Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status

Abstract

Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific αMHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77–90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-015-0489-2) contains supplementary material, which is available to authorized users.

Deciphering the Epigenetic Code of Cardiac Myocyte Transcription

“…Twelve-week-old male C57BL/6 N mice, 12-week-old female Myh6-H2B-mCherry transgenic mice 9 and Wistar rats were used. Experimental infarction by ligation of the left anterior descending coronary artery was performed as described.…”

Mending a Faltering Heart

“…To this end, a recently reported transgenic system composed of an α-myosin heavy chain positive:H2B-mCherry transgene and a CAG (a strong synthetic promoter)-eGFP (enhanced green fluorescent protein)-anillin transgene could be useful for unequivocal identification of cardiomyocyte nuclei and analysis of its cell cycle. 53 Compared with zebrafish hearts, mammalian hearts are of a more complex design in structure (4-chambered versus 2-chambered), tissue organization (fibroblasts account for most of the mammalian heart mass but are rare in the fish heart), mechanical properties (higher blood pressure in mammals), and electric properties to handle the higher metabolic demand of mammals. One may speculate that in such complex hearts, the large-scale dedifferentiation and electric decoupling of cardiomyocytes as seen during zebrafish heart regeneration may not be compatible with organismal survival (because of lethal arrhythmia or reduced ability to evade predation).…”

Caught Red-Handed

“…3 However, reliable detection of rare cell cycling events in adult cardiomyocytes proved to be difficult, mostly because of the tightly packed arrangement of multiple cell types in the myocardium 4 and the abundance of noncardiomyocytes, which constitute 60% of all cell nuclei in the neonatal mouse heart and ≈70% in young adult mice. 5 Pioneering research from Loren Field's group has used genetic labeling of cardiomyocytes in mice using an myosin heavy chain-nuclear localized p-galactosidase reporter transgene in combination with tritiated thymidine incorporation to overcome this problem, 3 whereas others have used antibodies against pericentriolar material 1 as an accurate marker to label human and murine cardiomyocyte nuclei. 6 However, such approaches are either cumbersome and time-consuming or rely on the use of multiple antibodies.…”

“…Therefore, other researchers have integrated different read-outs (ie, identification of a specific cell type and monitoring of cell cycle activity) into a single system by using cardiomyocyte-specific promoters driving the expression of cell cycle markers, which provided a detailed map of cell cycle dynamics during development in living cardiomyocytes. 5,7 Hirai at al 8 have now come up with another simple and robust system to monitor S-to M-phase cell cycle activity in murine cells. The approach relies on the conditional activation of a cyclinA2-enhanced green fluorescent protein (eGFP) fusion gene by expression of Cre-recombinase.…”

“…However, combination of the CAG-anillin:eGFP line with mice that express a fusion protein of human histone 2B and the red fluorescence protein mCherry under the control of the α-myosin heavy chain promoter allows trustworthy detection of dividing cardiomyocytes (Figure [B]). 12 Activation of the cyclinA2-eGFP reporter relies on the cell type-specific expression of Cre-recombinase, which makes it difficult to combine the technique with other Crerecombinase-based labeling approaches. For example, Kimura et al 13 have recently devised a clever system for fate mapping of hypoxic cardiomyocytes, which are present at low numbers in the adult heart but contribute widely to formation of new cardiomyocytes.…”