Acute Pressure Overload Could Induce Hypertrophic Responses in the Heart of Angiotensin II Type 1a Knockout Mice
Abstract—Increasing evidence has suggested that locally produced angiotensin II (Ang II) plays an important role in the development of cardiac hypertrophy through the Ang II type 1 receptor (AT1). We and others have recently reported that Ang II is critical for mechanical stress–induced hypertrophic responses in vitro. Using AT1a knockout (KO) mice, we examined whether Ang II is indispensable for pressure overload–induced cardiac hypertrophy in the present study. Reverse-transcriptase polymerase chain reaction analysis revealed that AT1 mRNA levels were <10% in the heart of KO mice compared with wild-type (WT) mice, but the Ang II type 2 receptor gene was expressed at almost the same levels in the hearts of both mice. Intravenous infusion of subpressor dose of Ang II induced c-fos gene expression in the hearts of WT mice but not KO mice. Acute pressure overload, however, induced expressions of immediate-early response genes and activations of mitogen-activated protein kinases in the hearts of KO mice as well as WT mice. Both basal and activated levels of all these responses were significantly higher in KO mice than in WT mice. Pressure overload markedly increased the heart weight–to–body weight ratio in both mice strains at 14 days after aortic banding. These results suggest that acute hypertrophic responses could be induced by pressure overload in the in vivo heart without AT1 signaling.
Since recent clinical studies have suggested that cardiac hypertrophy is an independent risk factor of cardiac morbidity and mortality,1 it has become even more important to clarify the mechanism of how cardiac hypertrophy is developed. Cardiac hypertrophy is induced by mechanical load2 3 4 and humoral factors, such as Ang II,5 ET-1,6 PHE,7 and peptide growth factors.8 Among them, Ang II has recently attracted great attention, not because of its potency (compared with ET-1 and PHE) but because of its established importance in vivo as well as in vitro.5 9 A growing body of evidence has suggested that locally produced Ang II, more than circulating Ang II, is a potent stimulator of cardiac hypertrophy and that hemodynamic overload induces cardiac hypertrophy by activating the local renin-angiotensin system.5 9 Baker et al10 have reported that the increase in left ventricular mass induced by constricting the abdominal aorta was completely prevented by an ACE inhibitor without decreasing the arterial pressure. Ang II directly induces hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts without an increase in vascular resistance or cardiac afterload.5 9 11 Expression of ventricular Ang II receptors has been reported to be enhanced during the development of cardiac hypertrophy.12 13 In addition, it has been demonstrated that mRNA levels of cardiac ACE,14 angiotensinogen,10 and local synthesis of Ang II14 are increased in the hypertrophied heart.
We and others have reported that mechanical stress stimulates the secretion of Ang II from cardiac myocytes and that Ang II induces cardiomyocyte hypertrophy through AT1.15 16 However, since AT1-specific antagonists only partially inhibit stretch-induced hypertrophic responses,16 signaling pathways other than Ang II may be involved in mechanical stress–induced hypertrophy. We have reported that ET-1 is also involved in stretch-induced cardiac myocyte hypertrophy.17 It has been demonstrated that passive load and Ang II evoke different responses in terms of gene expression and protein synthesis in cardiomyocytes.18 Therefore, it is not clear whether Ang II really plays a critical role in the development of cardiac hypertrophy induced by pressure overload. Moreover, we have recently observed that chronic pressure overload produced by constricting the abdominal aorta induces cardiac hypertrophy with the expression of fetal genes not only in WT mice but also in AT1a KO mice,19 in which signaling pathways through AT1a are genetically deleted, suggesting that Ang II is not required for the development of pressure overload–induced cardiac hypertrophy (authors’ unpublished data, 1998).
In the present study, to confirm that Ang II is not required for the development of pressure overload–induced cardiac hypertrophy and to gain insight into the mechanism by which pressure overload induces cardiac hypertrophy without Ang II−evoked signaling pathways, we examined acute hypertrophic responses in the hearts of AT1a KO mice by using a different pressure-overload model. Both RT-PCR analysis and intravenous infusion of Ang II showed that there was little functional AT1, if any, in KO hearts. Pressure overload produced by constricting the transverse aorta, however, induced the expression of immediate-early response genes, such as c-fos, c-jun, and BNP, and the activation of MAPKs in the hearts of KO mice as well as WT mice. The HW/BW ratio was also increased in both KO and WT mice in this pressure-overload model.
