Animal protocols
The experiments and procedures in this study were approved by the University of Illinois at Chicago (UIC) Institutional Animal Care and Use Committee (IACUC). Male mice from the C57BL/6J (B6), and MRL/MpJ (MRL), with an intact Fas gene, strains were housed in the same animal facility on a 14 h light, 10 h dark cycle with water ad libitum. At 3 weeks of age, mice were assigned either a chow (CD, 7012 Teklad LM-485 Mouse/Rat Sterilizable Diet, Harlan Laboratories, Indianapolis, IN, USA) or high fat diet (HFD, 60 % kcal from fat, D12492 Research Diets Inc, New Brunswick, NJ, USA). This ad libitum diet was maintained for 12 weeks through the end of the experiment. The mice were euthanized following UIC and nationally accepted protocols with prolonged carbon dioxide exposure followed by cervical dislocation. Tissue samples were harvested immediately. Samples designated for immunoblotting were flash frozen in liquid nitrogen and stored at −80 °C until processed. Tissue samples for immunofluorescence were placed in optimal cutting temperature (OCT) compound and then frozen in liquid nitrogen cooled isopentane.
Echocardiography
Echocardiography was performed by Dr. Robert Gaffin of the UIC Center for Cardiovascular Research. Mice were anesthetized using 3 % isoflurane, which was decreased to 2 % for anesthesia maintenance. Cardiac morphological, diastolic, and systolic parameters were assessed using the VisualSonics Vevo 770 (VisualSonics, Toronto, Canada) with a RMV707B (30 MHz) scanhead and the built-in echocardiography software.
Histology and immunofluorescence
The previously frozen tissues were sectioned at seven microns on a cryostat. Tissue samples for Picro Sirius Red were sectioned at 20 microns and processed by the UIC Research Resources Center (RRC) Histology Core. Heart sections from all four groups were placed on the same slide to minimize artifacts. ImageJ was utilized to quantify the percentage of red staining per image.
The slides for immunofluorescent staining were fixed for 20 min in −20 °C MetOH, washed 3 times in 4 °C PBS for 3 min, and blocked in 5 % FBS in PBS for 15 min at room temperature. The slides where incubated with anti-γ sarcoglycan (1:100, Novocastra Labs, Buffalo Grove, IL, USA), anti-Glut4 (1:50, EMD Millipore, Darmstadt, Germany) or anti-fibronectin (1:100, Sigma, St. Louis, MO, USA) in PBS with 5 % fetal bovine serum for 1 h at room temperature, then aspirated and washed three times for 15 min in 4 °C PBS. The secondary antibodies (Invitrogen) at 1:500 in 5 % FBS in PBS were placed onto the slide for 1 h at room temperature in the dark. After another series of washes, the slides were mounted using Vectashield mounting solution, with DAPI (Vector Laboratories, Burlingame, CA, USA). Heart sections from all four slides were affixed to a single slide to minimize artifacts. ImageJ was used to get the average intensity of staining across each section or along a line segment.
Cardiomyocyte cell size quantification
Cardiomyocyte cell size was quantified by highlighting the γ-sarcoglycan staining on the tissue images using a combination of GIMP (http://www.gimp.org/) and ImageJ (http://rsbweb.nih.gov/ij/). In brief, the stain was outlined and used to generate an image of uniformly black cells on a white background. The minimum diameter was then quantified in ImageJ and used to calculate area. One septal image (100× initial magnification) per animal, and at least four animals per group were quantified; the averages per group were then statistically compared.
Immunoblot analysis
Expression levels of specific proteins were assessed by immunoblotting. Each tissue sample was added to 250 μl lysis solution (20 mM HEPES pH 7.4, 10 mM NaF, 50 mM β-glycerol phosphate, 2 mM EGTA, 1 % Triton X100, 10 % glycerol, 2.5 μl of sodium orthovanadate, 2.5 μl of DTT, 2.5 μl of Halt Protease and Phosphatase Inhibitor Cocktail (100×, Thermo Scientific, Hanover Park, IL, USA). The mixture was homogenized on ice using a TissueRuptor (Qiagen, Venlo, Netherlands). The solution was then spun at 4 °C for 10 min. Supernatant protein concentrations were determined by Bradford assay (Thermo Scientific Coomassie, Bradford Protein Assay Kit). Fifty ug of protein were heated for 5 min at 100 °C and loaded onto 12 % acrylamide gels (Mini-Protean TGX, Biorad, Hercules, CA, USA). Gels were transferred onto PVDF membrane using a semi-dry transfer system (iBlot, Invitrogen, Camarillo, CA, USA). Five percent reconstituted non-fat dairy milk (NFDM) in tris-buffered saline with 0.1 % tween-20 (TBST) was used to block the gels for 15 min. The membranes were incubated at 4 °C overnight in 5 % NFDM/TBST with primary antibodies [anti-AMPK, anti-pAMPK, and PGC-1α (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-ACC, anti-pACC, fibronectin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HKII (1:5000, Millipore, Billerica, MA, USA); VDAC, Oxidative Phosphorylation antibody cocktail (1:1000, Abcam, Cambridge, MA, USA), Glut4 (1:500, EMD Millipore, Darmstadt, Germany), and γ-sarcoglycan (1:1000, Novocastra Labs, Buffalo Grove, IL, USA)]. Three 15-min washes with 1× TBST were performed after the primary antibody was removed. Blots were then incubated at room temperature for 1 h with horseradish peroxidase (HRP) conjugated species specific secondary antibodies (Invitrogen, 1:2500 in 1× TBST). Following another set of washes bound HRP was detected with ECL Prime (Amersham, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and a Chemidoc (Biorad Life Sciences Group, Hercules, CA, USA). Protein bands at the appropriate size from at least six animals per group were quantified using ImageJ. Ponceau staining was used to verify loading for each gel and γ-sarcoglycan, a membrane localized protein, immunoblotting of sister gels was used for normalization. To compare across gels, bands from each gel were normalized to the CD B6 average for that protein.
Electron microscopy
Freshly isolated heart apexes from one animal of each group were delivered to the electron microscopy facility of the UIC RRC. Micrographs were captured in a blinded manner at 11,600× and 34,400× magnifications.
Quantitative PCR
Previously published methods were used to quantify the amount of mitochondrial genome represented by subunit II of cytochrome C and using the nuclear gene succinate dehydrogenase subunit A as a control [13, 18].
Statistics
Student t tests were performed in Microsoft Excel comparing the appropriate groups. Values <0.05 were considered significant between the groups being examined.