Christine Des Rosiers

Correction of 13C Mass Isotopomer Distributions for Natural Stable Isotope Abundance

Metabolism of singly or multiply 13C-labeled substrates leads to the production of molecules that contain 13C atoms at various positions. Molecules differing only in the number of isotopic atoms incorporated are referred to as mass isotopomers. The distribution of mass isotopomers of many molecules can be measured by gas chromatography/ mass spectrometry after chemical derivatization. Quantification of metabolite mass isotopomer abundance resulting from biological processes necessitates correction of the measured mass isotopomer distribution of the derivatized metabolite for contributions due to naturally occurring isotopes of its elements. This correction must take into account differences in the relative natural abundance distribution of each mass isotopomer (skewing). An IBM-compatible computer program was developed which (i) calculates the natural abundance mass isotopomer distribution of unlabeled and labeled standards given the molecular formula of the derivatized molecule or fragment ion, and (ii) calculates the natural abundance mass isotopomer distribution of the singly and multiply labeled molecule or fragment via non-linear fitting to the measured mass isotopomer distribution of the unlabeled molecule or fragment. The output of this program is used to correct measured mass isotopomer distributions for contributions from natural isotope abundances and to verify measured values for theoretical consistency. Differences between predicted and measured unlabeled and 13C-labeled isotopomer distributions for hydroxamate di-t-butyl-dimethylsilyl (di-TBDMS) derivatized pyruvate were measured. The program was applied to the mass isotopomer distribution of glucose labeled from [U-13C3]glycerol and of fatty acids labeled from [U-13C6]glucose and either [2-13C2] acetate or [U-13C2]acetate. In some of these cases, the measured mass isotopomer distributions corrected by the program were different from those corrected by the classical technique. Implications of these differences including those on the calculation of glucose production due to gluconeogenesis in isolated perfused rat liver are discussed.

Dystrophin-deficient cardiomyocytes are abnormally vulnerable to mechanical stress-induced contractile failure and injury

Although absence of the cytoskeletal protein dystrophin leads to dilated cardiomyopathy in humans, the functional role of dystrophin in cardiac muscle remains undefined. We have addressed the hypothesis that dystrophin could help protect the heart against injury and contractile dysfunction induced by mechanical stress. In normal and dystrophin‐deficient (mdx) mice, cardiac mechanical stress was first manipulated ex vivo in a perfused working heart preparation. Despite an afterload level in the normal physiologic range, ex vivo perfused mdx hearts developed severe contractile dysfunction and nonischemic tissue damage, as is shown by excessive LDH release without a rise in coronary lactate. Injury to dystrophin‐deficient hearts was significantly correlated with cardiac work, and reducing the afterload level improved contractility and prevented injury in mdx hearts studied ex vivo. The response to mechanical stress in vivo was also assessed by using the vital dye Evans blue, which penetrates into cardiomyocytes with a disrupted sarcolemma. In the mdx group only, cardiomyocyte injury was increased markedly by acute elevations of mechanical stress induced by isoproterenol or brief aortic occlusion. Strikingly accelerated mortality and cardiac necrosis were also observed in the mdx group subjected to chronically increased cardiac mechanical stress via subtotal aortic constriction. Taken together, our results provide the first direct evidence that dystrophin serves to protect cardiomyocytes from mechanical stress and workload‐induced damage. Accordingly, reducing cardiac work in patients with dystrophin deficiency could be beneficial not only in treating established cardiomyopathy, but also in preventing the onset of cardiac disease.

Mitochondrial Dysfunction and Lipid Accumulation in the Human Diaphragm during Mechanical Ventilation

RATIONALE Mechanical ventilation (MV) is associated with adverse effects on the diaphragm, but the cellular basis for this phenomenon, referred to as ventilator-induced diaphragmatic dysfunction (VIDD), is poorly understood. OBJECTIVES To determine whether mitochondrial function and cellular energy status are disrupted in human diaphragms after MV, and the role of mitochondria-derived oxidative stress in the development of VIDD. METHODS Diaphragm and biceps specimens obtained from brain-dead organ donors who underwent MV (15-176 h) and age-matched control subjects were compared regarding mitochondrial enzymatic function, mitochondrial DNA integrity, lipid content, and metabolic gene and protein expression. In addition, diaphragmatic force and oxidative stress after exposure to MV for 6 hours were evaluated in mice under different conditions. MEASUREMENTS AND MAIN RESULTS In human MV diaphragms, mitochondrial biogenesis and content were down-regulated, with a more specific defect of respiratory chain cytochrome-c oxidase. Laser capture microdissection of cytochrome-c oxidase-deficient fibers revealed mitochondrial DNA deletions, consistent with damage from oxidative stress. Diaphragmatic lipid accumulation and responses of master cellular metabolic sensors (AMP-activated protein kinase and sirtuins) were consistent with energy substrate excess as a possible stimulus for these changes. In mice, induction of hyperlipidemia worsened diaphragmatic oxidative stress during MV, whereas transgenic overexpression of a mitochondria-localized antioxidant (peroxiredoxin-3) was protective against VIDD. CONCLUSIONS Our data suggest that mitochondrial dysfunction lies at the nexus between oxidative stress and the impaired diaphragmatic contractility that develops during MV. Energy substrate oversupply relative to demand, resulting from diaphragmatic inactivity during MV, could play an important role in this process.

