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In vitro studies suggested that glucose metabolism through the oxidative pentose phosphate pathway (oxPPP) can paradoxically feed superoxide-generating enzymes in failing hearts. We therefore tested the hypothesis that acute inhibition of the oxPPP reduces oxidative stress and enhances function and metabolism of the failing heart, in vivo. In 10 chronically instrumented dogs, congestive heart failure (HF) was induced by high-frequency cardiac pacing. Myocardial glucose consumption was enhanced by raising arterial glycemia to levels mimicking postprandial peaks, before and after intravenous administration of the oxPPP inhibitor 6-aminonicotinamide (80 mg/kg). Myocardial energy substrate metabolism was measured with radiolabeled glucose and oleic acid, and cardiac 8-isoprostane output was used as an index of oxidative stress. A group of five chronically instrumented, normal dogs served as control. In HF, raising glycemic levels from ∼ 80 to ∼ 170 mg/dL increased cardiac isoprostane output by approximately twofold, whereas oxPPP inhibition normalized oxidative stress and enhanced cardiac oxygen consumption, glucose oxidation, and stroke work. In normal hearts glucose infusion did not induce significant changes in cardiac oxidative stress. Myocardial tissue concentration of 6P-gluconate, an intermediate metabolite of the oxPPP, was significantly reduced by ∼ 50% in treated versus nontreated failing hearts, supporting the inhibitory effect of 6-aminonicotinamide. Our study indicates an important contribution of the oxPPP activity to cardiac oxidative stress in HF, which is particularly pronounced during common physiological changes such as postprandial glycemic peaks.

Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as when cellular energy needs and energy production from mitochondria fatty acid β-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific perilipin 5 (Plin5) overexpression (MHC-Plin5), a member of the perilipin protein family. Hearts from MHC-Plin5 mice expressed at least 4-fold higher levels of plin5 and exhibited a 3.5-fold increase in triglyceride content versus nontransgenic littermates. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of peroxisome proliferator-activated receptor (PPAR)α target genes, decreased mitochondria function, and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative-induced genes via NF-E2-related factor 2 antioxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and it promotes cardiac steatosis without major heart function impairment.

Recently we described an ischemic preconditioning induced by repetitive coronary stenosis, which is induced by 6 episodes of non-lethal ischemia over 3 days, and which also resembles the hibernating myocardium phenotype. When compared with traditional second window of ischemic preconditioning using cDNA microarrays, many genes which differed in the repetitive coronary stenosis appeared targeted to metabolism. Accordingly, the goal of this study was to provide a more in depth analysis of changes in metabolism in the different models of delayed preconditioning, i.e., second window and repetitive coronary stenosis. This was accomplished using a metabolomic approach based on liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques. Myocardial samples from the ischemic section of porcine hearts subjected to both models of late preconditioning were compared against sham controls. Interestingly, although both models involve delayed preconditioning, their metabolic signatures were radically different; of the total number of metabolites that changed in both models (135 metabolites) only 7 changed in both models, and significantly more, p<0.01, were altered in the repetitive coronary stenosis (40%) than in the second window (8.1%). The most significant changes observed were in energy metabolism, e.g., phosphocreatine was increased 4 fold and creatine kinase activity increased by 27.2%, a pattern opposite from heart failure, suggesting that the repetitive coronary stenosis and potentially hibernating myocardium have enhanced stress resistance capabilities. The improved energy metabolism could also be a key mechanism contributing to the cardioprotection observed in the repetitive coronary stenosis and in hibernating myocardium. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism"., (Copyright © 2012. Published by Elsevier Ltd.)

Background: Glucose 6-phosphate dehydrogenase (G6PD) is the most common deficient enzyme in the world. In failing hearts, G6PD is upregulated and generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) that is used by the glutathione pathway to remove reactive oxygen species but also as a substrate by reactive oxygen species-generating enzymes. Therefore, G6PD deficiency might prevent heart failure by decreasing NADPH and reactive oxygen species production., Methods and Results: This hypothesis was evaluated in a mouse model of human G6PD deficiency (G6PDX mice, ≈40% normal activity). Myocardial infarction with 3 months follow-up resulted in left ventricular dilation and dysfunction in both wild-type and G6PDX mice but significantly greater end diastolic volume and wall thinning in G6PDX mice. Similarly, pressure overload induced by transverse aortic constriction (TAC) for 6 weeks caused greater left ventricular dilation in G6PDX mice than wild-type mice. We further stressed transverse aortic constriction mice by feeding a high fructose diet to increase flux through G6PD and reactive oxygen species production and again observed worse left ventricular remodeling and a lower ejection fraction in G6PDX than wild-type mice. Tissue content of lipid peroxidation products was increased in G6PDX mice in response to infarction and aconitase activity was decreased with transverse aortic constriction, suggesting that G6PD deficiency increases myocardial oxidative stress and subsequent damage., Conclusions: Contrary to our hypothesis, G6PD deficiency increased redox stress in response to infarction or pressure overload. However, we found only a modest acceleration of left ventricular remodeling, suggesting that, in individuals with G6PD deficiency and concurrent hypertension or myocardial infarction, the risk for developing heart failure is higher but limited by compensatory mechanisms.

In the advanced stages of heart failure, many key enzymes involved in myocardial energy substrate metabolism display various degrees of down-regulation. The net effect of the altered metabolic phenotype consists of reduced cardiac fatty oxidation, increased glycolysis and glucose oxidation, and rigidity of the metabolic response to changes in workload. Is this metabolic shift an adaptive mechanism that protects the heart or a maladaptive process that accelerates structural and functional derangement? The question remains open; however, the metabolic remodelling of the failing heart has induced a number of investigators to test the hypothesis that pharmacological modulation of myocardial substrate utilization might prove therapeutically advantageous. The present review addresses the effects of indirect and direct modulators of fatty acid (FA) oxidation, which are the best pharmacological agents available to date for 'metabolic therapy' of failing hearts. Evidence for the efficacy of therapeutic strategies based on modulators of FA metabolism is mixed, pointing to the possibility that the molecular/biochemical alterations induced by these pharmacological agents are more complex than originally thought. Much remains to be understood; however, the beneficial effects of molecules such as perhexiline and trimetazidine in small clinical trials indicate that this promising therapeutic strategy is worthy of further pursuit.

There is a constant high demand for energy to sustain the continuous contractile activity of the heart, which is met primarily by the beta-oxidation of long-chain fatty acids. The control of fatty acid beta-oxidation is complex and is aimed at ensuring that the supply and oxidation of the fatty acids is sufficient to meet the energy demands of the heart. The metabolism of fatty acids via beta-oxidation is not regulated in isolation; rather, it occurs in response to alterations in contractile work, the presence of competing substrates (i.e., glucose, lactate, ketones, amino acids), changes in hormonal milieu, and limitations in oxygen supply. Alterations in fatty acid metabolism can contribute to cardiac pathology. For instance, the excessive uptake and beta-oxidation of fatty acids in obesity and diabetes can compromise cardiac function. Furthermore, alterations in fatty acid beta-oxidation both during and after ischemia and in the failing heart can also contribute to cardiac pathology. This paper reviews the regulation of myocardial fatty acid beta-oxidation and how alterations in fatty acid beta-oxidation can contribute to heart disease. The implications of inhibiting fatty acid beta-oxidation as a potential novel therapeutic approach for the treatment of various forms of heart disease are also discussed.

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.

