Glycolysis-Mediated Activation of v-ATPase by Nicotinamide Mononucleotide Ameliorates Lipid-Induced Cardiomyopathy by Repressing the CD36-TLR4 Axis.

BACKGROUND: Chronic overconsumption of lipids followed by their excessive accumulation in the heart leads to cardiomyopathy. The cause of lipid-induced cardiomyopathy involves a pivotal role for the proton-pump vacuolar-type H+-ATPase (v-ATPase), which acidifies endosomes, and for lipid-transporter CD36, which is stored in acidified endosomes. During lipid overexposure, an increased influx of lipids into cardiomyocytes is sensed by v-ATPase, which then disassembles, causing endosomal de-acidification and expulsion of stored CD36 from the endosomes toward the sarcolemma. Once at the sarcolemma, CD36 not only increases lipid uptake but also interacts with inflammatory receptor TLR4 (Toll-like receptor 4), together resulting in lipid-induced insulin resistance, inflammation, fibrosis, and cardiac dysfunction. Strategies inducing v-ATPase reassembly, that is, to achieve CD36 reinternalization, may correct these maladaptive alterations. For this, we used NAD+ (nicotinamide adenine dinucleotide)-precursor nicotinamide mononucleotide (NMN), inducing v-ATPase reassembly by stimulating glycolytic enzymes to bind to v-ATPase. METHODS: Rats/mice on cardiomyopathy-inducing high-fat diets were supplemented with NMN and for comparison with a cocktail of lysine/leucine/arginine (mTORC1 [mechanistic target of rapamycin complex 1]-mediated v-ATPase reassembly). We used the following methods: RNA sequencing, mRNA/protein expression analysis, immunofluorescence microscopy, (co)immunoprecipitation/proximity ligation assay (v-ATPase assembly), myocellular uptake of [3H]chloroquine (endosomal pH), and [14C]palmitate, targeted lipidomics, and echocardiography. To confirm the involvement of v-ATPase in the beneficial effects of both supplementations, mTORC1/v-ATPase inhibitors (rapamycin/bafilomycin A1) were administered. Additionally, 2 heart-specific v-ATPase-knockout mouse models (subunits V1G1/V0d2) were subjected to these measurements. Mechanisms were confirmed in pharmacologically/genetically manipulated cardiomyocyte models of lipid overload. RESULTS: NMN successfully preserved endosomal acidification during myocardial lipid overload by maintaining v-ATPase activity and subsequently prevented CD36-mediated lipid accumulation, CD36-TLR4 interaction toward inflammation, fibrosis, cardiac dysfunction, and whole-body insulin resistance. Lipidomics revealed C18:1-enriched diacylglycerols as lipid class prominently increased by high-fat diet and subsequently reversed/preserved by lysine/leucine/arginine/NMN treatment. Studies with mTORC1/v-ATPase inhibitors and heart-specific v-ATPase-knockout mice further confirmed the pivotal roles of v-ATPase in these beneficial actions. CONCLUSION: NMN preserves heart function during lipid overload by preventing v-ATPase disassembly.

CD36 (SR-B2) as master regulator of cellular fatty acid homeostasis.

PURPOSE OF REVIEW: Transmembrane glycoprotein cluster of differentiation 36 (CD36) is a scavenger receptor class B protein (SR-B2) that serves various functions in lipid metabolism and signaling, in particular facilitating the cellular uptake of long-chain fatty acids. Recent studies have disclosed CD36 to play a prominent regulatory role in cellular fatty acid metabolism in both health and disease. RECENT FINDINGS: The rate of cellular fatty acid uptake is short-term (i.e., minutes) regulated by the subcellular recycling of CD36 between endosomes and the plasma membrane. This recycling is governed by the activity of vacuolar-type H+-ATPase (v-ATPase) in the endosomal membrane via assembly and disassembly of two subcomplexes. The latter process is being influenced by metabolic substrates including fatty acids, glucose and specific amino acids, together resulting in a dynamic interplay to modify cellular substrate preference and uptake rates. Moreover, in cases of metabolic disease v-ATPase activity was found to be affected while interventions aimed at normalizing v-ATPase functioning had therapeutic potential. SUMMARY: The emerging central role of CD36 in cellular lipid homeostasis and recently obtained molecular insight in the interplay among metabolic substrates indicate the applicability of CD36 as target for metabolic modulation therapy in disease. Experimental studies already have shown the feasibility of this approach.

