. ... .fiu . n s, .1 . . : (in...) $4.“... in...“ if??? . . c . i! 3. . I .2 fmmn-.: . F). x x . c‘b!l.!.:.r.§ {I}. 1.x. .5"..|bv.\3.. ta, 1". :1 !.I(.t€!¢!.l :. 1 .. ..9I)..|..'§ . . V :.:4|unc.rharfil.uka.u o z. . 3. .. 2, 324. 51:3. .{I \ I". .t I: {Pisa ‘ in: its}. :91. nmmatu: N, . . I ,4 iii... ‘9‘ it it. lat}. v u . , £11.35, . 2..» I ‘au'u‘nd'p i 7 _ , 80K) ' _LlBRARY Michigan State University This is to certify that the dissertation entitled GRAPE PHYTOCHEMICAL INTAKE ALTERS HEART FAILURE PATHOGENESIS AND CARDIAC GENE TRANSCRIPTION/TRANSLATION presented by E Mitchell Seymour has been accepted towards fulfillment of the requirements for the Doctoral degree in Human Nutrition ' C Major Professor’s Signature / / — 30 ~— 5200 C] Date MSU is an Affirmative Action/Equal Opportunity Employer PLACE lN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:lPro)/Acc&Pres/ClRC/DaleDue.indd GRAPE PHYTOCHEMICAL INTAKE ALTERS HEART FAILURE PATHOGENESIS AND CARDIAC GENE TRANSCRIPTION/TRANSLATION By E Mitchell Seymour A DISSERTATION Submitted to Michigan State University In partial fulfillment of the degree requirements for the degree of DOCTOR OF PHILOSOPHY Human Nutrition 2009 GRAPE F Hi; morbidity H}penens: reduced b appraisals modeled it on blood p effect 01‘ di Ii i hli‘enensii Ofa Wm “WM imi Sah‘smsu lowered in gmlie-fed Bi b) Ethel-CC focus“ Or ABSTRACT GRAPE PHYTOCHEMICAL INTAKE ALTERS HEART FAILURE PATHOGENESIS AND CARDIAC GENE TRAN SCRIPTION/T RANSLATION By E Mitchell Seymour High blood pressure or hypertension is a prevalent and significant contributor to morbidity and mortality from heart failure. The DASH (Dietary Approaches to Stop Hypertension) clinical trials provided evidence that diets rich in fruits and vegetables reduced blood pressure. Animal models of hypertension may permit mechanistic appraisals of the interaction of diet and disease. One recent study in hypertensive rats modeled the DASH diet using added vitamins and minerals, but failed to detect an effect on blood pressure. Therefore, a whole foods model may be more appropriate to assess the effect of diet on hypertension, versus elevated vitamins and minerals, alone. It is currently unknown if intake of phytochemical-rich whole foods affects hypertension-associated heart failure. This proposal uses whole table grapes as a model of a phytochernical-rich food. We first tested the hypothesis that a grape-enriched diet would impact the development of hypertension-related cardiac pathology in the Dahl Salt-Sensitive (Dahl-SS) rat model. Whole table grape powder (3% of diet) significantly lowered blood pressure, cardiac hypertrophy and cardiac oxidative damage. In addition, grape-fed rats displayed improved diastolic function and cardiac output. However, cardiac-specific mechanisms of these effects remain unknown. Bioavailable grape phytochernicals may have reduced heart failure pathogenesis by altered cardiac cell signaling and gene transcription/translation. One project arm focuses on transcription factor peroxisome proliferator-activating receptor (PPAR). PPAR 3 in me could ul- an‘l-hyd. i’XREsi Bioai‘uil. transcript like AhR .‘lllR’XRI phyiocher confer a si For reSpetlire cardiac Pp BeprCSNii and glutdr bem'cen ] gripe pifiiv. l Dali-SS i Milled [0 I During heart failure pathogenesis, cardiac PPAR isoforms are down-regulated while cardiac pro-inflammatory transcription factor NFKB activity is elevated. Irnportantly, PPAR activation directly reduces NF-IcB activation. Phytochemicals can activate PPARs in varied experimental models. If grape powder diet altered cardiac PPAR activity, it could also limit NF-KB activity and thereby reduce inflammation and fibrosis. Bioavailable grape phytochemicals could also activate the phenol—responsive, aryl-hydrocarbon receptor (AhR), which binds to genomic xenobiotic response elements (XREs) and stimulates the transcription of mRNA related to antioxidant defense. Bioavailable grape phytochemicals could also activate NF-E2 p45-related factor (Ner), a transcription factor which binds genomic antioxidant-responsive elements (ARES) and like AhR, stimulates the transcription of genes related to antioxidant defense. While AhR/XRE and Nrf2/ARE interactions have been shown in vitro with select phytochemicals, it is uncertain if physiologically-relevant doses of grape powder could confer a similar effect in vi v0. Four groups were studied: low salt diet + grape, high salt + grape, and their respective low-salt and high-salt, carbohydrate-equivalent controls. Grape diets enhanced cardiac PPAR and reduced NF-KB activity, N F-KB-related mRN A, and TNF—u and TGF- [3 expression. Grape diets also enhanced cardiac AhR and Nrf2 activation, related mRNA, and glutathione dynamics. Irnportantly, the majority of these effects were conserved between low salt grape and high salt grape groups, indicating specific effects from the grape powder treatment. In summary, consumption of whole table grape powder reduced Dahl-SS rat cardiac hypertensive pathology, and altered gene transcription/translation related to inflammation, fibrosis, and glutathione antioxidant defense. Williams fortune '41 life. )nu ; Chronic i1 something To Siffngrhs hUman bci Ill IEhpfcr DEDICATION I wish to dedicate this effort to my friends and martial art instructors, Tammy Williams and Dan Tirnlin. You give selflessly, providing me with your patience, your guidance, a safe place to learn, and a soft place to fall. You exemplify a brave and determined pursuit of excellence, in times of both fortune and adversity. Dan, despite your diagnosis of heart failure in the prime of your life, you adapted and thrived. Your attitude continues to inspire me in my own life with chronic illness. In return, I hope that my research efforts in heart failure can contribute something of value - in your honor. Your faith in me is a gift that I will cherish always. You have honored my strengths and counseled my weaknesses, gently supporting the evolution of a better human being. You are teachers in the highest sense, and friends of the highest character. In respect and gratitude, forever yours in “Black Belt Excellence”. iv I u their Iim‘ Les Bourq wish to lh; Kdufmdn. “ixh to Ii facilitating lei pral‘exxion. ethic. his g Bennink h; mentor Oil Bennink h degree und Fir: I0 F€~enrtif ‘he Wnlir. aimed m Tfidfik \‘i , ACKNOWLEDGMENTS I wish to thank my Michigan State University doctoral committee members for their time and effort, and especially for their flexibility and their private counsel — Drs. Les Bourquin, Maija Zile, Kathleen Hoag, Stephanie Watts, and Maurice Bennink. I also wish to thank my University of Michigan lab colleagues Drs. Ara Kirakosyan and Peter Kaufman, who provided daily support, scientific mentorship, and camaraderie. Finally, I wish to thank my lab director Dr. Steven Bolling, for generously supporting and facilitating my continued education and professional development. I extend a special thanks to my advisor Maurice Bennink, who has provided both professional and personal inspiration through his intellectual curiosity, his balanced work ethic, his gentle patience, and his genuine concern for his students. As a role model, Dr. Bennink has not only impacted my own work, but also the way in which I supervise and mentor other young scientists. In this manner, he truly “touches beyond his reach”. Dr. Bennink has been a valued mentor and friend, and I am truly honored to pursue this degree under his guidance. Finally, I wish to thank my partner Kristin and my family. Kristin encouraged me to re-enroll in my PhD program following my earlier departure due to chronic illness, and she continues to provide encouragement and inspiration. And to my family, I’ve finally earned my long-time moniker, “Dr Buff”. I’m looking forward to that vanity plate. Thank you for your continued love and support! TABLE OF CONTENTS LIST OF TABLES ................................................................................. viii LIST OF FIGURES ................................................................................ ix KEY TO SYNIBOLS and ABBREVIATIONS ............................................ x-xii INTRODUCTION .................................................................................. 1 CHAPTER ONE LITERATURE REVIEW A. HEART FAILURE ...................................................................... 7 B. SALT-SENSITIVE HYPERTENSION .............................................. 12 _C. FRUIT AND VEGETABLE INTAKE AND HYPERTENSION PATHOLOGIES ........................................................................... 13 D. CARDIOVASCULAR EFFECTS OF GRAPE CONSUMPTION .............. 18 E. RATIONALE FOR THE CURRENT STUDIES ................................... 20 F. LITERATURE CITED ................................................................. 31 CHAPTER TWO CHRONIC INTAKE OF A PHYTOCHEMICAL-ENRICHED DIET REDUCES CARDIAC FIBROSIS AND DIASTOLIC DYSFUNCTION CAUSED BY PROLONGED SALT-SENSITIVE HYPTERTENSION A. ABSTRACT ............................................................................. 42 B. INTRODUCTION ...................................................................... 43 C. MATERIALS AND METHODS ..................................................... 45 D. RESULTS ............................................................................... 5 1 E. DISCUSSION ........................................................................... 59 F. LITERATURE CITED ................................................................. 66 CHAPTER THREE A PHYTOCHEMICAL-ENRICHED DEIT IMPACTS CARDIAC PPAR AND NF KB ACTIVITY, FIBROSIS, AND CYTOKINE EXPRESSION IN RATS WITH DIASTOLIC HEART FAILURE A. ABSTRACT ............................................................................. 72 B. INTRODUCTION ...................................................................... 73 C. MATERIALS AND METHODS ..................................................... 75 D. RESULTS ............................................................................... 79 E. DISCUSSION ........................................................................... 84 F. LITERATURE CITED ................................................................. 92 CHAPTER FOUR A PHYTOCHEMICAL-ENRICHED DIET IMPACTS CARDIAC AhR and Ner TRANSCRIPTION FACTOR ACTIVITY AND GLUTATHIONE DYNAMICS A. ABSTRACT ............................................................................. 99 vi mmchw l CHAPTF. SUBLU A. B. C. B. INTRODUCTION ...................................................................... 99 C. MATERIALS AND METHODS ................................................... 102 D. RESULTS .............................................................................. 106 E. DISCUSSION ......................................................................... l l 1 F. LITERATURE CITED ....................... - ........................................ 1 18 CHAPTER FIVE SUMMARY, ALTERNATIVE APPROACHES, AND FUTURE DIRECTIONS A. SUMMARY .................................................................................................. 127 B. ALTERNATIVE APPROACHES - GRAPE INTAKE MAY AFFECT PEROXYNITRITE FORMATION AND RENIN SYNTHESIS................127 C. FUTURE DIRECTION S DETERMINE CARDIAC BIOAVAILABILTY OF GRAPE PHYTOCHEMICALS” ...131 D. FUTURE DIRECTIONS— DETERMINE THE EFFECT OF DIET TIMING UPON DAHL- SS RAT HYPERTENSION PATHOLOGY ......................... 134 E. LITERATURE CITED ................................................................ 135 vii Table 1 Table l Table 1 Table l Table l. Table l. L 3 Table l.‘ Table 3.1 Table 4.1. LIST OF TABLES Table 1.1. Diets and estimated nutrient content ............................................................... 46 Table 1.2. Grape Powder Phytochemical Analysis ........................................................... 47 Table 1.3. Serial Changes in Cardiac Geometry ............................................................... 54 Table 1.4. Serial Changes in Diastolic Parameters ........................................................... 55 Table 1.5. Serial Changes in Systolic Parameters ............................................................. 56 Table 1.6. Changes in Organ Weight and Cardiac Fibrosis .............................................. 57 Table 1.7. Cardiac GSH/GSSH, Cardiac MDA, and Plasma Inflammation ..................... 58 Table 3.1. RT-PCR Results ............................................................................................... 82 Table 4.1. RT-PCR Results .......................................................................................................... 109 viii ' I " .-‘~ "AV'A‘VI‘ Figure Figure figure . I igu re L meE Figure 3 figures - Figure 4. Figures 4 LIST OF FIGURES Figure 1.1. Subclasses of table grape phytochemicals ..................................................... 20 Figure 2.1. Serial body weight .......................................................................................... 52 Figure 2.2. Systolic blood pressure ................................................................................... 53 Figure 3.1. Cardiac PPARa, PPARy and NF-KB Activity ................................................ 80 Figure 3.2. Masson—Trichrome Stain Determined Cardiac Fibrosis ................................. 83 Figure 3.3. Cardiac TNF-u and TGF-B ............................................................................. 84 Figures 4.1A-4.1B. Cardiac Ner and AhR Activity ...................................................... 107 Figure 4.2. Cardiac GHS/GSSG ..................................................................................... 1 10 Figures 4.3A-3B. Cardiac GR Activity and GPx Activity ............................................ l 11 ix AhR AIN CGNIP CYPIA] CIPlBl DASH DIIF BA EUR: [8 GCS GPX QM CST a - Alpha [3 — Beta 8 — Delta 7 — Gamma K — Kappa AhR AIN cGMP CYP1A1 CYPlBl DASH DHF E/A E Dec t FS GCS GPx GSH GST KEY TO SYMBOLS and ABBREVIATIONS SYNIBOLS ABBREVIATIONS Aryl hydrocarbon receptor American Institute of Nutrition Cyclic guanosine monophosphate Cytochrome P450 1A1 Cytochrome P450 lBl Dietary Approached to Stop Hypertension Diastolic Heart Failure E Wave to A wave E Wave Deceleration time Fractional Shortening y-glutamylcysteine synthetase Glutathione Peroxidase Reduced Glutathione Glutathione S-Transferase I B 4 1- man“: GSSG GR HS HSG HSH [CAM [KBu lL-lB [L-6 [\VHS IVRT Keapl [AC-3180‘ L-NAMI; LS LSG LV LVEDD U‘ESD LV/Bw MCP.1 MBA GSSG GR HS HSG HSH ICAM licBa IL-lfl IWHS IVRT Keapl LC-MS/MS L-NAME LS LSG LV LVEDD LVESD LV/BW MCP-l MDA MTS Oxidized Glutathion Glutathione Reductase High salt diet High salt diet + grape High salt diet + hydralazine [ntercellular Adhesion Molecule Inhibitor kappa Ba Interleukin- 1 B Interleukin-6 Iowa Women’s Health Study isovolumetric relaxation time Kelch-associated protein 1 liquid chromotography tandem mass spectroscopy L-Nfi-Nitroarginine methyl ester Low Salt Low Salt + Grape Diet Left Ventricle Left ventricular end-diastolic dimension Left ventricular end-systolic dimension Left ventricle mass/body weight Monocyte chemotactic protein-1 Malonyldialdehyde Masson-Trichrome Stain xi I a: . - ~ frat-.1. sea-"'5: I NIKKB KHAN NTHGX NQO-l NC) NTTIA PDILS PG(11u PPAR RAAS RAS RNS ROS RR‘u, SHF m.“ TGRfi ['01le XRES NF-KB NHANES III NIH-NHLBI NQO-l N O ner NYHA PDE-S PGC-la PPAR RAAS ROS RW th SHF TN F -a TGF-B UGT1A6 XREs Nuclear factor-KB National Health and Nutrition Examination Survey [11 National Institutes of Health — National Heart Lung and Blood Institute N ADPquuinone oxioreductase-l Nitric Oxide N F-E2 p45-related factor New York Heart Association Phosphodiesterase-S PPAR-y coactivator IO. Peroxisome proliferator-activating receptor Renin-angiotensin-aldosterone-system Renin-angiotensin-system Reactive nitrogen species Reactive oxygen species Relative wall thickness Systolic heart failure Tumor necrosis factor-a Transforming growth factor-[3 UDP-glucuronosyltransferase 1 A6 Xenobiotic response elements xii INTRODUCTION H. ability to chronic d period of simplest L death. CL Lnited St Heart fail] diagnosis in patient failure d1 assmiatcd 6"eran eAlanine i appI-OaChc Tl“. although alteration. progrflgi‘ 0 . f de‘ 81L)" mtram £113 k Heart failure syndromes are frequently fatal illnesses in which the heart loses its ability to pump effectively in response to the body's needs. Heart failure is typically a chronic disease, with progressive deterioration of ventricular function occurring over a period of years or even decades. Patients may become incapable of performing even the simplest activities of daily living, and are at very high risk of medical complications and death. Currently, there are more than six million patients living with heart failure in the United States, with over 750,000 new diagnoses and over 300,000 deaths each year. Heart failure treatment has a profound economic impact, as well. Heart failure is the #1 diagnosis in the Medicare system based upon patient volume, the #1 discharge diagnosis in patients over 62, and the #1 cause of hospital readmission(l). Over 90% of heart failure diagnoses are proceeded by prolonged hypertension, and hypertension is associated with two to three times higher risk for developing heart failure(2). Therefore, experimental models of hypertension-induced heart failure are of particular value to examine interventions which may alter heart failure pathogenesis — including dietary approaches. The influence of nutrition on heart failure phenotypes is poorly understood, although epiderniologic and experimental evidence has emerged that suggests that alterations in cardiac nutrient metabolism may play a critical role in the development and progression of the disease. This implies that dietary modifications might reduce the risk of developing heart failure, or might delay disease progression to a more severe and intractable condition. Little guidance is available at present regarding optimal nutritional approaches costet‘t‘ecti i5 kn0\\ 11 pathogenes Hal nutrients. Compared failure LII; compositi. failure p; mnnnon-r TIi Effect 0 f fruit and l approaches for advanced heart failure, but dietary management represents a potentially cost-effective means of improving clinical outcomes for these patients. In addition, little is known about the impact of nutrition upon the trajectory of early heart failure pathogenesis, which would be vital to informed, preventive cardiology approaches. Hypothesis-testing research is needed to identify the roles of specific foods, nutrients, and dietary patterns in the development and progression of heart failure. Compared with nutrition guidelines for other cardiovascular conditions, those for heart failure are minimal. More information is needed regarding dietary macronutrient composition, overall dietary patterns, and the usage of dietary supplements amongst heart failure patients. In addition, more research is needed to identify plausible benefits of nutrition-based interventions and their biological mechanisms of effect. The Dietary Approaches to Stop Hypertension (DASH) clinical trials assessed the effect of dietary patterns with higher intakes of fruits, vegetables, and low-fat dairy products on hypertension. Results revealed that intake of an averaged 8.6 servings/day of fruit and vegetables reduced blood pressure as compared to the control group, which averaged 2.6 servings/day. The addition of low—fat dairy products to the diet further reduced blood pressure(3-5). However, the effects of DASH diet adherence upon heart failure incidence or mortality are not adequately understood. A recent large, prospective, observational study assessed the correlation of heart failure incidence with the intake of a DASH-style diet(6). Of the over 36,000 participants, those in the top quartile of the DASH diet food guidelines had a 37% lower rate of heart failure. Importantly, this effect was sustained after multivariate adjustments for age and other risk factors for heart In I‘D‘lyb'": “'- failure. T diets coul Tl patholog} permit su [0 nutritit DASH c carbohydr Houe\en phueeher DASH di Vegetables COHIIOI g: fldl'imO-ne. Phililstert. mechanjgt P3111010ng Th its effect examine T failure. This important study provided the first compelling evidence that DASH—style diets could reduce the incidence of heart failure. The specific mechanisms of the DASH—style diet upon hypertension-related pathology and heart failure are unknown, but studies in relevant animal models may permit such assessments. One recent study in spontaneously hypertensive rats attempted to nutritionally model the DASH diet. With detailed effort to match the control and DASH diet macronutrient and micronutrient profiles (vitamins, minerals, fats, carbohydrates, proteins, and fiber), the treatment failed reduce blood pressure(7). However, the semi-purified diets were not based upon whole-foods, and lacked phytochemicals, which could support the importance of these non-nutritive factors in the DASH diet benefits. In the DASH clinical trials, phytochemical intake from fruits, vegetables, and grains was distinctly different between the DASH-diet group and the control group(8). Phytochemicals elevated in the DASH diet included flavonols, flavanones, flavan-3-ols, B-carotene, B—cryptoxanthin, lycopene, lutein, zeaxanthin, and phytosterols. Therefore, it may be necessary to use a whole food model to mechanistically examine the benefits of DASH-style diets for hypertension and its related pathologic sequelae. This dissertation is focused upon modeling a fruit and vegetable-enriched diet and its effects upon hypertension-related diastolic heart failure. Specifically, the studies examine the impact of diet upon key cardiac transcription factors and genes in both healthy and failing hearts. The approach includes the Dahl-Salt Sensitive rat as a model of hypertension and diastolic heart failure, and diets supplemented with table grapes as a model experit‘ model of a phytochemical-containing diet. Finally, the limitations of the chosen experimental approaches and possible directions of future research are discussed. W LITERATURE REVIEW band an L11 blood thri ventricles circulatio dioxide i: the left s2? pumped 1, T1 systolic b refill With mills. Th left side 0 [“0 atria period du Period duI Tl’ LS Under 1.' In a heal; A. HEART FAILURE A1. Normal Cardiac Geometry and Function The normal heart is a strong muscle that beats about 120,000 times a day to pump blood through the body. The heart itself is made up of four chambers - two atria and two ventricles. De-oxygenated blood returns to the right side of the heart via the venous circulation. It is pumped into the right ventricle and then to the lungs where carbon dioxide is released and oxygen is absorbed. The oxygenated blood then travels back to the left side of the heart into the left atria, then into the left ventricle, from where it is pumped into the aorta and arterial circulation. The pressure created in the arteries by the contraction of the left ventricle is the systolic blood pressure. Once the left ventricle has fully contracted, it begins to relax and refill with blood from the left atria. The pressure inthe arteries falls whilst the ventricle refills. This is the diastolic blood pressure. Blood travels from right side of the heart to left side of the heart via the lungs. However, the chambers themselves work together. The two atn'a contract simultaneously, and the two ventricles contract simultaneously. The period during ventricular relaxation and blood filling is known as diastole, while the period during ventricular contraction and blood ejection is termed systole. The cardiovascular system is made up of the heart, lungs, arteries and veins, and it is under the control of the autonomic nervous system (sympathetic and parasympathetic). In a healthy individual with a healthy heart, heart rate is dictated by the body's needs. When the body is at rest, the organs, muscles and tissues require a reduced amount of blood and oxygen, resulting in reduced blood pressure, heart rate and respirations. When “51511 the '0‘“ .12. dexm“ mmbrc 3:316 C‘ destroys work 1‘- aheroxcl lapeneni are of f puhogen‘ bugnm Ratfiu ptogrexsr and my; lDHfl b the body is active, then the organs, muScles and tissues require an increasing amount of blood and oxygen, resulting in increased blood pressure, heart rate and respirations. These responses are all involuntary, under the direct control of the autonomic nervous system. If the individual remains reasonably healthy with no cardiac complications, then the cardiovascular system will continue to meet the demands of the body. A2. Heart Failure Definition and Etiology Heart failure is defined by the inability of the heart to adequately meet oxygen demands of the body. Specifically, this failure is characterized by the inefficient systolic and/or diastolic actions of the heart chambers and valves. Heart failure can be initiated by acute cardiac injury, such as a heart—targeted immune response or infarction which ‘ destroys functional heart tissue. More commonly, heart failure develops from prolonged work load induced by hypertension and/or increased vascular resistance from atherosclerosis. Over 90% of heart failure diagnoses are proceeded by prolonged hypertension(2). Therefore, experimental models of hypertension-induced heart failure are of particular value to examine interventions which may alter heart failure pathogenesis, including dietary approaches. It is generally agreed that patients with chronic heart failure can be divided into two groups based on changes in cardiac structure and function. Patients with systolic heart failure (SHF) are characterized by eccentric remodeling of the left ventricle with progressive left ventricular dilation, and by predominant abnormalities in left ventricular and myocardial systolic properties. By contrast, patients with diastolic heart failure (DHF) often have normal systolic parameters, but are characterized by concentric can is“... , l I. l. ‘1'... remodelin. diastolic r it is TClLLXlT. may prodtl A associatio 75 years t better lop} These In. approacht far less 31 [he b60611 been deli: ‘13- II. A,\ significzfl, Patients l and o‘er impaq. (. Hem far; dlSchargr Heart f, - (1. remodeling of the left ventricle and by abnormalities in left ventricular and myocardial diastolic properties. The heart can contract normally, but is stiff and less compliant when it is relaxing and filling with blood. This change impedes blood filling into the heart, and may produce symptoms of heart failure. A distinction between DHF and SHF is important because DHF has a strong association with “normal aging”, and is far more common than SHF in patients older than 75 years of age, especially in women with hypertension. While DHF is associated with better long-term survival than SHF, morbidity and quality of life is still greatly impacted. These two forms of heart failure, which may co—exist, may require different therapeutic approaches. Despite the high prevalence and economic impact of DHF, it has received far less attention than its systolic counterpart. Numerous clinical trials have documented the benefits of treatment for SHF; however, the optimal treatment for DHF has not yet been defined. A3. Heart Failure Epidemiology As a chronic disease, heart failure compromises patient quality of life and significantly promotes morbidity and mortality. Currently, there are more than six million patients living with heart failure in the United States, with over 750,000 new diagnoses and over 300,000 deaths each year. Heart failure treatment also has a profound economic impact. Over $40 billion of the US health care budget is spent on heart failure annually. Heart failure is the #1 diagnosis in Medicare system based upon patient volume, the #1 discharge diagnosis in patients over 62, and the #1 cause of hospital readmission( 1). Heart failure is therefore a significant and growing public health burden. Once diasnosr lear I h—— - (I) F; pUbHC hi .44. I Heart Fr All. (L oxidatixc IHUOgen lfllildlltm generatin angiotens norepincr Adduion, aCIlVit'V O the 0pm". Dunne di fibrog; I C0”Uzi'ctii 500096nk Oligen 3. fuflCllOn diagnosed, heart failure shows a average five-year mortality rate of 60%(9). Therefore, it is clear that prevention of heart failure, rather than treatment, would provide the greatest public health impact. A4. Interactive Roles of Cardiac Oxidative Stress, Inflammation, and Fibrosis in Heart Failure. A4.1. Oxidative Stress. Heart failure involves both systemic and cardiac-specific oxidative stress. Oxidative stress caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) contributes to the initiation and progression of heart failure. In the initiation phase, aberrant pressure and volume adjustment by the vasculature involves the generation of free radicals. Intracellular oxidative stress is generated by increased renin— angiotensin-aldosterone system activation and from increased exposure to norepinephrine. Oxidative stress may also be generated from local inflammation. In addition, pressure overload increases cardiac metabolic demand and involves greater activity of mitochondrial respiratory chain enzymes. High mitochondrial flux increases the opportunity for lost free electrons which can then generate reactive oxygen species. During disease progression, extended pressure overload leads to cardiac hypertrophy and fibrosis. Increased fibrosis in the heart muscle reduces its compliance, which leads to contractile insufficiency, especially apparent under exertion. If not quenched by endogenous antioxidants, (catalase, superoxide dismutase, glutathione, etc.) reactive oxygen species damage local lipids, proteins, and DNA, leading to sub-optimal cardiac function and cell death. Accumulating evidence suggests that there is a significant 10 correl: exerci.» surprb with be All imobe radical. widely regulate inilmrm interieui. redeem: pmllxltlf resultant .44.