The increase in interstitial and perivascular collagen that occurs in experimental overload-induced hypertrophy (1-8) is a determinant of impaired cardiac function due to alteration of left ventricular mechanical properties (9,10). The role of hormonal factors such as the renin-angiotensin-aldosterone system in accumulation of components of the extracellular matrix is well established, mainly in experimental rat models (6-8,11-13). However, the relation between myocardial fibrosis and left ventricular function in vivo during chronic neurohumoral activation may differ between species according to their different mechanisms of cardiac excitation-contraction coupling and levels of blood pressure.
The purpose of our study was therefore to investigate the relations between in vivo left ventricular function and the development of cardiac fibrosis in adult guinea pigs undergoing long-term (3 months) aldosterone/salt administration. In comparison with the rat, myocardial calcium handling and contractility in the guinea pig are more dependent on external calcium (14,15), and Na+,K+-adenosine triphosphatase (ATPase) is more sensitive to digitalis-induced inhibition (16). These characteristics make guinea-pig heart closer to human heart than is rat heart. In this experimental model, we studied invasive and noninvasive hemodynamic parameters, plasma hormonal profile, and collagen expression (messenger RNA; mRNA) and total amount of collagen in left ventricle (LV) and right ventricle (RV) submitted to different loading conditions.
MATERIALS AND METHODS
Animal models and hemodynamic studies
Male tricolor guinea pigs weighing 250-300 g at the beginning of experiments were used in this study. Animals were anesthetized before surgery (ketamine hydrochloride, 43 mg/kg, + chlorpromazine, 0.65 mg/kg, intramuscularly, i.m). Then they underwent uninephrectomy before random assignment into one of three groups:
- Aldosterone/salt hypertensive group (n = 7): an osmotic minipump (ALZET, Charles River, Paris) was implanted subcutaneously and set to deliver 0.75 μg/h d-aldosterone (Sigma, St. Quentin Fallavier, France). Minipumps were changed every 15 days. Animals received 1% NaCl and 0.3% KCl in the drinking water.
- Control/salt operated group (n = 5): an osmotic minipump was implanted to infuse normal saline solution, and animals received salt in the drinking water.
- Control-operated group (n = 5): guinea pigs of the same age were used as controls and received no salt in the drinking water.
All animals were fed ad libitum and treated for 3 months. The investigation was conducted under the guidelines established by the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH publication no. 85-23, revised 1985).
To determine the geometry and function of the LV, noninvasive two-dimensional guided M-mode echocardiographic studies, including pulsed-wave Doppler spectra of mitral inflow and LV outflow velocities, were performed in all animals 48 h before killing date (i.e., at the end of month 3 of treatment). Animals were lightly anesthetized with ketamine hydrochloride (40 mg/kg) + chlorpromazine (0.5 mg/kg), i.m. The animal was placed prone and the ultrasound probe placed on a shaved area of the anterior chest wall. Imaging was performed by using a commercially available echocardiography machine equipped with a 7.5-MHz transducer (Kontron Instruments, France), with two-dimensional real time and M-mode acquisition. The best acoustic window was from the left parasternal position, which gave long- and short-axis views of the LV, equivalent to the left parasternal window in humans. M-mode tracings were recorded through the anterior and posterior LV walls at a paper speed of 50 mm/s. Anterior and posterior wall (end-diastolic and end-systolic) thickness and LV internal dimensions were measured by using the American Society for Echocardiography approved method from at least three consecutive cardiac cycles on M-mode tracings (17) and were averaged by a single blinded observer. Contractility was expressed as (a) LV fractional shortening, calculated as [(LVDD − LVSD)/LVDD] × 100, where LVDD is LV internal end-diastolic dimension, and LVSD is LV internal end-systolic dimension; and (b) fractional thickening of the anterior and posterior walls. Pulsed-wave Doppler spectra of mitral inflow were recorded from the apical four-chamber view, adjusted to the position at which velocity was maximal and the flow pattern laminar. LV outflow velocity was recorded from the apical five-chamber view, achieved by anterior angulation of the transducer. All Doppler spectra were recorded on paper at 50 mm/s and analyzed off-line, as previously described. All values represent the mean of at least three consecutive cardiac cycles. Cardiac output was calculated as Equation (1) where VTI is the velocity-time integral.
