Perturbations in lipid metabolism and gut microbiota composition precede cardiac dysfunction in a mouse model of thalassemia.

Cardiomyopathy is a major complication of thalassemia, yet the precise underlying molecular mechanisms remain unclear. We examined whether altered lipid metabolism is an early driving factor in the development of cardiomyopathy using the Th3/+ mouse model of thalassemia. At age 20 weeks, male and female Th3/+ mice manifested anemia and iron overload; however, only males displayed metabolic defects and altered cardiac function. Untargeted lipidomics indicated that the circulating levels of 35 lipid species were significantly altered in Th3/+ mice compared to wild-type controls: triglycerides (TGs) with saturated fatty acids (FAs; TG42:0 and TG44:0) were elevated, while TGs with unsaturated FAs (TG(18:2_20:5_18:2 and TG54:8)) were reduced. Similarly, phosphatidylcholines (PCs) with long chain FAs (palmitic (16:0) or oleic (18:1)) were increased, while PCs with polyunsaturated FAs decreased. Circulating PC(16:0_14:0), GlcCer(d18:1/24:0) correlated significantly with iron overload and cardiac hypertrophy. 16S rRNA gene profiling revealed alterations in the intestinal microbiota of Th3/+ mice. Differentially abundant bacterial genera correlated with PC(39:6), PC(18:1_22:6), GlcCer(d18:1/24:1) and CE(14:0). These results provide new knowledge on perturbations in lipid metabolism and the gut microbiota of Th3/+ mice and identify specific factors which may represent early biomarkers or therapeutic targets to prevent development of cardiomyopathy in ß-thalassemia.

Impact of obesity on day-night differences in cardiac metabolism.

An intrinsic property of the heart is an ability to rapidly and coordinately adjust flux through metabolic pathways in response to physiologic stimuli (termed metabolic flexibility). Cardiac metabolism also fluctuates across the 24-hours day, in association with diurnal sleep-wake and fasting-feeding cycles. Although loss of metabolic flexibility has been proposed to play a causal role in the pathogenesis of cardiac disease, it is currently unknown whether day-night variations in cardiac metabolism are altered during disease states. Here, we tested the hypothesis that diet-induced obesity disrupts cardiac "diurnal metabolic flexibility", which is normalized by time-of-day-restricted feeding. Chronic high fat feeding (20-wk)-induced obesity in mice, abolished diurnal rhythms in whole body metabolic flexibility, and increased markers of adverse cardiac remodeling (hypertrophy, fibrosis, and steatosis). RNAseq analysis revealed that 24-hours rhythms in the cardiac transcriptome were dramatically altered during obesity; only 22% of rhythmic transcripts in control hearts were unaffected by obesity. However, day-night differences in cardiac substrate oxidation were essentially identical in control and high fat fed mice. In contrast, day-night differences in both cardiac triglyceride synthesis and lipidome were abolished during obesity. Next, a subset of obese mice (induced by 18-wks ad libitum high fat feeding) were allowed access to the high fat diet only during the 12-hours dark (active) phase, for a 2-wk period. Dark phase restricted feeding partially restored whole body metabolic flexibility, as well as day-night differences in cardiac triglyceride synthesis and lipidome. Moreover, this intervention partially reversed adverse cardiac remodeling in obese mice. Collectively, these studies reveal diurnal metabolic inflexibility of the heart during obesity specifically for nonoxidative lipid metabolism (but not for substrate oxidation), and that restricting food intake to the active period partially reverses obesity-induced cardiac lipid metabolism abnormalities and adverse remodeling of the heart.

First characterization of glucose flux through the hexosamine biosynthesis pathway (HBP) in ex vivo mouse heart.