} H can bf L PTOlongg ft'mgdch. hi'Fel'ITttr C0”336.71 Correlation between oxidative stress and indices of functional capacity, such as peak exercise oxygen consumption(lO-13) and the severity of heart failure(14-16). Not surprisingly, biochemical markers of oxidative stress are markedly elevated in patients with heart failure(13,17). A4.2. Inflammation. In addition to oxidative stress, heart failure pathogenesis also involves progressive local and systemic inflammation. ROS such as superoxide, hydroxy radical, and hydrogen peroxide, and RNS such as nitric oxide and peroxynitrite are widely implicated in inflammatory processes. Oxidative stress can activate redox- regulated transcription factors, such as NFKB and AP-l, which regulate genes related to inflammation including tumor necrosis factor-a (TNF-a), interleukin-113 (IL-113) and interleukin-6 (IL-6). It is established that heart failure involves early and sustained redox-regulated transcription factor activation(18,19). In addition, long-term antioxidant provision to animal model of heart failure reduces cardiac redox factor activation and the resultant cardiac inflammation, cardiac hypertrophy and cardiac dysfunction(20,21). 114.3 Fibrosis. Prolonged oxidative stress also contributes to cardiac fibrosis. Fibrosis can be a beneficial short-terrn response to inflammation and wound healing, but prolonged fibrosis can result in various degrees of tissue remodeling. Abnormal cardiac remodeling is characterized by structural rearrangements that involve myocyte hypertrophy, fibroblast hyperplasia, and disproportionate increases in extracellular matrix collagen deposition which collectively lead to myocardial fibrosis(22). Extracellular matrix collagen is an important determinant of myocyte shape and alignment, and plays 11 regular {mix-ti S\-\[Oi il IACIOIS 81.1 I emirom disease pressure HUBCVCr 1mm DEC regulatory roles in transduction of cardiac contractile force. Thus, remodeling of myocardial collagen matrix is critical in the development of ventricular diastolic and systolic dysfunctions(23). As in inflammation, redox factor activation triggers growth factors and the expansion of the extracellular matrix. Reduced redox factor activation could reduce both inflammation and fibrosis, key players in heart failure pathogenesis. B. SALT-SENSITIVE HYPERTENSION AND HEART FAILURE BI. Salt-Sensitive Hypertension — Definition, Epidemiology and Etiology 81.1 Definition and Epidemiology. Excessive salt intake is one of the most important environmental contributors to the high prevalence of hypertension and cardiovascular disease in developed countries. In humans, the link between salt intake and blood pressure has been established in cross-sectional and longitudinal epidemiological studies. However, it is also true that the blood pressure response to changes in salt intake varies from one individual to another, a phenomenon known as “salt sensitivity”. Salt sensitivity affects approximately 50% of hypertensive patients and 20% of norrnotensive patients(24). The onset of salt sensitive hypertension increases with age. In addition, salt sensitive hypertension is more prevalent in blacks, a population at higher risk for disease related to hypertension including renal dysfunction, heart failure, and stroke(25). Greater understanding of the pathology and sequelae of salt-sensitive hypertension is critical to reducing the public health burden of hypertension. 12 {C‘s rfl ‘ [L v; 1.. '1 .I'I'st \l‘ju 81.3 11.11““ to play pressure as in ex are ultir likely tl impairer hxenen [8161111011 riot diree “Mild ra: SUFPlUS C leading l Pitssure. C. I P C1. Dis eQSe BIZ The Etiology of Salt-Sensitive Hypertension. The etiology of salt-sensitive hypertension is likely pleiotropic and is not completely understood. The kidneys appear to play a central role in the functional disturbances that link salt intake to arterial blood pressure(26). Earlier studies on renal transplantation in hypertensive patients(27), as well as in experimental animal models of hypertension(28), provide evidence that the kidneys are ultimately responsible for salt-sensitive increases in blood pressure. Furthermore, it is likely that renal injury resulting from prolonged hypertension further contributes to impaired natriuresis and plasma volume regulation. While it has long been accepted that sodium retention tends to be associated with hypertension, the mechanisms involved have been debated for some time. Sodium retention causes extracellular volume expansion. Extracellular volume expansion does not directly increase blood pressure, but it could serve to increase cardiac output, which would raise tissue perfusion to levels exceeding the metabolic needs(29,30). The relative surplus of blood supply may then trigger an ‘autoregulatory’ response in the tissues leading to vasoconstriction, increased peripheral vascular resistance, and higher blood pressure. C. FRUIT AND VEGETABLE INTAKE AND HYPERTENSION PATHOLOGIES C1. The Effects of Nutrients Versus Whole Foods on Hypertension and Heart Disease 13 . . . ‘ V '2 ”(2‘ vegetdl benefit and \e; studies consist: failed 1 disease 1116 Ct) "carding Of untior T effect or Pill’ducts find nuts. reVaried 3i {Ump- It is clear from observational population studies that the intake of fruits and vegetables is inversely related to cardiovascular morbidity and mortality. However, the benefits of additional nutrients alone cannot likely explain the benefits of increased fruit and vegetable intake. This hypothesis is supported by both observational and intervention studies showing that vitamin and mineral supplementation studies do not confer consistent benefit for reduced hypertension(31-34). In addition, clinical trials have largely failed to demonstrate a beneficial effect of antioxidant supplements on cardiovascular disease morbidity and mortality. At this time, the American Heart Association endorses the consumption of a diet high in food sources of antioxidants and other “cardioprotective” whole foods such as fruits, vegetables, whole grains, and nuts, instead of antioxidant supplements, to reduce risk of heart disease(35,36). The Dietary Approaches to Stop Hypertension (DASH) clinical trials assessed the effect of dietary patterns with higher intakes of fruits, vegetables, and low-fat dairy products on hypertension. In addition, the DASH diet includes whole grains, poultry, fish and nuts, while limiting fats, red meat, sweets, and sugar-containing beverages. Results revealed that a mean intake of 8.6 servings/day of fruit/vegetables reduced blood pressure as compared to the control group, which averaged 2.6 servings/day(3-5,37). The content of phytochemicals was distinctly different between the DASH-diet and control diet group(8), and may be a vital part of the DASH diet . benefit. Phytochemicals elevated in the DASH diet included flavonols, flavanones, flavan-3-ols, beta-carotene, beta-cryptoxanthin, lycopene, lutein+zeaxanthin, and phytosterols. The importance of these non-nutritive components may be reflected in one very relevant animal study in spontaneously hypertensive rats. In this study, DASH-style diets were 14 m a‘hieted “ Show 3 her The semi-j. importance 1.0011 mode h§perten>2 0r: aim the if: and morn. 10W Wot: 01 six ye; inversely diet. The: mortality L116 CQn-C‘ additional “'LiiSt [O l 3C11t'i[y_ .d 11631-1 dise- H _ | NHS 51'. Provided the agret achieved. with altered nutrients using semi-purified diets. However, the results failed to show a benefit for blood pressure reduction as compared to rats fed the control diet(7). The semi-purified diets employed lacked phytochemicals, which could support the importance of these non-nutritive factors. Therefore, it may be necessary to use a whole food model to mechanistically examine the benefits of fruits and vegetable intake for hypertension and its downstream pathologies. One observational, prospective study examined whether a greater concordance with the DASH diet was associated with reduced incidence of self-reported hypertension and mortality from cardiovascular disease(38). Subjects included 20,993 women in the Iowa Women’s Health Study (1W HS), initially aged 55 to 69 years, followed for a period of six years. Adjusted for age and energy intake, the incidence of hypertension was inversely and significantly associated with the degree of concordance with the DASH diet. There were also inverse associations between better DASH diet concordance and mortality from coronary heart disease and “all cause” cardiovascular disease. However, the correlation between DASH diet and reduced mortality was not sustained after an additional level of multivariate adjustment, which included education, body mass index, waist to hip ratio, smoking status and frequency, estrogen use, alcohol intake, physical activity, and multivitamin use. These results suggest that DASH diet benefits for reduced heart disease may be specific to select patient groups with defined risk factors. However, numerous clear and potentially important differences between the IWHS study and the DASH diet clinical trials need mention. The two-month DASH trial provided food to study participants. In contrast, the IWHS observational study assessed the agreement of the participants’ typical diet agreed with the DASH diet guidelines. 15 Also, many of the IWHS participants had normal blood pressure levels, not above-normal levels as did the DASH trial participants. It is likely that the long-terrn effect of the DASH diet differs by initial level of blood pressure. In addition, the IWHS cohort was predominantly Caucasian, whereas the DASH trials over-represented African Americans who are at greater risk for hypertension. Additional design differences may also have contributed to study outcomes. In the IWHS, dietary intake was measured by a single, semi-quantitative food frequency questionnaire, in which diet assessment is typically imprecise and energy intake is often underestimated. Another shortcoming of [W HS was the reliance upon self-reports of hypertension; blood pressure was not actually measured, which limits quantitative assessment of group differences. In summary, this study and its conclusions may not be a logical extension of the DASH trials to assess diet effects upon hypertension and eventual heart disease. Another recent, prospective study evaluated the association between the DASH- style diet and mortality in hypertensive adults(39). Subjects included 5,532 participants from the Third National Health and Nutrition Examination Survey (NHANES III), who were followed for eight years. Like the IWHS, diet was assessed using a single assessment method, though with 24-hour food recall versus a food frequency questionnaire. Unlike IWHS, the subjects were hypertensive, and were from a broader racial demographic. Only 7.1% of these freely living NHANES 111 subjects consumed a DASH-like diet as determined by their study-specific criteria. The subjects were thus divided and compared as two groups (DASH diet n=391, non-DASH diet n=5,141). While diastolic blood pressure was lower in the DASH group (p<0.05), systolic blood pressure was not (p=0.85). Results demonstrated inverse associations between the 16 DASH-style diet and mortality from coronary heart disease, stroke, and all cardiovascular disease. After multivariate analysis, a DASH-style diet was still associated with lower all- cause mortality, but specific mortality from cardiovascular disease, ischemic heart disease, and stroke no longer reached statistical significance. The results from these observational studies in freely living subjects suggest a cardioprotective effect, but the specific diet impact upon heart failure morbidity and mortality was not examined. C2. The DASH-style Diet and Heart Failure Despite the epidemiologic evidence for the benefits of fruit and vegetable intake, little is known about the effect of diet on heart failure pathogenesis. Instead, because this diet pattern reduces hypertension, it is assumed that this hypotensive effect could reduce hypertension-associated heart failure. To date, one study has assessed the correlation of a DASH-style diet and heart failure. The Swedish Mammography Cohort(6) is a prospective, observational study, whose database contains information on subject diet and disease. Of the 36,000 participants, those in the top quartile of the DASH diet food guidelines had a 37% lower rate of heart failure after multivariate adjustment for age, physical activity, energy intake, education status, family history of myocardial infarction, cigarette smoking, post- menopausal hormone use, living alone, hypertension, high cholesterol, body mass index, and incident myocardial infarction. This important study provided the first compelling evidence that DASH-style diets reduced the incidence of heart failure, across a broad scope of confounding variables. 