Diabetic Rat Hearts Show More Favorable Metabolic Adaptation to Omegaven Containing High Amounts of n3 Fatty Acids Than Intralipid Containing n6 Fatty Acids.

BACKGROUND: While Omegaven, an omega-3 (n3) fatty acid-based lipid emulsion, fosters insulin signaling in healthy hearts, it is unknown whether beneficial metabolic effects occur in insulin-resistant diabetic hearts. METHODS: Diabetic hearts from fructose-fed Sprague-Dawley rats were perfused in the working mode for 90 minutes in the presence of 11 mM glucose and 1.2 mM palmitate bound to albumin, the first 30 minutes without insulin followed by 60 minutes with insulin (50 mU/L). Hearts were randomly allocated to Intralipid (25 and 100 µM), Omegaven (25 and 100 µM), or no emulsion (insulin alone) for 60 minutes. Glycolysis, glycogen synthesis, and glucose oxidation were measured with the radioactive tracers [5-H]glucose and [U-C]glucose. Central carbon metabolites, acyl-coenzyme A species (acyl-CoAs), ketoacids, purines, phosphocreatine, acylcarnitines, and acyl composition of phospholipids were measured with mass spectrometry. RESULTS: Diabetic hearts showed no response to insulin with regard to glycolytic flux, consistent with insulin resistance. Addition of either lipid emulsion did not alter this response but unexpectedly increased glucose oxidation (ratio of treatment/baseline, ie, fold change): no insulin 1.3 (0.3) [mean (standard deviation)], insulin alone 1.4 (0.4), insulin + 25 µM Intralipid 1.8 (0.5), insulin + 100 µM Intralipid 2.2 (0.4), P < .001; no insulin 1.3 (0.3), insulin alone 1.4 (0.4), insulin + 25 µM Omegaven 2.3 (0.5) insulin + 100 µM Omegaven 1.9 (0.4), P < .001. Intralipid treatment led to accumulation of acylcarnitines as a result of the released linoleic acid (C18:2-n6) and enhanced its integration into phospholipids, consistent with incomplete or impaired ß-oxidation necessitating a compensatory increase in glucose oxidation. Accumulation of acylcarnitines was also associated with a higher nicotinamide adenine dinucleotide reduced/oxidized (NADH/NAD) ratio, which inhibited pyruvate dehydrogenase (PDH), and resulted in excess lactate production. In contrast, Omegaven-treated hearts showed no acylcarnitine accumulation, low malonyl-CoA concentrations consistent with activated ß-oxidation, and elevated PDH activity and glucose oxidation, together indicative of a higher metabolic rate possibly by substrate cycling. CONCLUSIONS: Omegaven is the preferred lipid emulsion for insulin-resistant diabetic hearts.

Lipid Emulsion Containing High Amounts of n3 Fatty Acids (Omegaven) as Opposed to n6 Fatty Acids (Intralipid) Preserves Insulin Signaling and Glucose Uptake in Perfused Rat Hearts.

BACKGROUND: It is currently unknown whether acute exposure to n3 fatty acid-containing fish oil-based lipid emulsion Omegaven as opposed to the n6 fatty acid-containing soybean oil-based lipid emulsion Intralipid is more favorable in terms of insulin signaling and glucose uptake in the intact beating heart. METHODS: Sprague-Dawley rat hearts were perfused in the working mode for 90 minutes in the presence of 11 mM glucose and 1.2 mM palmitate bound to albumin, the first 30 minutes without insulin followed by 60 minutes with insulin (50 mU/L). Hearts were randomly allocated to 100 µM Intralipid, 100 µM Omegaven, or no emulsion (insulin treatment alone) for 60 minutes. Glycolysis and glycogen synthesis were measured with the radioactive tracer [5-H]glucose, and glucose uptake was calculated. Phosphorylation of protein phosphatase 2A (PP2A), protein kinase Akt, and phosphofructokinase (PFK)-2 was measured by immunoblotting. Glycolytic metabolites were determined by enzymatic assays. Mass spectrometry was used to establish acylcarnitine profiles. Nuclear factor κB (NFκB) nuclear translocation served as reactive oxygen species (ROS) biosensor. RESULTS: Insulin-mediated glucose uptake was decreased by Intralipid (4.9 ± 0.4 vs 3.7 ± 0.3 µmol/gram dry heart weight [gdw]·min; P = .047) due to both reduced glycolysis and glycogen synthesis. In contrast, Omegaven treatment did not affect insulin-mediated glycolysis or glycogen synthesis and thus preserved glucose uptake (5.1 ± 0.3 vs 4.9 ± 0.4 µmol/gdw·min; P = .94). While Intralipid did not affect PP2A phosphorylation status, Omegaven resulted in significantly enhanced tyrosine phosphorylation and inhibition of PP2A. This was accompanied by increased selective threonine phosphorylation of Akt and the downstream target PFK-2 at S483. PFK-1 activity was increased when compared with Intralipid as measured by the ratio of fructose 1,6-bisphosphate to fructose 6-phosphate (Omegaven 0.60 ± 0.11 versus Intralipid 0.47 ± 0.09; P = .023), consistent with increased formation of fructose 2,6-bisphosphate by PFK2, its main allosteric activator. Omegaven lead to accumulation of acylcarnitines and fostered a prooxidant response as evidenced by NFκB nuclear translocation and activation. CONCLUSIONS: Omegaven as opposed to Intralipid preserves glucose uptake via the PP2A-Akt-PFK pathway in intact beating hearts. n3 fatty acids decelerate ß-oxidation causing accumulation of acylcarnitine species and a prooxidant response, which likely inhibits redox-sensitive PP2A and thus preserves insulin signaling and glucose uptake.

