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GROWTH, DIFFERENTIATION, AND APOPTOSIS
Cardiology Research Laboratory, Departments of Medicine and Physiology and Pharmacology, London Health Sciences Center, University of Western Ontario, London, Ontario, Canada
Submitted 25 February 2006 ; accepted in final form 23 June 2006
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ABSTRACT |
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proliferation; heart development
Cardiomyogenesis and cardiac development are characterized by the coordination of a number of cellular events, including proliferation, differentiation, and structural remodeling. During fetal and early neonatal development, cardiomyocytes actively proliferate by hyperplasia and hypertrophy. Studies have shown that cardiomyocyte DNA synthesis and proliferation in the mouse heart progressively declines during prenatal development and remains low immediately after birth (6, 28). Interestingly, cardiomyocytes grow by hyperplasia during early neonatal life and are followed by eventual cessation in the mature heart, where all remaining growth occurs by cardiomyocyte hypertrophy. The switch from hyperplastic to hypertrophic growth occurs 
1 wk after birth in rodents (19, 28). It is therefore apparent that cardiomyocyte proliferation contributes to heart development in early postnatal life.
Cardiomyocytes express eNOS starting at embryonic day 9.5 and throughout embryonic heart development (4). Our recent studies have shown that deficiency in eNOS results in congenital atrial and ventricular septal defects (7). Interestingly, most eNOS knockout (eNOS–/–) neonatal mice develop heart failure and die within a few days after birth (7), which is a critical stage for postnatal cardiomyocyte proliferation. It is unknown if NO production from eNOS regulates the proliferation and maturation of cardiomyocytes in the neonatal heart. We hypothesized that deficiency in eNOS decreases cardiomyocyte proliferation and delays maturation of the neonatal heart. Cardiomyocyte proliferation was assessed by direct cell counts and bromodeoxyuridine (BrdU) incorporation using both in vitro and in vivo approaches. To examine cardiac maturation, the expression of differentiation markers atrial natriuretic peptide (ANP) and 
-myosin heavy chain (-MHC) was analyzed.
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MATERIALS AND METHODS |
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Primary neonatal cardiomyocyte cell culture and isolation.
Neonatal ventricular cardiomyocytes were cultured as we described previously (23, 27). Briefly, ventricular tissues from WT or eNOS–/– mice were isolated and minced within 24 h after birth. Subsequently, cardiomyocytes were dispersed by incubation in a collagenase type II buffer solution (Worthington Biochemical, Lakewood, NJ) at a concentration of 0.5 mg/ml, and the cellular suspension was filtered through a polypropylene macroporous filter (mesh opening 105 µm, Spectra Mesh; Spectrum Medical Industries). The suspension was then centrifuged at 200 g for 5 min, and the cellular pellet was suspended in medium 199 (M199) with 10% FBS and penicillin-streptomycin (50 µg/ml; GIBCO-BRL). The cellular suspension was preplated for 1 h at 37°C in 5% CO2 to remove any noncardiomyocytes. Cell density was adjusted to one million cells per milliliter using M199 supplemented with 10% FBS, and cells were seeded (1 x 105) in polystyrene, nonpyrogenic 96-well culture plates (Becton Dickinson, Franklin Lakes, NJ) precoated with 1% gelatin. Cells were incubated in 5% CO2 at 37°C. To determine cardiomyocyte purity, cells were seeded on glass slides and cultured for 72 h. Cells were then fixed in 20% acetone-80% methanol, stained with a primary monoclonal antibody against cardiac -actinin, and followed by a rhodamine-conjugated secondary antibody. Nuclei were stained by Hoechst. Cells were examined under a Zeiss fluorescence microscope (x630). Cardiomyocytes stained by -actinin antibody showed characteristic striation.
Drug treatment. After 24 h in culture medium supplemented with 10% FBS, cardiomyocytes were either starved in serum-free medium or maintained with serum for different drug treatment. Cardiomyocytes were then stimulated with either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) at concentrations ranging from 10 to 100 ng/ml. To assess the relationship between NO and cardiomyocyte proliferation, WT cardiomyocytes were treated with a nonselective NOS inhibitor molecule Nw-nitro-L-arginine methyl ester (L-NAME), and eNOS–/– cardiomyocytes were treated with the NO donor diethylenetriamine NO (DETA-NO). All drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO) unless specified.
Cardiomyocyte cell count. The level of proliferation in cultured cardiomyocytes was first determined by cell counts. After cardiomyocytes were cultured for 24, 48, 72, and 96 h, cells were detached with 50 µl of 0.5% trypsin for 10–15 min. Cells were counted using a hemocytometer and an automated NucleoCounter (New Brunswick Scientific).
