CHAPTER 3. Quantification of fibre type regionalisation: an analysis of lower hindlimb muscles in the rat

Transcription

1 MEASURES OF FIBRE TYPE REGIONALISATION Quantification of fibre type regionalisation: an analysis of lower hindlimb muscles in the rat L.C.Wang and D.Kernell Department of Medical Physiology, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands Journal of Anatomy (in press) 31

2 Abstract Newly developed concepts and methods for the quantification of fibre type regionalisation were used for comparison between all muscles traversing the ankle of the rat lower hindlimb (n = 12). For each muscle, cross-sections from the proximodistal midlevel were stained for myofibrillar ATPase and classified as type I ( slow ) or II ( fast ). For the 11 fast muscles (i.e. all except soleus), the muscle outline and the position of each type I fibre were digitised for further computer processing. Two potentially independent aspects of type I fibre regionalisation were evaluated quantitatively: (1) the degree to which type I fibres were restricted to a limited portion of the total cross-section area ( area regionalisation ); (2) the extent and direction of the difference (if any) between the centre of the muscle cross-section and the calculated centre for the type I fibre cluster ( vector regionalisation ). Statistical analysis showed that type I fibres were vector regionalised in practically all the investigated muscles and area regionalised within most of them, the only consistent exceptions being peroneus brevis and peroneus digitorum 4, 5. In muscles with a high degree of area regionalisation the population of type I fibres also had a markedly eccentric intramuscular position (i.e. high vector regionalisation). A significant relationship was observed between the relative position of a muscle within the hindlimb (transverse plane) and the direction and degree of its type I fibre eccentricity. On average, the degree of type I fibre eccentricity was greater for muscles remote from the limb centre than for those situated more centrally. In addition, the intramuscular concentration of type I fibres was typically greatest towards the centre of the limb, the most striking exception being tibialis posterior. For the slow soleus muscle, which is centrally placed within the limb, our analysis concerned the type II fibres, which were found to be weakly vector regionalised but not significantly area regionalised. It is concluded that, within muscles of the rat s lower hindlimb, fibre type regionalisation is a general and graded phenomenon which may reflect differentiating (embryological?) mechanisms of a transmuscular significance. Furthermore, the analysis demonstrated the usefulness of our new methods and concepts for the quantification of fibre type regionalisation. Key words: Skeletal muscle; Muscle fibre type differentiation Introduction Skeletal muscles are markedly heterogeneous in composition, containing mixtures of fibers with different histochemical and physiological properties (e.g. slow and various categories of fast fibres; for reviews see Burke, 1981; Kernell, 1992). These various types of fibre have properties that are optimised for different motor tasks. Slow fibres are metabolically efficient for near isometric or slow concentric postural contractions and restricted movements whereas fast fi- 32

3 MEASURES OF FIBRE TYPE REGIONALISATION bres are needed for generating the high power and shorting velocity of rapid motor acts (e.g. Rome et al., 1988). Apart from the variation at the level of single fibres and motor units, there are regional differences within muscles with respect to their fibre and unit composition ( regionalisation ; for review see Kernell, 1998). However, although the phenomenon of fibre type regionalisation was noted a long time ago (e.g. Gordon & Phillips, 1953), it has so far attracted little systematic and quantitative study. In the adult rat it is well known that slow fibres tend to be more common in deep than in superficial (subcutaneous) regions of the large ankle extensors and flexors (e.g., gastrocnemius, tibialis anterior; Armstrong & Phelps, 1984) but it is still unclear how consistent this behaviour is across different muscles and in different species. An increased knowledge concerning adult features of fibre type regionalisation is important for practical as well as theoretical reasons. From practical points of view, as has been pointed out repeatedly (e.g. Pullen, 1977 a), it is obviously important to know about regional fibre type distributions in muscles when studying their properties using localised sampling techniques (e.g. fine-wire electromyography; muscle biopsy). Theoretically, knowledge about fibre type regionalisation is essential in relation to questions concerning the control of muscle fibre properties. Fibre type regionalisation must have arisen because of topographically organised factors that contribute to muscle fibre differentiation. Some of these factors are clearly already active during early embryological development; even in absence of motor innervation, hindlimb muscles may develop characteristic patterns of fibre type regionalisation (Condon et al b). Later during development and in adult life, the manner in which muscle fibres are innervated and used also has pronounced influences on their properties. For the further analysis of the combined results of the multiple (and partly regionalised) mechanisms for muscle fibre differentiation, a reliable quantification of the degree and intralimb direction of fibre type regionalisation in adult muscles is desirable. Such quantification methods should be appropriate for systematic comparisons of normal properties between muscles and/or for investigations concerning the effects of experimental interventions (e.g. denervation and reinnervation; altered work load; stretch; etc.). Within muscle cross-sections, the methods so far used have mainly been based on comparisons of fibre composition between a limited number of characteristic sampling areas ( deep vs. superficial ; e.g. Johnson et al. 1973). In the context of a continuously varying fibre type composition, such methods give only a coarse assessment of the direction of regionalisation (the sites of the characteristic sampling regions are typically chosen by eye) and they may produce highly uncertain estimates of the degree of fibre type regionalisation (large sampling errors are possible). In a more refined (and more laborious) version of such techniques, many sampling areas were systematically aligned across the muscle in a preset direction (e.g. medial < > lateral, superficial < > deep), resulting in a multisample profile of composition along the predetermined axis (Pullen, 1977 a). Also in this method there is an appreciable element of subjective choice 33

