Project 1 - Endogenous and Exogenous Mechanisms that Promote Myocardial Remuscularization

总结/摘要 （项目1） 内源性和外源性机制，促进 心肌梗死后左室重构的心肌血运重建 进行性心力衰竭的分子和细胞基础是受损和受损细胞不能正常工作的结果。 凋亡的心肌细胞被替换。虽然许多基于细胞和组织的疗法可以限制这种功能障碍， 移植后在给药部位存活超过几周的细胞比例为 非常低。因此，很少报道梗死区域的大量肌肉重建; 有限的肌肉再生已被报道，它经常伴随着潜在的致命的心室 机制不明的心律失常。该建议旨在通过以下方法使受损心室肌肉化： 定义调节和促进天然心肌细胞（CM）细胞周期的关键内源性因子， 和来自外源性移植的生物工程心肌补片（hCMP）， CM细胞周期的调节因子，并将纳入功能性血管网络，并概括一些关键的 天然心脏组织的微环境线索。我们最近建立了一种新的hiPSC细胞系， CM的关键调节因子CCND 2（hiPSC-MHC-CCND 2 OE）的过表达，其可以使损伤的肌肉化 啮齿动物模型中的心室。这项建议的中心目标是“逆转时钟”的肌细胞 循环，这将有助于心肌修复。将审查这一目标的具体目标包括： 确定心肌缺血后早期新生猪心脏中促进细胞周期活性的关键调节因子， 损伤我们将：1）使用最先进的命运绘图分子生物学和成像技术，以及单一的 细胞/细胞核RNA测序（scRNAseq或snRNAseq）技术证明了关键的调节因子/信号转导 控制肌细胞细胞周期的途径;和2）通过以下方式测试受损心室的肌再生： 使用靶向modRNA或AAV 9递送策略操纵关键调节子，以选择性地修饰 这些调节剂在成年猪心脏缺血损伤后。SA2a。设计包含CM的hCMP， 移植后能够增殖，其特征在于以前无法达到的大小和厚度 其功能成熟并为体内血管化做好准备。SA2b.为了评估我们的 用于心肌损伤的大动物（猪）模型中的心肌恢复和肌肉重建的hCMP构建体 损伤我们将使用最先进的技术，包括光学映射结合3- 三维壁内心脏标测，以描绘整个心脏的潜在心律失常机制。 透壁和左心室表面。这些研究将与其他项目协同作用，并作为前奏 治疗倡议的重点是受伤的人类心脏的肌肉再生。

SUMMARY / ABSTRACT (Project 1) Endogenous and Exogenous Mechanisms that Promote Myocardial Remuscularization in Postinfarction LV Remodeling The molecular and cellular basis for progressive heart failure is the result of the inability of damaged and apoptotic myocytes to be replaced. While a number of cell- and tissue-based therapies can limit this dysfunction, the proportion of cells that survive at the site of administration for more than a few weeks after transplantation is extremely low. As such, substantial remuscularization of the infarcted region has rarely been reported; and when limited remuscularization has been reported, it is frequently accompanied by potentially lethal ventricular arrhythmias of unknown mechanism. This proposal aims at remuscularization of the injured ventricle by the definition of key endogenous factors that regulate and promote the cell cycle of the native cardiomyocyte (CM), and from exogenous transplanted bioengineered cardiac muscle patch (hCMP) that overexpresses key regulators of CM cell cycle, and will incorporate a functional vascular network and recapitulate some of the key micro environmental cues of native heart tissue. We recently established a novel hiPSC cell line with MHC-driven overexpression of a key regulator of CMs: CCND2 (hiPSC-MHC-CCND2OE), which can remuscularize injured ventricle in rodent model. The central objective of this proposal is to “turn back the clock” of the myocyte cell cycle, which will facilitate myocardial repair. The Specific Aims (SA) that will examine this objective include: SA1: To identify the key regulators that promote cell-cycle activity in the hearts of early neonatal pigs after myocardial injury. We will: 1) using state-of-the-art fate-mapping molecular biology and imaging technologies, and the single cell/nucleus RNA sequencing (scRNAseq or snRNAseq) technology demonstrate the key regulators/signaling pathways that govern the myocyte cell cycle; and 2) test the remuscularization of the injured ventricle by manipulating the key regulators using either targeted modRNA or AAV9 delivery strategies to selectively modify these regulators in adult pig hearts following ischemic injury. SA2a. To engineer hCMPs containing CMs that are capable of proliferating after transplantation, and characterized by previously unattainable size and thickness that are functionally mature and primed for in-vivo vascularization. SA2b. To evaluate the effectiveness of our hCMP constructs for myocardial recovery and remuscularization in a large-animal (pig) model of myocardial injury. We will use state-of-the-art techniques that includes optical mapping in combination with the 3- dimensional intramural cardiac mapping to delineate the potential arrhythmia mechanisms over the entire transmural and left ventricular surface. These studies will synergize with the other projects and serve as a prelude for therapeutic initiatives focused on remuscularization of the injured human heart.