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(Circulation Research. 2003;92:1153.)
© 2003 American Heart Association, Inc.

Integrative Physiology

COX-2–Dependent Cardiac Failure in Gh/tTG Transgenic Mice

Zhibing Zhang, Roberta Vezza, Theodore Plappert, Peter McNamara, John A. Lawson, Sandra Austin, Domenico Praticò, Martin St-John Sutton, Garret A. FitzGerald

From the Center for Experimental Therapeutics (Z.Z., R.V., P.M., J.A.L., S.A., D.P., G.A.F.), University of Pennsylvania School of Medicine, Philadelphia, Pa; and the Division of Cardiovascular Medicine (M.S.S.), University of Pennsylvania School of Medicine Philadelphia, Pa.

Correspondence to Garret A. FitzGerald, MD, Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, 153 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia, PA 19104-6084. E-mail garret{at}spirit.gcrc.upenn.edu

	Abstract

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Gh is a GTP binding protein that couples to the thromboxane receptor (TP), but also functions as tissue transglutaminase II (tTG). A transgenic mouse model was generated in which Gh was overexpressed (GhOE) in ventricular myocytes under the control of the -myosin heavy chain promoter. Heart rate was elevated and both blood pressure and left ventricular ejection fraction were depressed in GhOEs. Left ventricular mass was increased, consistent with genetic and ultrastructural evidence of hypertrophy. Fibrosis and apoptosis were also augmented. Survival declined disproportionately in older GhOEs. Cardiomyocyte expression of COX-2, thromboxane synthase (TxS), and the receptors for TxA₂ (the TP), PGF₂ (the FP), and PGI₂ (the IP) were upregulated and urinary 8,12-iso-iPF₂-VI,2,3-dinor-6-keto-PGF₁ and 2,3-dinor-thromboxane B₂ were increased in GhOEs, reflecting increased lipid peroxidation and cyclooxygenase (COX) activation. Selective COX-2 inhibition, TP antagonism, and suppression of lipid peroxidation each rescued the cardiac phenotype. Infusion of an FP agonist exacerbated the phenotype, whereas administration of an IP agonist improved cardiac function. Directed cardiac overexpression of Gh/tTG causes both TG activation and increased TP/Gh-dependent signaling. The COX-2–dependent increase in TxA₂ generation augments cardiac hypertrophy, whereas formation of PGI₂ by the same isozyme ameliorates the phenotype. Oxidant stress may contribute, via regulation of COX-2 expression and/or ligation of the TP and the FP by isoprostanes. Gh/tTG activation regulates expression of COX-2 and its products may differentially modulate cardiomyocyte commitment to cell death or survival.

Key Words: cardiomyocytes • cyclooxygenase • thromboxane • tissue transglutaminase • G proteins

	Introduction

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Tissue transglutaminase (tTG), or calcium-sensitive TG II, catalyzes polymerization reactions thought integral to stabilization of the protein scaffold in apoptotic cells, preventing leakage of destructive intracellular components into the extracellular milieu and preparing them for phagocytosis.^1,2 Both colocalization of tTG expression with markers of apoptosis and the use of tTG inhibitors have supported a role for this enzyme in the apoptotic pathway.^3,4 The activity of tTG has also been implicated in the fibrogenesis that accompanies apoptosis in some models.⁵ Although deletion of tTG permits fetal survival,^6,7 it appears to modulate the apoptotic response to diverse stimuli ex vivo.⁷

Interestingly, tTG can also function as a GTP-binding protein, termed Gh.⁸ Indeed, we previously reported the cosolubilization and partial purification of the thromboxane receptor (TP) with a high molecular weight G protein (80 kDa), characteristic of Gh, from human platelets.⁹ TP isoforms and Gh coimmunoprecipitate from platelets and vascular tissues and TP signals, in an isoform-dependent manner, via Gh in an expression system.¹⁰ Ligation both of the _1B and _1D adrenoreceptor isoforms and of the oxytocin receptor engages Gh and, like the TP, increases intracellular calcium and/or inositol phosphates in expression systems,^11–13 apparently via phospholipase C ₁.^14,15 Both GTP and GDP readily bind tTG.¹⁶ Distinct GTP binding and TG domains have been identified within Gh/tTG,¹⁷ and the latter function may be negatively regulated by GTP binding.¹⁸ Thus, whereas receptor ligation may inactivate TG function by inducing GTP binding, a subsequent increase in intracellular calcium may enhance TG activity.

The role of prostaglandins and their free radical catalyzed isomers, the isoprostanes (iPs), in the modulation of cardiac function is poorly understood. However, cyclooxygenase (COX)-2 is upregulated in animal models of cardiac failure,^19,20 and its expression has been detected in cardiomyocytes in heart failure in humans.²¹ Deletion of the COX-2 gene may result in myocardial fibrosis.²² Interestingly, expression of COX-2 has been associated with both the initiation and prevention of apoptosis, perhaps reflective of variations in the cell-specific pattern of product formation.^23,24 Cardiac failure is also associated with increased generation of iPs, which may act as incidental ligands at prostaglandin receptors.^25–27 The present studies investigate the relationship between Gh/tTG and COX-2. A transgenic mouse model overexpressing Gh/tTG revealed an unexpected dependence of the cardiac phenotype on COX-2–derived thromboxane (Tx) A₂, which superseded the effects of the more conventional product of this enzyme in cardiomyocytes, prostacyclin (PGI₂).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of Transgenic Mice
Generation of transgenic mice is described in the expanded Materials and Methods in the online data supplement available at http://www.circresaha.org.

