Cardiovascular Research Advance Access originally published online on July 15, 2008
Cardiovascular Research 2008 80(2):219-226; doi:10.1093/cvr/cvn194
E2F2 expression induces proliferation of terminally differentiated cardiomyocytes in vivo
1 Department of Medicine III, University of Halle-Wittenberg, Ernst-Grube-Strasse 40, 06097 Halle, Germany
2 Max-Planck-Institute for Heart and Lung Research, Parkstrasse 1, 61231 Bad Nauheim, Germany
* Corresponding author. Tel: +49 345 557 2113; fax: +49 345 557 2684. E-mail address: henning.ebelt{at}medizin.uni-halle.de
Received 9 February 2008; revised 2 July 2008; accepted 10 July 2008
Time for primary review: 16 days
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionFundingReferences |
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Aims: In previous experiments we have demonstrated that expression of the transcription factors E2F2 and E2F4 is sufficient to induce proliferation of isolated primary cardiomyocytes from newborn rats and mice. We now wanted to analyse whether E2F2 or E2F4 are also able to promote cell cycle progression of adult cardiomyocytes in vivo, which unlike cardiomyocytes from newborn rodents lack the ability to undergo cell proliferation.
Methods and results: E2F2 or E2F4 was expressed in hearts of mice at different developmental stages using adenoviral vectors. Effects regarding proliferation, hypertrophy, and apoptosis were analysed on histological sections, and quantitative assessment of cell cycle regulatory genes was performed by real-time PCR (polymerase chain reaction) and western blot. We found that both E2F2 and E2F4 can stimulate hypertrophic cell growth of cardiomyocytes. However, only directed expression of E2F2 but not of E2F4 was sufficient to induce proliferation of cardiomyocytes. Expression of E2F2 in vivo did not increase the percentage of apoptotic cardiomyocytes but down-regulated the expression of the pro-apoptotic genes caspase-6 and apaf-1. Further analysis of the cell cycle regulatory machinery revealed that expression of E2F2 caused a strong induction of cyclin A and E while the expression of cyclin-dependent kinase inhibitors (CKIs) such as p21 was not affected.
Conclusion: We conclude that a limited induction of cardiomyocyte cell proliferation can be achieved by E2F2-mediated stimulation of cyclin A and E expression without a reduction of CKIs.
KEYWORDS Cardiomyocytes; E2F transcription factors; Cell cycle; Apoptosis; Cyclins; CKIs
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionFundingReferences |
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A broad spectrum of human heart diseases is based on the inability to replace dead or damaged cardiomyocytes during adulthood. In the last few years several attempts have been made to induce heart regeneration. The majority of experiments were based on stem cell transplantation strategies, which has resulted in clinical studies yielding partially encouraging outcomes.1–3 Other experimental approaches demonstrated that it is feasible to induce proliferation of cardiomyocytes by targeted interference with the mechanisms that normally maintain the blockade of the cell cycle. Pioneering experiments have shown that the activation of E2F transcription factors—either by oncoprotein-induced inhibition of pocket proteins4 or by directed expression of E2F15,6—is sufficient to induce (limited) proliferation of heart muscle cells. However, cell cycle re-entry was most often accompanied by induction of apoptosis thereby limiting a potential therapeutic value.6,7 In a previous study we revealed that it is feasible to induce proliferation of cardiomyocytes isolated from newborn rats and mice by targeted expression of E2F2 or E2F4 without stimulation of apoptosis.8 A clear limitation of our preceding study was the use of neonatal cardiomyocytes, which still show a certain level of proliferation since cardiomyocytes lose their ability to divide only within the first days after birth.9,10 Additionally, all results were obtained from cell culture experiments, which left open the validity of this approach for the intact heart. To overcome these obstacles we now analysed the effects of overexpression of E2F2 and E2F4 in the adult mouse heart in vivo. Here, we demonstrate that both E2F2 and E2F4 induce hypertrophic cell growth but only E2F2 not E2F4 is able to induce proliferation of terminally differentiated cardiomyocytes in vivo. E2F2 does not induce but inhibits apoptosis probably by reduction of the expression of the pro-apoptotic genes caspase-6 and apaf-1.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionFundingReferences |
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2.1 Adenoviral expression constructs
