Heparin and Heparan Sulfate Block Angiotensin IIInduced Hypertrophy in Cultured Neonatal Rat Cardiomyocytes
A Possible Role of Intrinsic Heparin-like Molecules in Regulation of Cardiomyocyte Hypertrophy
Background Heparan sulfate, one of the primary components of extracellular matrix, is a potent antigrowth factor in certain types of cells. To elucidate a possible role of endogenous heparin-like molecules in regulating cardiomyocyte hypertrophy, we investigated the effects of heparin and heparan sulfate on angiotensin (Ang) II–induced hypertrophy in cultured neonatal rat cardiomyocytes.
Methods and Results Competitive [3H]heparin binding assay showed that cardiomyocytes had specific binding sites for heparin. In situ [3H]heparin binding assay demonstrated that heparin, which rapidly bound to the cardiomyocyte surface, was subsequently accumulated around the nuclei, suggesting that heparin might work in the nucleus. Cotreatment with heparin (20 μg/mL) completely inhibited increased cell surface area by Ang II (10−6 mol/L). Increased [3H]leucine incorporation by Ang II was reduced by heparin dose-dependently. The inhibitory effect of heparin on Ang II–induced cardiomyocyte hypertrophy also was confirmed by Northern blot analysis: heparin dose-dependently inhibited skeletal α-actin and atrial natriuretic peptide gene expression, genetic markers for cardiomyocyte hypertrophy. Heparan sulfate showed similar inhibitory effects on cell surface area, [3H]leucine incorporation, and skeletal α-actin gene expression. Treatment with heparinase I or III, which specifically digests the disaccharide chains of endogenous heparin-like molecules, upregulated protein synthesis and skeletal α-actin and atrial natriuretic peptide gene expression in cardiomyocytes.
Conclusions Our findings in this study strongly suggest that heparin and heparan sulfate are potent inhibitors of cardiomyocyte hypertrophy and that endogenous heparin-like substances negatively regulate cardiomyocyte hypertrophy.
Hypertrophy of the heart may be regulated primarily by extracellular factors that provide growth-stimulatory and growth-inhibitory effects. Over the last decade, many growth factors and neurohumoral factors have been shown to induce cardiomyocyte hypertrophy.1 2 3 4 5 6 7 However, in contrast to hypertrophy-stimulating factors, little is known about the extracellular factors that negatively regulate the formation of cardiomyocyte hypertrophy.
Heparin and HS, referred to as glycosaminoglycans, are linear heteropolysaccharides that possess a characteristic disaccharide repeat sequence. Glycosaminoglycan chains are covalently attached to a core protein; the resulting macromolecule is called a proteoglycan.8 HS proteoglycan, which is one of the major components of extracellular matrix and plasma membranes, is ubiquitously found in all mammalian tissues.9 Heparin, a well-characterized anticoagulant, closely resembles HS in structure. Recently, HS proteoglycan and heparin were shown to inhibit cell growth and proliferation in a variety of cell types.10 11 12 13 14 The mechanisms of this antigrowth effect are unknown, but it is postulated that membrane and nuclear events, including the binding of glycosaminoglycans to transcription factors, are involved.
Ang II, a potent vasoconstrictive peptide, is a well-characterized hypertrophy-stimulating factor of cardiomyocytes in vitro and may play an important role in the mechanism of cardiac hypertrophy.2 15 16 17 Using an in vitro model for cardiomyocyte hypertrophy induced by Ang II, we addressed the following questions: Do heparin and HS inhibit cardiomyocyte hypertrophy? Do endogenous heparin-like substances have a role in negative regulation of cardiomyocyte hypertrophy?
Drugs and cDNA
Recombinant human Ang II was provided by Peptide Institute Inc. Heparin sodium salt from porcine intestinal mucosa (170 U/mg), HS sodium salt from bovine intestinal mucosa, heparinase I (EC 220.127.116.11), heparinase III (heparitinase, EC 18.104.22.168), and protease-free chondroitinase ABC (EC 22.214.171.124) were purchased from Sigma Chemical Co. CV11974 was a gift of Takeda Chemical Industries, Ltd. [3H(G)]-heparin sodium salt was purchased from Du Pont New Research Products. The cDNA probe for the 3′ untranslated region of rat skeletal α-actin (184 bp) was synthesized as previously described.5 The cDNAs for human GAPDH and rat ANP were kind gifts of Drs K. Webster (SRI International) and H. Matsuo (National Cardiovascular Institute), respectively.
