Avian Dystrophy Research Articles

Expression of fast myosin heavy chain transcripts in developing and dystrophic chicken skeletal muscle

The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dotblot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation.

Expression of fast myosin heavy chain transcripts in developing and dystrophic chicken skeletal muscle

The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dot-blot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation. Dev. Dyn. 208:491–504, 1997. © 1997 Wiley-Liss, Inc.

The Avian Low Score Normal Muscle Weakness Alters Decorin Expression and Collagen Crosslinking

Extracellular matrix development of chicken pectoral muscle was examined in the Low Score Normal (LSN) genetic muscle weakness and compared to both normal and avian muscular dystrophy (MD). At 20 days of embryonic development significant elevations were noted in LSN total glycosaminoglycan concentration and decorin, while at 14 days, LSN glycosaminoglycan and decorin levels were indistinguishable from the controls. Levels of a large skeletal muscle chondroitin sulfate proteoglycan (M-CSPG) appear to be unaffected. Morphologically, at 20 days, the extracellular matrix space between muscle fibers increased to a level characteristic to that observed in avian muscular dystrophy. At six weeks posthatch a marked increase in LSN collagen crosslinking relative to MD or control tissues was observed, while collagen concentration was not altered. By one year posthatch LSN collagen crosslink levels did not significantly differ from normal tissue. These data support the concept that the LSN muscle weakness is associated with changes in both proteoglycan and collagen characteristics.

Differential effects of methimazole and dexamethasone in avian muscular dystrophy

We showed previously that thyroid antagonists and glucocorticoids partially alleviated the impaired righting ability and abnormally high levels of plasma creatine kinase activity in genetically dystrophic chicks. The goals of the present study were: (1) to ascertain whether the beneficial effects of methimazole (MMI; thyroid antagonist) on muscle function and plasma creatine kinase (CK) activity in dystrophic thickens are correlated with significant reduction in plasma triiodothyronine (T3) and thyroxine (T4); (2) to assess whether the MMI-induced thyroid changes are accompanied by increased plasma corticosterone level and/or changes in muscle glucocorticoid receptors which might account partially for the beneficial effects of MMI; and (3) to determine if plasma T3 and T4 are reduced in dexamethasone (DEX) treated dystrophic chickens which might account at least partially for the beneficial effects of DEX (a potent glucocorticoid) on avian dystrophy. The data show that beneficial effects of MMI are associated with reduced plasma levels of thyroid hormones and increased circulating levels of corticosterone. In addition, DEX actually increases plasma T3 levels. these differential effects indicate that reduced plasma thyroid hormone levels do not represent a common mechanism of beneficial drug effects in avian muscular dystrophy. On the other hand, elevated plasma glucocorticoid levels accompany the beneficial effects of both severe hypothyroidism and DEX treatment. The data also show that MMI induces down-regulation of muscle cytosolic glucocorticoid receptors which are higher than normal in dystrophic muscles.

Thyroidal involvement in the expression of avian muscular dystrophy

We showed previously that propylthiouracil (PTU), a thyroid inhibitor, could alleviate several major signs of hereditary muscular dystrophy in chickens. The goals of the present investigation were to: (1) determine whether a nearly athyroid condition (achieved within two days after hatching by surgical thyroidectomy plus PTU) during an 11-day period beneficially affects the dystrophic condition when followed by triiodothyronine (T3) replacement to 33 days of age; (2) determine the beneficial effects on the expression of avian dystrophy when the thyroidectomized-PTU-treated chickens received a wide range of moderate to low T3 replacement doses beginning by two days after thyroidectomy; and (3) examine the thyroid hormone receptor system in dystrophic muscle for a possible abnormality. Thyroid deprivation increased muscle function (righting ability) and reduced plasma creatine kinase activity in dystrophic chickens. The major thyroid-related abnormality in dystrophic pectoralis muscles was an increased maximum binding capacity of solubilized nuclear T3 receptors.

Beneficial effects of anti-growth hormone antiserum in avian muscular dystrophy.

