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Aldosterone Antagonism Fails to Attenuate Age-Associated Left Ventricular Fibrosis

¹ Division of Kinesiology, Laboratory of Molecular Kinesiology, University of Michigan, Ann Arbor.
² Division of Kinesiology and Health
³ Department of Animal Science, University of Wyoming, Laramie.

Address correspondence to Marvin O. Boluyt, PhD, Division of Kinesiology, Laboratory of Molecular Kinesiology, University of Michigan, 1209 CCRB, 401 Washtenaw Avenue, Ann Arbor, MI 48109-2214. E-mail: boluytm{at}umich.edu

	Abstract

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Collagen accumulates disproportionately in cardiac remodeling induced by hypertension and associated with advancing age. Spironolactone (Spiro), an aldosterone antagonist, attenuates the accumulation of collagen induced by hypertension. It was hypothesized that Spiro would attenuate the age-associated increase in percent collagen in the heart. Female Fisher 344 rats at 3 months (Y), 12 months (M), and 21 months (O) of age were treated with Spiro (30 mg/kg/d) or vehicle (Veh) for 2 months, yielding six groups: Y-Veh, Y-Spiro, M-Veh, M-Spiro, O-Veh, and O-Spiro. Hearts were harvested for immunoblotting, RNA blotting, and biochemical analysis. Percent collagen in the left ventricle and septum was greatest in the oldest rats. Spiro did not significantly attenuate the age-associated increase in collagen fraction or the age-associated increases in expression of atrial natriuretic factor and ß-myosin heavy chain messenger RNA. Chronic aldosterone antagonism does not attenuate the age-associated increase in collagen fraction in the female Fisher 344 rat heart.

Extracellular matrix in heart tissue is composed primarily of collagen proteins, although a number of other proteins, such as fibronectin and osteopontin, play important roles in the complex structure of the interstitial space. Fibrillar Type I and Type III collagens make up the vast majority of the collagen protein in the heart, and provide the raw materials for the connective tissue "skeleton" of the heart (16,17). Collagen proteins assemble into a network of epimysial, perimysial, and endomysial fibers. These collagenous fibers function to maintain the alignment of myocytes and blood vessels, organize myocytes into bundles, and transduce force from myocytes to the chamber. Collagen fibers also provide passive stiffness, which prevents excessive diastolic enlargement and contributes to the Starling effect. Fibrillar collagens are large trimeric molecules that form triple helices and contain amino acid sequences that bind specific myocyte and fibroblast receptors (integrins), as well as other extracellular matrix molecules, such as fibronectin. Generally, Type I collagen predominates in the left ventricle (LV), although in the fetus and neonate Type III collagen levels are high and may exceed levels of Type I. Type III collagen is considered "youthful" collagen; with aging there is a gradual shift toward more Type I and less Type III collagen (18–20). Significantly, during periods of rapid tissue growth and collagen accretion, the Type III collagen fraction increases and the Type I fraction decreases (16). Such an increase in Type III collagen accretion is observed in several models of cardiac hypertrophy and is considered evidence of connective tissue remodeling of the myocardium.

Excessive fibrosis is a major determinant of impaired stiffness and pumping capacity in hypertrophied hearts. Its disproportionate accumulation accounts for a ventricular dysfunction that first appears during diastole, subsequently involves systole, and ultimately accelerates progression to CHF (16). Treatments that ameliorate the devastating effects of CHF and slow the progression to irreversible failure and death include agents that antagonize the action of aldosterone by blocking the mineralocorticoid receptor (MR), but treatments that reverse the negative functional and fibrotic characteristics of heart failure have not been identified (21). Furthermore, the mechanisms by which MR antagonists work are not entirely clear, and the impact of age on MR antagonism is virtually unknown.

