Am J Physiol Cell Physiol 292: C1591–C1598, 2007.
First published January 17, 2006; doi:10.1152/ajpcell.00133.2006.
Elevated resting [Ca2⫹]i in myotubes expressing malignant hyperthermia
RyR1 cDNAs is partially restored by modulation of passive calcium leak
from the SR
Tianzhong Yang,1 Eric Esteve,1 Isaac N. Pessah,2 Tadeusz F. Molinski,3 Paul D. Allen,1 and José R. López1
1
Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital,
Boston, Massachusetts; 2Department of Molecular Biosciences, School of Veterinary Medicine, University
of California, Davis, California; and 3Department of Chemistry and Biochemistry and Skaggs School
of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California
Submitted 23 March 2006; accepted in final form 15 December 2006
ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum
MALIGNANT HYPERTHERMIA (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. The
pathophysiology of this syndrome is not yet fully understood.
However, it is generally accepted that MH susceptibility
(MHS) is caused by abnormal intracellular Ca2⫹ homeostasis
in skeletal muscle cells (33). The ryanodine receptor gene on
chromosome 19q13.1 is the primary molecular locus for MHS
in humans. More than 60 mutations of type 1 ryanodine
receptor (RyR1) located in three hot spots (hot spot I,
Address for reprint requests and other correspondence: P. D. Allen, Dept. of
Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s
Hospital, 75 Francis St., Boston, MA 02115 (e-mail: [email protected].
harvard.edu).
http://www.ajpcell.org
35– 615 aa; hot spot II, 2163–2458 aa; and hot spot III,
4643– 4898 aa) have been linked to ⬎50% of all MHS
families (36). Numerous studies have confirmed that MHS
muscles exhibit increased sensitivity to direct RyR1 agonists
such as caffeine, halothane, and 4-chloro-m-cresol (4-CmC),
and it is thought that this increased sensitivity is likely to be the
primary underlying mechanism for triggering the clinical MH
syndrome (33).
Intracellular Ca2⫹ measurements obtained using Ca2⫹-selective microelectrodes and fluorescent indicators have demonstrated that there is a significantly elevated resting intracellular
Ca2⫹ concentration ([Ca2⫹]i) in MH adult muscle fibers possessing unidentified human MH mutations in swine expressing
the Arg614Cys MH mutation (24, 25, 27) and in cultured cells
expressing MH mutations (13, 51, 52). The chronically increased resting [Ca2⫹]i in MHS muscle cells has been suggested to be associated with the increased sensitivity of these
cells to caffeine and 4-CmC stimulation (26, 28). Thus these
changes in resting [Ca2⫹]i in MHS muscle cells and their
possible contribution to the pathophysiology of MH represent
another interesting aspect. On the other hand, other studies
using the same fluorescent indicators in adult swine (14, 15)
and dyspedic myotubes or human embryonic kidney (HEK)
cells transfected with RyR1 cDNAs possessing some MH
mutations (12, 48) have failed to measure such an elevation.
Furthermore, the mechanistic relationship between MHRyR1
mutations and the possible dysfunction of intracellular Ca2⫹
homeostasis seen in MH-susceptible individuals with these
mutations remain to be established.
In the current investigation, we have used double-barreled
Ca2⫹ microelectrodes to measure the resting myoplasmic
[Ca2⫹]i in myotubes that were transduced with herpes simplex
virus (HSV) virions to express wild-type RyR1 (WTRyR1) or
one of four common MH mutations (MHRyR1), including at
least one from each of the three mutation hot spots. The new
findings are as follows: 1) myotubes expressing any of the four
MH mutations had a higher resting [Ca2⫹]i than those expressing WTRyR1; 2) blocking of MHRyR1s or WTRyR1 with ryanodine or FLA 365 had little or no influence on resting [Ca2⫹]i
of myotubes; and 3) bastadin 5 alone or ryanodine and bastadin
5 together significantly lowered the resting [Ca2⫹]i of
myotubes expressing MHRyR1, and this effect was more proThe costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6143/07 $8.00 Copyright © 2007 the American Physiological Society
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Yang T, Esteve E, Pessah IN, Molinski TF, Allen PD, López
JR. Elevated resting [Ca2⫹]i in myotubes expressing malignant
hyperthermia RyR1 cDNAs is partially restored by modulation
of passive calcium leak from the SR. Am J Physiol Cell Physiol
292: C1591–C1598, 2007. First published January 17, 2006;
doi:10.1152/ajpcell.00133.2006.—Malignant hyperthermia (MH)
is a pharmacogenetic disorder of skeletal muscle triggered in
susceptible individuals by inhalation anesthetics and depolarizing
skeletal muscle relaxants. This syndrome has been linked to a
missense mutation in the type 1 ryanodine receptor (RyR1) in more
than 50% of cases studied to date. Using double-barreled Ca2⫹
microelectrodes in myotubes expressing wild-type RyR1
(WTRyR1) or RyR1 with one of four common MH mutations
(MHRyR1), we measured resting intracellular Ca2⫹ concentration
([Ca2⫹]i). Changes in resting [Ca2⫹]i produced by several drugs
known to modulate the RyR1 channel complex were investigated.
