calpain and Muscular-Atrophy

Muscle atrophy is an inevitable sequel of fasting, denervation, aging, exposure to microgravity, and many human diseases including, cancer, type-2 diabetes, and renal failure. During atrophy the destruction of the muscle's fundamental contractile machinery, the myofibrils, is accelerated leading to a reduction in muscle mass, weakness, frailty, and physical disability. Recent findings indicate that atrophy can be a major cause of death in affected individuals, and inhibition of muscle wasting is likely to prolong survival. Major advances in our understanding of the mechanisms for myofibril breakdown in atrophy include the discovery of biological pathways and key components that play prominent roles. On fasting or denervation, degradation of myofibrillar proteins requires an initial dissociation of the desmin cytoskeleton, whose integrity is critical for myofibril stability. This loss of desmin filaments involves phosphorylation, ubiquitination, and subsequent depolymerization by calpain-1, and appears to reduce myofibrils integrity and facilitate their destruction. Consequently, depolymerization of desmin filament in atrophy seems to be an early key event for overall proteolysis. A focus of this review is to discuss these new insights and the specific role of calpain-1 in promoting desmin filaments loss, and to highlight important key questions that merit further study.

Topics: Animals; Calpain; Desmin; Humans; Muscular Atrophy; Myofibrils; Polymerization; Ubiquitination

Calpains are cysteine proteases expressed in skeletal muscle fibers and other cells. Although calpain was first reported to act as a kinase activating factor in skeletal muscle, the consensus is now that calpains play a canonical role in protein turnover. However, recent evidence reveals new and exciting roles for calpains in skeletal muscle. This review will discuss the functions of calpains in skeletal muscle remodeling in response to both exercise and inactivity-induced muscle atrophy. Calpains participate in protein turnover and muscle remodeling by selectively cleaving target proteins and creating fragmented proteins that can be further degraded by other proteolytic systems. Nonetheless, an often overlooked function of calpains is that calpain-mediated cleavage of proteins can result in fragmented proteins that are biologically active and have the potential to actively influence cell signaling. In this manner, calpains function beyond their roles in protein turnover and influence downstream signaling effects. This review will highlight both the canonical and noncanonical roles that calpains play in skeletal muscle remodeling including sarcomere transformation, membrane repair, triad junction formation, regulation of excitation-contraction coupling, protein turnover, cell signaling, and mitochondrial function. We conclude with a discussion of key unanswered questions regarding the roles that calpains play in skeletal muscle.

Topics: Animals; Calpain; Cell Membrane; Exercise; Humans; Mitochondria, Muscle; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Oxidation-Reduction; Phosphorylation; Protein Isoforms; Proteolysis; Sarcomeres; Sedentary Behavior; Signal Transduction

Skeletal muscle atrophy is associated with a loss of muscle protein which may result from both increased proteolysis and decreased protein synthesis. Investigations on cell signaling pathways that regulate muscle atrophy have promoted our understanding of this complicated process. Emerging evidence implicates that calpains play key roles in dysregulation of proteolysis seen in muscle atrophy. Moreover, studies have also shown that abnormally activated calpain results muscle atrophy via its downstream effects on ubiquitin-proteasome pathway (UPP) and Akt phosphorylation. This review will discuss the role of calpains in regulation of skeletal muscle atrophy mainly focusing on its collaboration with either UPP or Akt in atrophy conditions in hope to stimulate the interest in development of novel therapeutic interventions for skeletal muscle atrophy.

Topics: Animals; Calpain; Humans; Hypertrophy; Muscle Proteins; Muscular Atrophy; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-akt; Receptor Cross-Talk; Signal Transduction; Ubiquitin

The enhancement of atrophied muscle recovery after coming back to normal motor activities (landing of the spacecraft, withdrawal of the cast etc) is very important problem of rehabilitation as well as space medicine. Along with the recovery of the gravity-dependent motor control system the regrowth of the muscle mass seems to be the key event. This regrowth cause recovery of the muscle performance. The present review is dedicated to the structural and functional events, observed during 7 days after exposure of an animal to gravitational unloading (mainly in experiments with the hindlimb suspension model). The state of the main signaling pathways in muscle fibers is also considered. The data presented in the review allow to imagine how the destructive and synthetic events do interact in the initial period of recovery. The work hypotheses on the key triggering signaling mechanisms are also put forward.

Topics: Animals; Calcium; Calpain; Cytoskeleton; Gravitation; Hindlimb Suspension; Muscle Fibers, Skeletal; Muscle Proteins; Muscular Atrophy; Proteolysis; Rats; Recovery of Function; Sarcolemma; Signal Transduction; Time Factors

To review current knowledge about the impact of prolonged mechanical ventilation on diaphragmatic function and biology.. Systematic literature review.. Prolonged mechanical ventilation can promote diaphragmatic atrophy and contractile dysfunction. As few as 18 hrs of mechanical ventilation results in diaphragmatic atrophy in both laboratory animals and humans. Prolonged mechanical ventilation is also associated with diaphragmatic contractile dysfunction. Studies using animal models revealed that mechanical ventilation-induced diaphragmatic atrophy is due to increased diaphragmatic protein breakdown and decreased protein synthesis. Recent investigations have identified calpain, caspase-3, and the ubiquitin-proteasome system as key proteases that contribute to mechanical ventilation-induced diaphragmatic proteolysis. The scientific challenge for the future is to delineate the mechanical ventilation-induced signaling pathways that activate these proteases and depress protein synthesis in the diaphragm. Future investigations that define the signaling mechanisms responsible for mechanical ventilation-induced diaphragmatic weakness will provide the knowledge required for the development of new medicines that can maintain diaphragmatic mass and function during prolonged mechanical ventilation.

Topics: Animals; Antioxidants; Calpain; Caspase 3; Critical Illness; Diaphragm; Enzyme Inhibitors; Humans; Muscle Contraction; Muscular Atrophy; Proteasome Endopeptidase Complex; Respiration, Artificial; Risk Factors; Ubiquitin; Ventilator Weaning

The calcium-dependent proteolytic system is composed of cysteine proteases named calpains. They are ubiquitous or tissue-specific enzymes. The two best characterised isoforms are the ubiquitously expressed mu- and m-calpains. Besides its regulation by calcium, calpain activity is tightly controlled by calpastatin, the specific endogenous inhibitor, binding to phospholipids, autoproteolysis and phosphorylation. Calpains are responsible for limited proteolytic events. Among the multitude of substrates identified so far are cytoskeletal and membrane proteins, enzymes and transcription factors. Calpain activity is involved in a large number of physiological and pathological processes. In this review, we will particularly focus on the implication of the calcium-dependent proteolytic system in relation to muscle physiology. Because of their ability to remodel cytoskeletal anchorage complexes, calpains play a major role in the regulation of cell adhesion, migration and fusion, three key steps of myogenesis. Calcium-dependent proteolysis is also involved in the control of cell cycle. In muscle tissue, in particular, calpains intervene in the regeneration process. Another important class of calpain substrates belongs to apoptosis regulating factors. The proteases may thus play a role in muscle cell death, and as a consequence in muscle atrophy. The relationships between calcium-dependent proteolysis and muscle dysfunctions are being further developed in this review with a particular emphasis on sarcopenia.

Topics: Animals; Calcium; Calpain; Humans; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies

Skeletal muscle wasting is a prominent feature of cachexia, a complex systemic syndrome that frequently complicates chronic diseases such as inflammatory and autoimmune disorders, cancer and AIDS. Muscle wasting may also develop as a manifestation of primary or neurogenic muscular disorders. It is now generally accepted that muscle depletion mainly arises from increased protein catabolism. The ubiquitin-proteasome system is believed to be the major proteolytic machinery in charge of such protein breakdown, yet there is evidence suggesting that Ca(2+)-dependent system, lysosomes and, in some conditions at least, even caspases are involved as well. The role of Ca(2+)-dependent proteolysis in skeletal muscle wasting is reviewed in the present paper. This system relies on the activity of calpains, a family of Ca(2+)-dependent cysteine proteases, whose regulation is complex and not completely elucidated. Modulations of Ca(2+)-dependent proteolysis have been associated with muscle protein depletion in various pathological contexts and particularly with muscle dystrophies. Calpains can only perform a limited proteolysis of their substrates, however they may play a critical role in initiating the breakdown of myofibrillar protein, by releasing molecules that become suitable for further degradation by proteasomes. Some evidence would also support a role for lysosomes and caspases in muscle wasting. Thus it cannot be excluded that different intracellular proteolytic systems may coordinately concur in shifting muscle protein turnover towards excess catabolism. Many different signals have been proposed as potentially involved in triggering the enhanced protein breakdown that underlies muscle wasting. How they are transduced to initiate the hypercatabolic response and to activate the proteolytic pathways remains largely unknown, however.

Topics: Animals; Calcium; Calpain; Humans; Models, Biological; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Myofibrils; Proteasome Endopeptidase Complex; Ubiquitins

Calpains are intracellular nonlysosomal Ca(2+)-regulated cysteine proteases. They mediate regulatory cleavages of specific substrates in a large number of processes during the differentiation, life and death of the cell. The purpose of this review is to synthesize our current understanding of the participation of calpains in muscle atrophy. Muscle tissue expresses mainly three different calpains: the ubiquitous calpains and calpain 3. The participation of the ubiquitous calpains in the initial degradation of myofibrillar proteins occurring in muscle atrophy as well as in the necrosis process accompanying muscular dystrophies has been well characterized. Inactivating mutations in the calpain 3 gene are responsible for limb-girdle muscular dystrophy type 2A and calpain 3 has been found to be downregulated in different atrophic situations, suggesting that it has to be absent for the atrophy to occur. The fact that similar regulations of calpain activities occur during exercise as well as in atrophy led us to propose that the calpains control cytoskeletal modifications needed for muscle plasticity.

Topics: Apoptosis; Calpain; Humans; Isoenzymes; Models, Biological; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Necrosis

Muscle wasting in sepsis is associated with increased expression of messenger RNA for several genes in the ubiquitin-proteasome proteolytic pathway, indicating that increased gene transcription is involved in the development of muscle atrophy. Here we review the influence of sepsis on the expression and activity of the transcription factors activator protein-1, nuclear factor-kappaB (NF-kappaB), and CCAAT/enhancer binding protein, as well as the nuclear cofactor p300. These transcription factors may be important for sepsis-induced muscle wasting because several of the genes in the ubiquitin-proteasome proteolytic pathway have multiple binding sites for activating protein-1, nuclear factor-kappaB, and CCAAT/enhancer binding protein in their promoter regions. In addition, the potential role of increased muscle calcium levels for sepsis-induced muscle atrophy is reviewed. Calcium may regulate several mechanisms and factors involved in muscle wasting, including the expression and activity of the calpain-calpastatin system, proteasome activity, CCAAT/enhancer binding protein transcription factors, apoptosis and glucocorticoid-mediated muscle protein breakdown. Because muscle wasting is commonly seen in patients with sepsis and has severe clinical consequences, a better understanding of mechanisms regulating sepsis-induced muscle wasting may help improve the care of patients with sepsis and other muscle-wasting conditions as well.

Topics: Apoptosis; Calcium; Calpain; Gene Expression Regulation; Glucocorticoids; Humans; Models, Biological; Muscular Atrophy; Proteasome Endopeptidase Complex; Sepsis; Transcription Factors; Ubiquitin

Skeletal muscle atrophy occurs as a consequence of injury, illness, surgery, and muscle disuse, impacting appreciably on health care costs and patient quality of life, particularly in the absence of appropriate rehabilitation. The molecular mechanisms that regulate muscle mass during atrophy and rehabilitation in humans have not been elucidated, despite several robust candidate pathways being identified. Here, we induced skeletal muscle atrophy in healthy volunteers using two weeks of limb immobilization, and then stimulated the restoration of muscle mass with six weeks of supervised exercise rehabilitation. We determined muscle mass and function and performed targeted gene expression analysis at prescribed time points during immobilization and rehabilitation. For the first time, we have identified novel changes in gene expression following immobilization-induced atrophy and during a program of rehabilitative exercise that restored muscle mass and function. Furthermore, we have shown that exercise performed immediately following immobilization induces profound changes in the expression of a number of genes in favor of the restoration of muscle mass, within 24 h. This information will be of considerable importance to our understanding of how immobilization and contraction stimulate muscle atrophy and hypertrophy, respectively, and to the development of novel therapeutic strategies aimed at maintaining or restoring muscle mass.

