The susceptibility of muscle cells to oxidative stress is independent of nitric oxide synthase expression.

The free radical, nitric oxide (NO.), has been implicated in the pathogenesis of muscular dystrophies because the enzyme, nitric oxide synthase (NOS), which produces NO., binds to the dystrophin-glycoprotein complex (DGC). In various studies of tissue samples from human and animal muscular dystrophies due to DGC defects, correlations between reductions of NOS activity and disease severity have been reported. To test for any direct effect of NOS expression on muscle cell susceptibility, we examined muscle cells in vitro under conditions of experimentally altered NOS activity. There were no differences in susceptibility to oxidative stress between differentiated myotube cultures from wild-type and from neuronal NOS (nNOS)-deficient mice. Likewise, pharmacological inhibition of NOS did not alter cellular susceptibility to oxidative challenges. Overexpression of NOS neither enhanced nor diminished cellular susceptibility to oxidative stress. Finally, we assessed the effect of NOS overexpression on myotube cultures from dystrophin-deficient (mdx) mice. NOS protein was localized to both membrane and cytosolic compartments in the transduced cells. Still, no difference in susceptibility to oxidative stress was found between the NOS-overexpressing cells and control cells. These data suggest that muscle cell susceptibility to oxidative challenges is independent of the level of NOS expression. Therefore, any role NO. may play in the pathogenesis of muscular dystrophies is likely to be independent of its effect on the redox state of the cell.

Dystrophin mutations predict cellular susceptibility to oxidative stress.

Mutations in the dystrophin gene that lead to the expression of truncated forms of the dystrophin protein cause muscular dystrophies of varying severities both in humans and in mice. We have shown previously that dystrophin-deficient muscle is more susceptible to oxidative injury than is normal muscle. In this report, we have used muscle cells derived from mdx mice, which express no dystrophin, and mdx-transgenic strains that express full-length dystrophin or truncated forms of dystrophin to explore further the relationship between dystrophin expression and susceptibility of muscle to oxidative injury. We show that, when differentiated into myotubes, the relative susceptibility of the cell populations to oxidative stress correlates with the severity of the dystrophy in the strain from which the cells were isolated. The most susceptible populations exhibited the greatest oxidative damage as assessed by protein oxidation. Thus, the relative efficacy of truncated dystrophin proteins to protect muscle from necrotic degeneration in vivo is predicted by their ability to protect muscle cells from free radical mediated injury. These findings support the hypothesis that the dystrophin protein complex may have important regulatory or signaling properties in terms of cell survival and antioxidant defense mechanisms.

Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides.

Chimeric RNA/DNA oligonucleotides ("chimeraplasts") have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated "MDX1") designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1-2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were "revertant fibers." Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription-PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.

Integrin-mediated muscle cell spreading. The role of protein kinase c in outside-in and inside-out signaling and evidence of integrin cross-talk.

Muscle cell survival depends upon the presence of various integrins with affinities for different extracellular matrix proteins. The absence of either alpha(5) or alpha(7) integrins leads to degenerative disorders of skeletal muscle, muscular dystrophies. To understand the cell survival signals that are mediated by integrin engagement with matrix proteins, we studied the early signaling events initiated by the attachment of muscle cells to fibronectin, an interaction that is mediated primarily by alpha(5) integrins. Cells that express alpha(5) integrin rapidly spread on fibronectin, and this process is associated with the phosphorylation of focal adhesion kinase (FAK). Cells deficient in alpha(5) integrin failed to spread or promote FAK phosphorylation when plated on fibronectin. For alpha(5)-expressing cells, both spreading and FAK phosphorylation could be blocked by inhibitors of protein kinase C (PKC), indicating that PKC is necessary for this "outside-in signaling" mediated by alpha(5) integrin. Surprisingly, activators of PKC could promote spreading and FAK phosphorylation in alpha(5)-deficient muscle cells plated on fibronectin. This PKC-induced cell spreading appeared to be due to activation of alpha(4) integrins ("inside-out signaling") since it could be blocked by peptides that specifically inhibit alpha(4) integrin binding to fibronectin. A model of integrin signaling in muscle cells is presented in which there is a positive feedback loop involving PKC in both outside-in and inside-out signaling, and the activation of this cycle is essential for cell spreading and downstream signaling to promote cell survival. In addition, the data indicate a cross-talk that occurs between integrins in which the outside-in signaling via one integrin can promote the activation of another integrin via inside-out signaling.

