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Contractile activation characteristics of single permeabilized fibres from levator palpebrae superioris, orbicularis oculi and vastus lateralis muscles from humans

Síun P. Campbell, David A. Williams, Bartley R. Frueh and Gordon S. Lynch

MS 9327 Received 2 March 1999; accepted after revision 26 May 1999.

	ABSTRACT

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1. We investigated the contractile activation characteristics of single membrane-permeabilized fibres from the following muscles from humans: the levator palpebrae superioris (LPS), an extraocular muscle; the orbicularis oculi (OO), a facial muscle; and the vastus lateralis (VL), a major muscle of the thigh.

2. Single permeabilized muscle fibres were isolated from each of the different muscles, attached to a sensitive force transducer and activated by rapid immersion in buffered solutions of varying [Ca²⁺] and [Sr²⁺]. Fibres were allocated into discrete populations based on their contractile characteristics, including their differential force responses during Ca²⁺ and Sr²⁺ activation.

3. With the exception of one fibre from the LPS, all 152 fibres sampled from the three different human muscles could be classified into either population I (slow, type I) or population II (fast, type II) based on their force-pCa(pSr) relations. The LPS muscle fibre which was unable to be classified into the two major fibre populations displayed a combination of the typical force-pCa(pSr) relations for mammalian fast and slow muscle fibres.

4. Although fibres from the LPS, OO and VL muscles had similar differential sensitivities to Ca²⁺and Sr²⁺, the steepness of the force-pCa(pSr) curves for fibres from the LPS and OO muscles were highly variable compared with those for fibres from the VL muscle. Specific forces (N cm^-2) of the smaller diameter fibres from the LPS and OO muscles were significantly lower than those of fibres from the VL muscle.

5. The differences in the contractile activation characteristics between fibres from the VL muscle and those of fibres from facial (OO) muscles and extraocular (LPS) muscles, reflect the differences in their fibre composition that are responsible for their functional specificity.

	INTRODUCTION

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The levator palpebrae superioris (LPS) muscle in humans is classified as an extraocular muscle (EOM), originating from the same superior condensation and exhibiting a close association in structure and function with the superior rectus muscle (Sevel, 1981). Histochemically, its fibre type characteristics resemble those of other EOM with the presence of two additional fast fibres in the LPS muscle (Kuwabara et al. 1975; Spencer & Porter, 1988; Porter et al. 1989; Lucas & Hoh, 1997). However, the LPS muscle does not have multiple innervated fibres, nor is it morphologically separated into orbital and global regions, which are both defining characteristics of EOMs (Dietert, 1965). Porter and colleagues (1989) proposed that these differences in the morphology and innervation patterns reflect the functional demands placed on the LPS muscle. The LPS muscle functions to raise the eyelid and keep it open with a tonic-like innervation to maintain lid position. It also provides for the rapid movements required during the blink reflex which equate to the vergence and saccadic demands placed on EOM for the co-ordinated movements of the eye and eyelid (Evinger et al. 1984; Evinger, 1995; Porter et al. 1995; Lucas & Hoh, 1997).

One advantage of studying the LPS muscle is that one can contrast its functional properties directly with those of another muscle that has similar functional demands. This muscle is the orbicularis oculi (OO), a facial muscle, the antagonist to the LPS muscle but which arises from a different embryological origin. The OO muscle is responsible for closure of the eyelid (McLoon & Wirtschafter, 1991). Morphologically, it is composed of some of the smallest diameter muscle fibres within all human skeletal muscles (Happak et al. 1988; Nelson & Blaivas, 1991), with a large majority of these fibres not extending the full length of the muscle (Wirtschafter et al. 1994; Happak et al. 1997). The OO muscle is composed of up to 90 % type II (fast) muscle fibres in both humans (Happak et al. 1988; Nelson & Blavias, 1991) and primates (Porter et al. 1989).

