Targeting the Receptor-Gq Interface to Inhibit in Vivo Pressure Overload Myocardial Hypertrophy

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Science  24 Apr 1998:
Vol. 280, Issue 5363, pp. 574-577
DOI: 10.1126/science.280.5363.574


Hormones and neurotransmitters may mediate common responses through receptors that couple to the same class of heterotrimeric guanine nucleotide–binding (G) protein. For example, several receptors that couple to Gq class proteins can induce cardiomyocyte hypertrophy. Class-specific inhibition of Gq-mediated signaling was produced in the hearts of transgenic mice by targeted expression of a carboxyl-terminal peptide of the α subunit Gαq. When pressure overload was surgically induced, the transgenic mice developed significantly less ventricular hypertrophy than control animals. The data demonstrate the role of myocardial Gq in the initiation of myocardial hypertrophy and indicate a possible strategy for preventing pathophysiological signaling by simultaneously blocking multiple receptors coupled to Gq.

Myocardial hypertrophy is an adaptive response to various mechanical and hormonal stimuli and represents an initial step in the pathogenesis of many cardiac diseases that ultimately progress to ventricular failure. The mechanisms by which cardiac hypertrophy is initiated and how this condition eventually progresses to heart failure are poorly understood. Several independent signaling pathways have been implicated in the activation of the hypertrophic response in vitro (1). The G protein Gq is thought to be important in this process because various ligands, such as phenylephrine, angiotensin II (AngII), and endothelin I, that activate Gq-coupled receptors can trigger hypertrophic responses in cultured myocytes (2). In vivo studies with Gq-coupled receptor antagonists have also implicated Gq-mediated signaling in pressure-overload ventricular hypertrophy (3), and transgenic mice with cardiac overexpression of either Gαq, α1-adrenergic receptors (ARs), or AngII receptors present with myocardial hypertrophy (4-6). Although these data indicate that chronic stimulation of Gq-coupled receptors is sufficient to induce myocardial hypertrophy, they do not define the contribution of Gq to the physiological hypertrophic response to ventricular pressure overload.

We examined the possibility of class-specific G protein inhibition through targeting the receptor-Gq interface, thereby simultaneously eliminating responses from multiple receptors that couple to Gq. This therapeutic strategy would potentially eliminate the need for multiple receptor antagonists in a variety of diseases including pressure overload hypertrophy. We designed a COOH-terminal peptide of Gαq that contains the region of the Gα subunit that interacts with the intracellular domains of agonist-occupied receptors (7) and created transgenic mice with myocardial-targeted overexpression of this peptide.

Cellular expression of the third intracellular domain (3i) of the α1B-AR antagonizes in vitro α1B-AR–mediated signal transduction, apparently through competition between the 3i peptide and the activated receptor for binding sites on Gαq (8). We sought to determine whether the expression of peptides derived from the COOH-terminus of Gαq would similarly antagonize receptor-mediated signaling. Two Gαq “minigene” constructs were created that correspond to the COOH-terminal peptide sequence of Gαq, residues 305 to 359, and the NH2-terminal peptide sequence of Gαq, residues 1 to 54. COS-7 cells were transiently transfected with plasmid DNA encoding the Gαq minigenes, and expression of these peptides was demonstrated by protein immunoblotting (Fig.1A). Coexpression of α1B-ARs with the intact Gαq subunit led to enhancement of epinephrine-stimulated inositol phosphate (IP) production compared with that in cells expressing equal numbers of receptors alone. In contrast, coexpression of Gαq(305-359) resulted in a marked inhibition (47.8 ± 4.4%) of maximal α1B-AR–mediated IP production (Fig.1B). Coexpression of the Gαq NH2-terminus [Gαq(1-54)] had no effect. Inhibition by Gαq(305-359) was apparently specific for Gq-coupled receptors because neither α2A-AR–mediated IP production (Gi-coupled) nor dopamine D1A receptor–mediated cAMP production (Gs-coupled) were inhibited, whereas signaling through both the Gq-coupled α1B-AR and M1 muscarinic acetylcholine receptor (AChR) were attenuated (Fig. 1C). Thus, the expression of Gαq(305-359) specifically uncouples Gq-coupled receptors.

