0022-1554/90/$3.30 The Journal of Histochemistry and Cytochemistry Copyright © 1990 by The Histochemical Society,
Vol.
LUISA
of General
Received
for
A novel
type
Pathology,
publication
University
March
ofmyosin
29,
heavy
ofPadova
1989
chain
and
and CNR
in revised
form
called
(MHC),
2X,
Unit for Muscle August
has
of skeletal
histochemistry.
1969).
muscle
A currently actomyosin
ofskeletal
1955;
Actomyosin
1 fibers,
used ATPase
fibers
Herman,
Bnooke
activity
is the based
demonstration on
the
1969;
is acid-stable and
and
Guth
accepted
38:257-265,
Samaha,
in type
2 fibers.
(Schiaffino
and
Pienobon-Bormiobi,
treating
muscle
into sections
type
to pH corresponds to differences in ATh isolated from slow and fast muscles (Guth
1969), and is also observed after extraction detergents that remove membrane-bound
subdivided
in
type with
1973). 2A,
type
buffers
Type
2B, and
at acid
ofthe 5ccenzymes
2 fibers type
pH (Brooke
can
be
2C by preand
Kaiser,
in pant by grants
2
Correspondence
University
from CNR and Ministero
Pubblica
Is-
of Padova,
to: Dr. Luisa Gorza, via Tnieste 75, 35132
Institute Padova,
of General Italy.
Pathology,
1989
(9A1655).
1990)
or with pH
paraformaldehyde
(Guth
and
followed
Samaha,
1970).
ben was demonstrated
by varying
buffering
pre-incubation
agents 1984;
ofthe
Golbnick
and
the
1981;
MHC
in these
by biochemical type
studies
2C fibers
Gauthier
composition
(Schiaffino
Recently, (MAb), this muscle
et
al.,
Libera
to be hybrid
Lowey,
to be expressed
composition and guinea
et al., fibers
1980). containing
The is con-
In conmix-
et al., 1980, and neonatal
1986).
in a subset
et al., study,
the GoIb-
1977).
populations
using a panel of antimyosin monocbonal laboratory has identified a novel MHC
(Schiaffino
and
by immunological heavy chains (MHC)
tunes oftype 1 and type 2A MHC (Pierobon-Bormioli 1981), or regenerating fibers containing embryonic MHC
and
(Matoba
and
two fiber
(Dalba
were found
2 fi-
of type
1984).
et al.,
trast,
ionic
can also be identified specific for myosin
existence firmed
at alka-
subtype
medium
(Pierobon-Bormioli of distinct
by incubation
Another
Matoba,
Type 2A and 2B fibers methods using antibodies
In the present
truzione.
29,
Italy.
Muscle
WORDS:
was found
1 Supported
August
Padova,
fiber typing; Histochemical myosin ATPase; Monoclonal anti-myosin antibodies; Immunohistochemistry; Rat skeletal muscle; Mouse skeletal muscle; Guinea pig skeletal muscle. KEY
nick,
(Padykand
and Samaha, tions with further
differential
alkali-labile
alkali-stable
of
and Physiopathology,
whereas it is low in guinea pig 2X fibers. Succinate dehydrogenase displays moderate to high activity in 2X fibers of all species. Taken together, these staining patterns allow this noiclfiber population to be distingUished from the other type 2 fibers using only enzyme histochemistry. Nevertheless, the combined use ofimmunoand enzyme histochemistry prevents incorrect fiber typing due to the interspecies variability of myosin AThise activity among the correspondent fiber types, and completely modifies the presently used classification ofmouse type 2 fibers. (JHistochcm Cytochem
line
by enzyme
and acid pre-incubations
and Kaiser,
it is acid-labile
This differential sensitivity ase activity ofactomyosin
obtained
method
to alkali
ATPase
whereas
is routinely
Biology
8, 1989;
1970)
typing
ula and
1990
in USA.
of a Novel Type 2 Fiber Population in Skeletal Muscle by Combined Use of Myosin ATPase and Anti..myosin Antibodies’
Introduction
response
257-265,
GORZA2
Institute
myofibnillar
pp.