Two days after the final echocardiogram, guinea pigs were anesthetized with ketamine + chlorpromazine, i.m. The left carotid artery was cannulated with a polyethylene catheter connected to a pressure transducer (Gould Electronic, Cleveland, OH, U.S.A.). Systolic and diastolic blood pressure were allowed to stabilize for ∼1 min, and pressure tracings were then recorded on a strip chart recorder at a paper speed of 100 mm/s (Gould 2000 series; Gould Electronic). Guinea pigs were allowed to breathe spontaneously during pressure recordings. At the end of the study, the transducer was removed, and blood samples were collected.
Hormone assays and electrolyte levels
Blood samples were collected in tubes containing 100 mM EDTA for plasma aldosterone, plasma renin activity (PRA), angiotensin I (AI), and angiotensin II (AII) levels and in tubes containing 43 lithium heparin U.S.P. units for Na+, K+, bicarbonate, epinephrine, norepinephrine, and dopamine concentrations. After centrifugation, the plasma samples were removed and stored at −20°C. Hormones of the renin-angiotensin-aldosterone system were measured by radioimmunoassay, as described previously by Schaison et al. (18). Plasma concentrations of norepinephrine, epinephrine, and dopamine were measured by a radioenzymatic method with addition of [3H]S-adenosyl-L-methionine (Amersham, Les Ulis, France) and are expressed in picograms per milliliter. Na+, K+ and bicarbonate concentrations were assayed by flame photometry.
Before animals were killed, they were intubated and mechanically ventilated. The chest was opened by median sternotomy, and the heart and lungs were removed. The hearts were rinsed in ice-cold saline solution and blotted. One equatorial coronal section was cut and rapidly fixed in Bouin's fixative for histologic analysis. The LV with septum and RV were separated, weighed, frozen in liquid nitrogen, and stored at −80°C until RNA extraction. The lungs were removed, gently blotted, and weighed.
Total RNA extraction
Total RNA from LV and RV (300 mg) was prepared according to the method of Chirgwin et al. (19). RNA concentrations were measured by 260-nm absorbance, assuming 40 μg/ml for 1 absorbance unit. The RNAs were resuspended in TRIS·HCl-EDTA (TE, pH 7.4), and aliquots were stored at −80°C until use.
Northern blots and slot blots
For Northern blots, samples of 20 μg of RNA were denatured in 50% formamide, 2.2 M formaldehyde, and 1× MOPS buffer (pH 8.0) and electrophoresed in a 1% agarose gel. Total RNA was then transferred to a Hybond-N membrane (Amersham). For slot-blot analysis, 2, 5, 10, and 15 μg RNA of each sample was spotted on the membranes. All blots were submitted to ultraviolet irradiation covalently to link the RNA samples.
Slots were hybridized with the following oligomers or complementary DNA (cDNA) probes: a rat collagen α1 (I) cDNA of 1,600 bp complementary to the carboxy terminal propeptide (20) and a rat glyceraldehyde-3 phosphate dehydrogenase (GAPDH) cDNA probe of 1,300 bp (Pst I, a gift from Dr. F. Moreau-Gachelin). This latter was used to normalize the amount of total RNA per lane in all blots. Hybridization with collagen and GAPDH cDNAs was carried out in 50% formamide, 5× Denhardt's solution, 5× standard saline phosphate ethylenediaminetetraacetic acid (SSPE), 0.1% sodium dodecyl sulfate (SDS), 200 μg/ml herring sperm DNA, and 20 μg/ml poly (A+) at 42°C. Slots were prehybridized in this solution for 12 h and hybridized for 24 h with added cDNA, radiolabeled by random primer extension by using an Amersham Megaprime DNA labeling system. α(32P)-dCTP (3,000 Ci/mmol, Du Pont-New England Nuclear, Boston, MA, U.S.A.) was incorporated to obtain a specific activity of 2-8 × 108 counts/μg. Washes were performed twice in 0.1 × SSC, 0.1% SDS at room temperature for 10 min, and twice more in the same solution at 50°C for 15 min. Northern blots were exposed to Amersham Hyperfilm at −80°C with Quanta III intensifying screens. Slot blots were used to quantify mRNA levels and were exposed to intensifying screens (Fuji imaging plate type BAS IIIS, Fuji Co., Tokyo, Japan) and then analysed in a Bio-Imaging Analyser System (BAS 1000 Mac BAS; Fuji Co.). Densitometric scores for specific collagen type I mRNA were normalized to GAPDH mRNA to correct for sampling loading.
