Two cardiac cell lines were applied for in-vitro experiments. Rat cardiomyoblasts, immature cells – H9C2 (obtained from the Lab of Department of Medical Biochemistry, Wroclaw Medical University) which were grown in Dulbecco's Modified Eagle's Medium glucose (Lonza) with the addition of 10% fetal bovine serum (FBS, Lonza), 2 mM Glutamine and 100x penicillin/streptomycin (Sigma). Primary human cardiac myocytes - HCM (PromoCell GmbH, Germany) were isolated from the ventricles of the adult heart and grown in Myocyte Growth Medium (C-22070, PromoCell GmbH, Germany) with the addition of Supplement Mix (C-39275, PromoCell GmbH, Germany). For experiments, the cells were removed by trypsinizing (0.25% Trypsin-EDTA solution, Sigma), and washed with PBS. The cells were maintained in a humidified atmosphere at 37°C and 5% CO2.
Custodiol HTK (Kӧhler Chemie GmbH, Germany) is intracellular ionic composed cardioplegia (low K⁺) with the addition of three amino acids (histidine, tryptophan, ketoglutarate) and based on Bretschneider’s solution introduced in 1975 . Custodiol HTK based on its buffering capacity, is often used as a single-dose product.
Plegisol (Hospira Inc, USA) based on STH N°2 (St. Thomas’ Hospital, London) extracellular solution (high K⁺) as a multi-dose solution is used from the ‘70s of the last century . Plegisol requires buffering with the NaHCO₃ just before administration. The compositions of both solutions are shown in Table 1.
Simulation of the cardioplegic arrest and OS
Cells were removed from medium and immersed in cooled (4-6°C) both cardioplegic solutions incubated for 0.5, 1, 2, and 4 hours in a humidified atmosphere at 37°C and 5% CO2. After that, the experiments were performed. The temperature of cardioplegic solutions and the specific period of incubation were similar to the intraoperative conditions. Thus, we simulated the ischemia, such as during the aorta cross-clamping (arrest of the coronary perfusion). However, the typical reperfusion injury was not possible to obtain in our in-vitro model we provoked OS. The imbalance of prooxidant and antioxidant levels resulting from ischemia resulted in the overproduction of reactive oxygen species (ROS). However, the estimation of ROS concentration cannot be measured, the deleterious effect of ROS affected cellular lipids, proteins, and cardiomyocytes viability was examined in viability assay, the concentration of malondialdehyde (MDA) as an evidence of lipid peroxidation and determination of carbonyl groups caused by proteins damage. The presence of OS was also proven by the changes in antioxidative enzyme activity (iNOS, MnSOD) and chaperone activity protein (HSP27) which are regarded as OS markers.
The viability assay and cytotoxicity
The viable cardiomyocytes contain NAD(P)H-dependent oxidoreductase (mitochondrial dehydrogenase) enzyme, which reduces MTT (tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to insoluble formazan crystals. According to the protocol procedure, the cellular viability and the cytotoxic impact of cardioplegic solutions were determined by the MTT assay (Sigma, In VITRO Toxicology Assay). Cells were seeded into 96-well microculture plates (Nunc, Biokom) and allowed to attach for 24h. Then the incubation with Custodiol HTK or Plegisol for 0.5h, 1h, 2h, and 4h was performed. The dissolved and colored formazan crystals were quantified spectrophotometrically by measuring the absorbance at 570 nm (Multiscan MS microplate reader, Theremo Fisher). The results were expressed as a percentage of the untreated control (percentage of control cells).
Protocol for cells preparation for lipid peroxidation and carbonyl groups
Cells were grown in 25 cm2 flasks (Nunc) to obtain full monolayer. Then they were incubated with Custodiol HTK or Plegisol for 0.5h, 1h, 2h, and 4h. After the treatment, cells with cardioplegia in-vitro were removed by trypsinizing and washed twice in PBS (IITD, PAN, Poland). Then cells suspensions (ca. 5 x106) were centrifuged 5 min at 1500 rpm (Centrifuge MPW Med. Instruments MPW-341 with stable rotor).
Cell samples were suspended in 200 µL of PBS. Then 200 µL of 15% TCA (trichloric acid; Roth) in 0.25 M HCl and 200 µl of 0.37% TBA in 0.25 M HCl were added. The control sample contained 200 µL of deionized water instead of the cell suspension. Then the samples were incubated for 20 min at 90°C (Termoblock TB-941 U). After incubation, the samples were centrifuged for 5 min at 5000 rpm. MDA, the final product of fatty acid peroxidation, reacts with TBA to form a colored complex. The level of TBARS was measured based on the absorbance at the wavelength of 535 nm. The concentration of malondialdehyde was quantified spectrophotometrically based on a set of MDA standards of known concentration. All measurements were performed on UV/Vis spectrophotometer (JASCO V-530, MEDSON).
Proteins’ damage – carbonyl groups
Oxidative damage to proteins was investigated by determining carbonyl groups based on the reaction with dinitrophenylhydrazine (DNPH) (Sigma). Briefly, proteins were precipitated by the addition of 20% trichloroacetic acid (Sigma) and dissolved in 10 mM DNPH, and the absorbance was read at 570 nm. The results were calculated using an extinction coefficient of ε = 21.01 mmol-1/cm-1 for aliphatic hydrazone. Experiments were repeated three times.
Immunocytochemistry (MnSOD, iNOS, HSP27)
Manganese superoxide dismutase (MnSOD) is a typical antioxidative enzyme located in mitochondria. It plays a crucial role in ROS scavenging and protecting mitochondria from oxidative damage. Inducible nitric oxide synthase (iNOS) catalyzes nitric oxide production, which prevents ROS formation and activates the synthesis of glutathione - another antioxidative factor. Heat shock protein 27 (HSP27) is regarded as a protein chaperone, antioxidant, and inhibits apoptosis. Immunocytochemistry was performed using the ABC method. Briefly, cultures were fixed and dehydrated using 4% paraformaldehyde and an ethanol gradient respectively. Samples were then permeabilized and blocked by incubation with 0.1% Triton X-100 (Sigma) in PBS. The expression of proteins was visualized with the mouse monoclonal antibody (1:100, Santa Cruz). For conventional bright-field microscopy (peroxidase-ABC labeling), the samples were incubated with the diaminobenzidine-H2O2 mixture (DAKO) to visualize the peroxidase label, counterstained with hematoxylin (Alchem, Poland) for 30 sec. Samples were examined on a simple Olympus microscope (BX41, Japan). Stained cell numbers were determined by counting 100 cells in 3 randomly selected fields. Two independent investigators performed the counting. The result was judged to be positive if staining was observed in more than 5% of cells. The intensity of immunocytochemical staining was evaluated as (–) negative (no reaction), (+) weak, (++) moderate, and (+++) strong. All experiments were repeated three times.
The normality of the continuous variables was checked by the Shapiro-Wilk test. The significance of the difference between mean values of different groups of cells (MTT test, lipid peroxidation, carbonyl groups concentration, MnSOD, iNOS and HSP27 expression) compared with the control group untreated cells) was assessed by Student’s t-test with value of p≤0.05 taken as statistically significant. Statistical analysis was performed using STATISTICA 10 software.