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Circulation Research. 2000;86:1069-1077

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(Circulation Research. 2000;86:1069.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Enhanced Cardiomyocyte DNA Synthesis During Myocardial Hypertrophy in Mice Expressing a Modified TSC2 Transgene

Kishore B. S. Pasumarthi, Hidehiro Nakajima, Hisako O. Nakajima, Shaoliang Jing, Loren J. Field

From the Wells Center for Pediatric Research and Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Ind.

Correspondence to Loren J. Field, Herman B Wells Center for Pediatric Research, James Whitcomb Riley Hospital for Children, 702 Barnhill Dr, Room 2600, Indianapolis, IN 46202-5225. E-mail ljfield{at}iupui.edu


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract—Tuberous sclerosis complex (TSC) is a rare genetic disorder characterized by the appearance of benign tumors in multiple organs, including the heart. Disease progression is accompanied by homozygous mutation at 1 of 2 loci (designated TSC1 or TSC2), leading to the suggestion that these genes function as tumor suppressors. In this study, we generated a series of TSC2 cDNAs in which one or more structural motifs were deleted, with the hope that expression of the modified gene product would override the growth-inhibitory activity of the endogenous TSC2 gene product. Several of the modified cDNAs enhanced growth rate, increased endocytosis, and promoted aberrant protein trafficking when expressed in NIH-3T3 cells, thereby mimicking phenotypes typical of TSC2-deficient cells. Surprisingly, targeted expression of the most potent TSC2 cDNA to the heart did not perturb cardiac development. However, the level of cardiomyocyte DNA synthesis in adult transgenic mice was elevated >35-fold during isoproterenol-induced hypertrophy compared with their nontransgenic siblings. These results suggest that alteration of TSC2 gene activity in combination with ß-adrenergic stimulation can reactivate the cell cycle in a limited number of terminally differentiated adult cardiomyocytes.


Key Words: tumor suppressor • cardiac regeneration


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Tuberous sclerosis complex (TSC) is characterized by the appearance of nonmalignant tumors in a wide spectrum of organs, including the brain, kidney, lung, skin, and heart. The familial form of the disease exhibits an autosomal dominant pattern of inheritance, and 2 disease-causing loci (designated TSC1 and TSC2) have been identified. The TSC1 gene, which encodes a 130-kD protein called hamartin, is located at chromosome 9q34.1 The TSC2 gene, which encodes a 198-kD protein called tuberin, is located at chromosome 16p13.3.2 Molecular analyses have demonstrated that tumor formation in TSC patients is frequently associated with loss of heterozygosity at either locus,3 4 confirming earlier predictions based on genetic analyses that the TSC genes function as tumor suppressors. This view is further supported by analyses of Eker rats, which carry a mutation at the TSC2 locus.5 6 Rats homozygous for the TSC2 mutation die in utero at embryonic day 13.5, whereas rats heterozygous for the TSC2 mutation are viable but highly prone to renal carcinoma. Tumor progression in the heterozygous Eker rats is accompanied by loss of heterozygosity at the TSC2 locus, and expression of a transgene encoding a wild-type TSC2 cDNA blocks renal carcinoma in this model.7

Primary myocardial tumors in the general population are extremely rare.8 In contrast, >50% of TSC patients exhibit primary myocardial tumors.9 These results suggest that the TSC gene products play an important role in the regulation of the cardiomyocyte cell cycle. This view is supported by analysis of embryonic day 12.5 fetuses derived from intercrosses of heterozygous Eker rats.10 Spontaneously contractile cardiomyocytes were observed after multiple passages of whole-embryo cultures prepared from fetuses homozygous for the mutant TSC2 allele but not from cultures prepared from heterozygous or wild-type fetuses. Immune cytological and electron microscopic analyses confirmed that the contractile cells in the homozygous mutant cultures were cardiomyocytes. Tritiated thymidine incorporation analyses indicated that these cells exhibited sustained DNA synthesis after as many as 8 passages. Although disruption of the TSC2 gene resulted in embryo death in mice, histological analyses suggested the presence of enhanced ventricular myocardial proliferation in the homozygous mutant fetuses.11 Thus, the TSC2 gene product is important for cardiomyocyte terminal differentiation.

Molecular analyses have identified several structural motifs that appear to be important for tuberin activity. For example, a small 38-amino-acid motif near the C-terminus of tuberin exhibits homology to the GTPase-activating protein rap1GAP,2 12 a protein that negatively regulates rap1. Rap1, a member of the ras superfamily, was originally isolated by its ability to diminish the transforming activity of the Ki-ras mutant.13 The observation that tuberin stimulated the GTPase activity of rap1a suggested that this motif is functionally active,14 a view supported indirectly by the colocalization of these two proteins in the Golgi apparatus.15 Other studies identified a 59-amino-acid motif located at the C-terminus of tuberin that binds to rabaptin-5.16 Rabaptin-5 is a direct effector of the small GTPase Rab5, a rate-limiting component of the endocytotic membrane docking apparatus.17 Tuberin possesses potent Rab5 GAP activity,16 leading these authors to propose that rabaptin-5 functions as an adaptor protein to recruit Rab5 for tuberin-mediated GAP activity. In support of this, abnormally high levels of endocytosis were observed in embryonic fibroblasts lacking tuberin. More recently, tuberin amino acid residues 346 to 371 were shown to be sufficient for binding to hamartin, the product of the TSC1 gene.18 Finally, an alternatively spliced region encoding a leucine zipper located at the N-terminus of the protein has also been identified.19

In this study, we generated modified TSC2 cDNAs in which one or more of these structural motifs were altered. The modified cDNAs were then screened for their ability to promote growth in NIH-3T3 cells, which express relatively high levels of the endogenous tuberin gene product. Cells expressing the modified cDNAs exhibited phenotypes typical of TSC2-deficient cells, including enhanced growth rates, increased endocytosis, and aberrant p27 trafficking. The cDNA exhibiting the greatest growth-promoting activity in cell culture was tested for its ability to alter cardiomyocyte cell cycle regulation in vivo. Although cardiac development in transgenic mice expressing the cDNA under the control of the {alpha}-cardiac myosin heavy chain (MHC) promoter appeared normal, the level of cardiomyocyte DNA synthesis during isoproterenol-induced hypertrophy was {approx}35-fold greater than that observed in nontransgenic siblings. These results are discussed within the context of promoting regenerative growth in the adult myocardium.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
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 down arrowDiscussion
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Recombinant DNA Protocols
Isolation and characterization of the wild-type murine tuberin cDNA was described previously.19 All recombinant DNA manipulations used standard methodologies,20 and construct fidelity was confirmed by diagnostic DNA sequencing. All animal experimentation was in accordance with institutional and federal guidelines.

