Shape and disposition of clefts, tubules, and sarcoplasmic reticulum in long and short sarcomere fibers of crab and crayfish.

The disposition of surface invaginations (clefts, Z and T tubules) and of the sarcoplasmic reticulum has been examined by electron microscopy at three accelerating voltages (100, 200 and 1000 kV) and by phase-contrast light microscopy in crustacean muscles infiltrated by the "Golgi stain." In long-sarcomere, tonic type fibers, an extensive system of invaginating clefts has been observed, along with both Z and T tubules. Z and T tubules form interconnections with each other, but only T tubules form specific contacts with the sarcoplasmic reticulum, which in these fibers forms an extended and continuously fenestrated network. In short-sarcomere, phasic type fibers, a ladder-like disposition of an abundant T network is found. Z tubules are absent in these fibers. The sarcoplasmic reticulum forms more frequent junctions with flattened areas of T tubules and with clefts, but has less extensive free surfaces than in the long-sarcomere fibers.

Potentially fatal cardiac dysrhythmia and hyperkalemic periodic paralysis.

An 11-year-old boy was evaluated for mild periodic muscular weakness exacerbated on separate occasions by disopyramide phosphate and procainamide. He and his mother both had bidirectional ventricular tachydysrhythmia (BVT), short stature, microcephaly, and clinodactyly. The mother, but not the child, had lingual myotonia. The two antiarrhythmic drugs worsened the muscular weakness without benefiting the cardiac dysrhythmia. Potassium loading produced skeletal muscle weakness and transient conversion of the BVT to normal sinus rhythm. Hypokalemia aggravated the BVT without causing weakness. Acetazolamide had no effect. The patient suffered a nonfatal cardiac arrest after several days of increased carbohydrate intake. Imipramine controlled the dysrhythmia without inducing weakness. Periodic paralysis should be considered as the diagnosis in children with BVT, a potentially fatal condition.

Benign infantile mitochondrial myopathy due to reversible cytochrome c oxidase deficiency.

A 2-week-old boy had profound generalized weakness, hypotonia, hyporeflexia, macroglossia, and severe lactic acidosis. The infant improved spontaneously: he held his head at 4 1/2 months, rolled over at 7 months, and walked by 16 months. At 33 months of age, he had mild proximal weakness. Macroglossia disappeared by age 4 months. Blood lactic acid declined steadily and was normal by 14 months of age. Histochemical and ultrastructural studies of muscle biopsy specimens obtained at 1 and 7 months of age showed excessive mitochondria, lipid, and glycogen; a third biopsy at age 36 months showed only atrophy of scattered fibers. Cytochrome c oxidase stain was positive in fewer than 5% of fibers in the first biopsy, in approximately 60% of fibers in the second biopsy, and in all fibers in the third biopsy. Biochemical analysis showed an isolated defect of cytochrome c oxidase activity, which was only 8% of the lowest control level in the first biopsy; the activity increased to 47% in the second biopsy and was higher than normal in the third. In contrast to that in the fatal infantile form of cytochrome c oxidase deficiency, the enzyme defect in this condition is reversible. The biochemical basis for this difference remains to be explained.

Structure of membranes in crayfish muscle: comparison of phasic and tonic fibres.

Membranes of two crayfish muscles with different contraction speeds were studied with freeze-fracture replicas and thin sections. A fast-contracting, short sarcomere phasic muscle, the tail flexor, and a slowly contracting long sarcomere tonic muscle, the carpopodite flexor, were chosen for this study. Membranes examined included the plasmalemma, clefts, T-system, Z-tubules and sarcoplasmic reticulum (SR). We found distinct differences in the distribution of these membranes: T-system and clefts are more elaborate in the tail flexor, while SR is more extensive in the leg flexor. The tail flexor apparently lacks Z-tubules. These differences were more obvious in freeze-fracture replicas than in thin sections. In freeze-fracture replicas, both junctional and non-junctional T-tubule membranes can be distinguished from Z-tubules by content of intramembranous particles. The junctional regions of T-system and surface membranes contain large (10-11 nm) intramembranous particles that are absent from non-junctional parts of these membranes. There is also a class of particles on the junctional SR fracture faces that differs from intramembranous particles on non-junctional SR. These junctional specialization are similar in long and short sarcomere fibres.

