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First published online November 30, 2007
Journal of Experimental Biology 210, 4298-4306 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.011114
Parasites, proteomics and performance: effects of gregarine gut parasites on dragonfly flight muscle composition and function
Department of Biology, 208 Mueller Lab, Pennsylvania State University, University Park, PA 16802, USA
* Author for correspondence at present address: School of Biological Sciences, 348 Manter Hall, University of Nebraska, Lincoln, NE 68588, USA (e-mail: rschilder2{at}unl.edu)
Accepted 9 October 2007
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155 kDa fragment of muscle myosin heavy chain (MHC;
206 kDa intact size). Insect MHC gene sequences contain evolutionarily
conserved amino acid motifs predicted for calpain cleavage, and we found that
calpain digestion of purified dragonfly MHC produced a peptide of 155
kDa. Thus, gut parasites in dragonflies are associated with what appears to be
a reduction in proteolytic degradation of MHC. MHC155 abundance showed a
strong negative relationship to muscle power output in healthy dragonflies but
either no relationship or a weakly positive relationship in infected
dragonflies. Troponin T (TnT) protein isoform profiles were not significantly
different between healthy and infected dragonflies but whereas TnT isoform
profile was correlated with power output in healthy dragonflies, there was no
such correlation in infected dragonflies. Multivariate analyses of power
output based on MHC155 abundance and a principal component of TnT protein
isoform abundances explained 98% of the variation in muscle power output in
healthy dragonflies but only 29% when data from healthy and infected
dragonflies were pooled. These results indicate that important, yet largely
unexplored, functional relationships exist between (pathways regulating)
myofibrillar protein expression and (post-translational) protein processing.
Moreover, infection by protozoan parasites of the midgut is associated with
changes in muscle protein composition (i.e. across body compartments) that,
either alone or in combination with other unmeasured changes, alter muscle
contractile performance.
Key words: dragonfly, infection, muscle performance, muscle protein composition, myosin heavy chain, troponin T
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;
Schilder and Marden, 2004) and
how this variation affects the outcome of aerial competition in nature
(Marden and Cobb, 2004).
Recently, we have found that infection of the dragonfly midgut by gregarine
parasites (Protozoa: Apicomplexa) is an important contributor to variability
in muscle performance. Gregarine infection causes decreased male territorial
and mating success in odonates (Plaistow
and Siva-Jothy, 1996; Marden
and Cobb, 2004) and causes impairment of in vitro muscle
lipid metabolism and power output
(Schilder and Marden, 2006).
Here, we explore molecular mechanisms underlying some of these phenotypes by
examining how gregarine infection affects flight muscle protein composition
and its relation to flight muscle contractile performance.
Previous mechanistic examinations of parasite effects on muscle have been
restricted to specialized parasites such as Trichinella spiralis that
directly infect muscle cells (Jasmer,
1990; Jasmer and Kwak,
2006), causing local pathology. Here, we take the different
approach of examining a systemic effect of non-invasive parasites residing in
the gut. Our results show that dragonfly skeletal muscle is sensitive and
responsive to the presence of parasites in other tissues, with specific
effects on muscle protein composition.
Effects of parasites on muscle performance are best examined in the context
of the dynamic and versatile nature of healthy skeletal muscle. Skeletal
muscle consists of cytoskeletal networks
(Clark et al., 2002) that
provide the foundation for contractile activity. Structural integrity and
quality of cytoskeletal networks are maintained through continuous protein
turnover – a balance between protein degradation and synthesis
(Rooyackers and Nair, 1997;
Mykles, 1998). In addition,
contractility depends on the types of myofibrillar proteins expressed in
sarcomeres (i.e. protein isoform composition). Both protein turnover and
isoform composition are sensitive to changes in nutritional status and
exercise intensity (White et al.,
2000; Ferrando et al.,
2002; Wackerhage and Rennie,
2006) and are affected by aging and disease (e.g.
