Abb e ia ions: ACS, acu e co ona y synd ome; ADHP, 10-ace yl-3,7-dihyd oxyphenoxazine; APF, 2-
(6-(4-aminophenoxy)-3-oxo-3H-xan en-9-yl)-benzoic acid; BSA, bo ine se um albumin; CAD,
co ona y a e y disease; CI, ca bonyla ion index; CV, ca dio ascula ; DNPH, 2,4-
dini ophenylhyd azine; DMF, dime hyl o mamide; DTDP, di hiodipy idine; DTNB, 5,5’-di hio-
bis(2-ni obenzoic acid); DTT, di hio ei ol; ECL, enhanced chemiluminescence; EGTA,
e hyleneglycol e aace ic acid; Fac i e, ca diomyocy e ac i e o ce; Fpassi e, ca diomyocy e passi e
o ce; HDL, high-densi y lipop o ein; HF, hea ailu e; HOCl, hypochlo ous acid; H2O2, hyd ogen
pe oxide; Iso, isola ing solu ion; LDL, low-densi y lipop o ein; LV, le en icula ; Me SO,
me hionine sul oxide; MHC, myosin hea y chain; MI, myoca dial in a c ion; MLC-1, myosin ligh
chain–1; MPO, myelope oxidase; MPO-I, MPO inhibi o (4-aminobenzhyd azide); MyBP-C, myosin-
binding p o ein C; N2B, s i i in iso o m; N2BA, complian i in iso o m; NAC, N-ace yl-L-
cys eine; NO, ni ic oxide; NOS, ni ic oxide syn hase; NTB, 2‐ni o‐5‐ hiobenzoic acid; PBS,
phospha e-bu e ed saline; pCa50, measu e o calcium sensi i i y; PMSF, phenylme hylsul onyl
luo ide; ROS, eac i e oxygen species; SDS, sodium dodecyl sulpha e; SH, sul hyd yl; Tm,
opomyosin.
*Co esponding au ho a : Di ision o Clinical Physiology, Ins i u e o Ca diology, Facul y
o Medicine, Uni e si y o Deb ecen, H-4032 Deb ecen, Mó icz Zsigmond k . 22.,
Hunga y, Telephone/Fax: +36 52 255928, E-mail: [email p o ec ed]
O iginal Con ibu ion 1
2
Myelope oxidase impai s he con ac ile unc ion in isola ed human ca diomyocy es 3
4
Judi Kalász, Enikő Pász o né Tó h, Miklós Fagyas, Ágnes Balogh, A ila Tó h, Vik ó ia 5
Csa ó, Is án Édes, Zol án Papp and A ila Bo bély* 6
7
Di ision o Clinical Physiology, Ins i u e o Ca diology, Facul y o Medicine, Uni e si y o 8
Deb ecen, Deb ecen, Hunga y 9
10
11
2
Abs ac 1
Pu pose: We se ou o cha ac e ize he mechanical e ec s o myelope oxidase (MPO) in 2
isola ed le en icula human ca diomyocy es. Oxida i e myo ilamen p o ein modi ica ions 3
(sul hyd yl (SH) g oup oxida ion and ca bonyla ion) induced by he pe oxidase and 4
chlo ina ing ac i i ies o MPO we e addi ionally iden i ied. The speci ici y o he MPO-5
e oked unc ional al e a ions was es ed wi h an MPO inhibi o (MPO-I) and he an ioxidan 6
amino acid Me . 7
Resul s: The combined applica ion o MPO and i s subs a e, hyd ogen pe oxide (H2O2), 8
la gely educed he ac i e o ce (Fac i e), inc eased he passi e o ce (Fpassi e) and dec eased 9
he Ca2+ sensi i i y o o ce p oduc ion (pCa50) in pe meabilized ca diomyocy es. H2O2 alone 10
had signi ican ly smalle e ec s on Fac i e and Fpassi e and did no al e pCa50. The MPO-I 11
blocked bo h he pe oxidase and chlo ina ing ac i i ies, while Me selec i ely inhibi ed he 12
chlo ina ing ac i i y o MPO. All o he MPO-induced unc ional e ec s could be p e en ed 13
by he MPO-I and Me . Bo h H2O2 alone and MPO+H2O2 educed he SH con en o ac in 14
and inc eased he ca bonyla ion o ac in and myosin-binding p o ein C o he same ex en . 15
Nei he he SH-oxida ion no he ca bonyla ion o he gian sa come ic p o ein i in was 16
a ec ed by hese ea men s. 17
Conclusions: MPO ac i a ion induces a ca diomyocy e dys unc ion by a ec ing Ca2+-18
egula ed ac i e and Ca2+-independen passi e o ce p oduc ion and myo ilamen Ca2+ 19
sensi i i y, independen ly o p o ein SH oxida ion and ca bonyla ion. The MPO-induced 20
dele e ious unc ional al e a ions can be p e en ed by he MPO-I and Me . Inhibi ion o MPO 21
may be a p omising he apeu ic a ge o limi myoca dial con ac ile dys unc ion du ing 22
in lamma ion. 23
Keywo ds: ca diomyocy e con ac ile unc ion, myelope oxidase, hyd ogen pe oxide, 24
oxida i e pos - ansla ional p o ein modi ica ions, an ioxidan s 25
3
In oduc ion 1
Oxida i e s ess- ela ed myo ilamen p o ein al e a ions ha e been shown o play key oles in 2
he impai ed ca diomyocy e con ac ili y in esponse o myoca dial in lamma ion, ischemia-3
epe usion inju y and le en icula (LV) emodeling ollowing a myoca dial in a c ion 4
(MI) [1, 2]. In pa icula , eac i e oxygen species (ROS) oxidize cellula componen s [3], 5
leading o ca diomyocy e con ac ile dys unc ion, myocy e apop osis o ca diac hype ophy 6
[4, 5]. 7
Myelope oxidase (MPO; EC 1.11.2.2) is a membe o he heme pe oxidase 8
supe amily, syn hesized by neu ophils, monocy es and mac ophages, s o ed in hei 9
azu ophilic g anules and eleased in subs an ial amoun upon leukocy e ac i a ion [6]. MPO 10
has bene icial e ec s in he inna e hos de ense mechanisms [7]. Conside able e idence has 11
eme ged o sugges , ha ROS o ma ion by MPO p omo es a ious dele e ious ac ion in he 12
ca dio ascula (CV) sys em and con ibu es o he de elopmen o CV diseases [6]. 13
Indi iduals wi h a o al o sub o al MPO de iciency (a de ec wi h a equency o ≈1 in e e y 14
2000 o 4000 Caucasians) a e p o ec ed om CV diseases [6]. An ele a ed le el o 15
ci cula ing MPO is a p ognos ic ma ke o mo ali y and p edic s he isks o subsequen 16
majo ad e se ca diac e en s in pa ien s wi h acu e co ona y synd ome (ACS) [8], 17
pa icula ly in associa ion wi h a low LV ejec ion ac ion [9]. MPO also con ibu es o 18
ad e se LV emodeling a e a MI [10]. MPO exe s ad e se e ec s on he ascula u e, 19
oxidizes low-densi y lipop o ein (LDL) [11], impai s he high-densi y lipop o ein (HDL) 20
unc ion [12] and educes he bioa ailabili y o ni ic oxide (NO) [13]. MPO can he e o e 21
se e as a aluable bioma ke o in lamma ion in co ona y a e y disease (CAD) and ACS 22
[14]. The se um le el o MPO co ela es posi i ely wi h he se e i y o he LV dys unc ion 23