Dystrophic muscle in mice chimeric for expression of alpha5 integrin.

alpha5-deficient mice die early in embryogenesis (). To study the functions of alpha5 integrin later in mouse embryogenesis and during adult life we generated alpha5 -/-;+/+ chimeric mice. These animals contain alpha5-negative and positive cells randomly distributed. Analysis of the chimerism by glucose- 6-phosphate isomerase (GPI) assay revealed that alpha5 -/- cells contributed to all the tissues analyzed. High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was lower than for the controls, however the subsequent life span of the survivors was only slightly reduced compared with controls (). Histological analysis of alpha5 -/-;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle degeneration-regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an increase in the number of mitochondria in some muscle fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant muscle fibers. All the alterations were already visible at late stages of embryogenesis. The number of altered muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric animals. Differentiation of alpha5 -/- ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was impaired. Our data suggest that a novel form of muscle dystrophy in mice is alpha5-integrin-dependent.

Muscle cells from mdx mice have an increased susceptibility to oxidative stress.

Several lines of evidence suggest that free radical mediated injury and oxidative stress may lead to muscle necrosis in the muscular dystrophies, including those related to defects in the dystrophin gene. We have examined muscle cell death using an in vitro assay in which the processes that lead to myofiber necrosis in vivo may be amenable to investigation in a simplified cell culture system. Using myotube cultures from normal and dystrophin-deficient (mdx) mice, we have examined the susceptibilities of the cells to different metabolic stresses. Dystrophin-deficient cells were more susceptible to free radical induced injury when compared to normal cells, but the two populations were equally susceptible to other forms of metabolic stress. The differential response appeared to be specifically related to dystrophin expression since undifferentiated myoblasts (which do not express dystrophin) from normal and mdx mice were equally sensitive to oxidative stress. Thus, the absence of dystrophin appears to render muscle specifically more susceptible to free radical induced injury. These results support the hypothesis that oxidative stress may lead to myofiber necrosis in these disorders. Elucidating the mechanisms leading to cell death may help to explain the variabilities in disease expression that are seen as a function of age, among different muscles, and across species in animals with muscular dystrophy due to dystrophin deficiency.

Evidence of oxidative stress in mdx mouse muscle: studies of the pre-necrotic state.

Considerable evidence indicates that free radical injury may underlie the pathologic changes in muscular dystrophies from mammalian and avian species. We have investigated the role of oxidative injury in muscle necrosis in mice with a muscular dystrophy due to a defect in the dystrophin gene (the mdx strain). In order to avoid secondary consequences of muscle necrosis, all experiments were done on muscle prior to the onset of the degenerative process (i.e. during the 'pre-necrotic' phase) which lasted up to 20 days of age in the muscles examined. In pre-necrotic mdx muscle, there was an induction of expression of genes encoding antioxidant enzymes, indicative of a cellular response to oxidative stress. In addition, the levels of lipid peroxidation were greater in mdx muscle than in the control. Since the free radical nitric oxide (NO*) has been shown to mediate oxidative injury in various disease states, and because dystrophin has been shown to form a complex with the enzyme nitric oxide synthase, we examined pre-necrotic mdx muscle for evidence of NO*-mediated injury by measuring cellular nitrotyrosine formation. By both immunohistochemical and electrochemical analyses, no evidence of increased nitrotyrosine levels in mdx muscle was detected. Therefore, although no relationship with NO*-mediated toxicity was found, we found evidence of increased oxidative stress preceding the onset of muscle cell death in dystrophin-deficient mice. These results lend support to the hypothesis that free radical-mediated injury may contribute to the pathogenesis of muscular dystrophies.

Stimulus-dependent subcellular localization of activated protein kinase C; a study with acidic fibroblast growth factor and transforming growth factor-beta 1 in cardiac myocytes.

Protein kinase C (PKC) isozymes regulate a number of cardiac functions including contractility, gene expression, and hypertrophy. There are at least six PKC isozymes in neonatal rat ventricular myocytes. We have shown previously that stimulation of cardiac myocytes in culture with norepinephrine (NE) or phorbol 12-myristate 13-acetate (PMA) results in translocation of each isozyme to distinct subcellular sites. In the present work, we demonstrated that PKC isozymes vary in their sensitivity to stimulation by acidic fibroblast growth factor (aFGF) and transforming growth factor-beta 1 (TGF-beta 1). Moreover, immunocytochemical studies indicated differences in the subcellular localization of activated isozymes following stimulation with each growth factor. These data suggest that the site of translocation and the resulting function of individual PKC isozymes are distinct for different PKC activators. Identification of the PKC isozymes that respond to aFGF and TGF-beta 1 and their subcellular localization may provide a molecular basis for the divergent cardiac functions mediated by these two growth factors.

