Protective role of Parkin in skeletal muscle contractile and mitochondrial function.

KEY POINTS: Parkin, an E3 ubiquitin ligase encoded by the Park2 gene, has been implicated in the regulation of mitophagy, a quality control process in which defective mitochondria are degraded. The exact physiological significance of Parkin in regulating mitochondrial function and contractility in skeletal muscle remains largely unexplored. Using Park2-/- mice, we show that Parkin ablation causes a decrease in muscle specific force, a severe decrease in mitochondrial respiration, mitochondrial uncoupling and an increased susceptibility to opening of the permeability transition pore. These results demonstrate that Parkin plays a protective role in the maintenance of normal mitochondrial and contractile functions in skeletal muscles. ABSTRACT: Parkin is an E3 ubiquitin ligase encoded by the Park2 gene. Parkin has been implicated in the regulation of mitophagy, a quality control process in which defective mitochondria are sequestered in autophagosomes and delivered to lysosomes for degradation. Although Parkin has been mainly studied for its implication in neuronal degeneration in Parkinson disease, its role in other tissues remains largely unknown. In the present study, we investigated the skeletal muscles of Park2 knockout (Park2-/- ) mice to test the hypothesis that Parkin plays a physiological role in mitochondrial quality control in normal skeletal muscle, a tissue highly reliant on mitochondrial content and function. We first show that the tibialis anterior (TA) of Park2-/- mice display a slight but significant decrease in its specific force. Park2-/- muscles also show a trend for type IIB fibre hypertrophy without alteration in muscle fibre type proportion. Compared to Park2+/+ muscles, the mitochondrial function of Park2-/- skeletal muscles was significantly impaired, as indicated by the significant decrease in ADP-stimulated mitochondrial respiratory rates, uncoupling, reduced activities of respiratory chain complexes containing mitochondrial DNA (mtDNA)-encoded subunits and increased susceptibility to opening of the permeability transition pore. Muscles of Park2-/- mice also displayed a decrease in the content of the mitochondrial pro-fusion protein Mfn2 and an increase in the pro-fission protein Drp1 suggesting an increase in mitochondrial fragmentation. Finally, Park2 ablation resulted in an increase in basal autophagic flux in skeletal muscles. Overall, the results of the present study demonstrate that Parkin plays a protective role in the maintenance of normal mitochondrial and contractile functions in normal skeletal muscles.

Vaginally Administered Danazol: An Overlooked Option in the Treatment of Rectovaginal Endometriosis?

Danazol has been used in the treatment of endometriosis and heavy menstrual bleeding for more than 40 years. This medication has both central antigonadotropic actions and direct atrophic effects on endometriotic tissue. Although it demonstrates a high-efficacy profile, the associated side effects have resulted in limited usage. Vaginal administration of the drug may prove favourable specifically in rectovaginal endometriosis. This targeted mode of delivery is associated with a significant reduction in both pain symptoms and nodule size. The relative persistence of these therapeutic benefits is likely related to the direct tissue effects after absorption through the vaginal mucosa. Vaginal administration would also limit systemic propagation of danazol and thus should minimize androgenic side effects. Use of vaginal danazol also improves heavy menstrual bleeding and may even restore fertility in some patients. In this review we provide a critical analysis of the existing literature on the use of vaginal danazol.

Le danazol est utilisé dans la prise en charge de l'endométriose et des saignements menstruels abondants depuis plus de 40 ans. Ce médicament exerce tant des effets antigonadotropes centraux que des effets atrophiques directs sur le tissu endométriotique. Bien qu'il présente un profil solide d'efficacité, les effets indésirables qui lui sont associés en ont limité l'utilisation. L'administration de ce médicament par voie vaginale pourrait s'avérer favorable, particulièrement dans les cas d'endométriose rectovaginale. Ce mode d'administration ciblée est associé à une atténuation significative des symptômes de douleur et de la taille des nodules. La persistance relative de ces avantages thérapeutiques est probablement associée aux effets tissulaires directs qui sont constatés à la suite de l'absorption au travers de la muqueuse vaginale. L'administration par voie vaginale permettrait également de limiter la propagation générale du danazol, ce qui devrait en minimiser les effets indésirables androgéniques. L'utilisation de danazol par voie vaginale entraîne également une atténuation des saignements menstruels abondants et pourrait même restaurer la fertilité chez certaines patientes. Dans le cadre de cet article, nous offrons une analyse critique de la littérature existante sur l'utilisation du danazol par voie vaginale.

