CD36 as a gatekeeper of myocardial lipid metabolism and therapeutic target for metabolic disease.

The multifunctional membrane glycoprotein CD36 is expressed in different types of cells and plays a key regulatory role in cellular lipid metabolism, especially in cardiac muscle. CD36 facilitates the cellular uptake of long-chain fatty acids, mediates lipid signaling, and regulates storage and oxidation of lipids in various tissues with active lipid metabolism. CD36 deficiency leads to marked impairments in peripheral lipid metabolism, which consequently impact on the cellular utilization of multiple different fuels because of the integrated nature of metabolism. The functional presence of CD36 at the plasma membrane is regulated by its reversible subcellular recycling from and to endosomes and is under the control of mechanical, hormonal, and nutritional factors. Aberrations in this dynamic role of CD36 are causally associated with various metabolic diseases, in particular insulin resistance, diabetic cardiomyopathy, and cardiac hypertrophy. Recent research in cardiac muscle has disclosed the endosomal proton pump vacuolar-type H+-ATPase (v-ATPase) as a key enzyme regulating subcellular CD36 recycling and being the site of interaction between various substrates to determine cellular substrate preference. In addition, evidence is accumulating that interventions targeting CD36 directly or modulating its subcellular recycling are effective for the treatment of metabolic diseases. In conclusion, subcellular CD36 localization is the major adaptive regulator of cellular uptake and metabolism of long-chain fatty acids and appears a suitable target for metabolic modulation therapy to mend failing hearts.

AICAR confers prophylactic cardioprotection in doxorubicin-induced heart failure in rats.

Doxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects, leading to heart failure (HF). Impaired mitochondrial function is thought to be key factor driving progression into HF. We have previously shown in a rat model of DOX-HF that heart failure with reduced ejection fraction correlates with mitochondrial loss and dysfunction. Adenosine monophosphate-dependent kinase (AMPK) is a cellular energy sensor, regulating mitochondrial biogenesis and energy metabolism, including fatty acid oxidation. We hypothesised that AMPK activation could restore mitochondrial function and therefore be a novel cardioprotective strategy for the prevention of DOX-HF. Consequently, we set out to assess whether 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR), an activator of AMPK, could prevent cardiac functional decline in this chronic intravenous rat model of DOX-HF. In line with our hypothesis, AICAR improved cardiac systolic function. AICAR furthermore improved cardiac mitochondrial fatty acid oxidation, independent of mitochondrial number, and in the absence of observable AMPK-activation. In addition, we found that AICAR prevented loss of myocardial mass. RNAseq analysis showed that this may be driven by normalisation of pathways associated with ribosome function and protein synthesis, which are impaired in DOX-treated rat hearts. AICAR furthermore prevented dyslipidemia and excessive body-weight loss in DOX-treated rats, which may contribute to preservation of myocardial mass. Though it is unclear whether AICAR exerted its cardioprotective effect through cardiac or extra-cardiac AMPK-activation or via an AMPK-independent effect, these results show promise for the use of AICAR as a cardioprotective agent in DOX-HF to both preserve cardiac function and mass.

Cardiomyocyte tetrahydrobiopterin synthesis regulates fatty acid metabolism and susceptibility to ischaemia-reperfusion injury.

