Discrimination and consistency of five myosin ATPase stains in human normal and Duchenne dystrophic muscle

Limitations in the ability of the human visual system to assess accurately the relative staining densities of individual fibers in muscle tissue stained for myosin. ATPase can complicate the objective evaluation of fiber type populations. In this study a novel approach is employed which utilizes human visual capabilities to provide accurate fiber classification. Using this approach, the ability of five ATPase staining techniques to discriminate fiber type categories in single samples of human normal and Duchenne dystrophic skeletal muscle is evaluated, as is the consistency of the fiber type classifications between stains. While no major discrepancies in fiber typing were observed in the sample of normal muscle, significant differences in classification, along with a decrease in the ability to discriminate fiber types were noted in the sample of Duchenne muscular dystrophy. For the most part, these discrepancies were resolved by a re-interpretation of the staining characteristics of fibers in one stain.     

Heat, mechanics, and myosin ATPase in normal and hypertrophied heart muscle

In this paper we review our previous work on the myothermic economy of isometric force production in compensated cardiac hypertrophy secondary to pulmonary artery constriction (pressure overload) and/or thyrotoxicosis (volume overload). Hypertrophy-induced changes in isotonic and isometric twitch mechanics are correlated with accompanying changes in actin-activated myosin ATPase and heat liberation. Heat measurements were made with rapid, high-sensitivity thermopiles on right ventricular papillary muscles from normal and hypertrophied rabbit hearts. Total activity-related heat was separated into initial and recovery heat. Initial heat was separated into a tension-dependent component (TDH) relating to cross-bridge activity, and a tension-independent component (TIH) relating to excitation-contraction coupling. There were oppositely directed changes in most parameters studied in pressure overload hypertrophy (P) as compared with thyrotoxic hypertrophy (T). Thus, in P there was depression (30-50% in the rate of isometric force production, mechanical Vmax, TDH and TDH rate, myosin ATPase, TIH, and prolongation in time-to-peak twitch tension, whereas in T all parameters were oppositely changed except for no change in TIH. Thyrotoxicosis following pressure overload reversed the P-induced changes in all parameters. There was a direct, linear relation between in vitro actin-activated myosin ATPase and in vivo TDH. However, TDH per unit twitch tension or tension-time integral varied inversely with ATPase, making force production more economical than normal in P muscles and less economical than normal in T muscles. These cellular changes beneficially equip P hearts for slow, high-pressure, economical pumping the T hearts for fast, high-volume, uneconomical pumping. The differences are similar to those between slow and fast skeletal muscle and between neonatal and adult skeletal muscle. The mechanism of these changes is discussed in terms of an enzyme kinetic scheme of chemomechanical coupling in actomyosin interaction.     

Arylamidase and cathepsin-A activity of normal and dystrophic human muscle.

Human skeletal muscle homogenate has been shown to contain enzymes that catalyze the hydrolysis of L-leucyl p-nitroanilide and carbobenzoxyglutamyl-L-tyrosine, known substrates, respectively, for arylamidase and cathepsin A. The muscle arylamidase was found to be inhibited by p-chloromercuribenzoate. Addition of Co2+ resulted in slight stimulation of its activity. Neither ethylenediamine tetraacetate nor thiol compounds had any appreciable effect on the enzyme. When compared to controls, no significant differences in muscle arylamidase levels were observed in patients with muscular dystrophies and certain selected neuromuscular diseases. Cathepsin A was, however, increased in muscles moderately affected by muscular dystrophy and denervating diseases.     

Calcium currents in normal and dystrophic human skeletal muscle cells in culture

Human muscle cells obtained from biopsy specimens were grown in a primary culture system and electrophysiologically studied. Whole cell patch-clamp recordings revealed the presence of two types of calcium currents: (i) a low-threshold (-60 mV) one (ICa, T) with fast activation and inactivation kinetics (time-to-peak: 39 ms at -30 mV); and (ii) a high-threshold (-10 mV) one (ICa,L) with slower kinetics (time-to-peak: 550 ms at 20 mV). These two types of calcium currents could be also distinguished by their pharmacological characteristics since ICa,L was sensitive to the antagonist and agonist dihydropyridine derivatives contrary to ICa,T which was completely resistant to these compounds. These functional calcium channels existed both in normal and Duchenne dystrophic (DMD) human skeletal muscle cells in culture. We discuss a possible role of these two types of calcium channels in the myoplasmic calcium accumulation observed in the Duchenne muscular dystrophy.     

