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§§
From the Institute of Pharmacology,*
Christian-Albrechts-University of Kiel, Kiel, Germany; the German
Institute for High Blood Pressure Research,
Heidelberg, Germany; the Institute of
Pathology,
Justus-Liebig-University, Giessen,
Germany; and the Department of Physiology,§
University of Florida, Gainesville, Florida
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the exact physiological role of the AT2 receptor still remains to be defined it has been widely accepted that these binding sites are involved in processes precipitated by tissue injury. For example, AT2 receptor expression is significantly increased after myocardial infarction3,4 and peripheral nerve injury.5 Further, AT2 receptor stimulation promotes optic nerve regeneration.6 However, AT2 receptors have not only been associated with cell protection but there is also strong evidence that Ang II acting through the AT2 receptor induces apoptosis.7-11 At present, it is not understood in which particular physiological or pathophysiological situation activation of the AT2 receptor leads to cell death or cell survival.
In the heart, angiotensin receptors are involved in a variety of processes. Stimulation of normal and postinfarcted ventricular myocytes with Ang II induces hypertrophy via AT1 receptors.12 Although AT1 receptor-dependent mechanisms mediate fibroblast and vascular smooth muscle cell proliferation13 as well as collagen synthesis,14 AT2 receptor activation inhibits cell proliferation.15-17 The expression of AT1 and AT2 receptors and the Ang II-induced growth or anti-growth effects in cardiomyocytes in culture18 depend on the developmental stage at which the cells are prepared and on the ratio between those receptor subtypes. Depending on experimental conditions, AT1 receptors have been demonstrated to mediate apoptosis in cultured cardiomyocytes19,20 or to promote hypertrophy12 via paracrine release of transforming growth factor-ß1 and endothelin-1.21
To investigate the angiotensin receptor expression in the heart, a variety of techniques such as receptor binding or reverse transcriptase-polymerase chain reaction (RT-PCR) on tissue homogenates have been used in the past. Altogether, these studies could not answer the question as to which specific cardiac cell type(s) express(es) the AT1 and AT2 receptor. In the present study, we applied a laser-assisted cell-picking technique and combined it with a single-cell RT-PCR for both angiotensin receptor subtypes. These experiments served to determine whether adult rat cardiomyocytes express AT1 and AT2 receptors under physiological conditions and 1 day after myocardial infarction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experiments were performed in male Wistar rats (250 to 300 g). Animals were randomly divided into three experimental groups. Myocardial infarction was induced by permanent ligation of the left descending coronary artery by a modified technique described by Johns and Olson.22 Briefly, after induction of anesthesia with an intraperitoneal injection of chloral hydrate, rats were intubated, artificially ventilated, and connected to an electrocardiogram (ECG) recorder for continuous monitoring during surgery. A left thoracotomy was carried out by cutting the third and fourth rib and a rib-spreading chest retractor was inserted. Then, the left descending coronary artery was ligated intrathoracically using sterile 6-0 suture material under a microstereoscope. Successful ligation of the coronary artery was verified by the occurrence of arrhythmia in the ECG and, visually, by the color change of the ischemic area. In rats with sham surgery, the ligation was placed beside the coronary artery. The thoracic cavity was closed during respiration hold, and analgesia was induced by a subcutaneous injection of buprenorphin-HCl (0.2 mg/kg). Finally, animals that were neither subjected to anesthesia nor to thoracotomy served as additional controls.
Isolation and Treatment of Tissues
Twenty-four hours after permanent coronary ligation, rats were sacrificed and the hearts were immediately excised. After removal of the atria and large vessels, the ventricles were cut in a standardized fashion into transversal slices from the apex to the basis. This was achieved using a special Plexiglas box, adapted to the hearts of rats, which contains slits for a microtome knife at 3-mm intervals. The five slices obtained were immediately frozen in isopentane on dry ice (-30°C) and stored at -80°C until further processing. The use of isopentane protected the tissue against freezing-induced bursts and damage of the histological ultrastructure, making it possible to separate the different cell types by laser-assisted cell picking.
Micromorphometrical Evaluation of Infarct Sizes
The morphometrical determination of infarct sizes was performed according to Klein et al.23
Laser-Assisted Cell Picking for Isolation of Nucleus-Free Single Cardiomyocytes
To obtain isolated, nucleus-free cardiomyocyte section profiles
from infarcted and noninfarcted areas of the adult rat heart,
a laser-assisted cell-picking technique (P.A.L.M., Bernried, Germany)
was applied.24
The dissection and cell harvesting (cell
picking) procedures are illustrated and explained in detail in the
legend to Figure 1
. Briefly, two to three
samples containing 10 cardiomyocyte profiles were harvested from the
noninfarcted myocardium of the posterior left ventricular wall of the
respective hearts after photolysing the entire interstitial tissue
between these cardiomyocytes. Microscopically visible nuclei of
cardiomyocytes were photolysed by UV laser to destroy genomic DNA.