Guidelines on models of diabetic heart disease.

Diabetes is a major risk factor for cardiovascular diseases, including diabetic cardiomyopathy, atherosclerosis, myocardial infarction, and heart failure. As cardiovascular disease represents the number one cause of death in people with diabetes, there has been a major emphasis on understanding the mechanisms by which diabetes promotes cardiovascular disease, and how antidiabetic therapies impact diabetic heart disease. With a wide array of models to study diabetes (both type 1 and type 2), the field has made major progress in answering these questions. However, each model has its own inherent limitations. Therefore, the purpose of this guidelines document is to provide the field with information on which aspects of cardiovascular disease in the human diabetic population are most accurately reproduced by the available models. This review aims to emphasize the advantages and disadvantages of each model, and to highlight the practical challenges and technical considerations involved. We will review the preclinical animal models of diabetes (based on their method of induction), appraise models of diabetes-related atherosclerosis and heart failure, and discuss in vitro models of diabetic heart disease. These guidelines will allow researchers to select the appropriate model of diabetic heart disease, depending on the specific research question being addressed.

Evaluation of the Interaction of Sex Hormones and Cardiovascular Function and Health.

PURPOSE OF REVIEW: Sex hormones drive development and function of reproductive organs or the development of secondary sex characteristics but their effects on the cardiovascular system are poorly understood. In this review, we identify the gaps in our understanding of the interaction between sex hormones and the cardiovascular system. RECENT FINDINGS: Studies are progressively elucidating molecular functions of sex hormones in specific cell types in parallel with the initiation of crucial large randomized controlled trials aimed at improving therapies for cardiovascular diseases (CVDs) associated with aberrant levels of sex hormones. In contrast with historical assumptions, we now understand that men and women show different symptoms and progression of CVDs. Abnormal levels of sex hormones pose an independent risk for CVD, which is apparent in conditions like Klinefelter syndrome, androgen insensitivity syndrome, and menopause. Moreover, sex hormone-based therapies remain understudied and may not be beneficial for cardiovascular health.

Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly-Rescue by Specific Amino Acid Supplementation.

The heart is metabolically flexible. Under physiological conditions, it mainly uses lipids and glucose as energy substrates. In uncontrolled diabetes, the heart switches towards predominant lipid utilization, which over time is detrimental to cardiac function. Additionally, diabetes is accompanied by high plasma ketone levels and increased utilization of energy provision. The administration of exogenous ketones is currently being investigated for the treatment of cardiovascular disease. Yet, it remains unclear whether increased cardiac ketone utilization is beneficial or detrimental to cardiac functioning. The mechanism of lipid-induced cardiac dysfunction includes disassembly of the endosomal proton pump (named vacuolar-type H+-ATPase; v-ATPase) as the main early onset event, followed by endosomal de-acidification/dysfunction. The de-acidified endosomes can no longer serve as a storage compartment for lipid transporter CD36, which then translocates to the sarcolemma to induce lipid accumulation, insulin resistance, and contractile dysfunction. Lipid-induced v-ATPase disassembly is counteracted by the supply of specific amino acids. Here, we tested the effect of ketone bodies on v-ATPase assembly status and regulation of lipid uptake in rodent/human cardiomyocytes. 3-ß-hydroxybutyrate (3HB) exposure induced v-ATPase disassembly and the entire cascade of events leading to contractile dysfunction and insulin resistance, similar to conditions of lipid oversupply. Acetoacetate addition did not induce v-ATPase dysfunction. The negative effects of 3HB could be prevented by addition of specific amino acids. Hence, in sedentary/prediabetic subjects ketone bodies should be used with caution because of possible aggravation of cardiac insulin resistance and further loss of cardiac function. When these latter maladaptive conditions would occur, specific amino acids could potentially be a treatment option.