Cardiomyocyte DNA synthesis in vitro. Cellular proliferation was also quantified via BrdU (Roche Molecular Diagnostics, Montreal, Quebec, Canada) incorporation into the DNA of proliferating cardiomyocytes isolated from both eNOS–/– and WT mice. BrdU incorporation into actively dividing cardiomyocytes was determined by immunoassay according to the manufacturer's instructions. Briefly, cells were grown for 18, 42, 66, and 90 h. After the respective growth periods, cells were labeled with the pyrimidine analog BrdU at a concentration of 10 µM for a period of 6 h. After BrdU incubation, cells were washed with PBS buffer solution (pH 7.4) and fixed with 200 µl of precooled 70% ethanol in HCl (vol/vol) at a final concentration of 0.5 M HCl. Subsequently, cells were washed three times with PBS and incubated with 100 µl exonuclease III (0.4 U/µl; MBI Fermentas, Burlington, Ontario, Canada) at 37°C for 30 min. Next, the cells were treated with the primary monoclonal antibody anti-BrdU conjugated with peroxidase (Roche Molecular Diagnostics) at 37°C for 30 min. Cells were incubated with the peroxidase substrate (2,2'azino-bis,3 ethylbenzthiazoline-6-sulfonic acid; ABTS) for 10 min at room temperature to allow color development. The absorbance of the samples was measured at 405 nm in a Bio-Rad microtiter plate reader (Bio-Rad Laboratories, Mississauga, Ontario, Canada).
Cardiomyocyte DNA synthesis in vivo. As an index of DNA synthesis, BrdU incorporation was measured in vivo using a BrdU-labeling and staining kit (Roche Molecular Diagnostics). BrdU (50 mg/kg) was administered to neonatal mice subcutaneously. Later (6 h), animals were killed, and hearts and small intestine (positive control) were obtained. Frozen tissue sections (10 µm) were stained for BrdU incorporation.
Briefly, cryosections were fixed in 70% ethanol in 2.4 mM HCl for 20 min at –20°C. Endogenous peroxidase activity was inhibited by incubating tissue sections in 0.3% H2O2-H2O (vol/vol). Tissue sections were treated with proteinase K (20 µg/ml; GIBCO-BRL, Burlington, Ontario, Canada) to expose the antigens. Sections were incubated with mouse anti-BrdU (1:10) for 1 h at 37°C in a humidified atmosphere. After incubation with the primary antibody, the slides were washed in PBS buffer solution (pH 7.4) three times and incubated with the secondary antibody, biotinylated horse anti-mouse IgG (1:50; Vectastain ABC Kit; Vector Laboratories, Burlingame, CA), for 30 min at room temperature. Tissue sections were then washed with PBS buffer solution (pH 7.4) and incubated with an avidin-biotin-peroxidase amplification system (Vector Laboratories) for 30 min at room temperature. BrdU incorporation into proliferating cells was detected after incubation with the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride at a concentration of 1 mg/ml (wt/vol) in PBS for 10 min at room temperature. Tissue sections were subsequently counterstained in Harris modified hematoxylin (Fischer Scientific, Fairlawn, NJ). Labeled nuclei cells with striation were quantified in 10 independent fields by microscopic analysis (Leitz) using a x50 objective with the BrdU-labeling index expressed as the ratio of BrdU positive cells to total nuclei.
Nitrite measurement. The formation of nitrite in culture medium was used as an indicator of NO production by neonatal cardiomyocytes. Culture medium was concentrated for five times before nitrite levels were measured by Griess reaction as we described previously (24, 27). The nitrite concentration was expressed as nanomoles per milligram cell protein.
RNA isolation and RT-PCR.
Total RNA was isolated from neonatal hearts of WT and eNOS–/– mice with TRIzol reagent (GIBCO-BRL) and subsequently reverse transcribed into first-strand cDNA using the Moloney murine leukemia virus RT system (MOP-64395; GIBCO-BRL). The mRNA expression of ANP and -MHC was determined by semiquantitative RT-PCR. Oligonucleotide primers were synthesized by GIBCO-BRL. The cDNA was amplified by PCR using a programmable thermal cycler. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified to ensure that an equal amount of RNA was present in all samples. Target gene accession numbers, reaction primer sequences, cycle numbers, and annealing temperatures are summarized in Table 1. PCR samples were then electrophoresed, separated on a 1.2% agarose gel containing ethidium bromide, and visualized under ultraviolet light to detect the expression of ANP and -MHC. Samples were analyzed via computer densitometry, and all densitometric data were standardized to GAPDH mRNA.
