Richard K. Entrikin

MAJOR PHYSIOLOGIC AND HISTOCHEMICAL CHARACTERISTICS OF INHERITED DYSTROPHY OF THE CHICKEN

Inherited muscular dystrophy of the chicken was first described more than 20 years ago.’ Since then. several genetic lines have been developed, some have even become extinct, and many studies have been undertaken of the morphologic, physiologic, and biochemical properties of muscles of birds with the disorder. Early research on the abnormality has been reviewed by Julian and Asmundson2 and Asmundson et ~ 1 . ~ This paper discusses some of the major characteristics of the dystrophic chicken that occur in the several lines developed and maintained at the University of California, at Davis, and describes how these properties have been investigated in our laboratory.

Effect of hind-limb suspension on young and adult skeletal muscle: I. Normal mice

The purpose of this study was to determine the effect of hind-limb suspension (HS) on morphometric, histologic, and contractile characteristics of fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles in adult and immature mice. Hind-limb suspension for 2 weeks was used to produce atrophy in two groups of mice, ages 4 and 12 weeks, with nonsuspended animals serving as controls. Young HS mice exhibited marked decreases in SOL weight, length, cross-sectional area (CSA), twitch and tetanic tensions, and rates of tension development and relaxation, with increases in fatigue resistance. HS reduced the diameter of both type I and IIA fibers, increased the percentage of type I fibers, and decreased the percentage of type IIA fibers in both young and adult SOL. Muscle weight, length, CSA, IIA and IIB fiber areas, and maximum rate of tetanic tension development were decreased in EDL of young HS mice; fatigue resistance and EDL half-relaxation times were increased. For most parameters evaluated, slow twitch muscle was more affected than fast twitch. HS affected contractile characteristics less than morphometric or histologic parameters. Rates of tension development and relaxation were the contractile parameters most affected by HS, and the time parameters of contraction were least affected. For all measurements young mice were more affected than adult mice.

Righting ability and skeletal muscle properties of phenytoin-treated dystrophic chickens.

Abstract Chickens with an inherited muscular dystrophy (line 413) and genetically related normal controls (line 412) were treated with phenytoin (diphenylhydantoin, DPH) on Days 1 through 40 ex ovo . DPH (20 mg/kg, intraperitoneally, b.i.d.) and a combination of DPH and intermittent “exercise” (twice-daily testing of righting ability) beneficially affected muscle function, morphology, and biochemistry. DPH dramatically improved the righting ability of dystrophic chicks, and the exhaustion scores of many treated birds were still as high as those of normal chicks after 30 days of treatment. Muscles from chicks killed after 27 to 40 days of treatment had decreased fiber diameters, increased lactic dehydrogenase (LDH) activity, and reduced acetylcholinesterase and butyrylcholinesterase activities. The effects of DPH were more pronounced in the posterior latissimus dorsi than in the pectoral muscle and were potentiated by exercise. Exercise alone produced a transient increase in righting ability and increased LDH activity of dystrophic muscles. The abnormal, rounded appearance of dystrophic muscle fibers was not altered by treatments. Research reported elsewhere showed that DPH partially corrected abnormal electromyographic activity of dystrophic chicken muscles. The data presented here show that other symptoms of avian dystrophy are also alleviated by DPH and suggest that abnormal membrane activity plays an important role in this myopathy.

Fast and slow skeletal muscles: Effect of ethanol on contractility of muscles from mice

We examined in vitro the effect of ethanol at four concentrations (0g%, 0.1g%, 0.2g%, and 0.4g%) on contractile parameters of 40 fast extensor digitorum longus (EDL) and 40 slow soleus muscles from healthy mice at 35C. Preparations were curarized to avoid the possible effect of ethanol on the terminal axons or skeletal neuromuscular junction. Contractile parameters measured included: (1) twitch and tetanic tension; (2) rate of tension development; (3) time to peak tension and half relaxation for twitch; (4) time to first evidence of relaxation in the tetanus; and (5) maximum rate of relaxation. The three lower concentrations of ethanol had no significant effect on muscle contractility; however, the 0.4g% dose reduced EDL twitch tension by 9%. High doses of ethanol (2.5g%) reduced the tetanic tension produced by the EDL and soleus muscles 31% and 26%, respectively. Ethanol at 2.5g% also reduced the twitch tension of the EDL and soleus by 50% and 38%, respectively. The data suggested that the 0.4g% is the highest dose of ethanol that should be used to dilute drugs in a solution that will bathe directly stimulated curarized muscle without confounding effects. In addition, it is highly unlikely that a direct effect of ethanol on muscle contractility in humans is related to an impairment in driving.

