First published online January 5, 2005
Journal of Experimental Biology 208, 409-417 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01375
Troponin T expression in trout red muscle correlates with muscle activation
David J. Coughlin*,
Nicholas D. Caputo,
Krista L. Bohnert and
Frances E. Weaver
Widener University, Department of Biology, One University Place,
Chester, PA 19013, USA
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Fig. 1. Contraction kinetics of red muscle in rainbow and brook trout. For rainbow
trout, activation and relaxation time increased significantly from anterior to
posterior (data are from Coughlin,
2000 ). For brook trout, new data on muscle activation and
relaxation are plotted for three body positions (N=10 for anterior
and posterior, N=3 for middle). Brook trout show a significant
increase in relaxation time from anterior to posterior, but there is no
difference in activation rate along the length of these fish. Asterisks
indicate statistically significant variation along the length of the fish
(P<0.05).
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Fig. 2. (A) Identification of troponin T (TnT) S1 and S2 isoforms in rainbow trout
using western blotting. Lane 1 is a pre-stained ladder of protein standards
(BioRad), and lane 2 is authentic mammalian troponin used as a control (Sigma
T-3515). Lane 3 is the 75 mmol l–1 phosphate hydroxy-apatite
column fraction that contains two isoforms of purified rainbow trout red
muscle TnT, identified as TnT S1 and S2. (B) Determination of sizes of TnT
isoforms using a silver-stained PAGE gel. Lane 1 is a protein ladder (BioRad),
and lane 2 is the purified TnT.
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Fig. 3. Troponin T (TnT) expression in a representative rainbow trout. Red muscle
TnT isoforms can be identified based on size using a silver-stained PAGE gel
(Top). Lanes 1 and 9 contain purified TnT. Lanes 2 to 8 contain purified
myofibrils from 25, 35, 45, 55, 65, 75 and 85% of total length from the snout.
TnT isoforms can be identified in these samples based on band position
relative to standards (Std.). The relative expression levels (as indicated by
staining intensity) for the lower band (TnT S2) decrease from anterior to
posterior of the fish. A sample densitometry profile is shown at the bottom
with two peaks that correspond to the two TnT isoforms.
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Fig. 4. Relative expression of troponin T (TnT) isoforms along the length of
rainbow trout. There is a significant shift in the percentage expression of
TnT S1 isoform relative to TnT S2 from anterior to posterior. The average
expression of TnT S1 increases from  75% in the anterior to 100% in the
posterior myotome. Double asterisks indicate statistically significant
variation along the length of the fish (P<0.01).
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Fig. 5. (A) Identification of troponin T (TnT) S1 and S2 isoforms in brook trout
based on hydroxy-apatite chromatography and a silver-stained PAGE gel. Lane 1
is a ladder of protein standards (BioRad), and lane 2 is the 75 mmol
l–1 phosphate hydroxy-apatite column fraction that contains
two isoforms of purified rainbow trout red muscle TnT, identified as TnT S1
and S2. (B) Myofibrillar proteins in a representative brook trout. Partially
purified myofibrillar proteins ranging in size from myosin heavy chain at the
top of the gel (largest protein visible) to putative parvalbumin at the bottom
of the gel (smallest protein visible). Lanes 1–3 contain purified
myofibrils from 35, 55 and 75% of total length from the snout. Lane 4 contains
a ladder of protein standards. There is some indication of longitudinal
variation in a protein at 33–34 kDa (tropomyosin?, see diamond-head
arrow). At the anterior position (35% TL) there is a doublet, but the
middle and posterior positions show only a single band. No other obvious
longitudinal variations are observed here or in other gels except for
variations in TnT. (C) TnT expression in a representative brook trout.
Magnification of the bands from B. Red muscle TnT isoforms were identified
based on size using a Sypro Ruby-stained PAGE gel. The two visible bands are
the brook trout red muscle TnT isoforms. In this individual, relative
expression levels (as indicated by staining intensity) for the lower band (TnT
2S) increase from anterior to posterior of the fish.
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Fig. 6. Relative expression of troponin T (TnT) isoforms along the length of brook
trout. There is no significant variation in the percentage expression of TnT
S1 isoform relative to TnT S2 from anterior to posterior. The mean average
expression of TnT S1 is 65% at all body positions.
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Fig. 7. Individual analysis of brook trout (BT) troponin T (TnT) expression (A),
muscle activation (B) and muscle relaxation (C). Three representative analyses
are shown, but of the ten fish analysed, six showed a decrease in the relative
TnT S1 expression from anterior to posterior (e.g. BT 8 and BT 9). The other
four fish showed an increase (e.g. BT 10). For eight of the fish, the pattern
of variation in TnT S1 agrees with the longitudinal pattern of muscle
activation (e.g. BT 8 and BT 10). All fish showed the identical pattern of
increasing muscle relaxation time from anterior to posterior.
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Fig. 8. Individual patterns of the relationship of troponin T (TnT) expression and
activation time in brook trout (BT). Individual points are the proportion of
TnT S1 and the activation time for 2–3 muscle samples per fish (ANT and
POST or ANT, MID and POST). For each fish, a line of best fit is included to
indicate the relationship of the two variables. For eight of the fish, the
evident trend is a higher proportion of TnT S1 is associated with a longer
time of activation (slower muscle). There were two exceptions (BT 1 and BT
9).
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© The Company of Biologists Ltd 2005
