Daniel R. Ball

Real-time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy

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). [2‐13C]Pyru‐ vate 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 13C‐labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of 13C‐ 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 [2‐13C]pyruvate.— Schroeder, M. A., Atherton, H. J., Ball, D. R., Cole, M. A., Heather, L. C., Griffin, J. L., Clarke, K., Radda, G. K., Tyler, D. J. Real‐time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy. FASEBJ. 23, 2529–2538 (2009)

Fumarate Is Cardioprotective via Activation of the Nrf2 Antioxidant Pathway

Summary 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 fumarates 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.

In vivo alterations in cardiac metabolism and function in the spontaneously hypertensive rat heart.

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.

Hyperpolarized butyrate: A metabolic probe of short chain fatty acid metabolism in the heart

Butyrate, 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.

Differential Translocation of the Fatty Acid Transporter, FAT/CD36, and the Glucose Transporter, GLUT4, Coordinates Changes in Cardiac Substrate Metabolism During Ischemia and Reperfusion

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.

Metabolic imaging of acute and chronic infarction in the perfused rat heart using hyperpolarised [1-13C]pyruvate.

Hyperpolarised 13C 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 13C 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 31P 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. 31P‐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.

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 13C 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.

REAL-TIME ASSESSMENT OF KREBS CYCLE METABOLISM WITH HYPERPOLARISED [2-13C]PYRUVATE

The Krebs cycle is fundamental to cardiac energy production, and is often implicated in energetic imbalances characteristic of heart disease. To date, Krebs cycle flux has been measured using 13C-magnetic resonance spectroscopy with isotopomer analysis; however, this approach is limited to the study of steady-state metabolism …

Hyperpolarized ketone body metabolism in the rat heart

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.

P9 Hyperpolarised ketone body metabolism in the rat 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.