Increasing cardiac pyruvate dehydrogenase flux during chronic hypoxia improves acute hypoxic tolerance.

KEY POINTS: The cardiac metabolic reprogramming seen in heart diseases such as myocardial infarction and hypertrophy shares similarities with that seen in chronic hypoxia, but understanding of how the hypoxic heart responds to further hypoxic challenge - hypoxic tolerance - is limited. The pyruvate dehydrogenase complex serves to control irreversible decarboxylation of pyruvate within mitochondria, and is a key regulator of substrate metabolism, potentially regulating hypoxic tolerance. Acute activation of the pyruvate dehydrogenase complex did not improve cardiac function during acute hypoxia; however, simultaneous activation of the pyruvate dehydrogenase complex during chronic hypoxic exposure improved tolerance to subsequent acute hypoxia. Activation of the pyruvate dehydrogenase complex during chronic hypoxia stockpiled cardiac acetylcarnitine, and this was used during acute hypoxia. This maintained cardiac ATP and glycogen, and improved hypoxic tolerance as a result. These findings demonstrate that pyruvate dehydrogenase complex activation can improve cardiac function under hypoxia. ABSTRACT: The pattern of metabolic reprogramming in chronic hypoxia shares similarities with that following myocardial infarction or hypertrophy; however, the response of the chronically hypoxic heart to subsequent acute injury, and the role of metabolism is not well understood. Here, we determined the myocardial tolerance of the chronically hypoxic heart to subsequent acute injury, and hypothesised that activation of a key regulator of myocardial metabolism, the pyruvate dehydrogenase complex (PDC), could improve hypoxic tolerance. Mouse hearts, perfused in Langendorff mode, were exposed to 30 min of hypoxia, and lost 80% of pre-hypoxic function (P = 0.001), with only 51% recovery of pre-hypoxic function with 30 min of reoxygenation (P = 0.046). Activation of the PDC with infusion of 1 mm dichloroacetate (DCA) during hypoxia and reoxygenation did not alter function. Acute hypoxic tolerance was assessed in hearts of mice housed in hypoxia for 3 weeks. Chronic hypoxia reduced cardiac tolerance to subsequent acute hypoxia, with recovery of function 22% of pre-acute hypoxic levels vs. 39% in normoxic control hearts (P = 0.012). DCA feeding in chronic hypoxia (per os, 70 mg kg-1  day-1 ) doubled cardiac acetylcarnitine content, and this fell following acute hypoxia. This acetylcarnitine use maintained cardiac ATP and glycogen content during acute hypoxia, with hypoxic tolerance normalised. In summary, chronic hypoxia renders the heart more susceptible to acute hypoxic injury, which can be improved by activation of the PDC and pooling of acetylcarnitine. This is the first study showing functional improvement of the chronically hypoxic heart with activation of the PDC, and offers therapeutic potential in cardiac disease with a hypoxic component.

On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury.

The role of peroxisome proliferator-activated receptor α (PPARα)-mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 wk before in vivo contractile function was measured using cine MRI. In isolated, perfused hearts, energetics were measured using (31)P magnetic resonance spectroscopy (MRS), and glycolysis and fatty acid oxidation were measured using [(3)H] labeling. Compared with a normoxic, chow-fed control mouse heart, hypoxia decreased PPARα expression, fatty acid oxidation, and mitochondrial uncoupling protein 3 (UCP3) levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations ([ATP]) and thereby, ejection fractions. A high-fat diet increased cardiac PPARα expression, fatty acid oxidation, and UCP3 levels with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high-fat diet, with the result that [ATP] and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in control mouse hearts were found to have occurred already in PPARα-deficient (PPARα(-/-)) mouse hearts and sustained function in hypoxia despite an inability for further metabolic remodeling. We conclude that decreased cardiac PPARα expression is essential for adaptive metabolic remodeling in hypoxia, but is prevented by dietary fat.-Cole, M. A., Abd Jamil, A. H., Heather, L. C., Murray, A. J., Sutton, E. R., Slingo, M., Sebag-Montefiore, L., Tan, S. C., Aksentijevic, D., Gildea, O. S., Stuckey, D. J., Yeoh, K. K., Carr, C. A., Evans, R. D., Aasum, E., Schofield, C. J., Ratcliffe, P. J., Neubauer, S., Robbins, P. A., Clarke, K. On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury.