A high-fat diet can increase adiposity, leptin secretion, and plasma fatty acid concentration. In hypertension, this scenario may accelerate cardiac hypertrophy and development of heart failure but could be protective by activating peroxisome proliferator-activated receptors and expression of mitochondrial oxidative enzymes. We assessed the effects of a high-fat diet on the development of left ventricular hypertrophy, remodeling, contractile dysfunction, and the activity of mitochondrial oxidative enzymes. Mice (n = 10-12/group) underwent transverse aortic constriction (TAC) or sham surgery and were fed either a low-fat diet (10% of energy intake as fat) or a high-fat diet (45% fat) for 6 wk. The high-fat diet increased adipose tissue mass and plasma leptin and insulin. Left ventricular mass and chamber size were unaffected by diet in sham animals. TAC increased left ventricular mass (approximately 70%) and end-systolic and end-diastolic areas (approximately 100% and approximately 45%, respectively) to the same extent in both dietary groups. The high-fat diet increased plasma free fatty acid concentration and prevented the decline in the activity of the mitochondrial enzymes medium chain acyl-coenzyme A dehydrogenase (MCAD) and citrate synthase that was observed with TAC animals on a low-fat diet. In conclusion, a high-fat diet did not worsen cardiac hypertrophy or left ventricular chamber enlargement despite increases in fat mass and insulin and leptin concentrations. Furthermore, a high-fat diet preserved MCAD and citrate synthase activities during pressure overload, suggesting that it may help maintain mitochondrial oxidative capacity in failing myocardium.

When recovering from heart failure (HF), the myocardium displays a marked plasticity and can regain normal gene expression and function; however, recovery of substrate oxidation capacity has not been explored. We tested whether cardiac functional recovery is matched by normalization of energy substrate utilization during post-HF recovery. HF was induced in dogs by pacing the left ventricle (LV) at 210-240 beats/min for 4 wk. Tachycardia was discontinued, and the heart was allowed to recover. An additional group was studied in HF, and healthy dogs served as controls (n = 8/group). Cardiac free fatty acids (FFAs) and glucose oxidation were measured with [3H]oleate and [14C]glucose. At 10 days of recovery, hemodynamic parameters returned to control values; however, the contractile response to dobutamine remained depressed, LV end-diastolic volume was 28% higher than control, and the heart mass-to-body mass ratio was increased (9.8 +/- 0.4 vs. 7.5 +/- 0.2 g/kg, P < 0.05). HF increased glucose oxidation (76.8 +/- 19.7 nmol.min(-1).g(-1)) and decreased FFA oxidation (20.7 +/- 6.4 nmol.min(-1).g(-1)), compared with normal dogs (24.5 +/- 6.3 and 51.7 +/- 9.6 nmol.min(-1).g(-1), respectively), and reversed to normal values at 10 days of recovery (25.4 +/- 6.0 and 46.6 +/- 6.7 nmol.min(-1).g(-1), respectively). However, similar to HF, the recovered dogs failed to increase glucose and fatty acid uptake in response to pacing stress. The activity of myocardial citrate synthase and aconitase was significantly decreased during recovery compared with that in control dogs (58 and 27% lower, respectively, P < 0.05), indicating a persistent reduction in mitochondrial oxidative capacity. In conclusion, cardiac energy substrate utilization is normalized in the early stage of post-HF recovery at baseline, but not under stress conditions.

The malate-aspartate (M-A) shuttle provides an important mechanism to regulate glycolysis and lactate metabolism in the heart by transferring reducing equivalents from cytosol into mitochondria. However, experimental characterization of the M-A shuttle has been incomplete because of limitations in quantifying cytosolic and mitochondrial metabolites. In this study, we developed a multi-compartment model of cardiac metabolism with detailed presentation of the M-A shuttle to quantitatively predict non-observable fluxes and metabolite concentrations under normal and ischemic conditions in vivo. Model simulations predicted that the M-A shuttle is functionally localized to a subdomain that spans the mitochondrial and cytosolic spaces. With the onset of ischemia, the M-A shuttle flux rapidly decreased to a new steady state in proportion to the reduction in blood flow. Simulation results suggest that the reduced M-A shuttle flux during ischemia was not due to changes in shuttle-associated enzymes and transporters. However, there was a redistribution of shuttle-associated metabolites in both cytosol and mitochondria. Therefore, the dramatic acceleration in glycolysis and the switch to lactate production that occur immediately after the onset of ischemia is mediated by reduced M-A shuttle flux through metabolite redistribution of shuttle associated species across the mitochondrial membrane.

Whole body protein synthesis is reduced during the fed-to-fasted transition and in cases of chronic dietary restriction; however, less is known about tissue-specific alterations. We have assessed the extent to which protein synthesis in cardiac muscle responds to dietary perturbations compared with liver and skeletal muscle by applying a novel (2)H(2)O tracer method to quantify tissue-specific responses of protein synthesis in vivo. We hypothesized that protein synthesis in cardiac muscle would be unaffected by acute fasting or food restriction, whereas protein synthesis in the liver and gastrocnemius muscle would be reduced when there is a protein-energy deficit. We found that, although protein synthesis in liver and gastrocnemius muscle was significantly reduced by acute fasting, there were no changes in protein synthesis in the left ventricle of the heart for either the total protein pool or in isolated mitochondrial or cytosolic compartments. Likewise, a chronic reduction in calorie intake, induced by food restriction, did not affect protein synthesis in the heart, whereas protein synthesis in skeletal muscle and liver was decreased. The later observations are supported by changes in the phosphorylation state of two critical mediators of protein synthesis (4E-BP1 and eIF2alpha) in the respective tissues. We conclude that cardiac protein synthesis is maintained in cases of nutritional perturbations, in strong contrast to liver and gastrocnemius muscle, where protein synthesis is decreased by acute fasting or chronic food restriction.

The heart adapts the rate of mitochondrial ATP production to energy demand without noticeable changes in the concentration of ATP, ADP and Pi, even for large transitions between different workloads. We suggest that the changes in demand modulate the cytosolic Ca2+ concentration that changes mitochondrial Ca2+ to regulate ATP production. Thus, the rate of ATP production by the mitochondria is coupled to the rate of ATP consumption by the sarcomere cross-bridges (XBs). An integrated model was developed to couple cardiac metabolism and mitochondrial ATP production with the regulation of Ca2+ transient and ATP consumption by the sarcomere. The model includes two interrelated systems that run simultaneously utilizing two different integration steps: (1) The faster system describes the control of excitation contraction coupling with fast cytosolic Ca2+ transients, twitch mechanical contractions, and associated fluctuations in the mitochondrial Ca2+. (2) A slower system simulates the metabolic system, which consists of three different compartments: blood, cytosol, and mitochondria. The basic elements of the model are dynamic mass balances in the different compartments. Cytosolic Ca2+ handling is determined by four organelles: sarcolemmal Ca2+ influx and efflux; sarcoplasmic reticulum (SR) Ca2+ release and sequestration (SR); binding and dissociation from sarcomeric regulatory troponin complexes; and mitochondrial Ca2+ flows. Mitochondrial Ca2+ flows are determined by the Ca2+ uniporter and the mitochondrial Na+Ca2+ exchanger. The cytosolic Ca2+ determines the rate of ATP consumption by the sarcomere. Ca2+ binding to troponin regulates the rate of XBs recruitment and force development. The mitochondrial Ca2+ concentration determines the pyruvate dehydrogenase activity and the rate of ATP production by the F(1)-F(0) ATPase. The workload modulates the cytosolic Ca2+ concentration through feedback loops. The preload and afterload affect the number of strong XBs. The number of strong XBs determines the affinity of troponin for Ca2+, which alters the cytosolic Ca2+ transient. Model simulations quantify the role of Ca2+ in simultaneously controlling the power of contraction and the rate of ATP production. It explains the established empirical observation that significant changes in the metabolic fluxes can occur without significant changes in the key nucleotide (ATP and ADP) concentrations. Quantitative investigations of the mechanisms underlying the cardiac control of biochemical to mechanical energy conversion may lead to novel therapeutic modalities for the ischemic and failing myocardium.

