Figure 1. Remifentanil preconditioning-mediated effects on metabolic activity (A), cell number (B), the levels of necrotic cells (C), troponin I release, a marker of myocardial damage (D) and the levels of apoptotic cells (E, F) during normoxic and hypoxic conditions in human cardiac myocytes (HCM). (A) The metabolic activity was assayed using MTT test. Metabolic activity at control normoxic conditions (CTR) is considered as 100%. A solvent action (0.9% NaCl) is also shown. Based on MTT results, the concentration of 8 ng/ml remifentanil was selected for further analysis. Cell number (B) and necrotic cell death (C) were evaluated using TC10™ automated cell counter. (C) Necrosis was analyzed using trypan blue exclusion assay. (D) Western blot analysis of the levels of cardiac troponin I. Cardiac troponin I levels in supernatants (Myocyte Growth Medium, MGM) were calculated per 10000 cells. Two biomarkers of apoptotic cell death were considered, namely phosphatidylserine externalization (E) and the activity of caspase 3/7 (F) using Muse® Cell Analyzer and Muse® Annexin V and Dead Cell Assay Kit and Muse® Caspase-3/7 Assay Kit, respectively. Representative dot-plots are also shown. Bars indicate SD, n = 3, ***p < 0.001, **p < 0.01, *p < 0.05 compared to normoxic control (CTR), ###p < 0.001, #p < 0.05 compared to hypoxic control (CTR) (ANOVA and Dunnett's a posteriori test). CTR, control; R, remifentanil preconditioning.