Regulation of extracellular matrix proteins in pressure-overload cardiac hypertrophy: effects of angiotensin converting enzyme inhibition.

OBJECTIVE: Left ventricular hypertrophy (LVH) is characterized by remodeling of both myocyte and interstitial compartments of the heart. The aim of this investigation was to study the effects of angiotensin converting enzyme (ACE) inhibition on alterations in the composition of the interstitium in chronic pressure-overload hypertrophy. DESIGN: LVH was induced in weanling rats by banding the ascending aorta. Animals with aortic banding received either vehicle (n = 20), hydralazine (20 mg/kg per day, n = 20), or the ACE inhibitor ramipril (10 mg/kg per day, n = 20) during weeks 6-12 after banding. RESULTS: Compared with sham-operated, untreated rats (n = 20), aortic-banded vehicle and hydralazine-treated rats displayed substantially increased left ventricular weights and myocyte diameters whereas ramipril significantly blunted the hypertrophic response at the myocyte level (each P < 0.001) as well as the increase in left ventricular weight (each P < 0.01). In addition, image analysis revealed a significant induction of perivascular and interstitial tissue accumulation in vehicle- and hydralazine-treated rats (2.5-fold, each P < 0.0001). In contrast, ramipril-treated rats displayed attenuated interstitial and perivascular fibrosis, both being significantly diminished compared with vehicle- and hydralazine-treated rats (each P< 0.001). Further, vehicle- and hydralazine-treated rats were characterized by elevated steady-state messenger (m)RNA levels of fibronectin (2.7- and 2.8-fold, P< 0.005), collagen I (2.0- and 1.8-fold, P < 0.0005), collagen III (both 2.2-fold, P < 0.001) and laminin B (1.6- and 1.6-fold, P < 0.005). In parallel, the corresponding immunohistochemical signals were markedly enhanced in these groups. In comparison, ramipril significantly blunted the induction of collagen I and III, laminin B and fibronectin at both the mRNA and protein levels. These morphological and molecular differences between the hydralazine and ramipril groups could not be attributed to differences in left ventricular-pressures, which were markedly elevated in all aortic stenosis rats (1.9-fold, each P < 0.001 versus sham). In fact, given that ramipril but not hydralazine blunted the hypertrophic response to pressure overload, the echocardiographic measurements revealed that left ventricular systolic wall stress was higher in the ramipril group (70 +/- 1 versus 34 +/- 0.7 kdyn/cm2; P < 0.02). CONCLUSIONS: ACE inhibition may limit both myocyte and interstitial remodeling despite ongoing cardiac pressure overload.

Reciprocal regulation of pulmonary and cardiac angiotensin-converting enzyme in rats with severe left ventricular hypertrophy.

OBJECTIVE: Numerous studies support the concept that cardiac angiotensin-converting enzyme (ACE) is involved in the pathophysiology of left ventricular hypertrophy. However, the pulmonary vasculature is considered to be the most prominent site of ACE expression. We thus examined the tissue specificity of ACE regulation in rats with severe cardiac pressure overload hypertrophy in transition to cardiac failure with secondary pulmonary hypertension. METHODS AND RESULTS: Rats were studied 12 weeks after banding of the ascending aorta (LVH, n = 20) that resulted in a 1.7-fold increase in left ventricular (LV) to body weight ratio. In addition, as compared to sham-operated rats (n = 20), we observed in LVH rats a 1.6-fold increase in right ventricular (RV) to body weight ratio, the development of pulmonary hypertension, and elevated plasma renin activities. Moreover, ACE mRNA and activity levels were more than 2-fold higher in both hypertrophied ventricles (P < 0.01, each). In contrast, pulmonary ACE mRNA and activity levels were markedly decreased in animals with LVH (more than 30%, respectively, P < 0.05 vs. sham), Interestingly, LV and RV ACE activity, as well as systolic pulmonary artery pressure and plasma renin activity, were all inversely related to pulmonary ACE activity. In order to differentiate the potential role of elevated renin in the down-regulation of pulmonary ACE, additional rats (n = 12) were treated with furosemide that resulted in a 8-fold rise in plasma renin activity, but only in a marginal decrease of pulmonary ACE mRNA levels and activity (-10% vs. sham (n = 8), P-value n.s.). CONCLUSIONS: The data indicate tissue specific reciprocal regulation of pulmonary and cardiac ACE in rats with cardiac pressure overload hypertrophy and pulmonary hypertension, a phenomenon that may potentially result in a partial shift of angiotensin II formation from the pulmonary to the cardiac circulation.

Development of heart failure following isoproterenol administration in the rat: role of the renin-angiotensin system.

