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Functional Characterization of a Central Core Disease RyR1 Mutation (p.Y4864H) Associated with Quantitative Defect in RyR1 Protein

Authors:
  • Claude Bernard University Lyon 1 & Hospices Civils de Lyon

Abstract and Figures

Background: Central Core Disease (CCD) is a congenital myopathy often resulting from a mutation in RYR1 gene. Mutations in RyR1 can increase or decrease channel activity, or induce a reduction in the amount of protein. The consequences of a single mutation are sometimes multiple and the analysis of the functional effects is complex. Objective: The consequences of the p.Y4864H mutation identified in a CCD patient have been studied regarding both RyR1 function and amount. Methods: The amount of RyR1 in human and mouse muscles was evaluated using qRT-PCR and quantitative Western blot, and calcium release was studied using calcium imaging on primary cultures. The results were compared between human and mouse. Results: The p.Y4864H mutation induced an alteration of calcium release, and in addition was associated to a reduction in the amount of RyR1 in the patient's muscle. This suggests two possible pathophysiological mechanisms: the alteration of calcium release could result from a modification of the channel properties of RyR1 or from a RyR1 reduction. In order to discriminate between the two hypotheses, we used the heterozygous RyR1 knockout (RyR1+/-) mouse model showing a comparable RyR1 protein reduction. No reduction in calcium release was observed in primary muscle culture from these mice, and no muscle weakness was measured. Conclusions: Because the reduction in the amount of RyR1 protein has no functional consequences in the murine model, the muscle weakness observed in the patient is most likely the result of a modification of the calcium channel function of RyR1 due to the p.Y4864H mutation.
Calcium release in immortalized patient cells. Calcium imaging performed on control CTRL cells (black circle), and on patient's Y4864H cells (white circle) differentiated for 7 to 8 days before calcium imaging. (A) Fluorescence variation curves induced by membrane depolarization (KCl 140 mM) applied during 60 s (black bar) in the presence of 2 mM external calcium, presented as mean (symbols) ± SEM. (B) Fluorescence variation curves induced by application of 4-Chloro-m-Cresol (CmC) 500 M during 60 s (black bar) in the presence of 2 mM external calcium, presented as mean (symbols) ± SEM. (C) Fluorescence variation curves induced by application of caffeine 40 mM plus thapsigargin 1 M (Caf+Thapsigargin) during 60 s (black bar) in the presence of 2 mM external calcium, presented as mean (symbols) ± SEM. (D) Fluorescence variation curves induced by application of caffeine 40mM plus thapsigargin 1 M in absence of extracellular calcium (Caf+Thapsigargin -Ca 2+ ) during 60 s (black bar) in the presence of Cd 2+ and La 3+ , presented as mean (symbols) ± SEM. (E) The maximal amplitude of the peak for each curve is presented in the bar plots, with the number of myotubes analyzed in each bar. * * * * p<0.0001, Student's t test comparisons between CTRL and Y4864H cells, for each stimulation. (F) The area under each curve (A.U.) has been calculated for each stimulation, in control myotubes (black bars) and Y4864H myotubes (white bars) and is presented as mean ± SEM of the number of myotubes indicated in each bar. Statistics : Student's t-test of Y4864H myotubes compared to control myotubes * * * * p < 0.0001, * * * p < 0.001, ns: non significant.
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Journal of Neuromuscular Diseases 2 (2015) 421–432
DOI 10.3233/JND-150073
IOS Press
421
Research Report
Functional Characterization of a Central
Core Disease RyR1 Mutation (p.Y4864H)
Associated with Quantitative Defect
in RyR1 Protein
Marine Cacheuxa,b, Ariane Bluma,b, Muriel S´
ebastiena,b, Anne Sophie Woznya,b,c,
Julie Brocarda,b, Kamel Mamchaouid, Vincent Moulyd, Nathalie Roux-Buissona,b,c,
John Rendua,b,c, Nicole Monniera,b,c, Ren´
ee Krivosice, Paul Allenf, Arnaud Lacourg,
Jo¨
el Lunardia,b,c, Julien Faur´
ea,b,cand Isabelle Martya,b,
aINSERM U836, Grenoble Institut des Neurosciences, Equipe Muscle et Pathologies, Grenoble, France
bUniversit´eJoseph Fourier, Grenoble, France
cCentre Hospitalier R´egional Universitaire de Grenoble, Hˆopital Michallon, Biochimie G´en´etique
et Mol´eculaire, Grenoble, France
dUMRS974 Inserm, UMR7215 CNRS, Institut de Myologie, GH Piti´eSalp´etri`ere, 47 bd de l’hˆopital, Paris, France
eD´epartement Anesth´esie-R´eanimation, Hˆopital Roger Salengro, CHRU de Lille, Lille, France
fDepartment of Molecular Biosciences, School of Veterinary Medicine, University of California at Davis,
Davis CA, USA
gService de Neurologie, Hˆopital Roger Salengro, CHRU de Lille, Lille, France
Abstract.
Background: Central Core Disease (CCD) is a congenital myopathy often resulting from a mutation in RYR1 gene. Mutations
in RyR1 can increase or decrease channel activity, or induce a reduction in the amount of protein. The consequences of a single
mutation are sometimes multiple and the analysis of the functional effects is complex.
Objective: The consequences of the p.Y4864H mutation identified in a CCD patient have been studied regarding both RyR1
function and amount.
Methods: The amount of RyR1 in human and mouse muscles was evaluated using qRT-PCR and quantitative Western blot, and
calcium release was studied using calcium imaging on primary cultures. The results were compared between human and mouse.
Results: The p.Y4864H mutation induced an alteration of calcium release, and in addition was associated to a reduction in the
amount of RyR1 in the patient’s muscle. This suggests two possible pathophysiological mechanisms: the alteration of calcium
release could result from a modification of the channel properties of RyR1 or from a RyR1 reduction. In order to discriminate
between the two hypotheses, we used the heterozygous RyR1 knockout (RyR1+/) mouse model showing a comparable RyR1
protein reduction. No reduction in calcium release was observed in primary muscle culture from these mice, and no muscle
weakness was measured.
Conclusions: Because the reduction in the amount of RyR1 protein has no functional consequences in the murine model, the
muscle weakness observed in the patient is most likely the result of a modification of the calcium channel function of RyR1 due
to the p.Y4864H mutation.
Keywords: Ryanodine receptor, Central Core Disease, Malignant Hyperthermia, calcium release
Correspondence to: Isabelle Marty, GIN-U836-Eq 4, Bat EJ
Safra – Chemin Fortun´
e Ferrini, 38700 La Tronche – France. Tel.:
+33 4 56 52 05 71; Fax: +33 4 56 52 05 72; E-mail: isabelle.marty@
ujf-grenoble.fr.
ISSN 2214-3599/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License.
422 M. Cacheux et al. / RyR1 Quantitative Defect
ABBREVIATIONS
CCD central core disease
CmC 4-chloro-m-cresol
CTRL control
He heterozygous
KO knockout
MH Malignant Hyperthermia
MHN MH Negative
MHS MH Susceptible
RyR ryanodine receptor
SERCA SarcoEndoplasmic reticulum Ca2+-ATPase
SR Sarcoplasmic reticulum
INTRODUCTION
The sarcoplasmic reticulum (SR) calcium channel
ryanodine receptor RyR1 is encoded by the RYR1 gene
(MIM#180901). In association with the voltage gated
calcium channel dihydropyridine receptor (DHPR) and
numerous regulating proteins, they form the skele-
tal muscle calcium release complex responsible for
the excitation-contraction coupling process in skeletal
muscle [1].
Mutations in the RYR1 gene have been associated
with congenital myopathies such as Central Core Dis-
ease (CCD; OMIM#117000) [2–4] and Multiminicore
Disease (MmD; OMIM# 255320) [5–7]. CCD has
been named after anatomo-pathological muscle anal-
ysis, and is characterized by the presence of cores in
type 1 muscle fibers, which are large areas of abnormal
myofibrillar architecture with sarcomeric disorganiza-
tion and absence of mitochondria [2, 8]. CCD presents
either an autosomal dominant or a recessive transmis-
sion pattern. Clinical presentations are heterogeneous
ranging from mild phenotype, with moderate hypo-
tonia during early childhood, delayed motor abilities,
and slowly progressive proximal muscle weakness, to
severe phenotype, including fetal akinesia, respiratory
insufficiency at birth and generalized muscle weakness
[9, 10].
Dominantly inherited RYR1 mutations have been
extensively studied to identify the pathophysiological
mechanism. Two mechanisms have been proposed so
far: either a gain of function of RyR1 (hyperactivity)
leading to a calcium leak or a loss of RyR1 function
with impaired calcium conductance [11–14].
