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Nebivolol Improves Diastolic Dysfunction and Myocardial Remodeling Through Reductions in Oxidative Stress in the Zucker Obese Rat

Originally publishedhttps://doi.org/10.1161/HYPERTENSIONAHA.109.145136Hypertension. 2010;55:880–888

Abstract

Insulin resistance is associated with obesity and may be accompanied by left ventricular diastolic dysfunction and myocardial remodeling. Decreased insulin metabolic signaling and increased oxidative stress may promote these maladaptive changes. In this context, the β-blocker nebivolol has been reported to improve insulin sensitivity, increase endothelial NO synthase activity, and reduce NADPH oxidase–induced superoxide generation. We hypothesized that nebivolol would attenuate diastolic dysfunction and myocardial remodeling by blunting myocardial oxidant stress and promoting insulin metabolic signaling in a rodent model of obesity, insulin resistance, and hypertension. Six-week–old male Zucker obese and age-matched Zucker lean rats were treated with nebivolol (10 mg · kg · day−1) for 21 days, and myocardial function was assessed by cine MRI. Compared with untreated Zucker lean rats, untreated Zucker obese rats exhibited prolonged diastolic relaxation time (27.7±2.5 versus 40.9±2.0 ms; P<0.05) and reduced initial diastolic filling rate (6.2±0.5 versus 2.8±0.6 μL/ms; P<0.05) in conjunction with increased homeostatic model assessment of insulin resistance (7±2 versus 95±21; P<0.05), interstitial and pericapillary fibrosis, abnormal cardiomyocyte histoarchitecture, 3-nitrotyrosine, and NADPH oxidase–dependent superoxide. Nebivolol improved diastolic relaxation (32.8±0.7 ms; P<0.05 versus untreated Zucker obese), reduced fibrosis, and remodeling in Zucker obese rats, in concert with reductions in nitrotyrosine, NADPH oxidase–dependent superoxide, and improvements in the insulin metabolic signaling, endothelial NO synthase activation, and weight gain (381±7 versus 338±14 g; P<0.05). Results support the hypothesis that nebivolol reduces myocardial structural maladaptive changes and improves diastolic relaxation in concert with improvements in insulin sensitivity and endothelial NO synthase activation, concomitantly with reductions in oxidative stress.

Obesity-induced cardiomyopathy is characterized by impaired left ventricular (LV) relaxation in association with insulin resistance (IR).1,2 This cardiomyopathy develops independent of blood pressure, ischemia, impaired systolic function, and age.1,2 Impaired insulin metabolic signaling and increased generation of reactive oxygen species (ROS) play important roles in maladaptive myocardial remodeling3–5 and impaired diastolic relaxation.6,7 Excessive ROS in the heart and vasculature, derived from several enzymatic sources, including NADPH oxidase,8 can lead to decreased bioavailable NO and reduced delivery of glucose and insulin to myocardial tissue.4,9

β-Adrenergic receptor blockers have clinical use in treating heart failure, but traditional β-blockers have been associated with weight gain and worsening of IR. Nebivolol, a third-generation β1-receptor blocker improves diastolic dysfunction10 and reduces mortality in elderly heart failure patients.10–12 Nebivolol does not have adverse effects on IR, nor does it cause weight gain.13–15 Potential beneficial effects of nebivolol on diastolic dysfunction associated with IR may be mediated through improvements in bioavailable NO, reductions in ROS, and improvements in insulin metabolic signaling.

We hypothesized that nebivolol would improve diastolic dysfunction in an IR rat through reductions in NADPH oxidase activity and improved insulin metabolic signaling. To address our hypothesis, we used young (6- to 7-week–old) Zucker obese (ZO) rats, an IR model that manifests increased myocardial oxidative stress and diastolic dysfunction.16–18 Thus, the ZO rat provides a unique model to investigate the effect of nebivolol on impaired myocardial diastolic relaxation as a result of oxidative stress, reduced bioavailable NO, and impaired insulin metabolic signaling.16

Methods

Animals

Animal procedures were approved by the University of Missouri animal care committees and housed according to National Institutes of Health guidelines.

Drug Preparation

Nebivolol was dissolved in 50% dimethyl sulfoxide/50% propylene glycol to a final concentration of 70 mg/mL and filter sterilized. The solution (or its vehicle) was loaded into a model 2004 Alzet pump and inserted subcutaneously behind the shoulder blades under brief isoflurane anesthesia.

Systolic Blood Pressure and Total Body Weight

Within a day or 2 of termination of the experiment, systolic blood pressures (SBPs) were measured in triplicate using the tail-cuff method as described previously.19

Homeostatic Model Assessment of IR

A venous blood sample was collected from a subset of fasting rats in each treatment group at the end of the study, and plasma was stored at −80°C. Glucose and insulin were measured by an automated hexokinase G-6-PDH assay and an ELISA kit specific for rat insulin, respectively. Homeostatic model assessment was calculated by taking the product of the glucose (in millimoles per liter) and insulin (in microunits per milliliter) values and dividing by 22.

Cine-MRI

MRI scans were performed on rats after 2 weeks of treatment with nebivolol or vehicle. For details describing procedures to determine LV functional parameters, please see the online Data Supplement at http://hyper.ahajounals.org.

Light Microscopic Analysis for Myocardial Interstitial Fibrosis

Fixed paraffin sections of the left ventricle were evaluated with Verhoeff-van Gieson stain, which stains elastin (black), nuclei (blue black), collagen (pink), and connective tissue (yellow), as described previously.19 For details of the morphometric analysis please see the online Data Supplement.

Ultrastructural Analysis With Transmission Electron Microscopy

Details of fixation, embedding, sectioning, and staining procedures have been described previously.19 A JOEL 1400-EX transmission electron microscope was used to view all of the samples.

