Effects of noradrenaline, serotonin, and selected antagonists on the vascular smooth muscle of normal and dystrophic chickens

The pathogenesis of the human muscular dystrophies is unknown, and several competing hypotheses have been proposed. The vascular hypothesis states that muscle fibre necrosis occurs in dystrophy as a result of transient muscle ischemia. Although abnormalities of the vascular system may be demonstrated in dystrophy, their role in pathogenesis remains obscure. The responses to serotonin (5-HT) and noradrenaline (NA) were examined in isolated ischiatic artery preparations from normal and genetically dystrophic chickens. The tension generated in response to 5-HT was greater in arteries from normal chickens than in arteries from dystrophic chickens, whereas responses to NA were similar. Analysis of the concentration-response relationships demonstrated that the dystrophic ischiatic artery was less sensitive to 5-HT than was the normal artery, although the sensitivity to NA was similar in both vessels. The results of this study are not consistent with the view that muscle fibre necrosis in avian dystrophy is a consequence of muscle anoxia. These data do demonstrate pharmacological differences between dystrophic avian arteries and arteries from normal chickens, but their presence may represent merely the expression of dystrophy in vascular smooth muscle.     

GSH system in relation to redox state in dystrophic skin fibroblasts

Glutathione and GSH-related enzymes were determined in human Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) skin fibroblasts in order to relate muscular dystrophy to the redox state of the cell. The analysis of GSH, GSSG and total GSH levels in normal and dystrophic-cultured fibroblasts shows no differences in normal growth condition. However, the specific activity of two GSH-related enzymes, glutathione S-transferases (GST) and gamma-glutamylcysteine synthetase (gamma-GCS), shows significant variations between normal and both types of dystrophic skin fibroblasts. These results suggest that even in normal growth condition some components of GSH metabolism may be altered. A condition of sublethal oxidation obtained by H(2)O(2) treatment was able to show a difference in the cellular response of GSH system components between normal and dystrophic cells. While in DMD cells there is a decrease of roughly 55% in GSH and of 30% in total GSH concentration, no changes are measured in normal and BMD cells. The remarkable increase in glutathione peroxidase (GPx) activity and decrease in GSH-reductase (GR) activity measured in DMD cells can in part explain these changes. These results indicate a different capacity of DMD cells to support oxidative stress with respect to BMD and normal cells, and suggest a possible role of the GSH-antioxidant system in dystrophic pathology.     

Spontaneous regeneration of older dystrophic muscle does not reflect its regenerative capacity

Young dystrophic (dy) murine muscle is capable of "spontaneous" regeneration (i.e., regeneration in the absence of external trauma); however, by the time the mice are 8 weeks old, this regeneration ceases. It has been suggested that the cessation of regeneration in dystrophic muscle may be due to exhaustion of the mitotic capability of myosatellite cells during the early stages of the disease. To test this hypothesis, orthotopic transplantation of bupivacaine treated, whole extensor digitorum longus muscles has been performed on 14 to 16-week-old 129 ReJ/++ and 129 ReJ/dydy mice. The grafted dystrophic muscle is able to produce and maintain for 100 days post-transplantation 356 +/- 22 myofibers, a number similar to that found in age-matched dystrophic muscle. The ability of old dystrophic muscle to regenerate subsequent to extreme trauma indicates that the cessation of "spontaneous" regeneration is due to factor(s) other than the exhaustion of mitotic capability of myosatellite cells. Moreover, there is no significant difference in myosatellite cell frequencies between grafted normal and dystrophic muscles (100 days post-transplantation). Myosatellite cell frequencies in grafted muscles are similar to those in age-matched, untraumatized muscles. While grafting of young dystrophic muscle modifies the phenotypic expression of histopathological changes usually associated with murine dystrophy, grafts of older dystrophic muscle show extensive connective-tissue infiltration and significantly fewer myofibers than do grafts of age-matched normal muscle. As early as 14 days post-transplantation, it is possible to distinguish between grafts of old, normal and dystrophic muscles. It is suggested that the connective tissue stroma, present in the dystrophic muscle at the time of transplantation, may survive the grafting procedure.     

