François Singh

Mitochondria: Mitochondrial participation in ischemia–reperfusion injury in skeletal muscle

Irrespective of the organ involved, restoration of blood flow to ischemic tissue is vital, although reperfusion per se is deleterious. In the setting of vascular surgery, even subtle skeletal muscle ischemia contributes to remote organ injuries and perioperative and long-term morbidities. Reperfusion-induced injury is thought to participate in up to 40% of muscle damage. Recently, the pathophysiology of lower limb ischemia-reperfusion (IR) has been largely improved, acknowledging a key role for mitochondrial dysfunction mainly characterized by impaired mitochondrial oxidative capacity and premature mitochondrial permeability transition pore opening. Increased oxidative stress triggered by an imbalance between reactive oxygen species (ROS) production and clearance, and facilitated by enhanced inflammation, appears to be both followed and instigated by mitochondrial dysfunction. Mitochondria are both actors and target of IR and therapeutic strategies modulating degree of ROS production could enhance protective signals and allow for mitochondrial protection through a mitohormesis mechanism.

Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study of ageing.

BackgroundIn contrast to mammalian erythrocytes, which have lost their nucleus and mitochondria during maturation, the erythrocytes of almost all other vertebrate species are nucleated throughout their lifespan. Little research has been done however to test for the presence and functionality of mitochondria in these cells, especially for birds. Here, we investigated those two points in erythrocytes of one common avian model: the zebra finch (Taeniopygia guttata).ResultsTransmission electron microscopy showed the presence of mitochondria in erythrocytes of this small passerine bird, especially after removal of haemoglobin interferences. High-resolution respirometry revealed increased or decreased rates of oxygen consumption by erythrocytes in response to the addition of respiratory chain substrates or inhibitors, respectively. Fluorometric assays confirmed the production of mitochondrial superoxide by avian erythrocytes. Interestingly, measurements of plasmatic oxidative markers indicated lower oxidative stress in blood of the zebra finch compared to a size-matched mammalian model, the mouse.ConclusionsAltogether, those findings demonstrate that avian erythrocytes possess functional mitochondria in terms of respiratory activities and reactive oxygen species (ROS) production. Interestingly, since blood oxidative stress was lower for our avian model compared to a size-matched mammalian, our results also challenge the idea that mitochondrial ROS production could have been one actor leading to this loss during the course of evolution. Opportunities to assess mitochondrial functioning in avian erythrocytes open new perspectives in the use of birds as models for longitudinal studies of ageing via lifelong blood sampling of the same subjects.

Reductive stress impairs myoblasts mitochondrial function and triggers mitochondrial hormesis

Even though oxidative stress damage from excessive production of ROS is a well known phenomenon, the impact of reductive stress remains poorly understood. This study tested the hypothesis that cellular reductive stress could lead to mitochondrial malfunction, triggering a mitochondrial hormesis (mitohormesis) phenomenon able to protect mitochondria from the deleterious effects of statins. We performed several in vitro experiments on L6 myoblasts and studied the effects of N-acetylcysteine (NAC) at different exposure times. Direct NAC exposure (1mM) led to reductive stress, impairing mitochondrial function by decreasing maximal mitochondrial respiration and increasing H₂O₂production. After 24h of incubation, the reactive oxygen species (ROS) production was increased. The resulting mitochondrial oxidation activated mitochondrial biogenesis pathways at the mRNA level. After one week of exposure, mitochondria were well-adapted as shown by the decrease of cellular ROS, the increase of mitochondrial content, as well as of the antioxidant capacities. Atorvastatin (ATO) exposure (100μM) for 24h increased ROS levels, reduced the percentage of live cells, and increased the total percentage of apoptotic cells. NAC exposure during 3days failed to protect cells from the deleterious effects of statins. On the other hand, NAC pretreatment during one week triggered mitochondrial hormesis and reduced the deleterious effect of statins. These results contribute to a better understanding of the redox-dependant pathways linked to mitochondria, showing that reductive stress could trigger mitochondrial hormesis phenomenon.

