Engineered Cell Alignment for Improved Beating in a Fibrin-Based Heart Patch

描述（由申请人提供）：心血管疾病可导致心肌梗死（Ml）和随后的心力衰竭。目前有许多旨在预防或治疗心肌梗死后心力衰竭的疗法。只有心脏移植替代梗死心肌来恢复心脏功能，但供体心脏缺乏，心血管疾病的发病率持续上升。一种新的创新选择是使用体外制造的“心脏贴片”（又名心肌等效物）在体内植入。这里提出的研究旨在解决与这种心脏贴片的功能和最终使用有关的关键问题-与天然组织相比，心肌等效物的发力能力较低。我们的假设是，细胞诱导的纤维蛋白凝胶心肌等效物的收缩不仅导致组织对齐，而且改善了兴奋-收缩耦合和更大的组织收缩力，而不仅仅是对齐收缩的心肌细胞。我们认为这些增强是两种机制的结果：1)与肌细胞排列相关的间隙连接形成增加，2)肌细胞-成纤维细胞-肌细胞传导途径增加。因此，提出的研究的具体目的如下：1)评估细胞介导的纤维蛋白凝胶的收缩和排列如何诱导肌细胞排列，从而促进间隙连接的形成，从而增加收缩力；2)确定心脏成纤维细胞在这些组织构建中肌细胞之间的兴奋信号转导中的作用。为了完成目标1，我们将通过将新生大鼠心肌细胞和心脏成纤维细胞（以其天然比例）包裹在两种不同几何形状的纤维蛋白凝胶中，即矩形板状和管状，来创建对齐和各向同性的结构。我们将通过免疫组织化学测量间隙连接的形成及其光学功能（染料转移，通过细胞内钙传导延迟）和结构收缩（即兴奋阈值，收缩力间隔）。为了完成Aim 2，我们将创建与Aim 1中方法相似的结构，然而，我们将通过预镀来去除成纤维细胞来改变心脏成纤维细胞的比例。然后，我们将预先标记成纤维细胞，以便在光学测量中区分它们，并创建具有不同比例的成纤维细胞和肌细胞的结构，记录两种细胞类型之间间隙连接的形成和功能。此外，我们将记录整个结构的收缩特性，并将其与成纤维细胞密度联系起来。我们期望细胞诱导的纤维蛋白凝胶的收缩、排列和重塑将导致更强的收缩结构，并且这种增强的细胞机制将被阐明。

DESCRIPTION (provided by applicant): Cardiovascular disease can lead to myocardial infarction (Ml) and subsequent heart failure. There are currently a number of therapies aimed at preventing or treating heart failure post-MI. Only heart transplantation replaces infarcted myocardium to restore heart function, but there is a paucity of donor hearts and the incidence of cardiovascular disease continues to rise. A new and innovative option is the use of "heart patches" (a.k.a. myocardial equivalents) created in vitro for implantation in vivo. The research proposed here aims to address a critical issue pertaining to the function and eventual use of such heart patches - the lower force generation capacity of myocardial equivalents when compared to native tissue. Our hypothesis is that cell-induced contraction of fibrin gel-based myocardial equivalents results not only in tissue alignment but also improved excitation-contraction coupling and greater tissue contraction force than what would be expected merely from aligning contracting cardiomyocytes. We propose that these enhancements are the result of two mechanisms: 1) increased gap junction formation associated with myocyte alignment, and 2) increased myocyte-fibroblast-myocyte conduction pathways. The specific aims of the proposed research are thus as follows: 1) Evaluate how cell-mediated contraction and alignment of a fibrin gel induces myocyte alignment, which promotes formation of gap junctions and, as a result, increases contraction force, and 2) Determine the role that cardiac fibroblasts play in transducing the excitation signal between myocytes in these tissue constructs. To complete Aim 1 we will create aligned and isotropic constructs by entrapping neonatal rat cardiomyocytes and cardiac fibroblasts (in their native proportions) in fibrin gels in two different geometries: rectangular slab and tubular. We will measure the formation of gap junctions via immunohistochemistry and their function both optically (dye transfer, conduction delay via intracellular Calcium) and in terms of contraction of the constructs (i.e. excitation threshold, contraction force interval). To complete Aim 2 we will create constructs similar to the methods in Aim 1, however, we will alter the proportion of cardiac fibroblasts by pre-plating to remove fibroblasts. We will then pre-label the fibroblasts to distinguish them in optical measurements and create constructs with varying ratios of fibroblasts to myocytes, recording the formation and function of gap junctions between the two cell types. In addition, we will record the contractile properties of the entire construct and correlate them with fibroblast density. We expect that cell-induced contraction, alignment, and remodeling of fibrin gel will result in a more contractile construct and that the cellular mechanism for this enhancement will be elucidated.