Myoblast senescence in muscular dystrophy

The limited proliferative capacity of normal diploid cells predicts that the utilization of cell divisions in vivo should reduce the lifespan of cells in culture. Because of the continuing demands for muscle regeneration in muscular dystrophy, myoblasts isolated from affected muscles should thus show a decrease in the number of cell divisions they are capable of expressing in culture. This hypothesis was tested by examining the proliferative capacity of myoblasts from different muscles for normal line 412 and dystrophic line 413 chickens of various ages. Prior to approx. 2 months of age, dystrophic myoblasts exhibited a relatively normal proliferative lifespan. By 5 months of age, myoblasts from the severely affected pectoralis major showed a 40% reduction in their proliferative potential, while myoblasts from the less affected posterior latissimus dorsi muscle showed a 25% decrease in their cultured lifespan. The time course of the appearance of a decreased proliferative capacity only after the disease has been clinically manifested strongly supports it representing a secondary response rather than it being an intrinsic property of dystrophic myoblasts. A hypothesis for manipulating the pattern of stem cell division in order to increase the mass of muscle produced from a constant number of cell divisions is presented. If myoblast senescence and the consequent failure of muscle regeneration is a contributing factor in the progressive deterioration of muscle function in the disease, then this hypothesis might provide an important therapeutic strategy for ameliorating the course of muscular dystrophy.     

Relaxing effect of atrial natriuretic factor on endothelin-precontracted vascular strips

Endothelin (ET), a peptide recently isolated from the supernatant of cultured porcine aortic endothelial cells, is a potent vasoconstrictor. On the other hand, atrial natriuretic factor (ANF) is a powerful vasorelaxant found in cardiocytes. Its effect was investigated in ET-precontracted rabbit vascular strips. ANF-induced a dose-dependent relaxation of maximally-precontracted mesenteric, renal and aortic strips. Mesenteric artery strips were more sensitive to ANF than either renal or aortic strips. The relaxant effect of ANF on ET-precontracted arteries was more potent than that of other vasorelaxant agents, such as isoproterenol and sodium nitroprusside. Renal and aortic arteries were more sensitive to the vasoconstrictor effect of ET than mesenteric strips. From these results, we conclude that ANF may play a role as a physiological antagonist of ET. The different sensitivity of vascular segments to ET could be due to varying vascular ET receptor densities.     

Abnormal collagen synthesis in skeletal muscle of dystrophic chicken

Specific molecular properties of skeletal muscle collagens from normal and dystrophic chickens have been compared. When dystrophy develops in skeletal muscle tissue there was an increase in the amount of total collagen and an increased proportion of Type III collagen in the tissue. The results from the cross-link study as well as the analysis of the solubility of collagen showed that skeletal muscle of dystrophic chicken produces more immature collagen fibers compared to normal chicken. These findings strongly indicate an important role of collagen in the pathogenesis of the extensive connective tissue prolipheration characteristic of muscular dystrophies.     

Elevation of the Level of Thiobarbituric Acid-Reactive Products in Hindleg Skeletal Muscle of Dystrophic Mice, but Non-Elevation in Tongue Muscle

In order to understand the pathogenesis of mouse muscular dystrophy, we investigated the levels of the thiobarbituric acid-reactive substances (TBARS), H₂O₂ and NADPH oxidase activity, which were relative to the acceleration of oxidative conditions, in tongue and hindleg skeletal muscles from C57BL/6J-dy mice. The TBARS content (702 nmol/g protein) in skeletal muscles from 2-months-old dystrophic mice was increased significantly over that (384 nmol/g protein) in muscles from age-matched normal mice. The H₂O₂ concentration in dystrophic skeletal muscles was 30% higher than that in normal ones. Microsomal NADPH oxidase activity which was related to the production of superoxide anions, was similar between dystrophic muscles (4.66 nmol/10 min/mg protein) and normal muscles (4.11 nmol/ 10 min/mg protein). These results indicate that oxidation is accelerated in the dystrophic muscles. However, the TBARS content in the tongues of dystrophic mice was identical to that of normal mice. This finding supports our bone-muscle growth imbalance hypothesis for the pathogenesis of mouse muscular dystrophy.     

