INSTITUTO NACIONAL DE PESQUISAS DA AMAZÔNIA – INPA PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA, CONSERVAÇÃO E BIOLOGIA EVOLUTIVA - GCBEv

EXPRESSÃO DO GENE HIF-1α DURANTE A EXPOSIÇÃO DO CICLÍDEO crassipinnis (Heckel, 1840) À HIPÓXIA

WALDIR HEINRICHS CALDAS

MANAUS/ AM

Junho de 2016

WALDIR HEINRICHS CALDAS

Expressão do gene HIF-1α durante a exposição do Ciclídeo (Heckel, 1840) à hipóxia

Orientador: Dra. Vera Maria Fonseca de Almeida-Val

Dissertação apresentada no Programa de Pós-Graduação em

Genética, Conservação e Biologia Evolutiva (INPA-PPG GCBEv) como parte dos requisitos para a

obtenção do título de mestre em Ciências Biológicas, Área de Concentração em Genética,

Conservação e Biologia Evolutiva .

MANAUS/ AM

Junho de 2016 vC145 Caldas, Waldir Heinrichs

Expressão do gene HIF-1a durante a exposição do ciclídeo Astronotus crassipinnis (Heckel, 1840) à hipóxia / Waldir Heirichs Caldas. --- Manaus: [s.n.], 2016.

ix, 38 f.: il.

Dissertação (Mestrado) --- INPA, Manaus, 2016.

Orientadora: Vera Maria Fonseca de Almeida e Val

Área de concentração: Genética, Conservação e Biologia Evolutiva

1. Acará-açu. 2. Hipóxia. 3. Expressão génica. I. Título.

CDD 597.5

SINOPSE: Este trabalho apresenta as variações na expressão do gene HIF-1α em dois tecidos do cichlideo Astronotus crasspinnis exposto à hipóxia, onde se observou um aumento na expressão no fígado, em relação ao controle, enquanto no músculo não houve variação. Estes resultados de expressão condizem com os observados no perfil metabólicos destes dois tecidos e com os resultados da respiração deste , demonstrando que este animal ativa seu metabolismo anaeróbico e reduz o aeróbico durante hipóxia, com fortes relações entre a expressão do gene HIF-1α e a inibição da atividade da LDH.

Palavras-chave: Hipóxia; Astronotus crasspinnis; Expressão gênica; Metabolismo; Atividade enzimática; LDH.

i

À minha mãe, Eva de Oliveira Heinrichs Caldas, eterna melhor amiga. ii

Agradecimentos

À professora Dra. Vera Val por sua orientação, atenção, direcionamento e por sempre trazer estímulo e motivação aos seus alunos.

Ao professor Dr. Adalberto Val por sua liderença e exemplo, sempre nos ensinando os caminhos necessários para sermos grandes pesquisadores.

À minha “mãezona”, Eva de Oliveira Heinrichs Caldas, por todo amor, companheirismo, ensinamento e incentivo, sendo fundamental para o meu crescimento pessoal e profissional.

Ao meu pai, Waldir Caldas Junior, por ser sempre meu amigo, me guiar e ensinar os caminhos da vida, sendo a todo momento um exemplo de “pai de família”, sempre presente nas minhas derrotas e vitórias.

À minha noiva e amiga, Allana Negreiros, minha princesa que tanto me incentiva e, com muita paciência, abranda todos meus temores acadêmicos. Sem você nada teria sentido.

Ao meu grande amigo, Derek Campos, por toda a ajuda no planejamento e na execução desse projeto, por todas nossas conversas de bar, onde sempre planejamos os trabalhos futuros que vão revolucionar a ciência.

À “Naza”, Nazaré Paula, por toda paciência, “puxões de orelha” e ajuda diária fundamental para a execução e conclusão desse trabalho.

À minha amiga, Susana Braz, que apesar de gostar de mim “só às vezes”, sempre é uma boa companheira e sempre traz uma grande contribuição intelectual.

À toda equipe do LEEM por todos os debates, seminários e conversas de bastidor, além de estar sempre disponível para ajudar com experimentos e análises.

A todos meus amigos envolvidos direta e indiretamente na realização desse projeto, incluindo os amigos da turma do GCBEv/2014.

Ao INPA por toda a estutura disponível e aos professores e à equipe da coordenação do curso de Genética, Conservação e Biologia Evolutiva, pelo constante interesse na manutenção e evolução do curso.

À CAPES, ao CNPq e à FAPEAM pelo financiamento do projeto ADAPTA que custeou esse projeto. iii

À FAPEAM pela concessão da bolsa de estudo durante a realização deste projeto.

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“As coisas são semelhantes: isto faz a ciência possível; as coisas são diferentes: isso faz a ciência necessária”

(Levins e Lewontin, 1985) v

HIF-1α expression during hypoxia exposure and recover of the Amazon Astronotus crasspinnis (Heckel, 1840)

ABSTRACT

The aquatic habitats of the present deep oxygen level variation, and many aquatic developed biochemical and physiological adaptations to survive such changes. The advanced teleost Astronotus crassipinnis is tolerant to hypoxia and has the ability to depress its metabolic rate (oxygen consumption) and to increase its anaerobic activity when exposed to hypoxia episodes. Hypoxia-inducible factor-1α (HIF-1α) is the first gene related to hypoxia-tolerance, being regulated under these circumstances to maintain regular cellular function and control anaerobic metabolism. In the present work we studied HIF-1α expression and correlated the gene expression with changes in the metabolism of Astronotus crassipinnis exposed to 1, 3, and 5 hours of hypoxia, followed by 3 hours of recovery. The results show that A. crassipinnis presents metabolic depression under hypoxia and increases its anaerobic metabolism, and differentially regulates this gene in each of the studied tissues, with positive relationship with its metabolic profile, suggesting that HIF-1α might be the main factor to regulate responses to hypoxia tolerance.

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Expressão do gene HIF-1α durante a exposição do ciclídeo Astronotus crassipinnis (Heckel, 1840) à hipóxia

RESUMO

Os ambientes aquáticos da Amazônia apresentam variações profundas, diárias e sazonais, nos níveis de oxigênio. Com isso, vários animais aquáticos desenvolveram adaptações bioquímicas e fisiológicas para sobreviver em tais condições. Astronotus crassipinnis é um teleósteo tolerante à hipóxia e possui a habilidade de diminuir o consumo de oxigênio e aumentar o seu metabolismo anaeróbico. Hypoxia-Inducible factor (HIF) é um gene altamente relacionado à sobrevivência à hipóxia, sendo regulado nessas circunstâncias com o objetivo de manter o funcionamento celular normal e controlar o metabolismo anaeróbico. No presente estudo, foram relacionados os efeitos da expressão do gene HIF-1α com mudanças no perfil metabólico de Astronotus crassipinnis expostos a 1, 3 e 5 horas de hipóxia, seguindo um período de 3 horas de recuperação. Os resultados mostraram que esse animal deprime o seu metabolismo aeróbico e ativa o anaeróbico, aumentando a atividade da enzima LDH, regulando diferencialmente o gene HIF-1α em cada tecido estudado, com relações com seu perfil metabólico, sugerindo que esse gene pode ser um dos principais fatores na regulação de respostas à tolerância à hipóxia.

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SUMÁRIO

LISTA DE FIGURAS ...... vii LISTA DE TABELAS ...... ix 1. Introdução ...... 1 1.1. A hipóxia em ambientes aquáticos ...... 1 1.2. Adaptações da ictiofauna à hipóxia ...... 2 1.3. HIF-1 ...... 4 1.4. Astronotus crassipinnis ...... 5

2. Objetivos ...... 8 2.1. Objetivo geral ...... 8 2.2. Objetivos específicos ...... 8

CAPÍTULO 1 ...... 9 1. INTRODUCTION …………………………………………………………………..…….. 10 2. MATERIAL AND METHODS ………………………………………………………...… 12 2.1. Study Animals and Acclimation ……………………………………………… 12 2.2. Critical Oxygen Tension and Opercular Movements …………………….... 12 2.3. Experiment and Metabolites ……….…………………………………….. 13 2.4. Enzymatic Assays …………………………………………………………….. 14 2.5. Total RNA Extraction and First-Strand (cDNA) Synthesis …………...…… 14 2.6. Real-time Quantitative PCR …………………………………………………. 15 2.7. Statistical Analysis ……………………………………………………………. 15 3. RESULTS ………………………………………………………………………………… 16 3.1. Respirometry and Opercular Movements ………………………………….. 16 3.2. Metabolites and Enzymatic Activity ………………………………………… 16 3.3. LDH Inhibition Rates ...... 16 3.4. Gene Expression ……………………………………………………………… 17 4. DISCUSSION …………………………………………………………………………….. 17 5. REFERENCES …………………………………………………………………………… 22

4. Referências ...... 32

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LISTA DE FÍGURAS

INTRODUÇÃO

Figura 1. Vista lateral do cichlidae Astronotus crassipinnis...... 6

CAPÍTULO 1.