We found that myotubes expressing any of the MHRyR1s had a 2.0to 3.7-fold higher resting [Ca2⫹]i than those expressing WTRyR1.
Exposure of myotubes expressing MHRyR1s to ryanodine (500
␮M) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365;
20 ␮M) had no effects on their resting [Ca2⫹]i. However, when
myotubes were exposed to bastadin 5 alone or to a combination of
ryanodine and bastadin 5, the resting [Ca2⫹]i was significantly
reduced (P ⬍ 0.01). Interestingly, the percent decrease in resting
[Ca2⫹]i in myotubes expressing MHRyR1s was significantly greater
than that for WTRyR1. From these data, we propose that the high
resting myoplasmic [Ca2⫹]i in MHRyR1 expressing myotubes is
due in part to a related structural conformation of MHRyR1s that
favors “passive” calcium leak from the sarcoplasmic reticulum.
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RESTING MYOPLASMIC [Ca2⫹] IN
nounced in myotubes expressing MHRyR1s than in myotubes
expressing WTRyR1. On the basis of these data, we propose that
the mechanism causing increased resting myoplasmic [Ca2⫹]i
in MHRyR1-expressing myotubes is not the result of a heightened gating activity of MHRyR1 release channels. Rather, the
chronic increase in resting [Ca2⫹]i results from a structural
conformation of MHRyR1 that favors a constant “passive” Ca2⫹
release from the SR that can be rescued by allosteric modulators of the RyR1 complex.
MATERIALS AND METHODS
AJP-Cell Physiol • VOL
RYR1 MYOTUBES
with a frequency of 1,000 Hz with AxoGraph (version 4.8; Axon
Instruments, Foster City, CA), and stored for further analysis.
Three criteria were used as key elements to accept or reject
individual measurements of resting [Ca2⫹]i: 1) the calibrations before
and after the intracellular Ca2⫹ determination had to agree with one
another within 2.5 mV; 2) an instantaneous change in potential (Vm
and VCa) had to occur during the cell impalement, followed by a stable
recording (⫾2–3 mV) for at least 40 s; and 3) the membrane potential
had to be more negative than ⫺60 mV. In experiments where resting
[Ca2⫹]i determinations were carried out twice in the same cell, before
and after drug treatment, cells were identified with grid labels attached
to the bottom of the plate to allow the identification of an individual
cell.
Statistics. All values are expressed as means ⫾ SE. Prism software
(version 4.0b) was used for statistical analysis (GraphPad Software, San
Diego, CA; www.graphpad.com). One-way ANOVA and Tukey’s multiple comparison tests were used to compare the resting [Ca2⫹]i values
among cells expressing different RyR1 constructs; paired t-tests were
used to compare the resting [Ca2⫹]i values in a single cell before and
after drug treatment.
RESULTS
Measurements of resting [Ca2⫹]i. Differentiated 1B5 myotubes were infected with HSV virion particles containing
WTRyR1 or MHRyR1 cDNAs at an MOI of 0.5 for 2 h.
Forty-eight hours after incubation with the virions, [Ca2⫹]i
measurements were performed on 10 –15 myotubes in each
well, using double-barreled Ca2⫹-selective microelectrodes.
Representative data traces of VCa and Vm measurements in
(untransduced) 1B5 myotubes and those expressing WTRyR1 or
T4825IRyR1 are shown in Fig. 1A. Phase-contrast images of the
same cells are pictured in Fig. 1B. To make certain that
measured cells were transduced and expressed no ryanodine
receptor, WTRyR1, or MHRyR1s, we performed immunostaining immediately after obtaining the measurements of resting
[Ca2⫹]i (Fig. 1C). Grid labels attached to the bottom of the
culture plate allowed identification of each cell before and after
immunostaining (see MATERIALS AND METHODS) and appeared as
the bar-shaped shadows in the background of panel B pictures.
