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Cardiovascular Research Advance Access originally published online on July 2, 2008
Cardiovascular Research 2008 80(2):181-190; doi:10.1093/cvr/cvn183
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Cardiomyocyte proliferation and protection against post-myocardial infarction heart failure by cyclin D1 and Skp2 ubiquitin ligase

Mimi Tamamori-Adachi1,{dagger},*, Hiromitsu Takagi2,{dagger}, Kimio Hashimoto1, Kazumichi Goto2, Toshinori Hidaka2, Uichi Koshimizu2, Kazuhiko Yamada1, Ikuko Goto1, Yasuhiro Maejima3, Mitsuaki Isobe3, Keiichi I. Nakayama4, Norio Inomata2 and Shigetaka Kitajima1,*

1 Department of Biochemical Genetics, Medical Research Institute and Laboratory for Gene Structure and Regulation, School of Biomedical Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
2 Biomedical Research Laboratories, Asubio Pharma Co., Ltd, 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan
3 Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
4 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

* Corresponding authors. Tel: +81 3 5803 5823; fax: +81 3 5803 0248. E-mail address: mtam.bgen{at}mri.tmd.ac.jp (M.T.-A.), kita.bgen{at}mri.tmd.ac.jp (S.K.)

Received 28 August 2007; revised 9 June 2008; accepted 26 June 2008

Time for primary review: 30 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Cyclins and other cell-cycle regulators have been used in several studies to regenerate cardiomyocytes in ischaemic heart failure. However, proliferation of cardiomyocytes induced by nuclear-targeted cyclin D1 (D1NLS) stops after one or two rounds of cell cycles due in part to accumulation of p27Kip1, an inhibitor of cyclin-dependent kinase (CDK). Thus, expression of S-phase kinase-associated protein 2 (Skp2), a negative regulator of p27Kip1, significantly enhances the effect of D1NLS and CDK4 on cardiomyocyte proliferation in vitro. Here, we examined whether Skp2 can also improve cardiomyocyte regeneration and post-ischaemic cardiac performance in vivo.

Methods and results: Wistar rats underwent ischaemia/reperfusion injury by ligation of the coronary artery followed by injection of adenovirus vectors for D1NLS and CDK4 with or without Skp2. Enhanced proliferation of cardiomyocytes in the presence of Skp2 was demonstrated by increased expression of Ki67, a marker of proliferating cells (1.95% vs. 4.00%), and mitotic phosphorylated histone H3 (0.24% vs. 0.58%). Compared with rats that received only D1NLS and CDK4, expression of Skp2 improved left ventricular function as measured by the maximum and minimum rates of change in left ventricular pressure, the left ventricle end-diastolic pressure, left ventricle end-diastolic volume index, and the lung/body weight ratio.

Conclusion: Expression of Skp2 enhanced the effect of D1NLS and CDK4 on the proliferation of cardiomyocytes and further contributed to improved post-ischaemic cardiac function. Skp2 might be a versatile tool to improve the effect of cyclins on post-ischaemic regeneration of cardiomyocytes in vivo.

KEYWORDS Cardiac regeneration; Cell cycle; Mitotic proliferation; Cyclin D1; Skp2


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Mammalian cardiomyocytes irreversibly withdraw from the cell cycle soon after birth and undergo terminal differentiation. DNA synthesis, karyokinesis, and cytokinesis do not occur in rat cardiomyocytes 3 weeks after birth.1–5 Therefore, cardiac injury causes permanent myocardial loss and cardiac dysfunction. Recent evidence suggests that isolation and amplification of extra- or intra-cardiac stem cells may contribute to improved myocardial function.6–11 In contrast, several laboratories including ours have proposed another strategy to induce the proliferation of cardiomyocytes by re-activation through the manipulation of cell-cycle regulators.3,5,12–19

Progression of the mammalian cell cycle is regulated by a combination of positive and negative regulators, cyclins and cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs).20,21 During the G1 phase, cyclin D1 and other D-type cyclins assemble with their catalytic partners, CDK4 and CDK6, and accumulate in the nuclei. We have previously shown that cyclin D1 is induced by mitogenic stimuli, but its nuclear import is impaired in terminally differentiated cardiomyocytes. The expression of nuclear localizing signal (NLS)-tagged cyclin D1 (D1NLS) and CDK4 triggers the cell cycle, indicating that the impairment of nuclear expression of cyclin D1 is one of the barriers of the cell cycle in cardiomyocytes.22 However, the cells cease to proliferate after only one or two cell cycles, at least in part, by accumulating the CDK inhibitor p27kip1 in the nuclei.23 CDKIs negatively regulate the progression of the cell cycle by inhibiting the activity of cyclin–CDK complexes. CIP/KIP family proteins, including p27, suppress the activities of cyclin A/CDK2 and cyclin E/CDK2, and mediate the exit from the cell cycle. p27 Protein accumulates in quiescent non-proliferating cells, but is ubiquitylated in proliferating cells by SCFSkp2 (Skp1–Cul1–Rbx1–Skp2) ubiquitin ligase complex and degraded, relieving the blockade of cell-cycle progression. Therefore, degradation of p27 by Skp2 plays a crucial role in the progression of the cell cycle.24,25 In contrast, we showed that the degradation of p27 is remarkably suppressed at least in part due to increased degradation of Skp2 in cardiomycytes, and additional expression of Skp2 on D1NLS/CDK4 promotes stable cardiomyocyte proliferation in vitro.23 However, it is still uncertain whether adult cardiomyocytes can divide in vivo to protect or prevent cardiac function by D1NLS/CDK4 and Skp2 expression.