EOMs exhibit the greatest diversity among mammalian skeletal muscles due to the large number of myosin heavy chain (MyHC) isoforms expressed (Stål et al. 1994; Brueckner et al. 1996). It is not surprising that many of the common muscle fibre classification schemes do not apply to EOMs (Porter et al. 1997). The structural complexity of EOMs and their diverse MyHC expression reflects the range of movements that these muscles are required to perform, including fast saccades, slow vergeance movements and fixation (Rushbrook et al. 1994). The contractile function of EOM has almost exclusively been studied on intact muscles from animals (Barmack et al. 1971; Close & Luff, 1974; Lennerstrand, 1974; Asmussen & Gaunitz, 1981; Frueh et al. 1994; Goldberg & Shall, 1997; Goldberg et al. 1997). There have been few studies that have investigated the contractile activation characteristics of EOM at the cellular (single fibre) level. Previously, we characterized the Ca²⁺- and Sr²⁺-activated contractile characteristics of single permeabilized fibre segments from the superior rectus and LPS muscles of the rabbit (Lynch et al. 1994a). That study highlighted the existence of a large population of fibres isolated from both muscles of the rabbit which displayed composite force-pCa(pSr) relations (Lynch et al. 1994a). The force-pCa(Sr) curves for these fibres were similar to those representative of fibres composed of mixed fast and slow contractile and regulatory proteins (Lynch et al. 1995), highlighting the functional and morphological complexity of EOM (Sartore et al. 1987; Jacoby et al. 1989).

Given the diversity in their functional requirements, it was of interest to compare and contrast the contractile activation characteristics of single fibres isolated from the LPS and OO muscles and also compare these properties with those of fibres isolated from the vastus lateralis (VL) muscles of the thigh. In this way, functional differences or similarities could be characterized for fibres from an EOM, a facial muscle, and a limb skeletal muscle, from humans. Although the majority of fibres from the LPS, OO and VL muscles could be classified into two discrete populations based on their differential sensitivity to Ca²⁺ and Sr²⁺, we found that the steepness of the force-pCa(pSr) curves for fibres from the LPS and OO muscles were highly variable compared with those for fibres from the VL muscle. In addition, the specific force-producing capacity of the smaller diameter fibres from the LPS and OO muscles was significantly lower than that of fibres from the VL muscle.

	METHODS

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Human EOM and OO muscle samples: patients and reasons for muscle removal

All experimental procedures were approved by the Human Experimentation Ethics Committee of The University of Melbourne. Extraocular muscle was obtained from both male and female patients aged between 8 and 93 years. Patients requiring surgical procedures that necessitated the removal of the OO or LPS muscles gave voluntary informed and written consent for these procedures. All samples were obtained under general anaesthetic. Portions of the excised muscle samples were isolated for testing and deemed by pathologists to have normal histological muscle features not altered or modified by any disease states.

All muscle samples were obtained immediately following excision by the surgeon and blotted on filter paper to remove excess blood and connective tissue. The muscles were tied at resting length to a capillary tube and placed immediately in a vial containing skinning solution of the following composition (mM): potassium propionate, 125; EGTA, 5; ATP, 2; MgCl₂, 2; imidazole, 20; and 50 % v/v glycerol, adjusted to pH 7·1 with 4 M KOH; and stored at -20°C for up to four months (Lynch et al. 1994a).

Percutaneous muscle biopsy of the vastus lateralis muscle

A percutaneous needle biopsy was obtained from a random sample of untrained and trained male and female subjects recruited primarily for other exercise studies within the Department of Physiology at The University of Melbourne. Voluntary informed and written consent was sought from all subjects prior to the procedure.

After location of the biopsy site in the mid-belly region of the vastus lateralis muscle, a small injection of a local anaesthetic (Xylocaine, 2 %) was administered. A small incision was made into the skin and surrounding fascia of the muscle, and a needle biopsy taken (Goldberger et al. 1978). The retrieved muscle sample was thoroughly blotted on filter paper, tied to small capillary tubes and then stored in the glycerol-based skinning solution at -20°C for up to four months.

