Single bout of running exercise changes LC3-II expression in rat cardiac muscle

Macroautophagy (autophagy) is an intracellular catalytic process. We examined the effect of running exercise, which stimulates cardiac work physiologically, on the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, an indicator of autophagy, as well as some autophagy-related proteins in rat cardiac muscle. The left ventricles were taken from rats immediately (0 h), and at 0.5 h, 1 h or 3 h after a single bout of running exercise on a treadmill for 30 min and also from rats in a rest condition. In these samples, we evaluated the level of LC3-II and p62, and the phosphorylation level of mammalian target of rapamycin (mTOR), Akt and AMP-activated protein kinase alpha (AMPKα) by Western blotting. The exercise produced a biphasic change in LC3-II, with an initial decrease observed immediately after the exercise and a subsequent increase 1 h thereafter. LC3-II then returned to the rest level at 3 h after the exercise. A negative correlation was found between the LC3-II expression and mTOR phosphorylation, which plays a role in inhibiting autophagy. The exercise increased phosphorylation of AMPKα, which stimulates autophagy via suppression of mTOR phosphorylation, immediately after exercise. The level of p62 and phosphorylated Akt was not altered significantly by the exercise. These results suggest for the first time that a single bout of running exercise induces a biphasic change in autophagy in the cardiac muscle. The exercise-induced change in autophagy might be partially mediated by mTOR in the cardiac muscle.     

Autophagic Signaling and Proteolytic Enzyme Activity in Cardiac and Skeletal Muscle of Spontaneously Hypertensive Rats following Chronic Aerobic Exercise

Hypertension is a cardiovascular disease associated with deleterious effects in skeletal and cardiac muscle. Autophagy is a degradative process essential to muscle health. Acute exercise can alter autophagic signaling. Therefore, we aimed to characterize the effects of chronic endurance exercise on autophagy in skeletal and cardiac muscle of normotensive and hypertensive rats. Male Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were assigned to a sedentary condition or 6 weeks of treadmill running. White gastrocnemius (WG) of hypertensive rats had higher (p<0.05) caspase-3 and proteasome activity, as well as elevated calpain activity. In addition, skeletal muscle of hypertensive animals had elevated (p<0.05) ATG7 and LC3I protein, LAMP2 mRNA, and cathepsin activity, indicative of enhanced autophagic signaling. Interestingly, chronic exercise training increased (p<0.05) Beclin-1, LC3, and p62 mRNA as well as proteasome activity, but reduced (p<0.05) Beclin-1 and ATG7 protein, as well as decreased (p<0.05) caspase-3, calpain, and cathepsin activity. Left ventricle (LV) of hypertensive rats had reduced (p<0.05) AMPKα and LC3II protein, as well as elevated (p<0.05) p-AKT, p-p70S6K, LC3I and p62 protein, which collectively suggest reduced autophagic signaling. Exercise training had little effect on autophagy-related signaling factors in LV; however, exercise training increased (p<0.05) proteasome activity but reduced (p<0.05) caspase-3 and calpain activity. Our results suggest that autophagic signaling is altered in skeletal and cardiac muscle of hypertensive animals. Regular aerobic exercise can effectively alter the proteolytic environment in both cardiac and skeletal muscle, as well as influence several autophagy-related factors in skeletal muscle of normotensive and hypertensive rats.     

Autophagic response to exercise training in skeletal muscle with age

Autophagy, a highly conserved quality control mechanism, is essential for the maintenance of cellular homeostasis and for the orchestration of an efficient cellular response to stress. During aging, the efficiency of autophagic degradation declines, and intracellular waste products accumulate. Therefore, in this study, we tested the hypothesis that skeletal muscle from old mice would have decreased autophagosome formation when compared to the muscle from young mice. We also examined whether autophagic regulatory events differ between muscle fiber types and in response to exercise in aged male mice. The extensor digitorum longus (EDL) and gastrocnemius muscles were studied in young and old ICR mice. Exercise was performed by allowing the mice to run on a treadmill with a 5° incline at 16.4 m/min for 40 min/day, 5 days/week for 8 weeks after a 1-week adaptation period. Our results indicated that the levels of microtubule-associated protein 1b light chain 3, a marker of autophagosome formation, were lower in both the EDL and the gastrocnemius muscle of old mice compared to those young mice. To identify the factors related to the changes observed, the expression of autophagy regulatory proteins was examined in the EDL and gastrocnemius muscles. Beclin-1, autophagy-related gene 7 (ATG7), and lysosome-associated membrane protein were found to be lower in the EDL and gastrocnemius muscles of old mice compared to those in the young mice, then Beclin-1, ATG7, and muscle-specific RING finger protein-1 upregulated after regular exercise. Moreover, the muscle weight/body weight was significantly increased only in the gastrocnemius muscle of the old trained mice. These data suggest that autophagy regulatory events are attenuated in old skeletal muscle. However, this effect is upregulated when animals are subjected to exercise training.     