Topics: Adolescent; Adult; Calpain; Cysteine Endopeptidases; Exercise; Gene Expression Profiling; Gene Expression Regulation; Humans; I-kappa B Kinase; Immobilization; Insulin-Like Growth Factor I; Isometric Contraction; Male; Multienzyme Complexes; Muscle, Skeletal; Muscular Atrophy; Muscular Disorders, Atrophic; Myostatin; Organ Size; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; RNA, Messenger; Signal Transduction; Time Factors; Transforming Growth Factor beta; Ubiquitin

Patients with cystic fibrosis (CF) often suffer from skeletal muscle atrophy, most often attributed to physical inactivity and nutritional factors. CF is also characterized by abnormally elevated systemic inflammation. However, it is unknown whether the lack of a functional CF transmembrane conductance regulator (CFTR) gene predisposes to exaggerated inflammation-induced muscle proteolysis. CF mice (

Topics: Animals; Calpain; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Diaphragm; Humans; Inflammation; Lipopolysaccharides; Mice; Muscle, Skeletal; Muscular Atrophy; Proteasome Endopeptidase Complex; Ubiquitins

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, an unintended consequence of prolonged MV is the rapid development of diaphragmatic atrophy and contractile dysfunction, known as ventilator-induced diaphragm dysfunction (VIDD). Although the mechanism(s) responsible for VIDD are not fully understood, abundant evidence reveals that oxidative stress leading to the activation of the major proteolytic systems (i.e., autophagy, ubiquitin-proteasome, caspase, and calpain) plays a dominant role. Of the proteolytic systems involved in VIDD, calpain has received limited experimental attention due to the longstanding dogma that calpain plays a minor role in inactivity-induced muscle atrophy. Guided by preliminary experiments, we tested the hypothesis that activation of calpains play an essential role in MV-induced oxidative stress and the development of VIDD. This premise was rigorously tested by transgene overexpression of calpastatin, an endogenous inhibitor of calpains. Animals with/without transfection of the calpastatin gene in diaphragm muscle fibers were exposed to 12 h of MV. Results confirmed that overexpression of calpastatin barred MV-induced activation of calpain in diaphragm fibers. Importantly, deterrence of calpain activation protected the diaphragm against MV-induced oxidative stress, fiber atrophy, and contractile dysfunction. Moreover, prevention of calpain activation in the diaphragm forstalled MV-induced mitochondrial dysfunction and prevented MV-induced activation of caspase-3 along with the transcription of muscle specific E3 ligases. Collectively, these results support the hypothesis that calpain activation plays an essential role in the early development of VIDD. Further, these findings provide the first direct evidence that calpain plays an important function in inactivity-induced mitochondrial dysfunction and oxidative stress in skeletal muscle fibers.

Topics: Animals; Calpain; Diaphragm; Humans; Mitochondria; Muscle Weakness; Muscular Atrophy; Respiration, Artificial

There is growing evidence about the ability of cyclic adenosine monophosphate (cAMP) signaling and nonselective phosphodiesterase (PDE) inhibitors on mitigate muscle atrophy. PDE4 accounts for the major cAMP hydrolyzing activity in skeletal muscles, therefore advances are necessary about the consequences of treatment with PDE4 inhibitors on protein breakdown in atrophied muscles. We postulated that rolipram (selective PDE4 inhibitor) may activate cAMP downstream effectors, inhibiting proteolytic systems in skeletal muscles of diabetic rats.. Streptozotocin-induced diabetic rats were treated with 2 mg/kg rolipram for 3 days. Changes in the levels of components belonging to the proteolytic machineries in soleus and extensor digitorum longus (EDL) muscles were investigated, as well as cAMP effectors.. Treatment of diabetic rats with rolipram decreased the levels of atrogin-1 and MuRF-1 in soleus and EDL, and reduced the activities of calpains and caspase-3; these findings partially explains the low ubiquitin conjugates levels and the decreased proteasome activity. The inhibition of muscle proteolysis may be occurring due to phosphorylation and inhibition of forkhead box O (FoxO) factors, probably as a consequence of the increased cAMP levels, followed by the activation of PKA and Akt effectors. Akt activation may be associated with the increased levels of exchange protein directly activated by cAMP (EPAC). As a result, rolipram treatment spared muscle mass in diabetic rats.. The antiproteolytic responses associated with PDE4 inhibition may be helpful to motivate future investigations about the repositioning of PDE4 inhibitors for the treatment of muscle wasting conditions.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Calpain; Caspase 3; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus, Experimental; Male; Muscle, Skeletal; Muscular Atrophy; Nerve Tissue Proteins; Phosphodiesterase 4 Inhibitors; Phosphorylation; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Rolipram; Signal Transduction

The size and shape of skeletal muscle fibers are affected by various physiological and pathological conditions, such as muscle atrophy, hypertrophy, regeneration, and dystrophies. Hence, muscle fiber cross-sectional area (CSA) is an important determinant of muscle health and plasticity. We adapted the Imaris software to automatically segment muscle fibers based on fluorescent labeling of the plasma membrane and measure muscle fiber CSA. Analysis of muscle cross sections by the Imaris semiautomated and manual approaches demonstrated a similar decrease in CSA of atrophying muscles from fasted mice compared with fed controls. In addition, we previously demonstrated that downregulation of the Ca

Topics: Animals; Automation, Laboratory; Calpain; Cell Size; Disease Models, Animal; Fasting; Fluorescent Antibody Technique; Image Processing, Computer-Assisted; Male; Mice, Inbred ICR; Microscopy, Confocal; Microscopy, Fluorescence; Muscle Fibers, Skeletal; Muscular Atrophy; Software

A non‑classical calpain, calpain 6 (CAPN6), can inhibit skeletal muscle differentiation and regeneration. In the present study, the role of CAPN6 in the regulation of the autophagy of myoblasts

Topics: Animals; Apoptosis; Autophagy; Calpain; Cell Differentiation; Cytokines; Disease Models, Animal; Humans; Inflammation; Male; Microtubules; Muscle Development; Muscle, Skeletal; Muscular Atrophy; Myoblasts; Rats; Rats, Sprague-Dawley; Renal Insufficiency, Chronic; Signal Transduction

To test the hypothesis that p38α-MAPK plays a critical role in the regulation of E3 ligase expression and skeletal muscle atrophy during unloading, we used VX-745, a selective p38α inhibitor. Three groups of rats were used: non-treated control (C), 3 days of unloading/hindlimb suspension (HS), and 3 days HS with VX-745 inhibitor (HSVX; 10 mg/kg/day). Total weight of soleus muscle in HS group was reduced compared to C (72.3 ± 2.5 vs 83.0 ± 3 mg, respectively), whereas muscle weight in the HSVX group was maintained (84.2 ± 5 mg). The expression of muscle RING-finger protein-1 (MuRF1) mRNA was significantly increased in the HS group (165%), but not in the HSVX group (127%), when compared with the C group. The expression of muscle-specific E3 ubiquitin ligases muscle atrophy F-box (MAFbx) mRNA was increased in both HS and HSVX groups (294% and 271%, respectively) when compared with C group. The expression of ubiquitin mRNA was significantly higher in the HS (423%) than in the C and HSVX (200%) groups. VX-745 treatment blocked unloading-induced upregulation of calpain-1 mRNA expression (HS: 120%; HSVX: 107%). These results indicate that p38α-MAPK signaling regulates MuRF1 but not MAFbx E3 ligase expression and inhibits skeletal muscle atrophy during early stages of unloading.

Topics: Animals; Calpain; Hindlimb Suspension; Interleukin-6; Male; Mitogen-Activated Protein Kinase 14; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Protein Biosynthesis; Protein Kinase Inhibitors; Proteolysis; Pyridazines; Pyrimidines; Rats; Rats, Wistar; SKP Cullin F-Box Protein Ligases; Tripartite Motif Proteins; Ubiquitin; Ubiquitin-Protein Ligases

Unloading leads to skeletal muscle atrophy via the upregulation of MuRF-1 and MAFbx E3-ligases expression. Reportedly, histone deacetylases (HDACs) 4 and 5 may regulate the expression of MuRF1 and MAFbx. To examine the HDAC-dependent mechanisms involved in the control of E3-ubiquitin ligases expression at the early stages of muscle unloading we used HDACs 4 and 5 inhibitor LMK-235 and HDAC 4 inhibitor Tasqinimod (Tq). Male Wistar rats were divided into four groups (eight rats per group): nontreated control (C), three days of unloading/hindlimb suspension (HS) and three days HS with HDACs inhibitor LMK-235 (HSLMK) or Tq (HSTq). Treatment with LMK-235 diminished unloading-induced of MAFbx, myogenin (MYOG), ubiquitin and calpain-1 mRNA expression (

Topics: Animals; Calpain; Hindlimb Suspension; Histone Deacetylases; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Myogenin; Rats; Rats, Wistar; RNA, Messenger; SKP Cullin F-Box Protein Ligases; Tripartite Motif Proteins; Ubiquitin; Ubiquitin-Protein Ligases

The effect of HDACs 4 and 5 on the level of atrophy, calpain-1 and titin content, and TTN gene expression in rat soleus after 7-day gravitational unloading (hindlimb suspension model) was studied. The development of atrophic changes induced by gravitational unloading in rat soleus was accompanied by an increase in the calpain-1 content, an increase in titin proteolysis, and a decrease in the mRNA content of the protein. Inhibition of HDACs 4 and 5 did not eliminate the development of unloading-induced atrophy but significantly prevented proteolysis of titin and the decrease in the TTN gene expression.

Topics: Animals; Benzamides; Calpain; Connectin; Disease Models, Animal; Gene Expression; Hindlimb Suspension; Histone Deacetylase Inhibitors; Histone Deacetylases; Male; Muscle, Skeletal; Muscular Atrophy; Proteolysis; Rats; Rats, Wistar

We examined the lateral gastrocnemius (LG), plantaris (PL), and extensor digitorum longus (EDL) muscles to determine whether differential activation of the calpain system is related to the degree of atrophy in these fast-twitch skeletal muscles during hibernation in Daurian ground squirrels (

Topics: Adaptation, Physiological; Animals; Calcium; Calcium-Binding Proteins; Calpain; Cytosol; Female; Hibernation; Male; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Sciuridae; Troponin T

A calpain-3 (CAPN3) gene heterozygous deletion (c.643_663del21) was recently linked to autosomal dominant (AD) limb-girdle muscular dystrophy. However, the possibility of digenic disease was raised. We describe 3 families with AD calpainopathy carrying this isolated mutation.. Probands heterozygous for CAPN3 c.643_663del21 were identified by targeted next generation or whole exome sequencing. Clinical findings were collected for probands and families. Calpain-3 muscle Western blots were performed in 3 unrelated individuals.. Probands reported variable weakness in their 40s or 50s, with myalgia, back pain, or hyperlordosis. Pelvic girdle muscles were affected with adductor and hamstring sparing. Creatine kinase was normal to 1,800 U/L, independent of weakness severity. Imaging demonstrated lumbar paraspinal muscle atrophy. Electromyographic findings and muscle biopsies were normal to mildly myopathic. Muscle calpain-3 expression was reduced.. This study provides further evidence for AD calpainopathy associated with CAPN3 c.643_663del21. No pathogenic variants in other genes known to cause myopathy were detected. Muscle Nerve 57: 679-683, 2018.

Topics: Adult; Aged; Calpain; Creatine Kinase; DNA Mutational Analysis; Electromyography; Female; Heterozygote; High-Throughput Nucleotide Sequencing; Humans; Male; Middle Aged; Muscle Proteins; Muscle Weakness; Muscular Atrophy; Muscular Dystrophies, Limb-Girdle; Mutation; Paraspinal Muscles; Pedigree; Sequence Analysis, DNA; Sequence Deletion

Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIβ (CaMKIIβ). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIβ signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIβ in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIβ signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and wild type mice to hindlimb unloading and reloading and assessed CaMKIIβ signaling and gene expression by RNA-sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIβ activation and expression of inflammatory and cellular stress genes were increased. C3KO muscles failed to activate CaMKIIβ signaling, did not activate the same pattern of gene expression and demonstrated impaired growth at 4 days of reloading. Moreover, C3KO muscles failed to activate inducible HSP70, which was previously shown to be indispensible for the inflammatory response needed to promote muscle recovery. Likewise, C3KO showed diminished immune cell infiltration and decreased expression of pro-myogenic genes. These data support a role for CaMKIIβ signaling in induction of HSP70 and promotion of the inflammatory response during muscle growth and remodeling that occurs after atrophy, suggesting that CaMKIIβ regulates remodeling in multiple contexts: endurance exercise and growth after atrophy.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calpain; Cell Line; HSP70 Heat-Shock Proteins; Immunohistochemistry; Male; Mice; Mice, Knockout; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy

Myofibril breakdown is a fundamental cause of muscle wasting and inevitable sequel of aging and disease. We demonstrated that myofibril loss requires depolymerization of the desmin cytoskeleton, which is activated by phosphorylation. Here, we developed a mass spectrometry-based kinase-trap assay and identified glycogen synthase kinase 3-β (GSK3-β) as responsible for desmin phosphorylation. GSK3-β inhibition in mice prevented desmin phosphorylation and depolymerization and blocked atrophy upon fasting or denervation. Desmin was phosphorylated by GSK3-β 3 d after denervation, but depolymerized only 4 d later when cytosolic Ca

Topics: Animals; Calcium; Calpain; Desmin; Down-Regulation; Gene Expression Regulation, Developmental; Glycogen Synthase Kinase 3 beta; Male; Mice; Muscular Atrophy; Myofibrils; Phosphorylation

