© 1999 American Heart Association, Inc.
Rapid Communication |
A Mammalian Myocardial Cell-Free System to Study Cell Cycle Reentry in Terminally Differentiated Cardiomyocytes
From the Department of Cardiology (F.B.E., L.H., R.D., R.v.H.), Franz Volhard Clinic, Humboldt University, and the Max Delbrück Center for Molecular Medicine (M.C.C., H.L.), Berlin, Germany.
Correspondence to Rüdiger v. Harsdorf, MD, Franz-Volhard-Klinik, Universitätsklinikum Charité, Medizinische Fakultät der Humboldt-Universität zu Berlin, Wiltbergstr 50, 13125 Berlin, Germany. E-mail rharsdo{at}mdc-berlin.de
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Abstract |
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Abstract—Cardiomyocytes withdraw from the cell cycle in the early neonatal period, rendering the adult heart incapable to regenerate after injury. In the present study, we report the establishment of a cell-free system to investigate the control of cell cycle reentry in mammalian ventricular cardiomyocyte nuclei and to specifically address the question of whether nuclei from terminally differentiated cardiomyocytes can be stimulated to reenter S phase when incubated with extracts from S-phase cells. Immobilized cardiomyocyte nuclei were incubated with nuclei and cytoplasmic extract of synchronized H9c2 muscle cells or cardiac nonmyocytes. Ongoing DNA synthesis was monitored by biotin-16-dUTP incorporation as well as proliferating cell nuclear antigen expression and localization. Nuclei and cytoplasmic extract from S-phase H9c2 cells but not from H9c2 myotubes induced DNA synthesis in 92% of neonatal cardiomyocyte nuclei. Coincubation in the presence of cycloheximide indicated that de novo translation is required for the reinduction of S phase. Similar results were obtained with adult cardiomyocyte nuclei. When coincubated with both cytoplasmic extract and nuclei or nuclear extracts of S-phase cells, >70% of adult cardiomyocyte nuclei underwent DNA synthesis. In conclusion, these results demonstrate that postmitotic ventricular myocyte nuclei are responsive to stimuli derived from S-phase cells and can thus bypass the cell cycle block. This cell-free system now makes it feasible to analyze the molecular requirements for the release of the cell cycle block and will help to engineer strategies for regenerative growth in cardiac muscle.
Key Words: cell cycle • cardiomyocyte • DNA synthesis
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Introduction |
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Mammalian cardiomyocytes lose their ability to proliferate and exit the cell cycle in vivo during the first 2 weeks of age,1 2 and thus the mammalian heart is incapable of regeneration after ischemic or other forms of injury. Under in vitro conditions, neonatal3 and even fetal4 5 cardiomyocytes lose their proliferative capacity as soon as they are taken into culture. Natural compensatory processes in the heart are limited to hypertrophy of the adjacent myocardium and replacement of necrotic regions with nonfunctional fibrotic scar tissue, resulting in a loss of contractility. In contrast, the cardiac muscle of amphibians is capable of regenerating up to 50% of the ventricle after mechanical excision. This process involves dedifferentiation of the remaining cardiac muscle cells, DNA synthesis, and subsequent mitosis of both cardiomyocytes and connective tissue cells.6 7 Thus, a thorough understanding of the mechanisms that regulate the withdrawal from the cell cycle and the development of approaches to reactivate proliferation and dedifferentiation of mammalian cardiomyocytes would be of tremendous therapeutic value.
