Immunohistochemical fiber typing of harbor seal skeletal muscle
1 Texas A&M University, Galveston, TX 77551, USA
2 Department of Kinesiology, Texas A&M University, College Station, TX
77840, USA
* Author for correspondence (e-mail: rrw6205{at}yahoo.com)
Accepted 6 August 2003
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Summary |
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Key words: harbor seal, Phoca vitulina, diving, skeletal muscle, muscle fiber, pinniped, fiber type, immunohistochemistry
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Introduction |
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There have been a few attempts to quantify fiber type composition in marine
mammal skeletal muscles. Histochemical ATPase staining of the swimming muscles
of seals has shown an average numerical composition of approximately 46% type
I (slow twitch, oxidative fibers), 46% type IIb (fast twitch, glycolytic
fibers), and the balance type IIa (fast twitch, oxidative fibers)
(Hochachka and Foreman, 1993;
Reed et al., 1994
). These
results appear to conflict with the suggestion that skeletal muscles of seals
are adapted for aerobic metabolism, since type IIb fibers characteristically
do not possess high concentrations of mitochondria or myoglobin. Although the
two studies measured the oxidative capacity of the fibers by staining for NADH
diaphorase and succinate dehydrogenase (SDH) activity, neither reported the
results. Fiber typing of biopsies taken from the locomotory (epaxial) muscles
of one Pacific white-sided dolphin Lagenorhynchus obliquidens and the
hypaxial and epaxial muscles of one live and one dead bottlenose dolphin
Tursiops truncatus showed approximately 50% fast twitch, glycolytic
fibers and 50% slow twitch, oxidative fibers
(Ponganis and Pierce, 1978
;
Bello et al., 1983
;
Goforth, 1983
). Of these, only
Goforth (1983
) performed SDH
staining and verified that fast twitch, oxidative-glycolytic fibers were rare
or absent due to the lack of staining overlap between SDH activity and fast
twitch fibers. Recently, Kanatous et al.
(2002
) performed metachromatic
histochemical staining of Weddell seal skeletal muscles and verified the
results of the stain with immunohistochemical (IHC) fiber typing. They found
that the epaxial muscles were composed of approximately 67% type I fibers, 33%
type IIa fibers and no type IIb fibers
(Kanatous et al., 2002
).
However, only 1-2 muscle samples from five animals were analyzed using
immunohistochemistry, and it is possible that the biopsy samples (ca. 0.5 g
each) were not representative of the entire musculature. Kanatous et al.
(2002
) provides the only
evidence to date to suggest a lack of type IIb fibers in a pinniped.
The purpose of this study was to collect multiple samples from the primary
(epaxial muscles) and secondary (M. pectoralis) swimming musculature of the
harbor seal Phoca vitulina and apply IHC techniques to determine the
fiber types present, quantify fiber type populations, and determine the
distribution of fiber types within the muscle. Based on previous studies of
enzyme activities, fiber typing and mitochondrial volume density, we
hypothesized that there would be a higher proportion of type I and type IIa
fibers than type IIb fibers. We also hypothesized that the fiber type
distribution within the muscles would be heterogeneous (i.e. we expected to
see fast twitch fibers located superficially and slow twitch fibers located
deeper in the muscle). Our results showed that all fibers in both of the
muscles sampled were either type I or type IIa, which supports the fiber
typing results of Kanatous et al.
(2002). The pectoralis muscle
possessed significantly more type IIa fibers than the epaxial muscles. In
addition, fiber type distribution within the locomotory muscle did not show
pronounced spatial heterogeneity.
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Materials and methods |
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Immunohistochemical analysis of muscle fiber types
Serial cross sections (7 µm thick) of frozen epaxial and pectoralis
muscle samples were cut on a cryostat microtome and mounted on glass slides.