Tetrahydrocannabinol Induces Brain Mitochondrial Respiratory Chain Dysfunction and Increases Oxidative Stress: A Potential Mechanism Involved in Cannabis-Related Stroke

Cannabis has potential therapeutic use but tetrahydrocannabinol (THC), its main psychoactive component, appears as a risk factor for ischemic stroke in young adults. We therefore evaluate the effects of THC on brain mitochondrial function and oxidative stress, key factors involved in stroke. Maximal oxidative capacities V max (complexes I, III, and IV activities), V succ (complexes II, III, and IV activities), V tmpd (complex IV activity), together with mitochondrial coupling (V max/V 0), were determined in control conditions and after exposure to THC in isolated mitochondria extracted from rat brain, using differential centrifugations. Oxidative stress was also assessed through hydrogen peroxide (H2O2) production, measured with Amplex Red. THC significantly decreased V max (−71%; P < 0.0001), V succ (−65%; P < 0.0001), and V tmpd (−3.5%; P < 0.001). Mitochondrial coupling (V max/V 0) was also significantly decreased after THC exposure (1.8±0.2 versus 6.3±0.7; P < 0.001). Furthermore, THC significantly enhanced H2O2 production by cerebral mitochondria (+171%; P < 0.05) and mitochondrial free radical leak was increased from 0.01±0.01 to 0.10±0.01% (P < 0.001). Thus, THC increases oxidative stress and induces cerebral mitochondrial dysfunction. This mechanism may be involved in young cannabis users who develop ischemic stroke since THC might increase patients vulnerability to stroke.

Statins Trigger Mitochondrial Reactive Oxygen Species-Induced Apoptosis in Glycolytic Skeletal Muscle.

AIMS Although statins are the most widely used cholesterol-lowering agents, they are associated with a variety of muscle complaints. The goal of this study was to characterize the effects of statins on the mitochondrial apoptosis pathway induced by mitochondrial oxidative stress in skeletal muscle using human muscle biopsies as well as in vivo and in vitro models. RESULTS Statins increased mitochondrial H2O2 production, the Bax/Bcl-2 ratio, and TUNEL staining in deltoid biopsies of patients with statin-associated myopathy. Furthermore, atorvastatin treatment for 2 weeks at 10 mg/kg/day in rats increased H2O2 accumulation and mRNA levels and immunostaining of the Bax/Bcl-2 ratio, as well as TUNEL staining and caspase 3 cleavage in glycolytic (plantaris) skeletal muscle, but not in oxidative (soleus) skeletal muscle, which has a high antioxidative capacity. Atorvastatin also decreased the GSH/GSSG ratio, but only in glycolytic skeletal muscle. Cotreatment with the antioxidant, quercetin, at 25 mg/kg/day abolished these effects in plantaris. An in vitro study with L6 myoblasts directly demonstrated the link between mitochondrial oxidative stress following atorvastatin exposure and activation of the mitochondrial apoptosis signaling pathway. INNOVATION Treatment with atorvastatin is associated with mitochondrial oxidative stress, which activates apoptosis and contributes to myopathy. Glycolytic muscles are more sensitive to atorvastatin than oxidative muscles, which may be due to the higher antioxidative capacity in oxidative muscles. CONCLUSION There is a link between statin-induced mitochondrial oxidative stress and activation of the mitochondrial apoptosis signaling pathway in glycolytic skeletal muscle, which may be associated with statin-associated myopathy.

High reactive oxygen species in fibrotic and nonfibrotic skin of patients with diffuse cutaneous systemic sclerosis

