Involvement of Tissue Transglutaminase in Endothelin 1–Induced Hypertrophy in Cultured Neonatal Rat Cardiomyocytes
A potential link between tissue-type transglutaminase (tTG) and cardiac hypertrophy was suggested recently. However, whether tTG is implicated in hypertrophic agonist-induced cardiac hypertrophy is not yet known. The purpose of this study was to investigate the effects of tTG on cardiomyocyte hypertrophy induced by endothelin (ET) 1. Real-time quantitative RT-PCR and Western blot analysis demonstrated that ET-1 increased the expression of tTG mRNA and protein in cardiomyocytes by activating ETA receptors. ET-1 failed to cause increases in cell size and [3H]leucine uptake, sarcomere reorganization, and gene induction of the atrial natriuretic factor when cardiomyocytes were treated with monodansylcadaverine, a competitive inhibitor of tTG. Furthermore, the effects of ET-1 on multifunctional activities of tTG were determined by evaluating the incorporation of [3H]putrescine into N,N′-dimethylated casein and charcoal absorption, respectively. The results showed that ET-1 did not influence the basal transglutaminase activity of cardiomyocytes but significantly inhibited the 0.1-mmol/L Ca2+-stimulated transglutaminase activity. Otherwise, ET-1 elevated the activity of GTPase in a concentration- and time-dependent manner. In vivo, right ventricular hypertrophy induced by 2 weeks of chronic hypoxia was depressed by the tTG inhibitor cystamine (10 to 30 mg/kg, 2 times per day, IP) in a dose-dependent manner. Taken together, our data strongly supported the notion that tTG may act as a positive regulator of the hypertrophic program in response to ET-1. This is probably attributable to the signaling activity of tTG rather than transglutaminase activity.
Cardiac hypertrophy is an adaptive response to pressure or volume stress, mutations of sarcomere (or other) proteins, or loss of contractile mass from a previous infarction. Although cardiac hypertrophy is initially compensatory for an increased workload, prolonged hypertrophy is a highly important risk factor for the development of heart failure. Regulation of cardiomyocyte hypertrophy occurs via complex mechanisms but to a large degree involves receptor-mediated intracellular processes initiated by paracrine, autocrine, and hormonal factors.1 It has been widely recognized that the hypertrophic agonists, including α1 adrenoceptor agonist phenylephrine, angiotensin II, and endothelin (ET) 1, bind to specific Gq protein–coupled receptors to initiate the intracellular response.2,3⇓ There was evidence of potential cross-talk among these agents, particularly regarding ET-1, as playing a central role in mediating the actions of other hypertrophic factors.
Tissue-type transglutaminase (tTG), also known as “Gh,” is a multifunctional GTP-binding protein that has been proposed to mediate both transglutaminase (TGase) and receptor-stimulated phospholipase C activation.4,5⇓ The physiological role of the enzyme is still being uncovered and includes cell adhesion, wound healing, apoptosis, and matrix reorganization.6 Recently, numerous studies have proposed that tTG was involved in the cardiac hypertrophic response. The initial finding showed upregulation of tTG mRNA in rat models of cardiac hypertrophy and failure.7 Subsequent studies showed alterations in tTG protein function and expression in ischemic and dilated cardiomyopathic human disease.8 Zhang et al9 demonstrated that cardiac tTG overexpression resulted in the hemodynamic, genetic, and ultrastructural changes of left ventricular hypertrophy. Furthermore, Small et al10 have used a transgenic mouse model to probe, finding that tTG overexpression resulted in a mild cardiac hypertrophy, elevated expression of β-myosin heavy chain and α-skeletal actin genes, and diffuse interstitial fibrosis. These results suggested a potential link between tTG and cardiac hypertrophy. Nevertheless, direct in vitro or vivo evidence that tTG is implicated in agonist-induced cardiac hypertrophy is still lacking. Accordingly, the present study was designed to investigate the potential involvement of tTG in cardiomyocyte hypertrophy induced by ET-1 and underlying mechanisms.
ET-1, BQ-123, BQ-788, MDC, and cystamine were purchased from Sigma Corporation; [3H]leucine (117 Ci/mmol), [3H]putrescine (117 Ci/mmol), and [γ-32P]GTP (3000 Ci/mmol) were from PerkinElmer Life Sciences; and rhodamine phalloidin was from Invitrogen Corporation. Other chemicals were of the highest grade available commercially.