Considerable effort has been invested to achieve mammalian cardiomyocyte proliferation. Various growth factors, viral oncoproteins, and cellular cell cycle activators were studied and shown to induce DNA synthesis in vitro, but none of them were able to establish sustained proliferation of adult cardiomyocytes.8 Recently, it has been shown that the overexpression of E1A4 9 or E2F-110 11 induced predominantly apoptosis in cardiomyocytes. In these instances, apoptosis could be prevented in part by the simultaneous overexpression of E1B or Bcl-24 9 10 or in the presence of the survival factor insulin-like growth factor I,11 leading to the induction of DNA synthesis in neonatal cardiomyocytes. Adenoviral delivery of E2F-1 in postmitotic adult cardiomyocytes resulted predominantly in apoptosis and to a lesser extent in the induction of S phase both in vitro and in vivo.12 Hearts of transgenic mice that overexpress cyclin D1 were characterized by multinucleation.13 Although these studies indicated the possibility of inducing DNA synthesis in cardiomyocytes, our understanding of the factor(s) and molecular mechanisms required is still very limited.
Classical cell fusion experiments and recent experiments using a human cell-free system have shown that S-phase nuclei can induce DNA synthesis in G1 phase but not in G2-phase nuclei.14 15 16 To determine, whether nuclei from terminally differentiated rat cardiomyocytes can be stimulated to reenter S phase when incubated with extracts from S-phase cells, we established a mammalian myocardial cell-free system, in which DNA synthesis in primary cardiomyocyte nuclei is efficiently induced, to complement currently used molecular genetic approaches. The present study demonstrates that initiation of DNA replication in vitro depends on both cytoplasmic and nuclear factors from S-phase cells and requires de novo protein synthesis. This new myocardial cell-free system is applicable to a variety of biochemical analyses aimed at the molecular dissection of cell cycle control in differentiated cardiomyocytes.
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Materials and Methods |
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Rat Primary Cardiomyocyte Cultures
The care and use of the animals in the present study were in accordance with approved guidelines of the Max-Delbrück-Center (Berlin). Ventricular cardiomyocytes from 2-day-old Wistar rats (Möllegard, Schönwalde, FRG) were isolated and cultivated as described with minor modifications.17 Briefly, hearts were dissected, minced, and trypsinized. For selective enrichment of cardiomyocytes, cells were then preplated for 1 to 2 hours. The resultant cell suspension (6x106 cells) was plated in 100-mm culture dishes. Cells were cultured for 24 hours in the presence of 0.1 mmol/L BrdU (Sigma) to prevent proliferation of nonmyocytes. More than 90% of the cells were myocytes, as evaluated by indirect immunofluorescence staining with an antibody to sarcomeric tropomyosin (CH1, Sigma).
Adult ventricular cardiomyocytes were isolated from 12- to 16-week-old male Wistar rats (250 to 350 g) following basically Powell's procedure.18
Cell Culture and Synchronization
H9c2 cells (American Type Culture Collection [Manassas, Va],
CRL 1446, a muscle cell line derived from embryonic rat heart tissue
and nonmyocytes (derived from preplating of neonatal
cardiomyocytes) were grown to 70% confluence in DMEM/F12
(Gibco) supplemented with 10% heat-inactivated FCS (PAA),
100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L
L-glutamine (all from Gibco). Cells were synchronized in S
phase using a single thymidine (2.5 mmol/L, Sigma) block in
culture medium for 18 hours, followed by a release into medium lacking
thymidine for 4 hours. Differentiated H9c2 myotubes were obtained by
incubation for 10 days without changing the medium.
Cell Cycle Analysis by Flow Cytometry
For detection of DNA synthesis, cells were pulse-labeled with
50 µmol/L BrdU for 4 hours and stained with FITC-conjugated
anti-BrdU antibody (B44; Becton Dickinson) and propidium iodide (Sigma)
(2.5 µg/mL) for DNA content according to the manufacturer's
instructions. Samples (104 cells) were
analyzed with a flow cytometer (Coulter Epics), and the
distribution of cells in the different cell cycle stages was determined
using Epics software and Multicycle software (Coulter).
