Troponin T expression in trout red muscle correlates with muscle activation
Widener University, Department of Biology, One University Place, Chester, PA 19013, USA
* Author for correspondence (e-mail: djcoughlin{at}mail.widener.edu)
Accepted 8 November 2004
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Summary |
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Key words: troponin T, rainbow trout, Oncorhynchus mykiss, brook trout, Salvelinus fontinalis, muscle activation
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Introduction |
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Fish vary in terms of degree of body bending during swimming, from
stiff-bodied thunniform and carangiform swimmers to animals that display
higher degrees of body curvature along their entire length, such as
anguilliform and salmoniform swimmers. Furthermore, these categories of
swimmers are loosely associated with different anterior-posterior patterns of
muscle contraction kinetics (Coughlin,
2002). For instance, the relatively stiff-bodied scup
Stenotomus chrysops shows limited variation in muscle kinetics, with
only relaxation rate differing from front to back
(Rome et al., 1993
).
Alternatively, rainbow trout (F. Salmonidae, Oncorhynchus mykiss
Walbaum), which swim with greater body curvature, display a greater level of
variation in contractile properties of their red muscle
(Fig. 1). In rainbow trout,
both relaxation rate and activation rate varies from anterior to posterior
(Coughlin, 2000
).
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This study examines the molecular correlates of longitudinal variation in
the contractile properties of the red muscle in fishes. The specific question
is, what is the molecular basis for variations in activation rate along the
length of the fish? One molecular candidate is the muscle protein troponin T
(TnT), because differences in Ca2+ sensitivity in isoforms of
troponin T result in variations in activation time
(Schachat et al., 1987;
Fitzhugh and Marden, 1997
). An
additional line of evidence that TnT is a powerful regulator of muscle
activation comes from examination of human muscle diseases such as
cardiomyopathy. Mutations in TnT affect Ca2+ sensitivity of muscle,
leading to certain forms of hypertrophic cardiomyopathy (e.g.
Sweeney et al., 1998
;
Robinson et al., 2002
).
The troponin complex consists of three subunits: troponin I (TnI) binds to
actin and inhibits the actin-myosin interaction; troponin C (TnC) binds
calcium ions and regulates TnI activity and troponin T (TnT) connects the
troponin complex to tropomyosin (Tm). The binding of Ca2+ by TnC
enhances the bonding of TnC to both TnI and TnT and weakens the TnI-actin
interaction (Gordon et al.,
2000). This leads to a conformational change in the troponin
complex that is transmitted by TnT to the tropomyosin. Changes in tropomyosin
position on the actin filament open up myosin binding sites on the actin and,
thereby, initiate contraction. TnT has several roles in Ca2+
regulation of muscle activity through its complex interactions with Tm, TnI,
TnC and actin (Gordon et al.,
2000
).
The relatively high number of isoforms of TnT (e.g.
Breitbart et al., 1985;
Smillie et al., 1988
;
Berchtold et al., 2000
) suggest
a wide range of contractile properties, including variations in
Ca2+ sensitivity and rates of activation of the myosin ATPase
(Gordon et al., 2000
).
Rostral-caudal variations in troponin T expression that correlate with
variation in activation rate of the anaerobic or white muscle in fishes have
been described in cod (Thys et al.,
1998
) and largemouth bass
(Thys et al., 2001
). The
possible contribution of TnT to the regulation in rainbow trout red muscle
activation is suggested by the identification of two isoforms of troponin T in
the red muscle (S1 and S2) (Waddleton et
al., 1999
). Differential expression of these isoforms along the
length of the fish could lead to variation in activation time between the
anterior and posterior muscle.
In the first part of this study, we test the hypothesis that the longitudinal shift in activation kinetics in rainbow trout aerobic muscle is a function of a shift in TnT isoform expression. Having found a correlation between activation time and TnT expression in rainbow trout, the second part of this study aimed to examine TnT and contraction kinetics in a second species of salmonid, brook trout or charr (F. Salmonidae, Salvelinus fontinalis Mitchill). For this species of trout, we measured contractile properties from anterior and posterior red muscle and we examined TnT expression in muscle samples from the same fish. The paired-sample nature of the physiological data and the analysis of protein expression in the sample fish permitted a rigorous examination of connections between muscle activation and troponin T.
