Optimizing resuscitation of the donation after circulatory death heart by mitochondrial protection in a female porcine model.

BACKGROUND: Due to the shortage of donor organs, an increasing number of transplant organs are harvested after circulatory arrest (donation after circulatory death, DCD). Using a translational porcine DCD model, we developed and evaluated a protocol based on cardioprotection by multi-drug postconditioning to optimize resuscitation of DCD hearts by ex situ heart perfusion (ESHP). METHODS: Hearts of female pigs (45.0±4.5 kg) were procured following a clinically identical DCD protocol, consisting of the termination of ventilator support and confirmation of circulatory arrest, followed by a 15-min standoff period. DCD hearts were randomly allocated to ESHP (38.4°C) in the absence (untreated, N=5) or presence (treated, N=5) of a postconditioning treatment added to the perfusate, consisting of Intralipid (1%), sevoflurane (2% v/v), and remifentanil (3 nM). All hearts were perfused with blood and Krebs-Henseleit solution (1:1) for 60 min in Langendorff mode and for additional 300 min in working mode for a total perfusion time of 6 hrs. Oxidative capacity and detailed left ventricular (LV) mechanical function under increasing workload (left atrial pressure 6-12 mmHg) were assessed hourly. LV tissue was snap-frozen at the end of ESHP and used for molecular analyses. RESULTS: LV inotropy (LVdP/dtmax) did not decline over time in treated DCD hearts and was significantly higher at the end of the protocol as compared with untreated DCD hearts (ΔLVdP/dtmax= 440 mmHg/s, p=0.009). Treated DCD hearts exhibited persistently higher LV stroke work index (LVSWI) during the 6-hr period of ESHP, whereas untreated DCD hearts displayed a significant decline (ΔLVSWI=-3.10 mL*mmHg/g, p(time within untreated group)<0.001). Treated DCD hearts displayed higher metabolic activity as measured by oxygen consumption (ΔO2=3.11 mL O2/min/100g; p=0.004), and released lower amounts of cell-free mitochondrial DNA into the perfusate, a marker of potential graft dysfunction. Treated hearts also used fatty acids from Intralipid as an energy source, while untreated DCD hearts showed glyceroneogenesis with triglyceride accumulation and depletion of tricarboxylic acid cycle intermediates, reduced mitochondrial complex I, II, and III activities with accumulation of mitochondrial NADH, and signs of ultrastructural damage. CONCLUSIONS: A translationally relevant protective ESHP protocol consisting of treatment with Intralipid, sevoflurane, and remifentanil markedly accelerated functional recovery and improved viability of DCD hearts.

Novel lipid emulsion for total parenteral nutrition based on 18-carbon n-3 fatty acids elicits a superior immunometabolic phenotype in a murine model compared with standard lipid emulsions.

BACKGROUND: While lipid emulsions in modern formulations for total parenteral nutrition (TPN) provide essential fatty acids and dense calories, they also promote inflammation and immunometabolic disruptions. OBJECTIVES: We aimed to develop a novel lipid emulsion for TPN use with superior immunometabolic actions compared with available standard lipid emulsions. METHODS: A novel lipid emulsion [Vegaven (VV)] containing 30% of 18-carbon n-3 fatty acids (α-linolenic acid and stearidonic acid) was developed for TPN (VV-TPN) and compared with TPN containing soybean oil-based lipid emulsion (IL-TPN) and fish-oil-based lipid emulsion (OV-TPN). In vivo studies were performed in instrumented male C57BL/6 mice subjected to 7-d TPN prior to analysis of cytokines, indices of whole-body and hepatic glucose metabolism, immune cells, lipid mediators, and mucosal bowel microbiome. RESULTS: IL-6 to IL-10 ratios were significantly lower in liver and skeletal muscle of VV-TPN mice when compared with IL-TPN or OV-TPN mice. VV-TPN and OV-TPN each increased hepatic insulin receptor abundance and resulted in similar HOMA-IR values, whereas only VV-TPN increased hepatic insulin receptor substrate 2 and maintained normal hepatic glycogen content, effects that were IL-10-dependent and mediated by glucokinase activation. The percentages of IFN-γ- and IL-17-expressing CD4+ T cells were increased in livers of VV-TPN mice, and liver macrophages exhibited primed phenotypes when compared with IL-TPN. This immunomodulation was associated with successful elimination of the microinvasive bacterium Akkermansia muciniphila from the bowel mucosa by VV-TPN as opposed to standard lipid emulsions. Assay of hepatic lipid mediators revealed a distinct profile with VV-TPN, including increases in 9(S)-hydroxy-octadecatrienoic acid. When co-administered with IL-TPN, hydroxy-octadecatrienoic acids mimicked the VV-TPN immunometabolic phenotype. CONCLUSIONS: We here report the unique anti-inflammatory, insulin-sensitizing, and immunity-enhancing properties of a newly developed lipid emulsion designed for TPN use based on 18-carbon n-3 fatty acids.

