Impact of prednisone on TGF-beta1 and collagen in diaphragm muscle from mdx mice.

The purpose of this study was to assess the impact of prednisone treatment for 8 weeks on the level of transforming growth factor-beta 1 (TGF-beta1), hydroxyproline (HYP) concentrations, and level of the mature, nonreducible collagen cross-link hydroxylysylpyridinoline (HP) in diaphragm muscle from 12-week-old mdx mice. Diaphragm muscle from untreated mdx mice had a significantly higher level of TGF-beta1, HYP, and HP cross-link compared with normal C57BL/10J (control) mice. Prednisone treatment significantly reduced the level of TGF-beta1 and HYP in diaphragm from mdx mice to values similar to control mice, but resulted in a higher level of the HP cross-link compared with untreated mdx mice. These findings indicate that short-term treatment of mdx mice with prednisone can attenuate the fibrotic response in diaphragm muscle, possibly by mediating the level of TGF-beta. Although prednisone was beneficial in preventing collagen accumulation, it resulted in a higher level of the HP cross-link, presumably by decreasing collagen turnover

Efficacy of drug regimen exceeds electrostimulation in treatment of avian muscular dystrophy.

Autosomal-recessive dystrophic chickens were treated in three experimental groups with an intraperitoneal multicomponent drug mixture (50 mg/kg Ep475, 20 mg/kg Cinanserin, 10 mg/kg stanazolol, 100 mg/kg leucine, 0.1 mg/kg insulin, 100 mg/kg glucose, and 50 mg/kg carnitine), percutaneous high-frequency electrostimulation of the pectoralis muscle, or a combination of both drug and electrostimulation treatments. Therapeutic efficacy was determined in each group by measurements of strength, righting ability, and histomorphometric analyses of the pectoralis musculature. Drug treatment alone was found to significantly improve muscular strength, function, and relative myofiber necrosis compared with sham-injected controls. The efficacy of drug treatment was found to be equal to or better than singular electrostimulation treatment; there was no apparent additive effect of electrostimulation. As a result, these findings support the use of drug treatment as a useful nongenetic approach to the management of human muscular dystrophy where there is the potential risk of injury from exercise usage.

Enhanced sensitivity of mdx mice to intramuscular injection of compound 48/80.

Species-specific differences in the inflammatory response, specifically with regard to mast cells, have been proposed to explain the phenotypic variation among dystrophin-deficient humans, and mdx mice (Gorospe et al., 1994). To test this hypothesis we have intramuscularly injected a mast cell secretogogue into both dystrophin-negative mdx and dystrophin-positive normal mice. Mast cell activity was determined by measuring the activity of mast cell tryptase, while creatine kinase activity was used to determine the course of muscle damage in vivo. Area of damage around the injection site was measured at autopsy, and used as an indication of relative sensitivity to the secretogogue effect of compound 48/80. Mdx mice exhibited more damage in response to intramuscular injection than normal control mice. In addition, mdx mice showed a substantial increase in plasma tryptase activity, followed by a large increase in muscle creatine kinase activity. On the other hand, dystrophin-positive normal controls injected with 48/80 liberated little CK or tryptase activity. These results are consistent with the hypothesis that species-specific differences in mast cell activity, or sensitivity to mast cell products could account for the variation in pathology seen in dystrophin-deficient animals.

Strength and endurance in the therapeutic evaluation of prednisolone-treated MDX mice.

The dystrophin-deficient, X-linked dystrophic mouse (mdx) was used to evaluate the efficacy of prednisolone treatment. A test protocol was used to take advantage of the quantifiable weakness and disability as well as molecular genetic defect shared with the X-linked Duchenne muscular dystrophy (DMD). Whole-body weakness and fatigue were determined by non-invasive force-transducer physiographic and variable-speed treadmill techniques, respectively. Other measurements included hind-limb muscle protein, calcium, and histomorphology. Subcutaneously-administered prednisolone elicited significant improvements in whole body strength throughout a two-month test period. Increases in strength were also accompanied by measurable increments in running endurance. In fact, prednisolone treatment appeared to protect mdx mice from the stressful effect of continuous running as determined by strength and muscle fiber diameter. Test results from this study support the limited therapeutic benefit observed previously in DMD patients treated with the glucocorticoid.

Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity.

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin. Basal levels of Ca2+-ATPase activity (no added calmodulin) were comparable in muscles of unaffected and affected animals, and the Ca2+ optima of the enzymes in normal and dystrophic muscle were identical. Purified SR vesicles, obtained by calcium phosphate loading and sucrose density gradient centrifugation, showed the same resistance of dystrophic Ca2+-ATPase to exogenous calmodulin as the SR-enriched muscle membrane fraction. Dystrophic muscle had increased Ca2+ content compared to that of normal animals (P less than 0.04) and has been previously shown to contain increased levels of immuno- and bioactive calmodulin and of calmodulin mRNA. The calmodulin resistance of the Ca2+-ATPase in dystrophic muscle reflects a defect in regulation of cell Ca2+ metabolism associated with elevated cellular Ca2+ and calmodulin concentrations.

Cell and fiber-type distribution of dystrophin.

Duchenne muscular dystrophy is the result of dystrophin deficiency. We have determined the cell types likely to express the pathogenic effects of this neuromuscular disease by determining the pattern of dystrophin expression in normal cells. We find that all physiological types of muscle cells express dystrophin at similar levels, and that the dystrophin content of various tissues correlates with the myogenic cell population of each tissue. The dystrophin content of brain and spinal cord, however, is found not to correlate with any type of muscle cell, and it is suggested that neurons express dystrophin. The potential involvement of striated muscle fibers, the vasculature, and the nervous system in the etiology of Duchenne muscular dystrophy makes it likely that the disease is a complex disorder of combined pathogenesis. We also find that the dystrophic chicken does not represent an animal model for dystrophin deficiency.

Effects of denervation on spectrin concentration in avian skeletal muscle.

The effect of denervation on avian muscle alpha-spectrin was examined in fast and slow muscles. Using immunofluorescence, the surgically denervated fast-twitch posterior latissimus dorsi (PLD) exhibited a significant increase in spectrin antigen associated with the sarcolemma and within the sarcoplasm compared with the contralateral innervated control muscle. Using gel electrophoresis followed by immunoblotting, we found a two- to three-fold increase in the levels of spectrin in the denervated PLD over that found in the innervated PLD. These levels were comparable to those found previously in slow and dystrophic muscle. The intrafiber distribution of spectrin is similar between the denervated PLD and the slow-tonic anterior latissimus dorsi (ALD). When spectrin was examined in dystrophic PLD muscle, denervation was found to have no effect. These results support our hypothesis that the concentration of spectrin within muscle fibers reflects the physiological state of those fibers. Changes in spectrin concentration may be a useful probe to study the various alterations in physical parameters found among fast, slow, dystrophic, and denervated fibers.

Early abnormal development of calmodulin gene expression and calmodulin-resistant Ca2+-ATPase activity in avian dystrophic muscle.

We have reported previously that the pectoralis muscle from three month-old dystrophic chickens with signs of myopathy exhibits increased calmodulin content, elevated calmodulin-specific mRNA (Biochem. Biophys. Res. Commun. 137:507-512, 1986), and reduced sarcoplasmic reticulum (SR) Ca2+-ATPase activity in response to calmodulin exposure in vitro (Clin. Res. 34: 725A, 1986). To determine the early time sequence for development of these abnormalities, we have studied muscle from embryos and post-hatched chickens at various ages. Quantitated by dot blot analysis, there was an approximate two-fold increase in calmodulin-specific mRNA in dystrophic muscle as early as 13 days ex ovo which was maintained throughout development up to three months ex ovo. Similarly, Ca2+-ATPase activity measured in SR membranes from chickens as early as 13 days post-hatch was also found to be resistant to stimulation in vitro by exogenous calmodulin, whereas the enzyme from normal muscle was calmodulin-stimulable. These findings suggest that the genetic lesion expressed in the avian dystrophic animal model involves the loss of normal control of intracellular calcium metabolism early in the maturation of the affected musculature and prior to appearance of disease signs.

