Cardiac Specific Overexpression of Transglutaminase II (Gh) Results in a Unique Hypertrophy Phenotype Independent of Phospholipase C Activation*

Tissue type transglutaminase (TGII, also known as Gh) has been considered a multifunctional protein, with both transglutaminase and GTPase activity. The role of the latter function, which is proposed as a coupling mechanism between α1-adrenergic receptors and phospholipase C (PLC), is not well defined. TGII was overexpressed in transgenic mice in a cardiac specific manner to delineated relevant signaling pathways and their consequences in the heart. Cardiac transglutaminase activity in the highest expressing line was ∼37-fold greater than in nontransgenic lines. However, in vivo signaling to PLC, as assessed by inositol phosphate turnover in [3H]myoinositol organ bath atrial preparations, was not increased in the TGII mice at base line or in response to α1-adrenergic receptor stimulation; nor was protein kinase Cα (PKCα) or PKCε activity enhanced in the TGII transgenic mice. This is in contrast to mice moderately (∼5-fold) overexpressing Gαq, where inositol phosphate turnover and PKC activity were found to be clearly enhanced. TGII overexpression resulted in a remodeling of the heart with mild hypertrophy, elevated expression of β-myosin heavy chain and α-skeletal actin genes, and diffuse interstitial fibrosis. Resting ventricular function was depressed, but responsiveness to β-agonist was not impaired. This set of pathophysiologic findings is distinct from that evoked by overexpression of Gαq. We conclude that TGII acts in the heart primarily as a transglutaminase, and modulation of this function results in unique pathologic sequelae. Evidence for TGII acting as a G-protein-like transducer of receptor signaling to PLC in the heart is not supported by these studies.

Activation of ␣ 1 AR expressed on myocytes by catecholamines stimulates PLC activity, increasing inositol phosphate turnover in the heart (20). Traditionally, this signal has been considered to be transduced by the ␣ subunit of the heterotrimeric G protein G q (21). Indeed, a number of other G protein-coupled receptors that signal via G q are expressed on myocytes, including those for endothelin and angiotensin II (22). Persistent activation of this pathway, by overexpression of receptor (23) or G ␣q (24,25) or continuous exposure to agonist (26 -29), results in a hypertrophic response in myocytes and intact hearts. This has prompted the notion that one or more elements of this pathway are sufficient to trigger the response; thus, this overexpression strategy in transgenic mice has provided a biochemical mechanism for evoking hypertrophy and ventricular dysfunction in the absence of the systemic effects of continuous agonist exposure or the hemodynamic loading evoked by vascular banding. Whether TGII also signals via PLC in the heart, as has been proposed from cell-based studies, is not known. Interestingly, a potential link between TGII and heart failure has been suggested by Iwai et al. (30), who showed up-regulation of TGII mRNA in rat models of cardiac hypertrophy and failure, and Hwang et al. (31), who observed alterations in TGII protein function and expression in ischemic and dilated cardiomyopathic human hearts.
The current study was undertaken to investigate the signaling of TGII to its proposed intracellular pathways of transglutamination and activation of the PLC-inositol phosphate-PKC cascade, in a relevant target tissue, the heart. Our recent creation of transgenic mice overexpressing G ␣q in the heart has provided an animal with well documented activation of PLCinositol phosphate-PKC for anatomic, biochemical, and physiologic comparison (24). To this end, the myosin heavy chain promoter was used to drive cardiac specific expression of the wild-type TGII gene in transgenic mice (32). Our results show that overexpression of TGII in mouse heart results in unique pathologic consequences and depressed systolic function without enhanced inositol phosphate turnover, ␣1AR function, or PKC activation. Thus, TGII in the heart appears to function primarily as a transglutaminase.