Sections of muscle were fixed with cold AFA (50 ml of 37% zinc formalin + 370
ml 95% ethanol + 25 ml glacial acetic acid) for 5 min and then hydrated for 10
min in PBS prior to blocking. PowerBlock (InnoGenex, San Ramon, CA, USA) was
added to the sections and incubated for 5 min at room temperature. Following
removal of excess blocker, primary antibodies to the myosin heavy chains, type
I (BA-D5), type IIa (SC-71) and type IIb (BF-F3) were added to the appropriate
sections, and the slides were incubated at 4°C overnight in a humid
chamber. Following incubation, slides underwent two 10 min washes in PBS with
gentle rotation. After washing, a biotinylated goat anti-mouse Ig secondary
antibody was added to the sections for 20 min at room temperature. After
washing the slides as described above, streptavidin-alkaline phosphatase
conjugate was added, and the sections were incubated for 20 min at room
temperature. The conjugate was removed by washing (as in prior steps), and a
solution of naphthol phosphate buffer and Fast Red dye was added. The sections
were then incubated until adequate color development was observed,
counterstained with Mayer's Hematoxylin and mounted with Glycergel
(DakoCytomation California Inc., Carpinteria, CA, USA). Fibers containing the
myosin heavy chains expressed a red color following exposure to the
immunohistochemical staining procedure. Samples were analyzed using a BIOQUANT
image analysis system (R&M Biometrics, Inc., Nashville, TN, USA). This
system consists of an Olympus BX-60 microscope (Olympus America Inc.,
Melville, NY, USA) with an attached Optronics (Goleta, CA, USA) DEI 470 camera
interfaced with a personal computer. All artifact-free fibers were counted at
a total magnification of 100x for each serial section (between 300-1500
fibers per section) and characterized as type I, type IIa, type IIb or
`unstained', as described by Schiaffino et al.
(1989). Cells that showed
inconsistent, light staining due to non-specific binding of the antibody (e.g.
Fig. 5B,C) were considered
`unstained'. The relative abundance of fiber types for each section was
determined and is presented as a percentage of the total number of fibers
counted. Serial muscle sections were also examined for IIx fibers (i.e. fibers
that expressed no staining following exposure to any of the heavy chain
antibodies).
|
Verification of antibody reactivity
We used a combination of mouse anti-rat primary antibody and goat
anti-mouse secondary antibody to differentiate between three myosin heavy
chain isoforms. Western blot analysis (data not shown) and SDS-PAGE
(Fig. 3) verified that the
fiber types of seals matched the electrophoretic properties of rat fiber
types. We have also verified antibody reactivity in serial sections of Weddell
seal skeletal muscle subjected to both IHC fiber typing and a traditional
histochemical staining procedure. Fiber type populations were similar for both
methodologies (Kanatous et al.,
2002).
|
Data analysis
We analyzed fiber type distribution along the length of the epaxial muscles
by comparing average fiber type percentages among the CR, MID and CA sections.
To analyze fiber type distribution with respect to proximity to the vertebrae,
we divided the seven samples of the CR, MID and CA sections into one of two
categories, `proximal' or `distal'. We then applied a 3-factor analysis of
variance (ANOVA) using Minitab statistical software. By using a 3-factor
ANOVA, we were able to analyze simultaneously the distribution of fibers in
both the lateral and longitudinal planes of the epaxial muscles. The fixed
factors were `section' (either CR, MID or CA) and `proximity', and the random
effects factor was the individual animal. For fixed factor `proximity', we
grouped the samples of each section into two categories in the following
manner. The grid used to identify the seven sample locations was divided into
two sections delineated by the equation y=-1x+0. Samples
that fell on either side of the line were pooled into two categories depending
on their location within the muscle in vivo: either `proximal' or
`distal' to the vertebral column (Fig.
4). Samples that fell on the line were discarded. Type I and type
IIa percentages were analyzed separately. Comparisons of mean percentages of
fiber types in the epaxial muscle and the pectoralis were analyzed using a
Student's paired t-test. Fiber type populations were also analyzed
with respect to seal sex and mass. All results are expressed as means ±
1 standard deviation (S.D.) and tested at a level of significance
of P<0.05.
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Results |
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The distribution of type IIa fibers was not significantly different with respect to the long axis of the epaxial muscle fibers (Table 1) or proximity to the vertebrae. The distribution of type I fibers was not significantly different with respect to the long axis of the epaxial muscle fibers (Table 1). However, statistics describing the distribution of type I fibers with respect to the vertebrae were inconclusive (3-factor ANOVA, P=0.05; paired t-test, P=0.06). Fiber type composition was not significantly different between females (N=8) and males (N=2), nor was there a mass-specific relationship. There was a significant difference between fiber type percentages of the epaxial muscles and the pectoralis. The pectoralis was composed of significantly less type I fibers and significantly more type IIa fibers when compared to the epaxial muscle (Table 1) (P<0.0001).
