Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.
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.
Institute of Physiology I, Life and Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, 53105, Bonn, Germany.
Alexandra
A
Raulf
Hannes
H
Horder
Laura
L
Tarnawski
Caroline
C
Geisen
Annika
A
Ottersbach
Wilhelm
W
Röll
Stefan
S
Jovinge
Bernd K
BK
Fleischmann
Michael
M
Hesse
D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology; D005434:Flow Cytometry; D006321:Heart / Q000196:embryology / Q000254:growth & development; D006657:Histones; D006801:Humans; D008164:Luminescent Proteins; D051379:Mice; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D061848:Optical Imaging / Q000379:methods*; D011993:Recombinant Fusion Proteins; D014162:Transfection; D000097573:Red Fluorescent Protein
D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein