abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC São Paulo, SP, Brazil dgQS5xq9hJm7Rd4THM9ZSpR 00010 S0066-782X2008000600010 S0066-782X(08)09000610 10.1590/S0066-782X2008000600010 Artigo de Revisão Fatores e mecanismos envolvidos na hipertrofia ventricular esquerda e o papel anti-hipertrófico do óxido nítrico Garcia José Antonio Dias Incerpi Erika Kristina Alfenas Universidade José do Rosário Vellano Brazil Alfenas Universidade Vale do Rio Verde Brazil 06 2008 90 6 443 450 30 10 2007 17 10 2007 02 08 2007 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

A hipertrofia ventricular esquerda (HVE) ocorre em reposta à sobrecarga hemodinâmica relatada em várias condições fisiológicas e patológicas. Entretanto, ainda não está completamente elucidado se o estímulo primário para a hipertrofia é o estiramento mecânico do coração, fatores neuro-humorais, ou mesmo a interação de ambos. Esses fatores são traduzidos no interior da célula como alterações bioquímicas que levam à ativação de segundos (citosólicos) e terceiros (nucleares) mensageiros que irão agir no núcleo da célula, regulando a transcrição, e finalmente determinarão a expressão gênica que induza HVE. A HVE é caracterizada por alterações estruturais decorrentes do aumento das dimensões dos cardiomiócitos, da proliferação do tecido conjuntivo intersticial e da rarefação da microcirculação coronariana. Nos últimos anos, o óxido nítrico (•NO) surgiu como um importante regulador do remodelamento cardíaco, especificamente reconhecido como um mediador anti-hipertrófico. Vários estudos têm demonstrado os alvos celulares, as vias de sinalização anti-hipertrófica e o papel funcional do •NO. Portanto, a HVE parece desenvolver-se em decorrência da perda do balanço entre as vias de sinalização pró e anti-hipertróficas. Esses novos conhecimentos sobre as vias de sinalização pró e anti-hipertróficas permitirão desenvolver novas estratégicas no tratamento das HVE patológicas.

 Hipertrofia ventricular esquerda óxido nítrico cardiomiócitos 25 06 2008

ARTIGO DE REVISÃO

Fatores e mecanismos envolvidos na hipertrofia ventricular esquerda e o papel anti-hipertrófico do óxido nítrico

Factors and mechanisms involved in left ventricular hypertrophy and the anti-hypertrophic role of nitric oxide

José Antonio Dias GarciaI; Erika Kristina IncerpiII

IUniversidade José do Rosário Vellano (UNIFENAS), Alfenas, Três Corações, MG - Brasil

IIUniversidade Vale do Rio Verde (UninCor), Alfenas, Três Corações, MG - Brasil

Correspondência

RESUMO

A hipertrofia ventricular esquerda (HVE) ocorre em reposta à sobrecarga hemodinâmica relatada em várias condições fisiológicas e patológicas. Entretanto, ainda não está completamente elucidado se o estímulo primário para a hipertrofia é o estiramento mecânico do coração, fatores neuro-humorais, ou mesmo a interação de ambos. Esses fatores são traduzidos no interior da célula como alterações bioquímicas que levam à ativação de segundos (citosólicos) e terceiros (nucleares) mensageiros que irão agir no núcleo da célula, regulando a transcrição, e finalmente determinarão a expressão gênica que induza HVE. A HVE é caracterizada por alterações estruturais decorrentes do aumento das dimensões dos cardiomiócitos, da proliferação do tecido conjuntivo intersticial e da rarefação da microcirculação coronariana. Nos últimos anos, o óxido nítrico (•NO) surgiu como um importante regulador do remodelamento cardíaco, especificamente reconhecido como um mediador anti-hipertrófico. Vários estudos têm demonstrado os alvos celulares, as vias de sinalização anti-hipertrófica e o papel funcional do •NO. Portanto, a HVE parece desenvolver-se em decorrência da perda do balanço entre as vias de sinalização pró e anti-hipertróficas. Esses novos conhecimentos sobre as vias de sinalização pró e anti-hipertróficas permitirão desenvolver novas estratégicas no tratamento das HVE patológicas.

Palavras-chave: Hipertrofia ventricular esquerda, óxido nítrico, cardiomiócitos.

Introdução

Segundo a Organização Mundial de Saúde (2005), as doenças cardiovasculares lideram as causas de morte no mundo. Dentre essas doenças, a hipertrofia ventricular esquerda (HVE) constitui um indicador de grande relevância no risco de morbidade e mortalidade cardiovascular. Segundo o Framingham Heart Study1, os indivíduos que apresentaram HVE, diagnosticada por alterações eletrocardiográficas, apresentaram risco de morte seis vezes maior que a população em geral.

O miocárdio dos mamíferos passa por um crescimento hipertrófico durante a maturação pós-nascimento, que é caracterizado por aumento no tamanho individual dos cardiomiócitos sem divisão celular2. Esse padrão de desenvolvimento hipertrófico pode ser reiniciado no coração adulto em resposta a alterações hemodinâmicas e/ou neuro-hormonais3.

O óxido nítrico (•NO) produzido no coração é apontado como inibidor endógeno da cascata de sinalização que induz a hipertrofia cardíaca mal-adaptada. As primeiras evidências de que o •NO pode apresentar efeitos anti-hipertróficos no coração foram obtidas em ratos espontaneamente hipertensos (SHR), sob tratamento crônico com L-arginina4. Posteriormente, tal papel do •NO foi confirmado em camundongos que hiperexpressam óxido nítrico sintase endotelial (NOSe), nos quais o •NO atenuou a hipertrofia cardíaca induzida pela infusão crônica de isoprenalina (ISO)5, indicando que o •NO endógeno atua como modulador negativo para a hipertrofia cardíaca.

O objetivo desta revisão é descrever os fatores hipertróficos e/ou proliferativos indutores da HVE, assim como os alvos celulares, as vias de sinalização e o papel funcional do •NO como molécula anti-hipertrófica.

Conceito e classificação da hipertrofia ventricular esquerda

A HVE constitui um conjunto de alterações estruturais decorrentes do aumento das dimensões dos cardiomiócitos, da proliferação do tecido conjuntivo intersticial e da rarefação da microcirculação coronariana6.

Quando o cardiomiócito recebe um estímulo hipertrófico, esse é traduzido no interior da célula, como alterações bioquímicas que levam à ativação de segundos (citosólicos) e terceiros (nucleares) mensageiros que irão agir no núcleo da célula, regulando a transcrição, e finalmente determinarão a expressão gênica que induz a HVE (fig. 1).

O crescimento dos cardiomiócitos na hipertrofia ventricular esquerda pode ocorrer pela adição de sarcômeros em série (sobrecarga de volume) ou em paralelo (sobrecarga de pressão) (fig. 2), permitindo que a célula aumente em comprimento ou em diâmetro, levando à hipertrofia excêntrica ou concêntrica, respectivamente7 (fig. 2).

Segundo Kempf e Wollert7, a hipertrofia causada por sobrecargas hemodinâmicas pode conduzir à hipertrofia adaptada (fisiológica) ou mal-adaptada (patológica). Hipertrofia fisiológica é aquela desenvolvida em decorrência da sobrecarga hemodinâmica transitória, como as observadas no crescimento cardíaco durante a adolescência e a gestação, e em resposta a exercícios regulares, enquanto a hipertrofia patológica é aquela decorrente de sobrecarga hemodinâmica persistente.

Fatores que induzem a hipertrofia ventricular esquerda

Fatores hemodinâmicos

Aumento da necessidade metabólica

A sobrecarga de trabalho é considerada o fator mais freqüentemente envolvido na HVE. O aumento na atividade cardíaca pode estar associado à maior demanda fisiológica, como no exercício físico8 e em estados de anemia crônica9. Assim, como conseqüência do aumento da necessidade de bombear mais sangue para a periferia, há uma adaptação adequada às novas exigências.

Sobrecarga de pressão e/ou volume

Condições patológicas, como hipertensão arterial, estenose ou coarctação da aorta, denominadas sobrecarga de pressão, ou as de insuficiência aórtica ou comunicação interatrial, denominadas sobrecarga de volume, podem promover a hipertrofia por aumento do volume dos cardiomiócitos, acompanhado de aumento e alteração na qualidade dos componentes da matriz colágena5,7.

O estiramento é capaz de ativar canais de Ca+2 tipo L (LTCC) (fig. 3), de Na+ e os trocadores de Na+/H+; inativar canais de K+; ativar adenilato-ciclase e fosfolipase C10 (fig. 3), além de estar associado ao acúmulo de inositóis de fosfato, os quais atuam como segundos mensageiros11. As alterações no funcionamento dos canais iônicos do sarcolema levam a variações da concentração iônica intracelular, o que pode representar um estímulo inicial para ativação de proteínas quinases ativadoras mitogênicas (MAPK). Dentre a superfamília das MAPK, a quinase regulada por sinal extracelular (ERK), a quinase c-jun NH2-terminal (JUNK) e a quinase p38 são mediadores da sinalização hipertrófica das células miocárdicas, pois induzem a transcrição de genes associados à hipertrofia12 (fig. 3). O estímulo mecânico também pode ativar os receptores integrinas, localizados na membrana celular entre a matriz extracelular (ECM) e o complexo de proteínas que formam a linha Z do sarcômero12. Nessa malha, posicionam-se proteínas sinalizadoras, como as tirosina-quinases coativadoras de receptores esteroidais (Src) e quinase de adesão focal (Fak), além de outras responsáveis pelo início do processo de sinalização ativado pelas integrinas13 (fig. 3).

O estímulo mecânico também induz a liberação local de fatores autócrinos e parácrinos pelas células miocárdicas, como endotelina 1 (ET1), fatores de crescimento e citocinas (fator de crescimento de fibroblatos (FGF), fator de crescimento transformador b (TGFb), fator de crescimento semelhante à insulina (IGF) e cardiotrofina-112. Esses fatores podem ligar-se aos receptores específicos de membrana e ativar cascatas intracelulares acopladas à calcineurina, fosfoquinase-C e à via da MAPK e dar início à cascata de eventos responsáveis pelo crescimento hipertrófico cardíaco (fig. 3 e 4).

Fatores neuro-humorais

Catecolaminas e sistema nervoso simpático

Os cardiomiócitos expressam receptores b-adrenérgicos (b-AR) e a1-adrenérgicos (a1-AR). A estimulação dos receptores b-adrenérgicos (b-AR) ativa a adenilato-ciclase pela interação com a proteína G estimulatória (Gs), a qual desencadeia cascatas intracelulares que ativam proteínas quinases A (PKA), estimulando também a p38-MPAK14. A estimulação crônica dos b-AR pela administração de isoproterenol15 induz o aumento da massa cardíaca, dos cardiomiócitos, da fibrose miocárdica e da disfunção progressiva, o que culmina com insuficiência cardíaca. Agudamente, a ativação dos a1-AR aumenta a contratilidade mediada pela ativação da proteína Gq. Esta provoca a ativação da fosfolipase C, que estimula hidrólise de fosfatidil inositóis da membrana, gerando dois segundos mensageiros, o diacilglicerol (DAG) e o inositol trifosfato (IP3). O IP3 estimula a liberação de Ca+2 do retículo sarcoplasmático, enquanto o DAG ativa a proteína quinase C (PKC) e, esta, por sua vez, induz a hipertrofia em cultura de miócitos neonatais16 (fig. 4).

Angiotensina II

Estudos de Lindpaintner e Ganten17 relatam a síntese de angiotensina II (Ang II) tanto em miócitos como em fibroblastos e miofibroblastos. Seus efeitos biológicos são mediados, basicamente, pelos receptores de membrana AT1 e AT2.

A Ang II interage com o receptor AT1, associado à proteína G, que estimula a fosfolipase C. Esta, por sua vez, induz a formação de trifosfato de inositol, assim como de diacilglicerol, o que provoca aumento da concentração citoplasmática de Ca+2, levando à ativação da proteína quinase C (PKC) e da adenilatociclase18. A Ang II, via receptor AT1, também é capaz de induzir uma cascata de ativação via tirosinas-quinases19. Essas tirosinas-quinases regulam vias efetoras intracelulares, incluindo a MAPK, que ativam vários fatores de transcrição de proteínas nos cardiomiócitos (fig. 4).

Os receptores AT2 apresentam estrutura transmembrana clássica de um receptor associado à proteína G20. Estudos de Senbonmatsu e cols.21 demonstraram que camundongos com deleção do receptor AT2 apresentaram atenuação da hipertrofia induzida por sobrecarga pressórica. Entretanto, animais com deleção de receptores AT1 não apresentaram atenuação da hipertrofia induzida por sobrecarga pressórica22, sugerindo que a deficiência de receptores AT1 pode ser compensada pelos receptores AT2, ou, ainda, que este subtipo AT2 pode ter papel predominante nos efeitos tróficos da Ang II em cardiomiócitos.

Insulina

A insulina liga-se à subunidade a do receptor pertencente à família dos receptores de membrana que possuem capacidade tirosina-quinase23, provoca uma mudança conformacional na subunidade b, que leva à sua autofosforilação em tirosina, e ativa sua capacidade tirosina-quinase. Uma vez ativado, o receptor de insulina (IR) é capaz de fosforilar diversos substratos intracelulares, entre eles os substratos do receptor de insulina (IRS-1-4), Shc (SRC Homology collagen) e Jak-224 (fig. 4). Essas proteínas fosforiladas recrutam e ativam diversos efetores intracelulares, com funções celulares diferentes23. A via ERK/MAPK está envolvida no controle do crescimento (fig. 4) e da mitogênese, enquanto a ativação fosfatidil inositol (PI) 3-quinase pelo IRS-1 está envolvida preferencialmente nas ações metabólicas da insulina23,25 (via IRS/PI3K/AKT/mTOR) (fig. 4). Em situações normais, a insulina ativa a produção de •NO em células endoteliais por um mecanismo dependente da fosfatidil inositol (PI) 3-quinase, levando ao aumento da produção local de •NO26 com conseqüente efeito vasodilatador e antiapoptótico27.

A insulina induz a fosforilação em tirosina do IRS-1, enquanto agentes que provocam resistência a insulina, tais com TNFa, ácidos graxos livres, estresse celular e hiperinsulinemia, induzem a ativação de quinases de serina/treonina que fosforilam o IRS-1 em serina, inibindo sua função28. Assim, a redução da ativação da via fosfatidil inositol (PI) 3-quinase/Akt e a ativação preservada da via ERK/MAPK, em situação de resistência a insulina e hiperinsulinemia, estão sendo consideradas como eventos fundamentais para o desenvolvimento da hipertrofia cardíaca29 (fig. 4).

Indiretamente, a insulina também pode induzir a hipertrofia cardíaca via aumento na expressão de RNAm dos receptores AT230 e na ativação do sistema nervoso simpático3.

Estresse oxidativo

O desequilíbrio entre a produção e a remoção de espécies reativas de oxigênio (ROS) e de nitrogênio (RNS) é denominado estresse oxidativo/nitrosativo, respectivamente. Várias situações fisiopatológicas e genéticas podem induzir o estresse oxidativo cardíaco, como aumento na concentração de Ang II31, hipercolesterolemia32, estresse mecânico no miocárdio33 e processos inflamatórios34.

