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1 Heart Institute (InCor), Department of Medicine-LIM13, University of São Paulo Medical School, São Paulo
2 Federal University of Espirito Santo, Vitoria, Espirito Santo, Brazil
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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50% upon 3- or 6-wk PO. Angiotensin II AT1 receptor blocker treatment produced a significant pressure independent decrease in the LVMI/SBP ratio. Unlike the one-copy group, PO resulted in a significant reduction in angiotensinogen and an increase in Ace mRNA expression accompanied by an increase in cardiac angiotensin II levels in the three-copy group. Similarly, the human ACE D gene variant influenced cardiac mass, estimated by Sokolov-Lyon index, in a sample of 1,507 individuals from an urban population only in individuals in the 4th quartile of the blood pressure distribution. Collectively, these data provide direct evidence that ACE gene dosage per se does not influence cardiac mass but upon a pathological stimulus, such as elevation in blood pressure, it modulates cardiac mass in both mice and humans. pressure overload; renin-angiotensin system; angiotensin converting enzyme polymorphism; hypertension
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INTRODUCTION |
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The activation of local components of the renin-angiotensin system (RAS) that accompanies the development of cardiac hypertrophy appears to play a role in this process (7, 14, 51). Left ventricle hypertrophy induced by aortic ligation results in a significant upregulation of RAS local components, while plasma renin levels remain unchanged (4, 41). Additionally, there have been clear beneficial effects on cardiac hypertrophy regression associated with antihypertensive pharmacological therapy based on angiotensin converting enzyme (ACE) inhibitors (37). These effects appear to be independent, at least in part, from decreasing hemodynamic load, suggesting a direct contribution of the RAS components (13). A number of studies have shown the association between functional gene variants of the RAS and cardiovascular disease. However, there is little evidence indicating that small quantitative changes in ACE activity is directly associated to development of cardiac hypertrophy. Using genetically modified animals, Tian et al. (47) demonstrated in rats that a 13- to 50-fold increase in cardiac Ace augments cardiac hypertrophy in response to PO but does not influence basal cardiac mass. Similarly, 100-fold increase in cardiac ACE levels in mice, while kidney or vascular endothelial ACE remained unchanged, resulted in enlargement of both atria and high incidence of sudden death with no changes in blood pressure and renal function (52). These results show that large increases in ACE per se or combined with a stress results in altered cardiac phenotype, but it does not properly address the possible effects of small perturbations in Ace, less than a fold change, similarly to plasma or tissue ACE levels observed with naturally occurring human ACE gene variants, e.g., insertion-deletion (I/D) polymorphism (6, 10, 33, 38, 48). In this regard, we have demonstrated that an increase or decrease of less than a fold in normal ACE levels does not influence the magnitude of physiological exercise-induced cardiac hypertrophy in mice (15).
In the present study we wanted to test the hypothesis that the same small ACE gene perturbation can indeed influence the magnitude of cardiac hypertrophy in response to a pathological stimulus, such as PO. First, genetic engineered mice carrying one, two, or three copies of the Ace gene at its endogenous locus, developed to achieve lower and above normal ACE levels (24), were submitted to PO. Then, the ACE I/D polymorphism, which is associated with different levels of plasma and tissue ACE, was tested for association with cardiac mass in a large sample from an urban population taking into account blood pressure levels.
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METHODS |
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PO-Induced Cardiac Hypertrophy
PO was produced by surgical constriction of the thoracic aorta. In brief, mice were anesthetized by intraperitoneal injection of a mixture of ketamine (70 mg/kg) and xylazine (4 mg/kg). Endotracheal intubation was performed via a blunt 20-gauge needle that was connected to a volume-cycled ventilator (model 683, Harvard Apparatus) with a volume of 1.0 ml and a respiratory rate of 90/min. The chest cavity was entered in the third intercostal space, and the thoracic aorta was isolated. We performed thoracic aorta constriction by tying the vessel with a 4.0 nylon suture ligature against a 27-gauge needle. The chest was closed with 6-0 nylon strings, the pneumothorax was evacuated, and mice were extubated and allowed to recover from the anesthesia.