EXPERIMENTAL PROCEDURES
Transgenic Mice-Cardiac specific expression of TGII in transgenic mice was achieved using the murine ␣-myosin heavy chain (MHC) promoter that directs expression to atria and ventricles. Briefly, the 2.1-kb coding region of the wild-type rat TGII gene (11) was blunt end-ligated into the SalI site of a plasmid containing the full-length (4.5-kb) murine ␣-MHC promoter (32). The resulting recombinant plasmid, ␣MHC-TGII, was confirmed by restriction mapping and sequencing. The transgene construct was digested with NotI to release an 8.3-kb fragment that was isolated, purified, and used for microinjection into the male pronuclei of FVB/N embryos. Injected embryos were implanted into oviducts of pseudopregnant females. Pups were screened for the presence of the transgene by Southern blot analysis performed on genomic DNA digested with BglII and probed with a 2.7-kb 32 P-labeled BglII fragment isolated from the ␣MHC-TGII transgene construct. Founders that were positive for the transgene were mated with nontransgenic animals to establish transgenic lines. Two lines (1.1 and 8.3) expressing TGII were propagated for further study. Second and third generation heterozygous mice between 12 and 16 weeks of age were used for all studies.
Transgene Expression-Mouse hearts were homogenized in 1 ml of 20 mM HEPES buffer (pH 7.4) containing 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 10% glycerol, and protease inhibitors (10 g/ml phenylmethylsulfonyl fluoride, 2 g/ml bacitracin, 100 g/ml benzamidine, 2 g/ml soybean trypsin inhibitor, and 20 g/ml antipain) and stored at Ϫ80°C until use. Western blots were performed as described previously (31) using a TGII-specific antibody, G ␣h7 (9). Transglutaminase activity was determined by evaluating the incorporation of [ 3 H]putrescine into N,NЈ-dimethylated casein in HSD buffer (20 mM HEPES (pH 7.4), 100 mM NaCl, 1 mM dithiothreitol) containing 0.05% sucrose monolaureate (33,34). Briefly, 5 g of heart lysate was preincubated with 2 mM MgCl 2 in a 100-l total volume at room temperature for 30 min. Samples were then transferred to an ice-water bath where 1% casein, 1 Ci of [ 3 H]putrescine, and 0.5 mM CaCl 2 (final concentrations) were added. Following a 30-min incubation at 30°C, reactions were iced and stopped by the addition of 100 l of cold 50% trichloroacetic acid containing 0.1% putrescine. The precipitates were trapped on GF/F glass fiber filters (Whatman), washed six times with ice-cold 5% trichloroacetic acid, and counted. The GTP-mediated inhibition of transglutaminase activity was determined under the same conditions after preincubation of the samples in the presence of 0.5 mM GTP (final) at room temperature for 30 min. The GTP binding activity of TGII was determined by direct photoaffinity labeling of [␣-32 P]GTP as described previously (7)(8)(9)(10)(11). Heart lysate preparations (10 g/assay) were incubated in HSD buffer containing 0.05% sucrose monolaureate in the presence of 10 Ci of [␣-32 P]GTP, 2 mM MgCl 2 , and 100 M AppNHp. After incubation at room temperature for 20 min, the samples were subjected to UV irradiation in an ice-water bath for 10 min. Samples were then fractionated by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography.
