(Circulation. 1996;94:1886-1893.)
© 1996 American Heart Association, Inc.
Articles |
Evidence for the Existence of a Functional Cardiac Renin-Angiotensin System in Humans
the Istituto di Clinica Medica Generale e Cardiologia, Center for Heart and Thrombosis Research, University of Florence, Italy.
Correspondence to G.G. Neri Serneri, Istituto di Clinica Medica Generale e Cardiologia, Viale Morgagni 85, 50134 Florence, Italy.
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Abstract |
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Background The presence of mRNA for the essential components of the renin-angiotensin system (RAS) has been found in animal and human hearts. The present study was designed to provide evidence for the existence of a (functional) cardiac RAS.
Methods and Results Twenty-four patients with atypical chest pain undergoing coronary angiography for diagnostic purposes were investigated. The cardiac production rate of angiotensins was estimated by measurement of the cardiac extraction of 125I-angiotensin I and 125I-angiotensin II associated with the determination of endogenous angiotensins in aortic and coronary sinus blood in normal, low, or high sodium diets. In a normal sodium diet, angiotensin I and II aorta–coronary sinus gradients were tendentially negative (-1.8±2.5 and -0.9±1.7 pg/mL, respectively), and the amounts of angiotensin I and II added by cardiac tissues were 6.5±3.1 and 2.7±1.3 pg/mL, respectively. The low sodium diet caused a significant increase in both plasma renin activity (PRA) and angiotensin I concentration in aortic but not in coronary sinus blood, resulting in a more negative aorta–coronary sinus gradient (-9.7±3.1 pg/mL, P<.01). Angiotensin formation by PRA in blood during transcardiac passage increased (P<.001), whereas angiotensin I formed by cardiac tissues decreased dramatically. Accordingly, in the low sodium diet, 125I-angiotensin II extraction did not change, the cardiac fractional conversion rate of 125I-angiotensin I to 125I-angiotensin II notably decreased (P<.01), and angiotensin II formation by cardiac tissues was undetectable. The high sodium diet caused a decrease in PRA and no changes in cardiac extraction of radiolabeled angiotensins; conversely, angiotensin I formed by cardiac tissues, cardiac Ang I fractional conversion rate, and angiotensin II formed during transcardiac passage significantly (P<.01 for all) increased.
Conclusions These results provide evidence for the existence of a functional cardiac RAS independent of but related to the circulating RAS.
Key Words: angiotensin • enzymes • renin
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Introduction |
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The RAS has been considered for many decades as only a circulating system that influences both directly or indirectly the regulation of blood pressure and fluid and electrolyte homeostasis.1 2 This classic concept has undergone important changes because of accumulating evidence indicating the synthesis and presence of components of RAS in many tissues, thus suggesting that in addition to the circulating system, locally synthesized angiotensin may be an important modulator of tissue function and structure.3 4 5 The presence of essential components of RAS has been reported in the heart of different animal species. In vitro studies have shown that freshly cultured neonatal rat ventricular myocytes and fibroblasts form relevant amounts of Ang I and Ang II from endogenously produced angiotensinogen.6 7 Ventricular ACE and angiotensinogen RNA expression are increased in rats by pressure-overloaded left ventricular hypertrophy,8 9 experimental infarction,10 or tachypacing-induced heart failure.11 Perfusion of isolated rat hearts with renin led to a concentration-dependent release of Ang I and Ang II, and perfusion with Ang I resulted in a release of Ang II.12
Studies in humans are very rare. Coexpression of gene coding for angiotensinogen, renin, and ACE has been reported in three human atrial appendage samples obtained at the time of cardiac bypass surgery.13 Expression of angiotensinogen and ACE mRNA has been detected in all atrial, valve, and ventricular samples from human hearts obtained from patients during heart surgery,14 whereas atrial renin mRNA was demonstrable in only 70% of the samples.14 Although the renin synthesis by cardiac tissues is still debated,15 these results, taken together, suggest that the heart may be a place of local angiotensin production.
