Effects of diuretic treatment on cardiac and circulating RASin chronic heart failure post-myocardial infarction in rats

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1 The European Journal of Heart Failure 5 (2003) Effects of diuretic treatment on cardiac and circulating RASin chronic heart failure post-myocardial infarction in rats a,1 b,1 c a d d Ute Seeland, Ichiro Kouchi, Oliver Zolk, Friedrich Jockenhovel, Gabi Itter, Wolfgang Linz, Michael Bohm a, * amed. Klinik und Poliklinik der Universitat des Saarlandes, Innere Medizin III, Kardiologie und Angiologie, HomburgySaar, Germany b Kinki University Nara Hospital, Cardiovascular Medicine, Otoda, Ikoma, Japan cinstitut fur Experimentelle und Klinische Pharmakologie und Toxikologie Universitat Erlangen-Nurnberg, Erlangen-Nurnberg, Germany d Aventis Pharma, DG Cardiovascular Research, FrankfurtyMain, Germany Abstract Received 27 March 2002; received in revised form 30 August 2002; accepted 2 September 2002 Background: Cardiac angiotensin converting enzyme (ACE) is activated by an increase in wall stress and is involved in remodeling processes. Heart failure is often treated with ACE inhibitors and diuretics although diuretic treatment could activate the renin angiotensin system (RAS). Aims: To examine the effects of diuretic treatment on cardiac and circulating RASin postinfarction chronic heart failure. Methods: Myocardial infarction was produced by coronary artery ligation in spontaneously hypertensive rats. The rats were randomly assigned to receive either ramipril (1 mgykgyday), furosemide (4 mgykgyday), or combination therapy for 6 weeks, commencing 2 weeks after infarction. Results: All three treatment protocols euivalently attenuated reactive hypertrophy of the right ventricle and ventricular septum and improved left ventricular systolic function. Both cardiac ACE mrna and activity were significantly increased in untreated rats. This increase was attenuated by both ramipril and furosemide and further depressed by the combination. The increase in activity was completely inhibited by either agent alone. Plasma renin activity was upregulated by ramipril or ramipril plus furosemide but not influenced by infarction or furosemide alone. Conclusions: Furosemide and ramipril significantly reduced cardiac ACE and remodeling. Diuretics work favorably and do not interfere with the effects of ACE inhibitors. Possibly, a reduction in wall stress due to decreased volume overload accounts for the effects of diuretics on cardiac ACE in the treatment of post-infarction remodeling in hypertensive hearts. These data suggest a new mechanism for the freuently observed beneficial effect of diuretics in heart failure European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved. Keywords: 1. Introduction Renin angiotensin system; Remodeling; Heart failure; ACE inhibitors; Diuretics Ventricular remodeling post-myocardial infarction is regarded as an initial step in the development of heart failure w1x. Accumulated evidence shows that renin angiotensin system (RAS) activation plays an important role in the remodeling process induced by pressure overload w2x or myocardial infarction w3x. Angiotensin Abbreviations: SHR, spontaneously hypertensive rats; RAS, renin angiotensin system; LV, left ventricle or left ventricular. *Corresponding author. Tel.: ; fax: addresses: boehm@med-in.uni-sb.de (M. Bohm), seeland@med-in.uni-sb.de (U. Seeland). 1 Contributed eually to the article. converting enzyme (ACE) inhibitors are effective in preventing remodeling in experimental models w4x and improve clinical outcome of patients with severe heart failure w5x. Loop diuretics are often used in combination with ACE inhibitors in the treatment of heart failure to prevent volume overload, although clinical trials to show their favorable effects on mortality are lacking. In addition, little information is available about the effects of diuretics on the remodeling process. Since it has been suggested that diuretics activate the RAS w6x their use could offset the favorable effects of ACE inhibitors when they are co-administered. The present study was conducted to examine the effects of loop diuretic treat /03/$ European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved. doi: /s ž

