Cardiomyocyte Differentiation Regulates Cardiac Function

项目摘要/摘要：与许多器官不同，心脏以前被认为是一种 终末分化的有丝分裂后器官。这导致了一种假设，即心脏包含固定数量的 如果损伤导致心肌细胞死亡，心肌将需要维持其功能 角色，减少单元格数量。而心脏起源的横纹肌细胞经历终末分化 哺乳动物出生后不久，已有研究表明，心肌损伤后会发生再生。这 这一概念引起了人们对心脏干细胞/祖细胞潜力的兴趣。内源性心脏祖细胞 细胞(CPC)能够在损伤部位有限地再生心肌，然而治疗潜力 外源性给药的祖细胞一直是一个激烈的研究领域，因为心力衰竭 对于数百万美国人来说，心肌损伤仍然是一个棘手而致命的问题。因此，这一行动的一个主要目标 应用是为了进一步了解决定CPC命运的途径(在发育过程中和 出生后进入成年)，并了解这些祖细胞分裂和分化为 有功能的心肌细胞。肌球蛋白轻链激酶(MLCK)是一类重要的蛋白质家族，对 肌细胞功能。这个家族的一个成员是纹状优先表达基因(SpeG)。《Speg》 基因座产生四种不同的基因亚型，SpeG和SpeG在纹状体中优先表达 肌肉(包括心脏起源的肌肉)。SpeG和SpeG与MLCK家族成员有同源性，以及 与Oblcurin-MLCK一样，都是MLCK家族中独特的成员，因为它们包含两个相互连接的排列 丝氨酸/苏氨酸激酶(MLCK)结构域。我们实验室的前期研究表明，SpeG亚型 (尤其是SpeG)是横纹肌分化的标志。然而，SpeG的功能意义 亚型尚不清楚。我们打乱了小鼠的SpeG基因座，发现纯合子 突变(SpeG-/-)心脏在出生后16.5天(DPC)开始增大，到18.5DPC表现出明显的 右、左心房和左心室扩张。这些扩张的SpeG突变心脏显示出功能不佳， 一种与扩张型心肌病一致的表型。SpeG-/-小鼠也经历了显著的新生 死亡率。有趣的是，SpeG-/-小鼠的心脏显示每毫米组织中的细胞数量减少 与SpeG+/+小鼠相比，透射电子显微镜(EM)显示分化程度较低， 提示心脏实质细胞的发育发生了改变。该应用程序的总体目标是 阐明SpeG在CPC的命运和分化为有功能的心肌细胞中的作用，并确定 SPG在心脏损伤、修复和功能中的重要性。为了实现这一目标，我们提出了以下建议 目的：1)研究SpeG在CPC命运、对心肌细胞谱系的承诺和心肌细胞中的作用 辨证；2)破译心脏功能不全的发生机制 缺乏SpeG；&3)确定SpeG在成年小鼠心脏损伤(压力超负荷)过程中的重要性。

PROJECT SUMMARY / ABSTRACT: Different from many organs, the heart previously was felt to be a terminally differentiated, postmitotic organ. This led to the assumption that the heart contained a fixed number of cells postnatally, and that if injury led to myocyte death, the myocardium would need to maintain its functional role with a reduced number of cells. While striated muscle cells of cardiac origin undergo terminal differentiation shortly after birth in mammals, it has been shown that myocardial regeneration occurs following injury. This concept led to an interest in the potential of stem/progenitor cells in the heart. Endogenous cardiac progenitor cells (CPCs) are capable of limited myocardial regeneration at sites of injury, however the therapeutic potential of exogenously administered progenitor cells has been an intense area of investigation, as heart failure due to myocardial injury remains a difficult and deadly problem for millions of Americans. Thus, a major objective of this application is to further understand pathways responsible for the fate of CPCs (both during development and postnatally into adulthood), and to understand the ability of these progenitor cells to divide and differentiation into functional cardiomyocytes. Myosin light chain kinases (MLCK) are a family of proteins that are important for myocyte function. One member of this family is the striated preferentially expressed gene (Speg). The Speg locus generates four different gene isoforms, with Speg¿ and Speg¿ being expressed preferentially in striated muscle (including muscle of cardiac origin). Speg¿ and Speg¿ share homology with MLCK family members, and along with obscurin-MLCK, are unique members of the MLCK family as they contain two tandemly arranged serine/threonine kinase (MLCK) domains. Prior investigations in our laboratory revealed that Speg isoforms (particularly Speg¿) are markers of striated muscle differentiation. However, the functional significance of Speg isoforms was yet to be elucidated. We disrupted the Speg gene locus in mice, and revealed that homozygous mutant (Speg-/-) hearts began to enlarge by 16.5 days post-coitum (dpc), and by 18.5 dpc showed a marked dilation of right and left atria and ventricles. These dilated Speg mutant hearts demonstrated poor function, and a phenotype consistent with a dilated cardiomyopathy. Speg-/- mice also experienced significant neonatal mortality. Interestingly, the hearts of Speg-/- mice showed a reduced number of cells per mm3 of tissue compared with Speg+/+ mice, and a less differentiated appearance by transmission electron microscopy (EM), suggesting an alteration in the development of cardiac parenchymal cells. The overall goal of the application is to elucidate the role of Speg in the fate and differentiation of CPCs into functional cardiomyocytes, and to ascertain the importance of Speg in cardiac injury, repair, and function. To achieve this goal, we propose the following Aims: 1) investigate the role of Speg in CPC fate, commitment to the cardiomyocyte lineage, and cardiomyocyte differentiation; 2) decipher the mechanisms responsible for the development of cardiac dysfunction in the absence of Speg; & 3) determine the importance of Speg during cardiac injury (pressure overload) in adult mice.