Short and Nair, 2001;
Nair, 2005;
Du et al., 2004). Proteomic
studies of muscle contractility to date have focused exclusively on protein
composition but not at all on proteolytic degradation of proteins, the latter
of which is a likely first-order indicator of cytoskeletal integrity and/or
turnover. In the present study, we examine metrics of both protein degradation
and isoform composition that, for healthy dragonfly flight muscle, provide
strong explanatory power for variability in contractile performance.
In vertebrates, fibers containing different myosin heavy chain (MHC)
protein isoforms have different metabolic and mechanical characteristics, and
their relative abundance in a muscle fiber determines speed of contraction and
metabolic profile [i.e. fiber type designation; slow or fast, glycolytic or
oxidative (Pette and Staron,
2001)]. Shortening velocity and Mg2+-ATPase activities
vary with MHC protein isoform composition
(Bottinelli et al., 1991;
Bottinelli et al., 1994) and,
for many muscles, fiber type distributions change in response to exercise,
with age and during disease.
In the best-studied insect, Drosophila, alternative mRNA splicing
of a single MHC gene gives rise to at least 14 mRNA transcripts
(Vigoreaux, 2001). However,
only one MHC protein isoform is expressed in Drosophila flight
muscles (Bernstein et al.,
1986) and little is known about MHC isoform composition of flight
muscles of any other insects; this raises the question of how insects regulate
and vary muscle performance.
One mechanism that insects use to accomplish plasticity of muscle
contractile performance is alternative splicing of mRNA coding for the
myofibrillar protein troponin T (TnT). Alternative splicing of TnT in
Libellula pulchella Drury dragonflies (Odonata: Anisoptera) produces
seven mRNA transcripts, six of which are found in flight muscles. The relative
abundance of particular TnT mRNA transcripts correlates positively with muscle
fiber Ca2+ sensitivity, in vitro muscle performance and
kinematic measures of in vivo flight performance
(Marden et al., 1999;
Marden et al., 2001), but the
relationship between TnT splice form variation and contractility has not yet
been examined quantitatively at the protein level.
In vertebrates, variation in TnT isoform composition of muscle fiber types
corresponds tightly with variation in MHC isoform identity, indicating that
MHC expression and TnT alternative splicing are co-regulated
(Galler et al., 1997). MHC and
TnT expression profiles have not been examined together in studies of insect
muscle contractile performance. Here, we use two-dimensional polyacrylamide
gel electrophoresis (2-D PAGE) and MALDI-TOF mass spectrometry to quantify
expression of TnT isoforms and the abundance of an identified product of MHC
proteolytic degradation in dragonfly flight muscle. We compare these metrics
in healthy and infected dragonflies and their relationship to in
vitro flight muscle performance.
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).
Directly after flight muscle performance measures (see below), muscle tissues
were stored in liquid nitrogen until further analyses were performed. For none
of the parameters examined here did we observe a dosage effect based on the
number of gregarines present in the midgut, which is why comparisons are
between healthy and infected groups.
2-D PAGE
For electrophoretic analyses, we chose a subset of basalar muscle samples
that spanned the variation observed in a previous study in which we measured
muscle power output and gregarine infection status
(Schilder and Marden, 2006). A
total of 17 muscle protein samples was prepared and separated using 2-D PAGE.
Sample preparation was performed according to instructions provided with a
MK-1 tissue preparation kit (Kendrick Labs, Madison, WI, USA). Basalar muscles
tested for contractile performance were dissected out and homogenized in
osmotic lysis buffer [10 mmol l–1 Tris, pH 7.4, 0.3% sodium
dodecyl sulfate (SDS)] containing 1% protease inhibitor cocktail [20 mmol
l–1 4-(2-aminoethyl) benzenesulfonyl fluoride, 1 mg
ml–1 leupeptin, 0.36 mg ml–1 E-64, 500 mmol
l–1 EDTA and 5.6 mg ml–1 benzamidine].
Homogenates were heated at 95°C in SDS boiling buffer (5% SDS, 5%
β-mercaptoethanol, 10% glycerol and 60 mmol l–1 Tris, pH
6.8). Samples were then shipped to Kendrick Labs for 2-D PAGE separation.
