Mitophagy Proteins Research Articles

Dietary Tributyrin Attenuates Intestinal Inflammation, Enhances Mitochondrial Function, and Induces Mitophagy in Piglets Challenged with Diquat.

The study evaluated the effects of butyric acid, in the form of tributyrin on the oxidative stress, inflammation, and mitochondrial function in diquat-challenged pigs. Twenty-four weaned pigs were allocated to four treatments in a 2 × 2 factorial arrangement with the main effects of tributyrin supplementation and diquat challenge. The results showed that supplemental tributyrin increased ( P < 0.05) average daily gain and average daily feed intake of diquat-challenged pigs. Tributyrin elevated ( P < 0.05) the activities of total antioxidant capacity and superoxide dismutase, reduced ( P < 0.05) malondialdehyde content, and increased ( P < 0.05) mRNA levels of copper and zinc superoxide dismutase and manganese-containing superoxide dismutase of diquat-challenged pigs. Tributyrin relieved ( P < 0.05) intestinal inflammation reflected by decreased mRNA abundances of tumor necrosis factor-α, interferon-γ, and interleukin-6 in the intestine. Tributyrin reduced ( P < 0.05) serum diamine oxidase activity and d-lactate content, increased ( P < 0.05) transepithelial electrical resistance, decreased paracellular flux of dextran (4 kDa), and prevented the diquat-induced decrease ( P < 0.05) in the expressions of claudin-1, occludin, and zonula occludens-1. Tributyrin alleviated ( P < 0.05) diquat-induced mitochondrial dysfunction shown by lowered reactive oxygen species, increased mitochondrial membrane potential, and increased adenosine triphosphate content. Furthermore, tributyrin increased ( P < 0.05) expressions of mitophagy proteins (PTEN-induced putative kinase 1 and Parkin), and ratio of light chain 3-II to light chain 3-I in intestine. Collectively, tributyrin attenuated oxidative stress and intestinal inflammation, improved mitochondrial function, and induced mitophagy in diquat-challenged pigs.

Intensified mitophagy in skeletal muscle with aging is downregulated by PGC-1alpha overexpression in vivo

Mitochondrial dysfunction plays an important role in the etiology of age-related muscle atrophy known as sarcopenia. PGC-1α is positioned at the center of crosstalk in regulating mitochondrial quality control, but its role in mitophagy in aged skeletal muscle is currently unclear. The present study investigated the effects of aging and PGC-1α overexpression via in vivo DNA transfection on key mitophagy protein markers, as well as mitochondrial dynamics related proteins, metabolic function and antioxidant capacity in mouse muscle. C57BL/6J mice at the age of 2 mo (young, Y; N = 14) and 24 mo (old, O; N = 14) were transfected in vivo with either PGC-1α DNA (OE, N = 7) or GFP (N = 7) into the tibialis anterior (TA) muscle followed by electroporation. PINK1 and Parkin protein contents were 3.6 and 1.4-fold higher (P < 0.01), whereas mitochondrial ubiquitination (Ub) increased 1.5-fold (P < 0.05), in O vs. Y mice. PGC-1 OE suppressed PINK and Parkin protein levels by 50–60% (P < 0.01), and decreased Ub by 20% (P < 0.05) in old mice. Aging significantly increased the protein content of LC3II (30%, P < 0.05), p62 (42%, P < 0.05), RheB (5.5-fold, P < 0.01), Beclin-1 (3-fold, P < 0.01) and Mfn2 (~4-fold, P < 0.01) in the TA muscle. However, these age-related increases in mitophagy markers were attenuated by PGC-1α OE. Furthermore, aging dramatically increased Fis-1 protein content by 14-fold (P < 0.01), along with a severe reduction of citrate synthase activity (64%, P < 0.01) and cytochrome c oxidase subunit IV (COXIV) protein content (85%, P < 0.01). PGC-1α OE mitigated the age effects on Fis-1 and Drp-1 (P < 0.05). Moreover, PGC-1α OE enhanced mitochondrial oxidative function and antioxidant enzyme activities, and decreased lipid peroxidation and inner membrane damage found in old mice (P < 0.01). In summary, our data demonstrate that mitophagy protein expression in skeletal muscle was enhanced at old age driven possibly by increased mitochondrial dysfunction, damage, and fission. PGC-1α OE was effective in ameliorating mitochondrial deficits but did not restore muscle fiber atrophy.

