DETAILDE DESCRIPTION OF THE METHODS
Preparation of perfusion solution
The Krebs-Henseleit buffer (KHB) used for initial perfusion consisted 120 mM
NaCl, 25 mM NaHCO
3
, 4.7 mM KCl, 1.2 mM KH
2
PO
4
, 1.2 mM MgCl
2
, 1.2 mM
CaCl
2
, 0.03 mM EDTA, 10 mM glucose and was gassed with 95% O
2
-5% CO
2
pH
7.4. For the infarction study and palmitate oxidation measurements the hearts were perfused with modified KHB containing for infarction study: 10 mM glucose, 0.3 mM sodium palmitate bound to 2% BSA, 2 mM lactate, 0.2 mM pyruvate, and 100 mU/ml insulin; for palmitate oxidation measurements: 5 mM glucose, 1.2 mM sodium palmitate bound to 2% BSA, 1 mM lactate, 0.1 mM pyruvate, and 10 mU/ml insulin.
For the palmitate oxidation measurements, the concentration of 1.2 mM palmitate was chosen, since it has been previously demonstrated that this concentration of palmitate can maximally inhibit glucose oxidation (Wall &Lopaschuk 1989, Saddik
&Lopaschuk 1991).
The detailed method of perfusate solution preparation was described previously
(Lopaschuk & Barr 1997). BSA was dissolved in approximately 1/3 of required initial
KHB (without glucose) volume at constant stirring and low heat. The appropriate amount of palmitate was first dissolved in water:ethanol mixture (60:40 vol/vol) containing Na
2
CO
3
(1:1.2 molar ratio of palmitate and Na
2
CO
3
). The mixture was heated with constant stirring. Once ethanol was evaporated, mixture was quickly poured into dissolved BSA solution continuing stirring to provide appropriate palmitate binding to BSA. The obtained solution was dialyzed overnight against 10 volumes of KHB solution (without glucose) using 12-14 000 mol wt cut-off
Spectra/Por dialysis membrane (Spectrum Medical Industries, Los Angeles, CA). The next day solution was filtered, and glucose, lactate, pyruvate and insulin were added just before use.
Infarction study in isolated rat heart
The infarction study was performed according to the Langendorff constant pressure recirculation technique using modified Krebs-Henseleit solution. Wistar rats were anesthetized with intraperitoneal injection of sodium pentobarbital (60 mg/kg), and heparin (1 IU/g) was administered concomitantly. Hearts were excised, placed in ice-cold Krebs-Henseleit solution, then quickly mounted and retrogradely perfused

with oxygenated (95% O
2
- 5% CO
2
) Krebs-Henseleit solution via the aorta at a constant pressure of 70 mmHg. Water-ethanol mixture (1:1)-filled balloon
(polyethylene wrapping film) connected to a physiological pressure transducer
(ADInstruments, Chalgrove, UK) was gently inserted into the left ventricle, and baseline end-diastolic pressure was set at 5–10 mm Hg. Heart rate, left-ventricle developed pressure, contractility and relaxation were recorded continuously using
PowerLab8/30 system from ADInstruments. Coronary flow was measured using an ultrasound flow detector system connected to PowerLab8/30 system. A 4-0 coated, braided silk suture (Sofsilk (Syneture, Dublin, Ireland)) was passed under the left anterior descending coronary artery (LAD) and threaded through a small elastic plastic tube to permit reversible occlusion of the LAD. Hearts were adapted for 20 minutes and occlusion was performed for 30 minutes by constricting threads through a plastic tube. Successful occlusion was confirmed by coronary flow decrease of about 40 percent (Liepinsh 2006, Kuka 2012). Reperfusion was achieved by releasing the threads. At the end of the 120-minute reperfusion, the LAD was re-occluded and the non-risk zone was stained with 5 ml of 0.1% methylene blue solution in Krebs-
Henseleit buffer infused via the aortic root at a constant pressure of 70 mmHg.
Hearts were sectioned transversely from the apex to the base in 4 slices of 2-mm thickness using coronal rat heart slicer matrix (Zivic Instruments, Pittsburgh, USA) and incubated in 1% triphenyl-tetrazolium chloride in phosphate buffer (pH 7.4, 37ºC) for 10 minutes to stain viable tissue red and necrotic tissue white. Computerized planemetric analysis of left-ventricle slices photographs (Sony A900, 100 mm Macro lens, 1:1 reproduction ratio) was performed using Image-Pro Plus v6.3 software to determine the area at risk (AR) and area of necrosis (AN), each expressed as a percentage of the left ventricle area. Obtained values were then used to calculate the infarct size (IS) as a percentage of the risk area, according to the formula IS =
AN/AR×100%.
To measure the LCFA profile, peroxisomal and mitochondrial function and mRNA levels, the LAD was occluded for 30 minutes as described above. The nonrisk area was delineated with 0.1% methylene blue solution as described above either at the end of ischemia or after 60 minutes of reperfusion. Samples from the non-risk area and the area at risk (Fig. 1) were immediately frozen in liquid nitrogen and stored at -80

C.

