Consequences of thyroxine treatment on diaphragm and EDL of normal and dystrophic hamsters

Previously administration of thyroxine (T4) to dystrophic hamsters improved ventilation and slowed the progression of the disease. We hypothesized that the normalization of ventilation in these animals was due to T4 improving structural and functional characteristics of the diaphragm. In the present study, contractile characteristics of the diaphragm and the extensor digitorum longus (EDL) from normal and dystrophic hamsters were evaluated after two months of T4 treatment. Compared to their placebo-treated counterparts, diaphragms and EDLs of T4-treated normal hamsters showed increased optimal muscle lengths and twitch tension, decreased contraction times and increased fatigability. T4-treatment in dystrophic hamsters showed only an increase in diaphragmatic twitch tension development. Force-frequency curves before treatment were generally higher for the normal compared to dystrophic diaphragms and EDLs. T4 administration only increased the force in normal diaphragms at the lower frequencies and in the EDLs at the higher frequencies. Although T4 serum levels were increased in both T4-treated groups, triiodothyronine (T3) was much lower in the dystrophic compared to normal hamsters, suggesting that conversion of T4 to T3 was reduced in dystrophic hamsters. We conclude that the limited functional changes in the diaphragms of T4-treated dystrophic hamsters cannot account for the marked improvement in ventilation previously reported.     

Age-related changes in protein turnover and ribonucleic acid of the diaphragm muscle of normal and dystrophic hamsters.

Diaphragm muscles of dystrophic hamsters were found to be larger than those of control animals at two of three ages studied. The additional growth of these afflicted muscles correlated with large increases in protein synthesis and concentrations of RNA. Protein breakdown was also increased in the dystrophic muscles, but to a smaller extent than synthesis.     

Protein synthesis in muscles from normal and dystrophic hamsters.

Homogenates of hindleg muscle were obtained from control and dystrophic male hamsters, 30 and 190 days of age, and were used to prepare the postmicrosomal pH5-supernatant fraction. The activity of this fraction in the incorporation of [14C]phenylalanyl-tRNA into peptides was increased in the dystrophic-muscle preparations. No such increase was found in brain or liver preparations from dystrophic hamsters. The increased capacity for aminoacyl-tRNA binding that was observed in preparations from dystrophic animals is discussed.     

Acid phosphatase activity in soleus and plantaris muscle fibres of normal and dystrophic hamsters

Summary The activity of acid phosphatase in skeletal muscle fibres of the plantaris and soleus of normal and dystrophic male hamsters was quantified using a histochemical post-coupling semipermeable membrane technique. Althoug the absolute levels of activity were found to vary widely from one animal to another, the ratio of the mean activities in the two muscles in each animal was virtually constant. In normal muscles, the ratio was about 0.73 and in dystrophic muscles, about 0.77. The activity in plantaris muscle fibres was always significantly lower than that in the corresponding soleus fibres, and in normal fibres compared to dystrophic ones. Another difference was that in normal fibres the mean activity declined to a constant level in mature animals older than about 3 months. In contrast, the activity in dystrophic muscles appeared to fall exponentially throughout life. The functional significance of these findings is discussed.In honour of Prof. P. van Duijn     

Halothane anaesthesia of normal and dystrophic hamsters.

Induction, carried out in a small clear-plastic box with 3-5% (v/v) halothane in 30:70 (v/v) oxygen: nitrous oxide, was quiet and rapid. Recovery was almost instantaneous. 2% halothane in the oxygen-nitrous oxide mixture was sufficient for maintenance anaesthesia. The anaesthetic mixture was given by face mask in an open circuit specially designed to function at low gas-flow rates. The halothane content of the muscle and blood after 25 min anaesthesia was estimated by gas chromatography of n-heptane extracts. The mean level (+/- s.e.m.) in blood was 22-8 +/- 2-7 mg/100 ml (n=4), and in dystrophic muscle 226 +/- 36-8 mg/100 g wet weight of tissue (n=4): there was a positive correlation (r=0-94) between them (p less than 0-02).     

