Fumarate Is Cardioprotective via Activation of the Nrf2 Antioxidant Pathway

Summary 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.

Table S1 related to Figure 4 Results for FT-ICR mass spectrometry metabolomics List of putative identities based on the mass spectrometry data of the top 350 discriminant peaks between wild type and Fh -/-mouse cardiac biopsies. Peaks were selected by a PLS-DA forward-selection approach based on minimising the classification error. Only peaks that could be ascribed an identity are displayed.  2-(beta-D-Glucosyl)-snglycerol, 3-beta-D-Galactosyl-sn-glycerol a) fold change in concentration of compound in knock out compared to wild type b) LV1 weights from a PLS-DA model built from variables that had been forward selected based on regression values to maximise the separation between groups

Supplemental Experimental Procedures
Two-dimensional echocardiography 2-D transthoracic echocardiography was performed under isoflurane anaesthesia (1-1.25%) using a Vevo 2100 Imaging System (VisualSonics, Toronto, Canada) with a 30 MHz linear array transducer on a bench-mounted adjustable heated platform with continual ECG monitoring.
Ventricular dimensions and function were assessed from averaged measurements of at least three cardiac cycles using images obtained from the parasternal short-axis and long-axis views in accordance with consensus guidelines (Schiller et al., 1989). Image analysis was performed blinded to genotype off-line using Vevo software.

Left ventricular haemodynamics
A representative sample of mice fulfilling the above criteria was anaesthetized with isoflurane (1.25-1.5%) for the measurement of haemodynamic indices using a 1.4 F Millar Mikro-tip catheter (SPR-671) inserted into the LV via the carotid artery. After 15 min of baseline recordings, contractile reserve was assessed by infusion of dobutamine via the jugular vein (4-16 ng/g body wt/min) (Ten et al., 2005).

Perfused Heart Experiments
Mice were anesthetized with pentobarbitone (140 mg/kg body wt with 150 IU heparin, IP). Hearts were rapidly excised, cannulated, and perfused in Langendorff mode at a constant pressure of 80 mm Hg, 37°C with a modified Krebs-Henseleit buffer gassed with 95% O2/5% CO2 (pH 7.4) containing the following (in mmol/L): NaCl 118.5, NaHCO3 25.0, KCl 4.75, KH2PO4 1.18, MgSO4 1.19, D-glucose 11.0, and CaCl2 1.41. Susceptibility of FH-/-hearts to ischemia/reperfusion injury was tested by subjecting isolated hearts to 40 minutes of no-flow ischemia and 1 hour of reperfusion. All hearts had to fulfill the following inclusion criteria: coronary flow between 1.5 and 4.5 ml/ min, time from thoracotomy to aortic cannulation <3 min and no persistent dysrhythmia during stabilization. Coronary flow was determined every 5 minutes by collecting effluent from the right ventricular outflow tract during 1 minute. After reperfusion hearts were stained with 1% 2,3,5 triphenyltetrazolium chloride (TTC) at 37˚C (pH 7.4), frozen and then sliced into 1 mm sections. Area of necrosis was assessed by computerised planimetry (Image-Pro Plus, MediaCybernetics, Bethesda, MD, USA), with measurements normalised against the weight of individual slices and final necrosis size expressed as a percentage of myocardial mass. To block heme oxygenase activity, 30 mg/kg zinc deuteroporphyrin 2,4-bis etylenglycol (ZnBG; Porphyrin Products) was administered to mice ip. once daily for four days preceding heart perfusion. ZnBG was dissolved in 50 mmol/l Na 2 CO 3 to a final concentration of 1.4 mg/ml. The blocker solution was sterile-filtered, protected from light and before use adsorption was checked to exclude photodegradation (Czibik et al., 2009). 31 P MRS experiments were conducted on an 11.7 T (500 MHz) MR system comprising a vertical bore magnet (Magnex Scientific, Oxford, UK) and a Bruker Avance console (Bruker Medical, Ettlingen, Germany). A birdcage coil with an inner diameter of 10 mm (Rapid Biomedical, Wurzburg, Germany) was used as a transmit/receive coil for both phosphorus and proton frequencies. Following cannulation, the heart was lowered into the centre of the magnet and the position verified with scout images. After shimming, the water resonance line width was less than 50 Hz. Initially a single fully relaxed phosphorus spectrum was acquired for baseline analysis and correction of the spectra subsequently acquired under partially saturated conditions. Acquisition parameters were TR 10s, 60 averages, flip angle 90, 10 kHz bandwidth and acquisition data size of 2048 points. Immediately following the completion of this scan, a series of partially saturated acquisitions were performed. Spectra were acquired every minute for 70 minutes. The acquisition parameters, chosen to optimize signal-to-noise ratio, included TR 0.25 sec, eight dummy scans, 232 averages, flip angle 35, 10 kHz bandwidth and acquisition data size of 2048 points. As described above, the hearts were initially perfused at a normal flow rate for 10 minutes, followed by 30 minutes of global ischemia and then 30 minutes of reperfusion at the original flow rate. All spectra were fitted in the time-domain using the AMARES algorithm within jMRUI (Naressi et al., 2001a;Naressi et al., 2001b), with fits performed for phosphocreatine (PCr), inorganic phosphate (Pi) and adenosine tri-phosphate (ATP). Prior knowledge used with the AMARES algorithm included specification of the J-coupling of the ATP peaks, approximations of the peak positions, and imposition of Lorentzian line shapes.

