Wei Chen1, Chalermchai Khemtong1, Weina Jiang1, Craig R. Malloy1, and A. Dean Sherry1
1Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, United States
Synopsis
A large prior literature on inter-conversion of β-hydroxybutyrate (β-HB) and acetoacetate (AcAc) indicates that the process is mitochondrial and the ratio reflects specifically mitochondrial redox state. Therefore the conversion of [1,3-13C]AcAc to [1,3-13C]β-HB is expected to be sensitive to redox. In this study, we explored the utility of using hyperpolarized [1,3-13C]AcAc to study the mitochondrial redox state in perfused rat hearts. Our results show that the production of HP β-HB from HP-AcAc was much higher in ischemic hearts, reflecting the increased concentration of NADH under this reduced state. The redox-dependent conversion between this metabolic pair in mitochondria may lead to the development of an imaging tool for redox imaging of the heart by hyperpolarized 13C MRI.
A large prior literature on inter-conversion of β-hydroxybutyrate (β-HB) and acetoacetate (AcAc) indicates that the process is mitochondrial and the ratio reflects specifically mitochondrial redox state. Therefore the conversion of [1,3-13C]AcAc to [1,3-13C]β-HB is expected to be sensitive to redox. In this study, we explored the utility of using hyperpolarized [1,3-13C]AcAc to study the mitochondrial redox state in perfused rat hearts.
Methods
[1,3-13C]-acetoacetate in 50% glycerol-water was polarized in a HyperSense polarizer in the presence of OX63 radical (15 mM) and ProHance (2 mM Gd). Hearts excised from male Sprague-Dawley rats were placed in an 18-mm NMR tube attached to a water-jacketed glass perfusion apparatus bubbled continuously with 95:5 O2/CO2 and placed inside the bore of a 9.4T vertical-bore magnet. The hearts were studied using standard Langendorff methods at 37°C in four groups: 1) normal perfusion heart (control heart); 2) global ischemia perfusion heart; 3) low perfusion pressure (LPP), 25 cm H2O; 4) normal perfusion pressure with rotenone (25 µM). All hearts were initially perfused at 100-cm water pressure with Krebs-Henseleit buffer containing 0.75% bovine serum albumin, 0.4 mM non-labeled free fatty acid, 5.5 mM glucose, 1 mM pyruvate, 0.1 mM lactate. After the heart had reached the metabolic steady state (~20 min), the heart was subjected to global ischemia by stopping perfusate delivery, low-flow ischemia by reducing the perfusion pressure to 25-cm water, or 25 μM rotenone, a mitochondrial complex I inhibitor, for 30 min. All hearts were monitored continuously by 31P NMR. Myocardial O2 consumption, heart rate, and coronary flow were also recorded to monitor functions of the hearts. After 50 min perfusion, HP [1,3-13C]AcAc was injected directly above the heart via a catheter. 13C NMR acquisition was initiated concomitantly with the HP injection. Hearts were freeze-clamped immediately after the NMR was complete high resolution NMR analyses. All 13C NMR spectra were acquired with decoupling using 20° pulses, 1-s acquisition time, and 1-s delay time. FIDs were zero-filled before Fourier transformation and relative peak areas in the phased spectra were measured by integration. The area of each metabolite resonance was normalized by the total area of all metabolite resonances and plotted as a function of time. The heart tissues were extracted with perchloric acid and analyzed by high resolution 1H NMR (600 MHz). The oxygen consumption measured in µmol/min/g dry weight of the hearts in each group 1 min before injection of the HP agent were 19.93±2.89 for control, 10.91±0.48 for global ischemia measured immediately after reperfusion, 2.67±2.13 for low flow ischemia and 2.07±1.56 for rotenone. 31P NMR of the hearts confirmed significant myocardial ischemia in the global ischemia, low flow ischemia, and rotenone-treated hearts as demonstrated by a marked increase in [Pi]/[ATP] ratios. In normoxic hearts, a low conversion of HP [1,3-13C]AcAc (HP-AcAc) to HP [1,3-13C]-β-HB (HP-β-HB) was observed following the injection of the HP agent. The production of HP β-HB was increased in low-flow ischemic (3.8-fold), global ischemic (1.8-fold), and rotenone-treated (1.5-fold) hearts. The apparent increased HP-AcAc to HP β-HB conversion agreed well with expected reduced mitochondrial redox state, thus increased [NADH], in these hearts. Inhibition of the electron transport chain in complex I enzyme resulted in excess mitochondrial NADH and therefore increased reduction of HP-AcAc. The increased β-HB as observed by HP 13C NMR was corroborated by high resolution 1H NMR of heart tissue extracts, where higher concentrations of β-HB were measured in the ischemic hearts (20±0.25, 61±0.67, 230±8.8, and 210±5.6 nM/g dry weight for control, global ischemia, low flow ischemia, and rotenone-treated hearts, respectively).
We demonstrate here the use of hyperpolarized 13C NMR to monitor 13C-acetoacetate/13C-β-hydroxybutyrate metabolism as an indicator for mitochondrial redox state in perfused rat hearts. The redox-dependent conversion between this metabolic pair in mitochondria may lead to the development of an imaging tool for redox imaging of the heart by hyperpolarized 13C MRI.
Acknowledgements
No acknowledgement found.References
Williamson DH, et al. (1967) Biochem J 103(2):514-527; Stein LR & Imai S (2012) Trends Endocrinol Metab 23(9):420-428; Khemtong C, et al. (2015) Magn Reson Med 74(2):312-319