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Expression of a fast myosin heavy chain mRNA in individual rabbit skeletal muscle fibers with intermediate oxidative capacity.

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THE ANATOMICAL RECORD 23052-56 (1991)
Expression of a Fast Myosin Heavy Chain mRNA
in Individual Rabbit Skeletal Muscle Fibers With
Intermediate Oxidative Capacity
DAVID J. DIX AND BRENDA RUSSELL EISENBERG
Department of Biochemistry, North Carolina State University, Box 7622, Raleigh, North
Carolina 27695-7622 (D.J.D.); Department of Physiology and Biophysics, University of
Illinois, Mail Code 901, Box 6998, Chicago, Illinois 60680 (B.R.E.)
ABSTRACT
In situ hybridization (ISH) of myosin heavy chain (MHC) mRNA,
immunofluorescent detection of MHC protein, and oxidative enzyme histochemistry were performed on the same fibers in serially sectioned rabbit skeletal muscle.
By combining these three techniques quantitatively, on a fiber-by-fiber basis, fibers that expressed mRNA complementary to a fast MHC cDNA pMHC24-79 of
unknown subtype (Maeda et al., 1987) were classified into fiber types with respect
to slow myosin expression and oxidative capacity. As expected, slow fibers had low
hybridization to pMHC24-79. Fast fibers were divided into three subtypes. mRNA
from the low oxidative fibers (fast-glycolytic, IIB) did not hybridize with pMHC2479. Fast fibers whose mRNA hybridized best to pMHC24-79 were mainly in the
intermediate range of oxidative capacity (probably IIX). The fast fibers with the
highest oxidative capacity had low hybridization to this MHC mRNA (probably
IIA). Thus, pMHC24-79 was identified as a clone of a fast isomyosin, tentatively
designated as the fast IIX with intermediate oxidative capacity. The expression of
more than a single species of fast and slow isomyosin mRNAs in classically defined
fiber type was considered in interpreting these results.
Seven distinct myosin heavy chain (MHC) genes
have been cloned and classified in rat (Mahdavi et al.,
1986).These include three adult skeletal isoforms: fastglycolytic (IIB); fast-oxidative (IIA); and slow-oxidative
(I), the same gene as p-cardiac). A fourth adult skeletal
muscle isoform has recently been reported, labelled IIX
by one group (Schiaffino et al., 1986, 1988, 1989) and
IId by another (Termin et al., 1989). This new discovery
necessitates the re-evaluation of all the classical subdivisions of fast fiber-types. In rat fibers expressing IIX
isomyosin the oxidative enzyme content is intermediate compared to fast-glycolytic (IIB) and fast-oxidative
(IIA) fibers. However, in mouse the IIX fibers have a
higher oxidative content than IIA fibers and IIA fibers
are the rarest of the three fast isoforms (Schiaffino et
al., 1991). Rabbit fibers are more similar to rat than
mouse in their isomyosin expression and the relative
oxidative enzyme content amongst these fibers.
Previously we have used a n a-MHC cDNA as a hybridization probe for slow MHC mRNA in skeletal
muscle fibers (Dix and Eisenberg, 1988). This was possible because of the high degree of similarity between
the a-cardiac and p-cardiac isomyosins. p-cardiac
MHC is identical to slow MHC in skeletal muscle in all
species studied to date. In the present work a subclone
of the rabbit fast skeletal MHC cDNA pMHC24-79
(Maeda et al., 1987) provides RNA probes for in situ
hybridization (ISH). By matching ISH results to MHC
immunofluorescence and oxidative enzyme histochemistry within the same fibers in serial sections, we were
able to determine which skeletal muscle fiber type ex0 1991 WILEY-LISS. INC.
presses this fast MHC mRNA. Thus, identifying which
fast isomyosin gene is represented by the pMHC24-79
cDNA.