Primary cultures of neonatal rat cardiomyocytes were prepared by the method originally described by Simpson and Savion18 with minor modifications.19 Briefly, hearts from 1- or 2-day-old Wistar rats (Japan Laboratory Animals, Tokyo) were minced and dissociated with 0.1% collagenase type II (Sigma Chemical Co). After dispersed cells were incubated in 100-mm culture dishes (Primaria, Falcon) for 15 minutes at 37°C in 5% CO2, nonattached viable cells were collected and seeded into 60-mm dishes (2×106 cells per dish) or 24-well plates (2×105 cells per dish). Cardiomyocytes were incubated in Eagle’s minimum essential medium (Flow Laboratories) supplemented with 5% calf serum (Flow Laboratories) for 48 hours and then replaced with serum-free minimum essential medium for 24 hours before each experiment. To reduce the ratio of contaminating nonmyocytes, cytosine arabinoside (Ara-C, 10−6 mol/L) was added to each dish 12 hours after cell seeding. The percentage of nonmyocytes was <10% at the onset of each experiment as we previously reported.19
Crystal Violet Assay
The toxicity of heparin was examined by crystal violet assay as previously described.20 This method is based on assaying the binding of crystal violet to cell nuclei21 and has been shown to give a reliable cell count compared with counting cells with a Coulter counter.22 Briefly, cardiomyocytes plated in 24-well plates and treated with various concentrations of heparin for 24 hours were fixed with 10% formaldehyde and stained with 0.1% crystal violet. After the excess dye was washed out with water, dye was extracted with 50% ethanol and 0.1 mol/L sodium citrate, and the absorption was measured at 570 nm by a spectrophotometer.
[3H]heparin Binding Experiments
Cardiomyocytes (2×105 cells per well) plated in 24-well plates were incubated with serum-free minimum essential medium for 24 hours before the binding experiment and were maintained at 4°C during the assay as described by Castellot et al.23 Time course experiments of [3H]heparin binding to cardiomyocytes were performed as follows. The cells were washed three times with ice-cold PBS and incubated with [3H]heparin (13 μg/mL, 2×106 cpm per well) for various time periods at 4°C. The cells were then washed four times with ice-cold PBS and processed for liquid scintillation counting. In the competitive binding assay, serial dilutions of unlabeled heparin followed by [3H]heparin (13 μg/mL, 2×106 cpm per well) were added to wells; the cells were incubated at 4°C for 60 minutes and then harvested.
Microautoradiography With [3H]heparin
Cardiomyocytes seeded into tissue culture chambers (Nunc Inc) were incubated with or without [3H]heparin (1 μCi/mL, 37 kBq) for 15 minutes or 2 hours; after the incubation period, cells were washed four times with PBS and fixed with 3.7% formaldehyde. Then the slides were dipped into autoradiographic emulsion NTB 3 (Kodak) and kept in light-tight boxes at 4°C for 3 weeks. After photographic development, cells were stained with hematoxylin-eosin.
Immunocytochemistry and Measurement of Cell Surface Area
Immunocytochemical study using anti-human sarcomeric α-actin antibody (Dakopatts) was performed by the ABC method with a Vectastain ABC Kit (Vector Laboratories). After cardiomyocytes plated on 35-mm culture dishes were fixed in 90% ethanol containing 10% acetic acid, cells were preincubated with 5% of normal goat serum for 10 minutes and incubated with primary antibody (anti-human sarcomeric α-actin antibody) diluted to 1:40 in PBS with 1% BSA overnight at 4°C. After they were washed three times in PBS, the cells were incubated with biotin-conjugated anti-mouse IgG diluted to 1:100 in PBS for 1 hour at room temperature. Peroxidase was visualized with chromogen 3,3′-diaminobenzine and H2O2.
