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

Alexandra Raulf,Caroline Geisen,Wilhelm Röll,Annika Ottersbach,Hannes Horder,Stefan Jovinge,Laura Tarnawski,Bernd K Fleischmann,Michael Hesse

doi:10.1007/s00395-015-0489-2

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.

Highlights

The mammalian heart is composed of different cell types such as endothelial cells, fibroblasts, smooth muscle cells, and cardiomyocytes (CMs)
To establish a system that allows unequivocal identification of CM nuclei in vitro and in vivo, we cloned a fusion cDNA consisting of human histone 2B (H2B) and mCh behind the cardiac-specific a-Myosin-heavy-chain promoter (Fig. 1a)
After dissolving of the nuclear membrane in M-phase, transgenic systems using a nuclear localization signal encounter a translocation of the fluorescence protein to the cytoplasm, which is hard to detect for cell tracking image analysis algorithms [27]

Summary

Introduction

The mammalian heart is composed of different cell types such as endothelial cells, fibroblasts, smooth muscle cells, and cardiomyocytes (CMs). The latter provide the contractile properties of the heart muscle and CM loss in pathologies, such as myocardial infarction or cardiomyopathy, leads to functional impairment and eventually heart failure [16]. Basic Res Cardiol (2015) 110:33 transgenic CM-specific overexpression of fluorescence proteins. In those sections, non-CM nuclei in close proximity to CM cytoplasm cannot be distinguished from CM nuclei leading to false positives [1]

Methods

Results

Conclusion