Defining process control parameters for cardiac reprogramming

定义心脏重编程的过程控制参数 项目摘要 充血性心力衰竭（CHF）的患病率近年来急剧上升，这是由于改善了对心脏的控制。 缺血性心脏病的临时管理，缺血性心脏病是全球死亡的主要原因。根本 然而，CHF发病机制下的未解决的问题是不可逆的心肌细胞（CM）损失。虽然 已经提出了用于心脏修复的各种策略，每种方法都具有特定的缺点， 而且，在一些情况下，人工翻译进行得很快，没有足够的机械特性， 提前定。近年来，GHMT直接将成纤维细胞重编程为诱导型CM样细胞（iCLMs）， （Gata 4，Hand 2，Mef 2c和Tbx 5）已经成为一种可行的替代再生策略。尽管高度 然而，尽管CM重编程在临床前模型中获得了令人鼓舞的结果，但CM重编程的效率仍然不理想。 因此，这项研究计划的长期目标是了解直接免疫的分子基础。 心脏重编程作为一种新的心脏再生和发育模式。的目的 建议是阐明心脏重编程的基本过程控制特征。基于 强有力的初步数据，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测试这一假设：1）确定生物化学 心脏重编程的构建块和2）定义心脏重编程的操作参数。 在特定目标#1中，我们将评估心脏重编程期间个体GHMT因子的必要性 并使用一系列全长、缺失和突变的连接子将它们的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 理想的加法顺序和关键的时间窗口，这是优化心脏重编程所必需的， 应用已建立的和新的重编程方法。我们的做法是创新的，因为它将uti- 通过稳健且经验证的单细胞测定，在功能性细胞形成过程中询问3个离散步骤， iCLMs：基因组重组、肌节组装和亚型多样性。这个项目意义重大，有- 因为它试图定义关键的生化输入和功能设计原则，这些原则是心血管疾病的基础。 AC重编程。总的来说，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，一种体外疾病建模系统，以及一种独特的 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.