Phase-contrast pictures of cultured cardiomyocytes (magnification ×40) were scanned and saved in a personal computer. Outlines of 100 cells positive for sarcomeric α-actin staining for each experimental condition were manually traced, and the cell surface area of cardiomyocytes was calculated by use of image analysis software (NIH Image).
Incorporation of [3H]leucine
Protein synthesis in cultured rat cardiomyocytes was evaluated by incorporation of [3H]leucine into the cells. Cardiomyocytes (2×105 cells per well) on a 24-well plate were pretreated with or without various doses of heparin (or HS) for 4 hours before the addition of Ang II (10−6 mol/L) and then incubated for 20 hours in serum-free medium with or without heparin (or HS) and/or Ang II. The cells treated with heparinase I or III received additional heparinase treatment at 12 hours because of its short half-life. After incubation for 20 hours, the cells were incubated for 4 more hours with 1 μCi [3H]leucine (Amersham). At the end of labeling, cultures were rinsed three times with ice-cold PBS, and incubated with 5% trichloroacetic acid on ice for 20 minutes. After the cells were washed twice with ice-cold 5% trichloroacetic acid, they were solubilized in 0.5N NaOH for 30 minutes at room temperature. An aliquot of trichloroacetic acid–insoluble materials was neutralized with 0.5N HCl, and radioactivity was counted with a liquid scintillation counter (model 460CD, Packard Instrument Co Inc).
Northern Blot Analysis
Total RNA of neonatal rat cardiomyocytes was isolated by the guanidinium thiocyanate–phenol–chloroform method.24 Ten micrograms of total RNA was size fractionated through a 1.4% agarose gel in 0.7 mol/L formaldehyde and 20 mmol/L morpholinopropanesulfonic acid/5 mmol/L sodium acetate/1 mmol/L EDTA. Northern blot hybridization was performed with a hybridization buffer containing 50% formamide, 5× Denhardt’s solution, 100 μg/mL salmon sperm DNA, and 5× SSPE (0.75 mol/L NaCl/0.05 mol/L Na2HPO4/0.005 mol/L EDTA). 32P-labeled cDNA probes were prepared by a random primer method. The membranes (Magnagraph Nylon, Micron Separations Inc) were washed twice with 5× SSPE/10% SDS at room temperature, once with 1× SSPE/10% SDS at 37°C, and once with 0.1× SSPE/10% SDS at 37°C. Autoradiography was performed on a Fuji RX film with an intensifying screen at −80°C. Autoradiograms were quantified by an image analyzer (BAS2000, Fuji Film Corp). Results were normalized to GAPDH gene expression.
One-way ANOVA with multiple comparison methods by Scheffé’s test was used for statistical analyses. A value of P<.05 was considered significant. Data are expressed as mean±SEM.
Heparin Binding and Internalization Into Cardiomyocytes
Treatment with heparin, up to 200 μg/mL for 48 hours, revealed no cytotoxicity as morphologically observed and no reduction of cell numbers as evaluated by crystal violet assay (OD570, 1.933±0.173 versus 1.827±0.046, untreated cells versus 200 μg/mL heparin-treated cells; n=6; P=NS).
Time course experiments of [3H]heparin binding showed that 85% of radiolabeled heparin bound to cardiomyocytes within 15 minutes and that the binding reached a plateau at 40 minutes (data not shown). Fig 1⇓ demonstrates competitive [3H]heparin binding at various concentrations of unlabeled heparin. Addition of Ang II (10−6 mol/L) did not alter the [3H]heparin binding to cardiomyocytes. To examine the subsequent fate of bound [3H]heparin, microautoradiography was carried out (Fig 2⇓). After a 15-minute incubation, grains of [3H]heparin were found diffusely on the whole cell; 2 hours later, grains were concentrated around the nuclei.
