Beneficial Effects of Dietary Flaxseed on Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), a significant cause of chronic liver disease, presents a considerable public health concern. Despite this, there is currently no treatment available. This study aimed to investigate dietary flaxseed in the JCR:LA-corpulent rat strain model of NAFLD. Both obese male and female rats were studied along with their lean counterparts after 12 weeks of ingestion of a control diet, or control diet with flaxseed, or high fat, high sucrose (HFHS), or HFHS plus flaxseed. Obese rats showed higher liver weight and increased levels of cholesterol, triglyceride, and saturated fatty acid, which were further elevated in rats on the HFHS diet. The HFHS diet induced a significant two-fold elevation in the plasma levels of both aspartate aminotransferase and alanine aminotransferase in the obese male and female rats. Including flaxseed in the HFHS diet significantly lowered liver weight, depressed the plasma levels of both enzymes in the obese male rats, and reduced hepatic cholesterol and triglyceride content as well as improving the fatty acid profile. In summary, including flaxseed in the diet of male and female obese rats led to an improved lipid composition in the liver and significantly reduced biomarkers of tissue injury despite consuming a HFHS chow.

Considerations for choosing an optimal animal model of cardiovascular disease.

The decision to use the optimal animal model to mimic the various types of cardiovascular disease is a critical one for a basic scientist. Clinical cardiovascular disease can be complex and presents itself as atherosclerosis, hypertension, ischemia/reperfusion injury, myocardial infarcts, and cardiomyopathies, amongst others. This may be further complicated by the simultaneous presence of two or more cardiovascular lesions (for example, atherosclerosis and hypertension) and co-morbidities (i.e., diabetes, infectious disease, obesity, etc). This variety and merging of disease states creates an unusually difficult situation for the researcher who needs to identify the optimal animal model that is available to best represent all of the characteristics of the clinical cardiovascular disease. The present manuscript reviews the characteristics of the various animal models of cardiovascular disease available today, their advantages and disadvantages, with the goal to allow the reader access to the most recent data available for optimal choices prior to the initiation of the study. The animal species that can be chosen, the methods of generating these models of cardiovascular disease, as well as the specific cardiovascular lesions involved in each of these models are reviewed. A particular focus on the JCR:LA-cp rat as a model of cardiovascular disease is discussed.

The Plasma Oxylipidome Links Smoking Status to Peripheral Artery Disease.

Peripheral artery disease (PAD) is prevalent among individuals with a history of tobacco smoking. Although oxidation of lipids may contribute to atherogenesis in vascular disease, enzymatically and nonenzymatically produced oxidized lipids can have varying and contrasting physiological effects. The underlying mechanisms of atherogenic vulnerability can be better elucidated with the recent advances in oxylipidome quantification using HPLC-MS/MS technology. In a randomized, controlled clinical trial, the plasma oxylipidome was analyzed in participants living with PAD by smoking status (n = 98) and in nonsmoking comparators without chronic disease (n = 20). Individuals with PAD had approximately a four-fold higher level of total plasma oxylipins versus the comparator. Cessation of smoking in individuals with PAD was associated with significantly lower levels of linoleic acid-derived TriHOMEs, greater levels of omega-3 fatty acid-derived oxylipins, and greater levels of nonfragmented oxidized phosphatidylcholines (OxPCs). Individuals living with PAD but without a history of smoking, exhibited higher levels of the putative atherogenic fragmented OxPCs versus individuals who currently or previously smoked. These data implicate the plasma oxylipidome in PAD and that smoking cessation is associated with a less inflammatory profile. Furthermore, fragmented OxPCs may play a more significant role in the pathophysiology of PAD in individuals without a history of smoking.

Bacteria and the growing threat of multidrug resistance for invasive cardiac interventions.

