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. Author manuscript; available in PMC: 2013 Apr 10.
Published in final edited form as: Circulation. 2012 Mar 2;125(14):1757–1764. doi: 10.1161/CIRCULATIONAHA.111.067801

The Polyphenols Resveratrol and S17834 prevent the Structural and Functional Sequelae of Diet-Induced Metabolic Heart Disease in Mice

Fuzhong Qin 1,*, Deborah A Siwik 1,*, Ivan Luptak 1, Xiuyun Hou 1, Lei Wang 1, Akiko Higuchi 1, Robert M Weisbrod 1, Noriyuki Ouchi 1, Vivian H Tu 1, Timothy D Calamaras 1, Edward J Miller 1, Tony J Verbeuren 1, Kenneth Walsh 1, Richard A Cohen 1, Wilson S Colucci 1
PMCID: PMC3354628  NIHMSID: NIHMS368092  PMID: 22388319

Abstract

Background

Diet-induced obesity is associated with metabolic heart disease characterized by left ventricular (LV) hypertrophy and diastolic dysfunction. Polyphenols such as resveratrol (RSV) and the synthetic flavonoid derivative S17834 exert beneficial systemic and cardiovascular effects in a variety of settings including diabetes and chronic hemodynamic overload.

Methods and Results

We characterized the structural and functional features of a mouse model of diet-induced metabolic syndrome, and used the model to test the hypothesis that the polyphenols prevent myocardial hypertrophy and diastolic dysfunction. Male C57BL/6J mice were fed a normal diet or a diet high in fat and sugar (HFHS) with or without concomitant treatment with S17834 or RSV for up to 8 months. HFHS diet-fed mice developed progressive LV hypertrophy and diastolic dysfunction with preservation of systolic function in association with myocyte hypertrophy and interstitial fibrosis. In HFHS-fed mice there was increased myocardial oxidative stress with evidence of oxidant-mediated protein modification via tyrosine nitration and 4-OH-2-nonenol (HNE) adduction. HFHS-fed mice also exhibited increases in plasma fasting glucose, insulin and HOMA-IR indicative of insulin resistance. Treatment with S17834 or RSV prevented LV hypertrophy and diastolic dysfunction. For S17834, these beneficial effects were associated with decreases in oxidant-mediated protein modifications and hyper-insulinemia, and increased plasma adiponectin.

Conclusions

RSV and S17834 administered concurrently with a HFHS diet prevent the development of LV hypertrophy, interstitial fibrosis and diastolic dysfunction. Multiple mechanisms may contribute to the beneficial effects of the polyphenols including a reduction in myocardial oxidative stress and related protein modifications, amelioration of insulin resistance and increased plasma adiponectin. The polyphenols RSV and S17834 may be of value in the prevention of diet-induced metabolic heart disease.

Keywords: left ventricular hypertrophy, diastolic dysfunction, 4-OH-2-nonenol, metabolic syndrome, oxidative stress

Introduction

The prevalence of diet-induced obesity, diabetes and the metabolic syndrome (MS) is increasing at an alarming rate and is now a major contributor to cardiovascular morbidity and mortality 1, 2, including heart failure 3. The MS, defined as the constellation of obesity, diabetes, hypertension and increased triglycerides 4 is associated with left ventricular (LV) hypertrophy and impaired diastolic function that can lead to heart failure with a preserved ejection fraction (HFpEF) 5. The mechanism responsible for myocardial hypertrophy and diastolic dysfunction in MS is incompletely understood.

Transgenic mouse models with inherent derangements in glucose and/or lipid handling have provided important insight regarding the pathobiology of diastolic dysfunction in metabolic heart disease 6, 7. However, since MS is often diet-induced, it is desirable to study the cardiovascular consequences in a model in which the syndrome is also diet-induced. The C57BL/6J mouse fed an “American” diet high in fat and sugar (HFHS) is a commonly used model of diet-induced obesity that is associated with diabetes, hypertension and increased serum trigylcerides 8–11. Very little is known about the cardiac phenotype of these mice, and there is no information about LV diastolic function. We theorized that HFHS feeding would cause a cardiac phenotype typical of metabolic heart disease with myocardial hypertrophy, diastolic dysfunction and preservation of systolic function. Accordingly, our first goal was to characterize the myocardial structural and functional features associated with a chronic HFHS diet.

