Iron Accumulation and Lipid Peroxidation in Cellular Models of Nemaline Myopathies

Academic Edi o : Johan Lindq is
Recei ed: 23 No embe 2024
Re ised: 27 Janua y 2025
Accep ed: 6 Feb ua y 2025
Published: 8 Feb ua y 2025
Ci a ion: López-Cab e a, A.;
Piñe o-Pé ez, R.; Ál a ez-Có doba, M.;
Cille os-Holgado, P.;
Gómez-Fe nández, D.; Reche-López,
D.; Rome o-González, A.;
Rome o-Domínguez, J.M.; de la Ma a,
M.; de Pablos, R.M.; e al. I on
Accumula ion and Lipid Pe oxida ion
in Cellula Models o Nemaline
Myopa hies. In . J. Mol. Sci. 2025,26,
1434. h ps://doi.o g/10.3390/
ijms26041434
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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condi ions o he C ea i e Commons
A ibu ion (CC BY) license
(h ps://c ea i ecommons.o g/
licenses/by/4.0/).
A icle
I on Accumula ion and Lipid Pe oxida ion in Cellula Models o
Nemaline Myopa hies
Alejand a López-Cab e a 1, Rocío Piñe o-Pé ez 1, Mónica Ál a ez-Có doba 1, Paula Cille os-Holgado 1,
Da id Gómez-Fe nández 1, Diana Reche-López 1, Ana Rome o-González 1, José Manuel Rome o-Domínguez 1,
Ma io de la Ma a 2, Rocío M. de Pablos 3,4, Susana González-G ane o 5, José Manuel Ga cía-Ve dugo 5
and José A. Sánchez-Alcáza 1,*
1
Cen o Andaluz de Biología del Desa ollo (CABD-CSIC-Uni e sidad Pablo de Ola ide), 41013 Se illa, Spain;
[email p o ec ed] (A.L.-C.); [email p o ec ed] (R.P.-P.); [email p o ec ed] (M.Á.-C.);
[email p o ec ed] (P.C.-H.); [email p o ec ed] (D.G.-F.); [email p o ec ed] (D.R.-L.);
[email p o ec ed] (A.R.-G.); jm [email p o ec ed] (J.M.R.-D.)
2Depa amen o de Fisiología, Facul ad de Ciencias de la Salud, Uni e sidad de G anada, 51001 Ceu a, Spain;
m delama a@ug .es
3Depa amen o de Bioquímica y Biología Molecula , Facul ad de Fa macia, Uni e sidad de Se illa,
41012 Se illa, Spain; [email p o ec ed]
4
Ins i u o de Biomedicina de Se illa (IBiS), Hospi al Uni e si a io Vi gen del Rocío (HUVR)-CSIC-Uni e sidad
de Se illa, 41013 Se illa, Spain
5Labo a o y o Compa a i e Neu obiology, Ca anilles Ins i u e o Biodi e si y and E olu iona y Biology,
Uni e si y o Valencia and CIBERNED-ISCIII, 46980 Valencia, Spain; susana.gonzalez@u .es (S.G.-G.);
j.manuel.ga cia@u .es (J.M.G.-V.)
*Co espondence: [email p o ec ed]
Abs ac : One o he mos p e alen ypes o congeni al myopa hy is nemaline myopa hy
(NM), which is ecognized by his opa hological examina ion o muscle ibe s o he p es-
ence o “nemaline bodies” ( ods). Mu a ions in he ac in alpha 1 (ACTA1) and nebulin (NEB)
genes esul in he mos p e alen ypes o NM. Muscle weakness and hypo onia a e he
main clinical cha ac e is ics o his disease. Un o una ely, he pa hogene ic mechanisms
a e s ill unknown, and he e is no cu e. In p e ious wo k, we showed ha ac in ilamen
polyme iza ion de ec s in pa ien -de i ed ib oblas s we e associa ed wi h mi ochond ial
dys unc ion. In his manusc ip , we examined he pa hophysiological consequences o
mi ochond ial dys unc ion in pa ien -de i ed ib oblas s. We analyzed i on and lipo uscin
accumula ion and lipid pe oxida ion bo h a he cellula and mi ochond ial le el. We
ound ha ib oblas s de i ed om pa ien s ha bo ing ACTA1 and NEB mu a ions showed
in acellula i on and lipo uscin accumula ion, inc eased lipid pe oxida ion, and al e ed
exp ession le els o p o eins in ol ed in i on me abolism. Fu he mo e, we showed ha
ac in polyme iza ion inhibi ion in con ol cells ecapi ula es he main pa hological al e -
a ions o mu an nemaline cells. Ou esul s indica e ha mi ochond ial dys unc ion is
associa ed wi h i on me abolism dys egula ion, leading o i on/lipo uscin accumula ion
and inc eased lipid pe oxida ion.
Keywo ds: nemaline myopa hy; i on accumula ion; lipid pe oxida ion
1. In oduc ion
Congeni al myopa hy is a gene ically he e ogeneous g oup o he edi a y muscle
diseases ca ego ized acco ding o he his ological cha ac e is ics iden i ied in muscle
biopsy [
1
,
2
]. Speci ically, nemaline myopa hy (NM) is he mos common sub ype o con-
geni al myopa hy, cha ac e ized by he p esence o inclusions in muscle ibe s known
In . J. Mol. Sci. 2025,26, 1434 h ps://doi.o g/10.3390/ijms26041434
In . J. Mol. Sci. 2025,26, 1434 2 o 26
as nemaline bodies. NM, a a e gene ic skele al muscle disease a ec ing 1 in 50,000 li e
bi hs [
3
,
4
], was i s desc ibed in 1963 by Conen e al. [
5
] and Shy e al. [
6
]. NM mani es s
clinical ea u es such as muscle weakness and hypo onia, dec eased espi a o y unc ion,
and impai ed mo o de elopmen .
Cu en ly, 14 genes ha e been iden i ied as esponsible o NM [
2
]. Mos o he
genes code o componen s o he hin ilamen o he sa come e o addi ional s uc u al
o egula o y componen s o he sa come e. The genes mos associa ed wi h NM a e
ACTA1 and NEB genes, which code o ac in alpha 1 (ACTA1) and nebulin (NEB) p o eins,
espec i ely [
7
]. I is es ima ed ha o e 50% o NM cases a e caused by mu a ions in he
NEB gene, while 15–20% can be a ibu ed o mu a ions in ACTA1 gene. Due o i s la ge
molecula weigh (600–800 kDa), NEB is o en e e ed o as he gian ac in-binding p o ein.
I is c ucial o main aining and con olling he leng h o he ac in ilamen . Ac in iso o m
ACTA1 is p edominan ly loca ed in he hin ilamen s o skele al muscles and is necessa y
o muscula con ac ion oge he wi h myosin [2,8].
The diagnosis o NM is mainly made by examining his opa hological indings in
muscle biopsies [
9
]. His opa hological al e a ions in NM a e cha ac e ized by he p esence
o nemaline bodies, which a e elec on-dense p o ein agg ega es ha a e dis ibu ed in he
o m o ods, which can be isualized wi hin he muscle ibe s o muscle biopsies om
pa ien s [
9
–
11
]. Diso ganized sa come e Z-disk p o eins, including ac in, opomyosin,
myo ilin,
γ
-phylamine, co ilin-2, e onin, nebulin, and ac in-like hin ilamen s, cons i u e
he p o ein agg ega es o hese nemaline bodies [
7
,
8
]. In addi ion, muscle enzyme le els
such as c ea inine kinase a e analyzed, which may show no mal o sligh ly ele a ed
alues. Howe e , he o igin o hese nemaline bodies is unce ain, al hough, because o he
accumula ed p o eins, hey a e belie ed o be de i ed om he Z-line o he sa come e [
12
].
Consequen ly, his opa hological indings ob ained wi h elec on mic oscopy a e he
main e idence o he diagnosis o he disease. In addi ion o he nemaline bodies, o he
peculia i ies can be ound in muscle biopsies, such as changes in he p opo ion and size
o muscle ibe s, since pa ien s wi h NM p esen a p edominance o ype I (slow wi ch)
muscle ibe s [7,10].
NM disease has been ela ed o mi ochond ia dys unc ion, pa icula ly complex I
dys unc ion o de iciency [
13
,
14
]. I has been desc ibed ha ACTA1 mu an s p esen a
“down egula ion” o mi ochond ial complex I due o he dis up ion o he in eg i y o he
hin ilamen s, impai ed muscle unc ion, and a educed p oduc ion o adenosine iphos-
pha e (ATP) [
14
]. Fu he mo e, ac in ilamen s a e c ucial o he co ec mi ochond ial
mo phology and unc ion [
15
–
17
]. The e o e, ac in ilamen s mainly con ol mi ochond ial
dynamics, me abolism, a icking, and au ophagy [18–21].
Thecloseconnec ionsbe weenmi ochond iadys unc ionandac in
cy oskele on—including
ac in ilamen depolyme iza ion, a de iciency o mi ochond ial bioene ge ics, and a down-
egula ion o he exp ession le els o mi ochond ial p o eins—we e desc ibed in a p e ious
in es iga ion by ou g oup [16].
Ele a ed in acellula i on le els a e associa ed wi h a wide ange o pa hological
condi ions [
22
,
23
]. I on is an essen ial elemen o se e al cellula p ocesses, se ing as a co-
ac o o enzymes in ol ed in oxygen me abolism (such as oxidases, pe oxidases, ca alases,
and hyd oxylases), elec on anspo (cy och omes), and oxygen deli e y (hemoglobin).
I also plays a i al ole in neu onal unc ion and he immune sys em [
24
]. Wi hin cells,
i on is p ima ily u ilized in he mi ochond ia, whe e i con ibu es o he biosyn hesis
o heme and i on–sul u clus e s [
22
]. I on o e load can pa icipa e in he gene a ion o
eac i e oxygen species (ROS) h ough Fen on eac ion, causing oxida i e s ess, dec easing
espi a o y unc ion, and damaging mi ochond ial DNA and cellula componen s such as
lipids [25–27].