Dissection and mounting of the muscle fibre to the force recording equipment

On the day of an experiment, a portion of the muscle was cut from the main bundle and placed in a dish with a Sylgard base (Dow-Corning, Midland MI, USA) that was filled with skinning solution at room temperature (22°C). Muscle fibres were isolated using fine forceps to carefully split the muscle portion into smaller bundles. Separating the fibres in this way also loosened the surrounding connective tissue. Removing only small bundles of fibres at a time allowed random sampling of fibres from different portions of the whole of the muscle sample, whilst maintaining the integrity and viability of the other portions of the muscle not being dissected.

The mounting of the isolated single muscle fibres to the force recording equipment has been described previously in detail (Lynch et al. 1994a). Briefly, a loop of fine surgical silk (9/0, Deknatel Inc., Fall River, MA, USA) was attached to the free end of the isolated fibre. The loop of silk thread was tied to a pin in direct contact with a sensitive force transducer (AE801, SensoNor, Horten, Norway). The other end of the fibre was clamped between the jaws of a pair of jeweller's forceps (No. 5, Dumont and Fils, Switzerland). Any portion of the fibre still attached to the muscle bundle was severed with fine iris scissors and excess suture was carefully trimmed. After mounting, the fibre was immersed in a small spectrophotometric vial filled with a high-relaxing solution, which contained 50 mM EGTA. The fibre was then slowly stretched to a just-taut length, and sarcomere length was estimated from the diffraction pattern resulting from a monochromatic He-Ne laser (Model 155A_SL, Spectra-Physics, Oregon, USA) directed onto the fibre at several locations along its length. Where possible, sarcomere length was adjusted to 2·7 µm, which is within the optimal range for maximum force production of mammalian skeletal muscle fibres (Stephenson & Williams, 1981). Details of fibre end compliance and sarcomere uniformity have been described (Lynch et al. 1994a). Measurement of the fibre diameter and length were resolved using a calibrated graticule in the eyepiece of a dissection microscope (Nikon, Japan). Contractile responses were recorded with, and data analysed on, a MacLab 8-channel data acquisition unit (ADInstruments, Castle Hill, NSW, Australia) coupled to a Macintosh IIci computer, running Chart software (v3.5.1, ADInstruments).

Contractile activation of permeabilized muscle fibres

The composition of solutions and procedures for Ca²⁺ and Sr²⁺ activation of permeabilized fibres were identical to those described previously (Lynch et al. 1994a). All experiments were performed at room temperature (22°C). Briefly, solutions containing varying concentrations of Ca²⁺ and Sr²⁺ were obtained by mixing, in varying proportions, a stock solution of EGTA^2- (50 mM) with either a stock of CaEGTA (50 mM) or SrEGTA (40 mM + 10 mM EGTA^2-) stock solutions with a high-relaxing (50 mM EGTA^2-) solution. A low-relaxing, pre-activating solution, used to wash off EGTA and prepare the fibre for rapid activation, was prepared by mixing 1,6 diaminohexane-N,N,N',N'-tetraacetic acid (HDTA) (49·9 mM) with a small amount of EGTA^2- (0·1 mM). All solutions contained (mM): Hepes, 60; K⁺, 17; Na⁺, 36; Mg²⁺, 1; NaN₃, 1; ATP, 8; and creatine phosphate, 10. The affinity constants (K_app) of Ca²⁺ and Sr²⁺ to EGTA at pH 7·10 in the presence of 1 mM Mg²⁺ were 4·78 × 10⁶ m^-1 and 1·91 × 10⁴ m^-1, as determined previously by Moisescu & Thieleczek (1978) and Stephenson & Williams (1981).

All fibres were activated in a stepwise fashion (from the lowest to highest [Ca²⁺] or [Sr²⁺]). Each fibre was first bathed for several minutes in the high-relaxing solution, then transferred to the low-relaxing solution for approximately 1 min before being transferred to the activating solutions. At the completion of the activation process, the fibre was returned to the high-relaxing solution. The complete Ca²⁺-and Sr²⁺-activation sequence was repeated twice.