Primary culture of neonatal rat cardiomyocytes was prepared from ventricles of 1-day–old Wistar rats (Institute of Jingfeng Medical Laboratory Animal, Beijing, China) as described by Simpson and Savion.11
Immunoblotting and Real-Time Quantitative RT-PCR
Cell lysates were separated by electrophoresis through a 10% to 12% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Primary antibodies were a rabbit polyclonal antibody to tTG (NeoMarkers, RB-060) in vitro and a mouse monoclonal antibody to tTG (NeoMarkers, MS-279) in vivo. A mouse monoclonal antibody to β-actin (1:1000, ab6276) was used as an internal control. Horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology) was used at a dilution of 1:1000. The blots were detected using enhanced chemiluminescence kits (ECL, Amersham Pharmacia Biotech.).
Real-time quantitative RT-PCR was carried out using a LightCycler rapid thermal cycler system (Roche Diagnostics), and SYBR Green was used for the detection of double-strand DNA. An expanded Methods section is available in the online Data Supplement (available at http://hyper.ahajournals.org).
In Vitro Experiments
Assessment of Cardiomyocyte Hypertrophy
Cell surface area was measured on F-actin–stained cardiomyocytes by confocal laser microscopy and Lim Image software, as reported previously.12 At least 100 cardiomyocytes in 25 to 30 fields at ×63 magnification were examined in 3 independent experiments. F-actin was detected with rhodamine-conjugated phalloidin (Molecular Probes), as described previously.13 The rate of protein synthesis was determined by incorporation of [3H]leucine into the cells, as described by Hirotani et al.14
TGase and GTPase Activity
tTG transamidation assay was performed as described previously15 with slight modifications. The TGase activity in cardiomyocyte lysate (40 μg of proteins per assay) was determined by valuating the incorporation of [3H]putrescine (1 millicurie) into N,N′-dimethylated casein 1% at 30°C for 30 minutes in Tris-HCl (pH 7.4) buffer containing 0.05% sucrose monolaurate in a 100-μL final volume. The GTP-mediated inhibition of TGase activity was determined under the same conditions after preincubation of the samples in the presence of 0.5 mmol/L GTP for 30 minutes at room temperature. GTP hydrolysis activity was measured by the quantization of radioactivity of released Pi from [γ-32P]GTP, as described previously.16
In Vivo Experiments
The protocol of chronic hypoxia was carried out according to our previous reports,17 and the experimental procedures used in this study have been approved by the local committee on animal care and use. The weight ratio of right ventricle/left ventricle+septum was used as an index for right ventricular hypertrophy. An expanded Methods section is available in the online Data Supplement.
Data were represented as mean±SEM, with “n” indicating the number of independent experiments or animals. ANOVA, followed by the Dunnett multiple comparison test, was used to analyze data. Values of P<0.05 were considered significant.
In Vitro Experiments
tTG mRNA and Protein Expression After ET-1 Stimulation in Cardiomyocytes
To determine whether tTG mRNA in cardiomyocytes was changed after stimulation with ET-1, the real-time RT-PCR analysis was performed. As shown in Figure 1A and 1B, the expression of tTG mRNA was significantly increased by treatment with 0.1 nmol/L of ET-1 for 24 hours (1.7±0.43-fold) or 10 nmol/L for 6 hours (1.58±0.17-fold). The increase reached maximum at 100 nmol/L of ET-1 for 24 hours or 10 nmol/L for 48 hours. An ET-1–induced increase in tTG mRNA expression could be significantly inhibited by BQ-123, a selective antagonist of the ETA receptor but not by the ETB receptor–selective antagonist BQ-788 (Figure S1). The observation suggested that ET-1 increased tTG mRNA expression via the ETA receptor. Western blot analysis revealed that ET-1 increased the expression of the tTG protein in a concentration- and time-dependent manner (Figure 2A and 2B).
Effects of tTG Inhibitor on Cardiomyocyte Hypertrophy Responses to ET-1
The hypertrophic response to ET-1 was determined by the induction of hypertrophic gene atrial natriuretic factor (ANF), protein synthesis, sarcomere reorganization, and increases in cardiomyocyte surface area. The protein synthesis was evaluated by incorporation of [3H]leucine into cultured cardiomyocytes. The results showed that ET-1 (10 nmol/L) increased [3H]leucine uptake (2.43±0.38-fold), and the increase was significantly inhibited by the tTG inhibitor monodansylcadaverine (MDC) in a concentration-dependent manner (Figure 3). The inhibitory effect of BQ-123 revealed the involvement of the ETA receptor in the ET-1–induced cardiomyocyte hypertrophy (Figure 3).
