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1.  Evaluation of Docosahexaenoic Acid in a Dog Model of Hypertension Induced Left Ventricular Hypertrophy 
Marine n-3 polyunsaturated fatty acids alter cardiac phospholipids and prevent cardiac pathology in rodents subjected to pressure overload. This approach has not been evaluated in humans or large animals with hypertension-induced pathological hypertrophy. We evaluated docosahexaenoic acid (DHA) in old female dogs with hypertension caused by 16 weeks of aldosterone infusion. Aldosterone-induced hypertension resulted in concentric LV hypertrophy and impaired diastolic function in placebo treated dogs. DHA supplementation increased DHA and depleted arachidonic acid in cardiac phospholipids, but did not improve LV parameters compared to placebo. Surprisingly, DHA significantly increased serum aldosterone concentration and blood pressure compared to placebo. Cardiac mitochondrial yield was decreased in placebo treated hypertensive dogs compared to normal animals, which was prevented by DHA. Extensive analysis of mitochondrial function found no differences between DHA and placebo groups. In conclusion, DHA did not favorably impact mitochondrial or LV function in aldosterone hypertensive dogs.
PMCID: PMC4793776  PMID: 24065618
Fish oil; n3 polyunsaturated fatty acids; heart failure; mitochondrial permeability transition
2.  A Metabolic Signature of Mitochondrial Dysfunction Revealed through a Monogenic Form of Leigh Syndrome 
Cell reports  2015;13(5):981-989.
A decline in mitochondrial respiration represents the root cause of a large number of inborn errors of metabolism. It is also associated with common age-associated diseases and the aging process. To gain insight into the systemic, biochemical consequences of respiratory chain dysfunction, we performed a case-control, prospective metabolic profiling study in a genetically homogenous cohort of patients with Leigh syndrome French Canadian variant, a mitochondrial respiratory chain disease due to loss-of-function mutations in LRPPRC. We discovered 45 plasma and urinary analytes discriminating patients from controls, including classic markers of mitochondrial metabolic dysfunction (lactate and acylcarnitines), as well as unexpected markers of cardiometabolic risk (insulin and adiponectin), amino acid catabolism linked to NADH status (α-hydroxybutyrate), and NAD+ biosynthesis (kynurenine and 3-hydroxyanthranilic acid). Our study identifies systemic, metabolic pathway derangements that can lie downstream of primary mitochondrial lesions, with implications for understanding how the organelle contributes to rare and common diseases.
Graphical abstract
PMCID: PMC4644511  PMID: 26565911
3.  Mitochondrial Vulnerability and Increased Susceptibility to Nutrient-Induced Cytotoxicity in Fibroblasts from Leigh Syndrome French Canadian Patients 
PLoS ONE  2015;10(4):e0120767.
Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC), a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX) and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol), or modulate fatty acid (L-carnitine) or Krebs cycle metabolism (propionate) are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol) exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise questions about the nature of the diets, particularly excess fat intake, as well as on the use of antioxidants in patients with LSFC and, possibly, other COX defects.
PMCID: PMC4383560  PMID: 25835550
4.  Propofol compared with isoflurane inhibits mitochondrial metabolism in immature swine cerebral cortex 
Anesthetics used in infants and children are implicated in the development of neurocognitive disorders. Although propofol induces neuroapoptosis in developing brain, the underlying mechanisms require elucidation and may have an energetic basis. We studied substrate utilization in immature swine anesthetized with either propofol or isoflurane for 4 hours. Piglets were infused with 13-Carbon-labeled glucose and leucine in the common carotid artery to assess citric acid cycle (CAC) metabolism in the parietal cortex. The anesthetics produced similar systemic hemodynamics and cerebral oxygen saturation by near-infrared spectroscopy. Compared with isoflurane, propofol depleted ATP and glycogen stores. Propofol decreased pools of the CAC intermediates, citrate, and α-ketoglutarate, while markedly increasing succinate along with decreasing mitochondrial complex II activity. Propofol also inhibited acetyl-CoA entry into the CAC through pyruvate dehydrogenase, while promoting glycolytic flux with marked lactate accumulation. Although oxygen supply appeared similar between the anesthetic groups, propofol yielded a metabolic phenotype that resembled a hypoxic state. Propofol impairs substrate flux through the CAC in the immature cerebral cortex. These impairments occurred without systemic metabolic perturbations that typically accompany propofol infusion syndrome. These metabolic abnormalities may have a role in the neurotoxity observed with propofol in the vulnerable immature brain.