Mouse Acute MI model
12 hours following the final gavage, mice were subject to an acute myocardial infarction (MI) as described previously (Ahmet et al., 2009).
Briefly mice were anesthetized with using isofluorane (3% v/v) and the chest cavity was opened. The left coronary artery was ligated at the level of left atrial apex. The chest and skin then was closed and the mouse was allowed to recover. 24 hours after the MI, the heart was perfused with 5% Evans Blue (Sigma), via an opening in the left ventricle, to distinguish the perfused area (blue staining) from the under-perfused area (nostaining). (Ahmet et al., 2009) Using a surgical microscope, the heart was isolated and cut into four pieces at the short axis and stained with 4% triphenyltetrazolium chloride (TTC, Sigma) for 30 min at 370C to distinguish the infarct area (unstained) from the area at risk (AAR; brick red stained) in the under-perfused area. The heart was then stored in 10% formalin and digitalized. All heart images were analyzed using NIH imagej software (http://rsb.info.nih.gov/ij/). The MI size was expressed as the percentage of dead area (unstained) to the total AAR (brick red). The surgeon and the MI assessor were blinded to the genotype and the treatments of the mice (the latter were randomised to DMF and vehicle). Data is expressed as the mean ± SEM. Comparisons were performed using a Students t-test, with p-values < 0.05 considered significant.

Microdialysis estimation of interstitial metabolite concentrations
In selected groups of the isolated perfused mouse heart study, we performed microdialysis for metabolic analysis. Following mounting of hearts in Langendorff mode, a microdialysis probe (membrane length 4 mm, cut-off 6 Da; AgnTho's AB, Sweden) was inserted into the LV free wall.
Following implantation, a 20 min stabilization period was allowed for metabolites to reach equilibrium in the perturbed tissue. Perfusion fluid consisted of KHB solution deoxygenated with 95% N2 and 5% CO2. Perfusion velocity was 1 μL/min and the sampling time for each fraction was 10 min. Following separation in a Waters ACQUITY™ Ultra-performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA, USA), metabolites were quantitated using a Waters Xevo™ triple quadrupole tandem mass spectrometer (Waters Corp., Manchester, UK) with a Z-spray electrospray ionization source operating both in the positive and negative ion mode. All analytes were verified using two daughter ions when possible and deuterated internal standards for selected components. The final interstitial concentrations were calculated by correction for relative recovery rate: (citrate 27%, isocitrate 13%, malate 26%, fumarate 67%, succinate 26%, pyruvate 35%, α-ketoglutarate 26%, lactate 37 %, adenosine 41%, inosine 38%, hypoxanthine 38%, and xanthine 58%) measured during in vitro experiments at a flow of 1 μL/min (Birkler et al., 2010).