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

Distinct responses of protein kinase C isozymes to c-erbB-2 activation in SKBR-3 human breast carcinoma cells.

We have studied the effect of activation of the c-erbB-2 receptor tyrosine kinase on protein kinase C (PKC) in cultured SKBR-3 human breast cancer cells. Treatment with the agonistic anti-receptor monoclonal antibody TAb 250 induces receptor autophosphorylation and stimulates phospholipase C-gamma 1 (L. K. Shawver et al. Cancer Res., 54: 1367-1373, 1994). TAb 250 induced a rapid and marked translocation of PKC histone phosphorylation activity to the particulate fraction of SKBR-3 cells. By immunoblot, however, this translocation was limited to specific PKC isozymes. beta PKC and zeta PKC translocated to the particulate fraction, whereas epsilon PKC underwent "partial reversed translocation" to the cell soluble fraction after receptor stimulation. Furthermore, beta PKC was rapidly degraded following TAb 250 treatment. By immunocytochemistry, beta IPKC translocated from the perinuclear area to the cytosol and into the nucleus, whereas zeta PKC translocated to the perinuclear region and into the nucleus. Consistent with the Western blot results, epsilon PKC translocated from the nucleus to the perinuclear area and the cytosol. These changes in the localization of PKC isozymes were not observed after addition of normal IgG1 or a nonagonistic anti-c-erbB-2 monoclonal antibody to SKBR-3 cells. alpha, beta II, or delta PKC present in these cells did not translocate following receptor stimulation. These data indicate that c-erbB-2 signal transduction may involve the activation of specific PKC isozymes. The biological role of these enzymes in the phenotype and cellular responses of c-erbB-2-overexpressing carcinoma cells remains to be studied.

Developmental expression of protein kinase C subspecies in rat brain-pituitary axis.

We have examined the neonatal developmental expression of protein kinase C subspecies (PKCs) in rat brain, pituitary glands and cells by enzymatic activity assays, immunohistochemistry and Western blot analysis with type-specific antibodies. A very large increase (455%) was noticed in brain PKC activity during the first week of life with the particulate fraction (22% of total enzyme activity on day 1) increasing dramatically (900%) during the first week to 50% of enzyme activity. In contrast, the pituitary gland showed high activity on day 1 that decreased progressively to reach the lowest levels at 1 year of age. Paradoxically, the number of pituitary cells immunolabeled for PKC increases as a function of age. Western blot analysis showed only small changes in PKC alpha, PKC beta and PKC epsilon when brains from 6-day-old and 3-month-old female rats were compared, whereas PKC tau and PKC delta increased markedly during this period. On the other hand, brain PKC zeta decreased between 6 days and 3 months of age. Western blot analysis showed no major changes in pituitary PKC alpha, PKC beta and PKC zeta when 6-day-old and 3-month-old female rats were compared, while PKC tau was not detected. The major band of pituitary PKC delta (76 kDa) decreased markedly between 6 days and 3 months of age whereas the minor band (68 kDa) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of protein kinase C isozymes in cardiac myocytes.

Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell-soluble fraction to the cell-particulate fraction, presumably near their protein substrates. Therefore, identifying the subcellular localization of each activated PKC isozyme may help to elucidate its role in cardiac functions. In the present work, we have determined the subcellular localization of six PKC isozymes (alpha, beta I, beta II, delta, epsilon, and zeta) in nonstimulated cardiac myocytes and in myocytes stimulated by norepinephrine (2 microM) or phorbol 12-myristate 13-acetate (100 nM). Activated PKC isozymes were localized in various subcellular compartments such as inside the nucleus and on myofibrils. The presence of serum in the growth medium also caused a redistribution of PKC isozymes in the cells distinct from that obtained with cells cultured in defined medium. We suggest that isozyme-specific localization may determine phosphorylation of different protein substrates present at these respective translocation sites and the resulting PKC-mediated cellular responses.

Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

Effects of calcium and calcium-channel blocker methoxyverapamil on the beta-adrenoceptors in myocardial cells in vitro.