Lipid emulsion rich in n-3 polyunsaturated fatty acids elicits a pro-resolution lipid mediator profile in mouse tissues and in human immune cells.

BACKGROUND: Lipid emulsions are a key component of total parenteral nutrition (TPN) and are administered to patients who are unable to ingest their daily required calories orally. Lipid emulsions rich with n-6 (ω-6) PUFAs are known to cause parenteral nutrition-associated liver disease and have inflammatory side effects, whereas n-3 PUFA-rich emulsions have favourable clinical outcomes. OBJECTIVES: The present study used targeted lipid mediator analysis to investigate the metabolism of a n-3 PUFA-rich lipid emulsion and a n-6 PUFA-rich lipid emulsion in a mouse model of TPN and in primary human monocyte-derived macrophages (MDMs) and CD4+ T cells. RESULTS: Mice given n-3 PUFA-based TPN for 7 d had a less proinflammatory lipid mediator profile compared with those receiving n-6 PUFA-based TPN. This was characterized by higher concentrations of specialized pro-resolving mediators (SPMs) and endocannabinoids, including resolvin D (RvD) 1, maresin (MaR) 1, MaR2, protectin D1 (PD1), protectin DX (PDX), and the endocannabinoids eicosapentaenoyl ethanolamide (EPEA) and docosahexaenoyl ethanolamide (DHEA) in the liver and RvD1, 17R-RvD1, RvD2, RvD3, RvD5, MaR1, MaR2, PD1, PDX, and EPEA and DHEA in the spleen. The spleen was identified as a source of high lipid mediator and SPM formation as lipid mediator concentrations were on average 25-fold higher than in the liver. Additionally, n-3 PUFA-treated primary human MDMs produced RvD5 and the endocannabinoids EPEA and DHEA, which was associated with an increased IL-10 secretion. In contrast, primary human CD4+ T cells showed only an increase in SPM precursors and an increase in the endocannabinoids EPEA and DHEA, which was associated with reduced cytokine expression. CONCLUSIONS: This demonstrates that lipid mediators, particularly SPMs and endocannabinoids from spleen, could play a key role in facilitating the favorable clinical outcomes associated with the use of n-3 PUFA-rich lipid emulsions in TPN.

Gut microbiome and circulating bacterial DNA ("blood microbiome") in a mouse model of total parenteral nutrition: Evidence of two distinct separate microbiotic compartments.

BACKGROUND & AIMS: Total parenteral nutrition (TPN) causes gut atrophy, dysbiosis and leakage of the gut barrier. This study aimed to characterize the gut microbiome in response to different TPNs and tested the hypothesis whether increased gut permeability in TPN would lead to changes in the circulating bacterial DNA ("blood microbiome"). METHODS: Male C57BL/6J mice were randomly allocated to the following groups for seven days (1) chow-fed control (C) without jugular vein catheter (JVC, n=6) (2) chow-fed with JVC and infusion of saline (S) (n = 6) (3) Intralipid-based TPN (n-6:n-3 ratio 7:1) (IL, n = 6) (4) Omegaven-based TPN (n-6:n-3 ratio 1:8) (OV, n = 6). Blood was collected by cardiac puncture and feces (stool pellet) were collected from the colon. Blood and stool samples were analyzed by 16S rRNA gene sequencing. RESULTS: TPN administration was associated with a compositional shift in the gut microbial community that involved the expansion of Bacteroidota along with a decrease in gut bacteria belonging to the Firmicutes phylum as compared to chow-fed mice. Gram-negative Verrucomicrobiota and Proteobacteria were also increased in the gut microbiome of mice receiving TPN. Gammaproteobacteria, namely Burkholderiales, were specifically increased in Intralipid-based TPN. On the other hand, Proteobacteria and Actinobacteriota were the dominant taxa in blood samples. The families Comamonadaceae and Burkholderiaceae (both from Burkholderiales order) were increased in the "blood microbiome" of mice with indwelling JVC when compared with chow-fed mice without JVC. The increase in Burkholderiaceae was more pronounced in Intralipid-based TPN. CONCLUSIONS: Profound changes in the gut microbiome of mice subjected to TPN occurred, which were not reflected in the "blood microbiome" suggesting that the gut and "blood microbiome" represent two rather distinct separate microbiotic compartments. The parenteral provision of n-3 fatty acids appears to protect against proinflammatory bacteria in the gut and against the increased presence of JVC-associated bacteria as measured by circulating bacterial DNA.

Quantitative profiling of inflammatory and pro-resolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS.