Materials and Methods
AT1a KO mice and WT mice (18 weeks old) from the same genetic background were used in the present study. Mice were housed under climate-controlled conditions with a 12-hour light/dark cycle and were provided with standard food and water ad libitum as described previously.20 All protocols were approved by local institutional guidelines.
Pressure overload was produced by transverse aortic constriction as described previously.21 22 Briefly, mice were anesthetized by intraperitoneal injection of a cocktail of ketamine (100 mg/kg) and xylazine (5 mg/kg), and respiration was artificially controlled with a tidal volume of 0.2 mL and a respiratory rate of 110 breaths/min. The transverse aorta was constricted with 7–0 nylon strings by ligating the aorta with a blunted 27-gauge needle, which was pulled out later. To monitor the hemodynamic effects of aortic constriction, both right and left carotid arteries were cannulated with flame-stretched PE-50 tubing, and pulse wave forms were simultaneously monitored by using a polygraph system (Nihon Koden Co). After aortic constriction, the chest was closed, and the mice were allowed to recover from anesthesia. At the end of the experiments, hearts were excised, weighed, and frozen in liquid nitrogen.
Infusion of Subpressor Doses of Ang II
Mice were anesthetized by intraperitoneal injection of a cocktail of ketamine and xylazine as described above. A flame-stretched PE-50 tube was placed in the carotid artery and the jugular vein. Arterial pressures were recorded continuously by using the polygraph system. After baseline was stabilized, a subpressor dose of Ang II (100 ng · kg−1 · min−1)23 was administered into the jugular vein for 10 minutes at a rate of 50 μL/min (total volume, 500 μL). As a control study, the same volume of saline alone was infused into the jugular vein. Thirty minutes after infusion, hearts were removed and frozen in liquid nitrogen for Northern blot analysis.
Quantitative RT-PCR Analysis
Total RNA was prepared from the hearts of mice by using RNA STAT-60 (TEL-TEST “B” Inc) and treated with DNase (Takara Shuzo) to eliminate contamination of genomic DNA.12 The RT-PCR analysis for AT1 and AT2 mRNA quantification was performed using the deletion-mutated cRNA as an internal control as described before.12 13 19 24 We have reported that the amplification efficiencies of target and competitor transcripts are equal under optimal concentrations of competitor transcripts. The oligonucleotide primers used for RT-PCR analysis are as follows12 13 19 24 : for AT1, 5′-GAGTCCTGTTCCACCCGATCACCGATCAC-3′ and 5′-GGATGACGCCCAGCTGAATCAGCACATCC-3′; for AT2, 5′-TTGCTGCCACCAGCAGAAAC-3′ and 5′-GTGTGGGCCTCCAAACCATTGCTA-3′. The sequence of these primers is identical to that of murine Ang II receptor cDNA.25 26 We also quantified both ACE mRNA and angiotensinogen mRNA levels by using basically the same method. The following PCR primers were designed from the cDNA sequence of mouse ACE27 and used for RT-PCR analysis: sense primer 1, 5′-CGGAGTCAATGCTGGAGAAA-3′; antisense primer 2, 5′-CATGGTCCAGTAGGCCGATT-3′. The 298-bp PCR product was subcloned into the pCR II vector (Promega Corp). To obtain deletion-mutated cDNA, we amplified this PCR product by the following PCR primers: sense primer 1, 5′-CGGAGTCAATGCTGGAGAAA-3′; antisense primer 3, 5′-CATGGTCCAGTAGGCCGATTGCTTAATCCCCGGAAGTCCT-3′. Since antisense primer 3 corresponds to two sequences (one is primer 2, and the other is the middle region of the first PCR product) that are 117 bp apart, the second PCR product was deleted by 117 bp. The deletion-mutated cRNA was synthesized using SP6 RNA polymerase (Promega Corp) after linearization with XhoI. Total RNA (1 μg) and the deletion-mutated