Limits...
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.

Show MeSH

Related in: MedlinePlus

Assessment of cardiac morphology in H2B-mCh mice and H2B-mCh expression during embryonic development. a Exclusion of hypertrophic effects of the transgene in Langendorff dissociated adult CMs. N = 3 animals per group; n > 110 CMs per group. b Comparison of heart weight to tibia length between 10-week-old male αMHC-H2B-mCh transgenic and wt littermates (n = 3). c Sections of H2B-mCh and wt hearts stained with Picrosirius red for collagen. Scale bar 100 µm. d Macroscopic pictures of E12.5, E15.5, and E18.5 H2B-mCh transgenic hearts illustrate the expression of the fusion protein in atrial and some ventricular CM nuclei. Scale bar 500 µm. e Section of an E15.5 H2B-mCh heart (left picture) displays strong expression in atrial CM nuclei (right upper picture) and weak expression in some ventricular trabecular CMs (right lower picture). Scale bars 200 µm (overview), 50 µm (close-ups)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4414935&req=5

Fig3: Assessment of cardiac morphology in H2B-mCh mice and H2B-mCh expression during embryonic development. a Exclusion of hypertrophic effects of the transgene in Langendorff dissociated adult CMs. N = 3 animals per group; n > 110 CMs per group. b Comparison of heart weight to tibia length between 10-week-old male αMHC-H2B-mCh transgenic and wt littermates (n = 3). c Sections of H2B-mCh and wt hearts stained with Picrosirius red for collagen. Scale bar 100 µm. d Macroscopic pictures of E12.5, E15.5, and E18.5 H2B-mCh transgenic hearts illustrate the expression of the fusion protein in atrial and some ventricular CM nuclei. Scale bar 500 µm. e Section of an E15.5 H2B-mCh heart (left picture) displays strong expression in atrial CM nuclei (right upper picture) and weak expression in some ventricular trabecular CMs (right lower picture). Scale bars 200 µm (overview), 50 µm (close-ups)

Mentions: H2B-mCh+ nuclei were easily detectable by flow cytometry and contributed to 25.0 ± 4.2 % of total heart nuclei (Fig. 2g). To assess the accuracy of the aforementioned nuclear CM markers, we exemplarily stained nuclei from H2B-mCh+ hearts for PCM-1. Flow cytometric analysis of the stained nuclei revealed a distinct CM-population for H2B-mCh, while the PCM-1 staining displayed a broad spreading (Fig. 2h); the correlation between the two markers was 90 %, (Fig. 2h). Staining for PCM-1 on tissue sections of adult mouse hearts also revealed a good overlap with the H2B-mCh signal (Fig. 2i). However, not all of the CM nuclei could be clearly identified by PCM-1 staining (Fig. 2i, arrow). To further exclude possible side effects of the transgene on CMs, we performed morphometric analysis on Langendorff dissociated adult CMs and excluded a hypertrophic effect of transgene expression (Fig. 3a). Also the percentage of binucleated adult CMs in H2B-mCh mice [89.5 ± 2 % (n = 4)] matched published numbers [21, 43]. Accordingly, 10-week-old transgenic mice did not show any obvious differences in heart weight (Fig. 3b) compared to their wildtype littermates. Transgene stability was monitored for 6 generations in mice from a mixed 129S6/SvEvTac × C57BL/6Ncr × CD-1 background and for 2 generations of back-crosses into C57BL/6 background. No changes in specificity or intensity of H2B-mCh expression were noted and no detrimental effects on cardiac function and hemodynamics were observed (Table 1); in addition, no fibrosis (Fig. 3c) could be detected.Fig. 3


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)

Assessment of cardiac morphology in H2B-mCh mice and H2B-mCh expression during embryonic development. a Exclusion of hypertrophic effects of the transgene in Langendorff dissociated adult CMs. N = 3 animals per group; n > 110 CMs per group. b Comparison of heart weight to tibia length between 10-week-old male αMHC-H2B-mCh transgenic and wt littermates (n = 3). c Sections of H2B-mCh and wt hearts stained with Picrosirius red for collagen. Scale bar 100 µm. d Macroscopic pictures of E12.5, E15.5, and E18.5 H2B-mCh transgenic hearts illustrate the expression of the fusion protein in atrial and some ventricular CM nuclei. Scale bar 500 µm. e Section of an E15.5 H2B-mCh heart (left picture) displays strong expression in atrial CM nuclei (right upper picture) and weak expression in some ventricular trabecular CMs (right lower picture). Scale bars 200 µm (overview), 50 µm (close-ups)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Assessment of cardiac morphology in H2B-mCh mice and H2B-mCh expression during embryonic development. a Exclusion of hypertrophic effects of the transgene in Langendorff dissociated adult CMs. N = 3 animals per group; n > 110 CMs per group. b Comparison of heart weight to tibia length between 10-week-old male αMHC-H2B-mCh transgenic and wt littermates (n = 3). c Sections of H2B-mCh and wt hearts stained with Picrosirius red for collagen. Scale bar 100 µm. d Macroscopic pictures of E12.5, E15.5, and E18.5 H2B-mCh transgenic hearts illustrate the expression of the fusion protein in atrial and some ventricular CM nuclei. Scale bar 500 µm. e Section of an E15.5 H2B-mCh heart (left picture) displays strong expression in atrial CM nuclei (right upper picture) and weak expression in some ventricular trabecular CMs (right lower picture). Scale bars 200 µm (overview), 50 µm (close-ups)
Mentions: H2B-mCh+ nuclei were easily detectable by flow cytometry and contributed to 25.0 ± 4.2 % of total heart nuclei (Fig. 2g). To assess the accuracy of the aforementioned nuclear CM markers, we exemplarily stained nuclei from H2B-mCh+ hearts for PCM-1. Flow cytometric analysis of the stained nuclei revealed a distinct CM-population for H2B-mCh, while the PCM-1 staining displayed a broad spreading (Fig. 2h); the correlation between the two markers was 90 %, (Fig. 2h). Staining for PCM-1 on tissue sections of adult mouse hearts also revealed a good overlap with the H2B-mCh signal (Fig. 2i). However, not all of the CM nuclei could be clearly identified by PCM-1 staining (Fig. 2i, arrow). To further exclude possible side effects of the transgene on CMs, we performed morphometric analysis on Langendorff dissociated adult CMs and excluded a hypertrophic effect of transgene expression (Fig. 3a). Also the percentage of binucleated adult CMs in H2B-mCh mice [89.5 ± 2 % (n = 4)] matched published numbers [21, 43]. Accordingly, 10-week-old transgenic mice did not show any obvious differences in heart weight (Fig. 3b) compared to their wildtype littermates. Transgene stability was monitored for 6 generations in mice from a mixed 129S6/SvEvTac × C57BL/6Ncr × CD-1 background and for 2 generations of back-crosses into C57BL/6 background. No changes in specificity or intensity of H2B-mCh expression were noted and no detrimental effects on cardiac function and hemodynamics were observed (Table 1); in addition, no fibrosis (Fig. 3c) could be detected.Fig. 3

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