Cardiomyocyte Differentiation Regulates Cardiac Function

项目总结/摘要：与许多器官不同，心脏以前被认为是一个 终末分化的有丝分裂后器官。这导致了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， 减少细胞数量的作用。而心脏起源的横纹肌细胞经历终末分化 在哺乳动物中，在出生后不久，已经显示在损伤后发生心肌再生。这 这一概念引起了人们对心脏干/祖细胞潜力的兴趣。内源性心脏祖细胞 细胞（CPC）能够在损伤部位进行有限的心肌再生，但其治疗潜力 外源性给予祖细胞的研究一直是一个激烈的研究领域，因为心力衰竭是由于 心肌损伤对于数百万美国人来说仍然是一个困难和致命的问题。因此，本报告的一个主要目标是 应用程序是为了进一步了解决定CPC命运的途径（无论是在开发过程中还是在 出生后进入成年期），并了解这些祖细胞分裂和分化为 功能性心肌细胞肌球蛋白轻链激酶（MLCK）是一个蛋白质家族，其对于肌球蛋白的合成和功能是重要的。 肌细胞功能这个家族的一个成员是条纹优先表达基因（Speg）。斯佩格 基因座产生四种不同的基因亚型，Speg <$和Speg <$在横纹肌中优先表达。 肌肉（包括心脏起源的肌肉）。Speg <$和Speg <$与MLCK家族成员具有同源性，并且 沿着的还有obscurin-MLCK，它们是MLCK家族的独特成员，因为它们含有两个串联排列的 丝氨酸/苏氨酸激酶（MLCK）结构域。我们实验室的先前研究显示，Speg亚型 （特别是Speg）是横纹肌分化的标志物。然而，斯佩格的功能意义 同种型尚待阐明。我们破坏了小鼠的Speg基因位点，并发现纯合子 突变型（Speg-/-）心脏在术后16.5天（dpc）开始增大，到18.5 dpc时， 左右心房和心室扩张。这些扩张的Speg突变心脏表现出功能低下， 一种与扩张型心肌病相符的表型Speg-/-小鼠也经历了显著的新生儿 mortality.有趣的是，Speg-/-小鼠的心脏显示出每mm3组织中细胞数量的减少， 与Speg +/+小鼠相比，透射电子显微镜（EM）显示分化程度较低， 提示心脏实质细胞发育的改变。应用程序的总体目标是 阐明Speg在CPC向功能性心肌细胞的命运和分化中的作用，并确定 Speg在心脏损伤、修复和功能中的重要性。为实现这一目标，我们提出以下建议 目的：1）研究Speg在CPC命运、向心肌细胞谱系定向和心肌细胞分化中的作用。 分化; 2）破译机制负责心功能不全的发展，在 不存在Speg; 3）确定Speg在成年小鼠心脏损伤（压力超负荷）期间的重要性。

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.