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

Cited

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the nature

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D (1989): Myosin types of rat muscle.

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isoforms in in press

Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies.

A novel type of myosin heavy chain (MHC), called 2X, has been recently identified in type 2 fibers of rat skeletal muscles using an immunochemical app...
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