Spironolactone is a competitive antagonist of the MR that has been available for decades. It is relatively selective for the MR, but does possess some progestational and antiandrogenic properties that produce unwanted side effects in a subset of patients (22). A clinical trial demonstrated efficacy of spironolactone therapy in human patients with heart failure (23), but an understanding of the mechanisms by which spironolactone exerts its effects requires additional animal model studies. Aldosterone production is elevated in failing ventricles of humans (24), and spironolactone reduces the levels of collagen fragments in the plasma (25), suggesting that spironolactone works, at least in part, by reducing fibrosis of the myocardium. Past and recent data from animal models support and amplify this concept (26,27). Spironolactone inhibits the production of collagen by cultured cardiac fibroblasts (28). In animal models of hyperaldosteronism and hypertension, spironolactone therapy is effective in preventing or attenuating myocardial fibrosis (29,30). Another rat study showed that spironolactone and angiotensin-converting enzyme inhibitors interact to reduce fluid volume, and suggests that fluid reduction may at least partially explain the benefits of MR antagonism in humans (31). Aldosterone antagonism with a new, slightly more selective agent, eplerenone, reduces or prevents fibrosis in the viable myocardium after myocardial infarction in rats and improves the outcomes of patients with LV dysfunction after myocardial infarction (32,33). Despite the remarkable success of MR antagonism in treating heart failure patients, there is much to learn about the mechanisms of aldosterone signaling and the effectiveness of aldosterone antagonism in various subpopulations of patients, as pointed out recently (34,35). In particular, the effects of age on the efficacy of MR antagonists are not known, but at least one clinical study suggests that MR antagonism may be less effective in the elderly population (33). To our knowledge, there are virtually no data regarding age-associated changes in the local cardiac aldosterone signaling system. Thus, there is a need to obtain basic information on important components required for the postulated effects of mineralocorticoid excess on the fibrosis of advancing age in the heart.

Collectively, these data strongly suggest that the current lack of information regarding age-associated differences in the cardiac aldosterone system and the impact of age on efficacy of MR antagonists should be addressed by basic and clinical studies. The female Fisher 344 (F344) rat has served as a model to study the mechanisms of these changes in the heart and their implications for the response of the heart to physiological and pathological challenges (9,10,14,36–39). The hypothesis that aldosterone antagonism by spironolactone would attenuate the disproportionate age-associated increase in collagen accumulation was tested. The effects of spironolactone on the expression of two genes that typically exhibit age-associated increases in the heart, atrial natriuretic factor, and ß-myosin heavy chain (ß-MHC) were also studied.

	MATERIALS AND METHODS

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Animals
Specific pathogen-free female Fisher 344 (F344) rats were obtained from the National Institute on Aging-sponsored colony at Harlan Industries (Indianapolis, IN). Rats were obtained at 3 (young; Y), 12 (middle-aged; M), and 21 (old; O) months of age and were treated with spirolactone (Spiro; 30 mg/kg/d) or vehicle (Veh; vegetable oil) orally for the next 2 months. Groups were as follows: Y-Veh (n = 6), Y-Spiro (n = 6), M-Veh (n = 6), M-Spiro (n = 6), O-Veh (n = 8), and O-Spiro (n = 6). Rats were housed 2–3 per cage and fed a standard laboratory diet 5001 (PMI Nutrition International, LLC, Richmond, IN) and water ad libitum, and were maintained on a 12-hour light/dark cycle. All animal protocols were approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan. All animal protocols conformed to the Guiding Principles in the Care and Use of Animals of the American Physiological Society. At designated times, the LVs, septum, and right ventricles (RVs) were quickly dissected free, weighed, and rapidly frozen by clamping with tongs cooled to the temperature of liquid nitrogen. Tissues were stored at –80°C for subsequent analysis.

