Dietary Fatty Acids, Redox Signaling, and the Heart - Springer

Dietary Fatty Acids, Redox Signaling, and the Heart

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Julianne Beam, Amy Botta, Rebekah Barendregt, and Sanjoy Ghosh

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endogenous Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Superoxide Dismutases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glutathione Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catalase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heart: Major Cell Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Why Fatty Acids? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dietary Fatty Acids: Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saturated Fatty Acids and Cardiac Redox Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Monounsaturated Fatty Acids and Cardiac Redox Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polyunsaturated Fatty Acids: Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Omega-3 PUFA and Cardiac Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Omega-6 PUFA and Cardiac Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular Changes Mediated by PUFA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Fluidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulating Endogenous Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

In Western societies, cardiac disease (CD) remains the primary reason for unexpected morbidity and mortality. A frequent accomplice that participates in many cardiac disorders is the omnipresence of aberrant oxidative stress in such disease processes. As more research suggests nutritional factors have an important influence on this mechanism, there is an increasing realization that dietary fats modulate cardiac redox signaling. Fatty acids are the most energy

J. Beam • A. Botta • R. Barendregt • S. Ghosh (*) Department of Biology, University of British Columbia-Okanagan, Kelowna, BC, Canada e-mail: [email protected]; [email protected] I. Laher (ed.), Systems Biology of Free Radicals and Antioxidants, DOI 10.1007/978-3-642-30018-9_44, # Springer-Verlag Berlin Heidelberg 2014

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dense macronutrients and therefore their signaling roles on cardiac cell types, during normal or disease physiology, may be crucial in strategizing therapeutic dietary interventions to attenuate cardiac oxidative damage. This chapter provides a summary of various dietary fatty acid classes, their impact on cardiac metabolism, and their role in either augmenting or attenuating cardiac oxidative stress. Keywords

Antioxidant • Heart • MUFA • Oxidative stress • PUFA • SFA

Introduction Oxidative stress represents an imbalance between the production of pro-oxidant species and the antioxidant defense mechanisms leading to cell, tissue, or organ damage (Avery 2011). This results from incraesed pro-oxidant production and/or decreased antioxidant defense, which often leads to inflammation (Matsunami et al. 2010) and apoptosis (Duvall and Wyllie 1986; Lennon et al. 1991) at the cellular level. These changes are believed to be linked to various disease processes (Roberts et al. 2009) and aging (Harman 1956). Pro-oxidant species in vivo are often referred to as reactive oxygen species (ROS) or reactive nitrogen species (RNS). ROS are either radical species such as superoxide anion (O2• ) and the hydroxyl radical (OH ) that contain unpaired valence electrons or non-radical species that are unstable and has a higher oxidizing potential like hydrogen peroxide (H2O2). An example of RNS is the peroxynitrite (ONOO ) radical, which is derived from nitric oxide (NO). In considering “oxidative stress” as a purely negative process, it is also important to appreciate its evolutionary and beneficial roles in homeostasis. It is well known that a small amount of ROS generation is required for phagocytosis, cell signaling, apoptosis, and other normal homeostatic mechanisms. For example, the immune system employs the respiratory burst from phagocytes to remove infectious agents and to heal injury. The respiratory burst depends upon the enzyme, NADPH oxidase, and occurs in cells such as neutrophils, eosinophils, and mononuclear phagocytes, which have potent bactericidal effects (Dahlgren and Karlsson 1999; Babior et al. 2002). ROS are also involved in antioxidant hormesis. For example, when produced at low levels, the O2• radical promotes the upregulation of antioxidants such as superoxide dismutases 1 and 2, and glutathione peroxidase in both skeletal muscle (Franco et al. 1999; Sreekumar et al. 2002) and erythrocytes (Robertson et al. 1991). Activities such as exercise that increase mild oxidative stress are thought to promote cardiovascular antioxidants in healthy, young humans (Ristow et al. 2009) but not in aging, older diseased animals (Laher et al. 2013). Albeit its beneficial roles, the general interest in oxidative stress in cardiac biology stems from the fact that aberrant oxidative stress is most often correlated with major diseases in the heart from atherosclerosis, cardiomyopathies, and heart failure. Being a necessary part of the human diet, both total and choice of dietary fats can alter cell metabolism and modulate redox signaling in the cardiovascular

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system. Therefore, the focus of this chapter will be to delineate the role of major dietary fatty acid classes in redox signaling in the normal and the diseased heart.

Endogenous Antioxidants Antioxidants are agents that either neutralize ROS (direct antioxidants) or diminish their effects (indirect antioxidants) to counteract oxidative stress. They accomplish this function by accepting or transferring electrons from ROS or other reactive intermediates, which results in the deactivation of free radical based reactions (Ungvari et al. 2011). Antioxidants can also be subdivided into two broad categories depending on their distribution: exogenous and endogenous. Exogenous antioxidants such as polyphenols, lipoic acid, and vitamins C and E are normally supplied in the diet. They usually work either as “chain-breaking” antioxidants (polyphenols, vitamin E), or by enhancing the efficacy or concentration of other endogenous antioxidants (lipoic acid). Alternatively, endogenous antioxidants are synthesized de novo and are generally more potent than exogenous antioxidants. Enzymatic endogenous antioxidants include superoxide dismutase, glutathione peroxidase, and catalase whereas nonenzymatic examples include thioredoxin and glutathione. These antioxidants are located throughout the cell including the cell membrane, mitochondria, nucleus, cytosol, and blood plasma (Maritim et al. 2003). The enzymatic antioxidants form the first line of defense against ROS, and their regulation depends mainly upon the oxidant status of the cell (Kumar and Jugdutt 2003).

