calpain and Muscle-Weakness

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

Topics: Aging; Animals; Calcium-Binding Proteins; Calpain; Longevity; Mice; Muscle Weakness; Muscle, Skeletal

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

Calpain contributes to infection-induced diaphragm dysfunction but the upstream mechanism(s) responsible for calpain activation are poorly understood. It is known, however, that cytokines activate neutral sphingomyelinase (nSMase) and nSMase has downstream effects with the potential to increase calpain activity. We tested the hypothesis that infection-induced skeletal muscle calpain activation is a consequence of nSMase activation. We administered cytomix (20 ng/ml TNF-α, 50 U/ml IL-1β, 100 U/ml IFN-γ, 10 μg/ml LPS) to C2C12 muscle cells to simulate the effects of infection in vitro and studied mice undergoing cecal ligation puncture (CLP) as an in vivo model of infection. In cell studies, we assessed sphingomyelinase activity, subcellular calcium levels, and calpain activity and determined the effects of inhibiting sphingomyelinase using chemical (GW4869) and genetic (siRNA to nSMase2 and nSMase3) techniques. We assessed diaphragm force and calpain activity and utilized GW4869 to inhibit sphingomyelinase in mice. Cytomix increased cytosolic and mitochondrial calcium levels in C2C12 cells (P < 0.001); addition of GW4869 blocked these increases (P < 0.001). Cytomix also activated calpain, increasing calpain activity (P < 0.02), and the calpain-mediated cleavage of procaspase 12 (P < 0.001). Procaspase 12 cleavage was attenuated by either GW4869 (P < 0.001), BAPTA-AM (P < 0.001), or siRNA to nSMase2 (P < 0.001) but was unaffected by siRNA to nSMase3. GW4869 prevented CLP-induced diaphragm calpain activation and diaphragm weakness in mice. These data suggest that nSMase2 activation is required for the development of infection-induced diaphragm calpain activation and muscle weakness. As a consequence, therapies that inhibit nSMase2 in patients may prevent infection-induced skeletal muscle dysfunction.

Topics: Aniline Compounds; Animals; Benzylidene Compounds; Calpain; Cell Line; Diaphragm; Enzyme Activation; Lipopolysaccharides; Mice; Muscle Strength; Muscle Weakness; Muscle, Skeletal; Proteolysis; Sepsis; Sphingomyelin Phosphodiesterase

Infections induce severe respiratory muscle weakness. Currently there are no treatments for this important clinical problem. We tested the hypothesis that β-hydroxy-β-methylbutyrate (HMB) would prevent sepsis-induced diaphragm weakness. Four groups of adult male mice were studied: controls (saline-injected), sepsis (intraperitoneal lipopolysaccharide), sepsis+HMB (injected intravenously), and HMB. Diaphragm force generation and indices of caspase 3, calpain, 20S proteasomal subunit, and double-stranded RNA-dependent protein kinase (PKR) activation were assessed after 24h. Sepsis elicited large reductions in diaphragm specific force generation at all stimulation frequencies. Endotoxin also activated caspase 3, calpain, the 20S proteasomal subunit and PKR in the diaphragm. HMB blocked sepsis-induced caspase 3, 20S proteasomal and PKR activation, but did not prevent calpain activation. Most importantly, HMB administration significantly attenuated sepsis-induced diaphragm weakness, preserving muscle force generation at all stimulation frequencies (p<0.01). We speculate that HMB may prove to be an important therapy in infected patients, with the potential to increase diaphragm strength, to reduce the duration of mechanical ventilation and to decrease mortality in this patient population.

Topics: Animals; Calpain; Caspase 3; Diaphragm; Disease Models, Animal; eIF-2 Kinase; Lipopolysaccharides; Male; Mice; Muscle Strength; Muscle Weakness; Neuromuscular Agents; Organ Size; Phosphorylation; Proteasome Endopeptidase Complex; Sepsis; Valerates