Abstract 1332: Autophagic clearance of protein aggregates is impaired in cancer cells with dysfunctional mitochondria

Abstract Mitophagy and aggrephagy are selective types of autophagy that degrade damaged mitochondria and protein aggregates, respectively. Dysfunctional mitochondria are known to undergo mitophagy and cause protein aggregation. How a cell can simultaneously coordinate the removal of dysfunctional mitochondria and protein aggregates via autophagy is unknown. Here, two agents, Mitoquinone (MitoQ) and MitoApocynin (MitoApo), known to induce mitochondrial dysfunction and mitophagy in MDA-MB-231 cells were used to investigate the accumulation of protein aggregates and the mechanisms that contribute to selective autophagy. Using a protein aggregate dye and a poly-ubiquitinated antibody, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), MitoQ, and MitoApo were identified as agents that caused an accumulation of poly-ubiquitinated protein aggregates in cancer cells by FACS analysis and confocal imaging. Microflow imaging of the cellular lysates confirmed a relative accumulation of large protein (&amp;gt;460 kDa) complexes. To establish the presence of aggrephagy, the aggregates were colocalized with LC3. Wild type and ATG7 knockout MEF cells were used to test whether protein aggregates accumulated in an autophagy-dependent manner. Our data currently demonstrates the kinetics of autophagic clearance of drug-induced protein aggregation and dysfunctional mitochondria. Citation Format: Thomas Biel, Ashutosh Rao. Autophagic clearance of protein aggregates is impaired in cancer cells with dysfunctional mitochondria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1332.

Loss of Insulin/IGF-1 Receptors in Muscle Coordinately Downregulates Mitochondrial Metabolism and Alters Mitophagy via Foxo Transcription Factors

Diabetes is associated with decreased muscle strength and energy production. There is a strong association between muscle insulin resistance and mitochondrial abnormalities, but cause/effect remains hotly debated. Recently, we showed that insulin receptor (IR) and IGF-1 receptor (IGF1R) signaling controls muscle atrophy by FoxO1/3/4-dependent regulation of autophagy. To directly test the roles of IR, IGF1R, and FoxO signaling on muscle metabolism and mitochondrial autophagy (mitophagy) we measured mitochondrial function in mice lacking IR (M-IR-/-), both IR/IGF1R (MIGIRKO), or IR/IGF1R/FoxO1/3/4 (QKO) in muscle. RNA seq analysis revealed decreases in multiple mitochondrial pathways from M-IR-/- and MIGIRKO muscle, including OXPHOS. Respiratory capacity and ATP production were decreased in mitochondria and soleus fibers isolated from M-IR-/- and MIGIRKO mice using complex I substrates. These decreases in OXPHOS and mitochondrial function were normalized when FoxOs were deleted in QKO mice. MIGIRKO muscle showed decreased PINK1 (mitophagy protein) and increased LC3II/I ratio, indicating changes in respiration and mitophagy occurred together. To determine if this was cell autonomous, C2C12 myotubes were pre-treated with insulin and/or CCCP (an uncoupler and mitophagy-inducer), and assayed for respiration. Insulin increased respiration and PINK1 levels after 24 hours. Four hour CCCP pre-treatment reduced basal respiration and increased LC3II/I which were partially, but significantly, rescued with insulin in C2C12 myotubes. PINK1 was also decreased in primary myotubes with acute deletion of IR/IGF1R. Thus, loss of IR/IGFR signaling in skeletal muscle directly reduces mitochondrial respiratory capacity and alters mitophagy, which is regulated by FoxO transcription factors. Disclosure G. Bhardwaj: None. C.M. Penniman: None. P.A. Suarez Beltran: None. C.M. Foster: None. B.T. O'Neill: None.

Skeletal Muscle Mitochondrial Fission Is Increased after an Overnight Lipid Infusion in Healthy Humans

