Published online before print August 10, 2006, doi: 10.1161/01.RES.0000240147.49390.61
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© 2006 American Heart Association, Inc.
Integrative Physiology |
An Angiotensin II Type 1 Receptor Mutant Lacking Epidermal Growth Factor Receptor Transactivation Does Not Induce Angiotensin II–Mediated Cardiac Hypertrophy
From the Cardiovascular Research Institute (P.Z., J.G., J.L., J.S.), Department of Cell Biology and Molecular Medicine, University of Medicine & Dentistry of New Jersey, New Jersey Medical School, Newark; and Oncology Research Institute (E.H., X.Y., T.W.), Greenville, SC.
Correspondence to Junichi Sadoshima, MD, PhD, Cardiovascular Research Institute, UMDNJ, New Jersey Medical School, 185 South Orange Ave, MSB G609, Newark, NJ 07103. E-mail sadoshju{at}umdnj.edu
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Abstract |
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We have shown previously that tyrosine 319 in a conserved YIPP motif in the C terminus of angiotensin II (Ang II) type 1 receptors (AT1Rs) is essential for transactivation of epidermal growth factor receptor (EGFR) in vitro. We hypothesized that the signaling mechanism mediated through the specific amino acid sequence in the G protein–coupled receptor plays an important role in mediating cardiac hypertrophy in vivo. Transgenic mice with cardiac-specific overexpression of wild-type AT1R (Tg-WT) and an AT1R with a mutation in the YIPP motif (Tg-Y319F) were studied. Tg-Y319F mice developed no significant cardiac hypertrophy, in contrast to the significant development of hypertrophy in Tg-WT mice. Expression of fetal-type genes, such as atrial natriuretic factor, was also significantly lower in Tg-Y319F than in Tg-WT mice. Infusion of Ang II caused an enhancement of hypertrophy in Tg-WT mice but failed to induce hypertrophy in Tg-Y319F mice. Left ventricular myocardium in Tg-Y319F mice developed significantly less apoptosis and fibrosis than that in Tg-WT mice. EGFR phosphorylation was significantly inhibited in Tg-Y319F mice, confirming that EGFR was not activated in Tg-Y319F mouse hearts. In contrast, activation/phosphorylation of protein kinase C, STAT3, extracellular signal-regulated kinase, and Akt and translocation of G
q/11 to the cytosolic fraction were maintained in Tg-Y319F hearts. Furthermore, a genetic cross between Tg-WT and transgenic mice with cardiac-specific overexpression of dominant negative EGFR mimicked the phenotype of Tg-Y319F mice. In conclusion, overexpression of AT1-Y319F in cardiac myocytes diminished EGFR transactivation and inhibited a pathological form of cardiac hypertrophy. The YIPP motif in the AT1R plays an important role in mediating cardiac hypertrophy in vivo.
Key Words: AT1 receptor • YIPP motif • transactivation • EGFR • hypertrophy
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Introduction |
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Cardiac hypertrophy has been considered to be an adaptive process for withstanding the increased afterload, while reducing load to individual sarcomeres and, hence, oxygen consumption, especially in an acute setting.1 On the other hand, the continued presence of cardiac hypertrophy in the presence of pressure and volume overload often leads to cardiac failure. Thus, the presence of cardiac hypertrophy itself could be a risk factor for congestive heart failure in patients. However, these concepts have recently been challenged because, at least in mouse models, some forms of hypertrophy remain adaptive,2 whereas inhibition of other forms of cardiac hypertrophy halts progression into heart failure despite the presence of markedly elevated wall stress.3,4 The emerging concept is that the underlying signaling mechanism, rather than the presence of hypertrophy alone, may determine the functional outcome of cardiac hypertrophy.1 Thus, it would be important to understand the signaling mechanism activated by each hypertrophic stimulus and its effect on cardiac phenotype.
