Z-line Width Research Articles

Analysis of Titin in Red and White Muscles: Crucial Role on Muscle Contractions Using a Fish Model.

Several studies have compared molecular components between red and white skeletal muscles in mammals. However, mammalian skeletal muscles are composed of mixed types of muscle fibers. In the current study, we analyzed and compared the distributions of titin, lipid, phosphate ions, and fatty acid levels in red and white muscles using a fish model (Tilapia), which is rich in red and white muscles, and these are well separated. Oil-red O staining showed that red muscle had more-abundant lipids than did white muscle. A time-of-flight secondary-ion mass spectrometric (TOF-SIMS) analysis revealed that red muscle possessed high levels of palmitic acid and oleic acid, but white muscle contained more phosphate ions. Moreover, elastica-van Gieson (EVG) and Mito-Tracker green FM staining showed that collagen and elastic fibers were highly, respectively, distributed in connective tissues and mitochondria in red muscle. An electron micrographic analysis indicated that red muscle had a relatively higher number of mitochondria and longer sarcomere lengths and Z-line widths, while myofibril diameters were thicker in white muscle. Myofibrillar proteins separated by SDS-PAGE showed that the major giant protein, titin, was highly expressed in white muscle than in red muscle. Furthermore, ratios of titin to myosin heavy chain (MHC) (titin/MHC) were about 1.3 times higher in white muscle than red muscle. We postulated that white muscle is fit for short and strong contractile performance due to high levels of titin and condensed sarcomeres, whereas red muscle is fit for low intensity and long-lasting activity due to high levels of lipids and mitochondria and long sarcomeres.

Relationship between PPARα mRNA expression and mitochondrial respiratory function and ultrastructure of the skeletal muscle of patients with COPD

ABSTRACTPeripheral muscle dysfunction is an important complication in patients with chronic obstructive pulmonary disease (COPD). The objective of this study was to explore the relationship between the levels of peroxisome proliferator-activated receptor α (PPARα) mRNA expression and the respiratory function and ultrastructure of mitochondria in the vastus lateralis of patients with COPD. Vastus lateralis biopsies were performed on 14 patients with COPD and 6 control subjects with normal lung function. PPARα mRNA levels in the muscle tissue were detected by real-time PCR. A Clark oxygen electrode was used to assess mitochondrial respiratory function. Mitochondrial number, fractional area in skeletal muscle cross-sections, and Z-line width were observed via transmission electron microscopy. The PPARα mRNA expression was significantly lower in COPD patients with low body mass index (BMIL) than in both COPD patients with normal body mass index (BMIN) and controls. Mitochondrial respiratory function (assessed by respiratory control ratio) was impaired in COPD patients, particularly in BMIL. Compared with that in the control group, mitochondrial number and fractional area were lower in the BMIL group, but were maintained in the BMIN group. Further, the Z-line became narrow in the BMIL group. PPARα mRNA expression was positively related to mitochondrial respiratory function and volume density. In COPD patients with BMIN, mitochondria volume density was maintained, while respiratory function decreased, whereas both volume density and respiratory function decreased in COPD patients with BMIL. PPARα mRNA expression levels are associated with decreased mitochondrial respiratory function and volume density, which may contribute to muscle dysfunction in COPD patients.

Z-Line Elongation Observed in Titin Labeled Myofibrils

A muscle myofibril (below) is composed of sarcomeres in series. These sarcomeres are made of myosin, actin and titin, representing the three major filamentous proteins in muscle. Titin links myosin in the A-band region, to actin 100nm from the Z-line. This particular junction where titin binds actin, is where T12 antibody localizes. Using single myofibrils isolated from rabbit psoas muscle, we labeled titin with T12 conjugated to quantum dots and stretched myofibrils between a cantilever and rigid glass needle. Preliminary data suggests that myofibrils display a broadening between these T12 bands when stretched, despite being considered mainly inelastic due to titin-actin interactions. Interestingly, densitometric analysis revealed the distance between flanking T12 bands increased, while the width of the fluorescent bands remained unchanged. Two alternative explanations could require the width of the Z-line to increase with stretch (Tonino et al., 2009), or titin could dislodge from actin to accommodate stretch. Given the longest average sarcomere length was 3.07μm, the actin dislodging seems less likely as the psoas muscle has not approached peak tension (∼3.9μm (Wang et al., 1991)). Thus, it appears that Z-line width increases with stretch to a greater extent than previously reported.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Human skeletal muscle glycogen utilization in exhaustive exercise: role of subcellular localization and fibre type

Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, VO2 max = 68 ± 5 ml kg-1 min-1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. .