It has been observed that recessive mutations in
RYR1 can also result in a reduction in the amount
of protein [15, 16], which leads to muscle weakness
and altered calcium release [17]. In the present work,
we studied the pathological mechanisms leading to a
moderate CCD in which a missense dominant muta-
tion in RyR1 was associated with a 25% decrease in
the amount of protein. Until now the effect of such a
RyR1 reduction has never been explored. Using an ani-
mal model presenting with a similar reduction in the
amount of RyR1, we showed no alteration of muscle
calcium release or of muscle strength. We concluded
that the consequence of this RyR1 mutation in the
patient’s muscle was not related to the reduction in
the amount of protein, but rather to the direct effect
of the mutation on the channel function. These results
provide new insights into the pathogenesis of RYR1-
related myopathies with RyR1 deficiency.
MATERIALS AND METHODS
Ethics statement
Investigations on patient material were performed
after signature of an informed consent according to
the French regulation and have received approval
from the local ethical committee (Comit´
e de Protec-
tion des Personnes-Sud-Est, France). All procedures
using animals were approved by the Institutional Ethics
Committee and followed the guidelines of the National
Research Council Guide for the care and use of labo-
ratory animals.
Muscle biopsy and IVCT
Part of the muscle biopsy realized in vastus
lateralis has been directly processed for in vitro
contracture test (IVCT) according to the Euro-
pean Malignant Hyperthermia Group Guidelines
(http://www.emhg.org/emhg/mh-diagnosis/). Another
part was used for cell culture and Western blot, and the
remaining muscle was frozen in liquid nitrogen cooled
isopentane for histological and mRNA analyses.
Molecular genetic studies
RYR1 mutations screening was performed using
cDNA obtained after reversetranscription of total RNA
extracted from muscle biopsy as previously described
[2]. The cDNA was amplified in overlapping frag-
ments. Sequencing reactions were analyzed on an ABI
3130 DNA Analyzer (Life Technologies, Saint Aubin,
France). The presence of the mutation identified in the
transcript was confirmed in genomic DNA by direct
M. Cacheux et al. / RyR1 Quantitative Defect 423
sequencing of the corresponding exon and intron-exon
junctions.
Mouse lines
RyR1 knock out mice (RyR1/), heterozygous
mice (RyR1+/) and wild type (RyR1+/+) were
obtained by crossing heterozygous Ryr1tm1Alle mice
(http://www.informatics.jax.org/allele/key/637575).
In this mouse line, the insertion of a neomycin selec-
tion cassette in the KPN site at nt840 in exon10 of
the RYR1 gene leads to disruption of gene expression
[27].
Mouse muscle homogenate preparation
Skeletal muscles were collected from the hind
limbs of adult (6-7 months old) male mice. Crude
homogenates were prepared by homogenization in 200
mM sucrose, 20 mM HEPES (pH 7.4), 0.4 mM CaCl2,
200 M phenylmethylsulfonyl fluoride, 1 mM diiso-
propyl fluorophosphate, as described previously [18].
Protein concentration was measured using a modified
Folin assay in presence of SDS.
Quantitative western blot analysis
The amount of RyR1 present in muscle samples
(20–40 g of muscle homogenate) was determined
by quantitative Western blot analysis using antibod-
ies directed against RyR1 [19] and normalized to the
amount of myosin heavy chain as described previ-
ously [16]. Briefly, after electrophoretic separation on
a 5–15% gradient acrylamide gel and electrotrans-
fer to Immobilon P (Biorad, Marnes la Coquette,
France) during 4h at 0.8A to ensure a complete trans-
fer of the loaded proteins, the membrane was incubated
with anti-RyR1 antibodies and then HRP-labelled sec-
ondary antibodies. Variation in protein loading or in
muscle protein content due to heterogeneity of the sam-
ple, e.g. muscle fibrosis, was evaluated as the amount of
myosin in each lane determined by Coomassie staining
of the Immobilon membrane after immunorevelation
(measured as the surface of the myosin band). The total
amount of RyR1 in each experiment (total signal on
the two or three bands) was thus corrected from the
amount of myosin and normalized to the amount of
RyR1 present in the control referred as 100%. Quantifi-
cation was also performed using anti-desmin antibody
(Dakocytomation, Les Ulis, France) for normalization
compared to the amount of desmin, and similar results
were obtained. Signal quantification was performed
using a ChemiDoc XRS apparatus (Biorad, Marnes
la Coquette, France) and the Quantity One software
(Biorad).
q RT-PCR
Human muscle
Total RNA was extracted from frozen muscle biopsy
using TRIzol reagent (Life Technologies, Saint Aubin,
France) as previously described [16]. First strand
cDNA synthesis from 500ng total RNA was realized
using random priming with the High Capacity cDNA
Reverse Transcription kit (Life Technologies, Saint
Aubin, France). Real time quantification of mRNAs
of target gene RYR1 and of reference genes (hRPL27
and ACTB) was performed with Power SYBR Green
PCR Master Mix (Life Technologies, Saint Aubin,
France) using a STEPONEPLUS (Life Technologies,
Saint Aubin, France) detection system. The following
primers were used (53): RYR1 pcr1 forward: CAT
GGC TTC GAG ACT CAC AC, RYR1 pcr1 reverse:
CTC CTG ACC CGT GTG TTC T, RYR1 pcr2 for-
ward: GAC TCA CAC GCT GGA GGA G, RYR1 pcr2
reverse: TCC AGA CAT AAG ACT CCT GAC C,
ACTB forward: CTC CTG AGC GCA AGT ACT CC,
ACTB reverse : TGT TTT CTG CGC AAG TTA GG,
hRPL27 forward : CGC AAA GCT GTC ATC GTG,
hRPL27 reverse : GTC ACT TTG CGG GGG TAG.
The following experimental protocol was used: denat-
uration 95C for 10 min and 40 cycles of 95C for
15 sec and 60C for 60 sec. Melting curve analysis
showed specific melting temperatures.
Mouse muscle
Total RNA from skeletal muscle (tibialis anterior)
of 2-month-old WT (RyR1+/+) and heterozygous
(RyR1+/) mice (3 animals in each group) was
extracted using TRIzol reagent (Life Technologies,
Saint Aubin, France) and PureLink RNA Mini Kit
(Life Technologies, Saint Aubin, France). First strand
cDNA was obtained using oligo(dT) primed reverse
transcription from 500ng of RNA. Real time quantifi-
cation of mRNAs of target gene RYR1 and of reference
gene (GAPDH) was performed by iQ SYBR Green
supermix (BioRad, Marnes la Coquette, France) using
an IQ iCycler (BioRad, Marnes la Coquette, France)
detection system. The following primers were used
(53): RYR1, forward - ATG ACC GTA GGG CTC
CTG GCC GTA G, reverse - GGG TCC TCG ATC
TCG TCC CCG A; GAPDH forward - GTA TGA CTC
CAC TCA CGG CAA A, reverse - TTC CCA TTC
TCG GCC TTG. The following experimental protocol
424 M. Cacheux et al. / RyR1 Quantitative Defect
was used: denaturation 95C for 3 min and 40 cycles
of 95C for 10 sec and 55C for 45 sec. Melting curve
analysis showed specific melting temperatures.
Data were analyzed with the comparative thresh-
old cycle (Ct) relative-quantification method. Relative
gene expression was quantified as follows: fold
change = 2(Ct) where Ct =Cttarget Ctreference
and (Ct) =Ctsample Ctcontrol. Ct is the frac-
tional cycle number at which the fluorescence passes
the fixed threshold. The target gene represents RYR1
gene, reference genes are the hRPL27 and ACTB gene
in human and the GAPDH gene in mouse, “sample”
refers to patient or heterozygous mice and “control” to
control human or wild type mice.
Production of human and mouse primary cultures
Human satellite cells were produced from a muscle
biopsy of a 25-year-old donor without neuromuscu-
lar disorder (CTRL cells) and from the biopsy of the
patient (Y4864H cells). These cells were immortalized
and cloned as previously described [20, 21]. Pri-
mary cultures of skeletal muscle from WT, RyR1+/
and RyR1/E19 mouse embryos were produced as
described previously [22].
Cell culture
Immortalized human satellite cells or mouse satel-
lite cells were amplified in proliferation medium
composed of Ham’s F-10 (Life technologies, Saint
Aubin, France) supplemented with 20% FBS (Life
technologies, Saint Aubin, France), 2% Ultroser G
(Pall Biosepra, St Germain en Laye, France) and
2% Penicillin-Streptomycin (Life technologies, Saint
Aubin, France). Differentiation into myotubes was
induced by a shift to differentiation medium: DMEM
(Life technologies, Saint Aubin, France) supplemented
with 2% Heat Inactivated Horse Serum (Life tech-
nologies) and 1% Penicillin-Streptomycin. Human
myotubes were cultured for seven to eight days and
mouse myotubes for two to three days before intracel-
lular calcium measurements.