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-ß-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.

Cost-effectiveness of atrial fibrillation screening in Canadian community practice.

Background: Contemporary guidelines recommend opportunistic screening for atrial fibrillation (AF). Objective: The objective of this study was to assess the cost-effectiveness of single time point opportunistic AF screening for patients 65 years and older by using the single-lead electrocardiogram. Methods: An established Markov cohort model was adapted by updating the background mortality estimates, epidemiology, screening efficacy, treatment patterns, resource use, and cost inputs to reflect a Canadian health care setting. Inputs were derived from a contemporary prospective screening study performed in Canadian primary care settings (screening efficacy and epidemiology) and the published literature (unit costs, epidemiology, mortality, utility, and treatment efficacy). The impact of screening and oral anticoagulant treatment on the cost and clinical outcomes was analyzed. A Canadian payer perspective over lifetime was used for analysis, with costs expressed in 2019 Canadian dollars. Results: Among the estimated screening-eligible population of 2,929,301 patients, the screening cohort identified an additional 127,670 AF cases compared with the usual care cohort. The model estimated avoidance of 12,236 strokes and incremental quality-adjusted life-years of 59,577 (0.02 per patient) over lifetime in the screening cohort. Cost savings were substantial because of improved health outcomes, reflecting screening being the dominant strategy (affordable and effective). Model results were robust across sensitivity and scenario analyses. Conclusion: Single time point opportunistic screening of AF using a single-lead electrocardiogram device in Canadian patients 65 years and older without known AF may provide improved health outcomes with cost savings from the perspective of a single payer health care environment.

Peroxisome proliferator-activated receptor γ coactivator1- gene α transfer restores mitochondrial biomass and improves mitochondrial calcium handling in post-necrotic mdx mouse skeletal muscle.

Alterations of mitochondrial function have been implicated in the pathogenesis of Duchenne muscular dystrophy. In the present study, mitochondrial respiratory function, reactive oxygen species (ROS) dynamics and susceptibility to Ca(2+)-induced permeability transition pore (PTP) opening were investigated in permeabilized skeletal muscle fibres of 6-week-old mdx mice, in order to characterize the magnitude and nature of mitochondrial dysfunction at an early post-necrotic stage of the disease. Short-term overexpression of the transcriptional co-activator PGC1α, achieved by in vivo plasmid transfection, was then performed to determine whether this intervention could prevent mitochondrial impairment and mitigate associated biochemical abnormalities. Compared with normal mice, mdx mice exhibited a lower mitochondrial biomass and oxidative capacity, greater ROS buffering capabilities, and an increased vulnerability to Ca(2+)-induced opening of the mitochondrial permeability transition pore complex. PGC1α gene transfer restored mitochondrial biomass, normalized the susceptibility to PTP opening and increased the capacity of mitochondria to buffer Ca(2+)(.) This was associated with reductions in the activity levels of the Ca(2+)-dependent protease calpain as well as caspases 3 and 9. Overall, these results suggest that overexpression of PGC1α in dystrophin-deficient muscles, after the onset of necrosis, has direct beneficial effects upon multiple aspects of mitochondrial function, which may in turn mitigate the activation of proteolytic and apoptotic signalling pathways associated with disease progression.

Cyclophilin-D is dispensable for atrophy and mitochondrial apoptotic signalling in denervated muscle.

