Dulce Marléne Martins Fontoura - …repositorium.sdum.uminho.pt/bitstream/1822/15966/1/Dulce...

Dulce Marléne Martins Fontoura

Outubro de 2010

Universidade do Minho

Escola de Ciências

Avaliação da Expressão Génica e Vias Subcelulares Neuroendócrinas e Metabólicas em Modelos Experimentais de Hipertensão Pulmonar

Dissertação de Mestrado Mestrado em Genética Molecular

Dulce Marléne Martins Fontoura

Outubro de 2010

Universidade do Minho

Escola de Ciências


Trabalho efectuado sob a orientação doProfessor Doutor Adelino F. Leite Moreirae daProfessora Doutora Maria João Sousa

DE ACORDO COM A LEGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTA TESE

Universidade do Minho, ___/___/______

Assinatura: ________________________________________________

iii

AGRADECIMENTOS

Agradeço a todos aqueles que contribuíram para a realização desta tese e me

ajudaram no caminho exigente da investigação em Fisiopatologia Cardiovascular.

Ao Professor Doutor Adelino F. Leite Moreira, orientador desta tese, que de forma

incansável me ensinou os conteúdos práticos e teóricos da Fisiopatologia Cardiovascular.

Agradeço igualmente pela sua disponibilidade permanente e dedicação durante a minha

formação como investigadora, pelos seus conselhos e orientação profissional, por ter

garantido todas as condições necessárias para o desenvolvimento e realização do projecto

científico presente nesta tese, assim como, pela oportunidade e privilégio que me deu em

colaborar com a sua equipa de investigação. Desta forma, manifesto a minha sincera

gratidão e admiração.

À Professora Doutora Maria João Sousa pelo seu incansável apoio e orientação ao

longo do Mestrado em Genética Molecular.

A todos os elementos do Serviço de Fisiologia, da Faculdade de Medicina do Porto,

o meu profundo reconhecimento pelo apoio que sempre souberam prestar. À Professora

Doutora Carmen Brás Silva pela sua preocupação, apoio e amizade. À Professora Doutora

Inês Pires pela disponibilidade e recente colaboração científica. O meu reconhecido

agradecimento ao Professor Doutor Amândio Rocha, ao Professor Doutor Roberto Roncon

de Albuquerque, ao Professor Doutor Tiago Henriques Coelho e ao Professor Doutor Paulo

Chaves. Ao Doutor André Lourenço pelo seu profissionalismo, entusiasmo, rigor,

persistência, exigência e dedicação, pela preocupação constante com o decorrer do meu

trabalho, por acreditar e incentivar todas as minhas actividades como investigadora, assim

como pela amizade que me tem confiado. À Dra. Antónia Teles pela atenciosa

disponibilidade durante os seus ensinamentos e por ser uma pessoa encantadora. À Marta

Oliveira e à Maria José pelo apoio técnico prestado. À Daniela Miranda, ao Francisco

Vasques Nóvoa, ao Vasco Sequeira, ao Rui Cerqueira, ao José Pedro Pinto, ao Pedro

Ferreira e à Carolina Rocha pelo convívio pessoal e saudável espírito de equipa. À D.

Francelina Marques, à D. Rosa Gonçalves, à D. Margarida, ao Sr. Armando, ao Sr. Alberto

e ao Pedro Leitão o meu reconhecido agradecimento.

iv

À Professora Doutora Cândida Lucas pela sua disponibilidade e dedicação. Ao

Professor Doutor Rui Oliveira pelo convívio científico, amizade e estímulo ao longo do meu

percurso profissional.

Aos meus pais pelo incansável apoio, amor, compreensão e paciência ao longo do

meu percurso de vida e por me terem privilegiado com formação de qualidade. Ao meu

irmão e à minha cunhada pelos momentos bem passados, pelas palavras de apoio e

verdadeira amizade. Aos meus avós pelo ensinamento dos seus valores e sabedoria. À

Professora Doutora Hercília Guimarães e ao Professor Doutor Jorge Areias pelo fantástico

apoio e amizade, assim como à Doutora Cristina Areias e ao Doutor José Castro. Ao Luís

Pinto Leite e seus familiares pelo privilégio da sua amizade, paciência e atitude positiva. À

Isis Alonso, ao Augusto Silva, à Filipa Neiva, à Manuela Araújo e à Paula Brilhante Simões

pela amizade e apoio que sempre me confiaram.

v

Avaliação da Expressão Génica e Vias Subcelulares Neuroendócrinas e Metabólicas

em Modelos Experimentais de Hipertensão Pulmonar (Resumo)

A hipertensão pulmonar (HP) é um síndrome de etiologia diversificada, que conduz à

hipertrofia e falência ventricular direita (VD), partilhando muitas características com a

insuficiência cardíaca (IC), que é um síndrome complexo caracterizado pelo

comprometimento da ejecção ou do preenchimento do miocárdio. HP e IC associam-se a

activação neuroendócrina e inflamatória e a manifestações sistémicas como a caquexia

cardíaca, que pode definir-se por perda ponderal superior a 6% num intervalo mínimo de 6

meses, e acompanha 50% dos casos de IC severa, constituindo um preditor de mau

prognóstico. Entre os mecanismos complexos subjacentes destacam-se alterações

metabólicas e activação imunitária e neuroendócrina. Regimes alimentares hipercalóricos

são um factor de risco cardiovascular estabelecido, no entanto, e paradoxalmente, a

obesidade tem sido associada a melhor prognóstico na IC e a suplementação nutricional,

na doença crónica, contraria a anorexia e a caquexia. Os efeitos de uma dieta hipercalórica

(DH) na HP com IC poderão ser, portanto, inteiramente distintos. Neste trabalho, avaliámos

as repercursões de uma DH, rica em gordura saturada e carbo-hidratos simples, na IC e

caquexia associadas à HP. Induzimos HP em ratos Wistar machos por injecção subcutânea

única de 60 mg.Kg-1 de monocrotalina (MCT), distribuindo aleatoriamente os animais por

regimes alimentares com DH (5,4 kcal.g-1) ou dieta normal (DN) (2,9 Kcal.g-1). A evolução

ponderal, ingestão calórica e mortalidade foram registadas. Às 5 semanas, procedeu-se à

avaliação hemodinâmica e morfométrica, à avaliação histológica pulmonar e miocárdica, da

apoptose miocárdica, da expressão génica da endotelina-1 (ET-1), interleucina-6 (IL-6),

factor de necrose tumoral-α (TNF-α) e de enzimas metabólicas, bem como à quantificação

sérica do TNF-α e miocárdica da actividade dos factores de transcrição nuclear -κB (NF-κB)

e do receptor activado pelo proliferador do peroxissoma-α (PPAR-α). Ratos injectados com

MCT alimentados com DN, desenvolveram HP e IC, com elevada mortalidade, bem como

remodelagem e apoptose miocárdica. Apresentaram menor ingestão calórica e ganho

ponderal. A HP foi acompanhada por maior actividade do factor de transcrição NF-κB,

sobre-expressão de ET-1, TNF-α e IL-6, e aumento de TNF-α circulante, com redução da

actividade do PPAR-α. A DH atenuou a IC e caquexia, melhorando a sobrevida, a função e

remodelagem miocárdicas e reduzindo a actividade inflamatória e neuroendócrina. Os

resultados incentivam a realização de ensaios clínicos com DH em pacientes com caquexia

cardíaca.

vii

Neuroendocrine and Metabolic Gene Expression and Subcellular Pathway Changes in

Experimental Pulmonary Hypertension (Abstract)

Pulmonary hypertension (PH), a syndrome of diverse etiology that leads to right

ventricular (RV) hypertrophy and failure, shares many features with heart failure (HF), a

syndrome characterized by impaired ability of the myocardium to fill or eject. PH and HF are

associated with neuroendocrine and inflammatory activation and systemic manifestations

such as cardiac cachexia, defined by a weight loss of more than 6% over 6 months.

Cachexia accompanies HF in up to 50% of severe cases and independently determines a

poor prognosis. As part of the complex and still incompletely revealed mechanisms

underlying cachexia associated with HF and PH are abnormalities of general metabolism as

well as of the immune and neuroendocrine systems. Hypercaloric diet (HD) regimens are

well-established cardiovascular risk factors, but since obesity has paradoxically been shown

to predict a better prognosis in HF and nutritional and energetic support have been shown to

partly counteract anorexia and cachexia in chronic diseases, the effects of a HD regimen

may have quite distinct effects in severe PH and terminal HF. Our study was designed to

evaluate the repercussions of a HD, rich in saturated fat and simple carbohydrates in HF

and cachexia associated with PH. We induced PH in male Wistar rats by subcutaneous

injection of 60 mg.Kg-1 monocrotaline (MCT) and randomly allocated rats to be fed with

either a 5.4 kcal.g-1 HD or a normal 2.9 Kcal.g-1 diet (ND). Calorie intake, weight gain and

mortality were recorded daily. Five weeks after, we evaluated RV and left ventricular

hemodynamics and morphometry, lung and myocardial histology, myocardial expression of

endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and metabolic

enzymes, plasma TNF-α levels as well as myocardial apoptosis and nuclear factor-κB (NF-

κB) and peroxisome proliferator-activated receptor-α (PPAR-α) transcription factor activities.

ND fed MCT-injected rats showed PH and HF, associated with high mortality, myocardial

remodeling and apoptosis, reduced calorie intake, and loss of body weight, lean and fat

mass that were attenuated by the HD. PH was accompanied in ND fed MCT-injected rats by

increased myocardial NF-κB transcription factor activity and ET-1, TNF-α and IL-6

overexpression, reduced PPAR-α activity, and increased circulating TNF-α, which were

offset by the HD. HD attenuated experimental PH with HF and cachexia, improving survival,

myocardial function and remodeling, and reduced inflammatory and neuroendocrine

activities. Our results encourage the conduction of clinical trials with HD regimens in patients

with cardiac cachexia.

ix

ÍNDICE

Agradecimentos.…………………………………………………………………...…………….…..iii

Resumo.………………………………………………………………………………………...….....v

Abstract.………………………………………………………………………………...………...….vii

Índice.……………………………………………………………………………………...………….ix

Lista de abreviaturas.……………………………………………………………………..……......xii

1. Introdução .......................................................................................................................... 1

1.1. Hipertensão Pulmonar ................................................................................................. 1

1.2. Diagnóstico, apresentação e avaliação clínica ............................................................ 2

1.3. Prognóstico .................................................................................................................. 3

1.4. Modelos experimentais ................................................................................................ 3

1.5. Fisiopatologia ............................................................................................................... 5

1.6. HPA como panvasculopatia ......................................................................................... 5

1.7. Mecanismos genéticos ................................................................................................ 6

1.8. Inflamação ................................................................................................................... 6

1.9. O miocárdio na HP ....................................................................................................... 7

1.10. Terapêutica ................................................................................................................ 8

1.11. IC e caquexia ............................................................................................................. 9

1.12. Fisiopatologia da caquexia cardíaca ........................................................................ 10

1.13. Remodelagem metabólica na progressão da IC ...................................................... 11

1.14. Nutrição na IC e caquexia ........................................................................................ 14

2. Objectivos………….. ........................................................................................................ 15

3. Metodologia ...................................................................................................................... 16

3.1. Modelos animais ........................................................................................................ 16

3.2. Estudos de biologia molecular ................................................................................... 17

3.2.1. Quantificação de DNA e conteúdo protéico do miocárdio do VD e VE ................ 17

3.2.2. Quantificação de mRNA ...................................................................................... 17

3.2.3. Quantificação das isoformas das cadeias pesadas de miosina ........................... 19

3.2.4. Quantificação do TNF-α no plasma ..................................................................... 20

3.2.5. Apoptose miocárdica ........................................................................................... 20

3.2.6. Quantificação do NF-κB e do PPAR-α ................................................................ 21

4. Resultados ....................................................................................................................... 23

x

A hipertensão pulmonar experimental associada à insuficiência cardíaca e caquexia é

atenuada pela dieta rica em gorduras e carbo-hidratos simples (trabalho original em

revisão na revista American Journal of Physiology Heart and Circulatory Physiology)....24

5. Discussão e Conclusões .................................................................................................. 52

Relevância Clínica e Perspectivas Futuras ...................................................................... 58

6. Anexo ............................................................................................................................... 59

Novos Conceitos Fisiopatológicos e Abordagem Terapêutica da Hipertensão Pulmonar

(trabalho de revisão submetido para a revista International Journal of Cardiology)… … 60

7. Referências ...................................................................................................................... 97

xi

LISTA DE ABREVIATURAS

ACSL1 Acil-CoA Ligase de Ácidos Gordos de Cadeia Longa tipo 1

AG Ácidos Gordos

BCC Bloqueadores dos Canais de Ca2+

BMP Proteínas de Morfogénese Óssea

BMPR2 Receptor das Proteínas de Morfogénese Óssea do tipo 2

BNP Peptídeo Natriurético do tipo B

CCD Cateterismo Cardíaco Direito

CoA Coenzima A

DC Débito Cardíaco

DPOC Doença Pulmonar Obstrutiva Crónica

ET-1 Endotelina-1

FO Fosforilação Oxidativa

GI Gastrointestinal

GLUT-4 Transportador de Glicose do tipo 4

HP Hipertensão Pulmonar

HPA HP Arterial

HPAf HPA familiar

HPAi HPA idiopática

HpnA HP não-Arterial

HRP Peroxidase de Rábano

IC Insuficiência Cardíaca

IL-6 Interleucina-6

I-κB Inibidor-κB

LCAD Desidrogenase de Acil-CoA de Cadeia Longa

LPS Lipopolissacarídeo

MCAD Desidrogenase de Acil-CoA de Cadeia Média

MCT Monocrotalina

NF-κB Factor de Transcrição Nuclear-κB

PAP Pressão(ões) da Artéria Pulmonar

PDH Desidrogénase do Piruvato

PDK Cínase da Desidrogénase do Piruvato

PDK4 PDK do tipo 4

PmAP Pressão(ões) médias da Artéria Pulmonar

xii

PPAR Receptor Activado pelo Proliferador do Peroxissoma

PPRE Elemento de Resposta do Proliferador do Peroxissoma

ROS Radicais Livres de Oxigénio

SDS-PAGE Electroforese em Gel de Poliacrilamida e Dodecil Sulfato de Sódio

TEP Tromboembolismo Pulmonar

TGF-β1 Factor de Transformação de Crescimento-β1

Tm Temperatura de fusão ou melting

TM6M Teste de Marcha durante 6 Minutos

TNF-α Factor de Necrose Tumoral-α

TUNEL Marcação dos Terminais Desoxiuridina Trifosfato pela Desoxinucleotidil

Transferase (terminal UDP nick-end labeling)

VD Ventrículo(ar) Direito(a)

VE Ventrículo(ar) Esquerdo(a)

VEGF Factor de Crescimento do Endotélio Vascular


1

1. Introdução

1.1. Hipertensão Pulmonar

A hipertensão pulmonar (HP) é um síndrome rapidamente progressivo e fatal, apesar

das alternativas terapêuticas actuais. É classicamente diagnosticada pela demonstração

hemodinâmica de pressões médias da artéria pulmonar (PmAP) superiores a 25 mmHg em

repouso, ou 30 mmHg durante o exercício (Fishman, 2004).

Na maioria dos casos de HP, esta acompanha doenças cardio-respiratórias comuns,

não existindo doença vascular pulmonar intrínseca. No entanto, a HP também se pode

manifestar, menos frequentemente, sob a forma de HP arterial (HPA), uma doença

vasoproliferativa que envolve as pequenas artérias pulmonares e que se define, em termos

hemodinâmicos, não só pela elevação das PmAP, mas também pela presença de pressões

de enchimento ventriculares esquerdas (VE) normais, ou seja pressões de encravamento

capilar pulmonar ou pressões de enchimento VE inferiores a 15 mmHg (Chin e Rubin,

2008). A HPA apresenta traços anatomopatológicos característicos, alguns dos quais,

nomeadamente as lesões plexiformes, não observáveis noutras formas de HP (Rabinovitch,

2008).

De modo a melhor traduzir os mecanismos fisiopatológicos e apresentação clínica, a

classificação etiológica da HP tem vindo a sofrer modificações. Resumidamente, a HP pode

ser classificada em HPA e HP não-arterial (HPnA), sendo esta última composta por vários

subtipos de doença, com causa estabelecida, indefinida ou multifactorial. A classificação

adoptada mais recentemente é sumariada na tabela 2 do trabalho de revisão anexado.

A incidência e prevalência estimadas da HPA, com base em vários estudos

populacionais, são cerca de 2,4 a 7,6 casos/milhão e 15 a 26 casos/milhão de habitantes

por ano, respectivamente (Humbert et al., 2006; Peacock et al., 2007). Com a excepção da

HPA idiopática (HPAi), não há estimativas precisas da incidência e prevalência da HP, mas,

uma vez que as manifestações clínicas são inespecíficas, qualquer previsão constituirá

uma subestimativa (Provencher et al., 2005). Nas últimas décadas, a HPnA tem sido

crescentemente reconhecida, nomeadamente a HP associada à doença pulmonar

obstrutiva crónica (DPOC), uma vez que, segundo a maioria das previsões, em 2020,

devido ao tabagismo, será a 3ª causa de morte (Murray e Lopez, 1997). De facto, a DPOC

é presentemente responsável por até 84% dos casos de cor pulmonale. A insuficiência

cardíaca (IC) direita é a sua complicação mais grave, sendo responsável por 10 a 30% dos

internamentos devido à IC descompensada (MacNee, 1994).


2

A HP e a IC direita subsequente constituem uma importante causa de mortalidade e

incapacidade a nível mundial (Humbert, 2009). No entanto, o impacto na saúde é

provavelmente maior do que o reconhecido, sobretudo pelas associações recentemente

estabelecidas com a hemodiálise (Fruchter e Yigla, 2008) e com o síndrome metabólico

(Robbins et al., 2009), assim como, com patologias infecciosas altamente prevalentes nos

países em desenvolvimento (Butrous et al., 2008).

As duas últimas décadas foram pródigas em estudos experimentais e clínicos que

permitiram desvendar um pouco dos seus complexos mecanismos fisiopatológicos e

tornaram disponíveis vários novos fármacos (Rabinovitch, 2008). No entanto, o prognóstico

HP continua a ser pouco satisfatório e muitos dos pacientes acabam por necessitar de

transplantação (Keogh et al., 2009).

1.2. Diagnóstico, apresentação e avaliação clínica

Os sintomas, como por exemplo a fadiga e a dispneia, são normalmente inespecíficos,

insidiosos e indefinidos, tornando o diagnóstico um desafio para os clínicos (Provencher et

al., 2005). A HP é sub-diagnosticada, sendo a maior parte dos doentes identificados apenas

após o aparecimento de manifestações como dor torácica, palpitações, edema, ascite e

síncope. Normalmente o diagnóstico é efectuado com 2 ou 3 anos de atraso, e muitas

vezes apenas após episódios de síncope, quando o débito cardíaco (DC) está já

comprometido e as pressões da artéria pulmonar (PAP) atingem níveis supra-sistémicos

(Nef et al., 2010; Rabinovitch, 2008). No entanto, o diagnóstico e tratamento precoce, em

estadios reversíveis, é fundamental para melhorar a resposta à terapêutica. Para facilitar o

diagnóstico, na definição mais recente foram incluídos novos critérios de diagnóstico, não-

invasivos e mais práticos, baseados na ecocardiográfica (Nef et al., 2010). Contudo, em

termos práticos, a ecocardiografia é normalmente utilizada para rastreio, uma vez que não

permite a quantificação do fluxo transpulmonar e das pressões venosas, sendo a

confirmação do diagnóstico feita pelo cateterismo cardíaco direito (CCD), que continua a

ser o gold standard (Chin e Rubin, 2008). O CCD também é essencial na selecção de

doentes que poderão beneficiar de terapia crónica com bloqueadores dos canais de Ca2+

(BCC) (McLaughlin et al., 2009; Sitbon et al., 2005). Vários exames auxiliares de

diagnóstico podem fornecer indicações úteis ao rastreio, diagnóstico definitivo e diferencial

(McLaughlin et al., 2009; Nef et al., 2010; van Wolferen et al., 2007). Outros exames

auxiliares são empregues para avaliar a capacidade funcional, como por exemplo a

espirometria, a difusão de monóxido de carbono e o teste de marcha durante 6 minutos

(TM6M) (Sun et al., 2003). A descrição detalhada destes não se encontra no âmbito da


3

presente tese, mas pode ser consultada no trabalho de revisão em anexo. O TM6M é um

indicador sensível e reprodutível no compromisso moderado do desempenho

cardiopulmonar, sendo vulgarmente empregue para quantificar a melhoria clínica após

intervenções terapêuticas em ensaios clínicos (Miyamoto et al., 2000). As orientações da

American Thoracic Society relativamente ao TM6M constituem uma perspectiva abrangente

sobre as suas indicações, contra-indicações, precauções de segurança, aspectos técnicos

e limitações (ATS statement, 2002).

1.3. Prognóstico

A raridade da HPA, a sua etiologia diversa e a constante evolução na abordagem

terapêutica tem dificultado as estimativas da taxa de mortalidade anual. Um registo

recolhido entre 1981 e 1985, estimou taxas de sobrevida de 68% e 48% aos 1 e 3 anos,

respectivamente na HPAi (McLaughlin et al., 2004). Contudo, registos mais recentes

apontam para uma melhoria no prognóstico, com sobrevidas de 83 a 88% após 1 ano e de

58 a 72% aos 3 anos (McLaughlin e Suissa, 2010). A progressão da HP nas formas de

HPnA é normalmente mais lenta e o prognóstico geralmente mais favorável. No entanto, o

desenvolvimento de HP tem um impacto importante na qualidade de vida e sobrevida

(Hoeper et al., 2009).

1.4. Modelos experimentais

Embora nenhum modelo animal recapitule completamente todas as características da

HPA humana, o estudo da fisiopatologia da HP tem sido levado a cabo essencialmente em

modelos animais de HPA. A combinação de diferentes agressões, de acordo com a

hipótese das agressões múltiplas, permitiu desenvolver fenótipos mais severos que melhor

mimetizam as características da HPA humana (Robbins, 2004).

O modelo experimental mais frequentemente utilizado pela sua reprodutibilidade e

facilidade de execução, é o modelo de HP induzida pela monocrotalina (MCT) no rato

(Brown et al., 1998; Jasmin et al., 2001; Lalich e Merkow, 1961; Lourenço et al., 2006). A

MCT é um alcalóide pirrolizidínico, isolado a partir da Crotalaria spectabilis, que após

desidrogenação pelo citocromo P450 hepático num pirrol reactivo, adquire acção tóxica no

endotélio, provocando uma endarterite obliterativa das arteríolas pulmonares. As lesões

histológicas pulmonares são semelhantes às da HP, sendo a MCT desprovida de acção

tóxica directa no miocárdio (Chen et al., 1998). Este modelo experimental contribuiu de

forma muito relevante para a evolução terapêutica recente na HP. Praticamente todos os


4

fármacos que tiveram sucesso na HPA humana também foram eficazes neste modelo, com

a excepção do beraprost (Stenmark et al., 2009). Adicionalmente, neste modelo a activação

neuro-humoral assemelha-se à da IC (Brunner, 1999; Leineweber et al., 2002), pelo que

tem sido empregue para estudar a hipertrofia miocárdica (Buermans et al., 2005) e

comparar alterações funcionais e moleculares entre os dois ventrículos (Kögler et al., 2003;

Leineweber et al., 2002; Schott et al., 2005; Seyfarth et al., 2000), o VE que é apenas

influenciado por mediadores neuroendócrinos e o ventrículo direito (VD) que é

simultaneamente sobrecarregado. Foi este modelo animal, que evolui rapidamente para IC

descompensada e fatal, com anorexia e caquexia acentuadas e perda de massa músculo-

esquelética (Ceconi et al., 1989; Hessel et al., 2006; Lourenço et al., 2006; Steffen et al.,

2008; Werchan et al., 1989) que seleccionamos para desenvolver o trabalho experimental

que levamos a cabo e que descrevemos nas secções de metodologia, resultados e

discussão.

