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 levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.     

Myogenic defect in muscular dystrophy of the chicken.

Inherited muscular dystrophy of the chicken is thought to arise from abnormal development of trophic regulation of skeletal muscles by their innervating nerves. To determine whether expression of muscular dystrophy in the chicken is a property of the nerves or of the muscles, wing limb buds were transplanted between normal and dystrophic chick embryos at $\text{3}\text{1}\text{2}$ days of incubation (stage 19–20). Muscles of donor limbs innervated by nerves of the hosts were compared to contralateral unoperated host limb muscles in chicks from 6 to 25 weeks after hatching. Expression of normal or dystrophic phenotype was determined by examination of five different properties which are altered in dystrophic chick muscle: electromyographic evidence of myotonia; fiber diameter; acetylcholinesterase activity, localization, and isozymes; lactic dehydrogenase activity; and succinic dehydrogenase activity. Genetically normal muscle innervated by nerves of normal or dystrophic hosts was phenotypically normal while genetically dystrophic muscle innervated by normal nerves was phenotypically dystrophic. The results suggest that inherited muscular dystrophy of the chicken arises from a defect of muscle rather than from a lesion in the nerves themselves.     

Glycogen phosphorylase activities in skeletal muscle and heart of genetically dystrophic Syrian hamster.

A simple, rapid and reliable procedure of tissue preparation was devised to estimate glycogen phosphorylase activity in cardiac and skeletal muscle of normal and genetically dystrophic Syrian hamsters of various ages. Total phosphorylase activities of dystrophic skeletal muscle, compared to normal, were reduced. Except for the case of heart from the younger dystrophic animals (45 days old), in which higher phosphorylase activity was noted, hearts from dystrophic hamsters, compared to normal, also showed reduced phosphorylase activities. There were, however, no significances in the ratios of phosphorylase alpha to total phosphorylase between the normal and dystrophic tissues.     

Calcium-related defects in cardiac and skeletal muscles of dystrophic mice

Ca2+ ATPase and calcium binding proteins were studied in cardiac and skeletal muscles of normal and dystrophic mice. In normal and dystrophic mice, Ca2+ ATPase was quite reduced in cardiac muscle compared to skeletal muscle and was, unlike skeletal muscle, insensitive to orthovanadate. Ca2+ ATPase in skeletal muscle of dystrophic mice was reduced as compared to normal mice. In both cases (normal and dystrophic), calcium binding proteins were the same (identical molecular weight). The effect of 2 drugs (Polymixine B and Bepridil) which decrease protein bound calcium was studied: the muscle proteins of dystrophic mice did not present the same sensitivity to Bepridil as controls. These findings suggest the existence of a calcium-related defect in skeletal and cardiac muscle of dystrophic mice.     

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 mM 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 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.     

Detection and isolation of a 30 kDa abnormal protein in avian dystrophic muscle

We have studied the protein composition of the pectoralis superficialis muscle of genetically dystrophic (New Hampshire line 413) and normal control (line 412) chickens by one- and two-dimensional gel electrophoresis. A protein, referred to hereafter as the 30 kDa abnormal protein, was specifically detected in the affected muscle. It was purified to homogeneity, and its molecular properties were studied. It is a monomer with a molecular mass of approximately 30 kDa and an isoelectric point of about pI 8.4. We have screened by Western blotting a variety of muscles from line 412 and line 413 chickens for the presence of the 30 kDa protein. While the pattern of total protein is very similar in all cases, the 30 kDa protein was not detected in the pectoralis superficialis muscle of line 412 chickens. However, the immunoreactive bands were detected in the sartorius muscle and the tensor fasciae latae muscle from dystrophic and normal chickens. Interestingly, the immunoreactive bands of normal skeletal muscles are smaller in molecular weight than those of dystrophic skeletal muscles. To determine the early time sequence of the appearance of the abnormal protein, we studied muscles from embryos and post-hatched chickens at various ages. The abnormal protein was detected in dystrophic muscles as early as 15 days ex ovo and occurred throughout development up to six months ex ovo. Although the implication of the dystrophy-associated appearance of the 30 kDa protein in the affected muscle is not clear at present, it would be of particular interest to elucidate the biochemical functions of the 30 kDa protein in the affected muscle (pectoralis superficialis muscle) of genetically dystrophic chicken.     