Adenoviruses encoding E2F2, E2F4, and EGPF have been described before.8 They were handled according to standard procedures with HEK293 as the packaging cell line. Viral titers were determined by plaque assay.8,11
2.2 Adenoviral transfection of mouse hearts in vivo
For all in vivo experiments transgenic mice carrying a nuclear localized LacZ-reporter gene under the control of the 
MHC-promotor12 were used to enable definitive identification of cardiomyocytes on histological sections (kindly provided by Dr. L.J. Field). Transfection of hearts of newborn mice has been described before in detail.11 Newborn mice were anaesthetized by cooling on ice for 
2 min and put in front of a cold light source to visualize the silhouette of the heart. Using a Hamilton syringe with a 26-gauge needle a total volume of 10 µL was injected into the thoracic cavity beside the heart at a left parasternal position. Finally, animals were re-warmed and put back to their mothers. After 13 days, the mice received a single i.p.-injection of BrdU [100 mg/kg body weight (BW)] and were then sacrificed the next day. The hearts were removed and either embedded for cryosectioning or shock-frozen in liquid nitrogen for RNA (ribonucleic acid) and protein isolation, respectively. Adult mice (>3 months) were anaesthetized with 2.5% isofluorane and mechanically ventilated, left-sided thoracotomy was performed in the fourth intercostal space. Under microscopic control the heart was gently uncovered and 10 µL of the virus suspension was injected two times in the left ventricular free wall and once in the interventricular septum.6,13,14 On day 3 and 4 after surgery, the mice received an i.p.-injection of BrdU (100 mg/kg BW) before they were finally sacrificed on day 5.
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
2.3 Histological analysis
Ten micro meter cryosections were obtained from hearts of neonatal and adult mice and stained either by X-gal or anti-LacZ to identify nuclei of cardiomyocytes. Immunoassays to detect BrdU incorporation (anti-BrdU; Vector Laboratories), phosphorylated histone-3 (anti-PhoshoH3; Upstate), AuroraB-kinase (anti-AuroraB, Abcam), and activated caspase-3 (anti-activ. casp3, Promega) were performed according to the manufacturer’s specifications. For terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labelling (TUNEL) cryosections were incubated with TUNEL labelling mixture (TdT and biotin-11-dUTP; Fermantas) at 37°C for 60 min. The resulting signal was visualized using VECTASTAIN Elite ABC Kit (Vector Laboratories).
For all quantifications using immunocytochemistry (ICC), a minimum of four slides containing cryosections from distinct regions of the heart were analysed. All hearts were embedded in long axis orientation to facilitate identification of right ventricular and left ventricular free walls and the interventricular septum after sectioning.
Expressions of E2F2 and E2F4 were confirmed by western blot and ICC (antibodies: sc-632 and sc-866, Santa Cruz Biotechnology). Hypertrophy of cardiomyocytes was determined by measuring myocyte cross-sectional area as described.15
2.4 Ribonucleic acid isolation and quantitative real-time polymerase chain reaction
Total RNA was isolated using Trizol according to standard techniques. Complementary deoxyribonucleic acid (cDNAs) were synthesized using oligo-dT-primers as described.8 Real-time polymerase chain reaction (PCR) was performed in 25 µL total volume containing 1 µL cDNA, 0.1U Taq-polymerase, 5 nmol forward primer, 5 nmol reverse primer, 10 µmol dNTPs, 0.25 µL x100 fluorescein, and 0.5 µL x10 SybrGreen I (Sigma) as described.8 Primer sequences and specific amplification conditions can be obtained from the authors upon request.
2.5 Protein isolation and western blot
For protein isolation, the hearts were lysed in RIPA-buffer [50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% sodium dodecyl sulphate (SDS), 1 mM ethylenediaminetetraacetic acid containing 1 mM phenylmethylsulphonyl fluoride], 5 µg/mL Aprotinin and 5 µg/mL Leupeptin (all from Sigma). Thirty microgram of protein lysates were separated on 10% SDS–PAGE(polyacrylamide gel electrophoresis) gels and transferred onto nitrocellulose membranes (Invitrogen Life Technologies, Groningen, The Netherlands). Immunoreactive proteins were visualized with corresponding horseradish peroxidase-conjugated secondary antibodies on Hyperfilm (GE Healthcare) using the SuperSignal West Pico or West Femto detection solutions (Perbio Science). Blots were scanned and analysed using ImageJ software (NIH). The following antibodies were used: anti-cyclin A (sc-751), anti-cyclin D2 (sc-593), and anti-cyclin E (sc-481; all from Santa Cruz Biotechnology); anti-cyclin D1, anti-cyclin D3, anti-cyclin-dependent kinase (CDK4), anti-p15INK4, and anti-p27Kip1(all from Cell Signaling Technology); anti-p21Waf (BD).