Enhanced myocardial protection in cardiac donation after circulatory death using Intralipid® postconditioning in a porcine model.

PURPOSE: Intralipid® (ILE), a clinically used lipid emulsion, reduces ischemia-reperfusion (IR) injury in healthy and infarct-remodelled rat hearts. We tested whether ILE is also cardioprotective in large porcine hearts in the context of the donation after circulatory death (DCD) model, where human hearts are procured for transplantation after cardiac arrest and thus are exposed to significant IR injury. METHODS: After induction of anesthesia, surgical preparation, termination of ventilator support, and cardiac arrest, hearts of female pigs were procured following a 15 min standoff period, with an optimized normokalemic crystalloid adenosine-lidocaine cardioplegia. Hearts were then randomly allocated to ex vivo reperfusion (38°C) in the absence (control) or presence of 1% ILE. All hearts were perfused with blood and Krebs-Henseleit solution (1:1) for 30 min in Langendorff mode and for an additional 30 min in working mode to assess mechanical function. Left ventricular (LV) biopsies were obtained after five minutes of reperfusion and LV tissue was preserved at the end of reperfusion for biochemical analyses and immunohistochemistry. RESULTS: Intralipid® postconditioning reduced cell membrane damage as assessed by the mean (standard deviation) leakage of myocardial glutathione disulfide (39 (9) nmol·mg-1 protein vs 19 (7) nmol·mg-1 protein; P = 0.006), protected LV tissue from protein carbonylation (3.4 [0.6] nmol·mg-1 protein vs 5.3 [0.9] nmol·mg-1 protein; P = 0.006), decreased myeloperoxidase activity (35 [8] nmol·min-1·mg-1 protein vs 75 [11] nmol·min-1·mg-1 protein; P < 0.001), and increased inotropy (maximum rate of rise of LV pressure 2001 [345] mmHg·sec-1vs 1584 [192] mmHg·sec-1; P = 0.044). Intralipid® postconditioning triggered reactive oxygen species signalling at early reperfusion and activated protection signalling (Akt, signal transducer and activator of transcription 3, and glycogen synthase kinase 3ß) in LV tissue, recapitulating all features of ILE-mediated protection reported in small rodent hearts. CONCLUSIONS: Our data show that ILE postconditioning elicits protection signalling in large mammalian hearts while mimicking clinical conditions, and is capable of enhancing protection of DCD hearts.

Differential Effects of Anesthetics and Opioid Receptor Activation on Cardioprotection Elicited by Reactive Oxygen Species-Mediated Postconditioning in Sprague-Dawley Rat Hearts.

