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（CHD）每年困扰着36，000名在美国出生的婴儿，幸存者在其缩短的生命中通常需要多次手术干预。尽管不断努力，导致冠心病的机制仍然很不清楚。在某种程度上，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 收缩和血液动力学都可能改变负责心脏发育的表达途径。然而，由于缺乏适当的成像工具，这些生物力学和电生理信号没有被完全识别或很好地理解，特别是在早期循环阶段，此时心脏缺陷的轨迹可以开始。在上一个资助期间，我们开发并展示了4-D光学相干断层扫描（OCT）技术，作为一种强大的工具，在早期循环心脏中成像收缩动力学和血流动力学。在这个拟议的项目期间，我们将补充光学相干断层扫描与光学测绘（OM）使用电位和钙敏感的荧光染料，以揭示心脏传导和钙信号的模式。有了一个集成的成像系统，可以捕捉收缩力学，血流动力学，钙瞬变，并在整个发育阶段的电传导，在此期间，心管是循环，我们将能够调查这些个别因素，它们之间的复杂的相互作用和它们的作用，在冠心病的出现。OCT和OM的集成将支持和实现新的技术和实验方法。我们将利用OCT来纠正OM成像过程中的运动，避免对兴奋-收缩（E-C）解耦药物的需要，从而使E-C耦合首次在发育中的心脏中直接和全面地研究。我们将开发3-D OM使用二次谐波产生电位染料将和光学相干显微镜，使歧视的径向不对称的胚胎心管的传导系统的3-D异质性。我们将通过感染鹌鹑胚胎，使它们在遗传上表达一种Ca++敏感蛋白来开发体内钙成像。这些新工具可以应用于许多实验模型，例如消融（例如神经嵴细胞，NCC），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）、NCC相关的CHD病因学及其从早期阶段影响心脏功能的能力之间的联系。基于文献和我们自己的初步研究结果，我们提出，乙醇暴露在胚胎模型在脆弱的阶段，破坏NCC的发展，并导致心脏缺陷，通过心脏功能的损害。我们还假设，叶酸/肌醇（FA/MI）的组合将减轻这些功能异常，并防止FAS相关的 冠心病我们提出的研究可能包括第一步，以制定新的治疗策略的基础上FA/MI预防出生缺陷，以造福公众健康。

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.