The role of mitochondria in insulin resistance and type 2 diabetes (T2D) is the subject of much debate. Recent evidence has suggested the presence of altered skeletal muscle mitochondrial dynamics, both increased fission and reduced fusion, in the etiology of insulin resistance and T2D, however, it is unclear what role, if any, changes in mitochondrial dynamics play in the induction of insulin resistance. We have proposed that increased mitochondrial fission may represent an early mechanistic link between the mitochondria and the onset of skeletal muscle insulin resistance. To test this hypothesis we recruited healthy humans to receive, in a randomized crossover design, an overnight saline (control) or lipid infusion (20% Intralipid) at 0.55 ml/kg/h, followed by a 2h, 90 mg/dL, 40 mU/m2 hyperinsulinemic-euglycemic clamp to assess insulin sensitivity. Changes to mitochondrial dynamics protein expression were assessed by western blot, ex vivo mitochondrial membrane potential (TMRM staining) by confocal microscopy, oxidative capacity by respirometry (Oroboros O2k), and mitochondrial morphology by transmission electron microscopy, in skeletal muscle biopsy samples following the overnight infusions. Preliminary data (n=8; 5M, 3F; Age: 26±1 years; BMI: 22.6±0.7; VO2max: 44.0±2.5) indicate that lipid infusion results in a tendency for reduced insulin sensitivity (P=0.08) and mitochondrial membrane potential (P=0.09), and an increase in Complex I state 3 respiration (P&amp;lt;0.05). Dynamin related protein 1 (Drp1) activation (P&amp;lt;0.05) by phosphorylation at serine 616—a key step in mitochondrial fission, and the Drp1 adaptor protein MiD49 (P=0.10) were increased after lipid infusion. No changes to mitochondrial fusion (Mfn1, Mfn2, OPA1), or mitophagy (Pink1, Parkin) proteins were observed following the lipid infusion. These data support our hypothesis that increased mitochondrial fission is an early adaptation to lipid induced insulin resistance in healthy humans. Disclosure C.E. Fealy: None. C.L. Axelrod: None. M.L. Erickson: None. E. Huang: None. A. Hari: None. A. Mulya: None. H. Fujioka: None. C.L. Hoppel: None. J.P. Kirwan: None.

Effect of exercise training intensity on mitochondrial dynamics and mitophagy in post myocardial infarction rats

Myocardial infarction (MI) is the most common type of heart disease. According to recent studies, mitochondrial dysfunction has been suggested as a central player in cardiac disease and evidences point out the association of mitochondrial morphology with development of heart diseases. Exercise training plays a protective role against cardiovascular disease. However, the role of exercise training on proteins involved in mitochondrial dynamics and mitophagy system are not well understood. Therefore, the aim of the present study was to investigate these on cardiac mitochondrial dynamic and mitophagy proteins in rats with myocardial infarction. The present study was post-test design experiment with the control group. after MI with ligation of the left anterior descending coronary artery (LAD) and ensuring the creation of MI by echocardiography, male rats were subjected to high intensity interval training (HIIT), moderate (MIIT), low (LIIT), sedentary myocardial infarction (SED-MI) and healthy control groups. After six weeks exercise, the levels of MFN2, DRP1, Parkin, P62 and PGC-1α proteins were measured by ELISA method. Data analysis showed that proteins levels of MFN2, PGC-1α, Parkin and P62 decreased significantly in SED-MI group compared to healthy control while DRP1 protein levels increased significantly (P≤0.05). Also, MFN2 and PGC-1α proteins increased in MIIT group compared with SED-MI group and DRP1 protein levels were significantly decreased (P≤0.05). Moderate-intensity interval training (MIIT) resulted to improve mitochondrial fusion and fusion proteins in rats with myocardial infarction. While high and low intensity interval training (HIIT, LIIT), despite increasing MFN2 and PGC-1α and reducing DRP1, failed to improve fusion and mitochondrial fission

Parkin Mediates Mitophagy to Participate in Cardioprotection Induced by Late Exercise Preconditioning but Bnip3 Does Not.

Late exercise preconditioning (LEP) is confirmed to have a protective effect on acute cardiovascular stress. However, the mechanisms by which mitophagy participates in exercise preconditioning (EP)-induced cardioprotection remain unclear. LEP may involve mitophagy mediated by the receptors PARK2 gene-encoded E3 ubiquitin ligase (Parkin) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) to scavenge damaged mitochondria. Our EP protocol involved four 10-minute periods of running, separated by 10-minute recovery intervals, plus a period of exhaustive running at 24 hours after EP. We assessed this late protective effect by injection of the autophagy inhibitor wortmannin, transmission electron microscopy, laser scanning confocal microscopy, and other molecular biotechnology methods; we simultaneously detected related markers, analyzed the specific relationships between mitophagy proteins, and assessed mitochondrial translocation. Exhaustive exercise (EE) causes serious injuries to cardiomyofibrils, inducing hypoxia-ischemia and changing the ultrastructure. EE fails to clear excessively generated mitochondria to link with LC3 accumulation. After EP, increased autophagy levels at 30 minutes were converted to mitophagy within 24 hours. We found that LEP significantly suppressed EE-induced injuries, which we confirmed by observing decreased levels of the mitochondria-localized proteins COX4/1 and TOM20. LEP to exhaustion caused mitochondrial degradation by increasing the efficiency of LC3-outer mitochondrial membrane translocation in a Parkin-mediated manner, in which activated protein kinase and TOM70 may play both key roles. However, we did not observe mitophagy to be associated with Bnip3 mediation in LEP-induced cardioprotection. However, Bnip3 may play a role in inducing mitochondrial LC3-II increases. Wortmannin had no effect on LC3 translocation; instead, it influenced LC3-I to convert to LC3-II. Thus, suppressing mitophagy led to the attenuation of EP-induced cardioprotection.