Inhibitory Action of Heparin and HS on Hypertrophy of Cardiomyocytes Induced by Ang II
Cardiomyocytes treated with Ang II (10−6 mol/L) for 48 hours showed obviously enlarged cytoplasm compared with control cells, whereas cardiomyocytes treated with Ang II plus heparin (20 μg/mL) were similar to untreated cells (Fig 3⇓, top). The surface area of cardiomyocytes treated with Ang II (10−6 mol/L) significantly increased (by 48%) over control (784±27 versus 1157±42 μm2, control versus Ang II; P<.0001; Fig 3⇓, bottom). CV11974, an angiotensin receptor antagonist, inhibited the increase in cell surface area induced by Ang II. The surface area of cells cotreated with heparin (20 μg/mL) in the presence of Ang II for 48 hours was almost equal to that of control cells (763±25 μm2, P=NS), suggesting that heparin blocked Ang II–induced cardiomyocyte hypertrophy. The morphology and cell surface area were not altered by the treatment with heparin alone (data not shown).
Treatment with 10−6 mol/L Ang II for 24 hours increased protein synthesis as evaluated by [3H]leucine incorporation into cultured cardiomyocytes by 38% to 62% over control. This increase was inhibited by CV11974 in a dose-dependent manner (10−9 to 10−5 mol/L). Cotreatment with heparin dose-dependently reduced the increase in protein synthesis by Ang II: 2 to 20 μg/mL of heparin was enough to reduce the protein synthesis to the control level (Fig 4A⇓ and 4B⇓).
Because it is well known that skeletal α-actin and ANP mRNAs increase in hypertrophied cardiomyocytes in vivo25 26 27 and in vitro,1 28 29 30 we evaluated mRNA levels of these muscle specific genes in cardiomyocytes as markers for cardiomyocyte hypertrophy. mRNA levels of skeletal α-actin in cardiomyocytes increased 6 hours after treatment with 10−6 mol/L Ang II. Cotreatment with heparin inhibited the increment of skeletal α-actin gene expression stimulated by Ang II in a dose-dependent manner (Figs 5⇓ and 6A⇓). Heparin also inhibited ANP gene expression upregulated by Ang II (Figs 5⇓ and 6B⇓). Treatment with heparin alone revealed no significant effects on basal levels of the skeletal α-actin or ANP gene expression.
HS showed similar inhibitory effects to those of heparin on Ang II–induced cardiomyocyte hypertrophy. HS blocked the increase of cell surface area (Fig 3A⇑ and 3B⇑) and dose-dependently inhibited [3H]leucine incorporation induced by Ang II (Fig 7⇓). Cotreatment with 20 μg/mL HS significantly reduced the skeletal α-actin gene expression (Fig 8⇓).
A Possible Role for Intrinsic Heparin-like Molecules in Regulating Cardiac Hypertrophy
To test the possibility that intrinsic heparin-like molecules may be involved in the negative regulation of cardiomyocyte hypertrophy, we examined the effect of heparinase I or III, which specifically digests the disaccharide chain of heparin and HS. Treatment with heparinase I (10 U/mL) or III (1 U/mL) for 24 hours significantly upregulated [3H]leucine incorporation by 77.4±25.1% and 66.6±9.2% over control, respectively (Fig 9⇓). Both heparinase I and III did not cause any morphological changes of the cells. Treatment with heparinase further upregulated the Ang II–induced increase in [3H]leucine incorporation (Table⇓). Chondroitinase ABC (protease free), which specifically digests the disaccharide chain of chondroitin sulfate, showed no effect on [3H]leucine incorporation (Table⇓). A higher dose of chondroitinase ABC (1 U/mL) downregulated [3H]leucine incorporation, indicating its toxicity at this dose in cultured cardiomyocytes (data not shown). Heparinase I (10 U/mL) or III (1 U/mL) also upregulated skeletal α-actin and ANP gene expression, whereas chondroitinase ABC (0.1 U/mL) did not alter the expression of these genes (Fig 10⇓).
In the present study, we have reported that heparin and HS inhibited the Ang II–induced hypertrophy of cardiomyocytes. In addition, we have shown that treatment with digestive enzymes for heparin-like molecules increased skeletal α-actin and ANP gene expression and protein synthesis in cardiomyocytes. These results suggest a negative regulatory role of an intrinsic heparin-like substance, which is a component of extracellular matrix and plasma membranes, in the pathophysiology of cardiomyocyte hypertrophy.