Recent work indicates that infections are a major contributor to diaphragm weakness in patients who are critically ill and mechanically ventilated, and that diaphragm weakness is a risk factor for death and prolonged mechanical ventilation. Infections activate muscle calpain, but many believe this is an epiphenomenon and that other proteolytic processes are responsible for infection-induced muscle weakness. We tested the hypothesis that muscle-specific overexpression of calpastatin (CalpOX; an endogenous calpain inhibitor) would attenuate diaphragm dysfunction in cecal ligation puncture (CLP)-induced sepsis. We studied 1) wild-type (WT) sham-operated mice, 2) WT CLP-operated mice, 3) CalpOX sham-operated mice, and 4) CalpOX CLP-operated mice (n = 9-10/group). Twenty-four hours after surgery, we assessed the diaphragm force-frequency relationship, diaphragm mass, and total protein content and diaphragm levels of talin and myosin heavy chain (MHC). CLP markedly reduced diaphragm-specific force generation (force/cross-sectional area), which was prevented by calpastatin overexpression (force averaged 21.4 ± 0.5, 6.9 ± 0.8, 22.4 ± 1.0, and 18.3 ± 1.3 N/cm(2), respectively, for WT sham, WT CLP, CalpOX sham, and CalpOX CLP groups, P < 0.001). Diaphragm mass and total protein content were similar in all groups. CLP induced talin cleavage and reduced MHC levels; CalpOX prevented these alterations. CLP-induced sepsis rapidly reduces diaphragm-specific force generation and is associated with cleavage and/or depletion of key muscle proteins (talin, MHC), effects prevented by muscle-specific calpastatin overexpression. These data indicate that calpain activation is a major cause of diaphragm weakness in response to CLP-induced sepsis.

Topics: Animals; Calcium-Binding Proteins; Calpain; Cecum; Diaphragm; Ligation; Mice; Muscle Proteins; Muscle Weakness; Muscles; Myosin Heavy Chains; Sepsis; Talin

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

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

Calpain activation occurs in skeletal muscle in response to infection, but it is unknown if calpain inhibition improves muscle functional capacity. We hypothesized that infection induces diaphragm calpain activation, that calpain activation results in cleavage of important diaphragm cytoskeletal proteins, and that inhibition of calpain attenuates infection-induced diaphragm dysfunction. Mice (n = 4-6/group) were given: (1) saline (intraperitoneal); (2) endotoxin (12 mg/kg intraperitoneal); (3) calpain inhibitor peptide III (12 mg/kg intraperitoneal); and (4) endotoxin (12 mg/kg) plus calpain inhibitor peptide III (12 mg/kg). At 24 hours, diaphragms were removed and the following determined: (1) calpain activity by fluorogenic assay; (2) calpain I and II protein levels; (3) talin protein levels; and (4) the force-frequency relationship. Endotoxin significantly increased diaphragm calpain activity (P < 0.001), active calpain I protein (P < 0.001), active calpain II protein (P < 0.01), levels of a calpain-specific cleavage talin degradation product (P < 0.003), and reduced diaphragm force (P < 0.001). Calpain inhibitor III administration prevented endotoxin-induced increases in calpain activity, reduced talin degradation, and attenuated reductions in diaphragm force. Diaphragm-specific force at 150 Hz stimulation was significantly higher in control, endotoxin plus calpain inhibitor III, and calpain inhibitor III alone groups (23 +/- 1, 20 +/- 1 and 23 +/- 1 N/cm(2), respectively) than in the endotoxin alone group (15 +/- 1 N/cm(2)) (P < 0.01). This model of sepsis results in significant diaphragm calpain activation and calpain-dependent diaphragm cytoskeletal protein cleavage. Moreover, calpain inhibition attenuates endotoxin-induced diaphragm weakness, suggesting that such inhibitors may be a potential treatment to improve respiratory muscle function in infected patients.

Topics: Animals; Calpain; Diaphragm; Endotoxins; Male; Mice; Mice, Inbred ICR; Models, Biological; Muscle Contraction; Muscle Strength; Muscle Weakness; Muscle, Skeletal; Sepsis; Superoxide Dismutase; Talin

Unloading skeletal muscle results in atrophy and weakness. Inhibition of calpain activity during unloading reduced atrophy, but the impact on force generation has not been determined. Our hypothesis was that inhibition of calpain, through muscle-specific overexpression of calpastatin, would prevent the disruption of sarcomere structure and decreased specific force (kN/m(2)) observed during unloading. Calpastatin-overexpressing (cp) and wild-type (wt) mice were subjected to 3, 9, or 14 days of hindlimb suspension (HS). Compared with soleus muscles of non-suspended control mice, soleus muscles of wt mice showed a 25% decline in mass after 14 days of HS while maximum isometric force (P(o)) decreased by 40%, resulting in a specific P(o) that was 35% lower than control values. Over the same time period, muscles of cp mice demonstrated 25% declines in both mass and P(o) but no change in specific P(o). Consistent with the preservation of specific force during HS, soleus muscles of cp mice also maintained a high degree of order in sarcomere structure, in contrast to wt muscles that demonstrated misalignment of z-lines and decreased uniformity of thick filament lengths. Susceptibility to lengthening contraction-induced injury increased with the duration of HS and was not different for muscles of cp and wt mice. We conclude that inhibition of calpain activity during unloading preserves sarcomere structure such that the isometric force-generating capability is not diminished, while the effects of unloading on lengthening contraction-induced injury likely occur through calpain-independent mechanisms.