RNA Blotting
RNA blotting was performed as described previously with modifications (35,36). RNA was isolated from the LV. Ten micrograms of total RNA were size-fractionated by electrophoresis through 1% agarose gels, transferred electrophoretically at 5 V/cm to nylon (Nytran-SPC; Whatman, Inc., Florham Park, NJ) membrane, and hybridized with ³²P-radiolabeled probes overnight at 68°C for complementary DNA probes and 42–45°C for oligonucleotide probes using PerfectHyb Plus (Sigma, St. Louis, MO). Hybridization intensity was quantified with a Personal PhosphorImager FX (Bio-Rad, Hercules, CA). Signals visualized on a computer screen were identified by position relative to 18S and 28S ribosomal RNA migration, delineated by rectangles, and quantified after background subtraction. Each blot was subsequently stripped and reprobed. The signal from each sample was normalized to the signal obtained with an oligonucleotide specific for the 3'-untranslated region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (40). The probe for ß-MHC was an oligonucleotide described previously (41). A complementary DNA probe for atrial natriuretic factor (ANF) was synthesized from a template (41) by the random prime method (Promega, Madison, WI).

Plasma Aldosterone
Plasma aldosterone levels were measured by radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA).

Collagen Type III Immunoblotting
Portions of the frozen LV were processed for immunoblotting as described (42). The relative levels of collagen Type III protein were measured using a monoclonal antibody directed at collagen Type III (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were quantified using enhanced chemiluminescence.

Hydroxyproline Assay
Hydroxyproline concentration was measured colorimetrically as described previously (10). Collagen concentration was calculated assuming that collagen weighs 7.25 times hydroxyproline and has a molecular weight of 300,000 daltons.

Statistics
Values are expressed as mean ± standard error (SE). Age differences were assessed with two-way analysis of variance (ANOVA), and group differences were determined with the Fisher least significant difference (LSD) procedure. Differences were considered significant if p <.10 (43).

	RESULTS

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Postmortem Analysis
Ontogenic growth accounts for the age-associated increases in body weight with advancing age (Table 1), as documented previously (38). Spiro treatment for 2 months had no effect on body weight at any of the ages studied. LV weight was increased with advancing age (p <.01), and there was a main effect of Spiro treatment to increase LV weight by an average of 5% (p <.05). There was also a main effect for Spiro treatment to increase the ratio of the LV weight to body weight by an average of 5% (p <.10). Wet weight to dry weight ratio of the LV did not differ among age groups or in response to Spiro treatment, indicating that there were no effects of age or Spiro treatment on hydration status. RV weight was increased with advancing age (p <.001), but not by Spiro treatment.

View larger version (11K): [in this window] [in a new window]   Figure 1. Level of plasma aldosterone in female Fisher 344 rats treated with spironolactone (Spiro) or vehicle (Veh). Y, young; M, middle-aged; O, old; ALDO, aldosterone. Data are expressed as mean ± standard error [SE] for n = 6–8 per group. Inset: p values for two-factor analysis of variance main effects and interactive effects of age and Spiro treatment. , p <.05 versus Veh of the same age group (Fisher least significant difference procedure)

View larger version (11K): [in this window] [in a new window]   Figures 2. Level of aldosterone (ALDO) in the left ventricle free wall of female Fisher 344 rats treated with spironolactone (Spiro) or vehicle (Veh). Y, young; M, middle-aged; O, old; NS, not significant. Data are expressed as mean ± standard error [SE] for n = 5–8 per group. Inset: p values for two-factor analysis of variance main effects and interactive effects of age and Spiro treatment. , p <.05 versus Veh of the same age group (Fisher least significant difference procedure)

View larger version (23K): [in this window] [in a new window]   Figure 3. Percent collagen in the left ventricle of rats treated with spironolactone (Spiro) or vehicle (Veh). A, Collagen fraction in the left ventricle free wall measured by biochemical assay as described in the Materials and Methods section. B, Representative Western blot of collagen III protein in the left ventricle free wall. C, Level of collagen III protein in the left ventricle free wall of rats treated with Spiro or Veh. Y, young; M, middle-aged; O, old; NS, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Data are expressed as mean ± standard error [SE] for n = 5–8 per group. Inset: p values for two-factor analysis of variance main effects and interactive effects of age and Spiro treatment. *, p <.05 versus Y of same treatment group; , p <.05 versus M of same treatment group; , p <.05 versus Veh of the same age group (Fisher least significant difference procedure)