Polyphenols exert pleiotropic actions that may be beneficial in MS, including anti-inflammatory and anti-oxidant effects 12, 13 and activation of sirtuins 14. Several studies have demonstrated beneficial effects of resveratrol (RSV) or the synthetic flavonoid derivative S17834 (6,8-diallyl 5,7-dihydroxy 2-(2-allyl 3-hydroxy 4-methoxyphenyl)1-H benzo(b)pyran-4-one) on systemic 15 and cardiovascular 12, 13 abnormalities associated with diabetes 16, 17, ischemia/reperfusion 18, pressure overload 19, 20, hypertension 21 and myocardial infarction 22. However, the ability of the polyphenols to ameliorate metabolic heart disease associated with diet-induced MS is not known. Accordingly, our second goal was to test the hypothesis that S17834 and RSV prevent LV hypertrophy and diastolic dysfunction in mice fed a HFHS diet. The mechanism responsible for the beneficial effect of the polyphenols on cardiac structure and function is not understood at the molecular level, and may vary depending on the underlying pathophysiology. HFHS feeding is associated with multiple metabolic abnormalities that may cause myocardial hypertrophy or otherwise adversely affect cardiac structure and function including oxidative stress and hyper-insulinemia 23. Accordingly, our third goal was to identify the potential role of these mechanisms in mediating the cardiac effects of polyphenols in this model of MS.

Methods

Experimental animals

Male C57BL/6J mice 8 weeks of age were fed a normal chow diet (Teklad Global 18% Protein Rodent Diet, Product # 2018, Harlan Laboratories) or a HFHS diet (Bio-Serv Product # F1850) containing 35.5% fat (primarily lard) and 36.3% carbohydrate (primarily sucrose). Some of the mice in the HFHS group also received S17834 (130 mg/kg/day) or RSV (130 mg/kg/day) compounded into the food for 8 months (S17834) or 4 months (RSV). S17834 was obtained from the Institut de Recherches Servier, and RSV was obtained from Orchid Chemicals & Pharmaceuticals (Nungambakkam, Chennai, India). The protocol was approved by the Institutional Animal Care and Use Committee at Boston University School of Medicine.

Measurement of plasma glucose, insulin, lipids and adiponectin

Blood was collected from the tail vein, centrifuged and the plasma was separated. Plasma glucose levels were measured by glucose meters (ACCU-CHEK, Roche Applied Science, Indianapolis, IN). Plasma insulin level was measured using ELISA kits (Crystal Chem, Downers Grove, IL). The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated using the following formula: [fasting plasma glucose (mg/dl) X fasting plasma insulin (μU/ml)]/405. Plasma triglyceride, cholesterol and free fatty acids were measured enzymatically using infinity reagents (Thermo DMA, Louisville, CO) according to the manufacturer’s instructions. Plasma adiponectin levels were determined with adiponectin ELISA kits (Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan).

Two-dimensional and M-mode echocardiography

LV dimensions and systolic function were measured in non-anesthetized mice using an Acuson Sequoia C-256 echocardiograph machine equipped with a 15 MHz linear transducer (model 15L8), as we have described 24. Briefly, the heartwas imaged in the 2-D parasternal short-axis view, and an M-mode echocardiogram of the mid-ventricle was recordedat the level of papillary muscles. Anterior wall thickness (AWT), posterior wall thickness (PWT), LV end-diastolic (EDD) and end-systolic (ESD) dimensions were measured from the M-mode image. LV fractional shortening (FS) was calculated as (EDD–ESD)/EDD × 100.