Calorie Restriction Prevents Hypertension and Cardiac Hypertrophy in the Spontaneously Hypertensive Rat

Because recent evidence demonstrated that calorie restriction (CR) has numerous beneficial cardiovascular effects, we investigated whether short-term CR could reduce hypertension and prevent cardiac hypertrophy inherent to the nonobese spontaneously hypertensive rat (SHR). After 5 weeks of either ad libitum feeding or short-term CR, SHRs subjected to short-term CR had lower systolic blood pressure (BP) and reduced left ventricular wall thickness as assessed by noninvasive tail-cuff BP measurements and echocardiography, respectively. In addition, ultrasound measurements of the femoral artery revealed that flow-mediated vasodilation was significantly improved in SHRs with CR compared to controls. Moreover, pressure myography of isolated mesenteric arteries and subsequent histological and biochemical analysis of these arteries demonstrated that short-term CR improved vascular compliance, increased endothelial nitric oxide synthase (eNOS) activity and nitric oxide bioavailability, and reduced vascular remodeling compared to ad libitum-fed SHRs. Although these effects are likely multifactorial, they were associated with elevated levels of the circulating adipokine, adiponectin, and enhanced AMP-activated protein kinase (AMPK) activity. To provide evidence that elevated adiponectin levels in the SHR is sufficient to prevent an increase in BP, adenoviral-mediated overexpression of adiponectin increased circulating levels of adiponectin, reduced BP, and activated the AMPK/eNOS pathway in the absence of CR. Overall, our findings provide compelling evidence that short-term CR exerts beneficial effects in the SHR via stimulation of an adiponectin/AMPK/eNOS signaling axis. As a result, CR may serve as an effective nonpharmacological treatment of hypertension, and targeting the adiponectin/AMPK/eNOS pathway may improve treatment of hypertension.

Abnormal in vivo myocardial energy substrate uptake in diet-induced type 2 diabetic cardiomyopathy in rats

The purpose of this study was to determine in vivo myocardial energy metabolism and function in a nutritional model of type 2 diabetes. Wistar rats rendered insulin-resistant and mildly hyperglycemic, hyperinsulinemic, and hypertriglyceridemic with a high-fructose/high-fat diet over a 6-wk period with injection of a small dose of streptozotocin (HFHFS) and control rats were studied using micro-PET (microPET) without or with a euglycemic hyperinsulinemic clamp. During glucose clamp, myocardial metabolic rate of glucose measured with [(18)F]fluorodeoxyglucose ([(18)F]FDG) was reduced by approximately 81% (P < 0.05), whereas myocardial plasma nonesterified fatty acid (NEFA) uptake as determined by [(18)F]fluorothia-6-heptadecanoic acid ([(18)F]FTHA) was not significantly changed in HFHFS vs. control rats. Myocardial oxidative metabolism as assessed by [(11)C]acetate and myocardial perfusion index as assessed by [(13)N]ammonia were similar in both groups, whereas left ventricular ejection fraction as assessed by microPET was reduced by 26% in HFHFS rats (P < 0.05). Without glucose clamp, NEFA uptake was approximately 40% lower in HFHFS rats (P < 0.05). However, myocardial uptake of [(18)F]FTHA administered by gastric gavage was significantly higher in HFHFS rats (P < 0.05). These abnormalities were associated with reduced Glut4 mRNA expression and increased Cd36 mRNA expression and mitochondrial carnitine palmitoyltransferase 1 activity (P < 0.05). HFHFS rats display type 2 diabetes complicated by left ventricular contractile dysfunction with profound reduction in myocardial glucose utilization, activation of fatty acid metabolic pathways, and preserved myocardial oxidative metabolism, suggesting reduced myocardial metabolic efficiency. In this model, increased myocardial fatty acid exposure likely occurs from circulating triglyceride, but not from circulating plasma NEFA.