Metabolic Interventions to Prevent Hypertrophy-Induced Alterations in Contractile Properties In Vitro.

(1) Background: The exact mechanism(s) underlying pathological changes in a heart in transition to hypertrophy and failure are not yet fully understood. However, alterations in cardiac energy metabolism seem to be an important contributor. We characterized an in vitro model of adrenergic stimulation-induced cardiac hypertrophy for studying metabolic, structural, and functional changes over time. Accordingly, we investigated whether metabolic interventions prevent cardiac structural and functional changes; (2) Methods: Primary rat cardiomyocytes were treated with phenylephrine (PE) for 16 h, 24 h, or 48 h, whereafter hypertrophic marker expression, protein synthesis rate, glucose uptake, and contractile function were assessed; (3) Results: 24 h PE treatment increased expression of hypertrophic markers, phosphorylation of hypertrophy-related signaling kinases, protein synthesis, and glucose uptake. Importantly, the increased glucose uptake preceded structural and functional changes, suggesting a causal role for metabolism in the onset of PE-induced hypertrophy. Indeed, PE treatment in the presence of a PAN-Akt inhibitor or of a GLUT4 inhibitor dipyridamole prevented PE-induced increases in cellular glucose uptake and ameliorated PE-induced contractile alterations; (4) Conclusions: Pharmacological interventions, forcing substrate metabolism away from glucose utilization, improved contractile properties in PE-treated cardiomyocytes, suggesting that targeting glucose uptake, independent from protein synthesis, forms a promising strategy to prevent hypertrophy and hypertrophy-induced cardiac dysfunction.

Endurance-Type Exercise Increases Bulk and Individual Mitochondrial Protein Synthesis Rates in Rats.

Physical activity increases muscle protein synthesis rates. However, the impact of exercise on the coordinated up- and/or downregulation of individual protein synthesis rates in skeletal muscle tissue remains unclear. The authors assessed the impact of exercise on mixed muscle, myofibrillar, and mitochondrial protein synthesis rates as well as individual protein synthesis rates in vivo in rats. Adult Lewis rats either remained sedentary (n = 3) or had access to a running wheel (n = 3) for the last 2 weeks of a 3-week experimental period. Deuterated water was injected and subsequently administered in drinking water over the experimental period. Blood and soleus muscle were collected and used to assess bulk mixed muscle, myofibrillar, and mitochondrial protein synthesis rates using gas chromatography-mass spectrometry and individual muscle protein synthesis rates using liquid chromatography-mass spectrometry (i.e., dynamic proteomic profiling). Wheel running resulted in greater myofibrillar (3.94 ± 0.26 vs. 3.03 ± 0.15%/day; p < .01) and mitochondrial (4.64 ± 0.24 vs. 3.97 ± 0.26%/day; p < .05), but not mixed muscle (2.64 ± 0.96 vs. 2.38 ± 0.62%/day; p = .71) protein synthesis rates, when compared with the sedentary condition. Exercise impacted the synthesis rates of 80 proteins, with the difference from the sedentary condition ranging between -64% and +420%. Significantly greater synthesis rates were detected for F1-ATP synthase, ATP synthase subunit alpha, hemoglobin, myosin light chain-6, and synaptopodin-2 (p < .05). The skeletal muscle protein adaptive response to endurance-type exercise involves upregulation of mitochondrial protein synthesis rates, but it is highly coordinated as reflected by the up- and downregulation of various individual proteins across different bulk subcellular protein fractions.

Putative Role of Protein Palmitoylation in Cardiac Lipid-Induced Insulin Resistance.

