Prior to treatment, M-mode echocardiographic measurements of the dorsal and ventral LV wall thickness were similar in control and treated rats (Fig. 2). A significant and progressive increase in the wall thickness was observed in CON and SPIRO SS/Mcwi and SS-16BN/Mcwi rats as treatment progressed. Captopril and combined therapy significantly attenuated the increase in the LV ventral and dorsal wall thickness at the first and second weeks of treatment in SS/Mcwi and SS-16BN/Mcwi rats. This effect was maintained throughout the experimental period.
After 4 weeks, the total LV wall thickness measured by histology showed a significant decrease in the total LV wall thickness in SS-16BN/Mcwi CAP and CMB groups compared with CON group (Fig. 3). The combined therapy offered additional reduction in the wall thickness compared with the monotherapy with captopril (Fig. 3). In SS/Mcwi rats, the evaluation of the wall thickness by histology showed that all treatments promoted a significant reduction in LV wall thickness when compared with control (Fig. 3). The only contrast between histological and echocardiographic measurements is that histology indicated a reduction in wall thickness due to spironolactone treatment in SS/Mcwi rats, whereas the echocardiographic measurements showed no effect.
Mean arterial pressure (MAP) increased significantly after 4 weeks in CON and SPIRO SS/Mcwi rats (115.30 ± 1.91 vs. 125.68 ± 3.51 and 112.00 ± 2.51 vs. 123.09 ± 1.68 mm Hg, respectively). Captopril alone or in combination with spironolactone suppressed the increase in MAP in SS/Mcwi rats and also decreased the MAP of SS-16BN/Mcwi rats (Table 1).
The LV functional analyses performed by echocardiography revealed that LV EDV and ESV, heart rate, cardiac output, stroke volume, and the LV FS did not differ significantly between the control and treated groups in either SS/Mcwi or SS-16BN/Mcwi rats. However, in both control SS/Mcwi and SS-16BN/Mcwi groups, the cardiac index (cardiac output/body weight) decreased significantly at the end of experimental period when compared with pretreatment measurements, and captopril or combined treatment prevented this decrease (Fig. 4). Interestingly, in SS-16BN/Mcwi rats treated with captopril or combined therapy, the cardiac index tended to be greater than pretreatment levels. At the end of experimental period, the cardiac index of SS-16BN/Mcwi CMB group was significantly higher than the CON group (0.63 ± 0.06 control vs. 0.85 ± 0.06 CMB mL/min/g, P < 0.05).
Collagen deposition was distributed heterogeneously throughout the myocardium, and fibrosis was significantly greater in the inner layer (endocardial side) than in the middle and outer layers in all groups (Fig. 5). Subendocardial collagen density was 17% greater in SS-16BN/Mcwi rats when compared with SS/Mcwi rats. The percent of fibrosis did not differ among SS/Mcwi control and treated groups. In SS-16BN/Mcwi rats, however, the percentage of cardiac fibrosis in all treated groups tended to be less than from the control group, although these differences did not reach statistical significance.
Although gene expression was not assessed in this study, it is interesting that the transfer of chromosome 16 produced the hypertrophic phenotype; a number of important cardiac regulatory genes are found on rat chromosome 16, including VEGF-c,30 NPY1r,31 NPY5r,32 and perhaps most interestingly Hand2.33 The HAND basic Helix-Loop-Helix (bHLH) transcription factors are essential for normal cardiac and extra-embryonic development.33 Induction of cardiac hypertrophy shows modulation of HAND expression, corresponding with observations in human cardiomyopathy.34 The downregulation of HAND expression observed in rodent hypertrophy and human cardiomyopathy may reflect a permissive role allowing cardiomyocytes to reinitiate the fetal gene program, which is associated with pathological cardiovascular remodeling.