A hipertensão e o estresse mecânico no miocárdio induzem o aumento das ROS nos cardiomiócitos, ativando a via MAPK33, que desempenha papel importante na hipertrofia cardíaca. Além disso, o desequilíbrio redox reduz a biodisponibilidade do •NO no sistema cardiovascular35 e altera o equilíbrio entre os fatores hipertróficos (e/ou proliferativos) e os fatores anti-hipertróficos (e/ou antiproliferativos) (fig. 5), desencadeando um remodelamento do miocárdio.

Hipercolesterolemia

A hipercolesterolemia, além de induzir o estresse oxidativo32, pode alterar a função e a expressão de canais de KATP em miocárdio36, provocando a hipertrofia cardíaca, pelo simples fato de que a ativação desses canais atenua a hipertrofia cardíaca por meio da inibição da 70- KDa S6 Kinase37, enzima que atua como gatilho para síntese protéica no remodelamento do miocárdio. Além disso, a hiperlipidemia está associada ao aumento na concentração plasmática de ET1, induzindo a alteração vasomotora38. Em adição à propriedade vasoconstritora, a endotelina ativa vias de sinalização hipertrófica nos cardiomiócitos: PKC e MAPK39.

Citocinas e fatores de crescimento induzidos pelo processo inflamatório

A trilogia composta por processo inflamatório40, disfunção do endotélio41 e estresse oxidativo31, no ambiente cardiovascular, é considerada o denominador comum dentre as condições que promovem e sustentam a hipertrofia cardíaca.

O CD40L (ligante) é uma proteína transmembrana expressa na superfície de linfócitos, das células endoteliais, das células da musculatura lisa vascular e de macrófagos. Essa proteína exerce um efeito pró-oxidante, e sua interação com seu receptor CD40 induz a resposta inflamatória, favorecendo a síndrome coronariana aguda42. A interação entre CD40 e CD40L ativa a via Nfkappab43 e promove a fosforilação do IKK (inibidor Kappab quinase), resultando na translocação do fator nuclear Kappa b (NFkappa b) para o núcleo, onde ativa genes envolvidos na inflamação e no crescimento celular44. A ativação do NFkappa b participa do desenvolvimento da hipertrofia cardíaca em camundongos, caracterizada por aumento do depósito de colágeno44.

A ativação dos linfócitos T resulta na produção de interferon-g que, por sua vez, aumenta a síntese de citocinas inflamatórias, como TNF-a e IL-1. Essas citocinas induzem a produção de grandes quantidades de IL-6, a qual estimula a produção de proteínas inflamatórias45, e à hipertrofia cardíaca em camundongos, pela sua interação com receptores de membrana gp-13046. O TNFa induz a hipertrofia cardíaca in vivo, por determinar aumento na síntese de proteínas estruturais e contráteis dos cardiomiócitos, além de provocar o aumento na expressão de receptores AT1, elevando o efeito mediado por angiotensina II em favor da fibrose cardíaca47.

Óxido nítrico/óxido nítrico sintase no coração

Síntese do óxido nítrico

O •NO é produzido por enzimas denominadas óxido nítrico sintases (NOS). Essa é uma família de enzimas complexas que catalisam a oxidação da L-arginina para produzir óxido nítrico e L-citrulina. Três isoformas de NOS foram inicialmente caracterizadas: a isoforma neuronal (NOSn = NOS1), identificada no cérebro; a isoforma induzida (NOSi = NOS2) em macrófagos; e a isoforma endotelial (NOSe = NOS3) em células endoteliais48 e cardiomiócitos49. A NOSn e NOSe apresentam expressão constitutiva (óxido nítrico sintases constitutivas (NOSc)) e produzem baixa quantidade de •NO, quando ativadas por cálcio (Ca+2)50. A NOSi é expressa somente em resposta a estímulos pró-inflamatórios e citocinas, e pode produzir grande quantidade de •NO50.

No miocárdio sadio, a NOSe encontra-se expressa principalmente no endotélio vascular coronariano e no endotélio cardíaco51, assim como nos cardiomiócitos49. Nessas células, a NOSe localiza-se nas cavéolas, ancorada pela caveolina 3 na membrana plasmática, próximo aos canais de Ca+2 tipo L7. A NOSn está presente nos gânglios nervosos intracardíacos, nas fibras nervosas atriais e em algumas fibras nervosas perivasculares dos ventrículos52. Sua expressão também foi detectada nos cardiomiócitos e nas células da musculatura lisa de pequenas e grandes artérias coronarianas de ratos53. O coração adulto não expressa normalmente NOSi. Essa é ativada por mediadores do processo inflamatório em muitos tipos de células, incluindo células endoteliais e cardiomiócitos54. Pode ser identificada no citosol7, mas já foi encontrada no espaço perinuclear, no aparelho de Golgi, na mitocôndria e na membrana plasmática55.

Óxido nítrico: molécula anti-hipertrófica

As duas principais substâncias endógenas envolvidas no papel anti-hipertrófico do coração são o peptídeo natriurético atrial (ANP) e o •NO. O ANP secretado de grânulos atriais, em resposta ao estiramento atrial agudo ou crônico, exerce uma função anti-hipertensiva, anti-hipervolêmica e anti-hipertrófica através da ativação da guanilato-ciclase e conseqüente aumento nos níveis da guanosina monofosfato cíclica (GMPc)56.

A produção de •NO nos cardiomiócitos é altamente compartimentalizada57, como resultado da localização das NOS. Em razão da alta reatividade natural do •NO, sua síntese na proximidade do seu alvo facilita sua acessibilidade aos processos intracelulares para uma sinalização coordenada. Independentemente da especificidade funcional das NOSc em cardiomiócitos, o •NO exerce um efeito anti-hipertrófico in vivo, já que camundongos deficientes em NOSn ou NOSe desenvolveram hipertrofia cardíaca espontânea e, ainda, os deficientes em ambas as enzimas desenvolveram hipertrofia mais acentuada58.

O alvo intracelular bem definido do •NO é a guanilato-ciclase solúvel (GCs), a qual possui um grupo heme que atua como aceptor de •NO e converte cataliticamente a guanosina trifosfato (GTP) em guanosina monofosfato cíclica (cGMP). Uma série de estudos demonstrou que o •NO promove efeito anti-hipertrófico por meio da modulação negativa das vias pró-hipertróficas7, e a via dependente de GMPc desempenha um papel central nesse efeito.

O aumento de GMPc no meio intracelular inibe, via enzima proteína quinase-G (PKG), a rede de sinalização das diferentes vias pró-hipertróficas que envolvem as MAPK59. Essa via •NO/GMPC/PKG desenvolve um importante papel na regulação negativa sobre a hipertrofia de cardiomiócitos induzida por Ang II, ET1, insulina e fatores de crescimento, inibindo a sinalização da MAPK/ERK e diminuindo a transcrição de genes associados à hipertrofia60.

A via •NO/GMPC/PKG, além de inibir MAPK, também reduz a resposta hipertrófica induzida pela sobrecarga mecânica e por citocinas, o que inibe os seguintes processos: 1) a via calcineurina/fator nuclear ativador de células T (NFAT), pela diminuição do influxo de cálcio pelos canais de cálcio tipo L (LTCC)59; 2) a expressão dos genes hipertróficos, como a ciclina D261; e 3) a ativação do fator de transcrição NFkappa b44. Essa via (•NO/GMPC/PKG) também interage de maneira inibitória com as vias de estimulação b-adrenérgicas. A PKG induz efeito inotrópico negativo no coração por: 1) fosforilar a troponina I, diminuindo a sensibilidade dos miofilamentos ao cálcio; 2) inibir a liberação de cálcio do retículo sarcoplasmático, por fosforilar e inibir os receptores de IP3 presentes em suas membranas62. Existe evidência de que o •NO bloqueia a ação da fosfolipase C, inibindo a liberação de cálcio mediada pelo trifosfato de inositol (IP3)63. Todos esses efeitos podem diminuir a concentração de mensageiros hipertróficos intracelulares ativados pela estimulação adrenérgica.