BACKGROUND: Despite an array of cardioprotective interventions identified in preclinical models of ischemia-reperfusion (IR) injury, successful clinical translation has not been achieved. This study investigated whether drugs routinely used in clinical anesthesia influence cardioprotective effectiveness by reducing effects of reactive oxygen species (ROS), upstream triggers of cardioprotective signaling. Effects of propofol, sevoflurane, or remifentanil were compared on postischemic functional recovery induced by ROS-mediated postconditioning with Intralipid. METHODS: Recovery of left ventricular (LV) work, an index of IR injury, was measured in isolated Sprague-Dawley rat hearts subjected to global ischemia (20 minutes) and reperfusion (30 minutes). Hearts were either untreated or were treated with postconditioning with Intralipid (1%, throughout reperfusion). Propofol (10 µM), sevoflurane (2 vol%), remifentanil (3 nM), or combinations thereof were administered peri-ischemically (before and during IR). The effects of anesthetics on ROS production were measured in LV cardiac fibers by Amplex Red assay under phosphorylating and nonphosphorylating conditions. RESULTS: Recovery of LV work (expressed as percentage of the preischemic value ± standard deviation) in untreated hearts was poor (20% ± 7%) and was improved by Intralipid postconditioning (58% ± 8%, P = .001). In the absence of Intralipid postconditioning, recovery of LV work was enhanced by propofol (28% ± 9%, P = .049), sevoflurane (49% ± 5%, P < .001), and remifentanil (51% ± 6%, P < .001). The benefit of Intralipid postconditioning was abolished by propofol (33% ± 10%, P < .001), but enhanced by sevoflurane (80% ± 7%, P < .001) or remifentanil (80% ± 9%, P < .001). ROS signaling in LV fibers was abolished by propofol, but unaffected by sevoflurane or remifentanil. We conclude that propofol abolishes ROS-mediated Intralipid postconditioning by acting as a ROS scavenger. Sevoflurane and remifentanil are protective per se and provide additive cardioprotection to ROS-mediated cardioprotection. CONCLUSIONS: These divergent effects of routinely used drugs in clinical anesthesia may influence the translatability of cardioprotective therapies such as Intralipid postconditioning.

Hearts lacking plasma membrane KATP channels display changes in basal aerobic metabolic substrate preference and AMPK activity.

Cardiac ATP-sensitive K+ (KATP) channels couple changes in cellular metabolism to membrane excitability and are activated during metabolic stress, although under basal aerobic conditions, KATP channels are thought to be predominately closed. Despite intense research into the roles of KATP channels during metabolic stress, their contribution to aerobic basal cardiac metabolism has not been previously investigated. Hearts from Kir6.2+/+ and Kir6.2-/- mice were perfused in working mode, and rates of glycolysis, fatty acid oxidation, and glucose oxidation were measured. Changes in activation/expression of proteins regulating metabolism were probed by Western blot analysis. Despite cardiac mechanical function and metabolic efficiency being similar in both groups, hearts from Kir6.2-/- mice displayed an approximately twofold increase in fatty acid oxidation and a 0.45-fold reduction in glycolytic rates but similar glucose oxidation rates compared with hearts from Kir6.2+/+ mice. Kir6.2-/- hearts also possessed elevated levels of activated AMP-activated protein kinase (AMPK), higher glycogen content, and reduced mitochondrial density. Moreover, activation of AMPK by isoproterenol or diazoxide was significantly blunted in Kir6.2-/- hearts. These data indicate that KATP channel ablation alters aerobic basal cardiac metabolism. The observed increase in fatty acid oxidation and decreased glycolysis before any metabolic insult may contribute to the poor recovery observed in Kir6.2-/- hearts in response to exercise or ischemia-reperfusion injury. Therefore, KATP channels may play an important role in the regulation of cardiac metabolism through AMPK signaling.NEW & NOTEWORTHY In this study, we show that genetic ablation of plasma membrane ATP-sensitive K+ channels results in pronounced changes in cardiac metabolic substrate preference and AMP-activated protein kinase activity. These results suggest that ATP-sensitive K+ channels may play a novel role in regulating metabolism in addition to their well-documented effects on ionic homeostasis during periods of stress.

Myocardial mechanical dysfunction following endotoxemia: role of changes in energy substrate metabolism.

Cardiovascular depression due to endotoxemia remains a major cause of mortality in intensive care patients. To determine whether drug-induced alterations in cardiac metabolism may be a viable strategy to reduce endotoxemia-mediated cardiac dysfunction, we assessed endotoxemia-induced changes in glucose and fatty acid metabolism under aerobic and post-ischemic conditions. Endotoxemia was induced in male Sprague-Dawley rats by lipopolysaccharide (Escherichia coli 0111:B4c, 4 mg/kg, i.p.) 6 h prior to heart removal for ex vivo assessment of left ventricular (LV) work and rates of glucose metabolism (glucose uptake, glycogen synthesis, glycolysis and glucose oxidation) and palmitate oxidation. Under aerobic conditions, endotoxemic hearts had impaired LV function as judged by echocardiography in vivo (% ejection fraction, 66.0 ± 3.2 vs 78.0 ± 2.1, p < 0.05) or by LV work ex vivo (2.14 ± 0.16 vs 3.28 ± 0.16, Joules min(-1) g dry wt(-1), p < 0.05). However, rates of glucose uptake, glycogen synthesis, glycolysis, and glucose oxidation were not altered. Palmitate oxidation was lower in endotoxemic hearts in proportion to the decreased workload, thus metabolic efficiency was unaffected. In hearts reperfused following global ischemia, untreated hearts had impaired recovery of LV work (52.3 ± 9.4 %) whereas endotoxemic hearts had significantly higher recovery (105.6 ± 11.3 %, p < 0.05). During reperfusion, fatty acid oxidation, acetyl CoA production and metabolic efficiency were similar in both groups. As impaired cardiac function appeared unrelated to depression of energy substrate oxidation, it is unlikely that drug-induced acceleration of fatty acid oxidation will improve mechanical function. The beneficial repartitioning of glucose metabolism in reperfused endotoxemic hearts may contribute to the cardioprotected phenotype.