Figure 1. Opercular movement of Astronotus crassipinnis exposed to graded hypoxia

(n= 7, mean ±SEM). MO2 of Astronotus crassipinnis exposed to graded hypoxia (n= 7, -1 mean ±SEM). Line shows expansion of opercular movements (1.8 mg O2 . L ± 0.036) -1 immediately before reaching PO2Crit (PO2crit = 1.26 mg.l ± 0.038). *indicates significantly difference between each O2 concentration (P<0.05)……………………….27

Figure 2. Astronotus crassipinnis MO2 during experimental conditions (n=8, -1 mean±SEM, T= 28.3°C). Letters indicate differences between normoxia (7.42 mgO2 .L -1 -1 ± 0,10), hypoxia (0.70 mgO2 .L ± 0.05) and recovery (7.56 mgO2 .L ± 0.19) (P<0.05). ………………………………………………………………………………………………….27

Figure 3. Plasma glucose levels in Astronotus crassipinnis exposed to normoxia (7.42 -1 -1 mgO2 .L ± 0,10) and hypoxia (0.70 mgO2 .L ± 0.05) for 5 hours and recovery (7.56 -1 mgO2 .L ± 0.19) for 3 hours (n=6, mean± SEM). Letters indicate difference between time, normoxia, hypoxia and recovery (P<0.05). …………………………………………28

Figure 4. Plasma lactate levels in Astronotus crassipinnis exposed to normoxia (7.42 -1 -1 mgO2 .L ± 0,10) and hypoxia (0.70 mgO2 .L ± 0.05) for 5 hours and recovery (7.56 -1 mgO2 .L ± 0.19) for 3 hours (n=6, mean± SEM). Letters indicate difference between time, normoxia, hypoxia and recovery (P<0.05).……………………………………..….. 28

Figure 5. Relative expression of HIF-1α in skeletal muscle of Astronotus crassipinnis -1 -1 exposed to normoxia (7.42 mgO2 .L ± 0.10) and hypoxia (0.70 mgO2 .L ± 0.05) for 5 -1 hours and hypoxia recovery (7.56 mgO2 .L ± 0.19) for 3 hours (n=6, mean± SEM). Letters indicate difference between time, normoxia, hypoxia and recovery (P<0.05). ……………………………………………………………………………………………....….29

Figure 6. Relative expression of HIF-1α in Liver of Astronotus crassipinnis exposed to -1 -1 normoxia (7.42 mgO2 .L ± 0.10) and hypoxia (0.70 mgO2 .L ± 0.05) for 5 hours and -1 hypoxia recovery (7.56 mgO2 .L ± 0.19) for 3 hours (n=6, mean± SEM). Letters indicate difference between time, normoxia, hypoxia and recovery (P<0.05). ………………………………………………………………………………………………… 29

Figure 7. Correlates between HIF-1α expression and pyruvate inhibition rates in liver (A) and skeletal muscle (B). …………………………………………………………..……30

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LISTA DE TABELAS

Table 1. Real-time PCR primers used in this study. ……………………………………. 28

Table 2. Enzyme activities of lactate dehydrogenase (LDH 1mM), LDH inhibition rate (L/H), malate dehydrogenase (MDH), pyruvate kinase (PK) and citrate synthase (CS) in skeletal muscle and liver of Astronotus crassipinnis (n=6, mean ± SEM) exposed to -1 -1 normoxia (7.42 mgO2 .L ± 0.10) and hypoxia (0.70 mg O2 . L ± 0.05) for 5 hours and -1 recovery (7.56 mgO2 .L ± 0.19) for 3 hours (n=6 , mean± SEM). Letters indicate differences between time, normoxia, hypoxia and recovery (P<0.05). ……………………………………………………………………..………………………... 28

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1. Introdução

1.1 A hipóxia em ambientes aquáticos

A diminuição nos níveis de oxigênio dissolvido acontece naturalmente ou por ação antrópica. Um ambiente em que o oxigênio dissolvido é 0% é conhecido como anóxico, enquanto um ambiente com uma concentração baixa, variando de 1 a 30%, é denominado como ambiente hipóxico. Salinidade, temperatura, camadas de gelo, vegetação, difusão da luz, decomposição de matéria orgânica e oxidação de íons metálicos são fatores ambientais que podem influenciar a concentração de oxigênio no meio aquático (Rabalais et al, 1999).

Hipóxia e anóxia podem ocorrer em ambientes marinhos, regiões de estuário e rios de água doce. Os oceanos costumam ser ambientes estáveis em relação ao oxigênio dissolvido, sendo as regiões mais profundas, entre 200 a 1000 metros, as mais afetadas por que a taxa de consumo de oxigênio nesses ambientes excede a produção fotossintética (Levin, 2003). Hipóxia é comum em ambientes costeiros, regiões estuarinas e baías, sendo decorrentes de causas antrópicas como, por exemplo, o uso excessivo de fertilizantes, ou por causas naturais, devido a ondas e consumo de oxigênio em recifes de corais (Conley et al., 2002; Rabalais, 2002; Nilsson e Ostlund-Nilsson, 2004).

Na Amazônia, o ciclo anual das águas é o fator ambiental de maior dimensão. Segundo informações do porto de Manaus, a variação do Rio Negro nos últimos 50 anos foi, em média, 10 metros. Este ciclo anual possui efeito direto nas características químicas, físicas e biológicas dos ambientes aquáticos, inclusive na concentração de oxigênio dissolvido nas águas, quando os níveis de oxigênio dissolvido podem chegar a zero durante as cheias (Forsberg et. al., 1995; Val e Almeida-Val, 1996). Nos diferentes ambientes aquáticos amazônicos, como rios, lagos, igarapés, praias, várzeas e igapós, a concentração de oxigênio é bem conhecida, variando de 15 a 30% na maioria dos lagos. Em um trabalho realizado por Junk e colaboradores (1983) no lago Camaleão foi observada a flutuação nos níveis de oxigênio durante esse ciclo, demonstrando um aumento durante o período de seca e uma diminuição drástica durante a cheia. Outros estudos também mostram tal variação, como observado por Val e Almeida-Val (1996) durante a cheia, há uma redução nos níveis de oxigênio, criando ambientes hipóxicos ou anóxicos, enquanto no período de seca, observa-se uma saturação nos níveis de oxigênio, com exceção de ambientes com acúmulo de macrófitas, fator crítico na determinação dos níveis de oxigênio dissolvido em florestas de várzea e igapós (Junk, 1984; Val e Almeida-Val, 1996). Essas alterações nas águas 2 do rio Amazonas são cíclicas e muito mais acentuadas que em rios de zonas temperadas. Apesar disso, as variações diárias de oxigênio podem ser mais críticas, variando de uma concentração supersaturada ao meio dia, a zero durante a noite (Junk et al., 1983; Val e Almeida-Val, 1995). Essas variações sazonais e diárias causam impactos profundos na distribuição e ecologia dos peixes, como por exemplo, a sua distribuição e abundância. Para enfrentar essas situações, esses animais desenvolveram adaptações ao longo do processo evolutivo, as quais ocorrem em níveis comportamentais, morfológicos, fisiológicos e bioquímicos (Val e Almeida-Val, 1995; Crampton, 1998; Rabalais et al, 1999; Anjos et al., 2008).

1.2 Adaptações da ictiofauna à hipóxia

Adaptações para sobreviver à hipóxia são comumente observadas em peixes, anfíbios e alguns répteis, que vivem em ambientes sujeitos a alterações temporais nos níveis de concentração de oxigênio. A escassez de oxigênio no ambiente leva a alterações no metabolismo normal dos animais, fazendo com que essas adaptações sejam necessárias para a sobrevivência em variados ambientes (Bickler e Leslie, 2007).

O surgimento dos peixes é datado no período Cambriano (há cerca de 500 milhões de anos atrás) e estes constituíram a base da evolução dos vertebrados (Shu et al., 1999). A diversidade desse grupo se deu por sucessivos eventos de duplicação genômica (poliploidização) originando novos genes e promovendo radiações adaptativas que fizeram parte da história evolutiva destes organismos, tornando-os o grupo de maior diversidade genética entre os vertebrados (Ohno, 1970).

