Morphological and physiological characterization of fiber types in the iliofibular muscle of Rana esculenta]

In both longitudinal and cross sections of the M. iliofibularis of Rana esculenta three types of muscle fibres are identified by means of light and electron microscopy. These fibretypes called A-, B- and C-fibres are according to the fibres of m. rectus abdominis of the frog. They can be compared with the fibres of the m. rectus abdominis of rat and mouse. But there is another distribution of the fibretypes A, B and C in the m. iliofibularis and in the m. rectus abdominis. The m. iliofibularis is divided into two parts called "Tonusbündel" and "nichttonischer Teil" by means of their reaction to acetylcholine. The surface of the "Tonusbündel" consists of A-, B- and C-fibres while its inside is onlyformed by A- and B-fibres. They continue the "Tonusbündel" in the "nichttonischer Teil". This part chiefly consists of A-fibres. In cross sections their myofibrils are larger in their extent than the A-fibres known before. Therefore the A-fibretype has to be distinguished into two A-fibres: A1 and A2. The new one is called A2-fibre. A1-fibre is described in the "Tonusbündel" and in further investigations. The difference between the two fibres can be understood as a greater manifestation of power of the A1-fibre. The surface of the "nichttonischer Teil" of the m. iliofibularis consists of A2-fibres which easily could be found opposite the "Tonusbündel". At this point in contrary to the "Tonusbündel" could be found a defined morphological substrate for physiological investigations. The different reactions of "Tonusbündel" and "nichttonischer Teil" to acetylcholine could only be explained by the sum of reactions of all fibretypes in each bundle in correspondence with the reaction of the fibres in the neighbour bundle. But their different behaviour by summer- and winterfrogs is unknown. Therefore it is to discuss whether it is allowed to refer generally the results to "muscle" or "musclefibre" got from frogs living in cooled rooms. It is known in literature that not all results of physiological investigations can be interpreted with the two fibre- theorie ("twitch" and "slow") of muscle. Those not interpretable physiological results could be associated to the B-fibre can not be explained by morphological methods but must be proofed by physiological investigations. In tables are summerised morphological criteria of the three types and it is tried to associate the physiological qualities known from literature. Besides there is summerised the usual nomenclature with the first citations.     

Quantitative electron-microscopy analysis of cranial nerves III, IV, and VI and of the extrinsic eye muscles in fog.

A comparative study of the quantitative data of the frog extraocular muscles and the cranial nerves that innervate them was performed. Oculorotatory muscles contain muscle fibres of at least 4 types which are arranged in heterogeneous layers. The zonal arrangement of the muscles does not occur on the cross-sections in the vicinity of muscle insertions. In these regions only two muscle fibre types are present and the total number of fibres is smaller by 70% than in the central region of the muscle. Most numerous are muscle fibres in the rectus inferior muscle, while the smallest number of fibres is found in rectus interior muscle. Three distinct types of nerve fibres are distinguished according to the following criteria: occurrence and thickness of myelin sheath, fibre diameter and ratio "g". The fibres with thin myelin sheaths indicate small diameters (1-5--6- mum) and their ratio "g" equals 0-82 +/- 0-08. They constitute about 30% of the myelinated fibres in the nerve supply of the oculorotatory muscles and about 14% in the supply of the retractor bulbi muscle. Both the value of the ratio "g" and a greater number of these fibres in the nerve supply of the muscles that contain slow contracting muscle fibres indicate that they are rather slow conducting nerve fibres. The range of the diameters of the fibres with thick myelin sheaths is greater (3-5--13-5 mum) and their "g" equals 0-66 +/- 0-06. These fibres constitute about 70% of the myelinated ones in the nerve supply of the oculorotatory muscles and 86% in the supply of the retractor bulbi muscles. The value of the ratio "g" in these fibres indicates that they are fast contracting ones. The smallest diameters are found in the myelinated fibres (0-5--1-7 mum). These fibres occur frequently in all the examined nerves; they constitute 36--47% of the total number of all the nerve fibres. The frog extraocular muscles are characterized by an abundal nerve supply which is reflected in the low innervation ratio (1:4--1:5). On the distal cross-section of nerves the number of nerve fibres is greater than on the proximal ones. Ganglionic neurons occur sporadically around the nerves; in the nerve III synaptic contacts between two neurons were observed.     

