.Journal of Muscle Research and Cell Motility
Interchain
12, 439-446
(1991)
and intra-specific
variation
and troponin
I composition
in myosin
light
in fast muscle
fibres from two species of fish (genus Oreochromis )
which have different
temperature-dependent
contractile
properties
T. CROCKFORDl,
T. P. JOHNSONl
1Gatty Marine
K. E. WOMMACKl*,
Laboratory,
2Institute of Aquaculture,
Department
University
A.
TOHNSTON1~.
of Biology and Preclinical Medicine,
of StirlinJ{, StirlinJ{ FK9 4LA,
B.
McANDREW2,
The University,
St Andrews,
G.
MUTUNGl1§
Fife, KY16
and
BLB, UK
UK
Received21 December 1990; revised and accepted21 March 1991
Summary
The contractile properties and myofibrillar protein composition of fast muscle have been characterized in pure strains of two
tropical fish Oreochromis niloticus and 0. andersoni. Single fast muscle fibres were isolated from the abdominal myotomes and
chemically skinned. The maximum tension-temperature relationships of fibres were similar at 25-300 C, but diverged below
17° C. At 10° C, maximum tension was around 60% higher in 0. andersoni (160:t 15 kN m-2) than 0. niloticus
(105 :t 13 kN m-2) (mean:t SD). The myofibrillar protein composition of fast fibres was investigated using one-dimensional and
two-dimensional gel electrophoresis and peptide mapping. The two Oreochromis species differed with respect to the
composition of myosin light chains, troponin land mysoin heavy chains (VB protease and chymotrypsin peptide maps). An
unexpected finding was the presence of two isoforms of myosin light chain 1 in 0. andersoni, with apparent molecular masses
of 27.5 kDa (LC1f1) and 26.9 kDa (LC1!2). Individuals with LC1f1 (n = 20) and LC1f1 + LC1!2 (n = 12) were represented in the
population studied. The myosin light chain 3 (LC3J content of fibres was similar in both cases. Breeding experiments
established that these intra-specific variations in isoform composition were heritable. Fast muscle from 0. niloticus and 0.
andersoni contain two isoforms of troponin I (TNIrl + TNIr2) which were both expressed in single fibres. The identity of TNI
was confirmed using a stationary phase troponin-C affinity column. Of the 20 0. niloticus studied seven contained only TNIrl.
The two Oreochromis species studied produce fertile F1 hybrids, are susceptible to ploidy manipulation, have a short
generation time and rapid growth rates. They therefore represent a good model for investigating the genetic mechanisms
underlying the inheritance of different force-generating capacities in fish.
Introduction
Maximum tension generation is significantly reduced at
low temperatures in muscle fibres from tropical fish
Oohnston & Brill, 1984; Johnston & Altringham, 1985;
Johnson & Johnston, 1991). In contrast, muscle fibres from
polar fish generate high forces at low temperature, but
become progressively inexcitable at higher temperatures
Oohnson & Johnston, 1991). At normal body temperatures
the maximum tension generated by fast muscles is broadly
comparable for species from different habitat temperatures
Oohnston & Altringham, 1988, 1989). In contrast to force
.T o whom correspondenceshould be addressed.
:I: Presentaddress:Department of Biology, University of Maryland,
Maryland, USA.
§ Presentaddress:Department of Animal Physiology, University of
Nairobi, Kenya.
0142-4319/91
$03.00+.12
generation, rate parameters, including force development,
relaxation and maximum contraction speed, only show a
limited degree of temperature adaptation and are significantly slower in polar than tropical fish (Johnston &
Brill, 1984; Johnson & Johnston, 1991).
The group of African fishes which are now commonly
called tilapia is made up of over 70 different species
assembled into four genera (Trewavas, 1983). The species
inhabit a wide range of aquatic habitats in tropical and
subtropical Africa from hotsprings, hypersaline lakes,
euryhaline estuarine and coastal areas to freshwater rivers
and lakes ranging in temperature from 14 to 42° C
(Trewavas, 1983). The adaptability of tilapia makes them
an ideal species for aquaculture and because of this one or
other species has now been introduced into every tropical
country in the world. Species from the maternal mouthbrooding genus Oreochromis are particularly important
@ 1991 Chapman and Hall Ltd
,u,
440
CROCKFORD, WOMMACK,
and have been widely studied. These fish are particularly
hardy and will tolerate a range of water conditions from
salt to freshwater, are easy to breed with a short generation
time { < 6 months), will form fertile F1 hybrids with other
species of the same genus and can be subject to a variety
of genetic manipulations {Hussain et al., 1990). They
therefore represent a good model for investigating the
genetic mechanisms underlying temperature adaptation.
