Myoca dial e ec o In e medin in
hype ophic hea :
mechanisms and in ol emen o
endo helial dys unc ion
Ma a And eia Ribei o de Pinho
Po o
2013
Myoca dial e ec o
In e medin in
hype ophic hea :
mechanisms and
in ol emen o
endo helial dys unc ion
Ma a And eia Ribei o de Pinho
Biologia Celula e Molecula
Depa amen o de Biologia
2013
O ien ado
Dou o a Ana Luísa Pi es
In es igado a Associada
Depa amen o de Obs e ícia e Ginecologia
Uni e sidade de Camb idge, Reino Unido
Todas as co eções de e minadas
pelo jú i, e só essas, o am e e uadas.
O P esiden e do Jú i,
Po o, ______/______/_________
Disse ação de candida u a ao g au de
Mes e em Biologia Celula e Molecula
subme ida à Faculdade de Ciências
da Uni e sidade do Po o.
O p esen e abalho oi desen ol ido sob a
o ien ação cien í ica da Dou o a Ana Pi es, na
Faculdade de Medicina das Uni e sidades do
Po o e de Coimb a.
Disse a ion o applying o a Mas e ’s Deg ee
in Cell and Molecula Biology, submi ed o he
Facul y o Sciences o he Uni e si y o Po o.
The p esen wo k was de eloped unde he
scien i ic supe ision o Doc o Ana Pi es and
was done a he Facul y o Medicine o he
Uni e si ies o Po o and Coimb a.
Lis o O iginal Publica ions
This disse a ion con ains esul s om he ollowing published a icle:
Pi es AL, Pinho M, Al es BS, Pinho S, Sena C, Seica RM, Lei e-Mo ei a AF.
Re e se myoca dial e ec s o in e medin in p essu e o e loaded a s: ole o
endo helial ni ic oxide syn hase ac i i y. J Physiol 2012.

“The ung o a ladde was ne e mean o es upon,
bu only o hold a man's oo long enough
o enable him o pu he o he somewha highe .”
Thomas Huxley
Acknowledgemen s
"Não é aos sal os que se sobe uma mon anha, mas a passos len os."
São G egó io Magno
Ao longo des a caminhada a é es a e apa, mui os o am aqueles que, de uma ou
de ou a o ma, me apoia am e incen i a am! Sob e udo, es es companhei os de iagem
es i e am semp e p esen es em cada uma dessas pequenas passadas que ui dando. A
odos eles, o meu since o ob igado!
Aos meus pais e a ós ag adeço a cons an e mo i ação. Não são p ecisos ges os
g andes ou p og amados: é na simplicidade do dia-a-dia e no exemplo de ida que semp e
me êm mos ado que a ida é uma lu a cons an e pelo sucesso e pela elicidade!
Aos meus i mãos, Ma co, So ia e E a ag adeço a boa-disposição, a aleg ia e a
paciência cons an es. O osso apoio incondicional o nou a conclusão des a e apa mais
ácil e possí el!
Ao Mano ag adeço a paciência, a comp eensão, o op imismo, a aleg ia e a escu a.
Nos momen os menos bons, o eu ab aço con o ou-me; nos momen os bons ele oi
exp essão de aleg ia e signi icou semp e mo i ação pa a con inua .
À Dou o a Ana Luísa Pi es ag adeço a since idade com que semp e exp essou
as suas opiniões, a comp eensão pelos e os que ui come endo, a mo i ação, o apoio e a
disponibilidade que semp e demons ou. Consigo ap endi, c esci e o nei-me mais
au ónoma!
À Dou o a C is ina Sena ag adeço os conhecimen os cien í icos que me
ansmi iu du an e as expe iências ealizadas em Coimb a.
À D a. Bá ba a Al es ag adeço a companhia, as con e sas, o abalho e o es o ço
nas mui as ho as que passamos jun as no labo a ó io.
À D a. Ma a Oli ei a ag adeço o apoio labo a o ial que semp e mos ou.
Às uncioná ias do Se iço de Fisiologia D. F ancelina, D. Ma ga ida e D.
Rosinha ag adeço o apoio p á ico que semp e me de am e a simpa ia com que semp e me
a a am.
Ao Dou o Adelino Mo ei a ag adeço o espaço e ma e ial disponibilizados sem os
quais es e abalho não pode ia e sido conc e izado.
Po im, mas igualmen e impo an e, ag adeço às minhas amigas Nádia Eusébio,
So ia Mo ei a e Ma ina San os o apoio incondicional, os conselhos e a comp eensão,
especialmen e nos momen os menos bons! A pa ilha de conhecimen os e expe iências oi
ambém essencial ao longo des e pe cu so.
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Resumo
A insu iciência ca díaca a ec a g ande pa e da população mundial de al o ma que
é já conside ada uma epidemia. Es a sínd ome ca ac e iza-se po al e ações es u u ais e
uncionais no co ação, que passa a bombea o sangue de o ma insu icien e. O es udo da
p og essão des a deso dem ca díaca é po an o essencial e os es udos ecen es isam
iden i ica po enciais al os e apêu icos que sejam mais e icazes. Recen emen e, oi
iden i icado um no o pép ido chamado in e medina (IMD), cujos ní eis de exp essão es ão
aumen ados em á ios modelos de pa ologias ca díacas, incluindo hipe ensão sis émica e
isquemia. No en an o, o seu papel nes as pa ologias ainda não es á comple amen e
es abelecido.
Assim, começamos po es uda o e ei o miocá dico da IMD em dois modelos de
insu iciência ca díaca induzida po sob eca ga de p essão: “TAC”, cuja sob eca ga esul a
do ape o da ao a; o modelo que denominamos po “L-NAME”, cuja sob eca ga de
p essão esul a da inibição da p odução de óxido ní ico, po adminis ação o al de L-
NAME, um inibido das sin ases de óxido ní ico. Nos seus espec i os con olos (“Sham” e
“C l”), a adição de IMD induziu um e ei o ino ópico nega i o. Apesa de semelhan e em
alo absolu o, o e ei o da IMD nos modelos de doença oi o almen e opos o: e i icamos
um e ei o ino ópico posi i o acompanhado po um aumen o da lusi opia. Ao ní el basal,
e i icamos que os dois modelos de doença ap esen am unção sis ólica inal e ada,
hipe o ia do en ículo esque do e s ess oxida i o aumen ado. Ao ní el p o eico,
e i icamos que, nos con olos, a adição de IMD iplicou a quan idade de eNOS os o ilada
no esíduo Se -1177, suge indo um aumen o de ac i idade des a p o eína. Além disso,
ambém obse amos um aumen o da os o ilação da oponina I, possi elmen e de ida à
ac i ação da ia de sinalização dependen e de óxido ní ico e cGMP. Po ou o lado, nos
modelos de doença, e i icamos que a os o ilação basal da eNOS es a a aumen ada e
que a adição de IMD não al e ou es es ní eis. Ve i icamos, con udo, que hou e um
aumen o da os o ilação da os olambam, possi elmen e de ida à ac i ação da ia de
sinalização dependen e de cAMP e PKA.
Assim, mos amos pela p imei a ez que a IMD induz e ei os opos os em
miocá dios saudá eis e doen es, que se de em à incapacidade de a IMD aumen a a
ac i idade da eNOS nos modelos de doença. Além disso, e dado que mecanismos
dis in os medeiam es es e ei os, es e abalho suge e que a dis unção endo elial ca díaca
desempenha um papel p eponde an e na pa o isiologia da insu iciência ca díaca po
sob eca ga de p essão e, ao mesmo empo, apon a a IMD como po encial ma cado da
dis unção endo elial ca díaca.
Pala as-cha e: insu iciência ca díaca, dis unção endo elial ca díaca, sin ase endo elial de óxido ní ico, in e medina.
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Abs ac
Hea ailu e a ec s many people a ound he wo ld and hen i is conside ed an
epidemic. This synd ome is cha ac e ized by s uc u al and unc ional changes in hea ,
which ine icien ly pumps blood. The s udy o p og ession o his ca diac diso de is hus
c ucial and ecen s udies aim o iden i y po en ial and mo e e ec i e he apeu ic a ge s.
Recen ly, a new pep ide called in e medin (IMD) was iden i ied, whose exp ession le els
a e augmen ed in se e al models o ca diac pa hologies, such as sys emic hype ension
and ischemia. Howe e , i s ole in hese pa hologies is no ye comple ely es ablished.
The e o e, we s a ed o s udy myoca dial e ec s o IMD in wo hea ailu e models
induced by p essu e-o e load: “TAC”, whose o e load esul s om ao ic banding; he
model which we named as “L-NAME”, whose p essu e-o e load esul s om inhibi ion o
ni ic oxide p oduc ion, by o al adminis a ion o L-NAME, an inhibi o o ni ic oxide
syn hases. In hei espec i e con ols (“Sham” and “C l”), addi ion o IMD elici ed a
nega i e ino opic e ec . Despi e being simila in absolu e alue, e ec o IMD in diseased
models was o ally opposi e: we e i ied a posi i e ino opic e ec , allied o an inc ease o
lusi opy. A he baseline, we obse ed hese wo diseased models p esen p ese ed
sys olic unc ion, le en icle hype ophy and inc eased oxida i e s ess. A he p o ein
le el, in con ols, IMD ipled con en o phospho yla ed eNOS a he Se -1177, which
sugges an inc ease o eNOS ac i i y. Mo eo e , we also e i ied an inc ease o
phospho yla ion o oponin I, possibly due o ac i a ion o signaling pa hway dependen o
ni ic oxide and cGMP. On he o he hand, in diseased models, basal phospho yla ed
eNOS was inc eased and IMD did no al e hose le els o phospho yla ion. Howe e , IMD
elici ed phospho yla ion o phospholamban, e en ually due o ac i a ion o cAMP/PKA
signaling pa hway.