Cell Culture Experiments
For colony growth assays, TSC2 cDNAs were subcloned into pRcCMV and transfected into NIH-3T3 cells.20 Cells were selected in G418 for 13 days (0.3 mg/mL), and the dishes were then stained with 0.1% gentian violet. Stable cell lines were isolated by long-term selection with G418. To monitor the effect of transgene expression on growth, stable cell lines were plated in triplicate at a density of 104 cells/100-mm dish and cultured for 2, 4, 6, and 8 days. Total cell number was determined at each time point with a hemocytometer. For tumorigenesis analysis in SCID mice, 106 cells of each line were injected into SCID/NOD mice. Tumor growth was scored as positive when the tumor mass reached a 2.5-cm diameter.

Western Blot Analyses
Cells or hearts were homogenized directly in NP40 buffer and processed as described previously.21 22 The TSC2 antibody used in this study was C-20 (Santa Cruz Biochemicals). Signal was visualized by the enzyme-linked chemiluminescence method according to the manufacturer’s protocol (Amersham).

Endocytosis Assay
Fluid-phase endocytosis was quantified with a horseradish peroxidase (HRP) uptake assay.23 HRP uptake was determined by measuring the absorbency values at 455 nm and normalized to the protein content in the sample.

p27 Immune Reactivity
Cells plated in chamber slides were serum-synchronized, fixed in ice-cold acetone and methanol (1:1), and processed for anti-p27 immune cytology (antibody sc-528, Santa Cruz Biochemicals) at a 1:100 dilution. p27 immune reactivity signal was visualized with a Vectastain ABC kit from Vector Laboratories.

Generation of the MHC{Delta}RL Transgenic Mice
The MHC{Delta}RL transgene was constructed by use of the transcriptional regulatory sequences of the mouse MHC gene24 and the TSC2 {Delta}RL cDNA. The SV40 early-region transcription terminator/polyadenylation site (nucleotide residues 2586 to 2452)25 was inserted downstream from the {Delta}RL cDNA insert. Transgene insert DNA was microinjected into zygotes by standard methodologies.26 Transgenic animals were identified by diagnostic polymerase chain reaction amplification as described.27

Cardiomyocyte DNA Synthesis Assays
Cardiomyocyte DNA synthesis was monitored with a thymidine incorporation assay and an MHC-nLAC reporter transgenic strain as described.28 29 Mice received a single injection of [3H]thymidine [200 µCi IP at 28 Ci/(mmol/L), Amersham] and were euthanized 4 hours later. Standard methods were used for cryosectioning.30 Cardiomyocyte DNA synthesis was scored by the colocalization of ß-galactosidase (ß-gal) activity (dark blue staining when visualized under bright-field illumination) and silver grains. For anti-tuberin immune cytology, heart sections were reacted with anti-tuberin antibody C-20, and signal was visualized with a Vectastain ABC kit from Vector Laboratories.

Myocardial Hypertrophy Model
Myocardial hypertrophy was induced by isoproterenol infusion with Alzet minipumps in adult mice as described previously.31

An expanded Materials and Methods section is available online at http://www.circresaha.org.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Colony Growth Screen for TSC2 Modifications With Growth-Promoting Activity
Previous analyses have identified several conserved motifs in human,2 rat,32 and mouse19 tuberin and to a lesser degree in puffer fish33 and Drosophila tuberin.34 These include a leucine zipper near the N-terminus of the molecule, as well as a rap1GAP homology motif and a rabaptin-5 binding motif near the C-terminus (Figure 1ADown). The high level of conservation suggests that these motifs may be important for tuberin tumor suppressor activity, a prediction that has been partially born out by functional studies (see Introduction). A series of TSC2 cDNA expression constructs in which the rap1GAP, leucine zipper, or rabaptin-5 motifs were deleted individually or in combination were generated (see Figure 1ADown) and tested for their ability to promote growth when expressed in cells with high levels of the endogenous TSC2 gene product. The expression constructs used the cytomegalovirus (CMV) promoter and also carried a neor expression cassette to facilitate selection of transfected cells.



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  		Figure 1. A, Schematic of the modified TSC2 cDNAs used in this study. The positions of the leucine zipper (L), rap1GAP (G), and rabaptin-5 (R) motifs are indicated. For the {Delta}G construct, amino acid residues 1617 through 1655 were deleted. For the {Delta}L construct, amino acid residues 81 through 102 were deleted. For the {Delta}R construct, a splice variant in which amino acid residues 1679 through 1742 were substituted was used. WT is the wild-type TSC2 cDNA. B, Colony growth assay of NIH-3T3 cells transfected with the various TSC2 expression constructs. Transfected cells were selected for 13 days in G418, and the dishes were fixed and stained with gentian violet. Transfection efficiencies were calculated by monitoring expression of a cotransfected CMV–ß-gal reporter gene at 48 hours after transfection. ß-gal activities (relative luminescence/mg protein, x105) were: CMV-null, 1.7±0.12; CMV{Delta}R, 1.7±0.12; CMV{Delta}G, 1.7±0.06; CMV{Delta}L, 1.7±0.06; CMV{Delta}RL, 1.7±0.06; CMV{Delta}RG, 1.6±0.15; CMV{Delta}RGL, 1.8±0.15; and CMV-WT, 1.7±0.66. There was no significant difference between groups by Kruskal-Wallis nonparametric ANOVA test.