Expression of MHRyR1s is associated with higher resting
[Ca2⫹]i than expression of WTRyR1s. Although our previous
studies clearly showed that human and porcine MH muscle
fibers had an elevated myoplasmic resting [Ca2⫹]i compared
with nonsusceptible individuals, the mechanism for this increase has never been systematically tested in situ in myotubes
expressing MHRyR1s. In the present study, WTRyR1 and four
MHRyR1s, R164C, R615C, R2163C, and T4825I, were packaged into HSV virions and used to transduce differentiated 1B5
myotubes(35). Measurements of membrane potential and resting [Ca2⫹]i in MHRyR1-expressing myotubes showed that expression of MHRyR1s and WTRyR1s caused no significant
change in resting membrane potential compared with untransduced 1B5 myotubes. However, cells expressing any of the
four MH mutations had a significantly (P ⬍ 0.001) increased
resting [Ca2⫹]i compared with cells expressing WTRyR1
(Fig. 2). The degree of elevation of resting [Ca2⫹]i varied
significantly among mutations, with expression of T4825I
causing the largest increase (418 ⫾ 10.89 nM, n ⫽ 23) and
R164C producing the smallest increase (242.3 ⫾ 11.45 nM,
n ⫽ 18) compared with the WTRyR1 (112 ⫾ 1.9 nM, n ⫽ 11).
The resting [Ca2⫹]i in T4825I-expressing myotubes was also
significantly higher (P ⬍ 0.001) than that of all other con-
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Construction and expression of MHRyR1 cDNAs. A detailed explanation of construction and expression of the MHRyR1s used in the
current study has been previously described (53). With the use of a
helper virus-free packaging system, all mutated and WTRyR1 cDNAs
were packaged into HSV1 virions, which were then used to transduce
1B5 myotubes (49) at a multiplicity of infection (MOI) of 0.5 for
2 h and then cultured for 48 h before measurement of [Ca2⫹]i at
room temperature. As described previously (53), 1B5 cells (RyR1,
RyR2, and RyR3 null) were cultured in growth medium (15% FBS
in low-glucose DMEM; GIBCO no. 11885-084) at 37°C to passages 5–10 and then plated on Matrigel (BD Bioscience, San Jose,
CA)-coated plates (96-well plates; Opticlear Costar 3614). Differentiation was started when cell density reached 20 –30% confluence by
changing growth medium to differentiation medium (0.2% horse
serum in low-glucose DMEM). Typical multinuclear myotubes as
shown in Fig. 1 were obtained by continued incubation of the cells in
differentiation medium at 37°C for 5–7 days.
Preparation of bastadin 5. The bromotyrosine derivative bastadin
5 was extracted and purified from the marine sponge Ianthella basta
as previously described (30, 40).
Immunostaining. Immunostaining for RyR1 expression was performed using monoclonal antibody 34C (Airey JA and Sutko JL,
ISHB, University of Iowa, Iowa City, IA) that recognizes both RyR1
and RyR3, and Cy3-conjugated goat anti-mouse secondary antibody
(Jackson ImmunoResearch, West Grove, PA). To help identify the
cells in which resting [Ca2⫹]i was measured, pictures were taken with
grid labels attached to the bottom of the plate before each experiment
and then matched to pictures taken with the same grid after immunostaining.
Ca2⫹-selective microelectrodes. Double-barreled Ca2⫹-selective
microelectrodes were prepared as described previously (28). They
were backfilled with the neutral carrier ETH 129 (Fluka,
Ronkonkoma, NY) and then with pCa 7. Each Ca2⫹-selective microelectrode was individually tested as described previously (23), and
only those with a linear relationship between pCa3 and pCa7 (Nernstian response, 28.5 mV per pCa unit at 24°C) and a response ⬎16 –20
mV between pCa 7 and pCa 8 were used experimentally. All electrodes were then recalibrated after taking measurements of resting
[Ca2⫹], and if the two calibration curves did not agree within 2.5 mV
in the relevant range of the calibration curve (pCa 6 – 8), the data from
that microelectrode was discarded. The calcium sensitivity of the
Ca2⫹ microelectrodes was not affected by any of the drugs used in the
present study, as determined by direct calibration.
Intracellular Ca2⫹ measurements. Single myotube impalements
were observed through an inverted microscope (Zeiss Axiovert 10)
fitted with a ⫻10 eyepiece and a ⫻40 dry objective. The potentials
from the 3 M KCl barrel (Vm) and the Ca2⫹ barrel (VCae) were
recorded via a high-impedance amplifier at ⬎1011 M⍀ (model FD223; WPI, Sarasota, FL). The potential of the voltage microelectrode
(Vm) was subtracted electronically from the potential of the Ca2⫹
electrode (VCae) to give the differential signal (VCa) that represents the
resting myoplasmic Ca2⫹ concentration. Vm and VCa potentials were
filtered using a low-pass filter (LPF-30; WPI) at 10 –30 kHz, acquired
MH
RESTING MYOPLASMIC [Ca2⫹] IN
structs tested; R2163C and R615C had similar [Ca2⫹]i values,
and in both of these constructs, [Ca2⫹]i was significantly
higher (P ⬍ 0.01) than in R164C.
