Maintenance of muscle mass and load‐induced growth in Muscle RING Finger 1 null mice with age

Age‐related loss of muscle mass occurs to varying degrees in all individuals and has a detrimental effect on morbidity and mortality. Muscle RING Finger 1 (MuRF1), a muscle‐specific E3 ubiquitin ligase, is believed to mediate muscle atrophy through the ubiquitin proteasome system (UPS). Deletion of MuRF1 (KO) in mice attenuates the loss of muscle mass following denervation, disuse, and glucocorticoid treatment; however, its role in age‐related muscle loss is unknown. In this study, skeletal muscle from male wild‐type (WT) and MuRF1 KO mice was studied up to the age of 24 months. Muscle mass and fiber cross‐sectional area decreased significantly with age in WT, but not in KO mice. In aged WT muscle, significant decreases in proteasome activities, especially 20S and 26S β5 (20–40% decrease), were measured and were associated with significant increases in the maladaptive endoplasmic reticulum (ER) stress marker, CHOP. Conversely, in aged MuRF1 KO mice, 20S or 26S β5 proteasome activity was maintained or decreased to a lesser extent than in WT mice, and no increase in CHOP expression was measured. Examination of the growth response of older (18 months) mice to functional overload revealed that old WT mice had significantly less growth relative to young mice (1.37‐ vs. 1.83‐fold), whereas old MuRF1 KO mice had a normal growth response (1.74‐ vs. 1.90‐fold). These data collectively suggest that with age, MuRF1 plays an important role in the control of skeletal muscle mass and growth capacity through the regulation of cellular stress.     

Expression of subunits of the 19S complex and of the PA28 activator in rat skeletal muscle

A precise knowledge of the role of subunits of the 19S complex and the PA28 regulator, which associate with the 20S proteasome and regulate its peptidase activities, may contribute to design new therapeutic approaches for preventing muscle wasting in human diseases. The proteasome is mainly responsible for the muscle wasting of tumor-bearing and unweighted rats. The expression of some ATPase (MSS1, P45) and non ATPase (P112-L, P31) subunits of the 19S complex, and of the two subunits of the PA28 regulator, was studied in such atrophying muscles. The mRNA levels for all studied subunits increased in unweighted rats, and analysis of MSS1 mRNA distribution profile in polyribosomes showed that this subunit entered active translation. By contrast, only the mRNA levels for MSS1 increased in the muscles from cancer rats. Thus, gene expression of the proteasome regulatory subunits depends on a given catabolic state. Torbafylline, a xanthine derivative which inhibits tumor necrosis factor production, prevented the activation of protein breakdown and the increased expression of 20S proteasome subunits in cancer rats, without reducing the elevated MSS1 mRNA levels. Thus, the increased expression of MSS1 is regulated independently of 20S proteasome subunits, and did not result in accelerated proteolysis.     

Identification of the Immunoproteasome as a Novel Regulator of Skeletal Muscle Differentiation

While many of the molecular details of myogenesis have been investigated extensively, the function of immunoproteasomes (i-proteasomes) in myogenic differentiation remains unknown. We show here that the mRNA of i-proteasome subunits, the protein levels of constitutive and inducible proteasome subunits, and the proteolytic activities of the 20S and 26S proteasomes were significantly upregulated during differentiation of skeletal muscle C2C12 cells. Knockdown of the i-proteasome catalytic subunit PSMB9 by short hairpin RNA (shRNA) decreased the expression of both PSMB9 and PSMB8 without affecting other catalytic subunits of the proteasome. PSMB9 knockdown and the use of i-proteasome-specific inhibitors both decreased 26S proteasome activities and prevented C2C12 differentiation. Inhibition of the i-proteasome also impaired human skeletal myoblast differentiation. Suppression of the i-proteasome increased protein oxidation, and these oxidized proteins were found to be more susceptible to degradation by exogenous i-proteasomes. Downregulation of the i-proteasome also increased proapoptotic proteins, including Bax, as well as cleaved caspase 3, cleaved caspase 9, and cleaved poly(ADP-ribose) polymerase (PARP), suggesting that impaired differentiation is likely to occur because of significantly increased apoptosis. These results demonstrate for the first time that i-proteasomes, independent of constitutive proteasomes, are critical for skeletal muscle differentiation of mouse C2C12 cells.     