In . J. Mol. Sci. 2025,26, 1434 3 o 26
The ela ionship be ween mi ochond ial dys unc ion and i on accumula ion was
ecen ly examined by ou esea ch g oup in se e al a e neu ogene ic diso de s unde
he classi ica ion o neu odegene a ion wi h b ain i on accumula ion (NBIA), such as
β
-p opelle p o ein-associa ed neu odegene a ion (BPAN) [
25
] and pan o hena e kinase-
associa ed neu odegene a ion (PKAN) [
28
]. Mo eo e , his connec ion has been also
demons a ed in a e mi ochond ial diseases induced by mu a ion in lipoyl ans e ase 1
(LIPT1) gene [29].
This s udy aimed o analyze in acellula i on con en and lipid pe oxida ion in
ib oblas s de i ed om wo pa ien s wi h ACTA1 and wo pa ien s wi h NEB pa hogenic
a ian s. Addi ionally, we examined he e ec o ac in depolyme izing agen s such as
ROCK (Rho-associa ed p o ein kinase) inhibi o s and Cy ochalasin D on con ol ib oblas s
o con i m whe he ac in depolyme iza ion mimics he physiopa hology o NM pa ien -
de i ed ib oblas s by analyzing in acellula i on con en , lipid pe oxida ion, and he
exp ession le els o p o eins in ol ed in i on me abolism, among o he s.
2. Resul s
2.1. NM Pa ien -De i ed Fib oblas s Show I on Accumula ion
As mi ochond ial dys unc ion can be associa ed wi h impai ed i on me abolism [
30
], we
i s examined in acellula i on accumula ion by P ussian Blue s aining in bo h con ol and
NM ib oblas s P1, P2, P3, and P4. The esul s e ealed a signi ican inc ease in i on s aining
in NM mu an cells compa ed o con ols. To se e as a nega i e con ol and alida e he
speci ici y o P ussian Blue s aining o i on, P1 ib oblas s we e ea ed wi h de e ip one
(De ), an i on-chela ing d ug (Figu es 1A,B and S1). To con i m he abno mal cellula i on
con en in NM ib oblas s, we nex de e mined in acellula i on le els by induc i ely coupled
mass spec ome y (ICP-MS). Mu an NM ib oblas s P1, P2, P3, and P4 showed a signi ican
inc ease in o al i on con en wi h espec o con ol cells (Figu e 1C).
In . J. Mol. Sci. 2025, 26 3
dec easing espi a o y unc ion, and damaging mi ochond ial DNA and cellula compo-
nen s such as lipids [25–27].
The ela ionship be ween mi ochond ial dys unc ion and i on accumula ion was e-
cen ly examined by ou esea ch g oup in se e al a e neu ogene ic diso de s unde he
classi ica ion o neu odegene a ion wi h b ain i on accumula ion (NBIA), such as β-p o-
pelle p o ein-associa ed neu odegene a ion (BPAN) [25] and pan o hena e kinase-asso-
cia ed neu odegene a ion (PKAN) [28]. Mo eo e , his connec ion has been also demon-
s a ed in a e mi ochond ial diseases induced by mu a ion in lipoyl ans e ase 1 (LIPT1)
gene [29].
This s udy aimed o analyze in acellula i on con en and lipid pe oxida ion in i-
b oblas s de i ed om wo pa ien s wi h ACTA1 and wo pa ien s wi h NEB pa hogenic
a ian s. Addi ionally, we examined he effec o ac in depolyme izing agen s such as
ROCK (Rho-associa ed p o ein kinase) inhibi o s and Cy ochalasin D on con ol ib o-
blas s o con i m whe he ac in depolyme iza ion mimics he physiopa hology o NM pa-
ien -de i ed ib oblas s by analyzing in acellula i on con en , lipid pe oxida ion, and
he exp ession le els o p o eins in ol ed in i on me abolism, among o he s.
2. Resul s
2.1. NM Pa ien -De i ed Fib oblas s Show I on Accumula ion
As mi ochond ial dys unc ion can be associa ed wi h impai ed i on me abolism [30],
we i s examined in acellula i on accumula ion by P ussian Blue s aining in bo h con-
ol and NM ib oblas s P1, P2, P3, and P4. The esul s e ealed a signi ican inc ease in
i on s aining in NM mu an cells compa ed o con ols. To se e as a nega i e con ol and
alida e he speci ici y o P ussian Blue s aining o i on, P1 ib oblas s we e ea ed wi h
de e ip one (De ), an i on-chela ing d ug (Figu es 1A,B and S1). To con i m he abno mal
cellula i on con en in NM ib oblas s, we nex de e mined in acellula i on le els by
induc i ely coupled mass spec ome y (ICP-MS). Mu an NM ib oblas s P1, P2, P3, and
P4 showed a signi ican inc ease in o al i on con en wi h espec o con ol cells (Figu e
1C).
Figu e 1. I on accumula ion in NM cells. (A) P ussian Blue s aining o con ol cells (C1) and NM
ib oblas s (P1, P2, P3, and P4) was ca ied ou as desc ibed in Sec ion 4, Ma e ials and Me hods. P1
Figu e 1. I on accumula ion in NM cells. (A) P ussian Blue s aining o con ol cells (C1) and NM
ib oblas s (P1, P2, P3, and P4) was ca ied ou as desc ibed in Sec ion 4, Ma e ials and Me hods.
P1 ib oblas s we e exposed o 100
µ
M de e ip one (De ), an i on chela ing agen , o 24 h as a
nega i e con ol. Images we e made in b igh ield by an Axio Ve A1 in e ed op ical mic oscope
(Zeiss, Obe kochen, Ge many) wi h a 40
×
objec i e and we e analyzed using Fiji-ImageJ so wa e
In . J. Mol. Sci. 2025,26, 1434 4 o 26
( e sion 2.9.0/1.53 ) (Na ional Ins i u e o Heal h, Be hesda, MD, USA). Scale ba = 20
µ
m. (B) Quan-
i ica ion o P ussian Blue s aining images was pe o med by he Image J so wa e ( e sion 1.54 ).
(C) I on con en de e mined by ICP-MS in NM ib oblas . ICP-MS was used o measu e he o al
i on con en o con ol and NM pa ien s as de ailed in he Ma e ial and Me hods. Da a ep esen he
mean ±SD
o h ee sepa a e expe imen s. * p< 0.05, ** p< 0.01, *** p< 0.001 be ween NM ib oblas s
and con ols;
aa
p< 0.01 be ween un ea ed and ea ed NM cells be ween he p esence and he
absence o de e ip one (De ). A.U., a bi a y uni s.
2.2. NM Pa ien -De i ed Fib oblas s P esen Lipo uscin-like Agg ega e Accumula ion
As abno mal accumula ion o lipo uscin has been epo ed in se e al cell ypes p esen -
ing i on o e load [
28
,
31
] and lipo uscin accumula ion can esul om lipid pe oxida ion,
a p ocess s imula ed by i on [
24
], we nex examined he p esence o lipo uscin by Sudan
Black s aining and au o luo escence analysis in con ol and NM ib oblas s. Compa ed
o con ol cells, NM mu an cells displayed signi ican ly highe le els o Sudan Black
s aining (Figu es 2A,B and S2). In addi ion, NM ib oblas s p esen ed highe le els o
au o luo escence (Figu e 3A,B), indica ing inc eased lipo uscin accumula ion.
In . J. Mol. Sci. 2025, 26 4
ib oblas s we e exposed o 100 µM de e ip one (De ), an i on chela ing agen , o 24 h as a nega i e
con ol. Images we e made in b igh ield by an Axio Ve A1 in e ed op ical mic oscope (Zeiss,
Obe kochen, Ge many) wi h a 40× objec i e and we e analyzed using Fiji-ImageJ so wa e ( e sion
2.9.0/1.53 ) (Na ional Ins i u e o Heal h, Be hesda, MD, USA). Scale ba = 20 µm. (B) Quan i ica ion
o P ussian Blue s aining images was pe o med by he Image J so wa e ( e sion 1.54 ). (C) I on
con en de e mined by ICP-MS in NM ib oblas . ICP-MS was used o measu e he o al i on con en
o con ol and NM pa ien s as de ailed in he Ma e ial and Me hods. Da a ep esen he mean ± SD
o h ee sepa a e expe imen s. * p < 0.05, ** p < 0.01, *** p < 0.001 be ween NM ib oblas s and con ols;
aa
p < 0.01 be ween un ea ed and ea ed NM cells be ween he p esence and he absence o de e -
ip one (De ). A.U., a bi a y uni s.
2.2. NM Pa ien -De i ed Fib oblas s P esen Lipo uscin-like Agg ega e Accumula ion
As abno mal accumula ion o lipo uscin has been epo ed in se e al cell ypes p e-
sen ing i on o e load [28,31] and lipo uscin accumula ion can esul om lipid pe oxida-
ion, a p ocess s imula ed by i on [24], we nex examined he p esence o lipo uscin by
Sudan Black s aining and au o luo escence analysis in con ol and NM ib oblas s. Com-
pa ed o con ol cells, NM mu an cells displayed signi ican ly highe le els o Sudan
Black s aining (Figu es 2A,B and S2). In addi ion, NM ib oblas s p esen ed highe le els
o au o luo escence (Figu e 3A,B), indica ing inc eased lipo uscin accumula ion.
Figu e 2. Lipo uscin accumula ion in NM ib oblas s. (A) Sudan black s aining o con ol cells (C1)
and NM ib oblas s (P1, P2, P3, and P4) was pe o med as desc ibed in Sec ion 4, Ma e ials and
Me hods. P1 ib oblas s we e exposed o 100 µM de e ip one (De ), an i on chela ing agen , o 24
h, as a nega i e con ol. Images we e made in b igh ield by an Axio Ve A1 in e ed op ical mi-
c oscope (Zeiss, Obe kochen, Ge many) wi h a 40× objec i e and we e analyzed using Fiji-ImageJ
so wa e ( e sion 2.9.0/1.53 ) (Na ional Ins i u e o Heal h, Be hesda, MD, USA). Scale ba = 20 µm.
