Angiotensin II Type 2 Receptor Antagonizes Angiotensin II Type 1 Receptor–Mediated Cardiomyocyte Autophagy
Autophagy has emerged as an important process in the pathogenesis of cardiovascular diseases, but the proximal triggers for autophagy are unknown. Angiotensin II plays a central role in the pathogenesis of cardiac hypertrophy and heart failure. In this study, we used angiotensin II type 1 (AT1) and type 2 (AT2) receptor–expressing adenoviruses in cultured neonatal cardiomyocytes to provide the first demonstration that neonatal cardiomyocyte autophagic activity is differentially modulated by AT1 and AT2 receptor subtypes. Angiotensin II stimulation (48 hours) of neonatal cardiomyocytes expressing the AT1 receptor alone (Ad-AT1; 10 multiplicities of infection) induced a significant increase in the number of HcRed-LC3 autophagosomes per cell (17.3±1.6 versus 33.3±4.1 autophagosomes per cell; P<0.05). Coexpression of a high ratio of AT2:AT1 (Ad-AT2:Ad-AT1 multiplicity of infection ratio: 20:5) receptors completely abrogated the AT1-mediated increase in autophagy (9.3±1.4 versus 33.3±4.1 autophagosomes per cell; P<0.05). Treatment with the AT2 receptor antagonist PD123319 did not reverse the AT2-mediated antiautophagic effect. AT1- and AT2-mediated autophagic responses were also assessed in cardiomyocytes from a genetic model that exhibits neonatal myocardial growth suppression. In these neonate myocyte cultures, AT1 receptor activation induced a marked increase in the number of myocytes containing cytoplasmic vacuoles compared with the control (22.7±4.1% versus 1.1±0.6%; P<0.001) and was characterized by a nonapoptotic autophagic phenotype. The incidence of cardiomyocyte autophagic vacuolization in this myocyte population decreased dramatically to only 0.4±0.2% in myocytes infected with a high ratio of Ad-AT2:Ad-AT1. This study provides the first description of reciprocal regulation of cardiomyocyte autophagic induction by the AT1 and AT2 receptor subtypes.
- angiotensin II
- angiotensin II type 1 receptor
- angiotensin II type 2 receptor
- hypertrophic heart rat
Angiotensin II (Ang II) plays an important role in cardiac development1 and the pathogenesis of cardiovascular diseases, including cardiac hypertrophy and heart failure.2 Most of the known physiological functions of Ang II are mediated by the Ang II type 1 (AT1) receptor.3 The Ang II type 2 (AT2) receptor is predominantly expressed in fetal and neonatal tissues,4 and its function is less well delineated. Normally present only at low levels in the adult myocardium, AT2 expression is upregulated in the pathological remodeling heart.3 AT2-mediated cardiovascular responses have been characterized as generally opposing AT1-mediated growth effects, but some studies indicate that the AT2 receptor is also involved in inducing left ventricular hypertrophy.5,6 We have also shown that the AT2 receptor can mediate myocyte hypertrophy independent of Ang II in isolated neonatal cardiomyocytes.7 Thus, the role of the AT2 receptor remains ambiguous.
Autophagy is an intracellular degradation process that involves the destruction of long-lived proteins and organelles. Recently, autophagy has been particularly recognized as important in the turnover of cytoplasmic constituents in the heart. Autophagic activity is commonly increased in the heart under conditions that also involve upregulation of the renin-angiotensin system. For example, high levels of autophagy are found in the heart after acute and chronic ischemia, heart failure, and neonatal starvation.8 There is emerging evidence that pressure overload, a major risk factor for cardiac hypertrophy and heart failure, is associated with an excess of autophagy, which is ultimately maladaptive under such conditions of hemodynamic stress.9 The proximal triggers for autophagy in the heart are not known.