No entanto, o modelo de HP induzida pela MCT apresenta algumas limitações

(Robbins, 2004). O espectro anatomopatológico mais amplo da HPAi só é reproduzido por

completo quando aos ratos tratados com MCT se associa a realização de pneumectomia.

Neste caso, pode observar-se a formação de neo-íntima e obliteração de pequenas

arteríolas pulmonares (Tanaka et al., 1996). A MCT também tem acção tóxica, embora

menos marcada, no endotélio renal e hepático, o que acarreta efeitos sistémicos. O modelo

é menos reprodutível no murganho, e mesmo estirpes diferentes de ratos apresentam

susceptibilidades diversas. Finalmente, há diferenças na gravidade do fenótipo atribuíveis

ao timming da injecção (Lalich e Merkow, 1961).

Outro modelo experimental utilizado frequentemente é o modelo de HP induzida pela

hipóxia. Este modelo experimental é mais relevante no estudo da HP associada à hipóxia e

doença respiratória e não tanto da HPA. Os animais são expostos a pressões alveolares de

oxigénio < 70 mmHg, de forma a desencadear vasoconstrição pulmonar. Na exposição

prolongada ocorre remodelagem dos ramos distais das artérias pulmonares, com

hiperplasia endotelial e miocitária (Fallon et al., 1998; Rhodes, 2005; Taraseviciene-Stewart

et al., 2001). Para além destes dois modelos principais, vários outros têm sido usados

ocasionalmente. Os ratos de capuz castanho, que têm um defeito plaquetário no

armazenamento de serotonina, desenvolvem HP espontaneamente, podendo o fenótipo ser

acelerado pela exposição à hipóxia (Stenmark et al., 2009). A administração de uma dose

única de semaxinib (SU5416), um antagonista do receptor do factor de crescimento do

endotélio vascular (VEGF) leva ao desenvolvimento de lesões oclusivas da neo-íntima em

ratos expostos à hipoxia (Taraseviciene-Stewart et al., 2001). O modelo de HP induzida


5

pela embolização com coágulos autólogos ou microsferas de diversos materiais (ex.

sefadex, polidextrano), possibilita o estudo das formas agudas e crónicas de HP associada

ao tromboembolismo pulmonar (TEP) (Shelub et al., 1984; Watts et al., 2006; Watts et al.,

2010). Adicionalmente, têm sido estudados também animais manipulados geneticamente

(Greenway et al., 2004; Ivy et al., 2005; Steiner et al., 2009).

1.5. Fisiopatologia

Como vimos, a HPA tem sido largamente estudada sobretudo recorrendo a modelos

animais. Paradoxalmente, poucos estudos têm sido realizados sobre a fisiopatologia das

formas mais comuns, a HPnA. No entanto, admite-se que a fisiopatologia destas formas

tem aspectos similares à HPA (Hoeper et al., 2009). De facto, sabe-se que na HP

associada a doenças respiratórias crónicas a vasoconstrição hipóxica não é um mecanismo

fundamental (Chaouat et al., 2005), sendo mais importantes a disfunção endotelial e a

activação neuroendócrina e inflamatória (Wright et al., 2005); que na HP associada ao TEP

crónico a elevação das resistências vasculares pulmonares não se deve apenas à oclusão

vascular pelos êmbolos, mas também a graus variáveis de arteriopatia de pequenos vasos

(Hoeper et al., 2006); e, finalmente, que na disfunção VE e patologia valvular, para além da

transmissão à circulação pulmonar de pressões de preenchimento VE elevadas também

ocorre disfunção endotelial e perturbação da produção de serotonina, tromboxano A2 e

angiotensina-II, que contribuem para o agravamento adicional da HP (Moraes et al., 2000).

1.6. HPA como panvasculopatia

A concepção mais prevalente nas últimas décadas admite que a HP resulta de um

desequilíbrio entre vasoconstritores e vasodilatadores pulmonares (Christman et al., 1992).

A síntese de prostaciclina e óxido nítrico, vasodilatadores e mediadores antiproliferativos,

está diminuída na HPA, enquanto a de endotelina-1 (ET-1), um vasoconstritor e mitógeneo

das células musculares lisas, está aumentada. Efectivamente, os prostanóides e outros

vasodilatadores pulmonares, têm constituído a base terapêutica na HPA. No entanto, sabe-

se actualmente que esta concepção é insuficiente para explicar a complexidade dos

mecanismos fisiopatológicos da HP. De facto, apesar dos avanços terapêuticos mais

recentes, o prognóstico continua a ser muito reservado (Rabinovitch, 2008). De acordo com

a maioria dos autores, a HPA é actualmente encarada como uma panvasculopatia, com

vários traços histológicos característicos identificáveis nas pequenas artérias e arteríolas

pulmonares (McLaughlin et al., 2009). Como hipótese fisiopatológica admite-se que após


6

apoptose endotelial persistente sejam seleccionados fenótipos endoteliais resistentes à

apoptose, que interagem com as células musculares lisas através da produção de factores

de crescimento, induzindo proliferação muscular lisa e transdiferenciação das células

endoteliais e dos fibroblastos em miócitos (Sakao et al., 2009). Adicionalmente, a activação

de metaloproteases conduz à perda de integridade da barreira da membrana basal, e ao

recrutamento de células inflamatórias que participam também na libertação de peptídeos

mitogénicos (Cowan et al., 2000).

Outra hipótese fisiopatológica sustenta que a HPA e o cancro partilham anomalias do

metabolismo mitocondrial, designadas por “fenótipo de Warburg”. Este consiste na

progressiva substituição da fosforilação oxidativa (FO) pela glicólise anaeróbia, mesmo em

condições de normóxia, que se associa a proliferação celular e inibição da apoptose e se

deve à alteração da sensibilidade mitocondrial ao O2 (Archer et al., 2008; Bonnet et al.,

2006). Uma descrição molecular detalhada das perspectivas fisiopatológicas mais recentes

sobre a HP é apresentada no trabalho de revisão em anexo.

1.7. Mecanismos genéticos

Em mais de 80% dos pacientes com HPA familiar (HPAf) é possível identificar

mutações inactivadoras do receptor das proteínas de morfogénese óssea do tipo 2

(BMPR2), um receptor activado pela superfamília do factor de transformação de

crescimento-β1 (TGF-β1), que inclui as proteínas de morfogénese óssea (BMP). Estas

mutações levam à inibição da via de sinalização da smad, e à menor diferenciação das

células musculares lisas, à proliferação celular e ao aumento do tono muscular (Morrell et

al., 2001; Yang et al., 2005), uma vez que a via das smads regula normalmente a

transcrição génica promovendo respostas citostáticas. Contudo, as mutações do BMPR2

constituem apenas 10 - 20% dos casos de HPA não familiar, e mesmo na HPAf a

penetrância é baixa (Newman et al., 2004), portanto, outros mecanismos genéticos deverão

estar envolvidos. Para alguns genes, foram identificados polimorfismos de nucleotídeos

singulares relacionados com predisposição para HPA (MacLean e Dempsie, 2009;

Provencher et al., 2005; Shapiro et al., 2007).

1.8. Inflamação

O estado pró-inflamatório dos vasos pulmonares tem, recentemente, sido apontado

como um evento primário e mecanismo de progressão da doença, alternativamente à visão

tradicional, que o encarava como mera consequência da doença. A presença de auto-

anticorpos circulantes e a infiltração de células inflamatórias nos pulmões são


7

características da HPA associada às doenças do tecido conjuntivo, mas também podem

surgir na HPAi (Dorfmuller et al., 2003). As citocinas, como o factor de necrose tumoral-α

(TNF-α) e a interleucina-6 (IL-6), que estão igualmente envolvidas na patogénese de várias

doenças crónicas, como neoplasias, IC e DPOC, parecem desempenhar uma função

importante na vasculopatia (Steiner et al., 2009).

1.9. O miocárdio na HP

A principal função do VD é conduzir sangue desoxigenado através da circulação

pulmonar, com baixa pressão de perfusão, até ao VE, possibilitando trocas gasosas

eficazes e adaptações rápidas de retorno venoso (Bogaard et al., 2009a). É mais

complacente e fino do que o VE e adapta-se facilmente quer a variações de pré-carga, quer

ao trabalho de ejecção de volume. Pelo contrário, não tolera elevações repentinas de pós-

carga, porque é incapaz de desenvolver agudamente pressões máximas superiores a 40

mmHg, quando uma sobrecarga de pressão repentina ultrapassa este valor, o volume de

ejecção diminui e o VD dilata (Blaise et al., 2003). Contudo, se a sobrecarga de pressão for

progressiva o VD desenvolve hipertrofia gradual e remodelagem que lhe permitem

desenvolver pressões mais elevadas, compensando a elevação de pós-carga (Ritchie et al.,

1993; van Wolferen et al., 2007). No entanto, tal como sucede na sobrecarga de pressão

VE, vários mecanismos acabam por levar à disfunção miocárdica (Bogaard et al., 2009a). A

activação neuroendócrina local contribui não só para a hipertrofia (Lourenço et al., 2006;

Miyauchi et al., 1993), mas também para fibrose, apoptose e rarefação capilar no

miocárdio. A sobrecarga prolongada associa-se igualmente a maior stress oxidativo,

activação de citoquinas inflamatórias e de factores de crescimento que também são

importantes determinantes de remodelagem adversa e disfunção (Bogaard et al., 2009a,

2009b). Adicionalmente, o miocárdio VD poderá adaptar-se, segundo alguns autores, a um

estado de hipoperfusão relativa e entrar num processo semelhante ao de hibernação

miocárdica, característico da doença isquémica, uma vez que, dadas a maior pressão e

massa do VD, é maior a resistência ao fluxo sistólico nas artérias coronárias (van Wolferen

et al., 2008). De facto, a sobrecarga e a hipertrofia VD reduzem a capacidade mitocondrial

de produção energética (Daicho et al., 2009) e tornam o miocárdio altamente dependente

do metabolismo anaeróbio da glicose (Piao et al., 2009).

As alterações morfofuncionais e a resposta miocárdica são determinantes fundamentais

da qualidade de vida e sobrevida dos pacientes com HP (van Wolferen et al., 2007). A

modulação da remodelagem e disfunção miocárdica constituirá um alvo terapêutico


8

promissor (Archer et al., 2010), o que é, ainda mais, reforçado pela reversibilidade potencial

da lesão miocárdica, como se verifica após transplante pulmonar (Ritchie et al., 1993).

1.10. Terapêutica

O tratamento da HPA evoluiu consideravelmente na última década, vários algoritmos

terapêuticos têm sido propostos. A terapêutica deve ser ajustada individualmente, em

função da gravidade da doença, das comorbilidades, dos efeitos laterais dos fármacos e da

experiência clínica (McLaughlin et al., 2009). O teste de vasoreactividade é efectuado em

pacientes com HPAi e hemodinâmica estável. Um teste positivo prevê a possibilidade de

resultados favoráveis com terapêutica crónica com BCC (Rich et al., 1992). Em caso

contrário, os antagonistas dos receptores da ET - 1 ou os inibidores da fosfodiestérase são

normalmente seleccionados como terapêutica de primeira linha, exceptuando os casos em

que a via oral não é praticável, os casos em classe IV da Organização Mundial de Saúde

ou que apresentam sinais clínicos evidentes de IC direita. Nestes, a primeira escolha são

os prostanóides administrados por via intravenosa (Barst et al., 1996). No entanto, com a

excepção dos doentes com respostas favoráveis aos BCC, a maior parte dos fármacos

apenas atinge reduções de 10 - 20% da PmAP e muitos dos doentes permanecem

sintomáticos. Neste caso, devem ponderar-se quer a terapêutica combinada,

particularmente quando surgem efeitos laterais, quer a inclusão em ensaios clínicos de

novos agentes farmacológicos. Todavia, antes que ocorra uma deterioração sistémica

irreversível, devem ponderar-se a septostomia interauricular (Reichenberger et al., 2003) e

o transplante. Nos casos refratários é crucial a referenciação precoce para transplante

(Keogh et al., 2009; McLaughlin et al., 2009). Apesar da mortalidade substancial associada

à transplantação, o resultado a longo prazo é favorável, sendo a sobrevida de 47% aos 5

anos (Trulock et al., 2007). No grupo de pacientes com HPnA é consensual que a

optimização da terapêutica dirigida à doença primária terá efeitos benéficos, mas apesar

desta abordagem, pode persistir HP e alguns pacientes têm mesmo HP desproporcionada,

isto é, um grau de HP que não é facilmente atribuível à condição médica subjacente. Em

muitos destes casos, os novos fármacos aprovados para a HPA têm sido utilizados com

algum sucesso (Hoeper et al., 2009). Para uma perspectiva mais completa sobre as

abordagens farmacológicas e cirúrgicas da HP, nas suas diversas etiologias, pode

consultar-se o trabalho de revisão em anexo.


9

1.11. IC e caquexia

A IC pode definir-se clinicamente como um síndrome complexo resultante de uma

perturbação cardíaca funcional ou estrutural que compromete a capacidade do ventrículo

para se preencher ou ejectar (Hunt et al., 2009). De forma sumária, após uma lesão, a

capacidade de bombeamento diminui, desencadeando-se um processo lento e progressivo

de deterioração funcional, inicialmente compensado por sistemas homeostáticos, como o

sistema adrenérgico e os sistemas de retenção de sal e água, assim como pela activação

de moléculas vasodilatadoras, incluindo peptídeos natriuréticos, que contrariam a

vasoconstrição excessiva. Os doentes podem manter-se assintomáticos durante anos, mas

acabam por descompensar devido quer à activação excessiva de citocinas e sistemas

neuro-humonais, quer às alterações de remodelagem (Mann e Bristow, 2005). Os

cardiomiócitos de pacientes com IC sofrem alterações progressivas que comprometem a

função contráctil, nomeadamente o aumento da expressão das isoformas β das cadeias

pesadas de miosina, a perda de miofilamentos, alterações na composição do citoesqueleto,

perturbação do acoplamento excitação-contração e menor sensibilidade à sinalização

adrenérgica (Dhalla et al., 2009; Lehnart et al., 2009). A prevalência e a incidência da IC

continuam a aumentar, em grande parte devido ao envelhecimento populacional, ao

aumento da prevalência de hipertensão arterial e aterosclerose, bem como aos avanços

tecnológicos e modificações de estilos de vida que contribuíram para a prevenção e

tratamento da doença coronária (Young, 2004). Os inibidores da enzima de conversão da

angiotensina e os antagonistas β-adrenérgicos impedem a progressão da disfunção

miocárdica e previnem a remodelagem, mas apesar destes avanços terapêuticos a IC

continua a ser uma doença crónica de prognóstico reservado (Hunt et al., 2009).

Outro síndrome, a caquexia cardíaca, acompanha até 50% dos casos de IC grave

(Filippatos et al., 2000) e constitui um preditor independente e importante de mau

prognóstico (Anker et al., 1997). O termo caquexia (do Grego: kakós – má; hexis -

condição) corresponde à perda de massa corporal que surge nos estadios avançados de

várias doenças crónicas. A primeira definição do síndrome foi avançada por Hipócrates “A

carne é consumida e transforma-se em água, ... o abdómen preenche-se com água, os pés

e as pernas edemaciam, os ombros, as clavículas, o tórax e as coxas definham ... A doença

é fatal” (Doehner e Anker, 2002). Apenas deve ser considerada a perda ponderal não

edematosa, ou seja, relativa ao peso corporal excluindo edemas. O limiar mais consensual

para perda ponderal significativa derivou da reanálise do estudo SOLVD, nesta o limiar que

melhor previa a mortalidade era 6%, durante um período de pelo menos 6 meses (Anker et

al., 2003). Mais recentemente, numa reunião de consenso, definiu-se caquexia como a


10

complicação de doença crónica que surge quando os pacientes perdem pelo menos 5% do

peso corporal em 12 meses, ou menos, ou têm índice de massa corporal inferior a 20, e

cumprem pelo menos 3 de 5 critérios (diminuição da força muscular, fadiga, anorexia, baixo

índice de massa gorda livre ou inflamação, anemia ou diminuição da albumina sérica)

(Evans et al., 2008).

1.12. Fisiopatologia da caquexia cardíaca

Tanto a IC como a HP são síndromes multi-sistémicos que afectam os sistemas

músculo-esquelético, renal, neuroendócrino e imunológico (Dorfmuller et al., 2003; Francis,

2001). Como parte do envolvimento sistémico, muitos doentes desenvolvem caquexia. Os

mecanismos envolvidos são a elevação do metabolismo basal, a redução do apetite e

perturbações gastrointestinais (GI) que limitam a ingestão calórica, assim como um

comprometimento geral do metabolismo e dos sistemas imunológico e neuroendócrino (von

Haehling et al., 2007).

A activação inflamatória e as perturbações neuroendócrinas e imunológicas

correlacionam-se fortemente quer com a perda de massa corporal quer com a sobrevida,

na IC (Anker et al., 2003). O TNF-α, em particular, parece estar implicado nas vias que

conduzem à caquexia e poderá constituir a via final comum de todas as suas etiologias.

Inicialmente isolado a partir de murganhos infectados com soro do bacilo de Calmette-

Guérin após tratamento com lipopolissacarídeo (LPS), foi designado “caquexina” porque

mimetizava a acção de necrose tumoral do LPS e inibia a lipase das lipoproteínas,

pressumindo-se que desempenharia um papel na caquexia cardíaca (Beutler et al., 1985).

Sabe-se hoje que os seus efeitos integram as principais alterações que acompanham a

progressão da IC, tais como a disfunção ventricular, a remodelagem ventricular, a apoptose

dos cardiomiócitos, o desenvolvimento de anorexia e caquexia, a redução do fluxo

sanguíneo muscular, a disfunção endotelial e a insulino-resistência (Meldrum, 1998).

A anorexia é um elemento comum a todas as formas de caquexia. Na caquexia

cardíaca os pacientes apresentam redução de apetite e da ingestão alimentar, o que

poderá dever-se parcialmente ao aumento da actividade da melanocortina e à sensação de

saciedade induzidas por citocinas pró-inflamatórias (Inui, 1999). No caso da caquexia

cardíaca, o tracto GI parece desempenhar um papel importante. O edema e hipoperfusão

GI contribuem para a má-absorção (Witte e Clark, 2002) e perda de proteínas,

particularmente na IC direita. Nos casos mais extremos, os pacientes apresentam o

síndrome de enteropatia com perda de proteínas e hipoproteinémia (Thorne et al., 1998).


11

Por outro lado, o edema da parede GI também facilita a translocação bacteriana e,

portanto, a endotoxemia. De facto, níveis elevados de endotoxina normalizam após o

tratamento diurético (Niebauer et al., 1999). A translocação bacteriana também é agravada

como consequência da hipoperfusão e isquemia da mucosa GI, que aumentam a

permeabilidade da parede intestinal (Krack et al., 2005). O TNF-α e outras citocinas são

induzidas nos monócitos (Rauchhaus et al., 2000) e nos cardiomiócitos pelas endotoxinas

circulantes (Wagner et al., 1998), embora uma hipótese alternativa considere que a

activação pró-inflamatória possa surgir na sequência da menor perfusão e hipóxia tecidual

(Tsutamoto et al., 1998).

O principal factor de transcrição subjacente quer à actividade celular quer à indução da

síntese do TNF-α é o factor de transcrição nuclear-κB (NF-κB). A activação do NF-κB tem

sido associada a vários processos patológicos que acompanham a progressão da IC,

incluindo a libertação de citocinas pelos cardiomiócitos, o stress oxidativo, a hipertrofia e a

remodelagem do miocárdio (Valen et al., 2001). A família de factores de transcrição NF-κB

é responsável pela regulação de mais de 150 genes alvo. Na maioria das células, os

complexos NF-κB estão presentes no citoplasma sob a forma inactiva, complexados com o

inibidor-κB (I-κB). Vários estímulos, entre os quais o LPS e citocinas, resultam na

fosforilação, ubiquitinação e degradação subsequente do I-κB, libertando um conjunto de

aminoácidos no NF-κB que promove a translocação nuclear e a ligação a elementos de

resposta específicos dos genes alvo nos cardiomiócitos (Valen et al., 2001).

Para além das alterações inflamatórias, várias modificações endócrinas acompanham

também a progressão da caquexia (Cicoira et al., 2003; Doehner e Anker, 2002; Leyva et

al., 1998; Nagaya et al., 2001).

1.13. Remodelagem metabólica na progressão da IC

O miocárdio saudável depende de um fornecimento constante de O2 para manter o seu

metabolismo. O oxigénio é essencial para a FO, que é responsável por aproximadamente

90% da produção de ATP (Ventura-Clapier et al., 2004). Vários metabolitos, como ácidos

gordos (AG), glicose e lactato, podem ser utilizados como substrato pela FO no miocárdio

(Bing et al., 1954). O metabolismo energético miocárdico e a taxa de hidrólise de ATP

ajustam-se às exigências inerentes ao trabalho cardíaco. Em condições de esforço

máximas, as vias metabólicas são capazes de produzir quantidades elevadas de ATP,

aumentando a actividade mitocondrial para cerca de 80 - 90% da sua capacidade máxima

(Mootha et al., 1997). Numa situação de repouso, a β-oxidação de AG é responsável por 60


12

- 90% do total de energia produzida (van der Vusse et al., 2002), enquanto a oxidação do

piruvato e do lactato, provenientes da glicólise, representam apenas 10 - 40% (Wisneski et

al., 1985). A preferência por substratos energéticos é baseada a curto prazo no equilíbrio

entre as necessidades e o fornecimento, sendo ditada por factores mecânicos e

endócrinos, dependendo principalmente de uma efectiva reciprocidade entre os substratos.

De facto, em quantidade suficiente, cada substrato inibe automaticamente as restantes vias

metabólicas. A rápida adaptação aos substratos metabólicos disponíveis e às exigências

variáveis do trabalho cardíaco são características primordiais do miocárdio saudável

(Stanley et al., 2005).

Nas formas avançadas da IC, o miocárdio torna-se incapaz de converter eficientemente

a energia química em trabalho contráctil e o seu conteúdo de ATP encontra-se

substancialmente diminuído, em boa parte devido ao comprometimento do metabolismo

oxidativo (Ormerod et al., 2008). Para além de uma menor eficiência metabólica global, as

vias metabólicas sofrem adaptações, entre as quais se destaca a substituição progressiva

da oxidação de AG pela glicólise anaeróbia como principal mecanismo de produção de

energia (Davila-Roman et al., 2002). Apesar das consequências da disfunção metabólica na

IC serem ainda pouco compreendidas, um número crescente de evidências apoia um papel

causal na génese da disfunção contráctil e na remodelagem do miocárdio. As perturbações

do metabolismo miocárdico que acompanham a progressão da IC têm vindo a ser

exploradas como potencial alvo terapêutico. Estas adaptações a longo prazo, dependem de

alterações na expressão génica e na actividade de transportadores ou de enzimas chave,

da fosforilação de proteínas e dos níveis de co-fatores que possibilitam alterações

pronunciadas nas vias metabólicas (Stanley et al., 2005). O factor de transcrição do

receptor activado pelo proliferador do peroxissoma (PPAR) tem sido implicado como

potencial responsável por parte destas adaptações.

