Published online before print December 2, 2002, doi: 10.1161/01.RES.0000049105.15329.1C
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© 2003 American Heart Association, Inc.
UltraRapid Communications |
Critical Role of Cyclin D1 Nuclear Import in Cardiomyocyte Proliferation
From the Department of Biochemical Genetics (M.T.-A., S.K.), Medical Research Institute, Department of Internal Medicine (H.I., S.A., M.H., F.M.), Section of Molecular embryology (P.S., M.-A.I.), and Cardiothoracic Surgery (M. Shimizu, J.K., M. Sunamori), Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
Correspondence to Masa-Aki Ikeda, Section of Molecular Embryology, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail mikeda.emb{at}tmd.ac.jp
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Abstract |
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Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.
Key Words: cardiomyocyte • cyclin D1 • CDK4 • nuclear localizing signals • cell cycle progression
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Introduction |
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Mammalian cardiomyocytes irreversibly withdraw from the cell cycle soon after birth.1,2 Recent studies, however, have shown that the adult heart contains a small population of cardiomyocytes that retain the proliferative capacity.3,4 For instance, a significant number (0.08%) of mitotic cardiomyocytes has been identified in regions adjacent to the infarcts.4 However, cardiomyocytes remaining in these regions cannot reconstitute damaged tissue, at least in part, due to the quite limited proliferative capacity of cardiomyocytes. Therefore, cardiomyocyte loss is irreversible and frequently leads to diminished cardiac function and severe heart failure. Recent studies have demonstrated that the engraftment of cells derived from embryonic or bone marrow stem cells may be useful for regenerating the functional myocyte mass in the adult heart.5–7 In addition to these approaches, evidence that the adult heart has limited but significant proliferative capacity raises the alternative possibility that increasing the number of the remaining cardiomyocytes by activating their proliferative potential could replace damaged myocardium. In this regard, investigating the inhibitory mechanism of cardiomyocyte proliferation might open new avenues of therapy with which to treat cardiovascular diseases that cause cardiomyocyte loss.
Progression of mammalian cell cycle is regulated by a family of cyclins and cyclin-dependent kinases (CDKs). During G1 phase, cyclin D1 and other D-type cyclins (D2 and D3) accumulate in response to mitogenic stimulation and assemble with their catalytic partners, CDK4 and CDK6. Cyclin D1 plays an important role for promoting G1-to-S phase progression by inactivating the action of the retinoblastoma protein (Rb) through phosphorylation. In addition to Rb phosphorylation, cyclin D/CDK complexes sequester CDK inhibitors such as p21Cip1 and p27Kip1 and thereby facilitate activation of the cyclin E/CDK2 and cyclin A/CDK2 complexes required for entry into and the progression of S phase.8,9 During cardiomyocyte differentiation, Rb phosphorylation and CDK activity are reduced in association with cell cycle arrest.10 However, postmitotic cardiomyocytes still retain the capacity to respond to mitogenic stimulation, which induces hypertrophic cell growth and upregulates expression of cyclins and CDKs, including cyclin D1 and CDK4.11,12 Thus, the mechanisms underlying cardiomyocyte withdrawal from the cell cycle have remained obscure. In this study, we studied the regulation of subcellular localization of cyclin D1 in neonatal cardiomyocytes. The results showed that the nuclear import of cyclin D1/CDK4 plays an important role in cardiomyocyte proliferation in vitro and in vivo.