For detection of the percentage of nonmyocyte cells, fixed cultures were permeabilized (15 minutes, 0.5% Triton X-100 in PBS), incubated for 30 minutes in 1% BSA/PBS, and subsequently incubated for 45 minutes with an antibody to sarcomeric tropomyosin (diluted 1:1000 in 1% BSA/PBS, CH1, Sigma). After washing with 0.1% Nonidet P40 and 1% BSA/PBS, cells were incubated with an FITC-conjugated goat anti-mouse IgG antibody (diluted 1:500 in 0.5% Tween and 1% BSA/PBS; Dianova), washed again, stained with propidium iodide (Sigma) (2.5 µg/mL) for DNA content, and analyzed as described above. All manipulations were carried out at room temperature.
Preparation of Nuclei and Extracts
For preparation of cytoplasmic extracts,
107 cells were washed twice in ice-cold hypotonic
buffer (0.01 mmol/L HEPES [pH 7.4] and 1.5 mmol/L
MgCl2) and allowed to swell for 10 minutes on
ice. After centrifugation (300g, 4°C, 5
minutes), the pellet was disrupted with 20 to 30 strokes in a Dounce
homogenizer using a tight-fitting pestle (Wheaton).
Nuclei were pelleted at 5000 rpm for 5 minutes and 4°C in an
Eppendorf 5415L centrifuge. The cytoplasmic extract was
recentrifuged for 30 minutes at 14 000 rpm and snap-frozen in
liquid nitrogen after adding protease inhibitors (0.6
µmol/L aprotinin, 4 µmol/L leupeptin, 3 µmol/L
pepstatin, and 2 mmol/L Pefabloc; all from Sigma) and phosphatase
inhibitors (25 mmol/L NaF and 1 mmol/L
Na3VO4; both from Sigma).
Cytoplasmic extract contained 5 to 7 µg/µL protein (determined by
BCA protein assay; Pierce).
Pelleted nuclei were resuspended in 5 mL hypotonic buffer with 5 strokes in a Dounce homogenizer and washed twice in PBS. Numbers and integrity of nuclei were determined by staining with Trypan Blue using a hemocytometer. For the preparation of nuclear extracts, pelleted nuclei were resuspended in PBS and sonicated (Bachhofer Sonoplus HD70, MS 72/D), centrifuged, and mixed with inhibitors as described above. Nuclear extracts contained 2 to 4 µg/µL protein.
For chemical isolation of nuclei, 250 µL lysis solution (3% glacial acetic acid and 5% ethylhexadecyldimethylammonium bromide in H2O; Sigma) per 5 mL of hypotonic buffer was added to washed cells, and tubes were shaken every minute for 5 to 10 minutes for cardiomyocytes and 1 to 2 minutes for H9c2 cells.19 The release of nuclei was examined by light microscopy.
DNA Synthesis Assay and Immunofluorescence
Unless specified otherwise, all solutions were prepared in PBS
with 0.68 mmol/L CaCl2 and 0.49 mmol/L
MgCl2, all antibodies were diluted in blocking
buffer as indicated, and all manipulations were carried out at room
temperature. Chemically isolated cardiomyocyte nuclei (1 to
1.5x105 neonatal, 0.5 to
1x105 adult) were immobilized onto
poly-L-lysine (Sigma)–coated coverslips by
centrifugation for 8 minutes at 600 rpm and 4°C in a
Beckman CS-6R centrifuge. Nuclei were then washed with ice-cold
PBS and subsequently covered with 30 µL cytoplasmic extract
(substituted with PBS for control reactions), 20 µL mechanically
isolated H9c2 nuclei (2 to 2.5x105 nuclei), or
30 µL nuclear extract and 20 µL of a buffered
nucleotide mix (40 mmol/L K-HEPES [pH 7.8], 7
mmol/L MgCl2, 3 mmol/L ATP; 0.1 mmol/L
each of GTP, CTP, UTP; 0.1 mmol/L each of dATP, dGTP, dCTP;
7.5 µmol/mL biotin-16-dUTP; 0.5 mmol/L DTT; 40 mmol/L
creatine phosphate, and 0.5 µg/µL phosphocreatine kinase; all from
Boehringer Mannheim) and incubated for 2 hours (neonatal) or 3
to 4 hours (adult) at 37°C in a humidified atmosphere of 95%
O2/5% CO2.
