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J Venom Res, 2011, Vol 2, 1-5
RESEARCH REPORT
TTX, cations and spider venom modify avian muscle tone
in vitro
Volker Herzig
α,
*, Wayne C Hodgson
β
and Edward G Rowan
γ
α
Division of Chemical and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Building 80,
Service Road, St Lucia, QLD 4072, Australia,
β
Monash Venom Group, Department of Pharmacology, Monash University,
Clayton, Victoria 3800, Australia,
γ
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde,
Glasgow, G4 0NR, United Kingdom
*Correspondence to: Volker Herzig, Email: schnakenstich@yahoo.com, Tel: +61 7 3346 2014, Fax: +61 7 3346 2101
Received 21 December 2010; Accepted 02 January 2011; Published online 02 January 2011
© Copyright The Author(s): Published by Library Publishing Media. This is an open access article, published under the
This license permits non-commercial use, distribution and reproduction of the article, provided the original work is
appropriately acknowledged with correct citation details.
ABSTRACT
Agents that reduce skeletal muscle tone may have a number of useful clinical applications,
e.g.
, for muscle
spasticity and other muscle disorders. Recently, we reported that the venoms of two species of Australian
theraphosid (Araneae, Theraphosidae) spiders (
Coremiocnemis tropix
and
Selenotholus foelschei
) reduced the
baseline tension of chick
biventer cervicis
nerve-muscle preparation. The purpose of this study was to deter-
mine the underlying physiology mediating the change in muscle tension, which was addressed by conducting
isometric tension experiments.
We found that MgCl
2
(20mM), CaCl
2
(20mM), tetrodotoxin (1µM) or
C. tropix
venom (2µl/ml) produced a similar decrease in baseline tension, whereas d-tubocurarine (100µM), gadolinium
(1mM), verapamil (10mM), an increase in osmotic pressure by the addition of glucose (40mM), or the pres-
ence/absence of electrical stimulation did not produce a signifi cant change in baseline tension.
We suggest that
mechanosensitive or muscle TTX-sensitive sodium channels are activated during muscle stretch. This may have
implications for the treatment of stretch induced muscle damage.
KEYWORDS:
Theraphosidae,
Coremiocnemis tropix
, venom, baseline muscle tension, chick
biventer cervicis
nerve-muscle preparation
INTRODUCTION
the venom of
Leiurus quinquestriatus
(Sontheimer, 2008;
Orndorff and Rosenthal, 2009). In common with scorpion
venoms spider venoms contain a myriad of peptide and non-
peptide ion channel toxins that can block or activate voltage
dependent ion channels (Estrada et al, 2007) and are recog-
nised as a source of bioactive molecules that can be used to
identify therapeutic targets, elucidate mechanisms of action
and design novel pharmaceutical drugs. In a recent study, we
reported that bath application of venoms from the Australian
theraphosid (Araneae, Theraphosidae) spiders
Coremiocne-
mis tropix
(Herzig and Hodgson, 2009) and
Selenotholus
foelschei
(Herzig and Hodgson, 2008) on the isolated chick
biventer cervicis
nerve-muscle preparation cause a signifi -
cant reduction in the resting (10mN) baseline muscle ten-
sion. This effect has not been reported before, and agents
that relax skeletal muscle have the potential to be of medical
value in the treatment of muscle spasticity and other muscle
disorders. The present study therefore aims to elucidate the
Animal venoms contain numerous pharmacologically active
peptides and enzymes that can disrupt the normal physiol-
ogy of cells. Snake venoms can affect the clotting cascade,
bind to receptors at the neuromuscular junction and have
the ability to target nerve terminals, alter the propagation of
the nerve terminal action potential and disrupt neurotrans-
mitter transmitter release (Kini, 2005; Hodgson et al, 2007;
Servent and Fruchart-Gaillard, 2009). Scorpion venoms
contain numerous peptides that interact with a large range
of ion channels and receptors (Billen et al, 2008; Prestipino
et al, 2009). A number of these peptides have been identifi ed
to selectively block insect ion channels (de Lima et al, 2007),
thus having the potential to be developed as novel insecti-
cides. Additionally scorpion venoms have been used therapeu-
tically to treat, and when conjugated to an appropriate ligand,
to visualise tumors of the CNS,
i.e.
, chlorotoxin isolated from
©The Author(s)
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1
mechanisms underlying the observed reduction in muscle
tone induced by
C. tropix
venom by mimicking the venom-
induced relaxation using other pharmacological or physi-
ological agents. Besides
C. tropix
venom, we have tested
gadolinium (antagonist of mechanosensitive channels),
verapamil (antagonist of voltage-gated calcium channels),
d-tubocurarine (d-TC, antagonist of nicotinic acetylcholine
receptors), tetrodotoxin (TTX, antagonist of voltage-gated
sodium channels), MgCl
2
and CaCl
2
(both to increase the
extracellular ion concentrations), and glucose (to increase
osmotic pressure). A separate control experiment was used
to examine whether the presence/absence of electrical stim-
ulation had any effect on the baseline muscle tension.
was turned on (n = 6) or off (n = 8). In case of the muscles
that received the electrical stimulation of the motor nerves,
only the baseline tension in the 10sec interval between
twitches was analysed to allow for comparability with the
non-stimulated muscle.
