Dystrophic Chickens Research Articles

Low frequency chronic electrical stimulation of normal and dystrophic chicken muscle.

The fast-twitch posterior latissimus dorsi muscle of normal and genetically dystrophic chickens was subjected to continuous indirect electrical stimulation at 10 Hz for periods of 4-8 weeks. To sustain this in vivo nerve stimulation an internally implantable miniature stimulator device was designed. This regime of stimulation caused complete fatigue of the normal muscle within 5 min of its initiation. The dystrophic muscles maintained a very small degree of contractile activity during this initial phase. Tangible twitching of the muscle returned in 5 week birds between 3 and 5 days and in 10 week birds between 11 and 16 days after implantation. After 4 weeks of stimulation, no significant change was measured in the time-to-peak of the isometric twitch response, nor in the half-relaxation time. The resistance to fatigue was significantly increased in the stimulated muscles when tested with a series of tetani at 40 Hz. The mean fibre area was decreased, in all muscles stimulated for longer than 3 weeks, in comparison to their contralateral controls, except where fibre splitting in dystrophic birds abnormally reduced the control value. The majority fibre type of the muscle was changed from type IIB to IIA. The histochemical reactions for both NADH-linked oxidation and phosphorylase were distinctly increased in the stimulated muscles. In normal muscle, stimulation increased somewhat the number of nuclei per unit area and changed their intracellular distribution, so that a greater proportion was found adjacent to the sarcolemma. The normal posterior latissimus dorsi muscle responded to chronic stimulation with increases of 3-6-fold in its acetylcholinesterase (AChE) activity. The maximum change in AChE occurred after 2 weeks stimulation; a steady level, 3 times that of the control unstimulated muscle, persisted at later times. Chronic stimulation suppressed the over-production of AChE that is characteristic of dystrophic chicken fast-twitch muscle, to attain a level comparable to the AChE activity in a stimulated normal muscle. Stimulation exerted a strong normalizing influence on dystrophic muscle, as assessed morphologically. The characteristic fibre rounding, fibre hypertrophy and myonuclear proliferation were reduced. This influence was most marked where the stimulation was initiated before the major pathological changes had occurred, but was also significant when commenced in strongly affected birds of 10-11 weeks.

The localization of a tcRNA102 gene near the 3' OH terminus of a fast myosin heavy chain gene. A comparison between normal and dystrophic chickens.

Two genomic fragments were isolated from a normal and a dystrophic library containing the 3'OH terminus of the fast isoform of myosin heavy chain gene. Restriction map analysis confirmed that the genes were similar. The sequences coding for myosin were defined and shown to be the same in each genomic fragment. However, using a cDNA clone for tcRNA102 and two specific oligomers for tcRNA102 sequences, we determined that only the genomic fragment from normal chick contained homologous sequences to tcRNA102. Dystrophic chick DNA did not contain these regions of homology. In addition, the normal genomic fragment transcribes tcRNA102 in vitro via RNA polymerase III while the corresponding fragment of DNA from dystrophic chick was inactive. These results suggest that there are detectable differences between the normal and dystrophic genomes in this regard.

Analysis of tcRNA102 associated with myosin heavy chain-mRNPs in control and dystrophic chick pectoralis muscle.

Translational control RNA (tcRNA102) is closely associated with nonpolysomal myosin heavy chain-mRNA in mRNP particles. The nucleotide sequence of tcRNA102 has revealed a heterogeneity at the 3' end. This heterogeneity is mostly with regard to an ambiguity between adenine and guanine residues. tcRNA102 (obtained from pectoralis muscle) runs as a single band on denaturing acrylamide gels. When this band is extracted and rerun on a native gel at low voltage, two individual bands appear (A the slower moving and B the faster moving). From the partial RNase U2 sequence analysis and our previous sequence determinations (McCarthy, T. L., Siegel, E., Mrockowski, B., and Heywood, S. M. (1983) Biochemistry 22, 935-941), we may now assign tcRNA102 (A) the 3'-terminal sequence ... GGUUGGACGG-3' and tcRNA102(B) and 3' terminal sequence ... GAUUAAGCAA-3'. Analysis of the tcRNA102s indicates that dystrophic pectoralis muscle contains much less tcRNA102 than a similar preparation from control muscle. The tcRNA102 found in dystrophic pectoralis muscle is of the "A" type while normal pectoralis muscle contains predominantly the "B" type. In addition, control leg muscle from dystrophic chick contains predominantly "B" type. These results suggest that the differences observed at the DNA level (see accompanying paper, Zezza, D. J., and Heywood, S. M. (1986) J. Biol. Chem. 261, 7455-7460) may be reflected in the RNA transcripts.

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.

