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(Circulation Research. 1996;79:716-726.)
© 1996 American Heart Association, Inc.

Articles

Insulin-like Growth Factor II Induces DNA Synthesis in Fetal Ventricular Myocytes In Vitro

Qingquan Liu, Huajun Yan, Nicola J. Dawes, Giuliano A. Mottino, Joy S. Frank, Hong Zhu

the Cardiovascular Research Laboratories, Department of Physiology, UCLA School of Medicine, Los Angeles, Calif.

Correspondence to Hong Zhu, PhD, University of California, Los Angeles, School of Medicine, 176022, 675 Circle Dr S, MRL-3645, Los Angeles, CA 90095-1760. E-mail hong@cvrl.ucla.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insulin-like growth factor II (IGF2) belongs to a family of growth factors that includes insulin and insulin-like growth factor I (IGF1). Although the accumulating evidence indicates that IGF1 is involved in regulating proliferation of ventricular myocytes, the role of IGF2 is less clear. To gain more insight into the functions of IGF2, rat ventricular expression of IGF2 mRNA at four developmental stages was examined by Northern analysis. An abundant IGF2 mRNA of 3.8 kb was detected in fetal ventricles. It was dramatically decreased in neonatal ventricles and became undetectable in juvenile and adult ventricles. Similar expression patterns of the mRNA encoding IGF1 receptor and IGF2 receptor were observed. Since the results of Northern analysis strongly suggest the importance of IGF2 in regulating proliferation of fetal rat ventricular myocytes, the effects of an exogenous IGF2 on DNA synthesis in cultured rat ventricular myocytes were determined. DNA synthesis, which was monitored by measuring 5-bromo-2'-deoxyuridine (BrdU) and [³H]thymidine incorporation, was increased by twofold to threefold in IGF2-stimulated fetal ventricular myocytes, whereas no change in BrdU or [³H]thymidine incorporation was observed in neonatal ventricular myocytes. Instead, IGF2 seemed to induce hypertrophy in neonatal ventricular myocytes. An antisense oligonucleotide against rat IGF2 mRNA was able to significantly reduce BrdU incorporation, and this effect was quantitatively reversed by the addition of exogenous IGF2. Reversion by exogenous IGF2 was abolished by a monoclonal antibody against IGF1 receptor. In conclusion, our results suggest that IGF2 directly regulates proliferation of fetal rat ventricular myocytes in a paracrine/autocrine fashion.

Key Words: insulin-like growth factor II • ventricular myocyte • proliferation • hypertrophy • antisense oligonucleotide

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insulin-like growth factor II belongs to a family of peptide growth factors that includes insulin and IGF1.¹ Both IGF1 and IGF2 induce mitotic proliferation of a number of cell types in culture by binding to specific cell surface receptors, such as IGF-1R. The cytoplasmic domain of IGF-1R has tyrosine kinase activity, which is activated by binding of ligands, including IGF1 and IGF2, to the extracellular domain of IGF-1R. Activation of the tyrosine kinase of IGF-1R initiates a cascade of protein phosphorylation reactions catalyzed by the cellular protein kinases composing the ras/mitogen-activated protein kinase pathway. The activation of the ras/mitogen-activated protein kinase pathway positively regulates progression through the G₁ phase in a number of mammalian cell types. However, in cultured neonatal rat ventricular myocytes, activated ras protein does not induce DNA synthesis.² Instead, the activated ras protein seems to induce hypertrophy in neonatal rat ventricular myocytes. In addition to numerous in vitro studies, the proliferation/promotion role of IGF2 is also strongly implicated in certain tumors. The IGF2 gene is a member of a small group of genomically imprinted genes whose expression is highly limited to one of two alleles.³ For instance, only the paternal allele of the IGF2 gene is expressed (maternally imprinted). However, a number of tumors are associated with the relaxation of IGF2 imprinting and the consequential elevation of IGF2 mRNA and protein levels.^{4 5 6 7 8 9}

Although both IGF2 and IGF1 can bind to IGF1 receptor with a high affinity and result in similar biological effects in a number of cell types,^{10 11} IGF2 possibly plays the dominant role during murine prenatal development for the following reasons: First, IGF2 mRNA and protein are expressed in mouse embryos as early as preimplantation, when the IGF1 and insulin genes are not expressed.¹² Second, IGF2 is expressed at a much higher level in most tissues, including heart, than IGF1 during prenatal development.^{13 14} Finally, IGF2 expression dramatically decreases while IGF1 expression increases during postnatal development.¹⁴ More direct evidence comes from studies of the effects of perturbing the expression of the endogenous IGF2 gene on embryonic and fetal growth. For instance, Rappolee et al¹² showed that an antisense oligonucleotide against the first six codons of the IGF2 coding region can specifically inhibit IGF2 translation and slow down embryonic growth. In addition, mouse embryos contained significantly more cells after being treated with exogenous IGF2 than did control embryos. Gene-targeting studies by DeChiara et al¹⁵ showed that mouse embryos or neonates lacking the functional IGF2 gene were smaller than their wild-type counterparts. Interestingly, these mutant mice all reached adulthood and were fertile.

IGF2 also binds to the IGF2/cation-independent mannose-6-phosphate receptor (IGF-2R), which is bound for lysosomes.^{16 17} Although it is unclear whether IGF-2R is involved in any forms of signal transduction, it may play a critical role in controlling local IGF2 levels by internalizing IGF2. The internalized IGF2 is then transported to lysosomes, where it is degraded by lysosomal protease. Transgenic mice lacking a functional IGF-2R gene are overgrown at the fetal stage and are perinatal lethal.¹⁸ The circulating level of IGF2 is significantly higher in these mutant fetuses than in wild-type fetuses, whereas the IGF2 mRNA level remains unchanged. Interestingly, the hearts of 19-day-old mutant fetuses are three times heavier than the wild-type fetal hearts, whereas the other tissues are only 11% to 20% heavier, which is proportional to the overall increase in body weight. Myocardial hyperplasia is observed in the mutant fetuses by histological studies. These results are consistent with the early observation that IGF2 mRNA is expressed at a very high level in fetal hearts of wild-type mice.¹ As a further proof of the functions of IGF-2R as an IGF2 scavenger receptor, the perinatal lethality caused by lack of functional IGF-2R can be rescued by a second mutation, which eliminates IGF2.¹⁹

