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 对人类心脏再生的研究和刺激将改善许多患者的生活。扩散 心肌细胞是心脏发育和再生所必需的。出生后， 心肌细胞的增殖能力下降，但新生哺乳动物的心脏被认为保持了 再生的能力，直到心肌细胞经历永久性的细胞周期停止，为 再生。刺激心肌细胞增殖的策略正在制定中，将是最 在心肌细胞永久退出细胞周期之前启动时有效。然而，这一点 人类心肌细胞的哪一种增殖停止是有争议的；一些研究认为这一点在出生前， 一些在出生后第一年，我们的数据显示心肌细胞增殖延伸到 童年，即人生的头10-20年。这幅图景因我们出版的和 未发表的结果表明，患有先天性心脏病(CHD)的婴儿数量减少 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一个重要问题。加深对心肌细胞的认识 增殖将是开发新的再生策略以预防和治疗心力衰竭的关键 青紫型先天性心脏病患者，最常见的出生缺陷影响0.3%的新生儿。我们将决定 当儿科患者的心肌细胞增殖减少时，通过调查两种主要的心脏病： 法洛四联症(ToF)，最常见的青紫型先天性心脏病(CHD)，影响 0.3%的新生儿，以及扩张型心肌病(DCM)，这是美国心脏移植最常见的原因 儿科年龄组。我们的中心假设是心肌细胞的增殖表现出一个年龄--和 疾病依赖性衰退。我们将招募新生儿、婴儿和儿童来量化心肌细胞 扩散。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 我们将用携带稳定同位素标记的无害胸腺嘧啶核苷(15N-胸腺嘧啶核苷和2H-胸腺嘧啶核苷)标记患者。 胸苷)。处于细胞周期的S期的细胞将胸腺嘧啶核苷结合到它们的dna中，它们的后代保留了 至少6个细胞分裂的标签。我们将用一种创新的方法将手术后组织样本中的同位素可视化 方法：用多同位素成像质谱仪(MIMS)定量检测心肌细胞的增殖和 差异化。我们第一个研究的ToF患者的初步数据提供了第一个人类结果和 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 This picture is further complicated by our published and unpublished results suggesting that infants with congenital heart disease (CHD) have decreased cardiomyocyte proliferation.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.