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

Raulf A, Horder H, Tarnawski L, Geisen C, Ottersbach A, Röll W, Jovinge S, Fleischmann BK, Hesse M - Basic Res. Cardiol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology I, Life and Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, 53105, Bonn, Germany.

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.

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Visualization of CM nuclei after cardiac infarction. a Macroscopic pictures of an αMHC-H2B-mCh heart 10 days after cryoinjury. Close-up depicts border zone region. Scale bars overview: 1 mm, close-up: 200 µm. b Section of a cryo-infarcted αMHC-H2B-mCh heart. H2B-mCh signals are only abundant in intact CMs and are lost in the infarcted region. Scale bar 200 µm. (c, d) αMHC-H2B-mCh+ heart section stained with either α-actinin (c) or cardiac Troponin T (d) to identify CMs. Good co-localization between the cardiac markers and H2B-mCh in the border zone. Scale bars 20 µm. e H2B-mCh expression facilitates the identification of CM nuclei in cross sections of the border zone. Arrows depict CM nuclei, which are not centered in the cell. Scale bar 20 µm. f H2B-mCh expression facilitates the identification of individual surviving CMs (arrows) within the lesioned area. Scale bar 20 µm
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Fig7: Visualization of CM nuclei after cardiac infarction. a Macroscopic pictures of an αMHC-H2B-mCh heart 10 days after cryoinjury. Close-up depicts border zone region. Scale bars overview: 1 mm, close-up: 200 µm. b Section of a cryo-infarcted αMHC-H2B-mCh heart. H2B-mCh signals are only abundant in intact CMs and are lost in the infarcted region. Scale bar 200 µm. (c, d) αMHC-H2B-mCh+ heart section stained with either α-actinin (c) or cardiac Troponin T (d) to identify CMs. Good co-localization between the cardiac markers and H2B-mCh in the border zone. Scale bars 20 µm. e H2B-mCh expression facilitates the identification of CM nuclei in cross sections of the border zone. Arrows depict CM nuclei, which are not centered in the cell. Scale bar 20 µm. f H2B-mCh expression facilitates the identification of individual surviving CMs (arrows) within the lesioned area. Scale bar 20 µm

Mentions: After experimental myocardial infarction, it is challenging to clearly identify and quantify infarct size and surviving CMs in the border zone and infarcted area. This is especially true for islands of surviving CMs within the infarct. Additionally, staining for antigens in the infarct zone is difficult due to high background issues. We thought that these difficulties could be addressed by the prominent in vivo labeling of CM nuclei and therefore analyzed cardiac sections 10 days after the injury (n = 2). The lesioned area and the native myocardial tissue were easy to discriminate macroscopically by H2B-mCh signals (Fig. 7a). Staining of sections from lesioned transgenic hearts revealed a good demarcation of the infarct area and the viable myocardium with H2B-mCh+ nuclei (Fig. 7b). Specificity of the H2B-mCh signal was corroborated by staining for α-actinin (Fig. 7c) and cardiac troponin T (Fig. 7d).Fig. 7


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

Raulf A, Horder H, Tarnawski L, Geisen C, Ottersbach A, Röll W, Jovinge S, Fleischmann BK, Hesse M - Basic Res. Cardiol. (2015)

Visualization of CM nuclei after cardiac infarction. a Macroscopic pictures of an αMHC-H2B-mCh heart 10 days after cryoinjury. Close-up depicts border zone region. Scale bars overview: 1 mm, close-up: 200 µm. b Section of a cryo-infarcted αMHC-H2B-mCh heart. H2B-mCh signals are only abundant in intact CMs and are lost in the infarcted region. Scale bar 200 µm. (c, d) αMHC-H2B-mCh+ heart section stained with either α-actinin (c) or cardiac Troponin T (d) to identify CMs. Good co-localization between the cardiac markers and H2B-mCh in the border zone. Scale bars 20 µm. e H2B-mCh expression facilitates the identification of CM nuclei in cross sections of the border zone. Arrows depict CM nuclei, which are not centered in the cell. Scale bar 20 µm. f H2B-mCh expression facilitates the identification of individual surviving CMs (arrows) within the lesioned area. Scale bar 20 µm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4414935&req=5

Fig7: Visualization of CM nuclei after cardiac infarction. a Macroscopic pictures of an αMHC-H2B-mCh heart 10 days after cryoinjury. Close-up depicts border zone region. Scale bars overview: 1 mm, close-up: 200 µm. b Section of a cryo-infarcted αMHC-H2B-mCh heart. H2B-mCh signals are only abundant in intact CMs and are lost in the infarcted region. Scale bar 200 µm. (c, d) αMHC-H2B-mCh+ heart section stained with either α-actinin (c) or cardiac Troponin T (d) to identify CMs. Good co-localization between the cardiac markers and H2B-mCh in the border zone. Scale bars 20 µm. e H2B-mCh expression facilitates the identification of CM nuclei in cross sections of the border zone. Arrows depict CM nuclei, which are not centered in the cell. Scale bar 20 µm. f H2B-mCh expression facilitates the identification of individual surviving CMs (arrows) within the lesioned area. Scale bar 20 µm
Mentions: After experimental myocardial infarction, it is challenging to clearly identify and quantify infarct size and surviving CMs in the border zone and infarcted area. This is especially true for islands of surviving CMs within the infarct. Additionally, staining for antigens in the infarct zone is difficult due to high background issues. We thought that these difficulties could be addressed by the prominent in vivo labeling of CM nuclei and therefore analyzed cardiac sections 10 days after the injury (n = 2). The lesioned area and the native myocardial tissue were easy to discriminate macroscopically by H2B-mCh signals (Fig. 7a). Staining of sections from lesioned transgenic hearts revealed a good demarcation of the infarct area and the viable myocardium with H2B-mCh+ nuclei (Fig. 7b). Specificity of the H2B-mCh signal was corroborated by staining for α-actinin (Fig. 7c) and cardiac troponin T (Fig. 7d).Fig. 7

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Physiology I, Life and Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, 53105, Bonn, Germany.

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.

Show MeSH
Related in: MedlinePlus