Although there is emerging evidence that IGF2 is involved in regulating fetal myocardial growth, direct evidence of IGF2 regulating proliferation of ventricular myocytes is still missing. In the present study, we present direct evidence that IGF2 induces DNA synthesis in fetal rat ventricular myocytes in vitro. First, the exogenous IGF2 increases both BrdU and [³H]thymidine labeling in cultured fetal rat ventricular myocytes. Second, an antisense oligonucleotide against rat IGF2 mRNA can specifically induce degradation of the endogenous IGF2 mRNA and therefore reduce BrdU labeling in fetal rat ventricular myocytes. Third, the inhibition by IGF2 antisense oligonucleotide can be quantitatively reversed by the addition of exogenous IGF2. The reversion by exogenous IGF2 was abolished by a monoclonal antibody against IGF-1R. However, DNA synthesis is not activated in neonatal rat ventricular myocytes by IGF2. This result is consistent with the Northern blot results, which showed that the IGF2 and IGF-1R genes are virtually switched off in neonatal ventricles. Our results support the hypothesis that IGF2 mediates fetal myocardial growth by inducing proliferation of fetal ventricular myocytes in a paracrine/autocrine fashion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fetal and Neonatal Rat Ventricular Myocytes and Cell Culture
The isolation and culture of fetal and neonatal rat ventricular myocytes have been described previously.²⁰ In the present study, Sprague-Dawley is the only rat species used for all the studies. Myocytes were dispersed from the ventricles of rat fetuses of 15-day gestation or from the ventricles of 2-day-old neonates by digestion with collagenase II (Worthington) and pancreatin (GIBCO BRL) at 37°C. Myocytes were further purified by a discontinuous Percoll (Pharmacia Biotechnology Inc) gradient to obtain myocardial cell cultures with >95% myocytes, as assessed by immunofluorescence with an antibody directed against MLC-2v (a gift from Dr Kenneth R. Chien, University of California, San Diego). Fetal and neonatal ventricular myocytes were plated on Lab-Tek plastic chamber slides coated with laminin. Ventricular myocytes were cultured in DMEM+10% FBS+20% horse serum (HS) at a density of 1.0x10⁵ per 4-cm² chamber for 16 hours and switched to a low-sera medium (DMEM+1% FBS+2% HS). Ventricular myocytes were stimulated with the human recombinant IGF2 (Promega) on the fourth day of culture at a final concentration of 50 ng/mL for 72 hours.

Northern Blot Analysis
Total RNA was isolated by the Chomczynski method,²¹ and 30 to 50 µg RNA of each sample was resolved on a 1% formaldehyde agarose gel. RNA was transferred from the agarose gel onto a nylon membrane by capillary blotting and fixed by UV cross-linking. Prehybridization and hybridization were carried out in 6x SSC+2x Denhardt's solution+0.1% SDS+0.1 mg/mL salmon sperm DNA at 68°C. The filters were washed once in 1x SSC+0.1% SDS at 25°C for 20 minutes and three times in 0.5x SSC+0.1% SDS at 68°C for 20 minutes. The filters were finally exposed to Kodak x-ray films. The IGF2 cDNA probe was obtained by RT-PCR using fetal rat ventricular RNA and primers derived from the first and third exons of the rat IGF2 gene. The IGF-1R cDNA and IGF-2R cDNA were kindly provided by Dr Derek LeRoith, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Md.

Monitoring DNA Synthesis
To monitor DNA synthesis, BrdU was added to the media at a final concentration of 20 µmol/L, and the BrdU labeling was continued for 24 hours. The incorporated BrdU was then detected by immunofluorescence staining as described below. Alternatively, myocytes were labeled with [³H]thymidine (2 µCi/mL) for 24 hours. Myocytes were harvested by scraping and lysed in 0.5 mL of 10 mmol/L Tris-HCl, pH 7.4, 1 mmol/L EDTA, 100 mmol/L NaCl, and 0.5% SDS. Lysates were precipitated with an equal volume of 20% TCA on ice for 10 minutes. The suspension was filtered onto glass microfiber disks and washed three times with 5% TCA and once with 95% ethanol. After drying at room temperature, the radioactivity was measured in a scintillation counter.

Indirect Immunofluorescence Staining
The indirect immunofluorescence assays were carried out as previously described with some minor modifications.²² Myocytes were rinsed with PBS and fixed for 15 minutes at 25°C in 2 mL of 3% paraformaldehyde in buffer A (10 mmol/L sodium phosphate, pH 7.4, 150 mmol/L NaCl, and 1 mmol/L MgCl₂). The cells were changed into 2 mL of 50 mmol/L NH₄Cl in buffer A and incubated at 25°C for 10 minutes. After they were washed twice with PBS, the cells were permeabilized with 2 mL of 0.2% Triton X-100 in buffer A at 25°C for 15 minutes, followed by three additional washes with PBS. The slides were blocked with 1% bovine serum albumin for 10 minutes at 25°C, incubated with antibodies against MLC-2v and BrdU (Amersham) for 60 minutes at 37°C, rinsed, and washed four times with PBS. The cells in chamber slides were then incubated with appropriate secondary antibodies at 37°C for 60 minutes. After they were rinsed once and washed four times with PBS, the slides were mounted on glass coverslips with 90% glycerol in 0.1 mol/L Tris (pH 9.4) and viewed by fluorescence microscopy.

Measuring Relative Rate of Protein Synthesis
To measure the rate of protein synthesis, myocytes were labeled with L-[2,3,4,5,6-³H]phenylalanine (5 µCi/mL, Amersham) for 24 hours. Myocytes were then harvested and lysed as described above, and total cellular proteins were precipitated with 10% TCA on ice for 30 minutes. The precipitates were washed three times with 10% TCA and solubilized in 0.5 mL of 1% SDS and subjected to scintillation counting.