In this study, cardiac hypertrophy was assessed by echocardiographic and histological measurements of the LV wall thickness and by heart to body weight ratio. The echocardiographic analysis of LV dorsal and ventral wall thickness as well as the heart to body weight ratio were not modified by spironolactone monotherapy. In contrast to our study, Tsybouleva et al10 showed that spironolactone (50 mg/kg/d) treatment for 10 weeks was able to reduce heart/body weight ratio in cardiomyophatic mice. The differences between these studies may be due to differences in the animal model. In contrast to our model, the hallmark of the mouse cardiomyopathic model is an increase in fibrosis and myocyte disarray rather than the clear cardiac hypertrophy observed in humans and in our rat model. The dose and duration of the treatment may be another factor that influenced the different responses; however, measurements of the plasma potassium as well as the aldosterone levels were not evaluated in the Tsybouleva et al study.
The measurements of the total LV wall thickness by histology at the end of the experimental period also offered a good assessment of the geometric changes in the left ventricle caused by the different treatments. The histological measurements of the LV wall thickness correlated well with the ventral and dorsal wall echocardiography measurements with one exception: The histological evaluation of the wall thickness in SS/Mcwi rats in different regions of the left ventricle showed that all treatments promoted a significant reduction in LV wall thickness when compared to control. Echocardiography did not show a benefit from spironolactone treatment. This indicates that in some cases ultrasound measurements may under estimate the global reduction in LV hypertrophy. This study illustrates the need for use of different methods of cardiac hypertrophy assessment to better evaluate the cardiac remodeling changes after drug treatment.
Associated with the significant reduction in the cardiac hypertrophy after captopril or the combined therapy, the echocardiographic LV functional analysis revealed that the cardiac index significantly deteriorated at the end of the experimental period in the control SS/Mcwi and SS-16BN/Mcwi groups as compared with the pretreatment measurements. Captopril or combined treatment preserved the cardiac index in SS-16BN/Mcwi rats. The most significant effect was observed in the CMB group; however, no changes were seen in the stroke volume, cardiac output, or FS between the SS/Mcwi and SS-16BN/Mcwi groups.
Studies indicate that the increase in perivascular and interstitial myocardial fibrosis by aldosterone is one of the principal mechanisms responsible for the impairment in the cardiac function.42,43 Studies have shown that aldosterone regulates tissue inflammatory responses and stimulates cytokine secretion, fibroblast growth, and collagen turnover.44 These phenomena in turn lead to myocardial fibrosis and adverse ventricular remodeling. The analysis of myocardial fibrosis in SS/Mcwi and SS-16BN/Mcwi rats by Masson's Trichrome staining showed that there was a slight decrease in the percentage of LV fibrosis in the SS-16BN/Mcwi rats that received captopril, spironolactone, or the combined therapy as compared with the control group; however, the decreases did not reach statistical significance. Perhaps a reduction in myocardial fibrosis in these animals might be demonstrable if higher doses of spironolactone were used or if captopril or combined therapy with spironolactone were continued for a longer period of time. Our studies, however, have not been extended to investigate the effects of high doses of spironolactone on cardiac hypertrophy or fibrosis. A wide range of doses of spironolactone as well as the duration of the treatment have been reported in the literature. In the bulk of the studies, spironolactone dose normally varied from 5 to 80 mg/kg/d, although some studies have reported the use of a very high dose of 200 mg/kg/d.45 Although the high doses of spironolactone have been proved to be efficient in reducing heart fibrosis and hypertrophy in experimental studies,46,47 it is not applicable to the clinical settings due to the severe side effects, including hyperkalemia and sexual-hormone related disorders, which are difficult to assess in animals. In our study, we administered a 20 mg/kg/d dose of spironolactone for 4 weeks, which has been considered safe and efficacious in reducing heart fibrosis and hypertrophy in different animal models of heart hypertrophy48,49 and tested the combined effect with ACE inhibitors to evaluate the efficacy in reducing heart fibrosis and hypertrophy in the SSBN-16 rats.
Recently, 2 major clinical trials, the Randomized Aldactone Evaluation Study (RALES)7 and the Eplerenone Neurohormonal Efficacy and Survival Study (EPHESUS),50 have strongly supported the efficacy of low doses of aldosterone antagonists in combination with the current therapy (ACE inhibitors, β-blocker, and diuretics) for the treatment of myocardial failure and selected cases of hypertension. Our findings provide new insights into the pathophysiology of HCM and reveal how early intervention with aldosterone antagonist and ACE inhibitors could attenuate changes in cardiac remodeling in a consomic rat model of HCM.
