Identification of Potential Muscle Biomarkers in McArdle Disease: Insights from Muscle Proteome Analysis

Ci a ion: Ga cía-Consueg a, I.;
Asensio-Peña, S.; Ga ido-Mo aga, R.;
Pinós, T.; Domínguez-González, C.;
San alla, A.; Nogales-Gadea, G.;
Se ano-Lo enzo, P.; And eu, A.L.;
A enas, J.; e al. Iden i ica ion o
Po en ial Muscle Bioma ke s in
McA dle Disease: Insigh s om
Muscle P o eome Analysis. In . J.
Mol. Sci. 2022,23, 4650. h ps://
doi.o g/10.3390/ijms23094650
Academic Edi o s: Jose Rena o Pin o
and Daniel Taillandie
Recei ed: 28 Decembe 2021
Accep ed: 18 Ap il 2022
Published: 22 Ap il 2022
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In e na ional Jou nal o
Molecula Sciences
A icle
Iden i ica ion o Po en ial Muscle Bioma ke s in McA dle
Disease: Insigh s om Muscle P o eome Analysis
Inés Ga cía-Consueg a 1,2,†, Sa a Asensio-Peña 1,† , Rocío Ga ido-Mo aga 1, Tomàs Pinós2,3,
C is ina Domínguez-González 1,2 , Al edo San alla 4, Gisela Nogales-Gadea 5, Pablo Se ano-Lo enzo 1,2 ,
An oni L. And eu 6, Joaquín A enas 1,2, JoséL. Zugaza 7,8 , Alejand o Lucia 1,9 and Miguel A. Ma ín1,2,*
1Mi ochond ial and Neu omuscula Diso de s G oup, Hospi al 12 de Oc ub e Heal h Resea ch
Ins i u e (imas12), 28041 Mad id, Spain; [email p o ec ed] (I.G.-C.); [email p o ec ed] (S.A.-P.);
ocioga ido [email p o ec ed] (R.G.-M.); [email p o ec ed]g (C.D.-G.);
[email p o ec ed] (P.S.-L.); joaquin.a [email p o ec ed]g (J.A.);
alejand o.lucia@uni e sidadeu opea.es (A.L.)
2Cen o de In es igación Biomédica en Red de En e medades Ra as (CIBERER), 28029 Mad id, Spain;
omas.pinos@ hi .o g
3Mi ochond ial and Neu omuscula Diso de s Uni , Vall d’Heb on Ins i u de Rece ca,
Uni e si a Au ònoma de Ba celona, 08193 Ba celona, Spain
4Depa men o Compu e and Spo Sciences, Uni e sidad Pablo de Ola ide, 41013 Se illa, Spain;
[email p o ec ed]
5G up de Rece ca en Malal ies Neu omuscula s i Neu opedià iques, Depa men o Neu osciences,
Ins i u d’In es igacio en Ciencies de la Salu Ge mans T ias i Pujol i Campus Can Ru i,
Uni e si a Au ònoma de Ba celona, 08916 Ba celona, Spain; [email p o ec ed]
6EATRIS, Eu opean In as uc u e o T ansla ional Medicine, 1019 Ams e dam, The Ne he lands;
[email p o ec ed]
7Achuca o Basque Cen e o Neu oscience, Science Pa k o he UPV/EHU, and Depa men o Gene ics,
Physical An h opology, and Animal Physiology, Facul y o Science and Technology, UPV/EHU,
48940 Leioa, Spain; [email p o ec ed]
8IKERBASQUE, Basque Founda ion o Science, Plaza Euskadi 5, 48009 Bilbao, Spain
9Facul y o Spo Sciences, Uni e sidad Eu opea de Mad id, 28670 Mad id, Spain
*Co espondence: [email p o ec ed]
† These au ho s con ibu ed equally o his wo k.
Abs ac :
Glycogen s o age disease ype V (GSDV, McA dle disease) is a a e gene ic myopa hy
caused by de iciency o he muscle iso o m o glycogen phospho ylase (PYGM). This esul s in a
block in he use o muscle glycogen as an ene ge ic subs a e, wi h subsequen exe cise in ole ance.
The pa hobiology o GSDV is s ill no ully unde s ood, especially wi h ega d o some ea u es such
as pe sis en muscle damage (i.e., e en wi hou p io exe cise). We aimed a iden i ying po en ial
muscle p o ein bioma ke s o GSDV by analyzing he muscle p o eome and he molecula ne wo ks
associa ed wi h muscle dys unc ion in hese pa ien s. Muscle biopsies om eigh pa ien s and eigh
heal hy con ols showing none o he ea u es o McA dle disease, such as equen con ac u es and
pe sis en muscle damage, we e s udied by quan i a i e p o ein exp ession using isoba ic ags o
ela i e and absolu e quan i a ion (iTRAQ) ollowed by a i icial neu onal ne wo ks (ANNs) and
opology analysis. P o ein candida e alida ion was pe o med by Wes e n blo . Se e al p o eins
p edominan ly in ol ed in he p ocess o muscle con ac ion and/o calcium homeos asis, such
as myosin, sa coplasmic/endoplasmic e iculum calcium ATPase 1, opomyosin alpha-1 chain,
oponin iso o ms, and alpha-ac inin-3, showed signi ican ly lowe exp ession le els in he muscle o
GSDV pa ien s. These p o eins could be po en ial bioma ke s o he pe sis en muscle damage in
he absence o p io exe ion epo ed in GSDV pa ien s. Fu he s udies a e needed o elucida e he
molecula mechanisms by which PYGM con ols he exp ession o hese p o eins.
Keywo ds:
PYGM; myophospho ylase; p o eomics; McA dle disease; GSDV; iTRAQ; skele al muscle;
me abolic myopa hy; p o ein bioma ke s
In . J. Mol. Sci. 2022,23, 4650. h ps://doi.o g/10.3390/ijms23094650 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 4650 2 o 18
1. In oduc ion
Glycogen s o age disease ype V (GSDV) (OMIM#232600), also known as McA dle
disease, is a a e au osomal ecessi e myopa hy caused by biallelic pa hogenic mu a ions
in he PYGM gene [
1
] ha esul in de iciency o he skele al muscle iso o m o glycogen
phospho ylase (o ‘myophospho ylase’, PYGM) [
2
]. GSDV has been epo ed o ha e an
es ima ed p e alence o 1 in 100,000—350,000 people [2–5].