View larger version (10K): [in this window] [in a new window]   Figure 4. Percent collagen in the septum of rats treated with spironolactone (Spiro) or vehicle (Veh). Y, young; M, middle-aged; O, old; NS, not significant. Data are expressed as mean ± standard error [SE] for n = 5–8 per group. Inset: p values for two-factor analysis of variance main effects and interactive effects of age and Spiro treatment. *, p <.05 versus Y of same treatment group; , p <.05 versus M of same treatment group (Fisher least significant difference procedure)

Gene Expression
To determine whether Spiro treatment altered the expression of other cardiac genes known to change with age, the level of ANF and ß-MHC mRNA were also studied. ANF mRNA expression was increased with advancing age as observed previously (44), but did not differ with Spiro treatment (Figure 5). ß-MHC mRNA expression also increased with advancing age, but was not affected by Spiro treatment (Figure 6). When normalized to 18S, the ANF and ß-MHC mean data exhibited a pattern similar to the GAPDH-normalized data and lead to identical conclusions (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 5. Level of atrial natriuretic factor (ANF) messenger RNA (mRNA) in the left ventricle of rats treated with spironolactone (S or Spiro) or vehicle (V or Veh). A, Representative Northern blot. B, Mean values. Y, young; M, middle-aged; O, old; NS, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Data are expressed as mean ± standard error [SE] for n = 4–8 per group. Inset: p values for two-factor analysis of variance main effects and interactive effects of age and Spiro treatment. *, p <.05 versus Y of same treatment group (Fisher least significant difference procedure)

View larger version (30K): [in this window] [in a new window]   Figure 6. Level of ß-myosin heavy chain (ß-MHC) messenger RNA (mRNA) in the left ventricle of rats treated with spironolactone (S or Spiro) or vehicle (V or Veh). A, Representative Northern blot. B, Mean values. Y, young; M, middle-aged; O, old; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, not significant. Data are expressed as mean ± standard error [SE] for n = 5–8 per group. Inset: p values for two-factor analysis of variance main effects and interactive effects of age and Spiro treatment. *, p <.05 versus Y of same treatment group (Fisher least significant difference procedure)

	DISCUSSION

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The lack of a Spiro effect raises the question of whether the Spiro treatment was effectively absorbed after oral administration. The markedly elevated levels of plasma aldosterone observed in Spiro-treated rats of each age group (Figure 1) and the significant effect of Spiro on cardiac aldosterone levels (Figure 2) provide indirect evidence that the Spiro was appropriately absorbed. Furthermore, the same dose of Spiro (30 mg/kg/d) administered in the same manner for 6 months to aorta-constricted rats was effective in reducing the percent collagen in the heart (our unpublished observations). Moreover, Brilla and coworkers (29,30) used a lower dose of Spiro (20 mg/kg/d) to effectively prevent cardiac fibrosis induced by either hyperaldosteronism or hypertension. Thus, it is unlikely that the failure of Spiro treatment to prevent cardiac fibrosis in the present study is due to insufficient bioavailability of the drug. Due to the relatively slow turnover of collagen, the possibility that a longer course of Spiro treatment might significantly reduce age-associated cardiac fibrosis cannot be ruled out by the present findings.

