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Cardiovascular Research 1998 38(2):340-347; doi:10.1016/S0008-6363(98)00015-7
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Copyright © 1998, European Society of Cardiology

Angiotensin II receptor subtype AT1 and AT2 expression after heart transplantation

Lars Gullestada,1,1, Guy Haywooda, Halfdan Aassb, Heather Rossa, Gail Yeea, Thor Uelandb, Odd Geiranb, John Kjekshusb, Svein Simonsenb, Nanette Bishoprica and Michael Fowlera,*

aFalk Cardiovascular Research Center, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5246, USA
bRikshospitalet University Hospital, Oslo, Norway

* Corresponding author. Tel.: +1 (415) 723 7846; Fax: +1 (415) 725 1599.

Received 20 October 1997; accepted 22 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Cardiac hypertrophy appears early after heart transplantation, and may represent a myocardial response to injury. Recent evidence suggests that angiotensin II (Ang II) may promote growth through the AT1 and inhibit growth through the AT2 receptor subtypes. We therefore asked whether hypertrophy after heart transplantation is characterized by alterations in Ang II receptor gene expression. Methods: The expression of Ang II receptor subtypes, AT1 and AT2, was analyzed in right ventricular endomyocardial biopsies taken from 10 human donor hearts prior to implantation (controls) and from 17 heart transplant recipients, 11 studied during annual evaluation (>1 year after transplantation) and 6 one week after transplantation. Competitive reverse transcription polymerase chain reaction (RT-PCR) was performed using synthetic RNA internal standards for both receptor subtypes. Results: AT1 and AT2 receptor mRNAs were detected in all samples. AT1 receptor mRNA decreased 4.5 fold (p<0.01) and AT2 receptor mRNA 4.2 fold (p<0.001) in transplant patients compared with controls. In the subgroup of patients examined one week after surgery AT1 was reduced relative to AT2 receptor mRNA, resulting in an altered ratio of AT1 to AT2 early after transplantation. There was no correlation between Ang II receptor levels and left ventricular wall thickness, and the decrease in receptor level did not correlate with any hemodynamic parameters, cyclosporine blood levels, or plasma renin, Ang II or pANP, except for a negative correlation between AT2 mRNA and plasma renin (r=–0.49,p=0.05). Conclusions: Contrary to our expectations, mRNA for both Ang II receptors was downregulated after heart transplantation. The cause of myocardial hypertrophy after heart transplantation is still unclear, but the hypertrophy does not appear to be driven by increased transcription of the AT1 receptor.

KEYWORDS Clinical; Heart; Molecular biology; Human; Angiotensin; Gene expression; Receptors; Transplantation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Cardiac hypertrophy has been demonstrated by echocardiography in approximately 40% of recipients one to two weeks after heart transplantation, with 25–30% exhibiting persistent hypertrophy [1, 2]. This is corroborated by myocyte hypertrophy in right ventricular biopsies in most patients as early as one week after transplantation and is sustained indefinitely in most recipients [3].

The mechanism for hypertrophy remains unclear. Although some reports suggest an association between acute cellular rejection and increased ventricular thickening after transplantation [4], this is not a consistent observation [1]. Hypertension has been suggested as a cause of left ventricular hypertrophy [5], but the issue is controversial since hypertrophy appears equal in hypertensive and normotensive recipients [6]. Increased sympathetic and renin—angiotensin system (RAS) activity may cause hypertension and cardiac hypertrophy [7], but the role of the RAS in myocardial remodelling following heart transplantation is unclear.

The RAS plays a major role in normal control of the cardiovascular system and in the pathophysiology of various cardiac diseases [8]. Angiotensin II (Ang II), the primary active compound of this system, mediates its effects through receptors that are widely distributed in the body. Two subtypes, AT1 and AT2, have so far been described in humans.

Most of the known effects of Ang II in adult tissues are mediated through the AT1 receptor, including myocyte hypertrophy and hyperplasia [9]. Less is known about the effect of the AT2 receptor, but its abundant expression during embryonic and neonatal life suggests involvement in the regulation of various growth processes. Recent studies have demonstrated that stimulation of the AT2 receptor inhibits growth on vascular smooth muscle [10]and endothelial cells [11], and stimulates apoptotic pathways [12]. Thus, it appears that the AT1 receptor promotes growth, whereas the AT2 receptor may have the opposite effect.