METHODS
Animals and Tissue
Governmental and institutional guidelines for animal care and use were followed a t all times. Two female New Zealand (NZ) white rabbits, approximately
2.5 kg body weight, provided medial gastrocnemius
muscles. Rabbits were anesthetized with a n intramuscular injection of Acepromazine maleate and ketamine
HC1 (0.8 mg and 35 mg, respectively, per kg body
weight) and muscles removed. Rabbits were euthanized by lethal injection. Blocks of tissue for ISH, immunofluorescence, and histochemistry were flashfrozen in isopentane cooled with liquid nitrogen and
then stored at -80°C in isopentane until use.
In Situ Hybridization
Hybridizations were performed as previously described (Dix and Eisenberg, 1988). RNA probes were
transcribed from a subcloned 377-bp Sau 3A fragment
of rabbit fast skeletal MHC cDNA pMHC24-79 (Maeda
et al., 1987). This fragment from the 3'-end of the cod-
Received July 10, 1990; accepted October 15, 1990.
Address reprint requests to Dr. Brenda Eisenberg, Department of
Physiology and Biophysics, University of Illinois, Mail Code 901, Box
6998, Chicago, IL 60680.
FAST MYOSIN mRNA AND OXIDATIVE CAPACITY
53
ing region was ligated into transcription vector Bluescribe M13 + (Stratagene, San Diego, CA) such th a t T3
RNA polymerase transcribes complementary antisense
RNA and T7 RNA polymerase transcribes sense RNA
(the kind gift of Dr. K. Maeda, Max Planck Institut,
Heidelberg, FRG). Biotin-1 1-UTP (Bethesda Research
Laboratories, Gaithersburg, MD) substituted for UTP
in transcription reactions. Hybridization with the fast
MHC probe was 52°C for 3 h. After hybridization, sections were treated with RNase A, washed, and then the
biotin label was detected with streptavidin-alkaline
phosphatase (SAP, Clontech, Palo Alto, CA). Sections
were viewed and photographed with a Nikon Microphot-FXA.
Histochemistry
NADH tetrazolium-reductase histochemistry to assess oxidative enzyme content was done as published
(Dubowitz and Brooke, 1973).
lmmunofluorescence
Immunofluorescent detection of slow MHC protein
was accomplished with the monoclonal antibody HPM7 at 1:2,000 dilution (Kennedy et al., 1986, a kind gift
of Dr. R. Zak of the University of Chicago). Slow specificity of HPM-7 in rabbit was confirmed by comparison
with ATPase-stained serial sections. Sections were
viewed and photographed with a Nikon MicrophotFXA with indirect immunofluorescence. The internal
microphotometer was used to record fluorescent intensities.
Video Image Analysis
The optical density (OD) of stain in individual fibers
following ISH and histochemistry were measured by
a n IBM-AT based video image analysis system (TM540 CCD camera, Pulnix America, Sunnyvale, CA;
PCVision plus frame-grabber, Imaging Technology,
Woburn, MA; ImageMeasure software, Microscience,
Federal Way, WA) added on to a Nikon Microphot-FXA
microscope. This sytem’s response was calibrated with
a Kodak photographic step tablet number 3 (OD range
0.05-3.05). Mean OD measurements were converted to
a linear relative scale (0-1.0) for ISH and histochemistry values.
All OD measurements were of individual fibers. This
allowed subtraction of background from appropriate serial sections on a fiber-by-fiber basis to provide the OD
of the signal due to the detection of the endogenous
mRNA. Mean OD values were total OD signal divided
by fiber cross-sectional area.
RESULTS
ISH with the pMHC24-79 RNA probe and subsequent histochemical detection resulted in a mosaic
staining pattern of muscle fibers in medial gastrocnemius (Fig. 1A). Control sections hybridized with the
sense RNA probe stained a t very low levels (not shown)
in agreement with our previous results (Dix and Eisenberg, 1988, 1990, 1991). Immunofluorescent detection
of slow MHC with the HPM-7 antibody identified slowoxidative fibers (Fig. 1B); this identification was confirmed by the ATPase reaction in serial sections.
