Chronic hypobaric hypoxia mediated skeletal muscle atrophy: role of ubiquitin–proteasome pathway and calpains

The most frequently reported symptom of exposure to high altitude is loss of body mass and decreased performance which has been attributed to altered protein metabolism affecting skeletal muscles mass. The present study explores the mechanism of chronic hypobaric hypoxia mediated skeletal muscle wasting by evaluating changes in protein turnover and various proteolytic pathways. Male Sprague-Dawley rats weighing about 200 g were exposed to hypobaric hypoxia (7,620 m) for different durations of exposure. Physical performance of rats was measured by treadmill running experiments. Protein synthesis, protein degradation rates were determined by (14)C-Leucine incorporation and tyrosine release, respectively. Chymotrypsin-like enzyme activity of the ubiquitin-proteasome pathway and calpains were studied fluorimetrically as well as using western blots. Declined physical performance by more than 20%, in terms of time taken in exhaustion on treadmill, following chronic hypobaric hypoxia was observed. Compared to 1.5-fold increase in protein synthesis, the increase in protein degradation was much higher (five-folds), which consequently resulted in skeletal muscle mass loss. Myofibrillar protein level declined from 46.79 ± 1.49 mg/g tissue at sea level to 37.36 ± 1.153 (P < 0.05) at high altitude. However, the reduction in sarcoplasmic proteins was less as compared to myofibrillar protein. Upregulation of Ub-proteasome pathway (five-fold over control) and calpains (three-fold) has been found to be important factors for the enhanced protein degradation rate. The study provided strong evidences suggesting that elevated protein turnover rate lead to skeletal muscle atrophy under chronic hypobaric hypoxia via ubiquitin-proteasome pathway and calpains.     

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway

The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.     

Role of ubiquitin‐proteasome‐dependent proteolytic process in degradation of muscle protein from diabetic rabbits

The activity of ATP, ubiquitin (Ub)dependent proteases partially purified from skeletal muscle (psoas) from alloxan diabetic rabbits was determined at different periods of insulin deficiency. Two days after alloxan injection, no change was observed in the activity of ATP, Ubdependent proteases, but this activity increased 3 and 5 days after diabetes induction, attaining 181% of control values on the 5th day. However, after this early rise, the activity of muscle ATP, Ubdependent proteases decreased, returning to values that did not differ significantly from controls 7 and 10 days after alloxan injection. After 15 days, the activity of these proteases was 57% lower than in muscle from control rabbits. Both the initial increase and the subsequent fall in the activity of the enzymes were prevented by insulin treatment of alloxan diabetic rabbits. The data suggest that Ubproteasomedependent proteolysis have an important role in the control of muscle protein degradation and may be regulated by insulin.     

Acute treatment with tumour necrosis factor-α induces changes in protein metabolism in rat skeletal muscle

Acute treatment of rats with recombinant tumour necrosis factor (TNF-) caused an enhanced proteolytic rate —measured as tyrosine released in the presence of cycloheximide — insoleus muscle (34%). The cytokine treatment also decreased the rate of protein synthesis in this muscle (22%) while it had no effect upon the same parameter inextensor digitorum longus (EDL) (26%) muscle. In addition, treatment of rats with TNF- increased amino acid uptake by transport system A in the incubated muscles both insoleus (45%) andEDL (99%) in the presence of insulin in the incubating medium. This effect was not associated with a direct action of TNF on muscle since the addition of different concentrations of the cytokine to the preparations did not alter the uptake of -(methyl)-aminoisobutyric acid by the incubated muscles. It can be concluded that acute TNF- treatment causes changes in protein metabolism in red-type muscles — suchsoleus — while little effects are seen in white-type muscles — such as EDL. The results presented may, to some extent, be related to the cachectic response associated with cancer and inflammation.     

The role of PGC-1α in the regulation of skeletal muscle metabolism

The purpose of this review was to provide an understanding of the role of PGC-1α in the regulation of skeletal muscle metabolism and to describe the results of studies on the association of the polymorphism gene PPARGC1A with human muscle performance.     