The eby, we showed o he i s ime IMD induces opposi e e ec s in heal hy and
diseased myoca dium, which a e due o inabili y o IMD o inc ease eNOS ac i i y in hese
la e models. Besides, and gi en ha dis inc mechanisms media e hose e ec s, his wo k
sugges s ca diac endo helial dys unc ion plays a p eponde an ole in pa hophysiology o
p essu e-o e load-induced hea ailu e and, a he same ime, poin s IMD as a po en ial
ma ke o ca diac endo helial dys unc ion.
Key wo ds: hea ailu e, ca diac endo helial dys unc ion, endo helial ni ic oxide syn hase, in e medin.
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x
Lis o Abb e ia ions
AM
Ad enomedullin
ATP
Adenosine iphospha e
BH4
Te ahyd obiop e in
CaM
Calmodulin
cAMP/cGMP
Cyclic adenosine/guanosine monophospha e
CGRP
Calci onin gene- ela ed pep ide
CRLR
Calci onin ecep o -like ecep o
cTnI
Ca diac oponin I
ECM
Ex acellula ma ix
eNOS
Endo elial ni ic oxide syn hase
FAD
Fla in adenine dinucleo ide
FMN
Fla in mononucleo ide
GTPCH1
Guanosine iphospha e cyclohyd olase 1
HF
Hea ailu e
IMD
In e medin
L-NAME
NG-ni o-L-a ginine me hyl es e
LTCC
L- ype calcium channels
LV
Le en icle/Le en icula
NADPH
nico inamide adenine dinucleo ide phospha e
NO
Ni ic oxide
NOS
Ni ic oxide syn hase
PKA/PKB/PKG
P o ein kinase A/B/G
PLB
Phospholambam
RAMP
Recep o ac i i y-modi ying p o eins
ROS/RNS
Reac i e oxygen/ni ogen species
Se
Se ine
SNS
Sympa he ic ne ous sys em
SR
Sa coplasmic e iculum
TAC
T ans e se ao ic cons ic ion
Th
Th eonine
Ty
Ty osine
β-ARs
Be a ad ene gic ecep o s

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I. In oduc ion
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1. Hea Physiology
1.1 Func ional ana omy.
Hea (Figu e 1A) is a i al and muscula o gan esponsible o blood pumping
o all body pa s. Ana omically, i has ou chambe s: wo au icles o a ia, which a e
esponsible o ecei ing blood, and wo en icles, whose main unc ion is pumping
blood. Hea is con ained in a sac called pe ica dium, which is di ided in ib ous and
pa ie al pe ica dium. Nex , he e is he pe ica dial ca i y con aining he se ous luid and
hen he e a e he epica dium, he myoca dium (composed en i ely by muscle cells
called ca diomyocy es) and endoca dium (composed by endo helial cells). All hese
laye s a e illus a ed in Figu e 1B. The ca diac muscle is an in olun a y con ac ion
muscle wi h a ype o esponse o a s imulus called “all-o -none esponse”.
Figu e 1 – A: S uc u e o he hea , om an e io pe spec i e. B: T ans e sal sec ion o he hea , showing i s di e en
laye s. F om h p://www.guwsmedical.in o/hea - ailu e/images/4415_27_47-pe ica dium-and-hea -wall.jpg .
B
A
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Ca diomyocy es a e sho b anching in e connec ing cells, a anged as a
syncy ium, which is a mul inuclea ed, p o oplasmic mass o cells. In ac , myoca dium
unc ions like a syncy ium since an applied s imulus in some pa o he muscle esul s
on con ac ion o whole muscle. Howe e , ana omically, myoca dium is no conside ed
a syncy ium since e ec i ely ca diomyocy es a e sepa a ed om each o he : la e ally,
by hei cellula memb anes (called sa colemmas) and a hei ends by dense
s uc u es called in e cala ed disks (Figu e 2). These disks ha e gap junc ions (Figu e
2), which acili a e he ca diac impulse conduc ion om a cell o ano he , and longe
desmosomes (called asciae adhe ens; Figu e 2), which keep no only he
ca diomyocy es oge he du ing con ac ion bu also he no mal elec ical coupling
be ween adjacen ca diomyocy es (Rasmussen, Palm eld e al., 2013).
Ca diomyocy es con ain a la ge numbe o mi ochond ia (called sa cossome; Figu e 2)
in o de o allow a quick subs a e oxida ion and adenosine iphospha e (ATP)
syn hesis (D ago, De S e ani e al., 2012), which suppo he myoca dial ene ge ic
equi emen s. In mos species, each one o ca diomyocy es ha e wo nuclei (W igh ,
Wu e al., 2012) (Figu e 2) and, on a e age, each one is associa ed o one capila ,
allowing a as exchange and mo emen o impo an molecules be ween
ca diomyocy e and capila . This ascula ne wo k p o ides a sui able a ailabili y o
oxygen and subs a es in o de o sus ain he me abolic machine y.
Figu e 2 – De ailed iew o disposi ion and s uc u al composi ion o ca diomyocy es, showing gap jun ions,
desmossomes, in e cala ed discs, nucleus, sa colemma, mi ochond ia and sa coplasmic e iculum. F om
h p://apb www5.apsu.edu/ hompsonj/Ana omy%20&%20Physiology/2010/2010%20Exam%20Re iews/Exam%203%20
Re iew/18-11_Ana Ca dMusc.JPG
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Inside ca diomyocy es, myo ib ils a e he con ac ile s uc u es cons i u ed by
housands o sa come es (G ego io and An in, 2000), which is de ined as he sec ion
be ween wo Z consecu i e discs (Figu e 3A). The sa come e is di ided in o wo main
bands (Figu e 3B): I band, which comp ises he hin (ac in) ilamen s segmen which
a e no o e lapped wi h hick (myosin) ilamen s; A band, which comp ises he hick
ilamen s, ei he hose o e lapped (zones o o e lap) and hose non-o e lapped (H
zone) wi h hin ilamen s. I ’s because o hose zones o o e lap ha ca diac muscle
has a s ia ed appea ance (Coope , 2000). The c oss-connec ing elemen s o
cy oskele on cons i u e he M line, which de ines he middle line o he sa come e
(Figu e 3A). As we can see in Figu e 3B, hin and hick ilamen s a e a anged in
pa allel (K ans, 2010). In addi ion o ac in and myosin, sa come e has o he p o eins
such as i in and nebulin (Figu e 3C), which a e gian p o eins ha , among o he
unc ions, a e esponsible o p o ec ing myo ib ils when hey a e o e s e ched
(Joseph, S ie e al., 2001) and o egula ion o muscle con ac ion (Labei , O enheijm
e al., 2011). T oponin complex a e ano he g oup o p o eins o sa come e, which is
associa ed o opomyosin and ac in ilamen s and includes ca diac oponins I, C and
T (cTnI, cTnC and cTnT). Pa icula ly, cTnI is suscep ible o be phospho yla ed in i s
Th eonine 144 (Th -144), Se ine 149 (Se -149), Se ine 43/45 (Se -43/45) and Se ine
23/24 (Se -23/24) esidues ( ela i ely o human sequence) (Layland, Sola o e al.,
2005). Phospho yla ion o his la e is media ed by p o ein kinase A (PKA) and
con ibu es o dec easing o myo ilamen Ca2+-sensi i y (Kooij, Saes e al., 2010).
A
B
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Figu e 3 – A: S uc u e o a myo ib il, showing i s e e ence poin s ( he M line and he Z discs) and he s ia ed pa e n
o sa come e. B: De ailed s uc u e o a sa come e, showing he i in ilamen s and he pa allel alignmen o hin (ac in)
and hick (myosin) ilamen s. Bands A and I, Z discs, M line and o e lap and H zones a e also ep esen ed. C: P o ein
composi ion o a sa come e, showing opomyosin, oponin complex (I, C and T), nebulin, C p o ein, myosin and i in.
F om h p://www. e espca diol.o g/ima ges/255/255 59n05/g ande/255 59n05-13088668 ig05.jpg and
h p://www.baileybio.com/plogge /images/ana omy___physiology/05._powe poin _-_muscula _sys em/sa come e.jpg .
Myo ib ils a e su ounded by sa coplasmic e iculum (SR; Figu e 2), a small-
diame e sa co ubules ne wo k, which is esponsible o s o ing in acellula Ca2+. Since
i s disco e y, SR has always been ou ed as playing an impo an ole in
con ac ion/ elaxa ion cycle (Fawce , 1961), which was con i med la e . The e is
ano he ubula sys em inside ca diomyocy es which is composed by a la ge numbe o
ans e sal ubules (T- ubules; Figu e 4), which a e in agina ions o he sa colemma
in o ca diomyocy es a he Z lines. This ubula sys em plays an impo an ole in
exci a ion-con ac ion coupling and p o ides o myo ib ils and mi ochond ia a eady
access o in e s i ial luid.
