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Differences in histochemical attributes between diaphragm and hindleg muscles of the rat.

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Differences in Histochemical Attributes Between
13iaphragm and Hindleg Muscles of the R a t
Laboratory of Neuropathology and Neuroanatomical Sciences,
National Institute o f Neurological Diseases and Stroke,
National Institutes of Health, Public Health Service,
U. S . Department of Health, Education and Welfare,
Bethesda, Maryland 20014
Muscle fibers of the diaphragm and hindleg muscles of the rat
were characterized histochemically by their mitochondria1 distribution ( succinic
dehydrogenase activity) as A, B and C (after Stein and Padykula, ’62),by their
myofibrillar, alkali-stabile ATPase activity as a, p and ap (after Samaha et al.,
’70), and by their acid-stabile ATPases (intermyofibrillar and myofibrillar) as
D (dark), M (moderate) and L (light). Comparisons of the differentially stained
serial sections revealed profound dissimilarities between the fibers of functionally disparate muscles. The majority of fibers of the hindleg muscles were distinguishable as the conjugates AapM, BBD and CaL (figs. 1-3), whereas those
of the diaphragm were principally AaM, BaL and CBD (figs. 4-6). Thus, no one
histochemical reaction could accurately predict the multiple attributes of fibers
of both muscle groups. Previous studies suggest that additional discrepancies in
histochemical appearance would arise were other specialized muscles, e.g., extraocular muscles (Yellin, ’69), compared with the more common muscles of body
and limb. Any extrapolation of data on the basis of limited histochemical analyses is not without risk, whether comparing the same muscle in different species
(Yellin and Guth, ’70), or different muscles in the same species.
Although the histochemical heterogeneity of mammalian skeletal muscle
fibers is now well established (Stein and
Padykula, ’62; Romanul, ’64; Yellin, ’67;
Schiaffino et al., ’70 j , i t is still widely held
that the many muscles of an animal are
composed of varying proportions of the
same two or three “fundamental fibertypes.” This notion derives largely from the
apparent invariabIe association of two or
more histochemical attributes in individual
imuscle fibers (Engel, ’70, table 1 j. This
impression can result in unwarranted extrapolation of data between species (Yelliin and Guth, ’70; Guth and Yellin, ’71)
rand, as will be shown, even between muscles of a single species.
Any analysis of the diaphragm can hope
ffor little better in the way of introductory
iremarks than those provided by Lee,
IGuenther, and Meleney in 1916, in their
icomprehensive study of this unique muslcle: “. . . . while most skeletal muscles in
the living body contract with varying deANAT. REC., 173: 333-340.
g e e s of intensity and at irregular intervals
between which occur long periods of rest,
the diaphragm from the time of birth to
that of death performs day and night a
continual succession of brief contractions
of a fairly regular rhythm and fairly uniform in extent, alternating with brief intervals of rest. Thus, this muscle, together
with other respiratory muscles, holds a
unique position among skeletal muscles
and suggests a crude analogy with the
heart. Like the heart too, the diaphragm
performs during the lifetime of the individual an incredibly huge amount of work,
probably more than any other skeletal
muscle .”
“In view of these facts it might be expected that a careful study of this important muscle would reveal physical and
chemical peculiarities which would distinguish it from other muscles.”
The present work illustrates the striking
differences in histochemical attributes beReceived Nov. 30, ’71. Accepted Feb. 18, ’72.
tween the muscle fibers of the diaphragm
and those of the hindleg of the rat. Furthermore, the resulting incongruities in
muscle fiber characterization stress the
need for a more inclusive and flexible
nomenclature than has hitherto been employed in histo-descriptive studies of
Mid-portions of selected hindleg muscles
and of the left hemidiaphragm were removed from adult Osborne-Mendel female
rats (250-275 gm) anesthetized with
chloral hydrate (400 mgm/Kg, ip). Both
sternal and costal portions of the diaphragm were studied to detect any regional
differences in the fiber population of that
muscle (see George and Susheela, '61').
The diaphragmatic segment and one or
another of the leg muscles were placed together, and immediately quenched in liquid
nitrogen for up to three minutes. The muscles were then allowed to warm to -17°C
in a cryostat, and transverse, 10 thick,
serial sections were cut and affixed to consecutive glass slides. The successive muscle
sections were processed histochemically to
reveal the presence of mitochondrial succinic dehydrogenase (SDH) activity (Pearse,
'61) as well as the alkali-stabile component
of the myofibrillar adenosine triphosphatase (ATPase) and the acid-stabile components of the intermyofibrillar and myofibrillar ATPase (Guth and Samaha, '70;
see Guth and Yellin, '71 for a description of
the cellular components revealed by the
method for acid-stabile ATPase (s).
