INTRODUCTION

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CARDIAC HYPERTROPHY is an in vivo adaptive response that allows the organism to maintain or to increase its cardiac output. Although the mechanism that triggers this cardiovascular dysfunction is still unclear, the involvement of hormones and paracrine substances related with blood pressure control, such norepinephrine and the renin-angiotensin system (RAS), has been reported (11, 24, 40). Activation of the local RAS by mechanical stress (32) or -adrenergic stimulation can provoke hypertrophy (9). The reversion of cardiac hypertrophy after angiotensin-converting enzyme (ACE) inhibitors and angiotensin II (ANG II) antagonist treatment (12, 45) suggests that ANG II plays an important role in the cardiac hypertrophy seen in most forms of clinical and experimental hypertension. In the past two decades, a wealth of information has been amassed about the role of tissue ANG II. In the heart, this peptide seems to mediate several important functions, such as vascular coronary tonus, positive inotropic and chronotropic effects (38), and also growth of the myocardium (13, 28). There is evidence that all the components of the RAS are present in the heart. Even though some authors speculate on the putative mechanism of renin uptake from the plasma into the heart (14), it is well described that renin synthesis in the heart is ~2% of that of the kidney (15). On the other hand, some studies have demonstrated that, in the human heart, ACE inhibitors are able to block only partially the formation of ANG II and that the major proteinase responsible for ANG II generation in the left ventricle (LV) is the serine proteinase chymase (46). The existence of alternative pathways for ANG II formation involving proteinases other than renin and ACE has been shown in several species, including humans (4, 39, 41). An enzyme that represents a good candidate to be involved in ANG II generation as an alternative route is the serine proteinase tonin, which is able to release ANG II directly from angiotensinogen (AG) (20). Tonin is present in many organs, such as the brain, kidney, prostate, and submandibular glands (1, 2, 10, 21, 29, 30). There is no information about the presence of tonin in the rat heart. Tonin is secreted from the submandibular gland to saliva and venous effluent by controversial mechanisms. In in vivo experiments, by intraperitoneal injection of the -adrenoceptor agonist isoproterenol, it was shown that tonin secretion is enhanced by -adrenergic stimulation (18, 19, 22). On the other hand, experiments using dispersed cells from the rat submandibular gland showed that the release of tonin is -adrenoceptor dependent (25, 31).

Chronic exposure to isoproterenol can induce cardiac hypertrophy (34). The myocardium growth induced by isoproterenol could be the result of either direct stimulation or mediated by secondary metabolic, hemodynamic, or endocrine alterations produced by adrenergic stimulation. In this model, the ANG II-generating system(s) is activated, and the progression and prevention of cardiac hypertrophy were associated with changes in cardiac tissue ANG II (42). In the present study, with the use of repeated administration of isoproterenol in subpressor dosages, we provide evidence for the participation of tonin in the development of isoproterenol-induced cardiac hypertrophy in the rat.

	METHODS

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Hypertrophy development and tissue extraction. All experimental procedures were conducted in accordance with our institutional guidelines. Cardiac hypertrophy was induced in male Wistar rats (n = 12) by treatment with isoproterenol (Sigma) for 7 days (5 mg · kg¹ · day¹ sc). The control group received saline injections (n = 12). On day 8, animals were killed by decapitation, and the heart was quickly removed, washed with 0.9% NaCl (wt/vol), and separated into atrial [right plus left atria (AT)], right ventricular (RV), and LV sections. Hypertrophy was monitored by a cardiac index (heart weight/body weight) and by atrial natriuretic peptide (ANP) expression in the LV of animals. The use of ANP mRNA measurement to confirm cardiac hypertrophy status has been used by other groups (5, 42, 47).

