Cardiomyocyte Differentiation Regulates Cardiac Function

描述（申请人提供）：与许多器官不同，心脏以前被认为是终末分化的有丝分裂后器官。这导致了这样的假设，即心脏在出生后含有固定数量的细胞，如果损伤导致心肌细胞死亡，心肌将需要以减少的细胞数量来维持其功能作用。虽然哺乳动物的心脏起源的横纹肌细胞在出生后不久就经历了终末分化，但已经表明心肌再生发生在损伤后。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（CPC）能够在损伤部位进行有限的心肌再生，然而外源性给予祖细胞的治疗潜力一直是研究的热点，因为心肌损伤导致的心力衰竭仍然是数百万美国人的难题和致命问题。因此，本申请的主要目的是进一步了解负责CPC命运的途径（在发育期间和出生后进入成年期），并了解这些祖细胞分裂和分化为功能性心肌细胞的能力。肌球蛋白轻链激酶（MLCK）是对肌细胞功能重要的蛋白质家族。这个家族的一个成员是条纹优先表达基因（Speg）。Speg基因座产生四种不同的基因亚型，Speg 1和Speg 2优先在横纹肌（包括心脏起源的肌肉）中表达。Speg 1和Speg 2与MLCK家族成员具有同源性，并且沿着有暗蛋白-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 1）是横纹肌分化的标志物。然而，Speg亚型的功能意义尚未阐明。我们破坏了小鼠的Speg基因位点，发现纯合子突变体（Speg-/-）心脏在交配后16.5天（dpc）开始扩大，到18.5 dpc时，右心房和左心室显着扩张。这些扩张的Speg突变心脏表现出功能差，表型与扩张型心肌病一致。Speg-/-小鼠也经历了显著的新生儿死亡率。有趣的是，与Speg+/+小鼠相比，Speg-/-小鼠的心脏显示出每mm 3组织的细胞数量减少，并且通过透射电子显微镜（EM）观察到分化程度较低的外观，这表明心脏实质细胞的发育发生了变化。本申请的总体目标是阐明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。为了实现这一目标，我们提出了以下目标：1）研究Speg在CPC命运，心肌细胞谱系的承诺和心肌细胞分化中的作用; 2）破译在Speg缺失的情况下导致心功能障碍发展的机制; 3）确定Speg在成年小鼠心脏损伤（压力超负荷）期间的重要性。 公共卫生相关性：80多年来，心血管疾病一直是美国的主要死亡原因，最近占美国所有死亡人数的35.3%。心力衰竭是一种非常普遍的疾病过程，其导致与心血管疾病相关的死亡，并且心力衰竭的发病率仍然极高，2006年在美国诊断出670，000例新病例。这一应用将为心肌细胞的分化和成熟提供新的见解，并确定这种分化过程对新生儿和成人心脏整体心脏功能的重要性。

DESCRIPTION (provided by applicant): 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 Speg1 and Speg2 being expressed preferentially in striated muscle (including muscle of cardiac origin). Speg1 and Speg2 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 Speg1) 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. PUBLIC HEALTH RELEVANCE: Cardiovascular diseases have been the leading cause of death in the United States for more than 80 years, recently accounting for 35.3% of all deaths in the United States. Heart failure is a very prevalent disease process that contributes to deaths associated with cardiovascular diseases, and the incidence of heart failure remains extremely high with 670,000 new cases diagnosed in the United States in 2006. This application will provide new insight into the differentiation and maturation of cardiac muscle cells, and determine the importance of this differentiation process on overall heart function in newborn and adult hearts that have been injured.