Wild type C57BL/6 (WT) and homozygous transgenic mice overexpressing the rat tonin enzyme [TGM'(rton)] were used in this study. Mice were housed in a temperature-controlled room (21 ± 1°C) under a 12:12-h dark light cycle, received standard laboratory chow and water ad libitum. The research ethics committee of the Federal University of São Paulo approved experimental protocols used in this study.

Transgenic TGM'(rTon) mice were generated by microinjection of the rat tonin transgene into zygotes as described by Ribeiro et al. (17, 18). TGM'(rTon) mice exhibited higher tonin activity in the brain, heart, kidney, liver, and bladder compared to WT (17).

Catheters filled with heparinized saline were implanted into carotid artery and jugular vein of the anesthetized mice (80 mg/kg ketamine and 12 mg/kg xylazine, i.p.) Animals were placed in individual clean cages with free access to food and water. A heating lamp was used to keep the animals warm until their full recovery. Forty-eight hours later if a mouse presented signs of decreased activity, reluctance to move, hunched posture, and ungroomed fur, it was withdrawn from the study. The animals that recovered well were connected to computerized acquisition systems by connecting the arterial cannula to an electromagnetic transducer (Kent Instruments) by a polyethylene tube. This transducer was linked to an amplifier (General Purpose Amplifier-Stemtech, Inc.) and connected to a computer with an analog to digital converter board (Windaq/DataQ). The venous catheter was tethered to a 25 cm polyethylene tube filled with heparinized saline. To allow for stabilization of their blood pressure, the animals remained connected for at least half an hour to the system. After the adaptation period, 30 min of real-time blood pressure and heart rate signals were recorded before and after a single injection of losartan (20 mg/Kg, iv) in conscious animals who were free to move. Losartan was injected through the venous catheter.

Analysis of blood pressure signals was performed using an algorithm implemented in acquisition system Windaq/DataQ. This software allowed the detection of the maximal value in the blood pressure curve, beat to beat, providing the values of systolic blood pressure (SBP). Heart rate was determined from the pulse interval (PI) between two systolic peaks. Heart rate variability was measured by linear methods in time and frequency domains. Temporal series of PI and SBP (30 min of baseline recording) were analyzed in time domain, obtaining the total variance of PI (VAR PI) and the variance of SBP (VAR SBP). Mean square root of differences between consecutives PI (RMSSD) was also evaluated. For the frequency domain, one spectrum was obtained for each segment and the oscillatory components were quantified in two different frequencies: low frequency (LF) from 0.1 to 1 Hz and high frequency (HF) from 1 to 5 Hz. The results are represented by absolute values (ms² and mmHg²), the percentage of total spectrum (%) and normalized units (nu) (percentage of LF and HF bands only).

Animals were euthanized by decapitation at 12–14 weeks old. Blood samples were collected and plasma was immediately separated from the whole blood by centrifugation at 1,500 rpm for 10 min and stored at −80°C. Hearts were rapidly removed, snap frozen in liquid nitrogen and stored at −80°C until experimental procedures were performed.

