Advancing Experimental Models to Study Intercellular Crosstalk of Cardiac Cells

说明(申请人提供)：心肌细胞(CM，占细胞总数的50%)和心脏成纤维细胞(CF，40%-60%)是心肌中高度分散的两种主要细胞类型，每个CM与一个或多个CF接壤。细胞间串扰被认为在确定正常的心脏功能和许多心血管疾病引起的心脏重塑反应中发挥核心作用，通常导致CM肥大、CF激活和ECM产生增加(纤维化)，并经常导致心力衰竭和心律失常。然而，对于CM-CF通信的功能重要性和机制的理解存在着根本的差距，部分原因是缺乏合适的实验模型。由于CF和CM错综复杂的相互作用使得原位研究它们的串扰变得极其困难，因此需要细胞培养系统以直接和可控的方式研究这两种细胞之间的功能相互作用。本应用的目的是促进CM和CF的体外共培养方法，并研究在生理和病理生理条件下细胞-细胞相互作用和旁分泌效应的贡献以及CF-到-CM串扰的功能意义。我们的长期目标是利用新的实验模型来发现新的途径，以调节心脏中CM和CF之间的相互作用，并有助于重塑反应。其具体目标是(1)开发具有明确同型和异型相互作用的微图案2D CM-CF共培养物，并确定CF对CM形态、基因表达和功能的影响；(2)以散布或紧凑的形式开发与CF共培养的3D CM-CF共培养物，并确定CF对三维微组织形态和综合功能反应的影响。将检查CM和CF的大小、表型、形态和胶原生成以及CM功能的变化(即动作电位传播和钙瞬变)。分子机制将通过激光捕获、显微解剖和电生理学研究，使用细胞类型选择的基因表达分析来研究。这项拟议的研究是创新的，因为我们的多学科方法将导致新的和补充的实验模型，加强对CM-CF相互作用的控制(2D模型)，并具有模拟心室肌的分布(3D模型)。这些模型将使研究在组织环境中错综复杂地联系在一起的细胞类型的交流成为可能。提出的研究具有重要意义，因为这些模型可以作为新的平台，无偏分子发现新的分子途径，调节和调节CM和CF之间的相互作用。这些新途径可以作为开发新策略的靶点，以对抗或防止心脏重构对血流动力学应激的反应。此外，我们预计将开发的模型和在该项目中获得的见解将促进细胞间的研究 其他类型的细胞在心脏领域和更远的领域之间的串扰。

DESCRIPTION (provided by applicant): Cardiac myocytes (CM, <50% of total cell number) and cardiac fibroblasts (CF, 40-60%) are the two major cell types in the myocardium that are highly interspersed, with one or more CF bordering each CM. Intercellular crosstalk is believed to play a central role in determining normal cardiac function and the cardiac remodeling response that ensues in response to many cardiovascular diseases, generally entails CM hypertrophy, CF activation and increased ECM production (fibrosis), and often leads to heart failure and arrhythmias. However, there is a fundamental gap in understanding of the functional importance and mechanisms of CM-CF communication, in part due to a lack of suitable experimental models. Since the intricate interspersion of CF and CM makes investigations of their crosstalk in situ extremely difficult, cell culture systems are required to investigate functional interactions between the two cell types in a direct and controllable manner. The objectives of this application are to advance in vitro co-culture approaches for CM and CF and to investigate the contributions of cell-cell interactions and paracrine effects as well as the functional significance of CF-to-CM crosstalk, both under physiological and pathophysiological conditions. Our long-term goal is to utilize the new experimental models to discover novel pathways that mediate and regulate the interaction between CM and CF in the heart and contribute to the remodeling response. The Specific Aims are (1) To develop micropatterned 2D CM-CF co-cultures with defined homo- and heterotypic interactions and to determine the effect of CF on CM morphology, gene expression and function; (2) To develop 3D CM-CF co-cultures with CF in interspersed or compact configuration and to determine the effect of CF on morphological and integrated functional responses of 3D microtissues. CM and CF size, phenotype, morphology and collagen production as well as CM functional changes (i.e., action potential propagation and calcium transients) will be examined. Molecular mechanisms will be investigated using cell-type-selected gene expression analysis via laser capture microdissection and electrophysiological studies. The proposed study is innovative because our multidisciplinary approach will lead to novel and complementary experimental models with enhanced control of CM-CF interactions (2D model) and with a distribution that mimics the ventricular myocardium (3D model). The models will enable investigations of the communication of cell types that are intricately linked in a tissue context in situ. The proposed research is significant because the models can serve as new platforms for unbiased molecular discovery of novel molecular pathways that mediate and regulate the interaction between CM and CF. the new pathways could serve as targets for the development of new strategies to counteract or prevent cardiac remodeling in response to hemodynamic stress. Furthermore, we anticipate that the models that will be developed and the insights gained in this project will facilitate research on intercellular crosstalk between other cell types in the cardiac field and beyond.