Article
been recently identified in type 2 fibers ofrat skeletal musdes using an immunochemical approach. In the present study, the same panel ofanti-MHC monodonal antibodies was used in immunohistochemistry combined with enzyme histochemistry to identify and compare type 2X fibers in hindlimb skeletal muscles of rat, mouse, and guinea pig. Immunohistochemistry shows that 2X MHC is localized in a barge subset of type 2 fibers and is co-expressed with 2A or 2B MHC in a small number offibers. Enzyme histochemistry shows that type 2X fibers display low myosin ATPase activity after pre-incubanion at pH 4.3 and high activity after paraformaldehyde pre-incubation at pH 10.4. After preincubation at pH 4.6, myosin ATPase shows intermediate and high activity in rat and mouse 2X fibers, respectively,
Fiber
No. 2,
Printed
Original
Identification Mammalian Histocheinical Monoclonal
38,
Inc.
of type
2 fibers
antibodies isoform that of rat skeletal
1989). the
expression
in different hindlimb pig were investigated
of 2X MHC
skeletal muscles using combined
and
the fiber
of rat, mouse, immunoand
257
Downloaded from jhc.sagepub.com at KAI NAN UNIV on February 18, 2015
GORZA
258
Downloaded from jhc.sagepub.com at KAI NAN UNIV on February 18, 2015
A
NOVEL
TYPE
enzyme
2 FIBER
in all the
MAMMAUAN
Type 2X fibers
histochemistry.
lation
IN
species
studied;
SKELETAl
represent
a major
in addition,
259
MUSCLE
fiber
in each
species
anti-MHC
three
ofactomyosin
type
in different
ATPase species,
can be obtained with anti-MHC skeletal
the
may vary for the same
precise
identification
fibers
in light
adopted
of the
in previous
studies
presented
here.
findings
fibers Monod onal BA-D5
SC-B
anthodics
SC-71
BF-F3
BF-32
RT-D9
BF-35
types Rat
analysis of mouse
must
muscle
fiber
of fiber
only by combined immunocytochemicab antibodies. In particular, fiber typing
muscle
dered
inactivation
immunohistochemical reactivity of antibodies with various types of
monoclonal
skeletal
subtypes oftype 2 fibers can be identified by myosin ATPase staining using two different pre-incubation assays. However, as the degree
1 . Comparative
Table
popu-
Type!
be reconsi-
+
-
-
-
+
-
+
2A
-
+
+
-
+
-
+
2B
-
+
-
+
-
+
+
2X
-
+
-
-
-
+
-
+
-
-
-
+
+
+
-
+
+
-
+
+
+
-
+
-
+
-
+
+
-
+
-
-
-
+
-
Mouse
Materials
and
Typel
Methods
2A 2B 2X
Tissue Source. Different hindlimb skeletal muscles (tibialis anterior, extensor digitorum longus, soleus, penoneus, gastnocnemius) from Wistar rats, Balb/c mice, and guinea pigs were excised and frozen in liquid nitrogen.
For interspecies
des
were
comparison,
collected
on
the
cryosections
same
glass
from
slide
and
corresponding
processed
Guinea
mus-
pig
Type!
Monocbonal Antibodies. The MAb used were produced by hybridomas obtained in different fusions and were found to display differential neactivity with four MHC isoforms in rat skeletal muscle (Schiaffino et al., 1989). In brief, SC-71 and
BA-D5 with
reacted
type
2A MHC;
types except
with
type
2A MHC;
RT-D9
1 MHC;
BF-F3
with
type
with
SC-75
type
2B and
with
2B MHC;
2X MHC;
all type BF-32
and
BF-35
with
type
pH
7.4,
for 4 mm
at room
temperature,
at room temperature (PFA-10.4) (Guth and Samaha, Succinate dehydrogenase activity was demonstrated dunes previously described (Nachlas et al., 1957). Immunohistochemistry dase im
techniques cryostat
mm at 37C 1:2-1:10
was performed
as previously sections
were
in a humid
in PBS,
pH
ringer; Mannheim, in PBS before
with
peroxidase
(Dakopatts;
0.5%
for 30
bovine
peroxide.
in 0.05
M Tnis-HCI,
Reaction
After
pH,
7.6 with
was performed
by repeated Balsam,
and
observed
140 in PBS,
of0.Ol%
in a Zeiss
-
+
-
-
-
+
+
2X
-
+
-
-
-
+
-
rinses in PBS bound
PA)dis-
of hydrogen
at room
tempera-
were dehydrated, IM 35 microscope.
of reactivity
types of three rat,
different
ter acid
pne-incubation.
anti-MHC
species
Type
appeared
dark
1 fibers after
the
was obtained
by com-
of myofibniblar were
acid
differ-
in the seine
1-3.