The effect of Spiro treatment to induce a small (3%–9%) but significant increase in LV weight was unexpected and was most evident in the oldest group (Table 1), indicating that Spiro treatment exacerbates age-associated cardiac hypertrophy. To our knowledge, no previous studies have reported a hypertrophic effect of Spiro treatment on the intact heart. In contrast, in rats with low-aldosterone hypertension, Nagata and coworkers (46) reported that Spiro treatment attenuates LV hypertrophy, without an antihypertensive effect. Exposure of cultured neonatal rat cardiac myocyte to aldosterone leads to myocyte hypertrophy mediated by genomic effects of the MR that are blocked by aldosterone antagonism (47,48). At present, no explanation for this hypertrophic effect of Spiro treatment on the LV is apparent, but it is noteworthy that Spiro treatment did not have a similar hypertrophic effect on the RV free wall (Table 1). The finding that Spiro induced a small but significant increase in LV mass was surprising. Although the magnitude of cardiac hypertrophy induced by Spiro in the present study was quite modest, it was statistically significant, and although the magnitude of hypertrophy did not differ by age, it was numerically greatest in the oldest group. In fact, if the oldest group were not included in the study, the cardiac hypertrophy would not have been statistically significant, and would therefore have escaped detection. Thus, modest cardiac hypertrophy may be a side effect of chronic Spiro treatment. To our knowledge, there are no published reports in which Spiro treatment causes cardiac hypertrophy; therefore, the mechanism is unknown. Because aldosterone induced cardiac myocyte hypertrophy in culture, it might have been predicted that Spiro would decrease, rather than increase LV mass. It is noteworthy that chronic Spiro treatment induced a large compensatory increase in circulating levels of aldosterone, and it is possible that circulating aldosterone induced the observed increase in LV mass by an MR-independent mechanism.

The two major limitations of the present study are that a treatment period longer than 2 months might be required to observe an effect of Spiro on the relatively long-lived collagen protein and the limited number of rats (n = 6–8) per group. The former limitation should be considered in light of the observations of Brilla and coworkers (29,30) that 2 months of Spiro treatment was effective in preventing fibrosis induced by hyperaldosteronism and hypertension, observations that were used to guide the design of the present study. The limitation imposed by the small number of rats per group is mitigated by the 2 x 3 design and the statistical robustness of the two-factor ANOVA procedure. The conclusions are bolstered by the fact that there are no obvious trends in the data from the Spiro groups to suggest that Spiro was having the hypothesized effect to attenuate the age-associated increase in collagen accumulation. Instead, with the exception of the collagen fraction in the septum, most trends are in the opposite direction (e.g., Figure 3A and C). Thus, the conclusion that 2 months of Spiro treatment did not favorably influence the age-associated increases in the percent collagen in the rat ventricle can be stated with confidence.

Another potential limitation is that peripheral blood pressure was not measured in the present study. Therefore, the possibility that Spiro may have exerted effects by modifying blood pressure cannot be ruled out. For example, when Spiro is administered at a high dose (200 mg/kg/d) to hypertensive rats, it has a modest effect to lower blood pressure (49). Based on studies of normotensive rats, however, administration of 30 mg/kg/d of Spiro would not be expected to influence blood pressure in normotensive rats (30). Indeed even a much higher dose of Spiro (200 mg/kg/d) failed to significantly alter blood pressure in normotensive 22-month-old rats (50). Thus, it seems unlikely that daily treatment of 30 mg/kg/d Spiro for 2 months influenced the blood pressure of the rats in the present study.

Because a disproportionate number of elderly patients suffer from CHF, cardiac fibrosis, and diastolic dysfunction (51), and because aldosterone antagonism is an important feature of pharmacotherapy for CHF, these findings have potential clinical implications: They suggest that aldosterone antagonism may be less effective at combating fibrosis and heart failure in elderly persons, a possibility also suggested by a clinical study of aldosterone antagonism (33). Further study of the effectiveness of aldosterone antagonism in heart failure models of younger and older animals is warranted.

	Acknowledgments

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This work was supported by an American Heart Association Midwest Affiliate Scientist Development Grant (to M.O.B.), a Claude Pepper Older Americans Independence Center Pilot Grant (AG08808; to M.O.B.), and a National Institutes of Health R03 Award (AG022625; to M.O.B.).