Quantitative Analysis of Mitochondrial Number, Enzyme Level, and Activity

Mitochondrial Quantification

Fixed samples were immunolabeled with an antibody to complex IV-1 and viewed under a laser confocal microscope (Bio-Rad) and a multiphoton confocal system. Images were captured with Laser-sharp software and the immunofluorescence quantified using MetaMorph software. For details please see the online Data Supplement.

Citrate Synthase Activity

Citrate synthase activity in mitochondrial fractions of LV tissue was determined as described previously.20 For details please see the online Data Supplement.

β-Hydroxyacyl-Coenzyme A Dehydrogenase Activity

β-Hydroxyacyl-coenzyme A dehydrogenase (β-HAD) activity was measured as described previously with modifications.21 For details please see the online Data Supplement.

Markers of Oxidative Stress

3-Nitrotyrosine Immunostaining

3-Nitrotyrosine (3-NT) was quantified as described previously.19,22

Superoxide Formation

Superoxide was determined by chemiluminescence, as described previously.19,22 Superoxide values were normalized to total protein in the whole homogenate and expressed as relative light units per second per milligram of protein.

NADPH Oxidase Activity

Activity was determined in plasma membrane fractions as described previously.19,22

Nox2, Nox4, Rac1, p47phox, Endothelial NO Synthase, and Ser1177 Endothelial NO Synthase Immunostaining

LV sections were immunostained and quantitated as described previously.19,22

Quantification of IRS-1, Ser473 Akt, and Ser1177eNOS by Western Blot Analysis

Protein concentrations of tissue homogenates were measured as described previously.19 Briefly, samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Reactive bands were detected by chemiluminescence, and images were recorded using a Bio-Rad ChemiDoc XRS image analysis system. Quantitation of protein band density, normalized to β-actin band density, was performed using Quantity One software (Bio-Rad). Data are reported as the normalized protein band density in arbitrary units.

Statistical Analysis

All of the values are expressed as mean±SE. Statistical analyses were performed with Sigma Stat (Aspire Software Intl) using Student t tests or ANOVA with the Fisher least significant difference test for post hoc comparisons. Significance was accepted as P<0.05.

Results

Nebivolol Effects on Body Weight and SBP

Both the ZO-control (C) and ZO-nebivolol (N) groups had higher body weights compared with the Zucker lean group (ZL-C; P<0.05), although there was a modest reduction in weight gain in the ZO-N rats compared with ZO-C rats (P<0.05; Table 1). Although SBP tended to be higher in ZO-C and normalized in ZO-N compared with ZL-C rats, the ANOVA main effect was not significant (P>0.05). A t test indicated a significant decrease in SBP in ZO-N compared with ZO-C rats (128±5 versus 156±12 mm Hg; P<0.05).

Table 1. Effects of Nebivolol on Body Weight, Blood Pressure, Fasting Plasma Glucose and Insulin, and Homeostatic Model Assessment-IR of 9-Week–Old ZL and ZO Rats

ParameterZL-CZL-NZO-CZO-N
Numbers in parentheses are sample sizes. HOMA indicates homeostatic model assessment. Data represent mean±SE.
*P<0.05 vs ZL-C.
P<0.05 vs ZO-C.
Body weight, g245±7 (6)240±7 (6)381±7 (6)*338±14 (6)*
SBP, mm Hg136±11 (5)133±10 (6)156±12 (6)128±5 (7)
Glucose, mmol/L7.3±0.5 (6)6.6±0.6 (2)11.4±1.2 (8)*7.1±0.4 (6)
Insulin, μU/mL21±5 (6)18±12 (2)177±32 (8)*134±29 (6)
HOMA-IR7.2±1.9 (6)5.6±4.1 (2)95±21 (8)*41±8 (6)*

Nebivolol Effects on IR

ZO-C rats had elevated fasting plasma insulin levels (Table 1) compared with ZL-C (177±32 versus 21±5 μU/mL; P<0.05); nebivolol treatment tended to blunt this hyperinsulinemia, but the trend was not significant (134±29 μU/mL; P>0.05 versus ZO-C). ZO-C rats were slightly hyperglycemic compared with ZL-C rats (11.4±1.2 versus 7.3±0.5 mmol/L; P<0.05), although glucose levels were normalized in ZO-N rats (7.1±0.4 μΜ; P<0.05 versus ZO-C). Homeostatic model assessment-IR indicated elevated IR in ZO-C compared with ZL-C (95±21 versus 7±2 mmol/L; P<0.05), and ZO-N exhibited improved insulin sensitivity (41±8; P<0.05 versus ZO-C).

Nebivolol Improves Diastolic Relaxation

LV diastolic relaxation time, diastolic peak filling rate, and initial filling rate, as well as septal wall thickness, ejection fraction, and stroke volume were determined via cine MRI (Figure 1A and 1B; Table 2). There were increases in septal wall thickness in ZO-C relative to ZL-C rats (P<0.05), and nebivolol tended to blunt the increased septal wall thickness in the ZO strain (P>0.05). LV diastolic relaxation time was prolonged in ZO-C compared with ZL-C rats (40.88±1.94 versus 27.68±2.50 ms; P<0.05). Nebivolol decreased diastolic relaxation time in the ZO rats (ZO-N: 32.77±0.73 ms; P<0.05 versus ZO-C). Diastolic initial filling rate was reduced in ZO-C versus ZL-C rats (P<0.05). Heart rate, diastolic peak filling rate, systolic ejection fraction, and stroke volume were similar among all of the groups (Table 2).