Drugs and chemicals
d-TC and TTX were obtained from Sigma Chemical Co
(St Louis, MO, USA). Gadolinium, verapamil, MgCl
2
(20mM), CaCl
2
(20mM), and glucose were supplied by
Sigma-Aldrich Company Ltd (Dorset, England). Stock
solutions of drugs were made up in Milli-Q water unless
otherwise stated.
MATERIALS AND METHODS
Statistical analysis
Sigma-Plot 11.0 (Systat Software Inc., San Jose, California,
USA) was used for all statistical analysis. The baseline
muscle tension values for control and treatment for each
group were compared by using either a t-test or (in cases
where the normality test failed) a Mann-Whitney rank sum
test. For all tests, signifi cance levels were set to 0.05 and
data are expressed as mean minus standard error of the
mean (S.E.M.).
Venom collection
The venom from female
C. tropix
spiders was collected by
applying electrostimulation to the venom apparatus using
a recently described method (Herzig and Hodgson, 2009).
Isolated chick biventer cervicis nerve-muscle
preparation
We used the chick
biventer cervicis
nerve-muscle prepara-
tion according to our previous method (Herzig and Hodgson,
2008) with some modifi cations. Briefl y,
biventer cervicis
muscles were removed from male chicks (8-15 days old),
mounted in glass (10ml) organ baths and maintained at
34ºC under 1g resting tension in a physiological saline
solution of the following composition: 118.4mM NaCl,
4.7mM KCl, 1.2mM MgSO
4
, 1.2mM KH
2
PO
4
, 2.5mM
CaCl
2
, 25mM NaHCO
3
, 11.1mM D-glucose and bubbled
continuously with 95% O
2
+ 5% CO
2
to maintain the pH
between 7.2 - 7.4. Isometric contractions were measured
via a Grass transducer (FTO3) connected via either one
or two Quad Bridge modules (ADInstruments, Australia)
to a Powerlab 4/20, MacLab/4s or MacLab/8e system (all
ADInstruments, Australia). Twitches (where applicable)
were evoked by stimulating the motor nerve (supramaxi-
mal voltage, duration 0.2ms, 0.1Hz) via silver electrodes
connected to a Grass S88 stimulator. Nerve-mediated
(indirectly evoked) twitches were confi rmed by the addi-
tion of d-TC (10µM) a blocker of postsynaptic nicotinic
acetylcholine receptors. Muscles that did not respond to
acetylcholine, carbachol and KCl were excluded from
this study.
RESULTS AND DISCUSSION
Physical stretch of muscle
We have previously shown that the venom from female
C. tropix
spiders
not only reduced the baseline muscle
tension but also produced an irreversible block of nerve-
evoked twitch tension on
biventer cervicis
nerve-muscle
preparations (Herzig and Hodgson, 2008). A possibility to
explain the reduced muscle tension therefore would be that
the omission of the electrically induced twitches resulted
in a relaxation of the muscle. Stretching of skeletal mus-
cles results in a number of physiological effects (
e.g.
, to
activate the contractile apparatus resulting in an increase of
the force of contraction or to increase transmitter release)
all whose end point is to elevate cytosolic calcium ions (see
Rosenberg, 2009
for a recent review on calcium entry in
skeletal muscle). Stretching of the muscle deforms both
the cytoskeleton and cell membrane and these changes in
shear forces are thought to cause ion channels and recep-
tors to undergo conformational changes and open or acti-
vate. By blocking the muscle from being stretched during
electrically induced twitches,
C. tropix
venom might have
induced a decrease in the cytosolic calcium levels, lead-
ing to a decrease in muscle tension. This hypothesis, how-
ever, is not substantiated by our data (Figure 1A and B) that
show that the absence of nerve-evoked twitches does not
induce the muscle to relax signifi cantly more than the time-
matched control that received the nerve-evoked twitches.
We also confi rm our earlier observation on avian nerve-
muscle preparations that
C. tropix
venom signifi cantly
reduces resting tension when the tissue was subjected to
a resting tension of about 10mN (Figure 1C) (Herzig and
Hodgson, 2009).