Antisense Oligonucleotide Transfection
The antisense oligonucleotide derived from the first 20 nucleotides of the rat IGF2 coding region (5'-UUCCCCACUGGGAUCCCCAU-3') was purchased from Retrogen. The oligonucleotide was synthesized as phosphorothioate derivatives in order to extend its half-life in the cells.²³ Random oligonucleotides with the same base composition as the IGF2 antisense oligonucleotide were used as controls. For transfection, fetal or neonatal rat ventricular myocytes were plated at a density of 1.0x10⁵ cells per each 4-cm² chamber. The mixture, containing 25 nmol oligonucleotide, 75 µg DOTAP (Boehringer Mannheim), and 1.0x10⁸ plaque-forming units of a replication-defective adenovirus (D1343, a gift from Dr Lynn Hendrick, University of California, Los Angeles), was incubated in 100 µL of 20 mmol/L HEPES, pH 7.3, at room temperature for 15 minutes before being added to 1 mL medium in one chamber. Transfection was continued for 3 hours in a 37°C CO₂ incubator. The cells were then washed with 2 mL of 10% dimethyl sulfoxide in 1x PBS and cultured in 2 mL fresh media. Transfection was carried out on the second day of myocyte culture and repeated 24 hours later, and the cells were cultured for 48 hours before BrdU labeling.

RT-PCR Southern Analysis
Total RNA was isolated from purified fetal ventricular myocytes cultured on chamber slides by the Chomczynski method.²¹ One microgram of total RNA was mixed with 0.1 µg oligo (dT)_12-18 (Stratagene) in 12 µL H₂O and heated to 70°C for 10 minutes. The solution was quickly chilled on ice, and then 4 µL of 5x first-strand buffer (GIBCO BRL), 2 µL of 0.1 mol/L dithiothreitol, and 1 µL of 25 mmol/L dNTP mix (25 mmol/L each dATP, dGTP, dCTP, and dTTP) were added. The resulting mixture was incubated at 42°C for 2 minutes before adding 1 µL of SuperScript II (reverse transcriptase from GIBCO BRL). RT was carried out at 42°C for 1 hour and then terminated by heating at 70°C for 15 minutes. The PCR was performed in 50 µL of a medium containing 5 µL of RT reaction, 0.5 µg forward primer, 0.5 µg reverse primer, 2 mmol/L MgCl₂, 5 µL of 10x PCR buffer (GIBCO BRL), 1 µL of 25 mmol/L dNTP mix, and 5 U Taq DNA polymerase (GIBCO BRL). The PCR program was designed as follows: one cycle at 90°C for 10 minutes; 15 cycles at 90°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; and one cycle at 72°C for 10 minutes. For the IGF2 mRNA, the sequence was 5'-GGAAGTCGATGTTGGTGCTT-3' for the forward primer and 5'-CGAATTTGAAGAACTTGCCC-3' for the reverse primer; for GAPDH mRNA, the sequence was 5'-CATCAAGAAGGTGGTGAAGC-3' for the forward primer and 5'-ACCCTGTTGCTGTAGCCATA-3' for the reverse primer. The two primers for IGF2 mRNA were from exon 1 and exon 3, respectively, so that the possible contaminating genomic DNA would generate a PCR product larger than the product amplified from the first-strand cDNA. Southern blot analysis was carried out according to the standard procedure.²⁴

Antibody Blocking
To block IGF-1R on the surface of fetal ventricular myocytes, a monoclonal antibody against IGF-1R (Oncogene Science) was added to the media (final, 15 µg/mL) on the fourth day of culture. This antibody has been shown to block the binding of IGF1 and IGF2 to IGF-1R and inhibit growth of MCF-7 cells in culture.^{25 26} IGF2 stimulation was performed 2 hours after preincubation with IGF-1R antibody. For the experiment of antisense oligonucleotide transfection, IGF-1R antibody was added to the cell at the end of oligonucleotide transfection. IGF2 stimulation of the transfected cells was then initiated after a 2-hour preincubation with the IGF-1R antibody.

Statistics
Values of experimental data were expressed as mean±SEM. Statistical analysis was performed using the Mann-Whitney U test. Mann-Whitney probability values (P) were for the significance of differences in the percentage of BrdU-positive ventricular myocytes between the control and stimulated fetal or neonatal myocyte preparations. Results were considered significant if P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Expression Patterns of the IGF2, IGF-1R, and IGF-2R Genes Are Correlated With Proliferative Status of Rat Ventricular Myocytes
Ventricular myocytes are mitotically active at the fetal stage and become permanently postmitotic shortly after birth.²⁷ Although the expression of IGF2 mRNA in various tissues during embryogenesis is well documented,^{13 14} the ventricular IGF2 mRNA expression during development is poorly understood. To establish any possible correlation of IGF2 mRNA expression with the proliferation status of ventricular myocytes, ventricular expression of IGF2 mRNA at four developmental stages was examined by Northern blot analysis. Total RNA was isolated from ventricles of fetal (day-15 postcoitum), neonatal (day-2 postpartum), juvenile (day-19 postpartum), and adult (2-month postpartum) rats and subjected to Northern blot analysis. As shown in Fig 1A, an IGF2 mRNA of 3.8 kb is quite abundant in fetal ventricles and becomes barely detectable in neonatal ventricles, suggesting that the downregulation of IGF2 expression may be involved in inducing ventricular myocytes to withdraw from the cell cycle. Furthermore, the expression of IGF2 mRNA is undetectable in both juvenile and adult rat ventricles, where most myocytes are postmitotic. It has been reported previously that rat IGF2 gene is transcribed from three distinct promoters in a number of fetal tissues, such as liver, to generate three mRNA species (3.6 kb, 3.8 kb, and 4.6 kb).^{28 29 30 31 32} In fetal rat ventricles, however, only one IGF2 mRNA band is detected. Based on its mobility, the ventricular IGF2 mRNA band could represent either the 3.8-kb or the 3.6-kb species or both, which migrate together in formaldehye-agarose gels.³² Therefore, unlike fetal liver, the promoter that directs the synthesis of 4.6-kb IGF2 mRNA seems inactive in fetal ventricles (Fig 1B). To determine whether IGF2 mRNA is expressed in ventricular myocytes, Northern blot analysis was performed with total RNA isolated from fetal rat ventricular myocytes, which were purified through the Percoll gradient. Usually, a ventricular myocyte purity of >95%, assessed by staining with a polyclonal antibody against ventricular specific MLC-2v, can be obtained by this method. The same IGF2 mRNA band was observed with RNA isolated from purified myocytes as was observed with ventricular RNA, which indicates that the IGF2 gene is expressed in ventricular myocytes (Fig 1C).