Histological, Biochemical, and Genetic Analyses
These are described in the expanded Materials and Methods (see online data supplement).

Analysis of 8,12-iso-iPF₂-VI, 2,3-dinor-6-keto PGF₁ and 2,3-dinor-thromboxane B₂
Urine collections (24-hour) from 7- to 10-month-old mice, corresponding to the time of evaluation of cardiac function, were analyzed as previously described.^28,29

Physiological Measurements
The hemodynamic and biochemical analyses are described in the expanded Materials and Methods (see online data supplement).

Statistical Analysis
Data are reported as mean±SEM (n). Statistical comparisons were performed by analysis of variance (ANOVA) and using two-tailed Student’s t test comparisons as appropriate. Values of P<0.05 were considered significant. Life table analysis was performed on the survival data.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals from line 53 expressed Gh at higher levels than animals from line 47 and were used for the majority of the experiments (Figures 1A and 1B). Similar results were obtained for Gh mRNA by Northern blotting. Analysis of protein extracted from lung, kidney, and liver revealed no difference in Gh expression between the transgenic animals and WT controls. Cardiac TGase activity was increased in a gene dose-dependent fashion in the GhOEs (online Figure 1, in the online data supplement available at http://www.circresaha.org). There was no difference in G_s, G_q, G_q/11, or Gß expression between the two transgenic lines and WT mice (data not shown), but G_i protein expression was higher in line 53 GhOEs, compared with both line 47 GhOEs and with WT mice (Figure 1C). Life table analysis revealed a delayed increase in mortality in line 53 GhOEs compared with WTs (Figure 1D). However, the difference only became pronounced in older (15 months) mice, after the emergence of the cardiac phenotype. No difference in survival was noted in line 47 GhOEs (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 1. Gh protein and RNA expression in the hearts of 9- to 10-month-old transgenic Gh-overexpressing (GhOE) and wild-type (WT) mice. A, Western blotting of Gh in cardiac tissue from WT mice and lines 47 and 53 GhOEs. Blot is representative of 3 similar experiments. B, Immunohistochemistry of Gh in cardiac tissue in WT and GhOE mice. Result presented is representative of 3 similar experiments. C, Gi protein is upregulated in line 53 GhOEs compared with WT mice. Western blot selected (left) is 1 of 3 that gave similar results. Group data are presented in the right panel. Gi expression was unaltered in line 47 GhOEs (data not shown). D, Life table analysis revealed a significant difference in survival between WT mice and line 53 GhOEs (*P<0.05, **P<0.01). This became evident at 15 months of age.

Fibrosis and Apoptosis
Staining for heart collagen was performed in 3- to 4-month and 9- to 10-month-old WT and line 53 GhOE mice. Fibrosis was not evident in any 3- to 4-month-old mice, but was increased at 9 to 10 months in the GhOEs (Figures 2A and 2B). TUNEL staining was performed in the same animals used for collagen staining. Again, no difference in the percentage of apoptotic cells was evident in 3- to 4-month-old mice. However, a higher percentage of apoptotic cells were apparent in 9- to 10-month-old GhOEs compared with their WT littermates (Figures 2C and 2D). Ultrastructural analysis of myocardium in 9- to 10-month old animals revealed myofibrillar disarray (Figure 2E) and disordered mitochondria (Figure 2F) in line 53 GhOEs. There was a gene dose dependent increase in expression of -skeletal actin, and ß myosin heavy chain, but not of ANF, in the transgenic animals (online Figure 2).