Superoxide Dismutases The superoxide dismutase (SOD) family is thought to protect the brain, lungs, and virtually all other tissues from oxidative stress (Mao et al. 1993). SOD enzymes play a major role in the antioxidant defense system of aerobic cells by catalyzing the dismutation of O2• to oxygen and H2O2. In fact, most of the H2O2 production during metabolic processes results from the breakdown of O2• by SOD (Mao et al. 1993). Three isoforms of SOD are currently known to exist: SOD1, SOD2, and SOD3 (Packer 2002). The first isoform, SOD1, is a soluble protein found in both cytoplasmic and mitochondrial intermembrane spaces where it functions as a homodimer to bind zinc and the copper ion. The next isoform, SOD2, is a member of the iron/manganese superoxide dismutase family and unlike SOD1 is found in the mitochondrial matrix and is most abundant in cardiac myocytes. Mutations in the gene for SOD2 have been associated with idiopathic cardiomyopathy (IDC), premature aging, sporadic motor neuron disease, and cancer (Xu et al. 2008). Lastly, SOD3 is a human isoform that is secreted into the extracellular space where it forms glycosylated homotetramers (Marklund 1982). These forms of SOD3 are anchored to the extracellular matrix (ECM) and cell surface through interaction with heparan sulfate, proteoglycan, and collagen (Fukai et al. 2002).

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Glutathione Peroxidase Glutathione peroxidase (GPX) is a cytoplasmic enzyme that functions in the scavenging and inactivation of H2O2 and lipid peroxides (Michiels et al. 1994; de Haan et al. 1998). GPX catalyzes the conversion of H2O2 to water and molecular oxygen, which requires the donation of reducing equivalents provided by glutathione (GSH). The levels of GPX in the body therefore correlate very closely with GSH levels, under most conditions (Hayes and McLellan 1999). GPX is critical in the protection of the myocardium and preservation of function. Deficiency of GPX1 in a murine model resulted in cardiac vascular and structural abnormalities as well as endothelial dysfunction (Forgione et al. 2002). Clinically, patients with lower GPX-1 activity demonstrate a higher risk for cardiac disease (CD) (Blankenberg et al. 2003). Similarly, very low levels or absence of GPX activity has been observed in atherosclerotic tissue (Lapenna et al. 1998), indicating that this enzyme is critical for progression of this disease process.

Catalase The other important endogenous antioxidant enzyme is catalase (CAT). Similarly to GPX, CAT catalyzes the conversion of two molecules of H2O2 to two molecules of water and molecular oxygen (Kang et al. 1996). GPX is the main agent in the detoxification of H2O2 (Jones et al. 1978), whereas CAT primarily functions in the detoxification of H2O2 formed in the peroxisomes by dehydrogenase reactions (Kang et al. 1996). CAT is present in almost all mammalian cells at varying concentrations; for example, CAT is highest in erythrocytes and the liver (Deisseroth and Dounce 1970) and lowest in the heart (Kang et al. 1996). This low activity in the heart is accompanied by a complete absence of CAT in human vascular smooth muscle cells and endothelial cells, both of which are important within the cardiac system (Shingu et al. 1985).

Heart: Major Cell Types The mammalian heart is an obligate aerobic organ composed of cardiac muscle and vasculature. The outer wall of the heart is comprised of three distinct layers of cells, each having their own function. The outer wall is known as the epicardium, and it is a protective layer encompassing the whole heart. The middle layer is termed the myocardium and is primarily involved in cardiac contractions and the inner layer or endocardium functions in direct contact with blood. The main cardiac cell types in all these three layers are fibroblasts, cardiomyocytes, endothelial cells, vascular smooth muscle, and invading macrophages from the circulatory system. Although cardiomyocytes occupy 75 % of the myocardial tissue volume (Moore et al. 1980; Vliegen et al. 1991), fibroblasts account for 70 % of the total number of cells in the myocardium (Nag 1980). Fibroblasts are found in connective tissue and

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are responsible for the creation of the extracellular matrix (ECM) and the secretion of collagen (Kanekar et al. 1998). Under normal physiological circumstances, the total deposited collagen in the heart is low, whereas under cardiac pathologies, there is increased deposition of fibrous substances or cardiac fibrosis, such as in heart failure (HF) and cardiomyopathy. As an example, in response to injury and cardiomyocyte cell death, collagen deposition increases dramatically, causing altered cardiac structure and function, leading to HF (Gurtner et al. 2008). Rather than purely an inotropic defect mediated by systolic insufficiency, a predominant feature in at least 50 % of HF patients is the evidence of progressive diastolic dysfunction where the heart muscle cannot relax properly to allow ventricular filling, thereby reducing cardiac output (Gary and Davis 2008). Cardiac fibroblasts contribute the majority of fibrillar collagen in the myocardium and promote stiffening and myocardial remodeling as a maladaptive phenomenon following injury. Increasingly, fibroblasts are becoming an interesting target for the possible prevention of HF (Chen et al. 2011). Cardiac myocytes (cardiomyocytes) are the functional units involved in the conduction of electrical signals and myocardial contraction (Walker and Spinale 1999). To allow for proper cardiac contraction, these cells contain large calcium (Ca2+) stores (Bers 2008), and up to 40 % of their volume is comprised of mitochondria to provide the required high levels of ATP (Voet and Voet 2005). The large proportion of mitochondria also serves to take up large amounts of Ca2+ ions, preventing Ca2+ overload in the cytosol and therefore improper contraction in the cardiomyocyte (Crompton 1990). Mitochondrial damage has been widely reported in the hearts of obese animals (Boudina et al. 2005; Dong et al. 2006; Bugger and Abel 2008) and in human patients (Ritov et al. 2005) with lipotoxicity or cardiomyopathy (Sebastiani et al. 2007). As an example, perfusion of isolated cardiac mitochondria from obese diabetic mice with fatty acids leads to increased oxygen consumption but reduction in ATP production. A fall in ATP/O ratio is indicative of uncoupling of oxidative phosphorylation from ATP production (Boudina et al. 2005). Mitochondria can be uncoupled due to persistent mitochondrial oxidative stress (Boudina et al. 2007). The mitochondrial-membrane-associated ETC is the primary source of ATP production for the beating heart. The ETC is comprised of four enzyme complexes termed as complex I to IV, through which electrons are transferred sequentially to activate ATP synthase and generate ATP. However, the process of electron transfer is not perfect. Often electrons “leak out” of the ETC and react with available molecular oxygen to generate ROS such as O2• (Betteridge 2000). An excess of un-esterified fatty acids directly or via its conversion to other non-oxidative metabolites such as ceramides, increases generation of mitochondrial O2• (Listenberger and Schaffer 2002). As a charged molecule, O2• has a short half-life and is not a good oxidizing agent. Excess O2• may generate H2O2, which is far more stable and has a greater oxidizing potential (Betteridge 2000). Apart from increased generation of ROS, mitochondrial oxidative stress can arise due to a deficit in mitochondrial antioxidants, which allow ROS to exist unchecked (Betteridge 2000). Increased mitochondrial ROS production can also damage mitochondria