Direct Blood Pressure Measurements
Systolic blood pressure (SBP) was directly monitored proximal to the aortic banding before the killing of the animals to determine an index of cardiac pressure load. A polyethylene cannula (PE-08) was implanted under anesthesia (pentobarbital sodium, 40 mg/kg ip) into the left carotid artery and tunneled to the back of the mice. Blood pressure was monitored 6 h later in awake animals. The analogical signal from the strain gauge transducer (Stathan P23Dd; Hato Rey, Puerto Rico) was amplified (GPA-4 model 2, Stemtech), converted to digital at 10 bytes (DataQ Instruments), and further recorded at 2,000 Hz.
Left Ventricular Mass Quantification
After blood pressure measurements, mice were killed by an overdose of pentobarbital sodium. The chest was opened, and left and right atria and left and right ventricles were dissected and weighed. Left ventricular hypertrophy index was calculated by the left ventricle weight-to-body weight ratio (mg/g).
mRNA Quantitation of the RAS Components
Renin, angiotensinogen (Agt), Ace, and Ang II type 1a receptor (AT1a) and type 2 (AT2) expression levels were evaluated on left ventricle tissues from 27 mice (1 copy + Control n = 06, 1 copy + PO n = 06, 3 copy + Control n = 10, 3 copy + PO n = 07) by the real-time RT-PCR method.
RNA extraction and cDNA synthesis.
Total RNA was isolated using TRIzol reagent according to the manufacturer's instruction (Life Technologies, Grand Island, NY). Samples were then quantified spectrophotometrically at 260 nm and checked for integrity by EtBr-agarose gel electrophoresis. RNA was primed with 0.5 µg/µl oligo dT(12–18) (Invitrogen Life Technologies) to generate first-strand cDNA. Reverse transcription (RT) was performed using SuperScript II Reverse Transcriptase (Invitrogen Life Technologies).
Real-time RT-PCR.
Before analyzing samples, we obtained a standard curve for each amplicon using serial dilutions of cDNA to determine amplification primer efficiency and the amount of material for each reaction. Primers were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). DNA sequence was obtained from GenBank, and primers were designed in contiguous exons to distinguish by size PCR products derived from cDNA from those derived from genomic DNA contaminants. The mRNA expression of RAS components was assessed by the following oligonucleotides: (Agt: 5'-TCTACCCTTTTGGGTGCTG-3' and 5'-CAAGGAGGATGCTGTTGAGA-3'; renin: 5'-CGGATCAGGGAGAGTCAAA-3' and 5'-CAGCATGAAAGGGATCAGG-3'; Ace: 5'-CAGGAACGTGGAACTTGGA-3' and 5'-CTTTGACGGAAGCATCACC-3'; AT1a: 5'-CACAACCCTCCCAGAAAGTG-3' and 5'-AGGGCCATTTTGTTTTTCTG-3'; AT2: 5'-GAGTCCGCCTTTAATTGCTC-3' and 5'-CACCTTTTTAGGGCCCTTTT-3'; and cyclophilin: 5'-AATGCTGGACCAAACACAAA-3' and 5'-CCTTCTTTCACCTTCCCAAA-3'). Real-time quantification of the target genes was performed with a SYBR Green PCR Master Mix (Applied Biosystems, PE) using ABI PRISM 7700 Sequence Detection System (Applied Biosystems, PE). The expression of cyclophilin A was measured as an internal control for sample variation in the RT reaction. An aliquot of the RT reaction was used for 50-cycle PCR amplification in the presence of SYBR green fluorescent dye according to a protocol provided by the manufacturer (PE Applied Biosystems). PCR product generation was monitored by measuring the increase in fluorescence caused by the binding of SYBR green to double-stranded DNA at each annealing phase. A dissociation curve was generated at the end of the reaction to verify that a single product was amplified. Each heart sample was analyzed in triplicate. Relative quantities of target gene expressions of the experimental groups were compared after normalization to the values of cyclophilin, and data were expressed as fold change compared with the mean values of the 1-copy+control group. Expression products requiring >35 PCR cycles were disregarded, thus cardiac renin and AT2 expression were not further analyzed.
