Identification of growth factors and cytokines regulating the postnatal cell cycle arrest of cardiomyocytes

在哺乳动物的宫内发育过程中，胚胎和胎儿心脏通过心肌细胞的增殖而生长，从而导致细胞数量的逐渐增加。然而，心肌细胞的细胞周期活动在出生后停止，因此，出生后的心脏主要通过增加现有细胞的大小来生长，而没有大量产生新的心肌细胞。这种出生后心肌细胞周期停滞被认为是成年哺乳动物心脏损伤后无法再生的主要原因。此外，由于早期宫内生长中断而导致的潜在心肌细胞缺陷在出生后无法正常化，这对早产儿童也有重大影响。调控出生后心肌细胞周期停滞的分子机制是复杂的，尚不完全清楚。在出生后的第一周，线粒体从糖酵解到氧化代谢的转变伴随着心肌细胞的逐渐成熟和分化，以满足与产前心脏相比增加的工作负荷。然而，围产期心肌细胞周期活动的调控尚不清楚。我们在老鼠身上的初步研究表明，心脏的增殖率在出生后几小时内下降。这与调节心脏生长和心肌细胞周期活动的各种信号通路的失活是平行的，这些信号通路对细胞外生长因子和细胞因子有反应。因此，我们假设，出生后暴露于子宫外环境后，立即诱导了出生后早期阶段的心肌细胞周期停滞。这可能是由心肌中生长因子和细胞因子的表达和可用性的快速变化引起的。本项目旨在精确计时和表征围产期小鼠心脏在出生前后5天内的心肌细胞增殖。同时，使用无偏倚的蛋白质组学筛选，将心肌和循环血液中的生长因子和细胞因子在出生前一天与出生后一天进行量化。细胞外因子通过该筛选发现了最显著的变化，随后将在体外和体内分析它们刺激新生小鼠和大鼠心肌细胞增殖的能力，以及它们维持胎儿心肌细胞细胞周期活性和延缓后者细胞周期停滞的潜力。该项目预期的数据将促进对早产后心脏生长和长期功能的理解，并可能对新生儿和潜在的成人心脏再生产生重大影响。

During intrauterine mammalian development the embryonic and fetal heart grows by proliferation of cardiomyocytes resulting in a progressive increase in cell number. Cardiomyocyte cell cycle activity ceases after birth, however, such that the postnatal heart primarily grows by increasing the size of existing cells without substantial generation of new cardiomyocytes. This postnatal cardiomyocyte cell cycle arrest is considered a main reason for the inability of the adult mammalian heart to regenerate after injury. It furthermore has major consequences for children born preterm, as a potential cardiomyocyte deficit due to the early interruption of intrauterine growth could not be normalized postnatally.The molecular mechanisms regulating postnatal cardiomyocyte cell cycle arrest are complex and incompletely understood. During the first postnatal week a switch from a glycolytic towards an oxidative metabolism in mitochondria accompanied by progressive maturation and differentiation of cardiomyocytes to meet the increasing workload of the post- compared to the prenatal heart are involved. The regulation of cardiomyocyte cell cycle activity in the immediate perinatal period, however, is less well understood. Our preliminary work in mice shows that proliferation rates in the heart drop within hours after birth. This is paralleled by inactivation of a variety of signaling pathways which regulate cardiac growth and cardiomyocyte cell cycle activity and are responsive to extracellular growth factors and cytokines. We therefore hypothesize that an early phase of postnatal cardiomyocyte cell cycle arrest is induced immediately after birth by the exposure to an extrauterine environment. This could be caused by rapid changes in the expression and availability of growth factors and cytokines in the myocardium.This project aims at precisely timing and characterizing cardiomyocyte proliferation in the perinatal mouse heart over a period of five days before and after birth. At the same time using an unbiased proteomics screen, growth factors and cytokines will be quantified in the myocardium and the circulating blood on the day before compared to the day after birth. Extracellular factors identified by this screen showing the most significant changes will subsequently be analyzed in vitro and in vivo for their ability to stimulate proliferation of neonatal mouse and rat cardiomyocytes as well as their potential to maintain cell cycle activity in fetal cardiomyocytes and delay cell cycle arrest in the latter. The data anticipated from this project will foster the understanding of postnatal cardiac growth and long-term function after preterm birth and might have major implications for regeneration of the neonatal and potentially the adult heart.