Biomedical subjects

J G Tidball

Publications and source records attributed to J G Tidball.

At least 19 recordsLinked to original sources

PubMed2004

Null mutation of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro.

The giant protein titin serves a primary role as a scaffold for sarcomere assembly; however, proteins that mediate this remodeling have not been identified. One potential mediator of this process is the protease calpain 3 (C3), the protein mutated in limb girdle muscular dystrophy type 2A. To test the hypothesis that C3 mediates remodeling during myofibrillogenesis, C3 knockout (C3KO) mice were generated. The C3KO mice were atrophic containing small foci of muscular necrosis. Myogenic cells fused normally in vitro, but lacked well-organized sarcomeres, as visualized by electron microscopy (EM). Titin distribution was normal in longitudinal sections from the C3KO mice; however, EM of muscle fibers showed misaligned A-bands. In vitro studies revealed that C3 can bind and cleave titin and that some mutations that are pathogenic in human muscular dystrophy result in reduced affinity of C3 for titin. These studies suggest a role for C3 in myofibrillogenesis and sarcomere remodeling.

Animals↗

PubMed2003

Administration of the non-steroidal anti-inflammatory drug ibuprofen increases macrophage concentrations but reduces necrosis during modified muscle use.

OBJECTIVE: To test the hypothesis that ibuprofen administration during modified muscle use reduces muscle necrosis and invasion by select myeloid cell populations. METHODS: Rats were subjected to hindlimb unloading for 10 days, after which they experienced muscle reloading by normal weight-bearing to induce muscle inflammation and necrosis. Some animals received ibuprofen by intraperitoneal injection 8 h prior to the onset of muscle reloading, and then again at 8 and 16 h following the onset of reloading. Other animals received buffer injection at 8 h prior to reloading and then ibuprofen at 8 and 16 h following the onset of reloading. Control animals received buffer only at each time point. Quantitative immunohistochemical analysis was used to assess the presence of necrotic muscle fibers, total inflammatory infiltrate, neutrophils, ED1+ macrophages and ED2+ macrophages at 24 h following the onset of reloading. RESULT: Administration of ibuprofen beginning 8 h prior to reloading caused significant reduction in the concentration of necrotic fibers, but increased the concentration of inflammatory cells in muscle. The increase in inflammatory cells was attributable to a 2.6-fold increase in the concentration of ED2+ macrophages. Animals treated with ibuprofen 8 h following the onset of reloading showed no decrease in muscle necrosis or increase in ED2+ macrophage concentrations. CONCLUSION: Administration of ibuprofen prior to increased muscle loading reduces muscle damage, but increases the concentration of macrophages that express the ED2 antigen. The increase in ED2+ macrophage concentration and decrease in necrosis may be mechanistically related because ED2+ macrophages have been associated with muscle regeneration and repair.

Animals↗

PubMed2001

A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice.

Dystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy.

Animals↗

PubMed2001

Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle.

Duchenne muscular dystrophy (DMD) and mdx mouse dystrophy result from mutations in the dystrophin gene. Although these mutations are primarily responsible for the defects that underlie the pathology of dystrophinopathies, other factors may contribute importantly to the pathology. In the present investigation, we tested whether T cells present in mdx muscles are activated and contribute significantly to the pathological process. Flow cytometric analyses showed a significantly higher frequency of activated CD44(high) T cells in the blood and muscle of mdx mice when compared to normal B10 mice. However, the frequency of activated T cells was not elevated in mdx lymph nodes, suggesting muscle-specific T cell activation. In vivo antibody-mediated depletions of CD4(+) T cells from mdx mice significantly reduced the amount of histologically discernible muscle pathology by 61% in mdx mice, while depletion of CD8(+) T cells resulted in a 75% decrease in pathology. Finally, adoptive transfer of mdx immune cells in combination with muscle extracts resulted in muscle pathology in healthy murine recipients. These results indicate that T cells promote the mdx pathology and suggest that immune-based therapies may provide benefit to DMD patients.

Adoptive Transfer↗

PubMed2001

Do immune cells promote the pathology of dystrophin-deficient myopathies?