Coronal ventricular sections were dehydrated with ethanol and xylol and embedded in paraffin. Six sequential, 5-μm-thick sections, containing a complete cross-sectional cut of both ventricles, were obtained from each heart: the first three were stained with hematoxylin-eosin, and the last three with the collagen-specific Sirius red stain (0.5% in saturated picric acid) and were studied blindly by a single examiner. Each field was digitized on a Macintosh IIfx by a gray-level camera (Hamamatsu, Japan) mounted on a light microscope (Leitz, Germany) at a magnification of ×100, and collagen was quantified by using image-analysis software (OPTILAB, Graftek, France). Interstitial collagen volume fraction (i.e., the ratio of interstitial collagen surface area to total ventricular surface area, expressed as a percentage) and perivascular collagen of the stained tissue were determined separately in both LV and RV. Perivascular collagen area was divided by the area of the corresponding arterial lumen, because a correlation exists between perivascular collagen and vessel luminal area in a given animal (4).
Results are expressed as mean ± SEM. Comparisons between groups were performed by the Kruskal-Wallis test, a nonparametric analysis of variance, and significant differences determined by using a Mann-Whitney test. A p level of <0.05 was considered significant. To assess relations between hemodynamic or collagen variables and hormonal data, linear correlations were performed for all animals and the Pearson coefficient r was determined. The normality of distribution of the different variables was tested (Kolmogorov-Smirnov method) to be able to perform such linear correlation on data pooled together from the different groups.
All animals included in the protocol survived surgery and the duration of treatment.
Compensated left ventricular hypertrophy in aldosterone/salt-induced hypertension
Left ventricular geometry and anatomic data. In aldosterone/salt-treated guinea pigs, LV internal end-diastolic dimension and the diastolic thickness of the anterior and posterior LV walls corrected for body weight (BW) were significantly increased (+17, +32, and +31%, respectively) compared with controls (p < 0.01; Table 1). These echocardiographic results of the study are in agreement with the anatomic data. The LV/BW and LVW/RVW ratios were increased by 60 ± 4% and 56 ± 3%, respectively, in the aldosterone/salt group compared with the control or control/salt groups (p < 0.01). There was no evidence of RV hypertrophy in the aldosterone/salt-treated guinea pigs. There were no differences in lung weights between animal groups and no evidence of heart failure at autopsy.
Left ventricular function. The indices of LV contractility measured by M-mode echocardiographic fractional shortening and percentage of anterior and posterior wall thickening did not differ between groups (Table 2). Doppler measurements of mitral inflow showed that aldosterone/salt treatment did not modify either early LV filling (E) or atrial filling velocity (A). Mitral regurgitation was not observed. Similarly stroke volume and cardiac output, assessed by using the aortic Doppler signal, did not differ between aldosterone/salt-treated and control groups. It should be noted that there was no difference in heart rate between groups.
Invasive hemodynamic measurement showed increase of systolic blood pressure in aldosterone/salt-treated guinea pigs compared with control and control/salt groups (p < 0.01). The data obtained in control guinea pigs are in agreement with those reported previously (3,5,21). The LV hypertrophy index measured in vivo (diastolic posterior wall thickness of the LV in M-mode echocardiography) and ex vivo (LVW/RVW ratio) correlated with systolic blood pressure (LV diastolic posterior wall thickness of the LV: r = 0.70; p = 0.008; LVW/RVW ratio: r = 0.88; p = 0.001).
