scholarly journals Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction

2016 ◽  
Vol 310 (10) ◽  
pp. L975-L984 ◽  
Author(s):  
Gerald S. Supinski ◽  
Alexander P. Alimov ◽  
Lin Wang ◽  
Xiao-Hong Song ◽  
Leigh A. Callahan
Keyword(s):  
Skeletal Muscle ◽  
Cecal Ligation ◽  
C2c12 Cells ◽  
Superoxide Generation ◽  
Diaphragm Dysfunction ◽  
Calpain Activation ◽  
Calcium Dependent ◽  
Mitochondrial Superoxide ◽  
Cecal Ligation Puncture ◽  
Diaphragm Weakness

Calpain activation contributes to the development of infection-induced diaphragm weakness, but the mechanisms by which infections activate calpain are poorly understood. We postulated that skeletal muscle calcium-dependent phospholipase A2 (cPLA2) is activated by cytokines and has downstream effects that induce calpain activation and muscle weakness. We determined whether cPLA2 activation mediates cytokine-induced calpain activation in isolated skeletal muscle (C2C12) cells and infection-induced diaphragm weakness in mice. C2C12 cells were treated with the following: 1) vehicle; 2) cytomix (TNF-α 20 ng/ml, IL-1β 50 U/ml, IFN-γ 100 U/ml, LPS 10 μg/ml); 3) cytomix + AACOCF3, a cPLA2 inhibitor (10 μM); or 4) AACOCF3 alone. At 24 h, we assessed cell cPLA2 activity, mitochondrial superoxide generation, calpain activity, and calpastatin activity. We also determined if SS31 (10 μg/ml), a mitochondrial superoxide scavenger, reduced cytomix-mediated calpain activation. Finally, we determined if CDIBA (10 μM), a cPLA2 inhibitor, reduced diaphragm dysfunction due to cecal ligation puncture in mice. Cytomix increased C2C12 cell cPLA2 activity ( P < 0.001) and superoxide generation; AACOCF3 and SS31 blocked increases in superoxide generation ( P < 0.001). Cytomix also activated calpain ( P < 0.001) and inactivated calpastatin ( P < 0.01); both AACOCF3 and SS31 prevented these changes. Cecal ligation puncture reduced diaphragm force in mice, and CDIBA prevented this reduction ( P < 0.001). cPLA2 modulates cytokine-induced calpain activation in cells and infection-induced diaphragm weakness in animals. We speculate that therapies that inhibit cPLA2 may prevent diaphragm weakness in infected, critically ill patients.

scholarly journals Neutral sphingomyelinase 2 is required for cytokine-induced skeletal muscle calpain activation

2015 ◽  
Vol 309 (6) ◽  
pp. L614-L624 ◽  
Author(s):  
Gerald S. Supinski ◽  
Alexander P. Alimov ◽  
Lin Wang ◽  
Xiao-Hong Song ◽  
Leigh A. Callahan
Keyword(s):  
Skeletal Muscle ◽  
Cecal Ligation ◽  
C2c12 Cells ◽  
In Vivo Model ◽  
Calpain Activity ◽  
Calpain Activation ◽  
Neutral Sphingomyelinase ◽  
Cecal Ligation Puncture

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.

scholarly journals MitoTEMPOL, a mitochondrial targeted antioxidant, prevents sepsis-induced diaphragm dysfunction

2020 ◽  
Vol 319 (2) ◽  
pp. L228-L238 ◽  
Author(s):  
Gerald S. Supinski ◽  
Lin Wang ◽  
Elizabeth A. Schroder ◽  
Leigh Ann P. Callahan
Keyword(s):  
Free Radical ◽  
Cell Size ◽  
Muscle Cell ◽  
Mitochondrial Function ◽  
Force Generation ◽  
Superoxide Generation ◽  
Diaphragm Dysfunction ◽  
Free Radical Generation ◽  
Radical Generation ◽  
Diaphragm Weakness