Invasive cardiovascular procedures which include heart transplantations, congenital heart surgery, coronary artery bypass grafts, cardiac valve repair and replacement, and interventional cardiac electrophysiology procedures represent common mechanisms to treat a variety of cardiovascular diseases across the globe. The majority of these invasive approaches employ antibiotics as a regular and obligatory feature of the invasive procedure. Although the growing incidence of bacterial resistance to currently used antibiotics threatens to curtail the use of all interventional surgical techniques, it remains an underappreciated threat within the arsenal of cardiovascular therapies. It is reasonable to expect that the continued overuse of antibiotics and the frequent management of coronavirus disease 2019 (COVID-19) infected patients with high doses of antibiotics will inevitably accentuate the rise of multidrug resistance. The purpose of this article is to heighten awareness of the role of bacterial infections in cardiovascular disease, the use of antibiotics in today's cardiovascular surgical theaters, the threat facing cardiovascular surgery should multidrug resistance continue to rise unabated, and the development of new antibiotic platforms to solve this problem.

Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB-NqrD subunit interface.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the major Na+ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na+-NQR. While the mechanisms underlying Na+-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na+-NQR. An in silico model of the NqrB-D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na+-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB-D interface underwent a non-physiological transition after 350 ns.

Dietary flaxseed reduces Myocardial Ischemic Lesions, improves cardiac function and lowers cholesterol levels despite the presence of severe obesity in JCR:LA-cp Rats.

Previous work has shown that dietary flaxseed can significantly reduce cardiac damage from a coronary artery ligation-induced myocardial infarction. However, this model uses healthy animals and the ligation creates the infarct in an artificial manner. The purpose of this study was to determine if dietary flaxseed can protect the hearts of JCR:LA-cp rats, a model of genetic obesity and metabolic syndrome, from naturally occurring myocardial ischemic lesions. Male and female obese rats were randomized into four groups (n = 8 each) to receive, for 12 weeks, either a) control diet (Con), b) control diet supplemented with 10% ground flaxseed (CFlax), c) a high-fat, high sucrose (HFHS) diet, or d) HFHS supplemented with 10% ground flaxseed (HFlax). Male and female JCR:LA-cp lean rats served as genetic controls and received similar dietary interventions. In male obese rats, serum total cholesterol and LDL-C were significantly lower in CFlax compared to Con.  Obese rats on HFHS exhibited increased myocardial ischemic lesions and diastolic dysfunction regardless of sex. HFlax significantly lowered the frequency of cardiac lesions and improved diastolic function in male and female obese rats compared to HFHS. Blood pressures were similar in obese and lean rats. No aortic atherosclerotic lesions were detectable in any group. Collectively, this study shows that a HFHS diet increased myocardial ischemic lesion frequency and abolished the protective effect of female sex on cardiac function. More importantly, the data demonstrates dietary flaxseed protected against the development of small spontaneous cardiac infarcts despite the ingestion of a HFHS diet and the presence of morbid obesity.

The Impact of Nutrition on the COVID-19 Pandemic and the Impact of the COVID-19 Pandemic on Nutrition.

The coronavirus disease 2019 (COVID-19) pandemic has had significant morbidity, mortality, social and financial implications for the global population. Despite this knowledge, we still know very little about how COVID-19 infection affects quality of life resulting from changes in nutritional behaviour and, conversely, how nutrition could modulate the epidemiology of COVID-19. In addition, the social isolation most have experienced due to the regulations imposed by governments during the COVID-19 pandemic may have also had effects on our nutritional behaviour. It is possible that nutritional interventions may have effects on the incidence of COVID-19 infection and mortality rates. The purpose of this review is to evaluate the current status of research on the topic of nutrition as it relates to the COVID-19 pandemic.

The Influence of Diet and Sex on the Gut Microbiota of Lean and Obese JCR:LA-cp Rats.