In the present study, we showed that cyclin D1 and Skp2 induce the expression of various genes regulating the cell cycle and DNA replication to effectively promote cardiomyocyte division in vivo and protect cardiac function in a rat myocardial infarction (MI) model. Our findings provide a novel insight into cell-cycle control mechanisms in adult cardiomyocytes and demonstrate that manipulation of positive and negative regulators could be a rational option for the treatment of ischaemic heart failure.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Animals
All animal procedures were approved by the Institutional Animal Care and Use Committee in Tokyo Medical and Dental University. 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).

2.2 Cell culture and cell-cycle analysis
Rat neonatal cardiomyocytes were isolated from 3-day-old postnatal Sprague–Dawley rats, purified by Percoll gradient centrifugation, and cultured as previously described.22,23,26 The cardiomyocytes were incubated in Minimum Essential Medium (MEM) supplemented with 5% calf serum for 24 h at 37°C, after which the medium was replaced with serum-free medium. Following a 48-h incubation, the cells were subjected to various analyses. Adult rat cardiomyocytes were isolated from 4-week-old Sprague–Dawley rats, using No Langendorf Perfusion Cardiomyocyte Isolation Kit (ac-7018, CELLUTRONTM Life Technologies). For the cell-cycle analysis, cells grown on glass coverslips were fixed in 70% ethanol, and stained with anti-tropomyosin antibody, followed by treatment with propidium iodide (50 µg/mL) and RNase A (500 µg/mL). The DNA content of cells positive for tropomyosin was analysed using a laser scanning cytometry (LSC 101, Olympus) as described previously.22,23

2.3 Adenoviruses and infection
Adenoviruses for expressing nuclear localization signal-tagged cyclin D1 (Ad-D1NLS), CDK4 (Ad-CDK4), Skp2 (Ad-Skp2), and LacZ (Ad-LacZ) as a control were regulated by CAG promoter and prepared as described previously.22,23 Cardiomyocytes in culture were infected with the indicated adenoviruses at 100 multiplicity of infection (m.o.i.).

2.4 RNA isolation and microarray analysis
Cells were infected with the indicated adenoviruses for 48 h. Total RNA was then isolated by acid-guanidinium method using a kit from Qiagen. After amplification and labelling of cRNA with Cy3 or Cy5, microarray analysis was performed using custom-made in situ-synthesized 60-mer oligo microarrays containing 22 500 features including controls (Agilent Technologies).

2.5 Real-time reverse transcription-polymerase chain reaction
Isolated total RNA was reverse-transcribed using the PrimeScript Reverse Transcriptase Kit (TaKaRa BIO). Primers used are shown in Supplementary material online, Table S1. Polymerase chain reactions (PCRs) were performed using ABI PRISM 7900HT Sequence detection systems (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems). The thermal cycling conditions composed of an initial denaturation step at 50°C for 2 min and 95°C for 10 min followed by 40 cycles of PCR under the following conditions: 95°C for 15 s, 60°C for 20 s, and 72°C for 30 s.

2.6 Ischaemia/reperfusion injury and gene transfer
We used 8-week-old, male Wistar rats. Under pentobarbital (50 mg/kg, i.p.) anaesthesia, the left main coronary artery of the rat was ligated for 30 min and then reperfused. Just before reperfusion, a combination of adenoviruses (50 µL volume) was injected directly into five different points of both the ischaemic and border zone using a 30 G syringe needle. The rats were divided into four groups, and were injected with the following combinations of adenoviruses: LacZ group, 4 x 109 pfu of Ad-LacZ; D1NLS group, 1 x 109 pfu each of Ad-D1NLS and Ad-CDK4, and 2 x 109 pfu of Ad-LacZ; and Skp2 group, 1 x 109 pfu each of Ad-D1NLS, Ad-CDK4, Ad-Skp2, and Ad-LacZ. The sham group underwent thoracotomy without coronary ligation. Two hours post-reperfusion, a blood sample was withdrawn from each rat, and the concentration of plasma cardiac troponin T (cTnT) was measured using a Cardiac Reader (Roche Diagnostics), to determine the extent of cardiac damage (Table 1). Echocardiography, hemodynamic analysis, and measurement of infarct size were performed 6 weeks after reperfusion.