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Discussion |
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Traditional histochemical staining procedures use acidic and alkaline
preincubations to selectively inhibit the ATPase of the different fiber types,
allowing for differentiation (Brooke and
Kaiser, 1970). This procedure is based on the correlation between
the velocity of muscle contraction and the concentration of actomyosin ATPase
within each fiber type and has been used reliably and extensively in both
research and clinical settings. However, under some applications, ATPase
staining may have limitations. Of primary concern is the inability of the
ATPase technique to reliably differentiate between the types of fast twitch
fibers (types IIa, IIb, IIc and IId/x) in some species and the variability of
optimal ATPase staining conditions from species to species
(Green et al., 1982
;
Gorza, 1990
;
Amann et al., 1993
;
Rivero et al., 1996
). ATPase
staining also misrepresents cases of fibers coexpressing two different fiber
types (hybrid fibers) because the most dominant isoform is histochemically
stained (Gorza, 1990
).
Finally, the technique itself is sensitive. Inaccuracies may result from small
changes in preincubation and incubation time, temperature, pH, preincubation
buffer type and the ionic composition of the preincubation medium
(Matoba and Gollnick, 1984
).
Taken together, these considerations potentially make the results of
actomyosin ATPase fiber typing difficult to qualify, variable and
irreproducible when making interspecies comparisons or fiber typing a species
for the first time (Green et al.,
1982
; Amann et al.,
1993
), and raise questions about the interpretation of the results
obtained from previous histochemical fiber typing of seal muscles
(Ponganis and Pierce, 1978
;
Bello et al., 1983
;
Goforth, 1983
;
Hochachka and Foreman, 1993
;
Reed et al., 1994
). The
exception is the aforementioned study by Kanatous et al.
(2002
), which used an
ATPase-based metachromatic stain in combination with IHC fiber typing on
Weddell seal skeletal muscles. Their results showed a fiber type profile
similar to what we have found in the harbor seals. However, based on the
variable staining intensity of the histochemically stained cells shown in the
figures, we believe that without the accompanying IHC fiber typing, the
differentiation of type II fibers may have been difficult
(Kanatous et al., 2002
). Thus,
to maximize the accuracy of histochemical staining techniques in novel or
controversial fiber typing applications, multiple staining protocols are
recommended (Braund et al.,
1978
; Snow et al.,
1982
; Amann et al.,
1993
; LaTorr et al., 1993;
Kanatous et al., 2002
).
There is strong evidence that myosin heavy chain (MyHC) composition
directly corresponds to the shortening velocity of muscle fibers, subsequent
ATPase activity, and thus, ATPase staining intensity
(Reiser et al., 1985;
Betto et al., 1986
;
Staron and Pette, 1986
;
Termin et al., 1989
;
Gorza, 1990
). To circumvent
potential ATPase staining difficulties, we used IHC fiber typing to
characterize the fiber composition of seal muscle. Since IHC fiber typing
utilizes the specific antigenicity of MyHC isoforms to differentiate between
the fiber types, antibody binding capacity is binary in pure fibers. Thus, IHC
staining eliminates the subjective determination of `stain intensity' to
separate fiber type, making quantification more accurate. IHC fiber typing may
be used on a wide range of mammalian species because MyHC genes in striated
muscle are highly conserved in a variety of animals spanning several phyla,
from nematodes to man (Nguyen et al.,
1982
). Furthermore, the MyHC genes themselves have a highly
conserved organization and primary structure
(Mahdavi et al., 1986
).
Although IHC fiber typing is not a new technique
(Arndt and Pepe, 1975
), it has
not been widely used other than on laboratory animal and human tissues. We are
confident that the IHC fiber typing technique demonstrates a lack of classical
type IIb fibers in the locomotory muscles of harbor seals. However, we cannot
rule out the presence of other type II MyHC isoforms, in particular type IId/x
and type IIc. SDS-PAGE of harbor seal and rat skeletal muscle yielded
corresponding bands for type I and type IIa fibers in the epaxial muscles, but
in the pectoralis muscle the correspondence for the type IIa band is not clear
and appears to be associated with the type IId/x MyHC isoform found in the rat
(Fig. 3). For this study, we
did not have an antibody capable of specifically differentiating the type
IId/x isoform and therefore we analyzed the type IId/x fiber population by
process of elimination. Based on the results of the electrophoresis, further
investigation on the presence of type IId/x fibers in the harbor seal
pectoralis is warranted. In addition, the presence of type IIc fibers, which
are considered `undifferentiated' or `transitional' fibers
(Betto et al., 1986
), were not
found nor analyzed electrophoretically. Type IIc fiber population as a
percentage of total fibers counted is usually small (<3%) and probably does
not contribute significantly to the total muscle fiber population
(Betto et al., 1986
;
Amann et al., 1993
).