(B) Quan i ica ion o Sudan Black in con ol and NM ib oblas s was pe o med by ImageJ so wa e
( e sion 1.54 ). Da a ep esen he mean ± SD o h ee sepa a e expe imen s. ** p < 0.01, *** p < 0.001
Figu e 2. Lipo uscin accumula ion in NM ib oblas s. (A) Sudan black s aining o con ol cells (C1)
and NM ib oblas s (P1, P2, P3, and P4) was pe o med as desc ibed in Sec ion 4, Ma e ials and
Me hods. P1 ib oblas s we e exposed o 100
µ
M de e ip one (De ), an i on chela ing agen , o
24 h, as a nega i e con ol. Images we e made in b igh ield by an Axio Ve A1 in e ed op ical
mic oscope (Zeiss, Obe kochen, Ge many) wi h a 40
×
objec i e and we e analyzed using Fiji-ImageJ
so wa e ( e sion 2.9.0/1.53 ) (Na ional Ins i u e o Heal h, Be hesda, MD, USA). Scale ba = 20
µ
m.
(B) Quan i ica ion o Sudan Black in con ol and NM ib oblas s was pe o med by ImageJ so wa e
( e sion 1.54 ). Da a ep esen he mean
±
SD o h ee sepa a e expe imen s. ** p< 0.01, *** p< 0.001
be ween NM ib oblas s and con ols;
aa
p< 0.01 be ween un ea ed and ea ed NM cells be ween
he p esence and he absence o de e ip one (De ). A.U., a bi a y uni s.
In . J. Mol. Sci. 2025,26, 1434 5 o 26
In . J. Mol. Sci. 2025, 26 5
be ween NM ib oblas s and con ols;
aa
p < 0.01 be ween un ea ed and ea ed NM cells be ween
he p esence and he absence o de e ip one (De ). A.U., a bi a y uni s.
Au o luo escence and Sudan Black s aining in pa ien ib oblas s we e signi ican ly
educed a e ea men wi h 100 µM de e ip one (De ) [28], sugges ing ha i on is con-
ibu ing o he accumula ion o he au o luo escence and Sudan Black-posi i e
lipo uscin-like ma e ial (Figu es 2A,B, 3A,B and S2). Fu he mo e, o con i m he
lipo uscin-like ea u es o he agg ega es, he luo escence spec al cha ac e is ics o
lipo uscin g anules in NM cells we e measu ed by con ocal lase scanning mic oscopy.
Unde exci a ion a 405 nm, lipo uscin g anules showed an emission peak a 520–540 nm
(Figu e 3C). These esul s a e consis en wi h he cha ac e is ics o lipo uscin g anules ob-
se ed in e inal pigmen epi helial cells [32] and in pan o hena e kinase-associa ed neu-
odegene a ion (PKAN) cellula models [28].
Figu e 3. Lipo uscin-like agg ega es in NM ib oblas s. (A) Rep esen a i e au o luo escence and
b igh ield (BF) images o con ol (C1) and NM ib oblas s (P1, P2, P3, and P4). Con ol cell and P1
we e ea ed wi h 100 µM de e ip one (De ) o 24 h as a nega i e con ol. Scale ba = 20 µm. (B)
Figu e 3. Lipo uscin-like agg ega es in NM ib oblas s. (A) Rep esen a i e au o luo escence and
b igh ield (BF) images o con ol (C1) and NM ib oblas s (P1, P2, P3, and P4). Con ol cell and
P1 we e ea ed wi h 100
µ
M de e ip one (De ) o 24 h as a nega i e con ol. Scale ba = 20
µ
m.
(B) Quan i ica ion o cell au o luo escence was de e mined by image analysis using he Fiji so wa e
( e sion 2.9.0/1.53 ). (C) The au o luo escence spec a o lipo uscin g anules we e measu ed by
con ocal lase scanning mic oscopy (Nikon A1R, Shinagawa, Tokyo, Japan) in con ol, NM ib oblas s
(P1, P2, P3, and P4), and nega i e con ols (con ol and P1 ea ed wi h 100
µ
M de e ip one (De )).
Exci a ion lase sou ce: 405 nm. The emission spec a we e eco ded in 20 la ge lipo uscin g anules in
20 cells. Resul s a e exp essed as mean
±
SD o au o luo escence in ensi y. ** p< 0.01, *** p< 0.001
be ween NM ib oblas s and con ols;
aa
p< 0.01 be ween he p esence and he absence o DEF in
con ols and Pa ien 1 cells. A.U., a bi a y uni s.
Au o luo escence and Sudan Black s aining in pa ien ib oblas s we e signi ican ly
educed a e ea men wi h 100
µ
M de e ip one (De ) [
28
], sugges ing ha i on is con ibu -
ing o he accumula ion o he au o luo escence and Sudan Black-posi i e lipo uscin-like
ma e ial (Figu e 2A,B, Figu e 3A,B and Figu e S2). Fu he mo e, o con i m he lipo uscin-
like ea u es o he agg ega es, he luo escence spec al cha ac e is ics o lipo uscin g an-

In . J. Mol. Sci. 2025,26, 1434 6 o 26
ules in NM cells we e measu ed by con ocal lase scanning mic oscopy. Unde exci a ion a
405 nm, lipo uscin g anules showed an emission peak a 520–540 nm (Figu e 3C). These
esul s a e consis en wi h he cha ac e is ics o lipo uscin g anules obse ed in e inal
pigmen epi helial cells [
32
] and in pan o hena e kinase-associa ed neu odegene a ion
(PKAN) cellula models [28].
The p esence o ele a ed in acellula lipo uscin-like g anules in NM ib oblas s was
alida ed using TEM analysis (Figu e 4A,B). In addi ion, he examina ion o mi ochond ial
al e a ions in NM cells showed mi ochond ial acuoliza ion and condensa ion o damaged
a eas o mi ochond ial memb anes, which e en ually we e ex uded o he cy osol, o ming
dense lipo uscin-like g anules (Figu e 4C).
In . J. Mol. Sci. 2025, 26 6
Quan i ica ion o cell au o luo escence was de e mined by image analysis using he Fiji so wa e
( e sion 2.9.0/1.53 ). (C) The au o luo escence spec a o lipo uscin g anules we e measu ed by con-
ocal lase scanning mic oscopy (Nikon A1R, Shinagawa, Tokyo, Japan) in con ol, NM ib oblas s
(P1, P2, P3, and P4), and nega i e con ols (con ol and P1 ea ed wi h 100 µM de e ip one (De )).
Exci a ion lase sou ce: 405 nm. The emission spec a we e eco ded in 20 la ge lipo uscin g anules
in 20 cells. Resul s a e exp essed as mean ± SD o au o luo escence in ensi y. ** p < 0.01, *** p < 0.001
be ween NM ib oblas s and con ols;
aa
p < 0.01 be ween he p esence and he absence o DEF in
con ols and Pa ien 1 cells. A.U., a bi a y uni s.
The p esence o ele a ed in acellula lipo uscin-like g anules in NM ib oblas s was
alida ed using TEM analysis (Figu e 4A,B). In addi ion, he examina ion o mi ochon-
d ial al e a ions in NM cells showed mi ochond ial acuoliza ion and condensa ion o
damaged a eas o mi ochond ial memb anes, which e en ually we e ex uded o he cy-
osol, o ming dense lipo uscin-like g anules (Figu e 4C).
Figu e 4. Elec on mic oscopy images o con ol and P2 (ACTA1) and P3 (NEB) ib oblas s. (A) Rep-
esen a i e elec on mic oscopy images o con ol (C1) and NM ib oblas s (P2 and P3). Red a ows
we e used o highligh he lipo uscin-like g anules. (B) Quan i ica ion o lipo uscin-like agg ega es.
(C) P2 cells showed mi ochond ial acuoliza ion, and condensa ion/la e aliza ion o mi ochond ial
memb anes (o ange a ow). Scale ba = 2 µm. Da a ep esen he mean ± SD o he examina ion o
50 cells pe condi ion. *** p < 0.001 be ween NM cells and con ols.
Figu e 4. Elec on mic oscopy images o con ol and P2 (ACTA1) and P3 (NEB) ib oblas s. (A) Rep e-
sen a i e elec on mic oscopy images o con ol (C1) and NM ib oblas s (P2 and P3). Red a ows
we e used o highligh he lipo uscin-like g anules. (B) Quan i ica ion o lipo uscin-like agg ega es.
(C) P2 cells showed mi ochond ial acuoliza ion, and condensa ion/la e aliza ion o mi ochond ial
memb anes (o ange a ow). Scale ba = 2
µ
m. Da a ep esen he mean
±
SD o he examina ion o
50 cells pe condi ion. *** p< 0.001 be ween NM cells and con ols.
2.3. Mi ochond ial I on Accumula ion in NM Fib oblas s
Nex , we examined he le els o mi ochond ial Fe
2+
by Mi oFe oG een s aining. This
assay was ca ied ou in con ol, mu an s’ ib oblas s P1 and P2 ha bo ing ACTA1 mu a ions,
and P3 and P4 ha bo ing NEB mu a ions (Figu e 5A). Mu an NM cells p esen ed ele a ed
le els o mi ochond ial Fe
2+
. Cellula models o pan o hena e kinase-associa ed neu ode-
gene a ion (PKAN) we e used as a posi i e con ol [
28
]. Addi ionally, PKAN-de i ed
In . J. Mol. Sci. 2025,26, 1434 7 o 26
ib oblas s we e ea ed wi h 100
µ
M de e ip one as a nega i e con ol. Colocaliza ion
analysis wi h Mi oT acke
TM
Deep Red FM, a mi ochond ia ma ke , demons a ed ha he
signal colocalizes wi h Mi oFe oG een s aining (Pea son co ela ion coe icien > 0.75).