Clinical studies indicate that sepsis-induced diaphragm dysfunction is a major contributor to respiratory failure in mechanically ventilated patients. Currently there is no drug to treat this form of diaphragm weakness. Sepsis-induced muscle dysfunction is thought to be triggered by excessive mitochondrial free radical generation; we therefore hypothesized that therapies that target mitochondrial free radical production may prevent sepsis-induced diaphragm weakness. The present study determined whether MitoTEMPOL, a mitochondrially targeted free radical scavenger, could reduce sepsis-induced diaphragm dysfunction. Using an animal model of sepsis, we compared four groups of mice: 1) sham-operated controls, 2) animals with sepsis induced by cecal ligation puncture (CLP), 3) sham controls given MitoTEMPOL (10 mg·kg−1·day−1 ip), and 4) CLP animals given MitoTEMPOL. At 48 h after surgery, we measured diaphragm force generation, mitochondrial function, proteolytic enzyme activities, and myosin heavy chain (MHC) content. We also examined the effects of delayed administration of MitoTEMPOL (by 6 h) on CLP-induced diaphragm weakness. The effects of MitoTEMPOL on cytokine-mediated alterations on muscle cell superoxide generation and cell size in vitro were also assessed. Sepsis markedly reduced diaphragm force generation. Both immediate and delayed MitoTEMPOL administration prevented sepsis-induced diaphragm weakness. MitoTEMPOL reversed sepsis-mediated reductions in mitochondrial function, activation of proteolytic pathways, and decreases in MHC content. Cytokines increased muscle cell superoxide generation and decreased cell size, effects that were ablated by MitoTEMPOL. MitoTEMPOL and other compounds that target mitochondrial free radical generation may be useful therapies for sepsis-induced diaphragm weakness.

scholarly journals Muscle-specific calpastatin overexpression prevents diaphragm weakness in cecal ligation puncture-induced sepsis

2014 ◽  
Vol 117 (8) ◽  
pp. 921-929 ◽  
Author(s):  
Gerald S. Supinski ◽  
Lin Wang ◽  
Xiao-Hong Song ◽  
Jennifer S. Moylan ◽  
Leigh Ann Callahan
Keyword(s):  
Protein Content ◽  
Total Protein ◽  
Cecal Ligation ◽  
Force Generation ◽  
Total Protein Content ◽  
Calpain Inhibitor ◽  
Force Frequency ◽  
Specific Force ◽  
Cecal Ligation Puncture ◽  
Diaphragm Weakness

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/cm2, 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.

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

2000 ◽  
Vol 278 (5) ◽  
pp. H1480-H1489 ◽  
Author(s):  
Nigel C. Gocan ◽  
Jeremy A. Scott ◽  
Karel Tyml
Keyword(s):  
Nitric Oxide ◽  
Skeletal Muscle ◽  
Control Level ◽  
Cecal Ligation ◽  
Calcium Dependent ◽  
Hindlimb Muscle ◽  
Vascular Responsiveness ◽  
Red Blood Cell Velocity ◽  
Neuronal Nos ◽  
Oxide Synthase

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity ( V RBC) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V RBC increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso- N-acetylpenicillamine or sodium nitroprusside reduced diameter and V RBC responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the “selective” iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.

scholarly journals Mitoquinone Mesylate (MitoQ) Prevents Sepsis Induced Diaphragm Dysfunction

2021 ◽  
Author(s):  
Gerald S. Supinski ◽  
Elizabeth A. Schroder ◽  
Lin Wang ◽  
Andrew J. Morris ◽  
Leigh Ann P. Callahan
Keyword(s):  
Free Radical ◽  
Cecal Ligation ◽  
Free Radical Scavenger ◽  
Force Generation ◽  
Radical Scavenger ◽  
Diaphragm Dysfunction ◽  
Mechanically Ventilated ◽  
Female Mice ◽  
Diaphragm Function ◽  
Cecal Ligation Puncture

