(FHL1) in Signaling Pathways of Cardiac Hypertrophy and Disease

描述（由申请人提供）：我们已经鉴定并表征了一组新的仅具有四个半LIM结构域（FHL）的LIM蛋白，其在横纹肌中富集。FHL 1在各种心脏肥大模型中显著上调，在心力衰竭模型中下调，表明它在这些疾病中起重要作用。与此一致，我们已经产生了FHL 1缺陷小鼠，并有初步数据表明，FHL 1缺陷小鼠心脏显示出对压力超负荷后心脏肥大的迟钝反应，而不会引起任何血液动力学损害。FHL 1缺陷小鼠也可以完全挽救心肌病Gq转基因小鼠模型中心脏质量的增加。虽然其机制仍有待确定，但酵母双杂交筛选FHL 1伴侣的结果显示，其中一个候选伴侣是Raf 1，这是一种先前被其他人证明对压力超负荷诱导的心脏肥大和功能障碍至关重要的基因。FHL 1也被证明与Raf-1的下游靶标Erk 2相互作用。总之，这些结果使我们的假设，FHL 1是一种新的支架蛋白选择性介导的信号转导的Raf 1-Mek 1/2-Erk 1/2信号通路和必要的压力超负荷介导的心脏肥大。因此，具体目标是：1。使用酵母双杂交和GST下拉方法确定FHL 1及其伴侣Raf 1、MEK 1和ERK 2内的最小相互作用结构域。2.评估FHL 1缺乏或其与Raf 1、MEK 1/2或ERK 1/2相互作用中断对新生大鼠和成年小鼠心室肌细胞ERK 1/2磷酸化和/或肥大的影响。还将检查该途径中每个组分的亚细胞定位。3.确定FHL 1缺陷或其与RAF 1、MEK 1/2或ERK 1/2相互作用的破坏对长期压力超负荷和Gq过表达诱导的肥大的体内影响。将使用FHL 1缺陷小鼠和表达阻断FHL 1与Raf 1、MEK 1或ERK 2相互作用的肽的转基因小鼠。FHL 1信号通路的识别和理解可能最终使我们能够开发出知情的治疗方法，以消除人类患者心脏肥大的发展，并对心脏功能产生有益的结果。

DESCRIPTION (provided by applicant): We have identified and characterized a new group of LIM-only proteins with four and one half LIM domains (FHL), which are enriched in striated muscle. FHL1 is dramatically upregulated in various models of cardiac hypertrophy and down-regulated in failing heart, suggesting that it plays an important role in these disease settings. Consistent with this, we have generated FHL1 deficient mice and have preliminary data demonstrating that FHL1 deficient mouse hearts display a blunted response to cardiac hypertrophy following pressure overload, without evoking any hemodynamic compromise. FHL1 deficient mice can also completely rescue the increase in cardiac mass in the cardiomyopathic Gq transgenic mouse model. Although the mechanism remains to be determined, yeast two-hybrid screening for FHL1 partners has revealed that one candidate partner is Raf1, a gene previously shown by others to be essential for cardiac hypertrophy and dysfunction induced by pressure overload. FHL1 has also been shown to interact with Erk2, the downstream target of Raf-1. Altogether, these results have led us to the hypothesis that FHL1 is a novel scaffolding protein selectively mediating signaling by the Raf1-Mek1/2-Erk1/2 signaling pathway and essential for pressure-overload mediated cardiac hypertrophy. Accordingly, the Specific Aims are: 1. To define minimal interaction domains within FHL1 and its partners Raf1, MEK1, and ERK2 using yeast- two-hybrid and GST pull-down approaches. 2. To assess effects of FHL1 deficiency or disruption of its interactions with Raf1, MEK1/2, or ERK1/2 on ERK1/2 phosphorylation and/or hypertrophy in both neonatal rat and adult mouse ventricular myocytes. Subcellular localization of each component in this pathway will also be examined. 3. To determine in vivo effects of FHL1 deficiency or disruption of its interactions with RAF1, MEK1/2, or ERK1/2 on long-term pressure overload and Gq overexpression induced hypertrophy. Both FHL1 deficient mice and transgenic mice expressing peptides to block interactions of FHL1 with Raf1, MEK1, or ERK2 will be utilized. The identification and understanding of the FHL1 signaling pathway may ultimately allow us to develop informed therapies to abolish the development of cardiac hypertrophy in human patients with a beneficial outcome for cardiac function.