Effect of training on skeletal muscle bioenergetic system in patients with mitochondrial myopathies: A computational study.

A computer model of the skeletal muscle bioenergetic system, involving the "Pi double-threshold" mechanism of muscle fatigue, was used to investigate the effect of muscle training on system kinetic properties in mitochondrial myopathies (MM) patients with inborn OXPHOS deficiencies. An increase in OXPHOS activity and decrease in peak Pi can account for the training-induced increase in V̇O2max, acceleration of the primary phase II of the V̇O2 on-kinetics, delay of muscle fatigue and prolongation of exercise at a given work intensity encountered in experimental studies. Depending on the mutation load and work intensity, training can bring the muscle from severe- to very-heavy- to moderate-exercise-like behavior, thus lessening the exertional fatigue and lengthening the physical activity of a given intensity. Training significantly increases critical power (CP) and slightly decreases the curvature constant (W') of the power-duration relationship. Generally, a mechanism underlying the training-induced changes in the skeletal muscle bioenergetic system in MM patients is proposed.

Successful treatment of a patient with mitochondrial myopathy with alirocumab.

A 48-year-old man presented to our lipid clinic with statin intolerance and elevated serum creatine kinase levels, being affected by mitochondrial myopathy because of heteroplasmic mitochondrial DNA missense mutation in MTCO1 gene (m.7671T>A). He had just been treated with a coronary artery bypass 4 years before because of acute coronary syndrome, and he had consistently high levels of both low-density lipoprotein cholesterol and triglycerides. Dyslipidemia was successfully treated using 75 mg of alirocumab subcutaneously every 2 weeks, 10 mg of ezetimibe daily, 2 g of marine omega-3 fatty acids daily, and 145 mg of micronized fenofibrate every 2 days. Although muscle weakness persisted, myalgia did not reoccur and serum creatine kinase levels remained almost stable over the time.

Rewiring of Glutamine Metabolism Is a Bioenergetic Adaptation of Human Cells with Mitochondrial DNA Mutations.

Using molecular, biochemical, and untargeted stable isotope tracing approaches, we identify a previously unappreciated glutamine-derived α-ketoglutarate (αKG) energy-generating anaplerotic flux to be critical in mitochondrial DNA (mtDNA) mutant cells that harbor human disease-associated oxidative phosphorylation defects. Stimulating this flux with αKG supplementation enables the survival of diverse mtDNA mutant cells under otherwise lethal obligatory oxidative conditions. Strikingly, we demonstrate that when residual mitochondrial respiration in mtDNA mutant cells exceeds 45% of control levels, αKG oxidative flux prevails over reductive carboxylation. Furthermore, in a mouse model of mitochondrial myopathy, we show that increased oxidative αKG flux in muscle arises from enhanced alanine synthesis and release into blood, concomitant with accelerated amino acid catabolism from protein breakdown. Importantly, in this mouse model of mitochondriopathy, muscle amino acid imbalance is normalized by αKG supplementation. Taken together, our findings provide a rationale for αKG supplementation as a therapeutic strategy for mitochondrial myopathies.

Triheptanoin versus trioctanoin for long-chain fatty acid oxidation disorders: a double blinded, randomized controlled trial.

BACKGROUND: Observational reports suggest that supplementation that increases citric acid cycle intermediates via anaplerosis may have therapeutic advantages over traditional medium-chain triglyceride (MCT) treatment of long-chain fatty acid oxidation disorders (LC-FAODs) but controlled trials have not been reported. The goal of our study was to compare the effects of triheptanoin (C7), an anaplerotic seven-carbon fatty acid triglyceride, to trioctanoin (C8), an eight-carbon fatty acid triglyceride, in patients with LC-FAODs. METHODS: A double blinded, randomized controlled trial of 32 subjects with LC-FAODs (carnitine palmitoyltransferase-2, very long-chain acylCoA dehydrogenase, trifunctional protein or long-chain 3-hydroxy acylCoA dehydrogenase deficiencies) who were randomly assigned a diet containing 20% of their total daily energy from either C7 or C8 for 4 months was conducted. Primary outcomes included changes in total energy expenditure (TEE), cardiac function by echocardiogram, exercise tolerance, and phosphocreatine recovery following acute exercise. Secondary outcomes included body composition, blood biomarkers, and adverse events, including incidence of rhabdomyolysis. RESULTS: Patients in the C7 group increased left ventricular (LV) ejection fraction by 7.4% (p = 0.046) while experiencing a 20% (p = 0.041) decrease in LV wall mass on their resting echocardiogram. They also required a lower heart rate for the same amount of work during a moderate-intensity exercise stress test when compared to patients taking C8. There was no difference in TEE, phosphocreatine recovery, body composition, incidence of rhabdomyolysis, or any secondary outcome measures between the groups. CONCLUSIONS: C7 improved LV ejection fraction and reduced LV mass at rest, as well as lowering heart rate during exercise among patients with LC-FAODs. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov NCT01379625.