NEW FINDINGS: What is the central question of this study? What are the physiological roles of cardiomyocyte-derived tetrahydrobiopterin (BH4) in cardiac metabolism and stress response? What is the main finding and its importance? Cardiomyocyte BH4 has a physiological role in cardiac metabolism. There was a shift of substrate preference from fatty acid to glucose in hearts with targeted deletion of BH4 synthesis. The changes in fatty-acid metabolic profile were associated with a protective effect in response to ischaemia-reperfusion (IR) injury, and reduced infarct size. Manipulating fatty acid metabolism via BH4 availability could play a therapeutic role in limiting IR injury. ABSTRACT: Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) synthases in which its production of NO is crucial for cardiac function. However, non-canonical roles of BH4 have been discovered recently and the cell-specific role of cardiomyocyte BH4 in cardiac function and metabolism remains to be elucidated. Therefore, we developed a novel mouse model of cardiomyocyte BH4 deficiency, by cardiomyocyte-specific deletion of Gch1, which encodes guanosine triphosphate cyclohydrolase I, a required enzyme for de novo BH4 synthesis. Cardiomyocyte (cm)Gch1 mRNA expression and BH4 levels from cmGch1 KO mice were significantly reduced compared to Gch1flox/flox (WT) littermates. Transcriptomic analyses and protein assays revealed downregulation of genes involved in fatty acid oxidation in cmGch1 KO hearts compared with WT, accompanied by increased triacylglycerol concentration within the myocardium. Deletion of cardiomyocyte BH4 did not alter basal cardiac function. However, the recovery of left ventricle function was improved in cmGch1 KO hearts when subjected to ex vivo ischaemia-reperfusion (IR) injury, with reduced infarct size compared to WT hearts. Metabolomic analyses of cardiac tissue after IR revealed that long-chain fatty acids were increased in cmGch1 KO hearts compared to WT, whereas at 5 min reperfusion (post-35 min ischaemia) fatty acid metabolite levels were higher in WT compared to cmGch1 KO hearts. These results indicate a new role for BH4 in cardiomyocyte fatty acid metabolism, such that reduction of cardiomyocyte BH4 confers a protective effect in response to cardiac IR injury. Manipulating cardiac metabolism via BH4 could play a therapeutic role in limiting IR injury.

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.

Rapid, B1 -insensitive, dual-band quasi-adiabatic saturation transfer with optimal control for complete quantification of myocardial ATP flux.

PURPOSE: Phosphorus saturation-transfer experiments can quantify metabolic fluxes noninvasively. Typically, the forward flux through the creatine kinase reaction is investigated by observing the decrease in phosphocreatine (PCr) after saturation of γ-ATP. The quantification of total ATP utilization is currently underexplored, as it requires simultaneous saturation of inorganic phosphate ( Pi ) and PCr. This is challenging, as currently available saturation pulses reduce the already-low γ-ATP signal present. METHODS: Using a hybrid optimal-control and Shinnar-Le Roux method, a quasi-adiabatic RF pulse was designed for the dual saturation of PCr and Pi to enable determination of total ATP utilization. The pulses were evaluated in Bloch equation simulations, compared with a conventional hard-cosine DANTE saturation sequence, before being applied to perfused rat hearts at 11.7 T. RESULTS: The quasi-adiabatic pulse was insensitive to a >2.5-fold variation in B1 , producing equivalent saturation with a 53% reduction in delivered pulse power and a 33-fold reduction in spillover at the minimum effective B1 . This enabled the complete quantification of the synthesis and degradation fluxes for ATP in 30-45 minutes in the perfused rat heart. While the net synthesis flux (4.24 ± 0.8 mM/s, SEM) was not significantly different from degradation flux (6.88 ± 2 mM/s, P = .06) and both measures are consistent with prior work, nonlinear error analysis highlights uncertainties in the Pi -to-ATP measurement that may explain a trend suggesting a possible imbalance. CONCLUSIONS: This work demonstrates a novel quasi-adiabatic dual-saturation RF pulse with significantly improved performance that can be used to measure ATP turnover in the heart in vivo.

Hyperpolarized magnetic resonance shows that the anti-ischemic drug meldonium leads to increased flux through pyruvate dehydrogenase in vivo resulting in improved post-ischemic function in the diabetic heart.