17 The specific mechanisms of the DASH-style diet upon hypertension-related pathology and heart failure are unknown, but studies in relevant animal models may permit such assessments. It is possible that fruit and vegetable intake would exert tissue- specific effects which may alter heart failure pathogenesis. Animal models of heart failure would allow specific diet manipulation and simultaneous assessment of tissue- specific effects. D. CARDIOVASCULAR EFFECTS OF GRAPE PRODUCT CONSUMPTION D1. Grape Intake and Cardiovascular Epidemiology Observational studies show that men and women with moderate alcohol consumption are substantially less likely to die of a heart attack than non-drinkers. Specifically, low cardiac mortality rates are observed in countries with a high wine consumption. However, these same populations also consume higher fat diets, exercise less, and smoke more cigarettes than neighboring countries. This seemingly illogical finding has been coined the ‘French Paradox’(40). Wine consumption, particularly red wine, is speculated to play a role in the protective association of the French Paradox. The cardioprotective constituents in wine are unknown, but numerous studies suggest that the polyphenolic compounds in grapes may play a causative role. The cardiovascular effects of grape product consumption are broad. Clinical trials and animal studies with purple grape juice suggest cardioprotective effects(41) through enhanced vasodilation(42-44), reduced platelet aggregation(45-47), reduced oxidation of ‘18 plasma. lipids(48-50), DNA(49), and protein(49), and enhanced plasma antioxidant capacity(49,50). The fact that both wine and grape juice experimentally confers cardiovascular benefits supports that some critical polyphenols are shared between the two grape products. The grape constituents responsible for the health benefits remain unclear. Furthermore, differences in polyphenol content exist between grape components (juice, pomace, seed), different varietals of grape, and different geographic regions of origin(5 1). DZ. Table Grape Phytochemicals Table grapes are the primary Vitis vinifera variety of whole grapes sold as produce. Table grapes are available in green, red, and black varietals, which vary in their ' phytochemical content depending on regional differences in growth conditions, including climatic and soil differences. Phenolic compounds in table grapes can arbitrarily be divided into four groups: simple phenols, flavonols and flavan-3-ols, anthocyanins, and stilbenes. Grape phytochemicals are often called polyphenols because of their common phenolic acid group. Differences in the degree of oxidation and hydroxylation of the phenolic rings lead to a large family of structures with essential differences in biological behavior, bioavailability, and efficacy. Grape polyphenols of potential interest for common dietary intake are found within the skins and seeds. Grape phenols and polyphenols exist as free compounds, as sugar polymers, or as part of larger molecular weight oligomeric chains or structures(52). The most common polyphenols in grape skin and pomace include phenolic compounds, including simple phenols, phenolic acids, cinnamic acids, stilbenes, flavonoids, flavans, l9 flavonols, and anthocyanins. Figure 1.1 illustrates these groups, and lists some representative compounds present in table grapes. GRAPE PHYTOCHEMICALS J l l ' l I [ PHENOLIC ] [ FLAVONOLS, ] [ANTHOCYANINS j [ STILBENES ] ACIDS FLAVAN-3-OLS Gallic Acid Catechin Malvidin Resveratrol Caffeic Acid Epicatechin Cyanidin Ellagic Acid Quercetin Delphinidin Figure 1.1. Subclasses of table grape phytochemicals Of interest to dietary intake, these components are found in various concentrations in fresh grapes, grape juice, wine, and grape skin extract. However, it remains unknown which grape constituents or their in vivo metabolites offer greater biologic/health effects, or if these components act synergistically. E. RATIONALE FOR CURRENT STUDIES E1 . Rationale for Grape Product Efficacy Against Hypertension-Related Pathology. This project will assess diet effects on heart failure using a whole foods model to simulate a phytochemical-rich diet. For modification of heart failure pathogenesis, the potential antioxidant, anti-inflammatory, and vasodilatory effects of grape product intake 20 are of great interest. The following in vitro and in vivo studies demonstrate that grape products can confer these biologic effects. E1.] Antioxidant Efi‘ects. Grape products limit tissue and systemic oxidative stress in several animal models. Proposed mechanisms of this effect include direct scavenging of free radicals and improved endogenous antioxidant defense. If able to provide an in vivo antioxidant effect in humans, grape phytochemical intake may modify diverse contributors to cardiovascular morbidity and mortality. The antioxidant effects of grape product ingestion can be measured directly by enhanced plasma antioxidant capacity, or indirectly by reduced plasma oxidative stress and/or reduced oxidative damage of biomolecules within tissues. Several grape products are capable of providing in vivo and ex vivo antioxidant effects; they have demonstrated in vivo capacity to enhance plasma antioxidant capacity(49,53,54), to decrease oxidation of LDL(53-58), and to lower 8- isoprostane(49,59), a lipid oxidation product which serves as a systemic marker of oxidative stress. In addition, effects were demonstrated in both healthy subjects and at- risk subjects. E1.2. Anti-Inflammatory Efi‘ects. Inflammation is a key contributor to the progression of many forms of heart disease. Local inflammation directly damages target tissue and generates reactive oxygen species which can damage neighboring tissue. In vitro studies and animal studies suggest that grape polyphenols have the potential to modulate eicosanoid metabolism(60). In particular, the 5-1ipoxygenase pathway is an important target because it is involved in the synthesis of leukotrienes which contribute to inflammation. In vitro, the polyphenols quercetin and resveratrol prove to be effective 21 inhibitors of pro-inflammatory lipoxygenase pathways(61). In addition, red wine extracts reduce adhesion of monocytes to the endothelial surface and block cytokine-induced expression of endothelial adhesion molecules(62). Red wine and select grape phytochemicals inhibit activation of nuclear factor-kB (NF-ch) activity and production of pro-inflammatory factors in endothelial cells and immune cells(62,63). Incubation of monocytes with catechin decreased their adhesion to endothelial cells(64). Relevant polyphenols also inhibit NF-KB activity in T lymphocytes(65,66). Resveratrol has also demonstrated anti-inflammatory effects, including inhibition of adhesion molecule expression and reduced responses to cytokines(67-71). Also, resveratrol inhibits the release of degradative enzymes by neutrophils and downregulates neutrophil surface expression of adhesion-dependent, pro-thrombogenic proteins(72). In vivo studies examining anti-inflammatory effects of grape products have been more lirnited(54,73-75). In humans, treatment with table grape powder for four weeks was associated with a reduction in plasma TNF-or, but not C-reactive protein or IL—6(59). Wine consumption for four weeks also reduced systemic markers of inflammation in healthy men(76). Collectively, these studies show that diverse grape products are able to lower systemic and local markers of inflammation. E13. Vasodilatory Efi‘ects. The vascular endothelium plays a central role in the regulation of vascular tone, thrombosis, local inflammation, and cell proliferation by producing paracrine factors that act on the arterial wall and on blood cells. In vitro studies demonstrate the favorable effects of grape products on endothelial function. In cultured endothelial cells, wine, grape juice, grape seed extract, and specific polyphenols increase 22 the activity of the endothelial isoforrn of nitric oxide synthase and stimulate nitric oxide (NO) production(77,78). NO is formed from the guanidine-nitrogen terminal of L- arginine by nitric oxide synthases. Endothelium-derived NO plays a crucial role in the homeostasis of the vascular tone by acting as a vasodilator. As a dynamic mediator of vascular compliance, NO is a freely diffusible gas that can act as an intracellular and intercellular messenger molecule. Most of the cellular actions of NO are explained by the activation of the cytosolic enzyme soluble guanylate cyclase, which catalyzes the formation of cyclic guanosine monophosphate (cGMP). Increased cGMP activates protein kinase G, which in turn phosphorylates a number of proteins involved in vasodilation. In addition to the depressor effect of vasodilation, NO inhibits platelet adherence to the endothelium. Enhanced NO production or availability would thereby enhance vasodilation and lower blood pressure. In vitro studies indicate that NO degradation by phosphodiesterase-S is delayed by exposure to grape phytochemicals,(79) which would prolong NO availability. In addition, incubating arterial rings in a tissue bath containing diluted grape juice increased endothelial-dependent vasorelaxation by a NO-dependent mechanism(80). In the short term, polyphenols stimulate endothelial NO synthase phosphorylation via protein kinases phsophatidylinositol—3-hydroxy kinase and Akt(81). Longer term exposure to red wine extracts or resveratrol increases nitric oxide synthase enzyme expression and activity(77,78). Human studies support a benefit of grape beverages on endothelial function(82). In patients with coronary artery disease, consumption of purple grape juice improved endothelium-dependent brachial artery flow(44,48). In healthy subjects, de- alcoholized wine also improved brachial artery flow-mediated dilation(83). 23 In addition to the effects on NO, grapes have important effects on other factors , that influence vascular function. For example, flavonoid-containing beverages increase endothelial production of prostacyclin and suppress production of endothelin-1, a potent endothelium-derived vasoconstrictor(84,85). Also, NO activity is negatively impacted by oxidative stress, because NO can be oxidized to non-dilatory peroxynitrite. An antioxidant effect of grape intake could reduce the conversion of NO to peroxynitrite and thus prolong NO action and vasodilation. In summary, these studies show that diverse grape products have the capacity to improve vascular reactivity, and perhaps lower blood pressure. It remains unknown if these temporal changes can then extend to sustained reductions in blood pressure, or confer greater resistance to hypertension-related pathologies. E2. The Disease Model - The Dahl Salt Sensitive Rat The model employed. in this proposal is the Dahl-Salt Sensitive (Dahl-SS) rat. Dahl-SS rats are used as a model of human salt-sensitive hypertension. This model was first characterized by Louis K. Dahl in the early 19705, as a spontaneous mutation in Sprague-Dawley rats conferring salt-sensitivity. Kidney transplant studies in Dahl-SS rats indicate that the kidneys are the primary source of pathology due to aberrant volume/solute regulation(86-88). Dahl-SS rats follow a very predictable course from hypertension and renal hypertrophy to cardiac hypertrophy, followed by cardiac insufficiency and diastolic heart failure. Dahl-SS rats develop only mild systolic impairment; the rats typically die of renal failure before the onset of overt systolic dysfunction. Diastolic heart failure develops 24 within 15-20 weeks of high-salt feeding, depending on the amount of salt provided. Unlike surgical models of heart failure induction, high-salt fed animals show excellent correlation with one another in disease course, enhancing statistical power. The NIH- NHLBI Program for Genomic Applications funded a study to characterize disease progression in the Dahl-SS rat, fed a 6.0% NaCl diet, the model and salt intake included in this proposal. Because of its highly reproducible and predictable pathogenesis, the Dahl-SS rat has been used for pharmaceutical studies of angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, B-blockers, and other common drugs for heart failure. However, the effect of diet on heart failure development is poorly understood. As with human heart failure, the Dahl-SS rat pathogenesis involves oxidative stress. In addition, Dahl-SS rats have compromised antioxidant defenses when given a high salt diet. Of significance for this proposal, salt-fed Dahl-SS rat hearts show significantly decreased glutathione peroxidase activity and reduced glutathione(89), which leads to sustained exposure to ROS. Synthetic antioxidants and concentrated antioxidant vitamins provide substantial benefit to the Dahl-SS rat, supporting the impact of oxidative stress upon pathogenesis. For example, daily treatment with superoxide dismutase mimetic Tempol improves Dahl-SS renal function, reduces oxidative stress, and improves heart function(90-98). Vitamin E limits Dahl-SS hypertension, hypertensive nephropathy, renal oxidative stress, and improves glomerular filtration rate and renal perfusion in the Dahl-SS rat. Taken together, these findings suggest that in the Dahl-SS rat, higher salt diets are associated with increased oxidative stress, and that antioxidant intake can counter these adverse effects and reduce hypertension-related 25 pathologies. However, it is unclear if an antioxidant-rich whole food would provide a similar benefit or alter cardiac pathology. While antioxidant treatment limits these early phenotypes of Dahl-SS hypertension, the current gap in knowledge concerns whether these accumulated effects of diet will affect the eventual development of heart failure. Antioxidant-rich diets may pay dividends beyond short-term depressor effects by limiting the degree of irreversible organ damage caused by prolonged hypertension. E3. The Diet Intervention Model - Whole Table Grape Powder For greater relevance to normal dietary intake, as opposed to dietary supplementation, this project will utilize a whole food model. The freeze-dried grape powder used in this study is a composite of green, red, and black table grapes grown in California (supplied by the California Table Grape Commission, processed and chemically characterized by National Food Laboratories, Inc). As detailed earlier, table grape powder is a source of diverse phytochemicals including phenols, simple phenolic acids, cinnamic acids, stilbenes, flavonoids, flavans, flavonols, and anthocyanins. E3.] Table Grape Powder — Demonstrated Efi‘icacy. Irnportantly, this standardized, whole grape powder has been shown by other investigators to reduce both plasma and tissue markers of oxidative stress in vivo(55,59,99). This protection was afforded by grape powder delivered by three different mechanisms [gavage(59), diet(99), drinking water(55)] and in multiple models [mouse(55), gerbil(99), human(59)]. Protection 26 occurred at both a tissue and a systemic level; protection was associated with reduced oxidative stress