Investigation of Troponin I-interacting kinase, a cardiomyocyte specific kinase, in the immune response to cardiac injury

 描述(由申请人提供)：病毒性心肌炎(VM)是导致心力衰竭的重要因素。VM是一种潜伏性疾病，可导致扩张型心肌病，通常需要心脏移植。此外，目前只有支持性疗法可以对抗VM。人们对这种疾病的发病机制知之甚少。越来越明显的是，过度的宿主免疫反应在很大程度上决定了VM患者心肌病的进展。基于生物信息学的分析揭示了肌钙蛋白I相互作用激酶(TNNI3K)在VM过程中潜在地调节免疫反应。TNNI3K是一种心肌细胞特异性的激酶，调节心脏对缺血和压力超负荷的反应。总体目标是确定TNNI3K作为对抗VM的治疗靶点的有效性。我们的初步发现首次表明，TNNI3K在VM期间加剧了免疫反应。虽然病毒病原体的清除需要免疫细胞的渗透，但急性炎性细胞渗透的严重程度与心功能不全的发展之间存在明显的有害关系。我们的初步发现显示：(1)在VM啮齿动物模型中，CM特异性的TNNI3K过表达导致心肌免疫细胞浸润增加；(2)CM特异性的TNNI3K缺失反过来限制了免疫细胞的渗透，我们假设抑制TNNI3K在急性VM的环境中将具有保护作用。此外，由于TNNI3K是一种CM特异性的激酶，它有助于成为合适的治疗靶点。有了1)表达野生型或激酶失活的TNNI3K的转基因小鼠，2)条件性CM特异的TNNI3K基因敲除小鼠(CKO)，以及3)药理上的TNNI3K抑制剂，我们感到有充分的准备来剖析TNNI3K在心脏损伤的免疫反应中的作用。利用这些工具，我们打算完成以下两个特定的目标：1)表征TNNI3K对VM免疫应答的影响。我们将使用流式细胞术和免疫组织化学来表征免疫细胞群和血流动力学，以评估患有VM的小鼠的功能结果。2)研究TNNI3K对心脏损伤免疫反应的调节机制。利用分离的成年VM小鼠CM，我们将利用分子生物学来研究底物之间的相互作用，以确定它们对免疫反应的影响。总体而言，这些发现将为CM对病毒的免疫反应提供洞察力，提供新的TNNI3K直接相互作用，并回答TNNI3K是否为对抗VM的合适治疗靶点。

 DESCRIPTION (provided by applicant): Viral myocarditis (VM) is a significant contributor to heart failure. VM is an insidious disease that results in dilated cardiomyopathy and often requires cardiac transplantation. Furthermore, only supportive therapies currently exist for combating VM. Very little is known about the pathogenesis of the disease. It is becoming apparent that an excessive host immune response largely defines the progression of cardiomyopathy in patients with VM. Bioinformatics based analyses revealed Troponin I-interacting kinase (TNNI3K) to potentially regulate the immune response during VM. TNNI3K is a cardiomyocyte (CM) specific kinase that regulates the cardiac response to ischemia and pressure overload. The overall goal is to determine the validity of TNNI3K as a therapeutic target for combating VM. Our preliminary findings are the first to show that TNNI3K exacerbates the immune response during VM. Although immune cell infiltration is required for elimination of viral pathogen, there is a clear detrimental relationship between severity of acute inflammatory cell infiltrate and the development of cardiac dysfunction. As our preliminary findings reveal (1) Overexpression of CM-specific TNNI3K in a rodent model of VM results in an increased myocardial immune cell infiltration and (2) CM-specific deletion of TNNI3K conversely limits immune cell infiltration, we hypothesize that inhibition of TNNI3K will be protective in a setting f acute VM. Furthermore, as TNNI3K is a CM-specific kinase, it makes for a suitable therapeutic target. With 1) transgenic mice expressing wild-type, or kinase in-active TNNI3K, 2) conditional CM-specific TNNI3K knockout mice (cKO), and 3) a pharmacological TNNI3K inhibitor, we feel well equipped to dissect the role of TNNI3K in the immune response to cardiac injury. Using these tools, we intend to complete the following two specific aims: 1) Characterize the effect of TNNI3K on the immune response to VM. We will use flow cytometry and immunohistochemistry to characterize immune cell populations and hemodynamics to assess functional outcomes in mice with VM. 2) Examine the mechanism of TNNI3K in regulation of the immune response to cardiac injury. Using isolated adult CM from mice with VM, we will use molecular biology to investigate substrate interactions to determine their effect on the immune response. Overall, these findings will provide insight on the CM immune response to virus, provide novel TNNI3K direct interactions, and answer if TNNI3K is a suitable therapeutic target for combating VM.