The Angiotensin-(1-7)/Mas Receptor Axis Is Expressed in Sinoatrial Node Cells of Rats

Abstract

The authors’ previous studies have indicated that angiotensin(Ang)-(1-7) protects the heart against reperfusion arrhythmias. The aim of this study was to determine whether a functional angiotensin-converting enzyme2 (ACE2)/Ang-(1-7)/Mas receptor axis is present in the sinoatrial node (SAN) of Wistar rats. SAN cells were identified by Masson’s trichrome staining, HCN4 expression, and lack of connexin43 expression. Immunohistochemistry technique was used to detect the expression of ACE2, Ang-(1-7), and Mas in the SAN. To evaluate the role of this axis in the SAN function, atrial tachyarrhythmias (ATs) were induced in isolated rat atria perfused with Krebs-Ringer solution (KRS) alone (control) or KRS containing Ang-(1-7). The specific Mas antagonist, A-779, was used to evaluate the role of Mas in the Ang-(1-7) effects. The findings showed that all components of the ACE2/Ang-(1-7)/Mas branch are present in the SAN of rats. Importantly, it was found that this axis is functional because perfusion of atria with Ang-(1-7) significantly reduced the duration of ATs. Additionally, this anti-arrhythmogenic effect was attenuated by A-779. No significant changes were observed in heart rate, contractile tension, or ±dT/dt. These observations demonstrate that the ACE2/Ang-(1-7)/Mas axis is expressed in SAN cells of rats. They provide the morphological support to the anti-arrhythmogenic effect of Ang-(1-7).

Keywords

angiotensin-converting enzyme 2 angiotensin II cardiac arrhythmias connexin43 heart renin–angiotensin system

Angiotensin(Ang)-(1-7) is a key member of the renin–angiotensin system (RAS) because many of its actions are opposite to those attributed to Ang II. Its biological relevance was substantiated by the recent discoveries of the main Ang-(1-7)–forming enzyme, angiotensin-converting enzyme 2 (ACE2) (Donoghue et al. 2000; Tipnis et al. 2000) and of the Ang-(1-7) receptor Mas (Santos et al. 2003). Since then, researchers have become aware of the existence of a counter-regulatory axis within the RAS composed of ACE2, Ang-(1-7), and Mas that balances the ACE/Ang II/AT₁ receptor axis.

Many reports have demonstrated that activation of the ACE2/Ang-(1-7)/Mas axis exerts protective effects in the heart. Indeed, administration of exogenous Ang-(1-7) (Ferreira et al. 2002) or Ang-(1-7) analogs [AVE 0991 and HPβCD/Ang-(1-7)] (Benter et al. 2006; Ferreira, Jacoby, et al. 2007; Ferreira, Oliveira, et al. 2007; Marques et al. 2011), overexpression of Ang-(1-7) in the heart (Ferreira et al. 2010; Mercure et al. 2008), and activation of endogenous ACE2 (Hernández Prada et al. 2008) induce beneficial and protective effects against cardiac injuries. Notably, most of these effects were abolished by the treatment with Mas antagonists, indicating not only that Ang-(1-7) is important but that the whole axis is involved in these cardioprotective actions.

This axis is apparently involved in the maintenance of the cardiac rhythm during ischemia/reperfusion (I/R) procedures. We have demonstrated that isolated hearts perfused with Ang-(1-7) present a significant reduction in the duration and incidence of I/R arrhythmias through the interaction of this peptide with Mas (Ferreira et al. 2001; Ferreira et al. 2010; Santos et al. 2004). Potential mechanisms by which Ang-(1-7) plays these effects include (1) activation of sodium pump, (2) induction cell membrane hyperpolarization, (3) reduction of the action potential duration, and (4) an increment of refractoriness (De Mello 2004; De Mello et al. 2007). However, the effects of Ang-(1-7) on heart rate (HR) are controversial, and both increases and decreases in HR have been reported in response to Ang-(1-7) (Braga et al. 2002; Ferreira et al. 2002; Santos et al. 2004). The aims of the current study were to identify the presence of the ACE2/Ang-(1-7)/Mas axis in the sinoatrial node (SAN) of rats and to verify whether this axis is functional using an isolated atria model. In addition, we took advantage of this model to direct evaluate the effect of Ang-(1-7) on HR.

