Effects of Dopamine β-Hydroxylase Inhibition With Nepicastat on the Progression of Left Ventricular Dysfunction and Remodeling in Dogs With Chronic Heart Failure
Background—Inhibition of dopamine β-hydroxylase (DBH) results in a decrease in norepinephrine synthesis. The present study was a randomized, blinded, placebo-controlled investigation of the long-term effects of therapy with the DBH inhibitor nepicastat (NCT) on the progression of left ventricular (LV) dysfunction and remodeling in dogs with chronic heart failure (HF).
Methods and Results—Moderate HF (LV ejection fraction [LVEF] 30% to 40%) was produced in 30 dogs by intracoronary microembolization. Dogs were randomized to low-dose NCT (0.5 mg/kg twice daily, n=7) (L-NCT), high-dose NCT (2 mg/kg twice daily, n=7) (H-NCT), L-NCT plus enalapril (10 mg twice daily, n=8) (L-NCT+ENA), or placebo (PL, n=8). Transmyocardial (coronary sinus–arterial) plasma norepinephrine (tNEPI), LVEF, end-systolic volume, and end-diastolic volume were measured before and 3 months after initiating therapy. tNEPI levels were higher in PL compared with NL (86±20 versus 13±14 pg/mL, P<0.01). L-NCT alone and L-NCT+ENA reduced tNEPI toward normal (28±4 and 39±17 pg/mL respectively), whereas HD-NCT reduced tNEPI to below normal levels (3±10 pg/mL). In PL dogs, LVEF decreased but was unchanged with L-NCT and increased with L-NCT+ENA. L-NCT and L-NCT+ENA prevented progressive LV remodeling, as evidenced by lack of ongoing increase in end-diastolic volume and end-systolic volume, whereas H-NCT did not
Conclusions—In dogs with HF, therapy with L-NCT prevented progressive LV dysfunction and remodeling. The addition of ENA to L-NCT afforded a greater increase in LV systolic function. NCT at doses that normalize tNEPI may be useful in the treatment of chronic HF.
Blockade of the renin-angiotensin and sympathetic nervous systems in heart failure (HF) elicits beneficial effects that underscore their importance in the pathogenesis of the disease and its progression.1 2 Enhanced sympathetic nervous system activity is manifested by increased plasma norepinephrine, whereas that of the renin-angiotensin system is manifested by increased circulating angiotensin-II. Modulation of these neurohormones with ACE inhibitors and β-adrenergic receptor blockers results in improvement in the clinical signs and symptoms of HF, a decrease mortality and morbidity,3 4 5 and modulation of ventricular remodeling,6 7 a global and cellular process deemed important in the development of and progression of HF.8
One possible approach aimed at directly countering the adverse effects of enhanced sympathetic drive in HF is inhibition of the biosynthesis and release of norepinephrine. This can be achieved by inhibiting dopamine β-hydroxylase (DBH), an enzyme that converts dopamine to norepinephrine within sympathetic nerve vesicles.9 The reduction in norepinephrine results in decreased stimulation of β1- and β2-adrenergic receptors and α1 receptors.10 At the same time, DBH inhibition will increase dopamine levels in the nerve terminal, which can have a beneficial effect on renal function in HF, and can act on prejunctional D1 receptors to inhibit norepinephrine release.10 The recent development of nepicastat (NCT), a novel and potent DBH inhibitor with a high degree of selectivity,11 has made it possible to study the efficacy of this therapeutic approach in HF. The purpose of the present study was to examine the effects of chronic therapy with NCT on the progression of left ventricular (LV) dysfunction and remodeling in dogs with HF. Because ACE inhibition is standard therapy in the treatment of HF, we also assessed the efficacy of NCT when combined with an ACE inhibitor.
