Cytoprotective Effects of Dinitrosyl Iron Complexes on Viability of Human Fibroblasts and Cardiomyocytes

Abstract

Nitric oxide (NO) is an important signaling molecule that plays a key role in maintaining vascular homeostasis. Dinitrosyl iron complexes (DNICs) generating NO are widely used to treat cardiovascular diseases. However, the involvement of DNICs in the metabolic processes of the cell, their protective properties in doxorubicin-induced toxicity remain to be clarified. Here, we found that novel class of mononuclear DNICs with functional sulfur-containing ligands enhanced the cell viability of human lung fibroblasts and rat cardiomyocytes. Moreover, DNICs demonstrated remarkable protection against doxorubicin-induced toxicity in fibroblasts and in rat cardiomyocytes (H9c2 cells). Data revealed that the DNICs compounds modulate the mitochondria function by decreasing the mitochondrial membrane potential (ΔΨ_m). Results of flow cytometry showed that DNICs were not affected the proliferation, growth of fibroblasts. In addition, this study showed that DNICs did not affect glutathione levels and the formation of reactive oxygen species in cells. Moreover, results indicated that DNICs maintained the ATP equilibrium in cells. Taken together, these findings show that DNICs have protective properties in vitro. It was further suggested that DNICs may be uncouplers of oxidative phosphorylation in mitochondria and protective mechanism is mainly provided by the leakage of excess charge through the mitochondrial membrane. It is assumed that the DNICs have the therapeutic potential for treating cardiovascular diseases and for decreasing of chemotherapy-induced cardiotoxicity in cancer survivors.

Keywords: cell viability; dinitrosyl iron complexes; donors nitric oxide; heart disease; membrane potential.

Figure 1

The time dependence of NO amount generated by DNIC#3 and DNIC#6 in aqueous solutions at pH 7 and T = 25 °C under anaerobic conditions. The DNICs concentration was (2 × 10^-4 M). The values presented are the means ± SD of three independent experiments, n = 3.

Figure 2

Effect of DNICs on the cell viability of human lung embryonic fibroblasts (cell line HLEF-104) (A) and rat cardiomyocytes (cell line H9c2) (B) for 60 min. DNICs (2 × 10^-4 M) were added to cells, then cell viability was tested in fibroblasts and rat cardiomyocytes by AlamarBlue^® Cell Viability Assay. *P ≤ 0.05 vs. control group, with ANOVAs followed by Tukey’s post hoc test. The values presented are the means ± SD of four independent experiments, n = 4.

Figure 3

Effect of DNICs on the cell viability of human lung embryonic fibroblasts (cell line HLEF-104) (A) and rat cardiomyocytes (cell line H9c2) (B) for 35 h. DNICs (2 × 10^-4 M) were added to cells, then cell viability was tested in fibroblasts and rat cardiomyocytes by AlamarBlue^® Cell Viability Assay. *P ≤ 0.05 vs. control group. The values presented are the means ± SD of four independent experiments, n = 4.

Figure 4

Effects of DNICs on cell viability of fibroblasts, treated with doxorubicin for 60 min (A) and 35 h (B). DNICs (2 × 10^-4 M) were added to HLEF-104 cells, then doxorubicin (1.4 × 10^-4 M, 20 min after DNICs treatment) was added, cell viability was tested in fibroblasts by AlamarBlue^® Cell Viability Assay after doxorubicin-induced toxicity. *P ≤ 0.05 vs. control group. The values presented are the means ± SD of four independent experiments, n = 4.

Figure 5

Effects of DNICs on the mitochondrial membrane potential of fibroblasts (cell line HLEF-104) (A) and rat cardiomyocytes (cell line H9c2) (B). Cells were treated with DNICs (2 × 10^-4 M) or without (control) for 15 min, then JC-1 was added. The ratio of red fluorescence (mitochondrial JC-1) to green fluorescence (cytoplasmic JC-1) was used to calculate the mitochondrial potential. *P ≤ 0.05 vs. control group. The values presented are the means ± SD of four independent experiments, n = 4.

Figure 6

Flow cytometric analysis of effect of DNICs on cell-cycle of HLEF-104 cells. The cells were treated with DNICs (2 × 10^-4 M) and untreated (control) for 24 h, samples were analyzed using FACS (Calibur Flow Cytometer, Becton Dickinson, USA). The cell cycle phase profile of HLEF-104: (A) Control, (B) DNIC#3, (C) DNIC#4. (D) Graphical presentation of distribution (%) of cells in different phases of cell cycle. The values in the bars indicates the percent population of cells in respective cell cycle phases. Results are presented as mean ± SD of three independent experiments.

Figure 7

Effect of DNICS on the level of glutathione in fibroblasts (A), in rat cardiomyocytes (B). HLEF-104 or H9c2 cells were either kept untreated or treated with DNICs (2 × 10^-4 M) for 15 min and intra cellular GSH was measured as described under Materials and Methods section. Results are presented as mean ± SD of three independent experiments. Differences between untreated control and DNICs treated cells are significant at *p ≤ 0.05 with ANOVAs followed by Tukey’s post hoc test.

Figure 8

Effect of DNICs on the level of ROS in human lung embryonic fibroblasts.HLEF-104 cells were either kept untreated or treated with DNICs (2 × 10^-4 M) and intra cellular ROS generation was measured [in terms of peroxide using dichlorofluorescein diacetate (DCF-DA)] as described under Materials and Methods section. Data are presented as mean ± SD of three independent experiments. Differences between control and DNICs treated cells are significant at *p ≤ 0.05 with ANOVAs followed by Tukey’s post hoc test.

Figure 9

Effect of DNICs at specific ATPase activity in lysates, prepared from fibroblasts. Lysates (1.5 mg/ml) were preincubated 15 min at 4 °C with no additions (1) or in the presence of DNIC#3, DNIC#4 or DNIC#6. Then the ATPase activity was measured as described under Materials and Methods section. Data are presented as mean ± SD of three independent experiments. Differences between control and DNICs treated cells are significant at *p ≤ 0.05 with ANOVAs followed by Tukey’s post hoc test.

Figure 10

Effect of DNICs on total ATP in fibroblasts cell lysates. HLEF-104 cells treated with DNICs (2 × 10^-4 M) or untreated (control) and incubated for 15 min at 37 °C, 5% CO₂. Then prepared cell lysates according to protocol and total ATP was measured as described under Materials and Methods section. Data are presented as mean ± SD of three independent experiments. Differences between control and DNICs treated cells are significant at *p ≤ 0.05 with ANOVAs followed by Tukey’s post hoc test.

Figure 11

The proposed mechanism of action of DNICs.