Atria and right and left ventricles were dissected for homogenization in 100 mM sodium phosphate, 240 mM sucrose, 300 mM NaCl, and the protease inhibitors: 1 mM EDTA (metalloprotease inhibitor), 1 μM o-phenanthroline (metalloprotease inhibitor), 120 μM pepstatin A (aspartyl protease inhibitor), 1 μM PMSF (serine protease inhibitor), and 1 mM 4-chloromercuribenzoic acid (cysteine protease inhibitor) at a ratio of 5 ml of buffer per gram of tissue. Homogenates were centrifuged at 20,000x g for 1 h at 4°C. In each resulting supernatant, 1 nmol of Sar¹-AngII was introduced as an internal standard. Samples were purified and concentrated using C18 Sep-Pak columns previously activated with methanol (5 ml), tetrahydrofuran (5 ml), hexane (5 ml), methanol (5 ml), and water (10 ml). After loading the samples into the column, purification was performed by washing the column with ultrapure water. Samples were eluted from the column using 5 ml of ethanol/acetic acid/water in the proportion (90:4:6). The eluates were lyophilized then rehydrated in 500 ul of mobile phase A (5% acetonitrile in 0.1% orthophosphoric acid. Each sample was filtered (0.22 um pore size filter) and injected into the HPLC. The peptides were separated on the reverse phase column Aquapore 300 ODS (250 x 4.6 mm 7 μ) connected to a high-performance liquid chromatography system (Shimatzu) with 214 nm UV detection. The peptide separation was performed with 20 min of linear gradient from 5 to 35% mobile phase B (95% acetonitrile in 0.1% orthophosphoric acid) after 5 min of isocratic gradient over, at constant flow rate of 1.5 ml/min. Sar¹-AngII, AngI, AngII, and Ang1-7 synthetic peptides (Sigma) were used as standards and the angiotensins in each sample were identified according to the angiotensin standards retention time. The concentrations were expressed in picomol per gram of tissue.

Hearts were dissected into atria, right ventricle, and left ventricle. These chambers were homogenized in 50 mM Tris/HCl, 0.5 mM ZnCl₂ buffer, pH 7.5, followed by centrifugation at 10,000 rpm at 4°C for 10 min. The resulting supernatants were separated from the pellet. The protein concentration of the supernatants was determined by Bradford (Biorad) method as recommended by the manufacture. Protein quantification was performed on the supernatants for further normalization of each peptide chromatographic-peak-area.

Tetradecapeptide (TDP) (10 nmol), a synthetic analog of angiotensinogen, was incubated in 1 ml of buffer (50 mM Tris/HCl, 0.5 mM ZnCl₂, pH 7.5) containing either 50 ul of plasma, 100 ul of atria or right or left ventricle homogenates at 37°C for 24 h. Aliquots were collected at 10, 20, 40, 60, 120, 240, 360, and 1,440 min into a tube containing 10% formic acid. Angiotensin I, Angiotensin II, and Angiotensin 1-7 were quantified by liquid chromatography/mass spectrometry.

All data are expressed as mean and standard error of mean. Significance level was determined by using two-way ANOVA with Bonferroni post-hoc test or with multiple t-test comparison. 95% confidence of interval was used and P-value < 0.05 was considered statistically significant.

Overexpression of tonin caused an increase in diastolic and systolic BP (p = 0.0464) without altering heart rate. Pharmacological blockade of AT1R reduced systolic and diastolic BP in both strains (p = 0.0036; Figure 1). However, when the delta value of BP was evaluated before and after losartan injection, it was observed that losartan induced greater reduction of BP in TGM'(rTon) than WT (21.46 vs. 11. 86 mmHg, respectively).

At baseline the TGM'(rTon) mice showed a reduction in total HR variability, represented by variance of pulse interval (VAR PI). Together a clear reduction in the sympathetic specific spectrum band (LF) was observed when analyzed the total (ms²), percentage (%), and normalized (nu). Interestingly, an elevation of parasympathetic modulation was observed when analyzing its specific spectrum band (HF). Consequently, a decreased sympathovagal balance was observed when compared to WT group (Figure 2). In the TGM'(rTon), blockade of AT1R caused further decrease and increase in the sympathetic and parasympathetic modulation, respectively. Both effects were greater in the TGM'(rTon) when compared to the WT group.

TGM'(rTon) at baseline and after angiotensin receptor blockage did not significantly alter the systolic BP variability represented by VAR SBP. Pharmacological blockade of AT1R caused reduction of sympathetic modulation in the controls, but it increased in the TGM'(rTon) (Figure 3). After losartan injection, TGM'(rTon) exhibited lower baroreflex sensitivity measured by the alpha index compared to WT group.