The canine model of chronic HF used in this study was previously described in detail.12 In this preparation, HF is produced by multiple sequential intracoronary embolizations with polystyrene Latex microspheres (70 to 102 μm in diameter), which results in loss of viable myocardium. Thirty healthy mongrel dogs, weighing between 20 to 25 kg, underwent coronary microembolizations to produce HF. Embolizations were performed 1 to 3 weeks apart and were discontinued when LV ejection fraction, determined angiographically, was between 30% and 40%. Microembolizations were performed during cardiac catheterization under general anesthesia and sterile conditions. Anesthesia consisted of a combination of intravenous injection of oxymorphone (0.22 mg/kg), diazepam (0.17 mg/kg), and sodium pentobarbital (150 to 250 mg) to effect. The study was approved by the Henry Ford Health System Care of Experimental Animals Committee and conformed to the “Position of the American Heart Association on Research Animal Use,” adopted by the Association in November 1984.
Study Design and End Points
The study was a randomized, blinded, placebo-controlled trial. Two weeks after the last embolization, dogs underwent a prerandomization left and right heart catheterization. One day later, dogs were randomized to 3 months oral therapy with low-dose NCT (0.5 mg/kg twice daily, n=7), high-dose NCT (2.0 mg/kg twice daily, n=7), combined low-dose NCT and enalapril (10 mg twice daily, n=8), or placebo (n=8). Treatments were based on body weight at the time of randomization. Hemodynamic, angiographic, and neurohormonal measurements were made at 2 weeks after the last embolization and before initiating therapy (pretreatment) and were repeated after completing 3 months of therapy (posttreatment). The primary study end points were (1) changes in LV ejection fraction determined angiographically and (2) changes in global LV remodeling based on changes in LV end-systolic and end-diastolic volumes, also determined angiographically. Secondary end points were (1) changes in transmyocardial (coronary sinus–arterial) plasma concentrations and LV tissue levels of norepinephrine and dopamine and (2) changes in histomorphometric measures of cellular remodeling, namely, changes of cardiomyocyte hypertrophy, replacement fibrosis, interstitial fibrosis, capillary density, and oxygen diffusion distance. Blood and LV tissue samples were obtained from 7 normal dogs for comparison.
Hemodynamic and Angiographic Measurements
Aortic pressure was measured with catheter-tipped micromanometers (Millar Instruments). Left ventriculograms were obtained with the dog placed on its right side and recorded on 35-mm cinefilm at 30 frames/s. Correction for image magnification was made with a radiopaque calibrated grid placed at the level of the LV. LV end-systolic and end-diastolic volumes were calculated by use of the area-length method.13 LV ejection fraction was calculated as previously described.12 The LV end-systolic sphericity index was calculated from ventriculograms as the ratio of the major to minor axis.14 As this index approaches unity, LV shape approaches that of a sphere.
At the end of 3 months of therapy, the heart was removed and placed in ice-cold cardioplegia solution. Three transverse slices, one each from the basal, middle, and apical thirds of the LV, ≈3 mm thick, were obtained. Transmural tissue blocks obtained from the free wall segment of the middle slice were mounted on cork with Tissue-Tek embedding medium (Miles Inc) and rapidly frozen in isopentane precooled in liquid nitrogen. Cryostat sections ≈8 μm thick were prepared and stained with fluorescein-labeled peanut agglutinin (Vector Laboratories Inc) and used to delineate the myocyte border and the interstitial space.15 Sections were double stained with rhodamine-labeled Griffonia simplicifolia lectin I to identify capillaries. Ten radially oriented, scar-free, microscopic fields (magnification ×100) were selected at random from each section and used to measure myocyte cross-sectional area.15 The surface area occupied by interstitial space and the surface area occupied by capillaries were measured from each field by means of computer-based video densitometry (JAVA, Jandel Scientific). The volume fraction of interstitial collagen was calculated as the percent total surface area occupied by interstitial space minus the percent total area occupied by capillaries. Capillary density was calculated by means of the index capillary per fiber ratio.16 The oxygen diffusion distance was measured as half the distance between two adjoining capillaries.16 The volume fraction of replacement fibrosis, namely, the proportion of scar to viable tissue, was calculated from trichrome-stained sections as the percent total surface area occupied by fibrosis.