Materials and Methods

Animals

Male Wistar rats weighting 200–250 g (3 months of age) were obtained from the animal facility of the Federal University of Minas Gerais (CEBIO, Brazil). The animals were housed in a climate-controlled environment under a 12-hr light/dark cycle with free access to rat chow and water. All the animal experiments were carried out in accordance with current guidelines for the care and humane use of laboratory animals and were authorized by the Ethics Committee of Federal University of Minas Gerais.

Histological Analysis

Under anesthesia with 10% ketamine/2% xylazine (4:3, 0.1 ml/100 g, intraperitoneally), the rats were killed, the thoraxes were opened, and the hearts were carefully dissected. Right atria were dissected and placed in 4% Bouin’s fixative for 24 hr at room temperature. The tissues were dehydrated by sequential washes with 70%, 80%, 90%, and 100% ethanol and embedded in paraffin. Serial transversal sections (5 µm) were cut and stained with Masson’s trichrome (n = 4 different animals). This staining identifies the collagenous connective tissue surrounding the SAN cells. An Olympus BX 41 (Irving, TX) microscope was used for visualization and imaging the SAN.

Immunohistochemical Analysis

Paraffin-embedded atrial sections (5 µm, n=4 different animals) were first incubated with 0.3% H₂O₂ in phosphate-buffered saline (PBS) for 15 min followed by incubation with 1.5% goat serum in PBS containing 0.3% Triton X100 for 1 hr. Sections were incubated overnight at 4C with one of the following antibodies diluted in PBS containing 0.3% Triton X100 and 0.3% BSA: rabbit polyclonal anti-ACE2 (1:500, GeneTex, Inc., Irvine, CA), rabbit polyclonal anti-Ang-(1-7) (1:600) (Ferreira et al. 2009), rabbit polyclonal anti-Mas (1:100, Abcam, Inc., Cambridge, MA), mouse polyclonal anti-Mas (1:100) (Becker et al. 2007), and rabbit polyclonal anti-connexin43 (1:1000, Santa Cruz Technologies, Inc., Santa Cruz, CA). After four or five rinses in PBS, biotinylated goat anti-rabbit IgG or biotinylated goat anti-mouse IgG secondary antibodies were added for 1 hr followed by incubation with avidin–biotin–peroxidase complex reagents for 1 hr, stained with diaminobenzidine solution for 4 min (Vector Laboratories, Burlingame, CA), and analyzed using an Olympus BX 41 microscope (Irving, TX). Each step was followed by washing the sections with PBS containing 0.3% Triton X100. Sections incubated without primary antibodies were used as negative controls.

Immunofluorescence Analysis

The ventricles (right and left) and left atrium were dissected free from the right atrium and discarded. Right atria were washed in PBS to remove excess blood, cryofixed in a −80C solution of 80% methanol and 20% dimethyl sulfoxide (DMSO), and then embedded in paraffin. Immunofluorescence-labeling technique was used to investigate the distribution of hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) in right atria. Heart sections (5 µm, n=2 different animals) were mounted on slides, deparaffinized with xylene, rehydrated through a graded series of ethanol to PBS, and then incubated in blocking solution (1% BSA and 0.1% Tween 20 in PBS) at room temperature for 1 hr. Sections were incubated overnight at 4C with rabbit polyclonal anti-HCN4 antibody (1:100, Alomone Labs, Jerusalem, Israel). The antibody was diluted with 1:10 diluted blocking solution. After four or five PBS rinses, donkey anti-rabbit IgG conjugated with Cy3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was added for 1 hr in the dark, at room temperature. Following washes with PBS, sections were mounted on 90% glycerin/10% 1 M Tris, pH 9.0, and viewed with a laser scanning confocal microscope (Zeiss 510Meta–CEMEL/UFMG). All confocal settings (aperture, gain, and laser power) were determined at the beginning of the imaging session, and these parameters were not changed. Images were captured at 12 bits using a gray scale, ranging from 0 to 255.