OBJECTIVES: Lipid mediators are bioactive lipids which help regulate inflammation. We aimed to develop an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify 58 pro-inflammatory and pro-resolving lipid mediators in plasma, determine preliminary reference ranges for adolescents, and investigate how total parenteral nutrition (TPN) containing omega-3 polyunsaturated fatty acid (n-3 PUFA) or n-6 PUFA based lipid emulsions influence lipid mediator concentrations in plasma. METHODS: Lipid mediators were extracted from plasma using SPE and measured using UHPLC-MS/MS. EDTA plasma was collected from healthy adolescents between 13 and 17 years of age to determine preliminary reference ranges and from mice given intravenous TPN for seven days containing either an n-3 PUFA or n-6 PUFA based lipid emulsion. RESULTS: We successfully quantified 43 lipid mediators in human plasma with good precision and recovery including several leukotrienes, prostaglandins, resolvins, protectins, maresins, and lipoxins. We found that the addition of methanol to human plasma after blood separation reduces post blood draw increases in 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE), 12S-hydroxyeicosatrienoic acid (12S-HETrE), 14-hydroxydocosahexaenoic acid (14-HDHA) and thromboxane B2 (TXB2). Compared to the n-6 PUFA based TPN, the n-3 PUFA based TPN increased specialized pro-resolving mediators such as maresin 1 (MaR1), MaR2, protectin D1 (PD1), PDX, and resolvin D5 (RvD5), and decreased inflammatory lipid mediators such as leukotriene B4 (LTB4) and prostaglandin D2 (PGD2). CONCLUSIONS: Our method provides an accurate and sensitive quantification of 58 lipid mediators from plasma samples, which we used to establish a preliminary reference range for lipid mediators in plasma samples of adolescents; and to show that n-3 PUFA, compared to n-6 PUFA rich TPN, leads to a less inflammatory lipid mediator profile in mice.

Nutritional Lipids and Mucosal Inflammation.

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation in the intestine. Given their role in regulation of inflammation, long-chain n-3 polyunsaturated fatty acids (PUFAs) represent a potential supplementary therapeutic approach to current drug regimens used for IBD. Mechanistically, there is ample evidence for an anti-inflammatory and pro-resolution effect of long-chain n-3 PUFAs after they incorporate into cell membrane phospholipids. They disrupt membrane rafts and when released from the membrane suppress inflammatory signaling by activating PPAR-γ and free fatty acid receptor 4; furthermore, they shift the lipid mediator profile from pro-inflammatory eicosanoids to specialized pro-resolving mediators. The allocation of long-chain n-3 PUFAs also leads to a higher microbiome diversity in the gut, increases short-chain fatty acid-producing bacteria, and improves intestinal barrier function by sealing epithelial tight junctions. In line with these mechanistic studies, most epidemiological studies support a beneficial effect of long-chain n-3 PUFAs intake on reducing the incidence of IBD. However, the results from intervention trials on the prevention of relapse in IBD patients show no or only a marginal effect of long-chain n-3 PUFAs supplementation. In light of the current literature, international recommendations are supported that adequate diet-derived n-3 PUFAs might be beneficial in maintaining remission in IBD patients.

Novel Strategies to Prevent Total Parenteral Nutrition-Induced Gut and Liver Inflammation, and Adverse Metabolic Outcomes.

Total parenteral nutrition (TPN) is a life-saving therapy administered to millions of patients. However, it is associated with significant adverse effects, namely liver injury, risk of infections, and metabolic derangements. In this review, the underlying causes of TPN-associated adverse effects, specifically gut atrophy, dysbiosis of the intestinal microbiome, leakage of the epithelial barrier with bacterial invasion, and inflammation are first described. The role of the bile acid receptors farnesoid X receptor and Takeda G protein-coupled receptor, of pleiotropic hormones, and growth factors is highlighted, and the mechanisms of insulin resistance, namely the lack of insulinotropic and insulinomimetic signaling of gut-originating incretins as well as the potentially toxicity of phytosterols and pro-inflammatory fatty acids mainly released from soybean oil-based lipid emulsions, are discussed. Finally, novel approaches in the design of next generation lipid delivery systems are proposed. Propositions include modifying the physicochemical properties of lipid emulsions, the use of lipid emulsions generated from sustainable oils with favorable ratios of anti-inflammatory n-3 to pro-inflammatory n-6 fatty acids, beneficial adjuncts to TPN, and concomitant pharmacotherapies to mitigate TPN-associated adverse effects.

Choice of Lipid Emulsion Determines Inflammation of the Gut-Liver Axis, Incretin Profile, and Insulin Signaling in a Murine Model of Total Parenteral Nutrition.