Synergistical effect of 20-HETE and high salt on NKCC2 protein and blood pressure via ubiquitin–proteasome pathway

We previously generated a cytochrome P450 4F2 (CYP4F2) transgenic mouse model and demonstrated that overexpressed CYP4F2 and overproduced 20-HETE in the kidneys contribute to the increase of blood pressure in the CYP4F2 transgenic mice with normal salt intake. We currently expect to elucidate a potential mechanism of salt-related hypertension whereby diverse levels of 20-HETE interact with dietary salt on Na⁺-K⁺-2Cl^? cotransporter, isoform 2 (NKCC2) in the kidneys of the transgenic and wild-type mice with high salt intake. High salt intake reduced about 85 % abundance of renal NKCC2 protein in the transgenic mice and about 24 % in the wild-type mice by Western blot. Furthermore, we first found that NKCC2 was ubiquitinated and interacted with Nedd4-2 by immunoprecipitation in the transgenic mice with high salt intake. In addition, inhibition of 20-HETE synthesis or proteasome activity reversed the reduction of NKCC2 expression induced by 20-HETE and high salt intake. These results suggest that 20-HETE and high salt intake synergistically decrease the expression of NKCC2 protein via Nedd4-2-mediated ubiquitin–proteasome pathway, and thereby modulate natriuresis and blood pressure. We propose that diverse levels of 20-HETE have diverse effects on blood pressure in different salt concentrations.     

Different patterns of protein turnover in skeletal and gastrointestinal smooth muscle and the production of Nτ-methylhistidine during fasting in the rat

During four days of fasting in rats skeletal muscle protein synthesis fell pro-gressively, whereas skeletal muscle protein breakdown was unchanged until the third and fourth days when it rose dramatically. In contrast, the synthetic rate of smooth muscle protein was unchanged during three days of fasting despite a loss of protein content, indicating an abrupt rise in protein breakdown in this tissue on the first day of fasting which was sustained thereafter. Urinary excretion of N-methylhistidine was significantly increased throughout fasting. The concentration of free N-methylhistidine in plasma and in muscle tissue was elevated throughout the period of fasting. This elevation was not caused by reduced renal clearance, but appears to have been mainly the result of increased breakdown of N-methylhistidine-containing proteins in tissues other than skeletal muscle.     

Chicken heat shock protein HSPB1 increases and interacts with αB-crystallin in aged skeletal muscle

International trading markets of meat require the animal’s age information to prevent cross-contamination of ineligible meat products. Individual livestock age is either evaluated from physiological features or verified by breeding history. However, it remains impossible to perform age verification on meat when a suspicion of error occurred in the importing country. To investigate an age-related protein in skeletal muscle of livestock, we compared protein expression among chicken pectoralis major of different ages. Results indicated that the level of expression of chicken HSPB1, one of the small heat shock proteins, was increased in aged muscles. On the other hand, other heat shock proteins, heat shock factors, and myosin heavy chain isoform did not change the expression levels in aged chicken muscle. In addition, we identified that αB-crystallin interacted with HSPB1 in aged chicken muscle. These results suggest that HSPB1 protein forms complexes with αB-crystallin in aged chicken muscle and suppose to become the candidate of age-related bio-marker for verifying the age of chicken meat.     

Calcineurin B subunit interacts with proteasome subunit alpha type 7 and represses hypoxia-inducible factor-1α activity via the proteasome pathway

The calcineurin (CN) B subunit (CNB) is the regulatory subunit of CN, which is the only serine/threonine-specific protein phosphatase regulated by Ca²⁺/CaM. It has been shown to have potential as an anticancer agent, and has a positive effect on the phagocytic index and coefficient. We report here that CNB binds to proteasome subunit alpha type 7 (PSMA7) and inhibits the transactivation activity of hypoxia-inducible factor-1α (HIF-1α) via the proteasome pathway. In addition, we show that CNB represses the expression of vascular endothelial growth factor (VEGF), which is regulated by HIF-1α. These results indicate that CNB modulates cellular proteasome activity via a specific interaction with PSMA7. This may provide a molecular basis for its anticancer and antiviral activities.     

