© 1996 American Heart Association, Inc.
Articles |
Mechanical Stretch Induces Enhanced Expression of Angiotensin II Receptor Subtypes in Neonatal Rat Cardiac Myocytes
the Department of Medicine II (K.K., H.M., S.M., K.M., Y.M., N.O., T.I., M.I.), Kansai Medical University, Osaka, Japan, and the Department of Medicine III (I.K., Y.Y.), Tokyo University School of Medicine, Tokyo, Japan.
Correspondence to Hiroaki Matsubara, MD, Department of Medicine II, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570, Japan.
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Abstract |
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Mechanical stress plays a pivotal role in the development of cardiac hypertrophy during hemodynamic overload, and angiotensin (Ang) II secreted from stretched myocytes plays an important role in mechanical stretch–induced hypertrophy. In the present study, we examined stretch-induced expression of Ang II receptors in an in vitro stretch model using 1-day-old rat myocytes. Both Ang II type 1 receptor (AT1-R) and type 2 receptor (AT2-R) mRNA levels were upregulated by myocyte stretching with similar time courses: significant increases were evident 6 hours after stretching, maximal levels (2.8- and 3.3-fold, respectively) were observed at 12 hours, and these were sustained for up to 18 hours. Ang II receptor expression in fibroblast-rich cultures was not affected by stretching. Conditioned medium in which myocytes were stretched for 12 hours significantly downregulated AT1-R and AT2-R mRNA levels in recipient myocytes, and this effect was almost completely blocked by AT1-R antagonists but not AT2-R antagonists. Stretch-induced expression of AT1-R and AT2-R mRNAs was further increased by 27% and 31%, respectively, after pretreatment with AT1-R antagonists, suggesting that Ang II secreted from stretched myocytes downregulates both AT1-R and AT2-R. Western blot and binding assays showed that the number of AT1-Rs and AT2-Rs increased by 2.4- and 2.6-fold, respectively, without affecting receptor affinities. Inositol phosphate response to 0.5 µmol/L Ang II was enhanced 2.1-fold in stretched myocytes. Nuclear runoff assays and treatment with actinomycin D revealed that stretch-induced upregulation of AT1-R was mainly due to increased transcription, whereas that of AT2-R resulted from a stabilizing effect on AT2-R mRNA metabolism. Stretch-induced changes in levels of Ang II receptors were inhibited by genistein but not by H-7, staurosporin, and protein kinase C depletion or by BAPTA-AM. Exposure to cycloheximide did not affect stretch-induced changes. These findings indicate that nonsecretory pathways activated by myocyte stretching upregulate the expression of Ang II receptor subtypes transcriptionally and posttranscriptionally through mechanisms involving stretch-activated tyrosine kinases independently of de novo protein synthesis and that the AT1-R–mediated action of Ang II is functionally enhanced in stretched cardiac myocytes.
Key Words: angiotensin II • receptor • myocyte stretch • cardiac hypertrophy • cardiomyocyte
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Introduction |
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Cardiac hypertrophy is not only a fundamental process of adaptation to workload but also an important cause of increased morbidity and mortality.1 Many lines of evidence have suggested that mechanical stress plays a pivotal role in development of cardiac hypertrophy during hemodynamic overload without the involvement of humoral and/or neural factors.2 We have previously reported that mechanical loading on cultured myocytes, using an in vitro stretch model, activates protein kinase phosphorylation cascades3 and induces the expression of specific genes and increases in protein synthesis.3 4 5 Mechanical stretch–induced events were reported to be completely dependent on Ang II secreted from myocytes,6 whereas we found that nonsecretory pathways are also involved in this response.7
Evidence for the presence of an endogenous renin-Ang system in the heart includes the demonstration of mRNAs encoding angiotensinogen and renin,8 ACE,9 and Ang II receptors10 11 12 and the detection of Ang I and II radioimmunoreactivities.13 Upregulation of left ventricular angiotensinogen,14 ACE,15 and Ang II receptor mRNAs10 11 12 has been described in association with cardiac hypertrophy and myocardial infarction. Moreover, an increase in left ventricular mass produced by pressure overload was completely prevented by the ACE inhibitor without any change in afterload or plasma renin activity.16 These findings suggest that the local Ang system plays a critical role in cardiac hypertrophy induced by pressure overload and that Ang II acts to promote the growth of cardiac myocytes by autocrine or paracrine mechanisms.
At least two main Ang II receptor subtypes, AT1-R and AT2-R, have been identified using receptor subtype–specific nonpeptide antagonists.17 Most of the well-known Ang II functions in the cardiovascular system are mediated by AT1-R; therefore, AT1-R antagonists have been widely used in patients with hypertension or heart failure. A cDNA clone encoding AT2-R has been isolated recently, and it was shown to have 32% homology with AT1-R at the protein level,18 19 whereas there is little information available regarding the physiological roles of AT2-R.20 Using mice lacking the AT2-R, Ichiki et al21 and Hein et al22 have recently found that the AT2-R is involved in the maintenance of systemic blood pressure and responsiveness of the cardiovascular system to Ang II. The AT2-R has an antiproliferative effect on the neointima after vascular injury23 and on coronary endothelial cells.24 In addition, it was shown that the AT2-R contributes to the induction of apoptosis25 and modifies phosphotyrosine phosphatase activity19 and voltage-sensitive ion currents.26 27 The AT2-R is abundantly and widely expressed in fetal tissues, declines immediately after birth, and is localized in limited regions in adults,20 28 29 and its expression is activated in skin wounds30 or neointima after vascular injury.23 These findings suggest an involvement of AT2-R in the control of the cardiovascular system as well as an important role in growth and development. Although the levels of expression of AT1-R and AT2-R are very low in adult rat hearts, we quantified these receptor subtypes at the transcription, mRNA, and protein levels and demonstrated that levels of both AT1-R and AT2-R expression are increased in neonatal rat hearts10 11 or in hearts that have undergone cardiac remodeling, such as seen with hypertrophy10 or infarction.12 Developmental changes and upregulation of Ang II receptors during cardiac remodeling are consistent with the findings of other studies,31 32 33 34 35 suggesting that Ang II plays an important role in cardiac development and the process of cardiac hypertrophy and that the neonatal cardiac myocyte is a relevant experimental model that is useful for examining the expression profiles of Ang II receptors in cardiac hypertrophy.