2 242 U. Seeland et al. / The European Journal of Heart Failure 5 (2003) Table 1 Infarct size, heart weight and hemodynamic data Sham C R F RyF n Infarct size (% oflv) 29"3 27"2 26"3 24"2 ** LV weight (g) 1.08" "0.03 * 1.17" " "0.03 ** RV weight (g) 0.20" "0.04 * 0.25"0.03 ** 0.24"0.02 ** 0.24"0.01 ** Body weight (g) 370" " " "7.96 ** "6.26 ** MAP (mmhg) 156"8 139"10 126"10 * 142"16 100"9 *,** Heart rate, (bpm) 386"9 338"14 * 334"33 368"12 309"21 * Mortality (%) ** ** Sham indicates sham-operated; C, untreated myocardial infarction; R, infarction treated with ramipril; F, infarction treated with furosemide; Ry F, infarction treated with furosemide and ramipril together; n, number of rats; LV, left ventricle; RV, right ventricle; MAP, mean arterial pressure. Values are expressed as mean"s.e.m. * P-0.05 vs. sham. ** P-0.05 vs. C. ment on remodeling and ACE inhibition in spontaneously hypertensive rats (SHR) with severe heart failure following myocardial infarction w7x. 2. Methods 2.1. Post-infarction remodeling model All experiments conform with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No , revised 1996). Severe chronic heart failure was induced in 16-week-old 110 male SHR, weighing g. The rats were anesthetized with intraperitoneal injection of ketamine (35 mgykg) plus xylazine (2 mgy kg) and mechanically ventilated. Left thoracotomy was performed and myocardial infarction was induced by permanent ligation of the left coronary artery at 2 3 mm beneath the origin. Sham operation consisted of the identical procedure without coronary ligation. Mortality within 24 h was less than 10% using xylocaine before and furosemide 3 days after surgery. Rats with myocardial infarction (20 40% determined by planimetry) were randomized into four groups as follows: (1) untreated (Cscontrol), (2) treated with ramipril (R; 1 mgykgyday), (3) treated with furosemide (F, 4 mgy kgyday), (4) treated with ramipril and furosemide together (RyF; 1 and 4 mgykgyday, respectively). All agents were administered via drinking water for 6 weeks, starting 2 weeks after coronary ligation. Body weight increased significantly following myocardial infarction (8 weeks) due to congestive heart failure (% related to body weight measured at the beginning of the study: sham 10.9"1.1, C 17.4"3; P-0.05) and decreased following treatment (Table 1). General symptoms of the SHR with chronic heart failure are impaired motion, dyspnoea and subcutaneous edema. Furthermore, nuclear magnetic resonance images show dilated left ventricle (LV) and pleura effusion in post-myocardial infarction SHR with severe congestive heart failure. Eight weeks after operation, the rats were reanesthetized and the right carotid artery was cannulated with a polyethylene catheter to monitor blood pressure. Blood samples were collected through the catheter for plasma renin activity measurement using a specific radioimmunoassay kit (GammaCoat, DiaSorin Inc.). The hearts were excised and mounted on Langendorff apparatus. The isolated heart was perfused in the working heart mode with a filling pressure of 15 mmhg and an afterload pressure of 60 mmhg as described previously w8x. After the evaluation of the external heart work, the heart was weighed and the LV was sectioned transversely into four slices. Photographs were taken from each slice and the tissues were stored at y80 8C. The area of infarction and the thickness of the ventricular septum were determined by planimetry Northern blot analysis of cardiac ACE Total RNA was isolated from noninfarcted LV myocardium and analyzed as described previously w9x. Twenty micrograms of total RNA was separated by gel electrophoresis, blotted onto nylon filters, and fixed by UV cross-linking. The blots were hybridized with a 32 random-primed P-radiolabeled cdna probe for ACE. The signals on the autoradiographs were uantified densitometrically. GAPDH was used as an internal control to normalize for differences in loading of RNA Cardiac ACE activity ACE activity was determined according to Cushman and Cheung w10x. Noninfarcted LV tissue was homogenized using a glass Teflon homogenizer in ice-cold potassium phosphate buffer. The homogenate was centrifuged at 600=g for 10 min and the resulting supernatant was centrifuged at =g for 60 min. The final pellet was resuspended in the same buffer and incubated at 37 8C for 2 h with 12 mmolyl Hip His Leu as substrate in 0.1 molyl potassium phosphate buffer

