Genetic Studies of Sarcomere-based Cardiac Diseases

项目摘要 肌动蛋白(TTN)截断变异体(TTNtvs)被发现是最常见的遗传因素 扩张型心肌病(DCM)。然而，也发现了TTNtv DCM的等位基因异质性，即TTNtv 在参考人群中，这些患者的诊断和治疗进展明显混乱。 致病TTNtv主要存在于C-末端A带区域(TTNtv-AS)，而在N-末端Z-AS中较少。 磁盘区(TTNtv-Zs)。此外，TTNtv DCM的病理信号通路在很大程度上仍不清楚。 在这里，我们的目标是利用斑马鱼遗传学带来的独特研究机会来破译潜在的 发病机制，发现病理信号通路，开发有效的治疗途径。我们的 初步研究表明，TTNtv DCM的AH可以在胚胎和成年斑马鱼中重现， 为活体AH的机制研究打开了大门。从我们的已知心肌病信号屏幕上 通路，我们确定mTOR、自噬、MAPK和PDE1作为候选信号通路可能是 用于治疗方面的好处。我们还建立了一种基于F0的遗传分析方法，使用微同源- 中介末端连接(MMEJ)基因组编辑技术，使我们能够快速发现新的信号 小路。基于这些初步研究，我们建议利用独特的遗传和化学遗传 斑马鱼工具证明斑马鱼是第一个TTNtv DCM等位基因异质性的活体动物模型， 可用于破译TTNtvs引起的原发损害，以发现顺序病理 信号通路，并开发基于机制的疗法。该提案分为3个具体的部分 目标。在具体目标1中，我们将通过研究一组ttntv突变体来破译ttntv dcm的等位基因异质性。 和ttn零突变体。在特定的目标2中，我们建议阐明自噬失调的分子基础。 Ttntv DCM并开发基于自噬的疗法。在具体目标3中，我们将确认MAPK和PDE1为 通过携带基于MMEJ的F0候选信号并发现额外的新基因和信号通路 屏幕。在完成提案后，我们预计将实现以下交付成果：1)提供活体证据 澄清为什么TTNtv-AS比TTNtv-Z更有可能导致DCM表型；2)获得关于 TNTV DCM中的自噬、MAPK和PDE信号通路，并确定基于机制的治疗 3)建立了一种基于F0的遗传筛选方法，该方法能够系统地 Ttntv DCM新基因的发现，为AND的机制研究打开了前所未有的机遇 遗传性心肌病。

Project Summary TITIN (TTN) truncating variants (TTNtvs) have been found to be the most common genetic factor for dilated cardiomyopathy (DCM). However, allelic heterogeneity (AH) of TTNtv DCM, i.e. TTNtvs are also found in reference populations, significantly confound diagnosis and therapeutic development of these patients. Pathogenic TTNtvs are mainly found in the C-terminal A-band region (TTNtv-As) but less in the N-terminal Z- disc region (TTNtv-Zs). Moreover, pathological signaling pathways for TTNtv DCM remain largely unknown. Here, we aim to leverage unique research opportunities enabled by zebrafish genetics to decipher underlying mechanisms of AH, discover pathological signaling pathways, and develop effective therapeutic avenues. Our preliminary studies showed that AH of TTNtv DCM can be recapitulated in both embryonic and adult zebrafish, opening the door for mechanistic studies of AH in vivo. From our screen of known cardiomyopathy signaling pathways, we identified mTOR, autophagy, MAPK and PDE1 as candidate signaling pathways that could be leveraged for therapeutic benefits. We also established a F0-based genetic assay using the Microhomology- mediated end joining (MMEJ) genome editing technology that enables us to rapidly discover new signaling pathways. Based on these preliminary studies, we proposed to leverage unique genetic and chemical genetic tools in zebrafish to prove that zebrafish is the first in vivo animal model for allelic heterogeneity of TTNtv DCM, which can be used to decipher primary damages incurred by TTNtvs, to discover sequential pathological signaling pathways, and to develop mechanism-based therapies. The proposal is organized into the 3 specific aims. In Specific Aim 1, we will decipher allelic heterogeneity of ttntv DCM via studying a panel of ttntv mutants and ttn null mutants. In Specific Aim 2, we propose to elucidate molecular basis of autophagy dysregulation in ttntv DCM and develop an autophagy-based therapy. In Specific Aim 3, we will confirm MAPK and PDE1 as candidate signalings and discover additional new genes and signaling pathways by carrying MMEJ-based F0 screens. Upon completion of the proposal, we anticipate the following deliverables: 1) provide in vivo evidence to clarify why TTNtv-As are more likely to cause DCM phenotypes than TTNtv-Zs; 2) obtain insights on autophagy, MAPK and PDE signaling pathways in ttntv DCM, and identify mechanism-based therapeutic avenues for ttntv DCM; 3) establish a F0-based genetic screening approach that is capable of systematically discovering new genes for ttntv DCM, opening an unprecedented opportunity for mechanistic studies of an inherited cardiomyopathy.