in plasma(55,59), in isolated leukocytes(55) and in tissues(99-101). E3.2 Table Grape Powder - Cardiac Bioavailability. Of specific interest for this project, the cardiac-specific benefits of table grape powder have already been demonstrated. Cui et a1. (100,101)employed the same table grape powder (provided by the California Table Grape Commission) that we use in the current studies. Healthy male rats were gavaged with table grape powder for three weeks. Afterwards, hearts were excised and perfused ex vivo with an oxygenated buffer. Beating hearts were then made ischemic for thirty minutes followed by two hours of oxygenated buffer reperfusion. In this blood—free model, grape powder intake provided significant cardioprotection as evidenced by improved post-ischemic ventricular recovery and reduced degree of myocardial infarction. Grape powder intake also reduced the post-reperfusion cardiac malonyldialdehyde content, indicating reduction of oxidative damage to cardiac lipids. The results demonstrate a direct cardioprotective role of regular intake of table grape powder; the model was saline-perfused and blood—free, so treatment effect was associated with a tissue-specific mechanism. In addition, the results are more reflective of the chronic effects of grape intake rather than acute exposure to plasma metabolites of the ingested table grape powder. In summary, numerous in vitro and in vivo studies suggest protective effects from grape products. Of interest to this proposal, ex vi v0 cardiac studies indicate that beneficial components and/or metabolites of the whole table grape powder provided greater resistance to cardiac oxidative stress(100). As such, bioavailable grape phytochemical 27 metabolites could confer health benefits against diseases which involve oxidative stress such as hypertension—associated heart failure. E4. Rationale and Specific Aims of Dissertation Bioavailable grape phytochemicals may reduce heart failure pathogenesis and alter cardiac cell signaling, resulting in reduced inflammation and fibrosis. The current project focuses on cardiac signaling related to the transcription factor peroxisome proliferator—activating receptor (PPAR). Although the effects of cardiac PPAR agonism are diverse, including altered metabolism and cell differentiation, the inverse relation between PPAR activity and nuclear factor kappa B (NF-KB)-related inflammation is of particular interest for this proposal. Cardiac PPAR isoforms are down-regulated with Dahl—SS rat heart failure, while cardiac pro-inflammatory transcription factor NF-KB activity is elevated. PPAR activation reduces N F-KB activation, because PPAR activation increases transcription of a cytoplasmic inhibitor of NF-KB activation. Phytochemical- rich extracts can activate PPARs in varied experimental models. If the grape diet alters cardiac PPAR activity, it could also limit NF-KB activity and thereby reduce inflammation and fibrosis. Bioavailable grape phytochemicals may also reduce heart failure pathogenesis by improved cardiac antioxidant defense. Grape phytochemicals could activate the phenol- responsive, aryl-hydrocarbon receptor (AhR), which binds to genomic xenobiotic response elements (XREs) and stimulates the transcription of mRNA related to antioxidant defense. Polyphenols can also interact with sulthydryl moieties on kinase proteins and alter their secondary structure and activity. This type of effect is observed in 28 NF—E2 p45-related factor (nrf2) activation, a transcription factor which binds genomic antioxidant-responsive elements (ARES) and like AhR, stimulates the transcription of genes related to antioxidant defense. While AhR/XRE and nrf2/ARE interactions have been shown in vitro, it is uncertain if physiologically-relevant doses of grape powder could confer a similar effect in vivo. The central hypothesis of this dissertation is that whole grape powder supplementation increases Dahl—SS rat cardiac PPAR-activity and decreases NF-KB activity and related genes/proteins, and increases the activation of cardiac AhR and nrf2 and the expression of antioxidant defense genes. The first phase of the doctoral project is to test the efficacy of the table grape powder against diastolic heart failure progression in the Dahl-SS rat, as described in Chapter Two. Five groups are studied: low salt diet + grape, high salt diet + grape, high salt diet + vasodilator drug hydralazine, and low-salt and high-salt, carbohydrate-equivalent controls. The second phase will then examine potential cardiac-specific mechanisms of effect, as detailed in Chapters Three and Four. Archived left ventricular tissue from the Chapter Two efficacy studies will be analyzed by the following two Aims: $1 Compare Diet Effect on Cardiac PPARs and Cardiac NF—kB signaling. The working hypothesis is that grape-fed rats have enhanced PPAR and reduced NF-KB activity and NF-IcB-related protein expression. Examine changes in cardiac nuclear PPAR-a, PPAR-y, and NF-KB DNA binding and downstream genomic targets and proteins related to inflammation and fibrosis. M2, Compare Diet Effect on Cardiac Endogenous Antioxidant Defense. The working hypothesis is that grape-fed animals have enhanced AhR and nrf2 activation and 29 related protein expression. Examine changes-in cardiac AhR and nrf2 DNA binding and downstream expression of select XRE and ARE-related genomic targets related to antioxidant defense. Several trials show that intake of grape juice and red wine cause transient vasodilation. However, it is unknown if and how chronic intake of physiologically relevant, phytochemical-rich grape products could affect chronic disease related to high blood pressure. 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Cui J, Juhasz B, Tosaki A, Maulik N, and Das DK. Cardioprotection with grapes. J Cardiovasc Pharmacol 2002;40:762-769. 40 CHAPTER TWO CHRONIC INTAKE OF A PHYTOCHEMICAL-ENRICHED DIET REDUCES CARDIAC FIBROSIS AND DIASTOLIC DYSFUNCTION CAUSED BY PROLONGED SALT-SENSITIVE HYPTERTENSION 41 A. ABSTRACT Salt-sensitive hypertension is common in the aged. Increased fruit and vegetable intake reduces hypertension, but its effect upon eventual diastolic dysfunction is unknown. This relationship is tested in the Dahl-Salt Sensitive (Dahl-SS) rat model of salt-sensitive hypertension and diastolic dysfunction. Table grape powder contains phytochemicals that are relevant to human diets. For 18 weeks, male Dahl-SS rats were fed one of five diets: Low Salt (LS), a Low Salt + grape powder (LSG), High Salt, or a High Salt + grape powder (HSG), or High Salt + vasodilator hydralazine (HSH). Compared to HS, HSG diet lowered blood pressure and improved cardiac function, reduced systemic inflammation, reduced cardiac hypertrophy, fibrosis, and oxidative damage, and increased cardiac glutathione. HSH similarly reduced blood pressure but did not reduce cardiac pathogenesis. LSG reduced cardiac oxidative damage and increased cardiac glutathione. In conclusion, physiologically relevant phytochemical intake reduced salt-sensitive hypertension and diastolic dysfunction. 42 B. INTRODUCTION In humans, the link between salt intake and blood pressure has been established in cross-sectional and longitudinal epidemiological studies. However, blood pressure response to changes in salt intake can vary from one individual to another, a phenomenon known as “salt sensitivity”. Salt sensitivity affects approximately 50% of hypertensive patients and 20% of norrnotensive patients(l), and its incidence increases linearly with age. Greater understanding of the pathology and sequelae of salt-sensitive hypertension is then critical to reducing the public health burden of hypertension and its associated pathologies. Prolonged hypertension frequently contributes to the development of heart failure. Heart failure is defined by the inability of the heart to adequately meet oxygen demands of the body, characterized by inefficient systolic and/or diastolic actions of the heart chambers and valves. Over 90% of heart failure cases are preceded by prolonged hypertension(2). Heart failure is a significant and growing problem in our aging population. Heart failure is the #1 diagnosis in the Medicare system based upon patient volume, the #1 discharge diagnosis in patients over 62, and the #1 cause of hospital readmission(3). As such, preventive approaches which address risk factors for heart failure could impact public health burden. Our group is focused on the effects of diet upon hypertension-associated cardiac pathogenesis. The Dietary Approaches to Stop Hypertension (DASH) clinical trial 1 revealed that diets rich in fruits and vegetables reduced blood pressure(4,5). One recent animal study carefully modeled the DASH diet nutrients to assess effects on hypertension in Spontaneously hypertensive rats. However, the findings failed to reveal an anti- 43 hypertensive effect(6). Importantly, this study did not include any non-nutritive phytochemicals contained in fruits and vegetables. The DASH diet phytochemical profile was distinctly different than the control group(7), and these compounds may be vital to the diet benefits. The Dahl Salt-Sensitive (Dahl-SS) rat is a model which provides insight into the pathology and treatment of salt-sensitive hypertension. When fed a higher salt diet, the Dahl-SS rat predictably and gradually develops the clinically relevant sequelae of hypertension, renal hypertrophy, renal dysfunction, cardiac hypertrophy, and diastolic dysfunction. Many animal models of induced heart failure depend upon infarct induction or surgical modification of the vasculature, which impdse rapid morbidity and higher mortality. In contrast, the hypertension-induced model described here develops pathology over several months, allowing one to serially test for the more gradual effects of diet modification. As such, we propose that the Dahl-SS rat is a valuable model of diet effects on aging-related salt sensitive hypertension and its associated cardiac pathologies. The current study examines the cumulative cardiac effects of a diet supplemented with phytochemical-rich whole table grape powder. Although the use of one food source may be a simplified approach, table grapes are a relevant model to hmnan diets because they are a widely available and affordable produce, and because they contain the major classes of commonly consumed, produce-derived flavonoids, including anthocyanins, flavanols (e.g. catechin, epicatechin, proanthocyanins), and flavonols (e.g. quercetin, kaempferol, isorhamnetin)(8). In addition, the table grape powder employed in this study has already been shown to reduce other pathologies(9-l3), evidence which supports the in vivo efficacy and bioavailability of the grape powder constituents. Using the Dahl-SS 44 rat model, we tested the hypothesis that table grape powder-enriched diets could lower hypertension-associated cardiac pathology and diastolic dysfunction. Furthermore, because grape product consumption is known to impart acute vasodilation(l4-18), we compared grape treatment effects to those of hydralazine, a well-characterized vasodilator which has been shown at the selected dose to lower blood pressure in the Dahl-SS rat(19-21). C. MATERIALS AND METHODS C1. Animal Care and Diets Five week old Dahl-Rapp Salt-Sensitive rats (Harlan, Indianapolis, IN) were acclimated for one week on AIN-76a powdered diet (Research Diets, New Brunswick, NJ). Afterwards, each rat was randomly assigned (11 = 12 each) to one of five treatments. Low Salt diet (LS, AIN-76a with 2.8% added carbohydrate, glucose:fructose 1:1), Low Salt diet + grape powder (LSG, AIN-76a with 3.0% w/w added grape powder), High Salt diet with 6% added NaCl (HS, AIN-76a with 2.8% w/w added carbohydrate), High Salt Diet + grape powder (HSG, AIN-76a with 3.0% w/w added grape powder), or High Salt Diet + hydralazine (20mg/kg body weight/day, in drinking water). Hydralazine dose was selected based upon previously published findings in the Dahl-SS rat(19-21), to obtain a similar % reduction in systolic blood pressure as that observed with our grape powder. Diet nutrient content is described in Table 2.1, while grape powder phytochemical content is described in Table 2.2. The freeze-dried table grape powder was obtained from the California Table Grape Commission as a composite of green, red, and black 45 California table grapes, processed and chemically characterized by the National Food Laboratory, Inc (Dublin, CA). Grape powder or added carbohydrate was mixed weekly into the AIN-76A base diet in-house using a commercial baking blender, and then stored in vacuum-sealed bags (Deni Magic Vac, Buffalo NY) at 4°C. Table 2.1. Diets and estimated nutrient content LS LSG HS HSG HSH Total Protein 20 21 20 21 20 Total Carb 68 68 68 68 68 Total Fat 5 5 5 5 5 Total Fiber 5 5 5 5 5 kcal/gram of diet 3.9 4 3.9 4 3.9 g/kg diet g/kg diet g/kg diet g/kg diet g/kg diet Casein 198 198 198 198 198 Protein from Grape 0 1-1 0 1-1 0 Corn Starch 150 150 150 150 150 Sucrose 500 500 500 500 500 Sugar from Grape 0 27.7 0 27.7 0 Dextrose 14 O 14 O 14 Fructose l4 0 l4 0 14 Cellulose 50 50 50 50 50 Fiber from Grape 0 0.02 0 0.02 0 Corn Oil 50 50 50 50 50 A1N76a Vitamin Mix 10 10 10 10 10 A1N76a Mineral Mix 35 35 35 35 35 Vitamin C (Grape) 0 0.001 0 0.001 0 Potassium (Grape) 0.3 0 0.3 0 Vitamin A (Grape) 0 99.6 [D 0 99.6 [U 0 Nutrient content of grape powder was analyzed by National Food Laboratory, Inc. (Dublin, CA) Nutrient content of the base A1N76a diet was provided by Research Diets, Inc. 46 Table 2.2. Grape Powder Phytochemical Analysis Grape Powder Analysis (per kg of grape powder) Anthocyanins Cyanidin 380.0 mg Malvidin 170.3 mg Peonidin 33.5 mg Monomeric Flavanols Catechin 19.1 mg Epicatechin 12.5 mg Flavonols Quercetin 49 mg Kaempferol 5.7 mg Isorharnnetin 4.4 mg Stilbenes Resveratrol 36.0 mg Phytochemical analysis (per kg of grape powder) by National Food Laboratories, Inc. Hydralazine-fortified drinking water was made every two days, with concentration adjusted dynamically based upon changing water intake and body weight over the course of the study. Animals were fed 20g of powdered diet/head/day. Ad libitum intake of AIN diet averages 19-21 grams of AIN powder/day in the Dahl-SS rat(22), so provision of 20 grams/day ensured complete daily consumption. For the high salt diets, NaCl was added directly to the food hopper and mixed carefully with the daily ration of powdered diet. Rats were housed three/cage in 12h light:12h dark cycles, and water was provided ad libitum. This project was approved by the Animal Care and Use Committee at the University of Michigan. 