NADH-tetrazolium reductase histochemistry assayed
oxidative enzyme content and helped differentiate the
Fig. 1. Serially sectioned medial gastrocnemius. A. ISH of fast skeletal MHC mRNA with biotinylated pMHC24-79 RNA probe. B. Immunofluorescence with antislow MHC HPM-7 identifies the slow-oxidative (SO) fibers. C. NADH-tetrazolium reductase histochemistry
differentiates fast-glycolytic (FG) and fast-oxidative (FO) fibers. Note
that fast-oxidative fibers vary in the ISH staining. Those fast-oxidative fibers that stain darkest following hybridization with pMHC2479 probe are mainly of intermediate oxidative capacity. Bar = 50 pm.
three fast fiber types. Fast-glycolytic fibers stain
lightly because of their low mitochondria1 content. The
moderate and darker stained fast fibers with greater
oxidative enzyme content in Figure 1C were the two
fast-oxidative fiber types (IIA and 11x1. After classifying individual fibers via the‘ results in Figs. 1B,C, it
was possible to go back to Figure 1A and note that
fibers staining the darkest with the pMHC24-79 probe
were mainly in the intermediate range of oxidative capacity.
The micrographs in Figure 1 are representative of
hundreds of fibers observed. Video image analysis of
100 fibers provided relative OD of stain within individ-
54
D.J. DIX AND B.R. EISENBERG
A
A
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0.2
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0.4
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NADH TETRAZOLIUM REDUCTASE
(RELATIVE OD)
Fig. 2. Scattergram of relative ODs from ISH with the fast MHC
mRNA probe vs. NADH-tetrazolium reductase histochemistry within
single fibers. Triangles denote fibers which reacted with the HPM-7
antibody (slow-oxidative fibers). Open circles are fast-glycolytic fibers. Filled circles are intermediate and high oxidative fast fibers.
The fibers hybridized with the greatest amounts of pMHC24-79 probe
are fast fibers with intermediate oxidative capacity.
ual fibers. A scattergraph of relative OD for ISH versus
NADH-histochemistry from these 100 fibers illustrates
the clustering of data points into three major groups
(Fig. 2). Fast-glycolytic fibers and slow-oxidative fibers
which stained lightly following ISH with pMHC24-79
probe, and fast-oxidative fibers, some of which stained
darker following ISH. Mean values of ISH and histochemistry relative OD for each of these fiber-types
are presented in Table 1.
Closer examination of the 48 fast-oxidative fibers revealed two subgroups within this classification (Table
1). The mean relative OD of slow-oxidative fibers following ISH with the fast MHC mRNA probe was considered as nonspecific staining of muscle fibers by the
probe. Thus 0.29 plus 0.40 (2 SD) or 0.69 provided a
reasonable lower confidence limit for legitimate mRNA
hybridization. There were 27 fast-oxidative fibers with
a n ISH relative OD over 0.69. The mean relative OD
for NADH-histochemistry of these fibers was 0.26 (0.09
SD). Twenty-one fast-oxidative fibers had a n ISH OD of
<0.69. The mean NADH OD of these fibers was 0.34
(0.22 SD). The difference in oxidative enzyme activity
of these two subgroups is probably significant (P<0.05
by small-sample t-test).
DISCUSSION
Hybridization with a subclone of pMHC24-79 on sectioned medial gastrocnemius indicated that this rabbit
fast skeletal MHC DNA is one of the fast-oxidative
isoforms. The ISH stain density for fast-oxidative fibers
was roughly twofold that of the other fiber types. Sequence divergence of the pMHC24-79 probe from the
mRNAs of slow skeletal (Sinha et al., 1984) and the
other fast isomyosins (Maeda et al., 1987) would account for this probe specificity under stringent hybridization conditions. Limited sequence comparisons to
MHC cDNAs from rat support the conclusion that
pMHC24-79 represents a fast-oxidative MHC, not the
fast-glycolytic (IIB) MHC (K. Maeda, Max-Planck-Institut, Heidelberg, FRG, personal communication).