View larger version (19K): [in this window] [in a new window]   Figure 1. Northern blot analysis. A, Total RNA (50 µg) isolated from the ventricles of fetal (F), neonatal (N), juvenile (J), and adult (A) rats was subjected to Northern blot analysis. The blot was probed with radiolabeled rat IGF2 cDNA. B, Northern blot analysis was performed with 50 µg of total RNA isolated from fetal rat liver (L) and ventricles (V). The blot was probed with IGF2 cDNA. C, Northern blot analysis was performed with 30 µg of total RNA isolated from purified fetal rat ventricular myocytes (M) and fetal rat ventricles (V). The blot was probed with IGF2 cDNA. D and E, Northern blot analysis was performed with 50 µg of total RNA isolated from the ventricles of fetal (F), neonatal (N), juvenile (J), and adult (A) rats. The RNA blots were probed with radiolabeled rat IGF-1R cDNA (D) and rat IGF-2R cDNA (E). 28S rRNA for normalization is shown at the bottom of each panel.

The mitogenic effects of IGF2 are mediated through IGF-1R in a number of cell types, whereas IGF-2R is a critical element for regulating the local IGF2 level. However, the developmental regulation of these two genes in ventricles is poorly characterized. Therefore, ventricular expression of both IGF-1R and IGF-2R genes at four developmental stages was assessed by Northern blot analysis as described above. Similar to the expression pattern of the IGF2 gene, both IGF-1R and IGF-2R genes are expressed in fetal rat ventricles, but the expression levels are dramatically decreased in postnatal development (Fig 1D and 1E). The correlation of the expression patterns of the IGF2, IGF-1R, and IGF-2R genes with the proliferation status of rat ventricular myocytes strongly suggests that IGF2 plays an important role in regulating fetal rat ventricular growth.

Exogenous IGF2 Induces DNA Synthesis in Fetal but Not Neonatal Rat Ventricular Myocytes in Culture
G₁ to S transition is highly regulated in a variety of mammalian cell types.³³ The IGFs stimulate cell proliferation in culture, probably by stimulating G₁ to S transition.^{34 35} To determine directly whether IGF2 can stimulate G₁ to S transition in fetal ventricular myocytes, the effects of stimulation with exogenous IGF2 on DNA synthesis were determined. Myocytes isolated from fetal rat ventricles were treated with 50 ng/mL of recombinant human IGF2 (Promega) on the fourth day of culture, and this treatment was continued for 72 hours. DNA synthesis was monitored by measuring the incorporation of either BrdU or [³H]thymidine, which was added to the media 24 hours before termination of IGF2 stimulation. Immunofluorescence staining or scintillation counting was performed at the end of 72-hour stimulation with IGF2. The BrdU molecules incorporated into genomic DNA through DNA synthesis were detected by staining with a BrdU monoclonal antibody (Amersham), whereas the incorporated [³H]thymidine molecules were detected by scintillation counting. Ventricular myocytes in culture were distinguished from the contaminating cardiac fibroblasts or atrial myocytes by staining with the polyclonal antibody against MLC-2v. Cells stained positive for both MLC-2v and BrdU were counted as ventricular myocytes that had replicated genomic DNA during the experiment.

During the first 3 days in culture, the fetal ventricular myocytes are mitotically inactive (data not shown). On the fourth day, fetal ventricular myocytes start to replicate DNA. In a low-sera medium (1% FBS and 2% HS), approximately one third of the fetal ventricular myocytes were stained positive with BrdU antibody (Fig 2A). After stimulation with 50 ng/mL of recombinant human IGF2 for 72 hours, the percentage of BrdU-positive ventricular myocytes was obviously increased (Fig 2B). Neonatal rat ventricular myocytes were stimulated with the recombinant human IGF2 in the same way as fetal ventricular myocytes were stimulated. Consistent with the early observation that myocytes exit from the cell cycle shortly after birth,²⁷ the percentage of BrdU-positive ventricular myocytes from neonatal rat ventricles was much lower than that of fetal rat ventricles (Fig 2C). The percentage of BrdU-positive neonatal ventricular myocytes was not obviously changed by stimulation with IGF2 (Fig 2D). However, both stimulated fetal and neonatal ventricular myocytes appear to be larger and contain more organized myofibrils than the corresponding control myocytes. To quantify the differences in size between the IGF2-stimulated and control myocytes, image analysis was performed. The photographs were scanned by HP ScanJet IIC scanner (Hewlett Packard) with Aldus Photostyler 2.0 software (Aldus Co). The areas of myocytes were measured by SigmaScan 3.0 software (Jandel Scientific Software) and used as the approximation of the cell size. For each sample, the areas of 200 myocytes were directly measured by this method. The results show that the areas of both IGF2-stimulated fetal and neonatal ventricular myocytes were twice as large as the corresponding control myocytes (Fig 3A), suggesting that IGF2 has a hypertrophic effect on fetal and neonatal ventricular myocytes. To confirm the activation of the hypertrophic pathway in neonatal ventricular myocytes, the expression of "embryonic markers" and constitutively expressed genes was first assessed by Northern blot analysis. As shown in Fig 3B, the steady state levels of atrial natriuretic factor, ß-myosin heavy chain, -skeletal actin, and MLC-2v mRNA were increased, whereas the level of sarcoplasmic reticulum Ca²⁺-ATPase mRNA was decreased in IGF2-stimulated neonatal ventricular myocytes. These results strongly suggest an activation of a hypertrophic pathway in IGF2-stimulated neonatal ventricular myocytes. To compare the relative rates of protein synthesis between IGF2-stimulated and control neonatal ventricular myocytes, [³H]phenylalanine incorporation into cellular proteins was measured. [³H]Phenylalanine was added to the media (5 µCi/mL) 24 hours before termination of IGF2 stimulation, and [³H]phenylalanine labeling was continued for 24 hours. The incorporated [³H]phenylalanine was quantified by TCA precipitation of cell lysates and scintillation counting. As shown in Fig 3C, [³H]phenylalanine incorporation was increased by 1.8-fold after IGF2 stimulation, which is statistically significant (P<.05). These results support the notion that IGF2 activates a hypertrophic pathway in neonatal ventricular myocytes.