View larger version (76K): [in this window] [in a new window]   Figure 2. Fibrosis and apoptosis in the hearts of 9- to 10-month-old WT and line 53 GhOEs. A, Fibrosis stains blue and is depicted in representative samples from WT mice and in GhOEs with and without treatment with the COX-2 inhibitor, nimesulide (n=4 in each group). Magnifications, x100 in A and x200 in B. Ten fields were analyzed in tissues obtained from each mouse. B, Collagen-positive area in cardiac ventricles were quantitated examined in 9- to 10-month-old WT and line 53 GhOE mice (n=4). Collagen increased significantly in the transgenic animals (**P<0.01). Treatment with nimesulide, a COX-2 inhibitor, reduced significantly collagen positivity in GhOEs (#P<0.05 vs untreated), but collagen remained elevated when compared with age-matched WT mice (*P<0.05). C, Apoptosis of cardiomyocytes. TUNEL (A) and PI staining (B) of heart sections from 9- to 10-month-old WT and GhOE mice with and without treatment with the COX-2 inhibitor, nimesulide (n=4 in each group). Arrows indicate apoptotic cells. D, Percentage of apoptotic cells in hearts of WT and GhOE mice at 3 to 4 months of age and at 9 to 10 months of age divided before (baseline) and after (treated) 4 weeks administration of chow alone (column 1, black bar), the COX-2 inhibitors, nimesulide (column 2, light shade), or MF tricyclic (column 3, dark shade) (n=4 for each group; *P<0.05). E, Low-magnification view of myocardium in 9- to 10-month-old WT (left) and GhOEs (right). WT myocardium shows regular organization of sarcomeres and mitochondria, which appear normal, with dense matrices. GhOE mouse heart, by contrast, displays apparent disarray of myofilaments and mild, but universal swelling of mitochondria. These features are consistently associated with myocardial hypertrophy. Magnification, x10 000. Bar=2 µm. Arrows indicate mitochondria. F, High-magnification view further to illustrate the ultrastructural features of myocardium in 9- to 10-month old WT (left) and GhOEs (right). Note the regular arrangement of sarcomeres and normal mitochondria with dense and parallel cristae in WT mice (left), and the obvious disarray of sarcomeres and mitochondrial swelling in the GhOEs (right). Length and width of the sarcomeres are increased in GhOEs. Magnification, x26 000. Bar=500 nm. Arrows indicate mitochondria.

Prostanoids
Expression of COX-2, but not COX-1 mRNA, was increased in the older GhOEs compared with WTs. Expression of TxS was also increased in GhOEs (Figures 3A and 3B). Western blotting confirmed increased expression of COX-2 and TP protein in the transgenic animals (Figure 3C). mRNA for the receptors for prostacyclin (PGI₂) and PGF₂, the IP and the FP, respectively, was also increased in the hearts of GhOEs (Figures 3A and 3B).

View larger version (45K): [in this window] [in a new window]   Figure 3. Expression of prostanoid biosynthetic enzymes and receptors in 9- to 10-month old GhOEs. A, Representative mRNA expression of the cyclooxygenase isoforms (COX-1 and COX-2), thromboxane synthase (TxS), and prostanoid receptors (TP, IP, and FP). B, Summary of expression data depicted in A; data from 4 separate experiments (*P<0.01). C, Expression of COX-2 and TP proteins in WT and line 53 GhOE hearts at 9 to 10 months of age. Representative Western blot is depicted in the left panel and the summary data from 4 separate experiments for the TP protein are shown in the right panel (*P<0.01).

Consistent with these observations, we found that excretion of 2,3-dinor-6-keto PGF₁ (PGI-M) and 2,3-dinor thromboxane B₂ (Tx-M), major urinary metabolites of PGI₂ and Tx, respectively, were elevated in 7- to 10-month-old GhOEs compared with WT animals (Table 1). Finally, urinary excretion of 8,12-iso-iPF₂-VI was also increased in older GhOEs.

View this table: [in this window] [in a new window]   Table 1. Urinary Prostanoid (ng/mg Creatine) in Wild-Type (WT) and Transgenic Mice (GhOE)

Physiological Studies
Analysis of variance revealed a dose response relationship (F=19.9; P<0.05) between Gh expression and cardiac function in 7- to 10-month-old mice (Table 2), but not in 3- to 4-month-old mice (data not shown). Subsequent pairwise comparison between the two transgenic lines only attained significance for left ventricular mass, in which the most marked alteration was evident. The heart weight/body weight ratio and heart rate were also increased and correspondingly, the ejection fraction and systolic blood pressure were decreased in line 53 GhOEs, when compared with WT mice (Table 2).

View this table: [in this window] [in a new window]   Table 2. Physiological Parameters in Wild-Type and Transgenic Mice

We next sought to clarify the origin and functional importance of the altered prostanoid formation in the GhOEs. First, we utilized two structurally distinct COX-2 inhibitors, nimesulide²⁹ and MF tricyclic.³⁰ Given that COX-2 is the dominant source of PGI₂ formation by cardiomyocytes in rodents³¹ and of PGI-M excretion in mice²⁹ and humans,³² it was unsurprising that both of these compounds depressed PGI-M excretion in both WT mice and in GhOEs (Table 1). However, inhibition of COX-2 also depressed completely the increment in Tx-M excretion in GhOEs, suggesting that COX-2 was also the source of increased Tx biosynthesis in the transgenic animals.

The functional importance of the increment in PGI₂ formation was assessed by evaluation of cardiac function in animals infused with the IP agonist, iloprost.³³ Both ejection fraction and heart rate were corrected in the transgenic animals by iloprost (Table 3). Activation of the FP by contrast³⁴ dose-dependently exacerbated the GhOE phenotype (Table 3).

View this table: [in this window] [in a new window]   Table 3. Effect of Iloprost and Fluprostenol on Ejection Fraction and Heart Rate in 9- to 10-Month-Old Wild-Type (WT) and Transgenic (GhOE) Mice

The depressed ejection fraction and reduction in SBP, together with the increases in both LVM and heart rate that were evident in the older line 53 GhOEs, were all corrected by treatment with nimesulide, a selective COX-2 inhibitor (Figure 4A). MF tricyclic, a structurally distinct COX-2 inhibitor, also rescued the cardiac phenotype in older GhOEs (Figure 4B). Nimesulide also decreased both the number of apoptotic cardiomyocytes and the degree of myocardial fibrosis in the transgenic animals (Figures 2A through 2D).

