Filamin C Truncation Mutations Are Associated With Arrhythmogenic Dilated Cardiomyopathy and Changes in the Cell-Cell Adhesion Structures

Abstract

Objectives: The purpose of this study was to assess the phenotype of Filamin C (FLNC) truncating variants in dilated cardiomyopathy (DCM) and understand the mechanism leading to an arrhythmogenic phenotype.

Background: Mutations in FLNC are known to lead to skeletal myopathies, which may have an associated cardiac component. Recently, the clinical spectrum of FLNC mutations has been recognized to include a cardiac-restricted presentation in the absence of skeletal muscle involvement.

Methods: A population of 319 U.S. and European DCM cardiomyopathy families was evaluated using whole-exome and targeted next-generation sequencing. FLNC truncation probands were identified and evaluated by clinical examination, histology, transmission electron microscopy, and immunohistochemistry.

Results: A total of 13 individuals in 7 families (2.2%) were found to harbor 6 different FLNC truncation variants (2 stopgain, 1 frameshift, and 3 splicing). Of the 13 FLNC truncation carriers, 11 (85%) had either ventricular arrhythmias or sudden cardiac death, and 5 (38%) presented with evidence of right ventricular dilation. Pathology analysis of 2 explanted hearts from affected FLNC truncation carriers showed interstitial fibrosis in the right ventricle and epicardial fibrofatty infiltration in the left ventricle. Ultrastructural findings included occasional disarray of Z-discs within the sarcomere. Immunohistochemistry showed normal plakoglobin signal at cell-cell junctions, but decreased signals for desmoplakin and synapse-associated protein 97 in the myocardium and buccal mucosa.

Conclusions: We found FLNC truncating variants, present in 2.2% of DCM families, to be associated with a cardiac-restricted arrhythmogenic DCM phenotype characterized by a high risk of life-threatening ventricular arrhythmias and a pathological cellular phenotype partially overlapping with arrhythmogenic right ventricular cardiomyopathy.

Keywords: Filamin C; arrhythmias; arrhythmogenic dilated cardiomyopathy; cardiovascular genetics; familial dilated cardiomyopathy; heart failure.

FIGURE 1. Pedigrees of DCM Families With FLNC Truncating Variants Displaying an Arrhythmogenic Phenotype

(A) Squares indicate males, circles indicate females, slashes indicate deceased individuals, black shading indicates a dilated cardiomyopathy (DCM) phenotype, and vertical lines indicate history of heart disease. The arrows indicate the proband. Carriers (+) and noncarriers (−) of a FLNC truncation variant are shown. (B) Electrocardiogram of subject TSSDC130 (II:3) shows sustained ventricular tachycardia. (C) Electrocardiogram depicts nonsustained ventricular tachycardia from individual II:1 (family DNFDC057) (9).

FIGURE 2. Structural Distribution of Truncating Variants in the Human FLNC Protein

Schematic of the FLNC immunoglobulin-like repeats labeled 1 to 24 using transcript NP_001449.3. Hinge 1 and 2 domains are labeled as H1 and H2, respectively. Red vertical lines indicate protein positions of the 6 FLNC truncation variants detected in our dilated cardiomyopathy (DCM) families. Black vertical lines represent previously reported hypertrophic cardiomyopathy (HCM) (7), restrictive cardiomyopathy (RCM) (8), DCM variants (12), and myofibrillar skeletal myopathies (MFM) (6) variants.

FIGURE 3. Cardiac Tissue Analysis ofFLNC Truncation Variant Carrier From Family DNFDC057 (II:2)

(A) Trichrome staining of the left ventricle shows subepicardial interstitial and focal replacement fibrosis and fatty infiltration (circle) in areas containing degenerating cardiac myocytes. Scale bar = 200 μm. (B) Fatty infiltration and mild fibrosis are seen in the right ventricle. Scale bar = 100 μm.

FIGURE 4. Electron Microscopy Images of Left Ventricular Cardiac Muscle From Family DNFDC057

(A) Representative images from individual II:2 show disarrayed Z-discs (white arrows). (B) Magnified image of the white box in A, showing a thickened Z-disc pattern. (C and D) Representative TEM images from individual II:1 exhibiting disarray of Z-discs (black arrows). No aggregates are seen in cardiac myocytes. Scale bars: (A) 1 μm, (B) 200 nm, (C and D) 1 μm.

FIGURE 5. Immunohistochemistry of Left Ventricular Myocardial Tissue

Immunostaining of the explanted heart of patient DNFDC057-II:2 carrying the G1891Vfs61X FLNC truncation. (A) Immunoreactive signals for plakoglobin and connexin 43 (Cx43) at intercalated discs are normal compared with control samples, whereas junctional signal for desmoplakin is reduced. N-cadherin is used as a tissue quality control and is normal in all samples. (B) SAP97 signal is depressed compared with control samples. GSK3β maintained its normal cytoplasmic distribution.

FIGURE 6. FLNC in Cardiac Tissue

Immunohistochemical staining of FLNC does not show significant presence of aggregates in cardiomyocytes, and the weaker staining in the patient (C) suggests a reduced amount of FLNC protein compared with control subjects (A and B), as previously shown by western blot (9).

FIGURE 7. Immunohistochemistry of Buccal Mucosa

Immunostaining of the buccal mucosa in patients DNFDC057-II:1 and II:2 shows normal signals for plakoglobin, and diminished signal for connexin 43 (Cx43), desmoplakin, and SAP97. E-cadherin staining is used as a cell quality control and is normal in all samples.