Human subjects and mice have been found to have a milder progression of muscular dystrophy when the disease is associated with genotypically determined dwarfism. In this paper we describe an experimental test for reducing growth hormone in dystrophic chickens that uses rabbit anti-chicken growth hormone anti-serum (anti-cGH). Antiserum was injected daily into dystrophic (line 413) male chickens from day 1 to day 8 after hatching. Dystrophic chickens injected with anti-cGH maintained a significantly higher score in the standardized test for righting ability (P less than 0.001-0.051) from 3 to 9 1/2 wk after hatching when compared with dystrophic controls. The observed prolongation of the functional ability of injected dystrophic animals suggests that growth hormone plays a role in potentiating the symptoms of dystrophy in chickens.

Corticosteroid therapy in avian muscular dystrophy: Evaluation by magnetic resonance relaxation times

Genetically dystrophic chicks (line 413) were administered 0.3 mg/kg dexamethasone (DEX), a corticosteroid, from 2 to 28 days ex ovo. Diluent-injected dystrophic and normal (line 412) chicks served as controls. The effects of DEX on muscle function were evaluated weekly by the exhaustion score (ES) method. Proton magnetic resonance relaxation times, T 1 and T 2, of excised pectoral muscles of all chicks were measured at 31 days of age. There were two measurable components of T 1 and T 2 relaxation times, all of which were significantly prolonged in control dystrophic muscles. DEX improved ESs of dystrophic chicks and these functional improvements were reflected in significant reductions in the short and long components of T 1 and T 2. This study suggests that magnetic resonance techniques, including imaging, can complement and extend the information obtained by other methods that are commonly used in therapeutic studies of muscle disorders.

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken ( M. T. Crow and F. E. Stockdale, 1986, Dev. Biol. 118, 333–342 ), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.

Dystrophic symptoms prevented by phenobarbital in avian muscular dystrophy

New Hampshire normal (line 412) and dystrophic (line 413) chickens were treated with central stimulants and central depressants, respectively, once a day from the 2nd to the 10th day after hatching, and effects of the treatment were examined on a number of muscle fibers, areas of muscle fibers, and the coefficient of variation of a muscle on the 13th day. Central depressants, especially phenobarbital, effectively prevented development of the dystrophic symptoms, whereas central stimulants made normal chickens “dystrophic”. The righting ability of the dystrophic chickens was normal after treatment with 10 mg/kg of phenobarbital during the early postnatal period from the 2nd to the 28th day after hatching.

Dystrophic symptoms prevented by phenobarbital in avian muscular dystrophy

New Hampshire normal (line 412) and dystrophic (line 413) chickens were treated with central stimulants and central depressants, respectively, once a day from the 2nd to the 10th day after hatching, and effects of the treatment were examined on a number of muscle fibers, areas of muscle fibers, and the coefficient of variation of a muscle on the 13th day. Central depressants, especially phenobarbital, effectively prevented development of the dystrophic symptoms, whereas central stimulants made normal chickens “dystrophic”. The righting ability of the dystrophic chickens was normal after treatment with 10 mg/kg of phenobarbital during the early postnatal period from the 2nd to the 28th day after hatching.

Electrophysiologic differences between normal and dystrophic avian muscle

In the avian model of muscular dystrophy, electrophysiologic studies have shown alterations in the action potential characteristics of dystrophic muscle in vitro, supporting the notion that a membrane defect exists in avian dystrophy. As neurogenic and vascular etiologies have also been proposed, we examined the characteristics of action potentials recorded in a novel in vivo preparation of the extensor digitorum communis muscle in 8-week-old normal and dystrophic chickens. To facilitate intracellular recording, dantrolene sodium was used to attenuate the muscle twitch. Results showed that although the resting membrane potential, action potential amplitude and the action potential maximum rate of rise were similar in normal and dystrophic cells, the action potential duration at half the maximum amplitude was increased in dystrophic cells. This observation has not been previously reported for dystrophic avian muscle and suggests that a defect in the sarcolemmal potassium conductance is an early change in dystrophic avian muscle.

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.

The dystrophic expressions retarded by atropine in avian muscular dystrophy.

The dystrophic expressions retarded by atropine in avian muscular dystrophy.

Myosin heavy chain in avian muscular dystrophy corresponds to the neonatal isozyme.