Abnormal properties of Mg2(+)-ATPase in transverse tubule membranes from dystrophic chicken

Purified transverse tubule membranes from normal and dystrophic chicken skeletal muscle were isolated by a calcium-loading procedure. Normal and dystrophic T-tubules were similar in cholesterol content and (Na+,K+)-ATPase and 5'-nucleotidase activities but a significant decrease of Mg2(+)-ATPase activity was observed in dystrophic membranes. A comparative analysis of the enzyme properties revealed that the kinetic parameters were altered in dystrophic T-tubules and the ATP-hydrolyzing activity was differently affected by the ionic strength. However, the influence of temperature and the regulatory effect of concanavalin A were the same as in normal T-tubules. Membrane fluidity was similar in both preparations as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium diphenylhexatriene. These results point to an impairment in the function of Mg2(+)-ATPase due to structural alterations of the enzyme.     

Adenyl cyclase and cyclic nucleotide phosphodiesterase activities in murine muscular dystrophy.

Adenyl cyclase and cyclic nucleotide phosphodiesterase activities were assayed in homogenates of hind leg skeletal muscle from dystrophic and normal mice. Adenyl cyclase activity was stimulated 2.5 times by epinephrine and 6 times by fluoride over the basal activity in both dystrophic and normal mice. The activity of adenyl cyclase from dystrophic muscle of mice was significantly higher than that of normal mice under all the conditions tested (i.e. basal, epinephrine and fluoride). Cyclic nucleotide phosphodiesterase from skeletal muscle of mice has two Km's (2.1 and 11 mumol/l) which suggests the existence of either two forms of enzyme or a single enzyme with negative cooperativity. The activity of this enzyme was significantly elevated in the skeletal muscle of dystrophic mice compared to the normal controls. The available evidence suggests that the same cyclic nucleotide phosphodiesterase is responsible for the hydrolysis of both cyclic AMP and cyclic GMP.     

Studies on mitochondria from dystrophic skeletal muscle of mice

Mitochondrial respiration and oxidative phosphorylation were compared in normal and dystrophic mouse skeletal muscles. To obtain the maximum respiration control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio from isolated muscle mitochondria, it is found that there is an advantage in having a low concentration of proteinase and EGTA present in the medium during preparation of mitochondria by centrifugation fractionation. Using pyruvate, acetylcarnitine, and palmitylcarnitine as substrates for oxidation, a highly significant reduction (40-60%) is shown in oxygen uptake by dystrophic muscle mitochondria as compared to normal muscle mitochondria. Studies of the integrity of the oxidative phosphorylation apparatus in these samples showed that there is a reduction of the RCR and ADP/O ratio in dystrophic muscle mitochondria as compared to normal muscle mitochondria.     

Connective tissue metabolism in muscular dystrophy. Amino acid composition of native types I, III, IV and V collagen isolated from the gastrocnemius muscle of embryonic chickens with genetic muscular dystrophy

The amino acid composition data on types I, III, IV and V collagen isolated from embryonic dystrophic skeletal muscle strongly indicate that alterations in collagen synthesis occur in intramuscular connective tissue of developing muscles in embryonic dystrophic chickens. The changes observed in the amino acid composition of dystrophic collagen were: (a) a selective removal of polar amino acids and substitution with non-polar amino acids; (b) significant decreases in basic (lysine, hydroxylysine and arginine) and hydroxylated (4-hydroxyproline and hydroxylysine) amino acids; and (c) significant increases in the amounts of glycine, proline and alanine. The amino acid substitutions suggest a genetic alteration in the collagen synthesizing process and a change in its structure. The variations in amino acid composition of collagen from dystrophic chickens could give rise to a decrease in both inter- and intramolecular cross-linking, thus decreasing the stability and functionality of newly formed collagen fibrils. The differences associated with the dystrophic collagen reported in this study are probably due to the differences in primary structure in terms of amino acid sequence rather than post-translational modifications. The structural differences noted would also lead to an alteration of the role collagen plays in regulating the differentiation of developing muscles. The changes in amino acid structure strongly suggest that the 'collagen' formed by dystrophic chickens should be considered a collagen-like protein or 'collagenoid'.     