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itself and perpetuate a vicious cycle of augmented ROS release. In order to defend against this production of ROS, mitochondria contain a SOD isoform, known as MnSOD or SOD2 (Weisiger and Fridovich 1973) and mitochondrial GSH, also called mtGSH (Griffith and Meister 1985). Mitochondrial oxidative damage can hinder mitochondrial b-oxidation of fatty acid in the heart and promote buildup of non-oxidative metabolites such as diacylglycerol and ceramide, leading to lipotoxicity in cardiac cells (Listenberger and Schaffer 2002). Cardiac cells contained in the vasculature include vascular smooth muscle cells, endothelial cells, and macrophages. Vascular smooth muscle cells (VSMCs) compose the muscle of coronary blood vessel walls and are involved in the constriction and dilation of blood vessels. The endothelium is the innermost layer of blood vessel walls of the vascular system and contains a single layer of metabolically active cells. The endothelium is the most important cell type concerning oxidative stress and its regulation by dietary fatty acids as bloodborne fatty acids come into direct contact with the endothelial cells first before any other cardiac cell type (Tsai et al. 2004). Extensive literature exists on the role of various fatty acid classes on either pro- or antioxidant signaling in endothelial cells (Egan et al. 2001; Hennig et al. 2001; Balakumar and Taneja 2012). It is well known that oxidative stress impairs vascular endothelial function (Ohara et al. 1993). The predominant pathway for such damage seems to be disturbances in the nitric oxide (NO) pathway, critical for vascular function (Palmer et al. 1988), vascular tone (Palmer et al. 1987), platelet activity (Radomski et al. 1987), and leukocyte adhesion (Suematsu et al. 1994). When oxidative stress is increased, NO is quenched, which contributes to vascular dysfunction and development of atherosclerosis and coronary heart diseases.

Why Fatty Acids? The cardiac muscle has the ability to utilize a variety of metabolic fuels including glucose, ketones, lactate, amino acids, and fatty acids. However, under normal healthy conditions, fatty acids are the preferred fuel to provide the required energy to fulfill all metabolic requirements. Fatty acids contribute up to 70 % of the total acetyl-CoA for the production of energy, while the remaining is provided by glucose (Liedtke 1981). As anaerobic pathways of fatty acid utilization do not exist, a constant supply of oxygen is crucial to sustain cardiac function and viability (Icardo 1988). Consequently, due to its high oxygen consumption, ETC activity, and high mitochondrial density, the heart is particularly susceptible to oxidative stress. During certain metabolic deficiencies such as insulin resistance, diabetes, or aging, when insulin signaling is impaired, approximately 90–95 % of cardiac energy is provided by fatty acids (Wall and Lopaschuk 1989). Therefore, it is hardly surprising that the impact of various fatty-acid-mediated redox signaling occurs under conditions of relative insulin resistance on cardiac physiology. Indeed, high levels of lipid peroxidation and oxidative DNA damage

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are characteristic of metabolically challenged or aged hearts (Miro´ et al. 2000). The fact that most cardiomyocytes are terminally differentiated and have lost all power of regeneration perpetuates the effect of such oxidative damage (Cai and Harrison 2000).

Dietary Fatty Acids: Background For most populations, dietary fats constitute around 30–35 % of total dietary energy. Beyond such levels, absorption of fats is limited due to a mismatch with bile availability. Based on saturation, dietary fatty acids are divided into two broad classes: saturated and unsaturated fatty acids. Saturated fatty acids (SFAs) have a saturated carbon chain and contain no double bonds, whereas unsaturated fatty acids have at least one double bond. SFAs are present predominantly in animal fat sources such as butter, which contains predominantly palmitic acid (PA: C16:0), and red meats such as beef, which contains large amounts of stearic acid (C18:0). Plant sources of SFAs include coconut oil, which is composed mainly of mediumchain saturated fatty acids such as lauric (C12:0) and myristic (C14:0) acids, and palm oil containing PA. All SFAs are waxy solids at room temperature whereas unsaturated fatty acids exist as oils at room temperature. Unsaturated fatty acids are further subdivided into two types: monounsaturated (MUFA), such as oleic acid (OA: C18:1n-9), which contains only one double bond, and polyunsaturated (PUFA) such as linoleic acid (LA: C18:2n-6), containing two or more double bonds (Moran et al. 1989). One of the richest naturally occurring sources of MUFA (for example OA) is olive oil. PUFAs are further classified according to the number of carbon atoms in the chain (medium- or long-chain PUFAs), and the position and number of double bonds in the carbon chain (Voet and Voet 2005). The positional classification of PUFAs is based on the location of the first double bond in the hydrocarbon chain, counted from the methyl (CH3) end of the molecule. For example, omega-3 PUFAs have the first double bond between the third and fourth carbons whereas the omega-6 PUFAs have a double bond between the sixth and seventh carbons (Ruxton et al. 2004). All of these fatty acid classes exert differential effects on cardiac redox signaling as enumerated in the next sections.