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 Ca2+ content in dystrophic muscle, the current findings of increased sarcoplasmic calmodulin suggest the latter is a cellular response to defective Ca2+ transport at the level of cell efflux or intracellular organelle (sarcoplasmic reticulum) uptake.

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.

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.

Characterization of lymphocyte interferons with different species specificities from normal and genetically dystrophic chickens.

Lymphocytes from thymus and spleen of normal (Line 412) and genetically dystrophic (Line 413) chickens produce two types of interferons (IFNs) with different host cell specificities. The first type, referred to as ChIFN-alpha, demonstrates antiviral activity on primary normal chicken embryo (CE) cells. This activity is stable at 60 degrees C for 1 h and, in this respect, ChIFN-alpha is similar to the standard ChIFN-beta. In contrast, the second type, referred to as ChIFN-alpha 1, demonstrates antiviral activity in human and simian cells but not in primary CE cells. This activity is labile at 60 degrees C for 1 h. The amount of these two types of IFNs produced in lymphocytes from the spleen of dystrophic chickens was fourfold greater than that produced from normal chickens under similar experimental conditions. In contrast to the lymphocytes from thymus and spleen, the lymphocytes from the bursa of both the normal and dystrophic chickens produced only one type of IFN, namely ChIFN-alpha 1. The development of antiviral state in human cells by ChIFN-alpha 1 requires host RNA synthesis. Although ChIFN-alpha 1 has antiviral properties similar to HuIFN-alpha in human cells, the two IFNs are not antigenically related.

In vivo effects of three calcium blockers on chickens with inherited muscular dystrophy.

Genetically homozygous Line 413 dystrophic chickens were given in separate trials daily i.p. injections of aqueous solutions of the calcium blocker drugs, diltiazem, verapamil, or nifedipine. At a dosage of 20 mg/kg/day, drug therapy in each case significantly prolonged the functional ability of the dystrophic chickens as quantitated regularly by a standardized test for righting ability. Enhanced functional ability, however, was not generally accompanied by a decrease in the usually high plasma creatine kinase activity. In addition, there was no change in the pectoralis muscle mass or protein with any of the drug treatments. Moreover, no significant reduction in the abnormally high total muscle calcium was found with calcium blocker treatment. Also, there was no marked change in the histopathology of muscle from the drug-treated dystrophic chickens. We concluded that drugs with calcium entry blocker activity offer only limited benefit in retarding dystrophic symptoms expressed in the chicken (viz., short-term enhancement in righting ability).

Limited benefit to genetically dystrophic chickens from a synthetic proteinase inhibitor: Ep475.

Chickens with inherited muscular dystrophy (Line 413) were treated in two separate trials with daily intraperitoneal injections of 10% DMSO-water solutions containing the proteinase inhibitors, Ep475 and E64. Drug therapy in each case significantly prolonged the functional ability of the treated chickens. Diluent control chickens around day 35 ex ovo characteristically reached a maximum ability to right from the supine position in a standardized functional test for muscle weakness. Subsequently, the control chickens were found to decline progressively in their ability to right. Treatment with the proteinase inhibitors had no effect on the typically elevated levels of plasma creatine kinase activity. In a histological analysis of the affected pectoralis major muscle, drug treatment had no effect on the relative distribution of degenerating, and vacuolated fibers, inflammatory cells, and abnormal fiber diameters. An exception was seen in decreased necrotic fibers of chickens treated with high doses of Ep475. Moreover, both inhibitors had positive effects on two biochemical abnormalities common to the dystrophic pectoralis muscle: increase in noncollagen protein, and reduction in total calcium.

Parenteral branched-chain amino acid treatment and avian dystrophy.

Genetically homozygous line 413 dystrophic chickens were given twice-daily intraperitoneal injections of solutions containing branched-chain amino acids (BCCA-leucine, valine, isoleucine) either alone or in combination; and their alpha-ketoacid analogs (alpha-ketoisocaproic and alpha-ketoisovaleric acids). Another trial consisted of an amino acid mixture containing BCAA. Amino acid supplementation in each case significantly prolonged righting ability measured regularly by a standardized flip-test procedure. Enhanced functional ability was not generally accompanied by a decrease in plasma creatine kinase activity. However, a measurable increase in the affected pectoralis major muscle mass and protein content (female chickens in particular) was found with BCAA therapy. Moreover, the increase in muscle bulk was attended in some cases by a reduction in the relative number of degenerating fibers quantitated microscopically. Contrariwise, the amino acid mixture caused a reduction in pectoralis muscle mass. It is concluded that parenteral BCAA therapy offers limited benefit in retarding dystrophic symptoms in the chickens.