To investigate such a hypothesis in humans, we undertook the present study by measuring the production rate of Ang I and Ang II by cardiac tissues. To this goal, we combined the measurement of endogenous Ang I and Ang II in aorta and coronary sinus blood with the study of the cardiac kinetics of 125I-Ang I and 125I-Ang II. The study of kinetics gives information about the plasma extraction and metabolism rate of a substance when the endogenous and the radiolabeled substances have the same plasmatic clearance.16 17 Indeed, highly purified monoiodinated 125I-Ang I and 125I-Ang II have been demonstrated to be appropriate for measurements of Ang I–to–Ang II conversion and Ang I and Ang II degradation in both dogs and humans.18 19
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Subjects
Twenty-four normotensive patients (9 men and 15 women; mean [±SD] age, 41±6 years) affected by atypical chest pain who underwent coronary angiography for diagnostic purposes were investigated. All patients gave written consent after a full explanation of the purposes and potential risks involved in participating in the study. No patient had ECG evidence of exercise-induced myocardial ischemia (ST-segment depression of
1 mm from baseline), and all patients had no significant coronary atherosclerotic lesions at the coronary angiography. Patients had received no medication for at least 1 week before the study. All subjects received 5 mL Lugol's solution per day from 2 days before to 4 days after the 125I-Ang I or II infusion.
Study Design
In all 24 patients, Ang I and Ang II were measured in aortic, coronary sinus, and peripheral venous blood, and PRA was assayed in peripheral venous blood after the patients had been on a normal sodium diet for 1 week (108 mEq/d).125I-Ang I kinetics was studied in 12 patients (group A), and 125I-Ang II kinetics was performed in the remaining 12 patients (group B). The patients of each group were randomly assigned to low sodium diet (20 mEq/d; n=6) and to high sodium intake (400 mEq/d; n=6) to study 125I-Ang I and 125I-Ang II kinetics after activation (low sodium diet) or inhibition (high sodium diet) of the plasma RAS. During the three study periods, subjects had the same basic diet containing a constant amount of protein (1.3 g/kg), calories (126 KJ/kg), and calcium (800 mg/d), while sodium intake varied. Each experimental period was followed by 1 week of normal sodium diet.
Experimental Procedure
All patients were studied in the morning and in an overnight fasting state. Tea, coffee, alcohol, and cigarettes were withheld for at least 24 hours before the study. Patients were moved in the supine position from their beds to the hemodynamic room and were premedicated with oral diazepam (10 mg) 1 hour before the study. Catheter positioning and the sampling of aortic, coronary sinus, and peripheral vein blood were performed according to the procedure previously described in detail.20 Coronary sinus flow was measured by thermodilution technique.20 After 20 minutes of supine rest and 10 minutes after insertion of the catheters, 125I-Ang I or 125I-Ang II was infused at 
3.5x106 cpm/min for 20 minutes into the antecubital vein of the right arm. Between 10 and 15 minutes after the start of the infusion, when arterial and venous (coronary sinus) levels of 125I-Ang I or 125I-Ang II reached a plateau and were constant (coefficient of variation <5%), blood samples (5 mL each) for endogenous and radiolabeled Ang I and Ang II assays were contemporaneously drawn from the aorta and the coronary sinus. Soon after blood sampling, CBF was measured twice at 5-minute intervals, and blood samples for determination of cardiac oxygen extraction (arterial–coronary sinus difference) were also obtained. Each reported value is the mean of three determinations. For each determination, CVR was calculated as the quotient between mean aortic pressure and CBF with the assumption of a mean right atrial pressure of 3 mm Hg. Routine catheterization of the left side of the heart, including coronary angiography, was then performed by conventional techniques, as previously described.20 The patients were continuously monitored with ECGs. When patients were restudied after a week on low or high sodium diet, coronary angiography and left ventriculography were not repeated.
Mean transcoronary transit time, determined in a preliminary phase of the study according to the method of Gorlin and Storaasli,21 was on average 6 seconds; this value was used for the determination of Ang I formed by circulating PRA during the transcardiac passage. The transit time from coronary sinus to brachial vein through the catheter was on average 13 seconds.