47 C2. Blood Pressure and Echocardiograph y Measures During the 18 week study, blood pressure was measured bi-monthly by the [ITC Mark 12 photoelectric/oscillometric tail cuff system (IITC Life Sciences, Woodland Hills, CA) using the unit and method we described in detail previously and validated against telemetric approaches(22). Using preconditioned, conscious, restrained animals, the first two of ten measures were universally discarded due to acclimation to tail cuff pressure and to operational noise. A run was accepted if at least six of the eight measures were adequate (having detectable pulses and free of gross artifacts). When the requisite determinations were obtained, the average was calculated and used as the mean heart rate and the mean systolic value for that session. Echocardiography of all animals was performed at 0, 8 and 18 weeks of diet treatment, following the predicted Dahl-SS rat development of compensated cardiac hypertrophy and of diastolic dysfunction, respectively. All measurements were made by a trained research animal sonographer who was unaware of treatment assignment. Animals were anesthetized by 4% isoflurane and maintained with 1% isoflurane. Two- dimensionally guided M-mode recordings and Doppler tissue imaging were acquired as we described previously(22). Equations for each derived parameter are as described by Boluyt(23), with the exception of mid-wall fractional shortening. In the Dahl-SS rat, endocardial fractional shortening overestimates LV systolic function; mid-wall fractional shortening has been determined to be a more appropriate index of LV systolic function(24-26). Mid-wall fractional shortening was calculated according to the two-shell cylindrical model of Shimizu et al(27). 48 C3. Terminal Plasma Analysis At sacrifice, conscious rats were decapitated and trunk blood was collected. Whole blood was collected into an EDTA vacutainer, (BD Vacutainer Systems, Franklin Lakes, NJ) then spun at 4°C, 5,000 x g for 20 minutes. The plasma was stored at -80°C until further analysis. Plasma TNF-a and IL-6 were measured by enzyme immunoassay kits (R&D Systems, Minneapolis, MN) according to manufacturers’ instructions. C4. Organ Weights and Cardiac Hydroxyproline Content At sacrifice, the heart, kidneys, and lungs were harvested, blotted and weighed. Organ weights were compared to tibial length rather than to body weight, due to the variable weight loss from cachexia. The left ventricle (LV) was isolated and minced, then flash frozen and stored in aliquots in liquid nitrogen. Collagen component hydroxyproline was measured in LV homogenates as a quantitative index of fibrosis. Frozen LV tissue was homogenized in ice-cold phosphate-buffered saline containing a Complete Protease Inhibitor Mini-Tab cocktail (Roche, Indianapolis, IN). The tissue was homogenized with a 30 second pulse of a Polytron (Brinkmann) tissue homogenizer and hydrolysis of the sample solution was carried out with 6 N HCl at 100°C for 24 hours. The hydrolyzed samples were dried under a stream of nitrogen. Hydroxyproline standard solutions were prepared in a range from 2.0-10.0 pg/mL, and 0.5 mL of each standard and cardiac homogenates were placed in glass tubes with 1.0 mL of isopropanol and vortexed. To this solution, 0.5 mL of oxidant (0.35 g chloramine T in 5.0 mL water and 20.0 mL citrate buffer) was added, vortexed, and allowed to stand at room temperature for 4 minutes. Next, 3.25 mL of Ehrlich's reagent (3.0 mL Ehrlich's reagent in 15.0 mL 49 isopropanol) was added, and the tubes were kept at 25°C for 18 hours. The intensity of red coloration was measured using a spectrophotometer at 560nm. Total protein content was assessed using the BCA assay (Pierce, Rockford, IL). The amount of hydroxyproline was calculated using the standard curve and expressed as pig/mg total protein. C5. Cardiac Histology Area of Fibrosis Four hearts from each group were utilized for histology determination of cardiac fibrosis. A transverse section of the left ventricle was fixed in 10% neutral buffered formalin. Tissue sections were prepped and mounted, then stained with Masson- Trichrome stain (MTS) for determination of fibrosis. Digital images were acquired with a Olympus BX40 digital microscope camera mounted on a Nikon DN100 light microscope. MTS-stained cross sections of the heart were captured at x200 magnification. The fibrotic areas stained blue with the MTS. True-color image analysis was performed using Bioquant image analysis software (BIOQUANT Life Science, Nashville, TN). Perivascular fibrosis was determined from ten random measures around ten distinct vessels. The area of fibrosis value was derived from the total area encompassing the vessel lumen plus the fibrotic ring divided by the area of the vessel lumen. C6. Cardiac Oxidative Damage and Cardiac GSH/GSSG Frozen LV tissue was homogenized in ice-cold phosphate-buffered saline containing a Complete Protease Inhibitor Mini-Tab cocktail (Roche), and 0.01% volume of antioxidant 0.1M butylated hydroxytoluene in acetonitrile to limit auto-oxidation during sample processing. The tissue was homogenized with a 30 second pulse of a tissue 50 homogenizer (Polytron), then centrifuged at 4°C for 10 minutes at 3,000 x g. The supernatant was collected and stored at -80°C until fiirther analysis. Total protein content was assessed using the BCA assay (Pierce). Malonyldialdehyde (MDA) detection was accomplished using the Biotech LPO-5 86 kit (Oxis Research, Portland, OR) according to manufacturers’ instructions and expressed relative to total protein. Determination of the cardiac reduced/oxidized glutathione (GSH/GSSG) ratio was performed by using the Bioxytech GSH/GSSG-412 kit (Oxis Research) according to manufacturers’ instructions. C7. Statistical Methods All results are expressed 21: SEM. Groups were compared using one-way ANOVA. If the interaction was significant, between-group comparisons were conducted by the Bonferroni post-hoc test. Analysis was conducted using GraphPad PRISM 4(La Jolla, CA). A p value <0.05 was considered statistically significant. D. RESULTS D1. Body Weight and Blood Pressure No significant differences were observed between LS and LSG groups in body weight gain during the course of the study. Cachexia is characteristic of human heart failure and is positively correlated with disease severity and mortality. Dahl-SS rats also develop cachexia as heart failure progresses, so it was expected that body weight would decrease in salt-fed groups at the later time points of the study. The results in Figure 2.1 show that by 18 weeks of diet, body weight fell 22% in the HS group and by 19% in the 51 HSH group relative to the LS group, but only fell 12% in the HSG group. However, this difference from HSG only approached significance (p<0.08). -8- LS A 400‘ -e— LSG 3 * * + HSG g: 300‘ + HSH 27 + HS 5 .9 o 200- 3 100 I I I I I I I I I I I 0 2 4 6 8 1O 12 14 16 18 20 Weeks Figure 2.1. Serial body weight. Each value given is the mean from 12 rats per group. Shown without error bars to preserve visualization. Both the high salt (HS) and high salt + vasodilator hydralazine (HSH) groups were significantly affected, * p < 0.05 vs low salt (LS) group. (LSG) low salt + grape powder diet group; (HSG) high salt + grape powder diet group. As shown in Figure 2.2, HSG did not prevent the development of hypertension, but the HSG diet significantly reduced systolic blood pressure relative to HS diet. For both HSG and HSH, the first statistically significant decrease versus HS was detected at six weeks of treatment. The LSG group trended to have slightly lower systolic blood pressure versus the LS group, but the difference was not statistically significant at any time point. 52 Systolic Blood Pressure (mmHg) § o 2 4 6 a 1'0 1'2 1'4 1'6 1'3 Weeks of Diet Figure 2.2. Systolic blood pressure. Each value given is an average from 12 rats per group :1: standard error of the mean. * At least p < 0.05 vs low salt (LS) group; ** p < 0.05 vs high salt (HS) group. (HSG) high salt + grape powder diet group; (HSH) high salt + vasodilator hydralazine diet group; (LSG) low salt + grape powder diet group. D2. Echocardiograph y Cardiac geometry and function were measured at study baseline (0 weeks), compensated hypertrophy (8 weeks), and diastolic dysfimction (18 weeks). For changes in cardiac geometry, Table 2.3 shows that HS rats showed greater left ventricular end diastolic dimension (LVEDD) at 18 weeks. This remodeling was reduced with the HSG group but not in the HSH group. In the Dahl-SS rat, increasing relative wall thickness (RWT, or 2 x posterior wall thickness during diastole/LVEDD) is found to correlate strongly with contractile failure, more so than increasing LV mass(28). HS diets increased wall thickness, changes first evident at the 8 week compensated hypertrophy stage When measured at 18 weeks, HSG reduced RWT and left ventricular mass/body weight. This effect was not observed in the HSH group at any time point. 53 Table 2.3. Serial Changes in Cardiac Geometry Cardiac G LS LSG HS HSG HSH eometry LVEDD 0 w 7.2 1 0.4 7.0 1 0.4 7.3 10.4 7.1 10.5 7.1 10.3 8 w 7.8 10.5 7.7 10.3 *7.2 10.3 7.6 10.5 *7.0 10.4 18 w 8.1 10.3 8.0 10.5 *8.9 10.5 8.3 10.3 *8.8 10.3 LVESD 0 w 3.8 10.2 3.7 10.2 3.7 10.1 3.5 10.3 3.8 10.2 8 w 4.0 10.2 4.2 10.3 3.8 10.2 3.6 10.2 3.7 10.3 18 w 4.7 10.3 4.6 10.3 4.9 10.3 4.9 10.4 4.8 10.3 RW th 0 w 0.3 10.01 0.3 10.03 0.3 10.02 0.3 10.02 0.3 10.03 8 w 0.3 10.02 0.3 10.03 *0.6 10.02 0.4 10.02 *0.6 10.04 18 w 0.4 10.02 0.4 10.03 *0.7 10.02 0.5 10.03 *0.8 10.03 LV Mass _ 0 w 2.5 10.1 2.4 10.1 2.5 10.1 2.4 10.2 2.6 10.1 8 w 2.6 10.1 2.5 10.2 *3.5 10.2 2.7 10.2 *3.6 10.3 18 w 2.8 10.1 2.7 10.2 *4.3 10.4 3.1 10.2 *4.4 10.3 Echocardiography measures at 0 weeks of diet (baseline), 8 weeks of diet, and 18 weeks of diet. LVEDD (left ventricular end-diastolic dimension, mm), LVESD (left ventricular end systolic dimension, mm), RW th (relative wall thickness, mm), and LV/BW (gram left ventricular mass/gram body weight). Mean 1SEM. n=12 per group. * at least p<0.01 vs LS, LSG, HSG. In addition to grape-associated changes in cardiac geometry, diastolic parameters were also positively affected by HSG diets (Table 2.4). Changes in diastolic parameters were assessed by using M-Mode echocardiography and Doppler tissue imaging. Mild or early diastolic dysfunction is commonly characterized by altered filling velocities, which are measured by the ratio of peak early filling velocity (E wave) to late filling velocity (A wave). The E/A ratio falls at compensated hypertrophy stage, indicating an abnormal relaxation pattern or early diastolic dysfunction. However, E/A sharply rises with cardiac decompensation, indicating increased LV end diastolic pressures and pseudonormalized mitral valve inflow. During compensated hypertrophy at 8 weeks, HS-fed rats displayed a lower E/A ratio indicating a relaxation abnormality. This effect was attenuated in the HSG group, but not in the HSH group. At 18 weeks, HS showed the expected E/A elevation which was also significantly attenuated by HSG, but not in the HSH group. Isovolumetric relaxation time (IVRT) of the left ventricle increased significantly between 8 and 18 weeks. Prolonged IV RT can be considered an indicator of increased myocardial stiffness due to fibrosis(29-32), but HSG significantly reduced IVRT. This effect was not observed in the HSH group. Collectively, these findings indicate that diastolic parameters are improved by the grape-containing diet, but not by the vasodilator hydralazine. Table 2.4. Serial Changes in Diastolic Parameters Diastolic . LS LSG HS HSG HSH Function E/A 0 w 2.7 10.2 2.7 10.3 2.6 10.2 2.6 10.3 2.4 10.3 8 w 2.9 10.1 2.8 10.3 *2.0 10.3 “12.4 10.2 *2.2 10.2 18 w 2.6 10.3 2.5 10.1 *6.2 10.3 **3.8 10.1 *5.8 10.3 E Dec time 0 w 43.6 13 44.0 14 43.3 16 43.2 15 43.1 14 8 w 42.1 13 41.7 15 *48.3 13 45.7 14 *47.2 14 18 w 44.0 12 43.0 14 *34.2 13 139.9 13 *35.6 13 IVRT 0 w 17.7 13 17.3 12 18.8 12 16.7 11 18.113 8 w 18.3 12 18.1 13 20.5 12 20.113 19.5 12 18 w 21.1 12 20.4 13 *31.5 13 124.3 11 *30.1 13 Echocardiography measures at 0 weeks of diet (baseline), 8 weeks of diet, and 18 weeks of diet. (FJA) E Wave to A wave, (E Dec t, in milliseconds) E Wave Deceleration time, (IVRT, in milliseconds) Isovolumetric Relaxation Time. Mean 1SEM. n=12 per group. *at least p<0.05 vs LS, LSG. Tp<0.05 vs LS, LSG, HS, HSH. Regarding systolic function (Table 2.5), the % mid-wall fractional shortening and ejection fraction were not significantly altered by high-salt feeding, which is expected in this rat model; the Dahl-SS rat is a model of diastolic dysfunction rather than systolic dysfunction. However, cardiac index reflects cardiac contractile efficiency by measuring the volume of blood moved per minute (stroke volume x heart rate), per unit of body 55 weight. As such, cardiac index can reflect both diastolic and systolic function. The 8 week measures in all groups did not show significantly impaired cardiac index, which is expected during compensated hypertrophy. However, at 18 weeks, cardiac index was significantly lower in the HS fed group but was significantly improved by HSG. This effect was not observed in the HSH group. Table 2.5. Serial Changes in Systolic Parameters Systolic F . LS LSG HS HSG HSH unctlon % Mid-Wall FS 0 w 21.2 12 21.2 12 22.2 12 20.3 13 21.7 13 8 w 20.4 13 20.4 13 18.3 12 21.2 12 17.9 12 18 w 19.2 12 19.2 12 17.112 18.3 13 16.9 12 % Ejection Fraction 0 w 73.0 14 73.0 14 71.3 15 72.2 16 72.1 14 8 w 75.0 18 75.0 18 69.4 15 70.3 fl 68.6 16 18 w 72.114 72.114 70.114 71.115 70.4 15 Cardiac Index 0 w 444 132 440 127 440 123 438 133 438 117 8 w 435 131 434 125 431 124 432 131 426 122 18 w 437 122 436 119 *333 126 T375 121 *339 123 Heart Rate 0 w 386 122 394 121 392 120 382 119 391 118 SW 397117 410119 412135 410121 417125 18 w 385 123 404 125 423 114 418 123 415 124 Echocardiography measures at 0 weeks of diet (baseline), 8 weeks of diet, and 18 weeks of diet. FS = fractional shortening. Cardiac index is ml of blood/minute/g body weight. Heart rate is beats/minute. Mean 1SEM. n: 12 per group. *p<0.05 vs LS and LSG. 'l'p<0.05 vs LS, LSG, HS, HSH. Heart rate was not affected by treatment, so changes in cardiac index likely reflect changes in cardiac geometry and functionality independent of sympathetic outflow. As observed with diastolic function values, LSG did not confer benefits for systolic function over the LS group. 