THE ANATOMICAL RECORD 300:16361642 (2017)

Segmental Distribution of Myosin

Heavy Chain Isoforms Within Single
Muscle Fibers
MING ZHANG* AND MAREE GOULD
Anatomy Department, University of Otago, Dunedin, 9054 New Zealand

ABSTRACT
Despite many studies looking at the distribution of myosin heavy
chain (MHC) isoforms across a transverse section of muscle, knowledge of
MHC distribution along the longitudinal axis of a single skeletal muscle
fiber has been relatively overlooked. Immunocytochemistry was per-
formed on serial sections of rat extensor digitorum longus (EDL) muscle
to identify MHC types I, IIA, IIX, IIY, and IIB. Sixteen fascicles which
contained a total of 362 fibers were randomly and systematically sampled
from the three EDL muscles. All MHC type I and type II isoforms were
expressed. Segmental expression occurred within a very limited segment.
MHC isoform expression followed the accepted traditional order from
I () IIA () IIX () IIB, however, in some samples expression of an iso-
form was circumvented from IIB to I or from I to IIB directly. Segmental
distribution of MHC isoforms along a single muscle fiber may be because
of the myonuclear domain. Anat Rec, 300:16361642, 2017. V C 2017

Wiley Periodicals, Inc.

Key words: segmental expression; MHC; EDL; myonuclear

domain

proportions of type MHC I, IIA, and IIB fibers within an

INTRODUCTION
Skeletal muscle is not a homogeneous tissue, but
rather it is composed of a heterogeneous collection of
muscle fibers types, which result from differences in
the ultrastructure and in the biochemical and physio-
logical properties within a single muscle fiber or
myofiber.
Each myofiber contains several hundred nuclei, called
myonuclei within a continuous cytoplasm. The region of
cytoplasm controlled by each individual myonucleus is
termed the myonuclear domain. Each myonucleus regu-
lates the gene products within the myonuclear domain
(Cheek, 1985) as the messenger RNA produced by a
nucleus is confined to the area immediately surrounding
that nucleus (Ralston and Hall, 1992).
The myonuclear domain ratio may be related to myo-
sin heavy chain (MHC) isoform expression (Allen et al.,
1996). The MHC is a major structural protein of the
muscle fibers. Muscle fibers are classified into type I,
IIA, IIX, and IIB fibers, which contain MHC I, MHC
IIA, MHC IIX, and MHC IIB isoforms, respectively, and
have different structural and functional properties. The
individual subject vary between muscles, (Johnson et al.,
1973) within regions of a single muscle, (Elder et al.,
1982) and even within a single muscle fascicle (Sjostrom
et al., 1986).
Specific stimuli applied to the muscle provokes a tran-
sition always in a specific order from I to IIB
(I () IIA () IIX () IIB), thus changing their pheno-
type (Schiaffino et al., 1985). Hybrid fibers amidst tran-
sition that express two or more myosin types can also be
*Correspondence to: Ming Zhang, Anatomy Department, Uni-
versity of Otago, P.O. Box 913, Dunedin 9054, New Zealand.
Fax: 16434797254 E-mail: ming.zhang@otago.ac.nz
The authors state that no competing interests are declared.
This research received no specific grant from any funding
agency in the public, commercial or not-for-profit sectors
Received 11 September 2016; Accepted 6 October 2016.
DOI 10.1002/ar.23578
Published online 18 February 2017 in Wiley Online Library
(wileyonlinelibrary.com).

C 2017 WILEY PERIODICALS, INC.