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Materials and methods |
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Rainbow trout protein analysis
To identify the troponin T isoforms from rainbow trout red muscle, red
muscle samples were dissected from three body positions of two adult rainbow
trout (total length TL=28.5 cm and 23.7 cm). Purified myofibrillar
proteins were separated using hydroxy-apatite chromatography, as described by
Thys et al. (1998,
2001
). The column fractions
(ranging from 5 to 200 mmol l1 phosphate) were examined
using protein electrophoresis (SDSPAGE). Protein concentration was
determined using a detergent-compatible protein assay (DC Protein Assay;
Bio-Rad; Hercules, CA, USA). PAGE samples were made from the column fractions
to a final protein concentration of 0.025 mg ml1 in Laemmli
buffer. PAGE gels (Bio-Rad Precast Tris-HCl Ready Gels: 16 cm x16 cm
0.15 cm; 4% stacking gel and 12% resolving gel) were loaded with 50 µl of
sample and run for 30 min at 16 mA and then for 34 h at 24 mA, all at
4°C in a TrisglycineSDS running buffer. Half of each gel was
silver stained, and the other half was used for western blot analysis. The
sample buffer, running buffer, silver stain kit and electrophoresis cell were
supplied by Bio-Rad. Immunoblotting of PAGE gels was carried using a Bio-Rad
semi-dry blot transfer system (TransBlot SD), PVDF membrane, Bio-Rad
colorimetric blot analysis (GAM-AP) and monoclonal anti-mouse troponin
antibody (Sigma, T-6277; St Louis, MO, USA). After pre-incubation in Towbin
buffer (25 mmol l1 Tris, 192 mmol l1
glycine, 20% methanol. pH 8.3), blot transfer was achieved using 30 V for 15
min. The membrane was blocked using 3% gelatin in Tris-buffered saline (TBS).
Primary antibody was applied in a 1:1000 dilution in 1.5% gelatin in TBS. The
membrane was shaken for 1 h, placed in the refrigerator overnight and then
shaken for 1 h the next day before continuing with secondary antibody. The
secondary antibody (goat anti-mouse; Sigma A-3688) was also diluted 1:1000 in
1.5% gelatin in TBS and was applied for 1 h.Subsequent colour development of
the membrane took approximately 1 h. Gels and blots were digitally
photographed, and the size of TnT isoforms were estimated using Kodak 1-D Gel
Analysis software.
After two isoforms of troponin T had been reliably identified based on
size, relative expression patterns of the two forms could be studied. Red
muscle samples were dissected from seven body positions (25% to 85% of
TL) along the length of three fish [TL=27.1±2.7 cm,
mass=215±22 g (mean ± S.D.)]. Each muscle sample was
taken bilaterally from a 10% strip of the fish's length. For instance, the
`35%' sample was taken from 3040% of TL. Myofibril isolation
was carried out following the method of Lutz et al.
(1998), as adapted from
Talmadge and Roy (1993
). Gels
were run as described above, but only silver stain was used. Densitometry
(Kodak 1-D Gel Analysis) was used to quantify relative expression of the two
isoforms of TnT.
Brook trout muscle physiology
A total of ten brook trout were used for the examination of contractile
properties and TnT expression (TL=26.5±2.14 cm,
mass=211.2±51.7 g). One of those fish (TL=28.8 cm, mass=194.5
g) was also used to identify TnT isoforms. In all fish, contractile properties
of the anterior and posterior muscle were measured, as well as relative
expression of TnT isoforms in muscle samples from both body positions.
For muscle mechanics experiments, brook trout were killed by spinal
transection and pithing. After removing the scales, 1.0 mm wide strips of
red muscle were extracted from just above and below the lateral line of the
fish. Muscle preparations were dissected from two longitudinal positions:
anterior (ANT, 2545% of TL) and posterior (POST, 6585%
of TL). For a few of the fish, muscle from the middle (MID, 45-65% of
TL) position was also examined. Subsequent dissection was carried out
at 4°C with the use of a stereomicroscope in the presence of physiological
saline (Altringham and Johnston,
1990
). Live bundles were the length of one myomere
(
2.54 mm) with a muscle fibre cross-sectional area of
0.25
mm2. The bundles were tied into a muscle mechanics system
comprising a servomotor (Cambridge Technology 300S; Cambridge, MA, USA) and a
force transducer (Aurora Scientific 404A; Aurora, Ontario, Canada).
Temperature in the apparatus was maintained at 10°C for all experiments;
the physiological saline was aerated gently to supply oxygen and to induce
circulation. Experimental control and data collection were carried out using a
PC, Keithley-Metrabyte DAS-1601 input/output board and custom software.