View larger version (164K): [in this window] [in a new window]   Figure 2. Indirect immunofluorescence staining of fetal and neonatal rat ventricular myocytes in culture. The primary antibodies used for immunofluorescence staining are a rabbit polyclonal antibody against rat ventricle-specific MLC-2v and a monoclonal antibody against BrdU. The secondary antibodies are fluorescein-labeled goat anti-rabbit IgG and rhodamine-labeled donkey anti-mouse IgG. A, Control fetal rat ventricular myocytes. B, Fetal rat ventricular myocytes stimulated with 50 ng/mL of recombinant human IGF2 for 72 hours. C, Control neonatal rat ventricular myocytes. D, Neonatal ventricular rat myocytes stimulated with 50 ng/mL of the recombinant human IGF2 for 72 hours. BrdU was added to the cells 24 hours before the termination of IGF2 stimulation. E and F, Fetal rat ventricular myocytes transfected with IGF2 antisense oligonucleotide on the second day of culture. F, Myocytes stimulated with exogenous IGF2 at a final concentration of 50 ng/mL for 72 hours after transfection. BrdU was added to the cells 24 hours before the termination of IGF2 stimulation. Magnification x45.

View larger version (100K): [in this window] [in a new window]   Figure 3. A, The areas of control and IGF2-stimulated ventricular myocytes were measured by SigmaScan 3.0 software (n=6). For each experiment, the areas of 200 myocytes were measured. The results are expressed as the mean area of the myocytes±SEM. B, Northern blot analysis was performed with 25 µg of total RNA isolated from the control (C) and IGF2-stimulated (S) neonatal rat ventricular myocytes. The RNA blot was probed with radiolabeled rat atrial natriuretic factor (ANF), ß-myosin heavy chain (beta-MHC), MLC-2v, -skeletal actin (alpha-SkA), and sarcoplasmic reticulum Ca2+-ATPase (SERCA2) cDNA. At the bottom, 28S rRNA shows the loading normalization. C, Neonatal rat ventricular myocytes were labeled with [3H]phenylalanine (5 µCi/mL) during the last 24 hours of 72-hour IGF2 stimulation (n=5). [3H]Phenylalanine incorporation was quantified by TCA precipitation and scintillation counting of the cell lysates. The mean value of [3H]phenylalanine incorporation in the control myocytes is arbitrarily set as 100% ±SEM. *P<.05 vs control myocytes.

To quantify the effects of IGF2 stimulation on DNA synthesis, the percentage of BrdU-positive ventricular myocytes was first compared between the control and IGF2-stimulated samples. For each experiment, 500 ventricular myocytes were counted, and the percentage of BrdU-positive myocytes was calculated. Fig 4A summarizes the results of six independent experiments with six different myocyte preparations. The percentage of BrdU-positive fetal ventricular myocytes was increased by IGF2 stimulation from 30±5.5% (mean±SEM) to 72±6.4% (mean±SEM). The difference between the control and IGF2-stimulated samples of fetal ventricular myocytes is statistically significant (P<.05). On the other hand, the percentage of BrdU-positive neonatal ventricular myocytes was lower (3.5±0.2%, mean±SEM) and was not significantly changed by IGF2 stimulation (2.8±0.5%, mean±SEM). To confirm the induction of DNA synthesis by exogenous IGF2 in fetal myocytes, the [³H]thymidine uptake by both fetal and neonatal ventricular myocytes was measured. [³H]Thymidine was added to the media (2 µCi/mL) 24 hours before the termination of IGF2 stimulation, and the labeling was continued for 24 hours. As shown in Fig 4B, [³H]thymidine uptake by fetal myocytes was increased by threefold after IGF2 stimulation, whereas no significant change in [³H]thymidine uptake by neonatal myocytes was observed. The above results seem to support the concept that IGF2 stimulates G₁ to S transition in cultured fetal ventricular myocytes.

View larger version (50K): [in this window] [in a new window]   Figure 4. Quantification of DNA synthesis in fetal and neonatal ventricular myocytes. A, Quantification of BrdU-positive ventricular myocytes. Results from six different ventricular myocyte preparations are presented as the mean±SEM percentage of BrdU-positive ventricular myocytes. Myocytes were stimulated with IGF2 for 72 hours, and BrdU was added to the cells 24 hours before the termination of IGF2 stimulation. For each myocyte preparation, 500 ventricular myocytes were counted, and the BrdU labeling index was calculated. B, Quantification of [3H]thymidine uptake (n=6). Myocytes were stimulated as described in panel A. [3H]Thymidine labeling (2 µCi/mL) was performed during the last 24 hours of the 72-hour IGF2 stimulation. The level of [3H]thymidine uptake by the control fetal or neonatal ventricular myocytes is arbitrarily set as 100% ±SEM. *P<.05 vs control fetal ventricular myocytes.