Isolated Right Atria

Male Wistar rats (200–250 g body weight) were decapitated 10–15 min after intraperitoneal injection of 400 IU of heparin. The thorax was opened and the heart was carefully dissected. Right atria were isolated and mounted on a 10-ml organ bath containing Krebs-Ringer solution (KRS) (composition in mmol/L: 118.4 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄˙ 7 H₂O, 2.5 CaCl₂˙ 2 H₂O, 11.7 glucose, and 26.5 NaHCO₃) saturated with 95% O₂/5% CO₂ at 37 ±1C. The upper extremity of the atrial appendage was connected to a force transducer (model FT3, Grass, West Warwick, RI) to record the contractile force (tension, g) on a computer through a data-acquisition system (Biopac System, Santa Barbara, CA), and the lower extremity was pinned by a bipolar platinum electrode used for electrogram detection and stimulation. Electrical stimulation and signal acquisition were controlled by a computer-based system. Electrogram and isometric tension signals were acquired via a customized acquisition system (Biopac System, Santa Barbara, CA). After application of 0.5 g of resting tension, an equilibration period of 20 min was permitted; during this period, right atria were allowed to beat spontaneously. Atrial tachyarrhythmias (ATs) were induced by electrical stimulation consisted of application of pulse trains (250 bipolar voltage pulses, amplitude ×1.5 threshold, 0.2 msec duration, 8 mV) at 66.7 Hz (Zafalon et al. 2004). Each application of a block of train was defined as a trial. In control conditions, 1–5 trains of 5 sec applied 0.5 min apart were sufficient to evoke arrhythmias at each trial. Right atria were perfused with KRS alone (control, n = 5) or KRS containing Ang-(1-7) (0.22 nmol/L, n = 7) or Ang II (0.22 nmol/L, n = 5). In a different group of rats, the specific Mas antagonist, A-779 (22 nmol/L, n = 5), was used to evaluate the role of Mas in the Ang-(1-7) effects. To obtain a quantitative measurement, ATs were graded arbitrarily according to their duration. Thus, the occurrence of ATs for up to 1.5 min was assigned the factor 2; 1.5–3 min was assigned the factor 4; 3–5 min was assigned the factor 6; 5–7.5 min was assigned the factor 8; 7.5–10 min was assigned the factor 10; 10–12.5 min was assigned the factor 11; and 12.5–15 min was assigned the factor 12. Consequently, a value of 0–12 was obtained in each experiment and denoted as arrhythmia severity index (ASI). ATs with duration of 15 min were considered as irreversible arrhythmias, and for each experimental group, we calculated the percentage of hearts that presented this kind of arrhythmia. HR and ±dT/dt were derived from the tension recordings (Ferreira et al. 2001). Ang-(1-7), Ang II, and A-779 concentrations used in this protocol were based on previous studies (Ferreira et al. 2001; Ferreira et al. 2002).

Statistical Analysis

All data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by the Newman-Keuls post test or paired or unpaired Student t-test (Prism 4.0 Graphpad software, Inc., La Jolla, CA). Statistical significance was accepted at p<0.05.

Results

The SAN cells were first identified by Masson’s trichrome staining. Figure 1A shows the localization of the SAN. The presence of collagenous connective tissue surrounding the SAN cells (staining in blue–white asterisk in Figure 1B and Figure 1C) is a feature of the SAN, and we used this to differentiate atrial muscle (black asterisk in Figure 1B) from SAN tissue. Additionally, it is possible to observe that the SAN cells are bigger than cardiomyocytes (SAN: 20.87 ± 0.42 µm, n=95 cells; atrial cardiomyocytes: 13.71 ± 0.26 µm, n=95 cells, p<0.05). It has been reported that cardiomyocytes, but not SAN cells, express connexin43 (Liu et al. 2007). Thus, to confirm the histological differences between SAN and atrial muscle, we used immunohistochemistry technique to detect connexin43 expression. Indeed, it was found that atrial cells exhibited specific immunolabeling for connexin43 in the intercalated disks (Figure 2A) and that SAN cells did not show any detectable immunolabeling for this protein (Figure 2B). Negative control was obtained when the primary antibody was omitted from the incubation procedure (Figure 2C). To further verify the histological differences between SAN and atrial muscle, we performed additional experiments using immunofluorescence for HCN4, a specific marker for SAN cells. It was observed that HCN4 is expressed in SAN cells (Figure 3A) but not in atrial muscle cells (Figure 3B). Thus, this experiment served as a positive marker for SAN. Again, negative control was obtained when the primary antibody was omitted from the incubation procedure (Figure 3C). Altogether, these results indicate that we were able to distinguish the SAN from the atrial muscle.

Figure 1.

Identification of the sinoatrial node (SAN) of rats. Masson’s trichrome staining was used to differentiate atrial muscle from SAN tissue. (A) Low magnification; (B) intermediate magnification; and (C) high magnification. The presence of collagenous connective tissue surrounding the SAN cells (in blue–white asterisk in B and C) is a characteristic of the SAN. In contrast, atrial muscle did not show this feature (black asterisk in B). The SAN cells are bigger when compared with atrial cardiomyocytes. Arrow: SAN. RA: right atrium; VC: vena cava.

Figure 2.