Systemic sclerosis (SSc) is a chronic multisystemic connective tissue disease characterized by progressive fibrosis affecting skin and internal organs. Despite serious efforts to unveil the pathogenic mechanisms of SSc, they are still unclear. High levels of reactive oxygen species (ROS) in affected patients have been shown, and ROS are suggested to play a role in fibrosis pathogenesis. In this study we evaluate ROS levels in nonfibrotic and fibrotic skin of patients with SSc and we compare them with those obtained from healthy controls. We enrolled nine SSc patients fulfilling the EULAR/ACR classification criteria and seven healthy controls. Patients included four men and five women with mean age of 46 ± 10 years. Controls were matched by sex and age. All patients were affected by the diffuse cutaneous form of SSc and the ANA pattern anti-Scl70. Mean disease duration was 7.5 ± 5 years. Skin involvement was evaluated by modified Rodnan skin score. Skin samples (4-mm punch biopsy) were taken from fibrotic skin and nonfibrotic skin of patients and from healthy controls as well. To detect ROS, specimens were analyzed immediately after sampling by electron paramagnetic resonance spectroscopy. Blood samples were drawn from all patients and controls to assess oxidative stress biomarkers. ROS levels (expressed as median and range, in nmol/L/min/mg of dry weight) were 24.7 (10.9-47.0) in fibrotic skin, 18.7 (7.3-34.0) in nonfibrotic skin, and 7.7 (3.5-13.6) in healthy control skin. ROS levels in fibrotic and nonfibrotic skin of SSc patients were significantly higher than in healthy controls (p = 0.002 and p = 0.009, respectively). ROS levels in fibrotic skin were raised in comparison to nonfibrotic skin, when samples related to each patient were compared (p = 0.01). ROS levels in fibrotic skin were correlated with forced vital capacity (r = -0.75, p = 0.02) and erythrocyte sedimentation rate (r = 0.70, p = 0.04). All other clinical and lab parameters showed no significant correlation. Compared to controls, blood from SSc patients showed lower ascorbate (vitamin C) levels (8 (3.8-9.8) vs 10.5 (9-19.1) mg/L, p = 0.004) and higher lipid peroxides (873.5 (342-1973) vs 422 (105-576) μmol/L, p = 0.004). Our results indicate the presence of high oxidative stress in both nonfibrotic skin and fibrotic skin of SSc patients, but with higher tendency in the latter. Raised ROS levels in nonfibrotic skin of SSc patients might be a hint of early involvement in skin fibrogenesis. However, a longitudinal prospective study is necessary for such proof.

Endothelial function does not improve with high-intensity continuous exercise training in SHR: implications of eNOS uncoupling.

Exercise training is a well-recognized way to improve vascular endothelial function by increasing nitric oxide (NO) bioavailability. However, in hypertensive subjects, unlike low- and moderate-intensity exercise training, the beneficial effects of continuous high-intensity exercise on endothelial function are not clear, and the underlying mechanisms remain unknown. The aim of this study was to investigate the impact of high-intensity exercise on vascular function, especially on the NO pathway, in spontaneous hypertensive rats (SHR). These effects were studied on WKY, sedentary SHR and SHR that exercised at moderate (SHR-MOD) and high intensity (SHR-HI) on a treadmill (1 h per day; 5 days per week for 6 weeks at 55% and 80% of their maximal aerobic velocity, respectively). Endothelial function and specific NO contributions to acetylcholine-mediated relaxation were evaluated by measuring the aortic ring isometric forces. Endothelial nitric oxide synthase (eNOS) expression and phosphorylation (ser1177) were evaluated by western blotting. The total aortic and eNOS-dependent reactive oxygen species (ROS) production was assessed using electron paramagnetic resonance in aortic tissue. Although the aortas of SHR-HI had increased eNOS levels without alteration of eNOS phosphorylation, high-intensity exercise had no beneficial effect on endothelium-dependent vasorelaxation, unlike moderate exercise. This result was associated with increased eNOS-dependent ROS production in the aortas of SHR-HI. Notably, the use of the recoupling agent BH4 or a thiol-reducing agent blunted eNOS-dependent ROS production in the aortas of SHR-HI. In conclusion, the lack of a positive effect of high-intensity exercise on endothelial function in SHR was mainly explained by redox-dependent eNOS uncoupling, resulting in a switch from NO to O2− generation.