A colony growth assay was used to monitor the impact of the modifications on cell growth. NIH-3T3 cells were transfected with the various constructs, and the resulting colonies were visualized by gentian violet staining after 13 days of G418 selection (Figure 1BUp). Small colonies were observed in dishes transfected with expression vector lacking a cDNA insert (CMV-null construct), indicative of the rate of proliferation under the conditions used. Smaller and fewer colonies were observed in dishes transfected with a full-length wild-type TSC2 construct (CMV-WT), consistent with the previously described growth-suppressing activity of this molecule. Colonies in dishes transfected with constructs in which the rap1GAP (CMV{Delta}G) or leucine zipper (CMV{Delta}L) motifs were disrupted grew to the same extent as those transfected with the CMV-null construct. Colonies in dishes transfected with constructs in which the rabaptin-5 motif was disrupted (CMV{Delta}R) exhibited a modest but reproducible increase in size relative to the CMV-null control.

Expression vectors encoding tuberin molecules with combinatorial mutations were then generated to determine whether synergistic effects could be obtained. Markedly increased colony growth was observed in dishes transfected with constructs encoding tuberin molecules lacking the rabaptin-5 and rap1GAP motifs (CMV{Delta}RG) or the rabaptin-5 and leucine zipper motifs (CMV{Delta}RL; see Figure 1BUp). Paradoxically, growth inhibition (similar to that obtained with the wild-type tuberin construct) was observed in cells transfected with a construct in which all 3 motifs were disrupted (CMV{Delta}RGL). These results were reproduced in 4 independent experiments using a minimum of 3 independent DNA preparations; moreover, no significant difference in transfection efficiency was observed between the different constructs.

Modified TSC2 cDNAs Promote Cell Growth In Vitro and In Vivo
Stable NIH-3T3 cell lines harboring the growth-promoting constructs were generated to determine the consequences of long-term transgene expression. Cell lines with the CMV-null and CMV-WT constructs were generated as controls. Western blot analyses indicated that each construct expressed similar amounts of recombinant protein (Figure 2ADown: data from representative cell lines are shown; the level of recombinant protein expression was {approx}2- to 3-fold greater than that of the endogenous tuberin gene product). Cells from each group were plated in triplicate at a density of 104 cells/100-mm dish and cultured for 2, 4, 6, and 8 days. The cultures were then trypsinized and the cell numbers determined (Figure 2BDown). In agreement with the colony growth assay, cells expressing the CMV{Delta}RL and CMV{Delta}RG constructs exhibited the highest rate of proliferation. Similar results were obtained when 6 independent cell lines for each construct were analyzed (not shown).



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  		Figure 2. A, Western blot analysis of tuberin expression in NIH-3T3 cells transfected with various control and TSC2 expression constructs. The molecular weight shift resulting from various modifications was of insufficient magnitude to resolve on a 7.5% polyacrylamide gel used in this study. B, Growth curves for representative cell lines stably transfected with various control and TSC2 expression constructs. Each time point was analyzed in triplicate, and the error bars indicate the SEM. Growth rate values at days 6 and 8 were significantly different from the CMV-null controls (CMV{Delta}RG, P<0.005; CMV{Delta}RL, P<0.001; ANOVA with Bonferroni multiple comparison test). C, Kaplan-Meier plot of tumor-free mice after injection of 106 cells expressing control or TSC2 constructs. D, Western blot analysis of tuberin expression in tumors recovered from transplanted SCID mice. Note that sustained tuberin expression was present.

A SCID mouse tumor assay was used to determine whether the enhanced growth observed with the CMV{Delta}RL and CMV{Delta}RG constructs in vitro persisted in vivo. Cells expressing these constructs were injected into NOD/SCID mice (106 cells/mouse; 8 mice per group), and the animals were sequestered and monitored for tumor formation. The CMV-null and CMV-WT cell lines were used as controls. Tumor formation was accelerated in cells expressing the CMV{Delta}RL and CMV{Delta}RG constructs compared with the CMV-null construct, confirming that the growth enhancement of these modified tuberin cDNAs persisted in vivo (Figure 2CUp). Western blot analyses revealed sustained tuberin expression in tumor material recovered from the mice (Figure 2DUp). Interestingly, no tumors were detected in mice injected with cells expressing the CMV-WT construct, which provides compelling proof of the growth-suppressing activity of wild-type tuberin (the experiment was terminated after 125 days). To determine whether this tumor suppressor activity was associated with apoptosis, CMV-null and CMV-WT cultures were processed for in situ end-labeling (ISEL) reactivity, which provides a sensitive assay for DNA fragmentation.35 Many cells in the CMV-WT cultures had markedly condensed chromatin (visualized via Hoechst staining, blue signal), which was also positive for ISEL reactivity (green signal, see Figure 3ADown). Further inspection under phase-contrast illumination confirmed that the cells were dying (Figure 3ADown). Quantitative analysis of the cultures revealed that cells expressing the CMV-WT construct were highly prone to apoptosis (Figure 3BDown).



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  		Figure 3. A, Cultured CMV-WT cells were stained with Hoechst to visualize chromatin (blue signal), processed for ISEL reactivity to identify nuclei with fragmented DNA (green signal), and visualized under phase contrast for morphological analysis. Arrow indicates a cell with markedly condensed chromatin and fragmented DNA that appears to be undergoing apoptosis. B, Quantitative assessment of ISEL-positive nuclei in CMV-null and CMV-WT cultures grown in the presence of 10% or 0.1% serum. There was a significantly higher incidence of ISEL-positive nuclei in the CMV-WT cultures than in the CMV-null cultures (P<0.005, unpaired t test). Similar rates of apoptosis were seen in unselected NIH-3T3 cells and CMV-null cells.

