Conditioning of Embryonic Myocardial Tubes in Vitro

描述(由申请人提供)：为响应PA-06-418探索性/发展性生物工程研究资助，提出了一项为期两年的实验计划，以研究早期胚胎心肌细胞在一种新颖且独特合适的培养几何结构、人工心肌管在正常或反向跨壁应变梯度下的体外生长。我们的主要假设是，心脏壁内不均匀的生理应变和由此产生的条件反射是心肌早期成熟的关键生理信号。目标1将阐明不均匀的壁张力对维持或刺激细胞增殖的影响，或者相反，对不分裂、完全分化状态的承诺。我们将挑战对照甜甜圈和外翻甜甜圈，组装成体外试管，在不同的压力刺激下，并遵循细胞动力学指标的增殖、分化和细胞死亡。我们将测量体外培养环的张开角，以测试外翻甜甜圈的残余壁应变在时间为零时明显更高的假设，随着重塑的发生，将恢复到正常水平。目标2将探索与目标1中建立的条件性范式相关的分子变化。我们将使用组织化学、免疫组织化学和蛋白质组学方法1)表征特定候选分子(生长因子、细胞骨架和基质分子)的动力学，以及2)发现与细胞分化和停止-或恢复-细胞周期的决定潜在相关的新的候选分子。目标3将系统地优化体外心肌管的生长和活力，将其与心外膜或神经脊供体组织共培养。目标1将研究生长和细胞动力学，目标2将探索分子变化。这一创新的实验方案的意义在于进一步了解对胚胎心肌、传导组织的区域生长和分化以及整个心壁的成熟和肥大反应的物理贡献。这些研究还应该有助于设计肌肉结构的新策略，用于再生治疗，并鼓励非增殖性肌细胞恢复增殖能力。公共卫生相关性：该项目与公共健康的相关性在于了解心脏和其他肌肉的胚胎形成和畸形、心脏传导组织的局部生长以及心壁的成熟对健康和疾病的物理贡献。这些研究还应该有助于设计肌肉结构的新策略，用于再生治疗，并鼓励不分割的肌肉恢复增殖能力。

DESCRIPTION (provided by applicant): A two-year experimental program is proposed, in response to PA-06-418 Exploratory/ Developmental Bioengineering Research Grants, to study in vitro growth of early embryonic cardiac myocytes in a novel and uniquely suitable culture geometry, artificial myocardial tubes under normal or reversed transmural strain gradients. Our principal hypothesis is that uneven physical strain within the heart wall and resultant conditioning are physical cues critical to early maturation of cardiac muscle. Aim 1 will elucidate effects of uneven wall strain upon maintenance or stimulation of cellular proliferation or, conversely, commitment to a non-dividing, fully differentiated state. We will challenge both control and everted donuts, assembled into in vitro tubes, with diverse pressure stimuli and follow cytokinetic indices of proliferation, differentiation and cell death. We will measure opening angles of cultured in vitro loops to test the hypothesis that residual wall strain of everted donuts, demonstrably higher at time zero, will return toward normal as remodeling takes place. Aim 2 will explore molecular changes associated with conditioning paradigms established in Aim 1. We will use histochemical, immunohistochemical and proteomics approaches to 1) characterize kinetics of specific candidate molecules (growth factors, cytoskeletal and matrix molecules) and 2) discover new candidates of potential relevance to the cellular decision to differentiate and stop - or to resume - cell cycling. Aim 3 will systematically optimize growth and vigor of in vitro myocardial tubes, co-culturing them with epicardial or neural crest donor tissues. Growth and cytokinetics will be studied as in Aim 1 and molecular changes explored as in Aim 2. Significance of this innovative program of experiments lies in further understanding of physical contributions to regional growth and differentiation of embryonic cardiac muscle, conduction tissues, and to maturation and hypertrophic responses of the entire heart wall. These studies should also contribute to novel strategies for engineering muscle constructs for regenerative therapies and for encouraging non- proliferating myocytes to regain proliferative capacity. PUBLIC HEALTH RELEVANCE: Relevance of this project to public health lies in understanding of physical contributions to the embryonic formation and malformation of the heart and other muscle, to regional growth of cardiac conduction tissues, and to maturation of the heart wall, in health and disease. These studies should also contribute to novel strategies for engineering muscle constructs for regenerative therapies and for encouraging non-dividing muscle to regain proliferative capacity.