To proliferate or not to proliferate.

Cardiomyocytes in mammalian hearts undergo significant reduction in their cell proliferation capacity soon after birth, which is not disputed. However, it remains uncertain whether the adult heart contains a limited capacity for cell proliferation. Through the use of immunohistochemical staining methods, it has been reported that adult mouse cardiomyocytes can re-enter the cell cycle and duplicate. However, the origin of the cell-cycle-positive cells was not determined. Others reported permanent inhibition of cell-cycle progression in adult cardiomyocytes in mice. With advances in lineage tracing technology, a recent genetic labelling study demonstrated that stem or precursor cells did not contribute significantly to the formation of cardiomyocytes during normal ageing up to 1 year in the mouse. In contrast, precursor cells may participate in the formation of new cardiomyocytes after cardiac injury. In humans, high myocyte proliferation rates that could result in the exchange of all cardiomyocytes within 5 years have been reported by employing immunostaining technology on sections of human patients who died of acute myocardial infarction. In contrast, using C dating analyses in humans, Bergmann et al. reported that cardiomyocytes are renewed with a gradual decrease from 1% turning over annually at the age of 25 to 0.45% at the age of 75. According to the best-fit models that allow turnover rates to change with age, at the age of 50, 55% of the cardiomyocytes remain from the time around birth and 45% have been generated later. To resolve these conflicting results, it is necessary to employ comprehensive approaches that complement each other and to analyse the data quantitatively. The study performed by Walsh et al. appearing in this issue describes thorough analyses of cell-cycle control to address this conflicting problem. Walsh et al. provide strong evidence that cardiomyocytes in mice withdraw from the cell cycle soon after birth, and, therefore, cardiomyocytes in the murine adult heart do not proliferate under normal conditions. To investigate the proliferative capacity of cardiomyocytes, Walsh et al. performed purification of cardiomyocyte by FACS sorting of cTroponinT (cTnT)-positive cells. The authors then extracted nuclei from sorted cells to measure DNA content in order to accurately measure DNA synthesis of cardiomyocytes by considering the binuclear nature of cardiomyocytes. FACS sorting and immunostaining for BrdU uptake or Ki-67 indicated that a range of 12–23% (depending on different methods) of embryonic cardiomyocytes were positive and 1–8% by neonatal day 7, which became almost non-detectable by day 14. These results demonstrate a complete block of cell-cycle entry to G1 and S phase, indicating a permanent cell-cycle withdrawal. This study also demonstrated that polyploidization of murine myocytes occurred predominantly in the first week of postnatal life. Mouse cardiomyocytes began to be binucleated soon after birth and reached nearly 98% of binucleation between postnatal days 5 and 8. These data indicate a dramatic increase in uncoupling of karyokinesis and cytokinesis shortly after birth, which supports the notion of complete postnatal cell-cycle arrest. Gene profiling experiments were performed to compare embryonic, neonatal, and adult hearts using purified cardiomyocytes isolated from a-MHC-eGFP transgenic mice. Multiple genes responsible for cell-cycle regulation were enriched in the neonatal hearts, which seems to correlate well with cell-cycle withdrawal. The strengths of this study are (i) the use of multiple methods for cell-cycle detection such as immunohistochemistry involving BrdU uptake and Ki-67 antibodies, (ii) the use of flow cytometry for better quantification, (iii) the use of a-MHC-eGFP transgenic mice to mark cardiomyocytes, (iv) the comparison of gene expression levels among embryonic, neonatal, and adult cardiomyocytes by gene profiling experiments, and (v) the use of isolated cardiomyocyte nuclei for accurate quantification, since the high percentage of adult mouse cardiomyocytes are binucleated. Moreover, to pave a clear path for cardiac regeneration, one should define whether adult cardiomyocytes are capable of proliferating. In this regards, it is advantageous to use cTnT-positive cells as a cardiac marker, which selects differentiated cardiomyocytes and eliminates precursor or stem cells that may not express cTnT. Gene profiling experiments comparing gene expression patterns among purified cardiomyocytes from embryonic, neonatal, and adult hearts would provide crucial information on cell-cycle regulation during transitions from a proliferative to a non-proliferative period. However, a potential cardiac regenerative capacity cannot be addressed in this study. By collecting cells that express cTnT prior to examining BrdU incorporation or Ki-67 labelling, potential cardiac progenitor/stem cells that do not express cTnT have been excluded. Although this study strongly supports permanent withdrawal of postnatal cardiomyocytes from the cell cycle, the next step would be to investigate whether this phenomenon is altered under pathological conditions using the same methods employed in the current study. In addition, further analysis of gene profiling data would be necessary to