Inhibition of IGF2 Expression by an Antisense Oligonucleotide Results in a Significant Decrease in DNA Synthesis in Fetal Rat Ventricular Myocytes
The observation that IGF2 mRNA is abundant in fetal rat ventricles suggests that IGF2 may function in a paracrine or autocrine fashion. To determine whether ventricular expression of the IGF2 gene is required for DNA synthesis in fetal ventricular myocytes, IGF2 mRNA was eliminated from cultured fetal ventricular myocytes by an antisense oligonucleotide. Subsequently, DNA synthesis was compared between the control and IGF2 antisense oligonucleotide–transfected fetal ventricular myocytes. The antisense oligonucleotide was derived from the first 20 nucleotides of rat IGF2 coding region, since the oligonucleotide derived from the same region of both rat and mouse IGF2 genes has been successfully used by a number of laboratories.^{12 36} A random oligonucleotide with the same base composition was used as a negative control. Transfection was performed on the second day of culture and repeated once on the next day. Myocytes were cultured for 2 days after transfection and then harvested for RNA isolation. To confirm the specificity and efficiency of the IGF2 antisense oligonucleotide in eliminating the endogenous IGF2 mRNA, RT-PCR/Southern blot analysis was performed to assess the IGF2 mRNA levels in the transfected myocytes. The mRNA encoding GAPDH was used as an internal control. The PCR products generated with primers specific for the IGF2 or GAPDH gene were subject to Southern blot analysis. As shown in Fig 5, the intensities of PCR-amplified GAPDH cDNA were comparable in the lanes of control myocytes, random oligonucleotide-transfected myocytes, and IGF2 antisense oligonucleotide–transfected myocytes. However, the intensities of PCR-amplified IGF2 cDNA were only comparable in the lanes of control myocytes and random oligonucleotide–transfected myocytes, suggesting that the IGF2 mRNA level was not affected by this random oligonucleotide. PCR-amplified IGF2 cDNA was not detected in the lane of IGF2 antisense oligonucleotide–transfected myocytes. Therefore, as shown in other cell types,^{12 36} the IGF2 antisense oligonucleotide can also specifically eliminate IGF2 mRNA in cultured fetal rat ventricular myocytes. Attempts in assessing the IGF2 protein levels were not successful, presumably because the quantities of materials were limited. Since the half-life of IGF2 is relatively short: 20 to 30 minutes for free IGF2 and 12 to 15 hours for IGF binding protein–bound IGF2,³⁷ the IGF2 synthesized before transfection is likely to be quickly degraded.

View larger version (39K): [in this window] [in a new window]   Figure 5. RT-PCR/Southern blot analysis. The first-strand cDNA was synthesized by RT using 1 µg of total RNA isolated from the control fetal ventricular myocytes and random oligonucleotide–transfected and IGF2 antisense oligonucleotide–transfected fetal ventricular myocytes. Subsequently, PCR was performed with an aliquot of the RT reaction and a pair of primers specific for either the IGF2 gene or GAPDH gene. The PCR products were then subjected to Southern blot analysis with radiolabeled IGF2 cDNA or GAPDH cDNA.

The observation that the GAPDH mRNA level was not affected by either random oligonucleotide or IGF2 antisense oligonucleotide suggests that these oligonucleotides probably do not cause serious cytotoxicity at the concentration used. This idea was further supported by the fact that no major morphological differences were observed between the control and oligonucleotide-transfected fetal ventricular myocytes (Fig 2A, 2E, and 2F). Moreover, no massive cell death was observed after oligonucleotide transfection. To assess the effects of eliminating the endogenous IGF2 mRNA on DNA synthesis, fetal ventricular myocytes were transfected for 2 days, cultured for another 2 days, and labeled with BrdU for 24 hours. After immunofluorescence staining, the percentage of BrdU-positive myocytes was compared between the IGF2 antisense oligonucleotide–transfected and the random oligonucleotide–transfected myocytes. As shown in Fig 6, the percentage of BrdU-positive myocytes after transfection with the random oligonucleotide was 32±5.3% (mean±SEM, n=5), which is not significantly different from the untransfected myocytes. However, the percentage of BrdU-positive ventricular myocytes after transfection with IGF2 antisense oligonucleotide was decreased to 13±2.0% (mean±SEM, n=5). The difference between random oligonucleotide–transfected and IGF2 antisense oligonucleotide–transfected myocytes is statistically significant (P<.05).

View larger version (31K): [in this window] [in a new window]   Figure 6. Quantification of BrdU-positive ventricular myocytes after oligonucleotide transfection. Results from five different ventricular myocyte preparations are presented as the mean±SEM percentage of BrdU-positive ventricular myocytes. Fetal rat ventricular myocytes were transfected on the second day of culture with either the random oligonucleotide or IGF2 antisense oligonucleotide as described in "Materials and Methods." Transfection was repeated once on third day of culture. BrdU labeling was started 48 hours after transfection and continued for 24 hours. For IGF2 rescue, exogenous IGF2 was added to the cells at the end of antisense oligo transfection, and IGF2 stimulation was continued for 72 hours. BrdU labeling was performed during the last 24 hours of the 72-hour IGF2 stimulation. For the antibody blocking experiment, a monoclonal antibody against IGF-1R (IGF-1R Ab) was added to the cells at a final concentration of 15 µg/mL on the fourth day of culture. After a 2-hour preincubation with IGF-1R Ab, myocytes were stimulated with IGF2 and labeled with BrdU as described in Fig 2. IGF-1R Ab was also added to the cells at the end of transfection and incubated with myocytes for 2 hours. The myocytes were then stimulated with exogenous IGF2 and labeled with BrdU as described in Fig 2. *P<.05 vs the random oligo-transfected samples.

To further confirm the specificity of IGF2 antisense oligonucleotide, it was determined whether exogenous IGF2 could restore DNA synthesis in the IGF2 antisense oligonucleotide–transfected fetal ventricular myocytes. The fetal ventricular myocytes were stimulated with the recombinant human IGF2 at a final concentration of 50 ng/mL after transfection, and the stimulation was continued for 72 hours. BrdU labeling was carried out during the last 24 hours of IGF2 stimulation. As shown in Fig 6, the percentage of BrdU-positive myocytes was increased to 70±5.7% (mean±SEM, n=5) in spite of the presence of IGF2 antisense oligonucleotide. These results support the belief that IGF2 antisense oligonucleotide inhibits DNA synthesis by eliminating the endogenous IGF2 mRNA. To further confirm this notion, antibody blocking experiments were performed. A monoclonal antibody against IGF-1R (Oncogene Science) was used to block the binding of IGF2 to IGF-1R on the surface of fetal ventricular myocytes. This antibody has been shown to block the binding of IGF1 and IGF2 to IGF-1R and inhibit growth of MCF-7 cells in culture.^{25 26} Fetal myocytes were preincubated with the IGF-1R antibody at a final concentration of 15 µg/mL for 2 hours on the fourth day of culture. Subsequently, IGF2 stimulation, BrdU labeling, and assessment of the percentage of BrdU-positive ventricular myocytes were performed as described above. The results show that not only did the IGF-1R antibody abolish induction of DNA synthesis by exogenous IGF2, but it also decreased the basal level of BrdU-positive ventricular myocytes (Fig 6). The effects of IGF-1R antibody on IGF2 rescue of DNA synthesis were then determined. After transfection with the IGF2 antisense oligonucleotide, fetal ventricular myocytes were incubated with the IGF-1R antibody at a final concentration of 15 µg/mL for 2 hours, which was followed by IGF2 stimulation and BrdU labeling. The percentage of BrdU-positive ventricular myocytes was determined as described above. As shown in Fig 6, rescue of DNA synthesis by exogenous IGF2 after antisense oligonucleotide transfection was abolished by preincubation with the IGF-1R antibody.