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  		Table 1 Plasma cTnT concentration and body weight

 
2.7 Immunohistochemistry
Cells cultured on glass coverslips were fixed with 4% paraformaldehyde. Adult rat hearts were fixed with 4% paraformaldehyde by perfusion and sectioned at 10 µm thickness. Staining was performed as described (see Supplementary material online, Table S2). Immune complexes were detected with Alexa 488, Alexa 568 (Molecular Probes, Invitrogen), or Ci5 (Jackson ImmunoResearch) conjugating second antibodies. In the case of triple staining, sections were stained with primary antibody covalently labelled with Alexa 555 using the Zenon One Mouse IgG1 Labelling Kit (Molecular Probes) after double staining. Nuclei were stained with 4', 6-diamino-2-phenilindole (DAPI). Images were obtained with the laser-scanning confocal image system (LSM510, ZEISS).

2.8 5-Bromo-2'-deoxyuridine incorpolation
Intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg, Roche) were given at 3 days after manipulation to direct DNA synthesis. Twenty-four hours after injections, hearts were fixed with 4% paraformaldehyde by perfusion. The sections of tissues were denatured by incubation in 1 mol/L hydrochloric acid at 65°C for 30 min and probed with anti-BrdU (Roche) and Troponin I antibodies. Immune complexes were detected by second antibodies conjugated with Alexa 488 and Alexa 568, respectively.

2.9 Terminal transferase-mediated dUTP nick end-labelling assay
For analysis for apoptotic cells, sections were subjected to terminal transferase-mediated dUTP nick end-labelling (TUNEL) assay using the in situ Apoptosis Detection Kit (TaKaRa BIO) and then stained with Troponin I antibody/anti-rabbit antibody conjugated with Alexa 568 and 1 µg/mL of DAPI.

2.10 Echocardiography
Images were recorded using a 10–12-MHz phased-array transducer (Toshiba Medical). End-diastolic and end-systolic diameters of the left ventricle (EDD and ESD, respectively) were derived from M-mode tracings, obtained from parasternal short-axis views. End-diastolic and end-systolic areas of the left ventricle (EDA and ESA, respectively) were obtained from the long-axis view. Ketamine (50 mg/kg, i. p.) plus xylazine (10 mg/kg, i. p.) were used for anaesthesia.

2.11 Haemodynamic analysis
The right carotid artery was cannulated with a micromanometer-tipped catheter (SPC 320, Millar Instruments) that advanced into the left ventricle via the aorta for recording pressures and dP/dt. Pentobarbital sodium (55 mg/kg, i. p.) was used for anaesthesia.

2.12 Passive pressure–volume relation curve
In vitro LV pressure–volume curve was measured as described previously,27 with some modifications. The heart was arrested by an injection of KCl and then quickly excised. A double-lumen catheter was inserted into the left ventricle, which was isolated by ligating the atrioventricular groove. Reproducible pressure–volume curves were generated over a pressure range of 0–30 mmHg by infusing saline at a speed of 0.72 mL/min. The volume index (volume/body weight ratio) of the respective LV end-diastolic pressure [LVEDP, indicated as operated left ventricle endo-diastolic volume index (LVEDVI)] was assessed.

2.13 Measurement of infarct size
Hearts were fixed with 10% formalin and sectioned from each of six equally spread levels (atrium through apex). Sections were stained with Masson's trichrome, digitally imaged, and infarct size was determined as the mean percentage of epicardial and endocardial circumference occupied by scar tissue.

2.14 Statistical analysis
Numerical values are expressed as mean ± SEM. Statistical analysis of histologic data was performed with the Student's t-test. Infarct size, echocardiographic, and haemodynamic parameters were analysed using the Turkey–Kramer method.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 D1NLS, CDK4, and Skp2 upregulate cell-cycle regulators in neonatal cardiomyocytes in vitro
Genome-wide transcriptional program during the cell cycle has been investigated in a wide range of organisms.28,29 Thus, rat neonatal cardiomyocytes in culture expressing D1NLS and CDK4 were subjected to comprehensive gene expression profiling by microarray analysis. Among 22 500 genes analysed, 283 (1.31%) and 138 (0.63%) were significantly (>2-fold changes) up- and down-regulated, respectively. As summarized in Table 2, a gene subset, the expression of which was temporally regulated at G1/S transition, S phase, G2/M transition, and M phase, was significantly activated by D1NLS/CDK4. In contrast, house-keeping genes and cardiac-specific genes were not affected (Table 2, Supplementary material online, Tables S3 and S4). The results of the microarray analysis were confirmed further by quantitative RT–PCR analysis of selected cell-cycle regulatory genes. As illustrated in Figure 1, expression of Ki67, cyclin A2 and E, and cdc2 was induced by D1NLS and CDK4. Orc6 and geminin, both essential factors for DNA replication in the S phase, were also up-regulated by D1NLS and CDK4. Activation of cell-cycle regulators correlated with the progression of the cell cycle since more pronounced effects were observed in cardiomyocytes expressing D1NLS/CDK4 and Skp2 than in those expressing D1NLS/CDK4 alone (Figure 1A–C). On the other hand, expression of the cell-cycle inhibitor p27 was not affected by D1NLS/CDK4, or Skp2, consistent with our previous report showing that p27 is regulated by a post-transcriptional mechanism.23 Intringuingly, however, expression of other cell-cycle inhibitors, including p18, p21, p57, and CDK inhibitor 3 (CDKN3), was concomitantly increased in cells expressing D1NLS/CDK4 and Skp2.