Because PYGM ca alyzes he i s a e-limi ing s ep o glycogen me abolism (i.e., e-
mo al o e minal alpha-1,4-glycosidic bonds om he ou e b anches o his molecule
o elease glucose-1-phospha e), de iciency o his enzyme leads o a block in he use o
glycogen as an ene gy sou ce o muscle con ac ion [
6
]. Typical clinical ea u es consis o
muscle ‘c ises’ o pain and a igue, oge he wi h achyca dia du ing he i s minu es o
dynamic exe cise (e.g., b isk walking) ha a e a enua ed a e 7–10 min ha e elapsed— he
so-called ‘second wind’ phenomenon [
2
,
7
]. These episodes o ea ly exe cise in ole ance a e
equen ly accompanied by se e e muscle con ac u es, po en ially leading o habdomyol-
ysis and subsequen myoglobinu ia, as e lec ed by ‘da k u ines’. Ye , ano he ea u e o
he disease is a pe sis en s a us o muscle damage—(as e lec ed by e y high ci cula ing
le els o in a-muscle p o eins such as c ea ine kinase [CK]), e en in he absence o physical
exe cise on he p e ious day(s) [8].
Mo e han 170 pa hogenic mu a ions (including missense, nonsense, in- ame, ameshi ,
and splicing a ian s) ha e been iden i ied in he PYGM gene ha cause McA dle dis-
ease [
9
,
10
]. Mos o hese mu a ions esul in a o al absence o PYGM ac i i y [
11
] in
he pa ien s’ muscle issue, excep o wo pa ien s ca ying deep-in onic mu a ions in
compound he e ozygosi y ha led o some esidual (~1% o no mal) enzyme ac i i y, wi h
subsequen amelio a ion in clinical pheno ype [
12
]. The e is no associa ion be ween he
PYGM geno ype and disease pheno ype, since pa ien s wi h he same mu a ion(s) can
show qui e di e en deg ees o clinical se e i y [
13
]. The pa hobiology o GSDV is no
ully unde s ood, bu i seems ha he po en ial molecula consequences o he lack o
glycogenoly ic–de i ed ATP in ol e no only he expec ed ene ge ic de ici o ac in-myosin
c oss b idging, bu also impai men s in memb ane pump unc ion, exci a ion–con ac ion
coupling, and sa colemmal exci abili y [14].
In an a emp o iden i y po en ial muscle p o ein bioma ke s and gain insigh in o
he pa hobiology o GSDV, we analyzed he a ge ed p o eome in skele al muscle biopsies
ob ained om bo h pa ien s wi h his ochemical and gene ic diagnoses o GSDV and heal hy
con ols. In his ega d, since skele al muscle is he only issue ha is clinically a ec ed
in all pa ien s wi h GSDV, he con ol issue was skele al muscle biopsies om aged and
sex-ma ched heal hy indi iduals wi h no mal PYGM ac i i y and no signs o he ypical
ea u es o McA dle disease, such as equen exe cise-induced con ac u es o pe sis en
muscle damage in he absence o p io exe ion.
We i s used isoba ic ags o ela i e and absolu e quan i a ion (iTRAQ) analysis [
15
]
o compa e he muscle p o ein exp ession in pa ien s s. con ols. This was ollowed by
a sys ems biology ne wo k-based app oach o iden i y key p o eins in ol ed in dis inc
pa hways ha could be ela ed o he GSDV pheno ype, such as he b eakdown o muscle
ibe s, muscle con ac u es, and impai men in calcium homeos asis o in o he physiologi-
cal p ocesses o he skele al muscle. To his end, we applied he he apeu ic pe o mance
mapping sys em (TPMS) machine lea ning-based echnology, pa icula ly by applying a i-
icial neu onal ne wo ks (ANNs) ha we e ‘ ained’ using he human p o ein ne wo k and
d ug-pa hophysiology knowledge [
16
,
17
]. This echnology has p o en use ul o iden i y
non-ob ious unc ional ela ionships o d ug epu posing pu poses [
18
–
20
] and biological
da a analysis and p io i iza ion o p o eins acco ding o documen ed ela ionships wi h
pa hophysiological p ocesses [
21
–
23
], especially in a e diseases o when sample sizes a e
limi ed. A e he p io i iza ion p ocess, le els o he selec ed candida e p o eins we e
analyzed using Wes e n blo analyses.
Ou esul s indica e ha in addi ion o PYGM, myosin 1 (MYH-1), opomyosin alpha-
1 chain (TPM1), sa coplasmic/endoplasmic e iculum calcium ATPase 1 (ATP2A1, also
In . J. Mol. Sci. 2022,23, 4650 3 o 18
abb e ia ed as SERCA1), oponin iso o ms ( oponin I2, as skele al ype [TNNI2] and
oponin T3, as skele al ype [TNNT3]), and alpha-ac inin-3 (ACTN3) show a ela ionship
wi h GSDV, wi h hei le els educed in he skele al muscle issue o GSDV pa ien s wi h
espec o heal hy con ols. Mos o hese p o eins a e in ol ed in muscle con ac u es
associa ed wi h al e ed calcium homeos asis.
2. Resul s
The main cha ac e is ics o he pa ien s a e shown in Tables 1and 2. Sex dis ibu-
ion (50% and 62.5% emale in pa ien s and con ols; Chi-squa e es ’s p= 0.625) and
mean (±SD)
age (pa ien s: 38
±
12 yea s; con ols: 40
±
9 yea s; Mann–Whi ney’s U
p= 0.711) did no di e be ween he wo g oups.
Table 1. Demog aphic and disease cha ac e is ics o he GSDV pa ien s.