cRNA (1 pg) were simultaneously mixed and assayed by competitive RT-PCR using primers 1 and 2. The following PCR primers28 were designed from the cDNA sequence of rat angiotensinogen and used for RT-PCR analysis: sense primer 1, 5′-GACCGCGTATACATCCACCCCTTTCATCTC-3′; antisense primer 2, 5′-GTCCACCCAGAACTCATGGAGCCCAGTCAG-3′. The 810-bp PCR product was subcloned into the pCR II vector. The resulting plasmid was cut with BstEII and HincII, blunt-ended with Klenow treatment, and self-ligated. A fragment of 434 bp should be synthesized by PCR from the deletion-mutated cRNA. Total RNA (1 μg) and the deletion-mutated cRNA (1 pg) were simultaneously mixed and assayed by competitive RT-PCR using primers 1 and 2. Denaturing, annealing, and extension reactions were performed 30 times at 94°C for 45 seconds, 58°C for 1 minute, and 72°C for 1 minute, respectively. The possibility of genomic DNA contamination in the RNA sample was excluded by performing PCR without the step of RT, in which no significant product was visible after 40 cycles. The range of concentrations of sample RNA and internal control–deleted cRNA, as well as the number of amplification cycles, was selected from within the exponential phase. To quantify mRNA levels, 5 μCi of [α-32P]dCTP was included in the PCR reaction mixtures. The bands of interest were excised from the agarose gel, and 32P incorporation was measured with a scintillation counter as described before.12 13 19 24
Northern Blot Analysis
Total RNA was extracted from hearts of mice at 0, 30, 60, or 120 minutes after aortic constriction. Total RNA (10 μg) was separated on a 1.2% agarose/formaldehyde gel and blotted onto a Hybond-N membrane (Amersham Co). cDNA of human c-fos and c-jun were obtained from the Japanese Cancer Research Resources Bank. Rat BNP cDNA was a gift from H. Ito (Tokyo Medical and Dental University).29 These cDNAs were labeled by random priming with [α-32P]dCTP. Quantification of hybridized bands was carried out using a FUJIX Bio-Imaging Analyzer BAS 2000 (Fuji Film Co).
MAPK activities were measured using MBP-containing gels as described previously.16 In brief, MAPKs were immunoprecipitated with polyclonal antibodies against MAPKs (αY91) in the presence of 0.15% SDS, and the immunoprecipitates were electrophoresed on an SDS-polyacrylamide gel containing 0.5 mg/mL MBP. Phosphorylation of MBP was assayed by incubating the gel with [γ-32P]dATP. After incubation, the gel was washed extensively, dried, and then subjected to autoradiography.
All results are expressed as mean±SEM. Multiple comparisons among three or more groups were carried out by two-way ANOVA and the Fisher exact test for post hoc analyses. Statistical significance was accepted at a value of P<.05.
Quantitative RT-PCR Analysis of AT1 and AT2 mRNA in Murine Hearts
We assessed mRNA levels of AT1 and AT2 by quantitative RT-PCR analysis (Figure 1⇓). RT-PCR analysis of cardiac mRNA revealed that the AT1 gene was expressed abundantly in WT hearts, whereas only a slight expression of AT1 was detected in KO hearts (Figure 1A⇓). mRNA levels of AT1 in KO hearts were <10% of those in WT hearts (Figure 1A⇓). Since the AT1a gene was completely deleted from the genome of KO mice, the slight expression of AT1 in KO hearts might be the transcript of the AT1b gene. There was no difference in AT2 mRNA levels between WT and KO hearts (Figure 1B⇓). Pressure overload for 30 minutes did not change mRNA levels of AT1 and AT2 in either WT or KO hearts (Figure 1A⇓ and 1B⇓). Although basal levels of angiotensinogen gene expression were increased in KO mice, mRNA levels of ACE and angiotensinogen were not changed by pressure overload for 30 minutes in both animal hearts (Figure 1C⇓ and 1D⇓).