The force produced by the single fibres in each of these solutions was expressed as a percentage of the maximal force level (P_o) obtained by the fibre when activated in saturated Ca²⁺ or Sr²⁺ solutions. These force levels were then plotted as a function of the pCa or pSr values to produce force-pCa or force-pSr curves and a sigmoidal curve was fitted to the experimental data. The values derived include the threshold for contraction for that particular ion, determined from the pCa(pSr) which corresponded to the 10 % relative force level (pCa₁₀, pSr₁₀). The sensitivity of the fibre to the activating ion was assessed from the pCa(pSr) which produced 50 % relative force (pCa₅₀, pSr₅₀). The steepness of the force-pCa (pSr) curve was determined from the Hill coefficient (n_HCa, n_HSr), which best fitted the experimental data. The n_H value is that represented in the Hill equation where:

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Ca²⁺- and Sr²⁺-activated contractile characteristics of single permeabilized muscle fibres

LPS muscle. Muscle fibres obtained from human LPS muscle samples were classified into two populations based on their contractile activation characteristics, as outlined in Table 1. The first fibre group, designated population I, consisted of fibres which exhibited activation characteristics which were fitted by shallow force-pCa (force-pSr) curves with low n_HCa (n_HSr) values, and low differential sensitivity (pCa₅₀ - pSr₅₀) values. The second group, designated population II, consisted of fibres with characteristics which were fitted with steeper force-pCa (force-pSr) curves and larger differential sensitivities.

Table 1. Fibre type classification criteria for human LPS, OO, and VL muscles fibres based on Ca²⁺- and Sr²⁺-activated contractile characteristics

The contractile activation characteristics of all population I and population II fibres sampled from the LPS muscle are presented in Table 2. Population I fibres had force-pCa (force-pSr) relations which were less steep than those of fibres from population II. No difference in the force output (P_o), or fibre diameter was evident between fibres of these two populations.

Table 2. Contractile characteristics from single membrane permeabilized human LPS, OO and VL muscle fibres

Muscle	LPS		OO			VL
Fibre populations	I	II	Mixed	I	II	I	II
No. of fibres	6	40	1*	1	63	22	19
Sarcomere length (µm)	2·53 ± 0·06	2·44 ± 0·04	2·44	2·36	2·52 ± 0·03	2·63 ± 0·06	2·63 ± 0·05
pCa50	5·98 ± 0·05	5·85 ± 0·02	6·07	6·01	5·99 ± 0·03 *	6·03 ± 0·04	6·03 ± 0·03 **
nHCa	1·68 ± 0·11	2·66 ± 0·09	-	1·40	2·32 ± 0·06 *	2·03 ± 0·12	2·55 ± 0·07
pSr50	5·43 ± 0·06	4·54 ± 0·02	4·82	5·55	4·73 ± 0·02 *	5·62 ± 0·04	4·79 ± 0·04 **
nHSr	1·58 ± 0·07	2·62 ± 0·09	-	1·30	2·30 ± 0·06 *	1·99 ± 0·11	2·44 ± 0·07
pCa5 - pSr50	0·54 ± 0·05	1·30 ± 0·01	1·25	0·46	1·28 ± 0·01	0·42 ± 0·04	1·24 ± 0·03 **
Fibre diameter (µm)	35·4 ± 1·04	37·5 ± 2·8	31·20	25·00	36·6 ± 1·2	62·2 ± 2·0	63·8 ± 1·80 **
Po (N cm-2)	8·39 ± 1·78	11·84 ± 0·96	8·06	12·74	12·45 ± 0·72	30·47 ± 1·99	32·64 ± 3·85 **

Population I fibres comprised 13 % (6/47) and population II 85 % (40/47) of the total fibre sample. One fibre displayed force-pCa(pSr) characteristics which prevented it from being assigned to either population I or population II (Table 2). This fibre displayed force-pCa and force-pSr relations that were intermediate to those of the two main fibre populations. In particular, the force-pSr curve displayed two degrees of steepness in the single curve, and as such the data could not be fitted by a curve with a single Hill coefficient. Myofibrillar force oscillations, sometimes evident when fibres are activated submaximally by Ca²⁺ and Sr²⁺ (Fink et al. 1990), were not observed in either population I or population II fibres from the LPS muscle.

