Cholesterol

All rats were bilaterally ovariectomized from a dorsal approach under general anesthesia with pentobarbital sodium (50 mg/kg, i.p.; Somnopentyl; Kyoritsu Pharmaceutical, Tokyo, Japan) and sevoflurane (2%; Sevofran; Maruishi Pharmaceutical, Osaka, Japan). After the ovariectomy, a silicon capsule filled with a mixture of 17β-E2 (Sigma-Aldrich Japan, Tokyo, Japan) and cholesterol (Sigma-Aldrich Japan, Tokyo, Japan) powders (E2:cholesterol = 1:4 by weight) was implanted subcutaneously in E2-replaced rats, and a capsule containing cholesterol alone was implanted in vehicle-treated rats through an interscapular incision. The capsule was made from silicon tubing (ID, 2 mm; OD, 3 mm; inner length, 20 mm; ARAM Co., Osaka, Japan), and both ends of each capsule were sealed with silicon polymer bulking agents (Bath Caulk N; Cemedine, Tokyo, Japan). Our preliminary experiment demonstrated that this E2 replacement procedure elevates the plasma E2 concentration to the level that is comparable to the level during proestrus, and induces a relatively strong hypophagic effect. Following surgery, all rats were housed individually in a plastic cage in a chamber with a controlled ambient temperature of 23°C and a relative humidity of 40% under a 12/12 h light/dark cycle in which the lights were switched on at 0700 h. The illumination intensity was ~250 lx on the bottom of the cages.

A dried lipid mixture was synthesized by combining 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Sigma Aldrich, St. Louis, MO, USA), cholesterol (Sigma Aldrich, St. Louis, MO), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP; Avanti Polar Lipids, Alabaster, AL, USA) as 10 mM solutions in chloroform (Sigma Aldrich, St. Louis, MO, USA) in a 5 : 3 : 2 volume ratio. DOTAP is a cationic lipid commonly used as a cellular transfection agent for nucleic acid delivery. Other lipid components tested included the neutral lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; Sigma Aldrich, St. Louis, MO, USA) or negatively charged diacetyl phosphate (DCP; Sigma Aldrich, St. Louis, MO, USA) in place of DOTAP, with DPPC and cholesterol in the same 5 : 3 : 2 ratio. chloroform was chosen as an initial step solvent due to its high solubility potential for various lipid classes. The mixture was allowed to dry by solvent evaporation under rotation for 5 minutes in a Rotovap (Heidolph) or under ventilation for 48 hours.

GP63 entrapment in liposome was done using a previously described procedure (Ghosh et al, 2013) with slight modifications. Briefly, 12 mg phosphatidylcholine (Avanti Polar Lipids) (100 mg/ml), 8.7 mg cholesterol (Sigma) (100 mg/ml) (1:1.5 of cholesterol to PC molar ratio) and 6 mg n‐octyl‐β‐D‐glucopyranoside (Sigma) were dissolved in chloroform. The lipid mixture was coated as a uniform layer in a round glass bottle and kept in the desiccator. The lipid layer was solubilized in PBS containing either 250 μg purified GP63 or in the absence of GP63 and resuspended. The solution was sonicated in a bath sonicator for 15 min on ice water. The liposomes were ultracentrifuged at 100,000 × g at 4°C for 1 h to remove unincorporated protein and lipid particles. The liposomal pellet was resuspended in normal saline for animal or cell treatment. Approximately 20 μg of GP63 entrapped liposome was administered to the animal (BALB/c female adult mice) via intracardiac route. Mice were sacrificed after 24 h of administration. Liver tissues were collected for Western blot analysis. It has been shown by Ghosh et al (2013) that most of the administered liposome gets accumulated in the liver. For RAW 264.7 cell treatment, 1 μg of GP63 entrapped liposome were used for 10⁶ cells.

After the 1 hour recovery period cardiomyocytes were gravity settled and then resuspended in fresh HEPES-buffered medium (1 mM calcium, pH 7.4), medium supplemented with 5 mM methyl-β-cyclodextrin (MβCD), or medium containing 5 mM MβCD complexed with cholesterol 125 μg/ml. The MβCD-cholesterol complex was made by initially dissolving cholesterol (Sigma Aldrich) in a 1:1 solution of chloroform and methanol. Seventy-five μl of this solution was dried down and then reconstituted in medium supplemented with warmed 5 mM MβCD. This solution was sonicated for 1 hour and then kept at 37°C overnight prior to use. After the 1 hour treatments myocytes were gravity settled and the MβCD or MβCD-cholesterol containing medium was removed. Cell yields that were used for signaling studies were immediately processed. Myocytes used for contractility studies were resuspended in fresh HEPES-buffered medium.

We purchased the four unique 29mer shRNA constructs in a lentiviral GFP vector (#TL501193; Origene). To prepare the liposome, dimethyldioctadecylammonium bromide (#D2779; Sigma-Aldrich) was dissolved in chloroform (#C606SK-1; Fisher Chemical) in a 1:1 M ratio with cholesterol (#228111; Millipore). In a Rotavapor (#R-300; Buchi), chloroform was evaporated at 37°C, 100 rpm for 20–30 min. The dried lipid film was resuspended in 5% dextrose (#D16–500; Thermo Fisher Scientific) in water and further sonicated in Branson 2510 Ultrasonic Cleaner (#Z244910; Sigma-Aldrich) for 30 min, followed by 0.45 μm filtration. Liposome–shRNA (a cocktail of four shRNAs) complex was prepared with a concentration of 50 μg DNA/100 μg liposome per mouse (Liu et al., 2019 (link)). The physical characterization of the prepared liposome-shRNA complex was performed by measuring the hydrodynamic size and the surface zeta potential by using Malvern Zetasizer. The mice were fully anesthetized with ketamine/xylazine and the liposomes were i.t. injected.