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Materials and Methods |
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Cell Culture
Cardiomyocytes were cultured as previously described13,14 with some modifications. Heart ventricles were isolated from 17-day fetal or 3-day-old postnatal Sprague-Dawley rats, trisected, and then digested 4 times with collagenase type II (1 mg/mL, Worthington) in Ads buffer (in mmol/L: 116 NaCl, 20 HEPES, 1 NaH2PO4, 5.5 glucose, 5.4 KCl, 0.8 MgSO4; pH 7.35) at 37°C for 20 minutes. The dispersed cells from each digestion were combined, washed, and then were purified by centrifugation through a discontinuous Percoll gradient of 1.050, 1.060, and 1.082 g/mL, respectively. The cells at the 1.060/1.082 interface were collected and used for cardiomyocyte cultures. The purified cardiomyocytes were plated on 60-mm dishes (2x106 cells per dish) or 25-mm collagen-coated coverslips in 6-well plates (2x105 cells per well) in minimum essential medium (MEM) supplemented with 5% calf serum (CS), penicillin (100 U/mL, GIBCO), and streptomycin (100 µg/mL, GIBCO). Cardiomyocyte cultures were incubated in serum-containing medium at 37°C for 24 hours in humidified air with 5% carbon dioxide, after which the medium was changed to serum-free MEM and incubated for another 24 hours. The purity of the cultures was determined by immunohistochemistry with anti-sarcomeric actin (M0874, DAKO) and FITC-conjugated anti-mouse antibodies (23799, Polysciences) 48 hours after plating. Only cultures that consist of >95% FITC-positive cells determined by counting 300 cells in 3 different fields were subjected to various analyses. Control REF52 cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma) containing 10% fetal calf serum (FCS).
Adenoviruses
Ad-Ras61L and Ad-GSKDN (gifts from J.R. Nevins, Duke University, Durham, NC),15 Ad-D1 (a gift from J.H. Albrecht, Hennepin County Medical Center, Minneapolis, Minn),16 Ad-LacZ,17 and Ad-p16 and Ad-p2112 have been described. To generate Ad-CDK4, a blunt-ended fragment encoding CDK4 was isolated from pCMV-CDK4 (a gift from S. van den Heuvel, Massachusetts General Hospital Cancer Center, Boston, Mass) and then cloned into the SwaI site of the cosmid pAxCAwt (Takara). To generate Ad-D1NLS, the fragment encoding mouse cyclin D1 was first cloned into pEF1 myc/nuc (Invitrogen) to fuse with NLSs (fusion to myc epitope and NLSs added 
5 kDa to the gene product). The resultant fragment encoding D1NLS of the resulting plasmid was blunt-ended and cloned into the same site of pAxCAwt. Recombinant adenoviruses were generated by in vitro homologous recombination in 293 cells using the Adenovirus Expression Vector Kit (Takara) according to the manufacturer’s instructions. Viruses were propagated in 293 cells, and virus stocks were prepared as described.18 Viral titers were determined by an indirect immunofluorescent assay by using anti-72K serum.18 Cells were infected at the indicated multiplicity of infection (MOI) in serum-free MEM for 60 minutes with brief agitation every 15 minutes. After infection, the medium was replaced with culture medium.
Western Blotting
Whole cell, cytoplasmic, and nuclear extracts were prepared as described.19 Extracts prepared from equal number of cells were denatured and separated by 6% or 11% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). The blots were probed with primary antibodies and bands were detected using horseradish peroxidase conjugated secondary antibodies (Amersham Pharmacia Biotech) and the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech). The following antibodies were used in this study: mouse monoclonal anti-cyclin D1 (Ab-3, Oncogene science), anti-Rb (14001A, PharMingen), anti-sarcomeric actin (M0874, DAKO), anti-PCNA (sc-056), and anti-BrdU (Roche) antibodies, and rabbit polyclonal anti-Ki-67 (PRO229, YLEM), anti-ß-galactosidase (ß-gal) (ICN), anti-cyclin D2 (sc-593), anti-cyclin D3 (sc-182), anti-cyclin A (sc-751), anti-cyclin E (sc-481), anti-p21 (sc-6246), anti-CDK4 (sc-260), and anti-CDK2 (sc-163) antibodies (Santa Cruz Biotechnology).