Intracellular calcium measurements
Changes in intracellular calcium were evaluated
using the calcium-dependent fluorescent dye Fluo-
4 AM (Life Technologies, Saint Aubin, France), as
described previously [23]. Calcium imaging was per-
formed in Krebs buffer (136 mM NaCl, 5 mM KCl, 2
mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 6
mM D-Glucose). To obtain a calcium-free Krebs solu-
tion, CaCl2was left out, while 1mM EGTA, 10 M
La3+, and 50 MCd
2+were added. Thapsigargin
(Life technologies, Saint Aubin, France) was diluted at
1M in this medium and applied simultaneously with
caffeine (40 mM) as described previously [24, 25]. 4-
chloro-m-cresol (4-CmC) (Sigma-Aldrich) was diluted
at 500 M in Krebs buffer and 140 mM KCl solution
was prepared in 2 mM CaCl2, 1 mM MgCl2,10mM
HEPES, pH 7.4 and 6 mM D-Glucose. Fluorescence
was measured by confocal laser scanning microscopy
using a Leica TCS-SPE microscope in the xyt mode.
Changes in intracellular Ca2+concentrations were pre-
sented as the ratio of fluorescence intensities with
respect to the initial fluorescence intensity prior to drug
addition (F/F0). Data are given as mean ±SEM, and
nrepresents the number of myotubes studied in each
condition.
Evaluation of muscle strength
Muscle strength was evaluated in patients using
manual muscle testing according to the Medical
Research Council scale [26]. Muscle strength was eval-
uated in mice using a hang test as described previously
[18]. Two months old male mice were positioned on
a cross-wired surface turned upside down, and time
before fall (up to 300 s) was measured.
Statistical analysis
Data were pooled over animals or cells within
the same group and are presented as means ±SEM.
Differences between CTRL and Y4864H cells were
assessed using Student’s t-test. Differences between
WT, RyR1+/(He) cells, and RyR1/(KO) cells
were assessed using Student’s t-test with Bonferroni
correction for multiple comparison, and GraphPad
Prism 6 software, assuming significance at p< 0.05.
RESULTS
Clinical and genetic reports
The proband (Figs. 1A, III:3) was a 28-year-
old female who presented with a moderate muscle
weakness since childhood, a slightly delayed motor
development and a stable axial muscle deficiency.
Muscle strength evaluation showed no distal defi-
ciency, no reduction in cervical or hamstring muscles
strength, a slight reduction in pectoralis major and
M. Cacheux et al. / RyR1 Quantitative Defect 425
Fig. 1. Case description. (A). Pedigree of the family. Circles rep-
resent females and squares males. Filled symbols indicate affected
individuals. The proband is indicated by the black arrow. Segrega-
tion of alleles is indicated below each individual. MH= Malignant
Hyperthermia; MHN = MH Negative; MHS = MH Susceptible. (B).
Quantitative analysis of RyR1 expression (protein and mRNA)in the
skeletal muscle of the proband (Individual III.3). Twenty g mus-
cle homogenate from control (CTRL, lane 1) or proband (Y4864H,
lane 2) were loaded on a 4–15% polyacrylamide gel. The amount
of RyR1 protein is expressed as the percentage of RyR1 present in
the control muscle, which relative expression compared to myosin
was set at 100%. The central bars graph presents RyR1 protein mean
amount ±SEM from seven different Western blots. The bar graph
on the right is the Q-RT-PCR analysis of levels of RyR1 mRNA
expressed as a percentage of control (which relative expression
compared to reference genes was set to 100%). The data are pre-
sented as mean ±SEM of 9–12 different amplifications. ∗∗p< 0.01,
∗∗∗∗p< 0.0001, Student’s t-test. (C) Representative Western blot of
different controls (C1-C4), non-affected women between 20 and
23 years.
deltoid muscles strength (level evaluated at 4/5), and
a marked reduction in abdominal muscles strength
(level evaluated at 2/5). Creatine kinase (CK) levels
were normal (75 U/L, Table 1, III:3). A muscle biopsy
Table 1
Analysis of patients
CK IVCT data
Hal 2% Caf 2 mM
III : 3 75 6N 5N
II : 2 168 10N 6N
II : 1 51 <2N <2N
CK levels and IVCT data of the proband (III.3) and two individ-
uals from the family (II.2 and II.1). CK = Creatine Kinase activity
is expressed in international units per liter (IU/L; normal <205).
IVCT = In Vitro Contracture Test. Hal= tension in Newton (N) at
2% halothane, the normal value being <2N. Caf = tension at 2 mM
Caffeine, the normal value being <2N.
demonstrated the presence of central cores using
NADH staining and an in vitro contracture test (IVCT)
indicated susceptibility to Malignant Hyperthermia
(MHS) (Table 1, III:3) because of hypersensitivity of
RyR1 to both caffeine and halotane. The genetic analy-
sis performed on the mRNA extracted from the muscle
biopsy identified the c.14590T>C; p.Tyr4864His vari-
ation at heterozygous state in the RYR1 gene, resulting
in the substitution of the tyrosine in position 4864 by a
histidine (p.Y4864H) in the RyR1 protein. A diagnosis
of CCD was proposed.
The proband’s father (Figs. 1A, II:2) presented with
a late onset moderate muscle weakness, and did not
report any muscle weakness during childhood and
adolescence. A clinical examination at age 60 demon-
strated a mild quadriceps amyotrophy, right scapular
winging and scoliosis. Muscle strength evaluation indi-
cated a slight reduction in biceps, pectoralis major
and hamstring muscles strength (evaluated at 4/5),
and an important axial deficiency (abdominal muscles
2/5, cervical muscles 3/5). His CK levels were nor-
mal (168 U/L, Table 1, II:2). Muscle biopsy displayed
an aspect of congenital myopathy with the presence of
atypical cores using NADH staining. He was also diag-
nosed as being susceptible to Malignant Hyperthermia
(MHS) by IVCT (Table 1, II:2), and a genetic analysis
confirmed that he was carrying the same RYR1 het-
erozygous variant c. 14590T>C; p.Tyr4864His as his
daughter.
The patient’s uncle (II:1) who was tested MH Nega-
tive (MHN) by IVCT did not present with any clinical
sign of myopathy and did not have the variant (Fig. 1A
and Table 1). Therefore, the two individuals of the
family carrying the p.Y4864H mutation were affected
by a moderate CCD associated to MH, suggesting a
dominant transmission.
Using quantitative RT-PCR, the amount of RYR1
transcripts in the muscle of the proband (III.3) was
evaluated at 52.6%±3.2% of the control muscle
426 M. Cacheux et al. / RyR1 Quantitative Defect
(Fig. 1B), pointing to either the absence of tran-
script produced from one of the two RYR1 alleles
or a reduction in both transcripts. The presence of
the c.14590T>C variation at a heterozygous state in
the mRNA transcripts detected by Sanger sequenc-
ing, suggested that most probably both transcripts were
reduced. High Resolution Melting (HMR) analysis on
cDNA of the patient confirmed that 2 transcripts were
present in equivalent amount when amplifying a region
encompassing the c.14590 position (data not shown).
No other variation in the RyR1 mRNA was found to
explain the global transcript reduction.
Using quantitative Western blot, we determined that
the amount of RyR1 present in the muscle of the
proband (Ind. III.3) was 75.3 ±6.4% (n=7)ofthe
amount of RyR1 in a control muscle from a 25 years old
female patient without any muscle disease (Fig. 1B). To
confirm that the amount of RyR1 in age-related control
biopsies do not present similar variation and to validate
our quantitative Western blot analysis, a quantitative
Western blot was performed on four different 20–23
years-old non affected women, with normal physical
activity (Fig. 1C). The quantification of the amount of
RyR1 compared to desmin in 11 blots performed from
these 4 controls result in a relative amount of RyR1 of
100% ±6.3%.
Consequences of the mutation
To determine the physiological effects of the muta-
tion, primary cultures were produced from the muscle
biopsy of the patient, and were immortalized by
double retroviral transduction using telomerase and
Cdk4 [20, 21]. Calcium imaging studies were per-
formed on myotubes produced from the proband’s
immortalized cells (Y4864H cells) or from immor-
talized cells of a volunteer of 25 years with no
muscle disease (CTRL cells), to assess their abil-
ity to release calcium after stimulation (Fig. 2). In
response to the membrane depolarization induced by
addition of 140 mM KCl in presence of extracellu-
lar calcium, Ca2+release was significantly reduced in
Y4864H myotubes (Fig. 2A, white circles) compared
to CTRL (Fig. 2A, black circles) (p< 0.0001). Simi-
larly calcium release induced by a direct stimulation
of RyR1 by 4-CmC (Fig. 2B) was also significantly
decreased (p< 0.0001). The amount of Ca2+in the
SR stores was evaluated after caffeine stimulation
in presence of thapsigargin with or without extra-
cellular calcium (Fig. 2C and D). In all cases, the
maximal amplitude of calcium released was signif-
icantly reduced in Y4864H myotubes compared to
CTRL myotubes (Fig. 2E, p< 0.0001). The area under
the curve (Fig. 2F) reflecting the amount of calcium
released was also significantly reduced except for the
caffeine stimulation in presence of thapsigargin and
extracellular calcium. The latter indicates that the
reduction in the free SR calcium rapidly releasable
upon stimulation could be compensated by an influx
of external calcium (Fig. 2C).
