NAV3 is a genetic determinant of myocardial recovery in dilated cardiomyopathy and attenuates cardiac fibrosis
Authors: Wang M , Liu D , Nguyen T , McNamara D , Barlera S , Pileggi S , Mestroni L , Merlo M , Sinagra G , Pinet F , Krejci J , Kilianova A , De Groote P , Weishilboum R , Pereira N 
Published: October 2022

Abstract

Background
A genome-wide association study (GWAS) assessing change in left ventricular ejection fraction (LVEF) in dilated cardiomyopathy (DCM), a surrogate marker of morbidity and mortality in heart failure (HF), had not been performed previously and could provide insight into novel biological pathways that could lead to the development of new drugs that might target myocardial recovery. The presence and extent of cardiac fibrosis in DCM is independently associated with myocardial recovery and cardiovascular mortality.

Purpose
To identify the biological relevance of genetic targets that are associated with change in LVEF in patients with DCM.

Methods
A GWAS was performed using DNA from 686 patients with recent onset DCM who were on standard HF therapy using change in LVEF at a median of 6 months after initial diagnosis. Cultured human cardiac fibroblasts (HCFs) were used as an in vitro model to study the functional and biological relevance of the gene target identified in the GWAS. Specifically, HCFs were transfected with siRNA by using the Lipofectamine™ RNAiMAX Transfection Reagent for gene knockdown (KD). RNA-seq was performed using the Illumina TruSeq protocol with expression analysis conducted with the EdgeR package. Ingenuity Pathway Analysis was used.

Results
A single-nucleotide polymorphism, rs11105445(G&gt;A), mapping to the neuron navigator 3 (NAV3) gene (rs11105445, p=2.37E-07; beta 2.74±0.53) was associated with improvement in LVEF. We performed a phenome-wide association study using data from the UK Biobank and demonstrated that genetic variation in NAV3 was significantly associated with HF mortality (p=3.2E-28), highlighting the potential importance of this gene in HF. Using GTEx data we demonstrated that in LV tissue the minor allele A was associated with ↓NAV3 expression (p=0.03) suggesting that ↓NAV3 expression might be associated with improvement in LVEF. We demonstrated that NAV3 KD significantly ↓TGF-β1 mediated HCF transdifferentiation into myofibroblasts, ↓α-smooth muscle actin (ACTA2) and ↓collagen I (COL1A1), therefore NAV3 KD was anti-fibrotic (see Figure 1), 1a. HCFs treated by vehicle/TGF-β1 after KD of NAV3/ctrl, and ACTA2 and COL1A1 were analyzed by qPCR; 1b. Representative immunofluorescence staining for α-SMA (in green), RNA-seq after NAV3 KD followed by pathway analysis suggested that NAV3 exerted its effect by regulating cell cycle related proteins (Figure 2), 2a. Volcano plot shows significant differentially expressed genes identified by RNA-seq analysis (down-regulated in blue, up-regulated in red); 2b. NAV3 KD significantly increased expression of cell cycle related proteins, which was validated by Western blot.

Conclusions
Decreased expression of NAV3 is associated with myocardial recovery in DCM, most likely due to its anti-fibrotic effect via direct regulation of cell cycle proteins. The role of NAV3 as a novel therapeutic target in DCM needs to be explored.

Funding Acknowledgement
Type of funding sources: Public Institution(s). Main funding source(s): Mayo Clinic Center for Individualized MedicineMayo Clinic Department of Cardiovascular Medicine