PMCID: PMC3948133  PMID: 24398942
anesthesia; energy metabolism; glucose; mitochondria; MR spectroscopy
5.  Triiodothyronine Facilitates Weaning From Extracorporeal Membrane Oxygenation by Improved Mitochondrial Substrate Utilization 
Extracorporeal membrane oxygenation (ECMO) provides a bridge to recovery after myocardial injury in infants and children, yet morbidity and mortality remain high. Weaning from the circuit requires adequate cardiac contractile function, which can be impaired by metabolic disturbances induced either by ischemia‐reperfusion and/or by ECMO. We tested the hypothesis that although ECMO partially ameliorates metabolic abnormalities induced by ischemia‐reperfusion, these abnormalities persist or recur with weaning. We also determined if thyroid hormone supplementation (triiodothyronine) during ECMO improves oxidative metabolism and cardiac function.
Methods and Results
Neonatal piglets underwent transient coronary ischemia to induce cardiac injury then were separated into 4 groups based on loading status. Piglets without coronary ischemia served as controls. We infused into the left coronary artery [2‐13C]pyruvate and [13C6, 15N]l‐leucine to evaluate oxidative metabolism by gas chromatography‐mass spectroscopy and nuclear magnetic resonance methods. ECMO improved survival, increased oxidative substrate contribution through pyruvate dehydrogenase, reduced succinate and fumarate accumulation, and ameliorated ATP depletion induced by ischemia. The functional and metabolic benefit of ECMO was lost with weaning, yet triiodothyronine supplementation during ECMO restored function, increased relative pyruvate dehydrogenase flux, reduced succinate and fumarate, and preserved ATP stores.
Although ECMO provides metabolic rest by decreasing energy demand, metabolic impairments persist, and are exacerbated with weaning. Treating ECMO‐induced thyroid depression with triiodothyronine improves substrate flux, myocardial oxidative capacity and cardiac contractile function. This translational model suggests that metabolic targeting can improve weaning.
PMCID: PMC4187495  PMID: 24650924
cardiac metabolism; congenital heart defects; extracorporeal circulation; thyroid hormone
6.  Circulating levels of linoleic acid and HDL-cholesterol are major determinants of 4-hydroxynonenal protein adducts in patients with heart failure☆ 
Redox Biology  2013;2:148-155.
Measurements of oxidative stress biomarkers in patients with heart failure (HF) have yielded controversial results. This study aimed at testing the hypothesis that circulating levels of the lipid peroxidation product 4-hydroxynonenal bound to thiol proteins (4HNE-P) are strongly associated with those of its potential precursors, namely n-6 polyunsaturated fatty acids (PUFA).
Methods and results
Circulating levels of 4HNE-P were evaluated by gas chromatography-mass spectrometry in 71 control subjects and 61 ambulatory symptomatic HF patients along with various other clinically- and biochemically-relevant parameters, including other oxidative stress markers, and total levels of fatty acids from all classes, which reflect both free and bound to cholesterol, phospholipids and triglycerides. All HF patients had severe systolic functional impairment despite receiving optimal evidence-based therapies. Compared to controls, HF patients displayed markedly lower circulating levels of HDL- and LDL-cholesterol, which are major PUFA carriers, as well as of PUFA of the n-6 series, specifically linoleic acid (LA; P=0.001). Circulating 4HNE-P in HF patients was similar to controls, albeit multiple regression analysis revealed that LA was the only factor that was significantly associated with circulating 4HNE-P in the entire population (R2=0.086; P=0.02). In HF patients only, 4HNE-P was even more strongly associated with LA (P=0.003) and HDL-cholesterol (p<0.0002). Our results demonstrate that 4HNE-P levels, expressed relative to HDL-cholesterol, increase as HDL-cholesterol plasma levels decrease in the HF group only.