Carbon monoxide increases inducible NOS expression that mediates CO-induced myocardial damage during ischemia-reperfusion

We investigated the role of inducible nitric oxide (NO) synthase (iNOS) on ischemic myocardial damage in rats exposed to daily low nontoxic levels of carbon monoxide (CO). CO is a ubiquitous environmental pollutant that impacts on mortality and morbidity from cardiovascular diseases. We have previously shown that CO exposure aggravates myocardial ischemia-reperfusion (I/R) injury partly because of increased oxidative stress. Nevertheless, cellular mechanisms underlying cardiac CO toxicity remain hypothetical. Wistar rats were exposed to simulated urban CO pollution for 4 wk. First, the effects of CO exposure on NO production and NO synthase (NOS) expression were evaluated. Myocardial I/R was performed on isolated perfused hearts in the presence or absence of S-methyl-isothiourea (1 μM), a NOS inhibitor highly specific for iNOS. Finally, Ca(2+) handling was evaluated in isolated myocytes before and after an anoxia-reoxygenation performed with or without S-methyl-isothiourea or N-acetylcystein (20 μM), a nonspecific antioxidant. Our main results revealed that 1) CO exposure altered the pattern of NOS expression, which is characterized by increased neuronal NOS and iNOS expression; 2) cardiac NO production increased in CO rats because of its overexpression of iNOS; and 3) the use of a specific inhibitor of iNOS reduced myocardial hypersensitivity to I/R (infarct size, 29 vs. 51% of risk zone) in CO rat hearts. These last results are explained by the deleterious effects of NO and reactive oxygen species overproduction by iNOS on diastolic Ca(2+) overload and myofilaments Ca(2+) sensitivity. In conclusion, this study highlights the involvement of iNOS overexpression in the pathogenesis of simulated urban CO air pollution exposure.

Cryopreservation with dimethyl sulfoxide prevents accurate analysis of skinned skeletal muscle fibers mitochondrial respiration

Impact of cryopreservation protocols on skeletal muscle mitochondrial respiration remains controversial. We showed that oxygen consumption with main mitochondrial substrates in rat skeletal muscles was higher in fresh samples than in cryopreserved samples and that this difference was not fixed but grow significantly with respiration rates with wide fluctuations around the mean difference. Very close results were observed whatever the muscle type and the substrate used. Importantly, the deleterious effects of ischemia-reperfusion observed on fresh samples vanished when cryopreserved samples were studied. These data demonstrate that this technic should probably be performed only extemporaneously.

Impaired Exercise Performance and Skeletal Muscle Mitochondrial Function in Rats with Secondary Carnitine Deficiency

Purpose: The effects of carnitine depletion upon exercise performance and skeletal muscle mitochondrial function remain largely unexplored. We therefore investigated the effect of N-trimethyl-hydrazine-3-propionate (THP), a carnitine analog inhibiting carnitine biosynthesis and renal carnitine reabsorption, on physical performance and skeletal muscle mitochondrial function in rats. Methods: Male Sprague Dawley rats were treated daily with water (control rats; n = 12) or with 20 mg/100 g body weight THP (n = 12) via oral gavage for 3 weeks. Following treatment, half of the animals of each group performed an exercise test until exhaustion. Results: Distance covered and exercise performance were lower in THP-treated compared to control rats. In the oxidative soleus muscle, carnitine depletion caused atrophy (–24%) and impaired function of complex II and IV of the mitochondrial electron transport chain. The free radical leak (ROS production relative to oxygen consumption) was increased and the cellular glutathione pool decreased. Moreover, mRNA expression of markers of mitochondrial biogenesis and mitochondrial DNA were decreased in THP-treated compared to control rats. In comparison, in the glycolytic gastrocnemius muscle, carnitine depletion was associated with impaired function of complex IV and increased free radical leak, whilst muscle weight and cellular glutathione pool were maintained. Markers of mitochondrial proliferation and mitochondrial DNA were unaffected. Conclusions: Carnitine deficiency is associated with impaired exercise capacity in rats treated with THP. THP-induced carnitine deficiency is associated with impaired function of the electron transport chain in oxidative and glycolytic muscle as well as with atrophy and decreased mitochondrial DNA in oxidative muscle.