The aim of this work was to investigate the use of 13C‐labelled acetoacetate and β‐hydroxybutyrate as novel hyperpolarized substrates in the study of cardiac metabolism. [1‐13C]Acetoacetate was synthesized by catalysed hydrolysis, and both it and [1‐13C]β‐hydroxybutyrate were hyperpolarized by dissolution dynamic nuclear polarization (DNP). Their metabolism was studied in isolated, perfused rat hearts. Hyperpolarized [1‐13C]acetoacetate metabolism was also studied in the in vivo rat heart in the fed and fasted states. Hyperpolarization of [1‐13C]acetoacetate and [1‐13C]β‐hydroxybutyrate provided liquid state polarizations of 8 ± 2% and 3 ± 1%, respectively. The hyperpolarized T1 values for the two substrates were 28 ± 3 s (acetoacetate) and 20 ± 1 s (β‐hydroxybutyrate). Multiple downstream metabolites were observed within the perfused heart, including acetylcarnitine, citrate and glutamate. In the in vivo heart, an increase in acetylcarnitine production from acetoacetate was observed in the fed state, as well as a potential reduction in glutamate. In this work, methods for the generation of hyperpolarized [1‐13C]acetoacetate and [1‐13C]β‐hydroxybutyrate were investigated, and their metabolism was assessed in both isolated, perfused rat hearts and in the in vivo rat heart. These preliminary investigations show that DNP can be used as an effective in vivo probe of ketone body metabolism in the heart.

Hyperpolarised Magnetic Resonance Spectroscopy (MRS) permits the real time determination of metabolic fluxes in the living heart. In contrast to conventional thermal-equilibrium MRS, the hyperpolarisation technique increases the signal-to-noise ratio of acquired spectra by many orders of magnitude, and therefore allows isotopically labelled probes to be injected into an organism and followed through their subsequent biochemical pathways. We show here that [1–13C]acetoacetate and [1–13C]β-hydroxybutyrate can be hyperpolarised and probe ketone body metabolism in both the ex vivo perfused and in vivo rat heart. Downstream metabolites were observed within the perfused heart, including acetylcarnitine, citrate, and glutamate. In the in vivo heart, a statistically significant increase in acetylcarnitine production from acetoacetate was observed in the fed state, as well as a potential reduction in glutamate, when compared to fasted controls. The metabolism of acetoacetate and β-hydroxybutyrate is known to be altered in various disease states, including diabetic cardiomyopathy, and this proof-of-principle study shows that hyperpolarisation can probe the role of ketone bodies in the diseased heart. The increased rate of acetylcarnitine production following feeding is consistent with its reported role as a store of acetyl moieties should they be abundant in a post-prandial state, into which ketone oxidation is directed. In the fasted state, apparent glutamate levels were higher, which is consistent with an increased flux of ketone bodies into the TCA cycle during fasting. Further work will aim to quantify these fluxes, and explore the role of ketone bodies in animal models of cardiac disease, such as diabetic cardiomyopathy.

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.

PurposeButyrate, a short chain fatty acid, was studied as a novel hyperpolarized substrate for use in dynamic nuclear polarization enhanced magnetic resonance spectroscopy experiments, to define the pathways of short chain fatty acid and ketone body metabolism in real time.MethodsButyrate was polarized via the dynamic nuclear polarization process and subsequently dissolved to generate an injectable metabolic substrate. Metabolism was initially assessed in the isolated perfused rat heart, followed by evaluation in the in vivo rat heart.ResultsHyperpolarized butyrate was generated with a polarization level of 7% and was shown to have a T-1 relaxation time of 20 s. These physical characteristics were sufficient to enable assessment of multiple steps in its metabolism, with the ketone body acetoacetate and several tricarboxylic acid cycle intermediates observed both in vitro and in vivo. Metabolite to butyrate ratios of 0.1-0.4% and 0.5-2% were observed in vitro and in vivo respectively, similar to levels previously observed with hyperpolarized [2-C-13]pyruvate.ConclusionsIn this study, butyrate has been demonstrated to be a suitable hyperpolarized substrate capable of revealing multi-step metabolism in dynamic nuclear polarization experiments and providing information on the metabolism of fatty acids not currently achievable with other hyperpolarized substrates. Magn Reson Med 71:1663-1669, 2014. (c) 2013 Wiley Periodicals, Inc.