Unraveling Interactions Between Anesthetics and the Endothelium: Update and Novel Insights.

The vascular endothelium is one of the largest organs in the body and consists of a single layer of highly specialized cells with site-specific morphology and functions. Endothelial cells play a vital role in the regulation of vascular tone in arterial, venous, microvascular, and lymphatic vascular beds. The endothelium also coordinates angiogenesis and controls cell adhesion, fluid homeostasis, and both innate and adaptive immunity. Fundamental research has shown that general and local anesthetics markedly modulate the biological activities of endothelial cells under aerobic and ischemia-reperfusion conditions, making the endothelium an important target of anesthetics in the cardiovascular system. Halogenated volatile anesthetics provide significant anti-inflammatory actions and protect the endothelium against ischemia-reperfusion injury, despite their inhibiting effects on endothelium-dependent vasorelaxation. They provide not only acute but also potential long-term, beneficial effects. Although many effects of IV anesthetics on endothelial function are controversial, or completely unexplored, propofol and opioids appear to have the most favorable profile with respect to the preservation of endothelial function. Some opioids and ketamine have stereoselective effects on the endothelium. Finally, there is experimental evidence to suggest important effects of anesthetics on the regulation of vascular permeability, proliferation of stem cells, including endothelial progenitor cells, and promotion or inhibition of tumor growth, potentially related to alterations in angiogenesis. However, most of these findings are from in vitro experiments and await confirmation in an in vivo setting. Thus, the clinical implications of these interactions remain uncertain.

Propofol (Diprivan®) and Intralipid® exacerbate insulin resistance in type-2 diabetic hearts by impairing GLUT4 trafficking.

BACKGROUND: The IV anesthetic, propofol, when administered as fat emulsion-based formulation (Diprivan) promotes insulin resistance, but the direct effects of propofol and its solvent, Intralipid, on cardiac insulin resistance are unknown. METHODS: Hearts of healthy and type-2 diabetic rats (generated by fructose feeding) were aerobically perfused for 60 minutes with 10 µM propofol in the formulation of Diprivan or an equivalent concentration of its solvent Intralipid (25 µM) ± insulin (100 mU•L). Glucose uptake, glycolysis, and glycogen metabolism were measured using [H]glucose. Activation of Akt, GSK3ß, AMPK, ERK1/2, p38MAPK, S6K1, JNK, protein kinase Cθ (PKCθ), and protein kinase CCßII (PKCßII) was determined using immunoblotting. GLUT4 trafficking and phosphorylations of insulin receptor substrate-1 (IRS-1) at Ser307(h312), Ser1100(h1101), and Tyr608(hTyr612) were measured. Mass spectrometry was used to determine acylcarnitines, phospholipids, and sphingolipids. RESULTS: Diprivan and Intralipid reduced insulin-induced glucose uptake and redirected glucose to glycogen stores in diabetic hearts. Reduced glucose uptake was accompanied by lower GLUT4 trafficking to the sarcolemma. Diprivan and Intralipid inactivated GSK3ß but activated AMPK and ERK1/2 in diabetic hearts. Only Diprivan increased phosphorylation of Akt(Ser473/Thr308) and translocated PKCθ and PKCßII to the sarcolemma in healthy hearts, whereas it activated S6K1 and p38MAPK and translocated PKCßII in diabetic hearts. Furthermore, only Diprivan phosphorylated IRS-1 at Ser1100(h1101) in healthy and diabetic hearts. JNK expression, phosphorylation of Ser307(h312) of IRS-1, and PKCθ expression and translocation were increased, whereas GLUT4 expression was reduced in insulin-treated diabetic hearts. Phosphatidylglycerol, phosphatidylethanolamine, and C18-sphingolipids accumulated in Diprivan-perfused and Intralipid-perfused diabetic hearts. CONCLUSIONS: Propofol and Intralipid promote insulin resistance predominantly in type-2 diabetic hearts.