AT1 receptor activation stimulates protein synthesis and protein turnover in cardiomyocytes. Intracellular protein aggregation is an important precursor event for cardiomyocyte autophagy.10 Thus, links among activation of the renin-angiotensin system, protein turnover stimulation, and autophagic upregulation may be identified in several pathophysiological myocardial conditions. Therefore, in this study we hypothesized that the AT1 and AT2 receptor subtypes play a previously unrecognized role in cardiomyocyte autophagic induction and/or regulation. In our investigation, we used AT1 and AT2 receptor–expressing adenoviruses in a well-defined neonatal rat cardiomyocyte culture model7,11 to undertake a systematic evaluation of the capacity of AT1 and AT2 receptors, alone and in concert, to influence cardiomyocyte growth and autophagic responses. The relationship between Ang II receptor subtype activation and autophagy induction was tested in a genetic model where an underlying abnormality of cardiomyocyte growth has been identified. Finally, we extrapolated these findings to an in vivo setting to consider the pathophysiological implications for neonatal heart growth. We provide the first demonstration that neonatal cardiomyocyte autophagic activity is contingent on and differentially modulated by AT1 and AT2 receptor subtypes.
For detailed methods please see the online data supplement at http://hyper.ahajournals.org.
Experimental Animals and Tissue Collection
All of the experiments were conducted according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes with the approval of the University of Melbourne Animal Experimentation Ethics Committee. Hearts were excised from embryonic day 18 and postnatal day 2 rats obtained from colonies developed/maintained at the University of Melbourne (Sprague-Dawley, normal heart rat [NHR], and hypertrophic heart rat [HHR]).12 Atrial tissues were dissected away from ventricles (septum intact), and ventricles were blotted and weighed before snap freezing in liquid nitrogen.
Neonatal Cardiomyocyte Culture and Adenoviral Infection
Adenoviruses were used to express AT1A or AT2 receptors. Infection levels were titrated to define cardiomyocyte expression levels as required and as described previously7,11 (Figure S1, available in the online Data Supplement). Neonatal cardiomyocytes were cultured from 1- to 2-day-old neonate rats, as described previously. Forty-eight hours after adenoviral infection, cardiomyocytes were stimulated with the following: Ang II (0.1 μmol/L), candesartan (AT1 antagonist: 1 μmol/L), PD123319 (AT2 antagonist: 1 μmol/L), PD98059 (extracellular signal–regulated kinase 1/2 inhibitor: 20 μmol/L), LY294002 (phosphatidylinositol 3-kinase [PI3K] inhibitor: 2 μmol/L), or 3-methyladenine (blocks autophagic induction: 10 mmol/L). All of the inhibitors were administered 30 minutes before Ang II stimulation.
Angiotensin Receptor Expression
An angiotensin receptor-binding assay was used to determine the degree of angiotensin receptor expression in cardiomyocytes 72 hours after adenoviral infection, as described previously.7,11
After 72 hours of Ang II stimulation in the presence/absence of inhibitors, hypertrophic growth was assessed from the protein (Lowry assay):DNA (Burton assay) ratio and using quantitative phase microscopy.
Cultured neonatal cardiomyocytes were transfected with HcRed-LC3 using Lipofectamine2000 (Invitrogen) 24 hours before adenoviral infection. After 48 hours of Ang II stimulation in the presence/absence of inhibitors, localization of HcRed-LC3 fluorescence was viewed at ×63 magnification using a Zeiss LSM 510 META confocal microscope (Carl Zeiss Inc). Cellular density of HcRed-LC3 autophagosomes was delineated for each treatment.
For quantification of cardiomyocytes displaying evidence of autophagic vacuolization, a cardiomyocyte vacuolization index was calculated by counting the number of vacuolar cells as a percentage of the total number of green fluorescent protein (GFP)-expressing cells (ie, adenoviral infected). Counting was performed at ×20 magnification.
Ex Vivo Ischemia Reperfusion and Autophagy Markers
NHR and HHR (12 weeks) were subjected to ex vivo ischemia-reperfusion injury, as described previously.13 At the end of the reperfusion protocol, ventricles were frozen rapidly in liquid nitrogen for subsequent protein analysis. Heart lysates were prepared as described previously.13 Total expression levels of the autophagic markers beclin-1 and LC3B were assessed by Western blot. Primary antibodies used included beclin-1 (1:500) and LC3B (1:500), which were purchased from Cell Signaling.
