Richard Godin

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Mitochondrial Dysfunction and Lipid Accumulation in the Human Diaphragm during Mechanical Ventilation

RATIONALE Mechanical ventilation (MV) is associated with adverse effects on the diaphragm, but the cellular basis for this phenomenon, referred to as ventilator-induced diaphragmatic dysfunction (VIDD), is poorly understood. OBJECTIVES To determine whether mitochondrial function and cellular energy status are disrupted in human diaphragms after MV, and the role of mitochondria-derived oxidative stress in the development of VIDD. METHODS Diaphragm and biceps specimens obtained from brain-dead organ donors who underwent MV (15-176 h) and age-matched control subjects were compared regarding mitochondrial enzymatic function, mitochondrial DNA integrity, lipid content, and metabolic gene and protein expression. In addition, diaphragmatic force and oxidative stress after exposure to MV for 6 hours were evaluated in mice under different conditions. MEASUREMENTS AND MAIN RESULTS In human MV diaphragms, mitochondrial biogenesis and content were down-regulated, with a more specific defect of respiratory chain cytochrome-c oxidase. Laser capture microdissection of cytochrome-c oxidase-deficient fibers revealed mitochondrial DNA deletions, consistent with damage from oxidative stress. Diaphragmatic lipid accumulation and responses of master cellular metabolic sensors (AMP-activated protein kinase and sirtuins) were consistent with energy substrate excess as a possible stimulus for these changes. In mice, induction of hyperlipidemia worsened diaphragmatic oxidative stress during MV, whereas transgenic overexpression of a mitochondria-localized antioxidant (peroxiredoxin-3) was protective against VIDD. CONCLUSIONS Our data suggest that mitochondrial dysfunction lies at the nexus between oxidative stress and the impaired diaphragmatic contractility that develops during MV. Energy substrate oversupply relative to demand, resulting from diaphragmatic inactivity during MV, could play an important role in this process.

Autophagy and Skeletal Muscles in Sepsis

Background Mitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles. Methodology/Principal Findings Adult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS) or E. coli lipopolysaccharide (LPS, 20 mg/kg) and sacrificed 24 h later. The tibialis anterior (TA), soleus (SOLD) and diaphragm (DIA) muscles were quickly excised and examined for mitochondrial morphological injury, Ca++ retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis) were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor. Conclusion/Significance We conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the ventilatory muscles and that autophagy in skeletal muscles during sepsis is regulated in part through the NFκB transcription factor.

Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124+/-47 vs. 30.4+/-6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121+/-60 vs. 40+/-10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56+/-4 vs. 111+/-11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.

The Mitochondrial Phenotype of Peripheral Muscle in Chronic Obstructive Pulmonary Disease Disuse or Dysfunction

RATIONALE Peripheral muscle alterations have been recognized to contribute to disability in chronic obstructive pulmonary disease (COPD). OBJECTIVES To describe the mitochondrial phenotype in a moderate to severe COPD population and age-matched controls. METHODS Three primary aspects of mitochondrial function were assessed in permeabilized locomotor muscle fibers. MEASUREMENTS AND MAIN RESULTS Respiration rates per milligram of fiber weight were significantly lower in COPD muscle compared with healthy age-matched control muscle under various respiratory states. However, when variations in mitochondrial volume were taken into account by normalizing respiration per unit of citrate synthase activity, differences between the two groups were abolished, suggesting the absence of specific mitochondrial respiratory impairment in COPD. H(2)O(2) production per mitochondrion was higher both under basal and ADP-stimulated states, suggesting that mitochondria from COPD muscle have properties that potentiate H(2)O(2) release. Direct assessment of mitochondrial sensitivity to Ca(2+)-induced opening of the permeability transition pore (PTP) indicated that mitochondria from patients with COPD were more resistant to PTP opening than their counterparts in control subjects. CONCLUSIONS Comparison of these results with those of studies comparing healthy glycolytic with oxidative muscle suggests that these differences may be attributable to greater type II fiber expression in COPD muscle, as mitochondria within this fiber type have respiratory function similar to that of mitochondria from type I fibers, and yet are intrinsically prone to greater release of H(2)O(2) and more resistant to PTP opening. These results thus argue against the presence of pathological mitochondrial alterations in this category of patients with COPD.

Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction

Mitochondrial quality control plays a vital role in the maintenance of optimal mitochondrial function. However, its roles and regulation remain ill-defined in cardiac pathophysiology. Here, we tested the hypothesis that PARK2/Parkin, an E3-ligase recently described as being involved in the regulation of cardiac mitophagy, is important for (1) the maintenance of normal cardiac mitochondrial function; and (2) adequate recovery from sepsis, a condition known to induce reversible mitochondrial injury through poorly understood mechanisms. Investigations of mitochondrial and cardiac function were thus performed in wild-type and Park2-deficient mice at baseline and at 2 different times following administration of a sublethal dose of E. coli lipopolysaccharide (LPS). LPS injection induced cardiac and mitochondrial dysfunctions that were followed by complete recovery in wild-type mice. Recovery was associated with morphological and biochemical evidence of mitophagy, suggesting that this process is implicated in cardiac recovery from sepsis. Under baseline conditions, multiple cardiac mitochondrial dysfunctions were observed in Park2-deficient mice. These mild dysfunctions did not result in a visibly distinct cardiac phenotype. Importantly, Park2-deficient mice exhibited impaired recovery of cardiac contractility and constant degradation of mitochondrial metabolic functions. Interestingly, autophagic clearance of damaged mitochondria was still possible in the absence of PARK2 likely through compensatory mechanisms implicating PARK2-independent mitophagy and upregulation of macroautophagy. Together, these results thus provide evidence that in vivo, mitochondrial autophagy is activated during sepsis, and that compensation for a lack of PARK2 is only partial and/or that PARK2 exerts additional protective roles in mitochondria.

Peroxisome proliferator-activated receptor γ coactivator 1-α gene transfer restores mitochondrial biomass and improves mitochondrial calcium handling in post-necrotic mdx mouse skeletal muscle

•  Mitochondria are increasingly implicated in the pathogenesis of Duchenne muscular dystrophy (DMD). However, the extent to which the multiple facets of mitochondrial function are altered remains uncertain due to the lack of detailed assessment. •  Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC1α) was recently shown to improve muscle pathology in dystrophic muscle. However, the mechanisms are not fully elucidated. •  This study provides novel information on mitochondrial dysfunction in DMD namely that (i) loss of mitochondrial biomass precedes overt respiratory abnormalities, (ii) mitochondrial H2O2 metabolism is improved at a time when no oxidative damage is detectable in the muscle, and (iii) susceptibility to permeability transition pore (PTP) opening is increased, which has only been inferred in the past, but never actually measured. •  This study also provides new mechanistic information regarding the beneficial effects of PGC1α overexpression upon dystrophic muscles, namely that PGC1α not only increases mitochondrial biomass but also reduces PTP opening, improves mitochondrial Ca2+ handling and reduces the activation of Ca2+ and mitochondria‐dependent proteases in muscles of mdx mice. These mechanisms were not examined in previous investigations, which had largely attributed the improved histopathology after PGC1α therapy to utrophin upregulation.

Stress-induced opening of the permeability transition pore in the dystrophin-deficient heart is attenuated by acute treatment with sildenafil.