OO muscle. Single fibres from the human OO muscle could be separated into two distinct fibre populations, similar to those described for LPS muscle fibres. The contractile characteristics of all fibres sampled from the OO muscle are listed in Table 2. All but one fibre (63/64, 98 %) could be classified as population II, with the remaining fibre being allocated to population I. For the OO muscle, myofibrillar force oscillations were not exhibited by either population I or population II fibres when activated submaximally.

VL muscle. Fibres sampled from the VL muscle could be separated into the two main fibre populations outlined in Table 1. Unlike population I fibres from the LPS and OO muscles, some population I fibres from the VL muscle exhibited myofibrillar force oscillations when activated submaximally. Force oscillations were not exhibited by population II fibres from the VL muscle. The activation characteristics of fibres from populations I and II are outlined in Table 2. There were no differences in the specific forces (N cm^-2) or diameters of individual fibres from the two populations.

Comparison of the contractile activation characteristics of fibres from the LPS and OO muscles

Comparisons were made between population II fibres only, due to the small number (n = 1) of population I fibres sampled from the OO muscle. Population II fibres from the OO muscle were more sensitive to Ca²⁺ and Sr²⁺ than those from the LPS muscle, as indicated by their higher pCa₅₀ and pSr₅₀ values. Population II fibres from the LPS muscle had steeper force-pCa (pSr) relations than those fibres from the OO muscle, indicative of a greater co-operativity in the activation of tension. No significant difference was evident in the differential sensitivity to the activating ions (pCa₅₀ - pSr₅₀). There was no difference in the force-producing capacity of permeabilized fibres from the two muscles.

Comparison of the contractile activation characteristics of membrane-permeabilized VL, LPS and OO muscle fibres

The contractile characteristics of fibres sampled from population II from the three different muscles were also compared. Only a comparison of population II fibre characteristics was feasible between the three muscles due to the low number (n = 1) of population I fibres sampled from the OO muscle.

The LPS and OO muscle fibres had lower specific forces than fibres from the VL muscle. Fibre diameters were also smaller in the LPS and OO muscle fibres compared with VL muscle fibres. The LPS muscle fibres had a lower sensitivity to both Ca²⁺ and Sr²⁺ than VL muscle fibres, but displayed a greater differential sensitivity to the activating ions (pCa₅₀ - pSr₅₀) than VL muscle fibres.

	DISCUSSION

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Although EOMs are amongst the most complex muscles in terms of their structure and composition, their functional properties at the cellular level remain one of the most understudied areas in muscle physiology. Knowledge of the structural and functional properties of EOMs is vital for the development of treatments for eye movement disorders (Porter et al. 1995). This study demonstrated that the Ca²⁺- and Sr²⁺-activated contractile characteristics of single fibres are different for extraocular, facial and limb skeletal muscles in humans. This indicates that there are specific differences in the fibre composition and contractile function of these distinct muscle groups that reflect their specific functional requirements.

The levator palpebrae superioris and orbicularis oculi muscles fibres

Fibres from the LPS and OO muscles were allocated into two distinct fibre-type populations based on defining features of their force-pCa(pSr) relations; the differential sensitivity to Ca²⁺ and Sr²⁺ and the relative steepness of the force-pCa(pSr) relationship. Approximately 85 % of the fibres sampled from the LPS muscle could be classified as population II fibres (see Table 2), which have previously been shown to correlate with histochemically-defined type II fibres (Fink et al. 1990). Our finding that the majority of sampled fibres were of the type II variety correlates well with previous histochemical investigations of the LPS muscle in primates (Porter et al. 1989).

Of the 64 fibres sampled from the OO muscle only one fibre had contractile activation characteristics which classified it as population I (type I). This fibre originated from the preseptal regions of the OO muscle. The other 63 fibres could all be classified as population II (type II). Histochemical studies of the OO muscle suggest that it is composed of approximately 10 % type I fibres, which are found mostly in the preseptal regions (McLoon & Wirtschafter, 1991), with the remaining 90 % being type II fibres. Thus, our physiological classification of LPS and OO muscle fibres as population II (type II), and population I (type I) and the relative proportions of these fibres within each muscle correlate well with previous histochemical studies (Porter et al. 1989; McLoon & Wirtschafter, 1991).