We wished to test the hypothesis that an upregulation of transcription of AT1 receptors might result in increased myocyte sensitivity to the RAS and contribute to the hypertrophic response. We therefore examined the expression of AT1 and AT2 receptor mRNA in myocardial biopsies from heart transplant recipients. Since the factors modulating receptor expression may change over time, we compared the expression of receptor mRNA taken one week after surgery with those taken more than one year after transplantation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Patients
Right ventricular endomyocardial biopsies were obtained during routine surveillance from 17 orthotopic heart transplanted patients, 6 one week after transplantation and 11 during annual evaluation (mean time after transplantation of 3.4±18 years). Tissue (taken at the same place in the right ventricular septum) obtained from 10 donor hearts at the time of implantation served as controls. Clinical characteristics are given in Table 1. The protocol was approved by The Regional Ethics Committee at the University of Oslo, and informed consent was obtained. The study was performed in accordance with the declaration of Helsinki. All recipients were immunosuppressed with cyclosporine A, prednisolone, and azathioprine. Eight of the 11 patients at annual evaluation required antihypertensive therapy; 2 received an ACE inhibitor, 4 a calcium antagonist, 2 an alpha blocker, and 4 diuretics (3 received two drugs). All patients studied during annual evaluation were clinically stable and none had histological evidence of rejection on biopsy, whereas three of the six patients studied one week after transplantation had cellular rejection, one with grade IIIA and two with grade II.


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  		Table 1 Clinical characteristics of the donors, and transplanted recipients studied one week after surgery (n=6) and during annual evaluation (n=11)

 
None of the donors had evidence of cardiac or hypertensive disease, and all received a low-dose infusion of dopamine for circulatory support.

2.2 Clinical methods
Catheterization of the left- (only at annual evaluation) and right heart was performed after an overnight fast using standard techniques. Blood samples for hormonal analysis were drawn from the right atrium. Plasma was immediately separated and frozen at –80°C, and later analyzed for proatrial natriuretic factor (pANF; ANF(1–98)), renin and Ang II using radioimmunoassay. Two-dimensional guided M-mode echocardiography was performed (using Vingmed 800, Vingmed Sound, Horten, Norway) for assessment of left ventricular dimension in the 6 patients studied one week after transplantation.

2.3 RNA preparation
The tissue samples were immediately frozen in liquid nitrogen and stored at –80°C until analyzed.

Total RNA was isolated from the tissue samples using Trizol reagent (Gibco BRL Life Technologies, Gaithersburg, MD), essentially as described [13]. Following extraction, the RNA was dried, resuspended and stored in diethyl pyrocarbonate treated water at –80°C until use. The ratio of absorption (260:280 nm) was between 1.7 and 2.0 for all samples.

2.4 Reverse transcription polymerase chain reaction (RT-PCR)
RT-PCR was performed exactly as previously described [13]. 0.5 µg of total RNA was reverse-transcribed using random primers as a primer, and 5 µl of the resultant cDNA mixtures was amplified by PCR using the primers and conditions described in Table 2. The sequences for the primers for AT1 were complementary for sequences in exon 5, and yield a product of 255 bp. Confirmation of the specificity of the product was assessed by restriction enzyme cuts using Ssp 1 which resulted in the expected bands of 83 and 172 bp, respectively (data not shown). The primers for AT2 were located in exon 1 and 2. PCR with these primers gives a product length of 293 bp. The PCR products amplified by the AT2 primer pair were subcloned into a vector using the TA Cloning Kit (Invitrogen) and sequenced to confirm they matched the cDNA sequence (unpublished data 1996).


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  		Table 2 Oligonucleotide primer sequences and reaction conditions for PCR reactions

 
β-actin was amplified in order to demonstrate the integrity of intact mRNA in each sample (Table 2). Atrial natriuretic peptide (ANP), which is normally not expressed in normal ventricular myocardium, was used as an additional marker of myocardial stress an remodelling [14, 15], see Table 2.