Immunoprecipitation and Kinase Assays
Whole cell extracts were immunoprecipitated with anti-CDK4 (sc-260) followed by Western blotting with anti-cyclin D1 (Ab-3) or anti-p21 antibodies. To assay CDK2 kinase activity, extracts were immunoprecipitated with anti-CDK2 antibody and then histone H1 kinase was assayed as described.11
Immunofluorescent Staining and Cell Cycle Analysis
To examine the subcellular localization of cyclin D1, cells plated on glass coverslips were fixed in 70% ethanol, double-stained with anti-cyclin D1 and anti-sarcomeric actin antibodies, and then immunostains were visualized using fluorescent tyramide reagent according to the manufacturer’s protocols (TSA-direct NEL-701, NEN). To analyze the cell cycle profile, fixed cells were stained with anti-sarcomeric actin and FITC-conjugated anti-mouse antibodies, followed by propidium iodide (50 µg/mL) containing RNase A (500 µg/mL). To determine the DNA content, the intensity of propidium iodide staining was analyzed on cell populations that were positive for FITC staining using a laser scanning cytometer (LSC 101) (Olympus). Confocal images were acquired using a confocal microscope (FV500) (Olympus).
In Vivo Study
All animal studies were in accordance with TMDU institutional guidelines. Apical injections of viruses into the myocardium were performed under direct visualization after thoracotomy of Wister rats (250 to 300 g). Virus mixture containing 1x1010 PFU of either Ad-D1NLS or Ad-D1, together with Ad-CDK4 (1x1010 PFU), was injected into hearts. To identify the infected area, Ad-LacZ (2x109 PFU) encoding ß-galactosidase was added into the virus mixture. For controls, Ad-LacZ (2x1010 PFU) was used. In some experiments, intraperitoneal injections of 5-bromo-2'-deoxyuridine (BrdU) (50 mg/kg, Roche) were given every 12 hours before euthanasia to detect DNA synthesis. Four days after injections, hearts were fixed with 4% paraformaldehyde by perfusion. The sections of tissues were probed with anti-Ki-67 and anti-ß-gal antibodies. To confirm viability of Ki67 positive cardiomyocytes, sections were subjected to terminal transferase-mediated dUTP nick end labeling (TUNEL) assay using the in situ Apoptosis Detection Kit (Takara) and then stained with anti-Ki67 antibody and 1 µg/mL of DAPI. For analysis of BrdU incorporation, fixed sections were denatured by incubation in 1 mol/L hydrochloric acid at 65°C for 30 minutes20 and then probed with anti-BrdU (Roche) and anti-ß-gal (ICN) antibodies. For detection of primary antibodies, sections were incubated with the appropriate secondary antibody mix containing anti-mouse and anti-rabbit antibodies conjugated with Alexa 488, Alexa 568 (Molecular Probes), or Ci5 (Jackson) for 1 hour at room temperature. Lastly, to identify cardiomyocytes, all sections were stained with anti-sarcomeric actin antibody covalently labeled with Alexa 488 or Alexa 555 using the Zenon One Mouse IgG1 Labeling Kit (Molecular Probes). Images were obtained with a confocal laser microscope (FV500) (Olympus) or the laser-scanning confocal image system (Zeiss LSM510).
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Results |
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We first examined the effects of mitogenic or hypertrophic stimuli on cyclin D1 expression and Rb phosphorylation in neonatal rat cardiomyocytes. Serum-starved cardiomyocytes isolated from 3-day old rats were stimulated with fetal calf serum (FCS) or phenylephrine (PE), an
-adrenergic receptor agonist, or infected with an adenovirus encoding activated ras (Ras61L), all of which induce hypertrophy in cardiomyocytes.12,21–23 Western blotting of whole cell extracts revealed that all of these stimuli induced cyclin D1 protein in cardiomyocytes but not the Rb phosphorylation apparent in control proliferating REF52 cells (Figure 1A).
