Collection of human samples
A total of 18 explanted human hearts from ischemic cardiomiopathy patients was collected and immediately processed. In particular, these patients were undergoing cardiac transplantation at Sant Pau Hospital, Barcelona and La Fe Hospital, Valencia. Clinical data, electrocardiogram, Doppler echocardiography, hemodynamic studies, and coronary angiography were available on all patients. All patients were functionally classified according to the New York Heart Association (NYHA) criteria, and were receiving medical treatment according to the guidelines of the European Society of Cardiology , with diuretics 90 %, angiotensin-converting enzyme inhibitors 87 %, β-blockers 50 %, aldosterone antagonists 70 %, digoxin 49 % and statins 80 %.
Eight non-diseased hearts were also obtained from donors with neurological death caused by traffic accident. The hearts were initially considered for cardiac transplantation but were subsequently deemed unsuitable for transplantation either because of blood type or size incompatibility. All donors had normal LV function and no history of myocardial disease or active infection.
Transmural samples were taken from the infarct border zone, and were immediately stored at −80 °C. The project was approved by the local Ethics Committee (Biomedical Ethics Committee of “La Fe, Valencia” and “Sant Pau, Barcelona”, Spain) and conducted in accordance with the guidelines of the Declaration of Helsinki. All patients gave written informed consent that was obtained according to our institutional guidelines.
Frozen ventricular tissues (25 mg) were pulverized using a mortar and a pestle in liquid nitrogen. Sample were then homogenized in TriPure isolation reagent (Roche Molecular Biochemicals) for total RNA and protein extraction according to manufacturer’s instructions.
HL-1 cardiomyocyte cell culture
The murine HL-1 cell line was generated by Dr. W.C. Claycomb (Louisiana State University Medical Centre, New Orleans, Louisiana, USA) and kindly provided by Dr. U Rauch (Charité-Universitätmedizin Berlin). These cells show cardiac characteristics similar to those of adult cardiomyocytes. LRP1-deficient cardiomyocytes were generated as previously described . Control and LRP1-deficient HL-1 cardiomyocytes were maintained in Claycomb Medium (JRH Biosciences, Lenexa, KS, USA) supplemented with 10 % fetal bovine serum (FBS) (Invitrogen Corporation, Carlsbad, CA, USA), 100 μM norepinephrine, 100 units/mL penicillin, 100 μg/mL streptomycin, and L-Glutamine 2 mM (Sigma Chemical Company, St. Louis, MO, USA) in plastic dishes, coated with 12.5 μg/mL fibronectin and 0.02 % gelatin, in a 5 % CO2 atmosphere at 37 °C.
VLDL and LDL preparation
Human VLDL (d1.019-d1.019 g/mL) and LDL (d1.009-d1.063 g/mL) were obtained from pooled sera of normocholesterolemic volunteers. VLDL and LDL preparations were less than 24 hours old and without detectable levels of endotoxin (Limulus Amebocyte Lysate test, Bio Whittaker). Aggregated LDL (agLDL) was prepared by vortexing LDL in PBS at room temperature. The formation of LDL aggregates was performed as previously described [21–23]. The ultrastructure of agLDL obtained by vortexing was similar to that of LDL modified by versican , one of the main chondroitin sulfate proteoglycans structuring the arterial intima.
Exposure of cardiomyocytes to VLDL and LDL under normoxic and hypoxic conditions
Cells were exposed to normoxia (21 % O2) in a Nirco gas incubator with gas mixtures consisting of 74 % N2 and 5 % CO2 or to hypoxia (1 % O2) in a Hypoxic/Anoxic Workstation: H35 (Don Whitley Scientific Ltd.) with 94 % N2 and 5 % CO2. Lipoproteins were added and maintained for the last 12 hours of exposure to normoxic or hypoxic conditions (24 hours). Cells were then harvested in TriPure Reagent (Roche) for PCR and Western blot analysis or in NaOH 0.1 M for lipid extraction and thin layer chromatography.
RNA extraction and cDNA synthesis
Total RNA was extracted from fresh frozen tissue or cultured HL-1 cardiomyocytes using TriPure isolation reagent (Roche Molecular Biochemicals) and the RNeasy mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Extracted RNA was eluded in 25 μL of nucleases-free water. RNA yield and quality were assessed by agarose electrophoresis and spectrophotometry, and then stored at −80°C until was used. RNA was digested with DNase I (Invitrogen). One μg of total RNA was used for cDNA synthesis according to the protocol provided with the HighCapacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Recombinant RNasin Ribonuclease Inhibitor (Applied Biosystems) was added to prevent RNase-mediated degradation. The cDNA was also stored at –20 C.
