Cellular Microbiology (2015) 17(9), 1320–1331

doi:10.1111/cmi.12436 First published online 30 March 2015

A systemic Pasteurella multocida toxin aggravates cardiac hypertrophy and fibrosis in mice Markus Weise,1 Christiane Vettel,2 Katharina Spiger,2 Ralf Gilsbach,3 Lutz Hein,3 Kristina Lorenz,4,5 Thomas Wieland,2 Klaus Aktories1,6 and Joachim H. C. Orth1* 1 Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Dept. I, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, Freiburg 79104, Germany. 2 Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany. 3 Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Dept. II, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany. 4 Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany. 5 Comprehensive Heart Failure Center, University of Würzburg, Würzburg, Germany. 6 BIOSS Centre for Biological Signalling Studies, Universität Freiburg, Freiburg, Germany. Summary Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gαq/11, Gα12/13 and Gαi family without interaction with G proteincoupled receptors (GPCRs). We show that PMT acts on heart tissue in vivo and on cardiomyocytes and cardiac fibroblasts in vitro by deamidation of heterotrimeric G proteins. Increased normalized ventricle weights and fibrosis were detected after intraperitoneal administration of PMT in combination with the GPCR agonist phenylephrine. In neonatal rat cardiomyocytes, PMT stimulated the mitogen-activated protein kinase pathway, which is crucial for the development of cellular hypertrophy. The toxin induced phosphorylation of the canonical phosphorylation sites of the extracellular-regulated kinase 1/2 and, additionally, caused phosphorylation of the recently recognized autophosphorylation site, which appears to be important for the development of cellular hypertrophy. Moreover,

Received 13 August, 2014; revised 20 February, 2015; accepted 6 March, 2015. *For correspondence. E-mail [email protected]; Tel. 49-761-2035299; Fax 49-761-2035311.

PMT stimulated the small GTPases Rac1 and RhoA. Both switch proteins are involved in cardiomyocyte hypertrophy. In addition, PMT stimulated RhoA and Rac1 in neonatal rat cardiac fibroblasts. RhoA and Rac1 have been implicated in the regulation of connective tissue growth factor (CTGF) secretion and expression. Accordingly, we show that PMT treatment increased secretion and expression of CTGF in cardiac fibroblasts. Altogether, the data indicate that PMT is an inducer of pathological remodelling of cardiac cells and identifies the toxin as a promising tool for studying heterotrimeric G proteindependent signalling in cardiac cells. Introduction Pasteurella multocida toxin (PMT) is a major virulence factor of P. multocida serotype D and A that induces characteristic syndromes such as atrophic rhinitis in animals and zoonotic diseases in humans. Most common are skin or soft tissue infections in humans by P. multocida caused by scratches or bites of domesticated animals such as cats or dogs (Wilson and Ho, 2013). However, the role of PMT in these infections is not clear, as the toxigenic status of the P. multocida is usually not tested. Moreover, systemic toxin effects induced during the infection are not reported, although it is known that PMT is one of the most potent mitogens (Rozengurt et al., 1990). Recent elucidation of the molecular mechanism of PMT provides novel insights into the pathology of P. multocida infections caused by strains which produce PMT. The toxin activates heterotrimeric G proteins by a covalent modification (Orth et al., 2009). The toxin deamidates a specific glutamine residue, which is essential for the GTPase reaction, that is hydrolysis of the bound nucleotide GTP. Consequently, the deamidated G protein is persistently activated. From the four main families of heterotrimeric G proteins, PMT activates the Gαq/11, Gα12/13 and Gαi family (Orth et al., 2013). Gαs is not a substrate of PMT. In addition, toxin activation of the α-subunit of heterotrimeric G proteins releases the βγ-subunits stimulating the βγ-specific effectors, for example the PI3Kγ (Preuss et al., 2009). Cardiac hypertrophy can be induced by various physiological and pathophysiological stimuli. Etiological factors might be hypertension, myocardial infarction, myocarditis or diabetes. As a transition state, cardiac hypertrophy can