The hexosamine biosynthesis pathway (HBP) branches from glycolysis and forms UDP-GlcNAc, the moiety for O-linked ß-GlcNAc (O-GlcNAc) post-translational modifications. An inability to directly measure HBP flux has hindered our understanding of the factors regulating protein O-GlcNAcylation. Our goals in this study were to (i) validate a LC-MS method that assesses HBP flux as UDP-GlcNAc (13C)-molar percent enrichment (MPE) and concentration and (ii) determine whether glucose availability or workload regulate cardiac HBP flux. For (i), we perfused isolated murine working hearts with [U-13C6]glucosamine (1, 10, 50, or 100 µm), which bypasses the rate-limiting HBP enzyme. We observed a concentration-dependent increase in UDP-GlcNAc levels and MPE, with the latter reaching a plateau of 56.3 ± 2.9%. For (ii), we perfused isolated working hearts with [U-13C6]glucose (5.5 or 25 mm). Glycolytic efflux doubled with 25 mm [U-13C6]glucose; however, the calculated HBP flux was similar among the glucose concentrations at ∼2.5 nmol/g of heart protein/min, representing ∼0.003-0.006% of glycolysis. Reducing cardiac workload in beating and nonbeating Langendorff perfusions had no effect on the calculated HBP flux at ∼2.3 and 2.5 nmol/g of heart protein/min, respectively. To the best of our knowledge, this is the first direct measurement of glucose flux through the HBP in any organ. We anticipate that these methods will enable foundational analyses of the regulation of HBP flux and protein O-GlcNAcylation. Our results suggest that in the healthy ex vivo perfused heart, HBP flux does not respond to acute changes in glucose availability or cardiac workload.

Reducing 14-3-3ζ expression influences adipocyte maturity and impairs function.

One of the primary metabolic functions of a mature adipocyte is to supply energy via lipolysis, or the catabolism of stored lipids. Adipose triacylglycerol lipase (ATGL) and hormone-sensitive lipase (HSL) are critical lipolytic enzymes, and their phosphorylation generates phospho-binding sites for 14-3-3 proteins, a ubiquitously expressed family of molecular scaffolds. Although we previously identified essential roles of the 14-3-3ζ isoform in murine adipogenesis, the presence of 14-3-3 protein binding sites on ATGL and HSL suggests that 14-3-3ζ could also influence mature adipocyte processes like lipolysis. Here we demonstrate that 14-3-3ζ is necessary for lipolysis in male mice and fully differentiated 3T3-L1 adipocytes, as depletion of 14-3-3ζ significantly impaired glycerol and free fatty acid (FFA) release. Unexpectedly, reducing 14-3-3ζ expression was found to significantly impact adipocyte maturity, as observed by reduced abundance of peroxisome proliferator-activated receptor (PPAR)γ2 protein and expression of mature adipocyte genes and those associated with de novo triglyceride synthesis and lipolysis. The impact of 14-3-3ζ depletion on adipocyte maturity was further examined with untargeted lipidomics, which revealed that reductions in 14-3-3ζ abundance promoted the acquisition of a lipidomic signature that resembled undifferentiated preadipocytes. Collectively, these findings reveal a novel aspect of 14-3-3ζ in adipocytes, as reducing 14-3-3ζ was found to have a negative effect on adipocyte maturity and adipocyte-specific processes like lipolysis.

mTORC1 is required for expression of LRPPRC and cytochrome-c oxidase but not HIF-1α in Leigh syndrome French Canadian type patient fibroblasts.