Individual histochemical methods usually demonstrate three categories of skeletal muscle fibers. However, the utilization
of several techniques, on consecutive specimens of transversely sectioned muscle, reveal still more varied histochemical profiles
among the fibers of many muscles (reviewed in Guth and Yellin, '71). The reader
is cautioned that in attempting to illustrate
the dissimilarities between fibers of the
diaphragm and the hindleg muscles, only
the predominant fiber varieties have been
The considerable attention already given
to muscle fibers of the rat's hindleg permit
their utilization as a standard for comparison with the fibers of the diaphragm
(Stein and Padykula, '62; Romanul, '64;
Yellin, '67; Yellin and Guth, '70). In the
tibialis anterior, gastrocnemius, extensor
digitorum longus and plantaris muscles,
the SDH reaction reveals large A fibers
with sparse, often linearly-arranged, small
formazan particles, small B fibers with uniformly distributed small particles and,
small C fibers with subsarcolemmal accumulations of large particles that cause
the centers of the fibers to appear less dense
(fig. 1) (after the classification of Stein
and Padykula, '62; mitochondrial localization substantiated electron-microscopically
by Padykula and Gauthier, '63, '67;
Gauthier, '69). The reaction for the alkalistabile (myofibrillar) ATPase results in
dark, moderate and lightly stained fibers
designated a , u p , and p, respectively (after
the classification of Samaha et al., '70).
In the rat hindleg (see interspecies differences, Yellin and Guth, ' 7 0 ) , the a fibers
conform to the C fibers, the ap fibers to the
A fibers, and the p fibers to B fibers (fig. 2).
Staining for the acid-stabile (intermyofibrillar and myofibrillar ) ATPases results
in a general reversal of those staining intensities obtained for the alkali-stabile enzyme (fig. 3 ) . These properties of hindleg
muscle fibers (figs. 1, 2, 3) as well as fiber
diameter (represented by muscle fibers of
the tibialis anterior muscle) are listed and
compared with those of the diaphragm
(figs. 4, 5, 6 ) in table 1.
The striking differences between fibers
of the diaphragm and the hindleg muscles,
as represented by the tibialis anterior, are
readily apparent. Though A, B and C fibers
are represented in the diaphragm (fig. 4),
their staining for the alkali-stabile ATPase
is totally dissimilar from that of the hindleg muscles; the a fibers are representative
of both A and B fibers rather than the C
fibers alone, and the p fibers conform to
the C fibers rather than the B fibers. The
a@ fibers are usually, though not always
conspicuous by their absence. (When
present the u p fibers are A fibers.) The
reaction for the acid-stabile ATPases also
results in appreciable differences. Although
the A fiber is moderately stained in both
diaphragm and tibialis anterior, the B and
C fibers exhibit a reversal of staining in-
ATPase 2
ATPase( s) 3
Mean fiber
1 Characterized on the basis of the qualitative distribution of mitochondria.
2 Characterized as theoretical dimeric combinations, the constituents of which have either similar
or dissimilar pH-labilities.
3 Characterized on the basis of intensity as L, (light); M, (moderate) and D, (dark).
tensity. The B fiber, commonly dark in the
hindleg muscles, is light in the diaphragm,
and the C fiber commonly light in the
hindleg muscles is dark in the diaphragm
(fig. 6 ) . That the latter reversal is not a
result of error in interpretation of the SDH
reaction is supported by the intracellular
arrangement of the myofibrillar and sarcoplasmic constituents of the C fibers, which
are similar in both muscles (compare
figs. 2, 6).
In one respect, the diaphragm is like
many other skeletal muscles in the rat; the
relative proportions of its three varieties
of muscle fibers differ markedly from region to region. The p ( C ) fibers, for example, may vary from as little as 20% to
as much as 45% of the local fiber population; in some instances this population
change occurring within a 1000 p extent of
the diaphragm's circumference. The large a
(A) and small a (B) fibers may be present
in like numbers or may vary inversely, each
ranging from 20% to 40% of the total
fiber population. The most overt differences
in populations of fibers often occur in the
same locale, across a fibrovascular septum
that intermittently divides the caudad and
cephalad layers of the diaphragm. Whether
these regional differences in fiber population within the diaphragm are constant
from animal to animal, in the manner of
fiber concentrations in hindleg muscles
i:Yellin, '69) was not readily apparent, as
(he fluctuations occur quite rapidly along
the circumference of the muscle. Throughout those regions of the diaphragm studied,
there was usually a good mixture of the
1.hree basic fiber varieties, although aggregates of p ( C ) fibers were occasionally observed. The small blood vessels, recognized
by a precipitate associated with the ATPase
reactions, were closely associated with all
muscle fibers, regardless of their histochemical attributes (Romanul, '65).
Muscle spindles were observed in the diaphragm and did not appear to differ histochemically from those observed in the hindleg muscles. (Yellin, '69a,b).