Within each WT and TGM'(rTon) group, levels of AngII and Ang1-7 did not differ among heart compartments, indicating that the steady-state levels of AngII and Ang1-7 levels follow similar regulation. Levels of Ang I in the atria were 2 times higher than in the right and left ventricle in WT group and 1 time higher in TGM'(rTon). When levels of angiotensins are compared between groups, TGM'(rTon) mice showed decreased levels of AngI in atria and higher levels of AngI in both right ventricle and left ventricle. Contrastingly, AngII levels were uniformly decreased in TGM'(rTon) heart chambers compared with WT group (Figure 4). This observation was accompanied by a large increase in Ang1-7. These results indicate changes in angiotensin processing, resulting in production of Ang1-7 instead of production of Ang II in TGM'(rTon) mice.

The proteolytic capacity of each compartment generating angiotensins were tested in vitro by a mass spectrometry-based assay. Extracts of atria, right ventricle and left ventricle of both WT and TGM'(rTon) mice were preincubated with tetradecapeptide and levels of Ang I, Ang II, and Ang1-7 were quantified by LC-MSMS in a time-dependent manner. Total atria extract were capable of rapidly consuming the TDP, generating Ang I in the first minute of incubation until a maximum concentration was reached between the 120 and 240 min for WT group and 360 min for TGM'(rTon) mice (Figure 5A). Corroborating the observation made using HPLC assay, atrial production of Ang I was shown to be higher in WT than TGM'(rTon)mice.

Levels of angiotensins generated by right ventricle extracts from tetradecapetide precursor were also measured. There was a rapid formation (0–40 min) of Ang I, AngII, and Ang1-7 in the TGM'(rTon) group compared to the WT group (120–360 min), contrasting with the delay in formation observed in the WT extracts. Interestingly, the highest peak of Ang I production corresponds to 20 min in TGM'(rTon) group; in contrast, the highest peak of Ang I formation was reached at 4 h in the WT mice (Figure 5B). A similar difference was obtained for Ang II (40 min for TGM'(rTon) group vs. 4 h for WT group; Figure 5C) and for Ang1-7 (60 min for TGM'(rTon) and 360 min for WT group, Figure 5D). It is noteworthy that the total formation of Ang1-7 was 2 times higher than WT group. These data suggest that RV extracts of TGM'(rTon) produce all angiotensins faster than WT mice.

The levels of Ang1-7 were also measured using left ventricle extracts using Ang II as precursor (Figure 5E). The levels of Ang1-7 was significantly higher for TGM'(rTon) compared to WT mice. Total Ang1-7 formation in the atria, RV and LV corroborate the observation of Ang1-7 content by HPLC, i.e., higher levels of Ang1-7. Similarly, total AngII formation in the RV corroborate the observation found by HLPC, lower levels of AngII in the heart.

The proteolytic capacity of plasmatic proteases generating angiotensins from tetradecapeptide were also analyzed. Plasmatic proteases were capable of rapidly consuming tetradecapeptide, generating high concentrations of Ang I in the first 10 min of incubation for both TGM'(rton) and WT mice (Figure 6A). However, the amounts of plasmatic Ang I produced by TGM'(rTon) group was 10 times lower than the amounts obtained by WT mice, but still higher than the amounts obtained using atrial and RV extracts. The amounts of AngII formed in the plasma from TDP as substrate were similar in both mice (Figure 6B). Again, the amount of Ang1-7 was higher in the TGM'(rTon) compared to WT group (Figure 6C).

The amounts of Ang II and Ang1-7 generated by plasmatic proteases using Ang I and Ang II as precursors were also measured. When the tested precursor was Ang I (Figures 7A,B), the level of Ang II and Ang 1-7 levels were significantly higher in TGM'(rTon) compared to control mice, reaching a maximum concentration after 40–60 min of incubation. Again, higher Ang 1-7 levels were obtained using Ang II as precursor in the TGM'(rTon) group when compared to the WT mice (Figure 7C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HU declared a non-overlapping institutional affiliation, though no other collaboration, with one of the authors, ZJ to the handling Editor.