Novel ketone diet enhances physical and cognitive performance.

Ketone bodies are the most energy-efficient fuel and yield more ATP per mole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high-fat, low-carbohydrate diets has been used for the treatment of drug-refractory epilepsy and for neurodegenerative diseases, such as Parkinson's disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)-3-hydroxybutyl (R)-3-hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a treadmill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palm oil (P < 0.05). Ketone-fed rats completed an 8-arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts from rats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [31P]-NMR spectroscopy. The novel ketone diet, therefore, improved physical performance and cognitive function in rats, and its energy-sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.-Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M. K., Atherton, H. J., Schroeder, M. A., Deacon, R. M. J., Kashiwaya, Y., King, M. T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance.

Increased oxidative metabolism following hypoxia in the type 2 diabetic heart, despite normal hypoxia signalling and metabolic adaptation.

KEY POINTS: Adaptation to hypoxia makes the heart more oxygen efficient, by metabolising more glucose. In contrast, type 2 diabetes makes the heart metabolise more fatty acids. Diabetes increases the chances of the heart being exposed to hypoxia, but whether the diabetic heart can adapt and respond is unknown. In this study we show that diabetic hearts retain the ability to adapt their metabolism in response to hypoxia, with functional hypoxia signalling pathways. However, the hypoxia-induced changes in metabolism are additive to abnormal baseline metabolism, resulting in hypoxic diabetic hearts metabolising more fat and less glucose than controls. This stops the diabetic heart being able to recover its function when stressed. These results demonstrate that the diabetic heart retains metabolic flexibility to adapt to hypoxia, but is hindered by the baseline effects of the disease. This increases our understanding of how the diabetic heart is affected by hypoxia-associated complications of the disease. ABSTRACT: Hypoxia activates the hypoxia-inducible factor (HIF), promoting glycolysis and suppressing mitochondrial respiration. In the type 2 diabetic heart, glycolysis is suppressed whereas fatty acid metabolism is promoted. The diabetic heart experiences chronic hypoxia as a consequence of increased obstructive sleep apnoea and cardiovascular disease. Given the opposing metabolic effects of hypoxia and diabetes, we questioned whether diabetes affects cardiac metabolic adaptation to hypoxia. Control and type 2 diabetic rats were housed for 3 weeks in normoxia or 11% oxygen. Metabolism and function were measured in the isolated perfused heart using radiolabelled substrates. Following chronic hypoxia, both control and diabetic hearts upregulated glycolysis, lactate efflux and glycogen content and decreased fatty acid oxidation rates, with similar activation of HIF signalling pathways. However, hypoxia-induced changes were superimposed on diabetic hearts that were metabolically abnormal in normoxia, resulting in glycolytic rates 30% lower, and fatty acid oxidation 36% higher, in hypoxic diabetic hearts than hypoxic controls. Peroxisome proliferator-activated receptor α target proteins were suppressed by hypoxia, but activated by diabetes. Mitochondrial respiration in diabetic hearts was divergently activated following hypoxia compared with controls. These differences in metabolism were associated with decreased contractile recovery of the hypoxic diabetic heart following an acute hypoxic insult. In conclusion, type 2 diabetic hearts retain metabolic flexibility to adapt to hypoxia, with normal HIF signalling pathways. However, they are more dependent on oxidative metabolism following hypoxia due to abnormal normoxic metabolism, which was associated with a functional deficit in response to stress.

Cardiac metabolism in a new rat model of type 2 diabetes using high-fat diet with low dose streptozotocin.

