Moderate Exercise Allows for shorter Recovery Time in Critical Limb Ischemia.

Whether and how moderate exercise might allow for accelerated limb recovery in chronic critical limb ischemia (CLI) remains to be determined. Chronic CLI was surgically induced in mice, and the effect of moderate exercise (training five times per week over a 3-week period) was investigated. Tissue damages and functional scores were assessed on the 4th, 6th, 10th, 20th, and 30th day after surgery. Mice were sacrificed 48 h after the last exercise session in order to assess muscle structure, mitochondrial respiration, calcium retention capacity, oxidative stress and transcript levels of genes encoding proteins controlling mitochondrial functions (PGC1α, PGC1ß, NRF1) and anti-oxidant defenses markers (SOD1, SOD2, catalase). CLI resulted in tissue damages and impaired functional scores. Mitochondrial respiration and calcium retention capacity were decreased in the ischemic limb of the non-exercised group (Vmax = 7.11 ± 1.14 vs. 9.86 ± 0.86 mmol 02/min/g dw, p < 0.001; CRC = 7.01 ± 0.97 vs. 11.96 ± 0.92 microM/mg dw, p < 0.001, respectively). Moderate exercise reduced tissue damages, improved functional scores, and restored mitochondrial respiration and calcium retention capacity in the ischemic limb (Vmax = 9.75 ± 1.00 vs. 9.82 ± 0.68 mmol 02/min/g dw; CRC = 11.36 ± 1.33 vs. 12.01 ± 1.24 microM/mg dw, respectively). Exercise also enhanced the transcript levels of PGC1α, PGC1ß, NRF1, as well as SOD1, SOD2, and catalase. Moderate exercise restores mitochondrial respiration and calcium retention capacity, and it has beneficial functional effects in chronic CLI, likely by stimulating reactive oxygen species-induced biogenesis and anti-oxidant defenses. These data support further development of exercise therapy even in advanced peripheral arterial disease.

Mitochondrial function following downhill and/or uphill exercise training in rats.

INTRODUCTION: The goal of this study was to compare the effects of downhill (DH), uphill (UH), and UH-DH exercise training, at the same metabolic rate, on exercise capacity and skeletal muscle mitochondrial function. METHODS: Thirty-two Wistar rats were separated into a control and 3 trained groups. The trained groups exercised for 4 weeks, 5 times per week at the same metabolic rate, either in UH, DH, or combined UH-DH. Twenty-four hours after the last training session, the soleus, gastrocnemius, and vastus intermedius muscles were removed for assessment of mitochondrial respiration. RESULTS: Exercise training, at the same metabolic rate, improved maximal running speed without specificity for exercise modalities. Maximal fiber respiration was enhanced in soleus and vastus intermedius in the UH group only. CONCLUSIONS: Exercise training, performed at the same metabolic rate, improved exercise capacity, but only UH-trained rats enhanced mitochondrial function in both soleus and vastus intermedius skeletal muscle. Muscle Nerve 54: 925-935, 2016.

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.

The transcriptional coregulator PGC-1ß controls mitochondrial function and anti-oxidant defence in skeletal muscles.

The transcriptional coregulators PGC-1α and PGC-1ß modulate the expression of numerous partially overlapping genes involved in mitochondrial biogenesis and energetic metabolism. The physiological role of PGC-1ß is poorly understood in skeletal muscle, a tissue of high mitochondrial content to produce ATP levels required for sustained contractions. Here we determine the physiological role of PGC-1ß in skeletal muscle using mice, in which PGC-1ß is selectively ablated in skeletal myofibres at adulthood (PGC-1ß((i)skm-/-) mice). We show that myofibre myosin heavy chain composition and mitochondrial number, muscle strength and glucose homeostasis are unaffected in PGC-1ß((i)skm-/-) mice. However, decreased expression of genes controlling mitochondrial protein import, translational machinery and energy metabolism in PGC-1ß((i)skm-/-) muscles leads to mitochondrial structural and functional abnormalities, impaired muscle oxidative capacity and reduced exercise performance. Moreover, enhanced free-radical leak and reduced expression of the mitochondrial anti-oxidant enzyme Sod2 increase muscle oxidative stress. PGC-1ß is therefore instrumental for skeletal muscles to cope with high energetic demands.

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 patient's vulnerability to stroke.

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.

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.

Dynein mutations associated with hereditary motor neuropathies impair mitochondrial morphology and function with age.

Mutations in the DYNC1H1 gene encoding for dynein heavy chain cause two closely related human motor neuropathies, dominant spinal muscular atrophy with lower extremity predominance (SMA-LED) and axonal Charcot-Marie-Tooth (CMT) disease, and lead to sensory neuropathy and striatal atrophy in mutant mice. Dynein is the molecular motor carrying mitochondria retrogradely on microtubules, yet the consequences of dynein mutations on mitochondrial physiology have not been explored. Here, we show that mouse fibroblasts bearing heterozygous or homozygous point mutation in Dync1h1, similar to human mutations, show profoundly abnormal mitochondrial morphology associated with the loss of mitofusin 1. Furthermore, heterozygous Dync1h1 mutant mice display progressive mitochondrial dysfunction in muscle and mitochondria progressively increase in size and invade sarcomeres. As a likely consequence of systemic mitochondrial dysfunction, Dync1h1 mutant mice develop hyperinsulinemia and hyperglycemia and progress to glucose intolerance with age. Similar defects in mitochondrial morphology and mitofusin levels are observed in fibroblasts from patients with SMA-LED. Last, we show that Dync1h1 mutant fibroblasts show impaired perinuclear clustering of mitochondria in response to mitochondrial uncoupling. Our results show that dynein function is required for the maintenance of mitochondrial morphology and function with aging and suggest that mitochondrial dysfunction contributes to dynein-dependent neurological diseases, such as SMA-LED.