Development and composition of skeletal muscle fibres in mouse oesophagus

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

Differences in myosin composition between human oro-facial,masticatory and limb muscles: enzyme-, immunohisto-and biochemical studies

Summary Immunohistochemistry was used to determine the myosin composition of defined fibre types of three embryologically different adult muscles, the oro-facial, masseter and limb muscles. In addition, the myosin composition in whole muscle specimens was analysed with biochemical methods. Both similarities and differences between muscles in the content of myosin heavy chains and myosin light chains were found. Nevertheless, each muscle had its own distinct identity. Our results indicated the presence of a previously undetected fast myosin heavy chain isoform in the oro-facial type II fibre population, tentatively termed fast F. The masseter contained aberrant myosin isoforms, such as foetal myosin heavy chain and -cardiac myosin heavy chain and unique combinations of myosin heavy chain isoforms which were not found in the limb or oro-facial muscles. The type IM and IIC fibres coexpressed slow and fast A myosin heavy chains in the oro-facial and limb muscles but slow and a fast B like myosin heavy chain in the masseter. While single oro-facial and limb muscle fibres contained one or two myosin heavy chain types, single masseter fibres coexpressed up to four different myosin heavy chain isoforms. Describing the fibres according to their expression of myosin heavy chain isozymes, up to five fibre types could be distinguished in the oro-facial and limb muscles and eight in the masseter. Oro-facial and limb muscles expressed five myosin light chains, MLC_1S, MLC_2S, MLC_1F, MLC_2F and MLC_3F, and the masseter four, MLC_1S, MLC_2S, MLC_1F, and, in addition, an embryonic myosin light chain, MLC_temb, which is usually not present in normal adult skeletal muscle. These results probably reflect the way the muscles have evolved to meet the specialized functional requirements imposed upon them and are in agreement with the previously proposed concept that jaw and limb muscles belong to two distinct allotypes.     

Adaptive range of myosin heavy chain expression in regenerating soleus is broader than in mature muscle

Summary In adult rat muscles experimentally exposed to various patterns of activation, expression of myosin heavy chain isoforms changes, but only within a certain adaptive range. It is characteristic and different in fast or slow muscles. This may be due either to different intrinsic properties of the myogenic cells of the two types of muscles or to extrinsic factors. To test these assumptions, either rat soleus or extensor digitorum longus muscles were injured and transplanted to the bed of the extensor digitorum longus muscle. They regenerated and were reinnervated by the extensor digitorum longus nerve. Expression of myosin heavy chain isoforms was demonstrated immunohistochemically and by in situ hybridization, and analysed by SDS-gel electrophoresis. Three months after cross-transplantation, regenerated soleus expressed all adult myosin heavy chain isoforms, including the myosin heavy chain-2B. The latter was detected in about 50% of muscle fibres and contributed about 10–20% of all myosin heavy chains. The same percentage of myosin heavy chain-2B was found in regenerated extensor digitorum longus. In this regard therefore, the adaptive range of the regenerated soleus muscle was not significantly different from that of the extensor digitorum longus regenerating under the same conditions. This indicates that restriction of the adaptive range in a mature soleus muscle is not due to intrinsic properties of its myogenic cells. It is probably imposed by an extrinsic factor leading to irreversible shut-down of individual myosin heavy chain genes. On the other hand, myosin heavy chain-1 expression was significantly greater in the regenerated soleus than in the extensor digitorum longus innervated by the same nerve. Myosin heavy chain-1 and myosin heavy chain-2B were co-expressed in some regenerated soleus muscle fibres.     