Mitochondrial bioenergetics deregulation caused by long-chain 3-hydroxy fatty acids accumulating in LCHAD and MTP deficiencies in rat brain: a possible role of mPTP opening as a pathomechanism in these disorders?

Long-chain 3-hydroxylated fatty acids (LCHFA) accumulate in long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. Affected patients usually present severe neonatal symptoms involving cardiac and hepatic functions, although long-term neurological abnormalities are also commonly observed. Since the underlying mechanisms of brain damage are practically unknown and have not been properly investigated, we studied the effects of LCHFA on important parameters of mitochondrial homeostasis in isolated mitochondria from cerebral cortex of developing rats. 3-Hydroxytetradecanoic acid (3 HTA) reduced mitochondrial membrane potential, NAD(P)H levels, Ca(2+) retention capacity and ATP content, besides inducing swelling, cytochrome c release and H2O2 production in Ca(2+)-loaded mitochondrial preparations. We also found that cyclosporine A plus ADP, as well as ruthenium red, a Ca(2+) uptake blocker, prevented these effects, suggesting the involvement of the mitochondrial permeability transition pore (mPTP) and an important role for Ca(2+), respectively. 3-Hydroxydodecanoic and 3-hydroxypalmitic acids, that also accumulate in LCHAD and MTP deficiencies, similarly induced mitochondrial swelling and decreased ATP content, but to a variable degree pending on the size of their carbon chain. It is proposed that mPTP opening induced by LCHFA disrupts brain bioenergetics and may contribute at least partly to explain the neurologic dysfunction observed in patients affected by LCHAD and MTP deficiencies.

NARP mutation and mtDNA depletion trigger mitochondrial biogenesis which can be modulated by selenite supplementation.

The importance of mitochondrial biogenesis in the pathogenesis of mitochondrial diseases has been widely recognised but little is known about it with regard to NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) syndrome. Since such knowledge would contribute to the understanding of the pathogenesis of this disease, we designed a study to provide comprehensive overview of mitochondrial biogenesis in cybrid cells harboring NARP mutation (8993T>G). We also used Rho0 cells with the same nuclear background to show that distinct mtDNA defects lead to distinct cellular responses irrespective of nuclear genome. Mitochondrial biogenesis is regulated by mitochondria-to-nucleus (retrograde) communication which depends on intracellular signaling pathways sensitive to ROS. Since we previously found that selenite lowered ROS in NARP cybrids, we hypothesised that selenite could also modulate mitochondrial biogenesis in these cells. Although the mitochondrial mass was not changed in NARP cybrids, we showed the compensatory upregulation of respiratory chain subunits which prompted us to investigate the transcription factors that regulate their expression such as PGC-1α, NRFs, and TFAM. Selenite supplementation increased the level of NRF1 and nuclear accumulation of NRF2, but we did not detect any major changes in the levels of investigated respiratory chain proteins. These subtle changes in mitochondrial biogenesis in response to selenite treatment support the hypothesis that selenite could be considered as a potential therapeutic agent of NARP syndrome due to its antioxidant properties. Moreover, it could also be tested with regard to other mitochondrial disorders associated with ROS overproduction.

From analgesia to myopathy: When local anesthetics impair the mitochondrion.

