RESEARCH ARTICLE

Isoform-­specific mutation in Dystonin-­bgene causes late-­onset protein aggregate
myopathy and cardiomyopathy
Nozomu Yoshioka1,2, Masayuki Kurose3, Masato Yano1, Dang Minh Tran1,
Shujiro Okuda4, Yukiko Mori-­Ochiai1, Masao Horie5, Toshihiro Nagai6,
Ichizo Nishino7, Shinsuke Shibata6,8, Hirohide Takebayashi1,9*1

Division of Neurobiology and Anatomy, Graduate School of Medical and Dental
Sciences, Niigata University, Niigata, Japan; 2Transdisciplinary Research Programs,
Niigata University, Niigata, Japan; 3Department of Physiology, School of Dentistry,
Iwate Medical University, Iwate, Japan; 4Medical AI Center, School of Medicine,
Niigata University, Niigata, Japan; 5Department of Nursing, Niigata College ofNursing, Jōetsu, Japan; 6Electron Microscope Laboratory, Keio University, Tokyo,
Japan; 7Department of Neuromuscular Research, National Institute of Neuroscience,
National Center of Neurology and Psychiatry, Tokyo, Japan; 8Division of Microscopic
Anatomy, Graduate School of Medical and Dental Sciences, Niigata University,
Niigata, Japan; 9Center for Coordination of Research Facilities, Niigata University,
Niigata, Japan*For correspondence:
takebaya@med.niigata-u.ac.jp
Competing interest: See page
29
Funding: See page 29
Received: 07 March 2022
Preprinted: 19 March 2022
Accepted: 18 July 2022
Published: 09 August 2022
Reviewing Editor: Christopher
Cardozo, Icahn School of
Medicine at Mount Sinai, UnitedStates‍ ‍Copyright Yoshioka et al. This
article is distributed under theterms of the Creative Commons
Attribution License, which
permits unrestricted use andredistribution provided that theoriginal author and source arecredited.

Abstract Dystonin (DST), which encodes cytoskeletal linker proteins, expresses three tissue-­selective isoforms: neural DST-­a, muscular DST-­b, and epithelial DST-­e. DST mutations cause
different disorders, including hereditary sensory and autonomic neuropathy 6 (HSAN-­VI) and epidermolysis bullosa simplex; however, etiology of the muscle phenotype in DST-­related diseases has
been unclear. Because DST-­b contains all of the DST-­a-­encoding exons, known HSAN-­VI mutations
could affect both DST-­a and DST-­b isoforms. To investigate the specific function of DST-­b in striatedmuscles, we generated a Dst-­b-­specific mutant mouse model harboring a nonsense mutation. Dst-­bmutant mice exhibited late-­onset protein aggregate myopathy and cardiomyopathy without neuropathy. We observed desmin aggregation, focal myofibrillar dissolution, and mitochondrial accumulation in striated muscles, which are common characteristics of myofibrillar myopathy. We also foundnuclear inclusions containing p62, ubiquitin, and SUMO proteins with nuclear envelope invaginations
as a unique pathological hallmark in Dst-­b mutation-­induced cardiomyopathy. RNA-­sequencing
analysis revealed changes in expression of genes responsible for cardiovascular functions. In silico
analysis identified DST-­b alleles with nonsense mutations in populations worldwide, suggesting thatsome unidentified hereditary myopathy and cardiomyopathy are caused by DST-­b mutations. Here,
we demonstrate that the Dst-­b isoform is essential for long-­term maintenance of striated muscles.
Editor's evaluation
The authors demonstrate that isoform-­specific Dystonin-­b (Dst-­b) mutant mice show significant
myopathy in skeletal and cardiac muscle at older ages without the peripheral neuropathy or postnatal lethality that are commonly observed by loss of function of the DST gene. The study provides
novel information about the role of the Dst-­b isoform in maintaining skeletal and cardiac muscle
health. In addition, the study suggests that isoform-­specific mutations in Dst-­b gene may causesome hereditary skeletal and cardiac myopathies.

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Introduction
Skeletal and cardiac striated muscle fibers consist of a complex cytoskeletal architecture, and maintenance of the sarcomere structure is essential for muscle contraction. Dystonin (DST), also calledbullous pemphigoid antigen 1 (BPAG1), is a cytoskeletal linker protein that belongs to the plakinfamily, which consists of DST, plectin, microtubule actin cross-­linking factor 1 (MACF1), desmoplakin,and other plakins (Boyer et al., 2010a; Künzli et al., 2016; Horie et al., 2017). The DST gene consistsof over 100 exons and generates tissue-­selective protein isoforms through alternative splicing anddifferent transcription initiation sites. At least three major DST isoforms exist, DST-­a, DST-­b, and DST-­e,which are predominantly expressed in neural, muscular, and epidermal tissues, respectively (Leunget al., 2001). Although DST-­a and DST-­b share most of the same exons, DST-­b contains five additional
isoform-­specific exons. Loss-­of-­function mutations in the DST locus have been reported to cause
neurological disorders, hereditary sensory and autonomic neuropathy type 6 (HSAN-­VI) (Edvardson
et al., 2012), and the skin blistering disease epidermolysis bullosa simplex (Groves et al., 2010).
DST-­a is considered the crucial DST isoform in the pathogenesis of HSAN-­VI because it is a neuralisoform, and transgenic expression of Dst-­a2 under the control of a neuronal promoter partially rescues
disease phenotypes of DstTg4 homozygous mice, which is a mouse model of HSAN-­VI (Ferrier et al.,2014). HSAN-­VI patients exhibit muscular and cardiac abnormalities, such as reduced muscular action
potential amplitude, muscle weakness, and disrupted cardiovascular reflexes (Edvardson et al., 2012;
Manganelli et al., 2017; Fortugno et al., 2019; Jin et al., 2020; Motley et al., 2020). Because allreported HSAN-­VI mutations could disrupt both DST-­a and DST-­b, it is unknown whether these muscle
manifestations are caused by cell-­autonomous effects of DST-­b mutation and/or secondary effects ofneurological abnormalities caused by DST-­a mutation. Thus, the impact of DST-­b deficiency in skeletaland cardiac muscles on the manifestations of HSAN-­VI patients has not been fully elucidated.
Dystonia musculorum (dt) is a spontaneously occurring mutant in mice (Duchen et al., 1964; Horieet al., 2016) that results in sensory neuron degeneration, retarded body growth, dystonic and ataxic
movements, and early postnatal lethality. The Dst gene was identified as a causative gene in dt mice
(Brown et al., 1995; Guo et al., 1995), and later, dt mice were used as mouse models of HSAN-­VI(Ferrier et al., 2014). Dt mice have been reported to display muscle weakness and skeletal muscle
cytoarchitecture instability (Dalpé et al., 1999). We have also reported masseter muscle weakness in
Dst gene trap (DstGt) mice, neurodegeneration of the trigeminal motor nucleus, which innervates themasseter muscle, and muscle spindle atrophy in the masseter muscle (Hossain et al., 2018). Thus,
known Dst mutant mice have mutations in both Dst-­a and Dst-­b, and exhibit abnormalities in neuraland muscular tissues.
To investigate cell-­autonomous functions of the Dst-­b isoform in skeletal and cardiac muscles, we
generated novel isoform-­specific Dst-­b mutant mice. Dst-­b mutant mice displayed late-­onset protein
aggregate myopathy in skeletal and cardiac muscles without peripheral neuropathy and postnatal
lethality. In this study, we first demonstrated the role of Dst-­b in skeletal and cardiac muscle maintenance and that a Dst-­b isoform-­specific mutation causes protein aggregate myopathy, which has
characteristics similar to myofibrillar myopathies (MFMs). We also observed conduction disorders in
the electrocardiograms (ECGs) of Dst-­b mutant mice. RNA sequencing (RNA-­seq) analysis revealedchanges in the expression of genes that are important for maintenance of cardiovascular structuresand functions. These results suggest that the myopathic manifestations of patients with HSAN-­VI may
be caused by disruption of DST-­b, in addition to neurological manifestations caused by DST-­a mutation. Furthermore, because we identified a variety of DST-­b mutant alleles with nonsense mutations
worldwide using in silico analysis, it is possible that some unidentified hereditary myopathies and/orheart diseases are caused by isoform-­specific mutations of the DST-­b gene.

Results
Generation of novel isoform-specific Dst-b mutant mice
Among the three major Dst isoforms (Figure 1A), the Dst-­a and Dst-­b isoforms share the most exons,
and the Dst-­b isoform has five additional isoform-­specific exons (Figure 1B). We generated a novel
Dst-­b mutant allele in which a nonsense mutation was introduced into a Dst-­b-­specific exon using
the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-­associated protein 9(Cas9) genome editing method. The Dst-­b mutant allele harbors a nonsense mutation followed by a

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A

B

C

100 bpladderDst-bE2610Ter
Het Homo

WT

500 bp400 bp300 bp200 bpHeart
WT Dst-bE2610Ter

Brain
Dst-bE2610Ter
WT

Dst
Gapdh
(36 kDa)

E

Dst-a

Dst-b
1.5

*

1.0
0.5
0

WT Dst-bE2610Ter
(n=3)
(n=3)

Relative mRNA

Skeletal muscle
Dst-bE2610Ter
WT

Relative mRNA

D

ns

1.5
1.0
0.5
0

WT Dst-bE2610Ter
(n=3)
(n=3)

Figure 1. Generation of Dst-­bE2610Ter mutant mouse line. (A) The protein structure of Dst isoforms. The Dst-­bE2610Ter allele has the mutation between theplakin repeat domain (PRD) and the spectrin repeats. The DstGt allele has the gene trap cassette within the actin-­binding domain (ABD) shared by Dst-­aand Dst-­b isoforms. EB-­BD, EB-­binding domain; EFh, EF hand-­calcium binding domains; GAR, growth arrest-­specific protein 2-­related domain; IFBD,
intermediate filament-­binding domain; MTBD, microtubule-­binding domain. (B) A schematic representation of the Dst transcripts. The part of Dst-­b-­specific exons is enlarged, showing the mutation sites of the Dst-­bE2610Ter allele. The nonsense mutation and XhoI recognition sequence are inserted
within the Dst-­b-­specific exon. The primer-­annealing sites are indicated by arrows. (C) PCR-­restriction fragment length polymorphism (RFLP) genotyping
to distinguish WT, Dst-­bE2610Ter heterozygotes (Het), and Dst-­bE2610Ter homozygotes (Homo). PCR products from Dst-­bE2610Ter alleles are cut by XhoI.
(D) Western blot analysis using the Dst antibody in lysates from the skeletal muscle (hindlimb muscle), heart, and brain (n = 3 mice, each genotype).
Truncated Dst bands (arrowheads) were detected in the skeletal muscle and heart of Dst-­bE2610Ter/E2610Ter mice. Dst bands in the brain were unchanged
between WT and Dst-­bE2610Ter/E2610Ter. Glyceraldehyde-­3-­phosphate dehydrogenase (Gapdh) was used as an internal control. (E) Quantitative PCR (qPCR)
data of Dst-­a and Dst-­b mRNAs in the heart (n = 3 mice, each genotype). * denotes statistically significant difference at p<0.05 (Dst-­b, p=0.0479) and ns
means not statistically significant (Dst-­a, p=0.4196), using Student’s t-­test. Data are presented as mean ± standard error (SE). Similar truncations of the
Dst protein in three independent Dst-­bE2610Ter mouse lines are shown in Figure 1—figure supplement 1. The expression level of three N-­terminal Dst
isoforms is shown in Figure 1—figure supplement 2. qPCR data of Dst isoforms in the soleus are shown in Figure 1—figure supplement 3.
The online version of this article includes the following figure supplement(s) for figure 1:
Figure supplement 1. Expressions of truncated Dst-­b from three Dst-­bE2610Ter alleles.
Figure supplement 2. Expressions for N-­terminal isoforms of Dst in the cardiac tissue.
Figure supplement 3. Quantification of mRNA levels in the soleus.

XhoI site (Figure 1B). Restriction fragment length polymorphism (RFLP) analysis of PCR products wasused to genotype mice (Figure 1C). Hereafter, this Dst-­b mutant allele is referred to as the Dst-­bE2610Ter
allele. In the heterozygous cross, approximately one-­fourth of the progeny were Dst-­b homozygous(Dst-­bE2610Ter/E2610Ter) mice, according to the Mendelian distribution. Western blotting was performed
using an anti-­Dst antibody that recognizes both Dst-­a and Dst-­b proteins. Dst-­b bands were detected
in tissue extracts from heart and skeletal muscle of wild-­type (WT) mice, whereas shifted bands wereobserved in those of Dst-­bE2610Ter homozygous mice (Figure 1D). We analyzed three independent Dst-­
bE2610Ter mouse lines, and similar truncations of the Dst protein were observed in the homozygotes

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(Figure 1—figure supplement 1). In brain tissue extracts, Dst-­a bands were equally detected between
WT and Dst-­bE2610Ter/E2610Ter mice. Quantitative PCR (qPCR) analyses indicated that Dst-­b mRNA wassignificantly reduced by approximately half in the cardiac tissues of Dst-­bE2610Ter/E2610Ter mice comparedwith those of WT mice, while Dst-­a mRNA expression was unchanged (Figure 1E). Among three
distinct isoforms of Dst-­b (Dst-­b1, Dst-­b2, and Dst-­b3) differing in their N-­terminus (Jefferson et al.,2006), the expression level of Dst isoform1 mRNA was remarkably reduced in the cardiac tissue of
Dst-­bE2610Ter/E2610Ter mice, whereas there was no significant change in the amounts of isoform 2 andisoform 3 mRNAs (Figure 1—figure supplement 2A). Similar changes in mRNA expression of Dst
isoforms were observed in the soleus (Figure 1—figure supplement 3A). The frequency of exon
usage was compared between cardiac and brain tissues (Figure 1—figure supplement 2B). Dst-­b-­specific exons were exclusively used in the heart and scarcely used in the brain. In the heart, the firstexons of Dst isoform 1 and isoform 3 were more frequently used than those in the brain. However, Dst
isoform 2 was predominantly used in the brain and weakly expressed in the heart. Thus, these findings
suggest that Dst isoforms are differentially expressed among tissues and that the Dst-­bE2610Ter allele
affects each Dst isoform to a different degree.

