Biomedical subjects

J G Tidball

Publications and source records attributed to J G Tidball.

At least 37 records · Page 2Linked to original sources

PubMed1998

Nitric oxide synthase inhibition reduces muscle inflammation and necrosis in modified muscle use.

The objective of this study was to determine the role of nitric oxide in muscle inflammation, fiber necrosis, and apoptosis of inflammatory cells in vivo. The effects of nitric oxide synthase (NOS) inhibition on the concentrations of neutrophils, ED1+ and ED2+ macrophages, apoptotic inflammatory cells, and necrotic muscle fibers in rats subjected to 10 days of hindlimb unloading and 2 days of reloading were determined. Administration of NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the concentrations of neutrophils, ED1+ and ED2+ macrophages, and necrotic fibers in soleus muscle relative to water-treated controls. The concentration of apoptotic inflammatory cells was also significantly lower for L-NAME-treated animals compared with water-treated controls. However, the proportion of the inflammatory cell population that was apoptotic did not differ between L-NAME-treated and control animals, suggesting that L-NAME treatment did not decrease inflammatory cell populations by increasing the frequency of apoptosis. Thus, nitric oxide or one of its intermediates promotes muscle inflammation and fiber necrosis during modified muscle use and plays no more than a minor role in the resolution of muscle inflammation by inducing apoptosis of inflammatory cells.

Animals↗

PubMed1997

Myonuclear apoptosis in dystrophic mdx muscle occurs by perforin-mediated cytotoxicity.

Myonuclear apoptosis is an early event in the pathology of dystrophin-deficient muscular dystrophy in the mdx mouse. However, events that initiate apoptosis in muscular dystrophy are unknown, and whether elimination of apoptosis can ameliorate subsequent muscle wasting remains a major question. We have tested the hypothesis that cytotoxic T-lymphocytes initiate myonuclear apoptosis in dystrophic muscle, and examined whether perforin-mediated cytotoxicity plays a role in the pathophysiology of muscular dystrophy. Mdx mice showed muscle invasion by cytotoxic T cells and helper T cells at the onset of histologically detectable muscle fiber pathology. At this time, perforin-expressing cells were also present at elevated concentration. Mdx mice depleted of CD8(+) cells showed a significant reduction of apoptotic myonuclei concentration and a reduction in necrosis, judged by macrophage invasion of muscle fibers. Double-mutant mice, deficient in dystrophin and perforin, showed nearly complete absence of myonuclear apoptosis, and a significant reduction in the concentration of macrophages in the connective tissue surrounding muscle fibers. However, muscle fiber invasion by macrophages was not reduced significantly in double mutant mice. Thus, cytotoxic T-lymphocytes contribute significantly to apoptosis and necrosis in mdx dystrophy, and perforin-mediated killing is primarily responsible for myonuclear apoptosis.

Animals↗

PubMed1997

Absence of calpain 3 in a form of limb-girdle muscular dystrophy (LGMD2A).

Antibodies that recognise the muscle-specific calpain 3 (CANP3) were used in a 'blind' study to label blots of skeletal muscle from 12 control subjects and from 12 patients with various muscle diseases. Calpain 3 was clearly detected in all control muscle samples analysed, even though some of the muscle had been at room temperature for over an hour before being dissected and snap-frozen. Calpain 3 was also detected in the muscle biopsies from non-LGMD2A patients, but was absent in samples from 3 patients with LGMD2A. These results show that (i) calpain 3 protein can be detected in whole extracts of human muscle, and (ii) that antibodies can be used to differentiate patients with LGMD2A from those with other muscle diseases. This represents an invaluable diagnostic aid since the limb-girdle dystrophies are very difficult to separate on clinical grounds alone. One possible function that was considered for calpain 3 was the post-translational cleavage of the 97 kDa dystroglycan precursor polypeptide into the mature alpha- and beta-dystroglycan proteins. The beta-dystroglycan band was the correct size on blots of LGMD2A muscle, indicating that calpain 3 is probably not involved in the post-translational processing of dystroglycan.

Animals↗

PubMed1997

Platelet-derived growth factor-stimulated secretion of basement membrane proteins by skeletal muscle occurs by tyrosine kinase-dependent and -independent pathways.

