The Role of Cypher and ENH in heart

描述(由申请人提供)：Cypher及其紧密同系物ENH包含单个N-末端PDZ结构域以及三个C-末端LIM结构域。Cypher和ENH缺陷小鼠都表现出心肌病。人类Cypher/Zasp基因的许多突变在不同形式的心肌病患者中被发现。最近，在部分肌原纤维肌病患者中也发现了Cypher突变，称为Zasopathy。我们已经确定了Cypher和ENH的多种剪接异构体，包括短亚型和长亚型。N-末端PDZ结构域是短和长异构体共有的，而三个LIM结构域是长异构体所独有的。Cypher和ENH通过其PDZ结构域与Z线蛋白a-Actinin、Calsarcin-1和Myotilin的C末端相互作用。我们的初步数据还表明，Cypher长的异构体，而不是Cypher短的异构体，通过C端的LIM结构域与b1整合素相互作用。我们还表明，Cypher长亚型缺失(Cypher L-/-)的小鼠会发生心肌病并经历过早死亡，而Cypher短亚型缺失(Cyphers-/-)的小鼠表现正常。为了进一步了解Cypher和ENH在心脏中的潜在作用，我们正在通过建立和研究Cypher和ENH(ENH-/-；Cypher-/-)双零的小鼠来研究Cypher和ENH之间的相互作用。ENH-/-；Cypher-/-小鼠在胚胎10.5天左右死亡，心脏表型异常。虽然ENH-Null；Cypher杂合子复合突变体(ENH-/-；Cypher)在出生后仍能存活，但上述结果表明，这些突变体可能比单独的ENH-/-小鼠表现出更严重的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。上述结果使我们推测，Cypher通过与α-Actinin 2、Calsarcin、Myotilin和b1整合素的相互作用，在Z线和胞间体部都是一个关键的连接蛋白。此外，我们认为Cypher和ENH在心脏发育和功能中具有多余但独特的作用。这项建议的总体目标是了解Cypher异构体及其同系物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：在小鼠体内消融Cypher或ENH会导致心肌病。人类Cypher基因的许多突变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理解Cypher及其同系物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.