Cardiomyocyte Lineage and Turnover by Metabolic Fate-Mapping

描述（由申请人提供）：尽管最近的研究表明，人类可以产生新的心脏细胞，但随着年龄的增长，更新的速度会急剧下降。由于心力衰竭通常发生在人的后半生，因此了解心脏细胞如何再生以及为什么这一过程会随着年龄的增长而衰退是一个关键的目标。在哺乳动物细胞的命运定位中，可诱导的cre-lox方法是一种广泛使用的方法。在这项提议中，我们展示了初步数据，揭示了小鼠心肌细胞基因标记的可诱导的cre-lox方法不适合研究心肌细胞的衰老过程。我们的初步数据显示，在没有诱导cre活性的情况下，心肌细胞中的年龄相关重组。因此，我们开发了一种全新的方法，利用细胞特异性代谢前体中的同位素来绘制命运图。这种代谢命运定位方法利用了一种同位素富集的代谢示踪剂，特别是肌酸，它被细胞吸收并在细胞质磷酸肌酸穿梭中被利用。已知细胞内肌酸池以每天约2-3%的速率翻转，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多同位素成像质谱法（MIMS）进行鉴定，这是一种高分辨率的定量方法。利用这种新的代谢命运图谱技术，本研究的具体目的是：1。为了验证这一假设，与MIMS一起，使用同位素标记的肌酸进行代谢命运测绘，将能够检测所有年龄组小鼠在正常衰老过程中的心肌细胞再生。我们预计再生速率将会很低，并且这种基底细胞分裂速率会随着年龄的增长而降低。2. 为了验证这一假设，与MIMS一起，使用同位素标记的肌酸进行代谢命运测绘，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，而不是先前存在的心肌细胞分裂，而且这种再生速度将随着年龄的增长而下降。这种代谢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，这种新方法不仅可以用于心肌，还可以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对人类受试者的再生活性进行长期临床研究。

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.