Luc Demaison

Fatty acid oxidation in the heart.

The heart is known for its ability to produce energy from fatty acids (FA) because of its important beta-oxidation equipment, but it can also derive energy from several other substrates including glucose, pyruvate, and lactate. The cardiac ATP store is limited and can assure only a few seconds of beating. For this reason the cardiac muscle can adapt quickly to the energy demand and may shift from a 100% FA-derived energy production (after a lipid-rich food intake) or any balanced situation (e.g., diabetes, fasting, exercise). These situations are not similar for the heart in terms of oxygen requirement because ATP production from glucose is less oxygen-consuming than from FA. The regulation pathways for these shifts, which occur in physiologic as well as pathologic conditions (ischemia-reperfusion), are not yet known, although both insulin and pyruvate dehydrogenase activation are clearly involved. It becomes of strategic importance to clarify the pathways that control these shifts to influence the oxygen requirement of the heart. Excess FA oxidation is closely related to myocardial contraction disorders characterized by increased oxygen consumption for cardiac work. Such an increased oxygen cost of cardiac contraction was observed in stunned myocardium when the contribution of FA oxidation to oxygen consumption was increased. In rats, an increase in n-3 polyunsaturated FA in heart phospholipids achieved by a fish-oil diet improved the recovery of pump activity during postischemic reperfusion. This was associated with a moderation of the ischemia-induced decrease in mitochondrial palmitoylcarnitine oxidation. In isolated mitochondria at calcium concentrations close to that reported in ischemic cardiomyocytes, a futile cycle of oxygen wastage was reported, associated with energy wasting (constant AMP production). This occurs with palmitoylcarnitine as substrate but not with pyruvate or citrate. The energy wasting can be abolished by CoA-SH and other compounds, but not the oxygen wasting. Again, the calcium-induced decrease in mitochondrial ADP/O ratio was reduced by increasing the n-3 polyunsaturated FA in the mitochondrial phospholipids. These data suggest that in addition to the amount of circulating lipids, the quality of FA intake may contribute to heart energy regulation through the phospholipid composition. On the other hand, other intervention strategies can be considered. Several studies have focused on palmitoylcarnitine transferase I to achieve a reduction in beta-oxidation. In a different context, trimetazidine was suggested to exert its anti-ischemic effect on the heart by interfering with the metabolic shift, either at the pyruvate dehydrogenase level or by reducing the beta-oxidation. Further studies will be required to elucidate the complex system of heart energy regulation and the mechanism of action of potentially efficient molecules.

Oxyphytosterols are present in plasma of healthy human subjects

The oxidised derivatives of phytosterols (oxyphytosterols) were identified in plasma samples from thirteen healthy human volunteers, using MS. All the samples contained noticeable quantities of (24R)-5beta,6beta-epoxy-24-ethylcholestan-3beta-ol (beta-epoxysitostanol) and (24R)-ethylcholestan-3beta,5alpha,6beta-triol (sitostanetriol) and also trace levels of (24R)-5alpha,6alpha-epoxy-24-ethylcholestan-3beta-ol (alpha-epoxysitostanol), (24R)-methylcholestan-3beta,5alpha,6beta-triol (campestanetriol) and (24R)-ethylch olest-5-en-3beta-ol-7-one(7-ketositosterol). The amounts of these oxyphytosterols in plasma varied from 4.8 to 57.2 ng/ml. There are two possibilities concerning the origin of these compounds. First, they could come from the small amounts of oxyphytosterols in food. Second, they could originate from the in vivo oxidation of phytosterols in plasma. Very few data actually exist concerning these compounds. Their identification in human samples suggests that further research is necessary in this field.

INFLUENCE OF THE PHOSPHOLIPID N-6/N-3 POLYUNSATURATED FATTY ACID RATIO ON THE MITOCHONDRIAL OXIDATIVE METABOLISM BEFORE AND AFTER MYOCARDIAL ISCHEMIA