Chronic rotator cuff (RC) tears are characterized by retraction, fat accumulation, and atrophy of the affected muscle. These features pose an intractable problem for surgical repair and subsequent recovery, and their prevention may be easier than reversal. Using an established ovine model, we tested the hypothesis that inhibition of the protease calpain mitigates m. infraspinatus atrophy by preservation of the myofibers' structural anchors in the sarcolemma (the costameres). Already 2 weeks of distal tendon release led to a reduction in muscle volume (-11.6 ± 9.1 cm

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Dipeptides; Female; Muscular Atrophy; Rotator Cuff Injuries; Sarcolemma; Sheep

Previous studies in our lab have shown that tetramethylpyrazine (TMP) could effectively attenuate disuse induced muscle atrophy. In order to screening out the optimal dose of tetramethylpyrazine (TMP) for protection against disuse induced muscle atrophy in hindlimb unloading (HLU) rats, in this study, we compared effects of 4 TMP doses on muscle wet weight (MWW), the ratios of muscle wet weight/body weight (MWW/BW) and muscle wet weight/dry weight (MWW/DW), fiber type composition, as well as cross-sectional area (CSA) in soleus (SOL) muscle. Consequently, we quantified optimal dose effects on both functional properties and protein expression (calpain-1, calpain-2, calpastatin and MuRF1) in SOL and extensor digitorum longus (EDL) muscles. Data indicated that the protective potential of TMP was dose-dependent: 60mg/kg TMP was most effective in terms of atrophy prevention. This dose reduced SOL MWW, MWW/BW and CSA muscle loss by 60, 60 and 54% (P<0.001), respectively. HLU-induced slow-to-fast fiber transition was reduced by 17% (P<0.01). 60mg/kg TMP also significantly lessened the decrease of contractile force, the increase of shorting velocity and fatigability induced by HLU. Besides, it also attenuated expressions of calpain-1 (SOL -8.6%, P<0.05; EDL -10.9%, P<0.05), calpain-2 (SOL -60%, P<0.001; EDL -32%, P<0.01) and MuRF1 expression (SOL -21%, P<0.001; EDL -10%, P<0.01), promoted the expression of calpastatin by 18% (P<0.05) in SOL muscle. Taken together, present study demonstrated that 60mg/kg body weight was the optimal dose of TMP against disuse induced muscle atrophy which effectively protected muscle function by inhibiting calpain-1, calpain-2 and MuRF1 expression, promoted calpastatin expression, especially in slow-twitch muscle.

Topics: Animals; Calpain; Dose-Response Relationship, Drug; Female; Hindlimb Suspension; Muscle Contraction; Muscle, Skeletal; Muscular Atrophy; Pyrazines; Rats; Rats, Sprague-Dawley; Vasodilator Agents

The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12 cells were examined, including mRNA expression of myogenic regulatory factors, such as MyoD and myogenin, inflammatory pathways, such as TNF-α and NF-kB, as well as proteolytic enzymes, such as μ-calpain and m-calpain. The pre-treatment of Sunphenon (50 μg/mL)/Polyphenon 60 (50 μg/mL) on H2O2-treated C2C12 cells significantly down-regulated the mRNA expression of myogenin and MyoD when compared to those treated with H2O2-induced alone. Additionally, the mRNA expression of μ-calpain and m-calpain were significantly(p<0.05) increased in H2O2-treated C2C12 cells, whereas pre-treatment with Sunphenon/Polyphenon significantly down-regulated the above genes, namely μ-calpain and m-calpain. Furthermore, the mRNA expression of TNF-α and NF-kB were significantly increased in H2O2-treated C2C12 cells, while pre-treatment with Sunphenon (50 μg/mL)/Polyphenon 60 (50 μg/mL) significantly (p<0.05) down-regulated it when compared to the untreated control group.Subsequent analysis of DNA degeneration and caspase activation revealed that Sunphenon (50 μg/mL)/Polyphenon 60 (50 μg/mL) inhibited activation of caspase-3 and showed an inhibitory effect on DNA degradation. From this result, we know that, in stress conditions, μ-calpain may be involved in the muscle atrophy through the suppression of myogenin and MyoD. Moreover, Sunphenon may regulate the skeletal muscle genes/promote skeletal muscle recovery by the up-regulation of myogenin and MyoD and suppression of μ-calpain and inflammatory pathways and may regulate the apoptosis pathways. Our findings suggest that dietary supplementation of Sunphenon might reduce inflammatory events in muscle-associated diseases, such as myotube atrophy.

Topics: Animals; Apoptosis; Calpain; Caspase 3; Cell Line; Enzyme Activation; Flavonoids; Hydrogen Peroxide; Inflammation; Mice; Muscle, Skeletal; Muscular Atrophy; MyoD Protein; Myogenin; NF-kappa B; Oxidative Stress; Phenols; RNA, Messenger; Tumor Necrosis Factor-alpha

Unloading causes rapid skeletal muscle atrophy due to increased protein degradation via activation of calpains and decreased protein synthesis. Our study elucidated role of calpain-1 in the regulation of ubiquitin proteasome pathway (UPP) and anabolic processes mediated by Akt-mTOR-p70S6K and MAPK-Erk (p90RSK) signaling. We hypothesized that blocking calpain will inhibit activation of UPP and decrease protein degradation resulting in reduction of unloading-induced skeletal muscle atrophy. Rats were divided into three groups: non-treated control (C), three day hindlimb suspension with (HSPD) or without (HS) treatment with calpain inhibitor PD150606. When compared with control PD150606 treatment during unloading: 1) attenuated loss of muscle mass, 2) prevented accumulation of calpain-1 (1.8-fold in HS vs 1.3-fold in HSPD) and ubiquitin (2.3-fold in HS vs 0.7-fold in HSPD) mRNA and ubiquitinated proteins (1.6-fold in HS vs 0.8-fold in HSPD), 3) prevented decrease in the pAkt (0.4-fold in HS vs 1-fold in HSPD) and pFOXO3 (0.2-fold in HS vs 1.2-fold in HSPD) levels, 4) prevented increase in MAFbx (3.8-fold in HS vs 1.3-fold in HSPD) and eEF2k (1.8-fold in HS vs 0.6-fold in HSPD) mRNA. Our study indicates that blocking of calpain during unloading decreases skeletal muscle atrophy by inhibiting UPP activation and preserving anabolic signaling.

Topics: Acrylates; Animals; Calpain; Immobilization; Male; MAP Kinase Signaling System; Muscle Proteins; Muscular Atrophy; Proteolysis; Rats; Rats, Wistar

Topics: Adult; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Calpain; Humans; Macrophages; Male; Muscle Proteins; Muscular Atrophy; Muscular Dystrophies, Limb-Girdle; Mutation; Neuromuscular Junction

Calpain 3 (CAPN3), also known as p94, is a skeletal muscle-specific member of the calpain family that is involved in muscular dystrophy; however, the roles of CAPN3 in muscular atrophy and regeneration are yet to be understood. In the present study, we attempted to explain the effect of CAPN3 in muscle atrophy by evaluating CAPN3 expression in rat gastrocnemius muscle following reversible sciatic nerve injury. After nerve injury, the wet weight ratio and cross sectional area (CSA) of gastrocnemius muscle were decreased gradually from 1-14 days and then recovery from 14-28 days. The active form of CAPN3 (~62 kDa) protein decreased slightly on day 3 and then increased from day 7 to 14 before a decrease from day 14 to 28. The result of linear correlation analysis showed that expression of the active CAPN3 protein level was negatively correlated with muscle wet weight ratio. CAPN3 knockdown by short interfering RNA (siRNA) injection improved muscle recovery on days 7 and 14 after injury as compared to that observed with control siRNA treatment. Depletion of CAPN3 gene expression could promote myoblast differentiation in L6 cells. Based on these findings, we conclude that the expression pattern of the active CAPN3 protein is linked to muscle atrophy and regeneration following denervation: its upregulation during early stages may promote satellite cell renewal by inhibiting differentiation, whereas in later stages, CAPN3 expression may be downregulated to stimulate myogenic differentiation and enhance recovery. These results provide a novel mechanistic insight into the role of CAPN3 protein in muscle regeneration after peripheral nerve injury.

Topics: Animals; Calpain; Cell Differentiation; Cell Line; Disease Models, Animal; Gene Expression Regulation; Gene Knockdown Techniques; Isoenzymes; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Myoblasts; MyoD Protein; Peripheral Nerve Injuries; Rats; Regeneration; RNA, Messenger; RNA, Small Interfering; Sciatic Neuropathy

The influence of electrical stimulation on calpain and ubiquitin-proteasome systems was examined in the denervated and unloaded tibialis anterior muscles of male Wistar rats. Animals were divided into 5 groups: control, denervation, denervation plus electrical stimulation, unloading, and hindlimb unloading plus electrical stimulation groups. Due to denervation and unloading for 14 days, muscle atrophy markedly occurred in the denervated and unloading animals, and the atrophy in the former was significantly more severe than that in the latter. In the denervated muscle, the atrophy was significantly attenuated by the electrical stimulation, but not in the unloaded muscle. Overexpression of calpain-2 and ubiquitinated proteins was observed only in denervated muscles. In the unloaded animals, though the expression level of calpain-2 appeared to be slightly higher than that in the control, the expression level of ubiquitinated proteins was almost the same as that in the control. The overexpression of calpain-1, calpain-2, and ubiquitinated proteins in the denervated muscle was inhibited by the electrical stimulation. However, there was no difference in these expressions between the unloaded and unloaded plus electrical stimulation groups. The mechanism of the preventive effect of the electrical stimulation on muscle atrophy might differ between the denervated and unloaded muscles.

Topics: Animals; Body Composition; Calpain; Electric Stimulation; Hindlimb Suspension; Male; Muscle, Skeletal; Muscular Atrophy; Proteasome Endopeptidase Complex; Rats; Rats, Wistar; Ubiquitin

We investigated changes in muscle mass, calpains, calpastatin and Z-disk ultrastructure in the soleus muscle (SOL) of Daurian ground squirrels (Spermophilus dauricus) after hibernation or hindlimb suspension to determine possible mechanisms by which muscle atrophy is prevented in hibernators. Squirrels (n=30) were divided into five groups: no hibernation group (PRE, n=6); hindlimb suspension group (HLS, n=6); two month hibernation group (HIB, n=6); two day group after 90±12 days of hibernation (POST, n=6); and forced exercise group (one time forced, moderate-intensity treadmill exercise) after arousal (FE, n=6). Activity and protein expression of calpains were determined by casein zymography and western blotting, and Z-disk ultrastructure was observed by transmission electron microscopy. The following results were found. Lower body mass and higher SOL muscle mass (mg) to total body mass (g) ratio were observed in HIB and POST; calpain-1 activity increased significantly by 176% (P=0.034) in HLS compared to the PRE group; no significant changes were observed in calpain-2 activity. Protein expression of calpain-1 and calpain-2 increased by 83% (P=0.041) and 208% (P=0.029) in HLS compared to the PRE group, respectively; calpastatin expression increased significantly by 180% (P<0.001) and 153% (P=0.007) in HIB and POST, respectively; the myofilaments were well-organized, and the width of the sarcomere and the Z-disk both appeared visually similar among the pre-hibernation, hibernating and post-hibernation animals. Inhibition of calpain activity and consequently calpain-mediated protein degradation by highly elevated calpastatin protein expression levels may be an important mechanism for preventing muscle protein loss during hibernation and ensuring that Z-lines remained ultrastructurally intact.

Topics: Animals; Calcium-Binding Proteins; Calpain; Female; Hibernation; Hindlimb; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Sciuridae

Skeletal muscle undergoes significant atrophy in Type 2 diabetic patients and animal models. We aimed to determine if atrophy of Zucker rat skeletal muscle was due to the activation of intracellular damage pathways induced by excess reactive oxygen species production (specifically those associated with the peroxidation of lipid membranes) and calpain activity. 14 week old obese Zucker rats and littermate lean controls were injected with 1% Evan's Blue Dye. Animals were anaesthetised and extensor digitorum longus and soleus muscles were dissected, snap frozen and analysed for ROS-mediated F2-isoprostane production and calpain activation/autolysis. Contralateral muscles were histologically analysed for markers of muscle membrane permeability and atrophy.. Muscle mass was lower in extensor digitorum longus and soleus of obese compared with lean animals, concomitant with reduced fibre area. Muscles from obese rats had a higher proportional area of Evan's Blue Dye fluorescence, albeit this was localised to the interstitium/external sarcolemma. There were no differences in F2-isoprostane production when expressed relative to arachidonic acid content, which was lower in the obese EDL and soleus muscles. There were no differences in the activation of either μ-calpain or calpain-3.. This study highlights that atrophy of Zucker rat skeletal muscle is not related to sarcolemmal damage, sustained hyperactivation of the calpain proteases or excessive lipid peroxidation. As such, establishing the correct pathways involved in atrophy is highly important so as to develop more specific treatment options that target the underlying cause. This study has eliminated two of the potential pathways theorised to be responsible.

