Diseased Human Muscle Research Articles

Expression of three calpain isoform genes in human skeletal muscles

Calpain is thought to be involved in muscular degradation in progressive muscular dystrophy (PMD), especially Duchenne and Becker muscular dystrophies. To assess the expression of calpain genes in skeletal muscles of patients with myopathies, we examined mRNA levels of three calpain isoforms by the quantitative reverse transcriptase–polymerase chain reaction method in biopsied muscles from control, PMD and amyotrophic lateral sclerosis (ALS) patients. There was a statistically significant increase in calpain 1 and calpain 2 mRNA levels in PMD and ALS patients as compared to controls. In contrast, there was a decrease in expression of calpain 3 mRNA in PMD, but it was not statistically significant. Expression of calpain 1 and calpain 2 positively correlated with each other, but not with calpain 3. These results indicate that expression of calpain 1 and calpain 2, but not calpain 3, are upregulated in diseased human muscles, likely playing a regulatory role in the process of myofibrillar degradation at the transcriptional as well as posttranslational level.

Electrical restitution in diseased human ventricular myocardium

Action potential configuration and electrical restitution were studied in diseased human ventricular muscle by comparing the characteristics of hypertrophic (HYP) and dilated (DIL) human ventricular preparations. Conventional microelectrode techniques were used to evaluate action potentials evoked at increasingly longer diastolic intervals. The steady-state action potential duration (APD90) was significantly longer in DIL than in HYP preparations (393 +/- 5 ms, n = 4 and 296 +/- 11 ms, n = 4, respectively; P < 0.001, mean +/- SEM). In the dilated preparations studied at long diastolic intervals, the initial period of rapid repolarization (phase 1) was absent, and the rate of final repolarization (phase 3) was reduced. Electrical restitution relations in these preparations were fitted as the sum of two exponentials. The time constant of the fast component was significantly longer in DIL than in HYP preparations (242 +/- 9 ms and 121 +/- 4 ms, respectively; P < 0.001). No difference was observed in the time constants for the slow component of restitution in the two groups. Electrical restitution was also studied in single human ventricular myocytes by using patch clamp techniques. The initial 600 ms period of restitution was fitted in these cells to a monoexponential function. The time constant for this period of the restitution relation was significantly longer, while the estimated amplitude of this early rising phase was significantly lower in human cells obtained from DIL hearts than the respective parameters obtained in the healthy canine and guinea pig cells also examined. The observed changes in the restitution kinetics of the dilated human heart are, likely, the consequence of alterations in the ionic currents that underlie the cardiac action potential.

ADP Recovery After a Brief Ischemic Exercise in Normal and Diseased Human Muscle — a31P MRS Study

The pattern of cytosolic ADP recovery after exercise has not been fully characterized in human skeletal muscle. ADP recovery after brief, ischemic exercise was studied by 31phosphorus magnetic resonance spectroscopy in calf muscles of 33 normal control subjects, four patients with McArdle's disease and 13 patients with mitochondrial myopathy. In normal muscle, the half-time for the initial ADP decline was 0.18 +/- 0.07 min and was unaffected by the pH or the metabolic state at the end of exercise. ADP decreased to below rest values during the second min of recovery in 27 out of 33 control subjects. There was a significant (p < 0.001) linear correlation for both the size (r = 0.65) and duration (r = 0.64) of this ADP undershoot with intracellular pH. Phosphocreatine resynthesis continued during the ADP undershoot. ADP undershoot was also found in patients with mitochondrial diseases (in 11 out of 13), but not McArdle's disease (six patients). Thus ADP recovery follows a complex time course that is partly dependent on pH. Only the initial ADP recovery is independent of pH, which makes it suitable for comparative assessment of muscle mitochondrial function in vivo. As phosphocreatine recovery continues during the ADP undershoot, mitochondrial regulation must be different from that at the onset of recovery. These observations are consistent with variable, changing regulators of mitochondrial metabolism in human skeletal muscle.