types
staining
with
illustrated
in Figures
of fiber
the histochemical
la) and
MAb
in l#{224}ble1 and
identification
with
ure
of seven
is summarized
panison
labeled
ATPase
by BA-D5
pre-incubations
pale
after
SC-71
pH 4.6 pre-incubation
(Figure
(not shown). ate staining
ic)
term
two discrete
BF-F3,
“type
by both BF-F3 by RT-D9 and 2X. These ATPase cinate
fibers,
RT-D9,
(Figures
lh) and
which
also
to the
labeled
by
1 fibers
type
showed intermedilh), could be subon the basis
1ble
Ig were were
subset
of reac-
Figures
id-if).
of fibers
labeled
1 and
and RT-D9, whereas the subset of fibers labeled unreactive with BF-F3 and BF-35 was called type
dehydnogenase
whereas
which (Figure
(see
were
labeled
subpopulations BF-35
2B” was restricted
two subtypes
assay,
fiber and
could
demonstration
the former
(Figure
by BF-32,
Conversely, type 2B fibers, after pH 4.6 pre-incubanion
into with
and
af (Fig-
and ii). Type 2 fibers were labeled by SC-75 (Figure ib) and bight after pH 4.3 pre-incubation (Figure ig). Type 2A fibers
The
conjugated
diluted
rinses in PBS. Sections
+
2B
In the
30
diluted
by repeated
rinses
for 15 mm
+
+
pattern
muscle
10-
(BSA)(Boeh-
antibodies
addition
+
+
ent fiber
tivity
for
supennatants albumin
and
+
-
divided
In brief,
incubated
with 1 mg/mlp-phenylenediaminePolyscience Inc. Warnington,
in the dark
ture and was stopped
1988).
and
repeated
the
proce-
immunopenoxi-
was removed
Denmark)
at 37C.
mounted
in Canada
serum
antibody
antibodies were revealed by incubation pyrocathechol(Hanker Yates Reagent; solved
et al.,
immunogbobulin
Glostrup,
mm
indirect
slides
by
for 15 mm
1970). following
with hybrydoma
0.5%
anti-mouse
with
(Gorza on glass
FRG). Unbound
with
using
described collected
chamber
7.2,
applying BSA
followed
10.4 in 100 mM aminometylisopropanobol
at pH
-
-
Results The
M cacodylate,
-
+
1
all MHC
2X.
pre-incubation
-
-
2 MHC;
with
Enzyme and Immunohistochemistry. Myofibnillar actomyosin ATPase activity was demonstrated using procedures previously described (Matoba et al., 1985; Pierobon-Bonmioli et al., 1981; Padykula and Herman, 1955). In brief, consecutive serial cryosections were processed using three different pre-treatments: (a) pre-incubation in 100 mM acetate buffer, pH 4.3, for 10 mm at room temperature (Brooke and Kaiser, 1970); (b) pre-incubation in 100 mM acetate buffer, pH 4.6 for 10 mm at room temperature (Brooke and Kaiser, 1970) or in 100 mM citrate buffer, pH 4.6, for 3 mm at room temperature (Matoba et al. 1985); (c) fixation in 4% paraformaldehyde in 0.2
+
2A
together.
PFA-10.4
reaction
(Figures
type type
be also distinguished
after
2X fibers 2B fibers
lh and
were were
using
myosin
and
the suc-
pre-incubation darkly moderately
ij and l#{224}ble 2). With stained,
like type
stained.
With
2A the
succinate dehydrogenase reaction, type 2B fibers displayed weak activity, whereas type 2X fibers displayed moderate to strong activity. As shown in Figures 2 and 3, type 2X fibers can also be identified
using
similar
criteria
in mouse
and guinea
pig hindlimb
mus-
Figure 1. Rattibialis anterior, deep portion. Serial cryostat sections stained for indirect immunoperoxidase with anti-MHC antibodies(a-f), myofibnillar actomyosin ATPase(g-l), and succinate dehydrogenase(j). (a) BA-D5, anti-type 1MHC; (b) SC-75, anti-type 2MHC; (C) SC-Ti, anti-type 2A MI-IC; (d) BF-F3, anti-type 28 MHC; (a) RT-D9, anti-type 2X and 2B MHC; (f) BF-35, anti-type 1, 2A, and 2B MHC; (g) myosin ATPase after pH 4.3 pro-incubation; (h) myosin ATPaSS after PFA.1O.4 pre-incubation; (I) myosin ATPase after pH 4.6 pro-incubation; (J) succinate dehydrogenase activity. Type 2 fIber subpopulations are identified as A (2A), B (2B), and X (2X). Bar = 63 tm.