A ictiofauna apresenta inúmeras adaptações para compensar a baixa disponibilidade de oxigênio, seja diminuindo o consumo de oxigênio ou aumentando o metabolismo anaeróbico (Val e Almeida-Val, 1995; Bickler e Leslie, 2007; Hochachka, 1992), embora haja estudos demonstrando que alguns animais podem apresentar as duas estratégias simultaneamente (Almeida-Val et al., 1993). Esses animais também podem apresentar diferentes estratégias de respiração, como respiração aérea obrigatória ou facultativa e respiração na superfície aquática (ASR). Em trabalho realizado por Saint-Paul (1984) com o caracídeo amazônico Colossoma macropomum (tambaqui), foi constatado que, quando em condições de hipóxia severa (<0,5 mg

O2/L), o peixe utiliza a superfície da água para captar a camada d’água mais rica em oxigênio e, também, em concentrações abaixo da crítica, a espécie apresenta um aumento no batimento opercular, tendo suas trocas gasosas facilitadas pelas brânquias aumentadas. A espécie Arapaima gigas, endêmica da região amazônica, 3 sobrevive em ambientes de baixa concentração de oxigênio por meio de respiração aérea, efetuada através da bexiga natatória modificada, que funciona como um pulmão (Stevens & Holeton, 1978). O oxigênio desempenha um papel fundamental na respiração celular aeróbica, sendo necessário para a glicólise, sequência metabólica produtora do substrato piruvato que é posteriormente utilizado no ciclo respiratório. Com o acúmulo de piruvato no tecido, há um aumento na atividade da enzima lactato desidrogenase (LDH) que metaboliza esse piruvato em lactato e também desempenha a função reversa em tecidos específicos, transformando lactato em glicose ou glicogênio quando a demanda por glicose ainda é alta ou quando o animal não está mais passando por situação hipóxica. Na falta de oxigênio para os peixes, várias adaptações são observadas em diferentes tecidos, viabilizando a sobrevivência do animal. Por exemplo, durante hipóxia ou atividade física, os músculos permanecem com sua alta atividade anaeróbica, utilizando suas próprias reservas de glicogênio ou de outras fontes (Jensen et al., 1993; Val e Almeida-Val, 1995). Outra adaptação se dá no fígado quando o animal é exposto à hipóxia: o fígado do animal pode acumular glicogênio para que possa suprir a glicólise anaeróbica, mantendo assim a produção de ATP e o poder redutor sem consumo de oxigênio em tecidos anaeróbicos, como músculo branco (Hochachka, 1988). Almeida-Val e colaboradores (1993) mostraram que o tambaqui exposto à hipóxia por duas horas sem acesso à superfície eleva os níveis de ácido lático no sangue, mostrando uma dependência da glicólise anaeróbica durante o episódio de hipóxia; quando o ambiente é reoxigenado, em duas horas o tambaqui tem seus níveis de ácido lático plasmático voltando aos níveis normóxicos. A ativação do metabolismo anaeróbico é dependente do tecido/órgão do animal e do tempo de exposição à hipóxia, sendo sua sobrevivência nessas situações totalmente dependente da habilidade da espécie em reduzir sua taxa metabólica a níveis abaixo dos normais, diminuindo sua demanda por energia (Almeida-Val et al., 2000; Chippari- Gomes et al., 2005). Em trabalho realizado por Almeida-Val e colaboradores (1995), foram observadas, no ciclídeo Cichlasoma amazonarum, mudanças na distribuição das isoformas da enzima LDH, a qual tem papel crucial na regulação do metabolismo anaeróbico (Hochachka e Somero, 1984) em diferentes tecidos. Na espécie estudada por Almeida-Val e colaboradores(1995), a isoforma A4 foi mais ativa no cérebro e no coração e a isoforma B4 mais ativa no fígado, permitindo que esse animal, quando em hipóxia, utilize ambas adaptações: metabolismo anaeróbico e supressão da demanda energética. Estas diferentes estratégias adotadas em diferentes tecidos aparentam ser reguladas de acordo com as condições ambientais e disponibilidade de oxigênio por onde esses animais passam. 4

1.3 O gene HIF-1

Muitos trabalhos possuem como foco a conexão entre as mudanças nos níveis de oxigênio e suas consequências nas funções normais de uma célula ou organismo. Esses estudos abordam, geralmente, doenças humanas como cirrose hepática, derrame, cardiopatias, câncer, onde mudanças nos níveis de oxigênio causam alterações fisiológicas e patológicas (Bing et al., 1975; Hess e Manson, 1984). O gene HIF-1, Hypoxia-inducible factor-1, está ligado a essas deficiências causadas pela falta de oxigênio, pelo fato de ser uma resposta imediata a essa condição de pouco oxigênio disponível (Maxwell et al.,1993). Esse gene foi descoberto por Semenza e Wang (1992) em um trabalho que estudava a regulação da expressão de eritropoietina (EPO) em células de mamíferos, quando os autores observaram que havia um fator adicional que atuava na transcrição do EPO, que seria o HIF-1.

Quando os níveis de oxigênio são limitantes, a célula sinaliza a produção de fatores indutores de hipóxia (HIF). O polipeptídeo HIF-1 é uma hélice-volta-hélice, estrutura característica de fatores de transcrição, formado pela união das subunidades HIF-1α e HIF-1ß, que se liga em regiões específicas do DNA para acelerar a transcrição de genes regulados por oxigênio. HIF-1α é a subunidade relacionada à hipóxia e possui uma região chamada domínio de degradação dependente de oxigênio (ODD). Durante o período de normóxia, a região ODD medeia a detecção do polímero HIF-1α pela proteína Von Hippel-Lindau que medeia a degradação da subunidade HIF- 1α (Chaudhary et al., 1999; Maxwell et al.,1993). Em condições de hipóxia, HIF-1α é preservada e transportada para o núcleo celular, onde se polimeriza com a subunidade HIF-1β produzindo a proteína HIF-1, que é a proteína de resposta imediata da célula cujo papel é regular diversos genes, incluindo aqueles ligados à degradação da glicose, ativando, também, genes importantes para a manutenção da homeostase do oxigênio, aumentando a vascularização em áreas mais afetadas pela hipóxia e mudando o metabolismo da célula de aeróbico para anaeróbico afim de manter a produção de energia em forma de ATP na ausência de oxigênio (Carmeliet et al., 1998; Vaupel, 2004; Nikinmaa & Rees, 2005; Semenza, 1999; Wenger, 2002; Bruick, 2003; Kajimura et al, 2005)

A subunidade HIF-1α é criticamente regulada pela tomada de oxigênio e promove o metabolismo anaeróbico (Semenza, 2000). Apesar dos peixes serem um modelo excelente para estudar esse gene, uma vez que vivem em ambientes com flutuações drásticas na concentração de oxigênio dissolvido, poucos estudos foram realizados sobre os padrões de expressão desse gene em peixes. Ainda que existam 5 poucos estudos, sabe-se que o HIF-1α apresenta uma expressão diferenciada para os tecidos, como por exemplo, retina, gônadas, fígado, músculo esquelético e cardíaco, cérebro, coração e rins (Soitamo et al., 2001; Bruick, 2003; Terova et al., 2008), demonstrando a especificidade desse gene para cada tecido, e de acordo com seus padrões, essa expressão também é específica entre espécies. Enquanto alguns estudos demonstram que a expressão desse gene varia quando animais são expostos à hipóxia (Law et al., 2006; Terova et al., 2008; Rimoldi et al., 2012; Kodama et al., 2012), em alguns casos, não há variação nos seus níveis (Shen et al., 2010), o que parece estar relacionado à história de vida desses animais.

1.4. Astronotus crassipinnis

A espécie Astronotus crassipinnis, popularmente conhecida como acará-açu, possui o corpo alto e oval, com a cabeça, os olhos e a boca grandes. O corpo possui uma coloração que varia entre marrom-escuro no dorso e amarelo-alaranjado na região ventral, como mostra a figura 1. Uma das suas principais características é a ausência de ocelos na nadadeira dorsal, o que o diferencia de seu congênere Astronotus ocellatus (Ferreira et al., 1998). Esta espécie pertence à família Cichlidae, ordem Perciformes, a qual possui aproximadamente 9.300 espécies, sendo a mais diversa de todas as ordens de peixes (Nelson, 1994). A ordem Perciformes é predominantemente marinha, com algumas famílias vivendo em água doce em todo o mundo. São considerados peixes ósseos avançados (Galvis et al., 2006). A família Cichlidae é de origem marinha e apresenta uma das maiores diversidades dentre os peixes ósseos, com aproximadamente 1300 espécies identificadas (Kullander, 1998). A família está distribuída tanto em regiões tropicais quanto em regiões subtropicais, sendo que a maior parte vive em água doce (Ferreira et al., 1998). A maioria dos ciclídeos neotropicais habita ambientes lênticos, dentro de rios, lagos e igarapés, sendo que apenas algumas poucas espécies apresentam hábitos migratórios (Kullander, 1998). A desova dos ciclídeos é parcelada e os indivíduos possuem hábitos territoriais. Algumas espécies apresentam dimorfismo sexual, cuidado parental, criam ninhos no período de desova e guardam sua cria recém desovada na boca, quando há indicação de perigo. São espécies de grande, médio e pequeno porte e algumas espécies possuem importância comercial, tanto para a subsistência quanto para o setor ornamental e a pesca esportiva (Kullander, 1986). 6

Figura 1 Vista lateral do cichlidae Astronotus crassipinnis (Fonte: oscarfish.com).

O acará-açu habita lagos de florestas alagadas, locais rasos e com pouca corrente nas margens, vivendo entre as galhadas e em bancos de macrófitas (Sánchez-Botero e Araújo-Lima, 2001). São peixes que formam casais e apresentam cuidado parental, com deposição de 600 a 700 ovos por desova (Galvis et al., 2006).