Susceptibility of cardiomyocytes to stress-induced damage has been implicated in the development of cardiomyopathy in Duchenne muscular dystrophy, a disease caused by the lack of the cytoskeletal protein dystrophin in which heart failure is frequent. However, the factors underlying the disease progression are unclear and treatments are limited. Here, we tested the hypothesis of a greater susceptibility to the opening of the mitochondrial permeability transition pore (PTP) in hearts from young dystrophic (mdx) mice (before the development of overt cardiomyopathy) when subjected to a stress protocol and determined whether the prevention of a PTP opening is involved in the cardioprotective effect of sildenafil, which we have previously reported in mdx mice. Using the 2-deoxy-[(3)H]glucose method to quantify the PTP opening in ex vivo perfused hearts, we demonstrate that when compared with those of controls, the hearts from young mdx mice subjected to ischemia-reperfusion (I/R) display an excessive PTP opening as well as enhanced activation of cell death signaling, mitochondrial oxidative stress, cardiomyocyte damage, and poorer recovery of contractile function. Functional analyses in permeabilized cardiac fibers from nonischemic hearts revealed that in vitro mitochondria from mdx hearts display normal respiratory function and reactive oxygen species handling, but enhanced Ca(2+) uptake velocity and premature opening of the PTP, which may predispose to I/R-induced injury. The administration of a single dose of sildenafil to mdx mice before I/R prevented excessive PTP opening and its downstream consequences and reduced tissue Ca(2+) levels. Furthermore, mitochondrial Ca(2+) uptake velocity was reduced following sildenafil treatment. In conclusion, beyond our documentation that an increased susceptibility to the opening of the mitochondrial PTP in the mdx heart occurs well before clinical signs of overt cardiomyopathy, our results demonstrate that sildenafil, which is already administered in other pediatric populations and is reported safe and well tolerated, provides efficient protection against this deleterious event, likely by reducing cellular Ca(2+) loading and mitochondrial Ca(2+) uptake.

Autophagic flux and oxidative capacity of skeletal muscles during acute starvation

Autophagy is an important proteolytic pathway in skeletal muscles. The roles of muscle fiber type composition and oxidative capacity remain unknown in relation to autophagy. The diaphragm (DIA) is a fast-twitch muscle fiber with high oxidative capacity, the tibialis anterior (TA) muscle is a fast-twitch muscle fiber with low oxidative capacity, and the soleus muscle (SOL) is a slow-twitch muscle with high oxidative capacity. We hypothesized that oxidative capacity is a major determinant of autophagy in skeletal muscles. Following acute (24 h) starvation of adult C57/Bl6 mice, each muscle was assessed for autophagy and compared with controls. Autophagy was measured by monitoring autophagic flux following leupeptin (20 mg/kg) or colchicine (0.4 mg/kg/day) injection. Oxidative capacity was measured by monitoring citrate synthase activity. In control mice, autophagic flux values were significantly greater in the TA than in the DIA and SOL. In acutely starved mice, autophagic flux increased, most markedly in the TA, and several key autophagy-related genes were significantly induced. In both control and starved mice, there was a negative linear correlation of autophagic flux with citrate synthase activity. Starvation significantly induced AMPK phosphorylation and inhibited AKT and RPS6KB1 phosphorylation, again most markedly in the TA. Starvation induced Foxo1, Foxo3, and Foxo4 expression and attenuated the phosphorylation of their gene products. We conclude that both basal and starvation-induced autophagic flux are greater in skeletal muscles with low oxidative capacity as compared with those with high oxidative capacity and that this difference is mediated through selective activation of the AMPK pathway and inhibition of the AKT-MTOR pathways.

Role of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in denervation-induced atrophy in aged muscle: facts and hypotheses

Aging-related loss of muscle mass, a biological process named sarcopenia, contributes to mobility impairment, falls, and physical frailty, resulting in an impaired quality of life in older people. In view of the aging of our society, understanding the underlying mechanisms of sarcopenia is a major health-care imperative. Evidence obtained from human and rodent studies demonstrates that skeletal muscle denervation/reinnervation cycles occur with aging, and that progressive failure of myofiber reinnervation is a major cause of the accelerating phase of sarcopenia in advanced age. However, the mechanisms responsible for the loss of myofiber innervation with aging remain unknown. The two major strategies that counteract sarcopenia, that is, caloric restriction and endurance training, are well known to protect neuromuscular junction (NMJ) integrity, albeit through undefined mechanisms. Interestingly, both of these interventions better preserve PGC-1α expression with aging, a transcriptional coactivator which has recently been shown to regulate key proteins involved in maintaining NMJ integrity. We therefore propose that the aging-related decline in PGC-1α may be a central mechanism promoting instability of the NMJ and consequently, aging-related alterations of myofiber innervation in sarcopenia. Similarly, the promotion of PGC-1α expression by both caloric restriction and exercise training may be fundamental to their protective benefits for aging muscle by better preserving NMJ integrity.