(FHL1) in Signaling Pathways of Cardiac Hypertrophy

描述（由申请人提供）：我们已经鉴定并表征了一组新的LIM-only蛋白，具有四个半LIM结构域（FHL），富集于横纹肌。FHL1在各种心脏肥厚模型中显著上调，在心力衰竭模型中下调，表明它在这些疾病中发挥重要作用。与此一致，我们产生了FHL1缺陷小鼠，并有初步数据表明，FHL1缺陷小鼠的心脏在压力过载后对心脏肥厚表现出迟钝的反应，而不会引起任何血流动力学损害。FHL1缺陷小鼠也能完全挽救Gq转基因心肌病小鼠模型中心脏质量的增加。虽然机制仍有待确定，酵母双杂交筛选FHL1伴侣已经揭示了一个候选伴侣是Raf1，一个基因先前被其他人证明是由压力过载引起的心脏肥大和功能障碍所必需的。FHL1也被证明与Erk2相互作用，Erk2是Raf-1的下游靶标。总之，这些结果使我们假设FHL1是一种新的支架蛋白，可以选择性地介导Raf1-Mek1/2-Erk1/2信号通路的信号传导，对压力过载介导的心脏肥厚至关重要。因此，具体目标是：1。使用酵母双杂交和GST下拉方法定义FHL1及其伙伴Raf1、MEK1和ERK2的最小相互作用域。2. 评估FHL1缺乏或其与Raf1、MEK1/2或ERK1/2相互作用中断对新生大鼠和成年小鼠心室肌细胞ERK1/2磷酸化和/或肥厚的影响。亚细胞定位的每个组件在这一途径也将被检查。3. 确定FHL1缺乏或其与RAF1、MEK1/2或ERK1/2相互作用中断对长期压力过载和Gq过表达诱导的肥大的体内影响。将利用FHL1缺陷小鼠和表达肽阻断FHL1与Raf1、MEK1或ERK2相互作用的转基因小鼠。对FHL1信号通路的识别和理解可能最终使我们能够开发出明智的治疗方法，以消除人类患者心脏肥厚的发展，从而对心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.