Probing the mechanism of the MHRyR1-induced increase in
myoplasmic [Ca2⫹]i: Ryanodine and FLA 365 do not alter
resting myoplasmic [Ca2⫹]i. The alkaloid ryanodine is the
classic ryanodine receptor channel modulator. At low concentrations, ryanodine can enhance the open probability of the
channel, and at high micromolar concentrations (such as the
conditions used in this study), it fully blocks channel conductance (8, 10, 41, 55) and prevents Ca2⫹ release from the
sarcoplasmic reticulum (SR) induced by depolarization, Ca2⫹,
caffeine, and 4-CmC. Using both fluo-4 and microelectrodes,
we have found that incubation of intact myotubes expressing
WTRyR1 or any of the four MHRyR1s with 500 ␮M ryanodine
for 45 min completely abolished caffeine (20 mM)-induced
Ca2⫹ responses, and this caffeine insensitivity is not a result of
significant SR store depletion (data not shown). FLA 365 has
been shown to directly inhibit SR Ca2⫹ release by interacting
with the RyR1 by a mechanism distinct from other Ca2⫹
release inhibitors such as ruthenium red or neomycin (9, 30).
Unlike ruthenium red, FLA 365 is membrane permeant, which
permits its use to block RyR-mediated responses in intact cells
without microinjection, and unlike ruthenium red, its block is
reversible.
To test the effects of ryanodine or FLA 365 on resting
[Ca2⫹]i, we made measurements with microelectrodes in 1B5
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RYR1 MYOTUBES
C1593
myotubes expressing WTRyR1 or one of the four MHRyR1s
(R164C, R615C, R2163C, and T4825I) before and after incubation with 500 ␮M ryanodine for 45 min or before and after
incubation with 20 ␮M FLA 365 for 5 min. As shown in Fig. 3,
treatment with ryanodine or FLA 365 did not have any significant effect on resting [Ca2⫹]i in myotubes expressing any
RyR1 construct.
Probing the mechanism of the MHRyR1-induced increase in
myoplasmic [Ca2⫹]i: Bastadin 5 alone or ryanodine and bastadin 5 in combination partially restore myoplasmic [Ca2⫹]i.
Investigations using bastadins in isolated junctional SR and
BC3H1 cells have suggested a pharmacological link between
ryanodine-sensitive actively gating Ca2⫹ channels and ryanodine-insensitive leak pathways (30, 40). In combination with
500 ␮M ryanodine, bastadin 5 nearly eliminates the Ca2⫹ leak
unmasked by thapsigargin without causing any Ca2⫹ store
depletion, whereas ryanodine alone does not exhibit such an
effect (40). To test whether these ryanodine-insensitive leak
pathways contribute to the increased resting myoplasmic
[Ca2⫹]i in myotubes expressing MHRyR1s, we measured the
resting [Ca2⫹]i in myotubes expressing WTRyR1 or MHRyR1s
after incubation with 10 ␮M bastadin 5 for 5 min. Interestingly,
bastadin 5 alone significantly decreased the resting [Ca2⫹]i in
both types of myotubes (Fig. 4). To test the hypothesis that the
effect of bastadin 5 allosterically changes the conformation of
RyR1 from a ryanodine-insensitive “RyR1 leak conformation”
into RyR1s in an actively gating conformation that is sensitive
to ryanodine block (see DISCUSSION for detailed description of
these proposed RyR1 conformations), we then measured myoplasmic resting [Ca2⫹]i in myotubes before and after incubation with 500 ␮M ryanodine for 45 min followed by incubation
with 10 ␮M bastadin 5 for 5 min. As shown previously in this
study (see Fig. 3), incubation with ryanodine alone did not
change the resting [Ca2⫹]i in myotubes expressing either
WTRyR1 or any of the MHRyR1s tested. However, incubation of
ryanodine-blocked myotubes in 10 ␮M bastadin 5 for 5 min
significantly lowered resting myoplasmic [Ca2⫹]i in all myo-
Fig. 2. Resting [Ca2⫹]i measured using double-barreled Ca2⫹-selective microelectrodes in 1B5 myotubes expressing WTRyR1 and four MH mutations.
Vm and [Ca2⫹]i were measured 48 h after differentiated 1B5 myotubes were
transduced with virions carrying WTRyR1 and MH mutants. Expression of
RyR1 in the measured cells was confirmed by immunostaining. Data are shown
as means ⫹ SE. *Significantly different from WTRyR1 (P ⬍ 0.01, one-way
ANOVA with Tukey’s t-test). ⫹Significantly different from all other constructs (P ⬍ 0.01, one-way ANOVA with Tukey’s t-test).
