Ouabain

Data were obtained using conventional whole cell patch-clamp techniques.
Micropipette fabrication and data acquisition were as described previously for
undiseased donor heart[85] (link). Axopatch 200 amplifiers, Digidata 1200 converters,
and pClamp software were used (Axon Instruments/Molecular Devices). Experiments
were performed at 37°C.
The standard bath solution contained, in mM: NaCl 144,
NaH₂PO₄ 0.33, KCl 4.0, CaCl₂ 1.8,
MgCl₂ 0.53, Glucose 5.5, and HEPES 5.0 at pH of 7.4, and pipette
solutions contained K-aspartate 100, KCl 25, K₂ATP 5,
MgCl₂ 1, EGTA 10 and HEPES 5. The pH was adjusted to 7.2 by KOH
(+15−20 mM K⁺).
For L-type Ca²⁺ current measurement, the bath solution contained
in mM: tetraethylammonium chloride (TEA-Cl) 157, MgCl₂ 0.5, HEPES 10,
and 1 mM CaCl₂, or BaCl₂, or SrCl₂ (pH 7.4 with
CsOH). The pipette solution contained (in mM) CsCl 125, TEA-Cl 20, MgATP 5,
creatine phosphate 3.6, EGTA 10, and HEPES 10 (pH 7.2 with CsOH).
For Na⁺/Ca²⁺ exchange current measurement, the
bath solution contained, (in mM): NaCl 135, CsCl 10, CaCl2 1, MgCl₂1, BaCl₂ 0.2, NaH₂PO₄ 0.33, TEACl 10, HEPES 10,
glucose 10 and (in µM) ouabain 20, nisoldipine 1, lidocaine 50, pH 7.4.
The pipette solution contained (in mM): CsOH 140, aspartic acid 75, TEACl 20,
MgATP 5, HEPES 10, NaCl 20, EGTA 20, CaCl2 10 (pH 7.2 with CsOH).

The rat blood plasma was obtained by centrifugation of whole blood and immediately used to determine biochemical markers of the functional state of the liver (the activity of alanine aminotransferase and aspartate aminotransferase, bilirubin concentration, and the thymol test) and lipid concentrations^{2 (link)}. ALT and AST activities were determined by Reitman-Fresnel method, total and direct bilirubin were determined by Endraschik method and thymol test was performed using thymol reagent checking the test kits (R&D enterprise Felicity-Diagnostics, Ukraine).
We determined the concentration of the following compounds in the blood plasma (mg%): phospholipids, cholesterol (CHOL), cholesterol esters (ECHOL), free fatty acids, triglycerides. Lipids were divided by the method of thin-layer chromatography⁴⁹ . Chromatographic separation of lipid components of plasma was carried out on “Silufol” plates. After treatment with an aqueous solution of phosphomolybdic acid, a quantitative assessment of the color intensity of each fraction was performed using a densitometer DO-1 M (“Shimadzu”, Japan, λ 620 nm)^{50 (link)}.
Blood cell mass was used to obtain erythrocyte plasma membrane preparations by the slightly modified Dodge’s method. Plasma membrane preparations were used to determine the ATPase activities of the primary active ion transport systems (total Mg²⁺, Na⁺, K⁺-ATPase, basal Mg²⁺-ATPase and Na⁺, K⁺-ATPase). The protein concentration in the preparations of the erythrocyte plasma membranes (PM) was determined by Lowry’s method^{51 (link)}. Total Mg²⁺, Na⁺, K⁺-ATPase activity was determined in the fraction of erythrocyte PMs in the standard incubation medium (in mM): 1 ATP, 3 MgCl₂, 125 NaCl, 25 KCl, 1 EGTA, 20 Hepes-Tris-buffer (pH 7.4), 1 NaN₃ (inhibitor of mitochondria ATPase), 0.1 µm thapsigargin (the selective inhibitor of Ca²⁺,Mg²⁺-ATPase of endoplasmatic reticulum) and 0.1% digitonin (the factor of PM perforation), at 37 °C. The Mg²⁺-ATPase activity was determined by the presence of a selective inhibitor Na⁺,K⁺-ATPase ouabain (1 mM) in the incubation medium. The Na⁺, K⁺-ATP activity was calculated as the difference between the total Mg²⁺, Na⁺, K⁺-ATPase and the ouabain-insensitive Mg²⁺-ATPase activity^{52 (link),53 (link)}.
This paper presents a statistical analysis of the experimental data obtained in the study and processed by the variation statistics methods using the Origin Pro 8 software. The samples were checked to belong to normally distributed general populations according to the Shapiro–Wilk criterion. The dispersion analysis was used to determine reliable differences between the mean values of samplings, and the post-test comparison was made using the Tukey test. In all cases, the results were reliable on the condition of the probability value p under 5% (p < 0.05). The obtained results were presented as the arithmetic mean ± standard error of the mean value, and the n value was determined by the total in the number of experiments.

The Cellular Membrane Potential Assay Kit (Abcam, Germany) was used according to the manual. Briefly, cells were plated in 100 µl growth medium on a black 96 well cell culture plate (Corning, USA) overnight. Subsequently, 100 µl of the MP dye-loading solution was added to the cells. The 96 well plate was incubated for 30 min at 37 °C and 5% CO₂. Before adding the compounds, the fluorescence signal was measured at EX/EM = 540/590 nm using a Fluostar plate reader (BMG, Germany). Afterwards, cells were treated with a deca-log dilution series of Ouabain using a Tecan nano drop dispenser (Tecan, Switzerland). Each condition was done in duplicate. As control, cells were treated with the solvent DMSO. After 2 h, 4 h, and 6 h, fluorescence was measured from the bottom of the wells.

Migration assay was done using Collagen I-precoated ORIS-96 well plates (AmsBio, USA) according to the manual. Briefly, cells were seeded in 150 µl growth medium on ORIS plates. After 24 h, stopper inserts were removed except for the zero controls. Medium was exchanged and 10 µl of test samples containing Ouabain and the reference SKI-606 in a concentration range of 10^–12 to 10^–5 M were added to the cells. After a migration phase of 24 h, medium was substituted with 75 µL DMEM w/o Phenol red containing 2 µg/ml Calcein-AM. Cells were incubated for 15 min at 37 °C and finally, fluorescent cells in the insert-defined area were detected by a fluorescence microplate reader (Fluostar, BMG, Germany) using FITC-settings (EX/EM = 485/520 nm). Subsequently fluorescence photographs of each well were taken at 40 × magnification. Each condition was done in duplicate.