The basement membrane of skeletal muscle is produced by the muscle cells it ensheathes and by nonmuscle cells located in the surrounding extracellular matrix. In this study, we have shown that platelet-derived growth factor (PDGF) stimulates secretion of three basement membrane components of skeletal muscle: laminin (70% increase), fibronectin (30%), and type IV collagen (70%). Furthermore, we have found using the signal transduction inhibitors, genistein (tyrosine kinase inhibitor), phorbol 12-myristate 13-acetate (protein kinase C (PKC) inhibitor), thapsigargin (depletes intracellular Ca2+ stores), and H89 (protein kinase A inhibitor), that PDGF-stimulated secretion of these proteins occurs through distinct signaling pathways. Densitometry of Western blots of L6 myoblast supernatant indicates that the PDGF-induced increase in secretion of laminin and type IV collagen is tyrosine kinase-dependent. The increase in type IV collagen secretion also shows dependence on PKC, as well as the release of intracellular Ca2+. Inhibition of either of these pathways reduces the increase in type IV collagen secretion to 20%. In contrast, the PDGF-induced increase in laminin secretion is unaffected by inhibition of either PKC or intracellular Ca2+ release. The increase in fibronectin secretion by PDGF uses yet a third set of signals. PDGF-induced fibronectin secretion is not dependent on tyrosine kinase activity but is dependent on protein kinase A as well as the release of intracellular Ca2+. These divergent signaling pathways provide for independent regulation of basement membrane protein secretion, allowing a muscle cell to modify both the quantity and composition of its basement membrane in response to its environment.

Alkaline Phosphatase↗

PubMed1997

Calpain II expression is increased by changes in mechanical loading of muscle in vivo.

In the present investigation, we have tested the hypothesis that calpain expression or activity in skeletal muscle is influenced by changes in mechanical loading in vivo. Muscle unloading for 10 days produced no change in the concentrations of calpain I, or II, and no change in calpain activation, as assessed by measurements of the proportion of calpain I or II isoforms that exhibited autoproteolytic modifications. However, muscle reloading for 2 days produced a 90% increase in calpain II concentration per unit wet weight of muscle relative to ambulatory controls. Although no change in the activation index for calpain I or II was identified for reloaded muscle, this index is an expression of the proportion of the total mass of each calpain isoform that is autoproteolyzed. Thus, there is also approximately a 90% increase in autolyzed calpain II in muscle experiencing increased loading than in controls. Northern analysis shows that the concentration of mRNA for calpain II is increased in reloaded muscle, but no change in calpain II mRNA concentration in unloaded muscle. In situ reverse transcription polymerase chain reaction was used to confirm that nearly all calpain II mRNA in reloaded muscle is located in muscle fibers, with very little detectable calpain II mRNA in non-muscle cells present in the tissue. Together, these findings show that increased muscle loading causes a selective increase in the expression of calpain II isoform, thereby indicating that its regulation is independent from other calpain isoforms.

Animals↗

PubMed1996

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Animals↗

PubMed1996

Calpain translocation during muscle fiber necrosis and regeneration in dystrophin-deficient mice.

Previous studies have shown that calpains are autolytically cleaved during the disease process of mdx dystrophy, a mouse model for Duchenne muscular dystrophy, indicating that calpains may be activated and play a role in proteolysis that occurs in muscular dystrophy (J. Biol. Chem. 270(18), 10909-10914, 1995). In the present study, we investigated the location of calpain in dystrophic muscle fibers over the course of mdx dystrophy, to relate the protease distribution to its state of activation, and to determine whether calpain translocation was an early event in mdx dystrophy. Immunolabeling of health, fully differentiated muscle fibers showed calpain present throughout the cytosol, but more concentrated near the plasma membrane. However, degenerating mdx fibers did not contain higher concentrations of calpain at the plasma membrane and showed only a homogeneous, cytosolic distribution. Calpain distribution was similarly diffuse in young myotubes and regenerating fibers with increased cytosolic concentration in early myotubes. Calpain distribution in adult mdx tissue was similar to that occurring in healthy, fully differentiated fibers, although adult mdx fibers displayed higher concentrations of membrane-associated calpain than those observed in C57 controls. The association of calpain with the plasma membrane was verified by immunoblots of isolated sarcolemmal membrane from adult mdx and control muscle which showed calpain present predominantly in the cytosol along with some membrane association. Thus, changes in calpain distribution coincide with changes in enzymatic cleavage over the course of mdx dystrophy shown previously. Furthermore, the stages of pathology at which calpain cleavage is least coincides with those stages when calpain is most concentrated at the cell membrane, suggesting that calpain is retained in an inactive form at the plasma membrane.

