Electrical Biophysical Forces Impact Cardiac Morphogenesis

描述（由申请人提供）：先天性心脏病（CHD）是人类最常见的出生缺陷，影响每一百个活产婴儿中的近一个，并导致绝大多数产前损失。虽然已知几个心脏基因调控程序在心脏发育中发挥重要作用，但我们对生物物理力如何单独、共同或与这些心脏转录程序协同作用以调节心脏形态发生的理解还远未完成。因此，阐明生物物理力如何影响心脏形态发生的潜在细胞、分子和生理机制的研究可能最终有助于冠心病易感患者的诊断和治疗。过去的研究表明，生物力学的力量，如心肌细胞的收缩力和心内血流动力学流量可能会调节这一过程在体内。尽管体外研究表明心肌细胞可以根据电传导方向性重新排列自己，但这些天然心脏传导电流是否可以产生足以指导心肌细胞形状和迁移以及随后的体内心脏形态发生的电场仍不清楚。最近的动物研究表明，电传导可能会影响发育中的脊椎动物心脏的整体结构;然而，评估电心脏传导、机械心脏收缩和血液动力学力对整体心脏形态发生的个体贡献是困难的，因为这些生物物理力通过兴奋-收缩耦合在完整心脏中紧密耦合。因此，为了在体内解开这些生物物理因素，我们最近利用了沉默的心脏斑马鱼心脏突变体，它显示nonbeating，但导电的心脏，由于在肌节基因，心肌肌钙蛋白T的突变，并发现，电传导排除收缩和血液动力学的力量可以直接参与在体内重塑和形态发生的脊椎动物心脏。因此，我们假设，心脏电力发挥关键作用，对维持心肌细胞形态和整体心脏形态发生在脊椎动物心脏发育过程中，通过调节心肌细胞钙瞬变模式/梯度。我们的具体目标是：1）研究电作用力对体内心脏形态发生的影响; 2）研究心脏电作用力是否可以调节参与心肌细胞形态的细胞蛋白;以及3）研究心脏电作用力对心肌细胞钙瞬变/闪烁的时空组织的影响。总的来说，我们的跨学科方法，包括利用遗传上易处理但光学透明的动物系统以及新的生物工程和成像工具，将提供体内机制的洞察力如何电力可能直接影响心脏形态发生。这些研究不仅可以证明对我们的CHD的理解是有益的，而且还可以为未来基于心脏细胞的治疗提供额外的见解，以优化植入的胚胎心肌细胞在损伤心肌中的整合和对齐。 公共卫生相关性：先天性心脏病（CHD）是人类最常见的出生缺陷，每一百个活产婴儿中就有一个受到影响，并且是造成绝大多数产前损失的原因。虽然已知几个心脏基因调控程序在心脏发育中发挥重要作用，但我们对生物物理力如何单独、共同或与这些心脏转录程序协同作用以调节心脏形态发生的理解还远未完成。因此，这些研究将阐明电力如何影响心脏形态发生的潜在细胞，分子和生理机制，从而最终可能有助于冠心病易感患者的诊断和治疗。

DESCRIPTION (provided by applicant): Congenital Heart Disease (CHD) is the most common birth defect in humans affecting nearly one out of every one-hundred live births and is responsible for the vast majority of prenatal losses. Although several cardiac gene regulatory programs are known to play an important role in cardiac development, our understanding of how biophysical forces act alone, together, or in concert with these cardiac transcriptional programs to modulate heart morphogenesis is far from complete. Thus, studies which illuminate the underlying cellular, molecular, and physiologic mechanisms of how biophysical forces impact cardiac morphogenesis may ultimately aid in diagnosis and treatment of patients predisposed for CHD. Past studies have shown that biomechanical forces such as cardiomyocyte contractility and intracardiac hemodynamic flow may regulate this process in vivo. Though in vitro studies suggest that cardiomyocytes can realign themselves according to electrical conduction directionality, it remains unclear as to whether these natural cardiac conduction currents can generate electric fields sufficient to direct cardiomyocyte cell shape and migration and subsequent cardiac morphogenesis in vivo. Recent animal studies have suggested that electrical conduction may influence overall architecture of the developing vertebrate heart; however, assessing the individual contributions of electrical cardiac conduction, mechanical cardiac contraction and hemodynamic forces to overall heart morphogenesis is difficult because these biophysical forces are intimately coupled in the intact heart through excitation- contraction coupling. As a result, to uncouple these biophysical factors in vivo, we have recently exploited the silent heart zebrafish cardiac mutant, which displays nonbeating, but electrically conducting hearts due to a mutation in the sarcomeric gene, cardiac troponin T, and discovered that electrical conduction exclusive of contractile and hemodynamic forces can directly participate in in vivo remodeling and morphogenesis of the vertebrate heart. Thus, we hypothesize that cardiac electrical forces play a critical role toward maintaining cardiomyocyte morphology and overall cardiac morphogenesis during vertebrate heart development through regulation of cardiomyocyte calcium transient patterns/gradients. Our specific aims are: 1) to investigate the impact of electrical forces on cardiac morphogenesis in vivo; 2) to investigate whether cardiac electrical forces may regulate cellular proteins involved in cardiomyocyte morphology; and 3) to investigate the influences of cardiac electrical forces on the spatiotemporal organization of cardiomyocyte calcium transients/flickers. Overall, our interdisciplinary approach including the utilization of a genetically tractable yet optically transparent animal system as well as novel bioengineering and imaging tools will provide in vivo mechanistic insight into how electrical forces may directly impact cardiac morphogenesis. These studies may not only prove rewarding towards our understanding of CHD, but also provide additional insight towards optimizing integration and alignment of engrafted embryonic cardiomyocytes in injured myocardium for future cardiac cell-based therapy. PUBLIC HEALTH RELEVANCE: Congenital Heart Disease (CHD) is the most common birth defect in humans affecting nearly one out of every one-hundred live births and is responsible for the vast majority of prenatal losses. Although several cardiac gene regulatory programs are known to play an important role in cardiac development, our understanding of how biophysical forces act alone, together, or in concert with these cardiac transcriptional programs to modulate heart morphogenesis is far from complete. Thus, these studies will illuminate the underlying cellular, molecular, and physiologic mechanisms of how electrical forces may impact cardiac morphogenesis and thus may ultimately aid in diagnosis and treatment of patients predisposed for CHD.