Table 2. Effects of Nebivolol on In Vivo Cardiac Functions in 8-Week–Old ZL and ZO Rats Evaluated by Cine-MRI

Parameter, Cine MRI DataZL-CZL-NZO-CZO-N
Data represent mean±SE.
*P<0.05 vs ZL-C.
P<0.05 vs ZO-C.
Sample size, n106106
Age, wk8.77±0.098.14±0.28.76±0.098.07±0.16
Heart rate, bpm368±7383±6376±10363±8
Stroke volume, μL372.5±20.1314.8±28350.4±19.2303.7±23.8
Ejection fraction, %80.9±1.575.6±2.482.6±1.980.2±1.0
End-diastolic septal wall thickness, mm1.41±0.041.37±0.031.78±0.09*1.61±0.04
Initial filling rate, μL/ms6.16±0.495.67±0.822.84±0.58*3.88±0.35
Peak filling rate, μL/ms8.07±0.617.80±0.918.61±0.987.06±0.51
Diastolic relaxation, ms27.68±2.5027.50±3.3340.88±1.95*32.77±0.73

Figure 1. Nebivolol improves diastolic relaxation, reduces myocardial fibrosis, and improves ultrastructural remodeling of myocardial capillaries. A, Representative cine-MRIs illustrate early diastole phases (frame 7 to 12 of 16 captured) in a cardiac cycle. The top row demonstrates prolonged diastolic relaxation time in ZO-C compared with reduced diastolic relaxation time and increased initial filling rate in ZO-N rats shown in the bottom row. B, Bar graph shows diastolic relaxation times for experimental groups. C, Light micrographs show representative LV sections stained with Verhoeff-van Gieson stain, which stains collagen pink. The bar graph below shows that nebivolol attenuates the increased interstitial fibrosis in the ZO myocardium. Scale bar: 50 μm. *P<0.001 vs ZL-C; †P<0.01 vs ZO-C. D, Representative transmission electron microscopy micrographs at ×400 demonstrate constricted capillaries in ZO-C rats that improved with nebivolol treatment in ZO-N (top). Small dark arrows point to a capillary, which is shown at higher magnification in the white boxes. Scale bar: 2 μm. The X in the center of the bottom left panel shows pericapillary fibrosis in the ZO-C heart, and the area inside the dark box is shown at higher magnification in the white box (scale bar: 0.1 μm). From bottom to top, each image shows the capillary lumen, a single endothelial cell layer composing the capillary wall, a prominent layer of pericapillary collagen, and cardiomyocytes. A pericapillary collagen layer was not observed in the ZO-N (bottom right). White arrows indicate an area of abundant endothelial cell transcytotic vesicles in the ZO-N.

Nebivolol Reduces Myocardial Interstitial Fibrosis

Interstitial fibrosis, as represented by the average grayscale intensity of Verhoeff-van Gieson staining (for collagen), was increased in ZO-C compared with ZL-C rats (P<0.001) and improved in ZO-N compared with ZO-C rats (P<0.01; Figure 1C). Coronary arterioles of ZO rats also exhibited perivascular fibrosis, which was attenuated by nebivolol (data not shown).

Nebivolol Improves Myocardial Capillary and Tissue Remodeling

On ultrastructural analysis, there were constricted capillaries with pericapillary fibrosis in the ZO-C myocardium, which improved with nebivolol treatment (Figure 1D). There was also an increase in endothelial cell transcytotic vesicles in ZO-N rats compared with ZO-C rats (Figure 1D).

Nebivolol Improves Mitochondrial Function and Structure

Mitochondrial complex IV subunit 1, the last enzyme in the respiratory electron transport chain and an index of mitochondrial number, was quantitated by protein immunofluorescence. Citrate synthase, a Kreb cycle enzyme used to assess aerobic capacity of mitochondria, and β-HAD, an enzyme involved in mitochondrial fatty acid β-oxidation, were also quantitated. Control ZO myocardium exhibited increased (P<0.05) mitochondrial numbers (increase in complex IV-1) without differences in citrate synthase and β-HAD (Figure 2A through C, respectively). Complex IV-1 signal in the ZO myocardium was reduced with nebivolol treatment (P<0.05). Normal levels of citrate synthase and β-HAD activities, coupled with the increased numbers of mitochondria in the ZO myocardium, suggest that it takes more mitochondria in the ZO myocardium to provide normal levels of ATP. Nebivolol treatment restored the enzyme activities and mitochondrial number in ZO rats to levels similar to those in the ZL-C and ZL-N myocardium. Compared with ZL-C, ZL-N, and ZO-N, ZO-C rats demonstrated increases in intermyofibrillar mitochondria and disorganized sarcomere structure (Figure 2D). ZO-C mitochondria exhibited swollen and disrupted cristae (Figure 2E). Nebivolol abrogated the increased mitochondrial biogenesis and improved mitochondrial sarcomere organization (Figure 2D and 2E).

Figure 2. Nebivolol improves myocardial mitochondrial function. A, Representative LV sections immunostained for mitochondrial complex IV-1 of treated and untreated ZL and ZO rats. The increased level of complex IV-1 immunostaining in the ZO-C myocardium indicates increased mitochondrial number compared with all of the other groups. Bar graph to the bottom shows quantification of converted signal intensities of complex IV-1 protein. B, Bar graphs show myocardial citrate synthase and (C) β-HAD activities. No differences were observed in enzyme activities among the groups (P>0.05). *P<0.05 vs ZL-C; †P<0.05 vs ZO-C. D, Representative myocardial transmission electron microscopy micrographs at ×1000. Compared with ZL-C (top left), ZO-C rats possess increased numbers of intermyofibrillar mitochondrial and a disorganized sarcomere structure (bottom left). An abrogation of the increased mitochondrial biogenesis was observed in ZO-N rats (bottom right). Scale bar: 1 μm. E, Representative transmission electron microscopy micrographs show intermyofibrillar mitochondria at ×6000. Compared with ZL-C (top left), the ZO-C myocardium (bottom left) shows multiple layers of mitochondria between myofibrils compared with a single layer arrangement of mitochondria in ZL-C, ZL-N, and ZO-N hearts. Large lipid droplets are more abundant in the ZO-C myocardium, which also shows abnormal mitochondrial structure (swollen and disrupted cristae and irregular open matrix). The appearance of mitochondria was improved in the ZO-N myocardium (bottom right). Scale bar: 200 nm. Arrows point to mitochondria, and arrowheads point to lipid droplets.