Release of creatine kinase and lactate dehydrogenase from frog skeletal muscles treated with imipramine.

Frog sartorius and extensor longus digiti IV ("toe") muscles were mounted in sample collecting units, isometric tension was recorded and the activities of creatine kinase and lactate dehydrogenase released into the bathing medium were measured. Addition of imipramine (greater than 0.2 mM) to the control medium induced muscle contractures and increased the rates of enzyme release. Electronmicrographs of muscles exposed to imipramine (0.5--1.0 mM) showed fibers in which the extracellular marked horseradish peroxidase was detected in the sarcoplasm. Both the enzyme-releasing and the contractile effects of imipramine were inhibited by procaine (but not by lidocaine) and were enhanced by replacement of external chloride ions with propionate. The data are consistent with the suggestion that events associated with tension development contribute greatly to the imipramine-induced loss of sarcoplasmic enzymes from frog skeletal muscles. The elevated Ca++ concentration in the sarcoplasm during the imipramine-induced contracture is thought to lead to changes in the plasma membrane that account for the increased enzyme loss and for the penetration of horseradish peroxidase into the sarcoplasm.

Calcium accumulation by the sarcoplasmic reticulum in two populations of chemically skinned human muscle fibers. Effects of calcium and cyclic AMP.

In previous efforts to characterize sarcoplasmic reticulum function in human muscles, it has not been possible to distinguish the relative contributions of fast-twitch and slow-twitch fibers. In this study, we have used light scattering and 45Ca to monitor Ca accumulation by the sarcoplasmic reticulum of isolated, chemically skinned human muscle fibers in the presence and absence of oxalate. Oxalate (5 mM) increased the capacity for Ca accumulation by a factor of 35 and made it possible to assess both rate of Ca uptake and relative sarcoplasmic reticulum volume in individual fibers. At a fixed ionized Ca concentration, the rate and maximal capacity (an index of sarcoplasmic reticulum volume) both varied over a wide range, but fibers fell into two distinct groups (fast and slow). Between the two groups, there was a 2- to 2.5-fold difference in oxalate-supported Ca uptake rates, but no difference in average sarcoplasmic reticulum volumes. Intrinsic differences in sarcoplasmic reticulum function (Vmax, K0.5, and n) were sought to account for the distinction between fast and slow groups. In both groups, rate of Ca accumulation increased sigmoidally as [Ca++] was increased from 0.1 to 1 microM. Apparent affinities for Ca++ (K0.5) were similar in the two groups, but slow fibers had a lower Vmax and larger n values. Slow fibers also differed from fast fibers in responding with enhanced Ca uptake upon addition of cyclic AMP (10(-6) M, alone or with protein kinase). Acceleration by cyclic AMP was adequate to account for adrenaline-induced increases in relaxation rates previously observed in human muscles containing mixtures in fast-twitch and slow-twitch fibers.

Release of sarcoplasmic enzymes from frog skeletal muscle.

Isolated, intact frog muscles bathed in control saline release creatine kinase (CK) and lactate dehydrogenase (LDH) at constant rates for several hours. The basal rates of release from "toe" muscles (CK 0.087%/min; LDH 0.105%/min) were one order of magnitude greater than those from semitendinosus muscles. This is attributed to differences in muscle mass and geometry, and to the smaller diameter of toe muscle fibers. Enzyme release rates were not affected by Na-free or Cl-free solutions, whereas LDH release rate doubled during exposure to Ca-free (EGTA-containing) saline or in the presence of isosmotic solutions containing 120 mM KCl or potassium propionate. Following mechanical injury or detergent treatment (Brij 58), the enzyme release rates into Ca-free medium reached peak values 4 and 16 times (toe muscle), and 16 and 20-30 times (semitendinosus), respectively, the control rates. The greater effect of detergent treatment is ascribed to a larger area of sarcolemmal damage plus possible changes in the state of the enzymes in the sarcoplasm.

Functional heterogeneity of the sarcoplasmic reticulum within sarcomeres of skinned muscle fibers.

