A Cypher/ZASP mutation associated with dilated cardiomyopathy alters the binding affinity to protein kinase C.

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 disease-causing 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 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).
Myocardial function is associated with the regulation or activation of cell signal kinases such as protein kinase C (PKC). For example, modification of myocardial proteins by PKC plays a key role in the regulation of contractility and the growth of cardiomyocytes (21), whereas alterations in the expression, activity, or localization of PKC are associated with cardiac hypertrophy and failure (22,23). Recently, a gene for the PDZ and LIM domain-containing cytoskeletal protein, Cypher/ZASP, was identified in mouse (Cypher) (24) and human (ZASP) (25). Cypher is also called Oracle (26). Cypher/ZASP is expressed in the striated muscles and encodes several isoforms (24 -27). The PDZ domain at the N terminus is required for binding to ␣-actinin in the Z-disc (25), and the LIM domains at the C terminus are involved in the binding to PKCs (24). In addition, Cypher/ZASP knockout mice display disorganized and fragmented Z-discs in both skeletal and cardiac muscles, leading to a severe form of congenital myopathy and cardiomyopathy (27,28). Moreover, human Cypher/ZASP is mapped on chromosome 10q22.3-q23.2 (University of California at Santa Cruz Human Genome Browser, July 2003; chromosome 10, 88093025-88159329) (25), overlapping with a DCM locus (16).
We report here a Cypher/ZASP gene mutation, associated with late-onset familial DCM, that increases the LIM-PKC interaction. This is the first report suggesting that an abnormality in the anchoring-protein of PKC may play an important role in the pathogenesis of DCM. Subjects-Patients and family members were evaluated as described previously (12). Thirty-four proband patients with familial DCM and 62 sporadic DCM cases were the subjects. These patients had been examined before and had no mutation in the genes for dystrophin, ␣-cardiac actin, desmin, lamin A/C, ␤-cardiac myosin heavy chain, cardiac troponin T, ␣-tropomyosin, vinculin, MLP, Tcap/ telethonin, and titin. When a sequence variation in Cypher/ZASP was found, family relatives and 400 unrelated healthy controls were examined. Blood samples were collected after obtaining informed consent from the subjects. All patients and controls were Japanese. The protocol for research on human materials was approved by the Ethics Reviewing Committee of the Medical Research Institute, Tokyo Medical and Dental University.
Genomic DNA Extraction and PCR-SSCP Analysis-DNA was extracted from peripheral blood leukocytes from each subject. Extracted DNA was subjected to PCR by using primer pairs specific to the analyzed regions. Sequences of primers are available upon request. The PCR products from the subjects were searched for sequence variations by the PCR-SSCP method (12). When an abnormal SSCP pattern was obtained, the PCR fragment was sequenced on both strands. To confirm the D626N mutation in exon 15, TspEI was used for detection of the mutation, and the digestion products were electrophoresed in a 2% agarose gel.
Plasmid Construction and Immunoprecipitation from 293 Cells-Expression vectors for PKC-␣, -␤, and -⑀ and wild type Cypher1 have been described previously (24). The mutant Cypher1 was constructed by PCR-based mutagenesis and sequenced to ensure that no PCR errors were introduced. Plasmids containing wild type, mutant Cypher, and distinct PKCs (5 g each) were transfected into 293 cells, which were plated in 10-cm dishes at 80% confluence via Superfect (Qiagen Inc.). Cells were harvested, and 100 g of total protein from PKC-transfected cells was mixed with 100 g of total protein from either wild type or mutant Cypher-transfected cells to perform immunoprecipitation assay as described (24).

Expression of Cypher/ZASP in Human
Tissues-Several different cDNA isoforms of Cypher/ZASP were reported in human and mouse striated muscle (24 -26). These isoforms are generated by alternative splicing of a single gene (Fig. 1A, Ensembl gene identification number ENSG00000122367). To investigate the alternative splice pattern in various human tissues, we performed RT-PCR analysis. As shown in Fig. 1B, human Cypher/ZASP expressed several transcripts. There were two different PCR products from exon 1 to 9; a short form (Cypher 2s; nomenclature is according to Ref. 27) was preferentially expressed in the skeletal muscle, whereas a long form (Cypher 2c) was abundant in the fetal heart. On the other hand, transcripts covering exons 4 -7 were expressed preferentially in the fetal heart. Two different transcripts spanning exons 6 -11 were generated; the longer form (corresponding to Cypher 1s) was expressed in the heart. Several other combinations of primers were used in addition to investigate the alternative splicing (for example, exons 4 -11; data not shown). The RT-PCR products were sequenced to confirm that there were at least six different splice variants in human (Fig. 1C) as was reported in mouse (27). On the other hand, ZASP variant 2, which was reported to have a deletion of 31 base pairs (25), presumably as a result of a splice to a minor acceptor site in exon 11, could not be identified in this study with any combinations of primers (data not shown).