In . J. Mol. Sci. 2025, 26 7
2.3. Mi ochond ial I on Accumula ion in NM Fib oblas s
Nex , we examined he le els o mi ochond ial Fe
2+
by Mi oFe oG een s aining. This
assay was ca ied ou in con ol, mu an s’ ib oblas s P1 and P2 ha bo ing ACTA1 mu a-
ions, and P3 and P4 ha bo ing NEB mu a ions (Figu e 5A). Mu an NM cells p esen ed
ele a ed le els o mi ochond ial Fe
2+
. Cellula models o pan o hena e kinase-associa ed
neu odegene a ion (PKAN) we e used as a posi i e con ol [28]. Addi ionally, PKAN-de-
i ed ib oblas s we e ea ed wi h 100 µM de e ip one as a nega i e con ol. Colocaliza-
ion analysis wi h Mi oT acke
TM
Deep Red FM, a mi ochond ia ma ke , demons a ed
ha he signal colocalizes wi h Mi oFe oG een s aining (Pea son co ela ion coefficien >
0.75).
Figu e 5. Mi ochond ial e ous i on (Fe
2+
) in con ol and NM cells. (A) Le els o mi ochond ial
e ous i on in con ol (C1) and NM ib oblas s (P1, P2, P3, and P4) we e assayed by Mi oFe oG een
s aining as desc ibed in Ma e ials and Me hods. Cellula models o pan o hena e kinase-associa ed
neu odegene a ion (PKAN) we e used as a posi i e con ol. PKAN cells we e exposed o 100 µM
de e ip one (De ), an i on chela ing agen , o 24 h as a nega i e con ol. (B) Mi oFe oG een s ain-
ing quan i ica ion was conduc ed by using Fiji so wa e ( e sion 2.9.0/1.53 ). Cells we e incuba ed
Figu e 5. Mi ochond ial e ous i on (Fe
2+
) in con ol and NM cells. (A) Le els o mi ochond ial
e ous i on in con ol (C1) and NM ib oblas s (P1, P2, P3, and P4) we e assayed by Mi oFe oG een
s aining as desc ibed in Ma e ials and Me hods. Cellula models o pan o hena e kinase-associa ed
neu odegene a ion (PKAN) we e used as a posi i e con ol. PKAN cells we e exposed o 100
µ
M
de e ip one (De ), an i on chela ing agen , o 24 h as a nega i e con ol. (B) Mi oFe oG een s aining
quan i ica ion was conduc ed by using Fiji so wa e ( e sion 2.9.0/1.53 ). Cells we e incuba ed wi h
Mi oT acke
TM
Deep Red FM o demons a e ha Mi oFe oG een signal colocalizes wi h a mi ochon-
d ial ma ke . The colocaliza ion o bo h ma ke s was assessed by he Del aVision so wa e ( e sion
so WoRx 7.0; Applied P ecision; Issaquah, Washing on (WA), Uni ed s a es (USA)) calcula ing he
Pea son co ela ion coe icien . Pea son co ela ion coe icien was >0.75 in NM ib oblas s and
con ol. Scale ba = 20
µ
m. *** p< 0.001 be ween con ol and NM ib oblas s;
aaa
p< 0.001 be ween he
p esence and he absence o DEF be ween ea ed and un ea ed PKAN pa ien cells. Da a ep esen
he mean ±SD o ou sepa a e expe imen s. A.U., a bi a y uni s.
In . J. Mol. Sci. 2025,26, 1434 8 o 26
2.4. Lipid Pe oxida ion in NM Fib oblas s
As i on accumula ion in NM cells can induce he oxida ion o in acellula lipids by
Fen on eac ion [
24
], we nex add essed cellula and mi ochond ial lipid pe oxida ion by
BODIPY
®
and MITOPeDPP s aining, espec i ely. Cellula lipid pe oxida ion was no ably
inc eased in mu an NM cells (Figu e 6A,B). In addi ion, mu an NM cells showed inc eased
le els o mi ochond ial lipid pe oxida ion (Figu e 7A,B). Con ol ib oblas s we e ea ed
wi h 500
µ
M Lupe ox (Te -bu yl hyd ope oxide) as a posi i e con ol o lipid pe oxida ion.
In . J. Mol. Sci. 2025, 26 8
wi h Mi oT acke
TM
Deep Red FM o demons a e ha Mi oFe oG een signal colocalizes wi h a
mi ochond ial ma ke . The colocaliza ion o bo h ma ke s was assessed by he Del aVision so wa e
( e sion so WoRx 7.0; Applied P ecision; Issaquah, Washing on (WA), Uni ed s a es (USA)) calcu-
la ing he Pea son co ela ion coefficien . Pea son co ela ion coefficien was >0.75 in NM ib oblas s
and con ol. Scale ba = 20 µm. *** p < 0.001 be ween con ol and NM ib oblas s;
aaa
p < 0.001 be ween
he p esence and he absence o DEF be ween ea ed and un ea ed PKAN pa ien cells. Da a ep-
esen he mean ± SD o ou sepa a e expe imen s. A.U., a bi a y uni s.
2.4. Lipid Pe oxida ion in NM Fib oblas s
As i on accumula ion in NM cells can induce he oxida ion o in acellula lipids by
Fen on eac ion [24], we nex add essed cellula and mi ochond ial lipid pe oxida ion by
BODIPY
®
and MITOPeDPP s aining, espec i ely. Cellula lipid pe oxida ion was no ably
inc eased in mu an NM cells (Figu e 6A,B). In addi ion, mu an NM cells showed in-
c eased le els o mi ochond ial lipid pe oxida ion (Figu e 7A,B). Con ol ib oblas s we e
ea ed wi h 500 µM Lupe ox (Te -bu yl hyd ope oxide) as a posi i e con ol o lipid pe-
oxida ion.
Figu e 6. Cellula lipid pe oxida ion in con ol and NM cells. (A) Le els o cellula lipid pe oxida-
ion in con ol (C1) and NM ib oblas s (P1, P2, P3, and P4) we e measu ed using BODIPY
®
s aining
as de ailed in Sec ion 4, Ma e ial and Me hods. (B) BODIPY
®
s aining quan i ica ion was pe o med
by using he Fiji so wa e ( e sion 2.9.0/1.53 ). Images we e made in by an Axio Ve A1 in e ed
op ical mic oscope (Zeiss, Obe kochen, Ge many) wi h a 40× objec i e. Con ol ib oblas s we e
ea ed wi h 500 µM Lupe ox (Te -bu yl hyd ope oxide) o 15 min as posi i e con ol o lipid
Figu e 6. Cellula lipid pe oxida ion in con ol and NM cells. (A) Le els o cellula lipid pe oxida ion
in con ol (C1) and NM ib oblas s (P1, P2, P3, and P4) we e measu ed using BODIPY
®
s aining as
de ailed in Sec ion 4, Ma e ial and Me hods. (B) BODIPY
®
s aining quan i ica ion was pe o med by
using he Fiji so wa e ( e sion 2.9.0/1.53 ). Images we e made in by an Axio Ve A1 in e ed op ical
mic oscope (Zeiss, Obe kochen, Ge many) wi h a 40
×
objec i e. Con ol ib oblas s we e ea ed wi h
500
µ
M Lupe ox (Te -bu yl hyd ope oxide) o 15 min as posi i e con ol o lipid pe oxida ion. Scale
ba = 20
µ
m. *** p< 0.001 be ween con ol and NM ib oblas s;
aaa
p< 0.001 be ween he p esence and
he absence o Lupe ox be ween ea ed and un ea ed Con ol cells. Da a ep esen he mean ±SD
o ou sepa a e expe imen s. A.U., a bi a y uni s.
In . J. Mol. Sci. 2025,26, 1434 9 o 26
In . J. Mol. Sci. 2025, 26 9
pe oxida ion. Scale ba = 20 µm. *** p < 0.001 be ween con ol and NM ib oblas s;
aaa
p < 0.001 be-
ween he p esence and he absence o Lupe ox be ween ea ed and un ea ed Con ol cells. Da a
ep esen he mean ± SD o ou sepa a e expe imen s. A.U., a bi a y uni s.
Figu e 7. Mi ochond ia lipid pe oxida ion in con ol and NM cells. (A) Le els o mi ochond ial lipid
pe oxida ion in con ol (C1) and NM ib oblas s (P1, P2, P3, and P4) we e assayed by Mi oPeDPP
s aining as de ailed in Sec ion 4, Ma e ial and Me hods. (B) Mi oPeDPP s aining quan i ica ion was
pe o med by using he Fiji so wa e ( e sion 2.9.0/1.53 ). Cells we e incuba ed wi h Mi oT acke
TM
Deep Red FM o demons a e ha Mi oPeDPP signal colocalizes wi h a mi ochond ial ma ke . Con-
ol ib oblas s we e ea ed wi h 500 µM Lupe ox (Te -bu yl hyd ope oxide) o 15 min as posi i e
con ol o lipid pe oxida ion. Colocaliza ion o bo h ma ke s Mi oPeDPP and Mi oT acke
TM
was
assessed by he Del aVision so wa e ( e sion so WoRx 7.0Applied P ecision; Issaquah,Washing-
on (WA), Uni ed s a es (USA)). Scale ba = 20 µm. ** p < 0.01, *** p < 0.001 be ween con ol and NM
ib oblas s;
aaa
p < 0.001 be ween he p esence and he absence o Lupe ox be ween ea ed and
Figu e 7. Mi ochond ia lipid pe oxida ion in con ol and NM cells. (A) Le els o mi ochond ial lipid
pe oxida ion in con ol (C1) and NM ib oblas s (P1, P2, P3, and P4) we e assayed by Mi oPeDPP
s aining as de ailed in Sec ion 4, Ma e ial and Me hods. (B) Mi oPeDPP s aining quan i ica ion was
pe o med by using he Fiji so wa e ( e sion 2.9.0/1.53 ). Cells we e incuba ed wi h Mi oT acke
TM
Deep Red FM o demons a e ha Mi oPeDPP signal colocalizes wi h a mi ochond ial ma ke . Con ol
ib oblas s we e ea ed wi h 500
µ
M Lupe ox (Te -bu yl hyd ope oxide) o 15 min as posi i e con ol
o lipid pe oxida ion. Colocaliza ion o bo h ma ke s Mi oPeDPP and Mi oT acke
TM
was assessed
by he Del aVision so wa e ( e sion so WoRx 7.0Applied P ecision; Issaquah, Washing on (WA),
Uni ed s a es (USA)). Scale ba = 20
µ
m. ** p< 0.01, *** p< 0.001 be ween con ol and NM ib oblas s;
aaa
p< 0.001 be ween he p esence and he absence o Lupe ox be ween ea ed and un ea ed con ol
cells. Da a ep esen he mean ±SD o ou sepa a e expe imen s. A.U., a bi a y uni s.
