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(Circulation. 2001;103:1453.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Cardiac Overexpression of a G_q Inhibitor Blocks Induction of Extracellular Signal–Regulated Kinase and c-Jun NH₂-Terminal Kinase Activity in In Vivo Pressure Overload

Giovanni Esposito, MD; Sathyamangla V. Naga Prasad, PhD; Antonio Rapacciuolo, MD; Lan Mao, MD; Walter J. Koch, PhD; Howard A. Rockman, MD

From the Department of Medicine and Cell Biology (G.E., S.V.N.P., A.R., L.M., H.A.R.) and the Department of Surgery (W.J.K.), Duke University Medical Center, Durham, NC.

Correspondence to Howard A. Rockman, MD, Department of Medicine and Cell Biology, Duke University Medical Center, DUMC 3104, Durham, NC, 27710. E-mail h.rockman{at}duke.edu

	Abstract

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Background—Understanding the cellular signals that initiate cardiac hypertrophy is of critical importance in identifying the pathways that mediate heart failure. The family of mitogen-activated protein kinases (MAPKs), including the extracellular signal–regulated kinases (ERKs), c-Jun NH₂-terminal kinase (JNK), and p38 MAPKs, may play specific roles in myocardial growth and function.

Methods and Results—To determine the mechanism of activation of MAPK pathways during the development of cardiac hypertrophy, we evaluated the induction of MAPK activity after aortic constriction in wild-type and in 2 types of cardiac gene-targeted mice: one overexpressing a carboxyl-terminal peptide of G_q that inhibits G_q-mediated signaling (TG GqI mouse) and another overexpressing a carboxyl-terminal peptide of ß-adrenergic receptor kinase-1 that inhibits Gß signaling (TG ßARKct mouse). Wild-type mice with pressure overload showed an acute induction of JNK, followed by the induction of p38/p38ß at 3 days and ERK at 7 days. Both JNK and p38 activity remained elevated at 7 days after banding. In TG GqI mice, hypertrophy was significantly attenuated, and induction of ERK and JNK activity was abolished, whereas the induction of p38 and p38ß was robust, but delayed. By contrast, all 3 MAPK pathways were activated by aortic constriction in the TG ßARKct hearts, suggesting a role for G_q, but not Gß.

Conclusions—Taken together, these data show that the induction of ERK and JNK activity in in vivo pressure-overload hypertrophy is mediated through the stimulation of G_q-coupled receptors and that non–G_q-mediated pathways are recruited to activate p38 and p38ß.

Key Words: receptors • kinases • genes • hypertrophy

	Introduction

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Although cardiac hypertrophy is thought to be an adaptive response of the heart to a variety of stimuli, it is associated with an increased mortality and increased incidence of heart failure in epidemiological studies.^{1 2} Understanding the cellular signals that initiate the hypertrophic response is of critical importance in identifying pathways that mediate this maladaptive deterioration. In vitro studies have suggested a pivotal role of G_q-coupled receptor signaling in promoting cardiomyocyte hypertrophy.^{3 4} Furthermore, cardiac overexpression of G_q-coupled receptors or G_q itself in transgenic mice leads to myocardial hypertrophy,^{5 6} apoptosis,^{6 7} and heart failure.^{6 7}

G protein–coupled receptors (GPCRs) are able to activate mitogen-activated protein kinases (MAPKs) and, under certain conditions, will lead to a mitogenic response.^{8 9} The MAPK superfamily includes 3 major pathways: the extracellular signal–regulated kinase (ERK)1/2 pathway and 2 stress-activated protein kinase pathways, c-Jun NH₂-terminal kinase (JNK) and p38 MAPK.¹⁰ Activation of MAPK pathways by growth factors, cytokines, and cell stress selectively mediates a variety of cellular responses ranging from cell growth and differentiation to apoptosis. Studies by Xia et al¹¹ in rat PC-12 pheochromocytoma cells have demonstrated that the dynamic balance between growth factor–activated ERK and stress-activated JNK-p38 pathways may determine whether a cell survives or undergoes apoptosis.