Cardiac troponin-I is not expressed in fetal and healthy or diseased adult human skeletal muscle tissue

Cardiac troponin-I (cTnI) is not found in sera of patients with skeletal muscle disease in the absence of myocardial injury. It is not known, however, whether trace amounts of cTnI are expressed in regenerating human skeletal muscle, as has been observed with creatine kinase MB. Using immunohistochemical and biochemical techniques, we investigated cTnI expression in various human muscle tissues: human heart tissue (n = 5), normal adult skeletal muscle (n = 3), and fetal heart (n = 3) and skeletal muscle (n = 3) obtained, respectively, during heart transplant, from autopsy, or from a tissue bank. Specimens from diagnostic tissue biopsies were used as diseased skeletal muscle: polymyositis (PM), n = 13; Duchenne muscular dystrophy (DMD), n = 6. Frozen sections 8 microns thick were stained immunohistochemically for either cTnI or TnI (cardiac or skeletal) by using monoclonal antibodies (MAb) 2B1.9 (cTnI specific) or 3C5.10 (reactive with all TnI isoforms), respectively. cTnI was measured in tissue homogenates by an immunofluorometric assay. Cardiac muscle was stained by both MAbs. Normal fetal and adult skeletal muscle, and samples from all of the PM and DMD patients, stained only with the nonspecific MAb (3C5.10), confirming the sole presence of skeletal TnI. No cTnI was detectable by immunoassay in any skeletal muscle sample. We conclude that cTnI is not expressed in human skeletal muscle during development or during regenerative muscle disease processes such as PM or DMD.

Applications of NMR to the study of in vivo human muscle metabolism

Nuclear Magnetic Resonance is one of the few non-invasive techniques for metabolic investigation. Its advantages and the kind of informations it provides are quickly explained. Examples are given of various contributions to the in vivo study of normal or diseased human muscle metabolism, mainly based on phosphorus 31 (31P) studies. The complementarity of informations provided by various nuclei is shown by the evaluation of muscle glycogen levels in myopathies by natural abundance carbon 13 NMR. Biochemical/functional information on exercising muscles can be obtained by magnetic resonance imaging (MRI) combined with 31P-NMR, which may have diagnostic and pathophysiological applications. The latter study has raised questions about the amount of perfusion in some muscle diseases which has been answered by a combined 31P-NMR and positron emission tomography (PET) study.

Beta-Amyloid precursor epitopes in muscle fibers of inclusion body myositis.

Sporadic inclusion body myositis (IBM) and hereditary inclusion body myopathy (hIBM) are severe and progressive muscle diseases, characterized pathologically by vacuolated muscle fibers that contain 15- to 21-nm cytoplasmic tubulofilaments (CTFs). Those vacuolated muscle fibers also contain abnormally accumulated ubiquitin and beta-amyloid protein (A beta), and they contain amyloid in beta-pleated sheets as indicated by Congo red and crystal violet positivity. Using several well-characterized antibodies, we have now demonstrated that, in addition to A beta, two other epitopes, N-terminal and C-terminal, of the beta-amyloid precursor protein (beta PP) are abnormally accumulated in IBM vacuolated muscle fibers and similarly in hIBM. At the light microscopy level, immunoreactivities of N- and C-epitopes of beta PP closely colocalized with A beta and ubiquitin immunoreactivities. However, by immunogold electronmicroscopy, even though N-, C-, and A beta epitopes of beta PP and ubiquitin colocalized at the amorphous and dense floccular structures, only A beta was localized to the 6- to 10-nm amyloid-like fibrils and only ubiquitin was localized to CTFs. beta PP immunoreactive structures were often in proximity to CTFs, but CTFs themselves never contained beta PP immunoreactivities. The fact that A beta but not C- or N-terminal epitopes of beta PP localized to the 6- to 10-nm amyloid-like fibrils suggests that free A beta might be generated during beta PP processing and, after aggregation, may be responsible for the amyloid present within IBM muscle fibers. Our study demonstrates that three epitopes of beta PP accumulate abnormally in diseased human muscle, and therefore this phenomenon is not unique to Alzheimer's disease, Down's syndrome brain, and Dutch-type cerebrovascular amyloidosis.

