Regulation of Angiotensin II Receptor Subtypes During Atrial Fibrillation in Humans
Background—Previous studies have suggested that atrial fibrillation (AF) is associated with the activation of the atrial angiotensin system. However, it is not known whether the expression of angiotensin II receptors changes during AF. The purpose of this study was to determine the atrial expression of angiotensin II type 1 and type 2 receptors (AT1-R and AT2-R) in patients with AF.
Methods and Results—Atrial tissue samples from 30 patients undergoing open heart surgery were examined. Eleven patients had chronic persistent AF (≥6 months; cAF), 8 patients had paroxysmal AF (pAF), and 11 patients were in sinus rhythm. AT1-R and AT2-R were localized in the atrial tissue by immunohistochemistry and quantified at the protein and mRNA level by Western blotting and quantitative polymerase chain reaction. Both types of AT-R were predominantly expressed in atrial myocytes in all groups. The amount of AT1-R was reduced to 34.9% during cAF (P<0.01) and to 51.7% during pAF (P<0.05) compared with patients in sinus rhythm. In contrast, AT2-R was increased during cAF (246%; P=NS) and pAF (505%; P<0.01). AT1-R/AT2-R mRNA content was similar in all groups.
Conclusions—AF is associated with the down-regulation of atrial AT1-R and the up-regulation of AT2-R proteins. These findings may help define the pathophysiological role of the angiotensin system in the structural remodeling of the fibrillating atria.
Recent reports suggest that atrial fibrillation (AF) is associated with the activation of the atrial angiotensin system. Pedersen et al1 provided the first evidence that angiotensin-converting enzyme (ACE) inhibitor therapy can reduce the occurrence of AF in patients with left ventricular dysfunction after myocardial infarction. In addition, Van den Berg et al2 observed that pretreatment with ACE inhibitors reduced the relapse rate of AF after electrical cardioversion. Thus, the inhibition of the cardiac angiotensin system by ACE inhibitors/angiotensin receptor antagonists might affect the pathophysiological substrate of AF and may offer a new therapeutic approach. However, the atrial expression of angiotensin II type 1 and type 2 receptors (AT1-R and AT2-R) has not been investigated in patients with AF.
The purpose of the present study was to localize atrial AT1-R/AT2-R and to quantify the expression of these receptors at the protein and mRNA level in patients with and without AF.
After written informed consent was obtained, right atrial appendages were obtained from patients undergoing cardiac bypass surgery or mitral/aortic valve replacement. Tissue samples were taken from 11 consecutive patients with chronic, persistent AF (≥6 months; cAF) and from 11 matched patients with no history of AF (sinus rhythm [SR]). In addition, 8 consecutive patients with documented episodes of paroxysmal AF (pAF; 3±2 AF episodes per month) were studied (Table⇓).
Tissue samples were homogenized in 2× RotiLoad (Roth) using an UltraTurrax. Aliquots of 300 μg were separated in 4% to 12% gradient SDS-polyacrylamide gels (Novex Electrophoresis) and transferred onto nitrocellulose membrane BA85 (Schleicher & Schüll). Rabbit anti-AT1-R and anti-AT2-R polyclonal antibodies (Biotrend), goat-anti-rabbit-peroxidase (New England Biolabs), and SuperSignal West Dura Extended Duration Substrate (Pierce) were used for immunodetection. The resulting images were densitometrically analyzed. The mean relative absorption units of the control group were compared with the corresponding means of the AF groups. Comparison of the different groups was only done on blots processed equally and exposed on the same x-ray film.
Quantitative Polymerase Chain Reaction
One microgram of total RNA, which was prepared using TRIZOL (Gibco BRL), was reverse-transcribed. A total of 5% of the cDNA mixture was used for quantitative polymerase chain reaction (PCR) by means of the Lightcycler LC24 (Idaho Technology). The 10-μL reaction mixture consisted of 1× reaction buffer with BSA (Idaho Technology), 3 mmol/L MgCl2, 200 μmol of desoxyribonucleotide mixture, 0.4 U of InViTaq polymerase (InViTec), 0.2 μL of SYBR-Green I (1:1000, Molecular Probes), and 0.5 μmol of the AT-R-specific primers (AT1-R: 5′-GACGCACAATGCTTGTAGCCA and 5′-CTGCAATTCTACAGTCACGTATG; AT2-R: 5′-GGGCTTGTGA-ACATCTCTGG and 5′-GTAAATCAGCCACAGCGAGG).
Amounts of 18S-mRNA were used to normalize cDNA contents. Initial denaturation at 95°C for 3 s was followed by 40 cycles with denaturation at 95°C for 0 s, annealing at 65°C (AT1-R) or 60°C (AT2-R) for 3 s, and elongation at 72°C for 15 s (AT1-R) or 9 s (AT2-R). The fluorescence intensity, which reflected the amount of actually formed PCR product, was read at the end of each elongation step. Initial amounts of template mRNA were calculated by determining the time point at which the linear increase of PCR product started, relative to the corresponding points of a standard curve.