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Fig. 1. Representative data traces of simultaneous measurements of membrane
potential and intracellular calcium concentration ([Ca2⫹]i) and immunostaining with ryanodine receptor antibody 34C to confirm expression of malignant
hyperthermia (MHRyR1) or wild-type type 1 ryanodine receptors (WTRyR1). A:
representative traces of membrane potential (Vm) and Ca2⫹ potential (VCa) in
control 1B5 myotubes and those expressing wild-type or T4825I RyR1. The
values shown are averages of the trace data during the ⬃45-s measurement.
Downward voltage deflection is the impalement of the cell with the microelectrodes, and the upward deflection is the Ca2⫹ microelectrode being
withdrawn from the myotube. B: bright-field images showing the cells from
which the measurements in A were recorded (arrows). The bar-shaped shadows
in the background are the labels attached to the bottom of the plate to identify
the cells from which recordings were taken, as explained in MATERIALS AND
METHODS. C: immunofluorescence images confirming the expression of RyR1
in the cells from which recordings were taken (arrows).
MH
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RESTING MYOPLASMIC [Ca2⫹] IN
MH
RYR1 MYOTUBES
tive. The ryanodine-sensitive conformation is largely accepted
to represent the high-conductance channel that is typically
reconstituted in bilayer lipid membranes. Under resting conditions, the population of ryanodine-insensitive MHRyR1s in the
RyR1 leak conformation is larger than the population of
WTRyR1 in the RyR1 leak conformation.
DISCUSSION
tubes studied (Fig. 5A). Furthermore, the degree of reduction in
resting [Ca2⫹]i induced by the two drugs in combination was
greater for all MHRyR1s than for WTRyR1 (P ⬍ 0.05) in both
absolute and percent values (Fig. 5B.). These data are consistent with a mechanism by which bastadin 5 converts RyR1s
that are in a ryanodine-insensitive RyR1 leak conformation
into the actively gating conformation that is ryanodine sensi-
Fig. 4. A: bastadin 5 (Bst) lowers resting [Ca2⫹]i in 1B5 myotubes expressing WT/MHRyR1s. Forty-eight hours after infection, myotubes were incubated with 10
␮M Bst for 3 min. Resting membrane potentials and [Ca2⫹]i were measured immediately before and after incubation, using the same microelectrodes. Expression
of WT/MHRyR1s in the measured cells was confirmed by immunostaining. Data are presented as means ⫹ SE. *Significantly different from the resting [Ca2⫹]i
for the same construct before treatment with Bst (P ⬍ 0.05, Student’s t-test). B: the decrease in resting [Ca2⫹]i ([Ca2⫹]i before Bst treatment ⫺ [Ca2⫹]i after
Bst treatment) in cells expressing WT/MHRyR1s after treatment with Bst. Cells expressing MHRyR1s had an almost twofold greater percent reduction in [Ca2⫹]i
after treatment with Bst than cells expressing WTRyR1. *Significantly different (P ⬍ 0.05) compared with WTRyR1.
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Fig. 3. Ryanodine (A) and FLA 365 (B) do not alter resting [Ca2⫹]i in 1B5
myotubes expressing WTRyR1 or any of the four MHRyR1s. Myotubes were
incubated in 500 ␮M ryanodine for 45 min or in 20 ␮M FLA 365 for 5 min
at 48 h after transduction. Resting membrane potentials and [Ca2⫹]i were
measured immediately before and after the incubation, using the same microelectrodes in the same myotubes. Expression of MH/WTRyR1s in the measured
cells was confirmed by immunostaining. Data are presented as means ⫹ SE.
No significant difference was found between [Ca2⫹]i values in any cell after
treatment with ryanodine or FLA 365 (P ⬎ 0.05, Student’s t-test).
Mutations of RyR1 (52) have been implicated in the pathophysiology of the MH syndrome in more than 50% of susceptible patients. In the present study we measured intracellular
resting [Ca2⫹]i in myotubes expressing either WTRyR1 or one
of four common missense MHRyR1s found in humans, using
double-barreled Ca2⫹ microelectrodes. The new findings in the
present study are as follows: 1) myotubes expressing any of the
four MHRyR1s (at least 1 from all 3 mutation hot spots) had a
higher resting [Ca2⫹]i than those expressing WTRyR1; 2) the
elevated resting [Ca2⫹]i observed in myotubes expressing the
four MHRyR1s varied among the individual mutations; 3)
treatment of myotubes expressing WT/MHRyR1s with ryanodine
or FLA 365 had no effect on resting [Ca2⫹]i; 4) incubation of
myotubes with bastadin 5 or ryanodine and bastadin 5 in
combination significantly lowered resting [Ca2⫹]i in myotubes
expressing either WTRyR1 and MHRyR1s; and 5) the percent
decrease in resting [Ca2⫹]i after treatment with bastadin 5 or
the combination of ryanodine and bastadin 5 in myotubes
expressing MHRyR1s was significantly greater than in myotubes expressing WTRyR1s. Each of these new findings is
discussed below.