In order to minimise variation, the same muscles were used
for control and treatment (consisting of various pharmaco-
logical or physiological agents). Hence, each muscle fi rst
received 10µl/ml Milli-Q water as control and was monitored
over 60min. Without receiving a washout, the baseline was
then manually re-adjusted to the initial starting value and
the respective treatment was applied immediately before the
muscle was monitored for another 60min. This treatment
schedule allowed each muscle to act as its own control. Each
muscle was only used for a single treatment. The applied
treatments consisted of
C. tropix
venom (2µg/ml, n = 5),
gadolinium (1mM, n = 7), verapamil (10mM, n = 4), d-TC
(100µM, n = 6), TTX (1µM, n = 6), MgCl
2
(20mM, n = 17),
CaCl
2
(20mM, n = 6), and glucose (40mM, n = 4). A sepa-
rate control experiment was carried out to test for effects on
the baseline muscle tension when the electrical stimulation
At frog neuromuscular junctions
it was demonstrated
that stretch of skeletal muscles induces an increase in
spontaneous and evoked transmitter release mediated by
mechanical tension on synaptically located membrane
©The Author(s)
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Figure 1.
Baseline muscle tension values (inclding S.E.M.) after various treatments as recorded using a chick
biventer cervicis
nerve-
muscle preparation. No effect of the stimulation of the motor nerve on the baseline muscle tension could be observed when tested under
the presence (
A
) and absence (
B
) of electrical stimulation of the motor nerve. In the case of
A
, only the baseline tension in between
twitches was analysed to allow for comparability with the non-stimulated muscle. All of the remaining treatments were carried out in
the absence of electrical stimulation. These treatments include
C. tropix
venom (2µg/ml,
C
), d-TC (100µM,
D
), gadolinium (1mM,
E
),
verapamil (10mM,
F
), TTX (1µM,
G
), MgCl
2
(20mM,
H
) and CaCl
2
(20mM,
I
), and glucose (40mM,
J
). Signifi cant differences are
indicated by *(P < 0.05) and **(P < 0.01).
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bound integrins (Chen and Grinnell, 1995). It has been sug-
gested that this effect is limited to the frog neuromuscular
junction (Grinnell et al, 2003),
as
transmitter release is not
signifi cantly changed when adult rat diaphragm neuromus-
cular junctions were subjected to stretch. The effects of
stretch on transmitter release on avian neuromuscular junc-
tions has not been adequately studied, but if stretch increased
transmitter release, it would be predicted to result in the acti-
vation of postsynaptic receptors and subsequent depolarisa-
tion of the multiply innervated muscle fi bres to generate a
sustained tonic contracture. As the nicotinic acetylcholine
receptor antagonist d-TC (Figure 1D) did not signifi cantly
reduce baseline tension, we assume that despite the evidence
for stretch-induced increased transmitter release in the frog,
block of augmented acetylcholine release from stretched
avian neuromuscular junctions cannot account for the reduc-
tion of baseline tension seen by
C. tropix
venom.
in baseline tension. However, the lack of effect of verapamil
(Figure 1F) would suggest that L-type calcium channels are
not implicated in the effect of
C. tropix
venom.
Voltage-gated sodium channels (VGSCs) are sensitive to
stretch, which can result in an increased permeability to
sodium ions (Wallace et al, 1998; Shcherbatko et al, 1999;
Tabarean et al, 1999). It has been suggested that stretch acti-
vates mechanosensitive sodium channels, which leads to
an increase in intracellular sodium (Wolf et al, 2001). This
increased sodium infl ux has two different effects: Firstly, it
induces the sodium-calcium exchanger to pump in ‘reverse-
mode’ (
i.e.
, calcium is pumped into the cell and sodium is
pumped out of the cell) and secondly it induces membrane
depolarization, which causes more sodium channels to open
and activates VGCCs. The resulting increase in intracellular
calcium activates the contractile apparatus, which results in
shortening of muscle sarcomeres and a subsequent increase
in baseline muscle tension. In all cases the effects of acti-
vating the sodium channel can be blocked by application of
the selective sodium channel blocker TTX. The reduction in
baseline tension induced by TTX (Figure 1G) and the lack of
action of d-TC would suggest a postsynaptic mechanism of
action for TTX,
i.e.
, acting through muscle sodium channels
as opposed to neuronal sodium channels. The reduction of
baseline muscle tension by excess Mg
2+
(Figure 1H) and Ca
2+
ions (Figure 1I) could further be explained by these cations
blocking VGSCs, as suggested by another study (Yamamoto
et al, 1984). The lack of effect of the osmotic equivalence
of these ions through adding 40mM glucose (Figure 1J)
to the physiological salt solution strongly suggests a phar-
macological role for these ions. We have not measured the
cation concentration from the venom of
C. tropix,
however,
a related theraphosid spider
Aphonopelma steindachneri
(previously named
Eurypelma californicum
according to
Platnick, 2010) is reported to contain approximately 100nM
of cations (Savel-Niemann and Roth, 1989), well below the
threshold that would affect the opening of sodium channels.
Stretch-activated ion channels (SACs)
The method used in the present study includes the applica-
tion of a pre-tension of 10mN to the chick biventer muscles.
Hence, there exists the possibility that this pre-tension (
i.e.