Tissue harvest
All mice were weighed, euthanized by cervical dislocation, the heart excised, briefly washed with ice-cold PBS, blotted, and weighed before snap freezing in liquid nitrogen and storage at −80°C for further processing as described below.

Gene expression arrays
Total RNA was extracted from snap-frozen and pulverized heart tissue using the RNeasy Mini Kit (Qiagen, UK) following the manufacturers recommendations. Subsequent steps in the microarray expression processing and analysis were carried out by the Genomics and Bioinformatics Core Facilities at the Wellcome Trust Centre for Human Genetics.

Microarray Data Analysis
Gene expression data were obtained by hybridising a total of 10 samples from two experimental groups (n=5 per group) to Illumina MouseWG-6 Expression BeadChips. Raw data were exported from the Illumina GenomeStudio software (v1.6.0) for further processing and analysis using R statistical software (v2.11) (http://www.R-project.org) and BioConductor packages (Gentleman et al., 2004). Raw signal intensities were background corrected using array-specific measures of background intensity based on negative control probes, prior to being transformed and normalised using the 'vsn' package (Huber et al., 2002). Quality control analyses did not reveal any outlier samples. The dataset was then filtered to remove probes not detected (detection score <0.95) in any of the samples, resulting in a final dataset of 24,078 probes.
Statistical analysis was performed using the Linear Models for Microarray Analysis (limma) package. Differential expression between the experimental groups was assessed by generating relevant contrasts corresponding to the relevant comparisons. Raw p-values were corrected for multiple testing using the false discovery rate controlling procedure of Benjamini and Hochberg, adjusted p-values below 0.05 were considered significant. Significant probe lists were then annotated using the relevant annotation file (MouseWG-6_V2_0_R0_11278593_A) that was downloaded from the Illumina website (http://www.illumina.com) for further biological investigation. Gene set enrichment analysis methods (GSEA) are as described by Subramaniam and colleagues (Subramanian et al., 2005) and by Mootha and coworkers (Mootha et al., 2003), and were performed using web-based software (www.broad.mit.edu/gsea/).

Gene expression analysis
Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect mRNA levels of select target genes. One microgram of total RNA was reverse transcribed with High Capacity cDNA Reverse transcription Kit (Applied Biosystems) prior to amplification reactions performed using Fast Universal Master Mix on a StepOnePlus system (Applied Biosystems). Samples were run in duplicates in a total reaction volume of 10 μl. All Taqman oligos were inventoried products of Applied Biosystems. The relative quantities of target gene mRNA levels were normalized against the endogenous control beta actin, and relative expression was quantified using the comparative C T method (User Bulletin #2; Applied Biosystems).

Immunoprecipitation of Keap-1
Immunoprecipitation of Keap1 was undertaken from whole myocardial lysates generated as previously described by homogenisation in RIPA buffer. Lysates were pooled by three in WT and KO groups. Preclearing of the lysates with irrelevant antibody (abcam ab6046 Rb IgG β-tubulin 0.01 μg/ml) and protein A sepharose (Sigma Aldrich, UK) 100 μl/ml, for 30 minutes under rotatory agitation, was undertaken to reduce unspecific protein A or immunoglobulin binding. Centrifugation followed for 10 min at 14000 g. The supernatant was recovered and Keap-1 antibody (Abcam ab66620, anti-Rb 1:10 dilution) added. Rotatory agitation followed for 1 hour. Protein A sepharose slurry was added at 70 μl/ml lysate, followed by rotatory agitation over night. The immunoprecipitate was centrifuged at 14000 g for 10 min and the supernatant discarded. The immunoprecipitate was resuspended in lysis buffer and subsequently centrifuged at. 14000g for 10 min. This was repeated three times. All steps until this point was performed on ice or in refrigerated spaces of ca 4˚C temperature. The final supernatant was discarded and the immunoprecipitate heated to 96˚C for 5 minutes in a single volume of double concentrated Laemmli loading buffer. The suspension was centrifuged at 14000 for 10 min and the supernatant recovered for SDS-PAGE.