In the heart, inhibition of the insulin cascade following lipid overload is strongly associated with contractile dysfunction. The translocation of fatty acid transporter CD36 (SR-B2) from intracellular stores to the cell surface is a hallmark event in the lipid-overloaded heart, feeding forward to intracellular lipid accumulation. Yet, the molecular mechanisms by which intracellularly arrived lipids induce insulin resistance is ill-understood. Bioactive lipid metabolites (diacyl-glycerols, ceramides) are contributing factors but fail to correlate with the degree of cardiac insulin resistance in diabetic humans. This leaves room for other lipid-induced mechanisms involved in lipid-induced insulin resistance, including protein palmitoylation. Protein palmitoylation encompasses the reversible covalent attachment of palmitate moieties to cysteine residues and is governed by protein acyl-transferases and thioesterases. The function of palmitoylation is to provide proteins with proper spatiotemporal localization, thereby securing the correct unwinding of signaling pathways. In this review, we provide examples of palmitoylations of individual signaling proteins to discuss the emerging role of protein palmitoylation as a modulator of the insulin signaling cascade. Second, we speculate how protein hyper-palmitoylations (including that of CD36), as they occur during lipid oversupply, may lead to insulin resistance. Finally, we conclude that the protein palmitoylation machinery may offer novel targets to fight lipid-induced cardiomyopathy.

Augmenting Vacuolar H+-ATPase Function Prevents Cardiomyocytes from Lipid-Overload Induced Dysfunction.

The diabetic heart is characterized by a shift in substrate utilization from glucose to lipids, which may ultimately lead to contractile dysfunction. This substrate shift is facilitated by increased translocation of lipid transporter CD36 (SR-B2) from endosomes to the sarcolemma resulting in increased lipid uptake. We previously showed that endosomal retention of CD36 is dependent on the proper functioning of vacuolar H+-ATPase (v-ATPase). Excess lipids trigger CD36 translocation through inhibition of v-ATPase function. Conversely, in yeast, glucose availability is known to enhance v-ATPase function, allowing us to hypothesize that glucose availability, via v-ATPase, may internalize CD36 and restore contractile function in lipid-overloaded cardiomyocytes. Increased glucose availability was achieved through (a) high glucose (25 mM) addition to the culture medium or (b) adenoviral overexpression of protein kinase-D1 (a kinase mediating GLUT4 translocation). In HL-1 cardiomyocytes, adult rat and human cardiomyocytes cultured under high-lipid conditions, each treatment stimulated v-ATPase re-assembly, endosomal acidification, endosomal CD36 retention and prevented myocellular lipid accumulation. Additionally, these treatments preserved insulin-stimulated GLUT4 translocation and glucose uptake as well as contractile force. The present findings reveal v-ATPase functions as a key regulator of cardiomyocyte substrate preference and as a novel potential treatment approach for the diabetic heart.

Statins Promote Cardiac Infarct Healing by Modulating Endothelial Barrier Function Revealed by Contrast-Enhanced Magnetic Resonance Imaging.

OBJECTIVE: The endothelium has a crucial role in wound healing, acting as a barrier to control transit of leukocytes. Endothelial barrier function is impaired in atherosclerosis preceding myocardial infarction (MI). Besides lowering lipids, statins modulate endothelial function. Here, we noninvasively tested whether statins affect permeability at the inflammatory (day 3) and the reparative (day 7) phase of infarct healing post-MI using contrast-enhanced cardiac magnetic resonance imaging (MRI). APPROACH AND RESULTS: Noninvasive permeability mapping by MRI after MI in C57BL/6, atherosclerotic ApoE-/-, and statin-treated ApoE-/- mice was correlated to subsequent left ventricular outcome by structural and functional cardiac MRI. Ex vivo histology, flow cytometry, and quantitative polymerase chain reaction were performed on infarct regions. Increased vascular permeability at ApoE-/- infarcts was observed compared with C57BL/6 infarcts, predicting enhanced left ventricular dilation at day 21 post-MI by MRI volumetry. Statin treatment improved vascular barrier function at ApoE-/- infarcts, indicated by reduced permeability. The infarcted tissue of ApoE-/- mice 3 days post-MI displayed an unbalanced Vegfa(vascular endothelial growth factor A)/Angpt1 (angiopoetin-1) expression ratio (explaining leakage-prone vessels), associated with higher amounts of CD45+ leukocytes and inflammatory LY6Chi monocytes. Statins reversed the unbalanced Vegfa/Angpt1 expression, normalizing endothelial barrier function at the infarct and blocking the augmented recruitment of inflammatory leukocytes in statin-treated ApoE-/- mice. CONCLUSIONS: Statins lowered permeability and reduced the transit of unfavorable inflammatory leukocytes into the infarcted tissue, consequently improving left ventricular outcome.