Inhibition of myocardial fatty acid oxidation can improve left ventricular (LV) mechanical efficiency by increasing LV power for a given rate of myocardial energy expenditure. This phenomenon has not been assessed at high workloads in nonischemic myocardium; therefore, we subjected in vivo pig hearts to a high workload for 5 min and assessed whether blocking mitochondrial fatty acid oxidation with the carnitine palmitoyltransferase-I inhibitor oxfenicine would improve LV mechanical efficiency. In addition, the cardiac content of malonyl-CoA (an endogenous inhibitor of carnitine palmitoyltransferase-I) and activity of acetyl-CoA carboxylase (which synthesizes malonyl-CoA) were assessed. Increased workload was induced by aortic constriction and dobutamine infusion, and LV efficiency was calculated from the LV pressure-volume loop and LV energy expenditure. In untreated pigs, the increase in LV power resulted in a 2.5-fold increase in fatty acid oxidation and cardiac malonyl-CoA content but did not affect the activation state of acetyl-CoA carboxylase. The activation state of the acetyl-CoA carboxylase inhibitory kinase AMP-activated protein kinase decreased by 40% with increased cardiac workload. Pretreatment with oxfenicine inhibited fatty acid oxidation by 75% and had no effect on cardiac energy expenditure but significantly increased LV power and LV efficiency (37 +/- 5% vs. 26 +/- 5%, P < 0.05) at high workload. In conclusion, 1) myocardial fatty acid oxidation increases with a short-term increase in cardiac workload, despite an increase in malonyl-CoA concentration, and 2) inhibition of fatty acid oxidation improves LV mechanical efficiency by increasing LV power without affecting cardiac energy expenditure.

The intracellular circadian clock consists of a series of transcriptional modulators that together allow the cell to perceive the time of day. Circadian clocks have been identified within various components of the cardiovascular system (e.g. cardiomyocytes, vascular smooth muscle cells) and possess the potential to regulate numerous aspects of cardiovascular physiology and pathophysiology. The present study tested the hypothesis that ischemia/reperfusion (I/R; 30 min occlusion of the rat left main coronary artery in vivo) alters the circadian clock within the ischemic, versus non-ischemic, region of the heart. Left ventricular anterior (ischemic) and posterior (non-ischemic) regions were isolated from I/R, sham-operated, and naïve rats over a 24-h period, after which mRNAs encoding for both circadian clock components and known clock-controlled genes were quantified. Circadian clock gene oscillations (i.e. peak-to-trough fold differences) were rapidly attenuated in the I/R, versus the non-ischemic, region. Consistent with decreased circadian clock output, we observe a rapid induction of E4BP4 in the ischemic region of the heart at both the mRNA and protein levels. In contrast with I/R, chronic (1 week) hypobaric chamber-induced hypoxia did not attenuate oscillations in circadian clock genes in either the left or right ventricle of the rat heart. In conclusion, these data show that in a rodent model of myocardial I/R, circadian clocks within the ischemic region become rapidly impaired, through a mechanism that appears to be independent of hypoxia.

Under resting conditions, the failing heart shifts fuel use toward greater glucose and lower free fatty acid (FFA) oxidation. We hypothesized that chronic metabolic abnormalities in patients with dilated cardiomyopathy (DCM) are associated with the absence of the normal increase in myocardial glucose uptake and maintenance of cardiac mechanical efficiency in response to pacing stress. In 10 DCM patients and 6 control subjects, we measured coronary flow by intravascular ultrasonometry and sampled arterial and coronary sinus blood. Myocardial metabolism was determined at baseline, during atrial pacing at 130 beats/min, and at 15 min of recovery by infusion of [(3)H]oleate and [(13)C]lactate and measurement of transmyocardial arteriovenous differences of oxygen and metabolites. At baseline, DCM patients showed depressed coronary flow, reduced uptake and oxidation of FFA, and preferential utilization of carbohydrates. During pacing, glucose uptake increased by 106% in control subjects but did not change from baseline in DCM patients. Lactate release increased by 122% in DCM patients but not in control subjects. Cardiac mechanical efficiency in DCM patients was not different compared with control subjects at baseline but was 34% lower during stress. Fatty acid uptake and oxidation did not change with pacing in either group. Our results show that in DCM there is preferential utilization of carbohydrates, which is associated with reduced flow and oxygen consumption at rest and an impaired ability to increase glucose uptake during stress. These metabolic abnormalities might contribute to progressive cardiac deterioration and represent a target for therapeutic strategies aimed at modulating cardiac substrate utilization.

Obesity results in marked alterations in cardiac energy metabolism, with a prominent effect being an increase in fatty acid uptake and oxidation by the heart. Obesity also results in dramatic changes in the release of adipokines, such as leptin and adiponectin, both of which have emerged as important regulators of cardiac energy metabolism. The link among obesity, cardiovascular disease, lipid metabolism, and adipokine signaling is complex and not well understood. However, optimizing cardiac energy metabolism in obese subjects may be one approach to preventing and treating cardiac dysfunction that can develop in this population. This review discusses what is presently known about the effects of obesity and the impact adipokines have on cardiac energy metabolism and insulin signaling. The clinical implications of obesity and energy metabolism on cardiac disease are also discussed.

Severe heart failure (HF) is characterized by profound alterations in cardiac metabolic phenotype, with down-regulation of the free fatty acid (FFA) oxidative pathway and marked increase in glucose oxidation. We tested whether fenofibrate, a pharmacological agonist of peroxisome proliferator-activated receptor-alpha, the nuclear receptor that activates the expression of enzymes involved in FFA oxidation, can prevent metabolic alterations and modify the progression of HF. We administered 6.5 mg/kg/day p.o. fenofibrate to eight chronically instrumented dogs over the entire period of high-frequency left ventricular pacing (HF + Feno). Eight additional HF dogs were not treated, and eight normal dogs were used as a control. [3H]Oleate and [14C]Glucose were infused intravenously to measure the rate of substrate oxidation. At 21 days of pacing, left ventricular end-diastolic pressure was significantly lower in HF + Feno (14.1 +/- 1.6 mm Hg) compared with HF (18.7 +/- 1.3 mm Hg), but it increased up to 25 +/- 2 mm Hg, indicating end-stage failure, in both groups after 29 +/- 2 days of pacing. FFA oxidation was reduced by 40%, and glucose oxidation was increased by 150% in HF compared with control, changes that were prevented by fenofibrate. Consistently, the activity of myocardial medium chain acyl-CoA dehydrogenase, a marker enzyme of the FFA beta-oxidation pathway, was reduced in HF versus control (1.46 +/- 0.25 versus 2.42 +/- 0.24 micromol/min/gram wet weight (gww); p < 0.05) but not in HF + Feno (1.85 +/- 0.18 micromol/min/gww; N.S. versus control). Thus, preventing changes in myocardial substrate metabolism in the failing heart causes a modest improvement of cardiac function during the progression of the disease, with no effects on the onset of decompensation.