Figure 1

Selective in vitro inhibition of Gq-receptor coupling by expression of the Gαq(305-359) peptide. (A) COS-7 cells were transiently transfected with plasmid DNA encoding either empty vector (nontransfected), intact Gαq(1-359), GαqI(305-359) (left panel), or Gαq(1-54) (right panel) (22, 23). Expression of the Gαqminigene products was determined by protein immunoblot analysis (24). (B) COS-7 cells were transiently transfected with plasmid DNA encoding the α1B-AR (0.01 to 1.0 μg of DNA per well) and either intact Gαq, Gαq(305-359), or Gαq(1-54) (2.0 μg of DNA per well) (23). Basal (circles) and epinephrine-stimulated (Epi, squares) IP production was determined as described (8) (solid lines). Responses from control cells transfected with the receptor plasmid plus empty vector are shown in each panel (dashed lines). Data are presented in arbitrary units such that one unit equals the basal amount of IPs measured in cells transfected with empty vector alone. Data shown represent mean ± SEM values for triplicate determinations in one of four separate experiments. (C) COS-7 cells were transiently transfected with plasmid DNA encoding the Gq/11-coupled α1B-AR, the M1 AChR, the Gi-coupled α2A-AR, or the Gs-coupled D1A dopamine receptor (0.1 μg of DNA per well), and either the Gαq(305-359) minigene (black bars) or empty vector (2.0 μg per well) (open bars). Basal and agonist-stimulated IP or adenosine 3′,5′-monophosphate (cAMP) production was determined (8). Data are presented in arbitrary units such that one unit equals the basal amount of IP or cAMP measured in unstimulated cells transfected with empty vector alone. Each panel represents mean ± SEM values for three separate experiments performed in triplicate. * P < 0.05 versus control stimulation [analysis of variance (ANOVA)].

To study the effects of this peptide on Gq-mediated signaling pathways in vivo, we created transgenic mice with cardiac-specific expression of Gαq(305-359). This Gq inhibitor transgene (GqI) was targeted to the myocardium by linking it with the murine α-myosin heavy chain (αMyHC) promoter (9, 10). Five founder lines that transmitted the transgene were established (TG GqI-8, -10, -11, -26, and -38). The TG GqI-10 line had the greatest transgene expression as shown by Northern (RNA) analysis (11), so we used heterozygous (+/−) animals of this line in all further studies. At 10 weeks of age, GqI peptide expression was documented by protein immunoblot analysis of myocardial extracts from the TG GqI-10 line (Fig.2A). These transgenic mice were normal in size, appearance, and behavior compared with nontransgenic littermate control (NLC) animals.

Figure 2

Myocardial expression and in vivo inhibitory activity of the GqI peptide. (A) Expression of Gαq(305-359) was determined by protein immunoblot analysis of myocardial extracts from an NLC (lane 1) or TG GqI (lane 2) heart, or from COS-7 cells expressing the GαqI(305-359) minigene product (lane 3) (24). (B) Lipid extraction was done from NLC (n = 5) and TG GqI (n = 5) left ventricles, and basal diacylglycerol (DAG) content was quantified as described (12). Data shown are means ± SEM.* P < 0.05 versus NLC (Student'st test). (C) Left ventricle injections of the Gq-coupled receptor agonists phenylephrine (PE) (n = 6) or AngII (n = 8) (100 μM), compared with saline injections, were performed in NLC (open bars) and TG GqI (black bars) mice, and myocardial MAP kinase activity toward myelin basic protein (MBP) was measured (13). Activity is expressed as the percent of NLC basal activity determined in saline-injected hearts. Data shown represent means ± SEM of phosphorylated MBP, quantified with a PhosphorImager. Also shown is a representative PE experiment done in two animals for each condition. *P < 0.02 versus NLC (Student'st test). (D) MAP kinase activation in response to carbachol, an agonist for Gi-coupled receptors (100 μM) (14) (n = 4). Carbachol-elicited responses in NLC (open bars) and TG GqI (black bars) mice were significantly elevated (P < 0.05) compared with basal (saline-injected) responses (Student's ttest).

Stimulation of Gq-coupled receptors leads to the activation of phospholipase C and the generation of the second messengers inositol trisphosphate and diacylglycerol. As a direct measurement of the state of endogenous Gq signaling in these mice, we measured basal left ventricular diacylglycerol content (12). The diacylglycerol content in the TG GqI mice was significantly depressed compared with that in NLC mice (Fig. 2B). This finding indicates that basal Gq signaling is decreased in the transgenic hearts, verifying the in vivo Gq-inhibitory properties of the transgene.

We also studied p42/44 mitogen-activated protein (MAP) kinase activity in response to endogenous myocardial Gq-coupled receptor stimulation. In anesthetized transgenic and NLC mice, we directly injected phenylephrine, AngII, or saline into the left ventricle (13). In the hearts of NLC animals, phenylephrine elicited an approximate threefold increase in MAP kinase activity, whereas very little stimulation of MAP kinase activity was caused by phenylephrine in TG GqI mice (Fig. 2C). AngII-stimulated myocardial MAP kinase activity in TG GqI mice was also significantly reduced compared with that in NLC mice (Fig. 2C). Similar results were also obtained with endothelin I (11). In all agonist studies, there was no difference in basal MAP kinase activity between TG GqI and NLC myocardial extracts (Fig. 2C). Thus, acute in vivo signaling through multiple Gq-coupled receptors is inhibited by the GqI peptide. To demonstrate specificity, we tested MAP kinase activation elicited by the Gi-coupled receptor agonist carbachol (14), and responses were the same in TG GqI and NLC mice (Fig. 2D). In addition, adenylyl cyclase activity in response to β-AR–Gs stimulation was the same in TG GqI and NLC myocardial membrane extracts (15) (Table1). Thus, the GqI peptide is specific for inhibiting Gq-coupled receptor signaling in vivo.