Heavy Load Exercise Causes Mitochondrial Dysfunction and Increases Mitophagy

PURPOSE: To observe the effect of heavy load exercise on mitochondrial structure and function in skeletal muscle of rats and analyze the change of mitophagy protein of PINK1 and Parkin and related proteins, and to explore the role of PINK1/Parkin pathway in exercise-induced mitochondrial damage in skeletal muscle. METHODS: Male Sprague-Dawley rats were divided into quiet control group (C group, n=8) and exercise group (E group, n=40). Rats in the E group performed a running on a treadmill down a 16°incline at 16m/min for 90 min, and those were further divided into 0h,12h,24h,48h and 72h sub-groups (n=8), and at each time point the soleus muscle was collected under anesthesia. Mitochondrial ultrastructural changes in skeletal muscle were observed by a transmission electron microscope. The content of quatitative enzyme citrate synthase (CS) and the activities of mitochondrial respiratory chain Complex II and IV were measured by ELISA. Protein expression of skeletal muscle cytochrome c oxidease subunit I (COX I),PTEN-induced putative kinase 1 (PINK1) and mitochondrial Parkin,microtubule-associated protein 1 light chain 3 (LC3) were determined by western blot. Mitochondrial co-localization with Parkin,ubiquitin (Ub),p62/Sequestosome 1 (p62) and LC3 was measured by the immunofluorescence double labeling technique. One-way ANOVA was used to evaluate statistical significance. RESULTS: After heavy load exercise, the mitochondrial structure appeared to be abnormal and formed a lot of mitophagosomes; the CS content and Complex II activity significantly decreased, whereas the Complex IV activity and COX I protein level remained unchanged; the expression of PINK1 (E12=2.552±0.141),Parkin (E24=2.535±0.100),Ub (E24=2.501±0.191) ,p62(E12=2.662±0.240) ,LC3 (E12=2.757±0.180) significantly increased (C=1.000, P<0.05 or P<0.01). CONCLUSION: A heavy load exercise may activate the PINK1/Parkin pathway and promote the combination of Ub,p62,LC3 and mitochondria, and result in mitophagy and mitochondrial damage within skeletal muscle. Supported by Natural Science Foundation of China (31471133).

Profiling Mitochondrial Quality Control in Human Myotubes Derived from Severely Obese Non‐diabetic and Type 2 Diabetic Patients

Mitochondrial networks are maintained via integration of mitochondrial quality control processes, including biogenesis, fusion/fission dynamics and mitophagy. There is evidence to suggest that mitochondrial quality control plays a critical role in the regulation of mitochondrial function and glucose metabolism in skeletal muscle, and disruptions of these processes accompany the presence of skeletal muscle insulin resistance and type 2 diabetes (T2D). However, whether disruptions of mitochondrial quality control processes are retained at the muscle cell level is largely unknown. The purpose of this study was to determine whether the expression of proteins involved in mitochondrial quality control processes were dysregulated in primary human myotubes derived from type 2 diabetic patients. Primary human skeletal muscle cells were isolated from muscle biopsies obtained from eight severely obese, non‐diabetic subjects (BMI = 52.4 kg/m2, fasting glucose = 98.9 mg/dl) and eight severely obese, type 2 diabetics (BMI = 49 kg/m2, fasting glucose 132.3 mg/dl), and were differentiated to myotubes. On day 7 of differentiation, myotubes were harvested for immunoblot analysis to assess the expression of proteins involved in each of the four mitochondrial quality control processes. Mitochondrial fusion protein expression (Optic Atrophy Protein 1 (Opa1) and Mitofusin 1 (Mfn1)) were significantly lower in myotubes derived from type 2 diabetic patients in comparison to the BMI‐matched, non‐diabetic subjects (0.038 ± 0.003 vs. 0.024 ± 0.002 and 0.440 ± 0.042 vs. 0.320 ± 0.038, respectively, P &lt; 0.05). In addition, expression of the mitophagic protein Parkin was significantly lower in primary myotubes derived from type 2 diabetic patients (0.0028 ± 0.0003 vs. 0.0018 ± 0.0002, P &lt; 0.05). No significant differences were found in expression of other fusion or mitophagy proteins (Mitofusin 2 (Mfn2), or PTEN‐Induced Putative Kinase 1 (PINK1)), or proteins related to mitochondrial fission (dynamin related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1)), and biogenesis (PGC‐1α). These data suggest that mitochondrial quality control is dysregulated in primary myotubes derived from type 2 diabetic patients in a manner that favors fission over fusion.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Roux‐en‐Y Gastric Bypass Surgery on Mitochondrial Quality Control Proteins in Human Myotubes Derived from Severely Obese Humans