Abnormal expression of the calmodulin gene in muscle from the dystrophic chicken

Compared to that of genetically-related normal chickens, pectoralis muscle from the dystrophic chicken contained increased calmodulin measured by radioimmunoassay. Determined by the dot blot procedure, expression of the calmodulin gene was enhanced in muscle from affected animals. The bioactivity of the gene product was normal. Together with previous studies reporting increased cell Ca 2+ content in dystrophic muscle, the current findings of increased sarcoplasmic calmodulin suggest the latter is a cellular response to defective Ca 2+ transport at the level of cell efflux or intracellular organelle (sarcoplasmic reticulum) uptake.

Microtubules are not altered in the dystrophic chicken

The association of an altered cytoplasmic microtubule complex in cells of the dystrophic chicken was investigated. Dystrophic chickens of lines 304 and 413 were compared with their genetically matched control, 412 (obtained from UC, Davis). Explants and trypsin-dissociated tissues were prepared from breast and heart muscles of chickens at 1, 3, 7, 14, 20, 40, 80 and 120 days ex ovo. The cells were cultured for 7 days and then processed for antitubulin immunofluorescence. Over 90% of the cells displayed an extensive cytoplasmic microtubule complex, although there was significant elevation of creatine phosphokinase in the dystrophic chickens after 20 days ex ovo. In both dystrophic and control preparations, one to two distinct functionally intact microtubule-organizing centers per cell were observed. Dystrophic and control chicken brain extracts demonstrated essentially the same extent of microtubule assembly as assayed by turbidity increase and protein in sedimentable polymer. SDS—PAGE revealed no significant differences in the microtubule proteins polymerized from the dystrophic and control brains. These results suggest that no significant alteration occurs in the structure, assembly or distribution of cytoplasmic microtubules in the cells of the dystrophic chicken.

Increased concentration of spectrin is observed in avian dystrophic muscle.

A significant increase in the concentration of spectrin has been observed in dystrophic chicken pectoralis major muscle when compared to normal fast-twitch muscle. In normal muscle, alpha-spectrin-specific immunofluorescence delineates each myofiber with a network pattern of staining at the sarcolemma with little staining within the cytoplasm. In dystrophic fibers, numerous intensely stained areas occur within the cytoplasm and staining at the sarcolemma is increased, thereby obscuring or eliminating the highly regular network arrangement of spectrin usually seen in this region. When immunofluorescence experiments are performed on microsomal vesicles isolated from normal and dystrophic tissues, only a small fraction of normal vesicles are stained, whereas most of the dystrophic vesicles are associated with spectrin. An increase in spectrin concentration is observed using immunoautoradiography of whole muscle and isolated microsomes, thus supporting the immunofluorescent observations described above. The early-age post-hatching when increases in spectrin concentration can be detected and the simplicity of the immunofluorescent technique make this observation useful as a new diagnostic parameter. This observation also shows that the distribution of spectrin and its concentration within nonerythroid cells can be modified by abnormal physiological states; this modification may contribute to subsequent symptoms, such as increased rigidity and abnormal calcium metabolism, that are observed in dystrophy.

Activities of antioxidant enzymes in muscle, liver and lung of chickens with inherited muscular dystrophy

An inherited form of muscular dystrophy in chickens has been used as a model of Duchenne muscular dystrophy. The pectoralis major muscle of chickens with this disease showed a significantly elevated activity of catalase (CAT) one day after hatching, and by 7 days showed elevated superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST) activities. Increases were also found in tissues of the dystrophic birds that, unlike the pectoralis muscle, are considered to be unaffected by the pathology of muscular dystrophy. The soleus muscle contained significantly increased levels of SOD and GPX in 1 and 7 day old chickens, and increased GST in 1, 14, and 28 day old birds. CAT was significantly increased in liver from 1 and 7 day old chickens, while GPX was increased in lung from 1, 7 and 14 day old birds. These results support the possibility that excess oxygen free-radicals or altered cellular antioxidant defenses play some role in the pathogenesis of muscular dystrophy.

Connective tissue metabolism in muscular dystrophy. Early amino acid changes in collagen types isolated from the gastrocnemius muscle of developing dystrophic chicken embryos

1. 1. The amino acid composition of all collagen types present in the gastrocnemius muscle of dystrophic chick embryos showed an altered profile at both day 14 and day 20 in ovo when compared with the controls. 2. 2. The changes observed at both day 14 and day 20 in ovo suggests that there is a removal of polar side-chains in dystrophic collagen and substitution with non-polar amino acids. 3. 3. The amino acid composition data between day 14 and day 20 indicated: (a) a decrease in hydroxylation (hydroxyproline and hydroxylysine) with a concurrent increase in proline and lysine and a decrease in the levels of arginine; (b) the levels of glycine and alanine did not change with age; and (c) the ratios of glycine to hydroxyproline and proline to hydroxyproline changed significantly in all dystrophic collagen types between day 14 and day 20. 4. 4. Contrast analysis results clearly showed that the changes in amino acid composition observed in each dystrophic type of collagen between day 14 and day 20 were not due to the effect of aging but to some other factor(s). 5. 5. This study provides more evidence that a problem lies in the biosynthesis of collagen present in developing muscles of dystrophic chick embryos, particularly with respect to the transcription or translation of procollagen genes and/or a failure in the processing and differentiation of collagen types.