Nebivolol Reduces Myocardial NADPH Oxidase Activity

NADPH oxidase activity was elevated in ZO-C compared with that of ZL-C rats (P<0.05; Figure 3A). ZO-N rats had lower NADPH oxidase activity versus ZO-C rats (P<0.05). There were also increases in NADPH oxidase subunit immunostaining of Nox2, Nox4, Rac1, and p47phox in ZO-C rats compared with ZL-C rats (P<0.05 for each; Figure 3C). Expression levels for all of these proteins were normalized after nebivolol therapy (P<0.05 versus ZO-C rats for each protein).

Figure 3. Nebivolol reduces NADPH oxidase-mediated oxidant stress. A, The bar graph demonstrates increased NADPH oxidase activity in the myocardium of ZO-C relative to ZL-C rats (P<0.05). Nebivolol reduces NADPH oxidase activity in the ZO myocardium compared with ZO-C rats (P<0.05). B, The bar graph demonstrates increased formation of superoxide in the myocardium of ZO-C relative to ZL-C rats (P<0.05). Nebivolol reduces superoxide formation in the ZO myocardium compared with ZO-C rats (P<0.05). C, Representative confocal images showing immunofluorescence for NADPH oxidase membrane-bound proteins Nox2 and Nox4. B, Bar graphs show average grayscale intensities for NADPH oxidase subunits Nox2, Nox4, p47phox, and Rac1. D, Representative photomicrographs showing 3-NT immunostaining in the myocardium of ZL and ZO rats. In the bar graph to the right, average grayscale intensity measures of the 3-NT staining show that nebivolol blunts the increase in 3-NT levels in the ZO myocardium. Scale bar: 50 μm. *P<0.05 vs ZL-C; †P<0.05 vs ZO-C.

Nebivolol Reduces Myocardial Oxidative Stress

There were increases in superoxide levels in ZO myocardium compared with ZL rats (P<0.05; Figure 3B). ZO-N rats had lower superoxide levels compared with ZO-C rats (P<0.05). There were increases in myocardial 3-NT content in the ZO-C (32.8±2.1 grayscale intensities) compared with ZL-C rats (19.2±2.1 grayscale intensities; P<0.05), and 3-NT was reduced in nebivolol-treated ZO rats (22.1±2.1 grayscale intensities; P<0.05; Figure 3D).

Nebivolol Modulates the IRS-1/Akt/eNOS Signaling Pathway

To explore whether nebivolol modulates myocardial insulin signaling, we examined the IRS-1/Akt/eNOS cascade by immunoblotting (Figure 4A through 4C). Although the protein levels of IRS-1 or the ratio of phosphorylated Akt at Ser473 to total Akt were similar in the ZL-C, ZL-N, and ZO-C groups, there was a 2.26-fold increase in IRS-1 content and a 2.05-fold increase in activated Akt, that is, the ratio of phosphorylated Akt at Ser473 to total Akt, in ZO rats treated with nebivolol compared with ZO controls (each P<0.05; Figure 4A and 4B).

Figure 4. Nebivolol improves insulin metabolic signaling and enhances coronary arteriolar eNOS activation. A, The bar graph shows a quantitative densitometric analysis for IRS-1 protein (normalized to β-actin) as a percentage of ZL-C (ie, fold increase). Representative protein bands for IRS-1 and β-actin are shown above the bar graph. Nebivolol increased IRS-1 protein level in the ZO myocardium compared with ZO-C. B, Representative Western blots show phosphorylated Ser473 Akt and total Akt, as well as their corresponding β-actin bands. The bar graph shows the ratio of phospho-Akt:total Akt expressed as a percentage of ZL-C control. C, Representative Western blots show phosphorylated eNOS at Ser1177 and total eNOS, as well as their corresponding β-actin bands. The bar graph displays the ratio of phospho-eNOS Ser1177:total eNOS expressed as a percentage of ZL-C control. D, Representative confocal micrographs show total eNOS (top row) and phospho-eNOS Ser1177 (bottom row) immunofluorescence in the myocardium and coronary arterioles of ZL and ZO rats. Scale bar: 50 μm. The bar graphs to the right show average grayscale intensity measures of total eNOS and phospho-eNOS Ser1177 immunofluorescence in the endothelium of coronary arterioles of ZL and ZO rats and indicate that nebivolol enhances eNOS phosphorylation at Ser1177. *P<0.05 vs ZL-C; †P<0.05 vs ZO-C.

To ascertain whether nebivolol induced eNOS activation in ZO heart tissue, Ser1177 phosphorylation of eNOS (activation) and total eNOS protein were measured using Western blots (Figure 4C). There were increases in Ser1177 eNOS (1.79-fold) and eNOS (1.59-fold) in ZO rats with nebivolol treatment compared with ZO controls (each P<0.05). The ratio of Ser1177 eNOS to total eNOS in the ZO-C rat was not different from ZL-C rats (P>0.05); however, the ratio of Ser1177 eNOS to total eNOS was increased in ZO-N compared with ZO-C rats (P<0.01).