Agonists for heterotrimeric G protein–coupled receptors (GPCRs), especially those coupled to Gq, including angiotensin II (Ang II), endothelin-1 (ET-1), and phenylephrine (Phe), play an important role in mediating cardiac hypertrophy in patients.5 Among them, increasing lines of evidence suggest that the AT1 receptor, a primary receptor mediating cardiac hypertrophy in response to Ang II, participates in many unconventional signaling mechanisms besides classical Gq-mediated signaling mechanisms.6 For example, we and others have shown that AT1 receptor mutants lacking heterotrimeric G protein coupling are able to activate several downstream signaling mechanisms, including Src and extracellular signal-regulated kinase (ERK), through heterotrimeric G protein–independent mechanisms.7–12 In addition, the cytoplasmic domains of the AT1 receptor directly interact with intracellular signaling molecules, including JAK2, SHP2, phospholipase C
, and AT1 receptor–associated protein,13–16 thereby positively or negatively regulating their functions. Such mechanisms, which rely on the amino acid sequence of the AT1 receptor, provide the AT1 receptor with a unique modality to communicate with downstream signaling mechanisms. Furthermore, stimulation of AT1 receptors also induces transactivation of epidermal growth factor receptor (EGFR), thereby regulating activation of ERK2 and cardiac hypertrophy in cultured cardiac myocytes in vitro.17,18 These novel signaling mechanisms may mediate the effect of Ang II on growth and death of cardiac myocytes in vivo as well, thereby affecting the pathogenesis of heart failure. However, the roles of these unconventional signaling mechanisms in regulating Ang II–induced cardiac hypertrophy and failure are not well understood in vivo.
Thus, the goal of this study was to elucidate the in vivo function of one of the signaling mechanisms critically dependent on the amino acid sequence of the AT1 receptor. We used AT1-Y319F, an AT1 receptor having a mutation in the conserved YIPP motif, as a model because we have previously shown that this mutant selectively lacks EGFR coupling in COS-7 cells.19 Considering the potential importance of EGFR coupling in mediating growth of cardiac myocytes, we speculated that the use of the AT1 receptor with "biased" signaling mechanisms would allow us to prove the importance of the sequence specific function of the AT1 receptor in vivo. We asked the following questions. (1) Does Y319 play a critical role in mediating downstream signaling mechanisms, such as transactivation of EGFR, in cardiac myocytes in vivo? (2) If so, does Y319 regulate growth and death of cardiac myocytes as well as global function of the heart in vivo?
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Materials and Methods |
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For an expanded Materials and Methods section, please refer to the online data supplement, available at http://circres.ahajournals.org.
All transgenic mice were generated on an FVB background, using the -myosin heavy chain promoter. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the New Jersey Medical School. Data are reported as mean±SEM. Statistical analyses between groups were performed by 1-way ANOVA, and differences among group means were evaluated using the Student–Newman–Keuls multiple comparison test. A probability value of <0.05 was considered significant.
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Results |
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Generation of Tg-WT and Tg-Y319F Mice
An AT1 receptor mutant in which the tyrosine 319 residue located in the C-terminal YIPP motif is replaced with phenylalanine (AT1-Y319F) lacks the ability to transactivate EGFR.19 To examine the importance of the signaling mechanism mediated through this amino acid residue in the AT1 receptor in vivo, we made transgenic mice with cardiac-specific overexpression of AT1-Y319F (Tg-Y319F). Cardiac tissue AT1 receptor expression was assessed by quantitative RT-PCR at the mRNA level and by ligand-binding assay at the protein level. We obtained 3 lines with distinct levels of AT1-Y319F mRNA expression in the order of lines 7>44>16 (Figure 1A). We have previously reported characterization of transgenic mice with cardiac-specific overexpression of wild-type AT1 receptors (Tg-WT), where 3 lines with distinct expression levels were available in the order of lines 15>11>8.20 The level of mRNA expression in Tg-Y319F line 7 was not significantly different from that in Tg-WT line 15. Receptor-binding assays also confirmed that AT1 receptor expression in line 7 of Tg-Y319F mice was not significantly different from that in line 15 of Tg-WT mice (Figure 1B).
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Tg-Y319F Mice Do Not Develop Cardiac Hypertrophy
We have previously shown that Tg-WT mice exhibit cardiac hypertrophy proportional to the level of AT1 receptor expression. We confirmed that left ventricular weight (LVW)/body weight (BW) and LVW/tibial length (TL) of Tg-WT mice (line 15) were significantly elevated compared with those of nontransgenic (NTg) littermates (Figure 2A and Table I in the online data supplement). By contrast, none of the 3 lines of Tg-Y319F mice exhibited significant increases in LVW/BW or LVW/TL compared with NTg (Figure 2A and supplemental Table I for line 7, the highest expression line, and data not shown). Left ventricular (LV) myocyte cross-sectional area was also significantly greater in Tg-WT, but not in Tg-Y319F, mice compared with that in NTg (Figure 2B). With increasing levels of AT1 receptor expression, there was a dose-dependent increase in mRNA expression of atrial natriuretic factor (ANF) and -skeletal actin (ASA), fetal-type genes, in Tg-WT mice, but there was a less-prominent increase in Tg-Y319F mice (Figure 2C and 2D). mRNA expression of ANF and ASA was significantly lower in Tg-Y319F (line 7) than in Tg-WT (line 15) mice.