These results could be explained either i) by a
reduction in the amount of calcium stored due to
the p.Y4864H mutation leading to a “leaky” RyR1
channel, as usually observed with MH mutations lead-
ing to RyR1 hypersensitivity or ii) by defects in the
RyR1-DHPR coupling leading to impaired calcium
conductance [11, 12], or iii) by the decreased quantity
of RyR1 protein [17].
RyR1 expression in RyR1+/heterozygous mice
So far, the effect on calcium release of a small
RyR1 protein decrease such as the one measured for
the patient has not been evaluated. To test this effect,
we used cells from heterozygous mice of a RyR1 KO
model [27]. Muscle homogenates were prepared from
WT and heterozygous RyR1+/mice to measure the
amount of RyR1 transcript and protein. Only one allele
of the RYR1 gene is expressed in RyR1+/heterozy-
gous mice, and we first confirmed using quantitative
RT-PCR that the mRNA of RyR1 was reduced by about
50% compared to WT (42.6% ±7% for RyR1+/mice
compared to 100% ±4.8% for WT, n= 3) (Fig. 3C).
Using quantitative Western blot, the amount of RyR1
at the protein level detected in RyR1+/mice muscles
was estimated to be 83.4 ±2.6% of WT muscle (n=9)
(Fig. 3A and B).
These results showed that both the amount of RyR1
transcript and protein were decreased in RyR1+/
heterozygous mice compared to WT mice, although
to a lesser extent at the protein level. The relative
protein amount of RyR1 measured in the patient
with the p.Y4864H mutation compared to control
(75.3 ±6.4%, n= 7) was not statistically different from
the one found in RyR1+/mice compared to WT mice
(83.4 ±2.6%, n= 9), and the relative amounts of tran-
script were also not different (52.6% ±3.2%, n=12
in the patient and 42.6% ±7%, n= 3 in the mouse).
In both case, the alteration in the amount of RyR1 at
the protein level is milder than the 50% lowering of
transcription. Therefore the RyR1+/mouse line con-
stitutes a good model to study the effect of such a RyR1
reduction.
M. Cacheux et al. / RyR1 Quantitative Defect 427
Fig. 2. Calcium release in immortalized patient cells. Calcium imaging performed on control CTRL cells (black circle), and on patient’s Y4864H
cells (white circle) differentiated for 7 to 8 days before calcium imaging. (A) Fluorescence variation curves induced by membrane depolarization
(KCl 140 mM) applied during 60 s (black bar) in the presence of 2 mM external calcium, presented as mean (symbols) ±SEM. (B) Fluorescence
variation curves induced by application of 4-Chloro-m-Cresol (CmC) 500 M during 60s (black bar) in the presence of 2 mM external calcium,
presented as mean (symbols) ±SEM. (C) Fluorescence variation curves induced by application of caffeine 40 mM plus thapsigargin 1M
(Caf+Thapsigargin) during 60 s (black bar) in the presence of 2 mM external calcium, presented as mean (symbols) ±SEM. (D) Fluorescence
variation curves induced by application of caffeine 40mM plus thapsigargin 1M in absence of extracellular calcium (Caf+Thapsigargin - Ca2+)
during 60 s (black bar) in the presence of Cd2+and La3+, presented as mean (symbols) ±SEM. (E) The maximal amplitude of the peak for each
curve is presented in the bar plots, with the number of myotubes analyzed in each bar. ∗∗∗∗p<0.0001, Student’s ttest comparisons between CTRL
and Y4864H cells, for each stimulation. (F) The area under each curve (A.U.) has been calculated for each stimulation, in control myotubes
(black bars) and Y4864H myotubes (white bars) and is presented as mean ±SEM of the number of myotubes indicated in each bar. Statistics :
Student’s t-test of Y4864H myotubes compared to control myotubes ∗∗∗∗p< 0.0001, ∗∗∗ p< 0.001, ns: non significant.
428 M. Cacheux et al. / RyR1 Quantitative Defect
Fig. 3. Expression of RyR1 in heterozygous RyR1+/mouse muscles. (A) Quantitative Western blot analysis of RyR1 expression in skeletal
muscle homogenates from WT mice (WT) or from heterozygous RyR1+/mice (He). (B) The relative amount of RyR1 at the protein level
compared to myosin was set to 100% in WT mice. The amount of RyR1 in He mice is presented as mean ±SEM of 9 experiments performed in
3 different mice. ∗∗∗p< 0.001 Student’s t-test between WT and He. (C) Q-RT-PCR analysis of levels of RyR1 mRNA expressed as a percentage
of WT mice (which relative expression compared to GAPDH was set to 100%). The data are presented as mean±SEM of 3 different mice.
∗∗p< 0.01 Student’s t-test between WT and He.
Measure of muscle strength in RyR1+/mice
To evaluate the overall muscle performance of
RyR1+/heterozygous mice, a hang test was per-
formed. Two-month-old male mice were allowed to
grip on a cross-wired surface placed upside down. Time
spent hanging on the surface before fall was measured
and no significant difference was observed between
WT (182 ±43 s; n= 7) and RyR1+/(168 ±36 s;
n= 9) mice (supplementary data, Figure S1). This hang
test thus showed that the decrease in RyR1 protein in
RyR1+/mice did not modify muscle strength.
Effect of RyR1 reduction on calcium fluxes
To check whether the decrease in the quantity of
RyR1 protein in RyR1+/mice induced defects on
the calcium release, calcium imaging studies were
performed on RyR1+/+(WT), RyR1+/(He) and
RyR1/(KO) myotubes (Fig. 4). In agreement with
some previous results [28] we observed that, com-
pared to WT (Fig. 4A and B, black circles) and He
myotubes (Fig. 4A and B, gray squares), the calcium
release was greatly depressed in KO myotubes (Fig. 4A
and B, white circle) whether it be after a membrane
depolarization (140mM KCl) or after a direct RyR1
stimulation (500 M 4-CmC). Both stimuli induced
calcium release in WT myotubes (Fig. 4A and B,
black circles) and He myotubes (Fig. 4A and B, gray
squares). The amplitude of the peak in WT and He
myotubes using RyR1 direct stimulation was simi-
lar (Fig. 4C). The peak was significantly increased
in He compared to WT when KCl stimulation was
used (Fig. 4C). These data demonstrated that the 16%
decrease in RyR1 protein observed in He mice did not
impair their ability to release calcium upon stimulation
compared to the WT mice, consistent with the lack of
muscle weakness observed during the hang test.
DISCUSSION
Functional studies of common dominant RYR1
mutations associated with CCD have suggested two
main mechanisms responsible for a disturbed func-
tion of the mutant RyR1 channel, namely either the
presence of a leaky and hyperactive calcium channel
associated with a reduction of SR calcium stores [13],
or an “uncoupled” channel with reduced permeabil-
ity of RyR1 to Ca2+[29]. In the present situation,
the p.Y4864H mutation resulted in the presence of a
calcium channel with altered properties which is asso-
ciated with a reduction of the protein amount. A 50%
reduction in the RyR1 mRNA in the patient biopsy
was also observed, but the mechanisms leading to in
this reduction in the RyR1 mRNA was not identi-
fied. In vitro calcium imaging showed that with all the
stimulations used the amount of calcium released in
Y4864H cells was significantly reduced. A reduction
in the amplitude of the calcium store was observed
using caffeine stimulation in the absence of external
M. Cacheux et al. / RyR1 Quantitative Defect 429
Fig. 4. Calcium release in mouse cells. Calcium imaging performed on WT satellites cells (black circle), on RyR1+/heterozygous mouse cells
(gray square) and on RyR1/KO mouse cells (white circle) differentiated for 2 to 3 days before calcium imaging. (A) Fluorescence variation
curves induced by membrane depolarization (KCl 140 mM) applied during 40 s (black bar) in the presence of 2 mM external calcium, presented
as mean (symbols) ±SEM. (B) Fluorescence variation curves induced by application of 4-Chloro-m-Cresol (CmC) 500 M during 40s (black
bar) in the presence of 2 mM external calcium, presented as mean (symbols) ±SEM. (C) The maximal amplitude of the peak for each curve is
presented in the bar plots, with the number of myotubes analyzed in each bar. ∗∗∗∗p< 0.0001, Student’s ttest followed by Bonferroni correction
for multiple comparison, compared to WT. (D) The area under each curve (A.U.) has been calculated for each stimulation, in WT myotubes
(black bars), He myotubes (gray bars) and KO myotubes (white bars) and is presented as the mean ±SEM of the number of myotubes indicated
in each bar. ∗∗∗∗p< 0.0001, Student’s ttest followed by Bonferroni correction for multiple comparison, compared to WT.