小鼠缺血性脑损伤模型急性期海马SIRT3与自噬相关蛋白表达水平的相关性分析

摘要 目的：观察小鼠缺血性脑损伤模型急性期海马区域沉默信息因子3(SIRT3)和自噬相关蛋白的表达,并探讨两者的相关性。方法：选择C57BL/6J小鼠,采用大脑中动脉阻塞(MCAO)法建立缺血性脑损伤模型,并将小鼠随机分为假手术组(sham)和模型组(MCAO)。缺血性脑损伤急性期(6h),采用间接免疫荧光法观察小鼠海马CA1、CA3和DG区域SIRT3的表达,应用蛋白印迹法检测SIRT3、自噬相关蛋白LC3 I/II和Beclin-1的表达,而后用Spearman相关性分析明确SIRT3和LC3-II、Beclin-1表达的相关性。结果：海马各区域SIRT3阳性细胞数量在损伤后明显增多(P<0.05),且SIRT3蛋白表达也相对上调(P<0.05);损伤后自噬相关蛋白LC3-II和Beclin-1表达亦增高(P<0.05);Spearman相关性分析发现SIRT3与自噬相关蛋白LC3-II、Beclin-1表达均呈显著正相关(P<0.05)。结论：小鼠缺血性脑损伤模型急性期海马区域SIRT3和自噬相关蛋白的表达具有显著相关性,SIRT3对海马区域自噬的调节可能有重要作用。     

Regional differences in expression of VEGF mRNA in rat gastrocnemius following 1 hr exercise or electrical stimulation

Background  

Vascular endothelial growth factor (VEGF) mRNA levels increase in rat skeletal muscle after a single bout of acute exercise. We assessed regional differences in VEGF₁₆₅ mRNA levels in rat gastrocnemius muscle using in situ hybridization after inducing upregulation of VEGF by treadmill running (1 hr) or electrical stimulation (1 hr). Muscle functional regions were defined as oxidative (primarily oxidative fibers, I and IIa), or glycolytic (entirely IIb or IId/x fibers). Functional regions were visualized on muscle cross sections that were matched in series to slides processed through in situ hybridization with a VEGF₁₆₅ probe. A greater upregulation in oxidative regions was hypothesized.     

Decreased expression of Beclin-1 and LC3 in human lung cancer

Autophagy, a conversed response to stress, has recently been studied in human cancers. Two important autophagic genes—Beclin-1 and LC3 are reported in several human cancers. However, the expressions of Beclin-1 and LC3 in lung cancer have not yet been investigated. In the present study, we investigated the expression of Beclin-1 and LC3, and the relationship between the expression profile and the clinical or pathological changes in human lung cancer. 40 primary lung cancer patients are involved in present study. mRNA expressions of Beclin-1 and LC3-II were detected by Real Time PCR and the protein levels were assessed by immunohistochemistry and western blot. Relative lower expressions of Beclin-1 and LC3-II mRNA were found in the lung cancer tissues compared to counterpart normal tissues. Consistently, the lower amount of Beclin-1 and LC3-II protein was found in lung cancer tissues. However, the expressions of Beclin-1 and LC3-II in lung cancer tissues were not affected by patients’ age, gender, smoking, histological type, lymph node metastasis and tumor-node-metastasis (TNM) stage. Both mRNA and protein levels of Beclin-1 and LC3-II were significantly decreased in lung cancer tissues which suggested that autophagy may be involved in the pathogenesis of lung cancer.     