Quanto aos canais de KATP e à hipertrofia ventricular, a via •NO/GMPC ativa os canais de KATP do sarcolema64, com provável inibição da 70-KDa S6 Kinase, atenuando a resposta hipertrófica em modelos hiperlipidêmicos 37.

Em adição a seus efeitos anti-hipertróficos, o •NO apresenta um efeito pró-apoptótico dose-dependente em cardiomiócitos. Assim, baixas concentrações de •NO inibem a hipertrofia do cardiomiócito, enquanto altas concentrações de •NO são requeridas para provocar a ativação de caspases, fragmentação de DNA e apoptose65. A apoptose estimulada pelo •NO em cardiomiócitos adultos está associada à alteração na expressão dos genes pró-apoptóticos da família Bcl-2, o Bax e o Bak, os quais desempenham papel crítico na determinação do destino da célula, em parte por alteração da permeabilidade da membrana mitocondrial66.

Há várias evidências de que o •NO é o efetor da apoptose mediada por citocinas e da ativação dos genes pró-apoptóticos, como: 1) a capacidade das citocinas de provocarem a produção do •NO em cardiomiócitos é proporcional à sua capacidade de ativar a morte celular programada (apoptose); 2) os antagonistas da NOSi previnem a produção de •NO e da apoptose e bloqueiam a expressão de Bcl-2 e o Bak67.

O •NO gerado pela NOSi tem sido o responsável pela indução de apoptose em diferentes tipos de células67, incluindo os cardiomiócitos68. Após a indução, a NOSi permanece ativada por 20 horas69, durante as quais sintetiza •NO em concentração 1.000 vezes maior que as NOSc70 e, em condições de substrato ou co-fatores deficientes, reduz oxigênio molecular a superóxido7. O superóxido e o peroxinitrito, formado pela interação de •NO com superóxido, são altamente tóxicos para os cardiomiócitos7. Ing e cols.68 e Arstall e cols.71 determinaram que apoptose de cardiomiócitos induzida pela NOSi parece ser independente da GCs e do GMPc, mas parece ser predominantemente pela formação de peroxinitrito. A citotoxidade mediada pela NOSi não foi somente confinada em miócitos neonatal, mas também em miócitos adulto, onde o •NO endógeno gerado pela NOSi, após a exposição à combinação das citocinas INFg e IL-1b, provoca apoptose71.

Moduladores da biodisponibilidade do óxido nítrico

Doadores de óxido nítrico

Entre os compostos que apresentam grande potencial como doadores de •NO, encontram-se os S-nitrosotióis (RSNOs) de baixo peso molecular. Os RSNOs são espécies endógenas que foram detectados em fluidos do revestimento das vias aéreas, nas plaquetas e em neutrófilos, onde atuam nos sistemas biológicos como carregadores de •NO, na forma de tióis livres ou em proteínas contendo cisteína72.

Os RSNOs exógenos são fármacos promissores a serem utilizados no tratamento de doenças que envolvem disfunções na biodisponibilidade de •NO, uma vez que oferecem vantagens sobre as outras drogas existentes porque não induzem a tolerância nas células vasculares73 como fazem o nitrato orgânico e o nitroprussiato de sódio.

Estudos clínicos mostraram que os RSNOs podem ser benéficos em uma série de desordens cardiovasculares74. Podem também ter acesso ao compartimento intracelular pela ação catalítica da disulfeto isomerase de membrana, associada com uma reação de nitrosilação74. Os membros dessa classe de composto incluem: S-nitroso-glutationa (GSN), S-nitroso-N-acetilpenicilamina (SNAP), S-nitroso-albumina e o S-nitroso-N-acetilcisteína (SNAC).

Inibidores da enzima conversora de angiotensina

A enzima conversora de angiotensina (ECA), além de gerar ANG II, também degrada bradicinina75. A bradicinina é um vasodilatador dependente do endotélio, estimulando o endotélio a produzir •NO. Assim, os inibidores da ECA potencializam a bradicinina e têm sido utilizados pelo seu efeito farmacológico benéfico em aumentar a biodisponibilidade do •NO no tecido cardiovascular76. Além disso, a ANG II pode estimular a produção de superóxido, o que reduziria a biodisponibilidade do •NO77, um evento que pode ser bloqueado pelos inibidores da ECA.

Inibidores da fosfodiesterase

Inibidores da fosfodiesterase como o sildenafil prolongam a ação sinalizadora do •NO por inibir a hidrólise da GMPc. A inibição da fosfodiesterase pelos inibidores da fosfodiesterase previne a degradação da GMPc em GMP5', o que prolonga o tempo de ação da GMPc, mantendo baixa a concentração intracelular de Ca++ na muscular lisa dos vasos e conseqüente vasodilatação78.

Conclusão

O aumento no estiramento dos cardiomiócitos é o principal fator indutor de crescimento hipertrófico, mas substâncias circulantes – como endotelina 1 (ET I), angiotensina II, insulina e catecolaminas –, fatores de crescimento, citocinas liberadas localmente pelas células miocárdicas e produtos do estresse oxidativo, como o ânion superóxido (O2-), também provocam o crescimento hipertrófico dos cardiomiócitos. Esses, por sua vez, ativam segundos mensageiros como a fosfolipase C (PLC), as proteínas quinases ativadoras mitogênicas (MAPK) e a calcineurina. Essas proteínas ativadas promovem alterações nos fatores nucleares e na regulação de genes hipertróficos. Assim sendo, parece que não há uma cascata de sinalização isolada para cada estímulo ou resposta, mas que múltiplas moléculas sinalizadoras ocorrem e podem formar uma rede de cascatas com numerosos elementos, facilitando o cruzamento entre elas. Assim, torna-se evidente que o efeito anti-hipertrófico do •NO é feito por meio da modulação negativa das vias pró-hipertróficas, já que a via dependente de GMPc desempenha um papel central nesse efeito. Contudo, o •NO não bloqueia apenas uma das vias de sinalização intracelular, o que não seria suficiente para prevenir eficientemente o crescimento hipertrófico ventricular. Portanto, a HVE parece desenvolver-se em decorrência da perda do balanço entre as vias de sinalização pró e anti-hipertróficas. Esses novos conhecimentos sobre as vias de sinalização pró e anti-hipertróficas permitirão desenvolver novas estratégicas no tratamento das HVE patológicas.

Potencial Conflito de Interesses

Declaro não haver conflitos de interesses pertinentes.

Fontes de Financiamento

O presente estudo não teve fontes de financiamento externas.

Vinculação Acadêmica

Este artigo é parte de tese de doutorado de José Antonio Dias Garcia pela Universidade Estadual de Campinas (Unicamp).

José Antonio Dias Garcia

Rua Antônio Esteves, 850, Jardim Aeroporto

37.130-000, Alfenas, MG - Brasil

E-mail:

jadgarcia@uol.com.br

Artigo recebido em 02/08/07; revisado recebido em 17/10/07; aceito em 30/10/07.