The expanding utilization of local anesthesia and analgesia revealed the occurrence of myopathies induced by local anesthetics. Such iatrogenic effect encouraged anesthesiologists to study the toxicity of local anesthetics and to reevaluate their protocols in order to reduce muscle pain and dysfunction. Studies performed in rats and human cells showed that bupivacaine induces muscle histological damages with sarcomers disruption along with structural alteration of mitochondria, the powerplant of the cell. Bupivacaine-induced myopathies (BIM) are underestimated as patients are not examined by the anesthesiologist after the surgery. Biochemical analyses indicate that BIM could be explained both by the alteration of mitochondrial energetics with consecutive oxidative stress and mitophagy, and the modification of sarcoplasmic reticulum activity with perturbations of calcium homeostasis. BIM is time-dependent, local anesthetic concentration-dependent, enhanced by preexisting metabolism alteration or young age, and could be prevented in part by antioxidant agents and rhEPO. These observations suggest that adapted changes in postoperative analgesia protocols, including the adjustment of LA concentration and volume, a more precise delivery of the drug and an adapted duration of analgesia, may prevent myopathies consecutive to local anesthesia.

Antioxidant defence systems and generation of reactive oxygen species in osteosarcoma cells with defective mitochondria: effect of selenium.

Mitochondrial diseases originate from mutations in mitochondrial or nuclear genes encoding for mitochondrial proteome. Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) syndrome is associated with the T8993G transversion in ATP6 gene which results in substitution at the very conservative site in the subunit 6 of mitochondrial ATP synthase. Defects in the mitochondrial respiratory chain and the ATPase are considered to be accompanied by changes in the generation of reactive oxygen species (ROS). This study aimed to elucidate effects of selenium on ROS and antioxidant system of NARP cybrid cells with 98% of T8993G mutation load. We found that selenium decreased ROS generation and increased the level and activity of antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Therefore, we propose selenium to be a promising therapeutic agent not only in the case of NARP syndrome but also other diseases associated with mitochondrial dysfunctions and oxidative stress.

Coenzyme Q10 is frequently reduced in muscle of patients with mitochondrial myopathy.

Coenzyme Q(10) (CoQ(10)) deficiency has been associated with an increasing number of clinical phenotypes. Whereas primary CoQ(10) defects are related to mutations in ubiquinone biosynthetic genes, which are now being unraveled, and respond well to CoQ(10) supplementation, the etiologies, and clinical phenotypes related to secondary deficiencies are largely unknown. The purpose of this multicenter study was to evaluate the frequency of muscle CoQ(10) deficiency in a cohort of 76 patients presenting with clinically heterogeneous mitochondrial phenotypes which included myopathy among their clinical features. A reliable diagnostic tool based on HPLC quantification was employed to measure muscle CoQ(10) levels. A significant proportion of these patients (28 over 76) displayed CoQ(10) deficiency that was clearly secondary in nine patients, who harbored a pathogenic mutation of mitochondrial DNA. This study provides a rationale for future therapeutic trials on the effect of CoQ(10) supplementation in patients with mitochondrial diseases presenting with myopathy among clinical features.

New indications and controversies in arginine therapy.

Arginine is an important, versatile and a conditionally essential amino acid. Besides serving as a building block for tissue proteins, arginine plays a critical role in ammonia detoxification, and nitric oxide and creatine production. Arginine supplementation is an essential component for the treatment of urea cycle defects but recently some reservations have been raised with regards to the doses used in the treatment regimens of these disorders. In recent years, arginine supplementation or restriction has been proposed and trialled in several disorders, including vascular diseases and asthma, mitochondrial encephalopathy lactic acidosis and stroke-like episodes (MELAS), glutaric aciduria type I and disorders of creatine metabolism, both production and transportation into the central nervous system. Herein we present new therapeutic indications and controversies surrounding arginine supplementation or deprivation.

Supplemental oxygen and muscle metabolism in mitochondrial myopathy patients.