Leigh syndrome French Canadian type (LSFC) is a mitochondrial disease caused by mutations in the leucine-rich pentatricopeptide repeat-containing (LRPPRC) gene leading to a reduction of cytochrome-c oxidase (COX) expression reaching 50% in skin fibroblasts. We have shown that under basal conditions, LSFC and control cells display similar ATP levels. We hypothesized that this occurs through upregulation of mechanistic target of rapamycin (mTOR)-mediated metabolic reprogramming. Our results showed that compared with controls, LSFC cells exhibited an upregulation of the mTOR complex 1 (mTORC1)/p70 ribosomal S6 kinase pathway and higher levels of hypoxia-inducible factor 1α (HIF-1α) and its downstream target pyruvate dehydrogenase kinase 1 (PDHK1), a regulator of mitochondrial pyruvate dehydrogenase 1 (PDH1). Consistent with these signaling alterations, LSFC cells displayed a 40-61% increase in [U-13C6]glucose contribution to pyruvate, lactate, and alanine formation, as well as higher levels of the phosphorylated and inactive form of PDH1-α. Interestingly, inhibition of mTOR with rapamycin did not alter HIF-1α or PDHK1 protein levels in LSFC fibroblasts. However, this treatment increased PDH1-α phosphorylation in control and LSFC cells and reduced ATP levels in control cells. Rapamycin also decreased LRPPRC expression by 41 and 11% in LSFC and control cells, respectively, and selectively reduced COX subunit IV expression in LSFC fibroblasts. Taken together, our data demonstrate the importance of mTORC1, independent of the HIF-1α/PDHK1 axis, in maintaining LRPPRC and COX expression in LSFC cells.

Loss of hepatic LRPPRC alters mitochondrial bioenergetics, regulation of permeability transition and trans-membrane ROS diffusion.

The French-Canadian variant of Leigh Syndrome (LSFC) is an autosomal recessive oxidative phosphorylation (OXPHOS) disorder caused by a mutation in LRPPRC, coding for a protein involved in the stability of mitochondrially-encoded mRNAs. Low levels of LRPPRC are present in all patient tissues, but result in a disproportionately severe OXPHOS defect in the brain and liver, leading to unpredictable subacute metabolic crises. To investigate the impact of the OXPHOS defect in the liver, we analyzed the mitochondrial phenotype in mice harboring an hepatocyte-specific inactivation of Lrpprc. Loss of LRPPRC in the liver caused a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency caused destabilization of polyadenylated mitochondrial mRNAs, altered mitochondrial ultrastructure, and a severe complex IV (CIV) and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency was not limited to OXPHOS, but also included impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H2O2 diffusion, which was traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. This study underscores the value of mitochondria phenotyping to uncover complex and unexpected mechanisms contributing to the pathophysiology of mitochondrial disorders.

Comprehensive and Reproducible Untargeted Lipidomic Workflow Using LC-QTOF Validated for Human Plasma Analysis.

The goal of this work was to develop a label-free, comprehensive, and reproducible high-resolution liquid chromatography-mass spectrometry (LC-MS)-based untargeted lipidomic workflow using a single instrument, which could be applied to biomarker discovery in both basic and clinical studies. For this, we have (i) optimized lipid extraction and elution to enhance coverage of polar and nonpolar lipids as well as resolution of their isomers, (ii) ensured MS signal reproducibility and linearity, and (iii) developed a bioinformatic pipeline to correct remaining biases. Workflow validation is reported for 48 replicates of a single human plasma sample: 1124 reproducible LC-MS signals were extracted (median signal intensity RSD = 10%), 50% of which are redundant due to adducts, dimers, in-source fragmentation, contaminations, or positive and negative ion duplicates. From the resulting 578 unique compounds, 428 lipids were identified by MS/MS, including acyl chain composition, of which 394 had RSD < 30% inside their linear intensity range, thereby enabling robust semiquantitation. MS signal intensity spanned 4 orders of magnitude, covering 16 lipid subclasses. Finally, the power of our workflow is illustrated by a proof-of-concept study in which 100 samples from healthy human subjects were analyzed and the data set was investigated using three different statistical testing strategies in order to compare their capacity in identifying the impact of sex and age on circulating lipids.

Protecting the heart through MK2 modulation, toward a role in diabetic cardiomyopathy and lipid metabolism.