Signaling Measurements-HPLC determination of total 3 H-labeled inositol phosphate production in intact mouse atria was measured as described in detail by Woodcock (37). Briefly, labeling was performed by incubating isolated atria from TGII-8.3 and G␣q-40 (24) transgenic and nontransgenic littermates in Kreb's buffer (pH 7.4) with 40 Ci/ml [ 3 H]myoinositol for 4 h at 37°C followed by the addition of 10 mM LiCl (final concentration) for 10 min. For agonist stimulation of ␣ 1 AR, atria were pretreated with the ␤AR antagonist propanolol for 10 min and then incubated with 100 M norepinepherine for 10 min. Atria were then washed two times with cold Kreb's solution, and each atrium was placed in 300 l of ice-cold 5% trichloroacetic acid buffer containing 2.5 mM EDTA and 1 mM phytic acid. Following rapid homogenization, samples were spun at 5000 rpm for 15 min at 4°C. Supernatants were saved, and pellets were re-extracted with 300 l of the trichloroacetic acid/EDTA/phytic acid solution. Combined supernatants were further extracted with diethyl ether, and pH was adjusted to ϳ7.0. Inositol phosphates were resolved by high performance anion exchange liquid chromatography and eluted with a continuous gradient of ammonium formate (0 -1 M). 3 H-Labeled inositol phosphates were quantitated by scintillation spectroscopy, calibrated using authentic standards. Twenty g each of AMP, ADP, and ATP were included as internal UV standards. Results are given as total inositol phosphates and represent the sum of all isomers. Typically, 1000 -5000 cpm of 3 H-labeled inositol phosphates were measured per sample, and the data are reported as total inositol phosphates/mg of atria. PKC isoform activation was assayed as the relative proportion of PKC in Triton X-100-extracted membrane (particulate) versus cytosolic ventricular fractions as determined in quantitative Western blots (38). Anti-PKC␣ and -⑀ antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and the anti-PLC␦1 antibody was from Upstate Biotechnology, Inc. Measurements of ␤AR receptor density and adenylyl cyclase activity were carried out as described previously (39).
Cardiac Gene Expression-RNA was extracted from heart tissue using Tri-reagent (Molecular Research Center, Inc.), and quantitative assessment of cardiac hypertrophy gene expression was performed by RNA dot blotting as described previously (40). Filters were scanned using a PhosphorImager (Molecular Dynamics, Inc.), and the radioactivity of each hybridizing signal was quantitated using ImageQuant software. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase expression.
Physiologic Measurements-Echocardiography was performed in sedated mice as described (24,41). Calculations for left ventricle (LV) mass and wall thickness/radius ratio (H/r) were as follows: LV mass ϭ ((EDD ϩ (SWT ϩ PWT) 3 Ϫ (EDD) 3 ) ϫ 1.06 and H/r ϭ PWT/(EDD/2). Closed chest invasive hemodynamic studies were performed on 12-16week-old nontransgenic and TGII-8.3 littermates as described previously (42). Briefly, animals were anesthetized with ketamine (50 g/g, body weight) and thiobutabarbitol (100 g/g, body weight). A tracheotomy was performed to maintain airway patency, and mice were maintained on a thermally controlled surgical table. Catheters were placed in the right femoral artery and vein for measurement of systemic arterial pressure and infusion of drugs. A Millar Mikrotip transducer was advanced to the LV via the right carotid artery for measurement of LV pressure and dP/dt. Incremental doses of dobutamine were infused for 3 min each, and steady-state measurements were made during the final 30 s of each dose. Pressure signals were monitored and recorded continuously using a MacLab data acquisition system.
Statistical Analysis-Data are reported as means Ϯ S.E. Statistical comparisons were performed with two-tailed Student's t tests comparing transgenic values to nontransgenic littermates. For in vivo hemodynamic measurements, data were analyzed by one factor (within) or by mixed, two-factor analysis of variance using SUPERANOVA software by Abacus. Differences between individual means were further analyzed using single degree-of-freedom contrasts. Differences were regarded as significant at the p Ͻ 0.05 probability level.