2.5. NM Pa ien -De i ed Fib oblas s Also P esen ed Al e ed Exp ession Le els o P o eins In ol ed
in I on Me abolism
We nex assessed i on me abolism dys egula ion by examining he exp ession le els
o key p o eins in ol ed in i on a icking, s o age, and egula ion, such as TFR1, DMT1,
IRP1, Fe i in, Mi o e i in, Mi o e in2, FXN, ISCU, M -ACP, and PANK2. NM mu an
ib oblas s showed inc eased exp ession le els o TFR1, DMT1, IRP1, and Fe i in espec o
In . J. Mol. Sci. 2025,26, 1434 16 o 26
2.10. Inc eased Lipid Pe oxida ion in Con ol Fib oblas s T ea ed wi h Y27632o Cy ochalasin D
Nex , we analyzed he e ec s o Y27632 o Cy ochalasin D ea men s on cellula
lipid pe oxida ion. Compa ing ea ed and un ea ed con ol ib oblas s, we obse ed ha
con ols ib oblas s ea ed wi h bo h inhibi o s showed ele a ed cellula lipid pe oxida ion
(Figu e 15A,B). Con ol ib oblas s we e also ea ed wi h 500
µ
M Lupe ox (Te -bu yl
hyd ope oxide) as a posi i e con ol o lipid pe oxida ion.
In . J. Mol. Sci. 2025, 26 16
Cy ochalasin D o 3 h. Images we e made in b igh ield by an Axio Ve A1 in e ed op ical mic o-
scope (Zeiss, Obe kochen, Ge many) wi h a 40× objec i e and we e analyzed using Fiji-ImageJ so -
wa e( e sion 2.9.0/1.53 ) (Na ional Ins i u e o Heal h, Be hesda, MD, USA). Scale ba = 20 µm. (B)
Quan i ica ion o Sudan Black s aining images was pe o med by he Image J so wa e ( e sion
1.54 ). Da a ep esen he mean ± SD o h ee sepa a e expe imen s. *** p < 0.001 be ween un ea ed
and ea ed con ol cells. A.U., a bi a y uni s.
2.10. Inc eased Lipid Pe oxida ion in Con ol Fib oblas s T ea ed wi h Y27632o Cy ochalasin D
Nex , we analyzed he effec s o Y27632 o Cy ochalasin D ea men s on cellula lipid
pe oxida ion. Compa ing ea ed and un ea ed con ol ib oblas s, we obse ed ha con-
ols ib oblas s ea ed wi h bo h inhibi o s showed ele a ed cellula lipid pe oxida ion
(Figu e 15A,B). Con ol ib oblas s we e also ea ed wi h 500 µM Lupe ox (Te -bu yl
hyd ope oxide) as a posi i e con ol o lipid pe oxida ion.
Figu e 15. Cellula lipid pe oxida ion in con ol cells ea ed wi h Y27632 o Cy ochalasin D inhibi-
o s. (A) Con ol cells (C1) we e ea ed wi h 10 µM Y27632 o 24 h, o wi h 20 µM Cy ochalasin D
o 3 h. The le els o cellula lipid pe oxida ion we e measu ed using BODIPY
®
s aining as de ailed
in he Ma e ial and Me hods. (B) BODIPY
®
s aining quan i ica ion was pe o med by using he Fiji
so wa e ( e sion 2.9.0/1.53 ). Con ol ib oblas s we e ea ed wi h 500 µM Lupe ox (LUP, Te -
bu yl hyd ope oxide) o 15 min as posi i e con ol o lipid pe oxida ion. Scale ba = 20 µm. ** p <
0.01, *** p < 0.001 be ween ea ed and un ea ed con ol ib oblas s. Da a ep esen he mean ± SD
o ou sepa a e expe imen s. A.U., a bi a y uni s.
3. Discussion
Nemaline myopa hy (NM) is a a e sub ype o congeni al myopa hy cha ac e ized
by muscle weakness, hypo onia, and he p esence o ods in he cy oplasm o muscle i-
be s, also known as nemaline bodies [7,8,10]. In his wo k, we analyzed i on accumula ion
and lipid pe oxida ion in NM cellula models using pa ien -de i ed ib oblas s ha bo ing
ACTA1 and NEB pa hogenic a ian s.
Figu e 15. Cellula lipid pe oxida ion in con ol cells ea ed wi h Y27632 o Cy ochalasin D inhibi o s.
(A) Con ol cells (C1) we e ea ed wi h 10
µ
M Y27632 o 24 h, o wi h 20
µ
M Cy ochalasin D o
3 h. The le els o cellula lipid pe oxida ion we e measu ed using BODIPY
®
s aining as de ailed in
he Ma e ial and Me hods. (B) BODIPY
®
s aining quan i ica ion was pe o med by using he Fiji
so wa e ( e sion 2.9.0/1.53 ). Con ol ib oblas s we e ea ed wi h 500
µ
M Lupe ox (LUP, Te -bu yl
hyd ope oxide) o 15 min as posi i e con ol o lipid pe oxida ion. Scale ba = 20
µ
m. ** p< 0.01,
*** p< 0.001
be ween ea ed and un ea ed con ol ib oblas s. Da a ep esen he mean
±
SD o ou
sepa a e expe imen s. A.U., a bi a y uni s.
3. Discussion
Nemaline myopa hy (NM) is a a e sub ype o congeni al myopa hy cha ac e ized by
muscle weakness, hypo onia, and he p esence o ods in he cy oplasm o muscle ibe s,
also known as nemaline bodies [
7
,
8
,
10
]. In his wo k, we analyzed i on accumula ion
and lipid pe oxida ion in NM cellula models using pa ien -de i ed ib oblas s ha bo ing
ACTA1 and NEB pa hogenic a ian s.
Recen ly, Piñe o e al. desc ibed he in e ac ions be ween ac in polyme iza ion de-
iciency and mi ochond ia unc ion in ACTA1 and NEB mu an s [
16
]. Pa ien -de i ed
ib oblas s s ained by Rhodamine–Phalloidin showed de ec s and uns uc u ed ac in poly-
me iza ion, as well as sho e leng h han con ol ib oblas s. The esul s ob ained om
he analysis o he exp ession le els o mi ochond ial p o eins such as NDUFA9 (complex
I), NDUFS4 (complex I), m ND1 (complex I), SDHB (complex II), UQCRC2 (complex III),
m CO2 (complex IV), COX4 (complex IV), ATP5A (complex V), and VDAC1 showed ha
mu an ib oblas s exhibi ed educed exp ession le els o hese mi ochond ial p o eins.

In . J. Mol. Sci. 2025,26, 1434 17 o 26
In addi ion, mi ochond ial mo phology and dys unc ion we e analyzed using
Mi o acke
TM
s aining, and mi ochond ial espi a o y unc ion was e alua ed by he
Mi os ess es assay in a XF24 ex acellula lux analyze [
16
]. The bioene ge ic pa ame e s
analyzed showed signi ican ly lowe le els o mi ochond ial espi a ion, basal and maximal
espi a ion, ese e espi a o y capaci y, and mi ochond ial ATP p oduc ion. Mo eo e ,
mi ochond ial dynamics we e also ound o be comp omised in NM mu an ib oblas s.
Two key p o eins in ol ed in mi ochond ial usion and ission, dynamin- ela ed p o ein 1
(DRP1) and op ic a ophy p o ein 1 (OPA1) [
17
,
35
], we e also analyzed [
16
]. The esul s
demons a ed ele a ed exp ession le els o DRP1 and educed le els o OPA1, sugges ing
an imbalance be ween he p ocesses o mi ochond ial usion and ission [16].
Pa icula ly ele an o unde s anding NM pa hophysiology is he ela ionship be-
ween mi ochond ial dys unc ion and i on me abolism. The mi ochond ion upholds he
syn hesis o i on–sul u clus e s (ISCs) and heme, he mos abundan i on-con aining
p os he ic g oups in a la ge a ie y o p o eins; he e o e, a ac ion o incoming i on
mus go h ough his o ganelle be o e eaching i s inal des ina ion [
30
]. Apa o de-
c eased ATP syn hesis, mi ochond ial dys unc ion also esul s in dec eased syn hesis o
ISCs and heme p os he ic g oups and as a consequence may induce a dys egula ion o
i on me abolism [
36
]. In u n, he mi ochond ial espi a o y chain is he sou ce o eac i e
oxygen species (ROS) de i ed om leaks in he elec on anspo chain. The co-exis ence
o bo h i on and ROS in he secluded space o he mi ochond ion makes his o ganelle
pa icula ly p one o hyd oxyl adical-media ed damage. Mi ochond ial dys unc ion/i on
o e load has long been associa ed wi h se e al neu odegene a i e diseases ha include
NBIA diso de s, Alzheime ’s disease (AD), Hun ing on’s disease (HD), Pa kinson’s disease
(PD), amyo ophic la e al scle osis (ALS), and F ied ich’s A axia (FA) [24,28,31,37–39].