OBJECTIVE:Left ventricular assist device placement is an increasingly common treatment for cardiac failure, resulting in cardiac unloading and potentially reversing the remodelling changes seen in heart failure. A popular animal model for human ventricular unloading is the rodent heterotopic non-working heart transplant; the volume loading status of this preparation is important to interpreting the resulting reverse remodelling yet has not been previously investigated. This study was designed to assess the variability of left ventricular volume loading in the rodent transplant model.METHODS:Heterotopic abdominal heart transplant was performed on syngeneic rats; high resolution cine magnetic resonance imaging was subsequently performed on the heterotopic transplanted hearts in anesthetised rats, after variable post-transplant recovery times, in order to assess ventricular loading status.RESULTS:Highly variable left ventricular volume loading status was demonstrated, with some hearts exhibiting considerable ventricular filling and ejection.CONCLUSIONS:These observations call into question the assumption that studies using this model are consistently examining fully unloaded ventricles, and indicate the desirability of in vivo imaging of such hearts to quantify the degree of ventricular loading.

Lisa C. Heather, Katharine M. Pates, Helen J. Atherton, Mark A. Cole, Daniel R. Ball, Rhys D. Ischemia and Reperfusion Transporter, GLUT4, Coordinates Changes in Cardiac Substrate Metabolism During Differential Translocation of the Fatty Acid Transporter, FAT/CD36, and the Glucose Print ISSN: 1941-3289. Online ISSN: 1941-3297 Copyright © 2013 American Heart Association, Inc. All rights reserved. 75231 is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Circulation: Heart Failure doi: 10.1161/CIRCHEARTFAILURE.112.000342 2013;6:1058-1066; originally published online August 12, 2013; Circ Heart Fail. http://circheartfailure.ahajournals.org/content/6/5/1058 World Wide Web at: The online version of this article, along with updated information and services, is located on the http://circheartfailure.ahajournals.org/circhf/suppl/2013/08/12/CIRCHEARTFAILURE.112.000342.DC1.html Data Supplement (unedited) at:

Hyperpolarised 13 C MRI can be used to generate metabolic images of the heart in vivo . However, there have been no similar studies performed in the isolated perfused heart. Therefore, the aim of this study was to develop a method for the creation of 13 C metabolite maps of the perfused rat heart and to demonstrate the technique in a study of acute and chronic myocardial infarction. Male Wistar rat hearts were isolated, perfused and imaged before and after occlusion of the left anterior descending (LAD) coronary artery, creating an acute infarct group. In addition, a chronic infarct group was generated from hearts which had their LAD coronary artery occluded in vivo . Four weeks later, hearts were excised, perfused and imaged to generate metabolic maps of infused pyruvate and its metabolites lactate and bicarbonate. Myocardial perfusion and energetics were assessed by first‐pass perfusion imaging and 31 P MRS, respectively. In both acute and chronically infarcted hearts, perfusion was reduced to the infarct region, as revealed by reduced gadolinium influx and lower signal intensity in the hyperpolarised pyruvate images. In the acute infarct region, there were significant alterations in the lactate (increased) and bicarbonate (decreased) signal ratios. In the chronically infarcted region, there was a significant reduction in both bicarbonate and lactate signals. 31 P‐derived energetics revealed a significant decrease between control and chronic infarcted hearts. Significant decreases in contractile function between control and both acute and chronic infracted hearts were also seen. In conclusion, we have demonstrated that hyperpolarised pyruvate can detect reduced perfusion in the rat heart following both acute and chronic infarction. Changes in lactate and bicarbonate ratios indicate increased anaerobic metabolism in the acute infarct, which is not observed in the chronic infarct. Thus, this study has successfully demonstrated a novel imaging approach to assess altered metabolism in the isolated perfused rat heart. © 2013 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd

Background—Fatty acid and glucose transporters translocate between the sarcolemma and intracellular compartments to regulate substrate metabolism acutely. We hypothesised that during ischemia fatty acid translocase (FAT/CD36) would translocate away from the sarcolemma to limit fatty acid uptake when fatty acid oxidation is inhibited. Methods and Results—Wistar rat hearts were perfused during preischemia, low-flow ischemia, and reperfusion, using 3H-substrates for measurement of metabolic rates, followed by metabolomic analysis and subcellular fractionation. During ischemia, there was a 32% decrease in sarcolemmal FAT/CD36 accompanied by a 95% decrease in fatty acid oxidation rates, with no change in intramyocardial lipids. Concomitantly, the sarcolemmal content of the glucose transporter, GLUT4, increased by 90% during ischemia, associated with an 86% increase in glycolytic rates, 45% decrease in glycogen content, and a 3-fold increase in phosphorylated AMP-activated protein kinase. Following reperfusion, decreased sarcolemmal FAT/CD36 persisted, but fatty acid oxidation rates returned to preischemic levels, resulting in a 35% decrease in myocardial triglyceride content. Elevated sarcolemmal GLUT4 persisted during reperfusion; in contrast, glycolytic rates decreased to 30% of preischemic rates, accompanied by a 5-fold increase in intracellular citrate levels and restoration of glycogen content. Conclusions—During ischemia, FAT/CD36 moved away from the sarcolemma as GLUT4 moved toward the sarcolemma, associated with a shift from fatty acid oxidation to glycolysis, while intramyocardial lipid accumulation was prevented. This relocation was maintained during reperfusion, which was associated with replenishing glycogen stores as a priority, occurring at the expense of glycolysis and mediated by an increase in citrate levels.

AIMS:The aim of this work was to use hyperpolarized carbon-13 ((13)C) magnetic resonance (MR) spectroscopy and cine MR imaging (MRI) to assess in vivo cardiac metabolism and function in the 15-week-old spontaneously hypertensive rat (SHR) heart. At this time point, the SHR displays hypertension and concentric hypertrophy. One of the cellular adaptations to hypertrophy is a reduction in β-oxidation, and it has previously been shown that in response to hypertrophy the SHR heart switches to a glycolytic/glucose-oxidative phenotype. METHODS AND RESULTS:Cine-MRI (magnetic resonance imaging) was used to assess cardiac function and degree of cardiac hypertrophy. Wistar rats were used as controls. SHRs displayed functional changes in stroke volume, heart rate, and late peak-diastolic filling alongside significant hypertrophy (a 56% increase in left ventricular mass). Using hyperpolarized [1-(13)C] and [2-(13)C]pyruvate, an 85% increase in (13)C label flux through pyruvate dehydrogenase (PDH) was seen in the SHR heart and (13)C label incorporation into citrate, acetylcarnitine, and glutamate pools was elevated in proportion to the increase in PDH flux. These findings were confirmed using biochemical analysis of PDH activity and protein expression of PDH regulatory enzymes. CONCLUSIONS:Functional and structural alterations in the SHR heart are consistent with the hypertrophied phenotype. Our in vivo work indicates a preference for glucose metabolism in the SHR heart, a move away from predominantly fatty acid oxidative metabolism. Interestingly, (13)C label flux into lactate was unchanged, indicating no switch to an anaerobic glycolytic phenotype, but rather an increased reliance on glucose oxidation in the SHR heart.

The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.