Alteration of 20S proteasome-subtypes and proteasome activator PA28 in skeletal muscle of rat after induction of diabetes mellitus

Insulin-dependent diabetes mellitus is known to go along with enhanced muscle protein breakdown. Since evidence has been presented that the ubiquitin-proteasome system is significantly involved in muscle wasting under this condition, we have investigated, whether this biological role goes along with alterations of the proteasome system in skeletal muscle of streptozotocin-diabetic rats. Previously, we have found a drop of overall proteasome activity in muscle extracts of rats after induction of diabetes but no change in total amount of 20S proteasome was detected. In the present investigation under the same diabetic conditions we have measured a significant decrease in the amount of proteasome activator PA28, a finding that explains the loss of total proteasome activity. Since increased mRNA levels of proteasome subunits have been measured in muscle tissue of rats after induction of diabetes, we have isolated and purified 20S proteasomes from muscle tissue of control and 6 days diabetic rats. The specific chymotrypsin-like, trypsin-like, and peptidylglutamylpeptide-hydrolysing activities of proteasomes from diabetic and control rats were found to be not significantly different. Therefore, we have fractionated 20S proteasomes into their subtypes and detected that induction of diabetes mellitus effects a redistribution of subtypes of all three proteasome populations but only the increase in subtype V (immuno-subtype) was statistically significant. This altered subtype pattern obviously meets the requirements to the system under wasting conditions. Since this process goes along with de novo biogenesis of 20S proteasomes, it most likely explains the phenomenon of elevated mRNA concentrations of proteasome subunits after induction of diabetes mellitus.     

The 20S/26S proteasomal pathway of protein degradation in muscle tissue

Similar to all other eukaryotic cells and tissues muscle tissue contains the proteolytic system of 20S/26S proteasomes with the 20S proteasome existing predominantly in a latent state. Unlike with the mammalian enzymein vitro transition from the latent to the activated state of the 20S proteasomes isolated from muscle of several fish species and from lobster can be achieved by heat shock. It is very likely that the activated state of the 20S proteasome corresponds to the physiologically active form of the enzyme since only that one is able to attack sarcoplasmic and myofibrillar proteins to any significant extent. As perfusion of rat hindquarters with presumptive low molecular mass activators like free fatty acids does not result in an activation of the muscle proteasome other — possibly protein activators — may serve this purposein vivo. The 26S proteasome complex may be regarded as such a proteasome/activator complex. The 26S proteasome complex has the ability to degrade protein (-ubiquitin-conjugates) by an ATP-consuming reaction. Since increased amounts of ubiquitinated proteins as well as an enhanced activity of the ATP (-ubiquitin)-dependent proteolytic system have been measured in rat muscle tissue during various catabolic conditions, it is not unlikely that this pathway is responsible for catalysis of muscle protein breakdown.Abbreviations Bz benzoyl - PGPH peptidylglutamylpeptide hydrolysing - Suc succinyl - Z benzyloxycarbonyl     

Sex-dependent Antioxidant Enzyme Activities and Lipid Peroxidation in Ageing Mouse Brain

We investigated whether oxidant status and antioxidant enzyme activities during ageing of mouse brain are regulated in sex-dependent manner. In the homogenate from the brain of 1, 4, 10 and 18 months old male and female CBA mice, lipid peroxidation (LPO), total superoxide dismutase (tSOD), catalase (CAT) and glutathione peroxidase (Gpx) were determined. LPO was age- and sex-related, favoring males over females throughout the lifespan with the peak in both sexes at 10 months of age. Throughout ageing, no difference in tSOD activity between male and female brains was observed, except in immature 1 month old mice. Gender-related difference in Gpx activity was observed, with higher level in females comparing to males, reaching statistical significance in senescent (18 months old) animals. CAT activity was drastically changed with ageing in both the male and female brain. We found different age associated trends in CAT activity in males and females: decreased with age in males and increased with age in females. Taken together, the present findings indicate that brains of female mice have lower oxidant and higher antioxidant capacity mostly related to CAT and to a lesser extent to Gpx activity.     