Neurohumoral and electrolyte profile
Plasma hormonal, catecholamine, and electrolyte concentrations from control, control/salt nonhypertensive, and aldosterone/salt hypertensive guinea pigs are shown in Table 3. Results in the two control groups did not differ, other than a decrease in aldosterone levels in the control/salt group (p < 0.01) compared with the control group. As expected, plasma aldosterone and Na+ concentrations were significantly increased in the aldosterone/salt-treated guinea pigs, whereas PRA, AI, and AII were significantly reduced. Furthermore, aldosterone/salt treatment was associated with a marked increase in plasma norepinephrine concentrations.
Cardiac collagen distribution after aldosterone/salt treatment
Histologic studies. No obvious qualitative difference was found between the three groups on histologic examination. There was no cellular or architectural distortion apparent with hematoxylin-eosin staining, and no evidence of vascular medial thickening or necrosis, inflammatory infiltration, or myocardial necrosis. Collagen fiber deposits were located mainly in the myocardial interstitial space (Fig. 1B and D) and the myocardial vascular adventitia (Fig. 1B and F) of aldosterone/salt-treated guinea pigs. There was no evidence of replacement fibrosis.
Collagen morphometry. As shown in Table 4, collagen density did not differ between the control and control/salt groups after 3 months of treatment. In contrast, an increase in collagen density was found in LV and RV from aldosterone/salt-treated guinea pigs, affecting total, interstitial, and perivascular collagen. This increase was the same in LV and RV but did not correlate with systolic blood pressure.
Ventricular type I collagen mRNA expression
As shown in Fig 2, Northern blot analysis revealed two bands for LV and RV RNA that corresponded in length to collagen type I. No signal was detected in brain RNA. Faint signals were detected in LV and RV RNA from control and control/salt hearts, and strong signals from aldosterone/salt hearts.
The results of quantitative slot-blot analysis after hybridization with specific rat α1-I collagen probes are shown in Fig. 3. The mRNA collagen type I/mRNA GAPDH ratio was not different in hearts from control and control/salt-treated guinea pigs. In contrast, this ratio was significantly increased in hypertrophied LV and non-hypertrophied RV from aldosterone/salt-treated guinea pigs compared with both control groups.
This study shows that long-term aldosterone/salt treatment in guinea pigs induces arterial hypertension, LV hypertrophy, and increases in collagen type I mRNA and total collagen deposition in LV and nonhypertrophied RV, without altering LV systolic or diastolic function. This steady-state LV hypertrophy is associated with sympathetic activation, which may contribute to hemodynamic adaptation to pressure overload. The extent of cardiac fibrosis did not correlate with the degree of hypertension or LV hypertrophy. These results confirm that aldosterone induces effects on the extracellular matrix in the guinea pig that are more dependent on neurohumoral influences than the induced pressure overload or degree of LV hypertrophy, because collagen deposition was increased to similar extent in both RV and LV, as previously reported in rats (4,6,8).
The increase in blood pressure induced by long-term aldosterone/salt administration to the guinea pig in our study (+35%) appears to be lower than that in previous rat studies (+100%; 6), which used similar doses of aldosterone. However, it induced a similar degree of cardiac hypertrophy, suggesting that guinea-pig heart is more sensitive to hypertension than is rat heart, or that additional factors interfere with the hypertrophy process. An alternative explanation could be the longer period of pressure overload in our experiments compared with the previous rat studies using 8 weeks of aldosterone (4,8) or 5 weeks of deoxycorticosterone acetate (DOCA) treatment (22). Lower arterial baroreceptor sensitivity in the guinea pig could also contribute to the development of cardiac hypertrophy by reducing the normal loss of sympathetic tone that occurs during pressure overload. In such cases, the lower baroreceptor sensitivity induces higher levels of sympathetic tone when systolic blood pressure increases.