Doppler echocardiography

LV diastolic function was assessed by transmitral and tissue Doppler echocardiography using a VisaulSonics Vevo 770 high-resolution imaging system (Toronto, Canada) equipped with a 30-MHz RMV-707B transducer 25, 26. Briefly, mice were anesthetized with isofluorane by a facemask at a concentration of 2.5% for induction and then 1.5% for maintenance. Pulsed-wave Doppler images were collected in the apical four-chamber view to record the mitral Doppler flow spectra. Peak early (E) and late (A) mitral inflow velocities, E/A ratio, deceleration time of early filing (DT) and isovolumetric relaxation time (IVRT) were measured. Tissue Doppler images were collected in the parasternal short-axis view. Myocardial peak early diastolic velocity (Em) was measured, and E/Em was calculated. Doppler spectra were recorded for 12–14 cardiac cycles, from which at least 5 consecutive cardiac cycles were selected and the values were averaged in accordance with the American Society of Echocardiography guidelines 27. Data analysis was performed offline with the use of a customized version of Vevo 770 Analytic software.

Organ weight and histology

The mice were sacrificed at the end of study. Heart and LV with septum were weighed, and LV samples were fixed in 10% buffered formalin, embedded with paraffin and sectioned. Myocyte cross-sectional area and fibrosis were measured as previously described 24. Briefly, sections were stained with hematoxylin and eosin and examined under a light microscopy (BX 40, Olympus). Five random fields from each of 4 sections per animal were analyzed and 60 myocytes per animal were measured. The quantification of myocyte diameter was determined using NIH ImageJ software. To assess fibrosis, sections were stained with Masson’s trichrome kit (Sigma) and examined under a light microscope (BX 40, Olympus).

Immunohistochemistry for 3-nitrotyrosine and 4-hydroxy-2-nonenal

Immunohistochemistry was performed as previously described 24. Briefly, LV tissue sections (4 μm) were blocked with 10% goat serum in phosphate-buffered saline, incubated with rabbit anti-3-nitrotyrosine polyclonal antibody or mouse anti-HNE monoclonal antibody, and incubated with goat biotin-conjugated anti-rabbit IgG or goat biotin-conjugated anti-mouse IgG (Vector Laboratory, Burlingame, CA). The sections were incubated with avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratory) and stained with 3-amino-9-ethylcarbazole (Vector Laboratory) and hematoxylin (Vector Laboratory). The samples were examined under a light microscope (BX 40, Olympus).

Immunoprecipitation and immunoblots

For immunoprecipitation of LKB-1, frozen LV were homogenized in 1X RIPA buffer (Cell Signaling) with 1 mM PMSF and 1% protease inhibitor set I (Calbiochem). Total protein (250μg) was incubated with mouse anti-LKB1 (Santa Cruz) overnight at 4°C. Protein A/G agarose beads were added and incubated for 1 hr at 4°C. After 3 washes, proteins were eluted in Laemmli buffer, separated by SDS-PAGE, and transferred to PVDF membranes. Blots were incubated with rabbit anti-HNE (Calbiochem), then goat anti-mouse IgG IRDye 800CW and quantified using the Odyssey Infrared Imaging System (LICOR Biosciences). Blots were stripped and reprobed with goat anti-LKB1 (Santa Cruz) then donkey anti-goat IgG IRDye 680. Immunoblots for AMPK were performed on frozen LV that was homogenized in tissue lysis buffer (Hepes pH 7.4 20mM, B-Glycerol phosphate 50 mM. EGTA 2mM, DTT 1mM, NaF 10mM, NaVO4 1mM, Triton-X 100 1%. Glycerol 10 %, and 1 protease inhibitor complete mini tablet-EDTA free/20 ml, RochTotal protein (25 μg) was separated by SDS-PAGE and transferred to PVDF membranes. Blots were incubated with rabbit anti-phosphoThr172-AMPK (Cell Signaling) and detected using the Licor Odyssey fluorescent system.