Saturated Fatty Acids and Cardiac Redox Mechanisms The relationship of SFAs to oxidative stress and heart disease has been studied over the last two decades (Renaud et al. 1983; Hertog et al. 1995; Ascherio et al. 1996; Hu et al. 1997; Micha and Mozaffarian 2010). Previous research has shown that fatty acids undergo b-oxidation at differing rates in vivo in humans and rats, with SFAs being oxidized at a slower rate than unsaturated fatty acids (Jones et al. 1985; DeLany et al. 2000). The current hypothesis on the “lipotoxicity” hypothesis states that when SFAs are consumed in excess, the shuttling of excess FA to white adipose tissue to be stored as

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benign triglyceride is overwhelmed, leading to “spillover” in the blood and other organs (hyperlipidemia) (Unger and Zhou 2001; Unger 2002). Under such high lipidemic conditions, excess SFAs infiltrate non-storage organs such as the heart and the pancreas, leading to ectopic fat depositions. Of the non-oxidative metabolism pathways, the ceramide (a form of sphingolipid) pathway is the most extensively studied in regard to excess lipid storage and oxidative stress in cardiomyocytes (Zhou et al. 2000). Sphingolipids are synthesized de novo from serine and PA (the most prevalent dietary SFA) by the enzyme serine palmitoyl transferase (SPT) (Hannun and Obeid 2008). In cells with decreased leptin, a hormone that limits adipocyte mass and reduces caloric intake, excess PA is related to high SPT and increased condensation of palmitoyl CoA and serine to form dihydrosphingosine, which is required for the first step in de novo ceramide biosynthesis (Weiss and Stoffel 1997; Shimabukuro et al. 1998). Elevated cellular ceramide levels have been positively correlated with an increase in ROS and apoptosis (Unger 2002). A ceramide-mediated increase in ROS production is known to inhibit the ETC (complex III), such that excess electrons at this complex “leak out” and form ROS (Garcıá-Ruiz et al. 1997; Suematsu et al. 2003). The cytokine TNF-a is capable of increasing cellular ceramide levels and generating ROS (Suematsu et al. 2003). TNF-a-induced ROS production has been implicated in structural and phenotypic changes in cardiomyocytes, causing cardiac hypertrophy, dilated cardiomyopathy (Kubota et al. 2001), and myocardial dysfunction (Machida et al. 2003). However, ceramide pathways alone cannot explain the increase in ROS that accompanies an increase in SFA. As such, it appears that SFA can act through ceramide-independent pathways, causing the generation of ROS and leading to endoplasmic reticulum (ER) stress and apoptosis (Listenberger et al. 2001; Wei et al. 2006). In skeletal muscle, it has been shown that PA causes an increase in the expression of inducible nitric oxide synthase (iNOS), which increases the production of NO and ONOO , leading to an increase in apoptosis (Slawik and Vidal-Puig 2006; Rachek et al. 2007; Samokhvalov et al. 2009). In cardiomyocytes, iNOS can be under regulatory control by PA and is linked to an increase in apoptosis (Slawik and Vidal-Puig 2006; Jung et al. 2009). As an experimental model of fatty acid overload both in vivo and in vitro, PA remains the most extensively studied fatty acid. High-fat feeding with PA can lead to an increase in ROS and further to the development of mitochondrial dysfunction (Bonnard et al. 2008; Mantena et al. 2008). Dietary PA has been shown to increase the uncoupling proteins (UCPs), which are mitochondrial inner membrane proteins that play a role in dissipating energy in the form of heat (Boudina et al. 2007). An increase in UCPs has been believed to cause both an increase as well as a decrease in pathological ROS production. This idea is still a controversial topic (Laskowski and Russell 2008). In cardiomyocytes, PA-induced cell death has been shown to occur because of disruption of mitochondrial transmembrane potential, which can be reversed with cyclosporin A (Kong and Rabkin 2000). Interestingly, the loss of mitochondrial function in cardiomyocytes due to PA can be prevented with the addition of either OA or a SOD mimetic. This would imply that PA does generate excess O2• in the heart, which is linked to mitochondrial impairment. However, as

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SFA

Direct Effects Ceramide

Ceramide Independent Pathways

iNOS

Mitochondria

ER Stress ROS Apoptosis

Indirect Effects

Uncoupling proteins Mitochondrial Dysfunction

White Adipose Tissue

TNFalpha IL-10

Inflammation

MAPK

p38 JNK ERK

Apoptosis

Fig. 66.1 Schematic of the primary effects of saturated fatty acids on inflammation and oxidative stress. Overview of the effects of SFAs on cellular processes with respect to oxidative stress. Abbreviations: SFA saturated fatty acid, iNOS inducible nitric oxide synthase, ROS reactive oxygen species, ER stress endoplasmic reticulum stress, MAPK mitogen-activated protein kinase, TNFa tumor necrosis factor alpha, IL-10 interleukin-10, JNK c-jun N-terminal kinase, ERK extracellular signaling regulating kinase

O2• is the starting ROS for most in vivo pro-oxidant pathways, whether it is O2• itself or other downstream mediators such as H2O2 or OH radicals that are responsible for such an effect cannot be determined. With similar actions as a SOD mimetic, OA clearly exhibits an antioxidant role (Gao et al. 2012). The known primary effects of SFAs in the myocardium are outlined in Fig. 66.1.