Pa ien
PYGM PYGM Age
Sex CK (U/L) Se e i y
Class 2
Muscle
Used o
Biopsy
PYGM PYGM
Allele 1 1Allele 2 1(Yea s) S ain
(Muscle)
Ac i i y
(Muscle)
1c.148C>T c.148C>T 19 F1250 2Biceps
b achii Absen NR
p.(R50*) p.(R50*)
2c.148C>T c.1366G>A 34 F969 2Biceps
b achii Absen NR
p.(R50*) p.(V457M)
3c.2262delA c.2262delA 32 F500 1Vas us
la e alis Absen NR
p.(K754N s*) p.(K754N s*)
4c.148C>T c.148C>T 24 M 533 2Vas us
la e alis Absen Absen
p.(R50*) p.(R50*)
5c.148C>T c.613G>A 52 F2328 2Biceps
b achii Absen NR
p.(R50*) p.(G205S)
6c.148C>T c.2111C>T 48 M4889 2Biceps
b achii Absen NR
p.(R50*) p.(A704V)
7c.148C>T c.347T>C 55 M1330 2Biceps
b achii Absen NR
p.(R50*) p.(L116P)
8c.2392T>C c.2392T>C 43 M1550 2Biceps
b achii Absen Absen
p.(W798R) p.(W798R)
1
PYGM e e ence sequence: NM_005609.4;
2
as de e mined wi h he mos commonly used pheno ype se e i y
scale o GSDV, he so-called ‘Ma inuzzi scale’ ( anging om 0 [lowes ] o 3 [highes ]) o his disease [
24
];
whe e: 0 = asymp oma ic o i ually asymp oma ic (mild exe cise in ole ance, bu no unc ional limi a ion in any
daily li e ac i i y); 1 = exe cise in ole ance, con ac u es, myalgia, and limi a ion o acu e s enuous exe cise, and
occasionally in daily li e ac i i ies; no eco d o myoglobinu ia, no muscle was ing o weakness; 2 = same as 1,
plus ecu en exe ional myoglobinu ia, mode a e es ic ion in exe cise, and limi a ion in daily li e ac i i ies;
3 = same
as 2, plus ixed muscle weakness, wi h o wi hou was ing and se e e limi a ions on exe cise and mos
daily li e ac i i ies.
Table 2. Main cha ac e is ics o he heal hy con ol g oup.
Con ol
Age
Sex
CK Muscle Used
o Biopsy
PYGM S ain
(Muscle)
(Yea s) (U/L)
1 41 M N.A. Biceps b achii No mal
2 27 F <200 Vas us la e alis No mal
3 35 F <200 Biceps b achii No mal
4 52 M N.A. Biceps b achii No mal
5 35 M <200 Biceps b achii No mal
6 40 F <200 Biceps b achii No mal
7 56 F <200 Biceps b achii No mal
8 34 F N.A. Vas us la e alis No mal
By quan i a i e p o eome analysis o skele al muscle biopsies ob ained om he
Biceps b achii o Vas us la e alis o eigh GSDV pa ien s and eigh heal hy con ols using
iTRAQ labeling ollowed by e e sed-phase liquid ch oma og aphy-mass spec ome y
(RP-LC-MS/MS), 178 p o eins we e iden i ied. The pa ien and heal hy con ol samples
we e sepa a ely pooled, and pa allel double labeling was pe o med o each pool, esul ing
In . J. Mol. Sci. 2022,23, 4650 4 o 18
in wo label alues pe g oup (113 and 115 o pa ien s and 114 and 116 o con ols); all
alues we e e e enced o he alues o he 113 pa ien s’ pool (Supplemen a y Table S1).
The pep ide alue dis ibu ion o each p o ein wi h pep ide numbe >10 was com-
pa ed be ween con ol and pa ien pool alues, espec i ely, o ob ain a o al o 21 p o eins
wi h compa able con ol pool alues, on he one hand, and di e ences be ween con ols
and pa ien s, on he o he (Table 3). These esul s we e used o se a con ol/pa ien alue
a io-based h eshold, conside ing he mean o his alue o hese 21 p o eins (= 1.675).
Nex , we de ec ed he mos di e en ially exp essed p o eins by calcula ing he con-
ol/pa ien alue a io o he global da a (i.e., o all 178 p o eins de ec ed [Supplemen a y
Table S1] ega dless o he numbe o pep ides measu ed) and iden i ied 15 p o eins wi h
con ol/pa ien alues a io >1.676 (Table 4).
To allow analyses wi h TPMS echnology, all da a we e mapped o 14 unique e iewed
SwissP o KB en ies (Table 4). Nine o he ou een p o eins exhibi ed a leas a wo- old
highe change in one o he con ol pools compa ed o he 113-labeled pa ien pool, which
was used as a e e ence o labeling he es o he p o eins (indica ed as bold alues in
Table 4).
The possible ela ionship be ween he mos di e en ially exp essed p o eins (Table 4)
and GSDV a ending o hei ‘molecula cha ac e iza ion’ was e alua ed by means o ANNs
(Table 5and Supplemen a y Table S3 show esul s conside ing GSDV as a whole o consid-
e ing he di e en mo i es sepa a ely, espec i ely). A ending o he associa ed p- alues,
we so ed he ANN anking sco e in o ou ca ego ies: ‘ e y s ong’ (p< 0.01), ‘s ong’
(p< 0.05)
, ‘medium-s ong’ (p< 0.25), and ‘weak’ (p> 0.25)
(Supplemen a y Table S4)
.
Th ee p o eins, ATP2A1, MYH1, and TPM1, showed a e y s ong ela ionship wi h GSDV
(Table 5). These h ee p o eins we e pa o he unc ional mo i ele a ed cy osolic calcium le -
els, and speci ically o he pe sis en con ac ion o muscle cell sub-mo i o MYH1 and TPM1
(Supplemen a y Tables S2 and S3). The oponin iso o ms, TNNI2 and TNNT3, displayed a
s ong ela ionship wi h GSDV (Table 5) and we e also e ec o s o he sub-mo i pe sis en
con ac ion o muscle cells. All he p o eins we e ela ed o muscle s uc u e and ac i i y
and showed a s onge ela ionship wi h GSDV de ini ion han he enzyme PYGM, he
de ec i e p o ein in GSDV, which p esen ed a medium-s ong sco e wi h GSDV molecula
cha ac e iza ion and was assigned as an e ec o o he glycogenoly ic pa hway.