Western blotting
SDS-PAGE and Western blotting were used for the detection of total or nuclear protein expression. Total protein extracts were prepared from approximately 20 mg of frozen, ground LV tissue. The pulverised heart was mixed with 300 µL of RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS supplemented with protease inhibitor cocktail and PhosSTOP tablets [Roche]) and 0.5 mM phenylmethyl sulfonyl fluoride and sonicated for 2x30 seconds followed by passing the lysate through a 23 G needle 6 times and incubating for 1 hour at 4°C under constant agitation. The extract was centrifuged at 13000 rpm for 15 min at 4°C and the supernatant analysed for protein concentration using the Bradford protein assay (Biorad). Nuclear extracts were prepared as described previously with minor modification (Czibik et al., 2008). Finally, the pellets were incubated for 30 minutes with a buffer containing 20 mM Hepes pH 7.5, 400 mM NaCl, 1 mM of each EDTA and EGTA plus the same protease inhibitors and they were centrifuged at 14000g for 5 minutes. The supernatant was the nuclear fraction.
Equal amounts of protein (50 µg) were loaded onto 10% SDS-PAGE gels, which were run at 120 V for 2 hrs followed by transfer onto a PVDF membrane using an iBLOT system (Invitrogen). The membrane was blocked for two hours with 5% ECLadvance blocking agent and probed with rabbit anti-FH (Autogen Bioclear), anti-Keap1 (Abcam), anti-Hmox1 (Abcam) or goat anti-Nrf2 (Santa Cruz) primary antibodies in 1:1000 dilution followed by goat anti-rabbit-HRP or rabbit anti-goat-HRP secondary antibodies in 1:4000 (Abcam), respectively. To confirm equal loading of protein, the membrane was stripped of antibodies by incubating for 45 min in stripping buffer (2% SDS, 62.5 mM Tris-HCl, pH 6.8, and 100 mM 2-mercaptoethanol) at 50°C, followed by staining with either rabbit anti-beta-tubulin (Abcam) or anti-H3 histone (Abcam) antibodies followed by goat anti-rabbit-HRP (Abcam). The antibodies were detected with either ECL or ECL Advance chemiluminescence kit (GE Healthcare, UK), visualized with a Biorad Chemi Doc XRS system and quantified with Quantity One software.

Biochemical analysis
Total creatine and total adenine nucleotide content was measured by HPLC as described previously with minor modifications (Neubauer et al., 1995) Frozen tissue was powdered with mortar and pestle in liquid nitrogen. 10-20 mg powder was homogenized in 0.4 N perchloric acid at 0°C and an aliquot of the homogenate was removed for protein determination. The homogenate was neutralized with KOH and centrifuged at 5500 rpm for 5 min at 4°C. The supernatant was used for the determination of high energy phosphates in relation to a standard.
Protein concentration was measured by the Lowry method. Metabolite concentrations are expressed as nmol/mg protein.

Histology
Mouse hearts were cannulated through the aorta, rinsed with ice cold PBS followed perfusion with 4% paraformaldehyde for 2 min. Hearts were embedded in OCT compound. For a general overview of cardiac morphology, 10µm cryosections were stained according to Masson (Sigma) and visualized with a wide-field inverted Nikon TE2000U fluorescence microscope. To stain cell membranes, sections were incubated with Texas red-conjugated wheat germ agglutinin (Invitrogen) and visualized with a Zeiss 510 Metahead confocal fluorescence microscope.

Metabolite extraction for metabolomics analyses
Methanol and water were HPLC grade and were from Scientific and Chemical Supplies Ltd (Bilston, UK). Precellys homogeniser tubes were bought ready-filled from Stretton Scientific (Stretton, UK) and ammonium acetate and HPLC grade chloroform were purchased from Fisher Scientific (Loughborough, UK). Cardiac tissue from both WT (n=9) and FH -/-(n=9) was extracted in chloroform/methanol/water using a two step protocol based on the method previously described by Wu et al.(Wu et al., 2008) Briefly, 60 mg (±5 mg) of cardiac tissue was taken for each mouse. Each sample was homogenised in 8 µl/mg methanol and 1.7 µl/mg water, using a Precellys-24 bead based homogeniser (Stretton Scientific, UK). Next, 8 µl/mg chloroform and 4 µl/mg water were added resulting in a final ratio of 2:2:1.6. The biphasic mixture was centrifuged and the upper polar layer removed and split into a 50 µl aliquot for mass spectrometry with the remainder retained for NMR analysis. After drying in a centrifugal concentrator (Thermo Savant, Holbrook, NY, US), all aliquots were stored at -80˚C prior to analysis.
Quality control (QC) samples were also prepared by mixing 50 µl of the polar extracts from 8 different samples, and drying this solution in 100 µl aliquots prior to storage at -80˚C.