Quantitative In Vitro Autoradiography
Cryosections (20 μm) from postnatal day 2 NHR and HHR hearts (midventricle) were analyzed for Ang II receptor binding using standard methods, with 125I-[Sar1,Ile8]Ang II as the radioligand, in the presence and absence of either candesartan (AT1 antagonist: 1 μmol/L) or PD123319 (AT2 antagonist: 1 μmol/L).
mRNA expression for AT1A, AT1B, and AT2 was measured by real-time quantitative RT-PCR. 18S ribosomal RNA was used as a housekeeping gene for all of the samples. Expression of the gene of interest was normalized to 18S expression using the 2−ΔΔCt method or a standard curve, as appropriate.
All of the data are presented as mean±SEM. All multigroup comparisons were made using 1-way or 2-way ANOVA followed by Tukey’s honestly significant differences test. Student unpaired t test was used for comparisons between 2 groups. A value of P<0.05 was considered significant.
Evaluation of AT1- and AT2-Mediated Hypertrophic and Autophagic Responses in Neonatal Cardiomyocytes
AT2 Modulation of Hypertrophic Growth
Ang II did not exert any significant biological effect in neonatal cardiomyocytes in the absence of adenoviral infection (data not shown). Expression of Ad-AT1 alone (≈150 fmol of receptor per milligram of protein; Figure S1) did not elicit a significant hypertrophic response (Figure 1). After the addition of Ang II to neonatal cardiomyocytes expressing AT1 receptors alone, a significant hypertrophic response was observed (121.3±1.3%), which was reversed by treatment with the AT1 receptor antagonist candesartan or the extracellular signal–regulated kinase 1/2 inhibitor PD98059 (Figure 1). In contrast, expression of Ad-AT2 alone (≈150 fmol of receptor per milligram of protein; Figure S1) elicited significant basal hypertrophy (119.5±5.7%), which was unaffected by the addition of Ang II or PD98059 (Figure 1 and data not shown). This AT2-mediated constitutive hypertrophic response was not attributable to a nonspecific effect of GFP overexpression, because infection with a control noncoding adenovirus (Ad-G0) did not significantly affect protein content under basal conditions (104.0±3.0%). Treatment of AT2-expressing neonatal cardiomyocytes with the PI3K inhibitor LY294002 completely reversed this AT2-mediated constitutive hypertrophic response (Figure 1). Coexpression of AT2 and AT1 receptors (Ad-AT2:Ad-AT1 multiplicity of infection [MOI] ratio: 20:5) induced significant basal hypertrophy (110.7±3.3%) and produced a markedly augmented hypertrophic response to Ang II that was also abolished by treatment with LY294002 (Figure 1). Thus, both exogenously expressed receptor subtypes mediate a hypertrophic response but use different signaling pathways, and only the AT1 receptor exhibits a requirement for ligand activation.
AT2 Modulation of AT1-Induced Autophagy
The capacity of AT1 and AT2 receptors to mediate or modulate cardiomyocyte autophagic responses was assessed. For this purpose, an HcRed-LC3 construct was generated to detect and quantify autophagic activity in live cells coexpressing the GFP-tagged receptor constructs. Ang II stimulation (48 hours) of neonatal cardiomyocytes expressing the AT1 receptor alone (Ad-AT1; 10 MOIs) induced a significant increase in the number of autophagosomes per cell (Figure 2). The Ang II–induced increase in autophagy was attenuated by treatment with the AT1 receptor antagonist candesartan and with 3-methyladenine, which inhibits autophagy in mammalian cells (Figure 2). Neonatal cardiomyocytes infected with Ad-AT2 alone (20 MOIs) or with both AT2 and AT1 receptors (Ad-AT2:Ad-AT1 MOI ratio: 20:5) exhibited significantly fewer autophagosomes per cell under basal conditions relative to cells infected with Ad-AT1 alone (Figure 2). Under conditions of AT2 coexpression, autophagic activity levels were similar to those seen in uninfected control cardiomyocytes (6.2±1.3 autophagosomes per cell). Ang II did not modulate autophagic activity in myocytes infected with Ad-AT2 alone or those coinfected with Ad-AT2 and Ad-AT1 (Figure 2). Furthermore, coexpression of AT2 and AT1 receptors completely abrogated the Ang II–induced increase in autophagy that was observed in myocytes expressing AT1 receptors alone (9.3±1.4 versus 33.3±4.1 autophagosomes per cell; P<0.05; Figure 2). Treatment with the AT2 receptor antagonist PD123319 did not reverse the AT2-mediated antiautophagic effect (Figure 2). Thus, the AT2 receptor constitutively augments AT1-induced growth and prevents autophagic induction in neonatal cardiomyocytes.