Topics: Animals; Calpain; Hindlimb Suspension; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle Contraction; Muscle Weakness; Muscle, Skeletal; Sarcomeres

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

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

Mutations in the gene encoding muscle-specific calpain 3 protease cause limb girdle muscular dystrophy type 2A. Calpainopathy is characterised by progressive symmetrical atrophy of pelvic, scapular and trunk muscles with an elevated creatine kinase. Most patients develop symptoms in childhood and lose the ability to walk by the age of 40 years. We describe a man who presented with foot drop at the age of 41 years, together with neurophysiological, histopathological and genetic data. This is the first report of calpainopathy presenting as foot drop, and widens the phenotype associated with this disease.

Topics: Adult; Blotting, Western; Calpain; Creatine Kinase; DNA; Electromyography; Exons; Foot; Humans; Magnetic Resonance Imaging; Male; Muscle Proteins; Muscle Weakness; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Neural Conduction; Neuromuscular Diseases; Spine

An 11-year-old girl with a calpain-3 gene (CAPN-3) mutation and eosinophilic myositis on muscle biopsy had high serum CK levels and eosinophil counts which showed spontaneous fluctuations. After commencement of immunosuppressive therapy reciprocal changes occurred in response to alterations in doses of the medications. Subacutely evolving and spreading muscle weakness developed during tapering of the immunosuppressive medications. These observations suggest that either the occurrence of eosinophilic myositis or the withdrawal of the immunosuppressive treatment may have accelerated the clinical course of the calpainopathy in this case. The positive effect of immunosuppressive therapy might have implications for the management of calpainopathy with an inflammatory component.

Topics: Azathioprine; Calpain; Child; Creatine Kinase; Disease Progression; Dose-Response Relationship, Drug; Drug Therapy, Combination; Eosinophilia-Myalgia Syndrome; Eosinophils; Female; Genetic Predisposition to Disease; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Leukocyte Count; Methylphenazonium Methosulfate; Muscle Proteins; Muscle Weakness; Muscle, Skeletal; Muscular Dystrophies, Limb-Girdle; Myositis; Prednisolone; Treatment Outcome

The cecal ligation perforation (CLP) model of sepsis is known to induce severe diaphragm dysfunction, but the cellular mechanisms by which this occurs remain unknown. We hypothesized that CLP induces diaphragm caspase-3 and calpain activation, and that these two enzymes act at the level of the contractile proteins to reduce muscle force generation. Rats (n = 4/group) were subjected to 1) sham surgery plus saline (intraperitoneal); 2) CLP; 3) CLP plus administration of calpain inhibitor peptide III (12 mg/kg ip); or 4) CLP plus administration of a caspase inhibitor, zVAD-fmk (3 mg/kg). At 24 h, diaphragms were removed, and the following were determined: 1) calpain and caspase-3 activities by fluorogenic assay; 2) caspase-3 and calpain I protein levels; 3) the intact diaphragm force-frequency relationship; and 4) the force generated by contractile proteins of single, permeabilized diaphragm fibers in response to exogenous calcium. CLP significantly increased diaphragm calpain activity (P < 0.02), caspase-3 activity (P < 0.02), active calpain I protein levels (P < 0.02), and active caspase-3 protein (P < 0.02). CLP also reduced the force generated by intact diaphragm muscle (P < 0.001) and the force generated by single-fiber contractile proteins (P < 0.001). Administration of either calpain inhibitor III or zVAD-fmk markedly improved force generation of both intact diaphragm muscle (P < 0.01) and single-fiber contractile proteins (P < 0.001). CLP induces significant reductions in diaphragm contractile protein force-generating capacity. This force reduction is mediated by the combined effects of activated caspase and calpain. Inhibition of these pathways may prevent diaphragm weakness in infected patients.

Topics: Animals; Calpain; Caspase 3; Diaphragm; Male; Muscle Weakness; Rats; Sepsis

Topics: Animals; Calpain; Caspase 3; Diaphragm; Male; Muscle Weakness; Rats; Sepsis

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

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

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