Altered contents of tocopherols in chickens with inherited muscular dystrophy

Oxidative damage has been hypothesized as the basis for some of the changes in enzymatic functions and physical properties of membranes in inherited muscular dystrophy. The contents of alpha- and gamma-tocopherol (vitamin E) and their oxidation products, the tocopheryl quinones, were measured at 1 to 4 weeks after hatching in the muscle and other tissues of chickens with inherited muscular dystrophy. Analyses at these early ages minimized the potential influence of pathological changes on the measured parameters. The affected muscle (pectoralis major) of dystrophic birds contained significantly higher levels of alpha-tocopheryl quinone and a decreased ratio of alpha- to gamma-tocopherol. Consistent changes in these parameters were not observed in other tissues. Although their basis remains unclear, these changes in the tocopherols are suggestive of oxidative stress in dystrophic muscle membranes. Lipid extracts of tissues of normal and dystrophic birds exhibited no significant differences in the content of conjugated dienes or lipofuscins, two other indices of oxidative stress. These data do not consistently support the hypothesis that oxidative stress plays a causal role in damage to dystrophic muscle, although it remains possible that free-radical damage is involved in the secondary alterations associated with muscular dystrophy.     

Newer drug treatment in dermatology

In a study of 58 patients with various diseases of muscle or of the neuromuscular system, the serum activity of various enzymes was measured. Abnormal elevation of serum activities of aldolase, lactic dehydrogenase and, to a lesser extent, glutamic-oxalacetic transaminase and phosphohexose isomerase, was an almost constant feature in patients with progressive muscular dystrophy. These elevations were very frequent in dermatomyositis, common in acute cerebral vascular accidents, and rarely seen in other neurological disorders. Abnormal serum activity of iso-citric dehydrogenase was not observed in the course of the present study. Supplementary protein feeding of patients with muscular dystrophy had no effect on serum enzyme activity, no consistent effect on urinary creatine excretion and no effect on the strength of the patient or the course of the disease. Dystrophic muscles from a dystrophic strain of mice showed a decrease in activity of lactic dehydrogenase and aldolase below that of control muscle and an increase of iso-citric dehydrogenase activity. These findings, taken with the differences in serum activities of lactic dehydrogenase, aldolase and isocitric dehydrogenase in the dystrophic animals, support the conclusion that dystrophic animals handle these soluble enzymes in quite different ways.     

The dynamics of the nitric oxide release-transient from stretched muscle cells

Potent nitric oxide (NO) signals are described for many forms of cell-cell communication. Although NO plays a significant role in skeletal muscle metabolism and contractility and in precursor activation during muscle formation and stretching, there is no direct evidence of stretch-induced NO release from muscle. Differentiated muscle cell cultures from normal and dystrophic mdx mice were preloaded with the NO-specific dye DAF-2 (diaminofluorescein-2) before stretching. NO release was detected by video-microscopy. NO was released rapidly from wild-type (WT) cells after stretch and intensity declined rapidly to a plateau. Mdx cells showed much less NO release. Direct observations of the time-course of stretch-induced NO release in WT cells is congruent with the hypothesis of NO-mediated stretch activation of satellite cells in normal skeletal muscle. Distinct differences in the time-course between normal and dystrophic cells indicate visualization methods for NO release will be a sensitive measure of NOS-1 restoration following diverse treatment approaches to muscular dystrophy.     

Myofibrillar creatine kinase in Duchenne and avian muscular dystrophy

The presence and activity of the fraction of creatine kinase (CK) which was associated with myofibrils and located in the M line of the sarcomeres was determined in normal and dystrophic avian muscle and in normal and dystrophic (Duchenne) human muscle. Myofibrils were isolated from homogenates of muscle and washed nine times so as to remove nonmyofibrillar CK. In myofibrils from dystrophic muscle the enzyme CK was localized to the M line using immunofluorescent techniques and was enzymatically active. These results suggest that in both avian and Duchenne muscular dystrophy, there is not a myofibrillar disorder of the phosphocreatine shuttle.     

Role of calcium in postjunctional supersensitivity.