© 2015 John Wiley & Sons Ltd

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Action of G protein-activating PMT on the heart 1321 finalize in chronic heart failure, a status of deficient capability of the heart to adequately pump blood. The primary stimuli to induce cardiac hypertrophy are stretch-sensitive and/or neurohumoral mechanisms. These neurohumoral mechanisms comprise the action of growth factors, cytokines, chemokines and hormones such as catecholamines. Increased release of catecholamines from the sympathetic nervous system restores the cardiac output by positive inotropic and chronotropic effects. However, longterm increase of catecholamines themselves leads to severe deregulation of heart function and remodelling of heart tissue, for example cardiomyocyte hypertrophy or fibrosis (Anker et al., 1997; Braunwald and Bristow, 2000; Gilsbach et al., 2010). Catecholamines act on β- and α-adrenoceptors, belonging to the family of G proteincoupled receptors (GPCR). β1-adrenoceptors increase the contractility of cardiomyocytes by stimulating heterotrimeric G proteins of the Gαs family, leading to enhanced activity of the adenylyl cyclase (AC) and in turn to increased cAMP levels. α1-adrenoceptors couple to Gαq/11, activating the phospholipase Cβ1 (PLCβ1) to increase Ca2+ levels and thereby increasing contractility of myocytes (Wettschureck and Offermanns, 2005). Finally, impaired cardiac function and hypertrophy are a consequence of a deregulated transcription programme (Heineke and Molkentin, 2006). Although the underlying pathways are still not completely understood, the contribution of G protein signalling on cardiac hypertrophy has been extensively studied over the last decades. As a result of these studies, various families of heterotrimeric G proteins have been implicated in the development of cardiac hypertrophy (Heineke and Molkentin, 2006; Tilley, 2011). Of special importance are the Ca2+dependent signalling and the transactivation of the mitogenactivated protein kinase (MAPK) pathway. The second messenger Ca2+ is canonical regulated by Gαq/11 via the PLCβ1 and the Gαs-cAMP pathway regulates intracellular Ca2+ levels via EPAC (exchange protein directly activated by cAMP) (Morel et al., 2005). The Gα12/13 family stimulates the small GTPase RhoA via specific RhoGEFs. Moreover, the small GTPase Rac1 has been implicated in cardiac hypertrophy (Satoh et al., 2006). Gβγ subunits of activated Gαi stimulate PI3K, which in turn stimulates the RacGEF Tiam1 leading to Rac1 activation and phosphorylation of the downstream extracellular-regulated kinase (ERK) (Vettel et al., 2012). The congruency of PMT-activated G proteins with those implicated in chronic heart failure, prompted us to study the toxin’s effect on the heart in vivo and in vitro. We show that PMT acts on heart tissue in vivo. To decipher the underlying mechanisms, different cardiac cell types, such as cardiomyocytes and cardiofibroblasts, were studied. We report that PMT induces cellular hypertrophy in cardiomyocytes and causes strong secretion of connective tissue growth factor (CTGF) in cardiofibroblasts. Pre© 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1320–1331

Fig. 1. PMT deamidates heterotrimeric G proteins in cardiac cells. PMT or the catalytically inactive mutant PMTC1165S was intraperitoneally injected in mice (500 ng kg−1 bw). After 72 h, the heart was prepared and tissue samples were tested for PMT-induced deamidation by immuno blotting using the deamidation-specific GαQE antibody. Equal loading was tested by Gαq/11 antibody. Each lane (#1–5) represents treatment of an individual mouse (n = 5).

viously, PMT was utilized to stimulate G-protein signalling in cardiomyocytes (Sabri et al., 2002; Obreztchikova et al., 2006), but at that time the molecular mechanism and the substrate specificity of PMT were not known and PMT was used as a specific activator of Gαq signalling. Accordingly, all cellular effects of PMT-like cellular hypertrophy were attributed to Gαq activity. Here, we investigated the involved signal transduction pathways for PMTinduced effects on the basis of the recently established molecular mechanism and its substrate specificity. Results Effect of PMT in vivo It is known that PMT activates heterotrimeric G proteins of the Gαq/11, Gα12/13 and Gαi family by deamidation of a specific glutamine residue of the respective α-subunits (Orth et al., 2013). These G protein families are clearly involved in pathological signal transduction resulting in cardiac hypertrophy (Tilley, 2011). Therefore, we analysed whether heart tissue is susceptible to the toxin action. Mice were intraperitoneally injected with PMT or a catalytically inactive mutant PMTC1165S. To verify the direct action of the toxin, we utilized a recently described antibody, which specifically detects the toxin-induced deamidation of heterotrimeric G proteins in the switch II region (Kamitani et al., 2011). As shown in Fig. 1, deamidated G proteins were detected in heart tissue samples prepared 72 h after PMT injection, whereas the catalytically inactive PMTC1165S did not induce deamidation. Next, we studied the PMT effects on the heart in vivo. PMT was intraperitoneally injected into mice. The PMT treatment was repeated on day 3 and hearts were analysed on day 8. This treatment was compared with repeated administration of the α1-adrenoceptor agonist phenylephrine (PE) and the combined treatment of PE with PMT. PE was used at a relatively low dose [15 mg kg−1 body weight (bw)] to allow for the detection of synergistic action of PMT treatment with α1-adrenoceptor stimulation. Analysing the ventricle weight revealed an increase in the

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Fig. 2. PMT effects on the heart in vivo. Mice were intraperitoneally injected with buffer control (con), PE (15 mg kg−1 bw) and/or PMT (100 ng kg−1 bw) as indicated. Treatment was repeated on day 4, 5 and 6 for PE and day 1 and 4 for PMT. After 1 week, hearts were prepared. A. Ventricle weight/bw ratio. B. Ventricle weight/tibia length ratio. C. Quantification of Sirius red staining presented as arbitrary units (AU). D. mRNA expression of CTGF in heart tissue calculated to untreated control. E. Representative transversal heart section (left side, scale bar 1 mm) and Sirius red staining (right side, scale bar 50 μm) of histological sections. Result are given as mean ± standard error of the mean (con, PMT + PE, PMT: n = 5; PE: n = 4).