The PDZ and LIM domain p o ein 7 (PDLIM7) and alpha-ac inin-3 (ACTN3) showed
a medium-s ong sco e, and he es o he e alua ed p o eins showed a low p obabili y o
being ela ed o GSDV in a molecula -dependen manne (Table 5), acco ding o he used
molecula cha ac e iza ion.
A e e alua ing he ela ionship be ween he mos di e en ially exp essed p o eins
and each GSDV mo i , as desc ibed by molecula cha ac e iza ion
(Supplemen a y Table S3)
,
we obse ed ha he mo i ha exhibi ed he highes p obabili y o a ela ionship wi h he
a ailable da a was ele a ed cy osolic calcium le els, and pa icula ly he submo i pe sis en
con ac ion o muscle cell. Mos o hese p o eins showed a s ong o medium-s ong p oba-
bili y o a ela ionship wi h he mo i and submo i . In ac , o all he candida e p o eins,
he highes p obabili y sco e was obse ed o ele a ed cy osolic calcium le els.
In . J. Mol. Sci. 2022,23, 4650 5 o 18
Table 3.
P o eins wi h pep ide numbe > 10, o wi h compa able pep ide alues’ dis ibu ion be ween con ol pool alues, o wi h di e en pep ide alue dis ibu ion
be ween con ol and GSDV pa ien pool alues, espec i ely.
UniP o ID P o ein Gene Con ol/Pa ien Values Ra io Con ols (114/113 s. 116/113)
FDR q-Values
Con ols s. Pa ien s
115/113 s. 114/113 115/113 s. 116/113
P11217 Glycogen phospho ylase, muscle iso o m PYGM 4.527 0.23 5.99 ×10−11 5.99 ×10−11
P20929-2 Nebulin NEB 1.597 0.25 7.31 ×10−71.97 ×10−7
Q14324 Myosin-binding p o ein C, as - ype MYPC2 2.035 0.22 3.39 ×10−55.50 ×10−6
P14618-2 Py u a e kinase, iso o m-1 PKM1 1.379 0.03 1.58 ×10−81.01 ×10−10
P08237 ATP-dependen 6-phospho uc okinase, muscle ype PFKM 1.521 0.26 8.77 ×10−58.77 ×10−5
P04075 F uc ose-bisphospha e aldolase A ALDOA 1.626 0.02 2.80 ×10−65.35 ×10−7
Q8WZ42-11 Ti in, iso o m-11 TTN 11 1.492 0.05 1.01 ×10−10 1.48 ×10−11
Q08043 Alpha-ac inin-3 ACTN3 2.265 0.26 1.91 ×10−72.92 ×10−8
P00558-2 Phosphoglyce a e kinase 1, iso o m 2 PGK1 1.578 0.04 3.39 ×10−53.39 ×10−5
O14983-2 Iso o m SERCA1A sa coplasmic/endoplasmic
e iculum calcium ATPase 1 ATP2A1 1.680 0.26 2.66 ×10−54.24 ×10−6
O60662 Kelch-like p o ein 41 KLHL41 1.469 0.26 2.12 ×10−52.12 ×10−5
P54296 Myomesin-2 MYOM2 1.465 0.45 2.12 ×10−52.12 ×10−5
P13929 β-enolase ENO3 1.542 0.03 3.39 ×10−55.50 ×10−6
P16615-2 Iso o m 2-sa coplasmic/endoplasmic e iculum
calcium ATPase 2 ATP2A2A 1.363 0.24 7.75 ×10−31.74 ×10−3
P12882 Myosin-1 MYH-1 1.972 0.04 8.49 ×10−10 1.46 ×10−10
P04406-2 Iso o m2-glyce aldehyde-3-phospha e dehyd ogenase GAPDH 1.422 0.04 2.29 ×10−42.29 ×10−4
P06576 ATP syn hase subuni be a, mi ochond ial ATP5F1B 1.157 0.13 1.63 ×10−22.69 ×10−3
P25705 ATP syn hase subuni alpha, mi ochond ial ATP5F1A 1.142 0.07 1.34 ×10−21.05 ×10−2
P17661 Desmin DES 1.200 0.14 1.54 ×10−32.83 ×10−5
P06732 C ea ine kinase M- ype CKM 1.517 0.05 6.43 ×10−91.12 ×10−9
Q14315-2 Iso o m 2- ilamin-C FLNC 1.253 0.05 5.83 ×10−51.20 ×10−5
Con ol/pa ien alues a io mean 1.676
The able also shows he con ol/pa ien alue a io (conside ing he mean be ween 114/113 and 116/113 alues as ‘con ol alue’ and 115/113 alue as ‘pa ien alue’), he lowes
q- alue ob ained o compa ison wi hin con ols, and he highes q- alue ob ained o compa ison be ween con ols and pa ien s. Tes s applied: S uden ’s - es , Wilcoxon ank-sum, o
one-way ANOVA.
Table 4. Mos di e en ially exp essed muscle p o eins in GSDV s. con ols.
