Aging and Myocardial Progenitor Recruitment

描述（由申请人提供）：心血管系统老化的特征是心脏功能下降，心血管疾病经常在后半生发作。许多实验结果表明，成年哺乳动物心肌中存在大量的心脏干细胞，这些细胞具有向心肌细胞和其他细胞类型（如内皮细胞和血管平滑肌细胞）分化的潜能。像其他干细胞一样，心脏干细胞可能会随着年龄的增长而衰老，可能会降低其再生潜力。我们的实验室已经开发了一种遗传细胞命运映射方法来量化心肌细胞的周转，我们已经使用这个系统来证明，在生命的前半期，在没有损伤的情况下，心肌细胞不会被干细胞/前体细胞取代;然而，在心肌梗死后，有一个干细胞/前体细胞库的激活。遗传命运图谱是一种强大的细胞跟踪方法，但它不能同时量化所识别的细胞群的细胞分裂速率。直到最近，用于跟踪细胞分裂的方法主要依赖于BrdU掺入或氚（3 H）标记;这些技术具有局限性，因为它们并不总是具有足够的分辨率来定量跟踪细胞分裂，并且它们在某些情况下可能是有毒的。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，允许定量评估细胞分裂历史。MIMS的灵敏度允许在很宽的时间范围内监测变化，因为标记脉冲是使用对动物和人类无毒的稳定的非放射性同位素产生的。MIMS可以在体外和体内监测细胞分裂，时间尺度可能从几分钟到几年不等，因为稳定的同位素不会衰变或发射辐射。在这个建议中，我们描述了实验，研究心肌祖细胞招募衰老小鼠使用的遗传细胞命运映射和MIMS，这在组合的互补技术将有助于定量跟踪不同的细胞群和它们的相对细胞分裂率。我们的具体目标是：目标1。为了验证假设，即在没有损伤的情况下，衰老小鼠中的心肌细胞不会被前体细胞显著更新。目标2.验证衰老降低了哺乳动物心肌在代表人类疾病的损伤后通过干细胞/前体细胞库更新心肌细胞的能力的假设。目标3：为了检验稳定同位素MIMS定量结合遗传命运作图揭示基础心肌细胞分裂速率低且在衰老期间成年哺乳动物心肌中前体池的细胞分裂没有可测量的贡献的假设。目标4：为了验证以下假设，即损伤后，心肌细胞主要由干细胞补充，而不是由预先存在的心肌细胞的细胞分裂补充，而与年龄无关。公共卫生相关性：心脏病经常在后半生发作，心脏组织的愈合和再生可能会因衰老过程而受损。该项目使用新的复杂方法来确定心脏的愈合过程是否随着小鼠年龄的变化而变化。这些实验将为美国的衰老过程和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.