To more specifically evaluate myocardial or coronary arteriolar changes in total eNOS and phosphorylated eNOS at Ser1177, we performed semiquantitative immunofluorescence analyses. Both total eNOS and Ser1177 eNOS immunofluorescent signals were detected in the myocardium and in the vascular wall of coronary arterioles (Figure 4D). Ser1177 eNOS was increased in ZO rats treated with nebivolol compared with ZO controls (Figure 4D; P<0.05).

Discussion

This investigation demonstrates that nebivolol improves LV diastolic function and insulin sensitivity; reduces myocardial NADPH oxidase activity, oxidative stress, and interstitial fibrosis; reduces capillary and mitochondria ultrastructural abnormalities; and enhances the IRS-1/Akt/eNOS signaling pathway. Finally, nebivolol, unlike traditional β-blockers, which promote weight gain,23 reduced weight gain in the ZO rat.

In metabolic heart disease, relaxation abnormalities often appear before the onset of contractile dysfunction. Diastolic dysfunction is characterized by a decrease in the ability of the left ventricle to fill with blood during the early diastolic filling.24 Using high-resolution cine-MRI, we observed delayed LV diastolic relaxation and decreased early diastolic filling in the ZO rat in the absence of measurable contractile dysfunction. Nebivolol treatment improved LV diastolic relaxation in concert with reductions in interstitial fibrosis.

There were substantive ultrastructural abnormalities of the coronary microvasculature and intermyofibrillar mitochondria in ZO hearts, which were reversed with nebivolol treatment. Capillaries were constricted, exhibited diffuse pericapillary fibrosis, and contained fewer transcytotic vesicles in endothelial cells. ZO hearts had marked increases in intermyofibrillar mitochondria, which resulted in disorganized sarcomere structure as observed previously in other rodent models of IR. 19,25–27 The increase in complex IV-1 in ZO-C rats, whereas indicative of an increase in number of mitochondria, may also reflect a compensatory response, which limits oxidative stress by diverting molecular oxygen toward the terminal steps in aerobic metabolism leading to ATP synthesis. Indeed, there is recent evidence that mitochondria in the untreated diabetic ZO heart produce excessive superoxide.28 There was a marked reduction in mitochondria in nebivolol-treated ZO hearts, yet they exhibited improved cristae structure and sarcomere organization.

The balance between ROS production and elimination plays a key role in preserving cardiac function; excessive myocardial ROS precipitates impairment of myocardial function and abnormalities in cardiac structure.29 The NADPH oxidase complex serves as a major source for the generation of superoxide in the cardiovascular system. ZO myocardium had increases in NADPH oxidase activity, superoxide, and 3-NT; these increases were blunted with nebivolol therapy. There were increases in NADPH subunits Nox2, Nox4, p47phox, and Rac1 in ZO-C hearts; however, the expression of all of these proteins was significantly reduced by nebivolol treatment. These data suggest that NADPH oxidase activation may be the primary mediator of increased superoxide production in the heart of the ZO rat. In diabetic fatty rats, the mitochondria may also be a significant source of ROS in the myocardium.28 Interestingly, these young ZO rats did not exhibit increases in mitochondrial citrate synthase activity or β-HAD, a marker of fatty acid β-oxidation. Excessive fatty acid oxidation can lead to increased mitochondrial superoxide synthesis. Others have shown increased fatty acid uptake in ZO hearts30,31 in the absence of increased fatty acid oxidation.32

IR is associated with endothelial dysfunction and impaired vasodilation, which may be partially dependent on excess generation of ROS.4,33 Superoxide may react with NO released by eNOS to generate peroxynitrite. The increased 3-NT staining in the ZO myocardium observed in this study is indirect evidence of formation of peroxynitrite. Peroxynitrite can contribute to endothelial dysfunction by reducing the bioavailability of NO,33,34 in part by promoting uncoupling of eNOS, which results in eNOS-derived increases in superoxide synthesis.4

eNOS activity is regulated by posttranslational modifications, including phosphorylation of specific sites and protein-protein interactions. Phosphorylation of eNOS at Ser1177 is associated with increased enzyme activity.3 The ZO rat exhibits impairments in endothelial function and endothelium-dependent vasodilation.35 In this study, both eNOS expression and eNOS phosphorylation at Ser1177 were increased in coronary arterioles of ZO rats with nebivolol treatment. Commensurate with these changes in eNOS, transmission electron microscopy measurements demonstrated constricted capillaries with decreased endothelial transcytotic vesicles in ZO rats that improved with nebivolol treatment. Our data suggest that nebivolol improves capillary endothelial function and remodeling in the ZO rat and that this may be because of a reduction in oxidative stress, promotion of NO bioavailability, and an increase in NO biosynthesis. This compliments a previous observation that nebivolol inhibits endothelial dysfunction via diminishing superoxide formation by NADPH oxidase in the heart of angiotensin II–treated rats.36 In the heart, among the 3 isoforms of NO synthase, eNOS is constitutively expressed in both endothelial cells and cardiomyocytes.37 NO produced by eNOS is not only a primary determinant of blood vessel tone but also a regulator of cardiac function. In this regard, eNOS-derived NO facilitates increased myocardial diastolic relaxation and decreased O2 consumption.38