Gene expression analyses by RT-PCR
Gene expression analyses of LRP1
VLDLR, and LDLR mRNA were performed at mRNA level by quantitative real-time reverse transcriptase-polymerase chain reaction (q-RT-PCR). Specific primer and fluorescent TaqMan probe for LRP1
VLDLR and LDLR were selected within a list of predesigned assays (Assays-on-Demand LRP1 (Hs00233999_m1), VLDLR (Hs01045922_m1) and LDLR (Hs00181192_m1) (Applied Biosystems). 18srRNA (4319413E) was used as a housekeeping gene. We mixed 5 μl of single-stranded cDNA (equivalent to 100 ng of total RNA) with 1 μl of 20x TaqMan Gene Expression Assays for each Assay-on-Demand, 10 μl of TaqMan Universal PCR Master Mix, and 4 μl of nucleases-free water. After gentle mixing, the mixture was transferred into a real-time PCR microplate. The Real-time PCR microplate was sealed, centrifuged, and then was placed in the sample block of an Applied Biosystems 7300 Real Time PCR System (Applied Biosystems). The thermal cycling conditions were 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Expression levels were measured in triplicate. The threshold cycle (Ct) values were normalized to the housekeeping gene [17, 18].
Total protein was extracted from fresh frozen tissue or HL-1 cell cultures using TriPure isolation reagent (Roche Molecular Biochemicals). Proteins were analyzed by Western blot analysis as previously described [17, 18]. Blots were incubated with monoclonal antibodies against human LRP1 (β-chain, clone 8B8 RDI 61067), VLDLR (Santa Cruz Biotechnology, Inc, D-17, sc-11823), HIF-1α (Santa Cruz Biotechnology, H-206, sc-10790), LDLR (Epitomics, EP1553Y, 1956–1), VEGF (Santa Cruz Biotechnology, Inc, A2611, sc-152) and mouse monoclonal anti-Troponin T (Thermo scientific MS-295). Equal protein loading in each lane was verified staining filters with Pounceau and also by incubating blots with monoclonal antibodies against β-actin (Abcam, ab8226).
Lipid extraction and semi-quantitative analysis of cholesteryl ester, free cholesterol and triglyceride content of cardiomyocytes and myocardium
HL-1 cardiomyocytes were exhaustively washed and harvested in NaOH 0.1 M following the lipoprotein incubation period. In the animal experimental model, one portion of myocardial tissue (5 mg) was also homogenized in NaOH 0.1 M. Lipids were extracted as previously described [17, 18] and CE, FC and TG content was analyzed by thin layer chromatography.
The organic solvent was removed under an N2 stream, the lipid extract redissolved in dichloromethometane and one aliquot was partitioned by thin layer chromatography (TLC). TLC was performed on silica G-24 plates. The different concentrations of standards (a mixture of cholesterol, cholesterol palmitate, triglycerides, diglycerides and monoglycerides) were applied to each plate. The chromatographic developing solution was heptane/diethylether/acetic acid (74:21:4, vol/vol/vol). The spots corresponding to cholesteryl esters (CE), triglycerides (TG) and free cholesterol (FC) were quantified by densitometry against the standard curve of cholesterol palmitate, triglycerides and cholesterol, respectively, using a computing densitometer.
Hearts were obtained from human transplant operations. Immediately after surgical excision, myocardium was cut in appropriated blocks. Myocardial tissues were immersed in fixative solution (4 % paraformaldehyde), embedded in paraffin, cut into 5 μm thick serial sections and placed on poly-L-lysine coated slides. The primary antibodies were rabbit monoclonal anti-LRP1 (Epitomics 2703 dilution 1:100), mouse monoclonal anti-Troponin T (Thermo scientific MS-295, dilution 1:100) and mouse monoclonal anti-HIF-1α (Novus NB100-105, dilution 1:50). Antigen retrieval was required before performing immunohistochemical staining of Troponin T and HIF-1α. In a set of experiments, before incubation with primary antibody (2 hours), sections were washed and endogenous peroxidase activity suppressed with H2O2. Non-specific binding was blocked with an appropiate serum. The primary antibodies were detected using the avidin-biotin immunoperoxidase technique. The sections were incubated with an appropriate biotinylated secondary antibody (1:200, Vector®). 3,3’-diaminobenzidine-haematoxylin chromogen was used for nuclear stain. The images were captured by Nikon Eclipse 80i microscope and digitized by Retiga 1300i Fast camera. Magnification (240X).
In other set of experiments, cryosections were subsequently incubated with a Cy3-conjugated secondary antibody (Jackson Immuno Research Laboratories) at 37 °C for 1 h. Slices were finally counterstained for 10 min with Hoechst 33342 (Sigma), and analyzed under a TCS SP5 confocal microscope (Leica).
Results are expressed as mean ± standard deviation (SD). Statistical significance between groups was analyzed by one-way analysis of variance (ANOVA) followed by a post-hoc Tamhane test. Correlation analysis was performed according to Pearson. Statistics were calculated using Statistical software package Statview (SPSS) for Windows. A value of P < 0.05 was considered significant.