V
 SEGMENTAL MHC EXPRESSION
found amongst these pure fibers (Tajsharghi et al.,
2004).
Single muscle fibers are classically identified based on
the molecular properties of their myosin heavy-chain as
these were shown to vary between fast glycolytic type 2
fibers and slow oxidative type 1 muscle fibers in mam-
mals (Luff and Atwood, 1972). Along the length of a
myofiber, one region may differ from another in their
kinetic properties. Fast and slow types of myosin have
been found to co-exist within individual human skeletal
muscle fibers (Gauthier and Lowey, 1979, Lutz et al.,
1979). The myosin isoenzyme pattern may also vary
from one region to another within the same fiber
(Edman et al., 1985) which perhaps is unsurprising
because a skeletal muscle fiber is in reality the fusion of
several myoblasts (Edman et al., 1988).
Single muscle proteomic studies have clearly estab-
lished that in individual fibers, various subsets of mito-
chondrial proteins show distinct fiber-type-specific
patterns of metabolic function (Murgia et al., 2015) and
the variations in tetanic force are determined by the var-
iations in myosin content (Buschman et al., 1997).
Hence, the phenomenon of heterogeneity of myosin
isoform expression dates back to 30 years ago, but there
are still many open questions. Although single muscle
fibers were isolated decades ago, only a few studies have
investigated the distribution of various MHC isoforms
within a single muscle fiber (Bortolotto et al., 2000). The
aim of this project, thus, was to use a novel approach to
investigate whether single muscle fibers segmentally
express different MHC isoforms.
MATERIALS AND METHODS
Animals
All experiments were approved by the University of
Otagos Committee on Ethics in the Care and Use of
Laboratory Animals (Animal ethic No 84/96). Three Wis-
tar rats were used; aged 3, 5, and 8 months old, respec-
tively. Animals were single housed in 12-hour light/12-hr
TABLE 1. Length of fascicles traced and muscles
No. fascicles Length of
Muscle traced Distance traceda muscle
#1 6 4.5 (7.512.0) mm 20.9 mm
#2 4 4.0 (15.519.5) mm 23.0 mm
#3 6 11.5 (15.527.0) mm 31.5 mm
a expressing each MHC isoform were counted. Proportions
Total distance traced (proximal starting point 2 distal fin-
ishing point).
1637
dark cycle with ad libitum access to food or water. After
cervical dislocation, the extensor digitorum longus (EDL)
muscles were dissected and frozen at their resting posi-
tions in melting isopentane and stored in a 2808C freez-
er until use.
Qualitative Analysis
Sections were cut with a cryostat (10 mm). Sets of four
adjacent sections were systematically selected from an
entire muscle. The distance between the sets of sections
was 500 mm.
Four adjacent sections at each level were labeled with
a mixture of anti-dystrophin antibody (DYS2; Novovas-
tra laboratories, New Castle, UK) to label the cell mem-
brane and one of four anti-MHC monoclonal antibodies
reactive with MHC I (NOQ, gift of R. Fitzsimons), MHC
IIA (SC71; gift of Dr. Schiaffino), MHC IIB (BFF3;
Regeneron Pharmaceuticals, Tarrytown, NY), and all
MHCs except MHC IIx (BF35; Dr. Schiaffino) (Zhang
et al., 1998). The fiber that is BF35 positive but NOQ,
BFF3, and SC71 negative was termed type IIY (Zhang
et al., 1998).
The procedure for immunohistochemistry was per-
formed as previously described (Zhang and McLennan,
1995). Briefly, the sections were fixed in 1% paraformal-
dehyde at 48C for 30 min, incubated overnight at 48C
with the above antibodies. The immunoreactivity was
then developed using biotinylated-anti-mouse IgG, strep-
tavidin biotinylated-horseradish peroxidase complex and
diaminobenzidine as the chromogen. Nonspecific immu-
nohistochemical staining was controlled for by replacing
the monoclonal antibody with nonimmunized mouse
IgG. The sections were examined using an Olympus
AX70 microscope.
Quantitative Analysis
In addition to those sections examined during qualita-
tive analysis, 16 fascicles were randomly selected from
three EDL muscles and longitudinally traced for a
length of 4.0, 4.5, or 11.5 mm, which were less than one-
third of the length of the muscles (Table 1). Images from
each level were assembled to form a montage of the
selected fascicles. The montage was used during exami-
nation of sections as a guide for the identification of dif-
ferent fiber types. A total of 362 fibers from 16 selected
fascicles were examined (Table 2). The numbers of fibers
of the fibers expressing one or more MHC isoforms were