In the present study, we examined the effects of mechanical stress on the expression of AT1-R and AT2-R subtypes in neonatal rat cardiac myocytes using an in vitro stretch model. Although it is shown that the AT1-R is downregulated by the addition of exogenous Ang II36 and that AT2-R expression is decreased by the stimulation of protein kinase C and intracellular Ca2+ mobilization,37 mechanical stretching unexpectedly caused increases in the expression of both AT1-R and AT2-R, resulting in enhancement of the Ang II–mediated inositol phosphate response. Medium conditioned by stretched myocytes inhibited both AT1-R and AT2-R expression, indicating the existence of a stretch-activated nonsecretory pathway(s) that causes an increase in Ang II receptor expression. The nonsecretory pathway(s) was closely involved in stretch-induced activation of tyrosine kinase but independent of protein kinase C and intracellular Ca2+ mobilization or de novo protein synthesis and appeared to regulate AT1-R and AT2-R genes by different mechanisms: AT1-R gene expression is mainly due to increased gene transcription, whereas the induction of AT2-R results from stretch-mediated stabilization of AT2-R mRNA.
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Materials and Methods |
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Cell Culture and Stretching of Cardiac Myocytes
Primary cultures of cardiac myocytes were prepared from ventricles of 1-day-old Wistar rats, and stretching of myocytes was conducted as described previously.11 Briefly, cardiomyocytes, after digestion with 0.1% collagenase, were collected and incubated for 60 minutes at 37°C, which allowed for selective attachment of nonmyocytes (mostly fibroblasts). Cardiomyocyte-enriched suspensions were removed from the culture dishes and plated at a density of 1x105 cells/mm2 on silicone rubber culture dishes. Cells were cultured for 24 hours in DMEM containing 10% FCS and antibiotics. Thereafter, culture medium was changed to DMEM consisting of 5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL selenium for 12 hours and stretched for an additional 12 hours in the same medium. The culture dishes containing attached fibroblasts were placed into an incubator and passaged 48 hours later with 0.25% trypsin and used as fibroblast-rich cultures.11 Uniaxial strain was applied by stretching the silicone sheet by 10%. Stretch and control experiments were carried out simultaneously with the same pool of cells in each experiment to match temperature, CO2 content, and pH of the medium for the stretched and control cells.2 3 4 5
Quantification of AT1-R and AT2-R mRNA Levels
Total RNA was isolated by means of guanidium isothiocyanate–cesium chloride centrifugation followed by digestion with DNase (Takara Shuzo) in the presence of RNase inhibitor (Takara Shuzo) to remove contaminating genomic DNA.10 11 12 AT1-R and AT2-R mRNA levels were quantified by competitive RT- PCR using deletion-mutated cRNAs, as described previously in detail.10 11 12 The amplification efficiencies of target and competitor transcripts were equivalent under optimal concentrations of competitor transcripts. Briefly, specific PCR primers for AT1-R were designed from the cDNA sequences of the coding region common to rat AT1a-R38 and AT1b-R39 : 5'-GGAAACAGCTTGGTGGTG-3' for sense and 5'-GCACAATCGCCATAATTATCC-3' for antisense. Primers for AT2-R were designed according to the coding region of the rat AT2-R gene18 19 : 5'-CTGACCCTGAACATGTTTGCA-3' for sense and 5'-GGTGTCCATTTCTCTAAGAG-3' for antisense. To obtain deletion-mutated cRNAs (
AT1-R and AT2-R), PCR products were subcloned into the pGEM-T vector (Promega Corp). The plasmids were cut with Msc I for AT1-R and Tth 111 I for AT2-R and self-ligated to contain inserts lacking Msc I–Msc I (288 bp, AT1-R) and Tth 111 I–Tth 111 I (280 bp, AT2-R). Sequencing of PCR products subcloned into pGEM-T confirmed that they originated from rat AT1-R and AT2-R mRNAs. Total RNA (1 µg) and deletion-mutated cRNA (0.1 to 
0.3 pg) were mixed and assayed by competitive RT-PCR. 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. To quantify the AT1-R and AT2-R mRNAs, a trace amount (5 µCi) of [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. The 32P counts in AT-R signals were normalized to both 32P counts in the deletion-mutated cRNA signals (to control for the efficiency of RT-PCR amplification against tube-to-tube variation) and GAPDH counts on Northern blots (8 µg of total RNA), measured by scanning densitometry (to control for the variability in the amount of input RNA). The normalized values in appropriate controls were expressed as 1 arbitrary unit for quantitative comparison. Native AT1-R and AT1-R should give 607- and 419-bp fragments, respectively. Native AT2-R and AT2-R should give 710- and 430-bp DNA fragments, respectively. AT1a-R or AT1b-R mRNA was amplified with the oligonucleotide primers specific for the respective 3'-noncoding sequences as reported previously.11 Denaturing, annealing, and extension reactions were performed 30 times at 94°C for 30 seconds, 58°C for 45 seconds, and 72°C for 1 minute. The PCR products were 385 bp for AT1a-R and 204 bp for AT1b-R.