Identification of a Missense Mutation in the Cypher/ZASP Gene in a DCM Family-Sequence variations in the Cypher/ ZASP gene were searched in 96 patients with DCM, and five different variations were identified (Fig. 1A). These include a T to C transition in an intron (Ϫ13 in intron 6) and four variations in the exons (GTC to ATC at codon 55 in exon 2, GTC to ATC at codon 588 in exon 14, GAT to AAT at codon 626 in exon15, and CAT to CAC at codon 644 in exon 15; codon numbers are from Cypher/ZASP 1c in Fig. 1C). Among the variations leading to amino acid replacement, V55I and V588I were polymorphisms because they were also found in unrelated healthy controls. In contrast, the D626N variation identified in a proband patient of familial DCM (designated II-6; Fig. 2, A and C) was not found in 400 unrelated healthy controls. A family study showed that the D626N mutation was present in all affected members tested (Fig. 2, B and D). The mutation was located at the fifth position next to a constant cystein (29) in the third LIM domain, and this position was occupied exclusively by acidic residues in Cypher/ZASP and other PDZ-LIM proteins and in Enigma and the Enigma homologue protein (ENH) from various species (Fig. 2E).
The patients in this family developed DCM after age 50 (in the early (II-1 and II-9) or late (II-5 and II-6) fifties in male cases and at age 69 in a female case (II-2)), suggesting that the mutation was associated with late-onset DCM ( Fig. 2A). Electrocardiogram findings of the affected individuals demonstrated no primary conduction defect. It was interesting to note that a sister (II-4) had the mutation but did not suffer from DCM, although she had been affected with cerebellar ataxia (CA). The CA was initially considered to be as a clinical conse-quence of her carrying the mutation; however, this possibility was ruled out because another brother (II-3) who also suffered from CA did not have the mutation. No sign of skeletal muscle involvement was noted in the DCM patients, although a muscle biopsy could not be performed because consent was not obtained.
On the other hand, the Y2H assays showed that the ␤-gal activity in the colonies of Cypher-MB and PKCA-P was significantly higher than that of Cypher-CB and PKCA-P (1.072 Ϯ 0.108 versus 0.747 Ϯ 0.094, p Ͻ 0.05). In addition, the ␤-gal activity obtained from the mutant LIM and PKC-⑀ interaction was significantly higher than that of the normal LIM and PKC-⑀ interaction (0.872 Ϯ 0.054 versus 0.562 Ϯ 0.036, p Ͻ 0.001). Similarly, the ␤-gal activity for the mutant LIM-PKCinteraction was significantly higher than that for the normal LIM-PKC-interaction (0.554 Ϯ 0.026 versus 0.325 Ϯ 0.010, p Ͻ 0.001) (Fig. 3B).
We investigated, through an independent approach, whether the mutation would affect the LIM-PKC interaction. In the pull-down experiments, a mutation equivalent to human D626N was introduced into mouse Cypher/ZASP (24). Western blot analysis of immunoprecipitates of wild type or mutant Cypher with HA-tagged PKCs (PKC-␣, PKC-␤, and PKC-⑀) revealed that, despite equal expression of genes, mutant Cypher had a higher affinity to the PKCs than the normal Cypher (1.96 Ϯ 0.03-fold, p Ͻ 0.01 for PKC-␣; 1.38 Ϯ 0.12-fold, p Ͻ 0.02 for PKC-␤; and 1.50 Ϯ 0.29-fold, p Ͻ 0.04 for PKC-⑀) (Fig. 3, C-E). DISCUSSION In this study, a Cypher/ZASP mutation in the third LIM domain was found in a DCM family, and the mutation altered the function of the LIM domain (i.e. a decrease in dimeric binding while the binding to PKCs was increased). All affected members had the mutation, but one female carrying the mutation did not suffer from DCM (II-4, 65 years old). She might develop DCM later in life, because the DCM phenotype was  1 and 4), PKC-␤ (lanes 2 and 5), or PKC-⑀ (lanes 3 and 6) were mixed with protein extracts from cells transfected with DNA containing either wild type (lanes 1-3) or mutant Cypher1c (lanes 4 -6), and an immune precipitation (IP) assay was performed. Substantially greater amounts of mutant Cypher1c than of wild type Cypher1c were detected in anti-HA-tagged immunoprecipitates with anti-Cypher antibody. D, substantially greater amounts of PKCs were detected in anti-Cypher immune precipitation with anti-HA antibody with mutant Cypher (lanes 4 -6) than wild type Cypher (lanes 1-3). E, Western blot with anti-Cypher antibody showed that comparable amounts of wild type (lane 1) and mutant (lane 2) Cypher were utilized for the immune precipitation experiments. late-onset and the eldest sister (II-2) developed the disease at the age of 69. Another reason why II-4 may not develop DCM might also be because she could not exercise for several years due to CA, since DCM due to the Z-disc abnormality can be exacerbated by cardiac stress, as demonstrated in MLP-deficient mice (13).