Impaired skeletal muscle glucose oxidation in response to insulin is a hallmark characteristic of humans with severe obesity and Type 2 Diabetes. Roux‐en‐Y gastric bypass (RYGB) surgery is an effective treatment of these metabolic diseases by markedly improving metabolic health, including enhanced glucose oxidation and insulin action in skeletal muscle. However, the underlying mechanisms responsible for these improvements are not fully understood. Skeletal muscle mitochondria are critical in preserving cellular health, but exhibit defective functions in severely obese humans. Recent studies have suggested that dysregulated mitochondrial dynamics are associated with mitochondrial dysfunction and insulin resistance. This study aims to determine the effects of severe obesity and RYGB surgery on mitochondrial dynamic proteins and its association with insulin sensitivity and glucose oxidation in human skeletal muscle. Human skeletal muscle cell culture model was utilized by isolating human skeletal muscle cells from muscle biopsies obtained from lean subjects (n=6, BMI = 22.7 ± 1.2 kg/m2, HOMA‐IR = 1.9 ± 0.4) and RYGB patients prior to, 1‐month and 7‐months following surgery (n=6, BMI = 47.3 ± 2.8 kg/m2, 41.0 ± 2.9 kg/m2, 33.7 ± 2.2 kg/m2, HOMA‐IR = 3.4 ± 0.4, 2.9 ± 0.9, 1.7 ± 0.2, respectively). Human skeletal muscle cells were differentiated to myotubes. On day 7 of differentiation, myotubes were harvested for immunoblot analysis in order to assess the expressions of mitochondria dynamics proteins. Myotubes derived from severely obese patients had a 35% increase in mitochondrial fusion protein Optic Atrophy 1 (Opa1) expression before surgery when compared to lean controls (p &lt;0.05). In addition, there was a trend towards a decrease of fusion proteins Opa1 and Mitofusin 1 (Mfn1) expressions in myotubes derived from the same patients at 7‐months following surgery (26% and 18%, p=0.065, respectively) in comparison to pre‐surgery state, and virtually no difference from lean controls. Furthermore, Opa1 expression negatively correlated with insulin‐stimulated glucose oxidation in myotubes (r = −0.614, p &lt;0.05), while Mfn1 is positively correlated with HOMA‐IR (r = 0.495, p &lt;0.05). Mitochondrial fission and mitophagy proteins observed no differences between lean, pre‐surgery, 1‐month and 7‐months following RYGB surgery. In conclusion, these data suggest that RYGB surgery corrected severe obesity‐induced dysregulation in mitochondrial fusion process in human myotubes, which may contribute to improved insulin sensitivity and skeletal muscle glucose oxidation in RYGB patients.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitophagy: A Mechanism for Plant Growth and Survival.

Mitophagy is a conserved cellular process that is important for autophagic removal of damaged mitochondria to maintain a healthy mitochondrial population. Mitophagy also appears to occur in plants and has roles in development, stress response, senescence, and programmed cell death. However, many of the genes that control mitophagy in yeast and animal cells are absent from plants, and no plant proteins marking defunct mitochondria for autophagic degradation are yet known. New insights implicate general autophagy-related proteins in mitophagy, affecting the senescence of plant tissues. Mitophagy control and its importance for energy metabolism, survival, signaling, and cell death in plants are discussed. Furthermore, we suggest mitochondrial membrane proteins containing ATG8-interacting motifs, which might serve as mitophagy receptor proteins in plant mitochondria.