Med19 is involved in chemoresistance by mediating autophagy through HMGB1 in breast cancer

Adriamycin (ADM)-based regimens are the most effective chemotherapeutic treatments for breast cancer. However, intrinsic and acquired chemoresistance is a major therapeutic problem. Our goal was to clarify the role of mediator complex subunit 19 (Med19) in chemotherapy resistance and to elucidate the related molecular mechanisms. In this study, ADM-resistant human cells (MCF-7/ADM) and tissues exhibited increased Med19 expression and autophagy levels relative to the corresponding control groups. Additionally, MCF-7/ADM cells showed changes in two selective markers of autophagy. There was a dose-dependent increase in the light chain 3 (LC3)-II/LC3-I ratio and a decrease in sequestosome 1 (P62/SQSTMl) expression. Furthermore, lentivirus-mediated Med19 inhibition significantly attenuated the LC3-II/LC3-I ratio, autophagy-related gene 3 (Atg3) and autophagy-related gene 5 (Atg5) expression, P62 degradation, and red fluorescent protein-LC3 dot formation after treatment with ADM or rapamycin, an autophagy activator. Furthermore, the antiproliferative effects of ADM, cisplatin (DDP), and taxol (TAX) were significantly enhanced after suppressing Med19 expression. Notably, the effects of Med19 on autophagy were mediated through the high-mobility group box-1 (HMGB1) pathway. Our findings suggest that Med19 suppression increased ADM chemosensitivity by downregulating autophagy through the inhibition of HMGB1 signaling in human breast cancer cells. Thus, the regulatory mechanisms of Med19 in autophagy should be investigated to reduce tumor resistance to chemotherapy.     

The promoting role of lysosome-localized c-Src in autophagosome-lysosome fusion

Src-family kinases (SFKs), such as c-Src, Lyn and Fyn, belong to non-receptor-type tyrosine kinases and play key roles in cell proliferation, adhesion, and migration. SFKs are anchored to the plasma membrane, Golgi membranes and lysosomal membranes through lipid modifications. Although the functions of SFKs being localized to the plasma membrane are intensively studied, those of SFKs being localized to organelle membranes are poorly understood. Here, we show that, among SFKs, c-Src in particular is involved in a decrease in the amount of LC3-II. c-Src and non-palmitoylated Lyn [Lyn(C3S) (cysteine-3 → serine-3)], which are localized onto lysosomes, decrease the amount of LC3-II and treatment with SFK inhibitors increases the amount of LC3-II, suggesting the importance of SFKs' lysosomal localization for a change of autophagic flux in a kinase activity-dependent manner. Colocalization of LC3-II with the lysosome-associated membrane protein LAMP1 shows that lysosome-localized SFKs promote the fusion of autophagosomes with lysosomes. Lysosome-localized SFKs play a positive role in the maintenance of cell viability under starvation conditions, which is further supported by knockdown of c-Src. Therefore, our results suggest that autophagosome-lysosome fusion is promoted by lysosome-localized c-Src, leading to cell survival under starvation conditions.     

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca^2 + properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca^2 + release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca^2 + properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca^2 + anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.     

Autophagy is decreased in mesenteric fat tissue but not in intestinal mucosae of patients with Crohn’s disease

Crohn??s disease (CD) is a chronic intestinal disease with a multifactorial etiology. Recently, a role for mesenteric fat has been proposed in CD pathophysiology, since fat hypertrophy is detected close to the affected intestinal area; however, there are few studies regarding autophagy and the hypertrophied mesenteric tissue in CD. To evaluate autophagy-related proteins in intestinal mucosae and mesenteric fat of patients with CD and controls, patients with ileocecal CD (CD Group) and with non-inflammatory disease (FC Group) selected for surgery were studied. Expression of LC3-II was determined by immunoblotting of protein extracts. In addition, beclin-1, LC3 and Atg16-L1 RNA levels were measured using RT-PCR. The expression of LC3-II was significantly lower in the mesenteric tissue and higher in intestinal mucosae of CD when compared to controls. However, mRNA expression of autophagy-related proteins was similar when comparing the mesenteric fat groups. These findings suggest a defect in autophagy activation in the mesenteric fat tissue of CD individuals, which could be involved in the maintenance of the inflammatory process.     

Long-term Effects of Recurrent Neonatal Seizures on Neurobehavioral Function and Related Gene Expression and Its Intervention by Inhibitor of Cathepsin B

Cathepsins are families of proteases that have been reported to play the key roles in neuroexcitotoxicity. The present study was sought to determine the effect of CBI, a cathepsin B inhibitor, in the prevention of neurobehavioral deficits after inhalant flurothyl-induced recurrent neonatal seizures in rats. We examined the expression pattern of autophagy-related genes at acute phase after the last seizures using western blot method, and evaluated behavioral deficits during postnatal day 28 (P28) to P35. The results showed improved neurological scores and learning ability in CBI-treated rats compared with the nontreated control. Flurothyl-induced increases in the ratio of LC3-II/LC3-I, Beclin-1 and Cathepsin-B were blocked by pre-treatment with CBI at 1.5, 3, 6 and 24 h after the last seizures in hippocampus and cerebral cortex by western blot analysis. Meanwhile, CBI also reversed flurothyl-induced down-regulation of Bcl-2 protein levels. Furthermore, in the long-term time point of 35 days (P35), PRG-1 mRNA and protein level in hippocampus and cerebral cortex of recurrent seizure group were up-regulated when compared to the control rats; meanwhile, the up-regulated expression of PRG-1 were robustly inhibited by CBI. These date demonstrated, for the first time, that lysosomal enzymes participate in neonatal seizure-induced brain damage and that modulation of cathepsin B may offer a new strategy for the development of therapeutic interventions for treatment of developmental seizure-induced brain damage.     