Precipitation of Ca oxalate in the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers caused an increase in light scattering which was proportional to the amount of Ca accumulated per unit fiber volume. The increase in scattering was used to measure net accumulation rates and steady-state Ca capacities of the sarcoplasmic reticulum in single fibers. The data obtained were qualitatively and quantitatively similar to those reported for isolated vesicle preparations. Under conditions in which Ca was not depleted from the medium, Ca accumulation was linear with time over much of its course. Steady-state capacities were independent of the Ca concentration; uptake rates were half-maximal at 0.5 microM Ca++ and saturated above about 1.0 microM. Both rate and capacity varied with the oxalate concentration, being maximal at oxalate concentrations greater than or equal to mM and decreasing in proportion to one another at lower concentrations, with a threshold near 0.25 mM. At the lower loads, electron micrographs showed many sarcoplasmic reticulum elements empty of precipitate alongside others that were full, whereas virtually all were filled in maximally loaded fibers. These data indicate that the Ca oxalate capacity of each fiber varies with the number and volume of elements in which Ca oxalate crystals can form at a given oxalate concentration, and that individual regions of the sarcoplasmic reticulum within each sarcomere differ in their ability to support Ca oxalate precipitation. Our working hypothesis is that this range in ability to form Ca oxalate crystals involves differences in ability to accumulate and retain ionized Ca inside the sarcoplasmic reticulum.

Isolation of influenza virus from muscle in myoglobinuric polymyositis.

We report the first isolation of influenza virus from muscle in a man with myoglobinuria and acute polymyositis. Influenza virus was isolated from cultures of Madin Darby bovine kidney and primary rhesus monkey kidney cells inoculated with muscle homogenates in the presence of trypsin; the virus was identified by neutralization and hemagglutination inhibition studies using influenza B/Lee antiserum. Viral plaque assay was performed with Madin Darby canine cells. Viral antigen was also detected by specific immunofluorescence in muscle, and myxovirus-like particles were seen in subsarcolemmal vacuoles by electronmicroscopy. The pathologic findings were similar to those of childhood dermatomyositis, except for a large proportion of necrotic muscle fibers. The evidence suggests that the pathogenesis of influenzal polymyositis in this patient involved direct viral infection of muscle.

Debrancher deficiency: neuromuscular disorder in 5 adults.

Five patients, 4 men and 1 woman, had adult-onset and slowly progressive weakness. There was distal wasting in 2, hepatomegaly in 3, and congestive heart failure in 2. Electromyography showed a mixed pattern with abundant fibrillations. Serum creatine phosphokinase was increased 5- to 45-fold. Blood glucose failed to respond to epinephrine or glucagon, and venous lactate did not rise after ischemic exercise. Muscle biopsy showed vacuolar myopathy affecting both fiber types. By electron microscopy the vacuoles corresponded to large pools of glycogen not limited by a membrane. Glycogen concentration was 3 to 5 times normal in muscle and 7 to 21 times normal in erythrocytes. In the presence of iodine, muscle glycogen showed a spectrum characteristic of phosphorylase-limit-dextrin. Debrancher activity was measured by a spectrophotometric assay and by a radioactive reverse reaction. The activity was lacking in muscle and erythrocytes of 4 patients according to both assays; in 1 patient the reverse reaction was not impaired. Though previously reported in only 5 patients, debrancher deficiency myopathy may not be rare and should be considered in the differential diagnosis of adult-onset hereditary myopathies.

Chemically skinned mammalian skeletal muscle. I. The structure of skinned rabbit psoas.

We studied the morphology of rabbit psoas muscle fixed at increasing intervals of time in a chemical skinning solution (Wood et al., 1975), or after skinning and storage for times up to 1 week. The storage solution, in which the chemically skinned muscled fibers were kept at -20 degrees C, had the same ionic composition as the skinning solution but was made with 50% (v/v) glycerol. Progressive structural changes occurred in fibers exposed to skinning solution. The structural changes were essentially complete after 24-48 hr in skinning solution and no further changes were detected in fibers stored for periods up to 1 week. Structural changes were: (i) holes or gaps in the plasma membrane; (ii) swelling of mitochondria and disorganization of their internal structure; (iii) slight swelling of the sarcoplasmic reticulum; (iv) disappearance of sarcoplasmic reticulum (SR) feet from triadic gaps. Other changes included loss of glycogen between fibrils and extraction of myoplasm, or the change of its staining properties. All architectural elements of the SR, except "feet", remained during skinning and storage, and the SR remained able to accumulate calcium. The morphology of the myofilaments during chemical skinning and during storage did not differ from control fibers. We conclude that chemical skinning alters the gross structure of the plasma membrane and mitochondria, but produces minimal changes in the sarcoplasmic reticulum and contractile proteins.