Tonin activity. After being weighed, heart structures (n = 9 each) were homogenized in 250 mM sucrose (pH 7.0) containing 20 mM EDTA, 10 mM o-phenanthroline, 16 mM dipyridyl, and 10 mM sodium tetrathionate for determination of tonin activity. After centrifugation, a fraction of each supernatant was incubated with synthetic renin substrate [(1-14)AG] in 0.1 M sodium phosphate buffer (pH 6.8) containing the inhibitors previously described, and the ANG II liberated was determined by radioimmunoassay (21). Alternatively, tissues were homogenized in sucrose solution without inhibitors and incubated with (1-14)AG also in the absence of inhibitors.

Gene expression. From a second group of treated (n = 3) and control animals (n = 3), the heart structures, submandibular gland, and kidney were used for extraction of total RNA using TRIzol reagent (Life Technologies; Eggenstein, Germany) according to the protocol of the manufacturer. Rat tonin, ANP, AG, renin, ACE, and -actin mRNA were identified by ribonuclease protection assay (RPA; AMS Biotechnology) or RT-PCR. For RPA, the specific probes were generated using as the template a DNA fragment cloned into the pGEM-T Easy vector (Promega). The probes were prepared by linearization of the plasmid and purification from agarose gel using the QIAEX II gel extraction kit (Qiagen). The labeled antisense RNA probes were synthesized by T7 RNA polymerase in the presence of [³²P]UTP using a RNA transcription kit (Stratagene) and purified on a 5% acrylamide-containing 8 M urea gel. Fifty micrograms of total RNA per sample were hybridized with ~32 nCi of the radiolabeled antisense probe (4). The -actin probe was used as a control for the amount of RNA. The hybridized fragments protected from ribonuclease A digestion were separated by electrophoresis on a denaturing gel (5% polyacrylamide and 8 M urea) and analyzed using a FUJIX BAS 2000 phosphorimager system (Fuji). The assays were repeated at least three times with similar results. The RT reaction was carried out using 1 µg of total RNA, oligo(dT) primers, and the reverse transcriptase Moloney Murine Leukemia Virus (Life Technologies), followed by a specific PCR utilizing the primers ton5 (5'-ACCTGATACCATGTGGCTCC-3') and ton3 (5'-CATGGTGGGTTTTATTGAGAC-3') for tonin, rACE5 (5'-GTCACCGCCGCTCTTGATGCTG-3') and rACE3 (5'-GGCTTCATTACTGAGGGCAG-3') for ACE, and A954-7 (5'-GTTCCGATGCCCCGAGGATCT-3') and A954-8 (5'-GCATTTGCGGTG- CACGATGGA-3') for rat -actin. -Actin cDNA was amplified as a control for the amount of RNA.

Other methods. Iodinated ANG II (35) was purified utilizing a reverse-phase column (Mino RPC) in a HPLC system. Protein (7) and peptide (26) concentrations were determined spectrophotometrically [molar extinction coefficient () = 1,379 M¹ · cm¹ for ANG II and  = 2,758 M¹ · cm¹ for (1-14)AG]. Data are given as means ± SD. Statistical comparisons were performed by one-way ANOVA and Student's t-test for unpaired data to evaluate differences between the control and experimental groups. A P value <0.05 was considered statistically significant.

	RESULTS

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In the present study, treatment with isoproterenol for 7 days induced significant hypertrophy in the rat heart. This effect was evaluated by the ratio between the weight of the heart or heart structures and the weight of the respective animal (Fig. 1) and by the expression level of ANP, which is a marker for hypertrophy, in the LV (Fig. 2, A and B). The hypertrophic effect of isoproterenol was observed by the increase in the heart ratio of the treated animal compared with the value obtained for the control animal (4.33 ± 0.24 vs. 3.25 ± 0.23) and for all the heart structures analyzed separately, however, with different ratios (RV: 1.14 ± 0.05 vs. 0.79 ± 0.03, LV: 2.79 ± 0.15 vs. 2.11 ± 0.11, and AT: 0.24 ± 0.01 vs. 0.17 ± 0.01). The expression of ANP in the LV, normalized by the expression of the housekeeping gene -actin, increased significantly after treatment (18.62 ± 1.38 vs. 10.51 ± 0.56).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Effect of isoproterenol (Iso) treatment on the weight of rat heart structures. AT, left plus right atrium; RV, right ventricle; LV, left ventricle. *P < 0.05 compared with control.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Expression levels of atrial natriuretic peptide (ANP) in the LV of control (C) and Iso-treated animals determined by ribonuclease protection assay (RPA). A: graphic representation of the ANP-to--actin mRNA ratio; B: radioactive image of the polyacrylamide gel electrophoresis. *P < 0.05 compared with control.