As shown in Figure 4A and 4B, the cardiomyocyte surface area was enlarged from 2570±895 μm2 (control group) to 6042±1700 μm2 (treated with 10 nmol/L of ET-1 for 24 hours). The enlargement was significantly depressed by 100 μmol/L of MDC (3891±1178 μm2).
Cardiomyocytes treated with ET-1 induced an apparent increase of the F-actin meshwork and a heavily striated appearance, reflecting the organization of this F-actin cytoskeleton into sarcomeric structures (Figure 4C). Treatment with MDC led to the elimination of the enhancement of the sarcomere organization. The real-time PCR analysis revealed a 3-fold increase in the ANF gene expression in cardiomyocytes stimulated with ET-1, and the increase was abrogated by the tTG inhibitor MDC (Figure 4D). MDC itself exhibited no effects on induction of the hypertrophic gene ANF, protein synthesis, or sarcomere reorganization (Figure 4A through 4D).
Changes in tTG Activities After ET-1 Stimulation in Cardiomyocytes
To test the effects of ET-1 on multifunctional activities of tTG in cultured neonatal rat cardiomyocytes, the activities of TGase and GTPase were determined by evaluating the incorporation of [3H]putrescine into N,N′-dimethylated casein and charcoal absorption, respectively. Ca2+ concentration–dependent TGase activity was measured by using 40 μg of protein from the cardiomyocyte extracts. The concentration-response relationship showed that half of the maximal activity of TGase was obtained at a concentration of 0.13 mmol/L of Ca2+ (Figure S2). A calcium concentration of 0.1 and 0.5 mmol/L was used to activate ≈30% and ≈90% of maximal Ca2+-stimulated TGase activity, respectively.
The basal cardiomyocyte TGase activity was low and did not change with increased concentration of ET-1 when CaCl2 was not added (Figure 5A and 5B). On Ca2+ stimulation, the TGase activity of tTG was markedly increased. This increase could be inhibited by ET-1 in a concentration- and time-dependent manner (Figures 5A and 5B and S3A and S3B). To evaluate the degree of participation of tTG in cardiomyocyte lysate activity, the inhibitory effect of GTP was tested. As shown in Figure 5A, 0.5 mmol/L of GTP blocked ≈95% of Ca2+-stimulated TGase activity. In contrast to the effect of ET-1 on TGase, ET-1 modestly increased the GTPase activity in a concentration- and time-dependent manner (Figure 6A and 6B). These results suggested that cardiomyocyte hypertrophy induced by ET-1 may depend on GTPase activation and not TGase.
In Vivo Experiments
To address the role of tTG in an in vivo model of heart hypertrophy, rats were subjected to a chronic hypoxia protocol. One to 4 weeks chronic hypoxia significantly induced hypertrophy of the right ventricle and increased the pulmonary arterial pressure and the heart tTG protein expression compared with the air control group (Figure S4). Administration of cystamine (10 to 30 mg/kg, 2 times per day, IP) did not affect the elevation of pulmonary arterial pressure but significantly lowered the increases in the index of right ventricular hypertrophy induced by hypoxia in a dose-dependent manner (Figure 7). A 30-mg/kg dose of cystamine did not influence the properties of rats in the air control group. These data showed that tTG was involved in the process described above. In addition, it was found that small pulmonary arterial (diameter: 50 to 100 μm) remodeling induced by 2 weeks of hypoxia was significantly attenuated by 30 mg/kg of cystamine (Figure S5A and S5B).
The present study demonstrated that tTG was partly involved in the process of ET-1–induced cardiomyocyte hypertrophy in a positive regulatory manner. This was based on our observation that the direct parameters related to cardiomyocyte hypertrophy were inhibited by a selective tTG inhibitor, MDC. Furthermore, our data demonstrated that the process of tTG involved in ET-1–induced cardiomyocyte hypertrophy may depend on GTPase activation by tTG and not TGase.
In the present experiments, we demonstrated that ET-1 increased the expressions of mRNA and protein levels of tTG in cardiomyocytes. To the best of our knowledge, this is the first report showing that the expressions of tTG mRNA and protein were upregulated by ET-1. It has been reported that cardiomyocytes predominantly expressed the ETA receptor, and the binding of ET to ETA receptors located on cardiomyocytes causes myocardial contraction and hypertrophy. In the present study, it was found that ET-1 increased mRNA levels of tTG via the ETA receptor but not the ETB receptor. These results suggested that tTG may play a role in regulating hypertrophic growth in response to ET-1.
