By Mario D. Cordero, Manuel de Miguel, PhD, Placido Navas, PhD, and Jos Antonio S¡nchez Alcazar, MD, PhD
Since mitochondrial dysfunction was first described in the 1960s, biomedical research has advanced in the understanding of the role that these cellular components play in health, disease, stress, and aging.
Along with inherited mitochondrial diseases, a wide range of seemingly unrelated disorders such as stress, schizophrenia, Alzheimers disease, epilepsy, migraine, stroke, neuropathic pain, Parkinsons disease, chronic fatigue syndrome, diabetes, hepatitis C, and fibromyalgia share the common pathophysiological mechanisms of production of mitochondrial reactive oxygen species (ROS), which results in mitochondrial dysfunction. As a consequence of these findings, antioxidant therapies hold promise of improving mitochondrial performance.
Reactive Oxygen Species
In addition to energy (see box on page 62), mitochondrial oxidative phosphorylation also generates reactive oxygen species, or ROS. When the mitochondrial respiratory chain (MRC) becomes highly reduced, excess electrons can be passed directly to oxygen to generate a superoxide anion. Superoxide is transformed to hydrogen peroxide by the detoxification enzymes manganese superoxide dismutase or copper/zinc superoxide dismutase, and then to water by catalase, glutathione peroxidase, or peroxidredoxin III. However, when these enzymes cannot convert ROS to water fast enough, oxidative damage occurs.
If hydrogen peroxide encounters a reduced transition metal or is mixed with superoxide, it can be further reduced to a hydroxyl radical, the most potent oxidizing agent among ROS. Additionally, nitric oxide is produced within the mitochondria by mitochondrial nitric oxide synthase, and also freely diffuses into the mitochondria from the cytosol. Nitric oxide reacts with superoxide to produce peroxynitrite. Together, these two radicalsâ€”as well as othersâ€”can do great damage to mitochondria and other cellular components.
Under normal physiological conditions, ROS production is highly regulated. However, if the respiratory chain is inhibited, or key mitochondrial components such as CoQ are deficient, then electrons accumulate on the MRC carriers, greatly increasing the rate of a single electron being transferred to oxygen to generate superoxide. Excessive mitochondrial ROS production can exceed the cellular antioxidant defense, and the cumulative damage can ultimately destroy the cell.
It has been proposed that ROS damage can induce mitochondria permeabilization by the opening of permeability transition pores in the mitochondrial inner membrane. This, in turn, leads to a simultaneous collapse of mitochondrial membrane potential and the elimination of dysfunctional mitochondria.
Degradation of excess or dysfunctional organelles is one of the major challenges to maintaining cell integrity and to adapting cellular activities to environmental changes. To solve this fundamental issue, cells utilize autophagy, which is a self-eating system that generates double-membrane vesicles called autophagosomes, sequesters cytoplasmic components as cargoes, and transports them to lysosomes for degradation. In the past decade, 30 autophagy-related genes (ATG) required for selective and/or nonselective autophagic functions have been identified.
Selective autophagy contributes to the control of both quality and quantity of organelles. It is conceivable that mitochondria are the primary targets of selective autophagy because they accumulate oxidative damage due to their own by-products, ROS. Consistent with this idea, autophagy-dependent clearance of dysfunctional mitochondria is important for organelle quality control. The term mitophagy refers to the selective removal of mitochondria by autophagy.
Mitochondrial Dysfunction and Fibromyalgia
Recently, several groups have described different degrees of mitochondrial dysfunction in muscle biopsies from FM patients, such as subsarcolemmal mitochondrial accumulation, abnormal mitochondria, a low number of mitochondria, electron-dense inclusions, lack of inner membrane in mitochondria, ragged red fibers, and fibers showing defects of cytochrome-c-oxidase, the complex IV of oxidative phosphorilation.
Moreover, several studies have shown high levels of oxidative stress markers in FM patients, suggesting that this process may have a role in the pathophysiology of this disease. In general, oxidative stress could be defined as an imbalance between the presence of high levels of ROS and reactive nitrogen species (RNS), and the antioxidant defense enzymes and molecules. It has been hypothesized that oxidative stress is linked to both the initiation and the progression of neurodegenerative disorders such as Parkinsons, Alzheimers, and Huntingtons diseases. Oxidative stress is also believed to aggravate the symptoms of many diseases, including haemolytic anemias and amyotrophic lateral sclerosis.