Blood Sampling
Blood for angiotensin measurements was rapidly (7 to 15 seconds) drawn with a plastic 5-mL syringe containing 0.5 mL inhibitor solution (0.125 mmol/L disodium EDTA; 0.025 mol/L 1,10-phenantroline; 1 mg/mL captopril22 ; and 100 mmol/L of the renin inhibitor RO 42-5892 [final concentration in blood]) and transferred into prechilled polystyrene tubes. The renin inhibitor RO 42-5892, proven to be a potent and specific inhibitor of human renin,23 was kindly provided by Dr Walter Fischli (La Roche, Basel, Switzerland). The blood samples were centrifuged at 2000g for 20 minutes at 4°C. Plasma was stored at -70°C, extracted within 5 days, and assayed within 2 weeks. Blood for PRA assay was collected into glass tubes containing EDTA (0.084 mL tripotassic EDTA in 7 mL blood; final concentration, 0.34 mol/L). The samples were centrifuged at room temperature at 3000g for 10 minutes, and the plasma was stored at -20°C.
Extraction and HPLC Separation of Angiotensins
Angiotensins were extracted from plasma by solid-phase extraction octadecasilyl-silica cartridges (Sep-Pak C18, Waters Chromatography Div) according to the method of Hermann et al.24 The dry residue was dissolved in 100 µL HPLC mobile phase and transferred into polyethylene tubes. This solution was used for application to the HPLC column.24
Separation of the angiotensin peptides in the plasma extracts was performed with HPLC according to the procedure of Nussberger et al25 with some modifications. More precisely, peptide separation was performed by reversed-phase HPLC (6000 A Waters pump, Waters SpA, equipped with a Nucleosil C 18 steel column, 250x4.6 mm, 10-µm particle size, Perkin Elmer C18 SIC-X-10). Elution was performed as follows: 65% of 0.085% orthophosphoric acid containing 0.02% sodium azide (pH 2.33; mobile phase A) and 35% methanol (mobile phase B) from 0 to 9 minutes, followed by a linear gradient to 40% A/60% B until 23 minutes. The flow was 1 mL/min, and the working temperature was 45°C. The vacuum-dried plasma extracts were dissolved in 100 µL HPLC solvent 65% A/35% B (vol/vol), centrifuged, and injected with a 100-µL syringe (Hamilton). The eluate was collected in 1-minute fractions into polystyrene tubes and evaporated in the concentrator before radioimmunoassay.
The retention times of Ang I, Ang II, Ang-(2-8) heptapeptide, Ang-(4-8) pentapeptide, Ang-(2-10) nonapeptide, and Ang-(3-8) hexapeptide were established with 100 ng of the respective UV-detectable human standard (Bakem Feinckemikalien); the high reproducibility of retention times allowed the recovery of Ang I and Ang II in picogram quantities from plasma extract in fractions corresponding to Ang I and Ang II peaks. The fractions containing Ang I and Ang II were cooled separately.
The extraction recoveries of radiolabeled angiotensins were determined by adding 125I-Ang I and 125I-Ang II (6000 cpm for each peptide) to 1-mL portions of six plasma samples counted in a gamma counter before and after extraction procedures. After Sep-Pak extraction, the recoveries were 90.6±4.3% for 125I-Ang I and 91.1±5.4% for 125I-Ang II, whereas after HPLC separation, the recoveries were 89.3±3.1% and 90.2±2.5% for 125I-Ang I and 125I-Ang II, respectively. Overall recoveries were 87.3±5.8% for labeled Ang I and 88.7±6.0% for labeled Ang II. For unlabeled angiotensins, 35 fmol of (Ile5)-Ang I and (Ile5)-Ang II were added to 1-mL portions of six plasma samples. After extraction, the recoveries were 89.8±5.4% for (Ile5)-Ang I and 90.3±5.8% for (Ile5)-Ang II; after HPLC separation, the recoveries were 90.4±3.5% and 90.8±3.1%, respectively. Overall recoveries were 86.7±6.0% for (Ile5)-Ang I and 87.8±5.4% for (Ile5)-Ang II. Results were not corrected for incomplete recovery.
