Mechanical regulation of CM proliferation and morphogenesis

项目摘要 该建议致力于阐明心肌细胞（CM）增殖的机械决定因素， 成熟和功能。我们专注于CM增殖的机械调节，因为我们希望 提高了我们对为什么与大多数细胞类型不同，CM在它们被激活后不能有意义地增殖的理解。 已经成熟。有趣的是，关键的细胞周期激活因子Yap 1和Erk 1/2在许多细胞中受到机械负荷的调节， 细胞类型，但在CM中的Yap 1和Erk 1/2的机械调节知之甚少。了解 CM增殖的机械阻滞，以及促进增殖的机械刺激， 促进CM增殖和心脏再生的策略。我们的研究还将检查细胞周期激活 导致病理性肥大，以确定为什么真正的细胞分裂不会发生，并将揭示 损伤后适应不良重塑的机制。 在心脏中，CM紧密连接并相互施加机械负荷。所以保持 这些相互作用对于有意义地理解CM功能的机械调节是必不可少的。我们有 工程化复杂的3D心脏组织，重现这些相互作用，并允许操纵特定的 机械力，包括应变、后负荷和基质刚度。这些组织也适合 以亚细胞分辨率成像。我们最近生产了多色荧光膜报告器， （“brainbow”）载体和人诱导干细胞（hiPSC）系，我们将使用它们来检查CM增殖率。 和形态测量学。此外，我们将研究 细胞外基质（ECM）水平对CM增殖和形态计量学的影响 体内ECM。因为Yap 1转录辅激活因子和Erk 1/2被认为是 机械力传感和非CM基因表达调控之间的中间体，我们将研究 在不同条件下，Yap 1和Erk 1/2核定位和靶基因表达在CM中是如何调节的 体外和体内小鼠模型中3D工程心脏组织的机械载荷。拟议的研究 对于理解机械应变、后负荷和刚度如何调节关键细胞周期至关重要 体内模型和组织工程方法中的机械传感器将克服数十年的困难 研究受到广泛作用损伤或2D培养物的体外伪影的混淆。

Project Summary Abstract This proposal strives to elucidate the mechanical determinants of cardiac myocyte (CM) proliferation, maturation, and function. We are focusing on mechanical regulation of CM proliferation because we wish to improve our understanding of why, unlike most cell types, CMs are unable to meaningfully proliferate after they have matured. Interestingly, key cell cycle activators Yap1 and Erk1/2 are regulated by mechanical load in many cell types, but the mechanoregulation of Yap1 and Erk1/2 in CMs is poorly understood. Understanding the mechanical blocks to CM proliferation, as well as mechanical stimuli that promote proliferation, may unlock new strategies of promoting CM proliferation and heart regeneration. Our study will also examine cell cycle activation that results in pathological hypertrophy to identify why true cell division does not occur, and will uncover mechanisms that underly maladaptive remodeling post injury. In the heart, CMs are tightly connected and exert mechanical load on each other. Therefore, maintaining these interactions is essential for meaningful understanding of mechanoregulation of CM functions. We have engineered complex 3D heart tissues that recapitulate these interactions and allow manipulation of specific mechanical forces, including strain, afterload, and matrix stiffness. These tissues are also amenable to live imaging with subcellular resolution. We recently generated multi-colored fluorescent membrane reporter (“brainbow”) vectors and human induced stem cell (hiPSC) lines we will use to examine CM proliferation rates and morphometry when specific mechanical stimuli are applied. In addition, we will study the effects of extracellular matrix (ECM) levels on CM proliferation and morphometry in transgenic mouse models with tunable ECM in vivo. Because the Yap1 transcriptional-coactivator and Erk1/2 have been implicated as key intermediates between mechanical force sensing and gene expression regulation in non-CMs, we will examine how Yap1 and Erk1/2 nuclear localization and target gene expression are modulated in CMs under various mechanical loads in 3D engineered heart tissues in vitro and in in vivo mouse models. The proposed studies are essential for understanding how mechanical strain, afterload, and stiffness regulate key cell cycle mechanotransducers in in vivo models and tissue engineering approaches that will overcome decades of research confounded by broad-acting injuries or in vitro artefacts from 2D cultures.