Myopathic Animals Research Articles

Defective membrane systems in dystrophic skeletal muscle of the UM-X7.1 strain of genetically myopathic hamster.

1. The function of mitochondria, sarcotubular membranes (heavy microsomes), sarcolemma and myofibrils from the hind-leg skeletal muscle of about 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters was examined. 2. The mitochondrial calcium uptake as well as mitochondrial phosphorylation and respiratory rates were lower in 60-day-old myopathic skeletal muscle, unlike 150-day-old myopathic animals, when pyruvate-malate and glutamate-malate were used as substrates. However, mitochondria from 150-day-old myopathic animals showed depressed glutamate-dependent respiratory and phosphorylation rates and succinate-supported initial rate of calcium uptake. 3. The microsomal calcium-uptake, but not calcium-binding, and Ca2+-stimulated adenosine triphosphatase (ATPase) activity of the 150-day-old myopathic skeletal muscle were lower than the control values. Although microsomal calcium-binding, calcium-uptake and ATPase activities of the 60-day-old myopathic muscle were not depressed significantly, the initial rate of calcium uptake was less than the control. 4. The sarcolemmal Ca2+-ATPase, but not Mg2+-ATPase or Na+ +K+-ATPase, activity was higher in 60-day-old myopathic muscle whereas the activities of all these enzymes from 150-day-old myopathic animals were higher than the control. On the other hand, the Na+ +K+-ATPase activities from 60- and 150-day-old myopathic animals were inhibited by ouabain to a lesser extent in comparison with the respective control values. 5. The myofibrillar Ca2+-ATPase and Mg2+-ATPase activities as well as inhibition of Mg2+-ATPase due to Na+ and K+ in myopathic muscle were no different from the control values. 6. The results reported here give further support to the view that different membrane systems of the dystrophic muscle are defective.

The synthesis of myofibrillar and soluble proteins in cell-free systems and in intact cultured muscle cells from newborn polymyopathic hamsters

Myofibrillar proteins from both hearts and skeletal muscles of control and myopathic hamsters, at different ages, were extracted and analysed electrophoretically. Significant quantitative differences were found, more or less uniformly affecting all the myofibrillar components, even of embryonic hearts. In an attempt to determine whether the observed differences become established as the result of defective biosynthesis or for some other reason, reconstituted cell-free systems were used to study the in vitro synthesis of some of the specific myofibrillar protein components. A generally defective biosynthesis of myosin, (both heavy and light chains) and actin was demonstrated, while gel-electrophoretic analysis of soluble proteins synthesized in vitro showed that the profile of radioactivity in systems derived from myopathic animals was also generally lower than in those from controls. In order to demonstrate that the defect in protein synthesis was present in intact beating heart cells, myopathic and control heart cells were treated with radioactive amino acids, and the soluble and myofibrillar proteins subsequently extracted and fractionated. The biosyntheses of both types of proteins were clearly impaired in the case of cells derived from myopathic animals.

Evidence for defective transfer ribonucleic acid in polymyopathic hamsters and its inhibitory effect on protein synthesis.

1. Different reaction steps involved in protein synthesis were studied in skeletal muscles from control and myopathic hamsters. 2. There was no difference between partially purified aminoacyl-tRNA synthetases from myopathic and control animals in yield or catalytic activity, as tested with exogenous deacylated tRNA. 3. However, isolated deacylated tRNA from myopathic muscle was aminoacylated by these synthetases to a lesser extent than that derived from control muscle. 4. Addition of deacylated tRNA isolated from control muscle improved the performance of pH5 enzymes from myopathic muscle in polypeptide synthesis on homologous polyribosomes; tRNA isolated from myopathic animals did not. 5. Preparation of extracts from both types of animals in the presence of the ribonuclease-absorbent bentonite led to an increased capacity of endogenous tRNA to accept amino acids in pH5 enzymes prepared from normal and abnormal tissue, but the difference between the two systems remained the same. 6. Total tRNA nucleotidyltransferase activity, tested with twice-pyrophosphorolysed rat liver tRNA, was identical in both extracts. 7. Added tRNA nucleotidyltransferase incorporated more AMP and CMP into endogenous tRNA with the pH5 enzyme from myopathic muscle than with that from control muscle. 8. Preincubation of deacylated tRNA from myopathic muscle with ATP, CTP and tRNA nucleotidyltransferase more than doubled its subsequent aminoacyl-acceptor activity, and halved the extent of the defect relative to aminoacylation of control tRNA similarly treated. Endogenous tRNA in pH5 enzyme preparations behaved likewise. 9. It is suggested that a 3'-exonuclease in myopathic muscles attacks tRNA molecules in such a way that some of them remain substrates for tRNA nucleotidyltransferase, which may incorporate into RNA not only AMP and CMP, but also GMP. 10. Cell-free protein synthesis in preparations from myopathic hamster muscles is limited by the supply of intact tRNA molecules.