There is an increased interest in the gut microbiota as it relates to health and obesity. The impact of diet and sex on the gut microbiota in conjunction with obesity also demands extensive systemic investigation. Thus, the influence of sex, diet, and flaxseed supplementation on the gut microbiota was examined in the JCR:LA-cp rat model of genetic obesity. Male and female obese rats were randomized into four groups (n = 8) to receive, for 12 weeks, either (a) control diet (Con), (b) control diet supplemented with 10% ground flaxseed (CFlax), (c) a high-fat, high sucrose (HFHS) diet, or (d) HFHS supplemented with 10% ground flaxseed (HFlax). Male and female JCR:LA-cp lean rats served as genetic controls and received similar dietary interventions. Illumine MiSeq sequencing revealed a richer microbiota in rats fed control diets rather than HFHS diets. Obese female rats had lower alpha-diversity than lean female; however, both sexes of obese and lean JCR rats differed significantly in ß-diversity, as their gut microbiota was composed of different abundances of bacterial types. The feeding of an HFHS diet affected the diversity by increasing the phylum Bacteroidetes and reducing bacterial species from phylum Firmicutes. Fecal short-chain fatty acids such as acetate, propionate, and butyrate-producing bacterial species were correspondingly impacted by the HFHS diet. Flax supplementation improved the gut microbiota by decreasing the abundance of Blautia and Eubacterium dolichum. Collectively, our data show that an HFHS diet results in gut microbiota dysbiosis in a sex-dependent manner. Flaxseed supplementation to the diet had a significant impact on gut microbiota diversity under both flax control and HFHS dietary conditions.

A Brief Review on the Biology and Effects of Cellular and Circulating microRNAs on Cardiac Remodeling after Infarction.

Despite advances in diagnostic, prognostic, and treatment modalities, myocardial infarction (MI) remains a leading cause of morbidity and mortality. Impaired cellular signaling after an MI causes maladaptive changes resulting in cardiac remodeling. MicroRNAs (miRNAs/miR) along with other molecular components have been investigated for their involvement in cellular signaling in the pathogenesis of various cardiac conditions like MI. miRNAs are small non-coding RNAs that negatively regulate gene expression. They bind to complementary mRNAs and regulate the rate of protein synthesis by altering the stability of their targeted mRNAs. A single miRNA can modulate several cellular signaling pathways by targeting hundreds of mRNAs. This review focuses on the biogenesis and beneficial effects of cellular and circulating (exosomal) miRNAs on cardiac remodeling after an MI. Particularly, miR-1, -133, 135, and -29 that play an essential role in cardiac remodeling after an MI are described in detail. The limitations that will need to be addressed in the future for the further development of miRNA-based therapeutics for cardiovascular conditions will also be discussed.

Oxidized phosphatidylcholines trigger ferroptosis in cardiomyocytes during ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) injury increases the generation of oxidized phosphatidylcholines (OxPCs), which results in cell death. However, the mechanism by which OxPCs mediate cell death and cardiac dysfunction is largely unknown. The aim of this study was to determine the mechanisms by which OxPC triggers cardiomyocyte cell death during reperfusion injury. Adult rat ventricular cardiomyocytes were treated with increasing concentrations of various purified fragmented OxPCs. Cardiomyocyte viability, bioenergetic response, and calcium transients were determined in the presence of OxPCs. Five different fragmented OxPCs resulted in a decrease in cell viability, with 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) having the most potent cardiotoxic effect in both a concentration and time dependent manner (P < 0.05). POVPC and PONPC also caused a significant decrease in Ca2+ transients and net contraction in isolated cardiomyocytes compared to vehicle treated control cells (P < 0.05). PONPC depressed maximal respiration rate (P < 0.01; 54%) and spare respiratory capacity (P < 0.01; 54.5%). Notably, neither caspase 3 activation or TUNEL staining was observed in cells treated with either POVPC or PONPC. Further, cardiac myocytes treated with OxPCs were indistinguishable from vehicle-treated control cells with respect to nuclear high-mobility group box protein 1 (HMGBP1) activity. However, glutathione peroxidase 4 activity was markedly suppressed in cardiomyocytes treated with POVPC and PONPC coincident with increased ferroptosis. Importantly, cell death induced by OxPCs could be suppressed by E06 Ab, directed against OxPCs or by ferrostatin-1, which bound the sn-2 aldehyde of POVPC during I/R. The findings of the present study demonstrate that oxidation of phosphatidylcholines during I/R generate bioactive phospholipid intermediates that disrupt mitochondrial bioenergetics and calcium transients and provoke wide spread cell death through ferroptosis. Neutralization of OxPC with E06 or with ferrostatin-1 prevents cell death during reperfusion. Our study demonstrates a novel signaling pathway that operationally links generation of OxPC during cardiac I/R to ferroptosis. Interventions designed to target OxPCs may prove beneficial in mitigating ferroptosis during I/R injury in individuals with ischemic heart disease.NEW & NOTEWORTHY Oxidized phosphatidylcholines (OxPC) generated during reperfusion injury are potent inducers of cardiomyocyte death. Our studies have shown that OxPCs exert this effect through a ferroptotic process that can be attenuated. A better understanding of the OxPC cell death pathway can prove a novel strategy for prevention of cell death during myocardial reperfusion injury.