Increased Endocytosis and Aberrant Protein Trafficking in Cells Expressing Modified TSC2 cDNAs
Recent studies have suggested that loss of tuberin activity results in increased endocytosis16 and aberrant trafficking of negative cell cycle–regulatory proteins.34 36 Endocytosis was measured in the CMV-null, CMV{Delta}RG, and CMV{Delta}RL cell lines with an HRP uptake assay.23 A 2- to 2.5-fold increase in endocytosis rates was observed in the CMV{Delta}RL and CMV{Delta}RG cell lines compared with the CMV-null control (Figure 4ADown). The subcellular localization of p27, a negative regulator of the S-phase cyclin-dependent kinases, was also monitored. Prominent nuclear p27 immune reactivity was seen in the CMV-null control cells (Figure 4BDown). In contrast, only weak and diffuse cytoplasmic p27 immune reactivity was observed in the CMV{Delta}RL cells (Figure 4CDown). Cytoplasmic p27 immune reactivity was also observed in the CMV{Delta}RG cells (data not shown).



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  		Figure 4. A, HRP endocytosis assay for CMV-null, CMV{Delta}RG, and CMV{Delta}RL cell lines. Results are normalized relative to the value for the CMV-null cells. Error bars indicate SEM. Each cell line was analyzed in triplicate. Endocytosis values were significantly different from the CMV-null controls (CMV{Delta}RG, P<0.0005; CMV{Delta}RL, P<0.0001; unpaired t test). B, Photomicrograph of a CMV-null cell processed for p27 immune reactivity (HRP-conjugated secondary antibody, black signal). Prominent nuclear p27 immune reactivity is seen. Of the cells expressing the CMV-null transgene, 97.1±0.52% exhibited nuclear p27 immune reactivity (n=500). C, Photomicrograph of a CMV{Delta}RL cell processed for p27 immune reactivity (HRP-conjugated secondary antibody, black signal). Diffuse cytoplasmic p27 immune reactivity is seen; no signal is apparent in the nucleus. Only 4.2±0.34% of the cells expressing the CMV{Delta}RL transgene exhibited nuclear p27 immune reactivity (n=500); the remainder of the cells had predominantly cytoplasmic immune reactivity.

Cardiac Development Is Normal in Transgenic Mice Expressing the TSC2{Delta}RL cDNA in the Heart
We next determined whether targeted expression of a modified TSC2 cDNA would alter cardiac development in transgenic mice. The TSC2{Delta}RL cDNA was tested, because this modification had the greatest effect on cell growth in NIH-3T3 cells. A transgene comprising the mouse {alpha}-cardiac MHC promoter and the {Delta}RL tuberin cDNA was produced (Figure 5ADown). The resulting transgene (designated MHC{Delta}RL) was microinjected into 1-cell embryos to produce transgenic animals. Seven of 29 mice born from the microinjected embryos were transgenic. These mice gave rise to 3 independent lineages, and transgene expression was stratified via Western blot analysis. MHC{Delta}RL line 1 expressed high levels of the transgene (Figure 5BDown) and was selected for subsequent analysis. Endocytosis was examined in primary cardiomyocyte cultures prepared from control and MHC{Delta}RL mice. Endocytosis was increased {approx}2.5-fold in the transgenic hearts (Figure 5CDown), consistent with the biological activity observed for the TSC2{Delta}RL cDNAs in cultured cells.



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  		Figure 5. A, Schematic of the MHC{Delta}RL transgene. The MHC promoter consisted of 4.5 kb of 5' flanking sequence and 1 kb of the gene including exon 1, exon 2, and a portion of exon 3. SV40 refers to the SV40 early region transcription terminator and polyadenylation sequences. B, Western blot analysis demonstrating recombinant tuberin expression in the MHC{Delta}RL transgenic heart. (-) indicates the level of tuberin expression in nontransgenic animals. C, HRP endocytosis assay of primary heart cultures prepared from nontransgenic and MHC{Delta}RL transgenic hearts in the absence or presence of isoproterenol. Results are normalized relative to the value for the nontransgenic hearts in the absence of isoproterenol treatment. Error bars indicate SEM. Endocytosis values for the transgenic hearts were significantly different from the nontransgenic controls (MHC{Delta}RL, P<0.0001; unpaired t test).

To monitor the consequences of transgene expression on cardiomyocyte DNA synthesis, the MHC{Delta}RL mice were crossed with MHC-nLAC transgenic mice,28 29 which express a nuclear localized ß-gal reporter under the regulation of the MHC promoter. Reporter gene activity permits rapid and unambiguous identification of cardiomyocyte nuclei in histological sections by simple X-Gal staining (cardiomyocyte nuclei appear dark blue as a result of the nuclear ß-gal activity). Progeny inheriting both the MHC-nLAC and MHC{Delta}RL transgenes were used as experimental animals, and those inheriting only the MHC-nLAC transgene were used as controls. There was no evidence of altered cardiac growth (TableDown; 11-week-old mice were examined). Histochemical analyses did not detect any overt consequences of transgene expression.


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  		Table 1. Cardiac Attributes in MHC{Delta}RL Transgenic Mice

To monitor the effect of transgene expression on adult cardiomyocyte DNA synthesis, mice received a single injection of tritiated thymidine, and the hearts were then harvested and cryosectioned. The resulting sections were stained with X-Gal and then processed for autoradiography. Cardiomyocyte DNA synthesis was scored by colocalization of ß-gal activity and silver grains in autoradiograms of histological sections. Approximately 100 000 cardiomyocyte nuclei were counted for the experimental and control animals (n=5 animals per group). No cardiomyocyte DNA synthesis was observed in any of the animals (see TableUp). Analyses of older MHC{Delta}RL transgenic mice revealed no evidence of myocardial tumorigenesis (18 months old) or cardiomyocyte DNA synthesis (12 months old, 60 000 nuclei screened).