Ang II Binding Assay for Cardiomyocytes
The Ang II binding assay for cardiomyocytes was described in detail in our previous study.11 Briefly, myocytes were washed with incubation buffer containing 0.01 mg/mL bacitracin and 0.25% BSA. Incubation buffer containing 0.05 nmol/L [125I-Sar1,Ile8]Ang II (New England Nuclear) and increasing concentrations of unlabeled ligands were added. Incubation proceeded at 22°C for 60 minutes and was terminated by aspirating the binding buffer. Bound radioactivity was removed by adding 1 mL of 0.25 µmol/L NaOH/0.5% SDS for 10 minutes. Specific binding was determined by subtracting the radioactivity bound in the presence of 1 µmol/L unlabeled Ang II. Binding to AT1-R and AT2-R was estimated by subtracting the nonspecific binding from the maximum saturation after preincubation with 10 µmol/L of CV11974 (Takeda Chemical Industries, Ltd) and PD123319 (Parke-Davis), respectively, for 30 minutes.
Immunoblotting of AT1-R Protein
For immunoblotting, 9% SDS-PAGE was performed, and proteins were electrotransferred on Immobilon-P membranes (Millipore), which were blocked in 5% skimmed milk in TBS-T (pH 7.6) containing 20 mmol/L Tris, 137 mmol/L NaCl, and 3.8 mL/L of 1N HCl for 1 hour and then incubated with a polyclonal antibody to AT1-R (a kind gift from Dr Mohan K. Raizada, Florida University)40 at a dilution of 1:2000 for 1 hour at room temperature. Membranes were washed with TBS-T and then incubated for a further hour with horseradish peroxidase–linked donkey anti-rabbit immunoglobulin F(ab')2 fragment (Amersham) at a dilution of 1:1000 in TBS-T. After a further washing with TBS-T, membranes were treated with ECL reagent (Amersham), and chemiluminescence was detected by exposure to Hyperfilm-ECL for 5 minutes. The intensity of the bands was quantified by laser densitometry (LKB 2222 UltraScan XL, Pharmacia Biotech).
Inositol Phosphate Assays
Cardiomyocytes were incubated with DMEM consisting of 5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL selenium for 12 hours in the presence of 1 µCi/mL [3H]myoinositol and stretched for an additional 12 hours in the same medium. Cells were then rinsed three times with DMEM containing 20 mmol/L HEPES (pH 7.4) and 10 mmol/L LiCl and subsequently incubated with Ang II in the same medium for 60 minutes at 37°C. The reactions were stopped by aspiration of the medium and addition of 0.5 mL of ice-cold 20 mmol/L formic acid. Cells were frozen and thawed three times and extracted with chloroform. The organic phase was dried, redissolved in scintillation fluid, and counted to determine label incorporation into the lipid pool. Separation of [3H]inositol phosphates was performed using Dowex columns by the method of Berridge et al.41
Transcript Stability Analysis and Nuclear Runoff Assay
Cardiomyocytes were stretched for 12 hours in the presence of DMEM containing 5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL selenium for 12 hours and then exposed to actinomycin D (5 µmol/L). After various incubation periods (0, 6, 12, and 24 hours), total RNA was isolated from individual dishes, and the changes in mRNA abundance were determined by RT-PCR without deletion-mutated cRNA.11 42 Nuclei were prepared from stretched or nonstretched myocytes, and a runoff assay was performed as previously reported.11 12 42 Nuclei were incubated for 20 minutes at 30°C in the presence of 50 mmol/L Tris (pH 7.9), 100 mmol/L KCl, 12.5% glycerol, 6 mmol/L MgCl, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 4 mmol/L of ATP, GTP, and CTP, 1 U/µL RNasin, and 200 µCi of [
-32P]UTP. After RNase-free DNase I and proteinase K digestion, the reaction products were extracted with guanidium isothiocyanate (4 mmol/L) and phenol/chloroform, and unincorporated [-32P]UTP was removed by trichloroacetic acid precipitation and filtration. The radiolabeled RNA (3 to 4x106 cpm) was hybridized at 42°C for 48 hours with linearized pGEM vector containing rat AT1a-R cDNA (20 µg), AT1b-R cDNA (20 µg), AT2-R cDNA (20 µg), or GAPDH cDNA (10 µg) fragments. These cDNA fragments were obtained by subcloning the RT-PCR product into pGEM. After washing the membrane in 2x SSC+0.1% SDS at 65°C for 1 hour, 0.2x SSC+0.1% SDS at room temperature for 30 minutes twice, and 0.2x SSC+10 µg/mL RNase at room temperature for 15 minutes, the bound radioactivity was determined by scintillation counting.
Reagents and Statistical Methods
All reagents were purchased from Sigma Chemical Co, unless otherwise indicated. The ET-1 receptor antagonist BQ123 was provided from Banyu Pharmaceutical Co, Ltd. The results are expressed as mean±SE. ANOVA and Dunnett's test were used for multigroup comparisons, with P<.05 considered significant.
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Results |
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Myocyte Stretching Causes Increases in AT1-R and AT2-R mRNA Levels
We and others have demonstrated that AT1-R–mediated signals of Ang II released from stretched myocytes play an important role in mechanical stretch–induced events,5 6 whereas stretch-induced changes in Ang II receptor expression have not yet been defined. Since the levels of AT1-R and AT2-R mRNAs in myocytes prepared from 1-day-old neonatal rat ventricles are too low to determine by Northern blotting, we quantified the levels of these transcripts by competitive RT-PCR. Fig 1
shows that there is a linear relationship between changes in input RNA amounts and values determined by the competitive RT-PCR and that small changes (25%) in RNA levels are detectable by this method, suggesting that mRNA analyses used in the present study are quantitative.