We have previously demonstrated, based on comparison of homologous amino acid sequences and of two-dimensional CNBr peptide gel patterns, that the myosin heavy chain in pectoralis muscles of Storrs, Connecticut dystrophic chickens is different from that of their normal controls (Huszar, G., Vigue, L., De-Lucia, J. Elzinga, M., and Haines, J. (1985) J. Biol. Chem. 260, 7429-7434). Others have shown, however, that genomic banks and mRNA complements of the control and dystrophic birds are not different. In the present studies, we have examined the hypothesis that the "dystrophic" myosin heavy chain is not a novel gene product, but is a developmental isozyme which is expressed in pectoralis muscles of adult chickens due to the dystrophic process. Two-dimensional maps of myosin heavy chain CNBr peptides were prepared from breast muscles of 17-day in ovo (embryonic), 25-day posthatch (neonatal), and adult birds of the Storrs dystrophic and of two control strains. Also, myosin and actomyosin ATPase enzymatic activities of the various preparations were determined in the pH range of 5.5 to 9.0. Analysis of the peptide maps demonstrates that the embyronic, neonatal, and control adult myosin heavy chain isozymes are distinctly different gene products with only minute variations between the respective developmental isozymes in dystrophic and control muscles. However, the pectoralis myosin heavy chain of adult dystrophic birds, which is a homogeneous isozyme population by amino acid sequences and gel patterns, corresponds to that of the neonatal-type myosin heavy chain. The ATPase properties of the embryonic, neonatal, or adult pectoralis myosins and actomyosins were not different, whether the level of specific activity or the pattern of pH activation is considered. Since the mobility of neonatal chicks (primarily neonatal-type isozymes) is not restricted, the differences in myosin heavy chain structures are part of the syndrome, but not the cause of avian muscular dystrophy.

Structure of myosin heavy chain in avian muscular dystrophy.

We have studied the structure of myosin heavy chain (MHC) in the pectoralis muscle of genetically dystrophic (Connecticut Strain) and White Leghorn chicks. MHC was alkylated with N-ethylmaleimide, purified by Sepharose-4B chromatography, and cleaved with cyanogen bromide. The MHC CNBr peptides were analyzed by one-dimensional and two-dimensional isoelectric focusing/sodium dodecyl sulfate gradient gels and by amino acid sequencing. Specific changes were detected in the gel patterns which could be correlated with the loss of muscle function as measured by the exhaustion score (the ability of chicks to rise from a reclining position) in three experimental groups (exhaustion scores: less than 3, 10-20, greater than 30). We have also examined the amino acid sequence of a 3-methyl-histidine-containing peptide which originates from the 20-kDa fragment of pectoralis muscle MHC in dystrophic chicks: Val-Leu-Asn-Ala-Ser-Ala-Ile-Pro-Glu-Gly-*Gln-Phe-*Ile-Asp-Ser-Lys-Lys- Ala-Ser-Leu-Gln-Lys-Leu-Gly-Ser-Ile-Asp-Val-(Asp, 3-methylhistidine, Gln). Comparison of the homologous MHC sequences shows two positions at which MHC from dystrophic chicks differs from that of the White Leghorn chicks *(Glu----Gln and Met----Ile). Thus, both the peptide map and sequence analyses demonstrate that in avian muscular dystrophy an abnormal pectoralis MHC is synthesized. It is not yet clear whether the "dystrophic" MHC is a variant MHC or if it arises from the abnormal expression of an earlier developmental form (embryonic or neonatal) of pectoralis muscle MHC.

Avian muscular dystrophy: Serum thyroid defect and limited improvement with methimazole and propylthiouracil

Serum concentrations of triiodothyronine (T 3) and thyroxine (T 4) were determined by radioimmunoassay in normal and genetically related muscular dystrophic chicks at 2 through 42 days ex ovo. There were no significant differences in T 4 concentrations, but T 3 concentrations were reduced about 35% below normal values in dystrophic birds at 14 to 42 days. The situation was reversed, however, on day 2, with T 3 concentrations about 50% greater in dystrophic than in normal serum. Administration of T 3 beginning on day 2 ex ovo did not alter phenotypic expression of dystrophic signs. Administration of the thyroid “antagonists,” methimazole and propylthiouracil, however, significantly increased righting ability and reduced serum creatine kinase activity in dystrophic chicks. None of the administered substances improved the histopathology of dystrophic pectoralis major muscles. The data indicate that serum T 3 concentrations may provide an early “marker” for avian dystrophy, and suggest that lowered serum T 3 concentrations in older chicks may represent a compensatory response to the elevated serum T 3 in newly hatched dystrophic chicks.