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To evaluate the direct effect of AT1-Y319F on cardiac myocyte hypertrophy, in vitro experiments were conducted using neonatal rat cardiac myocytes. Comparable levels of AT1-WT and AT1-Y319F were overexpressed, using adenovirus transduction (supplemental Figure IA). As reported previously,20 overexpression of AT1-WT enhanced Ang II–induced cardiac hypertrophy. In contrast, overexpression of AT1-Y319F, rather, inhibited Ang II–induced cardiac hypertrophy (Figure 2E).
Tg-Y319F Mice Have Preserved Left Ventricular Diastolic Function
The heart rates of Tg-WT (line 15) and Tg-Y319F (line 7) mice were significantly slower than those of corresponding NTg mice (Table 1). ECG showed that Tg-WT mice had second degree atrioventricular (AV) block, whereas Tg-Y319F mice exhibited first degree AV block with atrial arrhythmia (supplemental Figure II and supplemental Table II). Echocardiographically determined LV ejection fraction (LVEF) and percent fractional shortening (%FS) of Tg-Y319F mice (line 7) were not significantly different from those of NTg mice (Table 1). dP/dtmax, an index of the systolic function, was depressed in both Tg-WT and Tg-Y319F mice, with Tg-Y319F mice having a higher dP/dtmax than Tg-WT mice (Table 2). dP/dtmin, an index of the left ventricular diastolic function, was significantly decreased in Tg-WT compared with NTg mice. Although dP/dtmin of Tg-Y319F mice was slightly reduced compared with NTg mice, dP/dtmin of Tg-Y319F mice was significantly higher than that of Tg-WT (Table 2), indicating that LV diastolic dysfunction may be milder in Tg-Y319F than in Tg-WT mice. Similar results were obtained after ventricular pacing (500 per minute) (supplemental Figure III), suggesting that the difference in dP/dtmax and dP/dtmin between Tg-Y319F and Tg-WT mice is not attributed to the slower heart rate in Tg-WT mice. Tg-Y319F mice exhibited smaller lung weight per BW and LV end-diastolic pressure than Tg-WT mice after 4 weeks of transverse aortic constriction (supplemental Figure IV and supplemental Table III), suggesting that Tg-Y319F mice appear to have preserved cardiac function under stress compared with Tg-WT.
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Tg-Y319F Hearts Do Not Develop Fibrosis or Apoptosis
Picric acid Sirus red staining showed that interstitial fibrosis was increased in Tg-WT, but not in Tg-Y319F, mice compared with corresponding NTg mice (Figure 3A). Quantitative analyses showed that the level of interstitial fibrosis in Tg-Y319F was significantly less than that in Tg-WT mice (Figure 3B). To evaluate the frequency of cell death caused by apoptosis in the myocardium, TUNEL staining was performed using heart sections obtained from 10- to 12-month-old mice. Significantly more TUNEL-positive myocytes were observed in Tg-WT, but not in Tg-Y319F, mice compared with corresponding NTg littermates. The level of apoptosis in Tg-Y319F mice was significantly lower than that in Tg-WT (Figure 3C). Taken together, fibrosis and apoptosis are not increased in Tg-Y319F mice.
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Chronic Ang II Infusion Does Not Cause Cardiac Hypertrophy in Tg-Y319F Mice
Continuous infusion of Ang II (200 ng/kg/min) for 2 weeks did not significantly affect blood pressure and heart rates in Tg-WT, Tg-Y319F, or corresponding NTg littermates (data not shown). However, this treatment resulted in a significant increase in LVW/BW, LVW/TL, and myocyte cross-sectional area in NTg mice, which was significantly enhanced in Tg-WT mice, consistent with our previous observations.20 Interestingly, Ang II–induced increases in LVW/BW, LVW/TL, and myocyte size were abolished in Tg-Y319F mice (Figure 4A through 4C). Although Ang II infusion increased mRNA expression of ANF and ASA in NTg and Tg-WT mice, Ang II–induced increases in ANF and ASA expression were abolished in Tg-Y319F mice (Figure 4 DE). Ang II infusion significantly reduced LVEF and %FS in Tg-WT, but not in Tg-Y319F, mice (supplemental Table IV). These results suggest that expression of AT1-Y319F attenuates Ang II–induced cardiac hypertrophy and mild LV dysfunction observed in Tg-WT mice.