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Using this competitive RT-PCR, we found that stretching myocytes caused increases in both AT1-R and AT2-R mRNA accumulation with a similar time course: the increase was significant 6 hours after stretching, and the maximal increase (2.8-fold in AT1-R and 3.3-fold in AT2-R) was observed after 12 hours and was sustained for up to 18 hours. We and others have shown that serum deprivation or insulin treatment upregulates the AT2-R mRNA level.37 43 44 Since myocytes were incubated with serum-free medium containing insulin (0.87 µmol/L), transferrin (5 µg/mL), and selenium (5 ng/mL) (hereafter referred to as ITS medium) for 12 hours and subsequently stretched in the same medium, we examined the influence of incubation medium on AT2-R mRNA levels. Changes in AT2-R mRNA levels after 12-hour incubation with serum-free ITS medium were 15% to 32%, much lower than the increase observed in stretched myocytes (Fig 2). Therefore, subsequent experiments were performed by stretching myocytes for 12 hours in serum-free ITS medium.
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P<.05 vs values at time 0. MWM indicates molecular weight marker.
Because previous studies established that stretch-induced events mainly depend on the endogenous Ang II secreted from stretched myocytes,5 6 we next examined the effects of Ang II receptor antagonists on stretch-induced expression of Ang II receptors. It is notable that exposure to an AT1-R antagonist (CV11974) elevated the AT1-R (27%) and AT2-R (31%) mRNA levels in stretched myocytes, whereas exposure to the AT2-R antagonist (PD123319) had no such effect (Fig 3). These findings suggest that AT1-R–mediated signals have inhibitory effects on both AT1-R itself and AT2-R and that stretch-induced upregulation of Ang II receptors is caused by an undefined pathway(s) other than the Ang II–mediated pathway.
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We have previously shown that medium conditioned by stretching of myocytes induces mitogen-activated protein kinase activation and that ET-1 and Ang II secreted from stretched myocytes are involved in stretch-induced hypertrophic responses.45 To examine the effects of these secreted substances on Ang II receptors, incubation medium obtained from myocytes that were stretched for 12 hours was transferred to myocytes cultured on regular culture dishes. The addition of conditioned media significantly suppressed both AT1-R (22%) and AT2-R (27%) mRNA levels in recipient myocytes (Fig 4). Pretreatment of the recipient cells with CV-11974 almost completely blocked the changes induced by the addition of conditioned media, whereas pretreatment with PD123319, an AT2-R antagonist, did not affect the changes in AT1-R and AT2-R mRNA levels. Very recently, we found that ET-1 is also secreted from stretched myocytes and induces activation of Raf-1 kinase and mitogen-activated protein kinases through ET-1 type A receptors.45 Exposure of the recipient myocytes to the ET-1 type A antagonist BQ123 did not affect the changes in AT1-R and AT2-R mRNA levels induced by the addition of conditioned media (Fig 4). We have found that the concentration of Ang II (500 pmol/L) in conditioned medium is much higher (100-fold) than that of ET-1 (5 pmol/L)7 45 ; therefore, the difference in concentrations of Ang II and ET-1 may account for the lack of ET-1 effect on expression levels of Ang II receptors. Thus, Ang II secreted from stretched myocytes is the major molecule in conditioned medium that downregulates Ang II receptor levels, and secreted ET-1 contributes to the regulation mechanism to a lesser extent.
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AT1-R Expression in Cardiac Fibroblasts Is Not Responsive to Stretching
We and others have previously shown that fibroblast-rich cultures prepared from 1-day-old neonatal rat ventricles predominantly express AT1-R.11 46 47 Although immunochemistry revealed that contamination by fibroblasts in myocyte-rich cultures is <5% to 10%,11 we examined whether the AT1-R present in contaminating fibroblasts influences stretch-induced changes in myocyte-rich cultures. The results showed that the amount of AT1-R mRNA expressed in fibroblast-rich cultures was not affected by stretching (6 and 12 hours) (Fig 5A), suggesting that the effect of contamination by fibroblasts in myocyte-rich culture is negligible in terms of changes of AT1-R mRNA expression by myocyte stretching.
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AT1a-R, but Not AT1b-R, Is Responsive to Myocyte Stretching
AT1a-R and AT1b-R are minor subtypes of AT1-R.20 Since these subtypes are highly similar (96%) at the protein sequence level, it is possible to distinguish them only by using specific PCR primers designed from noncoding regions. The PCR primers used for quantification of AT1-R mRNA levels in the present study (Figs 1
to 4) were designed from common sequences between AT1a-R and AT1b-R and can detect expression of both receptors. Therefore, we examined the effects of myocyte stretching on AT1a-R and AT1b-R mRNA levels using specific PCR primers designed from 3' noncoding regions as described previously.11 As shown in Fig 5B, only AT1a-R mRNA was upregulated by stretching, whereas AT1b-R mRNA was not responsive to this mechanical stimulation. In this assay, we performed RT-PCR without deletion-mutated cRNAs specific for AT1-R minor subtypes because of the high degree of homology of the coding region at the nucleotide sequence level and the difficulty in obtaining DNA fragments from noncoding regions with sufficient lengths and useful restriction enzyme recognition sites. However, interassay variations between different samples (n=6) were 8.9% for AT1a-R and 7.7% for AT1b-R, indicating the validity of data shown in Fig 5B. These findings suggest that stretch-induced changes in AT1-R mRNA levels are mainly derived from AT1a-R mRNA, but not from AT1b-R mRNA.
