Nebivolol improves diastolic dysfunction and myocardial remodeling through reductions in oxidative stress in the Zucker obese rat

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 beta-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 x kg(-) x 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 microL/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.

Figure 1. Nebivolol improves diastolic relaxation, reduces myocardial fibrosis, and improves ultrastructural remodeling of myocardial capillaries

A, Representative cine-MRI images illustrate early diastole phases (frame 7–12 of 16 captured) in a cardiac cycle. The upper row demonstrates prolonged diastolic relaxation time in ZO-C compared to reduced diastolic relaxation time and increased initial filling rate in ZO-N shown in the lower 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 TEM micrographs at ×400 demonstrate constricted capillaries in ZO-C that improved with nebivolol treatment in ZO-N (upper panels). Small dark arrows point to a capillaris which are shown at higher magnification in the white boxes. Scale bar = 2μm. The X in the center of the lower 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 comprising the capillary wall, a prominent layer of pericapillary collagen, and cardiomyocytes. A pericapillary collagen layer was not observed in the ZO-N (lower right panel). White arrows indicate an area of abundant endothelial cell transcytotic vesicles in the ZO-N.

Figure 2. Nebivolol improves myocardial mitochondrial function

A, Representative left ventricular 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 to all other groups. Bar graph to the right 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 TEM micrographs at ×1,000. Compared to ZL-C (upper left) ZO-C rats possess increased numbers of intermyofibrillar mitochondrial and a disorganized sarcomere structure (lower left). An abrogation of the increased mitochondrial biogenesis was observed in ZO-N rats (lower right). Scale bar=1 μm. E, Representative TEM micrographs show intermyofibrillar mitochondria at ×6,000. Compared to ZL-C (upper left) the ZO-C myocardium (lower left) shows multiple layers of mitochondria between myofibrils compared to 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 (lower right). Scale bar=200nm. Arrows point to mitochondria and arrowheads point to lipid droplets.

Figure 3. Nebivolol reduces NADPH oxidase subunit protein expression

A, The bar graph demonstrates increased NADPH oxidase activity in the myocardium of ZO-C relative to ZL-C (P<0.05). Nebivolol reduces NADPH oxidase activity in the ZO myocardium compared to ZO-C (P<0.05). B, The bar graph demonstrates increased formation of superoxide in the myocardium of ZO-C relative to ZL-C (P<0.05). Nebivolol reduces superoxide formation in the ZO myocardium compared to ZO-C (P<0.05). C, Representative confocal images showing immunofluorescence for NADPH oxidase membrane bound proteins Nox2 and Nox4. B, Bar graphs show average gray scale intensities for NADPH oxidase subunits Nox2, Nox4, p47^phox and Rac1 as. D Representative photomicrographs showing 3-nitrotyrosine immunostaining in the myocardium of ZL and ZO rats. In the bar graph to the right average grayscale intensity measures of the 3-nitrotyrosine staining show that nebivolol blunts the increase in 3-nitrotyrosine levels in the ZO myocardium. Scale bar=50μm. *P<0.05 vs ZL-C; †P<0.05 vs ZO-C.

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 (i.e., 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 to 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 to 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 to total eNOS expressed as a percentage of ZL-C control. D, Representative confocal micrographs show total eNOS (upper row) and phospho-eNOS Ser¹¹⁷⁷ (lower row) immunofluorescence in the myocardium and coronary arterioles of ZL and ZO rats. Scale bar=50 μm. The bar graphs below show average grayscale intensity measures of total eNOS (left) and phospho-eNOS Ser¹¹⁷⁷ (right) immunofluorescence in the endothelium of coronary arterioles of ZL and ZO rats and indicate that nebivolol enhances eNOS phosphorylation at serine¹¹⁷⁷ *P<0.05 vs ZL-C; †P<0.05 vs ZO-C.