The influence of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on heart pump function and mitochondrial energy metabolism was investigated before and after ischemia. Weanling male Wistar rats were fed for 8 weeks a diet containing either 10% of sunflower seed oil (SSO group) or 10% of a 1:1 (w/w) mixture of fish oil and sunflower seed oil (FO group). The hearts were perfused according to the working mode for 15 min with a Krebs-Henseleit medium containing glucose (11 mM), insulin (10 IU/L) and caprylic acid (25 microM). They were then either maintained in normoxic conditions (70 min) or subjected to a global no-flow normothermic ischemia (20 min) followed by reperfusion (50 min). The aortic and coronary flows were monitored at 5-min intervals. The lactate dehydrogenase (LDH) release in the coronary effluent was evaluated in the control hearts and during ischemia/reperfusion. At the end of the perfusion, two subpopulations of mitochondria were prepared from each heart, by either mechanical or enzyme extraction (ME and EE mitochondria, respectively). The succinate dehydrogenase (SDH) activity was evaluated. Furthermore, the respiration parameters were assessed with either glutamate (20 mM) or palmitoylcarnitine (25 microM) as substrate. Substituting sunflower seed oil by fish oil in the diet provoked a large decrease in the n-6/n-3 PUFA ratio of cardiac phospholipids. The n-3 PUFA enrichment did not alter the coronary and aortic flows nor the LDH release in physiological conditions. Conversely, during post-ischemic reperfusion, the increased amount of n-3 PUFA improved the recovery of aortic flow and decreased the LDH release, without affecting significantly the coronary flow. In ME and EE mitochondria, the phospholipid n-6/n-3 PUFA ratio was similarly modified by the dietary manipulations. The analysis of total cardiac SDH activity suggested an ischemia-induced oedema, of similar magnitude in the two dietary groups. However, neither dietary manipulations nor ischemia influenced the mitochondrial extraction. Similarly, the parameters of glutamate oxidation were also unaffected. Conversely, with palmitoylcarnitine, post-ischemic reperfusion induced a decrease in both state III respiration rate and energy production which were more important in the EE mitochondria of the SSO group. These results suggest that the recovery of mitochondrial energy metabolism and myocardial pump function during reperfusion may be improved in n-3 PUFA-rich hearts. This could be related to a lower injury in n-3 PUFA-rich membranes. Since cardiac function in physiological conditions was not affected by the diet, fish oil could be considered as a beneficial factor to limit heart injury during ischemia and reperfusion.

Trimetazidine: In vitro influence on heart mitochondrial function

The mechanism of action of the antianginal trimetazidine (TMZ) remains largely unknown. In cultured rat ventricular myocytes in physiologic conditions, TMZ (5 x 10(-4) M) reduced the plateau potential level, the upstroke velocity, and the spontaneous action potential rate. When the cardiomyocytes were submitted to hypoxia (150 or 240 minutes) in a glucose-free medium, treatment with TMZ largely prevented the hypoxia-induced electromechanical alterations, i.e., the decrease in plateau amplitude, in resting membrane potential, in action potential duration, in rate, and in contractility. No hypoxia-induced arrhythmia was observed in the TMZ-treated cells. Moreover, the lactate dehydrogenase leakage was significantly reduced in the TMZ-treated cardiomyocytes (-58% and -36%, after 150 and 240 minutes of hypoxia, respectively). The drug was not efficient in reducing the hypoxia-induced decrease in adenosine triphosphate (ATP) content. The cellular ATP content was slightly lower in the TMZ-treated cells in normoxic conditions and in hypoxic conditions, but only in the glucose-free medium. To investigate further the relation between TMZ and energy metabolism, the respiration parameters were measured in heart mitochondria isolated from control and TMZ-treated rats (6 mg/kg/day, 7 days) with different substrates. This treatment resulted in a slight alteration of pyruvate oxidation, which was observed in the absence and in the presence of TMZ (10(-4) M) in the respiration medium. Conversely, a potent inhibition of palmitoylcarnitine oxidation was measured when TMZ was added to the respiration medium. Neither pretreatment of the rats, nor addition of TMZ to the medium affected the oxidation of glutamate or citrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities of mitochondrial functioning can partly explain the metabolic disorders encountered in sarcopenic gastrocnemius

Aging triggers several abnormalities in muscle glycolytic fibers including increased proteolysis, reactive oxygen species (ROS) production and apoptosis. Since the mitochondria are the main site of substrate oxidation, ROS production and programmed cell death, we tried to know whether the cellular disorders encountered in sarcopenia are due to abnormal mitochondrial functioning. Gastrocnemius mitochondria were extracted from adult (6 months) and aged (21 months) male Wistar rats. Respiration parameters, opening of the permeability transition pore and ROS production, with either glutamate (amino acid metabolism) or pyruvate (glucose metabolism) as a respiration substrate, were evaluated at different matrix calcium concentrations. Pyruvate dehydrogenase and respiratory complex activities as well as their contents measured by Western blotting analysis were determined. Furthermore, the fatty acid profile of mitochondrial phospholipids was also measured. At physiological calcium concentration, state III respiration rate was lowered by aging in pyruvate conditions (−22%), but not with glutamate. The reduction of pyruvate oxidation resulted from a calcium‐dependent inactivation of the pyruvate dehydrogenase system and could provide for the well‐known proteolysis encountered during sarcopenia. Matrix calcium loading and aging increased ROS production. They also reduced the oxidative phosphorylation. This was associated with lower calcium retention capacities, suggesting that sarcopenic fibers are more prone to programmed cell death. Aging was also associated with a reduced mitochondrial superoxide dismutase activity, which does not intervene in toxic ROS overproduction but could explain the lower calcium retention capacities. Despite a lower content, cytochrome c oxidase displayed an increased activity associated with an increased n−6/n−3 polyunsaturated fatty acid ratio of mitochondrial phospholipids. In conclusion, we propose that mitochondria obtained from aged muscle fibers display several functional abnormalities explaining the increased proteolysis, ROS overproduction and vulnerability to apoptosis exhibited by sarcopenic muscle. These changes appear to be related to modifications of the fatty acid profile of mitochondrial lipids.