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

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

Propofol ameliorates hyperglycemia-induced cardiac hypertrophy and dysfunction via heme oxygenase-1/signal transducer and activator of transcription 3 signaling pathway in rats.

OBJECTIVES: Heme oxygenase-1 is inducible in cardiomyocytes in response to stimuli such as oxidative stress and plays critical roles in combating cardiac hypertrophy and injury. Signal transducer and activator of transcription 3 plays a pivotal role in heme oxygenase-1-mediated protection against liver and lung injuries under oxidative stress. We hypothesized that propofol, an anesthetic with antioxidant capacity, may attenuate hyperglycemia-induced oxidative stress in cardiomyocytes via enhancing heme oxygenase-1 activation and ameliorate hyperglycemia-induced cardiac hypertrophy and apoptosis via heme oxygenase-1/signal transducer and activator of transcription 3 signaling and improve cardiac function in diabetes. DESIGN: Treatment study. SETTING: Research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: In vivo and in vitro treatments. MEASUREMENTS AND MAIN RESULTS: At 8 weeks of streptozotocin-induced type 1 diabetes in rats, myocardial 15-F2t-isoprostane was significantly increased, accompanied by cardiomyocyte hypertrophy and apoptosis and impaired left ventricular function that was coincident with reduced heme oxygenase-1 activity and signal transducer and activator of transcription 3 activation despite an increase in heme oxygenase-1 protein expression as compared to control. Propofol infusion (900 µg/kg/min) for 45 minutes significantly improved cardiac function with concomitantly enhanced heme oxygenase-1 activity and signal transducer and activator of transcription activation. Similar to the changes seen in diabetic rat hearts, high glucose (25 mmol/L) exposure for 48 hours led to cardiomyocyte hypertrophy and apoptosis, both in primary cultured neonatal rat cardiomyocytes and in H9c2 cells compared to normal glucose (5.5 mmol/L). Hypertrophy was accompanied by increased reactive oxygen species and malondialdehyde production and caspase-3 activity. Propofol, similar to the heme oxygenase-1 inducer cobalt protoporphyrin, significantly increased cardiomyocyte heme oxygenase-1 and p-signal transducer and activator of transcription protein expression and heme oxygenase-1 activity and attenuated high-glucose-mediated cardiomyocyte hypertrophy and apoptosis and reduced reactive oxygen species and malondialdehyde production (p < 0.05). These protective effects of propofol were abolished by heme oxygenase-1 inhibition with zinc protoporphyrin and by heme oxygenase-1 or signal transducer and activator of transcription 3 gene knockdown. CONCLUSIONS: Heme oxygenase-1/signal transducer and activator of transcription 3 signaling plays a critical role in propofol-mediated amelioration of hyperglycemia-induced cardiomyocyte hypertrophy and apoptosis, whereby propofol improves cardiac function in diabetic rats.

Adenosine-mediated inhibition of 5'-AMP-activated protein kinase and p38 mitogen-activated protein kinase during reperfusion enhances recovery of left ventricular mechanical function.

Attenuation of excessive rates of myocardial glycolysis limits proton production and Ca(2+) overload during reperfusion and improves recovery of post-ischemic left ventricular (LV) function. In order to elucidate mechanisms underlying glycolytic inhibition by adenosine (ADO), this study tested the hypothesis that the beneficial effects of ADO are due to Ser/Thr protein phosphatase (PP)-mediated inhibition of 5'-AMP-activated protein kinase (AMPK) and phosphofructokinase-2 (PFK-2). In isolated perfused working rat hearts subjected to global ischemia (GI) and reperfusion, ADO (500µmol/l), added 5min prior to the onset of GI and present throughout reperfusion, inhibits glycolysis and proton production during reperfusion and improves post-ischemic LV work. These metabolic effects of ADO are also evident during aerobic perfusion. Assays of glycolytic intermediates show that ADO-induced glycolytic inhibition occurs at the step catalyzed by PFK-1, an effect mediated by reduced activation of PFK-2 by AMPK. The PP1 and PP2A inhibitors, cantharidin (5µmol/l) or okadaic acid (0.1µmol/l), added 10min prior to ADO prevent ADO-induced inhibition of glycolysis and AMPK, as well as ADO-induced cardioprotection. ADO also inhibits p38 MAPK phosphorylation during reperfusion in a cantharidin-sensitive manner, and pharmacological inhibition of p38 MAPK (by SB202190, 10µmol/l) during reperfusion also reduces glycolysis and is cardioprotective. These results indicate that attenuation of glycolysis during reperfusion and cardioprotection can be achieved by inhibition of the stress kinases, AMPK and p38 MAPK.