calcium influx and in the absence of calcium re-uptake
in the sarcoplasmic reticulum (caffeine plus thapsigar-
gin, Cd2+and La3+, Fig. 2D). This reduction in the
amount of stored calcium, which could reflect a leaky
calcium channel, was compensated by an influx of
external calcium when present (caffeine stimulation in
presence of thapsigargin with Ca2+, Fig. 2C), leading
to equivalent amounts of calcium released in Y4864H
cells compared to control cells (Fig. 2F). This reduc-
tion in the amount of stored calcium could account for
the muscle weakness observed in the patient, but as it
can be compensated in some conditions, it can only be
a partial explanation. In order to identify another mech-
anism for the muscle weakness observed, we focused
our studies on the impact of the reduction of RyR1
quantity. As RyR1 deficiency has been proposed to
result in disruption of EC coupling [17], we studied
calcium release in a mouse model expressing a nor-
mal RyR1 but with a similar reduction in the amount
of protein. This model was the heterozygous RyR1+/
mouse line that has only one functional RYR1 allele. We
first confirmed the presence of RyR1 transcript at about
a 50% level of a WT mouse. Noticeably, the RyR1 pro-
tein level was higher, reaching 85% of a WT mouse,
suggesting a post-transcriptional regulation of the pro-
tein amount, identical to the situation in observed
in the patient. In the heterozygous mouse myotubes,
no reduction in the amount of released calcium was
observed either after KCl membrane depolarization
mimicking EC coupling or after direct RyR1 stimu-
lation by CmC. In addition, RyR1+/mice showed
no muscle weakness. Those results confirmed that this
degree of RyR1 reduction was not sufficient to result in
muscle weakness or to alter calcium release. Therefore,
430 M. Cacheux et al. / RyR1 Quantitative Defect
Fig. 5. Localization of the mutation in the structure of the pro-
tein. The localization of the mutation presented in this study,
p.Y4864H, as well as two close amino acids R4864 and I4898 both
involved in CCD, are reported on the structure of RyR1 recently
proposed from single particle electron cryomicroscopy [31, 32].
These amino acids are in the luminal loop between transmembrane
helixes S5 and S6, which contain the pore helix (P-helix) involved in
pore formation.
muscle weakness and alteration of calcium release
observed in the patient expressing the p.Y4864H muta-
tion is most probably not related to the reduction of
RyR1 amount, but rather to the presence of mutant
monomers into the tetrameric channel. As the mutation
induced a reduction in the amount of calcium stored in
the sarcoplasmic reticulum, and as this channel is most
probably hyperactive as observed by the hypersensitiv-
ity evaluated in IVCT, it could be hypothesized that this
mutation resulted in a hyperactive and leaky calcium
channel. Calcium leak would explain the reduction
in the stored calcium and muscle weakness, and the
hyperactive channel would explain the increased sen-
sitivity observed in IVCT leading to the MHS status of
the patient.
The 4864 position mapped within the last luminal
loop of RyR1 monomer, which is involved in the selec-
tivity pore for calcium permeation (P-loop), and is
close to several other sites already found mutated in
association with Central core Disease. A CCD fam-
ily has previously been described with a mutation at
the same position but leading to the substitution of
tyrosine for cysteine instead of histidine (p.Y4864C)
[30]. The affected patients of this family presented with
a moderate myopathy, not associated to MH. Note-
worthy, another mutation in position 4898 (p.I4898T),
localized only 34 amino acids further away in the
same intracellular loop, resulted in a severely uncou-
pled RyR1 [3]. It can therefore be postulated that the
Y4864 and the I4898 amino acids belong to different
functional domains although they are in close vicinity
along the primary sequence of RyR1. A steric inhi-
bition in the movement of the luminal loop has been
proposed as a consequence of the p.I4898T mutation,
but due to a different outcome on muscle physiology,
cannot account for the effect of p.Y4864H. Look-
ing at the structure of RyR1 recently published [31,
32] (Fig. 5), these two residues are on both side of
the so-called P-loop or pore-helix between transmem-
brane segments S5 and S6. Because of the alteration
of RyR1’s channel function found in the patient one
can hypothesize that the p.Y4864H mutation disrupts
a binding site specific of a modulator of RyR1 func-
tion. Triadin could be such a modulator, as 3 amino
acids in position 4878, 49707 and 49708 have been
shown to be involved in the RyR-triadin interaction
[33], D4878 being very close to Y4864. Nevertheless,
mutation for Ala of this single Asp in position 4878
induced a slight reduction in the RyR-triadin bind-
ing, but no modification in the calcium release [34].
Therefore the effect of a mutation in position 4864
should most probably not be related to an alteration of
the interaction RyR-triadin. Mutation on amino acid
4861 has also been frequently associated with CCD,
often as a neo-mutation [35], with no or only a slight
reduction in the amount of protein [35] and small alter-
ation in calcium release (reduction in the amount of
calcium stored, measured on immortalized B-cells,
[36]). The effects of mutation in position 4861 are
quite similar to those observed for mutation in posi-
tion 4864, and a similar physiopathological mechanism
could be hypothesized for mutations at these two posi-
tions.
Overall, our study reports a CCD case where the
effect of the RyR1 Y4864H mutation was explored.
The reduction of total RyR1 mRNA in the patient mus-
cle could not be explained but the fact that the amount
of RyR1 protein was at 75% of a control suggested the
presence of post transcriptional or epigenetic controls.
This hypothesis is strengthened by the study of mRNA
and protein in a RYR1 KO heterozygous mouse model,
that shows the same level of RyR1 produced with a
50% reduction of mRNA. Our work also showed that
the pathophysiological mechanisms linked to defects
in calcium release in cells of the patient could not be
attributed to a reduction in RyR1 protein levels, but
rather to a direct effect of the p.Y4864H mutation on
the channel properties.
M. Cacheux et al. / RyR1 Quantitative Defect 431
ACKNOWLEDGMENTS
We thank all family members for their contribution
to this study. This work was supported by grants from
the “Association Franc¸aise contre les Myopathies”
(AFM), the “Fondation Daniel Ducoin", the “Institut
National de la Sant´
e et de la Recherche M´
edicale”
(INSERM), and the “Vivier de la Recherche de la
Facult´
edeM
´
edecine de Grenoble “. We thank Dr A.F.
Delmas and Mrs. I. Stix for their help with the muscle
biopsies, and the Myocastor study’s group (MSG) for
fruitful discussions.
SUPPLEMENTARY MATERIAL
Supplementary Figure S1 is available in the
electronic version of this article: http://dx.doi.org/
10.3233/JND-150073.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
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... To dissect the mechanisms related to a reduction in RyR1 amount we have developed and characterized a mouse model with a conditional and inducible RYR1 knock out. In mice, the full deletion of the RYR1 gene is lethal at birth (Takeshima et al., 1994), and heterozygous deletion of a single RYR1 allele results only in a modest 15% reduction in RyR1 protein, with no functional consequences (Cacheux et al., 2015). In order to obtain a model with reduced amount of RyR1 specifically in skeletal muscles, we have developed a new mouse line RyR1 Flox/Flox ::HSA-Cre-ER T2 in which a decreased RYR1 expression is induced by tamoxifen injection. ...
... Our results suggest a direct correlation between the amount of RyR1 protein and the muscle strength. In our model, almost no muscle weakness was observed when RyR1 amount was above 80% of control, as already observed with the heterozygous whole body RyR1-KO mice that show a RyR1 protein amount of 85% of control and no functional alteration (Cacheux et al., 2015). ...
... Signal quantification was performed using a ChemiDoc Touch apparatus (Biorad, France) and the Image Lab software (Biorad). The amount of the chosen protein in each sample was corrected for differences in loading using either the amount of myosin, GAPDH or the total amount of proteins using the stain free system from Biorad, and normalized to the amount of the same protein present in the control, set to 100% as described previously (Cacheux et al., 2015). Ten human controls (muscle biopsy from individuals non-affected by neuromuscular disease) of different age have been used, from 3.5 years to 64 years. ...
Article
Full-text available
Mutations in the RYR1 gene, encoding the skeletal muscle calcium channel RyR1, lead to congenital myopathies, through expression of a channel with abnormal permeability and/or in reduced amount, but the direct functional whole organism consequences of exclusive reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Tamoxifen-induced recombination in the RYR1 gene at adult age resulted in a progressive reduction in the protein amount reaching a stable level of 50% of the initial amount, and was associated with a progressive muscle weakness and atrophy. Measurement of calcium fluxes in isolated muscle fibers demonstrated a reduction in the amplitude of RyR1-related calcium release mirroring the reduction in the protein amount. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution and membrane remodeling. An increase in the expression level of many proteins was observed, as well as an inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Dusty Core Disease patients (a subtype of Central Core Disease), pointing to common pathophysiological mechanisms related to RyR1 reduction.
... To dissect the mechanisms related to a reduction in RyR1 amount we have developed and characterized a mouse model with a conditional and inducible RYR1 knock out. In mice, the full deletion of the RYR1 gene is lethal at birth (Takeshima et al., 1994), and heterozygous deletion of a single RYR1 allele results only in a modest 15% reduction in RyR1 protein, with no functional consequences (Cacheux et al., 2015). In order to obtain a model with reduced amount of RyR1 specifically in skeletal muscles, we have developed a new mouse line RyR1 Flox/Flox ::HSA-Cre-ER T2 in which a decreased RYR1 expression is induced by tamoxifen injection. ...
