Hélène Lemieux

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism

HIF prolyl hydroxylases (PHD1–3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparα pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2α and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.

Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart.

Heart failure is a consequence of progressive deterioration of cardiac performance. Little is known about the role of impaired oxidative phosphorylation in the progression of the disease, since previous studies of mitochondrial injuries are restricted to end-stage chronic heart failure. The present study aimed at evaluating the involvement of mitochondrial dysfunction in the development of human heart failure. We measured the control of oxidative phosphorylation with high-resolution respirometry in permeabilized myocardial fibres from donor hearts (controls), and patients with no or mild heart failure but presenting with heart disease, or chronic heart failure due to dilated or ischemic cardiomyopathy. The capacity of the phosphorylation system exerted a strong limitation on oxidative phosphorylation in the human heart, estimated at 121 pmol O(2)s(-1)mg(-1) in the healthy left ventricle. In heart disease, a specific defect of the phosphorylation system, Complex I-linked respiration, and mass-specific fatty acid oxidation were identified. These early defects were also significant in chronic heart failure, where the capacities of the oxidative phosphorylation and electron transfer systems per cardiac tissue mass were decreased with all tested substrate combinations, suggesting a decline of mitochondrial density. Oxidative phosphorylation and electron transfer system capacities were higher in ventricles compared to atria, but the impaired mitochondrial quality was identical in the four cardiac chambers of chronic heart failure patients. Coupling was preserved in heart disease and chronic heart failure, in contrast to the mitochondrial dysfunction observed after prolonged cold storage of cardiac tissue. Mitochondrial defects in the phosphorylation system, Complex I respiration and mass-specific fatty acid oxidation occurred early in the development of heart failure. Targeting these mitochondrial injuries with metabolic therapy may offer a promising approach to delay the progression of heart disease.

Mitochondria in the elderly: Is acetylcarnitine a rejuvenator?

Endogenous acetylcarnitine is an indicator of acetyl-CoA synthesized by multiple metabolic pathways involving carbohydrates, amino acids, fatty acids, sterols, and ketone bodies, and utilized mainly by the tricarboxylic acid cycle. Acetylcarnitine supplementation has beneficial effects in elderly animals and humans, including restoration of mitochondrial content and function. These effects appear to be dose-dependent and occur even after short-term therapy. In order to set the stage for understanding the mechanism of action of acetylcarnitine, we review the metabolism and role of this compound. We suggest that acetylation of mitochondrial proteins leads to a specific increase in mitochondrial gene expression and mitochondrial protein synthesis. In the aged rat heart, this effect is translated to increased cytochrome b content, restoration of complex III activity, and oxidative phosphorylation, resulting in amelioration of the age-related mitochondrial defect.

Mitochondria in the human heart

The heart relies mainly on mitochondrial metabolism to provide the energy needed for pumping blood to oxygenate the organs of the body. The study of mitochondrial function in the human heart faces many obstacles and elucidation of the role of mitochondria in cardiac diseases has relied mainly on studies with animal models. Cardiac diseases are the leading cause of mortality worldwide. With the emergence of new therapies to treat and prevent heart disease, some aiming at metabolic modulation, a need for acquiring a better understanding of mitochondrial function in the human heart becomes apparent. Our review is aimed at specific evaluation of the human heart in terms of (1) methods to understand mitochondrial function, with particular emphasis on integrated function, (2) data on the role of mitochondrial dysfunction in cardiovascular disease, and (3) possible applications of this knowledge in the treatment of patients with cardiac disease.

Decrease in Mitochondrial Function in Rat Cardiac Permeabilized Fibers Correlates With the Aging Phenotype

We measured the loss of cardiac mitochondrial function related to aging in males of three rat strains presenting with different longevity and aging phenotypes: the Fischer 344 (F344), the Brown Norway (BN), and the hybrid F344×BN. The F344 rat has a short life span and a ∼45% decrease in coupled mitochondrial oxidation in the cardiac permeabilized fibers from the old rats compared with the young rats. Citrate synthase activity in the permeabilized fibers (mitochondrial content) did not change significantly with aging. The BN live longer compared with the F344 and have a 15%-18% loss of mitochondrial respiration in the aged rats compared with the young rats. The differences are not significant. In hybrids, more resistant to aging than are the BN and the F344, mitochondrial function is preserved during aging. The difference in longevity of the different strains is correlated with mitochondrial dysfunction in the heart, suggesting the importance of mitochondria in cardiac aging.

Physical fitness and mitochondrial respiratory capacity in horse skeletal muscle.

Background Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general. Methodology/Principal Findings We applied high-resolution respirometry and multiple substrate-uncoupler-inhibitor titration protocols to study mitochondrial physiology in small (1.0–2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses. Oxidative phosphorylation (OXPHOS) capacity (pmol O2•s−1•mg−1 wet weight) with combined Complex I and II (CI+II) substrate supply (malate+glutamate+succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained and competitive horses (N = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (N = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the CI+II substrate cocktail. Relative to this maximum OXPHOS capacity, Complex I (CI)-linked OXPHOS capacities were only 50% with glutamate+malate, 64% with pyruvate+malate, and 68% with pyruvate+malate+glutamate, and ∼78% with CII-linked succinate+rotenone. OXPHOS capacity with glutamate+malate increased with fitness relative to CI+II-supported ETS capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the CII/CI+II substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the CI- over CII-linked pathway capacity was reduced with physical fitness. Conclusions/Significance The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of CI-linked respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology.