To explore whether tTG acted in a critical role in the cardiomyocyte hypertrophy induced by ET-1, we examined the hypertrophic features of cardiomyocytes by several methods.18 In the current study, it was found that ET increased leucine incorporation and cell area and led to sarcomere reorganization. Moreover, ANF expression was markedly augmented by ET-1. These results indicated that ET-1 could induce pathological hypertrophy in cardiomyocytes. Furthermore, our data showed that MDC prevented all of the features of cardiomyocyte hypertrophy induced by ET-1. These results strongly supported the idea that the activation of tTG was involved in the above process in a positive regulation manner and indicated the importance of inhibiting the tTG signaling pathway to prevent cardiomyocyte hypertrophy. These results have prompted the notion that ≥1 element of this pathway was implicated in the process of ET-1–induced cardiomyocyte hypertrophy. Recently, emerging data showed that focal adhesion kinase was necessary for ET-1–induced cardiomyocyte hypertrophy.19 Cell surface tTG could activate intracellular focal adhesion kinase signaling cascades by forming a ternary complex with integrins and fibronectin,20 suggesting that focal adhesion kinase was involved in the downstream signaling of tTG activation.
The TGase activity and signaling activities of tTG were reciprocally regulated by Ca2+ and GTP.21,22⇓ TGase activity was inhibited by guanine nucleotides (GTPγS>GTP>GDP), whereas GTP photolabeling was inhibited by Ca2+.21 Ca2+ and GTP appeared to control the functional switch between the 2 activities of tTG. These facts showed the importance of understanding the multifunctional properties of tTG in defining its cellular and extracellular functions. Therefore, we examined the GTPase and TGase activities of tTG in hypertrophic cardiomyocytes. Our data showed that ET-1 significantly inhibited both ≈30% and ≈90% of maximal Ca2+-stimulated TGase activity but did not influence the basal TGase activity. In addition, ET-1 significantly elevated the activity of GTPase in a concentration- and time-dependent manner. These results provided strong support that tTG participated in cardiomyocyte hypertrophy, possibly through the capacity of tTG to signaling activity but not to its TGase-mediated protein cross-linking. Indeed, the involvement of tTG in membrane signaling appeared to be independent of its TGase activity, because it can be demonstrated at a physiological level of Ca2+ that was well below those required for enzyme activation.22 Moreover, it was reported that, at the physiological levels of the ATP and GTP, intracellular TGase activity was virtually 0, even in the presence of a high concentration of Ca2+ (10 mmol/L).23 In fact, only when Ca2+ was 100 mmol/L and nucleotide levels were low or absent could TGase activity be observed.
In the experiment, the causal relationship between tTG and cardiac hypertrophy was studied using a hypoxia experimental model in vivo. The results showed that chronic hypoxia increased the expression of heart tTG protein in rats in a time-dependent manner. Additional results proved that the tTG inhibitor cystamine did not alter the pulmonary hypertension exposed to chronic hypoxia, but right ventricular hypertrophy induced by hypoxia was significantly attenuated by cystamine in a dose-dependent manner. These data suggested that tTG could be involved in the development of cardiac hypertrophy evoked by hypoxia. Because cystamine has some other actions, such as antioxidant activity and inhibition of caspase 3, more experiments should be conducted to evaluate these effects of cystamine on the cardiac hypertrophy. There was evidence showing that ET-1 played a dominant role in the development of cardiac hypertrophy exposed to hypoxia.17,24⇓ In this present study, upregulation of heart tTG mRNA induced by hypoxia was significantly inhibited by ETP-508,17 a selective ETA receptor antagonist (Figure S6), suggesting that tTG may act as a regulator in the process of ET-1 involved cardiac hypertrophy associated with chronic hypoxia. Bakker et al25 have used several experimental models to probe whether small artery remodeling with chronic vasoconstriction depended on tTG. Interestingly, in the experiment, cystamine prevented pulmonary small arterial remodeling exposed to chronic hypoxia, and this result established the role of tTG in the small artery remodeling.
The current study provides an insight into the role of tTG in the development of cardiac hypertrophy. Our results suggest that tTG behaves as an endogenous, positive regulator of hypertrophic response. This is probably dependent on GTPase activation by tTG but not by TGase. In addition, our findings may provide a novel therapeutic target for cardiac hypertrophy.