Myocardial Hypertrophy Stimulates Cardiomyocyte DNA Synthesis in MHC{Delta}RL Mice
Cardiomyocyte DNA synthesis was also monitored during isoproterenol-induced cardiac hypertrophy. After 7 days of isoproterenol infusion, a similar hypertrophic response was observed in the control MHC-nLAC and experimental MHC-nLAC/MHC{Delta}RL animals (36±4.3% increase in HW/TL for control mice, 40±4.3% increase in HW/TL for experimental mice, see TableUp). In addition, a similar induction in ventricular atrial natriuretic factor mRNA expression was observed for the isoproterenol-treated control and experimental animals (not shown). The tritiated thymidine incorporation assay failed to detect any cardiomyocyte DNA synthesis in the MHC-nLAC mice after isoproterenol-induced hypertrophy (see TableUp; {approx}100 000 nuclei were counted). In contrast, DNA synthesis was reactivated in the ventricular myocardium of the MHC{Delta}RL transgenic mice during isoproterenol-induced hypertrophy (see TableUp). Examples of cardiomyocytes synthesizing DNA (as evidenced by colocalization of silver grains and nuclear ß-gal activity) are shown in Figure 6ADown and 6BDown. An overall cardiomyocyte tritiated thymidine labeling index of 0.018% was observed in the ventricles of MHC{Delta}RL transgenic mice after 7 days of isoproterenol infusion. Immune histological analyses confirmed that the cells synthesizing DNA were expressing the MHC{Delta}RL transgene (Figure 6CDown, arrow). Moreover, the level of transgene expression appeared to be similar to that in cardiomyocytes not synthesizing DNA (Figure 6CDown, arrowheads). Isoproterenol treatment had no effect on endocytosis in the transgenic hearts (Figure 5CUp), suggesting that ß-adrenergic stimulation did not directly alter transgene activity. Finally, infusion of phenylephrine failed to reactivate cardiomyocyte DNA synthesis in the MHC{Delta}RL transgenic mice, despite a marked hypertrophic response (52.0% increase in HW/BW ratio, 60 000 nuclei screened). This finding suggests that reactivation of cardiomyocyte DNA synthesis in MHC{Delta}RL mice is dependent on ß-adrenergic stimulation, rather than a simple response to hypertrophic myocardial growth.



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  		Figure 6. A and B, Examples of cardiomyocyte DNA synthesis in sections prepared from MHC-nLAC/MHC{Delta}RL transgenic animals with cardiac hypertrophy. After 7 days of isoproterenol infusion, the mice received a single injection of tritiated thymidine, and the hearts were harvested, sectioned, and processed for X-Gal staining and autoradiography. C, Analysis of tuberin immune reactivity and DNA synthesis in MHC{Delta}RL cardiomyocytes. Tuberin immune reactivity was detected via HRP assay (reddish brown signal); cardiomyocyte nuclei were visualized by X-Gal staining. Similar levels of transgene expression were seen in cardiomyocytes synthesizing DNA (arrow) and cardiomyocytes that were not synthesizing DNA (arrowheads). The photographic emulsion used for the autoradiograph in C has finer grain, hence the difference in silver grain size compared with A and B.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The experiments described above were based on the premise that expression of TSC2 cDNAs in which one or more structural motifs were altered might antagonize the activity of the endogenous gene product and thereby mimic TSC2 deficiency. We selected 3 structural motifs for which there was either direct experimental evidence attesting to functional importance (the rap1GAP and rabaptin-5 binding motifs), or alternatively, evidence for splice variants that directly altered or deleted the motif (the leucine zipper). We have not yet tested the consequence of deleting the hamartin-binding domain, which has only recently been identified.18

Cells Expressing the {Delta}RG and {Delta}RL cDNAs Mimic TSC2-Deficient Cells
Loss of tuberin activity is associated with enhanced rates of proliferation.37 Similarly, enhanced proliferation was observed in the CMV{Delta}RG and CMV{Delta}RL cell lines. A 2-fold increase in endocytosis was reported in homozygous mutant Eker rat embryonic fibroblasts compared with wild-type fibroblasts.16 A similar increase in endocytosis was observed in CMV{Delta}RL and CMV{Delta}RG cells. Finally, aberrant subcellular localization of the cyclin-dependent kinase inhibitor p27 was observed in tuberin-deficient cells36 as well as in the CMV{Delta}RG and CMV{Delta}RL cells. Thus, cells expressing the {Delta}RG or {Delta}RL cDNAs share multiple phenotypic attributes with tuberin-deficient cells.

Given that tuberin clearly interacts with multiple proteins (including hamartin, rap1a, and rabaptin-5), it is relatively easy to envision how the modified cDNAs could interfere with the activity of the endogenous gene product. For example, tuberin encoded by the {Delta}RL cDNA would lack the ability to bind rabaptin-5 and interact with other leucine zipper proteins but would nonetheless retain the capacity to interact with rap1 and potentially other G proteins. Expression of the {Delta}RL protein could effectively block the activity of the endogenous tuberin (and thereby mimic a loss of function mutation) by a simple titration mechanism. If a generalized scheme in which a disruption of protein/protein interactions underlies the observed cell cycle effects is correct, we would anticipate that some TSC2 mutations in TSC patients could act in a dominant negative manner (provided that the mutated protein was expressed at a high enough level to compete with the wild-type gene product). In that regard, it is of interest to note that a mutation encompassing an L717R substitution in tuberin resulted in the formation of multiple lung cysts in the absence of loss of heterozygosity, consistent with dominant negative activity.38

Cardiac Development Is Normal in MHC{Delta}RL Mice
Both clinical and experimental observations support the notion that tuberin activity is important for normal cardiomyocyte terminal differentiation. Given the effects of TSC2{Delta}RL cDNA expression in cell culture experiments, it was surprising that cardiac development was normal in the MHC{Delta}RL mice. No apparent morbidity or mortality was associated with myocardial transgene expression, nor was there evidence for either abnormal cardiac hypertrophy or sustained cardiomyocyte DNA synthesis in unperturbed adult transgenic mice. Additional studies failed to detect any differences in cardiomyocyte nucleation or gross histological appearance in fetal and adult transgenic mice compared with their nontransgenic sibs (K.B.S.P. and L.J.F., unpublished observations). It would thus appear that expression of the TSC2{Delta}RL cDNA did not perturb the normal process of cardiomyocyte terminal differentiation. This is in contrast to the situation in homozygous mutant Eker fetuses10 and in TSC2 patients,9 in whom there is clear evidence for increased cardiomyocyte proliferation in the absence of tuberin expression.