RESULTS AND DISCUSSION
Two transgenic mouse lines (TGII-1.1 and TGII-8.3) that stably expressed the ␣MHC-TGII transgene were established. Mice from both lines were viable and reproduced normally. To quantitate functional TGII expression, transglutaminase activity of TGII was measured in transgenic and nontransgenic heart lysates (Fig. 1A). In the absence of Ca 2ϩ , basal myocardial transglutaminase activity was similar between nontransgenic (0.43 Ϯ 0.04 pmol/min/mg), TGII-1.1 transgenic mice (0.38 Ϯ 0.08 pmol/min/mg), and TGII-8.3 transgenic mice (0.51 Ϯ 0.06 pmol/min/mg). Upon Ca 2ϩ stimulation, nontransgenic animals exhibited a modest 1.5-fold increase in transglutaminase activity over basal levels. Ca 2ϩ -stimulated activities of TGII-1.1 and TGII-8.3 lysates showed increases to 2.5 Ϯ 0.50 pmol/min/mg (ϳ6.6-fold over basal) and to 19 Ϯ 1.7 pmol/ min/mg (ϳ37-fold over basal), respectively (Fig. 1A), indicating functional overexpression of the transgene. As would be predicted by the proposed model (1,4), this Ca 2ϩ -stimulated transglutaminase activity was inhibited by the addition of 0.5 mM GTP in the assays from both transgenic mouse lines and nontransgenic littermates. Western blots showed migration of the overexpressed TGII at the expected molecular mass (ϳ74 kDa) of the mature protein (Fig. 1B). A previously described assay that was optimized to show TGII GTP-binding activity by photoaffinity labeling in vitro (31) was also utilized to confirm the integrity of the transgenic TGII protein. Increased radiolabeling of an ϳ74-kDa protein corresponding to TGII was found in heart lysates from both TGII-1.1 and TGII-8.3 transgenic lines showing 5.2-and 12-fold increases in GTP binding, respectively, compared with nontransgenic littermates (Fig. 1C).
In vitro reconstitution and coimmunoprecipitation studies have defined PLC␦1 as the effector of TGII G-protein signaling (10,12). Western blot data confirm that, in mice, TGII and PLC␦1 are indeed coexpressed in both the atria and ventricles of the heart and that overexpression of TGII does not appear to alter PLC␦1 expression in either tissue (Fig. 2). Therefore, to investigate whether overexpression of TGII enhanced PLC activation in live tissue, total inositol phosphate levels were examined in organ cultured atria from TGII-8.3-and G ␣q -overexpressing mice and their nontransgenic littermates (Fig. 3A). Despite abundant expression of PLC␦1 in the mouse heart, basal inositol phosphate levels from TGII-8.3 transgenic mice were similar to those of nontransgenic littermates (71.1 Ϯ 8.45 cpm/mg versus 92.1 Ϯ 19.6 cpm/mg, respectively). These results contrast with parallel studies using G ␣q transgenic mice atria in which basal inositol phosphate levels were increased 4.4-fold (p Ͻ 0.01) over those of nontransgenic littermates (Fig. 3A). Furthermore, with the addition of the full ␣ 1 AR agonist norepinepherine for 10 min (in the presence of the ␤AR antagonist propanolol), the increase in inositol phosphate levels with atria from TGII-8.3 mice to 191 Ϯ 6.00 cpm/mg (2.7-fold over basal) was the same as that observed with nontransgenic littermates, which increased to 236 Ϯ 23.6 cpm/mg (2.6-fold over basal). Taken together, these results do not support coupling of TGII to inositol phosphate hydrolysis in mouse heart.
We have recently shown that transgenic overexpression of G ␣q in the heart results in translocation of PKC⑀ from cytosolic to particulate fractions (24) and that total PKC activity is increased 2.6-fold in G ␣q hearts as well. 2 Thus, it appears that chronic cardiac PKC activation is an expected outcome of enhanced signaling via PLC in cardiomyocytes. Activation of PKC in TGII-8.3 and nontransgenic littermates was assessed by quantitating the expression of PKC⑀ and PKC␣ (the two dominant diacylglycerol-regulated isoforms in mouse heart) in cardiac particulate versus cytosolic fractions. As shown in Fig. 3B, neither PKC isoform expression nor distribution was altered by TGII overexpression. Thus, two measurements of PLC coupling (inositol phosphate hydrolysis and PKC activation) failed to show increases in TGII transgenic mice despite ϳ37-fold overexpression of transglutaminase activity. From these studies, then, it appears that TGII does not couple (or couples very 2 G. W. Dorn, unpublished data.