Immunological detection of m- and μ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca²⁺-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15^th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.Key words: m-calpain, µ-calpain, skeletal muscle, Marchigiana cattle, immunohistochemistry, Electron Microscopy.     

Different skeletal effects of the peroxisome proliferator activated receptor (PPAR)α agonist fenofibrate and the PPARγ agonist pioglitazone

Background

All the peroxisome proliferator activated receptors (PPARs) are found to be expressed in bone cells. The PPARγ agonist rosiglitazone has been shown to decrease bone mass in mice and thiazolidinediones (TZDs) have recently been found to increase bone loss and fracture risk in humans treated for type 2 diabetes mellitus. The aim of the study was to examine the effect of the PPARα agonist fenofibrate (FENO) and the PPARγ agonist pioglitazone (PIO) on bone in intact female rats.

Methods

Rats were given methylcellulose (vehicle), fenofibrate or pioglitazone (35 mg/kg body weight/day) by gavage for 4 months. BMC, BMD, and body composition were measured by DXA. Histomorphometry and biomechanical testing of excised femurs were performed. Effects of the compounds on bone cells were studied.

Results

The FENO group had higher femoral BMD and smaller medullary area at the distal femur; while trabecular bone volume was similar to controls. Whole body BMD, BMC, and trabecular bone volume were lower, while medullary area was increased in PIO rats compared to controls. Ultimate bending moment and energy absorption of the femoral shafts were reduced in the PIO group, while similar to controls in the FENO group. Plasma osteocalcin was higher in the FENO group than in the other groups. FENO stimulated proliferation and differentiation of, and OPG release from, the preosteoblast cell line MC3T3-E1.

Conclusion

We show opposite skeletal effects of PPARα and γ agonists in intact female rats. FENO resulted in significantly higher femoral BMD and lower medullary area, while PIO induced bone loss and impairment of the mechanical strength. This represents a novel effect of PPARα activation.     

Cancer–testis antigen HCA587/MAGE-C2 interacts with BS69 and promotes its degradation in the ubiquitin–proteasome pathway

HCA587, also known as MAGE-C2, belonging to the MAGE gene family which is characterized by a conserved MAGE Homology Domain, is active in various types of tumors and silent in normal tissues except in male germ-line cells. The biological function of HCA587 is largely unknown. To analyze it, we attempted to identify protein partners of HCA587. We immunopurified HCA587-containing complex from HEK293 cells and identified BS69, a potential tumor suppressor, as an associated protein by mass spectrometry, and the following Immunoprecipitation and GST pull-down assays confirmed HCA587 interaction with BS69. Interestingly, overexpression of HCA587 promoted ubiquitination and the proteasomal degradation of BS69 whereas knockdown of endogenous HCA587 increased the protein level of BS69. Consistent with a functional role for BS69 in negatively regulating LMP1-induced NF-κB activation, overexpression of HCA587 resulted in a significant enhancement of LMP1-induced IL-6 production. These data indicate that HCA587 is a new negative regulator of BS69.     

Coordinate induction of PPARα and SREBP2 in multifunctional protein 2 deficient mice

Mice with inactivation of the D-specific multifunctional protein 2 (MFP2), a crucial enzyme of peroxisomal β-oxidation, develop multiple pathologies in diverse tissues already starting in the postnatal period. Gene expression profiling performed on liver of 2-day-old pups revealed up-regulation of PPARα responsive genes in knockout mice. Surprisingly, also genes involved in cholesterol biosynthesis were markedly induced. Real-time PCR confirmed the induction of PPARα target genes and of HMGCR and SREBP2, both involved in cholesterol synthesis, in lactating and in adult MFP2 knockout mice. In accordance, the rate of cholesterol biosynthesis was significantly increased in liver of knockout mice but the hepatic cholesterol concentration was unaltered. In MFP2/PPARα double knockout mice, up-regulations of SREBP2 and HMGCR were markedly attenuated. These data demonstrate a tight interrelationship between induction of PPARα by endogenous ligands and up-regulation of genes of cholesterol biosynthesis through increased expression of SREBP2.     