,
stretch) caused the activation of SACs. Guharay and Sachs
(1984) already reported that stretch causes the activation of
SACs in chick skeletal muscle. It was found that the current
evoked by the stretch appears neither to be due to activation
of the nicotinic acetylcholine receptor ion channel complex
nor due to the opening of Ca
2+
activated K
+
channels. The
current was further shown to have a reversal potential of
around -30mV, to be cation (Ca
2+
, K
+
and Na
+
) selective, and
to only poorly discriminate between Na
+
and K
+
ions. Based
upon these biophysical properties, activation of this channel
will result in depolarisation of skeletal muscle cells, and if
the depolarisation is suffi ciently large, the contractile appa-
ratus would be activated through the subsequent infl ux of
calcium ions through excitation-coupled calcium entry.
Further studies on chick embryonic myoblasts (Shin et al,
1996)
demonstrated that stretch elevated intracellular Ca
2+
concentration suffi ciently, resulting in the activation of
Ca
2+
-activated K
+
channels, and these effects were com-
pletely blocked by 10µM gadolinium. The lack of effect of
gadolinium (up to 50µM) in our study (Figure 1E) would
either suggest that blocking calcium infl ux through SACs
does not account for the effect of
C. tropix
venom on base-
line tension or that the effect was caused by a toxin from the
venom that acts on a sub-type of SACs, which are insensi-
tive to gadolinium.
CONCLUSIONS
Our results show that excess Ca
2+
and Mg
2+
as well as TTX
and
C. tropix
venom reduces the baseline muscle tension in
the chick
biventer cervicis
nerve-muscle preparation. Based
on the lack of effect of verapamil and d-TC, we can exclude
that blocking voltage-gated calcium channels (VGCC) and
nicotinic acetylcholine receptors are responsible for the
reduced baseline tension. Our data strongly suggests that
muscle TTX-sensitive sodium channels are activated toni-
cally during muscle stretch and that blocking these channels
causes a reduction in baseline resting tension through an as
yet undefi ned mechanism The role of the sodium channel in
muscle disease has seen a new resurgence as mutations of
voltage-gated sodium channels have been recognised to be
involved in a number of myotonias (see Platt and Griggs,
2009 for a review). Our data would suggest that skeletal
muscle sodium channels (Na
v
1.4) play an indirect role in
activating the contractile apparatus. Further detailed elec-
trophysiological, and calcium imaging experiments will be
employed to explore this hypothesis.
Voltage-gated ion channels
Calcium entry in skeletal muscle requires the functioning of
L-type voltage-gated calcium channels (VGCCs). These ion
channels mediate excitation-contraction coupling by act-
ing as voltage sensors that trigger the opening of ryanodine
receptors and induce calcium release from the sarcoplasmic
reticulum. It was shown that the IVS6 segment of the L-type
channel is critical for the binding of phenylalkylamines (ver-
apamil) and accounts for the sensitivity of muscle contrac-
tion to these drugs (Schuster et al, 1996). As spider venoms
are a rich source of ion channel toxins (Herzig et al, 2011)
the venom of
C. tropix may
contain compounds that will
block L-type calcium channels and account for the change
The question that remains open is which of the possi-
ble mechanisms that we have shown to reduce the muscle
©The Author(s)
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Journal of Venom Research
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2011
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tension is employed by
C. tropix
venom. While Ca
2+
and Mg
2+
have been shown to be present in another theraphosid spi-
der venom (Savel-Niemann and Roth, 1989), their quantity
in the low nanomolar range would not have been suffi cient
to explain the present results induced by millimolar concen-
tration of these ions. Although gadolinium did not show any
effect on the muscle tension in the present experiments, there
still exists the possibility that a toxin from
C. tropix
venom
acted on a different sub-type of SACs, which is insensitive to
gadolinium. Whereas theraphosid spider venoms are known
to contain blockers of mechanosensitve channels such as
M-TRTX-Gr1a (new toxin name according to ArachnoServer
(Herzig et al, 2011), previously known as GsMTx4) from
the venom of the theraphosid spider
Grammostola rosea
(Suchyna et al, 2000), more detailed experiments would be
required to prove this assumption. Our most favourable expla-
nation is that
C. tropix
venom reduced the tension by blocking
muscle TTX-sensitive sodium channels. According to the spi-
der toxin database ArachnoServer (Herzig et al, 2011), VGSC
are among the main targets for spider toxins.
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Selenotholus foelschei
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pharmacological properties of
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(Araneae,
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ACKNOWLEDGEMENTS
VH was funded by fellowships from the DAAD (Deutscher
Akademischer Austauschdienst) and DFG (Deutsche Fors-
chungsgemeinschaft). VH and WCH were supported by the
ARC (Australian Research Council). We wish to thank Alan
Harvey for helpful comments on the manuscript.
STATEMENT OF COMPETING INTERESTS
None declared.
