Heterogeneity Of The Cardiomyocyte Population In Heart Development And Regeneration

For over a hundred years, heart regeneration was intensely studied in mammals and humans, driven by the ability of newts to regenerate body parts. Until recently, this interest was restricted to science fiction. During the past decade, great innovation in stem cell biology and its implications for heart regeneration revived the studies on 'cardiac regeneration medicine'. Surprisingly, the latest breaking study showed the regenerative plasticity of neonatal murine heart. This finding contradicts traditional views and leads to new hope for clinical applications. However, the mechanism of endogenous cardiac repair is unclear. In this study, we investigated the nature of neonatal cardiomyocytes regarding heart growth and regeneration. Our study uncovered, for the first time, genetic evidence that indicated heterogeneity in neonatal myocardial tissues, and that a particular subpopulation of cardiomyocytes contributes to cardiac regeneration more than other subpopulations, providing a new focus for the investigation of heart regeneration.Traditional concepts held that postnatal heart growth results from cardiomyocyte hypertrophy. However, our histological data contradict this view, because we found a nonlinear relationship between increased volume of heart mass and myocytes. With BrdU-mediated lineage tracing, we observed that up to 40% of the ventricular myocardium in juvenile heart was derived from pre-existing myocardial cell proliferation. Immunofluorescence data showed vigorousproliferation activity in the neonatal cardiomyocyte population, which expresses cell cycle markers and exhibits a radially condensed chromatin structure. Interestingly, major proliferating cardiomyocytes, termed mT-CMs, exhibited a phenotype in which troponin proteins were localized beneath the cell membrane. Molecular evidence supports this particular feature in the mT-CM population independent of the disorganization of the cytoskeleton in mitosis. The mT-CMs might be typical cardiomyocytes exhibiting mitosis or a proliferative subpopulation leading to the development of the neonatal ventricular myocardium. The former proposal implies that proliferation of myocardial cells is a random event; consequently, a dispersed cell cycle signal would be detected in cardiac tissue. However, lineage tracing experiments with BrdU pulse treatment showed myocyte clusters in the developing heart, which are usually composed of at least 5 neighboring cells in a single section, instead of labeled, dispersed single cells. This evidence implicated de novo single-cell-derived cardiomyocyte clusters in neonatal murine heart development. In addition, a subpopulation of neonatal cardiomyocytes, which were likely mT-CMs, proliferated to form myocyte clusters and were different from the others. To further confirm this hypothesis that BrdU labeling myocyte clustsers were derived from single cell, we used spatial-temporal inducible genetic lineage tracing. Our assay is sensitive enough for single cell labeling via pulsed tamoxifen treatment. Single cell lineage tracing experiments revealed the capacity for clone formation by proliferative neonatal cardiomyocytes. Generally, one cluster was composed of approximately ten cardiomyocytes, derived from a single labeled cardiomyocyte. Surprisingly, similar results were observed in juvenile hearts as well, when the murine heart shows much less regenerative capacity in response to acute injury. This evidence suggests that a particular cardiac myocyte subpopulation, which is likely mT-CMs, exhibited sustained proliferation in the heart, providing cellular resources for cardiac growth.In comparison to typical cardiomyocytes, mT-CMs also showed distinguishing molecular features. Cardiac progenitor markers and stem cell markers were not expressed by mT-CMs, indicating mT-CMs are not in interphase during the process of stem cell differentiation into cardiomyocytes. Western blot analysis revealed that nuclear ?-catenin was highly enriched in neonatal ventricular tissues and gradually diminished over time. Immunofluorescence imaging further confirmed that the nuclear P-catenin signal was specifically localized in the mT-CM population. However, we did not observe expression of a canonical Wnt signal downstream target. More evidence is required to clarify the effect of non-canonical Wnt signaling in the mT-CM proliferation. Interestingly, mT-CMs specifically expressed GATA4, the essential fetal gene in cardiogenesis. Genetic evidence further confirmed this observation. In our opinion, GATA4 is a potential marker for labeling of the mT-CM subpopulation in neonatal myocardium.Functional evidence further confirms the existence of a heterogeneous mT-CM subpopulation in neonatal cardiomyocytes. Analysis of heart regeneration revealed different cardiac contributions from mT-CMs compared to typical cardiomyocytes. With a genetically inducible long-term lineage tracing system, we spatially and temporally labeled neonatal proliferating cardiomyocytes (putatively mT-CMs) and studied their cellular behavior and lineage contribution in heart regeneration. During the process of heart repair, we observed a large portion of tagged myocytes revert to neonatal-proliferating status, in which troponin or cytoskeleton proteins were localized beneath the cell membrane. This phenotype was reported by other groups and in multiple animal models, and was considered as a characteristic of proliferation because of the linearship of it and cell division marker expression. Moreover, a significant increase in the number of labeled cells was identified during the repair of infarct tissue. A fraction of these cells was identified as cardiomyocytes by their organized sarcomere structure. Although pulsed term long term lineage tracing could not label all of the proliferating cardiomyocytes in neonatal heart, remarkably, statistical results revealed a significant contribution of traced lineage to de nova cardiac tissues. This evidence demonstrated that a labeled proliferative myocyte subpopulation provides a primary contribution to cardiac regeneration in comparison to other myocyte populations. In summary, we characterized a novel cardiomyocyte subpopulation, which shows higher proliferative activity than typical cardiac myocytes and acts primarily by contributing to neonatal heart growth and cardiac regeneration.