Unexpected challenges for the translation of research on food interventions to applications in the food industry: using flaxseed research as an example.

One of the primary purposes of the studies that life science researchers carry out is to translate their findings into demonstrable impacts in the lives of the general population. If we study the mechanism of heart disease, for example, it is our hope that new therapies or preventative strategies can be created from these mechanistic data. In the field of nutrition, it is the ultimate goal to translate research findings on the health benefits of functional foods and nutraceuticals into products consumed by the public that will benefit their health, improve quality of life, prevent disease, and prolong life. However, the pathway from research on the health benefits of specific foods or food products into industry applications is often a pathway with multiple, unexpected roadblocks for the unsuspecting scientist. The purpose of this article, therefore, is to identify these obstacles that have confronted industry translation in the past by using flaxseed research as an example. The ultimate goal of the review is to alert those in research and in the food industry of these translational hindrances to avoid them in the future and promote a more rapid and effective translation of food/health research into marketing success.

Cardioprotective Effects of Dietary Flaxseed Post-Infarction Are Associated with Changes in MicroRNA Expression.

MicroRNAs (miRNAs/miRs) such as miR-1, miR-133a, miR-133b, miR-135a, and miR-29b play a key role in many cardiac pathological remodeling processes, including apoptosis, fibrosis, and arrhythmias, after a myocardial infarction (MI). Dietary flaxseed has demonstrated a protective effect against an MI. The present study was carried out to test the hypothesis that dietary flaxseed supplementation before and after an MI regulates the expression of above-mentioned miRNAs to produce its cardioprotective effect. Animals were randomized after inducing MI by coronary artery ligation into: (a) sham MI with normal chow, (b) MI with normal chow, and (c-e) MI supplemented with either 10% milled flaxseed, or 4.4% flax oil enriched in alpha-linolenic acid (ALA), or 0.44% flax lignan secoisolariciresinol diglucoside. The feeding protocol consisted of 2 weeks before and 8 weeks after the surgery. Dietary flax oil supplementation selectively upregulated the cardiac expression of miR-133a, miR-135a, and miR-29b. The levels of collagen I expression were reduced in the flax oil group. We conclude that miR-133a, miR-135a, and miR-29b are sensitive to dietary flax oil, likely due to its rich ALA content. The cardioprotective effect of flaxseed in an MI could be due to modulation of these miRNAs.

The Cardioprotective Role of Flaxseed in the Prevention of Doxorubicin- and Trastuzumab-Mediated Cardiotoxicity in C57BL/6 Mice.