Characteristics of paravertebral muscles – fibre type distribution pattern in the pika, Ochotona rufescens (Mammalia: Lagomorpha)

The evolution of the locomotor apparatus in vertebrates is marked by major reorganizations in trunk's musculature. The hypothesized functions of mammalian back muscles in the literature are discussed under consideration of the distribution and proportion of oxidative, type‐I‐fibres, oxidative‐glycolytic, type‐IIa‐fibres and glycolytic, type‐IIb‐fibres in paravertebral muscles of a small mammal. The fibre type distribution was examined from a complete series of histological sections maintaining topographical relationships between the muscles as well as within the muscle, in order to establish the overall distribution pattern. The deep and short muscles showed the highest percentage of oxidative fibres. The larger, superficial paravertebral muscles contained the highest percentage of glycolytic fibres. Two muscles were intermediate in their proportion of fibre types. All epaxial muscles together can be interpreted as an antigravity muscle–complex counteracting enduringly against the rebound tendency caused by gravitation, comparable with antigravity muscles in limbs. A gradient from deep to superficial, or a clear regionalization of oxidative muscle fibres in central deep regions around a large intramuscular tendon was found in the m. spinalis and the m. quadratus lumborum, respectively. Concepts of the function of human back muscles as those of A. Bergmark (1989: Acta Orthop. Scand. 230 , 1) or S.G.T. Gibbons & M.J. Comerford (2001: Orthop. Division Rev. March/April, 21) were exposed to be more general within mammals. Functional specializations of different muscles and muscle parts are discussed under the consideration of evolutionary reorganization of the paravertebral musculature in tetrapods. Along the cranio‐caudal axis, the percentage of oxidative fibres was decreased in caudal direction within the same muscles, whereas the proportion of glycolytic fibres was increased. Therefore, classifications of muscles as ‘glycolytic’ or ‘oxidative’ based on biopsies or analyses of single cross‐sections may result in wrong interpretations. Changes in the proportions of the fibre type distribution pattern were mostly due to oxidative and glycolytic fibre types, whereas the percentage of oxidative‐glycolytic fibres had only minor influence. A significant positive correlation between the cross‐sectional area of the single fibre and its percentage in the area investigated were observed for oxidative fibres, whereby the size was positive correlated to the proportion of the oxidative fibres.     

A comparative microphotometric study of succinate dehydrogenase activity levels in type I,IIA and IIB fibres of mammalian and human muscles

Summary Activity levels of succinate dehydrogenase (SDH) were determined kinetically by means of comparative microphotometric measurements in situ. Activities were correlated with fibre types classified histochemically according to Brooke and Kaiser (1970). Analyses of tibialis anterior muscles in the mouse, rat, guinea pig, rabbit, cat and the human showed pronounced variations in the activity profiles of type I, type IIA and IIB fibres of these muscles. Large scattering of enzyme activity existed in the three fibre populations. Overlaps of varying extent were found for the SDH profiles between the different muscles. Type I fibres reveal species diffeences in aerobic oxidative capacity. Whereas the majority of the IIB fibres in rabbit muscle tended to be low in SDH activity, the main fraction of this fibre population was characterized by high activities in mouse muscle. Similarly, the IIA fibre populations revealed opposite properties in mouse and rabbit muscles. These extremes as well as intermediate activity patterns indicate that no general scheme exists according to which the histochemically assessable myosin ATPase is correlated with the aerobic oxidative capacity of muscle fibres in various mammalian muscles.     

The properties of the extraocular muscles of the frog. I. Mechanical properties of the isolated superior oblique and superior rectus muscles.