Characterization of synthesized protein in the dystrophic chicken sympathetic ganglion

Characterization of synthesized protein in the dystrophic chicken sympathetic ganglion

Elevation of calmodulin in avian muscular dystrophy

In an attempt to understand the mechanism of calcium accumulation in myopathies, changes in the major calcium-binding protein, calmodulin, was studied in genetically dystrophic chickens. Measurements by radio-immunoassay revealed an increase in the calmodulin concentration of dystrophic chicken muscles. Poly A-containing RNA(s) of fast and slow muscles from the normal and dystrophic chicks were hybridized with [ 32P]-labeled calmodulin cDNA probe by the dot-hybridization technique. Densitometric scan of the autoradiogram showed that the calmodulin mRNA levels of dystrophic fast muscles (pectoralis and posterior latissimus dorsi) were approximately two-fold higher than those of the corresponding normal muscles. No significant change in calmodulin and calmodulin messenger RNA of slow muscle (ALD) was found in dystrophic chickens. Our results suggest that increased calcium flux within the dystrophic muscle may be modulated by calmodulin.

Effects of percutaneous electrical stimulation on functional ability, plasma creatine kinase, and pectoralis musculature of normal and genetically dystrophic chickens

The breast musculature of genetically dystrophic Line 413 and genetically related normal Line 412 chickens were treated in three separate trials with high-frequency electrical stimulation (ES). Beginning on days 7 or 14 ex ovo, each bird received three ES treatments per week. Each stimulation cycle repeated five times per day consisted of 15 s “on” followed by 50 s “off.” In the third trial only, the birds were additionally treated beginning day 3 ex ovo with either leucine (100 mg/kg) or the proteinase inhibitor Ep475 (10 mg/kg). ES significantly delayed the onset of righting disability in the dystrophic chickens. However, this improvement was temporary and could be masked by single treatments of either leucine or Ep475. Plasma creatine kinase activities were increased generally in both the stimulated normal and dystrophic birds. In two trials ES increased the relative muscle mass, and in one trial increased protein. ES had little effect on normal muscle mass or protein. However, ES treatment together with either leucine or Ep475 appeared to improve both normal and dystrophic muscle mass and protein. Furthermore ES decreased dystrophic muscle calcium but not acetylcholinesterase activity. On the other hand, ES had no effect on the total normal muscle calcium but increased normal acetylcholinesterase values. In both normal and dystrophic muscle samples, ES treatment in combination with leucine appeared to increase the mean muscle fiber diameters and number of myonuclei, and in the case of the dystrophic muscle, appeared to decrease the relative proportion of vacuolated, degenerating, and intensely oxidative histochemical fibers. In general, stimulation (especially in combination with leucine) appears to alter in varying degrees the phenotypic expression of the muscle disease exhibited in the dystrophic chicken.

Endogenous lectins in normal and dystrophic muscle development.

Two endogenous lectin activities, one specific for beta-D-galactose (beta-D-Gal) residues and purified on asialofetuin-Sepharose and the second specific for iduronic acid containing glycosaminoglycans and purified on heparin-Sepharose, have been studied during myogenesis in both normal and dystrophic chickens. The Storrs strain, homozygous for muscular dystrophy, and the dystrophic strain 413 from the University of California at Davis were both used in this study. Strain 412 and local hatchery chickens were used as controls. The lectins derived from all sources appeared to be identical based on physical properties and carbohydrate specificity. Both normal and dystrophic adult chickens possessed similar lectin levels in lung, spleen, kidney, heart, and muscle tissue. No differences were noted in the temporal appearance of the heparin-binding lectin; however, the beta-D-Gal-binding lectin appeared earlier in the Storrs dystrophic strain than it did in the 413, 412, or hatchery embryos.