Measurements of Norepinephrine and Dopamine
Transmyocardial plasma norepinephrine (tNEPI) and dopamine (tDOP) concentrations were estimated by obtaining simultaneous blood samples from the ascending aorta and coronary sinus during cardiac catheterization. tNEPI and tDOP were calculated as the difference between the coronary sinus and arterial samples. Fresh-frozen tissue obtained from the LV free wall was used to measure tissue levels of norepinephrine and dopamine. Norepinephrine and dopamine were assayed in plasma and LV tissue by high-performance liquid chromatography, as previously described.11
For primary end points, tNEPI, tDOP, and sphericity index, intragroup comparisons between pretreatment and posttreatment measures were made by means of the Student’s paired t test, with a value of P<0.05 considered significant. Significance of treatment effect was examined by comparing the placebo group and each of the 3 treatment groups on the basis of the change (Δ) of each measure calculated as the difference between pretreatment and posttreatment values. Subsequent pairwise comparisons with placebo were performed by means of Fisher’s least significant differences test, with a Bonferroni correction on the pairwise probability value if the overall probability value was significant (P<0.05). The Bartlett test for homogeneity of variances was performed before the ANOVA, and if the Bartlett probability value was P<0.05, then the corresponding nonparametric analysis (Kruskal-Wallis test) was performed instead, with subsequent pairwise comparisons performed on the ranks. The statistical analysis for tissue levels of norepinephrine, dopamine, and their ratio and histomorphometric measures was conducted separately. The data were examined by a 1-way ANOVA, with α set at 0.05. If significance was achieved, pairwise comparisons were performed by means of the Student-Newman-Keuls test, with a value of P<0.05 considered significant. All data are reported as mean±SEM.
Catecholamine and Dopamine Concentrations in LV Tissue
In placebo dogs, the concentration of dopamine and norepinephrine in the LV tissue was lower compared with normal dogs (P<0.05) (Figure 1⇓). Treatment with low- and high-dose NCT resulted in a further decline in norepinephrine level and an increase in dopamine and dopamine-to-norepinephrine ratio (Figure 1⇓). Combined therapy of low-dose NCT and enalapril also resulted in an increase in dopamine and dopamine-to-norepinephrine ratio but did not significantly lower the norepinephrine concentration compared with placebo (Figure 1⇓).
Transmyocardial Plasma Levels of Norepinephrine and Dopamine
Levels of tNEPI and tDOP are depicted in Figure 2⇓. There was a near 6-fold increase in tNEPI in placebo-treated dogs compared with normal dogs (Figure 2⇓) (P<0.05). tNEPI difference was significantly reduced with low- and high-dose NCT and with low-dose NCT combined with enalapril. The reduction was most dramatic with the high dose NCT (97%) and was significantly lower than in normal. The reduction was more modest with the low dose NCT (67%) and with combined therapy (55%) and was not statistically different than in normal dogs. tDOP increased with all treatments, but the increase was not significant compared with placebo (Figure 2⇓).
Hemodynamic and Angiographic Findings
The pretreatment and posttreatment hemodynamic and angiographic measures are shown in Table 1⇓. Monotherapy with NCT and combination of NCT with enalapril had no effects on heart rate or mean arterial pressure (Table 1⇓). Placebo was associated with a significant reduction of LV ejection fraction and LV sphericity index and a significant increase of both LV end-diastolic and end-systolic volumes. In dogs treated with high-dose NCT, LV ejection fraction decreased and was accompanied by an increase in LV end-systolic and end-diastolic volumes, with no change in the sphericity index. These changes were nearly similar to placebo. In dogs treated with low-dose NCT, posttreatment values of LV ejection fraction, end-diastolic volume, end-systolic volume, and sphericity index were not different compared with pretreatment (Table 1⇓). In dogs treated with combined low-dose NCT and enalapril, LV ejection fraction and LV sphericity index increased significantly after 3 months of therapy, whereas LV volumes remained essentially unchanged (Table 1⇓).
Comparisons of Treatment Effect
The changes between pretreatment and posttreatment LV ejection fraction and end-systolic and end-diastolic volumes are shown in Figures 3⇓, 4⇓, and 5⇓. Intergroup comparisons of change showed that compared with placebo, low-dose NCT but not high-dose NCT prevented the decline in LV ejection fraction and the increase in LV end-diastolic and end-systolic volumes. Similarly, intergroup comparisons of change showed that combination therapy significantly improved LV ejection fraction and reduced LV end-systolic and end-diastolic volumes compared with placebo. In placebo dogs, the change in LV sphericity index was −0.10±0.03 and was not different from high-dose NCT (−0.03±0.06) but significantly different than low-dose NCT (0.04±0.02, P<0.05) and combination therapy (0.08±0.03, P<0.05).