ratio of ventricle/bw or ventricle/tibia length in the group of combined administration of PMT and PE, while treatment of PMT or PE alone did not increase normalized ventricle weight (Fig. 2A and B). Furthermore, we analysed histological sections of prepared hearts. A Sirius red staining to study fibrosis showed increased fibrosis in hearts after combined treatment with PMT and PE (Fig. 2C and E). Determination of the mRNA expression of CTGF revealed increased expression induced by PE and by the combined PE/PMT treatment (Fig. 2D). However, PMT or PE treatment alone did not increase the cell area of cardiomyocytes determined by WGA (wheatgerm agglutinin)-staining (data not shown). PMT acts on cardiomyocytes To decipher the PMT effect on the different cell types present in the heart, we utilized different cell culture models

of primary cells. Initially, we focused on the toxin effect on neonatal rat cardiomyocytes (NRCM) in vitro. The α1-adrenoceptor agonist PE induced cellular hypertrophy as measured by increasing protein synthesis. Also PMT, which activates the heterotrimeric G proteins downstream of the α1-adrenoceptor, but not the inactive mutant PMTC1165S stimulated proteins synthesis (Fig. 3A). Moreover, typical markers of cardiomyocyte hypertrophy were observed after PMT treatment (Fig. 3B). For example, mRNA of the atrial and brain natriuretic peptide (Nppa, Nppb) were elevated (Murakami et al., 2002; Ogawa and de Bold, 2014) and the mRNA of the K+-channel Kcnh2 was down-regulated (Hu et al., 2011; Zhao et al., 2012). NRCM were directly targeted by PMT as demonstrated by detection of deamidation of heterotrimeric G proteins (Fig. 4A). The MAPK pathway is one of the major importance for cardiac hypertrophy. Especially, the ERK1/2 has been extensively studied, revealing an ambiguous network of © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1320–1331

Action of G protein-activating PMT on the heart 1323 Fig. 3. PMT induces cellular hypertrophy of NRCM. A. NRCM were treated with PE (50 μM), PMT or inactive mutant PMTC1165S (each 10 nM) for 24 h. Afterwards protein synthesis was measured by (14C) isoleucine incorporation. Induction of protein synthesis is given relative to unstimulated control. B. NRCM were treated without or with PMT (10 nM, 24 h). Then mRNA was extracted and qPCR was performed for Nppa (natriuretic peptide A), Nppb (natriuretic peptide B) and Kcnh2 [potassium voltage-gated channel, subfamily H (ERG1), member 2] and normalized against HPRT. Result are given as mean ± standard error of the mean (n = 3).

downstream effects. It was shown that ERK1/2 activation contributes to cardiac hypertrophy but also protects the heart from cell death and ischaemic injury (Lorenz et al., 2009; Vidal et al., 2012). It is still discussed whether these distinct downstream effects are due to a specific phosphorylation status of ERK1/2. Beside the classical phosphorylation sites of ERK1 at Thr203 and Tyr205 (Thr183 and Tyr185 of Erk2), an autophosphorylation of ERK2 at Thr188 (Thr208 in ERK1; mouse/rat) has been suggested to specifically induce cardiac hypertrophy without affecting other cellular functions of ERK1/2 (Ruppert et al., 2013). We observed that PMT not only stimulated phosphorylation of ERK1 at Thr203 and Tyr205 (Thr183 and Tyr185 of Erk2) (Fig. 4A) but also induced autophosphorylation of Thr188 of ERK2 (Fig. 4B). These effects depended on the active deamidating toxin because the inactive mutant (PMTC1165S) had no effect on the phosphorylation status of ERK1/2. As expected, the α1adrenoceptor agonist PE also stimulated autophosphorylation of Thr188 (Fig. 4B). Additionally, we observed synergistic action of submaximal concentrations of PMT in combination with PE on ERK1/2 activity (Supporting Information Fig. S1). Small GTPases of the Rho family are also involved in hypertrophic signalling (Brown et al., 2006; Loirand et al., 2013). As shown in Fig. 4C, PMT and the GPCR agonist ET-1 stimulated RhoA activity in NRCM as measured by an effector pull-down assay. After 4 h of incubation, PMT, but not the inactive mutant PMTC1165S, led to an efficient effector-binding of RhoA. Moreover, the small GTPase Rac, which was recently implicated in α1-adrenoceptormediated cardiomyocyte hypertrophy (Vettel et al., 2012), was stimulated by PMT and ET-1 but not by PMTC1165S (Fig. 4D). ET-1 acts as a GPCR agonist. Therefore, downstream signalling events are already detectable after an incubation time of 1.5 min. The bacterial protein toxin PMT has to reach the cytosol before heterotrimeric G proteins can be activated to evoke downstream signalling. Subsequently, we used different approaches to investigate the contribution of the various signalling pathways © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1320–1331