For each bundle, activation conditions (muscle length, pulse length and
amplitude for twitch contractions, stimulus duration and frequency tetanic
contractions) were optimised to generate maximal tetanic force. Typical
stimulations for tetanus were: stimulus duration of 200250 ms composed
of 23 ms pulses at a frequency of 125 Hz. Each pulse had an amplitude
of 79 V. Isometric force was converted to stress after live fibre area
of the muscle bundles was determined. For tetanic contractions, time of
activation was defined as the time from 1090% of maximum isometric
stress, and time of relaxation was the time from 9010% of peak
isometric stress. For a limited number of fish (N=5), maximum
shortening velocity (Vmax) was also determined for
anterior and posterior muscle as previously described
(Coughlin et al., 2001).
The fibre area of the live muscle bundles was determined at the end of each
experiment (Coughlin, 2000).
Bundles were stained with Trypan Blue to identify dead tissue, embedded in
gelatin and frozen with liquid nitrogen. The frozen bundles were sectioned at
16 µm and stained with succinic dehydrogenase (SDH) for mitochondrial
content. Darkly stained areas indicated aerobic muscle fibres. The cross
section of live muscle fibres within the experimental muscle bundles could be
determined by excluding both dead fibres and connective tissue. This
cross-sectional area was used to calculate isometric tension for twitch and
tetanic contractions.
Brook trout protein analysis
Protein analysis on brook trout followed the same methods as for rainbow
trout. PAGE along with hydroxy-apatite chromatography was used to identify the
apparent size of two TnT isoforms. The relative expression of those isoforms
was quantified using PAGE and densitometry for three body positions: ANT, POST
and MID (4565% TL) for the same ten fish used in the
physiology experiments. In these experiments, silver staining was used to
determine the sizes of TnT isoforms in brook trout, while Sypro Ruby (BioRad)
was used to assess expression.
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Results |
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Brook trout contraction kinetics
Red muscle bundles from the two body positions of brook trout did not
differ in terms of force production, ratio of twitch force to tetanic force
production or Vmax
(Table 1). Brook trout red
muscle displays a longitudinal shift in muscle relaxation, but no shift in
muscle activation (Fig. 1). The
posterior red muscle displayed significantly longer relaxation times than the
anterior red muscle (Paired sample t-test, t=2.90, df=9,
P=0.018). All ten fish showed this same pattern (see below). However,
there was no rostral-caudal shift in muscle activation of brook trout red
muscle (Paired sample t-test, t=0.35, d.f.=9,
P=0.73). Furthermore, some fish showed a rostral-caudal increase in
activation time, as observed in rainbow trout, and others displayed a
rostral-caudal decrease in activation time.
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Brook trout TnT expression
The molecular mass of brook trout TnT estimated from the silver-stained
gels were 29.2 kDa for TnT S1 and 28.0 kDa for TnT S2
(Fig. 5A). As with rainbow
trout, troponin was found in the highest concentration in the 75 mmol
l1 phosphate concentration hydoxy-apatite column fraction
(data not shown). Brook trout display no consistent rostral-caudal pattern of
the relative expression of the two isoforms of TnT. Some fish show a shift of
increasing relative expression of TnT S2
(Fig. 5C), while others show
the opposite pattern. Overall, there was no significant relationship between
body position and the relative expression of TnT S1 and S2
(KruskalWallis test, H=0.258, P>0.05;
Fig. 6).
|
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Individual examination of TnT expression and muscle activation revealed intra-specific variation. All ten brook trout displayed the same pattern of increasing relaxation time from anterior to posterior (Fig. 7C). However, only six showed a rostral-caudal increase in activation time, while four showed a decrease (Fig. 7B). For eight of the ten fish, there is agreement in the predicted relationship of TnT isoform expression and activation time. Four of those fish showed the pattern observed in rainbow trout: slower activation in the posterior is associated with higher relative expression of TnT S1 (e.g. brook trout 10, Fig. 7). Four other fish showed the opposite but equivalent pattern: faster activation in the posterior is associated with relatively higher expression of TnT S2 (e.g. brook trout 8, Fig. 7). For two fish, the physiological and protein expression results do not agree with patterns observed in rainbow trout (e.g. brook trout 9, Fig. 7). In these two fish, the anterior muscle had faster rates of activation but also had relatively higher expression of the TnT S1 isoform. The binomial probability of two or fewer mismatches out of ten is 0.055. This indicates that the results observed (8 out of 10 fish matching the prediction) would occur by chance in at most 5.5% of repetitions of this experiment.