Fiber-type classification was based on the following
rationale. Slow-oxidative fibers were identified by im-
munof luorescent detection of slow MHC with the
HPM-7 monoclonal. Fast-type fibers were defined as
those which did not react with HPM-7. Fast-glycolytic
fibers were classified as fast fibers with a NADH relative OD <0.10. This determination was partially based
on mitochondria1 fraction volumes published for the
different fiber-types (Eisenberg, 1983). This classified
only 16% of the fibers as fast-glycolytics, much fewer
than the 40% fast-glycolytic reported earlier for rabbit
medial gastrocnemius (Dix and Eisenberg, 1988). This
conservative estimate of fast-glycolytic fibers precludes
the mistake of incorrectly designating fast-oxidative
fibers a s fast-glycolytics.
The 48 fast-oxidative fibers analyzed in this study
fell into two categories (Table 1). Twenty-seven fibers
had ISH relative OD significantly above background
(i.e., mean OD for slow-oxidative fibers plus 2 SD) and
21 fibers did not. The 27 fibers that hybridized the
pMHC24-79 probe had a significantly lower mean
NADH OD than the 21 fibers that did not hybridize.
This indicates that fast-oxidative fibers with a moderate oxidative enzyme content expressed the pMHC2479 mRNA. Thus, since rabbit muscle is like rat in
isomyosin expression, it is possible that these 27 fibers
were expressing the IIX MHC (Schiaffino et al., 1991).
The 21 fast-oxidative fibers which did not express
pMHC24-79 mRNA had the highest average oxidative
enzyme content in the fast fiber category and are therefore most likely IIA fibers.
The conclusion that pMHC24-79 represents a fastoxidative clone is brought into question by the interpretation of blot filter hybridizations of RNA extracts
from whole muscle (Brownson et al., 1988).This group
used clone pMHC13, from the same gene and group of
cDNAs as pMHC24-79 (Maeda et al., 1987). Probes
from pMHC13 seemed to have greater affinity for RNA
from tibialis anterior than masseter, soleus or extensor
digitorum longus. The conclusion drawn from these hybridizations was that pMHC13 represented the fastglycolytic MHC. These conclusions do not agree with
our results and warrent reinterpretation in light of two
complicating factors; the lack of 100% homogeneity of
MHC expression pattern in any anatomical muscle and
the presence of the IIX MHC isomyosin.
Tibialis anterior has equivalent fractions of fast-glycolytic and fast-oxidative fibers (Leberer and Pette,
19841, thus one would expect either isoform-specific
probe to hybridize RNA extracts from this muscle. The
uncertainty of IIA and IIX expression only adds to the
confusion. Extensor digitorum longus expresses a
greater proportion of fast-glycolytic MHC than tibilais
anterior (Leberer and Pette, 1984), indicating that the
pMHC13 did not have a greater affinity for IIB mRNA.
Masseter is known to express a unique superfast MHC
whose mRNA would not be expected to hybridize with
pMHC13 (Hoh and Hughes, 1988). The combination of
these facts makes it impossible to interpret hybridization results judiciously from whole muscle RNA extracts.
The distribution of MHC isoforms within classically
defined fiber types in rabbit muscle are not in perfect
agreement. Fibers often coexpress more than one isoform in numerous combinations and ratios (Betto et al.,
1986; Staron and Pette, 1987a,b; Termin et al., 1989).