in PMT-stimulated hypertrophy of NRCM. As shown in Fig. 4E, overexpression of ERK2T188A, which is a dominant-negative mutant for ERKThr188-downstream signalling, in NRCM attenuated the hypertrophic response towards PMT. Additionally, treatment of NRCM with the MEK inhibitor U0126 decreased PMT-induced hypertrophy, indicating that ERK1/2 activity is required (Fig. 4F). Furthermore, NSC23766, an inhibitor of the Rac-GEF Tiam1, diminished PMT-stimulated isoleucine incorporation into newly synthesized proteins (Fig. 4G). In addition, we show that PMT-induced ERK1/2 activation is not affected by NSC23766. However, pretreatment of cells with the MEK inhibitor U0126 (DeSilva et al., 1998) abolished ERK1/2 activation. These results exclude any unspecific effects of the Rac-GEF inhibitor on toxinstimulated ERK activity (Supporting Information Fig. S2). Cardiofibroblasts and PMT Besides myocytes, cardiofibroblasts are another important cardiac cell entity. There is growing evidence that cardiofibroblasts are substantially involved in pathological heart conditions (Banerjee et al., 2007; Fujiu and Nagai, 2014). Therefore, we tested neonatal rat cardiofibroblasts (NRCF) for their susceptibility towards PMT. A deamidation of heterotrimeric G proteins was detectable in the immunoblot analysis of PMT-treated NRCF but not after treatment with PMTC1165S (Fig. 5A). Activation of cardiofibroblasts can result in the expression and release of CTGF. Recently, it was demonstrated that in NRCF release and expression of CTGF are regulated by the small GTPases RhoA and Rac1 (Adam et al., 2010; Lavall et al., 2014). We observed that PMT strongly stimulated RhoA and Rac1 activity in NRCF after 1 h and 4 h (Fig. 5B and C). In contrast, PMTC1165S did not show any activity towards RhoA and Rac1. Moreover, the GPCR agonist angiotensin-II (AT-II) had only minor effects on RhoA and Rac1 activities. To investigate the PMT effect on CTGF in more detail, we determined the secretion and expression of CTGF by NRCF. As shown in Fig. 5D and E, PMT

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Fig. 4. PMT stimulates the MAPK pathway and the small GTPases RhoA and Rac1. A. PMT stimulates ERK1/2 phosphorylation at Thr202/Tyr204 (Thr185 and Tyr187 of Erk2). NRCM were treated without or with PMT, inactive mutant PMTC1165S (each 10 nM, 2 h) or calf serum (10%, 3 min). RIPA lysates were prepared and ERK1/2 phosphorylation was determined by phospho-specific ERK1/2 antibody. Moreover, deamidation by PMT was detected by GαQE antibody. Equal loading was verified by tubulin antibody. B. PMT stimulates ERK2 autophosphorylation at Thr188 (Thr208 in ERK1). NRCM were treated without (con) or with PE (4 mM, 6 h), PMT or inactive mutant PMTC1165S (each 10 nM, 6 h) (n = 10). Shown are representative immunoblots. Quantification was calculated using MultiGauge and demonstrated as fold induction normalized to untreated cells. C–D. PMT activates the small GTPases RhoA (C) and Rac1 (D) in NRCM. NRCM were treated without (con) or with endothelin (ET-1, 100 nM, 1.5 min), PMT or inactive mutant PMTC1165S (each 10 nM, for the indicated periods of time). Then an effector pull-down assay for RhoA and Rac1 was performed. Precipitated/activated G proteins and total content of G proteins was determined by immunoblot against RhoA and Rac1. E. ERK (Thr188) phosphorylation contributes to PMT-induced NRCM hypertrophy. NRCMs that were either transduced with a control virus encoding for green fluorescent protein (GFP) or a mutant of ERK2 that is dominant negative for ERKThr188 signalling (ERK2T188A, T188A) were stimulated with PMT (10 nM, 24 h) (n = 14). Protein synthesis was measured by (3H) isoleucine incorporation. Induction of protein synthesis is given relative to unstimulated control. F–G. PMT-induced protein synthesis is inhibited by blockade of MAPK signalling (F) or blockade of Rac1 (G). NRCM were serum starved and treated without or with PMT (10 nM, 24 h) and inhibitors as indicated (U0126, 5 μM; NSC, NSC23766, 100 μM). Induction of protein synthesis is given relative to unstimulated control. Result are given as mean ± standard error of the mean (n = 3).

© 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1324–1331

Action of G protein-activating PMT on the heart 1325 Fig. 5. PMT affects neonatal rat cardiofibroblasts to secrete CTGF. A. Susceptibility of NRCF for PMT was verified by detecting deamidated heterotrimeric G proteins. NRCF were treated without (con) or with PMT or PMTC1165S (each 1 nM, over night). Immunoblot analysis utilizing GαQE antibody showed PMT-induced deamidation. Equal loading was tested by anti-tubulin antibody. B–C. PMT activates the small GTPases RhoA (B) and Rac1 (C) in NRCF. NRCF were treated without (con) or with AT-II (100 nM, 0.5 min), PMT or inactive mutant PMTC1165S (each 10 nM, for the indicated periods of time). Then an effector pull-down assay for RhoA and Rac1 was performed. Precipitated/activated G proteins and total content of G proteins was determined by immunoblot against RhoA and Rac1. D–F. Expression and secretion of CTGF by PMT in NRCF. NRCF were treated without (con) or with AT-II (100 nM), PMT or inactive mutant PMTC1165S (each 10 nM) for 24 h. CTGF secretion was measured by immunoblot analysis of CTGF in the culture supernatant or expression was determined by determining cellular CTGF. Equal loading was tested by β-actin detection. A representative immunoblot of six performed is shown. (E, secretion; F, expression) Quantification of data shown in D. Values are normalized to β-actin and presented as mean ± standard error of the mean (n = 6), *P < 0.05, **P < 0.01, ***P < 0.001 versus con.