Many features of dystrophin-deficient muscle pathology are not clearly related to the loss of mechanical support of the muscle membrane by dystrophin. In the present review, evidence that supports a role for the immune system in promoting the pathology of dystrophinopathy is presented. The findings summarized here indicate that specific, cellular immune responses by cytotoxic T-lymphocytes and helper T-lymphocytes contribute to muscle pathology in dystrophin-deficient muscle, and that removal of specific lymphoid cell populations can reduce muscle pathology. In addition, innate immune responses may also promote dystrophinopathies by the tremendous infiltration of myeloid cell populations into the dystrophic muscle. Loss of normal redox homeostasis by dystrophin-deficient muscle may increase its sensitivity to free radical-mediated damage by myeloid cells. Collectively, the observations presented here suggest that the contribution of the immune system to dystrophinopathies may be significant, and that therapeutic approaches based upon immune interventions may ameliorate the pathological progression of dystrophin deficiency.

Animals↗

PubMed2001

Desmin integrates the three-dimensional mechanical properties of muscles.

Striated muscle is a linear motor whose properties have been defined in terms of uniaxial structures. The question addressed here is what contribution is made to the properties of this motor by extramyofilament cytoskeletal structures that are not aligned in parallel with the myofilaments. This question arose from observations that transverse loads increase muscle force production in diaphragm but not in the hindlimb muscle, thereby indicating the presence of structures that couple longitudinal and transverse properties of diaphragmatic muscle. Furthermore, we find that the diaphragms of null mutants for the cytoskeletal protein desmin show 1) significant reductions in coupling between the longitudinal and transverse properties, indicating for the first time a role for a specific protein in integrating the three-dimensional mechanical properties of muscle, 2) significant reductions in the stiffness and viscoelasticity of muscle, and 3) significant increases in tetanic force production. Thus desmin serves a complex mechanical function in diaphragm muscle by contributing both to passive stiffness and viscoelasticity and to modulation of active force production in a three-dimensional structural network. Our finding changes the paradigm of force transmission among cells by placing our understanding of the function of the cytoskeleton in the context of the structural and mechanical complexity of muscles.

Animals↗

PubMed2000

Dominant negative myostatin produces hypertrophy without hyperplasia in muscle.

Myostatin, a TGF-beta family member, is a negative regulator of muscle growth. Here, we generated transgenic mice that expressed myostatin mutated at its cleavage site under the control of a muscle specific promoter creating a dominant negative myostatin. These mice exhibited a significant (20-35%) increase in muscle mass that resulted from myofiber hypertrophy and not from myofiber hyperplasia. We also evaluated the role of myostatin in muscle degenerative states, such as muscular dystrophy, and found significant downregulation of myostatin. Thus, further inhibition of myostatin may permit increased muscle growth in muscle degenerative disorders.

Animals↗

PubMed2000

Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors.

Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

Adoptive Transfer↗

PubMed2000

Complement activation promotes muscle inflammation during modified muscle use.

Modified muscle use can result in muscle inflammation that is triggered by unidentified events. In the present investigation, we tested whether the activation of the complement system is a component of muscle inflammation that results from changes in muscle loading. Modified rat hindlimb muscle loading was achieved by removing weight-bearing from the hindlimbs for 10 days followed by reloading through normal ambulation. Experimental animals were injected with the recombinant, soluble complement receptor sCR1 to inhibit complement activation. Assays for complement C4 or factor B in sera showed that sCR1 produced large reductions in the capacity for activation of the complement system through both the classical and alternative pathways. Analysis of complement C4 concentration in serum in untreated animals showed that the classical pathway was activated during the first 2 hours of reloading. Analysis of factor B concentration in untreated animals showed activation of the alternative pathway at 6 hours of reloading. Administration of sCR1 significantly attenuated the invasion of neutrophils (-49%) and ED1(+) macrophages (-52%) that occurred in nontreated animals after 6 hours of reloading. The presence of sCR1 also reduced significantly the degree of edema by 22% as compared to untreated animals. Together, these data show that increased muscle loading activated the complement system which then briefly contributes to the early recruitment of inflammatory cells during modified muscle loading.

Animals↗

PubMed2000

Calpains and muscular dystrophies.

Calpains are a ubiquitous, well-conserved family of calcium-dependent, cysteine proteases. Their function in muscle has received increased interest because of the discoveries that the activation and concentration of the ubiquitous calpains increase in the mouse model of Duchenne muscular dystrophy (DMD), but null mutations of muscle specific calpain causes limb girdle muscular dystrophy 2A (LGMD2A). These findings indicate that modulation of calpain activity contributes to muscular dystrophies by disrupting normal regulatory mechanisms influenced by calpains, rather than through a general, nonspecific increase in proteolysis. Thus, modulation of calpain activity or expression through pharmacological or molecular genetic approaches may provide therapies for some muscular dystrophies.