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Relationship between inositol 1,4,5-trisphosphate receptor isoforms and subcellular Ca2+ signaling patterns in nonpigmented ciliary epithelia. Invest Ophthalmol Vis Sci. 1999; 40 (9): 2046-53. Relationship between inositol 1,4,5-trisphosphate receptor isoforms and subcellular Ca2+ signaling patterns in nonpigmented ciliary epithelia Invest Ophthalmol Vis Sci 1999 2046 53 9 40 Hirata K Nathanson MH Burgstahler AD Okazaki K Mattei E Sears ML 64. Baker JE, Contney SJ, Singh R, Kalyanaraman B, Gross GJ, Bosnjak ZJ.Nitric oxide activates the sarcolemmal K(ATP) channel in normoxic and chronically hypoxic hearts by a cyclic GMP-dependent mechanism.J Mol Cell Cardiol. 2001;33 (2): 331-41. Nitric oxide activates the sarcolemmal K(ATP) channel in normoxic and chronically hypoxic hearts by a cyclic GMP-dependent mechanism. J Mol Cell Cardiol 2001 331 41 2 33 Baker JE Contney SJ Singh R Kalyanaraman B Gross GJ Bosnjak ZJ 65. Wollert KC, Fiedler B, Gambaryan S, Smolenski A, Heineke J, Butt E, et al. Gene transfer of cGMP-dependent protein kinase I enhances the antihypertrophic effects of nitric oxide in cardiomyocytes. Hypertension. 2002; 39: 87-92. Gene transfer of cGMP-dependent protein kinase I enhances the antihypertrophic effects of nitric oxide in cardiomyocytes Hypertension 2002 87 92 39 Wollert KC Fiedler B Gambaryan S Smolenski A Heineke J Butt E 66. Haunstetter A, Izumo S. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res. 1998; 82 (11): 1111-29. Apoptosis: basic mechanisms and implications for cardiovascular disease Circ Res 1998 1111 29 11 82 Haunstetter A Izumo S 67. Xie K, Huang S, Wang Y, Beltran PJ, Juang SH, Dong Z, et al. Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis. Cancer Immunol Immunother.1996; 43: 109-15. Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis Cancer Immunol Immunother 1996 109 15 43 Xie K Huang S Wang Y Beltran PJ Juang SH Dong Z 68. Ing DJ, Zang J, Dzau VJ, Webster KA, Bishopric NH. Modulation of cytokine-induced cardiac myocyte apoptosis by nitric oxide, Bak, and Bcl-x. Circ Res. 1999; 84: 21-33. Modulation of cytokine-induced cardiac myocyte apoptosis by nitric oxide, Bak, and Bcl-x Circ Res 1999 21 33 84 Ing DJ Zang J Dzau VJ Webster KA Bishopric NH 69. Davies MG, Fulton GJ, Hagen PO. Clinical biology of nitric oxide. Br J Surg. 1995; 82: 1598-610. Clinical biology of nitric oxide Br J Surg 1995 1598 610 82 Davies MG Fulton GJ Hagen PO 70. Wong GKT, Marsdern PA. Nitric oxide synthases: regulation in disease. Nefron Dial transplant. 1996,11: 215-20. Nitric oxide synthases: regulation in disease Nefron Dial transplant 1996 215 20 11 Wong GKT Marsdern PA 71. Arstall MA, Sawyer DB, Fukazawa R, Kelly RA. Cytokine-mediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation. Circ Res. 1999; 85: 829-40. Cytokine-mediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation Circ Res 1999 829 40 85 Arstall MA Sawyer DB Fukazawa R Kelly RA 72. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, et al. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci USA. 1992; 89: 444-8. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds Proc Natl Acad Sci USA 1992 444 8 89 Stamler JS Simon DI Osborne JA Mullins ME Jaraki O Michel T 73. Jaworski K, Kinard F, Goldstein D, Holvoet P, Trouet A, Schneider YJ, et al. S-nitrosothiols do not induce oxidative stress, contrary to other nitric oxide donors, in cultures of vascular endothelial or smooth muscle cells. Eur J Pharmacol. 2001; 425 (1): 11-9. S-nitrosothiols do not induce oxidative stress, contrary to other nitric oxide donors, in cultures of vascular endothelial or smooth muscle cells Eur J Pharmacol 2001 11 9 1 425 Jaworski K Kinard F Goldstein D Holvoet P Trouet A Schneider YJ 74. Ignarro J, Napoli C, loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide. Cir Res. 2002; 90: 21-8. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide Cir Res 2002 21 8 90 Ignarro J Napoli C 75. Taylor-McCabe KJ, Erasahin C, Simmons WH. Bradykinin metabolism in the isolated perfused rabbit heart. J Hypertens. 2001; 19: 1295-9. Bradykinin metabolism in the isolated perfused rabbit heart J Hypertens 2001 1295 9 19 Taylor-McCabe KJ Erasahin C Simmons WH 76. Vanhoutte PM. Endothelial dysfunction and inhibition of converting enzyme. Eur Heart J. 1998; 19 (Suppl): J7-J15. Endothelial dysfunction and inhibition of converting enzyme Eur Heart J 1998 J7 J15 Suppl 19 Vanhoutte PM 77. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK. Angiotensin II-mediated hypertension in the rate increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations in vasomotor tone. J Clin Invest. 1996; 97: 1916-23. Angiotensin II-mediated hypertension in the rate increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations in vasomotor tone J Clin Invest 1996 1916 23 97 Rajagopalan S Kurz S Munzel T Tarpey M Freeman BA Griendling KK 78. Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med. 1992; 326: 90-4. Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission N Engl J Med 1992 90 4 326 Rajfer J Aronson WJ Bush PA Dorey FJ Ignarro LJ Review Article Factors and mechanisms involved in left ventricular hypertrophy and the anti-hypertrophic role of nitric oxide

The left ventricular hypertrophy (LVH) occurs in response to the hemodynamic overload in some physiological and pathological conditions. However, it has not been completely elucidated whether the primary stimulation for the hypertrophy is the mechanical stretching of the heart, neurohumoral factors, or even the interaction of both. These factors are translated inside the cell as biochemical alterations that lead to the activation of second (cytosolic) and third (nuclear) messengers that will act in the cell nucleus, regulating transcription, and will finally determine the genic expression that induces LVH. The LVH is characterized by structural alterations due to the increase in the cardiomyocyte dimensions, the proliferation of the interstitial connective tissue and the rarefaction of the coronary microcirculation. Recently, nitric oxide (•NO) has appeared as an important regulator of cardiac remodeling, specifically recognized as an anti-hypertrophic mediator. Some studies have demonstrated the cellular targets, the anti-hypertrophic signaling pathways and the functional role of •NO. Thus, the LVH seems to develop as a result of the loss of the balance between the pro and the anti-hypertrophic signaling pathways. This new knowledge about the pro and anti-hypertrophic signaling pathways will allow the development of new strategies in the treatment of pathological LVH.

 Hypertrophy, left ventricular nitric oxide myocytes, cardiac

REVIEW ARTICLE

Factors and mechanisms involved in left ventricular hypertrophy and the anti-hypertrophic role of nitric oxide

José Antonio Dias GarciaI; Erika Kristina IncerpiII

IUniversidade José do Rosário Vellano (UNIFENAS), Alfenas, Três Corações, MG - Brazil

IIUniversidade Vale do Rio Verde (UninCor), Alfenas, Três Corações, MG - Brazil

Mailing address

SUMMARY

The left ventricular hypertrophy (LVH) occurs in response to the hemodynamic overload in some physiological and pathological conditions. However, it has not been completely elucidated whether the primary stimulation for the hypertrophy is the mechanical stretching of the heart, neurohumoral factors, or even the interaction of both. These factors are translated inside the cell as biochemical alterations that lead to the activation of second (cytosolic) and third (nuclear) messengers that will act in the cell nucleus, regulating transcription, and will finally determine the genic expression that induces LVH. The LVH is characterized by structural alterations due to the increase in the cardiomyocyte dimensions, the proliferation of the interstitial connective tissue and the rarefaction of the coronary microcirculation. Recently, nitric oxide (•NO) has appeared as an important regulator of cardiac remodeling, specifically recognized as an anti-hypertrophic mediator. Some studies have demonstrated the cellular targets, the anti-hypertrophic signaling pathways and the functional role of •NO. Thus, the LVH seems to develop as a result of the loss of the balance between the pro and the anti-hypertrophic signaling pathways. This new knowledge about the pro and anti-hypertrophic signaling pathways will allow the development of new strategies in the treatment of pathological LVH.