Characterizing Ang II Receptor Involvement in Growth and Autophagy Induction in a Pathophysiological Setting
AT1 Receptor-Mediated Growth Responses in NHR/HHR Neonatal Cardiomyocytes
The HHR, a genetic model derived from the spontaneously hypertensive rat, displays neonatal cardiac growth suppression followed by cardiac hypertrophy at maturity in the absence of pressure loading.12–14 At embryonic day 18 and at postnatal day 2, HHR ventricular weights were significantly lower than those of the NHR (Table). The disparity between HHR and NHR is also marked when ventricular weights are normalized to body weight (ventricular weight index; Table). Thus, relative to body size, the HHR exhibits suppressed myocardial growth during prenatal and early postnatal development.
In vitro techniques were used to examine HHR and NHR neonatal cardiomyocyte trophic responses to identify possible intrinsic differences in growth and autophagy induction. NHR, but not HHR, neonatal cardiomyocytes exhibited an Ang II–dependent hypertrophic growth response when the AT1 receptor alone was expressed in culture (Figure 3A and 3B). NHR myocytes undergoing AT1-induced growth displayed characteristic hypertrophic morphology, with cell body enlargement and process extension associated with a total increase in cellular volume (123.4±6.1%; Figure 3B). The Ang II–dependent elevation in the protein:DNA ratio in NHR was fully suppressed by the AT1 receptor antagonist candesartan or by the extracellular signal–regulated kinase 1/2 inhibitor PD98059 (102.7±6.6% and 102.5±8.4%; n=4 each; data not shown).
Autophagic Responses to Cardiomyocyte Stress in the NHR/HHR
In Ad-AT1–infected HHR cultures, Ang II induced a marked increase in the number of myocytes containing cytoplasmic vacuoles compared with NHR cultures (P<0.001; Figure 4A). We sought to determine whether vacuolar morphology was associated with a defined pathway of cell death induction. Vacuolar cardiomyocytes did not stain positive for the apoptotic marker TUNEL (Figure 4B), but rather were characterized by the presence of HcRed-LC3 puncta, indicative of autophagy (Figure 4B). Furthermore, experiments with control Sprague-Dawley cultures confirmed that Ang II stimulation (48 hours) of neonatal cardiomyocytes expressing either AT1 receptors alone or a high ratio of AT2:AT1 failed to elicit a significant increase in the incidence of apoptosis (Figure S2). Thus, HHR neonatal cardiomyocytes display an increased susceptibility to AT1 receptor–mediated autophagic vacuolization in vitro.
Evidence of an intrinsic vulnerability to autophagy in the HHR at a more mature developmental stage was sought. Ischemia has been identified previously as a potent stimulus for autophagy in the heart,15 and we have earlier reported that, in young adult HHR male hearts exhibiting established hypertrophy, there is increased susceptibility to ischemia-reperfusion injury compared with the NHR.13 An assessment of autophagic activity in the adult HHR myocardial tissue postischemia reperfusion was undertaken using a molecular marker approach. Although the autophagic markers beclin-1 and LC3B were not different between NHRs and HHRs at 12 weeks of age under basal conditions (data not shown), expression levels of both beclin-1 and LC3B were significantly elevated in HHR hearts relative to NHR hearts after ischemia reperfusion (P<0.01; Figure 4C).
Hypertrophic and Autophagic Effects of a High AT2:AT1 Receptor Expression Ratio in Neonatal HHR Cardiomyocytes
We next examined whether a perturbed balance of Ang II receptor subtypes could modulate the hypertrophic and autophagic response of HHR neonatal cardiomyocytes to Ang II in vitro. In the HHRs, where the Ad-AT1–mediated Ang II–dependent growth response was found to be suppressed (Figure 3 discussed above), when Ad-AT2 was coexpressed with Ad-AT1 (Ad-AT2:Ad-AT1 MOI ratio: 20:5), a significant growth response was restored. Measured by both an increase in protein/DNA and cell volume, this response under coexpression conditions was comparable to the NHR in the presence and absence of Ang II (Figure 5A). Treatment with the AT2 receptor antagonist PD123319 did not suppress the hypertrophic response (data not shown).