Figu e 4 – De ailed iew o ca diomyocy e, e idencing he T- ubula sys em. Adap ed om
h p://www.cou s udy.com/wp-con en /uploads/2013/07/mic oscopic-ana omy-o -ca diac-muscle-sa colemma- asciae-
adhe ens-in e cala ed-discs-gap-junc ions- - ubules.jpg .
C

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1.2 Exci a ion-con ac ion coupling
Muscula con ac ion (Figu e 5) begins when an exci a ion wa e sp eads quickly
along he sa colemma and gap junc ions and in o ca diomyocy es ia T- ubules. A
sa colemmal le el, pe meabili y o Ca2+ inc eases, leading o Ca2+ en e ing on he cell
h ough sa colemmal Ca2+ channels, called L- ype Ca2+ channels (LTCC), whose
channel p o eins a e phospho yla ed by cAMP, and T- ubules. This ex acellula Ca2+ is
no su icien o se o myo ib ils con ac ion; indeed, i is jus a igge o Ca2+
eleasing by SR, which augmen s ee Ca2+ in cy oplasm. In u n, hese Ca2+ ions bind
o cTnC, o ming a complex which in e ac s wi h opomyosin, unlocking ac i e si es
be ween ac in and myosin ilamen s. Then, myo ib ils con ac and sys ole occu s. A
he end o sys ole, Ca2+ in lux s ops and SR is no mo e s imula ed. Cyclic adenosine
monophospha e (cAMP) p omo es phospholamban phospho yla ion (p-PLB), which
s imula es ATP-dependen Ca2+ pump (placed on SR), esul ing in pumping o Ca2+ in o
SR. Besides, cAMP also phospho yla es cTnI, inhibi ing he binding o Ca2+ o cTnC.
So, cTnI binds o ac in, keeping ac in/ opomyosin complex and p e en ing he myosin
connec ing o he ac in (Gomes, Po e e al., 2002). Dias ole occu s (Figu e 5) and
ex acellula Ca2+ is emo ed om cy oplasm h ough a Na+/Ca2+ exchange and an
ATP-dependen Ca2+ pump, bo h localized on sa colemma.
Figu e 5 – Sys ole and dias ole mechanisms. Numbe s indica e he succession o e en s esponsible o ca diac
con ac ion and elaxa ion.
F om h p:// acul y.pasadena.edu/dkwon/chap e %2014/Chap e %2014_ca diology_ iles/slide0059_image021.jpg .
Ac ion po en ial en e s om adjacen cells.
 Vol age-ga ed Ca2+ channels open. Ca2+ en e s
cell.
Ca2+ induces Ca2+ elease h ough yanodine
ecep o -channels (RyR).
 Local elease causes Ca2+ spa k.
 Summed Ca2+ spa ks c ea e a Ca2+ signal.
 Ca2+ ions bind o oponin o ini ia e con ac ion.
 Relaxa ion occu s when Ca2+ unbinds om
oponin.
 Ca2+ is pumped back in o he sa coplasmic
e iculum o s o age.
 ca2+ is exchanged wi h Na+.
 Na+ g adien is main ained by he Na+/K+
ATPase.










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2. Hea ailu e
2.1 De ini ion and gene al conside a ions
Hea ailu e (HF), also called conges i e hea ailu e (CHF), is de ined as a
clinical synd ome cha ac e ized by bo h s uc u al and unc ional al e a ions in hea
(F ancis and Tang, 2003). Tha condi ion incapaci a es hea , hus i does no pump
su icien blood o all pa s o body, leading o a mul isys em diso de . Nowadays, hea
ailu e is conside ed an epidemic disease, gi en ha many people a ound he wo ld
su e i : in ac , in Eu ope, i is he majo cause o dea h (Figu e 6). This ele a ed a e
is in ima ely associa ed wi h inc eased p e alence o isk ac o s, such as diabe es,
hype ension and obesi y (López-Sendón, 2011), a ising om unheal hy li es yles.
While a he socie y i is inc easingly impo an o aise awa eness o he p e en ion o
HF, a he scien i ic communi y he e is an e o o un a el p ocesses and mechanisms
esponsible o eme gence and p og ession o hea ailu e.
Figu e 6 – Majo causes o dea h in Eu ope. Ca dio ascula disease is he majo one in bo h gende s. F om
h p://www.mic omedc .com/eu/images/s o ies/majo _causes_dea h_eu ope-big.jpg.
Clinically, acco ding he New Yo k Hea Associa ion (NYHA), p og ession o
HF is di ided in o ou di e en classes, cha ac e ized by se e i y o symp oms (Table
1) and quali y o li e.
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Table 1 – Classi ica ion o hea ailu e and hei espec i e symp oma ology, acco ding wi h he New Yo k Hea
Associa ion.
HF may assume wo di e en o ms, since i can esul om a disabili y o he
en icles bo h con ac no mally (called hea ailu e wi h educed ejec ion ac ion o
sys olic hea ailu e, HF EF) o elax comple ely du ing dias ole (called hea ailu e
wi h p ese ed ejec ion ac ion o dias olic hea ailu e, HFpEF) (Bo laug and Paulus,
2011; Gu ie ez and Blancha d, 2004). Popula ion s udies ha e poin ed ou his la e
dys unc ion as he mos popula among elde ly people (Ki zman, 2000); mo eo e ,
HFpEF is conside ed a majo cause o mo bidi y and mo ali y (Bo laug and Paulus,
2011). Despi e no ha ing sys olic dys unc ion, hese pa ien s ha e a s i hea due o
ib osis, esul ing in a dec easing o dias olic illing. Hence, he amoun o blood
pumped is lowe du ing sys ole (Ki zman, 2000). Gi en i s impo ance and since i s
pa hophysiology is no e y well unde s ood ye (Bo laug and Paulus, 2011), dias olic
HF has been s udied by many g oups.
2.2 Animal Models
Al hough he e is no an animal model able o comple ely mimic he human HF
(Dogg ell and B own, 1998), in es iga o s ha e ied o de elop he bes one. Cu en ly,
a is he mos used animal model since i p esen s nume ous ad an ages, such as low
cos s and ad anced echnology, which has been op imized o his model (Pa en and
Hall-Po e , 2009). Howe e , his animal model also p esen s some limi a ions
especially conce ning i s myoca dial unc ion, which di e s om human in some
aspec s, such as he size o ac ion po en ial (Hasen uss, 1998). Rega ding HF, he e
a e many di e en a models, depending on he p ima y eason o ha ailu e and
wha he in es iga o s wan o s udy (Table 2) (Dogg ell and B own, 1998; Hasen uss,
1998). One o he mos used models is he ans e se ao ic cons ic ion (TAC), whe e
HF esul s om a p essu e o e load-induced hype ophy (Rockman, Ross e al.,
Class
Pa ien Symp oms
I (Mild)
No limi a ion o physical ac i i y. O dina y physical ac i i y does no cause undue a igue,
apid/i egula hea bea (palpi a ion) o sho ness o b ea h (dyspnea).
II (Mild)
Sligh limi a ion o physical ac i i y. Com o able a es , bu o dina y physical ac i i y
esul s in a igue, apid/i egula hea bea (palpi a ion) o sho ness o b ea h (dyspnea).
III (Mode a e)
Ma ked limi a ion o physical ac i i y. Com o able a es , bu less han o dina y ac i i y
causes a igue, apid/i egula hea bea (palpi a ion) o sho ness o b ea h (dyspnea).
IV (Se e e)
Unable o ca y ou any physical ac i i y wi hou discom o . Symp oms o a igue,
apid/i egula hea bea (palpi a ion) o sho ness o b ea h (dyspnea) a e p esen a es .
I any physical ac i i y is unde aken, discom o inc eases.
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1991). Acco ding o deAlmeida e al, TAC is a mo e ai h ul model o ca diac
hype ophy and esembles o human HF because o i s g adual de elopmen
(deAlmeida, an Oo e al., 2010). This model consis s in cons ic ion o ao a,
esul ing in a p essu e-o e loaded hea .
Table 2 – Some a models o hype ension, hype ophy and hea ailu e. This able was adap ed om Dogg ell e al
(Dogg ell and B own, 1998).