In the present study, we specifically determined whether the regulatory protein cyclophilin-D (CypD), and by extension opening of the permeability transition pore (PTP), is involved in the activation of mitochondria-derived apoptotic signalling previously described in skeletal muscle following loss of innervation. For this purpose, CypD-defficient (CypD-KO) mice and their littermate controls were submitted to unilateral sciatic nerve transection, and mitochondrial resistance to Ca2+-induced opening of the PTP, and muscle apoptotic signalling were investigated 14 days post-surgery. Denervation caused atrophy, facilitated Ca2+-induced opening of the PTP in vitro in permeabilized muscle fibres, and activation of the apoptotic proteolytic cascade in the whole muscle of both mouse strains. In CypD-KO mice, mitochondrial resistance to Ca2+-induced PTP opening was greater than in WT mice, in both the normal and the denervated state, indicating that lack of CypD desensitized to PTP opening. However, denervation in CypD-KO mice still resulted in a facilitation of PTP opening compared to normally innervated contralateral muscle, indicating that in vitro additional factors could poise mitochondria from denervated muscle toward PTP opening. At the whole muscle level, lack of CypD, despite conferring greater resistance to PTP opening, did not protect against atrophy, release of mitochondrial pro-apoptotic factors and activation of caspases following denervation. Altogether, these results provide direct evidence that CypD-dependent PTP opening is dispensable for atrophy and apoptotic signalling in skeletal muscle following denervation.

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.

Dose specific effectiveness and safety of DOACs in patients with non-valvular atrial fibrillation: A Canadian retrospective cohort study.

BACKGROUND: Direct oral anticoagulants (DOACs) have been proven to be effective and safe for prevention of ischemic stroke and systemic embolism in patients with non-valvular atrial fibrillation (NVAF). However, suboptimal adherence, variable dosing and use in patient populations that otherwise would have been excluded from clinical trials may impact the efficacy and safety profile of DOACs in a routine care setting. We compared stroke, bleeding, and mortality rates on and off therapy for standard and low-dose DOACs (apixaban, rivaroxaban, dabigatran) versus warfarin in a Canadian cohort. We also assessed persistence of DOACs compared to warfarin. METHODS: We conducted six 1-1 propensity-score matched retrospective cohort analyses using Quebec health administrative databases (2011-2017). NVAF patients (≥18 years) covered by the public medication insurance plan entered the cohort on the first OAC dispensation date. We excluded those with OAC use in the previous year or stroke or bleeding diagnoses in the previous two years. Follow-up ended at death, March 2017 or end of medication coverage by the public plan. Time-dependent Cox regression was applied. RESULTS: We evaluated 10,893 patients initiated on apixaban (7206 standard, 3687 low-dose), 10,190 on rivaroxaban (7396 standard, 2794 low-dose), 5884 on dabigatran (2756 standard, 3128 low-dose), and propensity score-matched warfarin users. Across standard-dose DOACs, compared to warfarin, stroke risks were similar; bleeding risks were lower with apixaban (hazard ratio 0.63; 95% confidence interval 0.52-0.77) and dabigatran (0.47; 0.35-0.64) but not rivaroxaban (0.93; 0.79-1.10); death risks were lower with all DOACs. For low-dose DOACs, rivaroxaban demonstrated higher stroke (1.79; 1.21-2.64) and bleeding risks (1.37; 1.09-1.73); other agents had stroke risks similar to warfarin and bleeding risks lower than warfarin; only low-dose dabigatran had lower death risk (0.59; 0.52-0.68). Treatment discontinuation was lower with DOACs versus warfarin with the exception of low-dose rivaroxaban. The risks of stroke were 2-4 folds higher during time off any OAC versus time on warfarin. The risks of death were higher, while the risks of bleeding were generally lower during times off any OAC. CONCLUSIONS: Standard-dose DOACs had similar stroke, better persistence and mortality profiles than warfarin. Only standard dose apixaban and dabigatran had better bleeding profiles than warfarin. Low-dose rivaroxaban had worse persistence, stroke and bleeding profiles than warfarin, while low-dose apixaban and dabigatran had similar stroke and better bleeding profiles. Real-world use of DOACs may explain some of the differences observed in Canadian routine care versus the phase III clinical trials.

Detection of atrial fibrillation in asymptomatic at-risk individuals.