In Ad-AT1–infected HHR cultures, Ang II stimulation induced a marked increase in the number of identifiable autophagic myocytes characterized by vacuolar appearance (Figure 5B). This Ang II–mediated induction of cardiomyocyte vacuolization in the HHR was suppressed by treatment with the AT1 receptor antagonist candesartan (Figure 5B). The incidence of cardiomyocyte vacuolization in the HHR myocyte population decreased dramatically from 22.7±4.1% in Ad-AT1–infected cells to only 0.4±0.2% in myocytes coinfected with Ad-AT2 and Ad-AT1 (Figure 5B). Treatment with the AT2 receptor antagonist PD123319 did not reverse the AT2-mediated inhibition of autophagic vacuolization in HHR myocytes (Figure 5B). Therefore, in myocytes genetically predisposed to a heightened AT1-mediated autophagic response, a high AT2:AT1 receptor ratio completely prevented autophagic induction.
Investigating the In Vivo Relevance of AT1 and AT2 Receptor Expression in the Neonatal Heart
To establish the in vivo relevance of AT2-mediated inhibition of AT1-induced neonatal cardiomyocyte autophagy, AT1 and AT2 receptor expression levels were assessed in HHR neonatal hearts. Total Ang II binding sites were markedly reduced in the HHR ventricle at postnatal day 2 (NHR: 210±8.05 dpm/mm2 versus HHR: 99.5±19.1 dpm/mm2; P<0.0001). AT1-specific binding sites (ie, Ang II binding in presence of the AT2-selective antagonist PD123319) were significantly reduced in the HHR neonate ventricle (P=0.01; Figure 6A). AT1A and AT1B transcriptions were assessed by real-time quantitative RT-PCR. AT1A mRNA expression levels were 41% lower in HHR neonatal hearts, whereas AT1B expression did not differ between the 2 strains (Figure 6B). Significant AT2 receptor binding sites (ie, Ang II binding in presence of the AT1-selective antagonist candesartan) could not be resolved in the NHR/HHR ventricle at postnatal day 2. However, AT2 receptor mRNA was readily detectable (Ct ≈20 to 22 cycles) in NHR/HHR neonatal ventricles by real-time RT-PCR. At postnatal day 2, HHR hearts exhibited significantly higher (2.5-fold) AT2 receptor mRNA expression levels compared with NHR hearts (Figure 6B). Thus, in HHR hearts, which display a heightened susceptibility to AT1-induced autophagy in vitro, reduced AT1 receptor expression levels are identified, and AT2 receptor mRNA expression is upregulated.
Autophagy has emerged as an important process in the pathogenesis of cardiovascular diseases. However, the proximal triggers for autophagic induction in the heart are, as yet, only partially understood. This study is the first to demonstrate that Ang II plays a causative role in the induction of cardiomyocyte autophagy. Using in vitro conditions that allow quantitative modulation of receptor expression levels, we showed that Ang II exerts a proautophagic effect mediated via the AT1 receptor subtype. A novel antiautophagic role for the AT2 receptor subtype was identified, involving a constitutive, nonligand-regulated mechanism. The pathophysiological in vivo relevance of AT1-induced cardiomyocyte autophagy was assessed in a genetic model that exhibits cardiac growth abnormality. Neonatal HHR cardiomyocytes displayed an impaired AT1-mediated growth response to Ang II and were found to be more susceptible to AT1-induced autophagy in vitro. Coexpression of the AT2 receptor in vitro “rescued” the growth responsiveness to Ang II in the HHR and completely prevented autophagic induction. These findings indicate that the AT1 and AT2 receptor subtypes reciprocally regulate cardiomyocyte autophagy. A heightened susceptibility to Ang II–induced autophagy during early development may play a previously unrecognized role in the pathogenesis of primary cardiac hypertrophy. A potential role for AT2 receptor activation conferring therapeutic benefit in the clinical setting of autophagic cardiomyopathies is suggested.