O PPAR, que apresenta três isoformas, todas elas activadas pelos AG, hetero-dimeriza

com o receptor retinóide X, sendo o complexo resultante translocado para o núcleo. No

núcleo, é activada a transcrição de genes após ligação ao elemento de resposta do

proliferador do peroxissoma (PPRE). O PPAR aumenta a expressão dos genes que

codificam as proteínas envolvidas na captação, transporte e β-oxidação dos AG (Finck e

Kelly, 2002) e diminui reciprocamente a utilização de glicose por modificação da expressão

génica de moduladores do metabolismo da glicose (Kodde et al., 2007). De facto, níveis

elevados de lípidos circulantes e de AG no citoplasma dos cardiomiócitos conduzem, por

activação transcricional mediada pelo PPAR-α, à sobre-expressão de cínase da

desidrogénase do piruvato (PDK). A PDK, por sua vez, fosforila e inactiva o complexo da


13

desidrogénase do piruvato (PDH), que é responsável pelo principal passo regulador da

oxidação da glicose. O complexo da PDH da membrana mitocondrial interna, transporta o

piruvato através da membrana e catalisa a transformação irreversível em acetil-Coenzima A

(CoA), sendo esta uma etapa fundamental da oxidação dos carbo-hidratos. Por outro lado,

a activação da oxidação de AG aumenta as razões acetil-CoA/CoA livre e NADH/NAD+ na

mitocôndria, activando a PDK. Reciprocamente, a redução da oxidação dos AG aumenta a

oxidação de glicose e de lactato pela desinibição da PDH (Stanley et al., 2005). A isoforma

α dos PPAR é a principal reguladora a nível cardíaco, mas a isoforma γ exerce uma

influência miocárdica indirecta por acção em adipócitos (Barak et al., 1999) e elevação dos

níveis circulantes de AG.

Na doença cardíaca prolongada, que conduz a hipertrofia e posteriormente a IC, a

expressão de PPAR-α diminui e, concomitantemente, o miocárdio diminui a sua capacidade

de metabolização de AG e aumenta a sua capacidade de utilização de glicose (Garnier et

al., 2003). O significado desta alteração metabólica não está completamente elucidado,

sendo ainda indefinido se terá uma acção adaptativa e benéfica ou desadaptativa e

prejudicial. Alguns estudos recentes verificaram que a activação do PPAR-α induz

disfunção contrátil moderada e acentua a hipertrofia em modelos experimentais de

hipertrofia induzida pela sobrecarga de pressão (Young et al., 2001) e de diabetes mellitus

(Kiec-Wilk et al., 2005), respectivamente, enquanto outros demonstraram efeitos benéficos

na hipertrofia cardíaca induzida pela sobrecarga de pressão em ratos após tratamento com

fenofibrato, que é um indutor dos PPAR (Ogata et al., 2002). Os resultados contraditórios

podem dever-se às diferenças existentes entre espécies, à duração das intervenções, à

idade dos animais e aos modelos experimentais.

Outra característica da remodelagem metabólica que acompanha a progressão da IC é

o desenvolvimento de insulino-resistência, mesmo em pacientes não diabéticos (Mamas et

al., 2010). O estudo RESOLV demonstrou que a insulino-resistência se correlaciona com a

severidade da IC (Suskin et al., 2000) e com os níveis plasmáticos de peptídeo natriurético

do tipo B (BNP) (Tassone et al., 2009). Para além disso, a insulino-resistência e as

alterações no metabolismo da glicose são preditores independentes de mortalidade

(Doehner et al., 2005). Contudo, ainda não estão definidos os mecanismos responsáveis

pela associação da insulino-resistência à IC ou o modo como cada uma destas condições

predispõe à outra. Nos cardiomiócitos, a insulina regula tanto o metabolismo dos carbo-

hidratos como o dos AG, na insulino-resistência, a entrada de glicose no miocárdio, através

do transportador de glicose do tipo 4 (GLUT-4), está comprometida e, consequentemente

também a sua via metabólica. Além do mais, os efeitos periféricos da insulino-resistência


14

levam ao aumento dos níveis de AG na circulação reforçando as vias metabólicas dos AG

e, suprimindo adicionalmente o metabolismo de glicose. Estas modificações parecem

associar-se a menor eficiência cardíaca e maior stress metabólico (Witteles e Fowler,

2008). De facto, o excesso de AG induz lipotoxicidade no miocárdio, maior formação de

radicais livres de oxigénio (ROS), contribuindo para o desenvolvimento da cardiomiopatia

(Pillutla et al., 2005).

1.14. Nutrição na IC e caquexia

O papel da nutrição como factor de risco cardiovascular está bem estabelecido. A

American Heart Association recomenda a redução do total de calorias ingeridas, a redução

da ingestão de gorduras, especialmente saturadas e colesterol, bem como o aumento da

ingestão de legumes, frutas e produtos lácteos com baixo teor em gordura (Azhar e Wei,

2006; Krauss et al., 2000). A avaliação clínica de períodos longos de restrição calórica em

pacientes não-obesos corrobora estas recomendações (Meyer et al., 2006). No entanto,

estas recomendações podem não se aplicar à IC grave, associada a perturbações do

metabolismo cardiomiocitário e caquexia.

Embora as dietas hipercalóricas, do tipo ocidental, sejam um factor de risco bem

estabelecido para o desenvolvimento de doença cardiovascular, paradoxalmente, estudos

epidemiológicos demonstraram que a obesidade é um factor de prognóstico favorável na IC

(Abel et al., 2008). Efectivamente, pacientes com IC que apresentem simultaneamente

excesso ponderal ou obesidade têm menor mortalidade (Oreopoulus et al., 2008) e o

suporte nutricional pode contrariar a anorexia das doenças crónicas e prevenir a caquexia

(Laviano et al., 2005). No entanto, a suplementação nutricional não pode reverter

completamente o processo de perda ponderal que acompanha a caquexia, porque a

anorexia, por si só, não explica toda a complexidade fisiopatológica da caquexia cardíaca.

A investigação experimental e clínica foca cada vez mais o papel que a nutrição pode

desempenhar na progressão da IC e caquexia (Akner e Cederholm, 2001; Sandek et al.,

2008). E as mais diversas abordagens têm sido testadas (Azhar e Wei, 2006).


15

2. Objectivos

O principal objectivo dos trabalhos levados a cabo foi testar os efeitos de um regime

alimentar hipercalórico, próximo da típica dieta Ocidental (rico em gordura animal e carbo-

hidratos simples) na sobrevida, no grau de HP, na função e remodelagem do miocárdio, na

IC e caquexia que acompanham o modelo experimental de HP severa, e rapidamente

progressiva, induzida pela MCT. Como objectivo adicional procuramos estudar os

mecanismos moleculares inerentes à remodelagem metabólica, às alterações

morfofuncionais do miocárdio e à caquexia que acompanham este modelo, bem como os

mecanismos pelos quais o regime alimentar hipercalórico modificou a sua evolução.


16

3. Metodologia

3.1. Modelos animais

Ratos Wistar, machos, com pesos corporais compreendidos entre 180 - 200 g e cerca

de 6 - 7 semanas de idade (Charles-River Barcelona, Espanha; n = 192), receberam

aleatoriamente uma única injecção subcutânea de 60 mg.kg-1 de MCT (Sigma Chemical, St.

Louis, MO) ou igual volume de veículo (soro fisiológico a pH neutro). Quarenta e oito horas

depois, os dois grupos passaram, aleatoriamente, a ser alimentados com uma dieta

hipercalórica, rica em lípidos e carbo-hidratos simples (F2685; BioServe Frenchtown; 5,4

Kcal.g-1, 35% de gordura animal, 35% de carbo-hidratos simples, 20% de proteínas e 0,4%

Na+) ou mantiveram uma dieta normal, de composição padrão (A04; Scientific Animal

Food&Engineering; 2,9 Kcal.g-1, 3% de gordura não-animal, 60% de carbo-hidratos

complexos, 16% de proteínas e 0,25% Na+). Registou-se diariamente o peso corporal, a

ingestão alimentar e a mortalidade. A insulino-resistência e a tolerância à glicose oral foram

avaliadas 20 - 23 dias após injecção. Os ratos foram colocados em gaiolas metabólicas

durante 24 horas, 24 dias após injecção. A avaliação hemodinâmica e a colheita de

amostras foram realizadas após um período de 24 horas com ração regular, dos 28 aos 32

dias. Os animais foram alojados em grupos de 5 por caixa, em ambiente controlado (ciclo

luminosidade-obscuridade 12:12 horas, a 22 ºC e dieta ad libitum). Foram respeitadas a lei

Portuguesa para o bem-estar animal e o National Institutes of Health Guide for the Care and

Use of Laboratory Animals (NIH Pub. No. 85 - 23, revista em 1996).

A avaliação metabólica, hemodinâmica e histológica padrão podem ser consultadas no

trabalho original submetido para publicação, que consta na secção resultados desta tese. A

avaliação estatística foi efectuada com auxílio de software (SigmaStat 3.5, Systat software,

Inc.) e pode ser consultada na mesma secção. Durante a realização dos trabalhos

experimentais, tive a oportunidade de aprender e executar as técnicas de biologia

molecular que foram empregues neste trabalho, assim como aprender e contribuir para a

análise e interpretação de dados e redacção do mesmo. Em seguida será apresentada de

forma detalhada a metodologia de biologia molecular.


17

3.2. Estudos de biologia molecular

3.2.1. Quantificação de DNA e conteúdo protéico do miocárdio do VD e VE

Foram analisados 10 mg das amostras de miocárdio do VD e do VE, provenientes de

sete animais de cada grupo, para quantificar DNA genómico e proteína total. As amostras

biológicas foram tratadas com uma solução tampão de lise composta por tiocianato de

guanidina, inactivando, deste modo, DNases, RNases e proteases e garantindo, portanto, o

isolamento de DNA, RNA e proteínas intactas. As amostras foram, então, homogeneizadas

com um rotor (PRO 200, Cat. No. 01 - 02200, Oxford, CT, USA), durante cerca de 30

segundos, para assegurar uma quantidade significativa de ácidos nucléicos livres. O

restante processo de extracção foi levado a cabo de acordo com as instruções do

fabricante por eluição sequencial em colunas (AllPrep® DNA/RNA/Protein Mini Kit, Cat. No.

80004, Qiagen, København, Dinamarca).

A concentração total de proteína foi testada pelo método do ácido bicinconínico

(Pierce® BCA Protein Assay Kit-Reducing Agent Compatible, Cat. No. 23250, Rockford, IL

USA). O ensaio consiste na redução de Cu+2 a Cu+ por proteínas, em meio alcalino,

apresentando elevada sensibilidade e especificidade na detecção e na quantificação

colorimétrica de proteínas. O efeito de redutores de cobre foi minimizado através de um

reagente de compatibilidade que altera os grupos dissulfito dos agentes redutores. A

absorvância das amostras foi quantificada por espectrofotometria com um comprimento de

onda de 562 nm e comparada com uma curva padrão obtida por diluição seriada de

padrões de albumina do soro bovino (µg/mL).

A concentração de DNA foi determinada por espectrofotometria, medindo a absorvância

a 260 nm (Eppendorf 6131000.012) após diluição das amostras numa solução tampão de

Tris-Cl com pH neutro. A pureza foi estimada pela razão das absorvâncias a 260 nm e 280

nm (A260/A280). O DNA puro tem uma razão A260/A280 de 1,7 - 1,9. Tanto o DNA como o

conteúdo protéico foram normalizados para a massa inicial do tecido do miocárdio.

3.2.2. Quantificação de mRNA

As amostras biológicas foram colhidas imediatamente após a eutanásia dos animais,

colocadas em reagente de estabilização de RNA (RNAlater) após fragmentação em

dimensões inferiores a 1 mm, e armazenadas, então, a -80 ºC. A extracção de RNA foi

efectuada a partir de 30 mg de tecido de cada amostra, por homogeneização em tampão de

lise com um rotor (PRO 200, Cat. No. 01 - 02200, Oxford, CT, USA), e eluição sequencial

em colunas utilizando diversos tampões de acordo com o método de guanidina-tiocianato e


18

ligação selectiva a uma membrana de gel de sílica seguindo as instruções do fabricante

(RNeasy® Mini Kit, Cat. No. 74124, Qiagen). Este método exclui RNA com extensão inferior

a 200 nucleótidos, como o rRNA e o tRNA, que em conjunto correspondem a 15 - 20% do

RNA total. A concentração e a pureza do RNA foram avaliadas por espectrofotometria

(Eppendorf 6131000.012), admitindo como RNA puro amostras com razões entre as

absorvâncias a 260 e 280 nm de 1,9 a 2,1, após diluição em tampão de Tris-Cl com pH de

7,5.

A quantificação de mRNA foi realizada, nas amostras de VD, VE e gordura mesentérica

provenientes de 7 animais de cada grupo, através da técnica RT-PCR em tempo real. No

miocárdio, avaliou-se a expressão relativa da ET-1, das citocinas TNF-α e IL-6, do PPAR-α

e das enzimas reguladoras do metabolismo, da PDK do tipo 4 (PDK4), da Acil-CoA ligase

de AG de cadeia longa tipo 1 (ACSL1), da desidrogenase de acil-CoA de cadeia média

(MCAD) e de cadeia longa (LCAD). A β-actina foi quantificada como gene de controlo

interno. Na gordura visceral quantificamos o TNF-α, a IL-6 e a adipocina adiponectina,

usando o desidrogenase do gliceraldeído-3-fosfato como gene de controlo interno, uma vez

que para a β-actina foram observadas diferenças significativas entre os grupos. Os pares

de primers específicos para o estudo destes genes foram desenhados com recurso a

software (DNAstar™, Lasergene) e são indicados no trabalho apresentado na secção

resultados, atendendo às seguintes características: comprimento dos primers até 18 - 21

nucleótidos; comprimento da sequência do produto de PCR com 100 - 150 pares de base;

conteúdo de guaninas e citosinas de 40 - 60%; temperatura de fusão, ou melting (Tm), do

transcrito 8 ºC superior à dos primers e temperaturas de fusão dos primers não diferindo

mais de 2 ºC; e, finalmente, escolha preferencial de sequências de primers intron spanning

para amplificação selectiva de RNA, ou, em alternativa, caso isto não seja possível, intron

flanking que possibilitam diferenciar amplificação de RNA da de DNA (caso seja transcrito

tem maior dimensão, uma vez que inclui uma sequência de um intrão).

A transcrição reversa foi realizada seguindo a metodologia de random primers. Num

volume total de reacção de 20 µL: 40 U/reacção (0,3 µL) de transcriptase reversa

(Superscript™ II, Invitrogen 18064 - 014), 20 U/reacção (0,5 µL) do inibidor de Rnases

(Promega N2515), 30 ng de random primes (Invitrogen 48190 - 011), solução tampão (50

mM Tris-HCl, 75 mM KCl; 3 mM MgCl2), 10 mM de ditiotreitol e 0,5 mM de

desoxiribonucleotídeos trifosfatados (MBI Fermentas R0192). A transcrição foi efectuada

num termociclador (Whatman Biometra 050 - 901) seguindo os seguintes passos: 10

minutos a 22 ºC para desnaturação do RNA, 50 minutos a 42 ºC para a transcrição reversa

e 10 minutos a 95 ºC para inactivação da enzima e destruição dos produtos de reacção. O


19

cDNA resultante foi armazenado a -20 ºC e posteriormente amplificado por PCR em tempo

real. A reacção de amplificação génica processou-se num volume final de 20 µL, com 500

nM de cada primer, mix com nucleotídeos, uma hot-start DNA polimerase e SYBR green

como marcador fluorescente de acordo com as instruções do fabricante

(QuantiTect™SYBR® Green PCR Kit, Cat. No. 204143, Qiagen). A reacção decorreu em

capilares de acordo com as instruções do fabricante (Lightcycler II, Roche), brevemente,

após 15 minutos a 95 ºC para activação da polimerase, sucederam-se 35 - 55 ciclos de 15

segundos a 94 ºC para a desnaturação, 20 - 30 segundos a 50 ºC para annealing entre

primers e DNA e 10 - 30 segundos a 72 ºC para extensão.

Criaram-se curvas padrão fazendo diluições seriadas (2,5; 5; 12,5; 25; 50; 100; 250 ng)

do RNA de uma amostra de um animal do grupo controlo. A transcrição reversa foi

efectuada, numa experiência única, incluindo a curva padrão e 25 ng de RNA de cada

amostra. Subsequentemente, os PCRs foram efectuados para cada gene, incluindo todas

as amostras e usando como referência a curva padrão. O SYBR green liga-se ao DNA de

dupla cadeia emitindo fluorescência, durante a amplificação exponencial para cada amostra

e padrão, o valor máximo da segunda derivada do traçado de fluorescência (início da fase

exponencial de amplificação) foi obtido automaticamente pelo software (Lightcycler II,

Roche). As experiências foram efectuadas em duplicado.

No final do PCR, para cada gene, o DNA foi desnaturado, por aquecimento lento de 65

a 95 ºC, e a fluorescência adquirida continuamente, por forma a obter traçados de melting,

assegurando a pureza do produto amplificado e a ausência de dímeros de primers. Para

uma amostra de cada gene correu-se uma electroforese com o DNA amplificado, num gel

de agarose a 2% corado com brometo de etídio (0,5 µg/mL) para reconfirmar a pureza do

produto de amplificação. A expressão génica foi normalizada para a expressão do gene de

controlo interno, que não variou entre os grupos experimentais. Os resultados são

apresentados relativamente ao valor médio do grupo injectado com veículo e alimentado

com dieta normal, que foi definido como unidade arbitrária.

3.2.3. Quantificação das isoformas das cadeias pesadas de miosina

A comutação de isoformas das cadeias pesadas de miosina do tipo α para o tipo β é

um traço característico da remodelagem do miocárdio que ocorre na progressão da IC

(Mann e Bristow, 2005). Em amostras de VD e VE, de seis animais de cada grupo

experimental, foram extraídas proteínas por lise tecidual. Posteriormente, 15 µg de proteína

total, diluídas 1:1 em tampão de Laemmli num volume final de 20 µL, e desnaturadas por

agitação durante 5 minutos a 95 ºC, foram separados por electroforese em gel de


20

poliacrilamida e dodecil sulfato de sódio (SDS-PAGE), juntamente com um marcador de

peso molecular. Resumidamente, prepararam-se dois géis, de separação (5% de

Acrilamida/Bisacrilamida) e de concentração (3% de Acrilamida/Bisacrilamida), preencheu-

se o sistema de electroforese com solução tampão de separação e a electroforese decorreu

a voltagem constante de 30 mA, durante 6 horas (PowerPac 200, Cat. No. 165 - 3301, BIO-

RAD). As isoformas das cadeias pesadas de miosina α e β visualizaram-se, no final, como

duas bandas distintas com um peso molecular aproximado de 200 KDa. Os géis foram

então corados, durante 15 - 20 minutos, com uma solução de azul brilhante de Coomassie

R-250. O gel foi digitalizado a 700 nm num sistema de aquisição de imagem (Odyssey, LI-

COR Biosciences, Lincoln, NE, USA). A percentagem da isoforma β, para cada amostra, foi

quantificada a partir da razão entre as absorvâncias da isoforma β e a total.

3.2.4. Quantificação do TNF-α no plasma

O plasma colhido no final da avaliação hemodinâmica em 7 animais por grupo, foi

utilizado para quantificar a concentração plasmática de TNF-α através de um imunoensaio

enzimático do tipo sandwich em fase sólida. Brevemente, neste ensaio altamente sensível,

que possibilita a detecção de concentrações mínimas de TNF-α (0,7 pg/mL), as amostras,

juntamente com a curva padrão e os controlos positivo e negativo, foram incubados com

anticorpos monoclonais específicos do TNF-α que revestem as microplacas (TNF-α (Rat)

Ultrasensitive EIA, 45-TNFRTU-E01, Alpco Diagnostics™, Salem, NH). Posteriormente,

adicionou-se sequencialmente anticorpo secundário biotinilado, estreptavidina-peroxidase

de rábano (HRP) e uma solução de substrato, o corante TMB, com passos de lavagem

intermédios. Após a reacção o substrato cromogénio amarelado muda de cor, para azul,

após oxidação, sendo adicionada solução de bloqueio. A intensidade da cor produzida é

directamente proporcional à concentração de TNF-α presente na amostra, de acordo com a

curva padrão. As absorvâncias no comprimento de onda de 450 nm (UVM-340

Monochromator based reader, ASYS Hitech GmbH, Eugendorf, Áustria) foram analisadas

(Microplate instrument analyser, versão 1.2.0.2). Todas as avaliações foram efectuadas em

duplicado.

3.2.5. Apoptose miocárdica

A morte celular programada, ou apoptose, é um dos mecanismos que acompanha a

remodelagem miocárdica na progressão da IC, determinando concomitantemente o

comprometimento funcional pela perda de unidades contrácteis. Na base desta, encontra-

se a degradação do DNA genómico por endonucleases específicas dependentes de cálcio.


21

O grau de apoptose miocárdica VD e VE foi avaliada por histoquímica, em secções

histológicas de 7 animais de cada grupo. Empregou-se a metodologia enzimática de

marcação com fluoresceína dos terminais desoxiuridina trifosfato pela desoxinucleotidil

transferase (TUNEL, terminal UDP nick-end labeling), segundo as instruções do fabricante

(TACS™ TdT In Situ Apoptosis Detection Kit, R&D Systems, Minneapolis, MN USA).

Sumariamente, cinco secções histológicas intervaladas de 100 µm foram obtidas

aleatoriamente e desparafinadas, a 57 ºC, num aquecedor de placas. As amostras de

tecido foram então fixadas, para evitar a perda de fragmentos de DNA de baixo peso

molecular, procedeu-se à rehidratação das amostras e, finalmente, adicionou-se proteinase

K para permeabilizar os tecidos. As lâminas histológicas foram, então, imergidas em

solução supressora da actividade peroxidásica e marcadas com solução tampão de

marcação. Após bloqueio da reacção com uma solução tampão, as lâminas são coradas

com HRP e marcador azul TACS. O resultado final foi um precipitado escuro insolúvel que

assinala zonas em que ocorreu fragmentação de DNA. As lâminas são, então, contra-

coradas com hematoxilina. Numa das secções adicionou-se TACS-Nuclease™ por forma a

gerar fragmentação do DNA na maioria das células, confirmando, como controlo positivo,

se a permeabilização e marcação decorreram adequadamente. Enquanto que, noutra

secção, não foi adicionado tampão de marcação

Dois observadores independentes, fizeram a contagem dos núcleos de cardiomiócitos

marcados, em 50 campos escolhidos ao acaso em cada secção histológica (400 x). A taxa

de apoptose foi expressa como percentagem de núcleos de cardiomiócitos apoptóticos pelo

número total de cardiomiócitos.

