Advancing Experimental Models to Study Intercellular Crosstalk of Cardiac Cells

描述（由申请人提供）：心肌细胞（CM，<50%的总细胞数）和心脏成纤维细胞（CF，40-60%）是心肌中高度散布的两种主要细胞类型，一个或多个CF与每个CM相邻。细胞间串扰被认为在确定正常心脏功能和心脏重塑反应中起核心作用，心脏重塑反应响应于许多心血管疾病而增强，通常需要CM肥大、CF激活和ECM产生增加（纤维化），并且通常导致心力衰竭和心律失常。然而，在理解CM-CF通信的功能重要性和机制方面存在根本性的差距，部分原因是缺乏合适的实验模型。由于CF和CM的复杂散布使得原位研究它们的串扰极其困难，因此需要细胞培养系统以直接和可控的方式研究两种细胞类型之间的功能相互作用。本申请的目的是推进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-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，介导和调节心脏中CM和CF之间的相互作用，并有助于重塑反应。具体目的是（1）开发具有确定的同型和异型相互作用的微图案化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）开发具有散在或紧凑构型的CF的3D CM-CF共培养物，并确定CF对3D微组织的形态和综合功能反应的影响。CM和CF的大小、表型、形态和胶原产生以及CM功能变化（即，动作电位传播和钙瞬变）。分子机制将通过激光捕获显微切割和电生理学研究使用细胞类型选择的基因表达分析进行研究。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互补的实验模型，增强控制CM-CF相互作用（2D模型）和模拟心室心肌的分布（3D模型）。该模型将使调查的通信的细胞类型是错综复杂的联系在组织背景下原位。该研究具有重要意义，因为该模型可以作为新的平台，用于无偏分子发现介导和调节CM和CF之间相互作用的新分子途径。这些新的途径可以作为开发新策略的目标，以抵消或预防响应于血液动力学应激的心脏重塑。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。 在心脏领域和更远的其他细胞类型之间的串扰。

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.