Radioimmunoassay of Angiotensins and PRA
Ang I and Ang II plasma concentrations were measured with commercial kits (Peninsula Labs, Inc, for Ang I; ITS, Technogenetics, for Ang II). The Ang I antiserum also reacted with Ang-(2-10) nonapeptide (98%) but did not react (<0.1%) with Ang II, Ang III, Ang-(3-8) hexapeptide, or Ang-(4-8) pentapeptide. The Ang II antiserum also reacted with Ang III (67%), Ang-(3-8) hexapeptide (70%), and Ang-(4-8) pentapeptide (91%) but virtually not at all with Ang I (0.1%) and Ang-(2-10) nonapeptide (0.2%). The concentrations were expressed in picograms per milliliter. The lower detection limit (2xSD difference from B0) was 1 pg/mL for both Ang I and Ang II. Overall intra-assay and interassay variation coefficients were 6.3% and 12.4% for Ang I and 7.7% and 13.6% for Ang II, respectively.
Measurement of PRA was performed by measuring the quantity of Ang I generated in vitro by radioimmunoassay with a commercial kit (Sorin Biomedica) and was expressed as nanograms of Ang I per 1 mL plasma per hour of incubation. The normal level of PRA in antecubital venous plasma of control subjects in the supine position (n=30) was 0.7±0.6 ng·mL-1·h-1.
Characteristics of Radiolabeled Ang I and Ang II
125I-Ang I and 125I-Ang II were obtained from Du Pont de Nemours, NEN Division. Immediately before each infusion, the 125I-Ang I or 125I- Ang II solution was sterilized by filtration through a 0.22-µm Millipore membrane filter (Waters). The specific radioactivity of the 125I-Ang I and 125I-Ang II preparations was 81.4 TBq/mmol. The purity of radiolabeled solutions assessed by injection of a sample into the HPLC column and counting of the collected fractions was >99%.
The elimination half-life of 125I-Ang I and 125I-Ang II was 0.72±0.17 minutes, which was not different from the elimination half-life (0.70±0.23 minutes) of the unlabeled angiotensins (Ang I and Ang II, Sigma Chemical Co) contemporaneously infused in the same subjects at 1.5 ng/min for 20 minutes. Urinary excretion of radioactivity was followed over a period of 96 hours. Eighty-eight percent of the administered radioactivity was excreted within 24 hours, and 98% was recovered in the 96-hour period. The calculated exposure to radioactivity was 0.24 µGy (or 0.6 mrad) from 125I-Ang I or 125I-Ang II infusion.
125I-Ang I and 125I-Ang II were extracted from plasma according to the procedure described for the noniodinated peptides; concentrations of 125I-Ang I and 125I-Ang II in collected chromatographic fractions were measured directly in a 12-channel gamma counter (Multigamma 1261 LBK-Wallak, EG&G) for 20 minutes.
Parameters for Evaluation of Ang I and Ang II Kinetics
For cardiac Ang I kinetics, the following parameters were considered: (1) Cardiac Extraction (Conversion Plus Degradation)=1-(125I-Ang I ven/125I-Ang I art); (2) Coronary Sinus Ang I Derived From Aortic Delivered Ang I=Aortic Blood Ang Ix(1-Cardiac Extraction); (3) Coronary Sinus Ang I Formed by Circulating PRA During Transcardiac Passage=PRAxTranscoronary Blood Transit Time; (4) Coronary Sinus Ang I Formed During Transcardiac Passage (De Novo Cardiac Ang I)=Ang I ven-[Ang I artx(1-Cardiac Extraction)]; (5) Coronary Sinus Ang I Derived From Cardiac Tissue Production=(De Novo Ang I)-(Ang I Formed by PRA); (6) Formation of Ang I and Ang II in Relation to Coronary Blood Flow (Output)=pg/mLxmL/min=pg/min; and (7) Cardiac Ang I Fractional Conversion Rate According to Schunkert et al26 After Subtraction of the Aortic Concentration of 125I-Ang I and 125I-Ang II to Levels Found in Coronary Sinus Blood=(125I-Ang II ven-125I-Ang II art)/125I-Ang I ven+(125I-Ang II ven-125I-Ang II art), where ven is coronary sinus blood and art is aortic blood.
