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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, opital 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, opital 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, opital 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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... Les modèles récapitulant le mieux la situation pathologique des patients sont ceux qui expriment la ou les mutations de ces patients dans des cellules musculaires. Il peut s'agir de cellules issues de biopsies de patients : ce type de cellules est souvent utilisé pour toutes les formes de RYR1-RM, y compris dans notre équipe (Rendu et al. 2013, Cacheux et al. 2015. Alternativement, l'ADNc de RYR1 porteur d'une mutation de patient peut également être transfecté dans des cellules musculaires dyspédiques murines qui n'expriment pas RYR1 mais expriment les autres protéines du CRC (Lawal et al. 2020). ...
... La seconde lignée, PLA, est issue d'une biopsie d'une patiente porteuse d'une mutation dominante, c.14590T>C (Y4864H ; Cacheux et al. 2015). Cette mutation est responsable de la déstabilisation de l'ARNm. ...
... Surprisingly, no modification in DHPR amount or in the T-tubule density was observed, although calcium influx through DHPR was reduced by 30%. This could reflect the so-called retrograde coupling [35] by which RyR1 controls DHPR function, as observed in the RyR1-KO cells with DHPR functional alteration [36]. ...
Thesis
La contraction musculaire est un processus complexe qui comprend plusieurs étapes. Une stimulation provenant du motoneurone provoque d’abord une dépolarisation de la membrane plasmique de la fibre musculaire. Cette dépolarisation se propage jusqu’à la triade, une structure constituée d’une invagination de la membrane plasmique (le tubule T) flanquée de deux citernes terminales de réticulum sarcoplasmique. La dépolarisation active un canal calcique situé dans le tubule T, le récepteur des dihydropyridines (DHPR). Celui-ci active un second canal calcique situé dans la membrane du réticulum, auquel il est mécaniquement couplé, le récepteur de la ryanodine (RyR1). L’ouverture du canal RyR1 conduit à un relâchement de calcium du réticulum vers le cytosol à l’origine du glissement des filaments contractiles au niveau des sarcomères et donc la contraction musculaire. Les mutations de RYR1, qui sont très nombreuses, provoquent des altérations de l’expression ou de la fonction de RyR1 qui résultent en une diminution du relâchement calcique et causent des pathologies musculaires appelées « myopathies liées à RYR1 » (ou RYR1-related myopathies, RYR1-RM). Le principal symptôme est un défaut de force musculaire de sévérité variable. Il n’existe actuellement aucun traitement pour ces pathologies, bien que plusieurs approches thérapeutiques soient en cours d’investigation. C'est dans ce contexte que nous avons ciblé les altérations du relâchement calcique, que l'on retrouve chez une majorité de patients, quelle que soit la mutation.L’objectif de ce travail de thèse était d’identifier des petites molécules chimiques capables de restaurer le relâchement calcique dans des cellules musculaires issues de patients atteints de RYR1-RM, et ce peu importe la mutation.Dans un premier temps, nous avons étudié l’effet du fulvestrant, un inhibiteur de la voie estrogénique, sur le relâchement calcique de cellules musculaires humaines et murines et sur la force musculaire dans un modèle murin de RYR1-RM développé par notre équipe. Le fulvestrant retarde l’évolution de la perte de force chez les souris mâles uniquement et améliore le relâchement calcique dans les cellules murines. Cependant, il n’a aucun effet sur les cellules humaines.Dans un second temps, nous avons mis au point une méthode de criblage permettant de tester rapidement un grand nombre de molécules et criblé une chimiothèque de 9 252 molécules sur une lignée immortalisée de cellules musculaires d’un patient myopathe. Les hits ont été validés statistiquement, et plusieurs d’entre eux sont efficaces sur une lignée immortalisée issue d’un second patient myopathe porteur d’une mutation différente, ainsi que sur des cellules primaires issues de notre modèle murin de RYR1-RM. Nous avons finalement sélectionné deux des molécules les plus prometteuses, 38D12 et 64E5, et initié la caractérisation de leur mécanisme d’action. Nous avons mis en évidence que 38D12 agit a priori en amont du couplage DHPR/RyR1, alors que 64E5 augmente les stocks calciques libérables du réticulum sarcoplasmique par le biais de l’augmentation de la recapture du calcium. Nous avons ensuite étudié les propriétés pharmacocinétiques de ces molécules. Nous nous dirigeons à présent vers une démarche de validation pré-clinique des molécules.