Preserved endothelium-dependent dilatation of the coronary microvasculature at the early phase of diabetes mellitus despite the increased oxidative stress and depressed cardiac mechanical function ex vivo

BackgroundThere has been accumulating evidence associating diabetes mellitus and cardiovascular dysfunctions. However, most of the studies are focused on the late stages of diabetes and on the function of large arteries. This study aimed at characterizing the effects of the early phase of diabetes mellitus on the cardiac and vascular function with focus on the intact coronary microvasculature and the oxidative stress involved.Materials and methodsZucker diabetic fatty rats and their lean littermates fed with standard diet A04 (Safe) were studied at the 11th week of age. Biochemical parameters such as glucose, insulin and triglycerides levels as well as their oxidative stress status were measured. Their hearts were perfused ex vivo according to Langendorff and their cardiac activity and coronary microvascular reactivity were evaluated.ResultsZucker fatty rats already exhibited a diabetic state at this age as demonstrated by the elevated levels of plasma glucose, insulin, glycated hemoglobin and triglycerides. The ex vivo perfusion of their hearts revealed a decreased cardiac mechanical function and coronary flow. This was accompanied by an increase in the overall oxidative stress of the organs. However, estimation of the active form of endothelial nitric oxide synthase and coronary reactivity indicated a preserved function of the coronary microvessels at this phase of the disease. Diabetes affected also the cardiac membrane phospholipid fatty acid composition by increasing the arachidonic acid and n-3 polyunsaturated fatty acids levels.ConclusionsThe presence of diabetes, even at its beginning, significantly increased the overall oxidative stress of the organs resulting to decreased cardiac mechanical activity ex vivo. However, adaptations were adopted at this early phase of the disease regarding the preserved coronary microvascular reactivity and the associated cardiac phospholipid composition in order to provide a certain protection to the heart.

Protective effects of trimetazidine on hypoxic cardiac myocytes from the rat

Summary— The electrophysiological effects of the antianginal drug trimetazidine (TMZ) were investigated in cultured rat ventricular myocytes using a substrate‐free hypoxia model of ischemia. The transmembrane potentials were recorded with glass microelectrodes and the contractions were simultaneously monitored with a video motion detector. The cardiomyocytes were treated with TMZ (1–5.10−4 M final concentration) in the bath. The untreated and the drug‐treated cells were submitted either to 150 min normoxia or to 150 min hypoxia followed by 90 min reoxygenation in the absence of oxidizable substrate. In normoxic conditions, TMZ did not affect the maximal diastolic potential (MDP) but significantly lowered the plateau potential level (OS) and decreased the upstroke velocity (Vmax) and the spontaneous action potential rate (APR). Conversely, TMZ significantly increased action potential duration at 80% repolarization (APD80). Under substrate‐free hypoxia, the untreated cells displayed a progressive contractile failure and an important decrease in OS and APD. In parallel, early postdepolarizations triggering high rate spikes were observed. Prolonging oxygen depletion led to the cessation of the spontaneous electrical activity and thereafter to a gradual decrease in MDP. Near normal rhythmic action potentials and contractions resumed after reoxygenation. Comparatively, the treatment by 5.10−4 M TMZ almost completely prevented the decrease in plateau amplitude, resting membrane potential, Vmax, APD80, and rate caused by substrate‐free hypoxia. Moreover, the hypoxia‐induced arrhythmias and the cessation of spontaneous electromechanical activities did not occur in the presence of TMZ (5.40−4 M). After reoxygenation, the TMZ‐treated cells exhibited a higher action potential amplitude than that of the untreated cells, although the TMZ‐induced depressive effects on the spontaneous frequency and the Vmax persisted. In conclusion, this study shows that TMZ (5.10−4 M) is efficient in protecting the isolated cardiac myocytes against the functional alterations induced by substrate‐free hypoxia and led thus to a better recovery upon reoxygenation. This cytoprotective action may be linked, at least in part, to apparent ion channel blocking effects of the drug, which appeared in basal conditions at concentrations used in this study.

