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Probing Human Myocardial Krebs Cycle Metabolism and Response to Glucose Challenge using Hyperpolarized [2-¹³C]Pyruvate MR Spectroscopy

Hsin-Yu Chen¹, Jeremy W. Gordon¹, Nicholas Dwork¹, Brian T. Chung¹, Andrew Riselli², Robert A. Bok¹, James B. Slater¹, M. Roselle Abraham³, Daniel B. Vigneron¹, and Peder E.Z. Larson¹
¹Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, United States, ²School of Pharmacy, University of California, San Francisco, San Francisco, CA, United States, ³Department of Medicine-Cardiology, University of California, San Francisco, San Francisco, CA, United States

Synopsis

This study explored the safety and feasibility to visualize real-time myocardial Krebs cycle energetics in healthy volunteers using hyperpolarized [2-¹³C]pyruvate MR spectroscopy, and to investigate the response to oral glucose challenge. Identified metabolic products included [2-¹³C]lactate, Krebs cycle-related intermediate [5-¹³C]glutamate, and [1-¹³C]acetylcarnitine, a key player in the “carnitine shuttle” of mitochondrial fatty acid oxidation. Upon oral glucose challenge, the levels of all three products increased, illustrating the metabolic flexibility of human heart to switch between fatty acid and carbohydrates.

Purpose

Despite advances in the medical management and research, heart diseases remain the top cause of death both in the US and globally¹. There is mounting evidence that shifts in metabolism and substrate selection are present at early stages of heart disease and cardiomyopathies, but it is challenging to non-invasively measure these shifts in vivo². Previous work has shown feasibility of hyperpolarized [1-¹³C]pyruvate in the human heart^3,4,5 and detection of [1-¹³C]lactate facilitated by lactate dehydrogenase (LDH) and ¹³C-bicarbonate facilitated by pyruvate dehydrogenase (PDH) as pyruvate enters the Krebs cycle. The goal of this study was to assess the feasibility of human studies probing myocardial Krebs cycle metabolism using hyperpolarized [2-¹³C]pyruvate which, in animal models, is able to detect additional Krebs cycle metabolites of glutamate, citrate and acetylcarnitine^6,7,8.

Methods

Hyperpolarized-¹³C Exam: GMP [2-¹³C]pyruvic acid was prepared and polarized in a 5T GE SpinLab (GE Healthcare, Chicago IL) polarizer for at least 2.5 hours. The dissolution yielded 202-245 mM(mean:228) pyruvate with 27.1-31.2%(mean:28.5) polarization and 0.6-2.2uM residual trityl radical. Two healthy volunteers (male, age:40-42 y/o, median:41) were enrolled in this study and received two injections of [2-¹³C]pyruvate. The subjects were on a > 4 hour fast at the baseline hyperpolarized scan. Another hyperpolarized scan was acquired 30 minutes after p.o. glucose challenge with sports drink containing approximately 29 grams of sugar. Serum glucose level was measured right before each hyperpolarized injection. The study was conducted on a clinical 3T system (MR750, GE Healthcare) using a clamshell ¹³C transmitter and an 8-channel paddle receiver array. As illustrated in Figure 1, the two paddles were positioned such that one paddle covers the top and the other the side of the chest wall. The study protocol was approved by the UCSF Institutional Review Board and an FDA IND.

Pulse Sequence: A multiband spectral-spatial RF pulse (Figure 1B) was used to excite frequencies for [2-¹³C]pyruvate, [5-¹³C]glutamate/[5-¹³C]glutamine/[1-¹³C]acetylcarnitine (ALCAR), and [2-¹³C]lactate with 5°, 20°, and 20°, respectively. Dynamic slab spectroscopy (TE = 3.7ms) was acquired at 3s intervals, targeting a 5cm-thick axial slab over the left ventricle.

Data Processing and Analysis: The spectroscopy data was processed using MATLAB (MathWorks, Natick MA) and Mestrenova (Mestrelab, Spain). Briefly, the array channels were combined using pre-whitened SVD as previously described⁹. The dynamic spectra were enhanced using SVD rank truncation¹⁰ with rank=10, chosen based on the number of distinct ¹³C species. This was followed by phase and baseline corrections, and peak quantification in Mestrenova.

