The Role of Cypher and ENH in heart

描述（由申请人提供）：Cypher及其近同源物ENH含有单个N-末端PDZ结构域以及三个C-末端LIM结构域。Cypher和ENH缺陷小鼠均显示心肌病。在不同形式的心肌病患者中，已经发现了人类Cypher/Zasp基因的许多突变。最近，Cypher突变也被发现在一部分患者的肌原纤维肌病，称为“Zaspopathy”。我们已经表征了Cypher和ENH的多种剪接亚型，包括短亚型和长亚型。N-末端PDZ结构域是短和长同种型共同的，而三个LIM结构域是长同种型所特有的。Cypher和ENH通过它们的PDZ结构域与Z线蛋白α-辅肌动蛋白、钙肌肽-1和Myotilin的C末端相互作用。我们的初步数据还表明，Cypher长亚型，但不是Cypher短亚型，通过C-末端LIM结构域与b1整联蛋白相互作用。我们还表明，Cypher长亚型（CypherL-/-）无效的小鼠发生心肌病并经历过早死亡，而Cypher短亚型（CypherS-/-）无效的小鼠表现出表型正常。为了进一步了解Cypher和ENH在心脏中的潜在作用，我们正在通过产生和研究Cypher和ENH双重无效（ENH-/-;Cypher-/-）的小鼠来研究Cypher和ENH之间的相互作用。ENH-/-;Cypher-/-小鼠在胚胎第10.5天左右死亡，具有异常心脏表型。虽然ENH-null; Cypher杂合复合突变体（ENH-/-;Cypher）在出生后存活，上述结果表明这些突变体可能表现出比单独的ENH-/-小鼠更严重的DCM。此外，虽然我们已经发现ENH-/-;CypherL-/-突变体在出生前死亡，但ENH-/-;CypherS-/-突变体在出生后存活。上述结果使我们假设Cypher是Z线和肋节处的关键接头蛋白，通过其与α-辅肌动蛋白2、钙肌肽、肌肽蛋白和β 1整联蛋白的相互作用。此外，我们认为Cypher和ENH在心脏发育和功能中具有冗余但独特的作用。该提案的总体目标是了解Cypher亚型及其同源物ENH在心脏发育和功能中的作用，并深入了解Cypher突变导致心肌病的机制。 公共卫生相关性：在小鼠中消融Cypher或ENH会导致心肌病。人类Cypher基因的许多突变已在不同形式的心肌病患者中被发现。我们提出的研究将有助于了解Cypher及其同源物ENH在心脏发育和功能中的作用，并深入了解Cypher突变导致心肌病的机制。

DESCRIPTION (provided by applicant): Cypher and its close homologue, ENH contain a single N-terminal PDZ domain as well as three C-terminal LIM domains. Both Cypher and ENH deficient mice display cardiomyopathy. Many mutations in the human Cypher/Zasp gene have been indentified in patients with different forms of cardiomyopathy. Recently, Cypher mutations were also found in a portion of patients with myofibrillar myopathy, termed "Zaspopathy". We have characterized multiple splice isoforms of Cypher and ENH, which include short and long subtypes. An N-terminal PDZ domain is common to both short and long isoforms, whereas three LIM domains are unique to long isoforms. Cypher and ENH interact with the C-termini of Z-line proteins a-actinin, Calsarcin-1 and Myotilin, through their PDZ domains. Our preliminary data also show that Cypher long isoforms, but not Cypher short isoforms, interact with b1 integrin via the C-terminal LIM domains. We have also shown that mice null for Cypher long isoforms (CypherL-/-) develop cardiomyopathy and experience premature mortality, while mice null for Cypher short isoforms (CypherS-/-) appear phenotypically normal. To further understand potential roles of Cypher and ENH in heart, we are studying interactions between Cypher and ENH by generating and studying mice which are doubly null for Cypher and ENH (ENH-/-;Cypher-/-). ENH-/-;Cypher-/- mice die around embryonic day (E) 10.5 with an abnormal cardiac phenotype. Although ENH-null;Cypher heterozygous compound mutants (ENH-/-;Cypher) survive postnatally, the foregoing results suggest that these mutants are likely to exhibit a more severe DCM than ENH-/- mice alone. Also, while we have found that ENH-/-;CypherL-/- mutants die before birth, ENH-/-;CypherS-/- mutants survive postnatally. The foregoing results have led us to the hypothesis that Cypher is a critical linker protein both at the Z- line and at the costamere, via its interactions with a-actinin 2, Calsarcin, Myotilin, and b1 integrin. Furthermore, we suggest that Cypher and ENH have redundant, yet unique, roles in cardiac development and function. The overall goals of this proposal are to understand the role of Cypher isoforms and its homologue, ENH, in cardiac development and function and to gain insight into mechanisms by which mutations in Cypher causes cardiomyopathy. PUBLIC HEALTH RELEVANCE: Ablation of Cypher or ENH in mice results in cardiomyopathy. Many mutations in the human Cypher gene have been identified in patients with different forms of cardiomyopathy. Our proposed studies will help to understand the role of Cypher and its homologue, ENH, in cardiac development and function and to gain insight into mechanisms by which mutations in Cypher cause cardiomyopathy.