Melatonin Balance the Autophagy and Apoptosis by Regulating UCP2 in the LPS-Induced Cardiomyopathy.

To explore the mechanism of mitochondrial uncoupling protein 2 (UCP2) mediating the protective of melatonin when septic cardiomyopathy. UCP2 knocked out mice and cardiomyocytes were used to study the effect of melatonin in response to LPS. Indicators of myocardial and mitochondria injury including mitochondrial membrane potential, mitochondrial permeability transition pore, calcium loading, ROS, and ATP detection were assessed. In addition cell viability and apoptosis as well as autophagy-associated proteins were evaluated. Melatonin was able to protect heart function from LPS, which weakened in the UCP2-knockout mice. Consistently, genipin, a pharmacologic inhibitor of UCP2, augmented LPS-induced damage of AC16 cells. In contrast, melatonin upregulated UCP2 expression and protected the cells from the changes in morphology, mitochondrial membrane potential loss, mitochondrial Ca2+ overload, the opening of mitochondrial permeability transition pore, and subsequent increased ROS generation as well as ATP reduction. Mitophagy proteins (Beclin-1 and LC-3β) were increased while apoptosis-associated proteins (cytochrome C and caspase-3) were decreased when UCP2 was up-regulated. In conclusion, UCP2 may play a protecting role against LPS by regulating the balance between autophagy and apoptosis of cardiomyocytes, and by which mechanisms, it may contribute to homeostasis of cardiac function and cardiomyocytes activity. Melatonin may protect cardiomyocytes through modulating UCP2.

Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells.

Although deletion of certain autophagy-related genes has been associated with defects in hematopoiesis, it remains unclear whether hyperactivated mitophagy affects the maintenance and differentiation of hematopoietic stem cells (HSCs) and committed progenitor cells. Here we report that targeted deletion of the gene encoding the AAA+-ATPase Atad3a hyperactivated mitophagy in mouse hematopoietic cells. Affected mice showed reduced survival, severely decreased bone-marrow cellularity, erythroid anemia and B cell lymphopenia. Those phenotypes were associated with skewed differentiation of stem and progenitor cells and an enlarged HSC pool. Mechanistically, Atad3a interacted with the mitochondrial channel components Tom40 and Tim23 and served as a bridging factor to facilitate appropriate transportation and processing of the mitophagy protein Pink1. Loss of Atad3a caused accumulation of Pink1 and activated mitophagy. Notably, deletion of Pink1 in Atad3a-deficient mice significantly 'rescued' the mitophagy defect, which resulted in restoration of the progenitor and HSC pools. Our data indicate that Atad3a suppresses Pink1-dependent mitophagy and thereby serves a key role in hematopoietic homeostasis.

Skeletal muscle autophagy and mitophagy in endurance-trained runners before and after a high-fat meal

ObjectiveWe tested the hypothesis that skeletal muscle of endurance-trained male runners would exhibit elevated autophagy and mitophagy markers, which would be associated with greater metabolic flexibility following a high-fat meal (HFM). MethodsMuscle biopsies were collected to determine differences in autophagy and mitophagy protein markers and metabolic flexibility under fasting conditions and 4 h following a HFM between endurance-trained male runners (n = 10) and sedentary, non-obese controls (n = 9). ResultsMaximal oxygen consumption (ml·kg·min−1) was approximately 50% higher (p < 0.05) in endurance-trained runners compared with sedentary controls (65.8 ± 2.3 and 43.1 ± 3.4, respectively). Autophagy markers were similar between groups. Mitophagy and mitochondrial dynamics protein markers were significantly higher in skeletal muscle of endurance-trained runners compared with sedentary controls in the fasted state, although unaffected by the HFM. Skeletal muscle metabolic flexibility was similar between groups when fasted (p > 0.05), but increased in response to the HFM in endurance-trained athletes only (p < 0.005). Key mitophagy markers, phospho-Pink1Thr257 and phospho-ParkinS65 (r = 0.64, p < 0.005), and phospo-ParkinSer65 and phospho-Drp1Ser616 (r = 0.70, p < 0.05) were correlated only within the endurance-trained group. Autophagy and mitophagy markers were not correlated with metabolic flexibility. ConclusionIn summary, mitophagy may be enhanced in endurance-trained runners based on elevated markers of mitophagy and mitochondrial dynamics. The HFM did not alter autophagy or mitophagy in either group. The absence of a relationship between mitophagy markers and metabolic flexibility suggests that mitophagy is not a key determinant of metabolic flexibility in a healthy population, but further investigation is warranted.