Various signaling pathways have been identified in the heart as important players during development, physiological adaptation or pathological processes. This includes the MAPK families, particularly p38MAPK, which is involved in several key cellular processes, including differentiation, proliferation, apoptosis, inflammation, metabolism and survival. Disrupted p38MAPK signaling has been associated with several diseases, including cardiovascular diseases (CVD) as well as diabetes and its related complications. Despite efforts to translate this knowledge into therapeutic avenues, p38 inhibitors have failed in clinical trials due to adverse effects. Inhibition of MK2, a downstream target of p38, appears to be a promising alternative strategy. Targeting MK2 activity may avoid the adverse effects linked to p38 inhibition, while maintaining its beneficial effects. MK2 was first considered as a therapeutic target in inflammatory diseases such as rheumatoid polyarthritis. A growing body of evidence now supports a key role of MK2 signaling in the pathogenesis of CVD, particularly ischemia/reperfusion injury, hypertrophy, and hypertension and that its inhibition or inactivation is associated with improved heart and vascular functions. More recently, MK2 was shown to be a potential player in diabetes and related complications, particularly in liver and heart, and perturbations in calcium handling and lipid metabolism. In this review, we will discuss recent advances in our knowledge of the role of MK2 in p38MAPK-mediated signaling and the benefits of its loss of function in CVD and diabetes, with an emphasis on the roles of MK2 in calcium handling and lipid metabolism. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.

Assessing Cardiac Metabolism: A Scientific Statement From the American Heart Association.

In a complex system of interrelated reactions, the heart converts chemical energy to mechanical energy. Energy transfer is achieved through coordinated activation of enzymes, ion channels, and contractile elements, as well as structural and membrane proteins. The heart's needs for energy are difficult to overestimate. At a time when the cardiovascular research community is discovering a plethora of new molecular methods to assess cardiac metabolism, the methods remain scattered in the literature. The present statement on "Assessing Cardiac Metabolism" seeks to provide a collective and curated resource on methods and models used to investigate established and emerging aspects of cardiac metabolism. Some of those methods are refinements of classic biochemical tools, whereas most others are recent additions from the powerful tools of molecular biology. The aim of this statement is to be useful to many and to do justice to a dynamic field of great complexity.

A high omega-3 fatty acid diet rapidly changes the lipid composition of cardiac tissue and results in cardioprotection.

The present study was designed to ascertain the effects of 3 diets with different omega-3/6 fatty acid ratios on infarct size and the modifications that these diets induce in the lipid composition of cardiac tissue. Sprague-Dawley rats were fed omega-3/6 fatty acid diets with 1:1, 1:5, or 1:20 ratios for at least 10 days, followed by occlusion of the left anterior descending artery for 40 min and 24 h of reperfusion. Infarct size was significantly smaller in the 1:1 group than in the other groups. Significantly higher concentrations of the omega-3 fatty acids eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid were found in the 1:1 group than in the other groups. Omega-6 polyunsaturated fatty acid levels were similar between groups, although they were higher in the 1:5 and 1:20 groups than in the 1:1 group. Margaric acid concentrations were higher in the 1:1 group than in the other groups. Docosahexaenoic acid levels in cardiac tissue and infarct size were significantly correlated with no other significant links being apparent. The present study indicated that a 1:1 omega-3/6 fatty acid ratio protected against ischemia and was associated with increased omega-3 fatty acid composition of cardiac tissue.

Circulating acylcarnitine profile in human heart failure: a surrogate of fatty acid metabolic dysregulation in mitochondria and beyond.