BACKGROUND: To study the pathogenesis of diabetic cardiomyopathy, reliable animal models of type 2 diabetes are required. Physiologically relevant rodent models are needed, which not only replicate the human pathology but also mimic the disease process. Here we characterised cardiac metabolic abnormalities, and investigated the optimal experimental approach for inducing disease, in a new model of type 2 diabetes. METHODS AND RESULTS: Male Wistar rats were fed a high-fat diet for three weeks, with a single intraperitoneal injection of low dose streptozotocin (STZ) after fourteen days at 15, 20, 25 or 30 mg/kg body weight. Compared with chow-fed or high-fat diet fed control rats, a high-fat diet in combination with doses of 15-25 mg/kg STZ did not change insulin concentrations and rats maintained body weight. In contrast, 30 mg/kg STZ induced hypoinsulinaemia, hyperketonaemia and weight loss. There was a dose-dependent increase in blood glucose and plasma lipids with increasing concentrations of STZ. Cardiac and hepatic triglycerides were increased by all doses of STZ, in contrast, cardiac glycogen concentrations increased in a dose-dependent manner with increasing STZ concentrations. Cardiac glucose transporter 4 protein levels were decreased, whereas fatty acid metabolism-regulated proteins, including uncoupling protein 3 and pyruvate dehydrogenase (PDH) kinase 4, were increased with increasing doses of STZ. Cardiac PDH activity displayed a dose-dependent relationship between enzyme activity and STZ concentration. Cardiac insulin-stimulated glycolytic rates were decreased by 17% in 15 mg/kg STZ high-fat fed diabetic rats compared with control rats, with no effect on cardiac contractile function. CONCLUSIONS: High-fat feeding in combination with a low dose of STZ induced cardiac metabolic changes that mirror the decrease in glucose metabolism and increase in fat metabolism in diabetic patients. While low doses of 15-25 mg/kg STZ induced a type 2 diabetic phenotype, higher doses more closely recapitulated type 1 diabetes, demonstrating that the severity of diabetes can be modified according to the requirements of the study.

Metabolic adaptation to chronic hypoxia in cardiac mitochondria.

Chronic hypoxia decreases cardiomyocyte respiration, yet the mitochondrial mechanisms remain largely unknown. We investigated the mitochondrial metabolic pathways and enzymes that were decreased following in vivo hypoxia, and questioned whether hypoxic adaptation was protective for the mitochondria. Wistar rats were housed in hypoxia (7 days acclimatisation and 14 days at 11% oxygen), while control rats were housed in normoxia. Chronic exposure to physiological hypoxia increased haematocrit and cardiac vascular endothelial growth factor, in the absence of weight loss and changes in cardiac mass. In both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from hypoxic hearts, state 3 respiration rates with fatty acid were decreased by 17-18%, and with pyruvate were decreased by 29-15%, respectively. State 3 respiration rates with electron transport chain (ETC) substrates were decreased only in hypoxic SSM, not in hypoxic IFM. SSM from hypoxic hearts had decreased activities of ETC complexes I, II and IV, which were associated with decreased reactive oxygen species generation and protection against mitochondrial permeability transition pore (MPTP) opening. In contrast, IFM from hypoxic hearts had decreased activity of the Krebs cycle enzyme, aconitase, which did not modify ROS production or MPTP opening. In conclusion, cardiac mitochondrial respiration was decreased following chronic hypoxia, associated with downregulation of different pathways in the two mitochondrial populations, determined by their subcellular location. Hypoxic adaptation was not deleterious for the mitochondria, in fact, SSM acquired increased protection against oxidative damage under the oxygen-limited conditions.

A high fat diet increases mitochondrial fatty acid oxidation and uncoupling to decrease efficiency in rat heart.