Figure 1
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  		Figure 1 Gene expression of cell-cycle regulators in neonatal rat cardiomyocytes in culture. Cells were infected with the indicated adenoviruses for 48 h. (A) Cell-cycle analysis using Laser scan cytometre. (B and C) Total RNA purified from cells was subjected to quantitative RT–PCR. (B) Specific expression of infected adenoviruse-encoded genes in cells. (C) Gene expression of cell-cycle regulators. Primers are shown in Table S1 (see Supplementary material online). Data were normalized by GAPDH. Data are average of three independent experiments; bars represent standard error (SE) between experiments. *P < 0.05 vs. LacZ cells. {dagger}P < 0.05 vs. D1NLS cells.

 


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  		Table 2 Change of cell-cycle regulatory genes expression

 
3.2 Immunocytological evidence of cell-cycle progression through mitosis in D1NLS/CDK4 or D1NLS/CDK4/Skp2 neonatal cardiomyocytes in culture
To test whether rat neonatal cardiomyocytes expressing D1NLS, CDK4, and Skp2 in vitro can activate the cell cycle, immunohistochemical analysis was carried out using antibodies against Ki67, phosphorylated histone H3 (H3P), and Aurora B together with cardiomyocyte-specific markers. Ki67 is a marker of proliferating cells.30 As shown in Figure 2A, the proportion of Ki67-positive cells among those expressing toropomyosin reached 90% at 48 h, but subsequently decreased at 72 h after transfection. The proportion of Ki67-positive cells was higher in cardiomyocytes infected with D1NLS/CDK4/Skp2 than in those infected with D1NLS/CDK4 (Figure 2A). Next, mitotic cardiomyocytes were detected by immunostaining mitotic markers, phosphorylated histone H3 (H3P), and Aurora B (Figure 2B–E).31,32 The percentage of H3P- or Aurora B-positive cardiomyocytes was higher in D1NLS/CDK4/Skp2-treated cells than in D1NLS/CDK4-treated cells at each time point (Figure 2B and E). We next examined whether D1NLS/CDK4/Skp2 has a positive effect on the proliferation of adult cardiomyocytes. The proportion of cardiomyocytes positive for Ki67 was about 80% 72 h after infection with D1NLS/CDK4 and subsequently decreased to 62% at 96 h. In D1NLS/CDK4/Skp2 cells, the proportion of Ki67-positive cells was significantly higher compared with that in D1NLS/CDK4 cells 120 h after infection (Figure 2F and G). Moreover, in adult cardiomyocytes treated with D1NLS/CDK4/Skp2, mitotic H3P-positive cells were observed (Figure 2H). Taken together, these data show that Skp2 had more pronounced effects on the stimulation and maintenance of cardiomyocyte proliferation.


Figure 2
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  		Figure 2 Immunohistochemical analysis of Ki67, H3P, and Aurora B in cultured rat cariomyocytes. Neonatal (A–E) or adult (F–H) rat cardiomyocytes in culture infected with LacZ (control), D1NLS, CDK4, and Skp2 adenoviruses were immunostained with Ki67, H3P, and Aurora B antibodies. Percentage of Ki67-positive (A), H3P-positive (B), and Aurora B-positive (E) neonatal cardiomyocytes. (C) Identification of cardiomyocytes undergoing mitosis. Upper: H3P (green); Tropomyosin (red). Lower: Aurora B (green); Troponin I (red); DAPI (blue). (D) Identification of cardiomyocytes undergoing cytokinesis. Arrowheads represent Aurora B. Aurora B (green); Troponin I (red); DAPI (blue). (F) Identification of Ki67-positive adult cardiomyocytes (arrowheads). Ki67 (green); Tropomyosin (red); DAPI (blue). (G) The graph represents the percentage of Ki67-positive cardiomyocytes. (H) Identification of H3P-positive adult cardiomyocytes. H3P (green); Tropomyosin (red); DAPI (blue). Scale bars represent 10 µm in (C), and 20 µm in (D), (F), and (H). Data are from 300 cardiac-specific factors (Tropomyosin or Troponin I) positive-cells, and represent mean ± SD, n = 5 fields/time point per group (A, B, E, and G).