Isoelectric focusing (IEF) was performed in a glass tube (inner diameter 2 mm)
using 2.0% pH 3.5–10 ampholines (Amersham Pharmacia Biotech, Piscataway,
NJ, USA) for 9600 Vh (volt-hours). After equilibration in a buffer containing
10% glycerol, 50 mmol l–1 dithiothreitol, 2.3% SDS and 0.0625
mol l–1 Tris, pH 6.8, the tube gel was sealed to the top of a
stacking gel that overlaid a 10% acrylamide slab gel (0.75 mm thick). SDS slab
gel electrophoresis was carried out for 4 h at 12.5 mA/gel. Molecular mass
standards varying from 14 to 220 kDa were added to the gel. Gels were stained
with Coomassie Brilliant Blue R-250 to allow protein identification and
quantification. For more details on methodology, refer to
http://www.kendricklabs.com/about2d.htm.
Protein spot identification and quantification
Protein spot profiles were analyzed from scanned 2-D gel images using
PD_Quest analysis software (Bio-Rad, Hercules, CA, USA). Matching of spots
across gels and measurement of spot size in relation to total size of all
spots within gels indicated that quantities for protein Spot 6
(Fig. 1), in particular, varied
strongly between muscle samples from healthy and infected groups. The identity
of Spot 6 was therefore examined using MALDI-TOF mass spectrometry followed by
peptide mass fingerprinting. Coomassie-stained protein spots were excised from
the gels, transferred to microcentrifuge tubes and destained by two
alternating washes of 50 mmol l–1 aqueous ammonium
bicarbonate and 100% acetonitrile. In-gel digestion was performed by addition
of 0.1 µg of trypsin (Promega, Madison, WI, USA), with digestion proceeding
at 25°C for 24 h. Digestion products were extracted by washing three times
with 1% formic acid in 50/50 (v/v) acetonitrile/water, and all extracts were
combined and concentrated nearly to dryness using a Savant SpeedVac (Savant
Instruments Inc., Farmingdale, NY, USA). The residue was diluted by addition
of 50 µl of 0.1% aqueous trifluoroacetic acid (TFA), and peptides were
desalted using a C18 ZipTip (Millipore, Billerica, MA, USA) and eluted
according to the manufacturer instructions. Digestion products were spotted
onto the MALDI plate using alpha-cyano-4-hydroxycinnamic acid as matrix.
Peptide mass mapping was performed on a Voyager-DE STR MALDI-TOF mass
spectrometer (Applied Biosystems, Foster City, CA, USA) in delayed-extraction
reflectron mode using trypsin autoproteolysis peaks as internal mass
references (D. Jones, personal communication).
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Tryptic digestion and MALDI-TOF analysis produced peptides whose masses
were compared against entries in database NCBI nr.12.16.2006 constrained to
D. melanogaster using MS-Fit routines within ProteinProspector
(http://prospector.ucsf.edu/)
(Clauser et al., 1999). One
missed cleavage was allowed, and peptide mass tolerance was set to 50 p.p.m.
Cysteines were assumed unmodified, and default amino acid modification mode
was used.
The location of 4-5 TnT protein isoforms on 2-D gels was identified based
on previous work that used a combination of gel electrophoresis and western
blotting with an antibody against TnT
(Marden et al., 1999). In the
present study, in addition to spot 6, we examined normalized quantities for
five TnT protein isoforms (Spots 1–5 in
Fig. 1).
Proteolytic digestion of purified dragonfly MHC
To further examine the identity of Spot 6 (a proposed MHC degradation
product; see Results), L. pulchella MHC was purified and digested
with mammalian calpain (rabbit skeletal muscle calpain 80 kDa subunit; P4533
Sigma-Aldrich, St Louis, MO, USA). Preparation of purified MHC was similar to
that described previously (Johnson and
Bennett, 1995). Briefly, basalar muscles were dissected out,
homogenized in 0.5 mol l–1 KCl, 0.03 mol l–1
NaHCO3, pH 7.0 and incubated for 20 min at 4°C. Homogenates
were centrifuged at 10 000 g for 10 min at 4°C, after
which the pellet was discarded. Supernatants were rapidly diluted in 1:15 v/v
ice-cold ddH2O. Suspensions were centrifuged at 10 000
g for 10 min, after which supernatants were discarded. Pellets
were suspended in 1 mol l–1 KCl and incubated for another 20
min at 4°C, centrifuged at 10 000 g for 10 min and rapidly
diluted in 1:15 v/v ddH2O. Suspensions were centrifuged one last
time at 10 000 g, for 10 min, after which the supernatants
were discarded and the pellet, now enriched in myofibrillar proteins, was
dissolved in 1x gel buffer.