UniP o ID 115/113 Value 114/113 Value 116/113 Value Con ol/Pa ien Value Ra io SwissP o KB ID 1P o ein Gene
P11217 0.924 4.056 4.315 4.527 P11217 Glycogen phospho ylase, muscle o m PYGM
H9KVA2 0.916 1.979 2.233 2.298 P45378 T oponin T, as skele al muscle TNNT3
C9JZN9 1.079 2.263 2.668 2.286
Q08043 0.933 2.002 2.224 2.265 Q08043 Alpha-ac inin-3 ACTN3
P09493 0.987 1.955 2.265 2.137 P09493 Fou and a hal LIM domains p o ein 3 FHL3
Q13642-1 1.050 1.942 2.408 2.072 Q13642 T opomyosin alpha-1 chain TPM1
Q14324 0.979 1.790 2.196 2.035 Q14324 Iso o m 1 o ou and a hal LIM domains p o ein 1 FHL1
P12882 1.018 1.863 2.151 1.972 P12882 Myosin-1 MYH1
Q13643 1.150 1.992 2.410 1.913 Q13643 Iso o m 6 o PDZ and LIM domain p o ein 7 PDLIM7
Q9NR12-6 1.027 1.844 1.919 1.832 Q9NR12 Myosin-binding p o ein C, as - ype MYBPC2

In . J. Mol. Sci. 2022,23, 4650 6 o 18
Table 4. Con .
UniP o ID 115/113 Value 114/113 Value 116/113 Value Con ol/Pa ien Value Ra io SwissP o KB ID 1P o ein Gene
Q96A32 1.078 1.712 2.079 1.759 Q96A32 Iso o m 1A o sa coplasmic/endoplasmic e iculum calcium
ATPase 1 ATP2A1
P15121 0.910 1.473 1.676 1.731 P15121 Myosin egula o y ligh chain 2, skele al muscle iso o m MYLPF
A0A087WXS0 0.961 1.686 1.614 1.718 P48788 T oponin I, as skele al muscle TNNI2
Q96DG6 0.942 1.606 1.592 1.697 Q96DG6 Ca boxyme hylenebu enolidase homolog CMBL
O14983-2 1.030 1.758 1.705 1.680 O14983 Aldose educ ase AKR1B1
1
P o eins showing a leas a wo- old change in one con ol pool compa ed wi h he pa ien 113 pool a e in bold ace. UniP o ID as mapped by he SEQUEST algo i hm. Values in
bold ace indica e pa ien alues wi h espec o 113-labeled con ol alues > 2. SwissP o KB ID indica es he co esponding e iewed SwissP o KB iden i ie associa ed wi h each
p o eome esul .
Table 5. Rela ionship be ween candida e p o eins and GSDV desc ibed by molecula cha ac e iza ion.
Gene 1Unip o ID ANN Sco e 2Sco e Ca ego y 3GSDV E ec o and Mo i 4In e ac o GSDV E ec o Genes
MYH (*) P12882 93 Ve y s ong Ele a ed cy osolic calcium le els -
Pe sis en con ac ion o muscle cell
ATP2A1 [25] O14983 93 Ve y s ong Ele a ed cy osolic calcium le els -
TPM1 (*) P09493 92 Ve y s ong Ele a ed cy osolic calcium le els MYH11;MYH6;MYH8;MYL1;MYL6;TNNC1;TNNI1;TNNI2;TNNT1;
TNNT3;TPM2;TPM3;TPM4
Pe sis en con ac ion o muscle cell
TNNI2 (*) P48788 85 S ong Ele a ed cy osolic calcium le els MYH6;MYH8;MYL1;TNNC1;TNNI1;TNNT1;TNNT3;TPM1;TPM2;
TPM3;TPM4
Pe sis en con ac ion o muscle cell
TNNT3 (*) P45378 83 S ong Ele a ed cy osolic calcium le els MYH6;MYH8;MYL1;TNNC1;TNNI1;TNNI2;TNNT1;TPM1;TPM2;
TPM3;TPM4
Pe sis en con ac ion o muscle cell
PYGM (**) P11217 75 Medium-S ong Glycogenolysis blockade GBE1 *; GYS1 *; PHKA1 *; TPM2
PDLIM7 Q9NR12 66 Medium-S ong - TPM2
ACTN3 Q08043 43 Medium-S ong - MYH6;MYH8;MYL1;TNNC1;TNNI1;TNNI2;TNNT1;TNNT3;TPM1;
TPM2;TPM3;TPM4
MYBPC2 Q14324 38 Weak - MYH6;MYH8;MYL1;TNNC1;TNNI1;TNNI2;TNNT1;TNNT3;TPM1;
TPM2;TPM3;TPM4
FHL3 Q13643 36 Weak - -
FHL1 Q13642 27 Weak - -
MYLPF Q96A32 18 Weak - MYH11;MYL6;MYLK2;SLC2A4 *; TPM1;TPM2;TPM3;TPM4
AKR1B1 P15121 17 Weak - -
CMBL Q96DG6 10 Weak - -
1
Supe sc ip nea he gene indica es he sou ce om which he in o ma ion o column ‘GSDV e ec o and mo i ’ was ob ained: (*) Molecula Cell Biology 4 h Edi ion, Tex book, ISBN-13,
978-0-7167-3706-3; (**) OMIM#232600, Online Mendelian Inhe i ed in Man, h ps://omim.o g (accessed on 15 Feb ua y 2021);
2
anking sco es o he p obabili y o ela ionship wi h he
whole cha ac e iza ion o GSDV by means o ANNs;
3
ca ego y o he ANN anking sco e (see Supplemen a y Table S2);
4
whe he p o ein was p e iously desc ibed as implica ed in
GSDV o p ocesses associa ed wi h muscle deg ada ion.
In . J. Mol. Sci. 2022,23, 4650 7 o 18
To u he unde s and he in e molecula ela ionships iden i ied by ANNs, we gene -
a ed a p o ein in e ac ome wi h he human p o ein ne wo k used o model cons uc ion
and based on publicly a ailable sou ces. This allowed us o iden i y he in e ac ion be ween
he mos di e en ially exp essed (‘candida e’) p o eins and he e ec o p o eins iden i ied
as impo an in GSDV molecula cha ac e iza ion (Table 5, Figu e 1). Mos o he candida es
showed an in e ac ion wi h e ec o s o he biological mo i es ele a ed cy osolic calcium le els-
pe sis en con ac ion o muscle cell (Supplemen a y Tables S3 and S5). Howe e , wo o he
mos di e en ially exp essed p o eins, he skele al muscle iso o m o he myosin egula o y
ligh chain 2 (MYLPF) and PYGM, in e ac ed wi h p o eins belonging o he modula ion o
al e na i e me abolic pa hways o ene gy ob ainmen -inc eased glucose up ake mo i . In addi ion,
acco ding o he da abases used (see opological analysis in he me hods sec ion), six o
he mos di e en ially exp essed p o eins (i.e., MYH1, ATP2A1, iso o m 1 o ou and a
hal LIM domains p o ein 1 (FHL1), ou and a hal LIM domains p o ein 3 (FHL3), aldose
educ ase (AKR1B1) and ca boxyme hylenebu enolidase homolog (CMBL)) did no di ec ly
in e ac wi h any o he GSDV e ec o s no wi h any o he mos di e en ially exp essed
p o eins (Table 5).