... Our results suggest a direct correlation between the amount of RyR1 protein and the muscle strength. In our model, almost no muscle weakness was observed when RyR1 amount was above 80% of control, as already observed with the heterozygous whole body RyR1-KO mice that show a RyR1 protein amount of 85% of control and no functional alteration (Cacheux et al., 2015). ...
... Signal quantification was performed using a ChemiDoc Touch apparatus (Biorad, France) and the Image Lab software (Biorad). The amount of the chosen protein in each sample was corrected for differences in loading using either the amount of myosin, GAPDH or the total amount of proteins using the stain free system from Biorad, and normalized to the amount of the same protein present in the control, set to 100% as described previously (Cacheux et al., 2015). Ten human controls (muscle biopsy from individuals non-affected by neuromuscular disease) of different age have been used, from 3.5 years to 64 years. ...
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Some mutations in the RYR1 gene lead to congenital myopathies, through reduction in this calcium channel expression level, but the functional whole organism consequences of reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Recombination in the RYR1 gene resulted in a progressive reduction in the protein amount and was associated with a progressive muscle weakness and atrophy. Calcium fluxes in isolated muscle fibers were accordingly reduced. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution, membrane remodeling, associated with increase in the expression level of many proteins and inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Central Core Disease patients, pointing to common pathophysiological mechanisms related to RyR1 reduction.
... If not treated quickly with the RYR1 antagonist dantrolene to lower intracellular Ca 2+ , it can be fatal (Lopez et al., 1987). Susceptibility to MHS is diagnosed by the standardized European in vitro contracture test, where a muscle biopsy is exposed to incremental doses of stimulants (halothane, caffeine, succinythane) to assess the threshold of muscle contraction (Cacheux et al., 2015;Gillard et al., 1992;Mickelson and Louis, 1996;Quane et al., 1993;Shuaib et al., 1987). If threshold contraction levels are lower than normal, the patient is deemed at risk of MHS. ...
... The lines were derived both from healthy donors and patients with 14 different muscle diseases, including Duchenne, facioscapulohumeral, and congenital muscular dystrophies (Mamchaoui et al., 2011). They also developed a CCD line carrying the p.Y4864H mutation that was used by Cacheux et al. (2015) to assess the effect of this patient mutation in 2D myotubes. The authors discovered that p.Y4864H altered RYR1channel properties and reduced protein expression, as observed in the patient. ...
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The core myopathies are a group of congenital myopathies with variable clinical expression - ranging from early-onset skeletal-muscle weakness to later-onset disease of variable severity - that are identified by characteristic 'core-like' lesions in myofibers and the presence of hypothonia and slowly or rather non-progressive muscle weakness. The genetic causes are diverse; central core disease is most often caused by mutations in ryanodine receptor 1 (RYR1), whereas multi-minicore disease is linked to pathogenic variants of several genes, including selenoprotein N (SELENON), RYR1 and titin (TTN). Understanding the mechanisms that drive core development and muscle weakness remains challenging due to the diversity of the excitation-contraction coupling (ECC) proteins involved and the differential effects of mutations across proteins. Because of this, the use of representative models expressing a mature ECC apparatus is crucial. Animal models have facilitated the identification of disease progression mechanisms for some mutations and have provided evidence to help explain genotype-phenotype correlations. However, many unanswered questions remain about the common and divergent pathological mechanisms that drive disease progression, and these mechanisms need to be understood in order to identify therapeutic targets. Several new transgenic animals have been described recently, expanding the spectrum of core myopathy models, including mice with patient-specific mutations. Furthermore, recent developments in 3D tissue engineering are expected to enable the study of core myopathy disease progression and the effects of potential therapeutic interventions in the context of human cells. In this Review, we summarize the current landscape of core myopathy models, and assess the hurdles and opportunities of future modeling strategies.
... To date, few studies have focused on the elucidation of the specific pathophysiological mechanisms of disease and in general, to disease treatment. Analysis of ryr1 +/− heterozygous mice showed that ablation of one RYR1 allele leads to a 15% decrease of RyR1 protein content (21). Such a modest decrease of RyR1 does not impact muscle function as determined by the hanging wire test, and does not affect peak calcium transients in cultured satellite-derived myotubes. ...
... In this context it should be pointed out that the reduction of protein content in muscle biopsies from patients carrying recessive RYR1 mutations (9) is 2-3-fold lower compared to that observed in the ryr1 +/− mice. In addition, the mutation of the ryr1 +/− mice investigated by Cacheux et al. (21) is not isogenic with mutations identified so far in patients. ...
Article
Here we characterized a mouse model knocked-in for a frameshift mutation in RYR1 exon 36 (p.Gln1970fsX16) that is isogenic to that identified in one parent of a severely affected patient with recessively inherited multiminicore disease. This individual carrying the RYR1 frameshifting mutation complained of mild muscle weakness and fatigability. Analysis of the RyR1 protein content in a muscle biopsy from this individual showed a content of only 20% of that present in a control individual. The biochemical and physiological characteristics of skeletal muscles from RyR1Q1970fsX16 heterozygous mice recapitulates that of the heterozygous parent. RyR1 protein content in the muscles of mutant mice reached 38% and 58% of that present in total muscle homogenates of fast and slow muscles from wild-type (WT) littermates. The decrease of RyR1 protein content in total homogenates is not accompanied by a decrease of Cav1.1 content, whereby the Cav1.1/RyR1 stoichiometry ratio in skeletal muscles from RyR1Q1970fsX16 heterozygous mice is lower compared to that from WT mice. Electron microscopy (EM) revealed a 36% reduction in the number/area of calcium release units accompanied by a 2.5-fold increase of dyads (triads that have lost one junctional sarcoplasmic reticulum element); both results suggest a reduction of the RyR1 arrays. Compared to WT, muscle strength and depolarization-induced calcium transients in RyR1Q1970fsX16 heterozygous mice muscles were decreased by 20% and 15%, respectively. The RyR1Q1970fsX16 mouse model provides mechanistic insight concerning the phenotype of the parent carrying the RYR1 ex36 mutation and suggests that in skeletal muscle fibres there is a functional reserve of RyR1.
... This protocol was applied to immortalized myoblasts from a healthy subject 15 (so-called HM cells, for human myoblasts), in which the RyR1 has been previously characterized 16 The best guides predicted with Crispor software were selected in order to have as few off-targets as possible, while keeping the best efficiency. We chose to use two guides at the same time in the same viral vector, to make sure that a major part of the cells will be knock-out, and to ease the detection of the deletion in edited clones using PCR. ...
Article
One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.
... The use of Tg to deplete ER/SR calcium stores is in skeletal muscle cells is well documented [55][56][57][58][59]. Many cellular model systems of RYR1-RM rely on the transfection of mutant RYR1 cDNA into HEK293 cells, which lack several components responsible for the regulation of RYR1 function [60]. ...
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Background Aberrations to endoplasmic/sarcoplasmic reticulum (ER/SR) calcium concentration can result in the departure of endogenous proteins in a phenomenon termed exodosis. Redistribution of the ER/SR proteome can have deleterious effects to cell function and cell viability, often contributing to disease pathogenesis. Many proteins prone to exodosis reside in the ER/SR via an ER retention/retrieval sequence (ERS) and are involved in protein folding, protein modification, and protein trafficking. While the consequences of their extracellular presence have yet to be fully delineated, the proteins that have undergone exodosis may be useful for biomarker development. Skeletal muscle cells rely upon tightly coordinated ER/SR calcium release for muscle contractions, and perturbations to calcium homeostasis can result in myopathies. Ryanodine receptor type-1 (RYR1) is a calcium release channel located in the SR. Mutations to the RYR1 gene can compromise calcium homeostasis leading to a vast range of clinical phenotypes encompassing hypotonia, myalgia, respiratory insufficiency, ophthalmoplegia, fatigue and malignant hyperthermia (MH). There are currently no FDA approved treatments for RYR1-related myopathies (RYR1-RM). Results Here we examine the exodosis profile of skeletal muscle cells following ER/SR calcium depletion. Proteomic analysis identified 4,465 extracellular proteins following ER/SR calcium depletion with 1,280 proteins significantly different than vehicle. A total of 54 ERS proteins were identified and 33 ERS proteins significantly increased following ER/SR calcium depletion. Specifically, ERS protein, mesencephalic astrocyte-derived neurotrophic factor (MANF), was elevated following calcium depletion, making it a potential biomarker candidate for human samples. Despite no significant elevation of MANF in plasma levels among healthy volunteers and RYR1-RM individuals, MANF plasma levels positively correlated with age in RYR1-RM individuals, presenting a potential biomarker of disease progression. Selenoprotein N (SEPN1) was also detected only in extracellular samples following ER/SR calcium depletion. This protein is integral to calcium handling and SEPN1 variants have a causal role in SEPN1-related myopathies (SEPN1-RM). Extracellular presence of ER/SR membrane proteins may provide new insight into proteomic alterations extending beyond ERS proteins. Pre-treatment of skeletal muscle cells with bromocriptine, an FDA approved drug recently found to have anti-exodosis effects, curbed exodosis of ER/SR resident proteins. Conclusion Changes to the extracellular content caused by intracellular calcium dysregulation presents an opportunity for biomarker development and drug discovery.