Results from this study emphasize the importance of considering changes in lipids and lipoproteins in the interpretation of measurements of lipid peroxidation products. Further studies appear warranted to explore the possibility that HDL-cholesterol particles may be a carrier of 4HNE adducts.
Graphical abstract
•Peroxidation of n-6 polyunsaturated fatty acids (PUFA) forms 4-hydroxynonenal (4HNE).•Heart failure (HF) patients have lower plasma levels of n-6 PUFA linoleic acid (LA).•Blood levels of 4HNE bound to proteins (4HNE-P) in HF patients are similar to controls.•4HNE-P levels are associated with those of LA in the entire population (p<0.02).•4HNE-P levels are strongly associated with LA and HDL-cholesterol in HF patients.
PMCID: PMC3909262  PMID: 24494189
4HNE, 4-hydroxynonenal; 4HNE-P, 4-hydroxynonenal bound to circulating thiol proteins; AA, arachidonic acid; CRP, C-reactive protein; DHA, docosahexanaenoic acid; eGFR, estimated glomerular filtration rate; EPA, eicosapentaenoic acid; GSH, reduced glutathione; GSSG, oxidized glutathione; HF, heart failure; HFC-MHI, heart failure clinic of the Montreal Heart Institute; HOMA-IR, homeostatic model assessment of insulin resistance; LA, linoleic acid; MDA, malondialdehyde; MPO, myeloperoxidase; NT-pro-BNP, N-terminal proB-type natriuretic peptide; NYHA, New York Heart Association; PUFA, polyunsaturated fatty acids; RAS, renin-angiotensin system; TBARS, thiobarbituric acid-reactive substances; TNF, tumor necrosis factor; 4-Hydroxynnonenal; Oxidative stress; Lipid peroxidation; Linoleic acid; Polyunsaturated fatty acids; Heart failure patients
7.  High intake of saturated fat, but not polyunsaturated fat, improves survival in heart failure despite persistent mitochondrial defects 
Cardiovascular Research  2011;93(1):24-32.
The impact of a high-fat diet on the failing heart is unclear, and the differences between polyunsaturated fatty acids (PUFA) and saturated fat have not been assessed. Here, we compared a standard low-fat diet to high-fat diets enriched with either saturated fat (palmitate and stearate) or PUFA (linoleic and α-linolenic acids) in hamsters with genetic cardiomyopathy.
Methods and results
Male δ-sarcoglycan null Bio TO2 hamsters were fed a standard low-fat diet (12% energy from fat), or high-fat diets (45% fat) comprised of either saturated fat or PUFA. The median survival was increased by the high saturated fat diet (P< 0.01; 278 days with standard diet and 361 days with high saturated fat)), but not with high PUFA (260 days) (n = 30–35/group). Body mass was modestly elevated (∼10%) in both high fat groups. Subgroups evaluated after 24 weeks had similar left ventricular chamber size, function, and mass. Mitochondrial oxidative enzyme activity and the yield of interfibrillar mitochondria (IFM) were decreased to a similar extent in all TO2 groups compared with normal F1B hamsters. Ca2+-induced mitochondrial permeability transition pore opening was enhanced in IFM in all TO2 groups compared with F1B hamsters, but to a significantly greater extent in those fed the high PUFA diet compared with the standard or high saturated fat diet.
These results show that a high intake of saturated fat improves survival in heart failure compared with a high PUFA diet or low-fat diet, despite persistent mitochondrial defects.
PMCID: PMC3243037  PMID: 21960686
Cardiomyopathy; Low-carbohydrate diet; Metabolism; Obesity
8.  Cardiac anaplerosis in health and disease: food for thought 
Cardiovascular Research  2011;90(2):210-219.
