ACTIVATION OF LIVER X RECEPTORS IN THE HEART LEADS TO ACCUMULATION OF INTRACELLULAR LIPIDS AND ATTENUATION OF ISCHEMIA-REPERFUSION INJURY

Basic Res Cardiol

P Lei, ABaysa, H Nebb, G Valen, T Skomedal, JB Osnes, Z Yang, F Haugen*

*Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway, P.O.box 1103 Blindern, 0317 Oslo, Norway;Email:

SUPPLEMENTARY MATERIAL

Detailed Methods Description

Gene expression analysis

Total RNA was isolated from tissues and cells using the RNeasy kit (Qiagen, Hilden, Germany). RNA concentration was measured using a Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, Roskilde, Denmark), and integrity evaluated using RNA Nano Chips (Agilent Technologies, Santa Clara, USA). Random-primed reverse transcription of 1 μg of total RNA was performed with superscript III reverse transcriptase in a reaction containing 100 mM DTT, 25mM dNTP, 1 U RNaseOUT (Life Technologies, Carlsbad, USA) on a thermal cycler: 1 cycle of 10 minutes at 25 °C, 50 minutes at 42 °C, and 4 minutes at 94 °C. Real-time PCR, in 20 μL reactions containing 25 ngcDNA, 0.25 µM forward primer, 0.25 µM reverse primer, and 10 μLl of SYBR Green master mix, was performed using an ABI Prism 7900 instrument (Life Technologies) programmed as follows: 1 cycle of 2 minutes at 56 °C and 10 minutes at 95 °C; followed by 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C. Primers (see Supplementary Table 1 below) were designed using the Primer Express software (Life Technologies). Relative changes were calculated by the comparative method using GAPDH as the reference gene. Gene expression relative to GAPDH was calculated following the standard formula: 2-ΔΔCT, where ΔΔCT= (CT Target sample − CT GAPDH sample) − (CT Target calibrator – CT GAPDH calibrator) according to the comparative CT method.

Isolation of primary cardiomyocytes

Cardiomyocytes were isolated and cultured following the procedure described by O’Connell et al. [1]. Hearts from sacrificed mice were isolated, cannulated via the aorta and subjected to retrograde perfusion in a Langendorff apparatus heated to 37 C. First, hearts were perfused for 4 minutes with a buffer [1] containing 120 mMNaCl, 14.7 mMKCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Na-Hepes, 5.5 mM Glucose, 4.6 mM NaHCO3, 30 mMTaurine, 10 mM BDM to remove blood. Hearts were perfused for 12 minutes with the same perfusion buffer containing 2.4 mg/mL Collagenase II (Worthington Biochemical, Lakewood, USA) to digest the extracellular matrix; the last 8 minutesin the presence of 40 µM CaCl2. The digested ventricles were cut from the perfusion apparatus, mechanically disrupted and suspended in the perfusion buffer with the addition of 12.5 µM CaCl2 and 10 % FCS (Lonza). The cardiomyocytes were purified by serial centrifugations for three minutes at ≤ 20 × g gradually increasing calcium to 1.2 mM. Cardiomyocytes were resuspended in Minimum Essential Medium with Hank’s balanced salt solution with 10 % FCS, 10 mM/L butanedionemonoxime, 100 Units/mL penicillin and 2 mM glutamine and plated on six-well plates coated with ultrapure mouse laminin (1µg/cm2) (BD Biosciences). The cells were were left to attach for 4 hours at 37 °C and 2 % CO2. To activate LXR, isolated cardiomyocytes were changed to fresh medium supplemented with 0.1 % DMSO ± 1 µM GW3965 after the attachment period, and incubated for 20 hours before harvest.

Non-cardiomyocytes present in the supernatant from the first low-speed centrifugation were transferred to a separate tube. This tube was centrifuged for 5 min at 2000 rpm, and the cells were resuspended in the Minimum Essential Medium with Hank’s balanced salt solution containing 10 % FCS, penicillin/streptomycin (100 U/mL; 100 mg/mL) and 2 mM L-glutamine, and plated on non-coated six-well plates incubated for 4 hours at 37 °C and 5 % CO2.

To assess the cellular content of the two populations, RNA was isolated from the cells, and qRT-PCR was conducted with primers specific for the following cell marker proteins: Troponin I as a cardiomyocyte marker, vimentin as a fibroblast marker, cadherin as an endothelial cell marker and smooth muscle alpha actin as a smooth muscle cell marker. Three individual experiments were repeated in duplicates.

