Life Sciences, Vol . 16, pp .775-778 Printed in the U .S .A .
Pergamon Press
THE DIAMETER AID MEAN SARCQ~RE LENGTH OF II~NIDUAL MUSCLE FIBRES A . Coral Hopper and James P . Hanrahan Departments of Anataay and Agricultural Biology, Ltniversity College, Dublin, Ireland . (Received is final form August 4, 1975) S++^a .+"+. - Studies of the coefficients of variation and the repeatability of the measurements indicate that a sample of 25 fibres is sufficient to provide an accurate estimate of the aeon fibre diameter sad sarcanera length of a auscle . There is a significant negative correlation (-0 .55) between the diameter and mean sarcamere length of an individual muscle fibre . Because they affect sarcomere length postmortem mechanical influences aunt be strictly standardised if fibre diameter is to be a reliable parameter of muscle groerth . Although affected by many variables fibre diameter is probably the most widely studied parameter of muscle growth . During active contraction the diameter of isolated ayofibrils increases wh¢n the sarcameres shorten (1,2) . This is presumably due to an increase in the centre-to centre distance between actin and ayosin filaments (the myofilasent It packing density) which varies imrersely with sarcamere length (3) . can prob :bly be assumed that fibre diameter is increased durinç active contraction although a search of the literature has failed to reveal a study in which this assumption has been confiraed by measuranent . Because of the difficulty of avoiding the contraction of at least sane fibres during exposure or romoval, the study of muscles which have entered rigor has been advocated (4) . However, even the fibre diameter of rigor muscles may be affected by their condition during the cruet of rigor sortis . The mean fibre diameter of muscle strips subjected to increased tension is reduced (5, 6) and similar findings were reported is a study of the muscles of bov-" ine carcasses which had been hung vertically during the onset of rigor mortis (7) . In the latter study the sear sarcamere length of fibres in a separate sample from such muscles xas also measured and a highly significant negative correlation between fibre diameter sad mean sarcamere length was found . Because these parameters were measured in separate samples the present study was performed to examine the relationship between the diameter and mean sarcanere length of individual fibres . In addition the results were analysed to determine whether a asmple of 25 fibres is sufficient to provide an accurate estimate of the diameter and mean sarcamere length of the constituent fibres of a auscle . MethQda Twenty adult male mice (Mus musculua) from Falconer s R strain were killed by ether anaesthesia . Iamediately after death the anisals 775
776
Fibre Diameter and Sarcomere Length
Vol. 17, No . 5
The overall means and standard errors for fibre diameter and mean sarcanere length are shown in Table 1 together with the within muscle and between-muscle variances of these parameters . Calculated on a within-muscle basis the coefficient of correlation between the diameter of a fibre and it+s mean sarcomere length is -0 .55, The coefficient a value which is statistically significant (PO .OS) . The repeatability and coefficient of variation of fibre diameter and mean sarcomere length are shown in Table 2 . TABLE 2 Repeatability and Coefficient of Variation of Fibre Diameter and Mean Sarcomere Length Fibre Diameter Repeatability Coefficient of variation
0 .80 10 .3096
Sarcamere Length 0 .84 7 .8096
Discussion The campariaon of two or more samples is an essential feature of Yet the value of such studies may be considmany biological studies . erably reduced by a lack of information about the statistical properties of the parameters involved . The coefficient of variation provides a useful indication of the precision of the measurement of a parameter and the values obtained in the present study confira the usefulness of relatively small samples in the evaluation of the mean fibre diameter snd sarccaere length of a muscle . Furthermore, the high repeatability values obtained indicate that the measurement of 25 fibres is sufficient to provide an accurate estimate of the mean value of these parameters in an individual muscle . Although anticipated by the results of an indirect study (?) the results of the present study .provide the first direct evidence of an association between the diameter and mean sarcamere length of rigor muscle fibres . This negative correlation would seem to confirm the basic morphological similarity that exists between the shortening of active contraction and the shortening which occurs during the onset of rigor mortis . For same time after death the actin and myosin filaments slide freely over oae another and some shortening occurs in the presence of residual adenosine triphosphate (ATP) . When the level of ATP falls below approximately 2096 of the resting level the cross-bridges between the filaments become "locked" and further sliding is not possible . The result is the well known muscular inextenaibility of rigor mortis . The sarcomere length of rigor muscle therefore depends on the relative position of the filaments when the amount of ATP in the fibres falls below this critical level . The mean sarcomere length found in the present study is comparable to the value of 2 .8~rm reported in another study of the mouse biceps