Monounsaturated Fatty Acids and Cardiac Redox Mechanisms Over the last few decades, most human, animal, and cellular studies have indicated that MUFA’s, such as OA and palmitoleic acid (C16:ln-7), are generally protective in the context of lipotoxicity. As discussed earlier, while PA is known to elicit primarily negative effects and increase ROS production, MUFAs such as OA have been shown to reverse these effects when coincubated with PA in cardiomyocytes (Miller et al. 2005; Cnop 2008; Coll et al. 2008; Gao et al. 2012). The greater toxicity of PA over OA has been attributed to less efficient esterification of excess PA into triglycerides compared to OA. Presence of OA helps PA to be channeled into the production of TGs (Listenberger et al. 2003). This is important because formation of triglycerides traps the reactive fatty acids with glycerol, thereby removing its potential to form secondary metabolites such as ceramide (Cnop et al. 2001). In rat cardiomyocytes, it has been shown that ceramide levels are

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significantly higher in SFA-fed animals over MUFA-fed animals (Okere et al. 2006). Another mechanism of the protective effect of OA could be its impact on slowing down the cellular uptake of PA (Huang et al. 2002). PA is also known to augment uptake of oxidized low-density lipoprotein (oxLDL) by monocytes, which are key in initiating events that trigger the process of atherosclerosis (Ishiyama et al. 2010). Furthermore, cells incubated with PA have reduced levels of the antioxidant, GSH, whereas in cells incubated with OA, these effects are not observed (Gao et al. 2012). Another benefit of OA is observed when coincubated with PA, where this MUFA completely inhibits PA-mediated activation of p38MAPK, ERK, and JNK, which are kinases linked to ROS generation and apoptosis in cardiomyocytes (Miller et al. 2005).

Polyunsaturated Fatty Acids: Background Linoleic acid (LA; C18:2n6) and a-linolenic acid (ALA; C18:3n3) are parent omega-6 and omega-3 PUFAs (called n-6 and n-3 PUFAs). They are required at 0.5–2 % of the total dietary energy for proper development and function and are considered “essential” fatty acids (Rustan et al. 1997; Ruxton et al. 2004). They need to be provided through the diet because unlike SFA or MUFA, these fatty acids cannot be synthesized de novo in mammals as they lack D-3 desaturase enzyme, which inserts the double bond (at the 3rd position from the carboxyl end) in a fatty acid (Ruxton et al. 2004). Currently, in Canada, the average consumption of LA is around 7 % of energy, which is far beyond nutritional needs, while intake of omega-3 PUFA remains low at approximately 0.7 % of energy (Kris-Etherton et al. 2002; Madden et al. 2009). These two classes of PUFAs are functionally and metabolically very different and often lead to biologically opposite effects (Simopoulos 2002). PUFAs exhibit important functions including regulation and formation of cellular membrane composition, regulation of signaling pathways through transcription factors and eicosanoid production pathways (Calder 2006). All cell membranes consist of a different lipid composition, which requires a tight regulation of the membrane PUFA composition, depending on its function. Such composition is often a direct reflection of dietary intakes of PUFA, and therefore, a balanced PUFA consumption is required to maintain the lipid composition (Trevizol et al. 2011) and specific properties (Safarinejad et al. 2010) of cell membranes including mitochondrial, plasma (Prasad et al. 2010), neuronal (Trevizol et al. 2011), and cardiac membranes (Balkova et al. 2009).

Omega-3 PUFA and Cardiac Signaling The main sources of ALA in the North American diet include canola oil and soybean oil, while flaxseed oil is another ALA-rich source (Kris-Etherton et al. 2002). In mammals, ALA is converted to long-chain, more biologically potent forms such as docosahexaenoic acid (DHA: C22:6n-3), and eicosapentaenoic acid

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(EPA: C20:5n-3) (Ruxton et al. 2004) via desaturation and elongation (Calder 2006). Fish are the major dietary source of EPA and DHA; yet, levels of these PUFAs vary depending upon the species and environmental factors. Fish with high EPA/DHA content include Atlantic farmed salmon, Chinook salmon, sardines, Pacific oyster, and herring (Kris-Etherton et al. 2003). Fish oil (FO) supplements also contain high amounts of EPA and DHA. Therefore, the American Heart Association (AHA) recommends daily intakes of FO contributing between 2 and 4 g/day of DHA + EPA as permissible under medical supervision to lower blood TGs. For more general use, AHA recommends up to 1 g/day of EPA + DHA to offset high LA intake (average intake of 18 g/day in USA) and to treat CD (Kris-Etherton et al. 2000; Kris-Etherton et al. 2002; Gebauer et al. 2006). DHA and EPA have potent anti-inflammatory properties. The immunomodulatory effects of omega-3 PUFAs are believed to occur through two modes of action: (1) interference with eicosanoid and autacoid production from phospholipids, and (2) eicosanoid-independent mechanisms which includes interference with intracellular signaling, production or activation of cytokines and transcription factors, and modulation of gene expression (Simopoulos 2002). The anti-inflammatory and antioxidant properties of omega-3 PUFAs are of great interest for potential treatment of many inflammatory and autoimmune diseases. Several studies have reported that fish in the diet or FO supplementation reduces the risk of coronary heart disease and CD in general (Kris-Etherton et al. 2003). In addition to reducing inflammation as previously described, consumption of long-chain omega-3 PUFAs from FO has been shown to lower serum TGs, blood pressure, thrombosis, cholesterol levels, and arrhythmias (James et al. 2000). Increasing the consumption of long-chain omega-3 PUFAs has also been shown to increase insulin sensitivity, which further contributes to the reduced risk of severe CD in diabetic patients (Nettleton and Katz 2005). Additionally, omega-3 PUFAs improve endothelial function, which contributes to decreased risk of atherosclerosis (Wang et al. 2012). Taken together, all of these factors contribute to omega-3 PUFAs’ anti-atherosclerotic nature and cardiovascular protective effects. However, based on the fact that long-chain omega-3 PUFAs such as DHA and EPA have six and five double bonds, respectively, there is the possibility of augmentation of the membrane unsaturation index, oxidative stress, and lipid peroxide formation when omega-3 PUFA is co-administered with high dietary omega-6 PUFA (Jude et al. 2003; Ghosh et al. 2007; Kabuto et al. 2009; Tanito et al. 2009). As an example, diets containing 8 % w/w of FO have demonstrated detrimental effects in various cell systems (Hammer and Wills 1978; Yuan and Kitts 2003). Although it is hard to overconsume fish and other natural sources of omega-3 PUFAs, one can speculate that current practices of widespread omega-3 PUFA supplementation in common foods such as eggs, bread, and yogurt in an unregulated fashion could provoke oxidative stress-related diseases in populations. However, it should be emphasized that upper tolerable limits of either omega-6 or omega-3 PUFAs for human consumption have never been established due to the lack of research.

