doi:10.

1093/brain/awu368 BRAIN 2015: 138; 246–268 | 246

REVIEW ARTICLE
Pathophysiological concepts in the congenital
myopathies: blurring the boundaries,
sharpening the focus
Gianina Ravenscroft,1 Nigel G. Laing1 and Carsten G. Bönnemann2

The congenital myopathies are a diverse group of genetic skeletal muscle diseases, which typically present at birth or in early
infancy. There are multiple modes of inheritance and degrees of severity (ranging from foetal akinesia, through lethality in the
newborn period to milder early and later onset cases). Classically, the congenital myopathies are defined by skeletal muscle
dysfunction and a non-dystrophic muscle biopsy with the presence of one or more characteristic histological features. However,
mutations in multiple different genes can cause the same pathology and mutations in the same gene can cause multiple different
pathologies. This is becoming ever more apparent now that, with the increasing use of next generation sequencing, a genetic
diagnosis is achieved for a greater number of patients. Thus, considerable genetic and pathological overlap is emerging, blurring
the classically established boundaries. At the same time, some of the pathophysiological concepts underlying the congenital
myopathies are moving into sharper focus. Here we explore whether our emerging understanding of disease pathogenesis and
underlying pathophysiological mechanisms, rather than a strictly gene-centric approach, will provide grounds for a different and
perhaps complementary grouping of the congenital myopathies, that at the same time could help instil the development of shared
potential therapeutic approaches. Stemming from recent advances in the congenital myopathy field, five key pathophysiology
themes have emerged: defects in (i) sarcolemmal and intracellular membrane remodelling and excitation-contraction coupling;
(ii) mitochondrial distribution and function; (iii) myofibrillar force generation; (iv) atrophy; and (v) autophagy. Based on numerous
emerging lines of evidence from recent studies in cell lines and patient tissues, mouse models and zebrafish highlighting these
unifying pathophysiological themes, here we review the congenital myopathies in relation to these emerging pathophysiological
concepts, highlighting both areas of overlap between established entities, as well as areas of distinction within single gene disorders.

1 Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia, Nedlands, Western
Australia, Australia
2 National Institute of Neurological Disorders and Stroke/NIH, Porter Neuroscience Research Centre, Bethesda, MD, USA
Correspondence to: Carsten G. Bönnemann, MD,
Neuromuscular and Neurogenetic Disorders of Childhood Section, Neurogenetics Branch
National Institute of Neurological Disorders and Stroke/NIH
Porter Neuroscience Research Centre 35 Convent Drive, Bldg 35, Room 2A-116
Bethesda, MD 20892-3705
E-mail: carsten.bonnemann@nih.gov

Keywords: congenital myopathy; pathophysiology; mitochondria; membrane remodelling; force generation; atrophy; autophagy;
triad; excitation-contraction coupling
Abbreviations: CNM = centronuclear myopathy; DHPR = dihydropyridine receptors; KI/KO = knock-in/knock-out;
NMJ = neuromuscular junction; SR = sarcoplasmic reticulum

Received May 6, 2014. Revised November 21, 2014. Accepted November 22, 2014. Advance Access publication December 31, 2014
Published by Oxford University Press on behalf of the Guarantors of Brain 2014. This work is written by US Government employees and is in the public domain in the US.
Pathophysiology of congenital myopathies
Introduction
The congenital myopathies are clinically defined by hypo-
tonia and skeletal muscle weakness and pathologically by
the presence of one or more histopathological or ultrastruc-
tural hallmarks on muscle biopsy (North, 2011; Romero
and Clarke, 2013). In extremely rare instances, congenital
myopathy may also be associated with hypertonia as will
be discussed below (Jain et al., 2012; C. G. Bönnemann
et al., unpublished data). There are three broad histopatho-
logical subtypes within the classical congenital myopathies:
core myopathies characterized by the presence of myofibres
with foci devoid of oxidative enzymes (Jungbluth et al.,
2011); centronuclear myopathies (CNMs) defined by the
presence of internally located myonuclei (Romero and
Bitoun, 2011) (which are now recognized as one of the
most common type of congenital myopathy, due to the
relatively high prevalence of RYR1 mutations that can
also be associated with this histological phenotype)
(Amburgey et al., 2011; Maggi et al., 2013); and nemaline
myopathy, defined by the presence of electron dense nema-
line bodies/rods within myofibres (Wallgren-Pettersson
et al., 2011). In addition, there are a number of other
structural abnormalities that can be observed in the con-
genital myopathies including actin aggregates, caps, cylin-
drical spirals, dystrophic-like features, fibre-type
disproportion, intranuclear rods, lobulated myofibres, neck-
lace myofibres and zebra bodies (Clarke, 2011; Goebel and
Blaschek, 2011). There are also frequently cases that pre-
sent with mixed morphologies, such as cores and nemaline
bodies (Romero et al., 2009; Olive et al., 2010) or cores
and internal nuclei (Wilmshurst et al., 2010).
It has been increasingly recognized that the histological
features observed within a muscle biopsy may represent
manifestations along spectra of possible abnormalities,
rather than discrete pathological entities (Sewry et al.,
2008; Nance et al., 2012). For example central nuclei are
classically associated with mutations in BIN1, DNM2,
MTM1, and SPEG however, more recently, prominent
central nuclei have been reported in cases resulting from
mutations in CFL2 (Ockeloen et al., 2012), KBTBD13
(Olive et al., 2010), RYR1 (Wilmshurst et al., 2010) and
TTN (Ceyhan-Birsoy et al., 2013; Palmio et al., 2014). On
the flip side, although congenital myopathies have trad-
itionally been classified purely by their defining histopath-
ology, with the discovery of the underlying genetic cause
for an increasing number of patients, the field has also
begun in parallel to classify patients based on the causative
disease gene, such that there is now, for instance, reference
to ACTA1-related myopathies (Nowak et al., 2013),
RYR1-related myopathies (Klein et al., 2012; Amburgey
et al., 2013; Bharucha-Goebel et al., 2013) and SEPN1-
related myopathies (Schara et al., 2008; Scoto et al.,
2011). Each of these gene-associated spectra has continued
to expand with multiple possible morphological appear-
ances per gene, which together with ever-greater genetic
BRAIN 2015: 138; 246–268 | 247
heterogeneity, through the continuous identification of
novel disease genes, has led to an increased blurring of
the established diagnostic boundaries between the subsets
of the classic congenital myopathies. Such novel genes pro-
viding new mechanistic aspects have included CCDC78 in
a family with autosomal dominant CNM with cores
(Majczenko et al., 2012), HACD1 (now known as
PTPLA) in a family with a non-specific autosomal recessive
congenital myopathy (Muhammad et al., 2013), STAC3 in
Native American myopathy (Horstick et al., 2013), STIM1
in tubular aggregate myopathy (although this is not strictly
a congenital myopathy) (Bohm et al., 2013a) but also TTN
mutation in patients presenting with myopathies character-
ized by internal nuclei and/or cores (Ceyhan-Birsoy et al.,
2013; Chauveau et al., 2014). In this review we have there-
fore focused on the emerging pathophysiological themes
across these entities, under the hypothesis that increased
understanding of these mechanistic aspects of disease will
not only help classification, but will ultimately facilitate the
preclinical development of therapeutic approaches for these
diseases (see Fig. 1 for a schematic guide to the cellular
context of the pathophysiological concepts discussed in
the review and table 1 for a correlation of individual
genetic entities and associated pathophysiological themes).
Given the focus of this review, we are not redescribing the
clinical entities but refer the reader to recent excellent re-
views (Nance et al., 2012; Maggi et al., 2013; Romero
and Clarke, 2013).
Membrane remodelling
defects
Membrane remodelling and the
phosphoinositide phosphatases
Membrane remodelling is crucial to a number of biological
processes including exocytosis, intracellular transport, syn-
aptic vesicle function, autophagy (see separate discussion
below), and membrane repair. These processes involve pro-
teins that regulate lipids; they act as lipid adaptor molecules
and function to regulate cytoskeletal organization.
Strikingly, a number of genes found to underlie congenital
myopathies characterized by the presence of internal myonu-
clei, are involved in such membrane remodelling [amphi-
physin 2/bridging integrator 1, BIN1 (MIM 601248);
dynamin 2, DNM2 (MIM 602378); myotubularin, MTM1
(MIM 300415) and myotubularin-related protein 14,
MTMR14 (MIM 611089)]; (reviewed in De Matteis and
Luini, 2011; Cowling et al., 2012) and most recently, striated
preferentially expressed gene, SPEG (Agrawal et al., 2014).
Myotubularin (MTM1), found to be the cause of X-
linked myotubular myopathy (XLMTM, MIM 310400),
is the canonical member of a family of phosphoinositide
phosphatases. The mammalian myotubularin family com-
prises MTM1 itself and 14 MTM1-related proteins
248 | BRAIN 2015: 138; 246–268 G. Ravenscroft et al.

Figure 1 Schematic representation of a muscle cell placing the general pathophysiological concepts discussed in this review
into the cellular context. (1) Sarcolemmal and intracellular membrane remodelling and excitation-contraction coupling; (2) mitochondrial
distribution and function; (3) myofibrillar force generation; (4) atrophy; and (5) autophagy.

(MTMR1–14). Phosphoinositide phosphatases are crucial
to the maintenance of phosphoinositides (PIs) and via the
regulation of these metabolites, are central to membrane
trafficking and signalling (Di Paolo and De Camilli, 2006;
Nicot and Laporte, 2008). The key metabolites regulated
by myotubularins are phosphatidylinositol 3-phosphate
(PI3P), which acts as a membrane address marker and
functions to recruit proteins to the membrane, and
PtdIns(3,5)P2 and PtdIns(5)P. MTM1 dephosphorylates
phosphoinositides, including PI3P [produced by PI
3-kinase (PI3K) activity] and PtdIns(3,5)P2 [PI3,5P2; pro-
duced by PI 5-kinase (PI5K) activity]. Mutations in
MTMR14 have been associated with myopathy in fish
(Dowling et al., 2010) and possibly CNM in man (Tosch
et al., 2006), although the evidence for the latter remains
incomplete. It is worth noting that mutation of MTMR2
and MTMR13 are responsible for some cases of autosomal
recessive Charcot–Marie–Tooth type hereditary motor and
sensory neuropathies (CMT4B), which is of interest be-
cause mutations in DNM2 are also seen in both CNM
and in autosomal dominant CMT (CMTDIB) (De Matteis
and Luini, 2011). How disrupted phosphoinositide regula-
tion leads to neuromuscular disease via possible
disturbance of membrane metabolism is still not completely
understood; however, studies in animal models are begin-
ning to shed light in this area, highlighting several potential
mechanisms that are addressed in the following sections.
Excitation-contraction coupling, transverse-tubule
and sarcoplasmic reticulum defects
The precise regulation of intracellular Ca2+ is crucial for
normal myogenesis and skeletal muscle function.