Topics: Animals; Blood Glucose; Body Weight; Calpain; Lipid Peroxidation; Male; Muscle, Skeletal; Muscular Atrophy; Obesity; Oxidative Stress; Rats; Rats, Zucker

The peculiar clinical features and the pathogenic mechanism related to calpain-3 deficiency (impaired sarcomere remodelling) suggest that the ubiquitin-proteasome degradation pathway may have a crucial role in Limb Girdle Muscular Dystrophy 2A (LGMD2A). We therefore investigated muscle atrophy and the role of the ubiquitin-proteasome and lysosomal-autophagic degradation pathways.. We selected 25 adult male LGMD2A patients (and seven controls), classified them using clinical severity score, analysed muscle fibre size by morphometry and protein and/or transcriptional expression levels of the most important atrophy- and autophagy-related genes (MuRF1, atrogin1, LC3, p62, Bnip3).. Muscle fibre size was significantly lower in LGMD2A than in controls and it was significantly correlated with patients' clinical disability score recorded at the time of biopsy, suggesting that functional and structural muscle impairment are dependent. The large majority of atrophic fibres originate from a mechanism different from regeneration, as assessed by neonatal myosin immunolabelling. As compared with controls, LGMD2A muscles have higher MuRF1 (but not atrogin1) protein and MuRF1 gene expression levels, and MuRF1 protein levels significantly correlated with both muscle fibre size and clinical disability score. LGMD2A muscles have slightly increased levels of LC3-II and p62 proteins and a significant up-regulation of p62 and Bnip3 gene expression.. In LGMD2A muscles the activation of the atrophy programme appeared to depend mainly upon induction of the ubiquitin-proteasome system and, to a lesser extent, the autophagic-lysosomal degradation pathway.

Topics: Adolescent; Adult; Calpain; Humans; Male; Middle Aged; Muscle Proteins; Muscular Atrophy; Muscular Dystrophies, Limb-Girdle; Proteasome Endopeptidase Complex; Regeneration; Ubiquitin; Up-Regulation; Young Adult

Prolonged skeletal muscle inactivity results in a rapid decrease in fiber size, primarily due to accelerated proteolysis. Although several proteases are known to contribute to disuse muscle atrophy, the ubiquitin proteasome system is often considered the most important proteolytic system during many conditions that promote muscle wasting. Emerging evidence suggests that calpain and caspase-3 may also play key roles in inactivity-induced atrophy of respiratory muscles, but it remains unknown if these proteases are essential for disuse atrophy in limb skeletal muscles. Therefore, we tested the hypothesis that activation of both calpain and caspase-3 is required for locomotor muscle atrophy induced by hindlimb immobilization. Seven days of immobilization (i.e., limb casting) promoted significant atrophy in type I muscle fibers of the rat soleus muscle. Independent pharmacological inhibition of calpain or caspase-3 prevented this casting-induced atrophy. Interestingly, inhibition of calpain activity also prevented caspase-3 activation, and, conversely, inhibition of caspase-3 prevented calpain activation. These findings indicate that a regulatory cross talk exists between these proteases and provide the first evidence that the activation of calpain and caspase-3 is required for inactivity-induced limb muscle atrophy.

Topics: Animals; Calpain; Caspase 3; Extremities; Female; Hindlimb Suspension; Mitochondria; Muscle Fibers, Slow-Twitch; Muscular Atrophy; Proteasome Endopeptidase Complex; Proteolysis; Rats; Rats, Sprague-Dawley

Muscle atrophy is a disease that is usually caused by denervation. The aim of the present study was to determine whether electrical stimulation by semi-implantable electrodes is capable of decreasing the levels of specific proteins associated with sciatic nerve injury-induced muscle atrophy. Male Sprague Dawley (SD) rats with damaged sciatic nerves were maintained on a 12‑h light/dark cycle. Thirty-two SD rats were randomly allocated into 4 groups (each group, n=8). The rats in group C received no electrical stimulation; the rats in groups D, N and DN received electrical stimulation by semi-implantable electrodes during the daytime alone, nighttime alone and both the daytime and nighttime, respectively. Immunoblot assays were performed to detect the expression of cellular proteins associated with muscle atrophy. The number of muscle satellite cells was determined using a microscope, indicating that electrical stimulation increased the number of muscle satellite cells. Immunoblot assay results showed that electrical stimulation reduced the expression levels of cathepsin L, calpain 1 and the ubiquitinated muscle ring finger‑1 (MuRF-1) protein. In conclusion, electrical stimulation by semi-implantable electrodes constitutes a potential method for the treatment of sciatic nerve injury-induced muscle atrophy. The decreased expression levels of the cellular proteins cathepsin L and calpain 1, as well as the ubiquitinated protein MuRF-1, are associated with the attenuation of sciatic nerve injury-induced muscle atrophy.

Topics: Animals; Body Weight; Calpain; Cathepsin L; Electric Stimulation; Electrodes, Implanted; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Organ Size; Peripheral Nerve Injuries; Rats; Satellite Cells, Skeletal Muscle; Sciatic Neuropathy; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Ubiquitinated Proteins

Long periods of skeletal muscle disuse result in muscle fiber atrophy, and mitochondrial production of reactive oxygen species (ROS) appears to be a required signal for the increase in protein degradation that occurs during disuse muscle atrophy. The experiments detailed here demonstrate for the first time in limb muscle that the inactivity-induced increases in E3 ligase expression and autophagy biomarkers result from increases in mitochondrial ROS emission. Treatment of animals with a mitochondrial-targeted antioxidant also prevented the disuse-induced decrease in anabolic signaling (Akt/mammalian target of rapamycin signaling) that is normally associated with prolonged inactivity in skeletal muscles. Additionally, our results confirm previous findings that treatment with a mitochondrial-targeted antioxidant is sufficient to prevent casting-induced skeletal muscle atrophy, mitochondrial dysfunction, and activation of the proteases calpain and caspase-3. Collectively, these data reveal that inactivity-induced increases in mitochondrial ROS emission play a required role in activation of key proteolytic systems and the downregulation of important anabolic signaling molecules in muscle fibers exposed to prolonged inactivity.

Topics: Animals; Antioxidants; Biomarkers; Calpain; Caspase 3; Down-Regulation; Female; Immobilization; Mitochondria; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Peptide Hydrolases; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Proteasome Endopeptidase Complex; Proteolysis; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Transcription Factors; Ubiquitin-Protein Ligases

Controlled mechanical ventilation (CMV) is known to result in rapid and severe diaphragmatic dysfunction, but the recovery response of the diaphragm to normal function after CMV is unknown. Therefore, we examined the time course of diaphragm function recovery in an animal model of CMV. Healthy rats were submitted to CMV for 24-27 h (n = 16), or to 24-h CMV followed by either 1 h (CMV + 1 h SB, n = 9), 2 h (CMV + 2 h SB, n = 9), 3 h (CMV + 3 h SB, n = 9), or 4-7 h (CMV + 4-7 h SB, n = 9) of spontaneous breathing (SB). At the end of the experiment, the diaphragm muscle was excised for functional and biochemical analysis. The in vitro diaphragm force was significantly improved in the CMV + 3 h SB and CMV + 4-7 h SB groups compared with CMV (maximal tetanic force: +27%, P < 0.05, and +59%, P < 0.001, respectively). This was associated with an increase in the type IIx/b fiber dimensions (P < 0.05). Neutrophil influx was increased in the CMV + 4-7 h SB group (P < 0.05), while macrophage numbers remained unchanged. Markers of protein synthesis (phosphorylated Akt and eukaryotic initiation factor 4E binding protein 1) were significantly increased (±40%, P < 0.001, and ±52%, P < 0.01, respectively) in the CMV + 3 h SB and CMV + 4-7 h SB groups and were positively correlated with diaphragm force (P < 0.05). Finally, also the maximal specific force generation of skinned single diaphragm fibers was increased in the CMV + 4-7 h SB group compared with CMV (+45%, P < 0.05). In rats, reloading the diaphragm for 3 h after CMV is sufficient to improve diaphragm function, while complete recovery occurs after longer periods of reloading. Enhanced muscle fiber dimensions, increased protein synthesis, and improved intrinsic contractile properties of diaphragm muscle fibers may have contributed to diaphragm function recovery.

Topics: Animals; Calpain; Carrier Proteins; Caspase 3; Diaphragm; Intracellular Signaling Peptides and Proteins; Male; Models, Animal; Muscle Contraction; Muscle Fibers, Skeletal; Muscle Proteins; Muscular Atrophy; Neutrophils; Oxidative Stress; Phosphoproteins; Proteolysis; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Recovery of Function; Respiration, Artificial; Time Factors; Ventilator Weaning

Vitamin D deficiency leads to muscle wasting in both animals and humans. A vitamin D-deficient rat model was created using Sprague Dawley male rats. We studied the involvement of the ubiquitin proteasome and other proteolytic pathways in vitamin D deficiency-induced muscle atrophy. To delineate the effect of hypocalcemia that accompanies D deficiency, a group of deficient rats was supplemented with high calcium alone. Total protein degradation in muscle was assessed by release of tyrosine; proteasomal, lysosomal, and calpain enzyme activities were studied using specific substrates by fluorometry, and E2 enzyme expression was assessed by Western blot analysis. Muscle histology was done by myosin ATPase staining method, whereas 3-methylhistidine in the urine was estimated using HPLC. Muscle gene expression was measured by semiquantitative RT-PCR. Total protein degradation in muscle and the level of 3-methylhistidine in urine were increased in the deficient group compared with the control group. Proteasomal enzyme activities, expression of the E2 ubiquitin conjugating enzyme, and ubiquitin conjugates were increased in the deficient group compared with controls. On the other hand, lysosomal and calpain activities were not altered. Type II fiber area, a marker for muscle atrophy, was decreased in the deficient muscle compared with control muscle. Muscle atrophy marker genes and proteasomal subunit genes were up-regulated, whereas myogenic genes were down-regulated in D-deficient muscle. From the results it appears that the ubiquitin proteasome pathway is the major pathway involved in vitamin D deficiency-induced muscle protein degradation and that calcium supplementation alone in the absence of vitamin D partially corrects the changes.

Topics: Animals; Body Composition; Body Weight; Calcium; Calpain; Gene Expression Regulation; Hypocalcemia; Lysosomes; Male; Muscle Fibers, Skeletal; Muscle Proteins; Muscular Atrophy; Random Allocation; Rats; Rats, Sprague-Dawley; Ubiquitin; Ubiquitin-Conjugating Enzymes; Vitamin D Deficiency

Muscle unloading due to long-term exposure of weightlessness or simulated weightlessness causes atrophy, loss of functional capacity, impaired locomotor coordination, and decreased resistance to fatigue in the antigravity muscles of the lower limbs. Besides reducing astronauts' mobility in space and on returning to a gravity environment, the molecular mechanisms for the adaptation of skeletal muscle to unloading also play an important medical role in conditions such as disuse and paralysis. The tail-suspended rat model was used to simulate the effects of weightlessness on skeletal muscles and to induce muscle unloading in the rat hindlimb. Our series studies have shown that the maximum of twitch tension and the twitch duration decreased significantly in the atrophic soleus muscles, the maximal tension of high-frequency tetanic contraction was significantly reduced in 2-week unloaded soleus muscles, however, the fatigability of high-frequency tetanic contraction increased after one week of unloading. The maximal isometric tension of intermittent tetanic contraction at optimal stimulating frequency did not alter in 1- and 2-week unloaded soleus, but significantly decreased in 4-week unloaded soleus. The 1-week unloaded soleus, but not extensor digitorum longus (EDL), was more susceptible to fatigue during intermittent tetanic contraction than the synchronous controls. The changes in K+ channel characteristics may increase the fatigability during high-frequency tetanic contraction in atrophic soleus muscles. High fatigability of intermittent tetanic contraction may be involved in enhanced activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and switching from slow to fast isoform of myosin heavy chain, tropomyosin, troponin I and T subunit in atrophic soleus muscles. Unloaded soleus muscle also showed a decreased protein level of neuronal nitric oxide synthase (nNOS), and the reduction in nNOS-derived NO increased frequency of calcium sparks and elevated intracellular resting Ca2+ concentration ([Ca2+]i) in unloaded soleus muscles. High [Ca2+]i activated calpain-1 which induced a higher degradation of desmin. Desmin degradation may loose connections between adjacent myofibrils and further misaligned Z-disc during repeated tetanic contractions. Passive stretch in unloaded muscle could preserve the stability of sarcoplasmic reticulum Ca2+ release channels by means of keeping nNOS activity, and decrease the enhanced protein level and activity of calpain to

Topics: Animals; Calcium Signaling; Calpain; Desmin; Muscle Contraction; Muscle Fatigue; Muscle, Skeletal; Muscular Atrophy; Myosin Heavy Chains; Rats; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Weightlessness Simulation

Previous workers have demonstrated that controlled mechanical ventilation results in diaphragm inactivity and elicits a rapid development of diaphragm weakness as a result of both contractile dysfunction and fiber atrophy. Limited data exist regarding the impact of pressure support ventilation, a commonly used mode of mechanical ventilation-that permits partial mechanical activity of the diaphragm-on diaphragm structure and function. We carried out the present study to test the hypothesis that high-level pressure support ventilation decreases the diaphragm pathology associated with CMV.. Sprague-Dawley rats were randomly assigned to one of the following five groups:1) control (no mechanical ventilation); 2) 12 hrs of controlled mechanical ventilation (12CMV); 3) 18 hrs of controlled mechanical ventilation (18CMV); 4) 12 hrs of pressure support ventilation (12PSV); or 5) 18 hrs of pressure support ventilation (18PSV).. We carried out the following measurements on diaphragm specimens: 4-hydroxynonenal-a marker of oxidative stress, active caspase-3 (casp-3), active calpain-1 (calp-1), fiber type cross-sectional area, and specific force (sp F). Compared with the control, both 12PSV and 18PSV promoted a significant decrement in diaphragmatic specific force production, but to a lesser degree than 12CMV and 18CMV. Furthermore, 12CMV, 18PSV, and 18CMV resulted in significant atrophy in all diaphragm fiber types as well as significant increases in a biomarker of oxidative stress (4-hydroxynonenal) and increased proteolytic activity (20S proteasome, calpain-1, and caspase-3). Furthermore, although no inspiratory effort occurs during controlled mechanical ventilation, it was observed that pressure support ventilation resulted in large decrement, approximately 96%, in inspiratory effort compared with spontaneously breathing animals.. High levels of prolonged pressure support ventilation promote diaphragmatic atrophy and contractile dysfunction. Furthermore, similar to controlled mechanical ventilation, pressure support ventilation-induced diaphragmatic atrophy and weakness are associated with both diaphragmatic oxidative stress and protease activation.