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Omega-6 PUFA and Cardiac Signaling The second important group of PUFAs is the omega-6 (n-6 or o-6) PUFAs, mainly consisting of LA, which contains 18 carbons and 2 double bonds. Omega-6 PUFAs are consumed in the North American diet from organ meats and vegetable oils (Catala 2010) such as corn, safflower (Prasad et al. 2010), maize, rapeseed (Czernichow et al. 2010), and soybean oils (Schmitz and Ecker 2008). LA is metabolized into arachidonic acid (AA: C20:4n-6) and dihomo-g-linolenic acid (DGLA: C20:3n-6) (Rustan et al. 1997). The omega-6 PUFAs have exhibited both beneficial and adverse effects on cardiac health and are currently a subject of raging controversy regarding the actual nature of these fatty acids, irrespective of current clinical dietary recommendation. Early research on omega-6 PUFAs demonstrated beneficial effects on CD in the prevention and development of atherosclerosis (Rudel et al. 1995), and arrhythmias (McLennan 1993). Recently, such beneficial effects of omega-6 PUFAs have been questioned and several earlier assumptions challenged. As an example, beneficial effects were reported when 5 % of daily energy intake from SFAs was replaced by omega-6 PUFAs, namely LA, which showed a risk reduction of 26 % for CD-linked deaths (Jakobsen et al. 2009), a risk reduction of 24 % for cardiovascular events, and a decrease in LDL levels (Gordon 1995). However, upon careful analysis, it becomes evident that the study described PUFA as a single entity and did not discriminate between omega-3 and 6 PUFAs. The tendency to report omega-3 and omega-6 PUFA as a single entity has been practiced in multiple clinical trials and analysis (Calder 2010). There is no doubt that omega-6 PUFA lowers LDL cholesterol levels (Hegsted et al. 1993). However, whether such a drop in LDL cholesterol is offset by an elevated inflammation or oxidative stress remains currently unclear. In fact, by directly analyzing postmortem samples of human atherosclerotic plaques, it was concluded that oxidative modification of the unsaturated LA in human LDL promoted their uptake into the vessel wall (Felton et al. 1994). An increase in dietary LA is commonly thought to be linked to inflammatory and thrombotic events (Harris 2010). LA is the precursor for AA, which produces potent inflammatory and arrhythmic molecules such as the prostaglandins (PG), PGI2 and PGE2 (Schmitz and Ecker 2008), the thromboxane (TX), TXA2 (Thomas et al. 2003), and the hydroxyeicosatetraenoic acid (HETE), 20-HETE (Hoff et al. 2011). Therefore, an increase in omega-6 PUFAs could lead to adverse health effects including CD by increasing inflammation. In a meta-analysis of omega-6 PUFA RCTs by Ramsden et al., omega-6 PUFAs exhibited no beneficial effects, but instead showed adverse effects and increased the risk of CD (Ramsden et al. 2010). It was demonstrated that the differential effects of omega-6 PUFA reported was due to the lack of a consistent diet, with many studies using a diet of mixed omega-6/omega-3 PUFA versus an omega-6 PUFA specific. Studies that used a specific omega-6 PUFA diet showed a 13 % increased risk of CD, which is vastly different from the 22 % risk reduction of CD seen in mixed omega-6/omega-3 PUFA diets (Ramsden et al. 2010). Indeed, apart from the cardiovascular system, a high omega-6, low omega-3 PUFA diet has been linked to several

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pro-inflammatory conditions such as insulin resistance (Ghosh et al. 2004a; Canete et al. 2007; Ramel et al. 2008), atherosclerosis (Reaven et al. 1991; Abbey et al. 1993), colorectal (Fernandez-Banares et al. 1996; Nkondjock et al. 2003) and pancreatic cancers (Nkondjock et al. 2005), and inflammatory bowel disease (Belluzzi et al. 1996; Innis and Jacobson 2007).

Cellular Changes Mediated by PUFA Membrane Fluidity Membranes are composed of a lipid bilayer, carbohydrates, and associated proteins. Membrane fluidity depends upon the degree of unsaturation of the constituent lipids. In general, as the degree of unsaturation increases, so does membrane fluidity, which is important for cell function and interactions between the lipid constituents and lipid-associated proteins (Voet and Voet 2005). Previous studies have shown that SFAs decrease membrane fluidity (Leaf and Xiao 2001), whereas PUFAs increase membrane fluidity (Villacara et al. 1989; Hashimoto et al. 1999). Omega-3 PUFAs increase EPA and DHA in aortic endothelial cell membranes (Hashimoto et al. 1999) and help in reducing cholesterol-induced decreases in the fluidity of membranes (Hashimoto et al. 2006). Possible benefits of increased incorporation of omega-3 PUFAs in the membrane may lie in their ability to suppress voltage-activated sodium and calcium channels, which could act in an anti-arrhythmic manner (Leaf and Xiao 2001). Most studies on membrane fluidity changes have focused on SFAs and omega-3 PUFAs but not on omega-6 PUFAs. Both excess omega-6 and omega-3 PUFAs can cause acute cytotoxicity due to their rapid incorporation into cell membranes (Zerouga et al. 1996; Prasad et al. 2010). However, the degree in change of membrane fluidity is higher with omega-6 PUFA, which is directly proportional to such cytotoxic effects (Ahs et al. 2011). Studies have demonstrated a greater increased membrane fluidity with omega-6 PUFAs, such as docosatetraenoic acid (DTA), and AA could lead to an increase in initial release of Ca2+ from the endoplasmic reticulum followed by ROS generation in endothelial cells (Ahs et al. 2011). Such an increase in fluidity can also provoke oxidative stress (Saraswathi et al. 2004). Excess omega-6 PUFAs can also cause increased cell damage/apoptosis (Sergent et al. 2005).