Sarcomeric-alpha-actinin defective in vinculin-binding causes Z-line expansion and nemaline-like body formation in cultured chick myotubes

The Z-line in each striated muscle has a precisely defined width that corresponds to muscle fiber type, and it can enlarge several fold in nemaline myopathy. To explore the mechanism(s) underlying Z-line width and structure maintenance, a series of sarcomeric-α-actinin mutants tagged with myc-epitope was transfected into cultured chick myotubes. By double-staining transfected myotubes with myc and myofibrillar protein antibodies, we found that alpha-actinin mutants with deletion of the region from the beginning of the fourth spectrin repeat to the start of the EF-hands resulted in expansion of Z-line width, often displayed a doublet staining pattern, and resulted in formation of nemaline-like bodies in older myotubes under fluorescence microscope. Yeast-two hybridization analysis demonstrated that this region was involved in vinculin binding, and for vinculin to bind alpha-actinin, residues 1–116 and 258–323 were required. Hence, we have defined a critical region of s-α-actinin that affects the width and integrity of the Z-line. This region is at least involved in the interaction with vinculin.

Reduced mitochondrial density in the vastus lateralis muscle of patients with COPD

Skeletal muscle dysfunction is a well-recognised hallmark of chronic obstructive pulmonary disease (COPD) leading to exercise intolerance. The vastus lateralis of COPD patients is characterised by reduced mitochondrial enzyme activity; however, this is not the case in the tibialis anterior. It is, however, unclear whether the compromised oxidative capacity in the vastus is due to reduced mitochondrial volume density. Muscle biopsies were obtained from the vastus lateralis of six COPD patients and four healthy age-matched controls, and from the tibialis anterior of another six COPD patients and six controls. Mitochondrial number, fractional area and morphometry, as well as Z-line width (as a surrogate marker of fibre type), were analysed using transmission electron microscopy. Mitochondrial number (0.34 versus 0.63 n.microm(-2)) and fractional area (1.95 versus 4.25%) were reduced in the vastus of COPD patients compared with controls. Despite a reduced mitochondrial number (0.65 versus 0.88 n.microm(-2)), the mitochondrial fractional area was maintained in the tibialis of COPD patients compared with controls. It can be concluded that the reduced mitochondrial fractional area is likely to contribute to the decreased oxidative capacity in the vastus of chronic obstructive pulmonary disease patients, whereas the maintained mitochondrial fractional area in the tibialis may explain the normal oxidative capacity.

Ectopic endplates induce localized changes in skeletal muscle ultrastructure.

To investigate the processes by which motoneurons control protein synthesis, and thus the ultrastructure of the muscle fibers they innervate, ectopic endplates were induced to form on adult mouse skeletal muscle fibers by transplantation of a foreign nerve onto the muscle. In the dually innervated muscle fibers thus created, we examined two ultrastructural parameters that correlate with the expression of distinct isoforms of the myofibrillar proteins alpha-actinin and titin, specifically, Z-line width and sarcomere length. It was found that Z-lines were significantly thinner (98 vs. 128 nm) and sarcomeres were significantly shorter (1.69 vs. 2.06 microm) near the ectopic than near the original endplates. Thus, ectopic endplate formation on adult skeletal muscle fibers induces a localized alteration in myofibrillar morphology. These results may help to elucidate the role played by motoneurons in the determination and maintenance of muscle fiber properties and the processes that occur following muscle reinnervation after nerve injury.

