Fenestrations in regenerating skeletal muscle capillaries

A new observation of fenestrae with diaphragms in the endothelial wall of regenerating skeletal muscle capillaries is described in seven-day-old wounds of the cremaster muscle of adult male guinea pigs. The fenestrations were 44 nm in diameter and contained diaphragms with a central dense structure. These findings suggest an additional route for physiological exchange mechanisms in wound capillaries.     

Fixation of human anti-actin autoantibodies on skeletal muscle fibres.

Sera from five patients with chronic aggressive hepatitis containing smooth muscle autoantibodies were tested by means of indirect immunofluorescence for their binding to isolated rabbit skeletal muscle myofibrils. In all cases, the immunofluorescent staining was sharply localized to I bands. After incubation of these sera with skeletal muscle troponin-torpomyosin complex, purified troponin or purified tropomyosin, no changes in immunofluorescent staining of myofibrils were noted. However, the staining was abolished after incubation of the sera with skeletal muscle actin. In double immunodiffusion experiments, a single precipitation line was obtained after diffusion of the sera against crude or purified actin. It is concluded that, at least for the sera examined, smooth muscle autoantibodies are anti-actin autoantibodies. The high titre of such autoantibodies and their availability in clinical immunology laboratories make them a useful tool to study actin distribution in muscular and non-muscular cells.     

Human skeletal muscle mitochondrial capacity

Under aerobic work, the oxygen consumption and major ATP production occur in the mitochondria and it is therefore a relevant question whether the in vivo rates can be accounted for by mitochondrial capacities measured in vitro. Mitochondria were isolated from human quadriceps muscle biopsies in yields of approximately 45%. The tissue content of total creatine, mitochondrial protein and different cytochromes was estimated. A number of activities were measured in functional assays of the mitochondria: pyruvate, ketoglutarate, glutamate and succinate dehydrogenases, palmitoyl-carnitine respiration, cytochrome oxidase, the respiratory chain and the ATP synthesis. The activities involved in carbohydrate oxidation could account for in vivo oxygen uptakes of 15-16 mmol O2 min-1 kg-1 or slightly above the value measured at maximal work rates in the knee-extensor model of Saltin and co-workers, i.e. without limitation from the cardiac output. This probably indicates that the maximal oxygen consumption of the muscle is limited by the mitochondrial capacities. The in vitro activities of fatty acid oxidation corresponded to only 39% of those of carbohydrate oxidation. The maximal rate of free energy production from aerobic metabolism of glycogen was calculated from the mitochondrial activities and estimates of the DeltaG or ATP hydrolysis and the efficiency of the actin-myosin reaction. The resultant value was 20 W kg-1 or approximately 70% of the maximal in vivo work rates of which 10-20% probably are sustained by the anaerobic ATP production. The lack of aerobic in vitro ATP synthesis might reflect termination of some critical interplay between cytoplasm and mitochondria.     

Visualization of capillaries in skeletal muscle by the ATPase reaction

Summary A simple and reliable technique for the visualization of capillaries in skeletal muscle of dogs, guinea pigs and rats is described. 10–20 μm frozen sections were incubated in a medium containing Ca²⁺ and ATP following acid preincubation (pH 3.8–4.2). The capillaries stained in black and were readily seen surrounding the muscle fibers. Serial sections were also treated for alkaline phosphatase. Values for capillary density using both methods were not different. Supported by USPHS NIH Grant HL 12679-HL 18145 Supported by a fellowship from the Rockefeller Fndn     

Exercise-induced metallothionein expression in human skeletal muscle fibres

Exercise induces free oxygen radicals that cause oxidative stress, and metallothioneins (MTs) are increased in states of oxidative stress and possess anti-apoptotic effects. We therefore studied expression of the antioxidant factors metallothionein I and II (MT-I + II) in muscle biopsies obtained in response to 3 h of bicycle exercise performed by healthy men and in resting controls. Both MT-I + II proteins and MT-II mRNA expression increased significantly in both type I and II muscle fibres after exercise. Moreover, 24 h after exercise the levels of MT-II mRNA and MT-I + II proteins were still highly increased and the MT-II mRNA expression reached a 15-fold increase. As expected, immunohistochemical detection of malondialdehyde (MDA) and nitrotyrosine (NITT) showed that formation of free radicals and oxidative stress were clearly increased in exercising muscle peaking shortly after the end of exercise in both type I and II muscle fibres. This is the first report demonstrating that MT-I + II are significantly induced in human skeletal muscle fibres following exercise. As MT-I + II are antioxidant factors that protect various tissues during pathological conditions, the MT-I + II increases post exercise may represent a mechanism whereby contracting muscle fibres are protected against cellular stress and injury.     