Activation of the IRS-1/Akt pathway plays a central role in the regulation of myocardial glucose metabolism, cell survival, and cardiac function.19 In particular, the IRS-1/Akt pathway activates eNOS by phosphorylation at the Serine1177 residue, which promotes NO production.39 Our laboratory has observed previously that there was a relationship between increased NADPH oxidase activity and diminished Akt activation in vivo.3,19 In this study, there were significant increases in phosphorylated Ser473 Akt, as well as total IRS-1 protein, in ZO rats with nebivolol treatment in the absence of significant differences in the expression of IRS-1, Akt phosphorylation, and eNOS, as well as eNOS phosphorylation between control ZO and ZL rats at 9 weeks of age. This somewhat surprising result suggests that there could be a compensatory mechanism under hyperinsulinemia in the young ZO rats. Despite the apparent normal state of eNOS activation in the untreated ZO myocardium observed in this study, it is likely that nebivolol acts to enhance the bioavailability of NO by reducing levels of ROS and promoting activation of the IRS-1/Akt/eNOS pathway. Indeed, nebivolol dilates the coronary arterial microvasculature via an agonist effect on endothelial β3-adrenoreceptors that promotes the release of NO, in part by dephosphorylating eNOS on Thr495.40 Such a scenario predicts improvements in cardiac function and structure.

The notion that traditional β-blockers promote modest weight gain may be attributed to several factors, including reductions in the metabolic rate and insulin sensitivity.23,41 However, our data suggest that nebivolol-treated ZO rats, on average, weighed less by 11% (P<0.05), were more insulin sensitive, and were normotensive compared with control ZO rats. This is consistent with observations in human trials wherein nebivolol has largely been found to be weight and metabolically neutral compared with more traditional agents.14,15 However, it is possible that this modest weight loss contributes to the overall improvements in insulin sensitivity, SBP, and diastolic function. In obese humans, modest weight loss can lead to improvements in insulin sensitivity, decreased blood pressure, and improved endothelial function. It is also possible that the β3 agonist properties of nebivolol mediate modest weight loss by inducing transdifferentiation of white adipose tissue into brown adipose tissue.42 Indeed, in rodents and humans, β3 receptor agonists stimulate oxidation of fats, reduce fat weight, improve insulin sensitivity, and spare lean body mass.43

Perspectives

This investigation indicates that nebivolol, a highly cardioselective β1-receptor blocker, improves myocardial remodeling and diastolic dysfunction, as well as IRS-1/Akt/eNOS signaling pathways, by inhibiting myocardial NADPH oxidase–mediated superoxide formation in an obese insulin-resistant rodent model. These findings suggest that nebivolol prevents or at least delays the development of cardiomyopathy associated with IR. Finally, nebivolol, unlike traditional β-blockers, which promote weight gain, reduced weight gain in the ZO rat. These data are highly clinically relevant as a first report using an animal model of obesity that ascribes potential mechanisms to explain observed human improvements in diastolic function and insulin sensitivity in patients on nebivolol.

Exceptional support was provided by the Veterans’ Affairs Biomolecular Imaging Center at the Harry S. Truman Veterans’ Affairs Hospital, as well as the Electron Microscope Core Center at the University of Missouri-Columbia for their help with tissue preparation of animal specimens.

Sources of Funding

This research was supported by the National Institutes of Health (R01 HL73101-01A1; to J.R.S.), the Veterans’ Affairs Merit System grants 0018 (to J.R.S.) and CDA-2 and VISN15 (to A.W.C.), and the Forest Research Institute.

Disclosures

J.R.S. received investigator-initiated support from the Forest Research Institute.

Footnotes

Correspondence to James R. Sowers, Department of Internal Medicine, University of Missouri, 1 Hospital Drive, Columbia, MO 65212. E-mail