For detection of DNA replication foci, chemically isolated immobilized nuclei were washed twice with ice-cold PBS, permeabilized twice at 4°C for 30 seconds with 0.5% Triton X-100 in CSK buffer (100 mmol/L NaCl, 300 mmol/L sucrose, 3 mmol/L MgCl2, and 10 mmol/L PIPES [pH 6.8]), and then fixed for 5 minutes in -20°C methanol. Nuclei were then incubated for 20 minutes in 5% goat serum/0.2% (vol/vol) Tween 20 and subsequently incubated with a mouse monoclonal IgG2a antibody to proliferating cell nuclear antigen (PCNA) for 1 hour (PC10, diluted 1:40; Dianova). Nuclei were washed 4 times with 0.1% (vol/vol) Nonidet P-40, incubated with an FITC-conjugated goat anti-mouse IgG antibody (diluted 1:50; Dianova) for 1 hour, washed 3 times, and mounted (13.3% [wt/vol] Mowiol 4-88, 34% [vol/vol] glycerol, and 2.5% [wt/vol] DABCO in Tris-HCl [pH 8.5]).20
To assess DNA synthesis, biotin-16-dUTP incorporation was used. Chemically isolated, immobilized nuclei were fixed with 3.7% (vol/vol) formaldehyde for 10 minutes. DNA was denatured with 2 N HCl/0.5% (vol/vol) Triton X-100 for 20 minutes and neutralized with 0.1 mol/L Na2B4O7 [pH 8.5]. Afterward, nuclei were blocked in 0.5% (vol/vol) Tween 20/1% (wt/vol) BSA for 30 minutes, incubated with Texas Red streptavidin (diluted 1:100 in 0.5% Tween 20; Amersham) for 45 minutes, and washed 3 times with PBS containing 0.1% (vol/vol) Nonidet P-40. For visualization of DNA, Hoechst 33258 was included in the last wash at a final concentration of 1 µg/mL. Mounted samples were examined, photographed, and scanned or directly collected as digital images (Olympus IX 70 or BX 60 fluorescence microscope). Digital images were assembled and annotated using Adobe Photoshop and PowerPoint software.
Statistical Analysis
All experiments were performed on 2 coverslips and repeated at
least 3 times. For the quantitative analyses, 200 nuclei were
counted in random fields. For the determination of the differences
between the cross-sectional area of neonatal cardiomyocyte
nuclei and H9c2 nuclei, 150 nuclei of each type were analyzed.
Diameters of nuclei were determined by an object micrometer
(Olympus), and areas were subsequently calculated. Statistical
significance was determined using unpaired t test (StatView
software).
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Results |
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Characterization of S-Phase Cells in Primary Rat Neonatal Ventricular Cardiomyocyte Cultures
Primary rat neonatal ventricular cardiomyocyte cultures were analyzed by flow cytometry using sarcomeric tropomyosin antibody and propidium iodide to determine the percentage of nonmyocytes and cardiomyocytes in S phase. In the unpurified cell population (without preplating), 13.6% of cells were in S phase, whereas only 6.3% were in S phase in the purified preparations (with preplating, Figure 1A
). By preplating,
the percentage of nonmyocytes could be reduced from 48.7% to
9.5% (Figure 1B). Around 4% of cardiomyocytes were
in S phase (Figure 1C).
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As a marker for S phase, we used indirect
immunostaining for PCNA and fluorescence
microscopy analysis. In proliferating cells, PCNA cycles
between a chromatin-bound, detergent-insoluble state in S phase and a
diffuse-soluble state when DNA is not being
synthesized.21 22 After the extraction of the Triton X-100
soluble fraction of PCNA, <1% of immobilized
cardiomyocyte nuclei were stained positively before (data
not shown) and after coincubation with their own cytoplasm (Figure 2A).