BACKGROUND: Although the combination of doxorubicin (DOX) and trastuzumab (TRZ) reduces the progression and recurrence of breast cancer, these anticancer drugs are associated with significant cardiotoxic side effects. OBJECTIVE: We investigated whether prophylactic administration of flaxseed (FLX) and its bioactive components, α-linolenic acid (ALA) and secoisolariciresinol diglucoside (SDG), would be cardioprotective against DOX + TRZ-mediated cardiotoxicity in a chronic in vivo female murine model. METHODS: Wild-type C57BL/6 female mice (10-12 wk old) received daily prophylactic treatment with one of the following diets: 1) regular control (RC) semi-purified diet; 2) 10% FLX diet; 3) 4.4% ALA diet; or 4) 0.44% SDG diet for a total of 6 wks. Within each arm, mice received 3 weekly injections of 0.9% saline or a combination of DOX [8 mg/(kg.wk)] and TRZ [3 mg/(kg.wk)] starting at the end of week 3. The main outcome was to evaluate the effects of FLX, ALA, and SDG on cardiovascular remodeling and markers of apoptosis, inflammation, and mitochondrial dysfunction. Significance between measurements was determined using a 4 (diet) × 2 (chemotherapy) × 2 (time) mixed factorial design with repeated measures. RESULTS: In the RC + DOX + TRZ-treated mice at week 6 of the study, the left ventricular ejection fraction (LVEF) decreased by 50% compared with the baseline LVEF (P < 0.05). However, the prophylactic administration of the FLX, ALA, or SDG diet was partially cardioprotective, with mice in these treatment groups showing an ∼68% increase in LVEF compared with the RC + DOX + TRZ-treated group at week 6 (P < 0.05). Although markers of inflammation (nuclear transcription factor κB), apoptosis [poly (ADP-ribose) polymerase-1 and the ratio of BCL2-associated X protein to B-cell lymphoma-extra large], and mitochondrial dysfunction (BCL2-interacting protein 3) were significantly elevated by approximately 2-fold following treatment with RC + DOX + TRZ compared with treatment with RC + saline at week 6, prophylactic administration of FLX, ALA, or SDG partially downregulated these signaling pathways. CONCLUSION: In a chronic in vivo female C57BL/6 mouse model of DOX + TRZ-mediated cardiotoxicity, FLX, ALA, and SDG were partially cardioprotective.

Dietary flaxseed protects against ventricular arrhythmias and left ventricular dilation after a myocardial infarction.

Dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been associated with a lower incidence of cardiovascular events and sudden cardiac death. Flaxseed is a rich plant source of n-3 PUFAs and can retard the progression and accelerate the regression of atherosclerotic plaques. The aim of the study was to examine the preventive and therapeutic effects of dietary flaxseed on arrhythmias and heart dysfunction that develops after a myocardial infarction (MI). The left anterior descending coronary artery was ligated in rats to induce the MI. Rats were randomized into five groups: sham MI with normal chow, MI with normal chow, MI with 10% milled flaxseed supplementation (flax), MI with 4.4% supplemented flax oil enriched in alpha-linolenic acid (ALA) and MI with flax lignan secoisolariciresinol diglucoside (SDG) supplementation (0.44%). Animals were fed with their respective diets for 2 weeks before and for 8 weeks after the surgery. Echocardiography and continuous electrocardiographic recordings were obtained after ligation to confirm the induction of the MI, to check for arrhythmias and to assess cardiac function. Histological examination was also performed to evaluate cardiac fibrosis. Dietary supplementation with flaxseed, ALA or SDG before and after the induction of the MI significantly reduced the incidence of arrhythmias and resulted in significantly smaller infarct size, less left ventricle dilation, and decreased myocardial fibrosis and tumor necrosis factor-α levels compared to the control MI group. Together, this study supports a beneficial effect of dietary flaxseed in patients for the prevention and treatment of arrhythmias and ventricular remodeling post-MI.

Phospholipid oxidation products in ferroptotic myocardial cell death.