In cell culture studies, it has recently been reported that although signaling through different G proteins (G_q, G_i, and G_s) can selectively activate MAPKs and promote cell differentiation, stimulation of only G_q-coupled receptors can equally activate all 3 major MAPK pathways.¹² Interestingly, in cultured rat neonatal cardiomyocytes, 2 isoforms of p38 kinase, p38 and p38ß, appear to have distinct functions¹³ : the p38ß isoform promotes a hypertrophic phenotype, and the p38 isoform tends to promote an apoptotic phenotype.¹³ Furthermore, the induction of ERK activity in cultured cardiac fibroblasts was found to be mediated by Gß signals, whereas in cultured cardiac myocytes, it was G_q-mediated.¹⁴

The important role of GPCRs and MAPK signaling in the development of cardiac hypertrophy has recently been shown by the generation of transgenic mice overexpressing G_q^{6 15} and the angiotensin type 1 receptor^{6 15} and by the adenovirus-mediated transfer of a dominant inhibitory mutant of an upstream activator of JNK.¹⁶ Furthermore, overexpression of a constitutively active mutant of the transforming growth factor-ß–activated kinase, a member of the MAPK kinase kinase family, leads to cardiac hypertrophy and dysfunction in transgenic mice.¹⁷ However, whether G_q-coupled receptor stimulation is required for the induction of MAPK pathways in in vivo hypertrophy remains unclear. In this regard, we have recently demonstrated that inhibition of G_q-coupled receptor signaling in transgenic mice significantly reduces the hypertrophic response to in vivo pressure overload.¹⁸ Inhibition of G_q signaling was achieved through overexpression of a carboxyl-terminal peptide of G_q that inhibits the heterotrimeric G_q interaction with agonist-occupied receptors. Although that study showed a critical role for G_q signals in mediating the hypertrophic phenotype, it did not identify the downstream cellular pathways involved.

To determine whether the mechanism for the induction of MAPK in cardiac hypertrophy is dependent on G_q- and/or Gß-mediated pathways, we evaluated ERK1/2, p38, p38ß, and JNK activity during the development of in vivo pressure overload in wild-type mice, G_q inhibitor transgenic mice (TG GqI), and transgenic mice overexpressing a peptide inhibitor of Gß signaling (TG ßARKct, where ßARK indicates ß-adrenergic receptor kinase).

	Methods

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Experimental Animals
Adult wild-type and transgenic mice, inbred on a C57/B6 background, were studied at 2 to 3 months of age. The transgenic mice used in the present study were (1) mice with cardiac-specific overexpression of a carboxyl-terminal peptide of G_q that inhibits G_q-mediated signaling (TG GqI mice)¹⁸ and (2) transgenic mice with cardiac-targeted overexpression of a peptide inhibitor of Gß-mediated signaling (TG ßARKct mice).¹⁹ The ßARKct peptide is composed of the last 194 amino acid residues of ßARK1 and contains the domain responsible for Gß binding, a process required for ßARK1 activation.¹⁹ The cardiac phenotypes of the TG GqI and TG ßARKct mice were previously described for 2 independent lines,^{18 19} which have remained consistent over numerous generations. The animals in the present study were handled according to approved protocols and animal welfare regulations by the Institutional Review Board at Duke University Medical Center.

Transthoracic Echocardiography
Transthoracic 2D guided M-mode echocardiography was performed in anesthetized mice (2.5% Avertin, 14 µL/g IP) before and 7 days after the induction of pressure-overload hypertrophy, with use of an HDI 5000 echocardiograph (Advanced Technology Laboratories) as previously described.²⁰ Parameters measured are shown in the Table.

View this table: [in this window] [in a new window]   Table 1. Physiological Parameters in Wild-Type, TG GqI, and TG ßARKct Mice

In Vivo Pressure-Overload Hypertrophy
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (2.5 mg/kg), and transverse aortic constriction (TAC) was performed as previously described.²¹ Sham-operated mice underwent the same operation except for aortic constriction.