View larger version (30K): [in this window] [in a new window]   Figure 4. Inhibition of COX-2 and cardiac function in 7- to 10-month-old line 53 GhOEs. A, Effects of 4 weeks of treatment with the COX-2 inhibitor nimesulide in 7- to 9-month-old WT and GhOE mice. PCAA is the percentage change in left ventricular cavity area, an estimate of the ejection fraction. LVM indicates left ventricular mass. Comparisons are between WT and the corresponding GhOE mice (*P<0.05: n=9 for each group). B, Effects of treatment with chow alone (open bar), the COX-2 inhibitor MF tricyclic (black bar), or with vitamin E (shaded bar), for 4 weeks in 9- to 10-month old GhOEs (n=8 for each group). Comparisons are made with the corresponding control animals (chow alone). **P<0.01 and *P<0.05 compared with the control).

TP expression was increased in GhOE and Gh coimmunoprecipitated from cardiac tissue of GhOEs with the TP (online Figure 3). Furthermore, there was evidence of Gh-dependent TP activation. Stimulation of cardiac tissue with a TP agonist resulted in augmented ERK activation in GhOEs and this effect was blocked by neutralization of Gh (online Figure 4). We utilized a TP antagonist, SQ 29,548,³⁵ to examine the functional relevance of increased Tx biosynthesis in GhOEs. We confirmed that the dosing regimen of SQ 29,548 had attained TP antagonism by measurement of a rightward shift in the TP agonist–induced platelet aggregation response ex vivo (data not shown) as previously described.²⁸ TP antagonism, like COX-2 inhibition, rescued the cardiac phenotype in older line 53 GhOE mice (Figures 5A and 5B). These mice also exhibit increased iP generation, which may activate the TP and/or the FP.^25,26 Suppression of iP generation with vitamin E (Table 1) also diminished the severity of the cardiac phenotype in GhOEs (Figure 4B).

View larger version (28K): [in this window] [in a new window]   Figure 5. Effect of TP antagonism on cardiac function in 9- to 10-month-old line 53 GhOEs. Echocardiography was performed before (baseline) and 10 minutes after SQ 29,548 (1 mg/kg) was given by intraperitoneal injection in 9- to 10-month-old GhOEs. A, Depressed ejection fraction (PCAA) in GhOEs is significantly improved by the TP antagonist SQ 29,548, although it remains below that in WT mice. TP antagonism did not significantly alter PCAA in WT mice (left). Increment in PCAA over baseline is significantly greater in GhOEs (right) (n=10 for each group; *P<0.05 for comparison between GhOE and corresponding WT control, #P<0.05 for comparison between baseline and SQ-treated GhOEs). B, Elevated heart rate in 9- to 10-month-old GhOEs is significantly reduced by treatment with the TP antagonist, SQ 29,548 (n=10 for each group; *P<0.05 for comparison between GhOE and corresponding WT control, #P<0.05 for comparison between baseline and SQ-treated GhOEs). TP antagonism does not alter heart rate in WT mice significantly (left). Decrement in heart rate is significantly greater in GhOEs than in WT mice (right).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cardiac hypertrophy and/or decompensation are phenotypic consequences of directed overexpression of several G proteins, including Gq, Gi, and Gh/tTG, in cardiomyocytes.^36–39 However, the mechanisms that result in these phenotypes are poorly understood. We focused on Gh/tTG, as it has been implicated in proapoptotic pathways (1, 2, and 7). Cardiomyocyte apoptosis is a feature of many forms of heart failure.⁴⁰ We were interested in the possible role of COX-2 in mediating the consequences of cardiac overexpression of Gh/tTG, given the contrasting and probably product-dependent roles reported for this enzyme in apoptosis in diverse tissues.²³ Both Gh and COX-2 are expressed in cardiomyocytes of patients with heart failure,^21,41,42 although the clinical importance of these observations is unknown.

We report that directed overexpression of Gh/tTG, under the control of the -MHC promoter, results in age-dependent left ventricular hypertrophy and cardiac decompensation, characterized by cardiomyocyte apoptosis and fibrosis and a delayed impact on survival. Elements of this phenotype were present in a transgenic line that modestly overexpressed Gh/tTG and more floridly in a line with more robust overexpression of the transgene (line 53). Whereas COX-1 mRNA was evident in cardiomyocytes of both the WT and GhOEs, expression of COX-2, TxS, and the TP were increased coincident with the emergence of the cardiac phenotype in the transgene. Biosynthesis of both Tx and PGI₂ was increased in the GhOEs and their marked suppression by two structurally distinct selective inhibitors of COX-2—nimesulide and MF tricyclic—suggest that this isozyme is the dominant source of their formation. Rescue of the cardiac phenotype by the COX-2 inhibitors places the regulation of COX-2 downstream of Gh/tTG overexpression in cardiomyocytes and implicates a COX-2 product in the emergence of the phenotype. Agonist activation of the TP is associated with increased signaling from the TP via Gh, resulting in ERK activation. Furthermore, tTG activity is also increased. This can result in amidation of RhoA, resulting in activation of mitogen-activated protein kinases (MAPKs).⁴³ Thus, activation of either or both of the Gh or tTG functions of the overexpressed protein may regulate COX-2 expression via recognized AP-1 motifs in its promoter.^19,44,45