Excitation-contraction (E–C) coupling exquisitely modu-
lates sarcoplasmic Ca2+ levels during muscle contraction
and depends on a number of specialized membranes and
structural components including components of the triad,
namely T tubules and the voltage-sensing dihydropyridine
receptors (DHPR) in the T tubule membrane, and the
sarcoplasmic reticulum (SR) and the Ca2+ release chan-
nels/ryanodine receptors (RYR1) in the sarcoplasmic reticu-
lum membrane. Structural defects of triads and abnormal
localization of both DHPR and RYR1 have been noted in
myofibres from patients with CNMs due to MTM1, BIN1
and CCDC78 (MIM 614666) mutations (Al-Qusairi et al.,
2009; Dowling et al., 2009; Toussaint et al., 2011;
Majczenko et al., 2012). Similar defects have also been
Pathophysiology of congenital myopathies BRAIN 2015: 138; 246–268 | 249

Table 1 Summary of the pathophysiological features associated with particular congenital myopathy disease genes

Gene Membrane defects Mitochondrial and energy Myofibrillar force Atrophy Autophagy
metabolism abnormalities defects defects defects
T tubules/SR NMJ
BIN1 3
DNM2 3 3 3
MTM1 3 3 3 3 3
MTMR14 3 3 3
RYR1 3 3 3
ACTA1 3 3
CCDC78 3
KBTBD13 3
KLHL40 3
MEGF10 3
MYH7 3
NEB 3 3
TPM2 3
TPM3 3 3 3
SEPN1 3 3

observed and more closely investigated in animal models of
the various CNMs.
Myotubularin
Studies of Drosophila myotubularin (mtm1) showed that
mtm1 is central to endolysosomal function, cortical actin
remodelling and is also involved in integrin-mediated myo-
fibre attachment (Velichkova et al., 2010). Mtm1 ablation
resulted in accumulation of integrin and PI3P on endo-
somes, suggesting that mtm1 is required for intracellular
integrin trafficking and recycling to the sarcolemma
(Ribeiro et al., 2011). The phenotype could be rescued
by depletion of class II PI3K, confirming that the abnorm-
alities observed are truly IP-related.
Knock-down of mtm1 in zebrafish resulted in delayed
hatching and an impaired touch-evoked escape contraction
(Dowling et al., 2009). Muscle from the mtm1 morphants
displayed centrally located and abnormally enlarged nuclei
and skeletal muscle hypotrophy, mimicking the human dis-
ease. Interestingly, the levels of PI3P were elevated in the
skeletal musculature of mtm1 morphants relative to con-
trols (similar to the Drosophila studies), but they were not
elevated in whole embryo extracts, suggesting that myotu-
bularin is the primary phosphoinositide phosphatase in
skeletal muscle (Dowling et al., 2009). The skeletal
muscle phenotype of the mtm1 morphants was rescued
by delivery of MTMR1 and MTMR2, indicating that
the primary defect in the morphants was due to a lack of
compensation by other phosphoinositide phosphatases
(Dowling et al., 2009). In zebrafish (Dowling et al.,
2009) and mice (Amoasii et al., 2013), myotubularin local-
izes to the T tubules as evidenced by co-localization with
antibodies to the DHPR. The perinuclear compartments of
mtm1 morphant myofibres displayed abnormal tubule-like
structures, ranging from dilated T tubule and sarcoplasmic
reticulum networks, to severe disorganization of the T tu-
bules and sarcoplasmic reticulum (Dowling et al., 2009).
The most well studied myotubularin model is the Mtm1
knock-out (KO) mouse. These mice develop a generalized
and progressive myopathy from 4 weeks of age with
increased internalized nuclei leading to death at 6–14
weeks of age (Buj-Bello et al., 2002). Analysis of Mtm1
null mouse muscle found that the sarcoplasmic reticulum
membranes were abnormally located within myofibres and
that the sarcoplasmic reticulum was dilated (Amoasii et al.,
2013). In order to study the SR/ER (endoplasmic reticulum)
in isolation, Amoasii and colleagues (2013) examined the
endoplasmic reticulum network in myoblasts from Mtm1
null mice and MTM1 patients. The myoblasts lacked triads
and T tubules and showed defects in the endoplasmic
reticulum network, suggesting that an absence of MTM1
resulted in sarcoplasmic reticulum defects (as the sarcoplas-
mic reticulum is a specialization of the endoplasmic reticu-
lum) independent of abnormalities of the T tubules and
triads. This suggests a general defect in the maintenance
of various membranous muscle organelles. In vivo modu-
lation of MTM1 lipid phosphatase activity in normal
mouse muscle using viral overexpression of wild-type or
mutant (phosphatase inactive) MTM1, resulted in altered
PI3P levels specifically at the junctional sarcoplasmic reticu-
lum as well as altered sarcoplasmic reticulum remodelling.
Importantly, this study suggests that dysregulation of the
MTM1/PI3P pathway, leading to altered modulation of the
sarcoplasmic reticulum structure, may represent a crucial
mechanism underlying disease pathogenesis in myotubular
myopathy.
This same group also showed that the phenotype of
the Mtm1 null mouse could be partially rescued by
adeno-associated viral delivery of the MTM1Cys375Ser or
MTM1Ser376Asn phosphatase-dead mutants, including a sig-
nificant increase in muscle force generation, suggesting that
250 | BRAIN 2015: 138; 246–268
many features of the disease are not purely due to the
enzymatic defect (muscle atrophy, myofibre hypotrophy,
abnormal organelle placement) (Amoasii et al., 2012).
Consistent with this is the consideration that MTM1 is
quite ubiquitously expressed with not only skeletal muscle
specific localization, whereas XL-CNM is largely (though
not exclusively) a skeletal muscle disease suggesting that
defects in interactions between MTM1 and muscle-specific
proteins could play a major role in disease causation. The
most evident extra-phosphatase activity for MTM1 might
be in protein–protein interactions and functioning as a scaf-
fold for the intermediate filament network (desmin in par-
ticular) (Hnia et al., 2011); which is involved in proper
organelle placement (Amoasii et al., 2012).
The mouse model generated by knocking-in the human
disease associated mutation p.Arg69Cys in Mtm1 is less
severe compared to the Mtm1 KO model, likely due to
some residual activity of the full length mutant myotubu-
larin (Pierson et al., 2012). Mtm1 p.Arg69Cys knock-in
(KI) mice are asymptomatic at birth and show normal ini-
tial skeletal muscle development. However, by 2 months of
age muscle atrophy and weakness is detected. These mice
had a median survival age of 66 weeks and died suddenly
and unexpectedly, consistent with cardiorespiratory failure.
Histologically, skeletal muscle showed many small myofi-
bres, central nuclei, staining suggestive of necklace fibres
similar to the human pathology, and altered staining pat-
terns with antibodies to RYR1 and DHPR. Reduced num-
bers of, and poorly formed, T tubules and triads were
identified at the ultrastructural level (Pierson et al.,
2012). These morphological abnormalities result in
excitation-contraction coupling defects, since for Mtm1
KO and p.Arg69Cys KI mice, electrically-mediated whole
muscle-specific force was greatly reduced, whereas there
was no change between specific force generated by wild-
type and Mtm1 chemically-skinned (in which T tubules and
triads are not functioning) myofibres (Lawlor et al., 2013).
Most recently, adeno-associated virus-mediated knock-
down of Mtm1 in adult mouse muscle resulted in
abnormally oriented T tubules that were more frequently
appearing as longitudinal (L-) tubules (Joubert et al., 2013)
highlighting that myotubularin is continuously required for
adult skeletal muscle organelle maintenance.
Beggs and colleagues (2010) reported a Labrador
Retriever line with a missense mutation in MTM1; the
affected male dogs displayed many of the clinical and histo-
logical hallmark features of human MTM1 related XL-
CNM, presenting with progressive limb weakness leading
to an inability to rise or walk without assistance between
10–19 weeks of age. Clinically, there was severe muscle
atrophy. Histologically there were numerous small central-
ly-nucleated myofibres, resembling myotubes, necklace
myofibres, abnormal localization of RYR1 and DHPR
and diminished and disturbed T tubule structures (Beggs
et al., 2010). Thus in both non-mammalian and mamma-
lian models (small and large) the pathophysiology of
MTM1-related disease is remarkably similar and consistent
G. Ravenscroft et al.
with functionally important abnormalities of triadic struc-
ture with consequences on excitation-contraction coupling.
MTMR14
MTMR14 is a phosphatase that shares similar enzymatic
activity to MTM1. Although two human MTMR14 vari-
ants have been described, and were shown to reduce the
enzymatic activity of MTMR14 (Tosch et al., 2006), they
are still awaiting further confirmation as a direct cause of
CNM or a disease modifier. Of the two variants described,
one de novo MTMR14 missense variant (p.Y462C) was
identified in an isolated proband in combination with a
de novo known DNM2 mutation (p.E368K), the
p.Y462C variant was also identified in a control sample.
The second proband harboured a p.R336Q variant in
MTMR14, this variant was also present in the unaffected
father (Tosch et al., 2006). Inactivation of Mtmr14 in mice
(Shen et al., 2009; Hnia et al., 2012) and fish causes a
myopathy characterized by excitation-contraction coupling
defects but without the presence of central or abnormally
located myonuclei. In zebrafish, knock-down of mtmr14
resulted in a functional deficit (reduced touch-evoked
escape responses and a lack of response to high frequency
stimulation) without obvious histopathological abnormal-
ities (Dowling et al., 2010). Hnia et al. (2012) showed
that muscle from Mtmr14 KO mice displayed significantly
swollen and elongated T tubules and suggested that these
structural abnormalities might contribute to the defects in
excitation-contraction coupling noted previously in
Mtmr14 KO mice (Shen et al., 2009) and morphant fish
(Dowling et al., 2010).
Amphiphysin 2 and dynamin 2
Altered T-tubular and sarcoplasmic reticulum systems have
also been noted due to mutation in DNM2 and BIN1,
which are both known to underlie forms of centronuclear
myopathy. Early studies showed that amphiphysin 2
(BIN1) was concentrated at the T tubules and was required
for T tubule biogenesis. Expression of BIN1 in non-muscle
cells resulted in tubular invaginations of the plasma mem-
brane (Lee et al., 2002).
The original study that showed that BIN1 mutations
resulted in autosomal recessive-CNM, also showed that
missense mutations resulted in impaired tubule formation
and pull-down assays showed that partial truncation of
BIN1 abrogated the interaction with DNM2 (Nicot et al.,
2007). The authors concluded that BIN1 mutation caused
CNM by interfering with remodelling of T tubules and that
interaction between BIN1 and DNM2 is required for
muscle function and myonuclei positioning. Most recently,
BIN1 has also been shown to interact directly with MTM1
(Royer et al., 2013).