Effects of Infused Ang II on Expression of c-fos Gene
Sequences of AT1a and AT1b are 96% identical at amino acid levels, and to date, they are pharmacologically indistinguishable from each other.30 Therefore, to determine the effect of the slightly expressed AT1b on Ang II signaling, we infused a subpressor dose of Ang II (100 ng · kg−1 · min−1) intravenously for 10 minutes at a rate of 50 μL/min and examined expression of the c-fos gene as one of the early hypertrophic responses, which have been reported to be induced by Ang II through AT1.15 16 Systolic blood pressure was unchanged in both mice receiving Ang II, consistent with a previous report.23 Subpressor doses of Ang II induced expression of the c-fos gene in the hearts of WT but not KO mice (Figure 2⇓), suggesting that hypertrophic responses evoked through AT1 were markedly reduced in KO hearts.
Hemodynamic Response to Aortic Constriction
Pressure overload was produced by constricting the transverse aorta under anesthesia as described previously.21 This model is a well-characterized in vivo model of pressure overload–induced hypertrophy.22 The blood pressure was monitored at bilateral carotid arteries. Although arterial pressure was somewhat variable, the peak-to-peak systolic pressure gradients across the stenosis were almost the same between WT and KO mice, 43.8±10.7 mm Hg in WT mice (Figure 3A⇓) and 43.3±8.8 mm Hg in KO mice (Figure 3B⇓). The pressure gradients were maintained constant throughout the subsequent 120 minutes. This finding was in good agreement with a previous report.21
Quantitative RT-PCR Analysis of ACE mRNA in Murine Hearts
The expression levels of ACE mRNA were examined in the left ventricles of WT mice before and after aortic constriction for 14 days. Quantitative RT-PCR analysis of cardiac mRNA revealed that ACE mRNA levels in the hearts of WT mice after transverse aortic banding were significantly higher than those of sham-operated WT mice (3.7-fold increase), suggesting that the intracardiac renin-angiotensin system was activated in this pressure-overload model.
Effects of Pressure Overload on Expression of c-fos, c-jun, and BNP Genes
Previous studies have reported that cardiac hypertrophy produced by transverse aortic banding is a good model for examining the induction of immediate-early response genes by mechanical stimuli.22 To determine the role of Ang II in pressure overload–induced gene expression in the heart, we examined the expression of immediate-early response genes, such as c-fos, c-jun, and BNP, which have been reported to be induced in the rat heart by pressure overload.2 As previously reported in rat hearts,2 acute pressure overload rapidly induced the expression of c-fos, c-jun, and BNP genes in WT mouse hearts (Figure 4A⇓). In KO hearts, basal levels of c-jun and BNP genes were higher than those of WT hearts, and expressions of all these genes were induced more abundantly in KO hearts than in WT hearts (Figure 4B⇓ and 4C⇓). Expressions of all these genes peaked at 30 minutes after aortic constriction in both WT and KO mice, and there was no difference in the time course of the gene induction between WT and KO mice. These results suggest that Ang II is not required for the induction of these immediate-early response genes by acute pressure overload.
Effects of Pressure Overload on MAPKs
Our and other laboratories have reported that mechanical stress activates MAPKs in cultured cardiac myocytes of neonatal rats.16 31 Recent studies have suggested that MAPKs were critical for a variety of hypertrophic responses.32 33 Thus, we next examined whether acute pressure overload induces activation of MAPKs in in vivo hearts of WT and KO mice. Pressure overload generated by transverse aortic constriction activated 42- and 44-kD MAPKs in the hearts of WT mice (Figure 5A⇓). In KO mice, some activities of MAPKs were detected in control hearts, and pressure overload markedly activated both 42- and 44-kD MAPKs (Figure 5A⇓). Both basal and stimulated levels of MAPKs were higher in KO hearts than in WT hearts (Figure 5B⇓). These results suggest that AT1 is not necessary for pressure overload–induced activation of MAPKs, which are critical for cardiac hypertrophy.
Ventricular Hypertrophy by Transverse Aortic Banding
To determine whether pressure overload not only induces acute hypertrophic responses but also induces cardiac hypertrophy in the absence of AT1 signaling, we examined the HW/BW ratio at 14 days after transverse aortic banding. Pressure overload increased the HW/BW ratio not only in WT mice but also in KO mice (Figure 6⇓). The degree of an increase in the HW/BW ratio was significantly higher in KO mice than in WT mice (WT, 37.7±5.2% increase; KO, 55.7±2.0% increase; P<.05).