Soluble cholinesterase of muscle from dystrophic and normal mice

The percentage of cholinesterase extractable by isotonic sucrose from dystrophic mouse muscle was greater than that found in normal muscle. Of the total cholinesterase found in normal and dystrophic muscle about 60% was specific AChE and 40% was non-specific cholinesterase. The extract from dystrophic muscle showed, on sucrose sedimentation, one major peak of AChE activity with a sedimentation constant of approximately 4.3 S. This was much higher than that from normal muscle.     

Satellite cells of normal and dystrophic muscle

The structure, the ultrastructure and the number of myonuclei and satellite cells in Duchenne's muscular dystrophy and in control muscles were compared in order to determine the possible changes in the satellite cells population. The bioptical fragments were obtained from 16 healthy (control) and from 16 dystrophic male children from 12 to 96 months of age. The biopsies were embedded in paraffin and in Durcupan and the sections were stained with ematossilin-eosin, P.A.S. for the light microscope observation and with uranil-acetate and lead-citrate for the electron microscope study. Moreover the semithin sections were stained according to the method of Ontell (1974) that is specific for the satellite cells identification. The morphological aspects of the dystrophic muscles are the same previously reported by other authors. The quantitative analysis of the myonuclei and satellite cells in control and dystrophic muscles was carried out on five random sections of each biopsy. The whole number of nuclei (myonuclei and satellite cell nuclei) and the number of the satellite cells nuclei were evaluated and the mean values in controls and dystrophic muscles were compared with the t Student test. The obtained results show that: 1) in the control muscles the satellite cells number is nearly the same in all ages considered; 2) in the dystrophic muscles the satellite cells number is in a statistically significant way greater than in control muscles and show a moderate trend to increase with aging; 3) in the dystrophic muscles the whole number of nuclei (myonuclei and satellite cells) is greater than in control in a statistically significant way and this increase is due to the number of satellite cells.     

Increased contractility in vascular smooth muscle of dystrophic hamsters

To investigate the "vascular" hypothesis of muscular dystrophy, the sensitivity and contractility of aortic spiral strips of dystrophic (BIO 14.6) and normal (FIB) hamsters have been determined to various smooth muscle agonists. The results obtained with cumulative dose-response curves show that there is no increase in the sensitivity of the dystrophic compared with the normal aorta to noradrenaline, phenylephrine, isoproterenol, histamine, or 5-hydroxytryptamine. However, there was a significant increase in the force generated by aortic strips of the dystrophic animals to all agonists. Determination of noncollagen and collagen protein showed that there was no difference in the relative proportions of these proteins in the aortas from the two strains. The results show that in this animal model of dystrophy an increased response to vasopressor amines occurs and is in accordance with that expected of the vascular hypothesis.     

Ultrastructure of diaphragm from dystrophic alpha-sarcoglycan-null mice

alpha-Sarcoglycan is a 50 kDa single-pass transmembrane glycoprotein exclusively expressed in striated muscle that, together with beta-, gamma-, and delta-sarcoglycan, forms a sub-complex at the muscle fibre cell membrane. The sarcoglycans are components of the dystrophin-associated glycoprotein (DAG) complex which forms a mechanical link between the intracellular cytoskeleton and extracellular matrix. The DAG complex function is to protect the muscle membrane from the stress of contractile activity and as a structure for the docking of signalling proteins. Genetic defects of DAG components cause muscular dystrophies. A lack or defects of alpha-sarcoglycan causes the severe type 2D limb girdle muscular dystrophy. alpha-Sarcoglycan-null (Sgca-null) mice develop progressive muscular dystrophy similar to the human disorder. This animal model was used in the present work for an ultrastructural study of diaphragm muscle. Diaphragm from Sgca-null mouse presents a clear dystrophic phenotype, with necrosis, regeneration, fibre hypertrophy and splitting, excess of collagen and fatty infiltration. Some abnormalities were also observed, such as centrally located nuclei of abnormal shape, fibres containing inclusion bodies within the contractile structure, and fibres with electron-dense material dispersed over almost the entire cell. Additionally, unusual interstitial cells of uncertain identity were detected within muscle fibres. The abnormal ultrastructure of the diaphragm from Sgca-null mice is discussed.     

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.