Expression of AT1-R and AT2-R Protein Is Induced by Myocyte Stretching
We established that myocytes prepared from 1-day-old neonatal rat ventricles have a single class of Ang II binding sites by receptor binding assay using [125I-Sar1,Ile8]Ang II as a ligand.11 To examine whether changes in AT1-R and AT2-R mRNA levels reflect their expression at the protein level, we performed binding assays and Western blot analyses using a polyclonal antibody against rat AT1-R (Table and Fig 5C). Myocytes contained a single class of Ang II receptors with high affinity, and myocyte stretching caused a 2.4-fold increase in Ang II receptor number without affecting the affinity (Table). Competition binding experiments using antagonists for AT1-R and AT2-R demonstrated that the proportions of AT1-R and AT2-R in total Ang II receptors in control cells were 62% and 36%, respectively, and that myocyte stretching caused significant increases in AT1-R (2.4-fold) and AT2-R (2.6-fold) numbers (Table). To further confirm the increase in AT1-R protein level, we performed Western blot analyses. AT1-R protein migrates at a position corresponding to 70 kD on Western blots,40 and the band was increased 2.2-fold by myocyte stretch (Fig 5C), in good agreement with the changes in AT1-R number determined by binding assays.
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Inositol Phosphate Response to Ang II Is Enhanced in Stretched Myocytes
To examine whether upregulation of AT1-R expression in stretched myocytes leads to enhanced AT1-R function, inositol phosphate production response to Ang II was measured in myocytes stretched for 12 hours. As shown in Fig 6A, myocyte stretching enhanced AT1-R function, as evident by inspection of the maximal inositol phosphate production response to 0.5 µmol/L Ang II, and in stretched myocytes, this enhancement was 2.1-fold that in nonstretched cells. Inositol phosphate production by 0.5 µmol/L Ang II was completely (n=3, P<.01) blocked by pretreatment of the AT1-R antagonist CV11974 (1 µmol/L) but not by that of the AT2-R antagonist PD123319 (1 µmol/L) (data not shown), indicating that inositol phosphate response is mediated through AT1-R and that AT1-R–mediated action of Ang II is functionally enhanced in stretched myocytes.
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Stretch-Induced Expression of AT1-R Is Caused by Enhancement of Transcriptional Activity and Stretch-Induced mRNA Stabilization Is Involved in Regulation of AT2-R
We determined whether myocyte stretching regulated the mRNA levels of Ang II receptors through a transcriptional or posttranscriptional mechanism. To investigate this, a nuclear runoff transcription assay was performed, and the stabilities of AT1-R and AT2-R mRNAs were examined as previously reported.11 12 42 As shown in Fig 6B, levels of AT1a-R, AT1b-R, and AT2-R gene transcription in nonstretched control cells were almost at the background level of the pGEM plasmid vector. The transcriptional rate of the AT1a-R gene relative to that of the GAPDH gene was increased 2.3±0.3-fold in myocytes stretched for 12 hours compared with that in control cells, whereas there was no apparent difference in the density of hybridizing signals for AT2-R transcript. The rate of transcription of the GAPDH gene was not altered in the stretched myocytes. The AT1a-R probe used in the runoff assay was designed from the 3' noncoding region of the AT1a-R gene and was less likely to hybridize with the AT1b-R gene transcript.11 In fact, when the AT1b-R probe was used in the runoff assay, no significant signals were detected in either stretched and control cells (Fig 6B).
Ang II receptor mRNA turnover was examined by inhibiting new mRNA transcription with actinomycin D (Fig 7). Half-lives for AT1a-R and AT2-R mRNAs in control myocytes (n=4) were 12 and 14.5 hours, respectively. Stretching myocytes for 12 hours caused a marked increase (2.0-fold) in the half-life of AT2-R mRNA (28 hours) (Fig 7), whereas that of AT1a-R mRNA was increased by 1.3-fold (16 hours). Taken together, these findings suggest that upregulation of AT1-R is mainly induced by enhancement of gene transcription, whereas a stretch-induced mRNA-stabilizing effect is involved in the increase in AT2-R expression.
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Activation of Tyrosine Kinase by Nonsecretory Pathway(s) Is Involved in Stretch-Induced Expression of Ang II Receptors Without Requirement of De Novo Protein
The results described above indicate that activation of the AT1-R causes the downregulation of both AT1-R and AT2-R and that there is a nonsecretory pathway(s) that upregulates Ang II receptor levels in the stretch-induced hypertrophic response. Since it is known that tyrosine kinases and protein kinase C are activated and that Ca2+ mobilization is induced in response to myocyte stretching,48 49 we examined the involvement of these cellular events in stretch-induced expression of Ang II receptors (Fig 8). Exposure of myocytes to genistein (20 µmol/L) (a tyrosine kinase inhibitor), to H-7 (1 µmol/L) or staurosporin (1 µmol/L) (general inhibitors of protein kinases), or to BAPTA-AM (100 µmol/L) (membrane-permeable Ca2+-chelating compound) did not affect the basal mRNA levels of Ang II receptors. We next examined the effects of these inhibitors on the stretch-induced changes. As shown in Fig 8, treatment with genistein inhibited changes in both AT1-R and AT2-R mRNAs in stretched myocytes, whereas H-7, staurosporin, and BAPTA-AM did not prevent the increases in AT1-R and AT2-R mRNA levels induced by myocyte stretching. A 24-hour pretreatment of myocytes with active phorbol ester (phorbol 12-myristate 13-acetate, 2 µmol/L) also did not affect the stretch-induced changes in Ang II receptors (data not shown). Genistein caused significant decreases in AT1-R (36%) and AT2-R (22%) mRNA levels in stretched myocytes, which may reflect the AT1-R–mediated inhibitory action on Ang II receptor expression in the absence of tyrosine kinase activation. Given that treatment with an AT1-R antagonist (Fig 2) and an ET-1 type A antagonist (data not shown) did not inhibit stretch-induced expression of Ang II receptors, these findings suggest that activation of tyrosine kinases mediated through a nonsecretory pathway(s), but not through AT1-R and ET-1 type A receptor, plays an important role in the mechanism of Ang II receptor upregulation in stretched myocytes.