For cardiac Ang II kinetics, the following parameters were considered: (1) Cardiac Extraction=1-(125I-Ang II ven/125I-Ang II art); (2) Coronary Sinus Ang II Derived From Aortic Delivered Ang II=Aortic Blood Ang IIx(1-Cardiac Extraction); and (3) Coronary Sinus Ang II Derived From De Novo Cardiac Formation=Ang II ven-[Ang II artx(1-Cardiac Extraction)].
Statistical Analysis
Data are presented as mean±SD. A paired Student's t test was used to compare Ang I and Ang II metabolism parameters found during normal sodium intake with those measured during low or high sodium intake. One-way ANOVA was used to assess differences in parameters of 125I-Ang I and 125I-Ang II kinetics among different sodium diets. Values are considered significantly different at P<.05.
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HPLC Separation of Angiotensin Peptides
Good separation was obtained among Ang I, Ang II, their metabolites, and radiolabeled angiotensins. Assessment of injection-to-injection and day-to-day variability of the retention times repeated 10-fold demonstrated very good stability of the chromatographic conditions (1.2% and 1.75%, respectively). Separation patterns for radiolabeled angiotensin plasma extracts from patients who had received an intravenous infusion of radiolabeled angiotensins demonstrated clear separation between the peak of 125I-Ang II and 125I-Ang I. The retention times of 125I-Ang II and 125I-Ang I were 12 and 21 minutes, respectively, and adequate separation from other metabolites was always obtained.
Transcardiac Gradients and Outputs of Ang I and Ang II
CBF was on average 74.7±5.7 (range, 68.2 to 90.1 mL/min), and CVR averaged 1.31±0.1 (range, 1.11 to 1.47 mm Hg·mL-1·min-1), respectively.
Ang I concentration showed a mean value of 31.6±5.7 pg/mL in aorta and 28.6±4.6 pg/mL in coronary sinus blood (P=.22, NS), resulting in tendentially negative aorta–coronary sinus gradient (-1.8±2.5 pg/mL) and cardiac Ang II output (Fig 1
). More precisely, 18 of 24 patients had negative gradients (from -6.2 to -0.2 pg/mL), and 6 patients had positive gradients (from 0.5 to 2.5 pg/mL).
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indicates patients who were restudied on low sodium intake; 
, those who were restudied on high sodium intake.
As for Ang I, both aorta–coronary sinus Ang II gradient (-0.9±1.7 pg/mL) and cardiac Ang II output were slightly negative (Fig 1
); namely, 18 of 24 patients had a negative aorta–coronary sinus gradient (Fig 1).
In peripheral venous blood, PRA was 0.87±0.23 ng·mL-1·h-1, and Ang I and Ang II concentrations were 28.9±4.9 and 13.2±6.4 pg/mL, respectively.
Kinetics of 125I-Ang I and 125I-Ang II
Kinetics of 125I-Ang I was investigated in 12 patients (group A), and the results are shown in Table 1. In all patients, 125I-Ang I was extracted during the aorta–coronary sinus passage (Table 1 and Fig 2). Because in these patients the mean Ang I concentration in aortic blood was 31.1±6.3 pg/mL, the Ang I delivered arterially (aortic) to coronary sinus blood after Ang I cardiac extraction would have been 21.4±5.2 pg/mL. This value is significantly lower than the Ang I level actually found in coronary sinus blood (29.1±4.9 pg/mL, P<.001). More precisely, the amount of de novo Ang I added during transcardiac passage was on average 7.8±2.9 pg/mL, 1.6±0.4 pg/mL formed by PRA during the transcardiac passage, and 6.5±3.1 pg/mL added de novo by cardiac tissues (Table 1 and Fig 2C and 2D).
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The fractional conversion rate of 125I-Ang I to 125I-Ang II during the transcardiac passage of blood from aorta to coronary sinus was 23.7±3.9% (Table 1 and Fig 2B).
Kinetics of 125I-Ang II was studied in 12 patients (group B). Like Ang I, Ang II underwent extraction during its transcardiac passage. After cardiac 125I-Ang II extraction, the amount of Ang II delivered from aorta to coronary sinus would have theoretically been 9.2±2.1 pg/mL, whereas the actual Ang II concentration found in coronary sinus blood was 12.1±2.4 pg/mL. This finding suggests an Ang II addition of on average 2.7 + 1.3 pg/mL by cardiac tissues during the transcardiac passage (Table 1), corresponding to a mean output of 198.1±82.1 pg/min (Fig 3).