Several hours to days after an animal is given reserpine its cardiovascular system becomes supersensitive to catecholamines. This phenomenon can be demonstrated for vascular tissue by in vitro experiments. This type of supersensitivity has been termed "nonspecific" because the tissue is supersensitive to varied agonists, including acetylcholine, calcium, potassium, and the catecholamines. Animals that have been treated with reserpine have been found to have a transient decrease in the calcium content of their vascular tissue. The responses to norepinephrine of aortic strips from reserpine-treated rabbits, even though of greater magnitude than those of untreated aortic strips, were less dependent on extracellular calcium than responses of strips from untreated rabbits. On the other hand, the responses to potassium were more dependent on extracellular calcium. In addition, when aortic strips from reserpine-pretreated animals are subjected to potassium in a calcium-free medium, they are not supersensitive to the ion. When aortic strips are placed in a calcium-free, depolarizing medium they are still supersensitive to norepinephrine and isoproterenol but not to acetylcholine. Tension decline and 45Ca efflux studies suggest that reserpine-treated tissues retain longer than untreated tissues a calcium fraction involved in contraction. It is concluded that reserpine alters binding or movement of calcium in at least two sites. The lack of supersensitivity to acetylcholine and potassium in a calcium-free medium indicates an effect of reserpine (or the loss of adrenergic transmitter) on the utilization of extracellular calcium, while some other site must be involved in at least part of the supersensitivity to the catecholamines.     

Synthesis of apolipoprotein A1 in skeletal muscles of normal and dystrophic chickens

We recently observed that, around the time of hatching, chick skeletal muscles synthesize and secrete apolipoprotein A1 (apo-A1) at high rates and that reinitiation of synthesis of this serum protein to high levels occurs in mature chicken breast muscle following surgical denervation (Shackelford, J. E., and Lebherz, H. G. (1983) J. Biol. Chem. 258, 7175-7180; 14829-14833). In the present work we investigate the effect of avian muscular dystrophy on the synthesis of apo-A1 in chicken muscles. The relative rate of synthesis of apo-A1 and levels of apo-A1 RNA in mature dystrophic breast (fast-twitch) muscle were about 6-fold higher than normal, while synthesis of apo-A1 in breast muscles derived from 2-day-old dystrophic chicks was close to normal. These observations suggest that the elevated apo-A1 synthetic rate in mature dystrophic breast muscle results from a failure of the diseased tissue to "shut down" apo-A1 synthesis to the normal level during postembryonic maturation. Apo-A1 synthesis in the "slow-twitch" lateral adductor muscle of dystrophic chickens was found to be normal. Our work is discussed in terms of the apparent similarities between the effects of surgical denervation and muscular dystrophy on the protein synthetic programs expressed by chicken skeletal muscles.     

Natural killer cell activity in murine muscular dystrophy. III. NK-sensitive myoblast cells and lack of NK activity in beige/dystrophic hybrid mice

The NK-susceptibility of dystrophic mouse myoblast cells was investigated. Spleen cells from 8- to 10-week-old normal (+/+) and dystrophic (dy2J/dy2J) male C57BL/6J mice were fractionated on Percoll density gradients and the cells at each density interface were incubated with either 51Cr-labeled YAC-1 or myoblast cells in a 6 hr 51Cr-release assay. Myoblast target cells were obtained from either heterozygous (+/dy2J) or homozygous (dy2J/dy2J) muscle cultures or a transformed tetraploid myoblast line (M14D2). The data indicate that the interface between the 50 and 60% (1.060-1.075 g/ml) Percoll density fractions of spleen cells from either normal or dystrophic mice contains the largest proportion of asialo GM-1 positive and NK-1 positive cells displaying NK activity. Myoblast cells from either heterozygous (phenotypically normal) or homozygous dystrophic mice were not significantly different in susceptibility to NK-mediated lysis by Percoll enriched normal or dystrophic mouse NK cells. However, dystrophic mouse spleen cells had the highest NK activity against both myoblast targets as compared with normal mouse spleen cells. The transformed myoblast cell line, M14D2, was significantly less susceptible to NK-mediated lysis by dystrophic mouse spleen cells when compared with freshly cultured myoblast target cells. Target cell binding studies revealed that conjugate forming cells from the 50% Percoll density interface of dystrophic mouse spleen cells were approximately twofold greater than that of normal mouse spleen cells against either heterozygous or homozygous dystrophic mouse myoblast targets. Cold target inhibition studies revealed that the natural killing of dystrophic mouse myoblast cells was due to a YAC-1 reactive NK cell. Breeding experiments between C57BL/6J homozygous "beige" (bgJ/bgJ) mutant mice and dystrophic (dy2J/dy2J) mice produced beige/dystrophic hybrid mice which displayed clinical symptoms of the dystrophy process by 3 to 4 weeks of age. Spleen cells from these hybrid mice showed no significant differences in NK activity against YAC-1 target cells when compared with homozygous beige mice. Taken together, these results demonstrate the first reported evidence that murine myoblasts are susceptible to NK-mediated lysis. In addition, the data indicate that although dystrophic mouse NK cells recognize myoblast cells as targets, the NK cell studies with the beige/dystrophic hybrid mice do not indicate a direct in vivo role for NK cells in the dystrophy process.     