strongly increased CTGF secretion. Notably, AT-II had only minor effects on CTGF secretion as compared with PMT. Besides increase in CTGF secretion, CTGF expression was also stimulated by PMT (Fig. 5D and F). The effects of PMT on secretion and expression were not further stimulated by additional AT-II treatment. Surprisingly, we observed a weak inhibitory effect of the inactive PMT mutant on the basal level of CTGF secretion, expression and AT-II-induced expression. As a control we utilized a heat-inactivated PMT, which was not able to stimulate CTGF secretion (Supporting Information Fig. S3). Discussion Neurohumoral mechanisms are important regulators of cardiac hypertrophy. Messengers such as catecholamines, AT-II or endothelin act on cardiomyocytes and cardiofibroblasts by binding to their respective GPCRs. These GPCRs bind to distinct families of heterotrimeric G proteins to stimulate various signal transduction pathways. From the four families of heterotrimeric G proteins, Gαs and Gαq/11 are known to induce positive inotropic and positive chronotropic effects. Via inhibition of AC and stimulation of Gβγ-dependent effectors, Gαi counteracts Gαs signalling, resulting in negative inotropic and chronotropic effects. However, all families of heterotrimeric G proteins, including Gα12/13 have been implicated in the development of cardiac hypertrophy. This was clearly established over the last decade by application of diverse genetic or pharmacological approaches (Adams et al., 1998; Wettschureck et al., 2001; Tilley, 2011; Takefuji et al., 2012; Vettel et al., 2012; Reuter et al., 2013). PMT is the causative agent of the atrophic rhinitis in animals. Atrophic rhinitis is characterized by the degradation of bone structures, especially nose turbinate bones © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1320–1331

1326 M. Weise et al. (Wilson and Ho, 2013). Moreover, in various in vivo studies, PMT application led to liver toxicity, pathological changes of bladder epithelium and/or pneumonia-like symptoms (Cheville et al., 1988; Cheville and Rimler, 1989; Chrisp and Foged, 1991; Ward et al., 1998). Because these studies were hampered by the inability to determine the direct toxin actions in cells or organs, it was not clear whether the observed pathological changes were secondary results. Ackermann et al. studied the distribution of iodinated PMT in rats (Ackermann et al., 1995). Surprisingly, higher concentrations of PMT were found in heart tissue as compared with bone (femur and humerus), which is the typical target site of PMT. In the present study, we used a deamidation-specific antibody, which directly detects the PMT-induced deamidation (Kamitani et al., 2011) to confirm the action of the toxin. Thereby, we detected the deamidation of heterotrimeric G proteins after intraperitoneal administration of PMT in the heart, an organ not yet connected to PMT pathogenesis. Studying the pathophysiological effects of PMT on the heart in vivo, we found an increased normalized ventricle mass and fibrosis when the toxin was co-administrated with a low dose regimen of the GPCR agonist PE. However, no cellular cardiomyocyte hypertrophy was detectable. Moreover, sole treatment with PMT or PE exhibited no effect under the experimental settings used, which were limited by 1-week observation time and a toxin dose, showing no general toxicity. The lack of any effect of PMT alone in the used in vivo model can be due to different reasons. For example, growing evidence suggests that GPCRs could also induce signal transduction independent of heterotrimeric G proteins (Sun et al., 2007). It would be interesting to study whether additional GPCR-induced signalling, besides the G protein-dependent signalling, enhances the development of cardiomyocyte hypertrophy in vivo. Because PMT acts on heterotrimeric G proteins without any GPCR interaction (Orth et al., 2007), it is assumed that PMT does not induce G protein-independent signalling. Prior to the development of cardiac hypertrophy, the expression of profibrotic genes increases (Kim et al., 2007). Therefore, the observed fibrosis is an indication for the early state of pathophysiological remodelling. Although PMT-induced fibrosis was detected, hypertrophy was not observed. The incubation time with the locally reached PMT concentration in heart muscle might, however, be too short to induce cardiomyocyte hypertrophy. The effect of combined treatment of PMT and PE on ventricle mass is in line with the known synergistic action of PMT with GPCR stimulation. This synergism was formerly demonstrated for GPCR agonists such as bombesin, thrombin and lysophosphatidic acid, which stimulate heterotrimeric G proteins of the Gαq/11 and Gαi family (Baldwin et al., 2003; Orth et al., 2007; 2008). Additionally,