Late sodium current inhibition alone with ranolazine is sufficient to reduce ischemia- and cardiac glycoside-induced calcium overload and contractile dysfunction mediated by reverse-mode sodium/calcium exchange.

Excessive reverse-mode (RM) sodium/calcium exchanger 1.1 (NCX1.1) activity, resulting from intracellular sodium accumulation caused by reduced Na+/K+-ATPase activity, increased Na-H exchanger 1 activity. The induction of the voltage-gated sodium channel late current component (late INa), is a major pathway for intracellular calcium (Ca2+i) loading in cardiac ischemia-reperfusion (IR) injury and cardiac glycoside toxicity. Inhibition of late INa with the antianginal agent ranolazine is protective in models of IR injury and cardiac glycoside toxicity. However, whether inhibition of late INa alone is sufficient to provide maximal protection or additional inhibition of RM NCX1.1 provides further benefit remains to be determined conclusively. Therefore, the effects of ranolazine were compared with the INa inhibitor lidocaine in models of IR injury and ouabain toxicity, RM NCX1.1-mediated Ca2+ overload, and patch-clamp assays of RM NCX1.1 currents. Ranolazine and lidocaine (10 µM) similarly reduced Ca2+i overload and improved left ventricle work recovery in whole-heart models of IR injury or exposure to ouabain (80 µM). Ranolazine (10 µM), but not lidocaine (10 µM), reduced RM NCX1.1-mediated Ca2+i overload in ventricular myocytes. Furthermore, ranolazine inhibited RM NCX1.1 currents (IC50 1.7 µM), without affecting forward mode currents, revealing that ranolazine has novel RM NCX1.1 inhibitory actions. However, because lidocaine provides similar protection to ranolazine in whole-heart models but does not inhibit RM NCX1.1, we conclude that induction of late INa is upstream of RM NCX1.1 activity and selective inhibition of late INa alone is sufficient to reduce Ca2+i overload and contractile dysfunction in IR injury and cardiac glycoside toxicity.

Choice of anesthetic combination determines Ca2+ leak after ischemia-reperfusion injury in the working rat heart: favorable versus adverse combinations.

BACKGROUND: There is a lack of studies investigating cardioprotection by common combinations of anesthetics. However, because a general anesthetic consists of a mixture of drugs with potentially interfering effects on signaling and cytoprotection, the most favorable combination should be used. METHODS: Working rat hearts were exposed to 20 min of ischemia and 30 min of reperfusion. Periischemic sevoflurane (2 vol-%), propofol (10 µM), or remifentanil (3 nM) (single treatments) and the three combinations thereof (combination treatments) were assessed for their ability to improve postischemic left ventricular work and to prevent intracellular Ca leak and overload. Beat-to-beat oscillations in intracellular [Ca] were measured using indo-1 AM. Phosphorylation of calcium/calmodulin-dependent protein kinase IIδ, ryanodine receptor-2, and phospholamban was determined. RESULTS: The single treatments with sevoflurane or remifentanil were highly protective with respect to functional recovery and Ca overload, but propofol, even at high concentrations, did not show similar protection. Sevoflurane combined with propofol completely lost its protection in the presence of low sedative propofol concentrations (≥1 µM), whereas remifentanil combined with propofol (10 µM) retained its protection. Propofol antagonism of sevoflurane protection was concentration-dependent and mimicked by the reactive oxygen species scavenger N-2-mercaptopropionyl-glycine. Addition of propofol to sevoflurane activated calcium/calmodulin-dependent protein kinase type IIδ and hyperphosphorylated the ryanodine receptor-2, consistent with causing a postischemic Ca leak from the sarcoplasmic reticulum. Remifentanil did not enhance sevoflurane protection. CONCLUSIONS: The choice of anesthetic combination determines the postischemic Ca leak and intracellular overload. The results from these experiments will help to design studies for optimizing perioperative cardioprotection in high-risk surgical patients.

Remote ischemic preconditioning applied during isoflurane inhalation provides no benefit to the myocardium of patients undergoing on-pump coronary artery bypass graft surgery: lack of synergy or evidence of antagonism in cardioprotection?