3.2.6. Quantificação do NF-κB e do PPAR-α

Procedeu-se à extracção de proteínas nucleares do miocárdio VD e do VE de 7 animais

por cada grupo experimental, segundo as instruções do fabricante (Nuclear Extraction Kit,

Cat. No. 11906 - 100, Marligen Biosciences, Inc., Ijamsville, MD, USA). Os extractos

nucleares foram quantificados pelo método de Bradford, com leitura de absorvâncias a 595

nm por espectrofotometria (Eppendorf 6131000.012). Nos extractos quantificou-se a

actividade dos factores de transcrição NF-κB e PPAR-α, de acordo com as instruções do

fabricante (NF-κB (p65) Transcription Factor Assay Kit e PPAR-α Transcription Factor

Assay Kit, Cat. No. 1007889 e Cat. No. 10006915, Cayman Chemical Company, Ann

Harbor, MI, USA, respectivamente). Brevemente, 20 µL de proteínas nucleares de cada

amostra foram adicionados aos poços de microplacas revestidos com sequências

específicas de DNA de dupla cadeia, o elemento de resposta NF-κB e o PPRE,


22

adicionando-se, de seguida, um anticorpo primário específico do NF-κB e do PPAR-α,

respectivamente (Cat. No. 10007442, Cayman Chemical Company, Ann Harbor, MI, USA).

Após incubação a 4 ºC, durante a noite, adicionou-se um anticorpo secundário conjugado

com um marcador infravermelho (IRDye 800CW Goat Anti-Rabbit Labeled Secondary

Antibody, Cat. No. 926 - 32211, Li-COR biosciences, Lincoln, NE, USA), diluído em solução

de bloqueio (1:1000) (Odyssey Blocking Buffer, Cat. No. 927 - 40000, Li-COR biosciences,

Lincoln, NE, USA) com 0,2% de Tween-20. A absorvância no comprimento de onda de 800

nm foi, então, lida num sistema de aquisição de imagem (Odyssey, LI-COR Biosciences,

Lincoln, NE, USA). As amostras foram avaliadas em duplicado, em conjunto com controlos

positivo e negativo, e um controlo em que se adicionou DNA de dupla cadeia que competiu

com o factor de transcrição para a ligação, por forma a confirmar a especificidade da

ligação.


23

4. Resultados

Experimental pulmonary hypertension with heart failure and cardiac cachexia

is attenuated by a high-fat high-simple carbohydrate diet

(A hipertensão pulmonar experimental associada à insuficiência cardíaca e

caquexia é atenuada pela dieta rica em gorduras e carbo-hidratos simples)

Artigo em revisão na revista American Journal of Physiology Heart and Circulatory Physiology,

submetido pelos autores Lourenço AP, Vasques-Nóvoa F, Fontoura D, Brás-Silva C, Roncon-

Albuquerque R, Jr., e Leite-Moreira AF, em Setembro de 2010.


24

Experimental pulmonary hypertension with heart failure and cardiac cachexia

is attenuated by a high-fat high-simple carbohydrate diet

André P. Lourenço, Francisco Vasques-Nóvoa, Dulce Fontoura, Carmen Brás-Silva,

Roberto Roncon-Albuquerque Jr, Adelino F. Leite-Moreira

Department of Physiology, Faculty of Medicine, University of Porto, Porto, Portugal

Running Head: Hypercaloric diet attenuates cardiac cachexia

Keywords: hypercaloric diet; cytokines; inflammation; neuroendocrine; myocardial function

Correspondence to:

Adelino F. Leite-Moreira, MD PhD

Department of Physiology

Faculty of Medicine

Al Prof. Hernâni Monteiro

4200 - 319

Porto, Portugal

Tel: +351225513644

Fax: +351225513646

Email: [email protected]


25

Abstract

To evaluate the repercussions of a hypercaloric diet in cardiac cachexia (CC) and heart

failure (HF), male Wistar rats randomly underwent (i) subcutaneous injection of 60 mg.Kg-1

monocrotaline (MCT) or vehicle (Ctrl) and (ii) feeding with either a 5.4 kcal.g-1, 35% simple

carbohydrate and 35% animal fat (hypercaloric diet, HD), or a 2.9 Kcal.g-1, 60% complex

carbohydrate and 3% vegetable fat (normal diet, ND). Calorie intake, weight gain and

mortality were recorded. Metabolic studies, insulin resistance and oral glucose tolerance

testing were performed. Five weeks after injection, we evaluated right (RV) and left

ventricular (LV) haemodynamics under general anesthesia. Morphometry, lung and

myocardial histology, plasma levels of tumor necrosis factor-α (TNF-α) as well as

myocardial apoptosis, nuclear factor κ-B (NF-κB) and peroxisome proliferator-activated

receptor-α (PPAR-α) transcription factor activation, and gene expression of endothelin-1

(ET-1), interleukin-6 (IL-6), TNF-α and metabolic enzymes were also assessed. MCT ND

showed pulmonary hypertension (PH) and RV and LV failure, associated with high mortality,

myocardial remodeling and apoptosis, reduced calorie intake, and loss of body weight, lean

and fat mass that were all attenuated in MCT HD. PH was accompanied by increased

myocardial NF-κB transcription factor activity and by ET-1, TNF-α and IL-6 overexpression,

as well as by increased circulating TNF-α, which were offset in MCT HD. PPAR-α activity,

however, was decreased in MCT ND but not in MCT HD. The hypercaloric diet attenuated

experimental PH associated with CC and HF, improving survival, myocardial function and

remodeling, and reduced inflammatory and neuroendocrine activities.


26

Introduction

Cardiac cachexia (CC), defined by a weight loss of more than 6% over 6 months (Anker et

al., 2003), accompanies heart failure (HF) in up to 50% of severe HF cases (Filippatos et al.,

2000) and independently determines a poor prognosis (Anker et al., 1997). As part of the

complex and still incompletely revealed mechanisms underlying CC are abnormalities of

general metabolism as well as of the immune and neuroendocrine systems (Anker and

Coats 1999). Besides increased basal metabolism, reduced appetite and gastrointestinal

derangements that compromise caloric intake (von Haehling et al., 2007), the failing heart

undergoes extensive and early metabolic changes, namely a myocardial shift from fatty acid

oxidation (FAO) to glycolysis and reduced mitochondrial oxidative capacity that partly

underlie functional disturbances and remodelling (Stanley et al., 2005). Caloric restriction

has been shown to reduce cardiovascular risk (Fontana et al., 2004) and improve overall

metabolism and organ function (Chou et al., 2010). American Heart Association nutritional

guidelines recommend avoiding simple carbohydrates and fat, particularly saturated, as

main energy sources in a balanced diet (Krauss et al., 2000). This may not apply however to

severe HF with disturbed cardiomyocyte metabolism and CC. Among the experimental

models of CC, monocrotaline (MCT) - induced pulmonary hypertension (PH) stands out by

rapidly progressive HF and cachexia (Dalla Libera et al., 2004, Steffen et al., 2008). Our

goal was to test the effects of a high-fat high-simple carbohydrate diet on survival, PH,

myocardial function and remodeling, neuroendocrine and inflammatory activities, and

cachexia, in severe MCT-induced PH.

Materials and Methods

Animal model

Seven-week old 180 - 200 g male Wistar rats (Charles-River, Barcelona, Spain; n = 192)

randomly received either a 60 mg.Kg-1 MCT (Sigma Chemical, St. Louis, MO) single

subcutaneous injection or an equal volume of vehicle. Both groups were randomly allocated,

48 h later, to be fed ad libitum with either a high-fat high-simple carbohydrate diet (F2685;

BioServe Frenchtown; 5.4 kcal.g-1, 35% animal fat, 35% simples carbohydrate, 20% protein,

and 0.4% Na+) or a regular diet (A04; Scientific Animal Food&Engineering; 2.9 Kcal.g-1, 3%

non-animal fat, 60% complex carbohydrate, 16% protein, and 0.25% Na+). Body weight

(BW), food ingestion and mortality were recorded daily (n = 136 for survival analysis). Insulin

resistance (IR) and oral glucose tolerance (OGT) were evaluated 20 - 23 days after


27

injection. Rats were then placed in metabolic cages for 24 h at the 24th day. Hemodynamic

evaluation and sample collection were carried out after 24 h of regular chow, from the 28th to

the 32nd day. Animals were housed in groups of 5 per cage with a controlled environment

under a 12:12 h light-dark cycle at a room temperature of 22 °C. Investigation conformed to

the Guide for the Care and Use of Laboratory Animals published by the US National

Institutes of Health (NIH Publication No. 85 - 23, revised 1996) and was approved by the

ethics committee of the Faculty of Medicine of the University of Porto. Methods are

presented in detail as supplemental data.

Metabolic studies

In 7 animals per group, IR and OGT were evaluated, after a 12 h fasting period, by

recording baseline, 15, 30, 45, 60, 90, and 120 min plasma glucose levels (Freestyle-Mini™)

after 0.5 U.Kg-1 intraperitoneal (ip) insulin injection and 1 g.Kg-1 glucose gavage,

respectively. Weight gain, diuresis, and calorie and water intake were measured during the

24 h metabolic cage stay (Techniplast, Buguggiate, Italy).

Haemodynamic evaluation

Briefly, 7 animals of each experimental group were sedated with 100 µg.Kg-1 ip fentanyl (50

µg.mL-1) and 5 mg.Kg-1 ip midazolam (5 mg.mL-1) and anaesthetized by inhalation of 8%

sevoflurane (Penlon Sigma Delta Anaesthetic Vaporizer). After endotracheal intubation and

mechanical ventilation, with respiratory rate set at 130.min-1, tidal volume adjusted for body

weight according to the manufacturer’s instructions (Harvard rodent ventilator model 683,

Harvard Apparatus, Holliston, MA) and positive end-expiratory pressure held at 4 cmH2O,

anaesthesia was maintained with 2.5 - 3% sevoflurane. Rats were placed on a heating pad.

The electrocardiogram was monitored, body temperature was kept at 38 ºC and 8 mL.Kg-

1.h-1 intravenous warm lactated Ringer solution was infused (Multi-Phaser, NE-1000, New

Era Pump Systems, Wantagh, NY). A left thoracotomy was performed under surgical

microscopy with animals in right lateral semidecubitus. Silk threads were passed around the

inferior vena cava (IVC). Pressure-volume (P-V) catheters were positioned along the long

axes of the left ventricle (LV) and right ventricle (RV) (models SPR-838, 2F, and PVR-1045,

1F, Millar Instruments, Houston, TX, respectively). Signals were continuously acquired

(MPVS 300, Millar Instruments, Houston, TX) and digitally recorded at 1000 Hz (ML880

PowerLab 16/30, ADinstruments Pty Ltd., Australia). A flow probe (200-367, Triton

Technology, San Diego, CA) positioned around the ascending aorta allowed cardiac output

(CO) measurement with an ultrasonic flowmeter (Active Redirection Transit Time

Flowmeter, System 6, Triton Technology, San Diego, CA). After stabilization for 15 minutes,


28

baseline and IVC occlusion recordings were obtained, with ventilation suspended at end-

expiration. Parallel conductance was calibrated by injection of 50 µL of 10% saline. Data

was analysed with the aid of software (PVAN 3.5, Millar Instruments, Houston, TX). Blood

was collected and stored at -80 ºC for subsequent analysis. After euthanasia with

intravenous pentobarbital 100 mg.Kg-1, heparinized blood was collected for volume

calibration with standard cuvettes (Part No. 910 - 1048, Millar Instr., Houston, TX), and the

lungs, heart and mesenteric fat were weighted. After careful dissection the RV free-wall and

the LV free-wall plus interventricular septum weights were determined. The right upper lung

lobe, RV and LV free-walls, and mesenteric fat were snap frozen in liquid nitrogen and

stored at -80 ºC for molecular biology studies. The weights of the liver, kidneys,

gastrocnemius muscle, perirenal and perigonadal fat, and the length of the tibia were

measured at necropsy.

Histology

In 7 additional animals per group, lungs were inflated and fixed with tracheal instillation of

ethanol. Lung and heart were immersed in 10% formalin. Transverse 4 µm-thick sections of

paraffin-embedded lung and myocardium (encompassing the RV, interventricular septum

and LV) were stained with H&E and Masson’s trichrome and photographed with a digital

camera (Leica DFC320). Sections were evaluated by two independent blinded observers.

For the myocardium, the shortest diameter of 50 transversally cut, randomly selected

cardiomyocytes from the RV and LV free wall myocardium (400 x) was measured at the

level of the nucleus in H&E sections, with image analyzer software (Leica IM-1000). We also

measured the percentage of blue in Masson’s trichrome staining as indicative of fibrosis in

10 randomly selected RV and LV myocardial fields (400 x) using the NIH Image Program

(Image J). For the lung, medial hypertrophy of pulmonary arterioles was assessed in 10

arterioles from randomly selected fields (400 x) as a percentage of the radius, by obtaining 8

orthogonal measurements of inner diameter (distance between internal elastic laminae) and

outer diameters (distance between external elastic laminae).

Quantification of LV and RV myocardial DNA and protein content

Total DNA and protein were assessed in 10 mg of RV and LV free-wall myocardial samples

from 7 animals of each group. Briefly, after tissue homogenation with a rotor stator (PRO

200, Cat. No. 01 - 02200, Oxford, CT, USA) samples were extracted according to the

manufacturer’s instructions (AllPrep® DNA/RNA/Protein Mini Kit, Cat. No. 80004, Qiagen,

København, Danmark). DNA concentration was assayed by spectrophotometry at 260 nm

(Eppendorf 6131000.012). Protein was dissolved in 5% Sodium-dodecyl sulphate and


29

concentration assayed by the bicinchoninic acid method (Pierce®, BCA Protein Asssay Kit-

Reducing Agent Compatible, Cat. No. 23250, Rockford, IL USA). DNA and protein contents

were normalized for the initial mass of myocardial tissue.

mRNA quantification

Briefly, two-step real-time reverse transcription (RT)-polymerase chain reaction (PCR) was

performed in mesenteric fat and LV and RV free-wall samples extracted from 7 animals per

group by the guanidium-thiocyanate selective silica-gel membrane-binding method (no.

74124; Qiagen). Concentration and purity were assayed by spectrophotometry (Eppendorf

6131000.012). After RT on a thermocycler (Whatman Biometra 050-901), with 40 U/reaction

reverse transcriptase (Invitrogen 18064-014), 20 U/reaction RNase inhibitor (Promega

N2515) and 30 ng/µl random primers (Invitrogen 48190-011), 0.5 mM nucleotide mix (MBI

Fermentas R0192) for a final volume of 20 µL, real-time PCR was performed using SYBR

green (Qiagen 204143) as fluorescent marker, according to the manufacturer’s instructions

(Lightcycler II, Roche). Standard curves were obtained with specific primer pairs for

endothelin-1 (ET-1; fw: 5’ – CGG GGC TCT GTA GTC AAT GTG - 3’ and rev: 5’ – CCA

TGC AGA AAG GCG TAA AAG - 3’), tumor necrosis factor-α (TNF-α; fw: 5’ – TGG GCT

ACG GGC TTG TCA CTC - 3’ and rev: 5’ – GGG GGC CAC CAC GCT CTT C - 3’),

interleukin-6 (IL-6; fw: 5’ – GAA GTT GGG GTA GGA AGG AC - 3’ and rev: 5’ – CCG TTT

CTA CCT GGA GTT TG - 3’), peroxisome proliferator activated receptor-α (PPAR-α; fw: 5’ –

TTC CAG CCC CTC CTC AGT CAG - 3’ and rev: 5’ – AGC CCT TGC AGC CTT CAC AT -

3’), pyruvate-dehidrogenase kinase type 4 (PDK4; fw: 5’ – GAG GCC ACC GTC GTC TTG -

3’ and rev: 5’ – ACA GGC GTT GGA GCA GTG G - 3’), long-chain-fatty-acid-CoA ligase 1

(ACSL1; fw: 5’ – CTA CAG GCA ACC CCA AAG GAG - 3’ and rev: 5’ – GGG CGA GAG

GCA AGA AAG ATA - 3’), medium chain acyl CoA dehydrogenase (MCAD; fw: 5’ – ACG

ATA AAA GCG GGG AAT ACC - 3’ and rev: 5’ – AGG CCA AGA CCA CCA CAA CTC - 3’),

long chain acyl CoA dehydrogenase (LCAD; fw: 5’ – CAT TTT CCG GGA GAG TGT AA - 3’

and rev: 5’ – CTT GCC AGC TTT TTC CCA GAG - 3’), adiponectin (fw: 5’ – GGG CTA CGG

GCT GCT CTG A - 3’ and rev: 5’ – TAT GGG GAA GGG GAC AAC AAT G - 3’), β-actin (fw:

5’ – ATC TGG GTC ATC TTT TCA CGG TTG G - 3’ and rev: 5’ – GAT TTG GCA CCA CAC

TTT CTA CA - 3’) and glyceraldehyde-3-phosphate dehydrogenase(GAPDH; fw: 5’ – CCG

CCT GCT TCA CCA CCT TCT - 3’ and rev: 5’ – TGG CCT TCC GTG TTC CTA CCC - 3’).

Equal amounts of mRNA from every sample underwent experiments in duplicate.

Thresholds for amplification were determined automatically by the second derivative

maximum method. Results are presented after normalization for β-actin and GAPDH internal


30

control genes, in the myocardium and adipose tissue, respectively, and setting the mean

value of the Ctrl ND group as arbitrary unit (AU).

Myosin-heavy chain (MHC) isoforms

In RV and LV samples from 6 animals of each experimental group, 15 µg of total protein

were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (3 and 5%

stacking and running gels, respectively) at 30 mA for 6 h (no. 165-3301, BIO-RAD), allowing

a clear separation of α and β myosin heavy-chain isoform bands (200 KDa). After staining

with Comassie Brilliant Blue R-250 gels were scanned at 700 nm by an image acquisition

system (Odyssey; LI-COR Biosciences, Lincoln, NE, USA). The percentage of β-isoform

was assessed as the ratio of β isoform absorbance to total absorbance.

Plasma TNF-α quantification

Plasma concentration of TNF-α was determined by solid phase sandwich quantitative

enzyme immunoassay with specific antibodies pre-coated onto microplates (45-TNFRTU-

E01, Alpco Diagnostics™, Salem, NH). Samples from 7 animals per group were run in

duplicates along with a standard curve and controls. A secondary biotinylated antibody,

streptavidin-peroxidase and a substrate solution were sequentially added with intermediate

washing steps and reading was done at a 450 nm wavelength (UVM-340 monochromator

based reader, ASYS Hitech GmbH, Eugendorf, Austria) and analysed (Microplate

instrument analyser, version 1.2.0.2).

Myocardial apoptosis

The extent of RV and LV myocardial apoptosis was assessed by immunohistochemical

terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) of

histological sections from 7 animals of each group. Briefly, 5 random sections from each

animal, obtained at 100 µm intervals, were deparaffinised, immersed in phosphate-buffered

saline, permeabilized with proteinase K, quenched for endogenous peroxidase activity with

5.0% hydrogen peroxide, and sequentially incubated in terminal deoxynucleotidyl

transferase (TdT) labeling mix, blocking buffer and streptavidin-HRP. Sections were then

developed with TACS Blue Label and counterstained with Nucler Fast Red according to the

manufacturer’s instructions (TdT In Situ Apoptosis Detection Kit, R&D Systems,

Minneapolis, MN USA). Positive and negative controls consisted of treatment with TACS-

Nuclease™ before labelling and a TdT-free labeling mix, respectively. TUNEL-positive

cardiomyocytes were counted by two independent blinded observers in 50 random fields


31

(400 x) of each section and apoptotic rate was expressed as percentage of apoptotic

cardiomyocytes nuclei per total number of cardiomyocytes.

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and PPAR-α

transcription factor assays

Nuclear protein extracts were prepared from frozen RV and LV free-wall myocardium

according to the manufacturer’s instructions (Cat. No. 11906 - 100, Marligen Biosciences,

Inc., Ijamsville, MD USA). Briefly, nuclear factor κ-light-chain-enhancer of activated B cells

(NF-κB) and peroxisome proliferator activated receptor-α (PPAR-α) activation was assayed

by adding 20 µg of nuclear protein sample to microplate wells with the specific dsDNA

response elements for NF-κB (p65) and PPAR-α immobilized onto the bottom (Cat. No.

1007889 and Cat. No. 10006915, Cayman Chemical Company, Ann Harbor, MI, USA,

respectively). Samples were run in duplicate and positive and negative controls were

included. After an overnight 4 ºC incubation, transcription factor binding was detected by

addition of a specific primary antibody (Cat. No. 10007442, Cayman Chemical Company,

Ann Harbor, MI, USA). Finally, infrared labelling was performed with an IRDye 800CW-

conjugated secondary antibody (Cat. No. 926 - 32211, Li-COR biosciences, Lincoln, NE,

USA) diluted (1:1000) in blocking buffer (Cat. no. 927 - 40000, Li-COR biosciences, Lincoln,

NE, USA) containing 0.2% Tween-20. Absorbance at 800 nm was read with an image

acquisition system (Odyssey; LI-COR Biosciences, Lincoln, NE, USA).

Statistical analysis

Data were analyzed by Kaplan-Meier survival analysis with Gehan-Breslow statistic, by two-

way repeated measures ANOVA for BW and caloric ingestion progression, and by two-way

ANOVA for all other purposes. Results are presented as mean ± SEM. Two-tailed P < 0.05

was considered significant.

Results

Survival, weight gain, caloric intake, body composition and metabolism

Mortality was decreased in MCT HD compared with MCT ND (Figure 1, Panel A). MCT ND

rats presented signs of overt HF, namely laboured breathing, ruffled hair, lethargy, ascites

and pleural effusion, while MCT HD appeared more active.


32

Figure 1. Survival (panel A), body weight and caloric ingestion (panel B) in controls and

monocrotaline (MCT) pulmonary hypertensives fed with either normal diet, Ctrl ND and MCT

ND, respectively, or hypercaloric diet, Ctrl HD and MCT HD, respectively.

MCT HD had reduced mortality compared with MCT ND (*P = 0.016). MCT-injected groups showed less weight

gain and calorie ingestion (†P < 0.001) compared with their respective controls. Both hypercaloric fed groups

showed increased weight gain (*P < 0.001), whereas only MCT HD showed increased calorie ingestion (*P =

0.005) compared with their normal diet fed littermates. Survival and follow-up evaluation were completed in 17

and 22 animals from Ctrl ND and Ctrl HD, respectively, and 53 and 44 animals from MCT ND and MCT HD,

respectively.

Throughout the study MCT-injected groups had a lower calorie intake and gained less BW

compared with Ctrl ND. MCT HD, however, presented a higher calorie intake and reached a

higher BW than MCT ND (Figure 1, Panel B). During metabolic cage studies, MCT ND was

the only group that lost weight, also presenting reduced diuresis and lower calorie ingestion

than MCT HD (Table 1). Furthermore, MCT ND presented reduced BW and tibial length (TL)

compared with their respective control as well as reduced gastrocnemius, liver and perirenal

and perigonadal fat pad weights normalized for TL. All but TL were offset in MCT HD (Table

2). Ctrl HD showed increased BW (Figure 1, Panel B), caloric intake (Table 1), and adiposity

compared with Ctrl ND (Table 2). BW was not increased in Ctrl HD, compared with Ctrl ND,

at the time of haemodynamic evaluation (Table 2). Hypercaloric diet (HD) fed control and

MCT-injected groups showed increased basal glycaemia levels and IR, which was also seen

in MCT ND compared with Ctrl ND (Table 1).


33

Table 1. Metabolic studies and glucose metabolism.