The increase in collagen mRNA content and myocardial interstitial and perivascular collagen deposition in our study confirm previous observations in rats during aldosterone administration (6,8). There is no evidence for any increase in RV wall thickness in hypertensive guinea pigs. This suggests that pressure in the pulmonary circulation is not increased and, thus, that increased pressure is not required for the increased fibrosis seen in the RV, contrary to the LV. This dissociation between the development of fibrosis and hypertrophy was explored in other models. By using the DOCA/salt model, Lattion et al. (23) observed an increase of atrial natriuretic peptide (ANP) mRNA both in atria and in the LV, but not in the RV. It is largely documented that ventricular overexpression of ANP reflects tissue stretch secondary to volume or pressure overload (24,25). In our team, Robert et al. (6) showed that ANP mRNA is strongly expressed in the LV of aldosterone-induced hypertensive rats but was absent from the RV of these animals. This result strengthens the idea that systemic hypertension is the main determinant for LV hypertrophy but not for cardiac fibrosis. On the other hand, the biventricular collagen increase suggests neurohumoral influences of collagen deposition in our study. In our model, total myocardial collagen content was positively correlated with plasma aldosterone and norepinephrine levels. In contrast, a negative correlation was found with PRA (which is downregulated by aldosterone administration and an increase in blood pressure). The respective roles of catecholamine and aldosterone in collagen synthesis cannot be determined from our experiments. Nevertheless, the positive correlation between collagen type I mRNA accumulation and norepinephrine levels is supported by previous reports of the effects of norepinephrine on cardiac collagen gene expression (26). Coadministration of α-adrenergic and β-adrenergic blockers could have allowed separation of the effects of catecholamines and aldosterone but would probably have resulted in a different pattern of pressure overload. Our experiments clearly show that plasma norepinephrine levels were greatly increased. The mechanism of this increase during aldosterone/salt hypertension is unclear, but several possibilities exist: (a) aldosterone-induced decrease in baroreceptor sensitivity (27), resulting in reduced sympathetic withdrawal during blood pressure increase; and (b) inhibition of postsynaptic norepinephrine uptake by aldosterone (28-30). Such inhibition increases norepinephrine concentration in the synaptic cleft and the amounts of plasma spillover. Consistent with this mechanism, Barr et al. (30) showed that mineralocorticoid receptor blockade by spironolactone increases myocardial norepinephrine uptake in heart failure. This potential aldosterone-induced increase in the synaptic cleft norepinephrine concentration could increase postsynaptic myocardial β-receptor stimulation with enhanced cardiac contractility and relaxation, contributing to the hemodynamic adaptation to aldosterone/salt hypertension. In our experiments, systolic and diastolic function were unaltered, despite the increase in myocardial collagen content. These results suggest that endogenous sympathetic activation results in a compensated hemodynamic profile without myocardial cells loss, because neither myocardial necrosis nor collagen scars were detected in this study. But the nonsignificant trends toward increase E/A ratio due to decrease of A-wave velocity may indicate onset of LV-chamber stiffness, which may increase with a longer period of aldosterone/salt administration.
In conclusion, this study clearly demonstrated that long-term aldosterone/salt administration induces cardiac collagen accumulation without changing systolic or diastolic function. These results emphasize the potential role of neurohumoral factors such as sympathetic stimulation in the cardiac functional response to structural modification. These long-term effects of hyperaldosteronism might exert a long-term influence on the time course of cardiac adaptation to overload and the transition toward heart failure.
Acknowledgment: This work was supported in part by a doctoral fellowship grant from "Ministère Français des Affaires Etrangères" and "Colciencias" (J.F.R.G.). We thank Dr. B. Prendergast for helpful discussion and text support, Dr. A. Carayon for plasma renin and angiotensin assays, Dr. F. Zoghby for plasma aldosterone assay, Dr. C. Landault for plasma catecholamine assays, and Françoise Moreau-Raillecove for technical assistance.