Inflammation Oxidative stress has been implicated as a causative factor in inflammation. As an example, excess ROS can lead to oxidation of LDLs and subsequent production of secondary inflammatory inducers (Huang and Glass 2010). The membrane omega-3 and omega-6 PUFAs are cleaved by phospholipase A2 (PLA2), which leads to production of “free” AA, EPA, and DHA. The three main enzymes responsible

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for the production of eicosanoids are cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP), which function to convert AA, EPA, and DHA into eicosanoids such as PGs, TXs, leukotrienes (LT) (Schmitz and Ecker 2008), HETEs, and epoxyeicosatrienoic acids (EETs) (Capdevila et al. 1981; Chen et al. 1999). AA is an omega-6 PUFA derived from LA that is required for cell growth, signaling, eicosanoid production (Liou et al. 2007), and is important both in the anti- and pro-inflammatory pathway (An et al. 2011). AA is the predominant precursor in the production of pro-inflammatory eicosanoids such as PGE2 and PGI2, TXA2 and TXB2, LTB4 (Schmitz and Ecker 2008), 20-HETE, and prostacyclins (Kris-Etherton et al. 2003; Calder 2006). The concentration of AA and its eicosanoid products must be balanced in order to maintain the pro-/anti-inflammatory milieu within specific organs. The omega-6 PUFA-derived eicosanoids are mainly pro-inflammatory in nature. PGs, such as PGI2 and PGE2, have proarrhythmic effects, while TXs, for example TXB2, function in vasoconstriction (Enzan et al. 1996) and platelet activation (Murray and FitzGerald 1989). LTs, such as LTB4, result in leukocyte chemotaxis, increased vascular permeability, and acts with PGE2 to potentiate the bradykinin response (Bray et al. 1981). The AA-derived LTs increase vascular permeability, production of the pro-inflammatory cytokines IL-1, IL-6, and TNFa, production of ROS (Schmitz and Ecker 2008), and leukocyte chemotaxis (Simopoulos 2002). Members of the CYP enzyme family (CYP2C and CYP2J) metabolize AA to form isoformspecific EETs, and other members (CYP4A and CYP4F) metabolize AA to produce 20-HETE. HETES and EETs play opposing roles. For example, EETs are antihypertensive (Fisslthaler et al. 1999), anti-inflammatory, and anti-apoptotic (Node et al. 1999; Dhanasekaran et al. 2008) whereas 20-HETE is involved in both the pro- and anti-hypertensive mechanisms. As an illustration, the overexpression of 20-HETE has demonstrated endothelial dysfunction and hypertension (Cheng et al. 2008; Ishizuka et al. 2008). EPA, derived from the omega-3 PUFA, ALA, functions mainly in the anti-inflammatory pathway (Schmitz and Ecker 2008). The anti-inflammatory and anti-arrhythmic molecules derived from EPA include PGE3 and PGI3, TXA3, and TXB3, and LTB5 (Schmitz and Ecker 2008). Eicosanoids produced by CYP enzymes that are derived from EPA are epoxyeicosatetraenoic acids (EETeTrs) and o/(o 1)-hydroxyeicosapentaenoic acids (19- and 20-HEPE) and DHA are epoxydocosapentaenoic acids (EDPs) and o/(o 1)hydroxydocosahexaenoic acids (21- and 22-HDoHE). EETs that are derived from omega-3 PUFAs have been shown to be more potent than EETs derived from AA. For example, EPA- and DHA-derived eicosanoids largely exceed the ability of EETs to activate sodium-potassium channels in cardiomyocytes (Lu et al. 2002). Benefits of omega-3 PUFAs in a balanced diet may occur due to the switch from the AA-derived 20-HETE and EETs to the more potent EPA- and DHA-derived EETeTrs and EDPs. With respect to CYP enzymes, EPA is the preferred substrate and DHA is metabolized at similar rates compared to AA. For example, the CYP2C isoform shows a 1.5–2-fold increase in activity when converting EPA instead of AA (Fer et al. 2008). CYP2J2 is the major EET-producing isoform in the heart, and shows preference for EPA (Arnold et al. 2010). A substitution of EPA and DHA for AA in

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the membranes may lead to increased CYP metabolites derived from EPA and DHA. This switch may be one of the various reasons for the perceived benefit of omega-3 PUFA on cardiovascular function. In addition to eicosanoids, the PUFAs such as AA, EPA, and DHA are precursors to autacoids (Schmitz and Ecker 2008), which are endogenous lipid mediators that are involved in mediating inflammation. Autacoids are agonists of specific membrane-bound G-protein-coupled receptors and can both limit granulocyte (neutrophils, eosinophils, and basophils) infiltration (counter-regulation) as well as promote tissue restoration by removing tissue-granulocyte infiltrates (resolution) (Bannenberg and Serhan 2010). The management of granulocytic infiltration is necessary to combat the adverse effects of inflammation on tissue structure and function. Recent research has also led to discoveries of the AA-derived lipoxin, resolvin, and protectin families, all of which carry out unique functions to promote resolution of inflammation (Schmitz and Ecker 2008). Finally, EPA and DHA can also reduce the production of pro-inflammatory cytokines such as TNFa and IL-1b (Lo et al. 1999). There is an inverse relationship between the content of EPA and the amount of TNFa and IL-1b produced in mononuclear cells (Calder 2006). This is caused by dietary PUFAs altering the expression of the genes encoding these cytokines. In one study, mice fed with FO showed no detectable levels of mRNA in the kidneys for IL-1b, IL-6, and TNF-a compared to mice fed with corn oil (high in omega-6 PUFA) (Chandrasekar and Fernandes 1994). Figures 66.2 and 66.3 outline the effects of omega-3 and omega-6 PUFAs.