Acetylcholinesterase in singly and multiply innervated muscles of normal and dystrophic chickens. II. Effects of denervation.

Neural regulation of mature normal fast twitch muscle of the chicken suppresses high activity, extrajunctional localization, and isozyme forms of acetylcholinesterase (AChE) characteristic of embryonic, denervated and dystrophic muscle. Normal adult slow tonic muscle ofthe chicken retains intermediate levels of activity and embryonic isozyme forms but not extrajunctional activity; it is not affected by muscular dystrophy. The hypothesis that neural regulation of the AChE system is lacking in slow tonic muscle and thus not affected by dystrophy was tested by denervating the fast twitch posterior latissimus dorsi and slow tonic anterior latissimus dorsi muscles of normal and dystrophic chickens. Extrajunctional AChE activity and embryonic isozyme forms increased, then declined, in both muscles. The results suggest that ocntrol of AChE is qualitatively similar in slow tonic and fast twitch muscle of the chicken.     

Comparison of the turnover patterns of total and individual muscle proteins in normal mice and those with hereditary muscular dystrophy

1. The incorporation of amino acids into hindleg muscle proteins of normal and dystrophic mice was measured (1/2)h to 16 days after administration of the radioactive pulse. 2. Dystrophic animals showed a faster initial rate of incorporation into total and soluble proteins in the first few hours after injection, but the extent of incorporation relative to the size of the amino acid pool was similar in both. There was little difference between the overall degradation rates although this started later in the dystrophic proteins. An initial fast phase of degradation reached a plateau after 3 days whereupon the residual label in the protein remained constant up to 16 days after injection. 3. Analyses of individual radioactive proteins fractionated by polyacrylamide-gel electrophoresis showed that the distribution of label was similar in all the soluble proteins from normal and dystrophic muscle. Time-course experiments revealed that in dystrophic mice the two major soluble proteins of the muscle, creatine kinase and adenylate kinase, initially incorporated 2-3 times more label relative to the initial size of the precursor pool. This label was then lost equally rapidly and the final plateau value was much less than that in normal mice. This initial peak of activity was not observed in normal mice. 4. A group of dehydrogenases showed similar initial turnover patterns in both dystrophic and normal mice but the final plateau value was much higher in the former. 5. The results provide support for the hypothesis that there is no obvious defect in the protein synthetic machinery of dystrophic muscle. However, certain proteins do show anomalous turnover patterns relative to those in normal animals. A single structural gene mutation giving rise to one particularly unstable and readily degradable muscle protein is excluded as the cause of the dystrophy.     

Heritable variation in erythrocyte membranes of mice with muscular dystrophy

Several physical and chemical characteristics of erythrocyte membranes from dystrophic mice differ from those of controls. In this study it is postulated that there is a heritable antigenic characteristic in erythrocyte membranes of dystrophic mice that is associated with muscular dystrophy. Accordingly, antisera were prepared in goats against erythrocyte membranes from dystrophic mice. These antisera discriminated between membranes from control mice and membranes from three different strains of dystrophic mice. Preliminary data on carrier detection are consistent with the hypothesis that the antigen is manifest in membranes of carriers.     