BIN1 is ubiquitously expressed and there are at least 12
different splice isoforms, those containing a phosphoinosi-
tide domain are found exclusively in skeletal muscle
isoforms, which all contain exon 11 (Toussaint et al.,
2011). Bohm et al. (2013b) identified the first mutation
Pathophysiology of congenital myopathies
affecting the muscle-specific BIN1 exon 11 in a consan-
guineous family presenting with a rapidly progressive and
fatal CNM. Mutation of the same acceptor-splice site in
exon 11 was identified to underlie canine inherited myop-
athy of Great Danes (IMGD). Cell-culture studies showed
that BIN1 constructs without exon 11 were unable to ini-
tiate membrane tubulation (Bohm et al., 2013b). Analysis
of patient and inherited myopathy of Great Danes muscle
showed central nuclei, fibre atrophy and lobulation, abnor-
mal triads, accumulations of membranes and vacuoles,
autophagic vacuoles and mitochondrial accumulations.
Proteins of the triad and sarcoplasmic reticulum (RYR1,
DHPR, SERCA1) and membrane-trafficking proteins
(MTM1, CAV3, DYSF) were also mislocalized in inherited
myopathy of Great Danes muscle.
DNM2-related dysfunction has been studied in zebrafish
and mice. Zebrafish overexpressing the p.Ser619Leu substi-
tution in DNM2 showed impaired motility, with smaller
myofibres, and dilated sarcoplasmic reticulum (Gibbs
et al., 2013). More recently it was shown that the
DNM2 p.Ser619Leu zebrafish accumulate aberrant ves-
icle material and show defects of excitation-contraction
coupling. Expression of the p.Ser619Leu mutant in
non-muscle cells resulted in defective BIN1-dependent tubu-
lation (Gibbs et al., 2014). Thus it seems that an under-
lying mechanism in the CNMs is defective membrane
tubulation.
Studies of a KI mouse model of the most frequently
occurring DNM2 mutation (p.Arg465Trp) revealed that
muscle from heterozygous KI mice contains increased tubu-
lar structures within the intermyofibrillar space and slightly
disorganized T tubule and sarcoplasmic reticulum struc-
tures, myonuclei were positioned normally (Durieux
et al., 2010). Homozygous mice displayed neonatal lethal-
ity. Cowling et al. (2011) generated mouse models that
overexpressed either wild-type or p.Arg465Trp DNM2
using intramuscular delivery of viral vectors. This study
showed that muscle-specific expression of mutant DNM2
resulted in a substantial loss of muscle mass, increased in-
ternal and central nuclei, reduced muscle strength and triad
defects (including abnormal T tubule morphology and
swollen sarcoplasmic reticulum). Swollen sarcoplasmic re-
ticulum were also observed in myofibres overexpressing
wild-type DNM2.
Cowling et al. (2014) observed that XLMTM patient
muscle and Mtm1 / mouse muscle contained elevated
levels of DNM2. Subsequently Mtm1 / mice that were
hemizygous for Dnm2 were generated. Reduction of
DNM2 levels, restored the lifespan of Mtm1 / mice, im-
proved whole-body strength and diaphragm function
(Cowling et al., 2014). Structural abnormalities including
myofibre atrophy, nuclear positioning and sarcomere and
triad disorganization, were also reduced in Mtm1 /
muscle when DNM2 levels were reduced. This study sug-
gests that MTM1 and DNM2 regulate skeletal muscle or-
ganization and function in a finely balanced system in
which MTM1 seems to be negative regulator of DNM2
BRAIN 2015: 138; 246–268 | 251
so that reduction of DNM2 restores this balance in
MTM1 deficiency. This approach could thus be beneficial
in XLMTM.
SPEG
Agrawal and colleagues (2014) identified SPEG as a novel
binding partner of MTM1 in a yeast two-hybrid screen and
subsequently showed that it likely localized to the terminal
cisternae of the sarcoplasmic reticulum. Recessive muta-
tions of SPEG were identified in patients with a severe
form of CNM, two cases also had cardiomyopathy
(Agrawal et al., 2014). Speg / mice display perinatal
death due to dilated cardiomyopathy (Liu et al., 2009),
evaluation of Speg / skeletal muscle revealed increased
central nuclei (Agrawal et al., 2014). While attractive to
consider, it remains to be seen whether loss of SPEG
leads to defects in membrane remodelling and tubulation,
as occurs in MTM1-, BIN1- and DNM2-related CNM.
Triadic Ca2+ release channel: ryanodine receptor
The so-called ryanodine receptor (RYR1) is the major Ca2+
release channel of the sarcoplasmic reticulum. It is located
at the triad, where it directly interacts with the voltage-
sensitive dihydropyridine receptor in the T tubule. Via
this connection it mediates excitation initiated Ca2+ release
into the sarcoplasm (Zorzato et al., 1990).
Myopathies resulting from autosomal dominant RYR1
(MIM 180901) mutations [including malignant hyperther-
mia susceptibility (MHS; MIM 145600), central core dis-
ease (MIM 117000), multi-minicore disease, congenital
fibre-type disproportion (CFTD), some subtypes of CNM
and King-Denborough Syndrome] are thought to arise due
to ‘leaky’ RyR1 channel activity, channel instability or
reduced Ca2+ conductance (Wilmshurst et al., 2010).
Recessive mutations may cause misolocalized RyR1 chan-
nels, or reduced RYR1 abundance (Zhou et al., 2013). KI
RYR1 p.Tyr522Ser (this mutation is responsible for MHS
in patients) mice exhibit malignant hyperthermia suscepti-
bility, (MHS)-like responses to halothane and heat stress.
Histologically the muscle is characterized by the step-wise
development of contracted sarcomeres and core-like re-
gions, secondary to mitochondrial dislocation and triad dis-
ruption (Boncompagni et al., 2009) (structured cores),
followed by sarcomeric disintegration in the core (unstruc-
tured cores). KI p.Tyr522Ser myofibres also display disor-
ganized T tubules and triad positioning (Boncompagni
et al., 2009). Another KI mouse model (KI p.Ile4898Thr)
of an excitation-contraction coupling RYR1 mutation (a
common central core disease mutation in patients) exhibits
slowly progressive myopathy with skeletal muscle weak-
ness, impaired mobility and hindlimb paralysis. The mice
also developed age-dependent structural lesions within their
muscle, sarcomeric misalignment and Z-disc streaming pro-
gressing to mini-cores, cores and then also nemaline bodies
(Zvaritch et al., 2009). Thus, this mouse model also
models the development of a ‘mixed’ congenital myopathy,
core-rod disease.
252 | BRAIN 2015: 138; 246–268
The pathogenesis of recessive RYR1 mutations has only
begun to be elucidated. Zhou et al. (2013) showed that
RYR1 protein levels are reduced in autosomal recessive
RYR1 patient muscle, as are DHPR levels, and that the
localization of the alpha-subunit of the DHPR is also per-
turbed in patient muscle and in cultured myotubes targeted
with siRNA to RYR1 (Zhou et al., 2013). These alter-
ations resulted in impaired excitation-contraction coupling
and Ca2+ handling. Unexpectedly, IP3 receptors (a down-
stream effector of PI3,5P2 which resides in the ER/SR and
upon IP3 binding mediates Ca2+ release) were more abun-
dant in patient muscle and cultured myotubes deficient for
RyR1, likely representing a secondary upregulation of the
IP3-Ca2+ axis. In cultured muscle cells, IP3 receptors are
expressed at the nuclear envelope and give rise to slow
nucleoplasmic Ca2+ transients that modulate gene expres-
sion (reviewed in Carrasco et al., 2004). While the IP3-
Ca2+ signalling pathway does not play a substantial role
in skeletal muscle excitation-contraction coupling under
normal conditions, it’s upregulation due to RyR1 deficiency
and potential effects on gene expression, may contribute to
the underlying pathophysiology of this condition and could
represent a potential therapeutic target (Zhou et al., 2013).
Excitation-contraction coupling and the sarcoplas-
mic reticulum in other congenital myopathies
Defects in sarcoplasmic reticulum function have also been
implicated in nebulin (NEB, MIM 161650) deficiency, usu-
ally associated with the histological phenotype of nemaline
myopathy. The sarcoplasmic reticulum Ca2+-ATPase pump
(SERCA) inhibitor, sarcolipin, is upregulated 70-fold in
muscle from Neb KO mice (Witt et al., 2006). Speed of
Ca2+ uptake by SERCA was significantly slower in sarco-
plasmic reticulum vesicles isolated from Neb KO myofibres
compared to wild-type, contributing to a slower speed of
muscle relaxation (Ottenheijm et al., 2008). Thus it ap-
pears that nebulin may also be involved in modulating
sarcoplasmic reticulum Ca2+ handling.
Most recently, mutation of the SH3 and cysteine-rich
domain 3 gene (STAC3; MIM 615521), encoding a novel
excitation-contraction coupling protein (Nelson et al.,
2013; Reinholt et al., 2013), has been found to underlie
Native American myopathy (Horstick et al., 2013). Native
American myopathy (MIM 255995) is characterized by
congenital muscle weakness, susceptibility to malignant
hyperthermia, joint contractures, dysmorphic features
including ptosis, and non-specific myopathic changes on
muscle biopsy (no rods, cores or internally-placed nuclei
were observed) (Stamm et al., 2008a, b). Thus both pri-
mary defects of excitation-contraction coupling (due to
RYR1 and STAC3 mutations) and secondary defects of
excitation-contraction coupling (arising from the structural
triad abnormalities seen in the CNMs due to mutations in
DNM2 and MTM1, but also NEB mutations) can be iden-
tified in the congenital myopathies. In this context, as noted
above, it is also of interest that dominantly acting muta-
tions in the sarcoplasmic Ca2+ sensor STIM1 have now
G. Ravenscroft et al.
been found to underlie a form of tubular aggregate myop-
athy (which although being associated with characteristic
morphological findings in the biopsy is not strictly speaking
a congenital myopathy) (Bohm et al., 2013a). Given the
crucial roles of the triad and sarcoplasmic reticulum in
muscle function and this apparently widespread perturb-
ation of these entities in the congenital myopathies, there
is an emerging view that impaired excitation-contraction
coupling and Ca2+ homeostasis are central to disease
pathogenesis and may thus represent an important thera-
peutic target across a number of the congenital myopathies.
Neuromuscular junction defects
The neuromuscular junction (NMJ) is a crucial, deeply in-
folded, specialization of the muscle membrane. Aberrant
NMJs and clinical overlap with the congenital myasthenic
syndromes were reported already in the earlier literature, as
a feature of then genetically uncharacterized CNMs (Sher
et al., 1967; Elder et al., 1983; Fidzianska and Goebel,
1994). In light of this and the previous discussion of the
role of membrane remodelling and shaping, the NMJ has
recently come into focus in our understanding of the patho-
genesis of the congenital myopathies, in particular for the
CNMs.
Myotubularin
Dowling and colleagues (2012b) re-examined two mouse
models of MTM1-associated disease (Mtm1 KO and KI
p.Arg69Cys) and found that both models showed features
consistent with a defect in neuromuscular transmission.