Os hábitats de sua preferência, lagos e florestas de várzea, sofrem mudanças bruscas, diárias e sazonais, na concentração de oxigênio dissolvido, passando por períodos de hipóxia e até mesmo anóxia (Junk, 1985). Para que seja possível a sobrevivência nesses locais, o Astronotus crassipinnis possui uma maior resistência a essas condições adversas. Chippari-Gomes e colaboradores (2005) observaram que após exposição gradual a baixas concentrações de oxigênio (<0,34 mgO2/L), essa espécie aumentou os batimentos operculares para compensar a tomada de oxigênio, aumentou os níveis de glicogênio no fígado como resultado da gliconeogênese, aumentando também os níveis de lactato e glicose no sangue, indicando um transporte de glicose para os seus tecidos e o transporte de lactato para ser metabolizado no fígado, mostrando que essa espécie é bastante adaptada a ambientes com baixas concentrações de oxigênio. Além disso, já foi observado que o congênere Astronotus ocellatus pode sobreviver aproximadamente quatro horas em anóxia, em temperatura ambiente (~28°C) (Muusze et al., 1998). Adicionalmente, essa espécie apresentou mudanças nos níveis (atividades) de diversas enzimas do metabolismo glicolítico e oxidativo em diferentes tecidos, mostrando que cada órgão tem diferentes modos de controlar seu metabolismo aeróbico e anaeróbico. No trabalho de Muusze e colaboradores (1998) foi observado que esses peixes são altamente resistentes à hipóxia, podendo sobreviver por mais de 16 horas em situações de hipóxia extrema, com concentrações de oxigênio dissolvido na água 7 menores que 0.4 mg/L, e até 4 horas em um ambiente anóxico em temperatura ambiente em torno de 28°C. Scott e colaboradores (2008) observaram que o A. crassipinnis regula seu metabolismo quando exposto à hipóxia progressiva e deprime esse metabolismo com respostas coordenadas em diferentes tecidos, ativando o metabolismo anaeróbico para a manutenção dos níveis de ATP. Essa alta tolerância está relacionada ao seu tamanho corporal; sua resistência à hipóxia aumenta durante o crescimento do animal graças ao aumento na sua capacidade de deprimir seu metabolismo e aumentar sua atividade anaeróbica (Almeida-Val et al., 2000). Esses estudos mostram que o A. crassipinnis é muito eficaz em equilibrar o suprimento de oxigênio com a demanda por ATP e, então, tolerar períodos prolongados de hipóxia, sendo capaz de visitar ambientes com baixas concentrações de oxigênio para alimentação, acasalamento, refúgio, etc. (Anjos et al., 2008). Ao estudar a expressão de dois genes relacionados ao metabolismo anaeróbico, HIF-1α e VEGF, Baptista e colaboradores (2016) observaram, na espécie congênere Astronotus ocellatus, um aumento da expressão dos dois genes e mostraram que essa resposta está relacionada à alta tolerância à hipóxia do animal. Apesar do HIF-1α estar altamente relacionado a respostas em baixas concentrações de oxigênio, esse ainda é o único estudo realizado com esse gene em peixes amazônicos. Estudos sobre os diferentes padrões de expressão do HIF-1α em diferentes tecidos são importantes para se entender como esses transcritos podem regular a depressão metabólica e o aumento do metabolismo anaeróbico desses animais altamente tolerantes à hipóxia quando expostos a baixas concentrações de oxigênio.

8

2. Objetivos

2.1. Objetivo Geral

Caracterizar a progressão da expressão do gene HIF-1α por meio de seus transcritos e relacioná-la às consequências metabólicas após a exposição à hipóxia no teleósteo altamente tolerante à hipóxia, Astronotus crassipinnis.

2.2. Objetivos específicos

(1) Investigar as mudanças na taxa metabólica de animais em diferentes concentrações de oxigênio, determinar a concentração de oxigênio na qual os animais mudam de estado regulador para estado conformista e medir a taxa metabólica durante exposição à hipóxia, seguida de recuperação;

(2) Investigar o perfil metabólico dessa espécie nas mesmas condições de hipóxia, no fígado e no músculo esquelético branco;

(3) Quantificar a expressão do HIF-1α nos músculos e no fígado de animais expostos à hipóxia, seguida de recuperação.

9

CAPÍTULO 1

METABOLIC RESPONSES OF Astronotus crassipinnis (Heckel, 1840) EXPOSED TO HYPOXIA AND RECOVER ARE RELATED TO CHANGES IN EXPRESSION OF HIF- 1α IN LIVER BUT NOT IN SKELETAL MUSCLE

Heinrichs-Caldas, W1*.; Campos, D. F.1; Paula-Silva, M. N.1; Almeida-Val, V. M. F.1;

1LEEM – Laboratório de Ecofisiologia e Evolução Molecular – Instituto Nacional de Pesquisas da Amazônia – Manaus, Amazonas

*Corresponding author

Phone: +55 92 3643-3188

E-mail address: [email protected] (Heinrichs-Caldas, W.)

ABSTRACT

The aquatic habitats of the Amazon basin present deep variation of oxygen level, and, to survive such changes, many aquatic animals developed biochemical and physiological adaptations. The advanced teleost Astronotus crassipinnis (Perciformes) is tolerant to hypoxia and has the ability to concomitantly depress its metabolic rate and increase its anaerobic activity when exposed to hypoxia episodes. Hypoxia-Inducible factor-1α (HIF-1α) is the first gene related to hypoxia-tolerance, being regulated under these circumstances to maintain regular cellular function and control anaerobic metabolism. In the present work we studied HIF-1α expression and correlated it with changes in the metabolism of Astronotus crassipinnis exposed to 1, 3 and 5 hours of hypoxia, followed by 3 hours of recovery. The results show that A. crassipinnis presents metabolic depression under hypoxia and increases its anaerobic metabolism, and differentially regulates HIF-1 gene in each tissue studied, with positive relations to its metabolic profile, suggesting that HIF-1α might be the main factor to regulate responses to hypoxia tolerance in this species.

Keywords: Amazon - Astronotus crassipinnis – Hypoxia – Respirometry – Enzyme activity – HIF-1expression.

10

1. INTRODUCTION

Aquatic habitats of the Amazon basin are characterized by daily and seasonal fluctuations in oxygen levels, thus having profound impacts on the ecology of species, such as distribution and abundance of (Crampton, 1998; Anjos et al., 2008). Temperature, vegetation, and water column are the main factors that influence the oxygen concentration (Rabalais et al, 1999; Val and Almeida-Val, 1996). Dissolved oxygen has a crucial role in survival and development of fishes, therefore these animals have their morphology, behavior and physiology adapted to face these conditions (Hochachka, 1986; Almeida-Val et al., 2006). Such adaptations resulted from the appearance of different respiration strategies, as obligatory and facultative air breathing, aquatic surface respiration (Saint-Paul, 1984; Braum and Junk, 1982; Val, 1995; Brauner and Val, 2006) and metabolic depression (Almeida-Val et al., 2000). Amazonian fishes can rely in two metabolic strategies to survive hypoxia: increasing anaerobic glycolysis or decreasing ATP demand by depression of aerobic metabolism throughout the reduction of oxygen consumption (Hochachka, 1992). Although not very common, some species may present both strategies, as described by Almeida-Val and colleagues (1993) for the cichlid Cichlasoma sp. In anaerobic metabolism, lactate dehidrogenase (LDH) has a crucial role in regulating the ATP production via pyruvate oxidation, avoiding its accumulation by producing lactate and regulating the levels of these metabolites in accordance with oxygen availability. This enzyme is responsible for lactate production and its oxidation back to pyruvate, which is further processed to replace the glycogen via gluconeogenesis (Hochachka and Somero, 1984). All this metabolic features improve survival of these fishes under acute or intermittent hypoxia.

When oxygen level is limiting, the cell signalizes the production of a hypoxia- inducible factor (HIF), a heterodimer composed by two subunits (α and β), that binds to specific site in the DNA to accelerate the transcription of O2 regulated genes (Bashan et al., 1992; Chaudhary et al., 1999). HIF-1β is the constitutive subunit and isn’t sensible to O2 variations; on the other hand, subunit HIF-1α and its nuclear accumulation is highly regulated by hypoxia (Uchida et al., 2004). HIF-1α has the basic helix-loop-helix (bHLH) structure and the oxygen-dependent degradation domain (ODD). Under normoxia, ODD site provides HIF-1α detection and ubiquitination by Von Hippel-Lindau protein, which insures proteasome-mediated degradation. When in hypoxia, HIF-1α is stable and enters the nucleus where it dimerizes with HIF-1β forming HIF-1 protein, allowing the activation of more than one hundred genes related to glycolysis, gluconeogenesis, iron metabolism, cell survival and proliferation and 11 muscle contraction (Semenza, 1999; Wenger, 2002; Bruick et al., 2003; Kajimura et al., 2005) improving hypoxia tolerance and survival time.