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionFundingReferences |
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3.1 Targeted expression of E2F2 counteracts cell cycle exit of cardiomyocytes and re-induces proliferation of adult cardiomyocytes
In a first series of experiments we determined whether expression of E2F2 or E2F4 counteracts the cell cycle exit of cardiomyocytes in the perinatal period. Adenoviral vectors encoding E2F2, E2F4, or a control vector (EGFP) were injected into the thoracic cavity of newborn mice, which results in a lasting and efficient gene expression in the heart.11 The directed expression of E2F2, E2F4, or EGFP was confirmed by ICC and western blot, respectively (Figure 1). Fourteen days after application of the viruses we found a significant increase in the heart weights from animals that received Ad-E2F2 (Table 1). Unexpectedly, this phenomenon was not observed after E2F4 expression although E2F4 is sufficient to induce S-phase entry in cultured cardiomyocytes from newborn rodents in vitro.8 In order to further explore the mechanisms responsible for the increase in heart weight induced by E2F2, we determined the number of proliferating and apoptotic cardiomyocytes in histological sections (Figure 2). Fourteen days after targeted expression of E2F2 we detected a significant increase in the number of cardiomyocytes within S-phase as demonstrated by incorporation of BrdU (Table 2). We also found a significant increase in the number of phosphorylated histon-H3 positive cardiomyocytes (Table 2) and cardiomyocytes expressing AuroraB-kinase (Table 2), which suggested that these cardiomyocytes completed the entire cell cycle including mitosis and cytokinesis. Expression of E2F4 resulted only in a slight increase in BrdU- and phoshorylated histon-H3 positive cardiomyocytes in comparison with untreated control animals and EGFP-treated mice, which was statistically not significant. We also investigated whether the E2F transcription factors have an impact on hypertrophic cell growth of cardiomyocytes. Interestingly, both E2F2- and E2F4-induced hypertrophy of cardiomyocytes resulting in enlarged cross-sectional areas of myocytes (Figure 3).
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P < 0.05 vs. control; #P < 0.05 vs. EGFP.
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Since forced induction of proliferation often coincides with increased rates of apoptotic cell death5–7 we determined the effects of E2F2 and E2F4 on apoptosis. Apopotic cells were visualized by staining of activated caspase-3, which identifies cells that are irrevocably committed to apoptosis.16 Histological analysis revealed that E2F2 significantly reduced the number of apoptotic cells in comparison with EGFP-expressing mice while directed expression of E2F4 led to a marked increase in apoptosis (Table 3). To further distinguish between apoptotic cardiomyocytes and non-muscle cells, we used the TUNEL assay in combination with cardiomyocyte specific X-gal staining. This approach revealed that pro- apoptotic effects of E2F4 were restricted to non-cardiomyocytes while cardiomyocytes were not adversely affected by E2F2 or E2F4 (Table 3). To define the molecular pathway that might control E2F4-mediated apoptosis we analysed the expression of apoptosis-related genes. Interestingly, we detected a clear increase in the mRNA expression of caspase-6 and apaf-1 in hearts infected with Ad-E2F4 and Ad-EGFP, which was not seen in E2F2-treated animals (Table 4). Furthermore, we found a repression of the pro-aopototic gene bax in hearts infected with adenoviruses encoding E2F-transcription factors and the EGFP-expressing control vector compared with uninfected controls animals (Table 4). Apparently, E2F2 specifically repressed transcription of the pro-apoptotic genes caspase-6 and apaf-1 while repression of the pro-apoptotic gene bax was due to an unspecific effect of the adenoviral backbone.
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So far, our results clearly demonstrated that E2F2 led to an increase in cardiomyocyte proliferation in 14-day-old mice without a concomitant increase in apoptosis. Although no signs of cardiomyocyte proliferation (neither S-phase activity nor mitosis) can be detected in hearts of untreated control mice at P14 (Table 2) it was not possible to distinguish whether the expression of E2F2 led to a delay of cell cycle exit or to a real re-induction of proliferation. Therefore, we turned to the adult situation and tested the effects of E2F2-expression on proliferation of cardiomyocytes in 3-month-old mice. As shown in Table 5, directed expression of E2F2 was sufficient to induce DNA-synthesis in terminally differentiated adult cardiomyocytes although the increase in the number of mitotic cells induced by E2F2 did not reach statistical significance. Similar to the situation in young mice at P14 we did not find signs of increased apoptosis induced by E2F2-activity.