Ctrl ND Ctrl HD MCT ND MCT HD

Weight gain, % 2.48 ± 1.29 4.50 ± 1.18 -0.08 ± 1.36* 3.81 ± 0.73†

Caloric intake, cal.g-1 0.246 ± 0.009 0.370 ± 0.031† 0.224 ± 0.024 0.326 ± 0.036†

Diuresis, µL.g-1 35.0 ± 3.4 33.1 ± 4.8 19.5 ± 2.8* 24.3 ± 4.5

Glicemia, mg.dL-1 107 ± 4 121 ± 5† 121 ± 5* 134 ± 3*†

OGT AUC, mg.min 21009 ± 2232 22370 ± 2074 21117 ± 1146 23674 ± 1002

IR AUC, mg.min 9735 ± 798 16442 ± 568† 12519 ± 1240* 16232 ± 1123†

Data collected during 24 h metabolic cage studies, oral glucose tolerance (OGT) and insulin resistance tests

(IR) from monocrotaline-injected (MCT) and vehicle injected rats (Ctrl) fed a normal (ND) or a hypercaloric

diet (HD). AUC, area under curve. *P < 0.05 vs Ctrl; †P < 0.05 vs similar group fed ND; n = 7 in each group.

Table 2. Morphometry


Weight, g 354 ± 6 361 ± 7 242 ± 7* 261 ± 7*†

Tibial length, cm 4.00 ± 0.03 3.93 ± 0.04 3.85 ± 0.02* 3.86 ± 0.04

Lung/tibia, mg.cm-1 383 ± 10 417 ± 12 650 ± 28* 520 ± 32*†

Gastrocnemius/tibia, mg.cm1 551 ± 14 513 ± 18 375 ± 21* 467 ± 10*†

Liver/tibia, mg.cm-1 3248 ± 186 3686 ± 328 2141 ± 125* 2844 ± 256*†

Perigonadal fat/tibia, mg.cm-1 804 ± 77 1155 ± 71† 473 ± 26* 633 ± 40*†

Perirenal fat/tibia, mg.cm-1 802 ± 77 1243 ± 158† 347 ± 45* 591 ± 36*†

Morphometric data from monocrotaline-injected rats fed either a normal (MCT ND) or a hypercaloric diet

(MCT HD) and their controls (Ctrl ND and Ctrl HD, respectively). *P < 0.05 vs Ctrl; †P < 0.05 vs ND; n = 7 in

each group.

Haemodynamics

Data are summarized in table 3.


34

Table 3. Haemodynamics


Baseline HR, min-1 375 ± 22 397 ± 10 284 ± 23* 359 ± 15†

CO, mL.min-1 57.8 ± 2.2 64.3 ± 7.2 28.3 ± 4.1* 43.6 ± 3.9*†

Right ventricle

SP, mmHg 38.2 ± 1.6 37.5 ± 3.8 73. 5 ± 4.5* 59.3 ± 5.0*†

EDP, mmHg 3.7 ± 0.3 4.2 ± 0.7 7.6 ± 2.9* 5.9 ± 0.6

EDV, µL 229.0 ± 15.6 241.4 ± 17.8 312.2 ± 36.4* 279.9 ± 41.2

EF, % 67.4 ± 2.5 66.5 ± 3.4 34.6 ± 5.1* 48.8 ± 4.6*†

τ log, ms 11.7 ± 1.5 12.0 ± 0.6 15.8 ± 1.3* 11.1 ± 1.6†

EA, mmHg.µL-1 0.23 ± 0.01 0.25 ± 0.05 0.81 ± 0.14* 0.55 ± 0.06*†

Left ventricle

SP, mmHg 128.7 ± 3.6 126.5 ± 4.8 95.2 ± 5.2* 110.6 ± 3.0*†

EDP, mmHg 5.0 ± 0.7 4.2 ± 0.4 4.7 ± 0.5 5.5 ± 0.6

EDV, µL 270.7 ± 19.0 258.2 ± 29.0 156.9 ± 26.9* 198.7 ± 20.1

EF, % 57.0 ± 4.8 61.7 ± 4.8 62.5 ± 2.9 58.0 ± 3.8

dP/dtmax, mmHg.s-1 10241 ± 733 11232 ± 1150 5779 ± 452* 8137 ± 594*†

τ log, ms 8.6 ± 0.8 7.6 ± 0.4 12.7 ± 1.1* 9.7 ± 0.6†

SW, mmHg.µL 13988 ± 1430 15919 ± 1824 6829 ± 761* 10802 ± 1334*†

EA, mmHg.µL-1 0.78 ± 0.05 0.76 ± 0.08 1.33 ± 0.27* 1.00 ± 0.11

IVC occlusion

Right ventricle

EDPVR, mmHg.µL-1 0.014 ± 0.004 0.016 ± 0.002 0.024 ± 0.002* 0.022 ± 0.006

ESPVR, mmHg.µL-1 0.196 ± 0.050 0.178 ± 0.070 0.733 ± 0.187* 0.563 ± 0.216

PRSW, mmHg 22.3 ± 4.8 18.9 ± 3.1 37.3 ± 4.1* 31.2 ± 7.1

dP/dtmax-EDV, mmHg.s-1.µL-1 9.08 ± 2.25 9.55 ± 1.51 17.08 ± 2.64* 14.97 ± 2.84

ME, mmHg.µL-1 0.64 ± 0.11 0.67 ± 0.15 1.24 ± 0.22* 0.88 ± 0.26

Left ventricle

EDPVR, mmHg.µL-1 0.038 ± 0.008 0.046 ± 0.008 0.099 ± 0.022* 0.066 ± 0.010

ESPVR, mmHg.µL-1 0.75 ± 0.18 0.69 ± 0.28 2.86 ± 0.87* 3.16 ± 1.23*

PRSW, mmHg 105 ± 18 113 ± 6 221 ± 111 145 ± 23

dP/dtmax-EDV, mmHg.s-1.µL-1 83 ± 15 77 ± 14 80 ± 19 105 ± 32

ME, mmHg.µL-1 2.91 ± 0.51 2.96 ± 0.66 6.84 ± 1.58* 5.99 ± 1.04*

HR, heart rate; CO, cardiac output; dP/dtmax, maximal rate of pressure rise; EA, arterial elastance; EF,

ejection fraction; IVC, inferior vena cava; ME, (time-varying) maximal elastance; EDP, end-diastolic

pressure; PRSW, preload recruitable stroke work slope; EDPVR, end-diastolic pressure-volume relationship

slope; ESPVR, end-systolic pressure-volume relationship slope; SW, stroke work; EDV, end-diastolic

volume; dP/dtmaxvs EDV, slope of the relationship between dP/dtmax and EDV; τ log, time-constant of

isovolumic relaxation by logarithmic regression; SP, systolic or maximum pressure. No differences were

observed for the intercepts of functional indexes described by linear regression. These are not presented for

the sake of simplicity. *P < 0.05 vs Ctrl; †P < 0.05 vs MCT ND; n = 7 in each group.

MCT ND showed increased pulmonary artery (PA) elastance, increased RV systolic

pressure (RVSP) and end-systolic P-V relationship (ESPVR) slope, along with reduced

ejection fraction (EF), prolonged time constant of isovolumetric relaxation, τ, and increased

RV end-diastolic volume (EDV), pressure (EDP) and end-diastolic P-V relationship (EDPVR)

slope, which were attenuated in MCT HD. Heart rate and CO fell in MCT ND compared with

Ctrl ND, and were also improved in MCT HD. As for the LV, MCT ND presented reduced LV


35

systolic pressure, maximal rate of pressure rise (dP/dtmax), and stroke work (SW) and

increased τ that were attenuated in MCT HD. MCT ND, but not MCT HD, also showed

increased arterial elastance, reduced LV EDV and elevated LV EDPVR slope.

Representative RV and LV P-V loops along with the respective EDPVR and ESPVR slopes

are presented in figure 2.

Figure 2. Representative left ventricular (LV, right panel) and right ventricular (RV,

left panel) pressure-volume loops and respective end-systolic (upper-left corner

of each loop) and end-diastolic relationship slopes (lower-right corner) obtained

from inferior vena cava occlusions.

Monocrotaline (MCT)-injected rats (black lines) and their respective controls (gray lines) fed with

either normal diet, Ctrl ND and MCT ND, respectively (solid lines), or hypercaloric diet, Ctrl HD and

MCT HD, respectively (dashed lines) are represented. For significant differences please refer to

table 3 of the manuscript.

Lung and myocardial morphometry and histology, myocardial apoptosis and MHC

isoforms

MCT ND showed increased medial hypertrophy of lung arterioles (Figure 3) and increased

lung to TL ratio (Table 2) that were attenuated in MCT HD.


36

Figure 3. Lung histology (200x) of control and monocrotaline (MCT)-injected rats fed

with either normal diet, Ctrl ND and MCT ND, respectively, or hypercaloric diet, Ctrl HD

and MCT HD, respectively (upper panel) and media layer thickness of pulmonary

arterioles (lower panel).

MCT ND showed increased media thickness (*P = 0.003 vs Ctrl ND) that was abrogated in their

pulmonary hypertensive MCT HD littermates (†P = 0.002 vs MCT ND). Histological sections from 7

animals per group were analysed.

As for the myocardium, both MCT groups presented increased RV cardiomyocyte diameter

whereas no changes were seen in LV free-wall myocytes (Table 4). Nevertheless, while RV

weight normalized for TL increased in both MCT groups, TL-normalized weight of LV +

interventricular septum (IVS) decreased only in MCT ND (Table 4).


37

Table 4. Myocardial morphometry, myocyte diameters and protein:DNA content


Morphometry

RV/tibia, mg.cm-1 47.8 ± 2.2 51.0 ± 3.0 84.0 ± 2.5* 85.7 ± 5.1*

LV+IVS/tibia, mg.cm-1 174.5 ± 7.7 177.2 ± 5.1 136.6 ± 5.9* 160.4 ± 4.2*†

Cardiomyocyte diameters

RV free-wall myocyte, µm 15.6 ± 0.7 16.8 ± 0.3 18.5 ± 0.6* 18.7 ± 0.5*

LV free-wall myocyte, µm 16.9 ± 0.2 17.1 ± 0.8 17.0 ± 0.3 16.3 ± 0.5

Protein:DNA content

RV protein content, µg/g 123.5 ± 5.1 119.1 ± 8.4 171.6 ± 14.2* 154.9 ± 15.2*

RV DNA content, ng/g 295.8 ± 10.2 310.2 ± 10.7 564.6 ± 65.4* 342.0 ± 15.4†

RV protein/DNA, µg/ng 0.42 ± 0.00 0.38 ± 0.02 0.30 ± 0.01* 0.47 ± 0.07†

LV protein content, µg/g 119.5 ± 12.0 120.2 ± 10.3 124.6 ± 5.0 130.4 ± 9.6

LV DNA content, ng/g 299.4 ± 14.9 274.2 ± 34.5 268.8 ± 26.2 261.0 ± 17.4

LV protein/DNA, µg/ng 0.40 ± 0.05 0.46 ± 0.06 0.49 ± 0.06 0.51 ± 0.04

IVS, interventricular septum; LV, left ventricle; RV, right ventricle. *P < 0.05 vs Ctrl; †P < 0.05 vs ND; n = 7 in

each group.

Myocardial fibrosis was observed in both the RV and LV myocardium of MCT ND and was

partly offset in MCT HD (Figure 4). MCT ND also presented increased apoptosis rate in both

ventricles compared with Ctrl ND, which was attenuated in MCT HD (Figure 5). Similarly,

β−MHC isoform percentage was increased in MCT ND and this change was attenuated in

MCT HD (Figure 6). No significant changes were observed in controls. Both MCT groups

presented increased RV myocardial protein content, but defiantly MCT ND showed a

decreased protein:DNA ratio which was due to increased DNA content. No differences were

noticed in the LV (Table 4).


38

Figure 4. Myocardial right ventricular (RV, left panel) and left ventricular (LV, right

panel) Masson’s trichrome staining (400 x) of control and monocrotaline (MCT)-

injected rats fed with either normal diet, Ctrl ND and MCT ND, respectively, or

hypercaloric diet, Ctrl HD and MCT HD, respectively (upper panel) and respective

percentage of fibrosis (lower panel).

MCT ND presented RV and LV fibrosis that was attenuated in MCT HD. *P<0.005 vs Ctrl ND;

†P=0.02

vs MCT ND; n = 7 in each group.

Figure 5. Right ventricular (RV, left panels) and left ventricular (LV, right panels)

myocardium terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling

(TUNEL) immunostaining (400 x) of rats with monocrotaline (MCT)-induced pulmonary

hypertension (PH) and their respective controls (Ctrl) fed either a normal or a

hypercaloric diet (ND and HD, respectively).

TUNEL-positive cardiomyocytes (dark staining nuclei) are expressed as a percentage of total

cardiomyocyte nuclei in the lower graph. Both MCT ND and MCT HD showed increased staining in the

RV and LV myocardium (*P < 0.001) compared with Ctrl ND and Cltr HD, respectively, which was

partly attenuated in MCT HD (†P < 0.05 vs MCT ND). Immunostaining was performed in 7 animals per

group.


39

Figure 6. Myosin-heavy chain (MHC) β isoforms in right ventricular (RV) and left ventricular (LV)

myocardium of monocrotaline (MCT)-injected and vehicle injected rats (Ctrl) fed either a normal

(ND) or a hypercaloric diet (HD).

Results are presented as a percentage of β isoforms from total MCH. Representative blots are shown above each

group’s bar. β−MHC isoform percentage was increased in MCT ND RV and LV (*P < 0.005) and was attenuated in

MCT HD (†P < 0.05 vs MCT ND). Six animals from each experimental group were studied.

Myocardium and visceral adipose tissue gene expression and plasma TNF-α

ET-1 expression was upregulated in both the RV and LV myocardium of MCT ND compared

with Ctrl ND which was lessened in MCT HD (Figure 7, Panel A). TNF-α and IL-6, however,

were selectively upregulated in the LV of MCT ND, changes which were also attenuated in

MCT HD (Figure 7, Panel A). PPAR-α expression was downregulated in both ventricles of

MCT ND but not in the LV of MCT HD. As for metabolic enzymes, both MCT groups showed

lower mRNA levels of ACSL1, MCAD, LCAD and PDK4 in the RV myocardium compared

with controls, while ACSL1, MCAD and LCAD LV expression was increased in Ctrl HD

compared with Ctrl ND, with no changes in MCT-injected groups. Distinctly, PDK4 mRNA

levels did not vary in the LV myocardium whereas they increased in the RV myocardium of

Ctrl HD (Figure 7, Panel B). As regards visceral adipose tissue, gene expression of TNF-α

and IL-6 cytokines was upregulated in MCT ND compared with controls, which was

alleviated in MCT HD. Adiponectin expression, likewise, increased less in MCT HD than in

MCT ND (Figure 8, Panel A). Plasma TNF-α increased ten-fold in MCT ND compared with

Ctrl ND, while MCT HD presented lower levels (Figure 8, Panel B).


40

Figure 7. Gene expression of endothelin-1 (ET-1), and tumor necrosis factor-α (TNF-α) and

interleukin-6 (IL-6) cytokines (panel A) and of peroxisome proliferator-activated receptor-α

(PPAR-α) and metabolism regulating enzymes pyruvate-dehidrogenase kinase-4 (PDK4), long-

chain-fatty-acid-CoA ligase 1 (ACSL1), medium chain acyl CoA dehydrogenase (MCAD), and

long chain acyl CoA dehydrogenase (LCAD) in the right ventricular and left ventricular

myocardium (panel B).

Ctrl ND expression, shown as a reference line, was set as arbitrary expression unit (AU). While monocrotaline

(MCT)-induced pulmonary hypertensive (PH) rats fed a normal diet (MCT ND) presented biventricular ET-1, and LV

overexpression of TNF-α and IL-6 compared with normal diet fed controls (Ctrl ND), their PH littermates fed a

hypercaloric diet (MCT HD) had abrogated gene activation. PPAR-α expression was downregulated in both

ventricles of MCT-injected groups, except for MCT HD LV, whereas metabolic enzymes ACSL1, MCAD and LCAD,

and PDK4 were downregulated in the RV but not in the LV. As for hypercaloric diet fed controls (Ctrl HD), no

changes were observed in ET-1, TNF-α or IL-6 expression while ACSL1, MCAD and LCAD were selectively

overexpressed in the LV and PDK4 in the RV, compared with Ctrl ND. *P < 0.05 vs respective vehicle-injected

group; †P < 0.05 vs similar group fed with normal diet; n = 7 in each group.

Figure 8. Mesenteric adipose tissue gene expression of tumor necrosis factor-α (TNF-α),

interleukin-6 (IL-6) and adiponectin (Panel A) and plasma TNF-α levels (Panel B) in vehicle-

injected and monocrotaline (MCT)-injected rats fed with normal (Ctrl ND and MCT ND,

respectively) and hypercaloric diets (Ctrl HD and MCT HD, respectively).

Ctrl ND gene expression is shown as a reference line and was set as arbitrary expression unit (AU) in panel A.

gene expression changes were normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA

levels. TNF-α, IL-6 and adiponectin were overexpressed in MCT ND compared with Ctrl ND. Gene expression

changes were attenuated in MCT HD. As for TNF-α plasma levels, these were markedly increased in MCT ND

compared with controls, which was attenuated in MCT DH. *P < 0.05 vs Ctrl ND; †P < 0.05 vs MCT ND; n = 7 in

each group.


41

Myocardial NF-κB and PPAR-α Transcription factor activity

MCT HD showed decreased PPAR-α transcription factor activity in the RV myocardium

compared with both its respective control group and MCT ND whereas in the LV

myocardium MCT ND denoted decreased activity which was reversed in MCT HD (Figure 9,

Panel A). As for NF-κB, MCT ND showed increased biventricular activity, which was tapered

in the RV and completely abrogated in the LV myocardium of MCT HD (Figure 9, Panel B).

Figure 9. Peroxisome proliferator activated receptor-α (PPAR-α, panel A) and nuclear

factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor activities

(panel B) in the right ventricular and left ventricular myocardium of rats with

monocrotaline-induced pulmonary hypertension (MCT) and their respective controls

(Ctrl) fed either a normal or a hypercaloric diet (ND and HD, respectively).

Examples of immune infrared fluorescent staining from representative samples are shown above the

bars of each group. While no significant changes were observed between control groups, MCT ND

showed LV downregulation (*P = 0.03) of PPAR-α transcription factor activity, as well as increased RV

and LV NF-κB transcription factor activities (*P < 0.05). These changes were partly attenuated or

completely restored to control levels in MCT HD (†P < 0.05). Data are presented in relative absorvance

(RA) compared with Ctrl ND. Transcription factor activities were assayed in 7 animals per group.


42

Discussion

A HD rich in animal fat and simple carbohydrates improved survival and attenuated PH and

CC in experimental MCT-induced PH. Both RV and LV dysfunction and remodeling were

attenuated, as well as neuroendocrine and inflammatory activities.

MCT-induced PH extensively activates neuroendocrine systems and rapidly evolves

towards fatal decompensated HF (Ceconi et al., 1989, Hessel et al., 2006, Lourenço et al.,

2006). Accordingly, MCT ND presented medial PA thickening, increased PA elastance, an

index of pulmonary vascular load, and PH, as assessed by RVSP. The overloaded RV

showed increased EDP and EDV, hypertrophy, fibrosis and increased apoptotic rate, along

with an increase in both DNA and protein content with lower protein:DNA ratio. EF and CO

were decreased, whereas τ and EDPVR slope were increased, denoting compromised

relaxation and diastolic dysfunction, respectively. Regression towards the fetal β-MHC

isoform and local overexpression of ET-1 were also observed, as previously reported

(Correia-Pinto et al., 2009, Lourenço, Roncon-Albuquerque, Bras-Silva, Faria, Wieland,

Henriques-Coelho, Correia-Pinto and Leite-Moreira 2006).

Besides overload and neuroendocrine activation, other mechanisms, namely mitochondrial

dysfunction, reactive oxygen species (ROS), apoptosis and extracellular matrix

modifications underlie RV myocardial compromise (Bogaard et al., 2009, Redout et al.,

2007). Not surprisingly, anti-inflammatory agents such as statins have been effectively

employed as experimental therapeutics (Sun and Ku 2008). Another successful anti-

inflammatory approach has been caloric restriction, it favourably alters energy metabolism

while reducing ROS and neuroendocrine activity (Chou, Lee, Huang, Wang, Yu and Lau

2010). Indeed, caloric restriction attenuated RV hypertrophy and polyamine synthesis 21

days after MCT injection (Hacker 1993). It also diminished inflammation and cachexia in the

slow developing salt-sensitive Dahl rat arterial hypertension model (Seymour et al., 2006).

MCT-induced PH, however, is a severe and rapidly progressive model that usually evolves

from compensated RV hypertrophy, up to 4 weeks after injection (Correia-Pinto, Henriques-

Coelho, Roncon-Albuquerque, Lourenço, Melo-Rocha, Vasques-Novoa, Gillebert and Leite-

Moreira 2009, Hessel, Steendijk, den Adel, Schutte and van der Laarse 2006), to

decompensated HF, accompanied by marked anorexia and cachexia, beyond 21 days after

injection (Dalla Libera, Ravara, Volterrani, Gobbo, Della Barbera, Angelini, Danieli Betto,

Germinario and Vescovo 2004, Steffen, Lees and Booth 2008). Our study was intentionally

conducted when rats presented overt HF, reduced diuresis and a high mortality rate, as well

as decreased calorie ingestion and extensive body wasting, encompassing both lean and fat

masses. At this point, animals also had decreased heart rate, SW and LVSP along with


43

decreased EDV and compromised LV diastolic function indexes, consonant with ventricular

interdependence.

Although no changes were observed in LV cardiomyocyte diameters we found decreased

LV+IVS mass, increased apoptosis rate and fibrosis, along with marked overexpression not

only of ET-1 but also of IL-6 and TNF-α. We have previously suggested that systemic

neuroendocrine activity could trigger intrinsic LV myocardial dysfunction and neuroendocrine

activation, namely of ET-1 (Lourenço, Roncon-Albuquerque, Bras-Silva, Faria, Wieland,

Henriques-Coelho, Correia-Pinto and Leite-Moreira 2006). Based on current results we

propose that LV myocardial cytokine activation might also be explained by cachexia and

unloading. Indeed we have previously described lower LV mass and TNF-α overexpression

in cachectic rats with nephrotic syndrome (Moreira-Rodrigues et al., 2007). Inflammatory

cytokines, that may have been partly responsible for LV dysfunction and remodeling

(Feldman et al., 2000), were also overexpressed in visceral adipose tissue, alongside

adiponectin, an adipokine marker of worse prognosis in cachexia (Kistorp et al., 2005).

Compared with MCT ND, MCT HD presented a higher calorie intake and was able to

preserve BW, lean and fat mass. Although rats were not force-fed by gavaging, the typical

“western” HD is highly palatable and it provided additional calorie content per mass

ingested. Substrate and energy supply are fundamental in critical illness (Martindale et al.,

2009). Although nutritional supplementation in HF is constrained by fluid and salt restriction

and cannot circumvent anorexia, early satiety, reduced nutrient absorption and increased

catabolism, some small studies have shown benefits in BW gain and muscle function after

energy supplementation, mainly by nasoenteric route, with few cases of decompensation

due to a refeeding-like syndrome (Akner and Cederholm 2001). Interestingly, RV

hypertrophy was not attenuated in MCT HD compared with MCT ND despite reduced PH.