Steady state concentrations of ATP and ADP in vivo are similar at low and high cardiac workloads; however, the mechanisms that regulate the activation of substrate metabolism and oxidative phosphorylation that supports this stability are poorly understood. We tested the hypotheses that (1) there is parallel activation of mitochondrial and cytosolic dehydrogenases in the transition from low to high workload, which increases NADH/NAD+ ratio in both compartments, and (2) this response does not require an increase in fatty acid oxidation (FAO). Anaesthetized pigs were subjected to either sham treatment, or an abrupt increase in cardiac workload for 5 min with dobutamine infusion and aortic constriction. Myocardial oxygen consumption and FAO were increased 3- and 2-fold, respectively, but ATP and ADP concentrations did not change. NADH-generating pathways were rapidly activated in both the cytosol and mitochondria, as seen in a 40% depletion in glycogen stores, a 3.6-fold activation of pyruvate dehydrogenase, and a 50% increase in tissue NADH/NAD+. Simulations from a multicompartmental computational model of cardiac energy metabolism predicted that parallel activation of glycolysis and mitochondrial metabolism results in an increase in the NADH/NAD+ ratio in both cytosol and mitochondria. FAO was blocked by 75% in a third group of pigs, and a similar increase in and the NAHD/NAD+ ratio was observed. In conclusion, in the transition to a high cardiac workload there is rapid parallel activation of substrate oxidation that results in an increase in the NADH/NAD+ ratio.

Background: Inhibition of myocardial fatty acid oxidation has been suggested as a therapeutic approach for improving cardiac function in chronic heart failure (HF). The novel piperazine derivative CVT-4325 was shown to inhibit fatty acid oxidation in cardiac mitochondria and in isolated perfused rat hearts. In the present study, we tested the hemodynamic and metabolic effects of acute intravenous CVT-4325 in dogs with HF., Methods and Results: HF (LV ejection fraction

1. Alterations in myocardial energy metabolism accompany pressure overload-induced hypertrophy. We previously described a novel model of catecholamine-induced hypertrophy in which A/J mice exhibit more robust cardiac hypertrophy than B6 mice. Accordingly, we assessed the influence of mouse strain on the activities of key myocardial metabolic enzymes and whether there are strain-related metabolic adaptations to short-term, high-dose isoproterenol (ISO) administration. 2. Thirty-nine male mice (19 A/J mice, 20 B6 mice), aged 12-15 weeks, were randomly assigned to receive either ISO (100 mg/kg, s.c.) or vehicle (sterile water) daily for 5 days. On Day 6, all hearts were excised, weighed, freeze clamped and assayed for pyruvate dehydrogenase (PDH), medium chain acyl-CoA dehydrogenase, carnitine palmitoyl transferase I and citrate synthase activities. Plasma fatty acids (FA) were also measured. 3. The ISO-treated A/J mice demonstrated greater percentage increases in gravimetric heart weight/bodyweight ratio than ISO-treated B6 mice (24 vs 3%, respectively; P < 0.001). All enzyme activities were significantly greater in vehicle-treated B6 mice than in A/J mice, illustrating a greater capacity for aerobic metabolism in B6 mice. Administration of ISO reduced PDHa (active form) activity in B6 mice by 47% (P < 0.001), with no significant change seen in A/J mice. Free FA levels were not significantly different between groups; thus, the differences in PDHa were not due to changes in FA. 4. The basal activity of myocardial metabolic enzymes is greater in B6 mice than in A/J mice and ISO alters myocardial PDH activity in a mouse strain-dependent manner. Compared with A/J mice, B6 mice demonstrate less ISO-induced cardiac hypertrophy, but greater activity of key enzymes regulating FA and carbohydrate oxidation, which may protect against the development of hypertrophy. The metabolic adaptations associated with ISO-induced hypertrophy differ from those reported with pressure overload hypertrophy.

In response to exercise, the heart increases its metabolic rate severalfold while maintaining energy species (e.g., ATP, ADP, and Pi) concentrations constant; however, the mechanisms that regulate this response are unclear. Limited experimental studies show that the classic regulatory species NADH and NAD+ are also maintained nearly constant with increased cardiac power generation, but current measurements lump the cytosol and mitochondria and do not provide dynamic information during the early phase of the transition from low to high work states. In the present study, we modified our previously published computational model of cardiac metabolism by incorporating parallel activation of ATP hydrolysis, glycolysis, mitochondrial dehydrogenases, the electron transport chain, and oxidative phosphorylation, and simulated the metabolic responses of the heart to an abrupt increase in energy expenditure. Model simulations showed that myocardial oxygen consumption, pyruvate oxidation, fatty acids oxidation, and ATP generation were all increased with increased energy expenditure, whereas ATP and ADP remained constant. Both cytosolic and mitochondrial NADH/NAD+ increased during the first minutes (by 40% and 20%, respectively) and returned to the resting values by 10-15 min. Furthermore, model simulations showed that an altered substrate selection, induced by either elevated arterial lactate or diabetic conditions, affected cytosolic NADH/NAD+ but had minimal effects on the mitochondrial NADH/NAD+, myocardial oxygen consumption, or ATP production. In conclusion, these results support the concept of parallel activation of metabolic processes generating reducing equivalents during an abrupt increase in cardiac energy expenditure and suggest there is a transient increase in the mitochondrial NADH/NAD+ ratio that is independent of substrate supply.

Acute inhibition of nitric oxide (NO) synthase causes a reversible alteration in myocardial substrate metabolism. We tested the hypothesis that prolonged NO synthase inhibition alters cardiac metabolic phenotype. Seven chronically instrumented dogs were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1) po) for 10 days to inhibit NO synthesis, and seven were used as controls. Cardiac free fatty acid, glucose, and lactate oxidation were measured by infusion of [(3)H]oleate, [(14)C]glucose, and [(13)C]lactate, respectively. After 10 days of L-NAME administration, despite no differences in left ventricular afterload, cardiac O(2) consumption was significantly increased by 30%, consistent with a marked enhancement in baseline oxidation of glucose (6.9 +/- 2.0 vs. 1.7 +/- 0.5 micromol.min(-1).100 g(-1), P < 0.05 vs. control) and lactate (21.6 +/- 5.6 vs. 11.8 +/- 2.6 micromol.min(-1).100 g(-1), P < 0.05 vs. control). When left ventricular afterload was increased by ANG II infusion to stimulate myocardial metabolism, glucose oxidation was augmented further in the L-NAME than in the control group, whereas free fatty acid oxidation decreased. Exogenous NO (diethylamine nonoate, 0.01 micromol.kg(-1).min(-1) iv) could not reverse this metabolic alteration. Consistent with the accelerated rate of carbohydrate oxidation, total myocardial pyruvate dehydrogenase activity and protein expression were higher (38 and 34%, respectively) in the L-NAME than in the control group. Also, protein expression of the constitutively active glucose transporter GLUT-1 was significantly elevated (46%) vs. control. We conclude that prolonged NO deficiency causes a profound alteration in cardiac metabolic phenotype, characterized by selective potentiation of carbohydrate oxidation, that cannot be reversed by a short-term infusion of exogenous NO. This phenomenon may constitute an adaptive mechanism to counterbalance cardiac mechanical inefficiency.

In the normal heart, there is loss of citric acid cycle (CAC) intermediates that is matched by the entry of intermediates from outside the cycle, a process termed anaplerosis. Previous in vitro studies suggest that supplementation with anaplerotic substrates improves cardiac function during myocardial ischemia and/or reperfusion. The present investigation assessed whether treatment with the anaplerotic medium-chain fatty acid heptanoate improves contractile function during ischemia and reperfusion. The left anterior descending coronary artery of anesthetized pigs was subjected to 60 min of 60% flow reduction and 30 min of reperfusion. Three treatment groups were studied: saline control, heptanoate (0.4 mM), or hexanoate as a negative control (0.4 mM). Treatment was initiated after 30 min of ischemia and continued through reperfusion. Myocardial CAC intermediate content was not affected by ischemia-reperfusion; however, treatment with heptanoate resulted in a more than twofold increase in fumarate and malate, with no change in citrate and succinate, while treatment with hexanoate did not increase fumarate or malate but increased succinate by 1.8-fold. There were no differences among groups in lactate exchange, glucose oxidation, oxygen consumption, and contractile power. In conclusion, despite a significant increase in the content of carbon-4 CAC intermediates, treatment with heptanoate did not result in improved mechanical function of the heart in this model of reversible ischemia-reperfusion. This suggests that reduced anaplerosis and CAC dysfunction do not play a major role in contractile and metabolic derangements observed with a 60% decrease in coronary flow followed by reperfusion.

