18-kDa Translocator Protein Ligands Protect H9C2 Cardiomyocytes from Cigarette Smoke-induced Cell Death: In Vitro Study

Abstract

Background: Cigarette smoke (CS) can induce cellular damage via alterations in 18 kDa translocator protein (TSPO)-related functions, leading to cardiovascular diseases. The current study focused on the possible protective effect of TSPO ligands against CS-induced damage to cardiac cells.

Materials and methods: H9C2 Cardiomyocyte cell line of rat origin was pre-treated with TSPO ligands. Cell death, TSPO binding, and TSPO protein expression levels were assessed following 30-min CS exposure with/without TSPO ligands.

Results: CS exposure of H9C2 cells significantly incensed cell death (by 26%, p<0.001). Pre-treatment with TSPO ligands at two concentrations prevented cell death. Neither CS nor ligands affected TSPO protein expression in H9C2 cells. CS led to increased cell death and reduced TSPO binding.

Conclusion: Reduced TSPO binding may have a role in CS-induced cell death, and TSPO ligand MGV-1 can prevent suppression of TSPO binding and corresponding cell death. These results may be relevant to treatment of cardiovascular diseases associated with CS.

Keywords: Cigarette smoke; H9C2; LDH; TSPO ligands; cardiomyocyte cell line.

Figure 1. The effect of exposure to cigarette smoke (CS) on death of H9C cardiomyocytes as measured by elevation in lactate dehydrogenase (LDH) level in the medium. A: Cell death level following 30 min of CS exposure. B: Cell death level following 60 min of CS exposure. The results are presented as mean O.D. values (arbitrary units)±S.E.M (n=12 in each group). ***Statistically significant differences at p<0.001 as compared to the control group.

Figure 2. The effect of pre-treatment with translocator protein (TSPO) ligand on death in H9C2 cardiomyocytes after exposure to cigarette smoke (CS) for 30 min. A: Comparison of H9C2 cell death in the CS group compared to the control group. The application of the TSPO ligand 2-Cl-MGV-1 at two different concentrations had a protective effect against CS-induced cell death. B: The protective effect of TSPO ligand MGV-1 against CS-induced death of H9C2 cells. The results are expressed as the mean O.D. (arbitrary units)±S.E.M (n=4 in each group). Significantly different at ***p<0.001 compared to the control group; #p<0.05, ##p<0.01, and ###p<0.001 compared to the CS group.

Figure 3. Protection by ligand MGV-1 (25 μM) against cigarette smoke (CS)-induced reduction in translocator protein (TSPO) binding of 3Hlabeled PK 11195 (6 nM) by H9C2 cardiomyocytes. The figure represents the mean percentage±S.E.M of the binding results of four experiments. Control: Cells not exposed to CS or ligand. Significantly different at p<0.05 compared to the *control group and #CS group.

Figure 4. Translocator protein (TSPO) expression in control and cigarette smoke (CS)-exposed cells. A: A representative western blot analysis of TSPO expression in the control group and H9C2 cardiomyocytes exposed for 30 min to CS. B: TSPO expression in the control group exposed to ambient air and H9C2 cells exposed to CS. Results are expressed in relative O.D. (arbitrary units) of TSPO expression normalized to that of β-actin±S.E.M.

Figure 5. Translocator protein (TSPO) expression in H9C2 cardiomyocytes represented by mean fluorescence intensity (MFI). A: Control group. B: Group treated with CS for 30 min. C: Cells pretreated for 24 h with TSPO ligand MGV-1 without exposure to CS. D: Cells pretreated for 24 h with 25 μM TSPO ligand 2-Cl-MGV-1 without exposure to CS. E: Cells pretreated for 24 h with 25 μM TSPO ligand MGV-1 prior to CS exposure. F: Cells pretreated for 24 h with 25 μM TSPO ligand 2-Cl-MGV-1. G: Quantitative data for TSPO level in the different experimental groups. Results are represented are the mean MFI±S.E.M (n=4 in each group).