Statistical analysis

Results are presented as mean ± SEM. The statistical significance of differences among groups or between two means was determined using analysis of variance and the Bonferroni adjustment for multiple comparisons. A Bonferroni-adjusted p-value <0.05 was considered significant. Pressure-volume curves were analyzed by repeated measures 2-way ANOVA.

Results

HFHS induces time-dependent LV hypertrophy (Figure 1)

Figure 1.

Figure 1

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Total wall thickness, relative wall thickness (RWT), LV end-diastolic (EDD) and end-systolic (ESD) dimensions, and LV fractional shortening (FS) in mice fed a normal chow diet (open bars), a HFHS diet (diagonal bars), or a HFHS diet + S17834 (hatched bars). Values are means ± SEM; n=3–4. Within each time point for each variable, a Bonferroni adjustment was applied to account for the 3 possible pairwise group comparisons. *P<0.05 vs. normal diet-fed mice. †P<0.05 vs. HFHS diet-fed mice.

In HFHS-fed mice, wall thickness was increased at 2 months and increased further at 5 and 8 months. The LV end-diastolic dimension likewise was increased at 2 months and increased further at 5 and 8 months. LV wall thickness relative to LV end-diastolic dimension (EDD) was unchanged at 2 and 5 months, but was increased at 8 months, indicating the development of concentric hypertrophy. LV fractional shortening (FS) was unchanged at any time, indicating that systolic function was preserved. Mice were sacrificed after 8 months of HFHS feeding. In HFHF-fed mice, heart and LV weights were increased 14% and 11%, respectively, relative to tibia length (Table 1), confirming the echocardiographic finding of LV hypertrophy.

Table 1.

Body and organ weights.

Normal HFHS HFHS+S17834
BW (g) 39.3 ± 2.3 45.0 ± 2.0* 48.0 ± 0.6*
TL (mm) 24.8 ± 0.5 24.7 ± 0.7 24.8 ± 0.3
HW (mg) 180.6 ± 16.2 208.6 ± 5.3* 184.9 ± 6.2†
HW/TL (mg/mm) 7.3 ± 0.5 8.5 ± 0.4* 7.5 ± 0.3†
LV W (mg) 126.1 ± 10.2 140.2 ± 8.9* 117.9± 6.1†
LV W/TL (mg/mm) 5.1 ± 0.3 5.7 ± 0.5* 4.8 ± 0.3†
RV W (mg) 21.2 ± 2.4 25.7 ± 1.2 24.0± 0.6
RW/TL (mg/mm) 0.9 ± 0.1 1.0 ± 0.1 1.0 ± 0.0
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BW: body weight; HW: heart weight; LV: left ventricular. W: weight; HFHS: high fat high sucrose. TL: Tibia length. N = 3–4. Values are mean ± SE.

*

P<0.05 vs. Normal.

†

P<0.05 vs. HFHS.

HFHS diet induces diastolic dysfunction

LV diastolic function was assessed after 8 months of HFHS feeding using transmitral and tissue Doppler echocardiography. There was prolongation of the isovolumetric relaxation time (IVRT) and deceleration time (DT), associated with a decrease in the E/A ratio (Figure 2). Em was decreased, indicative of slowed LV relaxation, and E/Em was increased, indicative of an increase in LA filling pressure. Taken together, these findings are internally consistent and demonstrate that HFHS feeding leads to impaired LV relaxation and filling 27.

Figure 2.

Figure 2

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Isovolumic relaxation time (IVRT), deceleration time (DT), the ratio of early-to-late diastolic mitral inflow velocity (E/A), myocardial peak early diastolic velocity (Em) and the ratio of peak early mitral inflow velocity to myocardial peak early diastolic velocity (E/Em) in mice fed a normal chow diet, a HFHS diet, or a HFHS diet + S17834 (S). Values are means ± SEM; n=3–4. *P<0.01 vs. normal diet-fed mice. †P<0.05 vs. HFHS diet-fed mice.