The mechanical properties of two extraocular muscles (superior oblique and superior rectus muscles) of the frog were studied and compared with those of a frog's skeletal muscle (iliofibularis muscle) which contains the same types of muscle fibres as the oculorotatory muscles. The extraocular muscles are very fast twitching muscles. They exhibit a smaller contraction time, a smaller half-relaxation time, a higher fusion frequency, and a lower twitch-tetanus ratio than the skeletal muscles. The maximum isometric tetanic tension produced per unit cross-sectional area is lower in the extraocular muscles than in skeletal muscles. However, the extraocular muscles show a higher fatigue resistance than the skeletal muscles. With respect to the dynamic properties there are some differences between the various oculorotatory muscles of the frog. The superior rectus muscle exhibits a faster time-course of the contraction, a higher fusion frequency, and a higher fatigability than the superior oblique muscle. An increase of the extracellular K+-concentration evokes sustained contractures not only in the extraocular muscles but also in the iliofibularis muscle; between these muscles there are no striking differences in the mechanical threshold of the whole muscle preparation. The mechanical threshold depends on the Ca++-concentration of the bathing solution and it is found in a range between 12.5 and 17.5 mM K+ in a normal Ringer solution containing 1.8 mM Ca++. The static-mechanical properties of the extraocular muscles of the frog and the dependence of the active developed tension on the muscle extension are very similar to those which are known to exist in the extraocular muscles of other vertebrates. In tetanic activated frog's oculorotatory muscles a linear relationship exists between length and tension. A variation of the stimulation frequency does not change the slope of this curve but causes parallel shifts of the curve. The peculiar properties of the extraocular muscles of the frog are discussed with respect to the muscle fibre types in these muscles and to the diameter of the muscle fibres.     

Electrophoretic analysis of proteins from single bovine muscle fibres.

A number of single fibres were isolated by dissection of four bovine masseter (ma) muscles, three rectus abdominis (ra) muscles and eight sternomandibularis (sm) muscles. By histochemical criteria these muscles contain respectively, solely slow fibres (often called type I), predominantly fast fibres (type II), and a mixture of fast and slow. The fibres were analysed by conventional sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the gels stained with Coomassie Blue. Irrespective of the muscle, every fibre could be classed into one of two broad groups based on the mobility of proteins in the range 135000-170000 daltons. When zones containing myosin heavy chain were cut from the single-fibre gel tracks and 'mapped' [Cleveland, Fischer, Kirschner & Laemmli (1977) J. Biol. Chem. 252, 1102-1106] with Staphylococcus proteinase, it was found that one group always contained fast myosin heavy chain, whereas the second group always contained the slow form. Moreover, a relatively fast-migrating alpha-tropomyosin was associated with the fast myosin group and a slow-migrating form with the slow myosin group. All fibres also contained beta-tropomyosin; the coexistence of alpha- and beta-tropomyosin is at variance with evidence that alpha-tropomyosin is restricted to fast fibres [Dhoot & Perry (1979) Nature (London) 278, 714-718]. Fast fibres containing the expected fast light chains and troponins I and C fast were identified in the three ra muscles, but in only four sm muscles. In three other sm muscles, all the fast fibres contained two troponins I and an additional myosin light chain that was more typical of myosin light chain 1 slow. The remaining sm muscle contained a fast fibre type that was similar to the first type, except that its myosin light chain 1 was more typical of the slow polymorph. Troponin T was bimorphic in all fast fibres from a ra muscles and in at least some fast fibres from one sm muscle. Peptide 'mapping' revealed two forms of fast myosin heavy chain distributed among fast fibres. Each form was associated with certain other proteins. Slow myosin heavy chain was unvarying in three slow fibre types identified. Troponin I polymorphs were the principal indicator of slow fibre types. The myofibrillar polymorphs identified presumably contribute to contraction properties, but beyond cud chewing involving ma muscle, nothing is known of the conditions that gave rise to the variable fibre composites in sm and ra muscles.     

Diversification of the myofibrillar M-band in rat skeletal muscle during postnatal development

Summary The fine structure of the M-band in soleus (SOL) and extensor digitorum longus (EDL) muscles in newborn and four-week-old rats was studied using electron-microscopic techniques. In newborn rats, all myotubes and fibres in both muscles had an identical myofibrillar appearance. A five-line M-band pattern was seen in longitudinal sections and distinct M-bridges in cross-sections. The Z-discs were of medium width. On the other hand, in four-week-old rats, different muscle fibre types were observed on the basis of their myofibrillar pattern. In SOL two fibre types were distinguished in longitudinal sections. One had a four-line M-band pattern and very broad Z-discs, whereas the other type had five lines in the M-band and broad Z-discs. In EDL, three different myofibrillar patterns were observed. The M-bands were composed of three, four or five lines. Fibres had either thin, broad or medium Z-disc widths, respectively. In cross-sections of the SOL muscle one group of fibres showed indistinct M-bridges, whereas distinct M-bridges were seen in the other fibres and in all observed EDL muscle fibres. We conclude that initially there seems to be a single intrinsic program for M-band genesis; this program becomes modified upon the induction of functionally differentiated fibres.     

