Identifying new regulators of cardiac fibrosis and inflammation using zebrafish

项目摘要/摘要 一些心脏疾病涉及心肌细胞的进行性或急性丧失，这些心肌细胞被 纤维组织。心脏纤维化是心力衰竭的主要病理因素之一，心力衰竭是一种进行性疾病。 这每年影响数百万人。尽管它的重要性得到了公认，但目前还没有有效的治疗方法 防止心脏纤维化的进展。最近的发现表明，自然产生的遗传变异 保护某些人免受心脏损伤后引发的不良变化，这转化为 导致心脏纤维化。改变疾病进展的基因之一是心肌细胞特异性的 激酶TnNI3K。取消TNNI3K的突变赋予对伤害的抵抗力，与高TNNI3K水平相关 功能迅速衰退和病理性重塑。TNNI3K是一种未被研究的激酶，其下游 它定义伤害后果的目标和具体机制尚不清楚。我们在这个项目中的目标是 确定TNNI3K水平与心肌纤维化之间的联系机制。在用斑马鱼进行的初步实验中 作为一个模型，我们发现高水平的TNNI3K在心脏损伤后不久就会导致纤维化沉积。相比之下， 对于他们的野生型兄弟姐妹来说，过度表达TNNI3K的动物表现出受损的纤维化消退，而新的 产生的TNNI3K突变体在损伤后表现出轻微的纤维化。转录图谱显示，高水平的 TnNI3K的表达与炎症反应加剧、T细胞浸润的标志物和 炎症小体的所有成分，它们都是心肌细胞诱导炎症的标志。我们 还确定了一个潜在的下游靶标itih5，此前已有报道称，该靶标在提取液中发挥作用。 皮肤中的扁平基质稳定。根据我们的初步结果，我们的中心假设是TNNI3K Itih5通过调节炎症在心脏纤维化中发挥了以前未被认识到的作用，而Itih5是一种中枢 这些影响的调停者。我们将在三个综合目标中检验我们的假设。在目标1中，我们将确定 炎症在高水平TNNI3K反应的纤维化发展中的作用。我们将利用一个收藏 以测试特定免疫群体对这种过度纤维化的贡献 回应。此外，我们将使用一种新的心肌细胞特异性Cas9株来鉴定触发- ING炎症反应高TNNI3K水平。在目标2中，我们将确定调节TNNI3K的效果 损伤前后对心脏纤维化和炎症的影响。我们将使用生成的新Tnni3kSWITCH行 这将使我们能够在不同的时间“关闭”TNNI3K的过度表达。在目标3中，我们将确定 TnNI3K下游瘢痕重塑的分子调控。我们将从分析Itih5如何监管开始 使用我们实验室为这一应用生成的新的得和失功能模型进行纤维化回归。 总的来说，这些研究将确立TNNI3K和Itih5作为心脏炎症和纤维化的调节因子。 斑马鱼。我们预计，这些基本知识将成为快速发现新目标的跳板。 治疗受损心脏的心脏纤维化。

Project Summary/Abstract Several cardiac diseases involve the progressive or acute loss of cardiomyocytes, which are replaced by fibrotic tissue. Cardiac fibrosis is one of the leading factors in the pathology of heart failure, a progressive disease that affects millions of people every year. Despite its recognized importance, there are no effective therapies to prevent the progression of cardiac fibrosis. Recent findings suggest that naturally occurring genetic variants protect certain individuals against the adverse changes triggered after cardiac injury, which translates into re- duced cardiac fibrosis. One of the genes that modifies the progression of disease is the cardiomyocyte-specific kinase Tnni3k. Mutations that abolish Tnni3k confer resistance to injury, and high Tnni3k levels are associated with rapid functional decline and pathological remodeling. Tnni3k is an understudied kinase, and its downstream targets and specific mechanisms by which it defines injury outcome are unknown. Our goal in this project is to identify the mechanisms linking Tnni3k levels to cardiac fibrosis. In preliminary experiments using the zebrafish as a model, we found that high levels of Tnni3k induce fibrosis deposition shortly after cardiac injury. In contrast to their wild-type siblings, animals overexpressing Tnni3k showed impaired fibrotic regression, while a newly generated tnni3k mutant exhibits minimal fibrosis after injury. Transcriptional profiling revealed that high levels of Tnni3k correlate with an exacerbated inflammatory response, markers of T-cell infiltration, and activation of all the components of the inflammasome, which are all landmarks of cardiomyocyte-induced inflammation. We also identified a potential downstream target, itih5, which has been previously reported to play a role in extracel- lular matrix stabilization in the skin. Based on our preliminary results, our central hypothesis is that Tnni3k plays a previously unappreciated role in cardiac fibrosis via modulation of inflammation, and that Itih5 is a central mediator of these effects. We will test our hypothesis in three integrative aims. In Aim 1, we will determine the role of inflammation in the development of fibrosis in response to high levels of Tnni3k. We will exploit a collection of immunocompromised zebrafish to test the contribution of specific immune populations to this excessive fibrotic response. Additionally, we will use a new cardiomyocyte specific Cas9 line to identify genes required for trigger- ing inflammation in response to high Tnni3k levels. In Aim 2, we will determine the effects of modulating Tnni3k levels pre- and post-injury on cardiac fibrosis and inflammation. We will use a new Tnni3kSWITCH line generated by our lab that will allow us to “turn off” the overexpression of Tnni3k at different times. In Aim 3, we will identify molecular regulators of scar remodeling downstream of Tnni3k. We will start by analyzing how Itih5 regulates fibrosis regression using new gain and loss of function models generated by our laboratory for this application. Collectively, these studies will establish Tnni3k and Itih5 as regulators of cardiac inflammation and fibrosis in zebrafish. We anticipate that this basic knowledge will serve as the springboard for rapid discovery of new targets to treat cardiac fibrosis in the injured heart.