The TP may be ligated by the COX products, TxA₂ and its PG endoperoxide precursor, PGH₂. Increased Tx biosynthesis, increased Gh-dependent signaling via the TP, and the effects of the TP antagonist all implicate TxA₂ as the dominant COX-2–derived mediator of the cardiac phenotype in GhOEs. Urinary excretion of an abundant iP, reflective of enhanced oxidant stress, was also increased in GhOEs. This is consistent with the finding of elevated generation of iPs in human heart failure.^27,46 The iPs (and, potentially, other products of lipid peroxidation) may activate the TP.²⁵ Such a mechanism may also have been relevant to activation of the FP.^26,47 Activation of the FP by a synthetic agonist, fluprostenol,⁴⁸ also exacerbates the phenotype in GhOEs. Oxidant stress may upregulate COX-2,²¹ acting via either of two NF-B binding motifs in its promoter,⁴⁹ independent of Gh. Suppression of iP generation by vitamin E rescues the phenotype, which is consistent with a role for oxidant stress both in induction of COX-2 and in the incidental activation of the TP. Consistent with the findings of Small et al,³⁶ we found that tTG was activated in the GhOEs. However, whereas they found no evidence of -adrenergic receptor–dependent Gh activation, TP-dependent signaling was increased in our transgenic model.

Upregulation of the IP, by contrast with the TP, appeared to subserve a compensatory response to the decline in cardiac function in GhOEs. This latter observation might be expected. Thus, expression of cardiomyocyte COX-2 is increased when cardiac failure is induced by doxorubicin in rats,¹⁹ and COX-2–dependent PGI₂ formation is cardioprotective in doxorubicin-treated rats in vivo.³¹ PGI₂ is similarly cardioprotective in rodent models of ischemia/reperfusion⁵⁰ and may, at least partly, mediate the beneficial effects of elevated expression of inducible nitric oxide synthase during the late phase of preconditioning.⁵¹ However, although PGI₂ biosynthesis is increased in GhOEs, COX-2 inhibition rescued, rather than exacerbated, the cardiac phenotype. In this case, the contrasting effects of another COX-2 product apparently superseded the "cardioprotective" effects of COX-2–dependent PGI₂ formation.

Overexpression of Gh/tTg also resulted in increased cardiac expression of Gi. Interestingly, Gh/tTG, like Gi, has been shown to inhibit adenylate cyclase-dependent signaling.^52,53 We did not address directly the contribution of Gi to the cardiac phenotype in GhOEs. However, Redfern et al,⁵⁴ using pharmacological activation of a modified opioid receptor, activated conditionally overexpressed Gi in the cardiomyocytes of 8-week-old mice. This results in a ventricular conduction delay and a rapidly lethal cardiomyopathy. Although this phenotype differs in its progress and histology from the cardiac phenotype in GhOEs, it is associated with a modest increase (1.5 fold) in expression of TP, but not in the mRNA for TxS or COX-2.⁵⁴ Activated TPs may potentially have engaged Gi⁵⁵ to modulate the phenotype in GhOEs. For example, TP agonists activate MAPK activity in vascular cells by coupling to Gi in a PKC-dependent manner and transactivating the epidermal growth factor receptor.⁵⁶ However, Gi is thought to have marginal influence in COX-2 regulation,⁵⁷ consistent with the findings of Redfern et al.⁵⁴ Rescue of the GhOE phenotype by COX-2 inhibitors argues against a major contribution from Gi to the alteration in cardiac function that we observed.

In summary, directed overexpression of Gh/tTG in cardiomyocytes results in activation of tTGase, increased expression of COX-2, and resultant TxA₂ formation. TP/Gh-dependent signaling is also augmented in the hearts of transgenic animals, and activation of the TP (and the FP) exacerbates the phenotype. COX-2–dependent PGI₂ formation, by contrast, subserves a homeostatic function. Coincident oxidant stress contributes to the phenotype, perhaps by increasing expression of COX-2 and/or by affording incidental ligands for the TP and the FP. These results suggest that the impact of COX-2 inhibitors in patients with heart failure may be complex and vary according to the pathophysiology that underlies their clinical condition.

	Acknowledgments

 
Grants HL-61364, HL-622250, and HL-57847 from the NIH supported this work. We thank Jian Yin and Lina X. Tang for their technical assistance. Dr FitzGerald is the Robinette Foundation Professor of Cardiovascular Medicine.