MHC purification from the enriched myofibrillar fraction was accomplished
by separation using SDS-PAGE (4% stacking, 7.5% separating gel). Gels were
briefly (30 s) stained with Coomassie Blue to visualize proteins and then
washed with ddH2O. MHC bands (200 kDa) were cut out using a
razor blade. Excised bands were finely chopped and homogenized in equal-volume
50 mmol l–1 Tris-HCl, 0.5% SDS, incubated at room temperature
for 1 h, centrifuged for 15 min (10 000 g), after which
supernatants were carefully removed.
Purified MHC samples were digested using a 0.3 mg ml–1
calpain [and 90 µl calpain activating solution; preparation according to
(Lakey et al., 1993)] solution
for 45 min at 25°C. Digestions were stopped by adding equal-volume sample
buffer and heating at 95°C for 2 min. Resulting peptides were resolved on
a 4–20% SDS-PAGE gel and silver stained (Bio-Rad).
Flight muscle performance
Animals were used in experiments within 4–6 h of capture. Maximum
muscle mass-specific power output of the first basalar muscle was determined
using a modification of the work-loop technique
(Josephson, 1985) described
previously (Marden et al.,
2001; Schilder and Marden,
2004). Importantly, maximum muscle power output represents
performance during imposed conditions that approximate in vivo
contraction regimes. Muscle power output results were reported in an earlier
publication (Schilder and Marden,
2006) and are used here to examine how power relates to muscle
protein composition.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Gelfi et al.,
2006). We therefore further examined the identity of Spot 6.
A group of Ca2+-dependent cysteine proteinases known as calpains
are implicated in the controlled degradation of myofibrillar proteins (e.g.
Kim et al., 2005). Pemrick and
Grebenau reported that proteolytic activity by calcium-activated neutral
protease (CANP; also known as calpain) in the head region of 200 kDa MHC
from rabbit muscle produced 180, 165 and 150 kDa sized MHC peptides
(Pemrick and Grebenau, 1984).
Similarly, digestion of 200 kDa Octopus MHC by endogenous
calpain-like proteinase CaDP results in a 155 kDa proteolytic product
(Hatzizisis et al., 2000). A
recent study indicated that protein digestion site preference of vertebrate
calpains is highest where the amino acids lysine (K) and serine (S) occur at
the P1 and P1' position (i.e. the amino acid positions between which
calpain digests a protein) across a wide range of proteins
(Tompa et al., 2004). In the
Drosophila muscle MHC head region, there are two such sites
(Lys559–Ser560 and
Lys611–Ser612) at which theoretical digestion by
calpain results in 161 kDa and 155 kDa (calculated using tools at
http://pir.georgetown.edu/pirwww)
proteolytic products, respectively (Fig.
2A). Interestingly, all 19 peptide matches obtained from MALDI-TOF
analyses of Spot 6 align within these two theoretical digestion products of
Drosophila MHC (i.e. the regions marked black in
Fig. 2). Moreover, the location
of the two calpain digestion sites in the MHC head region appears to be
conserved in insects (Fig. 2B).
This led us to hypothesize that Spot 6 on our 2-D gels represents a calpain
MHC degradation product.