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Effects of Low Sodium Diet on Cardiac Ang I and Ang II Extraction and Formation
Six patients from group A and 6 patients from group B were randomly allocated to low sodium intake. After 1 week of iponatriemic diet, PRA increased significantly (P<.001), and the concentration of Ang I in aortic blood was significantly higher than in the same 12 patients on normal sodium intake (P<.001; Table 2). On the contrary, Ang I concentration in coronary sinus blood did not change compared with the concentration found in the same patients on normal sodium intake, thus resulting in an increased negativity of Ang I transcardiac gradient compared with normal sodium diet (Table 2).
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In patients on low sodium diet, CBF slightly decreased and CVR increased (P<.01; Table 2). The transcardiac Ang I gradient expressed as output was markedly more negative than that found in the same patients on normal sodium intake (P<.001; Table 2 and Fig 1B).
125I-Ang I kinetics was again investigated in six patients from group A after 1 week on low sodium diet. Cardiac extraction of 125I-Ang I significantly decreased after low sodium intake (P<.01; Fig 2), whereas Ang I formation by PRA increased from 1.1±0.4 to 5.3±1.1 pg/mL (P<.01; Fig 2). On the contrary, Ang I formed de novo by cardiac tissues was dramatically decreased and was undetectable in all patients (Fig 2). Cardiac Ang I conversion rate was 8.2±3.5% (3% to 12%), significantly lower than the value observed during normal sodium intake (23.6±4.1%; P<.01; Fig 2).
After a week on low sodium intake, concentrations of Ang II in aorta and coronary sinus blood studied in 12 patients (6 from group A and 6 from group B) were significantly higher than during normal sodium diet (Table 3). However, the aorta–coronary sinus gradient was more negative than in baseline conditions (P<.001; Table 3). 125I-Ang II extraction investigated in 6 patients after 1 week on low sodium diet did not significantly differ from that found in the same patients on normal sodium diet (Fig 3). De novo cardiac formation of Ang II during the passage of blood from aorta to coronary sinus decreased markedly and was undetectable in all patients (Fig 3). Thus, despite the significantly higher amount of arterially delivered Ang II compared with that observed when patients were on normal sodium diet (20.6±5.2 versus 8.7±1.5 pg/mL, P<.01), the cardiac output of Ang II was more negative than the baseline Ang II output (P<.01versus normal sodium diet; Fig 1B and Table 3). Therefore, when Ang I and Ang II concentrations were elevated in aorta, as in patients on low sodium diet, cardiac formation of angiotensins was undetectable with consequent more negative aorto–coronary sinus gradient.
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Effects of High Sodium Diet on Cardiac Ang I and II Extraction and Formation
Six patients from group A and six patients from group B who had not been on hyponatremic diet were randomly allocated to high sodium diet. The increased sodium intake decreased PRA and Ang I concentrations in aortic blood and peripheral venous blood compared with levels in normal sodium intake (Table 2). Whereas Ang I concentration in aortic blood notably decreased compared with levels in normal sodium diet (P<.01), levels of Ang I in coronary sinus blood were similar to those found in the same patients on normal sodium intake, so the aorta–coronary sinus gradient became positive (P<.01; Table 2) and the cardiac output of Ang I notably increased in all patients (Table 2 and Fig 1C) in the absence of significant changes in CBF and CVR. 125I-Ang I extraction tendentially increased (from 29.5±4.4% in normal sodium diet to 31.8±3.1%), but the difference did not reach statistical significance (Fig 2).
Both Ang I formation by PRA during the transcardiac passage and the arterially delivered Ang I to coronary sinus decreased compared with that in normal sodium diet (0.8±0.2 versus 1.4±0.5 pg/mL, P<.01, and 17.9±2.7 versus 22.4±4.6 pg/mL, P<.01, respectively). Nevertheless, the amount of Ang I formed de novo by cardiac tissues significantly increased in all patients (8.4±1.1 instead of 6.2±2.9 pg/mL on normal sodium diet, P<.01). When the production rate of Ang I was expressed in relation to CBF, the increase became even more evident (Fig 1). Similarly, cardiac Ang I fractional conversion rate significantly increased in all patients (P<.05; Fig 2B).