and seems o be an essen ial ac o in he de elopmen and exace ba ion o hea ailu e (HF) 24
[15, 16]. In e es ingly, he MPO concen a ion was ea lie ound no o di e in ischemic and 25
4
non-ischemic ca diomyopa hy, sugges ing ha MPO has an independen pa hogenic ole in 1
he LV dys unc ion [17]. 2
MPO is known o gene a e nume ous eac i e oxidan s and di usible adical species 3
ia i s pe oxidase and chlo ina ing ac i i ies, which a e capable o p omo ing an a ay o 4
e e sible and i e e sible pos - ansla ional p o ein modi ica ions [18, 19]. The ela i e 5
concen a ions o chlo ide and he educing subs a e de e mine whe he MPO uses i s 6
subs a e hyd ogen pe oxide (H2O2) o pe oxida ion o chlo ina ion. MPO ampli ies he 7
oxida i e po en ial o H2O2 [20-22], which may o igina e om a numbe o sou ces in i o, 8
including leukocy e NADPH oxidases, xan hine oxidase and uncoupled NO syn hase (NOS) 9
[23, 24]. The pe usion o isola ed a hea s wi h H2O2 led o disul ide c oss-b idge 10
o ma ion in ac in and opomyosin (Tm) [25]. In one o ou p e ious s udies, he sul hyd yl 11
(SH) oxida ion o ac in and myosin ligh chain-1 (MLC-1) was sugges ed as he mechanism 12
in he H2O2-e oked dep essed ca diomyocy e con ac ili y [26]. 13
MPO is unique in i s abili y o c ea e hypochlo ous acid (HOCl, a po en an imic obial 14
agen ) h ough i s chlo ina ing ac i i y [22]. In e es ingly, he ca diac issue is highly 15
suscep ible o oxida ion e en by physiological concen a ions o HOCl [27]. Impo an ly, 16
HOCl is much mo e e ec i e han H2O2 in oxidizing p o eins in he myoca dium [27], i 17
causes SH oxida ion [28] and ca bonyla ion in myo ilamen p o eins [29], i dis u bs Ca2+ 18
homeos asis and Ca2+ handling [30], i inc eases he in acellula Ca2+ concen a ion in 19
isola ed a [31] and abbi [32] en icula ca diomyocy es, and i induces ca diomyocy e 20
dea h in a s [33]. I is also e y impo an o conside , how a H2O2 o HOCl can di use on 21
he cellula scale and whe he hese subs ances a e capable o pene a e he cell memb anes. 22
H2O2 is s able [34], memb ane pe meable [35], al hough, in i o concen a ion o H2O2 23
highly depends on i s gene a ion and consump ion a es [36, 37]. HOCl appea s o be mo e 24
oxic and eac i e and can also pene a e h ough cell memb anes, bu has a much sho e 25
5
li espan. An in i o s udy e ealed ha HOCl p oduc ion by neu ophils can be as high as 1
450 mM/h, which was shown o be less in an in i o model [38]. MPO gene a es HOCl in 2
mic o-mola concen a ion [39], bu in in lamma ic issue i is es ima ed o be as high as 5 3
mM [40]. 4
The an ioxidan amino acid Me ac s as a sca enge o HOCl and has been shown o 5
p e en he HOCl-induced mo phological changes and con ac ile dys unc ion in mu ine 6
myocy es [41]. Mo eo e , he ac ha MPO-de i ed chlo ina ing compounds can se e as 7
speci ic bioma ke s o disease p og ession has a ac ed conside able in e es in he 8
de elopmen o he apeu ically use ul MPO inhibi o s (MPO-Is) [42]. 9
Al hough he ole o MPO-de i ed oxidan s in he pa hogenesis o myoca dial 10
ischemia and HF is ela i ely well es ablished, only limi ed da a a e a ailable as conce ns he 11
exac cellula and subcellula mechanisms h ough which MPO could di ec ly a ec he 12
con ac ili y o he myoca dial cells, especially a he le el o he myo ib illa p o eins. In his 13
s udy, he e o e, we se ou (1) o cha ac e ize he unc ional e ec s o MPO and i s subs a e 14
H2O2 on single, pe meabilized human ca diomyocy es; (2) o iden i y he biochemical 15
al e a ions induced by he pe oxidase and chlo ina ing ac i i ies o MPO; (3) o in es iga e 16
he speci ici y o he MPO-induced con ac ile changes by using he MPO inhibi o (MPO-I) 17
4-aminobenzhyd azide and he an ioxidan amino acid Me ; and (4) o explo e he MPO-18
ela ed e e sible and i e e sible oxida i e myo ilamen p o ein modi ica ions in he human 19
LV myoca dium. 20
21
22
23

6
Ma e ials and me hods 1
2
I. Human myoca dial samples 3
LV myoca dial issue was ob ained om he hea s o ou gene al o gan-dono pa ien s (41- 4
and 46-yea -old women, and 53- and 57-yea -old men). All o hese pa ien s we e ee o any 5
ca diac abno mali ies and had no ecei ed any medica ion excep o plasma olume 6
expande s, dobu amine and u osemide. The cause o dea h included ce eb al con usion, 7
ce eb al hemo hage and suba achnoidal hemo hage. All biopsies we e anspo ed in 8
ca dioplegic solu ion (pH 7.4; in mM: NaCl 110, KCl 16, MgCl2 1.6, CaCl2 1.2, NaHCO3 5) 9
and we e ozen in liquid ni ogen and s o ed a -80 °C a he labo a o y. The expe imen s on 10
human issues complied in ull wi h he Helsinki Decla a ion o he Wo ld Medical 11
Associa ion and we e app o ed by he Hunga ian Minis y o Heal h (No. 323-8/2005-12
1018EKU) and by he Ins i u ional E hical Commi ee a he Uni e si y o Deb ecen, 13
Hunga y. 14
15
II. Fo ce measu emen s in pe meabilized ca diomyocy e p epa a ions 16
Fo ce measu emen s we e pe o med as desc ibed p e iously [43]. In b ie , ozen issue 17
samples we e i s de os ed and mechanically dis up ed in cell isola ion solu ion (Iso) (in 18
mM: KCl 100, e hyleneglycol e aace ic acid (EGTA) 2, MgCl2 1, Na2ATP 4, imidazole 10; 19
pH 7.0) con aining phenylme hylsul onyl luo ide (PMSF, 0.5 mM, Sigma-Ald ich, S . Louis, 20
MO, USA), leupep in (40 μM, Sigma, S . Louis, MO, USA) and E-64 (10 μM, Sigma-21
Ald ich, S . Louis, MO, USA) p o ease inhibi o s. The mechanically isola ed cells we e 22
skinned by incuba ion in Iso supplemen ed wi h 0.5% ( / ) T i on X-100 (Sigma-Ald ich, S . 23
Louis, MO, USA) o 5 min. T i on-X-100 was emo ed by washing a leas h ee imes in 24