Cell-free protein synthesis in heart and skeletal muscles from polymyopathic hamsters.

1. Cell-free protein synthesis was studied in striated and smooth muscles in an attempt to elucidate the primary genetic defect in polymyopathic hamsters. 2. When washed membrane-free polyribosomes from myopathic and control heart muscle were individually recombined with pH5 enzymes from both types of animals, the pH5 enzymes from myopathic muscle were less active in polypeptide synthesis than those from controls, irrespective of the source of polyribosomes. 3. The same defect was present in skeletal-muscle preparations. 4. Both the initial rate and the maximum extent of incorporation were affected in the defective preparations from myopathic muscle. 5. Concentration differences, with respect to total protein and RNA, were not responsible. 6. Preincubation of the pH5 enzymes resulted in a greater degree of inhibition. 7. The defect in the pH5 enzymes from myopathic muscle was also expressed in poly(U)-directed polyphenylalanine synthesis. 8. Acid proteinase activity in extracts of control and myopathic muscle was the same but general ribonuclease activity in the latter extracts was higher. 9. The defect was also present when both types of pH5 enzymes were prepared in the presence of the ribonuclease-asborbent bentonite. 10. pH5 enzymes from uterine smooth muscle, brains and livers of myopathic animals were similarly affected in homologous and heterologous combinations. 11. It is concluded that the general tissue defect is both qualitative and quantitative in nature, implying that there is a shortage of some essential soluble component in the pH5 fraction which is accompanied by the presence of an altered substituent. This prevents the attainment of extents of polypeptide synthesis in vitro obtained in control extracts from unaffected animals.

Myocardial contractility in relation to hypertrophy and failure in myopathic Syrian hamsters

Myocardial contractility of left ventricular papillary muscles from normal Syrian hamsters and those with hereditary cardiomyopathy has been studied. The animals were divided into five groups corresponding to different pathological stages and the mechanical parameters correlated with these stages. Developed force and dF dt corrected for cross-sectional area were depressed in all myopathic groups. However, developed force and dF dt , uncorrected for cross-sectional area, were restored to normal in the hypertrophied muscles in the myopathic animals at 220 and 320 days of age in the absence of congestive failure (i.e. without edema). In the early myolytic and healing stage and in the terminal edematous stages of the myopathy, force-velocity curves were depressed at all loads. However in hypertrophied and compensated hearts at 220 to 320 days, the force velocity curves were similar to controls when load was expressed as a fraction of P 0. Assuming developed force to reflect the number of contractile sites in parallel, it was concluded that the number of normally active contractile sites per unit area was reduced at all stages of the disease. In the hypertrophied, compensated phase, it was postulated that the qualitative nature of these sites remain normal despite their quantitative reduction.

Enzyme studies in the myopathic duck.

There is a correlation between the effect of ouabain upon membrane ATPase activity and muscle abnormality of the white Pekin duck. Membrane preparations (sarcoplasmic reticulum) and haemolyzed erythrocytes from normal animals showed a characteristic transport ATPase activity which was inhibited by 10-4M concentration of the cardiac glycoside, ouabain. Identical preparations from myopathic animals were stimulated rather than inhibited by the same concentration of ouabain. We have interpreted this observation to indicate that the myopathy has a generalized character recognizable in erythrocytes as well as in muscle cells and that the defect itself can be associated with membrane integrity which affects the properties of the transport enzyme. Aldolase levels have been found to be higher in myopathic animals than in normal animals in serum and subcellular fractions. Although hexokinase levels in serum were not different in experimental and control animals, the subcellular compartment activities were greatly different. Creatine phosphokinase levels in serum were identical in normal and experimental animals but the mitochondrial phosphokinase activity of the myopathic birds was greatly elevated. It appears that a pronounced abnormality of carbohydrate metabolism can be correlated with this form of myopathy. Relationships of the myopathy of this experimental animal to the human malady have been considered.

Enhanced muscle regeneration and increased strength in myopathic animals treated with ethylenedinitramine.

DIFFERENTIATION of skeletal muscle is activated as a function of replication1. This predicts that enhancement of growth in a myogenic system will increase the yield in new muscle. Excessive proliferation, however, can cancel muscle differentiation2. In employing the theory it is therefore necessary to stipulate that increased growth shall not reach deleterious levels (although just what these levels are cannot yet be specified). The properties of ethylenedinitramine (EDNA)3,4 seemed, intuitively, to satisfy this condition. EDNA enhanced the regeneration of muscle under several specific conditions.