4 (e.g. for the directions and positions of the analysis axes) and the results are not easily quantified in numerical terms that can be used for comparisons of the direction and degree of regionalisation between (differently treated) muscles. In this study, we present new methods and concepts for the quantification of fibre type regionalisation within muscle cross-sections. We have used the methods for a comparative study of the direction and degree of slow type I fibre regionalisation between all muscles traversing the ankle in the rat lower hindlimb. Large and gradual differences were found between the various muscles, and a coherent picture emerged with regard to the relationships between the degree and direction of fibre type regionalisation and intralimb muscle position. Methods The measurements were made on muscles from adult female Wistar rats, weighing g. Before the operations, the animals were kept in conventional plastic cages and fed ad libitum with standard laboratory food and tap water. General preparative procedures Before dissecting the muscles, the animals were anaesthetised with pentobarbitone (50 mg/kg i.p.). We studied the 12 muscles listed in Table 1. Flexor digitorum and hallucis longus (FD) was treated as a single muscle; these 2 muscles appeared fused and could not be separated without causing tissue damage. Furthermore, the 2 very thin muscles peroneus digiti quarti and quinti (PD) were also processed together to avoid undue tissue damage. Among the lower hindlimb muscles listed by Greene (1935), all were included in our study except popliteus, which is relatively short and does not cross the ankle joint, occupying a complex oblique position within the limb. Before cutting the tendons, the posterior and/or lateral aspects of each muscle were marked with water-insoluble coloured stains (yellow and blue), using Table 1. List of muscles and muscle abbreviations Name Abbreviation Plot symbol Extensor digitorum longus ED E Extensor hallucis longus EH H Flexor digitorum & hallucis longus FD F Gastrocnemius lateralis GL L Gastrocnemius medialis GM G Peroneus brevis PB B Peroneus digiti 4, 5 PD D Peroneus longus PE P Plantaris PL A Soleus SO S Tibialis anterior TA T Tibialis posterior TP I 34

5 MEASURES OF FIBRE TYPE REGIONALISATION a fine painter s brush. This technique for the labelling of rotational muscle position gave reproducible results for all muscles except for the very thin extensor hallucis longus ( ~ 1 mm thick) for which angular measurements were not included in Table 3. Tendons were cut close to the muscle tissue and each muscle was weighed (accuracy ± 1 mg), fixed to a metal clamp at its measured in situ length, and fixed by immersion in isopentane kept at its freezing point by liquid nitrogen. In a few cases, sections were cut from whole lower limbs (Fig. 4a). In these instances, the limbs were first dissected with the animal under general anaesthesia (pentobarbitone 50 mg/kg i.p.). At a level around the proximodistal middle of the lower hindlimb, the various muscles were gently separated from each other and stained with Alcian blue. In addition, at this level, a segment both of the fibula and tibia were freed from surrounding tissues and removed. After this the limb was fixed to a clamp and stretched to its original length, as measured between the knee and the ankle. These procedures led to a moderate degree of distorsion of muscle shape (loosened muscles retracting from each other, cf. Fig. 4a) but separating the muscles was needed in order to make it possible to recognise the borders between individual muscles in later cryostat sections. Following these initial preparations, the limb was severed above the knee and further freeze-fixed and processed with the same methods as Table 2. General muscle properties* Muscle n Mwt (mg) MArea (mm 2 ) EqD (mm) FibN FibD (mm -2 ) GL 7 787± ± ± ± ± 10.6 TA 6 633± ± ± ± ± 2.7 GM 7 713± ± ± ± ± 7.3 FD 8 392± ± ± ± ± 8.2 PL 7 297± ± ± ± ± 7.0 SO 8 129± ± ± ± ± 19.6 ED 7 152± ± ± ± ± 5.2 PE 8 145± ± ± ± ± 25.5 TP 8 185± ± ± ± ± 10.5 PB 8 117± ± ± ± ± 10.6 PD 7 56± ± ± ± ± 13.2 EH 7 13± ± ± ± ± 20.1 * Means ± S.D. n, number of measured muscles in each group; Mwt, muscle weight; MArea, muscle cross-section area; EqD, equivalent muscle diameter; FibN, number of target fibres (type II for SO, type I for all other muscles); FibD, target fibre density (fibres per mm 2 ). Muscle abbreviations as in Table 1. Muscles placed in order of size according to their cross-section area (MArea). 35