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Figu e1.In e ac omene wo kmapbe ween hemos di e en iallyexp essedp o einsand he
geneso GSDVe ec o s.The igu eshows hemos di e en iallyexp essedp o einsand hei in‐
e nal ela ionships,aswellas he ela ionshipswi h heGSDVe ec o s.Symbolsindica ewhe he 
hep o einisincludedasane ec o o eachGSDVpa hophysiologicalmo i .Themos di e en ially
exp essedp o einsa ehighligh edacco ding oacolo edg adien showing hecon ol/pa ien  al‐
ues a io.P o einswi hexp essionle elsa leas  wo‐ oldhighe inonecon ol haninpa ien 113
(i.e.,114/113o 116/113 alues)a ema kedbya hickbo de in heco espondingsymbol.GSDV
e ec o sno de ec edwi hin hemos di e en iallyexp essedp o einsa edepic edinpu plecolo .
Ne wo kbuil usingTPMShumanp o einne wo k[16,17]and isualizedusingCy oscape e sion
3.0.0.[26].
To alida e hep edic i e esul sob ainedusing heANNanalysiss a egy,skele al
musclele elso aselec edg oupo candida ep o einswe ealsoanalyzedbyWes e n
blo inGSDVpa ien sandheal hycon ols.Weselec edascandida es hemos di e en‐
iallyexp essedp o eins ha we eclassi iedin he‘ e ys ong’and‘s ong’ca ego ies
acco ding o hei  ela ionshipwi hGSDVasawhole(Table5):MYH1,ATP2A1,TPM1,
TNNI2,andTNNT3.Besides hesep o eins,ACTN3wasalsoconside edacandida eand
analyzeddespi ebeing ankedin hemedium‐s ongca ego y,due o wo ele an  ea‐
sons:(i)i hasbeendocumen ed oin e ac wi hPYGMandimplica edinal e edmuscle
calciumhandlingin heAc n3de icien (knockou )mousemodel[27],and(ii)a leas in
emalepa ien s,ACTN3geno ypesmigh con ibu e oexplainingindi idual a iabili y
in hepheno ypicmani es a iono  hisdiso de [28,29].Weshowed ha  heexp ession
le elso all es edcandida es(MYH1,ATP2A1,TPM1,TNNI2,TNNT3,andACTN3)we e
signi ican lylowe inpa ien s hanincon ols(Figu e2andSupplemen a yFigu eS1).
Figu e 1.
In e ac ome ne wo k map be ween he mos di e en ially exp essed p o eins and he genes
o GSDV e ec o s. The igu e shows he mos di e en ially exp essed p o eins and hei in e nal
ela ionships, as well as he ela ionships wi h he GSDV e ec o s. Symbols indica e whe he he
p o ein is included as an e ec o o each GSDV pa hophysiological mo i . The mos di e en ially
exp essed p o eins a e highligh ed acco ding o a colo ed g adien showing he con ol/pa ien
alues a io. P o eins wi h exp ession le els a leas wo- old highe in one con ol han in pa ien 113
(i.e., 114/113 o 116/113 alues) a e ma ked by a hick bo de in he co esponding symbol. GSDV
e ec o s no de ec ed wi hin he mos di e en ially exp essed p o eins a e depic ed in pu ple colo .
Ne wo k buil using TPMS human p o ein ne wo k [
16
,
17
] and isualized using Cy oscape e sion
3.0.0. [26].
F om he lis o he mos di e en ially exp essed p o eins, myosin ligh chain, phos-
pho yla able, as skele al muscle (MYLPF, Q96A32), and myosin binding p o ein (MYBPC2,
Q14324) we e no p edic ed o be ela ed o GSDV (weak ela ionship in Table 5); howe e ,
In . J. Mol. Sci. 2022,23, 4650 8 o 18
hey appea ed highly connec ed o p o eins wi hin he ele a ed cy osolic Ca
2+
le els mo i
(Figu e 1), which could explain he medium-s ong signal de ec ed be ween hese p o eins
and his mo i (Supplemen a y Table S3) despi e no being i s e ec o s.
To alida e he p edic i e esul s ob ained using he ANN analysis s a egy, skele al
muscle le els o a selec ed g oup o candida e p o eins we e also analyzed by Wes e n blo
in GSDV pa ien s and heal hy con ols. We selec ed as candida es he mos di e en ially
exp essed p o eins ha we e classi ied in he ‘ e y s ong’ and ‘s ong’ ca ego ies acco ding
o hei ela ionship wi h GSDV as a whole (Table 5): MYH1, ATP2A1, TPM1, TNNI2, and
TNNT3. Besides hese p o eins, ACTN3 was also conside ed a candida e and analyzed
despi e being anked in he medium-s ong ca ego y, due o wo ele an easons: (i) i has
been documen ed o in e ac wi h PYGM and implica ed in al e ed muscle calcium handling
in he Ac n3 de icien (knockou ) mouse model [
27
], and (ii) a leas in emale pa ien s,
ACTN3 geno ypes migh con ibu e o explaining indi idual a iabili y in he pheno ypic
mani es a ion o his diso de [
28
,
29
]. We showed ha he exp ession le els o all es ed
candida es (MYH1, ATP2A1, TPM1, TNNI2, TNNT3, and ACTN3) we e signi ican ly lowe
in pa ien s han in con ols (Figu e 2and Supplemen a y Figu e S1).