Received November 19, 2002; revision received April 2, 2003; accepted April 3, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Folk JE, Finlayson JS. The -(-glutamyl) lysine crosslink and the catalytic role of the transglutaminases. Adv Protein Chem. 1967; 31: 1–133.
2. Greenberg CS, Birckbichler PJ, Rice RH. Transglutaminases multifunctional cross-linking enzymes that stabilize tissues. FASEB J. 1991; 5: 3071–3077.[Abstract]
3. Kashima K, Yokoyama S, Daa T, Nakayama I, Iwaki T. Immunohistochemical study on tissue transglutaminase and copper-zinc superoxide dismutase in human myocardium: its relevance to apoptosis detected by the nick end labelling method. Virchows Arch. 1997; 430: 333–338.[CrossRef][Medline] [Order article via Infotrieve]
4. Thacher SM, Rice RH. Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell. 1985; 40: 685–695.[CrossRef][Medline] [Order article via Infotrieve]
5. Piacentini M, Farrace MG, Hassan C, Serafini B, Autuori F. ‘Tissue’ transglutaminase release from apoptotic cells into extracellular matrix during human liver fibrogenesis. J Pathol. 1999; 189: 92–98.[CrossRef][Medline] [Order article via Infotrieve]
6. De Laurenzi V, Melino G. Gene disruption of tissue transglutaminase. Mol Cell Biol. 2001; 21: 148–155.[Abstract/Free Full Text]
7. Nanda N, Iismaa SE, Owens WA, Husain A, Mackay F, Graham RM. Targeted inactivation of Gh/transglutaminase II. J Biol Chem. 2001; 276: 20673–20678.[Abstract/Free Full Text]
8. Nakaoka H, Perez DM, Baek KJ, Das T, Hussain A, Misono K, Im MJ, Graham RM. Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function. Science. 1994; 264: 1593–1596.[Abstract/Free Full Text]
9. Moran N, FitzGerald GA. Purification of an abundant guanine nucleotide binding (G) protein linked to human platelet prostaglandin (PGH₂)/thromboxane (TxA₂) receptors. Circulation. 1990; 82 (suppl 3): 461.Abstract.
10. Vezza R, Habib A, FitzGerald GA. Differential signaling by the thromboxane receptor isoforms via the novel GTP-binding protein, Gh. J Biol Chem. 1999; 274: 12774–12779.[Abstract/Free Full Text]
11. Chen S, Lin F, Iismaa S, Lee KN, Birckbichler PJ, Graham RM. 1-Adrenergic receptor signaling via Gh is subtype specific and independent of its transglutaminase activity. J Biol Chem. 1996; 271: 32385–32391.[Abstract/Free Full Text]
12. Singh US, Erickson JW, Cerione RA. Identification and biochemical characterization of an 80 kilodalton GTP-binding/transglutaminase from rabbit liver nuclei. Biochemistry. 1995; 34: 15863–15871.[CrossRef][Medline] [Order article via Infotrieve]
13. Park ES, Won JH, Han KJ, Suh PG, Ryu SH, Lee HS, Yun HY, Kwon NS, Baek KJ. Phospholipase C-1 and oxytocin receptor signaling: evidence of its role as an effector. Biochem J. 1998; 331: 283–289.[Medline] [Order article via Infotrieve]
14. Im MJ, Russell MA, Feng JF. Transglutaminase II: a new class of GTP-binding protein with new biological functions. Cell Signal. 1997; 9: 477–482.[CrossRef][Medline] [Order article via Infotrieve]
15. Feng JF, Rhee SG, Im MJ. Evidence that phospholipase 1 is the effector in the Gh (transglutaminase II). J Biol Chem. 1996; 271: 16451–16454.[Abstract/Free Full Text]
16. Prasanna Murthy SN, Lorand L. Nucleotide binding by the erythrocyte transglutaminase/G_h protein, probed with fluorescent analogs of GTP and GDP. Proc Natl Acad Sci U S A. 2000; 97: 7744–7747.[Abstract/Free Full Text]
17. Iismaa SE, Wu MJ, Nanda N, Church WB, Graham RM. GTP binding and signaling by G_h/transglutaminase II involves distinct residues in a unique GTP-binding pocket. J Biol Chem. 2000; 275: 18259–18265.[Abstract/Free Full Text]
18. Wu J, Liu SL, Zhu JL, Norton PA, Nojiri S, Hoek JB, Zern MA. Roles of tissue transglutaminase in ethanol-induced inhibition of hepatocyte proliferation and 1-adrenergic signal transduction. J Biol Chem. 2000; 275: 22213–22219.[Abstract/Free Full Text]
19. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem. 1999; 274: 5038–5046.[Abstract/Free Full Text]
20. Abassi Z, Brodsky S, Gealekman O, Rubinstein I, Hoffman A, Winaver J. Intrarenal expression and distribution of cyclooxygenase isoforms in rat experimental heart failure. Am J Renal Physiol. 2001; 280: F43–F53.[Abstract/Free Full Text]