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We next examined whether the effect of stretching on Ang II receptor expression is dependent on new protein synthesis. Cycloheximide treatment caused significant increases in the steady state expression of both AT1-R (1.4-fold) and AT2-R (1.7-fold) mRNAs compared with nonstretched control cells, whereas stretch-induced changes of these mRNA levels were not inhibited by coincubation with cycloheximide (Fig 8). Consistent with this result, Nickening and Murphy50 have also reported that AT1-R mRNA levels are increased by treatment with cycloheximide in rat vascular smooth muscle cells. These results suggest that newly synthesized protein plays an important role in the maintenance of steady state levels of AT1-R and AT2-R mRNA accumulation, whereas stretch-induced changes in levels of AT1-R and AT2-R were not regulated by de novo protein synthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mechanical stress exemplified by stretching myocytes stimulates the secretion of Ang II from myocytes, and the secreted Ang II evokes protein kinase activation and produces myocyte hypertrophy through the AT1-R.5 6 7 We have shown that nonsecretory factors other than Ang II are also involved in such mechanical stretch–induced events.7 45 In the present study, we focused on the regulation of Ang II receptor expression by myocyte stretching and found that mechanical stress results in increases in both AT1-R and AT2-R expression. Conditioned medium obtained from stretched myocytes downregulated both AT1-R and AT2-R, suggesting that a nonsecretory pathway(s) plays a crucial role in stretch-induced upregulation of Ang II receptor expression in myocytes.
Accumulating evidence suggests that humoral and/or neural factors as well as hemodynamic overload are involved in the development and regression of cardiac hypertrophy.51 52 Many animal and human studies have shown that the local renin-Ang system plays an important role in the pathogenesis of left ventricular hypertrophy.53 Increased myocardial angiotensinogen mRNA level and ACE activity are found in a rat model of hypertensive hypertrophy.54 We and others have previously shown that AT1-R expression is also increased in hypertrophied ventricles of SHR,10 in transgenic mice carrying renin and angiotensinogen,32 and after myocardial infarction.12 Anversa and colleagues33 34 have also reported that hypertrophied myocytes obtained from rat hearts after acute myocardial infarction contain increased levels of AT1-R protein. In addition, it was shown that a subpressor dose of ACE inhibitor and treatment with AT1-R antagonist caused regression of cardiac hypertrophy.16 55 These findings suggest that a local cardiac renin-Ang system is activated in response to hypertrophic stimulation in vivo and plays an important role in the development of ventricular hypertrophy.
In the present study, we found that both Ang II receptor subtypes are upregulated by as much as approximately threefold by myocyte stretching, which was consistent with the findings in vivo described above. Furthermore, we found that conditioned medium containing molecules secreted from stretched myocytes caused a decrease in the expression of Ang II receptor subtypes. These results suggest that a nonsecretory pathway(s) is activated by myocyte stretching, which is responsible for the induction of Ang II receptor expression. This observation is in good agreement with our previous suggestion that a nonsecretory pathway(s) independent of secreted Ang II is involved in the mechanical stretch–induced response.7 Since Ang II secreted from stretched myocytes into cultured medium is found to downregulate AT1-R and AT2-R expression, the mechanism of expression of Ang II receptors in myocytes appears to be quite distinct from other stretch-activated and Ang II–induced genes, such as c-fos, transforming growth factor-ß1, angiotensinogen, and atrial natriuretic factor.2 6 56 Interestingly, growth factors and cAMP analogues were reported to downregulate both rat AT1-R34 50 and AT2-R43 44 57 expression, and activation of the PKC-Ca2+ pathway caused a decrease in AT2-R expression.37 Substances or molecules that cause upregulation of AT1-R and AT2-R expression have not been identified, except for the glucocorticoid sensitivity of the AT1-R gene58 and the effect of insulin on AT2-R gene expression.44 In the present study, we found that activation of tyrosine kinases mediated through a nonsecretory pathway(s) plays a crucial role in the stretch-induced responses of AT1-R and AT2-R. Since Ang II36 and growth factors43 44 50 were reported to downregulate Ang II receptors, it is suggested that tyrosine kinases activated by a nonsecretory pathway(s) differ from those activated by Ang II and growth factors. Focal adhesion protein tyrosine kinases, such as p125FAK59 and paxillin,60 that are phosphorylated in response to cellular interactions with components of the extracellular matrix could be candidates for such a tyrosine kinase.
Stretch-induced hypertrophic responses or pressure overload in vivo are known to induce the expression of a number of immediate-early genes, such as c-fos, c-jun, c-myc, and Egr-1.60 After induction of immediate-early genes, the expression of several genes changes in response to pressure overload. These changes include induction of ß-myosin heavy chain, atrial natriuretic factor, skeletal muscle -actin, smooth muscle -actin, ß-tropomyosin, and ß-type creatine kinase as well as the downregulation of the sarcoplasmic reticulum Ca2+-ATPase.61 Since stretch-induced upregulation of Ang II receptors was evident after 6 hours of stretch and since increased expression of Ang II receptors is maintained during the chronic phase of hypertrophy in vivo,10 32 Ang II receptor genes are also classified into "stable late markers" of hypertrophy. The present study demonstrated that enhancement of transcriptional activity rather than a change in mRNA stability plays an important role in the stretch-induced response of the AT1-R gene. Sadoshima and Izumo62 have reported that the Ang II–responsive element of the c-fos gene, which is one of the first genes activated by myocyte stretch, is mapped to the serum response element,62 and computer-assisted search (TFD2) showed that there is no such sequence motif in the 5' flanking region of rat AT1a-R gene.20 63 64 We have characterized in detail cis-regulatory elements of the rat AT1-R gene by transient transfection using a reporter gene.63 64 Although we tried to identify the stretch-response element by transfecting AT1-R reporter gene constructs, including 2.2 kb of the 5' flanking region, to myocytes using lipofectamine, transfection efficiency was too low to analyze changes in promoter activities (data not shown). Further studies are required to define the mechanism of transcriptional regulation of the AT1a-R gene in this in vitro stretch model.