The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

Ultrastructural and metabolic characteristics of single muscle fibres belonging to the same type in various muscles in rats

Single muscle fibres from soleus (SOL) as well as extensor digitorum longus (EDL) muscles from Wistar male rats in relaxing solution were divided into three types by their histochemical features--slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), or fast-twitch glycolytic (FG) fibres. The relationship between ultrastructural profiles (mitochondrial volume, number, and Z-line width) and metabolic profiles (glycolytic and oxidative enzymes' activity) were analyzed using the same types of fibres dissected from different SOL and EDL muscles using stereological and biochemical techniques. The Z-line width is specialized in different fibre types. Fast-twitch (FG and FOG) fibres have narrow Z-line width compared to slow-twitch (SO) fibre in SOL and EDL muscles. A significant difference was observed between SOL muscle SO and FOG fibres and EDL muscle SO and FOG fibres. All glycolytic (lactate dehydrogenase (LDH), phosphofructokinase (PFK), pyruvate kinase (PK) and creatine kinase (CK) activities in FOG fibres from EDL muscles were significantly higher (p less than 0.01) than those dissected from SOL muscles. The oxidative enzyme (succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activity in SO and FOG fibres dissected from SOL muscle were significantly higher (p less than 0.01) than those dissected from EDL muscles. Mitochondrial volume and number in SO fibres dissected from SOL muscle were significantly higher (volume, p less than 0.01, number, p less than 0.01) than those dissected from EDL muscles. A significant difference was not observed in mitochondrial volume of FOG fibres between SOL and EDL muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Architecture of the pectoralis muscle of the Japanese quail (Coturnix japonica): histochemical and ultrastructural characterization, and distribution of muscle fiber types

The muscle fibers in the pectoralis muscle of the Japanese quail (Coturnix japonica) are classified as fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG), and slow-tonic on the basis of their histochemical and ultrastructural characteristics. There is an increasing proportion of FOG/FG fibers along a superficial to deep gradient throughout the entire belly of the muscle. Slow-tonic fibers are present in low numbers, and are restricted to a tiny area located in the deepest fasciculi of the cranial third of the muscle. This distribution of muscle fiber types is typical of those vertebrate muscles adapted to a locomotory function. The slow-tonic fibers are alkali-stable and acid-stable when preincubated for myofibrillar adenosine triphosphatase (mATPase) activity. Slow fibers in the chicken pectoralis and mouse soleus muscle, both types previously described as alkali-labile, acid-stable for mATPase activity, cannot be distinguished from each other or Japanese quail slow-tonic fibers on the basis of several ultrastructural characteristics: Z-line width, metabolic differences, or fusion of myofibrils. While mammalian slow fibers have one large motor end plate, all avian slow fibers have small multiple motor end plates. Mammalian slow fibers are slow-twitch, and avian slow fibers are probably slow-tonic. More complex secondary synaptic clefts distinguish mammalian from all avian fiber types.

The presence of two skeletal muscle alpha-actinins correlates with troponin-tropomyosin expression and Z-line width.

Two species of alpha-actinin from rabbit fast skeletal muscles were identified with a monospecific antisera. Designated alpha-actinin1f and alpha-actinin2f, their distribution in muscles does not correlate with histochemically defined fast fiber type. Rather, the presence of each correlates with Z-line width and with the expression of different thin filament Ca2+-regulatory complexes. alpha-Actinin1f is expressed with troponin T 1f-alpha beta tropomyosin, and alpha-actinin2f with troponin T 2f-alpha 2 tropomyosin. CNBr peptide maps show that the fast alpha-actinin species differ in primary structure. In contrast, the slow alpha-actinin is indistinguishable from alpha-actinin1f. Further evidence for the similarity of alpha-actinin1f and slow alpha-actinin comes from electron microscopic studies which show that fibers that express these species exhibit thick Z-lines. So, unlike other contractile proteins, the multiple forms of alpha-actinin do not reflect the distinction between fast- and slow-twitch muscles.

