Visualization of in vivo metabolic flows reveals accelerated utilization of glucose and lactate in penumbra of ischemic heart

Acute ischemia produces dynamic changes in labile metabolites. To capture snapshots of such acute metabolic changes, we utilized focused microwave treatment to fix metabolic flow in vivo in hearts of mice 10 min after ligation of the left anterior descending artery. The left ventricle was subdivided into short-axis serial slices and the metabolites were analyzed by capillary electrophoresis mass spectrometry and matrix-assisted laser desorption/ionization imaging mass spectrometry. These techniques allowed us to determine the fate of exogenously administered 13C6-glucose and 13C3-lactate. The penumbra regions, which are adjacent to the ischemic core, exhibited the greatest adenine nucleotide energy charge and an adenosine overflow extending from the ischemic core, which can cause ischemic hyperemia. Imaging analysis of metabolic pathway flows revealed that the penumbra executes accelerated glucose oxidation, with remaining lactate utilization for tricarboxylic acid cycle for energy compensation, suggesting unexpected metabolic interplays of the penumbra with the ischemic core and normoxic regions.

because at this time point, blood glucose concentration is high enough to trace metabolic pathways of in vivo organs by MS 2 . 13 C3-lactate (27 μg/g body weight, in saline) was administered by retro-orbital injection 1 min before LAD ligation 3 . We have confirmed the elevation of blood lactate concentration to 7-8 mM at this time point, which is within the physiological range ( Supplementary Fig. S5 online) 4 .

Fixation of heart metabolites by FMW
We used a laboratory microwave instrument (MMW-05 Muromachi Kikai, Tokyo) designed for the euthanasia of laboratory mice and rats ( Supplementary Fig. S1 online). This instrument differs from kitchen units, particularly in maximal power output (5 kW) and in having a tightly focused microwave beam. All units direct their microwave energy to a specific anatomical location on the animal. Mice were anesthetized with isoflurane, and placed into a transparent water-jacket holder (Muromachi-Kikai, MH28-HZ). The cone-parts of the holder were filled with ~1 mL of water to help elevate the temperature of the heart as uniformly as possible using microwave energy. Care was taken not to introduce air bubbles.
This was then inserted into the instrument in a position such that microwave irradiation was targeted on both brain and heart. For reliable fixation it is important to maintain the animal in the correct position; i.e., the mouse should be straight with its nose touching the top of the cone. The holder is set at the position shown in Supplementary Fig. S1, panel-D; the back end of the holder was set at 43 mm from the entrance of the insertion slot. We found this condition optimal for B6/J mice (8 week old males). Microwave irradiation at 5 kW for 0.96 s elevated the temperature of the heart to above 80°C which is sufficient to inactivate metabolic enzymes, such as acetylcholine esterase 5 .
To evaluate the effectiveness of the FMW fixation method, we compared it to two other procedures, i) rapid-freezing, in which hearts were isolated immediately after thoracotomy, then frozen in liquid N2 (total procedure takes ~20 s); ii) delayed freezing, in which hearts were isolated 10 min after cervical dislocation to allow postmortem degradation.

Preparation of tissue sections for metabolome and MALDI-imaging analyses
After FMW, hearts were dissected with a surgical knife at room temperature, embedded into a super cryo-embedding medium (SCEM, Section Lab Co. Ltd, Hiroshima, Japan), frozen in liquid N2, and stored at -80°C. We prepared five sets of short-axis (transverse) tissue sections where each set consisted of a thick 450 µm "block" for quantification of metabolites and an adjacent 8 µm thin "section" for MALDI imaging analyses (see Supplementary Fig. S6 online). The apical two thirds of the left ventricle in sham-operated hearts was subdivided into four short-axis blocks.
The thin sections were cut with a cryomicrotome (CM3050, Leica Microsystems) and thaw-mounted on an indium thin oxide-coated glass slide (BrukerDaltonics, Germany) at -16˚C. Heart tissues subjected to FMW tended to be more fragile than those treated by other methods, often making tissue sectioning challenging. However, embedding the tissue with SCEM medium, which did not interfere with the ionization efficiency of metabolites, helped achieve successful sectioning.