Synd ome
Model
Hype ension
 Sys emic
 Spon aneously Hype ensi e Ra s (SHRs)
 S oke-p one SHR (SHR-SP)
 Mine aloco icoids (DOCA–sal )
 NO syn hase inhibi ion (L-NAME adminis a ion)
 T ansgenics (TGR(mREN2)27 a s)
 Diabe ic hype ensi e a s (STZ-SHR, Zucke )
o Renal
o Renal a e y occlusion (1K1C, 2K1C)
 Pulmona y
 Monoc o aline
 Hypoxia (no moba ic, hypoba ic)
Hype ophy
- Spon aneously Hype ensi e Ra s (SHRs)
- Renal a e y occlusion
- P essu e loading (ao ic banding)
- Ca echolamines (no ad enaline, isop enaline)
- T ansgenics
Hea ailu e
 Sys emic hype ension
 Spon aneously Hype ensi e Ra s (SHRs-F)
 Hype ensi e HF p one a s
 Dahl/Rapp sal -sensi i e a s
o Ischaemic hea ailu e
o Non-occlusi e co ona y liga ion
 Myoca dial in a c ion
 Co ona y liga ion
 Mic oemboliza ion o co ona y essels
 Ca diomyopa hy
 Toxins (ad iamycin, e hanol)
o Myoca di is
o Au oimmune
o Chagas' disease
 Pulmona y
hype ension
 Monoc o aline
 Hypoxia
 Miscellaneous
 Ao aca al is ula (shun s)
Ano he ecognized p essu e-o e load model is L-NAME, which esul s om
ch onic adminis a ion o L-NAME (NG-ni o-L-a ginine me hyl es e , a ni ic oxide
syn hase inhibi o ) in d inking wa e , p omo ing sys emic hype ension. This model is
mos ly used in human hype ension s udies (Kopinco a, Puzse o a e al., 2012). Unde
hese condi ions, hea igge s a se o mechanisms esul ing in molecula , cellula and
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esponsible o deg ada ion and emo al o ECM molecules (Nagase, Visse e al.,
2006). In pigs wi h HF, MMPs abundance and ac i i y a e inc eased (Coke , Thomas e
al., 1998); u he mo e, unde hose condi ions also TIMPs le els a e al e ed, esul ing
in an imbalance be ween hese wo p o ein amilies (Moo e, Fan e al., 2012). Hence,
bo h MMPs and TIMPs a e p omising he apeu ic a ge s o disco e y o new d ugs,
which could be mo e e ec i ely in ea men o HF (Li, McTie nan e al., 2000).
2.3.4 Oxida i e s ess
E idence has also demons a ed ha in HF, oxida i e s ess, de ined as an
imbalance be ween ROS/RNS p oduced and cellula an ioxidan capaci y (Thannickal
and Fanbu g, 2000), is augmen ed and accompanied by an an ioxidan de ici (Belch,
B idges e al., 1991; Hill and Singal, 1996; Hill and Singal, 1997; Malla , Philip e al.,
1998). Despi e playing an impo an ole in many biological cellula p ocesses (Ana hy,
Robe son e al., 2012; Hancock, Desikan e al., 2001; Ma inez and And ian si ohaina,
2009; Simon, Haj-Yehia e al., 2000), bo h ROS and RNS cellula accumula ion is
dele e ious. To he molecula le el, oxida i e s ess is esponsible o damaging o
nucleic acids, lipids, p o eins and o he mac omolecules (Ma inez and
And ian si ohaina, 2009; Thannickal and Fanbu g, 2000) howe e , i can also p omo es
speci ic, e e sible/i e e sible oxida i e modi ica ions on p o eins, al e ing hei ac i i y
o unc ion (Finkel and Holb ook, 2000).
Wi hin he hea , ca diomyocy es, endo helial cells and neu ophils a e sou ces
o ROS, bu inside each ca diomyocy e he e a e many di e en sou ces esponsible
o ha p oduc ion, namely mi ochond ia, nico inamide adenine dinucleo ide phospha e
(NADPH) and xan hine oxidases and uncoupled NOS (Be e s, B aam e al., 2006;
Tsu sui, Kinugawa e al., 2011) (Figu e 10). Conce ning mi ochond ia, ecen da a
shows p og ession o HF is associa ed wi h a p og essi e loss o espi a o y ac i i y o
mi ochond ia, dec easing ATP p oduc ion and jeopa dising cellula p ocesses
esponsible o ca diac pump unc ion (Huss and Kelly, 2005). Many di e ences ha e
been disco e ed be ween mi ochond ia om no mal and ailing hea s, ei he a
mo phologic, unc ional and biochemical le els (Abel and Doens , 2011; Rosca and
Hoppel, 2010). Cu iously, despi e being a sou ce o ROS, mi ochond ia a e se e ely
a ec ed by hem since ROS a e esponsible o mi ochond ial DNA damage (Tsu sui,
Kinugawa e al., 2011). Recen ly, also endo helial dys unc ion was indica ed as a
po en ial s ong con ibu o o pa hophysiology and p og ession o HF (Lam and
B u sae , 2012) and is also associa ed wi h HF se e i y (Ma i, Gheo ghiade e al.,
2012). Gi en ha and since endo helial dys unc ion esul s om an imbalance be ween

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NO and ROS p oduc ion (Baue sachs and Scha e , 2004) and endo helial NOS
(eNOS) is he mos abundan o m o NOS exp essed in endo helium (Balligand, Fe on
e al., 2009), namely endoca dial endo helium (Kelly, Balligand e al., 1996), eNOS
migh play a cen al ole in endo helial dys unc ion and he e o e in p og ession o HF.
Acco dingly, some au ho s ha e epo ed he po en ial p o ec i e ole o endo helium-
de i ed NO in HF and o he ca diac dys unc ions, such as a hy hmia (Alb ech ,
S egeman e al., 2003; Bu ge and Feng, 2011; Fische , Rossa e al., 2005; Nishida,
Yu e al., 2009).
Figu e 10 – NADPH oxidase as he majo sou ce o ROS, unde di e en s imuli including p essu e-o e load. Also
mi ochond ia, xan hine oxidase and eNOS a e sou ces o ROS, which con ibu e o an inc easing o ROS/an ioxidan s
(AO) a io, esul ing in oxida i e s ess. This edox cellula imbalance leads o hea ailu e, h ough a se o ROS-
media ed al e a ions including apop osis and dys unc ion o ca diomyocy es, ib osis and hype ophy. F om Oca a ia e
al (Oc a ia, B unne -La Rocca e al., 2012).
Thus, unde s and how eNOS wo ks and is egula ed unde bo h physiologic and
pa hophysiologic condi ions will be an impo an help o disco e ing o new e icien
he apeu ic a ge s.
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2.3.4.1 eNOS
Endo helial NOS is cons i u i ely exp essed by endo helial cells and belongs o
a amily o enzymes called ni ic oxide syn hases (NOSs), which a e esponsible o NO
syn hesis (S ueh , 1999). This amily also comp ises wo o he NOS iso o ms: neu onal
and inducible NOS, nNOS and iNOS espec i ely (S ueh , 1999). In hea , bo h eNOS
and nNOS a e cons i u i ely exp essed by ca diomyocy es while iNOS is only
exp essed a e an in lamma o y s imuli (Moun , Kemp e al., 2007). S uc u ally, eNOS
has an N- e minal oxygenase domain con aining binding domains o heme, L-a ginine
and e ahyd obiop e in (BH4), a cen al egion enabling calmodulin (CaM) binding and
inally a C- e minal educ ase domain con aining binding si es o NADPH, la in
adenine dinucleo ide (FAD) and la in mononucleo ide (FMN) (S ueh , 1997) (Figu e
11). Endo helial NOS ac i e o m is an homodime ; howe e , since eNOS depends
p ima ily on Ca2+/CaM p esence (Ra iko , Fonseca e al., 2011), i should indeed be
conside ed a e ame (Alde on, Coope e al., 2001). eNOS syn he izes NO and L-
ci ulline om subs a e L-a ginine, equi ing molecula oxygen, i s dime o m and he
co ac o s: BH4, haem, FAD, FMN, CaM and NADPH (Fo s e mann and Munzel, 2006;
Ma le a, 1993). The co ac o BH4 is essen ial o eNOS no mal unc ion (Alp and
Channon, 2004): dec easing o BH4 bioa ailabili y, ei he by i s oxida ion o educ ion o
i s syn hesis, esul s in uncoupling o eNOS (Moens and Kass, 2006). Besides, BH4 is
esponsible o dime s abiliza ion and inc ease o subs a e a ini y o NOS, and has
some an ioxidan e ec s h ough sca enging o NO-de i ed RNS and ROS (Moens,
Kie adiso n e al., 2010). Despi e i s exogenous adminis a ion was shown o be
bene icial o ca diac unc ion, BH4 has po en ial limi a ions in clinical use (Moens,
Ke ne e al., 2011).
Figu e 11 – Endo helial NOS s uc u e, showing i s oxygenase and educ ase domains and i s CaM binding si e. This
p o ein is able o be phospho yla ed a y osine (Y), h eonine (T) and se ine (S) esidues, which a e numbe ed. O he
modi ica ions include my is oyla ion (M), palmi oyla ion (P), S-glu a hionyla ion (G) and S-ni osyla ion (N). Adap ed om
May (May, 2012).
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De no o syn hesis o BH4 is limi ed by guanosine iphospha e cyclohyd olase 1
(GTPCH1), whose ansc ip ion (Ka usic, S el e e al., 1998; Linscheid, Scha ne e
al., 1998), ac i i y (We ne -Felmaye , We ne e al., 1993) and phospho yla ion
(Lapize, Pluss e al., 1998) a e con olled by se e al ci okines, angio ensin-II and
pla ele -de i ed g ow h ac o . Deg ada ion o GTPCH1 was epo ed in ca dio ascula
diseases (Moens, Kie adiso n e al., 2010; Zhao, Zhu e al., 2012), such as diabe es
melli us (Xu, Wu e al., 2007) and hype ension (Xu, Wang e al., 2009).
