Integrated imaging of the form and function of the beating embryonic heart

描述(由申请人提供)：先天性心脏病(CHDS)每年困扰着在美国出生的3.6万名婴儿，幸存者在他们短暂的生命中通常需要几次手术治疗。尽管不断努力，导致CHD的机制在很大程度上仍然不清楚。在一定程度上，这是因为大多数发育心脏病学研究未能解决心脏功能改变在心脏发生中的影响作用。心肌兴奋，心肌细胞 收缩和血流动力学都可能改变负责心脏发育的表达途径。然而，由于缺乏合适的成像工具，这些生物力学和电生理信号并没有被完全识别或很好地理解，特别是在Loopin早期，那时心脏缺陷的轨迹可以开始。在之前的资助期间，我们开发和演示了4-D光学相干断层扫描(OCT)技术，作为一种强有力的工具来成像早期心脏循环的收缩动力学和血流动力学。在这个拟议的项目期间，我们将用光学标测(OM)来补充OCT，使用电位和钙敏感的荧光染料来揭示心脏传导和钙信号的模式。有了一个集成的成像系统，可以捕捉心脏导管环行期间整个发育阶段的收缩力学、血流动力学、钙瞬变和电传导，我们将能够调查这些单独的因素，它们之间的复杂相互作用，以及它们在冠心病发生中的作用。OCT和OM的集成将支持和启用新的技术和实验方法。我们将利用OCT来校正OM成像过程中的运动，省去了兴奋-收缩(E-C)去偶联药物的需要，从而使E-C偶联能够在发育中的心脏中首次得到直接和全面的研究。我们将使用二次谐波产生电位染料和光学相干显微镜来开发三维OM，从而能够区分径向不对称胚胎心管传导系统的三维异质性。我们将通过感染鹌鹑胚胎来开发体内钙成像，使它们在基因上表达一种钙敏感蛋白。这些新工具可以应用于许多实验模型，例如消融(例如神经脊细胞)、环境扰动(例如缺氧)或化学暴露(例如酒精)。我们选择关注与胎儿酒精综合征(Fas)相关的CHD的病因学和NCC之间的联系，以及它们在早期影响心功能的能力。根据文献和我们自己的初步发现，我们认为胚胎模型在脆弱阶段接触乙醇会扰乱NCC的发育，并通过心功能损害导致心脏缺陷。我们还假设，叶酸/肌醇(FA/MI)的联合应用将缓解这些功能异常，并防止Fas相关 CHDS。我们提出的研究可能包括开发基于FA/MI预防出生缺陷的新治疗策略的第一步，以造福于公众健康。

DESCRIPTION (provided by applicant): Congenital heart defects (CHDs) afflict 36,000 babies born in the US each year and survivors often require several surgical interventions in their shortened lifetime. Despite continuous efforts, the mechanisms leading to CHDs remain largely unclear. In part, this is because most developmental cardiology studies fail to address the influential role of altered cardiac function in cardiogenesis. Myocardial excitation, cardiomyocyte contraction and hemodynamics are all likely to modify expression pathways responsible for heart development. However, due to the lack of proper imaging tools, these biomechanical and electrophysiological signals are not fully identified or well understood especially at early loopin stages, when the trajectory to heart defects can begin. In the previous funding period, we developed and demonstrated 4-D optical coherence tomography (OCT) technology as a powerful tool to image contraction dynamics and hemodynamics in the early looping heart. In this proposed project period, we will complement OCT with optical mapping (OM) using potentiometric and calcium-sensitive fluorescent dyes to reveal patterns of cardiac conduction and calcium signaling. With an integrated imaging system that can capture contraction mechanics, hemodynamics, calcium transients, and electrical conduction throughout the developmental stages during which the heart tube is looping, we will be able to investigate these individual factors, the complex interplay between them and their role in the emergence of CHDs. The integration of OCT and OM will support and enable new technology and experimental methods. We will utilize OCT to correct motion during OM imaging, obviating the need for excitation-contraction (E-C) decoupling drugs, thus enabling E-C coupling to be studied directly and comprehensively for the first time in the developing heart. We will develop 3-D OM using second-harmonic generating potentiometric dyes will and optical coherence microscopy, enabling discrimination of the 3-D heterogeneity of the conduction system of the radially asymmetric embryonic heart tube. We will develop in vivo calcium imaging by infecting quail embryos causing them to genetically express a Ca++-sensitive protein. These new tools may be applied to numerous experimental models such as ablation (e.g. neural crest cells, NCCs), environmental perturbation (e.g. hypoxia), or chemical exposure (e.g. alcohol). We have chosen to focus on the connections between the etiology of CHDs associated with Fetal Alcohol Syndrome (FAS), NCCs, and their ability to influence cardiac function from an early stage. Based on the literature and our own preliminary findings, we propose that ethanol exposure in the embryonic model at a vulnerable stage disrupts NCC development, and leads to heart defects through the impairment of cardiac function. We also hypothesize that a combination of folate/myo-inositol (FA/MI) will alleviate these functional abnormalities, and prevent FAS- related CHDs. Our proposed studies could comprise a first step toward developing new therapeutic strategies based on FA/MI prevention of birth defects to benefit public health.