Sepsis-induced diaphragm dysfunction is a major contributor to respiratory failure in mechanically ventilated patients. There are no pharmacological treatments for this syndrome, but studies suggest that diaphragm weakness is linked to mitochondrial free radical generation. We hypothesized that administration of mitoquinone mesylate (MitoQ), a mitochondrially targeted free radical scavenger, would prevent sepsis- induced diaphragm dysfunction. We compared diaphragm function in 4 groups of male mice: (a) sham operated controls treated with saline (0.3 ml intraperitoneally, IP), (b) sham operated mice treated with MitoQ (3.5 mg/kg/day given IP in saline), (c) cecal ligation puncture (CLP) mice treated with saline, and (d) CLP mice treated with MitoQ. Forty-eight hours after surgery, we assessed diaphragm force generation, myosin heavy chain content, mitochondrial oxygen consumption (OCR) and aconitase activity. We also determined the effect of MitoQ administration in female mice with CLP sepsis and in mice with endotoxin-induced sepsis. CLP decreased diaphragm specific force generation and MitoQ prevented these decrements (e.g. maximal force averaged 30.2 ± 1.3, 28.0 ± 1.3, 12.8 ± 1.9 and 30.0 ± 1.0 N/cm2 for sham, sham + MitoQ, CLP, and CLP + MitoQ groups, p<0.001). CLP also reduced diaphragm mitochondrial OCR and MitoQ blocked this effect. Similar responses were observed in female mice, and in endotoxin-induced sepsis. Moreover, delayed MitoQ treatment was as effective as immediate treatment. These data indicate that MitoQ prevents sepsis-induced diaphragm dysfunction, preserving force generation and reducing fatigue. MitoQ may be a useful therapeutic agent to preserve diaphragm function in septic, critically ill patients.

Expression of avian Ca2+-ATPase in cultured mouse myogenic cells

1989 ◽  
Vol 9 (5) ◽  
pp. 1978-1986
Author(s):  
N J Karin ◽  
Z Kaprielian ◽  
D M Fambrough
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
C2c12 Cells ◽  
Cyanine Dye ◽  
Adult Rabbit ◽  
Base Pairs ◽  
Reading Frame ◽  
Cytoplasmic Vesicles ◽  
Fast Twitch ◽  
Chicken Skeletal Muscle

cDNA encoding Ca2+-ATPase was cloned from a chicken skeletal muscle library. The cDNA (termed FCa) comprised 3,239 base pairs, including an open reading frame encoding 994 amino acids which showed the highest degree of homology with the adult rabbit fast-twitch Ca2+-ATPase isoform (C. J. Brandl, S. de Leon, D. R. Martin, and D. H. MacLennan, J. Biol. Chem. 262:3768-3774, 1987). Radiolabeled FCa hybridized to a 3.2-kilobase transcript in chicken skeletal muscle RNA but not to cardiac muscle RNA, which confirmed its identity as encoding the fast Ca2+-ATPase isoenzyme. FCa was transfected into the mouse myogenic line C2C12, from which a protein of 100 kilodaltons was immunopurified by using a monoclonal antibody specific for the avian fast Ca2+-ATPase. Immunofluorescence microscopy of a line (designated C2FCa2) stably expressing the avian Ca2+-ATPase localized the protein to the nuclear envelope and a population of cytoplasmic vesicles. A similar pattern was observed when C2FCa2 cells were stained with DiOC6(3), a cyanine dye that labels endoplasmic reticulum and mitochondria (M. Terasaki, J. Song, J. R. Wong, M. J. Weiss, and L. B. Chen, Cell 38:101-108, 1984). We conclude that the avian Ca2+-ATPase fast isoform is expressed and correctly targeted to the endoplasmic reticulum in mouse C2C12 cells.

scholarly journals Upregulation of Heme Oxygenase-1 by Hemin Alleviates Sepsis-Induced Muscle Wasting in Mice

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Xiongwei Yu ◽  
Wenjun Han ◽  
Changli Wang ◽  
Daming Sui ◽  
Jinjun Bian ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Heme Oxygenase ◽  
Muscle Atrophy ◽  
Muscle Wasting ◽  
Cecal Ligation ◽  
Skeletal Muscle Atrophy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heme Oxygenase 1 ◽  
Protective Effects ◽  
Sod Activity

Hemin, an inducer of heme oxygenase-1 (HO-1), can enhance the activation of HO-1. HO-1 exhibits a variety of activities, such as anti-inflammatory, antioxidative, and antiapoptotic functions. The objective of this study was to investigate the effects of hemin on sepsis-induced skeletal muscle wasting and to explore the mechanisms by which hemin exerts its effects. Cecal ligation and perforation (CLP) was performed to create a sepsis mouse model. Mice were randomly divided into four groups: control, CLP, CLP plus group, and CLP-hemin-ZnPP (a HO-1 inhibitor). The weight of the solei from the mice was measured, and histopathology was examined. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were used to assess the expression levels of HO-1 and atrogin-1. Furthermore, we investigated the antioxidative effects of HO-1 by detecting malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity. CLP led to dramatic skeletal muscle weakness and atrophy, but pretreatment with hemin protected mice against CLP-mediated muscle atrophy. Hemin also induced high HO-1 expression, which resulted in suppressed proinflammatory cytokine and reactive oxygen species (ROS) production. The expression of MuRF1 and atrogin-1, two ubiquitin ligases of the ubiquitin-proteasome system- (UPS-) mediated proteolysis, was also inhibited by increased HO-1 levels. Hemin-mediated increases in HO-1 expression exert protective effects on sepsis-induced skeletal muscle atrophy at least partly by inhibiting the expression of proinflammatory cytokines, UPS-mediated proteolysis, and ROS activation. Therefore, hemin might be a new treatment target against sepsis-induced skeletal muscle atrophy.