Topics: Aldehydes; Animals; Calpain; Caspase 3; Cytokines; Diaphragm; Interactive Ventilatory Support; Muscle Contraction; Muscular Atrophy; Oxidative Stress; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Respiration, Artificial

Diaphragmatic weakness, due to both atrophy and contractile dysfunction, is a well-documented response following prolonged mechanical ventilation. Evidence indicates that activation of the proteases calpain and caspase-3 is essential for mechanical ventilation-induced diaphragmatic weakness to occur. We tested the hypothesis that a regulatory cross-talk exists between calpain and caspase-3 in the diaphragm during prolonged mechanical ventilation. To test this prediction, we determined whether selective pharmacological inhibition of calpain would prevent activation of caspase-3 and conversely whether selective inhibition of caspase-3 would abate calpain activation.. Animal study.. University Research Laboratory.. Female Sprague-Dawley rats.. Animals were randomly divided into control or one of three 12-hr mechanical ventilation groups that were treated with/without a selective pharmacological protease inhibitor: 1) control, 2) mechanical ventilation, 3) mechanical ventilation with a selective caspase-3 inhibitor, and 4) mechanical ventilation with a selective calpain inhibitor.. Compared to control, mechanical ventilation resulted in calpain and caspase-3 activation in the diaphragm accompanied by atrophy of type I, type IIa, and type IIx/IIb fibers. Independent inhibition of either calpain or caspase-3 prevented this mechanical ventilation-induced atrophy. Pharmacological inhibition of calpain prevented mechanical ventilation-induced activation of diaphragmatic caspase-3 and inhibition of caspase-3 prevented activation of diaphragmatic calpain. Furthermore, calpain inhibition also prevented the activation of caspase-9 and caspase-12, along with the cleavage of Bid to tBid, all upstream signals for caspase-3 activation. Lastly, caspase-3 inhibition prevented the mechanical ventilation-induced degradation of the endogenous calpain inhibitor, calpastatin.. Collectively, these results indicate that mechanical ventilation-induced diaphragmatic atrophy is dependent on the activation of both calpain and caspase-3. Importantly, these findings provide the first experimental evidence in diaphragm muscle that calpain inhibition prevents the activation of caspase-3 and vice versa and caspase-3 inhibition prevents the activation of calpain. These findings support our hypothesis that a regulatory calpain/caspase-3 cross-talk exists whereby calpain can promote caspase-3 activation and active caspase-3 can enhance calpain activity in diaphragm muscle during prolonged mechanical ventilation.

Topics: Animals; Calpain; Caspase 3; Caspase Inhibitors; Diaphragm; Enzyme Activation; Female; Muscular Atrophy; Proteolysis; Random Allocation; Rats; Rats, Sprague-Dawley; Respiration, Artificial; Signal Transduction; Time Factors

Deletion of muscle RING finger 1 (MuRF1), an E3 ubiquitin ligase, leads to sparing of muscle mass following denervation. The purpose of this study was to test the hypothesis that muscle sparing in mice with a deletion of MuRF1 is due to the selective inhibition of the ubiquitin proteasome system. Activities of the 20S and 26S proteasomes, calpain and cathepsin L, were measured in the triceps surae muscles of wild-type (WT) and MuRF1-knockout (KO) mice at 3 and 14 d following denervation. In addition, fractional protein synthesis rates and differential gene expression were measured in WT and KO muscle. The major finding was that 20S and 26S proteasome activities were significantly elevated (1.5- to 2.5-fold) after 14 d of denervation in both WT and KO mice relative to control, but interestingly, the activities of both the 20S and 26S proteasome were significantly higher in KO than WT mice. Further, mRNA expression of MAFbx was elevated after 14 d of denervation in KO, but not WT, mice. These data challenge the conventional dogma that MuRF1 is controlling the degradation of only contractile proteins and suggest a role for MuRF1 in the global control of the ubiquitin proteasome system and protein turnover.

Topics: Animals; Arabidopsis Proteins; Autophagy; Calpain; Cathepsin L; Female; Intramolecular Transferases; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Muscle Denervation; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Proteasome Endopeptidase Complex; SKP Cullin F-Box Protein Ligases; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Up-Regulation

Unloading in spaceflight or long-term bed rest induces to pronounced atrophy of anti-gravity skeletal muscles. Passive stretch partially resists unloading-induced atrophy of skeletal muscle, but the mechanism remains elusive. The aims of this study were to investigate the hypotheses that stretch tension might increase protein level of neuronal nitric oxide synthase (nNOS) in unloaded skeletal muscle, and then nNOS-derived NO alleviated atrophy of skeletal muscle by inhibiting calpain activity. The tail-suspended rats were used to unload rat hindlimbs for 2 weeks, at the same time, left soleus muscle was stretched by applying a plaster cast to fix the ankle at 35° dorsiflexion. Stretch partially resisted atrophy and inhibited the decreased protein level and activity of nNOS in unloaded soleus muscles. Unloading increased frequency of calcium sparks and elevated intracellular resting and caffeine-induced Ca(2+) concentration ([Ca(2+)]i) in unloaded soleus muscle fibers. Stretch reduced frequency of calcium sparks and restored intracellular resting and caffeine-induced Ca(2+) concentration to control levels in unloaded soleus muscle fibers. The increased protein level and activity of calpain as well as the higher degradation of desmin induced by unloading were inhibited by stretch in soleus muscles. In conclusion, these results suggest that stretch can preserve the stability of sarcoplasmic reticulum Ca(2+) release channels which prevents the elevated [Ca(2+)]i by means of keeping nNOS activity, and then the enhanced protein level and activity of calpain return to control levels in unloaded soleus muscles. Therefore, stretch can resist in part atrophy of unloaded soleus muscles.

Topics: Animals; Calcium; Calcium Channels, L-Type; Calcium Signaling; Calpain; Desmin; Hindlimb Suspension; Male; Muscle Fibers, Skeletal; Muscle Proteins; Muscle Stretching Exercises; Muscle Tonus; Muscle, Skeletal; Muscular Atrophy; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Protein Isoforms; Proteolysis; Rats; Rats, Sprague-Dawley; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

We hypothesized that pharmacological induction of HSP70 would attenuate soleus atrophy development under 3 d of rat hindlimb unloading. Male Wistar rats were divided into control (C; n=7), 3-d hindlimb unloading (HUL; n=7), HUL with HSP90 inducer administration, 17-allylamino-17-emethoxygeldanamycin (17-AAG; 60 mg/kg, HUL+17-AAG, n=8). The relative weight of soleus muscle to body weight [soleus wt (mg)/body wt (g)] in the HUL group was less than that of the C and HUL+17-AAG groups (P<0.05). We revealed HSP90, HSP70 mRNA decrease in the HUL group (but not the HUL+17-AAG group) vs. C (P<0.05). The unloading resulted in significant increases of μ-calpain and conjugated ubiquitin (Ub) levels (proteins as well as mRNAs) vs. the C group, whereas 17-AAG administration prevented these alterations (studied by SDS-PAGE and RT-PCR). pFOXO3 protein was decreased in the HUL group vs. C, but not in HUL+17-AAG. Content of E3-lygase (MuRF-1, MAFbx) mRNA was increased in both suspended groups. In summary, 17-AAG administration attenuates soleus muscle atrophy, μ-calpain, and Ub increases under hindlimb unloading as well as decrease of pFOXO3.

Topics: Animals; Benzoquinones; Blotting, Western; Calpain; Hindlimb; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Male; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Wistar; Ubiquitin; Ubiquitin-Protein Ligases

Controlled mechanical ventilation leads to diaphragmatic contractile dysfunction and atrophy. Since proteolysis is enhanced in the diaphragm during controlled mechanical ventilation, we examined whether the administration of a proteasome inhibitor, bortezomib, would have a protective effect against ventilator-induced diaphragm dysfunction.. Randomized, controlled experiment.. Basic science animal laboratory.. Anesthetized rats were submitted for 24 hrs to controlled mechanical ventilation while receiving 0.05 mg/kg bortezomib or saline. Control rats were acutely anesthetized.. After 24 hrs, diaphragm force production was significantly lower in mechanically ventilated animals receiving an injection of saline compared to control animals (-36%, p<.001). Importantly, administration of bortezomib improved the diaphragmatic force compared to mechanically ventilated animals receiving an injection of saline (+15%, p<.01), but force did not return to control levels. Compared to control animals, diaphragm cross-sectional area of the type IIx/b fibers was significantly decreased by 28% in mechanically ventilated animals receiving an injection of saline (p<.01) and by 16% in mechanically ventilated animals receiving an injection of bortezomib (p<.05). Diaphragmatic calpain activity was significantly increased in mechanically ventilated animals receiving an injection of saline (+52%, p<.05) and in mechanically ventilated animals receiving an injection of bortezomib (+36%, p<.05). Caspase-3 activity was increased after controlled mechanical ventilation with saline by 55% (p<.05), while it remained similar to control animals in mechanically ventilated animals receiving an injection of bortezomib. Diaphragm 20S proteasome activity was slightly increased in both ventilated groups, and the amount of ubiquitinated proteins was significantly and similarly enhanced in mechanically ventilated animals receiving an injection of saline and mechanically ventilated animals receiving an injection of bortezomib.. These data show that the administration of bortezomib partially protects the diaphragm from controlled mechanical ventilation-induced diaphragm contractile dysfunction without preventing atrophy. The fact that calpain activity was still increased after bortezomib treatment may explain the persistence of atrophy. Part of bortezomib effects might have been due to its ability to inhibit caspase-3 in this model.

Topics: Animals; Boronic Acids; Bortezomib; Calpain; Caspase 3; Diaphragm; Male; Muscle Contraction; Muscular Atrophy; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Rats; Rats, Wistar; Respiration, Artificial

Ullrich congenital muscular dystrophy (UCMD) is a common form of muscular dystrophy associated with defects in collagen VI. It is characterized by loss of individual muscle fibers and muscle mass and proliferation of connective and adipose tissues. We sought to investigate the mechanisms by which collagen VI regulates muscle cell survival, size, and regeneration and, in particular, the potential role of the ubiquitin-proteasome and calpain-proteolytic systems. We studied muscle biopsies of UCMD (n = 6), other myopathy (n = 12), and control patients (n = 10) and found reduced expression of atrogin-1, MURF1, and calpain-3 mRNAs in UCMD cases. Downregulation of calpain-3 was associated with changes in the nuclear immunolocalization of nuclear factor-κB. We also observed increased expression versus controls of regeneration markers at the protein and RNA levels. Satellite cell numbers did not differ in collagen VI-deficient muscle versus normal nonregenerating muscle, indicating that collagen VI does not play a key role in the maintenance of the satellite cell pool. Our results indicate that alterations in calpain-3 and nuclear factor-κB signaling pathways may contribute to muscle mass loss in UCMD muscle, whereas atrogin-1 and MURF1 are not likely to play a major role.