6 Tabel 3. Regionalisation parameters* Muscle C FR (%) FRh (%) FRs (%) VL (%) MLD (%) VA (deg) GL ± ± ± ± ± 6 TA ± ± ± ± ± 6 GM ± ± ± ± ± 12 FD ± ± ± ± ± 25 PL ± ± ± ± ± 12 SO ± 17.7 # 76.2± 19.3 # 7.0± ± ± 56 ED ± ± ± ± ± 24 PE ± 12.2 # 75.3± ± ± ± 19 TP ± ± ± ± ± 16 PB ± 2.8 # 84.6± 4.2 # 9.9± ± ± 12 PD ± 15.2 # 67.0± 16.8 # 11.2± ± ± 24 EH ± ± ± ± 6.2 n.i. * Means ± S.D. C FR, fibre region correction factor, i.e. percentage of muscle area (MArea) expected to be covered by target fibres in case of a uniform distribution (see Text); FRh, target fibre region calculated by convex hull method (% of MArea); FRs, target fibre region calculated by sector method (% of MArea); VL, length of target fibre vector (% of EqD); MLD, midline distribution (% of target fibres on target side of middle of muscle section); VA, angle of target fibre vector (degrees). Target fibres were type II in SO and type I in all other muscles. Number of muscles per group as in Table 1. Statistics (t tests, significance for P < 0.05 or better): All VL values significantly greater than zero. All MLD values significantly greater than 50%. FRh values significantly smaller than C FR for all except PB, PD, PE and SO (labelled # ). FRs values significantly smaller than C FR for all except PB, PD and SO (labelled # ). VA values for EH-muscle technically suspect and not included (n.i.). those used for individual muscles. After the end of the dissections, the animals were killed with an overdose of pentobarbitone (i.p.). Sectioning and histochemical procedures In the cryostat (maintained at -20 C), a transverse block with a length of 3-4 mm was cut from the middle of each muscle. Starting approximately at the proximodistal midlevel, µm serial sections were cut in a direction towards more distal muscle portions. The sections were mounted alternately onto 2 glass slides that had been freshly coated with 1% gelatin (to avoid loss or wrinkling of the sections). Both slides were processed according to standard staining procedures for myofibrillar ATPase (matpase), one after acid preincubation at ph 4.3 (ac-at- Pase; Brooke & Kaiser, 1970) and the other after prefixation with paraformaldehyde and alkaline preincubation at ph 10.3 (alk-atpase; Guth & Samaha, 1970); for details concerning our current 36

7 MEASURES OF FIBRE TYPE REGIONALISATION staining procedures, see Lind and Kernell (1991). Fibres classified as type I were consistently dark in ac-atpase and light in alk-atpase, and the reverse staining pattern was seen for the type II fibres (Fig. 1a-b). In the present investigation, we did not distinguish between the various subtypes of type II fibre (e.g., types IIA, IIB etc.; Brooke & Kaiser, 1970; Burke, 1981; Lind & Kernell, 1991). For the tracing of muscle fibre distribution, we used sections stained for ac-atpase. General measurement procedures In all mucles except soleus, the fraction of type I fibres was much smaller than that of the type II fibres. Therefore, in all muscles except soleus, fibre type regionalisation was assessed by determining the distribution of type I fibres (in soleus this was done for type II fibres). The anatomical measurements were made on a high-contrast photocopy of the respective muscle cross-section, using a graphic tablet connected to a PC and custom-made software. The photocopy was made directly from the stained histological section using a microfiche copying machine with exchangeable lenses. All ambiguous details on the photocopy were evaluated by examining the original section through a microscope at high magnification. The graphic tablet was used for tracing the outline of the muscle cross-section. Furthermore, across the whole muscle section, the position of each target fibre (type I fibre in all muscles except soleus) was digitised and stored in a computer file. On the graphic tablet, muscle images were routinely positioned with the posterior muscle face at the top and lateral towards the left (cf. Figs. 1-2). The tracing program could handle photocopies of any size; copies larger than the graphic tablet could be moved in steps across the measurement space. Custom-made software calculated the cross-sectional area of the muscle (MArea; substantial internal empty regions, e.g. tendon sheets, were not included). As a linear measure of muscle size, the equivalent muscle diameter (EqD) was calculated from MArea using the formula for a circle. The overall density of the target fibres (FibD) was expressed as the number of fibres per mm 2. Also, using the positions of all the target fibres, the program made calculations resulting in several measures for fibre type regionalisation. Measures of fibre type regionalisation Figures 1 and 2 show examples of how the target fibres were distributed in the 12 sampled muscles. As is evident from these illustrations, there were 2 ways in which the fibre type regionalisation was expressed. 1. Direction and degree of target fibre eccentricity: vector regionalisation. In most muscles, the population of target fibres was clearly located eccentrically. This was also evident in muscles for which the target fibre region covered most of the cross-section (e.g. PB, Fig. 2). We quantified this aspect of regionalisation by computing a target fibre vector, extending from the centre of the cross-section to the centre of the target fibre cluster (arrow, Fig. 1c). The geometric centre of the cross-section was found by calculating the centre of mass for a sheet with the same shape and a uniform thickness 37