The possible relationship between methoxyverapamil (D600) as a calcium-channel blocker and the beta-adrenoceptors was investigated on heart cells grown in culture, using [3H]CGP-12177 as a radioligand. Treatment with D600 (20 micrograms/mL) for 24 hr caused a decrease of 30% in the [3H]CGP-12177 binding sites. Scatchard analysis showed that the Bmax is similar in control and D600-treated cells, but the Kd in D600-treated cells increases. The effect of D600 on the isoproterenol-induced adenylate cyclase activation was examined and it was found that the D600 prevented the increase in cAMP obtained by isoproterenol treatment. These results indicate that the action of D600 on the beta-adrenoceptors is a competitive inhibition of the [3H]CGP-12177 binding sites. We investigated the effect of Ca2+ in the growth medium on the level of beta-adrenoceptors. Heart cells grown for 24 hr in Ca(2+)-free medium showed a decrease of 36% in the [3H]CGP-12177 binding sites without changing the dissociation constant. This decrease is probably a result of reduction in synthesis of the receptors. The level of receptors returned to control values following replenishment with normal growth medium. These results show that calcium is essential for the development of the beta-adrenoceptors in heart cells in vitro.

Regulation of beta-adrenoceptors by thyroid hormone and amiodarone in rat myocardiac cells in culture.

Hyperthyroidism is associated with elevation of heart cells sensitivity to catecholamines. We demonstrated that T3(10(-8) M) increased (30%) the number of beta-adrenoceptors in intact heart cells grown in vitro within 48 hr, without changing the affinity of the ligand [3H]CGP-12177. The increase in beta-adrenoceptors in T3-treated myocytes was not associated with an increase in receptor-mediated cAMP production. Amiodarone, an antiarrhythmic drug, reduces the sensitivity of the heart to catecholamines. To investigate this effect, we analysed the influence of amiodarone on the level of beta-adrenergic receptors. Ninety minute preincubation with amiodarone (5 x 10(-5) M) decreased the number of beta-adrenoceptors (35-50%) in intact heart cells and in heart membranes, without affecting the dissociation constants (Kd). Amiodarone inhibited isoproterenol induced cAMP production. These results indicate that the mechanism of action of amiodarone on the heart seems to be a non-competitive inhibition of catecholamine receptors.

Characterization of beta-adrenoceptors on rat skeletal muscle cells grown in vitro.

The binding properties of an hydrophilic beta-adrenergic receptor radioligand, (-)[3H](4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazolo-2-one ); ([3H]CGP-12177), were investigated in rat skeletal muscle cells in culture. The binding of [3H]CGP-12177 at 25 degrees was saturable, reversible and of high affinity (Kd = 1.3 +/- 0.3 nM). The maximal number of [3H]CGP-12177 binding sites was 30.6 +/- 3.2 fmol/dish (34 +/- 3.5 fmol/mg protein). beta-Adrenergic agonists and antagonists inhibited [3H]CGP-12177 binding. The competing ligand inhibition binding is a typical one for beta 2-adrenoceptors. The increase in beta-adrenoceptors was independent of cell fusion. Amiodarone (10(-5) M) decreased the beta-adrenoceptor number in skeletal muscle cells differentiated in vitro by 48%, while the affinity for [3H]CGP-12177 was not affected.

Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells.

Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16-BL6 melanoma; ESb T-lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monlayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intracellularly but it is instead found pericellularly and requires a continuous cell-matrix contact at the optimal pH for maintaining cell growth. The degradation of [35S]O4 = -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 micrograms/ml), arteparon (10 micrograms/ml), and heparin at a concentration of 3 micrograms/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo-beta-glucuronidase D-saccharic acid 1,4-lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases--alpha 2-macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)--failed to inhibit this degradation process, and only alpha 1-antitrypsin inhibited the heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 micrograms/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.

Evidence for a functional role of acetylcholinesterase in cultured chick myotubes.

This study was undertaken in order to assess the functional role of acetylcholinesterase (AChE) in cultures of chick skeletal muscle cells. Cultures of skeletal myotubes were prepared by mechanical dissociation of limb muscle removed from 11-day-old chick embryos and plating at a concentration of 0.8 X 10(6) cells/ml. Cultures incubated for 4-10 days were used for electrophysiological studies with intracellular microelectrodes. Individual myotubes differed with respect to the time course of repolarization following depolarization by acetylcholine (ACh), some cells repolarizing within 2-3 min and others only after 8-10 min. Physostigmine (10(-8)-10(-6) M) prolonged or sometimes completely prevented repolarization following ACh-induced depolarization. These results demonstrate that hydrolysis of ACh by AChE in cultured chick skeletal myotubes plays an important role in the repolarization of these cells following ACh-induced depolarization.