Figure 3 shows the tonin-specific (A) and total activities (B) determined in the heart structures. Tonin-specific activity was significantly higher in the AT compared with the other structures, and treatment with isoproterenol significantly increased the specific activity in this structure (0.91 ± 0.09 vs. 0.47 ± 0.07) but not in the RV and LV (Fig. 3A). The level of total tonin activity was shown to be higher in the LV; however, only in the AT did it significantly increase after isoproterenol treatment (0.83 ± 0.12 vs. 0.37 ± 0.05). With the use of the RPA, we evaluated tonin expression levels in the AT and LV structures (Fig. 4) and compared the values to that of the submandibular gland. In the untreated group, tonin expression was undetectable in the heart structures. Isoproterenol treatment induced tonin expression to levels detectable only in the AT. Evaluation of mRNA levels for AG in the AT and LV showed that this gene is expressed in the AT at higher levels than in the LV (Fig. 5). Isoproterenol treatment provoked a significant augmentation of AG expression only in the AT (1.8-fold). Renin expression was also evaluated in the heart structures and compared with the gene expression in the kidney. In the heart structures, renin is expressed at undetectable levels even after isoproterenol-induced hypertrophy (Fig. 6). Expression of ACE was determined by RT-PCR and, for comparison, tonin expression was also determined by the same method. Isoproterenol treatment did not change the levels of ACE expression in the AT and LV, and again we verified an increase of tonin expression in the AT (Fig. 7).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Tonin-specific (A) and total activities (B) in the heart structures. The activity was determined by incubating samples with (1-14)angiotensinogen (AG), and ANG II was quantified by radioimmunoassay. *P < 0.05 compared with control.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Expression levels of tonin determined by RPA in the AT and LV of the Iso-treated group (ATISO and LVISO, respectively) and the AT, LV, and submandibular gland (Sg) of the control group. A: graphic representation of the tonin-to--actin mRNA ratio; B: radioactive image of the polyacrylamide gel electrophoresis. *P < 0.05 compared with control.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Expression levels of AG determined by RPA in the AT and LV of the Iso-treated group and the AT and LV of the control group. A: graphic representation of the AG-to--actin mRNA ratio; B: radioactive image of the polyacrylamide gel electrophoresis. *P < 0.05 compared with control.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Expression levels of renin determined by RPA in the AT and LV of the Iso-treated group and the AT, LV, and kidney (K) of the control group. A radioactive image of the polyacrylamide gel electrophoresis is shown.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Expression levels of tonin (A) and angiotensin-converting enzyme (ACE; B) determined by RT-PCR in the AT and LV of the Iso-treated group and the AT and LV of the control group. *P < 0.05 compared with control.