8 Fig. 1. (a, b) Serial cross-sections from plantaris muscle, stained for myofibrillar ATPase after acid (a) and alkaline (b) preincubation. The slow type I fibres are black in a and white in b. (c, d) Digitised versions of section in panel a, showing muscle outline and the positions of all type I fibres. (c) An arrow ( type I fibre vector ) has been drawn from the centre of mass of the whole muscle section to the centre of mass for all the type I fibre sites (see Methods for calculations). In addition, a straight interrupted line has been drawn at right angles to the vector arrow and through the muscle centre (line used for midline distribution analysis, see Methods). The type I fibre region is enclosed by interrupted lines according to borders calculated using the convex hull method. In d, the region of type I fibres has been enclosed by interrupted lines as calculated using the sector method (20 sectors; interrupted lines). The fold in the lower-right part of the section represents a tendon sheat. and density. Correspondingly, the geometric centre of the target fibre population was found by computing the joint centre of mass for all the target fibre positions, each fibre site being represented by a point with equal mass; the measured interfibre distances were used for applying standard rules of moment arms in recursive calculations (see Appendix). One major advantage of this procedure is that a position of the population centre can be unequivocally computed for fibre distributions of 38

9 MEASURES OF FIBRE TYPE REGIONALISATION any shape, skewedness, size or extent. We expressed the size of the target fibre vector (VL) as a percentage of the equivalent muscle diameter (EqD, see above). The direction of the vector, the vector angle (VA), was given in degrees (medial direction 0 and 360, posterior 90, lateral 180, anterior 270 ). For comparisons between values straddling the 360 vs 0 transition, 360 were added to angles within the range Hence, in Fig. 4b some X-values exceed 360. As a supplementary parameter for the degree of target fibre eccentricity we also calculated the midline distribution pattern (MLD) i.e. the unevenness of fibre distribution across the middle of the the muscle cross-section. In this analysis an imaginary midline was drawn through the muscle at right angles to the target fibre vector and through the muscle centre of mass (Fig. 1c, interrupted straight line). The MLD parameter was equal to the percentage of target fibres situated on the same side of the midline as the centre of mass for the cloud of target fibres sites. 2. Size of target fibre region: area regionalisation. Muscles clearly differed with regard to the relative size of the region containing the type I fibres; in relation to the total crosssection area, this target fibre region (FR) was, for instance, relatively small in gastrocnemius medialis (GM, Fig. 2) and much larger in peroneus brevis (PB). We quantified this aspect of fibre type regionalisation by calculating the size of the target fibre region, expressed as a percentage of the whole cross-section area. In doing these calculations we used 2 alternative methods for delineating the target fibre region: the convex hull method (FRh) and the sector method (FRs). The convex hull method calculates the course of a line enclosing all the target points in a manner analogous to the course of a rubber band stretched along the outside of the whole cluster (Fig. 1c, interrupted line); the curvature of this line is outward convex all around. Algorithm for such calculations may be found in standard handbooks (e.g. Cormen et al. 1990). In fibre clusters with a shape including one or more substantial concavities, the area calculated using the convex hull method might include disturbingly large portions containing no target fibres. Therefore, as an alternative approach we developed the sector method, a procedure for regional circumscription that allows concave cluster-borders. In this method, the space surrounding the middle of the fibre population (i.e. its calculated centre of mass ) was subdivided into several equal-angle sectors and, within each sector, the fibre most remote from the population centre was identified. The outer border of the target fibre region was then defined by joining these most remote fibres with straight lines (Figs. 1d, 2; interrupted lines). Due to the manner in which these lines are calculated, occasional fibres may be lost and fall just outside the circumscribed area (cf. Fig. 1d). However, provided the number of sectors is adequate, the lost fibres will typically be very close to the regional border and such inaccuracies will not produce important quantitative errors in the area of the target fibre region. In the sector method, an adequate choice must be made with regard to the number of sectors used for the calculations: too few will 39

10 produce large errors (many lost fibres falling far outside the circumscribed region) and too many will produce very complex and unnatural looking clusterborders. In the present analysis, 20 sectors were used as a standard; a smaller number was typically used if the total number of muscle fibres was below about 60 and a larger one for fibre numbers exceeding (see Fig. 2, Legend). The choice was guided by visual inspection of the results, i.e. this method is less automatic than the convex hull method. When using the 2 measures for assessing the target fibre region (FRh and FRs) it is important to realise that a line just enclosing the target fibres would lie inside the muscle border; hence, even for a uniform fibre distribution the FR-value would be expected to be smaller than the area of the whole cross-section. The approximate extent of this discrepancy depends on the absolute number of the fibres. We calculated a fibre region correction factor (C FR, see Appendix) which gives an estimate of how much smaller than the total muscle cross-section area the circumscribed fibre region would be expected to be in case of a homogeneous fibre distribution. In Table 3, this correc- Fig.2. Digitised midlevel sections for the analysed muscles, all taken from the same rat. Type I fibres shown in all sections except soleus (SO, type II fibres indicated). Target fibre regions are enclosed by interrupted lines calculated according to the sector method (18 sectors for PD; 20 for ED, EH, PB, PE, PL, TA, TP; 30 for GM, GL, FD, SO). To facilitate comparisons of regionalisation features, all sections are shown at about the same absolute size; note differences in scale. Same rotational position for all sections (i.e. posterior up, medial right). Muscles arrranged in order of descending size (cf. Tables 2-3). Bars, 1mm. 40