Section profiles of these cells were collected with a syringe needle in
first strand buffer (see Reverse Transcription) and subjected to
RT-PCR for analysis of AT1 and
AT2-receptor mRNA expression.
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Reverse Transcription
Ten myocyte section profiles from each sample were transferred into 10 µl of First Strand Buffer (modified from Brady and Iscove25 ; 1% Igepal CA-630, 4% RNase inhibitor in 52 mmol/L Tris-HCl, pH 8.3, 78 mmol/L KCl, 3.1 mmol/L MgCl2). The samples were cooled on ice for 5 minutes and then immediately snap-frozen.
Preceding reverse transcription, the samples were heated (70°C for 10 minutes) and then cooled on ice (5 minutes). To confirm the absence of any genomic DNA contamination, each sample was divided into two aliquots of 5 µl each and then subjected to reverse transcription in the presence or absence of the enzyme. The cDNA synthesis was carried out using 1 µl dNTP (10 mmol/L each), 1 µl random hexamers (50 µmol/L), 0.5 µl (10 U) RNase inhibitor, and 1 µl (50 U) murine leukemia virus reverse transcription in a total volume of 17.5 µl. The reaction was performed at 20°C for 10 minutes, 43°C for 60 minutes, 99°C for 5 minutes, and finally for 5 minutes on ice.
Polymerase Chain Reaction
For the subsequent AT1 receptor PCR, the
following protocol was used: 4 µl buffer, 2 µl
MgCl2, 1 µl dNTP (10 mmol/L each), 1 µl of
each AT1 receptor-specific primer (20
µmol/L; for details see Table 1
), 8
µl cDNA, 0.5 µl (2.5 U) AmpliTaq Gold polymerase and 32.5
µl H2O. The PCR was performed at 94°C for
2.45 minutes, followed by 63 cycles at 94°C for 0.45 minute, 57°C
for 0.45 minute, and 72°C for 0.45 minute. After a final extension of
72°C for 7 minutes the PCR products were electrophoresed on a 1.8%
agarose gel and ethidium bromide stained. As internal positive control,
total RNA isolated from control hearts was used.
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), 5 µl cDNA, 0.25 µl (1.25 U)
AmpliTaq Gold polymerase and 13.15 µl H2O. The
PCR was carried out at 94°C for 5 minutes, followed by 70 cycles at
94°C for 0.45 minute, 63°C for 1.45 minutes, and 72°C for 2.10
minutes. After a final extension of 72°C for 10 minutes the PCR
products were electrophoresed on a 1.8% agarose gel and ethidium
bromide stained. Total RNA isolated from PC12W cells (passage <18)
which are known to abundantly express AT2
receptors served as internal control for RT-PCR amplification.
For amplification of the housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), PCR reaction mixtures were identical to
those for the AT1 receptor. Amplification
reactions were performed at 95°C for 6 minutes followed by a total of
45 three-temperature cycles (20 seconds at 95°C, 30 seconds at
61°C, and 30 seconds at 73°C) and a final extension at 73°C
for 5 minutes. The sequences of GAPDH PCR primers
used26,27
are given in Table 1
.
Materials
Gene-Amp RT-PCR kits and all reagents for RT-PCR were purchased from Perkin Elmer Applied Biosystems (Überlingen, Germany). One percent Igepal CA-630, Tris-HCl, KCl, and MgCl2 were obtained from Sigma (Deisenhofen, Germany). Specific primers for the rat AT1 and AT2 receptor gene were synthesized by Pharmacia (Erlangen, Germany). The UV laser microbeam (337-nm wavelength) was manufactured by P.A.L.M. (Bernried, Germany), and the inverted microscope Axiovert 135 was made by Zeiss (Jena, Germany). All RT-PCR reactions were performed on a GeneAmp PCR System 9600 manufactured by Perkin Elmer Applied Biosystems (Überlingen, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. For RT-PCR, 10 genomic DNA-free
section profiles, which equal approximately two entire cardiomyocytes,
were pooled in First Strand Buffer to form one sample. After dividing
samples into two identical aliquots, they were subjected to reverse
transcription with or without reverse transcriptase for subsequent PCR
amplification in a blinded fashion. Furthermore, as an additional
negative control RT-PCR, amplifications were always carried out in the
absence of cell material to exclude any possibility of sample
contamination. These reactions invariably proved to be negative for
both AT1 and AT2 receptors. AT1 Receptor Single-Cell PCR
PCR amplification of AT1 receptor mRNA
resulted in a single band of the predicted size (146 bp). As internal
positive control, total RNA isolated from whole control hearts was used
for AT1 receptor PCR (Figure 2)
.