LIST OF ABBREVIATIONS
d-TC; d-tubocurarine
SACs; stretch activated ion channels
TTX; tetrodotoxin
VGCCs; Voltage-gated calcium channels
VGSCs; Voltage-gated sodium channels
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ISSN: 2044-0324
J Venom Res, 2011, Vol 2, 1-5
RESEARCH REPORT
TTX, cations and spider venom modify avian muscle tone
in vitro
Volker Herzig
α,
*, Wayne C Hodgson
β
and Edward G Rowan
γ
α
Division of Chemical and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Building 80,
Service Road, St Lucia, QLD 4072, Australia,
β
Monash Venom Group, Department of Pharmacology, Monash University,
Clayton, Victoria 3800, Australia,
γ
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde,
Glasgow, G4 0NR, United Kingdom
*Correspondence to: Volker Herzig, Email: schnakenstich@yahoo.com, Tel: +61 7 3346 2014, Fax: +61 7 3346 2101
Received 21 December 2010; Accepted 02 January 2011; Published online 02 January 2011
© Copyright The Author(s): Published by Library Publishing Media. This is an open access article, published under the
This license permits non-commercial use, distribution and reproduction of the article, provided the original work is
appropriately acknowledged with correct citation details.
ABSTRACT
Agents that reduce skeletal muscle tone may have a number of useful clinical applications,
e.g.
, for muscle
spasticity and other muscle disorders. Recently, we reported that the venoms of two species of Australian
theraphosid (Araneae, Theraphosidae) spiders (
Coremiocnemis tropix
and
Selenotholus foelschei
) reduced the
baseline tension of chick
biventer cervicis
nerve-muscle preparation. The purpose of this study was to deter-
mine the underlying physiology mediating the change in muscle tension, which was addressed by conducting
isometric tension experiments.
We found that MgCl
2
(20mM), CaCl
2
(20mM), tetrodotoxin (1µM) or
C. tropix
venom (2µl/ml) produced a similar decrease in baseline tension, whereas d-tubocurarine (100µM), gadolinium
(1mM), verapamil (10mM), an increase in osmotic pressure by the addition of glucose (40mM), or the pres-
ence/absence of electrical stimulation did not produce a signifi cant change in baseline tension.
We suggest that
mechanosensitive or muscle TTX-sensitive sodium channels are activated during muscle stretch. This may have
implications for the treatment of stretch induced muscle damage.
KEYWORDS:
Theraphosidae,
Coremiocnemis tropix
, venom, baseline muscle tension, chick
biventer cervicis
nerve-muscle preparation
INTRODUCTION
the venom of
Leiurus quinquestriatus
(Sontheimer, 2008;
Orndorff and Rosenthal, 2009). In common with scorpion
venoms spider venoms contain a myriad of peptide and non-
peptide ion channel toxins that can block or activate voltage
dependent ion channels (Estrada et al, 2007) and are recog-
nised as a source of bioactive molecules that can be used to
identify therapeutic targets, elucidate mechanisms of action
and design novel pharmaceutical drugs. In a recent study, we
reported that bath application of venoms from the Australian
theraphosid (Araneae, Theraphosidae) spiders
Coremiocne-
mis tropix
(Herzig and Hodgson, 2009) and
Selenotholus
foelschei
(Herzig and Hodgson, 2008) on the isolated chick
biventer cervicis
nerve-muscle preparation cause a signifi -
cant reduction in the resting (10mN) baseline muscle ten-
sion. This effect has not been reported before, and agents
that relax skeletal muscle have the potential to be of medical
value in the treatment of muscle spasticity and other muscle
disorders. The present study therefore aims to elucidate the
Animal venoms contain numerous pharmacologically active
peptides and enzymes that can disrupt the normal physiol-
ogy of cells. Snake venoms can affect the clotting cascade,
bind to receptors at the neuromuscular junction and have
the ability to target nerve terminals, alter the propagation of
the nerve terminal action potential and disrupt neurotrans-
mitter transmitter release (Kini, 2005; Hodgson et al, 2007;
Servent and Fruchart-Gaillard, 2009). Scorpion venoms
contain numerous peptides that interact with a large range
of ion channels and receptors (Billen et al, 2008; Prestipino
et al, 2009). A number of these peptides have been identifi ed
to selectively block insect ion channels (de Lima et al, 2007),
thus having the potential to be developed as novel insecti-
cides. Additionally scorpion venoms have been used therapeu-
tically to treat, and when conjugated to an appropriate ligand,
to visualise tumors of the CNS,
i.e.
, chlorotoxin isolated from
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mechanisms underlying the observed reduction in muscle
tone induced by
C. tropix
venom by mimicking the venom-
induced relaxation using other pharmacological or physi-
ological agents. Besides
C. tropix
venom, we have tested
gadolinium (antagonist of mechanosensitive channels),
verapamil (antagonist of voltage-gated calcium channels),
d-tubocurarine (d-TC, antagonist of nicotinic acetylcholine
receptors), tetrodotoxin (TTX, antagonist of voltage-gated
sodium channels), MgCl
2
and CaCl
2
(both to increase the
extracellular ion concentrations), and glucose (to increase
osmotic pressure). A separate control experiment was used
to examine whether the presence/absence of electrical stim-
ulation had any effect on the baseline muscle tension.
was turned on (n = 6) or off (n = 8). In case of the muscles
that received the electrical stimulation of the motor nerves,
only the baseline tension in the 10sec interval between
twitches was analysed to allow for comparability with the
non-stimulated muscle.