AT1 Receptor Mediates Ang II–Induced Cardiomyocyte Autophagy
Increased autophagic activity has been identified as a prominent feature of the diseased myocardium. Autophagic cardiomyocytes have been reported in failing hearts from patients with dilated cardiomyopathy, valvular and hypertensive heart disease, and chronic ischemia.8 It appears that, whereas autophagy is initially beneficial and contributes to cell survival, chronic and/or excessive autophagic activity lead to cell death and may actively contribute to myocyte attrition responsible for the exacerbation of heart failure.8 Ang II plays a direct role in the pathogenesis of cardiac hypertrophy and heart failure. Cardiac-specific overexpression of angiotensinogen in the heart induces hypertrophy, heart failure, and premature mortality in transgenic mice.2 The current study indicates that Ang II exerts proautophagic effects in cardiomyocytes via the AT1 receptor subtype. To our knowledge, this is the first description of autophagic induction by a hormone known to be involved in the pathogenesis of heart failure.
Although angiotensin-converting enzyme inhibitors and AT1 receptor blockers are commonly prescribed to treat heart failure patients with lysosomal storage disorders16 and other vacuolar cardiomyopathies,17 the precise role of Ang II in the pathogenesis of these conditions is not known. Future studies should focus on the potential selective benefits of AT1 receptor blockade in the clinical setting of autophagic vacuolar cardiomyopathies.
AT2 Receptor Constitutively Antagonizes AT1-Mediated Autophagy
This study supports the contention that the AT2 receptor induces cardiomyocyte hypertrophy5–7,18 and reveals a novel role for this receptor in the regulation of cardiomyocyte autophagy. The present finding that the AT2 receptor induces cardiomyocyte growth and antagonizes cardiomyocyte autophagy is consistent with a PI3K-dependent signaling mechanism. Senbonmatsu et al19 demonstrated previously that the AT2 receptor interacts with the PLZF transcription factor, which increases transcription of the p85α regulatory subunit of class IA PI3K after AT2 receptor activation. Autophagy is inhibited by activation of class I PI3K.20 Physiological growth of the heart, as occurs during postnatal development and in response to regular physical activity or chronic exercise training, involves activation of PI3K and Akt. Although the impact of AT2 receptor knockout on cardiac remodeling in response to “physiological” hypertrophic stimuli has not been directly tested, it is noteworthy that cardiac overexpression of AT2 receptors improves left ventricular function at baseline and preserves function during postmyocardial infarction remodeling.21
Interestingly, we found that the AT2 receptor exerts a direct progrowth and antiautophagic action on neonatal cardiomyocytes, which is not modulated by Ang II or PD123319. Although it is not implausible to consider that the effects of AT2 receptor expression observed in the current study may be the result of an underlying interaction with an intracellular source of Ang II, our current observations are consistent with earlier observations from our laboratory7 and with other structural,22 pharmacological,23 and functional genomic24 analyses, indicating that the AT2 receptor is constitutively active. Therefore, many cellular effects of AT2 expression that antagonize AT1 receptor signaling and function may not be contingent on ligand interaction with this receptor. It has been proposed that the AT2 receptor antagonizes AT1 signaling through heterodimerization, and this physical interaction also does not appear to be regulated by Ang II.25 It is, therefore, possible that upregulation of the constitutively active AT2 receptor under pathological conditions allows the local renin-angiotensin system to escape regulatory control from its ligand by modulating AT1 receptor function through heterodimerization, independent of Ang II.