Animals↗

PubMed1996

Apoptosis of macrophages during the resulution of muscle inflammation.

We tested the hypothesis that apoptosis contributes to the depletion of macrophages expressing the ED1 or ED2 antigen during the resolution of rat muscle inflammation. Muscle inflammation was induced by subjecting rat soleus muscle to 10 days of unloading followed by periods of muscle reloading. Terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate (dUTP) labeling of apoptotic nuclei showed that apoptotic inflammatory cells increase in concentration within necrotic fibers and in the connective tissue at 2 days following muscle injury caused by increased loading. Four days following injury, the apoptotic cells within damaged fibers returned to control levels, and at 7 days following injury apoptotic cells in the connective tissue returned to control concentrations. No preferential, internucleosomal cleavage of DNA from inflamed muscle was observed, although there was greater fragmentation of DNA in inflamed muscle than in controls. Double labeling studies show that cells expressing either ED1 or ED2 antigen can undergo apoptosis in vivo. The time course of apoptosis and concentration of apoptotic cells within damaged muscle fibers indicates that apoptosis contributes to returning ED1+ cells to control concentration during the resolution of inflammation. However, apoptosis of ED2+ cells during the first week following injury is not sufficient to return ED2+ cell concentrations to control values.

Animals↗

PubMed1995

Calpains are activated in necrotic fibers from mdx dystrophic mice.

Death of dystrophin-deficient muscle purportedly results from increases in [Ca]in that cause the activation of calpains. We have tested whether calpains play a role in this process by assaying for changes in calpain concentration and activation in peak necrotic mdx mice (4 weeks of age) and in completely regenerated mdx mice (14 weeks of age). Biochemical fractionation and immunoblotting with epitope-specific antisera allowed measurement of the concentrations of m- and mu-calpains and the extent of autoproteolytic modification. Our findings show that total calpain concentration is elevated in both 4-week and 14-week mdx mice. This increase in concentration was shown to result primarily from a significant increase in m-calpain concentration at 4 weeks. Northern analysis demonstrated that neither m- nor mu-calpain mRNA concentrations differed between mdx and controls suggesting that the increased calpain concentration results from post-translational regulation. Immunoblotting with antibodies directed against amino-terminal peptides revealed an increase in autoproteolysis of mu-calpain, indicative of increased activation. The extent of autoproteolysis of mu-calpain returns to control levels during regeneration. This is not a consequence of increased calpastatin mRNA or protein. The findings reported here support a role for calpains in both the degenerative and regenerative aspects of mdx dystrophy.

Age Factors↗

PubMed1995

Site and mechanics of failure in normal and dystrophin-deficient skeletal muscle.

Partial or complete tearing of skeletal muscle occurs both in acute muscle injury and in some pathological muscle conditions. Despite the impact of such tearing on normal muscle function, few studies have examined the site or mechanism of muscle injury at cellular or subcellular levels of organization. The present study determines the ultrastructural location and mechanical conditions of tensile failure in normal mouse extensor digitorum longus muscles. All of these muscles failed near a myotendinous junction (MTJ), but within the muscle fibers, in a transverse plane coincident with the edge of an A-band. The breaking stress averaged 5.71 x 10(5) N/m2 for muscles stimulated tetanically during lengthening, and 5.01 x 10(5) N/m2 for unstimulated muscles. Breaking strain averaged 135%, and showed no dependence on the state of activation of the muscle. The energy absorbed by each muscle averaged 362.5 mJ/g in unstimulated muscle, and 613.2 mJ/g in the stimulated samples. In addition to the failure properties of normal muscle, the effect of dystrophin deficiency on the site and conditions of failure was determined using muscle from mdx mice, which lack dystrophin. The absence of dystrophin had no detectable effect on the stress, strain, or energy absorbed, regardless of the state of muscle activation. Unstimulated mdx muscle failed in the plane of an A-band, but tetanic stimulation produced failure in the reticular lamina of the tendon, just external to the MTJ, in 75% of the fibers in the mdx muscles. Although dystrophin's cytoplasmic location makes it unlikely that this unique mode of failure is due directly to the absence of the protein, failure in the reticular lamina may result from a difference in load distribution that accompanies the response of mdx muscle to dystrophin deficiency.