The neuroendocrine activation, involving specifically the renin-angiotensin system, has been extensively implicated in the progression of cardiac fibrosis and LV remodeling.5,51,52 In this study, spironolactone alone did not promote significant changes in the neuroendocrine profile in either SS/Mcwi or SS-16BN/Mcwi rats. The aldosterone levels increased almost 2-fold in the spironolactone-treated SS-16BN/Mcwi rats (85.57 ± 20.08 vs. 44.52 ± 4.7; P < 0.09); however, due to the high variability found on this measurement, it did not reach the statistical significance established in our study (P < 0.05). Captopril treatment did decrease plasma levels of angiotensin II, aldosterone, and ANF in both SS/Mcwi and SS-16BN/Mcwi rats as compared with control. Other studies have also shown that ACE inhibitors improve LV loading conditions, remodeling, and neurohormonal activation and reduce heart hypertrophy.53Although we observed a significant reduction in heart hypertrophy after the combined therapy with spironolactone, the plasma levels of ANF, angiotensin II, and aldosterone did not change in either SS/Mcwi or SS-16BN/Mcwi rats. Plasma atrial natriuretic peptide level has been used as a reliable index of cardiac hypertrophy and decompensation in chronic heart failure.54-56 However, studies have shown that in some cases ANF levels do not correlate with symptoms or echocardiographically derived indices of LV structure and diastolic function.57-59 It is still not clear whether the interaction between spironolactone and captopril affects the neurohormonal system independently of the reduction in the cardiac hypertrophy. Additional experiments are necessary to investigate this effect.
This work was supported by National Institutes of Health Grant HL-29587. The authors thank Christine Puza for expert technical assistance, Jennifer Labecki for echocardiography assistance, and Dr. James Southern for histological examination.
1. Young MJ, Funder JW. Mineralocorticoid receptors and pathophysiological roles for aldosterone in the cardiovascular system. J Hypertens
2. Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium: fibrosis and renin-angiotensin-aldosterone system. Circulation
. 1991;83: 1849-1865.
3. Rocha R, Chander PN, Khanna K, et al. Mineralocorticoid blockade reduces vascular injury in stroke-prone hypertensive rats. Hypertension
4. Wang W. Chronic administration of aldosterone depresses baroreceptor reflex function in the dog. Hypertension
5. Brilla CG. The cardiac structure-function relationship and the renin-angiotensin-aldosterone system in hypertension and heart failure. Curr Opin Cardiol
6. Cecchi F, Olivotto I, Montereggi A, et al. Prognostic value of non-sustained ventricular tachycardia and the potential role of amiodarone treatment in hypertrophic cardiomyopathy
7. Pitt B, Zannad F, Remme WJ, et al. The effect of spironolactone
on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med
8. Krum H, Nolly H, Workman D, et al. Efficacy of eplerenone added to renin-angiotensin blockade in hypertensive patients. Hypertension
9. Cicoira M, Zanolla L, Franceschini L, et al. Relation of aldosterone “escape” despite angiotensin-converting enzyme inhibitor administration to impaired exercise capacity in chronic congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol
10. Tsybouleva N, Zhang L, Chen S, et al. Aldosterone, through novel signaling proteins, is a fundamental molecular bridge between the genetic defect and the cardiac phenotype of hypertrophic cardiomyopathy
11. Seidman CE, Seidman JG. Molecular genetic studies of familial hypertrophic cardiomyopathy
. Basic Res Cardiol
12. Maron BJ, Gardin JM, Flack JM, et al. Prevalence of hypertrophic cardiomyopathy
in a general population of young adults: echocardiographic analysis of 4111 subjects in the CARDIA Study. Circulation
13. Maron BJ, Shirani J, Poliac LC, et al. Sudden death in young competitive athletes-clinical, demographic and pathological profiles. JAMA
14. Semsarian C, Maron BJ. Sudden cardiac death in the young. Med J
15. Semsarian C, Richmond DR. Sudden cardiac death in familial hypertrophic cardiomyopathy
: an Australian experience. Aust NZ J Med
16. Maron BJ, Bonow RO, Cannon III RO, et al. Hypertrophic cardiomyopathy
: interrelation of clinical manifestations, pathophysiology, and therapy. N Engl J Med
17. McKenna WJ, Harris L, Perez G, et al. Arrhythmia in hypertrophic cardiomyopathy
. II: Comparison of amiodarone and verapamil in treatment. Br Heart J
18. McKenna WJ, Oakley CM, Krikler DM, et al. Improved survival with amiodarone in patients with hypertrophic cardiomyopathy
and ventricular tachycardia. Br Heart J
19. Ostman-Smith I, Wettrell G, Riesenfeld T. A cohort study of childhood hypertrophic cardiomyopathy
: improved survival following high-dose β-adrenoceptor antagonist treatment. J Am Coll Cardiol
20. Wyatt HL, Meerbaum S, Heng MK, et al. Crosssectional echocardiography. III. Analysis of mathematic models for quantifying volume of symmetric and asymmetric left ventricles. Am Heart J
. 1980;100: 821-828.