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.
Three samples gained from sham-operated animals
(n = 4) were discarded because of positive
AT1 receptor PCR in the absence of reverse
transcriptase. Eight out of 24 remaining samples were
AT1 mRNA-positive (33%) (Figure 2)
.
Finally, 1 day after myocardial infarction (n =
4), the number of cardiomyocytes expressing AT1
receptor mRNA was not significantly changed. Because of a positive
signal in the absence of reverse transcriptase two samples had to be
excluded. Nine out of the remaining 20 samples (45%) were
AT1 receptor mRNA-positive (Figure 2)
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AT2 Receptor Single-Cell PCR
One single band of the expected size (217 bp) was gained from the
AT2 receptor PCR. Total RNA isolated from PC12W
cells (passage <18) was used as an internal positive control for the
AT2 receptor PCR in each experiment (Figure 3)
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.
In the sham-operated group (n = 4), positive
AT2 receptor mRNA signals were observed in three
out of 23 samples (13%) and no PCR signals were detected in the
absence of reverse transcriptase (Figure 3)
.
One day after myocardial infarction (n = 4), one
sample out of 22 had to be excluded from the study because of a
positive signal in the absence of reverse transcriptase. Three out of
the remaining 21 samples (14%) showed a strong
AT2 receptor mRNA signal (Figure 3)
revealing
that cardiomyocytes from adult rats do express
AT2 receptors. The RT-PCR data for
AT1 and AT2 receptors are
summarized in Table 2
.
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To demonstrate the general sensitivity of the described single-cell RT-PCR assay, the mRNA expression of the housekeeping gene GAPDH was investigated in cardiomyocyte section profiles gained from control animals. In none of the 54 samples studied, were PCR signals observed in the absence of reverse transcriptase. Out of these 54 samples, 52 were GAPDH-positive (96%) underlining the sensitivity and reproducibility of this assay (data not shown).
Determination of Infarct Sizes
To verify that myocardial infarctions were of similar degrees, the infarction sizes were micromorphometrically evaluated. These experiments revealed similar infarction sizes among the four animals of the myocardial infarction group (45.7%, 47.3%, 48.6%, and 49.1%, respectively) demonstrating the consistency of the procedure used (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrates for the first time that adult rat cardiomyocytes not only express the AT1 but also the AT2 receptor gene. Approximately 10 percent of cardiomyocytes isolated from adult rats expressed the AT2 receptor gene under physiological conditions as well as 1 day after myocardial infarction. Thus, in contrast to a previous study,28 we have demonstrated for the first time that adult rat cardiomyocytes, at least to a certain percentage, contain the AT2 receptor mRNA. Also, we could demonstrate that the number of cardiomyocytes that express the AT1 receptor mRNA is not increased after myocardial infarction.
In our study, a laser-assisted cell-picking technique24,29 was used to obtain cardiomyocyte samples without genomic DNA contamination. Because genomic DNA can serve as a template in RT-PCR reactions its absence must be verified to ensure that an observed gene expression indeed reflects mRNA levels of the respective genes. To avoid DNase treatments on tiniest amounts of total RNA obtained from single cells but to overcome this particular problem, microscopically guided UV laser photolysis of cell nuclei was applied. The cell-picking technique that was used here has previously been shown to be successful for the mRNA analysis of single cells by RT-PCR and has been proven to be highly effective in eliminating genomic DNA.30 However, to confirm the absence of genomic DNA in the samples used, different controls were performed for each sample. First, samples containing cell material were divided into two tubes and were subjected to RT-PCR, once in the presence and once in the absence of reverse transcriptase. Samples were only used for this study if they did not show any signal for both the AT1 and AT2 receptor in the absence of reverse transcriptase. Second, all negative controls, eg, samples without cell material were included in a blinded manner and did not elicit any signals for AT1 and AT2 receptors after RT-PCR.
The identification of cardiomyocytes was carried out by histological means and not via cardiomyocyte-specific gene expression because this approach is often not conclusive. This is particularly the case after a traumatic tissue injury like myocardial infarction (MI) that causes massive alterations in the gene expression patterns of different cell types. Even if a certain gene was exclusively expressed in cardiomyocytes before MI this would not necessarily be the case after MI resulting in possible false-positive identification. On the other hand, cardiomyocytes exhibit a characteristic and distinct morphology and are microscopically clearly distinguishable from cells such as neurons or fibroblasts which makes it possible to conclusively identify and isolate this particular cell type. Therefore, microdissection experiments combined with RT-PCR are the method of choice to isolate a certain cell type and to identify a cell type-specific gene expression.