Iso (1 ml in each washing s ep) and he skinned myocy es we e kep in cell Iso on ice un il 25
7
he measu emen s. A skinned single ca diomyocy e was moun ed be ween wo hin needles, 1
which we e a ached o a o ce ansduce elemen (SensoNo , Ho en, No way) and an 2
elec omagne ic mo o (Au o a Scien i ic Inc., Au o a, Canada) h ough he use o silicone 3
adhesi e (DAP, Bal imo e, MD, USA) o de e mina ion o he mechanical pa ame e s. The 4
measu emen s we e pe o med a 15°C on he s age o a ligh mic oscope. The a e age 5
sa come e leng h was adjus ed o 2.3 μm. 6
The composi ions o he elaxing and ac i a ing solu ions used du ing o ce 7
measu emen s we e calcula ed as desc ibed p e iously [43]. Bo h solu ions we e 8
supplemen ed wi h p o ease inhibi o s: leupep in (40 μM) and E-64 (10 μM). The pCa, i.e. 9
he -log10[Ca2+] alues o he elaxing and ac i a ing solu ions (pH 7.2), we e 9.0 and 4.75, 10
espec i ely. Solu ions wi h in e media e ee [Ca2+] le els we e ob ained by mixing 11
ac i a ing and elaxing solu ions [44]. Isome ic o ce p oduc ion was measu ed a e he 12
p epa a ion had been ans e ed om he elaxing solu ion o a se o Ca2+-con aining 13
solu ions. When a s eady o ce le el had been eached, he leng h o he myocy e was 14
educed by 20% wi hin 2 ms, and he myocy e was hen quickly es e ched ( elease- es e ch 15
maneu e ). As a esul , he o ce i s d opped om he peak isome ic le el o ze o 16
(di e ence = o al peak isome ic o ce, F o al) and hen s a ed o ede elop. Abou 6 s a e 17
he onse o o ce ede elopmen , he ca diomyocy e was e u ned o he elaxing solu ion, 18
whe e he leng h o he myocy e was again educed by 20% o 8 s o de e mine he Ca2+-19
independen passi e o ce componen (Fpassi e). The Ca2+-ac i a ed isome ic o ce (Fac i e) 20
was calcula ed by sub ac ing Fpassi e om F o al. Fac i e a submaximal le els o ac i a ion was 21
no malized o ha a maximal ac i a ion (pCa 4.75). The ea e , he no malized o ce alues 22
we e plo ed agains he Ca2+ concen a ion o he ac i a ing solu ions o c ea e a sigmoidal 23
cu e, in o de o de e mine he Ca2+ sensi i i y o o ce p oduc ion (pCa50). Maximal ac i e 24
8
o ce was also es ed a he end o he expe imen s a pCa 4.75. Expe imen s ha yielded a 1
alue below 80% o he ini ial alue we e disca ded. 2
To de e mine he mechanical consequences o myo ilamen p o ein oxida ion, 3
ca diomyocy es we e exposed o Iso supplemen ed wi h H2O2 (30 μM, Sigma-Ald ich, S . 4
Louis, MO, USA) o 15 min; MPO+H2O2 (8 U/l, Abcam, Camb idge, UK) o 15 min; 5
MPO+H2O2+MPO-I 4-aminobenzhyd azide (50 µM, Cayman Chemicals, Ann A bo , MI, 6
USA) o 15 min; o MPO+H2O2+Me (10 mM, Sigma-Ald ich, S . Louis, MO, USA) o 15 7
min a 15 °C. The e e sibili y o MPO+H2O2 e oked e ec s we e examined by he 8
applica ion o he educing agen di hio ei ol (DTT, Sigma-Ald ich, S . Louis, MO, USA, 10 9
mM, 30 min) o MPO+H2O2- ea ed ca diomyocy es. Fo ce-pCa ela ionships and pCa50 10
alues we e de e mined be o e and a e he applica ion o hese agen s. The e ec s o he 11
applied agen s on Fac i e and Fpassi e we e exp essed ela i e o hei con ol (un ea ed, be o e 12
applica ion o he agen a pCa 4.75 and pCa 9.0, espec i ely). Changes in Fac i e and Fpassi e 13
upon applica ion o he agen s we e compa ed wi h he o ce alues measu ed a e 14
incuba ion o he ca diomyocy es in Iso o 15 min ( ime con ol). 15
16
III. Measu emen s o MPO ac i i ies 17
MPO chlo ina ion and pe oxida ion assay ki s (Cayman Chemicals, Ann A bo , MI, USA) 18
we e used. The chlo ina ion ac i i y assay u ilizes a non luo escen subs a e (APF, 2-(6-(4-19
aminophenoxy)-3-oxo-3H-xan hen-9-yl)benzoic acid), which is clea ed by he MPO- 20
gene a ed hypochlo i e (OCl-) o p oduce highly luo escen luo escein. The pe oxidase 21
ac i i y assay uses a non luo escen subs a e (ADHP, 10-ace yl-3,7-dihyd oxyphenoxazine) 22
which is con e ed by MPO o he luo escen eso u in. Fluo escence was de ec ed wi h a 23
No oS a Mic opla e Reade (BMG Lab ech, O enbe g, Ge many) a λex 485 nm, λem 520 nm 24
in he chlo ina ion assay, and a λex 544 nm, λem 590 nm in he pe oxidase assay. The eac ion 25
9
solu ion con ained he non luo escen subs a e (APF (18 µM) o ADHP (45 µM)), assay 1
bu e (phospha e-bu e ed saline (PBS), pH 7.4) and H2O2 (30 µM), o MPO+H2O2 (38 U/l), 2
o MPO+H2O2+MPO-I (50 µM) o MPO+H2O2+Me (10 mM). Ac i i ies we e measu ed o 3
5 min a 24-s in e als. Fluo escence in ensi ies we e i ed by linea eg ession analysis 4
(be o e sa u a ion) and he slope o his ela ion was used o calcula e MPO ac i i ies. Values 5
we e co ec ed o he backg ound ( he ac i i y de e mined in he absence o MPO). 6
7
IV. Biochemical assays o he iden i ica ion o oxida i e p o ein modi ica ions 8
1. Ellman’s eac ion 9
O e all myo ilamen SH g oup con en was de e mined by Ellman’s eac ion. Skinned 10
ca diomyocy es we e ea ed wi h Iso ( ime con ol) o wi h Iso supplemen ed wi h H2O2 and 11
MPO as desc ibed o he mechanical expe imen s. Washing s eps ollowed he ea men s 12
and he ca diomyocy es we e hen incuba ed o 15 min in Ellman’s eagen (5,5’-di hio-13
bis(2-ni obenzoic acid), DTNB; Sigma-Ald ich, S . Louis, MO, USA), which eac s wi h 14
myo ilamen SH g oups and p oduces he yellow 2‐ni o‐5‐ hiobenzoic acid (NTB). The 15
abso bance o NTB was measu ed wi h No oS a Mic opla e Reade a 412 nm. N-Ace yl-L-16
cys eine (NAC, Sigma-Ald ich, S . Louis, MO, USA) was used o calib a e he NTB 17
abso bance in ela ion o he amoun o SH g oups. A known concen a ion o NAC was 18
eac ed wi h Ellman’s eagen and he abso bance a 412 nm, i ed wi h a single exponen ial, 19