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Both methods demonstrate that <4% of cardiomyocytes synthesize DNA under our conditions. The difference of S-phase cells as detected by propidium iodide and PCNA staining is relatively minor and could be due to the following reasons: (1) cardiomyocyte nuclei are more resistant to the isolation procedure than noncardiomyocytes, resulting in an additional enrichment of cardiomyocyte nuclei; (2) the threshold for detection by immunofluorescence is confounded by partial inactivation of the epitope by the isolation procedure and fixation; and (3) small populations are often overestimated by flow cytometry analysis.
Coincubation With Nuclei and Cytoplasmic Extract From S-Phase H9c2
Cells but Not From Differentiated H9c2 Myotubes Induces DNA Synthesis
in Neonatal Cardiomyocyte Nuclei
Studies using mammalian cell fusion experiments or a human
cell-free system have shown that S-phase nuclei induce DNA synthesis in
G1 phase but not G2 phase
nuclei.14 15 16 To test whether S phase could also be
induced in rat cardiomyocyte nuclei, we chose H9c2 cells, a
muscle cell line from the same species, to prepare S-phase cytoplasmic
extracts and nuclei. Synchronization of H9c2 cells was confirmed by
flow cytometry analysis of incorporated BrdU and propidium
iodide staining for DNA content. After a single thymidine block, 78%
of H9c2 cells were enriched in S phase (Figure 3). Immobilized
cardiomyocyte nuclei were coincubated with nuclei and
cytoplasm of S-phase H9c2 cells. Biotin-dUTP labeling and
immunofluorescence analysis showed that
92% of cardiomyocyte nuclei were induced to synthesize DNA
under these conditions (Figure 2B
and 2D, lane b). In contrast,
when incubated with cytoplasmic extract and nuclei of differentiated
H9c2 myotubes, <1% of cardiomyocyte nuclei underwent DNA
synthesis (Figure 2C and 2D, lane c).
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For a direct comparison of DNA replication patterns,
cardiomyocyte nuclei and S-phase H9c2 nuclei were
immobilized together and incubated with S-phase H9c2
cytoplasmic extract. The different types of nuclei can be distinguished
by their form and size. H9c2 nuclei are 3-fold bigger (cross-sectional
area) and oval, whereas neonatal cardiomyocytes are small
and round (areaH9c2=309.6±8.0
µm2,
areaneonatal=105.5±2.0
µm2, n=150 nuclei for each group,
P<0.0001). As depicted in Figure 4A and at higher magnification in Figure 4B, the subnuclear PCNA pattern of replication foci in
cardiomyocyte nuclei highly resembles that found in S-phase
H9c2 nuclei (Figure 4C). These results indicate that
cardiomyocyte nuclei undergo a normal S phase with PCNA
replication patterns indistinguishable from intact cycling cells.
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For the Induction of DNA Synthesis in Cardiomyocyte
Nuclei, De Novo Protein Synthesis in Conjunction With Both Nuclear and
Cytoplasmic Factors Is Necessary
To evaluate whether the translation machinery necessary of S-phase
H9c2 cytoplasm is required for DNA replication, we included
cycloheximide in the coincubation experiments. Cardiomyocyte nuclei and
S-phase H9c2 nuclei were immobilized together and incubated
with S-phase H9c2 cytoplasmic extract in the presence or absence of
cycloheximide (10 µg/mL). Under these conditions, DNA synthesis
proceeded normally in H9c2 nuclei, which had been synchronized in S
phase (Figure 5A and 5B, lanes g and h).
In contrast, the efficiency of initiation of DNA synthesis was reduced
to 20% in cardiomyocyte nuclei (Figure 5A and 5B,
lanes e and f).
