Experimental tools and mathematical models to study electrical-mechanical properties of spatial-temporal patterns in cultured cardiac cells

提出的研究旨在了解培养的心脏细胞的生物力学和电学特性之间的联系及其在自主电活动中的作用。心肌细胞（电活动的心肌细胞）动力学的高度复杂性需要多种方法和技术的整合，以揭示在培养中发生的多尺度变化以及在细胞和多细胞水平上对电活动的功能影响。在此，我们提出了一个涉及生物仪器开发、图像采集技术以及分析和建模的项目，以更好地了解细胞变形对生物电特性和时空电自组织的影响。在我们的生物反应器中开发一种结合的培养方法，并通过电和机械刺激获取数据，用于非终端实验和亚细胞电生理特性研究*我们开发了一种生物反应器，用于培养CMs，为细胞提供程序化的电和机械刺激。与此同时，一种记录荧光变化的采集系统（例如用fluo-4记录细胞内钙瞬态）已经被开发出来，以可视化电刺激和细胞在培养后的机械变形的影响。目前所提出的组合系统存在三个重要的局限性：拉伸时所研究的视场（FOV）的位移，线性步进电机控制引起的抖动，以及拉伸时无法获取荧光数据。我们建议通过集成反馈控制运动来稳定视场，并修改生物反应器伸展电子电路以实现微步进来纠正这些限制。弹性基质上培养的各向同性和图案心肌细胞单层的时空自主电活动研究：*我们小组的实验显示，在单个位点记录时，不同的时间依赖性行为。在急性电刺激或机械刺激后，颞叶活动可受到短暂或永久的影响。然而，很明显，局部观察到的变化在解释时空动态变化方面是有限的。因此，我们建议研究地形图案心肌细胞时空活动的稳定性。更准确地说，我们将研究电偶联CMs上的自组织活性及其稳定性在急性拉伸下的扰动，以了解细胞变形的作用。用数学模型评估自主电活动的本征频率的异质分散对有图案和无图案单层的整体活动的作用*用数学模型评估自主电活动的本征频率的异质分散对电自组织的作用。从新生儿心脏分离的CMs可以是自主细胞，也可以是非自主细胞。在细胞培养中，初始播种是随机的，因此这两个群体如何在单层内分布是未知的。我们建议基于一种新颖的数学建模方法来研究这两种种群混合对时空活动的影响，并研究细胞地形如何影响自主活动。CM动力学的高度复杂性需要整合这些创新的方法和技术，以揭示在培养中发生的多尺度变化以及在细胞和多细胞水平上对电活动的功能影响。

The proposed study is aimed at understanding the link between the biomechanical and electrical properties of cultured cardiac cells and their role on autonomous electrical activity. The high level of complexity of cardiomyocyte (electrically-active cardiac cells) dynamics needs integration of numerous approaches and techniques to uncover the multiscale changes occurring in culture and the functional impact on electrical activity at both the cell and multicellular levels. Here, we proposed a project involving bioinstrumentation development, image acquisition techniques as well as analysis, and modelling works summarized below to better understand the effects of cell deformation on bioelectric characteristics and spatio-temporal electrical self-organization.*1. Develop a combined approach for culture in our bioreactor and data acquisition with electrical and mechanical stimulations for non-terminal experiments and sub-cellular study of electrophysiological properties *We have developed a bioreactor for culture of CMs that provided programmed electrical and mechanical stimuli to cells. In parallel, an acquisition system to record fluorescence changes (for example for intracellular calcium transient with fluo-4) to visualize the effects of electrical stimulation and mechanical deformation of the cells in post-culture has been developed. Three important limitations of the proposed combined systems exist at this stage: displacement of the field of view (FOV) under study when stretched, jitter due to linear stepper motors control, and impossibility to acquire fluorescence data while stretching. We propose to correct these limitations by integrating a feedback control motion to stabilize the FOV and to modify the bioreactor stretching electronic circuit to microstepping.*2. Study of the spatial-temporal autonomous electrical activity of isotropic and patterned cardiomyocyte monolayers following culture on elastic substrates:*Experiments by our group present varying time-dependent behaviors when recording at a single site. The temporal activity can be transiently or permanently affected following acute electrical or mechanical stimulation. However, it is clear that the changes seen locally are limited to explain what could be variation in the spatial-temporal dynamics. We thus propose to study the stability of spatio-temporal activity of topographically patterned cardiomyocytes. More precisely we will study self-organized activity on electrically-coupled CMs and its stability is perturbed by acute stretch to understand the role of cell deformation.*3. Evaluate, with a mathematical model, the role of heterogeneous dispersion of intrinsic frequencies of autonomous electrical activity on global activity of patterned and unpatterned monolayers*Evaluate, with a mathematical model, the role of heterogeneous dispersion of intrinsic frequencies of autonomous electrical activity on electrical self-organization. CMs isolated from neonatal hearts can be either autonomous or non-autonomous cells. In cell culture, initial seeding is random such that how these two populations are distributed within the monolayer is unknown. We propose to look at the effects of having mixture of these two populations on spatio-temporal activity based on a novel approach of mathematical modeling and study how cell topography can influence the autonomous activity. The high level of complexity of CM dynamics needs integration of these innovative approaches and techniques to uncover the multiscale changes occurring in culture and the functional impact on electrical activity at both the cell and multicellular levels.