In his wo k, o he i s ime, we desc ibed he associa ion o mi ochond ial dys unc-
ion wi h i on accumula ion in cellula models o NM (Figu e 1and Figu e S1). Gi en ha
mu an cells showed inc eased au o luo escence spo s which a e posi i ely s ained wi h
Sudan black, i on is p esumably accumula ed in he o m o lipo uscin agg ega es (Figu e 2,
Figu e 3and Figu e S2). This hypo hesis is also suppo ed by he ac ha de e ip one ea -
men elimina ed bo h i on and au o luo escence/Sudan black spo s. Lipo uscin g anules
ha e been p e iously epo ed in diaph agm and ibialis an e io muscles in NM mouse
model ca ying he human Me 9A g mu a ion o alpha- opomyosin slow (Tpm3) [
40
]
and muscle biopsies o a pa ien ha bo ing a homozygous RYR1 c.8888T>C mu a ion [41].
Addi ionally, he TEM analysis shows he p esence o ele a ed in acellula lipo uscin-like
g anules in NM ib oblas s ha bo ing ACTA1 and NEB mu a ions (Figu e 4A,B).
As p e iously desc ibed, mi ochond ia play a c ucial ole in i on me abolism, since
hese o ganelles a e a signi ican loca ion o he use and accumula ion o i on. The e o e,
mi ochond ial i on le els should be s ic ly egula ed [
27
]. To analyze hem, we examined
he le els o mi ochond ial e ous i on (Fe
2+
) (Figu e 5) in NM mu an and con ols cells,
indica ing a dys unc ion o i on me abolism and mi ochond ial i on accumula ion. In addi-
ion, TEM analysis showed he condensa ion and la e aliza ion o damaged mi ochond ial
componen s which e en ually o med lipo uscin g anules (Figu e 4C). This mechanism
o mi ochond ial lipo uscinogenesis may p o ide cellula p o ec ion om mal unc ioning
mi ochond ia [25].
Lipids a e he p ima y s uc u al elemen s o cellula memb anes and subcellula
o ganelles and play a c ucial ole in biological p ocesses. Cell signaling, molecula anspo ,
p oli e a ion, ene gy s o age, sec e ion, and su i al a e among hei o he c ucial biological
oles [
24
,
42
]. Fu he mo e, he lipid composi ion o he inne and ou e mi ochond ial
memb ane is also essen ial o he p ope unc ioning o he elec on anspo chain, he
s abili y o espi a o y chain supe complexes, and ATP p oduc ion [
43
,
44
]. Excessi e lipid
In . J. Mol. Sci. 2025,26, 1434 18 o 26
oxida ion may al e p o eins and nucleic acids co alen ly and change he physicochemical
cha ac e is ics o biological memb anes. Lipid pe oxida ion and he oxida ion o espi a o y
chain p o eins a e he wo mechanisms ha may cause mi ochond ial dys unc ion by an
inc ease in ROS gene a ion [45,46].
One o he main causes o cellula and issue dys unc ion ha con ibu es o aging
and mos age- ela ed and oxida i e s ess- ela ed diso de s is he accumula ion o lipid
pe oxida ion (LPO) p oduc s in human issues [
45
,
47
]. I has been epo ed ha lipid
pe oxida ion in i on- ich o ganelles such as mi ochond ia may lead o i on accumula ion in
lipo uscin g anules, which in u n may inc ease lipid pe oxida ion [
24
,
31
]. This icious cycle
in which lipid pe oxida ion and i on accumula ion ein o ce each o he may pa icipa e in
NM pa homechanisms.
Con i ming his hypo hesis, we demons a ed ha i on o e load in NM mu an cells
was accompanied by inc eased lipid pe oxida ion. This in e connec ion be ween bo h
pa hological p ocesses can agg a a e he cellula damage, as ecen ly desc ibed by ou
g oup [
24
]. Co obo a ing i on handling dys egula ion, we ound ha NM pa ien -de i ed
ib oblas s p esen ed al e ed exp ession le els o p o eins ela ed o i on me abolism.
Figu e 6shows he exp ession le els o p o eins in ol ed in i on homeos asis: TFR1, DMT1,
IRP1, Fe i in, Mi o e i in, Mi o e in2, FXN, ISCU, M - ACP, and PANK2. The ele a ed
exp ession le els o TFR, DMT1, and Fe i in, p o eins esponsible o he i on anspo
and s o age wi hin cell, suppo he hypo hesis o a dys egula ion o i on me abolism in
NM mu an s. In addi ion, he exp ession le els o F a axin (FXN) and ISCU, mi ochond ial
p o eins in ol ed in he biosyn hesis o ISCs (i on–sul u clus e s), we e down egula ed
compa ed wi h con ol ib oblas . ISCs a e p os he ic g oups ha a e a ached o cy osolic
and mi ochond ial aconi ases, as well as mul iple subuni s o mi ochond ial espi a o y
complexes [
48
]. Consequen ly, a lack o p o eins in ol ed in ISC biogenesis may in e e e
wi h mi ochond ial unc ion, as p e iously desc ibed by ou g oup [
49
]. Al e a ions in
i on homeos asis a e ano he consequence o his de iciency ha may ul ima ely lead o
mi ochond ial i on excess. High i on le els in he mi ochond ia’s oxida i e en i onmen
can hen lead o a ise in ROS p oduc ion and mi ochond ial lipid pe oxida ion [50].
The pa adoxical esul s o dec eased LIP and i on accumula ion ha e been also e-
po ed in PKAN cells by ou esea ch g oup. In addi ional wo ks, PANK2 silencing by
siRNA in se e al human cell lines leads o a educed p oli e a ion a e, accompanied
by a pa adoxical i on de iciency and inc eased T R1 exp ession le els [
51
]. Conside ing
hese obse a ions, i has been p oposed ha he hypo hesis ha dys egula ion o i on
me abolism in mi ochond ia induces mi ochond ial i on o e load and cy osolic i on de i-
ciency. The esul is a icious cycle cha ac e ized by inc eased i on up ake due o inc eased
exp ession o Fe
2+
anspo e s and subsequen accumula ion in mi ochond ia and, inally,
in lipo uscin g anules [
28
,
52
]. This hypo hesis could explain he mi ochond ial dys unc ion
p esen in pa ien s’ cells.
In e es ingly, i on accumula ion, inc eased lipid pe oxida ion, and mi ochond ial
dys unc ion we e ep oduced in con ol cells ea ed wi h ac in depolyme izing agen s
(Figu es 10–15), co obo a ing he essen ial ole o ac in ilamen s in mi ochond ial unc ion
and consequen ly in i on me abolism and lipid pe oxida ion. The e a e se e al limi a ions
in his s udy: (1) Only ou pa ien s ha e been included in his wo k. (2) Fu he esea ch
is needed o de e mine i on/lipo uscin accumula ion and lipid pe oxida ion in animal
models and muscle biopsies om pa ien s.
In . J. Mol. Sci. 2025,26, 1434 19 o 26
4. Ma e ials and Me hods
4.1. Reagen s
The ollowing an ibodies we e pu chased om Abcam (Camb idge, Uni ed King-
dom, UK): alpha- ubuline (ab7291), MTFRN2 Mi o e in2 (ab80467), MTFRN1 Mi o e -
in 1 (ab56134) (an i-FXN F a axine (ab219414), and TFR T ans e in ecep o (ab84036).
The ollowing an ibodies we e acqui ed om San a C uz (Cali o nia (CA), Uni ed S a es
(USA)): IRP1 i on- esponsi e elemen -binding p o ein (sc-166022), Fe i in (sc-74513), and
PANK2 (sc-82288). ACP mi an ibody was pu chased om In i ogen The mo Fishe
Scien i ic (Whal ham, Massachuse s (MA), Uni ed S a es (USA)). DMT1 an ibody (Di alen
Me al T anspo e 1) ABS983 was acqui ed om EMD Millipo e. ISCU an ibody was ac-
qui ed om GeneTex (Cali o nia, (CA), Uni ed S a es (USA)). Pe l’s P ussian Blue, T ypsin,
dime hyl sul oxide (DMSO), saponin, T is base, 4
′
,6-diamidino-2-phenylindole (DAPI),
and e ame hyle hylenediamine (TEMED) we e pu chased om Sigma-Ald ich Chemical
Co. (S . Louis, Missou i (MO), Uni ed S a es (USA)). Dulbecco’s modi ied Eagle’s medium
(DMEM) wi h 4.5 g/L and 1 g/L glucose, L-glu amine, py u a e, penicillin–s ep omycin
(10,000:10,000), and e al bo ine se um (FBS) we e all sou ced om Gibco. Mowiol 4-88 Mw
was ob ained om Sigma Chemical Co. (S . Louis, Missou i (MO), Uni ed S a es (USA)),
while bo ine se um albumin (BSA) was pu chased om San a C uz Bio echnology (Paso
Robles, Cali o nia, (CA), Uni ed S a es (USA)). The Pie ce™ BCA P o ein Assay Ki was
p ocu ed om Fishe Scien i ic (Whal ham, Massachuse s (MA), Uni ed S a es (USA)).
Reagen s o e alua e he mi ochond ial ac i i y we e acqui ed om San a C uz Bio ech-
nology (Cali o nia, (CA), Uni ed S a es (USA)): oligomycin (sc-203342), ca bonyl cyanide
4-( i luo ome hoxy) phenylhyd azone (FCCP) (sc-203578), and an imycin A (sc-202467A).