Elevated levels of cardiac mitochondrial uncoupling protein 3 (UCP3) and decreased cardiac efficiency (hydraulic power/oxygen consumption) with abnormal cardiac function occur in obese, diabetic mice. To determine whether cardiac mitochondrial uncoupling occurs in non-genetic obesity, we fed rats a high fat diet (55% kcal from fat) or standard laboratory chow (7% kcal from fat) for 3 weeks, after which we measured cardiac function in vivo using cine MRI, efficiency in isolated working hearts and respiration rates and ADP/O ratios in isolated interfibrillar mitochondria; also, measured were medium chain acyl-CoA dehydrogenase (MCAD) and citrate synthase activities plus uncoupling protein 3 (UCP3), mitochondrial thioesterase 1 (MTE-1), adenine nucleotide translocase (ANT) and ATP synthase protein levels. We found that in vivo cardiac function was the same for all rats, yet oxygen consumption was 19% higher in high fat-fed rat hearts, therefore, efficiency was 21% lower than in controls. We found that mitochondrial fatty acid oxidation rates were 25% higher, and MCAD activity was 23% higher, in hearts from rats fed the high fat diet when compared with controls. Mitochondria from high fat-fed rat hearts had lower ADP/O ratios than controls, indicating increased respiratory uncoupling, which was ameliorated by GDP, a UCP3 inhibitor. Mitochondrial UCP3 and MTE-1 levels were both increased by 20% in high fat-fed rat hearts when compared with controls, with no significant change in ATP synthase or ANT levels, or citrate synthase activity. We conclude that increased cardiac oxygen utilisation, and thereby decreased cardiac efficiency, occurs in non-genetic obesity, which is associated with increased mitochondrial uncoupling due to elevated UCP3 and MTE-1 levels.

First-pass perfusion CMR two days after infarction predicts severity of functional impairment six weeks later in the rat heart.

BACKGROUND: In humans, dynamic contrast CMR of the first pass of a bolus infusion of Gadolinium-based contrast agent has become a standard technique to identify under-perfused regions of the heart and can accurately demonstrate the severity of myocardial infarction. Despite the clinical importance of this method, it has rarely been applied in small animal models of cardiac disease. In order to identify perfusion delays in the infarcted rat heart, here we present a method in which a T1 weighted MR image has been acquired during each cardiac cycle. METHODS AND RESULTS: In isolated perfused rat hearts, contrast agent infusion gave uniform signal enhancement throughout the myocardium. Occlusion of the left anterior descending coronary artery significantly reduced the rate of signal enhancement in anterior regions of the heart, demonstrating that the first-pass method was sensitive to perfusion deficits. In vivo measurements of myocardial morphology, function, perfusion and viability were made at 2 and 8 days after infarction. Morphology and function were further assessed using cine-MRI at 42 days. The perfusion delay was larger in rat hearts that went on to develop greater functional impairment, demonstrating that first-pass CMR can be used as an early indicator of infarct severity. First-pass CMR at 2 and 8 days following infarction better predicted outcome than cardiac ejection fraction, end diastolic volume or end systolic volume. CONCLUSION: First-pass CMR provides a predictive measure of the severity of myocardial impairment caused by infarction in a rodent model of heart failure.

Potential role for pyruvate kinase M2 in the regulation of murine cardiac glycolytic flux during in vivo chronic hypoxia.

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homoeostasis in the face of reduced O2 availability. As part of these in vivo adaptations during sustained hypoxia, the heart up-regulates and maintains a high glycolytic flux, but the underlying mechanism is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-3H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability. Following 3 weeks of in vivo hypoxia (11% oxygen), murine hearts were isolated and perfused in a retrograde mode with function measured via an intraventricular balloon and glycolytic flux quantified using [5-3H]-glucose labelling. At the end of perfusion, hearts were flash-frozen and central carbon intermediates determined via liquid chromatography tandem mass spectrometry (LC-MS/MS). The maximal activity of glycolytic enzymes considered rate-limiting was assessed enzymatically, and protein abundance was determined using Western blotting. Relative to normoxic hearts, CH increased ex vivo cardiac glycolytic flux 1.7-fold with no effect on cardiac function. CH up-regulated cardiac pyruvate kinase (PK) flux 3.1-fold and cardiac pyruvate kinase muscle isoenzyme M2 (PKM2) protein content 1.4-fold compared with normoxic hearts. CH also augmented cardiac pentose phosphate pathway (PPP) flux, reflected by higher ribose-5-phosphate (R5P) content. These findings support an increase in the covalent (protein expression) and allosteric (flux) control of PKM2 as being central to the sustained up-regulation of the glycolytic flux in the chronically hypoxic heart.

Ongoing dual-angle measurements for the correction of partial saturation in 31P MR spectroscopy.

