Myocardial effect of Intermedin in hypertrophic heart: mechanisms and involvement of endothelial dysfunction

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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion ii 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion iii 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 1 I. In oduc ion FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 2 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 3 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 4 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 5 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 6 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.           FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 7 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 8 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 9 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 16 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 17 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 18 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). FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 19 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; FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 20 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 21 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 22 (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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 23 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 24 II. Ma e ials & Me hods FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 25 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 32 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 . FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 33 III. Resul s FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 34 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 35 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 36 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 # * # * # * # * # * # * # * # * FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 37 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 38 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). FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 39 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 40 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 41 IV. Discussion FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 48 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 49 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 50 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 FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 51 V. Conclusion FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 52 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 53 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 54 VI. Fu u e Pe spec i es FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 55 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. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 56 VII. Re e ences The s yle used is based on Ame ican Jou nal o Physiology-Hea and Ci cula o y Physiology published by Ame ican Physiological Socie y. Adap a ions we e pe o med using EndNo e X4 So wa e. FCUP Myoca dial e ec o In e medin in hype ophic hea : mechanisms and in ol emen o endo helial dys unc ion 57 Abel ED, Doens T (2011). Mi ochond ial adap a ions o physiological s. pa hological ca diac hype ophy. Ca dio asc Res 90: 234-242. Ahmed A (2003). Myoca dial be a-1 ad enocep o down- egula ion in aging and hea ailu e: implica ions o be a-blocke use in olde adul s wi h hea ailu e. Eu J Hea Fail 5: 709-715. 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