Effect of treadmill exercise on PI3K/AKT/mTOR,autophagy, and Tau hyperphosphorylation in the cerebral cortex of NSE/htau23 transgenic mice

Purpose

Neurofibrillary tangles, one of pathological features of Alzheimer’s disease, are produced by the hyperphosphorylation and aggregation of tau protein. This study aimed to investigate the effects of treadmill exercise on PI3K/AKT/mTOR signal transmission, autophagy, and cognitive ability that are involved in the hyperphosphorylation and aggregation of tau protein.

Methods

Experimental animals (NSE/htau23 mice) were divided into non-transgenic control group (Non-Tg-Control; CON; n = 7), transgenic control group (Tg-CON; n = 7), and transgenic exercise group (Tg-Treadmill Exercise; TE; n = 7). The Tg-TE group was subjected to treadmill exercise for 12 weeks. After the treadmill exercise was completed, the cognitive ability was determined by conducting underwater maze tests. Western blot was conducted to determine the phosphorylation status of PI3K/AKT/mTOR proteins and autophagy-related proteins (Beclin-1, p62, LC3-B); hyperphosphorylation and aggregation of tau protein (Ser199/202, Ser404, Thr231, PHF-1); and phosphorylation of GSK-3β, which is involved in the phosphorylation of tau protein in the cerebral cortex of experimental animals.

Results

In the Tg-TE group that was subjected to treadmill exercise for 12 weeks, abnormal mTOR phosphorylation of PI3K/AKT proteins was improved via increased phosphorylation and its activity was inhibited by increased GSK-3β phosphorylation compared with those in the Tg-CON group, which was used as the control group. In addition, the expression of Beclin-1 protein involved in autophagosome formation was increased in the Tg-TE group compared with that in the Tg-CON group, whereas that of p62 protein was reduced in the Tg-TE group compared with that in the Tg-CON group. Autophagy was activated owing to the increased expression of LC3-B that controls the completion of autophagosome formation. The hyperphosphorylation and aggregation (Ser199/202, Ser404, Thr231, PHF-1) of tau protein was found to be reduced in the Tg-TE group compared with that in the Tg-CON group. Furthermore, in the underwater maze test, the Tg-TE group showed a reduced escape time and distance compared with those of the Tg-CON group, suggesting that learning and cognitive ability were improved.

Conclusion

These findings suggest that aerobic exercise such as treadmill exercise might be an effective approach to ameliorate the pathological features (or neurofibrillary tangles) of Alzheimer’s disease.     

WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.     

Inhibition of autophagy in the heart induces age-related cardiomyopathy

Constitutive autophagy is important for control of the quality of proteins and organelles to maintain cell function. Damaged proteins and organelles accumulate in aged organs. We have previously reported that cardiac-specific Atg5 (autophagy-related gene 5)-deficient mice, in which the gene was floxed out early in embryogenesis, were born normally, and showed normal cardiac function and structure up to 10 weeks old. In the present study, to determine the longer-term consequences of Atg5-deficiency in the heart, we monitored cardiac-specific Atg5-deficient mice for further 12 months. First, we examined the age-associated changes of autophagy in the wild-type mouse heart. The level of autophagy, as indicated by decreased LC3-II (microtubule-associated protein 1 light chain 3-II) levels, in the hearts of 6-, 14- or 26-month-old mice was lower than that of 10-week-old mice. Next, we investigated the cardiac function and life-span in cardiac-specific Atg5-deficient mice. The Atg5-deficient mice began to die after the age of 6 months. Atg5-deficient mice exhibited a significant increase in left ventricular dimension and decrease in fractional shortening of the left ventricle at the age of 10 months, compared to control mice, while they showed similar chamber size and contractile function at the age of 3 months. Ultrastructural analysis revealed a disorganized sarcomere structure and collapsed mitochondria in 3- and 10-month-old Atg5-deficient mice, with decreased mitochondrial respiratory functions. These results suggest that continuous constitutive autophagy has a crucial role in maintaining cardiac structure and function.     