The data presented here support the hypothesis that IGF2 directly induces DNA synthesis in fetal ventricular myocytes in a paracrine or autocrine fashion. Although IGF2 does not induce DNA synthesis in neonatal ventricular myocytes, it probably induces hypertrophy in these cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of DNA Synthesis in Cardiac Myocytes by Growth Factors
Although the regulation of G₁ to S transition has been extensively studied in a variety of cell types, it is poorly understood in cardiac myocytes. In the present study, we present the evidence that IGF2 stimulates DNA synthesis in cultured fetal rat ventricular myocytes, probably by a paracrine or autocrine mechanism. It has been previously reported by others that DNA synthesis in cardiac myocytes can be stimulated or reactivated by a number of growth factors. Speir et al³⁸ have reported that both aFGF and bFGF can induce a 2-fold increase in [³H]thymidine uptake by cultured adult rat cardiac myocytes.³⁸ Similarly, Pasumarthi et al³⁹ have shown that transient overexpression of a cDNA encoding bFGF leads to a 2- to 3-fold increase in both [³H]thymidine uptake and BrdU-positive neonatal rat ventricular myocytes in culture.³⁹ The same authors have also reported that transient overexpression of the bFGF cDNA in cultured embryonic chicken ventricular myocytes induces a 3-fold increase in both [³H]thymidine uptake and BrdU labeling.⁴⁰ The adult newt myocardium represents a unique model for studying cardiac myocyte proliferation because it is capable of regeneration after injury.⁴¹ By measuring [³H]thymidine uptake, Soonpaa et al⁴² have shown that DNA synthesis in cultured adult newt cardiac myocytes can be induced by a number of factors, such as aFGF (1.21-fold), bFGF (1.19-fold), a phorbol ester (TPA, 2.33-fold),⁴² and platelet-derived growth factor (1.2-fold).⁴³ On the other hand, DNA synthesis in the cultured adult newt cardiac myocytes is inhibited by TGF-ß, (38% of control).⁴² Interestingly, Sigel et al⁴⁴ have shown that TGF-ß₁ delivered by intravenous injection into rat heart can cause a moderate but statistically significant decrease in [³H]thymidine incorporation. However, in those studies, it is unknown whether the TGF-ß₁–induced decrease in [³H]thymidine incorporation actually occurs in cardiac myocytes. It has been shown by Palmer et al⁴⁵ that a cytokine, interleukin-1ß, can induce a 1.3-fold increase in [³H]thymidine uptake by cultured neonatal rat cardiac myocytes.

Nuclear oncoproteins prove to be useful tools to probe the nuclear factors involved in regulation of DNA synthesis in cardiac myocytes. For instance, Kirshenbaum and Schneider⁴⁶ have shown that overexpression of the adenoviral oncoproteins E1A and E1B in neonatal rat ventricular myocytes increases BrdU-positive myocytes from 15% to 80%. Similarly, Liu and Kitsis⁴⁷ have reported that overexpression of E1A in cultured fetal rat cardiac myocytes (day 20 postcoitum) increases BrdU-positive myocytes from nearly undetectable to 94%. Since E1A disrupts the functions of a number of growth suppressor proteins, known as pocket proteins, such as the retinoblastoma gene product, Rb, p107, and p130, the above results suggest that the pocket proteins play an important role in controlling G₁ to S transition in the nuclei of cardiac myocytes. Based on all the above observations, it is reasonable to assume that DNA synthesis in cardiac myocytes is regulated by multiple factors at various steps of various pathways. In contrast to aFGF, bFGF, TGF-ß₁, and interleukin-1ß, the in vitro effects of IGF2 on DNA synthesis seem to be restricted to those cardiac myocytes before terminal differentiation. After terminal differentiation of cardiac myocytes, IGF2 seems unable to reactivate DNA synthesis. These results imply that IGF2 stimulates DNA synthesis in mitotic fetal ventricular myocytes by shortening the progression through G₁ phase. Our results are consistent with the previous in vivo studies by Lau et al,¹⁸ which show that the local IGF2 concentration is critical for normal fetal myocardial growth and development. The elevation of the local IGF2 concentration above the normal level resulting from the lack of IGF-2R leads to fetal myocardial hyperplasia, malformation of certain cardiac structures, and perinatal lethality.

Pleiotropic Functions of IGF2
Mammalian skeletal and cardiac muscle are the two mesodermally derived striated muscle types that share many morphological, biochemical, and physiological properties. One long-observed difference between these two types of muscle in embryonic development is that the proliferation and differentiation processes are mutually exclusive for skeletal muscle but not for cardiac muscle.²⁷ The permanent withdrawal from cell cycle and differentiation into mature muscle tubes of skeletal myoblasts are regulated by a group of transcription factors (the MyoD family) that contain the common basic helix-loop-helix structure.^{48 49} These transcription factors activate transcription of two classes of genes: genes involved in terminating mitosis and genes responsible for muscle-specific phenotypes. The cultured skeletal myoblasts are able to fuse with each other and differentiate into muscle tubes in serum-depleted media. IGF2 seems to play a crucial role in controlling the spontaneous differentiation of cultured skeletal myoblasts into myotubes.³⁶ Elimination of IGF2 mRNA by an antisense oligonucleotide blocks the differentiation of cultured skeletal myoblasts into myotubes. Serum depletion induces the expression of IGF2 and IGF-1R in skeletal myoblasts. In addition, IGF2 has been shown to activate the expression of myogenin, one member of the MyoD family. Therefore, it is possible that IGF2 is involved in inducing permanent withdrawal of skeletal myoblasts from the cell cycle and differentiation into myotubes in vivo.