Metabolite analysis using FT-ICR mass spectrometry
Dried polar extracts were re-suspended in 150 µl (or 300 µl in the case of the QC sample) of an 80:20 methanol/water solution containing 20 mM ammonium acetate. The samples were analysed in negative ion mode using a hybrid 7-T Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT Ultra, Thermo Fisher Scientific, Bremen, Germany) with a chip-based direct infusion nanoelectrospray ionisation assembly (Triversa, Advion Biosciences, Ithaca, NY). Nano electrospray conditions consisted of a 200 nl/min flow rate, 0.3 psi backing pressure and -1.7 kV electrospray voltage controlled by ChipSoft software (version 8.1.0). Mass spectrometry conditions included an automatic gain control setting of 5 x 10 5 and a mass resolution of 100,000. Analysis time was 2.25 minutes (per technical replicate) using Xcalibur software (version 2.0, Thermo Fisher Scientific) and data was collected over a mass range of m/z 70-590 using the SIM-stitching FT-ICR method detailed by Southam et al.(Southam et al., 2007;Weber et al., 2011) Each sample was analysed in triplicate. Nine QC samples were analysed prior to the analysis and a QC sample was analysed in triplicate every fifth sample.
Transient data (in the time domain) was collected and processed as described previously (Southam et al., 2007) using custom written code in Matlab (version 7.9.0, The MathWorks). Specifically, SIM stitching code2 (version 1.16) was used to join together all the SIM windows, to reject peaks with a signal to noise ratio <3.5, and to internally calibrate the mass spectra using a list of 21 pre-defined metabolites. Next, a noise filtering algorithm (Payne et al., 2009) was applied such that only peaks that occurred in two (or more) of the three replicate measurements (per sample) were retained, and subsequently peaks were only retained if they were present in 50% (or more) of the cardiac samples. Finally, a Knearest neighbour approach was used to substitute missing values, and the data was normalised using the probabilistic quotient approach (Dieterle et al., 2006) before undergoing the generalised log transformation according to the approach reported by Parsons et al.(Parsons et al., 2007) This resulted in a final peak matrix that consisted of a list of m/z values and their intensities across the samples.
Multivariate (PCA and PLS-DA) analyses were conducted on the dataset. For the PLS-DA, to discover those mass spectral signals that maximally separated the FH -/-from the wild type mice, a forward selection strategy based on regression coefficients was used. This required ranking the signals according to their absolute regression coefficients from the initial PLS-DA model (i.e. built using all signals), and then sequentially adding one signal at a time to a new list, rebuilding the PLS model for each case, until the classification error was minimised.
Those signals contributing to the most discriminatory PLS-DA model were retained, and all other signals were rejected. The optimal PLS-DA model was cross validated using a venetian blinds approach with 4 splits. This approach was repeated 1000 times to achieve an "optimal model" classification error rate. To statistically evaluate this error rate and to be sure the model had not been overfitted, the class labels were randomly permutated and a new "permuted" model was built and cross validated in a similar way to give a "permuted" classification error rate. This permutation and model building process was also repeated 1000 times. Statistical significance of the prediction error for each class could then be calculated by comparing the optimal model classification error to the null distribution of permuted error rates. Specifically, a p value can be calculated by determining the number of instances where the permuted classification error is less than the optimal model error rate and dividing this value by the total number of permutations (in this case, 1000). A p value <0.05 was deemed to be of statistical significance.
35 µl of each sample was transferred into a 1.7 mm NMR tube using a Gilson 215 liquid handler, controlled by the PrepGilsonST software package (Bruker Biospin, v1.2.80B). All tubes were capped and kept refrigerated at ca. 4°C until analysis.