BACKGROUND: Undiagnosed atrial fibrillation (AF) exposes unsuspecting patients to elevated stroke risks. The optimal algorithm for identifying patients who should be screened for AF remains undetermined. The objective of this study is to determine the AF burden in an asymptomatic, at-risk population. We also sought to investigate potential predictors of undiagnosed AF. METHODS: This registry is a prospective observational study assessing continuous ECG monitoring in screening for AF using a wearable single lead 7-day continuous monitoring device. Patients included were asymptomatic individuals, at risk for AF as determined by either 1) ≥65 years of age with ≥1 high risk factor or; 2) ≥75 years of age and ≥2 moderate risk factors. A multivariable logistic regression was used to explore the predictive value of certain patient characteristics in identifying patients susceptible to have undiagnosed AF. RESULTS: Among the 942 patients included, 25 patients (2.7%) had evidence of AF detected. Only 8 patients had AF duration ≥24 h. History of perioperative AF (OR: 3.25, 95%CI: 1.08-9.79, p = 0.036), age over 85 (OR: 4.71, 95%CI: 1.31-16.92, p = 0.017) and absence of cardiovascular disease (CVD) (OR: 0.27, 95%CI: 0.10-0.76, p = 0.013) were found to be predictive of undiagnosed AF. CONCLUSION: This study demonstrates the feasibility of office-based AF screening in at-risk population. The low rate of AF detection suggests that the optimal algorithm for identifying asymptomatic patients who would benefit from continuous screening remains unclear. Advanced age, history of perioperative AF and absence of CVD are variables that could be explored further.

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.

Screening for Atrial Fibrillation Using a Mobile, Single-Lead Electrocardiogram in Canadian Primary Care Clinics.

BACKGROUND: Atrial fibrillation (AF), which affects 1% to 4% of the general population, is an important risk factor for stroke. New technologies for AF screening are now available, but their real-world application in clinical settings has not yet been thoroughly investigated. The aim of this project was to describe the introduction and feasibility of a mobile electrocardiogram (ECG) device for AF screening in a large-scale, undifferentiated population. METHODS: A total of 184 Canadian primary care physicians were provided with a KardiaMobile ECG device (AliveCor) for 3 months. Physicians were asked to obtain a single 30-second ECG recording of all patients seen in their daily practice who were ≥65 years old and not previously diagnosed with AF. Evaluation of the Kardia device by physicians was measured using a Likert scale-based questionnaire. RESULTS: One hundred thirty-three physicians (72%) reported their findings and completed the survey. A total of 7585 patients were screened (42% of eligible patients). AF was detected in 471 patients (6.2%). Anticoagulation therapy was initiated in 270 patients (57%). Physicians generally reported a high perceived clinical value (94%) and ease of integration (89%) of the device. CONCLUSIONS: Previously undiagnosed AF is common in older individuals attending primary care clinics. The KardiaMobile ECG device appears to be an effective screening tool for AF with high physician acceptability. More research on the feasibility of such novel technologies is warranted for future consideration of integration in population-based screening programs.

An opinion on the benefits of concomitant oral contraceptive therapy in premenopausal women treated with oral anticoagulants.

Women who are receiving an oral anticoagulant appear to be at higher risk of developing bleeding-related adverse events than men. Physiological bleeding related to the ovulatory cycle poses an ongoing risk for bleeding complications during anticoagulant therapy. Abnormal uterine bleeding and hemorrhagic ovarian cysts are risks specific to women of reproductive age who are treated with anticoagulants. The use of combined oral contraceptives can help minimize such adverse events and would also mitigate the risks of obstetrical complications related to thrombosis and anticoagulation, in addition to avoiding fetal exposure to potentially teratogenic anticoagulants. Clinicians tend to interrupt oral contraceptives in women who are receiving oral anticoagulants despite the absence of evidence that they increase thrombotic risks when taken concurrently. This letter of opinion aims to support the use of oral contraceptives, when clinically indicated, in women of reproductive age who also are receiving an oral anticoagulant.

Abnormal uterine bleeding in women receiving direct oral anticoagulants for the treatment of venous thromboembolism.

Abnormal uterine bleeding (AUB) is a common complication of anticoagulant therapy in premenopausal women affected with acute venous thromboembolism. AUB impacts quality of life, and can lead to premature cessation of anticoagulation. There is increasing data to suggest that the direct oral anticoagulants when used for the treatment of venous thromboembolism differ in their menstrual bleeding profile. This article aims to review the existing literature regarding the association between AUB and the direct oral anticoagulants and make practical recommendations.

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.

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.

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.

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.