The diabetic heart has a decreased ability to metabolize glucose. The anti-ischemic drug meldonium may provide a route to counteract this by reducing l-carnitine levels, resulting in improved cardiac glucose utilization. Therefore, the aim of this study was to use the novel technique of hyperpolarized magnetic resonance to investigate the in vivo effects of treatment with meldonium on cardiac metabolism and function in control and diabetic rats. Thirty-six male Wistar rats were injected either with vehicle, or with streptozotocin (55 mg/kg) to induce a model of type 1 diabetes. Daily treatment with either saline or meldonium (100 mg/kg/day) was undertaken for three weeks. in vivo cardiac function and metabolism were assessed with CINE MRI and hyperpolarized magnetic resonance respectively. Isolated perfused hearts were challenged with low-flow ischemia/reperfusion to assess the impact of meldonium on post-ischemic recovery. Meldonium had no significant effect on blood glucose concentrations or on baseline cardiac function. However, hyperpolarized magnetic resonance revealed that meldonium treatment elevated pyruvate dehydrogenase flux by 3.1-fold and 1.2-fold in diabetic and control animals, respectively, suggesting an increase in cardiac glucose oxidation. Hyperpolarized magnetic resonance further demonstrated that meldonium reduced the normalized acetylcarnitine signal by 2.1-fold in both diabetic and control animals. The increase in pyruvate dehydrogenase flux in vivo was accompanied by an improvement in post-ischemic function ex vivo, as meldonium elevated the rate pressure product by 1.3-fold and 1.5-fold in the control and diabetic animals, respectively. In conclusion, meldonium improves in vivo pyruvate dehydrogenase flux in the diabetic heart, contributing to improved cardiac recovery after ischemia.

Dimethyl-2-oxoglutarate improves redox balance and mitochondrial function in muscle pericytes of individuals with diabetes mellitus.

AIMS/HYPOTHESIS: Treatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype. METHODS: MPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography-mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte-endothelial interaction were assessed as functional readouts. RESULTS: D-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte-endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status. CONCLUSIONS/INTERPRETATION: These novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte-endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes. Graphical abstract.

Assessing the effect of hypoxia on cardiac metabolism using hyperpolarized 13 C magnetic resonance spectroscopy.

Hypoxia plays a role in many diseases and can have a wide range of effects on cardiac metabolism depending on the extent of the hypoxic insult. Noninvasive imaging methods could shed valuable light on the metabolic effects of hypoxia on the heart in vivo. Hyperpolarized carbon-13 magnetic resonance spectroscopy (HP 13 C MRS) in particular is an exciting technique for imaging metabolism that could provide such information. The aim of our work was, therefore, to establish whether hyperpolarized 13 C MRS can be used to assess the in vivo heart's metabolism of pyruvate in response to systemic acute and chronic hypoxic exposure. Groups of healthy male Wistar rats were exposed to either acute (30 minutes), 1 week or 3 weeks of hypoxia. In vivo MRS of hyperpolarized [1-13 C] pyruvate was carried out along with assessments of physiological parameters and ejection fraction. Hematocrit was elevated after 1 week and 3 weeks of hypoxia. 30 minutes of hypoxia resulted in a significant reduction in pyruvate dehydrogenase (PDH) flux, whereas 1 or 3 weeks of hypoxia resulted in a PDH flux that was not different to normoxic animals. Conversion of hyperpolarized [1-13 C] pyruvate into [1-13 C] lactate was elevated following acute hypoxia, suggestive of enhanced anaerobic glycolysis. Elevated HP pyruvate to lactate conversion was also seen at the one week timepoint, in concert with an increase in lactate dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac metabolism of pyruvate was comparable with that observed in normoxia. We have successfully visualized the effects of systemic hypoxia on cardiac metabolism of pyruvate using hyperpolarized 13 C MRS, with differences observed following 30 minutes and 1 week of hypoxia. This demonstrates the potential of in vivo hyperpolarized 13 C MRS data for assessing the cardiometabolic effects of hypoxia in disease.

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.

The 'Goldilocks zone' of fatty acid metabolism; to ensure that the relationship with cardiac function is just right.