Therefore, we addressed the question of whether Fibromyalgia is a mitochondrial disease, and whether mitochondria could be the origin of oxidative stress observed in FM. To answer these questions, we recruited 40 patients from the Sevillian Fibromyalgia Association (AFIBROSE) database. Patients were diagnosed by an experienced rheumatologist according to American College of Rheumatology criteria. The patients had not taken any drugs during the 15-day period before the collection of the blood samples, and had followed a standard balanced diet. Blood samples were collected from each patient; serum biochemical parameters were determined, and plasma and peripheral mononuclear cells were obtained from the blood.
First, we assessed oxidative stress markers in FM patients. Lipid peroxidation (LP), a common marker of oxidative stress, has been implicated in the etiology of FM symptoms. Therefore, we measured LP and protein carbonyls, another marker of oxidative stress, in the patients plasma. We found high levels of lipid peroxidation in these patients.
Next, we checked whether increased oxidative damage in plasma was associated with high levels of intracellular ROS. For this purpose, we cultured mononuclear blood cells blood cells from FM patients and determined ROS production. We found a significant increment of ROS in mononuclear cells from FM patients.
To further examine mitochondrial bioenergetics and antioxidant defenses, we analyzed CoQ levels in mononuclear cells and plasma from FM patients. We observed an altered distribution of CoQ levels between plasma and cells (high levels in plasma and low levels in cells). CoQ levels in blood cells have been reported to have good correlation with CoQ levels in other tissues, such as muscle. These results were published in Clinical Biochemistry in May 2009 (1).
As CoQ deficiency had been suggested to be useful as a mitochondrial dysfunction marker, we addressed mitochondrial dysfunction in blood cells from FM patients and examined whether mitochondrial disturbance could be involved in the pathophysiology of oxidative stress present in FM. We found that CoQ-deficient blood cells in FM patients showed a high level of mitochondrial ROS production and increased levels of lipid peroxidation.
To confirm oxidative stress in FM, blood cells of one representative patient were treated with three antioxidants (CoQ, vitamin E, and N-acetilcysteine), and mitochondrial ROS production was examined. Both CoQ10 and vitamin E, two well known lipophilic antioxidants, induced a significant reduction of ROS. These results suggest that ROS were produced in the lipophilic environment of mitochondrial membranes, and that CoQ deficiency may be involved in the oxidative stress observed in FM. In addition, blood cells of FM patients showed a decrease of 36 percent of mitochondrial membrane potential.
These results strongly support the hypothesis that oxidative stress in FM may be the consequence of a mitochondrial dysfunction. On the other hand, biochemical analysis of citrate synthase indicated a depletion of mitochondrial mass, suggesting selective mitochondrial degradation in blood cells from FM patients. These results were confirmed by electron microscopy that clearly showed autophagosomes where mitochondria are being degraded.
Autophagy can be beneficial for cells by eliminating dysfunctional mitochondria, but excess autophagy can promote cell injury and may contribute to the pathophysiology of FM. This study, published in Arthritis Research and Therapy in January 2010 (2), supports the hypothesis that mitochondrial dysfunction is the origin of oxidative stress in FM patients, and could help to understand the complex pathophysiology of this disorder. Both oxidative stress and extensive mitophagy can contribute to cell bioenergetics imbalance, compromising cell functionality.
An important problem in FM is the moderate effectiveness of pharmacological therapies for FM pain. In general, about half of all treated patients seem to experience a 30 percent decrease of symptoms, suggesting that many patients with FM would require additional therapies. But, in most cases, many side effects are induced by pharmacological therapy, and some drugs can induce mitochondrial damage. Therefore, the use of these drugs in patients with mitochondrial dysfunction could be counterproductive.
CoQ supplementation of blood cells from FM patients treated with CoQ showed a good response, with a decrease of ROS production. The protective effect of CoQ supplementation in blood cells is in agreement with the beneficial effects of CoQ administration in FM patients observed in a previous pilot study (3).