The presence of a single MHRyR1 missense mutation from
any and all of the three MH hot spots produces a chronically
elevated resting myoplasmic [Ca2⫹]i when compared with
myotubes expressing WTRyR1. These results strongly suggest
that an increased resting [Ca2⫹]i is a consistent phenotype of
myotubes expressing any MHRyR1. In addition, this finding is
in agreement with all of our previous studies showing increased resting [Ca2⫹]i in muscle cells in both MH patients and
swine (25–28) and with some recent studies using fluorescent
Ca2⫹ indicators in human and mouse myotubes expressing
other MH mutations [T2206M (51), V2168M (13), R2163H
(2), or R1086H in the dihydropyridine receptor (DHPR) (52)].
RESTING MYOPLASMIC [Ca2⫹] IN
MH
RYR1 MYOTUBES
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However, some other studies on the [Ca2⫹]i in MH muscle
cells have yielded conflicting results as to whether or not the
resting [Ca2⫹]i is higher in resting MHS skeletal muscle, and
studies using fluorescent indicators frequently have not been
able to discern a difference between WT and MH cells (12, 24,
29, 48, 51). At present we cannot explain the basis for why
different studies on the same expressed mutation yield different
results other than to suggest it is due to the difference in
experimental methods. It is important to note that there is a
broad variation in the reported absolute resting myoplasmic
[Ca2⫹]i for WTRyR1-expressing myotubes and fibers between
different studies using the same or different fluorescent Ca2⫹
indicators [⬃110 nM (48); ⬃50 nM (2); and 35– 40 nM (13)],
and there is an even larger variation in the absolute values from
cells studied in the same laboratory using this technique. We
propose that whatever is responsible for this broad variation
may be the underlying reason for the inconsistency among
studies. In general, the absolute resting [Ca2⫹]i in muscle fibers
and myotubes measured using fluorescent Ca2⫹ indicators is
always lower than that measured using electrodes. The most
probable explanation for this is the result of buffering of
intracellular Ca2⫹ by these fluorescent dyes, all of which are
derivatives of the Ca2⫹ chelator BAPTA, coupled with the
uncertainty about the accuracy of their calibration (Kd, temperature, viscosity, intracellular pH, ionic strength) (47).
One alternative suggestion that could be made is that the
resting membrane potential of our cells was low, and that the
increased Ca2⫹ was the result of an abnormal response to
partial depolarization. Although it is true that the membrane
potential observed in this study was less than resting membrane
potential recorded in myotubes and adult cells at 37°C (⫺80
mV), the value for the resting membrane potential found in all
types of myotubes studied are in accordance with the membrane potential of myotubes and adult cells recorded at temperatures ranging between 20 and 25°C by several other groups
(3, 16, 22, 32, 44). Furthermore, previous studies performed at
37°C in intact humans and pigs also had an higher than normal
resting [Ca2⫹]i, suggesting that the increased resting [Ca2⫹]i is
not simply an artifact caused by partial depolarization.
We cannot explain why some MHRyR1 mutations cause a
higher resting [Ca2⫹]i than others, as shown in Fig. 3. HowAJP-Cell Physiol • VOL
ever, the increased resting [Ca2⫹]i that we found in the present
study seems supportive of our previous results from [3H]
ryanodine binding analysis, which demonstrated similar differences in sensitivity to caffeine activation and to Ca2⫹ and
Mg2⫹ inhibition common among MH mutations (53). The high
resting [Ca2⫹]i associated with T4825I might suggest that like
COOH-terminal mutations of RyR1 associated with central
core disease, MH mutations in this region may be more
“leaky,” as previous studies have suggested (48), and related to
more significant functional changes than the mutations in the
other two hot spots.