Topics: Calpain; Child; Child, Preschool; Collagen Type VI; Female; Humans; Male; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies; NF-kappa B; Regeneration; Signal Transduction; Young Adult

Diaphragmatic function is a major determinant of the ability to successfully wean patients from mechanical ventilation (MV). Paradoxically, MV itself results in a rapid loss of diaphragmatic strength in animals. However, very little is known about the time course or mechanistic basis for such a phenomenon in humans.. To determine in a prospective fashion the time course for development of diaphragmatic weakness during MV; and the relationship between MV duration and diaphragmatic injury or atrophy, and the status of candidate cellular pathways implicated in these phenomena.. Airway occlusion pressure (TwPtr) generated by the diaphragm during phrenic nerve stimulation was measured in short-term (0.5 h; n = 6) and long-term (>5 d; n = 6) MV groups. Diaphragmatic biopsies obtained during thoracic surgery (MV for 2-3 h; n = 10) and from brain-dead organ donors (MV for 24-249 h; n = 15) were analyzed for ultrastructural injury, atrophy, and expression of proteolysis-related proteins (ubiquitin, nuclear factor-κB, and calpains).. TwPtr decreased progressively during MV, with a mean reduction of 32 ± 6% after 6 days. Longer periods of MV were associated with significantly greater ultrastructural fiber injury (26.2 ± 4.8 vs. 4.7 ± 0.6% area), decreased cross-sectional area of muscle fibers (1,904 ± 220 vs. 3,100 ± 329 μm²), an increase of ubiquitinated proteins (+19%), higher expression of p65 nuclear factor-κB (+77%), and greater levels of the calcium-activated proteases calpain-1, -2, and -3 (+104%, +432%, and +266%, respectively) in the diaphragm.. Diaphragmatic weakness, injury, and atrophy occur rapidly in critically ill patients during MV, and are significantly correlated with the duration of ventilator support.

Topics: Adult; Calpain; Diaphragm; Female; Humans; Male; Middle Aged; Muscle Weakness; Muscular Atrophy; Respiration, Artificial; Time Factors; Transcription Factor RelA; Ubiquitinated Proteins; Young Adult

Topics: Adrenergic beta-Agonists; Animals; Calpain; Ethanolamines; Formoterol Fumarate; Humans; Hypertrophy; Mice; Muscle, Skeletal; Muscular Atrophy; Organ Size; Protein Biosynthesis; Signal Transduction; Time Factors

Mechanical ventilation is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged mechanical ventilation is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during mechanical ventilation, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of mechanical ventilation- induced reactive oxygen species production in the diaphragm is important.. These experiments tested the hypothesis that elevated mitochondrial reactive oxygen species emission is required for mechanical ventilation-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm.. Cause and effect was determined by preventing mechanical ventilation-induced mitochondrial reactive oxygen species emission in the diaphragm of rats using a novel mitochondria-targeted antioxidant (SS-31).. None.. Compared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against mechanical ventilation-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against mechanical ventilation-induced myofiber atrophy and contractile dysfunction.. These results reveal that prevention of mechanical ventilation-induced increases in diaphragmatic mitochondrial reactive oxygen species emission protects the diaphragm from mechanical ventilation-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of reactive oxygen species production in the diaphragm during prolonged mechanical ventilation. These results could lead to the development of a therapeutic intervention to impede mechanical ventilation-induced diaphragmatic weakness.

Topics: Actins; Animals; Calpain; Caspase 3; Diaphragm; Female; Hydrogen Peroxide; Mitochondria, Muscle; Muscle Contraction; Muscle Fibers, Skeletal; Muscle Proteins; Muscle Weakness; Muscular Atrophy; Oligopeptides; Oxidative Stress; Rats; Rats, Sprague-Dawley; Respiration, Artificial; SKP Cullin F-Box Protein Ligases; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

High-load isometric exercise is considered an effective countermeasure against muscle atrophy, but therapeutic electrical stimulation for muscle atrophy is often performed without loading. In the present study, we investigated the combined effectiveness of electrical stimulation and high-load isometric contraction in preventing muscle atrophy induced by hindlimb unloading. Electrical stimulation without loading resulted in slight attenuation of muscle atrophy. Moreover, combining electrical stimulation with high-load isometric contraction enhanced this effect. In electrical stimulation without loading, inhibition of the overexpression of calpain 1, calpain 2, and MuRF-1 mRNA was confirmed. On the other hand, in electrical stimulation with high-load isometric contraction, inhibition of the overexpression of cathepsin L and atrogin-1 mRNA in addition to calpain 1, calpain 2, and MuRF-1 mRNA was confirmed. These findings suggest that the combination of electrical stimulation and high-load isometric contraction is effective as a countermeasure against muscle atrophy.

Topics: Animals; Calpain; Cathepsin L; Electric Stimulation Therapy; Hindlimb Suspension; Isometric Contraction; Male; Muscle Proteins; Muscular Atrophy; Physical Conditioning, Animal; Rats; Rats, Wistar; RNA, Messenger; SKP Cullin F-Box Protein Ligases; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Hibernators like bats show only marginal muscle atrophy during prolonged hibernation. The current study was designed to test the hypothesis that hibernators use periodic arousal to increase protein anabolism that compensates for the continuous muscle proteolysis during disuse. To test this hypothesis, we investigated the effects of 3-month hibernation (HB) and 7-day post-arousal torpor (TP) followed by re-arousal (RA) on signaling activities in the pectoral muscles of summer-active (SA) and dormant Murina leucogaster bats. The bats did not lose muscle mass relative to body mass during the HB or TP-to-RA period. For the first 30-min following arousal, the peak amplitude and frequency of electromyographic spikes increased 3.1- and 1.4-fold, respectively, indicating massive myofiber recruitment and elevated motor signaling during shivering. Immunoblot analyses of whole-tissue lysates revealed several principal outcomes: (1) for the 3-month HB, the phosphorylation levels of Akt1 (p-Akt1) and p-mTOR decreased significantly compared to SA bats, but p-FoxO1 levels remained unaltered; (2) for the TP-to-RA period, p-Akt1 and p-FoxO1 varied little, while p-mTOR showed biphasic oscillation; (3) proteolytic signals (i.e., atrogin-1, MuRF1, Skp2 and calpain-1) remained constant during the HB and TP-to-RA period. These results suggest that the resistive properties of torpid bat muscle against atrophy might be attained primarily by relatively constant proteolysis in combination with oscillatory anabolic activity (e.g., p-mTOR) corresponding to the frequency of arousals occurring throughout hibernation.

Topics: Animals; Arousal; Blotting, Western; Body Temperature; Calpain; Chiroptera; Electromyography; Forkhead Transcription Factors; Hibernation; I-kappa B Proteins; Male; Muscle Contraction; Muscle Proteins; Muscular Atrophy; Organ Size; Pectoralis Muscles; Periodicity; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins c-akt; Seasons; Shivering; Signal Transduction; TOR Serine-Threonine Kinases; Ubiquitin-Protein Ligases

Oxidative stress has been linked to accelerated rates of proteolysis and muscle fiber atrophy during periods of prolonged skeletal muscle inactivity. However, the mechanism(s) that links oxidative stress to muscle protein degradation remains unclear. A potential connection between oxidants and accelerated proteolysis in muscle fibers is that oxidative modification of myofibrillar proteins may enhance their susceptibility to proteolytic processing. In this regard, it is established that protein oxidation promotes protein recognition and degradation by the 20S proteasome. However, it is unknown whether oxidation of myofibrillar proteins increases their recognition and degradation by calpains and/or caspase-3. Therefore, we tested the hypothesis that oxidative modification of myofibrillar proteins increases their susceptibility to degradation by both calpains and caspase-3. To test this postulate, myofibrillar proteins were isolated from rat skeletal muscle and exposed to in vitro oxidation to produce varying levels of protein modification. Modified proteins were then independently incubated with active calpain I, calpain II, or caspase-3 and the rates of protein degradation were assessed via peptide mapping. Our results reveal that increased protein oxidation results in a stepwise escalation in the degradation of myofibrillar proteins by calpain I, calpain II, and caspase-3. These findings provide a mechanistic link connecting oxidative stress with accelerated myofibrillar proteolysis during disuse muscle atrophy.

Topics: Animals; Calpain; Caspase 3; Cells, Cultured; Diaphragm; Female; Muscle, Skeletal; Muscular Atrophy; Myofibrils; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Tissue Culture Techniques

Recent reports suggest numerous roles for cysteine proteases in the progression of skeletal muscle atrophy due to disuse or disease. Nonetheless, a specific requirement for these proteases in the progression of skeletal muscle atrophy has not been demonstrated. Therefore, this investigation determined whether calpains or caspase-3 is required for oxidant-induced C2C12 myotube atrophy. We demonstrate that exposure to hydrogen peroxide (25 microM H2O2) induces myotube oxidative damage and atrophy, with no evidence of cell death. Twenty-four hours of exposure to H2O2 significantly reduced both myotube diameter and the abundance of numerous proteins, including myosin (-81%), alpha-actinin (-40%), desmin (-79%), talin (-37%), and troponin I (-80%). Myotube atrophy was also characterized by increased cleavage of the cysteine protease substrate alphaII-spectrin following 4 h and 24 h of H2O2 treatment. This degradation was blocked by administration of the protease inhibitor leupeptin (10 microM). Using small interfering RNA transfection of mature myotubes against the specific proteases calpain-1, calpain-2, and caspase-3, we demonstrated that calpain-1 is required for H2O2-induced myotube atrophy. Collectively, our data provide the first evidence for an absolute requirement for calpain-1 in the development of skeletal muscle myotube atrophy in response to oxidant-induced cellular stress.

Topics: Animals; Calpain; Caspase 3; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Hydrogen Peroxide; Leupeptins; Mice; Muscle Proteins; Muscular Atrophy; Myoblasts, Skeletal; Oxidative Stress; RNA Interference; Sarcomeres; Superoxide Dismutase; Time Factors; Transfection

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, alpha-sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin-proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21(WAF1/CIP1), is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21(WAF1/CIP1) is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.

Topics: Animals; Biomarkers; Calpain; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Profiling; Male; Mice; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies; Nuclear Proteins; Proteasome Endopeptidase Complex; Repressor Proteins; Signal Transduction; Up-Regulation

At times, exercise accompanied by its anabolic effects is not a tractable countermeasure to muscle atrophy. Instead, training is often attempted after the affected muscle has atrophied greatly as a result of unloading. This study was designed to elucidate stress and signaling mechanisms underlying a process of muscle catch-up growth as a result of transitory exercise during unloading. Rats were exercised daily with a routine of 20- or 40-minute treadmill running (at 60% of maximum oxygen uptake) during the second week of a two-week hindlimb suspension. We examined the expression and activation of heat shock proteins and anabolic and proteolytic markers in the rat soleus muscle. Muscle mass relative to body mass decreased 2.4-fold in the unloaded group (HU) with respect to controls but decreased only 1.7-fold in the 40-min trained group (HT40) (P < 0.05) - equivalent to a 1.4-fold increase in the relative muscle mass over HU. Immunoblotting analyses on whole-tissue lysates demonstrated the following: (1) HSP72 and alphaB-crystallin were upregulated 7- and 2.5-fold, respectively, in HT40 versus HU; (2) phosphorylation of Akt1 and p70/S6K decreased only slightly in HU; (3) when compared to HU, HT40 phosphorylation of Akt1, S6K, and FoxO1 increased 1.4- to 3.0-fold while phosphorylation of FoxO3 was unchanged; and (4) activities of the ubiquitin E3 ligases, calpain 1 and caspase-3 increased 2- to 4-fold in the unloaded groups regardless of exercise duration. These results suggest that the significant upregulation of chaperones and anabolic markers (e.g., HSP72, p-Akt1, p-S6K) in HT40, along with the lack of the training effect on proteolytic activity, is likely crucial for muscle mass catch-up in the unloaded muscle.

Topics: Animals; Calpain; Caspase 3; Forkhead Transcription Factors; Heat-Shock Proteins; Hindlimb Suspension; Muscle, Skeletal; Muscular Atrophy; Nerve Tissue Proteins; Phosphorylation; Physical Conditioning, Animal; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Stress, Physiological; Ubiquitin-Protein Ligases

Catabolic stimuli induce a coordinate expression of the 20S proteasome subunits in skeletal muscles. However, contradictory data have been obtained for the 19S regulatory complex (RC) subunits, which could reflect differential regulation at the transcriptional and/or translational level. To address this point we used a well-established model of muscle atrophy (hindlimb suspension) and determined the mRNA levels for 19S subunits belonging to both the base (non-ATPase S1, ATPases S7 and S8) and the lid (S14) of the 19S RC. Concomitant increased mRNA levels were observed for all studied subunits in rat soleus muscles after 9 days of unloading. In addition, analysis of polysome profiles showed a similar proportion of actively translated mRNA (50%) in unloaded and control soleus muscle. Furthermore, the repressed pool of messenger ribonucleoparticles (mRNPs) was low in both control (14%) and unloaded (15%) animals. Our data show that representative 19S subunits (S7 and S8) were efficiently translated, suggesting a coordinate production of 19S RC subunits. The 19S RC is responsible for the binding of polyubiquitin conjugates that are subsequently degraded inside the 20S proteasome core particle. We observed that soleus muscle atrophy was accompanied by an accumulation of ubiquitin conjugates. Purification of ubiquitin conjugates using the S5a 19S subunit followed by deubiquitination identified telethonin as a 26S proteasome substrate. In conclusion, muscle atrophy induces a concomitant expression of 26S proteasome subunits. Substrates to be degraded include a protein required for maintaining the structural integrity of sarcomeres.