Ang II Formation Measurement
Tissue Ang II formation was estimated by immunohistochemistry in 16 mice (1 copy + control n = 04, 1 copy + PO n = 04, 3 copy + control n = 04, 3 copy + PO n = 04). Hearts were fixed with 10% formalin and embedded in paraffin, and transversal cross-sections of left ventricle were performed (4-µm thickness). An anti-Ang II rabbit antiserum (1:400; Peninsula, Belmont, CA) was used, and the secondary antibody specificity was tested in positive and negative control measurements as described elsewhere (17). Sections were stained for immunohistochemistry analysis. Images were obtained with a computer-assisted morphometric system (Leica Quantimet 500, Cambridge, UK), and analysis were performed counting the number of positive signals in cardiomyocytes marked by the fast red stain.
Population Study
A cross-sectional study of risk factors for cardiovascular diseases was performed in the urban population of Vitoria, Brazil, following the general guidelines of the WHO-MONICA project (51a). A sample of 2,044 individuals (from an eligible population of 137,330) of both sexes, aged 25–64 yr, was invited to participate in the study. The subjects were chosen after a random selection of domiciles. In each residence only one subject was invited according to the nearest birthday.
From this sample, 1,507 attended to the clinic visit and were evaluated for height, weight, smoking habits, blood pressure measurements, and use of medicines. Blood and urine samples for determination of plasma cardiovascular risk factors (blood glucose, total cholesterol, lipoprotein fractions, and triglycerides) were collected after a 12-h fasting period. All measurements were performed according to standard techniques.
During the clinic visit all subjects were also submitted to a ethnic classification according to a validated questionnaire for the Brazilian population (27, 29). Subjects were classified as European descent or African descent according to a set of phenotypic characteristics (skin color, hair texture, shape of the nose, aspect of the lip, and jaw position). On the basis of these characteristics mulattoes are considered ethnic mixed subjects.
The Institutional Review Board of the Espirito Santo Federal University and the University of São Paulo approved the study protocol, and all participants read and signed an approved informed consent.
Blood Pressure Phenotypes Determination
Trained technicians measured blood pressure with a standard mercury sphygmomanometer on the left arm after 5-min rest with the subject in the sitting position. The first and fifth phases of Korotkoff sounds were used for systolic and diastolic pressure, respectively. SBP and diastolic blood pressures were calculated from two readings taken by two different observers. The two measurements were obtained with a minimal interval of 10 min. Hypertension was defined as the mean SBP of 
140 mmHg and/or diastolic blood pressure of 90 mmHg (1). Pulse pressure was the difference between SBP and diastolic blood pressures. Data on antihypertensive drug use (16.7% of individuals) were not included in the definition of hypertensive individuals since only 30% of these treated subjects presented with normal blood pressure. Conservatively these were classified as normotensive, and the remaining ones were included as hypertensive individuals. We considered that adjusting a regression model for antihypertensive medication use and then selecting blood pressure quartiles would, under this situation, add more uncertainty to our model (different medications, regimens, and adherence).
Left Ventricle Hypertrophy Phenotype Determination (Sokolow-Lyon Index)
A 12-lead resting electrocardiography (ECG) was recorded for each subject, and left ventricle mass was determined by Sokolow-Lyon ECG criteria (44), calculating the sum of SV1 + RV5 or RV6. Cardiac mass was considered augmented when the sum of the voltage criteria was 35 mm. For the present analysis, Sokolow-Lyon ECG criteria were chosen in consideration of their general acceptance, recognized performance, and easy assessment of left ventricle hypertrophy in large sample analysis.
Assessment of ACE Gene Polymorphism Genotypes
The Ace gene I/D polymorphism was determined by means of a three-primer system previously described (16, 34, 35). Quality control for these assays was assessed by randomly selecting 50 samples to be regenotyped by three independent technicians.
Statistical Analysis
Left ventricle mass index (LVMI)/SBP relationships by a linear regression were evaluated and the slope of the curves was compared between groups. Human values have been adjusted for ethnicity using a dummy variable: 1-Caucasian, 2-Mulatto, and 3-African descent. This variable was categorized and added as a covariate to the ANOVA model used in all analysis. One-way ANOVA test for unpaired measurements was used to compare basal values of blood pressure, heart rate, and cardiac hypertrophy index between all genotypes groups. The linear regression slopes were compared between groups using an analysis of covariance. A P value of 
0.05 was considered significant. Data are reported as means ± SE.