se ed as calib a ion cu e. The SH con en s in 1-mg lyophilized myoca dial samples we e 20
calcula ed om he measu ed abso bance, he issue weigh and he calib a ion cu e. 21
Measu emen s we e pe o med in iplica es. 22
23
24
25
16
E ec s o MPO+H2O2 on he SH oxida ion and ca bonyla ion o myo ilamen p o eins 1
A emp s we e made o iden i y he changes in he oxida i e s a us o myo ilamen p o eins 2
con ibu ing o he MPO-induced ca diomyocy e dys unc ion in pa allel wi h he unc ional 3
measu emen s. Rela i e SH con en s we e de e mined in human LV skinned ca diomyocy es. 4
The baseline SH con en o myo ilamen p o eins in he dono hea samples a ied be ween 5
98.04.6% and 104.13.9% (p=0.35). Ellman’s eac ion e ealed a small, bu signi ican 6
dec ease in he o e all amoun o SH g oups in esponse o H2O2 ( o 90.4±1.5%, p<0.05, 7
n=3) o MPO+H2O2 ea men s ( o 86.7±4.0%, p<0.01, n=3) (Fig. 4A). An SH g oup 8
bio inyla ion assay was applied o iden i y indi idual myo ib illa p o eins a ec ed by MPO-9
media ed SH oxida ion. Samples ea ed wi h he oxida i e agen DTDP we e used as posi i e 10
con ols. H2O2 and MPO+H2O2 lowe ed he SH con en o ac in o simila ex en s ( o 11
75.9±7.1%, p<0.01, n=4, and 84.2±4.4%, p<0.05 s. ime con ol, espec i ely, n=9) (Fig. 12
4B). In con as , he SH con en s o myosin-binding p o ein C (MyBP-C, Fig. 4C) and he 13
mo e complian (N2BA) and s i e (N2B) iso o ms o he gian sa come ic p o ein i in we e 14
no a ec ed by hese ea men s (Fig. 4D-F). Using immunoblo s a Tm and an ac in 15
con aining complex was obse ed a on app oxima ely 90 kDa molecula weigh le el unde 16
non- educing condi ions (in a bu e no con aining β-ME), howe e , no inc ease in i s 17
in ensi y and ha o Tm and ac in could be de ec ed a e H2O2 and MPO+H2O2 ea men s 18
(Fig. 5). 19
P o ein ca bonyla ion assays e ealed a modes , bu signi ican inc ease in he 20
ca bonyla ion o ac in upon H2O2 ea men (CI=1.1±0.05, p<0.05 s. he ime con ol, n=5), 21
which was no u he a ec ed by he addi ion o MPO (CI=1.1±0.05, p=0.1 s. he ime 22
con ol, n=11) (Fig. 6A). Simila ly as o ac in, a sligh , bu signi ican inc ease in he 23
ca bonyl con en o MyBP-C was obse ed bo h a e H2O2 (CI=1.5±0.2, p<0.05 s. he ime 24
con ol, n=2) and a e MPO+H2O2 applica ion (CI=1.4±0.2, p<0.05 s. he ime con ol, 25

17
n=4) (Fig. 6B). The ex en o ca bonyl g oup o ma ion in he N2BA and N2B i in iso o ms 1
emained unal e ed a e H2O2 o MPO+H2O2 ea men (CI=0.9±0.2 and CI=1.0±0.2 o 2
N2BA; CI=1.0±0.1 and CI=0.9±0.1 o N2B, espec i ely) (Fig. 6C-E). 3
18
Discussion 1
2
This is he i s epo ed in es iga ion o he di ec e ec s o MPO on he con ac ile unc ion 3
o single, isola ed human myoca dial cells. The in i o model expe imen s e ealed ha (1) 4
MPO impai s Ca2+-dependen isome ic o ce gene a ion, inc eases he Ca2+-independen 5
Fpassi e and dec eases he Ca2+ sensi i i y o o ce p oduc ion; (2) he MPO-induced unc ional 6
changes can be p e en ed by an MPO-I and he an ioxidan Me ; (3) he le els o SH 7
oxida ion in ac in and o ca bonyla ion in ac in and MyBP-C a e inc eased by he applica ion 8
o MPO+H2O2 o H2O2 alone; (4) he MPO-e oked unc ional e ec s a e p obably media ed 9
by he chlo ina ing ac i i y o MPO. 10
Myoca dial in lamma ion and ischemia- epe usion inju y a e cha ac e ized by 11
enhanced ex en s o oxida i e s ess and con ac ile dys unc ion [46]. The applica ion o 12
MPO+H2O2 o human ca diomyocy es app eciably educed he Ca2+-ac i a ed Fac i e and 13
ma kedly dec eased pCa50. In con as , H2O2 (30 μM) alone induced a smalle dec ease in 14
Fac i e. Consis en wi h ou indings, a lowe concen a ion o H2O2 (10 μM) did no esul in a 15
dec ease in he maximal Ca2+-ac i a ed o ce in skinned a hea p epa a ions [47, 48]. This 16
sugges s ha he ac ion o H2O2 on con ac ile o ce gene a ion is concen a ion-dependen . 17
Lowe concen a ions ha e no measu able e ec s, whe eas highe concen a ions a ec he 18
ca diomyocy e con ac ili y. The dele e ious e ec on Fac i e can be explained by he MPO-19
media ed H2O2-de i ed p oduc ion o HOCl. In a p e ious s udy, HOCl ea men alone (10 20
μM and 50 μM o 1 min) e oked a signi ican dec ease in he maximum Ca2+-ac i a ed o ce 21
[47], simila ly o he esul o MPO+H2O2 ea men in he p esen s udy. In e es ingly, 22
nei he he H2O2- no he MPO-induced unc ional changes we e ela ed o any de e io a ion 23
in he c oss-s ia ion pa e n o he ca diomyocy es unde he ligh mic oscope. I is impo an 24
19
o no e, howe e , ha elec on mic oscopy has e ealed a myo ilamen la ice dis up ion a e 1
HOCl ea men [47]. 2
The sub le inc ease a e H2O2 applica ion and he ma ked ele a ion in he Ca2+-3
independen Fpassi e upon MPO+H2O2 ea men in he p esen s udy a e consis en wi h he 4
obse a ions ha H2O2 a low (<10 µM) concen a ion did no al e Fpassi e, while HOCl (10 5
μM and 50 μM) induced a signi ican ise in Fpassi e o skinned a abeculae [48]. I is well 6
es ablished ha he gian sa come ic p o ein i in plays a key ole in he de elopmen o 7
Fpassi e in pe meabilized ca diomyocy es by ac ing as a molecula sp ing in he sa come e 8
[49]. The ca diomyocy e Fpassi e can be modula ed by he i in iso o m swi ch (be ween he 9
sho and s i N2B and he longe and mo e complian N2BA iso o ms [50]) and by se e al 10
pos - ansla ional modi ica ions, including phospho yla ion [51], SH oxida ion [52] and 11
po en ially ca bonyla ion. One elegan s udy demons a ed ha he oxida i e s ess-induced 12
o ma ion o disul ide b idges wi hin he i in molecule (N2B unique sequence, N2B-Us) 13
educed he con ou leng h o he N2B-Us, leading o s i ening o he whole i in molecule 14