In addi ion, eNOS exp ession i sel is egula ed a ansc ip ional and pos -
ansc ip ional le els (Balligand, Fe on e al., 2009; Sea les, 2006). This la e includes
pos ansla ional modi ica ions such as S-ni osyla ion, S-glu a hionyla ion, binding o
CaM, palmi oyla ion, acyla ion and phospho yla ion (Chen, Wang e al., 2010;
Dudzinski and Michel, 2007). Pa icula ly, eNOS con ains many po en ial
phospho yla ion si es such as y osine, se ine and h eonine esidues (Alde on,
Coope e al., 2001) (Figu e 11). Thei phospho yla ion egula es NO p oduc ion,
depending on no only esidues ype bu also esidue pe se (Ful on, Chu ch e al.,
2005; Li, Ruan e al., 2007; Ri chie, Kohlhaas e al., 2010; Ruan, To es e al., 2011).
Ano he egula o y mechanism o NO p oduc ion by eNOS include p o ein-p o ein
in e ac ions: o ins ance, he binding o hea shock p o ein 90 (Hsp90) o eNOS is
needed o Ak (also called PKB)-media ed phospho yla ion o eNOS a Se -1177
(using he human sequence nomencla u e) (Moun , Kemp e al., 2007) while he
binding o ca eolin-1 o eNOS is esponsible o i s inac i a ion (Ju, Zou e al., 1997).
Also subcellula localiza ion is impo an o eNOS ac i i y (Sulli an and Pollock, 2003).
A he baseline, eNOS is associa ed wi h plasma memb ane (Chu ch and Ful on, 2006)
mainly due o i s my is oyla ion (Busconi and Michel, 1993; Michel, 1999). Howe e , in
hype ensi e a s, which p esen endo helial dys unc ion, au ho s e i ied eNOS
ansloca ion om memb ane o cy osol, sugges ing ha eNOS ansloca ion may
con ibu e o dec easing o NO bioa ailabili y and hus o wo sening o endo helial
dys unc ion and subsequen ly hype ension (Sulli an, Pollock e al., 2002).
As s a ed abo e, in he ailing hea he e is an o e p oduc ion o ROS om
a ious sou ces. Pa icula ly, gene a ed ●O2- eac wi h NO o o m pe oxyni i e
(ONOO-), which hen oxidizes BH4 (Balligand, Fe on e al., 2009), diminishing i s
bioa ailabili y. Consequen ly, eNOS su e s an enzyma ic change h ough al e a ion o
i s dime a chi ec u e as well as i s ca aly ic ac i i y, leading o he syn hesis o ●O2-
and/o hyd ogen pe oxide in i o (Moens and Kass, 2006) and e en ually o he di ec
p oduc ion o ONOO- (Balligand, Fe on e al., 2009). This p oduc ion o ●O2- ins ead o
NO is commonly designa ed as uncoupling o eNOS (Fo s e mann and Munzel, 2006;
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Massion, Fe on e al., 2003). A he p o ein le el, ONOO- eac s wi h y osine,
gene a ing 3-ni o y osine, which can be de ec ed by speci ic an ibodies (Daibe and
Munzel, 2012). Indeed, ni o y osine is e e ed as a ma ke o bo h ONOO- o ma ion
and p esence o oxida i e s ess (Sun, Ca e e o e al., 2005). Acco dingly, i s le els
a e inc eased in CHF pa ien s (H yniewicz, Yasskiy e al., 2004).
Endo helial NOS-de i ed NO is hough o be coupled o cyclic guanosine
monophospha e/p o ein kinase G (cGMP/PKG) pa hway (Hammond and Balligand,
2012). Ac i a ion o PKG is able o p omo e phospho yla ion o a ious p o eins such
as PLB, LTCC and cTnI (Tsai and Kass, 2009).
Th oughou his 2.3 sec ion, we men ioned some o he easons ha explain he
ansi ion be ween he compensa ed s a e and HF. Howe e , o now, o he
mechanisms and in e enien s a e s ill unde exhaus i e s udy and in es iga o s
con inue o sea ch o answe s (Keha and Molken in, 2010). Nex yea s, his so
complex ansi ion could be mo e highligh ed, b inging new po en ial and mo e e icien
he apeu ic a ge s.
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3. In e medin
The calci onin/calci onin gene- ela ed pep ide (CGRP) amily pep ide includes
pep ides such as calci onin, CGRP, amylin and AM (Chang, Roh e al., 2004). Bu in
2004, he numbe o membe s o his amily augmen ed, when a new pep ide – IMD –
was independen ly disco e ed by wo dis inc g oups (Roh, Chang e al., 2004; Takei,
Hyodo e al., 2004). I is o ha eason IMD is also known as AM2. The human IMD
p ep o ein is cons i u ed by 148 amino acids (p ep oIMD) bu , h ough p o eoly ic
clea age a he N- e minus and amyda ion o C- e minus (Yang, Jia e al., 2005), i is
able o gene a e wo dis inc ma u e o ms cons i u ed by 47 (IMD1-47 o IMDL) (Bell and
McDe mo , 2008) o 40 amino acids (IMD8-47 o IMDS). In addi ion, i is hough he e is
ano he IMD o m wi h 53 amino acids (IMD1-53) esul an om clea age o p ep oIMD
a he A ginine 93-A gine 94 si es (Yang, Jia e al., 2005). E en ually, IMD1-53 would be
deg aded endogenously bu migh ha e an impo an bioac i e ac ion (Yang, Jia e al.,
2005). Alignmen o ma u e IMD1-47 shows ha human and oden aminoacid
sequences a e 87% iden ical (Roh, Chang e al., 2004) (Figu e 12).
Figu e 12 – Alignmen o amino acids o IMD long o m (IMD 1-47) om human (hIMD), mouse (mIMD) and a ( IMD).
Adap ed om Roh e al (Roh, Chang e al., 2004).
IMD is dis ibu ed all o e he mammalians body, namely kidneys,
gas oin es inal ac , b ain, lungs, spleen, o a ies, pi ui a y, among many o he s
(Takahashi, Mo imo o e al., 2011). In a hea , IMD is ound in ca diomyocy es, wi h
g ea e le els a e ound in hose neona al when compa ed wi h adul ones (Pan, Yang
e al., 2005), which sugges s IMD plays an impo an ole du ing emb yonic
de elopmen (Bell and McDe mo , 2008).
The biological ac ions o IMD a e media ed by h ee di e en ecep o s, which
a e he e odime s cons i u ed by a calci onin ecep o -like ecep o (CRLR) and one o
he h ee ecep o ac i i y-modi ying p o eins (RAMP1-3) (Nao and Co nish, 2008).
Mo eo e , hese RAMP p o eins a e essen ial o ligand selec i i y since CRLR alone
shows a dec eased a ini y o i s ligands (P eil, Aslam e al., 2009). The CRLR/RAMP1
complex (o CGRP ecep o ) o ms he CGRP ecep o while he CRLR/RAMP2-3
complexes (also called AM1 and AM2 ecep o s, espec i ely) o m he AM ecep o s

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(Hay, Walke e al., 2011). IMD binds non-speci ically o all h ee CRLR/RAMP
complexes (Takahashi, Mo imo o e al., 2011). Despi e ha , he e is a deg ee o
binding a ini y (Figu e 13), since, o example, AM is also capable o bind o CGRP
ecep o (Zhao, Bell e al., 2006).
Figu e 13 – Binding a ini y o CGRP, AM and IMD (AM2) o CGRP, AM1 and AM2 ecep o s and hei cons i u ion. Ligh
g ey cylinde s ep esen calci onin ecep o -like ecep o (CRLR). ECD means ex acellula domain; TM means
ansmemb ane; ICD means in acellula domain. Adap ed om Hay e al (Hay, Walke e al., 2011).
3.1 Ca dio ascula e ec s
Conce ning ca dio ascula e ec s, IMD1-47 elici s an inc eased hea a e in a
media ed by ac i a ion o SNS (Taylo , Bagley e al., 2005). Besides, i s in a enous
adminis a ion p omo es a nega i e ino opic ac ion sugges ed by a decline o blood
p essu e (Bell and McDe mo , 2008). Howe e , a posi i e ino opic e ec elici ed by
IMD1-53 and IMD1-47 was epo ed bo h in isola ed pe used hea om a (Yang, Jia e
al., 2005) and en icula ca diomyocy es om mouse (Dong, Taylo e al., 2006),
espec i ely. This appa en con adic ion was ecen ly cla i ied by Pi es e al, who
es ablished a nega i e ino opic e ec o IMD on myoca dium, using a le en icula
(LV) papilla y muscles (Pi es, Pinho e al., 2012). Since AM also has di e en ino opic
ac ions, dependen on bo h species and me hodology used (Fon es-Sousa, Pi es e al.,
2009; Iha a, Ikeda e al., 2000; Ikenouchi, Kangawa e al., 1997; Szokodi, Kinnunen e
al., 1998), hese e idences show and ein o ce he complex biological ac ion o
membe s o his pep ide amily.