11 MEASURES OF FIBRE TYPE REGIONALISATION Fig.3. Statistics for measures of type I fibre regionalisation. (a) Type I fibre vector length (%, parameter VL in Table 4) vs type I fibre hull-region (%, FRh). (b) Type I fibre vector length (%, VL) vs midline distribution parameter (%, MLD). (c) Type I fibre sector-region (%, FRs) vs type I fibre hull-region (%, FRh). (d) Type I fibre hull-region (%, FRh) vs muscle weight (g, Mwt). Linear regression lines in a-d calculated by method of least squares (see Table 4 for correlations). tion factor is expressed as a percentage of MArea (i.e. multiplied by 100). Statistics Whenever applicable, mean values are given ± S.D. Pearson correlation coefficients were calculated for analysing the degree of covariation between different variables. Differences in properties between different groups of muscles were analysed using standard t test procedures. Calculations were made using Excel (Microsoft) or the software package SYSTAT. Cases with P < 0.05 were considered statistically significant. Results The 11 fast muscles will first be considered together; unless stated otherwise, all statistical calculations and illustrations concern these 11 muscles. General muscle dimensions and type I fibre densities The muscles varied considerably in weight and in their midlevel cross-sectional area (MArea, Mwt in Table 2). There was a high degree of correlation between weight and midlevel dimensions: 41

12 Fig.4. Analysis of how the intralimb position of muscles was related to the direction and degree of type I fibre regionalisation. (a) Digitised cross-section of whole lower hindlimb through level close to mid proximodistal region between knee and ankle. Outline and calculated centre of mass (small circle) shown for each muscle. Lines drawn from the calculated centre of mass for the whole limb cross-section to the centre of mass for each muscle. The length and angle of each such line represent the polar coordinates for the respective muscle, as seen from the limb centre. (b, c) Charts showing the relationship between polar muscle coordinates within the limb and the direction and extent of type I fibre vector regionalisation within each muscle. For the plot in b, type I fibre vector angles (VA parameter, cf. Table 3) were plotted vs inverse values of the respective polar muscle coordinates (i.e. actual coordinate ; muscle vs limb angle ). The continuous line is the calculated regression line (r = , n = 10, P < 0.001) and the interrupted line is the unity line (x = y). Plot symbols B and T overlap (muscles PB and TA). In c values for type I fibre vector length (%; parameter VL, Table 3) are plotted against the lengths of the corresponding polar muscle coordinates (%, normalised vs limb equivalent diameter; muscle vs limb eccentricity ). Calculated regression line, r = +0.85, n = 10, P < The plots of b and c include all studied fast muscles except extensor hallucis longus for which no reliable measurements of type I fibre vector angle were available. 42

13 MEASURES OF FIBRE TYPE REGIONALISATION heavy muscles were also thick (Table 4). The muscle with the largest weight and midlevel cross-section area was gastrocnemius lateralis (787 mg and 41.6 mm 2 respectively); the one with the smallest weight and midlevel cross-section area was extensor hallucis longus (13 mg and 0.81 mm 2 respectively). When calculated for the total crosssectional area of each fast muscle, there was a wide variation in the mean overall density of type I fibres, ranging from 9.2 fibres/mm 2 for tibialis anterior up to fibres/mm 2 for extensor hallucis longus (Table 2). When calculated for all individual muscles together, a significant negative correlation was obtained between type I fibre density and measures of muscle size (e.g. r = , n = 80, P < for relationship with equivalent muscle diameter): i.e., on average, large hindlimb muscles tended to be faster than the smaller. When calculated for mean values from each muscle species, these relationships did not attain full significance (Table 4). Type I fibre regionalisation One of our main aims was to determine which of the 11 fast muscles were truly regionalised with regard to their type I fibres and, if so, how the muscles differed in the direction and degree of this regionalisation. We looked at 2 diffferent aspects of regionalisation that might, potentially, exist indepently of each other: (1) the area regionalisation, meaning that the type I fibres were distributed within an area significantly smaller than that of the whole cross-section; and (2) the vector regionalisation, meaning that the type I fibres were eccentrically placed within the muscle. A centrally placed target fibre cloud might have an extent much less than that of the crosssection but still have a target fibre vector length (VL) equal to zero. Conversely, target fibres might be found across the whole muscle section (i.e. close to 100% FR) but with different densities of distribution in different directions, delivering a non-zero vector-length value. Area-regionalisation.For a significant degree of area-regionalisation we required that the mean value for either one of the target fibre region parameters (FRh, FRs) was significantly smaller than the value expected for a uniform fibre distribution (i.e. smaller than the fibre region correction factor, C FR ; Table 3). Table 3 lists all the mean values for FRh, FRs and C FR (the latter values calculated, for each musclegroup, from the respective mean number of target fibres (FibN); see Appendix). The parameter FRs varied over a more than 2-fold range from about 32% for the gastrocnemii and tibialis anterior up to about 85% for peroneus brevis (Table 3, cf. Fig. 2). As might be expected (see Methods) the parameter FRh was consistently somewhat larger than FRs (mean ratio for all, including soleus: 1.09 ± 0.06, n = 88), and the 2 parameters showed a very high degree of correlation (Fig. 3c, Table 4). The statistical analysis (t tests) showed that the mean values for FRh were significantly smaller than C FR for all fast muscles except the three peroneal ones (peroneus longus, brevis and digitorum 4&5; PE, PB, PD in Table 3). Very similar and partly overlapping results were obtained using the parameter FRs: the type I fibre regions were significantly smaller than C FR for all fast muscles 43