Use of a repetition time similar to, or shorter than, metabolite T(1)s is common in NMR spectroscopy of biological samples to improve the signal-to-noise ratio. Conventionally, the partial saturation that results from this is corrected using saturation factors. However, this can lead to erroneous results in the presence of chemical exchange or nonconstant T(1)s. We describe an alternative approach to correction for saturation, based on ongoing dual-angle T(1) measurement. Using (31)P magnetic resonance spectroscopy of the perfused rat heart undergoing ischemia-reperfusion, we demonstrate that signal alternations in the data acquired by the dual-angle approach are eliminated by the ongoing dual-angle T(1) measurement correction scheme, meaning that metabolite concentration and T(1) measurement can be made throughout the course of the ischemia-reperfusion protocol. Simulations, based on parameters pertinent to the perfused rat heart, demonstrate that accurate saturation correction is possible with this method except at times of rapid concentration change. Additionally, compared to the conventional saturation factor correction method, the ongoing dual-angle T(1) measurement correction scheme results in improved accuracy in determining the [phosphocreatine] recovery time constant. Thus, the ongoing dual-angle T(1) measurements procedure permits accurate monitoring of metabolite concentrations even in the setting of chemical exchange and T(1) changes and allows more accurate analysis of bioenergetic status.

Real-time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy.

The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2-(13)C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of (13)C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of (13)C-labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2-(13)C]pyruvate.

Increased mitochondrial uncoupling proteins, respiratory uncoupling and decreased efficiency in the chronically infarcted rat heart.

Heart failure patients have abnormal cardiac high energy phosphate metabolism, the explanation for which is unknown. Patients with heart failure also have elevated plasma free fatty acid (FFA) concentrations. Elevated FFA levels are associated with increased cardiac mitochondrial uncoupling proteins (UCPs), which, in turn, are associated with decreased mitochondrial respiratory coupling and low cardiac efficiency. Here, we determined whether increased mitochondrial UCP levels contribute to decreased energetics in the failing heart by measuring UCPs and respiration in mitochondria isolated from the viable myocardium of chronically infarcted rat hearts and measuring efficiency (hydraulic work/O(2) consumption) in the isolated, working rat heart. Ten weeks after infarction, cardiac levels of UCP3 were increased by 53% in infarcted, failing hearts that had ejection fractions less than 45%. Cardiac UCP3 levels correlated positively with non-fasting plasma FFAs (r=0.81; p<0.01). Mitochondria from failing hearts were less coupled than those from control hearts, as demonstrated by the lower ADP/O ratio of 1.9+/-0.1 compared with 2.5+/-0.2 in controls (p<0.05). The decreased ADP/O ratio was reflected in an efficiency of 14+/-2% in the failing hearts when perfused with 1 mM palmitate, compared with 20+/-1% in controls (p<0.05). We conclude that failing hearts have increased UCP3 levels that are associated with high circulating FFA concentrations, mitochondrial uncoupling, and decreased cardiac efficiency. Thus, respiratory uncoupling may underlie the abnormal energetics and low efficiency in the failing heart, although whether this is maladaptive or adaptive would require direct investigation.

Fatty acid transporter levels and palmitate oxidation rate correlate with ejection fraction in the infarcted rat heart.

OBJECTIVES: Cardiac fatty acid uptake occurs predominantly via sarcolemmal transporter proteins; fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm) and fatty acid transporter proteins (FATP) 1 and 6. We hypothesised that levels of the fatty acid transporters would be reduced in the chronically infarcted rat heart, in parallel with reduced dependence on fatty acid utilisation. METHODS AND RESULTS: In vivo left ventricular ejection fractions, measured using echocardiography, were 36% lower in rats six months after coronary artery ligation than in sham-operated control rats. In isolated, perfused, infarcted hearts, 3H-palmitate oxidation was 30% lower, and correlated with in vivo ejection fractions. As myocardial lipid incorporation was also reduced by 25%, total palmitate utilisation was 29% lower in the infarcted rat heart. The protein levels of the cardiac fatty acid transporters were reduced in the infarcted rat heart; FAT/CD36 by 36%, FABPpm by 12%, FATP6 by 21% and FATP1 by 26%, and the cytosolic fatty acid binding protein (cFABP) was 47% lower than in sham-operated rat hearts. Fatty acid transporter levels correlated with both palmitate oxidation rates and cardiac ejection fractions. CONCLUSIONS: Reductions in fatty acid oxidation and lipid incorporation rates were accompanied by downregulation of the cardiac fatty acid transporters. The metabolic shift away from fatty acid utilisation was proportional to the degree of functional impairment in the chronically infarcted rat heart.