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Connective tissue metabolism in muscular dystrophy. Levels of collagen and mucopolysaccharides in embryonic chickens with genetic muscular dystrophy

There were marked differences between the levels of collagen (measured as hydroxyproline) and mucopolysaccharides (measured as hexosamine) found in embryonic chicks with genetic muscular dystrophy and their normal controls. The chief differences were that the dystrophic tissues (gastrocnemius muscle and tendon, pectoralis major and skin) had: (a) greater amounts of hexosamine early in embryonic development; (b) hydroxyproline levels that rose at a faster rate, yielding different slopes than their normal controls; (c) relatively greater amounts of hydroxyproline than hexosamine later in embryonic life (day 20). Connective tissue systems in muscles were preferentially affected. The connective tissue system associated with dystrophic tissues appeared to lag behind the normal rhythm pattern of embryological development. The changes in connective tissue metabolism observed in dystrophic chicks suggested that the collagen from dystrophic embryonic chicks may be of a different structure or composition than that found in the normals.     

Aberrant glycosylation of alpha-dystroglycan causes defective binding of laminin in the muscle of chicken muscular dystrophy

Dystroglycan is a central component of dystrophin-glycoprotein complex that links extracellular matrix and cytoskeleton in skeletal muscle. Although dystrophic chicken is well established as an animal model of human muscular dystrophy, the pathomechanism leading to muscular degeneration remains unknown. We show here that glycosylation and laminin-binding activity of alpha-dystroglycan (alpha-DG) are defective in dystrophic chicken. Extensive glycan structural analysis reveals that Galbeta1-3GalNAc and GalNAc residues are increased while Siaalpha2-3Gal structure is reduced in alpha-DG of dystrophic chicken. These results implicate aberrant glycosylation of alpha-DG in the pathogenesis of muscular degeneration in this model animal of muscular dystrophy.     

The amino acid sequence of the peptide containing the thiol group of creatine kinase from normal and dystrophic chicken breast muscle. Comparison of some of the immunological properties of the antibodies developed in rabbits against these enzymes

The major (14)C-labelled peptides from creatine kinase from normal and dystrophic chicken muscle obtained by carboxymethylating the reactive thiol groups with iodo[2-(14)C]acetic acid and digestion with trypsin were purified by ion-exchange chromatography on Dowex-50 (X2) and by paper electrophoresis. The chromatographic characteristics of the (14)C-labelled peptides, their electrophoretic mobilities at pH6.5, and their amino acid compositions were identical for the two enzymes. The sequence of amino acids around the essential thiol groups of creatine kinase from normal and dystrophic chicken muscle was shown to be Ile-Leu-Thr-CmCys-Pro-Ser-Asn-Leu-Gly-Thr-Gly-Leu-Arg (CmCys, carboxymethylcysteine). This sequence is almost identical with that for the creatine kinases in human and ox muscle and bovine brain and is very similar to that of arginine kinase from lobster muscle. Antibodies to the enzymes were raised in rabbits and their reaction with the creatine kinase from normal and dystrophic muscles in interfacial, immunodiffusion and immunoelectrophoretic experiments was studied. The cross-reaction between normal muscle creatine kinase and antisera against the dystrophic muscle enzyme (or vice versa) observed by immunodiffusion and by immunoelectrophoretic experiments further suggests that the enzymes from normal and dystrophic chicken muscle are similar in structure. The results of the present study, the identical amino acid sequence of the peptides containing the reactive thiol group from both the normal and dystrophic chicken muscle enzymes and the immunological similarities of the two enzymes are in accord with the similarity of the two enzymes observed by Roy et al. (1970).     