SCOPE: The aim of this study is to test whether the choice of the lipid emulsion in total parenteral nutrition (TPN), that is, n-3 fatty acid-based Omegaven versus n-6 fatty acid-based Intralipid, determines inflammation in the liver, the incretin profile, and insulin resistance. METHODS AND RESULTS: Jugular vein catheters (JVC) are placed in C57BL/6 mice and used for TPN for 7 days. Mice are randomized into a saline group (saline infusion with oral chow), an Intralipid group (IL-TPN, no chow), an Omegaven group (OV-TPN, no chow), or a chow only group (without JVC). Both TPN elicite higher abundance of lipopolysaccharide binding protein in the liver, but only IL-TPN increases interleukin-6 and interferon-γ, while OV-TPN reduces interleukin-4, monocyte chemoattractant protein-1, and interleukin-1α. Insulin plasma concentrations are higher in both TPN, while glucagon and glucagon-like peptide-1 (GLP-1) were higher in IL-TPN. Gluconeogenesis is increased in IL-TPN and the nuclear profile of key metabolic transcription factors shows a liver-protective phenotype in OV-TPN. OV-TPN increases insulin sensitivity in the liver and skeletal muscle. CONCLUSION: OV-TPN as opposed to IL-TPN mitigates inflammation in the liver and reduces the negative metabolic effects of hyperinsulinemia and hyperglucagonemia by "re-sensitizing" the liver and skeletal muscle to insulin.

The ER chaperone calnexin controls mitochondrial positioning and respiration.

Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.

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.

Alterations in fatty acid metabolism and sirtuin signaling characterize early type-2 diabetic hearts of fructose-fed rats.

Despite the fact that skeletal muscle insulin resistance is the hallmark of type-2 diabetes mellitus (T2DM), inflexibility in substrate energy metabolism has been observed in other tissues such as liver, adipose tissue, and heart. In the heart, structural and functional changes ultimately lead to diabetic cardiomyopathy. However, little is known about the early biochemical changes that cause cardiac metabolic dysregulation and dysfunction. We used a dietary model of fructose-induced T2DM (10% fructose in drinking water for 6 weeks) to study cardiac fatty acid metabolism in early T2DM and related signaling events in order to better understand mechanisms of disease. In early type-2 diabetic hearts, flux through the fatty acid oxidation pathway was increased as a result of increased cellular uptake (CD36), mitochondrial uptake (CPT1B), as well as increased ß-hydroxyacyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase activities, despite reduced mitochondrial mass. Long-chain acyl-CoA dehydrogenase activity was slightly decreased, resulting in the accumulation of long-chain acylcarnitine species. Cardiac function and overall mitochondrial respiration were unaffected. However, evidence of oxidative stress and subtle changes in cardiolipin content and composition were found in early type-2 diabetic mitochondria. Finally, we observed decreased activity of SIRT1, a pivotal regulator of fatty acid metabolism, despite increased protein levels. This indicates that the heart is no longer capable of further increasing its capacity for fatty acid oxidation. Along with increased oxidative stress, this may represent one of the earliest signs of dysfunction that will ultimately lead to inflammation and remodeling in the diabetic heart.

Postconditioning with Intralipid emulsion protects against reperfusion injury in post-infarct remodeled rat hearts by activation of ROS-Akt/Erk signaling.

The clinically used lipid emulsion Intralipid (ILE) reduces ischemia reperfusion injury in healthy rodent hearts. We tested whether ILE is cardioprotective in postinfarct remodeled hearts. Post-infarct remodeled and sham Sprague-Dawley rat hearts were perfused in working mode and subjected to ischemia (15 minutes) and reperfusion (30 minutes). Left ventricular (LV) work was measured in hearts that were untreated or that received ILE (1%) postconditioning administered at the onset of reperfusion, or the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)-glycine (10 µM) alone or in combination with ILE. Mitochondrial O2 consumption was measured in LV muscle fibers. Acetyl CoA production was calculated from the oxidation of [U-14C]glucose and [9,10-3H]palmitate. ROS production was assessed by loss of aconitase activity as well as by release of hydrogen peroxide. Phosphorylation of Akt, Erk1/2, and STAT3 were used to evaluate protection signaling. Remodeled hearts exhibited LV dysfunction and signs of hypertrophy consistent with significant postinfarct remodeling. ILE postconditioning enhanced the recovery of postischemic LV function in remodeled hearts, preserved energy metabolism in mitochondria, accelerated palmitate oxidation and acetyl CoA production, and activated Akt/Erk/STAT3 in a ROS-dependent manner. Protection by ILE postconditioning evolved rapidly within the first minutes of reperfusion without evidence of additional cardiotonic effects due to provision of supplementary energy substrates potentially released from ILE during reperfusion. ILE represents a novel and clinically feasible cardioprotective strategy that is highly effective in remodeled hearts. Our data provide a rationale for the clinical evaluation of ILE postconditioning where ILE is administered as a bolus at the onset of reperfusion.