We are grateful to Scott Peshick and Agdas Mikkor for help with technical aspects of the project.

	Footnotes

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Decision Editor: Huber R. Warner, PhD

Received June 15, 2006

Accepted October 5, 2006

	References

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1. Ahmed A. American College of Cardiology/American Heart Association chronic heart failure evaluation and management guidelines: relevance to the geriatric practice. J Am Geriatr Soc. 2003;51:123-126.[Medline]
2. Huysman JAN, Vliegen HW, VanderLaarse A, Eulderinnk F. Changes in nonmyocyte tissue composition associated with pressure overload of hypertrophic human hearts. Pathol Res Pract. 1989;184:577-581.[Medline]
3. Pearlman ES, Weber KT, Janicki JS, Pietra G, Fishman AP. Muscle fiber orientation and connective tissue content in the hypertrophied human heart. Lab Invest. 1982;46:158-164.[Medline]
4. Beltrami CA, Finato N, Rocco M, et al. The cellular basis of dilated cardiomyopathy in humans. J Mol Cell Cardiol. 1995;27:291-305.[Medline]
5. Marino TA, Kent RL, Uboh CE, Fernandez E, Thompson EW, Cooper G, 4th. Structural analysis of pressure versus volume overload hypertrophy of cat right ventricle. Am J Physiol. 1985;249:H371-H379.[Medline]
6. Annoni G, Luvara G, Arosio B, et al. Age-dependent expression of fibrosis-related genes and collagen deposition in the rat myocardium. Mech Ageing Dev. 1998;101:57-72.[Medline]
7. Besse S, Robert V, Assayag P, Delcayre C, Swynghedauw B. Nonsynchronous changes in myocardial collagen mRNA and protein during aging. Effect of DOCA-salt hypertension. Am J Physiol. 1994;267:H2237-H2244.[Medline]
8. Boluyt MO, Lakatta EG. Cardiovascular aging in health. In: Bittar EE, Altschuld RA, Haworth RA, eds. Advances in Organ Biology: Heart Metabolism in Failure. Vol. 4B. Stamford, CT: JAI Press; 1998.
9. Thomas DP, McCormick RJ, Zimmerman SD, Vadlamudi RK, Gosselin LE. Aging- and training-induced alterations in collagen characteristics of rat left ventricle and papillary muscle. Am J Physiol. 1992;263:H778-H783.[Medline]
10. Thomas DP, Zimmerman SD, Hansen TR, Martin DT, McCormick RJ. Collagen gene expression in rat left ventricle: interactive effect of age and exercise training. J Appl Physiol. 2000;89:1462-1468.[Abstract/Free Full Text]
11. Vanpee D, Swine CH. Elderly heart failure patients with drug-induced serious hyperkalemia. Aging (Milan). 2000;12:315-319.
12. Yao J, Eghbali M. Decreased collagen gene expression and absence of fibrosis in thyroid hormone-induced myocardial hypertrophy. Response of cardiac fibroblasts to thyroid hormone in vitro. Circ Res. 1992;71:831-839.[Abstract/Free Full Text]
13. Vasan RS, Benjamin EJ, Levy D. Prevalence, clinical features and prognosis of diastolic heart failure: an epidemiologic perspective. J Am Coll Cardiol. 1995;26:1565-1574.[Abstract]
14. Boluyt MO, Opiteck JA, Devor ST, White TP. Age effects on the adaptive response of the female rat heart following aortic constriction. J Gerontol Biol Sci. 2000;55A:B307-B314.[Abstract/Free Full Text]
15. Spurgeon H, Thorne P, Yin F, Shock N, Weisfeldt M. Increased dynamic stiffness of trabeculae carneae from senescent rats. Am J Physiol. 1977;232:H373-H380.[Medline]
16. Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium. Fibrosis and renin-angiotensin-aldosterone system. Circulation. 1991;83:1849-1865.[Abstract/Free Full Text]
