Cardiomyocyte Lineage and Turnover by Metabolic Fate-Mapping

描述（由申请人提供）：虽然最近的研究表明，人类可以产生新的心脏细胞，但更新速度随着年龄的增长而急剧下降。由于心力衰竭通常发生在生命的后半段，因此了解心脏细胞如何再生以及为什么这个过程会随着年龄的增长而下降是一个关键目标。可诱导的cre-lox方法是用于哺乳动物中细胞的命运作图的广泛使用的方法。在这个提议中，我们展示了初步的数据，揭示了一种诱导型cre-lox方法来遗传标记小鼠心肌细胞不适合研究心肌的衰老过程。我们的初步数据显示，在没有cre活性诱导的情况下，心肌细胞中存在与年龄相关的重组。因此，我们开发了一种全新的方法，使用细胞特异性代谢前体中的同位素进行命运映射。这种代谢命运映射方法利用同位素富集的代谢示踪剂，特别是肌酸，其被细胞摄取并用于细胞质磷酸肌酸穿梭。已知细胞内肌酸池以每天约2-3%的速率周转，使其成为理想的心肌细胞特异性代谢标记。肌酸阳性的细胞可以通过使用多同位素成像质谱（MIMS）（一种高分辨率定量方法）进行鉴定。利用这种新的代谢命运图技术，本研究的具体目的是：1。为了检验这一假设，即与MIMS一起，使用同位素标记的肌酸的代谢命运图将能够检测所有年龄组的小鼠在正常衰老期间的心肌细胞再生。我们预计再生率将很低，并且这种基础细胞分裂率将随着年龄的增长而下降。 2.为了检验这一假设，即与MIMS一起，使用同位素标记的肌酸的代谢命运图将能够检测所有年龄组小鼠损伤后的心肌细胞再生。我们预计，再生将主要来自干细胞分化，而不是通过预先存在的心肌细胞分裂，并且这种再生率将随着年龄的增长而下降。 这种代谢性的命运映射方法将使心肌细胞衰老相关再生的研究成为可能。此外，这种新方法不仅可用于心肌，还可用于其他类型的组织，如骨骼肌、脑和脂肪细胞。最后，由于这种方法可以应用于稳定的非放射性同位素，因此可以通过这种方法实现人类受试者中再生活性的长期临床研究。

DESCRIPTION (provided by applicant): Although recent studies demonstrate that humans can generate new heart cells, the rate of renewal dramatically decreases with age. Since heart failure often strikes in the second half of life, understanding how heart cells regenerate and why this process declines with age is a critical goal. The inducible cre-lox approach is a widely-used method for fate-mapping of cells in mammals. In this proposal, we show preliminary data revealing that an inducible cre-lox approach to genetically label cardiomyocytes in mice is not suitable for studying the aging process in the myocardium. Our preliminary data reveal age-related recombination in cardiomyocytes in the absence of induction of the cre activity. Thus, we have developed an entirely new approach to fate-mapping using isotopes in a cell-specific metabolic precursor. This metabolic fate-mapping approach utilizes an isotope-enriched metabolic tracer, specifically creatine, which is taken up by cells and utilized in the cytoplasmic phosphocreatine shuttle. The intracellular creatine pool is known to turn over at a rate of approximately 2-3% per day, making it an ideal cardiomyocyte-specific metabolic label. Cells that are creatine positive can be identified via use of Multi-Isotope Imaging Mass Spectrometry (MIMS), a high resolution quantitative approach. Using this new metabolic fate-mapping technique, the specific aims for this study are: 1. To test the hypothesis that, together with MIMS, metabolic fate-mapping using isotope-labeled creatine will enable the detection of cardiomyocyte regeneration during normal aging for mice of all age groups. We anticipate that the rate of regeneration will be low, and this basal cell division rate will decrease as a function of age. 2. To test the hypothesis that, together with MIMS, metabolic fate-mapping using isotope-labeled creatine will enable the detection of cardiomyocyte regeneration after injury for mice of all age groups. We anticipate that regeneration will primarily be from stem cell differentiation and not by pre-existing cardiomyocyte cell division, and this rate of regeneration will decrease as a function of age. This metabolic fate-mapping approach will enable the study of aging-related regeneration of cardiomyocytes. Furthermore, this new approach can be used not only for the myocardium but in other types of tissues such as skeletal muscle, brain and adipocytes. Finally, because this approach can be applied with stable, non-radioactive isotopes, long-term clinical studies of regenerative activity in human subjects may be enabled by this method.