Mtm1 KI mice displayed reduced treadmill-running endur-
ance and increased grip fatigue compared to wild-type
mice, indicating exercise intolerance and fatigable muscle
weakness: two clinical indicators of defective neuromuscu-
lar transmission. Grip fatigue was attenuated following
administration of neostigmine [an acetylcholine esterase
(AChE) inhibitor], supporting the presence of a NMJ
defect. Evaluation of Mtm1 KO and KI p.Arg69Cys
muscle highlighted an increase in the area of individual
NMJs and a reduction in the complexity of the postsynap-
tic junctional folds (Dowling et al., 2012b). The authors
showed that trafficking of AChR from the endosomal com-
partment to the sarcolemma was impaired in Mtm1 KO
myotubes and propose that aberrant regulation of junc-
tional membrane turnover and abnormal NMJ trafficking
may underlie the altered ultrastructural appearance and
consequently abnormal function of the NMJs (Dowling
et al., 2012b). Treatment of Mtm1 KI p.Arg69Cys mice
with pyridostigmine (another AChE inhibitor) also resulted
in an improvement in motor function (Dowling et al.,
2012b).
Similarly, in mtm1 morphant zebrafish (Dowling et al.,
2009), disorganized NMJs were noted (Robb et al., 2011)
and movement and provoked swimming behaviour were
dramatically improved upon treatment with an AChE
inhibitor (edrophonium) (Robb et al., 2011).
Pathophysiology of congenital myopathies
Dynamin 2
In a zebrafish model overexpressing the p.Ser619Leu muta-
tion of DNM2, a human mutation associated with severe
CNM presenting in the neonatal period, severe motor de-
fects were observed (Gibbs et al., 2013). Disorganized
NMJs, including abnormal clustering of acetylcholine re-
ceptors and a reduced number of endplates per myofibre,
were a striking feature of this model. Treatment with edro-
phonium improved motor function (escape response) of
p.Ser619Leu larvae, but not uninjected controls or wild-
type larvae, indicating that AChE inhibition improved the
phenotype caused by the presence of the mutant dynamin 2
(Gibbs et al., 2013).
Studies of patient biopsies showed abnormal endplate
morphology (irregular and less complex post-junctional
folds) (Fidzianska and Goebel, 1994) and abnormal
AChE staining (Elder et al., 1983). Later, examination of
five patients harbouring either the DNM2 p.Ser619Leu or
p.Glu368Lys substitutions, found that two of the patients
had electrophysiological recordings suggestive of impaired
neuromuscular transmission (Gibbs et al., 2013).
Importantly, Gibbs et al. (2013) and others have reported
anecdotally that treatment of MTM1, DNM2 and genetic-
ally-unresolved CNM patients with pyridostigmine, re-
sulted in improved muscle strength and function, and
reduced fatigability (Liewluck et al., 2011; Robb et al.,
2011). Thus, modulation of neuromuscular transmission
through AChE inhibition or enhanced acetylcholine release
from the presynaptic nerve terminal (as proposed by
Dowling et al., 2012b) may represent a viable therapeutic
approach to attenuate the disease severity in patients with
CNMs, that may lead to an improvement in quality of life.
This approach will now need to be evaluated in a con-
trolled clinical trial in patients with CNM.
The neuromuscular junction in other congenital
myopathies
NMJ abnormalities may also occur in other congenital
myopathies. Munot et al. (2010) presented two unrelated
cases of congenital fibre-type disproportion, due to two
different heterozygous slow a-tropomyosin (TPM3, MIM
191030) mutations, which mimicked myasthenic syn-
dromes. Single fibre EMG recordings for both cases
showed a jitter consistent with impaired neuromuscular
transmission, however detailed histological assessment of
the NMJ was not performed. Pyridostigmine administration
resulted in a modest functional improvement in the only
patient that undertook treatment (Munot et al., 2010).
Thus, the link between TPM3 mutations and confirmed
abnormalities on neuromuscular transmission has to
remain tentative for now.
Most recently, it was shown that pyridostigmine admin-
istration resulted in clinical improvement in two siblings
with recessive RYR1-related congenital myopathy asso-
ciated with striking fatigable ptosis (Illingworth et al.,
2014). This study extended the phenotype of RYR1-related
BRAIN 2015: 138; 246–268 | 253
myopathy to include a myasthenic-like phenotype.
Pyridostigmine treatment resulted in improved facial
expression and stamina and decreased ptosis. Again, a
detailed analysis of neuromuscular transmission was not
possible in these patients leaving the final link open.
All in all, it seems to be worthwhile to investigate the
NMJ, histologically, ultrastructurally, and physiologically,
in models of other congenital myopathies, to assess whether
there might be a role for aberrant NMJ function beyond
the CNMs, and to what extent modulation of neuromus-
cular transmission may also represent a therapeutic option
for these patients.
Mitochondrial abnormalities
Abnormal distributions of mitochondria are a well-
recognized hallmark feature of the congenital myopathies
that are characterized by the presence of cores or core-like
regions within myofibres. In fact, cores are defined by being
devoid of mitochondria, recognizable by the appropriate
stains. Abnormal accumulations, localization and/or ultra-
structure of mitochondria have been reported in central
core disease, multi-minicore disease, centronuclear myop-
athy, and nemaline myopathy (Sanoudou et al., 2006;
Hnia et al., 2011; Dowling et al., 2012a; Mokbel et al.,
2013) with the possibility that this abnormal localiza-
tion will also lead to abnormal function regionally in
muscle.
Ryanodine receptor
A recessive ryr1 zebrafish model (due to a spontaneous
mutation in the fast-muscle specific ryanodine receptor
gene, ryr1b, that results in a premature stop codon)
showed reduced levels of ryr1 (5–10% of wild-type), de-
fective excitation-contraction coupling and a severe motor
phenotype (Hirata et al., 2007). The oxidative stress path-
way was found to be impaired in this model and it was
confirmed that ryr1 zebrafish had elevated basal oxidant
activity arising from mitochondrial-derived reactive
oxygen species, which were notably not present in dys-
trophic zebrafish models (Dowling et al., 2012a).
Furthermore, cultured RYR1 patient-derived myotubes
also displayed features consistent with increased oxidative
stress (Dowling et al., 2012a). The same authors showed
that treatment of RYR1 patient myotubes and ryr1 zebra-
fish with the antioxidant N-acetylcysteine (NAC) corrected
the oxidative stress and restored zebrafish motor function
and muscle pathology. These data implicate mitochond-
rially-mediated oxidative stress as a contributing factor in
the pathophysiology of RYR1-related myopathies and pro-
vide important preclinical evidence in support of antioxi-
dant therapy as a potential treatment intervention in these
diseases.
254 | BRAIN 2015: 138; 246–268
Selenoprotein N1
Autosomal recessive mutations in selenoprotein N1
(SEPN1, MIM 606210) underlie some cases of multi-
minicore disease and congenital fibre-type disproportion;
in addition to congenital muscular dystrophy with rigid
spine (MIM 602771) and myopathy with Mallory body-
like inclusions (Scoto et al., 2011). SEPN1 is an endoplas-
mic reticulum glycoprotein that seems more abundant in
foetal than adult tissues (Petit et al., 2003). SEPN1 is
thought to be involved in Ca2+ homeostasis and protection
from oxidative stress (Arbogast and Ferreiro, 2010; Castets
et al., 2012).
Sepn1 / mice show normal growth and lifespan and
only minor defects of muscle morphology and function
under basal conditions. However, under physical challenge
(e.g. forced swimming) Sepn1 / mice developed an obvi-
ous phenotype characterized by reduced mobility, body ri-
gidity and curvature of the spine - thus recapitulating
aspects of the human disease (Rederstorff et al., 2011).
Despite this phenotype, no cores or core-like regions were
observed in Sepn1 / muscle, and mitochondria were
ultrastructurally normal (Rederstorff et al., 2011).
Similar to RYR1 mutant myotubes, cultured SEPN1-
deficient patient myotubes also have a higher basal oxidative
activity and patient-derived fibroblasts are more susceptible
to H2O2 (Arbogast et al., 2009). This elevated sensitivity to
H2O2 could be mitigated by pretreatment with NAC
(Arbogast et al., 2009). It has been suggested therefore that
selenoprotein SEPN1 may be involved in buffering reactive
oxygen species and/or repairing damaged proteins (Castets
et al., 2012).
RYR1 activity is regulated by its oxidative status (Oba
et al., 2002) and both RYR1 and SEPN1 are enriched in
sarcoplasmic reticulum terminal cisternae fractions (Jurynec
et al., 2008), which resulted in the hypothesis that SEPN1
might modulate RYR1 activity. In support of such a con-
nection, Ca2+ handling was altered in the sepn1 morphant
zebrafish (Jurynec et al., 2008) and patient myotubes
(Arbogast et al., 2009).
However, it remains to be fully explained how SEPN1
might modulate RYR1 given that SEPN1 is barely detect-
able in adult myofibres (Castets et al., 2012). Nonetheless,
there is emerging evidence that links SEPN1 to RYR1 regu-
lation, and both RYR1 and SEPN1 to increased oxidative
stress, perhaps via mitochondria.
Tropomyosins
In striated muscle, there are three principal tropomyosin
isoforms: a-tropomyosinfast (TPM1), a-tropomyosinslow
(TPM3) and b-tropomyosin (TPM2). Tropomyosins inter-
act with the troponin complex to regulate actin-myosin
interactions. Mutations of TPM2 and TPM3 cause congeni-
tal myopathies with nemaline bodies, caps, or fibre-type
disproportion (reviewed in Romero and Clarke, 2013).
Sanoudou et al. (2006) reported enlarged mitochondria
G. Ravenscroft et al.
with abnormal structures and elevated levels of lipofusin
indicative of oxidative stress, in muscle from nemaline my-
opathy mice harbouring a p.Met9Arg mutation in TPM3.
Most recently, subsarcolemmal mitochondrial accumulations
and a greater variation in the abundance of respiratory chain
complexes and porin proteins has been described in muscle
biopsies from some patients harbouring the p.Lys7del muta-
tion in TPM2 compared with healthy individuals (Davidson
et al., 2013; Mokbel et al., 2013), indicating a contribution
of mitochondrial dysregulation and cellular stress to the
pathogenesis of some TPM2-related myopathies.
Myotubularin
Abnormal mitochondrial localization has also been re-
ported in MTM1 patient cells (Buj-Bello et al., 2008) and
the mtm1 morphant zebrafish (Dowling et al., 2009). In-
depth examination of MTM1 patient muscle and Mtm1
deficient mouse muscle and cells, found that there was a
collapse of the mitochondrial network, with the presence of
enlarged mitochondria containing fewer cristae (Hnia
et al., 2011). Mtm1-deficient cells also had reduced cyto-
chrome c oxidase activity and lower ATP levels than
wild-type cells (Hnia et al., 2011), suggesting altered mito-
chondrial function as a contributing factor to the patho-
physiology of MTM1-related CNM. Hnia et al. (2011)
identified that in skeletal muscle, the intermediate filament
protein desmin regulates mitochondrial dynamics and
morphology through direct interaction with MTM1.
These studies found that Mtm1 KO mouse muscle, Mtm1
siRNA-treated C2C12 and MTM1 patient myoblasts, con-
tained elevated levels of the intermediate filament protein
desmin, and abnormal desmin aggregates. MTM1 muta-
tions that cause CNM and are located within the critical
region for desmin binding abolished the MTM1-desmin
interaction. These data suggest that in the absence of
MTM1, desmin aggregates impair intermediate filament
function and contribute to aberrant internal organelle pos-
itioning and functioning (including mitochondria and
nuclei). In this context it is of note that deletion of
desmin in mice also results in altered distribution, morph-
ology and activity of mitochondria (Milner et al., 2000).
