Remifentanil preconditioning protects against hypoxia-induced senescence and necroptosis in human cardiac myocytes in vitro

Abstract

Remifentanil and other opioids are suggested to be protective against ischemia-reperfusion injury in animal models and coronary artery bypass surgery patients, however the molecular basis of such protection is far from being understood. In the present study, we have used a model of human cardiomyocytes treated with the hypoxia-mimetic agent cobalt chloride to investigate remifentanil preconditioning-based adaptive responses and underlying mechanisms. Hypoxic conditions promoted oxidative and nitrosative stress, p21-mediated cellular senescence and the activation of necroptotic pathway that was accompanied by a 2.2-, 9.6- and 8.2-fold increase in phosphorylation status of mixed lineage kinase domain-like pseudokinase (MLKL) and release of pro-inflammatory cytokine IL-8 and cardiac troponin I, a marker of myocardial damage, respectively. Remifentanil preconditioning was able to lower hypoxia-mediated protein carbonylation and limit MLKL-based signaling and pro-inflammatory response to almost normoxic control levels, and decrease hypoxia-induced pro-senescent activity of about 21% compared to control hypoxic conditions. In summary, we have shown for the first time that remifentanil can protect human cardiomyocytes against hypoxia-induced cellular senescence and necroptosis that may have importance with respect to the use of remifentanil to diminish myocardial ischemia and reperfusion injury in patients undergoing cardiac surgery.

Keywords: cardiomyocytes; hypoxia; necroptosis; remifentanil; senescence.

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.

Figure 2

HIF-1α upregulation (A) and nuclear translocation (B) upon stimulation with hypoxia-mimetic agent cobalt chloride and the effect of remifentanil preconditioning in HCM cells. (A) Western blot analysis of the levels of HIF-1α. Data were normalized to β-actin. (B) Immunofluorescence analysis of cellular localization of HIF-1α (red). Representative microphotographs are shown, objective 10×, scale bars 10 μm. F-actin staining (green) and nucleus staining (blue) were also considered. Nuclear immuno-signals of HIF-1α were calculated [%]. Bars indicate SD, n = 3, ^***p < 0.001 compared to normoxic control (CTR), ^###p < 0.001 compared to hypoxic control (CTR) (ANOVA and Dunnett's a posteriori test). CTR, control; R, remifentanil preconditioning.

Figure 3

Hypoxia-induced oxidative stress (A, B), nitrosative stress (C), adaptive oxidative stress, heat shock/chaperone and autophagy-based responses (D, E), and the effect of remifentanil preconditioning in HCM cells. (A) Superoxide levels were measured using Muse^® Cell Analyzer and Muse^® Oxidative Stress Kit. Representative histograms are presented. (B) Protein carbonylation was investigated using OxyBlot^™ Protein Oxidation Detection Kit. A negative control without DNPH derivatization (lane DNPH(-)) and a positive control with a mixture of standard proteins with attached DNP residues (lane M) are also shown. The levels of oxidative protein damage were normalized and protein carbonylation during normoxic control conditions was considered as 1. (C) Nitric oxide levels were investigated using Muse^® Cell Analyzer and Muse^® Nitric Oxide Kit. Representative dot-plots are also shown. (D) Western blot analysis of the levels of LC3B, SOD1, HSP70 and HSP90. Data were normalized to β-actin. (E) Immunofluorescence analysis of cellular localization of LC3B (red). Representative microphotographs are shown, objective 10×, scale bars 15 μm. F-actin staining (green) and nucleus staining (blue) were also considered. Bars indicate SD, n = 3, ^***p < 0.001, ^**p < 0.01, ^*p < 0.05 compared to normoxic control (CTR), ^###p < 0.001, ^##p < 0.01, ^#p < 0.05 compared to hypoxic control (CTR) (ANOVA and Dunnett's a posteriori test). CTR, control; R, remifentanil preconditioning.

Figure 4

Remifentanil preconditioning protects against hypoxia-induced senescence in HCM cells. (A) Senescence-associated β-galactosidase (SA-β-gal) activity. Representative microphotographs are shown. Scale bars 50 μm, objective 20×. The levels of SA-β-gal positive (blue) and negative (no blue) cells were calculated [%]. (B) Western blot analysis of the levels of cell cycle inhibitor p21. Data were normalized to β-actin. (C) Western blot analysis of the levels of pro-inflammatory cytokine IL-8. IL-8 levels in supernatants (Myocyte Growth Medium, MGM) were calculated per 10000 cells. Bars indicate SD, n = 3, ^***p < 0.001 compared to normoxic control (CTR), ^###p < 0.001, ^##p < 0.01 compared to hypoxic control (CTR) (ANOVA and Dunnett's a posteriori test). CTR, control; R, remifentanil preconditioning.

Figure 5

Remifentanil preconditioning protects against hypoxia-induced necroptosis in HCM cells. (A, B) Western blot analysis of the levels of RIP1, phospho-RIP1, RIP3, phospho-RIP3, MLKL and phospho-MLKL. Data were normalized to β-actin. The levels of phospho-RIP1, phospho-RIP3 and phospho-MLKL are also presented as a ratio of phospho-RIP1 to RIP1, phospho-RIP3 to RIP3 and phospho-MLKL to MLKL, respectively. (B) Bars indicate SD, n = 3, ^***p < 0.001, ^**p < 0.01 compared to normoxic control (CTR), ^###p < 0.001 compared to hypoxic control (CTR) (ANOVA and Dunnett's a posteriori test). CTR, control; R, remifentanil preconditioning.

Figure 6

Remifentanil preconditioning protects against hypoxia-induced cellular senescence and necroptosis in human cardiac myocytes that is achieved by remifentanil preconditioning-mediated decrease in the levels of cell cycle inhibitor p21, secretion of IL-8 proinflammatory cytokine as a part of senescence-associated secretory phenotype (SASP) and phospho-MLKL-based necroptotic signaling. Cellular senescence and necroptosis may be due to hypoxia-mediated oxidative stress (not shown). Moreover, necroptotic cells may release some proinflammatory signals (e.g., IL-8) that may also promote cellular senescence and then senescent cells may further stimulate the occurrence of other senescent cells by IL-8 signaling (black arrows). All these adverse effects can be reversed by remifentanil preconditioning.