Content and synthesis of glycolytic enzymes and creatine kinase in skeletal muscles and normal and dystrophic chickens

A number of workers have reported that avian muscular dystrophy causes alterations in the levels of certain enzyme activities in “fast-twitch” muscle fibers but has little effect on enzyme activities in “slow-twitch” muscle fibers. In the present work, the effects of this disease on the content and relative rates of synthesis of a number of glycolytic enzymes and the skeletal muscle-specific MM isoenzyme of creatine kinase in chicken muscles was investigated. It was shown that (i) the approximate 50% reductions in steady-state concentrations of three glycolytic enzymes (aldolase, enolase, and glyceraldehyde-3-P dehydrogenase) in dystrophic breast (fast-twitch) muscle result predominantly from decreases in relative rates of synthesis, rather than accelerations in relative rates of degradation, of these proteins in the diseased tissue; (ii) in contrast to the situation with the glycolytic enzymes, muscular dystrophy has only minor effects (25% or less) on the content and relative rate of synthesis of MM creatine kinase in breast muscle fibers; (iii) the muscular dystrophy-associated alterations in content and synthesis of the glycolytic enzymes in breast muscle fibers become apparent only during postembryonic maturation of this tissue; and (iv) as expected, muscular dystrophy has no significant effect on the content or relative rates of synthesis of glycolytic enzymes in slow-twitch lateral adductor muscles of the chicken. These results are discussed in terms of the apparent similarities between the effects of muscular dystrophy and surgical denervation on the protein synthetic programs expressed by mature fast-twitch muscle fibers.

Distribution of slow myosin in dystrophic chicken muscle.

Avian muscular dystrophy is a disorder that preferentially affects the development of fast twitch muscles (215,218). Previous research using anatomical, histochemical, and biochemical methods have demonstrated clear dystrophic abnormalities in the posterior latisslmus dorsi (PLD) while the anterior latissimus dorsi (ALD), a slow tonic muscle, is unaffected by the disease (216,219). Most of this research has been performed on normal and dystrophic chickens of different genetic backgrounds. While avian muscular dystrophy is caused by a single autosomal recessive gene, the dystrophic gene causes relatively rapid fiber atrophy when placed on a New Hampshire background (217,220). These effects are most likely due to differences in the rate of muscle growth in the two breeds. Since inbred lines of normal and dystrophic chickens now exist that contain greater than 90% of their genes in common, these strains (normal line 03 and dystrophic line 433) were used for the current study to ensure that the differences observed between normal and dystrophic muscles were due to the disease.KeywordsMyosin Heavy ChainMedial GastrocnemiusDystrophic MuscleIntrafusal FiberSlow MyosinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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.

The interaction of unregulated actin and myosin in avian muscular dystrophy.

The presence of an altered form of the heavy chain component of myosin subfragment-1 (S-1) in avian dystrophic pectoral muscle was confirmed by Triton-urea-acetic acid polyacrylamide gel electrophoresis. The potential functional significance of this altered form of S-1 was evaluated by measuring the ATPase activity of the unregulated acto-S-1 complex using all possible pairwise combinations of actin and S-1 from normal (N) and dystrophic (D) muscle. (NN, DD, ND, DN, where the first letter designates the actin and the second letter the S-1). With conventionally purified actin and S-1, NN not equal to DD not equal to ND not equal to DN, implying both N actin not equal to D actin and N S-1 not equal to D S-1 functionally. An alternate purification scheme for actin resulted in preparations from normal and dystrophic muscles of actin Mg-polymers with the same rheology (viscosity vs shear rate) and critical concentration for polymerization. When these actins were combined with more highly purified preparations of S-1, the adenosine triphosphatase (ATPase) activity of the acto-S-1 complex did not vary with changes in the pairwise composition and responded similarly to variation of the actin or adenosine triphosphate (ATP) concentration. In experiments with actin activation of intact myosin, no differences were observed between myosin from normal vs dystrophic muscle. The different isozymes of myosin present in normal and dystrophic chicken pectoral muscles are functionally equivalent as ATPases in their interactions with unregulated actin.