R
A
M
P
1
R
A
M
P
2
R
A
M
P
3
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Behind IMD myoca dial e ec s, he e is a se o signalling pa hways,
messenge s and media o s in ol ed. Howe e , he e is no di ec e idence p o ing ha
and conclusions ha e o en been d awn by ex apola ing o da a om o he membe s
o his pep ide amily and om s udies abou IMD ascula e ec s. Ne e heless, i is
hough IMD nega i e ino opic e ec and asodila o ac ion is endo helium-dependen
and media ed by NO/cGMP pa hway, whe eas posi i e ino opic e ec is media ed by
cAMP/PKA pa hway (Bu ak Kandilci, Gumusel e al., 2006; Dong, Taylo e al., 2006;
Pi es, Pinho e al., 2012). Bu mos impo an ly, IMD is now ecognized as a po en ial
impo an pep ide in ca diac pa hology since i s exp ession le els a e much g ea e in
se e al models o ailing hea s han in heal hy ones (Bell, Zhao e al., 2008; Hi ose,
To sune e al., 2008; Zeng, Yuan e al., 2009; Zhang, Jiang e al., 2009; Zhao, Bell e
al., 2006). Fu he mo e, some s udies ha e epo ed he po en ial p o ec i e ole o IMD
(Holmes, Campbell e al., 2013) as o example in p e en ion o myoca dial apop osis
media ed by mi ochond ia ia Ak /glycogen syn hase kinase 3 be a (GSK-3β) signalling
pa hway (Song, Teng e al., 2009) and induc ion o au ophagy o ca diomyocy es ia
cAMP/PKA and mi ogen-ac i a ed p o ein kinases/ex acellula signal- egula ed
kinases (MAPK/ERK) signalling pa hways, p e en ing bo h apop osis and hype ophy
o ca diomyocy es (Chen, Wang e al., 2013). Ne e heless, he e is much wo k o do
in o de o explain he pa hophysiologic impac o he augmen ed le els o IMD.
4. Aims
The gene al goal o his hesis was o s udy he myoca dial esponse o IMD in
ch onic p essu e-o e loaded a s, i s in acellula mechanisms and impac o ca diac
endo helial dys unc ion.
In o de o acili a e he unde s anding o he wo k, we di ided i in o h ee
di e en poin s:
1) To cla i y and show di e ences be ween he wo well-known p essu e-o e load
models TAC and L-NAME, ega ding hei mo phome ic, haemodynamic and
con ac ile pa ame e s.
2) To s udy myoca dial e ec s o IMD in bo h con ol and p essu e-o e load
animal models and un eil he po en ial in acellula signalling pa hways
in ol ed.
3) To in es iga e he in luence o eNOS in di e en ial esponse o myoca dium o
IMD.
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II. Ma e ials
&
Me hods
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1. E hical app o al
The animal handling and expe imen s we e pe o med in acco dance wi h
Po uguese law animal wel a e and con o m o he guidelines es ablished by he
Facul y o Medicine o he Uni e si y o Po o (Po o, Po ugal), animal wel a e
commi ee and p inciples o UK egula ions, as desc ibed by D ummond (D ummond,
2009).
2. Animals
2.1 TAC a s
T ans e se ao ic cons ic ion (TAC) was pe o med simila ly o Li win e al (Li win,
Ka z e al., 1995). Se en weeks-old male Wis a -Han a s (Ra us no egicus) we e
anes he ized wi h se o lu ane (2%) and placed on a empe a u e-con olled su gical able.
A e acheal in uba ion, a s we e connec ed o a oden en ila o (Topo, small animal
en ila o , Ken Scien i ic). A small incision a he le el o he second le in e cos al space
was pe o med, he di e en muscle laye s we e cu and ho acic ca i y was accessed.
A e iden i ica ion and isola ion o he ao ic a ch, a 3-0 non-abso bable silk su u e was
placed a ound ao a be ween he b achiocephalic and le ca o id a e ies (Figu e 14) and
ied oge he wi h a 25G needle, in o de o s anda dize he cons ic ion. A e ying he
su u e, he needle was immedia ely emo ed o p oduce an ao a wi h a s eno ic lumen.
Then he ho acic ca i y was closed using a 4-0 polyp opylene su u e, all laye s o muscle
we e eposi ioned and skin was closed wi h 4-0 silk su u e (TAC, n=14). The con ol
animals a e called “Sham” and hey we e subjec ed o a simila su ge y wi hou he
igh ening o ao a (Sham, n=12). Twen y one weeks a e su ge y, a s unde wen
hemodynamic e alua ion o papilla y muscles p o ocols, as desc ibed in Figu e 15A.
Figu e 14 – Diag am o ao a adap ed om an online quiz (h p://quizle .com/14064108/mini2-ana omy- lash-ca ds/).
The blue boxes iden i y he b achiocephalic and le common ca o id a e ies and he black line in a ch o ao a
ep esen s he local o he cons ic ion.
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9. Chemicals and solu ions
All chemicals we e ob ained om Sigma Chemical Co, S Louis, wi h he
excep ion o : IMD, which co esponds o he ac i e o m o 47 amino-acid (IMD1-47), ha
was ob ained om Bachem (Bubendo , Swi ze land); eNOS ac i i y ki , ob ained om
S a agene (La Jolla, Cali o nia, USA); [3H]-a ginine, ob ained om Pe ken Elme
(Massachuse s, USA). An ibodies an i-eNOS and an i-cTnI we e ob ained om Cell
Signaling Technology Inc., (Massachuse s, USA) and an ibodies an i-PLB we e
ob ained om Abcam (Camb idge, UK). The s ock solu ions, including IMD, we e
p epa ed in dis illed wa e and s o ed as ozen aliquo s a -20ºC un il use.
10. S a is ical analysis
G aphPad P ism 5.0 so wa e was used o da a analysis. Values a e p esen ed
as mean ± s anda d e o o mean (SEM) and n ep esen s he numbe o animals o
papilla y muscles. Di e ences in ea men e ec s among he di e en animal g oups
we e e alua ed using a wo-way ANOVA. When signi ican di e ences we e de ec ed
wi h any o he ANOVA es s, he Dunne es was selec ed o pe o m pai wise mul iple
compa isons, wi h P<0.05 being conside ed signi ican . In all o he expe imen s,
s a is ical signi icance was assessed using a wo- ailed S uden 's unpai ed es wi h
P<0.05 being conside ed signi ican .

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III. Resul s
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1. Cha ac e iza ion o animal models
In o de o cha ac e ize he animal models, we pe o med mo phome ic and
hemodynamic measu es, which a e summa ized in Table 4. Bo h TAC and L-NAME
animals p esen ed a signi ican inc ease in hea weigh /body weigh a io, compa ed o
hei espec i e con ols (Sham and C l). These esul s sugges TAC and L-NAME
animals de eloped LV hype ophy. Simila ly, le en icula sys olic p essu e (LVSP)
is signi ican ly inc eased in hose models ela i ely o hei con ols; howe e a
signi ican ly highe le en icula end-dias olic p essu e (LVEDP) was eco ded jus in
he TAC animals. Fu he mo e, hese animals showed a slowe le en icula
elaxa ion, sugges ed by a g ea e au alue.
Table 4 – Summa y o mo phome ic (7-8 animals) and hemodynamic (4-7 animals) measu es pe o med in Sham,
TAC, C l and L-NAME animals. All da a a e p esen ed as mean SEM (s anda d e o mean). HW means hea weigh ;
BW means body weigh; LVSP means le en icula sys olic p essu e; LVEDP means le en icula end-dias olic
p essu e; dPd max means maximum eloci y o p essu e ise; dPd min means maximum eloci y o p essu e decline;
means au. * P<0.05 s Sham; #P<0.05 s C l.
Sham
TAC
C l
L-NAME
HW/BW
(g/kg)
2.9±0.1
3.4±0.2*
2.3±0.1
2.7±0.2#
Hea Ra e
(bpm)
403±11
386±17
410±19
378±21
LVSP
(mmHg)
120±5
158±9*
110±3
147±12#
LVEDP
(mmHg)
4.0±0.4
10.0±2.0*
3.7±0.4
5.6±1.0
dPd max
(mmHg/sec)
7559±143
7234±629
6612±430
7131±900
dPd min
(mmHg/sec)
-9436±474
-8065±948
-8714±2
-9382±1583
8±0.3
12±1.0*
9±2.0
10±1.0
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2. E ec o IMD on con ac ile and
elaxa ion pa ame e s
Be o e he beginning o IMD p o ocol, con ac ile pa ame e s o s abilized le
en icula papila y muscles we e eco ded and a e summa ized in Table 5. Resul s
indica e he e a e no any s a is ical di e ences be ween he di e en animal models
excep o ime o hal elaxa ion ( HR), which is signi ican ly highe in L-NAME animals.
This pa icula esul was al eady expec ed since i is known NO p omo es has ening o
elaxa ion (Paulus, Van impon e al., 1994).
Table 5 – Summa y o con ac ile and elaxa ion pa ame e s a baseline o LV papilla y muscles om he di e en
animal models. All da a a e p esen ed as mean SEM (s anda d e o mean). AT means ac i e ension; dT/d max
means maximum eloci y o ension ise; dT/d min means maximum eloci y o ension decline; PS means passi e
ension; dL/d max means maximum eloci y o sho ening; dL/d min means maximum eloci y o leng hening; HR
means ime o hal elaxa ion. #P<0.05 s C l.