In vivo effects of protease inhibitors on chickens with hereditary muscular dystrophy.

Beginning on day 4 ex ovo, and every 3 d thereafter, genetically dystrophic Line 413 chickens were given intraperitoneal injections (4 mg/kg body wt) of a protease inhibitor, leupeptin, pepstatin, or antipain. Experimental chickens received protease inhibitors dissolved in a water:ethanol:dimethyl sulfoxide solution (50:40:10, vol:vol:vol). Control untreated animals received diluent injections. Untreated dystrophic chickens typically reach around day 30 ex ovo a maximum ability to right from the supine position in a standardized functional test for muscle weakness. After day 30 ex ovo, the dystrophic chickens are found to decline progressively in their ability to right, compared with normal, nondystrophic controls, which have an unimpaired ability to right. Concomitantly, dystrophic chickens exhibit characteristically high levels of plasma creatine phosphokinase enzyme activity. In addition, an increased frequency of degenerating, regenerating, and vacuolated myofibers, and inflammatory cells appear in the affected pectoralis major muscles from the dystrophic chicken. Throughout the duration of the trial, there was no major enhancement in the functional righting ability of dystrophic chickens receiving any one of the protease inhibitors tested. However, there was a significant reduction in the abnormally high levels of plasma creatine phosphokinase in the treated chickens. Also, there was an apparent reduction in the mean number of vacuolated fibers in the pectoralis muscle from the protease inhibitor-treated birds. No significant reductions were observed in the relative frequency of degenerating and regenerating myofibers or inflammatory cells. In addition to the plasma creatine phosphokinase decrease, however, therapeutic benefit was seen in 31.0, 30.5, and 14.8% increases in the wet weight (and total noncollagen protein) of pectoralis muscle from dystrophic chickens receiving leupeptin, pepstatin or antipain, respectively.

Screening of antiserotoninergic drugs with the genetically dystrophic chicken.

Line 413 early-onset, genetically homozygous dystrophic chickens were given twice-daily intraperitoneal injections of the antiserotoninergic drugs p-chlorophenylalanine hydrochloride, fluoxetine hydrochloride, ergonovine maleate, nortriptyline hydrochloride, methiothepin maleate, and methysergide bimaleate in combination with penicillamine. Except in one case, treatment with drugs significantly prolonged the righting ability of the treated dystrophic chickens, as measured by a periodic standardized flip-test procedure. Abnormally high levels of plasma creatine phosphokinase, lactate dehydrogenase, and SGOT were found in the untreated dystrophic chickens. However, of the drug-treated dystrophic chickens, in some cases the plasma enzyme activities were reduced whereas in others they were enhanced. In agreement with previous findings, the blood serotonin levels of the dystrophic chickens were found at all age groups to be significantly higher than those in the corresponding normal chickens. This phenomenon may in part account for the improvement in righting ability demonstrated in the dystrophic chickens receiving treatment with antiserotoninergic drugs.

Delayed functional disability in dystrophic chickens receiving chemotherapy.

Line 413 early-onset, genetically homozygous dystrophic chickens were given twice-daily intraperitoneal injections of the antiserotoninergic drug cinanserin, alone or in combination with methysergide. Other trials consisted of penicillamine treatment in combination with either methysergide or cyproheptadine. Chemotherapy significantly prolonged the righting ability of treated dystrophic chickens, as measured by a periodic standardized flip-test procedure. Plasma creatine kinase activities were not affected by any of the various drug treatments. However, the blood serotonin levels of the dystrophic chickens (grand mean 1.47 microgram serotonin/ml blood) were found to be significantly higher (p less than 0.001) than those in the corresponding normal chickens (0.99 microgram/ml). This finding may partially account for the antiserotoninergic drug enhancement in righting ability that was demonstrated in the drug-treated dystrophic chickens.