Myosin heavy chain composition of single fibres and their origins and distribution in developing fascicles of sheep tibialis cranialis muscles

Summary The myosin heavy chain (MHC) composition of single muscle fibres in developing sheep tibialis cranialis muscles was examined immunohistochemically with monoclonal antibodies to MHC isozymes. Data were collected with conventional microscopy and computerized image analysis from embryonic day (E) 76 to postnatal day (PN) 20, and from adult animals. At E76, 23% of the young myofibres stained for slow-twitch MHC. The number of these fibres considerably exceeded the number of primary and secondary myotubes. By E100, smaller fibres, negative for slow-twitch MHC, encircled each fibre from the initial population to form rosettes. A second population of small fibres appeared in the unoccupied spaces between rosettes. Small fibres, whether belonging to rosettes or not, did not initially express slow-twitch MHC, expressing mainly neonatal myosin instead. These small fibres then diverged into three separate groups. In the first group most fibres transiently expressed adult fast myosin (maximal at E110–E120), but in the adult expressed slow myosin. This transformation to the slow MHC phenotype commenced at E110, was nearing completion by 20 postnatal days, and was responsible for approximately 60% of the adult slow twitch fibre population. In the other two groups expression of adult fast MHC was maintained, and in the adult they accounted for 14% (IIa MHC) and 17% (IIb MHC) of the total fibre numbers. We conclude that muscle fibre formation in this large muscle involves at least three generations of myotube. Secondary myotubes are generated on a framework of primary myotubes and both populations differentiate into the young myofibres which we observed at E76 to form rosettes. Tertiary myotubes, in turn, appear in the spaces between rosettes and along the borders of fascicles, using the outer fibres of rosettes as scaffolds.     

Identification of alpha-cardiac myosin heavy chain mRNA and protein in extraocular muscle of the adult rabbit

Summary Extraocular muscles contain both fast-twitch and multiply-innervated, tonic-contracting fibres. In rat, these fibres collectively express numerous myosin heavy chain isoforms including fast-type embryonic and neonatal, adult slow twitch type I and fast twitch type II, and a fast isoform unique to extraocular muscle. Immunocytochemical and Western blotting results are presented which suggest that, in rabbit, an additional species, the -cardiac myosin heavy chain, is present. The immunoreactive species is found in all rabbit extraocular muscles and in the rotatory extraocular muscles is expressed in almost all fibres which do not contain a fast myosin heavy chain. Positive identification of this isoform as the -cardiac myosin heavy chain was obtained by sequencing a cloned PCR product derived from extraocular muscle mRNA unique to the 3-end of rabbit -cardiac myosin heavy chain mRNA. This is the first unequivocal demonstration of -cardiac myosin heavy chain expression in extraocular muscle.     

Complex three-dimensional patterns of myosin isoform expression: differences between and within specific extraocular muscles

Because complex structural differences in adult extraocular muscles may have physiological and pathophysiological significance, the three-dimensional pattern of myosin heavy chain (MHC) isoform expression within the orbital and global layers of the muscle bellies compared with the distal tendon ends was quantitatively assessed. Three of the six extraocular muscles of adult rabbits were examined for immunohistologic expression of all fast, fast IIA/X, slow, neonatal and developmental MHC isoforms. The percentages of myofibers positive for each of these 5 myosin isoforms were determined in the orbital and global layers. There were relatively similar patterns of fast and slow MHC expression in the orbital and global layers of each of the three muscles examined. There were high levels of developmental MHC in the orbital layers, but significantly fewer developmental MHC positive myofibers in the global layer. The most variable expression was found with the neonatal MHC. There were significant differences between the longitudinal expression of the various isoforms in the middle of each muscle compared with the tendon end. In the orbital layer of all three muscles examined, the large numbers of fibers positive for fast MHC in the middle of the muscle dramatically decreased at the tendon end, with a concomitant increase in expression of slow myosin. There was a greater number of developmental MHC-positive myofibers at the tendon end than in the middle of the muscle in all three muscles examined. In the global layer, the IIA/X-positive myofibers comprised only half of the total number of fast-positive myofibers whereas in the orbital layer they comprised all or almost all of the fast positive myofibers. The configuration of the extraocular muscles is more complex than might be indicated by previous studies. The lateral rectus muscle had the most individual pattern of MHC expression when compared with the inferior rectus and inferior oblique muscles. Together with dramatic cross-sectional MHC fiber type differences between the orbital and global layers of the muscles, there are pronounced longitudinal differences in the proportions of myofibers expressing these five MHC isoforms in the middle region of the muscles and those in the distal tendon ends. This longitudinal progression appears to occur both within single myofibers, as well as within the series of myofibers that comprise the length of the muscle. We also confirm that the number of myofibers is reduced at the tendonous end while the cross-sectional area of each of the remaining myofibers is proportionally increased with regard to those in the muscle belly. Future studies may yet require two additional schemes for anatomic classification of the named extraocular muscles. One will be based on immunohistochemical features of their constituent myofibers as a supplement to classifications based on their electron microscopic appearance, innervation patterns or relative position with regard to the globe and orbit. Another will be based on the proportional length and longitudinal position of individual myofibers within an individual extraocular muscle.     