Samples were analysed using a Bruker Avance III 600 MHz NMR spectrometer operating at 600.13 MHz 1 H resonance frequency, equipped with a 1.7 mm cryoprobe and SampleJet refrigerated automatic sample charger (Bruker Biospin, Coventry, UK). Automated tuning and matching was performed for each sample following 5 minutes post-insertion temperature equilibration at 298 K. 1-D 1H NMR spectra were collected using the standard Bruker pulse sequence noesygppr1d, which features NOESY pre-saturation water suppression during the relaxation delay, and spoil gradients. Pulse durations were optimised automatically for each experiment, with a typical 90° pulse duration being 9.4 µs.
Other acquisition parameters include a spectral width of 7.2 kHz, relaxation delay of 3 s and 96 transients collected into 32,768 data points, requiring a 10 min acquisition time. Datasets were zero-filled to 65,536 points, and 0.3 Hz exponential line-broadening applied before Fourier transformation. All spectra were phased manually, baseline-corrected using a low frequency filter to remove the residual water signal, and calibrated (TMSP at 0.0 ppm) using TopSpin (version 3.0, Bruker). Processed spectra were imported into the Chenomx NMR suite software package (version 6.1, Chenomx, Edmonton, Canada) and further baseline correction was performed by fitting a cubic spline function through a series of defined spectral point, consistent for all samples, deemed to be baseline through visual inspection. For all samples, the concentrations of metabolites giving rise to assignable NMR signals were calculated using Chenomx and its associated 600 MHz spectral library. These concentrations were corrected by multiplying each sample's concentrations by a normalisation scaling factor, obtained using Probabilistic Quotient Normalization4 (PQN) and custom-written Matlab scripts (Viant, 2003). Finally, t-tests were conducted within Matlab on each quantified metabolite.
For the isotopomer studies, isolated hearts were perfused with modified Krebs-Henseleit buffer as above with 1mM of [3-13 C] glutamate at 37°C. At the end of 45 minutes of perfusion, hearts were snap frozen in liquid nitrogen and metabolites extracted using the Precellys 24 system (Bertin Technologies). Samples were filled into 1.7mm NMR tubes using a Gilson 215 liquid handler as described previously. 1 H-13 C-HSQC spectra were recorded at 600MHz proton frequency employing a Bruker Avance II 600MHz spectrometer equipped with a crygenically cooled 1.7mm probe. 4096 increments were recorded for a sweep width of 160ppm in order to resolve the J-couplings between adjacent 13 C-atoms. In the direct dimension 1024 points were acquired, the sweep width was 13ppm. The retention time was 1.5s, 8 scans were acquired per increment yielding a total recording time of 7.5h for each HSQC spectrum. All 2D-spectra were analysed and signals integrated using the MetaboLab software (Ludwig and Gunther, 2011). MetaboLab facilitates the analysis of positional label incorporation by a spectra library in order to plot signals of individual atoms in metabolites, providing signal intensities for each metabolite. Signal intensities can be compared between the different atoms of each metabolite. We also compared with spectra of unlabelled control samples.
Individual bundles were transferred into respiration chamber containing air-saturated, continuously stirred respiration medium at 37°C.
Following the measurement of basal respiration (state 2), maximal ADP-stimulated (state 3) respiration was measured after addition of 2mM ADP. Postoligomycin (uncoupled, state 4) respiration was evaluated following the addition of oligomycin (1µg/ml) to inhibit ATP synthase (Lehman et al., 2008) or antimycin A in case of succinate stimulated respiration (Kuznetsov et al., 2008). Subsequently, fibers were removed, blotted and dried for 24h at 37ºC and dry weight recorded. The Strathkelvin 782 System v 4.4 software was used for data acquisition and analysis. Respiration rates are expressed as nanomoles of O2 per minute per milligram dry weight of fibers. Respiratory control ratios (RCR) were calculated as a ratio of state 3/state 4 respiration (Murray et al., 2008).