Myosin types in human skeletal muscle fibers

By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Myosin types in human skeletal muscle fibers

Summary By combining enzyme histochemistry for fiber typing with immunohistochemistry for slow and fast myosin a correlation between fiber type and myosin type was sought in human skeletal muscle. Fiber typing was done by staining for myofibrillar ATPases after preincubation at discriminating pH values. Myosin types were discriminated using type specific anti-rabbit myosin antibodies shown to cross-react with human myosin and were visualized by a protein A-peroxidase method. Type I fibers were shown to contain slow myosin only, type IIA and IIB fibers fast myosin only, and type IIC fibers both myosins in various proportions. When muscle biopsies from well-trained athletes were investigated essentially the same staining pattern was observed. However, rarely occurring type I fibers with high glycolytic activity were detected containing additional small amounts of fast myosin and occasional type IIA fibers had small amounts of slow myosin. Based on the observation of various fiber types in which slow and fast myosin coexist we propose a dynamic continuum of fibers encompassing all fiber types.     

Influence of spaceflight on rat skeletal muscle

The size, succinate dehydrogenase (SDH) and alpha-glycerolphosphate dehydrogenase (GPD) activities, and alkaline myofibrillar adenosinetriphosphatase (ATPase) staining properties were determined from quantitative histochemical analyses of single fibers from five hindlimb muscles of six male rats exposed to a 7-day National Aeronautics and Space Administration spaceflight mission (SL-3). These same properties were determined in a group of ground-based control rats housed under simulated environmental conditions. The wet weight of each of the flight muscles was significantly reduced relative to control. However, the loss of mass varied from 36% in the soleus to 15% in the extensor digitorum longus. The cross-sectional areas of fibers in the flight muscles also were reduced, except for the dark ATPase fibers in the medial gastrocnemius. The greatest relative fiber atrophy occurred in the muscles with the highest proportion of light ATPase fibers. An increase in the percentage of dark ATPase fibers also was observed in flight muscles with a predominance of light ATPase fibers. Also, there was an increase in the biochemically determined myofibrillar ATPase activity of tissue sections of the flight soleus. No changes in histochemical or biochemical measures of ATPase activity were observed in the flight extensor digitorum longus. In general, the SDH activity of flight muscles was maintained, whereas GPD activity either was maintained or increased. Based on a metabolic profile of ATPase, SDH, and GPD, there was an increase in the proportion of fast oxidative-glycolytic fibers in some muscles.     

Glucose transporter expression in human skeletal muscle fibers

The present study was initiated to investigate GLUT-1 through -5 expression in developing and mature human skeletal muscle. To bypass the problems inherent in techniques using tissue homogenates, we applied an immunocytochemical approach, employing the sensitive enhanced tyramide signal amplification (TSA) technique to detect the localization of glucose transporter expression in human skeletal muscle. We found expression of GLUT-1, GLUT-3, and GLUT-4 in developing human muscle fibers showing a distinct expression pattern. 1) GLUT-1 is expressed in human skeletal muscle cells during gestation, but its expression is markedly reduced around birth and is further reduced to undetectable levels within the first year of life; 2) GLUT-3 protein expression appears at 18 wk of gestation and disappears after birth; and 3) GLUT-4 protein is diffusely expressed in muscle cells throughout gestation, whereas after birth, the characteristic subcellular localization is as seen in adult muscle fibers. Our results show that GLUT-1, GLUT-3, and GLUT-4 seem to be of importance during muscle fiber growth and development. GLUT-5 protein was undetectable in fetal and adult skeletal muscle fibers. In adult muscle fibers, only GLUT-4 was expressed at significant levels. GLUT-1 immunoreactivity was below the detection limit in muscle fibers, indicating that this glucose transporter is of minor importance for muscle glucose supply. Thus we hypothesize that GLUT-4 also mediates basal glucose transport in muscle fibers, possibly through constant exposure to tonal contraction and basal insulin levels.     

Adaptations of skeletal muscle grafts to chronic changes of physical activity

After grafting, many structural and functional characteristics of skeletal muscle change with time until reaching a stable value. For several characteristics, these stable values are less than those observed in control skeletal muscle. Characteristics such as mass and fiber cross-sectional area are influenced by chronic changes in physical activity. The extent of our understanding of the dimensions and mechanisms of activity-induced adaptations of grafts is limited and based solely on experiments with rats. Improvements in mass, protein content, oxidative capacity, and glycogen concentration have been documented with conditioning by running of sufficient intensity and duration. The growth and development of soleus muscle grafts are severely impaired when normal weight-bearing activity is removed. The mass and maximum tension development of grafts are increased with ablation of muscles that function synergistically to the grafts, but are diminished after chronic electrical stimulation. Chronic electrical stimulation does increase oxidative capacity, capillarity, and the resistance to fatigue. Much remains to be learned about the mechanisms by which activity-related adaptations occur in skeletal muscle grafts.     