Sarcotubular development in dystrophic skeletal muscle cells

Summary Dilations of the sarcotubular system and misaligned myofilaments have been reported as early indicators of muscular dystrophy in skeletal muscle. Since the developing tubular component is believed instrumental in initial myofilament alignment during myogenesis, tubular development is evaluated using normal and dystrophic chick embryo skeletal muscle and cultures of normal and dystrophic embryonic pectoral muscle incubated in the presence of horse spleen ferritin. Comparisons of the findings show that periodic tubules are absent from dystrophic somitic muscle and that invaginating tubules from the sarcolemma are found in fewer, randomly located areas of dystrophic pectoral muscle cells. The results indicate that the tubular component is not involved in the bizarre vesiculations seen in mature dystrophic muscle, however, the malalignment of dystrophic myofilaments is probably the result of the poorer development of the T system in this muscle.     

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.     

Angiotensin II receptor type 1 blockade decreases CTGF/CCN2-mediated damage and fibrosis in normal and dystrophic skeletal muscles

Connective tissue growth factor (CTGF/CCN-2) is mainly involved in the induction of extracellular matrix (ECM) proteins. The levels of CTGF correlate with the degree and severity of fibrosis in many tissues, including dystrophic skeletal muscle. The CTGF overexpression in tibialis anterior skeletal muscle using an adenoviral vector reproduced many of the features observed in dystrophic muscles including muscle damage and regeneration, fibrotic response and decrease in the skeletal muscle strength. The renin-angiotensin system is involved in the genesis and progression of fibrotic diseases through its main fibrotic components angiotensin-II and its transducer receptor AT-1. The use of AT-1 receptor blockers (ARB) has been shown to decrease fibrosis. In this paper, we show the effect of AT-1 receptor blockade on CTGF-dependent biological activity in skeletal muscle cells as well as the response to CTGF overexpression in normal skeletal muscle. Our results show that in myoblasts ARB decreased CTGF-mediated increase of ECM protein levels, extracellular signal regulated kinases 1/2 (ERK-1/2) phosphorylation and stress fibres formation. In tibialis anterior muscle overexpressing CTGF using an adenovirus, ARB treatment decreased CTGF-mediated increase of ECM molecules, α-SMA and ERK-1/2 phosphorylation levels. Quite remarkable, ARB was able to prevent the loss of contractile force of tibialis anterior muscles overexpressing CTGF. Finally, we show that ARB decreased the levels of fibrotic proteins, CTGF and ERK-1/2 phosphorylation augmented in a dystrophic skeletal muscle from mdx mice. We propose that ARB is a novel pharmacological tool that can be used to decrease the fibrosis induced by CTGF in skeletal muscle associated with muscular dystrophies.     

Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle.

Phospholipid incorporation of 32P by primary myotube cultures and the tissue activity of sarcolemmal Na+/K(+)-transporting ATPase were studied to determine whether the absence of dystrophin from dystrophic (mdx) muscle would affect membrane lipid synthesis and membrane function. The incorporation of 32P by phospholipid as a ratio with total protein was greater in cultured dystrophic cells compared with control cells. The mdx cells also incorporated more 32P than control cells into phosphatidylethanolamine, which is thought to increase prior to myoblast fusion, and less into phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. There was no difference in total protein content or [3H]leucine or 32P incorporation into the aqueous fraction of dystrophic and control cells, although dystrophic cells incorporated less [35S]methionine into protein than controls. Isolated sarcolemma from mdx skeletal muscle tissue demonstrated a consistently greater specific activity of ouabain-sensitive Na+/K(+)-transporting ATPase than sarcolemmal preparations from control skeletal muscle. These observations suggest that cytoskeletal changes such as dystrophin deficiency may alter the differentiation of membrane composition and function.     

Cholinesterase in muscle of dystrophic hamsters (Bio-40.54)

Isozyme patterns of cholinesterase (ChE) from heart, tongue, and skeletal muscle of normal and dystrophic hamsters are presented. Two principal bands, bands 1 and 2, were evaluated. Band 1 migrates faster towards the anode than does band 2. While bands 1 and 2 stain for AChE and were found in control muscles, only band 2 was stained by a pseudocholinesterase (BuChE) and was decreased in samples from dystrophic hamsters. The decrease in BuChE was most pronounced in dystrophic heart muscle. The low level of BuChE measured for dystrophic animal tissue was similar to isozyme patterns found in embryonic tissue and in denervated muscle. BuChE obtained by acrylamide gel electrophoresis along with 16S AchE appears to be a useful biochemical marker of nerve-muscle interactions.