[52]. In he p esen s udy, nei he SH oxida ion no ca bonyla ion o he N2B and N2BA i in 15
iso o ms was ound o be a ec ed by MPO o H2O2 ea men . This may be explained by he 16
dis inc sensi i i ies o he i in N2B iso o m, ac in and MyBP-C o oxida i e changes based 17
on he di e ences in hei ul as uc u es and SH g oup con en s. Ou esul s indica e ha 18
modi ica ions o he han i in SH oxida ion o ca bonyla ion migh be esponsible o he 19
ma ked ele a ion in Fpassi e a e MPO ea men in human ca diomyocy es. 20
The signi ican dec ease obse ed in pCa50 a e MPO+H2O2 in his s udy is in ma ked 21
con as wi h he p e ious inding o an inc ease in pCa50 in skinned a abeculae in 22
esponse o HOCl ea men [48]. This appa en ly con lic ing esul migh be explained by (1) 23
he di e en concen a ion o HOCl p oduced by he MPO unde ou expe imen al 24
condi ions; (2) a di e ence in suscep ibili y o he myo ilamen s o HOCl be ween he wo 25
20
species; and (3) he di e ence in he expe imen al se ing, pe meabilized, single 1
ca diomyocy es p esen ing a negligible di usion obs acle in compa ison wi h abeculae. 2
Fu he , he p onounced MPO-induced dec ease in pCa50 sugges s ha di e en myo ilamen 3
p o ein modi ica ions occu and con ibu e o pCa50 in he cou se o MPO and H2O2
4
ea men s. Unde hese expe imen al condi ions H2O2 mo e p obably induced a s uc u al, 5
a he han a egula o y al e a ion in he con ac ile appa a us because pCa50 was no a ec ed. 6
The dele e ious e ec on he maximal Fac i e and he modes inc ease in Fpassi e upon H2O2 7
adminis a ion implies ha he H2O2-induced con ac ile al e a ions could be explained by a 8
educ ion in he numbe o o ce-gene a ing c oss-b idges due o he diminished longi udinal 9
ansmission o o ce along he sa come es. These indings a e consis en wi h he 10
obse a ions o MacFa lane e al., who exposed he supe oxide anion ( om which H2O2 11
o med endogenously h ough spon aneous o supe oxide dismu ase-ca alyzed dismu a ion) 12
o chemically skinned a ca diac muscles. They also ound a dose-dependen educ ion in he 13
maximal Fac i e wi hou any al e a ion in he pCa50 and concluded ha some aspec o he 14
c oss-b idge beha io is pa icula ly ulne able o supe oxide [53]. 15
A subs an ial numbe o da a indica e ha he inhibi ion o MPO may well be use ul 16
in CV pa hologies cha ac e ized by ele a ed MPO le els (myoca dial in lamma ion, 17
ischemia- epe usion inju y and acu e MI). Thus, despi e he ac ha MPO-Is may ha e 18
ad e se e ec s on he unc ion o MPO in he inna e hos -de ense mechanisms, po en ial 19
he apeu ic in e en ions h ough which o inhibi MPO ha e a oused conside able in e es 20
[42]. In he p esen s udy, bo h he MPO-I 4-aminobenzhyd azide (50 µM) and he 21
an ioxidan amino acid Me (10 mM) we e equally able o p e en all o he MPO-e oked 22
dele e ious con ac ile e ec s in skinned human ca diomyocy es, he la e po en ially by 23
sca enging he HOCl gene a ed by MPO. MPO ac i i y assays sugges ed ha he Me -24
inhibi ed chlo ina ing ac i i y is esponsible o he MPO-e oked unc ional changes. HOCl 25
21
eac s mos apidly wi h he sul u -con aining esidues (Me and Cys) [54]. I is likely, 1
he e o e, ha he high concen a ion o Me used in his s udy diminished he HOCl-e oked 2
oxida i e capaci y. The oxida ion o Me esidues esul s in he gene a ion o Me -sul oxide 3
(Me SO), a p ocess ha may be e e sed by Me SO educ ase [55]. Me is he e o e 4
conside ed o play a p o ec i e ole agains he dele e ious e ec s o p o ein oxida ion [28]. 5
In e es ingly, he incomple e e e sion and oxida ion o physiologically ele an Me esidues 6
has been shown o con ibu e o he impai ed unc ion o p o eins [56], including ac in [57]. I 7
is impo an o no e, ha o he HOCl sca enging subs ances han Me (e.g. glu a hione, 8
au ine and L-asco bic acid) we e also es ed ecen ly in HOCl sca enging assays [58]. Gi en 9
he apid eac ion a es o HOCl wi h biological ma e ials, howe e , much highe doses o L-10
asco bic acid and hiols we e equi ed o e ec i ely p o ec agains he di ec oxida i e 11
damage induced by HOCl. This la e sugges s ha inhibi ing he gene a ion o HOCl may be 12
a be e choice han sca enging HOCl a e i s gene a ion, o amelio a ion o HOCl induced 13
biological damage. 14
The dis inc e ec o he educing agen DTT on Fac i e and Fpassi e a e MPO+H2O2
15
ea men ound in his s udy migh be explained by di e en modi ica ions on he s uc u al 16
con o ma ion o unc ional ac i i y o he con ac ile and egula o y myo ilamen p o eins. 17
The p ecise na u e o he edox-dependen unc ional changes upon H2O2 and MPO+H2O2
18
ea men is complex and de e mined also by he ype and si e o he induced pos -19
ansla ional modi ica ions on indi idual p o eins wi hin he sa come e [59]. SH esidues o 20
Cys can unde go bo h e e sible and i e e sible modi ica ions. The eac ion be ween he Cys 21
hiola e anion and H2O2 esul s in o ma ion o in a- o in e molecula disul ide bonds, which 22
is e e sible, bu u he oxida ion can gene a e sul inic o sul onic acid, which a e 23
conside ed i e e sible al e a ions [60]. The HOCl-induced p o ein ca bonyla ion is hough 24

22
o be i e e sible, while me hionine oxida ion can be e e sed by Me SO- educ ase [28] o 1
can lead o an i e e sible p oduc (me hionine-sul one) [55]. 2
The ex en o o e all SH oxida ion obse ed a e MPO ea men in his s udy was 3
compa able o ha in hea issue slices exposed o high-dose HOCl [27]. The e is 4
biochemical e idence ha oxida i e modi ica ions modula e he a chi ec u e o he 5
myo ilamen p o ein ac in [61] and myosin [62]. In i o exposu e o pe meabilized human 6
LV ca diomyocy es o he oxida i e agen DTDP esul ed in a dec ease in maximal Ca2+-7
