Nesprin-1 mutations in human and murine cardiomyopathy

Abstract

Mutations in LMNA, the gene encoding the nuclear membrane proteins, lamins A and C, produce cardiac and muscle disease. In the heart, these autosomal dominant LMNA mutations lead to cardiomyopathy frequently associated with cardiac conduction system disease. Herein, we describe a patient with the R374H missense variant in nesprin-1alpha, a protein that binds lamin A/C. This individual developed dilated cardiomyopathy requiring cardiac transplantation. Fibroblasts from this individual had increased expression of nesprin-1alpha and lamins A and C, indicating changes in the nuclear membrane complex. We characterized mice lacking the carboxy-terminus of nesprin-1 since this model expresses nesprin-1 without its carboxy-terminal KASH domain. These Delta/DeltaKASH mice have a normally assembled but dysfunctional nuclear membrane complex and provide a model for nesprin-1 mutations. We found that Delta/DeltaKASH mice develop cardiomyopathy with associated cardiac conduction system disease. Older mutant animals were found to have elongated P wave duration, elevated atrial and ventricular effective refractory periods indicating conduction defects in the myocardium, and reduced fractional shortening. Cardiomyocyte nuclei were found to be elongated with reduced heterochromatin in the Delta/DeltaKASH hearts. These findings mirror what has been described from lamin A/C gene mutations and reinforce the importance of an intact nuclear membrane complex for a normally functioning heart.

Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Nesprin-1 R374H in an individual with nonischemic dilated cardiomyopathy

(A) Schematic representation of the gene structure (top) and protein structure (bottom) of nesprin-1α, an isoform of nesprin-1 highly expressed in cardiac and skeletal muscle. The vertical lines indicate the positioning of nesprin-1α exons within the gene. A sequence variant (g1316a) was identified in a patient with dilated cardiomyopathy (red). The variant maps to a conserved region between a spectrin repeat and the LEM-like domain in the protein. Variants previously identified by Zhang and colleagues are shown in black [17]. (B) The R374H variant is conserved across vertebrate species.

Figure 2. Dilated cardiomyopathy with nesprin-1α R374H

(A) Sequence analysis from the proband's DNA revealed a heterozygous G to A change at nucleotide 1316 in nesprin-1α. The G1361A change is predicted to result in an amino acid change, R374H, in nesprin-1α. (B) Pedigree from proband carrying the R374H variant. The proband and his father were both affected (black). (C) Human fibroblasts from the nesprin-1α R374H patient and a control cell line (CTL) were analyzed by immunofluorescence microscopy using antibodies for nesprin-1 and lamin A/C. DAPI shows nuclei. Scale bar=10μm (D) Immunoblotting was performed on human fibroblasts from the nesprin-1α R374H and control (CTL) using antibodies to nesprin-1 and lamin A/C. Ponceau staining is shown as loading control. (E) Densitometry was performed. The ratio of LINC components nesprin1-α and lamins A and C were normalized to loading control and averaged over 3 experiments, p

Figure 3. Δ/ΔKASH mice have prolonged refractory times affecting the atrial and ventricles compared to wildtype littermates

(A) Representative surface ECG (top) and corresponding intracardiac electrophysiologic (bottom) tracing from Δ/ΔKASH mouse. The pacing stimulus is labeled S, the atrial response A and the ventricular response V. At 60ms, the Δ/ΔKASH heart has no atrial response to an early stimulus, shown by the asterisk. (B) Graphs of the AERP and VERP in young (52 weeks) Δ/ΔKASH mice and wildtype littermates. Both young and old Δ/ΔKASH mice have a longer AERP (p=0.01 and p=0.006, respectively) and a slightly longer VERP (p=0.06) than wildtype littermates. Black bars represent wildtype mice, gray bars represent Δ/ΔKASH mice. Number of mice in each study are listed below the corresponding bars. ms=milliseconds

Figure 4. Δ/ΔKASH mice have reduced fractional shortening compared to wildtype littermates

(A) Representative m-mode echocardiography from an old (>52 weeks) Δ/ΔKASH mouse and wildtype littermate showing reduced fractional shortening. (B) Graphs of fractional shortening in 52 week old (bottom) Δ/ΔKASH mice (gray bar) and wildtype littermates (black bar). Number of animals tested shown at bottom under bar.

Figure 5. Δ/ΔKASH mice have abnormally shaped cardiomyocyte nuclei compared to wildtype littermates

(A) Histological analysis of coronal sections of hearts from >52 week old Δ/ΔKASH mice and wildtype littermates. Hematoxylin and eosin stained wildtype sections have rounded nuclei, while Δ/ΔKASH sections have elongated cardiomyocyte nuclei. Insets with white arrows demonstrate the measurement of nuclear length. Scale bar: 10μm. (B) Quantification of nuclear elongation in 52 week old mice (bottom) Δ/ΔKASH (gray bar) and wildtype littermates (black bar). Δ/ΔKASH mice in both age groups have elongated cardiomyocyte nuclei (p=0.0001). Three animals each genotype, each age were counted, approximately 100 nuclei per animal.

Figure 6. The Δ/ΔKASH cardiomyocyte nucleus is abnormally shaped and has thinner heterochromatin layer

(A) Electron microscopy was performed on longitudinal sections of hearts from