Role of AT2-Mediated Inhibition of Autophagy in the Neonate
Autophagic activity is elevated shortly after birth in neonatal tissues, including the heart.26 Autophagy is speculated to have several important roles in the neonate, including the recycling of amino acids in response to starvation, glycogen degradation, and programmed cell remodeling during the fetal-to-neonatal transition and in response to physiological oxidative stress that occurs at birth. HHR fetal and neonatal myocardial growth is suppressed and characterized by reduced expression levels of the AT1 receptor subtype relative to NHR. We have shown recently that there are fewer myocytes in the HHR heart at birth.14 HHR neonatal cardiomyocytes display a heightened sensitivity to AT1-induced autophagic vacuolization. The AT1 expression downregulation in the HHR neonate may be an adaptive response to protect against excess autophagy but may also be disadvantageous in conferring resistance to Ang II growth stimulation. We also found that adult HHR hearts display an increased susceptibility to autophagy in response to ex vivo ischemia-reperfusion injury. Although further experiments are required to determine the mechanistic basis for increased autophagic susceptibility at the cellular level in HHR adult hearts, these findings indicate that an underlying intrinsic susceptibility to autophagic cell death in the HHR contributes to cardiomyocyte loss in this model. A heightened autophagic response may have particularly significant ramifications during the neonatal starvation period,26 which occurs shortly after birth.
The precise contribution of elevated AT2 receptor gene expression to cardiac remodeling in the HHR neonate merits further resolution. Although AT2 receptor mRNA was readily detectable in NHR/HHR neonatal ventricles by real-time RT-PCR, AT2 receptor binding sites were below the detection limits of autoradiography. In this study, the in vitro AT2 coexpression level was manipulated to be relatively high (compared with AT1) to reflect pathological states where elevated AT2 expression has been reported previously. Interestingly, the expression analyses of neonatal HHR myocardium indicated a marked upregulation of AT2 mRNA coincident with downregulation of AT1A expression levels. It should, however, be noted that, in previous studies where high levels of AT2 receptor expression have been observed in the neonatal heart using autoradiography, vascular rather than myocardial localization may have predominated, and the cardiomyocyte expression levels achieved by adenoviral infection in the in vitro experiments detailed in this study may be higher than those encountered in vivo. Further experiments are required to determine the precise role of Ang II in modulating the HHR autophagic phenotype and to examine AT2 receptor–constitutive actions in vivo at low-level expression. Although the current findings suggest that overexpression of the AT2 receptor in the postnatal myocardium could potentially be exploited as an antiautophagic strategy in pathophysiological contexts, the potential impact of coincident hypertrophy induction would require consideration in a therapeutic setting.
In conclusion, this study provides novel evidence demonstrating that the AT2 receptor antagonizes AT1 receptor–mediated cardiomyocyte autophagy. This is the first study to demonstrate that Ang II regulates autophagy. We report that the AT2 receptor constitutively antagonizes AT1-mediated autophagy and that this effect is Ang II independent. In a model of neonatal myocardial growth suppression, an increased vulnerability to AT1-induced cardiomyocyte autophagic vacuolization is observed in vitro, and a high ratio of AT2:AT1 receptor expression is shown to rescue this autophagic state. These findings reveal a previously unrecognized role for the AT2 receptor in the regulation of cardiomyocyte autophagy and provide a lead for investigation of novel therapeutic strategies to enhance cell survival in congenital and adult autophagic cardiomyopathies. Future experiments will be necessary to experimentally evaluate whether AT2 receptor transgenic and knockout mice have altered myocardial autophagic activity under basal conditions and in response to stresses, eg, neonatal starvation and myocardial infarction. Given the X chromosome location of the AT2 receptor gene (Agtr2), the potential role of this receptor in congenital forms of X-linked autophagic vacuolar cardiomyopathy should also be considered.
We acknowledge William Meeker and Jaspreet Bassi for excellent technical contributions.
Sources of Funding
This work was supported by the National Health and Medical Research Council of Australia, the Australian Research Council, and the Wenkart Foundation. Career support (to E.R.P.) was provided by an Australian Postgraduate Award and a Baker Foundation Postgraduate Award (Henry Cooper Scholarship).
- Received December 22, 2008.
- Revision received January 14, 2009.
- Accepted April 12, 2009.
Domenighetti AA, Wang Q, Egger M, Richards SM, Pedrazzini T, Delbridge LM. Angiotensin II-mediated phenotypic cardiomyocyte remodeling leads to age-dependent cardiac dysfunction and failure. Hypertension. 2005; 46: 426–432.
Ichihara S, Senbonmatsu T, Price E Jr, Ichiki T, Gaffney FA, Inagami T. Angiotensin II type 2 receptor is essential for left ventricular hypertrophy and cardiac fibrosis in chronic angiotensin II-induced hypertension. Circulation. 2001; 104: 346–351.