56 D3. Cardiac Hypertrophy, Histolog and H ydroxyproline Content Cardiac hypertrophy correlates with increased blood pressure, increased fibrosis and collagen deposition, and reduced cardiac function. Irnportantly, cardiac hypertrophy precedes the development of more advanced, irreversible pathogenesis such as heart failure. Compared to H8 diet, HSG was associated with significantly lower cardiac and renal hypertrophy (Table 2.6). Strikingly, the HSG cardiac weights were similar in weight to that of LS and LSG group. HSH reduced renal weight but did not reduce cardiac weight. LSG did not impact cardiac weight relative to the LS group. LSG trended to reduce kidney weight relative to LS group, but the difference was not statistically significant. Hydroxyproline is a component of collagen and a quantitative index of fibrosis. Collagen accumulation occurs in the heart during heart failure and contributes to stiffening of the heart walls, impaired relaxation, impaired filling, and reduced cardiac output. As shown in Table 2.6, HSG had significantly reduced cardiac hydroxyproline and perivascular area of fibrosis content relative to HS, which was not observed in the HSH group. Table 2.6. Changes in Organ Weight and Cardiac Fibrosis LS LSG HS HSG HSH Heartl 0.34 10.01 0.33 10.02 *0.4210.l 103210.01 *0.4310.3 Kidneyl 0.71 10.04 0.65 10.04 *0.9810.05 108210.04 108310.06 Lungl 0.42 10.04 0.40 10.04 05410.06 04310.05 0.52 10.04 Hydoxproline 5.2 10.3 5.1 10.2 *9.210.2 17.4103 *9.1 10.3 Area F ibrosis2 1.2 10.04 1.2 10.02 *1.4510.1 “11.3 10.03 *l.40 10.4 lOrgan weights in grams/cm tibial length. Hydroxyproline is lag/mg total protein. Mean 1SEM. n=12 per group. 2Determined from ten random measures around ten distinct vessels *at least p<0.05 vs LS and LSG, Tp<0.05 vs LS, LSG, HS. 57 D4. Cardiac GSH/GSSG, Oxidative Damage, and Plasma Inflammation Malonyldialdehyde (MDA) is a by-product of the oxidation of lipids, and serves as a marker of oxidative stress. Data in Table 2.7 indicates that HSG diet was associated with significantly lower MDA content relative to HS, but HSH did not provide this effect. Although MDA was relatively low in the healthy LS rat hearts, LSG still conferred a significant treatment effect versus LS. Cardiac reduced glutathione (GSH) is decreased in the salt-fed Dahl-SS rat heart relative to oxidized glutathione (GSSG)(33,34). HSG diet significantly improved the GHS/GSSG ratio over HS group (Table 2.7), reflecting improved antioxidant defense. This effect was not observed in the HSH group. Interestingly, this effect was also observed in LSG rats as compared to LS rats, indicating that grape powder provision improved cardiac antioxidant defense even in the absence of concurrent disease. Also in Table 2.7, ELISA for plasma markers of inflammation indicated that HSG diet was associated'with significantly reduced plasma IL-6 and TNF- a relative to the HS group. This effect was not observed in the HSH group. While LSG trended to reduce IL-6 and TNF-01, the results were not statistically significant. Table 2.7. Cardiac GSH/GSSH, Cardiac MDA, and Plasma Inflammation LS LSG HS HSG HSH Cardiac * * * GSH/GSSG 148 1 7 184 1 9 45 1 3 175 1 5 53 1 6 ' 11.0 1 *1.3 1 * Cardiac MDA 0.4 1 0.02 0.3 1 0.02 1.4 1 0.1 0. 0 6 0.2 * plasma TNF-a 1.4 1 0.02 1.1 1 0.03 *68 1 0.3 14.3 1 0.3 ‘31: * plasma IL-6 0.9 1 0.02 0.7 1 0.02 *5.4 1 0.3 13.7 1 0.2 011$ GSH in 11M relative to GSSG in 11M. MDA is expressed as mg/per g total protein. TNF-a and IL-6 are in pg/mL. Mean 1SEM. n=12 per group. * at leastJK 0.05 vs LS, T p<0.05 vs LS, LSG, HS, HSH. 58 E. DISCUSSION The current results demonstrate the broad effects of a phytochemical-enriched diet on the gradual development of hypertension-associated diastolic dysfunction. The focus on diastolic pathogenesis is of great significance in the aged. Systolic dysfunction primarily concerns the heart’s reduced ejection capacity, where diastolic dysfunction concerns the heart’s reduced filling capacity. While systolic failure has a higher mortality rate, diastolic heart failure has a strong association with normal aging and is more common than systolic heart failure in the elderly(35,36). lmportantly, numerous clinical trials have documented the benefits of pharmacologic treatment for systolic heart failure; however, the optimal treatment for diastolic heart failure has not yet been defined. Diastolic dysfunction develops over a prolonged period of time and is largely reversible, so the effects of diet patterns on disease course are of great interest for both preventive and interventional cardiology. The descriptive approach employed here is intended to reveal the breadth of phytochemical-rich diet effects on many phenotypes relevant to hypertension and to diastolic heart failure pathogenesis. The mechanisms behind the treatment effects are likely complex and involve the interaction amongst a number of organ systems. For example, grape-related benefits may be derived in part from reduced blood pressure, and blood pressure is regulated dynamically by interactions amongst the kidney, brain, vasculature, and heart. In the HSG group, reduced systolic blood pressure was observed early and was sustained throughout the study. The lack of depressor effect in the LSG group as compared to LS suggests that grape-related depressor effects are largely observed in hypertensive as opposed to norrnotensive animals. The mechanisms of grape-associated vasodilation are 59 not completely understood, but some studies have indicated that grape-product consumption may improve the availability of the vasodilator nitric oxide. However, hydralazine afforded a similar reduction in systolic blood pressure throughout the study, yet failed to impact eventual cardiac pathology, suggesting that reduced hypertension alone is not sufficient for cardioprotection. Hydralazine limits calcium release from smooth muscle sarcoplasmic reticulum, resulting in arterial and arteriolar relaxation. In the Dahl-SS rat and in other hypertensive rat models, hydralazine consistently reduces arterial pressure but does not impact cardiac hypertrophy or fibrosis(19-21), results which support our current findings. Hydralazine can elicit a reflex sympathetic stimulation at higher doses, but the dose provided here did not cause elevated heart rate or cardiac output. It is possible that differing routes of vasodilation lead to different protective phenotypes, but it is clear that reduced blood pressure alone does not protect against cardiac fibrosis or hypertrophy in this model. The current results therefore imply that additional mechanisms beyond vasodilation are participating in grape-mediated cardioprotection. Hypertension contributes to cardiac oxidative stress, and grape-enrichment may confer antioxidant effects. In the heart, unquenched reactive oxygen and reactive nitrogen species damage local lipids, proteins, and DNA, leading to cardiomyocyte death and sub-optimal cardiac function. Although a specific relationship between oxidative stress and ventricular performance has not been clearly established, there is considerable association between oxidative stress and underlying components of cardiac pathogenesis including systemic inflammation, cardiomyocyte apoptosis, cardiac remodeling, mechanoenergetic uncoupling, and endothelial dysfunction. Furthermore, accumulated evidence suggests a 60 significant correlation between oxidative stress and clinical indexes of cardiac functional capacity, such as New York Heart Association class and peak exercise oxygen consumption(37,3 8). By reducing oxidative stress, antioxidant-rich diets may thus impact the pathogenesis or severity of cardiac dysfunction. Direct antioxidant effects in the heart tissue would require cardiac bioavailability of the grape phytochemicals. The predominant bioavailable components would likely include enterohepatic metabolites such as sulfate conjugates, glucuronides, and 0- methylated forms, with very low levels of non-conjugated, parent compounds. However, these enterohepatic and intracellular metabolites have a reduced ability to donate hydrogen, and are less effective scavengers of radicals as compared to their parent compounds. Also, concentrations of these metabolites in the plasma or in tissues are lower (nanomolar, low micromolar) than those recorded in vivo antioxidants such as ascorbate, uric acid, glutathione, and Vitamin E(39). Consequently, bioavailable grape phytochemicals are unlikely to supersede these antioxidants for radical scavenging effects, and thus direct antioxidant action of grape metabolites in cardiac tissue may be relatively minor. Instead, accumulating evidence suggests that the tissue antioxidant effects of phytochemicals may be mediated indirectly by their interactions with intracellular signaling cascades and with altered gene expression. For example, bioavailable phytochemicals may stimulate the transcription and translation of endogenous antioxidants in the heart. Polyphenols like those found in grape can activate response elements in the genome which regulate the transcription of glutathione-regulating enzymes. In the current study, both LSG and HSG were associated with elevated 61 GSH/GSSG relative to their controls LS and HS, respectively. Bioavailable grape phenolic phytochemicals may activate cardiac genes which modify glutathione dynamics, like glutathione peroxidase and glutathione-S-transferase(40). Finally, grape—related benefits may derive in part from indirect effects on reduced cachexia and systemic inflammation. Cachexia is a catabolic state characterized by weight loss and muscle wasting and occurs frequently in patients with heart failure, and is a strong independent risk factor for heart failure-related mortality(41). Cachexia is also characteristic of Dahl-SS pathogenesis, and appeared in our rats after 14 weeks of diet. The onset of cachexia is associated with elevations in pro-inflammatory mediators, including IL-6 and TNF-a, both of which correlate with advancing heart failure(22,42). Grape diet effect on body weight loss approached significance, and significantly reduced plasma TNF- a and IL-6. Thus, limited cachexia may contribute to grape-associated benefits. The phytochemical model presented here is limited by the use of only one fruit. We expect that treatment effect could be amplified were we to use a more complex mix of phytochemical-containing whole foods. Rationale for grape selection is supported from several studies showing a depressor effect of grape juice and wine consumption(14- 18). Results found here may not eXtend to the dietary supplement grape seed extract, which contains higher levels of high molecular weight tannins of questionable bioavailability, and which lacks anthocyanins. Instead, table grape powder derived from grape skin, flesh and seed contains a broader phytochemical profile that is more relevant to that observed in fruit/vegetable-rich human diets. Our intent was to use a model food that has modest antioxidant potential and has demonstrated efficacy. This standardized, 62 whole grape powder used here has been shown by other investigators to reduce both plasma and tissue markers of oxidative stress in vivo and ex vivo(9-13). These studies indicate that beneficial components of the whole table grape powder are bioavailable to tissues, and could confer health benefits against diseases which involve oxidative stress such as hypertension-associated heart failure. The simplified model presented here may thus serve as a precursor to studies which model more complex dietary patterns and their effects upon cardiac pathogenesis. With regards to grape “dose” justification, allometric scaling or the bioequivalence between rodents and man is unknown. As such, the dose of grape powder given per day was made relative to body weight. One human serving of fresh grapes is % cup, or approximately 126 grams. With loss on drying, one human serving of freeze dried whole grape powder equals 23 grams. The rat body weight equivalent of nine servings of grapes/day then averaged 600 milligrams/day, or 3% of the daily diet. In this manner, the dose used here attempted to model the nine servings/day of fruit/vegetables in the DASH diet trials(4). Other methods of estimating bioequivalence could lead to different dose justifications, including adjustments made relative to metabolic rate, food intake, food intake relative to body weight, differences in body surface area, or target organ weight relative to body weight. Regardless of the approach to estimate bioequivalence, the level of whole fruit powder employed here is likely to be physiologically relevant to human diets. Although we controlled for macronutrient and calorie intake in the current design, we cannot conclusively exclude any benefit from the additional micronutrients from grape (as described in Table 2.1). However, the 3% w/w grape powder enrichment 63 supplied a modest six milligram increase in potassium and a 0.02 milligram increase in vitamin C intake per day. Previous studies in the Dahl-SS rat suggest that greater potassium (43) (5-10x higher than provided here) and greater Vitamin C (44) (5,000x higher than provided here) is required to reduce blood pressure. Further studies may be warranted to ascertain the specific contribution of micronutrients in the absence and presence of phytochemicals. Clinical trials in patients with heart failure have failed to detect benefits from antioxidant micronutrient supplementation and indeed, some have observed adverse effects(45,46). In contrast to dietary supplements, whole food models allow synergistic interaction between micronutrients and phytochemicals which may improve their bioavailability or potency. Therefore, whole foods approaches may confer both increased efficacy and safety versus dietary supplements for the prevention or treatment of heart failure. In summary, the diet incorporation of grape-derived phytochemicals improved cardiac glutathione reserve and reduced experimental hypertension-induced cardiac fibrosis and diastolic dysfunction in the Dahl-SS rat. This benefit correlated with reduced cardiac oxidative damage and improved cardiac antioxidant reserve. The findings support the efficacy of phytochemical-enriched diets against hypertension-associated cardiac pathology. This may have particular importance to our aging population, which has reduced intake of both fruit and vegetables. The 2000 edition of the Dietary Guidelines for Americans(47) revealed that in individuals over age 60, only 35% of women and 39% of men met the two servings/day objective for fruits, and only 6% of both women and men met the three servings/day objective for vegetables. Because grape supplementation occurred at the onset of the study, this model examines preventive 64 effects versus interventional effects. 