At 7 hours, 3 days, or 7 days after surgery, sham-operated and banded mice from either the wild-type, TG GqI, or TG ßARKct groups were anesthetized and intubated, and after bilateral vagotomy the trans-stenotic gradient was assessed by recording the simultaneous measurement of right and left carotid artery pressures. Hearts were then excised, and chambers were dissected free, weighed, and then frozen in liquid N₂ within 25 seconds from harvesting. Despite identical surgical techniques, a broad range in the ratio of left ventricular (LV) weight to body weight (LVW/BW) is found after TAC, which varies directly with the level of systolic pressure gradient.¹⁸ Therefore, to avoid experimental bias, hearts for the MAPK assay from all groups were chosen from animals with a trans-stenotic pressure gradient between 45 and 100 mm Hg, thereby eliminating the high and low extremes.

Immunoblotting
Immunodetection of myocardial levels of MAPKs was performed on cytosolic extracts from LVs after immunoprecipitation using polyclonal antibodies to total ERK2-p42/ERK1-p44, p38, p38ß, and JNK1-p46/JNK3 (Santa Cruz Biotechnology). The kinases were detected with secondary antibodies conjugated with horseradish peroxidase (ECL, Amersham Pharmacia Biotech).

MAPK Assays
MAPK assay was performed as previously described.¹⁸ Briefly, 2 mg of clarified LV extract in 2 mL of RIPA (150 mmol/L NaCl, 50 mmol/L Tris-Cl [pH 8.0], 5 mmol/L EDTA [pH 8.0], 1% v/v Nonidet P-40, 0.5% w/v deoxycholate, 10 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 100 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, and 2 µg/mL leopeptin) was immunoprecipitated at 4°C for 2 hours with the use of antibodies to ERK2-p42/ERK1-p44, p38, p38ß, and JNK1-p46/JNK3 (Santa Cruz Biotechnology) and protein A–agarose or protein G–agarose (Boehringer-Mannheim). The immunoprecipitates were pelleted and washed twice with 1 mL of RIPA and twice with 1 mL of kinase assay buffer. Samples were then resuspended in 40 µL of kinase buffer with 20 µmol/L ATP, [-³²P]ATP (20 µCi/mL), and myelin basic protein (0.25 mg/mL) or glutathione S-transferase (GST)-c-Jun (10 µg) and incubated at 30°C for 20 minutes.

Reactions were terminated by adding 40 µL of 2x Laemmli loading buffer, and 30 µL of each reaction was electrophoresed through a 15% polyacrylamide/Tris-glycine gel. Phosphorylated myelin basic protein and GST-c-Jun on dried gels were quantified with a PhosphorImager (Molecular Dynamics).

Statistical Analysis
Data are expressed as mean±SEM. One-way ANOVA was used to evaluate the echocardiographic measurements, heart weight, and kinase activity data before and after aortic constriction and among wild-type, TG GqI, and TG ßARKct mice. Post hoc testing was performed with a Scheffé test. For all analyses, a value of P<0.05 was considered significant.

	Results

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Physiological Response to In Vivo Pressure Overload in Wild-Type and TG GqI Mice
To evaluate the hypertrophic response after TAC, we measured LVW/BW in wild-type and TG GqI mice at 7 hours, 3 days, and 7 days after surgical pressure overload (Figure 1 and Table). Three days after TAC, wild-type mice developed a small but nonsignificant 16% increase in LVW/BW compared with sham-operated mice. Seven days after TAC, wild-type mice developed significant LV hypertrophy with a 54% increase LVW/BW compared with sham-operated mice. No hypertrophy was detected at 7 hours after TAC.

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of in vivo pressure overload on LV mass in wild-type and TG GqI mice. A, Significant increase in LVW/BW was observed only 7 days after TAC in wild-type mice (*P<0.0005 vs sham). A 16% increase in LVW/BW was observed 3 days after TAC (P=NS). B, Despite similar trans-stenotic systolic pressure gradient, there was significantly blunted hypertrophic response in TG GqI mice 7 days after TAC compared with wild-type mice. No increase in LVW/BW was observed 3 days after TAC. In all groups, no significant difference in trans-stenotic systolic pressure gradient was observed (*P<0.01 vs sham; P<0.01 vs 7-day TAC wild type).