Histomorphometric results are shown in Table 2⇓. Volume fraction of replacement fibrosis was significantly lower in all treatment groups compared with placebo and was lowest with combination therapy. Volume fraction of interstitial fibrosis was significantly higher in placebo compared with normal dogs. It decreased in all 3 treatment groups when compared with placebo but was only significant in the low-dose NCT group and in the combined therapy, with the latter showing the most reduction. Cardiomyocyte cross-sectional area was significantly larger in placebo-treated dogs compared with normal dogs. It tended to be smaller in all 3 treatment groups compared with placebo, but the change reached significance only in dogs treated with combined low-dose NCT and enalapril. Capillary density was significantly lower in placebo dogs compared with normal dogs. It increased in all 3 treatment groups compared with placebo but reached significance in only the low-dose NCT group and the combination low-dose and enalapril (Table 2⇓). Oxygen diffusion distance was significantly longer in placebo dogs compared with normal dogs. It was significantly shorter in all 3 treatment groups and was shortest in the combined therapy group when compared with placebo.
This is the first study to assess the effects of DBH inhibition on the progression of LV dysfunction and remodeling in dogs with HF. As expected, inhibition of DBH resulted in a reduction of tNEPI levels with an increase, albeit not significant, of tDOP levels. Low-dose NCT normalized tNEPI, prevented progressive LV dysfunction, and attenuated LV remodeling. In contrast, high-dose NCT, which reduced tNEPI to levels below normal, had no beneficial effects on LV function and global LV remodeling compared with placebo. The addition of enalapril to low-dose NCT attenuated global LV remodeling to an extent similar to that of low-dose NCT alone, but, unlike low-dose NCT alone, the combination therapy significantly increased LV ejection fraction. The observed changes in global LV remodeling were associated with important structural/cellular changes. Low-dose NCT was associated with a lower volume fraction of replacement fibrosis, a lower volume fraction of interstitial fibrosis, reduced cardiomyocyte hypertrophy, increased capillary density, and improved oxygen diffusion distance compared with placebo, findings that favor prevention or attenuation of progressive LV remodeling. The improvements were even more pronounced with combination therapy of low-dose NCT and enalapril. These structural/cellular improvements tended to be present, but to a lesser extent, with high-dose NCT (Table 2⇑), a treatment regimen that failed to show benefits in LV function and global remodeling when compared with placebo.
The beneficial effects of NCT on the failing heart are likely due to the decrease in norepinephrine release from cardiac sympathetic nerves and subsequently to a lesser stimulation of α- and β-adrenergic receptors. Placebo-treated dogs had elevated tNEPI levels and lower cardiac tissue norepinephrine concentration compared with normal dogs, indicating a high rate of norepinephrine turnover in cardiac sympathetic nerve endings. In dogs, NCT produces a concentration-dependent inhibition of DBH and has negligible affinity for other enzymes and neurotransmitters11 and has been shown to cause a dose-dependent decrease in norepinephrine, increase in dopamine, and an increase in the dopamine/norepinephrine ratio in the LV.11 Studies performed with radioisotope spillover techniques concluded that the increase in cardiac norepinephrine spillover observed in HF was a result of an increase in norepinephrine release in the setting of normal neuronal uptake.17 In the present investigation, HF resulted in increased tNEPI difference, which was reduced by NCT in a dose-dependent manner. Interestingly, the large decrease in tNEPI to below normal levels with high-dose NCT did not improve ventricular function, suggesting that over-suppression of cardiac sympathetic activity, even in the setting of HF, can have an adverse effect on the cardiac performance. This observation offers a potential explanation for the increase in adverse events seen in the Moxonidine in Congestive Heart Failure Trial (MOXCON). One possibility is that moxonidine, a selective imidazole I1-receptor agonist with central sympatholytic effect, may have been given in too high a dose, which led to excessive reduction in plasma norepinephrine.18 Also of interest is the observation that NCT did not reduce heart rate despite its marked effects on tNEPI. The underlying cause of this finding remains to be elucidated.