Myocardial fatty acid oxidation is regulated by carnitine palmitoyltransferase I (CPT I), which is inhibited by malonyl-CoA. Increased cardiac power causes a fall in malonyl-CoA content and accelerated fatty acid oxidation; however, the mechanism for the decrease in malonyl-CoA is unclear. Malonyl-CoA is formed by acetyl-CoA carboxylase (ACC) and degraded by malonyl-CoA decarboxylase (MCD); thus a fall in malonyl-CoA could be due to activation of MCD, inhibition of ACC, or both. This study assessed the effects of increased cardiac power on malonyl-CoA content and ACC and MCD activities. Anesthetized pigs were studied under control conditions and during increased cardiac power in response to dobutamine infusion and aortic constriction alone, under hyperglycemic conditions, or with the CPT I inhibitor oxfenicine. An increase in cardiac power was accompanied by increased myocardial O(2) consumption, decreased malonyl-CoA concentration, and increased fatty acid oxidation. There were no differences among groups in activity of ACC or AMP-activated protein kinase (AMPK), which physiologically inhibits ACC. There also were no differences in V(max) or K(m) of MCD. Previous studies have demonstrated that AMPK can be inhibited by protein kinase B (PKB); however, PKB was activated by dobutamine and the elevated insulin that accompanied hyperglycemia, but there was no effect on AMPK activity. In conclusion, the fall in malonyl-CoA and increase in fatty acid oxidation that occur with increased cardiac work were not due to inhibition of ACC or activation of MCD, suggesting alternative regulatory mechanisms for the work-induced decrease in malonyl-CoA concentration.

The alterations in myocardial energy substrate metabolism that occur in heart failure, and the causes and consequences of these abnormalities, are poorly understood. There is evidence to suggest that impaired substrate metabolism contributes to contractile dysfunction and to the progressive left ventricular remodeling that are characteristic of the heart failure state. The general concept that has recently emerged is that myocardial substrate selection is relatively normal during the early stages of heart failure; however, in the advanced stages there is a downregulation in fatty acid oxidation, increased glycolysis and glucose oxidation, reduced respiratory chain activity, and an impaired reserve for mitochondrial oxidative flux. This review discusses 1) the metabolic changes that occur in chronic heart failure, with emphasis on the mechanisms that regulate the changes in the expression of metabolic genes and the function of metabolic pathways; 2) the consequences of these metabolic changes on cardiac function; 3) the role of changes in myocardial substrate metabolism on ventricular remodeling and disease progression; and 4) the therapeutic potential of acute and long-term manipulation of cardiac substrate metabolism in heart failure.

Nitric oxide (NO) inhibits myocardial glucose transport and metabolism, although the underlying mechanism(s) and functional consequences of this effect are not clearly understood. We tested the hypothesis that NO inhibits the activation of AMP-activated protein kinase (AMPK) and translocation of cardiac glucose transporters (GLUTs; GLUT-4) and reduces lactate production. Ischemia was induced in open-chest dogs by a 66% flow reduction in the left anterior descending coronary artery (LAD). During ischemia, dogs were untreated (control) or treated by direct LAD infusion of (i) nitroglycerin (NTG) (0.5 microg.kg(-1).min(-1)); (ii) 8-Br-cGMP (50 microg.kg(-1).min(-1)); or (iii) NO synthase inhibitor L-nitro-argininemethylester (40 microg.kg(-1).min(-1); n = 9 per group). Cardiac substrate oxidation was measured with isotopic tracers. There were no differences in myocardial blood flow or oxygen delivery among groups; however, at 45 min of ischemia, the activation of AMPK was significantly less in NTG (77 +/- 12% vs. nonischemic myocardium) and 8-Br-cGMP (104 +/- 13%), compared with control (167 +/- 17%). Similarly, GLUT-4 translocation was significantly reduced in NTG (74 +/- 7%) and 8-Br-cGMP (120 +/- 11%), compared with control (165 +/- 17%). Glucose uptake and lactate output were 30% and 60% lower in NTG compared with control. Inhibition of NO synthesis stimulated glucose oxidation (67% increase compared with control) but did not affect AMPK phosphorylation, GLUT-4 translocation and glucose uptake. Contractile function in the ischemic region was significantly improved by NTG and L-nitro-argininemethylester. In conclusion, in ischemic myocardium an NO donor inhibits glucose uptake and lactate production via a reduction in AMPK stimulation of GLUT-4 translocation, revealing a mechanism of metabolic modulation and myocardial protection activated by NO donors.

Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of free fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,10-(3)H]oleate, [U-(14)C]glucose, and [1-(13)C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity for malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomenon.

The primary effect of ischemia is reduced aerobic adenosine triphosphate (ATP) formation in mitochondria. This triggers accelerated glycolysis and reduced cell pH, Ca(2+) accumulation, K(+) efflux, adenosine formation, and the clinical signs of ischemia: chest pain and a shift in the ST segment. Traditional therapies for angina are aimed at either decreasing the need for ATP by suppressing heart rate, blood pressure, and cardiac contractility, or at increasing oxygen delivery to the mitochondria, or both. An additional approach to treating angina is to suppress myocardial fatty acid oxidation, increase pyruvate oxidation, and reduce anaerobic glycolysis. High fatty acid levels result in oxygen wasting and inhibit the oxidation of pyruvate in the mitochondria. In experimental models, the partial inhibition of myocardial fatty acid oxidation with agents such as oxfenicine, ranolazine, and trimetazidine stimulates glucose oxidation and reduces lactate production during ischemia. Clinical studies demonstrate that this approach is as effective as traditional hemodynamic therapies at improving exercise tolerance and reducing the frequency of angina. Moreover, because these agents do not suppress heart rate, blood pressure, or contractility, they are effective as add-on therapy to Ca(2+)-channel and beta-adrenergic receptor antagonists.

This study tested the robustness of our computational model of myocardial metabolism by comparing responses to two different inputs with experimental data obtained in pigs under similar conditions. Accordingly, an abrupt and a gradual reduction in coronary flow of similar magnitude were implemented and used as model input. After flow reductions reached 60% from control values, ischemia was kept constant for 60 min in both groups. Our hypotheses were that: (1) these two flow-reduction profiles would result in different transients (concentrations and flux rates) while having similar steady-state values and (2) our model-simulated responses would predict the experimental results in an anesthetized swine model of myocardial ischemia. The two different ischemia-induction patterns resulted in the same decrease in steady-state MVO2 and in similar steady-state values for metabolite concentrations and flux rates at 60 min of ischemia. While both the simulated and experimental results showed decreased glycogen concentration, accumulation of lactate, and net lactate release with ischemia, the onset of glycogen depletion and the switch to lactate efflux were more rapid in the experiments than in the simulations. This study demonstrates the utility of computer models for predicting experimental outcomes in studies of metabolic regulation under physiological and pathological conditions.

The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH(13)CO(3). This turnover is a direct measurement of the flux of acetyl-CoA carboxylation in the intact heart. It also reflects the rate of malonyl-CoA decarboxylation, i.e. the only known fate of malonyl-CoA in the heart. Conditions were selected to result in stable malonyl-CoA concentrations ranging from 1.5 to 5 nmol.g wet weight-(1). The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol.min(-) (1).g wet weight(-1) (slope = 0.98, r(2) = 0.94). The V(max) activities of acetyl-CoA carboxylase and of malonyl-CoA decarboxylase exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. However, conditions of perfusion that increased acetyl-CoA supply resulted in higher turnover and concentration, demonstrating that malonyl-CoA turnover is regulated by the supply of acetyl-CoA. The only condition where the activity of malonyl-CoA decarboxylase regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, resulting in increased malonyl-CoA concentration and decreased turnover. Our data show that, in the absence of enzyme inhibitors, the rate of acetyl-CoA carboxylation is the main determinant of the malonyl-CoA concentration in the heart.