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Dragonfly (muscle) calpain has not been characterized, but muscle calpains
share significant identity across phyla [i.e. invertebrates and mammals
(Kim et al., 2005)]. We
therefore used a mammalian skeletal muscle calpain (see Materials and methods;
this rabbit skeletal muscle calpain 80 kDa catalytic subunit aligns with up to
43% amino acid identity and 62% similarity over its entire 422 amino acid
length with the deduced amino acid sequence of a number of insect and
crustacean calpains; tBLASTn, E value=1x10–92) to
digest purified dragonfly MHC and examine the size of peptides generated. The
collection of peptides obtained after digestion of purified dragonfly MHC with
rabbit calpain is visualized in Fig.
3B. Three of the bands are likely to be in vitro calpain
degradation products of purified dragonfly MHC, with a prominent 156 kDa
band showing molecular mass similarity to our Spot 6
(Fig. 1). The numerous lower
molecular mass bands observed in Fig.
3B appear to be the result of calpain autodegradation, as observed
previously (Pemrick and Grebenau,
1984).
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Myofibrillar protein expression comparison
We found significantly higher mean values (P<0.001) for the
(log10-transformed) abundance (normalized quantities) of MHC155 for
healthy than for infected individuals (0.60±0.22 and
–0.69±0.34, respectively)
(Fig. 4A). This finding
indicates that infection is associated with a significant decrease in
dragonfly flight muscle MHC degradation.
Quantitative variation in TnT isoform composition was not affected by infection to the degree that MHC155 was, as we found no significant infection-related differences (P=0.36) between the means of the first principal component (PC1) of variation in relative abundance of TnT protein isoforms (1.05±1.59 and 0.51±0.81 for healthy and infected, respectively) (Fig. 4B). Rather, TnT isoform composition appears to vary widely (as demonstrated by relatively large standard deviations) within both healthy and infected groups.
Flight muscle performance
We found previously that there is a significant difference in maximum
flight muscle in vitro mechanical power output between healthy and
infected dragonflies (148.6±23.1 and 117.1±32.0 W
kg–1, respectively; P=0.0002, N=52)
(Schilder and Marden, 2006).
In the current study, we used a subset of those muscles that spanned the range
in observed power output. Among these 17 individuals, muscle power output
varied from 88 to 184 W kg–1 (N=6) in healthy
dragonflies and from 93 to 164 W kg–1 (N=11) in
infected dragonflies. A Student's t-test revealed no significant
difference for the contraction frequency at which maximum muscle power output
was achieved for healthy versus infected dragonflies (40.3±1.5
Hz and 40.8±1.1 Hz, respectively; P=0.80).
Relating protein expression to muscle performance
Flight muscle performance of healthy individuals was to a large extent
predicted by both MHC155 (Fig.
5A) and PC1 TnT (Fig.
5B). Within the healthy group, both MHC155
(r2=0.85, P=0.0089) and PC1 TnT
(r2=0.79, P=0.018) were negatively correlated
with muscle performance. This was not true for infected individuals. No
significant correlation was found between muscle performance and either MHC155
(Fig. 5C)
(r2=0.26, P=0.106) or PC1 TnT
(Fig. 5D)
(r2=0.01, P=0.73) for infected individuals.
When data for healthy and infected individuals were combined, we found a significant convex relationship (r2=0.38, P=0.03) between muscle performance and MHC155 abundance (Fig. 5E). Low-powered healthy dragonflies showed increased muscle MHC155 whereas low-powered infected individuals had decreased MHC155 abundance. This result suggests that, with respect to flight muscle performance, there is an optimum MHC degradation rate. However, because our methods require destructive sampling, we lack time series data (i.e. muscle power vs MHC155 abundance over time) necessary to evaluate that hypothesis.
For healthy and infected individuals combined (Fig. 5F), PC1 TnT explained 31% of the variation in muscle performance (P=0.021). Although data for TnT expression failed to explain variation in muscle performance among infected individuals separately (Fig. 5D), they fit along the same trend found for healthy individuals. Thus, flight muscle performance declines with increasing PC1 TnT and this relationship was not significantly affected by infection, other than to perhaps increase variation around the central tendency.