There has been a resurgence of interest for the field of cardiac metabolism catalysed by the increased need for new therapeutic targets for patients with heart failure. The primary focus of research in this area to date has been on the impact of substrate selection for oxidative energy metabolism; however, anaplerotic metabolism also has significant interest for its potential cardioprotective role. Anaplerosis refers to metabolic pathways that replenish the citric acid cycle intermediates, which are essential to energy metabolism; however, our understanding of the role and regulation of this process in the heart, particularly under pathophysiological conditions, is very limited. Therefore, the goal of this article is to provide a foundation for future directions of research on cardiac anaplerosis and heart disease. We include an overview of anaplerotic metabolism, a critical evaluation of current methods available for its quantitation in the intact heart, and a discussion of its role and regulation both in health and disease as it is currently understood based mostly on animal studies. We also consider genetic diseases affecting anaplerotic pathways in humans and acute intervention studies with anaplerotic substrates in the clinics. Finally, as future perspectives, we will share our thoughts about potential benefits and practical considerations on modalities of interventions targeting anaplerosis in heart disease, including heart failure.
PMCID: PMC3078802  PMID: 21398307
Heart failure; Energy metabolism; Mitochondria; NMR; GCMS
9.  The Cardioprotective Effects of Fish Oil During Pressure Overload Are Blocked by High Fat Intake 
Hypertension  2009;54(3):605-611.
Supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil may prevent development of heart failure through alterations in cardiac phospholipids that favorably impact inflammation and energy metabolism. A high-fat diet may block these effects in chronically stressed myocardium. Pathological left ventricle (LV) hypertrophy was generated by subjecting rats to pressure overload by constriction of the abdominal aorta. Animals were fed: (1) standard diet (10% of energy from fat), (2) standard diet with EPA+DHA (2.3% of energy intake as EPA+DHA), (3) high fat (60% fat); or (4) high fat with EPA+DHA. Pressure overload increased LV mass by ≈40% in both standard and high-fat diets without fish oil. Supplementation with fish oil increased their incorporation into cardiac phospholipids, and decreased the proinflammatory fatty acid arachidonic acid and urine thromboxane B2 with both the standard and high-fat diet. Linoleic acid and tetralinoloyl cardiolipin (an essential mitochondrial phospholipid) were decreased with pressure overload on standard diet, which was prevented by fish oil. Animals fed high-fat diet had decreased linoleic acid and tetralinoloyl cardiolipin regardless of fish oil supplemention. Fish oil limited LV hypertrophy on the standard diet, and prevented upregulation of fetal genes associated with heart failure (myosin heavy chain-β and atrial natriuetic factor). These beneficial effects of fish oil were absent in animals on the high-fat diet. In conclusion, whereas treatment with EPA+DHA prevented tetralinoloyl cardiolipin depletion, LV hypertrophy, and abnormal genes expression with pressure overload, these effects were absent with a high-fat diet.
PMCID: PMC3103889  PMID: 19597033
Omega-3 fatty acids; cardiac hypertrophy; heart failure; cardiolipin; phospolipids
10.  Acute Regulation of Cardiac Metabolism by the Hexosamine Biosynthesis Pathway and Protein O-GlcNAcylation 
PLoS ONE  2011;6(4):e18417.
The hexosamine biosynthesis pathway (HBP) flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc) levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization.
Isolated rat hearts were perfused with glucosamine (0–10 mM) to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by 13C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36.
Glucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation.
These data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism.
PMCID: PMC3073970  PMID: 21494549
11.  Dietary ω-3 Fatty Acids Alter Cardiac Mitochondrial Phospholipid Composition and Delay Ca2+-Induced Permeability Transition 
Consumption of ω-3 fatty acids from fish oil, specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases risk for heart failure and attenuates pathologic cardiac remodeling in response to pressure overload. Dietary supplementation with EPA+DHA may also impact cardiac mitochondrial function and energetics through alteration of membrane phospholipids. We assessed the role of EPA+DHA supplementation on left ventricular (LV) function, cardiac mitochondrial membrane phospholipid composition, respiration, and sensitivity to mitochondrial permeability transition pore (MPTP) opening in normal and infarcted myocardium. Rats were subjected to sham surgery or myocardial infarction by coronary artery ligation (n=10–14), and fed a standard diet, or supplemented with EPA+DHA (2.3% of energy intake) for 12 weeks. EPA+DHA altered fatty acid composition of total mitochondrial phospholipids and cardiolipin by reducing arachidonic acid content and increasing DHA incorporation. EPA+DHA significantly increased calcium uptake capacity in both subsarcolemmal and intrafibrillar mitochondria from sham rats. This treatment effect persisted with the addition of cyclosporin A, and was not accompanied by changes in mitochondrial respiration or coupling, or cyclophilin D protein expression. Myocardial infarction resulted in heart failure as evidenced by LV dilation and contractile dysfunction. Infarcted LV myocardium had decreased mitochondrial protein yield and activity of mitochondrial marker enzymes, however respiratory function of isolated mitochondria was normal. EPA+DHA had no effect on LV function, mitochondrial respiration, or MPTP opening in rats with heart failure. In conclusion, dietary supplementation with EPA+DHA altered mitochondrial membrane phospholipid fatty acid composition in normal and infarcted hearts, but delayed MPTP opening only in normal hearts.