Topics: Animals; Calpain; Hindlimb Suspension; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Proteasome Endopeptidase Complex; Protein Subunits; Random Allocation; Rats; Rats, Wistar; RNA, Messenger; Ubiquitin

Calpains have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. However, limited data are available about the specific involvement of each calpain in the early stages of muscle atrophy. The aims of this study were to determine whether calpains 1 and 2 are autolyzed after a short period of muscle disuse, and, if so, where in the myofibers the autolyzed products are localized. In the rat soleus muscle, 5 days of immobilization increased autolyzed calpain 1 in the particulate and not the soluble fraction. Conversely, autolyzed calpain 2 was not found in the particulate fraction, whereas it was increased in the soluble fraction after immobilization. In the less atrophied plantaris muscle, no difference was noted between the control and immobilized groups whatever the fraction or calpain. Other proteolytic pathways were also investigated. The ubiquitin-proteasome pathway was activated in both skeletal muscles, and caspase 3 was activated only in the soleus muscle. Taken together, our data suggest that calpains 1 and 2 are involved in atrophy development in slow type muscle exclusively and that they have different regulation and protein targets. Moreover, the activation of proteolytic pathways appears to differ in slow and fast muscles, and the proteolytic mechanisms involved in fast-type muscle atrophy remain unclear.

Topics: Animals; Autolysis; Calpain; Caspase 3; Disease Models, Animal; Enzyme Activation; Hindlimb Suspension; Male; Muscle Fibers, Fast-Twitch; Muscle Fibers, Skeletal; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Muscular Atrophy; Myofibrils; Phenotype; Proteasome Endopeptidase Complex; Rats; Rats, Wistar; Time Factors; Ubiquitin

Topics: Animals; Antioxidants; Calpain; Disease Models, Animal; Humans; Immobilization; Metallothionein; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Oxidative Stress; Peptide Hydrolases; Proteasome Endopeptidase Complex; Spinal Cord Injuries; Time Factors; Ubiquitin; Ubiquitin-Protein Ligases

Overexpression of the UCP3 gene in both murine and human myotube cell cultures leads to a significant activation of the different proteolytic systems involved in muscle myofibrillar protein breakdown. Thus, lysosomal (cathepsin B) and non-lysosomal (m-calpain and ubiquitin-proteasome) mRNA content was significantly increased in the different cell culture systems used. Interestingly, the overexpression of the UCP3 gene was not associated with any changes in apoptosis. Although the function of the UCP3 protein is not completely understood (uncoupling, oxidative stress), these results suggest a possible relation between these main mechanisms involved in muscle wasting during cancer.

Topics: Animals; Calpain; Carrier Proteins; Cathepsin B; Cells, Cultured; Enzyme Activation; Gene Expression; Humans; Ion Channels; Mice; Mitochondrial Proteins; Muscle Fibers, Skeletal; Muscular Atrophy; Neoplasms; Peptide Hydrolases; Proteasome Endopeptidase Complex; Transfection; Uncoupling Protein 3

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dexamethasone; Dipeptides; Gene Expression; Glycoproteins; Hydrogen Peroxide; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Myoblasts, Skeletal; Pepstatins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Sepsis; SKP Cullin F-Box Protein Ligases; Transfection; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Age-related decreases in muscle mass have been associated with the loss of myonuclei, possibly through a mechanism involving mitochondria. It is unclear if age-related apoptotic mechanisms vary by fiber type. Here we investigate indices of apoptosis along with the regulation of apoptotic mediators in the extensor digitorum longus (EDL) and soleus of adult (6 month), old (30 month), and very old (36 month) Fischer 344/NNiaHSD x Brown Norway/BiNia (F344/N x BN) rats. Compared to 6-month muscles, aged muscles exhibited decreases in muscle mass along with increases in the number of nuclei staining positively for DNA fragmentation. The expression of Bax, Bcl-2, caspase-3 and caspase-9 was regulated differently with aging between muscle types and in a manner not consistent with mitochondria-mediated apoptosis. To investigate the potential of calpain involvement in age-related myonuclear loss, the calpain-dependent cleavage of alpha-fodrin was examined. The proteolytic cleavage of alpha-fodrin by calpains was increased in both muscles with only the 36-month soleus exhibiting increased caspase-dependent alpha-fodrin cleavage. Taken together, these data suggest that apoptotic regulatory events differ between fiber types in the aging F344/N x BN and that mitochondrial-dependent apoptosis pathways may not play a primary role in the loss of muscle nuclei with aging.

Topics: Aging; Animals; Apoptosis; Calpain; Cell Nucleus; Male; Mitochondria, Muscle; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Inbred BN; Rats, Inbred F344

We tested the hypothesis that estrogen administration would retard immobilization-induced muscle atrophy in adult male rats. The rats were injected for 24 days with either estrogen (40 microg/kg(-1), beta-estradiol 3-benzoate in olive oil vehicle), or vehicle alone. At day 14 of estrogen treatment, the hindlimb muscles of one leg were immobilized in plantar flexion position by the use of a plaster cast. Following 10 days of immobilzation, the atrophic and the contralateral soleus muscles were both removed and analyzed to determine the level of muscle atrophy along with the measurement of the protein levels of Cu-Zn-superoxide dismutase (Cu-Zn-SOD), heat shock protein 72 (HSP72), and selected proteases. Compared to placebo animals, estrogen treatment significantly reduced (-35%) muscle atrophy. Further, estrogen significantly abridged the expression of the calcium-activated protease, calpain, in the atrophied hindlimb muscle. In contrast, estrogen treatment did not alter the protein levels of HSP72 in the immobilized soleus muscle. These results support the postulate that estrogen attenuates the rate of disuse muscle atrophy, partly because of reductions in immobilization-induced calcium-activated protease levels.

Topics: Animals; Antioxidants; Calpain; Estrogens; HSP72 Heat-Shock Proteins; Male; Muscle, Skeletal; Muscular Atrophy; Oxidative Stress; Rats; Rats, Wistar; Restraint, Physical; Superoxide Dismutase

Calpains are calcium regulated proteases involved in cellular functions that include muscle proteolysis both ante- and postmortem. Here, we describe the molecular characterization of the rainbow trout catalytic subunits of the mu- and m-calpains, respectively. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: I (propeptide), II (cysteine catalytic site), III (electrostatic switch), and IV (contains five EF-hands). Because starvation induces muscle wasting, the hypothesis of this study was that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1 (2.2-fold increase, P < 0.01), Capn2 (6.0-fold increase, P < 0.01), and calpastatins (1.6-fold increase, P < 0.05) as measured by quantitative real-time RT-PCR. The mRNA changes led to a 1.23-fold increase in the calpain catalytic activity. The results suggest a potential role of calpains in protein mobilization as a source of energy under fasting condition.

Topics: Amino Acid Sequence; Animals; Calpain; Cloning, Molecular; DNA, Complementary; Fish Proteins; Molecular Sequence Data; Muscular Atrophy; Oncorhynchus mykiss; RNA, Messenger; Sequence Homology, Amino Acid; Starvation; Tissue Distribution

Limb girdle muscular dystrophy 2A is a common variant secondary to mutations in the calpain 3 gene. A proportion of patients has early and severe contractures, which can cause diagnostic difficulties with other conditions. We report clinical and muscle magnetic resonance imaging findings in seven limb girdle muscular dystrophy 2A patients (four sporadic and three familial) who had prominent and early contractures. All patients showed a striking involvement of the posterior thigh muscles. The involvement of the other thigh muscles was variable and was related to clinical severity. Young patients with minimal functional motor impairment showed a predominant involvement of the adductors and semimembranosus muscles while patients with restricted ambulation had a more diffuse involvement of the posterolateral muscles of the thigh and of the vastus intermedius with relative sparing of the vastus lateralis, sartorius and gracilis. At calf level all patients showed involvement of the soleus muscle and of the medial head of the gastrocnemius with relative sparing of the lateral head. MRI findings were correlated to those found in two patients with the phenotype of limb girdle muscular dystrophy 2A without early contractures and the pattern observed was quite similar. However, the pattern observed in limb girdle muscular dystrophy 2A is different from that reported in other muscle diseases such as Emery-Dreifuss muscular dystrophy and Bethlem myopathy which have a significant clinical overlap with limb girdle muscular dystrophy 2A once early contractures are present. Our results suggest that muscle MRI may help in recognising patients with limb girdle muscular dystrophy 2A even when the clinical presentation overlaps with other conditions, and may therefore, be used as an additional investigation to target the appropriate biochemical and genetic tests.

Topics: Adolescent; Adult; Age of Onset; Calpain; Contracture; Disease Progression; Humans; Isoenzymes; Leg; Magnetic Resonance Imaging; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies, Limb-Girdle; Phenotype

We report on two siblings with late-onset, limb-girdle muscular dystrophy (LGMD) inherited in an autosomal recessive manner. The LGMD was characterized by many rimmed vacuoles and reduced expression of the laminin beta1 chain in skeletal muscle. Both patients developed a progressive wasting and weakness of limb-girdle muscles in the late forties or early fifties; their facial, ocular, bulbar, and cardiac muscles were not involved. Histopathology of skeletal muscles biopsies showed typical dystrophic changes with many rimmed vacuoles. The immunoreactivity of the laminin beta1 chain was reduced in the muscle fibers, while dystrophin, sarcoglycans, beta-dystroglycan, dysferlin, and other laminin components were normally expressed. A mutation search revealed that no mutation existed in the coding region of the calpain 3, telethonin and UDP-N-acetylglucosamine 2-epimerase/N-acetylmanosamine kinase (GNE) genes. We conclude that this autosomal recessive LGMD is unknown and characterized by its late onset, rimmed vacuoles and reduction of the laminin beta1 chain in muscle fibers.

Topics: Actin Cytoskeleton; Biopsy; Calpain; Chromosome Aberrations; Connectin; Consanguinity; Creatine Kinase; Cytoplasm; DNA Mutational Analysis; Female; Gait Apraxia; Genes, Recessive; Genetic Markers; Humans; Inclusion Bodies; Isoenzymes; L-Lactate Dehydrogenase; Microscopy, Electron; Microscopy, Fluorescence; Multienzyme Complexes; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophies; Neurologic Examination; Pedigree; Reverse Transcriptase Polymerase Chain Reaction; Tomography, X-Ray Computed; Vacuoles

A cDNA (1977 bp) encoding a crustacean calpain (Ha-CalpM; GenBank accession no. AY124009) was isolated from a lobster fast muscle cDNA library. The open reading frame specified a 575-amino acid (aa) polypeptide with an estimated mass of 66.3 kDa. Ha-CalpM shared high identity with other calpains in the cysteine proteinase domain (domain II; aa 111-396) and domain III (aa 397-575), but most of the N-terminal domain (domain I; aa 1-110) was highly divergent. Domain II contained the cysteine, histidine and asparagine triad essential for catalysis, as well as two conserved aspartate residues that bind Ca(2+). In domain III an acidic loop in the C2-like region, which mediates Ca(2+)-dependent phospholipid binding, had an expanded stretch of 17 aspartate residues. Ha-CalpM was classified as a non-EF-hand calpain, as it lacked domain IV, a calmodulin-like region containing five EF-hand motifs. Northern blot analysis, relative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that Ha-CalpM was highly expressed in skeletal muscles, but at much lower levels in heart, digestive gland, intestine, integument, gill, nerve cord/thoracic ganglion and antennal gland. An antibody raised against a unique N-terminal sequence recognized a 62 kDa isoform in cutter claw and crusher claw closer muscles and a 68 kDa isoform in deep abdominal muscle. Ha-CalpM was distributed throughout the cytoplasm, as well as in some nuclei, of muscle fibers. Purification of Ha-CalpM showed that the 62 kDa and 68 kDa isoforms co-eluted from gel filtration and ion exchange columns at positions consistent with those of previously described Ca(2+)-dependent proteinase III (CDP III; 59 kDa). Ha-CalpM mRNA and protein did not change during the moulting cycle. The muscle-specific expression of Ha-CalpM and the ability of Ha-CalpM/CDP III to degrade myofibrillar proteins suggest that it is involved in restructuring and/or maintaining contractile structures in crustacean skeletal muscle.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Calpain; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation, Enzymologic; Immunohistochemistry; Molecular Sequence Data; Molting; Muscle, Skeletal; Muscular Atrophy; Nephropidae; Phylogeny; Sequence Analysis, DNA; Sequence Homology, Amino Acid

An analytic method based on simulation and modeling of long-term 45Ca(2+) efflux data was used to estimate steady-state Ca(2+) contents (nmolCa(2+)g(-1)tissuewetwt.) and exchange fluxes (nmolCa(2+)min(-1)g(-1)tissuewetwt.) for extracellular and intracellular compartments in in vitro resting diaphragm from congestive heart failure (CHF, n=12) and sham-operated (SHAM, n=10) rats. Left hemidiaphragms were excised from experimental animals, loaded with 45Ca(2+) for 1h, and washed out with 45Ca(2+)-free perfusate for 8h. Tissue from the right hemidiaphragm was used to assess single-fiber cross-sectional area (CSA) as well as the relative proteolytic activity of Ca(2+)-dependent calpain. Kinetic analysis of 45Ca(2+) efflux data revealed that CHF was associated with increased Ca(2+) contents of extracellular and intracellular compartments as well as increased Ca(2+) exchange fluxes for all compartments. This accounted for the model prediction of a 250% increase in total diaphragm Ca(2+). Furthermore, single-fiber CSA was decreased 12% and proteolytic activity of calpain was increased twofold in CHF diaphragm relative to SHAM.. The kinetic data are consistent with the hypothesis that diaphragm Ca(2+) overload in CHF required all intercompartmental Ca(2+) fluxes to increase. The potential relationships among Ca(2+) overload, increased activity of calpain, and wasting of the diaphragm in CHF are discussed.