For healthy individuals, a multiple linear regression model including both MHC155 abundance and PC1 TnT explained 97.9% of variation in flight muscle power output (Fig. 6A) (P=0.0014). Both predictors significantly contributed to the explanatory power of this model (see Table 1; log10 %MHC155, P=0.0061; PC1 TnT, P=0.010). Although still significant (adjusted r2=0.29, P=0.034), this model lost much of its explanatory power (98.7% to 38.3%) when data for infected individuals were included (Fig. 6B), and log10 %MHC155 was no longer a significant predictor (see Table 1; P=0.21). Because infection did not significantly affect TnT protein isoform composition (Fig. 4B, Fig. 5F), we conclude that infection reduces flight muscle contractile function primarily by affecting MHC degradation.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Our results indicate that an important relationship exists between
dragonfly flight muscle MHC degradation and TnT protein isoform composition.
For healthy dragonflies, these two parameters combined to explain almost all
(i.e. 98%) variation in muscle contractile performance (albeit in a small
sample). Thus, combining metrics of different aspects of muscle cytoskeletal
composition and quality may provide higher explanatory power than limiting
studies to only protein isoform expression, or to only protein turnover and
cytoskeletal integrity.
It is important to note that we cannot be certain that the observed changes in MHC155 abundance and TnT protein isoform expression are the causative mechanisms for the observed variation in muscle power output, as these proteins may be markers for changes in a suite of co-regulated genes and/or proteins, including large sarcomeric proteins such as projectin that are not resolved on standard protein gels. However, we observed no apparent suite of correlated spot changes on our 2-D gels. Both MHC155 abundance and TnT isoform composition varied over a wide range, independently of each other and other proteins (Figs 1, 4).
MHC degradation
Based on 2-D gel spot identification by means of MALDI-TOF mass
spectrometry, western blotting using MHC antibody, and calpain digestion
experiments, we determined that MHC155 is most likely a degradation product of
MHC and therefore a possible indicator of MHC turnover in dragonfly flight
muscle. However, since we have not identified or examined any process in
vivo relating to changes in MHC turnover, this conclusion and the
following discussion are necessarily somewhat speculative.
Low-powered healthy individuals exhibited greater amounts of MHC155 (see
Fig. 5A) and therefore perhaps
a higher myosin degradation rate than high-powered healthy individuals. One
hypothesis is that the amount of MHC degradation may be related to the
nutritional status of dragonflies. Low nutritional status may induce increased
MHC proteolysis to mobilize amino acids for use as energy substrates. This
could create a net loss of MHC in the flight muscles and a reduction in
contractile performance. Indeed, low-energy (fasting) conditions have been
associated with increases in muscle proteolysis in rats
(Kettelhut et al., 1994) and
increased expression levels of calpains responsible for myofibrillar
degradation in rainbow trout (Salem et
al., 2005).
Alternatively, variation in abundance of MHC155 may be a result of age
differences within the healthy group of dragonflies. Aging vertebrate muscle
is commonly characterized by the loss of muscle function
(Stuerenburg et al., 2006;
Karakelides and Sreekumaran Nair, 2005) and imbalances between myofibrillar
protein synthesis and degradation (e.g.
Yarasheski, 2003). The
observed changes in MHC155 abundance were an unanticipated result and
therefore we did not obtain measures of nutritional status or age for the
individuals used in this study. We highlight these possibilities for the
purpose of shaping future hypotheses rather than as well-supported
explanations of the present results.
TnT isoform expression
Variability in TnT protein isoform composition is at least partly
determined by alternative splicing of the troponin-t gene. This
mechanism drives variability in the relative abundance of distinct
troponin-t mRNA transcripts, which has previously been shown to
correlate with muscle contractile and flight performance
(Fitzhugh and Marden, 1997;
Marden et al., 1999;
Marden et al., 2001). The
relationship between troponin-t splicing and muscle performance is
strengthened by the present results, which demonstrate for the first time a
quantitative relationship between muscle power output and TnT isoform
composition at the protein level (Fig.
5B,F).