Immunocytochemical analysis of the perinatal development of cat masseter muscle using anti-myosin antibodies

Summary The developmental changes in myosin gene expression in the masseter muscle of embryonic and juvenile kittens were examined immunocytochemically using anti-myosin heavy chain antibodies of various specificities. In the mature cat, this muscle contains only two phenotypes, the majority of fibres are superfast, the rest being slow fibres. In foetal tissues, the histological appearance of bundles of myotubes, comprising a large central myotube surrounded by a rosette of smaller myotubes, strongly suggest the existence in the jaw muscle of primary and secondary fibres during development. Immunocytochemical data are consistent with the hypothesis that there are four types of fibre; two types of primary fibre as well as two types of secondary fibre. (1) Slow primaries stain strongly with an anti-slow myosin antibody throughout the period under study. These fibres transiently express embryonic but not foetal myosin. (2) Superfast primaries stain for embryonic/foetal and slow myosins in the perinatal period but progressively replace these myosins with superfast myosin during postnatal development. (3) Superfast secondaries initially express embryonic/foetal myosins, but later, beginning around the time of birth progressively replace these myosins with superfast myosin. These fibres do not express slow myosin. (4) Slow secondaries, which initially also express embryonic/foetal myosins, but which postnatally express slow or slow and superfast myosins and express only slow myosin in the adult. These four types of fibres are homologous to the four isotypes of limb muscle fibres and may be derived from distinct lineages of myoblasts.     

Immunocytochemical and electrophoretic analyses of changes in myosin gene expression in cat posterior temporalis muscle during postnatal development

Summary Changes in myosin gene expression during the postnatal development of the homogeneously superfast kitten posterior temporalis muscle were examined using immunocytochemical techniques supplemented by pyrophosphate gel electrophoresis and gel electrophoresis-derived enzyme linked immunosorbent assay (GEDELISA) of myosin isoforms. The antibodies used were polyclonals directed against the heavy chains of superfast and foetal myosins and monoclonals against the heavy chains of slow and fast myosins. The fibres of the posterior temporalis in the newborn kitten stained almost uniformly with the anti-foetal myosin antibody and the largest of these fibres stained strongly for superfast myosin. A subpopulation of fibres staining for superfast myosin also stained lightly for slow myosin. These slow staining fibres were evenly distributed in the centres of muscle fibre bundles, reminiscent of primary fibres in limb fast muscle. During subsequent development, slow myosin staining disappeared and superfast myosin replaced foetal myosin so that by 50 days the muscle was virtually homogeneously superfast as in the adult. Fast myosin was never expressed at any stage. It is proposed that fibres staining transiently for slow myosin are superfast primary fibres which are homologous to fast primary fibres recently described in regions of limb muscles devoid of slow fibres in the matured animal. Other jaw-closing muscles have significant populations of slow fibres in the mature animal and it is postulated that there exists in these muscles a second class of jaw primary fibres, the slow primary fibres, in which slow myosin synthesis would be sustained in the adult. It is suggested that the myogenic cells of jaw-closing and limb muscles are of two distinct types preprogrammed to express different muscle genes.     

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures

Summary We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.     

Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle

Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis , a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity.     