Sham
(n=6)
TAC
(n=7)
C l
(n=6)
L-NAME
(n=5)
AT
(mN/mm2)
15±2
16±4
18±3
23±8
dT/d max
(
mN/mm2/sec)
141±35
198±53
199±20
230±81
dT/d min
(mN/mm2/sec)
-71±10
-99±20
-112±9
-115±39
PS
(%Lmax)
0.1±0.01
0.08±0.01
0.08±0.01
0.1±0.01
dL/d max
(Lmax/sec)
1.5±0.1
1±0.1
1±0.1
1±0.1
dL/d min
(Lmax/sec)
-1.7±0.3
-1.3±0.2
-1.2±0.2
-1.2±0.2
HR
(msec)
292±34
257±16
265±21
318±19#
Conce ning IMD p o ocols, we e i ied ha IMD addi ion in LV papilla y muscles om
Sham and C l a s induced a simila dec ease in AT (11±5% and 12±4%, espec i ely)
and dT/d max (5±4% and 7±4%, espec i ely) while elaxa ion pa ame e s did no
p esen any s a is ical changes (Figu e 17, A, B and C). On he o he hand, in le
en icula papilla y muscles om TAC and L-NAME a s, IMD induced an inc ease o
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AT (11±3% and 13±3%, espec i ely) and dT/d max (20±3% in bo h). Fu he mo e, in
hese animal g oups, IMD induced an inc ease in he a e o elaxa ion (dT/d min;
23±4% and 31±6%, TAC and L-NAME a s espec i ely), sugges ing an has ening
e ec on elaxa ion. In sho , hese da a showed a di e en IMD myoca dial e ec ,
p omo ing a nega i e ino opic e ec in bo h con ol g oups (Sham and C l) bu he
opposi e e ec in TAC and L-NAME a s, in which i induced a posi i e ino opic and
lusi opic e ec s.
Figu e 17 – E ec o inc easing concen a ions (10-10 o 10-6) o IMD on isola ed le en icula papilla y muscles om Sham (n=6),
TAC (n= 7), C l (n= 6) and L-NAME (n= 5) a s, in A) Ac i e Tension (AT), B) maximum eloci y o ension ise (dT/d max) and C)
maximum eloci y o ension decline (dT/d min). *P<0.05 s Sham; #P<0.05 s C l.
A
B
C
#
*
#
*
#
*
#
*
#
*
#
*
#
*
#
*
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3. Ca diac endo helial dys unc ion in TAC a s
Since IMD has an eNOS-media ed e ec on myoca dium, and so i is
dependen on NO bioa ailabili y (Pi es, Pinho e al., 2012), we sea ched o endo helial
dys unc ion in LV hipe ophy a models (Figu e 18).
Figu e 18– Sea ch o endo elial dys unc ion on le en icle hipe ophy animal models. A) Wes e n blo analysis o
exp ession o o al eNOS ( -eNOS), no malized o be a-ac in (β-ac in), whose g aphic ep esen a ion shows a
s a is ical simila i y be ween Sham and TAC a s. B) Immunohis ochemical s aining o le en icle samples om
Sham, C l, TAC and L-NAME a s (n= 5-6). The g aphic ep esen a ion shows bo h TAC and L-NAME a s ha e an
inc eased le el o ni o y osine ela i ely o hei espec i e con ols, Sham and C l. C) GTPCH-1 mRNA exp ession
in Sham and TAC a s, whose le els a e s a is ically simila in bo h g oups. D) Le els o con e sion o L-a ginine o L-
ci uline, which a e signi ican ly g ea e in Sham a s han in TAC a s, sugges ing a diminished eNOS ac i i y in hese
la e . *P<0.05 s Sham; #P<0.05 s C l.
A
C
B
#
*
D
*
140 KDa
45 KDa
TAC
L-NAME
S
C
Sham
C l

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To do ha , we s a ed by accessing eNOS p o ein exp ession in TAC a s,
which was s a iscally simila o ha in Sham a s (Figu e 18A). Since oxida i e s ess is
a main cause o endo helial dys unc ion, we immunohis ochemically s ained le
en icle samples om all animal models o de ec ny o i osine, whose le els we e
inc eased in TAC and L-NAME a s (Figu e 18B), sugges ing he p esence o high
le els o ONOO-, a esul an p oduc o eac ion be ween supe oxide and NO. On he
o he hand, dec ease o BH4 is ano he possible cause o eNOS uncoupling so we
in es iga ed ansc ip le els o GTPCH1, whose exp ession ansla es in p oduc ion o
he a e-limi ing enzyme o de no o syn hesis o BH4. Howe e , hose ansc ip le els
a e no s a is ically di e en in TAC model (Figu e 18C), indica ing ha BH4 p oduc ion
is unchanged. Despi e ha , TAC a s p esen ed a lowe con e sion o L-a ginine in o L-
ci uline, showing a diminished baseline eNOS ac i i y, ela i ely o Sham a s (Figu e
18D).
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4. Ac i a ion o eNOS by IMD1-47
Phospho yla ion o eNOS a Se -1177, which is associa ed wi h ac i a ion o
eNOS, is s a is ically g ea e in TAC a s when compa ed wi h Sham a s, a baseline
(Figu e 19A). In papilla y muscles om Sham a s, IMD addi ion ipled he eNOS
phospho yla ion le els, while in hose om TAC a s IMD addi ion was no able o
enhance hose le els (Figu e 19A). A simila end was obse ed in papilla y muscles
om C l and L-NAME a s (Figu e 19B). Thus, in TAC and L-NAME a s, he e is an
impai men o IMD-induced eNOS ac i a ion, since IMD is no able o inc ease baseline
le els o phospho yla ed eNOS in hose animal models.
5. IMD ea men -induced phospho yla ion o
egula o y p o eins
Acco ding wi h ou p e ious epo (Pi es, Pinho e al., 2012), addi ion o IMD
inc easing concen a ions in papilla y muscles om Sham and C l a s inc eased c-TnI
phospho yla ion ela i ely o hose muscles wi hou IMD addi ion (Figu e 20, A and C).
Howe e , IMD ea men in TAC and L-NAME a s had no s a is ical e ec in p-cTnI
*
*
Figu e 19 – Wes e n blo analysis o phospho yla ed eNOS a Se -1177 (p-eNOS) and o al eNOS ( -eNOS) le els in
papilla y muscles om A) Sham and TAC a s and B) C l and L-NAME a s. Papilla y muscles om Sham and C l
a s showed inc easing le els o p-eNOS upon IMD addi ion (IMD) ela i ely baseline le els (con ol), while in TAC
and L-NAME a s IMD addi ion ailed o inc ease hose le els. *P<0.05 s Sham con ol; #P<0.05 s C l con ol.
A
B
#
#
*
*
140 KDa
140 KDa
45 KDa
140 KDa
140 KDa
45 KDa
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le els (Figu e 20, A and C). Conce ning p-PLB, i s le els inc eased in bo h L-NAME
and TAC a s bu i was s a is ically signi ican only in hese la e (Figu e 20, B and D).
These esul s sugges ha IMD ac i a es di e en signalling pa hways in con ol and
p essu e-o e loaded animal models, wi h phospho yla ion o di e en p o eins.
Figu e 20 – Wes e n blo analysis o phospho yla ed ca diac oponin I (p-cTnI) and phospho yla ed phospholambam
(p-PLB) in papilla y muscles om Sham and TAC a s (A and B) and C l and L-NAME a s (C and D), wi hou
(con ol) and wi h IMD addi ion (IMD). Upon IMD addi ion, in bo h Sham and C l a s, cTnI phospho yla ion inc eased,
while in TAC (A) and L-NAME (C) a s his e ec was blun ed. Conce ning p-PLB, s a is ical inc easing was ound only
in TAC a s upon IMD addi ion (B), despi e a simila end is e i ied in L-NAME a s (D). *P<0.05 s Sham con ol;
**P<0.05 s TAC con ol; #P<0.05 s C l con ol.
A
B
*
**
C
D
#
28 KDa
28 KDa
6 KDa
6 KDa
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IV. Discussion
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con ibu ing o ele a ion o bo h ac i e ension and maximum eloci y o o ce
gene a ion (Figu e 23). In e es ingly, in HF a s, eloci y o o ce gene a ion is
dec eased due o bo h down egula ion o α-MHC (myosin hea y chain) and
up egula ion o β-MHC mRNA exp ession le els (Huang, Liu e al., 2001). The e o e,
IMD seems o play an impo an ole a MHC exp ession le els, e e sing α/β a io and
con ibu ing o inc easing o o ce gene a ion eloci y.