In contrast, 3 days after TAC, TG GqI mice developed a minimal 5% increase in LVW/BW compared with that in sham-operated mice (Figure 1, P=NS). Similar to our previous result,¹⁸ the banded TG GqI mice developed a significantly blunted increase in LVW/BW compared with that in wild-type mice (22% versus 54%, respectively, P<0.001; Table and Figure 1) despite a similar trans-stenotic pressure gradient 7 days after TAC (74.4±3.6 mm Hg; Table and Figure 1). No hypertrophy was detected at 7 hours after TAC. Echocardiography in TG GqI mice showed no change in LV size or percent fractional shortening after TAC, suggesting the preservation of cardiac function despite the blunted hypertrophic response (Table). Postsurgical mortality in banded TG GqI mice was not different from that in wild-type mice (19% and 20%, respectively).

Induction of MAPK Activity With In Vivo Pressure Overload in Wild-Type and TG GqI Mice
We evaluated JNK, ERK, p38, and p38ß activity 7 hours, 3 days, and 7 days after aortic constriction in wild-type and TG GqI mice. Data are represented as fold induction in TAC hearts compared with sham-operated hearts for each of the groups.

JNK Activity
A significant induction of JNK activity, compared with that after sham surgery, was observed during the initial phase of pressure overload, as early as 7 hours after TAC, showing the sensitive nature of JNK activation to acute stress (Figure 2a and 2b). Interestingly, compared with JNK activity after the sham operation, JNK activity remained elevated after 3 days and 7 days of pressure overload during the period of early cardiac hypertrophy to established cardiac hypertrophy (Figure 2a and 2b). To determine whether G_q-mediated pathways are involved in the induction of MAPK activity, we measured the time course of JNK activity in TG GqI mice after TAC. As shown in Figure 2c and 2d, the induction of JNK activity was completely abolished after acute pressure overload (7 hours) and remained abolished up to 7 days after pressure overload in the hearts of TG GqI mice.

View larger version (27K): [in this window] [in a new window]   Figure 2. JNK activity in LV extracts from wild-type (a and b) and TG GqI (c and d) hearts. a, Increased phosphorylation of substrate is evident at 7 hours, 3 days, and 7 days after TAC in wild-type mice. b, Mean data show significant induction of JNK activity observed after 7 hours, 3 days, or 7 days of pressure overload (n=7 for each group except for the 7-day time point, during which n=13 for sham and n=11 for TAC). *P<0.01 for TAC vs sham at each time point. c and d, Inhibiting Gq-coupled receptors in TG GqI mice abolished induction of JNK activity acutely (7 hours) and in later phases of pressure overload (3 and 7 days) (n=7 for each group except for the 7-day time point, during which n=9 for sham and n=8 for TAC). Activities were measured by capacity to phosphorylate GST-c-Jun in sham-operated and banded wild-type and TG GqI mice. Mean data represent fold induction in activity of TAC-operated hearts compared with sham-operated hearts from 7 to 13 animals and were calculated by dividing activity from each sham sample by mean for sham-operated group. Standard errors in hearts from sham-operated mice represent variability of basal MAPK activity studied within group of sham-operated hearts.

ERK Activity
The pattern of ERK activity after pressure overload in wild-type mice differed from the pattern of JNK activity, as shown in Figure 3a and 3b. Compared with the corresponding sham-operated hearts, hearts from wild-type mice showed a small nonsignificant (1.3-fold) increase in ERK activity at 3 days of TAC that became markedly increased after 7 days of pressure overload. Similar to the pattern observed for JNK, the banded TG GqI mice showed that the induction of ERK activity was completely blocked early (3 days) and later (7 days) after TAC (Figure 3c and 3d).

