of RCAN1.4 and loading control Vinculin in the LV and RV of two DCM patients. (B) mRNA and protein levels of RCAN1.4 in the LV and RV myocardial tissue of DCM patients (n = 25). (C) Correlation between RCAN1.4 mRNA and protein levels (adjusting for gel with partial correlation). (D) Correlation between the Wnt5a/sFRP3 ratio and RCAN1.4 mRNA and protein levels (adjusting for gel with partial correlation). (E) Correlations between RCAN1.4 and NT-proBNP (n = 25), LA diameter (n = 20), LVEF (n = 23), PASP (n = 24) and PCWP (n = 24) in the LV and RV of DCM patients. (F) NFAT luciferase activity in neonatal mouse cardiomyocytes after treatment with endothelin-1 (ET-1) and increasing concentrations of recombinant Wnt5a. (G) A relative sFRP3 release in culture medium from F. (H) NFAT luciferase activity after treatment with recombinant sFRP3, Wnt5a or both. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control (CTR), ^#p < 0.05, ^##p < 0.01 vs 1 nM Wnt5a, ^†1 µM endothelin-1 (ET-1). DCM, dilated cardiomyopathy; LA, left atrial; LV, left ventricle, LVEF, left ventricular ejection fraction; NFAT, nuclear factor of activated T-cells; NT-proBNP, N-terminal pro-brain natriuretic peptide; PASP, pulmonary artery systolic pressure; PCWP, pulmonary capillary wedge pressure; RCAN1.4, regulator of calcineurin 1 isoform 4; RV, right ventricle; sFRP3, secreted frizzled-related protein 3.