... The use of Tg to deplete ER/SR calcium stores is in skeletal muscle cells is well documented [55][56][57][58][59]. Many cellular model systems of RYR1-RM rely on the transfection of mutant RYR1 cDNA into HEK293 cells, which lack several components responsible for the regulation of RYR1 function [60]. ...
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Aberrations to endoplasmic/sarcoplasmic reticulum (ER/SR) calcium concentration can result in the departure of endogenous proteins in a phenomenon termed exodosis. Redistribution of the ER/SR proteome can have deleterious effects to cell function and cell viability, often contributing to disease pathogenesis. Many proteins prone to exodosis reside in the ER/SR via an ER retention/retrieval sequence (ERS) and are involved in protein folding, protein modification, and protein trafficking. While the consequences of their extracellular presence have yet to be fully delineated, the proteins that have undergone exodosis may be useful for biomarker development. Skeletal muscle cells rely upon tightly coordinated ER/SR calcium release for muscle contractions, and perturbations to calcium homeostasis can result in myopathies. Ryanodine receptor type-1 (RYR1) is a calcium release channel located in the SR. Mutations to the RYR1 gene can compromise calcium homeostasis leading to a vast range of clinical phenotypes encompassing hypotonia, myalgia, respiratory insufficiency, ophthalmoplegia, fatigue and malignant hyperthermia (MH). There are currently no FDA approved treatments for RYR1-related myopathies (RYR1-RM). Here we examine the exodosis profile of skeletal muscle cells following ER/SR calcium depletion. Proteomic analysis identified 4,465 extracellular proteins following ER/SR calcium depletion with 1280 proteins significantly different than vehicle. A total of 54 ERS proteins were identified and 33 ERS proteins significantly increased following ER/SR calcium depletion. Specifically, ERS protein, mesencephalic astrocyte-derived neurotrophic factor (MANF), was elevated following calcium depletion, making it a potential biomarker candidate for human samples. Despite no significant elevation of MANF in plasma levels among healthy volunteers and RYR1-RM individuals, MANF plasma levels positively correlated with age in RYR1-RM individuals, presenting a potential biomarker of disease progression. Selenoprotein N (SEPN1) was also detected only in extracellular samples following ER/SR calcium depletion. This protein is integral to calcium handling and SEPN1 variants have a causal role in SEPN1-related myopathies (SEPN1-RM). Extracellular presence of ER/SR membrane proteins may provide new insight into proteomic alterations extending beyond ERS proteins. Pre-treatment of skeletal muscle cells with bromocriptine, an FDA approved drug recently found to have anti-exodosis effects, curbed exodosis of ER/SR resident proteins. Changes to the extracellular content caused by intracellular calcium dysregulation presents an opportunity for biomarker development and drug discovery.
... Main observed features were muscle atrophy, abnormal mitochondrial distribution, fiber remodeling, inhibition of autophagy, and increased protein expression of proteins implicated in calcium handling and muscle structure including STIM1 and desmin. Of note, the Ryr1 +/− mice did not present muscle weakness or reduced Ca 2+ release, although only a 15% decrease in protein was achieved [149]. Overall, it seems that the reduction in RyR1 protein to at least 50% is necessary to observe functional defects in mice. ...
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Centronuclear myopathies (CNM) are rare congenital disorders characterized by muscle weakness and structural defects including fiber hypotrophy and organelle mispositioning. The main CNM forms are caused by mutations in: the MTM1 gene encoding the phosphoinositide phosphatase myotubularin (myotubular myopathy), the DNM2 gene encoding the mechanoenzyme dynamin 2, the BIN1 gene encoding the membrane curvature sensing amphiphysin 2, and the RYR1 gene encoding the skeletal muscle calcium release channel/ryanodine receptor. MTM1, BIN1, and DNM2 proteins are involved in membrane remodeling and trafficking, while RyR1 directly regulates excitation-contraction coupling (ECC). Several CNM animal models have been generated or identified, which confirm shared pathological anomalies in T-tubule remodeling, ECC, organelle mispositioning, protein homeostasis, neuromuscular junction, and muscle regeneration. Dynamin 2 plays a crucial role in CNM physiopathology and has been validated as a common therapeutic target for three CNM forms. Indeed, the promising results in preclinical models set up the basis for ongoing clinical trials. Another two clinical trials to treat myotubular myopathy by MTM1 gene therapy or tamoxifen repurposing are also ongoing. Here, we review the contribution of the different CNM models to understanding physiopathology and therapy development with a focus on the commonly dysregulated pathways and current therapeutic targets.
... While a ∼ 50% reduction in RYR1 mRNA was observed in muscle from both Ryr1 Indel/+ and Ryr1 TM/Indel mice, ∼ 80% reduction in RyR1 protein was observed only in Ryr1 TM/Indel mice. Our finding of a ∼ 50% reduction in RYR1 transcript with a minimal reduction in RYR1 protein in Ryr1 Indel/+ mice is similar to that reported recently in mice with a single RYR1 null allele (∼60% reduction in RYR1 transcript with only a ∼ 15% reduction in RYR1 protein) (32). The precise reason for the marked (∼80%) Downloaded from https://academic.oup.com/hmg/advance-article-abstract/doi/10.1093/hmg/ddz105/5489941 by Guilford College user on 19 July 2019 reduction in RYR1 protein level in Ryr1 TM/Indel mice will require additional experimentation. ...
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RYR1 related myopathies (RYR1 RM) are a clinically and histopathologically heterogeneous group of conditions that represent the most common subtype of childhood onset non-dystrophic muscle disorders. There are no treatments for this severe group of diseases. A major barrier to therapy development is the lack of an animal model that mirrors the clinical severity of pediatric cases of the disease. To address this, we used CRISPR/Cas9 gene editing to generate a novel recessive mouse model of RYR1 RM. This mouse (Ryr1TM/Indel) possesses a patient relevant point mutation (T4706M) engineered into one allele and a 16 base pair frameshift deletion engineer into the second allele. Ryr1TM/Indel mice exhibit an overt phenotype beginning at 14 days of age that consists of reduced body/muscle mass and myofibre hypotrophy. Ryr1TM/Indel mice become progressively inactive from that point onward and die at a median age of 42 days. Histopathological assessment shows myofibre hypotrophy, increased central nuclei, and decreased triad number, but no clear evidence of metabolic cores. Biochemical analysis reveals a marked decrease in RYR1 protein levels (20% of normal) as compared to only a 50% decrease in transcript. Functional studies at end stage show significantly reduced electrically-evoked Ca2+ release and force production. In summary, Ryr1TM/Indel mice exhibit a post-natal lethal recessive form of RYR1 RM that pheno-copies the severe congenital clinical presentation seen in a subgroup of RYR1 RM children. Thus, Ryr1TM/Indel mice represent a powerful model for both establishing the pathomechanisms of recessive RYR1 RM and pre-clinical testing of therapies for efficacy.
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Background: Pathogenic variations in the gene encoding the skeletal muscle ryanodine receptor (RyR1) are associated with malignant hyperthermia (MH) susceptibility, a life-threatening hypermetabolic condition and RYR1-related myopathies (RYR1-RM), a spectrum of rare neuromuscular disorders. In RYR1-RM, intracellular calcium dysregulation, post-translational modifications, and decreased protein expression lead to a heterogenous clinical presentation including proximal muscle weakness, contractures, scoliosis, respiratory insufficiency, and ophthalmoplegia. Preclinical model systems of RYR1-RM and MH have been developed to better understand underlying pathomechanisms and test potential therapeutics. Methods: We conducted a comprehensive scoping review of scientific literature pertaining to RYR1-RM and MH preclinical model systems in accordance with the PRISMA Scoping Reviews Checklist and the framework proposed by Arksey and O'Malley. Two major electronic databases (PubMed and EMBASE) were searched without language restriction for articles and abstracts published between January 1, 1990 and July 3, 2019. Results: Our search yielded 5049 publications from which 262 were included in this review. A majority of variants tested in RYR1 preclinical models were localized to established MH/central core disease (MH/CCD) hot spots. A total of 250 unique RYR1 variations were reported in human/rodent/porcine models with 95% being missense substitutions. The most frequently reported RYR1 variant was R614C/R615C (human/porcine total n = 39), followed by Y523S/Y524S (rabbit/mouse total n = 30), I4898T/I4897T/I4895T (human/rabbit/mouse total n = 20), and R163C/R165C (human/mouse total n = 18). The dyspedic mouse was utilized by 47% of publications in the rodent category and its RyR1-null (1B5) myotubes were transfected in 23% of publications in the cellular model category. In studies of transfected HEK-293 cells, 57% of RYR1 variations affected the RyR1 channel and activation core domain. A total of 15 RYR1 mutant mouse strains were identified of which ten were heterozygous, three were compound heterozygous, and a further two were knockout. Porcine, avian, zebrafish, C. elegans, canine, equine, and drosophila model systems were also reported. Conclusions: Over the past 30 years, there were 262 publications on MH and RYR1-RM preclinical model systems featuring more than 200 unique RYR1 variations tested in a broad range of species. Findings from these studies have set the foundation for therapeutic development for MH and RYR1-RM.