View larger version (28K): [in this window] [in a new window]   Figure 3. ERK activity in LV extracts from wild-type (a and b) and TG GqI (c and d) hearts. a, Increased phosphorylation of substrate is evident only at 3 days and 7 days after TAC in wild-type mice. b, Summary data show fold induction of ERK activity in wild-type mice (n=7 for each group except for the 7-day time point, during which n=14 for both sham and TAC). *P<0.01 for TAC vs sham. c and d, In contrast, induction in ERK activity in TG GqI mice was completely abolished throughout time course of in vivo pressure overload (n=7 for each group except for the 7-day time point, during which n=14 for sham and n=16 for TAC).

p38 and p38ß Activity
In wild-type mice, a different pattern of activation for p38 and p38ß by pressure overload was observed compared with that seen for JNK and ERK. In this case, strong induction of p38 and p38ß activity was seen as early as 3 days, which remained elevated at 7 days. In contrast, p38 and p38ß were significantly activated in wild-type hearts compared with sham-operated TG GqI hearts, but only after 7 days of in vivo pressure overload (Figure 4b and 4d).

View larger version (45K): [in this window] [in a new window]   Figure 4. p38 and p38ß activity was measured in LV extracts from wild-type (a and c) and TG GqI (b and d) hearts. At top of each panel, increased phosphorylation of substrate is evident at 3 days and 7 days after TAC in wild-type mice and at 7 days in the TG GqI mice. At bottom of each panel, mean data show significant induction of p38 and p38ß at each time point. In contrast to wild-type mice, induction of both p38 and p38ß activity in TG GqI mice was delayed and significantly induced only after 7 days of pressure overload (n=7 for each group except for the 7-day time point, during which n=8 for both sham and TAC). *P<0.01 for TAC vs sham.

In all extracts examined, no significant differences were found in total MAPK protein levels between sham operation and TAC for both wild-type and TG GqI mice, as assessed by Western blot, suggesting only a modulation of kinase activities after TAC (data not shown).

MAPK Activation in TG ßARKct Mice
To determine whether a mechanism for the induction of MAPK activity in cardiac hypertrophy involves Gß subunits released from either G_q-coupled receptors or from other GPCRs, experiments were performed in TG ßARKct mice. The ß subunits of G proteins (Gß) have been shown to activate signaling pathways in a variety of cells,^{22 23} including phosphoinositide 3-kinase (PI3K) in in vivo pressure-overload hypertrophy.²⁴ Thus, we tested whether activation of MAPK in hypertrophied hearts involves Gß. As shown in the Table, banded TG ßARKct mice develop cardiac hypertrophy in response to pressure overload to the same level as found in wild-type mice.

Heart extracts from sham-operated and 7-day banded TG ßARKct mice were used to measure MAPK activity. As shown in Figure 5, a statistically significant increase in activity for all the MAPKs tested was observed in the banded TG ßARKct hearts at 7 days after TAC compared with sham-operated TG ßARKct hearts. Importantly, the level of induction of all 3 MAPK pathways in the TG ßARKct TAC hearts was similar to that seen in the wild-type TAC hearts. Taken together, these data suggest a role for G_q, but not Gß, in the activation of MAPK pathways with pressure-overload cardiac hypertrophy.

View larger version (24K): [in this window] [in a new window]   Figure 5. Significant induction in all MAPK activities was observed in hearts of TG ßARKct mice 7 days after TAC (n=11 for each group). *P<0.01 for TAC vs sham.

Basal MAPK activity was evaluated in heart extracts from sham-operated wild-type, TG GqI, and TG ßARKct mice (n=5 for each group). No significant difference was found in the basal ERK, JNK, or p38 ( or ß) activity among the 3 groups.