Similar abnormalities are seen in patients with DES-related
myofibrillar myopathy (MIM 601419) (Reimann et al.,
2003; Bar et al., 2007).
It remains to be seen whether mitochondrial dysfunction
and oxidative stress are more widely applicable to the dis-
ease process in multiple congenital myopathies and how
much they are able to influence disease severity and
whether they could represent a more widely applicable
therapeutic target across various disorders.
Myofibrillar force generation
Defects of sarcomeric function and myofibrillar force gen-
eration are the most obvious consequence to consider in
Pathophysiology of congenital myopathies
congenital myopathies arising due to defects in genes
encoding thin filament proteins or proteins interacting
with or regulating such proteins (Nowak et al., 1999,
2013; Pelin et al., 1999; Laing and Nowak, 2005;
Ottenheijm et al., 2012). However, as we will review, the
exact effects on myofibrillar force generation may differ
from gene to gene and even between different mutations
in the same gene.
Nebulin
Myofibrillar structure and function has been most exten-
sively studied in patients harbouring mutations in NEB and
the Neb KO (Ottenheijm and Granzier, 2010) and
NebDExon55 mouse models (Ottenheijm et al., 2013).
Studies of nebulin-deficient mice (Neb), zebrafish (neb)
and NEB patient muscle, have revealed a greatly impaired
force-generating capacity (Bang et al., 2006; Witt et al.,
2006; Lawlor et al., 2011a; Ottenheijm et al., 2012,
2013; Telfer et al., 2012). Nebulin is thought of as a mo-
lecular ruler determining the length of the actin thin fila-
ment, thus it is perhaps not surprising that a unifying
feature of nebulin-deficient muscle is a greater range in
thin filament length, with many sarcomeres containing
shorter thin filaments, that result in reduced maximal
active tension (Bang et al., 2006; Witt et al., 2006;
Ottenheijm et al., 2009, 2013; Telfer et al., 2012).
Nebulin-deficient myofibrils also have a reduced Ca2+ sen-
sitivity of force production (Witt et al., 2006), a slower
rate of tension redevelopment and an elevated tension
cost, meaning more ATP has to be expended to generate
a given tension. This suggests that there is a reduced frac-
tion of force-generating cross-bridges, which, in turn, con-
tributes to muscle weakness (Ottenheijm et al., 2010;
Lawlor et al., 2011a). Recently studies have shown that
in vitro treatment of nebulin-deficient myofibres with a
fast skeletal muscle troponin activator (CK-2066260)
enhanced force production at submaximal activating Ca2+
levels (de Winter et al., 2013; Lee et al., 2013; Ottenheijm
et al., 2013). This compound belongs to a class of fast
skeletal muscle troponin activators that were recently
shown to augment force development by slowing the rate
of Ca2+ dissociation from troponin C (TnC) and enhancing
cross-bridge formation at a given Ca2+ concentration
(Russell et al., 2012). These fast myofibre specific troponin
activators may be applicable to a range of skeletal muscle
disorders, as they have also been shown to be effective in
myasthenic patients (Russell et al., 2012). In the context of
the congenital myopathies this class of compounds might of
course be particularly beneficial in patients with disorders
of the thin filament with abnormal contractility such as is
the case in some nemaline myopathies. In addition it has
also been suggested that they would be applicable in dis-
orders that manifest defects of excitation-contraction cou-
pling, including patients with core and central nuclear
myopathies, where it could be possible to maximize the
force produced from the reduced Ca2+ signalling during
BRAIN 2015: 138; 246–268 | 255
muscle contraction. It would of course only strengthen
fast twitch type fibres, which in congenital myopathies
are frequently fewer in number, but larger (as seen in
fibre-type disproportion, a common histological pattern
across various congenital myopathies). However, as
described later, such treatments could prove to be problem-
atic in cases where mutations result in increased Ca2+ sen-
sitivity of the myofibrillar apparatus, where such a
treatment could lead to worsening of the condition.
Skeletal muscle alpha-actin
Over 200 different mutations have been identified in the
skeletal muscle a-actin gene (ACTA1, MIM 102610), the
major component of the thin filament. The majority of
these disease-associated mutations act in a dominant
manner, causing significant weakness and hypotonia
(Laing et al., 2009), except for a case notably presenting
with muscle stiffness and hypertonia (Jain et al., 2012).
Rare recessive loss-of-function cases exist and are compat-
ible with survival because of compensatory upregulation of
cardiac a-actin in skeletal muscle (Nowak et al., 2007). A
range of structural histological lesions in muscle are asso-
ciated with ACTA1 mutations, including actin aggregates,
nemaline bodies (sarcoplasmic and intranuclear), caps,
core-like regions, cytoplasmic bodies, dystrophic features,
fibre-type disproportion and zebra bodies, while some
cases only show minimal changes within the muscle
biopsy (reviewed in Nowak et al., 2013). It remains un-
clear how the presence of mutant ACTA1 protein (in dom-
inant cases) resulting in hypotonic or hypertonic
phenotypes and the absence of ACTA1 (in the recessive
‘null’ cases) (Nowak et al., 2007) can give rise to some
of the same pathologies, such as nemaline bodies. In this
context it is of note that TPM3-domaint as well as null
mutations can both give rise to nemaline bodies
(Lehtokari et al., 2008). This, together with the fact that
nemaline bodies can also be seen in seemingly unrelated
genetic entities such as in RYR1 mutations, suggests that
the development of the nemaline bodies is a result of dys-
function of the sarcomere rather than due to a direct ef-
fect of the presence of mutant proteins or muscle weakness
per se.
Studies of transgenic mice expressing the nemaline myop-
athy-associated ACTA1 mutation p.Asp286Gly have re-
vealed that there is severe muscle weakness resulting in
early lethality in some mice, mimicking the severity seen
in patients (Ravenscroft et al., 2011). Studies of isolated
myofibres and whole muscle found a reduced Ca2+ sensi-
tivity compared to wild-type (Ravenscroft et al., 2011),
likely due to impaired actin–actin interactions within the
filamentous actin (Ochala et al., 2012). A similar decrease
in the force-frequency relationship (indicative of decreased
Ca2+ sensitivity) has been found in muscle from another
mouse model of nemaline myopathy (Ravenscroft, unpub-
lished data), the KI Acta1H40Y (p.His40Tyr) line (Nguyen
et al., 2011). In vitro studies of other ACTA1 mutations,
256 | BRAIN 2015: 138; 246–268
p.Met132Val and p.Phe352Ser, have shown an increase in
steady-state isometric force production associated with the
substitutions (Marston et al., 2004; Lindqvist et al., 2012).
Drosophila with ACTA1 mutations, in the indirect flight
muscles, were flightless and their muscle showed myofibril-
lar disorganization (Sevdali et al., 2013). One mutation,
p.Asp292Val, which causes an almost complete absence
of Ca2+ activated contraction (Clarke et al., 2007), resides
within a region of actin likely to bind tropomyosin and was
shown to rescue the hypercontractile phenotype of troponin
mutant flies (Sevdali et al., 2013). Jain et al. (2012) re-
ported a patient with nemaline myopathy that presented
with congenital hypertonia and stiffness, due to a de
novo p.Lys328Asn substitution. It was shown experimen-
tally, using the in vitro motility assay, that the mutant actin
extracted from the patient’s muscle biopsy displayed a sub-
stantially increased Ca2+ sensitivity (Jain et al., 2012). Thus
Ca2+ sensitizers, such as the troponin activators, may be
applicable to some ACTA1 mutations where there is
reduced Ca2+ sensitivity or reduced steady-state force pro-
duction, but not others. While the Jain et al. (2012) report
is the only published example of a hypertonic actin myop-
athy, this clinical entity is increasingly recognized and may
also be associated with other nemaline myopathy asso-
ciated gene mutations (C.G. Bönnemann et al., unpub-
lished data), thus a congenital myopathy should be
considered even in cases presenting with hypertonia and
stiffness.
Tropomyosins
Given the central role of tropomyosin as the crucial Ca2+-
regulated inhibitor of actin-myosin interactions, it was
thought that tropomyosin mutations would alter sarco-
meric force production and Ca2+ sensitivity. However,
there does not seem to be a simple unifying mechanism
associated with these mutations. The most widely studied
tropomyosin mutation is the slow a-tropomyosin (a-Tm,
TPM3) p.Met9Arg substitution (Laing et al., 1995;
Corbett et al., 2001, 2005). Expression of a-Tmp.Met9Arg
in cardiomyocytes resulted in a greatly reduced Ca2+ sensi-
tivity compared with cardiomyocytes expressing a-TmWT
(Michele et al., 1999). Analysis of muscle biopsies from
individuals with nemaline myopathy, or congenital fibre-
type disproportion, due to dominant or recessive mutations
in TPM3, revealed that changes in cross-bridge cycling kin-
etics underlie the muscular weakness (Ottenheijm et al.,
2011). Myofibres from all five TPM3 patients examined
in this study, showed a significantly lower rate of force
redevelopment and higher tensions costs (as measured by
ATP consumption rates per force production) suggesting
that the proportion of force-generating cross-bridges is di-
minished in patient myofibres. A striking feature of these
TPM3 myofibres was a greatly increased Ca2+ sensitivity,
resulting in significantly higher pCa50 values (Ottenheijm
et al., 2011). Notably, this increased sensitivity does not
lead to muscle stiffness but is rather thought to represent
G. Ravenscroft et al.
a compensatory mechanism counteracting some of the
effect of the diminished force generated by the cross-
bridges. The sum of this may then underlie the compara-
tively mild phenotype of some of these patients, since force
generation at submaximal Ca2+ concentrations was com-
parable between patients and healthy controls.
A similar mechanism to the altered cross-bridge cycling
kinetics described for the TPM3 mutations was also
observed in muscle expressing the p.Arg133Trp substitu-
tion in b-tropomyosin (TPM2) (Ochala et al., 2007).
Marttila et al. (2012) studied nemaline and cap myop-
athy-associated mutations in the b-tropomyosin gene
(TPM2) and showed that some mutations resulted in
altered affinity for actin (three showed reduced affinity,
one showed increased affinity) while others showed reduced
Ca2+ sensitivity of contractility (p.Glu117Lys and
p.Glu41Lys). Previously, studies of the TPM2 mutant
p.Glu41Lys also showed a reduced Ca2+ sensitivity of
force production, which could be mitigated by treatment
of myofibres with a Ca2+ sensitizer (EMD 57033)
(Ochala et al., 2008). A reduced actin binding affinity
has also previously been observed with the TPM2
p.Arg91Gly substitution associated with a distal arthrogry-
posis phenotype (Robinson et al., 2007). Most recently,
studies of the recurrent TPM2 p.Lys7del mutation asso-
ciated with a distal arthrogryposis and potentially worsen-
ing muscle contracture phenotype (Davidson et al., 2013;
Mokbel et al., 2013) have shown that myofibres harbour-
ing the mutant b-Tm produce comparable specific forces to
control myofibres but that the Ca2+ sensitivity of contrac-
tion is greatly increased (Mokbel et al., 2013). Davidson
et al. (2013) also showed, by expressing the p.Lys7del mu-
tation in zebrafish, that head-to-tail oligomerization of
b-tropomyosin polymers was likely to be impaired, that
the mutant tropomyosin failed to localize properly within
the thin filament and that sarcomere length was altered.
