A Cypher/ZASP Mutation Associated with Dilated Cardiomyopathy Alters the Binding Affinity to Protein Kinase C

Abstract

Dilated cardiomyopathy is characterized by ventricular dilation with systolic dysfunction of cardiac muscle. Recent genetic studies have revealed that mutations in genes for cytoskeleton proteins distributed in the Z-disc and/or intercalated discs of the cardiac muscle are major predictors of cardiomyopathy. However, as mutations in these genes can account for only a part of the patient population, there should be another diseasecausing gene(s) for cardiomyopathy. Cypher/ZASP appears to be an ideal candidate for the cardiomyopathy causative gene, because Cypher/ZASP encodes a Z-disc associated protein, and recent studies have demonstrated that Cypher/ZASP knock-out mice develop cardiomyopathy. In this study, we searched for sequence variations in Cypher/ZASP in 96 unrelated Japanese patients with dilated cardiomyopathy. A D626N mutation located within the third LIM domain was identified in a familial case but not found in the unrelated controls. A family study of the patient showed that all affected siblings tested had the same mutation. Clinical information of the affected family members suggested that the mutation was associated with late onset cardiomyopathy. To reveal the biochemical changes due to the mutation, we performed a yeast two-hybrid assay and a pull-down assay. It was demonstrated by both assays that the D626N mutation of Cypher/ZASP increased the affinity of the LIM domain for protein kinase C, suggesting a novel biochemical mechanism of the pathogenesis of dilated cardiomyopathy. Dilated cardiomyopathy (DCM)1 is characterized by ventricular dilation accompanied by systolic dysfunction in the absence of known causes that affect the cardiac function. Family studies of DCM patients have demonstrated that 20 -35% of DCM is caused by inherited gene mutations (familial DCM) (1). Although familial DCM can be transmitted as autosomal recessive, X-linked, or through mitochondrial traits, it is evident that autosomal dominant inheritance occurs the most (2, 3). Missense mutations in genes for cardiac ␣-actin, desmin, lamin A/C, ␦-sarcoglycan, ␤-cardiac myosin heavy chain, cardiac troponin T, ␣-tropomyosin, metavinculin, titin, muscle LIM protein (MLP), Tcap/telethonin, and phospholamban are all associated with autosomal dominant DCM (4 -15). However, mutations in the known causative genes can be found only in a part of the patient population. In addition, linkage studies in multiplex families have suggested that there are several other disease loci of which the gene responsible for causing DCM remains to be identified, including one mapped on 10q21-q23 (16).Abnormalities in Z-disc-related cytoskeletal proteins are associated with DCM also in animal models. For example, mice lacking MLP display many characteristic features of human DCM (17). Similarly, loss of ␦-sarcoglycan results in DCM in a hamster model (18), and a mouse knock-out for the actininassociated LIM protein develops DCM (19). These observations suggest that abnormalities in cytoskeletal proteins expressed in cardiac muscle cause DCM (20).Myo...

Clinical, pathological, and genetic mutation analysis of sporadic inclusion body myositis in Japanese people

“…Because of splice variants, not all exons are transcribed in cardiac and skeletal muscles, and 3 transcripts appear in skeletal muscle [16]. Exon 15 is present only in the 2 long transcripts; the mutation in exon 15 (D626N) increases the affinity of the LIM domain for protein kinase C [27]. The novel mutation described here is found in a highly evolutionarily conserved domain in exon 15.…”

“Z”eroing in on the Role of Cypher in Striated Muscle Function, Signaling, and Human Disease

“…Cypher mutations were first identified in patients with human familial and sporadic dilated cardiomyopathy (Arimura et al 2004, Vatta et al 2003, a disease that has been linked to the disruption of the cytoskeletal architecture (Vatta et al 2003). Mutations were identified in several domains within Cypher/ZASP, and found to be associated with a spectrum of different diseases.…”

“…A mutation in exon 2 (V55I), which is common to all isoforms, was independently shown to be associated with INLVM in a Japanese population (Xing et al 2006, Figure 2). Other independent studies identified Cypher/ZASP mutations (D626N) in a long isoform-specific exon (exon 15; within the third LIM domain) in Japanese patients exhibiting familial DCM and INLVM (Arimura et al 2004, Xing et al 2006, Figure 2). (Arimura et al 2004).…”

“…The binding of Cypher long isoforms to PKC suggests that Cypher may have RICK or RACK-like functions and play a signaling role. Interestingly, a D626N mutation identified in Japanese DCM patients is located in exon 15, which encodes the third LIM domain (Arimura et al 2004 and also see below). It has been found that the mutant Cypher protein has an increased affinity to PKC (Arimura et al 2004), further suggesting that Cypher plays a signaling role in the heart.…”

Molecular mechanisms of mechanosensing in muscle development

“…1). A mutation in the third LIM domain of Cypher causes dilated cardiomyopathy and increases Cypher affinity for PKC compared with the wild type protein, demonstrating the importance of PKC recruitment in vivo (Arimura et al, 2004). Apart from Enigma family proteins, other Z-disk-associated proteins were identified as potential anchors of PKC.…”