The function of HIF-1α is to critically regulate oxygen uptake and intermediate anaerobic metabolism (Semenza, 2000). Although is the perfect model to evaluate this gene, since it experiences wide fluctuations in oxygen tensions in its natural environment, few published reports are available on specific patterns of tissue gene expression in animals exposed to hypoxia. Even with few collected records, its known that the gene HIF-α is differently expressed in many fish tissues, such as eyes, gonad, liver, brain, muscle, heart, spleen and kidney (Soitamo et al., 2001; Bruick, 2003; Terova et al., 2008), suggesting that this gene is tissue specific, and also species- specific. While some authors suggest that the mRNA levels can increase when animals are exposed to hypoxia (Law et al., 2006; Terova et al., 2008; Rimoldi et al., 2012; Kodama et al., 2012), in some cases, there is no variation in expression (Shen et al., 2010), which seems to be related to the life history of these animals.

Astronotus crassipinnis is an Amazon hypoxia-tolerant teleost inhabiting rivers, lakes and flooded forests with preference for lentic environments (Kullander, 1998; Santos et al., 1984), and it is a great model for studies about cellular signalization and molecular adaptations, since it shows unique features to tolerate hypoxia. During low oxygen levels, this animal increases its opercular movements as a ventilatory compensation, regulates enzyme activities to maintain anaerobic pathways and survives long-term hypoxia episodes (Chippari-Gomes et al., 2005; Muusze et al., 1998). The hypoxia tolerance for this species is related to body size; the resistance to hypoxia tends to increase as the animal grows due to the increase in the capacity to suppress metabolic rate and rely on anaerobic energy supply (Sloman et al., 2006; Almeida-Val et al., 2000). Scott and colleagues (2008) observed that this animal regulates its metabolism under progressive hypoxia and depresses the metabolism with coordinated responses in many tissues, recruiting anaerobic metabolism to maintain ATP supply. These studies identified A. crassipinnis’ amazing capacity to match cellular O2 supply and requirement and, then, its tolerance during prolonged periods of hypoxia, being capable to visit environments with low oxygen concentration for feeding, breeding, etc (Anjos et al., 2008; Almeida-Val et al., 1995). Studying the expression of two genes related to anaerobic metabolism, HIF-1α and VEGF, Baptista and colleagues (2016) observed, in the congeneric species Astronotus ocellatus, an increase in expression of both genes in liver, relating them to fish metabolic responses and how these traits can be the key to enhance hypoxia tolerance in extremely tolerant species. Although HIF-1α has this main role in determining hypoxia tolerance, as far as 12 we know, this is the unique study realized with amazonian fishes as regard as HIF-1α expression.

Studies in the expression patterns of HIF-1α in different tissues are important to understand how its transcripts regulates the metabolic depression and the increase of anaerobic metabolism in these hypoxia-tolerant animals when exposed to low oxygen tension. Accordingly, the main objective of this study is to characterize the progression of HIF-1α expression and how it is related to the metabolic consequences of hypoxia exposure in this hypoxia-tolerant Amazon fish, Astronotus crassipinnis. To reach this goal, we aimed (1) to investigate the changes in A. crassipinnis’ metabolic rates in different oxygen concentrations to determine PO2crit value, and also verify metabolic depression during hypoxia exposure; (2) to investigate the metabolic responses of this species under hypoxia and recovery in muscle and liver; and (3) to quantify HIF-1α transcripts in fish exposed to hypoxia, followed by hours recovery in white skeletal muscle and liver.

2. MATERIALS AND METHODS

2.1. Animals and Acclimation

Juvenile of Astronotus crassipinnis were purchased from a commercial supplier (Fish Farm Santo Antônio, AM 010, Km 113, Ramal do Procópio, Km 1, Rio Preto da Eva, AM, Brazil) and transferred to the Laboratory of Ecophysiology and Molecular Evolution at INPA (Amazon National Research Institute), Manaus (AM), for acclimation. The animals were held outdoors in 250 L tanks under constant aeration at room temperature and regular water changes, under natural light, during 3 months. These fishes were fed daily with commercial pelleted food (37% protein) until 24 hours prior to the experiments. The use of these animals is in accordance with CONCEA Brazilian Guide for Animals Use and Care under INPA´s authorization (protocol #001/2016).

2.2. Critical Oxygen Tension and Opercular Movement

These experiments were conducted in the Laboratory of Ecophysiology and

Molecular Evolution (INPA). The critical oxygen tension (PO2crit) was determined prior to the following experiments to settle the oxygen concentration for hypoxia experiments. The PO2crit methodology is the same used in previous works (Campos et. al., 2016) as first described by Steffensen (1989). Eight individuals (45.3 g ± 3.5) were 13 initially kept for 3 hours in respirometry chambers (70 ml cylindrical glass) to recover from handling with continuously water flush and controlled temperature (28°C ± 0.5) inside a bath aquarium. An automated apparatus DAQ-M (Loligo System, Tjele, Denmark), which works in recirculating cycles, was used to measure oxygen consumption in the following steps: wait, flush, and measurement, with time between them to be determined, with this, it is determined the duration of a ‘loop’. The recirculating cycle is controlled by AutoResp software (Loligo System). During the flush phase, peristaltic pumps were used to exchange the water of the chambers with the aquarium. The oxygen measurement of the chambers occurred through optical cables connected to OXY-4 or Witrox-4 (Loligo System) and to sensors spot attached inside the chambers. The determination of PO2crit was obtained by suppressing the flush phase, so the PO2 decreased as the oxygen was consumed inside the chambers. The oxygen consumption rate was calculated, and PO2crit was determined as the point where the PO2 regression line of the oxygen regulation intersected the oxygen conforming, initiating the suppressed metabolic rate by segmented linear regression using the software SegReg (www.waterlog.info) (De Boeck et al., 2013). During the

PO2crit experiment, we measured the opercular movement. The opercular movements were counted directly for 5 min in each concentration and expressed in beats.min-1.

-1 -1 After determining the PO2crit, the routine metabolic rate, MO2 (mg O2. kg . h ), was measured simulating the main experiment set up. The animals (41.2 g ± 5.8) were kept in the same situation as above described, except this time fish was exposed to a full loop in the respirometry chamber (180 s flush + 90 s wait + 360 s measurement). -1 The experiment consisted of 5 hours in normal oxygen concentration (7.33 mg O2.L ± -1 0.12), followed by 5 hours in hypoxia (0.70 mg O2.L ± 0.05), where the oxygen was decreased slowly by dislocating O2 with nitrogen gas directly into the water. After the -1 hypoxic period, the oxygen level was returned to normal concentrations (7.33 mg O2.L

± 0.12) through full aeration, and the MO2 was measured for 3 hours. The MO2 was calculated as MO2 = -∆O.Vresp.B-1, where ∆O is the rate of change in oxygen -1 concentration (mgO2. h ), Vresp is the volume of the respirometry chamber, and B is the mass of the individual (kg).

2.3. Experiment and Hematological Assays

Forty eight animals (twenty four for control and twenty four for hypoxic treatment), weighting 48.6 g ± 4.7, were individually separated in 40 liters glass aquaria, filled with 30 liters water each, covered with plastic bubble to ensure oxygen from air did not diffuse into the water. The animals were transferred to the aquaria 24 14 hours prior to the experiment for acclimation. During this period the water temperature -1 was kept at 28°C ± 0.2 with constant aeration (O2 concentration: 7.42 mg O2.L ± 0.10).

The PO2 level was decreased slowly by N2 pumping directly in the water and the animals in hypoxic treatment were exposed for a period of 1 hour, 3 hours and 5 hours -1 hypoxia (0.71 mg O2.L ± 0.09) and 3 hours of recovery, when the PO2 was taken back -1 to normal concentrations (7.53 mg O2.L ± 0.15). Dissolved oxygen concentration was monitored with an oximeter. For each period of exposition, six animals (n=6) were sampled and blood was immediately drawn from the caudal vein into heparinized syringes. After blood sampling the animals were weighted and euthanized by head concussion followed by a cut in the spine cord. Then, white skeletal muscle and liver were excised and stored at -80°C until the assay of enzyme activity and gene expression. Plasma glucose determination was performed according to manufactory instructions with the Glucose Liquicolor Kit (InVitro), an enzymatic colorimetric quantitative method. Lactate concentration was determined according to Sigma Chemical Co. protocol.