In .J.Mol.Sci.2022,23,xFORPEERREVIEW9o 18
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Figu e2.Wes e nblo analysisinskele almuscle issueo  hecandida ep o eins ha we eclassi ied
in he‘ e ys ong’and‘s ong’ca ego iesin he‘molecula cha ac e iza ion’o GSDVbymeanso 
a i icialneu alne wo kanalysis.Uppe panel:Rep esen a i eblo s omGSDVpa ien sand
heal hycon ols(allpa ien s’andcon ols’Wes e nblo s o eachcandida ep o eindisplayedin
Supplemen a yFigu eS1);alpha‐ ubulinwasusedasloadingcon ol.Bo ompanel:P o einle els
inGSDVpa ien s(P,n=7)compa ed oheal hycon ols(C,n=6).*p<0.05.**p<0.01,***p<0.001
usingMann–Whi ney es .Eachdo  ep esen s hemeano  woquan i ica ions(i.e., wo echnical
eplica es).Dispe sionlinesindica emean±SD.
3.Discussion
GSDVisame abolicmyopa hy ypicallycha ac e izedbyexe cisein ole ance(i.e.,
musclepainandea lyexe ional a igue).I  heexe cises essisno  educedo hal ed,
se e emusclecon ac u es(beyond heusualbaselines a eo ‘pe sis en ’musclecon ac‐
ionanddamage)ande en ual habdomyolysismigh occu ,whichinsomecases,could
esul inacu e enal ailu e[4,30].Al hough heknowledgeo  hemolecula andpa ho‐
physiologicmechanismso GSDVhasimp o eddu ing helas  wodecades,pa icula ly
wi hinsigh sp o idedbyclinical,molecula ,o physiologicals udiesinpa ien s
[4,14,24,31–35],aswellasbys udiesinp eclinicalmodels[21,34,36–40], he eiss illno
explana ion(a leas a  hemolecula le el) o some ecognizedclinical ea u eso  he
disease,no ably hepe sis en muscledamagein heabsenceo p e iousphysicalexe cise
[8].
We he e o eaimeda in es iga ingindep h hemusclep o eomeand hemolecula 
ne wo ksassocia edwi hmuscledys unc ioninGSDVpa ien sinana emp  oiden i y
keymusclep o einsasbioma ke s ha couldhelp ounde s and heunde lyingmolecu‐
la mechanismso muscledys unc iono damage.To hebes o ou knowledge, his
ques ionhasno beenexplo edp e iously.Inacase‐con oldesignwi hmusclebiopsies
omhis ochemicalandgene icallyp o enGSDVpa ien sand omheal hycon ols,we
assessedquan i a i ep o einexp essionusing heiTRAQ echniqueand henpe o med
asys emsbiology‐baseds a egy,pa icula lyapplyingANNsand opologyin e ac ome
ne wo ks oiden i y hebes candida es.Ou analysissugges ed ha someo  heiden i‐
iedcandida ep o einsa e ela ed oGSDVdiseasep edominan ly h ough hemo i pe ‐
sis en con ac iono musclecellsdue oele a edcy osoliccalciumle els,wi h hep o eins
ACTN3,ATP2A1,MYH1,TNNT3,TPM1,andTNNI2showing hehighes p edic i e al‐
uesamongall hep o einse alua ed.Fu he mo e, he opologicalanalysisindica ed ha 
hecandida ep o einsiden i iedin hiss udyin e ac wi hp o einsin ol edin hepe ‐
sis en con ac iono musclecellsdue oele a edcy osoliccalciumle elsand hemodu‐
la iono al e na i eme abolicpa hways o ene gyob ainmen .
Thele elso ACTN3,ATP2A1,MYH1,TNNT3,TPM1,andTNNI2p o einswe e
signi ican lylowe in heskele almuscleo pa ien scompa edwi hheal hycon ols.
MYH1isaskele almusclep o ein ha ,incoo dina ionwi hac in,playsanessen ial ole
in hegene a iono ene gy o musclecon ac ion h oughATPhyd olysis[41].ATP2A1,
hesa coplasmic/endoplasmic e iculumcalciumATPase1(p e iouslyknownas
SERCA1),isamemb anep o ein ha is esponsible o  he anspo o calcium om he
Figu e 2.
Wes e n blo analysis in skele al muscle issue o he candida e p o eins ha we e classi ied
in he ‘ e y s ong’ and ‘s ong’ ca ego ies in he ‘molecula cha ac e iza ion’ o GSDV by means
o a i icial neu al ne wo k analysis. Uppe panel: Rep esen a i e blo s om GSDV pa ien s and
heal hy con ols (all pa ien s’ and con ols’ Wes e n blo s o each candida e p o ein displayed in
Supplemen a y Figu e S1); alpha- ubulin was used as loading con ol. Bo om panel: P o ein le els
in GSDV pa ien s (P, n= 7) compa ed o heal hy con ols (C, n= 6). * p< 0.05. ** p< 0.01,*** p< 0.001
using Mann–Whi ney es . Each do ep esen s he mean o wo quan i ica ions (i.e., wo echnical
eplica es). Dispe sion lines indica e mean ±SD.
3. Discussion
GSDV is a me abolic myopa hy ypically cha ac e ized by exe cise in ole ance (i.e., mus-
cle pain and ea ly exe ional a igue). I he exe cise s ess is no educed o hal ed, se e e
muscle con ac u es (beyond he usual baseline s a e o ‘pe sis en ’ muscle con ac ion and
damage) and e en ual habdomyolysis migh occu , which in some cases, could esul in
acu e enal ailu e [
4
,
30
]. Al hough he knowledge o he molecula and pa hophysiologic
mechanisms o GSDV has imp o ed du ing he las wo decades, pa icula ly wi h insigh s
p o ided by clinical, molecula , o physiological s udies in pa ien s [
4
,
14
,
24
,
31
–
35
], as well
as by s udies in p eclinical models [
21
,
34
,
36
–
40
], he e is s ill no explana ion (a leas a he
molecula le el) o some ecognized clinical ea u es o he disease, no ably he pe sis en
muscle damage in he absence o p e ious physical exe cise [8].