In contrast to β-adrenergic blockade, which has a rapid physiological effect on LV function, DBH inhibits norepinephrine synthesis over a number of days and even then only attenuates rather than totally abolishes preganglionic and tyramine-induced cardiovascular responses.11 Drugs that preferentially inhibit norepinephrine synthesis, such as NCT, affect the release of norepinephrine from tissues that have a low storage rate and a high turnover, which is the case in HF.19 The reduction of norepinephrine leads to diminished activation of not only β1 and β2 receptors but also α1 receptors. Inactivation of α1 receptors may have clinical importance in HF by decreasing ventricular arrhythmias and potentially the incidence of sudden death. Inhibition of DBH also increases dopamine production and release, which can promote urinary sodium excretion by activating dopamine receptors in the renal tubules.20
In addition to attenuating progressive global LV remodeling, low-dose NCT reduced replacement fibrosis, a measure of ongoing cell death, reduced reactive interstitial fibrosis, attenuated myocyte hypertrophy, and improved oxygen diffusion, alterations deemed beneficial in the treatment of HF. Enhanced and sustained norepinephrine release can cause cardiomyocyte hypertrophy by increasing protein synthesis.21 In a study of LV remodeling produced by repeated DC shock in dogs, the α1-adrenoceptor blocker terazosin attenuated early LV dilation and hypertrophy.22 The fact that DBH inhibitors deactivate both α1- and β-adrenergic pathways may explain the beneficial effect on cardiomyocyte hypertrophy observed with NCT therapy in this study. In contrast to low-dose NCT, high-dose NCT had no effect on global LV remodeling and a lesser effect on cellular components of LV remodeling despite greater adrenergic inhibition. Although not fully understood, one can only speculate that excessive inhibition of adrenergic drive in HF may also adversely impact cellular remodeling.
Combination of enalapril with low-dose NCT also resulted in attenuation of LV remodeling, along with a significant increase in LV ejection fraction. The increase in ejection fraction was due to a decrease in LV end-systolic volume, suggesting improved contractile performance. Combination therapy also resulted in decreased LV chamber sphericity, increased capillary density, and reduced oxygen diffusion distance; factors important in overall myocardial energetics. The increase in LV ejection fraction, therefore, could be interpreted to be due to improved cardiomyocyte energetics. Angiotensin II is closely linked to myocyte hypertrophy and fibroblast growth23 and therefore inhibiting its formation with an ACE inhibitor is likely to attenuate myocyte hypertrophy and interstitial fibrosis. Angiotensin II also has a direct effect on interstitial fibrosis through its action on fibroblasts.24 In the present study, combination therapy with enalapril elicited additional improvements in all morphological features examined compared with low-dose NCT alone. A limitation of this study, however, is the lack of an enalapril arm only. This would have been useful in determining whether the addition of NCT was beneficial to the underlying effects of the ACE inhibitor or whether the effects of NCT and the ACE inhibitor were additive.
In conclusion, results of the present study indicate that NCT in doses that normalize tNEPI attenuates LV remodeling and prevents progressive LV systolic dysfunction. This beneficial effect of NCT is lost, however, when doses that result in near depletion of tNEPI are used, suggesting that some threshold of sympathoadrenergic drive is necessary even in the setting of HF. Finally, when combined with enalapril, low-dose NCT elicits an improvement in LV ejection fraction that is similar to that seen with β-blockers in HF when used with ACE inhibitors.4 5 This observation argues in favor of enhanced and sustained sympathoadrenergic drive as a common denominator responsible, in part, for the abnormalities of LV function and LV chamber remodeling that are characteristic of the HF state.
This study was supported in part by research grants from Roche Bioscience and the National Heart, Lung, and Blood Institute (HL-49090-06).
Guest Editor for this article was Marc A. Pfeffer, MD, PhD, Brigham and Womens Hospital, Boston, Mass.
- Received March 17, 2000.
- Revision received May 19, 2000.
- Accepted May 25, 2000.
- Copyright © 2000 by American Heart Association
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