calpain and Atrophy

Skeletal muscles comprise the largest organ system in the body and play an essential role in body movement, breathing, and glucose homeostasis. Skeletal muscle is also an important endocrine organ that contributes to the health of numerous body organs. Therefore, maintaining healthy skeletal muscles is important to support overall health of the body. Prolonged periods of muscle inactivity (e.g., bed rest or limb immobilization) or chronic inflammatory diseases (i.e., cancer, kidney failure, etc.) result in skeletal muscle atrophy. An excessive loss of muscle mass is associated with a poor prognosis in several diseases and significant muscle weakness impairs the quality of life. The skeletal muscle atrophy that occurs in response to inflammatory diseases or prolonged inactivity is often associated with both oxidative and nitrosative stress. In this report, we critically review the experimental evidence that provides support for a causative link between oxidants and muscle atrophy. More specifically, this review will debate the sources of oxidant production in skeletal muscle undergoing atrophy as well as provide a detailed discussion on how reactive oxygen species and reactive nitrogen species modulate the signaling pathways that regulate both protein synthesis and protein breakdown.

Topics: Atrophy; Calpain; Humans; Muscle, Skeletal; Oxidation-Reduction; Oxidative Stress; Proteolysis; Reactive Nitrogen Species; Reactive Oxygen Species; Signal Transduction

Mechanical ventilation (MV) is a clinical tool that provides respiratory support to patients unable to maintain adequate alveolar ventilation on their own. Although MV is often a life-saving intervention in critically ill patients, an undesired side-effect of prolonged MV is the rapid occurrence of diaphragmatic atrophy due to accelerated proteolysis and depressed protein synthesis. Investigations into the mechanism(s) responsible for MV-induced diaphragmatic atrophy reveal that activation of the calcium-activated protease, calpain, plays a key role in accelerating proteolysis in diaphragm muscle fibers. Moreover, active calpain has been reported to block signaling events that promote protein synthesis (i.e., inhibition of mammalian target of rapamycin (mTOR) activation). While this finding suggests that active calpain can depress muscle protein synthesis, this postulate has not been experimentally verified. Therefore, we tested the hypothesis that active calpain plays a key role in the MV-induced depression of both anabolic signaling events and protein synthesis in the diaphragm muscle. MV-induced activation of calpain in diaphragm muscle fibers was prevented by transgene overexpression of calpastatin, an endogenous inhibitor of calpain. Our findings indicate that overexpression of calpastatin averts MV-induced activation of calpain in diaphragm fibers and rescues the MV-induced depression of protein synthesis in the diaphragm muscle. Surprisingly, deterrence of calpain activation did not impede the MV-induced inhibition of key anabolic signaling events including mTOR activation. However, blockade of calpain activation prevented the calpain-induced cleavage of glutaminyl-tRNA synthetase in diaphragm fibers; this finding is potentially important because aminoacyl-tRNA synthetases play a central role in protein synthesis. Regardless of the mechanism(s) responsible for calpain's depression of protein synthesis, these results provide the first evidence that active calpain plays an important role in promoting the MV-induced depression of protein synthesis within diaphragm fibers.

Topics: Atrophy; Calpain; Diaphragm; Humans; Respiration, Artificial; TOR Serine-Threonine Kinases

To investigate the effect of calpeptin on diaphragmatic injury and atrophy under controlled mechanical ventilation in rats.. A total of 24 SPF Sprague-Dawley (SD) rats were randomly divided into anesthetized control group (CON group), 24-hour controlled mechanical ventilation group (CMV group), and 24-hour CMV + treatment with calpeptin group (CMVC group), with 8 rats in each group. Animals in the CON group received an intraperitoneal injection of pentobarbital sodium without CMV and continuous infusion of pentobarbital sodium. A small-animal ventilator was used for 24 hours in rats of CMV group. Rats of CMVC were treated with a specific calpain inhibitor calpeptin (4 mg/kg). The drug was injected subcutaneously 2 hours before and 8, 15 and 23 hours after mechanical ventilation. Changes in diaphragm ultrastructure, light microscopic picture, and myosin heavy chain (MHC) expression were observed.. (1) Alignment of myofilaments and normal Z-band, and the shape of mitochondria were maintained in CON group as revealed by electron microscope. The signs of misalignment of myofibrils, disruption of Z-band and vacuolar mitochondria were found in CMV group, and they were obviously improved in CMVC group. The density of muscle injury (× 10⁻²/μm²) in CMV group was significantly higher than that in control group (36.8 ± 13.7 vs. 6.4 ± 6.3, t=6.373, P=0.001), and that in CMVC group was significantly lowered (17.6 ± 9.1 vs. 36.8 ± 13.7, t=3.694, P=0.002).(2) In CON group, the diaphragm fibers appeared regular in cross section without pathologic change under light microscopy. Fuzzy muscle striations, irregular muscle fibers, centralized nuclei and swelling of capillary endothelial cells were observed in CMV group, while pathological changes in the CMVC group were milder significantly. (3) In CMV group, the density of MHCslow and MHCfast was lower compared with that of CON group, and the gray value was lowered by 61.1% (t=8.138, P=0.001) and 77.1% (t=8.844, P=0.001), respectively, especially in MHCfast. However, the gray values of MHCslow and MHCfast were increased by 1.51 folds (t=4.601, P=0.010), and 1.33 folds (t=2.859, P=0.011), respectively, after treatment with calpeptin, and the elevation was more significantly in MHCslow.. Diaphragmatic injury and atrophy were found after CMV for 24 hours. Calpeptin could reverse the detrimental effects of CMV, and it suggested that calpain plays an important role in modulating the ventilator-induced dysfunction of the diaphragm.