Vol . 17, No . 5
Fibre Diameter and Sarcomas Length
777
were placed supine on a slab of foam polystyrene and pinned out with the limbo in full extension . The carcasses were left in this position at room teapersture (18oC) for four hours to allow the onset of rigor mortis . The skin was then removed from the carcasses and the right biceps brachü muscles were excised . The widest portion of the belly of this muscle contains all itts constituent fibres (8) . A transverse section of the entire muscle 1-2 ams . thick was excised with a scalpel and placed in a flat-bottomed tube (1 inch diameter) containing 1 ml calcium-free Ringer Locke solution . A hamogenizer consisting of a shaft containing two blunt blades surrounded by protective guards and capable of being rotated at 11,500 revolutions per minute was used to separate unfixed individual fibre fragments . This technique has been described in detail by Hegarty and Naude (9) . After separation the fibre fragmenta form a suspension in the Ringer Locke solution . 15 ml of this suspension was then transferred to the well of a hanging-drop slide with a Pasteur pipette and a cover slip placed over it . The iaage of the fibres was projected from a monocular microscope on to a sheet of white card at a aagnification of X750 by means of a mirror attached to the eye-piece . Measurements were then made on 25 fibres Pram each muscle . Short fragments were excluded as were fragments whose entire length was not in fxus and which consequently were not in the plane of focus . The number of A bands along a 100~m length of fibre was counted anu the aeon sarcameré length of the fibre was calculated from this . the diameter of the fibre was measured at the mid-point of this 100um length with a micrameter'callipers . Thus the diameter and mean sarconere length of 500 fibres xas determined . The overall and within-muscle means for fibre diameter and mean aarcomere length were calculated and their standard errors were deter mined . The coefficient of correlation between the disaster and mean ssrcamere length of individual fibres " was calculated on a within-muscle basis . The coefficient of correlation between the muscle means of these parameters was also calculated . A simple hierarchical analysis of varianç~e was used to estimate the between-muscle (6m ) and within-muscle (Cf ) sources of variation for fibre diameter a~ sarcamere number . These estimates were used to compute the repeatability of the measurements and th~ co~ffici~nt of variati~n of heir means defined respectively as (6m /4m ~r- dw /25) and (~ + Qw /25)~/overall aeon . Results TART-F 1
Overall Mean, Standard Error and Variances of Fibre Diameter and Mean Sarcamere Length
Mean (Wm) Standard error (gym) Between-muscle variance Within-muscle variance
Fibre Diameter 31 .5 0 .3 4 .7 21,3
Sarcamere Length 2 .91 0 .02 p .pq 0,27
778
Fibre Diameter and Sarcomere Length
Vol . 17, No . 5
brachü in which the muscle was allowed to enter rigor with the limb pinned out in full extension (10) . These values are considerably greater than the values found in four mouse muscles which entered rigor free of external mechanical restraint (4) . Subjecting a muscle to mechanical restraint or increased tension will reduce the degree of filament overlap which occurs during the onset of rigor mortis . Muscle subjected to such a aechanical force until after the advent of inextensibility will contain longer sarcomeres . Because of the negative correlation between the two parameters this increase in sarcomere length will be accompanied by a decrease in fibre diameter . It world therefore seam that the reduction in fibre diameter found when a muscle enters rigor under tension (5, 6, 7) is primarily due to the effect of tension on the sarcaaere length of the muscle . There is considerable variation in the mechanical forces exerted on individual muscles . Comparisons of fibre diameter in muscles of different species, in different muscles of the same species and even in different muscles of the same animal must therefore be made with caution . Mechanical factors such as limb position may even influence the fibre diameter of a particular muscle in different animals of the acme species . Such post-mortem mechanical factors moat be strictly standardised if fibre diameter is to be a reliable parameter of muscle growth . ACknawl edgemQnts We thank Miss Ellen Shiel for technical assistance . Ref4re_zçes 1 . J . ARONS~i, J . Cell Biol . 19 107-114 (1963) . 2 . J . HAN90C~T, J . biophya . biochem . Cytol . _2 691-710 (1956) . 3 . G .F . EL,LIOT, J . LOWY and B .M . MILLMAN, J . moles . Biol . 25 31-45 (1967) . 4 . P .V .J . HEGARTY and A .C . FFOOPER, J . Anat . 110 249-257 (1971) . 5 . E .M . BLACK and D .L . BLACK, J . Food Sci . 32 539-543 (1967) . 6 . W .A . GILLIS and R .L . iII3~1DRICXSOi~(, J . Food Sci . 34 375-377 (1969) . 7 . H .K . E~RRIIQG, R .G . CASSENS and E .J . BRISKEY, J . Food Sci . 30 1049-1054 (1965) . 8 . G . GOL.DSPIIVK, Proc . R . ir . Aced . 62 B 135-150 (1962) . 9 . P .V .J . I~GARTY and R .T . NALIDE, Lab . Pract . 19 161-164 (1970) . 10 . G . GOLDSPINK, J . Cell Sci . 3 539-548 (1968)