Structure of motor nerve terminals in chickens with hereditary muscular dystrophy

The structure and size of 1-week to 1-year-old normal (line 412) and dystrophic (line 413) chicken motor nerve terminals were studied using combined pre- and postsynaptic histologic endplate staining. The main result is that adult dystrophic terminals have abnormal structure and are significantly smaller than normal. These differences occurred progressively during development. At 1 week ex ovo, dystrophic motor nerve terminals were similar to normals in size and appearance. By 8 weeks, differences between normal and dystrophic terminal size and structural organization began to emerge. Qualitatively, beginning at 8 weeks and becoming more frequent by 1 year of age (the endpoint of this study), dystrophic motor endplates differed from normal in having: generally smaller synaptic boutons, often separated by extremely thin branching interconnectives; increasing incidence of multiple innervation; and frequent occurrences of apparent partial or total denervation, terminal sprouting, and reinnervation.

Altered membrane association of glycogen phosphorylase in the dystrophic chicken.

The subcellular distribution of glycogen phosphorylase in pectoralis muscle from normal and dystrophic chickens was determined as a function of age. A substantially larger proportion of the total activity was associated with membranous cellular organelles, both mitochondria and sarcoplasmic reticulum, in preparations from dystrophic birds. The difference could be detected as early as 2 weeks ex ovo. Interaction of phosphorylase with cellular membranes may provide a probe for the underlying membrane defect in this dystrophy model.

Developmental neuron cell death in dystrophic and normal chickens

The numbers of motoneurons in the brachial lateral motor columns were determined from the 6th embryonic day through the 5th week posthatching in age-matched dystrophic and normal chickens. The overall magnitude of cell loss was not significantly different between the dystrophic and normal strains. However there was a trend toward more motoneurons in the brachial lateral motor columns of dystrophic embryos and posthatch chickens which was found to be statistically significant at the 8th and 9th days of embryonic life. These observations are taken to suggest that, in addition to strain differences unrelated to the dystrophic gene, there may exist a temporal delay in developmental neuron cell death in dystrophic chick embryos that may be secondary to the primary defect within the dystrophic muscle.

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT ( p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36–44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 m m creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20–50 m m P i are reduced significantly. Finally, increasing concentrations of P i displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent K D , indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.

Stiffness and contractile properties of avian normal and dystrophic muscle bundles as measured by sinusoidal length perturbations

Both tension and stiffness as a function of muscle length were measured under relaxing conditions on isolated small bundles of chemically skinned myofibers from normal and dystrophic chicken pectoral muscles. It was shown that the dystrophic muscle was stiffer than normal muscle and developed more tension for the same amount of stretch. A fraction of stiffness was not removed by extraction with either 0.6 M KI or with 5 M guanidine HCl mixed with 1% mercaptoethanol. The stiffness of dystrophic muscle was also unaffected by treatment with bacterial collagenase under conditions that destroyed the stiffness of tendon. Nyquist plots of normal and dystrophic muscles during calcium-activated isometric contraction were very similar and were characteristic of fast-twitch muscle, as evidenced by three clear exponential processes. The normal appearance of the Nyquist plot of dystrophic muscle demonstrates that cross-bridge function is not altered, and the characteristic slowing of contraction and relaxation is not a consequence of a fast-to-slow transformation of muscle types. The increased stiffness of dystrophic muscle may be a very fundamental change in the biomechanics of dystrophy. We postulate that the stiffness is mediated by an altered form of collagen, which is collagenase-resistant by virtue of excessive crosslinking.

Altered cholesterol ester proportions in embryonic tissues of dystrophic chicken.

The concentrations of cholesterol esters in tissues of dystrophic chicken embryos are altered from normal. These changes are accompanied by significant changes in the proportions of the esterified fatty acids (the fatty acid profile). In serum and pectoral muscles there is a shift to a higher proportion of unsaturated fatty acids (in particular 18:1). Thigh muscle esters are little changed and in liver and brain the proportion of unsaturated fatty acids decreases.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activities in early stages of development in dystrophic chickens

Glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activities were assayed in superficial pectoral muscles of hereditary dystrophic chickens, 1 week, 2 weeks, 4 weeks and 4 months after hatching. In control chickens, activities of G6PDH and 6PGDH were very low at 4 months of age; however, at 1 week of age, they were much higher than those at 4 months of age. Activities of G6PDH and 6PGDH were significantly higher in dystrophic chickens compared with those in the controls at all the stages of development studied. These findings suggest that considerable activities of G6PDH and 6PGDH are present within the pectoral muscle cells at early stages of development, at least in dystrophic chickens. GAPDH activity was significantly lower in dystrophic chickens at 2 weeks, 4 weeks and 4 months of age compared with those in control chickens. These findings together with our previous studies (Mizuno 1984a,b) in which increased activities of superoxide dismutases, catalase, glutathione peroxidase and glutathione reductase were reported in dystrophic chickens, indicate the presence of an increased capacity for the turnover of oxygen-free radicals within muscle cells in dystrophic chickens, and that oxygen-free radicals and the related activated oxygen species may be playing a role in inducing cellular damage.