Heart infarction development during ischemia-reperfusion.
Representative heart slices after 30 min of ischemia (A), 60 min of reperfusion (B) and 120 min of reperfusion (C).
Tissue samples from the non-risk area (dark blue) and the area at risk (red) were taken after
30 min of ischemia and 60 min of reperfusion as shown with yellow circles. Tissue slices after 120 min of reperfusion (C) were used for measurements of infarct size.
FAO measurements in isolated rat heart
Oxidation of radiolabeled palmitate was measured as previously described
(Lopaschuk & Barr 1997) with some modifications as indicated below. Palmitate oxidation was determined by measuring the 3 H
2
O released from 9,10-[ 3 H]palmitate
(radiotracer specific activity 60 Ci/mmol). 9,10-[ 3 H]palmitate was prebound to BSA using the same procedure described in Preparation of perfusion solution following the step in which the unlabeled palmitate is dissolved.
3
H
2
O was separated from 9,10-
[
3 H]palmitate by subsequently mixing 100 μl of the perfusate sample with 500 μl of chloroform:methanol (1:2 vol/vol), 200 μl of chloroform and 200 μl of a 2 M KCl/0.4
M HCl solution. The samples were centrifuged at 1150 g for 15 min to separate them into polar and nonpolar phases, and a 50 μl aliquot from the polar phase was taken and counted for
3
H using a scintillation cocktail. The palmitate oxidation rates were calculated from the
3
H
2
O production, and the dilution factor incurred from the process of separating
3
H
2
O from 9,10-[
3
H]palmitate was taken into account.
Measurements of peroxisomal proliferation and fatty acid oxidation
Peroxisomes were isolated according to the method of Harrison 1988 from the non-risk area and the area at risk after LAD occlusion for 30 min with some modifications. Briefly, tissues were homogenized in 10 vol. of the medium containing

250 mM sucrose, 10 mM HEPES and 1 mM EGTA, and pH 7.4 at 4°C. The homogenate was centrifuged for 5 minutes at 750 g. Then supernatant was centrifuged for 10 min at 6800 g to sediment the mitochondrial fraction. The peroxisomal pellet was obtained by centrifuging the resulting supernatant for 20 min at 48000g. The obtained pellet was resuspended in the buffer containing 180 mM KCl, 20 mM Tris-
HCl, and pH 7.2 at 4°C, and was used for peroxisomal fatty acid oxidation measurements.
To determine peroxisomal proliferation (mg per g of heart) the protein and catalase activity was measured in the heart tissue homogenate and in isolated peroxisomal fraction as previously described (Wheeler et al., 1990). The determination of catalase activity is based on the reaction of the enzyme with methanol in the presence of H
2
O
2
, where formaldehyde produced is measured with
Purpald as the chromogen. Samples were incubated with 9M methanol and 5 mM
H
2
O
2
in PBS, for 20 min at room temperature. The reaction was terminated with 1 M
NaOH.Then the reaction mixture was incubated with purpald for 10 min at room temperature. To obtain a coloured compound, the product of the reaction between formaldehyde and purpald was oxidised by potassium periodate. The absorbance was measured at 540 nm. Peroxisomal proliferation (mg per g of heart) was calculated by dividing the activity of catalase expressed per g of heart (using homogenate) by the activity of enzyme expressed per mg of protein of isolated peroxisomal fraction.
The peroxisomal rates of palmitate oxidation were determined as described previously (Degrace 2004) with the exception that 9,10-[
3
H]palmitate was used for the assay. The reaction mixture contained 30 mM KCl, 75mM Tris-HCl, 10mM
KH
2
PO
4
, 0.1mM EGTA, 5 mM MgCl
2
, 1 mM NAD, 5 mM ATP, 100 µM CoA,. To inhibit potential mitochondrial β-oxidation activity, the samples were preincubated for
10 min with 75 µM antimycin, 10 µM rotenone and 250 µM KCN. The reaction was started by the addition of 120 µM palmitate (supplemented with 1 µCi 9,10-
[ 3 H]palmitate, radiotracer specific activity 60 Ci/mmol) bound to fatty-acid-free BSA.
After a 10-min incubation at 37°C, the samples were treated using a similar method as that for the FAO measurements in isolated rat heart. The peroxisomal rate of palmitate oxidation was calculated per milligram of protein.
Mitochondrial respiration measurements