21. Wong SC, Fukuchi M, Melnyk P, Rodger I, Giaid A. Induction of cyclooxygenase-2 and activation of nuclear factor-B in myocardium of patients with congestive heart failure. Circulation. 1998; 98: 100–103.[Abstract/Free Full Text]
22. Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD, Covington MB, Contel NR, Eng VM, Collins RJ, Czerniak PM. Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature. 1995;1995: 378: 406–409.[CrossRef][Medline] [Order article via Infotrieve]
23. Moore BC, Simmons DL. COX-2 inhibition, apoptosis, and chemoprevention by nonsteroidal anti-inflammatory drugs. Curr Med Chem. 2000; 7: 1131–1144.[Medline] [Order article via Infotrieve]
24. Gao Y, Yokota R, Tang S, Ashton AW, Ware JA. Reversal of angiogenesis in vitro, induction of apoptosis, and inhibition of AKT phosphorylation in endothelial cells by thromboxane A₂. Circ Res. 2000; 87: 739–745.[Abstract/Free Full Text]
25. Audoly LP. Rocca B, Fabre J-E, Koller BH, Thomas D, Loeb A, Coffman TM, FitzGerald GA. Cardiovascular responses to the isoprostanes iPF₂-III and iPE₂-III are mediated via the thromboxane A₂ receptor in vivo. Circulation. 2000; 101: 2833–2840.[Abstract/Free Full Text]
26. Kunapuli P, Lawson JA, Rokach JA, Meinkoth JL, FitzGerald GA. Functional characterization of the ocular prostaglandin F₂ (PGF₂) receptor: activation by the isoprostane, 8,12-iso-PGF₂. J Biol Chem. 1997; 272: 27147–27154.[Abstract/Free Full Text]
27. Li H, Lawson JA, Reilly M, Adiyaman M, Hwang SW, Rokach J, FitzGerald GA. Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F₂-isoprostanes in human urine. Proc Natl Acad Sci U S A. 1999; 96: 13381–13386.[Abstract/Free Full Text]
28. Rocca B, Loeb AL, Strauss JF, Vezza R, Habib A, Li H, FitzGerald GA. Directed vascular expression of the thromboxane A₂ receptor results in intrauterine growth retardation. Nat Med. 2000; 6: 219–221.[CrossRef][Medline] [Order article via Infotrieve]
29. Praticò D, Cyrus T, Li H, Rokach J, FitzGerald GA. Acceleration of atherogenesis by COX-1 dependent prostanoid formation in LDL receptor knockout mice. Proc Natl Acad Sci U S A. 2001; 98: 3358–3363.[Abstract/Free Full Text]
30. Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, Trzaskos JM, Evans JF, Taketo MM. Suppression of intestinal polyposis in Apc 716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell. 1996; 87: 803–809.[CrossRef][Medline] [Order article via Infotrieve]
31. Dowd NP, Scully M, Adderley SR, Cunningham AJ, Fitzgerald DJ. Inhibition of cyclooxygenase-2 aggravates doxorubicin-mediated cardiac injury in vivo. J Clin Invest. 2001;2001: 108: 585–590.[CrossRef][Medline] [Order article via Infotrieve]
32. McAdam BF, Catella-Lawson F, Mardini IA, Kapoor S, Lawson JA, FitzGerald GA. Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2. Proc Natl Acad Sci U S A. 1999; 96: 272–277.[Abstract/Free Full Text]
33. Kerins DM, Murray R, FitzGerald GA. Prostacyclin and PGE₁: molecular mechanisms and therapeutic utility. Prog Hemost. 1990; 10: 311–341.
34. Woodward DF, Lawrence RA, Fairbairn CE, Shan T, Williams LS. Intraocular pressure effects of selective prostanoid receptor agonists involve different receptor subtypes according to radioligand binding studies. J Lipid Mediat. 1993; 6: 545–553.[Medline] [Order article via Infotrieve]
35. Ogletree ML, Harris DN, Greenberg R, Haslanger MF, Nakane M. Pharmacological actions of SQ 29,548, a novel selective thromboxane antagonist. J Pharmacol Exp Ther. 1985; 234: 435–441.[Abstract/Free Full Text]
36. Small K, Feng J-F, Lorenz J, Donnelly ET, Yu A, Im M-J, Dorn GW II, Liggett SB. Cardiac specific overexpression of transglutaminase II (G_h) results in a unique hypertrophy phenotype independent of phospholipase C activation. J Biol Chem. 1999; 274: 21291–21296.[Abstract/Free Full Text]
37. Iwase M, Uechi M, Vatner DE, Asai K, Shannon RP, Kudej RK, Wagner TE, Wight DC, Patrick TA, Ishikawa Y. Cardiomyopathy induced by cardiac G_s overexpression. Am J Physiol. 1997; 272: H585–H589.[Medline] [Order article via Infotrieve]
38. Vatner DE, Asai K, Iwase M, Ishikawa Y, Wagner TE, Shannon RP, Homcy CJ, Vatner SF. Overexpression of myocardial G_s prevents full expression of catecholamine desensitization despite increased ß-adrenergic receptor kinase. J Clin Invest. 1998; 101: 1916–1922.[Medline] [Order article via Infotrieve]