Sarcolemmal ion channels in dystrophin-deficient skeletal muscle fibres

Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene and characterized by progressive skeletal muscle degeneration. A current hypothesis suggests that degeneration of dystrophin-deficient skeletal muscle results from a chronic intracellular Ca²⁺ overload. Ca²⁺ handling in skeletal muscle is tightly controlled by the membrane potential which is set by sarcolemmal ion channels activity. Also, with regard to the subsarcolemmal localization of dystrophin, it is reasonable to enquire if the distribution and function of ion channels might be affected by the absence of dystrophin. This paper briefly summarizes the current knowledge of the properties of sarcolemmal ion channels in fully differentiated dystrophin-deficient skeletal muscle fibres.     

"Giant" muscle fibres in skeletal muscle of normal pigs

Observations made during a growth and development study of the semitendinosus and trapezius muscles of 49 purebred Large White pigs between birth and 128 days of age revealed the presence of giant fibres. The occurrence, histochemical and ultrastructural properties of these giant fibres were investigated. A high proportion of the pigs (85 per cent) contained giant fibres in their muscles but these giant fibres usually represented less than 1 per cent of the total myofibre population. Giant fibres possessed enhanced adenosine triphosphatase activity and a high capacity for oxidative metabolism (indicated by succinate dehydrogenase activity) which was reflected ultrastructurally by the greatly heightened electron density of myofibrils and by an abnormally high percentage of mitochondria and lipid droplets. These deviations from normal muscle fibre composition, together with the reduced percentage volume of sarcoplasmic reticulum, were consistent with changes seen in functionally over-loaded muscle. It appears that giant fibre anomalies occur through increased activity stimulated in occasional muscle fibres, perhaps by a structural defect, such as an inadequate amount of sarcoplasmic reticulum, which causes hyper-contractile activity within the fibres and associated compensatory adaptations. Giant fibres did not appear to represent fibres undergoing degenerative changes.     

Induction of mitochondrial alterations ex vivo in skeletal muscle.

The ultrastructural alterations of mitochondria were studied, following a fixation delay of 45 min, in the normal type II red muscle fibres of pigeon semimetapatagialis muscle. These muscle fibres, were devoid of myofibrillar degeneration; however, their mitochondria showed derangement. The latter included irregular orientation and loss of cristae, and clearing of the matrix. Some mitochondria showed myelin figures of varying complexity in the subsarcolemmal areas as well as in the interior of muscle fibres. These alterations bear striking resemblance to that seen during some lysosomal formation. The significance of these observations is discussed.     

Relative volume of functioning capillaries in skeletal muscle in regional hypotension

Experiments on rats showed that 1 month after lowering the pressure in the blood vessels in the posterior half of the body by constricting the abdominal aorta the number of functioning capillaries in the gastrocnemius and soleus muscles was virtually the same as in the control. This suggests that a decrease in the hydraulic resistance of the resistive vessels in a region of chronic local arterial hypotension is not attributable to an increase in the number of simultaneously functioning vessels.Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Czechoslovakia. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 156–157, August, 1977.     

Development and composition of skeletal muscle fibres in mouse oesophagus

The development of skeletal muscle in mouse oesophagus was investigated by studying the expression of skeletal muscle type myosin heavy chain (MHC), troponin I (TnI) and tropoinin T (TnT) using immunocytochemical and immunoblotting procedures. Both slow and fast muscle fibres were first detected in outer layer muscularis externa of cranial oesophagus at 14 days gestation. The fast MHC was present in all skeletal muscle fibres of oesophagus while the slow MHC was restricted to only a subset of myotubes during foetal development, indicating that slow and fast fibres emerged during early stages of myogenesis. A small number of cells expressed both slow and fast MHCs in the caudal region of adult mouse oesophagus, suggesting that some muscle fibres did not differentiate fully even in the adult. The conversion of some muscle fibre types, from slow to fast, was apparent during postnatal development. This was indicated by a gradual reduction in the number of slow MHC positive fibres during postnatal growth. The complete suppression of slow MHC was observed in cranial oesophagus by 4 weeks of age. However, the persistence of some slow MHC in the caudal oesophagus was apparent even in the adult. The conversion of muscle fibres from slow to fast type was also evidenced by immunoblotting study of fast and slow TnI. The expression level of slow TnI decreased while that of fast TnI increased during neonatal growth period. Compared with the limb skeletal muscles, the onset of the adult fast TnT isoform expression was delayed in mouse oesophagus and its developmental isoforms were not completely suppressed in the adult, although their expression level was reduced.     

Effects of diethyl-stilboestrol on single fibres of frog skeletal muscle.