Effects on skeletal muscle fibres of diabetes and Ginkgo biloba extract treatment

Combined cytophotometric and morphometric analysis of muscle fibre properties and myosin heavy chain electrophoresis were performed on extensor digitorum longus and soleus muscles from healthy rats and rats with streptozotocin-induced diabetes. Moreover, the protective effect of Ginkgo biloba extract, a potent oxygen radical scavenger, on diabetic muscles was investigated. Changes in fibre type-related enzyme activities, fibre type distribution, fibre cross areas and myosin isoforms were found. In muscles of diabetic rats, a metabolic shift was measured mainly in fibres with oxidative metabolism. Fast-oxidative glycolytic fibres showed a shift to more glycolytic metabolism and about a third transformed into fast-glycolytic fibres. Slow-oxidative fibres became more oxidative. Fibre atrophy was measured in diabetic muscles dependent on fibre type and muscle. Different fibre types atrophied to a different degree. Therefore, a decreased area percentage of slow fibres and an increased area percentage of fast fibres of the whole muscle cross section in both muscles were found. This is supported by reduced slow and increased fast myosin heavy chain isoforms. These alterations of diabetic muscle fibres could be due to less motion of diabetic rats and diabetic neuropathy. After treatment with Ginkgo biloba extract, enzyme activities were increased mainly in oxidative fibres of diabetic muscles, which was interpreted as protective effect. Generally, the soleus muscle with predominant oxidative metabolism was more vulnerable to diabetic alterations and Ginkgo biloba extract treatment than the extensor digitorum longus muscle with predominant glycolytic metabolism.     

Myogenic and neurogenic regulation of myosin gene expression in cat jaw-closing muscles regenerating in fast and slow limb muscle beds

Summary Immunocytochemical techniques were used to study changes in myosin gene expression during the regeneration of the cat posterior temporalis muscle transplanted into the bed of either the fast extensor digitorum longus (EDL) or the slow soleus muscle. Strips of the posterior temporalis, a homogeneously superfast muscle, were treated with Marcaine and then transplanted into limb muscle beds which had been completely cleared of host muscle fibres. The regenerates were examined 6 to 224 days after surgery. Early regenerates in both muscle beds reacted with antibodies against the heavy chain of foetal, slow and superfast myosins, but not with antibodies against fast myosin. In the long-term, regenerates innervated by the EDL nerve expressed only superfast myosin whereas in the regenerates innervated by the soleus nerve most fibres expressed only slow myosin and only a few fibres reacted exclusively with the anti-superfast myosin antibody even after 210 days. In contrast, EDL and soleus muscles regenerating in their own beds expressed foetal, slow and fast myosin, but did not express superfast myosin. The isometric contraction times of the various types of regenerates reflected the types of myosin synthesized. It is concluded that jaw and limb muscle cells exist as two distinct allotypes, each having a distinct repertoire for the expression of adult isomyosins, and that within that repertoire isomyosin gene expression can be modulated by the nerve. Thus, myosin gene expression in skeletal muscle fibres is regulated by both myogenic and neurogenic mechanisms.     

Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat

This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca²⁺ activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.     