Gross phenotypes of Dst-bE2610Ter/E2610Ter mice
Dst-­bE2610Ter/E2610Ter mice had a normal appearance and seemed to have a normal life span. In the tailsuspension test, 1-­month-­old Dst-­bE2610Ter/E2610Ter mice maintained normal postures, while gene trap
mutant (DstGt/Gt) mice with the dt phenotype displayed hindlimb clasping and dystonic movement(Figure 2A). Next, histological analysis was performed on the muscle spindles and dorsal root ganglia
of 1-­month-­old mice, which are the main affected structures in dt mice. Dst-­bE2610Ter/E2610Ter mice exhibited normal muscle spindle structure, although the muscle spindles were markedly atrophied in DstGt/Gt
mice (Figure 2B). Similarly, neurofilament (NF) accumulation and induction of ATF3, a neural injury
marker, were observed in the dorsal root ganglia of DstGt/Gt mice but not in those of WT and Dst-­
bE2610Ter/E2610Ter mice (Figure 2C–E). We also found that parvalbumin (Pvalb)-­positive proprioceptive
neurons were normally observed in Dst-­bE2610Ter/E2610Ter mice (Figure 2F and G) but were markedly
decreased in DstGt/Gt mice as previously described in other dt mouse lines (Carlsten et al., 2001). We
next investigated the gross phenotypes of Dst-­bE2610Ter/E2610Ter mice over an extended time. Dst-­bE2610Ter/
E2610Ter
mice normally gained body weight until 1 year of age; then, they became lower body weightcompared with WT mice after 1 year of age (Figure 2H). Some Dst-­bE2610Ter/E2610Ter mice exhibitedkyphosis (data not shown). We have reported that dt mice display impairment of motor coordinationas assessed by the rotarod test and wire hang test (Horie et al., 2020); however, WT and Dst-­bE2610Ter/
E2610Ter
mice older than 1 year exhibited normal motor coordination (Figure 2I). These data also support
the idea that deficiency of the Dst-­a isoform, but not the Dst-­b isoform, is causative of dt phenotypes,
including sensory neuron degeneration, abnormal movements, and postnatal lethality.

Late-onset skeletal myopathy and cardiomyopathy in Dst-bE2610Ter/E2610Ter
mice
To further examine the skeletal muscle phenotype in aged Dst-­bE2610Ter/E2610Ter mice, we performed
histological analyses. In the soleus muscle of Dst-­bE2610Ter/E2610Ter mice, small-­caliber muscle fibers
and centrally nucleated fibers (CNFs), which indicate muscle degeneration and regeneration, were
frequently observed in mice at 16–23 months of age (Figure 3A) but were rare in 3–4-­month-­old
Dst-­bE2610Ter/E2610Ter mice (Figure 3—figure supplement 1A). The percentage of CNFs was significantly increased in the soleus, gastrocnemius, and erector spinae muscles, with different severities(Figure 3B, Figure 3—figure supplement 2). Muscle mass of soleus normalized by body weight
was not significantly different between control and Dst-­bE2610Ter/E2610Ter mice (control: 0.258 ± 0.012[mg/g body weight] vs. Dst-­bE2610Ter/E2610Ter: 0.279 ± 0.015 [mg/g body weight]; control, n = 6 muscles
from three mice; Dst-­bE2610Ter/E2610Ter, n = 6 muscles from three mice; p=0.3056, Student’s t-­test). Distribution of cross-­sectional area in the soleus showed that small-­caliber myofibers were abundant in
Dst-­bE2610Ter/E2610Ter mice compared with WT mice (Figure 3C). Masson’s trichrome staining indicatedskeletal muscle fibrosis and, particularly, a marked increase in the connective tissue surrounding small-­caliber muscle fibers (Figure 3D and E) in the soleus muscle of Dst-­bE2610Ter/E2610Ter mice. Cardiac fibrosis
was also observed in Dst-­bE2610Ter/E2610Ter mouse hearts (Figure 3D and E) but was not observed in thehearts of 3–4-­month-­old Dst-­bE2610Ter/E2610Ter mice (Figure 3—figure supplement 1B).

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WT

Dst-bE2610Ter

Dst Gt/Gt

C

WT

Dst-bE2610Ter

Dst Gt/Gt

F

25

***

WT

Dst-bE2610Ter

Dst Gt/Gt

WT

Dst-bE2610Ter

Dst Gt/Gt

50Body weight (g)

H

20
15
10
5
0

WT Dst-bE2610Ter Dst Gt/Gt
(n=3)
(n=6)
(n=3)

40

*

* **

WT (n=6)
Dst-bE2610Ter (n=7)

10

0

1

4

7

10

Months of Age

13

I

16

80

5

0

WT Dst-bE2610Ter Dst Gt/Gt
(n=3)
(n=3)
(n=6)

Rotarod test
ns

60
40

20
0

WT
(n=11)***

10

WT Dst-bE2610Ter Dst Gt/Gt
(n=3)
(n=3)
(n=6)

30

20

***

***
15

Wire hang test
Latency to fall (sec)10

G

Number of PV-positive cells***

Latency to fall (sec)E

Number of ATF3-positive cells

***

15

Number of cellswith NF accumulation

DPV

NF/ATF3

H&E

B

Dst-bE2610Ter(n=10)

ns

50

25

0

WT
(n=11)

Dst-bE2610Ter(n=10)

Figure 2. Characterizations of gross phenotypes of Dst-­bE2610Ter homozygous mice. (A) Tail suspension test of WT, Dst-­bE2610Ter/E2610Ter, and DstGt/Gt micearound 1 month old. WT and Dst-­bE2610Ter/E2610Ter mice exhibited normal posture, whereas the DstGt/Gt mice showed hindlimb clasping. (B) Muscle spindle
structure on the cross sections of soleus with H&E staining. Muscle spindles of Dst-­bE2610Ter/E2610Ter mice appeared normal, while DstGt/Gt mice showed
atrophy of the intrafusal muscle fiber in muscle spindles around 1 month old. (C) Double immunohistochemistry (IHC) of neurofilament (NF) and ATF3
on the sections of dorsal root ganglia (DRG) around 1 month old. In only DstGt/Gt mice, NF was accumulated in some DRG neurons, and the neural
injury marker, ATF3 was expressed. (D, E) Quantitative data of numbers of NF-­accumulating cells (D) and ATF3-­positive cells (E) in DRG of WT, Dst-­
bE2610Ter/E2610Ter, and DstGt/Gt mice (n = 6 WT mice; n = 3 Dst-­bE2610Ter/E2610Ter mice; n = 3 DstGt/Gt mice). *** denotes statistically significant difference at p<0.005(NF, WT vs. DstGt/Gt, p=0.0000; Dst-­bE2610Ter/E2610Ter vs. DstGt/Gt, p=0.0001; ATF3, WT vs. DstGt/Gt, p=0.0001; Dst-­bE2610Ter/E2610Ter vs. DstGt/Gt, p=0.0002), using
ANOVA. (F) Parvalbumin (PV) ISH on the section of DRG around 1 month old. PV-­positive proprioceptive neurons were greatly decreased in DRG of
DstGt/Gt mice but not in that of Dst-­bE2610Ter/E2610Ter mice. Dotted lines indicate the edge of DRG. (G) Quantitative data of number of PV-­positive cells in
DRG of WT, Dst-­bE2610Ter/E2610Ter, and DstGt/Gt mice (n = 6 WT mice; n = 3 Dst-­bE2610Ter/E2610Ter mice; n = 3 DstGt/Gt mice). *** denotes statistically significant
difference at p<0.005 (PV, WT vs. DstGt/Gt, p=0.0000; Dst-­bE2610Ter/E2610Ter vs. DstGt/Gt, p=0.0002), using ANOVA. (H) Male Dst-­bE2610Ter/E2610Ter mice normallygained body weight until 1 year old and then became lighter than male WT mice (n = 6 WT mice; n = 7 Dst-­bE2610Ter/E2610Ter mice; two-­way ANOVA,
genotype effect: p=0.1213; age effect: p=0.0000; genotype × age interaction: p=0.0398). * and ** denote statistically significant difference at p<0.05(13 months old, p=0.0197; 15 months old, p=0.0296) and p<0.01 (16 months old, p=0.0073). (I) Behavior tests to assess motor coordination and grip
strength around 1 year old. Dst-­bE2610Ter/E2610Ter mice showed normal motor coordination and grid strength (n = 11 WT mice; n = 10 Dst-­bE2610Ter/E2610Ter mice).
ns means not statistically significant (rotarod test, p=0.4126; fire hang test, p=0.0612), using Student’s t-­test. Data are presented as mean ± standard
error (SE). Scale bars: (B) 5 μm; (C, D) 50 μm.

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WT

Dst-bE2610Ter

H&E
SoleusA

Soleus***Gastrocnemius

*

10
5

WT Dst-bE2610Ter WT Dst-bE2610Ter WT Dst-bE2610Ter
(n=5)
(n=5)
(n=3) (n=3)
(n=6)
(n=5)

10
0

100-199 200-399 400-599 600-799 800-999

0-99

1000-

cross-sectional area (μm2)

WT

E

Dst-bE2610Ter

20

Soleus

15

*

Heart

***

10
5
0

LV

RV
LV

LA

RV
LV

Nppb

WT

Nppb

Dst-bE2610Ter

J

(ms)

*

4

ns

150

0

2
0

WT Dst-bE2610Ter

Tgfb2

*

4

Ctgf

**

6

4

2

2

0

(ms)

0

WT Dst-bE2610Ter

QRS
ns

20

WT Dst-bE2610Ter

QT
***

(ms)
50
40

90

30

10

60

20

30

K

WT Dst-bE2610Ter

8

120

0

**

6
4

2

H

RR

Nppb

Nppa
6

LV

Dst-bE2610Ter WT Dst-bE2610Ter
(n=5)
(n=5)
(n=5)

Relative mRNARA

G

Dst-bE2610Ter
Nppa

WT
(n=5)

Relative mRNA

WT

Relative mRNA

Nppa

LA

I

20

Heart

Dst-bE2610Ter*

30

SoleusWT

Dst-bE2610Ter (n=5)

Fibrosis area (%)

15

WT (n=5)40

Relative mRNA

Masson’s trichrome

F

***

20

0

D

C 50

Erector spinae

Percentage of myofibers (%)

Percentage of CNFs (%)

B

10

WT

Dst-bE2610Ter

0

WT

0

Dst-bE2610Ter

WT

Dst-bE2610Ter

PVC

Dst-bE2610Ter

1s

100 msFigure 3. Pathological alterations in skeletal and cardiac muscles of Dst-­bE2610Ter mice. (A) H&E-­stained cross soleus
sections of WT and Dst-­bE2610Ter/E2610Ter mice at 23 months of age. Dotted boxes in the soleus of Dst-­bE2610Ter/E2610Ter
include small-­caliber and centrally nucleated fibers (CNFs, arrows) shown as insets. Histological analysis of soleus
of Dst-­bE2610Ter/E2610Ter mice at 3–4 months of age are shown in Figure 3—figure supplement 1. HistologicalFigure 3 continued on next page

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Figure 3 continued

analysis of gastrocnemius and erector spinae is shown in Figure 3—figure supplement 2. (B) Quantitative data
representing percentages of CNFs in the soleus (n = 5 mice, each genotype), gastrocnemius (n = 5 WT mice; n= 6 Dst-­bE2610Ter/E2610Ter mice), and erector spinae (n = 3 WT mice; n = 3 Dst-­bE2610Ter/E2610Ter mice). * and *** denotestatistically significant difference at p<0.05 (gastrocnemius, p=0.0118), and p<0.005 (soleus, p=0.0009; erector
spinae, p=0.0044), using Student’s t-­test. (C) Distribution of cross-­sectional area in the soleus of WT and Dst-­
bE2610Ter/E2610Ter mice (n = 5 mice, each genotype, at 16–23 months old; two-­way ANOVA; genotype effect: p=0.0133;
area effect: p=0.0000; genotype × area interaction: p=0.2032). (D) Masson’s trichrome staining showed a fibrosis in
the soleus and heart of Dst-­bE2610Ter/E2610Ter mice at 16 months of age (arrowheads). (E) Quantitative data of the extent
of fibrosis in soleus and heart of WT and Dst-­bE2610Ter/E2610Ter mice (n = 5 mice, each genotype, at 16–23 months old).
* and *** denote statistically significant difference at p<0.05 (p=0.0109) and p<0.005 (p=0.0045), using Student’st-­test. (F) Nppa and Nppb ISH in the heat at 16 months of age. Nppa mRNA was upregulated in the myocardiumof left ventricle (LV) of Dst-­bE2610Ter/E2610Ter mice, but not in the right ventricle (RV). Dotted areas are shown as insets.
Nppa mRNA was strongly expressed in the myocardium of left atrium (LA) and right atrium (RA). Insets below
represents Nppb mRNA the in same regions. (G, H) qPCR analyses on cardiac stress markers (G) and profibrotic
cytokines (H) (n = 3 mice, each genotype, at 20–25 months old). * and ** denote statistically significant differenceat p<0.05 (Nppa, p=0.0119; Tgfb2, p=0.0149) and p<0.01 (Nppb, p=0.0053; Ctgf, p=0.0095), using Student’s t-­test.
(I) Representative electrocardiogram (ECG) images of P-­QRS-­T complex. (J) Intervals of RR, QRS, and QT were
quantified (n = 5 WT mice; n = 9 Dst-­bE2610Ter/E2610Ter mice, at 18–25 months old). *** denotes statistically significant
difference at p<0.005 (QT, p=0.0044) and ns means not statistically significant (RR, p=0.2000; QRS, p=0.8964), using
Student’s t-­test. (K) Premature ventricular contractions (PVC, arrows) were recorded in short-­range (left panel) andlong-­range (right panel) ECG images from a Dst-­bE2610Ter/E2610Ter mouse. Data are presented as mean ± SE. Scalebars: (A (left images), D) 50μm; (A (right magnified images)) 5 μm; (F) 1mm; (F (magnified images)) 200 μm.
The online version of this article includes the following figure supplement(s) for figure 3:
Figure supplement 1. Histological analysis in the soleus and heart of young Dst-­bE2610Ter mice.
Figure supplement 2. Histological analysis in gastrocnemius and erector spinae of Dst-­bE2610Ter mice.