Capillary electrophoresis-electrospray ionization (CE-ESI)-MS
Quantitative metabolome analysis was performed using CE-MS 6 . Briefly, to extract metabolites from the tissue, the frozen tissue block embedded in SCEM medium together with internal control compounds (see below) was homogenized in ice-cold methanol (500 μL) using a manual homogenizer (Finger Masher (AM79330); Sarstedt, Tokyo, Japan), followed by the addition of an equal volume of chloroform and 0.4 times the volume of ultrapure water (LC/MS grade; Wako). The suspension was then centrifuged at 15,000 g for 15 min at 4°C. After centrifugation, the aqueous phase was ultra-filtered using an ultrafiltration tube (Ultrafree-MC, UFC3 LCC NB; Human Metabolome Technologies, Tsuruoka, Japan). The filtrate was concentrated with a vacuum concentrator (SpeedVac; Thermo, Yokohama, Japan); this condensation process helps quantitate trace levels of metabolites. The concentrated filtrate was dissolved in 50 µL of ultrapure water and used for CE-MS.
All CE-MS experiments were performed using an Agilent CE System equipped with an air pressure pump, an Agilent 6520 Accurate Q-Tof mass spectrometer, an Agilent 1200 series isocratic high-performance LC pump, 7100 CE-system, a G1603A Agilent CE-MS adapter kit, and a G1607A Agilent CE-MS sprayer kit (Agilent Technologies).
Isomeric species, such as glucose 1-phosphate, glucose 6-phosphate, and fructose 6phosphate, can be hard to distinguish by MS/MS per se. However, we took advantage of the CE/ESI/MS system 6 , in which these species are eluted at a different retention time by capillary electrophoresis due to their differential mobility and/or chemical properties. The system separates isobaric or isomeric compounds effectively.

Electrospray interface and MS conditions
ESI-MS was conducted in negative ion mode, and the capillary voltage was set at 3.5 kV.
The nitrogen nebulizer pressure was set at 10 psi, and a flow rate of drying nitrogen gas (heater temperature 240°C) was maintained at 4 L/min. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards; namely, TFA anion (m/z 112.985587) and HP-0921 compound anion (m/z 1033.988109) (G1969-8500, API-TOF Reference Mass Solution Kit, Agilent Technologies). We confirmed that the mass error was within 10 ppm for the targeted metabolites. Exact mass data were acquired at a rate of 1.4 spectra/s over a 61−1050 m/z range (0.713 s duty cycle).

Quantification of metabolites by internal and external standards
We used both internal (added to the tissue before extraction) and external (used to produce calibration curves for each compound) standard compounds for concentration calculation.
The detailed method is as follows:

Internal standard (IS) compounds
We used 2-morpholinoethanesulfonic acid (MES) and 1,3,5-benzenetricarboxylic acid (trimesate) as ISs for anionic metabolites. These compounds are not present in the tissues; thus, they serve as ideal standards. Loss of endogenous metabolites during sample preparation was corrected by calculating the recovery rate (%) for each sample measurement.

External standard (ES) compounds
An external calibration curve was used to calculate the absolute abundance of metabolites.
Before sample measurement, we measured the mixture of authentic compounds of target metabolites at three different concentrations (50, 10 and 5 µM for adenine nucleotides, and 500, 100 and 50 µM for lactate) in ultrapure water to generate calibration curves.
Quantification (amount of metabolites, nmol/mg tissue or nmol/mg protein) was performed by comparing the IS-normalized peak areas against the calibration curves.

Liquid chromatography-tandem mass spectrometry
The amount of non-labeled and 13 C 6 -glucose in the heart was quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Briefly, a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source (LCMS-8030; Shimadzu Corporation, Kyoto, Kyoto, Japan) was used in the negative-ESI and multiple reaction monitoring (MRM) modes. The samples were resolved on the PC-HILIC S3 column (150 × 2.0 mm, i.d., 4 μm particle), and separated using mobile phase A (water) and mobile phase B (acetonitrile) at a flow rate of 0.8 mL/min and a column temperature of 40°C. Nonlabeled-and 13 C6-labeled-glucose were quantified by ion transitions from m/z 179 to m/z 89, and m/z 185 to m/z 92 respectively.