3 U. Seeland et al. / The European Journal of Heart Failure 5 (2003) Fig. 1. Thickness of ventricular septum determined in SHR with sham operation (sham), untreated infarction (Cscontrol), infarction treated with ramipril (R), infarction treated with furosemide (F), infarction treated with R and F together (RyF). ns10 15 each. *P-0.05 vs. Sham, P-0.05 vs. C. (ph 8.3) containing 0.3 molyl sodium chloride. A second set of reaction mixture was preincubated with 10 mmolyl enalapril to define specific ACE activity. Reaction was terminated by addition of 1 N HCl. The hippuric acid produced by the reaction was extracted with ethyl acetate and measured with a spectrophotometer at 280 nm Statistical analysis All data are described as mean"s.e.m. Statistical significance was estimated with Student s t-test for unpaired observations or a one-way ANOVA with Fisher s least significant difference as the post hoc test. A value of P-0.05 was considered significant. 3. Results 3.1. Structural and functional alterations Ventricular remodeling-related parameters are summarised in Table 1 and Fig. 1. In rats with untreated infarction, reactive hypertrophy was observed in the noninfarcted right ventricle. Right ventricular heart weight increased significantly in SHR with heart failure (C) compared to sham and decreased following treatment. Marked thinning of the infarcted LV free wall was measured because of large infarcted areas (20 40% of LV mass). LV peak positive dpydt was diminished in untreated animals and significantly (P-0.05) improved in animals treated with either protocol (Fig. 2) Cardiac ACE mrna and activity There were significant increases in both ACE mrna (3.9-fold) and activity (2.5-fold) levels in untreated remodeling hearts compared with sham-operated hearts (Fig. 3a and Fig. 4a). Linear regression analysis indicated a close correlation between infarct size and ACE mrna (rs0.77, P-0.01) or activity (rs0.89, P- 0.01) (Fig. 3b and Fig. 4b). The increase in ACE mrna induced by infarction was partially inhibited by either ramipril or furosemide alone and depressed by the combined treatment (Fig. 3a). The increase in ACE activity was completely blocked by all three treatment protocols to a level similar to the sham-operated hearts (Fig. 4a). There were no differences between ramipril and furosemide in the inhibitory effects on cardiac ACE mrna and activity Plasma renin activity There were no significant differences in plasma renin activity among sham-operated, untreated infarction and furosemide-treated groups (Fig. 5). However, the activity was raised by ramipril and further augmented by the concomitant administration of furosemide Mortality The 35-day mortality rate was reduced in rats treated with ramipril alone and in combination with furosemide. 4. Discussion We have shown that both the loop diuretic and the ACE inhibitor reversed the reactive hypertrophy and cardiac ACE activation induced by myocardial infarction in hypertensive rats. These effects were euivalent and enhanced rather than blunted by combined administration. The presented data suggest that diuretic treatment is beneficial to cardiac ACE inhibition in the treatment Fig. 2. Myocardial contractility measured by LV peak positive pressure (LVdpydt) in the same animal groups as in Fig. 1. ns10 15 each. *P-0.05 vs. Sham, P-0.05 vs. C.

4 244 U. Seeland et al. / The European Journal of Heart Failure 5 (2003) Fig. 3. (a) Representative autoradiograms and mean values of Northern blot analysis of LV ACE mrna from SHR in the same animal groups as in Fig. 1. GAPDH was used as an internal control. ns10 15 each. *P-0.05 vs. Sham, P-0.05 vs. C; (b) Linear regression analysis of infarct size (%) and ACEyGAPDH mrna (OD): ns10 of controls. rs0.77, P of post-infarction remodeling in rats. It has been well demonstrated that local rather than circulating RASis important in the pathogenesis of ventricular remodeling and subseuent heart failure development w11x. In our remodeling model of SHR, as shown in the standard remodeling model w3x, cardiac ACE expression was increased in proportion to infarct size which correlates with the degree of ventricular remodeling and dysfunction w12x. Although diuretics are widely used in the treatment of heart failure, surprisingly there are no studies evaluating their influence on cardiac RAS. In general, diuretics stimulate circulating RASand the humoral interaction leads to stimulation of the sympathetic nervous system w6x, whereas ACE inhibitors inactivate both systems. Diuretics, in this context, have been argued to counteract the effects of ACE inhibitors. Fig. 4. (a) LV ACE-activity measured in the same animal groups as in Fig. 1; (b) Linear regression analysis of infarct size (%) and ACE activity (mmolyminymg protein) ns7 of controls. rs0.89, P Fig. 5. Plasma renin activity (ngymlyh) measured in the same animal groups as in Fig. 1. ns10 15 each, *P-0.05 vs. Sham, P-0.05 vs. C.

