TNNI3K: A cardiac-specific kinase regulating ischemic injury and fibrotic remodel

描述(由申请人提供)：心力衰竭通常由缺血性或高血压疾病引起，迫切需要新的策略来解决世界范围内不断扩大的心力衰竭患者群体。在过去的两年里，我们探索了一种新的心脏特异性蛋白激酶-肌钙蛋白I3型相互作用激酶(基因名TNNI3K)在调节缺血损伤、心肌梗死后重塑和压力超负荷诱导重塑中的作用。我们采用了许多策略，包括1)表达野生型、结构性活性或非激活型TNNI3K的转基因小鼠，2)有条件的心脏特异性缺失TNNI3K(CKO小鼠)，以及3)我们在葛兰素史克-史密斯-克莱恩的合作者生产的能够在体外和体内使用的新型TNNI3K小分子抑制剂。我们的所有数据都支持两个关键结论：1)TNNI3K的抑制作用很强 在缺血/再灌注(I/R)损伤中，TNNI3K的保护和激活是非常有害的，2)TNNI3K似乎是心脏纤维化重塑的中央调节因子。我们现在建议确定TNNI3K调节这些过程的分子机制，从而扩大我们对缺血损伤和修复机制的理解。在我们的特定目标中，我们建议从基因组分析中识别TNNI3K靶标，并且已经确定了TNNI3K在调节与纤维化重塑有关的基因表达方面的深刻作用。这与我们在接受小分子抑制剂之一治疗的胸主动脉狭窄小鼠中观察到的纤维化显着减少是一致的。我们还将使用Shokat和他的同事开创的模拟敏感激酶等位基因(ASKA)方法，利用蛋白质组学方法来鉴定TNNI3K的直接底物。最后，我们将侧重于目标2中确定的特定底物的作用，并将确定其在缺血损伤和梗死后重构中的作用。鉴于我们在体内成功地使用了针对TNNI3K的小分子抑制剂，以及在我们的临床前模型中这种方法被证明是安全的，我们相信，如果我们的假设得到证实，就可以相当快地将其转化为临床试验。 公共卫生相关性：我们已经确定了TNNI3K在调节心脏对缺血损伤和纤维化重塑的反应中的一个新的关键角色，TNNI3K是一种心脏特异的蛋白激酶，功能尚不清楚。在这里，我们将确定这些影响的机制。

DESCRIPTION (provided by applicant): Heart failure commonly results from ischemic or hypertensive disease, and novel strategies are sorely needed to address the ever-expanding population of patients with heart failure world-wide. Over the past two years we have explored the role of a novel cardiac-specific protein kinase, troponin I type 3 interacting kinase (gene name TNNI3K) in regulating ischemic injury, post-MI remodeling, and pressure overload-induced remodeling. We have employed a number of strategies including 1) transgenic mice expressing either wild-type, constitutively active, or kinase-inactive TNNI3K, 2) conditional cardiac-specific deletion of TNNI3K (CKO mice), and 3) novel small molecule inhibitors of TNNI3K generated by our collaborators at Glaxo-Smith-Kline that are able to be used both in vitro and in vivo. All of our data support two key conclusions: 1) Inhibition of TNNI3K is strongly protective, and activation of TNNI3K is highly detrimental, in the setting of ischemia/reperfusion (I/R) injury, and 2) TNNI3K appears to be a central regulator of fibrotic remodeling in the heart. We now propose to determine the molecular mechanisms by which TNNI3K regulates these processes, thereby expanding our understanding of mechanisms of ischemic injury and repair. In our Specific Aims we propose to identify TNNI3K targets from genomic analyses and have already identified a profound role of TNNI3K in regulating expression of genes implicated in fibrotic remodeling. This is consistent with the significant reduction in fibrosis we observe in mice subjected to thoracic aortic constriction that were treated with one of the small molecule inhibitors. We will also employ proteomic approaches to identify direct substrates of TNNI3K using the analog sensitive kinase allele (ASKA) approach pioneered by Shokat and co-workers. Finally, we will focus on the role of select substrates identified in Aim 2, and will determine ther role in ischemic injury and post-infarct remodeling. Given our successful use of small molecule inhibitors targeting TNNI3K in vivo, and the demonstrated safety of this approach in our pre-clinical models, we believe that the stage could be set for fairly rapid translation to clinical trals if our hypotheses are borne out. PUBLIC HEALTH RELEVANCE: We have identified a novel critical role of TNNI3K, a cardiac-specific protein kinase of poorly understood function, in regulating the response to ischemic injury and fibrotic remodeling in the heart. Herein we will identify mechanisms of these effects.