Endocytosis in skeletal muscle fibers

Defining the organization of endocytic pathway in multinucleated skeletal myofibers is crucial to understand the routing of membrane proteins, such as receptors and glucose transporters, through this system. Here we analyzed the organization of the endocytic trafficking pathways in isolated rat myofibers. We found that sarcolemmal-coated pits and transferrin receptors were concentrated in the I band areas. Fluid phase markers were taken up into vesicles in the same areas along the whole length of the fibers and were then delivered into structures around and between the nuclei. These markers also accumulated beneath the neuromuscular and myotendinous junctions. The recycling compartment, labeled with transferrin, appeared as perinuclear and interfibrillar dots that partially colocalized with the GLUT4 compartment. Low-density lipoprotein, a marker of the lysosome-directed pathway, was transported into sparsely distributed perinuclear and interfibrillar dots that contacted microtubules. A majority of these dots did not colocalize with internalized transferrin, indicating that the recycling and the lysosome-directed pathways were distinct. In conclusion, the I band areas were active in endocytosis along the whole length of the multinucleated myofibers. The sorting endosomes distributed in a cross-striated fashion while the recycling and late endosomal compartments showed perinuclear and interfibrillar localizations and followed the course of microtubules.     

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

The biological mechanisms underlying decline in muscle power and fatigue with age are not completely understood. The contribution of alterations in the excitation-calcium release coupling in single muscle fibers was explored in this work. Single muscle fibers were voltage-clamped using the double Vaseline gap technique. The samples were obtained by needle biopsy of the vastus lateralis (quadriceps) from 9 young (25–35 years; 25.9 ± 9.1; 5 female and 4 male) and 11 old subjects (65–75 years; 70.5 ± 2.3; 6 f, 5 m). Data were obtained from 36 and 39 fibers from young and old subjects, respectively. Subjects included in this study had similar physical activity. Denervated and slow-twitch muscle fibers were excluded from this study. A significant reduction of maximum charge movement (Q_max) and DHP-sensitive Ca current were recorded in muscle fibers from the 65–75 group. Q_max values were 7.6 ± 0.9 and 3.2 ± 0.3 nC/F for young and old muscle fibers, respectively (P < 0.01). No evidences of charge inactivation or interconversion (charge 1 to charge 2) were found. The peak Ca current was (–)4.7 ± 0.08 and (–)2.15 ± 0.11 A/F for young and old fibers, respectively (P < 0.01). The peak calcium transient studied with mag-fura-2 (400 m) was 6.3 ± 0.4 m and 4.2 ± 0.3 m for young and old muscle fibers, respectively. Caffeine (0.5 mm) induced potentiation of the peak calcium transient in both groups. The decrease in the voltage-/ Ca-dependent Ca release ratio in old fibers (0.18 ± 0.02) compared to young fibers (0.47 ± 0.03) (P < 0.01), was recorded in the absence of sarcoplasmic reticulum calcium depletion. These data support a significant reduction of the amount of Ca available for triggering mechanical responses in aged skeletal muscle and, the reduction of Ca release is due to DHPR-ryanodine receptor uncoupling in fast-twitch fibers. These alterations can account, at least partially for the skeletal muscle function impairment associated with aging.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association, and National Institutes of Health (2-P60AG18484-06)     

Decreased thin filament density and length in human atrophic soleus muscle fibers after spaceflight.

Soleus muscle fibers were examined electron microscopically from pre- and postflight biopsies of four astronauts orbited for 17 days during the Life and Microgravity Sciences Spacelab Mission (June 1996). Myofilament density and spacing were normalized to a 2. 4-microm sarcomere length. Thick filament density ( approximately 1, 062 filaments/microm(2)) and spacing ( approximately 32.5 nm) were unchanged by spaceflight. Preflight thin filament density (2, 976/microm(2)) decreased significantly (P < 0.01) to 2,215/microm(2) in the overlap A band region as a result of a 17% filament loss and a 9% increase in short filaments. Normal fibers had 13% short thin filaments. The 26% decrease in thin filaments is consistent with preliminary findings of a 14% increase in the myosin-to-actin ratio. Lower thin filament density was calculated to increase thick-to-thin filament spacing in vivo from 17 to 23 nm. Decreased density is postulated to promote earlier cross-bridge detachment and faster contraction velocity. Atrophic fibers may be more susceptible to sarcomere reloading damage, because force per thin filament is estimated to increase by 23%.     