PMCID: PMC2783943  PMID: 19703463
eicosapentaenoic acid; docosahexaenoic acid; myocardial infarction; mitochondrial permeability transition pore
12.  ω-3 Polyunsaturated fatty acids prevent pressure overload-induced ventricular dilation and decrease in mitochondrial enzymes despite no change in adiponectin 
Pathological left ventricular (LV) hypertrophy frequently progresses to dilated heart failure with suppressed mitochondrial oxidative capacity. Dietary marine ω-3 polyunsaturated fatty acids (ω-3 PUFA) up-regulate adiponectin and prevent LV dilation in rats subjected to pressure overload. This study 1) assessed the effects of ω-3 PUFA on LV dilation and down-regulation of mitochondrial enzymes in response to pressure overload; and 2) evaluated the role of adiponectin in mediating the effects of ω-3 PUFA in heart.
Wild type (WT) and adiponectin-/- mice underwent transverse aortic constriction (TAC) and were fed standard chow ± ω-3 PUFA for 6 weeks. At 6 weeks, echocardiography was performed to assess LV function, mice were terminated, and mitochondrial enzyme activities were evaluated.
TAC induced similar pathological LV hypertrophy compared to sham mice in both strains on both diets. In WT mice TAC increased LV systolic and diastolic volumes and reduced mitochondrial enzyme activities, which were attenuated by ω-3 PUFA without increasing adiponectin. In contrast, adiponectin-/- mice displayed no increase in LV end diastolic and systolic volumes or decrease in mitochondrial enzymes with TAC, and did not respond to ω-3 PUFA.
These findings suggest ω-3 PUFA attenuates cardiac pathology in response to pressure overload independent of an elevation in adiponectin.
PMCID: PMC2939588  PMID: 20819225
13.  Applications of metabolomics and proteomics to the mdx mouse model of Duchenne muscular dystrophy: lessons from downstream of the transcriptome 
Genome Medicine  2009;1(3):32.
Functional genomic studies are dominated by transcriptomic approaches, in part reflecting the vast amount of information that can be obtained, the ability to amplify mRNA and the availability of commercially standardized functional genomic DNA microarrays and related techniques. This can be contrasted with proteomics, metabolomics and metabolic flux analysis (fluxomics), which have all been much slower in development, despite these techniques each providing a unique viewpoint of what is happening in the overall biological system. Here, we give an overview of developments in these fields 'downstream' of the transcriptome by considering the characterization of one particular, but widely used, mouse model of human disease. The mdx mouse is a model of Duchenne muscular dystrophy (DMD) and has been widely used to understand the progressive skeletal muscle wasting that accompanies DMD, and more recently the associated cardiomyopathy, as well as to unravel the roles of the other isoforms of dystrophin, such as those found in the brain. Studies using proteomics, metabolomics and fluxomics have characterized perturbations in calcium homeostasis in dystrophic skeletal muscle, provided an understanding of the role of dystrophin in skeletal muscle regeneration, and defined the changes in substrate energy metabolism in the working heart. More importantly, they all point to perturbations in proteins, metabolites and metabolic fluxes reflecting mitochondrial energetic alterations, even in the early stage of the dystrophic pathology. Philosophically, these studies also illustrate an important lesson relevant to both functional genomics and the mouse phenotyping in that the knowledge generated has advanced our understanding of cell biology and physiological organization as much as it has advanced our understanding of the disease.
PMCID: PMC2664943  PMID: 19341503

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