RETRACTED ARTICLE: A Chinese herbal formula, Jian-Pi-Yi-Shen decoction, improves muscle atrophy via regulating mitochondrial quality control process in 5/6 nephrectomised rats

Muscle atrophy is one of the serious complications of chronic kidney disease (CKD). Dysregulation of mitochondrial quality control (MQC) process, including decrease mitochondrial biogenesis, impair mitochondrial dynamics and induce activation of mitophagy, play an important role in mediating muscle wasting. This study aimed to observe effects of Jian-Pi-Yi-Shen (JPYS) decoction on muscle atrophy in CKD rats and explore its possible mechanism on regulation of MQC processes. The 5/6 nephrectomised rats were randomly allocated into 2 groups: CKD group and JPYS group. Besides, a sham-operated rats as sham group. All rats were treated for 6 weeks. Results showed that administration of JPYS decoction prevented body weight loss, muscle loss, muscle fiber size decrease, muscle protein degradation, and increased muscle protein systhesis. In addition, JPYS decoction increased the mitochondrial content and biogenesis proteins, and down-regulated the autophagy and mitophagy proteins. Furthermore, JPYS decoction increased mitochondrial fusion proteins, while decreased mitochondrial fission proteins. In conclusion, JPYS decoction increased mitochondrial content and biogenesis, restore the balance between fission and fusion, and inhibited autophagy-lysosome pathway (mitophagy). Collectively, our data showed that JPYS decoction to be beneficial to muscle atrophy in CKD, which might be associated with the modulation of MQC process.

Increase in proteins involved in mitochondrial fission, mitophagy, proteolysis and antioxidant response in type I endometrial cancer as an adaptive response to respiratory complex I deficiency

Pathogenic mtDNA mutations associated with alterations of respiratory complex I, mitochondrial proliferation (oncocytic-like phenotype) and increase in antioxidant response were previously reported in type I endometrial carcinoma (EC).To evaluate whether in the presence of pathogenic mtDNA mutations other mitochondrial adaptive processes are triggered by cancer cells, the expression level of proteins involved in mitochondrial dynamics, mitophagy, proteolysis and apoptosis were evaluated in type I ECs harboring pathogenic mtDNA mutations and complex I deficiency. An increase in the fission protein Drp1, in the mitophagy protein BNIP3, in the mitochondrial protease CLPP, in the antioxidant and anti-apoptotic protein ALR and in Bcl-2 as well as a decrease in the fusion protein Mfn2 were found in cancer compared to matched non malignant tissue. Moreover, the level of these proteins was measured in type I EC, in hyperplastic (the premalignant form) and in non malignant tissues to verify whether the altered expression of these proteins is a common feature of endometrial cancer and of hyperplastic tissue. This analysis confirmed in type I EC samples, but not in hyperplasia, an alteration of the expression level of these proteins. These results suggest that in this cancer mitochondrial fission, antioxidant and anti-apoptotic response may be activated, as well as the discharge of damaged mitochondrial proteins as adaptation processes to mitochondrial dysfunction.

Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations.

Objective:To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy controls.Methods:Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes.Results:Fibroblasts from 3 biallelic OPA1(−/−) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)–depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA–treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts.Conclusions:We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion.