The first principal component of TnT protein isoform composition had a
strong positive correlation with the two most abundant TnT spots (i.e. Spots 2
and 3), as indicated by their component loading scores
(Table 2). The most abundant
TnT spot (i.e. Spot 2) showed a negative correlation with muscle power output
(results not shown), which is similar to findings for RNA level analyses in
which the troponin-t transcript that had the highest relative
abundance also had a negative relationship with muscle power
(Marden et al., 2001). As
explained in that paper, relative abundances of isoforms are interrelated,
with increases in relative abundance of particular isoforms necessarily
accompanied by decreases in relative abundance of others. For that reason, it
remains difficult to determine if variation in contraction is affected by
increases in isoforms that promote contractility or by decreases in isoforms
that inhibit contractility.
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Effects of gregarine infection
Natural infection by gregarines of the dragonfly midgut impairs flight
muscle contractile performance and disrupts the apparent relationship between
MHC155 abundance and muscle power output. This infection-associated change in
muscle protein composition appears to be a specific effect on myosin rather
than general effects across all muscle proteins, as evidenced by the overall
similarity of 2-D gel spot patterns and the fact that PC1 TnT does not
markedly differ between healthy and infected individuals, whereas MHC155
abundance decreases radically (Fig.
4A).
We previously reported that gregarine infection causes chronic inflammation
of L. pulchella flight muscles and metabolic disturbances that
include a switch in substrate utilization towards glycolytic metabolism, loss
of lipid oxidation ability and deposition of lipid around flight muscles, and
an impaired response to exogenous insulin
(Schilder and Marden, 2006).
These findings are particularly interesting with respect to the decrease in
MHC degradation found for infected individuals. Mammalian obese and
insulin-resistant phenotypes that are commonly associated with chronic
inflammation (Wisse, 2004)
have recently been linked to a decreased expression of calpain 3
(Walder et al., 2002).
Calpain 3 expression is specific to skeletal muscle and is thought to
be involved in myofibrillar protein degradation during muscle remodeling
(Kramerova et al., 2005) and
metabolic signaling pathways (Walder et
al., 2002). In light of these mammalian symptoms, and based on the
assumption that MHC155 is a calpain degradation product, we tentatively
hypothesize that decreased expression of a calpain ortholog in dragonflies
underlies the observed decrease in MHC degradation that is associated with
infection-induced metabolic disease.
Alternatively, the decrease in MHC155 in muscle of infected dragonflies
might be a result of parasite-induced changes in behavior and muscle usage.
Flight muscles of L. pulchella males infected with gregarines are
unable to oxidize lipids and they tend to switch from energetically costly
territorial behavior to a cheaper (much less flying) `satellite lifestyle'
(Marden and Cobb, 2004). This
behavioral change may be directly linked to the muscle's inability to use
lipid fuels (Schilder and Marden,
2006), and reduced flight duration and intensity may decrease the
rate of myosin turnover. In this light, the differences in MHC degradation
between healthy and infected individuals could be an effect of training or
recent experience.
Perspective
Skeletal muscle is an intricate and precise machine that is capable of
modulating its performance to very subtle changes in physiology
(Clark et al., 2002). An
emerging picture from our research is that, in addition to changes in TnT
splicing that serve to adjust muscle contractile performance
(Marden et al., 2001),
dragonfly flight muscles also show functionally important variation in MHC
processing that varies according to parasite infection and combines with TnT
isoform profile to explain most of the variation in muscle performance in
healthy dragonflies. Presence of non-invasive protozoans in the midgut lumen
causes dramatic changes in muscle energy metabolism, protein composition, a
molecular marker of inflammation, and behavior. Metabolic pathologies of
infected dragonflies are highly similar to those associated with mammalian
metabolic syndrome and obesity (Schilder
and Marden, 2006), and in the present study we find another
parallel – a decrease in myosin degradation that may be caused by a
reduction in calpain expression, as has been reported in obese and
insulin-resistant mammals (Walder, 2002).
The effect of parasites on insect muscle is a nascent avenue of research with many uncertainties yet to be resolved, but an emerging picture is that the pathologies observed are highly congruent to those that occur in mammalian metabolic diseases. Invertebrates provide accessible systems for studying basic biology of muscle and yield results and hypotheses that may compliment, inform and stimulate research on muscle biology and metabolic diseases in general.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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