References

  • 1 Alpert MA, Lambert CR, Terry BE, Cohen MV, Mukerji V, Massey CV, Hashimi MW, Panayiotou H. Interrelationship of left ventricular mass, systolic function and diastolic filling in normotensive morbidly obese patients. Int J Obes Relat Metab Disord. 1995; 19: 550–557.MedlineGoogle Scholar
  • 2 Lauer MS, Anderson KM, Kannel WB, Levy D. The impact of obesity on left ventricular mass and geometry: the Framingham Heart Study. JAMA. 1991; 266: 231–236.CrossrefMedlineGoogle Scholar
  • 3 Wei Y, Whaley-Connell AT, Chen K, Habibi J, Uptergrove GM, Clark SE, Stump CS, Ferrario CM, Sowers JR. NADPH oxidase contributes to vascular inflammation, insulin resistance, and remodeling in the transgenic (mRen2) rat. Hypertension. 2007; 50: 384–391.LinkGoogle Scholar
  • 4 Munzel T, Daiber A, Ullrich V, Mulsch A. Vascular consequences of endothelial nitric oxide synthase uncoupling for the activity and expression of the soluble guanylyl cyclase and the cGMP-dependent protein kinase. Arterioscler Thromb Vasc Biol. 2005; 25: 1551–1557.LinkGoogle Scholar
  • 5 Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840–844.CrossrefMedlineGoogle Scholar
  • 6 Sawyer DB, Siwik DA, Xiao L, Pimentel DR, Singh K, Colucci WS. Role of oxidative stress in myocardial hypertrophy and failure. J Mol Cell Cardiol. 2002; 34: 379–388.CrossrefMedlineGoogle Scholar
  • 7 Sorescu D, Griendling KK. Reactive oxygen species, mitochondria, and NAD(P)H oxidases in the development and progression of heart failure. Congest Heart Fail. 2002; 8: 132–140.CrossrefMedlineGoogle Scholar
  • 8 Griendling KK, Ushio-Fukai M. Reactive oxygen species as mediators of angiotensin II signaling. Regul Pept. 2000; 91: 21–27.CrossrefMedlineGoogle Scholar
  • 9 Takimoto E, Kass DA. Role of oxidative stress in cardiac hypertrophy and remodeling. Hypertension. 2007; 49: 241–248.LinkGoogle Scholar
  • 10 Nodari S, Metra M, Dei CL. β-blocker treatment of patients with diastolic heart failure and arterial hypertension: a prospective, randomized, comparison of the long-term effects of atenolol vs. nebivolol. Eur J Heart Fail. 2003; 5: 621–627.CrossrefMedlineGoogle Scholar
  • 11 Ghio S, Magrini G, Serio A, Klersy C, Fucili A, Ronaszeki A, Karpati P, Mordenti G, Capriati A, Poole-Wilson PA, Tavazzi L. Effects of nebivolol in elderly heart failure patients with or without systolic left ventricular dysfunction: results of the SENIORS echocardiographic substudy. Eur Heart J. 2006; 27: 562–568.CrossrefMedlineGoogle Scholar
  • 12 van Veldhuisen DJ, Cohen-Solal A, Bohm M, Anker SD, Babalis D, Roughton M, Coats AJ, Poole-Wilson PA, Flather MD. β-blockade with nebivolol in elderly heart failure patients with impaired and preserved left ventricular ejection fraction: data from SENIORS (Study of Effects of Nebivolol Intervention on Outcomes and Rehospitalization in Seniors With Heart Failure). J Am Coll Cardiol. 2009; 53: 2150–2158.CrossrefMedlineGoogle Scholar
  • 13 Manrique C, Whaley-Connell A, Sowers JR. Nebivolol in obese and non-obese hypertensive patients. J Clin Hypertens (Greenwich). 2009; 11: 309–315.CrossrefMedlineGoogle Scholar
  • 14 Celik T, Iyisoy A, Kursaklioglu H, Kardesoglu E, Kilic S, Turhan H, Yilmaz MI, Ozcan O, Yaman H, Isik E, Fici F. Comparative effects of nebivolol and metoprolol on oxidative stress, insulin resistance, plasma adiponectin and soluble P-selectin levels in hypertensive patients. J Hypertens. 2006; 24: 591–596.CrossrefMedlineGoogle Scholar
  • 15 Rizos E, Bairaktari E, Kostoula A, Hasiotis G, Achimastos A, Ganotakis E, Elisaf M, Mikhailidis DP. The combination of nebivolol plus pravastatin is associated with a more beneficial metabolic profile compared to that of atenolol plus pravastatin in hypertensive patients with dyslipidemia: a pilot study. J Cardiovasc Pharmacol Ther. 2003; 8: 127–134.CrossrefMedlineGoogle Scholar
  • 16 Kurtz TW, Morris RC, Pershadsingh HA. The Zucker fatty rat as a genetic model of obesity and hypertension. Hypertension. 1989; 13: 896–901.LinkGoogle Scholar
  • 17 Poornima IG, Parikh P, Shannon RP. Diabetic cardiomyopathy: the search for a unifying hypothesis. Circ Res. 2006; 98: 596–605.LinkGoogle Scholar
  • 18 Vincent HK, Powers SK, Stewart DJ, Shanely RA, Demirel H, Naito H. Obesity is associated with increased myocardial oxidative stress. Int J Obes Relat Metab Disord. 1999; 23: 67–74.CrossrefMedlineGoogle Scholar
  • 19 Whaley-Connell A, Govindarajan G, Habibi J, Hayden MR, Cooper SA, Wei Y, Ma L, Qazi M, Link D, Karuparthi PR, Stump CS, Ferrario CM, Sowers JR. Angiotensin-II mediated oxidative stress promotes myocardial tissue remodeling in the transgenic TG (mRen2) 27 Ren2 rat. Am J Physiol Endocrinol Metab. 2007; 293: E355–E363.CrossrefMedlineGoogle Scholar
  • 20 Srere PA. Citrate synthase. Methods Enzymol. 1969; 13: 3–5.CrossrefGoogle Scholar
  • 21 Bass A, Brdiczka D, Eyer P, Hofer S, Pette D. Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur J Biochem. 1969; 10: 198–206.CrossrefMedlineGoogle Scholar
  • 22 Whaley-Connell A, Habibi J, Cooper SA, DeMarco VG, Hayden MR, Stump CS, Link D, Ferrario C, Sowers JR. Effect of renin inhibition and AT1R blockade on myocardial remodeling in the transgenic Ren2 rat. Am J Physiol Endocrinol Metab. 2008; 295: E103–E109.CrossrefMedlineGoogle Scholar
  • 23 Messerli FH, Bell DS, Fonseca V, Katholi RE, McGill JB, Phillips RA, Raskin P, Wright JT Jr, Bangalore S, Holdbrook FK, Lukas MA, Anderson KM, Bakris GL. Body weight changes with β-blocker use: results from GEMINI. Am J Med. 2007; 120: 610–615.CrossrefMedlineGoogle Scholar