Insulin resistance, abnormal energy metabolism and increased ischemic damage in the chronically infarcted rat heart.

OBJECTIVE: Many patients with heart failure have whole-body insulin resistance and reduced cardiac fluorodeoxyglucose uptake, but whether these metabolic changes have detrimental effects on the heart is unknown. Here, we tested whether there is a link between insulin resistance and ischemic damage in the chronically infarcted Wistar rat heart, postulating that the heart would have decreased insulin sensitivity, with lower GLUT4 glucose transporter protein levels due to high circulating free fatty acid (FFA) concentrations. A decreased capacity for glucose uptake would lower glycolytic adenosine triphosphate (ATP) production and thereby increase ischemic injury in the infarcted heart. METHODS AND RESULTS: In vivo left ventricular ejection fractions, measured using echocardiography, were 40% lower in rats 10 weeks after coronary artery ligation than in sham-operated control rats. Insulin-stimulated D[2-3H]glucose uptake was 42% lower in isolated, perfused, infarcted hearts. Myocardial GLUT4 glucose transporter protein levels were 28% lower in the infarcted hearts and correlated negatively with ejection fractions and with fasting plasma FFA concentrations. Compared with controls, chronically infarcted hearts had 46% lower total glucose uptake and three-fold faster ATP hydrolysis rates, measured using phosphorus-31 nuclear magnetic resonance spectroscopy, during 32-min ischemia at 0.4 ml/min/gww. During reperfusion, recovery of left ventricular developed pressure in infarcted hearts was 42% lower than in control hearts. CONCLUSIONS: Glucose uptake, in response to insulin or ischemia, was lower in the chronically infarcted rat heart and associated with increased circulating FFA concentrations and decreased GLUT4 levels. Thus, infarcted hearts had greater ATP depletion, and consequently incurred greater damage, during ischemia.

Thiazolidinedione treatment normalizes insulin resistance and ischemic injury in the zucker Fatty rat heart.

Obesity is associated with risk factors for cardiovascular disease, including insulin resistance, and can lead to cardiac hypertrophy and congestive heart failure. Here, we used the insulin-sensitizing agent rosiglitazone to investigate the cellular mechanisms linking insulin resistance in the obese Zucker rat heart with increased susceptibility to ischemic injury. Rats were treated for 7 or 14 days with 3 mg/kg per os rosiglitazone. Hearts were isolated and perfused before and during insulin stimulation or during 32 min low-flow ischemia at 0.3 ml small middle dot min(-1) small middle dot grams wet wt(-1) and reperfusion. D[2-(3)H]glucose was used as a tracer of glucose uptake, and phosphorus-31 nuclear magnetic resonance spectroscopy was used to follow energetics during ischemia. At 12 months of age, obese rat hearts were insulin resistant with decreased GLUT4 protein expression. During ischemia, glucose uptake was lower and depletion of ATP was greater in obese rat hearts, thereby significantly impairing recovery of contractile function during reperfusion. Rosiglitazone treatment normalized the insulin resistance and restored GLUT4 protein levels in obese rat hearts. Glucose uptake during ischemia was also normalized by rosiglitazone treatment, thereby preventing the greater loss of ATP and restoring recovery of contractile function to that of lean rat hearts. We conclude that rosiglitazone treatment, by normalizing glucose uptake, protected obese rat hearts from ischemic injury.

The PPARgamma-activator rosiglitazone does not alter remodeling but increases mortality in rats post-myocardial infarction.