Pergamon Press
THE DIAMETER AID MEAN SARCQ~RE LENGTH OF II~NIDUAL MUSCLE FIBRES A . Coral Hopper and James P . Hanrahan Departments of Anataay and Agricultural Biology, Ltniversity College, Dublin, Ireland . (Received is final form August 4, 1975) S++^a .+"+. - Studies of the coefficients of variation and the repeatability of the measurements indicate that a sample of 25 fibres is sufficient to provide an accurate estimate of the aeon fibre diameter sad sarcanera length of a auscle . There is a significant negative correlation (-0 .55) between the diameter and mean sarcamere length of an individual muscle fibre . Because they affect sarcomere length postmortem mechanical influences aunt be strictly standardised if fibre diameter is to be a reliable parameter of muscle groerth . Although affected by many variables fibre diameter is probably the most widely studied parameter of muscle growth . During active contraction the diameter of isolated ayofibrils increases wh¢n the sarcameres shorten (1,2) . This is presumably due to an increase in the centre-to centre distance between actin and ayosin filaments (the myofilasent It packing density) which varies imrersely with sarcamere length (3) . can prob :bly be assumed that fibre diameter is increased durinç active contraction although a search of the literature has failed to reveal a study in which this assumption has been confiraed by measuranent . Because of the difficulty of avoiding the contraction of at least sane fibres during exposure or romoval, the study of muscles which have entered rigor has been advocated (4) . However, even the fibre diameter of rigor muscles may be affected by their condition during the cruet of rigor sortis . The mean fibre diameter of muscle strips subjected to increased tension is reduced (5, 6) and similar findings were reported is a study of the muscles of bov-" ine carcasses which had been hung vertically during the onset of rigor mortis (7) . In the latter study the sear sarcamere length of fibres in a separate sample from such muscles xas also measured and a highly significant negative correlation between fibre diameter sad mean sarcamere length was found . Because these parameters were measured in separate samples the present study was performed to examine the relationship between the diameter and mean sarcanere length of individual fibres . In addition the results were analysed to determine whether a asmple of 25 fibres is sufficient to provide an accurate estimate of the diameter and mean sarcamere length of the constituent fibres of a auscle . MethQda Twenty adult male mice (Mus musculua) from Falconer s R strain were killed by ether anaesthesia . Iamediately after death the anisals 775
776
Fibre Diameter and Sarcomere Length
Vol. 17, No . 5
The overall means and standard errors for fibre diameter and mean sarcanere length are shown in Table 1 together with the within muscle and between-muscle variances of these parameters . Calculated on a within-muscle basis the coefficient of correlation between the diameter of a fibre and it+s mean sarcomere length is -0 .55, The coefficient a value which is statistically significant (PO .OS) . The repeatability and coefficient of variation of fibre diameter and mean sarcomere length are shown in Table 2 . TABLE 2 Repeatability and Coefficient of Variation of Fibre Diameter and Mean Sarcomere Length Fibre Diameter Repeatability Coefficient of variation
0 .80 10 .3096
Sarcamere Length 0 .84 7 .8096
Discussion The campariaon of two or more samples is an essential feature of Yet the value of such studies may be considmany biological studies . erably reduced by a lack of information about the statistical properties of the parameters involved . The coefficient of variation provides a useful indication of the precision of the measurement of a parameter and the values obtained in the present study confira the usefulness of relatively small samples in the evaluation of the mean fibre diameter snd sarccaere length of a muscle . Furthermore, the high repeatability values obtained indicate that the measurement of 25 fibres is sufficient to provide an accurate estimate of the mean value of these parameters in an individual muscle . Although anticipated by the results of an indirect study (?) the results of the present study .provide the first direct evidence of an association between the diameter and mean sarcamere length of rigor muscle fibres . This negative correlation would seem to confirm the basic morphological similarity that exists between the shortening of active contraction and the shortening which occurs during the onset of rigor mortis . For same time after death the actin and myosin filaments slide freely over oae another and some shortening occurs in the presence of residual adenosine triphosphate (ATP) . When the level of ATP falls below approximately 2096 of the resting level the cross-bridges between the filaments become "locked" and further sliding is not possible . The result is the well known muscular inextenaibility of rigor mortis . The sarcomere length of rigor muscle therefore depends on the relative position of the filaments when the amount of ATP in the fibres falls below this critical level . The mean sarcomere length found in the present study is comparable to the value of 2 .8~rm reported in another study of the mouse biceps