	Discussion

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In the present study, we report the time course of induction of all 3 major MAPK pathways (ERK, JNK, and p38) with the development of in vivo cardiac hypertrophy and establish the essential role for G_q signaling in this process. We show that there are 3 phases to the induction of MAPK activity during in vivo hypertrophy. In the first phase (acute pressure overload, 7 hours), only JNK activity is increased. The second phase (early cardiac hypertrophy, 3 days) is associated with robust induction of p38, p38ß, and JNK activity but with a minimal increase in ERK activity. The final phase (established hypertrophy, 7 days) is associated with strong and sustained induction of all the MAPK pathways. This pattern of MAPK activation is markedly altered in the pressure-overloaded TG GqI mice. In this case, JNK and ERK activity are completely abolished, whereas p38 and p38ß activation occurs but is delayed in appearance, suggesting recruitment of other non–G_q-mediated pathways for p38 activation.

In cultured cells, activation of the MAPKs is thought to be mediated by both and ß subunits of the heterotrimeric G protein after GPCR stimulation.^{25 26 27 28} However, among subunits, only G_q stimulation has been shown to activate all MAPK pathways.¹² In the rat, Yano et al²⁹ have recently shown that angiotensin II–induced cardiac hypertrophy is associated only with the early and transient activation of JNK. Although the study by Yano et al suggests that stimulation of the JNK pathway can induce a hypertrophic response, it did not assess the mechanism for activation in response to in vivo pressure overload. In the present study, we used the physiological stimulus of pressure overload and show a specific pattern of MAPK activation with early and persistent JNK activation, followed by both p38 and p38ß and then ERK. Furthermore, we show that all MAPKs are activated by signals originating from G_q-coupled receptors and that later recruitment of non–G_q-coupled receptor pathways can eventually lead to p38 activation once hypertrophy is established. Finally, it is possible that the strong induction in p38 MAPK activity in the TG GqI mice, which we show is non–G-protein (G_q or Gß)–mediated, is responsible for the mild hypertrophy that develops in these animals with pressure overload.

A recent study has reported that inhibition of JNK in the heart by expression of a dominant inhibitory activator mutant can inhibit the development of cardiac hypertrophy after banding in the rat.¹⁶ The present study adds to those findings by showing that the development of cardiac hypertrophy is associated with the induction of all 3 MAPK pathways and that the increase in activity is mediated through G_q-coupled receptors. Furthermore, the late induction of p38 and p38ß MAPK activity in banded TG GqI mice suggests that non–G_q-mediated pathways or signaling cascades can be recruited to activate p38 in the pressure-overloaded heart.³⁰ The non–G_q-mediated induction of p38 is consistent with a recent study by Zhang et al¹⁷ showing that overexpression of a constitutively active mutant of transforming growth factor-ß–activated kinase, a mediator of transforming growth factor-ß signaling, results in the activation of p38 but not ERK or JNK MAPK. Also consistent with our findings is the in vitro study by Sabri et al³¹ showing that in mouse cardiomyocytes, p38 MAPK activation was coupled to ß-adrenergic but not ₁-adrenergic receptor stimulation.

We have previously shown that in TG ßARKct mice, Gß dimers released from stimulation of G_q-coupled receptors can activate PI3K²⁴ and that PI3K can activate MAPK.³² Therefore, we tested whether released Gß subunits from either G_q receptors or other GPCRs play a role in the increase in MAPK activity with hypertrophy. We studied MAPK activity 7 days after TAC in mice overexpressing the ßARKct, and we have determined that JNK activity and ERK activity are significantly increased in hearts from TG ßARKct mice. Although we show that G_q-coupled receptor stimulation is required for the induction of JNK and ERK activity, by performing TAC in the TG ßARKct mice, we also demonstrate that Gß subunits arising from G_q heterotrimers (or for that matter from any other GPCR) do not significantly contribute to the induction of the 3 MAPK pathways. Nonetheless, because the ßARKct transgene is driven by the myosin heavy chain promoter, we cannot exclude a role for cardiac fibroblasts in the induction of MAPK activity in cardiac hypertrophy.¹⁴

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants HL-56687 (Dr Rockman) and HL-61690 (Dr Koch). We gratefully acknowledge Debbie Colpitts for her expert secretarial assistance.

Received July 21, 2000; revision received September 14, 2000; accepted September 19, 2000.

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