Actin-b-Tm co-sedimentation assays found that the actin
binding affinity of the mutant b-Tm was 6-fold lower
than wild-type b-Tm (Mokbel et al., 2013). A study by
Marston et al. (2013) showed that all tested TPM2 or
TPM3 mutations residing within repeating structural
motifs that bind actin caused a gain-of-function through
increased Ca2+ sensitivity, while mutations outside of
those regions resulted in reduced Ca2+ sensitivity. It will
be important to also correlate the other mutations modelled
in this study with clinical observations. Interestingly, some
TPM2 p.Lys7del patients presented with contractures at
birth or developed contractures postnatally (Davidson
et al., 2013; Mokbel et al., 2013) and one case was
noted to have generalized muscle hypertonia (Mokbel
et al., 2013), thus increased Ca2+ sensitivity associated
with specific TPM2 mutations may lead to muscle stiffness
with resulting contractures, adding to the differential diag-
nosis of this phenomenon (Sung et al., 2003; Robinson
et al., 2007; Jain et al., 2012; Mokbel et al., 2013). As
alluded to previously, Ca2+ sensitizers (Ochala, 2010) are
unlikely to be of therapeutic benefit in patients where there
Pathophysiology of congenital myopathies
already is enhanced sarcomeric Ca2+ sensitivity and may
even exacerbate the development of stiffness and contrac-
tures. Thus, knowledge of the exact physiological conse-
quences of a mutation will be essential in order to select
potential treatments for a given patient.
Myosin
The first skeletal muscle-specific MHC gene associated with
disease was MYH2. There are a handful of myopathy cases
in the literature arising due to autosomal dominant or re-
cessive mutation of the myosin heavy chain IIA gene
(MYH2; MIM 160740) (Martinsson et al., 2000;
Tajsharghi et al., 2005, 2010; D’Amico et al., 2013). The
phenotype of MYH2 cases can vary greatly with some cases
presenting with multiple joint contractures (Martinsson
et al., 2000; de Winter et al., 2013) and severe respiratory
distress at birth (D’Amico et al., 2013) to cases of child-
hood or adult onset of mild muscle weakness and myalgia
(Tajsharghi et al., 2005, 2010). In all cases the disease was
either non-progressive or was slowly progressive in the
third to fifth decade of life, ophthalmoparesis from birth
was a prominent feature in all cases. In the more rarely
occurring autosomal recessive cases, an absence of MHC
IIA myofibres has been demonstrated (Tajsharghi et al.,
2010). The surprisingly mild skeletal muscle phenotype of
the recessive MYH2 null patients is likely due to compen-
sation by the other MHC isoforms and contrasts with the
profound severity observed in patients with null mutations
of other sarcomeric genes [e.g. ACTA1 (Nowak et al.,
2007), CFL2 (Agrawal et al., 2007; Ockeloen et al.,
2012) and NEB (Ottenheijm et al., 2009)]. The proportion
of MHC IIA myofibres increases with age and it is thus
hypothesized that the progression of disease later in life is a
direct result of the increasing proportion of MYH2 mutant
protein (p.E706K) in the autosomal dominant cases
(Tajsharghi et al., 2002). Thus, similarly to the autosomal
dominant ACTA1 mutations, the mutant protein load
seems to be critical to disease severity.
Autosomal dominant mutations in the slow b-myosin
heavy chain gene (MYH7; MIM 160760) are known to
cause two congenital myopathies: hyaline body (myosin
storage) myopathy (Bohlega et al., 2004) and multi-mini-
core disease (Cullup et al., 2012; Clarke et al., 2013), in
addition to Laing distal myopathy (MIM 160500) and car-
diomyopathies. A recent report suggests that MYH7 muta-
tions may be more frequent in the congenital myopathies
than previously suggested (Clarke et al., 2013). Typically
mutations altering amino acid residues within the globular
head domain cause a cardiomyopathy whereas those alter-
ing amino acids within the tail rod domain cause a skeletal
muscle phenotype (Armel and Leinwand, 2009, 2010);
however, in recent years this distinction has become less
obvious (Muelas et al., 2010; Homayoun et al., 2011;
Lamont et al., 2014). The pathophysiology of MYH7-
related cardiomyopathies has begun to be elucidated, the
maximum force generating capacity of single cardiac
BRAIN 2015: 138; 246–268 | 257
myocytes and isolated myofibrils harbouring MYH7 muta-
tions were lower than controls, even when normalized to
cross-sectional area. Cardiomyocytes from patients har-
bouring MYH7 mutations were hypocontractile at maximal
and submaximal Ca2+ concentrations (Witjas-Paalberends
et al., 2013). Furthermore, cardiomyocytes from MYH7
patients show increased Ca2+ sensitivity, likely due to hypo-
phosphorylation of PKA and smaller length-dependent ac-
tivation (Sequeira et al., 2013); however these features
were observed in all cases of hypertrophic cardiomyopathy
(HCM) and are thus likely to be a generalized response to
the cellular remodelling that occurs in hypertrophic cardio-
myopathy rather than a direct result of the MYH7 gene
defect. Similar studies now need to be done for the
MYH7 mutations associated with skeletal muscle disease.
Autosomal dominant mutations in two genes (MYH3
and MYH8), encoding developmental MHC isoforms, are
responsible for some presentations of congenital distal
arthrogryposis, which postnatally is not progressive,
thus suggesting a largely developmental mode of action
(reviewed in Tajsharghi and Oldfors, 2013).
Atrophy
Skeletal muscle bulk is determined by the net effect of
hypertrophy and atrophy processes: protein synthesis and
degradation (comprising atrophy and autophagy) (reviewed
in Schiaffino and Mammucari, 2011). Atrophy and dis-
ordered autophagy are common in a wide variety of
muscle disorders so that it can be challenging to determine
how specific or non-specific these phenomena are in any
given disorder. In the congenital myopathies, gross skeletal
muscle atrophy is frequently observed upon clinical exam-
ination and, at the level of the myofibre, a common sec-
ondary or primary histological abnormality is type I (slow)
myofibre atrophy (Romero and Clarke, 2013; Wang and
Pessin, 2013). An emerging theme, therefore, is the role of
skeletal muscle protein synthesis and degradation molecular
pathways in the congenital myopathies; however, it must be
cautioned again that there is little direct evidence to sup-
port a causative link between the congenital myopathies
and skeletal muscle atrophy.
Protein degradation pathways
There are two main proteolytic systems that control protein
turnover in skeletal muscle: the ubiquitin-proteasome path-
way (atrophy) and the autophagy-lysosome machinery.
Atrophy and autophagy, while distinct programmes, are
both coordinately regulated via the transcription factors
FoxO3 and mammalian target of rapamycin (mTOR).
The ubiquitin-proteasome pathway is responsible for deg-
radation of short-lived soluble proteins and myofibrillar
proteins. Major players in the ubiquitin-proteasome path-
way are the E3 ubiquitin ligases (also known as atrogenes),
these include: F-box protein atrogin-1 (also known as
258 | BRAIN 2015: 138; 246–268
MAFbx, now known as FBXO32) and striated muscle spe-
cific tripartite motif (TRIM) proteins, also known as
MURF (muscle ring finger) proteins (reviewed in Perera
et al., 2012). Of particular interest to the congenital myo-
pathies is the finding that the sarcomeric contractile appar-
atus, dysfunction of which is so central to many congenital
myopathies, is directly involved in regulating atrophy sig-
nalling. For example, the mechanosensitive M-line titin
kinase domain is known to recruit MURF2 to the sarco-
mere (reviewed in Voelkel and Linke, 2011). Thus it is
conceivable, although not yet proven, that changes in the
tension generation on the sarcomere will influence these
atrophy pathways.
Atrophy in centronuclear myopathy
The role of atrophy in congenital myopathies has been best
characterized in the centronuclear myopathies. In the Mtm1
p.Arg69Cys mouse, skeletal muscle atrophy preceded the
onset of muscle weakness (Pierson et al., 2012), leading
the authors to suggest that the atrophy in this model was
not a consequence of muscle inactivity but was associated
intrinsically with the genetic defect. Joubert and colleagues
(2013) found that intramuscular delivery of an adeno-
associated virus designed to deplete MTM1 resulted in a
myotubular myopathy phenotype that included skeletal
muscle atrophy. The pathway by which MTM1 deficiency
leads to atrophy has begun to be elucidated. Elegant studies
by Razidlo et al. (2011) showed that in MTM1-depleted
HeLa cells and myotubes there were decreased levels of
phosphorylated Akt (activated, Akt-P), and identified that
this was mediated by an increase in PI3P levels. Decreased
levels of Akt-P resulted in greater activity of FoxO1 and
decreased activity of proteins (S6K and 4E-BP1) regulated
by mammalian target of rapamycin complex 1 (mTORC1)
of the growth axis (Razidlo et al., 2011). Thus aberrant
mTORC1 and FOXO1 signalling may underlie some of
the pathology of MTM1 deficiency. Indeed mTORC1-
deficient mice show skeletal muscle atrophy with numerous
centrally nucleated myofibres (Bentzinger et al., 2008),
mimicking some of the hallmark features of MTM1
deficiency.
In a Mtmr14 KO mouse model, it was found that age-
related muscle wasting, sarcopenia, occurred precociously
(Romero-Suarez et al., 2010). It was also noted that
Mtmr14 expression and MTMR14 protein levels were
decreased in old wild-type muscle, suggesting a role for
MTMR14 in sarcopenia (Romero-Suarez et al., 2010).
The force deficits and altered Ca2+ handling were similar
for mature Mtmr14 / (12–14 months of age) and old
wild-type muscle (22–24 months of age), suggesting that
muscle atrophy in both conditions (Mtmr14 deficiency
and sarcopenia) may be due to altered Ca2+ signalling
and feedback from the sarcomere.
KI mice, heterozygous for the p.Arg465Trp DNM2 muta-
tion, develop skeletal muscle weakness prior to the onset of
structural abnormalities and muscle atrophy (Durieux et al.,
G. Ravenscroft et al.
2010); homozygous mice died within 24 h of birth.
Expression of atrophy- and autophagy-related genes:
FOXO3, MURF1 and Gabarap11 were elevated in 2-
month-old heterozygous KI muscle relative to wild-type.
This study also showed upregulation of FOXO3 and
Gabarap11 in skeletal muscle from a single patient with a
DNM2 mutation (Durieux et al., 2010). Skeletal muscle
atrophy was also observed in mouse muscle in which wild-
type or p.Arg465Trp mutant DNM2 was overexpressed
using adeno-associated virus vectors (Cowling et al., 2011).