Still, myocardial DNA content did not increase, whilst myocardial apoptosis rate, fibrosis and

β-MHC isoform switch were attenuated, compared with MCT ND. We speculate the normal

DNA content compared with MCT ND could be due to a lower apoptosis rate, as suggested

for afterload-induced hypertrophy (Paquette et al., 2008). The higher substrate supply may

have accounted for differences in RV myocardial remodeling and hypertrophy. Additionally,

RV and LV myocardial function as well as CO were improved in MCT HD, along with

improved survival and attenuated inflammatory and neuroendocrine activities. These

findings are somewhat surprising but can be supported by several lines of evidence. Firstly,

observational studies suggest obesity and hypercholesterolemia improve survival in HF

(Curtis et al., 2005). The reverse epidemiology of cardiovascular risk factors (“obesity

paradox”) has been attributed not only to the fact that long term cardiovascular risk factors


44

may not behave as such in a poor prognosis condition such as severe HF (Kalantar-Zadeh

et al., 2004), but also to the lipoprotein-endotoxin hypothesis, which states lipoproteins,

which are increased in obesity and high-fat-feeding, neutralize bacterial lipopolysaccharides

(LPS) therefore protecting against inflammation (Rauchhaus et al., 2000). Chronic

inflammation accompanying HF partly results from intestinal bacterial translocation and

activation of cytokine production mostly from mononuclear cells but also from

cardiomyocytes (Anker and Coats 1999). Accordingly, the higher myocardial NF-κB activity,

and increased cytokine expression and plasma levels of TNF-α seen in MCT ND , were

mitigated in MCT HD. NF-κB signalling has been linked to cytokine release, oxidative stress,

hypertrophy, and remodeling in the failing myocardium (Valen et al., 2001). LPS or cytokines

activate inhibitor-κB (I-κB) kinase-mediated phosphorylation and degradation of I-κB,

unmasking a sequence within NF-κB that promotes nuclear translocation and binding to

response elements in target genes of failing cardiomyocytes (Valen, Yan and Hansson

2001). Some of these genes are cytokines, such as IL-6 and TNF-α, that underlie not only

many of the HF progression mechanisms (Feldman, Combes, Wagner, Kadakomi, Kubota,

Li and McTiernan 2000) but also IR, muscle wasting and cachexia (Sharma and Anker

2002), due to their pronounced effects in both cellular metabolism and PPAR-α function (Ye

2008). Decreased expression and activity of PPAR-α in the non-overloaded LV of MCT ND

might partly be attributed to TNF-α overexpression (Ye 2008).

Substantial metabolic adaptations accompany HF progression, triggering, and sustaining

functional and structural remodeling. PPAR plays a pivotal role in the transcriptional

regulation of genes involved in both FAO and glucose metabolism (Gulick et al., 1994).

Features such as impaired conversion of chemical energy into mechanical work, decline in

high-energy-phosphate content, mitochondrial dysfunction and loss of metabolic flexibility,

with a switch of energy source from FAO to anaerobic glycolysis are typically seen in the

failing myocardium (Ingwall 2009). Inactivation of PPAR-α during cardiac hypertrophy might

partly underlie this switch by transcriptional inactivation of FAO enzymes and PDK4 (Barger

et al., 2000). PDK4 phosphorylates and inhibits pyruvate dehydrogenase, an enzyme in

control of glycolysis-derived pyruvate oxidation (Stanley, Recchia and Lopaschuk 2005).

Although glucose metabolism increases the ATP production to O2 consumption ratio thereby

increasing energy efficiency (Stanley, Recchia and Lopaschuk 2005), this may not be

entirely adaptive, since more ATP is produced per mole of fatty acids (FA) (Barger, Brandt,

Leone, Weinheimer and Kelly 2000, van Bilsen et al., 2009). Indeed, cardiac work and

efficiency in human HF are definitely very dependent on FA (Tuunanen et al., 2006). The

combination of FA and carbohydrates in the HD regimen might have contributed to a better


45

preservation of myocardial function, as seen in MCT HD. MCT ND showed decreased RV

expression of PPARα, FAO enzymes and PDK4. LV PPAR-α expression and activity were

also decreased in MCT ND but not in MCT HD, which could be partly explained by PPAR-α

induction by the lipids in the HD (Gulick, Cresci, Caira, Moore and Kelly 1994). PPAR

activation normalizes cardiac substrate metabolism in RV hypertrophy and HF (Jucker et al.,

2007). Albeit we did not find LV FAO or PDK4 expression changes, these are not solely

regulated at the transcriptional level and expression levels do not translate enzymatic

activity (Stanley, Recchia and Lopaschuk 2005). Indeed, a high fat diet did not worsen

cardiac hypertrophy, in experimental LV pressure overload, and noticeably prevented the

decline in FAO enzyme activity and mitochondrial oxidative capacity (Chess et al., 2009).

Additionally, medium chain triglyceride diet enrichment in spontaneously hypertensive rats

preserved PPAR-α transcriptional activity, prevented FAO enzyme downregulation and

improved myocardial function (Iemitsu et al., 2008). Mitochondrial dysfunction, reduced

energy producing ability and increased production of ROS are known to accompany the

progression towards RV dysfunction in MCT-induced PH (Daicho et al., 2009, Redout,

Wagner, Zuidwijk, Boer, Musters, van Hardeveld, Paulus and Simonides 2007). Finally, we

must point out that recent evidence suggests PPAR agonists can attenuate PH (Hansmann

et al., 2007) and thus the high lipid content in the HD might have induced PPAR and

attenuated MCT-induced PH.

In summary, in a rapidly evolving experimental model of PH, HF and CC a HD regiment rich

in saturated fat and simple carbohydrates, which is a well-established nutritional

cardiovascular risk factor, attenuated PH, improved survival and myocardial function with

concomitant attenuation of neuroendocrine and inflammatory activities, possibly due to

beneficial changes in myocardial metabolism and transcription factor activity. Nevertheless,

we must acknowledge that a longer period of high-fat feeding could have led to cardiac

triglyceride accumulation and a decline in ventricular function (Akki and Seymour 2009).

Whether patients with CC and decompensated HF may profit from HD regimens remains to

be settled.


46

Acknowledgments

The authors would like to thank Dra. Antónia Teles for her excellent technical support.

Carmen Brás-Silva’s current affiliation in Faculty of Nutrition and Food Sciences, University

of Porto, Porto, Portugal.

Grants

This work was supported by the Portuguese Foundation for Science and Technology (grant

PTDC/SAU/65793/2006).

Disclosures

None to declare.


47

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5. Discussão e Conclusões

A dieta hipercalórica, do tipo ocidental, rica em gordura animal e carbo-hidratos

simples, melhorou a sobrevida e atenuou a HP e a caquexia cardíaca no modelo

experimental de HP severa e rapidamente progressiva, induzida pela MCT. A disfunção e a

remodelagem foram significativamente atenuadas no VD e no VE, assim como as

actividades neuroendócrina e inflamatória.

No grupo de animais injectados com MCT e alimentados com dieta normal, de acordo

com o esperado neste modelo experimental, observámos espessamento da camada média

das arteríolas pulmonares, HP, disfunção VD, com redução da fracção de ejecção, do DC e

elevação do volume e pressão telediastólicas, e remodelagem miocárdica, incluindo

hipertrofia, fibrose e apoptose. Acompanhando estas alterações, observámos igualmente

alterações moleculares típicas da regressão para o fenótipo cardiomiocitário fetal, que

usualmente acompanha a progressão da IC, nomeadamente a comutação para as

isoformas do tipo β, com actividade ATPásica lenta, das cadeias pesadas de miosina e a

maior expressão local de ET-1, como relatámos anteriormente (Correia-Pinto et al., 2009;

Lourenço et al., 2006). Para além da sobrecarga e da activação neuroendócrina, outros

mecanismos, designadamente a disfunção mitocondrial, a maior produção de ROS, a

apoptose e as modificações da matriz extracelular, podem contribuir para a disfunção do

miocárdio VD na HP (Bogaard et al., 2009a, 2009b; Redout et al., 2007). Não constitui

surpresa, portanto, que agentes anti-inflamatórios, como as estatinas, e o enriquecimento

alimentar com óleo de peixe (Baybutt et al., 2002) tenham sido bem sucedidos como terapia

experimental (Sun e Ku, 2008). Outra abordagem anti-inflamatória bem sucedida é a

restrição calórica, que modifica favoravelmente o metabolismo energético, reduz a

formação de ROS, a actividade neuroendócrina (Chou et al., 2010) e a degradação proteíca

(Redman et al., 2008; Weindruch et al., 2001). De facto, a restrição calórica atenuou a

hipertrofia e a síntese de poliaminas, 21 dias após a injecção de MCT (Hacker, 1993) e

atenuou a inflamação e a caquexia no modelo de hipertensão arterial sensível ao sal em

ratos Dahl, caracterizado pelo desenvolvimento lento de hipertensão arterial, IC e caquexia

(Seymour et al., 2006). No entanto, na avaliação do efeito da restrição calórica na HP

induzida pela MCT só foi estudada a hipertrofia ventricular direita e a síntese de poliaminas

(Hacker, 1993). Além disso, os animais foram estudados precocemente após a injecção,

quando geralmente apresentam um fenótipo de hipertrofia VD compensada sem sinais de

IC ou caquexia (Hessel et al., 2006; Werchan et al., 1989). Por outro lado, o modelo de

hipertensão arterial em ratos Dahl tem uma evolução muito lenta, de várias semanas, até à

ocorrência de caquexia e IC. O nosso estudo foi realizado 5 semanas após a injecção com


53

MCT, quando os animais apresentavam já mortalidade elevada e sinais clínicos evidentes

de IC, como a diminuição da diurese e edemas generalizados, e deterioração sistémica,

incluindo anorexia e caquexia. De facto, na avaliação dos animais do grupo injectado com

MCT e alimentado com dieta normal observamos para além de diminuição de ingestão

calórica, perda ponderal marcada, incluindo a massa magra e a massa gorda, mas também

bradicardia, diminuição do trabalho de ejecção e da pressão sistólica VE. No VE

observámos igualmente diminuição do volume telediastólico e comprometimento dos

índices de função diastólica, em consonância com a perturbação do preenchimento

decorrente da interdependência ventricular (Menzel et al., 2000). Contudo, não foram

observadas alterações nos diâmetros dos cardiomiócitos VE, apesar de se ter registado

uma diminuição da massa do conjunto constituído pelo VE e pelo septo interventricular e

aumento quer da taxa de apoptose quer da fibrose miocárdicas. No miocárdio VE,

observamos ainda sobre-expressão acentuada não só da ET-1, mas também de citocinas

inflamatórias, como a IL-6 e o TNF-α. Num trabalho anterior, demonstramos que a

actividade neuroendócrina sistémica poderia desencadear activação neuroendócrina VE,

nomeadamente de ET-1, e disfunção miocárdica intrínseca (Lourenço et al., 2006). Com

base nos resultados deste trabalho experimental propomos que a activação de citocinas no

miocárdio possa também dever-se à caquexia e à redução da pré-carga (unloading) VE. De

facto, descrevemos anteriormente num modelo experimental de caquexia associada ao

síndrome nefrótico diminuição de massa VE e sobre-expressão de TNF-α (Moreira-

Rodrigues et al., 2007). A expressão de citocinas inflamatórias, cuja acção miocárdica pode

ter sido parcialmente responsável pela disfunção e remodelagem (Feldman et al., 2000),

não aumentou apenas no VE, mas também no tecido adiposo visceral, e os níveis

circulantes destas encontram-se seguramente elevados, como confirmamos para o TNF-α.

No tecido adiposo observamos ainda maior expressão de adiponectina, uma adipocina que,

apesar de desempenhar funções cardiovasculares benéficas, é, paradoxalmente, um

marcador de mau prognóstico na caquexia cardíaca (Kistorp et al., 2005).

Quanto ao grupo injectado com MCT e alimentado com dieta hipercalórica, rica em

gordura animal e carbo-hidratos simples, apesar dos ratos não terem sido alimentados por

gavagem, e, portanto, terem ingerido calorias ad libitum, observamos um maior consumo

calórico, com preservação do peso corporal, e das massas magra e gorda. Este resultado

está de acordo com a elevada palatabilidade deste tipo de ração e com a maior composição

em calorias e substratos metabólicos energéticos por massa ingerida. Parte das melhorias

encontradas na evolução ponderal pode ter-se devido ao maior aporte energético e

calórico, visto que a manutenção do equilíbrio energético e metabólico é essencial no


54

doente crítico (Martindale et al., 2009). Curiosamente, apesar destas diferenças e da

redução no nível de HP, a hipertrofia VD não foi atenuada comparativamente com o regime

alimentar normal. De qualquer modo, a taxa de apoptose miocárdica VD e VE, a fibrose e a

comutação para a isoforma β das cadeias pesadas de miosina foram atenuadas. No grupo

injectado com MCT e alimentado com dieta hipercalórica observamos também melhor

sobrevida e melhoria do perfil hemodinâmico. Assim, os animais deste grupo,

comparativamente com os alimentados com dieta normal, não apresentaram dilatação VD,

e registaram melhorias significativas no DC e nos índices de função VD e VE.

Paralelamente, registamos uma atenuação da actividade inflamatória e neuroendócrina. Os

nossos resultados são originais e algo surpreendentes, face aos efeitos bem estabelecidos

deste tipo de regime alimentar como factor de risco cardiovascular. No entanto, podem ser

interpretados atendendo a várias linhas de evidência. Com efeito, apesar da suplementação

nutricional na caquexia ser limitada pela necessidade de restrição de fluídos e sal e de não

ser possível contornar a anorexia, saciedade precoce, redução da absorção de nutrientes e

o aumento do metabolismo que acompanham a IC (Azhar e Wei, 2006), pequenos estudos

têm relatado benefícios na manutenção do peso corporal e da função muscular após a

suplementação energética, principalmente por via nasoentérica, com um número reduzido

de casos de descompensação associada a um síndrome semelhante ao síndrome de

realimentação (Akner e Cederholm, 2001). Para além destes pequenos estudos clínicos,

diversos estudos observacionais de larga escala constituem evidência epidemiológica de

que a obesidade e a hipercolesterolémia são factores de bom prognóstico e melhoram a

sobrevida na IC (Curtis et al., 2005). Esta epidemiologia reversa dos factores de risco

cardiovascular, denominada por “paradoxo da obesidade”, tem sido atribuída não só à

possibilidade de que factores de risco a longo prazo possam não se comportar como tal

numa situação de rápida evolução e mau prognóstico, como a IC terminal (Kalantar-Zadeh

et al., 2004), mas também à hipótese lipoproteína-endotoxina. Segundo esta hipótese, as

lipoproteínas ligam-se e neutralizam os LPS circulantes, protegendo o organismo da

resposta inflamatória (Rauchhaus et al., 2000). A IC é uma patologia multissistémica que se

faz acompanhar cronicamente de activação inflamatória, esta activação parece ser devida

parcialmente à translocação bacteriana a nível intestinal. Os produtos bacterianos

circulantes serão activadores fundamentais da produção de citocinas (Anker e Coats,

1999). Esta hipótese é concordante com os resultados obtidos. Com efeito, no grupo de

ratos injectado com MCT e posteriormente alimentado com dieta normal, verificamos maior

actividade miocárdica do NF-κB, maior expressão de citocinas no miocárdio VE e no tecido

adiposo visceral, e níveis circulantes aumentados de TNF-α. Estas alterações foram


55

atenuadas pelo regime alimentar hipercalórico. As citocinas, como o TNF-α, para além de

contribuírem para mecanismos de progressão da IC como a remodelagem, apoptose e

disfunção do miocárdio (Feldman et al., 2000), também induzem insulino-resistência, sub-

nutrição energético-protéica, perda de massa muscular e caquexia (Sharma e Anker, 2002).

Estes efeitos dever-se-ão essencialmente à sua influência marcada no metabolismo celular

e na função do PPAR-α. A diminuição da expressão e da actividade do PPAR-α observada

no VE dos ratos injectados com MCT e alimentados com dieta normal, pode ser

parcialmente atribuída à maior expressão e actividade do TNF-α (Ye, 2008). Em larga

medida, a inflamação crónica parece ser o elo principal entre IC e caquexia (von Haehling

et al., 2007). Para além dos pontos anteriormente apontados, a progressão da IC também

se acompanha de adaptações metabólicas substanciais, que poderão desencadear ou

facilitar a remodelagem estrutural e funcional. O PPAR-α parece desempenhar um papel

fundamental neste contexto. É um dos principais reguladores da transcrição de genes que

codificam enzimas envolvidas na oxidação dos AG e no metabolismo da glicose (Gulick et

al., 1994). No miocárdio insuficiente são típicas alterações metabólico-energéticas como

uma conversão ineficiente de energia química em trabalho mecânico, uma diminuição do

conteúdo de grupos fosfato armazenadores de energia química, a disfunção mitocondrial e

uma perda da flexibilidade metabólica com maior dependência energética da glicólise

anaeróbia em detrimento da oxidação de AG (Ingwall, 2009). A inactivação do PPAR-α

durante a hipertrofia cardíaca pode, em parte, ser responsável pela inactivação da

transcrição das enzimas responsáveis pela oxidação dos AG e da isoforma 4 da PDK

(Barger et al., 2000). A PDK4 fosforila e inibe a PDH, a principal enzima reguladora da

capacidade oxidativa de carbo-hidratos, que converte o produto final da glicólise, o piruvato,

em acetil-CoA, possibilitando a sua metabolização no ciclo de Krebs (Stanley et al., 2005).

Admite-se que a preferência metabólica pelos carbo-hidratos possa ter um papel adaptativo

nas circunstâncias em que as necessidades energéticas do miocárdio estão aumentadas,

como é o caso da hipertrofia e da IC, porque a contractilidade miocárdica melhora quando o

miocárdio oxida preferencialmente glicose e lactato, em detrimento dos AG (Burkhoff et al.,

1991), ao passo que concentrações plasmáticas elevadas de AG livres diminuem a

eficiência mecânica (Hutter et al., 1984), provavelmente por desacoplamento da FO

(Schrauwen et al., 2003). Mais, o metabolismo da glicose tem uma maior razão entre a

produção de ATP e o consumo de O2, o que lhe confere maior eficiência energética

comparativamente com os AG (Stanley et al., 2005), e as enzimas glicolíticas

compartimentalizam-se nas imediações do retículo sarcoplasmático e do sarcolema (Pierce

e Philipson, 1985), sendo fundamentais à captação de Ca2+ pelo retículo endoplasmático


56

(Entman et al., 1977) e ao relaxamento do miocárdio durante situações de stress, como a

isquemia (Kusuoka e Marban, 1994). Contudo, o consumo preferencial de glicose pode não

ser completamente adaptativo, uma vez que por cada mole de AG é gerado mais ATP

(Barger et al., 2000; van Bilsen et al., 2009). De facto, nos doentes com IC, o trabalho e a

eficiência cardíaca são muito dependentes dos AG (Tuunanen et al., 2006). Nos animais

alimentados com dieta hipercalórica a conjugação de AG e carbo-hidratos poderá, deste

modo, ter contribuído para uma melhor preservação da função miocárdica

comparativamente com o grupo alimentado com dieta normal. Em consonância com as

observações anteriormente mencionadas, que relatam diminuição da actividade no PPAR-α

na progressão da IC (Barger et al., 2000), a expressão e actividade do PPAR-α diminuiu no

miocárdio VE dos animais do grupo injectado com MCT que manteve o regime alimentar

normal. O mesmo não se verificou, no entanto, no grupo alimentado com dieta

hipercalórica, o que poderá ser parcialmente explicado pela indução do PPAR-α pelos

lípidos oriundos da dieta (Gulick et al., 1994). Em concordância com os nossos resultados,

a activação de PPAR-α normalizou o metabolismo na hipertrofia e falência VD experimental

(Jucker et al., 2007). Apesar de não termos verificado alterações da expressão génica das

enzimas envolvidas na oxidação de AG e da isoforma 4 da PDK, devemos salvaguardar

que estas não são apenas reguladas a nível transcricional e que os seus níveis de

expressão nem sempre traduzem a actividade enzimática (Stanley et al., 2005). Contudo,

podemos pressupor que as alterações na expressão e actividade do PPAR-α induzidas

pela dieta hipercalórica tiveram muito provavelmente um efeito metabólico benéfico, uma

vez que dietas ricas em lípidos preveniram o declínio da actividade das enzimas

responsáveis pela oxidação dos AG e da actividade oxidativa mitocondrial, sem agravarem

a hipertrofia, quer num modelo experimental de sobrecarga de pressão VE (Chess et al.,

2009), quer em ratos espontaneamente hipertensos. Concomitantemente, a actividade de

transcrição do PPAR-α e a função mitocondrial e miocárdica foram preservadas (Iemitsu et

al., 2008). A disfunção mitocondrial, maior produção de ROS e a menor capacidade de

produção energética acompanham a progressão da disfunção do VD na HP induzida pela

MCT (Daicho et al., 2009; Redout et al., 2007). A atenuação da HP pela dieta hipercalórica

é aparentemente um resultado surpreendedor, no entanto, evidências recentes sugerem

que os agonistas do PPAR podem de facto atenuar a HP (Hansmann et al., 2007) e,

portanto, o elevado conteúdo lipídico da dieta hipercalórica ingerida pelos ratos injectados

com MCT poderá ter atenuado a HP simplesmente por indução do PPAR.

Concluindo, num modelo experimental de HP severa, com rápida progressão para IC e

caquexia, demonstramos que um regime alimentar hipercalórico, rico em gorduras


57

saturadas e carbo-hidratos simples, que normalmente é tido como um factor nutricional de

risco cardiovascular, atenuou a HP, melhorou a sobrevida e a função miocárdica,

possivelmente devido a alterações benéficas no metabolismo miocárdico e à atenuação

concomitante da actividade neuroendócrina e inflamatória, o que poderá ter-se devido, em

parte, à modificação da actividade de factores de transcrição. No entanto, devemos

salvaguardar que um período mais prolongado de alimentação hipercalórica poderia ter

induzido acumulação cardíaca de triglicerídeos e comprometido a função miocárdica (Akki e

Seymour, 2009).


58

Relevância Clínica e Perspectivas Futuras

Neste trabalho produzimos evidências experimentais e fisiopatológicas que apoiam os

achados epidemiológicos paradoxais de melhor prognóstico nos insuficientes cardíacos

mais obesos (“paradoxo da obesidade”).

Os resultados apresentados sugerem que pacientes com caquexia cardíaca e IC

descompensada poderão beneficiar de regimes alimentares hipercalóricos.

Para confirmar esta hipótese, será importante reproduzir estes achados em modelos

adicionais de caquexia cardíaca e, posteriormente, através de ensaios clínicos

randomizados, avaliar o efeito de regimes alimentares similares em pacientes com

caquexia cardíaca.

Se as hipóteses levantadas forem confirmadas, a atitude médica perante a nutrição dos

pacientes com IC terminal e caquexia poderá sofrer uma evolução importante, com

potencial melhoria dos cuidados de saúde prestados.


59

6. Anexo

Current Pathophysiological Concepts and

Management of Pulmonary Hypertension

(Novos Conceitos Fisiopatológicos e

Abordagem Terapêutica da Hipertensão Pulmonar)

Artigo de revisão submetido para peer review na revista International Journal of Cardiology, pelos

autores Lourenço AP, Fontoura D, Henriques-Coelho T, e Leite-Moreira AF, em Setembro de 2010.