Drugs and chemicals
d-TC and TTX were obtained from Sigma Chemical Co
(St Louis, MO, USA). Gadolinium, verapamil, MgCl
2
(20mM), CaCl
2
(20mM), and glucose were supplied by
Sigma-Aldrich Company Ltd (Dorset, England). Stock
solutions of drugs were made up in Milli-Q water unless
otherwise stated.
MATERIALS AND METHODS
Statistical analysis
Sigma-Plot 11.0 (Systat Software Inc., San Jose, California,
USA) was used for all statistical analysis. The baseline
muscle tension values for control and treatment for each
group were compared by using either a t-test or (in cases
where the normality test failed) a Mann-Whitney rank sum
test. For all tests, signifi cance levels were set to 0.05 and
data are expressed as mean minus standard error of the
mean (S.E.M.).
Venom collection
The venom from female
C. tropix
spiders was collected by
applying electrostimulation to the venom apparatus using
a recently described method (Herzig and Hodgson, 2009).
Isolated chick biventer cervicis nerve-muscle
preparation
We used the chick
biventer cervicis
nerve-muscle prepara-
tion according to our previous method (Herzig and Hodgson,
2008) with some modifi cations. Briefl y,
biventer cervicis
muscles were removed from male chicks (8-15 days old),
mounted in glass (10ml) organ baths and maintained at
34ºC under 1g resting tension in a physiological saline
solution of the following composition: 118.4mM NaCl,
4.7mM KCl, 1.2mM MgSO
4
, 1.2mM KH
2
PO
4
, 2.5mM
CaCl
2
, 25mM NaHCO
3
, 11.1mM D-glucose and bubbled
continuously with 95% O
2
+ 5% CO
2
to maintain the pH
between 7.2 - 7.4. Isometric contractions were measured
via a Grass transducer (FTO3) connected via either one
or two Quad Bridge modules (ADInstruments, Australia)
to a Powerlab 4/20, MacLab/4s or MacLab/8e system (all
ADInstruments, Australia). Twitches (where applicable)
were evoked by stimulating the motor nerve (supramaxi-
mal voltage, duration 0.2ms, 0.1Hz) via silver electrodes
connected to a Grass S88 stimulator. Nerve-mediated
(indirectly evoked) twitches were confi rmed by the addi-
tion of d-TC (10µM) a blocker of postsynaptic nicotinic
acetylcholine receptors. Muscles that did not respond to
acetylcholine, carbachol and KCl were excluded from
this study.
RESULTS AND DISCUSSION
Physical stretch of muscle
We have previously shown that the venom from female
C. tropix
spiders
not only reduced the baseline muscle
tension but also produced an irreversible block of nerve-
evoked twitch tension on
biventer cervicis
nerve-muscle
preparations (Herzig and Hodgson, 2008). A possibility to
explain the reduced muscle tension therefore would be that
the omission of the electrically induced twitches resulted
in a relaxation of the muscle. Stretching of skeletal mus-
cles results in a number of physiological effects (
e.g.
, to
activate the contractile apparatus resulting in an increase of
the force of contraction or to increase transmitter release)
all whose end point is to elevate cytosolic calcium ions (see
Rosenberg, 2009
for a recent review on calcium entry in
skeletal muscle). Stretching of the muscle deforms both
the cytoskeleton and cell membrane and these changes in
shear forces are thought to cause ion channels and recep-
tors to undergo conformational changes and open or acti-
vate. By blocking the muscle from being stretched during
electrically induced twitches,
C. tropix
venom might have
induced a decrease in the cytosolic calcium levels, lead-
ing to a decrease in muscle tension. This hypothesis, how-
ever, is not substantiated by our data (Figure 1A and B) that
show that the absence of nerve-evoked twitches does not
induce the muscle to relax signifi cantly more than the time-
matched control that received the nerve-evoked twitches.
We also confi rm our earlier observation on avian nerve-
muscle preparations that
C. tropix
venom signifi cantly
reduces resting tension when the tissue was subjected to
a resting tension of about 10mN (Figure 1C) (Herzig and
Hodgson, 2009).