ac i a ed o ce p oduc ion wi h a pa allel educ ion in he SH con en o ac in and MLC-1 8
[26]. Consis en wi h his, in he p esen s udy H2O2 dec eased he SH con en o ac in. 9
Howe e , despi e he ma ked educ ion in Fac i e, no addi ional dec ease in his pa ame e was 10
de ec ed a e MPO+H2O2 applica ion, sugges ing ha SH oxida ion may no be he main 11
con ibu o o he MPO-e oked dec ease in Fac i e unde hese expe imen al condi ions. 12
Mo eo e , o ma ion o an ac in and a Tm con aining p o ein complex obse ed in his s udy 13
is also unlikely o be esponsible o he con ac ile changes obse ed in he ca diomyocy es 14
a e H2O2 and MPO+H2O2 adminis a ion. The possible unc ional consequences o he 15
obse ed p o ein complexes equi e u he examina ions. 16
In a mouse model o expe imen al MI, we ecen ly iden i ied he inc eased 17
ca bonyla ion o ac in and myosin hea y chain (MHC) in he in a c ed a ea [2]. Simila ly o 18
MPO, in i o Fen on-based myo ilamen ca bonyla ion dec eased pCa50, i espec i ely o he 19
phospho yla ion s a us o he myo ilamen s. Mo eo e , pCa50 co ela ed s ongly wi h he 20
myo ilamen ca bonyla ion le els. In acco d wi h his, a ma ked (3- old) inc ease in ca bonyl 21
g oup o ma ion in ac in was obse ed a e 1 mM, bu no a e 0.1 mM H2O2 ea men [25]. 22
The applica ion o H2O2 o ca diomyocy es a a concen a ion highe han 0.1 mM was 23
hinde ed by i s inhibi o y e ec on he ac i i y o MPO [42]. 30 μM H2O2 lowe ed Fac i e in 24
pa allel wi h a sligh , bu signi ican inc ease in he ca bonyla ion o ac in and MyBP-C. 25
23
Simila ly o SH oxida ion, ca bonyla ion o hese myo ilamen p o eins was no u he 1
a ec ed by he addi ion o MPO, despi e i s no ewo hy e ec s on ca diomyocy e ac i e and 2
passi e o ce p oduc ion. This implies ha he physiological e ec s o MPO-ca alyzed 3
oxida i e p ocesses a e independen o SH g oup oxida ion o ca bonyla ion o human 4
myoca dial p o eins. 5
Oxida i e modi ica ions in he myoca dium p ima ily ha e been conside ed o esul 6
in educed o ce gene a ion, as also demons a ed in he p esen s udy. Howe e , ecen 7
e idence sugges s a mo e complex pic u e. Reac i e oxygen and ni ogen species can ac i a e 8
p o ec i e mechanisms and signaling pa hways ( edox egula ion) [60] o e en inc ease 9
ca diac pe o mance [63]. Mild oxida i e s ess induced S-ni osyla ion a speci ic Cys 10
esidues was shown o be ca diop o ec i e [64]. Sub le inc eases in ROS p oduc ion may 11
e en enhance ca diac con ac ili y unde physiological condi ions [65]. Indeed, ce ain 12
oxida i e myo ilamen modi ica ions can lead o posi i e unc ional consequences, such as 13
ni oxyl (HNO), a eac i e ni ogen species ela ed o ni ic oxide, induces o ma ion o ac in-14
Tm he e odime s, which co ela es wi h he inc ease in Ca2+ sensi i i y and dime ic o ms o 15
MHC and MLC-1, which a e associa ed wi h inc eased o ce gene a ion [63]. HNO was also 16
shown o inc ease maximum ension and Ca2+ sensi i i y o abeculae sa come es 17
unc ioning in si u [66]. These esul s s ongly sugges ha he bene icial o dele e ious 18
unc ional ou come is likely dic a ed by he s eng h and he na u e o he oxidizing agen and 19
he edox milieu o he myo ilamen compa men . 20
Since isola ion o ca diomyocy es and assessmen o myo ilamen p ope ies was 21
pe o med on LV biopsies o unused dono hea s, possible changes in he phospho yla ion 22
and oxida i e s a us o he myo ilamen p o eins occu ing be o e o du ing issue sampling 23
may ha e been in e e ed wi h he esul s o his s udy. In addi ion, ac i a ion o he β-24
ad ene gic signaling and a ious oxida i e pa hways migh also in luence he baseline 25
24
mechanical and biochemical cha ac e is ics o he ca diomyocy es. We ha e checked he 1
baseline unc ional pa ame e s o he cells in he s udy and ound no majo di e ences in he 2
ca diomyocy e mechanical p ope ies. Mo eo e , he baseline myo ilamen SH con en s we e 3
also simila in he LV samples used o he ca diomyocy e isola ion. These obse a ions a e 4
in line wi h hose ound in ou p e ious s udy, in which he educing agen DTT did no a ec 5
Fac i e and pCa50 o ca diomyocy es de i ed om human dono hea s [26]. 6
In his s udy LV hea samples we e ozen and hei unc ional and biochemical 7
p ope ies we e e alua ed upon hawing. To alida e he use o de os ed biopsy samples, in 8
one o ou p e ious s udies [67] o ce eco dings o ca diomyocy es isola ed om a biopsy 9
sample immedia ely a e p ocu emen we e compa ed o hose o ca diomyocy es isola ed 10
om a de os ed biopsy o he same pa ien . These o ce eco dings yielded iden ical esul s. 11
In addi ion, he ex en o issue he e ogenei y was also add essed in p e ious s udies using 12
explan ed hea s [68, 69] o su gically p ocu ed biopsies [70]. In hese s udies he a iabili y 13
o o ce measu emen s o ca diomyocy es isola ed om di e en po ions o he hea was 14
always less han 5%. 15
I is also impo an o no e ha se e al addi ional MPO-sensi i e p ocesses, such as 16
p o ein halogena ion [71], p o ein ni a ion [72], Me oxida ion, sul onic acid gene a ion 17
(Cys), [73] o p o ein deg ada ion [28], migh be esponsible o he obse ed unc ional 18
al e a ions. Fu he s udies a e clea ly equi ed o elucida e he ela i e con ibu ions o hese 19
p ocesses o he o e all pump unc ion du ing human ca diac pa hologies associa ed wi h 20
ele a ed MPO le els. 21
22
Conclusion 23
MPO-de i ed oxidan s con ibu e o myoca dial con ac ile dys unc ion by dec easing 24
he ca diomyocy e o ce p oduc ion and he myo ilamen Ca2+ sensi i i y and inc easing 25
25
Fpassi e in human ca diomyocy es. These e ec s could be p e en ed by MPO inhibi ion and 1