Early mitochondrial dysfunction in glycolytic muscle, but not oxidative muscle, of the fructose-fed insulin-resistant rat

Although evidence that type 2 diabetes mellitus (T2DM) is accompanied by mitochondrial dysfunction in skeletal muscle has been accumulating, a causal link between mitochondrial dysfunction and the pathogenesis of the disease remains unclear. Our study focuses on an early stage of the disease to determine whether mitochondrial dysfunction contributes to the development of T2DM. The fructose-fed (FF) rat was used as an animal model of early T2DM. Mitochondrial respiration and acylcarnitine species were measured in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscle. Although FF rats displayed characteristic signs of T2DM, including hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, mitochondrial content was preserved in both muscles from FF rats. The EDL muscle had reduced complex I and complex I and II respiration in the presence of pyruvate but not glutamate. The decrease in pyruvate-supported respiration was due to a decrease in pyruvate dehydrogenase activity. Accumulation of C14:1 and C14:2 acylcarnitine species and a decrease in respiration supported by long-chain acylcarnitines but not acetylcarnitine indicated dysfunctional β-oxidation in the EDL muscle. In contrast, the soleus muscle showed preserved mitochondrial respiration, pyruvate dehydrogenase activity, and increased fatty acid oxidation, as evidenced by overall reduced acylcarnitine levels. Aconitase activity, a sensitive index of reactive oxygen species production in mitochondria, was reduced exclusively in EDL muscle, which showed lower levels of the antioxidant enzymes thioredoxin reductase and glutathione peroxidase. Here, we show that the glycolytic EDL muscle is more prone to an imbalance between energy supply and oxidation caused by insulin resistance than the oxidative soleus muscle.

The mechanism of Intralipid®-mediated cardioprotection complex IV inhibition by the active metabolite, palmitoylcarnitine, generates reactive oxygen species and activates reperfusion injury salvage kinases.

Background Intralipid® administration at reperfusion elicits protection against myocardial ischemia-reperfusion injury. However, the underlying mechanisms are not fully understood. Methods Sprague-Dawley rat hearts were exposed to 15 min of ischemia and 30 min of reperfusion in the absence or presence of Intralipid® 1% administered at the onset of reperfusion. In separate experiments, the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)-glycine was added either alone or with Intralipid®. Left ventricular work and activation of Akt, STAT3, and ERK1/2 were used to evaluate cardioprotection. ROS production was assessed by measuring the loss of aconitase activity and the release of hydrogen peroxide using Amplex Red. Electron transport chain complex activities and proton leak were measured by high-resolution respirometry in permeabilized cardiac fibers. Titration experiments using the fatty acid intermediates of Intralipid® palmitoyl-, oleoyl- and linoleoylcarnitine served to determine concentration-dependent inhibition of complex IV activity and mitochondrial ROS release. Results Intralipid® enhanced postischemic recovery and activated Akt and Erk1/2, effects that were abolished by the ROS scavenger N-(2-mercaptopropionyl)glycine. Palmitoylcarnitine and linoleoylcarnitine, but not oleoylcarnitine concentration-dependently inhibited complex IV. Only palmitoylcarnitine reached high tissue concentrations during early reperfusion and generated significant ROS by complex IV inhibition. Palmitoylcarnitine (1 µM), administered at reperfusion, also fully mimicked Intralipid®-mediated protection in an N-(2-mercaptopropionyl)-glycine -dependent manner. Conclusions Our data describe a new mechanism of postconditioning cardioprotection by the clinically available fat emulsion, Intralipid®. Protection is elicited by the fatty acid intermediate palmitoylcarnitine, and involves inhibition of complex IV, an increase in ROS production and activation of the RISK pathway.

Dietary fatty acids and oxidative stress in the heart mitochondria

Our study compared the effects of different oils on oxidative stress in rat heart mitochondria, as well as on plasma parameters used as risk factors for cardiovascular disease. The rats were fed for 16 weeks with coconut, olive, or fish oil diet (saturated, monounsaturated, or polyunsaturated fatty acids, respectively). The cardiac mitochondria from rats fed with coconut oil showed the lowest concentration of oxidized proteins and peroxidized lipids. The fish oil diet leads to the highest oxidative stress in cardiac mitochondria, an effect that could be partly prevented by the antioxidant probucol. Total and LDL cholesterols decreased in plasma of rats fed fish oil, compared to olive and coconut oils fed rats. A diet enriched in saturated fatty acids offers strong advantages for the protection against oxidative stress in heart mitochondria.

Thermal sensitivity of oxidative phosphorylation in rat heart mitochondria: Does pyruvate dehydrogenase dictate the response to temperature?

To identify the most temperature-sensitive steps in the energy production pathways, we measured the thermal sensitivity of mitochondrial oxidative phosphorylation (OXPHOS), as well as that of the individual steps in this process in rat heart mitochondria. OXPHOS measured in the presence of pyruvate+malate as substrates have an unusually high thermal sensitivity between 5 and 15°C. Furthermore, the thermal sensitivity of OXPHOS correlates with the thermal sensitivity of pyruvate dehydrogenase between 5 and 35°C. Pyruvate dehydrogenase is a potential control point for pyruvate-supported mitochondrial respiration below physiological temperature in rat heart.