Cell death is an important component of the pathophysiology of any disease. Myocardial disease is no exception. Understanding how and why cells die, particularly in the heart where cardiomyocyte regeneration is limited at best, becomes a critical area of study. Ferroptosis is a recently described form of nonapoptotic cell death. It is an iron-mediated form of cell death that occurs because of accumulation of lipid peroxidation products. Reactive oxygen species and iron-mediated phospholipid peroxidation is a hallmark of ferroptosis. To date, ferroptosis has been shown to be involved in cell death associated with Alzheimer's disease, Huntington's disease, cancer, Parkinson's disease, and kidney degradation. Myocardial reperfusion injury is characterized by iron deposition as well as reactive oxygen species production. These conditions, therefore, favor the induction of ferroptosis. Currently there is no available treatment for reperfusion injury, which accounts for up to 50% of the final infarct size. This review will summarize the evidence that ferroptosis can induce cardiomyocyte death following reperfusion injury and the potential for this knowledge to open new therapeutic approaches for myocardial ischemia-reperfusion injury.

Overcoming the Bitter Taste of Oils Enriched in Fatty Acids to Obtain Their Effects on the Heart in Health and Disease.

Fatty acids come in a variety of structures and, because of this, create a variety of functions for these lipids. Some fatty acids have a role to play in energy metabolism, some help in lipid storage, cell structure, the physical state of the lipid, and even in food stability. Fatty acid metabolism plays a particularly important role in meeting the energy demands of the heart. It is the primary source of myocardial energy in control conditions. Its role changes dramatically in disease states in the heart, but the pathologic role these fatty acids play depends upon the type of cardiovascular disease and the type of fatty acid. However, no matter how good a food is for one's health, its taste will ultimately become a deciding factor in its influence on human health. No food will provide health benefits if it is not ingested. This review discusses the taste characteristics of culinary oils that contain fatty acids and how these fatty acids affect the performance of the heart during healthy and diseased conditions. The contrasting contributions that different fatty acid molecules have in either promoting cardiac pathologies or protecting the heart from cardiovascular disease is also highlighted in this article.

Dietary Flaxseed as a Strategy for Improving Human Health.

Flaxseed is a rich source of the omega-3 fatty acid, alpha linolenic acid, the lignan secoisolariciresinol diglucoside and fiber. These compounds provide bioactivity of value to the health of animals and humans through their anti-inflammatory action, anti-oxidative capacity and lipid modulating properties. The characteristics of ingesting flaxseed or its bioactive components are discussed in this article. The benefits of administering flaxseed or the individual bioactive components on health and disease are also discussed in this review. Specifically, the current evidence on the benefits or limitations of dietary flaxseed in a variety of cardiovascular diseases, cancer, gastro-intestinal health and brain development and function, as well as hormonal status in menopausal women, are comprehensive topics for discussion.

The Influence of Diet on MicroRNAs that Impact Cardiovascular Disease.

Food quality and nutritional habits strongly influence human health status. Extensive research has been conducted to confirm that foods rich in biologically active nutrients have a positive impact on the onset and development of different pathological processes, including cardiovascular diseases. However, the underlying mechanisms by which dietary compounds regulate cardiovascular function have not yet been fully clarified. A growing number of studies confirm that bioactive food components modulate various signaling pathways which are involved in heart physiology and pathology. Recent evidence indicates that microRNAs (miRNAs), small single-stranded RNA chains with a powerful ability to influence protein expression in the whole organism, have a significant role in the regulation of cardiovascular-related pathways. This review summarizes recent studies dealing with the impact of some biologically active nutrients like polyunsaturated fatty acids (PUFAs), vitamins E and D, dietary fiber, or selenium on the expression of many miRNAs, which are connected with cardiovascular diseases. Current research indicates that the expression levels of many cardiovascular-related miRNAs like miRNA-21, -30 family, -34, -155, or -199 can be altered by foods and dietary supplements in various animal and human disease models. Understanding the dietary modulation of miRNAs represents, therefore, an important field for further research. The acquired knowledge may be used in personalized nutritional prevention of cardiovascular disease or the treatment of cardiovascular disorders.