To further characterize LV function, hearts were subjected to Langendorff perfusion using the isovolumic, balloon-in-LV technique to allow assessment of LV function over a range of LV volumes 28. For any given LV volume, end-diastolic pressure was higher in HFHS-fed mice (Figure 3). LV systolic pressure was likewise shifted leftward, while LV developed pressure was similar to normal diet-fed mice, albeit at a smaller LV volume. These data indicate impaired LV filling with preserved systolic function, thus confirming the echocardiographic findings.

Figure 3.

Figure 3

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Isovolumic balloon-in-LV Langendorff measurements of LV peak systolic pressure (Panel A), end-diastolic pressure (Panel B) and developed pressure (Panel C) in mice fed a normal chow diet (triangles) or a HFHS diet (diamonds). The x-axis depicts LV balloon volume in μL/mg of LV weight. Values are means ± SEM; n=3. *P<0.001 vs. normal diet-fed mice by repeated measures 2-way ANOVA.

S17834 and RSV prevent LV hypertrophy and diastolic dysfunction in HFHS-fed mice

In HFHS-fed mice, the addition of S17834 to the diet prevented the increases in LV wall thickness (Figure 1), and heart and LV weights (Table 1). These effects were associated with improvement in diastolic function as assessed by Doppler echocardiography. The E/A ratio, DT and IVRT measured by transmitral Doppler were normalized, as was Em measured by tissue Doppler, and the ratio of E/Em (Figure 2). In HFHS-fed mice, RSV also prevented LV hypertrophy (Supplemental Figure 1) and improved diastolic function (Supplemental Figure 2). These effects were qualitatively and quantitatively similar to those observed with S17834.

S17834 prevents myocyte hypertrophy and interstitial fibrosis in HFHS-fed mice

Myocyte diameter was increased in HFHS-fed mice compared with mice fed a normal diet (Figure 4A, B). In HFHS-fed mice there was increased interstitial and perivascular fibrosis visualized by Masson trichrome staining (Figure 4C, D). Myocardial concentrations of glycogen and triglyceride were not increased in HFHS-fed mice (Supplemental Figure 3). Both myocyte hypertrophy and interstitial fibrosis were prevented by treatment with S17834 (Figure 4).

Figure 4.

Figure 4

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Effects of HFHS diet and S17834 (S) on myocyte diameter and cardiac fibrosis in left ventricular myocardium. Panel A shows photomicrographs of left ventricular tissue sections stained by hematoxylin and eosin. Panel B shows the quantification of myocyte diameter measured by NIH ImageJ. Panel C is representative photomicrographs of Masson trichrome staining for cardiac fibrosis showing myocardium in red and fibrosis in blue. Panel D shows the quantification of cardiac fibrosis measured by NIH ImageJ. The bar indicates 25 μm. Values are means ± SEM; n=3–4. *P<0.05 vs. normal diet-fed mice. †P<0.05 vs. HFHS diet-fed mice.

S17834 prevents oxidant-mediated post-translational protein modifications

Myocardial oxidative post-translational protein modifications were assessed histochemically using antibodies to 3-nitrotyrosine (NY) and the lipid peroxidation product 4-OH-2-nonenol (HNE). NY and HNE were markedly increased diffusely over myocytes in HFHS-fed mice, and the accumulation of both was prevented by treatment with S17834 (Figure 5A–D).

Figure 5.

Figure 5

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Effects of HFHS diet and S17834 (S) on myocardial nitrotyrosine and 4-hydroxy-2-nonenal (HNE) staining, and HNE-adducts of LKB. Shown are representative photomicrographs of myocardial nitrotyrosine staining (Panels A, B) and HNE staining (Panels C, D). Panel E shows representative immnoprecipitation (IP) and immunoblot (IB) data for HNE-adducts of LKB. Panel F shows group densitometry analysis. The bar indicates 25 μm. Values are means ± SEM; n=3–4. *P<0.05 vs. normal diet-fed mice. †P<0.05 vs. HFHS diet-fed mice.