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Transactivation of EGFR Is Missing in Tg-Y319F Mice
We examined the effect of AT1-Y319F overexpression on downstream signaling mechanisms in the mouse heart in vivo. Immunoblot analyses with anti–phospho (Y1173)–EGFR antibody showed that the level of EGFR phosphorylation was significantly increased in Tg-WT (line 15) compared with NTg littermates. However, no significant increase in Y1173 EGFR phosphorylation was observed in Tg-Y319F mice (line 7) (Figure 5A). Ang II infusion increased Y1173 phosphorylation of EGFR in NTg and Tg-WT, but not in Tg-Y319F, mice, further confirming the notion that EGFR is not activated in the heart overexpressing AT1-Y319F (Figure 5B). Similar results were obtained in cardiac myocytes where AT1-WT or AT1-Y319F were overexpressed (supplemental Figure IAB). In Cos7 cells, Ang II–induced activation of EGFR coincides with phosphorylation of Y319 in the AT1 receptor.19 Ang II–induced increases in phosphorylation of Y319 were enhanced in Tg-WT, but not in Tg-Y319F, mice (Figure 5C). To determine whether the JAK-STAT pathway is affected in Tg-Y319F mice, immunoblot analyses with anti–phospho-STAT3 (S727 or Y705) antibodies were performed. STAT3 phosphorylation at both residues was enhanced in Tg-WT and Tg-Y319F mouse hearts, suggesting that the JAK-STAT pathway is stimulated equally in Tg-WT and Tg-Y319F mice (Figure 5D). To determine whether the Gq-dependent signaling is activated in Tg-Y319F mice, subcellular localization of Gq/11 was determined, because redistribution of Gq into the cytosolic fraction has been shown to be an indirect marker of Gq activation.20 In both Tg-WT and Tg-Y319F mice, Gq/11 was translocated from the particulate to the cytosolic fraction to a similar extent (Figure 5E), suggesting that Gq/11 is activated equally in Tg-WT and Tg-Y319F mice. Consistent with this observation, comparable levels of translocation of protein kinase C (PKC) isoforms (, 
, and ) from the cytosolic to the particulate fraction were observed in Tg-WT and Tg-Y319F mice (Figure 5F through 5H).
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Overexpression of AT1-Y319F in cultured cardiac myocytes also enhanced Ang II–induced total inositol phosphate (IPx) production to a similar extent as that of AT1-WT (supplemental Figure IC), confirming the presence of Gq coupling in AT1-Y319F. Phosphorylated forms of ERK, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and Akt were increased similarly in Tg-WT and Tg-Y319F mice compared with NTg (Figure 5I through 5L). Taken together, whereas Gq coupling and activation/phosphorylation of PKC, STAT3, ERK, and Akt were maintained, activation of EGFR was impaired in Tg-Y319F mice.
Cardiac Hypertrophy in Tg-WT Mice Is Suppressed by Dominant Negative EGFR In Vivo
To further elucidate the role of EGFR coupling in cardiac hypertrophy induced by AT1 receptors in vivo, 3 independent lines of transgenic mice were generated in which dominant negative (DN)-EGFR was expressed specifically in the heart (Tg-DN-EGFR). Thus far, no obvious baseline cardiac phenotype has been observed in these mice after up to 1 year of follow-up (supplemental Tables V and VI). We characterized line 23, in which activation of endogenous EGFR by intraperitoneal injection of EGF was confirmed to be abolished (Figure 6A). Interestingly, basal phosphorylation of the EGF-ErbB family receptor tyrosine kinases, such as ErbB2 and ErbB4, was maintained in Tg-DN-EGFR (Figure 6B). To examine the effect of DN-EGFR on cardiac hypertrophy induced by AT1-WT mice, we crossed Tg-WT with Tg-DN-EGFR mice. EGFR was activated in Tg-WT but not in the bigenic mice (Figure 6C). In contrast, activation of PKC isoforms was maintained in the bigenic mice (Figure 6D). The bigenic mice exhibited smaller LVW/BW and LVW/TL than Tg-WT mice (Figure 6E). Echocardiographic measurement showed that LV dimensions in the bigenic mice were slightly smaller than those in Tg-WT mice (supplemental Table VI). Furthermore, the upregulation of fetal-type genes, including ANF and ASA, observed in Tg-WT mice was completely abolished in the bigenic mice (Figure 6F). In addition, continuous infusion of Ang II (200 ng/kg/min) failed to induce cardiac hypertrophy, increases in myocyte cross-sectional area, or expression of fetal-type genes in Tg-DN-EGFR (Figure 6G through 6I). These results are consistent with the notion that EGFR plays an essential role in mediating the development of cardiac hypertrophy induced by the AT1 receptor.