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The subcellular localization of cyclin D1 is regulated during the cell cycle.24–28 We next examined whether the subcellular localization of cyclin D1 might contribute to its inability to phosphorylate Rb in neonatal cardiomyocytes. Nuclear and cytoplasmic extracts from cells stimulated as described above were analyzed by Western blotting and immunostaining. The vast majority of cyclin D1 and CDK4, as well as other D-type cyclins (D2 and D3), localized to the cytoplasm, whereas Rb predominantly localized in the nucleus (Figures 1B and 1C). Moreover, even when cyclin D1 and CDK4 were overexpressed using recombinant adenoviruses in neonatal cardiomyocytes cultured in the presence of 5% calf serum (CS), exogenous cyclin D1 did not appear to accumulate in the nucleus (Figures 1D and 1E). In contrast, exogenous cyclin D1 accumulated in the nucleus in proliferating cardiomyocytes isolated from 17-day-old fetal hearts (Figure 1D). Finally, separation of nuclear and cytoplasmic fractions was confirmed by localization of proliferating-cell nuclear antigen (PCNA) and sarcomeric -actin, which were predominantly detected in the nucleus and cytoplasm, respectively. Thus, the inability of cyclin D1 and CDK4 to phosphorylate Rb may be due to their cytoplasmic sequestration in cardiomyocytes.
We next examined whether nuclear targeting of cyclin D1 and CDK4 could induce the phosphorylation of endogenous Rb, thereby activating downstream events that are regulated by Rb in cardiomyocytes. To efficiently target cyclin D1 to the nucleus, we constructed an adenovirus encoding a variant of cyclin D1 (D1NLS) in which the C-terminal end of cyclin D1 was fused with triplicate monopartitie NLS derived from the SV40 large T antigen. Overexpression of D1NLS and CDK4 by the recombinant adenoviruses revealed that D1NLS protein accumulated in the nucleus, whereas exogenous wild-type cyclin D1 predominantly localized in the cytoplasm, even though these proteins were expressed at comparable levels (Figures 2A and 2B). Furthermore, D1NLS expression caused significant accumulation of CDK4 protein in the nucleus (Figures 2C, lane 4, bottom). To address the complex formation, nuclear and cytoplasmic extracts (used in Figure 2B) were immunoprecipitated by anti-CDK4 antibody followed by Western blotting with cyclin D1 antibody. Figure 2D shows that D1NLS associated with CDK4 in the nucleus, whereas the wild-type cyclin D1/CDK4 complex was predominantly detected in the cytoplasm. Functionally, coexpression of D1NLS and CDK4 converted a majority of Rb into the hyperphosphorylated form (ppRb) (Figure 2E). Furthermore, D1NLS and CDK4 induced cyclin A and E expression and the CDK2 kinase activity, both of which are activated after Rb phosphorylation during the cell cycle (Figures 2F and 2G). Finally, that the Rb phosphorylation and CDK2 kinase activity depend on the kinase activity of D1NLS and CDK4 was confirmed by inhibition of these effects by coexpression of CKIs, p16INK4a, and p21Cip1 (Figures 2E and 2G).
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The above results indicate that the nuclear import of cyclin D1/CDK4 can activate the Rb regulatory pathway that is required for cell cycle progression. Therefore, we tested using laser scanning cytometry (LSC) whether the nuclear import of cyclin D1/CDK4 could cause the reentry of postmitotic cardiomyocytes into the cell cycle. Serum-starved cardiomyocytes plated on coverslips were infected with adenoviruses or stimulated with FCS, then double-stained with propidium iodide (PI) and sarcomeric actin, a marker for cardiomyocytes. LSC analysis of sarcomeric actin–positive cells revealed that the overexpression of D1NLS and CDK4 caused a significant induction of cell cycle progression, as revealed by an increase of cells with S and G2/M DNA content, whereas FCS stimulation had little effect (Figure 3A). In addition, in the presence of 5% CS, D1NLS/CDK4 potentiated the activity, whereas coexpression of wild type cyclin D1 and CDK4 had no effect. Notably, D1NLS/CDK4 expression induced no apparent apoptosis as indicated by the absence of cells with sub-G1 DNA content in the infected cells. Furthermore, blocking the cells in G2/M phase with nocodazole revealed that 95% of the cells were in the S and G2/M fraction, whereas cells expressing wild-type cyclin D1 and CDK4 remained in the G1 fraction (Figure 3B). In addition, the G1 fraction reappeared after release from nocodazole in cells expressing D1NLS/CDK4. These results demonstrated that virtually all of the cells expressing D1NLS/CDK4 entered the cell cycle, leading to cell division.