However, IGF2 does not seem to play any role in inducing cardiac myocytes to withdraw from the cell cycle. The IGF2 gene is expressed at a relatively high level in fetal rat ventricles when ventricular myocytes are actively proliferating. Cardiac myocytes remain proliferative until myocardial terminal differentiation occurs, shortly after birth, when cardiac myocytes become postmitotic. Although it is unclear whether IGF2 is required for cardiac myogenesis, it is probably not involved in inducing permanent withdrawal of cardiac myocytes from the mitotic cell cycle. Instead, both in vivo and in vitro evidence indicate that IGF2 is a key factor in regulating proliferation of fetal ventricular myocytes. The downregulation of the IGF2 gene as well as the IGF-1R and IGF-2R genes in neonatal ventricles may be associated with the withdrawal of these myocytes from the cell cycle. Thus, IGF2 could play distinct roles in the embryonic development of skeletal and cardiac muscle through distinct signaling pathways in skeletal myoblasts and fetal cardiac myocytes.

Interestingly, the data in the present study suggest that the effects of IGF2 on ventricular myocyte development are stage dependent. Exogenous IGF2 can induce DNA synthesis in fetal rat ventricular myocytes but not in neonatal rat ventricular myocytes. Instead, exogenous IGF2 induces hypertrophy in cultured neonatal ventricular myocytes. These results seem to contradict the previous report by Kajstura et al,⁵⁰ which suggests that the activation of the IGF1–IGF-1R system induces a proliferative but not a hypertrophic response in neonatal ventricular myocytes. However, the present study clearly indicates that the expression of IGF-1R gene in neonatal rat ventricular myocytes is below the detectable level. Thus, it is less likely that IGF-1R plays a major role in regulating proliferation of neonatal ventricular myocytes. It is probable that IGF-1R is expressed in a relatively small number of neonatal ventricular myocytes that are not yet terminally differentiated. In fact, the DNA synthesis activated by the IGF1–IGF-1R system in neonatal ventricular myocytes observed by Kajstura et al occurred in a relatively small percentage of myocytes. The present data show that IGF2-induced hypertrophy of neonatal ventricular myocytes is observed in the majority of the myocyte population. Our data seem to agree with the results of Ito et al,⁵¹ which indicate that IGF1 induces hypertrophy of cultured neonatal rat ventricular myocytes. Thus, it is possible that certain redundancy between IGF1 and IGF2 may exist in stimulating the onset of cardiac cell hypertrophy.

The distinct responses of fetal and neonatal rat ventricular myocytes to exogenous IGF2 stimulation suggest the possibility of separate signaling pathways in the two cell populations. An intriguing hypothesis would be that the functions of IGF2 are mediated by distinct receptors in fetal and neonatal ventricular myocytes. Shier and Watt⁵² reported the isolation of a human insulin receptor–related receptor that was structurally related to the insulin receptor and IGF-1R. Although the functions of the insulin receptor–related receptor remain unknown, it is expressed in adult heart, kidney, liver, skeletal muscle, and pancreas. Furthermore, it has been shown recently that IGF2 also interacts with another unknown receptor in mouse embryos and fetuses.⁵³ Therefore, it cannot be ruled out that receptors other than IGF-1R mediate hypertrophy of neonatal rat ventricular myocytes in response to IGF2 stimulation.

IGF Signaling Pathway and Other Signaling Pathways
The effects of IGF2 are strikingly similar to the effects of -adrenergic agonists (Q. Liu et al, unpublished data, 1996). The -adrenergic agonist, L-phenylephrine, induces a twofold increase in BrdU-positive fetal rat ventricular myocytes and threefold increase in myocyte number. However, as previously reported, L-phenylephrine induces hypertrophy in neonatal ventricular myocytes. Evidence from various cell types indicates that the cellular ras protein is required for both IGF2 and L-phenylephrine signaling pathways.^{1 2} However, the L-phenylephrine signaling pathway requires certain types of GTP-binding proteins, whereas the IGF signaling pathway is not dependent on heterotrimeric GTP-binding proteins.^{2 54 55} Since both IGF1 and insulin can stimulate the expression of a subtype of -adrenergic receptor in vascular smooth muscle cells,⁵⁶ the IGF signaling pathway may influence the -adrenergic agonist signaling pathway in those cells. As a result, the IGF1 or insulin-stimulated vascular smooth muscle cells are more responsive to -adrenergic agonists.⁵⁶ Since the IGF2 gene is expressed at a very early embryonic stage, it is likely that IGF2 is involved in regulating the expression of -adrenergic receptors and therefore modulating the responsiveness of ventricular myocytes to adrenergic innervation.

	Selected Abbreviations and Acronyms

 

aFGF, bFGF = acidic and basic fibroblast growth factor BrdU = 5-bromo-2'-deoxyuridine IGF-1R, IGF-2R = IGF1 and IGF2 receptor IGF1, IGF2 = insulin-like growth factor (IGF) I and II MLC-2v = myosin light chain-2 PCR = polymerase chain reaction RT = reverse transcription TCA = trichloroacetic acid TGF-ß = transforming growth factor-ß

	Acknowledgments

 
The authors would like to thank Dr Kenneth R. Chien for providing the rat ANF cDNA, the polyclonal antibody against rat MLC-2v; Dr Derek LeRoith for providing the rat IGF-1R cDNA and rat IGF-2R cDNA; and Dr Lynn Hendrick for providing the adenovirus (D1343). Dr Liu is a recipient of a postdoctoral fellowship from American Heart Association Greater Los Angeles Affiliate, Inc (A95-0495-00). Drs Yan, Dawes, and Zhu are supported by the Theodore C. Laubisch Research Fund (University of California, Los Angeles) and a Grant-in-Aid from the American Heart Association, National Center (A94-2457A-00). Drs Mottino and Frank are supported by the National Heart, Lung, and Blood Institute (R01 HL-28791-12 and P01 HL-30568).

Received January 26, 1996; accepted July 25, 1996.

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