Further examination of the results in Table I shows
55
FAST MYOSIN mRNA AND OXIDATIVE CAPACITY
TABLE 1. In situ hybridization of a fast isomyosin mRNA in
medial gastrocnemius
Fiber
type1
Slow-oxidative
I
In situ hybridization
Relative OD2
0.29 2 0.03
NADH histochemistry
Relative OD2
N
0.69 t 0.03
36
Fast-oxidative
IIA
IIX
0.68 2 0.03
<0.6g3
>0.6g3
0.30 t 0.02
0.34 0.22
0.26 2 0.09
*
48
21
27
Fast-glycolytic
IIB
0.38 t 0.04
0.05 t 0.01
16
'Fiber type determined as slow or fast with McAb HPM-7, NADH of 50.10 were designated fast-glycolytic fibers.
'Relative optical density (OD, darkest stain = 1.0) given as mean values rSE for N fibers from two
rabbits. Student's t-test indicates P<0.05 for all possible comparisons of fiber types to one another.
30.69 is 2 SD above the stain OD of slow fibers and values above this are assumed to be specific
hybridization of the fast MHC mRNA.
that the ISH staining density is significantly higher in
fast-glycolytic fibers than it is in slow fibers. This supports the conclusion that pMHC24-79 is a clone of IIX
MHC cDNA because individual fibers do coexpress IIB
and IIX MHC more often than they coexpress slow and
IIX in rabbit muscles (Schiaffino et al., 1991).
An alternative explanation for fast-glycolytic fibers
staining darker than slow-oxidatives is greater crosshybridization between the fast MHC probe and fast
MHC species of mRNA (as compared with cross-hybridization with slow MHC mRNA). The nucleotide sequence of the pMHC24-79 probe is 88% similar to the
published rabbit fast MHC sequence, while its similarity to slow MHC is 73% (Maeda et al., 1987; K. Maeda,
Max-Planck-Institut, Heidelberg, FRG, personal communication). These sequence dissimilarities cause differences in the thermal stability of hybrids formed by
RNA probe and mRNAs, which could result in varying
degrees of cross-hybridization. We consider it more
likely that there is greater expression of the pMHC2479 MHC mRNA in fast-glycolytic fibers than there is in
slow oxidative fibers.
Earlier results with a slow MHC mRNA probe in
medial gastrocnemius can also be interpreted in terms
of isomyosin coexpression within individual fibers (Dix
and Eisenberg, 1988). In this previous study none of
the fast-glycolytic fibers were darkly stained, 26% of
the fast-oxidative fibers were darkly stained and 89%
of the slow fibers were darkly stained following ISH. It
is possible that the fast-oxidative fibers which stained
darkly coexpressed slow MHC. Slow MHC protein may
have been overlooked in these intermediate fibers because of its low expression levels. Intermediate type
fibers that express both fast-oxidative and slow MHC
have been reported in normal rabbit muscles (Staron
and Pette, 1987a,b; Termin et al., 1989).
It was reassuring to note that staining density from
ISH with enzymatically detected biotinylated probes
does not always coincide with the oxidative enzyme
content of the fibers. Hybridization with this fast MHC
probe did not result in intense staining of the slowoxidative fibers. Nonspecific staining as a result of the
biotin-streptavidin alkaline phosphatase protocol has
been a n ongoing concern (Brooke and Engel, 1966;
Bussolati and Gugliotta, 1983), but these results and
other controls overcome these concerns.
CONCLUSIONS
On the basis of ISH results, pMHC24-79 is concluded
to be clone of one of the fast-oxidative isomyosins. I t is
possible that pMHC24-79 represents the recently described IIX MHC isoform, rather than IIA. Individual
skeletal muscle fibers appear to coexpress more than
one isoform of MHC mRNA, as has been reported for
the MHC proteins.
ACKNOWLEDGMENTS
We thank Dr. K. Maeda for providing the pMHC2479 subclone and for participating in early experiments
in our laboratory during the Summer of 1988. Dr. S.
Schiaffino was helpful in discussion and in providing
manuscripts still in press. This work was supported by
the Muscular Dystrophy Association and NIH HL
40880. DJD was supported in part by National Research Service Award 5 T32 HL 07320.
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