Histochemistry and Immunohistochemistry
Histochemistry and immunohistochemistry for localization of AT-Rs were performed in a total of 15 tissue specimens (4 SR, 6 cAF, 5 pAF). Sections from formalin-fixed and paraffin-embedded specimens were stained with hematoxylin and eosin. Immunostaining was performed with antibodies, as specified above, directed against AT1-R or AT2-R (dilution 1:20) following standard protocols. The specificity was controlled by omitting the primary antibody.
All values are expressed as mean±SD. Differences between the 3 groups of patients were evaluated using 1-way ANOVA. P<0.05 was considered statistically significant.
AT1-R and AT2-R
The relative amount of AT1-R in atrial tissue was significantly reduced in patients with cAF (34.9±40.9%; n=11; P<0.01) and pAF (51.7±26.6%; n=8; P<0.05) compared with patients with SR (100±58.1%; n=11). The difference between pAF and cAF was not significant (Figure⇓, A). The amount of AT1-R correlated neither with AF duration nor with left ventricular ejection fraction (r2=0.03 and r2=0.02, respectively; P=NS). AT2-R was increased in patients with pAF (505±331.6%; n=11; P<0.01) compared with patients with SR (100±108.8%; n=11). The observed increase in patients with cAF (246±323.2%; n=11) did not reach statistical significance.
AT1-R and AT2-R mRNA
The amount of AT1-R mRNA was not different in patients with cAF (85.7±80.4%; n=11) or pAF (65±36%; n=8) compared with patients in SR (100±86.5%; n=11). AT2-R mRNA content was not significantly increased during cAF (179.9±156.8%; n=11) or pAF (126.2±101.4%; n=8) compared with patients with SR (100±60.5%; n=11; P=0.4).
The action of angiotensin II is initiated by binding to AT1-R/AT2-R.3 4 Stimulation of AT1-R induces myocardial hypertrophy and the accumulation of extracellular matrix proteins; it can also affect atrial contractility.3 4 In contrast, stimulation of AT2-R inhibits proliferative processes.5
Our study describes, for the first time, the regulation of atrial AT1-R/AT2-R expression in patients with AF. pAF and cAF were associated with the down-regulation of AT1-R. An up-regulation of AT2-R was observed during pAF. In contrast, at the mRNA level, AT1-R/AT2-R expression was not significantly altered.
AF is associated with progressive structural changes of the atria, resulting in atrial dilation and loss of transport function.1 2 A previous study demonstrated that the atrial expression of ACE is increased in patients with AF, possibly leading to angiotensin II–dependent progressive atrial fibrosis.6 Increased angiotensin II tissue levels during AF may trigger the observed down-regulation of AT1-R. A reduction of AT1-R and an increase of AT2-R may, therefore, be compensatory to inhibit the progression of angiotensin II–dependent interstitial fibrosis.
The atrial expression of ACE and AT-R subtypes during AF resembles changes seen in patients with terminal left ventricular failure.3 4 6 Rogg et al3 showed that the atrial expression of AT1-R is positively correlated with left ventricular ejection fraction, whereas left ventricular ejection fraction and AT2-R are inversely related. In the present study, however, none of the patients had terminal heart failure. One can, therefore, hypothesize that the regulatory changes seen during AF characterize the presence of an “end-stage atrial myopathy.” This is supported by other studies,1 2 6 which have shown the development of severe morphological/functional atrial abnormalities during cAF.
The observed changes in amounts of AT-R proteins, despite the unaltered corresponding mRNA levels, suggest that the expression of AT-R subtypes is regulated by post-transcriptional mechanisms. Underlying regulatory processes may encompass inefficient AT-R mRNA translation or decreased AT-R stability.7
In conclusion, this study shows that cAF and pAF are associated with significant changes in the atrial expression of AT1-R/AT2-R. Further studies must define possible therapeutic consequences.
Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 387) and the Kultusministerium Sachsen-Anhalt (contract grant number 822A10318223). The authors thank Bianca Schultze, Karin Frank, Christine Wolf, and Ruth Hilde Hädicke for their excellent technical assistance.
- Received February 17, 2000.
- Revision received April 17, 2000.
- Accepted April 17, 2000.
- Copyright © 2000 by American Heart Association
Pederson OD, Bagger H, Kober L, et al. Trandolapril reduces the incidence of atrial fibrillation after myocardial infarction in patients with left ventricular dysfunction. Circulation. 1999;100:376–380.
Rogg H, de Gasparo M, Graedel E, et al. Angiotensin II-receptor subtypes in human atria and evidence for alterations in patients with cardiac dysfunction. Eur Heart J. 1996;17:1112–1120.
Asano K, Dutcher DL, Port JD, et al. Selective downregulation of the angiotensin II AT1-receptor subtype in failing human ventricular myocardium. Circulation. 1997;95:1193–1200.
Matsubara H. Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases. Circ Res. 1998;83:1182–1191.
Krishnamurthi K, Verbalis JG, Zheng W, et al. Novel cytosolic proteins binding 5′-leader sequence cis elements in the angiotensin AT1 receptor mRNA regulate receptor expression. Nucleic Acids Symp Ser. 1997;36:38–40.