Key words: Hypertrophy, left ventricular; nitric oxide; myocytes, cardiac.

Introduction

According to the World Health Organization (2005) cardiovascular diseases are the leading cause of death in the world. Among these diseases, the left ventricular hypertrophy (LVH) constitutes a very important indicator of cardiovascular morbimortality risk. According to the "Framingham Heart Study" 1, the individuals who present LVH, diagnosed through electrocardiographic alterations, presented a six-fold higher risk of death when compared to the general population.

The myocardium of the mammals goes through a hypertrophic growth phase during the post-birth maturation period, which is characterized by an increase in the individual size of the cardiomyocytes without cell division2. This pattern of hypertrophic development can be re-initiated in the adult heart in response to hemodynamic and/or neurohormonal alterations3.

The nitric oxide (•NO) produced in the heart is deemed an endogenous inhibitor of the signaling cascade that induces the heart hypertrophy maladaptation. The first evidence that the •NO can present anti-hypertrophic effects in the heart were obtained in spontaneously hypertensive rats (SHR) under chronic treatment with L-arginine4. Subsequently, such role of the •NO was confirmed in mice that hyper-expressed endothelial nitric oxide synthase (eNOS), in which the •NO attenuated the heart hypertrophy induced by the chronic infusion of isoprenaline (ISO)5, indicating that the endogenous •NO acts as a negative modulator for heart hypertrophy.

The objective of this review is to describe the LVH-inducing hypertrophic and/or proliferative factors, as well as the cell targets, the signaling pathways and the functional role of the •NO as an anti-hypertrophic molecule.

Concept and classification of the left ventricular hypertrophy

The LVH constitutes a set of structural alterations caused by the increased dimensions of the cardiomyocytes, the proliferation of the interstitial conjunctive tissue and the rarefaction of the coronary circulation6.

When the cardiomyocyte receives a hypertrophic stimulus, this is translated within the cell as biochemical alterations that lead to the activation of second (cytosolic) and third (nuclear) messengers that will act inside the cell, regulating transcription and will finally determine the genic expression that induces LVH (Figure 1).

The growth of the cardiomyocytes in the left ventricular hypertrophy can occur by the addition of sarcomeres in series (volume overload) or in parallel (pressure overload) (Figure 2), allowing the cell to increase in length or in diameter, leading to eccentric or concentric hypertrophy, respectively7 (Figure 2).

According to Kempf and Wollert7, the hypertrophy caused by hemodynamic overload can lead to adapted (physiological) or maladapted (pathological) hypertrophy. The physiological hypertrophy is the one developed due to a transient hemodynamic overload, such as those observed during cardiac growth at the adolescence, pregnancy and in response to regular exercises, whereas the pathological hypertrophy is the one caused by persistent hemodynamic overload.

Factors that induce left ventricular hipertrophy

Hemodynamic factors

Increase of the metabolic necessity

The work overload is considered the most frequently involved factor in LVH. The increase in the heart activity can be associated to a higher physiological demand, such as when performing physical exercises8 and chronic anemia states9. Thus, as a consequence of the increased necessity to pump more blood to the peripheral sites, there is an adequate adaptation to the new demands.

Pressure and/or volume overload

Pathological conditions, such as arterial hypertension, aorta stenosis or coarctation, called pressure overload; or conditions such as aortic failure or interatrial communication, called volume overload, can promote hypertrophy due to the increased cardiomyocyte volume, together with an increase in size and alteration in the quality of the collagen matrix components5,7.

The stretching is capable to activate L-type Ca+2 channels (LTCC) (Figure 3), Na+ channels and Na+/H+ exchangers; to inactivate K+ channels; to activate adenylate-cyclase and phospholipase C10 (Figure 3), as well as being associated to the accumulation of phosphate inositols, which act as second messengers11. The alterations in the functioning of the ionic channels of the sarcolemma lead to variations in the intracellular ionic concentration, which can represent an initial stimulation to activate the mitogen-activated protein kinase (MAPK). Among the MAPK superfamily, the extracellular-signal regulated kinase (ERK), the c-jun NH2-kinase (JUNK) and the p38 kinase are mediators of the hypertrophic signaling of the myocardial cells, as they induce the gene transcription associated to hypertrophy12 (Figure 3). The mechanical stimulation can also activate the integrin receptors, located in the cell membrane between the extracellular matrix (ECM) and the protein complex that form the sarcomere Z line12. Signaling proteins, such as the steroid receptor coactivator (Src) tyrosine-kinase and focal adhesion kinase (Fak) are located in this mesh, in addition to others responsible for the start of the integrin-activated signaling process13 (Figure 3).

The mechanical stimulation also induces the local release of autocrine and paracrine factors through the myocardial cells, such as endothelin 1 (ET1), growth factors and cytokines [fibroblast growth factor (FGF), transforming growth factor b (TGFb), insulin-like growth factor (IGF) and cardiotrophin-1]12. These factors can bind to specific membrane receptors and activate calcineurin-, phosphokinase-C and MAPK pathway-coupled intracellular cascades and initiate the cascade of events responsible for the hypertrophic growth of the heart (Figures 3 and 4).

Neurohumoral factors

Catecholamines and sympathetic nervous system

The cardiomyocytes express b-adrenergic (b-AR) and a1-adrenergic (a1-AR) receptors. The stimulation of the b-adrenergic receptors (b-AR) activates the adenylate-cyclase through the interaction with the stimulatory G protein (Gs), which triggers intracellular cascades that activate protein kinase A (PKA), also stimulating p38-MPAK14. The chronic stimulation of the b-AR by the administration of isoproterenol15 induces the increase of cardiac mass, cardiomyocytes, myocardial fibrosis and progressive dysfunction, which culminates in heart failure. Acutely, the activation of a1-AR increases the contractility mediated by the Gq protein activation. The latter induces the phospholipase C activation, which stimulates the hydrolysis of the Phosphatidyl inositol present in the membrane, generating two second messengers, the diacylglycerol (DAG) and the inositol triphosphate (IP3). The IP3 stimulates the release of Ca+2 from the sarcoplasmatic reticulum, whereas the DAG activates the protein kinase C (PKC), and the latter induces hypertrophy in culture of neonatal myocytes16 (Figure 4).

Angiotensin II

Studies by Lindpaintner and Ganten17 report the synthesis of angiotensin II (Ang II) in myocytes as well as in fibroblasts and myofibroblasts. Its biological effects are basically mediated by the membrane receptors AT1 and AT2.

The Ang II interacts with the AT1 receptor, associated to the G protein, which stimulates phospholipase C. The latter induces the formation of inositol triphosphate as well as of diaglycerol, which causes the increase in the cytoplasmic concentration of Ca++, leading to the activation of protein kinase C (PKC) and of the adenylate-cyclase18. The Ang II, through the AT1receptor, is also capable to inducing an activation cascade through the tyrosine-kinases19. These tyrosine-kinases regulate intracellular effector pathways, including MAPK, which activate several protein transcription factors in the cardiomyocytes (Figure 4).