In vivo myocardial ischemia

Mice were placed in a chamber ventilated with a mixture of > 5 % isoflurane and oxygen. When the animals were deeply anesthetized, they were intubated and connected to a rodent ventilator (Model 874092; B. Braun,Melsungen, Germany) with 1.5 % isoflurane in oxygen.With animals on a heating plate, a left-sided thoracotomy was performed in the fourth intercostal space. The pectoral muscles were retracted, the pericardium opened, and myocardial ischemia was induced with a polypropylene monofilament suture (Surulene USP 8–0; Suru, Mumbai, India) placed around the left coronary artery directly underneath the left auricle in the interventricular groove (n=9). Myocardial bleaching was observed to evaluate that the artery was occluded. Sham mice (n=9) were operated likewise, except the ligature was not tightened. The thoracotomy and skin incision were closed by polypropylene sutures (5–0). The mice were extubated when spontaneous respiration resumed, a few minutes after skin closure. The animals were given subcutaneous injection of Temgesic (0.1 mg/kg; RB Pharmaceuticals, Berkshire, UK) for post-operative analgesia, and kept postoperatively in a regulated environment at 37 °C for 2 hours to prevent heat loss. Twenty-four hours later the animals were sacrificed as described above. The apex of the heart was sampled for mRNA isolation and gene expression analyses.

Isolated heart perfusion

Langendorff perfusion was performed as described previously [2] with gassed (5% CO2, 95% O2) Krebs-Henseleit buffer at 37 ºC and a constant pressure (70 mmHg). The perfusion flow rate was monitored using a tubing flowmeter (T402-TT; ADInstruments, Oxford, UK) equipped with in-line flow sensors (ME2PXN; ADInstruments). A small self-made polyethylene balloon was inserted into the left ventricle via the left atrium for isovolumetric pressure registration using a PCLAB system (AstraZeneca, Mölndal, Sweden). The data were integrated into left ventricular systolic pressure (LVSP), end-diastolic pressure (LVEDP), heart rate (HR), and coronary flow (CF) using the PharmLab version 3.0 software (AstraZeneca, Mölndal, Sweden). Left ventricular developed pressure (LVDP) was calculated (LVDP = LVSP − LVEDP). During the stabilization period, the balloon was filled with water until left ventricular end-diastolic pressure (LVEDP) reached approximately 5 mmHg, and hearts were excluded at the end of the stabilization period if they did notmeet the inclusion criteria: HR > 200 beats/minute andLVSP 50 mmHg. Global ischemia was achieved by clamping the inflow tubing for 40 minutes, followed by reperfusion for 60 minutes. After perfusion, the hearts were cut into 1 mm transverse sections on an acrylic matrice (AL-1130 Small rat coronal; Roboz Surgical Instrument, Gaithersburg, USA) using a grid of 10 razor blades glued together. The sections were incubated in 1 % triphenyltetrazolium chloride (TTC; Sigma-Aldrich) for 15 minutes at 37 ºC and placed between two glass plates for digital imaging (Coolpix 5400; Nikon, Tokyo, Japan). Infarct area was calculated using planimetry by a blinded person in Adobe Photoshop CS (Adobe systems, San Jose, USA).

Lipid droplets analyses

All images were taken at 60X magnification using an Olympus IX81 confocal microscope. Fluorescence intensity and area were quantified using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; The whole region of the image was analyzed. A 1.0 pixel Gaussian filter was used to identify fluorescent lipid droplets. The Analyze Particles function in ImageJ was used to count the number of lipid droplets. The FociPicker3D function was used to quantify the fluorescence intensity and area of lipid droplets. The intensity values represent the average pixel density in each lipid droplet. The background level was set to automatic; the tolerance setting was set to 40; and minimum pixels number in the focus was set to 10. The area of the foci projection and mean intensity of the foci were reported. A detailed analysis of the experiment is shown in Supplemental Figure 1.