CD24, a signal-transducing molecule expressed on human B lymphocytes, is a marker for human regenerating muscle.

The expression of the CD24 molecule, a glycoprotein expressed at the surface of most B lymphocytes and differentiating neuroblasts, was studied in developing nerve and muscle (after 16 weeks of gestation), normal adult and various diseased human muscles using immunohistochemistry and Western blot analysis. Immunohistochemical studies demonstrated that: (1) in developing muscles, fibers did not express CD24, whereas only some mesenchymal areas, also expressing neural cell adhesion molecule (N.CAM) and vimentin, and developing nerves were positive; (2) in normal adult muscles, CD24 immunoreactivity was observed only in some unmyelinated nerve fibers--intra and extra fusal muscle fibers, satellite cells and neuromuscular junctions were negative; and (3) in all diseased muscles studied here, CD24 expression was always associated with a subpopulation of regenerative fibers. These fibers also expressed vimentin, desmin, developmental myosin heavy chain, N.CAM and its polysialylated isoforms (PSA-N.CAM). The number of CD24-positive fibers was always lower than that of PSA-N.CAM-positive fibers. Denervated fibers and vacuolated muscle fibers never expressed CD24. Western blot analysis indicated that the apparent molecular mass of CD24 antigen was different between muscle and developing nervous tissues, suggesting that CD24 glycosylation is tissue specific. Since the molecule was not expressed in developing human muscle fibers, it strongly suggests that regenerative and fetal myotubes are different with respect to the CD24 molecule expression.

Localized in vivo 1H spectroscopy of human skeletal muscle: Normal and pathologic findings

To obtain high signal to noise ratio in small volume elements (8 cm 3), in vivo 1H NMR spectroscopy of normal and diseased human skeletal muscle was performed using a double spin-echo localization method on a 1.5-T whole body system. High resolved spectra of normal calf muscle show the well known resonances of lipids (methyl, methylene, olefinic, and other fatty acid resonances), creatine/phosphocreatine, choline/carnitine, taurine, and histidine with good intraindividual reproducibility. Pronounced intraindividual differences in the lipid range were found between different upper thigh muscle groups. On pathologic conditions like myopathia, myositis or irradiation damage the spectral lipid content was increased. Three months after local irradiation of the medial vastus muscle (50 Gy), the localized 1H NMR spectrum showed a complete loss of the choline and creatine signals. In a case of M. Behçet with muscular involvement the relative reduction of the choline signal may provide an insight in the pathobiochemistry. The results of our investigations in nine healthy volunteers and three patients are presented in detail including relaxation times of the metabolites.

Expression of various isoforms of neural cell adhesive molecules and their highly polysialylated counterparts in diseased human muscles

Antibodies directed against neural cell adhesive molecules (NCAM) and in particular a monoclonal antibody recognizing polysialylated isoforms, were used to characterize the expression of these molecules in normal and diseased human muscles. Normal subjects as well as patients with inflammatory, dystrophic and denervating diseases were examined. By immunohistochemistry the main observations were (1) satellite cells expressed the non-sialylated form of NCAMs; (2) regenerative fibers strikingly expressed NCAMs and their sialylated isoforms both on membranes and in the cytoplasm; (3) in denervated muscles, fibers in atrophic groups and some fibers in acute denervation expressed NCAMs on their membrane but not the highly sialylated form; (4) finally, some fibers in myotonic dystrophy and fibers with rimmed vacuoles also expressed NCAMs. Biochemical approaches, using enzymes such as endoglycosidase N and phosphatidylinositol phospholipase C combined with immunoblot analysis allowed visualization of the nature of the expressed isoforms. We have shown that non activated cells, i.e. satellite cells and denervated fibers do not express polysialylated NCAMs. This post-translational modification may be only observed in activated or regenerating fibers. This would parallel the sequence of NCAM expression occurring in normal myogenic pathways.