17. Boluyt MO, Bing OH. The lonely failing heart: a case for ECM genes. Cardiovasc Res. 1995;30:836-840.[Medline]
18. Medugorac I. Collagen type distribution in the mammalian left ventricle during growth and aging. Res Exp Med. 1982;180:255-262.[Medline]
19. Mukherjee D, Sen S. Collagen phenotypes during development and regression of myocardial hypertrophy in spontaneously hypertensive rats. Circ Res. 1990;67:1474-1480.[Abstract/Free Full Text]
20. Yang CM, Kandaswamy V, Young D, Sen S. Changes in collagen phenotypes during progression and regression of cardiac hypertrophy. Cardiovasc Res. 1997;36:236-245.[Abstract/Free Full Text]
21. Lorell BH. Transition from hypertrophy to failure. Circulation. 1997;96:3824-3827.[Medline]
22. Pitt B, Zannad F, Remme W, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med. 1999;341:709-717.[Abstract/Free Full Text]
23. The RALES. Investigators. Effectiveness of spironolactone added to an angiotensin-converting enzyme inhibitor and a loop diuretic for severe chronic congestive heart failure (The randomized aldactone evaluation study [RALES]). Am J Cardiol. 1996;78:902-907.[Medline]
24. Mizuno Y, Yoshimura M, Yasue H, et al. Aldosterone production is activated in failing ventricle in humans. Circulation. 2001;103:72-77.[Abstract/Free Full Text]
25. Zannad F, Alla F, Dousset B, Perez A, Pitt B. Limitation of excessive extracellular matrix turnover may contribute to survival benefit of spironolactone therapy in patients with congestive heart failure: insights from the randomized aldactone evaluation study (RALES). Rales Investigators. Circulation. 2000;102:2700-2706.[Abstract/Free Full Text]
26. Lijnen P, Petrov V. Induction of cardiac fibrosis by aldosterone. J Mol Cell Cardiol. 2000;32:865-879.[Medline]
27. Rocha R, Stier T. Pathophysiological effects of aldosterone in cardiovascular tissues. Trends Endocrinol Metab. 2001;12:308-314.[Medline]
28. Brilla CG, Zhou G, Matsubara LS, Weber KT. Collagen metabolism in cultured adult rat cardiac fibroblasts: response to angiotensin II and aldosterone. J Mol Cell Cardiol. 1994;26:809-820.[Medline]
29. Brilla CG, Matsubara LS, Weber KT. Anti-aldosterone treatment and the prevention of myocardial fibrosis in primary and secondary hyperaldosteronism. J Mol Cell Cardiol. 1993;25:563-575.[Medline]
30. Brilla CG, Matsubara LS, Weber KT. Antifibrotic effects of spironolactone in preventing myocardial fibrosis in systemic arterial hypertension. Am J Cardiol. 1993;71:12A-16A.[Medline]
31. Bauersachs J, Fraccarollo D, Ertl G, Gretz N, Wehling M, Christ M. Striking increase of natriuresis by low-dose spironolactone in congestive heart failure only in combination with ace inhibition. Circulation. 2000;102:2325-2328.[Abstract/Free Full Text]
32. Delyani J, Robinson E, Rudolph A. Effect of a selective aldosterone receptor antagonist in myocardial infarction. Am J Physiol Heart Circ Physiol. 2001;50:H647-H654.
33. Pitt B, Remme W, Zannad F, et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med. 2003;348:1309-1321.[Abstract/Free Full Text]
34. Bouvy ML, Heerdink ER, Herings RM. Long-term therapy with spironolactone. Pharm World Sci. 2001;23:132-134.[Medline]
35. Bozkurt B, Agoston I, Knowlton AA. Complications of inappropriate use of spironolactone in heart failure: when an old medicine spirals out of new guidelines. J Am Coll Cardiol. 2003;41:211-214.[Abstract/Free Full Text]