The AT2 receptors present a classical transmembrane structure of a G-protein associated receptor20. Studies by Senbonmatsu et al 21 have demonstrated that mice with AT2 receptor deletion presented hypertrophy attenuation induced by pressure overload. However, animals with AT1 receptor deletion did not present hypertrophy attenuation induced by pressure overload22, suggesting that the deficiency of AT1 receptors might be compensated by the AT2 receptors, or that the AT2 subtype can have a predominant role on the trophic effects of Ang II on cardiomyocytes.

Insulin

The insulin binds to the subunit a of the receptor that belongs to the membrane receptor family that has tyrosine-kinase capacity23, causes a conformational change in the b subunit, which leads to its autophosphorylation. in tyrosine and activates its tyrosine-kinase capacity. Once activated, the insulin receptor (IR) is capable of phosphorylating several intracellular substrates and among them, the insulin-receptor substrates (IRS-1-4), Shc (SRC Homology collagen) and Jak-224 (Figure 4). These phosphorylated proteins recruit and activate several intracellular effectors, with different cellular functions23. The ERK/MAPK pathway is involved in the control of growth (Figure 4) and of mitogenesis, whereas the activation of phosphatidyl inositol (PI) 3-kinase by the IRS-1 is preferably involved with the metabolic actions of insulin23,25 (via IRS/PI3K/AKT/mTOR) (Figure 4). In normal situations, the insulin activates the production of •NO in endothelial cells through a phosphatidyl inositol (PI) 3-kinase-dependent mechanism, leading to the increase of the local production of •NO26 with a consequent vasodilating and anti-apoptotic effect27.

The insulin induces the phosphorylation in tyrosine of the IRS-1, whereas agents that lead to insulin resistance, such as TNFa, free fatty acids, cellular stress and hyperinsulinemia induce the activation of kinases of serine/threonine that phosphorylate IRS-1 in serine, inhibiting its function28. Thus, the reduction in the activation of the phosphatidyl inositol (PI) 3-kinase/Akt pathway in parallel to the preserved activation of the ERK/MAPK pathway in a situation of insulin resistance and hyperinsulinemia are being considered as vital events for the development of cardiac hypertrophy29 (Figure 4).

Indirectly, insulin can also induce cardiac hypertrophy through the increase in the expression of the mRNA of the AT2 receptors30 and in the activation of the sympathetic nervous system3.

Oxidative stress

The imbalance between the production and removal of the reactive oxygen species (ROS) and reactive nitrogen species (RNS) is denominated oxidative/nitrosative stress, respectively. Several physiopathological and genetic situations can induce cardiac oxidative stress, such as the increase in the concentration of AngII31, hypercholesteremia32, mechanical stress in the myocardium33 and inflammatory processes34.

Hypertension and the mechanical stress in the myocardium induce the increase of ROS in the cardiomyocytes, activating the MAPK pathway33, which has an important role in cardiac hypertrophy. Additionally, the redox imbalance reduces the bioavailability of •NO in the cardiovascular system35, altering the balance between the hypertrophic factors (and/or proliferative) and the anti-hypertrophic factors (and/or anti-proliferative) (Figure 5), triggering a myocardial remodeling.

Hypercholesterolemia

The hypercholesterolemia, in addition to inducing the oxidative stress32, can alter the function and expression of the KATP channels in the myocardium36, inducing cardiac hypertrophy, simply due to the fact that the activation of these channels attenuates the cardiac hypertrophy through the inhibition of 70- KDa S6 Kinase37, an enzyme that acts as a trigger for protein synthesis in the myocardium remodeling.

Moreover, the hyperlipidemia is associated with the increase in the plasma concentration of ET1, leading to vasomotor alterations38. In addition to the vasoconstrictor property, the endothelin activates the hypertrophic signaling pathways in the cardiomyocytes: PKC and MAPK39.

Cytokines and growth factors induced by the inflammatory process

The trilogy consisting of the inflammatory process40, endothelial dysfunction41 and oxidative stress31, in the cardiovascular environment, is considered the common denominator among the conditions that promote and support cardiac hypertrophy.

The CD40L (ligand) is a transmembrane protein expressed on the surface of lymphocytes, endothelial cells, vascular smooth musculature cells and macrophages. This protein has a pro-oxidative effect and its interaction with its receptor CD40 induces the inflammatory response, favoring the acute coronary syndrome42. The interaction between CD40 and CD40L activates the Nfkappab43 pathway, promoting the phosphorylation of IKK (inhibitor of Kappa-b kinase), resulting in the translocation of the nuclear factor Kappa b (NFkappa b) to the nucleus, where it activates the genes involved in cell growth and inflammation44. The activation of the NFkappa b participates in the development of cardiac hypertrophy in mice, characterized by the increase in collagen deposit44.

The activation of the T lymphocytes results in the production of interferon-g which, in turn, increases the synthesis of inflammatory cytokines, such as TNF-a and IL-1. These cytokines induce the production of large amounts of IL-6, which stimulates the production of inflammatory proteins45 and cardiac hypertrophy in mice, through its interaction with gp-130 membrane receptors46. TNFa induces cardiac hypertrophy in vivo by determining the increase in the synthesis of structural and contractile proteins of the cardiomyocytes, as well as by inducing an increase in the expression of AT1 receptors, increasing the effect mediated by angiotensin II in favor of cardiac fibrosis47.

Nitric oxide/ nitric oxide synthase in the heart

Nitric oxide synthesis

The •NO is produced by enzymes called nitric oxide synthases (NOS). This is a family of complex enzymes that catalyze the oxidation of L-arginine to produce nitric oxide and L-citrulline. Three isoforms of NOS were initially characterized: the neuronal isoforms (nNOS = NOS1), identified in the brain; the induced isoform (iNOS=NOS2) in macrophages; and the endothelial isoform (eNOS=NOS3) in endothelial cells48 and cardiomyocytes49. nNOS and eNOS present constitutive expression [constitutive nitric oxide synthases] and produce low amounts of •NO, when activated by calcium (Ca+2)50. The iNOS is expressed only in response to pro-inflammatory stimuli and cytokines and can produce large amounts of •NO50.

In the healthy myocardium, the eNOS is mainly expressed in the coronary vascular endothelium and the cardiac endothelium51, as well as in the cardiomyocytes49. In these cells, the eNOS is located in the caveoli, anchored by caveolin-3 in the plasmatic membrane, near the L-type Ca+2 channels7. The nNOS is present in the intracardiac nervous ganglia, in the atrial nervous fibers and in some perivascular nervous fibers of the ventricles52. Its expression has also been detected in cardiomyocytes and cells from the smooth musculature of small and large coronary arteries of rats53. The adult heart does not normally express iNOS. The latter is activated by inflammatory process mediators in many types of cells, including endothelial cells and cardiomyocytes54. It can be identified in the cytosol7, but it has been found in the perinuclear space, Golgi apparatus, mitochondria and plasma membrane55.

Nitric oxide: anti-hypertrophic molecule

The two main endogenous substances involved in the anti-hypertrophic role of the heart are the atrial natriuretic peptide (ANP) and •NO. The ANP secreted by atrial granules in response to acute or chronic atrial stretching, has an anti-hypertensive, anti-hypervolemic and anti-hypertrophic function through the activation of guanylate-cyclase and the consequent increase in the levels of cyclic guanosine monophosphate (cGMP)56.

The production of •NO in the cardiomyocytes is highly compartmentalized57, as the result of the location of NOS. Due to the high natural reactivity of •NO, its synthesis close to its target aids its accessibility to the intracellular processes for a coordinated signaling. Regardless of the functional specificity of the cNOS in the cardiomyocytes, the •NO has an anti-hypertrophic effect in vivo, considering that nNOS- or eNOS-deficient mice developed spontaneous cardiac hypertrophic and that those animals with both nNOS and eNOS deficiency developed a more severe hypertrophy58.