Effects of LY83583, nordihydroguaiaretic acid, and quinacrine on cyclic GMP elevation and inhibition of tension by muscarinic agonists in rabbit aorta and left atrium

Elevation of cyclic GMP by muscarinic agonists has been suggested to be responsible for the negative inotropic effects of these agents in cardiac muscle, and for the endothelium-dependent relaxation caused by these agents in vascular smooth muscle. These relationships were studied by monitoring the effects of muscarinic agonists on tension and cyclic GMP levels in rabbit left atrial strips and aortic rings, in the presence and absence of the cyclic GMP lowering agent, LY83583. LY83583 completely blocked both the cyclic GMP increase and the relaxation caused by acetylcholine in rabbit aortic rings with intact endothelial cells. Acetylcholine-induced cyclic GMP elevation and relaxation in these preparations were also blocked by quinacrine and nordihydroguaiaretic acid (NDGA), but neither response was blocked by the 5-lipoxygenase inhibitor U-60257. In the experiments with rabbit left atrium, LY83583 blocked the acetylcholine-induced cyclic GMP elevation but did not block the negative inotropic effects of the drug. Quinacrine, NDGA, and a guanylate cyclase inhibitor, methylene blue, failed to block either the cyclic GMP increase or the decrease in contractile force caused by carbachol in atrial strips. These results support the suggestion that an increase in cyclic GMP may be responsible for the endothelium-dependent relaxation of rabbit aorta by muscarinic agonists, but not for the direct negative inotropic effects of these drugs in rabbit atrium. Muscarinic agents appear to increase cyclic GMP levels in rabbit atrium and aorta by different mechanisms. Although both are blocked by LY83583, they differ not only in their requirements for endothelial cells, but also in their susceptibility to other blocking agents.     

Parathyroid hormone-induced changes in cyclic nucleotide levels during relaxation of the rabbit [correction of rat] aorta

Parathyroid hormone is a potent vasodilator in vivo and relaxes vascular tissue in vitro. Since parathyroid hormone action in kidney and bone is thought to be mediated by stimulation of cellular cyclic AMP production, the present study was designed to monitor changes in cyclic AMP and cyclic GMP in vascular tissue during relaxation by parathyroid hormone. Rabbit aortic strips were quick-frozen at various times after exposure to parathyroid hormone and the percent relaxation and cyclic nucleotide levels were determined. Cyclic AMP concentrations were elevated about 3-fold within 30 seconds after treatment with hormone. This corresponded to a 10% relaxation of the norepinephrine-contracted tissue. After five minutes, cyclic AMP was still elevated 2-fold above basal and the relaxation response was maximal (36%). The cyclic AMP and relaxation responses to parathyroid hormone were markedly potentiated by forskolin or methylisobutylxanthine. Parathyroid hormone produced a small but significant increase in cyclic GMP concentrations only at early time points whereas sodium nitroprusside substantially increased cyclic GMP and relaxed the strips at all times studied. The increase in cyclic AMP levels after exposure to parathyroid hormone occurred prior to or coincident with the onset of relaxation of the aortic strips. These findings are supportive of the hypothesis that the vascular actions of parathyroid hormone involve cyclic AMP.     

Intracellular protein degradation in cultures of dystrophic muscle cells and fibroblasts

We have analysed protein degradation in primary cultures of normal and dystrophic chick muscle, in fibroblasts derived from normal and dystrophic chicks, and in human skin fibroblasts from normal donors and from patients with Duchenne muscular dystrophy (DMD). Our results indicate that degradative rates of both short- and long-lived proteins are unaltered in dystrophic muscle cells and in dystrophic fibroblasts. Longer times in culture and co-culturing chick fibroblasts with the chick myotubes do not expose any dystrophy-related abnormalities in protein catabolism. Furthermore, normal and dystrophic muscle cells and fibroblasts are equally able to regulate proteolysis in response to serum and insulin. We conclude that cultures of chick myotubes, chick fibroblasts, and fibroblasts derived from humans afflicted with DMD are not appropriate models for studying the enhanced protein degradation observed in dystrophy.