This white paper discusses prospects for advancing hyperpolarization technology to better understand cancer metabolism, identify current obstacles to HP (hyperpolarized) 13C magnetic resonance imaging's (MRI's) widespread clinical use, and provide recommendations for overcoming them. Since the publication of the first NIH white paper on hyperpolarized 13C MRI in 2011, preclinical studies involving [1-13C]pyruvate as well a number of other 13C labeled metabolic substrates have demonstrated this technology's capacity to provide unique metabolic information. A dose-ranging study of HP [1-13C]pyruvate in patients with prostate cancer established safety and feasibility of this technique. Additional studies are ongoing in prostate, brain, breast, liver, cervical, and ovarian cancer. Technology for generating and delivering hyperpolarized agents has evolved, and new MR data acquisition sequences and improved MRI hardware have been developed. It will be important to continue investigation and development of existing and new probes in animal models. Improved polarization technology, efficient radiofrequency coils, and reliable pulse sequences are all important objectives to enable exploration of the technology in healthy control subjects and patient populations. It will be critical to determine how HP 13C MRI might fill existing needs in current clinical research and practice, and complement existing metabolic imaging modalities. Financial sponsorship and integration of academia, industry, and government efforts will be important factors in translating the technology for clinical research in oncology. This white paper is intended to provide recommendations with this goal in mind.

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.

Iron is essential to the cell. Both iron deficiency and overload impinge negatively on cardiac health. Thus, effective iron homeostasis is important for cardiac function. Ferroportin (FPN), the only known mammalian iron-exporting protein, plays an essential role in iron homeostasis at the systemic level. It increases systemic iron availability by releasing iron from the cells of the duodenum, spleen, and liver, the sites of iron absorption, recycling, and storage respectively. However, FPN is also found in tissues with no known role in systemic iron handling, such as the heart, where its function remains unknown. To explore this function, we generated mice with a cardiomyocyte-specific deletion of Fpn. We show that these animals have severely impaired cardiac function, with a median survival of 22 wk, despite otherwise unaltered systemic iron status. We then compared their phenotype with that of ubiquitous hepcidin knockouts, a recognized model of the iron-loading disease hemochromatosis. The phenotype of the hepcidin knockouts was far milder, with normal survival up to 12 mo, despite far greater iron loading in the hearts. Histological examination demonstrated that, although cardiac iron accumulates within the cardiomyocytes of Fpn knockouts, it accumulates predominantly in other cell types in the hepcidin knockouts. We conclude, first, that cardiomyocyte FPN is essential for intracellular iron homeostasis and, second, that the site of deposition of iron within the heart determines the severity with which it affects cardiac function. Both findings have significant implications for the assessment and treatment of cardiac complications of iron dysregulation. Significance The iron-exporting protein ferroportin is recognized as central to systemic iron regulation, but its role in tissues other than those involved in iron handling is unknown. This study shows that ferroportin expression in cardiomyocytes is essential to intracellular iron homeostasis and to normal cardiac function. It also demonstrates that the site of iron accumulation in the iron-overloaded heart depends on whether ferroportin is expressed in the cardiomyocytes. It further shows that the functional significance of cardiac iron overload is highly dependent upon the site of iron accumulation. These findings change our understanding of intracellular iron homeostasis and have significant implications for the clinical management of cardiac dysfunction associated with iron imbalance.

The advent of hyperpolarized ¹³ C 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- ¹³ C]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 (H ¹³ CO ₃ ⁻ ), a byproduct of the PDH-catalyzed conversion of [1- ¹³ C]pyruvate to acetyl-CoA. By noninvasively observing a 74% decrease in H ¹³ CO ₃ ⁻ production in fasted rats compared with fed controls, we have demonstrated that hyperpolarized ¹³ C MR is sensitive to physiological perturbations in PDH flux. Further, we evaluated the ability of the hyperpolarized ¹³ C MR technique to monitor disease progression by examining PDH flux before and 5 days after streptozotocin induction of type 1 diabetes. We detected decreased H ¹³ CO ₃ ⁻ 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 ¹³ C MR.

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]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 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.— 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)