Background: A computational model of myocardial energy metabolism was used to assess the metabolic responses to normal and reduced myocardial blood flow. The goal was to examine to what extent glycolysis and lactate formation are controlled by the supply of glycolytic substrate and/or the cellular redox (NADH/NAD+) and phosphorylation (ATP/ADP) states., Methods: Flow was reduced over a wide range and for a sufficient duration in order to investigate the sequence of events that occur during the transition to a new metabolic steady state., Results: Simulation results indicated multiple time-dependent controls over both glycolysis and lactate formation., Conclusions: Changes in phosphorylation state and glucose uptake only significantly affect the initial phase of the glycolytic response to ischemia, while glycogen breakdown exerts control over glycolysis during the entire duration of ischemia. Similarly, changes in the redox state affect the rates of lactate formation and release primarily during the initial transient phase of the response to the reductions in blood flow, while the rate of glycolysis controls the rate of lactate formation throughout the entire period of adaptation.

Abnormally high rates of fatty acid oxidation and low rates of glucose oxidation are important contributors to the severity of ischemic heart disease. Malonyl coenzyme A (CoA) regulates fatty acid oxidation by inhibiting mitochondrial uptake of fatty acids. Malonyl CoA decarboxylase (MCD) is involved in the decarboxylation of malonyl CoA to acetyl CoA. Therefore, inhibition of MCD may decrease fatty acid oxidation and protect the ischemic heart, secondary to increasing malonyl CoA levels. Ex vivo working rat hearts aerobically perfused in the presence of newly developed MCD inhibitors showed an increase in malonyl CoA levels, which was accompanied by both a significant decrease in fatty acid oxidation rates and an increase in glucose oxidation rates compared with controls. Using a model of demand-induced ischemia in pigs, MCD inhibition significantly increased glucose oxidation rates and reduced lactate production compared with vehicle-treated hearts, which was accompanied by a significant increase in cardiac work compared with controls. In a more severe rat heart global ischemia/reperfusion model, glucose oxidation was significantly increased and cardiac function was significantly improved during reperfusion in hearts treated with the MCD inhibitor compared with controls. Together, our data show that MCD inhibitors, which increase myocardial malonyl CoA levels, decrease fatty acid oxidation and accelerate glucose oxidation in both ex vivo rat hearts and in vivo pig hearts. This switch in energy substrate preference improves cardiac function during and after ischemia, suggesting that pharmacological inhibition of MCD may be a novel approach to treating ischemic heart disease.

Evidence indicates that nitric oxide (NO) suppresses myocardial oxygen consumption (MVO(2)) and regulates myocardial substrate oxidation, however data from in vivo and isolated heart preparations are conflicting. In addition, cardiac endothelin (ET-1) release has been shown to increase with inhibition of NO synthase (NOS), however the effects of ET-1 on myocardial energetics is not clear. We employed the isolated rat heart model to assess the role of NO and ET-1 on myocardial function and metabolism. Oxidation of glucose and FFA was measured using [U-(14)C]glucose and [9,10-(3)H]palmitate. NOS inhibition with N(G)-methyl-L-arginine acetate salt (L-NMMA, 50 microM), resulted in an increase in MVO(2) at a given rate of myocardial external workload, and no change in myocardial glucose or FFA oxidation. ET-1 (25 pM), which caused coronary vasoconstriction similar to that produced by L-NMMA, also increased MVO(2) without an effect on cardiac workload, or substrate oxidation, suggesting a role for ET-I in the regulation of myocardial energetics. We assessed also the effect of ET(A)/ET(B) receptor blockade (tezosentan; 5 nM) on MVO(2) and glucose and FFA oxidation and observed no effect, suggesting that basal ET-1 production does not play a role in regulating MVO2 or substrate selection. In conclusion, inhibition of NOS or the addition of ET-1 resulted in an increase in MVO2, but did not affect glucose or FFA oxidation.

This study tested the hypothesis that an acute infusion of beta-hydroxybutyrate inhibits myocardial fatty acid uptake and oxidation in vivo. Anesthetized pigs were untreated (n = 6) or treated with an intravenous infusion of fat emulsion (n = 7) to elevate plasma free fatty acid levels. A third group received fat emulsion plus an intravenous infusion of beta-hydroxybutyrate (25 micromol.kg-1.min-1; n = 7) for 60 min. All animals received a continuous infusion of [3H]palmitate, and myocardial fatty acid oxidation was measured from the cardiac production of 3H2O. Plasma free fatty acid concentrations were elevated in the fat emulsion group (0.77 +/- 0.11 mM) compared with the untreated group (0.15 +/- 0.03 mM), which resulted in greater myocardial free fatty acid oxidation. In contrast, the group receiving beta-hydroxybutyrate in addition to fat emulsion had elevated beta-hydroxybutyrate concentration (0.87 +/- 0.11 vs. 0.04 +/- 0.01 mM), but suppressed fatty acid oxidation (0.053 +/- 0.013 micromol.g-1.min-1) (P < 0.05) compared with the fat emulsion group (0.116 +/- 0.029 micromol.g-1.min-1). There were no differences among the three groups in the tissue content for malonyl-CoA, acetyl-CoA, or free CoA or the activity of acetyl-CoA carboxylase; thus the inhibition of fatty acid oxidation by elevated beta-hydroxybutyrate did not appear to be due to malonyl-CoA inhibition of carnitine palmitoyl transferase-I or to an increase in the acetyl-CoA-to-free CoA ratio. In conclusion, fatty acid uptake and oxidation is blocked by an infusion of beta-hydroxybutyrate; this effect was not due to elevated myocardial malonyl-CoA content.

While a number of studies underline the importance of anaplerotic pathways for hepatic biosynthetic functions and cardiac contractile activity, much remains to be learned about the sites and regulation of anaplerosis in these tissues. As part of a study on the regulation of anaplerosis from propionyl-CoA precursors in rat livers and hearts, we investigated the degree of reversibility of the reactions of the propionyl-CoA pathway. Label was introduced into the pathway via NaH13CO3, [U-13C3]propionate, or [U-13C3]lactate + [U-13C3]pyruvate, under various concentrations of propionate. The mass isotopomer distributions of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA revealed that, in intact livers and hearts, (i) the propionyl-CoA carboxylase reaction is slightly reversible only at low propionyl-CoA flux, (ii) the methylmalonyl-CoA racemase reaction keeps the methylmalonyl-CoA enantiomers in isotopic equilibrium under all conditions tested, and (iii) the methylmalonyl-CoA mutase reaction is reversible, but its reversibility decreases as the flow of propionyl-CoA increases. The thermodynamic dis-equilibrium of the combined reactions of the propionyl-CoA pathway explains the effectiveness of anaplerosis from propionyl-CoA precursors such as heptanoate.

