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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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Assay for screening the effects of cell cycle modifying substances on CMs. a Scheme depicts the cross-breeding of αMHC-H2B-mCh mice with the CAG-eGFP-anillin proliferation indicator mouse. Depending on the cell cycle status, CMs (H2B-mCh+ nuclei) express eGFP-anillin in different subcellular localizations. b Fluorescence pictures of dissociated αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts (P2), transfected with the cell cycle-modifying miR-199 and a miR-NC. Scale bars 100 µm. c Quantification of eGFP-anillin expression in miR-treated CMs 72 h after transfection (n ≥ 3). d Examples of CMs with cytokinesis-indicating eGFP-anillin localizations (arrows). Scale bars 100 µm. e Staining of eGFP-anillin/H2B-mCh CMs with the proliferation marker AURKB Aurora B kinase. Note the overlap between the M-phase specific localization of eGFP-anillin (green) contractile ring and Aurora B kinase (white). Scale bar 10 µm. f Analysis of different eGFP-anillin localizations in CMs after miR-treatment (n ≥ 3). g Portion of binuclear CMs 72 h after miR-transfection (n ≥ 3)
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Fig8: Assay for screening the effects of cell cycle modifying substances on CMs. a Scheme depicts the cross-breeding of αMHC-H2B-mCh mice with the CAG-eGFP-anillin proliferation indicator mouse. Depending on the cell cycle status, CMs (H2B-mCh+ nuclei) express eGFP-anillin in different subcellular localizations. b Fluorescence pictures of dissociated αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts (P2), transfected with the cell cycle-modifying miR-199 and a miR-NC. Scale bars 100 µm. c Quantification of eGFP-anillin expression in miR-treated CMs 72 h after transfection (n ≥ 3). d Examples of CMs with cytokinesis-indicating eGFP-anillin localizations (arrows). Scale bars 100 µm. e Staining of eGFP-anillin/H2B-mCh CMs with the proliferation marker AURKB Aurora B kinase. Note the overlap between the M-phase specific localization of eGFP-anillin (green) contractile ring and Aurora B kinase (white). Scale bar 10 µm. f Analysis of different eGFP-anillin localizations in CMs after miR-treatment (n ≥ 3). g Portion of binuclear CMs 72 h after miR-transfection (n ≥ 3)

Mentions: A potentially very interesting strategy to regenerate lost heart muscle is based on re-induction of proliferation in pre-existing CMs. For this purpose preferentially neonatal stages are used, as at this stage some proliferative activity is still present in CMs and these cells appear to be more amenable to cell cycle-modulating interventions. To establish an in vitro and in vivo screening system for such factors and substances, we crossed the αMHC-H2B-mCh mice with mice expressing the CAG-eGFP-anillin transgene [15], which visualizes cell cycle activity with high resolution of M-phase. In this double transgenic mouse line, CMs can be identified by nuclear expression of H2B-mCh and their cell cycle status by localization of the eGFP-anillin fusion protein (Fig. 8a). As proof of concept, we used postnatal CMs derived from double transgenic mice to test the influence of substances on cell cycle activity and proliferative behavior in vitro. We chose P2 mice for the experiment, as this age demarcates the time frame in which CMs are still proliferative, but will soon undergo the transition to binucleation, starting around P4. As the experiment lasts 3 days, which corresponds to P3–P5, both regular proliferation as well as cell cycle variations will take place, thereby providing a challenge for the detection system to correctly distinguish between the two states. Ventricles from P2 mice were dissociated and the cells were transfected with either microRNA 199, (miR-199) which was reported to strongly enhance the rate of proliferation in postnatal CMs [10] or scramble miRs as a negative control. By counting eGFP-anillin expressing CMs, which could be identified according to their H2B-mCh fluorescence, increased cell cycle activity after treatment with miR-199 compared to the scramble miR control could be directly detected without further stainings (Fig. 8b). While in transfections with scramble miRs, a basal cell cycle activity (6.6 %) could be determined, this was significantly (p = 0.0015) enhanced (19.1 %) after transfection with miR-199 (Fig. 8c). Importantly, the fraction of non-nuclear localizations of the eGFP-anillin signal, such as contractile rings and midbodies (Fig. 8d), which are indicative for cell division [15] and were verified by staining for Aurora B kinase (Fig. 8e), was increased after miR-199 treatment compared to the control (Fig. 8f). Further we noticed an increase in the fraction of binucleated CMs in miR-199-treated CMs (Fig. 8g). As the strength of our system is live-tracking of cell cycle progression, we performed video microscopy, which revealed both CMs’ divisions (Suppl. Video 2) as well as binucleation (Suppl. Videos 3 + 4) taking place. Binucleation was determined as 17.7 % of total CMs for miR-199 treatment and only 9.8 % for controls. This experiment demonstrates that the αMHC-H2B-mCh/CAG-eGFP-anillin system not only enables the identification of proliferation, but also of cell cycle variations such as binucleation. Therefore, it is ideally suited for screening of cell cycle-modifying substances in CMs.Fig. 8