One of the most investigated non-pharmacological therapies in the treatment of FM is moderate aerobic exercise, which has been demonstrated to improve some of the symptoms related to FM. Interestingly, exercise therapies have also been shown to provide significant benefits in patients with mitochondrial diseases by inducing mitochondrial biogenesis, which is proposed as an alternative therapeutic strategy in these conditions, and may explain to some extent the improvement in patients with FM.
Mitochondrial dysfunction opens a large field of research for a new therapeutic perspective in FM patients. Mitochondrial medicine, a relatively new area, deals with diseases that are related to mitochondrial dysfunction due to a number of causes, from free radical damage to genetic mutation. Early on, the well-recognized role of mitochondria in energy production gave rise to strategies designed to enhance electron transfer through the MRC. More recently, mitochondrial involvement in cell death programs, autophagy, and apoptosis has been demonstrated.
Attempts to prevent programmed cell death by blocking mitochondria participation are currently being investigated. Finally, oxidative stress is seen in many diseases. Since oxidative stress may either arise as a consequence of mitochondrial dysfunction or else interfere with mitochondrial function, reducing oxidative stress emerges as a form of mitochondrial medicine that could be beneficial in FM patients.
According to our results, CoQ deficiency would represent a biochemical marker for supporting the diagnosis of FM. Currently, we are collecting blood cells from FM patients to expand patient number and assess the percentage of patients with CoQ deficiency and mitochondrial dysfunction in blood cells. We will also study CoQ deficiency and mitochondrial dysfunction in other tissues from FM patients.
If oxidative damage plays a role in FM through the activation of mitochondrial permeabilization and mitophagy, then therapeutic strategies that reduce ROS may ameliorate the pathological process. But what is the relationship between oxidative stress and FM symptoms? Recent studies have shown that oxidative stress can cause peripheral and central sensitization and alter nociception, resulting in hyperalgesia mediated by both local and spinal oxidant mechanisms.
Superoxide plays a major role in the development of pain through direct peripheral sensitization, the release of various cytokines, the formation of peroxynitrite, and activation of PARP, a protein involved in a number of cellular processes. In addition, studies on depression, a typical symptom in FM patients, have elucidated the possible link between depression and lipid peroxidation.
Our study supports the hypothesis that CoQ deficiency, mitochondrial dysfunction, and extensive mitophagy can contribute to cell bioenergetics imbalance, compromising cell functionality in blood cells of FM patients. Abnormal blood cell performance can promote oxidative stress and may contribute to altered nociception in FM.
Cordero MD, Moreno-Fernandez AM, de Miguel M, Bonal P, Campa F, Jimenez-Jimenez LM, Ruiz-Losada A, Sanchez-Dominguez B, Sanchez Alcazar JA, Salviati L et al: Coenzyme Q10 distribution in blood is altered in patients with fibromyalgia. Clinical biochemistry 2009, 42(7-8):732-735.
Cordero MD, de Miguel M, Moreno Fernandez AM, Carmona Lopez IM, Garrido Maraver J, Cotan D, et al. Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implication in the pathogenesis of the disease. Arthritis Res Ther 2010; In press.
Lister RE. An open, pilot study to evaluate the potential benefits of coenzyme Q10 combined with Ginkgo biloba extract in fibromyalgia syndrome. J Int Med Res 2002; 30:195-9.
This work was supported by grants FIS PI080500 and FIS EC08/00076 from the Ministerio de Sanidad, Spain. This group is funded by the Centro de Investigacin Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII. The authors thank Victor and Heather Rice for their collaboration in this manuscript, and wish to dedicate this manuscript to FM patients and AFIBROSE (AsociaciÃ³n de Fibromialgia de Sevilla) for their unconditional help.
Mario D. CorderoÂ is a medical student at the Universidad de Sevilla and a lab technician at the Universidad Pablo de Olavides Centro Andaluz de Biolog a del Desarrollo in Seville, Spain. He became interested in fibromyalgia because his father suffered from chronic pain.
Dr. Manuel de Miguel is professor of histology at the medical school of Universidad de Sevilla.
Dr. Placido Navas is professor of enzimology and cell biology at the Universidad Pablo de Olavide in Seville, Spain.
Dr. Jose Antonio S¡nchez Alc¡zar is professor of cell biology at the Universidad Pablo de Olavide in Seville, Spain.