In order to minimise variation, the same muscles were used
for control and treatment (consisting of various pharmaco-
logical or physiological agents). Hence, each muscle fi rst
received 10µl/ml Milli-Q water as control and was monitored
over 60min. Without receiving a washout, the baseline was
then manually re-adjusted to the initial starting value and
the respective treatment was applied immediately before the
muscle was monitored for another 60min. This treatment
schedule allowed each muscle to act as its own control. Each
muscle was only used for a single treatment. The applied
treatments consisted of
C. tropix
venom (2µg/ml, n = 5),
gadolinium (1mM, n = 7), verapamil (10mM, n = 4), d-TC
(100µM, n = 6), TTX (1µM, n = 6), MgCl
2
(20mM, n = 17),
CaCl
2
(20mM, n = 6), and glucose (40mM, n = 4). A sepa-
rate control experiment was carried out to test for effects on
the baseline muscle tension when the electrical stimulation
At frog neuromuscular junctions
it was demonstrated
that stretch of skeletal muscles induces an increase in
spontaneous and evoked transmitter release mediated by
mechanical tension on synaptically located membrane
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Figure 1.
Baseline muscle tension values (inclding S.E.M.) after various treatments as recorded using a chick
biventer cervicis
nerve-
muscle preparation. No effect of the stimulation of the motor nerve on the baseline muscle tension could be observed when tested under
the presence (
A
) and absence (
B
) of electrical stimulation of the motor nerve. In the case of
A
, only the baseline tension in between
twitches was analysed to allow for comparability with the non-stimulated muscle. All of the remaining treatments were carried out in
the absence of electrical stimulation. These treatments include
C. tropix
venom (2µg/ml,
C
), d-TC (100µM,
D
), gadolinium (1mM,
E
),
verapamil (10mM,
F
), TTX (1µM,
G
), MgCl
2
(20mM,
H
) and CaCl
2
(20mM,
I
), and glucose (40mM,
J
). Signifi cant differences are
indicated by *(P < 0.05) and **(P < 0.01).
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bound integrins (Chen and Grinnell, 1995). It has been sug-
gested that this effect is limited to the frog neuromuscular
junction (Grinnell et al, 2003),
as
transmitter release is not
signifi cantly changed when adult rat diaphragm neuromus-
cular junctions were subjected to stretch. The effects of
stretch on transmitter release on avian neuromuscular junc-
tions has not been adequately studied, but if stretch increased
transmitter release, it would be predicted to result in the acti-
vation of postsynaptic receptors and subsequent depolarisa-
tion of the multiply innervated muscle fi bres to generate a
sustained tonic contracture. As the nicotinic acetylcholine
receptor antagonist d-TC (Figure 1D) did not signifi cantly
reduce baseline tension, we assume that despite the evidence
for stretch-induced increased transmitter release in the frog,
block of augmented acetylcholine release from stretched
avian neuromuscular junctions cannot account for the reduc-
tion of baseline tension seen by
C. tropix
venom.
in baseline tension. However, the lack of effect of verapamil
(Figure 1F) would suggest that L-type calcium channels are
not implicated in the effect of
C. tropix
venom.
Voltage-gated sodium channels (VGSCs) are sensitive to
stretch, which can result in an increased permeability to
sodium ions (Wallace et al, 1998; Shcherbatko et al, 1999;
Tabarean et al, 1999). It has been suggested that stretch acti-
vates mechanosensitive sodium channels, which leads to
an increase in intracellular sodium (Wolf et al, 2001). This
increased sodium infl ux has two different effects: Firstly, it
induces the sodium-calcium exchanger to pump in ‘reverse-
mode’ (
i.e.
, calcium is pumped into the cell and sodium is
pumped out of the cell) and secondly it induces membrane
depolarization, which causes more sodium channels to open
and activates VGCCs. The resulting increase in intracellular
calcium activates the contractile apparatus, which results in
shortening of muscle sarcomeres and a subsequent increase
in baseline muscle tension. In all cases the effects of acti-
vating the sodium channel can be blocked by application of
the selective sodium channel blocker TTX. The reduction in
baseline tension induced by TTX (Figure 1G) and the lack of
action of d-TC would suggest a postsynaptic mechanism of
action for TTX,
i.e.
, acting through muscle sodium channels
as opposed to neuronal sodium channels. The reduction of
baseline muscle tension by excess Mg
2+
(Figure 1H) and Ca
2+
ions (Figure 1I) could further be explained by these cations
blocking VGSCs, as suggested by another study (Yamamoto
et al, 1984). The lack of effect of the osmotic equivalence
of these ions through adding 40mM glucose (Figure 1J)
to the physiological salt solution strongly suggests a phar-
macological role for these ions. We have not measured the
cation concentration from the venom of
C. tropix,
however,
a related theraphosid spider
Aphonopelma steindachneri
(previously named
Eurypelma californicum
according to
Platnick, 2010) is reported to contain approximately 100nM
of cations (Savel-Niemann and Roth, 1989), well below the
threshold that would affect the opening of sodium channels.
Stretch-activated ion channels (SACs)
The method used in the present study includes the applica-
tion of a pre-tension of 10mN to the chick biventer muscles.
Hence, there exists the possibility that this pre-tension (
i.e.