This observation suggests that expression of the TSC2{Delta}RL cDNA in cardiomyocytes is not functionally equivalent to disruption of the tuberin gene. It is possible that the relative level of transgene expression was insufficient to completely antagonize the activity of the endogenous tuberin. It is nonetheless clear that the TSC2{Delta}RL cDNA is biologically active, as evidenced by markedly increased cardiomyocyte endocytosis in the transgenic animals. The absence of a developmental phenotype might also be attributable to the timing of transgene expression. In the case of the Eker rat studies, the homozygous mutant embryos never express tuberin. Consequently, tuberin deficiency preceded cardiomyogenic differentiation in that model. In contrast, the TSC2ÄRL cDNA was under regulation of the MHC promoter, and as such, transgene expression was not induced until after cardiomyogenic differentiation in the atria and not until birth in the ventricles.24 If a developmental cardioproliferative phenotype requires compromised tuberin activity during cardiomyogenic differentiation or early cardiac development, the transgenic model could not recapitulate this phenotype. It is difficult to directly compare the transgenic mouse model with clinical observations, because it is not clear when in cardiac development myocardial cells (or their precursors) become tuberin-deficient in TSC patients. Finally, as with all transgenic mouse models, we cannot rule out the possibility that sustained transgene expression has altered the normal developmental program, thereby making the myocardium less sensitive to targeted expression of the TSC2{Delta}RL cDNA.

Potential Mechanism for the Reactivation of Cardiomyocyte DNA Synthesis After ß-Adrenergic Stimulation in MHC{Delta}RL Mice
Given that cardiomyocyte terminal differentiation was normal in the MHC{Delta}RL mice, it was surprising that isoproterenol-induced hypertrophy reactivated cardiomyocyte DNA synthesis in adult animals. A cardiomyocyte DNA synthesis rate of 0.018% was observed in the MHC{Delta}RL mice after a single injection of isotope. Although no DNA synthesis was detected in the control MHC-nLAC hearts in the present study, previous experiments have established a maximum cardiomyocyte DNA synthesis rate of {approx}0.0005% in both unperturbed adult mice28 and in isoproterenol-treated animals.31 It should be noted that there is some discrepancy in the literature regarding the rates of cardiomyocyte DNA synthesis in normal and injured adult hearts (reviewed in References 39 and 40 ). Regardless of the differences observed in different species and with different models of cardiac injury, the salient observation in the present study is that transgene expression does not effect cardiomyocyte DNA synthesis in normal adult hearts but results in a 35-fold increase in cardiomyocyte DNA synthesis during hypertrophic myocardial growth.

The persistent cytoplasmic sequestration of p27 in mutant Eker embryonic fibroblasts36 and M-phase cyclins in TSC2-deficient Drosophila embryos34 suggest that anomalous protein trafficking might underlie the altered cell cycle regulation observed in tuberin-deficient cells. Expression of the TSC2{Delta}RL cDNA also perturbed p27 subcellular localization in transfected NIH-3T3 cells. Reactivation of cardiomyocyte DNA synthesis during cardiac hypertrophy in the MHC{Delta}RL transgenic mice may result from a similar mechanism. In vitro, hypertrophic stimulation of cultured neonatal cardiomyocytes (for example, angiotensin II treatment or mechanical stretch) is associated with a transient increase in a number of positive cell cycle–regulatory proteins, including immediate early genes, proto-oncogenes, and such G1/S regulatory proteins as D-type cyclins, CDK4, and hyperphosphorylated RB.41 42 Increased expression of many of these same gene products has been documented during cardiac hypertrophy in vivo.43 Although induction of these growth-promoting gene products during hypertrophy is insufficient to reactivate cardiomyocyte DNA synthesis in nontransgenic hearts (see TableUp),28 induction in a genetic background in which negative growth-regulatory pathways are compromised could easily result in reactivation of cardiomyocyte DNA synthesis. Importantly, the activities of many of these gene products are directly antagonized by p27 and other CDK inhibitors, which appear to be preferentially affected by anomalous protein trafficking in TSC2-deficient fibroblasts.

Expression of the MHC{Delta}RL transgene resulted in a 35-fold increase in the level of cardiomyocyte DNA synthesis during isoproterenol-induced hypertrophy compared with the nontransgenic animals. However, the absolute number of cardiomyocytes synthesizing DNA was low. Regional variability in the level of expression of CDK inhibitors throughout the myocardium,44 coupled to a global increase in cyclin and CDK expression observed in some instances during myocardial hypertrophy,45 may explain why only a small fraction of the cardiomyocytes expressing the MHC{Delta}RL transgene reactivate DNA synthesis. Importantly, isoproterenol had no effect on endocytosis in the transgenic cardiomyocytes, suggesting that ß-adrenergic stimulation did not directly alter transgene activity. Although several studies46 47 48 49 support a potential role for p27 in the cardiomyocyte DNA synthesis seen in hypertrophic MHC{Delta}RL hearts, preliminary efforts have failed to detect cytoplasmic sequestration of this protein in the transgenic hearts (K.B.S.P., unpublished observations). Thus, if aberrant protein trafficking does underlie the phenotype in the MHC{Delta}RL hearts, it most likely results from dysregulation of another protein(s). Given the pivotal role of Rab5 in regulating endocytosis and vesicle membrane docking,17 it is likely that intracellular protein trafficking is compromised to some degree in the MHC{Delta}RL hearts. At present, it is not clear whether those cardiomyocytes synthesizing DNA ultimately undergo karyokinesis and/or cytokinesis. Finally, it appears that the rap1GAP activity encoded by TSC2 is not important for adult cardiomyocyte cell cycle reactivation, because no DNA synthesis was detected in normal or hypertrophic hearts from mice expressing an MHC{Delta}RG transgene (K.B.S.P. and L.J.F., unpublished observations).