Fatty acids (FA) are the main fuel used by the healthy heart to power contraction, supplying 60-70% of the ATP required. FA generate more ATP per carbon molecule than glucose, but require more oxygen to produce the ATP, making them a more energy dense but less oxygen efficient fuel compared with glucose. The pathways involved in myocardial FA metabolism are regulated at various subcellular levels, and can be divided into sarcolemmal FA uptake, cytosolic activation and storage, mitochondrial uptake and ß-oxidation. An understanding of the critical involvement of each of these steps has been amassed from genetic mouse models, where forcing the heart to metabolize too much or too little fat was accompanied by cardiac contractile dysfunction and hypertrophy. In cardiac pathologies, such as heart disease and diabetes, aberrations in FA metabolism occur concomitantly with changes in cardiac function. In heart failure, FA oxidation is decreased, correlating with systolic dysfunction and hypertrophy. In contrast, in type 2 diabetes, FA oxidation and triglyceride storage are increased, and correlate with diastolic dysfunction and insulin resistance. Therefore, too much FA metabolism is as detrimental as too little FA metabolism in these settings. Therapeutic compounds that rebalance FA metabolism may provide a mechanism to improve cardiac function in disease. Just like Goldilocks and her porridge, the heart needs to maintain FA metabolism in a zone that is 'just right' to support contractile function.

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

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

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

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

The von Hippel-Lindau Chuvash mutation in mice alters cardiac substrate and high-energy phosphate metabolism.

Hypoxia-inducible factor (HIF) appears to function as a global master regulator of cellular and systemic responses to hypoxia. HIF pathway manipulation is of therapeutic interest; however, global systemic upregulation of HIF may have as yet unknown effects on multiple processes. We used a mouse model of Chuvash polycythemia (CP), a rare genetic disorder that modestly increases expression of HIF target genes in normoxia, to understand what these effects might be within the heart. An integrated in and ex vivo approach was employed. Compared with wild-type controls, CP mice had evidence (using in vivo magnetic resonance imaging) of pulmonary hypertension, right ventricular hypertrophy, and increased left ventricular ejection fraction. Glycolytic flux (measured using [(3)H]glucose) in the isolated contracting perfused CP heart was 1.8-fold higher. Net lactate efflux was 1.5-fold higher. Furthermore, in vivo (13)C-magnetic resonance spectroscopy (MRS) of hyperpolarized [(13)C1]pyruvate revealed a twofold increase in real-time flux through lactate dehydrogenase in the CP hearts and a 1.6-fold increase through pyruvate dehydrogenase. (31)P-MRS of perfused CP hearts under increased workload (isoproterenol infusion) demonstrated increased depletion of phosphocreatine relative to ATP. Intriguingly, no changes in cardiac gene expression were detected. In summary, a modest systemic dysregulation of the HIF pathway resulted in clear alterations in cardiac metabolism and energetics. However, in contrast to studies generating high HIF levels within the heart, the CP mice showed neither the predicted changes in gene expression nor any degree of LV impairment. We conclude that the effects of manipulating HIF on the heart are dose dependent.

Simultaneous in vivo assessment of cardiac and hepatic metabolism in the diabetic rat using hyperpolarized MRS.

Understanding and assessing diabetic metabolism is vital for monitoring disease progression and improving treatment of patients. In vivo assessments, using MRI and MRS, provide non-invasive and accurate measurements, and the development of hyperpolarized 13 C spectroscopy in particular has been demonstrated to provide valuable metabolic data in real time. Until now, studies have focussed on individual organs. However, diabetes is a systemic disease affecting multiple tissues in the body. Therefore, we have developed a technique to simultaneously measure metabolism in both the heart and liver during a single acquisition. A hyperpolarized 13 C MRS protocol was developed to allow acquisition of metabolic data from the heart and liver during a single scan. This protocol was subsequently used to assess metabolism in the heart and liver of seven control male Wistar rats and seven diabetic rats (diabetes was induced by three weeks of high-fat feeding and a 30 mg/kg injection of streptozotocin). Using our new acquisition, we observed decreased cardiac and hepatic pyruvate dehydrogenase flux in our diabetic rat model. These diabetic rats also had increased blood glucose levels, decreased insulin, and increased hepatic triglycerides. Decreased production of hepatic [1-13 C]alanine was observed in the diabetic group, but this change was not present in the hearts of the same diabetic animals. We have demonstrated the ability to measure cardiac and hepatic metabolism simultaneously, with sufficient sensitivity to detect metabolic alterations in both organs. Further, we have non-invasively observed the different reactions of the heart and liver to the metabolic challenge of diabetes.

Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart.

Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l(-1) NaCl (as control) or 0.7 mmol l(-1) NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.