... 20 This has been further confirmed in mice heterozygous for an RYR1-KO allele that show no phenotype. 21 In addition, allele-specific gene silencing using siRNA has demonstrated a functional benefit in two mouse models with dominant RYR1 mutations resulting in CCD and MH. 12 The goal of the project was therefore to show that switching-off the mutant RYR1 allele while preserving the normal one was beneficial in muscle cells of a patient with a dominant form of RyR1-RM. The allele knockdown was induced using the SpCas9 nuclease, and a double cleavage that would result in the deletion of an essential part of the RYR1 gene associated to frameshift was designed. ...
... Specifically, heterozygous animals from an RyR1 KO mouse line showed that protein production with a single allele reaches 85% of the normal amount produced in WT animals. 21 Moreover, mice with an RyR1 amount of 70% exhibited no muscle strength deficit, as observed in an inducible RyR1-KO mouse model. 26 In humans, the presence of a null allele along with a normal RYR1 allele has not been associated with muscle weakness, as seen in parents of patients with a recessive RyR1-RM. ...
Article
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More than 700 pathogenic or probably pathogenic variations have been identified in the RYR1 gene causing various myopathies collectively known as “RYR1-related myopathies.” There is no treatment for these myopathies, and gene therapy stands out as one of the most promising approaches. In the context of a dominant form of central core disease due to a RYR1 mutation, we aimed at showing the functional benefit of inactivating specifically the mutated RYR1 allele by guiding CRISPR-Cas9 cleavages onto frequent single-nucleotide polymorphisms (SNPs) segregating on the same chromosome. Whole-genome sequencing was used to pinpoint SNPs localized on the mutant RYR1 allele and identified specific CRISPR-Cas9 guide RNAs. Lentiviruses encoding these guide RNAs and the SpCas9 nuclease were used to transduce immortalized patient myoblasts, inducing the specific deletion of the mutant RYR1 allele. The efficiency of the deletion was assessed at DNA and RNA levels, and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study provides in cellulo proof of concept regarding the benefits of mutant RYR1 allele deletion, in the case of a dominant RYR1 mutation, from both a molecular and functional perspective, and could apply potentially to 20% of all patients with a RYR1 mutation.
... Cell culture and HTT-KO cell line production Immortalized human satellite cells (myoblasts) produced from a 25-yr control individual have been previously described and characterized (Mamchaoui et al., 2011;Cacheux et al., 2015; so called CTRL-HM for control human myoblasts in this study). The myoblasts were amplified in a proliferation medium composed of Ham's F-10 (Life Technologies) supplemented with 20% FBS (Life Technologies), 2% Ultroser G (Pall-Sartorius), and 2% penicillin-streptomycin (Life Technologies). ...
Article
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The expression of the Huntingtin protein, well known for its involvement in the neurodegenerative Huntington’s disease, has been confirmed in skeletal muscle. The impact of HTT deficiency was studied in human skeletal muscle cell lines and in a mouse model with inducible and muscle-specific HTT deletion. Characterization of calcium fluxes in the knock-out cell lines demonstrated a reduction in excitation–contraction (EC) coupling, related to an alteration in the coupling between the dihydropyridine receptor and the ryanodine receptor, and an increase in the amount of calcium stored within the sarcoplasmic reticulum, linked to the hyperactivity of store-operated calcium entry (SOCE). Immunoprecipitation experiments demonstrated an association of HTT with junctophilin 1 (JPH1) and stromal interaction molecule 1 (STIM1), both providing clues on the functional effects of HTT deletion on calcium fluxes. Characterization of muscle strength and muscle anatomy of the muscle-specific HTT-KO mice demonstrated that HTT deletion induced moderate muscle weakness and mild muscle atrophy associated with histological abnormalities, similar to the phenotype observed in tubular aggregate myopathy. Altogether, this study points toward the hypotheses of the involvement of HTT in EC coupling via its interaction with JPH1, and on SOCE via its interaction with JPH1 and/or STIM1.
... In line with previous findings, RYR1 expression was either unchanged or reduced in muscles of CCD patients carrying dominant RYR1 mutations. 49,50 This variable expression may be linked to the type of mutation present in the proband, which may influence the stability of the transcript. It should also be mentioned that most CCD-linked RYR1 mutations strongly influence the biophysical properties of the RyR1 calcium channel resulting in less calcium released during ECC. ...