Topics: Animals; Atrophy; Calpain; Diaphragm; Dipeptides; Disease Models, Animal; Male; Myosin Heavy Chains; Rats; Rats, Sprague-Dawley; Respiration, Artificial

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.

Topics: Animals; Atrophy; Calpain; Carbon Radioisotopes; Hypoxia; Male; Motor Activity; Muscle, Skeletal; Myofibrils; Pressure; Proteasome Endopeptidase Complex; Proteins; Proteolysis; Rats; Rats, Sprague-Dawley; Ubiquitin

Prolonged mechanical ventilation (MV) results in diaphragmatic weakness due to fiber atrophy and contractile dysfunction. Recent work reveals that activation of the proteases calpain and caspase-3 is required for MV-induced diaphragmatic atrophy and contractile dysfunction. However, the mechanism(s) responsible for activation of these proteases remains unknown. To address this issue, we tested the hypothesis that oxidative stress is essential for the activation of calpain and caspase-3 in the diaphragm during MV. Cause-and-effect was established by prevention of MV-induced diaphragmatic oxidative stress using the antioxidant Trolox. Treatment of animals with Trolox prevented MV-induced protein oxidation and lipid peroxidation in the diaphragm. Importantly, the Trolox-mediated protection from MV-induced oxidative stress prevented the activation of calpain and caspase-3 in the diaphragm during MV. Furthermore, the avoidance of MV-induced oxidative stress not only averted the activation of these proteases but also rescued the diaphragm from MV-induced diaphragmatic myofiber atrophy and contractile dysfunction. Collectively, these findings support the prediction that oxidative stress is required for MV-induced activation of calpain and caspase-3 in the diaphragm and are consistent with the concept that antioxidant therapy can retard MV-induced diaphragmatic weakness.

Topics: Aldehydes; Animals; Antioxidants; Atrophy; Calpain; Caspase 3; Chromans; Diaphragm; Electric Stimulation; Enzyme Activation; Female; Isometric Contraction; Lipid Peroxidation; Microfilament Proteins; Muscle Weakness; Oxidative Stress; Protein Carbonylation; Rats; Rats, Sprague-Dawley; Respiration, Artificial; Vesicular Transport Proteins

Two siblings originating from Reunion Island were affected by a limb-girdle muscular dystrophy (LGMD) type 2A and carried the same two mutations in the calpain gene: 946-1 AG-->AA, affecting a splice site, and S744G. They demonstrated the clinical variability possible with calpain-3 mutations. Onset was around 20 years of age in each of them. The girl's symptoms mimicked a metabolic myopathy, while her brother, at the same age, presented a classical phenotype of LGMD in an advanced functional stage.

Topics: Adult; Atrophy; Calpain; Family Health; Female; Humans; Male; Muscle Weakness; Muscle, Skeletal; Muscular Dystrophies; Nuclear Family; Phenotype; Reunion

To clarify the significance of intracellular lysosomal (cathepsins B, L and H) and extralysosomal (calpain) proteolytic systems in the process of muscle fiber degradation in inflammatory myopathies, biopsied muscle specimens were examined from patients with polymyositis (PM) and dermatomyositis (DM). Generally, in specimens from patients with PM and DM, but not in those from normal controls, muscle fibers surrounding inflammatory infiltrates or in the perifascicular regions, and occasionally mononuclear cell infiltrates demonstrated positive immunostaining for calpain and cathepsins B, L, and H. In addition, enzyme activities of cathepsins B and L increased in specimens with inflammatory myopathy. These results suggest that calpain and cathepsins play a significant role in the process of muscle fiber destruction in inflammatory myopathy.