 
3.3 D1NLS, CDK4, and Skp2 activate cell cycle of rat cardiomyocytes in vivo
To examine whether D1NLS, CDK4, and Skp2 promote cell cycle in adult cardiomyocytes in vivo, a rat ischaemia/reperfusion MI model was employed. To assess the degree of tissue damage, we monitored the plasma concentration of cTnT. There was a positive correlation between the levels of plasma cTnT 2 h and the infarct size 24 h after reperfusion (see Supplementary material online, Figure S1). Animals that had plasma cTnT <5 (each one in the LacZ, D1NLS, and Skp2 groups), however, were excluded from further studies since infarcts in these animals were too small to develop cardiac dysfunction later. As shown in Table 1, there was no significant difference in the cTNT level among the three MI groups, suggesting that these animals had similar ischaemic/reperfusion insults. To evaluate the number of cells in the cell cycle, we compared the expression of Ki67 in cells positive for myc-tag attached to the C-terminal end of D1NLS. As shown in Supplementary material online, Figure S2A, the proportion of LacZ-positive cells in the control group was 5% in the infarct and border zones (area inside the orange line), representing the efficiency of adenovirus infection. The LacZ expression defined the border zones. The expression of Ki67 was hardly detectable in the control group after 4 days, despite LacZ being detectable even after 7 days (Figure 3A–C, and Supplementary material online, Figure S2B and E). In contrast, Ki67-positive cells were significantly increased at Day 4 in the D1NLS group (Figure 3D–F) and the Skp2 group (Figure 3G–I). Mostly, Ki67-positive cells in these groups stained positive for myc-tag at the C-terminal end of cyclin D1NLS, suggesting that cardiomyocytes infected with D1NLS were driven into the cell cycle. The frequency of Ki67-positive cells in myc-positive cells was 48 ± 3.8 and 72 ± 3.0% in the D1NLS and Skp2 groups, respectively (Figure 3P and Supplementary material online, Table S5), supporting the notion that Skp2 enhances the effect of D1NLS. The efficiency of D1NLS adenovirus infection, as approximated by the ratio between the proportion of Ki67-positive cells in myc-D1NLS positive cells and the proportion of Ki67-positive cells among the total population, was 1.95/48 = 0.04 and 4.00/72 = 0.05 in the D1NLS and Skp2 groups, respectively, and was similar to that of LacZ as above (Figure 3Q and Supplementary material online, Table S5). To further assess DNA synthesis directly, we measured the incorporation of BrdU in cardiomyocytes. BrdU-positive cells were observed in the D1NLS group, and more significantly in the Skp2 group, but rarely in the LacZ group (Figure 3R–X and Supplementary material online Table S5). Taken together, these data suggest that adenovirus-mediated gene delivery of D1NLS induces entry into the cell cycle of cardiomyocytes, and is further enhanced by the addition of Skp2. It should be noted, however, that these effects were only transient because the number of Ki67-positive cardiomyocytes decreased dramatically in both the D1NLS and Skp2 groups 7 days after infection (Figure 3J–Q, and Supplementary material online, Table S5).


Figure 3
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  		Figure 3 Immunostaining of Ki67- or BrdU- postitive cardiomyocytes in vivo. (A–Q) The expression of Ki67 and myc-tag-D1NLS in cardiac cells injected with adenoviruses for LacZ (A–C), D1NLS/CDK4 (D–F, J–L) or D1NLS/CDK4/Skp2 (G–I, M–O) was examined at Day 4 (A–I), and Day 7 (J–O) after reperfusion. Ki67 (green); Tropomyosin (red); myc-tag (white). Scale bars represent 100 µm. Arrow heads represent Ki67-positive (in D, G, J, and M), myc-tag-positive (in E, H, K, and N), and Ki67/myc-tag positive (in F, I, L, and O) cardiomyocytes. (P and Q) Percentage of Ki67-positive cardiomyocytes in myc-tag-positive cardiomyocytes (P), and in total cardiomyocytes in the area surrounded by the orange line in Supplementary material online, Figure S2A (Q). Data are mean ± SEM., n = 6 rats per group. *P < 0.05 vs. LacZ group. {dagger}P < 0.05 vs. D1NLS group (4 days after manipulation). {dagger}{dagger}P<0.05 vs. D1NLS group (7 days after manipulation). #P < 0.05 vs. Skp2 group (4 days after manipulation). (R–X) BrdU incorporation in cardiomyocytes in LacZ (R and U), D1NLS (S and V), and Skp2 (T and W) groups was examined 4 days after manipulation. BrdU (green), Troponin I (red), DAPI (white). Scale bars represent 100 µm. (X) Percentage of BrdU-positive cardiomyocytes. Data are mean ± SEM, n = 6 rats per group. **P < 0.05 vs. LacZ group. {dagger}P < 0.05 vs. D1NLS group.