1. Anversa P, Olivetti G, Melissari M, Loud AV. Morphometric study of myocardial hypertrophy induced by abdominal aortic stenosis. Lab Invest
2. Thiedemann KU, Holubarsch C, Medugorac IRJ. Connective tissue content and myocardial stiffness in pressure overload hypertrophy: a combined study of morphologic, morphometric, biochemical and mechanical parameters. Basic Res Cardiol
3. Siri FM, Nordin C, Factor SM, Sonnenblick E, Aronson R. Compensatory hypertrophy and failure in gradual pressure-overload guinea pig
heart. Am J Physiol
4. Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT. Remodeling of the rat right and left ventricles in experimental hypertension. Circ Res
5. Randhawa AK, Singal PK. Pressure overload-induced cardiac hypertrophy with and without dilatation. J Am Coll Cardiol
6. Robert V, Thiem NV, Cheav SL, Mouas C, Swynghedauw B, Delcayre C. Increased cardiac types I and III collagen mRNAs in aldosterone-salt hypertension. Hypertension
7. Young M, Fullerton M, Dilley R, Funder F. Mineralocorticoids, hypertension, and cardiac fibrosis. J Clin Invest
8. Robert V, Silvestre JS, Charlemagne D, et al. Biological determinants of aldosterone-induced cardiac fibrosis in rats. Hypertension
9. Weber KT, Pick R, Jalil JE, Janicki JS, Carroll EP. Patterns of myocardial fibrosis. J Mol Cell Cardiol
10. Conrad CH, Brooks WW, Hayes JA, Sen S, Robinson KG, Boh L. Myocardial fibrosis and stiffness with hypertrophy and heart failure in the spontaneously hypertensive rat. Circulation
11. Young MJ, Funder JW. The renin-angiotensin-aldosterone system in experimental mineralocorticoid-salt-induced cardiac fibrosis. Am J Physiol
12. Zhou G, Kandala JC, Tyagi SC, Katwa LC, Weber KT. Effects of angiotensin II and aldosterone on collagen gene expression and protein turnover in cardiac fibroblasts. Mol Cell Biochem
13. Brilla CG, Weber KT. Reactive and reparative myocardial fibrosis in arterial hypertension in the rat. Cardiovasc Res
14. Lecarpentier Y, Waldenström A, Clergue M, et al. Major alterations in relaxation during cardiac hypertrophy induced by aortic stenosis in guinea pig
. Circ Res
15. Terracciano CMN, Mac Leod KT. Measurements of Ca2+
entry and sarcoplasmic reticulum Ca2+
content during the cardiac cycle in guinea pig
and rat ventricular myocytes. Biophys J
16. Katz AM. Increased Ca++
entry during the plateau of the action potential: a possible mechanism of cardiac glycoside action. J Mol Cell Cardiol
17. Litwin SE, Katz SE, Morgan JP, Douglas PS. Serial echocardiographic assessment of left ventricular geometry and function after large myocardial infarction in the rat. Circulation
18. Schaison FH, Ramirez-Gil JF, Ciferri S, et al. Acute and long-term dose-response study of quinapril on hormonal profile and tissue angiotensin-converting enzyme in wistar rats. J Cardiovasc Pharmacol
19. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter RW. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry
20. Genovese C, Rowe D, Kream B. Construction of DNA sequences complementary to rat α1 and α2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihy-droxyvitamin D. Biochemistry
21. Lechat P, Schmitt H. Interactions between the autonomic nervous system and the cardiovascular effects of ouabain in guinea pigs. Eur J Pharmacol
22. Karam H, Heudes D, Hess P, et al. Respective role of humoral factors and blood pressure in cardiac remodeling of DOCA hypertensive rats. Cardiovasc Res
23. Lattion AL, Michel JB, Arnauld E, Corvol P, Soubrier F. Myocardial recruitment during ANF mRNA increase with volume overload in the rat. Am J Physiol
24. Lang RE, Tholken H, Ganten D, Luft FC, Ruskoaho H, Unger T. Atrial natriuretic factor: a circulating hormone stimulated by volume loading. Nature
25. Mercadier JJ, Samuel JL, Michel JB, et al. Atrial natriuretic factor gene expression in rat ventricle during experimental hypertension. Am J Physiol
26. Bhambi B, Eghbali M. Effect of norepinephrine on myocardial collagen gene expression and response of cardiac fibroblasts after norepinephrine treatment. Am J Pathol
27. Wang W. Chronic administration of aldosterone depresses baroreceptor reflex function in the dog. Hypertension
28. Koehler MF, Webb RC, Jones AW, Bohr DF. In vitro effects of desoxycorticosterone on vascular smooth muscle. Am J Physiol
29. Weber MA, Purdy RE, Drayer JIM. Interactions of mineralocorticoids and pressor agents in vascular smooth muscle. Hypertension
30. Barr CS, Lang CC, Hanson J, Arnott M, Kennedy N, Struthers AD. Effect of adding spironolactone to an ACE inhibitor in congestive heart failure secondary to coronary artery disease. Am J Cardiol