To assess the influence of the Dst-­b mutation on cardiomyocytes, expression of the heart failure
markers natriuretic peptide A (Nppa) and natriuretic peptide B (Nppb) (Sergeeva and Christoffels,
2013) was investigated. Nppa mRNA was markedly upregulated in the myocardium of the left ventricle
in 16-­month-­old Dst-­bE2610Ter/E2610Ter mice and was not increased in that of WT mice (Figure 3F). Nppb
mRNA was also upregulated in the left ventricle myocardium in Dst-­bE2610Ter/E2610Ter mice (Figure 3F).
qPCR analysis also demonstrated increased expression of Nppa and Nppb mRNAs in the cardiac tissue
of Dst-­bE2610Ter/E2610Ter mice (Figure 3G). Expression of profibrotic cytokines, including transforming
growth factor beta-­2 (Tgfb2) and connective tissue growth factor (Ctgf) mRNAs, was also remarkably
increased in Dst-­bE2610Ter/E2610Ter mice (Figure 3H). At 3–4 months of age, the Nppa transcript was slightly
upregulated in Dst-­bE2610Ter/E2610Ter mouse hearts without obvious cardiac fibrosis (Figure 3—figure
supplement 1B). To assess the cardiac function, ECGs were recorded from anesthetized WT and
Dst-­bE2610Ter/E2610Ter mice at 18–25 months of age (Figure 3I). Dst-­bE2610Ter/E2610Ter mice exhibited remarkably prolonged QT intervals compared with those of WT mice (Figure 3J), which suggests abnormal
myocardial repolarization. However, the RR interval and QRS duration were not different between WT
and Dst-­bE2610Ter/E2610Ter mice (Figure 3J). In addition, premature ventricular contractions (PVCs) wererecorded in two of nine Dst-­bE2610Ter/E2610Ter mice analyzed (Figure 3K). Individual Dst-­bE2610Ter/E2610Ter miceharboring PVCs showed severe cardiac fibrosis and remarkable upregulation of Nppa mRNA in theventricular myocardium (data not shown). Such ECG abnormalities were not observed in 3-­month-­old
Dst-­bE2610Ter/E2610Ter mice (RR of WT mice: 134.2 ± 26.6 ms vs. Dst-­bE2610Ter/E2610Ter mice: 138.8 ± 33.5 ms;
p=0.8234, Student’s t-­test; QRS of WT mice: 17.3 ± 2.0 ms vs. Dst-­bE2610Ter/E2610Ter mice: 17.4 ± 1.2
ms; p=0.9514, Student’s t-­test; QT of WT mice: 31.7 ± 4.3 ms vs. Dst-­bE2610Ter/E2610Ter mice: 31.2 ± 2.3
ms; p=0.8599, Student’s t-­test; WT, n = 4 mice; Dst-­bE2610Ter/E2610Ter, n = 5 mice). These data indicatethat the Dst-­b isoform-­specific mutation causes late-­onset cardiomyopathy and cardiac conduction
disturbance.

Protein aggregations in myofibers of Dst-bE2610Ter/E2610Ter mice
MFM is a type of hereditary myopathy that is defined on the basis of common pathological features,such as myofibril disorganization beginning at the Z-­disks and ectopic protein aggregation in myocytes(De Bleecker et al., 1996; Nakano et al., 1996). Desmin is an intermediate filament protein located in

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Z-­disks, and aggregation of desmin and other cytoplasmic proteins is a pathological hallmark of MFM(De Bleecker et al., 1996; Clemen et al., 2013; Batonnet-­Pichon et al., 2017). First, we performed
desmin immunohistochemistry (IHC) and found abnormal desmin aggregation in the skeletal muscleof Dst-­bE2610Ter/E2610Ter mice at 16–23 months of age (Figure 4A and B). In the soleus of Dst-­bE2610Ter/
E2610Termice, desmin massively accumulated in the subsarcolemmal region, whereas in that of WT
mice, desmin protein was predominantly located underneath the sarcolemma (Figure 4A). Desmin
aggregates were also observed in the cardiomyocytes of Dst-­bE2610Ter/E2610Ter mice older than 16 months(Figure 4—figure supplement 1). In longitudinal sections of Dst-­bE2610Ter/E2610Ter soleus muscle, localization of desmin to the Z-­disks was mostly preserved; however, displacement of Z-­disks was occasionallyobserved in the muscle fibers bearing subsarcolemmal desmin aggregates (Figure 4C). At 3–4 monthsof age, desmin aggregates were scarcely observed in Dst-­bE2610Ter/E2610Ter mouse soleus (Figure 3—
figure supplement 1A).
Next, we investigated the molecular composition of the protein aggregates in Dst-­bE2610Ter/E2610Ter
mouse muscle fibers. We found that αB-­crystallin was co-­aggregated with desmin in the subsarcolemmal regions of the Dst-­bE2610Ter/E2610Ter soleus (Figure 4D). αB-­crystallin is a small heat shockprotein that binds to desmin and actin and is known to accumulate in muscles of patients with
MFMs (Clemen et al., 2013). In addition, plectin had also accumulated in desmin-­positive aggregates of the Dst-­bE2610Ter/E2610Ter soleus (Figure 4E). Plectin is a cytoskeletal linker protein that isnormally located in the Z-­disks. Because protein aggregates in muscles affected by MFM often
contain the Z-­disk protein encoded by the causative gene itself, we examined subcellular distribution of myotilin that is a Z-­disk component encoded by a causative gene for MFM. We foundthat myotilin was accumulated in some myofibers of the Dst-­bE2610Ter/E2610Ter soleus (Figure 4—figure
supplement 2). Furthermore, we evaluated the subcellular distribution of truncated Dst proteins in
the Dst-­bE2610Ter/E2610Ter soleus. As expected, truncated Dst proteins accumulated in subsarcolemmal
aggregates of desmin in the Dst-­bE2610Ter/E2610Ter soleus (Figure 4F). In addition, some small-­caliber
myofibers exhibited cytoplasmic accumulation of truncated Dst proteins surrounding nuclei. Dst
proteins were also distributed on Z-­disks (Figure 4G), as reported in previous studies (Boyeret al., 2010a; Steiner-­Champliaud et al., 2010). Truncated Dst proteins were also localized innormally shaped Z-­disks in the Dst-­bE2610Ter/E2610Ter soleus and were dispersed from disorganizedZ-­disks (Figure 4G). Taken together, these data indicate that protein aggregates in Dst-­bE2610Ter/
E2610Ter
mouse muscles are composed of a variety of proteins, similar to aggregates found in muscles
affected by other MFMs.

Mitochondrial structure and gene expression alterations in striatedmuscles of Dst-bE2610Ter mice
Mitochondrial abnormalities have also been reported in muscles of patients with MFMs and animalmodels (Joshi et al., 2014; Bührdel et al., 2015; Winter et al., 2016). We investigated the mitochondrial distribution using cytochrome C and Tom20 IHC and found abnormal accumulation of mitochondria underneath the sarcolemma in some fibers of Dst-­bE2610Ter/E2610Ter soleus muscles (Figure 5A).
Double staining with cytochrome C and desmin revealed that mitochondrial accumulation occurredin the same region as desmin aggregates (Figure 5C). We also found a strong phosphorylated PERK
signal, an organelle stress sensor, in the subsarcolemmal spaces with accumulated mitochondria(Figure 5D).
Next, we quantified the mRNA expression levels of several genes responsible for oxidative phosphorylation (Ndufb8, Sdha, Uqcrc2, Cox4i1, and Atp5a1) in the soleus (Figure 5E). The expressionof several genes, including Ndufb8, Sdha, and Cox4i1, was significantly reduced in Dst-­bE2610Ter/
E2610Termice, suggesting mitochondrial dysfunction in the skeletal muscle of Dst-­bE2610Ter/E2610Ter mice.
Changes in genes responsible for oxidative phosphorylation suggest the possibility of muscle
fiber type switch and/or oxidative stress (Bonnard et al., 2008; Zhang et al., 2017). However,quantification of expression levels of muscle fiber-­type marker genes and oxidative stress-­related
genes showed no changes in the soleus of Dst-­bE2610Ter/E2610Ter mice (Figure 1—figure supplement
3B and C). At 3–4 months of age, mitochondrial accumulation was already observed in soleus of
Dst-­bE2610Ter/E2610Ter mice before the appearance of CNF and desmin aggregates (Figure 3—figure
supplement 1A). Therefore, mitochondrial abnormalities may precede protein aggregation andmuscle degeneration.

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B

Dst-bE2610Ter

Percentage of myofibers
with desmin aggregates (%)

WTSoleusC

Dst-bE2610Ter

30

**

20

10

0

WT (n=5)

Dst-bE2610Ter (n=5)

WT

Dst-bE2610Ter

DesminPlectin

MergedDesminDst

MergedDesmin/α-Actin

DesminA

DesminαB-crystallin

MergedDst-bE2610Ter

Dst-bE2610Ter

WT

G

Dst-bE2610Ter

Dst

WT

Desmin/DAPI

Dst-bE2610Ter

F

E
WT

WT

D

Figure 4. Dst-­bE2610Ter mutation leads to protein aggregation myopathy. (A) Desmin immunohistochemistry (IHC) on the cross sections of soleus from
WT and Dst-­bE2610Ter/E2610Ter mice at 23 months of age using anti-­desmin antibody (RD301). Dotted boxes in Dst-­bE2610Ter/E2610Ter soleus indicate desmin
aggregates underneath the sarcolemma (arrowheads) and in the sarcoplasmic region (arrow) shown as insets. Desmin IHC on heart of Dst-­bE2610Ter/E2610Ter
mice is shown in Figure 4—figure supplement 1. (B) Quantitative data of the percentage of myofibers with desmin aggregates in soleus of WT and
Figure 4 continued on next page

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Figure 4 continued

Dst-­bE2610Ter/E2610Ter mice (n = 5 mice, each genotype, at 16–23 months old). ** denotes statistically significant difference at p<0.01 (p=0.0051), using
Student’s t-­test. Data are presented as mean ± SE. (C) Immunofluorescent images of longitudinal soleus sections labeled with anti-­desmin (rabbit IgG)
and anti-­alpha-­actin antibodies. Muscle fibers harboring subsarcolemmal desmin aggregates (arrowheads) showed the Z-­disk displacement (arrows). (D,E) Double IHC using anti-­desmin (RD301) and anti-αB-­crystallin antibodies (D) or anti-­desmin (Rabbit IgG) and anti-­plectin antibodies (E) on the crosssections of soleus. In Dst-­bE2610Ter/E2610Ter soleus, αB-­crystallin and plectin were accumulated in subsarcolemmal regions with desmin (white arrowheads).
Images of myotilin, a Z-­disk component, are shown in Figure 4—figure supplement 2. (F) Double IHC using anti-­desmin antibody and anti-­Dst
antibody on cross sections of WT and Dst-­bE2610Ter/E2610Ter soleus. Dst protein accumulated in subsarcolemmal desmin aggregates (white arrowheads).
Dst protein was also accumulated around myonuclei-­labeled with DAPI (arrows). Insets show localizations of desmin and Dst proteins around nuclei.
(G) Double IHC of desmin and Dst. In longitudinal soleus sections, Dst protein was distributed in a striped pattern at desmin-­positive Z-­disks. In Dst-­
bE2610Ter/E2610Ter soleus sections, Dst protein was dispersed around displaced Z-­disks (arrows). Scale bars: (A, D, E, F) 50μm; (A (magnified images), C, F
(insets), G) 5μm.
The online version of this article includes the following figure supplement(s) for figure 4:
Figure supplement 1. Desmin immunohistochemistry (IHC) on the sections of heart from WT and Dst-­bE2610Ter/E2610Ter mice at 23 months of age using anti-­
desmin antibody (RD301).
Figure supplement 2. Myotilin immunohistochemistry (IHC) on the cross sections of soleus from WT and Dst-­bE2610Ter/E2610Ter mice.

Ultrastructural changes in skeletal and cardiac muscles of Dst-bE2610Ter/
E2610Ter
mice
Transmission electron microscope (TEM) analysis was performed to further investigate the ultrastructural features of skeletal and cardiac muscles of Dst-­bE2610Ter/E2610Ter mice. In the Dst-­bE2610Ter/E2610Ter soleus,
focal dissolution of myofibrils and Z-­disk streaming were observed at 22 months of age (Figure 6A).
Subsarcolemmal regions of the Dst-­bE2610Ter/E2610Ter soleus were often filled with accumulated mitochondria and granulofilamentous materials including focal accumulation of glycogen granules, electron-­dense disorganized Z-­disks, and electron-­pale filamentous materials (Figure 6B). These observations
have been reported as typical features of MFMs (Batonnet-­Pichon et al., 2017). Furthermore, we
found dysmorphic nuclei in the soleus of Dst-­bE2610Ter/E2610Ter mice (Figure 6C).
Focal myofibrillar dissolution and mitochondrial accumulation were observed in Dst-­bE2610Ter/E2610Ter
mouse cardiomyocytes (Figure 6D). We often observed an invaginated nuclear envelope and a structure similar to the nucleoplasmic reticulum (Bezin et al., 2008; Malhas et al., 2011) with cytoplasmic
components in the nucleus (Figure 6E). Interestingly, inclusions containing crystalline structures were
frequently observed in dysmorphic nuclei, which were a unique feature of Dst-­bE2610Ter/E2610Ter cardiomyocytes. Only a limited number of reports have described myopathy with intranuclear inclusions(Oteruelo, 1976; Oyer et al., 1991; Weeks et al., 2003; Ogasawara et al., 2020).