5 U. Seeland et al. / The European Journal of Heart Failure 5 (2003) In the present study, circulating plasma renin activity was induced by ramipril treatment alone and further increased in the combined treatment group with furosemide. At doses which effectively reduced edema formation in this model, furosemide did not increase plasma renin activity and effectively reduced cardiac ACE activity. In contrast to the stimulatory effects of ramipril and furosemide on circulating RAS, both drugs similarly demonstrated inhibitory effects on cardiac ACE activity. It should be noted that alterations in cardiac ACE correlated with a reduction in septum thickness and right ventricular weights. Therefore, the present results demonstrate that inhibition of cardiac RASby ramipril and furosemide, is important for the remodeling process, especially based on hypertrophic mechanisms. Hypertrophy of myocytes is stimulated by neurohormonal factors, in which locally produced angiotensin II is involved w13x. This process often leads to mechanical deterioration. A regression or prevention of reactive hypertrophy by RASinhibition is generally associated with an improvement in ventricular performance. It has been demonstrated that hypertensive hearts with preexisting hypertrophy undergo more intense remodeling after infarction compared with normal hearts w7,14x. A previous hemodynamic study has shown that SHR with infarction benefit from ACE inhibitors w14x. In agreement with this, our 35-day mortality rate was significantly reduced in the groups treated with ramipril. This reduction in mortality rate was not apparent following furosemide treatment alone, despite the reduction in cardiac ACE, presumably because of increased heart rate due to sympathetic activation. Hypertrophy is also stimulated by mechanical load, therefore it is difficult to determine whether the beneficial effects of ACE inhibitors are mainly attributed to the suppression of RASor reduction of afterload. However, furosemide as well as ramipril improved major parameters related to remodeling. The reduction of right ventricular weights, data reflecting LV filling pressure as a reliable index of the overloading state, suggest that the unloading effect is comparable with either treatment. An explanation for the similar effects of diuretics and ACE inhibitors is that mechanical load, independent from circulating RAS, should be a primary determinant of activation of cardiac RASand thus progression of remodeling. The increase in ventricular wall stress can be a direct stimulus for local RASactivation. Therefore, diuretic treatment could reduce wall stress by preventing volume overload, thereby inhibiting cardiac ACE activation. However, the slightly more pronounced reduction in blood pressure in the combination RyF group cannot be excluded. There was a variation in infarct size, depending on the place of ligation, and therefore it has to be considered that mean infarct size in the RyF group was smaller compared with the control (C) group. Based upon this fact, a positive influence on survival in the RyF group cannot be ruled out. On the other hand the positive remodeling results may be dependant on two mechanisms; first by reducing cardiac ACE through direct inhibition of local ACE activity by ramipril, second by unloading with reduction of wall stress by furosemide. Nevertheless, it is important that furosemide did not worsen the beneficial effects of ramipril on cardiac ACE, regression of hypertrophy and hemodynamics. Interestingly the recently published LIVE study w15x compared the efficacy of enalapril and indapamide (a diuretic) in the reduction of LV mass index in 411 patients with LV hypertrophy. The LIVE results showed regression of hypertrophy by indapamide, which was even more pronounced than enalapril in LV mass index reduction after 12 months treatment. These findings in patients are in line with the experimental results reported herein. Diuretics can therefore be proposed as an effective treatment for hypertension, reducing myocardial hypertrophy without blunting the beneficial effects of ramipril when administered as combined treatment, as shown in hypertensive rats with post-infarction remodeling. The main benefit appears to result from hemodynamic unloading-dependent ACE activation in the heart in post-infarction remodeling of hypertensive rats. Impairment of the effects of ACE inhibitors by the coadministration of loop diuretics is unlikely. These findings shed new light on the myocardial effects of diuretics and suggest mechanisms for the beneficial clinical effects of diuretics in cardiac overload as shown in the LIVE study. 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