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RESULTS |
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-actinin, which stained Z-lines and showed typical striation of cardiomyocytes. Purity of cardiomyocytes from WT and eNOS–/– cultures was 94.6 ± 0.8 and 93.4 ± 2.1%, respectively [n = 3/group, P = not significant (NS)]. Proliferation of cultured neonatal cardiomyocytes was determined by cell count and BrdU incorporation. Results obtained through cell count revealed that 80% of the 100,000 cardiomyocytes initially seeded survived after 24 h in the cell culture model system employed (Fig. 1A). WT cardiomyocytes showed an increase in cell number and the level of BrdU incorporation over a 96-h time course (Fig. 1, A and B). Interestingly, cardiomyocytes deficient in eNOS displayed significantly fewer cells and lower BrdU incorporation compared with the WT cardiomyocytes after 72 and 96 h in culture (P < 0.05; Fig. 1, A and B).
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To investigate if the effects of NO on cardiomyocyte proliferation were mediated by cGMP, WT cardiomyocytes were treated with the selective guanylate cyclase inhibitor ODQ (100 µM) for 96 h. ODQ significantly decreased cell number in WT cardiomyocytes (129,875 ± 1,120 vs. 89,542 ± 1,142 cells/well; n = 4 independent experiments, P < 0.01). These results suggest that the effects of NO on cardiomyocyte proliferation are mediated by cGMP.
Effect of VEGF on the proliferation of eNOS–/– and WT cardiomyocytes. The ability of VEGF to stimulate cardiomyocyte proliferation was evaluated over time. WT cardiomyocytes treated with VEGF (30 ng/ml) displayed increased levels of BrdU incorporation over the unstimulated controls at 24, 48, 72, and 96 h (P < 0.05; Fig. 2A). On the basis of this time course, 48 h of VEGF treatment were chosen for the subsequent experiment. The effects of VEGF on BrdU incorporation were examined with and without serum. In the presence of serum, WT cardiomyocytes treated with VEGF displayed increased levels of BrdU incorporation in a dose-dependent manner, with maximal stimulation observed at a concentration of 100 ng/ml (P < 0.05; Fig. 2B). Conversely, VEGF did not significantly alter the level of BrdU incorporation in eNOS–/– cardiomyocytes (P = NS; Fig. 2B). The effects of VEGF (30 and 100 ng/ml) on BrdU incorporation were significantly decreased in eNOS–/– compared with WT cardiomyocytes (P < 0.05; Fig. 2B). Interestingly, in the absence of serum, VEGF did not significantly alter the level of BrdU incorporation in either WT or eNOS–/– cardiomyocytes (P = NS; Fig. 2B).
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-MHC mRNA in neonatal hearts.
The mRNA of ANP from neonatal hearts of WT and eNOS–/– mice was determined by semiquantitative RT-PCR using GAPDH as a loading control. PCR products of ANP and GAPDH obtained from both WT and eNOS–/– hearts are shown in Fig. 6A. The densitometric ANP-to-GAPDH ratios were significantly decreased in postnatal day 7 hearts of WT mice compared with postnatal day 1 hearts (P < 0.05; Fig. 6B). Cardiac -MHC mRNA expression was also evaluated in the hearts of WT and eNOS–/– mice by semiquantitative RT-PCR. PCR products generated from both WT and eNOS–/– neonatal mouse hearts are shown in Fig. 6C. The densitometric -MHC-to-GAPDH ratios were significantly decreased at postnatal days 5 and 7 in eNOS–/– compared with WT mice (P < 0.05; Fig. 6D).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Recent studies from our group and others support a role for eNOS in heart development (7, 18). However, mechanisms by which eNOS affects cardiac development are not fully understood. Interestingly, eNOS has been shown to regulate proliferation of several cell types, including endothelial cells and vascular smooth muscle cells (9, 22). However, the role of NO in cardiomyocyte proliferation has not previously been investigated. In the present study, cultured eNOS–/– cardiomyocytes displayed significantly decreased cell numbers and lower BrdU incorporation compared with WT cardiomyocytes. Treatment with the NO donor DETA-NO increased proliferation of cultured eNOS–/– cardiomyocytes. In our study, DETA-NO did not fully restore eNOS–/– cardiomyocyte proliferation to the WT level. This suggests that genetic deletion of eNOS gene may result in changes that also affect cardiomyocyte proliferation and cannot be corrected by an exogenous NO donor. On the other hand, inhibition of NOS activity using L-NAME in WT cardiomyocytes inhibited proliferation similar to that seen in the eNOS–/– population. Furthermore, in vivo BrdU labeling indexes in the neonatal myocardium at postnatal days 1 and 5 were significantly decreased in eNOS–/– compared with WT mice. These data suggest that eNOS modulates cardiomyocyte proliferation in vitro and in vivo.
