The typical remodelling process after cardiac injury is scarring and compensatory hypertrophy. The limited regeneration potential of the adult heart is due to the post-mitotic status of cardiomyocytes (CMs), which are mostly binucleated. Nevertheless, there is evidence for CM turnover in the adult heart, but its extent is still under debate. One technical limitation of quantitations is the unequivocal identification of CMs and of CM cell division.
In order to enable a clear identification of CM nuclei in-vivo, we have developed a transgenic mouse line in which a fusion protein of the human histone 2B and the red fluorescence protein mCherry is specifically and persistently expressed in CM nuclei (αMHC-H2B-mCh). The fluorescence label allowed the investigation of CM percentages in native tissue slices, which properly reflect the cellular composition of the heart. We focused on regional (atrium versus left and right ventricle, apical versus basal slices) and developmental-stage dependent changes in the percentage of CM nuclei and binuclearity. We therefore analyzed time-points before/during and after terminal differentiation of CMs (postnatal day 3 (P3), P7 and 9 weeks). In addition, we also investigated endoreduplication and acytokinetic mitosis using time lapse microscopy in postnatal CMs to better understand cell biological mechanisms leading to terminal differentiation.
As currently huge efforts are invested for the search of substances that increase the regeneration potential of the heart, we established a novel screening assay for cell-cycle modifying substances in isolated, postnatal CMs. We crossed the αMHC-H2B-mCh with the CAG-eGFPanillin mouse line, which marks cell-cycle activity with a high resolution of M-phase. Analysis of binuclearity and of different eGFPanillin subcellular localizations will be helpful to understand, whether CMs complete cytokinesis. As a proof of principle we investigated the effects of cell cycle activating micro RNAs199 and 590.
Thus, our double transgenic mouse line will be useful to examine the plasticity of mono- and binuclear CMs and to unravel cell biological mechanisms leading to terminal differentiation of CMs.