Quantitative succinate-dehydrogenase histochemistry. II. A comparison between visual and quantitative msucle fibre typing.

Most muscles exhibit a mosaic pattern of staining intensities of their muscle fibres after the histochemical reaction for succinate dehydrogenase (SDH). Visually these muscle fibres are usually classified into three groups: with low (A-fibres), intermediate (B-fibres), and high (C-fibres) enzymatic activity (staining intensity). Cytospectrophotometric methods were employed to investigate whether discrete groups of muscle fibres could be discerned, comparable to those found after the visual classification. The classifications were based on quantitative parameters of the total absorbance per cell and the distribution of the coloured endproduct over the fibre cross area.     

Relationships between mitochondrial content and glycogen distribution in porcine muscle fibres.

Serial sections of longissimus dorsi and rectus femoris muscles from 15 Yorkshire breed pigs (live weights 24-46 and 49-139 kg) were stained for glycogen (PAS) and a mitochondrial enzyme (NAD tetrazolium reductase). Muscle fibres with a low mitochondrial content in both muscles were more frequently PAS-positive than fibres with a high or intermediate mitochondrial content. However, some pigs had all their muscle fibres PAS-positive while one pig with a high post-mortem muscle pH had all rectus femoris fibres PAS-negative. Relative to lighter weight pigs, longissimus dorsi muscles of heavy pigs tended to have less fibres with a high mitochondrial content and less fibres with a positive PAS reaction. Compared to longissimus dorsi muscles, rectus femoris muscles had more fibres with a high mitochondrial content and less with a positive PAS reaction. All fibres in both muscles became PAS-negative with an accompanying decrease in pH by 24 hr post-mortem. Fibres from longissimus dorsi muscles frequently had PAS-positive sarcoplasmic cores between their myofibrils. Heavy pigs tended to have larger cores (up to a mean maximum diameter of 13.4 mum), more fibres with cores, and more cores per fibre. The pigs involved exhibited no other ante- or post-mortem muscle abnormalities.     

The extracellular space of voluntary muscle tissues

The volume occupied by the extracellular space has been investigated in six types of voluntary muscles: sartorius (frog), semitendinosus (frog), tibialis anticus longus (frog), iliofibularis (frog), rectus abdominis (frog), and diaphragm (rat). With the aid of four types of probe material, three of which are conventionally employed (inulin, sorbitol, sucrose) and one of which is newly introduced (poly-L-glutamate), and a different experimental method, we have demonstrated that the "true" extracellular space of frog sartorius, semitendinosus, tibialis anticus longus, and iliofibularis muscle and of rat diaphragm muscle is equal to, or probably less than, 8-9% (v/w) of the tissue. The frog rectus muscle shows a somewhat higher ceiling value of 14%.     

The presynaptic active zones in three different types of fibres in frog muscle

Presynaptic active zones were studied in slow, fast and intermediate types of frog muscle fibres in freeze-fracture replicas. In fast fibres, the double rows of paired particles are present on active zone ridges perperdicular to the longitudinal axis of the nerve whereas in slow fibres active zone ridges are rudimentary or absent and double rows of particles occur in all directions, mostly paired, sometimes single. In the intermediate type of muscle fibres both types of active zone deployment coexist on a single muscle fibre.     

Fine structure of myomyous junctions in the mouse skeletal muscles

The reaction product of acetylcholinesterase (AChE) activity is known to be specifically localized at a neuromuscular junction and a muscle-tendon junction of the striated skeletal muscles. In addition to the two junctions, we recently found some linear precipitates due to AChE activity running transversely across a fibre of the semitendinosus, rectus abdominis, gastrocnemius, tibialis anterior and diaphragm muscles in mice. Under an electron microscope, the linear precipitates were seen at the extracellular side of the muscle fibre endings. Most of the endings contacted each other to form a junction, which has been called the 'myomyous junction (M-Mj)'. The patterns of the M-Mj were grouped into three types: (1) a junction in which all contacts were firm, without any connective tissue, and invaginated deeply; (2) the ones in which numerous collagen fibres were visible in the space between the two separate opposing muscle fibres; (3) an intermediate type between (1) and (2), i.e. a junction with partial contacts. The muscle fibre ending forming M-Mj was constructed of finger-like processes like that of a muscle-tendon junction. However, the processes of a M-Mj adhered so closely to each other that no collagen fibrils could penetrate into their folds.     