D'Amore A, Black MJ, Thomas WG. The angiotensin II type 2 receptor causes constitutive growth of cardiomyocytes and does not antagonize angiotensin II type 1 receptor-mediated hypertrophy. Hypertension. 2005; 46: 1347–1354.
Tannous P, Zhu H, Nemchenko A, Berry JM, Johnstone JL, Shelton JM, Miller FJ Jr, Rothermel BA, Hill JA. Intracellular protein aggregation is a proximal trigger of cardiomyocyte autophagy. Circulation. 2008; 117: 3070–3078.
Thomas WG, Brandenburger Y, Autelitano DJ, Pham T, Qian H, Hannan RD. Adenoviral-directed expression of the type 1A angiotensin receptor promotes cardiomyocyte hypertrophy via transactivation of the epidermal growth factor receptor. Circ Res. 2002; 90: 135–142.
Harrap SB, Danes VR, Ellis JA, Griffiths CD, Jones EF, Delbridge LM. The hypertrophic heart rat: a new normotensive model of genetic cardiac and cardiomyocyte hypertrophy. Physiol Genomics. 2002; 9: 43–48.
Bell JR, Porrello ER, Huggins CE, Harrap SB, Delbridge LM. The intrinsic resistance of female hearts to an ischemic insult is abrogated in primary cardiac hypertrophy. Am J Physiol Heart Circ Physiol. 2008; 294: H1514–H1522.
Porrello ER, Bell JR, Schertzer JD, Curl CL, McMullen JR, Mellor KM, Ritchie RH, Lynch GS, Harrap SB, Thomas WG, Delbridge LM. Heritable pathologic cardiac hypertrophy in adulthood is preceded by neonatal cardiac growth restriction. Am J Physiol Regul Integr Comp Physiol. 2009; 96: R672–R680.
Matsui Y, Takagi H, Qu X, Abdellatif M, Sakoda H, Asano T, Levine B, Sadoshima J. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res. 2007; 100: 914–922.
Linhart A, Elliott PM. The heart in Anderson-Fabry disease and other lysosomal storage disorders. Heart. 2007; 93: 528–535.
Saijo M, Takemura G, Koda M, Okada H, Miyata S, Ohno Y, Kawasaki M, Tsuchiya K, Nishigaki K, Minatoguchi S, Goto K, Fujiwara H. Cardiomyopathy with prominent autophagic degeneration, accompanied by an elevated plasma brain natriuretic peptide level despite the lack of overt heart failure. Intern Med. 2004; 43: 700–703.
Yan X, Price RL, Nakayama M, Ito K, Schuldt AJ, Manning WJ, Sanbe A, Borg TK, Robbins J, Lorell BH. Ventricular-specific expression of angiotensin II type 2 receptors causes dilated cardiomyopathy and heart failure in transgenic mice. Am J Physiol Heart Circ Physiol. 2003; 285: H2179–H2187.
Senbonmatsu T, Saito T, Landon EJ, Watanabe O, Price E Jr, Roberts RL, Imboden H, Fitzgerald TG, Gaffney FA, Inagami T. A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy. EMBO J. 2003; 22: 6471–6482.
Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P. Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem. 2000; 275: 992–998.
Yang Z, Bove CM, French BA, Epstein FH, Berr SS, DiMaria JM, Gibson JJ, Carey RM, Kramer CM. Angiotensin II type 2 receptor overexpression preserves left ventricular function after myocardial infarction. Circulation. 2002; 106: 106–111.
Miura S, Karnik SS. Ligand-independent signals from angiotensin II type 2 receptor induce apoptosis. EMBO J. 2000; 19: 4026–4035.
Falcon BL, Veerasingham SJ, Sumners C, Raizada MK. Angiotensin II type 2 receptor-mediated gene expression profiling in human coronary artery endothelial cells. Hypertension. 2005; 45: 692–697.
AbdAlla S, Lother H, Abdel-tawab AM, Quitterer U. The angiotensin II AT2 receptor is an AT1 receptor antagonist. J Biol Chem. 2001; 276: 39721–39726.