The primary result of myocardial ischaemia is reduced oxygen consumption and adenosine triphosphate (ATP) formation in the mitochondria, and accelerated anaerobic glycolysis, lactate accumulation and cell acidosis. Classic pharmacotherapy for demand-induced ischaemia is aimed at restoring the balance between ATP synthesis and breakdown by increasing the oxygen delivery (i.e. with long acting nitrates or Ca2+ channel antagonist) or by decreasing cardiac power by reducing blood pressure and heart rate (i.e. with beta-blocker or Ca2+ channel antagonist). Animal studies show that fatty acids are the primary mitochondrial substrate during moderate severity myocardial ischaemia, and that they inhibit the oxidation of carbohydrate and drive the conversion of pyruvate to lactate. Drugs that partially inhibit myocardial fatty acid oxidation increase carbohydrate oxidation, which results in reduced lactate production and a higher cell pH during ischaemia. Trimetazidine (1-[2,3,4-trimethoxibenzyl]-piperazine) is the first and only registered drug in this class, and is available in over 90 countries world-wide. Trimetazidine selectively inhibits the fatty acid beta-oxidation enzyme 3-keto-acyl-CoA dehydrogenase (3-KAT), and is devoid of any direct haemodynamic effects. In double-blind placebo-controlled trials trimetazidine significantly improved symptom-limited exercise performance in stable angina patients when used either as monotherapy or in combination with beta-blockers or Ca2+ channel antagonists. Given available evidence, trimetazidine is an excellent alternative to classic haemodynamic agents, and is unique in its ability to reduce symptoms of angina when used in patients resistant to a haemodynamic treatment as vasodilators, beta-blockers or Ca2+ channel antagonists.

Normal cardiac metabolism requires continuous replenishment (anaplerosis) of catalytic intermediates of the citric acid cycle. Little is known about the quantitative aspects of propionate as a substrate of in vivo anaplerosis; therefore, we measured the rate of propionate entry into the citric acid cycle in hearts of anesthetized pigs. [U-(13)C(3)]propionate (0.25 mM) was infused in a coronary artery branch for 1 h via an extracorporeal perfusion circuit, and cardiac biopsies were analyzed for the mass isotopomer distribution of citric acid cycle intermediates. Infusion of propionate did not affect myocardial oxygen consumption, heart rate, or contractile function. In the infused territory, propionate infusion did not affect uptake of glucose and lactate but decreased free fatty acid uptake by one-half (P < 0.05). Propionate extraction and uptake were 57.4 +/- 3.3% and 0.078 +/- 0.009 micromol x min(-1) x g(-1). Anaplerosis from propionate, calculated from the mass isotopomer distribution of succinate, accounted for 8.9 +/- 1.3% of the citric acid cycle flux. Propioylcarnitine release accounted for only 0.033 +/- 0.002% of propionate uptake. Methylcitrate did not accumulate. Thus administration of a low concentration of propionate appears to be a convenient and safe way to boost anaplerosis in the heart.

The main objective of this study is to determine the transmural distribution of extracellular matrix (ECM) collagen and maturation in non-diabetic and diabetic hearts. The Yucatan miniature swine heart ECM was analyzed in eight streptozotocin (STZ) induced diabetic pigs (Diabetic-Swine) and age matched normal control pigs (Nondiabetic-Swine). After 12 weeks of STZ induced diabetes, transmural biopsies were obtained from the left ventricular free wall divided into subendocardial, mid- and subepicardial layers. Collagen concentration and maturation were measured by RP-HPLC determination of hydroxyproline (Hyp) and content of hydroxylysylpyridinoline (HP) cross-links, respectively. Results showed a significant elevation in arterial glucose (P<0.05) and reduction in arterial plasma insulin levels in the Diabetic-Swine. Hyp concentration was significantly greater (P<0.05) in the subendocardial layers in both the Diabetic and Nondiabetic animals. The HP cross-link content was significantly greater (17%) in the Diabetic-swine subendocardial layer compared to Nondiabetic-Swine (P<0.05), but not in other layers. In summary, the accumulation and/or increase in HP cross-link content in the Diabetic-Swine subendocardial layer suggests that myocardial fibrosis may be greater in this specific region.

Background: The nuclear receptors peroxisome proliferator-activated receptor-alpha (PPARalpha) and retinoid X receptor alpha (RXRalpha) stimulate the expression of key enzymes of free fatty acid (FFA) oxidation. We tested the hypothesis that the altered metabolic phenotype of the failing heart involves changes in the protein expression of PPARalpha and RXRalpha., Methods and Results: Cardiac substrate uptake and oxidation were measured in 8 conscious, chronically instrumented dogs with decompensated pacing-induced heart failure and in 8 normal dogs by infusing 3 isotopically labeled substrates: 3H-oleate, 14C-glucose, and 13C-lactate. Although myocardial O2 consumption was not different between the 2 groups, the rate of oxidation of FFA was lower (2.8+/-0.6 versus 4.7+/-0.3 micromol x min(-1) x 100g(-1)) and of glucose was higher (4.6+/-1.0 versus 1.8+/-0.5 micromol x min(-1) x 100g(-1)) in failing compared with normal hearts (P<0.05). The rates of lactate uptake and lactate output were not significantly different between the 2 groups. In left ventricular tissue from failing hearts, the activity of 2 key enzymes of FFA oxidation was significantly reduced: carnitine palmitoyl transferase-I (0.54+/-0.04 versus 0.66+/-0.04 micromol x min(-1) x g(-1)) and medium chain acyl-coenzyme A dehydrogenase (MCAD; 1.8+/-0.1 versus 2.9+/-0.3 micromol x min(-1) x g(-1)). Consistently, the protein expression of MCAD and of RXRalpha were significantly reduced by 38% in failing hearts, but the expression of PPARalpha was not different. Moreover, there were significant correlations between the expression of RXRalpha and the expression and activity of MCAD., Conclusions: Our results provide the first evidence for a link between the reduced expression of RXRalpha and the switch in metabolic phenotype in severe heart failure.

Partial fatty acid oxidation inhibition is effective therapy for the treatment of chronic stable angina and is particularly useful in patients with persistent angina despite optimal traditional therapy. The heart derives most of its energy from the oxidation of fatty acids. Fatty acid oxidation strongly inhibits pyruvate oxidation in the mitochondria and the uptake and oxidation of glucose. The primary effect of demand-induced ischaemia is impaired aerobic formation of ATP in the mitochondria, resulting in activation of non-oxidative glycolysis and lactate production, despite a relatively high residual myocardial oxygen consumption and continued reliance on fatty acid oxidation. Traditional drugs for chronic stable angina act by reducing the use of ATP through suppression of heart rate and blood pressure or by increasing aerobic formation of ATP by increasing coronary blood flow. Partial inhibition of fatty acid oxidation increases glucose and pyruvate oxidation and decreases lactate production, resulting in higher pH and improved contractile function during ischaemia. These agents do not affect heart rate, coronary blood flow or arterial blood pressure. Clinical trials with ranolazine or trimetazidine, either alone or in combination with a Ca2+ channel antagonist or a beta-adrenergic receptor antagonist, have demonstrated reduced symptoms of exercise-induced angina.

The chronically failing heart has been shown to be metabolically abnormal, in both animal models and in patients. Little data are available on the rate of myocardial glucose, lactate and fatty acid metabolism and oxidation in heart failure patients, thus at present, it is not possible to draw definitive conclusions about cardiac substrate preference in the various stages and manifestations of the disease. Normal cardiac function is dependent on a constant resynthesis of ATP by oxidative phosphorylation in the mitochondria. The healthy heart gets 60-90% of its energy for oxidative phosphorylation from fatty acid oxidation, with the balance from lactate and glucose. There is some indication that compensated NYHA Class III heart failure patients have a significantly greater rate of lipid oxidation, and decreased glucose uptake and carbohydrate oxidation compared to healthy age-matched individuals, and that therapies that acutely switch the substrate of the heart away from fatty acids result in improvement in left ventricular function. Clinical studies using long-term therapy with beta-adrenergic receptor antagonists show improved left ventricular function that corresponds with a switch away from fatty acid oxidation towards more carbohydrate oxidation by the heart. These findings suggest that chronic manipulation of myocardial substrate oxidation toward greater carbohydrate oxidation and less fatty acid oxidation may improve ventricular performance and slow the progression of left ventricular dysfunction in heart failure patients. At present, this intriguing hypothesis requires further evaluation.