Ultrastructure of frog muscle fiber thick filaments at rest and during potassium contracture]

Isolated slow and intermediate frog muscle fibres were fixed in the rest state and under potassium contracture (50-100 mM KC1). The longitudinal and cross sections of two types of fibres were investigated. It was shown that at the rest the thick filaments of different fibres had similar length (1.6-1.65 mum), diameter (160-165 A) and the amount of subunits (12-13). Under potassium contracture the length of the thick filaments of both fibre types was shortened by 25-30% of the rest-length, the diameter of the slow fibres increased to 180-185 A, the diameter of the intermediate fibres to 200-220 A. The amount of subunits increased to 14-15 in slow fibres and to 17-18 in intermediate fibres. We believe that the ultrastructural changes observed in the thick filaments are a result of molecular transformation in these filaments, which seems to be important for maintaining the contracture.     

The immunohistochemical location of cathepsin L in rabbit skeletal muscle. Evidence for a fibre type dependent distribution

The immunohistochemical location of cathepsin L in rabbit soleus, plantaris and psoas muscles was investigated using the peroxidase-anti-peroxidase (PAP) technique. The amount of enzyme detected varied according to the fibre type, which were identified by histochemical staining of serial sections for succinate dehydrogenase and alkali-stable myosin ATPase. In the three muscles studied labelling was strongest in the highly oxidative fibres and weaker in the other fibre types with least staining in the fast white fibres. Immunoreactive cathepsin L appeared to be most concentrated at the periphery of muscle fibres, especially near to the nuclei, although some staining was seen throughout the fibres.     

Parvalbumin in mouse muscle in vivo and in vitro

Parvalbumin is a cytosolic calcium-binding protein found in adult fast-twitch mammalian muscle. Using an antibody to paravalbumin, we have shown that its distribution in adult mouse muscles is associated with certain fibre types. It is absent from slow-twitch type 1 fibres, is absent or at low levels in fast-twitch type 2A fibres, but is present at moderate or high levels in fast-twitch type 2B fibres. When adult mouse muscle is cultured with embryonic mouse spinal cord, the regenerated fibres become innervated, express the adult fast isoform of myosin heavy chain and appear histochemically as fast-twitch fibres. We therefore investigated whether these apparently mature fibres also contained parvalbumin. Parvalbumin was not found in any fibres of twenty mature cultures, suggesting that neurotrophic activity in the absence of specific adult nerve activity patterns was insufficient to cause the expression of parvalbumin in the cultures.     

The properties of the extraocular muscles of the frog. II. Pharmacological properties of the isolated superior oblique and superior rectus muscles.

The pharmacological properties of the superior oblique and the superior rectus muscles of the frog's eye were investigated in comparison with those of a skeletal muscle (iliofibularis muscle) of the same animal. Acetylcholine causes sustained contractures of the extraocular muscles; this effect is increased by physostigmine and decreased or abolished by d-tubocurarine. Also the applications of succinylcholine, choline or caffeine are able to evoke contractures. There are no striking differences in pharmacological properties between extraocular and skeletal muscles of the frog. The time-course of the contractures and the sensitivity of the muscle preparations to the drugs which evoke contractures are identical in extraocular and iliofibularis muscles. In comparison with skeletal muscles there is no higher sensitivity of the extraocular muscles against curare-like drugs. The existence of adrenergic receptors could not be found neither in extraocular nor in skeletal muscles of the frog. It is concluded that in frogs no pharmacological differences exist between the muscle fibre types which compose the extraocular and the skeletal muscles.     

Localization of hyaluronan in various muscular tissues

Summary The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analysed in various types of muscles in the rat by use of a hyaluronan-binding protein (HABP) and the avidin-biotin/peroxidase complex staining procedure. Microwave-aided fixation was used to retain the extracellular location of the glycosaminoglycan. In skeletal muscles, HYA was detected in the connective tissue sheath surrounding the muscles (epimysium), in the septa subdividing the muscle fibre bundles (perimysium) and in the connective tissue surrounding each muscle fibre (endomysium). HYA was heterogeneously distributed in all striated muscles. In skeletal muscles with small fibre dimensions (e.g., the lateral rectus muscle of the eye and the middle ear muscles), HYA was predominantly accumulated around the individual muscle fibres. Perivascular and perineural connective tissue formations were distinctly HYA-positive. In cardiac muscles, HYA was randomly distributed around the branching and interconnecting muscle fibres. In comparison, smooth muscle tissue was devoid of HYA.     