To investigate this possibility further we have characterized the force-temperature relationship and myofibrillar protein composition of muscle fibres from genetically pure strains of two species with different
temperature tolerances.
Materials
and methods
Fish
Oreochromis niloticus (n = 20) of 15-25 cm standard length and
Oreochromis andersoni (n = 32) of 15-22 cm standard length
were obtained from the Tilapia Reference Collection, Institute
of Aquaculture, University of Stirling. The purity of strains was
verified by starch gel electrophoresis of 25 enzyme loci from
liver and muscle (McAndrew and Majumdar, 1983). the 0.
niloticus studied were half sibs from three families (three females
and one male), and the offspring of particular crosses were not
identified. Three families of 0. andersoni were used, (three
females and one male). The offspring from each cross was kept
separate until large enough to be Panjet marked (Alcian Blue)
after which they were kept as a single group. Dab (Limanda
limanda L.) were caught locally at St Andrews and held in the
marine aQuarium before use.
Measurement
of contractile properties
Small strips of fast muscle were dissected from anterior
abdominal myotomes and single fibres isolated under silicone
oil (BDH MS 550) at 0-4 ° C, Fibres (2-3 mm length, 90-100 ~m
diameter) were mounted between two stainless steel hooks
using Plexiglass / acetone glue and immersed in the first of three
water-jacketed chambers (controlled to :t 0.2° C) (Altringham
& Johnston, 1982), The fibres were chemically skinned in
relaxing solution containing 1% Brij 58 (polyoxyethylene 20
cetyl ether) for 10-15 min, Relaxing solution contained
(mmoll-l):
l,4-piperazine-bis (ethanesulphonic acid) (PIPES),
25; ethyleneglycol-bis(fJ-aminoethylether)N,N-tetraacetic acid
(EGTA), 15; MgCI2, 6.8; ATP, 6; phosphocreatine, 27.5; 25-50
units mI-l creatine phosphokinase (pH was adjusted to 7.2 at
20° C using KOH and allowed to vary freely with temperature ;
-0.0085 pH units per ° C), Fibres were transferred to the
second bath containing relaxing solution (5 min) and sarcomere
length was determined by laser diffraction and set to 2.3 ~m,
Fibre length and diameter were measured in situ using a
graticule and high-power microscope. Activating solution contained in the third bath was made by addition of 15.3 mmoll-l
CaCI2, Free ion concentrations were calculated using an iterative
computer program incorporating corrections for temperature
(Nico!, 1985). The main ionic species were at the following concentrations:
pMg 2.96-3.02,
pMgATP 2.28-2.26,
pCa 4.66-3.80,
pH 7.37-7.12,
and ionic strength 0.180.19 mol 1-1 (values Quoted are for 0° C and 30° C, respec-
JOHNSTON,
McANDREW,
MUTUNGI
and JOHNSON
tively). Preliminary experiments established that the calcium
concentration was sufficient to give maximal activations at each
temperature and that force was insensitive to changes in pH
over this range (see Mutungi & Johnston, 1988). Force was
measured with a silicon beam strain gauge (AME 801,
Horten, Norway), with a sensitivity of 0.5 mN y-l and baseline
stability of 5 mY h-i.
Preparation of proteins for electrophoresis
Myofibrils
were prepared from fast myotomal muscle as
described by Johnston and colleagues (1977). Triton X-IOO (I %)
was included in the first washing buffer to aid the removal of
membrane-bound proteins. The protein concentration of myofibrils was estimated by dissolving samples in an equal volume
of 10% sodium dodecyl sulphate (SDS) at 70° C for 5 min, and
the absorbance read at 280 nm, using bovine serum albumin as
a standard.