scholarly journals Caveolin-3 Associates with Developing T-tubules during Muscle Differentiation

1997 ◽  
Vol 136 (1) ◽  
pp. 137-154 ◽  
Author(s):  
Robert G. Parton ◽  
Michael Way ◽  
Natasha Zorzi ◽  
Espen Stang
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Muscle Cells ◽  
Membrane System ◽  
C2c12 Cells ◽  
Double Labeling ◽  
Specific Antibodies ◽  
T Tubules ◽  
Α1 Subunit

Caveolae, flask-shaped invaginations of the plasma membrane, are particularly abundant in muscle cells. We have recently cloned a muscle-specific caveolin, termed caveolin-3, which is expressed in differentiated muscle cells. Specific antibodies to caveolin-3 were generated and used to characterize the distribution of caveolin-3 in adult and differentiating muscle. In fully differentiated skeletal muscle, caveolin-3 was shown to be associated exclusively with sarcolemmal caveolae. Localization of caveolin-3 during differentiation of primary cultured muscle cells and development of mouse skeletal muscle in vivo suggested that caveolin-3 is transiently associated with an internal membrane system. These elements were identified as developing transverse-(T)-tubules by double-labeling with antibodies to the α1 subunit of the dihydropyridine receptor in C2C12 cells. Ultrastructural analysis of the caveolin-3– labeled elements showed an association of caveolin-3 with elaborate networks of interconnected caveolae, which penetrated the depths of the muscle fibers. These elements, which formed regular reticular structures, were shown to be surface-connected by labeling with cholera toxin conjugates. The results suggest that caveolin-3 transiently associates with T-tubules during development and may be involved in the early development of the T-tubule system in muscle.

scholarly journals TWIST1 and TWIST2 regulate glycogen storage and inflammatory genes in skeletal muscle

2015 ◽  
Vol 224 (3) ◽  
pp. 303-313 ◽  
Author(s):  
Jonathan M Mudry ◽  
Julie Massart ◽  
Ferenc L M Szekeres ◽  
Anna Krook
Keyword(s):  
Skeletal Muscle ◽  
Metabolic Diseases ◽  
Glycogen Synthesis ◽  
C2c12 Cells ◽  
Acid Oxidation ◽  
Diabetic Patients ◽  
Mrna Levels ◽  
Intact Mouse ◽  
Mouse Muscle ◽  
The Impact

TWIST proteins are important for development of embryonic skeletal muscle and play a role in the metabolism of tumor and white adipose tissue. The impact of TWIST on metabolism in skeletal muscle is incompletely studied. Our aim was to assess the impact of TWIST1 and TWIST2 overexpression on glucose and lipid metabolism. In intact mouse muscle, overexpression of Twist reduced total glycogen content without altering glucose uptake. Expression of TWIST1 or TWIST2 reducedPdk4mRNA, while increasing mRNA levels ofIl6,Tnfα, andIl1β. Phosphorylation of AKT was increased and protein abundance of acetyl CoA carboxylase (ACC) was decreased in skeletal muscle overexpressing TWIST1 or TWIST2. Glycogen synthesis and fatty acid oxidation remained stable in C2C12 cells overexpressing TWIST1 or TWIST2. Finally, skeletal muscle mRNA levels remain unaltered inob/obmice, type 2 diabetic patients, or in healthy subjects before and after 3 months of exercise training. Collectively, our results indicate that TWIST1 and TWIST2 are expressed in skeletal muscle. Overexpression of these proteins impacts proteins in metabolic pathways and mRNA level of cytokines. However, skeletal muscle levels of TWIST transcripts are unaltered in metabolic diseases.

Sign in / Sign up

Export Citation Format

Share Document