39. D’Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, Dorn GW II. Transgenic Gq overexpression induces cardiac contractile failure in mice. Proc Natl Acad Sci U S A. 1997; 94: 8121–8126.[Abstract/Free Full Text]
40. Heinke MY, Yao M, Chang D, Einstein R, dos Remedios CG. Apoptosis of ventricular and atrial myocytes from pacing-induced canine heart failure. Cardiovasc Res. 2001; 49: 127–134.[Abstract/Free Full Text]
41. Iwai N, Shimoike H, Kinoshita M. Genes up-regulated in hypertrophied ventricle. Biochem Biophys Res Commun. 1995; 82: 1254–1259.
42. Hwang KC, Gray CD, Sweet WE, Moravec CS, Im MJ. ₁-Adrenergic receptor coupling with G_h in the failing human heart. Circulation. 1996; 94: 718–726.[Abstract/Free Full Text]
43. Singh US, Pan J, Kao YL, Joshi S, Young KL, Baker DM. Tissue-transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. J Biol Chem. 2002; 278: 391–399.[Medline] [Order article via Infotrieve]
44. Barry OP, Kazanietz MG, Praticò D, FitzGerald GA. Arachidonic acid in platelet microparticles upregulates COX-2 dependent prostaglandin formation via a PKC/MAPK pathway. J Biol Chem. 1999; 274: 7545–7556.[Abstract/Free Full Text]
45. Guo YS, Hellmich MR, Wen XD, Townsend CM Jr. Activator protein-1 transcription factor mediates bombesin-stimulated cyclooxygenase-2 expression in intestinal epithelial cells. J Biol Chem. 2001; 276: 22941–22947.[Abstract/Free Full Text]
46. Mallat Z, Philip I, Lebret M, Chatel D, Maclouf J, Tedgui A. Elevated levels of 8-iso-prostaglandin F₂ in pericardial fluid of patients with heart failure: a potential role for in vivo oxidant stress in ventricular dilatation and progression to heart failure. Circulation. 1998; 97: 1536–1539.[Abstract/Free Full Text]
47. Kunapuli P, Lawson JA, Rockach J, Meinkoth JJ, FitzGerald GA. Prostaglandin F₂ (PGF₂) and the isoprostane, 8, 12-iso-isoprostane F₂-III, induce cardiomyocyte hypertrophy. J Biol Chem. 1998; 273: 22442–22452.[Abstract/Free Full Text]
48. Lai J, Jin H, Yang R, Winer J, Li W, Yen R, King KL, Zeigler F, Ko A, Cheng J. Prostaglandin F₂ induces cardiac myocyte hypertrophy in vitro and cardiac growth in vivo. Am J Physiol. 1996; 271: H2197–H21208.[Medline] [Order article via Infotrieve]
49. Yamamoto K, Arakawa T, Ueda N, Yamamoto S. Transcriptional roles of nuclear factor-B and nucleus factor-interleukin-6 in the tumor necrosis factor -dependent induction of cyclo-oxygenase-2 in MC3T3-E1 cells. J Biol Chem. 1995; 270: 31315–31320.[Abstract/Free Full Text]
50. Xiao CY, Hara A, Yuhki K, Fujino T, Ma H, Okada Y, Takahata O, Yamada T, Murata T, Narumiya S, Ushikubi F. Roles of prostaglandin I₂ and thromboxane A₂ in cardiac ischemia-reperfusion injury: a study using mice lacking their respective receptors. Circulation. 2001; 104: 2210–2215.[Abstract/Free Full Text]
51. Shinmura K, Xuan YT, Tang XL, Kodani E, Han H, Zhu Y, Bolli R. Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning. Circ Res. 2002; 90: 602–608.[Abstract/Free Full Text]
52. Porta R, De Santis A, Esposito C, Draetta GF, Di Donato A, Illiano G. Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts. Biochem Biophys Res Commun. 1986; 138: 596–603.[CrossRef][Medline] [Order article via Infotrieve]
53. Gentile V, Porta R, Chiosi E, Spina A, Valente F, Pezone R, Davies PJ, Alaadik A, Illiano G. tTGase/Gh protein expression inhibits adenylate cyclase activity in Balb-C 3T3 fibroblasts membranes. Biochim Biophys Acta. 1997; 1357: 115–122.[Medline] [Order article via Infotrieve]
54. Redfern CH, Degtyarev MY, Kwa AT, Salomonis N, Cotte N, Nanevicz T, Fidelman N, Desai K, Vranizan K, Lee EK. Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy. Proc Natl Acad Sci U S A. 2000; 97: 4826–4831.[Abstract/Free Full Text]
55. Brass LF, Woolkalis MJ, Manning DR. Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase. J Biol Chem. 1988; 263: 5348–5355.[Abstract/Free Full Text]
56. Gao Y, Tang S, Zhou S, Ware JA. The thromboxane A₂ receptor activates mitogen-activated protein kinase via protein kinase C–dependent Gi coupling and Src-dependent phosphorylation of the epidermal growth factor receptor. J Pharmacol Exp Ther. 2001; 296: 426–433.[Abstract/Free Full Text]
57. Reddy ST, Wadleigh DJ, Herschman HR. Transcriptional regulation of the cyclooxygenase-2 gene in activated mast cells. J Biol Chem. 2000; 275: 3107–3113.[Abstract/Free Full Text]

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