Conditioning of Embryonic Myocardial Tubes in Vitro

描述（由申请人提供）：为响应PA-06-418探索性/开发性生物工程研究赠款，拟定了一项为期两年的实验计划，以研究在正常或反向透壁应变梯度下，早期胚胎心肌细胞在新型和独特合适的培养几何结构（人工心肌管）中的体外生长。我们的主要假设是，心脏壁内不均匀的物理应变和由此产生的条件反射是心肌早期成熟的关键物理线索。目的1将阐明维持或刺激细胞增殖或相反，致力于非分裂，完全分化状态的不均匀壁应变的影响。我们将挑战控制和外翻甜甜圈，组装成体外管，与不同的压力刺激，并遵循增殖，分化和细胞死亡的细胞动力学指数。我们将测量体外培养环的张开角，以检验以下假设：外翻甜甜圈的残余壁应变（在零时明显较高）将随着重塑的发生而恢复正常。目标2将探讨与目标1中建立的条件反射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来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，2）发现与细胞决定分化和停止-或恢复-细胞周期潜在相关的新候选分子。目的3系统优化体外心肌管的生长和活力，将其与心外膜或神经嵴供体组织共培养。将按照目标1研究生长和增殖，按照目标2探索分子变化。这一创新性实验方案的意义在于进一步了解胚胎心肌、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。这些研究也将有助于新的策略，工程肌肉结构的再生治疗和鼓励非增殖肌细胞恢复增殖能力。公共卫生相关性：该项目与公共卫生的相关性在于了解身体对心脏和其他肌肉的胚胎形成和畸形、心脏传导组织的区域生长以及心脏壁成熟的贡献。这些研究也应该有助于工程肌肉结构的再生疗法和鼓励非分裂肌肉恢复增殖能力的新策略。

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.