Results and Discussions

All [2-¹³C]pyruvate injections were well tolerated without adverse events. The serum glucose levels were 81 and 119(mg/dL) in Volunteer 1 at the time of baseline and post-glucose C2 pyruvate injections, and 76 and 104 in Volunteer 2, respectively.

The substrates and products in the spectra (Figure 2) were assigned [5-¹³C]glutamate(182.5ppm), [1-¹³C]acetylcarnitine(ALCAR, 173.6ppm), [2-¹³C]lactate(70.8, 66.2ppm), [1,2-¹³C]pyruvate(171.8, 169.7ppm), and [2-¹³C]hydrate(94.2ppm), respectively, referencing to [2-¹³C]pyruvate (206ppm). The substrate/product assignment was consistent with their kinetic profiles (Figures 3&4). In contrast to a prior work looking at [2-¹³C]pyruvate in healthy human brain¹¹, ALCAR was notably present in the heart, whereas [5-¹³C]glutamine/[1-¹³C]citrate (temporarily assigned, 179.6ppm) was low/absent. The summed apparent SNR of pyruvate, lactate, glutamate, and ALCAR were 1979, 71, 24, and 22 after enhancement, respectively.

The metabolic product-to-pyruvate ratios before and after glucose challenge were summarized in Figure 4B Table. The baseline lactate and ALCAR levels were notably comparable between the two individuals. Also consistent were the percentage changes, with 40.9% increase in lactate/pyruvate, 24.2% in glutamate/pyruvate, and 114% in ALCAR/pyruvate. Interestingly, Volunteer 2 had lower fasting glutamate, and the slight variability in intersubject glutamate % changes are likely attributed to SNR. Figure 4C Table summarized the conversion rate constants, calculated to a first-order approximation using an “input-less” model¹². The increase of metabolic products [2-¹³C]lactate and [5-¹³C]glutamate upon glucose challenge was consistent with a prior study⁴ reporting the significant elevation of [1-¹³C]bicarbonate and slight increase of [1-¹³C]lactate in a normal volunteer cohort, indicative of the metabolic flexibility of the heart dictated by Randle cycle¹³.

The presence of [1-¹³C]ALCAR highlights its essential role in the carnitine shuttle as a part of fatty acid β-oxidation, which typically fuels ~70% of the energetics in a healthy heart. An ostensible explanation for the elevation of ALCAR in response to glucose challenge is the increased mitochondrial acetyl-CoA labeling being transferred onto carnitine via carnitine acetyltransferase (Figure 5). However, its real mechanism is likely more complex and remains of great interest for future studies.

In summary, it is safe and feasible to interrogate Krebs cycle energetics in the healthy human heart using hyperpolarized [2-¹³C]pyruvate. The level of Krebs cycle intermediates and acetylcarnitine labeling increased upon oral glucose challenge. This proof-of-concept [2-¹³C]pyruvate spectroscopy study of human heart warranted future development of imaging-based acquisition techniques on a dedicated cardiac MR software platform¹⁴ and investigation of Krebs cycle modulation in disease processes^4,5.

Conclusions

For the first time, hyperpolarized [2-¹³C]pyruvate MRS was applied for the real-time visualization of Krebs cycle metabolism in healthy human heart. The identification of downstream glutamate and acetylcarnitine was consistent with the Krebs cycle intermediates and fatty acid oxidation in myocardium.

Acknowledgements

This work was supported by grants from the UCSF Resource Allocation Program, Myokardia Inc. Myoseeds program, NIH (P41EB013598), and a postdoctoral fellowship from the American Heart Association.

References

[1] World Health Organization; https://www.who.int/data/gho/data/themes/mortality-and-global-health-estimates

[2] Timm KN et al., Cardiac applications of hyperpolarised magnetic resonance. Prog Nucl Magn Reson Spectrosc 2018; 106, 66-87

[3] Cunningham CH et al., Hyperpolarized ¹³C metabolic MRI of the human heart: initial experience. Circulation Research 2016; 119(11), 1177-1182

[4] Rider, OJ et al., Noninvasive in vivo assessment of cardiac metabolism in the healthy and diabetic human heart using hyperpolarized ¹³C MRI. Circulation Research 2020; 126(6), 106617.

[5] Park, JM et al., Effect of Doxorubicin on Myocardial Bicarbonate Production from Pyruvate Dehydrogenase in Women with Breast Cancer. Circulation Research 2020; 127, 1568-1570.

