Damian J. Tyler

In vivo assessment of pyruvate dehydrogenase flux in the heart using hyperpolarized carbon-13 magnetic resonance.

The advent of hyperpolarized 13C magnetic resonance (MR) has provided new potential for the real-time visualization of in vivo metabolic processes. The aim of this work was to use hyperpolarized [1-13C]pyruvate as a metabolic tracer to assess noninvasively the flux through the mitochondrial enzyme complex pyruvate dehydrogenase (PDH) in the rat heart, by measuring the production of bicarbonate (H13CO3−), a byproduct of the PDH-catalyzed conversion of [1-13C]pyruvate to acetyl-CoA. By noninvasively observing a 74% decrease in H13CO3− production in fasted rats compared with fed controls, we have demonstrated that hyperpolarized 13C MR is sensitive to physiological perturbations in PDH flux. Further, we evaluated the ability of the hyperpolarized 13C MR technique to monitor disease progression by examining PDH flux before and 5 days after streptozotocin induction of type 1 diabetes. We detected decreased H13CO3− production with the onset of diabetes that correlated with disease severity. These observations were supported by in vitro investigations of PDH activity as reported in the literature and provided evidence that flux through the PDH enzyme complex can be monitored noninvasively, in vivo, by using hyperpolarized 13C MR.

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.

Hyperpolarized (13)C magnetic resonance reveals early- and late-onset changes to in vivo pyruvate metabolism in the failing heart.

Impaired energy metabolism has been implicated in the pathogenesis of heart failure. Hyperpolarized 13C magnetic resonance (MR), in which 13C‐labelled metabolites are followed using MR imaging (MRI) or spectroscopy (MRS), has enabled non‐invasive assessment of pyruvate metabolism. We investigated the hypothesis that if we serially examined a model of heart failure using non‐invasive hyperpolarized [13C]pyruvate with MR, the profile of in vivo pyruvate oxidation would change throughout the course of the disease.

Measuring intracellular pH in the heart using hyperpolarized carbon dioxide and bicarbonate: a 13C and 31P magnetic resonance spectroscopy study.

Aims Technological limitations have restricted in vivo assessment of intracellular pH (pHi) in the myocardium. The aim of this study was to evaluate the potential of hyperpolarized [1-13C]pyruvate, coupled with 13C magnetic resonance spectroscopy (MRS), to measure pHi in the healthy and diseased heart. Methods and results Hyperpolarized [1-13C]pyruvate was infused into isolated rat hearts before and immediately after ischaemia, and the formation of 13CO2 and H13CO3− was monitored using 13C MRS. The HCO3−/CO2 ratio was used in the Henderson–Hasselbalch equation to estimate pHi. We tested the validity of this approach by comparing 13C-based pHi measurements with 31P MRS measurements of pHi. There was good agreement between the pHi measured using 13C and 31P MRS in control hearts, being 7.12 ± 0.10 and 7.07 ± 0.02, respectively. In reperfused hearts, 13C and 31P measurements of pHi also agreed, although 13C equilibration limited observation of myocardial recovery from acidosis. In hearts pre-treated with the carbonic anhydrase (CA) inhibitor, 6-ethoxyzolamide, the 13C measurement underestimated the 31P-measured pHi by 0.80 pH units. Mathematical modelling predicted that the validity of measuring pHi from the H13CO3−/13CO2 ratio depended on CA activity, and may give an incorrect measure of pHi under conditions in which CA was inhibited, such as in acidosis. Hyperpolarized [1-13C]pyruvate was also infused into healthy living rats, where in vivo pHi from the H13CO3−/13CO2 ratio was measured to be 7.20 ± 0.03. Conclusion Metabolically generated 13CO2 and H13CO3− can be used as a marker of cardiac pHi in vivo, provided that CA activity is at normal levels.

Hyperpolarized Magnetic Resonance A Novel Technique for the In Vivo Assessment of Cardiovascular Disease

Cardiovascular disease is associated with high morbidity, mortality, and financial burden to healthcare services.1–3 In the United States, cardiovascular disease is the leading cause of death in both men and women, accounting for 1 in every 2.9 deaths in 2006, with coronary disease accounting for 1 in every 6 deaths.2 Noninvasive cardiac imaging increasingly plays a fundamental role in diagnosing, assessing prognosis, and monitoring therapy response in cardiovascular disease.1,4,5 Two-dimensional echocardiography is the most commonly used imaging modality to measure heart function because of its low cost and widespread accessibility. Computed tomography (CT), single photon emission CT, and positron emission tomography (PET) expose patients to ionizing radiation but have been used successfully for clinical assessment of coronary arteries, myocardial perfusion, and viability, respectively. Cardiovascular magnetic resonance (CMR) applies no ionizing radiation and is now considered the gold standard in assessing cardiac anatomy, function, and mass.1 CMR has also shown great potential for evaluating perfusion and viability with gadolinium-based contrast agents. MR spectroscopy (MRS) and MR-based molecular imaging methods have shown promise for evaluating cardiac metabolism. For example, phosphorus-31 MRS assesses high-energy phosphate content and energy reserve in the human heart (reviewed elsewhere6). Other implementations of multinuclear MRS, including oxygen-17, carbon-13, sodium-23, and proton MRS, have described measurement of oxygen consumption,7 substrate selection, and rates of metabolic flux,8 postinfarct sodium accumulation,9 and lipid accumulation,10 respectively, in ex vivo and in vivo experimental models of disease. MR-based molecular imaging of particles labeled with fluorine-19 nuclei has been used to study tracer and drug pharmacokinetics and metabolism.11 Combined PET–MR imaging (MRI) methods have been demonstrated in preclinical and noncardiac applications to assess cardiac parameters in an infarct mouse model12 and for structural, functional, and molecular imaging …