An interesting example of how a sarcomeric protein can
directly influence muscle protein turnover is supplied by
titin and its kinase domain, which regulates a signalling
complex by interacting with Nbr, which regulates
MURF2 via p62/SQSTM1, thus providing a link to trans-
late sensing of mechanical load to regulation of muscle
protein turnover (Lange et al., 2005). A mutation in one
recessive titinopathy patient with arthrogryposis, cardiomy-
opathy and also muscle atrophy has been shown to com-
pletely abolish this kinase activity (Chauveau et al., 2014).
It will be interesting to see how this pathway will be af-
fected in other patients and animal models.
Atrophy in nemaline myopathy
Mutation of genes encoding BTB-Kelch proteins, known to
bind cullin 3 to form E3 ubiquitin ligases (Sambuughin
et al., 2012; Canning et al., 2013), has been shown to
cause nemaline myopathies [KBTBD13 (Sambuughin
et al., 2010), KLHL40 (Ravenscroft et al., 2013b),
KLHL41 (Gupta et al., 2013)] and an early-onset distal
myopathy (KLHL9) (Cirak et al., 2010); suggesting a
direct link between impairment of the ubiquitin-proteasome
system and skeletal muscle myopathies. Recently, Garg
et al. (2014) showed that Klhl40 KO mice presented with
a lethal nemaline myopathy-like disease. They also identi-
fied nebulin and leiomodin 3 (LMOD3) as KLHL40-bind-
ing proteins and showed that loss of KLHL40 resulted in
reduced abundance of NEB and LMOD3 protein in Klhl40
KO muscle. NEB and LMOD3 were also decreased in
muscle biopsies from some KLHL40 patients.
Intriguingly, co-expression of KLHL40 with either NEB
or LMOD3, resulted in increased abundance of these pro-
teins, in the absence of a change in transcript levels. The
increase in LMOD3 abundance (in the presence of
KLHL40) was similar to that observed when a proteasome
inhibitor was added and it was shown that KLHL40
decreased polyubiquitination of LMOD3 (Garg et al.,
2014). Thus despite the widely-held view that BTB-Kelch
proteins mediate protein degradation, it appears that
KLHL40 actually stabilizes the thin-filament proteins NEB
and LMOD3, by inhibiting proteasome-mediated degrad-
ation. The absence of NEB in some KLHL40 patient
muscle may explain the severity of KLHL40 disease, as
patients with two null alleles of NEB show a similarly
severe phenotype (Wallgren-Pettersson et al., 2002).
Pathophysiology of congenital myopathies
These observations of the multifaceted involvement of
atrophy have lead to preclinical investigations of treatment
counteracting atrophy in a number of congenital myopathy
models. In a mouse model of nemaline myopathy (KI
Acta1H40Y), upregulation of two hypertrophic factors
[IGF1 and four and a half LIM domains protein 1
(FHL1; Cowling et al., 2008)] or delivery of -tyrosine,
improved skeletal muscle strength and pathology (Nguyen
et al., 2011). Downregulation of components of the ubi-
quitin-proteasome pathway (based on microarray analysis
of transcript levels) were observed in skeletal muscle from
nemaline myopathy patients suggesting altered protein
turnover (Sanoudou et al., 2003), the functional signifi-
cance of these alterations remains unclear. There is anec-
dotal and some experimental evidence from the TPM3
Met9Arg mouse that exercise may be beneficial in nemaline
myopathy (Joya et al., 2004; Wallgren-Pettersson et al.,
2011); presumably by minimizing susceptibility to disuse
atrophy (Wallgren-Pettersson, 1989; Wallgren-Pettersson
et al., 1990; North et al., 1997).
The potential benefits of controlled exercise in individuals
with congenital myopathies clearly warrant further investi-
gation. Similarly it remains to be investigated more
systematically whether targeted disruption of the ubiqui-
tin-proteosomal pathway might represent a potential thera-
peutic target for the congenital myopathies.
Protein synthesis pathways
One of the most potent known activators of skeletal hyper-
trophy is IGF1 (insulin-like growth factor 1) (Musaro
et al., 1999). This hypertrophy requires myofibrillogenesis.
Until more recently the mechanism whereby IGF1 mediated
myofibrillogenesis remained completely obscure. Takano
et al. (2010) showed that IGF1 produces its hypertrophic
effects via activation of PI3K-Akt signalling that results in
complex formation of N-WASP and nebulin. Nebulin-N-
WASP complexes were found to mediate actin filament nu-
cleation from the Z-disc (Takano et al., 2010). Thus IGF1-
induced actin filament formation (via nebulin-N-WASP
complexes) is required for muscle maturation and
hypertrophy.
The most potent negative regulator of skeletal muscle
hypertrophy is myostatin, a member of the TGF beta-like
growth factor family, signalling through activin receptors
(Amthor and Hoogaars, 2012). Naturally-occurring and
genetically-engineered animal models that are myostatin
nulls (Mstn / ) present with a ‘double-muscled’ phenotype.
Counterintuitively, despite the plethora of naturally occur-
ing animal models (most notably Belgium Blue and
Piedmontese cattle, sheep, whippet and mouse) (Stinckens
et al., 2011) there is only a single-reported isolated case of
a MSTN partial loss-of-function patient (Schuelke et al.,
2004). Myostatin is a negative regulator of Akt and thereby
inhibits protein synthesis. In addition to this direct role on
protein synthesis, myostatin also signals through SMAD2
and SMAD3 to activate FOXO3, an inducer of both
BRAIN 2015: 138; 246–268 | 259
atrophy (through upregulation of atrogenes and MURF
proteins) and autophagy.
Approaches to therapy targeting
hypertrophy/atrophy pathways
Given the prominence of myostatin in regulating myofibre
atrophy, modulation of myostatin signalling has been vig-
orously pursued as a potential therapeutic avenue for skel-
etal muscle diseases (particularly the muscular dystrophies;
reviewed in Amthor and Hoogaars, 2012); however, some
studies suggest that myostatin inhibition may be detrimen-
tal to skeletal muscle function (Amthor et al., 2007;
Giannesini et al., 2013), perhaps in particular in a muscu-
lar dystrophy in which the muscle is inherently fragile. This
may suggest that non-dystrophic myopathies with promin-
ent muscle atrophy may be a better target for myostatin
modulation. Inhibition of the activin receptor IIb in
Mtm1 / mice improved muscle strength and lifespan
(Lawlor et al., 2011b). Thus modulation of the myostatin
pathway might be of benefit in some congenital myopa-
thies. It seems that this approach should be considered on
a disease-by-disease basis and should take into account all
of the different mechanisms resulting in skeletal muscle
atrophy.
Satellite cells defects
Although satellite cells play a major role during myogenesis
and muscle regeneration, their role in postnatal skeletal
muscle maintenance and hypertrophy is controversial. In
a limited number of recent studies a potential role for sat-
ellite cells has been implicated in the pathobiology of some
congenital myopathies, but again, the disease specificity of
these phenomena will need to be further investigated.
Similar to MTM1 patients, the early stages of myogenesis
in the Mtm1 KO mouse model seems normal - this has led
to the suggestion that there is defective skeletal muscle
maintenance or regeneration associated with MTM1 defi-
ciency (Buj-Bello et al., 2002). Reduced satellite cell num-
bers were observed in muscle from Mtm1 KO mice,
determined by measuring PAX7 abundance and by flow
cytometry to quantify the relative number of myogenic
cells (CD31 /CD45 /CD106 + ) isolated from the muscle.
This study also showed that the number of resident satellite
cells declined with age in the Mtm1 KO muscle. The Mtm1
KO progenitor cells showed reduced proliferative capacity
in vitro and in in vivo transplantation experiments and
were more susceptible to apoptosis (Lawlor et al., 2012).
The authors hypothesized that the altered Ca2+ homeostasis
due to MTM1 deficiency may cause mitochondrial stress
and thereby activate the mitochondrial-apoptotic pathway.
In mouse skeletal muscle depleted of MTM1 postnatally, a
90% reduction in the number of resident satellite cells
was observed (Joubert et al., 2013), these studies suggest
that exhaustion of the satellite cell population may
260 | BRAIN 2015: 138; 246–268
contribute to the disease progression associated with
MTM1 deficiency.
Exhaustion of the satellite cell population was also a fea-
ture of the Sepn1 null mouse (Castets et al., 2012); reduced
satellite cell numbers have also been observed in SEPN1
patient muscle and likely contributes to the muscle atrophy
observed (Castets et al., 2012) suggesting that SEPN1 plays
an essential role in maintenance of these skeletal muscle
progenitor cells.
Recessive mutations in MEGF10 (MIM 612453) were
associated with early onset myopathy, areflexia, respiratory
distress and dysphagia (EMARDD; MIM 614399) (Logan
et al., 2011; Boyden et al., 2012). MEGF10 is expressed in
quiescent and activated satellite cells and knock-down of
Megf10 in mouse muscle resulted in satellite cell depletion
(Holterman et al., 2007). EMARDD patient muscle
showed reduced mean myofibre diameter and lacked
PAX7 + nuclei (Logan et al., 2011).
Elevated satellite cell numbers were observed in skeletal
muscle from patients with nemaline myopathy (Sanoudou
et al., 2003) and TPM3 p.Met9Arg transgenic mice
(Sanoudou et al., 2006), suggesting that satellite cell-
activated muscle regeneration might be a feature in some
subtypes of nemaline myopathy. The role of satellite cells in
the pathophysiology of nemaline myopathy has not been
investigated further.
Myotubes and skeletal myofibres are extremely resistant
to apoptosis, and this is thought to be due to expression of
survival proteins: Akt, PI3K and p21 while satellite cells
express anti-apoptotic Bcl-2 as their primary survival
factor (reviewed in Schwartz, 2008). Recently it was
shown that myotubularin regulates Akt-mediated cell sur-
vival signalling via elevated levels of PI3P (Razidlo et al.,
2011). siRNA silencing of MTM1, in HeLa cells, resulted in
a robust activation of caspase-dependent pro-apoptotic sig-
nalling that resulted in DNA fragmentation and cell death.
Knock-down of myotubularin in cultured human myotubes
also resulted in inhibition of Akt phosphorylation, caspase-
dependent pro-apoptotic signalling and DNA fragmenta-
tion (Razidlo et al., 2011). Thus, there may also be a
role for aberrantly active apoptosis in the pathogenesis of
congenital myopathies.
Autophagy effected by
membrane targeting and
turnover
The role of autophagy in skeletal muscle homeostasis
(reviewed in Sandri, 2010) is less well understood than at-
rophy. Autophagy lysosomes regulate the turnover of long-
lived proteins and organelles (Sandri, 2010) and involves
BNIP3, Gabarap11, LC3 (now knwon as MAP1LC3A) and
p62, as well as a host of other players. Autophagy and
atrophy are inextricably linked; counterintuitively, autop-
hagy blockade in Atg7 / mouse muscle resulted in
G. Ravenscroft et al.
profound skeletal muscle atrophy and force deficits
(Masiero et al., 2009; Masiero and Sandri, 2010).