Myofibrils (3 mg mI-l) were prepared for SDS polyacrylamide gel electrophoresis (SDS-PAGE) by boiling for 3 min in
(final concentrations); Tris HCl, 60 mM, pH 6.75 at 20° C; SDS,
2 %; glycerol, 10%; 2-mercaptoethanol, I %; and Bromophenol
Blue, 0.002%. Samples of myofibrils (5 mg mi-l) for alkali-urea
polyacrylamide gel electrophoresis (AU-PAGE) were prepared
by dissolving myofibrils for 60 min at 25° C in (final concentrations) : urea, 8 M; Tris-glycine, 50 mM, pH 9 at 20° C; 2mercaptoethanol, I %; Bromophenol Blue, 0.002%; and either
5 mM CaCI2 or 10 mM EGT A.
One-dimensional electrophoresis
SDS-PAGE was carried out according to Laemmli (1970) using
a vertical slab gel apparatus. Resolving gels were either 7.5 %,
15% or 10-22% linear gradient acrylamide, while stacking gels
were always 5% acrylamide. BIS was used as crosslinker at a
concentration of 3.3%. AU-PAGE was performed by a modification of the method of Focant and colleagues (1976). The
electrode and gel buffers used were 50 mM Tris-glycine,
5 mM CaCI2, pH 9.0 at 20° C.
Apparent molecular masses were estimated using molecular
weight marker kits covering the range 14.2 to 205 kDa (MWSDS-200, MW-SDS-70L; Sigma).
T wo-dimensional
electrophoresis
First dimension (AU-PAGE) gels were rapidly stained with
Coomassie Blue and washed in several changes of water. The
lanes of interest were cut out and equilibrated to Tris HCl,
60 mM; SDS, 2%; glycerol, 10%; 2-mercaptoethanol1%;
and
Bromophenol Blue, 0.002%, pH 6.75 at 20° C, for 30 min at
25° C. The first dimension gel strip was held in position above
the second dimension gel (SDS-PAGE) by a 5% acrylamide
stacking gel. The second dimension was a 15% SDS-PAGE
resolving gel.
Peptide mapping
Myosin heavy chains (MHC) were purified by electrophoresis
on 7.5% SDS-PAGE gels. The gels were stained rapidly with
Coomassie Blue and washed in water. The MHC bands were cut
from the gel, finely chopped and equilibrated for 60 min to Tris
441
Fish contractile proteins
HCl, 50 mM, pH 7.4; SDS, 1 %. The MHCs were eluted and
concentrated electrophoretically using a tank buffer of Tris HCl,
50 mM, pH 7.5; SDS, 0.1% (Allington et al., 1978). MHCs were
digested, using either Staphylococcusaureus V8 protease or type
1-S chymotrypsin from bovine pancreas (both Sigma), with one
unit of protease per 50 ~l MHC sample. Digestion was carried
out at 25° C, for various periods, then stopped by the addition
of 2-mercaptoethanol to 2% and SDS to 2% and heating for
2 min at 100° C. The peptides produced were resolved on
10-22% linear ~radient SDS-PAGE gels.
both LCls both isofonns were expressed in single fast
muscle fibres (Fig. 6). The distribution of LCl isofonns in
0. andersoniwas studied in 3'2 individual fish. It was found
that 20 only had LClfl while the remainder expressed
both isofonns in equal proportions as detennined by
densitometric measurements. The total amounts of LCl
were similar in both groups. Breeding experiments, involving one male and three females, established that these
intra-specific variations in light chain composition were
heritable. One cross resulted in individuals with only
LClfl (ten offspring sampled). Offspring with either LClfl
Staining techniques
Gels were stCtined in Coomassie Brilliant Blue (R-250) 0.25%,
methanol 40% and acetic acid 7%, for 2 hand destained with
methanol 40% and acetic acid 7%. Silver staining was carried
out using the Sigma silver stain kit. Troponin C was identified
using the' Stains-all ' technique of Campbell and colleagues
(1983). A Shimadzu CS-9000 densitometer was used to analyse
Coomassie Blue-stained gels at 550 nm.
200
N
E
,
!
160
TNI
was isolated
affinity
column
using
a stationary
phase rabbit
troponin
C
(Syska et a/.' 1974).