60

Current Pathophysiological Concepts and Management of Pulmonary Hypertension

Authors

André P. Lourenço1, Dulce Fontoura1, Tiago Henriques-Coelho1, Adelino F. Leite-Moreira1

1Department of Physiology, Faculty of Medicine, University of Porto, Porto, Portugal

Word count: 4998 (abstract+manuscript+figure legends)

Address for correspondence:

Prof. Dr. Adelino F. Leite-Moreira

Departments of Physiology and Cardio-Thoracic Surgery

Faculty of Medicine, University Hospital São João

Alameda Professor Hernâni Monteiro

4200 - 319 Porto, PORTUGAL

Tel: +351225513644; Fax: +351225513646

Email: [email protected]


61

Abstract

Pulmonary hypertension (PH), increasingly recognized as a major health burden, remains

underdiagnosed due to unspecific symptoms and the need for right-heart catheterization.

Pulmonary artery hypertension (PAH) has been extensively investigated. Pathophysiological

knowledge derives mostly from experimental models. Paradoxically, common non-PAH PH

forms remain largely unexplored. Drugs targeting lung vascular tonus became available

during the last two decades, notwithstanding disease progresses in many patients. The aim

of this review is to summon recent advances in epidemiology, pathophysiology and

management with particular focus on associated myocardial and systemic compromise and

experimental therapeutic possibilities. PAH, currently viewed as a panvasculopathy, is due

to a crosstalk between endothelial and smooth muscle cells, inflammatory activation and

altered subcellular pathways. Cardiac cachexia and right ventricular compromise are

fundamental determinants of PH prognosis. Combined vasodilator therapy is already

mainstay for refractory cases, but drugs directed at these new pathophysiological avenues

may constitute a significant advance.


62

Abbreviations

5-HT, serotonin

5-HT2A, serotonin type 2A receptor

6MWT, 6-minute walk test

AC, adenylate cyclase

AS, atrial septostomy

BMP, bone morphogenetic protein

BMPR1, bone morphogenetic protein receptor 1

BMPR2, bone morphogenetic protein receptor 2

BNP, type B natriuretic peptide

CaL, L-type Ca2+

-channel

CC, cardiac cachexia

CCB, Ca2+

-channel blocker

CCR2, chemokine receptor 2

CCR5, chemokine receptor 5

CCT, chest computerized tomography

CDK, cyclin-dependent kinase

cGMP, cyclic guanosine monophosphate

CHD, congenital heart disease

CHLT, combined heart-lung transplantation

CO, cardiac output

CO-A/R, co-repressors or activators

COPD, chronic obstructive pulmonary disease

CTD, connective tissue disease

CTEPH, chronic thromboembolic pulmonary hypertension

CVC, central venous catheter

CX3CR1, chemokine receptor 1

CXCR4, α-chemokine receptor

DCA, dichloroacetate

DLCO, carbon monoxide diffusion

e-, electron

ECM, extracellular matrix

EF, ejection fraction

EGFR, epidermal growth factor receptor

EPC, endothelial progenitor cells

ERA, endothelin-1 receptor antagonists

ET-1, endothelin-1

ETA, endothelin-1 type A receptor

ETC, electron transport chain

FDA, Food and Drug Administration

fPAH, familial pulmonary arterial hypertension

GC, guanylate cyclase

Gq, protein Gq

HF, heart failure

HIF-1α, hypoxia-inducible factor-1α

HIV, human immunodeficiency virus

Id, inhibitor of DNA binding proteins

IL-6, Interleukin-6

IP1, prostaglandin receptor

IP3, inositol 3-phosphate

iPAH, idiopathic pulmonary arterial hypertension

iv, intravenous

Kv1.5, O2-sensitive K+-channels

LHD, left-heart disease

LV, left ventricular

LT, lung transplantation

MCP-1, monocyte chemotactic protein-1

MLC, myosin light-chain

MLCK (-P), myosin light-chain kinase, and respective phosphorylated

form

MMP, matrix metalloproteinases

mPAP, mean pulmonary artery pressure

NFAT, nuclear factor of activated T lymphocytes

NIH, National Institutes of Health

NO, nitric oxide

NRCT, non-randomized clinical trial

O2

.-, superoxide anion

PA, pulmonary arterial

PAP, pulmonary artery pressure

PAH, pulmonary arterial hypertension

PASMC, pulmonary artery smooth muscle cell

PEA, pulmonary endarterectomy

PCH, pulmonary capillary haemangiomatosis

PDE, phosphodiesterases

PDE5, type 5 phosphodiesterase

PDEi, phosphodiesterase inhibitors


63

PDGF, platelet derived growth factor

PDGFR, platelet derived growth factor receptor

PDH (-P), pyruvate dehydrogenase, and respective phosphorylated form

PDK, pyruvate dehydrogenase kinase

PGI2, prostacyclin

PH, pulmonary hypertension

PKA, protein-kinase A

PKG, protein-kinase G

PTE, pulmonary thromboembolism

PVOD, pulmonary veno-occlusive disease

PVR, pulmonary vascular resistance

QOL, quality of life

RANTES, regulated upon activation, normal T expressed and secreted

RCT, randomized clinical trials

RHC, right-heart catheterisation

ROS, reactive oxygen species

RV, right ventricular

RVAD, right ventricular assist device

sc, subcutaneous

SDF-1, stromal cell-derived factor-1

SLE, systemic lupus erythematosus

SOD, superoxyde dismutase

SPAP, systolic pulmonary artery pressure

SR, sarcoendoplasmic reticulum

TCW, time to clinical worsening

TGF-β, transforming growth factor -β

TGF-βR, transforming growth factor -β receptor

TP, ThromboxaneA2 receptor

TnC, tenascin C

TNF-α, tumor necrosis factor-α

Trp, transient receptor potential

TxA2, thromboxane A2

US, United States

VEGF, vascular endothelial growth factor

VEGR, vascular endothelial growth factor receptor

WHO, World Health Organization


64

Introduction

Pulmonary hypertension (PH), classically diagnosed by mean pulmonary arterial (PA)

pressure (mPAP) elevation above 25 mmHg at rest.1 In most cases, PH accompanies

cardio-respiratory conditions and does not involve the pulmonary vasculature. However,

more rarely it may present itself as pulmonary arterial hypertension (PAH), defined

additionally by normal left ventricular (LV) filling pressure.2 PAH is viewed as a

vasoproliferative disease with characteristic pathological abnormalities, such as arteriolar

plexiform lesions. Initial symptoms, mainly fatigue and dyspnea, are usually vague and

insidious, thus most cases are diagnosed when cardiac output (CO) is already low.3 Right

ventricular (RV) failure due to PH is an important cause of death4 whose complex

pathophysiological mechanisms are just beginning to be understood. The last two decades

have been fertile in experimental and clinical studies. Several new drugs have become

available.3 Nevertheless, the prognosis remains poor, and many patients require

transplantation.5 The present review aims to summarize much of what has recently been

published on the epidemiology, pathophysiology, diagnosis and management of PH.6,7

Aetiology and classification

Several conferences on PH have been fostered by the World Health Organization (WHO). A

classification was proposed in 1973 and then modified at Evian in 1988 to better reproduce

pathophysiology and clinical presentation. At Venice in 2003, the term primary PH was

substituted for idiopathic PAH (iPAH) and pulmonary veno-occlusive disease (PVOD) and

pulmonary capillary haemangiomatosis (PCH) were grouped under a single PAH

subcategory. In 2008, the 4th World Symposium held in Dana Point (Table 1) endorsed the

expression “non-PAH PH” to address categories other than PAH.


65

Table 1. New classification for pulmonary hypertension (PH) from the 4th World Symposium on

PH (Dana Point, 2008).

Pulmonary arterial

hypertension (PAH)

Non-PAH pulmonary hypertension (PH)

Well defined cause Unclear or

multifactorial

PAH (1)

Idiopathic

Hereditary

Drug/toxin induced

Disease associated:

CTD

HIV infection

Portal hypertension

Systemic-pulmonary

shunts

Schistosomiasis

Chronic haemolytic

anaemia

Subclass of PAH (1’)

PVOD and PCH

Left-heart disease (2)

Systolic dysfunction

Diastolic dysfunction

Valvular Disease

Lung diseases /hypoxia (3)

COPD

Interstitial lung disease

Sleep-disordered breathing

Chronic exposure to high

altitude

Broncho pulmonary dysplasia

Developmental abnormalities

CTEPH (4)

Unclear/multifactorial

mechanisms (5)

Haematologic disorders

Myeloproliferative disorders,

etc.

Systemic disorders

Vasculitis, sarcoidosis,

neurofibromatosis, etc.

Metabolic disorders

Glycogen storage disease,

thyroid disorders, etc.

Congenital heart disease (Other than systemic-pulmonary shunt)

Other

Fibrosing mediastinitis,

Chronic renal failure on

dialysis, etc.

Classes are presented between parenthesis. CTD, connective tissue disease; HIV, human

immunodeficiency virus; PVOD, pulmonary veno-occlusive disease; PCH, pulmonary capillary angiomatosis;

COPD, chronic obstructive pulmonary disease; CTEPH, chronic thromboembolic PH.

Diagnosis

During the 4th Conference exercise values were excluded as a criteria for diagnosis since

the increase in mPAP during exercise frequently exceeds 30 mmHg among the elderly.8

Additionally, non-invasive echocardiographic criteria of systolic tricuspid regurgitant velocity

were contemplated (Table 2).9 Nevertheless, transpulmonary flow and pulmonary venous

pressure are not reliably measured by echocardiography thus right-heart catheterisation

(RHC) remains the gold standard while echocardiography is usually a screening exam. RHC

is mandatory for the selection of patients that may benefit from Ca2+-channel blockers

(CCB), the positive responders during vasoreactivity test, those in whom mPAP drops more

than 10 mmHg or to values bellow 40 mmHg with normal or increased CO, after


66

administration of a short-acting vasodilator, such as nitric oxide (NO),10 epoprostenol or

adenosine.7

Table 2. New diagnostic criteria for pulmonary hypertension (PH) from the 4th World Symposium

on PH (Dana Point, 2008)

Method Normal Borderline Clear

mPAP (mmHg) < 21 21 - 25 > 25

systolic tricuspid regurgitation (m.s-1) < 2.5 2.5 - 2.8 > 2.8

mPAP, mean pulmonary artery pressure.

Epidemiology

The incidence and prevalence of PAH were estimated to be 2.4 - 7.6 cases/million/year and

15 - 26 cases/million, respectively, in large population studies.11, 12 Worldwide prevalence is

hard to appraise, but it is surely underdiagnosed13 and its onus is likely greater than

recognized, given the newly revealed associations with haemodialysis,14 the metabolic

syndrome,15 and developing world diseases, such as human immunodeficiency virus (HIV)

infection, schistosomiasis, and sickle cell disease.16 Apart from iPAH no precise estimates

of incidence or prevalence are available. Nevertheless, non-PAH PH is increasingly

recognized as a major health burden. Not only up to 60% of patients with severe LV systolic

dysfunction but also 70% of those with heart failure (HF) and normal ejection fraction (EF)17

develop PH.18, 19 Moreover, PH afflicts 70% of patients with rheumatic heart disease.20 Many

patients develop chronic thromboembolic PH (CTEPH) after pulmonary thromboembolism

(PTE)20 or PH during the progression of chronic obstructive pulmonary disease (COPD).

Prevalence ranges from 35 to 90% according to stage,21, 22 mostly limited to systolic PAP

(SPAP) values of 25 - 35 mmHg, though severe PH is not uncommon in advanced COPD.23

Some patients develop disproportionate PH and warrant particular attention,21 but even

modest PH has a strong impact on quality of life (QOL) and survival.22 Right HF, its most

severe complication, is responsible for 10 - 30% of admissions due to decompensated HF.24

Presently COPD is already responsible for 84% of cor pulmonale cases and, due to

smoking, will be the 3rd cause of death by 2020.23


67

Clinical Presentation and Workup

Severe disease may present with chest pain, palpitation, oedema, ascites, and syncope9 but

earlier treatment, at reversible stages, is fundamental. Diagnosis is challenging, a delay of 2

to 3 years is common and a high suspicion level is needed.13 The clinician may find RV

hypertrophy on the electrocardiogram and hilar PA prominence on the chest X-ray.

Echocardiography, generally undertaken after a suspicion, may show increased SPAP,

estimated by the velocity of tricuspid regurgitation jet, and/or increased RV outflow tract

acceleration time. It is fundamental to evaluate valve or primary myocardial disease, as well

as the degree of RV hypertrophy and dysfunction.9 Magnetic resonance imaging techniques

have also been used.25 Regarding differential diagnosis, patients with suspicion of PTE

should undergo the highly sensitive ventilation-perfusion (V-Q) scan. Nevertheless, final

staging and surgical indication rely on chest computerized tomography (CCT) and/or

angiography with RHC. High-resolution CCT is useful to assess PVOD or PCH and to

diagnose interstitial lung or connective tissue disease (CTD).7, 9 Finally antinuclear

antibodies, autoimmune disease markers, HIV and viral hepatitis screening, coagulation

disorder markers (eg, protein S and C, lupus anticoagulants, von Willebrand factor) and type

B natriuretic peptide (BNP) may be carried out for differential diagnosis.7, 9 Functional

respiratory evaluation relies on spirometry and carbon monoxide diffusion (DLCO).

Spirometry may be markedly altered in lung disease, whereas minor changes are found in

iPAH. DLCO impairment correlates with lung vascular surface area and PAH severity.26 The

6-minute walk test (6MWT), a common clinical trial end-point, evaluates moderate to severe

heart or lung disease (Table 3). It correlates well with CO, PVR, O2 consumption, QOL, and

predicts mortality in PAH.27 Nevertheless, since it depends on many individual variables, it is

not a reliable marker of disease progression.7 Additionally, its validity has been questioned

for CTD.28


68

Table 3. The 6-minute walk test (6MWT) in pulmonary hypertension (PH).

Features

Submaximal exercise test

Correlates well with activities of daily living (useful for moderately severe functional impairment)

Non-specific (evaluates the response of all systems)

Well tolerated (nevertheless, appropriate response to an emergency should be available)

Measurements

Dyspnoea and fatigue self-rating at the beginning and end (according to the Borg scale, see legend)

Distance walked

Demographic and anthropometric determinants

Gender, age and ethnicity

Height and weight

Advantages

Practical and inexpensive to perform (no equipment or specially trained technicians needed)

Reproducible (estimated coefficient of variability of 8%)

Ongoing monitoring of cardiopulmonary disease progression

Evaluation of response to therapy

Limitations

Merely a rough estimate of the general functional status (does not discard specific assessment tools)

Lack of validation for connective tissue disease associated PAH (musculoskeletal involvement)

“Ceiling effect” for patients with better baseline capacity

Biases: disturbed cognition, motivational factors, test repetition, musculoskeletal limitations, etc.

A comprehensive perspective on the 6MWT including indications, contraindications, safety precautions,

technical aspects, biases, can be found in the guidelines from the American Thoracic Society.119

The 6MWT

measures the distance that a patient can walk on a flat surface in a period of 6 minutes, patients are allowed

to stop and rest. The normal walked distance for healthy 60 year-old men and women of average constitution

is approximately 630 and 550 m, respectively,120 whereas idiopathic PAH patients on World Health

Organization functional class IV usually walk less than 200 m.27 A clinically important improvement in walking

distance for the PAH patient is generally 44 - 76 m.89

Borg scale: (0) nothing at all, (0.5) just noticeable, (1) very slight, (2) slight,(3) moderate, (4) somewhat

severe, (5) severe, (7) very severe, and, finally, (10) maximal.121


69

Pathophysiology

Although no animal model completely recapitulates human PAH, combining multiple insults,

according to the multiple-hit hypothesis, yielded severe phenotypes that closely mimic it.29

Pathophysiological knowledge, derived mostly from these animal studies, once viewed PH

as an imbalance between pulmonary vasoconstrictors and vasodilators.30 While prostacyclin

(PGI2) and NO normally govern vascular tone, endothelin-1 (ET-1), thromboxane A2 (TxA2)

and serotonin (5-HT) take over in PH. Not surprisingly, lung vasodilators have been the

mainstay of therapy (Figure 1).3 Nevertheless, recent research showed this view to be highly

incomplete.

PAH as panvasculopathy

PAH is currently viewed as a panvasculopathy, accompanied by histological features as

intimal hyperplasia, medial hypertrophy, and arteriolar occlusion by thrombosis, infiltration

by inflammatory cells or angioproliferative plexiform lesions (Figure 1).7 It is hypothesized

that apoptosis generates apoptosis-resistant endothelial cell phenotypes that cross-talk with

PA smooth muscle cell (PASMC) through growth factors such as transforming growth factor-

β (TGF-β), that are involved in endothelial cell and fibroblast transdifferentiation and PASMC

proliferation.31 Metalloproteinase activation leads to the disruption of the basement

membrane enabling inflammatory cell recruitment and further generation of mitogenic

peptides.32 The main mechanisms involved in inflammation, endothelial progenitor cell

(EPC) recruitment, growth factor activity and extracellular matrix remodeling are

summarized in figure 1. PAH shares a mitochondrial-metabolic abnormality with cancer, the

“Warburg phenotype”, a shift from oxidative phosphorylation to glycolysis (despite adequate

O2 supply) that enhances proliferation and prevents apoptosis (Figure 2). Hyperpolarization

of the mitochondrial membrane, reduced production of reactive oxygen species (ROS),

normoxic-activation of hypoxia inducible factor-1α, overexpression of pyruvate

dehydrogenase kinase (PDK) and decreased expression of O2-sensitive K+ channels

(Kv1.5) have been postulated to underlie changes in mitochondrial O2 sensing.33 PDK

activation suppresses aerobic glucose metabolism and decreased Kv1.5 conductance

depolarizes the membrane. Dichloroacetate (DCA), a mitochondrial PDK inhibitor and Kv1.5

channel opener, improved PAH33 both by activating pyruvate dehydrogenase (PDH) and

aerobic metabolism and by restoring membrane potential and ROS production.34


70

Figure 1. Pulmonary artery smooth muscle cell (PASMC) constriction and proliferation mechanisms.

The major sites of action of lung vasodilator drug classes are shown

in the upper panel, namely Ca2+

-channel blockers (CCB),

endothelin-1 (ET-1) receptor antagonists (ERA), phosphodiesterase

inhibitors (PDEi) and prostanoids. Myosin light-chain (MLC) kinase

(MLCK) is inactivated upon phosphorylation (MLCK-P). Other

mechanisms are presented in the lower panel. Several cytokines,

beyond interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α),

mostly produced by fibroblasts, such as stromal cell-derived factor-1

(SDF-1), monocyte chemotactic protein-1 (MCP-1), fractalkine,

RANTES (regulated upon activation, normal T expressed and

secreted), and vascular endothelial growth factor (VEGF) are

upregulated and induce PASMC proliferation and monocyte

recruitment, while monocytes upregulate the α-chemokine receptor

(CXCR4) and chemokine receptors 1, 2 and 5 (CX3CR1, CCR2

and CCR5, respectively).40 Elastase, early activated in PH, triggers

growth factors release from the extracellular matrix (ECM) and

induces tenascin C (TnC) through the activation of matrix

metalloproteinases (MMP). When TnC binds surface integrins on

PASMCs cell-survival signals are generated and growth factor

receptors are further activated. Serotonin (5-HT) induces

proliferation of PASMCs by stimulation of S100A4/Mts1, a S100

Ca2+-binding protein family member with metastasis-inducing

ability.3 Endothelial progenitor cells (EPC) may participate in vessel

repair, but on the other hand also take part in plexiform lesions.6

Other abbreviations: TxA2, thromboxane A2; Gq, protein Gq; IP3,

inositol 3-phosphate; SR, sarcoendoplasmic reticulum; ETA, ET-1

type A receptor; TP, TxA2 receptor; 5-HT2A, 5-HT type 2A receptor;

CaL, Type L Ca2+-channel; GC, guanylate cyclase; AC, adenylate

cyclase; IP1, prostacyclin receptor; PDE5, phosphodiesterase type

5; PKG, protein-kinase G; PKA, protein-kinase A; VEGFR, VEGF

receptor; PDGF, platelet derived growth factor; TGF-β, transforming

growth factor-β; PDGFR, PDGF receptor; TGFβR, TGF-β receptor;

EGFR, epidermal growth factor receptor.


71

Figure 2. Reactive oxygen species (ROS), disturbed O2 sensing, and mitochondrial dysfunction

in pulmonary arterial hypertension (PAH).

During oxidative phosphorylation, electrons (e-) are conveyed by the electron transport chain (ETC) from donors

(NADH and FAH) to O2, but minor side reactions also generate by-products, as superoxide anion (O

2

.-) that must be

detoxified to H2O2 by superoxyde dismutase (SOD).133

Under normoxia H2O2 constitutively opens plasma membrane O2-

sensitive K+-channels (Kv1.5) and inhibits hypoxia-inducible factor-1α (HIF-1α) activity, whereas during hypoxic

vasoconstriction, ROS and H2O2 production are decreased, Kv1.5 channels close, the plasma membrane depolarizes, Ca2+

enters the cell and myocytes contract. In PAH, mitochondrial abnormalities, most notably pyruvate dehydrogenase kinase

(PDK) activation, shift metabolism toward anaerobic glycolysis and impair the ETC. Reduced ROS production, nuclear

translocation of HIF-1α, and decreased expression of Kv1.5 ultimately lead to sustained membrane depolarization, L-type

Ca2+

-channel (CaL) activation and hypertrophy by Ca2+

-calcineurin-dependent activation of the nuclear factor of activated T

lymphocytes (NFAT).6 PDH, pyruvate dehydrogenase, and respective phosphorylated form (-P); ET-1, endothelin-1; VEGF,

vascular endothelial growth factor.


72

Genetics of PAH

Mutations in bone morphogenetic protein (BMP) receptor-2 (BMPR2), a constitutively active

receptor responsive to TGF-β superfamily (including BMP), are seen in more than 80% of

familial PAH (fPAH) cases, leading to loss of smad signaling (Figure 3) and therefore to

increased proliferation and decreased differentiation of PASMC 35, 36. Still, penetrance is low

and the mutation is seen only in 10 to 20% of non-fPAH 37. Other genetic mechanisms

predispose to PAH, namely single-nucleotide polymorphisms of Kv1.5,18 transient receptor

potential (trp) channels,13 and serotonin transporters.38 Trp channels regulate contractility

and cell proliferation by intracellular Ca2+.39 Elevated 5-HT levels and 5-HT transport have

been implicated in PAH pathogenesis.38

Figure 3. Growth-promoting pathways in bone morphogenetic protein (BMP) receptor type 2 (BMPR-2)

mutations.

Bone morphogenetic protein (BMP) receptor type 1 (BMPR-1)

and -2 dimerize upon activation by BMP and initiate a cytosolic

receptor-activated Smad protein-signaling cascade. Smads

(homology with drosophila’s mothers against decapentaplegic –

MAD - and Caenorhabditis elegans’ small phenotype - sma -

proteins) ultimately complex with common partner Smad4 and

translocate to the nucleus. The weak Smad-DNA interaction

requires co-repressors or activators (Co-A/R).134 Signal

disrupting mutations in BMPRII can be found in the ligand-

binding domain, in the kinase domain or in the cytoplasmic tail.