Ectopic overexpression of swine PPARγ2 upregulated adipocyte genes expression and triacylglycerol in skeletal muscle of mice

Peroxisome proliferator-activated receptor ??2 (PPAR??2) is a key regulator of adipocyte differentiation, fatty acid uptake and storage in mammals. The primary goal of the present study was to investigate the consequences of PPAR??2 overexpression in the muscle. A swine muscle creatine kinase promoter was used to drive swine PPAR??2 (sPPAR??2) overexpression in the muscle of a transgenic mice model. The results showed that the mRNA of multiple adipocyte genes was increased in the skeletal muscle, as evidenced by the up-regulation of fatty acid synthase (2.11-fold, P?<?0.05), lipoprotein lipase (2.08-fold, P?<?0.01), fatty acid-binding protein 4 (14.30-fold, P?<?0.01), and CD36 antigen (5.50-fold, P?<?0.01). Meanwhile, skeletal muscle triacylglycerol was increased (P?<?0.01) and the fatty acid profile of muscle fat was changed in that more polyunsaturated fats acid were augmented. The present study may further serve to develop transgenic pigs with higher intramuscular fat content and improved pork quality.     

Exercise training changes IL-10/TNF-α ratio in the skeletal muscle of post-MI rats

Heart failure (HF) is associated with changes in the skeletal muscle (SM) which might be a consequence of the unbalanced local expression of pro- (TNF-α) and anti- (IL-10) inflammatory cytokines, leading to inflammation-induced myopathy, and SM wasting. This local effect of HF on SM may, on the other hand, contribute to systemic inflammation, as this tissue actively secretes cytokines. Since increasing evidence points out to an anti-inflammatory effect of exercise training, the goal of the present study was to investigate its effect in rats with HF after post-myocardial infarction (MI), with special regard to the expression of TNF-α and IL-10 in the soleus and extensor digitorum longus (EDL), muscles with different fiber composition. Wistar rats underwent left thoracotomy with ligation of the left coronary artery, and were randomly assigned to either a sedentary (Sham-operated and MI sedentary) or trained (Sham-operated and MI trained) group. Animals in the trained groups ran on a treadmill (0% grade at 13–20 m/min) for 60 min/day, 5 days/week, for 8–10 weeks. The training protocol was able to reverse the changes induced by MI, decreasing TNF-α protein (26%, P < 0.05) and mRNA (58%, P < 0.05) levels in the soleus, when compared with the sedentary MI group. Training also increased soleus IL-10 expression (2.6-fold, P < 0.001) in post-MI HF rats. As a consequence, the IL-10/TNF-α ratio was increased. This “anti-inflammatory effect” was more pronounced in the soleus than in the EDL, suggesting a fiber composition dependent response.     

Correlation of individual skeletal muscle fibres from “semithin” sections stained with p-phenylene-diamine and histochemical sections incubated for myofibrillar ATP-ase

Summary Muscle fibres in transverse semithin sections of osmium-fixed and plastic embedded muscle tissue stained with p-phenylene-diamine has been classified into three types (M, INT and H) and correlated to the same fibres in neighboring sequential preincubated myofibrillar ATP-ase sections. A close correlation, but no equality was found between the fibre types classified according to the ATP-ase and the p-phenylene-diamine methods. On the average, the content of mitochondria and fat droplets was highest in the ATP-ase classified type I fibres (dominated by M fibres), and higher in the endurance trained than in the untrained subjects.