Downloaded from jhc.sagepub.com at KAI NAN UNIV on February 18, 2015
260
GORZA
B
x
C
Figure 2. Mouse tibialis anterior, deep portion. Serial cryostat sections stained for indirect immunoperoxidase with anti-MHC antibodies (a-.), myofibrillar actomyosin ATPase(g-h) and succinate dehydrogenase(l). (a) BA-D5, anti-type 1MHC; (b)SC-75, anti-type 2MHC; (C) SC-Ti, anti-type 2A MHC; (d) BF-F3 anti-type 2B MHC; (a) BF-35, anti-type 1, 2A, and 2B MHC; (f) myosin ATPSSe after pH 4.3 pro-incubation; (g) myosin ATPaSe after PFA-10.4 pro-incubation; (h) myosin ATPaSe after pH 4.6 pro-incubation; (I) succinate dehydrogenase activity. Type 2 fiber subpopulations are identified as A (2A), B (2B), and X (2X). Bar = 40 m.
Downloaded from jhc.sagepub.com at KAI NAN UNIV on February 18, 2015
A NOVEL
TYPE
2 FIBER
IN
MAMMAliAN
SKELETAl
MUSCLE
261
.
I
.-
:;:
1’
-
:‘
,.,F,, ,.
I.,
.
.
,0
E ,.:.‘
‘: ‘,
rCfl ‘
,
..
(er-
Figure
3. Guinea
pig tibialis
anterior,
superficialis
portion.
Serial
cryostat
sections
stained
actomyosin ATPase(e-9), and succinate dehydrogenase(h). (a) BA-D5, anti-type 1 MHC; anti-type 1, 2A, and 2B MHC; (e) myosin ATPase after pH 4.3 pro-incubation; (f) myosin pre-incubation;
(h) succinate
dehydrogenase
activity.
Type
2 fiber
subpopulations
are
for indirect immunoperoxidase with anti-MHC MAb (a-d), myofibrillar SC-75, anti-type 2MHC;(c) BF-32, anti-type 1 and 2A MHC; (d) BF-35, ATPase after PFA-10.4 pro-incubation; (g) myosin ATPase after pH 4.6 identified as A (2A), B (2B), and X (2X). Bar 65 sm. (b)
Downloaded from jhc.sagepub.com at KAI NAN UNIV on February 18, 2015
262
GORZA
Table
2.
Comparative
and actomyosin pre-incubation
succinate
A TPase assays
dehydrogenase
staining
offiber
activity
types
after
Myofibrillar ATPasc Fiber
type
SDH
pH 4.3
(SDH)
ous in the Schiaffino,
different
actomyosin staining
pH 4.6
Micnoheterogeneity
1
Interns
Dark
Dark
Light
Dark Light Interm/dark
Light Light Light
Light Interm Interm
Dark Interm Dark
1
Interm
Dark
Dark
Light
2A 2B
Dark Light Dark
Light Light Light
Light Interm Dank
Dark Intenm Dark
oftype
Dark Light
Dank Light
Light Intenm
tivity.
2A
Interm Dark
2B
Light
Light
Interm
Interm
2X
Intenm/dark
Light
Light
Dark
2X
Guinea
des.
1
Slight
different
differences fiber
in the
types
the guinea
pig,
anti-2B
unreactive,
but
three
guished tive
cal and
species
MHC
BF-F3
type
in this species
with
BF-32
identified
and
reactivity
of these
2 fiber
as reactive
with
antibodies
and
anti-2A
MHC
2A fibers with
SC-75
were
BA-D5,
and
type
unreactive
with
the
as reacwere
BF-35,
and
type
2B fibers were identified by exclusion. More pronounced interspecies differences were found in the pattern of inhibition of myosin ATPase staining after different preincubations (see Table 2). In the guinea pig, type 2X fibers could not be distinguished from the type 2A subpopulation, when classified
solely
2X
fibers,
2X MAb
together
RT-D9,
2A fibers; pression
with
2X
MHC
because
this
of2B
MHC
other
MHC
types
isoforms.
other
found
in rat and
MHC
was evident
were
was evaluated
by comparison
muscles.