2.5 Quantitative RT-PCR
In order to calculate the absolute level of target receptor, competitive RNA synthetic internal standards (rcRNA) were synthesized using a modification of the method of Heuvel et al. [16], as previously described [13]. The internal standard is identical to target mRNA but contains a short, but detectable, deletion. The length of the products produced by these primers were 137 and 254 bp for the AT1 and AT2 internal standards, respectively.

Detailed methods for competitive PCR using these primers have been published elsewhere [13]. In brief, a fixed amount of receptor RNA was added to 10 fold dilution series of the internal standard rcRNA, which thereafter was subjected to reverse-transcription to cDNA and PCR amplification.

To determine the point of equivalence (where the amount of target and internal standard competitor are equal), relative band intensities of the PCR products on ethidium stained gels were quantified using videodensitometry (Applied Imaging, Santa Clara, CA). To correct for differences in molecular weight, densitometric values from target AT1 and AT2 bands were multiplied by 137/255 and 254/293, respectively. The point of equivalence was thereafter calculated by extrapolation from the intersection of the curves. An estimate of receptor RNA was then made by reference to known concentrations of the internal rcRNA standard.

2.6 Control RT-PCR experiments
Three different methods were employed to document the absence of genomic DNA contamination: (1) a subgroup of the samples were treated with RNAse, or (2) reverse transcriptase was omitted from the reverse transcription mixture, and (3) in addition the samples were amplified with primers for the promotor region of apo (a) related gene C, a non-transcribed region of genomic DNA [17]. The primers were designed only to amplify genomic DNA, and the samples were run together with genomic DNA as positive controls.

We did several additional experiments to validate the method. First, to ensure equal amplification efficiency two samples and each internal standard was amplified for 28, 31, 34 and 37 cycles. Linear parallel increases in videodensitometric values were observed for target mRNA and internal standards (data not shown). Hence, the amplification seems to be equal despite differences in length between internal standard and target gene, as previously reported [18]. Second, additional experiments confirmed that the method was reproducible in both the high and low concentration range and sensitive to as little as 2.5 fold changes in target mRNA concentrations (not shown). Finally, in order better to compare the amount of mRNA for AT1 and AT2, we devised a second set of experiments in which we compared the two internal standards for AT1 and AT2 using a new synthetic RNA internal standard containing terminal sequences for the forward and reverse primers of both the AT1 and AT2 receptor subtypes as previously described [13]. Based on this approach appropriate corrections were made.

2.7 Statistical analysis
The results are expressed as mean±SD. Analysis of variance was used to compare the three groups. If ANOVA indicated an effect, the data were analyzed using unpaired Mann—Whitney U test. The Bonferoni method was used to adjust for multiple comparisons. Coefficients of correlations were calculated by the Spearman rank test. p<0.05 Was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Clinical and hemodynamic data
Clinical characteristics and hemodynamic data are outlined in Table 1. The donor age was significantly lower than in the two transplant groups, but there was considerable overlap. The ischemic time of donor hearts was 217±44 min. Echocardiography performed one week after transplantation demonstrated evidence of hypertrophy with interventricular septum (IVS) and left ventricular posterior wall thickness (PV) of 13±2 (reference value 10±1.2 mm) and 12±2 mm (reference value 10±1.1 mm), respectively, with 4 out of six hearts with IVS greater or equal to 13 mm.

3.2 Blood samples
Both renin and pANF were significantly increased one week and at annual control after transplantation compared with controls, while Ang II was not significantly different for the three groups (Table 1). Cyclosporine blood levels were 328±96 and 115±32 ng/ml at one week and during annual control, respectively. There was a significant correlation between plasma renin and PV (r=0.83, p<0.05), but not between Ang II and PV (r=0.46, p=0.30) or IVS (r=0.46, p=0.35) during one week after transplantation.

3.3 RT-PCR results
β-actin was present in all samples, confirming the integrity of mRNA. mRNA for ANF was expressed in all biopsies from the transplant patients, which contrast no visible band in the controls as previously reported by our group [15].