454 - Alveolar Cell Mitophagy in Staphylococcus Aureus Pneumonia in Mice

The lung’s antimicrobial defenses involve many innate and adaptive immune responses, but highly necrotizing (oxidizing) pathogenic bacteria like Staphylococcus aureus ( SA ) are controlled by incompletely understood mechanisms. A redox-regulated response to pneumonia involves mitophagy, or the removal of damaged mitochondria from cells that opposes inflammasome formation. In order to identify and localize mitophagy in the lung after SA infection, we administered 1×10 8 CFU of SA by nasal inoculation to C57BL/6 mice and harvested lungs at days 0, 1, 2, 3 for bronchoalveolar lavage (BAL), Western blot analysis, and qRT-PCR. Mitophagy and mitophagy proteins were localized by confocal immunofluorescence. In C57BL/6 mice, pneumonia-induced pulmonary inflammasome activation was detected by increases in IL-1β and NLRP3 protein levels at 1 day along with increased BAL cell count and protein. Overall lung resistance increased and lung compliance decreased post-infection. The LC3-II/LC3I ratio increased, and there was loss of p62, a redox-regulated scaffolding protein of the autophagosome. Those findings supported that SA infection promoted mitochondrial clearance in lung cells. Moreover, SA infection caused an increase in PTEN-induced kinase 1 (PINK1) and Parkin. Confocal microscopy showed increased PINK1 and Parkin with fragmented mitochondria in alveolar type 2 cells and alveolar macrophages. Because mitochondrial clearance is highly regulated and intimately linked with mitochondrial fission and fusion proteins, we checked key proteins Mff, its ligand Drp1, and Mfn2 and found an increase in the former at day 1 and the latter by day 2-3 post SA inoculation. Furthermore, alveolar mitophagy appeared to depend mainly on activation of the non-canonical mitophagy pathway and correlated with decreases in levels of TLR2 and 9 in alveolar macrophage, suggesting mitophagy as a protective mechanism in macrophages by playing a role in preventing over-responses to TLR2 and 9 ligands perhaps by lowering mitochondrial reactive oxygen species production. Thus, we propose activation of mitophagy in the alveolar region as a critical resolution mechanism for pulmonary inflammation caused by SA infection.

ERK2 regulation of mitophagy is important for T cell activation

Abstract Critical signals necessary for T cell activation are transduced into the cell, in part, by the MAPK pathway, for which extracellular regulated kinase 2 (ERK2) is a key member. Upon activation, changes to mitochondria location and metabolism mediate T cell priming; however the signaling pathways that modulate mitochondria in T cells are largely unknown. A primary mechanism regulating mitochondria is a form of selective autophagy termed mitophagy. Importantly, ERK2 is required for mitophagy in other cell types and has been shown to co-localize at the autophagosome during formation. These data suggest that ERK2 may have a unique role regulating mitophagy in T cells. Notably, dysregulation of mitochondria in T cells can result in impaired immunity and has also been associated with autoimmune disease. Here, we investigated the role of ERK2 for mitophagy in CD4+ T cells using a mouse model with T cell-specific deletion of ERK2. Our results show that Erk2−/− CD4+ T cells have impaired autophagosome formation and increased cell death upon activation, a phenotype consistent with mitophagy blockade. Further, we are working to identify key mitophagy proteins that are regulated by ERK2 and to characterize mitochondria signaling metabolites during T cell activation. By elucidating the signaling pathways that connect extracellular immune stimuli with mitochondria regulation, we will obtain important information that may lead to new therapeutic targets for T cell mediated diseases and dysfunction.

Parkin‐Mediated Mitophagy in Skeletal Muscle with Aging and Exercise

In skeletal muscle, the progressive age‐related loss of muscle mass and strength is known as sarcopenia. Alterations in skeletal muscle mitochondria are considered to be a contributing factor in this process. When mitochondria are no longer able to sustain the energetic requirements of the cell, selective autophagy (mitophagy) pathways are induced to promote the clearance of damaged mitochondria. In aged muscle, dysfunctional mitochondria generate increased amounts of reactive oxygen species (ROS) and can impair quality control mechanisms that facilitate their removal. It remains unclear if mitophagy mediates this reduced response in aged muscle. Parkin is an ubiquitin ligase involved in mitophagy, but little is known about its function with aging. To evaluate mitophagic signaling in aged muscle, young (3 months) and aged (18 months) Parkin deficient (KO) mice and wild‐type (WT) animals were assigned to three groups: control, acute exercise, or acute exercise plus 2 hours of recovery. Muscle mass was reduced by 46% and 65% respectively in aged Parkin KO and WT mice when compared to their young counterparts. Furthermore, aged WT animals displayed a 25% decrease in whole muscle cytochrome c oxidase activity. This was accompanied by a 40% reduction in State 3 (active) intermyofibrillar mitochondrial respiration in aged WT animals. In response to exercise, both aged Parkin KO and WT mice exhibited ~30% declines in running performance, and increased lactic acidosis by 2.1‐ and 1.4‐fold, respectively. Interestingly, basal mitophagy flux and subsarcolemmal mitochondrial localization of LC3II, p62 and ubiquitin were augmented in both young and aged Parkin KO muscle. Acute exercise increased mitochondrial flux in young animals, but reduced flux in older muscle. Our findings indicate that the expression of mitophagy proteins and their localization to mitochondria are not decreased in aged muscle, even in the absence of Parkin. However, attenuation of exercise‐induced mitophagy in response to aging may contribute to mitochondrial accumulation and dysfunction in aged muscle.Support or Funding InformationSupported by NSERC.