Introduction: Non-invasive cardiac imaging increasingly plays a fundamental role in diagnosing, assessing prognosis, and monitoring therapy response in cardiovascular disease. Magnetic resonance imaging allows for detailed study of cardiac structure, contractility and viability but with the recent advances in the sensitivity of the C nucleus via the Dynamic Nuclear Polarization (DNP) process, it is now possible to obtain cardiac metabolic images. Combining DNP with Chemical Shift Imaging (CSI) provides a potential method with which to assess the metabolic changes related to cardiovascular disease and to examine the heterogeneity of any such metabolic alterations. The aim of this work was to demonstrate the use of a DNP-CSI protocol in the isolated perfused heart to image metabolism in the setting of both acute and chronic myocardial ischemia. Materials and Methods: To assess the metabolic effects of acute ischemia, hearts from male Wistar rats (n=5) were perfused in the Langendorff mode and, following baseline metabolic assessment, a stitch was tied around the left descending coronary artery to restrict the flow of buffer to the antero-lateral portion of the left ventricle. To assess chronic ischemia, an in vivo myocardial infarction model was used, in which male Wistar rats (n=4) were anaesthetised and a thoracotomy was performed to allow a stitch to be placed around the left descending coronary artery. Four weeks later the hearts were excised for Langendorff perfusion and DNP-CSI performed. Heart perfusion: Hearts were perfused in the Langendorff mode with Krebs-Henseleit buffer containing 10 mM glucose and 2.5 mM pyruvate and oxygenated with 95% O2/5% CO2. The hearts were placed in the bore of an 11.7 T MRI system (Bruker-Biospin, Germany) for spectral assessment. MR Assessment Protocol: An initial P spectrum was acquired to assess the energetic status of the whole heart, followed by a first pass gadolinium contrast protocol to assess cardiac perfusion. This was followed by a combined hyperpolarized DNP dissolution and CSI protocol, as previously described, which yielded images of the infused pyruvate and its metabolic conversion to lactate and bicarbonate (TR = 0.5 s, FA = 45, slice thickness = 5 mm, FOV = 24 x 24 mm, Matrix = 8 x 8, Averages = 2). In the acute ischemia experiments, the heart was then removed from the magnet, a stitch placed around the left descending coronary artery, the heart replaced in the centre of the magnet and the entire protocol repeated. Results and Discussion: The first pass gadolinium contrast acquisition demonstrated that both models of ischemia led to reduced perfusion in the area of myocardium supplied by the left anterior coronary descending artery (infarct region). In both the acute and chronic models, reduced pyruvate signal was seen in the infarct region, confirming the reduction in perfusion. However, in the acute ischemia model, there was also a significant increase in the relative production of lactate and a concomitant decrease in the relative production of bicarbonate in the infarct region (Figure 1), despite no alteration in cardiac energetics. This would suggest a demonstrable change in metabolism in the acute ischemia model consistent with a reduction in aerobic oxidation and a shift to anaerobic metabolism. In the chronic ischemia model, there was a significant reduction in cardiac energetics (PCr/ATP) when compared to control hearts and a significant reduction in metabolism in the infarcted region which was proportional to the reduction in perfusion. Conclusion: In this work, a DNP-CSI protocol was successfully applied to identify infarcted areas of the perfused heart in two different models of ischemia. A switch towards anaerobic metabolism was observed in the setting of acute ischemia. Future work will investigate the translation of these techniques to in vivo rodent models of ischemia. References [1] [Golman et al, Magn Reson Med, 2008, 59, 1005-13], [2] [P. Lee et al, ISMRM 2010], [3] [Carr et al, Am J Physiol Heart Circ Physiol. 2008 Aug;295(2):H533-42], [4] [Stuckey et al, J Cardiovasc Magn Reson. 2011 Aug 3;13:38] Acknowledgements: This study was supported by the Medical Research Council, the British Heart Foundation and Oxford Instruments Molecular Biotools.

Introduction: The isolated perfused heart provides an ideal model system to investigate cardiovascular disease due to its reproducibility, and its ability to yield metabolic and physiological information independent of the variables found in vivo. When combined with the recently developed technique of Dynamic Nuclear Polarization (DNP), the perfused heart can provide useful metabolic information through the application of hyperpolarized C labelled metabolites, particularly C pyruvate. To ensure that a perfused heart preparation is as physiological as possible, fatty acids should ideally be provided in the perfusion buffer. However, previous work has shown that the method of using bovine serum albumin (BSA) to solubilise long chain fatty acids (FA) significantly reduces the observed signal from hyperpolarized pyruvate due to binding of the pyruvate by albumin. The aim of this work was therefore to find alternative ways of providing the perfused heart with a source of FA, without compromising the hyperpolarized signal intensity. To achieve this goal, we provided the perfused heart with either Intralipid (a triglyceride emulsion) or butyrate (a short chain, soluble FA) and assessed the effect of these fatty acid sources on the signal from hyperpolarized pyruvate. The metabolic and energetic effects of these different fatty acid sources were also assessed. Materials and Methods: Heart perfusion: Hearts from 10 male Wistar rats (Harlan, UK) in two groups (Intralipid perfusion N = 5, butyrate perfusion N = 5) were perfused in the Langendorff mode using Krebs Henselheit (KH) buffer oxygenated with 95% O2/5% CO2. A polyethylene balloon was placed into the left ventricle in order to measure contractile function. The hearts were placed in the bore of an 11.7 T MRI system (Bruker-Biospin, Germany) for spectral assessment. Spectroscopy: The hearts were initially perfused with KH buffer containing only 10 mM glucose and 2.5 mM pyruvate. After locating the heart in the centre of the magnet and shimming to reduce the proton linewidth to less than 50 Hz, a hyperpolarized pyruvate study was performed as previously described. Briefly, a 2.5 mM hyperpolarized [1-C]pyruvate solution was delivered to the heart as a bolus over 120 seconds, and a C pulse-acquire experiment commenced (TR = 1 s, FA = 30°, BW = 180 ppm, 8192 pts). Following acquisition, the ratio of the maximum bicarbonate signal to the maximum pyruvate signal was used as a measure of flux through the pyruvate dehydrogenase (PDH) enzyme. The KH buffer was subsequently switched to one containing glucose (10 mM), pyruvate (2.5 mM) and either Intralipid (0.4 mM) or butyrate (0.4 mM) and following a period of stabilization (~30 minutes) a repeat hyperpolarized pyruvate dissolution was carried out. Throughout the protocol, contractile function was recorded from the polyethylene balloon inside the left ventricle and P spectroscopy was performed to assess cardiac energetics.