Connexin43 expression in atria. (A) Connexin43 is expressed in intercalated disks of atrial cardiomyocytes (arrows). The arrowhead shows an atrial cardiomyocyte cut transversally. (B) Sinoatrial node (SAN) cells did not express connexin43. The asterisk shows SAN cells. (C) Negative control was obtained when the primary antibody was omitted from the incubation procedure. The arrowhead shows an atrial cardiomyocyte and the asterisk shows SAN cells.

Figure 3.

HCN4 expression in atria. Immunofluorescence technique for HCN4 revealed that this protein is expressed (A) in sinoatrial node (SAN) cells but (B) not in atrial muscle cells. Thus, this experiment served as a positive marker for SAN. Arrows indicate SAN cells, and the asterisk shows atrial cardiomyocytes. (C) Negative control was obtained when the primary antibody was omitted from the incubation procedure.

Figure 4.

Expression of the angiotensin-converting enzyme 2 (ACE2)/angiotensin(Ang)-(1-7)/Mas axis in the sinoatrial node (SAN). The ACE2/Ang-(1-7)/Mas axis is widely expressed in cells of the SAN. (A) ACE2, intermediate magnification, (B) ACE2, high magnification, (C) Ang-(1-7), intermediate magnification, (D) Ang-(1-7), high magnification, (E) Mas receptor, intermediate magnification, and (F) Mas receptor, high magnification. (G and H) The presence of Mas in SAN cells was further confirmed using a second anti-Mas antibody. This antibody was produced in Mas knockout mice, and the data obtained also showed that Mas is expressed in SAN cells. (G) Mas receptor, intermediate magnification and (H) Mas receptor, high magnification. Inserts represent negative controls obtained when the primary antibody was omitted from the incubation procedure. Arrows: positive immunolabeling.

To investigate whether the ACE2/Ang-(1-7)/Mas axis expressed in SAN cells is functional, we examined the participation of this axis in the duration and severity of ATs induced by electrical stimulation of isolated right atria. We observed that perfusion of isolated atria with Ang-(1-7) significantly reduced the duration of ATs (11.2 ± 0.8 arbitrary units in control atria vs. 4.8 ± 1.4 arbitrary units in Ang-(1-7)–treated atria, p<0.05, Figure 5) as well as the severity of ATs, because only 14% of the atria perfused with Ang-(1-7) presented irreversible arrhythmias, that is, arrhythmias with more than 15 min of duration [control atria: 67% and Ang-(1-7)–perfused atria: 14%]. This anti-arrhythmogenic effect of Ang-(1-7) was attenuated by the Mas antagonist A-779 (Figure 5). Sixty percent of the atria perfused with Ang-(1-7) plus A-779 presented irreversible arrhythmias. No significant changes were observed in HR, systolic and diastolic tensions, and ±dT/dt (Table 1). Ang II at the same concentration of Ang-(1-7) (0.22 nmol/L) was unable to induce significant alterations in the duration of ATs (11.2 ± 0.8 arbitrary units in control atria vs. 9.8 ± 1.9 arbitrary units in Ang II-treated atria, p>0.05, unpaired Student t-test).

Figure 5.

Effects of activation of the angiotensin-converting enzyme 2 (ACE2)/angiotensin(Ang)-(1-7)/Mas axis on atrial tachyarrhythmias (ATs) induced by electrical stimulation of isolated right atria. Ang-(1-7) perfusion significantly reduced the duration of ATs. The specific Mas antagonist, A-779 (22 nmol/L), attenuated the Ang-(1-7) effects on the duration of ATs. The data are shown as mean ± SEM. *p<0.05 vs. control and ^# p<0.05 vs. Ang-(1-7).

Table 1.

Effects of Angiotensin(Ang)-(1-7) on Atrial Function

	Control (n=5)		Ang-(1-7) (n=7)
	Before	After	Before	After
Systolic tension, g	0.71 ± 0.02	0.70 ± 0.02	0.78 ± 0.04	0.77 ± 0.04
Diastolic tension, g	0.47 ± 0.01	0.45 ± 0.01	0.43 ± 0.02	0.45 ± 0.01
+dT/dt, g/sec	22.3 ± 1.4	23.3 ± 1.1	24.6 ± 1.7	24.5 ± 1.4
−dT/dt, g/sec	21.9 ± 1.2	22.3 ± 1.1	22.9 ± 1.4	23.1 ± 1.3
Heart rate, bpm	199.1 ± 13.9	198.8 ± 11.4	242.3 ± 17.1	234.0 ± 20.9

Data are shown as mean ± SEM. No significant changes were observed in contractile tension, ±dT/dt, or heart rate of isolated atria when the periods before and after perfusion with vehicle (Krebs-Ringer solution) or Ang-(1-7) were compared. Before: Period before the addition of vehicle or Ang-(1-7) into the bath; After: Period after the addition of vehicle or Ang-(1-7) into the bath (paired Student t-test).