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)

Assay for screening the effects of cell cycle modifying substances on CMs. a Scheme depicts the cross-breeding of αMHC-H2B-mCh mice with the CAG-eGFP-anillin proliferation indicator mouse. Depending on the cell cycle status, CMs (H2B-mCh+ nuclei) express eGFP-anillin in different subcellular localizations. b Fluorescence pictures of dissociated αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts (P2), transfected with the cell cycle-modifying miR-199 and a miR-NC. Scale bars 100 µm. c Quantification of eGFP-anillin expression in miR-treated CMs 72 h after transfection (n ≥ 3). d Examples of CMs with cytokinesis-indicating eGFP-anillin localizations (arrows). Scale bars 100 µm. e Staining of eGFP-anillin/H2B-mCh CMs with the proliferation marker AURKB Aurora B kinase. Note the overlap between the M-phase specific localization of eGFP-anillin (green) contractile ring and Aurora B kinase (white). Scale bar 10 µm. f Analysis of different eGFP-anillin localizations in CMs after miR-treatment (n ≥ 3). g Portion of binuclear CMs 72 h after miR-transfection (n ≥ 3)
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Fig8: Assay for screening the effects of cell cycle modifying substances on CMs. a Scheme depicts the cross-breeding of αMHC-H2B-mCh mice with the CAG-eGFP-anillin proliferation indicator mouse. Depending on the cell cycle status, CMs (H2B-mCh+ nuclei) express eGFP-anillin in different subcellular localizations. b Fluorescence pictures of dissociated αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts (P2), transfected with the cell cycle-modifying miR-199 and a miR-NC. Scale bars 100 µm. c Quantification of eGFP-anillin expression in miR-treated CMs 72 h after transfection (n ≥ 3). d Examples of CMs with cytokinesis-indicating eGFP-anillin localizations (arrows). Scale bars 100 µm. e Staining of eGFP-anillin/H2B-mCh CMs with the proliferation marker AURKB Aurora B kinase. Note the overlap between the M-phase specific localization of eGFP-anillin (green) contractile ring and Aurora B kinase (white). Scale bar 10 µm. f Analysis of different eGFP-anillin localizations in CMs after miR-treatment (n ≥ 3). g Portion of binuclear CMs 72 h after miR-transfection (n ≥ 3)
Mentions: A potentially very interesting strategy to regenerate lost heart muscle is based on re-induction of proliferation in pre-existing CMs. For this purpose preferentially neonatal stages are used, as at this stage some proliferative activity is still present in CMs and these cells appear to be more amenable to cell cycle-modulating interventions. To establish an in vitro and in vivo screening system for such factors and substances, we crossed the αMHC-H2B-mCh mice with mice expressing the CAG-eGFP-anillin transgene [15], which visualizes cell cycle activity with high resolution of M-phase. In this double transgenic mouse line, CMs can be identified by nuclear expression of H2B-mCh and their cell cycle status by localization of the eGFP-anillin fusion protein (Fig. 8a). As proof of concept, we used postnatal CMs derived from double transgenic mice to test the influence of substances on cell cycle activity and proliferative behavior in vitro. We chose P2 mice for the experiment, as this age demarcates the time frame in which CMs are still proliferative, but will soon undergo the transition to binucleation, starting around P4. As the experiment lasts 3 days, which corresponds to P3–P5, both regular proliferation as well as cell cycle variations will take place, thereby providing a challenge for the detection system to correctly distinguish between the two states. Ventricles from P2 mice were dissociated and the cells were transfected with either microRNA 199, (miR-199) which was reported to strongly enhance the rate of proliferation in postnatal CMs [10] or scramble miRs as a negative control. By counting eGFP-anillin expressing CMs, which could be identified according to their H2B-mCh fluorescence, increased cell cycle activity after treatment with miR-199 compared to the scramble miR control could be directly detected without further stainings (Fig. 8b). While in transfections with scramble miRs, a basal cell cycle activity (6.6 %) could be determined, this was significantly (p = 0.0015) enhanced (19.1 %) after transfection with miR-199 (Fig. 8c). Importantly, the fraction of non-nuclear localizations of the eGFP-anillin signal, such as contractile rings and midbodies (Fig. 8d), which are indicative for cell division [15] and were verified by staining for Aurora B kinase (Fig. 8e), was increased after miR-199 treatment compared to the control (Fig. 8f). Further we noticed an increase in the fraction of binucleated CMs in miR-199-treated CMs (Fig. 8g). As the strength of our system is live-tracking of cell cycle progression, we performed video microscopy, which revealed both CMs’ divisions (Suppl. Video 2) as well as binucleation (Suppl. Videos 3 + 4) taking place. Binucleation was determined as 17.7 % of total CMs for miR-199 treatment and only 9.8 % for controls. This experiment demonstrates that the αMHC-H2B-mCh/CAG-eGFP-anillin system not only enables the identification of proliferation, but also of cell cycle variations such as binucleation. Therefore, it is ideally suited for screening of cell cycle-modifying substances in CMs.Fig. 8

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