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The ryanodine receptors (RyRs) are high-conductance intracellular Ca(2+) channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5,000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryomicroscopy. Three previously uncharacterized domains, named central, handle and helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative-charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity-filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.
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Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca 21) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation–contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å . We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended a-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the a-solenoid scaffold, suggesting a mechanism for channel gating by Ca 21 . Ryanodine receptors (RyRs) are intracellular Ca 21 release channels on the sarcoplasmic and endoplasmic reticula required for fundamental cellular functions in most tissues, including skeletal and cardiac muscle excitation–contraction coupling, synaptic transmission and pancre-atic beta cell function 1 . The type 1 ryanodine receptor (RyR1) mediates excitation–contraction coupling in skeletal muscle. It is a homotetra-mer of four ,565-kilodalton (kDa) channel-forming protomers, as well as regulatory subunits, enzymes and their respective targeting/ anchoring proteins, forming a macromolecular complex that exceeds 3,000,000 daltons 2 . In most tissues, RyRs are activated by the inward flow of Ca 21 via plasma-membrane Ca 21 channels, resulting in a mas-sive and rapid release of Ca 21 from intracellular stores (a process known as Ca 21
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In skeletal muscle, excitation-contraction coupling is the process whereby the voltage-gated dihydropyridine receptor (DHPR) located on the transverse tubules activates calcium release from the sarcoplasmic reticulum by activating ryanodine receptor (RyR1) Ca(2+) channels located on the terminal cisternae. This subcellular membrane specialization is necessary for proper intracellular signalling and any alterations in its architecture may lead to neuromuscular disorders. In this study we present evidence that patients with recessive RYR1-related congenital myopathies due to primary RyR1 deficiency also exhibit down-regulation of the alfa 1 subunit of the DHPR and show disruption of the spatial organization of the excitation-contraction coupling machinery. We created a cellular RyR1 knock-down model using immortalized human myoblasts transfected with RyR1 siRNA and confirm that knocking down RyR1 concomitantly down-regulates not only the DHPR but also the expression of other proteins involved in excitation-contraction coupling. Unexpectedly, this was paralleled by the up-regulation of inositol-1,4,5-triphosphate receptors; functionally however, up-regulation of the latter Ca(2+) channels did not compensate for the lack of RyR1-mediated Ca(2+) release. These results indicate that in some patients, RyR1 deficiency concomitantly alters the expression pattern of several proteins involved in calcium homeostasis and that this may influence the manifestation of these diseases.
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Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.
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Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.
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The type 1 isoform of the ryanodine receptor (RYR1) is the Ca(2+) release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation-contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1(I4898T) mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca(2+) content, and RYR1 Ca(2+) release channel function using adult heterozygous Ryr1(I4895T/+) knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca(2+) content, both electrically evoked and 4-chloro-m-cresol-induced Ca(2+) release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4-6-mo-old IT/+ mice. The sensitivity of the SR Ca(2+) release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca(2+) permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca(2+) release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca(2+) ion permeation.
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Triadin is a multiple proteins family, some isoforms being involved in muscle excitation-contraction coupling, and some having still unknown functions. To obtain clues on triadin functions, we engineered a triadin knock-out mouse line and characterized the physiological effect of triadin ablation on skeletal muscle function. These mice presented a reduced muscle strength, which seemed not to alter their survival and has been characterized in the present work. We first checked in these mice the expression level of the different proteins involved in calcium homeostasis and observed in fast muscles an increase in expression of dihydropyridine receptor, with a large reduction in calsequestrin expression. Electron microscopy analysis of KO muscles morphology demonstrated the presence of triads in abnormal orientation and a reduction in the sarcoplasmic reticulum terminal cisternae volume. Using calcium imaging on cultured myotubes, we observed a reduction in the total amount of calcium stored in the sarcoplasmic reticulum. Physiological studies have been performed to evaluate the influence of triadin deletion on skeletal muscle function. Muscle strength has been measured both on the whole animal model, using hang test or electrical stimulation combined with NMR analysis and strength measurement, or on isolated muscle using electrical stimulation. All the results obtained demonstrate an important reduction in muscle strength, indicating that triadin plays an essential role in skeletal muscle function and in skeletal muscle structure. These results indicate that triadin alteration leads to the development of a myopathy, which could be studied using this new animal model.
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The ryanodine receptor (RYR1) is an essential component of the calcium homeostasis of the skeletal muscle in mammals. Inactivation of the RYR1 gene in mice is lethal at birth. In humans only missense and in-frame mutations in the RYR1 gene have been associated so far with various muscle disorders including malignant hyperthermia, central core disease and the moderate form of multi-minicore disease (MmD). We identified a cryptic splicing mutation in the RYR1 gene that resulted in a 90% decrease of the normal RYR1 transcript in skeletal muscle. The 14646þ2.99 kb A!G mutation was associated with the classical form of MmD with ophthalmoplegia, whose genetic basis was previously unknown. The mutation present at a homozygous level was responsible for a massive depletion of the RYR1 protein in skeletal muscle. The mutation was not expressed in lymphoblastoid cells, pointing toward a tissue specific splicing mechanism. This first report of an out-of-frame mutation that affects the amount of RYR1 raised the question of the amount of RYR1 needed for skeletal muscle function in humans.
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. CCS embryonic stem (ES) cells possessing two mutant alleles (ry1r−/ry1r−) for the skeletal muscle ryanodine receptor (RyR) have been produced and injected subcutaneously into severely compromised immunodeficient mice to produce teratocarcinomas in which Ry1R expression is absent. Several primary fibroblast cell lines were isolated and subcloned from one of these tumors that contain the knockout mutation in both alleles and exhibit a doubling time of 18–24 h, are not contact growth inhibited, do not exhibit drastic morphological change upon serum reduction, and possess the normal complement of chromosomes. Four of these fibroblast clones were infected with a retrovirus containing the cDNA encoding myoD and a puromycin selection marker. Several (1–2 μg/ml) puromycin-resistant subclones from each initial cell line were expanded and examined for their ability to express myoD and to form multinucleated myotubes that express desmin and myosin upon removal of mitogens. One of these clones (1B5 cells) was selected on this basis for further study. These cells, upon withdrawal of mitogens for 5–7 d, were shown by Western blot analysis to express key triadic proteins, including skeletal triadin, calsequestrin, FK506-binding protein, 12 kD, sarco(endo)plasmic reticulum calcium–ATPase1, and dihydropyridine receptors. Neither RyR isoform protein, Ry1R (skeletal), Ry2R (cardiac), nor Ry3R (brain), were detected in differentiated 1B5 cells. Measurements of intracellular Ca2+ by ratio fluorescence imaging of fura-2–loaded cells revealed that differentiated 1B5 cells exhibited no responses to K+ (40 mM) depolarization, ryanodine (50–500 μM), or caffeine (20–100 mM). Transient transfection of the 1B5 cells with the full-length rabbit Ry1R cDNA restored the expected responses to K+ depolarization, caffeine, and ryanodine. Depolarization-induced Ca2+ release was independent of extracellular Ca2+, consistent with skeletal-type excitation–contraction coupling. Wild-type Ry1R expressed in 1B5 cells were reconstituted into bilayer lipid membranes and found to be indistinguishable from channels reconstituted from rabbit sarcoplasmic reticulum with respect to unitary conductance, open dwell times, and responses to ryanodine and ruthenium red. The 1B5 cell line provides a powerful and easily managed homologous expression system in which to study how Ry1R structure relates to function.
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Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca(2+) from intracellular stores during excitation-contraction coupling in both cardiac and skeletal muscle. RyRs are the largest known ion channels (> 2MDa) and exist as three mammalian isoforms (RyR 1-3), all of which are homotetrameric proteins that interact with and are regulated by phosphorylation, redox modifications, and a variety of small proteins and ions. Most RyR channel modulators interact with the large cytoplasmic domain whereas the carboxy-terminal portion of the protein forms the ion-conducting pore. Mutations in RyR2 are associated with human disorders such as catecholaminergic polymorphic ventricular tachycardia whereas mutations in RyR1 underlie diseases such as central core disease and malignant hyperthermia. This chapter examines the current concepts of the structure, function and regulation of RyRs and assesses the current state of understanding of their roles in associated disorders.