he an ioxidan Me . The associa ed unc ional and biochemical al e a ions may p o ide a 2
pha macological ool o he p e en ion and/o e e sion o MPO-induced con ac ile p o ein 3
al e a ions, which could ha e he apeu ic implica ions in ca diac pa hologies cha ac e ized by 4
ele a ed MPO le els. 5
6
7
8
9
10
11
32
[44] Fabia o, A.; Fabia o, F. Calcula o p og ams o compu ing he composi ion o he 1
solu ions con aining mul iple me als and ligands used o expe imen s in skinned muscle 2
cells. J Physiol (Pa is) 75:463-505; 1979. 3
[45] Can on, M.; Menazza, S.; Shee an, F. L.; Pol e ino de Lau e o, P.; Di Lisa, F.; Pepe, 4
S. Oxida ion o myo ib illa p o eins in human hea ailu e. J Am Coll Ca diol 57:300-309; 5
2011. 6
[46] Vin en-Johansen, J. In ol emen o neu ophils in he pa hogenesis o le hal 7
myoca dial epe usion inju y. Ca dio asc Res 61:481-497; 2004. 8
[47] Mille , D. J.; MacFa lane, N. G. In acellula e ec s o ee adicals and eac i e 9
oxygen species in ca diac muscle. J Hum Hype ens 9:465-473; 1995. 10
[48] MacFa lane, N. G.; Mille , D. J. E ec s o he eac i e oxygen species hypochlo ous 11
acid and hyd ogen pe oxide on o ce p oduc ion and calcium sensi i i y o a ca diac 12
myo ilamen s. P luge s A ch 428:561-568; 1994. 13
[49] an Hee ebeek, L.; F anssen, C. P.; Hamdani, N.; Ve heug , F. W.; Somsen, G. A.; 14
Paulus, W. J. Molecula and cellula basis o dias olic dys unc ion. Cu Hea Fail Rep 15
9:293-302; 2012. 16
[50] LeWin e , M. M.; G anzie , H. Ca diac i in: a mul i unc ional gian . Ci cula ion 17
121:2137-2145; 2010. 18
[51] Bo bely, A.; an Hee ebeek, L.; Paulus, W. J. T ansc ip ional and pos ansla ional 19
modi ica ions o i in: implica ions o dias ole. Ci c Res 104:12-14; 2009. 20
[52] G u zne , A.; Ga cia-Manyes, S.; Ko e , S.; Badilla, C. L.; Fe nandez, J. M.; Linke, 21
W. A. Modula ion o i in-based s i ness by disul ide bonding in he ca diac i in N2-B 22
unique sequence. Biophys J 97:825-834; 2009. 23

33
[53] MacFa lane, N. G.; Mille , D. J. Dep ession o peak o ce wi hou al e ing calcium 1
sensi i i y by he supe oxide anion in chemically skinned ca diac muscle o a . Ci c Res 2
70:1217-1224; 1992. 3
[54] Szuchman-Sapi , A. J.; Pa ison, D. I.; Ellis, N. A.; Hawkins, C. L.; Da ies, M. J.; 4
Wi ing, P. K. Hypochlo ous acid oxidizes me hionine and yp ophan esidues in myoglobin. 5
F ee Radic Biol Med 45:789-798; 2008. 6
[55] Vog , W. Oxida ion o me hionyl esidues in p o eins: ools, a ge s, and e e sal. 7
F ee Radic Biol Med 18:93-105; 1995. 8
[56] Oien, D. B.; Canello, T.; Gabizon, R.; Gasse , M.; Lundquis , B. L.; Bu ns, J. M.; 9
Mosko i z, J. De ec ion o oxidized me hionine in selec ed p o eins, cellula ex ac s and 10
blood se ums by no el an i-me hionine sul oxide an ibodies. A ch Biochem Biophys 485:35-11
40; 2009. 12
[57] Dalle-Donne, I.; Rossi, R.; Gius a ini, D.; Gagliano, N.; Di Simplicio, P.; Colombo, 13
R.; Milzani, A. Me hionine oxida ion as a majo cause o he unc ional impai men o 14
oxidized ac in. F ee Radic Biol Med 32:927-937; 2002. 15
[58] Zhongwei Liu, Y. Y., Suhua Wang, Wei-Yi Ong, Choon Nam Ong, Dejian Huang. 16
Assaying myelope oxidase inhibi o s and hypochlo ous acid sca enge s in HL60 cell line 17
using quan um do s. Ame ican Jou nal o Biomedical Sciences 5:140-153; 2013. 18
[59] S einbe g, S. F. Oxida i e s ess and sa come ic p o eins. Ci c Res 112:393-405; 19
2013. 20
[60] Chung, H. S.; Wang, S. B.; Venka aman, V.; Mu ay, C. I.; Van Eyk, J. E. Cys eine 21
oxida i e pos ansla ional modi ica ions: eme ging egula ion in he ca dio ascula sys em. 22
Ci c Res 112:382-392; 2013. 23
[61] Milzani, A.; DalleDonne, I.; Colombo, R. P olonged oxida i e s ess on ac in. A ch 24
Biochem Biophys 339:267-274; 1997. 25
34
[62] Wilson, G. J.; dos Remedios, C. G.; S ephenson, D. G.; Williams, D. A. E ec s o 1
sulphyd yl modi ica ion on skinned a skele al muscle ib es using 5,5'-di hiobis(2-2
ni obenzoic acid). J Physiol 437:409-430; 1991. 3
[63] Gao, W. D.; Mu ay, C. I.; Tian, Y.; Zhong, X.; DuMond, J. F.; Shen, X.; S anley, B. 4
A.; Fos e , D. B.; Wink, D. A.; King, S. B.; Van Eyk, J. E.; Paolocci, N. Ni oxyl-media ed 5
disul ide bond o ma ion be ween ca diac myo ilamen cys eines enhances con ac ile 6
unc ion. Ci c Res 111:1002-1011; 2012. 7
[64] Koh , M. J.; E angelis a, A. M.; Fe li o, M.; S eenbe gen, C.; Mu phy, E. S-8
ni osyla ion o TRIM72 a cys eine 144 is c i ical o p o ec ion agains oxida ion-induced 9
p o ein deg ada ion and cell dea h. J Mol Cell Ca diol 69:67-74; 2014. 10
[65] Kubin, A. M.; Skoumal, R.; Ta i, P.; Konyi, A.; Pe jes, A.; Leskinen, H.; Ruskoaho, 11
H.; Szokodi, I. Role o eac i e oxygen species in he egula ion o ca diac con ac ili y. J 12
Mol Cell Ca diol 50:884-893; 2011. 13
[66] Dai, T.; Tian, Y.; Tocche i, C. G.; Ka o i, T.; Mu phy, A. M.; Kass, D. A.; Paolocci, 14
N.; Gao, W. D. Ni oxyl inc eases o ce de elopmen in a ca diac muscle. J Physiol 15
580:951-960; 2007. 16
[67] Bo bely, A.; an de Velden, J.; Papp, Z.; B onzwae , J. G.; Edes, I.; S ienen, G. J.; 17
Paulus, W. J. Ca diomyocy e s i ness in dias olic hea ailu e. Ci cula ion 111:774-781; 18
2005. 19
[68] an De Velden, J.; Klein, L. J.; Za emba, R.; Boon je, N. M.; Huyb eg s, M. A.; 20
S ooke , W.; Eijsman, L.; de Jong, J. W.; Visse , C. A.; Visse , F. C.; S ienen, G. J. E ec s o 21
calcium, ino ganic phospha e, and pH on isome ic o ce in single skinned ca diomyocy es 22
om dono and ailing human hea s. Ci cula ion 104:1140-1146; 2001. 23
[69] an de Velden, J.; Papp, Z.; Za emba, R.; Boon je, N. M.; de Jong, J. W.; Owen, V. 24