Modulating Endogenous Antioxidants Excess levels of ROS lead to cell damage and apoptosis if they are not neutralized by antioxidants (Connor 2000). The concentration of endogenous antioxidants such as SOD, CAT, and GPX can be altered with changes in dietary intake of omega-3 and omega-6 PUFAs. It has been shown that supplements containing EPA and DHA can reduce the production of ROS by human neutrophils and monocytes (Calder 2006) as well as increase the activity of SOD, CAT, and GPX enzymes. In this way, omega-3 PUFAs can be considered important antioxidants with the potential to reduce and prevent the damage caused by oxidative stress. Unlike omega-3 PUFAs, increased dietary intake of LA decreases concentrations of endogenous antioxidant concentrations. Excess dietary intake of omega-6 PUFAs decreases CAT activity in the liver (Benson and Devi 2009), and decreases both CAT and SOD activity in the heart (Diniz et al. 2004), indicating that omega-6 PUFAs could play a role in the excess production of ROS as well as oxidative stress.

Conclusion Due to the prevalence of fatty acids in our diet, there has been a large body of research conducted to look at the effects of SFA, MUFA, and PUFA on cardiac

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Direct Effects

Indirect Effects

Membrane Fludity Cholesterol-induced decrease of fluidity Fluidity of aortic endothelium

Eicosanoids

Autacoids

Cytokines

Transcription Factors

PPAR

PGE3, PGI3 TXA3 TXB3, LTB5

D-series P E-series Rv

TNFalpha II-l beta

NFkappaB activity IkappaB activity

Cell proliferation NFkappaB

Altered membrane function Possible increased susceptibility to oxidative stress

Anti-carcinogenic

Inflammation

Fig. 66.2 Schematic of the effects of omega-3 polyunsaturated fatty acids on inflammation and oxidative stress. Overview of the effects of omega-3 PUFAs on cellular processes with respect to oxidative stress. Abbreviations: PUFA polyunsaturated fatty acid, PG prostaglandin, TX thromboxane, LT leukotriene, P protectin, Rv resolvin, TNF tumor necrosis factor, IL interleukin, NFkappaB nuclear factor kappa B, IkappaB inhibitor of NFkappaB Omega-6 PUFA

Indirect Effects

Direct Effects Membrane Fluidity

Lipid Peroxidation

Microviscosity Lipid lateral diffusion

4-HNE (from LA)

Eicosanoids PGI2, PGE2 TXB2 LTB4

Cytokines TNFalpha IL-1beta

ROS

ROS production Oxidative Damage Apoptosis

ROS production

Inflammation Leukocyte chemotaxis

Fig. 66.3 Schematic of the effects of omega-6 polyunsaturated fatty acids on inflammation and oxidative stress. Overview of the effects of omega-6 PUFAs on cellular processes with respect to oxidative stress. Abbreviations: PUFA polyunsaturated fatty acid, ROS reactive oxygen species, PG prostaglandin, TX thromboxane, LT leukotriene, TNF tumor necrosis factor, IL interleukin

health. The possible causes of CD and other related inflammatory diseases are the type and amount of dietary fat consumed. SFAs and cholesterol have been used as typical markers of CD. For this reason, the excess intake of SFAs has been blamed for the prevalence of these diseases. In an attempt to reduce the incidence of CD, there was a push to reduce the amount of SFA in the diet and as such SFA was

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replaced with carbohydrates, MUFA, and PUFA in the diet (Micha and Mozaffarian 2010). Taken together, this research appeared to provide support that SFA increased the risk of CD (Hegsted et al. 1965; Micha and Mozaffarian 2010). Yet, decades later with a modified diet of increased PUFAs, the prevalence of CD is not reduced. Studies have shown that rats fed with the same caloric content of a PUFA diet versus a SFA diet have an increased weight gain and final weight (Diniz et al. 2004). Diets rich in PUFAs, more specifically omega-6 PUFAs, for a prolonged period have been implicated to induce cardiac dysfunction and increased production of ROS (Ghosh et al. 2006). This may predispose the cardiac mitochondria to oxidative damage, resulting in decreased cellular activity in the heart and possibly apoptosis (Ghosh and Rodrigues 2006) or necrosis (Ghosh et al. 2004b). Although the majority of the research on most fatty acids with respect to its effect on CD tends to be conflicting, the research on the effects of MUFA with respect to CD generally tends to be positive. For example, replacement of partially hydrogenated vegetable oil with high oleic sunflower oil caused a significant decrease in the risk of CD (Mozaffarian and Clarke 2009). As well, research into the Mediterranean diet, which contains a large amount of OA, has shown a decreased risk of CD (Michel de Lorgeril et al. 1999; Trichopoulou et al. 2003; Parikh et al. 2005). Significant gaps remain in our understanding of the effects of various fatty acids on cardiac health. It has been argued that genetic factors and evolution of an individual could determine if a type of fatty acid will increase the risk of CD. Therefore, a combination of genetic, evolutionary, and biochemical approaches is needed to determine if a fatty acid poses an increased risk of CD for specific populations (Ramsden et al. 2009). Current research suggests that although SFA may cause an increased risk in atherosclerosis and CD in general, replacement of SFA with PUFA, while it lowers LDL, can also increase the risk of CD and other chronic diseases such as insulin resistance (Canete et al. 2007; Ramel et al. 2008), atherosclerosis (Reaven et al. 1991; Abbey et al. 1993), colorectal (Fernandez-Banares et al. 1996; Nkondjock et al. 2003) and pancreatic cancers (Nkondjock et al. 2005), and inflammatory bowel disease (Belluzzi et al. 1996; Innis and Jacobson 2007). Therefore, while this controversy waits to be resolved, as an alternate choice, replacement of SFAs and PUFAs with MUFAs such as OA seems to be the best option in preventing CD and maintaining redox homeostasis to the heart. Acknowledgments A Scholar award and Grant-In-Aids from the Canadian Diabetes Association and University Start-up Funds to SG supported this work. A Masters award from NSERC funds JB and AB is funded by a CIHR Doctoral Award.

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