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RESULTS |
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50% in the three-copy group compared with one-copy group when cardiac PO was applied for a 6-wk period (Fig. 1, C and D). It took twice as long for one-copy mice to achieve the same increase in cardiac mass compared with three-copy animals since the slopes of the curves from the one-copy 6-wk mice displayed the same slope as the three-copy 3-wk animals. To assess the role of the AT1 receptor in this response, we induced a 6-wk period PO in animals treated with an AT1 receptor blocker (ARB: losartan, 50 mg/ml). The slopes of the LVMI/SBP relationship curves decreased significantly in both one- and three-copy groups by 42 and 32%, respectively (Fig. 1, E and F). Interestingly, the linear regression curve from three-copy animals treated with AT1 receptor antagonist was not different from the one-copy nontreated mice, that is, ARB treatment made the three-copy mice behave similarly to the one-copy mice.
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DISCUSSION |
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Renin release is traditionally considered the rate-limiting step of Ang II generation in plasma while in the tissue milieu other components of the system may play a role. In the cardiac tissue, Ang II levels are higher than in the circulation, suggesting that local production of the peptide may be important (9). ACE I/D genotypes are associated to significant but less than a fold change in serum and tissue ACE levels (10, 38) and even cardiac Ang II levels (11), which raises the possibility that cardiovascular phenotypes including development of left ventricle hypertrophy may be influenced by this polymorphism. This is consistent with data showing that Ang II can directly stimulate cardiac protein synthesis (39) or influence cardiovascular phenotypes through a variety of other actions (37). Present evidence show that the ACE I/D polymorphism can influence the conversion of Ang I to Ang II (5, 49) even though this is not the case when lower doses of Ang I were infused (25). On the other hand, increased degradation of bradykinin was also associated to the ACE D allele (31).
In contrast to these results, genetic perturbations designed to produce pharmacological increases in components of the RAS has shown that activation of the system is sufficient to alter cardiac phenotypes or make it more susceptible to stressors. Transgenic models with 13- to 100-fold increase in cardiac Ace levels lead to higher sudden cardiac death rate associated to enlargement of both atria in mice, whereas in rats, basal cardiac mass and Ang II immunoreactivity remained unchanged and increased cardiac hypertrophy ensued in response to aortic banding (47, 52). Similar findings were observed when the Ang II AT 1 receptor gene was upregulated predominantly in the rat cardiac tissue (19). Mazzolai et al. (28) demonstrated that mice overexpressing the rat Agt gene restricted to the myocardium developed cardiac hypertrophy in the absence of elevated blood pressure. Moreover, different lines of transgenic mice engineered to directly produce the peptide Ang II in the cardiac tissue exhibited either cardiac fibrosis with unchanged hemodynamics or hypertension accompanied by cardiac hypertrophy when there was a spillover of Ang II into the circulation (50). In contrast to these data, Harada et al. (18) demonstrated that mice lacking Ang II AT1 receptors submitted to pressure overload developed cardiac hypertrophy similarly to wild-type mice, underscoring the fact that cardiac hypertrophy is influenced by the RAS but is not exclusively dependent on this pathway. Although informative, none of these findings directly addressed the potential role of discrete changes in Ace for altering basal or stimulus-induced cardiac phenotypes as seem in humans harboring different genotypes of the ACE I/D polymorphism.