14 Table 4. Correlation between various muscle properties and measures of type I fibre regionalisation* Mwt EqD EqD FibD ns (-0.562) FibD FRh ns FRh FRs ns FRs MLD (-0.590) MLD VL VL VA ns ns ns ns ns ns ns * Correlation coefficients for pairwise comparisons, calculated for average values of the indicated parameters for the 11 fast muscles. For measured parameters, see Tables 2, 3; ns, not significant (P > 0.1). Values in brackets indicate suggested significance (0.1 > P > 0.05). except peroneus brevis and peroneus digitorum 4, 5 (PB, PD; Table 3). Thus, according to our criteria for arearegionalisation, only 2 of the 11 fast muscles (PB and PD), consistently failed to show a significant degree of arearegionalisation. Vector-regionalisation.For a significant degree of vector regionalisation we required that the target fibre vector (arrow, Fig. 1c) should have a length significantly greater than zero and show a consistent behaviour with regard to its direction. As a minimum requirement with regard to vector direction we demanded a standard deviation for parameter VA of not more than 90 (i.e., total range of variation at least less than about half a circle). Table 3 lists all the mean values for the vector parameters. The vector length parameter (VL, normalised in relation to muscle diameter) varied over a 5-fold range, from about 5 % for extensor hallucis longus up to about 28 % for gastrocnemius medialis. The mean value of VL was significantly greater than zero in all cases and the vector angle (VA) had a S.D. of 25 or less for all the fast muscles with reliable angular measurements. With regard to the vector angle, strikingly variable results were obtained, probably for technical reasons (see Methods), from the tiny extensor hallucis longus (EH; SD of ± 117 ). Thus, in summary, all 10 fast muscles from which reliable measurements were available (including peroneus brevis (PB) and peroneus digitorum 4, 5 (PD)), showed a significant degree of vector-regionalisation. Relationships between different regionalisation measures Our 2 main measures of regionalisation were strongly correlated with each other, 44

15 MEASURES OF FIBRE TYPE REGIONALISATION i.e. in muscles with a small target fibre region (small FR) the type I fibres also had a highly eccentric localisation, lying towards one side of the muscle (large VL; see Table 4 for statistics, Fig. 3a for graphic display). No correlation was found between either one of these measures and the direction of eccentricity (VA; see Table 4). As a supplementary eccentricity-parameter MLD varied from > 95% for highly regionalised muscles (GL, GM, TA) down to close to 60% for the least regionalised examples (e.g. EH; Table 3). As might be expected, the 2 measures for target fibre eccentricity were highly correlated, i.e. they measured similar aspects of muscle fibre distribution (MLD vs. VL, Fig. 3b, Table 4). The coefficient of variation (S.D. / mean) was, however, significantly lower for the MLD parameter than for VL (MLD 8.6 ± 4.7 vs. VL 32.4 ± 18.2 (S.D.) %, n=11, P < 0.001). For all 11 fast muscles, the MLD parameter was significantly greater than 50 % (i.e. greater than the chance value; P < 0.02 or better). Intralimb muscle position and regionalisation Figure 4a shows a digitised display of a cross-section through the whole lower hindlimb, cut at a level close to the middle between knee and ankle. For most of the individual muscles, such a section would occur close to their mid proximodistal extent (less true for the more distally placed SO and EH than for the others). In 4 hindlimbs, we used digitised sections such as that of Fig. 4a for measuring the crosssectional position of each muscle within the limb. Our standard centre of mass calculations (see Methods) were employed for determining the centre of each whole-limb cross-section and that for each one of the component muscles. For each muscle represented in the cross-section, the position was defined by its polar coordinates, i.e. by the distance and direction of the muscle centre as seen from the limb centre. In Fig. 4b-c, relationships are plotted between mean measures of intralimb muscle position and intramuscular type I fibre regionalisation. The extent of intramuscular vector regionalisation (i.e. the degree of target fibre eccentricity) was significantly related to the distance of a muscle from the limb centre: more eccentrically placed muscles also had more eccentrically placed type I fibres (Fig. 4c). Furthermore, the angular direction of type I fibre regionalisation was, on average, clearly related to intralimb muscle position such that the type I fibre vectors tended to point roughly toward the limb centre (Fig. 4b, see interrupted unity line). The most evident deviation from this rule concerned tibialis posterior; within midlevel cross-sections of this muscle, the greatest concentration of type I fibres was superficial rather than deep (plot-symbol I in Fig. 4b; cf. TP in Figs. 2, 4a). In 3 hindlimbs we also mapped out the point of nerve entry for the various investigated muscles. No systematic relation was found between the angular position of nerve entry around the muscle circumference and the mean direction of type I fibre eccentricity. The difference between the nerve entry angle and that for the mean type I fibre vector averaged 90.7 ± 54.0 (n = 18; range ), which is very close to the mean difference expected for a random relationship (90 ; possible 45