2-Arachidonoylglycerol ameliorates inflammatory stress-induced insulin resistance in cardiomyocytes.

Several studies have linked impaired glucose uptake and insulin resistance (IR) to functional impairment of the heart. Recently, endocannabinoids have been implicated in cardiovascular disease. However, the mechanisms involving endocannabinoid signaling, glucose uptake, and IR in cardiomyocytes are understudied. Here we report that the endocannabinoid 2-arachidonoylglycerol (2-AG), via stimulation of cannabinoid type 1 (CB1) receptor and Ca2+/calmodulin-dependent protein kinase ß, activates AMP-activated kinase (AMPK), leading to increased glucose uptake. Interestingly, we have observed that the mRNA expression of CB1 and CB2 receptors was decreased in diabetic mice, indicating reduced endocannabinoid signaling in the diabetic heart. We further establish that TNFα induces IR in cardiomyocytes. Treatment with 2-AG suppresses TNFα-induced proinflammatory markers and improves IR and glucose uptake. Conversely, pharmacological inhibition or knockdown of AMPK attenuates the anti-inflammatory effect and reversal of IR elicited by 2-AG. Additionally, in human embryonic stem cell-derived cardiomyocytes challenged with TNFα or FFA, we demonstrate that 2-AG improves insulin sensitivity and glucose uptake. In conclusion, 2-AG abates inflammatory responses, increases glucose uptake, and overcomes IR in an AMPK-dependent manner in cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance.

Patients with type 2 diabetes (T2D) and/or insulin resistance (IR) have an increased risk for the development of heart failure (HF). Evidence indicates that this increased risk is linked to an altered cardiac substrate preference of the insulin resistant heart, which shifts from a balanced utilization of glucose and long-chain fatty acids (FAs) towards an almost complete reliance on FAs as main fuel source. This shift leads to a loss of endosomal proton pump activity and increased cardiac fat accumulation, which eventually triggers cardiac dysfunction. In this review, we describe the advantages and disadvantages of currently used in vitro models to study the underlying mechanism of IR-induced HF and provide insight into a human in vitro model: human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Using functional metabolic assays we demonstrate that, similar to rodent studies, hESC-CMs subjected to 16h of high palmitate (HP) treatment develop the main features of IR, i.e., decreased insulin-stimulated glucose and FA uptake, as well as loss of endosomal acidification and insulin signaling. Taken together, these data propose that HP-treated hESC-CMs are a promising in vitro model of lipid overload-induced IR for further research into the underlying mechanism of cardiac IR and for identifying new pharmacological agents and therapeutic strategies. 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.

A new leptin-mediated mechanism for stimulating fatty acid oxidation: a pivotal role for sarcolemmal FAT/CD36.

Leptin stimulates fatty acid oxidation in muscle and heart; but, the mechanism by which these tissues provide additional intracellular fatty acids for their oxidation remains unknown. We examined, in isolated muscle and cardiac myocytes, whether leptin, via AMP-activated protein kinase (AMPK) activation, stimulated fatty acid translocase (FAT/CD36)-mediated fatty acid uptake to enhance fatty acid oxidation. In both mouse skeletal muscle and rat cardiomyocytes, leptin increased fatty acid oxidation, an effect that was blocked when AMPK phosphorylation was inhibited by adenine 9-ß-d-arabinofuranoside or Compound C. In wild-type mice, leptin induced the translocation of FAT/CD36 to the plasma membrane and increased fatty acid uptake into giant sarcolemmal vesicles and into cardiomyocytes. In muscles of FAT/CD36-KO mice, and in cardiomyocytes in which cell surface FAT/CD36 action was blocked by sulfo-N-succinimidyl oleate, the leptin-stimulated influx of fatty acids was inhibited; concomitantly, the normal leptin-stimulated increase in fatty acid oxidation was also prevented, despite the normal leptin-induced increase in AMPK phosphorylation. Conversely, in muscle of AMPK kinase-dead mice, leptin failed to induce the translocation of FAT/CD36, along with a failure to stimulate fatty acid uptake and oxidation. Similarly, when siRNA was used to reduce AMPK in HL-1 cardiomyocytes, leptin failed to induce the translocation of FAT/CD36. Our studies have revealed a novel mechanism of leptin-induced fatty acid oxidation in muscle tissue; namely, this process is dependent on the activation of AMPK to induce the translocation of FAT/CD36 to the plasma membrane, thereby stimulating fatty acid uptake. Without increasing this leptin-stimulated, FAT/CD36-dependent fatty acid uptake process, leptin-stimulated AMPK phosphorylation does not enhance fatty acid oxidation.