In cardiac myocytes HNE-adducts have been shown to inhibit the activity of LKB, an upstream kinase for AMPK, thereby leading to increased down-stream hypertrophic signaling via mTOR/p70S6 kinase 29. To test for HNE-adducts of LKB, myocardium was immunoprecipitated with an antibody directed against HNE-lysine adducts and immunoblotted for LKB. LKB-HNE adducts were increased in HFHS-fed mice, and the increase was prevented by treatment with S17834 (Figure 5E, F). Although LKB is a regulator of AMPK, AMPK activity was not affected by HFHS-feeding or S17834 treatment (Supplemental Figure 4).

S17834 improves insulin sensitivity and increases plasma adiponectin level (Table 2)

Table 2.

Metabolic parameters.

Normal HFHS HFHS+S17834
Fasting plasma glucose (mg/dL) 87 ± 7 126 ± 10* 92± 12
Fasting plasma insulin (uIU/mL) 8.5 ± 0.5 15.8 ± 2.7* 10.6 ± 1.0
HOMA-IR 33 ± 4 93 ± 22 ** 44 ± 7
Plasma triglycerides (mg/dL) 86 ± 10 96 ± 10 101 ± 11
Plasma free fatty acid (mEq/L) 1.5 ± 0.1 1.4 ± 0.1 1.6 ± 0.1
Plasma cholesterol (mg/dL) 88 ± 4 126 ± 10 ** 145 ± 12
Plasma adiponectin (ug/mL) 14.1 ± 1.3 13.6± 1.1 20.7 ± 1.8†
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Plasma was obtained from mice fed normal diet, HFHS diet or HFHS diet plus S17834 for 8 weeks for triglycerides, free fatty acids, cholesterol and adiponectin (n = 9 – 14), or 5 weeks for fasting glucose, insulin and homeostasis model assessment of insulin resistance (HOMA-IR) index (n = 6). Values are mean ± SEM.

*

P < 0.05 vs. Normal,

**

P < 0.01 vs. Normal,

†

P < 0.05 vs. HFHS.

Consistent with prior reports 23, 30, fasting glucose, insulin and HOMA-IR index were increased in HFHS-fed mice. Treatment with S17834 decreased fasting glucose, insulin and HOMA-IR, suggesting improved insulin sensitivity. Plasma cholesterol was increased in HFHS-fed mice, but was not affected by S17834. Free fatty acids were not increased by HFHS, and were not affected by S17834. The plasma adiponectin level was not different in HFHS- vs. normal diet-fed mice, but was increased by treatment with S17834.

Discussion

This study provides several new findings with regard to the pathophysiology and treatment of metabolic heart disease. First, we demonstrate in mice that diet-induced obesity is associated with metabolic heart disease characterized by myocardial hypertrophy, diastolic dysfunction, myocyte hypertrophy, interstitial fibrosis, oxidant-mediated protein and lipid products, hyper-insulinemia and insulin resistance. Second, we show that treatment with S17834 or RSV prevents the cardiac structural and functional consequences of MS. Third, we show that treatment with S17834 exerts multiple actions that may account for the beneficial structural and functional effects including a) decreases in oxidative stress and oxidant-mediated protein modifications, b) amelioration of hyper-insulinemia/insulin resistance and c) an increase in plasma adiponectin.

LV hypertrophy and diastolic dysfunction in HFHS-fed mice

HFHS-induced obesity was associated with LV hypertrophy. HFHS-feeding caused a progressive increase in heart size with wall thickening and chamber growth leading to concentric hypertrophy. Heart and LV weights confirmed LV hypertrophy, and histological analysis revealed that organ growth was associated with increases in myocyte size and interstitial fibrosis. Myocardial triglyceride and glycogen were not increased in HFHS diet-fed mice, indicating that myocardial hypertrophy in this model is not due to accumulation of triglyceride or glycogen. LV hypertrophy was associated with impaired diastolic function. Doppler assessment of transmitral flow demonstrated prolongation of DT and IVRT times in association with a decrease in the ratio of the E/A wave measured by transmitral Doppler. Tissue Doppler further demonstrated a decrease in Em indicative of slowed LV relaxation, and an increase in the ratio of E/Em, reflective of an increase in left atrial pressure. All of these findings are indicative of impaired LV relaxation 27 and are typical of patients with metabolic syndrome 5. Diastolic dysfunction was further confirmed by isovolumic Langendorff perfusion, which demonstrated an upward shift in the diastolic pressure-volume relationship. In contrast, systolic function was preserved, as evidenced by normal fractional shortening on echocardiography and a normal developed pressure by Langendorff perfusion.