Comparison of the kinetics of AT1-R and AT2-R mRNA expression by treatment with actinomycin D revealed that a stretch-induced mRNA stabilization process is the principal mechanism whereby AT2-R mRNA is upregulated by stretching, with a partial contribution to AT1-R mRNA regulation. A general hypothesis for the mechanism of this mRNA stabilization is that stabilizing factors bind to recognition domains in mRNA and either suppress RNA degradation specifically by this interaction or enhance the RNA stability through inhibition of constitutively expressed RNase-like activities.65 Since stretch-induced changes in levels of AT1-R and AT2-R mRNAs were not influenced by pretreatment with cycloheximide, it is unlikely that myocyte stretching induces de novo protein synthesis, whose products function to stabilize AT1-R and AT2-R mRNAs. Given that AT1-R expression is mainly regulated at the transcriptional level in this stretch model, it is conceivable that constitutively expressed factors are activated by myocyte stretching, possibly through tyrosine phosphorylation, which in turn induces the expression of Ang II receptors transcriptionally or posttranscriptionally.
We have shown that mechanical stress exemplified by stretching stimulates the secretion of Ang II and ET-1 from myocytes and that Ang II and ET-1 participate in activation of the protein kinase cascade and the production of myocyte hypertrophy.7 45 Since Ang II is the major molecule secreted from stretched myocytes6 45 and Ang II increases the level of expression of transforming growth factor-ß as well as angiotensinogen genes in cardiac myocytes,66 Ang II is the initial mediator of the stretch response and triggers the subsequent autocrine/paracrine production of other secondary growth factors, which may act in concert to produce hypertrophic responses. In the present study, we demonstrated that a nonsecretory pathway(s) activated by myocyte stretching is responsible for the upregulation of Ang II receptors and that inositol phosphate response by Ang II is enhanced in stretched myocytes. Given that the angiotensinogen gene is also upregulated by this stretch model,6 these findings suggest that the cardiac renin-Ang system is activated in mechanical stress–induced hypertrophy, in which the AT1-R–mediated action of Ang II is functionally enhanced. Although the physiological role of AT2-R and its transduction signal remains to be determined, recent evidence suggested that AT2-R has antiproliferative effects23 24 and is involved in the maintenance of systemic blood pressure21 22 and that AT2-R contributes to the induction of apoptosis25 and modifies voltage-sensitive ion currents.26 27 Although the expression of AT2-R in adult rat hearts is very low, it is upregulated in cardiac hypertrophy of spontaneously hypertensive rats10 and after myocardial infarction.12 Thus, in vivo data regarding AT2-R regulation in hypertrophied ventricles appear to be in good agreement with the present observations of the stretch-induced response in vitro. However, the present data are informative for a situation specific to neonatal rat myocytes, and further studies will be required before applying them to distinct situations involving adult animals or human beings.
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Acknowledgments |
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This study was supported in part by research grants from the Ministry of Education, Science, and Culture, Japan; the Study Group of Molecular Cardiology; The Naito Foundation; the Clinical Pharmacology Foundation of Japan; the Japan Medical Association; and the Japan Smoking Foundation.
Received May 24, 1996; accepted August 16, 1996.
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S. Gunasegaram, R. S. Haworth, D. J. Hearse, and M. Avkiran Regulation of Sarcolemmal Na+/H+ Exchanger Activity by Angiotensin II in Adult Rat Ventricular Myocytes : Opposing Actions via AT1 Versus AT2 Receptors Circ. Res., November 12, 1999; 85(10): 919 - 930. [Abstract] [Full Text] [PDF]
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K. C Wollert and H. Drexler The renin-angiotensin system and experimental heart failure Cardiovasc Res, September 1, 1999; 43(4): 838 - 849. [Abstract] [Full Text] [PDF]
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R. Malhotra, J. Sadoshima, F. C. Brosius III, and S. Izumo Mechanical Stretch and Angiotensin II Differentially Upregulate the Renin-Angiotensin System in Cardiac Myocytes In Vitro Circ. Res., July 23, 1999; 85(2): 137 - 146. [Abstract] [Full Text] [PDF]
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Y. Moriguchi, H. Matsubara, Y. Mori, S. Murasawa, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Shibasaki, Y. Tanaka, T. Nakajima, et al. Angiotensin II–Induced Transactivation of Epidermal Growth Factor Receptor Regulates Fibronectin and Transforming Growth Factor-ß Synthesis via Transcriptional and Posttranscriptional Mechanisms Circ. Res., May 14, 1999; 84(9): 1073 - 1084. [Abstract] [Full Text] [PDF]
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V. Robert, C. Heymes, J.-S. Silvestre, A. Sabri, B. Swynghedauw, and C. Delcayre Angiotensin AT1 Receptor Subtype as a Cardiac Target of Aldosterone : Role in Aldosterone-Salt–Induced Fibrosis Hypertension, April 1, 1999; 33(4): 981 - 986. [Abstract] [Full Text] [PDF]