21. Rieder MJ, Roman RJ, Greene AS. Reversal of microvascular rarefaction and reduced renal mass hypertension. Hypertension
22. Raff H, Hong JJ, Oaks MK, et al. Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone, StAR, and PBR. Am J Physiol Regul Integr Comp Physiol
23. Perreault T, Baribeau J, Gosselin R, et al. Reduced vasodilator response to ANF in hypoxia-induced pulmonary hypertension in the newborn piglet. Am J Physiol
24. Kalkman EA, van Haren P, Saxena PR, et al. Early captopril prevents myocardial infarction-induced hypertrophy but not angiogenesis. Eur J Pharmacol
25. Schoemaker RG, Debets JJ, Struyker-Boudier HA, et al. Delayed but not immediate captopril therapy improves cardiac function in conscious rats, following myocardial infarction. J Mol Cell Cardiol
26. Qi XL, Stewart DJ, Gosselin H, et al. Improvement of endocardial and vascular endothelial function on myocardial performance by captopril treatment in postinfarct rat hearts. Circulation
27. Fraccarollo D, Galuppo P, Hildemann S, et al. Additive improvement of left ventricular remodeling and neurohormonal activation by aldosterone receptor blockade with eplerenone and ACE inhibition in rats with myocardial infarction. J Am Coll Cardiol
28. Kambara A, Holycross BJ, Wung P, et al. Combined effects of low-dose oral spironolactone
and captopril therapy in a rat model of spontaneous hypertension and heart failure. J Cardiovasc Pharmacol
29. Yoshiyama M, Omura T, Yoshikawa J. Additive improvement of left ventricular remodeling by aldosterone receptor blockade with eplerenone and angiotensin II type 1 receptor antagonist in rats with myocardial infarction. Nippon Yakurigaku Zasshi
30. Aharinejad S, Schafer R, Hofbauer R, et al. Impact of cardiac transplantation on molecular pathology of ET-1, VEGF-C, and mitochondrial metabolism and morphology in dilated versus ischemic cardiomyopathic patients. Transplantation
31. Nicholl SM, Bell D, Spiers J, et al. Neuropeptide Y Y(1) receptor regulates protein turnover and constitutive gene expression in hypertrophying cardiomyocytes. Eur J Pharmacol
32. Bell D, Allen AR, Kelso EJ, et al. Induction of hypertrophic responsiveness of cardiomyocytes to neuropeptide Y in response to pressure overload. J Pharmacol Exp Ther
33. Thattaliyath BD, Livi CB, Steinhelper ME, et al. HAND1 and HAND2 are expressed in the adult-rodent heart and are modulated during cardiac hypertrophy. Biochem Biophys Res Commun
34. Natarajan A, Yamagishi H, Ahmad F, et al. Human eHAND, but not dHAND, is down-regulated in cardiomyopathies. J Mol Cell Cardiol
35. Tanabe A, Naruse M, Hara Y, et al. Aldosterone antagonist facilitates the cardioprotective effects of angiotensin receptor blockers in hypertensive rats. 1: J Hypertens
36. Bauersachs J, Fraccarollo D, Ertl G, et al. Striking increase of natriuresis by low-dose spironolactone
in congestive heart failure only in combination with ACE inhibition: mechanistic evidence to support RALES. Circulation
37. Sato A, Hayashi K, Saruta T. Antiproteinuric effects of mineralocorticoid receptor blockade in patients with chronic renal disease. Am J Hypertens
38. Rossing K, Schjoedt KJ, Smidt UM, et al. Beneficial effects of adding spironolactone
to recommended antihypertensive treatment in diabetic nephropathy: a randomized, double-masked, cross-over study. Diabetes Care
39. Macdonald JE, Kennedy N, Struthers AD. Effects of spironolactone