OBJECTIVE: Peroxisome proliferator-activated receptor gamma (PPARgamma) activators may be beneficial in heart failure due to their metabolic and antihypertrophic effects, but these agents can cause oedema. We hypothesized that, on balance, the PPARgamma activator rosiglitazone would be beneficial in heart failure post-myocardial infarction. METHODS AND RESULTS: Rosiglitazone (3 mg/kg/day p.o.) given to male Wistar rats for 14 days, caused a 31% increase in left ventricular (LV) dP/dt(max) (P<0.05 vs. placebo). A separate group of rats was subjected to sham (SH) or coronary artery ligation and randomised to: untreated (UT); rosiglitazone 3 mg/kg/day (R); captopril, 2 g/l in drinking water (C); captopril+rosiglitazone (C+R). Mean LV infarct sizes were similar for all groups at 40+/-2%. After 8 weeks, echocardiographic ejection fractions were 82+/-3, 40+/-3, 50+/-2*, 49+/-2, 50+/-3% for SH, UT, R, C and C+R groups, respectively (*P<0.05 vs. UT). Captopril prevented LV dilatation, but rosiglitazone did not. In vivo hemodynamics showed that only UT had significantly elevated LV end-diastolic pressures and reduced +dP/dt(max), with R partially, and C and C+R almost completely preventing the increase in LVEDP. Captopril, but not rosiglitazone, significantly reduced LV hypertrophy [LV/bw; 1.97+/-0.09 (SH), 2.15+/-0.04 (UT), 2.10+/-0.05 (R), 1.81+/-0.04* (C), 1.88+/-0.07 (C+R); *(P<0.05 vs. UT)]. Rosiglitazone increased 8-week mortality, which was 26% for R and 19% for C+R compared with 0% for UT and C (P=0.03 vs. UT). CONCLUSIONS: Rosiglitazone did not modulate LV remodeling, but was associated with increased mortality post-myocardial infarction (MI) in rats. The mechanisms require further study, but these results caution against use of PPARgamma activators in post-MI heart failure in non-diabetics.

Differential translocation of the fatty acid transporter, FAT/CD36, and the glucose transporter, GLUT4, coordinates changes in cardiac substrate metabolism during ischemia and reperfusion.

BACKGROUND: Fatty acid and glucose transporters translocate between the sarcolemma and intracellular compartments to regulate substrate metabolism acutely. We hypothesised that during ischemia fatty acid translocase (FAT/CD36) would translocate away from the sarcolemma to limit fatty acid uptake when fatty acid oxidation is inhibited. METHODS AND RESULTS: Wistar rat hearts were perfused during preischemia, low-flow ischemia, and reperfusion, using (3)H-substrates for measurement of metabolic rates, followed by metabolomic analysis and subcellular fractionation. During ischemia, there was a 32% decrease in sarcolemmal FAT/CD36 accompanied by a 95% decrease in fatty acid oxidation rates, with no change in intramyocardial lipids. Concomitantly, the sarcolemmal content of the glucose transporter, GLUT4, increased by 90% during ischemia, associated with an 86% increase in glycolytic rates, 45% decrease in glycogen content, and a 3-fold increase in phosphorylated AMP-activated protein kinase. Following reperfusion, decreased sarcolemmal FAT/CD36 persisted, but fatty acid oxidation rates returned to preischemic levels, resulting in a 35% decrease in myocardial triglyceride content. Elevated sarcolemmal GLUT4 persisted during reperfusion; in contrast, glycolytic rates decreased to 30% of preischemic rates, accompanied by a 5-fold increase in intracellular citrate levels and restoration of glycogen content. CONCLUSIONS: During ischemia, FAT/CD36 moved away from the sarcolemma as GLUT4 moved toward the sarcolemma, associated with a shift from fatty acid oxidation to glycolysis, while intramyocardial lipid accumulation was prevented. This relocation was maintained during reperfusion, which was associated with replenishing glycogen stores as a priority, occurring at the expense of glycolysis and mediated by an increase in citrate levels.

Changes in cardiac substrate transporters and metabolic proteins mirror the metabolic shift in patients with aortic stenosis.