Topics: Animals; Calcium; Calcium Signaling; Calpain; Cell Compartmentation; Chronic Disease; Diaphragm; Disease Models, Animal; Extracellular Space; Heart Failure; Homeostasis; Intracellular Fluid; Kinetics; Male; Models, Biological; Muscle Fibers, Skeletal; Muscular Atrophy; Rats; Rats, Wistar; Respiration Disorders; Up-Regulation

Topics: Actin Cytoskeleton; Arm; Calpain; Creatine Kinase; Creatine Kinase, MM Form; Humans; In Situ Nick-End Labeling; Isoenzymes; Male; Middle Aged; Muscle Proteins; Muscle Weakness; Muscular Atrophy; Muscular Diseases; Myosins; Remission, Spontaneous

Under microgravity conditions similar to those in space, it is known that various nutritional and physiological changes in the body are induced. Especially in the aspect of nutrition, muscle atrophy is a characteristic phenomenon accompanying weightlessness. This study was conducted to investigate the ameliorated effect of muscle atrophy caused by suspension hypokinesia by using the soy protein isolate (SPI) as the protein source in comparison with casein. Male Wistar strain rats (8 wk old) were divided into two groups, each suspended with a suspension harness, and fed on a 20% SPI diet or a 20% casein diet for 10 d. The body weights of the suspended rats fed casein or SPI decreased similarly. The weight of the gastrocnemius and soleus muscle were decreased by suspension hypokinesia; however, the degree of the decrease of the muscle weights, especially soleus muscles, of rats fed the SPI diet was smaller than that of rats fed the casein diet. Serum Ntau-methylhistidine concentration was significantly lower in rats fed the SPI diet than in rats fed the casein diet. Similarly, the activities of muscle protein-degrading enzymes such as calpain and proteasome were significantly lower in rats fed the SPI diet than in rats fed the casein diet. Cathepsin B+L activities were not affected by the SPI or the casein diet. Therefore it is suggested that SPI caused a reduction of the proteolysis of myofibrillar protein in skeletal muscles through a reduction of calpain and proteasome activities, in consequence to ameliorate the muscle atrophy.

Topics: Animals; Calpain; Caseins; Cathepsins; Cysteine Endopeptidases; Dietary Proteins; Hypokinesia; Male; Methylhistidines; Models, Animal; Multienzyme Complexes; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Proteasome Endopeptidase Complex; Random Allocation; Rats; Rats, Wistar; Soybean Proteins; Weightlessness Simulation

Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo. We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro. Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease.

Topics: Animals; Calcium-Binding Proteins; Calpain; Female; Gene Expression; Hindlimb Suspension; Humans; Male; Mice; Mice, Transgenic; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Myosins; Phenotype; Protein Isoforms; Transgenes

Topics: Animals; Calpain; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endopeptidases; Enzyme Precursors; Hindlimb Suspension; Leucine; Ligases; Models, Biological; Multienzyme Complexes; Muscle, Skeletal; Muscular Atrophy; Myosin Heavy Chains; Proteasome Endopeptidase Complex; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Space Flight; Ubiquitins; Weightlessness; Weightlessness Simulation

In a previous report we suggested that muscle fibers in distal myopathy with rimmed vacuoles (DMRV) were degraded by both lysosomal proteolysis (cathepsins) and Ca2+-dependent, nonlysosomal proteolysis (calpain). Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, we examined the role of the ATP-ubiquitin-dependent proteolytic pathway (proteasomes) in myofiber degradation in this myopathy. Immunohistochemically, proteasomes (26S) were located in the cytoplasm in normal human muscle, but the staining intensity was weak. Quantitative analysis showed more reactivity for proteasomes in DMRV muscles and, to a lesser extent, in muscles from muscular dystrophy, polymyositis, and amyotrophic lateral sclerosis patients. In DMRV, proteasomes often were located within or on the rim of rimmed vacuoles, and in the cytoplasm of atrophic fibers. Ubiquitin accumulation was marked within rimmed vacuoles and was seen less extensively in the cytoplasm of atrophic fibers. The latter proteins colocalized well. In other diseased muscles, proteasomes and ubiquitin showed a positive reaction in the atrophic or necrotic fibers. The results indicate increased proteasome and ubiquitin in these muscle fibers as well as in other diseased muscle fibers. We suggest that the ATP-ubiquitin-proteasome proteolytic pathway as well as the nonlysosomal calpain and the lysosomal proteolytic pathway may participate in the muscle fiber degradation in DMRV.

Topics: Adenosine Triphosphate; Adult; Biopsy; Calpain; Cysteine Endopeptidases; Female; Humans; Lysosomes; Male; Middle Aged; Multienzyme Complexes; Muscle Fibers, Skeletal; Muscle Proteins; Muscular Atrophy; Muscular Diseases; Phagocytosis; Proteasome Endopeptidase Complex; Ubiquitins; Vacuoles

The muscles of IL-6 transgenic mice suffer from atrophy. Experiments were carried out on these transgenic mice to elucidate activation of proteolytic systems in the gastrocnemius muscles and blockage of this activation by treatment with the anti-mouse IL-6 receptor (mIL-6R) antibody. Muscle atrophy observed in 16-wk-old transgenic mice was completely blocked by treatment with the mIL-6R antibody. In association with muscle atrophy, enzymatic activities and mRNA levels of cathepsins (B and L) and mRNA levels of ubiquitins (poly- and mono-ubiquitins) increased, whereas the mRNA level of muscle-specific calpain (calpain 3) decreased. All these changes were completely eliminated by treatment with the mIL-6R antibody. This IL-6 receptor antibody could, therefore, be effective against muscle wasting in sepsis and cancer cachexia, where IL-6 plays an important role.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Body Weight; Calpain; Cathepsin B; Cathepsin L; Cathepsins; Cysteine Endopeptidases; Endopeptidases; Gene Expression; Humans; Interleukin-6; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multienzyme Complexes; Muscle, Skeletal; Muscular Atrophy; Organ Size; Proteasome Endopeptidase Complex; Rats; Rats, Wistar; Receptors, Interleukin; Receptors, Interleukin-6; RNA, Messenger; Ubiquitins

Atrophy of skeletal muscles is a serious problem in a microgravity environment. It is hypothesized that the unloading of postural muscles, which no longer must resist gravity force, causes an accelerated breakdown of contractile proteins, resulting in a reduction in muscle mass and strength. A crustacean model using the land crab, Gecarcinus lateralis, to assess the effects of spaceflight on protein metabolism is presented. The model is compared to a developmentally-regulated atrophy in which a premolt reduction in muscle mass allows the withdrawal of the large claws at molt. The biochemical mechanisms underlying protein breakdown involves both Ca(2+)-dependent and multicatalytic proteolytic enzymes. Crustacean claw muscle can be used to determine the interactions between shortening and unloading at the molecular level.

Topics: Actin Cytoskeleton; Adenosine Triphosphate; Animals; Brachyura; Calcium; Calpain; Disease Models, Animal; Molting; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Peptide Hydrolases; Research Design; Space Flight; Ubiquitins; Weightlessness

Inhibition of calpains in skeletal muscle by the tripeptide, leupeptin, after median-nerve transection in the mid-forearm and a delayed nerve repair of 3-weeks duration, was studied in a primate (Cebus apella) model. Results indicated that leupeptin facilitates axon regrowth and neuromuscular recovery after delayed nerve repair. Toxicologic testing showed that leupeptin, administered at 18 mg/kg intramuscularly, twice daily for 24 weeks after delayed nerve repair, did not adversely affect hematology, clotting, blood chemistry, or echocardiogram profiles. These data indicate that leupeptin is an effective and safe adjunct to delayed nerve repair.

Topics: Animals; Axons; Calpain; Cebus; Leupeptins; Median Nerve; Muscles; Muscular Atrophy; Nerve Regeneration; Neural Conduction; Time Factors

The purpose of this study was to investigate the possible role of calcium-activated neutral protease in the disorganization and dissolution of the myofibrils of the rat soleus that occurs following tenotomy. Rats were killed 3, 5, 7, 14, 21, and 42 days after tenotomy of the soleus, and the muscles were removed and assayed for calcium-activated protease activity. Maximal protease activity occurred 1 week after tenotomy, at the time when myofibril organization is completely disrupted. Activity was still high 2 and 3 weeks after the operation, but returned to normal levels by 6 weeks, when muscle histology had returned to normal. The time course of the calcium-activated protease activity corresponded closely to the time course of the morphological changes. Thus, calcium-activated neutral protease may play a major role in myofibrillar proteolysis following tenotomy and in making the myofibril susceptible to proteolytic attack by other, less specific proteases.

Topics: Animals; Calpain; Disease Models, Animal; Female; Hydrogen-Ion Concentration; Muscles; Muscular Atrophy; Muscular Dystrophy, Animal; Rats; Rats, Inbred Strains; Tendons

The reduction of protein content in skeletal muscle undergoing disuse-induced atrophy is correlated with accelerated rates of protein degradation and reduced rates of protein synthesis. It is not known in what manner myofibers are partially disassembled during disuse atrophy to fibers of smaller diameter; nor is it known which proteases are responsible for this morphological change in contractile protein mass. Dayton and colleagues have suggested that the Ca(2+)-activated protease (CaP) may initiate myofibril degradation. The discovery of a form of CaP that is activatable by nanomolar concentrations of Ca2+ indicates that CaP activity may be regulated by physiological concentrations of Ca2+. The enhancement of proteolysis by the Ca2+ ionophore A23187, reported by Etlinger, is consistent with a significant role for CaP in protein degradation. It was of interest, therefore, to measure the levels of CaP activity and the CaP inhibitor in extracts obtained from skeletal muscles of rat and chicken limbs undergoing disuse atrophy or stretch hypertrophy, respectively.

Topics: Animals; Calcium; Calpain; Chickens; Hindlimb; Hypertrophy; Immobilization; Ions; Male; Muscle, Skeletal; Muscular Atrophy; Protease Inhibitors; Rats; Rats, Sprague-Dawley

Rat soleus muscle Z-lines and Z-line anomalies induced by neostigmine methyl sulfate (NMS) and cat soleus muscle Z-lines and Z-line anomalies induced by tenotomy were examined by electron microscopy before and after dissection of muscle fibers with Ca2+-activated neutral protease (CAF) to elucidate structural properties of Z-lines and related Z-line-type structures. In both normal and treated muscles, interdigitation of thin (6-7 nm) filaments, which were continuous with I-filaments (actin) from adjacent sarcomeres, was observed at the Z-line in longitudinal section. Both neostigmine methyl sulfate and tenotomy treatments induced muscle atrophy associated with Z-line degradation, streaming, and irregular distribution and accumulation of Z-line material and Z-rod formation. Tenotomized muscle also was characterized by the presence of N-line-like bands and I-Z-I brushes. CAF digestion removed the electron-dense covering material from Z-rods and revealed a backbone of actin filaments. The origin of Z-rods, their structural similarity to Z-lines in longitudinal and cross section, and their susceptibility to CAF indicate that Z-rods are directly related to native Z-lines and are probably lateral polymers of a basic Z-line unit. The regular square net alignment (22 nm) of I-filaments (actin) in cross sections of I-Z-I brushes which contain no N-lines suggests that the I-square net arrangement near the Z-line is determined by Z-filament-actin filament interaction rather than by the N-line or other factors. The results suggest that I-filaments (actin) penetrate the mammalian Z-line and are Z-line constituents and that the width of Z-lines and the length of Z-rods are determined by the amount of overlap of actin filaments. The perpendicular periodicity of Z-rods and the zigzag-oblique arrowheadlike appearance seen in longitudinal sections of Z-lines are attributed to alpha-actinin.

Topics: Animals; Calpain; Cats; Endopeptidases; Hindlimb; Muscle Proteins; Muscles; Muscular Atrophy; Myofibrils; Myosin Subfragments; Neostigmine; Rats; Tendons