Quantification of the decline of heart muscle cell proliferation and its reversal in pediatric patients

摘要： 心力衰竭仍然是死亡率和发病率的主要原因。了解基本机制 研究人类心脏再生的可能性并刺激它们将改善许多患者的生活。增殖 心肌细胞（心肌细胞）对于心脏发育和再生是必不可少的。生后 心肌细胞的增殖能力下降，但新生哺乳动物的心脏被认为可以维持心肌细胞的增殖能力。 再生能力，直到心肌细胞经历永久性细胞周期停滞，建立一个屏障， 再生刺激心肌细胞增殖的策略正在开发中， 当在心肌细胞永久退出细胞周期之前启动时有效。然而，在 心肌细胞增殖在人类中何时停止是有争议的;一些研究将这一点放在出生前， 一些在出生后的第一年，我们的数据表明心肌细胞增殖延伸到 童年，即，生命的前10 - 20年。这一情况进一步复杂化， 未发表的结果表明，患有先天性心脏病（CHD）的婴儿减少了 心肌细胞增殖解决年龄和心脏病如何改变心肌细胞的时间模式 细胞周期退出是心脏生物学中的基本问题。提高对心肌细胞的认识 增殖对于开发新的再生策略以预防和治疗心力衰竭至关重要， 紫绀型CHD患者，最常见的出生缺陷影响0.3%的新生儿。我们将确定 当儿科患者的心肌细胞增殖减少时，通过研究两种主要的心脏疾病： 法洛四联症（ToF）是紫绀型先天性心脏病（CHD）的最常见形式， 0.3%的新生儿和扩张型心肌病（DCM），心脏移植的最常见原因， 儿童年龄组。我们的中心假设是心肌细胞增殖显示了年龄-和 疾病依赖性下降。我们将招募新生儿、婴儿和儿童， 增殖我们组建了一个由儿科心脏病专家和心脏外科医生组成的研究团队， 病理学家和基础科学家我们方法的关键要素是使用高度创新的方法 其中我们将用携带稳定同位素标记物（15 N-胸苷和2 H- 胸苷）。处于细胞周期S期的细胞将胸苷结合到它们的DNA中，并且它们的后代保留胸苷。 标记至少6次细胞分裂。我们将用一种创新的方法， 方法：多同位素成像质谱（MIMS）定量心肌细胞增殖， 分化来自我们第一个ToF研究患者的初步数据提供了第一个人体结果， 验证我们的方法。

ABSTRACT: Heart failure continues to be a leading cause of mortality and morbidity. Understanding the basic mechanisms of heart regeneration in humans and stimulating them would improve the lives of many patients. Proliferation of heart muscle cells (cardiomyocytes) is essential for heart development and regeneration. After birth, the proliferative potential of cardiomyocytes declines, but neonatal mammalian hearts are thought to maintain the ability to regenerate until cardiomyocytes undergo permanent cell cycle arrest, establishing a barrier for regeneration. Strategies for stimulating cardiomyocyte proliferation are under development and would be most effective when initiated before cardiomyocytes withdraw permanently from the cell cycle. However, the point at which cardiomyocyte proliferation ceases in humans is controversial; some studies place this point before birth, some in the first year after birth, and our data demonstrate cardiomyocyte proliferation extending well into childhood, i.e., the first 10 – 20 years of life. This picture is further complicated by our published and unpublished results suggesting that infants with congenital heart disease (CHD) have decreased cardiomyocyte proliferation. Resolving how age and heart disease alter the temporal pattern of cardiomyocyte cell cycle withdrawal is an essential problem in cardiac biology. Improved understanding of cardiomyocyte proliferation will be critical for developing new regenerative strategies to prevent and treat heart failure in patients with cyanotic CHD, the most common birth defect affecting 0.3% of newborns. We will determine when cardiomyocyte proliferation decreases in pediatric patients by investigating two leading cardiac diseases: tetralogy of Fallot (ToF), the most common form of cyanotic congenital heart disease (CHD), which affects 0.3% of newborns, and dilated cardiomyopathy (DCM), the most common cause for heart transplantation in the pediatric age group. Our central hypothesis is that cardiomyocyte proliferation shows an age- and disease-dependent decline. We will enroll neonates, infants, and children to quantify cardiomyocyte proliferation. We have formed a research team consisting of pediatric cardiologists and cardiac surgeons, pathologists, and basic scientists. The critical element of our approach is the use of a highly innovative method in which we will label patients with innocuous thymidine carrying stable isotope markers (15N-thymidine and 2H- thymidine). Cells in S-phase of the cell cycle incorporate thymidine into their DNA, and their offspring retain the label for at least 6 cell divisions. We will visualize the isotope in post-surgical tissue samples with an innovative method: Multi-isotope imaging mass spectrometry (MIMS) to quantify cardiomyocyte proliferation and differentiation. Preliminary data from our first study patient with ToF provide first-in-human results and validate our approach.