Regulation of the subcellular trafficking of CD36, a major determinant of cardiac fatty acid utilization.

Myocardial uptake of long-chain fatty acids largely occurs by facilitated diffusion, involving primarily the membrane-associated protein CD36. Other putative fatty acid transporters, such as FABPpm, FATP1 and FATP4, also play a role, but their quantitative contribution is much smaller or their involvement is rather permissive. Besides its sarcolemmal localization, CD36 is also present in intracellular compartments (endosomes). CD36 cycles between both pools via vesicle-mediated trafficking, and the relative distribution between endosomes versus sarcolemma determines the rate of cardiac fatty acid uptake. A net translocation of CD36 to the sarcolemma is induced by various stimuli, in particular hormones like insulin and myocyte contractions, so as to allow a proper coordination of the rate of fatty acid uptake with rapid fluctuations in myocardial energy needs. Furthermore, changes in cardiac fatty acid utilization that occur in both acute and chronic cardiac disease appear to be accompanied by concomitant changes in the sarcolemmal presence of CD36. Studies in various animal and cell models suggest that interventions aimed at modulating the sarcolemmal presence or functioning of CD36 hold promise as therapy to rectify aberrant rates of fatty acid uptake in order to fight cardiac metabolic remodeling and restore proper contractile function. In this review we discuss our current knowledge about the role of CD36 in cardiac fatty acid uptake and metabolism in health and disease with focus on the regulation of the subcellular trafficking of CD36 and its selective modulation as therapeutic approach for cardiac disease. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Post-translational modifications of CD36 (SR-B2): Implications for regulation of myocellular fatty acid uptake.

The membrane-associated protein CD36, now officially designated as SR-B2, is present in various tissues and fulfills multiple cellular functions. In heart and muscle, CD36 is the main (long-chain) fatty acid transporter, regulating myocellular fatty acid uptake via its vesicle-mediated reversible trafficking (recycling) between intracellular membrane compartments and the cell surface. CD36 is subject to various types of post-translational modification. This review focusses on the role of these modifications in further regulation of myocellular fatty acid uptake. Glycosylation, ubiquitination and palmitoylation are involved in regulating CD36 stability, while phosphorylation at extracellular sites affect the rate of fatty acid uptake. In addition, CD36 modification by O-linked N-acetylglucosamine may regulate the translocation of CD36 from endosomes to the cell surface. Acetylation of CD36 has also been reported, but possible effects on CD36 expression and/or functioning have not yet been addressed. Taken together, CD36 is subject to a multitude of post-translational modifications of which their functional implications are beginning to be understood. Moreover, further investigations are needed to disclose whether these post-translational modifications play a role in altered fatty acid uptake rates seen in several pathologies of heart and muscle. This article is part of a special issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck and Jan F.C. Glatz.

Dietary nitrate does not reduce oxygen cost of exercise or improve muscle mitochondrial function in patients with mitochondrial myopathy.