2.4. Enzymatic Assays

Lactate dehydrogenase (LDH; E.C. 1.1.1.27), malate dehydrogenase (MDH; E.C.1.1.1.37), citrate sintase (CS; E.C. 4.1.3.7) and pyruvate kinase (PK; E. C.2.7.1.40), were measured in the white skeletal muscle and liver following a well- established protocol for amazonian fish tissues (Driedzic and Almeida-Val, 1996). For muscle and liver, 0.01 g of tissue was homogenized in a 4x imidazole buffer (50 mM imidazole, 1 mM EDTA, and 1 mM DTT at pH 7.4) and centrifuged at 10.000xg in a Refrigerate Centrifuge 5430 R (Eppendorf, Hamburgo, GE) for 15 min at 4°C. The enzyme levels were measured according to the oxidation of NADH at 340 nm (mM extinction coefficient = 6.22) for LDH, MDH and PK, and the oxidation of DTNB at 412 nm (mM extinction coefficient = 13.6) for CS. LDH, MDH and PK assays were performed in 0.2 ml plate containing 0.15 mM NADH, 1 mM KCN and 50 mM imidazole, pH 7.4 at 28°C. The LDH reactions started with 1 and 10 mM pyruvate. The pyruvate inhibition rates were calculated to obtain the LDH inhibition values. LDH 1/10 mM ratios indicate predominance of aerobic metabolism (>1) or anaerobic metabolism (<1) (Hochachka and Somero, 2002). MDH reaction was initiated with 0.5 mM oxalacetate (OAA). PK reaction was initiated with 2,5 mM phosphoenolpyruvate. CS assays were performed in a 0.2 mL plate with 0.25 mM DTNB and 75 mM Tris base, and the reactions were initiated with 0.04 mM acetyl Co-A and 5 mM oxaloacetate. The enzymatic activities were determined at 25°C using the plate spectrophotometer SpectraMax Plus 384 (Molecular Devices, Sunnyvale, CA, USA). 15

2.5. Total RNA extraction and first-strand (cDNA) synthesis

Total RNA was isolated from liver and skeletal muscle using TRIzol Reagent (Life Technologies, CA, USA). Approximately 50 mg of tissue was used following manufacturer’s instruction. Non-denaturing agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) were used to guarantee the integrity and concentration of total RNA. A260/280 values were above 2 and electrophoresis showed that 28S and 18S rRNA were intact. After the extraction a DNase (Invitrogen, CA, USA) treatment was done according to manufacturer’s instructions. cDNA synthesis was obtained using 1 g of total with Revertaid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to manufacturer’s protocol. The purity and concentration of all cDNA samples were verified through NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA)

2.6. Real-time quantitative PCR

1000 ng of cDNA from the experimental samples in triplicate were used for real- time PCR measurements, which were performed using Fast SYBR® Green Master Mix (Applied Biosystems, Foster City, CA, USA) on a Viia 7 Dx PCR-System (Applied Biosystem). The primer pairs of HIF-1α and reference genes 28S, 18S and β-actin were specified for the congeneric specie Astronotus ocellatus and are listed in table 1. The amplification of reference genes was constant during the experiment, qualifying them as strong housekeeping for hypoxia. The efficiencies of these four genes are all approximately 100%, which ensures that the ΔΔCt equation is used correctly. The total 10 μl PCR mixture contained 5 μl Fast SYBR® Green Master Mix (Applied Biosystems), 2 μl nuclease free water, 1 μl sample cDNA, 1 pmol of each primer, forward and reverse, in triplicate for each sample. The PCR was performed under the following conditions: pre-denaturation at 95°C for 20 s, then 40 cycles: 95°C, 1 s; 60°C for 20 s, 95°C 15s, with data collection after each cycle. Next, the melting/dissociation curve analysis was performed with 95°C, 15 s; 60°C, 60 s; 95°C 15 s. The relative quantification was calculated by using 2- ΔΔCt method (Livak and Schmittgen, 2001)

2.7. Statistical analysis

Data are expressed as mean ± SEM. The normality and homogeneity were checked before parametric testing. Relative quantity values of gene expression, enzyme activities, and hematological parameters were examined by two-way analysis of variance (two-way ANOVA) testing for time of exposition between normoxia, 16 hypoxia, and recovery. Metabolic rate and opercular movements were tested by one- way ANOVA, testing between oxygen concentrations. Statistical analysis were done in SigmaStat (v. 3.5) and graphics were done in SigmaPlot software (v. 11.0)

3. RESULTS

3.1. Respirometry and opercular movements

When exposed to progressive hypoxia, Astronotus crassipinnis decreased MO2 in relation to normoxia (P<0.0073 ; F=2.24) and increased opercular movements (P<0.00002; F=6.38). The critical oxygen tension was 1.26 ± 0.038 mg.L-1. The increase in ventilation rate coincided with the PO2crit with an expansion in movements -1 noted at 1.8 ± 0.036 mgO2. L (Fig. 1). During the hypoxia experiment we observed a reduction in MO2 levels at low oxygen concentration during the hole exposure, which turned back to normal levels after 3 hours recovery (P<0.0081; F=2.76) (Fig. 2).

3.2. Metabolites and Enzymatic activity

In hypoxia, plasma glucose (P<0.0002; F=4.26) and lactate (P<0.012; F=3.16) levels increased in relation to control. Plasma lactate concentration increased during hypoxia exposition with a peak at 5 hours. Both parameters turned back to normal levels after 3 hours recovery (Fig. 3 and 4).

Enzymatic activities showed different responses in muscle and liver, reflecting the MO2 at various treatments. White skeletal muscle increased LDH activity after 3 hour hypoxia, returning to normal levels after recovery (P<0.047; F=3.68) while in liver LDH activity increased after 5 hour hypoxia, decreasing after recovery (P<0.006; F=3.34). MDH showed no difference between normoxia and hypoxia, both in white skeletal muscle (P<0.00081; F=6.00) and liver (P<0.015; F=2.86), except for a peak after hypoxia recovery in white skeletal muscle. CS and PK followed a pattern as similar as the metabolic rates. In white skeletal muscle (CS: P<0.016; F=2.91; PK: P<0.00005; F=6.32) and liver (CS: P<0.0012; F=4.28), these enzymes decreased in activity during hypoxia and increased after recovery returning to normoxic values (table 2).

3.3. LDH inhibition rates

The pyruvate inhibition ratios (1/10 mM) were used to calculate the lactate dehydrogenase inhibition ratios (LDH L/H). After 5 hours of hypoxia, inhibition ratios 17 were lower than 1.0 in liver, and in skeletal muscle, after 3 and 5 hours of hypoxia the values were below 1.0. According to Hochachka and Somero (2002), LDH 1/10 mM ratios below or close to 1.0 indicate anaerobic metabolism (LDH enzyme activation) and values higher than 1.0 indicate aerobic metabolism (LDH enzyme inhibition).

3.4. Gene expression

Skeletal muscle and liver showed contrasting results for HIF-1α mRNA expression. In white skeletal muscle there was no difference in expression (P<0.031; F = 2.60) between treatments (Fig 5). However, in liver, HIF-1α mRNA transcripts increased significantly (P<0.005; F = 3.67) after 3 and 5 hours hypoxia, with highest values at 5 hours, returning to normoxic levels after recovery (Fig 6).

4. DISCUSSION

Regular oxygen variations tend to happen in Amazon aquatic systems; these variations occur daily and seasonally, when the animals are exposed to more than five hours of hypoxia in várzea lakes and igapós (both flooded areas). This environmental oscillation drifted fishes to adapt their morphology, physiology and biochemistry, not only to survive theses changes but also to better perform their life needs in these conditions, such as reproduce and feed (Val and Almeida-Val, 1996). The increase in opercular movements during hypoxia is well described for Amazonian fishes (Rantin et al., 1992; de Salvo Souza et al., 2001; Oliveira et al., 2004; Leite et al., 2007, Chippari- Gomes et al., 2005) as a response to an increase in ventilation frequency. In the present work, the critical oxygen tension, which occurs when the animal respiration shifts from being independent of environmental PO2 (regulator) to being dependent on - environmental PO2 (conformer) (Pörtner and Grieshaber, 1993), occurred at 1.26 mg.L 1 . There is a controversy between authors regarding the PO2crit as a hypoxia tolerance indicator for fish. Although Scott and colleagues (2008) compared the PO2crit between species and suggested that this might not be a reliable indicator of hypoxia tolerance in fishes, McBryan and colleagues (2016) suggested that it is a reliable indicator of hypoxia tolerance once the Fundulus heteroclitus population with lower PO2crit presents higher tolerance to hypoxia. Similar results in Amazon ornamental tetras (Paracheirodon) where described by Campos and co-workers (2016) showing that species with lower PO2crit are more tolerant to hypoxia than its congeneric species. Considering the recent advances in respirometry technology, we suggest that further investigations to correlate the PO2crit values with hypoxia tolerance should be done. 18

The present work showed that A. crassipinnis presents two strategies to face hypoxia, as pointed out by Almeida-Val and colleagues (1993) for another Amazon cichlid. Herein, increased anaerobic metabolism and concomitantly depressed energy demands by decreasing the rates of oxidative metabolism, as observed by decreased oxygen consumption rates and oxidative enzyme activities during hypoxia exposure. In fact, the decrease in enzyme activities could be related with the metabolic rates decrease, resulting in a lower aerobic power and a higher anaerobic power during hypoxia in muscle, as shown by the increased anaerobic enzyme LDH activity, and the lower activities of the oxidative and glycolytic enzymes CS and PK, both in skeletal muscle and liver. The increased LDH activity in liver after 5 hours hypoxia is probably a strategy to regulate the anaerobic accumulation of pyruvate during low oxygen availability. Since LDH is the enzyme responsible for both lactate production, and its reoxidation to pyruvate, allowing the back conversion to glycogen (Hochachka and Somero, 1984), several works show that oxidative organs have the predominance of