Innervation, functional environment and
hormones are felt to play prominent roles
in determining the varied enzyme histochemical characteristics of mammalian
skeletal muscle fibers (Romanul and Van
Der Meulen, '67; Yellin, '67; Edgerton et
al., '69; Gutmann et al., '70; Guth and
Yellin, '71; Gutmann et al., '71). Consequently, it is not unreasonable to expect
some diversification in the histochemical
properties of muscle fibers in muscles subserving disparate functions, as has already
been described in extraocular and intrafusal muscle fibers (Yellin, '69b). A comparison of the functional characteristics
of the diaphragm and the hindleg muscles
suggests appreciable differences in (1) patterns and frequencies of activity, ( 2 ) nature of the work load, ( 3 ) extent of stretch
imparted by load and antagonists and ( 4 )
the mode of work translation (i.e., for the
diaphragm, in part, via central tendon and
contracting muscles of the contralateral
hemidiaphragm). The present study did in
fact disclose striking differences between
the histochemical attributes of muscle fibers of the diaphragm and the hindleg as
summarized in table 1.
Although mitochondria1 SDH activity appears to be related to the fatiguability of
motor units (Burke et al., '71), the sig-
nificance of the histochemically elicited
pH-dependent ATPases is more uncertain,
but may be related to speed of contraction
and duration of the active state (Sandow,
’70). It seems plausible that the discrepancies in fiber histochemical attributes of diaphragm and hindleg muscles are indicative
of real metabolic differences. Indeed, the
distinctive histochemical attributes of the
diaphragmatic fibers may reflect some of
the metabolic properties which led Lee et
al. (’16) to state, “Most of these facts indicate that the diaphragm possesses much
more efficient muscular tissue than do the
other muscles-in
other words, it is a
superior physiological mechanism. This is
exactly what might be expected when the
unique and superior role of the diaphragm
among skeletal muscles is considered. Here
seems to be a striking instance of physiological adaptation to physiological requirements.”
The present results serve to re-emphasize
the need for more inclusive and flexible
means of characterizing muscle fibers
(Schiaffino et al., ’70; Ashmore and Doerr,
’71; Barnard et al., ’71; Bass et al., ’71).
It is obvious from the findings that none
of the three techniques employed could accurately predict the results of the other two
methods. Thus, indiscriminate extrapolation of data on the basis of limited studies
is fraught with risk. The question remains,
how can the diverse findings, obtained from
a battery of histochemical assays, be expressed in a relatively simple and meaningful manner? Several of the muscle fiber
‘‘classifications’’ currently in vogue do,
when properly employed, conceptualize
specific, qualitative differences between
skeletal muscle fibers. Distinctions in mitochondrial distribution and myofibrillar
(actomyosin) ATPase pH-stability (see results and figures) are accurately conveyed
by the designations A, B, C and a, p, ap
respectively (Stein and Padykula, ’62;
Samaha et al., ’70). Other histochemical
differences, presumably reflecting levels of
enzymatic activity and identified by differences in coloration or intensity of the
reaction product, have been conveyed by
such labels as L (light), M (moderate),
D (dark), or some variation thereof. (Padykula and Gauthier, ’67 and Gauthier, ’69
have also suggested the terms “red,”
“white,” and “intermediate” on the basis of
“mitochondria1 content”. ) In the present
study, the designations L, M, and D have
been applied to the reaction demonstrating
the acid-stabile ATPase(s). Thus, if the
techniques employed and the distinctions
they make are briefly, but adequately described, the appropriate conjugation of the
several designations (there could easily be
more than three) enables the distinction(s)
between fibers to be expressed succinctly
(e.g., in discriminating the AapM, BpD and
CaL fibers of the hindleg from the AaM,
BaL and C p D fibers of the diaphragm in
the rat). Finally, these conjugations are
purely descriptive terms, and may be
readily matched to the functional properties
of fibers as the latter information becomes
available (Burke et al., ’71).
The author wishes to express his appreciation for the technical assistance
given by Mrs. Janina Ziemnowicz, and the
assistance in manuscript preparation provided by Miss Hilda Malcolm and Mrs.
Nancy Yellin.
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Mitochondrial succinic dehydrogenase activity in muscle fibers of the
tibialis anterior muscle. The fibers designated A, B and C illustrate the
qualitatively different distributions of the mitochondria.
Myofibrillar, alkali-stabile adenosine-triphosphatase (ATPase) activity
in the same three muscle fibers depicted in figure 1; designated ap, p
and a, respectively.
Intermyofibrillar and myofibrillar, acid-stabile ATPase activity in the
same fibers depicted in figures 1 and 2; designated as M (moderate),
D (dark) and L (light), respectively.
Mitochondrial succinic dehydrogenase activity in muscle fibers of the
respiratory diaphragm. The A, B and C fibers are similar though of
smaller diameter than those of the tibialis anterior muscle.
Myofibrillar, alkali-stabile ATPase activity in the same three fibers depicted in figure 4; designated a, a and p respectively.
Intermyofibrillar and myofibrillar, acid-stabile ATPase activity in the
same fibers depicted in figures 4 and 5; designated M (moderate),
L (light) and D (dark) respectively. All x 800.
Herbert Yellin
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muscle, hindleg, differences, diaphragm, rat, attributed, histochemical
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