BACKGROUND: Two preconditioning stimuli should induce a more consistent overall cell protection. We hypothesized that remote ischemic preconditioning (RIPC, second preconditioning stimulus) applied during isoflurane inhalation (first preconditioning stimulus) would provide more protection to the myocardium of patients undergoing on-pump coronary artery bypass grafting. METHODS: In this placebo-controlled randomized controlled study, patients in the RIPC group received four 5-min cycles of 300 mmHg cuff inflation/deflation of the leg before aortic cross-clamping. Anesthesia consisted of opioids and propofol for induction and isoflurane for maintenance. The primary outcome was high-sensitivity cardiac troponin T release. Secondary endpoints were plasma levels of N-terminal pro-brain natriuretic peptide, high-sensitivity C-reactive protein, S100 protein, and short- and long-term clinical outcomes. Gene expression profiles were obtained from atrial tissue using microarrays. RESULTS: RIPC (n = 27) did not reduce high-sensitivity cardiac troponin T release when compared with placebo (n = 28). Likewise, N-terminal pro-brain natriuretic peptide, a marker of myocardial dysfunction; high-sensitivity C-reactive protein, a marker of perioperative inflammatory response; and S100, a marker of cerebral injury, were not different between the groups. The incidence for the perioperative composite endpoint combining new arrhythmias and myocardial infarctions was higher in the RIPC group than the placebo group (14/27 vs. 6/28, P = 0.036). However, there was no difference in the 6-month cardiovascular outcome. N-terminal pro-brain natriuretic peptide release correlated with isoflurane-induced transcriptional changes in fatty-acid metabolism (P = 0.001) and DNA-damage signaling (P < 0.001), but not with RIPC-induced changes in gene expression. CONCLUSIONS: RIPC applied during isoflurane inhalation provides no benefit to the myocardium of patients undergoing on-pump coronary artery bypass grafting.

Metabolic profiling of hearts exposed to sevoflurane and propofol reveals distinct regulation of fatty acid and glucose oxidation: CD36 and pyruvate dehydrogenase as key regulators in anesthetic-induced fuel shift.

BACKGROUND: Myocardial energy metabolism is a strong predictor of postoperative cardiac function. This study profiled the metabolites and metabolic changes in the myocardium exposed to sevoflurane, propofol, and Intralipid and investigated the underlying molecular mechanisms. METHODS: Sevoflurane (2 vol%) and propofol (10 and 100 microM) in the formulation of 1% Diprivan (AstraZeneca Inc., Mississauga, ON, Canada) were compared for their effects on oxidative energy metabolism and contractility in the isolated working rat heart model. Intralipid served as a control. Substrate flux through the major pathways for adenosine triphosphate generation in the heart, that is, fatty acid and glucose oxidation, was measured using [H]palmitate and [C]glucose. Biochemical analyses of nucleotides, acyl-CoAs, ceramides, and 32 acylcarnitine species were used to profile individual metabolites. Lipid rafts were isolated and used for Western blotting of the plasma membrane transporters CD36 and glucose transporter 4. RESULTS: Metabolic profiling of the hearts exposed to sevoflurane and propofol revealed distinct regulation of fatty acid and glucose oxidation. Sevoflurane selectively decreased fatty acid oxidation, which was closely related to a marked reduction in left ventricular work. In contrast, propofol at 100 microM but not 10 microM increased glucose oxidation without affecting cardiac work. Sevoflurane decreased fatty acid transporter CD36 in lipid rafts/caveolae, whereas high propofol increased pyruvate dehydrogenase activity without affecting glucose transporter 4, providing mechanisms for the fuel shifts in energy metabolism. Propofol increased ceramide formation, and Intralipid increased hydroxy acylcarnitine species. CONCLUSIONS: Anesthetics and their solvents elicit distinct metabolic profiles in the myocardium, which may have clinical implications for the already jeopardized diseased heart.

High rates of residual fatty acid oxidation during mild ischemia decrease cardiac work and efficiency.