36. Thomas DP, Cotter TA, Li X, McCormick RJ, Gosselin LE. Exercise training attenuates aging-associated increases in collagen and collagen crosslinking of the left but not the right ventricle in the rat. Eur J Appl Physiol. 2001;85:164-169.[Medline]
37. Boluyt MO, Opiteck JA, Esser KA, White TP. Cardiac adaptations to aortic constriction in adult and aged rats. Am J Physiol. 1989;257:H643-H648.[Medline]
38. Boluyt MO, Devor ST, Opiteck JA, White TP. Age-associated changes in the regional variation of cardiac myosin isoforms of female rats. J Gerontol Biol Sci. 1999;54:B313-B317.[Abstract]
39. Boluyt MO, Converso K, Hwang HS, Mikkor A, Russell MW. Echocardiographic assessment of age-associated changes in systolic and diastolic function of the female F344 rat heart. J Appl Physiol. 2004;96:822-828.[Abstract/Free Full Text]
40. Boluyt MO, Li ZB, Loyd AM, Scalia AF, Cirrincione GM, Jackson RR. The mTOR/p70S6K signal transduction pathway plays a role in cardiac hypertrophy and influences expression of myosin heavy chain genes in vivo. Cardiovasc Drugs Ther. 2004;18:257-267.[Medline]
41. Boluyt MO, O'Neill L, Meredith AL, et al. Alterations in cardiac gene expression during the transition from stable hypertrophy to heart failure: marked upregulation of genes encoding extracellular matrix proteins. Circ Res. 1994;75:23-32.[Abstract/Free Full Text]
42. Cirrincione GM, Boluyt MO, Hwang HS, Bleske BE. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibition and extracellular matrix gene expression in the pressure-overloaded rat heart. J Cardiovasc Pharmacol 2006;47:521-530.[Medline]
43. Curran-Everett D, Benos DJ. Guidelines for reporting statistics in journals published by the American Physiological Society. Am J Physiol. 2004;287:R247-R249.
44. Younes A, Boluyt MO, O'Neill L, Meredith AL, Crow MT, Lakatta EG. Age-associated increase in rat ventricular ANP gene expression correlates with cardiac hypertrophy. Am J Physiol. 1995;269:H1003-H1008.[Medline]
45. McCormick RJ, Thomas DP. Collagen crosslinking in the heart: relationship to development and function. Basic Appl Myol. 1998;8:143-150.
46. Nagata K, Obata K, Xu J, et al. Mineralocorticoid receptor antagonism attenuates cardiac hypertrophy and failure in low-aldosterone hypertensive rats. Hypertension. 2006;47:656-664.[Abstract/Free Full Text]
47. Yamamuro M, Yoshimura M, Nakayama M, et al. Direct effects of aldosterone on cardiomyocytes in the presence of normal and elevated extracellular sodium. Endocrinology. 2006;147:1314-1321.[Abstract/Free Full Text]
48. Okoshi MP, Yan X, Okoshi K, et al. Aldosterone directly stimulates cardiac myocyte hypertrophy. J Card Fail. 2004;10:511-518.[Medline]
49. Benetos A, Lacolley P, Safar ME. Prevention of aortic fibrosis by spironolactone in spontaneously hypertensive rats. Arterioscler Thromb Vasc Biol. 1997;17:1152-1156.[Abstract/Free Full Text]
50. Lacolley P, Safar ME, Lucet B, Ledudal K, Labat C, Benetos A. Prevention of aortic and cardiac fibrosis by spironolactone in old normotensive rats. J Am Coll Cardiol. 2001;37:662-667.[Abstract/Free Full Text]
51. Thom T, Haase N, Rosamond W, et al.,. American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart Disease and Stroke Statistics: 2006 Update: A Report From the American Heart Association Statistics Committee and Stoke Statistics Subcommittee. Circulation. 2006;113:(6): e85-151.[Free Full Text]

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