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To further confirm that the nuclear import of cyclin D1/CDK4 can induce mitosis and cell division of cardiomyocytes, we examined the morphology of cardiomyocytes expressing D1NLS/CDK4. LSC analysis allowed the DNA content of each cell in a defined microscopic field to be determined. Figure 4A shows many mitotic cells (Figure 4A, area vi; 15, 16, 18, 21) and cells immediately after mitosis (Figure 4A, area i; 4, 5, 7, 8, 9, 10), as indicated by high peaks of PI fluorescence, in sarcomeric actin–positive cardiomyocytes. In addition, daughter cells that appeared shortly after cell division resumed beating as parental cardiomyocytes (data not shown). Furthermore, the number of cardiomyocytes coinfected with these viruses tripled 5 days after infection (Figure 4B). We therefore conclude that the nuclear import of cyclin D1/CDK4 can induce the complete cell cycle progression of primary neonatal cardiomyocytes.
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Finally, we examined the effects of nuclear import of cyclin D1/CDK4 on adult cardiomyocyte proliferation in vivo. We injected the cyclin D1NLS virus or cyclin D1WT virus, together with the CDK4 virus, into the apical regions of rat hearts and fixed 4 days after infection. In addition, to identify the infected area, Ad-LacZ was coinfected with these viruses. We used hearts infected with only the LacZ virus for controls. Because the Ki-67 nuclear protein is expressed in proliferating cells in all phases of the cell cycle and associated with cell division,29 we first stained infected heart tissues with anti-Ki-67 antibody, followed by staining with anti-ß-gal and sarcomeric actin antibodies to identify infected cardiomyocytes. Ki-67 expression was detected in a number of nuclei in ß-gal–positive cardiomyocytes and nonmyocytes, such as interstitial cells, in the regions coinjected with the cyclin D1NLS and CDK4 viruses (Figures 5A i and j, and 5B a). In contrast, the expression was not detected in cardiomyocytes infected only with the LacZ virus, or with the wild-type cyclin D1/CDK4 viruses (Figure 5A d and e; data not shown). Although recent studies have shown that a small population of cardiomyocytes that appear not to be terminally differentiated exists in the adult heart, the morphology of the Ki-67–positive cardiomyocytes was indistinguishable from that of adjacent fully differentiated cardiomyocytes in the uninfected regions.
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Previous experiments have shown that adenoviral delivery of E2F1, a downstream effector of the cyclin D1 pathway, leads to an apoptotic response in adult cardiomyocytes.30 Therefore, we examined the viability of Ki-67–positive cardiomyocytes produced by the infection of the D1NLS and CDK4 viruses. We performed in situ TUNEL staining to detect apoptotic cells in the infected regions. Figure 5B shows that, however, no TUNEL-positive nuclei was detected in the Ki-67–positive cardiomyocytes. Furthermore, nuclear staining with DAPI revealed that nuclear morphology of these cells retained normal shapes and no blebbing and shrunken nuclei, which are characteristic in apoptotic cells, were detected. In contrast, we observed various patterns of Ki-67 subnuclear localization, which changes drastically during the cell cycle,29 in cells infected with the D1NLS and CDK4 viruses.