KO animals afford novel opportunities to determine the role of molecules of interest in in vivo physiological situations. The aim of the present study was to clarify whether Ang II is really necessary for the induction of acute hypertrophic responses produced by pressure overload. We examined acute hypertrophic responses, such as the expression of immediate-early response genes and activation of MAPKs, in the hearts of AT1a null mutant mice, where almost no signal through AT1 was recognized. Acute pressure overload fully elicited acute hypertrophic responses, such as induction of c-fos, c-jun, and BNP gene expression and MAPK activation, in KO hearts as well as in WT hearts. Moreover, pressure overload increased the HW/BW ratio not only in WT mice but also in KO mice.
It has been suggested that in addition to neurohumoral factors, mechanical stress induces cardiac hypertrophy.2 15 16 However, it is still largely unknown how mechanical stress is converted into biochemical signals leading to cardiac hypertrophy. Our and other laboratories have shown that mechanical stretch induces a variety of hypertrophic responses, including activation of MAPKs, expression of specific genes, such as immediate-early response genes and fetal genes, and an increase in protein synthesis in cardiac myocytes through AT1.2 4 15 16 However, since AT1 antagonists only partially inhibited these stretch-induced cardiac events, factors other than Ang II may be involved. We have recently reported that secretion of ET-1 is also stimulated by mechanical stress and that ET-1, as well as Ang II, is involved in stretch-induced hypertrophic responses.17 Thus, vasoactive peptides may generally mediate mechanical stress–induced cardiac hypertrophy. However, various stretch-induced events were not completely inhibited, even in the presence of both Ang II and ET-1 antagonists.17 It has been reported that Ang II is stored in secretory granules in cardiac myocytes.15 Mechanical stress should first evoke some signals to induce the secretion of Ang II and ET-1 from cardiac myocytes. In addition, all previous results were obtained mainly from experiments using pharmacological agents,10 15 16 17 which may have nonspecific effects. Therefore, in the present study, we examined the role of Ang II in pressure overload–induced cardiac hypertrophy using genetically AT1a-deleted mice.
The development of cardiac hypertrophy produced by constricting thoracic14 34 35 or abdominal5 10 36 aortas has often been attributed to activation of the intracardiac renin-angiotensin system. Baker et al10 and Linz et al36 have reported that pressure overload produced by constricting the abdominal aorta induces cardiac hypertrophy by activating the cardiac renin-angiotensin system, because the administration of the ACE inhibitor attenuated the development of ventricular hypertrophy. Schunkert and colleagues14 34 35 have reported that ACE content, mRNA levels of ACE, and angiotensinogen are increased in the hypertrophic myocardium after ascending aortic banding. We constricted the transverse aorta because this pressure overload model has the advantage of examining the effects of the exact hemodynamic load on the heart.21 22 A previous study has reported that cardiac hypertrophy produced by transverse aortic banding is not prevented by the ACE inhibitor,37 suggesting that this form of pressure overload induces cardiac hypertrophy independent of the activation of the intracardiac renin-angiotensin system. However, there is a possibility that the amount of ACE inhibitor they used in the experiment was not enough to completely suppress the renin-angiotensin system in the heart. In the present study, we observed a 3.7-fold increase in ACE mRNA levels in WT hearts after transverse aortic banding, suggesting that the renin-angiotensin system is also activated by transverse aortic banding. Since these results are in good agreement with recent studies using the model of left ventricular hypertrophy produced by constricting the ascending aorta,14 34 35 we believe that our experimental model is appropriate for investigating the role of Ang II in pressure overload–induced cardiac hypertrophy.
It has been reported that two subtypes of AT1, AT1a and AT1b, are expressed in rat hearts.30 The expression ratio of AT1a and AT1b is different among tissues and developmental stages,12 30 and it has been unknown in murine hearts. Although we did not measure AT1a and AT1b mRNA levels separately in the present study, since there should be no AT1a mRNA in the KO heart,20 the slight amount of AT1 expressed in KO hearts might be transcripts of the AT1b gene. To evaluate the expression of AT1b in KO hearts, we intravenously infused the subpressor dose of Ang II. Although the infusion of Ang II markedly induced expressions of c-fos in WT hearts, expression of the c-fos gene was not induced in KO hearts. These results suggest that expression levels of AT1b are so low that response to Ang II through AT1 is markedly reduced in KO hearts.