Implications for Regenerative Myocardial Growth
Although a limited number of cardiomyocytes in the MHC{Delta}RL mice retained the capacity to reenter the cell cycle in response to specific stimuli, the absence of DNA synthesis in unperturbed transgenic hearts suggests that developmental cell cycle withdrawal proceeds normally in these animals. This is in contrast to other transgenic models (as exemplified by mice expressing cyclin D1 in the myocardium),50 in which adult cardiomyocyte DNA synthesis appeared to result from a failure of the terminal differentiation process. This distinction is extremely important if the gene (or pathway) under study is to be exploited for therapeutic intervention in diseased hearts. Many forms of cardiovascular disease are accompanied by cardiomyocyte loss, and it is generally accepted that the regenerative capacity of the adult myocardium is limited.39 40 The ability to reactivate the cell cycle in the adult heart, thereby effecting regenerative growth, could thus be of considerable therapeutic utility. Targeted expression of the {Delta}RL tuberin cDNA, in conjunction with isoproterenol stimulation, appears to have reactivated the cell cycle in a limited number of adult cardiomyocytes. Identification of the pathway(s) activated in the responsive cells or alternatively, synergistic interaction with other regulatory pathways might provide a means to increase cell cycle reactivation to a point sufficient for regenerative cardiomyocyte growth.


*    Acknowledgments
 
This study was supported by the NHLBI and a grant from Bristol-Myers Squibb (Dr Field). Dr Pasumarthi was supported by a postdoctoral fellowship from the American Heart Association, Indiana Affiliate. We thank D. Field for excellent technical assistance and M. Soonpaa, S.-C. Tsai, and Brett Zani for comments on the manuscript.

Received November 18, 1999; accepted March 30, 2000.