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J Am Diet Assoc 2000;100:769-774. 70 CHAPTER THREE A PHYTOCHEMICAL-ENRICHED DIET IMPACTS CARDIAC PPAR AND NF- KB ACTIVITY, FIBROSIS, AND CYTOKINE EXPRESSION IN RATS WITH DIASTOLIC HEART FAILURE 71 A. ABSTRACT Prolonged hypertension is the leading cause of heart failure. Failing hearts show reduced peroxisome proliferator-activating receptor (PPAR) activity and enhanced nuclear factor kappa B (NF-KB) activity, which modify cardiac inflammation and fibrosis. In vitro studies suggest that phytochemicals alter PPAR and NF-KB activity, but effects of a phytochemical-rich diet are less understood. Grapes contain an array of commonly consumed dietary phytochemicals and may serve as a model of a phytochemical-rich diet. In Dahl-Salt Sensitive hypertensive rats, we previously showed(1) that dietary provision of 3% wzw whole table grape powder for 18 weeks reduced blood pressure, cardiac hypertrophy, and diastolic dysfunction. The working hypothesis is that in rats, phytochemical provision from whole grape powder impacts cardiac PPAR and NF -xB activity and their related gene transcripts. In rat hearts, we measured PPAR and NF-KB DNA binding activity, mRNA related to PPAR and NF-xB activation, NF-KB target TNF-a and TGF-Bl expression, and histology-determined perivascular fibrosis. Grape-fed groups had enhanced PPAR-01 and PPAR-y activity and reduced NF -KB activity. RT-PCR indicated grape-associated up-regulation of PPAR-01 mRNA, PPAR-y co-activator-l, PPAR-y, and NF-KB inhibitor IxBa. RT-PCR also indicated significant, grape-associated down-regulation of tumor necrosis factor-a (TNF- a) and transforming grth factor-[31 (TGF -|31). Finally, grape intake was associated with significantly reduced cardiac TNF-a and TGF-Bl protein expression and reduced perivascular fibrosis. In the Dahl-SS rat, chronic intake of grapes altered cardiac transcripts related to PPAR and NF-xB, which may be significant to the observed diet- associated cardioprotection. 72 B. INTRODUCTION Prolonged hypertension is a prevalent and significant contributor to morbidity and mortality from heart failure. The DASH (Dietary Approaches to Stop Hypertension) clinical trials provided evidence that diets rich in fruits and vegetables reduced blood pressure(2,3). Animal models of hypertension may permit mechanistic appraisals of the interaction of diet and disease. One recent study in hypertensive rats modeled the DASH diet using added nutrients, but failed to show an effect on blood pressure(4). Therefore, a whole foods approach rather than altered nutrients alone may be more appropriate to assess the effect of diet on hypertension and related pathologies. Previous studies by Seymour et al(l). showed that in the Dahl Salt-Sensitive (DSS) rats fed a high salt diet, the incorporation of freeze-dried whole table grape powder (3% of diet) significantly lowered blood pressure, cardiac hypertrophy and cardiac, lipid peroxide formation. In addition, grape-fed rats displayed improved diastolic function and cardiac output. Furthermore, the benefits were not entirely related to blood pressure reduction, because comparable blood pressure reduction by hydralazine failed to match the cardioprotective effects of grape diet(l). However, cardiac-specific mechanisms of these effects remain unknown. The current project focuses on cardiac cell signaling related to transcription of PPAR and NF-KB. PPARs are nuclear receptor transcription factors which intimately impact cell metabolism, cell differentiation, and inflammation. PPAR agonist drugs are used clinically to address hyperlipidemia and/or insulin resistance, but ancillary anti- inflammatory affects have also been observed. lmportantly, PPARs are deficient in failing hearts(5,6), and PPAR agonism is beneficial in experimental heart failure 73 models(7-10). The risks and benefits of PPAR-targeted drugs in human heart failure are controversial(1 1-13) and are of current clinical interest given the widespread use of PPAR agonist drugs in cardiac patients. Transcription factor NF-KB is exquisitely sensitive to oxidative stress, and engages in inflammatory responses by activating the transcription of various pro- inflarnmatory genes including cell adhesion molecules, inflammatory cytokines, and chemokines. Cardiac NF-KB activity is positively correlated with heart failure progression(14-16), and inhibition of NF-KB limits heart failure progression(l7-21). The relation between PPAR activity and NF-KB-related inflammation can been described as bidirectional antagonism. That is, PPAR activation reduces NF-KB activation, and NF-KB reduces PPAR/DNA binding. In heart failure models, PPAR agonism with drugs experimentally reduces cardiac NF -xB activity and reduces morbidity and mortality(22,23). However, the effect of diet upon cardiac PPAR and NF-KB activity is poorly understood. In the current research model, bioavailable grape phytochemicals may have altered cardiac PPAR and/or NF-KB activity. Grapes are a source of diverse phytochemicals, but are particularly rich in pigment-conferring anthocyanins. In vitro studies show that anthocyanin-rich extracts can activate PPARs in varied experimental models(24-30). If the grape diet altered cardiac PPAR activity, it could also limit cardiac NF-KB activity and associated cardiac inflammation and fibrosis. This project then tests the hypothesis that 3% dietary grape powder supplementation, which reduces Dahl-SS rat diastolic heart failure pathogenesis( 1), is also associated with increased cardiac PPAR 74 activity and decreased NF-KB activity, and with reduced cardiac expression of cytokines and growth factors relevant to heart failure pathogenesis. C. MATERIALS AND METHODS CI. Animal Care and Diets Five week old Dahl-Rapp Salt-Sensitive rats (Harlan, Indianapolis, IN) were acclimated for one week on AIN-76a powdered diet (Research Diets, New Brunswick, NJ). Afterwards, each rat was randomly assigned (11 = 12 each) to one of four treatments. Low Salt diet (LS, AIN-76a with 2.8% added carbohydrate, glucose:fructose 1:1), Low Salt diet + grape powder (LSG, AIN-76a with 3.0% w/w added grape powder), High Salt diet with 6% added NaCl (HS, AIN-76a with 2.8% w/w added carbohydrate), or High Salt Diet + grape powder (HSG, AIN-76a with 3.0% w/w added grape powder). Grape powder content, diet preparation, and diet storage are as presented in Chapter Two, Section G]. Animals were fed 20 grams of powdered diet/head/day. Ad libitum intake of AIN diet averages 19-21 grams of AIN powder/day in the Dahl-SS rat(31), so provision of 20 grams/day ensured complete daily consumption. For the high salt diets, NaCl was added directly to the food hopper and mixed carefully with the daily ration of powdered diet. Rats were housed three/cage in 12h light:12h dark cycles, and water was provided ad libitum. This project was approved by the Animal Care and Use Committee at the University of Michigan. C2. Cardiac Tissue Sub-cellular Fractionation and Western Blot. 75 Rats were sacrificed by guillotine, then hearts were harvested, washed in phosphate-buffered saline (pH 7.4), blotted, and weighed. The left ventricle was minced and flash frozen in liquid nitrogen, then stored at -80°C until further use. Frozen cardiac tissue was fractionated to obtain nuclear and cytosolic homogenates using the method of Li et al.(32) with modifications, using a NE-PER Nuclear Extraction Kit (Pierce, Rockford, IL). Frozen cardiac fragments were powdered using a mortar and pestle cooled by a cocoon of dry ice. The powder was then added to kit buffer cytoplasmic extraction reagent I (CER I) at 50 mg powder/ml CER I buffer, and subjected to four, lO-second, low speed pulses of a Brinkmann Polytron homogenizer (Kinematica, Bohemia, NY). The resulting homogenate was incubated on ice for ten minutes, followed by the addition of cytoplasmic extraction reagent Buffer II (CERII, 55 pl/ml of CER I buffer volume). The sample was briefly vortexed and centrifuged for five minutes at 14,000 x g (at 4°C). The resulting supernatant was considered the cytosolic fraction. The pellet was then resuspended in the nuclear extraction reagent (N ER, 500pl/ml of CER I buffer volume), and incubated on ice for a total of 40 minutes with brief vortexing (10 seconds) at 10- minute intervals. The sample was then centrifuged for 10 minutes at 16,000 x g (at 4°C). The resulting supernatant was considered the nuclear fraction. Nuclear and cytosolic fractions (50 ug each) were mixed with SDS sample buffer, denatured for five minutes at 95°C, resolved on pre-cast NuPAGETM 10% Bis-Tris polyacrylarnide gels (Invitrogen, Carlsbad, CA, USA) by electrophoresis using a Novex Mini-Cell (Invitrogen), and subsequently transferred onto PVDF membranes. Blocking and antibody incubation steps were accomplished using the vacuum-based, SNAP i.d. Protein Detection System (Millipore, Billerica, MA, USA) using the ECL-specific 76 blocking reagents and ECL-chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). Successful fractionation was verified by immunodetection of 01- tubulin (1° antibody 1:1000, Santasz Biotechnology, CA) and lamin B (1° antibody 1:200, Santasz Biotechnology) for cytosolic and nuclear fractions, respectively. Membranes were exposed to CL-XPosure film (Pierce, Rockford, IL, USA) and band densities were analyzed using UN-SCAN-IT Gel software version 6.1 (Silk Scientific, Orem, UT, USA). C3. Transcription Factor DNA Binding Assays. Once successful fractionation was confirmed, PPAR-01, PPAR-y, and NF-KB activity were determined in nuclear extracts using Transcription Factor DNA Binding assays (Cayman Chemical, Ann Arbor MI) according to manufacturers’ instructions. A specific, proprietary oligonucleotide containing PPAR response elements (PPREs) or KB responsive elements was immobilized onto the bottom of the wells of a 96-well plate. If present in the nuclear extract (loaded at lOug/well), PPAR isoforms and NF-xB-element p65 bind to the well-bound oligonucleotide PPREs or chs, respectively. Binding was then detected by addition of specific primary antibodies directed against the individual PPAR isoforms or against p65 subunit of NF-KB. A secondary antibody conjugated to horseradish peroxidase was added to enable colorimetric detection by reaction with substrate TMB/hydrogen peroxide and measurement of color development at 450 .nm. Values were expressed as optical density relative to total protein in the respective nuclear extract as determined by the BCA Assay(Pierce). 77 C4. RT-PCR Total RNA from minced left ventricle was isolated with the RNeasyTM Fibrous Tissue Midi Kit (Qiagen, Valencia CA, USA) following the manufacturer’s protocol. RNA quality and quantity (260/280 ratio >2.0, prominent 18S and 28S bands) were verified using the Agilent 2100 BioAnalyzer(Agilent Technologies, USA). From the twelve animals per group, four representative RNA samples were obtained by randomly combining equirnolar amounts of RNA from three rats. First strand cDNA synthesis was accomplished with the RT2 First Stand Kit (SABiosciences, Frederick MD). cDNA was then added to the RT2 qPCR Master Mix, which contains SYBR Green and a reference dye. The relative abundance of eleven mRNA transcripts was then compared using a RT2 Profiler PCR ArrayTM (SABiosciences). Relative expression was determined by the AACT method as described by Livak(33), normalized relative to the average ACt of four housekeeping genes (Pl large ribosomal protein, hypoxanthine guanine phosphoribosyl transferase, ribosomal protein L13A, and lactate dehydrogenase). C5. Histology Determined Fibrosis Four hearts from each group were utilized for histology determination of cardiac fibrosis. A transverse section of the left ventricle was fixed in 10% neutral buffered formalin, and sections were stained with Masson-Trichrome stain for determination of fibrosis. The fibrotic areas stain blue/purple, and the non-fibrotic areas stain red. Digital- images were acquired with a Olympus BX40 microscope camera mounted on a Nikon DN100 light microscope. True-color image analysis was performed using Bioquant image analysis software (BIOQUANT Life Science, Nashville, TN). 78 C6. T NF -a and T GF -fl Enzyme-Linked Immunosorbent Assays (ELISA) ELISAs were conducted on cytosolic homogenates derived from frozen left ventricle. Total protein content was assessed using the BCA assay (Pierce). Cardiac TNF- a and TGF-B were measured using commercial kits (R&D Systems, Minneapolis, MN) according to manufacturers’ instructions. Results are expressed relative to total protein (BCA Assay, Pierce). C7. Statistics mRNA transcript pair-wise comparisons are determined 1 SD using the AACT method as described by Livak(33), using the PCR Array data analysis web portal of SABiosciences (http://www.sabiosciences.com/pcr/arrayanalysis.php). All other endpoints were expressed :1: SEM and compared using a two-way ANOVA with salt and grape as independent factors. Given significant interaction, pair-wise comparisons were accomplished with Bonferonni post-hoc tests. Analysis was conducted with SPSS, version 16.0 (SPSS, Chicago, IL). For all measures, a p value < 0.05 was considered statistically significant. D. RESULTS DI. Transcription Factor DNA Binding ELISA Results for the PPAR isoforms are in Figure3.lA and 3.1B. In the LSG group, both PPAR-a and PPAR-y activity were increased as compared to the LS group. In contrast, the HS group showed reduced PPAR-a and PPAR-y activity. Finally, compared 79 to HS, the HSG group showed enhanced PPAR-01 and PPAR-y activity. The conserved increase in nuclear extract PPAR binding in both LSG and HSG groups relative to their respective salt controls could suggest a specific effect of bioavailable grape phytochemicals and/or their metabolites upon PPAR activity. Results for NF-KB activity are shown in Figure 3.2C. Compared to LS rats, the LSG group showed reduced NF-KB activity. In contrast, the HS group showed sharply increased NF-KB activity. Finally, compared to HS, the HSG group showed reduced NF -1