Abnormal nuclear structures in Dst-bE2610Ter cardiomyocytes
The observation of unique nuclear inclusions in dysmorphic nuclei in heart tissue prompted us toperform detailed histological analyses of Dst-­bE2610Ter/E2610Ter mouse cardiomyocytes. Upon H&E
staining, eosinophilic structures were observed in Dst-­bE2610Ter/E2610Ter cardiomyocyte nuclei (Figure 7A).
Dysmorphic nuclei were clearly observed by lamin A/C IHC, showing a deeply invaginated nuclear
envelope devoid of DNA (Figure 7B). In TEM observations, nuclear crystalline inclusions and cytoplasmic organelles, such as lysosomes surrounded by the nuclear envelope, were sometimes observed
within one nucleus (Figure 7C). Crystalline inclusions were observed as lattice structures (Figure 7D).
Some subcellular organelles, such as mitochondria, were frequently observed within the nucleus of
Dst-­bE2610Ter/E2610Ter cardiomyocytes as a result of nuclear invaginations (Figure 7E and F) because they
were surrounded by the nuclear membrane. Moreover, vacuoles that seemed to contain liquid wereobserved in the nucleus. In some cases, endoplasmic reticulum and ribosomes were observed at
higher densities within the nucleus of Dst-­bE2610Ter/E2610Ter cardiomyocytes (Figure 7G), which is similar to
the findings for the nucleoplasmic reticulum (Bezin et al., 2008; Malhas et al., 2011). Next, we investigated the molecular components of nuclear inclusions and found that the autophagy-­associatedprotein p62 was deposited in Dst-­bE2610Ter/E2610Ter cardiomyocyte nuclei (Figure 7H). The percentage
of cell nuclei harboring p62-­positive structures was remarkably increased in Dst-­bE2610Ter/E2610Ter cardiomyocytes compared with that in WT cardiomyocytes (Figure 7I, WT mice: 0.55 ± 0.25% vs. Dst-­
bE2610Ter/E2610Ter mice: 14.3% ± 1.9%; n = 3 WT mice: 871 total nuclei, n = 4 Dst-­bE2610Ter/E2610Ter mice:
860 total nuclei). We observed co-­localization of p62 and ubiquitinated protein inside the nucleus

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A

WT

B

Tom20

Percentage of myofibers withmitochondria accumulations (%)

Cytochrome C

Dst-bE2610Ter

C

WT

D

50
40
30
20
10
0

WT
(n=5)

Dst-bE2610Ter
(n=5)
T
Dst-b
Dst bE2610Ter

WT

Phospho-PERK

Cytochrome C

MergedDesminMerged0

WT Dst-bE2610Ter

0.8

0.4
0

**

1.2

WT Dst-bE2610Ter

0.8
0.4
0

ns

*

1.2

WT Dst-bE2610Ter

0.8

0.4
0

1.2

Relative mRNA

0.4

1.2

Relative mRNA

0.8*
Relative mRNA

1.2

Atp5a1

Cox4i1

Uqcrc2

Sdha

Ndufb8Relative mRNAE
Relative mRNA

60

Cytochrome C

Dst-bE2610Ter*

70

WT Dst-bE2610Ter

ns

0.8

0.4

0

WT Dst-bE2610Ter

Figure 5. Dst-­bE2610Ter mutation leads mitochondrial alterations in skeletal muscle fibers. (A) Cytochrome C and Tom20 immunohistochemistry (IHC)
on the serial cross sections of the soleus at 23 months of age. In WT soleus, mitochondria were mainly stained underneath the sarcolemma. Dst-­
bE2610Ter/E2610Ter soleus showed accumulated mitochondria in subsarcolemmal regions (arrowheads). (B) Quantitative data of the percentage of myofibers
with accumulated mitochondria in soleus of WT and Dst-­bE2610Ter/E2610Ter mice (n = 5 mice, each genotype, at 16–23 months old). * denotes statisticallyFigure 5 continued on next page

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Figure 5 continued

significant difference at p<0.05 (p=0.0109), using Student’s t-­test. (C) Double IHC of cytochrome C and desmin in cross sections of the soleus. Each
protein was stained in subsarcolemmal spaces of WT soleus. In Dst-­bE2610Ter/E2610Ter soleus, cytochrome C-­positive mitochondria were accumulated at
the same positions with desmin aggregates (arrowheads). (D) Double IHC of cytochrome C and phospho-­PERK. Phospho-­PERK signals were strongly
detected in subsarcolemmal regions in which cytochrome C-­positive mitochondria accumulated (arrowheads). (E) qPCR analysis of genes responsible
for oxidative phosphorylation in the soleus (n = 3 mice, each genotype, 21 months of age). * and ** denote statistically significant difference at p<0.05(Ndufb8, p=0.0191; Cox4i1, p=0.0335) and p<0.01 (Sdha, p=0.0001), respectively, and ns means not statistically significant (Uqcrc2, p=0.2546; Atp5a1,
p=0.0769), using Student’s t-­test. Data are presented as mean ± SE. Scale bars: (A, C, D) 50 μm.

using super-­resolution microscopy (Figure 7J and Video 1). Small ubiquitin-­related modifier (SUMO)
proteins are major components of intranuclear inclusions in some neurological diseases, including
neuronal intranuclear inclusion disease (NIID) (Mori et al., 2012; Pountney et al., 2003). SUMO
proteins were also detected in the intranuclear structures of Dst-­bE2610Ter/E2610Ter cardiomyocytes using
anti-­SUMO-­1 and anti-­SUMO-­2/3 antibodies (Figure 7—figure supplement 1A and B). Because
desmin and αB-­crystallin were absent from the intranuclear structures, protein aggregates in the
nuclei were distinct from those in some molecular components of the cytoplasm (Figure 7—figure
supplement 1C and D). The p62-­positive structures inside nuclei were also devoid of LC3, which is an
autophagy-­associated molecule, whereas p62 and LC3 were co-­localized in the cytoplasm (Figure 7—
figure supplement 1E). Lamin A/C IHC also confirmed that the p62- and ubiquitin-­positive structures
were surrounded by or adjacent to lamin A/C-­positive lamina (Figure 7—figure supplement 1F and
G). Desmin IHC also confirmed cytoplasmic desmin in the space surrounded by highly invaginatednuclear membrane (Figure 7—figure supplement 1H). These results revealed that Dst-­b gene mutations lead to dysmorphic nuclei and formation of nuclear inclusions containing p62, ubiquitinatedproteins, and SUMO proteins in the Dst-­bE2610Ter/E2610Ter myocardium.

Next-generation sequencing analysis of heart tissues from Dst-bE2610Ter/
E2610Termice
To determine the molecular etiology of pathophysiological features of Dst-­bE2610Ter/E2610Ter mousehearts, we performed RNA-­seq analysis of transcripts obtained from the ventricular myocardium of
14–19-­month-­old Dst-­bE2610Ter/E2610Ter mice and age-­matched WT mice. Principal component analysis(PCA) and hierarchical clustering of RNA-­seq data showed that transcriptomic characteristics between
WT and Dst-­bE2610Ter/E2610Ter mouse hearts were different (Figure 8—figure supplement 1A and B).
RNA-­seq identified 728 differentially expressed genes with a false discovery rate (FDR) < 0.1 and
p<0.001 (Figure 8A), including 481 upregulated genes and 247 downregulated genes in Dst-­bE2610Ter/
E2610Ter
mouse hearts compared with those from WT mice. A volcano plot demonstrated that upregulated genes were associated with pro-­fibrotic pathways (e.g., Ctgf, Tgfb2, Crlf1), cytoskeletal regulation (e.g., Acta2, Cenpf, Ankrd1, Mybpc2, Myom2, Nefm), and metabolic control (Ucp2, Nmrk2,
Tbx15) and that downregulated genes were associated with transmembrane ion transport (e.g.,
Scn4b, Cacng6, Ano5, Tmem150c, Cacna1s, Scn4a). The reliability of RNA-­seq data was validated byusing qPCR analysis (Figure 8—figure supplement 1C). The differentially expressed genes included
several causative genes of congenital heart defects, hypertrophic cardiomyopathy, congenital long-­QT
syndrome, muscular dystrophy, and dilated cardiomyopathy (e.g., Acta2, Ankrd1, Scn4b, Ano5, Rpl3l,
Cenpf) (Figure 8B). Gene Ontology (GO) analysis confirmed the following terms specific to the skeletal muscle and heart: biological process (e.g., upregulated: muscle contraction, heart development,
skeletal muscle tissue development, sarcomere organization, and regulation of heart rate; downregulated: muscle organ development) and cellular components (e.g., upregulated: Z-­disc, sarcolemma,
myofibril, neuromuscular junctions, sarcoplasmic reticulum) (Figure 8C and D, Supplementary file
1A–E). Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway analysis also indicated abnormal
muscle pathways (e.g., upregulated: dilated cardiomyopathy, hypertrophic cardiomyopathy; downregulated: cardiac muscle contraction) (Figure 8E, Supplementary file 1C and F). These data provide
a comprehensive landscape for cardiomyopathy in Dst-­bE2610Ter/E2610Ter mice.

Mosaic analysis using conditional DstGt mice
Because truncated Dst-­b proteins expressed from the Dst-­bE2610Ter allele harbor an actin-­binding
domain (ABD) and a plakin domain, it is possible that they have a gain-­of-­function effect on the

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Figure 6. Transmission electron microscopy (TEM) analyses on the soleus and heart of Dst-­bE2610Ter mice. (A–C) TEM images on longitudinal soleus
ultrathin sections of WT and Dst-­bE2610Ter/E2610Ter mice at 22 months of age (n = 2 mice, each genotype). (A) Normal myofibril structure with well-­aligned Z-­
disks (z) and mitochondria (m) in WT soleus. In Dst-­bE2610Ter/E2610Ter soleus, Z-­disk streaming (arrows) was observed as displacements of Z-­disks (less severecase) and focal myofibrillar dissolution (severe case). Dotted box in Dst-­bE2610Ter/E2610Ter soleus indicates the severely disrupted myofibrillar structuresassociated with abnormally thickened Z-­disks (arrows) shown as the inset. (B) Subsarcolemmal accumulations of mitochondria and granulofilamentous
material (gfm) in Dst-­bE2610Ter/E2610Ter soleus. Gfm consists of glycogen granule deposits (arrowheads) and filamentous materials with electron-­dense Z-­
disk streaming (arrows) and filamentous pale area (asterisk). Accumulated mitochondria (ma) are adjacent to the gfm. Dotted box shows the enlarged
structure of gfm. (C) Nuclear invaginations in Dst-­bE2610Ter/E2610Ter soleus. Dotted box in the cell nucleus (Nu) indicates the deeply invaginated nuclear
Figure 6 continued on next page

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Figure 6 continued

envelope (NE, arrow) involving lysosomes (L) shown as the inset. (D, E) TEM images on heart ultrathin sections (n = 2 mice, each genotype). (D) Normal
myofibril structure with well-­aligned Z-­disks (z) and mitochondria (m) in WT cardiomyocytes (left panel). In Dst-­bE2610Ter/E2610Ter cardiomyocytes, focal
myofibrillar dissolution (double asterisks) was observed and accumulated mitochondria (ma) were evident there (right panel). (E) Abnormal shape of
nucleus (Nu) and intranuclear inclusions were frequently observed in Dst-­bE2610Ter/E2610Ter cardiomyocytes. Dotted box indicates that the NE (arrow) deeply
invaginated and involved cytoplasmic components such as lysosomes (L) to form nucleoplasmic reticulum (NR) shown as inset. Dysmorphic nucleus
harbored the crystalline inclusions in intranuclear and perinuclear regions (arrowheads). Scale bars: (A-­E) 1μm; (B (right magnified images)) 400 nm.

pathogenesis of Dst-­bE2610Ter/E2610Ter mice. Therefore, we analyzed conditional DstGt mice (Figure 9A),
in which both Dst-­a and Dst-­b are trapped within the N-­terminal ABD and lose their function in a
genetically mosaic manner. We crossed female DstGt-­inv/Gt-­inv mice with male β-actin (Actb)-­iCre; DstGt-­
DO/wt
mice to generate Actb-­iCre; DstGt-­DO/Gt-­inv mice (mosaic mice, Figure 9B). More than half of themosaic mice survived over several months and showed mild dt phenotypes, such as smaller body size
and impairment of motor coordination (Figure 9C). Western blotting analysis indicated that the Dst-­b
protein was almost absent in cardiac extract from surviving mosaic mice; however, the Dst-­a protein
was detected at variable levels in brain extract (Figure 9D). Cardiac fibrosis was observed in heartsof 10-­month-­old mosaic mice (Figure 9E). In addition, increases in Nppa mRNA expression and p62
deposition were evident in the left ventricular myocardium of mosaic mice compared with control
mice (Figure 9F). These data suggest that myopathy in Dst-­bE2610Ter/E2610Ter mice is caused by a loss-­of-­function mutation of Dst-­b.

DST mutant alleles with nonsense mutations in DST-b-specific exons
Next, we performed in silico screening for DST mutant alleles with nonsense mutations within DST-­b-­specific exons in a database containing normal human genomes. In a search of the dbSNP database, which contains information on single-­nucleotide variations in healthy humans, we identified
58 different types of nonsense mutations (94 alleles total) in all five DST-­b-­specific exons (Table 1,
Figure 10A). The alleles with nonsense mutations were found in different populations (European,
Asian, African, and American, Figure 10B). In Japan, four alleles with nonsense mutations were foundamong 16,760 alleles in the ToMMo 8.3KJPN database (Tadaka et al., 2021), suggesting that homozygous or compound heterozygous mutants may exist in one per approximately 16 million people.
We further investigated DST-­b mutations in Japanese patients diagnosed with myopathy (Nishikawa
et al., 2017). Although nonsense mutations were not identified in those patients, we identified twopatients with myopathy who harbored compound heterozygous DST variants (Supplementary file1G). All variants were predicted to be variants of uncertain significance (VUS); therefore, the involvement of these variants in myopathic manifestations needs to be carefully interpreted. Taken together,
these data suggest that unidentified familial myopathies and/or cardiomyopathies caused by DST-­bmutant alleles exist in all populations worldwide.

Discussion
In this study, we established the isoform-­specific Dst-­b mutant mouse as a novel animal model for
late-­onset protein aggregate myopathy. Myopathic alterations in Dst-­b mutants were similar to thoseobserved for MFMs. We also found nuclear inclusion as a unique pathological hallmark of Dst-­bmutant cardiomyocytes, which provided molecular insight into pathophysiological mechanisms ofcardiomyopathy. Because the Dst-­b-­specific nonsense mutation resulted in myopathy without sensory
neuropathy, and mutant DST alleles with nonsense mutations in DST-­b-­specific exons exist, our datasuggest that unidentified human myopathy may be caused by these DST-­b mutant alleles. Our dataalso indicated that the sensory neurodegeneration, movement disorders, and lethality in dt mice andpatients with HSAN-­VI, which are caused by mutations of both the Dst-­a (DST-­a) and Dst-­b (DST-­b)isoforms, are actually caused by deficiency of the Dst-­a (DST-­a) isoform.