The well-defined intracellular target of the •NO is the soluble guanylate-cyclase (sGC), which has a heme group that acts as an acceptor of •NO and catalytically converts the guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). Several studies have shown that the •NO promotes an anti-hypertrophic effect through the negative modulation of the pro-hypertrophic pathways7, with the cGMP-dependent pathway having a central role in this effect.

The increase in the cGMP in the intracellular environment inhibits, through the protein-kinase G (PKG) enzyme, the signaling network of the different pro-hypertrophic pathways that involve MAPK59. This •NO/cGMP/PKG pathway develops an important role in the negative regulation on cardiomyocyte hypertrophy induced by AngII, ET1, insulin and growth factors, inhibiting the MAPK/ERK signaling, decreasing the transcription of genes associated to hypertrophy60.

The •NO/cGMP/PKG pathway, in addition to inhibiting MAPK, also reduces the hypertrophic response induced by the mechanical overload and cytokines, inhibiting the following processes: 1) the calcineurin/nuclear factor of activated T cells (NFAT) pathway, through the decrease in the calcium inflow through the L-type calcium channels (LTCC)59; 2) the expression of the hypertrophic genes, such as cyclin D61; and 3) the activation of the NFkappa b transcription factor44. This pathway (•NO/cGMP/PKG) also interacts in an inhibitory way with the b-adrenergic stimulation pathways. The PKG induces a negative inotropic effect in the heart by: 1) phosphorylating troponin I, decreasing the sensitivity of the myofilaments to calcium; 2) inhibiting the calcium release from the sarcoplasmatic reticulum through the phosphorylation and inhibition of the IP3 receptors present in its membranes62. There is evidence that the •NO blocks the action of phospholipase C, inhibiting the release of calcium mediated by inositol triphosphate (IP3)63. All these effects can decrease the concentration of intracellular hypertrophic messengers activated by the adrenergic stimulation.

As for the KATP channels and ventricular hypertrophy, the •NO/cGMP pathway activates the KATP channels of the sarcolemma64, with probable inhibition of the 70-KDa S6 Kinase, attenuating the hypertrophic response in hyperlipidemic models37.

In addition to its anti-hypertrophic effects, the •NO has a dose-dependent pro-apoptotic effect on cardiomyocytes. Thus, low •NO concentrations inhibit the cardiomyocyte hypertrophy, whereas high •NO concentrations are required to induce the activation of caspases, DNA fragmentation and apoptosis65. The apoptosis stimulated by •NO in adult cardiomyocytes is associated to the alteration in the expression of pro-apoptotic genes of the Bcl-2 family, Bax and Bak, which have a crucial role in the determination of a cell’s destiny, partly due to the alteration in the permeability of the mitochondrial membrane66.

There is abundant evidence that •NO is the effector of cytokine-mediated apoptosis and the activation of pro-apoptotic genes, such as, for instance: 1) the capacity of the cytokines to induce the •NO production in cardiomyocytes is proportional to their capacity to activate programmed cell death (apoptosis); 2) iNOS antagonists prevent the production of •NO, of apoptosis and block the expression of Bcl-2 and Bak67.

The •NO generated by iNOS has been responsible for the induction of apoptosis in different types of cells67, including cardiomyocytes68. After the induction, the iNOS remains activated for 20 hours69, during which it synthesizes •NO at concentrations that are 1,000 higher than the cNOS70and, under conditions of deficient substrate or cofactors, it reduces molecular oxygen to superoxide7. The superoxide and the peroxynitrite, formed by the interaction of •NO with superoxide, are highly toxic to the cardiomyocytes7. Ing et al68 and Arstall et al71 determined that the apoptosis of cardiomyocytes induced by iNOS seems to be independent from sGC and cGMP, but it seems to be predominantly through the formation of peroxynitrite. The iNOS-mediated cytotoxicity was not exclusively confined to neonatal myocytes, but also in adult myocytes, where the endogenous •NO generated by the iNOS after the exposition to the combination of the cytokines INFg and IL-1b induces apoptosis 71.

Modulators of the bioavailability of nitric oxide

Nitric oxide donors

Among the compounds that present high potential as •NO donors are the low-molecular weight S-nitrosothiols (RSNOs). The RSNOs are endogenous species that were detected in the airway-lining fluid, platelets and neutrophils, where they act in the biological systems as •NO carriers, as free thiols or proteins containing cystein72.

The exogenous RSNOs are promising drugs to be used in the treatment of diseases that involve dysfunctions in the •NO bioavailability, as they offer advantages on the currently used drugs, due to the fact that they do not induce to tolerance in vascular cells73, as do the organic nitrate and sodium nitroprussiate.

Clinical studies have shown that the RSNOs can be beneficial in a series of cardiovascular disorders74. They can also have access to the intracellular compartment through the catalytic action of the membrane disulphide isomerase, associated to a nitrosylation reaction74. The members of this compound class include: S-nitrous-glutathione (GSN), S-nitro-N-acetylpenicillamine (SNAP), S-nitrous-albumin and S-nitrous-N- acetylcysteine (SNAC).

Angiotensin-converting enzyme inhibitors (ACEI)

The angiotensin-converting enzyme (ACE), in addition to generating ANGII, also degrades bradykinin75. The bradykinin is an endothelium-dependent vasodilator, stimulating the endothelium to produce •NO. Thus, the ACE inhibitors enhance the bradykinin and have been used due to their beneficial effect of increasing the bioavailability of •NO in the cardiovascular tissue76. Additionally, the ANGII can stimulate the production of superoxide, which would reduce the bioavailability of •NO77, an event that can be blocked by the ACE inhibitors.

Phosphodiesterase inhibitors

Phosphodiesterase inhibitors such as sildenafil, prolong the signaling action of •NO through the inhibition of cGMP hydrolysis. The inhibition of phosphodiesterase by phosphodiesterase inhibitors prevents the degradation of cGMP into GMP5', which prolongs the time of action of cGMP, keeping the intracellular concentration of Ca++ low in the smooth musculature of vessels and consequent vasodilation78.

Conclusion

The increase in the cardiomyocyte stretching is the main factor that induces the hypertrophic growth, but circulating substances, such as endothelin 1 (ET1), angiotensin II, insulin and the catecholamines, as well as growth factors and cytokines released locally by the myocardial cells and oxidative stress products, such as the superoxide anion (O2-), also induce the hypertrophic growth of the cardiomyocytes. These, in turn, activate second messengers such as phospholipase C (PLC), the mitogen-activated protein kinases (MAPK) and calcineurin. These activated proteins promote alterations in the nuclear factors and in the regulation of the hypertrophic genes. Thus, it seems that there is no isolated signaling cascade for each stimulus or response, but that multiples signaling molecules occur and can form a network of cascades with numerous elements, facilitating their crossing-over. Therefore, it becomes evident that the anti-hypertrophic effect of •NO is achieved through the negative modulation of the pro-hypertrophic pathways and that the cGMP-dependent pathway has a central role in this effect. However, •NO does not block just one of the intracellular signaling pathways, which would not be enough to effectively prevent hypertrophic ventricular growth. Therefore, the LVH seems to develop due to the loss of balance between the pro- and anti-hypertrophic signaling pathways. This new knowledge on the pro- and anti-hypertrophic signaling pathways will allow the development of new strategies for the treatment of pathological LVH.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Sources of Funding

There were no external funding sources for this study.

Study Association

This article is part of the thesis of doctoral submitted by José Antonio Dias Garcia from Universidade Estadual de Campinas (Unicamp).

References