Redefining Diabetic Cardiomyopathy: Perturbations in Substrate Metabolism at the Heart of Its Pathology.

Cardiovascular disease represents the leading cause of death in people with diabetes, most notably from macrovascular diseases such as myocardial infarction or heart failure. Diabetes also increases the risk of a specific form of cardiomyopathy, referred to as diabetic cardiomyopathy (DbCM), originally defined as ventricular dysfunction in the absence of underlying coronary artery disease and/or hypertension. Herein, we provide an overview on the key mediators of DbCM, with an emphasis on the role for perturbations in cardiac substrate metabolism. We discuss key mechanisms regulating metabolic dysfunction in DbCM, with additional focus on the role of metabolites as signaling molecules within the diabetic heart. Furthermore, we discuss the preclinical approaches to target these perturbations to alleviate DbCM. With several advancements in our understanding, we propose the following as a new definition for, or approach to classify, DbCM: "diastolic dysfunction in the presence of altered myocardial metabolism in a person with diabetes but absence of other known causes of cardiomyopathy and/or hypertension." However, we recognize that no definition can fully explain the complexity of why some individuals with DbCM exhibit diastolic dysfunction, whereas others develop systolic dysfunction. Due to DbCM sharing pathological features with heart failure with preserved ejection fraction (HFpEF), the latter of which is more prevalent in the population with diabetes, it is imperative to determine whether effective management of DbCM decreases HFpEF prevalence.

Targeting glucose metabolism with dichloroacetate (DCA) reduces zika virus replication in brain cortical progenitors at different stages of maturation.

The underlying threat of new Zika virus (ZIKV) outbreaks remains, as no vaccines or therapies have yet been developed. In vitro research has shown that glycolysis is a key factor to enable sustained ZIKV replication in neuroprogenitors. However, neither in vivo nor clinical investigation of glycolytic modulators as potential therapeutics for ZIKV-related fetal abnormalities has been conducted. Accordingly, we tested the therapeutic potential of metabolic modulators in relevant in vitro systems comprising two pools of neuroprogenitors (NPCs), which resemble early and late stages of pregnancy. Effective doses of metabolic modulators [3.0 µM] dimethyl fumarate (DMF), [3.2 mM] dichloroacetate (DCA), and [6.3 µM] VER-246608 were determined for these cells by their effect on lactate release, pyruvate dehydrogenase (PDH) activity and cell survival. The drugs were used in a 24h pre-treatment and kept throughout ZIKV infection of NPCs. Drug effects and ZIKV replication were assessed at 24- and 56-h post-infection. In early NPCs treated with DMF, DCA and VER-246608, there was a significant reduction in the extracellular release of ZIKV potentially by PDH-mediated increased mitochondrial oxidation of glucose. Out of the three drugs, only DCA was observed to reduce viral replication in late NPCs treated with DCA. Altogether, our findings suggest that reduction of anaerobic glycolysis could be of therapeutic potential against ZIKV-related fetal abnormalities and that clinical translation should consider the use of specific glycolytic modulators over different trimesters.

A practical guide to metabolomic profiling as a discovery tool for human heart disease.

Metabolomics has refreshed interest in metabolism across biology and medicine, particularly in the areas of functional genomics and biomarker discovery. In this review we will discuss the experimental techniques and challenges involved in metabolomic profiling and how these technologies have been applied to cardiovascular disease. Open profiling and targeted approaches to metabolomics are compared, focusing on high resolution NMR spectroscopy and mass spectrometry, as well as discussing how to analyse the large amounts of data generated using multivariate statistics. Finally, the current literature on metabolomic profiling in human cardiovascular disease is reviewed to illustrate the diversity of approaches, and discuss some of the key metabolites and pathways found to be perturbed in plasma, urine and tissue from patients with these diseases. This includes studies of coronary artery disease, myocardial infarction, and ischemic heart disease. These studies demonstrate the potential of the technology for biomarker discovery and elucidating metabolic mechanisms associated with given pathologies, but also in some cases provide a warning of the pitfalls of poor study design. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".

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

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