In previous studies it has been demonstrated that myocardial infarction evokes a dramatic up-regulation of both angiotensin receptor subtypes. Whereas Zhu et al4 observed a pronounced increase in the AT1 and AT2 receptor gene expression as soon as 24 hours after myocardial infarction, Nio et al3 reported a maximal expression after 7 days. Both studies used tissue homogenates and, therefore, did not discriminate between different cell types. By applying a single-cell RT-PCR technique, we show here that cardiomyocytes in vivo express AT1 and AT2 receptor mRNA under physiological conditions but that the number of cells expressing these receptors is not increased after myocardial infarction. However, with respect to the study performed by Nio et al3 the possibility cannot be excluded that a greater number of cardiomyocytes may express the AT2 receptor gene at a later time point.
With regard to the RT-PCR data presented the previously reported increase in AT1- and AT2-receptor mRNA levels after myocardial infarction might be explained in two different ways but it should be noted that the assay used in this study was not carried out under quantitative conditions: first, the AT1 and AT2 receptor mRNA expression is increased in those cardiomyocytes which already express these receptors under physiological conditions. However, the assay used in our study cannot be applied to address this particular question. Under the experimental conditions used here for the detection of both angiotensin receptor genes in single cells, saturation phases of PCR are reached making it extremely difficult to quantitate the gene expression. A second explanation is that other cell types within the heart such as fibroblasts, neurons, or endothelial cells are responsible for the observed AT1- and AT2-receptor mRNA up-regulation. Unfortunately, compared to cardiomyocytes these cell types are much smaller. Using the laser-assisted cell-picking technique, it is not yet possible to photolyse the nuclei of these cells without photolysing the entire cell. Therefore, this method is, at least at present, restricted to the larger cardiomyocytes. To determine the angiotensin receptor gene expression in other cell types, other improved experimental approaches have to be used in the future. Also, these studies will have to address the question whether adult rat cardiomyocytes do express the AT2 receptor protein or just the AT2 receptor mRNA.
The question which specific cell type is responsible for the AT1 and AT2 receptor expression in the heart is of major importance because Ang II plays an important role in cardiovascular function. In neonatal rats, AT2 receptor protein is detectable in cardiac myocytes but not in fibroblasts.31 Mechanical stretch of myocytes evokes increases in both AT1- and AT2-receptor mRNA levels32 indicating that nonsecretory pathways activated by myocyte stretching are involved in angiotensin receptor regulation. In the human heart, the level of expression of AT1 receptor genes seems to decrease in the failing ventricle whereas the level of AT2 receptor expression is unaffected.33 Finally, after heart transplantation, both angiotensin receptors are down-regulated.34
Several studies elucidated the physiological role of AT2 receptors in the heart but the available data are still controversial. Angiotensin receptors have both been demonstrated to induce apoptosis in different cell types. With respect to the heart, it has been reported that AT1 but not AT2 receptor stimulation induces programmed cell death in cardiomyocytes19,20 by activating p53.35 On the other hand, Ang II is capable of promoting both apoptosis and neuronal regeneration6,10 via its AT2 receptor. In PC12W cells, AT2 receptor-mediated apoptosis is paralleled by inactivation of Bcl-28 as well as generation of ceramides in PC12W cells.10,11 Thus, the observation that adult cardiomyocytes not only express AT1 but also AT2 receptors may have important consequences for the heart, under physiological and pathophysiological conditions.
The data presented here clearly indicate that cardiomyocytes in the adult organism are capable of expressing AT2 receptors. Therefore, when evaluating Ang II effects on the heart, AT2 receptor-mediated effects have to be taken into account. This is, in particular, the case when discussing potential side effects of AT1 receptor antagonists. One major concern about the use of these compounds for the treatment of hypertension is that they engender an indirect overstimulation of the unopposed AT2 receptor by increasing Ang II plasma levels. Because AT2 receptors have been identified as a receptor being able to induce apoptosis it was argued that one potential serious consequence of AT1 receptor blockade might be the AT2 receptor-mediated cell death of cardiomyocytes. Because our studies clearly demonstrate that cardiomyocytes express AT2 receptor mRNA under physiological and pathophysiological conditions the possibility of an AT1 receptor antagonist-induced and AT2 receptor-mediated apoptosis does exist.
In summary, we have shown that the AT2 receptor gene is expressed in ~10% of adult rat cardiomyocytes before and 1 day after myocardial infarction. Further, AT1 receptor mRNA is present in approximately every second cardiomyocyte with the number of cells expressing this receptor being not increased by tissue injury. Future studies will have to determine whether the observed angiotensin receptor mRNA expression in cardiomyocytes directly translates into protein expression and whether other cardiac cell types also express AT2 receptors. Also, they will have to assess whether the AT1 receptor antagonist-evoked increase in Ang II plasma levels indeed induces AT2 receptor-mediated side effects in the heart.
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Footnotes |
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S. B. and S. G. contributed equally to these studies.
Accepted for publication April 27, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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