Role of the F-Bar Protein CIP4 in Cardiac Hypertrophy

 描述(申请人提供)：心脏对慢性应激的反应包括激活信号转导网络，诱导心肌细胞肥大，在疾病中促进心肌细胞凋亡和间质纤维化，从而促进心力衰竭的发展。磷酸酶钙调神经磷酸酶(CaN)在这些过程中起着核心作用。在心肌细胞中存在两种不同的CaN催化亚基，Aα和Aβ，然而心肌细胞肥大只需要Aβ。由于CaN异构体具有相似的底物特异性并共享共同的结合伙伴，因此目前尚不清楚为什么同工酶之间会有功能上的差异。我们最近对CANAβ特异性结合伙伴进行了筛选。CIP4是一种与CANA特异结合的蛋白质，它是膜相关蛋白F-bar家族的成员。除了我们最近发表的文章显示CIP4是培养的新生儿心肌细胞肥大所必需的，F-bar蛋白在心脏中还没有被研究过。在这一应用中，我们检验了一种假设，即作为CANAβ支架，CIP4有助于调节心肌细胞肥大。我们认为，CIP4结合使CANAβ与上游受体、离子通道和/或其他信号分子共定位，从而特异性地赋予CANAβ在病理性心脏重塑中的功能。具体目标1：在活体心脏重塑中对CIP4的要求。根据我们的体外研究结果，我们建议对一种新的有条件的、心脏特异的CIP4基因敲除小鼠的心脏表型进行表征。这些小鼠将在无应激和慢性横状主动脉收缩、β肾上腺素能和血管紧张素II受体刺激的情况下进行研究。我们预计，CIP4基因敲除的小鼠在无应激时将具有相对正常的心功能，并将受到保护，不受压力超负荷和神经内分泌刺激引起的心脏重构的影响。特定目标2：心肌细胞中CIP4-CANA信号的潜在机制。使用新型FRET生物传感器检测CIP4复合体上的钙离子和CaN活性，我们建议研究CIP4结合的CANAβ在培养的成人和新生心肌细胞中的调节。我们将研究CIP4和CANAβ在心肌细胞中的共定位，并测试是否像在其他类型的细胞中一样，CIP4的定位可能受到生长因子和G蛋白偶联受体刺激以及Rho家族GTP酶的调节。此外，我们还将确定受CaN效应基因NFATc和MEF2调控的基因表达是否需要在体内表达CIP4。具体目标3：体内锚定CANAβ的治疗中断。这一应用的中心假设是CANAβ在病理性心肌细胞肥大中的独特作用是由于CANAβ通过特定的支架锚定。我们建议有选择地在心肌细胞中表达CANAβ锚定干扰肽，以测试CANAβ锚定的中断是否可以防止长期压力超负荷和儿茶酚胺输注儿茶酚胺所致的病理重塑。

 DESCRIPTION (provided by applicant): The cardiac response to chronic stress involves the activation of a signal transduction network that induces myocyte hypertrophy and that in disease promotes myocardial apoptosis and interstitial fibrosis contributing to the development of heart failure. The phosphatase calcineurin (CaN) plays a central role in these processes. In cardiac myocytes there are two different isoforms of the CaN catalytic subunit, Aα and Aβ, and yet only Aβ is required for myocyte hypertrophy. Because CaN isoforms have similar substrate specificity and share common binding partners, it is unclear why there would be functional differences between the isoenzymes. We recently performed a screen for CaNAβ-specific binding partners. One protein that bound CaNA specifically is CIP4, a member of the F-BAR family of membrane-associated proteins. With the exception of our recent publication showing that CIP4 is required for the hypertrophy of cultured neonatal ventricular myocytes, F-BAR proteins have not been studied in the heart. In this application, we test the hypothesis that by serving as a CaNAβ scaffold, CIP4 contributes to the regulation of cardiac myocyte hypertrophy. We propose that CIP4 binding co-localizes CaNAβ with upstream receptors, ion channels and/or other signaling molecules that specifically confer CaNAβ function in pathologic cardiac remodeling. Specific Aim 1: The Requirement for CIP4 in Cardiac Remodeling In Vivo. In light of our in vitro findings, we propose to characterize the cardiac phenotype of a new conditional, cardiac-specific CIP4 knock-out mouse. The mice will be studied both when unstressed and when subject to chronic transverse aortic constriction, β-adrenergic, and angiotensin II receptor stimulation. We anticipate that the CIP4 knock-out mouse will have relatively normal cardiac function when unstressed and will be protected from the cardiac remodeling induced by pressure overload and neuroendocrine stimulation. Specific Aim 2: Mechanisms underlying CIP4-CaNA signaling in myocytes. Using novel FRET biosensors that will detect Ca2+ and CaN activity at CIP4 complexes, we propose to study the regulation of CIP4-bound CaNAβ in cultured adult and neonatal myocytes. We will study the co-localization of CIP4 and CaNAβ in the myocyte and test whether, as in other cell types, CIP4 localization may be regulated by growth factor and G-protein coupled receptor stimulation and by Rho family GTPases. In addition, we will determine whether gene expression regulated by the CaN effectors NFATc and MEF2 requires CIP4 expression in vivo. Specific Aim 3: Therapeutic Disruption of CaNAβ Anchoring In Vivo. The central hypothesis of this application is that the unique role of CaNAβ in pathological myocyte hypertrophy is due to CaNAβ anchoring by specific scaffolds. We propose to express selectively in the cardiac myocyte anchoring disrupter peptides for CaNAβ in order to test whether disruption of CaNAβ anchoring can prevent pathological remodeling in response to long-term pressure overload and catecholamine infusion.