we demonstrate a synergistic effect of PMT and PE on ERK1/2 phosphorylation in vitro in NRCM (Supporting Information Fig. S1). Taken the low dose of PE used in our in vivo study into account, it might be speculated that an enhanced activation of the sympathetic nervous system during P. multocida infection will also result in a synergistic stimulation of hypertrophy inducing signalling cascades by PMT-catalyzed deamidation in heart tissue. Our in vitro cell culture model of NRCM and NRCF clearly show that these major constituent cells of the heart muscle are susceptible for PMT as demonstrated by deamidation of heterotrimeric G proteins. Because of the covalent modification of the α-subunit, G protein activation by PMT is a long-lasting effect. Recently, deamidated/ activated G proteins were detectable up to 1 week after a single treatment with PMT (Orth et al., 2013). Therefore, the functional outcome of PMT treatment should be comparable with continuous stimulation with GPCR agonists probably without desensitization effects of GPCRs. Actually, PMT treatment of NRCM provoked hypertrophy of NRCM detected by increase in protein synthesis and activation of a specific gene transcription programme. Similar results were reported recently (Sabri et al., 2002; Obreztchikova et al., 2006). Three of four heterotrimeric G protein families are activated by PMT, resulting in stimulation of a multiplicity of signal transduction pathways. One well-established model of GPCR-induced hypertrophic signalling is the transactivation of the MAPK pathway (Blaukat et al., 2000). Essential signal factors of the MAPK pathway are ERK1/2, which were causally associated with hypertrophic signalling (Bueno et al., 2000). Moreover, the contribution of distinct heterotrimeric G protein families in GPCRinduced ERK1/2 activation in cardiomyocytes has been reported (Snabaitis et al., 2005), demonstrating that Gαq/11, Gαi and Gα12/13 are capable of stimulating this signal transduction pathway. In congruence to these studies, we show that PMT stimulates the phosphorylation of ERK1/2. Recently, a new phosphorylation site at Thr188 of ERK2 (Thr208 in ERK1) was identified, which is suggested to trigger ERK1/2-mediated hypertrophic signalling (Lorenz et al., 2009). We demonstrate also that PMT induces this autophosphorylation of ERK. Whereas the canonical phosphorylation of ERK2 at Thr183/Tyr185 depends on the trans-activation of the MAPK pathway via α-subunits of heterotrimeric G proteins, autophosphorylation of Thr188 depends on βγ-subunits, which act as a scaffold. This perfectly fits to the action of PMT, which directly activates Gα thereby releasing the βγ-subunits. Subsequently, both factors (α and βγ subunits) induce their specific signalling cascade resulting in NRCM in the Gα-MAPKMEK-dependent phosphorylation of ERK1/2 and Gβγdependent autophosphorylation of Thr188 of ERK2. The essential role of PMT-induced MAPK/ERK1/2 activation © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1320–1331

Action of G protein-activating PMT on the heart 1327 was verified by inhibition of this pathway resulting in hampered PMT-dependent hypertrophic effects. The finding that the effect of the T188A mutant is small indicates the involvement of additional PMT-induced pathways. Small G proteins of the Rho family are recognized to be involved in hypertrophic signalling (Brown et al., 2006). Here, we show the activation of the small GTPase RhoA and Rac1 in NRCM by PMT. PMT-induced RhoA stimulation is a downstream event of the Gαq/11 and Gα12/13 families, which couple to distinct RhoGEF proteins to stimulate RhoA activity (Aittaleb et al., 2010). Although activation of Rac1 via Gαi-released βγ-subunits was reported (Gonzalez et al., 2006), stimulation of Rac1 by PMT was not recognized so far. It is supposed that Gβγ stimulate the RacGEF Tiam1 in a PI3K-dependent manner. Moreover, this pathway has been implicated in the hypertrophic response in NRCM towards α1-adrenoceptor signalling (Vettel et al., 2012) and Rac1 was associated with ERK activation in NRCM (Clerk et al., 2001; Vettel et al., 2012). As PMT also targets the Gαi family, we assume the same signalling pathway leading to Rac1 activation and supporting hypertrophic response via released Gβγ from PMT-activated Gαi. This hypothesis is supported by the finding that blockade of the RacGEF Tiam1 by NSC23766 resulted in inhibition of cellular hypertrophy. Cardiac fibroblasts contribute to the pathophysiological remodelling of the heart at different levels, that is secretion of growth factors and/or of extracellular matrix components appear to play crucial roles (Fujiu and Nagai, 2014). One factor released by cardiofibroblasts is CTGF, which is expressed upon GPCR activation by for example AT-II, ET-1 or PE (Daniels et al., 2009; Gu et al., 2012). Interestingly, PMT also deamidated heterotrimeric G proteins in NRCF. Moreover, like in NRCM, PMT stimulated the small GTPases RhoA and Rac1 in NRCF. Recently, these GTPases have been implicated in the regulation of expression and secretion of CTGF in NRCF (Adam et al., 2010; Lavall et al., 2014). Whereas RhoA activity was assigned to control of the secretion of CTGF, Rac1 activity was linked to regulation of CTGF expression (Adam et al., 2010). In congruence to these studies, we measured an increase in CTGF expression and secretion by PMT treatment of NRCM. CTGF released from cardiomyocytes and cardiofibroblasts may induce fibrosis and/or cardiomyocyte hypertrophy (Hayata et al., 2008; Panek et al., 2009; Koshman et al., 2013). The induction of fibrosis is in line with the observed fibrosis of the heart after combined treatment with PMT and the GPCR agonist PE. Actually, the role of pathological heart remodelling in the pathogenesis of infection with toxigenic P. multocida strains is not yet established. This might be due to limitations to detect the toxin effect directly in specific tissues, which was only recently overcome by the development of the deamidation specific Gα antibody. However, to provide © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1320–1331

a casual link between P. multocida infection and pathological heart remodelling, further studies are necessary. In summary, comparable with various neurohumoral hormones and transmitters, which target cardiac GPCRs, PMT stimulates heterotrimeric G protein-dependent signalling pathways. The persistent activation of these Gα- and Gβγ-dependent pathways by PMT-catalyzed deamidation of the G proteins induces major pathophysiological remodelling. As PMT-induced actions are independent of GPCRs, receptor-dependent desensitization processes are not involved, therefore PMT might be an extremely useful tool to study long-term activation of heterotrimeric G protein activation without affecting GPCR regulation.