- Received February 2, 2009.
- Revision received February 28, 2009.
- Accepted June 30, 2009.
- ↵Omura T, Yoshiyama M, Yoshida K, Nakamura Y, Kim S, Iwao H, Takeuchi K, Yoshikawa. Dominant negative mutant of c-Jun inhibits cardiomyocyte hypertrophy induced by endothelin 1 and phenylephrine. Hypertension. 2002; 39: 81–86.
- ↵Booz GW, Baker KM. Role of type 1 and type 2 angiotensin receptors in angiotensin II-induced cardiomyocyte hypertrophy. Hypertension. 1996; 28: 635–640.
- ↵Greenberg CS, Birckbichler PJ, Rice RH. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J. 1991; 5: 3071–3077.
- ↵Hwang KC, Gray CD, Sweet WE, Moravec CS, Im MJ. α1-Adrenergic receptor coupling with Gh in the failing human heart. Circulation. 1996; 94: 718–726.
- ↵Zhang Z, Vezza R, Plappert T, McNamara P, Lawson JA, Austin S, Pratico D, Sutton MS-J, FitzGerald GA. COX-2–dependent cardiac failure in Gh/tTG transgenic mice. Circ Res. 2003; 92: 1153–1161.
- ↵Small K, Feng JF, Lorenz J, Donnelly ET, Yu A, Im MJ, Dorn GW II, Liggett SB. Cardiac specific overexpression of transglutaminase II (Gh) results in a unique hypertrophy phenotype independent of phospholipase C activation. J Biol Chem. 1999; 274: 21291–21296.
- ↵Simpson P, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells: crossstriations,ultrastructure, and chronotropic response to isoproterenol. Circ Res. 1982; 50: 101–116.
- ↵Morel E, Marcantoni A, Gastineau M, Birkedal R, Rochais F, Garnier A, Lompré AM, Vandecasteele G, Lezoualc'h F. cAMP-binding protein Epac induces cardiomyocyte hypertrophy. Circ Res. 2005; 97: 1296–1304.
- ↵Hirotani S, Otsu K, Nishida K, Higuchi Y, Morita T, Nakayama H, Yamaguchi O, Mano T, Matsumura Y, Ueno H, Tada M, Hori M. Involvement of nuclear factor-κB and apoptosis signal-regulating kinase 1 in G-protein-coupled receptor agonist-induced cardiomyocyte hypertrophy. Circulation. 2002; 105: 509–515.
- ↵Antonyak MA, Miller AM, Jansen JM, Boehm JE, Balkman CE, Wakshlag JJ, Page RL, Cerione RA. Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin induced apoptosis in human breast cancer cells. J Biol Chem. 2004; 279: 41461–41467.
- ↵Frey N, Katus HA, Olson EN, Hill JA. Hypertrophy of the heart: a new therapeutic target? Circulation. 2004; 109: 1580–1589.
- ↵Eble DM, Strait JB, Govindarajan G, Lou J, Byron KL, Samarel AM. Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase. Am J Physiol Heart Circ Physiol. 2000; 278: 1695–1707.
- ↵Akimov SS, Krylov D, Fleischman LF, Belkin AM. Tissue transglutaminase is an integrin binding adhesion coreceptor for fibronectin. J Cell Biol. 2000; 148: 825–838.
- ↵Achyuthan KE, Greenberg CS. Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase: role of GTP and calcium ions in modulating activity. J Biol Chem. 1987; 262: 1901–1906.
- ↵Nakaoka H, Perez DM, Baek KJ, Das T, Husain A, Misono K, Im MJ, Graham RM. Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function. Science. 1994; 264: 1593–1596.
- ↵Smethurst PA, Griffin M. Measurement of tissue transglutaminase activity in a permeabilized cell system: its regulation by Ca2+ and nucleotides. Biochem J. 1996; 313: 803–808.
- ↵Galié N, Manes A, Branzi A. The endothelin system in pulmonary arterial hypertension. Cardiovasc Res. 2004; 61: 227–237.
- ↵Bakker EN, Buus CL, Spaan JA, Perree J, Ganga A, Rolf TM, Sorop O, Bramsen LH, Mulvany MJ, Vanbavel E. Small artery remodeling depends on tissue-type transglutaminase. Circ Res. 2005; 96: 119–126.