Patients with mitochondrial myopathy (MM) have a reduced capacity to perform exercise due to a reduced oxidative capacity. We undertook this study to determine whether skeletal muscle metabolism could be improved with oxygen therapy in patients with MM. Six patients with MM and six controls, matched for age, gender and physical activity, underwent (31)P-magnetic resonance spectroscopy ((31)P-MRS) examination. (31)P-MR spectra were collected at rest and in series during exercise and recovery whilst breathing normoxic (0.21 O(2)) or hyperoxic (1.0 O(2)) air. At rest, MM showed an elevated [ADP] (18 +/- 3 micromol/l) and pH (7.03 +/- 0.01) in comparison to the control group (12 +/- 1 micromol/l, 7.01 +/- 0.01) (P < 0.05) consistent with mitochondrial dysfunction. Oxygen supplementation did not change resting metabolites in either MM or the control group (P > 0.05). Inferred maximal ATP synthesis rate improved by 33% with oxygen in MM (21 +/- 3 vs. 28 +/- 5 mmol/(l min), P < 0.05) but only improved by 5% in controls (40 +/- 3 vs. 42 +/- 3 mmol/(l min), P > 0.05). We conclude that oxygen therapy is associated with significant improvements in muscle metabolism in patients with MM. These data suggest that patients with MM could benefit from therapies which improve the provision of oxygen.

Aerobic exercise and muscle metabolism in patients with mitochondrial myopathy.

Exercise therapy improves mitochondrial function in patients with mitochondrial myopathy (MM). We undertook this study to determine the metabolic abnormalities that are improved by exercise therapy. This study identified metabolic pathology using (31)P-magnetic resonance spectroscopy and magnetic resonance imaging (MRI) in a group of patients with MM compared to a control group matched for age, gender, and physical activity. We also observed the effect of exercise therapy for 12 weeks on muscle metabolism and physical function in the MM group. During muscle activity, there was impaired responsiveness of the mitochondria to changes in cytosolic adenosine diphosphate concentration, increased dependence on anaerobic energy pathways, and an adaptive increase in proton efflux in patients with MM. Following exercise therapy, mitochondrial function and muscle mass improved without any change in proton efflux rate. These metabolic findings were accompanied by improvements in functional ability. We conclude that there are significant metabolic differences between patients with MM and a control population, independent of age, gender, and physical activity. Exercise therapy can assist in improving mitochondrial function in MM patients.

Progression despite replacement of a myopathic form of coenzyme Q10 defect.

The authors report 7 years of follow-up evaluation of a patient with coenzyme Q10 (CoQ10) deficiency. Initial symptoms of exercise intolerance and hyperlactatemia improved markedly with substitutive treatment. However, CoQ(10) supplementation did not prevent the onset of a cerebellar syndrome. A switch to idebenone treatment resulted in clinical and metabolic worsening, which disappeared with subsequent CoQ10 treatment. CoQ10 defects may cause progressive neurologic disease despite supplementation.

Attenuation of free radical production and paracrystalline inclusions by creatine supplementation in a patient with a novel cytochrome b mutation.

Mitochondrial cytopathies are associated with increased free radical generation and paracrystalline inclusions. Paracrystalline inclusions were serendipitously found in a young male athlete with a very high respiratory exchange ratio during steady-state exercise; he also had an unusually low aerobic capacity. Direct sequencing of the mitochondrial DNA (mtDNA) coding regions revealed a novel missense mutation (G15497A) resulting in a glycine-->serine conversion at a highly conserved site in the cytochrome b gene in the subject, his mother, and sister. Cybrids, prepared by fusion of the subject's platelets with either U87MG rho degrees or SH-SY5Y rho degrees cells, generated higher basal levels of reactive oxygen species (ROS), had a lower adenosine triphosphate (ATP) content, and were more sensitive to oxygen and glucose deprivation and peroxynitrite generation compared to control cybrids with wild-type mtDNA. Cell survival was significantly enhanced with 50 mmol/L creatine monohydrate (CM) administration. The subject was also treated with CM (10 g/d) for a period of 5 weeks and a repeat muscle biopsy showed no paracrystalline inclusions. The results suggest that the development of exercise-induced paracrystalline inclusions may be influenced by the G15497A mtDNA mutation, and that CM mitigates against the pathological consequences of this mutation.

Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy.