Deletion of the peroxisome proliferator-activated receptor alpha (PPARa) gene in mice results in abnormal cardiac substrate metabolism and PPARa2/2 hearts have impaired function at high workload and increased post-ischaemic infarct size.We hypothesised that PPARa2/2 mouse hearts would be intolerant to chronic hypoxia,

Introduction: Alterations in cardiac metabolism and substrate selection underlie many diseases of the heart. The advent of cardiac hyperpolarized magnetic resonance spectroscopy (MRS), via dynamic nuclear polarization (DNP), has enabled a greater understanding of the in vivo metabolic changes seen as a consequence of myocardial infarction, hypertrophy and diabetes [1]. However, all studies performed to date have focused on rats and larger animals, whereas more information could be gained through the study of transgenic mouse models of heart disease. Translation from the rat to the mouse is challenging, due in part to the reduction in heart size (1/10) and the 50% increase in heart rate. In this study, we demonstrate for the first time a slice selective approach to investigate the in vivo metabolism of [1-C]pyruvate in the murine heart. To validate the sensitivity of the method to detect alterations in PDH flux, mice were fasted overnight, as this has previously been shown to reduce in vivo PDH flux in rats [1].

Introduction Spontaneously hypertensive rats (SHRs) are used as a model of hypertension and insulin resistance. In response to hypertension and several genetic defects, including loss-of-function mutations in CD36 (a long-chain fatty acid transporter), these animals develop pressure-overload concentric hypertrophy [1]. One of the cellular responses to hypertrophy is a reduction in fatty acid oxidation to reduce oxygen consumption. This reduction is further exacerbated in the SHR model by the loss of function in CD36, resulting in a hypothesised switch to a glycolytic phenotype. The aim of this work was to assess in vivo metabolism in the hypertensive rat heart using hyperpolarized [1-C] and [2-C]pyruvate and to observe whether there is a switch to a glycolytic phenotype in vivo.

Introduction : The recent development of liquid state Dynamic Nuclear Polarization (DNP) techniques has dramatically increased the signal available from C MRS experiments and has opened up new possibilities for metabolic imaging of the heart [1]. Oxidative decarboxylation of DNP hyperpolarized [1-C]pyruvate, mediated by the pyruvate dehydrogenase (PDH) complex, is a critical reaction that produces acetyl-CoA for ATP synthesis and also produces the byproducts NADH and [1C]carbon dioxide (CO2). CO2 is in pH-dependent equilibrium with bicarbonate (HCO3) and observation of HCO3 production has been shown to be an effective biomarker of real-time, in vivo PDH flux [2]. Further, reduction of hyperpolarized [1-C]pyruvate to [1-C]lactate and transamination to [1-C]alanine can be monitored and it has been shown that [1-C]lactate accumulation is a metabolic indicator of cardiac ischaemia [3]. Thus, the capability to visualize these metabolites in vivo allows us to probe metabolic processes that are critical to energy production and the physiological condition of the heart [1]. However, to fully understand the changes observed in vivo it is useful to be able to study a model system where the exact parameters of the system can be carefully controlled. The perfused rat heart has been used as such a model system for many years, but the limitations of a small heart size and the resulting requirement for high spatial resolution have so far prevented metabolic imaging of the perfused heart with hyperpolarized [1-C]pyruvate. In this study, we aimed to demonstrate the feasibility of mapping the spatial distribution of hyperpolarized pyruvate, lactate, alanine and bicarbonate in the perfused rat heart via the implementation of a rapid, high spatial resolution, chemical shift imaging (CSI) approach.

The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2-(13)C]Pyruvate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of (13)C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of (13)C-labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2-(13)C]pyruvate.