Representative gels of the competitive RT-PCR for the AT1 and AT2 reactions are presented in Fig. 1. mRNA for both AT1- and AT2 receptor subtypes were expressed in both donor and transplant right ventricular endomyocardial biopsies. Comparisons of the AT1 and AT2 receptor mRNA levels between donor controls and transplant recipients are shown in Fig. 2. In the donor hearts, the point of equivalence for AT1 mRNA was estimated to 371±343 fg, whereas AT2 expression was 80±61 fg (Fig. 2). In the transplanted patients the AT1 level decreased significantly 4.5 fold to 82±92 fg (p<0.001) and the AT2 receptor mRNA 4.2 fold to 19±16 fg (p<0.001) during annual evaluation (Fig. 2). The decrease was even more pronounced for the AT1 mRNA early after transplant (10±8 fg), resulting in significantly lower levels at this time point compared with annual transplants (p<0.01, Fig. 2). The differences in AT1 and AT2 mRNA receptor levels following transplantation resulted in a change in the relative percentages of the receptor expression. In the donors, about 82% of the receptor mRNA was of the AT1 subtype and 18% AT2, which contrasted with approximately equal amount of the two receptor mRNAs one week after transplantation. During later time points, however, the ratio of AT1 and AT2 mRNA resembled that seen in donor hearts (81% AT1).


Figure 1
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  		Fig. 1 Representative gel for one of the competitive RT-PCR experiments with rcRNA internal standard and target gene RNA, AT1 left and AT2 right, for donor (A) and transplanted (B) hearts. A constant amount of total RNA was mixed with an increasing number of AT1 or AT2 internal standard rcRNA. The PCR product of the internal standard (137) and target mRNA (255 bp) were run on a 1.5% agarose gel and stained with ethidium bromide. * denotes the point of equivalence as seen by eye.

 

Figure 2
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  		Fig. 2 Angiotensin II receptor subtype AT1 and AT2 mRNA in donor controls and transplanted hearts evaluated after one week (Tx-1W) and during annual evaluation (Tx-An). * denotes values significantly different from controls.

 
There was no correlation between any of the measured cardiac function parameters, blood cyclosporine levels or left ventricular myocardial thickness and AT1 or AT2 mRNA. In the whole transplant group (n=17) there was a negative correlation between mRNA for AT2 and renin (r=–0.49, p=0.05) or Ang II (r=–0.42, p=0.10), while no significant correlations were observed between mRNA AT1 and renin or Ang II.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this study we report downregulation of both AT1 and AT2 mRNA receptor levels at two different time points after transplantation. mRNA levels for both Ang II receptor subtypes decreased early after heart transplantation and remained downregulated later. Our results are in accordance with a recent study demonstrating reduced levels of AT1 mRNA in the transplanted heart [19]. Thus the hypertrophic response to cardiac transplantation is unlikely to be driven by upregulation of the AT1 receptor.

4.1 Possible role of Ang II receptors in the hypertrophied transplanted heart
A high prevalence of myocardial hypertrophy has been observed after heart transplantation in humans [1, 2]. The observation of expression of ANP in all transplant samples, which can be considered a biological marker for altered gene expression and a hallmark of hypertrophy [14], suggests that the transplanted myocardium undergoes a remodelling process at the molecular level. The exact mechanisms implicated in the pathogenesis of post-transplant hypertrophy are unknown, however.

The RAS has been linked to growth processes in the myocardium. In vitro studies have demonstrated that Ang II increases protein synthesis and cell size in cultured cardiac myocytes [20], and produces left ventricular hypertrophy (LVH) in animal models [9]. ACE inhibitors, even in doses that do not affect blood pressure, induces regression of LVH both in experimental animal models [21]and in hypertensive patients [22]. These effects of Ang II appear to be mediated through the AT1 receptor [9, 23], Several components of the RAS, including angiotensinogen, ACE, and Ang II receptors are upregulated in the myocardium during the remodelling process following volume- or pressure overload and myocardial infarction [24–27]. Hence, we had expected to find upregulation of the AT1 receptor mRNA in the present study. Our finding of decreased levels of mRNA AT1 receptor levels, and no correlation between receptor levels and left ventricular hypertrophy, therefore does not support a primary role for this receptor subtype in the hypertrophic response after heart transplantation.

We cannot, however, completely rule out a role for the AT1 receptor in the remodelling process following transplantation, as it could be argued that decreased AT1 mRNA is a reflection of agonist induced receptor downregulation [28]. In the present study we observed increased activity of the RAS system with increased plasma renin levels. The modifying role of circulating components of the RAS system on Ang II receptor levels is, however, unclear. For example, recent studies in dog models have demonstrated that local factors may be more important than systemic factors in the expression of components of the RAS system in the heart [29]. Thus, although our data do not support a role of Ang II receptors in the hypertrophic response following heart transplantation in humans, a possible role of the local RAS cannot be ruled out, and the importance of this system needs to be addressed using specific blockers.