FIG. 2. Western blot analysis confirms coexpression of TGII and PLC␦1 in atria and ventricle. Western analysis of TGII (top)
and and PLC␦1 (bottom) in atrial and ventricular lysates isolated from nontransgenic (Ϫ) and TGII-8.3 (ϩ) mice shows coexpression of TGII (74 kDa) and PLC␦1 (revealed as a 90-and 100-kDa doublet) in both chambers of the heart. In the TGII Western blot, 100 g of protein from each nontransgenic lysate as well as 50 and 10 g each from atrial and ventricular lysate from TGII-8.3 mice was analyzed, respectively, and for PLC␦1, 50 g of protein from each lysate was analyzed.

FIG. 3. Cell signal activation in TGII-8.3 mice. A, isolated atria were incubated in a physiologic organ bath in the presence of [ 3 H]myoinositol.
Total [ 3 H]inositol phosphate production was subsequently measured by HPLC anion exchange chromatography as described under "Experimental Procedures." Inositol phosphate production per mg of atria weight is shown for NTG and transgenic littermates from TGII-8.3 and G ␣q transgenic mouse lines. Each bar represents the average Ϯ S.E. for three mice. *, p Ͻ 0.05 difference from NTG (control) mice. B, Western analysis of PKC␣ and PKC⑀ in membrane and cytosolic fractions (50 g each) was performed using ventricular extracts from nontransgenic (lanes 1-3) and TGII-8.3 mice (lanes 4 -6) (see "Experimental Procedures"). Bands were quantitated using ScanAnalysis software. For cytosolic PKC⑀, the top band corresponding to this isoform was quantitated. No significant differences in PKC expression or the ratios of PKC in cardiac membranes/cytosol were found for either isoforms between TGII 8.3 mice and nontransgenic littermates. inefficiently) to PLC in the heart. This conclusion is particularly compelling given that only ϳ5-fold overexpression of G ␣q , a known activator of PLC, clearly enhances inositol phosphate hydrolysis and PKC activation in the heart.
Thus, the anatomic, biochemical, and functional consequences, if any, of overexpressing TGII in the heart would be expected to have little similarity to those of transgenic mice, such as those overexpressing G ␣q , where PLC activation is indeed enhanced. TGII-8.3 transgenic mice showed a mild cardiomegaly with an ϳ10% increase in whole heart weight indexed to body weight compared with nontransgenic littermates (6.79 Ϯ 0.30, n ϭ 14 versus 6.06 Ϯ 0.15, n ϭ 16, p Ͻ 0.03). Lung/body weight ratios were not different. Histologic examination of 12-16-week-old mice revealed normal cardiomyocyte morphology, with a moderate degree of diffuse interstitial fibrosis (Fig. 4). Further examination of a limited number of 7-month-old mice suggested that neither the cardiomegaly nor the interstitial fibrosis appears to worsen with age (data not shown). Since animal models of hypertrophy and the human disease are associated with increased expression of fetal cardiac genes, such as atrial naturietic factor, ␣-skeletal actin, and the ␤ isoform of the myosin heavy chain (␤-MHC) (43)(44)(45)(46)(47)(48), we considered that the mice with increased transglutaminase activity might be associated with increased levels of these genes, with potentially a different pattern than observed with models where PLC signaling is enhanced. TGII-8.3 mice indeed showed some modulation in fetal gene expression, with a 4.7and a 22-fold increase in ␤-MHC and ␣-skeletal actin expression, respectively (Fig. 5). Significantly, atrial naturietic factor gene expression, which is elevated in most models of hypertrophy, was not increased in TGII-8.3 mice. This pattern of fetal gene expression contrasts with that of G ␣q transgenic mice in which increased ␤-MHC and ␣-skeletal actin expression is accompanied by a dramatic (55-fold) increase in atrial naturietic factor expression (24).