Adaptability of ultrastructure in the mammalian muscle.

All the skeletal muscle fibres taken from an adult mammal do not look alike. The structural differences are a result of adaptations which allow gradations in mechanical output to be achieved. The anatomy is described and the amounts of the subcellular components are measured by stereological techniques from electron micrographs. A population of normal, adult fibres is classified by the Z-line width, by the amounts of the mitochondria, T-system and terminal cisternae (TC), and by the isoforms of contractile proteins present. Classification of fibres by some of these ultrastructural components gives clusters named fast-twitch and slow-twitch types, but classification by other components gives a continuum of overlapping properties. Transformation from the fast- to the slow-twitch type or vice versa follows a specific alteration in the use of the fibre. The mechanical demand on the fibre is modified by changing the frequency of stimulation in the nerve with an implanted electrode. The time course of the changes in subcellular composition in the fibre during adaptation is followed for many weeks. Changes in the membrane systems begin within hours and are complete in days. Changes in the contractile proteins and metabolic systems begin in days and are complete in weeks. During these transitional phases of adaptation the fibres have an unusual complement of components never seen in a normal adult fibre. Extreme alterations, such as myofibril disassembly or supranormal amounts of mitochondria also result during some adaptive transitions. The aberrant appearance in the transitional fibres may be a result of doing the required mechanical work with a less than optimal set of proteins. At the end of the fibre type transformation, the fibre ultrastructure is indistinguishable from normal.

Extensibility of the myofilaments in vertebrate skeletal muscle as revealed by stretching rigor muscle fibers.

The extensibility of the myofilaments in vertebrate skeletal muscle was studied by stretching glycerinated rabbit psoas muscle fibers in rigor state and examining the resulting extension of sarcomere structures under an electron microscope. Although stretches applied to rigor fibers produced a successive yielding of the weakest sarcomeres, the length of the remaining intact sarcomeres in many myofibrils was fairly uniform, being definitely longer than the sarcomeres in the control, nonstretched part of rigor fibers. The stretch-induced increase in sarcomere length was found to be taken up by the extension of the H zone and the I band, whereas the amount of overlap between the thick and thin filaments did not change appreciably with stretches of 10-20%. The thick filament extension in the H zone was localized in the bare regions, whereas the thin filament extension in the I band appeared to take place uniformly along the filament length. No marked increase in the Z-line width was observed even with stretches of 20-30%. These results clearly demonstrate the extensibility of the thick and thin filaments. The possible contribution of the myofilament compliance to the series elastic component (SEC) in vertebrate skeletal muscle fibers is discussed on the basis of the electron microscopic data and the force-extension curve of the SEC in rigor fibers.

Increase of muscle mitochondrial content with age in murine muscular dystrophy

Comparison of morphological features of gastrocnemius muscle fibers in normal and dystrophic (dy2J) mice during development was undertaken to determine the time course of increased oxidative capacity in dystrophic fibers. Measurements of mitochondrial volume percent and of Z-line width were made in superficial fast-twitch fibers using electron microscopy and stereological techniques. Dystrophic fibers develop a progressively higher mitochondrial volume percent than normal fibers after 1 month of age. Z-line width is positively correlated with mitochondrial volume percent. The results support the hypothesis that progressive changes in muscle fiber properties result from abnormal neural activity (pseudomyotonia) in dystrophic animals.

Does tenotomy of skeletal muscle alter Z-line width in older animals?

Results of unilateral achilles tenotomy on older (350–410 g) male rats, showed that the general width of Z-lines in the tenotomized muscles was not significantly altered by this procedure. Streaming of the Z-lines and rod formations was still present, as in younger rats.