 
3.4 D1NLS, CDK4, and Skp2 promotes mitosis of adult cardiomyocytes in vivo
To determine whether adult cardiomyocytes can undergo cell division in vivo, we performed H3P and Aurora B analysis. H3P-positive cardiomyocytes with characteristics from early prophase through telophase were identified in the D1NLS and Skp2 groups 4 days after reperfusion/infection, whereas no mitotic cardiomyocytes were found in the LacZ group (Figure 4A–K, Supplementary material online, Videos S1 and S2). These observations indicate that D1NLS and Skp2 promote karyokinesis of adult cardiomyocytes in situ. Quantitative analysis shows that additional expression of Skp2 had more significant effect at 4 days after operation, and that the number of mitotic cells decreased at 7 days in the D1NLS and Skp2 groups, as well as Ki67 (Figure 4K, Supplementary material online, Table S5). Furthermore, as illustrated in Figure 4L (white arrow-head), Aurora B is expressed in cardiomyocytes, and co-localized with DAPI, indicating that these cells are in the early phase of mitosis. We also identified a cardiomyocyte at anaphase, expressing Aurora B in a cleavage furrow (Figure 4L, yellow arrow-head and M). In some cardiomyocytes of the D1NLS and Skp2 groups, Aurora B and Survivin were detected on the midzone between the two daughter cells (Figure 4O, and Supplementary material online, Video S3), indicating that these cells were undergoing cytokinesis. Finally, breakage of the midbody resulted in two daughter cells with an Aurora B/ Survivin-positive remnant, representing a cell in late cytokinesis (Figure 4Q, and Supplementary material online, Video S4). It is important to note that these daughter cells were also positive for tropomyosin, showing that they retained the cardiac phenotype.


Figure 4
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  		Figure 4 Immunostaining of H3P, and Aurora B in infarcted heart. (A–K) H3P was immunostained in cardiomyocytes injected with adenoviruses for LacZ (A), D1NLS/CDK4 (B), and D1NLS/CDK4/Skp2 (C) at Day 4. Tropomyosin (red), H3P (green). Scale bars in A–C, 100 µm. (D–J) Identification of cardiomyocytes in mitosis. H3P (green); Tropomyosin (red); DAPI (white). Scale bars in (D–J), 20 µm. (K) Percentage of H3P-positive cardiomyocytes in the area surrounded by the orange line in online Supplementary material, Figure S2A. Data are mean ± SEM, n = 6 rats per group. **P < 0.01 vs. LacZ group. {dagger}P < 0.05 vs. D1NLS group (4 days after manipulation). ##P < 0.01 vs. Skp2 group (4 days after manipulation). (L–Q) The Aurora B expression in the peri-infarcted area of the Skp2 group. (L) White arrow heads and a yellow arrow-head represent cardiomyocytes in early mitosis and anaphase, respectively. (M) A large magnification view of cardiomyocyte surrounded by a white square in L. Troponin I (red); Aurora B (green); DAPI (blue). (N and P) Cardiomyocytes undergoing cytokinesis, expressing Aurora B and Survivin in D1NLS/CDK4 hearts (N), and D1NLS/CDK4/Skp2 (P). (O and Q) Large magnification view of the white square area in (N) and (P), respectively. White arrow heads represent midzones, and yellow represent daughter cells. Tropomyosin (red), Aurora B (green); Survivin (purple); DAPI (blue). Scale bars, 50 µm in (L, N), and (P); 10 µm in (M, O, and Q).

 
3.5 D1NLS, CDK4, and Skp2 increase a density of capillary vessels and reduce TUNEL-positive cardiomyocytes in vivo
Adenovirus vectors used in this study are regulated by CAG promoter, which supports strong and tissue non-specific gene expression. Therefore, cyclin D1NLS, CDK4, and Skp2 transgenes can be expressed in not only cardiomyocytes but also other cells such as vascular endothelial cells. Thus, we examined expression of von Willebrand factor (vWF), one of the capillary vessel markers. Immunohistochemical staining of vWF showed higher microvessel density in the scar and borderzone of infarct in the D1NLS and Skp2 groups both 4 and 7 days after manipulation (Figure 5A–E). These were more significantly increased at Day 7 in Skp2 group (Figure 5E). Further TUNEL assay showed that a number of apoptotic cardiomyocytes was significantly reduced in the D1NLS and Skp2 groups (Figure 5F). These data suggest that D1NLS and Skp2 induce not only cardiomyocyte mitosis, but also neovascularization and cardiomyocyte survival.


Figure 5
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  		Figure 5 Analysis of vWF and TUNEL assay. The expression of vWF in the borderzone and scar injected with adenoviruses for LacZ (A), D1NLS/CDK4 (B), or D1NLS/CDK4/Skp2 (C and D) was examined 4 days after reperfusion. (D) A large magnification view of a white square area in (C). vWF (green), Tropomyosin (red), DAPI (blue). Scale bars represent 100 µm (A–C) and 10 µm (D). (E) The data represent vWF-positive capillary density (per mm2) in the area surrounded by orange line in online Supplementary material, Figure S2A. Data are mean ± SEM, n = 6 rats per group. #P < 0.05 vs. Skp2 group (4 day after manipulation). {dagger}{dagger}P < 0.05 vs. D1NLS group (7 day after manipulation). (F) Quantitative analysis of percentage of TUNEL-cardiomyocytes in the area surrounded by the orange line in online Supplementary material, Figure S2A 4 days after manipulation. Data are mean ± SEM, n = 6 rats per group. **P < 0.05 vs. LacZ group.