Experimental procedures Cultivation of NRCM and NRCF The NRCM were isolated from 1- to 3-day-old Wistar rats as described previously (Will et al., 2010) with modifications. Briefly, hearts were minced and subjected to serial digestion in a mixture of 0.5 mg ml−1 collagenase type II (Cell Systems) and 0.6 mg ml−1 pancreatin (Sigma-Aldrich) to release single cells. The obtained cell suspension was then placed on top of a Percoll™-gradient (GE Healthcare) to separate cardiomyocytes from fibroblast (NRCF). The cardiomyocyte fraction were plated on poly-Llysine-coated well plates with Dulbecco’s modified Eagle medium (DMEM) containing 10% foetal calf serum (FCS), penicillin/ streptomycin and 97.69 nM 5-bromo-2′-deoxyuridine to avoid growth of remaining non-cardiomyocytes. The NRCMs were cultivated for at least 3 days after isolation. The NRCF were plated on 10 cm culture dishes and cultured in DMEM GlutaMaxTM with 4.5 g l−1 glucose, 10% FCS, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 1% non-essential amino acids until confluency was reached. NRCF were then splitted on 6-well plates and cultured for another 3–4 days before use (Passage 1, P1). Purity of cell preparations and cellular heterogeneity was analysed by histology and qPCR analysis of cell type specific marker genes.

Animal procedures and ethics statement C57BL/6 mice were used for in vivo experiments. Mice were intraperitoneally injected with indicated buffers and euthanized after indicated time periods. All animal experiments were performed in compliance with the German animal protection law (TierSchG). The animals were housed and handled in accordance with good animal practice as defined by FELASA (http://www.felasa.eu/) and the national animal welfare body GV-SOLAS (http://www.gv-solas.de/). The animal welfare committees of the University of Freiburg as well as the local authorities (Regierungspräsidium Freiburg) approved all animal experiments.

Histology Hearts were fixed with 4% paraformaldehyde in phosphatebuffered saline (PBS), embedded in paraffin, cut into 3 μm slices

1328 M. Weise et al. and stained with haematoxylin–eosin and Sirius red as previously described (Gilsbach et al., 2010; Schneider et al., 2011). Histological analysis was performed using AxioVision Rel.4.5 software (Carl Zeiss AG, Heidenheim, Germany) and Photoshop CS5 (Adobe, Basel, Switzerland).

Plasmids and cloning Flag-tagged murine Erk2 (T188T) and Flag-Erk2T188A (T188A) were subcloned into the Gateway vector pAd/CMV/V5-Dest vector using the transfer vector pDONR211 (Invitrogen) or the pAdTrack-CMV vector (Clontech). The identity of all constructs was confirmed by DNA sequencing.

RhoGTPase activation assay Serum-starved NRCM or NRCF (105 cells per cm2) were stimulated with agonist as indicated or treated with PMT or its inactive mutant for the indicated periods of time. The cells were lysed in ice-cold GST-fish buffer as described before (Clerk et al., 2001) and GTP-bound RhoGTPases were precipitated with either the Rho-binding domain of Rhotekin or the Rac-binding domain of PAK1 coupled to glutathione sepharose. The amounts of activated and total GTPases were then determined by immunoblot analysis.

Measurement of intracellular and secreted CTGF P1 NRCF were cultured in 6-well plates, serum starved for 24 h and stimulated with either 100 nM Ang II, 10 nM PMT or PMTC1165S for 24 h. Conditioned media were taken for analysis of secreted CTGF and cells were lysed in GST-fish buffer. The amount of secreted and intracellular CTGF was analysed by immunoblot.

Isoleucine incorporation The leucine incorporation was performed as described previously (Vettel et al., 2012) with the following modifications: The NRCMs (0.96 × 105 cm−2) were treated with serum-free DMEM, 0.2 μCi ml−1 L-[14C]-Isoleucine or -[3H]-Isoleucine (Perkin Elmer) and 10 nM PMT for 24 h at 37°C. For protein precipitation, a solution of 10% trichloroacetic acid (TCA) was used. After washing with PBS-buffer, a solution of 1 M NaOH with 0.01% sodium dodecyl sulfate (SDS) was added for 2 h at 37°C to dissolve the proteins. Five hundred microlitres of each sample was mixed with 3 ml Ultima Gold™ (Perkin Elmer, Cologne, Germany) and analysed in a Tri-Carb 2900 TR Liquid Scintillation Analyzer.