We examined the expression of c-fos, c-jun, and BNP genes as an early genetic marker of cardiac hypertrophy. Although the role of proto-oncogenes in cardiac hypertrophy is not fully understood, they may induce reprogramming of gene expression in the heart at later stages.2 BNP is a natriuretic peptide produced in the heart and has been recently reported to be one of the immediate-early response proteins.38 We measured the activity of MAPKs as another marker of acute hypertrophic response. Many lines of evidence have suggested that MAPKs function as integrators for mitogenic and differentiation signals in many cell types.39 In cardiac myocytes, activation of MAPKs is also required for the PHE-induced expression of specific genes, such as atrial natriuretic factor, c-fos, and myosin light chain-2 genes.32 Although activation of MAPKs may not be sufficient to fully promote cardiac hypertrophy,40 41 recent evidence using an antisense oligodeoxynucleotide has shown that MAPKs play a critical role in PHE-induced sarcomerogenesis and increased cell size.33 In the present study, pressure overload fully induced these hypertrophic responses and increased the HW/BW ratio of KO mice as well as WT mice. Moreover, although the reason is not clear at present, basal and stimulated levels of these hypertrophic responses were higher in KO hearts than in WT hearts. It is difficult to completely rule out the involvement of the slightly expressed AT1b in pressure overload–induced hypertrophic responses in KO mice; however, these results strongly suggest that pressure overload evoked hypertrophic responses not through AT1. Interestingly, ventricular weights at 14 days after aortic constriction were also higher in KO mice than in WT mice.
It has been reported that left ventricular hypertrophy induced by constricting the abdominal aorta was completely prevented by an ACE inhibitor.10 In vitro studies using cultured cardiac myocytes have also shown that mechanical stress–induced hypertrophic responses are inhibited by AT1 antagonists.15 16 Moreover, it has been demonstrated that Ang II is stored in granules of cardiac myocytes and that the secretion of Ang II is induced by mechanical stress.15 In the present study, however, mechanical stress fully induced hypertrophic responses and ventricular hypertrophy in hearts lacking AT1 signaling. We cannot explain the reason for the discrepancy between present and previous results at this moment; however, there are several possibilities, as follows: Ang II may actually play an important role in pressure overload–induced cardiac hypertrophy in vivo, and other factors may fully compensate its role in KO hearts. However, it may be difficult to explain the higher basal and poststimulated levels of hypertrophic responses in KO hearts by simple compensation of other factors. Some unknown factors, which induce hypertrophic responses, may be activated by the absence of signals from AT1a. Another possibility is that previous results based mainly on pharmacological agents may be wrong or oversimplified. ACE inhibitors and AT1 antagonists may inhibit or prevent the development of cardiac hypertrophy not by inhibiting local action of Ang II in the heart but by decreasing hemodynamic load in vivo. It has been reported that passive load and Ang II evoke different responses of gene expression and protein synthesis in cardiac myocytes.18 Ang II may not be a major mediator or at least may not be indispensable for mechanical stress–induced cardiac hypertrophy. These results suggest that the simple scheme, mechanical stretch→Ang II secretion→cardiac hypertrophy, may need to be reconsidered. Further studies elucidating how pressure overload induces hypertrophic responses in the hearts of KO mice may reveal the molecular mechanism of mechanical stress–induced cardiac hypertrophy.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1||=||Ang II type 1 receptor|
|AT2||=||Ang II type 2 receptor|
|BNP||=||brain natriuretic peptide|
|HW/BW ratio||=||heart weight–to–body weight ratio|
|MAPK||=||mitogen-activated protein kinase|
|MBP||=||myelin basic protein|
|PCR||=||polymerase chain reaction|
This study was supported by a grant-in-aid for scientific research, developmental scientific research, and scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Japan Heart Foundation/Pfizer Pharmaceuticals (grant for research on coronary artery diseases), Tanabe Medical Frontier, and the Sankyo Life Science and Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan (to Dr Komuro).
This manuscript was sent to Howard E. Morgan, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
- Received December 2, 1997.
- Accepted February 2, 1998.
- © 1998 American Heart Association, Inc.
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