80
E
"
E
.x
~
b~9
1
40
0 I
-5
I
0
I
5
Results
CONTRACTILE
PROPERTIES
The maximum Ca2+-activated tension generated (P0) by
fast muscle fibres from 0. andersoni and 0. niloticus were
similar at 25° C (Fig. 1). However, the force-temperature
relationship of fibres in the two species diverged below
17° C, such that over the range 10-00 C maximum tension
was 60% higher (P < 0.01) in 0. andersoni,the more coldtolerant species (Fig. 1). The QlOS for force generation
over the range 0-17° C were 1.4 in 0. andersoni and 1.7
in n nilnticus.
!
c 120
2
u
.-=
E
o
.~
of TNI
./'
c
0
.0
...,
IsoIation
T
'-9
T
I
10
I
15
I
20
I
25
i
30
Temperature C"C)
Fig. 1. The relationship between maximum isometric tension
generation and temperature for skinned fibres isolated from the
fast muscle of 0. andersoni ( .) and 0. niloticus ( 0).
1
2
MYOSINSUBUNITCOMPOSITION
Heavy chains (MHC)
Electrophoretically purified MHC ran as a single band on
re-electrophoresis. One-dimensional peptide maps of
myosin heavy chains produced by digestion with either
chymotrypsin or V8 protease were different for the two
species (Figs 2 and 3).
-4--
Light chains (LC)
An unexpected finding was evidence for intra-specific
variation in LClf from 0. andersoni {Fig. 4). Both of these
LCls had slightly different mobilities on SDS-P AGE gels
than LClf in 0. niloticus {molecular mass 26.5 kDa; Fig. 4).
The two LClf isoforms in 0. andersoni {LClfl and LClf2)
differed in both apparent relative molecular mass (molecular masses 27.5kDa
and 26.9kDa)
and isoelectric
point on two-dimensional
gels {Fig. 5). In individuals with
Fig. 2. Silver-stained one-dimensional peptide map of fast
muscle myosin heavy chain from 0. andersoni and 0. niloticus
digested with chymotrypsin. Lane 1, 0. andersoni; lane 2, 0.
niloticus ; arrows indicate variation between the two species. The
scale indicates the apparent molecular mass in kDa.
,0-
~
442
CROCKFORD. WOMMACK.
2
MUTUNGI
and JOHNSON
kDa
4
3
JOHNSTON. McANDREW.
60
30
LC2f
~
LC3f .
15
-'
Fig. 3. Silver-stained one-dimensional peptide map of fast
muscle myosin heavy chain from 0. andersoniand 0. niloticus
digested with V-8 protease (5. aureus).Lanes 1 and 3, 0.
andersoni;lanes 2 and 4, 0. niloticus; arrows indicate variation
between the two species. The scale indicates the apparent
molecular mass in kDa.
or LC1fl + LC1f2 were produced in two further crosses in
the ratios 5: 5 and 5: 7 (n = 22).
Myosin light chains LC2f and LC3f in the two species
had slightly different mobilities on SDS-PAGE gels (Fig.
4). The estimated relative molecular masses of LC2f were
18.3 kDa (0. andersoni) and 18.7 kDa (0. niloticus). LC3,
1
2
Fig. 5. Two-dimensional silver-stained gel of myofibrils from an
individual 0. andersoni containing both LCln and LClr2. The
first dimension was AU-PAGE in the presence of EGTA.
Abbreviations are given in the caption to Fig. 4.
had a molecular mass of about 15 kDa in both cases.Thus
fast muscles from the two species contained distinct forms
of all the myosin lip;ht chains.
I-FILAMENTPROTEINS
Tropomyosin (TM) (34-35 kDa) and troponin T (TNT)
(32.5-33.5 kDa) were resolved into double bands in both
species with similar molecular masses and pIs (Fig. 4).
Troponin C (TNC) was present as a single isoform
(confirmed by' Stains-all " gel not shown) of molecular
3
4
5
-~
LC1f
TNlf1
=
TM
=
TNT
-
-
LC2f
-
LC3f
-
Fig. 4. SDS-PAGE (10-22 %) linear gradient gel of fast muscle myofibrils from 0. andersoni and 0. niloticus stained with Coomassie
Blue. Lanes 1-5 represent different individual fish; 1, 3, and 5, 0. andersoni; 2 and 4, 0. niloticus. A, actin; TM, tropomyosin; TNT,
troponin T; LCln, fast muscle myosin light chain 1, isoform 1; LClf2' fast muscle myosin light chain 1, isoform 2; LC2f, fast muscle
myosin light chain 2; LC3f, fast muscle myosin light chain 3; TNIrl' troponin 1, isoform 1; TNlr2' troponin 1, isoform 2; TNC,
troponin C. The labels on the right refer to 0. andersoni and those on the left to 0. niloticus (where different to 0. andersoni).