Finally, to directly assess cell cycle reentry of adult cardiomyocytes by D1NLS and CDK4, we examined DNA synthesis of infected cardiomyocytes. Infected rats were injected BrdU every 12 hours and subjected to BrdU incorporation assay 4 days after infection. Figure 5C shows that a number of BrdU-positive cardiomyocytes were detected in the regions infected with the D1NLS/CDK4 viruses. In contrast, only sarcomeric -actin negative–cells were stained in areas infected with the control LacZ virus. Taken together, these results indicate that the nuclear import of cyclin D1/CDK4 could promote cell cycle entry of adult cardiomyocytes in vivo.
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Discussion |
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In the present study, we show that the nuclear import of the cyclin D1/CDK4 complex is tightly prevented in postmitotic cardiomyocytes. Furthermore, overcoming of this inhibitory mechanism is sufficient to induce cell cycle progression leading to cell division of neonatal cardiomyocytes. In addition, the daughter cells after cell division resumed beating and expressed sarcomeric actin, a marker for cardiomyocytes. The critical role of cyclin D1–dependent kinase activity in cell cycle progression of quiescence or terminally differentiated cells is supported by the recent studies using other types of cells, albeit the cyclin D1 activity is not sufficient to promote cell division in these cases.31–33 In this regard, previous studies have shown that activation of the Rb pathway by forced expression of adenovirus E1A or E2F1, a downstream target of Rb, can induce DNA synthesis but resulted in apoptotic cell death in cardiomyocytes.34,35 Thus, our findings indicate that the nucleocytoplasmic transport machinery of cyclin D1 plays a critical role for determining proliferative capacity of cardiomyocytes.
The precise mechanism preventing cyclin D1 nuclear accumulation remains unclear. Nucleocytoplasmic transport pathways of proteins are selectively regulated by various transport receptors and signals, such as NLSs and nuclear export signals (NESs), and are involved in various cell functions including cell cycle control.36,37 D-type cyclins and CDK4 lack consensus NLSs, and p21Cip1 and p27Kip1, which contain NLSs, promote the assembly and nuclear localization of the cyclinD1/CDK4 complex.9,27,28 However, studies on primary mouse embryonic fibroblasts (MEFs) lacking both p21Cip1 and p27Kip1 have demonstrated that cyclin D1 by itself is able to localize in the nucleus without detectable assembly with CDK4. Furthermore, these MEFs still retain some cyclin D–dependent kinase activity and exhibit no apparent effects on the cell cycle.27 In postmitotic cardiomyocytes, we show that the cyclin D1/CDK4 complex was formed but remained in the cytoplasm (Figure 2D). In addition, the cytoplasmic cyclin D1/CDK4 complex associated with p21Cip1 and ectopic expression of p21Cip1 or p27Kip1 did not promote nuclear localization of cyclin D1/CKD4 (unpublished data, 2002). On the other hand, the subcellular localization of cyclin D1 is also regulated by the phosphorylation of cyclin D1 at threonine286 by glycogen synthase kinase 3ß (GSK3ß) that promotes the nuclear export and degradation of cyclin D1 during S phase.24–26,38 Inhibition of endogenous GSK3ß activity by an adenovirus encoding a dominant negative mutant of GSK3ß (GSKDN) resulted in some increase in cyclin D1 accumulation in the nucleus (unpublished data). Therefore, it is possible that the GSK3ß-mediated nuclear export of cyclin D1 may contribute, at least to some extent, to the cytoplasmic sequestration of cyclin D1 in postmitotic cardiomyocytes. Additionally, although addition of efficient NLSs promotes nuclear localization of cyclin D1, the majority of D1NLS protein was still detected in the cytoplasm (Figures 2B and 2C). This may also support the notion that postmitotic cardiomyocytes have machinery positively preventing cyclin D1 nuclear localization, resulting in significant sequestration of D1NLS protein in the cytoplasm.