Enhancement of K+ conductance improves in vitro the contraction force of skeletal muscle in hypokalemic periodic paralysis

An abnormal ratio between Na+ and K+ conductances seems to be the cause for the depolarization and paralysis of skeletal muscle in primary hypokalemic periodic paralysis. Recently we have shown that the "K+ channel opener" cromakalim hyperpolarizes mammalian skeletal muscle fibers. Now we have studied the effects of this drug on the twitch force of muscle biopsies from normal and diseased human skeletal muscle. Cromakalim had little effect on the twitch force of normal muscle whereas it strongly improved the contraction force of fibers from patients suffering from hypokalemic periodic paralysis. Recordings of intracellular K+ and Cl- activities in human muscle and isolated rat soleus muscle support the view that cromakalim enhances the membrane K+ conductance (gK+). These data indicate that "K+ channel openers" may have a beneficial effect in primary hypokalemic periodic paralysis.

The effect of xamoterol in failing human myocardium

As the dual pharmacological action of partial beta 1-adrenoceptor agonists should improve left ventricular function while also protecting the myocardium against excessive sympathetic stimulation they may be useful in the treatment of heart failure. Therefore, we studied the pharmacological effects of xamoterol (Corwin, ICI 118, 587), a compound with mixed agonist and antagonistic properties at cardiac beta-adrenoceptors in electrically driven human papillary muscle strips from failing human hearts. Specimens were obtained from patients with different grades of myocardial failure who underwent mitral valve replacement (NYHA II-III) or heart transplantation (NYHA IV). Xamoterol (0.0001-100 mumol l-1) produced only negative inotropic effects, as measured by changes in isometric force of contraction in diseased human papillary muscle strips. However, isoprenaline (0.0001-10 mumol l-1) and ouabain (0.01-0.3 mumol l-1) enhanced force of contraction in the same hearts. Prestimulation with noradrenaline (3 mumol l-1) augmented the negative inotropic effect of xamoterol. It is concluded that xamoterol exerts primarily beta-adrenoceptor antagonistic activity in the failing human myocardium.

Expression of muscle-specific isozymes of phosphorylase and creatine kinase in human muscle fibers cultured aneurally in serum-free, hormonally/chemically enriched medium

Primary cultures of muscle cells derived from a biopsied adult human skeletal muscle were grown up to 6 weeks in a hormonally/chemically enriched serum-free medium. The expression of musclespecific isozymes of creatine kinase, glycogen phosphorylase, and phosphoglycerate mutase, indicative of muscle cell maturation, was studied after 1,4 and 6 weeks of growth. The maturation of muscle fibers cultured in serum-free medium was comparable to that achieved by muscle fibers cultured in medium containing 10% serum and supplemented with growth factors (insulin, epidermal growth factor, and fibroblastic growth factor) and was greater than that achieved in medium containing 10% serum only.Our study demonstrates that adult human muscle can be cultured aneurally for a long period of time in a serum-free medium, and that it can achieve a high degree of maturation. This study provides an important basis for investigations related to: 1.(1) assessment of the influence of individual components of the medium on human muscle maturation in culture;2.(2) studies of regulation of abnormal gene expression in diseased human muscle cultured in serum-free medium.

(Immuno) electron microscopy of mitochondrial crystals in diseased human skeletal muscle fibres

(Immuno) electron microscopy of mitochondrial crystals in diseased human skeletal muscle fibres

The use of Fura-2 to estimate myoplasmic [Ca 2+] in human skeletal muscle

Fura-2 was used to estimate myoplasmic [Ca 2+] in intact fibers and fiber segments from normal and diseased human muscles. Small muscle bundles (20–50 fibers) were loaded with the membrane-permeant form of the dye (Fura-2 AM). High-performance liquid chromatography was utilized to study the ability of these cells to hydrolyze Fura-2 AM. Immediately after the 30 min loading period, Fura-2 (the Ca 2+ indicator) was the predominant form of the dye in all preparations and the concentration within these fibers remained stable for over 4 1 2 hours. In addition, the resting myoplasmic [Ca 2+] in fiber segments from normal subjects and those susceptible to malignant hyperthermia were the same. However, halothane administration (1.5%) induced correlated increases in myoplasmic [Ca 2+] and force only in fibers from the susceptible patients. In contrast, caffeine administration causes correlated increases in myoplasmic [Ca 2+] and force in both types of muscle, but lower concentrations were needed to do so in the fibers from the susceptible patients. The effects of halothane and caffeine were reversible. We conclude that Fura-2 can be used successfully to estimate resting levels and changes in myoplasmic [Ca 2+] in human skeletal muscle.