~
443
Fish contractile proteins
1
1
2
TN If2 TNI f1 -
TNlf2
TNlf1
2
3
-
-TNI
~~~-Fig. 6. Silver-stained 15% SDS-PAGE gel of single fast muscle
fibres isolated from 0. andersoni. Lane 1 is of an individual
containing only LC1n; lane 2 is of an individual containing
LC1n and LC1r2. Note that both TNIr isoforms are expressed
in single fibres from both individuals.
Fig. 8. Silver-stained 15% SDS-PAGE gel of TNIr purified by
affinity chromatography. Lane 1, 0. andersoni; lane 2, 0.
niloticus; lane 3, Limanda limanda. Abbreviations are given in the
caption to Fig. 4.
-'
1
,~y
2
3
4
LC1f
TNlf2
TNlf1
-TNlf2
-T
Fig. 7. Two-dimensional silver-stained gel of myofibrils from an
individual 0. niloticus. The use of a AU-P AGE gel in the
presence of calcium in the first dimension, allowed the troponin
I bands to be seen in the second dimension (compare with Fig.
5). Abbreviations are given in the caption to Fig. 4.
mass 18 kDa in both species (Fig. 4). Two-dimensional
gels with AU-P AGE gels in the presence of Ca2+ as the
first dimension, allowed TNI to be visualized in the
second dimension (Fig. 7; compare with Fig. 5). Each
species was found to contain two isoforms of troponin I
(TNlfl and TNlf2) (Figs 4 and 7). This unusual finding was
confirmed by affinity chromatography (Fig. 8) and both
OreochromisTNI isoforms were co-expressed in single fast
muscle fibres (Fig. 6). In contrast TNI isolated from fast
muscle of dab ran as a single band (Fig. 8). The molecular
masses of TNlfl) were 20.5 kDa in 0. niloticus and
20.9 kDa in 0. andersoni. TNlf2 had a molecular mass of
22.4 kDa in both species (Fig. 4). The composition of TNI
isoforms showed intra-specific variation in 0. niloticus
(Fig. 9) Multiple samples prepared from the same indi-
N I f1
Fig. 9. SDS-PAGE (10-22 %) linear gradient gel of fast muscle
myofibrils from 0. niloticus stained with Coomassie Blue. Lanes
1-4 represent different individual fish. Abbreviations are given
in the caption to Fig. 4. .
vidual confirmed that the banding patterns seen were not
the result of variatioh in experimental procedure. Of 20
individuals examined seven had only TNIfl and 13 had
TNIfl + TNIr2. In four of 13 individuals another faint band
was observed which may correspond to an additional TNI
isoform, but this could not be positively identified.
Discussion
All the components of the tropomyosin-troponin complex
occur as isoforrns which are expressed in a tissue and/ or
developmental stage-specific manner (Wilkinson & Grand,
1978; Dhoot et al., 1978, 1979; Nakamura et al., 1989). In
444
CROCKFORD, WOMMACK,
chicken, for example, there are distinct isoforms of TNT
in slow, fast and cardiac muscle, and additional isoforms in
the fast muscle of the breast and leg (Wilkinson, 1978).
Fast myotomal muscle in Oreochromisspecies also contain
two isoforms of TNT with different molecular masses
(Fig. 4). Imai and colleagues (1986) reported around 40
isoforms of TNT to be present in the leg muscles of the
chicken. It has been shown that alternate RNA splicing of
the rat TNT gene has the potential to produce a maximum
of 64 different TNT mRNAs (Breitbart et al., 1985). In
contrast, each TNI isoform is probably encoded by a
separate gene, regulated primarily by transcriptional
mechanisms (Wilkinson & Grand, 1978; Bucher et al.,
1988). In adults TNI is thought to be specific to a
particular muscle type (Hallauer et al., 1988). Co-expression
of fast, slow and cardiac TNI isoforms are seen in the
newborn rat (Dhoot & Perry, 1980), the chicken embryo
(Toyota & Shimada, 1981) and in adult muscle following
changes in innervation (Amphlett et al., 1975; Dhoot et al.,
1981; Dhoot & Perry, 1982). An unusual finding in the
present study was the co-expression of two TNIs in single
fast muscle fibres (Fig. 6). Neither of these bands corresponded to those found in slow muscle fibres (unpublished data).