Induction of amino acid transporters expression by endurance exercise in rat skeletal muscle

We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28 m/min for 90 min. Immediately after the exercise, we observed that expression of mRNAs encoding l-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles.     

The attenuation of Th1 and Th17 responses via autophagy protects against methicillin-resistant Staphylococcus aureus-induced sepsis

Whether autophagy affects methicillin-resistant Staphylococcus aureus (MRSA)-induced sepsis and the associated mechanisms are largely unknown. This study investigated the role of autophagy in MRSA-induced sepsis. The levels of microtubule-associated protein light chain 3 (LC3)-II/I, Beclin-1 and p62 after USA300 infection were examined by Western blotting and immunohistochemical staining. Bacterial burden analysis, hematoxylin-eosin staining, and Kaplan–Meier analysis were performed to evaluate the effect of autophagy on MRSA-induced sepsis. IFN-γ and IL-17 were analyzed by ELISA, and CD4⁺ T cell differentiation was assessed by flow cytometry. Our results showed that LC3-II/I and Beclin-1 were increased, while p62 was decreased after infection. Survival rates were decreased in the LC3B^−/− and Beclin-1^+/− groups, accompanied by worsened organ injuries and increased IFN-γ and IL-17 levels, whereas rapamycin alleviated organ damage, decreased IFN-γ and IL-17 levels, and improved the survival rate. However, there was no significant difference in bacterial burden. Flow cytometric analysis showed that rapamycin treatment decreased the frequencies of Th1 and Th17 cells, whereas these cells were upregulated in the LC3B^−/− and Beclin-1^+/− groups. Therefore, autophagy plays a protective role in MRSA-induced sepsis, which may be partly associated with the alleviation of organ injuries via the downregulation of Th1 and Th17 responses. These results provide a nonantibiotic treatment strategy for sepsis.     

Response of mitochondrial fusion and fission protein gene expression to exercise in rat skeletal muscle

The purpose of this study was to investigate the changes in the gene expression of Mitofusion (Mfn) 1 and 2 and Fission 1 (Fis1) and mitochondrial energy metabolism in response to altered energy demand during prolonged exercise in rat skeletal muscle. Male Sprague–Dawley rats were subjected to an acute bout of treadmill running at various durations and killed immediately or during recovery. Mfn1/2 and Fis1 mRNA and protein contents, reactive oxygen species (ROS) generation, state 3 and state 4 respiration rates, trans-innermembrane potential and ATP synthase activity were measured in isolated muscle mitochondria. We found that (1) Mfn1/2 mRNA contents were progressively decreased during 150 min of exercise, along with decreased Mfn 1 protein levels. Fis1 mRNA and protein contents showed significant increases after 120–150 min of exercise. These changes persisted through the recovery period up to 24 h. (2) Mitochondrial ROS generation and state 4 respiration showed progressive increases up to 120 min, but dropped at 150 min of exercise. (3) State 3 respiration rate and respiratory control index were unchanged initially but decreased at 150 and 120 min of exercise, respectively, whereas ATP synthase activity was elevated at 45 min and returned to resting level thereafter. Our data suggested that the gene expression of mitochondrial fusion and fission proteins in skeletal muscle can respond rapidly to increased metabolic demand during prolonged exercise, which could significantly affect the efficiency of oxidative phosphorylation.     

Echovirus 7 Entry into Polarized Caco-2 Intestinal Epithelial Cells Involves Core Components of the Autophagy Machinery

Echovirus 7 enters polarized Caco-2 intestinal epithelial cells by a clathrin-mediated endocytic process and then moves through the endosomal system before releasing its genome into the cytoplasm. We examined the possible role in virus entry of core components of the autophagy machinery. We found that depletion of Beclin-1, Atg12, Atg14, Atg16, or LC3 with specific small interfering RNAs inhibited echovirus 7 infection upstream of uncoating but had little or no effect on virus attachment to the cell surface. These data indicate that multiple autophagy-related proteins are important for one or more events that occur after the virus has bound its receptor on the cell surface but before RNA is released from the virus capsid. Although we have not determined the mechanism by which each protein contributes to virus entry, we found that stable depletion of Atg16L1 interfered with virus internalization from the cell surface rather than with intracellular trafficking. Autophagy gene products may thus participate in the endocytic process that moves virus into polarized Caco-2 cells.