Sex-dependent antioxidant enzyme activities and lipid peroxidation in ageing mouse brain

We investigated whether oxidant status and antioxidant enzyme activities during ageing of mouse brain are regulated in sex-dependent manner. In the homogenate from the brain of 1, 4, 10 and 18 months old male and female CBA mice, lipid peroxidation (LPO), total superoxide dismutase (tSOD), catalase (CAT) and glutathione peroxidase (Gpx) were determined. LPO was age- and sex-related, favoring males over females throughout the lifespan with the peak in both sexes at 10 months of age. Throughout ageing, no difference in tSOD activity between male and female brains was observed, except in immature 1 month old mice. Gender-related difference in Gpx activity was observed, with higher level in females comparing to males, reaching statistical significance in senescent (18 months old) animals. CAT activity was drastically changed with ageing in both the male and female brain. We found different age associated trends in CAT activity in males and females: decreased with age in males and increased with age in females. Taken together, the present findings indicate that brains of female mice have lower oxidant and higher antioxidant capacity mostly related to CAT and to a lesser extent to Gpx activity.     

Proteasome subunits are regulated and expressed in comparable concentrations as a functional cluster

The proteasome is the main proteolytic enzyme that functions in the ubiquitin-proteasome system. The 26S proteasome has multi-subunit protease complexes consisting of 20S subunits composed of four seven-numbered rings with two outer rings containing α subunits and two central rings composed of β subunits, and 19S caps of 6 ATPase and 11 non-ATPase subunits; however, it is unclear how these subunits are regulated and the 26S proteasomes assembled. To verify whether each subunit’s mRNA expression is associated with the mRNA expression of other proteasome subunits, we carried out expression analysis of 34 proteasome subunits mRNA on peripheral blood from 75 subjects. The expression of proteasome subunits mRNA was comparable in each individual of the studied population and the mRNA expression has been investigated in each 20S or 19S proteasome. Our results suggest that each type of subunit is regulated by respectively common factors, and that the 20S and 19S proteasomes are regulated by different systems.     

Enzymatic properties of the 20S proteasome in wheat endosperm and its biochemical characteristics after seed imbibition

The 20S proteasome from wheat ( Triticum aestivum L., Yangmai 158) endosperm was purified to apparent homogeneity by three sequential centrifugations and gradient PAGE (GPAGE). The purified 20S proteasome clearly cleaved peptidyl-arylamide bonds in the model synthetic substrates Z-GGL-AMC and Z-GGR-AMC, which are used to reflect chymotrypsin-like and trypsin-like activity, respectively. For both substrates, the optimum pH was 8.0, but the optimum temperatures for chymotrypsin-like and trypsin-like activity were 55 °C and 37 °C, respectively. Both enzyme activities were clearly inhibited by MG115 and PMSF. Polyubiquitinated proteins remained constant from 0 to 7 days after seed imbibition, but caseinolytic activity and the amount of the 20S proteasome associated with the aleurone layer decreased from 1 to 2 days after imbibition (DAI), then increased from 2 to 4 DAI, and reached a maximum at 4 DAI that was retained until 7 DAI. An increase was seen in the mRNA level of the β5 subunit of the 20S proteasome from 2 DAI, and caseinolytic activity and the amount of the 20S proteasome increased from 3 DAI onwards. In addition, the main storage proteins of the wheat endosperm could not be hydrolyzed by the 20S proteasome. The evidence suggests that the main role of the 20S proteasome may not be to degrade massive proteins of the wheat endosperm after seed imbibition.     

The ubiquitin-proteasome proteolytic pathway in heart vs skeletal muscle: effects of acute diabetes

The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal subunits, the amount of free ubiquitin and the proteasome chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were significantly greater than in skeletal muscle (P 相似文献   

White muscle 20S proteasome activity is negatively correlated to growth rate at low temperature in the spotted wolffish Anarhichas minor

The effect of temperature and mass on specific growth rate (G) was examined in spotted wolffish Anarhichas minor of different size classes (ranging from 60 to 1500 g) acclimated at different temperatures (4, 8 and 12° C). The relationship between G and 20S proteasome activity in heart ventricle, liver and white muscle tissue was then assessed in fish acclimated at 4 and 12° C to determine if protein degradation via the proteasome pathway could be imposing a limitation on somatic growth. Cardiac 20S proteasome activity was not affected by acclimation temperature nor fish mass and had no correlation with G. Hepatic 20S proteasome activity was higher at 12° C but did not show any relationship with G. Partial correlation analysis showed that white muscle 20S proteasome activity was negatively correlated to G (partial Pearson's r = ?0·609) but only at cold acclimation temperature (4° C). It is suggested that acclimation to cold temperature involves compensation of the mitochondrial oxidative capacity which would in turn lead to increased production of oxidatively damaged proteins that are degraded by the proteasome pathway and ultimately negatively affects G at cold temperature.     