16 range ). General muscle properties and type I fibre regionalisation A general overview of how different muscle and regionalisation measures were interrelated is included in Table 4, which demonstrates that there was a tendency for vector regionalisation to be more marked in large and heavy muscles than in small and light ones (see also Fig. 3d). However, this might have been related to the eccentric intralimb position of the large muscles rather than to factors directly related to their size. The largest muscles tended have their midpoints relatively far removed from the limb centre (Fig. 4a); this was partly an effect of the thickness of these muscles (intramuscular midpoint far from muscle margin) but partly also reflecting genuine differences in intralimb position between large and smaller muscles. Also when analysed for all the fast muscles of Figure 4c except the 3 largest ones (GM, GL, TA), a significant correlation was still found between the intramuscular degree of vector regionalisation and the intralimb position of the muscles (r = +0,78, n=7, P < 0.05). Among these remaining 7 muscles, no significant correlation was found between the degree of vector regionalisation and muscle weight (P > 0.6). When performing the calculations for all the individual fast muscles together, moderate but statistically significant correlations were obtained between the overall density of type I fibres (FibD) and measures for the extent or eccentricity of type I fibre regionalisation (FR, VL). These results indicated that muscles with a high type I fibre density tended to be less regionalised than those with smaller densities of slow fibres (e.g., type I fibre density (FibD) vs type I fibre vector length (VL), r = , n = 80, P < 0.001; FibD vs type I fibre sector-region (FRs), r = , n = 80, P < 0.001; cf. also Table 4). These observations are in accordance with the finding that the large and highly regionalised muscles had a moderate tendency for being relatively type I fibre poor (see above; Table 2) Soleus As this slow muscle is clearly so different from the rest, it merits a separate treatment. In order to keep the number of counted and analysed fibres within managable limits, the fast type II fibres were the targets for our analysis of fibre type regionalisation in this muscle. However, generally speaking the observations on soleus were consistent with the conclusions based on the 11 fast muscles. The central position of soleus within the limb was in accordance with a marginal degree of target fibre regionalisation, here as seen for the type II fibres. There was no significant area regionalisation; for neither of the 2 fibre regional parameters (FRh, FRs) was the target fibre region significantly smaller than it would have been for a uniform fibre distribution (i.e., not smaller than C FR, Table 3). There was a small but statistically significant degree of vector regionalisation (parameter VL significantly larger than zero, P < 0.05) but the vector angle had a larger degree of variability (S.D. 56 ) than that for the fast muscles with reliable measurements (S.D. 25 or less, Table 3). As seen for the type I fibres soleus 46

17 MEASURES OF FIBRE TYPE REGIONALISATION was, of course, clearly not area regionalised, its type I fibre region covering 100% of the muscle cross-section area. Discussion Fibre type regionalisation Although it has long been known that fibre types may be regionalised within skeletal muscles, the present investigation is the first one performed with our new methods and concepts for the quantification of this phenomenon. Furthermore, for many of the lower hindlimb muscles of adult rats, the regionalisation properties had apparently not yet been studied by any method (e.g. FD, PB, PD, PE, TP). The main new findings and conclusions are as follows. (1) Within the rat lower hindlimb, all investigated fast muscles were significantly vector regionalised, i.e. their type I fibres tended to be eccentrically distributed across the muscle crosssection (Figs 2-4, Table 3). (2) Most of the fast muscles of the lower hindlimb (9 out of 11) were also significantly area regionalised, i.e. their type I fibres occupied only part of the total cross-sectional area of the muscle (Figs 2-4, Table 3). (3) The 2 aspects of regionalisation were strongly correlated: muscles with a high degree of vector regionalisation also tended to show a high degree of area regionalisation (Fig. 3a, Table 4). (4) The average degree and direction of type I fibre eccentricity (vector regionalisation) were both significantly related to the positions of the target muscles within the limb. Within individual muscles the type I fibre populations were typically concentrated towards the limb centre (Fig. 4b). Muscles close to the centre were less regionalised than those more remotely located (Fig. 4c). Our observations underline that, at least within the rat lower hindlimb, fibre type regionalisation is a highly significant and general feature of intra and intermuscular organisation; it is not a property restricted to only a few large and conspicuous muscles. The high correlation between the degrees of vector and area regionalisation (Fig. 3a) suggests that possibly similar, eccentrically organised mechanisms might underlie both aspects of regionalisation. It should be noted that it is not self-evident that there should be any correlation between the degree and the eccentricity of regionalisation: a concentrated population of type I fibres might conceivably have a central localisation within a muscle (for an example of a rather centralised regionalisation, see m. iliofibularis in the turtle, Laidlaw et al. 1995). Our findings concerning the correlation between the degree of intramuscular regionalisation and intralimb muscle position are new (Fig. 4c); this relationship has not been commented upon in preceding publications and has to be taken into account when trying to explain how regionalisation arises. The observed intralimb directions of the vector angles (Fig. 4b) are generally in accordance with preceding observations that, at least for many large-sized muscles of various mammals, type I fibres often tend to accumulate towards deep muscle regions (e.g. Pullen, 1977 a, b; Armstrong, 1980; Armstrong & Phelps, 1984; Totland & Kryvi, 1991). However, some of the results also provide an im- 47