The
type
In the rat, using
detected in a small number antibody also recognizes 2B MHC,
in mouse
with
coexistence
in rat sobeus (Figure 4), where It is interesting to note that
exceptional.
than
the
of type the ex-
was
tivity. Using such an approach, fibers labeled by both BF-F3, and therefore co-expressing 2A and 2B MHC,
BF-F3
reac-
SC-71 and were never
of 2X and
2A
pure type 2X fibers the ATPase activity
of these type 2A/2X fibers did not differ significantly from that ofthe pure type 2A, probably because ofthe low amount of2X MHC. Because of the back of a monoclonal antibody specific for 2X MHC,
ing 2X and F3 and ing
difficult
it was
2B MHC.
for BF-35
was evaluated
ied (‘I#{224}ble 3). Type type population des (EDL and
However,
were occasionally
to fibers that The percentage
fibers
to identify
co-expressed ofeach fiber in the tibialis 2X fibers
in this penoneus
unambiguously fibers
that
observed,
the fibers stained probably
express-
weakly
for BF-
correspond-
2X and 2B MHC. type population and of intermediate anterior
were found
ofthe
three
to represent
tongue
(S.
Pette,
1988;
Thus, can
the
identification
be obtained
either
Bnooke
and
are more ATPase
Termin
The
et
present
Samaha
study
Kaiser
(1970)
shows
type
2 fiber
using
(1970)
ac-
popula-
a combination appli-
of myofibniblar actomyosin by the pre-incubation con-
demonstrated
that
type
2 oxida-
sensitive to pH 4.6 inactivation of myofibnillar than type 2 glycolytic fibers; in addition, Guth showed
that
buffered paraformaldehyde, hibits myosin ATPase activity can
1989;
MAb on with the simultaneous histochemical stainings.
ditions. tive fibers actomyosin
novel
independently
anti-myosin different
histochemical demonstration can be profoundly influenced
This
,
1987).
recently identified and called type in a large subpopubation
The ATPase
and
et al. et al.,
of this
that
be identified
a brief
followed oftype
three
distinct
combining
the
pre-incubation
in 4%
by alkali pre-incubation, 1 and type 2 glycolytic subpopulations
different
infibers.
of type
patterns
2 fibers
of inhibition
of myofibnillar actomyosin ATPase after pH 4.6 and PFA-10.4 preincubation assays, as illustrated in Table 2; in addition, the use of specific monocbonal anti-MHC antibodies confirmed that distinct MHC types are expressed in these type 2 fiber subpopubations. studies
have
reported
controversial
results
in quantita-
tive analysis offiber type composition ofmammalian skeletal musdes based on the use of a single histochemical staining (Green et al., 1984; crepancy, in part
Pullen, because common
Nevertheless, lation might hydrogenase
1977). The present results histochemical properties to type
2B fibers
the possible be evinced activity
existence by those
and
can explain such a disof type 2X fibers are
in part
to type
of another type studies where
was performed
in association
2A fibers.
2 fiber popusuccinate deto myosin
ase. For instance, in the rat the distribution of metabolic was found heterogeneous with respect to 2A fiber type, using myosin ATPase after PFA-10.4 or with respect to 2B fiber type, incubation
(Nemeth
et al.,
1979).
pre-incubation identified Recently,
using
ATh
enzymes identified
(Pullen, 1977), pH 4.6 pre-
Matoba
and
Gollnick
(1984), modifying the ionic composition and the buffering agent of the acid pre-incubation medium as well as the time of the preincubation,
obtained
brillar which
actomyosin were called could
also
different
stud-
types
fiber
sensitivity of the copper (Gollnick
myofibnillar and Matoba,
with
patterns
the staining
degrees
of inhibition
ATPase activity in three Al, A2, and A3 (Matoba
species
musnumer-
Leung
when analyzed with different pre-incubation moderate to high succinate dehydrogenase
a major
muscle as well as in other hindlimb superficialis), and were especially
muscles
2 fibers in all the three species examined. Fibers expressing display a unique pattern ofstaining in histochemical my-
MHC
Previous
using the pH 4.6 pre-incubation. can be expressed also in fiber
MHC
in the
of rat skeletal
(Schiaffino
shows that the MHC isoform is expressed as the only MHC
lion
were
2X fibers
analyses
Bar and
osin ATPase reaction, protocols, and show
be distin-
identified
electnophoretic
study
2X
1. In
SC-71
could
and
composition
1989;
of type-specific cation of many
with
in Table
subpopulations
also. Type unreactive
of the
are illustrated
in MHC
al., 2X
pig
Type
shown)
predominantly composed of type 2 fibers has recently been demonstrated in this and in other laboratories using both immunochemi-
2A 2B 2X Mouse Type
(results not communication).