Table 1

Myocardial sarcolemmal membrane adenylyl cyclase activity. ISO, isoproterenol. Activity is presented as picomoles of cAMP per minute per milligram of protein and is the mean ± SEM ofn = 6 for each group.

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To assess the in vivo physiological role of Gq-coupled receptor signaling in the development of pressure overload hypertrophy, we subjected TG GqI and NLC mice to pressure overload by surgical transverse aortic constriction (TAC) (16). In this model, left ventricular hypertrophy can be seen 7 days after surgery (17) by using the left ventricle weight to body weight ratio (LVW/BW) as an index of myocardial mass. There was no difference in LVW/BW between sham-operated TG GqI and NLC mice (Table2). In the TAC group, LVW/BW in NLC animals increased by 36% compared with that in sham-operated animals. In contrast, 7 days after TAC the TG GqI mice had a significantly smaller increase (14%) in LVW/BW compared with sham-operated TG GqI mice (P < 0.01). The mean systolic pressure gradient created by TAC, an index of the load placed on the ventricle, was not different between the two groups: 66.4 ± 7.4 mm Hg for TAC in NLC animals and 62.3 ± 6.8 mm Hg for TAC in TG GqI animals (P, not significant). Across a wide range of systolic pressure gradients measured, LVW/BW was lower for the TG GqI mice compared with that in NLC mice (Fig. 3). Therefore, it appears that cardiac Gq-coupled receptors play a critical role in triggering left ventricular hypertrophy after the mechanical stimulus of hemodynamic stress.

Figure 3

Hypertrophic response to pressure overload. The index of left ventricular mass (LVW/BW) is plotted against the systolic pressure gradient produced by TAC for each NLC (n = 12) and TG GqI (n = 20) animal (open and black circles, respectively). The slopes of the linear regressions for NLC [y = 0.025x + 3.61, r = 0.85 (r is the correlation coefficient)] and TG GqI (y = 0.011x + 3.61, r = 0.60) animals were significantly different (P < 0.0005, ANOVA).

Table 2

Physiological parameters in response to pressure overload. Data are expressed as mean ± SEM. The systolic pressure gradient (SPG) is the difference between right and left carotid arterial systolic pressure, an index of load placed on the left ventricle.

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Myocardial hypertrophy is associated with enhanced Gqsignaling and accompanied by reactivation of ventricular embryonic genes including those for atrial natriuretic factor (ANF), skeletal α-actin, and β-myosin heavy chain (1). Similar findings have been reported in vitro after stimulation of Gq-coupled receptors, particularly α1-ARs (18). We therefore measured ventricular ANF mRNA in TG GqI and NLC mice 7 days after sham-operation or TAC. Basal ventricular ANF mRNA was nearly undetectable and not different between the two groups (Fig.4). However, after the stimulus of pressure overload, ventricular ANF mRNA increased almost sevenfold in the NLC group but only about twofold in TG GqI mice (Fig.4). Furthermore, after TAC, left ventricular diacylglycerol content was increased in NLC mice (73%) but not in TG GqI mice (11).

Figure 4

Left ventricle ANF mRNA quantitation. Total RNA (15 μg) was isolated (25) from the left ventricles of NLC (hatched bars) and TG GqI (black bars) hearts that underwent sham-operation or TAC. Northern blots were generated and probed with a mouse ANF cDNA followed by a glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA (4). The signals from the ANF blots were quantified with a PhosphorImager and normalized to the GAPDH signal. Data shown are the means ± SEM for n = 5 in each group. *P < 0.05 TG GqI TAC versus NLC TAC (ANOVA).

Because the depression of basal diacylglycerol content in TG GqI mice did not affect LVW/BW ratios in the absence of pressure overload (Table 2), it appears that Gq-mediated signals do not influence the normal growth of myocytes. In fact, no phenotype is evident in these animals until stress is placed on the heart. Transgenic mice with cardiac-specific expression of a constitutively active mutant α1B-AR have a hypertrophic phenotype (4). Transgenic mice with cardiac AngII receptor overexpression show even greater myocardial hypertrophy (5). Also, a transgenic mouse model with cardiac overexpression of Gαq itself exhibits myocardial hypertrophy (6).

Antagonists of AngII or endothelin I can attenuate ventricular hypertrophy and heart failure in response to pressure overload in animal models (3, 19). However, because these drugs act on vascular receptors to alter afterload, the direct involvement of these myocardial receptors in the hypertrophic response has not been established. Our approach was to block signaling from multiple receptors coupled to a single class of G proteins. TG GqI mice after TAC have the opportunity to use multiple mechanisms for initiating compensatory hypertrophy except signaling through Gq-coupled receptors present on cardiomyocytes. Our results indicate that Gq is a critical molecule in the initiation of myocardial hypertrophy. Targeting the receptor–G protein interface may point the way to the development of therapies that have the potential advantage over traditional receptor antagonists of dampening an entire class of receptor signals (those coupled to a particular G protein) rather than those derived from only a single type of receptor.

  • * To whom correspondence should be addressed. E-mail: koch0002{at}


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