We he e o e aimed a in es iga ing in dep h he muscle p o eome and he molecula
ne wo ks associa ed wi h muscle dys unc ion in GSDV pa ien s in an a emp o iden i y
key muscle p o eins as bioma ke s ha could help o unde s and he unde lying molecula
mechanisms o muscle dys unc ion o damage. To he bes o ou knowledge, his ques ion
has no been explo ed p e iously. In a case-con ol design wi h muscle biopsies om
his ochemical and gene ically p o en GSDV pa ien s and om heal hy con ols, we assessed
In . J. Mol. Sci. 2022,23, 4650 9 o 18
quan i a i e p o ein exp ession using he iTRAQ echnique and hen pe o med a sys ems
biology-based s a egy, pa icula ly applying ANNs and opology in e ac ome ne wo ks o
iden i y he bes candida es. Ou analysis sugges ed ha some o he iden i ied candida e
p o eins a e ela ed o GSDV disease p edominan ly h ough he mo i pe sis en con ac ion
o muscle cells due o ele a ed cy osolic calcium le els, wi h he p o eins ACTN3, ATP2A1,
MYH1, TNNT3, TPM1, and TNNI2 showing he highes p edic i e alues among all he
p o eins e alua ed. Fu he mo e, he opological analysis indica ed ha he candida e
p o eins iden i ied in his s udy in e ac wi h p o eins in ol ed in he pe sis en con ac ion
o muscle cells due o ele a ed cy osolic calcium le els and he modula ion o al e na i e
me abolic pa hways o ene gy ob ainmen .
The le els o ACTN3, ATP2A1, MYH1, TNNT3, TPM1, and TNNI2 p o eins we e
signi ican ly lowe in he skele al muscle o pa ien s compa ed wi h heal hy con ols. MYH1
is a skele al muscle p o ein ha , in coo dina ion wi h ac in, plays an essen ial ole in he
gene a ion o ene gy o muscle con ac ion h ough ATP hyd olysis [
41
]. ATP2A1, he
sa coplasmic/endoplasmic e iculum calcium ATPase 1 (p e iously known as SERCA1), is
a memb ane p o ein ha is esponsible o he anspo o calcium om he sa coplasm
back in o he sa coplasmic e iculum a e each sa come ic con ac ion, and whose unc ion
is dependen on he ene gy deli e ed by ATP hyd olysis. Likewise, ATPA21 con ibu es o
he exci a ion/con ac ion balance in ol ed in muscle ac i i y [
42
]. A dec ease in ATP2A1
le els would esul in an impai men in he eup ake o calcium back in o he sa coplas-
mic e iculum a e each con ac ion, wi h subsequen accumula ion o his ion in he
sa coplasm and impai men o muscle ibe elaxa ion— ha is, pe manen muscle con ac-
ion and muscle con ac u es. In e es ingly, besides he associa ion o p ima y pa hogenic
gene ic a ian s in he ATP2A1 gene wi h B ody myopa hy (OMIM#601003, a a e au oso-
mal ecessi e diso de cha ac e ized by painless muscle c amping and exe cise-induced
impai ed muscle elaxa ion) [
43
], o he condi ions linked wi h aging, neu odegene a ion,
and muscula dys ophy also dep ess ATP2A1 unc ion wi h he po en ial o impai in a-
cellula calcium homeos asis and con ibu e o muscle a ophy and weakness [
42
]. The e
is some con o e sy on how o assess calcium homeos asis in di e en human diseases
since mos esea ch has been pe o med in mu ine models [
44
–
47
]. On he o he hand, he
s abili y o ac in ilamen s in he muscle ibe s is ensu ed by he unc ion o opomyosin
(TPM1), which, in associa ion wi h he oponin complex (TNNI2 and TNNT3), plays a key
ole in he egula ion o calcium-dependen in e ac ions du ing muscle con ac ion [
48
].
In addi ion, ACTN3 plays an impo an ole in he s abili y o he con ac ile appa a us
a he Z-line, whe e his p o ein c oss-links and ancho s ac in ilamen s [
49
]. The e o e,
ou indings sugges ha dec eased exp ession o he a o emen ioned p o eins in GSDV
could be associa ed, a leas in pa , wi h he al e ed muscle con ac ile unc ion and a
p obable al e a ion o muscle calcium kine ics in his diso de . On he o he hand, PYGM
could also be in ol ed no only in ene gy gene a ion om glycogen b eakdown, bu also in
he O-linked
β
-N-ace ylglucosamine (O-GlcNa)c pos - ansla ional modi ica ions o some
p o eins [
6
,
50
]. In his e ec , O-GlcNAcyla ion plays an impo an ole in se e al skele al
muscle unc ions, including op imal modula ion o calcium homeos asis in ibe s [51,52].
Ou s udy is limi ed by he small sample size, al hough we belie e his is jus i iable
in he con ex o a a e condi ion such as McA dle disease. We also ailed o collec all he
samples om he same muscle, al hough he as majo i y o samples co esponded o he
Biceps b achii, and he p opo ion o muscle ype (i.e., 6/2 o Biceps b achii/Vas us la e alis)
was iden ical in pa ien s and heal hy con ols. Impo an ly, ou app oach also lacked a
compa ison g oup o pa ien s wi h simila ea u es o hose o McA dle disease, such as
muscle con ac u es—al hough we a e no awa e o any neu omuscula condi ion whe e
muscle con ac u es a e as equen o pe sis en as in McA dle disease—and he e o e we
canno add ess i he de ec ed di e en ially exp essed p o eins a e p ima ily o seconda ily
egula ed. In addi ion, i mus be kep in mind ha wi h ega d o po en ial bioma ke s
o McA dle disease, ou indings mus be iewed as mechanis ic—hope ully p o iding
use ul insigh s and amewo k o u u e esea ch— a he han p ac ical ones since muscle
In . J. Mol. Sci. 2022,23, 4650 16 o 18
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