Alterations to autophagy and/or lysosomal function are
observed in many skeletal muscle diseases, including: colla-
gen VI-related muscular dystrophies (in which autophagy
seems to underperform), Danon disease, Duchenne muscu-
lar dystrophy, Pompe disease and X-linked myopathy with
excessive autophagy (Bonaldo et al., 1998; Grumati et al.,
2010; De Palma et al., 2012; Ramachandran et al., 2013)
and have also recently been implicated in myofibrillar my-
opathy (Kley et al., 2012, 2013). Thus both impaired
autophagy and excessive autophagy can result in skeletal
muscle disease, highlighting the need for the right balance
between protein synthesis and degradation processes.
Recently, Castets et al. (2013) have shown that sustained
activation of mTORC1 results in impaired autophagy (via
inhibition of Ulk1, despite FOXO3 activation) and pro-
duced a late onset myopathy in the mouse. Conversely,
inhibition of mTORC1, via depletion of Raptor, induced
autophagy regardless of FOXO inhibition. These novel
data suggest that mTORC1 is, in fact, the major regulator
of skeletal muscle autophagy (Castets et al., 2013).
Within the congenital myopathies, autophagy has been
most studied in the CNMs. Given that autophagy is essen-
tially a membrane-driven process and that the primary
defect in the CNMs involves membrane remodelling, de-
fective autophagy in the CNMs may be a direct conse-
quence of the underlying genetic defect.
Centronuclear myopathies
A clear link between MTM1 deficiency and autophagy is
tenuous. PI3P is required for the initiation of autophagy
and PI3,5P2 is required for its progression. Muscle-specific
deletion of PIK3C3 (the primary kinase responsible for the
production of PI3P) causes a lethal dystrophic phenotype
in mice and a severe defect of the autophagolysosomal
pathway (Reifler et al., 2014). Thus turnover of PI3P by
myotubularins may be required for autophagy progression
and it is possible that loss of MTM1 could directly impair
autophagy. However, a study examining the role of myo-
tubularins in autophagy showed that MTM1 was only a
weak activator of the process (Vergne et al., 2009).
Levels of some autophagy-associated transcripts
(Gabarap11 and LC3b) are increased in Mtm1 KO
mouse muscle (Fetalvero et al., 2013; Joubert et al.,
2013). In Cre-induced Mtm1 depletion in adult skeletal
muscle, levels of LC3-I, LC3-II and p62 proteins were ele-
vated (Joubert et al., 2013), furthermore elevated levels of
phosphorylated S6, 4EBP1, Akt and phosphorylated Akt
were observed. Similarly, LC3-I, LC3-II, p62 and LAMP2
proteins were more abundant in Mtm1 / muscle than
wild-type; levels of phosphorylated S6 and Akt were also
elevated in Mtm1 / muscle (Fetalvero et al., 2013).
Elevated levels of LC3, p62, polyubiquitinated proteins
and dysfunctional mitochondrial are also a feature of
Atg7 / null mice (Masiero et al., 2009; Masiero and
Pathophysiology of congenital myopathies
Sandri, 2010). This suggests that mTORC1-mediated
blockade of normal autophagic degradation may contribute
to the impaired autophagy observed in Mtm1 depleted
muscle and partially contributes to the muscle pathology
associated with MTM1 deficiency (Fetalvero et al., 2013;
Joubert et al., 2013). However, the autophagic defect in
Mtm1 deficient mouse muscle may be secondary to the
triad dysfunction, the sarcoplasmic reticulum is a major
membrane donor to autophagy, or the general poor
health of these animals.
The link between autophagy and MTMR14 is much
clearer, where failed regulation likely of PI3,5P2 results in
impaired regulation of autophagic flux. Vergne and col-
leagues (2009) showed that siRNA knock-down of
MTMR14 and loss of its enzymatic activity, in C2C12
myoblasts, resulted in the formation of autophagosomes
and also facilitated their maturation into autolysosomes.
LC3-II levels were unchanged following MTMR14 siRNA
treatment in the absence of Atg5 (autophagy-related 5),
showing that MTMR14 negatively regulates autophagy in
an Atg5-dependent manner. In support of this, overexpres-
sing MTMR14 resulted in an increase of p62 aggregates in
transfected C2C12 myoblasts suggesting impaired autop-
hagy (Vergne et al., 2009). MTMR14 was found to co-
localize with ATG12, ATG16 and LC3-II in early autopha-
gic organelles and autophagasomes in C2C12 myoblasts.
Furthermore knock-down of MTMR14 resulted in
increased recruitment of the autophagic PI3P-binding pro-
tein WIPI1 (the mammalian homologue of yeast ATG18)
(Xie and Klionsky, 2007) to autophagosomes. Finally,
Vergne et al. (2009) showed that overexpression of a
CNM MTMR14 variant (p.Arg336Gln) within the phos-
phatase domain did not promote accumulation of p62
puncta, whereas wild-type and p.Tyr462Cys (a mutant out-
side of the catalytic domain) MTMR14, did increase p62
aggregation (Vergne et al., 2009). These data suggest that
the CNM p.Arg336Gln substitution renders MTMR14
unable to regulate autophagy.
Dowling et al. (2010) found elevated induction of autop-
hagy in mtm1/mtmr14 double morphant zebrafish, as
determined by increased LC-II levels and vacuolated mem-
branes, with severe effects on gross embryogenesis and
extensive autophagic defects. Although accumulation of ab-
normal membranous structures had previously been re-
ported for mtm1 morphant fish, autophagy was not
altered in these fish (Dowling et al., 2009). Thus, these
authors hypothesize that the phenotype of the double mor-
phants results from dysregulated organelle and membrane
breakdown and overloading of the autophagic system pri-
marily due to loss of mtmr14 (Dowling et al., 2010).
Skeletal muscle and cultured embryonic fibroblasts from
knock-in Dnm2 p.Arg465Trp (KI-Dnm2R465W) mice show
elevated levels of LC3, Gabarap11 and p62 transcripts and
greater protein abundance (Durieux et al., 2012). The
number of p62-positive accumulations and early phagosomes
were also elevated in KI- Dnm2R465W embryonic fibroblasts.
These data suggest that impaired autophagy contributes
BRAIN 2015: 138; 246–268 | 261
to the phenotype of KI-Dnm2R465W mice, as opposed to upre-
gulated autophagy observed with MTMR14 mutation.
Other possible therapies for
congenital myopathies
As we have pointed out throughout the discussion so far,
understanding the pathomechanisms active in the congeni-
tal myopathies is important in defining targets against
which to develop therapies, such as compounds that can
ameliorate the downstream consequences of the underlying
genetic defect/s that could modulate diseases. However, tar-
geting the primary cause of disease, i.e. the genetic muta-
tion and their immediate consequences, while challenging,
obviously represents a major goal for arriving at effective
treatments for these conditions.
Enzyme replacement therapy
Enzyme replacement therapy has been a therapeutic strat-
egy that has been pursued in a number of diseases resulting
from enzyme deficiencies (reviewed in Valayannopoulos,
2013). Notably, enzyme replacement therapy is now in
clinical use for a skeletal muscle disease, glycogen storage
disease II/Pompe disease resulting from deficiency of GAA
(Thurberg et al., 2006; Gungor et al., 2013). Lawlor et al.
(2013) have shown that intramuscular delivery of myotu-
bularin-fused with a skeletal muscle penetrating-antibody
protein (3E10Fv-MTM1) resulted in improved muscle con-
tractility and pathology and improved gait and ambulation
of Mtm1 KO mice. Recently, AAV8-mediated MTM1 gene
therapy was explored in Mtm1 KO mice and dogs carrying
a naturally occurring Mtm1 mutation (Childers et al.,
2014). It was shown that a single intravascular injection
was sufficient to increased muscle strength, reduce muscle
pathology and extend survival (Childers et al., 2014).
These studies demonstrate the efficacy of enzyme replace-
ment therapy and pave the way for clinical trials for myo-
tubular myopathy. Similarly, enzyme replacement therapy
may also be expected to be of therapeutic benefit in models
of MTMR14-related congenital myopathies although this
has not been attempted.
Upregulation therapy
Evidence from patients (Nowak et al., 2007; Ravenscroft
et al., 2008) and experimental data, concur that mutant
actin load determines disease severity in dominant
ACTA1 disease (Drummond et al., 1991; Domazetovska
et al., 2007; Ravenscroft et al., 2008; 2011, 2013a;
Haigh et al., 2010; Sevdali et al., 2013) and thus modula-
tion of the amount of mutant ACTA1 represents a target
for dominant ACTA1 patients. Furthermore, upregulation
of the foetal isoform of skeletal muscle a-actin (cardiac
a-actin; ACTC) in patients with recessive loss of ACTA1
moderates the disease allowing survival of affected children.
262 | BRAIN 2015: 138; 246–268
Upregulation of ACTC is thus a valid target for upregula-
tion or even gene transfer (Nowak et al., 2009;
Ravenscroft et al., 2013a) as it would be less likely to
cause an immune response to the introduced protein as
compared with re-introducing ACTA1.
Conclusions
Here we have attempted to highlight emerging patho-
physiological concepts spanning across different genetic
and structural entities that traditionally comprise the con-
genital myopathies, with the goal of pointing out avenues
for therapeutic developments that are based on disease
mechanisms. It remains to be more precisely determined
which of the pathophysiological concepts explored herein
are identified in which of the congenital myopathies, and to
what degree. For instance, in the most well characterized
models, those for CNMs, there appears to be a role for all
five of the key themes in the pathobiology of the disease.
It is perhaps necessary, given the wave of animal models
generated and studied for the congenital myopathies and
the studies of patient biopsies and cell lines, that standard
operating procedures be developed and implemented for
the systematic characterization of these models and tissues,
to allow for greater intermodel interpretation and for com-
parison of the efficacies of various therapeutic strategies.
This has been recognized and implemented for models of
muscular dystrophies (Grounds et al., 2008; Nagaraju and
Willmann, 2009) and spinal muscular atrophy (Willmann
et al., 2011) and is recognized by the presence of standard
operating procedures on the TREAT-NMD webpage
(http://www.treat-nmd.eu/research/preclinical/). It would
be desirable to expand this approach to models of the con-
genital myopathies.
Ultimately it seems likely that treatment of the congenital
myopathies will encompass multimodal regimes: e.g. gene
transfer therapy, enzyme replacement therapy, upregulation
of alternative genes, anti-atrophy approaches, in addition
to perhaps AChE inhibitors, and/or Ca2+ sensitizers and/or
anti-oxidants. The considerable overlap of pathobiological
mechanisms between the various genetic and structural sub-
groups of the congenital myopathies might indicate that
some of the potential therapies may be effective in a
broad range of the congenital myopathies and beyond.
Acknowledgements
We thank our anonymous reviewers for most helpful com-
mentary and advice.
Funding
G.R. is supported by a National Health and Medical
Research Council of Australia (NHRMC) Early Career
Researcher Fellowship (APP1035955) and N.G.L. by a
G. Ravenscroft et al.
NHMRC Research Fellowship (APP1002147). C.G.B. is
supported by intramural funds of NINDS/NIH.
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