Heart failure (HF) is associated with metabolic perturbations, particularly of fatty acids (FAs), which remain to be better understood in humans. This study aimed at testing the hypothesis that HF patients with reduced ejection fraction display systemic perturbations in levels of energy-related metabolites, especially those reflecting dysregulation of FA metabolism, namely, acylcarnitines (ACs). Circulating metabolites were assessed using mass spectrometry (MS)-based methods in two cohorts. The main cohort consisted of 72 control subjects and 68 HF patients exhibiting depressed left ventricular ejection fraction (25.9 ± 6.9%) and mostly of ischemic etiology with ≥2 comorbidities. HF patients displayed marginal changes in plasma levels of tricarboxylic acid cycle-related metabolites or indexes of mitochondrial or cytosolic redox status. They had, however, 22-79% higher circulating ACs, irrespective of chain length (P < 0.0001, adjusted for sex, age, renal function, and insulin resistance, determined by shotgun MS/MS), which reflects defective mitochondrial ß-oxidation, and were significantly associated with levels of NH2-terminal pro-B-type natriuretic peptide levels, a disease severity marker. Subsequent extended liquid chromatography-tandem MS analysis of 53 plasma ACs in a subset group from the primary cohort confirmed and further substantiated with a comprehensive lipidomic analysis in a validation cohort revealed in HF patients a more complex circulating AC profile. The latter included dicarboxylic-ACs and dihydroxy-ACs as well as very long chain (VLC) ACs or sphingolipids with VLCFAs (>20 carbons), which are proxies of dysregulated FA metabolism in peroxisomes. Our study identified alterations in circulating ACs in HF patients that are independent of biological traits and associated with disease severity markers. These alterations reflect dysfunctional FA metabolism in mitochondria but also beyond, namely, in peroxisomes, suggesting a novel mechanism contributing to global lipid perturbations in human HF.NEW & NOTEWORTHY Mass spectrometry-based profiling of circulating energy metabolites, including acylcarnitines, in two cohorts of heart failure versus control subjects revealed multiple alterations in fatty acid metabolism in peroxisomes in addition to mitochondria, thereby highlighting a novel mechanism contributing to global lipid perturbations in heart failure.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/acylcarnitines-in-human-heart-failure/.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake.

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with [13C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation reverses leucine's action, suggesting acetylation involvement in this phenomenon.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/leucine-metabolism-inhibits-cardiac-glucose-uptake/.

Ivabradine and metoprolol differentially affect cardiac glucose metabolism despite similar heart rate reduction in a mouse model of dyslipidemia.

While heart rate reduction (HRR) is a target for the management of patients with heart disease, contradictory results were reported using ivabradine, which selectively inhibits the pacemaker If current, vs. ß-blockers like metoprolol. This study aimed at testing whether similar HRR with ivabradine vs. metoprolol differentially modulates cardiac energy substrate metabolism, a factor determinant for cardiac function, in a mouse model of dyslipidemia (hApoB+/+;LDLR-/-). Following a longitudinal study design, we used 3- and 6-mo-old mice, untreated or treated for 3 mo with ivabradine or metoprolol. Cardiac function was evaluated in vivo and ex vivo in working hearts perfused with 13C-labeled substrates to assess substrate fluxes through energy metabolic pathways. Compared with 3-mo-old, 6-mo-old dyslipidemic mice had similar cardiac hemodynamics in vivo but impaired (P < 0.001) contractile function (aortic flow: -45%; cardiac output: -34%; stroke volume: -35%) and glycolysis (-24%) ex vivo. Despite inducing a similar 10% HRR, ivabradine-treated hearts displayed significantly higher stroke volume values and glycolysis vs. their metoprolol-treated counterparts ex vivo, values for the ivabradine group being often not significantly different from 3-mo-old mice. Further analyses highlighted additional significant cardiac alterations with disease progression, namely in the total tissue level of proteins modified by O-linked N-acetylglucosamine (O-GlcNAc), whose formation is governed by glucose metabolism via the hexosamine biosynthetic pathway, which showed a similar pattern with ivabradine vs. metoprolol treatment. Collectively, our results emphasize the implication of alterations in cardiac glucose metabolism and signaling linked to disease progression in our mouse model. Despite similar HRR, ivabradine, but not metoprolol, preserved cardiac function and glucose metabolism during disease progression.

PCSK9 Induces CD36 Degradation and Affects Long-Chain Fatty Acid Uptake and Triglyceride Metabolism in Adipocytes and in Mouse Liver.

OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor thereby elevating plasma low-density lipoprotein cholesterol levels and the risk of coronary heart disease. Thus, the use of PCSK9 inhibitors holds great promise to prevent heart disease. Previous work found that PCSK9 is involved in triglyceride metabolism, independently of its action on low-density lipoprotein receptor, and that other yet unidentified receptors could mediate this effect. Therefore, we assessed whether PCSK9 enhances the degradation of CD36, a major receptor involved in transport of long-chain fatty acids and triglyceride storage. APPROACH AND RESULTS: Overexpressed or recombinant PCSK9 induced CD36 degradation in cell lines and primary adipocytes and reduced the uptake of the palmitate analog Bodipy FL C16 and oxidized low-density lipoprotein in 3T3-L1 adipocytes and hepatic HepG2 cells, respectively. Surface plasmon resonance, coimmunoprecipitation, confocal immunofluorescence microscopy, and protein degradation pathway inhibitors revealed that PCSK9 directly interacts with CD36 and targets the receptor to lysosomes through a mechanism involving the proteasome. Importantly, the level of CD36 protein was increased by >3-fold upon small interfering RNA knockdown of endogenous PCSK9 in hepatic cells and similarly increased in the liver and visceral adipose tissue of Pcsk9(-/-) mice. In Pcsk9(-/-) mice, increased hepatic CD36 was correlated with an amplified uptake of fatty acid and accumulation of triglycerides and lipid droplets. CONCLUSIONS: Our results demonstrate an important role of PCSK9 in modulating the function of CD36 and triglyceride metabolism. PCSK9-mediated CD36 degradation may serve to limit fatty acid uptake and triglyceride accumulation in tissues, such as the liver.

Differential effects of octanoate and heptanoate on myocardial metabolism during extracorporeal membrane oxygenation in an infant swine model.

Nutritional energy support during extracorporeal membrane oxygenation (ECMO) should promote successful myocardial adaptation and eventual weaning from the ECMO circuit. Fatty acids (FAs) are a major myocardial energy source, and medium-chain FAs (MCFAs) are easily taken up by cell and mitochondria without membrane transporters. Odd-numbered MCFAs supply carbons to the citric acid cycle (CAC) via anaplerotic propionyl-CoA as well as acetyl-CoA, the predominant ß-oxidation product for even-numbered MCFA. Theoretically, this anaplerotic pathway enhances carbon entry into the CAC, and provides superior energy state and preservation of protein synthesis. We tested this hypothesis in an immature swine model undergoing ECMO. Fifteen male Yorkshire pigs (26-45 days old) with 8-h ECMO received either normal saline, heptanoate (odd-numbered MCFA), or octanoate (even-numbered MCFA) at 2.3 µmol·kg body wt(-1)·min(-1) as MCFAs systemically during ECMO (n = 5/group). The 13-carbon ((13)C)-labeled substrates ([2-(13)C]lactate, [5,6,7-(13)C3]heptanoate, and [U-(13)C6]leucine) were systemically infused as metabolic markers for the final 60 min before left ventricular tissue extraction. Extracted tissues were analyzed for the (13)C-labeled and absolute concentrations of metabolites by nuclear magnetic resonance and gas chromatography-mass spectrometry. Octanoate produced markedly higher myocardial citrate concentration, and led to a higher [ATP]-to-[ADP] ratio compared with other groups. Unexpectedly, octanoate and heptanoate increased the flux of propionyl-CoA relative to acetyl-CoA into the CAC compared with control. MCFAs promoted increases in leucine oxidation, but were not associated with a difference in protein synthesis rate. In conclusion, octanoate provides energetic advantages to the heart over heptanoate.

Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model.

Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we tested the hypothesis that prolonged systemic pyruvate supplementation activates pyruvate oxidation in an immature swine model in vivo. Twelve male mixed-breed Yorkshire piglets (age 30-49 days) received systemic infusion of either normal saline (group C) or pyruvate (group P) during the final 6 h of 8 h of ECMO. Over the final hour, piglets received [2-(13)C] pyruvate, as a reference substrate for oxidation, and [(13)C6]-l-leucine, as an indicator for amino acid oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of the citric acid cycle intermediates. An increase in anaplerotic flux through pyruvate carboxylation in group P occurred compared with no change in pyruvate oxidation. Additionally, pyruvate promoted an increase in the phosphorylation state of several nutrient-sensitive enzymes, like AMP-activated protein kinase and acetyl CoA carboxylase, suggesting activation for fatty acid oxidation. Pyruvate also promoted O-GlcNAcylation through the hexosamine biosynthetic pathway. In conclusion, although prolonged pyruvate supplementation did not alter pyruvate oxidation, it did elicit changes in nutrient- and energy-sensitive pathways. Therefore, the observed results support the further study of pyruvate and its downstream effect on cardiac function.

Mouse strain differences in metabolic fluxes and function of ex vivo working hearts.

In mice, genetic background is known to influence various parameters, including cardiac function. Its impact on cardiac energy substrate metabolism-a factor known to be closely related to function and contributes to disease development-is, however, unclear. This was examined in this study. In commonly used control mouse substrains SJL/JCrNTac, 129S6/SvEvTac, C57Bl/6J, and C57Bl/6NCrl, we assessed the functional and metabolic phenotypes of 3-mo-old working mouse hearts perfused ex vivo with physiological concentrations of (13)C-labeled carbohydrates (CHO) and a fatty acid (FA). Marked variations in various functional and metabolic flux parameters were observed among all mouse substrains, although the pattern observed differed for these parameters. For example, among all strains, C57Bl/6NCrl hearts had a greater cardiac output (+1.7-fold vs. SJL/JCrNTac and C57Bl/6J; P < 0.05), whereas at the metabolic level, 129S6/SvEvTac hearts stood out by displaying (vs. all 3 strains) a striking shift from exogenous FA (~-3.5-fold) to CHO oxidation as well as increased glycolysis (+1.7-fold) and FA incorporation into triglycerides (+2-fold). Correlation analyses revealed, however, specific linkages between 1) glycolysis, FA oxidation, and pyruvate metabolism and 2) cardiac work, oxygen consumption with heart rate, respectively. This implies that any genetically determined factors affecting a given metabolic flux parameter may impact on the associated functional parameters. Our results emphasize the importance of selecting the appropriate control strain for cardiac metabolic studies using transgenic mice, a factor that has often been neglected. Understanding the molecular mechanisms underlying the diversity of strain-specific cardiac metabolic and functional profiles, particularly the 129S6/SvEvTac, may ultimately disclose new specific metabolic targets for interventions in heart disease.

Metabolic effects of glutamine on the heart: anaplerosis versus the hexosamine biosynthetic pathway.

Glutamine, the most abundant amino acid in plasma, has attracted considerable interest for its cardioprotective properties. The primary effect of glutamine in the heart is commonly believed to be mediated via its anaplerotic metabolism to citric acid cycle (CAC) intermediates; however, there is little direct evidence to support this concept. Another potential candidate is the hexosamine biosynthetic pathway (HBP), which has recently been shown to modulate cardiomyocyte function and metabolism. Therefore, the goal of this study was to evaluate the contribution of anaplerosis and the HBP to the acute metabolic effects of glutamine in the heart. Normoxic ex vivo working rat hearts were perfused with (13)C-labeled substrates to assess relevant metabolic fluxes either with a physiological mixture of carbohydrates and a fatty acid (control) or under conditions of restricted pyruvate anaplerosis. Addition of a physiological concentration of glutamine (0.5mM) had no effect on contractile function of hearts perfused under the control condition, but improved that of hearts perfused under restricted pyruvate anaplerosis. Changes in CAC intermediate concentrations as well as (13)C-enrichment from [U-(13)C]glutamine did not support a major role of glutamine anaplerosis under any conditions. Under the control condition, however, glutamine significantly increased the contribution of exogenous oleate to ß-oxidation, 1.6-fold, and triglyceride formation, 2.8-fold. Glutamine had no effect on malonyl-CoA or AMP kinase activity levels; however, it resulted in a higher plasma membrane level of the fatty acid transporter CD36. These metabolic effects of glutamine were reversed by azaserine, which inhibits glucose entry into the HPB. Our results reveal a metabolic role of physiological concentration of glutamine in the healthy working heart beyond anaplerosis. This role appears to involve the HBP and regulation of fatty acid entry and metabolism via CD36. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".