We used Ang II as a stimulator of cardiomyocyte hypertrophy because this vasoconstrictive peptide is thought to play an important pathophysiological role in cardiac hypertrophy.3 4 15 16 31 In the present study, 10−6 mol/L Ang II induced cardiomyocyte hypertrophy, increased protein synthesis, and enhanced skeletal α-actin and ANP gene expression, both of which are considered genetic markers for cardiomyocyte hypertrophy in vivo25 26 27 and in vitro.1 28 29 30 Treatment with heparin specifically and significantly reduced the changes caused by Ang II in a dose-dependent manner. This inhibitory effect probably is not caused by cytotoxicity because changes in cell numbers and morphology of cardiomyocytes were not seen after exposure to heparin alone at the doses used in this study. Therefore, our results are in accord with our hypothesis that heparin inhibits Ang II–induced cardiomyocyte hypertrophy.
A binding assay using radiolabeled heparin demonstrated that [3H]heparin bound rapidly to cardiomyocytes and that the binding was competitively inhibited by unlabeled heparin, suggesting that cardiomyocytes have specific binding sites for heparin. These data are compatible with the observation by Castellot et al,23 who described the presence of a specific, high-affinity binding site and the initial rapid uptake of [3H]heparin in vascular smooth muscle cells. Furthermore, heparin bound to the cardiomyocyte surface was subsequently internalized and accumulated around the nuclei as indicated by our in situ binding assay. Taken together, our results suggest that heparin elicits its inhibitory action on cardiomyocyte hypertrophy through entering cardiomyocytes. Heparin or HS is well known to have a high affinity to basic fibroblast growth factor; therefore, one of the possible mechanisms of the biological action of heparin is postulated to be through altering the binding of basic fibroblast growth factor to its cell surface receptors.32 We did not approach the contribution of this extracellular mechanism in the present study; however, our findings that radiolabeled heparin accumulated in the nuclei immediately after binding to cell surface support the notion that there may be intracellular mechanisms in the action of heparin in cardiomyocytes.
The precise intracellular mechanism of the growth inhibitory action of heparin has remained unclear. Busch et al,33 however, reported that heparin selectively inhibits AP-1–mediated gene expression in vascular smooth muscle cells, transformed Hela cells, and nondifferentiated F9 teratocarcinoma cells. Au et al34 recently reported that heparin decreases AP-1 binding by posttranslational modification of Jun B, which is one of the components of AP-1, in baboon smooth muscle cells. These recent notions suggest that heparin seems to elicit its antigrowth action by inhibiting AP-1–regulated transcription. Because AP-1–mediated transcriptional regulation is considered crucial in the mechanism of cardiomyocyte hypertrophy, it can be postulated that the inhibitory action of heparin on hypertrophy of cardiomyocytes also may be related to this mechanism.
Heparin is not a physiological glycosaminoglycan. Therefore, we examined the effect of HS, which is very similar to heparin in structure and ubiquitously expressed in nearly all mammalian tissues. In the present study, HS inhibited cell surface area, protein synthesis, and skeletal α-actin gene expression induced by Ang II in a similar fashion to heparin. Furthermore, treatment with heparinase I or III specifically and dose-responsively upregulated protein synthesis and the expression of marker genes for hypertrophy in cardiomyocytes. These effects of heparinase do not seem to be caused by contaminating proteases because we have obtained similar results on [3H]leucine incorporation and Northern blot analysis using heparinase I and III from ICN Biomedicals Inc, which confirmed that the enzyme preparations were substantially free from protease activity (data not shown). In addition, heparinase additively upregulated the Ang II–induced increase in [3H]leucine incorporation, suggesting the specificity of this effect of heparinase. These findings support our hypothesis that endogenous heparin-like molecules in cardiomyocytes, possibly HS, may be involved in the negative regulation of cardiomyocyte hypertrophy.
Selected Abbreviations and Acronyms
|ANP||=||atrial natriuretic peptide|
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan, and by grants from the Japan Promotion Society for Cardiovascular Diseases and the Study Group of Molecular Cardiology, Tokyo.
- Received July 12, 1995.
- Revision received September 14, 1995.
- Accepted October 2, 1995.
- Copyright © 1996 by American Heart Association
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