Discussion

The major finding of our present study is that the SAN of rats expresses the ACE2/Ang-(1-7)/Mas axis of the RAS. More important, we showed that this axis is functional because Ang-(1-7) reduced the duration and incidence of ATs induced by electrical stimulation of right atria. It has been reported that Ang-(1-7) possesses anti-arrhythmogenic effects during I/R procedures in rats mediated by Mas activation (Ferreira et al. 2001). These effects were confirmed by genetically modified rats that present elevated plasma levels of Ang-(1-7) (Santos et al. 2004) or overexpress this heptapeptide in the heart (Ferreira et al. 2010). In addition, a recent study showed that Ang-(1-7) prevents changes in ionic currents induced by ATs in dogs (Liu et al. 2010). In line with these findings, we found that isolated right atria perfused with Ang-(1-7) presented a significant reduction in the duration and incidence of ATs. Furthermore, this anti-arrhythmogenic property of Ang-(1-7) was attenuated by co-administration of A-779, indicating that the Mas receptor is involved in these effects. Thus, our current data showing the presence of a functional ACE2/Ang-(1-7)/Mas axis in the SAN provide a direct morphological support to the anti-arrhythmogenic effect of Ang-(1-7).

The role of Ang-(1-7)/Mas in HR is not fully established. Both tachycardia and bradycardia effects have been reported in response to Ang-(1-7)/Mas activation (Braga et al. 2002; Ferreira et al. 2002; Neves et al. 1997; Santos et al. 2004). For instance, the increased HR observed in transgenic rats that present an approximately 2.5-fold augmentation in plasma Ang-(1-7) concentration (Santos et al. 2004) contrasts to the slight but significant bradycardia viewed in Wistar rats infused with Ang-(1-7) for 7 days (Braga et al. 2002). Moreover, Mas deletion caused a significant reduction in HR, suggesting that the basal interaction between Ang-(1-7) and Mas is important to maintain the cardiac rhythm (Santos et al. 2006). However, other studies failed to demonstrate any significant effect of Ang-(1-7) on HR (Ferreira et al. 2002; Neves et al. 1997). In the present study, we also did not observe any major change in the HR of isolated right atria after Ang-(1-7) perfusion. Strain differences and short- and long-term administration of the peptide and different methods may contribute to these divergent observations. Our preparation offered the possibility of investigating the direct actions of Ang-(1-7) on the SAN without the influence of others factors such as autonomic nervous system, ventricular mass, and circulating hormonal substances.

It has been reported that transgenic mice overexpressing ACE2 in myocardial cells present cardiac rhythm disturbances (Donoghue et al. 2003). However, nowadays it is generally accepted that ACE2 holds beneficial effects in the heart and that ACE2 genetic deficiency aggravates heart failure. The possible explanation for these opposite phenotypes in these transgenic models (overexpression vs. deficiency) may be related to an undefined role of ACE2 during development. It is reasonable that chronic overexpression of ACE2 and/or Ang-(1-7) may induce developmental abnormalities in transgenic mice. Another likely explanation for the deleterious effects viewed in ACE2 transgenic mice is the ability of Ang-(1-7), at high concentrations, to induce rhythm disturbances in rodents (De Mello et al. 2007). Finally, these effects could be tissue specific because Rentzsch et al. (2008) reported that overexpression of ACE2 in the vasculature reduces blood pressure and improves endothelial function in hypertensive rats. One limitation of our study is that we did not evaluate the functional role of ACE2 in the cardiac rhythm using specific inhibitors or the novel ACE2 activator XNT (Hernández Prada et al. 2008). However, we advanced the field by showing for the first time the presence of this enzyme in the SAN cells. Future experiments are obviously needed to demonstrate the functional role of ACE2 in the control of the cardiac rhythm.

In summary, we showed that the ACE2/Ang-(1-7)/Mas axis is expressed in the SAN cells of rats, providing the morphological support to the anti-arrhythmogenic effect of Ang-(1-7).

Footnotes

The author(s) declared no potential conflicts of interest with respect to the authorship and publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported in part by FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) (Grant No. APQ-00138-08).

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