  • 24 Nishimura RA, Tajik AJ. Evaluation of diastolic filling of left ventricle in health and disease: Doppler echocardiography is the clinician’s Rosetta Stone. J Am Coll Cardiol. 1997; 30: 8–18.CrossrefMedlineGoogle Scholar
  • 25 Nishio Y, Kanazawa A, Nagai Y, Inagaki H, Kashiwagi A. Regulation and role of the mitochondrial transcription factor in the diabetic rat heart. Ann N Y Acad Sci. 2004; 1011: 78–85.CrossrefMedlineGoogle Scholar
  • 26 Duncan JG, Fong JL, Medeiros DM, Finck BN, Kelly DP. Insulin-resistant heart exhibits a mitochondrial biogenic response driven by the peroxisome proliferator-activated receptor-α/PGC-1α gene regulatory pathway. Circulation. 2007; 115: 909–917.LinkGoogle Scholar
  • 27 Stas S, Whaley-Connell A, Habibi J, Appesh L, Hayden MR, Karuparthi PR, Qazi M, Morris EM, Cooper SA, Link CD, Stump C, Hay M, Ferrario C, Sowers JR. Mineralocorticoid receptor blockade attenuates chronic overexpression of the renin-angiotensin-aldosterone system stimulation of NADPH oxidase and cardiac remodeling. Endocrinology. 2007; 148: 3773–3780.CrossrefMedlineGoogle Scholar
  • 28 Serpillon S, Floyd BC, Gupte RS, George S, Kozicky M, Neito V, Recchia F, Stanley W, Wolin MS, Gupte SA. Superoxide production by NAD(P)H oxidase and mitochondria is increased in genetically obese and hyperglycemic rat heart and aorta before the development of cardiac dysfunction: the role of glucose-6-phosphate dehydrogenase-derived NADPH. Am J Physiol Heart Circ Physiol. 2009; 297: H153–H162.CrossrefMedlineGoogle Scholar
  • 29 Ritchie RH, Delbridge LM. Cardiac hypertrophy, substrate utilization and metabolic remodelling: cause or effect? Clin Exp Pharmacol Physiol. 2006; 33: 159–166.CrossrefMedlineGoogle Scholar
  • 30 Luiken JJ, Arumugam Y, Dyck DJ, Bell RC, Pelsers MM, Turcotte LP, Tandon NN, Glatz JF, Bonen A. Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats. J Biol Chem. 2001; 276: 40567–40573.CrossrefMedlineGoogle Scholar
  • 31 Coort SL, Hasselbaink DM, Koonen DP, Willems J, Coumans WA, Chabowski A, Van d V, Bonen A, Glatz JF, Luiken JJ. Enhanced sarcolemmal FAT/CD36 content and triacylglycerol storage in cardiac myocytes from obese zucker rats. Diabetes. 2004; 53: 1655–1663.CrossrefMedlineGoogle Scholar
  • 32 Young ME, Guthrie PH, Razeghi P, Leighton B, Abbasi S, Patil S, Youker KA, Taegtmeyer H. Impaired long-chain fatty acid oxidation and contractile dysfunction in the obese Zucker rat heart. Diabetes. 2002; 51: 2587–2595.CrossrefMedlineGoogle Scholar
  • 33 Katakam PV, Tulbert CD, Snipes JA, Erdos B, Miller AW, Busija DW. Impaired insulin-induced vasodilation in small coronary arteries of Zucker obese rats is mediated by reactive oxygen species. Am J Physiol Heart Circ Physiol. 2005; 288: H854–H860.CrossrefMedlineGoogle Scholar
  • 34 Laight DW, Anggard EE, Carrier MJ. Investigation of basal endothelial function in the obese Zucker rat in vitro. Gen Pharmacol. 2000; 35: 303–309.CrossrefMedlineGoogle Scholar
  • 35 Russo I, Del MP, Doronzo G, Mattiello L, Viretto M, Bosia A, Anfossi G, Trovati M. Resistance to the nitric oxide/cyclic guanosine 5′-monophosphate/protein kinase G pathway in vascular smooth muscle cells from the obese Zucker rat, a classical animal model of insulin resistance: role of oxidative stress. Endocrinology. 2008; 149: 1480–1489.CrossrefMedlineGoogle Scholar
  • 36 Oelze M, Daiber A, Brandes RP, Hortmann M, Wenzel P, Hink U, Schulz E, Mollnau H, von SA, Kleschyov AL, Mulsch A, Li H, Forstermann U, Munzel T. Nebivolol inhibits superoxide formation by NADPH oxidase and endothelial dysfunction in angiotensin II-treated rats. Hypertension. 2006; 48: 677–684.LinkGoogle Scholar
  • 37 Massion PB, Dessy C, Desjardins F, Pelat M, Havaux X, Belge C, Moulin P, Guiot Y, Feron O, Janssens S, Balligand JL. Cardiomyocyte-restricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates β-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction. Circulation. 2004; 110: 2666–2672.LinkGoogle Scholar
  • 38 Massion PB, Feron O, Dessy C, Balligand JL. Nitric oxide and cardiac function: ten years after, and continuing. Circ Res. 2003; 93: 388–398.LinkGoogle Scholar
  • 39 Kadi A, Moby V, de IN, Lacolley P, Menu P, Stoltz JF. Signalling transduction pathways implicated in Nebivolol-induced NO production in endothelial cells. Biomed Mater Eng. 2008; 18: 303–307.MedlineGoogle Scholar
  • 40 Dessy C, Saliez J, Ghisdal P, Daneau G, Lobysheva II, Frerart F, Belge C, Jnaoui K, Noirhomme P, Feron O, Balligand JL. Endothelial β3-adrenoreceptors mediate nitric oxide-dependent vasorelaxation of coronary microvessels in response to the third-generation β-blocker nebivolol. Circulation. 2005; 112: 1198–1205.LinkGoogle Scholar
  • 41 Manrique C, Johnson M, Sowers JR. Thiazide diuretics alone or with β-blockers impair glucose metabolism in hypertensive patients with abdominal obesity. Hypertension. 2010; 55: 15–17.LinkGoogle Scholar
  • 42 Solak Y, Atalay H. Nebivolol in the treatment of metabolic syndrome: making the fat more brownish. Med Hypotheses. In press.Google Scholar
  • 43 Arch JR. The discovery of drugs for obesity, the metabolic effects of leptin and variable receptor pharmacology: perspectives from β3-adrenoceptor agonists. Naunyn Schmiedebergs Arch Pharmacol. 2008; 378: 225–240.CrossrefMedlineGoogle Scholar