OBJECTIVE: High dosages of catecholamines induce cardiomyocyte necrosis and interstitial fibrosis in rats. We investigated whether this initial damage is followed by the development of heart failure and assessed the particular role of the renin-angiotensin system using ramipril. METHODS AND RESULTS: Following the administration of 0 mg or 150 mg isoproterenol/kg 6 groups of Wistar rats were followed for 2 or 16 weeks: Sham, isoproterenol, isoproterenol + ramipril. Isoproterenol induced significant increases of echocardiographically measured left ventricular end-diastolic posterior wall thickness and dimension, whereas ramipril treatment significantly attenuated these changes. Left ventricular end-diastolic pressure was markedly increased in isoproterenol-treated rats and normalized following ramipril. Isoproterenol rats were further characterized by hormonal activations including transient elevations of plasma renin activity, aldosterone and cardiac angiotensin converting enzyme activity. Histomorphological characterization of isoproterenol-treated hearts demonstrated cardiomyocyte necrosis and reparative fibrosis. Ramipril treatment only slightly reduced the amount of necrosis as well as the expression of extracellular matrix proteins. CONCLUSIONS: In rats, a toxic dosage of isoproterenol caused characteristic myocardial damage that subsequently resulted in mild heart failure. Ramipril administration following isoproterenol was highly effective to attenuate hemodynamic and hormonal alterations as well as the development of left ventricular hypertrophy, but had only little influence on the expression of extracellular matrix proteins. Since angiotensin converting enzyme inhibition had no impact on the initial myocardial injury, the development of heart failure in this model seems to require functional integrity of the renin-angiotensin system.

Functional activity and expression of the myocardial postreceptor adenylyl cyclase system in pressure overload hypertrophy in rat.

OBJECTIVE: The aim of the present study was to investigate the functional regulation of the myocardial postreceptor adenylyl cyclase (AC) system in compensated left ventricular hypertrophy (LVH) and the effect of long-term angiotensin converting enzyme (ACE) inhibition. METHODS: Pressure overload LVH was induced in rats by supravalvular aortic banding for 12 weeks. At 12 weeks left ventricular function and inner diameters were analyzed by echocardiography of anesthetized animals, and responsiveness to forskolin (systolic developed pressure) was determined in isolated perfused hearts. Functional activities of AC and the stimulatory G protein Gs were measured as well as mRNA expression (quantitative slot blot analyses) of AC type V, isoforms of Gs alpha and Gi alpha 2. G protein alpha-subunits were also quantified by immunoblotting. Rats were treated with ramipril (Ram, 10 mg/kg per day p.o.) during weeks 7 to 12 to induce regression of LVH or with vehicle (Veh, tap water). RESULTS: Pressure overload induced severe LVH (3.2 +/- 0.09 g/kg in Veh vs. 1.8 +/- 0.03 in sham; P < 0.05) which was significantly reduced by ramipril (2.7 +/- 0.09; P < 0.05 vs. Veh). In-vivo left ventricular function and diameters were unchanged in LVH. In contrast, in hearts with LVH, responsiveness of left ventricles to forskolin was attenuated and basal, GTP gamma S and forskolin as well as manganese chloride-stimulated adenylyl cyclase activity was significantly downregulated by approximately 40% (basal 20.8 +/- 1.9 pmol cAMP/mg per min vs. 34.0 +/- 2.2 in sham; P < 0.01). However, no significant changes of AC type V mRNA were found in hypertrophied left ventricles. Functional activity of the stimulatory G protein Gs was reduced in LVH (48 +/- 7 pmol cAMP/mg per min in Veh vs. 68 +/- 3 in sham), whereas mRNA expression of long and short Gs alpha-isoforms was not altered and that of Gi alpha 2 was only slightly increased in ramipril-treated animals. Western analysis showed no significant differences of Gs alpha or Gi alpha 2 subunits. Long-term blockade of the renin-angiotensin system had no effect on the activity of the adenylyl cyclase system. CONCLUSIONS: Functional desensitization of adenylyl cyclase and stimulatory G protein occurred in rat adaptive LVH prior to the onset of severe left ventricular dysfunction which was not restored by ACE-inhibitor treatment. The desensitization seems not to be mediated by significant changes of mRNA expression of AC type V or abundance of regulatory G proteins.

Angiotensin II receptor gene expression in hypertrophied left ventricles of rat hearts.

