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# Actin Mutations in Dilated Cardiomyopathy, a Heritable Form of Heart Failure

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Science  01 May 1998:
Vol. 280, Issue 5364, pp. 750-752
DOI: 10.1126/science.280.5364.750

## Abstract

To test the hypothesis that actin dysfunction leads to heart failure, patients with hereditary idiopathic dilated cardiomyopathy (IDC) were examined for mutations in the cardiac actin gene (ACTC). Missense mutations in ACTC that cosegregate with IDC were identified in two unrelated families. Both mutations affect universally conserved amino acids in domains of actin that attach to Z bands and intercalated discs. Coupled with previous data showing that dystrophin mutations also cause dilated cardiomyopathy, these results raise the possibility that defective transmission of force in cardiac myocytes is a mechanism underlying heart failure.

Heart failure is a major medical problem that affects 700,000 individuals per year in the United States and accounts for annual costs of $10 to$40 billion (1). Heart failure is the primary manifestation of dilated cardiomyopathy, a group of disorders characterized by cardiac dilation and pump dysfunction. Half of patients with dilated cardiomyopathy are diagnosed with idiopathic dilated cardiomyopathy (IDC), isolated heart failure of unknown etiology (affecting 5 to 8 in 100,000 individuals) (2). Cardiac transplantation is the only definitive treatment for end-stage disease.

IDC is hereditary in at least 20% of cases (3), indicating that genetic factors are important in its pathogenesis. In both familial and nonfamilial IDC, disease onset is delayed (mean age at diagnosis = 45 ± 17 years), and the 5-year mortality rate is 50% after symptoms develop (3, 4). Consequently, few multigeneration IDC families with many affected, living individuals have been identified. Although chromosomal loci for IDC (1p1-q1, 1q32, 3p22-p25, 9q13-q22, and 10q21-q23) have been identified by genetic linkage analysis in rare families (5), these families are too small for positional cloning of IDC genes. Furthermore, these loci do not identify all potential candidate genes, like cardiac actin (ACTC) on chromosome 15q14. As an alternative strategy, we used a candidate gene approach in small IDC families.

We studied two unrelated families with autosomal dominant IDC, one of German ancestry and the other of Swedish-Norwegian ancestry (Fig.1). Families were phenotypically characterized by echocardiography (3, 6). IDC was defined as left ventricular (LV) end-diastolic dimension >95th percentile for age and body surface area, and shortening fraction < 28% (7). The results of phenotypic evaluation are shown in Table 1. Individuals in both families had variable age at diagnosis (1 to 41 years), similar to other IDC families, with age at diagnosis differing by as much as 20 to 50 years (5, 8). Heart biopsy specimens from the proband of each family revealed histopathologic findings consistent with IDC (Fig. 2). Neither family had phenotypic features of hypertrophic cardiomyopathy (9).

Table 1

Phenotypic data for IDC families with missense mutations in ACTC. The 95th percentiles for LV end-diastolic dimension, based on body surface area and age, are indicated in parentheses. Normal shortening fraction is ≥28%. Abnormal values are indicated in bold type. Individuals <20 years of age with normal values were classified as uncertain, on the basis of ∼5 to 10% disease penetrance in this age group (26). Phenotypic data were obtained before DNA analyses. BSA, body surface area; LV, left ventricle.

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Actin is essential for normal structure and function of cardiac myocytes. During development, five of the six actin isoforms encoded by separate genes are expressed in myocytes. In mature cardiac myocytes, however, only cardiac and skeletal actin are expressed, and cardiac actin is the major isoform (∼80%) (10, 11). To test the hypothesis that actin dysfunction leads to heart failure, we investigated the cardiac actin gene (ACTC) on chromosome 15q14 as a candidate for IDC. Oligonucleotide primers complementary to flanking intron sequence were developed for the six exons ofACTC (12), and single-strand conformation polymorphism (SSCP) analyses (13) were performed.