In addi ion, pa icula ly in TAC a s, we canno exclude he in ol emen o
neu ohumo al ac i a ion- ela ed mechanisms, since p og ession o hea ailu e is
in ima ely associa ed wi h neu ohumo al ac i a ion (Middlekau and Ma k, 1998),
namely inc easing o plasma ca echolamines le els (Vique a , Daly e al., 1985). This
eleasing esul s in a ch onic ca echolamine s ess, which leads o a desensi iza ion
and down egula ion o β1-AR and β2-AR/adenylyl cyclase signaling pa hway
(Okumu a, Va ne e al., 2007), accompanied wi h up egula ion o β3-AR (Monio e,
Kobzik e al., 2001; Mo imo o, Hasegawa e al., 2004; Zhao, Wu e al., 2007), which
media es a NO-dependen nega i e ino opic e ec (Gau hie , Leblais e al., 1998).
Howe e , i seems IMD elici s ac i a ion o bo h β1 and β2-ARs, which a e able o
induce cAMP/PKA pa hway, leading o phospho yla ion o LTCC (Kamp and Hell,
2000). This modi ica ion p omo es he Ca2+ en e ing on he cell (Zipes and Jali e, 2009),
con ibu ing o he posi i e ino opic e ec (by enhancing ac i e ension) and o he
inc easing o eloci y o ension ise. Also he in ol emen o cyclic nucleo ide
phosphodies e ases (PDEs) canno be excluded since he e a e 11 mammalian PDE
amilies esponsible o speci ic cAMP and/o cGMP hyd olysis (Omo i and Ko e a,
2007). Some o hem a e epo ed as playing impo an ole in hea ailu e: o
example, PDE3A exp ession is dec eased in human samples (Ding, Abe e al., 2005)
while PDE5 inhibi ion is no only p esc ibed o ea men o e ec ile dys unc ion and
pulmona y hype ension (Kuma , F ancis e al., 2009) bu ecen ly also o he apeu ic
managemen o HF (Guazzi, 2008). Conce ning L-NAME a s, phospho yla ion o PLB
p esen s a posi i e end, howe e i was no s a is ically signi ican . This esul
sugges s he e a e o he possible egula o y p o eins in ol ed, so mo e esea ches a e
needed. The e o e, we a e awa e ha cascade o e en s which media es IMD
myoca dial e ec s in p essu e-o e load a s is much mo e complex. Ne e heless, we
cons uc ed Figu e 23 whe e we include bo h ou esul s and wha is al eady known
om li e a u e, c ea ing a simple illus a ion wi h wha we hink ha is he mos likely
sequence o e en s which leads o IMD myoca dial e ec s, unde physiologic and
pa hophysiologic condi ions.

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peNOS
cGMP
PKG ac i a ion
pTnI
Ca2+ sensi i i y
Nega i e
ino opic e ec
A
Legend:
IMD IMD ecep o s
NO SERCA2a complex
Ca2+ PLB
Oxida i e s ess
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Figu e 23 – Cascade o e en s which media ed myoca dial e ec s o IMD bo h unde physiological (A) and pa hophysiological (B) condi ions. In p esence o NO (A), cGMP/PKG signaling pa hway
is ac i a ed, esul ing in phospho yla ion o cTnI, dec ease o myo ilamen Ca2+-sensi i i y and he eby nega i e ino opic e ec . On he o he hand, in p esence o endo helial dys unc ion (B) he e is
no NO p oduc ion, hus IMD di ec ly ac i a es cAMP/PKA signaling pa hway, leading o posi i e ino opic and lusi opic e ec s media ed by phospho yla ion o PLB and inc ease o SR Ca2+ up ake.
This igu e was done using Se ie Medical A .
cAMP
PKA ac i a ion
pPLB
Posi i e ino opic
and lusi opic
e ec s
SR
Endo helial dys unc ion
B
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V. Conclusion
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The p esen wo k p o ides new insigh s abou he myoca dial e ec s o IMD
and mechanisms behind hese e ec s, in p essu e-o e load-induced HF. Besides, he
s udy o in ol emen o ca diac endo helial dys unc ion in hose e ec s poin IMD as a
po en ial ma ke o ca diac endo helial dys unc ion.
Fi s ly, haemodynamic and mo phome ic analysis showed ha he wo disease
models TAC and L-NAME a e qui e simila , wi h bo h showing LV hype ophy and a
p ese ed sys olic unc ion. Conce ning con ac ile and elaxa ion pa ame e s a he
baseline, also bo h models did no p esen any s a is ical di e ence, excep o au
which was inc eased in L-NAME, as we expec ed. Despi e hese simila i ies, o he
subsequen analysis, we ook in o accoun di e ences be ween he wo models, since
p essu e-o e load is ob ained h ough e y di e en ways: one by ao ic cons ic ion
(TAC), he o he one by sys emic hype ension (L-NAME).
Nex , adminis a ion o IMD in LV papilla y muscles om he ou di e en
animal g oups showed, o he i s ime, ha IMD elici s wo comple ely opposi e
e ec s: in diseased myoca dium i p omo es posi i e ino opic and lusi opic e ec s,
whe eas in no mal myoca dium da a sugges s a dec ease o ino opy. This obse a ion
emphasizes he pu a i e ca diop o ec i e ole o IMD in HF.
One o he easons which could explain hese so di e en beha io s is
endo helial dys unc ion, since IMD myoca dial e ec s a e media ed by endo helium and
HF is associa ed o i s dys unc ion. Despi e eNOS exp ession was unchanged a he
baseline, ac i i y assay showed eNOS unc ion was diminished. This ac allied o he
p esence o oxida i e s ess sugges s eNOS uncouling hus endo helial dys unc ion,
which is no due o he deple ion o BH4 bu p obably is pa ly due o a dec ease o i s
bioa ailabili y, since GTPCH1 exp ession emains unal e ed. In e es ingly, we e i ied
phospho yla ion o eNOS a Se -1177 was augmen ed a he baseline in diseased
myoca dium and IMD was no able o inc ease hose le els, con a y o wha we
obse ed in no mal myoca dium. We hypo hesize his inc eased phospho yla ion le els
a e likely due o Ak ch onic ac i a ion. Taken oge he , esul s sugges ha
phospho yla ion o eNOS a Se -1177 1) is a g ea con ibu o o endo helial
dys unc ion in TAC a s, by p omo ing he “pa hologic” ac i i y o eNOS and 2) explains
he opposi e myoca dial e ec s o IMD.
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Finally, and based on exp ession esul s o egula o y p o eins, we hypo hesize
IMD e ec s a e media ed by 1) ac i a ion o cAMP/PKA signaling pa hway and
inc ease o phospho yla ion o cTnI, in diseased myoca dium and 2) NO-dependen
ac i a ion o cGMP/PKG signaling pa hway and inc ease o phospho yla ion o PLB, in
no mal myoca dium. Since IMD adminis a ion elici s di e en e ec s which a e
media ed by dis inc mechanisms, his wo k also sugges ha 1) endo helial
dys unc ion plays a cen al ole in pa hophysiology o HF and 2) IMD is a po en ial
ma ke o ca diac endo helial dys unc ion.

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VI. Fu u e
Pe spec i es
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This wo k p o ides new insigh s abou myoca dial e ec s o in e medin and
signaling pa hways in ol ed, ein o cing i s pu a i e ca diop o ec i e ole in
ca dio ascula disease. Howe e , mo e s udies a e needed in o de o p o e his
hypo hesis. Fi s ly, i would be in e es ing d aw a p o ocol o IMD ch onic
adminis a ion on ch onic p essu e-o e loaded a s, namely in TAC model, since i is
he mos close model o human HF. Recen ly, IMD ch onic adminis a ion was done in
SHR model and esul s seem o be e y p omising (Yuan, Wang e al., 2012).
Ano he in e es ing opic would be o in es iga e he ole o o he pos -
ansla ional modi ica ions o eNOS such as S-glu a hionyla ion in HF. Indeed, ecen ly
Chen e al showed ha unde oxida i e s ess eNOS is S-glu a hionyla ed in
endo helial cells and his modi ica ion is esponsible o p oduc ion o ●O2- by eNOS
(Chen, Wang e al., 2010).
Gi en he impo ance o s udy o hea ailu e, many au ho s ha e in es iga ed
di e en playe s in his so complex ca diac dys unc ion. One o hose playe s is
oxida i e s ess. Con a y o expec ed, ea men s using an ioxidan s ha e shown no
clinical bene i s (Hennekens, Bu ing e al., 1996; Yusu , Dagenais e al., 2000). Fo his
eason, s udies ha e ocus on sou ces o ROS: eNOS ecoupling and inc ease
endogenous le els o an ioxidan s a e po en ial solu ions (Takimo o and Kass, 2007).
One o hese s udies ecoupled eNOS h ough adminis a ion o bo h BH4 and
apocynin, an inhibi o o NADPH oxidases, bu he s udy was pe o med only in
hype ensi e ao as (Cai, Laude e al., 2006).
Finally, in my opinion, he mos in e es ing s udy line is miRNAs. Se e al
miRNAs ha e been iden i ied as impo an modula o s o ca dio ascula de elopmen
and ca diac disease and ha e eme ged as a good al e na i e o d ugs (Small and
Olson, 2011). Howe e , c oss alk o many miRNAs is poo ly s udied, which is an
impo an obs acle o he apeu ics, since silencing o one miRNA could be no e ec i e.
Besides, a miRNA has no only one a ge , so i is e y impo an s udy and iden i y
speci ic miRNAs.
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VII. Re e ences
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