A morphometric analysis of human muscle fibers with relation to fiber types and adaptations to exercise

Muscle biopsies were obtained from the vastus lateralis of 14 male subjects: 3 long distance runners, 2 world class power lifters and 9 active, although not highly trained, individuals used as controls. The fibers were investigated by electron microscopy and the mitochondrial volume percent, lipid volume percent and Z-line width were analyzed morphometrically. With the combined data a direct correlation was found between mitochondrial volume percent and lipid volume percent, lipid volume percent and Z-line width and mitochondrial volume percent and Z-line width. The muscle fibers were classified as slow-twitch oxidative (SO), fast-twitch-oxidative-glycolytic (FOG) and fast-twitch-glycolytic (FG) based on relationships found in the data and well established properties of muscle fiber types. Although no distinct patterns emerged, a good approximation of fiber type characteristics was obtained, and values for volume percent of central mitochondria, volume percent lipid and Z-line width are reported. The fibers classified as SO were characterized by having wide Z-lines, a high mitochondrial volume percent and high lipid volume percent. The fast-twitch fibers (fibers with narrow Z-lines) were separated into 2 groups, those with high mitochondrial volume percent (FOG) and those with low mitochondrial volume percent (FG). No distinction could be made between the fast-twitch subgroups with regard to Z-line width. The fibers from distance runners differed from those from controls by exhibiting a greater capacity for aerobic activity as evidenced by the increased volume percent of mitochondria and lipid in both slow- and fast-twitch fibers. The high strength, anaerobic activity of the world class power lifters was reflected by the low mitochondrial volume percent of many fast-twitch fibers (FG) and the decreased lipid stores in all fibers.

Ultrastructural identification of muscle fiber types by immunocytochemistry.

In a fast-twitch muscle, three types of fibers (red, intermediate, and white) can be distinguished on the basis of mitochondrial content. Red fibers, identified by abundant mitochondria, can be further differentiated on the basis of a positive or negative response to antibodies specific for white ("fast") myosin. Because there is also a difference in Z-line width among fibers of the same muscle, the possibility existed that the two red fibers, which differ in type of myosin, might also differ in dimensions of the Z line. We therefore examined, with the electron microscope, fibers which had been exposed to antibody against white myosin. In those fibers which react with the antibody, an electron-opaque band is evident in the H-band region, thereby distinguishing reactive from unreactive fibers. The red fiber can now be subdivided on the basis of a positive or negative response to anti-white myosin visualized directly with the electron microscope. Both categories of red fibers ("fast" and "slow") have wide Z lines, and thus are distinguished from white and intermediate fibers, which react with the antibody but which have narrow Z lines. On the basis of combined immunocytochemical and ultrastructural characteristics, four types of fibers can be recognized in a single muscle. Moreover, it is demonstrated here that a wide Z line does not necessarily imply a slow speed of contraction.

Influence of acute hypoxia on Z-line width of cardiac muscle

Isolated guinea pig papillary muscles were exposed to 30 min of acute hypoxia. The preparations were then fixed for electron microscopy. The width of 435 control and 386 hypoxic Z-lines were measured. No statistical difference occurred between the two groups. We conclude, contrary to a recent report, that 30 min of acute hypoxia does not alter the Z-line width in heart muscle.

Fingerprint inclusions: Ultrastructural demonstration of muscle fiber type specificity

Fingerprint inclusions were identified in the skeletal muscle of a child with severe hypotonia. The mean Z-line and M-line widths from each of 19 muscle fibers containing the fingerprint inclusions were calculated. The mean Z-line widths were 85–99 nm, and the mean M-line widths were 75–101 nm. According to our present system of fiber typing, all of these fibers would be classified as Type I. The possible pathogenesis and fiber type specificity of the fingerprint inclusion is discussed.

Concentric laminated bodies: Ultrastructural demonstration of muscle fiber type specificity

Concentric laminated bodies were identified in the skeletal muscle of 3 children affected by muscle weakness and hypotonia with probable cerebral involvement. The mean Z-line and M-line widths from each of 11 muscle fibers containing the concentric laminated bodies were calculated. The mean Z-line widths were 61–81 nm and the mean M-line widths from 7 out of 11 fibers were 54–65 nm. According to our present system of fiber typing, the majority of these fibers would be classified as subtype IIB. The possible pathogenesis and fiber type specificity of the concentric laminated body is discussed.