LDH B4 isoform, which is adapted to “regulate” the reverse reaction from lactate to pyruvate and rapidly forward it back to glycogen by pursuing a high pyruvate inhibition rates (very low Km values) in order to avoid the toxicity of both pyruvate and lactate accumulation in oxidative tissues such as heart and liver (Hochachka and Somero, 1973; Almeida-Val and Val, 1993). The high levels of MDH during recovery in muscle can complement this explanation, since it increases the oxidative power capacity in Krebs cycle and supports gluconeogenesis during recovery (Almeida-Val et al., 2000; Chippari-Gomes et al., 2005). According to Chippari-Gomes and colleagues (2005) high concentrations of plasma glucose are due to hepatic glycogenolysis, while the high concentration of plasma lactate is the result of anaerobic glycolysis. These changes suggest that this species has metabolic adjustments to maintain anaerobic production of ATP while depressing oxygen consumption under hypoxia to decrease ATP demands.

In the present study we observed the liver and skeletal muscle levels of HIF-1α mRNA after different periods of fish exposure to acute hypoxia. Liver showed an increase in HIF-1α expression in fish after 3 and 5 hours under hypoxia, while these levels were rapidly reversed upon recovery. Fish white muscle, instead, showed no changes in mRNA levels of HIF-1α, even though the amount of mRNA transcripts was higher in fish muscle under all experimental groups when compared to fish liver, suggesting that the HIF-1 gene expression is tissue specific. Higher amounts of HIF- 1α transcripts in white skeletal muscle may be related to the prompt need for anaerobic metabolism during burst exercises, which are common in territorial and aggressive fish 19 species such as the Oscar. Baptista and colleagues (2016) measured HIF-1α expression in Oscar’s liver and showed that mRNA transcript levels varied during hypoxia and recovery, suggesting that up-regulation and stabilization of this gene is the mechanism by which Oscar regulates its homeostasis. Herein, we also observed that liver increases HIF-1 transcripts according to time of hypoxia exposure and decreases during recovery, while white skeletal muscle remains constant throughout all experimental procedure. This duality in the two analyzed tissues may be related to its different physiological function. In fact, white skeletal muscle consumes less oxygen than liver, due to the predominance of anaerobic metabolism in this tissue (Val and Almeida-Val, 1996). Actually, we could observe higher LDH activities in skeletal muscle, comparing with liver. Skeletal muscle’s anaerobic capacity can be related to HIF-1 protein concentration. As an anaerobic tissue, there is no need for up-regulation of HIF-1α expression once the tissue is uploaded with the peptide HIF-1 protein under a normoxic and hypoxic situations and can activate other genes regulated by post translational mechanism of HIF-1 protein (Soitamo et al., 2001). Zebrafish up-regulated HIF-1α expression after 2 hours hypoxia, suggesting that the early transcripts serve to up-regulate VEGF and EPO, both genes activated by HIF-1 protein that regulates the development and hypoxia-tolerance (Ding et al., 2013). Up-regulated HIF-1α expression in liver was also observed in Perca fluviatilis exposed to severe hypoxia, with no regulation in skeletal muscle expression (Rimoldi et al, 2012). In grass carp, a fish tolerant to hypoxia, HIF-1α was abundantly expressed in kidneys and eyes (Law et al, 2006). On the other hand, Megalobrama amblycephala showed no differences in HIF-1α expression in liver, suggesting this fish is not able to regulate glycolytic metabolism and is hypoxia-sensitive, dying after a short period of hypoxia (Shen et al, 2010). All of these findings could explain A. crassipinnis’ hability to tolerate short- and long-term periods of hypoxia once this species up-regulates HIF-1α expression in liver to control oxidative and glycolytic capacity in this organ, and keeps high levels of HIF- 1α transcripts in white skeletal muscle.

In this study, pyruvate inhibition rates indicated the activation of anaerobic metabolism (<1.0) after 3 and 5 hours of exposition to hypoxia in skeletal muscle, and 5 hours in liver. Even though the values for pyruvate inhibition were higher than 1.0 under normoxia, these values were low when compared to other fishes (Hochachka and Hulbert, 1978; Almeida-Val et al., 1991), which indicates a stronger LDH-A4 (muscle type LDH) activity in liver and skeletal muscle for A. crasspinnis. This change in inhibition ratio is related to a shift in isozymes, activating LDH-A4 in liver and muscle after 3 and 5 hours of hypoxia for skeletal muscle and 5 hours for liver. In fact, LDH-A4, 20 a skeletal muscle predominant isoform, was already described in heart, liver and brain of hypoxia tolerant and non-tolerant fishes, particularly Cichlidae (Almeida-Val et al., 1995). Val and colleagues (1998) also described the adjustment in LDH isoforms in different tissues of the Characiform rhombeus exposed to hypoxia and hyperoxia, showing the activation of LDH-A4 in heart and liver. In this study we found a correlation between pyruvate inhibition ratios (LDH L/H) and HIF-1α expression in muscle and liver. Our study reveal that HIF-1α expression is related to pyruvate inhibition rates in liver (P<0.00005) but is not related in skeletal muscle (P<0.34) (Fig 7). These results indicate that HIF-1α expression in liver is related to hypoxia responses and tolerance, once it showed to be highly related to cell survival, by keeping gycolytic pathways, under hypoxia. On the other hand, skeletal muscle may keep high levels of anaerobic metabolism constantly and, for that, HIF-1α transcripts are always at higher levels but do not change as oxygen varies in the water.

The expression of HIF-1 is highly related to animal’s life history, being species specific. Kodama and colleagues (2012) observed an increase in liver HIF-1α expression of Callionymus valenciennei and a low expression of this gene in muscle from animals collected directly from Tokyo Bay during periods of environmental hypoxia. Moreover, when testing hypoxia in controlled laboratory studies, they observed an increase in HIF-1α mRNA after seven days in the same tissues, indicating that these changes in expression are due to hypoxia exposure. The marine teleost, Atlantic croaker, also showed an increase in HIF-α transcripts in specific tissues when collected from hypoxic areas in an estuary and in a coastal region of the northern Gulf of Mexico (Thomas et al., 2007). These results are in accordance with those obtained in the present work, since A. crassipinnis lives in flooded forests, an environment with severe periods of hypoxia and, sometimes, anoxia. Further studies of this gene in A. crassipinnis collected in the field shall be done to corroborate its role in controlling hypoxia tolerance in natural habitats.

In conclusion, the present results suggest that an increase in the amount of HIF-1α transcripts is due to adaptations to environmental exposure to regular hypoxia, life history and evolution of this species, since HIF-1α expression proved to be related to oxygen demands, playing a crucial role in controlling anaerobic metabolism and being responsible by activating genes related to glycolysis, gluconeogenesis and cell survival, helping to keep the production energy and control the demand for ATP under hypoxic situations. In A. crassipinnis, the gene HIF-1α revealed to be tissue-specific. We suggest that the lack of variation in mRNA levels in skeletal muscle under hypoxia is due to this tissue’s high anaerobic capacity, since it can rely in different pathways to 21 maintain the ATP production. Thus, changes in the expression of HIF-1α are not the most important factor for this tissue, while keeping high expression levels of transcripts in all situations are more profitable. The increase in HIF-1α expression in liver of the animals exposed to hypoxia is probably related to higher energy demands and the need to activate anaerobic glycolysis for short periods. Accordingly, LDH activity in liver increased after five hours hypoxia, a late response to HIF-1α expression, when we believe there was a demand for pyruvate conversion to lactate in order to maintain liver energetic demand and thus, other tissues anaerobic metabolism. These results show HIF-1α differential expression has an important role in determining Oscar’s survival and tolerance to hypoxia.

22

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Anjos, M., Oliveira, R. R., Zuanon, J. (2008). Hypoxic environments as refuge against predatory fish in the Amazon floodplains. Brazilian Journal of Biology 68, 45-50.

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Hochachka, P. W., Hulbertm W. C. (1978). Glycogen “seas”, glycogen bodies, and glycogen granules in heart and skeletal muscle of two air-breathing, burrowing fishes. Can. J. Zool. 56, 774-786.

Hochachka, P. W., Somero, G. N. (1984). Biochemical Adaptation. Princenton University Press, Princeton.

Hochachka, P. W., Somero, G. N. (2002). Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, New York.

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Semenza, G. L., (2000). HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J. Appl. Physiol. 88, 1474–1480.

