RESUMO
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.
Assuntos
Divisão do Núcleo Celular , Núcleo Celular/fisiologia , Proliferação de Células , Miócitos Cardíacos/fisiologia , Animais , Aurora Quinase B/metabolismo , Biomarcadores/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Regeneração , Fatores de TempoRESUMO
Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.