,
stretch) caused the activation of SACs. Guharay and Sachs
(1984) already reported that stretch causes the activation of
SACs in chick skeletal muscle. It was found that the current
evoked by the stretch appears neither to be due to activation
of the nicotinic acetylcholine receptor ion channel complex
nor due to the opening of Ca
2+
activated K
+
channels. The
current was further shown to have a reversal potential of
around -30mV, to be cation (Ca
2+
, K
+
and Na
+
) selective, and
to only poorly discriminate between Na
+
and K
+
ions. Based
upon these biophysical properties, activation of this channel
will result in depolarisation of skeletal muscle cells, and if
the depolarisation is suffi ciently large, the contractile appa-
ratus would be activated through the subsequent infl ux of
calcium ions through excitation-coupled calcium entry.
Further studies on chick embryonic myoblasts (Shin et al,
1996)
demonstrated that stretch elevated intracellular Ca
2+
concentration suffi ciently, resulting in the activation of
Ca
2+
-activated K
+
channels, and these effects were com-
pletely blocked by 10µM gadolinium. The lack of effect of
gadolinium (up to 50µM) in our study (Figure 1E) would
either suggest that blocking calcium infl ux through SACs
does not account for the effect of
C. tropix
venom on base-
line tension or that the effect was caused by a toxin from the
venom that acts on a sub-type of SACs, which are insensi-
tive to gadolinium.
CONCLUSIONS
Our results show that excess Ca
2+
and Mg
2+
as well as TTX
and
C. tropix
venom reduces the baseline muscle tension in
the chick
biventer cervicis
nerve-muscle preparation. Based
on the lack of effect of verapamil and d-TC, we can exclude
that blocking voltage-gated calcium channels (VGCC) and
nicotinic acetylcholine receptors are responsible for the
reduced baseline tension. Our data strongly suggests that
muscle TTX-sensitive sodium channels are activated toni-
cally during muscle stretch and that blocking these channels
causes a reduction in baseline resting tension through an as
yet undefi ned mechanism The role of the sodium channel in
muscle disease has seen a new resurgence as mutations of
voltage-gated sodium channels have been recognised to be
involved in a number of myotonias (see Platt and Griggs,
2009 for a review). Our data would suggest that skeletal
muscle sodium channels (Na
v
1.4) play an indirect role in
activating the contractile apparatus. Further detailed elec-
trophysiological, and calcium imaging experiments will be
employed to explore this hypothesis.
Voltage-gated ion channels
Calcium entry in skeletal muscle requires the functioning of
L-type voltage-gated calcium channels (VGCCs). These ion
channels mediate excitation-contraction coupling by act-
ing as voltage sensors that trigger the opening of ryanodine
receptors and induce calcium release from the sarcoplasmic
reticulum. It was shown that the IVS6 segment of the L-type
channel is critical for the binding of phenylalkylamines (ver-
apamil) and accounts for the sensitivity of muscle contrac-
tion to these drugs (Schuster et al, 1996). As spider venoms
are a rich source of ion channel toxins (Herzig et al, 2011)
the venom of
C. tropix may
contain compounds that will
block L-type calcium channels and account for the change
The question that remains open is which of the possi-
ble mechanisms that we have shown to reduce the muscle
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tension is employed by
C. tropix
venom. While Ca
2+
and Mg
2+
have been shown to be present in another theraphosid spi-
der venom (Savel-Niemann and Roth, 1989), their quantity
in the low nanomolar range would not have been suffi cient
to explain the present results induced by millimolar concen-
tration of these ions. Although gadolinium did not show any
effect on the muscle tension in the present experiments, there
still exists the possibility that a toxin from
C. tropix
venom
acted on a different sub-type of SACs, which is insensitive to
gadolinium. Whereas theraphosid spider venoms are known
to contain blockers of mechanosensitve channels such as
M-TRTX-Gr1a (new toxin name according to ArachnoServer
(Herzig et al, 2011), previously known as GsMTx4) from
the venom of the theraphosid spider
Grammostola rosea
(Suchyna et al, 2000), more detailed experiments would be
required to prove this assumption. Our most favourable expla-
nation is that
C. tropix
venom reduced the tension by blocking
muscle TTX-sensitive sodium channels. According to the spi-
der toxin database ArachnoServer (Herzig et al, 2011), VGSC
are among the main targets for spider toxins.
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ACKNOWLEDGEMENTS
VH was funded by fellowships from the DAAD (Deutscher
Akademischer Austauschdienst) and DFG (Deutsche Fors-
chungsgemeinschaft). VH and WCH were supported by the
ARC (Australian Research Council). We wish to thank Alan
Harvey for helpful comments on the manuscript.
STATEMENT OF COMPETING INTERESTS
None declared.
LIST OF ABBREVIATIONS
d-TC; d-tubocurarine
SACs; stretch activated ion channels
TTX; tetrodotoxin
VGCCs; Voltage-gated calcium channels
VGSCs; Voltage-gated sodium channels
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