Va ious eagen s we e acqui ed om Bio-Rad Labo a o ies Inc. (He cules, Cali o nia
(CA), Uni ed S a es (USA)), including Ac ylamide 37.5:1 solu ion, Cla i y™ Wes e n ECL
subs a e, elec opho esis bu e (TGS), sodium dodecyl sul a e (SDS), T i on X-100, blo
bu e (TG), Tween 20, and DC P o ein Assay Reagen s A, B, and S. Addi ional chemicals
such as 2-p opanol, e hanol, me hanol, sodium chlo ide (NaCl), ammonium pe sul a e
(APS), glacial ace ic acid, po assium hyd oxide (KOH), and po assium chlo ide (KCl) we e
supplied by Pan eac (Ba celona, Spain). The P o ease Inhibi o Cock ail was ob ained om
Roche (F. Ho mann-La Roche L d., Basel, Swi ze land). Y27632 (sc-3536) and pa a o malde-
hyde (PFA; sc-25326B) we e pu chased om San a C uz Bio echnology (Cali o nia (CA),
Uni ed S a es (USA)).
4.2. Pa ien s and Cell Cul u es
Two con ols lines o p ima y human skin ib oblas s we e pu chased om ATCC,
and ou lines o ib oblas s de i ed om pa ien skin biopsies om he Pedia ic Depa -
men o Hospi al Uni e si a io Vi gen del Rocío, Se illa, Spain. Pa ien 1 (P1) p esen s a
he e ozygous pa hogenic mu a ion c.133G>T (p.Val45Phe) in ACTA1 ha causes a missense
a ian . The second pa ien (P2) is also he e ozygous, ca ying changes in posi ion c.760A>T
(p.Asn254Ty ) in ACTA1. The hi d pa ien (P3) p esen s he e ozygous pa hogenic a i-
an s c.10321A>C (p.Th 3441P o) and c.13669C>T (pA g4557*) in NEB. The ou h pa ien
(P4) p esen s he e ozygous pa hogenic a ian s c.24407_24410dup (p.Leu8137Phe s*18)
c.8425C>T (p.A g2809*) in NEB.
Con ol alues we e ep esen ed as means
±
SD o h ee con ol ib oblas cell lines.
Fib oblas s we e main ained in Dulbecco’s modi ied Eagle’s medium DMEM (Gibco™,
The moFishe Scien i ic, Wal ham, Massachuse s (MA), Uni ed S a es (USA)) supple-
men ed wi h 10% FBS (Gibco™, The moFishe Scien i ic, Massachuse s (MA), Uni ed
S a es (USA)), 100 mg/mL penicillin/s ep omycin. Fib oblas s we e cul u ed a 37
◦
C and
In . J. Mol. Sci. 2025,26, 1434 20 o 26
5% CO
2
. All he expe imen s we e pe o med wi h ib oblas s’ cell cul u es wi h a passage
numbe < 10.
4.3. De e mina ion o I on and Lipo uscin Accumula ion
I on o e load was de e mined by P ussian Blue s aining and quan i ied in a mi-
c opla e eade (Pola S a Omega, BMG Lab ech, O enbe g, Ge many) and by b igh ield
mic oscopy. Images we e aken by ligh and luo escence Axio Ve A1 mic oscope (Zeiss,
Obe kochen, Ge many) wi h a 20
×
and 40
×
objec i e. A quan i ica ion analysis was
pe o med by using he Image J so wa e ( e sion 1.54 ). Mo eo e , i on con en in cell
ex ac s was also measu ed by induc i ely coupled plasma mass spec ome y (ICP-MS).
Cell cul u e ex ac s we e ob ained by acid diges ion wi h HNO
3
. I on concen a ion was
exp essed as ng Fe
2+
/
µ
g p o ein. Da a we e p esen ed as means
±
SD (s anda d de ia ion),
n= 3 in all cases.
Lipo uscin g anules we e isualized by Sudan Black B (SBB) s aining as p e iously
desc ibed [
53
]. The quan i ica ion o SSB s aining also was pe o med wi h he ligh and
luo escence Axio Ve A1 mic oscope (Zeiss, Obe kochen, Ge many) wi h a 20
×
and
40
×
objec i e. The cell’s au o luo escence was measu ed by luo escence mic oscopy
(exci a ion 366 nm; emission 420–600 nm). Con ocal lase scanning mic oscopy (Nikon
A1R, Shinagawa, Tokyo, Japan) was used o ob aining he emission spec a o lipo uscin
g anules as p e iously desc ibed [
28
]. The emission spec a we e eco ded in 20 lipo uscin
g anules in 20 cells. All images we e analyzed by Fiji-ImageJ so wa e ( e sion 2.9.0/1.53 ).
In bo h de e mina ions, ib oblas s we e ea ed wi h 100
µ
M de e ip one (De ) [
28
],
an i on chela ing d ug which we e used as a nega i e con ol o co obo a e he speci ici y
o he conduc ed s aining’s and echniques.
4.4. TEM Analysis
T ansmission elec on mic oscopy was pe o med ollowing he p o ocol p e iously
desc ibed by ou g oup [
28
,
54
]. Con ol and pa ien s’ cells we e seeded on 8-well Pe manox
chambe slides (Nunc, The mo Scien i ic) and we e subsequen ly ixed in empe ed 3.5%
glu a aldehyde in 0.1 M phospha e bu e (PB) o 5 min a 37
◦
C and 55 min o 4
◦
C. Cells
we e pos ixed in 2% OsO4 o 1 h a oom empe a u e, insed, dehyd a ed, and embedded
in Du cupan esin (Fluka, Sigma-Ald ich). Semi hin sec ions (1.5
µ
m) we e cu wi h a
diamond kni e and s ained ligh ly wi h 1% oluidine blue. La e , ul a- hin (70 nm) sec ions
o he cells we e cu wi h a diamond kni e, s ained wi h lead ci a e (Reynolds solu ion),
and examined unde a ansmission elec on mic oscope (FEI Tecnai G2 Spi i BioTwin)
wi h a Xa osa (20 Megapixel esolu ion) digi al came a using Radius image acquisi ion
so wa e ( e sion 2.1, EMSIS GmbH, Müns e , Ge many).
4.5. Measu emen o Mi ochond ial I on Accumula ion (Mi o-Fe oG een)
Mi ochond ial i on le els we e measu ed by Mi o-Fe oG een s aining, a no el luo-
escen p obe o he de ec ion o e ous ion (Fe
2+
) in mi ochond ia whe e Fe-S clus e s
and heme p o eins a e syn hesized and enables li e cell luo escen imaging o in acellula
Fe
2+
. Mi o-Fe oG een was ob ained om he Dojindo Labo a o y (Kumamo o, Japan).
Fib oblas s we e washed h ee imes wi h HBSS supplemen ed wi h Ca
2+
and Mg
2+
and
incuba ed o 30 min a 37
◦
C wi h 5
µ
M Mi o-Fe oG een and 100 nM Mi oT acke ™ Deep
Red FM o 45 min a 37
◦
C. Then, cells we e washed h ee imes wi h HBSS supplemen ed
wi h Ca
2+
and Mg
2+
. Images we e aken using a Del aVision sys em ( e sion so WoRx 7.0;
Applied P ecision; Issaquah, Washing on (WA), Uni ed S a es (USA)) wi h an Olympus
IX-71 luo escence mic oscope (Olympus Co po a ion, Shinjuku, Tokyo, Japan) wi h a 40
×
oil objec i e and analyzed by Fiji-ImageJ so wa e ( e sion 2.9.0/1.53 ). To co obo a e
he speci ic Mi o-Fe oG een p esence in he mi ochond ia, cells we e co-s ained wi h
In . J. Mol. Sci. 2025,26, 1434 21 o 26
Mi oT acke ™ Deep Red FM, and he Pea son co ela ion coe icien was calcula ed using
he JaCoP plugin om Fiji-ImageJ so wa e ( e sion 2.9.0/1.53 ). A posi i e co ela ion was
conside ed when Pea son coe icien >0.75. Mi ochond ial i on le els we e de e mined
by quan i ica ion o he Mi o-Fe oG een luo escence in ensi y om 30 cells. Da a we e
p esen ed as means ±SD (s anda d de ia ion), n= 3 in all cases.
4.6. Inmunoblo ing
Wes e n blo ing was pe o med using s anda d me hods. A e p o ein ans e , he
memb ane was incuba ed wi h a ious p ima y an ibodies dilu ed 1:1000 and hen wi h
he co esponding seconda y an ibody coupled o ho se adish pe oxidase a a 1:10,000
dilu ion. Speci ic p o ein complexes we e iden i ied using he Immun-S a HRP subs a e
ki (Bio ad Labo a o ies Inc., He cules, Cali o nia (CA), Uni ed S a es (USA)).
4.7. Measu emen o Labile I on Pool
Labile i on pool (LIP) de e mina ion was ca ied ou as a sligh ly modi ied e sion o
wha has been p e iously desc ibed [
33
]. B ie ly, cells we e seeded in 96-well pla es. Con ol
and pa ien s’ ib oblas s we e incuba ed in he medium supplemen ed wi h 1 mg/mL BSA
and 0.25
µ
M calcein-AM o 30 min a 37
◦
C. A e wo washes wi h Hank’s Balanced Sal
Solu ion (HBSS), cells we e main ained in HBSS supplemen ed wi h 5 mM glucose, 20 mM
HEPES, and 15 mM NaCl o 10 min a 37
◦
C. Basal luo escence was measu ed using a
Pola S a Omega Mic opla e Reade a 485 nm (exci a ion) and 535 nm (emission). Cells
we e hen supplemen ed wi h Salicyladehyde Isonico inoyl Hyd azone (0.1 mM), a speci ic
i on chela o , o 15 min. Fluo escence was moni o ed du ing incuba ion wi h he chela o ,
and when a pla eau was eached, ha alue was he LIP alue. The inal de e mina ion o
he LIP le el is ca ied ou using he a io LIP alue/basal measu emen , and he esul s
we e no malized o he p o ein con en (mg o p o ein). The con ol cells we e p e iously
ea ed wi h 100
µ
M de e ip one as a nega i e con ol. Da a we e p esen ed as means
±
SD
(s anda d de ia ion), n= 3 in all cases.