TABLE 2. Percentage of fibers expressing MHC isoforms

Expression Fiber type Muscle 1 Muscle 2 Muscle 3 Subtotal (%)
Sole slow I 2 3 6 11 3.0
IIA 16 9 17 42 11.6
Sole fast IIX 23 9 36 68 18.8
IIB 86 16 51 153 42.3
I IIA 7 0 10 17 4.7
IIA X 14 2 2 18 5.0
Transition IIX A X 18 0 18 36 9.9
IIX Y 1 0 4 5 1.4
IIY B 0 1 11 12 3.3
Total 167 40 155 362 100
1638 ZHANG AND GOULD
determined by the number of the fibers divided by the
total number of the counted fibers.
RESULTS
Qualitative Analysis
In addition to a combination of type I 1 IIA, IIA 1 IIX,
IIX 1 IIY, or IIY 1 IIB, some single fibers switched their
MHC expression from IIB to I or from I to IIB. As shown
in Figure 1, a single fiber was traced at four levels with-
in a distance of 3 mm. At levels 1 and 4, it expressed
MHC IIB isoform alone. At level 2, the fiber co-
expressed strong MHC I and weak MHC IIB Immunos-
taining, and at Level 3 showed positive staining for only
MHC I. These results showed that in a single fiber MHC
expression transitioned through a series of MHC iso-
forms IIB to I 1 IIB to I and then to IIB alone.
Type I, IIA, IIX, IIY, and IIB fibers were observed in
the muscles examined in this study. Single muscle fibers
simultaneously and segmentally expressed different
MHC isoforms. The combination pattern of MHC co-
expression varied. As shown in Figure 2, two single mus-
cle fibers were traced and examined at two levels within
a distance of 1.5 mm and showed that one fiber transi-
tioned from MHC type IIY to type IIB, whereas another
fiber transitioned from MHC type IIX to type IIB.
Quantitative Analysis
To quantify the proportions of various combinations of
MHC co-expression, 16 fascicles which contained a total
of 362 fibers were randomly and systematically sampled
from the three EDL muscles. All the fibers within each
fascicle were traced for a distance of 4, 4.5, or 11.5 mm,
respectively. The results show that 76% of the fibers of
the adult rat EDL muscle expressed only a single MHC
isoform, the majority of which were type II fibers (73%)
but none of them were type IIY fibers (Table 2). About
24% of the fibers segmentally expressed two or more
MHC isoforms. Combinations of type I 1 IIA (5%),
IIA 1 IIX (15%), IIX 1 IIY (1%), and IIY 1 IIB (3%) were
seen. No other combinations were observed in the fas-
cicles examined for quantitative analysis (Table 2).
DISCUSSION
The results of this study provide evidence of the seg-
mental expression of different MHC isoforms along a
single fiber in the EDL muscle in the adult rat. As far
as the authors could ascertain, this is the first study on
the regional variability of the distribution of MHC iso-
forms along the longitudinal axis of a single skeletal
muscle fiber. Although the MHC transition of hybrid
segments of single fibers is commonly in an order
I () IIA () IIX () IIB, segmental expression of MHC
isoforms in some hybrid fibers does not follow the tradi-
tional order. The mechanism behind this phenomenon is
not clear.
Single Muscle Fibers Simultaneously and
Segmentally Express Different MHC Isoforms
Within a Very Limited Sequence
The finding that single muscle fibers can simulta-
neously and segmentally express different MHC isoforms
indicates the diversity of muscle fiber properties that
exist within a single fiber. It also provides an opportuni-
ty to understand the mechanisms of the heterogeneity
and plasticity of skeletal muscle fibers.
Muscle fiber types are determined by both intrinsic
factors, e.g. myotubal and myonuclear origins (Zhang
and McLennan, 1999) and extrinsic factors, which
include changes in neuromuscular activity, mechanical
loading, altered hormone levels, and aging. Neuromus-
cular activity establishes and maintains the specific
skeletal muscle isoforms, and the number of nerve ter-
minal and endplate branches increase as the fiber type
progresses from type I, IIa, IIx to IIb (Prakash et al.,
1996).
Of the fibers examined, 3% were MHC I (slow twitch
fibers) whereas 72% expressed isoforms IIA (11.6%), IIX
(18.8%), and IIB (42.3%). The fibers within the TA mus-
cle are predominantly type II. Moreover, in addition to
the pure fibers containing one MHC isoform, a number
of hybrid fibers co-expressing various amounts of two
MHC isoforms were seen. The switching of MHC expres-
sion from one isoform to another occurred within a short
segment and in a large number of fibers 24% of the
fibers examined co-expressed two MHC isoforms. Per-
haps a mechanism that would explain the change in the
fibers described in this study may be in a state of transi-
tion between two isoforms. Hybrid fibers could reflect a
state of transition from one isoform to another (William-
son et al., 2000).
After periods of changed physical activity or in old
age, the prevalence of hybrid muscle fibers containing
more than one isoform tends to increase (Williamson
et al., 2000). Hybrid muscles are also found in young
people (Klitgaard et al., 1990) indicating that changes in
workload may be sufficient to induce fiber-type changes
that lead to the co-existence of different MHC isoforms
in a single fiber.
A regional variability of the distribution of MHC iso-
forms has been seen along the length of the global and
orbital layers of human extraocular muscles, where
MHC IIA and MHC I were most abundant on the proxi-
mal and distal ends (Park et al., 2012). This was also
seen in the jaw and leg muscle from rabbits, where dif-
ferent parts of the fiber expressed different fiber type
compositions and thus, different contractile properties
(Korfage et al., 2016). Under certain circumstances (such
as after spaceflight), single muscle fibers can simulta-
neously express as many as five MHC isoforms (Allen
et al., 1996). However, we did not find any single muscle
fibers that segmentally expressed more than two MHC
isoforms.
SDS-PAGE gels combined with scanning densitome-
try can separate and quantify the relative proportion
of MHC isoforms co-expressed in a single muscle EDL
fiber (Bortolotto et al., 2000). However, one limitation
is that these techniques do not provide information on
the intracellular distribution of isoforms and relation-
ship with surrounding fibers and that overall there is a
high degree of variability with respect to the effective-
ness of separation of MHC isoform bands. Further-
more, the method described here provides a superior
overview of the location of the individual fibers within
the muscle providing information on the local
microenvironment.
 SEGMENTAL MHC EXPRESSION 1639