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Factors from S-phase H9c2 cells needed for the induction of DNA
synthesis in cardiomyocytes could be located in either the
nucleus or the cytoplasm. To address this question,
cardiomyocyte nuclei were incubated with either S-phase
H9c2 nuclei and PBS or S-phase H9c2 cytoplasmic extract alone. In
neither case could initiation of DNA synthesis be observed in
cardiomyocyte nuclei (Figure 5B, lanes a and b).
These data indicate that the induction of DNA synthesis in
cardiomyocyte nuclei requires components from both nuclei
and cytoplasmic extracts. However, the possibility that factors are
leached out of the nucleus during the preparation of cytoplasmic
extracts cannot be ruled out.
Next, neonatal cardiomyocyte nuclei were coincubated with
S-phase H9c2 nuclei and cytoplasmic extracts from different cell cycle
stages. Coincubation with S-phase H9c2 cytoplasmic extract resulted in
the efficient induction of DNA synthesis in 92% of
cardiomyocyte nuclei (Figure 5B, lane c).
Coincubation with differentiated H9c2 myotube cytoplasmic extract
failed to do so (Figure 5B, lane d). These data indicate that
S-phase–specific factors in addition to the translation machinery are
required for the reinitiation of DNA synthesis in
cardiomyocyte nuclei.
Induction of DNA Synthesis in Adult Cardiomyocyte Nuclei
Neonatal cardiomyocytes are not fully differentiated.
Furthermore, in vivo, they are still able to undergo DNA synthesis and
mitosis, giving rise to binucleated myocytes. Therefore, we wanted to
test whether fully differentiated adult cardiomyocytes
could be induced to reenter S phase or had irreversibly lost the
ability to synthesize DNA. Coincubation of adult rat
cardiomyocyte nuclei with their own cytoplasm (Figure 6A and 6D, lane a) or with nuclei and
cytoplasmic extract of differentiated H9c2 myotubes (Figure 6B and 6D, lane b) did not induce DNA synthesis. However, in 72% of adult
cardiomyocyte nuclei, DNA synthesis was induced upon
coincubation with nuclei and cytoplasmic extract from S-phase H9c2
cells (Figure 6C and 6D, lane c).
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Coincubation With Nuclei and Cytoplasmic Extract From S-Phase
Primary Rat Cardiac Nonmyocytes Induces DNA Synthesis in Adult
Cardiomyocyte Nuclei
H9c2 myocytes are immortalized cells that may express factors not
present in normal primary cells. To test whether factors
present in primary cells suffice to induce S phase, we
analyzed the effect of extracts from primary proliferating
cells on isolated cardiomyocyte nuclei in our coincubation
system. Cardiac fibroblasts that represented the vast
majority of nonmyocytes attaching to the bottom of culture
dishes during the preplating procedure were isolated and synchronized
in S phase (Figure 7). Seventy-six
percent of adult cardiomyocyte nuclei underwent DNA
synthesis when coincubated with nuclear and cytoplasmic extract from
S-phase nonmyocytes, but neither the nuclear nor the
cytoplasmic extract alone exhibited a significant induction of DNA
synthesis (Figure 8A and 8B). The
induction of biotin-16-dUTP incorporation correlated well with the
appearance of the characteristic PCNA replication–associated pattern
(Figure 8C), as it was observed before in neonatal rat
cardiomyocytes using H9c2 S-phase nuclei and cytoplasmic
extracts (Figure 4A and 4B).
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Discussion |
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In the present study, we describe the establishment and characterization of a mammalian myocardial cell-free system to investigate the cell cycle control of DNA replication in nuclei from differentiated cells. We show that nuclei and cytoplasmic extract from S-phase H9c2 cells but not from differentiated H9c2 myotubes efficiently initiate DNA synthesis in terminally differentiated cardiomyocyte nuclei. Furthermore, we demonstrate that the induction of DNA synthesis in cardiomyocyte nuclei depends on both nuclear and cytoplasmic factors in conjunction with de novo protein synthesis.