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3.2 E2F2 induces expression of cyclins D3, A, and E but does not affect expression of CDK inhibitors
In principle, it was possible that induction of cell cycle activity in cardiomyocytes after E2F2-expression was due to stimulation of pro-proliferative cyclins or to suppression of CDK inhibitors. Especially p21WAF has been reported to act as one of the major mediators of cell cycle arrest under physiological conditions in cardiomyocytes.17 p21WAF is also a known downstream target of p5318, which is of particular importance since E2F transcription factors induce expression of p53 in various settings.19
To determine the relative ratios of pro- and anti-proliferative effectors in cardiomyocytes we measured the mRNA expression levels of several cyclins in E2F-infected and control animals. We found that several cyclins were induced after directed expression of E2F2 (Table 6). Interestingly, we also found a slight increase in cyclins A, E, and D3 after injection of the control vector – although the changes induced by Ad-EGFP were clearly lower compared with Ad-E2F2 and did not reach statistical significance.
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Western blot analysis revealed that the protein concentrations of cyclin A, D3, and E were significantly elevated in hearts of E2F2-expressing mice (Figures 4 and 5) while no clear differences in the expression of any cyclin-dependent kinase inhibitors (CKIs) (p15, p21, p27) were found. Taken together our results clearly argue for a stimulation of cyclin gene expression by E2F2 and against an active repression of inhibitory cell cycle regulators (Figure 5).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionFundingReferences |
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Our experimental data clearly demonstrate that E2F2 is able to stimulate de novo proliferation of adult cardiomyocytes, which might have an impact on the therapeutic strategies to stimulate cardiac regeneration. Conceptually, our approach differs from previous studies using neonatal cells8 or transgenic mice, which constantly express pro-proliferative effectors and hence might generate populations of continuously cycling cardiomyocytes, which have never ceased cell division. Yet, a de novo induction of cell proliferation in the adult heart appears to be instrumental for any therapeutic intervention.
Previously, we reported that E2F1, E2F2, E2F3, and E2F4 were all able to stimulate S-phase entry in primary cultures of neonatal cardiomyocytes although induction of DNA synthesis was accompanied by increased apoptosis in case of E2F1 and E2F3 but not E2F2 and E2F4.8 These findings raised the question whether E2F2 or E2F4 might be used in vivo to unlock the cell cycle in adult cardiomyocytes. Surprisingly, we found that only E2F2 was sufficient to induce proliferation in postmitotic cardiomyocytes while E2F4 was not. None of the parameter that were increased after expression of E2F2 such as elevated DNA-synthesis, enhanced expression of mitotic markers on histological sections and increase in heart weight was seen in E2F4-treated animals. In parallel, expression of E2F2 did not induce apoptosis of cardiomyocytes but on the contrary led to the mRNA down-regulation of caspase-6 and apaf-1, which resembled the situation in cultured cardiomyocytes.
In our analyses, we have used nuclear antigens to determine both the number of apoptotic (TUNEL) and proliferating (BrdU, phosphoH3, AuroraB) cardiomyocytes (Table 3). Since we have used transgenic reporter mice in which the nuclei of all cardiomyocytes were labelled by the LacZ gene (MHC-nlsLacZ) we were able to unequivocally differentiate between cardiomyocytes and non-muscle cells, which is indispensable to enable a reliable analysis of the cell cycle activity of cardiomyocytes. It is interesting to note that we detected a similar percentage of cardiomyocytes which have passed through S-phase in control samples (control: 0.002% at day 14; <0.002% in adult mice) as the group of L.J. Field who has established this transgenic reporter mouse strain (<0.003% in adult mice20). In contrast, other groups that relied only on the less strict histological identification of cardiomyocytes occasionally reported mitotic indices several magnitudes higher (
2% at day 14;21 see also22).
