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  • br Mechanisms of cardiomyocyte regeneration pose challenges

    2018-11-12


    Mechanisms of cardiomyocyte regeneration pose challenges for investigation Cardiomyocyte generation in the adult mammalian heart is a slow process compared with the blood, skin, and the digestive system, which makes it difficult to characterize turnover dynamics. In situ time lapse imaging of the beating mammalian heart at cellular resolution is technically challenging (Hashimoto et al., 2014; Hesse et al., 2012). Myocardial biopsies from patients provide only a small amount of tissue for analysis. The cellular heterogeneity and close spatial packing of cells in the myocardium can obscure the identification of cardiomyocyte nuclei from neighboring non-cardiomyocytes (Soonpaa and Field, 1997; Soonpaa et al., 2012). Perhaps the most challenging aspect is that cardiomyocytes exhibit non-proliferative cell cycles that increase the DNA content without cell division. Despite these intrinsic biological features that complicate studying regeneration, several new methods have enabled the advance of our understanding of cardiomyocyte proliferation.
    Classical and new methods have advanced cardiac regeneration research Labeled thymidine and thymidine analogs are stably incorporated into the genome during DNA synthesis and can be used to record purchase PHA-793887 events. The use of tritiated thymidine is limited by potential radioactive toxicity due to long-term administration and by lower spatial resolution of visualization by autoradiography, compared with confocal microscopy (Soonpaa and Field, 1997). The early manual analysis of autoradiographs of tritiated thymidine in isolated cardiomyocytes fits with recent studies almost 2 decades later (Soonpaa and Field, 1997; Li et al., 1996; Senyo et al., 2013; Soonpaa et al., 1996). Thymidine analogs such as bromo-deoxyuridine (BrdU) have also been administered for up to several weeks for detecting cardiomyocyte cell cycle activity. Though high concentrations of BrdU can affect cell proliferation and differentiation, adverse effects were not reported with long-term labeling (Wilson et al., 2008; Malliaras et al., 2013). Similar to Soonpaa et al. with a tritium label, Li et al., using a single BrdU injection, observed progressive loss of cardiomyocyte-DNA synthesis in rats within ten days of birth (Li et al., 1996; Soonpaa et al., 1996). However, it should be noted that using Ki67 as a marker, Walsh et al. detected cell cycle activity in cardiomyocyte nuclei in mice up to 21days of age (Walsh et al., 2010). Naqvi et al. detected BrdU uptake and markers of mitosis and cytokinesis in cardiomyocytes in 13–15day-old mice (Naqvi et al., 2014). In the adult heart, Soonpaa et al. examined mice 2h after administration of tritiated thymidine (3 injections, 12-hour intervals) and determined a 0.0005% labeling frequency of cardiomyocytes expressing a transgenic marker from the α-MHC-promoter (Soonpaa and Field, 1997). More recently, the use of long term BrdU labeling demonstrated a low basal activity of cardiomyocyte proliferation though an order higher than previously reported (Li et al., 1996; Malliaras et al., 2013). Antibodies raised against markers of the cell cycle can be used for visualization by microscopy (e.g. Ki67 for G1, S, G2 and M-phase, phospho-histone3 for M-phase, Aurora B kinase for cytokinesis) (Senyo et al., 2013; Malliaras et al., 2013; Ellison et al., 2013; Mollova et al., 2013). Antibody-based detection has the advantage of broad applicability, but is potentially susceptible to false-positives and -negatives. These issues can be addressed with appropriate negative controls. It should be noted that Ki-67 is present in G1-, S-, G2-, and M-phase of the cell cycle. Thus, for mechanistic studies, Ki-67 assays should be combined with other, more specific cell cycle markers. Automated systems provide an unbiased approach for sampling large numbers of cardiomyocytes, but so far, they require dispersed cells. Flow cytometry (FACS) and laser scanning cytometry (LSC) offer high throughput methods to detect and quantify antibody labeling of isolated cardiomyocytes or nuclei (Malliaras et al., 2013; Walsh et al., 2010; Mollova et al., 2013). Recently, we examined cardiomyocyte cycling in humans using the M-phase marker phospho-histone3 and used LSC for read-out. This demonstrated a decrease in cardiomyocyte proliferation between birth and 20years of life in humans (Mollova et al., 2013). We compared the LSC results with visual quantification of H3P-positive cardiomyocyte nuclei on stained cryosections, which reproduced the pattern of decreasing H3P-activity between birth and 20years (Mollova et al., 2013). LSC in humans and FACS in mice showed agreement in that the frequency of cardiomyocyte cell cycle activity decreases after birth (Walsh et al., 2010; Mollova et al., 2013).