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When the activation time is plotted against the expression level of TnT S1 for each fish, the same eight fish show a positive correlation higher levels of TnT S1 are associated with longer activation times (Fig. 8). The same two fish show the opposite pattern. An ANOVA, with activation time as the dependent variable, fish identity as a random effects independent variable and the expression level of TnT S1 as covariate, was carried out for all ten fish. The expression data, which are proportions, were log-transformed to compensate for the skewed nature of percentages. However, neither fish identity nor TnT expression level significantly affect activation time (for TnT S1 expression, F=2.110, P=0.172; for fish identity, F=1.781, P=0.174). Eighteen of 23 data points fall are clustered along a positive correlation between TnT S1 expression level and activation time (Fig. 8).
|
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Discussion |
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In brook trout, there is no consistent shift in muscle activation, the mean value for muscle activation time is the same for both anterior and posterior red muscle. Similarly, there is no shift in TnT expression. However, the story is complicated by intra-specific variations in the patterns of muscle physiology and TnT expression in brook trout. Although mean values show no consistent longitudinal pattern of variation in activation time and TnT expression, individuals show a variety of patterns of activation and TnT expression. Some fish show a 3040% increase in muscle activation time from anterior to posterior and a similar increase in the relative expression of TnT S1 in the same muscle (e.g. brook trout 10, Fig. 7). Others show >40% decrease in muscle activation time from anterior to posterior and a similar decrease in the relative expression of TnT S1 in the same muscle (e.g. brook trout 8, Fig. 7).
Two of the eight fish show a distinctly different relationship between TnT S1 expression and activation time, with a negative correlation between TnT S1 and activation time (e.g. brook trout 1 and 9, Fig. 8). We cannot explain this observation with our current hypothesis but can only suggest that the variation in expression in various other muscle proteins may underlie observed variations in muscle contractile properties. Our laboratory is currently examining other muscle proteins in rainbow and brook trout, including myosin light chain 2 and parvalbumin, to assess intra-individual, intra-specific and inter-specific variations in these proteins.
Besides variations in longitudinal patterns of muscle activation and TnT
expression, brook and rainbow trout display other rostral-caudal variations in
muscle properties. At least for the hatchery strains of fish examined in this
and previous research in this laboratory, there are inter-specific differences
in muscle recruitment. At low and moderate steady swimming speeds, rainbow
trout recruit primarily their posterior red myotome, while brook trout recruit
their entire myotome (anterior to posterior) with an even level of recruitment
intensity (Coughlin et al.,
2004). Differences in muscle recruitment correlated with another
inter-specific variation in trout muscle. During steady swimming, rainbow
trout generate significantly more power in their posterior myotome than in the
anterior (Coughlin, 2000
),
which correlates with the preferential recruitment of muscle that produces
greater mechanical power at lower swimming speeds when not all of the red
myotome is needed (Coughlin et al.,
2004
). Alternatively, brook trout display no longitudinal
variation in red muscle power production during steady swimming
(McGlinchey et al., 2001
) and
correspondingly display no longitudinal variation in muscle recruitment. The
addition of inter-specific variation in longitudinal patterns of red muscle
protein expression is perhaps expected, as some underlying variation in muscle
composition (i.e. form) should be predicted to underlie variations in muscle
function.
Troponin T
A number of studies have demonstrated a role for troponin T in the
modulation of muscle activation, particularly in fishes. Cod Gadus
morhua show longitudinal variation in the activation of their white
muscle (Davies et al., 1995).
Thys et al. (1998
)
demonstrated that this shift in muscle contractile properties correlates with
a longitudinal pattern of expression of two isoforms of TnT. Similarly, Thys
et al. (2001
) showed a
significant rostralcaudal shift in both muscle activation and TnT
expression in the white muscle of largemouth bass. In both cod and bass, the
anterior muscle is kinetically faster and expresses relatively greater amounts
of an isoform of TnT that migrates faster on PAGE gels. (TnT-2; Thys et al.,
1998
, 2002). For both white
(cod and bass) and red (rainbow and brook trout) muscle in fishes, the
kinetically faster isoform of TnT appears to be the physically smaller isoform
found in each type of muscle. Other fish, such as saithe Pollachius
virens (Altringham et al.,
1993
), show rostral-caudal variation in the activation time of
swimming muscle and can be predicted to show variations in troponin T
isoforms.
The present study and those cited above for white muscle do not
definitively establish the role of TnT in modulating muscle activation in
fishes, but strong evidence exists in the literature that variations in TnT
isoform do affect contractile properties of muscle
(Schachat et al., 1987;
Fitzhugh and Marden, 1997
).