Monospecific antibodies against the three mammalian fast limb myosin heavy chains

Skeletal muscle fibres in mammalian limb muscles are of four types: slow, 2A, 2X, and 2B, each characterized by a distinct myosin heavy chain (MyHC) isoform. Existing monoclonal antibodies (mabs) against fast MyHCs lack fibre-type specificity across species and could not positively identify 2X fibres. In this work, mabs were raised against each of the fast MyHCs. These mabs were shown to be monospecific by Western blots and immunohistochemistry in the rat. The advantages of using these mabs for identifying the three fast fibre types and hybrid fibres expressing multiple isoforms were illustrated using rat tibialis anterior muscle. Immunohistochemical analyses confirmed the monospecificity of these mabs in the following additional species: mouse, guinea pig, rabbit, cat, and baboon. 2B fibres were absent in limb muscles of the cat and baboon. These mabs constitute a set of powerful tools for studying muscle fibre types and their transformations.     

Evidence of fibre hyperplasia in human skeletal muscles from healthy young men? A left-right comparison of the fibre number in whole anterior tibialis muscles

Cross-sections (thickness 10 microns) of whole autopsied left and right anterior tibialis muscles of seven young previously healthy right-handed men (mean age 23 years, range 18-32 years) were prepared for light-microscope enzyme histochemistry. Muscle cross-sectional area and total number of fibres, mean fibre size (indirectly determined) and proportion of the different fibre types (type 1 and type 2 on basis of myofibrillar adenosine triphosphatase characteristics), in each muscle cross-section were determined. The analysis showed that the cross-sectional area of the left muscle was significantly larger (P less than 0.05), and the total number of fibres was significantly higher (P less than 0.05), than for the corresponding right muscle. There was no significant difference for the mean fibre size or the proportion of the two fibre types. The results imply that long-term asymmetrical low-level daily demands on muscles of the left and the right lower leg in right-handed individuals provide enough stimuli to induce an enlargement of the muscles on the left side, and that this enlargement is due to an increase in the number of muscle fibres (fibre hyperplasia). Calculations based on the data also explain why the underlying process of hyperplasia is difficult, or even impossible, to detect in standard muscle biopsies.     

In vitro cleavage of eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate system

The vertebrate muscle Z-band organizes and tethers antiparallel actin filaments in adjacent sarcomeres and hence propagates the tension generated by the actomyosin interaction during muscular contraction. The axial width of the Z-band varies with fibre and muscle type: fast twitch muscles have narrow (approximately 30-50 nm) Z-bands, while slow-twitch and cardiac muscles have wide (approximately 100-140 nm) Z-bands. In electron micrographs of longitudinal sections of fast fibres like those found in fish body white muscle, the Z-band appears as a characteristic zigzag layer of density connecting the mutually offset actin filament arrays in adjacent sarcomeres. Wide Z-bands in slow fibres such as the one studied here (bovine neck muscle) show a stack of three or four zigzag layers. The variable Z-band width incorporating variable numbers of zigzag layers presumably relates to the different mechanical properties of the respective muscles. Three-dimensional reconstructions of Z-bands reveal that individual zigzag layers are often composed of more than one set of protein bridges, called Z-links, probably alpha-actinin, between oppositely oriented actin filaments. Fast muscle Z-bands comprise two or three layers of Z-links. Here we have applied Fourier reconstruction methods to obtain clear three-dimensional density maps of the Z-bands in beef muscle. The bovine slow muscle investigated here reveals a Z-band comprising six sets of Z-links, which, due to their shape and the way their projected densities overlap, appear in longitudinal sections as either three or four zigzag layers, depending on the lattice view. There has been great interest recently in the suggestion that Z-band variability with fibre type may be due to differences in the repetitive region (tandem Z-repeats) in the Z-band part of titin (also called connectin). We discuss this in the context of our results and present a systematic classification of Z-band types according to the numbers of Z-links and titin Z-repeats.