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28

FIGURES

140 MO2 80 Opercular … * 70

120

1 -

*

) 60 1 - 100

.h * 1 - 50 80 40 60 *

(mg O2.kg (mg 30 2 40 MO * 20 * h . movement Opercular 20 10

0 0 7,5 7 6,5 6 5,5 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 -1 Oxygen concentration (mg.L )

Fig 1. Opercular movement of Astronotus crassipinnis exposed to graded hypoxia (n=

7, mean ±SEM). MO2 of Astronotus crassipinnis exposed to graded hypoxia (n= 7, -1 mean ±SEM). Line shows opercular movement’s expansion (1.8 mg O2 . L ± 0.036) -1 right before PO2Crit (PO2 crit= 1.26 mg.l ± 0.038). * indicates significantly difference between each O2 concentration (P<0.05).

Normoxia 160 Hypoxia a 140 a a 1 a a

- 120 1.h - 100

80 b b b 60

40 MO2 (mg O2.kgMO2(mg 20

0 1 3 5 Recovery Time (hr)

Fig 2. Astronotus crassipinnis MO2 during experimental setup (n=8, ±SEM, T= 28.3°C). -1 Letters indicate difference between normoxia (7.42 mg O2 . L ± 0,10), hypoxia (0.70 -1 -1 mg O2 . L ± 0.05) and recovery (7.56 mg O2 . L ± 0.19) (P<0.05). 29

250 Normoxia Hypoxia b 200 b

b

1) 150 c - ac a a 100 a

Glucose(mg.dL 50

0 1 3 5 Recovery Time (hr)

Fig. 3 Plasma glucose levels in Astronotus crassipinnis exposed to normoxia (7.42 mg -1 -1 O2 . L ± 0,10) and hypoxia (0.70 mg O2 . L ± 0.05) for 5 hours and recovery (7.56 mg -1 O2 . L ± 0.19) for 3 hours (n=6 ± SEM). Letters indicate difference between time, normoxia, hypoxia and recovery (P<0.05).

70 Normoxia c 60 Hypoxia

50 1)

- bc

40 a ab 30 a a

Lactate(mg.dL 20 a a 10

0 1 3 5 Recovery Time (hr)

Fig. 4 Plasma lactate levels in Astronotus crassipinnis exposed to normoxia (7.42 mg -1 -1 O2 . L ± 0,10) and hypoxia (0.70 mg O2 . L ± 0.05) for 5 hours and recovery (7.56 mg -1 O2 . L ± 0.19) for 3 hours (n=6 ± SEM). Letters indicate difference between time, normoxia, hypoxia and recovery (P<0.05).

30

Normoxia

1,20 Hypoxia

1,00

0,80

0,60

0,40

0,20 Relative mRNA expression level expression mRNA Relative

0,00 1 h 3 h 5 h Recovery Time (hr)

Fig. 5 Relative expression of HIF-1α in skeletal muscle of Astronotus crassipinnis -1 -1 exposed to normoxia (7.42 mg O2 . L ± 0.10) and hypoxia (0.70 mg O2 . L ± 0.05) for -1 5 hours and hypoxia recovery (7.56 mg O2 . L ± 0.19) for 3 hours (n=6 ± SEM). Letters indicate difference between time, normoxia, hypoxia and recovery (P<0.05).

1,6 Normoxya c 1,4 Hypoxia 1,2

1 b

0,8 a a a 0,6 a a

0,4 a

0,2 Relative mRNA expression level expression mRNA Relative

0 1 h 3 h 5 h Recovery Time (hr)

Fig. 6 Relative expression of HIF-1α in Liver of Astronotus crassipinnis exposed to -1 -1 normoxia (7.42 mgO2 .L ± 0.10) and hypoxia (0.70 mgO2 .L ± 0.05) for 5 hours and -1 hypoxia recovery (7.56 mg O2 .L ± 0.19) for 3 hours (n=6 ± SEM). Letters indicate difference between time, normoxia, hypoxia and recovery (P<0.05). 31

2,5 R² = 0.4779 A ρ= 0.61

2

1,5

1

1α Relative 1α Expression -

HIF 0,5

0 0 0,5 1 1,5 2 2,5 LDH L/H

2,5 B R² = 0.0234

ρ= 0.34 2

1,5

1

1α Relative 1α Expression -

0,5 HIF

0 0 0,5 1 1,5 2 2,5 LDH L/H

Fig. 7 Correlates between HIF-1α expression and pyruvate inhibition rates in liver (A) and skeletal muscle (B).

32

Table 1 Real-time PCR primers used in this study.

Gene Primer sequence (5’-3’) HIF-1α (F) CTCTGGACACCAAGACCTTTCTC (R) CGTCACAATACGTGAACTTCATG 28S (F) TCGTTTGCGTTACCGCACTG (R) GGTCCAAGCCCCTAAACTTG 18S (F) AACGATGCCAACTAGCGATC (R) CGGAACCCAAAGACTTTGGT β-actin (F) CAACGTAGCACAGCTTCTCC (R) CTCCATCAGACACTCCAGTG

Table 2 Enzyme activities of lactate dehydrogenase (LDH), LDH inhibition rate (LDH 1mM/10mM), malate dehydrogenase (MDH), pyruvate - kinase (PK) and citrate synthase (CS) in skeletal muscle and liver of Astronotus crassipinnis (n=6 ± SEM) exposed to normoxia (7.42 mg O2 . L 1 -1 -1 ± 0.10) and hypoxia (0.70 mg O2 . L ± 0.05) for 5 hours and recovery (7.56 mg O2 . L ± 0.19) for 3 hours (n=6 ± SEM). * the enzyme activities were expressed as µmol.min-1.mg prot-1.Letters indicate differences between time, normoxia, hypoxia and recovery (P<0.05).

Skeletal Muscle Liver LDH 1mM LDH MDH CS PK LDH 1mM LDH MDH CS PK 1mM/10mM 1mM/10mM Normoxia 1 h 1.11 ±0.12ª 1.60 ±0.82ª 0.33 ±0.01ª 11.3 ±0.82ª 3.41 ±0.25ª 0.90 ±0.27ª 1.53 ±0.04ª 0.47 ±0.07 3.40 ±0.23ª 3.41 ±0.76ª 3 h 0.99 ±0.09ª 1.65 ±0.31a 0.40 ±0.05ª 8.89 ±1.51ª 3.37 ±0.45ª 0.77 ±0.28ª 1.59 ±0.21ª 0.42 ±0.04 3.49 ±0.50ª 3.37 ±0.66ª 5 h 0.83 ±0.08ª 1.50 ±0.12a 0.44 ±0.03ª 9.00 ±1.12ª 2.73 ±0.31ª 0.81 ±0.19ª 1.44 ±0.41a 0.47 ±0.07 3.25 ±0.43a 2.73 ±0.70a Rec 0.88 ±0.06ª 1.58 ±0.34a 0.34 ±0.01ª 8.18 ±0.29ª 2.85 ±0.22ª 0.76 ±0.19ª 1.31 ±0.10a 0.46 ±0.03 3.73 ±0.63ª 2.85 ±0.45ª

Hypoxia 1 h 1.10 ±0.15ª 1.59 ±0.02ª 0.36 ±0.05ª 6.81 ±2.13b 1.10 ±0.44b 1.08 ±0.18ª 1.18 ±0.33ª 0.45 ±0.05 3.28 ±0.54ª 1.10 ±0.07b 3 h 1.72 ±0.08b 0.88 ±0.12b 0.39 ±0.02ª 6.83 ±1.30b 1.44 ±0.37b 1.01 ±0.13ª 1.05 ±0.02ª 0.41 ±0.01 1.74 ±0.20b 1.44 ±0.18b 5 h 1.78 ±0.04b 0.46 ±0.09b 0.38 ±0.03ª 6.26 ±0.51b 1.41 ±0.18b 1.21 ±0.13b 0.80 ±0.62b 0.48 ±0.03 2.17 ±0.31b 1.41 ±0.10b Rec 0.99 ±0.01ª 0.92 ±0.54ª 0.49 ±0.03b 9.22 ±0.75a 3.85 ±0.48a 0.92 ±0.17ab 1.23 ±0.08a 0.48 ±0.01 3.91 ±0.48a 3.85 ±0.30ª

33

REFERÊNCIAS

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Almeida-Val, V. M. F., Farias, I. P., Paula-Silva, M. N., Duncan, W. P., Val, A. L. 1995. Biochemical adjustments to hypoxia by Amazon cichlids. Braz. J. Med. Biol. Res. 28, 1257-1263.

Almeida-Val, V. M. F., Val, A. L., Duncan, W. P., Souza, F. C. A., Paula-Silva, M. N., Land, S. 2000. Scaling effects on hypoxia tolerance in the Amazon fish Astronotus ocellatus (Perciformes: Cichlidae): contribution of tissue enzyme levels. Comp. Biochem. Physiol. 125 B, p. 219-226.

Anjos, M., Oliveira, R. R., Zuanon, J. 2008. Hypoxic environments as refuge against predatory fish in the Amazon floodplains. Brazilian Journal of Biology 68, 45-50.

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