Thymidine kinase 2 (TK2) is a mitochondrial (mt) pyrimidine deoxynucleoside salvage enzyme involved in mtDNA precursor synthesis. The full-length human TK2 cDNA was cloned and sequenced. A discrepancy at amino acid 37 within the mt leader sequence in the DNA compared with the determined peptide sequence was found. Two mutations in the human TK2 gene, His-121 to Asn and Ile-212 to Asn, were recently described in patients with severe mtDNA depletion myopathy (Saada, A., Shaag, A., Mandel, H., Nevo, Y., Eriksson, S., and Elpeleg, O. (2001) Nat. Genet. 29, 342-344). The same mutations in TK2 were introduced, and the mutant enzymes, prepared in recombinant form, were shown to have similar subunit structure to wild type TK2. The I212N mutant showed less than 1% activity as compared with wild type TK2 with all deoxynucleosides. The H121N mutant enzyme had normal K(m) values for thymidine (dThd) and deoxycytidine (dCyd), 6 and 11 microm, respectively, but 2- and 3-fold lower V(max) values as compared with wild type TK2 and markedly increased K(m) values for ATP, leading to decreased enzyme efficiency. Competition experiments revealed that dCyd and dThd interacted differently with the H121N mutant as compared with the wild type enzyme. The consequences of the two point mutations of TK2 and the role of TK2 in mt disorders are discussed.

Functional characterization of novel mutations in the human cytochrome b gene.

The great variability of the human mitochondrial DNA (mtDNA) sequence induces many difficulties in the search for its deleterious mutations. We illustrate these pitfalls by the analysis of the cytochrome b gene of 21 patients affected with a mitochondrial disease. Eighteen different sequence variations were found, five of which were new mutations. Extensive analysis of the cytochrome b gene of 146 controls found 20 supplementary mutations, thus further demonstrating the high variability of the cytochrome b sequence. We fully evaluated the functional relevance of 36 of these 38 mutations using indirect criteria such as the nature of the mutation, its frequency in controls, or the phylogenetic conservation of the mutated amino acid. When appropriate, the mtDNA haplotype, the heteroplasmic state of the mutation, its tissue distribution or its familial transmission were also assessed. The molecular consequences of the mutations, which appeared possibly deleterious in that first step of evaluation, were evaluated on the complex III enzymological properties and protein composition using specific antibodies that we have generated against four of its subunits. Two original deleterious mutations were found in the group of seven patients with overt complex III defect. Both mutations (G15150A (W135X) and T15197C (S151P)) were heteroplasmic and restricted to muscle. They had significant consequences on the complex III structure. In contrast, only two homoplasmic missense mutations with dubious clinical relevance were found in the patients without overt complex III defect.

Mitochondrial medicine--molecular pathology of defective oxidative phosphorylation.

Different tissues display distinct sensitivities to defective mitochondrial oxidative phosphorylation (OXPHOS). Tissues highly dependent on oxygen such as the cardiac muscle, skeletal and smooth muscle, the central and peripheral nervous system, the kidney, and the insulin-producing pancreatic beta-cell are especially susceptible to defective OXPHOS. There is evidence that defective OXPHOS plays an important role in atherogenesis, in the pathogenesis of Alzheimer's disease, Parkinson's disease, diabetes, and aging. Defective OXPHOS may be caused by abnormal mitochondrial biosynthesis due to inherited or acquired mutations in the nuclear (n) or mitochondrial (mt) deoxyribonucleic acid (DNA). For instance, the presence of a mutation of the mtDNA in the pancreatic beta-cell impairs adenosine triphosphate (ATP) generation and insulin synthesis. The nuclear genome controls mitochondrial biosynthesis, but mtDNA has a much higher mutation rate than nDNA because it lacks histones and is exposed to the radical oxygen species (ROS) generated by the electron transport chain, and the mtDNA repair system is limited. Defective OXPHOS may be caused by insufficient fuel supply, by defective electron transport chain enzymes (Complexes I - IV), lack of the electron carrier coenzyme Q10, lack of oxygen due to ischemia or anemia, or excessive membrane leakage, resulting in insufficient mitochondrial inner membrane potential for ATP synthesis by the F0F1-ATPase. Human tissues can counteract OXPHOS defects by stimulating mitochondrial biosynthesis; however, above a certain threshold the lack of ATP causes cell death. Many agents affect OXPHOS. Several nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit or uncouple OXPHOS and induce the 'topical' phase of gastrointestinal ulcer formation. Uncoupled mitochondria reduce cell viability. The Helicobacter pylori induces uncoupling. The uncoupling that opens the membrane pores can activate apoptosis. Cholic acid in experimental atherogenic diets inhibits Complex IV, cocaine inhibits Complex I, the poliovirus inhibits Complex II, ceramide inhibits Complex III, azide, cyanide, chloroform, and methamphetamine inhibit Complex IV. Ethanol abuse and antiviral nucleoside analogue therapy inhibit mtDNA replication. By contrast, melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III. Oral Q10 supplementation is effective in treating cardiomyopathies and in restoring plasma levels reduced by the statin type of cholesterol-lowering drugs.