SSCP analyses of ACTC in kindred 1453 (K-1453) identified an anomalous conformer for exon 5 that cosegregated with IDC (14). Sequencing (15) of the conformer revealed a G-to-A substitution in codon 312 (Arg312His) (Fig. 1A). We confirmed this alteration by testing for a new Bcl I restriction site. It was inherited by three individuals with IDC (ages 36, 5, and 2) and a 15-year-old who has not developed IDC.

Analysis of kindred 9695 (K-9695) revealed an anomalous conformer forACTC exon 6 that cosegregated with IDC (Fig. 1B). DNA sequence analysis demonstrated an A-to-G substitution in codon 361 (Glu361Gly). This alteration was inherited by two individuals with IDC (ages 41 and 14) in addition to a 34-year-old with a dilated heart and a 9-year-old with borderline heart size.

To eliminate the possibility that these substitutions were polymorphisms within the normal population, we tested 435 unrelated control individuals (870 chromosomes). No anomalous SSCP conformers were identified for ACTC exons 5 and 6 in these controls. In addition, sequence comparisons revealed that both substitutions affect amino acids that are invariant in all human actin isoforms and actin in mice, Drosophila, yeast, and rice (14,16). Arg312His and Glu361Gly substitutions have not been evaluated in functional studies of mutant actin. However, an Arg312Ala substitution causes reduced viability of haploid yeast (17). Thus, the ACTC variants described here are likely to be IDC-associated mutations rather than rare polymorphisms.

ACTC is one of six actin genes in humans (18), none of which thus far have been implicated in human disease. In cardiac myocytes, cardiac actin is the main component of the thin filament of the sarcomere. One end of the polarized actin filament forms cross-bridges with myosin, and the other end is immobilized, attached to a Z band or an intercalated disc (11,19). Thus, actin transmits force between adjacent sarcomeres and neighboring myocytes to effect coordinated contraction of the heart. The mutations we identified occur in subdomains 1 and 3 of the actin monomer (Fig. 3), which form the immobilized end of the actin filament. Moreover, the Glu361Gly substitution is within a common binding domain for actinin, a protein comprising Z bands and intercalated discs, and dystrophin, a protein linking myofibrils to the extracellular matrix (20).

In addition to our data, several lines of evidence support the hypothesis that relatively subtle molecular defects in force-transmitting proteins, like actin, lead to myocyte dysfunction and heart failure. First, missense mutations throughout the actin gene in Drosophila result in abnormal structure and function of flight muscle (17). Second, transgenic expression of a noncardiac actin in cardiac actin–deficient mice causes heart enlargement and dysfunction, resembling human IDC (21). Third, missense mutations in dystrophin have been identified in X-linked dilated cardiomyopathy (22). In mice, heterozygous disruption of ACTC is not associated with heart abnormalities (21). Thus, the missense mutations inACTC defined here likely lead to altered actin function rather than loss of function.

Hypertrophic cardiomyopathy (HCM) is characterized by hypertrophy of the heart, in contrast to IDC, which leads to chamber dilation and heart failure. The genes implicated in HCM all encode proteins involved in generation of force (β-myosin heavy chain, cardiac troponin T, α-tropomyosin, myosin-binding protein C, and essential and regulatory myosin light chains) (23). This has led to the hypothesis that HCM is caused by chronic reduction of force generation, which stimulates secondary myocyte hypertrophy (24). The cellular mechanism underlying IDC, however, may not involve force generation. Actin provides a scaffold for force generation by interacting with myosin, but the mutations we identified are not in regions that interact with myosin (25). Instead of generating force, actin transmits force to adjacent sarcomeres and myocytes and, like dystrophin, transmits force to the extracellular matrix. Further evidence that IDC does not result from a primary defect in force generation is that pathologic features of HCM are not observed during the course of IDC. We propose that IDC results from an episodic defect in force transmission. This defect may predispose affected myocytes to mechanical injury and cumulative cell death, secondary interstitial fibrosis, and cardiac dilation, a degenerative process that may take decades to develop.

• * To whom correspondence should be addressed. E-mail: timo{at}howard.genetics.utah.edu