In the hypertrophied human heart, fatty acid metabolism is decreased and glucose utilisation is increased. We hypothesized that the sarcolemmal and mitochondrial proteins involved in these key metabolic pathways would mirror these changes, providing a mechanism to account for the modified metabolic flux measured in the human heart. Echocardiography was performed to assess in vivo hypertrophy and aortic valve impairment in patients with aortic stenosis (n = 18). Cardiac biopsies were obtained during valve replacement surgery, and used for western blotting to measure metabolic protein levels. Protein levels of the predominant fatty acid transporter, fatty acid translocase (FAT/CD36) correlated negatively with levels of the glucose transporters, GLUT1 and GLUT4. The decrease in FAT/CD36 was accompanied by decreases in the fatty acid binding proteins, FABPpm and H-FABP, the ß-oxidation protein medium chain acyl-coenzyme A dehydrogenase, the Krebs cycle protein α-ketoglutarate dehydrogenase and the oxidative phosphorylation protein ATP synthase. FAT/CD36 and complex I of the electron transport chain were downregulated, whereas the glucose transporter GLUT4 was upregulated with increasing left ventricular mass index, a measure of cardiac hypertrophy. In conclusion, coordinated downregulation of sequential steps involved in fatty acid and oxidative metabolism occur in the human heart, accompanied by upregulation of the glucose transporters. The profile of the substrate transporters and metabolic proteins mirror the metabolic shift from fatty acid to glucose utilisation that occurs in vivo in the human heart.

Adenosine monophosphate-activated protein kinase activation, substrate transporter translocation, and metabolism in the contracting hyperthyroid rat heart.

Thyroid hormones can modify cardiac metabolism via multiple molecular mechanisms, yet their integrated effect on overall substrate metabolism is poorly understood. Here we determined the effect of hyperthyroidism on substrate metabolism in the isolated, perfused, contracting rat heart. Male Wistar rats were injected for 7 d with T(3) (0.2 mg/kg x d ip). Plasma free fatty acids increased by 97%, heart weights increased by 33%, and cardiac rate pressure product, an indicator of contractile function, increased by 33% in hyperthyroid rats. Insulin-stimulated glycolytic rates and lactate efflux rates were increased by 33% in hyperthyroid rat hearts, mediated by an increased insulin-stimulated translocation of the glucose transporter GLUT4 to the sarcolemma. This was accompanied by a 70% increase in phosphorylated AMP-activated protein kinase (AMPK) and a 100% increase in phosphorylated acetyl CoA carboxylase, confirming downstream signaling from AMPK. Fatty acid oxidation rates increased in direct proportion to the increased heart weight and rate pressure product in the hyperthyroid heart, mediated by synchronized changes in mitochondrial enzymes and respiration. Protein levels of the fatty acid transporter, fatty acid translocase (FAT/CD36), were reduced by 24% but were accompanied by a 19% increase in the sarcolemmal content of fatty acid transport protein 1 (FATP1). Thus, the relationship between fatty acid metabolism, cardiac mass, and contractile function was maintained in the hyperthyroid heart, associated with a sarcolemmal reorganization of fatty acid transporters. The combined effects of T(3)-induced AMPK activation and insulin stimulation were associated with increased sarcolemmal GLUT4 localization and glycolytic flux in the hyperthyroid heart.

Abnormal function and glucose metabolism in the type-2 diabetic db/db mouse heart.

This study examined cardiac function and glucose metabolism in the 6-month-old db/db mouse, a model of type-2 diabetes. Cine magnetic resonance spectroscopy (MRI) was used to measure cardiac function in vivo. The db/db mice had decreased heart rates (17%, p<0.01) and stroke volumes (21%, p<0.05) that resulted in lower cardiac output (35%, p<0.01) than controls. Although there was no difference in ejection fraction between the 2 groups, db/db mouse hearts had a 35% lower maximum rate of ejection (p<0.01) than controls. In a protocol designed to assess maximal insulin-independent glucose uptake, hearts were isolated and perfused in Langendorff mode and subjected to 0.75 mL.min(-1).(g wet mass)(-1) low flow ischemia for 32 min. Glucose uptake during ischemia was 21% lower than in controls, and post-ischemic recovery of cardiac function was decreased by 30% in db/db mouse hearts (p<0.05). Total cardiac GLUT 4 protein was 56% lower (p<0.01) in db/db mice than in controls. In summary, the db/db mouse has abnormal left ventricular function in vivo, with impaired glucose uptake during ischemia, leading to increased myocardial damage.