The findings of mild cardiomegaly, increased molecular markers of pathologic hypertrophy, and interstitial fibrosis in TGII-8.3 transgenics suggested the potential for physiological consequences of TGII overexpression. This was assessed in vivo using echocardiography and catheterization-derived hemodynamic measurements. Ventricular systolic function, as determined by echocardiographical fractional shortening, was significantly decreased in TGII-8.3 transgenic mice compared with nontransgenic littermates (32 Ϯ 1%, n ϭ 5, versus 51 Ϯ 3%, n ϭ 13, p Ͻ 0.0001, Fig. 6A). Calculated left ventricular mass was also significantly increased in TGII-8.3 mice (Table  I), in agreement with the increased heart/body weight ratios found in these mice. Furthermore, H/r, the ratio of wall thickness to heart radius at diastole was not different from that observed in nontransgenic (NTG) mice, suggesting an eccentric pattern of enlargement (Table I). Consistent with the echocardiographic data, invasive hemodynamic studies also showed depressed ventricular function. Base-line (resting) left ventricular contractility (dP/dt max , Fig. 6B) was depressed ϳ30% in TGII-8.3 mice as compared with nontransgenic littermates (6400 Ϯ 870 versus 9400 Ϯ 390 mm Hg/s, n ϭ 5 each, p Ͻ 0.04); however, the magnitude of the dP/dt max responses to dobutamine was not different between TGII transgenic and nontransgenic littermates. Similarly, base-line left ventricular re-laxation (dP/dt min ) was less in the TGII mice, while the dP/ dt min responses were not significantly different between the two lines (Fig. 6C). Heart rates and left ventricular systolic and diastolic pressures were the same at base line and in response to dobutamine (data not shown).
In many forms of heart failure or in hypertrophy with ventricular dysfunction, cardiac ␤AR function is impaired (35). Given that a subtle alteration might not be evident in the in vivo agonist infusion studies, expression and coupling of ␤AR was examined in the TGII-8.3 mice. In membranes from TGII-8.3 and nontransgenic mice (n ϭ 4 each), ␤AR density was not different (29 Ϯ 4 versus 32 Ϯ 8 fmol/mg), nor were basal (36 Ϯ 5 versus 30 Ϯ 2 pmol/min/mg) or maximal isoproterenol-stimulated adenylyl cyclase activities (71 Ϯ 5 versus 68 Ϯ 7 pmol/ min/mg). Again, these data are in marked contrast with the impaired cardiac function in G ␣q -overexpressing mice, which exhibit an absence of in vivo ␤AR stimulation of cardiac contractility and depressed receptor coupling to adenylyl cyclase, probably due to, among other mechanisms, ␤AR phosphorylation by PKC. 2 In summary, we have overexpressed TGII in cardiomyocytes of transgenic mice to delineate both biochemical and physiologically applicable signaling pathways in the heart. With such overexpression, we were unable to detect enhanced basal or agonist-stimulated coupling of ␣ 1 AR to inositol phosphate hydrolysis; nor was there evidence of cardiac PKC activation. This finding strongly suggests that TGII signaling, if present at all, represents a minor regulator of PLC activity in the heart. The TGII overexpressing mice do have, however, cardiac hypertrophy, diffuse interstitial fibrosis, and depressed ventricular function at rest with normal responsiveness to ␤AR stimulation. The remodeling observed is consistent with the transglutaminase activity of TGII, which in other systems has been implicated in cell growth (15), matrix formation (36), and tissue repair processes (18,19). As has been suggested by studies in heart failure (30,31), alterations in expression or function of TGII may play a role in pathogenesis of the syndrome; based on the current body of work, we conclude that this is probably due to the transglutaminase activity of TGII rather than signaling through PLC.
FIG. 6. In vivo cardiac function in TGII transgenic mice. A, echocardiography shows depressed fractional shortening in TGII-8.3 mice. Bars represent mean values Ϯ S.E. for nontransgenic (n ϭ 5) and TGII-8.3 (n ϭ 13) mice. B and C, catheterization-derived hemodynamic measurements show cardiac contractility (dP/dt max ) and relaxation (dP/ dt min ) of the TGII 8.3 mice are depressed at base line. The responses to dobutamine were not impaired. Shown are results from experiments conducted with five mice in each group. *, p Ͻ 0.05 difference from NTG (control) mice.