Phenytoin, methysergide, and penicillamine in hereditary muscular dystrophy of the chicken

Abstract Chickens with an inherited muscular dystrophy (line 413) were treated from days 1 through 90 ex ovo with either phenytoin, methysergide, or d -penicillamine. Under the conditions of the study, phenytoin and methysergide were equally effective in improving the righting ability of dystrophic birds, but penicillamine had little or no effect. Phenytoin and penicillamine decreased the abnormally high creatine kinase activity in dystrophic plasma, but only phenytoin reduced the high acetylcholinesterase activity in plasma and fast-twitch muscles of dystrophic birds. Beneficial effects of methysergide and phenytoin were also shown in trials as short as 25 days. None of the treatments, improved the abnormal histological appearance of 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.

Glucocorticoids in muscular dystrophy: beneficial effects of dexamethasone on avian myopathy.

A corticosteroid with mixed glucocorticoid‐mineralo‐corticoid actions was previously shown to improve neuromuscular function in muscular dystrophic chickens. The significance of that finding was recently underscored by reports that a mixed‐action corticosteroid improved muscle function in Duchenne dystrophy patients, albeit at high doses. In the present study a pure glucocorticoid improved function and retarded muscle histopathology in the chicken, but a pure mineralocorticoid did not. These observations suggest that elucidation of mechanisms by which glucocorticoids beneficially affect dystrophic muscle could lead to development of more effective therapies.— Entrikin, R. K.; Abresch, R. T; Bradford, D. P.; Larson, D. B.; Longley, K.J.; Wilson, B. W. Glucocorticoids in muscular dystrophy: beneficial effects of dexamethasone on avian myopathy. FASEB J. 2: 2722‐2725; 1988.

Baclofen, procainamide, verapamil, and prenylamine in hereditary muscular dystrophy of the chicken

Abstract Two “myotonia antagonists” (baclofen and procainamide) and two “calcium antagonists” (verapamil and prenylamine) were evaluated for their effects on hereditary muscular dystrophy of the chicken. Righting ability of dystrophic chicks was improved only by baclofen and procainamide. Plasma creatine kinase activity of dystrophic chicks was reduced 45% after chronic administration of baclofen, but was still nearly nine-fold greater than activity in plasma of normal chicks. Baclofen and verapamil both reduced acetylcholinesterase activity in pectoralis major muscles of dystrophic chicks by about 40%, but these values were still significantly greater than those measured in muscles of normal chicks. The data provide further support for the concept that impaired righting ability of young dystrophic chickens is associated with the presence of myotonia in affected muscles, but do not rule out the possibility that certain of the biochemical features of the avian dystrophy may involve calcium.

Effect of hind-limb suspension on young and adult skeletal muscle. II. Dystrophic mice.

Disuse atrophy induced by limb immobilization reportedly protects dystrophic mouse muscle from histopathological changes. This study was conducted to determine whether disuse atrophy induced by hind-limb suspension (HS) limits the histopathology and contractile abnormalities typically observed in the dystrophic mouse. Two weeks of hind-limb suspension were applied to dystrophic mice (line 129B6F1) at two ages, 4 weeks (6 mice) and 12 weeks (8 mice). Thirty-one untreated dystrophics served as controls. In general, HS exaggerated the dystrophic signs, especially in the younger mice; it reduced animal weight, muscle weight, maximum tetanic and twitch tensions, and rates of tetanic and twitch tension development. HS further slowed the contractile properties of soleus (SOL) and extensor digitorum longus (EDL) muscles, and increased their fatigue resistance. HS reduced the size of type I and IIA fibers in the 6-week SOL and EDL, but not in the 14-week muscles. HS produced a preferential atrophy of SOL type I fibers, with a parallel increase in type IIA fibers. However, it did not alleviate the fiber size variability, degree of necrosis, central nucleation, inflammation, or muscle fibrosis in dystrophic muscles. These data demonstrate that disuse by hind-limb suspension does not prevent the histopathological deterioration or loss of muscle function in 6- and 14-week dystrophic mice.

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 chickens 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.