Improved brain and muscle mitochondrial respiration with CoQ. An in vivo study by 31P-MR spectroscopy in patients with mitochondrial cytopathies.

We used in vivo phosphorus magnetic resonance spectroscopy (31P-MRS) to study the effect of CoQ10 on the efficiency of brain and skeletal muscle mitochondrial respiration in ten patients with mitochondrial cytopathies. Before CoQ, brain [PCr] was remarkably lower in patients than in controls, while [Pi] and [ADP] were higher. Brain cytosolic free [Mg2+] and delta G of ATP hydrolysis were also abnormal in all patients. MRS also revealed abnormal mitochondrial function in the skeletal muscles of all patients, as shown by a decreased rate of PCr recovery from exercise. After six-months of treatment with CoQ (150 mg/day), all brain MRS-measurable variables as well as the rate of muscle mitochondrial respiration were remarkably improved in all patients. These in vivo findings show that treatment with CoQ in patients with mitochondrial cytopathies improves mitochondrial respiration in both brain and skeletal muscles, and are consistent with Lenaz's view that increased CoQ concentration in the mitochondrial membrane increases the efficiency of oxidative phosphorylation independently of enzyme deficit.

Low brain intracellular free magnesium in mitochondrial cytopathies.

The authors studied, by in vivo phosphorus magnetic resonance spectroscopy (31P-MRS), the occipital lobes of 19 patients with mitochondrial cytopathies to clarify the functional relation between energy metabolism and concentration of cytosolic free magnesium. All patients displayed defective mitochondrial respiration with low phosphocreatine concentration [PCr] and high inorganic phosphate concentration [Pi] and [ADP]. Cytosolic free [Mg2+] and the readily available free energy (defined as the actual free energy released by the exoergonic reaction of ATP hydrolysis, i.e., deltaG(ATPhyd)) were abnormally low in all patients. Nine patients were treated with coenzyme Q10 (CoQ), which improved the efficiency of the respiratory chain, as shown by an increased [PCr], decreased [Pi] and [ADP], and increased availability of free energy (more negative value of deltaG(ATPhyd)). Treatment with CoQ also increased cytosolic free [Mg2+] in all treated patients. The authors findings demonstrate low brain free [Mg2+] in our patients and indicate that it resulted from failure of the respiratory chain. Free Mg2+ contributes to the absolute value of deltaG(ATPhyd). The results also are consistent with the view that cytosolic [Mg2+] is regulated in the intact brain cell to equilibrate, at least in part, any changes in rapidly available free energy.

Phosphorus magnetic resonance spectroscopy in the evaluation of mitochondrial myopathies: results of a 6-month therapy study with coenzyme Q.

31P magnetic resonance spectroscopy (MRS) was used to study an open therapeutic trial of coenzyme Q10 (CoQ) in mitochondrial encephalomyopathies. Eight patients were treated with 150 mg CoQ per day for 6 months. 31P MRS spectra of calf muscle were recorded at rest, during exercise and in the immediate postexercise recovery period. Although there was an improvement of the mean ratio of phosphocreatine (PCr) to inorganic phosphate during the post-exercise recovery period after 3 months of treatment, this finding was mainly due to a single therapy responder and did not reflect a beneficial effect on the whole group. Improved repletion of PCr persisted after 6 months of therapy. Our study identified a single responder to this therapy, whose response could not be predicted on the basis of clinical, biochemical or molecular data. These findings suggest that therapeutic trials of CoQ should be performed under close metabolic monitoring in order both to identify responders for subsequent long-term treatment and to evaluate possible mechanisms of this supportive therapy.