In general, we found that the fiber type population within the epaxial
muscles matched the myoglobin and enzyme activity data of tissue samples
collected from the same seals and from the same locations in the transverse
muscle sections. Harbor seal myoglobin (Mb) concentrations, citrate synthase
activities (CS; an indicator of aerobic metabolism) and ß-hydroxyacyl CoA
dehydrogenase activities (HOAD; an indicator of fatty acid metabolism) were
either the same or elevated compared to rat and dog (L. K. Polasek et al.,
manuscript in preparation). Moreover, in a separate study, mitochondria volume
density in harbor seal swimming muscle was elevated compared to the density in
locomotory muscles of sedentary terrestrial mammals of comparable size
(Kanatous et al., 1999). These
results are consistent with the characteristics of type I and type IIa fibers,
which are both oxidative. In addition, Polasek et al. (manuscript in
preparation) likewise did not find pronounced spatial heterogeneity of enzyme
activities or Mb concentrations within the CR, MID or CA transverse sections.
Since the cross sections of the harbor seal epaxial muscles were observed to
be a uniform deep red color during dissection, these results are not
surprising. However, Polasek et al. (manuscript in preparation) found
significantly higher CS and LDH activities in the CA and the MID transverse
sections compared to the CR section. These results indicate that a
longitudinal gradient for the physiological indices of aerobic capacity exists
in the harbor seal epaxial muscles, but it may not be manifest in the fiber
type distribution.
Fiber type distribution in the primary locomotory muscles (epaxial muscles)
of the seal was significantly different from the secondary locomotory muscle
(pectoralis). Whereas the epaxial muscles were composed of approximately 50%
type I fibers and 50% type IIa fibers, the pectoralis possessed approximately
15% type I fibers and 85% type IIa fibers
(Table 1). Seals swim using
lateral undulations of their hind flippers to propel themselves through the
water, and are characterized as thunniform swimmers
(Fish et al., 1988). The
foreflippers, which act as rudders and are used during burst swimming, do not
significantly contribute to forward propulsion. Thus, to generate force during
swimming, the epaxial muscles are alternately contracted and stretched.
Stretch-shortening cycles in the locomotory muscles of some terrestrial
mammals and fish maximize muscle force and power output during each stroke by
absorbing and storing potential energy during the lengthening phase of the
cycle for utilization during the shortening phase
(Altringham and Johnson, 1990
;
Curtin and Woledge, 1993
;
Lou et al., 1999
;
Lindstedt et al., 2002
). In
mammals, muscles that undergo active stretch (eccentric) contractions and
isometric contractions generally have more type I fibers than muscles that
perform concentric contractions (Armstrong
and Phelps, 1984
; Delp and
Duan, 1996
). Seal pectoralis may perform mostly concentric
contractions during foreflipper movement and therefore contain fewer type I
fibers than the epaxial muscles, which perform eccentric contractions during
the stretch-shortening cycle of thunniform locomotion.
The physiological profile of the harbor seal skeletal muscle appears to be
similar to that of terrestrial mammals adapted for sustained, aerobic exercise
(e.g. horses and dogs). This physiological profile includes an elevated
mitochondrial volume density, increased enzymatic capacity to oxidize fatty
acids, elevated tricarboxylic acid cycle enzyme capacity, and a fiber type
distribution of primarily type I and type IIa fibers in locomotory muscles.
However, in seals, routine metabolic rate during diving is generally less than
twice the resting, predive levels.
(Castellini et al., 1992;
Davis et al., 1985
; T. M.
Williams et al., manuscript in preparation). Additionally, behavioral studies
indicate that seals are not active swimmers and may not maximize their aerobic
capability in vivo. Rather, seals use energy-saving locomotory
strategies. Recent evidence shows that when seals dive, they often alternate
between an active stroke phase and a passive glide phase to conserve energy
and oxygen stores, a pattern that is demonstrated in a variety of diving
mammals (Williams et al.,
2000
; Davis et al.,
2001
). Consequently, this behavioral information coupled with
physiological data suggests that the elevated mitochondrial volume density
found in seal skeletal muscle may have a primary function of decreasing the
diffusion distance of oxygen stores in myoglobin to the site of oxidation at
the mitochondria (Kanatous et al.,
1999
).
Our fiber typing results show that harbor seal skeletal muscle is made exclusively of slow-twitch and fast-twitch oxidative fibers. These results are consistent with seal behavioral data and the theory that diving in marine mammals is an aerobic activity. Fiber type distribution did not show pronounced spatial heterogeneity along the dorso-ventral and medio-lateral axes of the epaxial muscles. Finally, differences in fiber type distribution in the epaxial muscles vs. the pectoralis muscle may be related to contraction velocity and ability to store elastic energy.
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Acknowledgments |
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