As already observed for Ang I, Ang II concentration in aortic blood significantly decreased after a week of high sodium diet compared with the concentration observed in the same patients on normal sodium intake (P<.01), whereas Ang II in coronary sinus blood decreased less than in aorta (from 12.1±2.7 to 11.1±2.1 pg/mL, P<.05). As a consequence of these changes, the transcardiac gradient became positive in all patients (P<.01 versus normal sodium diet), and the cardiac Ang II output markedly increased (P<.01; Table 3 and Fig 1). At the end of the week of high sodium diet, the cardiac 125I-Ang II extraction was not significantly different from that observed during normal sodium intake (Fig 3); however, because of the reduction of aortic blood Ang II levels, the Ang II delivered arterially to coronary sinus blood was lower than that found under baseline conditions (7.6±1.9 versus 9.1±1.7 pg/mL, P<.01). On the contrary, de novo cardiac Ang II production was higher than that found in the same patients during normal sodium intake, so the cardiac Ang II output increased significantly (P<.01; Fig 3). Therefore, the inhibition of circulating RAS, as in conditions of high sodium diet, was associated with an increased cardiac formation of both Ang I and Ang II, and the aorta–coronary sinus gradient of angiotensins became positive.
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Discussion |
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The present study addressed the question of whether cardiac tissues were able to form Ang I and Ang II. Combining 125I-Ang I and 125I-Ang II kinetics with HPLC and radioimmunoassay of plasma angiotensins in different conditions of sodium intake enabled the precise measurement of the metabolism, ie, conversion degradation and formation of the angiotensins during the transcardiac passage. Our results provide evidence for the existence of cardiac formation both of Ang I and Ang II.
Artifacts and nonspecific measures of Ang I and Ang II may lead to incorrect measurements of the real angiotensin formation.25 27 In the present study, however, HPLC separation of Ang I and Ang II caused the formation of very distinct peaks of the two angiotensins and their metabolites, and subsequently a very sensitive radioimmunoassay was used. 125I-Ang I and 125I-Ang II also were well differentiated by HPLC, and the retention times of 125I-Ang I and 125I-Ang II differed from the retention times of the other metabolites. Thus, the methods used seem adequate for the goal of the present study.
During the transcardiac passage, Ang I underwent important metabolic changes. Approximately 30% of the Ang I passing through the heart was extracted, partly converted to Ang II, and partly degraded by the angiotensinases into smaller inactive peptides. Despite this extraction, the Ang I concentration in coronary sinus blood was not different from that in aortic blood, thus indicating a cardiac formation of Ang I. Because the Ang I formation by PRA during the transcardiac passage was very low (<1.5 pg/mL, ie, 5% of the total amount of Ang I present in the coronary sinus blood), the remaining amount of Ang I present in coronary sinus blood after Ang I extraction was produced de novo by cardiac tissues. The possibility that Ang I was formed in the catheter27 during the passage of blood from the coronary sinus blood to the syringe (about 15 seconds) in a 100-cm-long catheter cannot be ruled out. The amount of Ang I that could have been formed in the catheter was about 2.5 to 3 pg/mL. However, this formation appears to be very unlikely because under conditions of high PRA, as occurred when patients were on low sodium diet, the amount of Ang I formed during the passage of blood from aorta to syringe was markedly lower than under conditions of normal PRA (normal sodium intake). Likewise, the formation of Ang I in the tube is highly unlikely because of the presence of a potent renin inhibitor. Therefore, the amount of Ang I found in coronary sinus blood exceeding the sum of arterially delivered Ang I plus that formed by PRA is added by cardiac tissues.