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Hemodynamic measurements indicated that Tg-DN-EGFR had normal baseline cardiac function, suggesting that the baseline activity of EGFR was not essential to the maintenance of LV function (Table 2). Tg-WT mice exhibited significantly reduced baseline dP/dtmax and dP/dtmin, consistent with our previous results.20 The heart rates of the bigenic mice remained suppressed, suggesting that reduction in heart rates may be mediated by EGFR-independent mechanisms. In contrast, the bigenic mice exhibited significantly higher baseline dP/dtmax and dP/dtmin than those of Tg-WT mice, and thus the LV function was reverted toward the control level (Table 2). These results suggest that the AT1 receptor causes cardiac dysfunction through EGFR-dependent signaling mechanisms.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results suggest that AT1-Y319F fails to stimulate cardiac hypertrophy despite the fact that it maintains G
q-coupling mechanisms. This suggests that besides conventional Gq-dependent mechanisms, an additional mechanism mediated through Y319, most likely EGFR coupling, is needed for the AT1 receptor to mediate cardiac hypertrophy. In other words, a mechanism critically dependent on the amino acid sequence of the AT1 receptor is required for cardiac hypertrophy caused by stimulation of the AT1 receptor. This relatively uncharacterized signaling mechanism may confer the specificity and the modulatory mechanism to the AT1 receptor.
Cardiac-specific overexpression of Gq is sufficient to induce pathological hypertrophy.21 Thus, it is surprising to see that Tg-Y319F mice failed to induce cardiac hypertrophy despite the fact that Gq is activated in these mice. It is possible that the extent of Gq activation in Tg-Y319F mice could be much less than that in Gq overexpression mice. However, judging from the complete lack of hypertrophy in Tg-Y319F mice, a more probable explanation could be that AT1-Y319–mediated cell signaling may work as an essential mediator of Gq signaling. Consistent with this notion, EGFR activation is required for cardiac hypertrophy by PKC activation in cultured cardiac myocytes (supplemental Figure V).
Transactivation of EGFR by the AT1 receptor is mediated by several mechanisms. The first is through modulation of intracellular diffusible signaling molecules, including Ca2+, PKC, and Src, whose activities are regulated by both Gq-dependent and -independent mechanisms,22–25 as well as by reactive oxygen species.26 The second is through activation of metalloproteinases, such as ADAM17,27 and subsequent cleavage/liberation of a diffusible factor, heparin-binding EGF (HB-EGF), into the extracellular space.28 Finally, the AT1 receptor and other GPCRs transiently associate with EGFR in a ligand-dependent manner, either in an endosomal complex containing ß-arrestin29 or in caveolin-enriched lipid rafts containing Src and c-Abl, nonreceptor tyrosine kinases.30,31 These mechanisms may not be mutually exclusive, because complex formation between the AT1 receptor and EGFR may facilitate activation of EGFR through the generation/liberation of either intracellular or extracellular diffusible factors by the AT1 receptor located nearby. We have previously shown that Y319 of the AT1 receptor is essential for physical interaction between the AT1 receptor and EGFR through SHP2 in COS7 cells.19 The present study suggest a similarity between in vitro and in vivo observations.
It has been shown that inhibition of EGFR activation by AG1478, as well as metalloproteinase inhibitor BB94, significantly attenuated Ang II–induced cardiac hypertrophy in cultured cardiac myocytes in vitro.17 Metalloproteinase inhibitors and antisense oligonucleotide against EGFR inhibited Ang II–induced cardiac hypertrophy in the mouse heart in vivo.32,33 However, neither systemic effects of these interventions nor nonspecific effects of the chemical inhibitors can be excluded. In fact, a significant reduction in blood pressure was noted in mice treated with antisense oligonucleotide against EGFR, which could secondarily affect the extent of cardiac hypertrophy.33 Our results show that specific inhibition of EGFR restricted to the heart inhibited cardiac hypertrophy induced by AT1 receptor expression, as well as by Ang II infusion, providing convincing evidence of the involvement of EGFR transactivation in Ang II–induced cardiac hypertrophy in vivo.
