Marie A. Schroeder

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)

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 …

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

The Cycling of Acetyl-Coenzyme A Through Acetylcarnitine Buffers Cardiac Substrate Supply A Hyperpolarized 13C Magnetic Resonance Study

Background— Carnitine acetyltransferase catalyzes the reversible conversion of acetyl-coenzyme A (CoA) into acetylcarnitine. The aim of this study was to use the metabolic tracer hyperpolarized [2-13C]pyruvate with magnetic resonance spectroscopy to determine whether carnitine acetyltransferase facilitates carbohydrate oxidation in the heart. Methods and Results— Ex vivo, following hyperpolarized [2-13C]pyruvate infusion, the [1-13C]acetylcarnitine resonance was saturated with a radiofrequency pulse, and the effect of this saturation on [1-13C]citrate and [5-13C]glutamate was observed. In vivo, [2-13C]pyruvate was infused into 3 groups of fed male Wistar rats: (1) controls, (2) rats in which dichloroacetate enhanced pyruvate dehydrogenase flux, and (3) rats in which dobutamine elevated cardiac workload. In the perfused heart, [1-13C]acetylcarnitine saturation reduced the [1-13C]citrate and [5-13C]glutamate resonances by 63% and 51%, respectively, indicating a rapid exchange between pyruvate-derived acetyl-CoA and the acetylcarnitine pool. In vivo, dichloroacetate increased the rate of [1-13C]acetylcarnitine production by 35% and increased the overall acetylcarnitine pool size by 33%. Dobutamine decreased the rate of [1-13C]acetylcarnitine production by 37% and decreased the acetylcarnitine pool size by 40%. Conclusions— Hyperpolarized 13C magnetic resonance spectroscopy has revealed that acetylcarnitine provides a route of disposal for excess acetyl-CoA and a means to replenish acetyl-CoA when cardiac workload is increased. Cycling of acetyl-CoA through acetylcarnitine appears key to matching instantaneous acetyl-CoA supply with metabolic demand, thereby helping to balance myocardial substrate supply and contractile function.

Simultaneous investigation of cardiac pyruvate dehydrogenase flux, Krebs cycle metabolism and pH, using hyperpolarized [1,2-(13)C2]pyruvate in vivo.

13C MR spectroscopy studies performed on hearts ex vivo and in vivo following perfusion of prepolarized [1‐13C]pyruvate have shown that changes in pyruvate dehydrogenase (PDH) flux may be monitored non‐invasively. However, to allow investigation of Krebs cycle metabolism, the 13C label must be placed on the C2 position of pyruvate. Thus, the utilization of either C1 or C2 labeled prepolarized pyruvate as a tracer can only afford a partial view of cardiac pyruvate metabolism in health and disease. If the prepolarized pyruvate molecules were labeled at both C1 and C2 positions, then it would be possible to observe the downstream metabolites that were the results of both PDH flux (13CO2 and H13CO3‐) and Krebs cycle flux ([5‐13C]glutamate) with a single dose of the agent. Cardiac pH could also be monitored in the same experiment, but adequate SNR of the 13CO2 resonance may be difficult to obtain in vivo. Using an interleaved selective RF pulse acquisition scheme to improve 13CO2 detection, the feasibility of using dual‐labeled hyperpolarized [1,2‐13C2]pyruvate as a substrate for dynamic cardiac metabolic MRS studies to allow simultaneous investigation of PDH flux, Krebs cycle flux and pH, was demonstrated in vivo. Copyright

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.