Isoform-specific Dst-b mutants result in late-onset protein aggregate
myopathy
Isoform-­specific Dst-­b mutant mice exhibited late-­onset protein aggregate myopathy with cardiomyopathy, which could be termed ‘dystoninopathy.’ Protein aggregates observed in the striatedYoshioka et al. eLife 2022;11:e78419. DOI: https://doi.org/10.7554/eLife.78419

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Figure 7. Defects in nuclear structure and intranuclear inclusions in cardiomyocytes of Dst-­bE2610Ter mice. (A) H&E staining of cardiomyocyte nuclei.
Eosinophilic structures (arrows) were observed inside the nucleus of Dst-­bE2610Ter/E2610Ter cardiomyocytes. (B) Nuclear lamina was immunolabeled with anti-­
lamin-­A/C antibody. In dysmorphic nuclei of Dst-­bE2610Ter/E2610Ter cardiomyocytes, invaginated nuclear lamina did not contain DAPI signals (arrowheads).
(C–G) Transmission electron microscope (TEM) images of cardiomyocyte nuclei of Dst-­bE2610Ter/E2610Ter mice (n = 2 mice, each genotype). (C) Crystalline
inclusions (asterisk) and cytoplasmic organelles such as lysosomes (L) surrounded by the nuclear envelope (NE, arrow) were observed inside nucleus(Nu). (D) Crystalline inclusions are lattice structure. (E) The NE surrounded various components such as organelles (arrow) or presumably liquid(arrowheads). There was a vacuole (V) seemed to contain liquid. (F) Dotted box (E) indicates mitochondria (m) surrounded by a membrane. (G) An
organelle surrounded by the NE (arrow) contained endoplasmic reticulum and ribosomes. Such structure was discriminated as nucleoplasmic reticulum(NR). (H) Immunofluorescent images of heart sections labeled with anti-­p62 and anti-­lamin A/C antibodies. p62 was deposited inside nuclei surroundedby lamin A/C-­positive lamina in Dst-­bE2610Ter/E2610Ter cardiomyocytes (arrowheads). (I) Quantitative data representing percentages of cardiomyocyte nuclei
harboring p62-­positive inclusions (n = 3 WT mice; n = 4 Dst-­bE2610Ter/E2610Ter mice). ** denotes statistically significant difference at p<0.01 (p=0.0062), using
Student’s t-­test. Data are presented as mean ± SE. (J) Super-­resolution microscopy images showed co-­localization of p62 and ubiquitin in the nucleus
Figure 7 continued on next page

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Figure 7 continuedof Dst-­bE2610Ter/E2610Ter cardiomyocytes. Immunohistochemistry (IHC) analysis of molecular features of nuclear inclusions are shown in Figure 7—figure
supplement 1. Scale bars: (A, B, H (insets), J) 5μm; (C, G) 1μm; (D) 100nm; (E) 2μm; (F) 600 nm; (H) 50μm.
The online version of this article includes the following figure supplement(s) for figure 7:
Figure supplement 1. Histological features of intranuclear structures in Dst-­bE2610Ter/E2610Ter cardiomyocytes.

muscles of Dst-­b mutant mice were composed of desmin, αB-­crystallin, plectin, and truncated Dst-­b.
MFMs are major groups of protein aggregate myopathies and are caused by mutations of genes
encoding Z-­disk-­associated proteins (e.g., DES, CRYAB, MYOT, ZASP, FLNC, BAG3, PLEC) (Keduka
et al., 2012; Clemen et al., 2013; Batonnet-­Pichon et al., 2017). It is plausible that Dst proteins arealso aggregated in muscles affected by other MFM types. Because Dst-­b encodes cytoskeletal linker
proteins that are localized in the Z-­disks, it is possible that mutations in Dst-­b lead to Z-­disk fragility,
and repeated contraction may lead to myofibril disruption and induction of muscle regeneration. Inline with this notion, we observed Z-­disk streaming and CNFs, which reflect muscle degeneration andregeneration.
It is possible that protein quality control impairment through the unfolded protein response, autophagy, and the ubiquitin proteasome system is involved in protein aggregation in Dst-­b mutant mousemuscles. Moreover, several genes responsible for the unfolded protein response, including heat shockproteins, were upregulated in Dst-­b mutant mouse hearts. We also observed the accumulation of the
autophagy-­associated molecules p62 and ubiquitinated proteins in the muscles of Dst-­b mutant mice.
Co-­aggregation of p62 and ubiquitinated proteins has been reported in autophagy-­deficient neuronsof Atg7 conditional knockout mice (Komatsu et al., 2007). In the sensory neurons of dt mice, loss of
Dst was reported to lead to disruption of the autophagic process (Ferrier et al., 2015; Lynch-­Godrei
et al., 2021). Autophagy was also reported to be involved in the pathogenesis of other types of
MFMs; the CryAB R120G mutation (CryABR120G) increases autophagic activity, and genetic inactivationof autophagy aggravates heart failure in CryABR120G mice (Tannous et al., 2008), while enhanced
autophagy ameliorates protein aggregation in CryABR120G mice (Bhuiyan et al., 2013). It would beinteresting to investigate the involvement of autophagy and other protein degradation machinery in
the process of protein aggregation myopathy in Dst-­bE2610Ter/E2610Ter mutant mice.
Accumulated evidence strongly suggests that myofibrillar integrity is essential for the maintenanceof mitochondrial function (Vincent et al., 2016). Mitochondrial dysfunctions have also been well recognized in some animal models of MFMs, such as desmin (Des), αB-­crystallin (Cryab), and plectin (Plec)mutants (Winter et al., 2015; Winter et al., 2016; Diokmetzidou et al., 2016; Alam et al., 2020).
Abnormal accumulation of mitochondria in the subsarcolemmal space was observed in Dst-­bE2610Ter/
E2610Ter
mutant muscles. Mitochondrial dysfunction in Dst-­bE2610Ter/E2610Ter mutant mouse cardiomyocytes
was also suggested by RNA-­seq data, including altered expression of genes responsible for oxidative
phosphorylation and other metabolic processes. The altered heart functions characterized by long QTintervals and PVCs in Dst-­b mutant mice may be
explained by mitochondrial dysfunction, as well
as altered expression of genes involved in transmembrane ion transport and muscle contraction.

Nuclear abnormalities in Dst-b
mutation-induced myopathy andcardiomyopathy

Video 1. Super-­resolution microscopy image showingco-­localization of p62 and ubiquitin in the nucleus of
Dst-­b mutant cardiomyocytes.
https://elifesciences.org/articles/78419/figures#video1

Yoshioka et al. eLife 2022;11:e78419. DOI: https://doi.org/10.7554/eLife.78419

Deep invaginations of the nuclear membrane
were also a characteristic feature of Dst-­b mutant
muscle fibers. The linker of nucleoskeleton andcytoskeleton (LINC) complex is a protein complex
that spans the inner and outer nuclear membrane
to bridge the cytoskeleton in the cytoplasm to
the nuclear lamina underlying the inner nuclear
membrane (Stroud et al., 2014). Mutations
in genes encoding LINC complex proteins are

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A

B-log10 P value (Dst-bE2610Ter vs WT)

45

Total 13003 genes
in heart tissues
Dst-bE2610Ter
vs WT

P value < 0.01
FDR < 0.1

Up-regulation:
481 genes
Down-regulation:
247 genes

Ctgf Sec14l5

35

Acta2
Ano5

25
Cacng615
5Helt

Sult4a1

Pde1c Tgfb2
2610028H24Rik
Ankrd1
Ubxn10
Scn4b
Rp1 Crip3
Nmrk2
Ucp2
Mybpc2
Tbx15 Nefm Slc35f3
Rpl3l
Cd44
Grin2c

Mylk4

Dok5
Crlf1
Ccl5
Enkur

Rtn4r

Mgat5b-5

DGO biological process
3.0

4.0

5.0

6.0

4

-2
2
0
Log2 fold-change (Dst-bE2610Ter vs WT)

E

GO cellular component-log10 P value
2.0

Ephx4

A530016L24Rik

Tmem150c
Amtn
Adcy8

-4

C

Cenpf

6

KEGG pathway-log10 P value
7.0

8.0

cell adhesion
muscle contraction

2.0
extracellular matrix
Z discsarcolemma

4.0

6.0

8.0

10.0

-log10 P value
12.0

positive regulation of neuron projection development

skeletal muscle tissue development

focal adhesion
PI3K-Akt signaling pathwaycGMP-PKG signaling pathway

focal adhesion
lamellipodium
neuromuscular junction
collagen trimer
M bandI bandactin cytoskeleton
cell projection
axon
extracellular exome
neuronal cell bodyneuron projection
myelin sheathsarcomere
sarcoplasmic reticulum
extracellular region
myosin filament
growth cone
cell junction
stress fiber
cell surface
ciliumintercalated discpostsynaptic density
cell bodycytosol

positive regulation of ERK1 and ERK2 cascade
cellular response to fibroblast growth factor stimulus

sarcomere organization
positive regulation of cardiac muscle contraction
cellular response to mechanical stimulus
peptidyl-tyrosine dephosphorylation
skeletal muscle contraction
actin filament organization
endodermal cell differentiation
striated muscle contraction
positive regulation of apoptotic processcell adhesion mediated by integrin
response to hypoxia
cardiac muscle contraction
cardiac muscle tissue morphogenesis
regulation of actin cytoskeleton organization
regulation of membrane potential
regulation of heart rate
response to heat

6.0

calcium signaling pathway

perinuclear region of cytoplasm

positive regulation of cell-substrate adhesion

5.0

hypertrophic cardiomyopathy

cytoskeleton
basement membrane
myofibrilfilopodium

heart development

4.0

3.0

dilated cardiomyopathy

proteinaceous extracellular matrix

response to unfolded protein

2.0
ECM-receptor interaction

protein digestion and absorption
arrhythmogenic right ventricular cardiomyopathy

oxytocin signaling pathway
vascular smooth muscle contraction

intracellular signal transduction
response to wounding-log10 P value-log10 P value
2.0
cell adhesion
metabolic process
fatty acid metabolic process

2.5

3.0

3.5

4.0

4.5

5.0

2.0

3.0

extracellular exosome

regulation of ion transmembrane transport

integral componentof plasma membranemuscle organ development

mitochondrion

angiogenesis
positive regulation of endothelial cell proliferation

4.0

5.0

-log10 P value
2.0

6.0

2.5

3.0

metabolic pathways
Alzheimer’s diseasecardiac muscle contraction
focal adhesion

Figure 8. RNA-­seq-­based transcriptome of hearts from Dst-­bE2610Ter mice. RNA-­seq analysis was performed in ventricular myocardium tissues from 14- to
19-­month-­old Dst-­bE2610Ter/E2610Ter mice and WT mice (n = 3, each genotype). Principal component analysis (PCA) and hierarchical clustering of RNA-­seq
data are shown in Figure 8—figure supplement 1. (A) Among the 13,003 genes expressed in the heart, 481 genes were upregulated and 247 genes
were downregulated. Thresholds were set at p<0.01 and false discovery rate (FDR) < 0.1, respectively. (B) Volcano plot shows differentially expressed
Figure 8 continued on next page

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Figure 8 continued

genes. Red and blue dots represent genes upregulated and downregulated, respectively. Dots of highly changed genes were labeled with genesymbols. Changes of gene expressions were validated by qPCR in Figure 8—figure supplement 1. Bar graphs show Gene Ontology (GO) biologicalprocess (C), GO cellular component (D), and Kyoto Encyclopedia of Genes and Genomics (KEGG) pathway (E) enriched in genes that are upregulated
and downregulated in the heart of Dst-­bE2610Ter/E2610Ter mice. Red and blue bars represent genes upregulated and downregulated, respectively. Items in
red words are specific to skeletal muscle and heart. List of genes resulting from GO analysis and KEGG pathway analysis is shown in Supplementary file
1A–F. RNA-­seq data can be accessed from the Gene Expression Omnibus under accession # GSE184101.
The online version of this article includes the following figure supplement(s) for figure 8:
Figure supplement 1. Transcriptomes of hearts from WT and Dst-­bE2610Ter mice.

known to cause skeletal myopathy and cardiomyopathy, called Emery–Dreifuss muscular dystrophy(Heller et al., 2020), and defects in nuclear structure are often observed in mice with mutations of
LINC and LINC-­associated molecules such as lamin A (Lmna), nesprin, ezrin, and Des (Nikolova et al.,
2004; Banerjee et al., 2014; Wada et al., 2019; Heffler et al., 2020). Because Dst can associate withnesprin-­3α via the ABD (Young and Kothary, 2008), and we demonstrated that Dst-­b can localize
around nuclei of muscle fibers, Dst-­b may be involved in the molecular bridge between the LINC
complex and cytoskeleton and maintain the shape of nuclei.
Furthermore, we found that nuclear inclusion was a unique hallmark of cardiomyopathy in Dst-­bmutant mice. The nuclear inclusions were different from the cytoplasmic protein aggregates in
autophagy-­deficient mice because nuclear inclusions lacked LC3 protein. Intranuclear inclusions arealso observed in NIID, which is a neurological disease caused by GGC repeat expansion of theNOTCH2NLC gene (Sone et al., 2019), which has also been reported to be associated with oculopharyngodistal myopathy with intranuclear inclusions (Ogasawara et al., 2020). The intranuclear
inclusions of NIID predominantly include p62, ubiquitinated proteins, and SUMOylated proteins(Mori et al., 2012; Pountney et al., 2003). In our observations, SUMOylated proteins were alsopresent in the nuclear inclusions of Dst-­b mutants. Thus, nuclear inclusions of Dst-­b mutant cardiomyocytes have common features with those of NIID in terms of molecular components (Sone et al.,2011). It would be interesting to further investigate the molecular components and pathogenesis of
nuclear inclusions in the cardiomyopathy of Dst-­b mutant mice and compare them with those foundin NIID.

Role of the Dst-b isoform in DST-related diseases
A wide variety of genetic diseases have been reported to be caused by DST mutations. Loss-­of-­function mutations in both DST-­a and DST-­b result in HSAN-­VI, and loss-­of-­function mutations of DST-­eresult in the skin blistering disease epidermolysis bullosa simplex (Groves et al., 2010; Edvardson
et al., 2012). Recently, DST was reported as a candidate gene of pulmonary atresia, a rare congenital heart defect (Shi et al., 2020). According to this study, it is possible that unidentified hereditary
myopathies and heart disease caused by human DST-­b-­specific mutations exist. Because the causative
gene is unidentified in almost half of MFM cases, it would be worthwhile to perform candidate genescreening on DST-­b-­specific exons in patients with MFMs.
Recently, HSAN-­VI has been proposed to be a spectrum disease because of its varying disease
severity and the diverse deficiency of DST isoforms (Lynch-­Godrei and Kothary, 2020). DST-­a2 seems
to be a crucial isoform for HSAN-­VI pathogenesis because DST-­a2-­specific mutations result in adult-­
onset HSAN-­VI (Manganelli et al., 2017; Fortugno et al., 2019), and neuronal expression of Dst-­a2
partially rescues the dt phenotype (Ferrier et al., 2014). We suggest that Dst-­a1 and Dst-­a2 play
redundant roles because DstGt mice, in which both Dst-­a1 and Dst-­a2 are trapped, show a severe
phenotype (Horie et al., 2014). Among three Dst-­b isoforms, remarkable reduction of Dst-­b1, but not
Dst-­b2 or Dst-­b3, was observed in Dst-­bE2610Ter/E2610Ter mice, suggesting that reduced Dst-­b1 expression
is involved in myopathic phenotypes in Dst-­bE2610Ter/E2610Ter mice. It is possible that DST isoforms 1 and2 may be important to different extents in neural and muscular tissues. Regarding this point of view,
it would be interesting to compare muscle phenotypes between patients harboring DST-­a1, -­a2, -­b1,
and -­b2 isoform mutations and patients with DST-­a2 and -­b2 isoform-­specific mutations (Manganelli
et al., 2017; Fortugno et al., 2019; Motley et al., 2020).