Influence of phospholipid polyunsatured fatty acid composition on some metabolic disorders induced in rat cardiomyocytes by hypoxia and reoxygenation

The influence of membrane polyunsaturated fatty acid (PUFA) composition on lactate production, energy status, enzyme leakage and cell defences against oxygen free radical production was studied in cultured rat ventricular myocytes during hypoxia and reoxygenation. After 4 days in a conventional serum-supplemented medium, the cardiomyocytes were incubated for 24 h in synthetic media containing either linoleate and arachidonate (SM6 Medium) or linolenate and eicosapentaenoate (SM3 Medium) as unique source of PUFA. The fatty acid n-6/n-3 ratio of phospholipid was 13.1 in SM6 cells and 0.9 in SM3 cells. Hypoxia induced an increase in lactate production, severe decreases in ATP and ADP, leakage of cellular lactate dehydrogenase and reduction of superoxide dismutase and glutathione peroxidase activities. Reoxygenation of hypoxic cells reduced lactate production to normal aerobic values and allowed slight resynthesis of ATP from AMP. However, lactate dehydrogenase release was not stopped by reoxygenation, and decreases in superoxide dismutase and glutathione peroxidase activities were not avoided. The majority of the biochemical parameters measured during normoxia, hypoxia and reoxygenation were not significantly affected by changes in the fatty acid composition of membrane phospholipids, except for reduced superoxide dismutase activity which appeared earlier in SM3 cells during hypoxia. We conclude that the sarcolemmal PUFA composition of cultured rat ventricular myocytes does not significantly influence altered cell metabolism elicited by hypoxia and reoxygenation.

Fatty acid oxidation and related gene expression in heart depleted of carnitine by mildronate treatment in the rat

The metabolic and genic effects induced by a 20-fold lowering of carnitine content in the heart were studied in mildronate-treated rats. In the perfused heart, the proportion of palmitate taken up then oxidized was 5–10% lower, while the triacylglycerol (TAG) formation was 100% greater than in controls. The treatment was shown to increase the maximal capacity of heart homogenates to oxidize palmitate, the mRNA level of carnitine palmitoyltransferase I (CPT-I) isoforms, the specific activity of CPT-I in subsarcolemmal mitochondria and the total carnitine content of isolated mitochondria. Concomitantly, the increased mRNA expression of lipoprotein lipase, fatty acid translocase and enzymes of TAG synthesis was associated with a 5- and 2-times increase in serum TAG and free fatty acid contents, respectively. The compartmentation of carnitine at its main functional location was expected to allow the increased CPT-I activity to ensure in vivo correct fatty acid oxidation rates. All the inductions related to fatty acid transport, oxidation and esterification most likely stem from the abundance of blood lipids providing cardiomyocytes with more fatty acids.

Middle age aggravates myocardial ischemia through surprising upholding of complex II activity, oxidative stress, and reduced coronary perfusion

Aging compromises restoration of the cardiac mechanical function during reperfusion. We hypothesized that this was due to an ampler release of mitochondrial reactive oxygen species (ROS). This study aimed at characterising ex vivo the mitochondrial ROS release during reperfusion in isolated perfused hearts of middle-aged rats. Causes and consequences on myocardial function of the observed changes were then evaluated. The hearts of rats aged 10- or 52-week old were subjected to global ischemia followed by reperfusion. Mechanical function was monitored throughout the entire procedure. Activities of the respiratory chain complexes and the ratio of aconitase to fumarase activities were determined before ischemia and at the end of reperfusion. H2O2 release was also evaluated in isolated mitochondria. During ischemia, middle-aged hearts displayed a delayed contracture, suggesting a maintained ATP production but also an increased metabolic proton production. Restoration of the mechanical function during reperfusion was however reduced in the middle-aged hearts, due to lower recovery of the coronary flow associated with higher mitochondrial oxidative stress indicated by the aconitase to fumarase ratio in the cardiac tissues. Surprisingly, activity of the respiratory chain complex II was better maintained in the hearts of middle-aged animals, probably because of an enhanced preservation of its membrane lipid environment. This can explain the higher mitochondrial oxidative stress observed in these conditions, since cardiac mitochondria produce much more H2O2 when they oxidize FADH2-linked substrates than when they use NADH-linked substrates. In conclusion, the lower restoration of the cardiac mechanical activity during reperfusion in the middle-aged hearts was due to an impaired recovery of the coronary flow and an insufficient oxygen supply. The deterioration of the coronary perfusion was explained by an increased mitochondrial ROS release related to the preservation of complex II activity during reperfusion.