on endothelial function, vascular angiotensin converting enzyme activity, and other prognostic markers in patients with mild heart failure already taking optimal treatment. Heart
40. Farquharson CA, Struthers AD. Spironolactone
increases nitric oxide bioactivity, improves endothelial vasodilator dysfunction, and suppresses vascular angiotensin I/angiotensin II conversion in patients with chronic heart failure. Circulation
41. MacFadyen RJ, Barr CS, Struthers AD. Aldosterone blockade reduces vascular collagen turnover, improves heart rate variability and reduces early morning rise in heart rate in heart failure patients. Cardiovasc Res
42. Brilla CG. Aldosterone and myocardial fibrosis in heart failure. Herz
43. Schmidt BM, Schmieder RE. Aldosterone-induced cardiac damage: focus on blood pressure independent effects. Am J Hypertens
44. Weber KT. Aldosterone in congestive heart failure. N Engl J Med
45. Brilla CG, Matsubara LS, Weber KT. Antifibrotic effects of spironolactone
in preventing myocardial fibrosis in systemic arterial hypertension. Am J Cardiol
46. Benetos A, Lacolley P, Safar ME. Prevention of aortic fibrosis by spironolactone
in spontaneously hypertensive rats. Arterioscler Thromb Vasc Biol
47. Lal A, Veinot JP, Leenen FH. Prevention of high salt diet-induced cardiac hypertrophy and fibrosis by spironolactone
. Am J Hypertens
48. Goineau S, Pape D, Guillo P, et al. Combined effects of metoprolol and spironolactone
in dilated cardiomyopathic hamsters. J Cardiovasc Pharmacol
49. Silvestre JS, Heymes C, Oubenaissa A, et al. Activation of cardiac aldosterone production in rat myocardial infarction: effect of angiotensin II receptor blockade and role in cardiac fibrosis. Circulation
50. Pitt B, Remme W, Zannad F, et al. Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study Investigators. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med
51. Dzau VJ. Contributions of neuroendocrine and local autocrine-paracrine mechanisms to the pathophysiology and pharmacology of congestive heart failure. Am J Cardiol
52. Francis GS, Benedict C, Johnstone DE, et al. Comparison of neuroendocrine activation in patients with left ventricular dysfunction with and without congestive heart failure. A substudy of the Studies of Left Ventricular Dysfunction (SOLVD). Circulation
53. Khalil ME, Basher AW, Brown EJ Jr, et al. A remarkable medical story: benefits of angiotensin-converting enzyme inhibitors in cardiac patients. J Am Coll Cardiol
54. Francis GS. Neuroendocrine activity in congestive heart failure. Am J Cardiol
55. Nakaoka H, Imataka K, Amano M, et al. Plasma levels of atrial natriuretic factor in patients with congestive heart failure. N Engl J Med
56. Yasumoto K, Takata M, Ueno H, et al. Relation of plasma brain and atrial natriuretic peptides to left ventricular geometric patterns in essential hypertension. Am J Hypertens
57. Fahy GJ, McCreery CJ, O'Sullivan F, et al. Plasma atrial natriuretic peptide is elevated in patients with hypertrophic cardiomyopathy
. Int J Cardiol
58. Tomoda H. [Relations of intracardiac dimensions as measured by echocardiography and plasma atrial natriuretic peptide levels in various cardiovascular diseases]. J Cardiol
59. Pawlicki L, Irzmanski R, Rozalski S, et al. Correlation between left ventricular mass and the resting and post-exercise release of ANP in healthy men. Med Sci Monit