Vol . 17, No . 5
Fibre Diameter and Sarcomas Length
777
were placed supine on a slab of foam polystyrene and pinned out with the limbo in full extension . The carcasses were left in this position at room teapersture (18oC) for four hours to allow the onset of rigor mortis . The skin was then removed from the carcasses and the right biceps brachü muscles were excised . The widest portion of the belly of this muscle contains all itts constituent fibres (8) . A transverse section of the entire muscle 1-2 ams . thick was excised with a scalpel and placed in a flat-bottomed tube (1 inch diameter) containing 1 ml calcium-free Ringer Locke solution . A hamogenizer consisting of a shaft containing two blunt blades surrounded by protective guards and capable of being rotated at 11,500 revolutions per minute was used to separate unfixed individual fibre fragments . This technique has been described in detail by Hegarty and Naude (9) . After separation the fibre fragmenta form a suspension in the Ringer Locke solution . 15 ml of this suspension was then transferred to the well of a hanging-drop slide with a Pasteur pipette and a cover slip placed over it . The iaage of the fibres was projected from a monocular microscope on to a sheet of white card at a aagnification of X750 by means of a mirror attached to the eye-piece . Measurements were then made on 25 fibres Pram each muscle . Short fragments were excluded as were fragments whose entire length was not in fxus and which consequently were not in the plane of focus . The number of A bands along a 100~m length of fibre was counted anu the aeon sarcameré length of the fibre was calculated from this . the diameter of the fibre was measured at the mid-point of this 100um length with a micrameter'callipers . Thus the diameter and mean sarconere length of 500 fibres xas determined . The overall and within-muscle means for fibre diameter and mean aarcomere length were calculated and their standard errors were deter mined . The coefficient of correlation between the disaster and mean ssrcamere length of individual fibres " was calculated on a within-muscle basis . The coefficient of correlation between the muscle means of these parameters was also calculated . A simple hierarchical analysis of varianç~e was used to estimate the between-muscle (6m ) and within-muscle (Cf ) sources of variation for fibre diameter a~ sarcamere number . These estimates were used to compute the repeatability of the measurements and th~ co~ffici~nt of variati~n of heir means defined respectively as (6m /4m ~r- dw /25) and (~ + Qw /25)~/overall aeon . Results TART-F 1
Overall Mean, Standard Error and Variances of Fibre Diameter and Mean Sarcamere Length
Mean (Wm) Standard error (gym) Between-muscle variance Within-muscle variance
Fibre Diameter 31 .5 0 .3 4 .7 21,3
Sarcamere Length 2 .91 0 .02 p .pq 0,27
778
Fibre Diameter and Sarcomere Length
Vol . 17, No . 5
brachü in which the muscle was allowed to enter rigor with the limb pinned out in full extension (10) . These values are considerably greater than the values found in four mouse muscles which entered rigor free of external mechanical restraint (4) . Subjecting a muscle to mechanical restraint or increased tension will reduce the degree of filament overlap which occurs during the onset of rigor mortis . Muscle subjected to such a aechanical force until after the advent of inextensibility will contain longer sarcomeres . Because of the negative correlation between the two parameters this increase in sarcomere length will be accompanied by a decrease in fibre diameter . It world therefore seam that the reduction in fibre diameter found when a muscle enters rigor under tension (5, 6, 7) is primarily due to the effect of tension on the sarcaaere length of the muscle . There is considerable variation in the mechanical forces exerted on individual muscles . Comparisons of fibre diameter in muscles of different species, in different muscles of the same species and even in different muscles of the same animal must therefore be made with caution . Mechanical factors such as limb position may even influence the fibre diameter of a particular muscle in different animals of the acme species . Such post-mortem mechanical factors moat be strictly standardised if fibre diameter is to be a reliable parameter of muscle growth . ACknawl edgemQnts We thank Miss Ellen Shiel for technical assistance . Ref4re_zçes 1 . J . ARONS~i, J . Cell Biol . 19 107-114 (1963) . 2 . J . HAN90C~T, J . biophya . biochem . Cytol . _2 691-710 (1956) . 3 . G .F . EL,LIOT, J . LOWY and B .M . MILLMAN, J . moles . Biol . 25 31-45 (1967) . 4 . P .V .J . HEGARTY and A .C . FFOOPER, J . Anat . 110 249-257 (1971) . 5 . E .M . BLACK and D .L . BLACK, J . Food Sci . 32 539-543 (1967) . 6 . W .A . GILLIS and R .L . iII3~1DRICXSOi~(, J . Food Sci . 34 375-377 (1969) . 7 . H .K . E~RRIIQG, R .G . CASSENS and E .J . BRISKEY, J . Food Sci . 30 1049-1054 (1965) . 8 . G . GOL.DSPIIVK, Proc . R . ir . Aced . 62 B 135-150 (1962) . 9 . P .V .J . I~GARTY and R .T . NALIDE, Lab . Pract . 19 161-164 (1970) . 10 . G . GOLDSPINK, J . Cell Sci . 3 539-548 (1968)