Discussion
PFA-pHlO.4
Rat Type
diaphragm personal
be distinguished
Downloaded from jhc.sagepub.com at KAI NAN UNIV on February 18, 2015
on the
of the myofi-
type 2 fiber populations, et al., 1985). These subbasis
of the
differential
myosin ATPase of each fiber type to 1984). Comparison of their results
described
in the present
work
reveals
that
-
*
c ,;,,
.
6
#{149}/‘
. .
-
,
,,.
!!d
;;‘
C
-
.
L__
-
.d
* C
a,
, ,; S . .
‘
..f
;
#{149}f
fl.,.
#{149}
\ ,
-,,
.
. .
.
Figure4. Ratsoleus. Serial cryostatsectionsstainedfor indirect immunoperoxidasewith anti-MHC MAb(a-f), myofibrillaractomyosin ATPase(g-l), and succinate dehydrogenase (j). (a) BA-D5, anti-type 1MHC; (b) SC-75, anti-type 2MHC; (C) SC-Ti, anti-type 2A MHC; (d) BF-F3, anti-type 2B MHC; (e) RT-D9, anti-type 2X and 2B MHC; (f) BF-35, anti-type 1, 2A, and 2B MHC; (g) myosin ATPase after pH 4.3 pre-incubation; (h) myosin ATPaSe after PFA-10.4 pre-incubation; (I) myosin ATPase after pH 4.6 pre-incubation; (j) succinate dehydrogenase activity. Type 2C fibers are identified as C. Closed circle indicates type 2A fibers containing 2X MHC. Asterisks indicate type 2C fibers containing 2X MHC. Bar = 54 sm.
263
Downloaded from jhc.sagepub.com at KAI NAN UNIV on February 18, 2015
GORZA
264
Table Fiber
Percentage
3. type
Rat (n
1
values
offiber Mouse
597)
=
types
(a
2.0%
in tibia/is Guinca
729)
=
anteriora (a
pig
0% 1.2%
2B
48.2%
42.6%
28.9%
2X
17.9%
51.3%
30.1%
29.3%
2C
2.0%
0%
0%
2A/2X’’
4.2%
2.3%
2.1%
2B12X’
5.8%
2.6%
7.5%
number
of fibers
counted
in 4 to 6 fields
of tibialis anterior muscle. weakly reactive in the rat with
portions
b Fibers
from
MAb SC-71
both
is low, in accordance
of type
the fiber
type
2B fibers
with
of other
previously
after
identified
both
pH
4.6
and
and
in a small number for 2X MHC, such of the degree less, no fiber
but unreactive
with BF-F3 ; in the mouse they were weakly reactive with SC-71 and unreactive with BF-F3; in the guinea pig they were weakly reactive with BF-32 and BF-35 and unreactive with BA-D5. C Fibers weakly reactive in the rat and the mouse with both BF-F3 and BF-35; in the guinea pig they were weakly reactive with BF-35 and unreactive with both BF-32 and BA-D5.
these
2B must
Finally,
be classified
MAb and the demonstratype 2X fibers appear dark
pre-incubations.
with type-specific are co-expressed
offibers. evidence
low oxidative species.