Animals↗

PubMed1995

Apoptosis precedes necrosis of dystrophin-deficient muscle.

The current view that death of dystrophin-deficient muscle fibers is a necrotic process relies primarily upon the histological appearance of the tissue after the degenerative process is well advanced. Here, we tested this view by examining the possibility that apoptosis is a component of dystrophin-deficient muscle cell death. Three assays for apoptosis were employed in analyzing prenecrotic, peak necrotic and regenerated hindlimb muscle of mdx mice: (1) terminal deoxynucleotidyl transferase (TdT) mediated end-labeling of DNA in nuclei in tissue sections; (2) assays for DNA ladders; and (3) electron microscopic assays for the presence of organelles undergoing structural changes characteristic of apoptosis. At all ages sampled, mdx muscle contained apoptotic nuclei, according to TdT-mediated dUTP labeling of tissue sections. Nuclei in regenerated mdx muscle fibers did not display apoptosis. dUTP-labeled nuclei in control C57 muscles were rare or absent at all ages sampled. DNA from 4-week-old mdx mice was found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. Electron microscopic analysis showed that organelle structural changes indicating apoptosis appear before pathological changes diagnostic of necrosis. For example, condensed mitochondria, fragmented sarcoplasmic reticulum and nuclei with chromatin condensations resembling apoptosis appear in fibers that otherwise possess normal morphology. Together, the findings show that apoptosis precedes any detectable necrotic change in mdx muscle, and that apoptotic events continue into the stage of dystrophic pathology that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in dystrophin-deficient muscle which is followed secondarily by necrotic processes.

Animals↗

PubMed1995

Inflammatory cell response to acute muscle injury.

Nonmuscle cells play central roles in muscle repair and regeneration during the inflammation that follows muscle injury, although many aspects of the mechanisms by which inflammatory cells are attracted to injury sites and activated are unknown. Current evidence indicates that substances released from injured muscle can act as "wound hormones" that initiate inflammation. Most evidence supports the view that mononucleated cells that normally reside in muscle are activated by the injury, and then provide the chemotactic signal to circulating inflammatory cells. Three subsequent stages of inflammation can be identified, according to differences in the populations of inflammatory cells. First, neutrophils rapidly invade the injury site and promote inflammation by releasing cytokines that can attract and activate additional inflammatory cells. In at least some muscle injuries, neutrophils may further damage the injured muscle by releasing oxygen-free radicals that can damage cell membranes. Next, there is an increase in macrophages that invade damaged muscle fibers and phagocytose debris. Finally, there is an increase in a second subpopulation of macrophages that are associated with muscle regeneration. Although many of the potential mediators that underlie the proliferation, invasion, and activation of these inflammatory cell populations are known, few have been demonstrated conclusively to function in injured muscle in vivo.

Acute-Phase Reaction↗

PubMed1995

PTH-mediated osteoblast retraction: possible participation of the calpain pathway.

Parathyroid hormone (PTH)-induced osteoblast retraction may play a pivotal role a role in bone resorption by providing osteoclasts direct access to mineralized bone surface. We have been working on the hypothesis that the calpains participate in this retractile response through a calcium-dependent process. We have first demonstrated the presence of calpain activities in MC3T3-E1 osteoblastic cells and that these activities can be stimulated by PTH. Second, we have demonstrated that the PTH-induced osteoblast retraction is dramatically attenuated by two different cysteine protease inhibitors. Finally, initial immunofluorescent cytochemical studies suggest that this PTH-induced osteoblastic retraction is mediated through a calpain-dependent, proteolytic modification of the cytoskeletal organization.

Amino Acid Sequence↗

PubMed1994

Assembly of myotendinous junctions in the chick embryo: deposition of P68 is an early event in myotendinous junction formation.