The extent to which these small changes influence a complex phenotype such as cardiac mass in humans remains a matter of debate even in the presence of additional stressors. Ortlepp et al. (32) found no association among five genetic polymorphisms of components of the RAS (Agt, ACE, Ang II AT1 receptor, cardiac chymase A, and aldosterone synthase genes) and left ventricle hypertrophy in patients with aortic stenosis. In contrast, Kim et al. (21) showed a synergistic effect of the ACE gene D allele and the Agt gene T allele on cardiac hypertrophy only in male patients with cardiovascular disease. No association was described between these two polymorphisms in normal healthy individuals. On the other hand, Dellgren et al. (12) showed that the DD genotype was associated with larger preoperative left ventricle hypertrophy in patients with aortic stenosis. Upon surgery for aortic valve replacement, PO normalized similarly, and, more important, the regression of cardiac hypertrophy reached the same degree regardless of the ACE genotype. These findings are the mirror image of the results depicted in Fig. 1A in mice with different copies of the Ace gene submitted to aortic banding. The DD genotype also influenced the phenotypic expression of the left ventricle hypertrophy in patients with hypertrophic cardiomyopathy specially in members of the same pedigree (26). Taken together, the present evidence is consistent with our findings in mice and men, suggesting that ACE can have a small but measurable influence on cardiac hypertrophy when additional genetic or hemodynamic stressors are present.
The RAS has circulating and local tissue components that are uniquely controlled in response to different stimuli. To predict outcomes in response to physiological or pathological stimuli when one or more components are specially affected, considering the existence of short and long control feedback loops, is virtually impossible. Unique genetic perturbations on a fixed background have been informative in this regard. Kim et al. (22) described that the cardiovascular homeostasis in mice with genetically decreased angiotensinogen is primarily compensated by an increased number of renin-producing cells. Moreover, Sumida et al. (45) showed that the Ang II receptor density, especially Ang II AT1 receptor, was significantly greater in Agt-deficient mice, illustrating the adjustments that may occur to compensate for changes in specific components of the RAS. Similarly, there are epidemiological data indicating that increases in plasma angiotensinogen observed in subjects carrying the angiotensinogen T235 allele may be compensated by lower plasma renin and prorenin levels (8). We have recently demonstrated that a physiological stimulus, such as exercise training, lead to compensatory cardiac hypertrophy of the same magnitude regardless of the Ace genotype in mice harboring one to four copies of the Ace gene (15). The increases or decreases in ACE were counterbalanced by reversible changes in plasma renin activity, while cardiac Ang II immunoreactive levels remained the same in mice harboring one and three copies of the Ace gene, before or after the exercise training. Thus, it appears that small genetic change in one component of the RAS is often compensated by reciprocal changes in other components. In addition, Takahashi et al. (46) has provided evidence from experimental as well as modeling data that modest increases in ACE activity may affect the steady-state plasma concentration of its substrate (e.g., lowers Ang I and bradykinin) but not of its products (e.g., Ang II). These adjustments may also contribute for the lack of correlation between cardiac mass and mice genotype under basal conditions observed in the present study or between the slopes of the LVMI/SBP curves between one- vs. two-copy mice. Furthermore, mice harboring one and two copies of the Ace gene failed to show changes in the magnitude of volume overload-induced cardiac mass hypertrophy by aortocaval fistula, which is consistent with the same cardiac Ang II levels observed despite higher cardiac ACE activity in the two-copy animals (36).
The results of the present study indicate that: 1) basal blood pressure and cardiac mass are not influenced by small different levels of ACE in transgenic mice; 2) small ACE levels indeed influence the magnitude of PO-induced cardiac hypertrophy; 3) this response appears to be mediated, at least in part, by the Ang II AT1 receptor; and 4) we also provide evidence that the ACE I/D polymorphism influences cardiac mass in the general population only in individuals on the fourth quartile of the SBP distribution.
Taken together, these data provide direct evidence that Ace gene dosage per se does not influence cardiac mass but upon a pathological stimulus, increased blood pressure, it modulates cardiac mass in both mice and humans. Importantly, these results indicate that stepwise changes in the normal diploid complement of genes involved in cardiovascular homeostasis may alter the endogenous response to physiological stimuli, potentially triggering disease processes. To that end, it has recently been recognized that copy number polymorphisms represent a major source of genetic variation among individuals (20, 42) and are likely determinants of phenotypical variation and disease susceptibility (3) through stepwise changes in gene complement similar to the ones evaluated in our study.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
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  S. Lacchini, A. S. Heimann, F. S. Evangelista, L. Cardoso, G. J. J. Silva, and J. E. Krieger Cuff-induced vascular intima thickening is influenced by titration of the Ace gene in mice Physiol Genomics, May 13, 2009; 37(3): 225 - 230. [Abstract] [Full Text] [PDF]
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