Midbody Positioning and Distance Between Daughter Nuclei Enable Unequivocal Identification of Cardiomyocyte Cell Division in Mice

Abstract

Rationale: New strategies in the field of cardiac regeneration are directed at identifying proliferation-inducing substances to induce regrowth of myocardium. Current screening assays utilize neonatal cardiomyocytes and markers for cytokinesis, such as Aurora B-kinase. However, detection of cardiomyocyte division is complicated because of cell cycle variants, in particular, binucleation. Objective: To analyze the process of cardiomyocyte binucleation to identify definitive discriminators for cell cycle variants and authentic cardiomyocyte division. Methods and Results: Herein, we demonstrate by direct visualization of the contractile ring and midbody in Myh6 (myosin, heavy chain 6)-eGFP (enhanced green fluorescent protein)-anillin transgenic mice that cardiomyocyte binucleation starts by formation of a contractile ring. This is followed by irregular positioning of the midbody and movement of the 2 nuclei into close proximity to each other. In addition, the widespread used marker Aurora B-kinase was found to also label binucleating cardiomyocytes, complicating the interpretation of existing screening assays. Instead, atypical midbody positioning and the distance of daughter nuclei on karyokinesis are bona fide markers for cardiomyocyte binucleation enabling to unequivocally discern such events from cardiomyocyte division in vitro and in vivo. Conclusions: The 2 criteria provide a new method for identifying cardiomyocyte division and should be considered in future studies investigating cardiomyocyte turnover and regeneration after injury, in particular in the postnatal heart to prevent the assignment of false positive proliferation events.

Length‐independent telomere damage drives post‐mitotic cardiomyocyte senescence

“…Based on our observations, we would suggest that function is maintained as a result of compensatory mechanisms that include CM renewal and replacement, as indicated by the increased frequency of EdU-and Ki-67-positive mononuclear CMs Data information: Asterisks denote a statistical significance using two-tailed t-test (B, C, K) or Mann-Whitney test (D, J). ***P < 0.001; **P < 0.01; *P < 0.05. and the observation of CMs expressing Aurora B symmetrically between nuclei (Hesse et al, 2018). Following senescence clearance, we also identified an increase in CM karyokinesis.…”

“…As CMs may undergo karyokinesis in the absence of cytokinesis resulting in bi-or multi-nucleation, EdU alone is not a marker of cell division. Expression of Aurora B, a component of the contractile ring required for cytoplasmic separation during cell division, was only observed in CMs from hearts where senescent cells had been cleared, where it was located in mid-body between nuclei, indicative of cytokinesis (Hesse et al, 2018; Fig 8H). Our data indicate senescent-cell clearance is accompanied by a significant increase of both multi-and mono-nucleated CMs (Fig 8H-J).…”

“…We next evaluated the percentage of CMs positive for proliferation marker Ki-67 and found a similar increase following navitoclax treatment and in aged INK-ATTAC mice treated with AP ( Figs EV5H and 8K). Expression of Aurora B, a component of the contractile ring required for cytoplasmic separation during cell division, was only observed in CMs from hearts where senescent cells had been cleared, where it was located in mid-body between nuclei, indicative of cytokinesis (Hesse et al, 2018; Fig 8H). Altogether, our data support that senescent CMs are involved in age-associated cardiac hypertrophy and fibrosis and that their clearance may induce a compensatory CM regeneration.…”

Isolation of cardiomyocytes undergoing mitosis with complete cytokinesis

“…[54][55][56][57] The contractile actomyosin ring is formed during CM binucleation, which constricts the central spindle resulting in midbody formation. [57][58][59] While SPG20 plays a role in midbody abscission, 28 we tested whether our dual-MB strategy was capable of distinguishing cell divisions from binucleated events. P3 rat-CMs were treated with serum and an increase in the percentage of binucleated CMs was observed ( Fig.…”

Interventions in WNT Signaling to Induce Cardiomyocyte Proliferation: Crosstalk with Other Pathways

“…It is important to note that degradation of the contractile apparatus of the CM is required for the completion of the cell cycle with cytokinesis to yield two daughter cells (Jopling et al, 2010;Szibor et al, 2014). Novel methods have recently been proposed to identify true CM proliferation, including midbody positioning combined with a correct spacing of the daughter nuclei (Hesse et al, 2018), CM-specific expression of fluorescence ubiquitination-based cell cycle indicator (Alvarez et al, 2019) and multiple-isotope mass spectrometry (Senyo et al, 2013). Using the latter technique, Senyo et al calculated that ∼3.2% of the CMs around the infarct area had undergone division at 8 weeks post-MI in mice (Senyo et al, 2013), indicating that proliferation of these cells actually can occur in response to cardiac ischemia.…”