The molecular mechanisms responsible for the arrest of
cardiomyocyte proliferation during the postnatal period are
largely unknown. Earlier studies have shown that the activity of
cardiomyocyte DNA polymerase 
decreases during the first
postnatal weeks.23 24 25 Recently, cyclin-dependent kinase
(cdk) activities in cardiomyocytes were also reported to
decline during the early neonatal period.26 27 28 In
particular, the phosphotransferase activity of cdk4 governing the
G1 phase of cell cycle is detectable until only
day 1, whereas the cdk2 activity, regulating the
G1–S-phase transition, decreases markedly after
day 2. Although FCS, for example, upregulates G1
and G1–S-phase cyclins, activates cdk4,
cdk2, and cdc2, and induces phosphorylation of the
retinoblastoma protein in cultured neonatal cardiomyocytes,
it failed to stimulate DNA synthesis in these cells.3
Taken together, these and other studies suggest that
cardiomyocytes lose their ability to undergo DNA synthesis
during the early neonatal phase.
Initiation of DNA replication is a key step in the cell division cycle. Both intracellular and extracellular signals are involved in mid-G1 phase, influencing the decision to either withdraw from the cell cycle or proliferate. Therefore, entry into S phase is a crucial initial step for the induction of regenerative growth. In our study, DNA synthesis could be achieved very efficiently in neonatal and adult rat cardiomyocyte nuclei when they were coincubated with nuclei and cytoplasmic extract of primary or immortalized S-phase cells.
Our results indicate that, in principle, the cell cycle block in cardiomyocytes is reversible. They also suggest that no dominant inhibitory factors are present, although we cannot exclude the possibility that they are titrated out. The isolation procedure could also affect the level and/or functional status of inhibitors in the nuclei, which by itself, however, is not sufficient to enter S phase. The induction of DNA synthesis in this system could also be due to the factors that are missing for the execution of DNA replication are directly supplied by the addition of S-phase nuclei and cytoplasmic extract.
The basic components of the DNA replication machinery have been mostly identified by use of an in vitro viral DNA replication system.29 One of the central proteins of the DNA replication machinery is PCNA. Our results show that PCNA is present at very low to undetectable levels in cardiomyocyte nuclei, which is in agreement with previous studies in which PCNA protein was not found in adult cardiomyocytes.30 Most replisome components are organized into megadalton complexes and tightly tethered to the nuclear matrix during S phase.31 32 Therefore, it is unlikely that replication factors such as PCNA, which are localized exclusively in the nucleus, would translocate from the S-phase H9c2 nuclei to cardiomyocyte nuclei. The importance of de novo translation for the induction of DNA synthesis is supported by our experiments using cycloheximide. PCNA accumulation was blocked and initiation of DNA synthesis was inhibited in cardiomyocyte nuclei, but elongation phase of DNA replication was not influenced in nuclei from H9c2 cells, which were already in S phase and had already accumulated the necessary proteins. However, we cannot exclude the possibility that PCNA or other cell cycle regulators were transcribed in the S-phase nuclei, translated by the cytoplasmic extract, and then translocated to the cardiomyocyte nuclei.
The mammalian myocardial cell-free system described in the present study now makes it feasible to study the transition of nuclei from the differentiated state into S phase and to analyze the requirements with a variety of biochemical techniques, such as fractionation of cellular extracts, addition of specific inhibitors, immunodepletion of candidate proteins, and extract mixing experiments.
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Acknowledgments |
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This work was supported in part by the Deutsche Forschungsgemeinschaft Grant Le721/2-1 (M.C.C., H.L.) and by the Max Delbrück Center for Molecular Medicine (R.v.H., M.C.C.). The authors wish to thank Marlies Grieben for her excellent technical assistance.
Received May 20, 1999; accepted June 11, 1999.
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