Muscle weakness and exercise intolerance negatively affect the quality of life of patients with mitochondrial myopathy. Short-term dietary nitrate supplementation has been shown to improve exercise performance and reduce oxygen cost of exercise in healthy humans and trained athletes. We investigated whether 1 wk of dietary inorganic nitrate supplementation decreases the oxygen cost of exercise and improves mitochondrial function in patients with mitochondrial myopathy. Ten patients with mitochondrial myopathy (40 ± 5 yr, maximal whole body oxygen uptake = 21.2 ± 3.2 ml·min-1·kg body wt-1, maximal work load = 122 ± 26 W) received 8.5 mg·kg body wt-1·day-1 inorganic nitrate (~7 mmol) for 8 days. Whole body oxygen consumption at 50% of the maximal work load, in vivo skeletal muscle oxidative capacity (evaluated from postexercise phosphocreatine recovery using 31P-magnetic resonance spectroscopy), and ex vivo mitochondrial oxidative capacity in permeabilized skinned muscle fibers (measured with high-resolution respirometry) were determined before and after nitrate supplementation. Despite a sixfold increase in plasma nitrate levels, nitrate supplementation did not affect whole body oxygen cost during submaximal exercise. Additionally, no beneficial effects of nitrate were found on in vivo or ex vivo muscle mitochondrial oxidative capacity. This is the first time that the therapeutic potential of dietary nitrate for patients with mitochondrial myopathy was evaluated. We conclude that 1 wk of dietary nitrate supplementation does not reduce oxygen cost of exercise or improve mitochondrial function in the group of patients tested.

Preservation of myocardial fatty acid oxidation prevents diastolic dysfunction in mice subjected to angiotensin II infusion.

RATIONALE: Diastolic dysfunction is a common feature in many heart failure patients with preserved ejection fraction and has been associated with altered myocardial metabolism in hypertensive and diabetic patients. Therefore, metabolic interventions to improve diastolic function are warranted. In mice with a germline cardiac-specific deletion of acetyl CoA carboxylase 2 (ACC2), systolic dysfunction induced by pressure-overload was prevented by maintaining cardiac fatty acid oxidation (FAO). However, it has not been evaluated whether this strategy would prevent the development of diastolic dysfunction in the adult heart. OBJECTIVE: To test the hypothesis that augmenting cardiac FAO is protective against angiotensin II (AngII)-induced diastolic dysfunction in an adult mouse heart. METHODS AND RESULTS: We generated a mouse model to induce cardiac-specific deletion of ACC2 in adult mice. Tamoxifen treatment (20mg/kg/day for 5days) was sufficient to delete ACC2 protein and increase cardiac FAO by 50% in ACC2 flox/flox-MerCreMer+ mice (iKO). After 4weeks of AngII (1.1mg/kg/day), delivered by osmotic mini-pumps, iKO mice showed normalized E/E' and E'/A' ratios compared to AngII treated controls (CON). The prevention of diastolic dysfunction in iKO-AngII was accompanied by maintained FAO and reduced glycolysis and anaplerosis. Furthermore, iKO-AngII hearts had a~50% attenuation of cardiac hypertrophy and fibrosis compared to CON. In addition, maintenance of FAO in iKO hearts suppressed AngII-associated increases in oxidative stress and sustained mitochondrial respiratory complex activities. CONCLUSION: These data demonstrate that impaired FAO is a contributor to the development of diastolic dysfunction induced by AngII. Maintenance of FAO in this model leads to an attenuation of hypertrophy, reduces fibrosis, suppresses increases in oxidative stress, and maintains mitochondrial function. Therefore, targeting mitochondrial FAO is a promising therapeutic strategy for the treatment of diastolic dysfunction.

Cardiac diastolic dysfunction in high-fat diet fed mice is associated with lipotoxicity without impairment of cardiac energetics in vivo.

Obesity is often associated with abnormalities in cardiac morphology and function. This study tested the hypothesis that obesity-related cardiomyopathy is caused by impaired cardiac energetics. In a mouse model of high-fat diet (HFD)-induced obesity, we applied in vivo cardiac (31)P magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) to investigate cardiac energy status and function, respectively. The measurements were complemented by ex vivo determination of oxygen consumption in isolated cardiac mitochondria, the expression of proteins involved in energy metabolism, and markers of oxidative stress and calcium homeostasis. We also assessed whether HFD induced myocardial lipid accumulation using in vivo (1)H MRS, and if this was associated with apoptosis and fibrosis. Twenty weeks of HFD feeding resulted in early stage cardiomyopathy, as indicated by diastolic dysfunction and increased left ventricular mass, without any effects on systolic function. In vivo cardiac phosphocreatine-to-ATP ratio and ex vivo oxygen consumption in isolated cardiac mitochondria were not reduced after HFD feeding, suggesting that the diastolic dysfunction was not caused by impaired cardiac energetics. HFD feeding promoted mitochondrial adaptations for increased utilization of fatty acids, which was however not sufficient to prevent the accumulation of myocardial lipids and lipid intermediates. Myocardial lipid accumulation was associated with oxidative stress and fibrosis, but not apoptosis. Furthermore, HFD feeding strongly reduced the phosphorylation of phospholamban, a prominent regulator of cardiac calcium homeostasis and contractility. In conclusion, HFD-induced early stage cardiomyopathy in mice is associated with lipotoxicity-associated oxidative stress, fibrosis, and disturbed calcium homeostasis, rather than impaired cardiac energetics.