Animals↗

PubMed2000

Modulation of myostatin expression during modified muscle use.

Previous findings have provided strong evidence that myostatin functions as a negative regulator of muscle mass during development and growth. In the present study, we test the hypothesis that myostatin may serve a similar function in fully differentiated muscle experiencing modified loading. Our findings show that myostatin expression can be modulated in fully differentiated, nonpathological skeletal muscle in a manner that is inversely related to changes in muscle mass. Atrophy of rat hind limb muscles induced by 10 days of unloading resulted in a 16% decrease in plantaris mass, a 110% increase in myostatin mRNA, and a 37% increase in myostatin protein. Immunohistochemical observations showed a detectable increase in myostatin concentration at myotendinous junctions during muscle unloading. The concentration of myostatin mRNA and protein returned to values not significantly different from ambulatory controls after 4 days of reloading, during which time plantaris mass also returned to control values. However, the results also show that periods of 30 min of daily muscle loading during the unloading period were sufficient to prevent significant losses of muscle mass caused by unloading, although myostatin mRNA still showed a 55% increase in concentration. Thus, significant increases in myostatin expression are not sufficient for muscle mass loss, although muscle mass loss during unloading is accompanied by increases in myostatin.

Animals↗

PubMed2000

Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells.

We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

Calcimycin↗

PubMed1999

Nitric-oxide synthase is a mechanical signal transducer that modulates talin and vinculin expression.

Mechanical stimuli can cause changes in muscle mass and structure which indicate that mechanisms exist for transducing mechanical stimuli into signals that influence gene expression. Myotendinous junctions show adaptations to modified muscle loading which suggest that these are transcriptionally distinct domains in muscle fibers that may experience local regulation of expression of structural proteins that are concentrated at these sites. Vinculin and talin are cytoskeletal proteins that are highly enriched at myotendinous junctions that we hypothesize to be subject to local transcriptional regulation. Our findings show that mechanical stimulation of muscle cells in vivo and in vitro causes an increase in the expression of vinculin and talin that is mediated by nitric oxide. Furthermore, nitric oxide-stimulated increases in vinculin and talin expression occur through a protein kinase G-dependent pathway and therefore differ from other mechanisms through which nitric oxide has been shown previously to modulate transcription. Analysis of vinculin mRNA distribution in mechanically stimulated muscle fibers shows that the mRNA is highly concentrated at myotendinous junctions, which supports the hypothesis that myotendinous junctions are distinct domains in which the expression of cytoskeletal proteins is modulated by mechanical stimuli through a nitric oxide and protein kinase G-dependent pathway.

Alkaloids↗

PubMed1999

Nitric oxide synthase inhibitors reduce sarcomere addition in rat skeletal muscle.

1. Mechanical stimuli are thought to modulate the number of sarcomeres in series (sarcomere number) in skeletal muscle fibres. However, the mechanisms by which muscle cells transduce mechanical signals into serial sarcomere addition have not been explored. In this study, we test the hypothesis that nitric oxide positively modulates sarcomere addition. 2. The soleus muscle was cast-immobilized in a shortened position in 3-week-old female Wistar rats. After 4 weeks, the casts were removed, creating a period of rapid sarcomere addition. During the remobilization period, nitric oxide synthase (NOS) inhibitors or substrate were administered. 3. Rats treated with the non-isoform-specific NOS inhibitor L-nitro-arginine methyl ester during 3 weeks of remobilization had smaller soleus sarcomere numbers than control rats. Rats treated with 1-(2-trifluoromethyl-phenyl)-imidazole, which has greater specificity for the neuronal isoform than for the endothelial isoform of NOS, also had smaller soleus sarcomere numbers than control rats. These results suggest that inhibition of the neuronal isoform of NOS reduces sarcomere addition during remobilization. 4. Rats treated with L-arginine, the substrate for NOS, during 1 week of remobilization had soleus sarcomere numbers for the immobilized-remobilized muscle which were closer to that for the contralateral, non-immobilized muscle than did rats that were not treated with L-arginine. 5. These results support the hypothesis that nitric oxide derived from the neuronal isoform of NOS positively modulates sarcomere addition.

Animals↗

PubMed1999

Macrophage invasion does not contribute to muscle membrane injury during inflammation.