 
3.6 D1NLS, CDK4, and Skp2 reduce infarct size, and prevent cardiac dysfunction and heart failure
To evaluate whether D1NLS/CDK4 and Skp2 induced myocardial regeneration in the ischaemic heart, we measured infarct size 6 weeks after reperfusion/infarction and adenoviral infection. Masson's trichrome staining showed that the infarct size was reduced in the D1NLS group compared with the LacZ group (Figure 6A–E). The effect was more pronounced in the Skp2 group.


Figure 6
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  		Figure 6 Size of myocardial infarction and cardiac function 6 weeks after ischaemia/reperfusion. (A–E) Cross-sections of heart were stained with Masson's trichrome in the sham (A), LacZ (B), D1NLS/CDK4 (C), and D1NLS/CDK4/Skp2 group hearts (D). (E) The infarct size in each group is shown. (F–J) Cardiac function was evaluated by echocardiography. Left ventricular wall movement was obtained from a short-axis view (F–I) and fractional shortening (J). (K–O) The haemodynamic parameters were obtained by cardiac catheterization. LVEDP, left ventricle end-diastolic pressure; LVEDVI, left ventricle endo-diastolic volume index. (P) Lung/body weight ratio was measured in several groups. Data are mean ± SEM, n = 11–12 rats per group. *P < 0.05 vs. sham group; {ddagger}P < 0.05 vs. LacZ group.

 
Next, to further assess the effect of D1NLS, CDK4, and Skp2 on cardiac function, echocardiographic and haemodynamic analysis was carried out 6 weeks after manipulation. Echocardiographical analysis revealed that the percentage LV fractional shortening (% FS) of the LacZ group was smaller than that of the control sham group. In contrast, % FS of D1NLS and Skp2 groups was significantly higher than that of the LacZ group (Table 3, Figure 6F–J). A similar analysis of haemodynamics showed that heart failure developed in the control LacZ group, as indicated by attenuation of the maximum and minimum rates of change in LV pressure (dP/dt), increase in the LV end-diastolic pressure (LVEDP) and the LVEDVI, and the right-shifted passive pressure–volume curve. These parameters were improved in the D1NLS group, compared with the LacZ group, and more pronounced improved in the Skp2 group (Figure 6K–O). Moreover, the lung/body weight ratio was increased in the LacZ group, suggesting the development of heart failure. In contrast, the lung/body weight ratio was reduced slightly in the D1NLS group, and was more pronounced in the Skp2 group (Figure 6P). Taken together, these results strongly indicate that the expression of D1NLS, CDK4, and Skp2 has protective effects on cardiac dysfunction and heart failure in a rat MI model.


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  		Table 3 Echocardiography at 6 weeks after reperfusion

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
In the present study, we demonstrated that a combination of cell-cycle drivers (cyclin D1/CDK4) and removal of a cell-cycle brake (Skp2) induces efficient and stable proliferation of adult cardiomyocytes in situ, and prevents development of ischaemic heart failure. Importantly, we provided unequivocal evidence of cytokinesis in cardiomyocytes, supporting our hypothesis that an improvement in cardiac performance is mediated by an increase in the number of cardiomyocytes.

Several studies have reported that the delivery of cell-cycle regulators induces myocardial regeneration and enhances cardiac function.14,17,33 Pasumarthi et al.14 demonstrated that cyclin D2 causes efficient DNA synthesis of cardiomyocytes in a transgenic mice model,14 and Woo et al.17 showed that the expression of cyclin A2 induces myocardial regeneration and protects cardiac function in ischaemic heart failure. Furthermore, Engel et al. demonstrated that adult cardiomyocytes were stimulated to re-enter the cell cycle by fibroblast growth factor 1 (FGF1) and p38 MAPK inhibitor both in vitro and in vivo accompanied with high expression of cyclin D2 and A2, and rescue cardiac function in a rat MI model.33,34 These reports, along with the findings in the present study, indicate that the proliferation of cardiomyocytes in situ by the expression of cell-cycle regulators could be a rational approach to cardiac regeneration. Among these approaches, however, the present study was unique in that NLS-tagged cyclin D1 was expressed to ensure its nuclear localization. We previously reported that nuclear expression of cyclin D1 is impaired in the rat neonatal heart.22,26 Consistent with this finding, Pasumarthi et al. observed that cyclin D1 is localized in the cardiomyocyte cytoplasm of injured hearts of cyclin D1 transgenic mice,14 and Ledda-Columbano et al.35 reported that cyclin D1 is retained in the cytoplasm of rat cardiomyocytes and is translocated into the nucleus upon T3. NLS-tagged cyclin D1 might have overcome the impedance of nuclear expression. In addition, Skp2 ubiquitin ligase was used to degrade the cell-cycle inhibitor p27, since our previous study suggested that p27 accumulates at a very early phase after forced stimulation of the cell cycle in rat cardiomyocytes.23 In the current study, we confirmed that overexpression of Skp2 mediates p27 degradation and in vitro ubiquitylation activity (see Supplementary material online, Figure S3A and B) in neonatal rat cardiomyocytes. Further, we showed that p27 accumulated in the nuclei of cardiomyocytes expressing myc-D1NLS in the D1NLS group but not in the Skp2 group (see Supplementary material online, Figure S3C and D), implying that p27 acts as a physiological brake of the cell cycle in adult cardiomyocytes in vivo as well as neonatal cells. In fact, more significant regeneration and protection of cardiac function were observed in the Skp2 group, indicating that a combination of cell-cycle acceleration by cyclin D1 and release of cell-cycle brake by Skp2 is a rational strategy to overcome specific impedance of cardiomyocyte regeneration. It is also possible that Skp2 might enhance cyclin D2-, or cyclin A2-induced activity of cardiomyocyte regeneration. We are conducting experiments on the effect of skp2 on other cell cycle regulators, including cyclin-induced cardiac regeneration in vitro and in vivo.