A moderate reduction in coronary blood flow results in decreased myocardial oxygen consumption, accelerated glycolysis, decreased pyruvate oxidation, and lactate accumulation. To quantitatively understand cardiac metabolism during ischemia, we have developed a mechanistic, mathematical model based on biochemical mass balances and reaction kinetics in cardiac cells. By numerical solution of model equations, computer simulations showed the dynamic responses in glucose, fatty acid, glucose-6-phosphate, glycogen, triglyceride, pyruvate, lactate, acetyl-CoA, and free-CoA as well as CO2, O2, phosphocreatine/creatine, nicotinamide adenine dinucleotide (reduced form)/nicotinamide adenine dinucleotide (oxidized form) (NADH/NAD+), and adenosine diphosphate/adenosine triphosphate (ADP/ATP). When myocardial ischemia was simulated by a 60% reduction in coronary blood flow, the model generated myocardial concentrations, uptakes, and fluxes that were consistent with experimental data from in vivo pig studies. After 60 min of ischemia the concentrations of glycogen, phosphocreatine, and ATP were decreased by 60%, 75%, and 50%, respectively. With the onset of ischemia, myocardial lactate concentration increased and the myocardium switched from net consumer to net producer of lactate. Our model predicted a rapid 13-fold increase in NADH/NAD+, but only a twofold increase in the ratio of acetyl-CoA to free-CoA. These findings are consistent with the concept that pyruvate oxidation is inhibited during ischemia partially by the rise in NADH/NAD+.

To test whether the acute reduction of nitric oxide (NO) synthesis causes changes in cardiac substrate metabolism and in the activity of key enzymes of fatty acid and glucose oxidation, we blocked NOS by giving N(omega)-nitro-L-arginine methyl ester (L-NAME; 35 mg/kg iv two times) to nine chronically instrumented dogs. [3H]oleate, [14C]glucose, and [13C]lactate were infused to measure the rate of cardiac substrate uptake and oxidation. Glyceraldehyde-3-phosphate dehydrogenase, acetyl-CoA carboxylase, and malonyl-CoA decarboxylase activities were measured in myocardial biopsies. In eight control dogs, ANG II was infused (20-40 ng x kg(-1) x min(-1)) to mimic the hemodynamic effects of L-NAME. After L-NAME, significant changes occurred for fatty acid oxidation (from 9.8 +/- 0.8 to 7.1 +/- 1.2 micromol/min), glucose uptake (from 12.9 +/- 5.5 to 45.0 +/- 14.2 micromol/min), and oxidation (from 4.4 +/- 1.2 to 19.9 +/- 2.3 micromol/min). ANG caused only a significantly lower increase in glucose oxidation. Lactate uptake increased by more than twofold in both groups. The enzyme activities did not differ significantly between the two groups. In conclusion, the acute inhibition of NO synthesis causes marked metabolic alterations that do not involve key rate-controlling enzymes of fatty acid oxidation nor glyceraldehyde-3-phosphate dehydrogenase.

Cardiac work is supported by high rates of combustion of carbon fuel and oxygen consumption. Fatty acids are the main fuel for the healthy heart, supplying approximately 60-80% of the energy. The balance of the energy comes from the oxidation of glucose and lactate. ATP is broken down to fuel contractile work. ATP is resynthesized in the mitochondria using energy from the oxidation of fatty acids, glucose, and lactate. Myocardial ischaemia dramatically alters fuel metabolism. Ischaemia occurs when the coronary blood flow is insufficient to supply enough oxygen to combust carbon fuels and resynthesize ATP at the normal rate. During partial reductions in coronary blood flow (30-60% of normal) there is a proportional decrease in the rates of oxygen consumption and production of ATP, and an increase in uptake of glucose by the heart. However, unlike under normal aerobic conditions, the glucose taken up by the ischaemic myocardium is not readily oxidized in the mitochondria, but rather is converted to lactate and there is a switch from uptake of lactate by the heart to lactate production. This causes a dramatic disruption in cell homeostasis: ATP content decreases; there is accumulation of lactate and H+, a fall in intracellular pH and a decrease in contractile work. Paradoxically, the ischaemic tissue continues to derive most of its energy (50-70%) from the oxidation of fatty acids despite there being a high rate of lactate production. This ischaemia-induced disruption of cardiac metabolism can be minimized by metabolic agents that decrease oxidation of fatty acids and increase the rates of combustion of glucose and lactate, resulting in clinical benefit to the ischaemic patient.

The goal of this study was to measure flux through pyruvate carboxylation and decarboxylation in the heart in vivo. These rates were measured in the anterior wall of normal anesthetized swine hearts by infusing [U-(13)C(3)]lactate and/or [U-(13)C(3)] pyruvate into the left anterior descending (LAD) coronary artery. After 1 h, the tissue was freeze-clamped and analyzed by gas chromatography-mass spectrometry for the mass isotopomer distribution of citrate and its oxaloacetate moiety. LAD blood pyruvate and lactate enrichments and concentrations were constant after 15 min of infusion. Under near-normal physiological concentrations of lactate and pyruvate, pyruvate carboxylation and decarboxylation accounted for 4.7 +/- 0.3 and 41.5 +/- 2.0% of citrate formation, respectively. Similar relative fluxes were found when arterial pyruvate was raised from 0.2 to 1.1 mM. Addition of 1 mM octanoate to 1 mM pyruvate inhibited pyruvate decarboxylation by 93% without affecting carboxylation. The absence of M1 and M2 pyruvate demonstrated net irreversible pyruvate carboxylation. Under our experimental conditions we found that pyruvate carboxylation in the in vivo heart accounts for at least 3-6% of the citric acid cycle flux despite considerable variation in the flux through pyruvate decarboxylation.

This review examines the in vivo techniques that are available for evaluation of the metabolic effects and efficacy of agents intended for the treatment of myocardial ischemia. Energy substrate metabolism is complex, and requires simultaneous measurement of a variety of processes in order to obtain a thorough understanding of the biochemical mechanisms underlying any functional response. Small animals (from the mouse to the rabbit) are generally not very useful in the study of cardiac metabolism in vivo because it is not possible to sample the coronary venous drainage and measure the rate of substrate uptake or metabolite efflux. Anesthetized open-chest swine or dog models allows simultaneous serial measurement of myocardial substrate use, and repeated tissue sampling for the activities and contents of key enzymes and metabolites. The swine model is particularly good because pigs, like humans, lack innate collateral vessels, thus one can induce regional myocardial ischemia in the left anterior descending coronary artery and sample the venous effluent from the anterior interventricular vein. In this review the biochemical and physiological methods that can be used in conjunction with this preparation are described.

The effects of diabetes on myocardial metabolism are complex in that they are tied to the systemic metabolic abnormalities of the disease (hyperglycemia and elevated levels of free fatty acid and ketone bodies), and changes in cardiomyocyte phenotype (e.g., down-regulation of glucose transporters and PDH activity). The cardiac adaptations appear to be driven by the severity of the systemic abnormalities of the disease. The diabetes-induced changes in the plasma milieu and cardiac phenotype both cause impaired glycolysis, pyruvate oxidation, and lactate uptake, and a greater dependency on fatty acids as a source of acetyl CoA. Studies in isolated hearts suggest that therapies aimed at decreasing fatty acid oxidation, or directly stimulating pyruvate oxidation would be of benefit to the diabetic heart during and following myocardial ischemia.