OBJECTIVE: To examine the expression of angiotensin II AT1a, AT1b and AT2 receptor genes in the left ventricles of rats subjected to ventricular pressure overloading induced by aortic banding for 6 weeks and then 6 weeks medical treatment. RESULTS: Aortic banding was related to an increase in relative weight of the left ventricle from 1.73 +/- 0.06 (sham-operated) to 2.81 +/- 0.25 g/kg, an increase in beta-myosin: alpha-myosin messenger RNA (mRNA) ratio from 0.30 +/- 0.02 to 1.94 +/- 0.55 and an 18-fold increase in left ventricular atrial natriuretic peptide mRNA levels. In contrast, left ventricular pressure overload hypertrophy was not related to a significant change in the abundance of AT1a and AT1b mRNA, which were expressed in a relative ratio of 5:1. Similarly, the abundance of AT2 mRNA was not significantly changed in hypertrophied ventricles. In rats receiving the angiotensin II AT, receptor antagonist losartan (40 mg/kg) for 6 weeks after banding, relative heart weights were 2.39 +/- 0.14 g/kg, the beta-myosin: alpha-myosin ratio was 1.04 +/- 0.20 and atrial natriuretic peptide mRNA levels displayed a blunted increase (11-fold over sham-treated controls), documenting a significant amelioration of left ventricular hypertrophy by blockade of the AT1 receptor. CONCLUSION: Losartan treatment in parallel did not affect AT1a, AT1b and AT2 receptor mRNA levels, which were not different from those in vehicle-treated or sham-treated controls. These findings confirm that left ventricular hypertrophy in the rat is associated with increased ventricular expression of beta-myosin and of atrial natriuretic peptide and with reduced expression of alpha-myosin. Despite these significant changes in cardiac gene expression no alteration was observed in AT1a, AT1b and AT2 receptor mRNA levels.

Alteration of growth responses in established cardiac pressure overload hypertrophy in rats with aortic banding.

We examined the acute effects of elevated wall stress, norepinephrine, and angiotensin II on cardiac protein synthesis as well as protooncogene expression in hearts with established pressure overload left ventricular hypertrophy. Isolated rat hearts with chronic hypertrophy (LVH) were studied 12 wk after ascending aortic banding when systolic function was fully maintained. New protein synthesis (incorporation of [3H]phenylalanine [Phe]) was analyzed in isolated perfused rat hearts after a 3-h protocol; c-fos, c-jun, c-myc, and early growth response gene-1 (EGR-1) mRNA levels (Northern blot) were studied over a time course from 15 to 240 min of perfusion. Under baseline conditions (i.e., before mechanical or neurohormonal stimulation), [3H]-Phe-incorporation (280 nmoles/gram protein/h) and protooncogene mRNA levels were similar in age-matched control and LVH hearts. However, hearts with chronic LVH were characterized by a markedly blunted or absent [3H]-Phe-incorporation after acute imposition of isovolumic systolic load (90 mmHg/gram left ventricle), as well as norepinephrine (10(-6)M), or angiotensin II infusion (10(-8)M plus prazosin 10(-7)M) compared with nonhypertrophied control hearts. Similarly, stimulation of LVH hearts with acute systolic load or norepinephrine was associated with a significantly blunted increase of protooncogene mRNA levels relative to control hearts. The blunted induction of c-fos mRNA in LVH hearts was not due to feedback inhibition, since cycloheximide perfusion of hearts exposed to elevated wall stress further increased the differences between age-matched control and LVH hearts. The data suggest that acute molecular growth responses to mechanical or neurohormonal stimulation are altered in rat hearts with established LVH relative to nonhypertrophied control hearts. This alteration of molecular adaptations in hearts with compensatory hypertrophy may prevent inappropriate excess cardiac growth in response to mechanical and neurohormonal stimuli.

Blockade of the renin-angiotensin system in cardiac pressure-overload hypertrophy in rats.

Left ventricular hypertrophy in response to pressure overload may be modified by neurohumoral activation. To investigate the contribution of the renin-angiotensin system, we studied rats after banding of the ascending aorta that developed severe left ventricular hypertrophy associated with normal plasma renin but elevated cardiac angiotensin-converting enzyme (ACE) levels. Rats were treated with vehicle, ACE inhibitor (ramipril), angiotensin II type 1 receptor antagonist (losartan), or vasodilator (hydralazine) during weeks 7 through 12 after aortic banding. A significant regression of left ventricular mass index as determined by serial echocardiography was observed in ramipril- and losartan-treated groups during weeks 9 through 12 after banding, whereas hypertrophy further increased in vehicle- and hydralazine-treated groups. Twelve weeks after banding, relative left ventricular weights and myocyte widths were markedly increased in vehicle- and hydralazine-treated groups, whereas ramipril and losartan significantly reduced these parameters. In addition, molecular adaptations in left ventricular hypertrophy, such as upregulation of left ventricular atrial natriuretic peptide and downregulation of sarcoplasmic reticulum Ca(2+)-ATPase mRNA levels, were blunted by ramipril or losartan treatment. Hypertrophic regression was associated with reduced mortality in rats treated with ramipril (11%) and losartan (13%) versus hydralazine (20%) and vehicle (31%). Thus, the renin-angiotensin system may be involved in the maintenance of chronic left ventricular hypertrophy. Blockade of the system may result in regression of the hypertrophic phenotype and improve survival in rats despite persistent pressure overload.