It should be mentioned here that not only cell-cycle activators but also inhibitors were induced in D1NLS/CDK4/Skp2 cells (Figure 1). Actually, the number of Ki67-positive cardiomyocytes was significantly decreased at Day 4 in vitro (Figure 2A and G), and Day 7 in vivo (Figure 3J–Q) after gene delivery during the period of adenoviral expression (Figure 3K and N, and Supplementary material online, Figure S2B–G). It is likely that cell-cycle inhibitors other than p27, such as those found to be elevated in our microarray and expression analysis, play a role in suppressing cell-cycle progression, which is a merit in a clinical context since unregulated proliferation of cardiomyocytes might result in tumours. Increased expression of cell-cycle inhibitors might reflect a unique property of terminally differentiated cardiomyocytes that were forced to enter and proceed in the cell cycle. Such a hypothesis awaits further investigation.

In the current study, we have shown evidence that expression of D1NLS, CDK4, and Skp2 significantly alleviates cardiac dysfunction caused by ischaemia/reperfusion injuries. However, some limitations of the present study should be mentioned. As we showed a good correlation between plasma cTnT concentration and MI size 24 h after manipulation (see Supplementary material online, Figure S1B), cTnT indicates the degree of tissue damage. On the basis of the infarct size affected by multiple factors (neovascularizaion, rate of apoptosis, paracrine effects, and remodelling), therefore, it is hard to insist that MI were made completely equivalent in this experiment. Next, although cell proliferation, as determined by Ki67 and H3P expression, correlates well with the improvement of cardiac function (Supplementary material online, Figure S4), it is questionable whether cardiomyocyte proliferation per se is sufficient for protecting or improving cardiac performance, or whether preferential regeneration of cardiomyocytes in the infarct and border zones at an early stage is beneficial. The observed cardio-protective effects are likely to be mediated by multiple parameters. It is the most possible mechanism that regenerated cardiomyocytes or other types of cells may produce cardio-protective and/or angiogenic factors, such as HGF, bFGF, and VEGF. We have also addressed this issue by analysing apoptosis using TUNEL assay. Indeed, the rate of apoptotic cardiomyocytes was significantly decreased in the D1NLS and Skp2 groups 4 days after manipulation. The existence of cells under cytokinesis in both the D1NLS and Skp2 groups suggests an increase in the number of cardiomyocytes after 4 days. Thus, some apoptotic factors secreted from the regenerated cardiomyocytes and fibroblasts may effect on cardiomyocytes in autocrine and paracrine manner, resulting in protection from apoptosis at an early stage. Furthermore, an increase in the vascular density estimated by expression of vWF in the scar and borderzone of the infarct was observed in the D1NLS and more significantly in the Skp2 groups, suggesting that D1NLS and Skp2 induce neovascularization. If neovascularization occurs and supply with oxygen and nutrients in the borderzone of infarct, not only newly regenerated cardiomyocytes but also cells undergoing to die can survive. Thus, D1NLS and Skp2 may support long-term recovery of the heart function also through angiogenesis and anti-apoptosis.

Finally, in this study, we found that additional expression of Skp2 to positive cell-cycle regulators D1NLS/CDK4 effectively re-activates the proliferation of cardiomyocytes in vivo and protects against cardiac dysfunction and heart failure in an ischaemia/reperfusion-induced acute MI model. Our results provide a novel insight into cell-cycle control mechanisms in adult cardiomyocytes and are also an important step towards the development of therapeutic strategies for preventing and treating heart failure.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology of Japan, a grant from the Ground-based Research for Space Utilization promoted by Japan Space Forum, and a grant from Collaborative Development of Innovative Seeds from Japan Science and Technology Agency, Japan.


   Acknowledgements
 
The authors thank Dr. Y. Tanaka for critical discussion.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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Manipulating myocyte cell cycle control for cardiac repair
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