J.; Bu on, P. B.; Goldmann, P.; Jaque , K.; S ienen, G. J. Inc eased Ca2+-sensi i i y o he 25
35
con ac ile appa a us in end-s age human hea ailu e esul s om al e ed phospho yla ion o 1
con ac ile p o eins. Ca dio asc Res 57:37-47; 2003. 2
[70] an de Velden, J.; Klein, L. J.; an de Bijl, M.; Huyb eg s, M. A.; S ooke , W.; 3
Wi kop, J.; Eijsman, L.; Visse , C. A.; Visse , F. C.; S ienen, G. J. Isome ic ension 4
de elopmen and i s calcium sensi i i y in skinned myocy e-sized p epa a ions om di e en 5
egions o he human hea . Ca dio asc Res 42:706-719; 1999. 6
[71] Da ies, M. J. Myelope oxidase-de i ed oxida ion: mechanisms o biological damage 7
and i s p e en ion. J Clin Biochem Nu 48:8-19; 2011. 8
[72] Yan, Z.; Liang, F.; Guo, L.; Wang, J.; Wang, X. L.; Cheng, X. L.; Ma, X. L.; Liu, H. 9
R. Myelope oxidase inc eased ca diomyocy e p o ein ni a ion in mice subjec ed o nonle hal 10
mechanical auma. Biochem Biophys Res Commun 393:531-535; 2010. 11
[73] Hawkins, C. L.; Mo gan, P. E.; Da ies, M. J. Quan i ica ion o p o ein modi ica ion 12
by oxidan s. F ee Radic Biol Med 46:965-988; 2009. 13
14 15
16
36
Figu e cap ions 1 2
FIG. 1. Myelope oxidase (MPO) and hyd ogen pe oxide (H2O2) impai he o ce 3
gene a ion o human pe meabilized ca diomyocy es. (A) A single ca diomyocy e (isola ed 4
om a human le en icle myoca dium) moun ed be ween a sensi i e o ce ansduce and 5
an elec omagne ic mo o . (B) O iginal o ce eco dings o maximal Ca2+-ac i a ed ac i e 6
(Fac i e) and Ca2+-independen passi e (Fpassi e) o ce componen s be o e (le panel) and a e 7
MPO+H2O2 ea men ( igh panel) a pCa (i.e -log10[Ca2+]) 4.75 and pCa 9.0, espec i ely. 8
MPO + H2O2 we e applied in Iso o 15 min. (C) pCa- o ce ela ionships de e mined be o e 9
and a e H2O2 o MPO+H2O2 ea men s (numbe o ca diomyocy es, n=7 and 12, 10
espec i ely). Fo ce le els a e exp essed ela i e o he alues measu ed be o e he 11
ea men s. (* s. Be o e H2O2, # s. Be o e MPO+H2O2,  s. A e H2O2; *,#,p<0.05) (D) 12
Changes in Fpassi e measu ed in he p esence o Iso and a e sequen ial applica ions o H2O2 13
o MPO+H2O2. (E) Signi ican igh wa d shi (i.e. dec ease in he Ca2+ sensi i i y o o ce 14
p oduc ion (pCa50)) in he no malized pCa- o ce ela ionships in esponse o MPO+H2O2, bu 15
no change a e H2O2 ea men (F). (Da a a e exp essed as mean±SEM.) 16
17
FIG. 2. Simila e ec s o he MPO inhibi o (MPO-I), bu dis inc ac ions o me hionine 18
(Me ) on he chlo ina ing and pe oxidase ac i i ies o myelope oxidase (MPO). Me 19
inhibi s he chlo ina ing (A), bu no he pe oxidase (B) ac i i y o MPO. Values a e 20
exp essed ela i e o he MPO ac i i y measu ed in he p esence o Iso and hyd ogen 21
pe oxide (H2O2). (Da a a e exp essed as mean±SEM, *p<0.05). 22
23
FIG. 3. The myelope oxidase inhibi o (MPO-I) and me hionine (Me ) p e en he 24
MPO-induced changes in isome ic o ce p oduc ion o human ca diomyocy es. 25
Maximal (pCa 4.75) Ca2+-dependen ac i e (Fac i e) (A) and Ca2+-independen (pCa 9) passi e 26
37
(Fpassi e) o ce (B) in le en icula ca diomyocy es ea ed in isola ing solu ion (Iso) 1
supplemen ed wi h hyd ogen pe oxide (H2O2) o myelope oxidase (MPO)+H2O2, MPO-I o 2
Me . Fo ces a e exp essed ela i e o he alues measu ed be o e he subsequen ea men s. 3
The MPO-I (C) and Me (D) p e en he MPO-e oked igh wa d shi in he no malized pCa-4
o ce ela ionships. Dashed lines indica e o ce-pCa ela ionships de e mined in Iso. (E) 5
Changes in he Ca2+ sensi i i y o o ce p oduc ion (pCa50) upon H2O2, MPO+H2O2, MPO-I 6
o Me ea men s. (Da a a e exp essed as mean±SEM, *p<0.05) 7
8
FIG. 4. Myelope oxidase (MPO) and hyd ogen pe oxide (H2O2) simila ly al e 9
sul hyd yl (SH) g oup oxida ion in myo ilamen p o eins. (A) SH g oup oxida ion in a 10
ca diomyocy e suspension ea ed in isola ing solu ion (Iso) supplemen ed wi h H2O2 and 11
MPO (Ellman’s eac ion). (B-E) Rep esen a i e examples o SH con en de e mina ion in 12
ac in (B), myosin-binding p o ein C (MyBP-C) (C), N2BA (D, E) and N2B (D, F) i in 13
iso o ms a e H2O2 o MPO+H2O2 ea men s h ough use o a p o ein bio inyla ion assay. 14
T2 indica es he i in deg ada ion p oduc . Samples exposed o di hiodipy idine (DTDP, 2.5 15
mM, o 2 min) we e used as posi i e con ol. To al p o ein amoun was de e mined wi h he 16
Syp o Ruby P o ein Blo S ain. Values a e exp essed ela i e o he SH g oup con en 17
de e mined in Iso ( ime con ol). (Da a a e exp essed as mean±SEM, *p<0.05 s. Iso.) 18
19
FIG. 5. No addi ional disul ide c oss-b idge o ma ion a e hyd ogen-pe oxide (H2O2) 20
and myelope oxidase (MPO) ea men . Le en icula myoca dial samples solubilized in 21
non- educing (-β-me cap oe hanol (β-ME)) o educing (+β-ME) sample bu e s and p obed 22
wi h an i- opomyosin (Tm) (le panel) and an i-ac in ( igh panel) an ibodies a e 23
immunoblo ing. (P o ein amoun was de e mined wi h he Syp o Ruby P o ein Blo S ain, 24
MW - molecula weigh .) 25

38
1
FIG. 6. Myelope oxidase (MPO) and hyd ogen pe oxide (H2O2) inc ease he 2
ca bonyla ion o ac in and myosin-binding p o ein C (MyBP-C), bu no ha o i in. 3
Rep esen a i e examples and measu emen o ca bonyl g oup o ma ion in ac in (A), MyBP-4
C (B), N2BA (C, D) and N2B (C, E) i in iso o ms ea ed wi h isola ing solu ion (Iso) 5
supplemen ed wi h H2O2 o MPO+H2O2. Le en icula myoca dial samples ea ed wi h 6
Fen on eagen (FeSO4, H2O2 and asco bic acid) se ed as posi i e con ol. P o ein 7
ca bonyla ion is exp essed as ca bonyla ion index (CI) (CI=1, ca bonyl g oup con en 8
measu ed in Iso). To al p o ein amoun was de e mined wi h he Syp o Ruby P o ein Blo 9
S ain. (Da a a e exp essed as mean±SEM, *p<0.05) 10
11