Several studies from the laboratory of James Marden have elucidated the
effects of subtle shifts in the relative expression TnT isoforms in dragonfly
Libellula pulchella flight muscle on Ca2+ sensitivity and
flight performance (Fitzhugh and Marden,
1997
; Marden et al.,
1999
,
2001
). Up to six TnT isoforms,
identified by Marden et al.
(1999
) as splice variants, are
found in the flight muscle of dragonflies. Differences between individuals in
terms of the relative expression of these isoforms leads to an order of
magnitude variation in Ca2+ sensitivity in skinned muscle fibres
and significant variation in the maximum oscillatory power output of muscle
(Marden et al., 2001
).
Furthermore, there are developmental shifts in TnT isoform expression that
correlate with changes in flight performance between newly emerged adult and
mature adult dragonflies (Fitzhugh and
Marden, 1997
), suggesting that TnT expression has a direct
relationship to behavioural performance.
Other animals show developmental shifts in TnT expression that may also
correlate with muscle function. For instance, there is a developmental shift
in TnT expression from an embryonic to adult form in rat hearts
(Jin and Lin, 1988). In
rainbow trout and brook trout, variations in TnT may well affect performance,
but that has not yet been clearly established. For a start, as mentioned
above, previous research has shown longitudinal variation in oscillatory power
output during swimming in rainbow trout
(Coughlin, 2000
) but not brook
trout (McGlinchey et al.,
2001
). In corresponding fashion, TnT expression varies along the
length of rainbow trout but not brook trout, suggesting that TnT expression
relates to function of muscle during swimming.
The present work cannot rule out that variation in TnT S1 merely correlates
with muscle kinetics and that other muscle proteins are responsible for
observed variations in muscle activation time in both rainbow and brook trout.
A superficial examination of all the contractile proteins from these fish
suggest that other proteins do not show marked longitudinal patterns, although
there may be some variation in another, unidentified protein
(Fig. 5B). In addition,
previous work using protein analysis has shown no longitudinal variation in
myosin heavy chain along the length of the red muscle in trout
(Coughlin et al., 2001).
Current work in our lab using quantitative real-time PCR to examine
developmental shifts in myosin light chain 2 expression have not shown any
longitudinal patterns of expression of this regulatory light chain in rainbow
trout. The absence of observations of longitudinal variation in other muscle
proteins and prior research demonstrating the functional role of TnT in
regulating activation in other species (e.g. dragonflies) taken together
strongly suggest that the relative expression of TnT S1 and S2 in trout red
muscle does have a regulatory effect on muscle activation.
Trout in the wild
A future direction of this work is the examination of protein expression in
wild populations of rainbow and brook trout. The present work was carried out
with hatchery strains raised under identical conditions at a trout hatchery
operated by the Commonwealth of Pennsylvania. Little is know about the
physiological properties and less about variations in muscle composition in
wild populations in these fish species. Marden et al.
(2001) reported `broad
intraspecific' variation in TnT expression and muscle contractile properties
in flight muscle from two distinct populations of dragonflies, similar to the
results reported here for the hatchery brook trout. Do wild populations of
brook trout also show such intraspecific variation in TnT expression? Given
that adult brook trout vary widely in size and body morphology in the
extensive variety of natural habitats in which they are found
(Karas, 1997
), such a
prediction can be readily made.
More important than simply finding variation in TnT expression is
determining whether or not patterns of TnT expression affect muscle
contractile properties. Is there a relationship between TnT expression and
muscle activation in wild-run fish? Do differences between rainbow trout and
brook trout described here and in previous studies in this lab (e.g.
Coughlin, 2000;
McGlinchey et al., 2001
;
Coughlin et al., 2004
) have any
bearing on fitness in the wild? To answer these questions will require a
comparative study of diverse populations of trout using muscle mechanics
experiments combined with analysis of TnT expression patterns to assess the
protein correlates of variations in muscle contractile properties.
Conclusions
In this study, variations in troponin T expression was described for the
first time in the red muscle of fish. Rainbow trout display a significant
longitudinal shift in the relative expression of two isoforms of TnT, and this
shift correlates with rostral-caudal differences in muscle activation. Another
species of salmonid, brook trout, was shown to not display the same pattern of
TnT expression and muscle activation. Instead, this species showed
considerable intraspecific variation in the role of TnT in modulating muscle
properties.
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Acknowledgments |
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