In our experiments, the percentage of double-stained cardiomyocytes (e.g. all nuclei double-positive for LacZ and BrdU in relation to all LacZ-positive nuclei) describes the amount of cardiomyocytes which underwent S-phase (BrdU), mitosis (phoshoH3), cytokinesis (AuroraB), or apoptosis (TUNEL). The unequivocal identification of cardiomyocytes, which undergo cell cycle progression and apoptosis, represents a clear advantage compared with studies that used less robust antibody labelling techniques and cytoplasmatic antigens such as activated caspase-3 to detect mitotic and apoptotic cardiomyocytes, respectively. On histological sections the majority of nuclei of cardiomyocytes are not in the same plane as the cytoplasm as pointed out before.20 This leads to the effect that significantly higher numbers of individual cardiomyocytes will be counted when cyoplasmic rather than nucleic markers are used (factor: 3.4 in Ref.20). This phenomenon makes it difficult to calculate reliably the ‘net effect’ of (nuclear) proliferation and (cytoplasmic) apoptosis. We have eliminated this restriction by using exclusively nuclear parameters to quantify all aspects of cell cycle activity and apoptosis. Hence it became possible to calculate the ‘net effect’ between proliferation and cell death indicating that expression of E2F2 favours proliferation of cardiomyocytes to a certain extent.
Another example for complex interaction of cellular processes is the increase in heart weight after expression of E2F2 but not E2F4. It seems likely that the increase in heart weights was caused by increased proliferation of both cardiomyocytes and non-muscle cells in combinaton with hypertrophy of individual cardiomyocytes since the relatively low increase in the number of cardiomyocytes was not sufficient to explain the total increase in heart mass. In the case of E2F4 this effect was apparently neutralized by enhanced apoptosis although it is possible that the observed differences in apoptosis between E2F2 and E2F4 may indicate variations in the immune response against the viral infections. In this context it seems noteworthy that we also found signs of increased apoptosis after injection of the control Ad-EGFP virus, which might be due to certain toxicity of the adenoviral vector and to the cytopathogenic effect of EGFP, which is able to cause cardiomyopathy after continous high-level expression in transgenic models.23
Cell cycle progression depends on the activation and inactivation of cyclin-dependent kinases, which in turn are affected by the concerted induction and degradation of regulatory cyclins. Proliferation of isolated cardiomyocytes does not appear to depend strictly on the induction of D-cyclins since directed expression of E2Fs was sufficient to overcome the restriction point even in the absence of a noteworthy stimulation of cyclin D-expression8 – a finding which had also been described in other cells and settings before.24 In the in vivo experiments presented here, E2F2 induced a strong increase in cyclin D3 expression both on the mRNA and protein level in postmitotic hearts. Nevertheless it is difficult to say whether this stimulation of D-cyclins was indispensable for the induction of proliferation of cardiomyocytes. Interestingly, we again observed a striking correlation between the ability of individual E2Fs to induce proliferation of cardiomyocytes and the expression levels of cyclin A and E as described in our cell culture experiments before. Our results suggest that the increased expression of cyclins A, D, and E and not a repression of CKIs are responsible for the E2F-induced proliferation of cardiomyocytes since we did not find evidence for a reduced expression of p21WAF, which is considered to be one of the major components responsible for cell cycle arrest in cardiomyocytes.17 This was a somewhat surprising finding because p21WAF is a known downstream target of E2F transcription factors25 and of p53,18 which can be induced and activated by E2Fs.19 However, it is not possible to confine these results solely to cardiomyocytes since our protein lysates also contain material from cardiac non-muscle cells.
Taken together our results indicate that E2F2 is an appropriate candidate to stimulate proliferation of cardiomyocytes in vivo in adult hearts. E2F2 does not only induce proliferation of cardiomyocytes but also reduces expression of pro-apoptotic genes such as caspase-6 and apaf-1. These features clearly favour E2F2 for potential therapeutic strategies in comparison with other S-phase inducing genes described so far.
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Funding |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionFundingReferences |
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This work was supported by the Deutsche Forschungsgemeinschaft (EB419/1-1 to H.E.), the Excellence Cluster Cardio-Pulmonary System, the Max-Planck-Society, and the Wilhelm-Roux-Program of the Martin-Luther-University (NBL3-5/41 to H.E.).
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Acknowledgements |
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The authors thank Nicole Glaubitz for skilful technical assistance. They wish to thank Dr L.J. Field (Wells Centre for Paediatric Research, Indianapolis, IN, USA) for the generous gift of
MHC-nlsLacZ mice. They are also indebted to J.R. Nevins (Howard Hughes Medical Institute, Durham, USA) for the donation of the E2F viruses. Conflict of interest: The authors wish to declare that there are no real or apparent conflicts of interests associated with this work.
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References |
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