The plasticity of motor units in change of the activity pattern by electric stimulation--electrostimulation and its possible clinical applications

Motoneuron and muscle fibers interact on the motor unit level, whereby discharge characteristics from the neuron imposed on the muscle seem to play a major role. Within the unit all muscle fibers are biochemically homogeneous and display a high degree of plasticity under different functional demands. To distinguish the existing different units rationals are listed that classify the units by physiological and histochemical parameters. Furthermore the review summarizes the available knowledge on the importance of activity patterns--as a biological principle--involved in the control of phenotypic expression of innervated and denervated muscle. The sequelae are shown of electrical stimulation on innervated and denervated animal muscles. In extent to these findings the consequences are discussed for stimulation procedures that can be imposed on normal and diseased human muscles as a therapeutic tool.

Cromakalim, pinacidil and RP 49356 activate a tolbutamide-sensitive K+ conductance in human skeletal muscle fibres.

S. Quasthoff 1, A. Spulerl, F. Lehmann-Horn 2 and P. Grafe 1 1Deparlment of Physiology, University of Munich 2Department of Neurology, Technical University of Munich, Munich, F.R.G. Introduction Cromakalim (BRL 34915), pinacidil and RP 49356 belong to a new class of drugs characterized as "K+ channel openers" (Weston and Abbott 1987). The present study was concerned with two experimental questions. (a) Do K + channel openers influence electrophysiological parameters of human skeletal muscle? In particular, effects on diseased human muscle would be of interest and may lead to new therapeutic strategies. (b) How do these drugs activate the K+ conductance of skeletal muscle? To answer these questions we performed electrophysiological recordings from human skeletal muscle biopsies maintained in vitro (Iaizzo and Lehmann-Horn 1988; Spuler et al. 1989). Methods Fibre segments (4-6 cm long) from human skeletal muscle biopsies were placed into a perspex chamber (volume 2 ml, flow rate 6-8 ml/min) and superfused at 36~ with a Bretag solution (Bretag 1969). Intracellular -6~,,7 recordings were performed with conventional - microelectrodes. Drugs were applied via the bathing solution. - 801 -90 Results and discussion Resting membrane potentials in fibres from human skeletal muscle biopsies varied between -55 and -85 mV. However, in spite of this wide range in resting potential, an uniform effect was seen when cromakalim, pinacidil, or RP 49356 at a concentration of 100 lxM were applied via the bathing solution. The effect consisted of a membrane hyperpolarization and an increase in membrane conductance. The change in membrane potential was dependent on the difference between resting and K+ equilibrium potential. Only small effects were seen in fibres with resting potentials around -85 mV. Furthermore, current-voltage relationships were determined before and during the action of K+ channel openers. These measurements revealed a reversal potential close to the K+ equilibrium potential. All these observations indicate an increase in membrane K§ conductance. In another series of experiments, the effects of the K+ channel blocker tolbutamide were explored. It was found that tolbutamide strongly antagonized the effects of cromakalim, pinacidil and RP 49356. Fig. 1 illustrates this interaction on three different muscle fibres (upper and lower traces from a patient with myotonic dystrophy, middle trace from a patient with hypokalaemic periodic paralysis). Tolbutamide on its own had no effect on membrane potential or conductance (Spuler et al. 1989).