mRNA isolation/quantitative PCR NRCM (0.78 × 105 cm−2) were serum starved for 24 h and treated as indicated. The preparation of the mRNA was performed with the RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was prepared with the QuantiTect reverse transcription kit (Qiagen). All kits were used following the manufacturer’s manual. Quantitative PCR was performed using the GoTaq qPCR Master Mix (Promega, Mannheim, Germany). The expression level of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was

used for an internal control and fold changes were calculated using the ΔΔCt method. Values are shown as 2−ΔΔCt. The following primer pairs were used for the analysis: HPRT: forwardgaccggttctgtcatgtcg, reverse-acctggttcatcatcactaatcac; Nppa: forward-cacagatctgatggatttcaaga, reverse-cctcatcttctaccggcatc; Nppb: forward-gtcagtcgcttgggctgt, reverse-ccagagctggggaaaga ag; Kcnh2: forward-gatcgccttctaccggaaa, reverse-cccatcctcattc ttcacg; All primers have a rat origin and were made by Apara (Denzlingen, Germany).

RIPA lysates Confluent 10 cm dishes with 1–3-day-old NRCM were treated as indicated for 24 h at 37°C. Cell lysates were prepared using RIPA buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% Nonidet P-40, 0.5% (w/v) deoxycholate and 0.1% (w/v) SDS] containing complete protease inhibitor (Roche, Mannheim, Germany). The harvested cells with RIPA buffer were lysed during overhead rotation for 1 h at 4°C.

Immunoblot analysis The proteins were subjected to SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membrane. pERK1/2 (Thr202/Tyr204 of ERK1; Thr185 and Tyr187 of Erk2) was from Cell Signaling Technology. Anti-RhoA (26C4, 1:200) and anti-CTGF (1:200) were from Santa Cruz Biotechnology. Anti-Rac1 (610650, 1:1000) was from BD Transduction. Antitubulin and anti-β-actin antibodies were purchased from SigmaAldrich. pERK (Thr188) specific antibody was used as described before (Lorenz et al., 2009). Deamidation specific antibody antiGαq Q209E was purified from a 3G3 hybridoma cell line, which was a grateful gift of Dr. Y. Horiguchi (Osaka University, Japan) (Kamitani et al., 2011). The GαQE antibody detects deamidated α-subunits of heterotrimeric G proteins, which have a molecular size between 39 and 44 kDa. Therefore multiple bands can appear in immunoblot analysis. Chemoluminescence reaction [100 mM Tris-HCl (pH 8.0), 1 mM luminol (Sigma-Aldrich, Taufkichen, Germany), 0.2 mM p-coumaric acid, and 3 mM H2O2] was used to detect binding of the second horseradish peroxidase-coupled antibody with the imaging system LAS-3000 (Fujifilm, Dusseldorf, Germany). Quantifications of immunoblots were performed using MultiGauge V 3.0 (Fujifilm, Düsseldorf, Germany).

Statistics Results are presented as means ± standard error. Significance was assessed by paired Student’s t-test. P-values < 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, n.s. non-significant). Multiple group comparisons were analysed by analysis of variance followed by Student’s t-test.

Acknowledgement We thank Dr. Y. Horiguchi (Research Institute for Microbial Diseases, Osaka University, Japan) for deamidation specific antibody, Petra Bartholomé and Silke Fieber for excellent technical assistance. The study was financially supported by the Deutsche Forschungsgemeinschaft (DFG, OR218 and SFB746). Conflict of interest statement: No conflicts declared. © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1320–1331

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Action of G protein-activating PMT on the heart 1331 Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Synergistic effect of PMT and PE on ERK1/2 activation. NRCM were serum starved and treated without (con), with PMT (100 pM), PE (100 nM) or PMT and PE for 3 h. RIPA lysates were prepared and ERK1/2 phosphorylation at Thr202/Tyr204 (Thr185 and Tyr187 of Erk2) was determined by phospho-specific ERK1/2 antibody. Equal loading was verified by tubulin antibody. Shown are representative immunoblots. Quantification was calculated using MultiGauge and demonstrated as percentage of maximum effect. Arrow indicates the calculated additive effect of PMT and PE-induced ERK phosphorylation. Fig. S2. PMT-induced ERK1/2 activation is inhibited by U0126 but not NSC23766. NRCM (A) or NRCF (B) were serum starved

© 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 1320–1331

and treated without or with PMT (10 nM, 24 h) and inhibitors as indicated (U0126, 10 μM; NSC, NSC23766, 100 μM). RIPA lysates were prepared and ERK1/2 phosphorylation at Thr202/ Tyr204 (Thr185 and Tyr187 of Erk2) was determined by phosphospecific ERK1/2 antibody. Equal loading was verified by tubulin antibody. Shown are representative immunoblots. Quantification was calculated using MultiGauge and demonstrated as percentage of maximum effect. Fig. S3. Heat-inactivated PMT does not effect CTGF secretion. NRCF were treated without (con), with PMT or heat-inactivated (95°C, 10 min) PMT (each 10 nM, over night). CTGF secretion was measured by immunoblot analysis of CTGF in the culture supernatant. Equal loading was tested by tubulin detection. A representative immunoblot is shown.

A systemic Pasteurella multocida toxin aggravates cardiac hypertrophy and fibrosis in mice.

Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gαq/11 , Gα12/13 and Gαi family without interaction with G p...
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