The HFHS-fed mouse has been used extensively to study the metabolic consequences of obesity 8–11. Despite the popularity of this model, the cardiac phenotype has not been characterized, and in particular, diastolic function has not been assessed. Our findings indicate that the cardiac phenotype of the HFHS-fed mouse is very similar to that in humans with metabolic heart disease 5.

An important mechanism of diastolic dysfunction is impaired myocardial relaxation due to abnormal calcium handling 31. As an initial approach to assessing the role of altered calcium handling in this model, we measured mRNA levels for several key calcium regulatory proteins including sarcoplasmic reticulum calcium ATPase, the ryanodine receptor, the sodium-calcium exchanger and the L-type calcium channel - all of which were unaffected by HFHS feeding (Supplemental Figure 5). While these data exclude a role for transcriptional dysregulation of calcium handling proteins in this model, it remains possible that there are alterations in protein function due to changes in protein turnover and/or post-translation modifications.

S17834 and RSV prevent LV hypertrophy and diastolic dysfunction

Both S17834 and RSV effectively prevented the development of LV hypertrophy and diastolic dysfunction. These effects were associated at the cellular level with prevention of cardiac myocyte hypertrophy and interstitial fibrosis. Prior studies with RSV have demonstrated beneficial effects on cardiac function in a variety of pathologic models. In hypertensive SHR rats, RSV prevented LV hypertrophy and improved diastolic function 21, 29. Likewise, RSV improved diastolic function in mice with type 1 diabetes due to STZ 17 or in db/db mice with type 2 diabetes 16. In contrast, RSV did not alleviate the extent of LV remodeling after myocardial infarction 32. Our report is the first demonstration of the cardiac effects of the synthetic flavonoid derivative S17834 in any condition. Prior studies have shown that S17834 can inhibit atherosclerosis in diabetic LDL receptor-deficient mice 13.

Mechanism of anti-hypertrophic effect of S17834

A prominent effect of S17834 and RSV was to prevent cardiac hypertrophy induced by HFHS feeding. Accordingly, we assessed mechanisms that are associated with hypertrophic signaling in cardiac myocytes. First, since we 33 and others 34 have shown that oxidant signaling can stimulate myocyte growth, we examined whether HFHS feeding was associated with increased oxidative stress in the myocardium, and if so, whether the increase was prevented by S17834. Immunohistochemistry showed generalized increases in 3-nitrotyrosine (NY) and the lipid peroxidation product, 4-OH-2-nonenol (HNE), indicative of oxidative stress in the myocardium. Furthermore, we found increased HNE adducts of LKB, a signaling molecule that has been implicated in the regulation of myocardial growth. In hypertensive SHR rats, Dolinsky et al 29 demonstrated increased LKB-HNE adducts in the myocardium that were associated with decreased activity of LKB and its downstream substrate AMPK, leading to de-inhibition of hypertrophic signaling via the mTOR-p70S6 kinase pathway. They further demonstrated that RSV prevented the increase in LKB-HNE adducts, restored LKB and AMPK activities, and inhibited hypertrophic signaling via mTOR-p70S6 kinase 29. In contrast, in HFHS-fed mice the increase in LKB-HNE adduct was not associated with a decrease in AMPK activity, and was not affected by S17834 treatment. Thus, although S17834 decreased myocardial oxidative stress as reflected by generalized decreases in nitrotyrosine and HNE, and prevented the oxidant-mediated lipid modification of at least one specific protein (LKB) implicated in the regulation of myocyte growth, myocyte hypertrophy in this model cannot be attributed to a decrease in LKB activity leading to a decrease in AMPK activity.

