Aging and Myocardial Progenitor Recruitment

描述（申请人提供）：心血管系统老化的特点是心功能下降，心血管疾病多发于后半生。许多实验结果表明，成年哺乳动物心肌中存在一群具有心肌细胞和其他细胞类型（如内皮细胞和血管平滑肌细胞）分化潜力的常驻心脏干细胞。像其他干细胞一样，心脏干细胞可能会随着年龄的增长而发生衰老变化，可能会降低其再生潜力。我们的实验室已经开发了一种基因细胞命运定位方法来量化心肌细胞的更替，我们已经使用该系统来证明，在生命的前半期，心肌细胞在没有损伤的情况下不会被干细胞/前体细胞取代；然而，心肌梗死后，干细胞/前体池被激活。遗传命运图谱是一种强大的细胞追踪方法，但它不能同时量化所鉴定细胞群的细胞分裂率。直到最近，跟踪细胞分裂的方法主要依赖于BrdU掺入或氚（3H）标记；这些技术有局限性，因为它们并不总是有足够的分辨率来定量地跟踪细胞分裂，而且在某些情况下它们可能是有毒的。一种名为多同位素成像质谱（MIMS）的新技术有可能克服这些缺点，允许对细胞分裂历史进行定量评估。MIMS的灵敏度允许在很长时间内监测变化，因为标记脉冲是使用对动物和人类无毒的稳定、非放射性同位素产生的。由于稳定的同位素不会衰变或发射辐射，MIMS可以监测体外和体内的细胞分裂，其时间尺度可能从几分钟到几年不等。在本文中，我们描述了使用遗传细胞命运定位和MIMS互补技术研究衰老小鼠心肌祖细胞募集的实验，这两种技术的结合将有助于定量跟踪不同的细胞群及其相对细胞分裂率。我们的具体目标是：目标1。为了验证在没有损伤的情况下，衰老小鼠的心肌细胞没有被前体细胞显著更新的假设。目标2。为了验证衰老降低哺乳动物心肌在具有代表性的人类疾病损伤后通过干细胞/前体细胞池更新心肌细胞的能力的假设。目的3：验证稳定同位素MIMS定量与遗传命运定位相结合的假设，揭示了成年哺乳动物心肌在衰老过程中基底心肌细胞分裂率低，前体池细胞分裂没有可测量的贡献。目的4：验证假设，损伤后心肌细胞主要由干细胞补充，而不是由已有心肌细胞的细胞分裂，与年龄无关。公共卫生相关性：心脏病经常在生命的后半期发作，心脏组织的愈合和再生可能因衰老过程而受损。这个项目使用新的复杂方法来确定心脏的愈合过程是否会随着老鼠的年龄而改变。这些实验将为了解衰老过程和美国最常见的死亡原因提供新的见解。

DESCRIPTION (provided by applicant): Aging of the cardiovascular system is characterized by a reduction in cardiac function, and cardiovascular disease frequently strikes in the latter half of life. Many experimental findings suggest that adult mammalian myocardium has a population of resident cardiac stem cells with differentiation potential for cardiomyocytes and other cell types like endothelial cells and vascular smooth muscle cells. Like other stem cells, cardiac stem cells may be subject to senescent changes with increasing age, possibly reducing their regenerative potential. Our laboratory has developed a genetic cell fate-mapping approach to quantify cardiomyocyte turnover, and we have used this system to demonstrate that in the first half of life, cardiomyocytes are not replaced by stem/precursor cells in the absence of injury; however, after myocardial infarction, there is activation of a stem/precursor pool. Genetic fate-mapping is a powerful approach for cell tracking, but it cannot simultaneously quantify cell division rates of the identified cell populations. Until recently, methods for tracking cell division relied mainly on BrdU incorporation or tritium (3H) labeling; these techniques have limitations because they do not always have sufficient resolution to track cell divisions quantitatively, and they can be toxic under certain circumstances. A new technology called Multi-Isotope Imaging Mass Spectrometry (MIMS) has the potential to overcome these drawbacks, permitting quantitative assessment of cell division history. The sensitivity of MIMS allows changes to be monitored over a wide range of times because a labeling pulse is generated using stable, nonradioactive isotopes that are non-toxic for animals and humans. MIMS can monitor cell division in vitro and in vivo with time scales potentially ranging from minutes to years, since stable isotopes do not decay or emit radiation. In this proposal, we describe experiments to study myocardial progenitor recruitment in aging mice using the complementary techniques of genetic cell fate-mapping and MIMS, which in combination will facilitate quantitative tracking of different cell populations and their relative rates of cell division. Our specific aims are: Aim 1. To test the hypothesis that in the absence of injury, cardiomyocytes are not significantly refreshed by precursor cells in the aging mouse. Aim 2. To test the hypothesis that aging reduces the capacity of mammalian myocardium to refresh cardiomyocytes by the stem/precursor cell pool following injuries representative of human diseases. Aim 3: To test the hypothesis that stable isotope MIMS quantification combined with genetic fate mapping reveals a low rate of basal cardiomyocyte cell division with no measurable contribution of cell division from a precursor pool in adult mammalian myocardium during aging. Aim 4: To test the hypothesis that, following injury, cardiomyocytes are replenished primarily by stem cells and not by cell division of pre-existing cardiomyocytes irrespective of age. PUBLIC HEALTH RELEVANCE: Heart disease frequently strikes in the latter half of life, and healing and regeneration of heart tissue may be impaired by the aging process. This project uses new sophisticated methods to determine if the healing process of the heart changes as mice age. These experiments will provide new insight into the aging process and the most common causes of death in the United States.