Defining process control parameters for cardiac reprogramming

确定心脏重编程的过程控制参数 项目总结 近年来，充血性心力衰竭(CHF)的患病率急剧上升，这是由于心脏状况的改善。 暂时处理缺血性心脏病，这是全球主要的死亡原因。最基本的 然而，CHF发病机制中尚未解决的问题是不可逆转的心肌细胞(CM)丢失。虽然 已经提出了各种心脏修复策略，每种方法都有特定的缺点， 而且，在一些情况下，人工翻译进展迅速，没有足够的机械特征-- 预先规格化。最近，GHMT将成纤维细胞直接重编程为诱导的CM样细胞(ICLM) (Gata4、Hand2、MEF2C和Tbx5)已成为一种可行的替代再生战略。尽管高度 然而，在临床前模型中有希望的结果，CM重新编程的效率仍然不是最优的。 因此，该研究计划的长期目标是了解DIRECT的分子基础。 心脏重编程作为一种新的心脏再生和发育范例。这样做的目的是 建议阐明心脏重编程的基本过程控制特征。基于 强大的初步数据，我们的中心假设是特定的GHMT蛋白结构域通过精确的 影响心脏重编程过程的动力学。在这里我们概述了一套全面的实验 旨在通过追求以下两个具体目标来检验这一假设：1)确定生化 心脏重编程的构建块以及2)定义心脏重编程的操作参数。 在具体目标#1中，我们将评估心脏重编程过程中单个GHMT因子的必要性 并使用一系列全长、缺失和突变的CON将其功能分配给特定的蛋白质结构域。 我们自己获得或生成的结构。在具体目标2中，我们将系统地确定 通过以下方式优化心脏重编程所必需的理想相加顺序和关键时间窗口 采用既定的和新颖的重新编制方案的方法。我们的方法是创新的，因为它将使用- 使我们强大和有效的单细胞分析在功能形成过程中询问3个离散的步骤 ICLM：基因组重组、肌节组装和亚型多样性。这个项目意义重大，在那里- 因为它寻求定义作为心脏基础的关键生化输入和功能设计原则- 交流重新编程。总而言之，这项研究计划的总体影响是充分利用 心脏重编程作为一种治疗干预，一个用于体外疾病建模的系统，以及一个独特的 了解心肌发生的平台。

Defining process control parameters for cardiac reprogramming PROJECT SUMMARY The prevalence of congestive heart failure (CHF) has risen dramatically in recent years due to improved con- temporary management of ischemic heart disease, the leading cause of death worldwide. The fundamental unresolved issue underlying CHF pathogenesis, however, is irreversible cardiomyocyte (CM) loss. Although various strategies for cardiac repair have been proposed, each approach possesses particular shortcomings, and, in several instances, human translation has proceeded rapidly without adequate mechanistic characteri- zation beforehand. Recently, direct reprogramming of fibroblasts into induced CM-like cells (iCLMs) by GHMT (Gata4, Hand2, Mef2c, and Tbx5) has emerged as a viable, alternative regenerative strategy. Despite highly promising results in preclinical models, however, the efficiency of CM reprogramming remains suboptimal. Therefore, the long-term goal of this research program is to understand the molecular underpinnings of direct cardiac reprogramming as a novel cardiac regenerative and developmental paradigm. The objective of this proposal is to elucidate the essential process control characteristics of cardiac reprogramming. Based on strong preliminary data, our central hypothesis is that specific GHMT protein domains function through precise kinetics to influence the cardiac reprogramming process. Here we outline a comprehensive set of experiments designed to test this hypothesis by pursuing the following two Specific Aims: 1) Determine the biochemical building blocks of cardiac reprogramming and 2) Define the operating parameters for cardiac reprogramming. In Specific Aim #1, we will evaluate the necessity of individual GHMT factors during cardiac reprogramming and assign their function to particular protein domains using a series of full-length, deletion, and mutant con- structs that we have obtained or generated ourselves. In Specific Aim #2, we will systematically determine the ideal order-of-addition and critical temporal windows that are necessary to optimize cardiac reprogramming by applying established and novel reprogramming methodologies. Our approach is innovative because it will uti- lize our robust and validated single-cell assays to interrogate 3 discrete steps during formation of functional iCLMs: genome reorganization, sarcomere assembly, and subtype diversity. This project is significant, there- fore, because it seeks to define the key biochemical inputs and functional design principles that underlie cardi- ac reprogramming. Taken together, the overall impact of this research program is to harness the full potential of cardiac reprogramming as a therapeutic intervention, a system for in vitro disease modeling, and a unique platform for understanding cardiomyogenesis.