Matrix coating and MALDI-IMS acquisition
Prior to matrix coating, the tissue slices were placed in desiccant for 10 min and allowed to equilibrate to room temperature. We used 9-aminoacridine as a matrix (10 mg/mL, dissolved in 80% ethanol) and manually spray-coated tissues sections with the solution using an artistic air-brush (Procon Boy FWA Platinum 0.2-mm caliber airbrush, Mr. Hobby, Tokyo, Japan). We maintained a distance of ~5 cm between the air-brush and the target during matrix coating, and allowed sections to dry between coating cycles to minimize delocalization of target compounds.
MALDI-IMS was performed using an Ultra Flextreme MALDI-time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Leipzig, Germany) equipped an Nd:YAG laser. Accurate MS and MS/MS analyses were performed with a prototype "Mass microscope" (Shimadzu Corporation, Kyoto, Japan). For both instruments, the laser power was optimized to minimize in-source decay of phosphate nucleotides. Data were acquired in the negative reflectron mode with raster scanning using a pitch distance of 100 μm. Each mass spectrum was the result of 300 laser shots at each data point. Signals between m/z 50 and 1000 were collected. Image reconstruction was performed using FlexImaging 4.0 software (Bruker Daltonics). Peaks of specific metabolite molecules were assigned by accurate MS analyses with an ion trap TOF instrument (see Supplementary Table S2  It is not possible to separate 13 C2-glu and 13 C3-gln by MALDI-imaging. To solve this problem, we utilized CE/ESI/MS analyses to determine 13 C2-glu and 13 C3-gln content in adjacent sections from the same sample. By doing so, we confirmed that the level of 13 C3-gln was below the level of detection. This led us to conclude that the m/z value, 149.13, in negative ion mode represents 13 C2-glu and that the sample is less likely to be contaminated with 13 C3-gln.

MALDI imaging of metabolites in tissue sections normalized by CE-MS based quantitative data
To construct apparent content maps for a specific metabolite, we modified a previously reported method 8 . Briefly, an apparent content of a specific metabolite at the i th spot of tissue ( ) was estimated as follows: where denotes the mean value of a metabolite content determined using CE/ESI/MS in corresponding tissue block, is the maximum intensity of the mass spectra within a specified range at the i th spot, and is the median of the maximum intensities of the metabolite from all the spots.

Statistical analysis
Measurements are reported as mean ± SEM. For single comparisons, we performed an unpaired two-tailed Student's t-test; for multiple comparisons, we used an analysis of variance (ANOVA) followed by Tukey's correction for post hoc comparisons. Significance was considered at P < 0.05. Statistical analyses were performed using SPSS® software (SPSS Inc., Chicago, IL, USA).

Animal welfare
According to the American Veterinary Medical Association Recommendations (AVMA Guidelines for the Euthanasia of Animals: 2013 Edition), high-energy microwave irradiation is a humane method for euthanizing small laboratory rodents where unconsciousness is achieved in less than 100 ms with a complete loss of brain function in less than 1 sec.
During mouse heart fixation, heartbeat completely ceased within 1 sec. Therefore, we consider FMW-fixation of the heart to be humane and satisfies the criteria provided by an ethical review at each research institute. (v)

Figure S2. Quantitative imaging of cardiac metabolites by Q-IMS
Application of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), combined with focused microwave irradiation (FMW) to rapidly fix tissue metabolism, accurately and reproducibly visualized regional contents of in vivo endogenous metabolites and spatial differences of metabolites in mice. MALDI-IMS reconstructed by capillary electrophoresis-mass spectrometry (CE-MS)-based data could evaluate the fluctuation of metabolites between different mouse tissue slices more quantitatively.

H&E-stained
Remnant blood in the left ventricle Figure S3.

Sections of heart tissues
Sections of heart tissue stained with hematoxylin and eosin following focused microwave irradiation at 5 kW for 0.94 sec, rapid-freezing and delayed-freezing methods.

Figure S7. Tandem MS analysis to identify glutamate and NADH
The chemical structures show the assignments of the diagnostic fragments. Comparisons of tissue MS/MS spectra with ion peaks at m/z 146 (negative-ion mode) and m/z 664 (negative-ion mode) obtained from tissue or glutamate and NADH standards, respectively. The similarity of the two spectra was used to assign the metabolites as glutamate and NADH.   Figure S11. Pathway-tracing analysis using 13 C 6 -glucose in the hearts 5 blocks obtained from LAD-ligated hearts were used for capillary electrophoresis-mass spectrometry (CE-MS) quantification of 13 C-containing metabolites. Data obtained from different blocks were represented with pie charts.
Two sets of results obtained from independent experiments are shown.

Table S1
List of metabolites that can be visualized by capillary electrophoresis-mass spectrometry (CE-MS) or matrixassisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). Note that the number of metabolites that can be visualized by IMS is smaller than that measurable by CE-MS.

Table S1
List of metabolites that can be visualized by capillary electrophoresis-mass spectrometry (CE-MS) or matrixassisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). Note that the number of metabolites that can be visualized by IMS is smaller than that measurable by CE-MS.