Myosin isoform transitions in four rabbit muscles during postnatal growth

Summary Four rabbit muscles (i.e. semimembranosus proprius, psoas major, biceps femoris and longissimus lumborum), differing in their fibre type composition in the adult, were investigated during postnatal development. Muscle samples were taken at 1, 7, 14, 21, 28, 35, 49 and 77 days of age. Complementary techniques were used to characterize myosin heavy chain (MHC) isoform transitions, i.e. SDS-PAGE, immunocytochemistry and conventional histochemistry. Good accordance was found between electrophoretic and immunocytochemical techniques. Our results show that rabbit muscles were phenotypically immature at birth. At 1 day of age, perinatal isoform represented 70–90% of the total isoform content of the muscles. Two generations of myofibres could be observed on the basis of their morphology and reaction to specific antibodies. In all muscles, primary fibres expressed slow MHC. In contrast, secondary generation of fibres never expressed slow MHC in future fast muscles, while half of them expressed slow MHC in the future slow-twitch muscle, the semimembranosus proprius. During the postnatal period, all muscles displayed a transition from embryonic to perinatal MHC isoforms, followed by a transition from perinatal to adult MHC isoforms. These transitions occured mainly during the first postnatal month. The embryonic isoform was no longer expressed after 14 days, except in longissimus where it disappeared after 28 days. On the contrary, large differences were found in the timing of disappearance of the perinatal isoform between the four muscles. The perinatal isoform disappeared between 28 and 35 days in semimembranosus proprius and 35 and 49 days in psoas and biceps femoris. Interestingly, the perinatal isoform was still present in 6% of the fibres in longissimus at 77 days, the commercial slaughter age, denoting a great delay in the maturation. Fate of each generation of fibres differed between muscles.     

A quantitative study of cholinesterase in myoneural junctions from rat and guinea-pig extraocular muscles

1. Cholinesterase staining of rat and guinea-pig extraocular muscle shows focally and multiply innervated fibres.2. The distribution of cholinesterase activities in the populations of focal endings (from singly innervated fibres) and fine motor endings (from multiply innervated fibres) was determined by a sensitive radiochemical method.3. Focal endings had a greater cholinesterase activity than fine motor endings.4. It is suggested that this difference in enzyme activities is related to the functional difference between twitch and slow fibres in extraocular muscle.     

Calcium sensitivity and myofibrillar protein isoforms of rat skinned skeletal muscle fibres

We investigated the calcium sensitivity for tension generation of different fibre types and the possible correlation between calcium sensitivity and the presence of distinct regulatory protein and myosin light chain (MLC) isoforms in rat skinned skeletal muscle fibres. Fibre types 1, 2A and 2B were identified by electrophoretic analysis of myosin heavy chain (MHC) isoforms. Fibres showing more than one MHC isoform were discarded. Type 1 fibres from the soleus showed a higher pCa (–log₁₀ [Ca], where [ ] denotes concentration) threshold and a lower slope of pCa/tension curve than type 2 extensor digitorum longus (EDL) fibres; between type 2 fibres, type 2B showed the higher slope of pCa/tension curve. Type 1 fibres from different muscles showed similar calcium sensitivities when containing only the slow set of regulatory proteins and MLC; when both slow and fast isoforms were present, calcium sensitivity shifted toward fast type fibre values. Type 2A fibres from different muscles showed a similar calcium sensitivity, independently of the set (purely fast or mixed) of regulatory proteins and MLC. It is suggested that when both fast and slow isoforms of regulatory proteins and of MLC are present in a muscle fibre, calcium sensitivity is dictated mainly by the fast isoforms.     

Stretch activation and myosin heavy chain isoforms of rat,rabbit and human skeletal muscle fibres

The underlying mechanism of stretch-induced delayed force increase (stretch activation) of activated muscles is unknown. To assess the molecular correlate of this phenomenon, we measured stretch activation of single, Ca2+-activated skinned muscle fibres from rat, rabbit and the human and analysed their myosin heavy chain complement by SDS gradient gel electrophoresis. Stretch activation kinetics was found to be closely correlated with the myosin heavy chain isoform complement (I, IIa, IId/x and IIb). In hybrid fibres containing two myosin heavy chain isoforms (especially IId and IIb), the kinetics of stretch activation depended on the percentage distribution of the two isoforms. Muscle fibres of the same type but originating from different mammalian species exhibited similar kinetics of stretch activation. Considering the differing unloaded shortening velocities of these fibres, the time-limiting factors for stretch activation and unloaded shortening velocity appear not to be the same. The stretch activation kinetics of the fibre types IIB, IID and IIA more likely seemed to follow a Normal Gaussian distribution than that of type I fibres. Several type I fibres had extraordinarily slow kinetics. This observation corroborates biochemical data indicating the possible existence of more than one slow myosin heavy chain isoform