4.2 Ang II receptor downregulation and possible mechanisms
In accordance with Asano et al. [30]we found mRNA levels for the AT1 receptor subtype to be considerably higher than for AT2 in ventricular biopsies from donor hearts. In contrast Regitz-Zagrosek et al. [31]found a predominance of the AT2 receptor in the atrium. However, both differences in control subjects and regional variation in the expression of the Ang II receptors with variation in translation efficiency between the two receptor subtypes [32], may account for the discrepant result.

Ang II receptor regulation is complex, and many factors including hormonal, pharmacological (i.e. cyclosporine, glucocorticoids, antihypertensives) and physiological factors (i.e. hypertension, denervation) may modify its expression after heart transplantation. We are not able from the present study to clearly identify the mechanisms for decreased AT1 and AT2 mRNA. Studies in cell cultures have demonstrated Ang II receptor downregulation during Ang II infusion [28]. However, in whole animal models Ang II infusion results in either up-regulation or no change of AT1 receptor levels in different tissues [33, 34], which are in line with the present result showing no correlation between plasma renin or Ang II and AT1 mRNA. We observed a negative correlation between plasma renin and AT2 mRNA, but no correlation between renin and AT1 mRNA, supporting previous observations that the two receptor subtypes are independently and differentially regulated in a tissue specific manner [13, 29, 30, 35]. Glucocorticoids [36]and cyclosporine [37]have been found to induce upregulation of ang II receptors. However in the present study, although all patients received treatment with these agents, no upregulation took place suggesting little net effect of these drugs in this setting.

Although local factors such as stress may modify local components of the RAS [38], we observed no correlation between any of the measured hemodynamic parameters, including EDP, and Ang II receptors. We cannot exclude the possibility that some of the increased expression of AT1 late compared with early after transplantation could be due to sympathetic reinnervation [39, 40], but renal denervation appears to have no effect of renal AT1 gene expression in lambs [41].

4.3 Limitations
The mRNA levels for the receptor measured in the present study do not necessarily reflect the protein product of the target gene. Ideally, measurement of mRNA levels should be followed by simultaneous use of another method such as radioligand binding studies, immunohistochemistry, or in situ hybridization, but the limited quantity of tissue available made this impossible. There is evidence that the AT1 receptor is alternatively spliced, and that inclusion of exon 1 and 2 inhibits translation [32]. In such cases there would be a discrepancy between the mRNA and protein levels. However, previous studies have shown a good correlation between AT1 mRNA levels and maximal AT1 receptor density as assessed by binding studies,[27, 42]and during heart failure both receptor density and mRNA levels are equally downregulated [13, 30, 31].

Difference in age between donors and recipients also did not appear to confound our results. We found no significant correlation between age and AT1 or AT2 mRNA receptor levels in a group of 12 donors, consistent with previous results using binding studies [43].

Another potential limitation is that control hearts were obtained from donors exposed to states of stress. Previous studies have, however, found no difference in ß-receptor density in biopsies from donor hearts and from the non-failing left ventricle of hearts removed from patients with right heart failure due to pulmonary hypertension [44].

4.4 Conclusions
The present study provides evidence for downregulation of both AT1 and AT2 mRNA receptor levels after heart transplantation. The decrease occurs early after transplantation and results in a change in the ratio between the receptors whereas later the ratio returns towards normal. We conclude that the hypertrophic response to heart transplantation is not associated with upregulation of the Ang II AT1 receptor subtype.

Time for primary review 22 days.


   Acknowledgements
 
We thank the surgical staff at Stanford, in particular Herman Reichenspurner and Kwok Yun, who assisted us in the collection of the myocardial samples. We thank Hannah A. Valantine, MD MRCP, for critical review of the paper. Lars Gullestad was supported by the Research Council of Norway.


   Notes
 
1 Medical Department B, Rikshospitalet, 0027 Oslo, Norway. Tel.: +47 22 867010, Fax: +47 22 868357. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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