Previous observations have shown that neutrophil invasion precedes macrophage invasion during muscle inflammation and that peak muscle injury is observed at the peak of ED1+ macrophage invasion. We tested the hypothesis that neutrophil invasion causes subsequent invasion by ED1+ macrophages and that ED1+ macrophages then contribute significantly to muscle membrane injury during modified muscle use. Rat hindlimbs were unloaded for 10 days followed by reloading by normal ambulation to induce inflammation. Membrane injury was measured by assaying Evans blue-bound serum protein influx through membrane lesions. Muscle neutrophil populations increased significantly during the first 2 h of reloading but ED1+ macrophages did not increase until 24 h. Neutrophil invasion was uncoupled from subsequent macrophage invasion by reloading rat hindlimbs for 2 h to cause neutrophil invasion, followed by resuspension for hours 2-24. This produced similar increases in neutrophil concentration as measured in muscles continuously reloaded for 24 h without causing an increase in macrophages. However, resuspension did not reduce the extent of muscle damage compared with that occurring in muscles that were reloaded continuously for 24 h. Thus, muscle invasion by neutrophils is not sufficient to cause invasion by ED1+ macrophages. In addition, muscle membrane injury that occurs during reloading is independent of invasion by ED1+ macrophages.

Animals↗

PubMed1998

Sparing of mdx extraocular muscles from dystrophic pathology is not attributable to normalized concentration or distribution of neuronal nitric oxide synthase.

Previous findings have led to speculations that decreased concentration of nNOS (neuronal nitric oxide synthase) may underlie some aspects of the pathophysiology of dystrophic muscle. We have tested whether the sparing of extraocular muscles (EOM) in muscular dystrophy is attributable to the presence of normal nNOS concentration and distribution in these muscles. Measurements of total nNOS concentration in control muscle showed that total nNOS comprises approximately 0.05% of total muscle protein, indicating a molar stoichiometry of approximately 60 and 20 to total dystrophin and syntrophin, respectively. Thus, most muscle nNOS is either not associated with the dystrophin complex, or binds to yet unidentified sites in the complex. nNOS concentration was at least two-fold greater in C57 EOM and tibialis anterior (TA) compared with mdx samples. No significant differences in nNOS concentration in EOM versus TA in either mdx or C57 mice were observed, nNOS was concentrated at the sarcolemma of all C57 samples, while mdx nNOS displayed a cytosolic distribution, except in fibers that reverted to express dystrophin. These data show that mdx EOM are spared by a mechanism other than normalized concentration and location of nNOS.

Aging↗

PubMed1998

Mechanical loading regulates NOS expression and activity in developing and adult skeletal muscle.

The hypothesis that changes in muscle activation and loading regulate the expression and activity of neuronal nitric oxide (NO) synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNA concentration in soleus muscles, which returned to control concentrations after return to weight bearing. Similarly, the concentration of nNOS in cultured myotubes increased by application of cyclic loading for 2 days. NO release from excised soleus muscles was increased significantly by a single passive stretch of 20% or by submaximal activation at 2 Hz, although the increases were not additive when both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on the presence of extracellular calcium. Cyclic loading of cultured myotubes also resulted in a significant increase in NO release. Together, these findings show that activity of muscle influences NO production in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression.

Aging↗

PubMed1998

Mechanical loading regulates expression of talin and its mRNA, which are concentrated at myotendinous junctions.

The hypothesis that mechanical loading regulates talin expression in developing and adult muscle was tested using in vitro and in vivo models. Talin was selected for study because it is a key structural link between the cytoskeleton and cell membrane. In the in vitro model, C2C12 myotubes were subjected to cyclic strains for 48 h. In the in vivo model, rat hindlimb muscles were unloaded for 10 days, then reloaded for 2 days. Cyclic loading of myotubes resulted in significant increases in the quantity of talin (68%) and its 190-kDa proteolytic fragment (70%), as well as talin mRNA (180%), relative to unloaded myotube cultures. Similarly, talin concentration and its mRNA increased by 68 and 136%, respectively, in soleus muscles reloaded for 2 days relative to ambulatory controls. Immunohistochemistry and in situ RT-PCR showed that talin and its mRNA are concentrated and colocalized at myotendinous junctions. Thus these findings indicate that increased mechanical loading promotes talin synthesis, which occurs principally at myotendinous junctions, according to talin mRNA distribution.

Animals↗