Mitochondria were isolated from the heart tissues, and mitochondrial respiration was measured at 37ºC using a Clark-type electrode (Microelectrodes Inc., Bedford,
USA) as previously described (Baliutyte 2010), with some modifications (Kuka
2012). Heart tissues were homogenized 1:10 in the medium containing 180 mM KCl,
10 mM Tris-HCl, 1 mM EGTA and pH 7.7 at 4°C. The homogenate was centrifuged at 750 g for 5 min, and the supernatant was centrifuged at 6800 g for 10 min. The obtained mitochondrial pellet was resuspended in the medium containing 180 mM
KCl, 20 mM Tris-HCl, and pH 7.2 at 4°C. Respiration measurements were performed in the incubation medium containing 150 mM KCl, 1 mM MgCl
2
, 10 mM Tris-HCl, 5 mM KH
2
PO
4
, and pH 7.2 at 37°C. To determine the CPT-I-independent metabolism of long-chain fatty acids, 36 µM palmitoylcarnitine was used as a substrate for the respiration measurements. Citrate synthase activity was measured as previously reported (Srere 1963) and used to standardize the mitochondrial protein amount.
To assess mitochondrial function after LAD occlusion for 30 min, left ventricular cardiac fibers from the area at risk and the non-risk area (Fig. 1) were prepared as previously described (Toleikis 2005, Kuka 2012) and ADP-stimulated respiration rates (STAte 3) with pyruvate/malate were measured. The bundles of cardiac fibers were permeabilized with 50 µg/ml saponin for 30 min at 4°C in buffer
A containing (20 mM imidazole, 20 mM taurine, 50 mM MES, 7.1 mM MgCl
2
, 0.5 mM dithiothreitol, 5 mM ATP, 15 mM phosphocreatine, 2.6 mM CaK
2
EGTA, 7.4 mM K
2
EGTA, pH 7.0 at 0°C. The fibers were washed twice for 10 min in buffer B containing 20 mM imidazole, 0.5 mM dithiothreitol, 20 mM taurine, 1.61 mM
MgCl2, 100 mM MES, 3 mM KH2PO4, 2.9 mM CaK
2
EGTA, 7 mM K
2
EGTA, pH
7.1 at 37°C. Respiration rates of cardiac fibers were measured at 37°C with Clarktype electrode in buffer B added with 2 mg/ml BSA using 6 mM pyruvate/6 mM malate and 1 mM ADP as substrates. mRNA isolation and quantitative RT-PCR
Total RNA from heart tissue was isolated using the TRI Reagent (Sigma, St.
Louis, USA) according to the manufacturer's protocol. The quality and quantity of extracted total RNA were examined by loading 5 μg of each sample on a denaturing agarose gel.
First-strand cDNA was synthesized using a High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems
TM
, Foster City, USA) following the

manufacturer’s instructions. Quantitative RT-PCR analysis for genes was performed by mixing synthesized cDNA, appropriate primers, and SYBR® Green
Master Mix
(Applied Biosystems
TM
, Foster City, USA) and run in the Applied Biosystems Prism
7500 according to the manufacturer’s protocol. The transcript levels for the constitutive housekeeping gene product β-actin were quantitatively measured for each sample, and PCR data were reported as the number of transcripts per number of βactin mRNA molecules. To avoid genomic DNA contamination, the primers were designed to span an intron. The primer sequences used for the quantitative RT-PCR analysis are available upon request.
Western blot of cytosolic and nuclear extracts
Heart tissue from the non-risk area and the area at risk were homogenized by an
Ultra-Turrax
®
homogenizer (IKA, Germany) at a ratio of 1:10 (w/v) at 4ºC in a buffer containing 100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 5 mM MgCl
2
, 1 mM glycerol
3-phosphate, 1 mM NaF and protease inhibitors (10 μM leupeptin, 1 μM pepstatin, 1
μM aprotinin, and 100 μM PMSF). The cytosolic extracts for western blot (WB) analysis and FA measurement were obtained by centrifugation of the homogenate at
6000 x g for 10 minutes at 4°C. The nuclear pellet was resuspended in ice-cold buffer, and purification of the nuclei was performed as described previously (10). Briefly, resuspended homogenate was twice filtered through nylon sieve mesh (50 μm), and the filtrate was centrifuged at 1500 x g for 10 min at 4ºC. The nuclear pellet was then washed twice and resuspended in ice-cold buffer.
PAGE and WB analysis of cytosolic and nuclear extracts were performed as described by Liepinsh et al. (10). Protein samples (40 µg) were separated on 8% SDS-
PAGE gels for 1 h at 100 V and electrotransferred onto a PVDF membrane
(Immobilon, Merck Millipore, USA). Membranes were blocked with 4% BSA
(Sigma) in PBS for 1 h at room temperature and then incubated for 1 h at room temperature with rabbit polyclonal antibodies for PPARα, NFκB (p65) NLS (Cayman chemical, USA), PGC1α, P AMPK, and mouse monoclonal AMPK (Abcam, UK) (1
μg/ml). After being washed with PBS, the blots were incubated for 30 min at room temperature with 0.2 μg/ml peroxidase-coupled goat anti-rabbit or anti-mouse IgG and then washed again in PBS. The blots were developed using chemiluminescence reagents (Merck Millipore, USA). The same blot was stripped (30 min at 50°C in 62.5 mM Tris base, 2% SDS, and 100 mM β-mercaptoethanol) and restained with 0.2

μg/ml monoclonal β-actin (Abcam, UK) or TATA-binding protein (QED Bioscience
Ich., USA) antibody as internal control. Western blot images were scanned and then analyzed by Gel-Pro Analyzer 6.0 (Media Cybernetics, USA) software.
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