Overexpression of AT1-Y319F in the mouse heart abolished cardiac hypertrophy by chronically infused Ang II (Figure 4A through 4E). Ang II–induced activation of EGFR was also abolished by AT1-Y319F (Figure 5B). Such dominant-negative–like function of AT1-Y319F against endogenous AT1 receptors is limited to the signaling mechanism related to EGFR, because overexpression of AT1-Y319F enhances Ang II–induced increases in inositol phosphates (supplemental Figure IC) and activation of MAP kinases (Figure 5I through 5K). Interestingly, AT1-Y319F failed to abolish cardiac hypertrophy induced by pressure overload (supplemental Figure IV), which may be also mediated by AT1 receptor– or EGFR-independent mechanisms.
Another important finding in this report is that the mutation of Y319 and suppression of EGFR activation inhibit not only cardiac hypertrophy but also the accompanying pathological phenotype caused by stimulation of the AT1 receptor, including fetal-type gene expression, fibrosis, and cardiac myocyte apoptosis. Although the results of our conventional hemodynamic analyses suggest that Tg-Y319F mice may have slightly better LV function than Tg-WT mice at baseline and in response to stress, this notion should be substantiated by determining end-systolic and end-diastolic pressure volume relationships in future experiments. The cardiac phenotype observed in Tg-WT mice was also significantly attenuated by overexpression of DN-EGFR in bigenic mice. Thus, it is likely that EGFR mediates the pathological phenotype of Ang II–induced cardiac hypertrophy. Some members of the EGFR family, such as human EGFR-2 (HER2), appear to be protective for the heart, because patients undergoing chemotherapy with Herceptin, a humanized anti-HER2 antibody, develop cardiomyopathy.34 Furthermore, conditional deletion of HER2 in adult mouse heart results in dilated cardiomyopathy.35 Although HER2 can be transactivated via the HB-EGF by Ang II,36 other members of the EGFR family, most likely EGFR/HER1, may mediate pathological hypertrophy. In fact, phosphorylation of ErbB2 and ErbB4 was not affected in our Tg-DN-EGFR mice.
It should be noted that AT1-Y319F, lacking transactivation of EGFR, is not entirely inactive in mediating cardiac phenotype, because Tg-Y319F mice exhibited a significant reduction in heart rate, with first degree AV block. Development of more severe AV conduction abnormality caused by overexpression of AT1-WT was not normalized by overexpression of DN-EGFR in bigenic mice. Thus, this cardiac phenotype seems to be mediated by EGFR-independent signaling mechanisms.
Although the Gq-dependent signaling mechanism is a major signaling mechanism that many agonists use to induce cardiac hypertrophy, recent evidence suggests that Gq-independent signaling mechanisms also mediate cardiac hypertrophy.20 Our preliminary results showed that EGFR activation was missing in the heart of transgenic mice with cardiac-specific overexpression of an AT1 receptor mutant lacking heterotrimeric G protein coupling (supplemental Figure VI), which exhibited severe but well-compensated cardiac hypertrophy whose cardiac phenotype is distinct from that of Tg-WT mice.20 Thus, EGFR transactivation by the AT1-WT requires heterotrimeric G protein coupling. We speculate that cardiac hypertrophy mediated by heterotrimeric G protein–independent mechanisms may be mediated by EGFR- or Y319-independent mechanisms (supplemental Figure VII).
Using a "biased receptor," a strategy similar to that used in this work, we have previously shown that an AT1 receptor mutant that does not couple to Gq or Gi stimulates cardiac hypertrophy with less apoptosis and fibrosis but severe bradycardia.20 Here we have shown that another biased AT1 receptor, lacking signaling mechanisms through Y319, eliminates the detrimental effects of the AT1 receptor on cardiac hypertrophy in vivo. It has been suggested that either naturally occurring or engineered ligands for the GPCR can either differentially or exclusively modulate one or another signaling mechanism within the receptor, a phenomenon termed "biased agonism" or "ligand-direct signaling."37 A recent review succinctly summarized the possibility of developing a "biased agonist" to modulate a targeted function of a given GPCR, thereby enabling specialized treatment without interfering with other important housekeeping functions of the receptor.38 In this regard, we expect that a biased ligand for the AT1 receptor specifically abolishing signaling through Y319 would be promising for treatment of heart failure.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants HL 33107, HL 59139, HL67724, HL67727, HL69020, and HL 73048 and by American Heart Association Grant 0340123N. P.Z. is supported by NIH grant 1 F32 HL080861.
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this study.
Original received March 2, 2006; revision received July 7, 2006; accepted July 27, 2006.
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