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.     

Immunohistochemical localization of smooth muscle myosin in normal human tissues

Immunohistochemical localization of smooth muscle myosin, an immunologically distinct contractile protein, was achieved using rabbit anti-human uterine smooth muscle myosin antibodies. In immunodiffusion studies and in cryostat sections, these antibodies were highly specific and reacted with smooth muscle myosin but not with platelet, skeletal muscle, or cardiac muscle myosin. To evaluate comprehensively the structural profile of smooth muscle elements in normal human tissues, an indirect immunoperoxidase technique (peroxidase-antiperoxidase) was applied to a wide variety of specimens. Parallel studies comparing cryostat sections with fixed (10% formalin, B5, Bouin's, or Zenker's solution) paraffin-embedded tissues revealed optimal immunoreactivity, sensitivity, and specificity of staining for smooth muscle myosin using frozen tissues. Strong immunoreactivity was present in muscular tissues such as blood vessels and the muscularis of gastrointestinal and genitourinary tracts. Distinct delineation of smooth muscle elements, including individual smooth muscle cells, and their specific patterns of alignment and organization, were observed, e.g., cells comprising the muscularis mucosae and extending into the lamina propria of the gastrointestinal tract, and myoepithelial cells of skin, exocrine glands, and breast. This method provides excellent morphologic preservation and readily permits unambiguous identification of individual cells containing smooth muscle myosin.     

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle

Summary Dystrophin is the product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin-related protein (utrophin), an autosomal homologue of dystrophin, was studied in skeletal muscle from normal fetuses aged 9–26 weeks and one stillbirth of 41 weeks' gestation, and compared with low- and high-risk DMD fetuses aged 9–20 weeks. Utrophin was present at the sarcolemma from before 9 weeks' gestation, although there was variability in intensity both within and between myotubes. Sarcolemmal immunolabelling became more uniform, and levels of utrophin increased to a maximum at approximately 17–18 weeks. Levels then declined, until by 26 weeks sarcolemmal labelling was negligible and levels were similar to adult control muscle. By 41 weeks there was virtually no sarcolemmal labelling, although immunolabelling of capillaries was bright. Similar results were obtained with normal and DMD fetal muscle. Utrophin is therefore expressed in the presence and absence of dystrophin and down-regulated before birth in normal fetal muscle fibres. Samples were not available to determine whether or when, utrophin levels decline in DMD fetal muscle. On Western blots, utrophin was shown to have a smaller relative molecular mass than adult dystrophin, but similar to the fetal isoform. Blood vessels were brightly immunolabelled at all ages, although utrophin immunolabelling of peripheral nerves increased with gestational age.     

Smooth muscle myosin isoform distribution and myosin ATPase in hypertrophied urinary bladder.

Hypertrophy of the urinary bladder was produced in rabbit by partial ligation of the urethra. Electrophoresis of the bladder smooth muscle myosin on highly porous (3.5-7% gradient) SDS-polyacrylamide gel revealed two heavy chain isoforms, SM-1 and SM-2 with approximate molecular weights of 204,000 and 200,000, respectively. The ratio of the SM-2 to SM-1 heavy chain is 3:1 for myosin isolated from normal bladder smooth muscle, and this ratio changes to about 1:1 in hypertrophied bladder. Despite a change in the ratio of SM-2 to SM-1, the myosin ATPase and the actin-activated ATPase activities are not altered in response to hypertrophy.     

Correlation between myofibrillar ATPase activity and myosin heavy chain composition in single human muscle fibers

Summary Single human muscle fibers were analysed using a combination of histochemical and biochemical techniques. Routine myofibrillar adenosine triphosphatase (mATPase) histochemistry revealed a continuum of staining intensities between the fast fiber types IIA and IIB (type IIAB fibers) after preincubation at pH 4.6. Electrophoretic analysis of single, histochemically-identified fibers demonstrated a correlation between the staining intensity and the myosin heavy chain (MHC) composition. All fibers classified as type I contained exclusively MHCI and all type IIA fibers contained only MHCIIa. Type IIAB fibers displayed variable amounts of both MHCIIa and MHCIIb; the greater the staining intensity of these fibers after preincubation at pH 4.6, the greater the percentage of MHCIIb. Those fibers histochemically classified as type IIB contained either entirely MHCIIb or, in addition to MHCIIb, a small amount of MHCIIa. These data establish a correlation between the mATPase activity and MHC content in single human muscle fibers.     

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.