[6] Schroeder MA et al., Real‐time assessment of Krebs cycle metabolism using hyperpolarized C magnetic resonance spectroscopy. The FASEB journal 2018; 23(8), 2529-2538

[7] Schroeder MA et al., Hyperpolarized ¹³C magnetic resonance reveals early‐and late‐onset changes to in vivo pyruvate metabolism in the failing heart. Eur J Heart Fail 2013; 15(2), 130-140

[8] Schroeder MA et al., The cycling of acetyl-coenzyme A through acetylcarnitine buffers cardiac substrate supply: a hyperpolarized ¹³C magnetic resonance study. Circ Cardiovasc Imaging 2012; 5(2), 201-209

[9] Vareth M et al., A comparison of coil combination strategies in 3D multi‐channel MRSI reconstruction for patients with brain tumors. NMR Biomed 2019; 31(11), e3929

[10] Brender, JR et al., Dynamic Imaging of Glucose and Lactate Metabolism by 13C-MRS without Hyperpolarization. Scientific Reports 2019; 9(1), 3410.

[11] Chung, BT et al., First hyperpolarized [2-¹³C] pyruvate MR studies of human brain metabolism. J Magn Reson Imaging 2019; 309, 106617.

[12] Larson PEZ et al., Investigation of analysis methods for hyperpolarized ¹³C‐pyruvate metabolic MRI in prostate cancer patients. NMR Biomed 2018; 31(11), e3997.

[13] Tyler, DJ et al., Demonstrating the Randle cycle in vivo: assessment of physiological alterations in human cardiac metabolism using hyperpolarised ¹³C MR spectroscopy. Proceedings of the ISMRM, 2017

[14] Tang, S et al., A regional bolus tracking and real‐time B1 calibration method for hyperpolarized ¹³C MRI. Magn Reson Med 2019; 81(2), 839-851.

Figures

Figure 1. A) Fiducials showing the placement of the 8-channel paddle receivers. One was located over the chest wall, whereas the other was on the side. The coil coverage encompasses the level of atrium to the liver dome in the superior-inferior direction. An axial 5-cm slab was targeted over the left ventricle. B) The specialized multi-band spectral-spatial RF excites the substrate [2-¹³C]pyruvate with 5° flip and products [2-¹³C]lactate, [5-¹³C]glutamate, and [1-¹³C]acetylcarnitine with 20° flip.

Figure 2. Representative summed spectra (Volunteer 1) reflecting baseline Krebs cycle energetics in human heart probed with hyperpolarized C2 pyruvate. The substrates and products in the spectra were assigned [5-¹³C]glutamate(182.5ppm), [1-¹³C]acetylcarnitine(ALCAR, 173.6ppm), [2-¹³C]lactate(70.8, 66.2ppm), [1,2-¹³C]pyruvate(171.8, 169.7ppm), and [2-¹³C]hydrate(94.2ppm), respectively.

Figure 3. Representative dynamic spectra over time (Volunteer 1) showing baseline metabolism of C2 pyruvate in human heart. The substrates ([2-¹³C]pyruvate, [1,2-¹³C]pyruvate, [2-¹³C]hydrate) and products ([5-¹³C]glutamate, [2-¹³C]lactate, [1-¹³C]acetylcarnitine) assignment was consistent with their kinetic profiles. The spectra was enhanced with SVD rank truncation as previously described¹⁰.

Figure 4. A) Temporal evolution of the injected [2-¹³C]pyruvate and downstream metabolites [2-¹³C]lactate, [5-¹³C]glutamate and [1-¹³C]acetylcarnitine(ALCAR) in Volunteer 1 before and 30-minutes after oral glucose challenge. Time was calculated from the start of injection. B) A table showing the metabolites ratios normalized to pyruvate. The levels of lactate, glutamate and ALCAR were elevated after oral glucose compared to baseline. C) Rate constants of pyruvate-to-product conversion were calculated to a first-order approximation.

Figure 5. A diagram representing the flux of [2-¹³C]pyruvate label into the Krebs cycle and the carnitine shuttle, showing the propagation of C13 labeling in this study. The carnitine group is used to “shuttle” the long chain fatty acid into the mitochondria for β oxidation.

Proc. Intl. Soc. Mag. Reson. Med. 29 (2021)

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