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A

B

DstGt
(Dst–a/b
Trapped)DstGt-inv(Untrapped)
DstGt-DO(Dst–a/b
Trapped)*1M 2M 6M

E

Rotarod test*

Latency to fall (sec)Ctrl (n=5)
Mosaic (n=5)

Body weight (g)CConditional gene-trap (cGT)D

Tailsuspension

Brain

Heart
Ctrl

Mosaic

Ctrl

MosaicDst
Gapdh(36 kDa)
Ctrl
(n=8)

Masson's trichrome

Mosaic(n=5)

Mosaic mouse

F

p62

Nppa

Ctrl

Ctrl

LV

MosaicMosaic

LV

Figure 9. Cardiomyopathy in conditional Dst conditional gene trap (cGT) mice. (A) Scheme of mosaic analysis by cGT of Dst-­a/b. The gene trap
cassette contains splice acceptor (SA) sequence, the reporter gene βgeo, and poly-­A (pA) termination signal. The gene trap cassette is flanked by pairsof inversely oriented target sites of FLP recombinase (Frt and F3: half circles) and Cre recombinase (loxP and lox5171: triangles). FLP- and Cre-­mediatedrecombination induce irreversible inversion from mutant DstGt allele to untrapped DstGt-­inv allele and DstGt-­inv allele to mutant DstGt-­DO allele, respectively.
(B) For generation of Actb-­iCre; DstGt-­DO/Gt-­inv (mosaic) mice, female DstGt-­inv/Gt-­inv mice were mated with male Actb-­iCre; DstGt-­DO/WT mice. (C) Body weight of
Figure 9 continued on next page

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Figure 9 continued

male mosaic mice reduced than Ctrl mice in several months of age (n = 5 mice, each genotype; two-­way ANOVA; genotype effect: p=0.0493; age effect:p=0.0000; genotype × age interaction: p=0.9126). Impairment of motor coordination in mosaic mice is shown by rotarod test (n = 8 Ctrl mice, n = 5
mosaic mice). * denotes statistically significant difference at p<0.05 (p=0.0110), using Student’s t-­test. Mosaic mice displayed hindlimb clasping and twist
movements during tail suspension. (D) Representative data of Western blot analysis showed a deletion of Dst band in heart lysates from mosaic mice,
while residual Dst bands were detected in those brain lysates (n = 2 mice, each genotype, 5–7 months of age). (E) Masson’s trichrome staining showed
extensive fibrosis in heart sections of mosaic mice at 10 months of age. (F) Nppa mRNA and p62-­positive depositions (arrowheads) were evident in theleft ventricular myocardium of mosaic mice than Ctrl mice. Data are presented as mean ± SE. Scale bars: (E, F) 50 μm.

Roles of plakin family proteins in muscles and other tissues
Members of the plakin protein family other than Dst, such as plectin and desmoplakin, are known to
be expressed and play crucial roles in maintaining muscle integrity (Leung et al., 2002; Boyer et al.,2010b; Horie et al., 2017). Plectin is a Dst-­associated protein and one of the most investigatedmembers of the plakin protein family (Castañón et al., 2013). There are four alternative splicing
isoforms of plectin in striated muscle fibers, which are localized in differential subcellular compartments: plectin 1 in nuclei, plectin 1b in mitochondria, plectin 1d in Z-­disks, and plectin 1f in the sarcolemma (Mihailovska et al., 2014; Staszewska et al., 2015; Winter et al., 2015). Plec deficiency islethal in mice at the neonatal stage, and these mice exhibit skin blistering and muscle abnormalities(Andrä et al., 1997). Conditional deletion of Plec in muscle fibers leads to progressive pathologicalalterations, such as aggregation of desmin and chaperon protein, subsarcolemmal accumulation of
mitochondria, and an abnormal nuclear shape (Konieczny et al., 2008; Staszewska et al., 2015;
Winter et al., 2014; Winter et al., 2015), some of which are also observed in the muscles of aged
Dst-­b mutant mice.
Dst and plectin also have common features in other cell types. In keratinocytes, plectin and Dst-­e
localize to the inner plaque of hemidesmosomes and anchor keratin intermediate fibers to hemidesmosomes (Künzli et al., 2016). Conditional deletion of Plec from epidermal cells causes epidermal
barrier defects and skin blistering (Ackerl et al., 2007), both of which is more severe than thatobserved in dt mice carrying Dst-­e mutations (Guo et al., 1995; Yoshioka et al., 2020). Furthermore, we recently demonstrated that conditional deletion of Dst from Schwann cells in the peripheral
nervous system leads to disorganization of the myelin sheath (Horie et al., 2020), similar to thatobserved for Plec-­deficient Schwann cells (Walko et al., 2013). These studies suggest that Dst andplectin, which are plakin family proteins, have overlapping roles in different cell types.

Materials and methods
Animals
Dst-­bE2610Ter mice, DstGt(E182H05) mice (MGI number: 3917429; Horie et al., 2014), and Actb-­iCre mice
(Zhou et al., 2018) were used in this study. DstGt(E182H05) allele was abbreviated as DstGt. Homozygous Dst-­bE2610Ter and DstGt mice were obtained by heterozygous mating. For mosaic analysis, female
DstGt-­inv/Gt-­inv mice were crossed with male Actb-­iCre;DstGt-­DO/wt mice. Dst-­bE2610Ter mutant line was
C57BL/6J. The DstGt mutant line was backcrossed to C57BL/6NCrj at least 10 generations. Mice
were maintained in groups at 23°C ± 3°C, 50% ± 10% humidity, 12 hr light/dark cycles, and food/water availability ad libitum. Both male and female mice were analyzed in this study. Genotyping
PCR for the DstGt allele was performed as previous described (Horie et al., 2014). For genotyping
PCR of the Dst-­bE2610Ter allele, the following primer set was used to amplify 377 bp fragments: Dst-­b
forward: 5′-TGA GCG ATG GTA GCG ACT TG-­3′ and Dst-­b reverse: 5′-GCG ACA CAC CTT TAG TTG
CC-­3′. PCR was performed using Quick Taq HS DyeMix (Toyobo, Osaka, Japan) and a PCR Thermal
Cycler Dice (TP650; Takara Bio Inc, Shiga, Japan), with the following cycling conditions: 94°C for
2 min, followed by 32 cycles of 94°C for 20 s, 61°C for 30 s, and 72°C for 30 s. PCR products were
cut with XhoI, which produced two fragments (220 bp and 157 bp) for the mutant Dst-­bE2610Ter allele.
For genotyping PCR for Actb-­iCre knockin allele, the following primer set was used to amplify 540 bp
fragments: iCre-­F: CTC AAC ATG CTG CAC AGG AGA T-­3′ and iCre-­R: 5′-­ACC ATA GAT CAG GCG
GTG GGT-­3′.

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Table 1. List of identified nonsense mutations in DST-­b.
No.

SNP_ID

DST-­b-­specificexon(Ex40-­Ex44)

Base substitution(NM_001374736.1)

Amino acidsubstitution(NP_001361651.1)

Database

Global frequency

Specific population frequency

1780727375

Ex40

p.Ser1839_
c.5513_5514insTTAGA, Ly1a840insTer

ExAC

1/120712

European: 1/73330

2775037762

Ex40

c.5581C>T

p.Gln1861Ter

ExAC

1/120756

European: 1/73346

3267601090

Ex40

c.6106C>T

p.Gln2036Ter

None

None

None

5/248332
1/140170
1/120410
0/10680

Asian: 3/48548; American:
2/34434,
European: 1/75902
American: 1/11450
None

4763489373

Ex40

c.6199C>T

p.Arg2067Ter

GnomAD_exome
GnomAD
ExAC
ALFA

5

980428529

Ex40

c.6413T>A

p.Leu2138TerTOPMEDALFA

1/264690
1/35428

None
European: 1/26584

62098511415

Ex40

c.6846T>A

p.Cy1b282Ter

ALFA

2/21326

European: 2/16854

71416256967

Ex40

c.6907G>T

p.Glu2303Ter

TOPMEDALFA

1/264690
0/14050

None
None

8

1563150977

Ex40

c.6952C>T

p.Gln2318Ter

GnomAD_exome

1/248356

European: 1/133910

92098509730

Ex40

c.7045C>T

p.Gln2349Ter

GnomADALFA

1/140156
0/10680

African: 1/42028
None

1/264690
3/222812
1/139940
3/32028
1/2922
1/998

None
European: 3/118158
African: 1/41914
European: 1/23832; Other:
2/4554
KOREAN: 1/2922
None

10

536128073

Ex40

c.7120C>T

p.Arg2374TerTOPMED
GnomAD_exome
GnomADALFA
KOREAN
GoNL

11747917821

Ex40

c.7171C>T

p.Gln2391Ter

GnomAD
ExAC

1/140100
2/83458

African: 1/41990
Asian: 1/19128; African: 1/7454

p.Ser2405Ter

TOPMED
GnomAD_exome
ALFA

1/264690 (C>A)
1/245012 (C>G)
0/10680 (C>A)

None
European: 1/131624
None

p.Leu2439Ter

GnomAD_exome
ExAC

1/248436
1/120426

Asian: 1/48550
Asian: 1/25104

1/247718
1/119556
1/5008Asian: 1/48546
Asian: 1/25084
South Asian: 1/978

121261702898

Ex40

c.7214C>G,
c.7214C>A,

13

757004287

Ex40

c.7316T>G

14

559852499

Ex40

c.7459C>T

p.Gln2487Ter

GnomAD_exome
ExAC
1000G

15

1437052580

Ex40

c.7510C>T

p.Gln2504Ter

GnomAD_exome
ALFA

1/247692
1/8988Asian: 1/48526
Asian: 1/56

16751368429

Ex40

c.7531_7534delp.Leu2510_
Asn2511insTer

GnomAD_exome
ExAC

1/247498
1/120056

American: 1/34358
American: 1/11378

171563144635

Ex40

c.7447_7534delp.Ile2482_Gly2483insTer GnomAD_exome

1/247498

Asian: 1/48522

18747767227

Ex40

c.7552C>T

p.Gln2518Ter

None

None

None

19

1243608666

Ex40

c.7578C>G

p.Tyr2526Ter

GnomAD_exome

1/247554

European: 1/133118

p.Gln2543TerTOPMED
GnomAD_exome
ALFA

1/264690
1/247988
0/10680

None
African: 1/15478
None

2/264690
2/248730
1/120578
0/14050
0/38541/3708None
European: 2/134142
European: 1/73284
None
None
TWIN COHORT: 1/3708

20

1190095913

Ex40

c.7627C>T

21

756643045

Ex40

c.8014C>T

p.Gln2672Ter

TOPMED
GnomAD_exome
ExAC
ALFA
ALSPACTWINSUK

22

1208663117

Ex40

c.8294G>A

p.Trp2765Ter

GnomAD_exome
ALFA

1/245286
1/21368

European: 1/131776
European: 1/16886

23

2098496754

Ex40

c.8374G>T

p.Glu2792Ter

GnomADALFA

1/140098
0/10680

African: 1/42014
None

Table 1 continued on next page
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Table 1 continued

No.

24

SNP_ID

1314301705

DST-­b-­specificexon(Ex40-­Ex44)

Ex40

Amino acidsubstitution(NP_001361651.1)

Database

Global frequency

Specific population frequency

c.8485C>T

p.Gln2829Ter,

TOPMED
GnomADALFA

1/264690
1/139898
0/14050

None
Ashkenazi Jewish: 1/3318
None

Base substitution(NM_001374736.1)

251563133123

Ex40

c.8623delp.Arg2874_
Val2875insTer

None

None

None

26

2098493114

Ex40

c.8635C>T

p.Gln2879Ter

8.3KJPN

1/16760

JAPANESE: 1/16760

27

1458968582

Ex40

c.8900C>G

p.Ser2967Ter

None

None

None

28

1048157544

Ex40

c.9076C>T

p.Gln3026Ter

TOPMEDALFA

1/264690
0/14050

None
None

29

910403635

Ex40

c.9172G>T

p.Gly3058TerTOPMEDALFA

2/264690
0/14050

None
None

30

747173454

Ex40

c.9202G>T

p.Glu3068Ter

GnomAD_exome
ExAC

1/248348
1/120120

European: 1/133958
European: 1/73002

31751807675

Ex40

c.9227_9237delp.Leu3075_
Leu3076insTer

ExAC

1/119230

European: 1/72390

32749282620

Ex40

c.9439A>T

p.Ly1c147Ter

GnomADALFA

1/140058
0/10680

African: 1/42014
None

2/264690
1/247036
0/10680

None
European: 1/132884
None

33

1301999896

Ex40

c.9549C>G

p.Tyr3183Ter

TOPMED
GnomAD_exome
ALFA

34

1411974489

Ex40

c.9580A>T

p.Ly1c194Ter

GnomAD_exome
ALFA

1/247422
1/8988Ashkenazi Jewish: 1/10018
European: 1/6062

35

2098482258

Ex40

c.9586delp.Asp3195_
Val3196insTer

None

None

None

36

972168431

Ex40

c.9818C>G,
c.9818C>A

p.Ser3273TerTOPMEDALFA

2/264690 (C>A)
0/14050 (C>A)

None
None

37

200867945

Ex40

c.9824T>A

p.Leu3275Ter

None

None

None

1/264690
2/247418
1/33212None
European: 2/133356
European: 1/24496

38

1346974625

Ex40

c.10045C>T

p.Gln3349TerTOPMED
GnomAD_exome
ALFA

39

1229343851

Ex40

c.10114G>T

p.Glu3372Ter

None

None

None

40

2098476192

Ex40

c.10166T>A

p.Leu3389Ter

TOPMEDALFA

1/264690
0/10680

None
None

41

2098474844

Ex40

c.10271C>A

p.Ser3424Ter,

TOPMEDALFA

1/264690
0/10680

None
None

42

2098473553

Ex40

c.10391delp.Glu3463_
Leu3464insTer

TOPMEDALFA

1/264690
0/10680

None
None

43

1249289191

Ex40

c.10516G>T

p.Glu3506Ter

8.3KJPN

1/16760

JAPANESE: 1/16760

1/264690
1/139986
0/11862

None
European: 1/75810
None

1/2922KOREAN: 1/2922

441428617557

Ex40

c.10570G>T

p.Glu3524TerTOPMED
GnomADALFA

45

1586342297

Ex40

c.10633G>T

p.Glu3545Ter

KOREAN

46

2098467261

Ex41c.10807C>T

p.Gln3603Ter

GnomAD

2/140068

European: 2/75876

47

1586330106

Ex41

c.10815T>A

p.Cy1c605Ter

Korea1K

1/1832KOREAN: 1/1832

48

2098463511

Ex42

c.10958_10959insTTA

p.Leu3653delinsPheTer

ALFA

0/11862None

49

2098462327

Ex42c.11071C>T

p.Gln3691Ter

GnomADALFA

1/139940
0/10680

American: 1/13576
None

50

1245541628

Ex43c.11204C>G

p.Ser3735Ter

GnomADALFA

1/139898
0/10680

European: 1/75804
None

Table 1 continued on next page

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Table 1 continued

No.