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K. Harada, I. Komuro, T. Sugaya, K. Murakami, and Y. Yazaki Vascular Injury Causes Neointimal Formation in Angiotensin II Type 1a Receptor Knockout Mice Circ. Res., February 5, 1999; 84(2): 179 - 185. [Abstract] [Full Text] [PDF]
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H. Matsubara Pathophysiological Role of Angiotensin II Type 2 Receptor in Cardiovascular and Renal Diseases Circ. Res., December 14, 1998; 83(12): 1182 - 1191. [Abstract] [Full Text] [PDF]
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Y. Tsutsumi, H. Matsubara, N. Ohkubo, Y. Mori, Y. Nozawa, S. Murasawa, K. Kijima, K. Maruyama, H. Masaki, Y. Moriguchi, et al. Angiotensin II Type 2 Receptor Is Upregulated in Human Heart With Interstitial Fibrosis, and Cardiac Fibroblasts Are the Major Cell Type for Its Expression Circ. Res., November 16, 1998; 83(10): 1035 - 1046. [Abstract] [Full Text] [PDF]
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S. Murasawa, Y. Mori, Y. Nozawa, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Moriguchi, Y. Shibasaki, Y. Tanaka, T. Iwasaka, et al. Role of Calcium-Sensitive Tyrosine Kinase Pyk2/CAKß/RAFTK in Angiotensin II–Induced Ras/ERK Signaling Hypertension, October 1, 1998; 32(4): 668 - 675. [Abstract] [Full Text] [PDF]
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K. Tamura, S. Umemura, N. Nyui, K. Hibi, T. Ishigami, M. Kihara, Y. Toya, and M. Ishii Activation of angiotensinogen gene in cardiac myocytes by angiotensin II and mechanical stretch Am J Physiol Regulatory Integrative Comp Physiol, July 1, 1998; 275(1): R1 - R9. [Abstract] [Full Text] [PDF]
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Z.-Q. Wang, A. F. Moore, R. Ozono, H. M. Siragy, and R. M. Carey Immunolocalization of Subtype 2 Angiotensin II (AT2) Receptor Protein in Rat Heart Hypertension, July 1, 1998; 32(1): 78 - 83. [Abstract] [Full Text] [PDF]
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S. Murasawa, Y. Mori, Y. Nozawa, N. Gotoh, M. Shibuya, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Moriguchi, Y. Shibazaki, et al. Angiotensin II Type 1 Receptor–Induced Extracellular Signal–Regulated Protein Kinase Activation Is Mediated by Ca2+/Calmodulin-Dependent Transactivation of Epidermal Growth Factor Receptor Circ. Res., June 29, 1998; 82(12): 1338 - 1348. [Abstract] [Full Text] [PDF]
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Y. Liu, A. Leri, B. Li, X. Wang, W. Cheng, J. Kajstura, and P. Anversa Angiotensin II Stimulation In Vitro Induces Hypertrophy of Normal and Postinfarcted Ventricular Myocytes Circ. Res., June 15, 1998; 82(11): 1145 - 1159. [Abstract] [Full Text] [PDF]
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C. Heymes, J.-S. Silvestre, C. Llorens-Cortes, F. Marotte, B. Chevalier, B. I. Levy, B. Swynghedauw, and J.-L. Samuel Cardiac Senescence Is Associated with Enhanced Expression of Angiotensin II Receptor Subtypes Endocrinology, May 1, 1998; 139(5): 2579 - 2587. [Abstract] [Full Text] [PDF]
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K. Harada, I. Komuro, Y. Zou, S. Kudoh, K. Kijima, H. Matsubara, T. Sugaya, K. Murakami, and Y. Yazaki Acute Pressure Overload Could Induce Hypertrophic Responses in the Heart of Angiotensin II Type 1a Knockout Mice Circ. Res., April 20, 1998; 82(7): 779 - 785. [Abstract] [Full Text] [PDF]
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H. Senzaki, Y. A. Gluzband, P. H. Pak, M. T. Crow, J. S. Janicki, and D. A. Kass Synergistic Exacerbation of Diastolic Stiffness From Short-term Tachycardia–Induced Cardiodepression and Angiotensin II Circ. Res., March 9, 1998; 82(4): 503 - 512. [Abstract] [Full Text] [PDF]
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Y. Sumida, S. Umemura, K. Tamura, M. Kihara, S.-i. Kobayashi, T. Ishigami, M. Yabana, N. Nyui, H. Ochiai, A. Fukamizu, et al. Increased Cardiac Angiotensin II Receptors in Angiotensinogen-Deficient Mice Hypertension, January 1, 1998; 31(1): 45 - 49. [Abstract] [Full Text]
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N. Ohkubo, H. Matsubara, Y. Nozawa, Y. Mori, S. Murasawa, K. Kijima, K. Maruyama, H. Masaki, Y. Tsutumi, Y. Shibazaki, et al. Angiotensin Type 2 Receptors Are Reexpressed by Cardiac Fibroblasts From Failing Myopathic Hamster Hearts and Inhibit Cell Growth and Fibrillar Collagen Metabolism Circulation, December 2, 1997; 96(11): 3954 - 3962. [Abstract] [Full Text]
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N. Makino, M. Sugano, S. Otsuka, and T. Hata Molecular Mechanism of Angiotensin II Type I and Type II Receptors in Cardiac Hypertrophy of Spontaneously Hypertensive Rats Hypertension, October 1, 1997; 30(4): 796 - 802. [Abstract] [Full Text]
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K. Tamura, Y. E. Chen, M. Lopez-Ilasaca, L. Daviet, N. Tamura, T. Ishigami, M. Akishita, I. Takasaki, Y. Tokita, R. E. Pratt, et al. Molecular Mechanism of Fibronectin Gene Activation by Cyclic Stretch in Vascular Smooth Muscle Cells J. Biol. Chem., October 27, 2000; 275(44): 34619 - 34627. [Abstract] [Full Text] [PDF]
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