Supression of Smad signalling partly underlies the hypertrophic

and proliferative phenotype of pulmonary artery smooth muscle

cells (PASMC).135 In normal PASMC, BMP stimulates

transcriptional activation of cyclin-dependent kinase (CDK)

inhibitors and repression of c-myc. CDK inhibitor prevents

progression in cell cycle, while c-myc encodes a transcriptional

activator responsible for growth and proliferation.136 Inhibitor of

DNA (Id) binding proteins, a family deprived of DNA-binding

domain that acts by inhibition of transcription factors, are also

major targets. Failure to induce Id genes makes PASMC

unresponsive to the growth suppressive effects of BMPs.137

Prostanoids, by cyclic adenosine monophosphate and a direct

effect on the Id promoter, drive the expression of Id proteins.134


73

Inflammation

The inflammatory state of the vessel wall has recently gained interestas primary event,

rather than mere consequence of the disease.40 Autoantibodies and infiltration by

inflammatory cells are common in PAH associated with CTD but are also seen in iPAH.40

Increased levels of cytokines and their receptors have been demonstrated, particularly in

iPAH patients,41 who also present heightened expression of inflammatory cell-associated

nuclear factor of activated T lymphocytes (NFAT).42 Cytokines involved in the pathogenesis

of chronic inflammatory diseases and cancer, such as tumor necrosis factor-α (TNF-α) and

IL-6, may play a role in PAH vasculopathy.43 Our group has tested an anti-inflammatory

approach in experimental models of PH with variable success44, 45. Inflammatory activation

may also underlie systemic manifestations, for instance cardiac cachexia (CC). CC is

characterized not only by neuroendocrine and inflammatory activation but also by

suppressed appetite and nutritional derangements46 and poses a significant prognostic

burden on HF patients.47 CC accompanies the progression of PH, indeed, patients with

severe PH have exaggerated and early post-prandial satiety hormone response.48

The myocardium in PH

The RV effectively serves as a thin, compliant reservoir for blood returning to the LV whose

primary function of is to deliver deoxygenated blood to the lungs, while maintaining low-

pressure perfusion.49 It is thus best suited for volume work and unable to suddenly generate

mPAP > 40mmHg. Sudden afterload decreases stroke volume and dilates the RV50,

whereas progressive overload allows gradual hypertrophy, remodeling and substantial

increases in mPAP. Curiously, although RV response partly determines the outcome,25, 51

despite the fact that the RV can be targeted therapeutically in PH,52 and even though RV

remodeling is potentially reversible, as seen after lung transplantation (LT),53 little is known

about the mechanisms underlying RV dysfunction.49 Many have shown neuroendocrine

activation can contribute to RV hypertrophy,54, 55 fibrosis and apoptosis, as well as to

oxidative stress, and activation of inflammatory cytokines and growth factors.49, 56 A state of

myocardial hibernation has been proposed based on systolic flow impediment to coronary

arteries which is proportional to RV pressure and mass.57 In contrast to the normal flexible

metabolism, in RV hypertrophy the myocardium relies solely on anaerobic glucose

metabolism partly due to PDK activation58 and possibly impaired mitochondrial energy-

producing ability.59 Moreover, changes in cardiomyocyte redox state can underlie


74

electrophysiological instability and remodeling, by mechanisms similar to those already

described for pulmonary vessels.60 Experimental findings and clinical observations suggest

that elevated mPAP cannot be the single driver for RV failure,56 therefore, targeting the RV

may be a promising approach.6 As for the LV myocardium, echocardiography shows

compromised LV function in various aetiologies of PH,61 mainly due to ventricular

interdependence and impaired filling.62 Nevertheless, myocardial abnormalities partly

underlie LV dysfunction. Indeed, despite immediate restoration of LV geometry and RV

function, LV filling is only normalized 1 year after single-LT in severe PH,63 and combined

heart-lung transplantation (CHLT) is favored if LV function is impaired because the LV may

not recover after LT alone.64 We have confirmed intrinsic LV myocardial dysfunction and

neuroendocrine activation experimentally.55, 65

Pathophysiology of non-PAH PH

Contrarily to PAH, and paradoxically, few data are available on the pathophysiology of the

far more common non-PAH PH. Regarding chronic pulmonary disease, several mechanisms

are potentially responsible. Hypoxia, such as is found at high altitude, is known to induce

PH, but low arterial O2 is not an independent predictor of mPAP, therefore after the Evian

Conference COPD-associated PH was no longer classified as ‘associated with

hypoxemia’.26 Pulmonary vessels in COPD consistently develop intimal fibro-elastic

thickening and overall muscularisation but this does also not provide a consistent

explanation.66 Endothelial dysfunction and inflammation, are currently viewed as the key to

vascular remodeling.67 Findings strongly suggest an involvement of vasoactive mediators

and cytokines.66 Plasma IL-6 correlates with mPAP and certain IL-6 genotypes are

associated with PH development in COPD.68 Symptomatic CTEPH affects 3.8% of patients

within 2 years of initial PTE accompanied by variable degrees of PAH-like vasculopathy.69

As for left-heart disease (LHD), increased filling pressures are transmitted to the pulmonary

circulation and generate, initially, pulmonary venous hypertension, but, later on, also PVR

increase, which leads to endothelial dysfunction and disturbances of 5-HT, TxA2 and

angiotensin-II production that further aggravate PH.70


75

Prognosis

Although PAH has been most extensively studied, its rarity, diverse etiology and changing

therapeutics preclude an estimation of yearly mortality rates. An early registry followed 194

patients with iPAH from 1981 to 1985 and estimated a median survival of 2.8 years, with 1-,

3-, and 5-year survival rates of 68, 48, and 34%, respectively.71 Present-day registries,

however, reveal a better prognosis, with 1 year survival ranging 83 to 88% and 3 year

survival 58 to 72%.72 A risk-prediction equation could be derived from multivariate analysis,

including gender, 6MWT, and CO at diagnosis as covariates.73 Four variables were

associated with increased 1-year survival: WHO functional class I, 6MWT ≥ 440 m, BNP <

50 pg/mL, and DLCO ≥ 80% of predicted.73 The progression in non-PAH PH is generally

slower and the overall prognosis is better. Still, there is a substantial impact on QOL and

survival.22, 74 The level of PAP is a good indicator of prognosis in COPD and a 50% 5-year

survival rate has been reported with PH.75 Regarding CTEPH, survival changed

dramatically. Before the advent of pulmonary endarterectomy (PEA) patients who had

mPAP higher than 30 mmHg steadily progressed to PH and 2 year-survival was lower than

20% after it reached 50 mmHg.76 Currently, in experienced centers and carefully selected

patients, PEA provides remarkable haemodynamic and clinical improvement with low

procedural mortality rate.5 In severe HF, the EF of the RV is the most important determinant

of short-term prognosis among hemodynamic variables.77 Although increased mPAP is

frequently coupled with reduced RV function, exceptions must be taken in account during

prognostic stratification.19

Therapeutics

Treatment of PAH has evolved considerably over the past decade, many treatment

algorithms have been proposed, mainly based on studies conducted in patients with iPAH

and PAH associated with CTDs. In Table 4 we summarize the major therapeutic studies on

PAH.


Table 4. Summary of clinical therapeutic studies on pulmonary arterial hypertension (PAH).

Class Drug Year Author Study Type Sample Patients Follow-up Positive outcomes

Epoprostenol 1996 Barst83

RCT (not blind) iPAH (WHO III-IV) 81 12w Haemodynamics, QOL, WHO class, survival

2002 Sitbon122

NRCT iPAH (WHO III-IV) 178 3m Haemodynamics, 6MWT, WHO class, survival

(Historical controls)

2002 McLaughlin84

NRCT iPAH (WHO III-IV) 162 Up to 3y Haemodynamics, exercise tolerance, WHO

class, survival (NIH prediction)

2003 Kuhn123

Observational iPAH and APAH (WHO

III-IV) 91 2.4y Survival (NIH prediction)

Treprostinil (sc) 2002 Simmoneau85

RCT iPAH, CTD and CHD

(WHO II-IV) 470 12w Haemodynamics, 6MWT, clinical evaluation

Iloprost (inh) 2002 Olschewski86

(AI

R) RCT iPAH, CTD and CTEPH

(WHO III-IV) 203 12w Haemodynamics, 6MWT, QOL, WHO class

2005 Opitz124

NRCT iPAH (WHO II-IV) 76 18m Haemodynamic, exercise tolerance

Beraprost 2002 Galiè125

(ALPHA

BET) RCT iPAH, CTD, CHD, portal

hypertension and HIV (WHO II-III)

130 12w Exercise tolerance, 6MWT, clinical evaluation

2003 Barst126

RCT iPAH, CTD and CHD (WHO II-III)

116 1y Exercise tolerance, 6 MWT, TCW,

ERA Bosentan 2002 Rubin89

(BREAT

HE1) RCT iPAH, CTD or SLE (WHO

III-IV) 213 16 -28w Exercise tolerance, 6MWT, WHO class, TCW;

2005 McLaughlin127

RCT (not blind) iPAH (WHO III-IV) 169 3y Survival (NIH prediction)

2005 Sitbon91

NRCT (vs epoprostenol records)

iPAH (WHO III) 485 (139 vs 346)

Up to 2y Similar survival with first line Bosentan

2006 Galiè128

(BREATHE5)

RCT CHD (WHO III) 54 16w Haemodynamics, 6 MWT

2008 Galiè92

(EARLY) RCT iPAH, CTD, CHD, HIV

(WHO II) 185 6m Haemodynamics, NT-pro-BNP and TCW

2008 Jais108

(BENEFIT) RCT CTEPH (WHO II-IV) 157 16w Haemodynamics

Ambrisentan 2005 Galiè129

RCT (dose ranging) iPAH, CTD, HIV and

anorexigen (WHO II-III)

64 12+12w (not-

blind) Haemodynamics, 6 MWT, clinical evaluation

2008 Galiè130

(ARIES

1&2) RCT iPAH, CTD, HIV and

anorexigen

202+192 (parts 1&2)

12w 6MWT, WHO class, QOL, TCW, NT-pro-BNP


2009 Oudiz94

(ARIES

1,2&E) RCT iPAH, CTD, HIV and

anorexigen

383 2y 6MWT, TCW and survival (combined

outcome)

Sitaxsentan 2004 Barst131

(STRIDE

1) RCT iPAH, CTD and CHD

(WHO II-IV) 178 12w Haemodynamics, 6MWT, WHO class

2006 Barst132

(STRIDE

2) RCT iPAH, CTD and CHD

(WHO II-IV) 245 18w 6MWT, WHO class

PDEi Sildenafil 2005 Galiè96

(SUPER1

) RCT iPAH, CTD and CHD

(WHO II-IV) 278 12w Haemodynamics, 6MWT, WHO class

Combined Bosentan +

Iloprost (inh)

2006 McLaughlin99

(S

TEP) RCT iPAH, APAH (WHO III) 67 12w Haemodynamics, WHO class, TCW

Epoprostenol +

sildenafil

2008 Simmoneau100

(

PACES) RCT iPAH and CTD 267 16w Haemodynamics, exercise tolerance, QOL,

TCW

The most relevant clinical studies on PAH therapeutics are summarized according to drug class, drug type and publication date. Study acronyms are presented when

applicable. Studies enrolling less than 50 patients as well as studies involving specific PAH groups, namely HIV-related and portal hypertension-related were excluded. RCT,

randomized controlled clinical trials; iPAH, idiopathic pulmonary arterial hypertension; WHO, world health organization; QOL, quality of life, NRCT, non-randomized controlled

clinical trials; 6MWT, 6 minute walk test; NIH, national institute of health; APAH, disease associated pulmonary arterial hypertension; CTD, connective tissue disease APAH;

CHD, congenital heart disease APAH; CTEPH, chronic thromboembolic pulmonary hypertension; HIV, human immunodeficiency virus APAH; TCW, time to clinical worsening;

SLE, systemic lupus erythematosus APAH; NT-pro-BNP, N terminal fragment of pro-type B natriuretic peptide. Study acronyms stand for: AIR, Aerosolized Iloprost

Randomized; ALPHABET, Arterial Pulmonary Hypertension and Beraprost European Study Group; BREATHE, Bosentan Randomized trial of Endothelin Antagonist THErapy

Study Group; EARLY, Endothelin Antagonist tRial in miLdlY symptomatic PAH patients; BENEFiT, Bosentan Effects in iNopErable Forms of chronic Thromboembolic

pulmonary hypertension; ARIES, Ambrisentan in pulmonary hypertension, randomized, Double Blinded, Placebo controlled, Multicenter, efficacy studies); STRIDE,

Sitaxsentan To Relieve ImpaireD Exercise; SUPER, Sildenafil Use in Pulmonary Arterial Hypertension Study Group; STEP, Safety and pilot efficacy Trial in combination with

bosentan for Evaluation in Pulmonary arterial hypertension; PACES, Pulmonary Arterial hypertension Combination study of Epoprostenol and Sildenafil.


78

Several general measures can be recommended. Regarding exercise practice, patients

should practice low level aerobic exercise, such as walking, whereas strenuous efforts

should be avoided. Some patients may not tolerate high altitudes, for instance during

airplane flights, and therefore require O2. Dietary sodium restriction is advised (≤ 2.4 g.d-1)

particularly in RV failure. Immunization against common respiratory pathogens is

recommended.7 Pregnancy should be avoided or early terminated due to the high mortality

rate.78

Despite the lack of randomized controlled trials (RCT), the initial therapeutic, and largely

supportive, approaches to the treatment of PAH were anticoagulation, diuretics, O2 therapy

and digoxin. Observational studies suggested improved survival after anticoagulation in

patients with iPAH, therefore most experts recommend anticoagulation (titrated to

international normalized ratio of 1.5 - 2.5). This is only advised for severe non-iPAH.7

Diuretics are used to manage HF symptoms. O2 therapy in hypoxemia is employed strictly to

avoid vasoconstriction. Based on a short-term effect study, digoxin may be used in patients

with low CO particularly with supraventricular arrhythmias.79 During the last two decades

substantial RCT and pharmacological research have yielded several new and more effective

alternatives to treat PH. The main pharmacological classes will be briefly presented. Most of

the studies are small scaled and short-termed, not suitable for survival analysis, but a recent

meta-analysis found an overall benefit in mortality.80 Nevertheless, most therapies reduce

mPAP by only 10 - 20%, with the exception of strong responders to CCB. Despite all the

advancement, many patients still remain symptomatic, with a suboptimal QOL and warrant

combined therapy or even invasive or surgical procedures.

Ca2+ channel blockers

Rich et al.81 have shown a marked improvement in survival rates, for patients with iPAH and

a positive vasoreactivity test, after long-term high-dose CCB therapy. Long acting nifedipine,

diltiazem, or amlodipine are more commonly used. If there is no recovery to functional

classes I or II patients are deemed as non-responders and should discontinue CCB. True

responders are rare in non-iPAH.82 Indiscriminate use is not recommended, due to systemic

vasodilation and negative inotropic effects.9


79

Prostanoids

There are presently several commercially available prostanoid formulations. Intravenous (iv)

epoprostenol was the first shown to improve functional class, hemodynamics and survival in

a 12 week follow-up period in patients with iPAH of classes III and IV.83 These beneficial

effects were reproduced in long-term observational comparisons with historical controls.84

Moreover, epoprostenol was also evaluated in CTD associated PAH and other forms of non

iPAH with favourable outcomes. Presently, because of the complex administration and

cumbersome follow-up, epoprostenol use is mainly confined to highly experienced centers.

Patients must keep a central venous catheter (CVC) and handle drug preparation and

infusion. Dosing must be carefully titrated. Most patients do well with an initial in-hospital

dose of 2 ng/kg/min and a dose range between 25 and 40 ng.kg-1.min-1. Unfortunately,

substantial side-effects have been reported, namely flushing, headache, and sudden death

after abrupt discontinuation, as well as risk of infection related to CVC.7 Treprostinil, a longer

half-life prostanoid, amenable to administration by subcutaneous (sc) route, circumventing

the need for CVC, showed minor beneficial effects in patients with functional classes II - IV

of idiopathic, CTD and (CHD) associated PAH.85 The United States (US) Food and Drug

Administration (FDA) approved it for functional classes II - IV also by iv route, when the sc

route is not tolerated due to pain or erythema. On another attempt to facilitate

administration, iloprost was developed for inhalation by aerosol device. After a 12-weeks

administration, iloprost improved the 6MWT and functional class in a multicenter RCT

enrolling patients with PAH of different aetiologies.86

Endothelin receptor antagonists

We have reviewed the role of ET-1 and its antagonists (ERA) in cardiovascular

pathophysiology.87 Briefly, after a small magnitude RCT had shown improvement in the

6MWT, mPAP and CO with the non-selective ERA bosentan,88 a larger scale study

conducted in patients with idiopathic or CTD associated PAH, reproduced these findings

and reported improvement in time to clinical worsening (TCW), a secondary endpoint

defined as a composite of mortality, LT, hospitalization, discontinuation due to lack of

recovery or need for epoprostenol or atrial septostomy (AS).89 As an important side-effect,

bosentan dose-dependently altered hepatic function. Anemia can also occur and the US

FDA therefore recommends liver function test and haematocrit surveillance.7 Long-term


80

evaluation as a first-line drug in functional class III patients also revealed good results,

although many patients demanded prostanoids.90 In fact, improved survival was only

demonstrated by comparison with historical data from epoprostenol treated iPAH WHO

class III patients, and unfortunately the two cohorts were not comparable.91 By now,

bosentan has also been tested in CHD, HIV-associated PAH and CTEPH with favourable

results. Moreover, it has been successfully used in a large sample of mildly symptomatic,

class II, multiple cause-PAH patients improving haemodynamics and TCW.92 Sixtasentan a

selective ETA ERA initially raised concerns due to fatal hepatitis but was later shown to have

comparable effects to bosentan in iPAH and PAH associated with CTD or CHD, with a

similar side-effect profile, with lower doses 93. It is currently approved in the European Union

but not in the US. Ambrisentan, another selective ETA ERA, also improved the 6MWT and

TCW, which was reproducible in long-term studies.94 It is approved by the FDA since 2007

for PAH patients in functional classes II and III.

Phosphodiesterase inhibitors

Phosphodiesterases (PDE) degrade cyclic guanosine monophosphate (cGMP) therefore

PDE inhibitors (PDEi) potentiate the effects of cGMP generated by NO activation of

guanylate cyclase. NO and NO donors have been extensively used as a rescue therapy to

mitigate mPAP in the perioperative period and in the critically ill patient, particularly in

children.95 Sildenafill, the first used PDEi, was shown to improve 6MWT, WHO functional

class and mPAP in idiopathic, CTD or CHD associated PAH, but there were no differences

in TCW.96 The FDA approved sildenafil in low doses for patients with PAH although there is

some debate as to whether higher doses might confer additional benefits.97 Other PDEi are

currently under study.

Combination therapy

The possibility to combine distinct drug classes that target different molecular pathways in

order to improve clinical efficacy and minimize side-effects is an attractive perspective. After

an initial attempt to combine bosentan and epoprostenol in a small scale and underpowered

trial conducted on patients with either iPAH or PAH associated to CTD that proved

unsuccessful,98 another trial that combined inhaled iloprost with bosentan in patients who


81

remained symptomatic showed improvement in functional class, TCW and

haemodynamics.99 More recently, the addition of sildenafil to PAH patients who remained

symptomatic on a stable dose of iv epoprostenol improved the 6MWT, as well as mPAP,

CO, and TCW.100

Invasive and surgical strategies in PAH

These include AS and LT or CHLT. Other possibilities, such as the RV mechanical assist

devices (RVAD) are still poorly investigated. AS creates a right-to-left shunt that unloads the

RV, decreases mPAP, and improves LV filling. The increase in CO offsets the shunting of

deoxygenated blood and ameliorates O2 delivery. Increased CO allows bridging to

transplantation in up to 40% of patients.101 Nevertheless, it is merely palliative and

procedural mortality is still high therefore it is just a last resort for patients on maximal

medical therapy and inotropic support. Improved techniques are being currently explored to

reduce procedural risk.5 Currently, PAH is responsible for approximately 4% of LT and

CHLT, and although there is a substantial procedural related mortality, the long-term

outcome is favourable, with a 47% survival after 5 years.102 The type of transplant is still a

matter of debate and highly related to the experience of each centre. Generally CHLT is

preferred either when patients have intractable HF or are dependent on inotropic support or

if PH is secondary to CHD or LHD.64

Therapeutic algorithm

Management must be tailored to each patient according to disease severity, comorbid

conditions, drug side-effects and each centre’s experience. Vasoreactivity test is usually

reserved for iPAH patients with stable hemodynamics, otherwise first line therapy should

consist of ERA or PDEi, unless the oral route is not available, patients are in functional class

IV or present overt RV failure. In these cases, the first choice is an iv prostanoid. Moreover,

combination therapy should always be kept in mind, particularly when side-effects arise or

patients are not responding. Enrolment in clinical trials with newer pharmacological agents

may be an option but AS and transplantation should be considered before systemic

deterioration. Early referral for transplantation is crucial particularly for refractory cases.7 A

simplified therapeutic algorithm is suggested in Figure 4.


82

Figure 4. Algorithm for pulmonary arterial hypertension (PAH) management.

CCB, calcium channel blockers; WHO, World Health Organization; ERA, endothelin-1

receptor antagonists; PDEi, phosphodiesterase inhibitors; RCT, randomized clinical

trials; iv, intravenous; sc, subcutaneous.

Non-PAH PH

Patients will benefit from medical optimization of their primary disease, but significant PH

may persist. Some patients actually present disproportionate PH not easily attributable to

the underlying condition. Particularly in these cases new PAH drugs have been used with

success.

In left-heart disease prostanoids, with the exception of inhaled route, are usually

contraindicated due to systemic vasodilation.103 ERA trials have been interrupted

prematurely due mainly to side-effects and absence of clinical benefit, even with reduced

dose,104 but selected cases may benefit from short trials as a bridge to transplantation.105 As

for PDEi short-term hemodynamic benefits, as well as long-term improvements have been

documented.106

Mild levels of PH are amenable to optimization of medical therapy in COPD. If PH is

disproportionate, and other PH causes have been ruled out, many centres are routinely

employing vasodilators despite V-Q mismatch.107 CTEPH is potentially curable with PEA

(usually with concomitant inferior vena cava filter placement).5 Yet, many patients are not


83

candidates so they remain anticoagulated and on diuretics. Many centres are promptly using

new PAH drugs off-label if there is associated vasculopathy.108

Recent progresses and future targets in PH

Based upon the most recent experimental findings, clinical trials targeting altered metabolic

and signalling pathways are warranted. DCA and Kv1.5 channel gene transfer have been

successful in experimental studies,33 as well as trp channel inhibitors, growth factor receptor

inhibitors and intracellular kinase inhibitors.3, 109 Inflammatory response modulation has also

been a major research topic. After several animal studies demonstrating beneficial effects of

statins,109 possibly due to pleiotropic effects, a human study disappointingly showed no

long-lasting improvement.110 Other immunomodulatory agents have been successful in

animal experiments,44, 111 but beneficial effects are mainly confined to CTD associated

PAH.112 We have also reported disturbances in endogenous endocrine and paracrine

systems113, 114 that may be targeted. Another tempting possibility is the recruitment or

infusion of EPC. The number and function of EPCs predicts prognosis, and most currently

used drugs increase circulating EPC numbers.115 Circulating EPCs home to sites of

endothelial injury, promote revascularization and improve vascular homeostasis,116

endothelial dysfunction may be related to the lack of EPCs.115 Finally, we must bear in mind

that RV failure is the final and most severe complication of PH. Agents such as

levosimendan that vasodilate lung vessels but are also positive inotropes are predictably

good therapeutic tools. Still, the clinical efficacy of these drugs has only just started to be

evaluated.117, 118

Acknowledgements

The authors would like to thank Daniela Silva, José Pinto, Francisco Vasques-Nóvoa, Rui

Cerqueira and Duarte Pinto for their contribution to the manuscript.

Conflict of Interest

None declared.


84

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