anti-myosin antibodwith 2A or 2B MHC
Because ofthe lack ofan MAb specific was obtained indirectly by comparison
of the stainings with the other antibodies. Neverthewas found to co-express 2A and 2B MHC. Adding
new data
antibodies
known
as type
PFA-l0.4
Immunohistochemistry ies showed that 2X MHC
the
mammalian
2X, as shown using anti-MHC of myosin ATPase activity. Mouse
tion
superficial
and RT-D9
fibers
as type
19.9%
a
545)
=
2.9%
2A
deep
of these potential
to previous
that
did
observations
not
2X
MHC
(Pierobon-Bormioli
et al.,
fibers
with
multiple
MHC
tween
type
2A
2B, as illustrated
and
obtained
discriminate 1981),
using
MHC
the
2X
immunoreactivity
2A and
fiber
can by the
polyclonal
from
type
2B
and
the
be inserted
following
be-
scheme:
i;
in the
rat muscle
the
the type 2A, 2X, lished observations). The
degree
tomyosin
and
A2,
and
oftype
on the basis
precise by the
(L. Gorza,
inactivation
varies
ofmyofibnillar
using after
pH
actomyosin
of each fiber demonstration
pear
unpubac-
among
differ-
ofsuccinate
nespecwere
ATPase;
how-
type could be obtained of distinct MHC iso-
the
the
combined
use
of enzyme
and
demonstration
4.6 pre-incubation,
dehydrogenase
and
activity,
to weakly
activity. Conversely, hydrogenase activity,
stained
type 2B at variance
ing widely
in size and
and
1984). exclusively
must MHC
type
With
2B fibers
detected
in rare type
isk),
never
but
in
fiber
2C
ofsoleus
fibers
2A MHC,
in pre-existing
a result
formed
might
directly
type
suggest
into 2A
represents
or whether
muscle
of tibialis
with high-frequency chronic that 2X MHC is co-expressed
Such
sequence
it might
by the intrinsic characteristics of the occasional expression of 2X MHC was
2C fibers
type
this plasticity
Experiments dc showed
correspond
low-
However, when this with the demonstra-
mouse and
to the
type
2A fibers
occasionally
show
identified ATPase
aphigh
fibers,
ATPase
activity
ase activity
oftype
2A fibers.
is more The
type
type
(Schiaffino
soleus
type
2X fibers,
2A/2X
4, aster-
(not
shown).
stimulation ofsoleus muswith 1 MHC, but not with
1 fibers
that
(see Figure anterior
1 fibers
et al.,
1988).
can
trans-
be
as illustrated:
2X
2X/2B
2B
which
arelightly
con-
stained
as type 2A because of their activity after 4.6 pne-incubation, they after
inhibited succinate
react PFA-l0.4
than
:;
same
study
85%
that
i
l/2X
demonstrated
2X MHC
is intermediate
and
that
showed
between
the
an intrinsic
the shortening
stimulated
solei
speed
of shortening
velocity
ofa
con-
predomi-
nantly type 2B MHC muscle and a type 1 MHC muscle (Schiaffino et aL, 1988). Therefore, it is possible that type 2X fibers influence muscle
physiology
of fiber fiber
type
like the other
percentage
represents
a major
Previous studies des in normal
fiber
in tibialis population
on fiber type and pathological
types.
Quantitative
anterior
showed
in the
three
composition conditions,
a single histochemical assay, must of the findings presented here.
species
ofvarious obtained
therefore
analysis that
type
2X
studied.
skeletal using
be reconsidered
musonly
in light
(Reichmann 2A fibers
after pH 4.6 pre-incubation and activity. In contrast, the barge-
as type 2B fibers: antibody BF-F3 and
the myosin
small
approach,
The tamed
of actomyosin
appearing
the present
to the small-sized
previously myosin
be considered monoclonal
and changed For example,
whether
in muscle
1/2A
fibers display high succinate dewith the properties oftype 2B fibers
for myofibrillar actomyosin ATPase display high succinate dehydrogenase sized fibers, termediate-low
be influenced muscle fiber.
immuno-
oberved in other mammalian species [see Figures ic and id in Reichmann and Pette (1984)]. In addition, mouse type 2A and 2B fibers are also unorthodox with respect to their size, type 2A fibers vary-
respond
pathway
of new classification criteria for type type 2A and 2B fibers are presently
histochemical
intermediately
Pette,
to be established
an obligatory
4t
and high-activity type 2 fibers, respectively. histochemical reaction is used in combination tion
It remains
to
of myofibrillar
subpopulations
histochemistry led to adoption 2 skeletal fibers. In this species, ATPase
correspond
and pH 4.6 pre-incubations, distinct type 2 fiber populations
identification immunological
expression. In the mouse,
identified
types
For instance, in guinea pig the type 2A and 2X fibers rat and mouse type 2A and 2X fibers in the degree
distinguished
form
A3 fiber respectively
alkali
2 fiber
of inhibition after PFA-l0.4 tively. In such a case, three ever, only
and
2B fibers,
of acid
ATPase
ent species. differ from
Al,
the myosin
The
author
criticism M.
wishes
to thank
during
Moretto
for
ProfS.
the preparation technical
Schiaffino ofthe
for
helpful
manuscript
and
discussion Mr A.
Buso
and and
assistance.
in-
with the anti-2B pre-incubation
dehydrogenase
Acknowledgments
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