SNP_ID

DST-­b-­specificexon(Ex40-­Ex44)

Base substitution(NM_001374736.1)

Amino acidsubstitution(NP_001361651.1)

Database

Global frequency

Specific population frequency

1/264690
1/139886
0/14050

None
African: 1/41950
None

51

1363675987

Ex43

c.11210C>G

p.Ser3737TerTOPMED
GnomADALFA

52

2098459417

Ex43

c.11217G>A

p.Trp3739Ter

ALFA

0/10680None

531230102996

Ex43

c.11222C>G

p.Ser3741Ter

GnomADALFA

2/139836
0/10680

European: 2/75762
None

54

2098459238

Ex43

c.11251G>T

p.Glu3751Ter

None

None

None

55

1305040869

Ex44

c.11392C>T

p.Gln3798Ter

None

None

None

56

2098446552

Ex44c.11419C>T

p.Gln3807Ter

8.3KJPN

1/16760

JAPANESE: 1/16760

57

1467862852

Ex44

c.11521C>T

p.Gln3841Ter

8.3KJPN
ALFA

1/16760
0/14050

JAPANESE: 1/16760
None

58

776397027

Ex44

c.11536C>T

p.Gln3846Ter

GnomAD_exome
ExAC

1/205888
1/34048

European: 1/107654
European: 1/18438

Generation of Dst-bE2610Ter mice by CRISPR-Cas9 system
We attempted to induce G-­to-­T (Glu to Stop) point mutation in the Dst-­b/Bpag1b using a previously
described procedure (Sato et al., 2018). The sequence (5′-­GCT ATC AGG AAA GAA CAC GG-­3′) was
selected as guide RNA (gRNA) target. The gRNA was synthesized and purified by GeneArt Precision
gRNA Synthesis Kit (Thermo Fisher Scientific, MA) and dissolved in Opti-­MEM (Thermo Fisher Scientific). In addition, we designed a 102-­nt single-­stranded DNA oligodeoxynucleotide (ssODN) donor
for inducing c.8058 G>T of the Dst-­b1 (accession # NM_134448.4); the nucleotide T was placed
between 5′- and 3′-­homology arms derived from positions 8013–8057 and 8066–8114 of the Dst-­b1
coding sequence, respectively. This ssODN was ordered as Ultramer DNA oligos from Integrated DNA
Technologies (IA, USA) and dissolved in Opti-­MEM. The pregnant mare serum gonadotropin (fiveunits) and the human chorionic gonadotropin (five units) were intraperitoneally injected into female
C57BL/6J mice (Charles River Laboratories, Kanagawa, Japan) with a 48 hr interval, and unfertilized
oocyte were collected from their oviducts. We then performed in vitro fertilization with these oocytes
and sperm from male C57BL/6J mice (Charles River Laboratories, Kanagawa, Japan) according tostandard protocols. 5 hr later, the gRNA (5 ng/μl), ssODN (100 ng/μl), and GeneArt Platinum Cas9
Nuclease (Thermo Fisher Scientific) (100 ng/μl) were electroplated to zygotes by using NEPA21 electroplater (Nepa Gene, Chiba, Japan) as previously reported (Sato et al., 2018). After electroporation,
fertilized eggs that had developed to the two-­cell stage were transferred into oviducts in pseudopregnant ICR female and newborns were obtained. To confirm the G-­to-­T point mutation induced by
CRISPR/ Cas9, we amplified genomic region including target sites by PCR with the same primers used
for PCR-­RFLP genotyping. The PCR products were cut with XhoI for genome editing validation, andthen, sequenced by using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). We
analyzed three independent Dst-­b lines in this study: line numbers #1, # 6, and #7.

Western blotting
Frozen heart, gastrocnemius muscle, and brain tissues were homogenized using a Teflon-­glass
homogenizer in ice-­cold homogenization buffer (0.32 M sucrose, 5 mM EDTA, 10 mM Tris-­HCl, andpH 7.4, phosphatase inhibitor cocktail tablet; Roche, Mannheim, Germany), centrifuged at 4500 rpm
for 10 min at 4°C and the supernatants were collected. The protein concentration was determinedusing the bicinchoninic acid Protein Assay Reagent (Thermo Fisher Scientific). Lysates were mixed
with an equal volume of 2× sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris-­HCl, pH 6.8, 4%
SDS, 20% glycerol, and 0.002% bromophenol blue) for a final protein concentration of 2 μg/μl anddenatured in the presence of 100 mM dithiothreitol at 100°C for 5 min. SDS-­polyacrylamide gel electrophoresis (PAGE) was performed with 20 μg per lane on 5–20% gradient gels (197-­15011; SuperSep
Ace; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) running at 10–20 mA for 150 min. The
gels were blotted onto an Immobilon-­P transfer membrane (Millipore, Billerica, MA). After blocking

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Figure 10. Nonsense mutations in the DST-­b-­specific exons. (A) Locations of nonsense mutations in the DST-­b-­specific exons on human chromosome 6(NM_001374736.1). DST-­b-­specific exons are indicated blue rectangles. Nonsense mutations identified in the dbSNP database were distributed in all five
DST-­b specific exons. (B) Pie chart shows the frequency distribution of nonsense mutations of DST-­b-­specific exons in different populations. The colors
indicate different populations. The numbers in the pie chart indicate the frequency of identified mutations in each population. DST variants identified inJapanese patients with myopathy are shown in Supplementary file 1G.

with 10% skim milk for 3 hr, blotted membranes were incubated with the following primary antibodies:
rabbit polyclonal anti-­Dst antibody (gifted from Dr. Ronald K Leim; 1:4000; Goryunov et al., 2007)
that recognizes the plakin domain of Dst, and mouse monoclonal anti-­glyceraldehyde-­3-­phosphate
dehydrogenase (Gapdh) antibody (016-­25523; clone 5A12, 1:10,000, Wako). Each primary antibody

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was incubated overnight at 4°C. Then membranes were incubated with peroxidase-­conjugatedsecondary antibodies for 1 hr at room temperature: anti-­rabbit immunoglobulin G (IgG) (AB_2099233;
Cat# 7074, 1:2000, Cell Signaling Technology), anti-­mouse IgG (AB_330924; Cat# 7076, 1:2000, Cell
Signaling Technology). Tris-­buffered saline (10 mM Tris-­HCl, pH 7.5, 150 mM NaCl) containing 0.1%
Tween-­20 and 10% skim milk was used for the dilution of primary and secondary antibodies, and Tris-­buffered saline containing 0.1% Tween-­20 was used as the washing buffer. Immunoreactions werevisualized with ECL (GE Healthcare, Piscataway Township, NJ) or ImmunoStar LD (FUJIFILM Wako
Pure Chemical) and a Chemiluminescent Western Blot Scanner (C-­Digit, LI-­COR, Lincoln, NE). The
signal intensity of each band was quantified using Image Studio software version 5.2 (LI-­COR).

RNA extraction and real-time PCR
Total RNA was extracted from the heart, soleus, and brain using the RNeasy Mini Kit (QIAGEN, Hilden,
Germany) including DNase digestion. 100 ng of RNA template was used for cDNA synthesis with oligo(dT) primers. Real-­time PCR was performed using a StepOnePlus Real-­Time PCR system (Thermo
Fisher Scientific) and the following cycling conditions: 95°C for 2 min, followed by 40 cycles of 95°C for
15 s, 60°C for 40 s, and 95°C for 15 s. Gene expression levels were analyzed using the ΔΔCT method.
Actb or Gapdh were used as internal controls to normalize the variability of expression levels. The
primers used for real-­time PCR are listed in Table 2.

RNA-seq analysis
RNA-­seq analysis was performed as described in the previous study with slight modification (Bizen
et al., 2022; Hayakawa-­Yano et al., 2017; Hayakawa-­Yano and Yano, 2019). mRNA libraries weregenerated from total RNA 5 μg/sample extracted from the heart using illumine TruSeq protocols forpoly-­A selection, fragmentation, and adaptor ligation. Multiplexed libraries were sequenced as 150-­nt
paired-­end runs on an NovaSeq6000 system. Sequence reads were mapped to the reference mouse
genome (GRCm38/mm10) with Olego. Expression and alternative splicing events were quantified with
Quantas tool. Integrative Genomics Viewer (IGV) was used as visualization of alignments in mouse
genomic regions (Thorvaldsdóttir et al., 2013). Statics of differential expression was determinedwith edseR (Robinson et al., 2010). Differentially expressed genes were corrected with threshold
set at p<0.01 and FDR < 0.1. GO analysis and KEGG pathway analysis were performed using DAVID
Bioinformatics Resources (Huang et al., 2009) (https://david.ncifcrf.gov/summary.jsp). The thresholdof GO analysis and pathway analysis was set at p<0.01. Three highly changed genes, Xist, Tsix, and
Bmp10, were excluded because the differences were due to extreme outliers in only one individual.
In GO analysis, the terms of cytoplasm and membrane that contained more than 190 genes were
excluded from the list. PCA and hierarchical clustering were performed and visualized with R software using the RNA-­seq datasets obtained from three independent WT and Dst-­bE2610Ter. PCA was
conducted in the prcomp function in RStudio (version 2021.09.1 build 372). RNA-­seq data can beaccessed from the Gene Expression Omnibus under accession, GSE184101.

Behavioral tests
The rotarod test and wire hang test (O’Hara & Co., Tokyo, Japan) were performed to evaluate motor
coordination as described in the previous study with slight modification (Horie et al., 2020). In therotarod test, the latency to fall from a rotating rod (30 mm diameter) with an acceleration from 10 to
150 rpm was measured. Each trial was conducted for a period of 3 min. In each mouse, two trials wereconducted in a day. In the wire hang test, mouse was placed on the top of the wire lid. The lid wasslightly shaken several times to force the mouse to grip the wires. The lid was turned upside down.
The latency to fall was measured.

Histological analysis
For tissue preparations, mice were euthanized via intraperitoneal injection with pentobarbital sodium(100 mg/kg body weight), and then perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate-­
buffered (PB) solution (pH 7.4). The tissues were fixed by cardiac perfusion with 0.01 M phosphate-­
buffered saline (PBS) followed by ice-­cold 4% PFA in 0.1 M PB (pH 7.4). Dissected tissues were
immersed in the same fixative overnight. To cut spinal cord and DRG sections, the specimens wererinsed with water for 10 min and decalcified in Morse solution (135-­17071; Wako, Osaka, Japan)

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Table 2. Primer list for qPCR.
Gene nameForward (5′ to 3′)

Reverse (5′ to 3′)

Acta2

GTCCCAGACATCAGGGAGTAATCGGATACTTCAGCGTCAGGAActb

GGCTGTATTCCCCTCCATCG

CCAGTTGGTAACAATGCCATGT

Ano5

TCCAAAGAGACCAGCTTTCTCAGTCGATCTGCCGGATTCCAT

Atp5a1

TCTCCATGCCTCTAACACTCG

CCAGGTCAACAGACGTGTCAG

Cenpf

GCACAGCACAGTATGACCAGG

CTCTGCGTTCTGTCGGTGAC

Cox4i1

ATTGGCAAGAGAGCCATTTCTACCACGCCGATCAGCGTAAGT

Ctgf

GGGCCTCTTCTGCGATTTCATCCAGGCAAGTGCATTGGTA

Dst-­a

AACCCTCAGGAGAGTCGAAGGTTGCCGTCTCCAATCACAAAG

Dst-­bACCGGTTAGAGGCTCTCCTG

ATCACACAGCCCTTGGAGTTT

Dst isoform1

TCCAGGCCTATGAGGATGTCGGAGGGAGATCAAATTGTGC

Dst isoform2

AATTTGCCCAAGCATGAGAGCGTCCCTCAGATCCTCGTAG

Dst isoform3

CACCGTCTTCAGCTCACAAA

AGTTTCCCATCTCTCCAGCA

Gapdh

AGGTCGGTGTGAACGGATTTG

TGTAGACCATGTAGTTGAGGTCA

Gpx1

CCACCGTGTATGCCTTCTCCAGAGAGACGCGACATTCTCAAT

Gpx4

GCCTGGATAAGTACAGGGGTT

CATGCAGATCGACTAGCTGAG

Hmox1

AAGCCGAGAATGCTGAGTTCAGCCGTGTAGATATGGTACAAGGA

Hspa1lTCACGGTGCCAGCCTATTTCCGTGGGCTCATTGATTATTCTCA

Hspb1

CGGTGCTTCACCCGGAAATA

AGGGGATAGGGAAAGAGGACAMyh7

ACTGTCAACACTAAGAGGGTCATTGGATGATTTGATCTTCCAGGG

Mylk4

GGGCGTTTTGGTCAGGTACATACGCTGATCTCGTTCTTCACA

Ndufb8TGTTGCCGGGGTCATATCCTAAGCATCGGGTAGTCGCCATA

Nppa

GCTTCCAGGCCATATTGGAG

GGGGGCATGACCTCATCTT

Nppb

CATGGATCTCCTGAAGGTGC

CCTTCAAGAGCTGTCTCTGG

Nqo1

AGCGTTCGGTATTACGATCCAGTACAATCAGGGCTCTTCTCG

Rpl3l

GAAGGGCCGGGGTGTTAAAGAGCTCTGTACGGTGGTGGTAA

Scn4a

AGTCCCTGGCAGCCATAGAACCCATAGATGAGTGGGAGGTT

Scn4b

TGGTCCTACAATAACAGCGAAACACTCTCACCTTAGGGTCAGACSdhaGGAACACTCCAAAAACAGACCTCCACCACTGGGTATTGAGTAGAA

Sod1

AACCAGTTGTGTTGTCAGGAC

CCACCATGTTTCTTAGAGTGAGG

Tgfb2

TCGACATGGATCAGTTTATGCG

CCCTGGTACTGTAGATGGA

Tnni1

ATGCCGGAAGTTGAGAGGAAATCCGAGAGGTAACGCACCTT

Tnni2

AGAGTGTGATGCTCCAGATAGCAGCAACGTCGATCTTCGCA

Tnnt1

CCTGTGGTGCCTCCTTTGATTTGCGGTCTTTTAGTGCAATGAG

Tnnt3

GGAACGCCAGAACAGATTGGTGGAGGACAGAGCCTTTTTCTT

Uchl1

AGGGACAGGAAGTTAGCCCTA

AGCTTCTCCGTTTCAGACAGA

Uqcrc2

AAAGTTGCCCCGAAGGTTAAAGAGCATAGTTTTCCAGAGAAGCA

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Table 3. Primary antibodies for immunohistochemistry.
Antigen name