The capacity of cardiac tissues to synthesize renin is still being debated.15 However, for Ang I to form, it is not necessary that renin be locally synthesized; it is sufficient that renin be locally taken up from plasma.28 It is worth pointing out that the possible uptake of renin from plasma by the cardiac tissues seems to be not a passive phenomenon but a finely regulated mechanism proposed to maintain the homeostasis between the local and systemic formation of Ang I. This is clearly demonstrated by the opposite changes between the increase or decrease in PRA and local formation of Ang I in relation to the changes in sodium intake. It is very difficult to understand why the formation of angiotensin during the transcardiac passage dramatically decreased when PRA markedly increased during the low sodium diet and increased when the patients were on high sodium intake. Thus, even if the present findings are not able to support a cardiac synthesis of renin, they strongly indicate the capacity of cardiac tissues to produce de novo Ang I independently from the circulating RAS.
Ang II also is formed during the transcardiac passage. Ang II formation showed a pattern similar to that of Ang I, was undetectable in conditions of low sodium intake when PRA was enhanced, and was increased when PRA was low, as under the conditions of high sodium diet. In the presence of nonsignificant changes of 125I-Ang II extraction during transcardiac passage, the higher concentration of Ang II in coronary sinus than in aortic blood indicates increased de novo formation of Ang II by cardiac tissues.
Besides changes in cardiac Ang I and Ang II formation, the conversion of 125I-Ang I to 125I-Ang II during transcardiac passage also was notably changed in relation to different sodium intakes. More particularly, fractional conversion of 125I-Ang I to 125I-Ang II was notably reduced in low sodium diet (when PRA was high) compared with that found in normal sodium intake. Conversely, fractional conversion increased when PRA was depressed as a consequence of high sodium intake, thus suggesting that cardiac-generated Ang II is essentially formed through the renin-angiotensin pathway. A recent investigation performed in vivo and in solubilized human heart membrane preparations showed that ACE mediated about 85% to 90% of the conversion of Ang I to Ang II.29
The changes of cardiac Ang II formation in relation to the different sodium intakes suggest both a physiological role of the cardiac RAS and the existence of a cardiac mechanism that controls the formation of angiotensins. Even if plasma concentrations of Ang II formed by cardiac tissues were of few picograms per milliliter (from undetectable in low sodium diet to 5.8 pg/mL in high sodium intake compared with 2.7 pg/mL in normal sodium diet), these differences are from 3 to 14 times greater than the intra-assay variability, and most importantly, the changes in cardiac Ang II formation induced by diet modifications were homogeneous in all subjects. Moreover, Ang II concentration in coronary sinus represents the amount of Ang II that has not bound to cardiac Ang II receptors.30 31 32 Because cardiac Ang II receptors of human heart are at high affinity,30 the actual quantity of Ang II produced by cardiac tissues is presumably much higher than that found in coronary sinus. Likewise, the amount of Ang II is not at all negligible when related to the CBF (from undetectable to 473 pg/min), and finally, cardiac Ang II may operate in a paracrine or autocrine fashion.28
The changes in cardiac angiotensin production as opposed to that of circulating RAS suggest both that the cardiac RAS participates in the formation of the plasma pool of circulating angiotensins and, above all, that it contributes to maintain the angiotensin homeostasis for cardiac tissues. The selective and independent upregulation or downregulation of the mechanism(s) that control Ang I and Ang II formation in the heart to maintain the local concentration of Ang II constant seems to operate as a feedback mechanism in relation to the Ang II concentration in aortic blood. Ang II has been reported to exert negative feedback regulation on renin gene transcription and renal renin secretion,33 34 35 36 as well as on ACE mRNA levels and activity in the lungs and testis but not in serum.26
In conclusion, the present results provide consistent evidence for the existence in humans of a functional cardiac RAS independent from but related to the circulating RAS. Because of the capacity of Ang II to operate in an autocrine, paracrine, or even intracrine fashion, local formation of Ang II, even in small amounts, may have important implications in pathophysiological conditions. The cardiac angiotensin system has to be viewed mainly as a locally operating system rather than as a blood pressure regulatory system, and its altered function could have important consequences for cardiac function and remodeling, cardiac atherosclerosis, and myocardial ischemia.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grant 04 01 000045 from Ministero della Ricerca Scientifica e Tecnologica, Rome, Italy.
Received December 11, 1995; revision received April 10, 1996; accepted April 15, 1996.
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