A 13C Mass Isotopomer Study of Anaplerotic Pyruvate Carboxylation in Perfused Rat Hearts

Anaplerotic pyruvate carboxylation was examined in hearts perfused with physiological concentrations of glucose, [U-13C3]lactate, and [U-13C3]pyruvate. Also, a fatty acid, [1-13C]octanoate, or ketone bodies were added at concentrations providing acetyl-CoA at a rate resulting in either low or substantial pyruvate decarboxylation. Relative contributions of pyruvate and fatty acids to citrate synthesis were determined from the13C labeling pattern of effluent citrate by gas chromatography-mass spectrometry (see companion article, Comte, B., Vincent, G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem. 272, 26117–26124). Precision on flux measurements of anaplerotic pyruvate carboxylation depended on the mix of substrates supplied to the heart. Anaplerotic fluxes were precisely determined under conditions where acetyl-CoA was predominantly supplied by β-oxidation, as it occurred with 0.2 or 1 mm octanoate. Then, anaplerotic pyruvate carboxylation provided 3–8% of the OAA moiety of citrate and was modulated by concentrations of lactate and pyruvate in the physiological range. Also, the contribution of pyruvate to citrate formation through carboxylation was equal to or greater than through decarboxylation. Furthermore, 13C labeling data on tissue citric acid cycle intermediates and pyruvate suggest that (i) anaplerosis occurs also at succinate and (ii) cataplerotic malate decarboxylation is low. Rather, the presence of citrate in the effluent perfusate of hearts perfused with physiological concentrations of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate efflux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondrial citrate efflux. In view of the proposed feedback regulation of glycolysis by cytosolic citrate, such a link would support a role of anaplerosis and cataplerosis in metabolic signal transmission between mitochondria and cytosol in the normoxic heart.

Dietary ω-3 fatty acids alter cardiac mitochondrial phospholipid composition and delay Ca2+-induced permeability transition

Consumption of omega-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.

Effects of N-acetylcysteine in the rat heart reperfused after low-flow ischemia: Evidence for a direct scavenging of hydroxyl radicals and a nitric oxide-dependent increase in coronary flow

The capacity of N-acetylcysteine to directly scavenge hydroxyl radical produced by rat hearts reperfused after 90 min of low-flow ischemia was assessed by the hydroxylation of 4-hydroxybenzoate into 3,4-dihydroxybenzoate using a gas chromatography-mass spectrometric assay. Reperfused hearts showed a massive release of 3,4-dihydroxybenzoate, lactate dehydrogenase, and total glutathione, contained less reduced and oxidized glutathione, but maintained spontaneous beating and coronary flow rates close to preischemic values. Compared to untreated hearts: reperfused hearts treated with N-acetylcysteine from the start of ischemia (i) released four times less 3,4-dihydroxybenzoate, but similar amounts of lactate dehydrogenase or glutathione, (ii) showed a nitric oxide-dependent increase in coronary flow rate, and (iii) contained less oxidized glutathione, but similar amounts of reduced glutathione. Reperfused hearts receiving N-acetylcysteine since the last 5 min of ischemia had also a four-times lower 3,4-dihydroxybenzoate release, but their coronary flow rate response was similar to that of untreated hearts. These results indicate that N-acetylcysteine can directly scavenge hydroxyl radicals produced by reperfused ischemic hearts, although this effect is not associated with any protective effects as indicated by the lactate dehydrogenase and glutathione release and cannot explain the nitric oxide-dependent reperfusion hyperemia.

Cardiac anaplerosis in health and disease: food for thought

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.

NADPH oxidase activation by hyperglycaemia in cardiomyocytes is independent of glucose metabolism but requires SGLT1

AIMS Exposure to high glucose (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase in cardiomyocytes, but the underlying mechanism remains elusive. In this study, we have dissected the link between glucose transport and metabolism and NADPH oxidase activation under hyperglycaemic conditions. METHODS AND RESULTS Primary cultures of adult rat cardiomyocytes were exposed to HG concentration (HG, 21 mM) and compared with the normal glucose level (LG, 5 mM). HG exposure activated Rac1GTP and induced p47phox translocation to the plasma membrane, resulting in NADPH oxidase (NOX2) activation, increased ROS production, insulin resistance, and eventually cell death. Comparison of the level of O-linked N-acetylglucosamine (O-GlcNAc) residues in LG- and HG-treated cells did not reveal any significant difference. Inhibition of the pentose phosphate pathway (PPP) by 6-aminonicotinamide counteracted ROS production in response to HG but did not prevent Rac-1 upregulation and p47phox translocation leading to NOX2 activation. Modulation of glucose uptake barely affected oxidative stress and toxicity induced by HG. More interestingly, non-metabolizable glucose analogues (i.e. 3-O-methyl-D-glucopyranoside and α-methyl-D-glucopyranoside) reproduced the toxic effect of HG. Inhibition of the sodium/glucose cotransporter SGLT1 by phlorizin counteracted HG-induced NOX2 activation and ROS production. CONCLUSION Increased glucose metabolism by itself does not trigger NADPH oxidase activation, although PPP is required to provide NOX2 with NADPH and to produce ROS. NOX2 activation results from glucose transport through SGLT1, suggesting that an extracellular metabolic signal transduces into an intracellular ionic signal.