Cardiac steatosis potentiates angiotensin II effects in the heart - PubMed (original) (raw)

Cardiac steatosis potentiates angiotensin II effects in the heart

Denis J Glenn et al. Am J Physiol Heart Circ Physiol. 2015.

Abstract

Lipid accumulation in the heart is associated with obesity and diabetes and may play an important role in the pathogenesis of heart failure. The renin-angiotensin system is also thought to contribute to cardiovascular morbidity in obese and diabetic patients. We hypothesized that the presence of lipid within the myocyte might potentiate the cardiomyopathic effects of ANG II in the cardiac diacylglycerol acyl transferase 1 (DGAT1) transgenic mouse model of myocyte steatosis. Treatment with ANG II resulted in a similar increase in blood pressure in both nontransgenic and DGAT1 transgenic mice. However, ANG II in DGAT1 transgenic mice resulted in a marked increase in interstitial fibrosis and a reduction in systolic function compared with nontransgenic littermates. Lipidomic analysis revealed that >20% of lipid species were significantly altered between nontransgenic and DGAT1 transgenic animals, whereas 3% were responsive to ANG II administration. ROS were also increased by ANG II in DGAT1 transgenic hearts. ANG II treatment resulted in increased expression of transforming growth factor (TGF)-β2 and the type I TGF-β receptor as well as increased phosphorylation of Smad2 in DGAT1 transgenic hearts. Injection of neutralizing antibodies to TGF-β resulted in a reduction in fibrosis in DGAT1 transgenic hearts treated with ANG II. These results suggest that myocyte steatosis amplifies the fibrotic effects of ANG II through mechanisms that involve activation of TGF-β signaling and increased production of ROS.

Keywords: angiotensin; diacylglycerol acyl transferase 1; fibrosis; lipotoxixity; transforming growth factor-β.

Copyright © 2015 the American Physiological Society.

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Figures

Fig. 1.

Fig. 1.

Blood pressure in non-transgenic (NTg) and α-myosin heavy chain promoter of diacylglycerol acyltransferase 1 (MHC-DGAT1) transgenic (Tg) mice. Mice were treated with either saline (sham) or ANG II (500 ng·kg−1·min−1) delivered by osmotic minipump for 14 days. A–C: systolic blood pressure (BP; A), diastolic BP (B), and mean arterial pressure (MAP; C) were assessed in NTg and MHC-DGAT1 Tg mice before treatment and on day 13 postsurgery. n = 6 NTg, NTg + ANG II, and MHC-DGAT1 Tg mice and 9 MHC-DGAT1 Tg + ANG II mice. Significance is indicated. NS, not significant.

Fig. 2.

Fig. 2.

Cardiac hypertrophy and fibrosis in MHC-DGAT1 Tg mice treated with ANG II. A–C: representative left ventricular (LV) micrographs in sham and ANG II-treated NTg and MHC-DGAT1 Tg mice stained with hematoxylin and eosin (A), oil red O (B), where red fluorescence indicates lipid accumulation, and Masson's trichrome stain (C), where blue-green stain indicates fibrosis. Bars = 50 μm. D and E: calculated heart weight-to-body weight ratios (HW/BW; D) and heart weight-to tibia-length ratios (HW/TL; E). F: atrial natriuretic peptide (ANP) gene expression normalized to GAPDH expression and compared with the NTg sham group as assessed by quantitative PCR. G: quantification of fibrotic area, expressed as a percentage of tissue area. H and I: gene expression analysis of collagen type 1A1 (Col1a1; H) and type 3A1 (Col3A1; I) normalized to GAPDH expression. n = 6 NTg, NTg + ANG II, and MHC-DGAT1 Tg mice and 9 MHC-DGAT1 Tg + ANG II mice.

Fig. 3.

Fig. 3.

Cardiac function is impaired in MHC-DGAT1 Tg mice treated with ANG II. A: representative transmitral blood flow Doppler images. Mitral inflow early filling velocity (E), mitral inflow late filling (atrial contraction) velocity (A), and deceleration time (DT) are shown in the first image and quantified as the _E_-to-A ratio (E/A; C) and DT (D). B: representative M-mode echocardiographs from NTg and MHC-DGAT1 Tg sham and ANG II-treated mice. Intaventricular septal (IVS), LV posterior wall (LVPW), and LV internal diameter (LVID) in systole (s) and diastole (d) are shown in the first image. E and F: fractional shortening (FS; E) and LV ejection fraction (EF; F) were quantified. n = 6 NTg, NTg + ANG II, and MHC-DGAT1 Tg mice and 9 MHC-DGAT1 Tg + ANG II mice.

Fig. 4.

Fig. 4.

Role of ROS in MHC-DGAT1 Tg hearts treated with ANG II. A: fresh frozen LV sections stained with the ROS-sensitive dye dihydroethidium (DHE). Representative fluorescent images are shown. B: DHE-stained images were quantified and normalized to NTg control fluorescence intensity (n = 4 for each group). C: lipid peroxidation as assessed by the quantification of 4-hydroxynonenal (HNE) content in LV tissue (n = 6 for each group). D: quantification of 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA oxidative stress, in LV samples (n = 6 for each group). E: gene expression analysis of SOD2. F: SOD2 activity normalized to milligrams of soluble protein (n = 6 for each group). G: Gene expression analysis of NADPH oxidase (Nox)4. Gene expression results were normalized to GADPH expression and compared with the NTg sham group. n = 6 NTg, NTg + ANG II, and MHC-DGAT1 Tg mice and 9 MHC-DGAT1 Tg + ANG II mice.

Fig. 5.

Fig. 5.

Role of transforming growth factor (TGF)-β in MHC-DGAT1 Tg hearts. A–C: gene expression analysis of TGF-β2 (A), TGF-β receptor (TGFBR)1 (B), and TGFBR2 (C). Results were normalized to GADPH expression and compared with the NTg sham group (n = 6 for each group). D: representative Western blot analysis of TGFBR1 expression (top) from membrane preparations of NTg and MHC-DGAT1 Tg hearts. Expression of β-tubulin is shown as a loading control (bottom). E: results were quantified, and TGFBR1 expression was normalized to β-tubulin (n = 4 for each group). F: representative Western blot analysis of phosphorylated (p-)Smad2 (top) and total Smad2 (bottom). G: results were quantified and normalized to total Smad2 (n = 4 for each group). H: representative Masson's trichrome-stained LV micrographs in sham and ANG II-treated NTg and MHC-DGAT1 Tg mice treated with IgG or TGF-β-neutralizing antibody (NAb), where blue-green stain indicates fibrosis. Bar = 50 μm. I: quantification of the fibrotic area, expressed as a percentage of tissue area (n = 4 for each group). J: Western blot analysis of p-Smad2 (top) and total Smad2 (bottom) in MHC-DGAT1 Tg hearts treated with ANG II and either IgG or TGF-β NAb.

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