Article
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Congenital myopathies are a group of early onset muscle diseases of variable severity often with characteristic muscle biopsy findings and involvement of specific muscle types. The clinical diagnosis of patients typically relies on histopathological findings and is confirmed by genetic analysis. The most commonly mutated genes encode proteins involved in skeletal muscle excitation contraction coupling, calcium regulation, sarcomeric proteins and thin-thick filament interaction. However, mutations in genes encoding proteins involved in other physiological functions (for example mutations in SELENON and MTM1, which encode for ubiquitously expressed proteins of low tissue specificity) have also been identified. This intriguing observation indicates that the presence of a genetic mutation impacts the expression of other genes whose product is important for skeletal muscle function. The aim of the present investigation was to verify if there are common changes in transcript and microRNA expression in muscles from patients with genetically heterogeneous congenital myopathies, focusing on genes encoding proteins involved in excitation-contraction coupling and calcium homeostasis, sarcomeric proteins, transcription factors and epigenetic enzymes. Our results identify RYR1, ATPB2B and miRNA-22 as common transcripts whose expression is decreased in muscles from congenital myopathy patients. The resulting protein deficiency may contribute to the muscle weakness observed in these patients. This study also provides information regarding potential biomarkers for monitoring disease progression and response to pharmacological treatments in patients with congenital myopathies.
... Mutations causing a reduction in RYR1 protein content: related mouse models A third mechanism of disease for RYR1-related myopathies is associated with a decrease in the overall RYR1 protein levels; this is usually correlated with the presence of compound heterozygous mutations, where the first mutation causes a premature termination codon and the second is a missense mutation (Monnier et al., 2008;Bevilacqua et al., 2011;Cacheux et al., 2015;Brennan et al., 2019). Disease severity is linked to the nature of the mutation present in the expressed second allele as well as on the residual expression of the first hypomorphic allele (Brennan et al., 2019;Elbaz et al., 2019). ...
Article
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In skeletal muscle, Ca²⁺ necessary for muscle contraction is stored and released from the sarcoplasmic reticulum (SR), a specialized form of endoplasmic reticulum through the mechanism known as excitation–contraction (E-C) coupling. Following activation of skeletal muscle contraction by the E-C coupling mechanism, replenishment of intracellular stores requires reuptake of cytosolic Ca²⁺ into the SR by the activity of SR Ca²⁺-ATPases, but also Ca²⁺ entry from the extracellular space, through a mechanism called store-operated calcium entry (SOCE). The fine orchestration of these processes requires several proteins, including Ca²⁺ channels, Ca²⁺ sensors, and Ca²⁺ buffers, as well as the active involvement of mitochondria. Mutations in genes coding for proteins participating in E-C coupling and SOCE are causative of several myopathies characterized by a wide spectrum of clinical phenotypes, a variety of histological features, and alterations in intracellular Ca²⁺ balance. This review summarizes current knowledge on these myopathies and discusses available knowledge on the pathogenic mechanisms of disease.
... 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
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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]. ...
Article
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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]. ...
Preprint
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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. ...
Article
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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.
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The type 1 ryanodine receptor (RyR1) is a Ca²⁺ release channel in the sarcoplasmic reticulum that is essential for skeletal muscle contraction. RyR1 forms a channel with six transmembrane segments, in which S5 is the fifth segment and is thought to contribute to pore formation. However, its role in channel gating remains unclear. Here, we performed a functional analysis of several disease-associated mutations in S5 and interpreted the results with respect to the published RyR1 structures to identify potential interactions associated with the mutant phenotypes. We demonstrate that S5 plays a dual role in channel gating: the cytoplasmic side interacts with S6 to reduce the channel activity, whereas the luminal side forms a rigid structural base necessary for S6 displacement in channel opening. These results deepen our understanding of the molecular mechanisms of RyR1 channel gating and provide insight into the divergent disease phenotypes caused by mutations in S5.
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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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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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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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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.
Article
Central core disease (CCD) is an autosomal dominant congenital myopathy. Diagnosis is based on the presence of cores in skeletal muscles. CCD has been linked to the gene encoding the ryanodine receptor (RYR1) and is considered to be an allelic disease of malignant hyperthermia susceptibility. However, the report of a recessive form of transmission together with a variable clinical presentation has raised the question of the genetic heterogeneity of the disease. Analyzing a panel of 34 families exclusively recruited on the basis of both clinically and morphologically expressed CCD, 12 different mutations of the C-terminal domain of RYR1 have been identified in 16 unrelated families. Morphological analysis of the patients’ muscles showed different aspects of cores, all of them associated with mutations in the C-terminal region of RYR1. Furthermore, we characterized the presence of neomutations in the RyR1 gene in four families. This indicates that neomutations into the RyR1 gene are not a rare event and must be taken into account for genetic studies of families that present with congenital myopathies type ‘central core disease’. Three mutations led to the deletion in frame of amino acids. This is the first report of amino acid deletions in RYR1 associated with CCD. According to a four-transmembrane domain model, the mutations concentrated mostly in the myoplasmic and luminal loops linking, respectively, transmembrane domains T1 and T2 or T3 and T4 of RYR1.