The predominance of population II (fast, type II) fibres within LPS and OO muscles reflects the functional requirements of these muscles for rapid and forceful movements during the blink reflex (Evinger, 1995). The higher proportion of population I (slow, type I) fibres within the LPS muscle relates to the tonic-like innervation required to hold the eyelids open during wakeful hours against the constant passive downward elastic forces generated by the ligaments and insertion points of the LPS muscles (Aramideh et al. 1994). Since these muscles also perform delicate lid saccades for positional changes in response to noxious influences or altered visual demands, resolution of fibre type may only partially explain the functional movements of these muscles. Neural influences such as the size of the motor units, their recruitment patterns, and the elastic components within the muscle should also be considered (Goldberg et al. 1998).

Only one fibre from the LPS muscle exhibited contractile characteristics which placed it in the mixed category of fibre types, i.e. those with characteristics that can only be fitted by composite force-pCa(pSr) relations. This is interesting considering that in our previous investigation of the contractile characteristics of single fibres from the LPS and superior rectus muscles of the rabbit (Lynch et al. 1994a), we found that a large proportion (35 %) of the sampled fibres exhibited composite force-pCa(pSr) relations. Such mixed fibres, representing those with a co-expression of fast and slow contractile and/or regulatory proteins within a single functioning single unit, have also been sampled from the limb muscles of boys with Duchenne muscular dystrophy (Fink et al. 1990), muscles from new born rats and from regenerating muscles following bupivacaine injection (Takagi, 1981), muscles from marsupials (Wilson & Stephenson, 1990), and from fast and slow muscles of rats subjected to endurance training from two weeks of age (Lynch et al. 1995).

The difference in the number of mixed fibres that we have sampled from rabbit superior rectus and LPS muscles compared with human LPS muscles makes the functional role of mixed fibres in EOM unclear. Jacoby et al. (1989), suggested that the mixture of slower myosins in fibres in the flanking and not the central regions of the muscle may have a dampening effect on the total force generation of these fibres, and that this would act to enhance the precision of the movements performed. It is likely that mixed fibres represent those multiply innervated fibres which predominate in the orbital layer of EOM expressing different MyHCs at the ends of the fibre compared with the mid-belly region (Porter et al. 1995). The LPS muscle in humans does not have multiply innervated fibres, nor is it morphologically separated into orbital and global regions (Dietert, 1965).

Although only one fibre could be identified as being of the mixed variety, the force-pCa (pSr) relations of population II fibres from the LPS muscle, showed a wide variation in the n_HCa (n_HSr) values for a relatively narrow range of pCa₅₀ - pSr₅₀ values. This indicates that many LPS muscle fibres exhibit similar differential sensitivities to Ca²⁺ and Sr²⁺ but display differing degrees of co-operative interactions within the activated thin filament. Co-operativity describes the process whereby force-producing cross-bridges act to enhance the activation and the formation of more force-producing cross-bridges (Brandt et al. 1982; Campbell, 1997). The expression of combined fast and slow troponin isoforms along the length of muscle fibres has been demonstrated in rabbit EOMs (Briggs et al. 1988). Differences in troponin isoform expression along the length of these fibres is possible, although this would likely alter the differential sensitivity of the fibre to Ca²⁺ and Sr²⁺. Functionally, the presence of fibres with different co-operative interactions may influence the force response to a given Ca²⁺ release. Depending on their location within the muscle, these fibres could generate higher or lower forces as required for lid saccades or blink reflexes.