Proteasome response to interferon-gamma is altered in senescent human fibroblasts

We have investigated immunoproteasomes in human fibroblasts during replicative senescence. Unlike levels of constitutive proteasome catalytic subunits and 26S proteasome regulatory subunits, levels of immunosubunits did not decrease dramatically in senescent cells. However, the induction of immunosubunits by interferon-gamma (IFN-gamma) was lost in senescent cells. In contrast, levels of the 11S proteasome regulator, PA28, were increased by IFN-gamma even in senescent cells, and both immunosubunits and PA28 increased with the reversible growth arrest in confluent cell cultures. The results highlight differences in the mechanisms of regulation of immunoproteasomes compared to constitutive proteasomes and in the irreversible growth arrest of senescent cells compared to reversible contact-induced growth arrest.     

Age-dependent declines in proteasome activity in the heart.

The proteasome is a major intracellular proteolytic system involved in the removal of oxidized and ubiquitinated protein and the induction of certain stress response pathways. In this study, age-dependent alterations in proteasome function were investigated to gain insight into potential factors which contribute to increased susceptibility to various forms of heart disease during aging. Proteasome activity in cellular extracts prepared from Fisher 344 rat hearts was found to decrease with age. These declines in activity were associated with a decreased 20S proteasome content and loss of specific activities. As determined by two-dimensional gel electrophoresis of purified 20S proteasome, the distribution and silver staining intensities of enzyme subunits were found to vary with age, suggesting that alterations in proteasome subunit composition and/or structure are involved in age-related declines in proteasome activity. In addition, age-dependent increases in the levels of oxidized and ubiquitinated proteins, known substrates of the proteasome, were observed. Thus, loss in proteasome function may impair the ability of myocytes to mount an appropriate response to stress, thereby enhancing the susceptibility of the aging heart to cardiovascular disease.     

Redox regulation of diaphragm proteolysis during mechanical ventilation

Prevention of oxidative stress via antioxidants attenuates diaphragm myofiber atrophy associated with mechanical ventilation (MV). However, the specific redox-sensitive mechanisms responsible for this remain unknown. We tested the hypothesis that regulation of skeletal muscle proteolytic activity is a critical site of redox action during MV. Sprague-Dawley rats were assigned to five experimental groups: 1) control, 2) 6 h of MV, 3) 6 h of MV with infusion of the antioxidant Trolox, 4) 18 h of MV, and 5) 18 h of MV with Trolox. Trolox did not attenuate MV-induced increases in diaphragmatic levels of ubiquitin-protein conjugation, polyubiquitin mRNA, and gene expression of proteasomal subunits (20S proteasome alpha-subunit 7, 14-kDa E2, and proteasome-activating complex PA28). However, Trolox reduced both chymotrypsin-like and peptidylglutamyl peptide hydrolyzing (PGPH)-like 20S proteasome activities in the diaphragm after 18 h of MV. In addition, Trolox rescued diaphragm myofilament protein concentration (mug/mg muscle) and the percentage of easily releasable myofilament protein independent of alterations in ribosomal capacity for protein synthesis. In summary, these data are consistent with the notion that the protective effect of antioxidants on the diaphragm during MV is due, at least in part, to decreasing myofilament protein substrate availability to the proteasome.     

Altered proteasome function and subunit composition in aged muscle

Myofibrillar protein degradation is mediated through the ubiquitin-proteasome pathway. To investigate if altered proteasome activity plays a role in age-related muscle atrophy, we examined muscle size and proteasome function in young and aged F344BN rats. Significant age-related muscle atrophy was confirmed by the 38% decrease in cross-sectional area of type 1 fibers in soleus muscle. Determination of proteasome function showed hydrolysis of fluorogenic peptides was equivalent between ages. However, when accounting for the 3-fold increase in content of the 20S catalytic core in aged muscle, the lower specific activity suggests a functional loss in individual proteins with aging. Comparing the composition of the catalytic beta-subunits showed an age-related 4-fold increase in the cytokine-inducible subunits, LMP2 and LMP7. Additionally, the content of the activating complexes, PA28 and PA700, relative to the 20S proteasome was reduced 50%. These results suggest significant alterations in the intrinsic activity, the percentage of immunoproteasome, and the regulation of the 20S proteasome by PA28 and PA700 in aged muscle.