Augmenting muscle diacylglycerol and triacylglycerol content by blocking fatty acid oxidation does not impede insulin sensitivity.

A low fat oxidative capacity has been linked to muscle diacylglycerol (DAG) accumulation and insulin resistance. Alternatively, a low fat oxidation rate may stimulate glucose oxidation, thereby enhancing glucose disposal. Here, we investigated whether an ethyl-2-[6-(4-chlorophenoxy)hexyl]-oxirane-2-carboxylate (etomoxir)-induced inhibition of fat oxidation leads to muscle fat storage and insulin resistance. An intervention in healthy male subjects was combined with studies in human primary myotubes. Furthermore, muscle DAG and triacylglycerol (TAG), mitochondrial function, and insulin signaling were examined in etomoxir-treated C57bl6 mice. In humans, etomoxir administration increased glucose oxidation at the expense of fat oxidation. This effect was accompanied by an increased abundance of GLUT4 at the sarcolemma and a lowering of plasma glucose levels, indicative of improved glucose homeostasis. In mice, etomoxir injections resulted in accumulation of muscle TAG and DAG, yet improved insulin-stimulated GLUT4 translocation. Also in human myotubes, insulin signaling was improved by etomoxir, in the presence of increased intramyocellular lipid accumulation. These insulin-sensitizing effects in mice and human myotubes were accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Our results show that a reduction in fat oxidation leading to accumulation of muscle DAG does not necessarily lead to insulin resistance, as the reduction in fat oxidation may activate AMPK.

Lack of UCP3 does not affect skeletal muscle mitochondrial function under lipid-challenged conditions, but leads to sudden cardiac death.

UCP3's exact physiological function in lipid handling in skeletal and cardiac muscle remains unknown. Interestingly, etomoxir, a fat oxidation inhibitor and strong inducer of UCP3, is proposed for treating both diabetes and heart failure. We hypothesize that the upregulation of UCP3 upon etomoxir serves to protect mitochondria against lipotoxicity. To evaluate UCP3's role in skeletal muscle (skm) and heart under lipid-challenged conditions, the effect of UCP3 ablation was examined in a state of dysbalance between fat availability and oxidative capacity. Wild type (WT) and UCP3(-/-) mice were subjected to high-fat feeding for 14 days. From day 6 onwards, they were given either saline or etomoxir. Etomoxir treatment induced an increase in markers of lipotoxicity in skm compared to saline. This increase upon etomoxir was similar for both, WT and UCP3(-/-) mice, suggesting that UCP3 does not play a role in protection against lipotoxicity. Interestingly, we observed 25 % mortality in UCP3(-/-)s upon etomoxir administration vs. 11 % in WTs. This increased mortality in UCP3(-/-) compared to WT mice could not be explained by differences in cardiac lipotoxicity, apoptosis, fibrosis (histology, immunohistochemistry), oxidative capacity (respirometry) or function (echocardiography). Electrophysiology demonstrated, however, prolonged QRS and QTc intervals and greater susceptibility to ventricular tachycardia upon programmed electrical stimulation in etomoxir-treated UCP3(-/-)s versus WTs. Isoproterenol administration after pacing resulted in 75 % mortality in UCP3(-/-)s vs. 14 % in WTs. Our results argue against a protective role for UCP3 on skm metabolism under lipid overload, but suggest UCP3 to be crucial in prevention of arrhythmias upon lipid-challenged conditions.