The opening and closing of the Ca2⫹ release channels
present within the terminal cisternae of SR are basic events in
the Ca2⫹ release process in skeletal muscle. Ryanodine is a
neutral plant alkaloid that is frequently used as a specific ligand
for characterizing the gating mechanism of ryanodine receptor
(31, 42). It has been shown that at high concentrations
(⬎200 ␮M), ryanodine irreversibly inhibits the ability of the
RyRs to release Ca2⫹ without changing the passive electrical
properties of the bilayer (8, 41). Likewise, FLA 365 reversibly
inhibits the Ca2⫹ release channel at the SR in both skeletal and
cardiac muscle cells (9, 11). As shown in RESULTS, treatment of
myotubes expressing either WTRyR1 or MHRyR1s with ryanodine (500 ␮M) for 45 min or with FLA 365 (20 ␮M) for 5 min
did not significantly modify resting [Ca2⫹]i. This suggests that,
similar to cells expressing WTRyR1, the mechanism for the
increased resting [Ca2⫹]i seen in cells expressing MHRyR1s is
not due to an increase in the spontaneous activity of these
channels as a result of the missense mutation leading to
enhanced efflux of Ca2⫹ from the SR. In fact, the current
results support previous conclusions that RyR1 in its ryanodine-sensitive actively gating conformation does not contribute
to the filling state of the SR stores (40). This is not surprising
with regard to the data of Ward et al. (50), who demonstrated
that the frequency of Ca2⫹ sparks (which represent spontaneous active gating) in intact myotubes expressing WTRyR1 was
very low. In addition, RyR1 gating states are in strong negative
control by their association with the DHPR, further reducing
the possible contribution that actively gating channels would
have on resting Ca2⫹ levels (21, 54).
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Fig. 5. A: the combination of ryanodine and bastadin 5 (Rya⫹Bst) lowers resting [Ca2⫹]i in 1B5 myotubes expressing WT/MHRyR1s. Forty-eight hours after
infection, myotubes were incubated in 500 ␮M ryanodine for 45 minutes, followed by incubation with 10 ␮M bastadin 5 for 3 min. Resting membrane potentials
and [Ca2⫹]i were measured immediately before and after incubation, using the same microelectrodes. Expression of WT/MHRyR1s in the measured cells was
confirmed by immunostaining. Data are presented as means ⫹ SE. *Significantly different from the resting [Ca2⫹]i for the same construct before treatment with
Rya⫹Bst (P ⬍ 0.05, Student’s t-test). B: the decrease in resting [Ca2⫹]i ([Ca2⫹]i before Rya⫹Bst treatment ⫺ [Ca2⫹]i after Rya⫹Bst treatment) in cells
expressing WT/MHRyR1s after treatment with Rya⫹Bst. Cells expressing MHRyR1s had an almost twofold greater percent reduction in [Ca2⫹]i after treatment with
Rya⫹Bst (36, 38, 35, and 42%, respectively) than cells expressing WTRyR1 (20%) *Significantly different (P ⬍ 0.05) compared with WTRyR1.
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RESTING MYOPLASMIC [Ca2⫹] IN
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channel when the cytoplasmic control domains are uncoupled
from the channel domain of the receptor; however, the mechanism for this uncoupling is as yet unknown (46).
The evidence for the existence of a RyR1 leak conformation
from our previous study was that in the presence of bastadin 5,
maximum binding for ryanodine binding of SR vesicles could
be increased more than twofold while Kd remained constant.
Likewise, Ca2⫹ uptake in SR vesicles treated with bastadin 5
increased nearly 2.5-fold in the presence of ryanodine or
ryanodine receptor (RR) (20) compared with untreated vesicles
or vesicles treated with ryanodine or RR alone. Our present
data represent the first direct pharmacological demonstration
that RyR1s set the resting [Ca2⫹]i in wild-type myotubes and
contribute significantly to the chronic elevation in resting
[Ca2⫹]i associated with MH. The current study also establishes
a relationship between resting [Ca2⫹]i and changes in the
filling capacity of the SR store previously reported. Both rely
on the RyR1 leak conformation. It is interesting to note that
with ryanodine and bastadin 5 treatment, the reduction in the
elevated resting [Ca2⫹]i in muscle cells expressing MHRyR1s is
not only absolutely greater but also significantly greater when
measured as a percentage of the resting value before treatment.
This would imply that expression of MHRyR1s is associated
with an absolute increase in the percentage of RyR1s that
assume the ryanodine-insensitive RyR1 leak conformation.
However, the fact that resting [Ca2⫹]i did not fall to normal
values in myotubes expressing MHRyR1s after cotreatment
with ryanodine and bastadin 5 suggests that this is not the only
mechanism contributing to the elevated resting [Ca2⫹]i. The
remaining elevation could come from the possibility that we
did not use sufficient concentrations of bastadin 5 to convert all
of the MHRyRs from their RyR1 leak conformation to an
actively gating conformation, since these actions are dose
dependent, and/or, more likely, that other mechanisms are also
involved.