Fig. 1. Segmental transition of MHC I and MHC IIB across a single
muscle fiber. A single muscle fiber (asterisk) was longitudinally traced
at four levels (a/e; b/f; c/g; and d/h) and two adjacent sections at
each level were labeled for MHC I (a-d) (NOQ-positive) and MHC IIB
(eh) (BFF-positive) isoforms, respectively. The interval between the
levels was 500 mm except that the interval between c/g and d/h was
1,000 mm. The traced single fiber expressed sole MHC IIB isoform at
level a/e, co-expressed strong MHC I and weak MHC IIB isoforms at
level b/f, and expressed sole MHC I isoform at level c/g and sole
MHC IIB isoform at level d/h. All sections were counterstained anti-
dystrophin antibody for cell membrane staining. Bars 5 40 lm.
1640 ZHANG AND GOULD

Fig. 2. A single muscle fiber expresses more than one MHC isoform.
Two single muscle fibers (1 and 2) were traced and examined at two
levels (ad and eh) with an interval of 1,500 mm. Four adjacent sec-
tions at each level were labeled for MHC I (a and e) (NOQ-positive),
MHC IIA (b and f) (SC71-positive), MHC IIX (c and g) (BF35-negative),
and MHC IIB (d and h) (BFF3-positive) isoforms, respectively. Fiber 1
did not express MHC I, IIA, IIX, and IIB isoforms at levels ad but
expressed sole MHC IIB isoform at levels eh, indicating a state of
transition from type IIY to type IIB. Fiber 2 expressed sole MHC IIX
isoform at levels ad and MHC IIB at levels eh, indicating a state of
transition from type IIX to IIB. Bars 5 40 lm.
 SEGMENTAL MHC EXPRESSION
Expression of MHC Isoforms Does Not
Necessarily Follow the Traditional Activation
Order
During transition from slow to fast phenotype, MHC
genes in adult rat skeletal muscle activate in a graded
and orderly sequential manner from type I () IIA
() IIX () IIB (Danieli Betto et al., 1986). In this
study, however, we observed that the fibers switched
their MHC expression from IIB to I or from I to IIB
directly (Fig. 2). This indicated that the fibers either cir-
cumvented the expression of distinct sequential MHC
isoforms altogether, or the expression of the missing iso-
forms was transient and not seen. Other researchers
have seen this after a spinal cord transection when a
small number of fibers in the rat soleus muscle bypassed
MHC IIA expression and converted directly from MHC I
expression to expressing MHC IIX (Talmadge et al.,
1995).
Myonuclei of transforming fibers may not work syn-
chronously, (Staron et al., 1987) reflecting the much lon-
ger turnover of the MHC proteins compared with that of
the corresponding mRNA transcripts indicating that
these fibers may be in a transitional state (Andersen
and Schiaffino, 1997). Fibers post bed-rest contained
only MHC I at the protein level, but expressed only
mRNA for MHC IIX indicating a conversion from type I
to type IIX fibers with only a transient or without a type
IIA phase (Talmadge et al., 1995). Overall, there was an
increase in fibers in a transitional state from phenotypic
type I () IIA and IIA () IIX in subjects on bed-
rest. These findings add a new dimension to the concept
of skeletal muscle cell heterogeneity and further sup-
ports concept of the myonuclear domain.
Each muscle cell has many myonuclei (Zhang and
McLennan, 1998) and each nucleus has jurisdiction over
a certain volume of cytoplasm. In order that muscle
fibers can coordinate their behavior, for example, in
response to neuro-input from the neuromuscular junc-
tion or loading at ends of the fiber, they must be able to
communicate with each other. Thus, the presence of
interaction between neighboring nuclei is of crucial
importance. Heterotypic myotubes formed from embry-
onic and fetal rat myoblasts in vitro had a regionalized
expression of MHC isoforms around individual myonu-
clei (Zhang et al., 1998). This suggests that myonuclei
with different embryonic origins may make the contribu-
tion towards segmental expression of different MHC iso-
forms within single muscle fibers.
CONCLUDING REMARKS
In this study, we have demonstrated at the protein
level the distributions of different MHC isoforms along a
short segment within a single fiber of the rat EDL mus-
cle. Expression was seen to transition from MHC IIB to
I or from I to IIB directly. The role of the myonuclear
domain and its influence on the segmental distribution
of the MHC isoforms remains unknown.
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