Although it would be possible to study functional
differences between the tropomyosin-troponin
complex
in the two Oreochromis species, e.g. the pCa-tension
relationships, the similar electrophoretic characteristics of
TNT, TNC and tropomyosin would make any genetic
analysis impractical.
Studies of different muscle fibre types have shown that
maximum force generation (P0) and contraction speed
(V max) are largely determined by the composition of
myosin heavy chains (Reiser et al., 1985; Lannergren,
1987), although the alkali light chains may have a
modulating role (Greaser et al., 1988). In the common carp
(Cyprinus carpio L.) both Po and V maxare increased at low
temperature following several weeks of cold acclimation
(Johnston et al., 1985; 1990). This is associated with
changes in the composition of both MHCs (Gerlach et al.,
1990) and myosin LCs (Crockford & Johnston, 1990). In
fish the maximum tensions generated at low temperature
are strongly correlated with habitat temperature (Johnston
& Brill, 1984; Johnston & Altringham, 1985). The forcetemperature relationships of fast muscle fibres in the two
Oreochromis species were found to diverge at low temperatures (Fig. 1). The higher tensions generated by fibres
from 0. andersoni below 17° C correlate with the better
survival of this species at lower temperature in the natural
habitat and in aquaculture (Trewavas, 1983).
In the roach (Rutilus rutilus) three isomyosins can be
distinguished in fast muscles by electrophoresis under
non-denaturing conditions (Karasinski & Kilarski, 1989).
This suggests there is a single isoform of MHCf which can
combine with the alkali light chains to give either LClf
and LC3f homodimers or the heterodimer (Karasinski &
Kilarski. 1989). Robert and colleaQues {1985). found no
JOHNSTON,
McANDREW,
MUTUNGI
and JOHNSON
evidence for genetic linkage between myosin light chains
betweeri two mouse species. On this basis F1 hybrids
between 0. andersoni and 0. niloticus could theoretically
contain a maximum of 18 different isomyosins.
However, an unexpected finding in the present study
was the presence of intra-specific variation in myosin
LC1r isoforms in 0. andersoni. The primary structures of
myosin alkali light chains are highly homologous, differing
only with respect to an N-terminal peptide of 41
residues rich in proline, lysine and alanine which is specific
to LC1r (Frank & Weeds, 1974). The mRNAs for LC1r and
LC3r are derived from the same gene by a process of
alternate promotor utilization and differential splicing of
two primary RNA transcripts (Nabeshima et al., 1984;
Periasamy et al., 1984). RNA splicing is catalysed by a
multi component spliceosome which contains both proteins and small RNA molecules (Maniatis & Reed, 1987).
Multiple LC1s in 0. andersoni could therefore have arisen
by allelic variation of the LC gene or within the
spliceosome. Whatever the mechanisms this would increase the number of isomyosins theoretically possible to
36. At this stage it is not known whether this intra-specific
variation in LC1r composition has any physiological
and/ or adaptive significance.
Myosin heavy and light chains from these two
Oreochromis species are readily differentiated using standard electrophoretic techniques. Using a needle biopsy
technique, sufficient tissue can be obtained to determine
both myosin composition and the contractile properties of
single muscle fibres (McAndrew, 1981). This makes
possible the use of breeding experiments to establish the
heritability of force-generating characteristics. As F1
hybrids are fertile, new strains can be produced by
backcrossing with the parental species or among hybrids
to produce strains fixed for a specific gene. This would
give rise to the possibility of investigating the contribution
of single genes or combinations of genes to the contractile
properties under study.
Acknowledgements
This work was supported by a Marine Genetics Special
Topics Grant from the Natural Environment
Research
('nuncil
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