Normal cardiac development is dependent on the coordinated expression and interaction of numerous growth factors and hormones. The expression of VEGF, for example, is tightly controlled such that either conditional deletion or overexpression of VEGF leads to cardiovascular malformation and embryonic lethality (8, 11). Although several growth factors have shown to exert mitogenic effects on cultured cardiomyocytes (16, 20), the role of NO in mediating this process is still not clear. In the present study, the effects of exogenous VEGF and bFGF on myocyte proliferation were explored. VEGF stimulated the proliferation of WT cardiomyocytes in a dose- and time-dependent manner. However, this effect was blunted in eNOS–/– cardiomyocytes, suggesting that VEGF-induced proliferation occurs through an NO-dependent mechanism. However, bFGF stimulated proliferation of WT and eNOS–/– cardiomyocytes to a similar extent, indicating that bFGF-induced proliferation occurs via an NO-independent pathway. Studies have shown that VEGF and bFGF have a synergistic effect on angiogenesis (25). To test whether there is any interaction between VEGF and bFGF on cardiomyocyte proliferation, WT cardiomyocytes were concurrently treated with VEGF and bFGF. Our results showed that VEGF and bFGF did not have a synergistic effect on cardiomyocyte proliferation.
ANP and MHC are differentiation markers expressed during heart development. Myocardial ANP expression decreases as the neonatal heart is maturing, and its expression eventually ceases in the mature ventricular myocardium (13, 14). MHC expression is also a differentiation marker for cardiac maturation. During neonatal development, there is a switch of MHC isoforms expressed in the myocardium. Although both -MHC and 
-MHC are expressed in the neonatal myocardium, the predominant MHC isoform is -MHC in the mature myocardium (21, 29). In the present study, ANP mRNA expression progressively decreased after birth in the WT hearts. However, ANP expression remained elevated in eNOS–/– hearts during the 1st wk of neonatal development. In addition, myocardial -MHC expression was significantly decreased at postnatal days 5 and 7 in eNOS–/– compared with WT mice. These results suggest that myocardial maturation of eNOS–/– mice is delayed.
The present study suggests that NO produced from eNOS is important for cardiomyocyte proliferation and maturation during neonatal heart development. NO has also been shown to promote cardiomyogenesis (4, 15). These effects of NO may in part contribute to the congenital heart defects and heart failure we previously reported in eNOS–/– mice (7). Deficiency in eNOS also leads to limb defects in mice (10). Interestingly, TBX5, a T-box-containing transcriptional factor expressed during early heart development, stimulates cardiomyogenesis and cardiomyocyte proliferation (12). Mutation of TBX5 in humans and deficiency in TBX5 in mice result in congenital heart and limb defects, the Holt-Oram syndrome (2, 3, 5). It is not known if there is any interaction between NO and TBX5. However, these studies and the present data do support the notion that factors regulating cardiomyogenesis and cardiomyocyte proliferation are important to fetal and neonatal heart development.
In summary, the present study demonstrated that proliferation is decreased in cultured eNOS–/– cardiomyocytes and in the hearts of neonatal eNOS–/– mice in vivo. The effect of VEGF on cardiomyocyte proliferation is NO dependent, whereas bFGF stimulates cardiomyocyte proliferation via NO-independent mechanisms. Furthermore, myocardial ANP expression is prolonged and -MHC expression is decreased in eNOS–/– mice, suggesting that cardiac maturation is delayed. We conclude that NO derived from eNOS plays an important role in cardiomyocyte proliferation and maturation during early neonatal heart development.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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P < 0.01). Treatment with N
-nitro-L-arginine methyl ester (L-NAME, 500 µM) significantly decreased the cell number in WT cardiomyocytes (*P < 0.05). Diethylenetriamine NONOate (DETA-NO) treatment (2 µM) significantly increased the cell number in eNOS–/– cardiomyocytes (*P < 0.05). B: BrdU incorporation. A time-dependent increase in BrdU incorporation was observed in WT cardiomyocytes, whereas this response was significantly attenuated in eNOS–/– cardiomyocytes (
