Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease

Abstract

Introduction: Fibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia.

Methods: We studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q10 levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells.

Results: We found reduced levels of coenzyme Q10, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy.

Conclusions: These findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.

Figure 1

Coenzyme Q₁₀ levels and mitochondrial membrane potential (ΔΨm) in blood mononuclear cells (BMCs) from fibromyalgia (FM) patients and healthy control subjects. (a) CoQ₁₀ levels were measured with high-performance liquid chromatography, as described in Materials and Methods. Data represent the mean ± SD of three separate experiments. (b) Mitochondrial membrane potential was analyzed in BMCs from control subjects and FM patients with flow cytometry, as described in Materials and Methods. Data represent the mean ± SD of three separate experiments. *P < 0.001 between controls and FM patients.

Figure 2

Reactive oxygen species (ROS) production and lipid peroxidation in fibromyalgia (FM) patients. (a) ROS production was analyzed in BMCs from control subjects and FM patients with flow cytometry, as described in Materials and Methods. Lipid peroxidation (MDA levels) in blood mononuclear cells (BMCs) (b) and plasma (c) from control subjects and FM patients were determined as described in Materials and Methods. Data represent the mean ± SD of three separate experiments. *P < 0.001 between controls and FM patients.

Figure 3

Effect of antioxidants on reactive oxygen species (ROS) generation. Blood mononuclear cells (BMCs) of representative fibromyalgia (FM) patients were treated with 10 μmol/L CoQ₁₀, 30 μmol/L α-tocopherol (α-toc), and 10 μmol/L N-acetylcysteine (N-Acet) for 24 h. Data represent the mean ± SD of three separate experiments. *P < 0.001 between controls and FM patients; **P < 0.005 between the absence or presence of CoQ₁₀ and α-toc treatment.

Figure 4

Autophagic markers in blood mononuclear cells (BMCs) from fibromyalgia (FM) patients. (a) Quantification of acidic vacuoles in control and patient BMCs by LysoTracker fluorescence and flow-cytometry analysis. (b) Reduction of LysoTracker fluorescence in BMCs from FM patients under CoQ₁₀ supplementation (100 μmol/L) for 24 h. Data represent the mean ± SD of three separate experiments. *P < 0.001 between controls and FM patients.

Figure 5

Autophagic genes expression. Expression levels of BECLIN 1 (a) and MAP-LC3 (b) transcripts in blood mononuclear cells (BMCs) from control and fibromyalgia (FM) patients were assessed with real-time polymerase chain reaction (PCR), as described in Materials and Methods. Data represent the mean ± SD of three separate experiments. *P < 0.001 between controls and FM patients. (c) Correlation of CoQ₁₀ levels and BECLIN 1 and MAP-LC3 expression levels in BMCs from FM patients.

Figure 6

Mitophagy in fibromylagia (FM) patients. (a) Decreased mitochondrial mass in blood mononuclear cells (BMCs) from FM patients. Citrate synthase specific activity in BMCs from control and FM patients was performed, as described in Materials and Methods. Data represent the mean ± SD of three separate experiments. *P < 0.001 between control and FM patients. (b) Ultrastructure of BMCs from FM patients. The control BMCs show mitochondria with a typical ultrastructure. Autophagosomes with mitochondria (arrows) were present in BMCs from a representative FM patient (P6); Bar = 1 μm.