Histochemical features of tubular aggregates in diseased human skeletal muscle fibres

This communication presents the results obtained in tubular aggregates of 24 enzyme histochemical techniques for demonstrating activity of oxidoreductases, transferases, hydrolases and isomerases. The activity characteristics of the tubular aggregates in m. gluteus medius of 18 patients with diseases of the neuromuscular system were almost identical. A high activity of the mitochondrial enzymes, NADPH: tetrazolium oxidoreductase, NADH:tetrazolium oxidoreductase and cytochrome c oxidase, could be shown in the pathological structures, whereas the activity of the mitochondrial enzymes, glycerol-3-phosphate:menadione oxidoreductase, succinate:PMS oxidoreductase, malate:NAD + oxidoreductase and isocitrate:NAD + oxidoreductase, and the partial mitochondrial enzymes, malate:NADP + oxidoreductase and isocitrate:NADP + oxidoreductase, was very slight or even absent. There was a moderate to strong activity of the glycolytic enzymes lactate : NAD + oxidoreductase, glyceraldehyde-3-phosphate : NAD + oxidoreductase, phosphofructokinase, phosphoglucomutase and glucose phosphate isomerase. In contrast, the activity of α-glucan phosphorylase was slight. The activity of phosphogluconate : NADP + oxidoreductase, glucose-6-phosphate : NADP + oxidoreductase and 5′-nucleotidase was slight, whereas there was no activity of myosine ATPase and mitochondrial ATPase, acid phosphatase or alkaline phosphatase. The high activity of AMP-deaminase was very striking. The activity of peroxidase was moderate. Results obtained with adsorption studies point to adsorption of some of the enzymes studied to the tubular aggregates in vivo and this phenomenon very probably determined the histochemical characteristics of these structures.

Binding of Ricinus communis I lectin to developing dystrophic muscle in human fetus

In previous studies it was shown that a d-galactose-specific lectin, Ricinus communis I (RCA I), does not bind to the plasma membrane of muscle fibres from patients with Duchenne muscular dystrophy (DMD) in contrast to normal muscle. We have now studied RCA I binding to the membranes of developing human fetal muscle in fetuses at 95% risk of DMD (n = 6) and normal controls (n = 5) with a developmental range of 12–20 weeks of gestation. The results were compared to the membrane appearance with conventional ultrastructure. Binding of RCA I to the muscle basement membrane was consistently strong from the early stages of myogenesis, such as in fusing myoblasts/myocytes. RCA I binding to the plasma membrane was weak but detectable in both DMD and normal fetuses at 12–14 weeks of gestation. Both the normal and diseased condition showed an increase of RCA I labelling of the muscle plasma membrane at 15–17 weeks and strong labelling at 18–20 weeks of gestation. No difference was observed in the RCA I localization of normal and diseased human fetal muscle plasma membrane. It is concluded that (a) the plasma membrane in developing fetal muscle undergoes a maturation process between 12 and 20 weeks gestational age leading to an increase in expression of RCA I binding carbohydrate moieties; and (b) that the absence of RCA I binding glycoprotein in mature DMD muscle plasma membrane reflects a change acquired during the course of disease.

Duchenne Muscular Dystrophy Gene Expression in Normal and Diseased Human Muscle

A probe for the 5' end of the Duchenne muscular dystrophy (DMD) gene was used to study expression of the gene in normal human muscle, myogenic cell cultures, and muscle from patients with DMD. Expression was found in RNA from normal fetal muscle, adult cardiac and skeletal muscle, and cultured muscle after myoblast fusion. In DMD muscle, expression of this portion of the gene was also revealed by in situ RNA hybridization, particularly in regenerating muscle fibers.

Histochemical localization of carbonic anhydrase in normal and diseased human muscle.

A method is described for histological localization of carbonic anhydrase (CA) in sections of frozen human muscle using the rapid and inexpensive histochemical technique of Hansson. Results obtained in normal subjects indicate clearly that CA reactive fibers are of type 1. Similarly, abnormalities seen with CA in the muscle biopsy of a patient presenting with type 1 fiber hypotrophy and preponderance duplicated almost exactly those observed with the actinomyosine adenosine triphosphatase and the reduced nicotinamide adenine dinucleotide dehydrogenase reactions. Observations of grouped CA-positive muscle fibers in a case of chronic neurogenic atrophy suggest that, like other enzymes, CA expression in muscle is under neurogenic control.