Calsequestrin targeting to sarcoplasmic reticulum of skeletal muscle fibers

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C₂C₁₂ myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HA24_NH2, CS-HA2D, CS-HA3D, and CS-HAHT, a double mutant of the NH₂ terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C₂C₁₂ myotubes, as judged by immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C₂C₁₂ myoblasts, and only CS-HA3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH₂ terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation. protein-protein interactions; oligomerization; intracellular sorting     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Induction of GLUT-1 protein in adult human skeletal muscle fibers

Prompted by our recent observations that GLUT-1 is expressed in fetal muscles, but not in adult muscle fibers, we decided to investigate whether GLUT-1 expression could be reactivated. We studied different stimuli concerning their ability to induce GLUT-1 expression in mature human skeletal muscle fibers. Metabolic stress (obesity, non-insulin-dependent diabetes mellitus), contractile activity (training), and conditions of de- and reinnervation (amyotrophic lateral sclerosis) could not induce GLUT-1 expression in human muscle fibers. However, regenerating muscle fibers in polymyositis expressed GLUT-1. In contrast to GLUT-1, GLUT-4 was expressed in all investigated muscle fibers. Although the significance of GLUT-1 in adult human muscle fibers appears limited, GLUT-1 may be of importance for the glucose supplies in immature and regenerating muscle.     

Adaptations in the activation of human skeletal muscle induced by short-term isometric resistance training.

This study employed longitudinal measures of evoked spinal reflex responses (Hoffman reflex, V wave) to investigate changes in the activation of muscle and to determine if there are "linked" neural adaptations in the motor pathway following isometric resistance training. Twenty healthy, sedentary males were randomly assigned to either the trained (n = 10) or control group (n = 10). The training protocol consisted of 12 sessions of isometric resistance training of the plantar flexor muscles over a 4-wk period. All subjects were tested prior to and after the 4-wk period. To estimate changes in spinal excitability, soleus Hoffman (H) reflex and M wave recruitment curves were produced at rest and during submaximal contractions. Recruitment curves were analyzed using the slope method (Hslp/Mslp). Modulation of efferent neural drive was assessed through evoked V wave responses (V/Mmax) at 50, 75, and 100% maximal voluntary contraction (MVC). After 4 weeks, MVC torque increased 20.0 +/- 13.9% (mean +/- SD) in the trained group. The increase in MVC was accompanied by significant increases in the rate of torque development (42.5 +/- 13.3%), the soleus surface electromyogram (60.7 +/- 30.8%), voluntary activation (2.8 +/- 0.1%), and the rate of activation (48.7 +/- 24.3%). Hslp/Mslp was not altered by training; however, V/Mmax increased 57.3 +/- 34.2% during MVC. These results suggest that increases in MVC observed in the first few days of isometric resistance training can be accounted for by an increase in the rate of activation at the onset of muscle contraction. Augmentation of muscle activation may be due to increased volitional drive from supraspinal centers.     

Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers

Skeletal muscle-mass loss with age has severe health consequences, yet the molecular basis of the loss remains obscure. Although mitochondrial DNA (mtDNA)-deletion mutations have been shown to accumulate with age, for these aberrant genomes to be physiologically relevant, they must accumulate to high levels intracellularly and be present in a significant number of cells. We examined mtDNA-deletion mutations in vastus lateralis (VL) muscle of human subjects aged 49-93 years, using both histologic and polymerase-chain-reaction (PCR) analyses, to determine the physiological and genomic integrity of mitochondria in aging human muscle. The number of VL muscle fibers exhibiting mitochondrial electron-transport-system (ETS) abnormalities increased from an estimated 6% at age 49 years to 31% at age 92 years. We analyzed the mitochondrial genotype of 48 single ETS-abnormal, cytochrome c oxidase-negative/succinate dehydrogenase-hyperreactive (COX-/SDH++) fibers from normal aging human subjects and identified mtDNA-deletion mutations in all abnormal fibers. Deletion mutations were clonal within a fiber and concomitant to the COX-/SDH++ region. Quantitative PCR analysis of wild-type and deletion-containing mtDNA genomes within ETS-abnormal regions of single fibers demonstrated that these deletion mutations accumulate to detrimental levels (>90% of the total mtDNA).     

Catalase in skeletal muscle fibers.

Catalase has been localized immunocytochemically with anti-bovine catalase in long thin filament structures in aerobic type I fibers in the skeletal muscles of normal and genetically dystrophic hamsters. The filaments range in length from 1 to 60 micron, are orientated regularly along the long axis of the fibers, and also seem to surround and project from muscle nuclei. The enzyme thus appears to be more prominent in the sarcoplasmic reticulum than in peroxisomes, and in this situation is suitably placed for destroying toxic hydrogen peroxide which may be continously generated in aerobic fibers.     

Calcium sparks in skeletal muscle fibers

Ca(2+) sparks monitor transient local releases of Ca(2+) from the sarcoplasmic reticulum (SR) into the myoplasm. The release takes place through ryanodine receptors (RYRs), the Ca(2+)-release channels of the SR. In intact fibers from frog skeletal muscle, the temporal and spatial properties of voltage-activated Ca(2+) sparks are well simulated by a model that assumes that the Ca(2+) flux underlying a spark is 2.5 pA (units of Ca(2+) current) for 4.6 ms (18 degrees C). This flux amplitude suggests that 1-5 active RYRs participate in the generation of a typical voltage-activated spark under physiological conditions. A major goal of future experiments is to estimate this number more precisely and, if it is two or more, to investigate the communication mechanism that allows multiple RYRs to be co-activated in a rapid but self-limited fashion.