The effects of diethyl-stilboestrol (DES), one of the most potent estrogens, were studied on single muscle fibres of the frog. In relatively low concentrations (5 muM), DES greatly potentiates the twitch response of the fibre without significantly affecting the amplitude of the tetanus response. The twitch potentiation is accompanied by an increase in time to peak tension and marked prolongation of the relaxation phase. In tetanic response the half decay time after the last stimulus is also prolonged by DES. DES causes no changes in the resting or action potential of the muscle fibre. The S-shaped curve relating peak contracture tension and caffeine concentration is shifted towards lower caffeine concentrations by DES. Dantrolene greatly suppresses the DES potentiated twitch. It is concluded that DES potentiates the twitch response and prolongs the relaxation time by inhibition of the calcium re-uptake by the sarcoplasmic reticulum.     

Charge conservation in intact frog skeletal muscle fibres in gluconate-containing solutions.

1. The conservation of intramembrane charge was investigated in intact voltage-clamped frog skeletal muscle fibres under conditions that minimized time-dependent ionic currents and so facilitated precise determination of capacitative charge. 2. Prolonged (q gamma) transients were demonstrated in 3,4-diaminopyridine and tetraethyl-ammonium gluconate-containing low [Ca2+] solutions in response to 125 ms pulses that explored the voltage range -90 to -20 mV. The tetracaine-sensitive, q gamma, component then accounted for a significant proportion (over 50%) of available charge. 3. Both delayed 'on' q gamma currents and 'off' current tails decayed to steady direct current (DC) baselines without significant residual ionic current slopes in the chosen extracellular solutions. This suggested that the current transients represented capacitative decays. It was also compatible with the precise determination of effective charge by integration. 4. The advent of 'on' q gamma current was accompanied by increased 'off' charge. Thus, charge was conserved through all 'on' and 'off' steps and through test voltages that extended from the threshold appearance of q gamma as a slow transient to its full merger with the earlier q beta decay at stronger depolarizations. 5. Charge conservation persisted through a wide range of 'on' pulse durations between 60 and 370 ms and was therefore independent of the interval following the q gamma decay. 6. The quantity of q gamma charge remained a monotonic single-valued function of test voltage, whether this potential was reached directly from the -90 mV holding potential or following a prepulse to -10 mV. 7. These findings suggest that the q gamma charge movement represents the electrical signature of an intramembrane entity whose transitions are primarily driven by, and therefore conserved with, the steady-state potential.     

Resistance to fatigue of individual Xenopus single skeletal muscle fibres is correlated with mitochondrial volume density

The purpose of the present study was to compare the individual fatigue characteristics of isolated single skeletal muscle fibres with their mitochondrial volume density (MVD), using direct histological morphometry. Single muscle fibres (n= 14) were microdissected from lumbrical muscle of adult female Xenopus laevis, and force was measured while fibres were stimulated (tetanic contractions of 200 ms trains with 70 Hz stimuli at 9 V) at progressively increasing frequencies (2 min each at 0.25, 0.33, 0.5 and 1 contractions s(-1)) until fatigue (<50% initial maximal force) had been established. Following the end of the fatigue protocol, MVD was determined by electron microscopy. Time to fatigue varied among the individual fibres from 3.3 to 10 min. MVD of individual fibres ranged from 3.0 to 9.2% and was positively correlated (r= 0.93) with time to fatigue of corresponding fibres. These results, using direct histological measurements of MVD: (1) support on a single cell basis the notion that oxidative capacity is a major determinant of muscle fatigue resistance; and (2) show that the fatigue profile of a single cell can be used to predict oxidative capacity.     

Slow recovery of force in single skeletal muscle fibres

After a bout of intense exercise, especially in untrained persons, recovery of muscle force is often slow. Force depression is much more marked at low frequencies of stimulation than at high frequencies (recovery is also seen in single muscle fibres from frog and mouse after fatigue induced by repeated, brief contractions. Evidence from our own and other laboratories indicates that the impairment is unlikely to result from metabolic changes and points to a defect in excitation–contraction coupling. We demonstrate that the likely site of failure is in the coupling between t-tubule depolarization and release of Ca²⁺ from the SR. The causative agent appears to be a localized increase in cytoplasmic Ca²⁺ which initiates some disruptive process, which can, however, be fully reversed, albeit slowly. Our experimental evidence does not support the involvement of Ca²⁺-activated proteases. Attempts to clarify the possible role of Ca²⁺-activated lipases (phospholipase A₂) and Ca²⁺/calmodulin have been hampered by side-effects of available inhibitors. Efforts to clarify how Ca²⁺ exerts its effects are continuing.