Ginseng Berry Extract Rich in Phenolic Compounds Attenuates Oxidative Stress but not Cardiac Remodeling post Myocardial Infarction.

The cardioprotective effects of ginseng root extracts have been reported. However, nothing is known about the myocardial actions of the phenolic compounds enriched in ginseng berry. Therefore, this study was undertaken to investigate the effects of American ginseng berry extract (GBE) in an experimental model of myocardial infarction (MI). Coronary artery ligation was performed on Sprague⁻Dawley male rats to induce MI after which animals were randomized into groups receiving either distilled water or GBE intragastrically for 8 weeks. Echocardiography and assays for malondialdehyde (MDA) and TNF-α were conducted. Flow cytometry was used to test the effects of GBE on T cell phenotypes and cytokine production. Although GBE did not improve the cardiac functional parameters, it significantly attenuated oxidative stress in post-MI rat hearts. GBE treatment also resulted in lower than control levels of TNF-α in post-MI rat hearts indicating a strong neutralizing effect of GBE on this cytokine. However, there was no effect of GBE on the proportion of different T cell subsets or ex-vivo cytokine production. Taken together, the present study demonstrates GBE reduces oxidative stress, however no effect on cardiac structure and function in post-MI rats. Moreover, reduction of TNF-α levels below baseline raises concern regarding its use as prophylactic or preventive adjunct therapy in cardiovascular disease.

Reduction of myocardial ischaemia-reperfusion injury by inactivating oxidized phospholipids.

Aims: Myocardial ischaemia followed by reperfusion (IR) causes an oxidative burst resulting in cellular dysfunction. Little is known about the impact of oxidative stress on cardiomyocyte lipids and their role in cardiac cell death. Our goal was to identify oxidized phosphatidylcholine-containing phospholipids (OxPL) generated during IR, and to determine their impact on cell viability and myocardial infarct size. Methods and results: OxPL were quantitated in isolated rat cardiomyocytes using mass spectrophotometry following 24 h of IR. Cardiomyocyte cell death was quantitated following exogenously added OxPL and in the absence or presence of E06, a 'natural' murine monoclonal antibody that binds to the PC headgroup of OxPL. The impact of OxPL on mitochondria in cardiomyocytes was also determined using cell fractionation and Bnip expression. Transgenic Ldlr-/- mice, overexpressing a single-chain variable fragment of E06 (Ldlr-/--E06-scFv-Tg) were used to assess the effect of inactivating endogenously generated OxPL in vivo on myocardial infarct size. Following IR in vitro, isolated rat cardiomyocytes showed a significant increase in the specific OxPLs PONPC, POVPC, PAzPC, and PGPC (P < 0.05 to P < 0.001 for all). Exogenously added OxPLs resulted in significant death of rat cardiomyocytes, an effect inhibited by E06 (percent cell death with added POVPC was 22.6 ± 4.14% and with PONPC was 25.3 ± 3.4% compared to 8.0 ± 1.6% and 6.4 ± 1.0%, respectively, with the addition of E06, P < 0.05 for both). IR increased mitochondrial content of OxPL in rat cardiomyocytes and also increased expression of Bcl-2 death protein 3 (Bnip3), which was inhibited in presence of E06. Notably cardiomyocytes with Bnip3 knock-down were protected against cytotoxic effects of OxPL. In mice exposed to myocardial IR in vivo, compared to Ldlr-/- mice, Ldlr-/--E06-scFv-Tg mice had significantly smaller myocardial infarct size normalized to area at risk (72.4 ± 21.9% vs. 47.7 ± 17.6%, P = 0.023). Conclusions: OxPL are generated within cardiomyocytes during IR and have detrimental effects on cardiomyocyte viability. Inactivation of OxPL in vivo results in a reduction of infarct size.