It is unknown what effects high levels of fatty acids have on energy metabolism and cardiac efficiency during milder forms of ischemia. To address this issue, isolated working rat hearts perfused with Krebs-Henseleit solution (5 mM glucose, 100 muU/mL insulin, and 0.4 (Normal Fat) or 1.2 mM palmitate (High Fat)) were subjected to 30 min of aerobic perfusion followed by 30 min of mild ischemia (39% reduction in coronary flow). Both groups had similar aerobic function and rates of glycolysis, however the High Fat group had elevated rates of palmitate oxidation (150%), and decreased rates of glucose oxidation (51%). Mild ischemia decreased cardiac work (56% versus 40%) and efficiency (29% versus 11%) further in High Fat hearts. Palmitate oxidation contributed a greater percent of acetyl-CoA production during mild ischemia in the High Fat group (81% versus 54%). During mild ischemia glycolysis remained at aerobic levels in the Normal Fat group, but was accelerated in the High Fat group. Triglyceride, glycogen and adenine nucleotide content did not differ at the end of mild ischemia, however glycogen turnover was double in the High Fat group (248%). Addition of the pyruvate dehydrogenase inhibitor dichloroacetate to the High Fat group resulted in a doubling of the rate of glucose oxidation and improved cardiac efficiency during mild ischemia. We demonstrate that fatty acid oxidation dominates as the main source of residual oxidative metabolism during mild ischemia, which is accompanied by suppressed cardiac function and efficiency in the presence of high fat.

Suppression of 5'-AMP-activated protein kinase activity does not impair recovery of contractile function during reperfusion of ischemic hearts.

Activation of 5'-AMP-activated protein kinase (AMPK) may benefit the heart during ischemia-reperfusion by increasing energy production. While AMPK stimulates glycolysis, mitochondrial oxidative metabolism is the major source of ATP production during reperfusion of ischemic hearts. Stimulating AMPK increases mitochondrial fatty acid oxidation, but this is usually accompanied by a decrease in glucose oxidation, which can impair the functional recovery of ischemic hearts. To examine the relationship between AMPK and cardiac energy substrate metabolism, we subjected isolated working mouse hearts expressing a dominant negative (DN) alpha(2)-subunit of AMPK (AMPK-alpha(2) DN) to 20 min of global no-flow ischemia and 40 min of reperfusion with Krebs-Henseleit solution containing 5 mM [U-(14)C]glucose, 0.4 mM [9, 10-(3)H]palmitate, and 100 microU/ml insulin. AMPK-alpha(2) DN hearts had reduced AMPK activity at the end of reperfusion (82 +/- 9 vs. 141 +/- 7 pmol.mg(-1).min(-1)) with no changes in high-energy phosphates. Despite this, AMPK-alpha(2) DN hearts had improved recovery of function during reperfusion (14.9 +/- 0.8 vs. 9.4 +/- 1.4 beats.min(-1).mmHg.10(-3)). During reperfusion, fatty acid oxidation provided 44.0 +/- 2.8% of total acetyl-CoA in AMPK-alpha(2) DN hearts compared with 55.0 +/- 3.2% in control hearts. Since insulin can inhibit both AMPK activation and fatty acid oxidation, we also examined functional recovery in the absence of insulin. Functional recovery was similar in both groups despite a decrease in AMPK activity and a decreased reliance on fatty acid oxidation during reperfusion (66.4 +/- 9.4% vs. 85.3 +/- 4.3%). These data demonstrate that the suppression of cardiac AMPK activity does not produce an energetically compromised phenotype and does not impair, but may in fact improve, the recovery of function after ischemia.

H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation.

Reactive oxygen species (ROS) and intracellular Ca(2+) overload play key roles in myocardial ischemia-reperfusion (IR) injury but the relationships among ROS, Ca(2+) overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H(2)O(2) impairs LV function by causing Ca(2+) overload by increasing late sodium current (I(Na)), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca(2+) concentrations (d[Ca(2+)](i) and s[Ca(2+)](i)) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H(2)O(2) (100 microM, 30 min) increased d[Ca(2+)](i) and s[Ca(2+)](i). LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca(2+)](i) and s[Ca(2+)](i). LV work increased transiently before declining gradually to 17%. Ouabain (80 microM) exerted similar effects to ATX-II. Late I(Na) inhibitors, lidocaine (10 microM) or R56865 (2 microM), reduced effects of ATX-II on [Ca(2+)](i) and LV function, but did not alter effects of H(2)O(2). The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H(2)O(2)-induced LV dysfunction, but did not alter [Ca(2+)](i). Paradoxically, further increases in [Ca(2+)](i) by ATX-II or ouabain, given 10 min after H(2)O(2), improved function. The failure of late I(Na) inhibitors to prevent H(2)O(2)-induced LV dysfunction, and the ability of MPG to prevent H(2)O(2)-induced LV dysfunction independent of changes in [Ca(2+)](i) indicate that impaired contractility is not due to Ca(2+) overload. The ability of further increases in [Ca(2+)](i) to reverse H(2)O(2)-induced LV dysfunction suggests that Ca(2+) desensitization is the predominant mechanism of ROS-induced contractile dysfunction.