The Cypher/ZASP isoforms have a PDZ domain and three LIM domains. These structures are highly homologous to other PDZ-LIM proteins such as Enigma and ENH (30,31). The ability of Cypher/ZASP isoforms to bind to ␣-actinin through the PDZ domain and various PKC subtypes via the LIM domains in the Z-discs of the cardiac muscle imply that Cypher/ ZASP might play a role in stretch response. The LIM domain consists of 50 -60 amino acid residues and participates in protein-protein interactions (29). The mutant LIM showed increased binding affinity to PKCs in both Y2H assays and pulldown assays, although the extent of the increased affinity was different between these assays. This could be because one human LIM domain was used in the Y2H assays, and three mouse LIM domains were used for the pull-down assays. Nevertheless, our results demonstrated that the interaction of the Cypher/ZASP LIM domain to PKC, especially the ⑀ subtype, was augmented by the mutation.
PKCs localize in nucleus, perinucleus, cytosol, and Z-discs in the cardiomyocytes (32,33). Upon activation by lipid-derived second messengers, PKCs are known to translocate from one cell component to another via the function of anchoring proteins, and the proteins anchoring the inactivated PKCs are referred to as RICKs (receptors for inactivated protein kinase C), whereas those anchoring the activated PKCs are referred to as RACKs (receptors for activated protein kinase C), and each PKC isozyme associates with specific anchoring proteins to mediate isozyme-specific PKC functions (34). We have shown here, as reported previously (24), that Cypher/ZASP interacts with PKCs. Although Cypher/ZASP is not a bona fide RICK or RACK, because it can bind multiple PKC isozymes it will be of interest to determine whether Cypher/ZASP has RICK-or RACK-like functions. On the other hand, it has been shown that the expression of PKC-⑀ and its translocation from a cytosol fraction to a membrane fraction is increased during the transition from compensated hypertrophy to congestive heart failure (23). Several studies have demonstrated that the Nterminal part of PKC-⑀ interacts selectively with a specific isotype of RACKs, RACK2 (32,33), and it was reported that PKC-⑀⅐RACK2 interactions played a key role in cardioprotection against ischemic stress (35,36). In contrast, disruption of PKC-⑀⅐RACK2 interaction has been shown to inhibit cardiac cell contraction (37) and accelerate cell death (38). It is likely that the translocation of PKC-⑀ in the heart may be involved in myocardial remodeling and ultimately lead to heart failure. Because the N-terminal part of PKC-⑀ interacts with both Cypher/ZASP and RACK2, an interesting hypothesis may be that the augmentation of binding affinity between Cypher/ ZASP and PKC-⑀ by the Cypher/ZASP mutation might, in turn, reduce the amount of a PKC-⑀⅐RACK2 complex and cause early progression to heart failure. This hypothesis should be tested in future studies.
In summary, we identified a Cypher/ZASP mutation associated with DCM that increased the LIM-PKCs interaction. These observations suggest an association between DCM and the inherited abnormality involved in signal transduction, which is consistent with the findings of mutations in phospholamban and MLP. Although the molecular mechanisms of DCM due to the Cypher/ZASP mutation remain to be elucidated, and other DCM patients should be screened for Cypher/ZASP mutation to formally confirm the causality of the mutation in DCM, our observations imply that the cardiac dysfunction might be associated not only with the alteration in each sarcomeric interaction but also with the altered recruitment of molecules participating in intracellular signaling.