18 portant modification of the traditional view: in one of the muscles (tibialis posterior, TP) the type I fibres were not accumulated towards the limb centre but rather towards more superficial limb portions (cf. Figs. 2, 4a). This apparent exception from a depth rule implies that, whatever the mechanisms underlying type I fibre regionalisation, adult muscles are not forced to (re)organise themselves such that their type I fibres occur deep rather than superficial. The reasons for the deviant behaviour of TP constitutes one of several interesting problems for further study (e.g., direction of TP regionalisation distorted during development?). In a recent reanalysis of the extensive human data published by Johnson et al. (1973), about half of the 11 muscles for which quantitative information was available failed to show a statistically significant deep vs superficial average regionalisation of type I fibres (Kernell, 1998). This lack of consistency might, however, have been partly or largely due to methodological problems. The present procedures for determining the existence, degree and direction of target fibre eccentricity should be more sensitive than pointwise determinations of fibre type frequencies within deep and superficial regions. However, it must also not be forgotten that the kinesiology and patterns of muscle use are very different for humans as compared with rats. For the further understanding of the functional meaning of fibre type regionalisation, thorough comparative studies between different animal species are highly desirable. The low degree of fibre type regionalisation within soleus (Fig. 2, Table 3) is in agreement with earlier observations of this muscle in rats (Pullen, 1977 b; Narusawa et al. 1987). Meaning and mechanisms One of the intriguing aspects of fibre type regionalisation is that, although the phenomenon is of common occurrence and, sometimes, very striking (Fig. 2, upper row), its functional meaning and the underlying mechanisms are still obscure. Our present investigations were made because, as a background for further investigations, we wished to learn more about how this enigmatic phenomenon manifests itself in the adult animal. With regard to the mechanisms involved it is known that, among the large rat hindlimb muscles, (part of) the characteristic type I fibre regionalisation will appear during early embryological development even in the absence of muscle innervation (Condon et al b). Thus, the regionalisation does not primarily (or only) develop as a consequence of how the muscles are innervated and used. During initial muscle development the emergence of fibre type regionalisation is related to the successive appearance of different generations of myotubes, and to time and region dependent switches in their properties (Narusawa et al. 1987; Condon et al a, b). In the hindlimb muscles of the rat, the first generation myotubes all start off as slow but, in the most clearly regionalised muscles, those located more superficially switch to become fast at later stages of development. The second generation myotubes emerge around the primary ones, using them as scaffolding. Most of the second generation myotubes start off as fast and remain so. It has been speculated that a 48

19 MEASURES OF FIBRE TYPE REGIONALISATION regionalised switch of myosin isoform among the primary myotubes might have been caused by a gradient of a slow morphogen originating from deep within the limb (or alternatively, a gradient of fast morphogen originating from the surface) (Condon et al a); the nature of this morphogen is still unknown. It remains an interesting question whether such topographically organised morphogens might exert an effect on fibre differentiation also in later, adult life. Even though fibre type regionalisation emerges already at an early stage of development, its features may become further modified by (regionalised) aspects of innervation and use; in some muscles regionalisation features which are visible during development may even disappear in the adult (Brandstetter et al. 1997). During early development, ingrowing slow and fast motor axons are apparently guided to make contact, ultimately, with the correct type of muscle fibre (for review, see Jansen & Fladby, 1990). In later life the properties of muscle fibres may become very drastically altered by a change of innervation and/or by altered use (for reviews, Kernell, 1992; Gordon, 1995); also purely mechanical aspects of muscle fibre use, such as the degree of stretch or active force production, may affect the properties and myosin composition of the muscle fibres (Goldspink, 1999). It remains a relevant question whether the presence of a regionalised fibre type distribution gives the adult animal an essential functional advantage, or whether the accumulation of slow fibres in deep portions of adult regionalised muscles may be mainly interpreted as vestiges of the original muscle primordium (Narusawa et al. 1987). It has been suggested that a deep localisation of the well-circulated type I fibres is useful for, for instance, the conservation of heat (Loeb, 1987). Furthermore, it should be further investigated whether a central position confers biomechanical advantages for the postural function of slow fibres or, conversely, whether the biomechanical functions of fast fibres are improved by their typically more superficial sites within single muscles. With regard to the possible importance of heat conservation it is interesting to consider the manner in which the fibre type regionalsation manifests itself in poikilothermic animals. While a preferentially deep localisation of slower fibres has also been found for several amphibian hindlimb muscles (e.g., Lännergren et al. 1982; Rowlerson & Spurway, 1988), a variety of preferential localisations was reported for hindlimb muscles of turtles (Laidlaw et al. 1995) and a preferentially superficial localisation of slow fibres is commonly present for trunk musculature of fish (e.g. Rome et al. 1988). This apparent variability among the cold-blooded animals might suggest that, in the absence of needs for heat conservation, other functional requirements (biomechanical factors?) become more dominant with regard to the possible advantages of fibre type regionalisation. The analysis of the present paper was limited to the traditional viewpoint in muscle histochemistry: fibre composition as seen in cross-sections somewhere through the mid proximodistal extent of the muscle. However, many mammalian muscles are pennate and, hence, longer than their component muscle fibres. 49

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