The inhibition of cyclin D1 nuclear accumulation is likely to be a critical barrier for maintaining cardiomyocytes tightly in the postmitotic state. Indeed, overexpression of wild-type cyclin D1 and CDK4 in reversibly quiescence fibroblasts, as well as proliferating fetal cardiomyocytes, caused nuclear accumulation of these proteins (M. Tamamori-Adachi, P. Sumrejkanchanakij, M.-A. Ikeda, unpublished observations, 2002) and has been shown to induce DNA synthesis.31–33 Thus, prevention of their nuclear import is unlikely to be a common feature in cells reversibly arrested in G0 phase. Rather, it appears to be a characteristic of postmitotic cardiomyocytes, which may play a critical role in switching from a proliferating state to a terminally differentiated state in cardiomyocytes and, possibly, in other terminally differentiated cells. A recent study reported that cyclin D1 overexpression can induce the reentry into the cell cycle from the terminally differentiated state in some types of established cell lines.33 We show, however, that ectopically expressed cyclin D1 was unable to localize in the nucleus and to induce cell cycle progression in neonatal cardiomyocytes. Thus, it is likely that postmitotic cardiomyocytes have a unique property in the nucleocytoplasmic transport of cyclin D1.
Finally, we show that the nuclear import of cyclin D1/CDK4 induces the cell cycle reentry of a number of cardiomyocytes in the adult heart as demonstrated by expression of the Ki-67 nuclear antigen and BrdU, markers associated with cell cycle entry. Furthermore, we did not detect apoptotic induction in these cardiomyocytes. Although recent evidence has demonstrated that the adult heart contains cardiomyocytes that have proliferative capacity,4 Ki-67 expression was only detected in a small population of myocytes in regions adjacent to the infracts. Therefore, our results suggest that the nuclear import of cyclin D1/CDK4 may potentiate the ability of cardiomyocytes that retain proliferative capacity. It is also possible that their nuclear import might promote the cell cycle reentry of a population of cardiomyocytes that otherwise do not enter into the cell cycle in the adult heart. Nevertheless, previous experiments using transgenic mice carrying wild-type cyclin D1 driven by -cardiac myosin heavy chain (MHC) promoter have shown that deregulated wild-type cyclin D1 expression causes an increase in cardiomyocyte number and cardiomyocyte DNA synthesis in the adult heart.39 In this study, however, we show that transient expression of cyclin D1 did not promote cell cycle progression in both neonatal and adult cardiomyocytes. Given that the -cardiac MHC gene begins to express during late stages of embryogenesis when cardiomyocytes have proliferative capacity and allow cyclin D1 nuclear accumulation (Figure 1D), it is possible that deregulated wild-type cyclin D1 expression might prevent cell cycle withdrawal of terminally differentiated cardiomyocytes, which normally occurs soon after birth. Alternatively, we cannot rule out the possibility that constitutive cyclin D1 expression might cause cell cycle reentry of the adult heart for long period of time in the transgenic model. Investigation of the molecular mechanism preventing cyclin D1 nuclear import in postmitotic cardiomyocytes will provide findings important to the development of strategies for regenerating cardiomyocytes. Although the engraftment of cells derived from embryonic or bone marrow stem cells may be useful for regenerating the functional myocyte mass in the adult heart,5–7 we believe that our results provide an important step toward the development of an alternative therapeutic application.
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Acknowledgments |
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This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, the Japan Society for the Promotion of Science, and a grant from the Atsuko Ouchi Memorial Fund. We thank Joseph. R. Nevins, Jeffrey. H. Albrecht, and Sander. van den Heuvel for materials, and Mieko Goto and Shiho Hashimoto for excellent technical assistance. We are also grateful to and Yoshimasa. Kiyomatsu for LSC analysis and Joseph. R. Nevins for critical reading of the manuscript.
Received June 19, 2002; revision received November 1, 2002; accepted November 12, 2002.
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