4.8. Measu emen o Memb ane Lipid Pe oxida ion
Lipid pe oxida ion was de e mined using 4,4-di luo o-5-(4-phenyl-1,3-bu adienyl)-
4-bo a-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY
®
581/591 C11) (D3861, The -
moFishe Scien i ic), a lipophilic luo escen dye [
25
,
55
]. Cells we e incuba ed wi h 5
µ
M
BODIPY
®
581/591 C11 o 30 min a 37
◦
C. Lupe ox
®
TBH70X (458139, Sigma-Ald ich) a
500
µ
M o 15 min was used as posi i e con ol o lipid pe oxida ion. Lipid pe oxida ion
in ib oblas s was e alua ed by an Axio Ve A1 luo escence mic oscope wi h a 40
×
ob-
jec i e. All images we e analyzed by Fiji-ImageJ so wa e ( e sion 2.9.0/1.53 ). Da a we e
p esen ed as means ±SD (s anda d de ia ion), n= 3 in all cases.
4.9. Measu emen o Mi ochond ial Lipid Pe oxida ion (Mi oPeDPP)
Mi ochond ial lipid pe oxida ion was e alua ed using a [3-(4-phenoxyphenylpy eny
lphosphino) p opyl] iphenylphosphonium iodide luo escen p obe (Mi oPeDPP
®
) de-
eloped by Shioji K., e al. [
56
]. Localiza ion o he Mi oPeDPP signal in mi ochond ia
was add essed by a colocaliza ion analysis wi h Mi oT acke ™ Deep Red FM, an
in i o
mi ochond ial dye. Con ol and pa ien s’ cells we e ea ed wi h 300 nM Mi oPeDPP® o
15 min a 37
◦
C and 100 nM Mi oT acke ™ Deep Red FM o 45 min a 37
◦
C. Lupe ox
®
TBH70X (458139, Sigma-Ald ich) a 500
µ
M o 15 min was used as posi i e con ol o
lipid pe oxida ion. Images we e aken by Del aVision ( e sion so WoRx 7.0; Applied
P ecision; Issaquah, Washing on (WA), Uni ed s a es (USA)) sys em wi h an Olympus IX-71
luo escence mic oscope(Olympus Co po a ion, Shinjuku, Tokyo, Japan) wi h a 40
×
oil

In . J. Mol. Sci. 2025,26, 1434 22 o 26
objec i e and analyzed by Fiji-ImageJ so wa e ( e sion 2.9.0/1.53 ). Da a we e p esen ed
as means ±SD (s anda d de ia ion), n= 3 in all cases.
4.10. Analysis o Mi ochond ial Ne wo k and Cy oskele al F-Ac in
In o de o e alua e he e ec o ac in inhibi o s Y27632 and Cy ochalasin D on he
s a e o ac in ilamen s and he mi ochond ial ne wo k, con ol and pa ien s’ ib oblas s
we e s ained wi h 1
µ
g/mL Rhodamine–Phalloidin o 30 min and 100 nM Mi oT acke ™
Deep Red FM o 45 min a 37
◦
C. E alua ion o mi ochond ial ne wo k and cy oskele al
F-ac in was conduc ed ollowing ou p e iously desc ibed p o ocols [
16
]. All images we e
aken by a Del aVision ( e sion so WoRx 7.0; Applied P ecision; Issaquah, Washing on
(WA), Uni ed s a es (USA)) sys em wi h an Olympus IX-71 luo escence mic oscope wi h a
40×oil objec i e and analyzed by Fiji-ImageJ so wa e ( e sion 2.9.0/1.53 ).
4.11. Bioene ge ics and Oxida i e S ess Analysis
An XF24 ex acellula lux analyze (Seaho se Bioscience, Bille ica, Massachuse s
(MA), Uni ed S a es (USA)) was used o pe o m a Mi os ess es expe imen o assess he
mi ochond ial espi a o y pe o mance o un ea ed and ea ed con ol wi h inhibi o s
Y27632 and Cy ochalasin D. In XF24 cell cul u e pla es, cells we e cul i a ed a a densi y
o 15,000 cells pe well wi h 150
µ
L o g ow h media (DMEM supplemen ed wi h 20%
FBS) a 37
◦
C and 5% CO
2
. Only 50
µ
L o media emained a e he g ow h medium om
each well was emo ed a e he incuba ion pe iod o 24 h. Following wo ounds o cell
washing wi h 1 mL o p e-wa med assay medium (XF base media supplemen ed wi h
10 mM glucose, 1 mM glu amine, and 1 mM sodium py u a e; pH 7.4), 450
µ
L o assay
medium (500
µ
L o al) was added o each well. Fib oblas s p e-equilib a e wi h he es
media by being cul u ed o an hou a 37
◦
C wi hou CO
2
. Fou di e en compounds ha
impac bioene ge ics we e injec ed sequen ially o es mi ochond ial ac i i y. The inal
doses o hese ou compounds we e adminis e ed as ollows: 1
µ
M oligomycin, 2
µ
M
FCCP (ca bonyl cyanide-4- i luo ome hoxy-phenylhyd azone), and 2.5
µ
M an imycin
A/ o enone.
To de e mine he ideal cell seeding densi y and he ideal dose o each inhibi o and
uncouple , p elimina y es s we e pe o med. A minimum o i e wells ha e been used o
each ea men in each expe imen . Impo an mi ochond ial cha ac e is ics, including ATP
syn hesis, spa e espi a o y capaci y, and basal and maximal espi a ion, may be es ima ed
wi h his es . The XF24 analyze ’s esul s we e s anda dized acco ding o he 15,000 cells
ha we e plan ed. The BioTek
TM
Cy a ion
TM
1 Cell Imaging Mul i-Mode Reade was used
o coun he cells in each well bo h be o e and a e he expe imen o e alua e i he numbe
o cells emained cons an .
4.12. S a is ics
S a is ical analysis was conduc ed in acco dance wi h ou esea ch g oup’s p e ious
desc ip ion [
16
]. We used non-pa ame ic s a is ics, whe e he e we e ew e en s (n < 30),
ha do no ha e any dis ibu ional assump ion, gi en he low eliabili y o no mali y es ing
o small sample sizes used in his wo k. In hese cases, mul iple g oups we e compa ed
using a K uskal–Wallis es . We used pa ame ic es s when he numbe o e en s was
g ea e (n> 30). In hese ins ances, a one-way ANOVA was used o compa e mul iple
g oups. S a is ical analyses we e conduc ed using he G aphPad P ism 9.0 (G aphPad
So wa e, San Diego, CA, USA). All esul s we e p esen ed as he mean
±
SD alues
o as an example om 3 independen expe imen s, and p- alues o less han 0.05 we e
conside ed signi ican .
In . J. Mol. Sci. 2025,26, 1434 23 o 26
5. Conclusions
In conclusion, ou indings demons a e ha ib oblas s de i ed om pa ien s wi h NM
a e use ul cellula models o s udy disease pa hophysiology. Fu he mo e, we con i m he
close ela ionship be ween he ac in cy oskele on, mi ochond ia unc ion, i on me abolism,
and lipid pe oxida ion. Pa ien -de i ed cellula models may complemen ACTA1 and
NEB mouse and zeb a ish models o unde s and disease pa homechanisms and enable he
e alua ion o genomic o pha macological he apies.
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps://
www.mdpi.com/a icle/10.3390/ijms26041434/s1.
Au ho Con ibu ions: Concep ualiza ion, J.A.S.-A., A.L.-C. and R.P.-P.; me hodology, R.P.-P., M.Á.-C.,
D.G.-F., P.C.-H., J.M.R.-D., A.R.-G., D.R.-L., M.d.l.M., R.M.d.P., S.G.-G., J.M.G.-V. and A.L.-C.; w i ing—
o iginal d a p epa a ion, A.L.-C., R.P.-P. and M.Á.-C.; w i ing— e iew and edi ing, J.A.S.-A., A.L.-C.
and R.P.-P.; unding acquisi ion, J.A.S.-A. All au ho s ha e ead and ag eed o he published e sion
o he manusc ip .
Funding: This wo k was suppo ed by FIS PI19/00377 and PI22/00142 g an s, Ins i u o de Salud
Ca los III, Spain, and Fondo Eu opeo de Desa ollo Regional (FEDER-Unión Eu opea), P oyec os
de In es igación de Excelencia de la Jun a de Andalucía CTS-5725 and PY18-850, and UPO-FEDER
2018 (UPO-1380614). J.M.G.-V. and S.G.-G., we e suppo ed by he Valencian Council o Educa ion,
Cul u e, Uni e si y and Employmen (CIPROM/2023/053).
Ins i u ional Re iew Boa d S a emen : The s udy was conduc ed in acco dance wi h he Decla a ion
o Helsinki and app o ed by Coo dina ing Commi ee o E hics o Biomedical Resea ch o Andalusia
(p o ocol code MYO-CURE 3, 11 July 2019).
In o med Consen S a emen : In o med consen was ob ained om all subjec s in ol ed in he s udy.
Da a A ailabili y S a emen : Da a suppo ing he indings o his s udy a e no openly a ailable due
o easons o sensi i i y and o p o ec he p i acy o indi iduals; howe e , da a a e a ailable om
he co esponding au ho upon easonable eques . Da a a e loca ed in con olled access da a s o age
a Pablo de Ola ide Uni e si y (h ps://jazmin.upo.es/bscw/bscw.cgi).
Acknowledgmen s: We acknowledge he unding suppo o he Spanish pa ien associa ion “Yo
Nemalínica”. We ex end ou g a i ude o Pila Bu gos Domenech om Ins i u o de Recu sos Na u ales
y Ag obiología de Se illa (IRNAS) o he assis ance in IPC-MS assay.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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