References

1.O'Connell TD, Rodrigo MC, Simpson PC (2007) Isolation and culture of adult mouse cardiac myocytes. Methods Mol Biol 357:271-296 doi:1-59745-214-9:271 [pii]10.1385/1-59745-214-9:271

2.Ruusalepp A, Czibik G, Flatebo T, Vaage J, Valen G (2007) Myocardial protection evoked by hyperoxic exposure involves signaling through nitric oxide and mitogen activated protein kinases. Basic Res Cardiol 102:318-326

Supplementary Figures and Tables

SupplementaryFigure 1 Distribution of lipid droplet staining intensity and lipid droplet size in HL-1 cells.HL-1 cells cultured on 8-well Labtek II slides were incubated for 48 hours in Claycomb medium in the presence of fatty acid (FA; 500 µM oleic acid;) or the vehicle only (BSA control; 200 µM BSA), and in the presence of LXR agonist (GW3965; 1 µM) or the vehicle only (DMSO control; 0.1 %). Lipid droplets were stained with Bodipy 493/503 and confocal 2-D images aquired. The lipid droplets were identified using ImageJ. Upper 4 panels show the signal intensity emitted from the lipid droplets. The lower 4 panels show the size (area) of the lipid droplets. For every experiment a single image was analyzed from 3 replicate wells. Numbers represent cumulative values from 3 independently repeatedexperiments (n = 9, in total)

Supplementary Table 1: Effects of LXR agonist GW3965 on triglyceride fatty acid profile in mouse ventricles
Fatty Acid / Control / GW3965
C14:0 / 1.6 ± 1.2 / 1.2 ± 0.8
C15:0 / 0.7 ± 0.5 / 0.5 ± 0 .3
C16:0 / 31.2 ± 3.0 / 25.8 ± 3.6*
C16:1 / 1.0 ± 0.4 / 1.4 ± 0.3*
C17:0 / 1.3 ± 0.5 / 1.0 ± 0.5
C17:1 / 0.6 ± 1.2 / 0.4 ± 0.5
C18:0 / 9.3 ± 8.3 / 5.6 ± 1.3
C18:1(n-9) / 43.5 ± 8.8 / 45.7 ± 8.6
C18:1(n-13) / 3.7 ± 1.4 / 4.3 ± 0.7
C18:2(n-6) / 4.0 ± 5.9 / 11.4 ± 15.6
C20:0 / 0.6 ± 0.28 / 0.5 ± 0.28
C18:3(n-3) / 2.5 ± 1.3 / 2.3 ± 1.0
Values (means ± SEM; n = 4–6 in each group) represent relative distribution (%) of different fatty acids in the triglyceride fraction isolated from murine ventricles. after injection i.p. with GW3965 (20 mg/kg) or vehicle alone (Control; PBS + 10 % DMSO). *p < 0.05 vs. Control.
Supplementary Table 2: Sequence of primers used for qRT-PCR
Primer / Sequence 5’ - 3’
LXRα
FP / CGACAGAGCTTCGTCCACAA
RP / GCTCGTTCCCCAGCATTTT
LXRβ
FP / CGTGCCTGGGAATGGTTCT
RP / AGTCTCCTGCCCCTCTTCCTT
ABCA1
FP / CGTTTCCGGGAAGTGTCCTA
RP / GCTAGAGATGACAAGGAGGATGGA
SREBP-1c
FP / GGAGCCATGGATTGCACATT
RP / GCTTCCAGAGAGGAGGCCAG
SCD1
FP / ATGACGTGTACGAATGGGC
RP / GAATGACGTGTACGAATGGGC
ACSL3
FP / CAGTGCAGGCTCTAGGAGTGAA
RP / GTGGACCCACTTGTGTACATGATT
GPAM
FP / TCACAAGGGTCAACTCGAGATG
RP / GTGCACCGGCAGAAACAAG
DGAT2
FP / TGGAACACGCCCAAGAAAG
RP / CACACGGCCCAGTTTCG
ABHD5
FP / TGGCTGGCCTCAGGATTG
RP / GGCCTCAAACGCTGCACTAG
PNPLA2
FP / CGCCTCTCGAAGGCTCTCT
RP / GTCCATCTCTGTAGCCCTGTTTG
PLIN2
FP / CAGCCAACGTCCGAGATTGT
RP / CACATCCTTCGCCCCAGTT
PLIN5
FP / GGGAAACTGAGGCAGAGCAA
RP / TGCCCCTCTCTGCATATGC
FP: forward primer; RP: reverse primer; LXRα and -ß: Liver X receptor -α and –ß; SREPB-1c: Sterol Regulatory Element-Binding Protein 1c; ABCA1: ATP-binding Cassette Transporter A1; SCD1: Stearoyl-Coenzyme A Desaturase 1; ACSL3: acyl-CoA synthetase long-chain family member 3; GPAM: glycerol-3-phosphate acyltransferase, mitochondrial; DGAT2: diacylglycerol O-acyltransferase 2; ABHD5: abhydrolase domain containing 5; PNPLA2: patatin-like phospholipase domain containing 2; PLIN2: perilipin 2; PLIN5: perilipin 5.