Topics: Adult; Atrophy; Biopsy; Calpain; Cathepsin B; Cathepsin H; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Dermatomyositis; Endopeptidases; Female; Humans; Inflammation; Lysosomes; Male; Middle Aged; Muscle Fibers, Skeletal; Muscle, Skeletal; Necrosis; Polymyositis

Nine days of hindlimb suspension resulted in atrophy (55%) and loss of protein (53%) in rat soleus muscle due to a marked elevation in protein breakdown (66%, P < 0.005). To define which proteolytic system(s) contributed to this increase, soleus muscles from unweighted rats were incubated in the presence of proteolytic inhibitors. An increase in lysosomal and Ca 2+-activated proteolysis (254%, P < 0.05) occurred in the atrophying incubated muscles. In agreement with the measurements in vitro, cathepsin B, cathepsins B + L and m-calpain enzyme activities increased by 111%, 92% and 180% (P < 0.005) respectively in the atrophying muscles. Enhanced mRNA levels for these proteinases (P < 0.05 to P < 0.001) paralleled the increased enzyme activities, suggesting a transcriptional regulation of these enzymes. However, the lysosomal and Ca 2+-dependent proteolytic pathways accounted for a minor part of total proteolysis in both control (9%) and unweighted rats (18%). Furthermore the inhibition of these pathways failed to suppress increased protein breakdown in unweighted muscle. Thus a non-lysosomal Ca 2+-independent proteolytic process essentially accounted for the increased proteolysis and subsequent muscle wasting. Increased mRNA levels for ubiquitin, the 14 kDa ubiquitin-conjugating enzyme E2 (involved in the ubiquitylation of protein substrates) and the C2 and C9 subunits of the 20 S proteasome (i.e. the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates) were observed in the atrophying muscles (P < 0.02 to P < 0.001). Analysis of C9 mRNA in polyribosomes showed equal distribution into both translationally active and inactive mRNA pools, in either unweighted or control rats. These results suggest that increased ATP-ubiquitin-dependent proteolysis is most probably responsible for muscle wasting in the unweighted soleus muscle.

Topics: Adenosine Triphosphate; Animals; Atrophy; Calcium; Calpain; Cathepsin B; Cathepsin D; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Endopeptidases; Enzyme Activation; Hindlimb; Lysosomes; Male; Muscle, Skeletal; Polyribosomes; Protease Inhibitors; Rats; Rats, Wistar; Time Factors; Transcription, Genetic; Ubiquitins

The effects of repeated biopsy sampling on muscle morphology was qualitatively and quantitatively assessed in strength-trained and untrained men and women. College-age men (13) and women (8) resistance trained twice a week for 8 weeks. A progressive resistance-training program was performed consisting of squats, leg presses, and leg extensions. Nontraining men (7) and women (5) served as controls. Muscle biopsy specimens and fasting bloods were obtained at the beginning and every 2 weeks and histochemical, biochemical, and ultrastructural methods were employed to assess the type and amount of damage. Except for a few scattered atrophic fibers in 2 of the 33 biopsy samples, all initial specimens were normal. In contrast, many of the subsequent biopsy samples from both untrained and resistance-trained men and women contained evidence of damage. Ultrastructural analysis confirmed that degenerative-regenerative processes were occurring in both groups. However, training subjects had a four-fold greater number of damaged fibers than nontraining subjects (8.53% vs 2.08%). In addition, only biopsy samples from training individuals contained fibers with internal disorganization (e.g., Z-line streaming, myofibrillar disruption). Calpain II levels in the biopsy samples and serum creatine kinase activity were not significantly affected supporting the light and electron microscopic observations that most of the damaged fibers were normal in appearance except for their small diameter. In summary, focal damage induced by the biopsy procedure is not completely repaired after 2 weeks and could affect the results, particularly cross-sectional area measurements. Moreover, resistance training appears to cause additional damage to the muscle and may delay repair of the biopsied region.

Topics: Adult; Atrophy; Calpain; Creatine Kinase; Female; Humans; Isoenzymes; Male; Microscopy, Electron; Muscles; Nerve Degeneration; Physical Education and Training

The effect of the protease inhibitor leupeptin on flight muscle histolysis in queen fire ants was studied by electron microscopy. In untreated animals artificially inseminated, muscle involution was apparent at 6 hr post-insemination and complete by 24 hr post-insemination. However, in animals pre-treated with leupeptin and subsequently artificially inseminated, no morphologic evidence of flight muscle breakdown was seen at any interval between 6 and 24 hr post-insemination. Such information appears to indicate that one or more proteases are involved in the process of insemination-induced muscle atrophy in fire ants. The most likely candidate is a soluble, calcium-activated myofilament-associated protease.

Topics: Animals; Ants; Atrophy; Calpain; Cytoskeleton; Endopeptidases; Female; Insemination; Leupeptins; Muscle Proteins; Muscles; Oligopeptides; Protease Inhibitors; Wings, Animal