*    References
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  1. van Slegtenhorst M, de Hoogt R, Hermans C, Nellist M, Janssen B, Verhoef S, Lindhout D, van den Ouweland A, Halley D, Young J, Burley M, Jeremiah S, Woodward K, Nahmias J, Fox M, Ekong R, Osborne J, Wolfe J, Povey S, Snell RG, Cheadle JP, Jones AC, Tachataki M, Ravine D, Kwiatkowski DJ, et al. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science. 1997;277:805–808.[Abstract/Free Full Text]
  2. The European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell. 1993;75:1305–1315.[Medline] [Order article via Infotrieve]
  3. Green AJ, Johnson PH, Yates JR. The tuberous sclerosis gene on chromosome 9q34 acts as a growth suppressor. Hum Mol Genet. 1994;3:1833–1834.[Abstract/Free Full Text]
  4. Green AJ, Smith M, Yates JR. Loss of heterozygosity on chromosome 16p13.3 in hamartomas from tuberous sclerosis patients. Nat Genet. 1994;6:193–196.[Medline] [Order article via Infotrieve]
  5. Yeung RS, Xiao GH, Jin F, Lee WC, Testa JR, Knudson AG. Predisposition to renal carcinoma in the Eker rat is determined by germ-line mutation of the tuberous sclerosis 2 (TSC2) gene. Proc Natl Acad Sci U S A. 1994;91:11413–11416.[Abstract/Free Full Text]
  6. Kobayashi T, Hirayama Y, Kobayashi E, Kubo Y, Hino O. A germline insertion in the tuberous sclerosis (Tsc2) gene gives rise to the Eker rat model of dominantly inherited cancer. Nat Genet. 1995;9:70–74.[Medline] [Order article via Infotrieve]
  7. Kobayashi T, Mitani H, Takahashi R, Hirabayashi M, Ueda M, Tamura H, Hino O. Transgenic rescue from embryonic lethality and renal carcinogenesis in the Eker rat model by introduction of a wild-type Tsc2 gene. Proc Natl Acad Sci U S A. 1997;94:3990–3993.[Abstract/Free Full Text]
  8. Braunwald E. Heart Disease. Philadelphia, Pa: WB Saunders; 1992.
  9. Watson GH. Cardiac rhabdomyomas in tuberous sclerosis. Ann N Y Acad Sci. 1991;615:50–57.[Abstract]
  10. Pajak L, Jin F, Xiao GH, Soonpaa MH, Field LJ, Yeung RS. Sustained cardiomyocyte DNA synthesis in whole embryo cultures lacking the TSC2 gene product. Am J Physiol. 1997;273:H1619–H1627.[Abstract/Free Full Text]
  11. Kobayashi T, Minowa O, Kuno J, Mitani H, Hino O, Noda T. Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice. Cancer Res. 1999;59:1206–1211.[Abstract/Free Full Text]
  12. Maheshwar MM, Cheadle JP, Jones AC, Myring J, Fryer AE, Harris PC, Sampson JR. The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis. Hum Mol Genet. 1997;6:1991–1996.[Abstract/Free Full Text]
  13. Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M. A ras-related gene with transformation suppressor activity. Cell. 1989;56:77–84.[Medline] [Order article via Infotrieve]
  14. Wienecke R, Konig A, DeClue JE. Identification of tuberin, the tuberous sclerosis-2 product: tuberin possesses specific Rap1GAP activity. J Biol Chem. 1995;270:16409–16414.[Abstract/Free Full Text]
  15. Wienecke R, Maize JC Jr, Shoarinejad F, Vass WC, Reed J, Bonifacino JS, Resau JH, de Gunzburg J, Yeung RS, DeClue JE. Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus. Oncogene. 1996;13:913–923.[Medline] [Order article via Infotrieve]
  16. Xiao GH, Shoarinejad F, Jin F, Golemis EA, Yeung RS. The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J Biol Chem. 1997;272:6097–6100.[Abstract/Free Full Text]
  17. Stenmark H, Vitale G, Ullrich O, Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell. 1995;83:423–432.[Medline] [Order article via Infotrieve]
  18. van Slegtenhorst M, Nellist M, Nagelkerken B, Cheadle J, Snell R, van den Ouweland A, Reuser A, Sampson J, Halley D, van der Sluijs P. Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Human Mol Genet. 1998;7:1053–1057.[Abstract/Free Full Text]
  19. Kim KK, Pajak L, Wang H, Field LJ. Cloning, developmental expression, and evidence for alternative splicing of the murine tuberous sclerosis (TSC2) gene product. Cell Mol Biol Res. 1995;41:515–526.[Medline] [Order article via Infotrieve]
  20. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
  21. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.[Medline] [Order article via Infotrieve]
  22. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76:4350–4354.[Abstract/Free Full Text]
  23. Stenmark H, Bucci C, Zerial M. Expression of Rab GTPases using recombinant vaccinia viruses. Methods Enzymol. 1995;257:155–164.[Medline] [Order article via Infotrieve]
  24. Gulick J, Subramaniam A, Neumann J, Robbins J. Isolation and characterization of the mouse cardiac myosin heavy chain genes. J Biol Chem. 1991;266:9180–9185.[Abstract/Free Full Text]
  25. Reddy VB, Thimmappaya B, Dhar R, Subramanian KN, Zain BS, Pan J, Ghosh PK, Celma ML, Weissman SM. The genome of simian virus 40. Science. 1978;200:494–502.[Abstract/Free Full Text]
  26. Hogan B. Manipulating the mouse embryo. Plainview, NY: Cold Spring Harbor Laboratory Press; 1994.
  27. Steinhelper ME, Cochrane KL, Field LJ. Hypotension in transgenic mice expressing atrial natriuretic factor fusion genes. Hypertension. 1990;16:301–307.[Abstract/Free Full Text]
  28. Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol. 1997;272:H220–H226.[Abstract/Free Full Text]
  29. Soonpaa MH, Koh GY, Klug MG, Field LJ. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science. 1994;264:98–101.[Abstract/Free Full Text]
  30. Bullock GR, Petrusz P. Techniques in Immunocytochemistry. New York, NY: Academic Press; 1982.
  31. Soonpaa MH, Field LJ. Assessment of cardiomyocyte DNA synthesis during hypertrophy in adult mice. Am J Physiol. 1994;266:H1439–H1445.[Abstract/Free Full Text]
  32. Kobayashi T, Nishizawa M, Hirayama Y, Kobayashi E, Hino O. cDNA structure, alternative splicing and exon-intron organization of the predisposing tuberous sclerosis (Tsc2) gene of the Eker rat model. Nucleic Acids Res. 1995;23:2608–2613.[Abstract/Free Full Text]
  33. Maheshwar MM, Sandford R, Nellist M, Cheadle JP, Sgotto B, Vaudin M, Sampson JR. Comparative analysis and genomic structure of the tuberous sclerosis 2 (TSC2) gene in human and pufferfish. Hum Mol Genet. 1996;5:131–137.[Abstract/Free Full Text]
  34. Ito N, Rubin GM. gigas, a Drosophila homolog of tuberous sclerosis gene product-2, regulates the cell cycle. Cell. 1999;96:529–539.[Medline] [Order article via Infotrieve]
  35. Fujita K, Ohyama H, Yamada T. Quantitative comparison of in situ methods for detecting apoptosis induced by x-ray irradiation in mouse thymus. Histochem J. 1997;29:823–830.[Medline] [Order article via Infotrieve]
  36. Soucek T, Yeung RS, Hengstschlager M. Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2. Proc Natl Acad Sci U S A. 1998;95:15653–15658.[Abstract/Free Full Text]
  37. Soucek T, Pusch O, Wienecke R, DeClue JE, Hengstschlager M. Role of the tuberous sclerosis gene-2 product in cell cycle control: loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase. J Biol Chem. 1997;272:29301–29308.[Abstract/Free Full Text]
  38. Zhang H, Yamamoto T, Nanba E, Kitamura Y, Terada T, Akaboshi S, Yuasa I, Ohtani K, Nakamoto S, Takeshita K, Ohno K. Novel TSC2 mutation in a patient with pulmonary tuberous sclerosis: lack of loss of heterozygosity in a lung cyst. Am J Med Genet. 1999;82:368–370.[Medline] [Order article via Infotrieve]
  39. Soonpaa MH, Field LJ. Survey of studies examining mammalian cardiomyocyte DNA synthesis. Circ Res. 1998;83:15–26.[Free Full Text]
  40. Anversa P, Kajstura J. Ventricular myocytes are not terminally differentiated in the adult mammalian heart. Circ Res. 1998;83:1–14.[Free Full Text]
  41. Sadoshima J, Aoki H, Izumo S. Angiotensin II and serum differentially regulate expression of cyclins, activity of cyclin-dependent kinases, and phosphorylation of retinoblastoma gene product in neonatal cardiac myocytes. Circ Res. 1997;80:228–241.[Abstract/Free Full Text]
  42. Sadoshima J, Izumo S. The cellular and molecular response of cardiac myocytes to mechanical stress. Annu Rev Physiol. 1997;59:551–571.[Medline] [Order article via Infotrieve]
  43. Hefti MA, Harder BA, Eppenberger HM, Schaub MC. Signaling pathways in cardiac myocyte hypertrophy. J Mol Cell Cardiol. 1997;29:2873–2892.[Medline] [Order article via Infotrieve]
  44. Koh KN, Kang MJ, Frith-Terhune A, Park SK, Kim I, Lee CO, Koh GY. Persistent and heterogenous expression of the cyclin-dependent kinase inhibitor, p27KIP1, in rat hearts during development. J Mol Cell Cardiol. 1998;30:463–474.[Medline] [Order article via Infotrieve]
  45. Li JM, Poolman RA, Brooks G. Role of G1 phase cyclins and cyclin-dependent kinases during cardiomyocyte hypertrophic growth in rats. Am J Physiol. 1998;275:H814–H822.[Abstract/Free Full Text]
  46. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell. 1996;85:733–744.[Medline] [Order article via Infotrieve]
  47. Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell. 1996;85:721–732.[Medline] [Order article via Infotrieve]
  48. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY. Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell. 1996;85:707–720.[Medline] [Order article via Infotrieve]
  49. Poolman RA, Li JM, Durand B, Brooks G. Altered expression of cell cycle proteins and prolonged duration of cardiac myocyte hyperplasia in p27KIP1 knockout mice. Circ Res. 1999;85:117–127.[Abstract/Free Full Text]
  50. Soonpaa MH, Koh GY, Pajak L, Jing S, Wang H, Franklin MT, Kim KK, Field LJ. Cyclin D1 overexpression promotes cardiomyocyte DNA synthesis and multinucleation in transgenic mice. J Clin Invest. 1997;99:2644–2654.[Medline] [Order article via Infotrieve]



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