Duchenne dystrophy: abnormal generation of tension and Ca++ regulation in single skinned fibers.

Skinned, single-fiber preparations from the quadriceps or gastrocneumius muscles of four ambulatory male children with Duchenne dystrophy were tested for theri ability to generate tension and to regulate CA++. To determine the intrinsic strength (P0) of the contractile material, the maximum Ca++ -activated tensions were normalized to the fiber diameters. Sixty-four percent of the Duchenne fibers had P0 values below 1.0 kg per square centimeter--the lowest value observed in control muscle--and the average P0 values of fibers from each Duchenne biopsy were significantly (p less than 0.01) below the average P0 values for control muscle fibers and for muscle fibers obtained from one obligatory carrier of the Duchenne gene. The low tensions in the Duchenne muscle fibers could not be ascribed to altered Ca++ regulation or to substrate sensitivity of the contractile proteins in the fibers, since these were normal. However, ultrastructural abnormalities of the myofilaments, which might reduce the ability of the contractile system to develop tension, were observed. Furthermore, Ca++ regulation by the sarcoplasmic reticulum (SR) was impaired in most of those muscle fibers, from both carriers and Duchenne patients, that did develop normal tension. These results suggest that in Duchenne muscle a functional disorder in the SR may precede loss of the ability of the contractile proteins to generate tension. However, since muscle fibers from Duchenne-gene carriers developed significantly greater tensions than fibers from Duchenne-patients, while yet having similar defects in Ca++ regulation, the SR disorder may not be exclusively responsible for abnormal contractile protein function.

Unusual thick and thin filament packing in a crustacean muscle.

The proximal accessory flexor (PAF) of the myochordotonal organ (MCO) in the meropodite of crayfish walking legs contains two populations of muscle fibers which are distinguishable by their diameters. The large accessory (LA) fibers are 40-80 micrometer in diam and are similar in ultrastructure to other slow crustacean fibers. The small accessory (SA) fibers are 1-12 micrometer in diam and have a unique myofilament distribution at normal body lengths. There is extensive double overlap of thin filaments at these lengths, and some of them form bundles that may extend the length of the sarcomere. In the middle of the sarcomeres, thick and thin filaments are totally segregated from each other. When the fibers are stretched to lengths beyond double overlap length, the myofilament patterns are conventional. The segregated pattern is reestablished when stretched fibers are allowed to shorten passively. The length-tension relationship of the SA fibers is described by a linear ascending branch, a plateau, and a linear descending branch. The ascending branch encompasses normal body lengths from slack length (Ls) with maximum double overlap to the length at which double overlap ceases (1.8 X Ls). The descending phase is comparable to that of other skeletal muscles. That is, tension decreases in proportion with the reduction in thick-thin filament interdigitation (2 X Ls to 3 X Ls).

Sites of action of D2O in intact and skinned crayfish muscle fibers.

The effect on tension development of replacing 90% of the H2O of the bathing saline with D2O was studied on intact single fibers, and on skinned fibers before and after the latter were treated so as to eliminate Ca-accumulation by the sarcoplasmic reticulum (SR). Excitation-contraction coupling (ECC) of intact fibers is not abolished, but is depressed by D2O so that higher depolarizations are required to elicit a given tension. The reduction in tension at a given level of depolarization is not due to inhibition of the contractile system. The latter showed an enhanced Ca sensitivity; that is, skinned fibers respond to Ca concentrations that are 1-2 orders of magnitude smaller in D2O than in H2O saline. When bathed in D2O saline, intact fibers or skinned fibers with functional SR can still accumulate and release Ca in sufficient quantities to allow repeated induction of maximum tensions. Relaxation is slowed in all three types of preparation, perhaps because of an increased affinity of troponin to Ca in D2O salines.