Bone marrow-derived stromal cells home to and remain in the infarcted rat heart but fail to improve function: an in vivo cine-MRI study

Basic and clinical studies have shown that bone marrow cell therapy can improve cardiac function following infarction. In experimental animals, reported stem cell-mediated changes range from no measurable improvement to the complete restoration of function. In the clinic, however, the average improvement in left ventricular ejection fraction is around 2% to 3%. A possible explanation for the discrepancy between basic and clinical results is that few basic studies have used the magnetic resonance (MR) imaging (MRI) methods that were used in clinical trials for measuring cardiac function. Consequently, we employed cine-MR to determine the effect of bone marrow stromal cells (BMSCs) on cardiac function in rats. Cultured rat BMSCs were characterized using flow cytometry and labeled with iron oxide particles and a fluorescent marker to allow in vivo cell tracking and ex vivo cell identification, respectively. Neither label affected in vitro cell proliferation or differentiation. Rat hearts were infarcted, and BMSCs or control media were injected into the infarct periphery (n = 34) or infused systemically (n = 30). MRI was used to measure cardiac morphology and function and to determine cell distribution for 10 wk after infarction and cell therapy. In vivo MRI, histology, and cell reisolation confirmed successful BMSC delivery and retention within the myocardium throughout the experiment. However, no significant improvement in any measure of cardiac function was observed at any time. We conclude that cultured BMSCs are not the optimal cell population to treat the infarcted heart.

Role of Pyruvate Dehydrogenase Inhibition in the Development of Hypertrophy in the Hyperthyroid Rat Heart A Combined Magnetic Resonance Imaging and Hyperpolarized Magnetic Resonance Spectroscopy Study

Background— Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. Methods and Results— Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg · kg−1 · d−1). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022±0.0002 versus 0.0055±0.0005 second−1; P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-13C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100±20 versus 200±30 mg; P=0.04) despite no change in the increase observed in cardiac output. Conclusions— This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic flexibility of the hyperthyroid heart and reduce the level of hypertrophy that develops while maintaining the increased cardiac output required to meet the higher systemic metabolic demand.Background Hyperthyroidism increases heart rate, contractility and cardiac output, as well as metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate utilisation. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase (PDK), thereby inhibiting glucose oxidation via pyruvate dehydrogenase (PDH). Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy (MRS) to investigate the rate and regulation of in vivo pyruvate dehydrogenase (PDH) flux in the hyperthyroid heart, and to establish whether modulation of flux through PDH would alter cardiac hypertrophy.Background— Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. Methods and Results— Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg · kg−1 · d−1). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022±0.0002 versus 0.0055±0.0005 second−1; P =0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-13C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100±20 versus 200±30 mg; P =0.04) despite no change in the increase observed in cardiac output. Conclusions— This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic flexibility of the hyperthyroid heart and reduce the level of hypertrophy that develops while maintaining the increased cardiac output required to meet the higher systemic metabolic demand. # Clinical Perspective {#article-title-52}

Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised 13C MRS.

Many diseases of the heart are characterised by changes in substrate utilisation, which is regulated in part by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information on the metabolic mechanisms of cardiac diseases. Hyperpolarised [1‐13C]pyruvate, detected using MRS, is a novel technique for the noninvasive evaluation of PDH flux. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarised MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDHa). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of pyruvate infusion during hyperpolarised MRS experiments. Hyperpolarised MRS measurements of PDH flux correlated significantly with ex vivo measurements of PDHa, confirming that PDH activity influences directly the in vivo flux of hyperpolarised pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarised MRS experiments was approximately 250 µM, equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. The concentrations of other metabolites, including lactate, glucose and β‐hydroxybutyrate, did not vary during the 60 s following pyruvate infusion. Hence, during the 60‐s data acquisition period, metabolism was minimally affected by pyruvate infusion. Copyright

A high-fat diet impairs cardiac high-energy phosphate metabolism and cognitive function in healthy human subjects

BACKGROUND High-fat, low-carbohydrate diets are widely used for weight reduction, but they may also have detrimental effects via increased circulating free fatty acid concentrations. OBJECTIVE We tested whether raising plasma free fatty acids by using a high-fat, low-carbohydrate diet results in alterations in heart and brain in healthy subjects. DESIGN Men (n = 16) aged 22 ± 1 y (mean ± SE) were randomly assigned to 5 d of a high-fat, low-carbohydrate diet containing 75 ± 1% of calorie intake through fat consumption or to an isocaloric standard diet providing 23 ± 1% of calorie intake as fat. In a crossover design, subjects undertook the alternate diet after a 2-wk washout period, with results compared after the diet periods. Cardiac (31)P magnetic resonance (MR) spectroscopy and MR imaging, echocardiography, and computerized cognitive tests were used to assess cardiac phosphocreatine (PCr)/ATP, cardiac function, and cognitive function, respectively. RESULTS Compared with the standard diet, subjects who consumed the high-fat, low-carbohydrate diet had 44% higher plasma free fatty acids (P < 0.05), 9% lower cardiac PCr/ATP (P < 0.01), and no change in cardiac function. Cognitive tests showed impaired attention (P < 0.01), speed (P < 0.001), and mood (P < 0.01) after the high-fat, low-carbohydrate diet. CONCLUSION Raising plasma free fatty acids decreased myocardial PCr/ATP and reduced cognition, which suggests that a high-fat diet is detrimental to heart and brain in healthy subjects.