	DISCUSSION

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The structural basis of cardiac hypertrophy and the participation of ANG II in this process has been extensively studied, and several experimental data suggest that the process is dependent on a direct effect of ANG II on the myocytes (6, 8, 48). Calcineurin, a Ca²⁺/calmodulin-dependent phosphatase, has a pivotal role as a cellular target for a variety of Ca²⁺-dependent signaling pathways and is involved in the signal transduction of ANG II-induced cardiomyocyte hypertrophy and fibroblast hyperplasia (17). Transgenic animal models have been generated with the aim to evaluate the importance of the RAS in cardiac hypertrophy. A new hypertensive mouse model lacking AG expression in the heart presented increased cardiac weight and collagen synthesis compared with that of the normotensive control mouse (3), showing that local synthesis of AG is involved but not essential in the development of cardiac hypertrophy. On the other hand, isoproterenol increases LV weight and ANG II concentration in this tissue even in nephrectomized rats (34), suggesting that the activation of local systems releasing ANG II in the heart may contribute to isoproterenol-induced hypertrophy. The abnormal accumulation of fibrillar collagen in the extracellular space of the hypertrophied heart was already observed by Weber and colleagues (47). The main site of AG synthesis in the heart is the AT (37). After 8 days of treatment with isoproterenol, AG mRNA levels in the heart were markedly induced (23). We also verified that AG is present in the AT and that its expression level in this tissue increases by treatment with isoproterenol. In the LV, AG was detected at very low levels without a significant difference after treatment. Even though renin mRNA has been detected by others in minute amounts in the AT, ventricles, and isolated cardiomyocytes (16, 49), we were not able to detect it in both the AT and LV in control or treated animals. The presence of renin in the heart is controversial. The general consensus indicates that renin is present in the heart in ~2% of the amount synthesized in the kidney; however, it is not yet clear whether it is locally synthesized or taken up from the plasma. In regard to ACE, our results show that its levels do not change in heart tissue after isoproterenol treatment, in contrast to the data of Ocaranza and colleagues (36). These authors observed that long-term administration of isoproterenol in the rat induces the expression of ACE, in that LV ACE activity correlated with ACE mRNA levels and paralleled development of LV hypertrophy. In the heart, ACE is mainly localized on fibroblasts and in the endothelium (50), and there is some controversy about its participation in the heart hypertrophy. It has been suggested that increased local ACE expression contributes to the development of pressure overload-induced cardiac fibrosis but is not responsible for hypertrophy in the rat (27) and that mechanisms different from activation of the RAS may play a decisive role in the maintenance of hypertrophy, particularly in the model of volume hemodynamic overload (44). Our results show for the first time that tonin is present in the heart. Furthermore, tonin activity and expression in the AT were stimulated by isoproterenol treatment. However, tonin mRNA could not be detected by RPA before treatment. This could be explained by the results showing low levels of tonin-specific activity in the tissue homogenate, which were significantly altered only in the AT. Tonin-specific activity in the AT increased almost 2-fold after treatment and became 6.5- and 13-fold higher than that in the LV and RV, respectively. Total activity in the AT increased 2.2-fold, whereas in the LV and RV the augmentation was proportional to the mass increase. Taking into account that the levels of tonin mRNA and activity increased significantly in the AT after treatment, tonin may represent one of these ANG II-releasing systems that contributes to isoproterenol-induced hypertrophy. Because tonin and AG are present in greater amounts in the AT, the cardiac interstitium could probably be the pathway by which tonin-generated ANG II can reach the ventricles (48). Tonin in the AT can also participate in the metabolism of other peptides such as substance P and in the processing of ANP. Tonin can cleave synthetic peptides that reproduce the sequence of rat pro-ANP in the region of the activation site (32). So far, no enzyme(s) that could process pro-ANP in vivo has been clearly identified. As ANP is stored as a large protein precursor in cardiac myocytes, tonin may be involved in the activation of this peptide in vivo.

In conclusion, our data suggest that tonin may be an important pathway for the generation of ANG II in the AT and ventricles, causing a new dynamic in heart function, with an increase in positive inotropic and chronotropic effects and remodeling of the myocardium. Therefore, the generation of drugs aiming to modulate tonin activity may be used as a new therapy to treat cardiovascular diseases. ACE inhibitors are important tools for reversing cardiac hypertrophy; however, a question that arises from these data is: how altered are the expression levels of tonin in cardiac tissue after treatment with these drugs?