Myocardial oxidative metabolism and protein synthesis during mechanical circulatory support by extracorporeal membrane oxygenation.

Extracorporeal membrane oxygenation (ECMO) provides essential mechanical circulatory support necessary for survival in infants and children with acute cardiac decompensation. However, ECMO also causes metabolic disturbances, which contribute to total body wasting and protein loss. Cardiac stunning can also occur, which prevents ECMO weaning, and contributes to high mortality. The heart may specifically undergo metabolic impairments, which influence functional recovery. We tested the hypothesis that ECMO alters oxidative metabolism and protein synthesis. We focused on the amino acid leucine and integration with myocardial protein synthesis. We used a translational immature swine model in which we assessed in heart 1) the fractional contribution of leucine (FcLeucine) and pyruvate to mitochondrial acetyl-CoA formation by nuclear magnetic resonance and 2) global protein fractional synthesis (FSR) by gas chromatography-mass spectrometry. Immature mixed breed Yorkshire male piglets (n = 22) were divided into four groups based on loading status (8 h of normal circulation or ECMO) and intracoronary infusion [(13)C(6),(15)N]-L-leucine (3.7 mM) alone or with [2-(13)C]-pyruvate (7.4 mM). ECMO decreased pulse pressure and correspondingly lowered myocardial oxygen consumption (∼40%, n = 5), indicating decreased overall mitochondrial oxidative metabolism. However, FcLeucine was maintained and myocardial protein FSR was marginally increased. Pyruvate addition decreased tissue leucine enrichment, FcLeucine, and Fc for endogenous substrates as well as protein FSR. The heart under ECMO shows reduced oxidative metabolism of substrates, including amino acids, while maintaining 1) metabolic flexibility indicated by ability to respond to pyruvate and 2) a normal or increased capacity for global protein synthesis.

Marine n3 polyunsaturated fatty acids enhance resistance to mitochondrial permeability transition in heart failure but do not improve survival.

Mitochondrial dysfunction in heart failure includes greater susceptibility to mitochondrial permeability transition (MPT), which may worsen cardiac function and decrease survival. Treatment with a mixture of the n3 polyunsaturated fatty acids (n3 PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is beneficial in heart failure patients and increases resistance to MPT in animal models. We assessed whether DHA and EPA have similar effects when given individually, and whether they prolong survival in heart failure. Male δ-sarcoglycan null cardiomyopathic hamsters were untreated or given either DHA, EPA, or a 1:1 mixture of DHA + EPA at 2.1% of energy intake. Treatment did not prolong survival: mean survival was 298 ± 15 days in untreated hamsters and 335 ± 17, 328 ± 14, and 311 ± 15 days with DHA, EPA, and DHA + EPA, respectively (n = 27-32/group). A subgroup of cardiomyopathic hamsters treated for 26 wk had impaired left ventricular function and increased cardiomyocyte apoptosis compared with normal hamsters, which was unaffected by n3 PUFA treatment. Evaluation of oxidative phosphorylation in isolated subsarcolemmal and interfibrillar mitochondria with substrates for complex I or II showed no effect of n3 PUFA treatment. On the other hand, interfibrillar mitochondria from cardiomyopathic hamsters were significantly more sensitive to Ca(2+)-induced MPT, which was completely normalized by treatment with DHA and partially corrected by EPA. In conclusion, treatment with DHA or EPA normalizes Ca(2+)-induced MPT in cardiomyopathic hamsters but does not prolong survival or improve cardiac function. This suggest that greater susceptibility to MPT is not a contributor to cardiac pathology and poor survival in heart failure.