Other possible mechanisms that could contribute to the
remaining intracellular Ca2⫹ dysfunction observed in muscle
cells expressing MHRyR1s could include MHRyR1-related mitochondrial dysfunction (6) that could cause a change in
energetics. Such a change, in turn, could lead to an increased
resting [Ca2⫹]i by changing SR Ca2⫹ pump efficiency, causing
a possible dysfunction of normal sarcolemmal Ca2⫹ entry, or
by causing a possible dysfunction in Ca2⫹ extrusion mechanisms. A change in basal energetics in MH-susceptible individuals, demonstrated using 31P NMR spectroscopy as a increase in the Pi/phosphocreatine ratio, was suggested (37–39),
but later studies were unable to confirm this abnormality (4,
34). Furthermore, none of the studies of muscle metabolism
that have measured skeletal muscle lactate production using
microdialysis have shown any measurable change in baseline
values between MH and wild-type humans or pigs (1, 7, 45).
Thus the theory that a defect in metabolism could be the
mechanism for the unresolved increase in resting [Ca2⫹]i
seems unlikely or at best is highly controversial. However, it is
possible that neither of these two techniques are sensitive
enough to account for the change in metabolism that would be
sufficient to cause the small non-RyR-related increase in resting [Ca2⫹]i that we observed. Further work is required to
resolve this issue and answer the question.
Finally, it is important to note that the mechanisms discussed
above and the results of this study are only relevant to MH
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What may be the most exciting result of this study, the effect
of exposure to bastadin 5 alone or the simultaneous exposure to
ryanodine and bastadin 5 on the resting [Ca2⫹]i in myotubes
expressing WT/MHRyR1, appears to shed some light on the
possible mechanism(s) for the chronically elevated resting
[Ca2⫹]i in MH muscle cells. Bastadin 5 is a natural product
isolated from the marine sponge I. basta, which selectively
modulates RyR1’s conformation through an allosteric mechanism requiring an intact FKBP12/RyR1 complex. In previous
studies (30, 40), our group has shown that RyRs appear to exist
in two conformations. The first is an active gating conformation. In the actively gating “channel” conformation, RyR1 has
a finite but variable number of rapid transitions from a closed
state to one or more high-conductance open states (⬃25–100
pS for Ca2⫹). The “gating channel” is readily measured in
bilayer lipid membrane reconstitutions, where closed and open
states and substates can be clearly defined. The probability that
the channel is in an open state (Po) when the Ca2⫹ concentration is in the physiological range in the myoplasm (60 –300 nM)
is very low (⬍0.01). When Ca2⫹ is increased to 100 ␮M, the Po
of channels nominally increases to a maximum of ⬃0.4 – 0.8,
making it possible to see this high-conductance channel rapidly
open and close. When a RyR1 in the channel conformation is
in the open state, it can bind ryanodine and can be blocked by
FLA 365 and ruthenium red. The present studies with bastadin
5 confirm previous results with isolated junctional SR showing
that, in addition to the gating channel conformation, which can
be completely blocked by all three compounds, an alternate
RyR1 leak conformation exists that is insensitive to these drugs
and is responsible for setting the filling state of the SR Ca2⫹
store (30, 40). The activity of the RyR1 leak conformation is
not easily measured in bilayer studies, because based on the
rates of efflux that have been previously measured (40), it is
likely to have a much smaller conductance than actively gating
channels; and although never directly measured, the RyR1 leak
conformation is likely to have a high Po with few transitions to
its closed state. Thus it would make only a small contribution
to the large background currents typically incorporated in
bilayer reconstitutions.
Thus, if incorporated into bilayers, the RyR1 leak conformation would likely make only a small contribution to the
relatively large offset current typically incorporated with gating channels. RyR1s in this conformation would be ignored as
baseline current. RyR1s in this RyR1 leak conformation cannot
and do not bind ryanodine with high affinity. Similarly RyR1
channel blockers such as FLA 365 or ruthenium red cannot and
do not act as blockers of the RyR1 leak conformation.
The concept of a channel switching from a leak conformation to one that actively gates is not unique to RyR1. In fact,
RyR1 is just one of several in a class of channels that can exist
in both conformations. The archetypical channels in this class
are the two-pore domain potassium (K2P) channels (for recent
reviews, see Refs. 5, 20). More than sixteen K2P channel genes
have been identified in the mammalian genome. All have
properties of background or leak K⫹ channels and play a
crucial role setting the resting membrane potential and regulating cell excitability. Some of these channels are the targets
of G proteins or AKAP150 and can be converted to an actively
gating state (17–19, 43). RyR1’s smaller “sister” endoplasmic
reticulum calcium release channel, the inositol trisphosphate
receptor, also has been shown to function as a Ca2⫹ leak
MH
RESTING MYOPLASMIC [Ca2⫹] IN
caused by RyR1 mutations and that the mechanisms causing
MH resulting from other causes or mutations independent of
RyR1 may be entirely different.
GRANTS
This work was partially supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grants AR46513 and AR052354 (to P. D.
Allen and I. N. Pessah) and fellowship grant SPE20040901554 (to E. Esteve)
from the Fondation Recherche Medical, Paris, France.
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