Host

Dilution

Clone nameSource, Cat#, or reference

α-Actin

Mouse

1:200

Alpha Sr-­1

BioLegend, MMS-­467S

αB-­Crystallin

Rabbit1:1000

BioLegend, PRB-­105P

ATF3

Rabbit1:1000Santa Cruz Biotechnology, sc-­188

Cytochrome C

Mouse

1:500

A-­8

Santa Cruz Biotechnology, sc-­13156

Desmin

Mouse

1:100

RD301

Santa Cruz Biotechnology, sc-­23879

DesminRabbit1:1000Novus Biologicals, NBP1-­85549

Dst

Rabbit1:1000Dr. Ronald K Liem, Goryunov et al., 2007

Lamin A/C

Mouse

1:100

E-­1

Santa Cruz Biotechnology, sc-­376248

LC3A/B

Rabbit1:1000D3U4C

Cell Signaling Technology, #12741

Myotilin

Rabbit

1:500

NF-­M

Mouse

1:200

1C8

Dr. Katsuhiko Ono, Horie et al., 2014

p62

Mouse

1:200

1B5.H9

BioLegend, MMS-­5034

p62

Rabbit1:400

ABclonal, A19700

Phospho-­PERK (phospho T982)

Rabbit1:500

Abcam, ab192591

Plectin

Mouse

1:100

10F6

Santa Cruz Biotechnology, sc-­33649

SUMO-­1

Mouse

1:200

D-­11

Santa Cruz Biotechnology, sc-­5308

SUMO-­2/3

Rabbit1:200

ABclonal, A5066

Tom20

Rabbit1:1000Santa Cruz Biotechnology, sc-­11415

Ubiquitin

Rabbit1:1000

Dako; Agilent Technologies Z0458

ProteinTech, 10731-­1-­APovernight. Tissues were then dehydrated using an ascending series of ethanol and xylene washes,
and then embedded in paraffin (P3683; Paraplast Plus; Sigma-­Aldrich, St. Louis, MO). Consecutive
10-μm-­thick paraffin sections were cut on a rotary microtome (HM325; Thermo Fisher Scientific),
mounted on MAS-­coated glass slides (Matsunami Glass, Osaka, Japan), and air-­dried on a hot plate
overnight at 37°C. Paraffin sections were deparaffinized in xylene, rehydrated using a descending
series of ethanol washes, and then rinsed in distilled water. H&E staining and Masson’s trichrome
staining were performed by standard protocols.
For IHC, deparaffinized sections were treated with microwave irradiation in 10 mM citric acid buffer,
pH 6.0 for 5 min, and incubated overnight at 4°C with the primaries listed in Table 3. All primary antibodies were diluted in 0.1 M PBS with 0.01% Triton X-­100 (PBST) containing 0.5% skim milk. Sections
were then incubated in horseradish peroxidase-­conjugated secondary antibody (1:200; MBL, Nagoya,Japan) diluted in PBST containing 0.5% skim milk for 60 min at 37°C. Between each step, sections
were rinsed in PBST for 15 min. After rinsing sections in distilled water, immunoreactivity was visualized in 50 mM Tris buffer (pH 7.4) containing 0.01% diaminobenzidine tetrahydrochloride and 0.01%
hydrogen peroxide at 37°C for 5 min. Sections were then dehydrated through an ethanol-­xylene
solution and placed on coverslips with Bioleit (23-­1002; Okenshoji, Tokyo, Japan). Digital images
were taken with a microscope (BX53; Olympus, Tokyo, Japan) equipped with a digital camera (DP74,
Olympus), and the TIF files were processed with Photoshop software (Adobe, San Jose, USA).
For immunofluorescent staining, sections were incubated in mixtures of Alexa488- or Alexa594-­
conjugated antibodies (1:200; Invitrogen, CA) for 60 min at 37°C. Cell nuclei were counterstained withDAPI (1:2000; Dojindo, Kumamoto, Japan) for 10 min at room temperature. Mounted sections wereair-­dried and coverslipped. Sections were observed and digital images were taken using a confocal
laser scanning microscopy (FV-­1200, Olympus). TIF files were processed with Adobe Photoshop software. Super-­resolution images were recorded on the confocal laser scanning microscopy LSM 980
equipped with Airyscan 2 (Leica, Germany).

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ISH was performed on paraffin sections as described in previous studies (Takebayashi et al.,2000) using following mouse probes: PV, also known as parvalbumin (Pvalb, GenBank accession,
NM_013645, nt 92-885), Nppa, also known as atrial natriuretic peptide (ANP, GenBank accession,
BC089615, nt 124-­529), and Nppb, also known as brain natriuretic peptide (BNP, GenBank accession,
D16497, nt 42-­752, without intron sequence) were used.

Electron microscope analysis
The detailed procedure for TEM analysis was described previously (Shibata et al., 2015). Briefly,
the muscle samples for TEM were primary fixed with 2.5% glutaraldehyde for 24 hr at 4°C. Samples
were washed in 50 mM HEPES buffer (pH 7.4) and were postfixed with 1.0% osmium tetroxide (TAAB
Laboratories, England, UK) for 2 hr at 4°C. Samples were dehydrated with a series of increasing
concentrations of ethanol (two times of 50, 70, 80, 90, 100% EtOH for 20 min each), soaked withacetone (Sigma-­Aldrich) for 0.5 hr, and with n-­butyl glycidyl ether (QY-­1, Okenshoji Co. Ltd., Tokyo,
Japan) two times for 0.5 hr, graded concentration of epoxy resin with QY-­1 for 1 hr, and with 100%
epoxy resin (100 g Epon was composed of 27.0 g MNA, 51.3 g EPOK-­812, 21.9 g DDSA, and 1.1 ml
DMP-­30, all from Okenshoji Co. Ltd.) for 48 hr at 4°C, and were polymerized with pure epoxy resin for
72 hr at 60°C. Resin blocks with tissues were trimmed, semi-­thin sliced at 350-­nm-­thickness stainedwith toluidine blue, and were ultrathin-­sectioned at 80-­nm thickness with ultramicrotome (UC7, Leica)
by diamond knife (Ultra, DiATOME, Switzerland). The ultrathin sections were collected on the copper
grids and were stained with uranyl acetate and lead citrate. The sections were imaged with TEM (JEM-­
1400 Plus, JEOL, Japan) at 100 keV.

Measurements of ECG signal
Under anesthesia with 2–3% isoflurane (Pfizer Inc, NY), ECG signals were recorded. Three needle electrodes were inserted into right and left forelimbs as recording, and right hindlimb as grounding. The
ECG signals were amplified using AC amplifier (band pass: 0.1–1 kHz), and the signals were digitizedwith A/D converter (Power 1401, Cambridge Electronic Design Ltd., Cambridge, UK).

Quantification and statistical analysis
Morphometric analysis was performed at least three sections per mouse. Quantifications of cross-­
sectional area and fibrosis were performed with MetaMorph software (Meta Series Software version
7.10.2; Molecular Devices, San Jose, CA). Unless otherwise noted, sample size (n) is the numberof animals in each genotype. For statistical analysis, Student’s t-­test and ANOVA were carried out.
ANOVA was performed using ANOVA4 on the Web (https://www.hju.ac.jp/~kiriki/anova4/).

In silico screening of DST-b mutations
In silico screening was performed using dbSNP database (Sherry et al., 2001) (https://www.ncbi.nlm.​
nih.gov/snp/?cmd=search). After downloading the results of a search using the keyword "DST" indbSNP in TSV format, we extracted the entries whose "function_class" was "stop_gained".

Compliance with ethical standards and study approval
All animal experiments were performed in accordance with the guidelines of the Ministry of Education, Culture, Sports, Science and Technology of Japan and were approved by the Institutional
Animal Care and Use Committees of Niigata University (permit number: SA00521 and SA00621) andTsukuba University (permit number: 17-­078). Human study was approved by the ethical committeesof the NCNP (permit number: A2019-­123). The human materials used in this study were obtained for
diagnostic purposes. The patients or their parents provided written informed consent for use of thesamples for research.

Acknowledgements
We thank Dr. Seiya Mizuno and Dr. Satoru Takahashi (Laboratory Animal Resource Center, Tsukuba
University) for their contribution to generate Dst-­b mutant mice, Dr. Kenji Sakimura and Dr. Manabu
Abe for the provision of Actb-­iCre-­IRES-­GFP mice, Dr. Kensuke Yamamura for electrophysiologicalapparatus, Dr. Ronald K H Liem for anti-­Dst antibody, Dr. Keisuke Watanabe for Pvalb plasmid, and
Dr. Riuko Ohashi and Mr. Kenji Oyachi (Histopathology Core Facility, Niigata University) for Masson’s

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trichrome staining. We also thank Dr. Norihisa Bizen for helpful discussion, Dr. Tomoko Shindo, Dr.
Li Zhou, Mr. Yuya Imada, Ms. Satoko Yamagiwa, Ms. Yumi Kobayashi, Mr. Seiji Takahashi, Ms. Jitrapa
Pinyomahakul, Ms. Aoba Shiina, and Mr. Osamu Arai for technical assistances. We thank Lisa Kreiner,
PhD, from Edanz (https://www.jp.edanz.com/ac) for editing a draft of this manuscript. This work
was supported by grants from JSPS (15H04667, 18H02592, 21H02652 to HT, 20K15912 to YN), The
Uehara Memorial Foundation (HT), Nagai N-­S Promotion Foundation for Science of Perception (HT),The Nakatomi Foundation (NY), Setsuro Fujii Memorial, Osaka Foundation for Promotion of Fundamental Medical Research (NY), LEGEND Research Grant from BioLegend (NY), and Niigata University
Interdisciplinary Research Grant (NY).

Additional information
Competing interests
Ichizo Nishino: IN held grants from Sanofi, Daiicji Sankyo, Medical & Biological Laboratories, CYTOO,
and Avidity Biosciences and holds a holds a patent on "Differentiating markers for inflammatory
myopathies and methods to differentiate inflammatory myopathies" (Patent number 6531306). IN has
received consultancy fees from Astellas, Sarepta, UCB Japan, Nobelpharma and Dyna Therapeutics,
and has received honoria payments for lectures from Sanofi, Japan Blood Products Organization,
Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, Kyowa Kirin, Biogen, Asahi Kasei Pharma
and Nihon Pharma. IN has participated either on the Advisory Board, Vice President or on the Executive board of Horizon Therapeutics, Astellas, World Muscle Society, Asian Oceanian Myology Center,
Japan Muscle Society, Japanese Society of Neuropathology, Japanese Society of Neurotherapeutics,
and Japanese Society of Neurology. The author has no other competing interests to declare. The
other authors declare that no competing interests exist.
Funding
FunderGrant reference number

AuthorJapan Society for the
Promotion of Science

15H04667

Hirohide Takebayashi

Japan Society for the
Promotion of Science

18H02592

Hirohide Takebayashi

Japan Society for the
Promotion of Science

21H02652

Hirohide Takebayashi

Japan Society for the
Promotion of Science

20K15912

Nozomu Yoshioka

Uehara Memorial
Foundation

Research grant

Hirohide Takebayashi

Nagai N-S Promotion
Foundation for Science ofPerception

Research grant

Hirohide Takebayashi

Nakatomi Foundation

Research grantNozomu Yoshioka

Setsuro Fujii Memorial,
Research grant
Osaka Foundation for
Promotion of Fundamental
Medical Research

Nozomu Yoshioka

BioLegend

LEGEND Research Grant

Nozomu Yoshioka

Niigata University

Interdisciplinary Research
Grant

Nozomu Yoshioka

The funders had no role in study design, data collection and interpretation, or thedecision to submit the work for publication.

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Author contributions
Nozomu Yoshioka, Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation,
Investigation, Visualization, Writing - original draft, Project administration; Masayuki Kurose, Shujiro
Okuda, Yukiko Mori-­Ochiai, Toshihiro Nagai, Ichizo Nishino, Shinsuke Shibata, Investigation; Masato
Yano, Data curation, Software, Investigation, Writing - review and editing; Dang Minh Tran, Validation,
Investigation; Masao Horie, Resources; Hirohide Takebayashi, Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration
Author ORCIDs
Nozomu Yoshioka ‍ ‍http://orcid.org/0000-0002-4425-5457
Shujiro Okuda ‍ ‍http://orcid.org/0000-0002-7704-8104
Hirohide Takebayashi ‍ ‍http://orcid.org/0000-0003-4493-6604
EthicsHuman study was approved by the ethical committees of the NCNP (Permit Number: A2019-­123).
The human materials used in this study were obtained for diagnostic purposes. The patients or theirparents provided written informed consent for use of the samples for research.
All animal experiments were performed in accordance with the guidelines of the Ministry of Education, Culture, Sports, Science and Technology of Japan and were approved by the Institutional Animal
Care and Use Committees of Niigata University (Permit Number: SA00521 and SA00621) and Tsukuba
University (Permit Number: 17-­078).
Decision letter and Author response
Decision letter https://doi.org/10.7554/eLife.78419.sa1
Author response https://doi.org/10.7554/eLife.78419.sa2

Additional files
Supplementary files•  Supplementary file 1. (A) List of upregulated genes of Gene Ontology (GO) biological process,(B) list of upregulated genes of GO cellular component, (C) list of upregulated genes of Kyoto
Encyclopedia of Genes and Genomics (KEGG) pathway, (D) list of downregulated genes ofGO biological process, (E) list of downregulated genes of GO cellular component, (F) list of
downregulated genes of KEGG pathway, and (G) variants of DST gene identified in Japanesepatients with myopathy.
•  Transparent reporting form
Data availability
RNA-­seq data can be accessed from the Gene Expression Omnibus under accession, GSE184101.
The following dataset was generated:
Author(s)Year

Dataset title

Dataset URL

Database and Identifier

Yoshioka N, Yano M,
Takebayashi H

2021

mRNAseq in control(WT)
and Dst-­b mutant mouseheart

https://www.​ncbi.​
nlm.​nih.​gov/​geo/​
query/​acc.​cgi?​acc=​
GSE184101

NCBI Gene Expression
Omnibus, GSE184101

Database and Identifier

The following previously published dataset was used:
Author(s)Year

Dataset title

Dataset URL

Hayakawa-­Yano Y,
Yano M2019

mRNAseq in control (WT)and Nes:Cre_Qk cKO

https://​metadataplus.​ NCBI Gene Expression
biothings.​io/​geo/​
Omnibus, GSE123927
GSE123927

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