As predicted by Briggs and colleagues (1988), the force- pCa(pSr) curves of EOM fibres are shallow, the data being fitted by curves with lower Hill coefficients than for VL muscle fibres as sampled from both the present study and from that of Lynch et al. (1994b). The shallow force-pCa(pSr) relations, coupled with the dependence of tension generation on the firing rate and the small number fibres per motor unit, are likely to contribute to the precise control of eye movement (Briggs et al. 1988). Population II fibres from OO muscle had n_HCa (n_HSr) values similar to those of population II fibres from the LPS muscle (Table 2 ). This indicates a similarity in regulatory protein composition of fibres from facial muscles and EOMs that is likely to be responsible for the functional differences between these fibres and those from limb (VL) skeletal muscles.

Previous investigations of the contractile activation characteristics of typical population I (type I, slow) and population II (type II, fast) fibres have shown that fast fibres have steeper force-pCa(pSr) relations and lower sensitivities to Ca²⁺ and Sr²⁺ than slow fibres (Mizusawa et al. 1982; Fink et al. 1990; Lynch et al. 1994b). Comparison of the contractile characteristics of population II fibres from the LPS and OO muscles indicate that OO muscle fibres are more sensitive to Ca²⁺ and Sr²⁺ whereas the LPS muscle fibres exhibit greater co-operativity.

The diameters of OO muscle fibres are among the smallest of all mammalian muscles, and are smaller than any found in limb skeletal muscle and EOM (Happak et al. 1988). In the present study, we found no difference in the mean estimated fibre diameter or force output of population II fibres sampled from the OO and LPS muscles. The diameter of population II fibres from both the LPS and OO muscles was significantly smaller than that of population II fibres from the VL muscle. This suggests that for the imposed work loads, the muscles of the eyelid do not require large diameter, high force-producing fibres. Rather, fine control through activation of many discrete bundles of low force-producing muscle fibres is necessary to ensure delicate and precise movements.

The finding that LPS and OO muscle fibres produce less force than limb skeletal muscle fibres is in accordance with previous studies of intact mammalian muscle which have demonstrated lower specific (normalized) forces in EOM than limb skeletal muscle (Close & Luff, 1974; Luff, 1981; Asmussen & Gauntiz, 1981; Frueh et al. 1994). These investigators speculated that the lower normalized force of EOM may be due to either an overestimation of the amount of contractile tissue within the muscle, an inability to excite all contractile tissue resulting in incorrect normalized force values, or a decrease in the tension generation per actomyosin interaction. In the present study, comparisons of force output between EOM, facial and limb skeletal muscles were made at the single fibre level such that there could be no underestimation of the force output due to the presence of connective tissue surrounding the fibre, nor due to differences in the force contributions of many fibres. We found that the force-producing capacity of permeabilized fibres from the LPS and OO muscles was lower than that of fibres from the VL muscle. In our previous study on the contractile activation characteristics of permeabilized fibres from rabbit EOM, we found that absolute force production was lower in EOM fibres than in limb muscle fibres (of the rat), but when corrected for cross-sectional area, the force producing capacity was similar for individual fibres of the different muscles (Lynch et al. 1994a). In the present study, we have made direct comparisons with limb muscle fibres from the same species (i.e. human) and have sampled many more fibres from the LPS muscle. The existence of many more mixed fibres in the LPS and superior rectus muscles of the rabbit compared with the limited number of mixed fibres sampled from the LPS muscle from humans (present study) makes direct comparisons difficult. We cannot rule out the possibility that a greater variation in fibre composition exists within skeletal muscles of individual humans compared with the limited variation in fibre composition within muscles of in-bred rabbits.

	REFERENCES

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Acknowledgements

This study was supported by the National Health and Medical Research Council (NHMRC) of Australia and the Benign Essential Blepharospasm Research Foundation (USA). G. S. L. was supported by a C.J. Martin Research Fellowship from the NHMRC.

Corresponding author

G. S. Lynch: Department of Physiology, The University of Melbourne, Parkville, Victoria, 3052, Australia.

Email: g.lynch{at}physiology.unimelb.edu.au

Author's present address

S. P. Campbell: School of Biomedical Science, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.

Author's permanent address

B. R. Frueh: W. K. Kellogg Eye Center, Department of Ophthalmology, The University of Michigan, Ann Arbor, MI 48104, USA.

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