Myotendinous junctions (MTJs) are specialized sites at the surface of muscle fibers where force is transmitted between muscle and tendon. Morphogenesis of MTJs in the latter half of embryonic chick development is characterized by formation of basement membrane, appearance of subsarcolemmal densities, association of myofibrils with the membrane, and then membrane folding. In the present investigation, the time of appearance of proteins involved in force transmission at MTJs is compared to those previously described structural specializations occurring during MTJ development. In addition, P68, a recently discovered collagen-binding protein that is shown to be identical or closely related to laminin-binding lectin is also demonstrated at MTJs, and an improved technique for P68 purification is presented. P68 accumulates on the surface of collagen fibers that lie near the ends of myotubes in chick hindlimb muscle prior to the appearance of ultrastructurally discernible basement membrane. At Day 10, the stage when MTJ basement membrane is first discernible by electron microscopy, laminin and the beta 1 subunit of integrin are detectable at the MTJ. At this stage, laminin distribution coincides with P68 distribution and no laminin is detectable at sites other than the forming MTJs. Fibronectin is detectable at the MTJ at Day 12, but is not enriched at the MTJ relative to other sites on the myotubes. At Day 13, the stage at which subsarcolemmal densities first appear, talin is demonstrable at the MTJ and fibronectin concentration at the MTJ is enriched. Collagen type IV cannot be discerned at the MTJ until Day 15. Thus, the earliest demonstrable specializations at sites of MTJ formation are: (1) the appearance of P68 on collagen fibers that lie at the ends of myotubes, which occurs prior to ultrastructurally discernible specializations at the MTJ and (2) the accumulation of laminin at the MTJ with a distribution identical to that of P68 at the stage of MTJ formation in which basement membrane first appears. These findings indicate that P68 and laminin may mediate early events in MTJ assembly.

Animals↗

PubMed1994

Modifications in myotendinous junction surface morphology in dystrophin-deficient mouse muscle.

Single muscle fibers from mdx mouse muscle, which is deficient in dystrophin, and control mouse muscle, containing dystrophin, were compared by scanning electron microscopy. In particular, comparisons were made of the surface morphology at myotendinous junctions and costameres, sites at the muscle cell surface that are enriched in dystrophin and where force is transmitted across the cell membrane. Muscle fibers from 4- and 6-week-old controls display nearly uniform surface morphology characterized by numerous digit-like processes at the myotendinous junction and nonjunctional surface membrane possessing distinct grooves at sites corresponding to underlying costameres. Mdx fibers at this stage showed blunted myotendinous junctions with few digit-like processes, infrequent indistinct costameric markings, and holes in the cell membrane. Cells from peak regenerating mdx muscle (6 weeks) showed surface morphology similar to 4-week mdx fibers, although the proportion of fibers displaying extensive structural defects was reduced at 6 weeks. Completely regenerated mdx fibers (23 weeks) were indistinguishable from fibers of 6-week-old mdx mice. In control mice, only approximately 6% of the fibers examined from 4- or 6-week-old mice showed any of the structural defects characteristic of the majority of mdx fibers. However, fibers from 23-week-old control mice displayed an increased frequency of cells with poorly defined junctional processes and surface striations. These findings indicate that the fibers displaying extensive disruption of surface features, which are most commonly observed in 4-week mdx mice at peak necrosis, are necrotic fibers. Specific defects, such as the reduction in myotendinous junction folding, loss of costameres, and increased occurrence of membrane holes, are observed in the majority of mdx fibers at all ages. Thus, these defects are more directly attributable to dystrophin's absence because their frequency of occurrence is independent of the stage of necrosis and regeneration.

Animals↗

PubMed1994

Differential response of macrophage subpopulations to soleus muscle reloading after rat hindlimb suspension.