Secondly, we found that S17834 treatment a) ameliorated hyper-insulinemia/insulin resistance, and b) increased plasma adiponectin. Hyper-insulinemia, which may contribute to myocardial hypertrophy in type 2 diabetes 35, has been noted previously in this model 23, 30. Our finding that S17834 decreased plasma insulin is consistent with similar observations showing that RSV decreases plasma insulin in other models of type 2 diabetes 36. A decrease in plasma insulin levels might oppose myocardial hypertrophy by decreasing the stimulation of the PI3K/Akt/mTOR/p70S6 pathway 37. Finally, we found that S17834 treatment is associated with an increase in plasma adiponectin. The increase in plasma adiponectin with S17834 treatment occurred in the absence of weight loss, suggesting a mechanism that is independent of weight change. In this regard, RSV has been shown to up-regulate adiponectin in cultured adipocytes 38. Since we 39 and others 40 have demonstrated that adiponectin exerts an anti-hypertrophic effect in myocardium and cardiac myocytes, an increase in adiponectin provides a third mechanism by which S17834 might inhibit hypertrophic signaling in this model.

Implications

The HFHS-fed mouse provides a valuable model of diet-induced myocardial hypertrophy and diastolic dysfunction which should prove useful in elucidating the pathobiology and treatment of metabolic heart disease. This model is associated with increased myocardial oxidative stress and systemic hyper-insulinemia/insulin resistance, both of which may promote myocardial hypertrophy. The polyphenols exert multiple effects in this model that may contribute to decreased myocardial hypertrophy and improved diastolic function including a) a decrease in myocardial oxidative stress, b) a decrease in oxidant-mediated protein modifications, c) an improvement in hyper-insulinemia/insulin sensitivity and d) an increase in plasma adiponectin (Figure 6). The ability of the HFHS-fed mouse to reproduce the cardinal myocardial abnormalities of metabolic heart disease observed in humans, and the ability of RSV and S17834 to prevent the structural and functional consequences of diet-induced heart disease in this model, suggests that these polyphenols could be of value in the treatment of metabolic heart disease in humans.

Figure 6.

Figure 6

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A schema that summarizes the observed mechanisms by which polyphenols may exert beneficial effects in the HFHS-fed mouse. HFHS feeding is associated with oxidative stress and oxidant-mediated protein modifications as evidenced by generalized immunohistochemical staining for nitrotyrosine and 4-hydroxy-2-nonenal (HNE) staining, and specific HNE-adducts of LKB that are associated with decreased enzyme activity and increased hypertrophic signaling. Modifications of other proteins (e.g., calcium handling or sarcomeric proteins) may contribute to impaired relaxation. HFHS feeding is also associated with insulin resistance and hyper-insulinemia, which may promote hypertrophic growth. Finally, oxidative stress can lead to interstitial fibrosis. Together, these and possibly other mechanisms may contribute to diastolic dysfunction, which is common in metabolic heart disease. By decreasing oxidative stress and inflammation, treatment with S17834 a) may decrease the generation of oxidative protein modifications, b) interstitial fibrosis, and c) insulin resistance/hyper-insulinemia. S17834 also increases plasma adiponectin, which may inhibit hypertrophic signaling.

Supplementary Material

1

Acknowledgments

Funding Sources: Supported by NIH grants HL-061639 (WSC), HL-064750 (WSC), HL031607 (RAC, XYT), PO1 HL 068758 (RAC, KW), the NHLBI-sponsored Boston University Cardiovascular Proteomics Center (Contract No. N01-HV-28178, RAC and WSC), and a Strategic Alliance between Servier and the Vascular Biology Section, Boston University Medical Center (RAC).

Footnotes

Conflict of Interest Disclosures: This work was performed as part of a Strategic Alliance between the Vascular Biology Section, Boston University Medical Center (RAC) and Servier, which provided the S17834. RAC is a consultant for Servier, and TJV is an employee of Servier.

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