The hypothesis that distinct populations of macrophages are associated with muscle necrosis and regeneration was examined in Wistar rat soleus muscle after 10 days of hindlimb suspension and 2, 4, and 7 days after the resumption of weight bearing. Necrosis was identified using histological features, such as muscle fiber infiltration, and regeneration was identified using immunohistochemical techniques for developmental myosin heavy chain (dMHC). Light-microscopic observations show that necrotic fibers in 2-day reloaded soleus muscle were invaded by ED1+ and Ia+ macrophages. The number of invaded fibers in muscles reloaded for 2 days increased to 2.8/mm2 compared with 0.2/mm2 in age-matched normal muscle but returned to control values by the 4th day of resumed weight bearing. In the interstitial spaces of 2-day recovery muscle, ED1+ and Ia+ macrophages numbered 369 and 332/mm2, respectively, compared with 12 and 72/mm2, respectively, in control soleus. After 7 days of reloading, the number of ED1+ cells was similar to that of control. Ia+ macrophages decreased to 240/mm2 at 4 days but after 7 days rose above control values to 429/mm2. ED2+ macrophages in 4- and 7-day reloaded soleus increased 70-80% in the interstitial spaces compared with control but were not observed to infiltrate necrotic muscle fibers at any time points. Immunohistochemistry and immunoblots using a monoclonal anti-dMHC antibody demonstrate a greater proportion of myofibers expressing dMHC isoforms after 4 and 7 days of reloading. These findings indicate that macrophage subpopulations are associated with distinct stages during the recovery process from hindlimb suspension: ED1+ macrophages are associated with muscle necrosis, whereas ED2+ cells are associated with muscle regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗

PubMed1994

Talin, vinculin and DRP (utrophin) concentrations are increased at mdx myotendinous junctions following onset of necrosis.

Duchenne muscular dystrophy (DMD) and the myopathy seen in the mdx mouse both result from absence of the protein dystrophin. Structural similarities between dystrophin and other cytoskeletal proteins, its enrichment at myotendinous junctions, and its indirect association with laminin mediated by a transmembrane glycoprotein complex suggest that one of dystrophin's functions in normal muscle is to form one of the links between the actin cytoskeleton and the extracellular matrix. Unlike Duchenne muscular dystrophy patients, mdx mice suffer only transient muscle necrosis, and are able to regenerate damaged muscle tissue. The present study tests the hypothesis that mdx mice partially compensate for dystrophin's absence by upregulating one or more dystrophin-independent mechanisms of cytoskeleton-membrane association. Quantitative analysis of immunoblots of adult mdx muscle samples showed an increase of approximately 200% for vinculin and talin, cytoskeletal proteins that mediate thin filament-membrane interactions at myotendinous junctions. Blots also showed an increase (143%) in the dystrophin-related protein called utrophin, another myotendinous junction constituent, which may be able to substitute for dystrophin directly. Muscle samples from 2-week-old animals, a period immediately preceding the onset of muscle necrosis, showed no significant differences in protein concentration between mdx and controls. Quantitative analyses of confocal images of myotendinous junctions from mdx and control muscles show significantly higher concentrations of talin and vinculin at the myotendinous junctions of mdx muscle. These findings indicate that mdx mice may compensate in part for the absence of dystrophin by increased expression of other molecules that subsume dystrophin's mechanical function.

Age Factors↗

PubMed1994

Macrophage activation and muscle remodeling at myotendinous junctions after modifications in muscle loading.

Modifications in muscle loading have been reported previously to result in increased numbers of mononucleated cells and changes in myofibril organization at myotendinous junctions (MTJs). The goals of this study were to determine the identity of those mononucleated cells and to examine the relationships between changes in their structure, location, and number with structural aspects of remodeling at MTJs experiencing modified loading. Soleus muscles from rats subjected to 10 days of hindlimb suspension were analyzed 0, 2, 4, and 7 days after return to weight bearing. Immunohistochemistry showed that ED1+, ED2+ and Ia+ macrophages were present at the MTJ and microtendon of control muscle. After reloading, ED2+ macrophages increased in number and size at MTJs and microtendons, indicating their activation. ED1+ cells showed no change in size or number whereas Ia+ cells were increased in size at day 7 of reloading. Electron microscopic observations showed that mononucleated cells near MTJs of control or suspended muscle were not highly active in protein synthesis or secretion. However, in reloaded muscle, mononucleated cells were found to be in close proximity to MTJs and to contain a high concentration of organelles associated with protein secretion. During these stages of reloading, extensive remodeling of myofibril-membrane associations occurred and nascent sarcomeres appeared in the MTJ regions of muscle fibers. Immunohistochemistry showed that during these stages of nascent sarcomere formation, there was renewed expression of developmental myosin heavy chain at MTJs, with this heavy chain appearing most prominently at the MTJ at day 7 of reloading. The activation and increased numbers of macrophages at MTJs and the close apposition of secretory cells to the MTJ membrane during remodeling lead us to propose that macrophage-derived factors may influence remodeling of MTJs in muscles experiencing modified loading.

Animals↗